Citrobacter

Zhonghua Yi Xue Za Zhi, 2004 Nov 17, 84(22), 1872 - 5
{A new member of CMY type cephalosporinase prevailing in Escherichia coli.}; Guan XZ et al.; OBJECTIVE: To study the resistant phenotype and molecular biology character of plasmid mediated high AmpC-producing clinical isolates of Escherichia coli and to find new AmpC genotype . METHODS: The cefoxitin highly resistant clinical isolates of Escherichia coli were studied by K-B method, three-dimensional method, Isoelectric Focusing (IEF) and the MIC of these strains were examined by micro-dilution method . The conjugation experiment, multiplex PCR and DNA sequencing methods were used in further study . RESULTS: Above 719 strains studied, there are 6 isolates were showed as high AmpC-producing by three-dimensional method and IEF found they could produce a beta-Lactamase which PI was 8.9 and could be inhibited by cloxacillin but not by clavulnate . The strains were resistant to most of third generation cephalosporins, but were susceptible to cefepime, meropenem and imipenem . The experiment also showed that the gene which express this AmpC like beta-Lactamase could be transferable . Multiplex PCR indicated they belong to Citrobacter freundii family . Sequencing of corresponding DNA revealed 99% identities of the deduced amino acid sequence with CMY-2 and CMY-7 respectively . It is a new CMY type cephalosporinase . CONCLUSION: A new CMY type cephalosporinase has been found in clinical strains of Escherichia coli in our hospital . It was resistant to many antibiotics and its resistance could be transferred horizontally.

J Comp Pathol, 2005 Jan, 132(1), 1 - 26
Attaching-effacing Bacteria in Animals; Wales AD et al.; Enteric bacteria with a demonstrable or potential ability to form attaching-effacing lesions, so-called attaching-effacing (AE) bacteria, have been found in the intestinal tracts of a wide variety of warm-blooded animal species, including man . In some host species, for example cattle, pigs, rabbits and human beings, attaching-effacing Escherichia coli (AEEC) have an established role as enteropathogens . In other host species, AE bacteria are of less certain significance . With continuing advances in the detection and typing of AE strains, the importance of these bacteria for many hosts is likely to become clearer . The pathogenic effects of AE bacteria result from adhesion to the intestinal mucosa by a variety of mechanisms, culminating in the formation of the characteristic intimate adhesion of the AE lesion . The ability to induce AE lesions is mediated by the co-ordinated expression of some 40 bacterial genes organized within a so-called pathogenicity island, known as the "Locus for Enterocyte Effacement" . It is also believed that the production of bacterial toxins, principally Vero toxins, is a significant virulence factor for some AEEC strains . Recent areas of research into AE bacteria include: the use of Citrobacter rodentium to model human AEEC disease; quorum-sensing mechanisms used by AEEC to modulate virulence gene expression; and the potential role of adhesion in the persistent colonization of the intestine by AE bacteria . This review of AE bacteria covers their molecular biology, their occurrence in various animal species, and the diagnosis, pathology and clinical aspects of animal diseases with which they are associated . Reference is made to human pathogens where appropriate . The focus is mainly on natural colonization and disease, but complementary experimental data are also included.

FEMS Microbiol Lett, 2005 Jan 15, 242(2), 319 - 24
Detection and characterization of beta-lactamase genes in subgingival bacteria from patients with refractory periodontitis; Handal T et al.; Fifty-three beta-lactamase-producing strains of oral bacteria isolated from patients with refractory periodontitis in Norway and USA were screened for the presence of the bla(TEM), bla(SHV), bla(OXA), bla(ampC), bla(cfxA), and bla(cepA/cblA) genes by the polymerase chain reaction (PCR) . The PCR products were characterized by direct sequencing of the amplified DNA . Thirty-four of the 53 enzyme-producing strains (64%) were positive in one of the PCR assays . All beta-lactamase-producing Prevotella and Capnocytophaga spp . were CfxA positive . TEM-type beta-lactamases were identified in one strain each of Escherichia coli and Neisseria sp., and one strain of Citrobacter freundii possessed an AmpC-type beta-lactamase . Screening for gene cassettes and genes known to be associated with integrons did not reveal the presence of integrons in these oral bacteria . Sequence analyses showed that most CfxA positive Prevotella and Capnocytophaga isolates from patients with refractory periodontitis harboured variants of the CfxA2 and CfxA3 enzyme . The present study also showed that many different genetic determinants of beta-lactamase production are found in bacteria isolated from refractory periodontitis, many of which remain to be characterized.

Eur J Immunol, 2005 Jan, 35(1), 180 - 8
Vaccine-induced protection against gastrointestinal bacterial infections in the absence of secretory antibodies; Uren TK et al.; Secretory IgA (SIgA) is widely held to be responsible for the defense of the mucosae against pathogenics and other potentially harmful agents . In this study, polymeric Ig receptor (pIgR) knockout mice, which lack secretory antibodies (SAb), were used to investigate the role of vaccine-elicited SAb in protection against gastrointestinal bacterial infections . An essential role for specific SAb in protection against Vibrio cholerae was evident from experiments showing that vaccinated pIgR(-/-) mice, but not vaccinated C57BL/6 mice, were susceptible to cholera toxin challenge . Vaccination of C57BL/6 mice with Salmonella typhimurium elicited strong antigen-specific, mucosal responses, which blocked in vitro invasion of epithelia . However, vaccinated C57BL/6 and pIgR(-/-) mice were equally resistant to challenge infection with virulent S . typhimurium . Finally, we investigated the importance of SIgA in protection against recurrent infections with Citrobacter rodentium . Although higher numbers of bacteria were detected early after challenge infection in feces of vaccinated pIgR(-/-) mice compared with vaccinated C57BL/6 mice, both mouse strains showed complete clearance after 9 days . These results suggested that, in immune animals, SIgA is crucial for the protection of gastrointestinal surfaces against secreted bacterial toxins, may inhibit early colonization by C . rodentium, but is not essential for protection against re-infection with S . typhimurium or C . rodentium.

Curr Microbiol . 2004 Nov 8; {Epub ahead of print}
Quantification of Salmonella by 5'-Nuclease Real-Time Polymerase Chain Reaction Targeted to fimC Gene; Piknova L et al.; A new 5'-nuclease real-time polymerase chain reaction (PCR) system for the quantification of Salmonella enterica was developed, with primers and the probe oriented to a Salmonella-specific region of the fimC gene . The PCR system was specific and sensitive, its inclusivity was 100% (determined by the analysis of 53 strains of Salmonella belonging to 38 serovars) and its exclusivity was 100% (determined by the analysis of 49 non-Salmonella strains) . For quantification purposes, calibration lines were constructed for three Salmonella strains belonging to three serotypes . These calibration lines were linear (r >/= 0.99) in the range from 10(3) to 10(7 )CFU/mL and practically identical in terms of very similar slopes and x-intercepts . Escherichia coli (10(6 )CFU/mL) and Citrobacter freundii (10(6) CFU/mL) had no effect on Salmonella quantification by the system.

J Clin Microbiol, 2004 Dec, 42(12), 5923 - 4
Histidine decarboxylase in Enterobacteriaceae revisited; Wauters G et al.; With a modification of Taylor's decarboxylation broth, histidine decarboxylase was detected in Enterobacter aerogenes, Morganella morganii, Raoultella ornithinolytica, and some strains of Citrobacter youngae and Raoultella planticola . This method provides a useful confirmatory test for identification of E . aerogenes strains.

Biotechnol Lett, 2004 Aug, 26(16), 1307 - 11
Improved borate method for the rapid distinction of glucosamine and galactosamine in an exopolysaccharide produced by Citrobacter sp; Hia HC et al.; An improved borate method for the quantitative distinction of glucosamine (GlcN) and galactosamine (GalN) in a mixture is presented which is based on the Elson-Morgan method with addition of sodium borate to differentiate colour formation by the two hexosamines . The r2 value and maximum deviation of the method based on calculations derived in this study were 0.9979 and 5.1 %, respectively . Using this method, the GlcN/GalN ratio in an exopolysaccharide (EPS) produced by Citrobacter sp . was found to change with time during the production process, with a maximum value at 9.8:1.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4589 - 96
In vitro and in vivo activities of novel 6-methylidene penems as beta-lactamase inhibitors; Weiss WJ et al.; Novel penem molecules with heterocycle substitutions at the 6 position via a methylidene linkage were investigated for their activities and efficacy as beta-lactamase inhibitors . The concentrations of these molecules that resulted in 50% inhibition of enzyme activity were 0.4 to 3.1 nM for the TEM-1 enzyme, 7.8 to 72 nM for Imi-1, 1.5 to 4.8 nM for AmpC, and 14 to 260 nM for a CcrA metalloenzyme . All the inhibitors were more stable than imipenem against hydrolysis by hog and human dehydropeptidases . Piperacillin was combined with a constant 4-microg/ml concentration of each inhibitor for MIC determinations . The combinations reduced piperacillin MICs by 2- to 32-fold for extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Klebsiella pneumoniae strains . The MICs for piperacillin-resistant (MIC of piperacillin, >64 microg/ml) strains of Enterobacter spp., Citrobacter spp., and Serratia spp . were reduced to the level of susceptibility (MIC of piperacillin, < or =16 microg/ml) when the drug was combined with 4, 2, or 1 microg of these penem inhibitors/ml . Protection against acute lethal bacterial infections with class A and C beta-lactamase- and ESBL-producing organisms in mice was also demonstrated with piperacillin plus inhibitor . Median effective doses were reduced by approximately two- to eightfold compared to those of piperacillin alone when the drug was combined with the various inhibitors at a 4:1 ratio . Pharmacokinetic analysis after intravenous administration of the various inhibitors showed mean residence times of 0.1 to 0.5 h, clearance rates of 15 to 81 ml/min/kg, and volumes of distribution between 0.4 and 2.5 liters/kg . The novel methylidene penem molecules inhibit both class A and class C enzymes and warrant further investigation for potential as therapeutic agents when used in combination with a beta-lactam antibiotic.

J Food Prot, 2004 Nov, 67(11), 2613 - 6
Identification of Enterobacteriaceae from washed and unwashed commercial shell eggs; Musgrove MT et al.; To evaluate the effect of processing on the safety and quality of retail shell eggs, a storage study was conducted with unwashed and commercially washed eggs . This work demonstrated that commercial processing decreased microbial contamination of eggshells . To know which species persisted during storage on washed or unwashed eggs, Enterobacteriaceae isolates were selected and identified biochemically . For each of three replications, shell eggs were purchased from a commercial processing plant, transported back to the laboratory, and stored at 4 degrees C . Once a week for 6 weeks, 12 eggs for each treatment (washed and unwashed control) were rinsed in sterile phosphate-buffered saline . A 1-ml aliquot of each sample was plated onto violet red bile glucose agar with overlay and incubated at 37 degrees C for 24 h . Following incubation, plates were observed for colonies characteristic of the family Enterobacteriaceae . A maximum of 10 isolates per positive sample were streaked for isolation before being identified to the genus or species level using commercially available biochemical strips . Although most of the isolates from the unwashed control eggs belonged to the genera Escherichia or Enterobacter, many other genera and species were identified . These included Citrobacter, Klebsiella, Kluyvera, Pantoea, Providencia, Rahnella, Salmonella, Serratia, and Yersinia . Non-Enterobacteriaceae also recovered from the unwashed egg samples included Xanthomonas and Flavimonas . Very few washed egg samples were contaminated with any of these bacteria . These data provide useful information on the effectiveness of processing in removing microorganisms from commercial shell eggs.

Mol Nutr Food Res, 2004 Dec, 48(7), 522 - 31
Occurrence of antibiotic-resistant enterobacteria in agricultural foodstuffs; Boehme S et al.; Antibiotic-resistant bacteria or their corresponding resistance determinants are known to spread from animals to humans via the food chain . We screened 20 vegetable foods for antibiotic-resistant coliform bacteria and enterococci . Isolates were directly selected on antibiotic-containing selective agar (color detection) . Thirteen "common vegetables" (tomato, mushrooms, salad) possessed 10(4)-10(7) cfu/g vegetable of coliform bacteria including only few antibiotic-resistant variants (0-10(5) cfu/g) . All seven sprout samples showed a some orders of magnitude higher contamination with coliform bacteria (10(7)-10(9) cfu/g) including a remarkable amount of resistant isolates (up to 10(7) cfu/g) . Multiple resistances (up to 9) in single isolates were more common in sprout isolates . Resistant bacteria did not originate from sprout seeds . The most common genera among 92 isolates were: 25 Enterobacter spp . (19 E . cloacae), 22 Citrobacter spp . (8 C . freundii), and 21 Klebsiella spp . (9 K . pneumoniae) . Most common resistance phenotypes were: tetracycline (43%), streptomycin (37%), kanamycin (26%), chloramphenicol (29%), co-trimoxazol (9%), and gentamicin (4%) . The four gentamicin-resistant isolates were investigated in molecular details . Only three (chloramphenicol) resistant, typical plant-associated enterococci were isolated from overnight enrichment cultures . In conclusion, a contribution of sprouts contaminated with multiresistant, Gram-negative enterobacteria to a common gene pool among human commensal and pathogenic bacteria cannot be excluded.

J Biol Chem . 2004 Nov 8; {Epub ahead of print}
Targeting of enteropathogenic Escherichia coli EspF to host mitochondria is essential for the bacterial pathogenesis: critical role of the 16th leucine residue in EspF; Nagai T et al.; The attachment of enteropathogenic Escherichia coli (EPEC) to host cells and the induction of attaching and effacing (A/E) lesions are prominent pathogenic features . EPEC infection also leads to host cell death and damage to the intestinal mucosa, which is partly dependent upon EspF, one of the effectors . In this study, we demonstrate that EspF is a mitochondrial import protein with a functional mitochondrial targeting signal (MTS), since the EspF activity to import into mitochondria was abrogated by MTS deletion mutants . Substitution of the 16th leucine with glutamic acid (EspF(L16E)) completely abolished the EspF activity . Infection of HeLa cells with wild type but not the espF mutant (DespF) decreased mitochondrial membrane potential (DYm), leading to cell death . The DYm decrease and cell death were restored in cells infected with DespF/pEspF but not DespF/pEspF(L16E), suggesting the 16th leucine in the MTS to be a critical amino acid for EspF function . To demonstrate the impact of EspF in vivo, we exploited Citrobacter rodentium by infecting C3H/HeJ mice with DespFCR, DespFCR/pEspFCR or DespFCR/pEspF(L16E)CR . The results indicate that EspF activity contributes to bacterial pathogenesis, as judged by murine lethality and intestinal histopathology, and promotion of bacterial colonization of the intestinal mucosa.

Biochemistry, 2004 Nov 16, 43(45), 14412 - 9
Role of lysine-256 in Citrobacter freundii tyrosine phenol-lyase in monovalent cation activation; Phillips RS et al.; Tyrosine phenol-lyase (TPL) from Citrobacter freundii is dependent on monovalent cations, K(+) or NH(4)(+), for high activity . We have shown previously that Glu-69, which is a ligand to the bound cation, is important in monovalent cation binding and activation {Sundararaju, B., Chen, H., Shillcutt, S., and Phillips, R . S . (2000) Biochemistry 39, 8546-8555} . Lys-256 is located in the monovalent cation binding site of TPL, where it forms a hydrogen bond with a structural water bound to the cation . This lysine residue is highly conserved in sequences of TPL and the paralogue, tryptophan indole-lyase . We have now prepared K256A, K256H, K256R, and E69D/K256R mutant TPLs to probe the role of Lys-256 in monovalent cation binding and activation . K256A and K256H TPLs have low activity (k(cat)/K(m) values of 0.01-0.1%), are not activated by monovalent cations, and do not exhibit fluorescence emission at 500 nm from the PLP cofactor . In contrast, K256R TPL has higher activity (k(cat)/K(m) about 10% of wild-type TPL), is activated by K(+), and exhibits fluorescence emission from the PLP cofactor . K256A, K256H, and K256R TPLs bind PLP somewhat weaker than wild-type TPL . E69D/K256R TPL was prepared to determine if the guanidine side chain could substitute for the monovalent cation . This mutant TPL has wild-type activity with S-Et-L-Cys or S-(o-nitrophenyl)-L-Cys but has no detectable activity with L-Tyr . E69D/K256R TPL is not activated by monovalent cations and does not show PLP fluorescence . In contrast to wild-type and other mutant TPLs, PLP binding to E69D/K256R is very slow, requiring several hours of incubation to obtain 1 mol of PLP per subunit . Thus, E69D/K256R TPL appears to have altered dynamics . All of the mutant TPLs react with inhibitors, L-Ala, L-Met, and L-Phe, to form equilibrating mixtures of external aldimine and quinonoid intermediates . Thus, Lys-256 is not the base which removes the alpha-proton during catalysis . The results show that the function of Lys-256 in TPL is in monovalent cation binding and activation.

J Clin Microbiol, 2004 Nov, 42(11), 5368 - 70
Identification and phylogeny of Enterobacter sakazakii relative to Enterobacter and Citrobacter Species; Iversen C et al.; The phylogenetic relationships of Enterobacter sakazakii strains were investigated using 16S ribosomal DNA (rDNA) and hsp60 sequencing . Each analysis distributed E . sakazakii strains among four clusters, indicating substantial taxonomic heterogeneity . The E . sakazakii type strain 16S rDNA sequence was 97.8% similar to that of Citrobacter koseri but 97.0% similar to that of Enterobacter cloacae.

Int J Food Microbiol, 2004 Dec 1, 97(1), 63 - 9
Antimicrobial effect of water extract of sumac (Rhus coriaria L.) on the growth of some food borne bacteria including pathogens; Nasar-Abbas SM et al.; The antimicrobial effect of water extracts of sumac (Rhus coriaria L.) at concentrations of 0.1%, 0.5%, 1.0%, 2.5% and 5.0% (w/v), non-neutralized and after neutralization to pH 7.2+/-0.1, was studied on the growth of 12 bacterial strains (six Gram positive strains and six Gram negative strains), mostly food borne including pathogens . It was found to be effective against all the test organisms with Gram positive strains being more sensitive than Gram negative strains . Significant differences (P<0.01) were found among the bacteria and between the non-neutralized and neutralized extracts with non-neutralized being more effective against all the bacteria . The minimal inhibitory concentration (MIC) of the extract for each bacterial strain was studied by a gradient plate method . Among the Gram positive organisms, Bacillus species (Bacillus cereus, Bacillus megaterium, Bacillus subtilis, and Bacillus thuringiensis) were found to be the most sensitive showing MICs of 0.25-0.32% (after 24 h incubation) followed by Staphylococcus aureus (0.49%), while Listeria monocytogenes was found to be the least sensitive demonstrating a MIC of 0.67% . Of the Gram negative organisms, Salmonella enteritidis was found to be the most resistant with a MIC of 0.67% followed by Escherichia coli Type I, E . coli O157:H7, Proteus vulgaris and Hafnia alvei having MICs of 0.63%, 0.60%, 0.55% and 0.45%, respectively; whereas Citrobacter freundii was found to be the least resistant surviving up to 0.42% . Some loss of antimicrobial activity was, however, observed after incubation for 3 days . Bacteriostatic/bactericidal effects of sumac, as studied by enumerating survival by the viable count technique after 1 h direct contact of each microorganism with various concentrations of sumac extract, revealed a 4-5 log cycle reduction in Bacillus spp . and 2-3 log cycle reduction in other bacteria tested with 1.0% sumac extract.

Drug Metabol Drug Interact, 2004, 20(3), 185 - 91
Detection of ticarcillin-clavulonic acid susceptibility with microdilution method in Citrobacter, Hafnia, Proteus and some gram negative bacteria; Uraz G et al.; The broth dilution method has been regarded as a good alternative test for detection of susceptibilities to antimicrobial agents . In this study, the antimicrobial activity of ticarcillin-clavulonic acid (TIM) was investigated by the minimal inhibitory concentration (MIC) method on strains of Aeromonas, Citrobacter, Hafnia, Morganella, Proteus, Pseudomonas and gram negative bacteria isolated from raw milk . The isolate collection included 91 gram negative strains . Fifty-one (56.04%) isolates were found sensitive (MIC < or = 8 microg/ml), 12 (13.19%) isolates were found intermediately sensitive (MIC 16-32 microg/ml), and 28 (30.77%) isolates were found resistant (MIC > or = 64 microg/ml) to TIM.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4435 - 7
Community-onset disease caused by Citrobacter freundii producing a novel CTX-M beta-lactamase, CTX-M-30, in Canada; Abdalhamid B et al.; Strains of Citrobacter freundii intermediate to cefotaxime but sensitive to ceftazidime were isolated from four different patients in Canada . Sequencing of PCR products by use of CTX-M-specific primers revealed a new combination of four amino acid substitutions . This new gene was designated bla(CTX-M-30) and was encoded on a 3-kb plasmid . The pI of CTX-M-30 was 8.0.

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4136 - 43
Four variants of the Citrobacter freundii AmpC-Type cephalosporinases, including novel enzymes CMY-14 and CMY-15, in a Proteus mirabilis clone widespread in Poland; Literacka E et al.; Twenty-nine Proteus mirabilis isolates from 17 Polish hospitals were analyzed . The isolates were resistant to a variety of antimicrobials, and their patterns of resistance to beta-lactams resembled those of the constitutive class C cephalosporinase (AmpC) producers . Indeed, beta-lactamases with a pI of approximately 9.0 were found in all of the isolates, and they were subsequently identified as four AmpC-type cephalosporinases, CMY-4, -12, -14, and -15, of which the two last ones were novel enzyme variants . The enzymes were of Citrobacter freundii origin and were closely related to each other, with CMY-4 likely being the evolutionary precursor of the remaining ones . The bla(CMY) genes were located exclusively in chromosomal DNA, within EcoRI restriction fragments of the same size of approximately 10 kb . In the CMY-12- and -15-producing isolates, an additional fragment of approximately 4.5 kb hybridized with the bla(CMY) probe as well, which could have arisen from a duplication event during the evolution of the genes . In all of the isolates, the ISEcp1 mobile element, which most probably is involved in mobilization of the C . freundii ampC gene, was placed at the same distance from the 5' ends of the bla(CMY) genes, and sequences located between them were identical in isolates carrying each of the four genes . These data suggested that a single chromosome-to-chromosome transfer of the ampC gene from C . freundii to P . mirabilis could have initiated the spread and evolution of the AmpC-producing P . mirabilis in Poland . The hypothesis seems to be confirmed by pulsed-field gel electrophoresis typing, which revealed several cases of close relatedness between the P . mirabilis isolates from distant centers and showed an overall similarity between the majority of the multiresistant isolates.

Rev Assoc Med Bras, 2004 Jul-Sep, 50(3), 268 - 71 Epub 2004 Oct 21.
{Effects of splenectomy on peritonitis produced by a colonic injury: study in rats}; Nassif LS et al.; BACKGROUND: Study the effects of splenectomy on the intra-abdominal infection by bowel flora, consequent to a colonic injury in Wistar rats . METHODS: We used 64 animals, 20 for Group A1 (normal with colon lesion left open), 22 for Group A2 (normal with colon lesion sutured) and 22 for Group B (spleen removed) . The animals were submitted to a laparatomy through a midline abdominal incision and peritonitis was induced by a colonic lesion in the colon previously distended with 2 ml of saline introduced in the rectum . Bacteriological studies of the abdominal wash obtained with a sterilized swab and microscopic studies of samples of the segment of the sutured colon obtained at 48 hrs, 96 hrs and on the 12 post op day, were made in each group . All rats were submitted to an autopsy on the day of death or on the 12th post op day when the survivors were sacrificed . RESULTS: Similar bacteria were found in the three groups . E . coli (100%); Enterococcus fecalis (97%); Klebsiela pneumoniae (70%); Citrobacter freundi (70%) and Enterobacter aglomerans (63%) . In the first 96 hours the rats without spleen had a lesser inflammatory reaction when compared to the group with spleen . The leading cause of death was generalized peritonitis in the first 96 hours . There was a significant statistical difference in the mortality rate between Group B (80%), Group A2 (no mortality) and Group A1 (35%) . CONCLUSION: There was a significant statistical difference in the mortality rate caused by peritonitis between the groups with splenectomy when compared to the group with no splenectomy.

Int J Med Microbiol, 2004 Sep, 294(2-3), 115 - 21
Shiga toxin-encoding bacteriophages--genomes in motion; Herold S et al.; Shiga toxins (Stx) represent a group of bacterial toxins that are involved in human and animal disease . Stx are mainly produced by Escherichia coli isolated from human and non-human sources, Shigella dysenteriae type 1, and sporadically, by Citrobacter freundii, Enterobacter cloacae and Shigella flexneri . The genes encoding Stx are encoded in the genome of heterogeneous lambdoid prophages (Stx-converting bacteriophages; Stx-phages) . They are located in a similar position in the late region of the prophage genome and stx is under control of phage genes . Therefore, induction of Stx-converting prophages triggers increased production of Stx . Following induction, Stx-phages can infect other bacteria in vivo and in vitro . Stx-phages may be considered to represent highly mobile genetic elements that play an important role in the expression of Stx, in horizontal gene transfer, and hence in genome diversification.

Biochemistry, 2004 Oct 19, 43(41), 13037 - 45
Phosphoenolpyruvate- and ATP-dependent dihydroxyacetone kinases: covalent substrate-binding and kinetic mechanism; Garcia-Alles LF et al.; Dihydroxyacetone (Dha) kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants, and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system (PTS) in most bacteria . Here, we compare the PTS-dependent kinase of Escherichia coli and the ATP-dependent kinase of Citrobacter freundii . They display 30% sequence identity . The binding constants of the E . coli kinase for eleven short-chain carbonyl compounds were determined by acetone precipitation of the enzyme-substrate complexes . They are 3.4 microM for Dha, 780 microM for Dha-phosphate (DhaP), 50 microM for D,L-glyceraldehyde (GA), and 90 microM for D,L-glyceraldehyde-3-phosphate . The k(cat) for Dha of the PTS-dependent kinase is 290 min(-1), and that of the ATP-dependent kinase is 1050 min(-1) . The Km for Dha of both kinases is <6 microM . The X-ray structures of the enzyme-GA and the enzyme-DhaP complex show that substrates as well as products are bound in hemiaminal linkage to an active-site histidine . Quantum-mechanical calculations offer no indication for activation of the reacting hydroxyl group by the formation of the hemiaminal . However, the formation of the hemiaminal bond allows selection for short-chain carbonyl compounds and discrimination against structurally similar polyols . The Dha kinase remains fully active in the presence of 2 M glycerol, and phosphorylates trace impurities of carbonyl compounds present in glycerol.

Comp Biochem Physiol A Mol Integr Physiol, 2004 Sep, 139(1), 65 - 9
Digestion of cellulose and xylan by symbiotic bacteria in the intestine of the Indian flying fox (Pteropus giganteus); Prem Anand AA et al.; Bats (Order Chiroptera) are a widely distributed group of mammals . Pteropus giganteus belongs to the Suborder Megachiroptera . This bat consumes fruits and leaves as their major food . Cellulose and xylan are the major composition of leaves . As they consume leaves in their diet, their digestive tract must contain cellulolytic and xylanolytic bacteria which help in the digestion of cellulose and xylan . The cellulolytic and xylanolytic bacteria were isolated and screened on Berg's agar containing cellulose and xylan . The bacteria isolated were characterized biochemically and found to be Proteus vulgaris, Proteus mirabilis, Citrobacter freundii, Serratia liquefaciens and Klebsiella oxytoca . These bacteria help in digestion of cellulose and xylan in the diet of the bat, P . giganteus . Here we show that leaves are also used as a carbohydrate source by these bats . An insectivorous bat, Hipposideros fulvus, was used as a control and does not possess cellulolytic and xylanolytic bacteria.

Indian J Pathol Microbiol, 2004 Jan, 47(1), 82 - 4
Correlation of extended spectrum beta lactamases production with cephalosporin resistance in gram negative bacilli; Ghatole M et al.; Beta lactamase production is an important mechanism of developing resistance to beta lactam group of antibiotics . Cephlosporins with extended spectrum of activity and stability were introduced to overcome this resistance, but soon production of extended spectrum beta lactamases (ESBLs), which are inducible in nature was reported . In this study Klebsiella aerogenes--166, Escherichia coli--120, Citrobacter spps--116, Pseudomonas spps--50, Proteus spps--32 and S . typhi--16, strains were subjected to sensitivity testing against various generations of cephalosporins by disc diffusion method and for the production of ESBLs using disc approximation method . Klebsiella aerogenes, Escherichia coli, Citrobacter spps and Pseudomonas spps showed statistically significant difference in the resistance pattern to all three generations of cephalosporins and ESBLs production.

J Immunol, 2004 Oct 15, 173(8), 5171 - 9
Impaired immunity to intestinal bacterial infection in stromelysin-1 (matrix metalloproteinase-3)-deficient mice; Li CK et al.; Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease . Matrix metalloproteinases (MMPs) have been shown to mediate matrix remodeling and cell migration during tissue injury and repair in the intestine . We have previously shown enhanced pathology in infected TNFRp55-/-, IL-12p40-/-, and IFN-gamma-/- mice, and here we show that this is associated with an increase in stromelysin-1 (MMP3) transcripts in colonic tissues . We have therefore investigated the role of MMP3 in colonic mucosal hyperplasia and the local Th1 responses using MMP3-/- mice . In MMP3-/- mice, similar mucosal thickening was observed after infection as in wild-type (WT) mice . Colonic tissues from MMP3-/- mice showed a compensatory increase in the expression of other MMP transcripts, such as MMP7 and MMP12 . However, MMP3-/- mice showed delayed clearance of bacteria and delayed appearance of CD4+ T lymphocytes into intestinal lamina propria . CSFE-labeled mesenteric lymph node CD4+ T lymphocytes from infected WT mice migrated in fewer numbers into the mesenteric lymph nodes and colon of MMP3-/- mice than into those of WT mice . These studies show that mucosal remodeling can occur in the absence of MMP3, but that MMP3 plays a role in the migration of CD4+ T lymphocytes to the intestinal mucosa.

Comp Biochem Physiol C Toxicol Pharmacol, 2004 Jun, 138(2), 139 - 48
Bacterial infection and tissue-specific Hsp72, -73 and -90 expression in western painted turtles; Ramaglia V et al.; Heat shock proteins (Hsps) are molecular chaperones that assist intracellular folding, assembly and translocation of proteins in prokaryotic and eukaryotic cells . A variety of stresses including hyperthermia, radiation, heavy metals, ischemia, anoxia and reoxygenation have been shown to increase the expression of Hsps . Likewise, bacterial infection represents a stress for the host cell . In this study, expression of the constitutive (Hsp73) and inducible (Hsp72) isoforms of Hsp70 and Hsp90 was monitored in brain, heart, liver and skeletal muscle from the western painted turtle Chrysemys picta bellii diagnosed with Septicemic Cutaneous Ulcerative Dermatitis (SCUD) . This disease is caused by a gram-negative bacterium probably belonging to the Citrobacter spp . The expression of Hsp73 increased 1.8-fold in brain and liver, 2.2-fold in heart but did not change in skeletal muscle; Hsp72 expression increased 5.5-fold in brain and 3-fold in liver but did not change in heart or skeletal muscle; Hsp90 expression increased 9-fold in brain, 2.7-fold in heart and 2.4-fold in skeletal muscle but did not change in liver . These results suggest a tissue-specific Hsp response during bacterial infection and a role for Hsps in immunopathological events in reptiles.

Adv Perit Dial, 2004, 20, 52 - 7
Changes in the organisms of resistant peritonitis in patients on continuous ambulatory peritoneal dialysis; Nakamoto H et al.; Peritonitis is one of the most serious complications of continuous ambulatory peritoneal dialysis (CAPD) . Approximately 20% of peritonitis infections have been reported to be resistant to initial therapy, either failing to resolve with appropriate antibiotics or relapsing within 2 weeks of antibiotic discontinuation . To investigate the current situation with regard to resistant peritonitis, we retrospectively examined the incidence and the organisms of resistant peritonitis in our unit over a period of 9 years . From January 1, 1994, to January 1, 2003, we introduced 325 patients onto CAPD . At January 1, 2003, we followed up 172 patients who were receiving CAPD in our unit . During 1994-1996, 12 cases of peritonitis and 3 cases of resistant peritonitis (25%) occurred among our patients . Microbiologic examination revealed that the organisms involved in peritonitis were alpha-streptococcus (17%), coagulase-negative staphylococcus {CNS (17%)}, and methicillin-sensitive Staphylococcus aureus {MSSA (17%)} . The organisms of resistant peritonitis were methicillin-resistant S . aureus {MRSA (67%)} and methicillin-resistant CNS (33%) . During 1997-1999, 39 cases of peritonitis and 13 cases of resistant peritonitis (33%) occurred among our patients . The most frequently cultured organisms in peritonitis were CNS (26%), Candida species (13%), and alpha-streptococcus (10%) . The organisms of resistant peritonitis were methicillin-resistant CNS (46%) and Candida species (38%) . In the most recent 3-year period (2000-2002), our patients experienced 57 cases of peritonitis and 24 cases of resistant peritonitis (42%) . The organisms of peritonitis were alpha-streptococcus (46%), CNS, MSSA, Escherichia coli, and Candida species (38%) . However, the organisms of resistant peritonitis were methicillin-resistant CNS (13%), Candida species (21%), Pseudomonas aeruginosa (13%), Serratia (13%), Citrobacter (13%), and Corynebacterium (13%) . In the last several years, methicillin-resistant CNS has come to be main organism of resistant peritonitis . In addition, opportunistic infection has become a serious peritonitis problem . Given these data, we conclude that the incidence of resistant peritonitis is increasing and that the organisms of resistant peritonitis are changing.

J Bacteriol, 2004 Oct, 186(19), 6536 - 43
Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens; Samuel G et al.; Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E . coli O55 and S . enterica O50 . The O-antigen gene cluster sequences for E . coli O157 and E . coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages . We determined sequences for S . enterica O30 and C . freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E . coli O157 cluster . We also determined the sequence of the S . enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E . coli O55 cluster . For both the S . enterica O30-C . freundii F90-E . coli O157 group and the S . enterica O50-E . coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations . The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes . Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis . We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.

Int J Food Microbiol, 2004 Nov 1, 96(2), 133 - 9
A selective differential medium for Enterobacter sakazakii, a preliminary study; Iversen C et al.; Enterobacter sakazakii can cause fatal invasive infection of neonates associated with the presence of this organism in powdered infant milk formula . A new chromogenic medium (Druggan-Forsythe-Iversen agar, DFI) is described for the selective detection of this emergent pathogen . The medium is based on the alpha-glucosidase reaction which is detected using 5-bromo-4-chloro-3-indolyl-alpha,D-glucopyranoside (XalphaGlc) . Ent . sakazakii hydrolyses this substrate to an indigo pigment, producing blue-green colonies on this medium . DFI was compared with the current method of detection on violet red bile glucose agar (VRBGA) followed by pigment production on tryptone soy agar (TSA) after 48-72 h at 25 degrees C and subsequent biochemical profile determination using Biomerieux API20E . Ninety-five clinical and food strains of Ent . sakazakii were detected on the DFI chromogenic medium 2 days sooner than the alternative method . The characteristics of 148 strains representing 17 genera of non-Ent . sakazakii Enterobacteriaceae were compared using the two methods . Only 16/18 Escherichia vulneris strains, 2/3 strains of Pantoea spp . and 1/8 Citrobacter koseri strains gave false positive results on DFI agar . Eight alpha-glucosidase positive strains were identified as Pantoea using their API20E biochemical profile, but had higher percentage identification as Ent . sakazakii using ID32E . Therefore the DFI medium enables the detection of Ent . sakazakii within mixed cultures of Enterobacteriaceae, whereas the organism could be missed when using VRBGA since the latter is a general Enterobacteriaceae selective medium . In addition, the common use of API20E to check yellow pigmented colonies on TSA may lead to false negative results and consequently the acceptance of a batch of infant formula milk (IFM) that contains Ent . sakazakii.

Vet Rec, 2004 Aug 7, 155(6), 169 - 74
Digestive pathology of sea turtles stranded in the Canary Islands between 1993 and 2001; Oros J et al.; Digestive lesions were observed in 84 of 136 sea turtles (128 Caretta caretta, four Chelonia mydas and four Dermochelys coriacea) stranded in the Canary Islands between January 1993 and December 2001 . In the oral cavity ulcerative and necropurulent stomatitis were the most frequently observed lesions, and in the oesophagus ulcerative and fibrinous oesophagitis, and traumatic oesophageal perforation were most frequently observed; all these lesions were mainly associated with the ingestion of fishing hooks . Different histological types of gastritis were observed in 35 of the turtles; necropurulent and fibrinous gastritis were associated with bacterial infections caused mainly by Proteus species, Vibrio alginolyticus, and Staphylococcus species, and larval nematodes of the genus Anisakis were responsible for a form of parasitic gastritis observed in 16 of the turtles . Different histological types of enteritis, including catarrhal, fibrinous, necropurulent and necrotising enteritis, affected 36 turtles; a wide range of gram-negative and gram-positive bacteria, including Bacillus species, Escherichia coli, Pasteurella species, Proteus species, Staphylococcus species, Streptococcus species and V . alginolyticus, were isolated from these lesions . All the cases of necrotising enteritis were associated with intestinal intussusception caused by the ingestion of monofilament fishing lines . Necrotising and/or multifocal granulomatous hepatitis were the lesions most commonly observed in the liver; they affected 29 of the turtles and were associated with Aeromonas hydrophila, Citrobacter species, E . coli, Proteus species, Staphylococcus species and V . alginolyticus infections . According to the stranding reports and the gross and histological lesions observed, 33 of the turtles had digestive lesions associated with the ingestion of hooks and monofilament lines, and two had lesions associated with the ingestion of crude oil.

J Microbiol, 2004 Jun, 42(2), 139 - 42
Isolation of Citrobacter sp . mutants defective in decolorization of brilliant green by transposon mutagenesis; Jang MS et al.; To identify genes involved in the decolorization of brilliant green, we isolated random mutants generated by transposon insertion in brilliant green-decolorizing bacterium, Citrobacter sp . The resulting mutant bank yielded 19 mutants with a complete defect in terms of the brilliant green color removing ability . Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants and these mutants appeared to have insertions at different sites of the chromosome . Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined . By comparing these with a sequence database, putative protein products encoded by bg genes were identified as follows: bg 3 as a LysR-type regulatory protein; bg 11 as a MalG protein in the maltose transport system; bg 14 as an oxidoreductase; and bg 17 as an ABC transporter . The sequences deduced from the three bg genes, bg 2, bg 7 and bg 16, showed no significant similarity to any protein with a known function, suggesting that these three bg genes may encode unidentified proteins responsible for the decolorization of brilliant green.

Cell Microbiol, 2004 Oct, 6(10), 963 - 72
Organ specificity, colonization and clearance dynamics in vivo following oral challenges with the murine pathogen Citrobacter rodentium; Wiles S et al.; Citrobacter rodentium belongs to a family of human and animal enteric pathogens that includes the clinically significant enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E . coli (EPEC) . These pathogens use attaching and effacing (A/E) lesions to colonize the host gastrointestinal tract . In this study we have used bioluminescence imaging (BLI) to investigate the organ specificity, dynamics of colonization and clearance of mice by C . rodentium in situ and in real time . The bioluminescent C . rodentium derivative, strain ICC180, expresses the luxCDABE operon from the entemopathogenic nematode symbiont Photorhabdus luminescens and light levels accurately reflect bacterial numbers both in vitro and in vivo . We have demonstrated that primary colonization of the mouse by C . rodentium takes place within the caecum, specifically within the specialized patch of lymphoid tissue known as the caecal patch . Following colonization of the caecum C . rodentium established a colonic infection . Clearance of C . rodentium ICC180 parallels the colonization dynamics, i.e . the caecum was first to be cleared followed by the colon . A bioluminescent eae (encoding the outer membrane adhesin intimin) C . rodentium mutant failed to establish long-term colonization, although low levels of bacteria could be recovered for up to 3 days post challenge from the caecum.

Appl Microbiol Biotechnol, 2004 Nov, 65(6), 678 - 85 Epub 2004 Aug 20.
Isolation and characterisation of bacterial strains containing enantioselective DMSO reductase activity: application to the kinetic resolution of racemic sulfoxides; Luckarift HR et al.; The kinetic resolution of racemic sulfoxides by dimethyl sulfoxide (DMSO) reductases was investigated with a range of microorganisms . Three bacterial isolates (provisionally identified as Citrobacter braakii, Klebsiella sp . and Serratia sp.) expressing DMSO reductase activity were isolated from environmental samples by anaerobic enrichment with DMSO as terminal electron acceptor . The organisms reduced a diverse range of racemic sulfoxides to yield either residual enantiomer depending upon the strain used . C . braakii DMSO-11 exhibited wide substrate specificity that included dialkyl, diaryl and alkylaryl sulfoxides, and was unique in its ability to reduce the thiosulfinate 1,4-dihydrobenzo-2, 3-dithian-2-oxide . DMSO reductase was purified from the periplasmic fraction of C . braakii DMSO-11 and was used to demonstrate unequivocally that the DMSO reductase was responsible for enantiospecific reductive resolution of racemic sulfoxides.

Infect Immun, 2004 Sep, 72(9), 5298 - 307
A Salmonella fim homologue in Citrobacter freundii mediates invasion in vitro and crossing of the blood-brain barrier in the rat pup model; Hess P et al.; From the invasive Citrobacter freundii strain 3009, an invasion determinant was cloned, sequenced, and expressed . Sequence analysis of the determinant showed high homology with the fim determinant from Salmonella enterica serovar Typhimurium . The genes of the invasion determinant directed invasion of recombinant Escherichia coli K-12 strains into human epithelial cell lines of the bladder and gut as well as mannose-sensitive yeast agglutination and were termed fim(Cf) genes . Expression of the Fim(Cf) proteins was shown by (35)S labeling and/or Western blotting . In the infant rat model of experimental hematogenous meningitis, C . freundii strain 3009 and the in vitro invasive recombinant E . coli K-12 strain harboring the fim(Cf) determinant reached the cerebrospinal fluid, in contrast to the case for the control strain . The fim determinant was also necessary for efficient in vitro invasion by C . freundii, because a deletion mutant was strongly reduced in its invasion efficiency . The mutation could be complemented in trans by the corresponding genes . Invasion by C . freundii could be blocked only by d-mannose, GlcNAc, and chitin hydrolysate and not by other carbohydrates tested . In contrast, yeast agglutination was not affected by GlcNAc or chitin hydrolysate . This finding indicated mannose residues to be essential for both yeast agglutination and invasion, whereas GlcNAc (oligomer) residues of host cells are involved exclusively in invasion . These results showed the fim determinant of C . freundii to be responsible for d-mannose- and GlcNAc-dependent in vitro invasion without being assembled into pili and for crossing of the blood-brain barrier in the infant rat model.

Infect Immun, 2004 Sep, 72(9), 5115 - 25
SseK1 and SseK2 are novel translocated proteins of Salmonella enterica serovar typhimurium; Kujat Choy SL et al.; Salmonella enterica is a gram-negative, facultative intracellular pathogen that causes disease symptoms ranging from gastroenteritis to typhoid fever . A key virulence strategy is the translocation of bacterial effector proteins into the host cell, mediated by the type III secretion systems (TTSSs) encoded in Salmonella pathogenicity island 1 (SPI-1) and SPI-2 . In S . enterica serovar Typhimurium LT2, we identified the protein products of STM4157 and STM2137 as novel candidate secreted proteins by comparison to known secreted proteins from enterohemorrhagic Escherichia coli and Citrobacter rodentium . The STM4157 and STM2137 proteins, which we have designated SseK1 and SseK2, respectively, are 61% identical at the amino acid level and differ mainly in their N termini . Western analysis showed that in vitro accumulation and secretion of these proteins in serovar Typhimurium were affected by mutations in the two-component systems SsrA/B and PhoP/Q, which are key mediators of intracellular growth and survival . SPI-2 TTSS-dependent translocation of recombinant SseK1::Cya was evident at 9 h postinfection of epithelial cells, while translocation of SseK2::Cya was not detected until 21 h . Remarkably, the translocation signal for SseK1 was contained within the N-terminal 32 amino acids . Fractionation of infected epithelial cells revealed that following translocation SseK1 localizes to the host cytosol, which is unusual among the currently known Salmonella effectors . Phenotypic analysis of DeltasseK1, DeltasseK2, and DeltasseK1/DeltasseK2 mutants provided evidence for a role that was not critical during systemic infection . In summary, this work demonstrates that SseK1 and SseK2 are novel translocated proteins of serovar Typhimurium.

Ground Water, 2004 Jul-Aug, 42(4), 526 - 33
Colonization by aerobic bacteria in karst: laboratory and in situ experiments; Personne JC et al.; Experiments were carried out to investigate the potential for bacterial colonization of different substrates in karst aquifers and the nature of the colonizing bacteria . Laboratory batch experiments were performed using limestone and PVC as substrates, a natural bacterial isolate and a known laboratory strain (Escherichia coli {E . coli}) as inocula, and karst ground water and a synthetic formula as growth media . In parallel, fragments of limestone and granite were submerged in boreholes penetrating two karst aquifers for more than one year; the boreholes are periodically contaminated by enteric bacteria from waste water . Once a month, rock samples were removed and the colonizing bacteria quantified and identified . The batch experiments demonstrated that the natural isolate and E . coli both readily colonized limestone surfaces using karst ground water as the growth medium . In contrast, bacterial colonization of both the limestone and granite substrates, when submerged in the karst, was less intense . More than 300 bacterial strains were isolated over the period sampled, but no temporal pattern in colonization was seen as far as strain, and colonization by E . coli was notably absent, although strains of Salmonella and Citrobacter were each observed once . Samples suspended in boreholes penetrating highly fractured zones were less densely colonized than those in the borehole penetrating a less fractured zone . The results suggest that contamination of karst aquifers by enteric bacteria is unlikely to be persistent . We hypothesize that this may be a result of the high flow velocities found in karst conduits, and of predation of colonizing bacteria by autochthonous zooplankton.

Infez Med, 2004 Jun, 12(2), 108 - 12
{Incidence of extended spectrum beta-lactamase (ESBL)-producing Enterobacteria in the area of}; Bellissima P et al.; The authors report the incidence of extended spectrum b-lactamases (ESBL)- producing strains in 1,602 enterobacteria consecutively isolated, from April 2002 to December 2003, in Caltagirone (CT) Hospital, and their in vitro susceptibility to several antimicrobial agents . The incidence of ESBL-producing Enterobacteria was equal to 1.5%, mainly among Escherichia coli, Klebsiella spp and Citrobacter koseri, isolated by emergency and medicine wards . In vitro, the enterobacteria were fully susceptible to carbapenems and highly susceptible to third-generation cephalosporin, aztreonam, piperacillin, amikacin, and ciprofloxacin.

J Clin Microbiol, 2004 Aug, 42(8), 3805 - 8
Molecular epidemiology of Enterobacteriaceae isolates producing extended-spectrum beta-lactamases in a French hospital; Lavigne JP et al.; In 2002, 80 isolates of Enterobacteriaceae producing extended-spectrum beta-lactamases (ESBLs) were collected from infected patients in our hospital . Enterobacter aerogenes was the most common bacterium isolated from all specimens (36.5%) . The ESBLs were predominantly (90%) TEM derivatives (TEM-24, TEM-3) . Pulsed-field gel electrophoresis highlighted that E . aerogenes, Klebsiella pneumoniae, and Citrobacter koseri had a clonal propagation.

J Antimicrob Chemother, 2004 Sep, 54(3), 634 - 9 Epub 2004 Jul 28.
Frequency and diversity of Class A extended-spectrum beta-lactamases in hospitals of the Auvergne, France: a 2 year prospective study; De Champs C et al.; OBJECTIVES: To evaluate the frequency and diversity of extended-spectrum beta-lactamases (ESBLs) produced by Enterobacteriaceae and Pseudomonas aeruginosa in one French region . METHODS: During 2001-2002, all the non-duplicate isolates of P . aeruginosa resistant to ceftazidime and of Enterobacteriaceae intermediate or resistant to ceftazidime and/or cefotaxime and/or aminoglycosides with an AAC(6') I phenotype were collected in nine hospitals of the area . ESBL isoelectric points were determined, bla genes were amplified and sequenced and epidemic isolates were genotyped with ERIC2-PCR . RESULTS: ESBLs were observed in 297 Enterobacteriaceae (0.8%) . The most frequent were TEM-3 like (n=152; 51.2%) and TEM-24 (n=115; 38.7%) . Four new enzymes were observed, TEM-112 (pI 5.4), TEM-113 (pI 6.3), TEM-114 (pI 5.9) and TEM-126 (pI 5.4) . Other TEMs were TEM-8, TEM-12, TEM-16, TEM-19, TEM-20, TEM-21, TEM-29 and TEM-71 . The other ESBLs were SHV-4, SHV-5 and SHV-12, CTX-M-1, CTX-M-3, CTX-M-14 and CTX-M-15 . In 37 P . aeruginosa (0.7%) only one ESBL was observed, PER-1 . Five epidemic strains were detected, Serratia marcescens TEM-3 and four observed in several hospitals, Enterobacter aerogenes TEM-24, Citrobacter koseri TEM-3, Proteus mirabilis TEM-3 and P . aeruginosa PER-1 . CONCLUSION: ESBL frequency was lower than in 1998, and CTX-M-type frequency higher (2.1% of ESBLs in 2001, 4.9% in 2002) . This long-term survey detected new sporadic enzymes (TEM-112, TEM-113, TEM-114 and TEM-126) and interhospital epidemic strains while avoiding any overestimation of ESBL frequency that may otherwise have occurred because of acute epidemics.

Clin Experiment Ophthalmol, 2004 Aug, 32(4), 445 - 7
Three cases of post-traumatic endophthalmitis caused by unusual bacteria; Essex RW et al.; Three cases of post-traumatic endophthalmitis caused by unusual bacteria are presented . The pathogens identified were: (i) Bacillus cereus and Citrobacter freundii; (ii) Pseudomonas fluorescens; and (iii) Chryseobacterium meningosepticum and Stenotrophomonas maltophilia . Two of these pathogens have not previously been reported to cause endophthalmitis . The available literature regarding the individual cases is summarized and a brief discussion of post-traumatic endophthalmitis is presented, with reference to a recently published large series at the authors' institution.

Antimicrob Agents Chemother, 2004 Aug, 48(8), 3172 - 4
CMY-13, a novel inducible cephalosporinase encoded by an Escherichia coli plasmid; Miriagou V et al.; An IncN plasmid (p541) from Escherichia coli carried a Citrobacter freundii-derived sequence of 4,252 bp which included an ampC-ampR region and was bound by two directly repeated IS26 elements . ampC encoded a novel cephalosporinase (CMY-13) with activity similar to that of CMY-2 . AmpR was likely functional as indicated in induction experiments.

Southeast Asian J Trop Med Public Health, 2004 Mar, 35(1), 202 - 9
Incidence of enteric bacteria and Staphylococcus aureus in day care centers in Akwa Ibom State, Nigeria; Itah AY et al.; The incidence of enteric bacteria and Staphylococcus aureus in four day care centers in Akwa Ibom State was studied using culture techniques . The percentage frequencies of the isolates from 124 samples were Staphylococcus aureus (33.9), Escherichia coli (19.0), Klebsiella sp (14.4), Citrobacter sp (12.5) and Proteus mirabilis (7.4) . The sources of contamination were floors, chairs, skin, bed linen, door handles, fans, children's tables, walls, windows, ceiling, headmistress's table and chairs, drinking water and wash water . Cultures from Aunty Chimmy's Day Care and Nursery School, Eket and Ideal Day Care and Nursery School, Eket yielded more organisms than those from Trinity International Nursery School, Ikot Ekpene and Adiaha Obong Day Care Center in Uyo . The results revealed the insanitary conditions in these day care centers . The enforcement of an effective public health enlightenment program is advocated in order to attract sufficient attention of the proprietors of these establishments to the role of fomites as reservoirs of pathogenic microorganisms.

J Food Prot, 2004 Jul, 67(7), 1335 - 43
An improved PCR primer pair based on 16S rDNA for the specific detection of Salmonella serovars in food samples; Lin CK et al.; Salmonella serovars are some of the major bacterial pathogens that can cause sporadic cases and outbreaks of foodborne illness . Based on the sequence data in the V3 region of the 16S rRNA gene, two PCR primer pairs have been designed for the detection of all serovars of Salmonella . However, none of these primers were specific for Salmonella because complete sequence homology with certain non-Salmonella strains has been found within each of them . Thus, the specificities of these two primer pairs could not rely on only one of the two primers . In this study, we modified our previous 16SFI primer by extending one base at the 5' end and three bases at the 3' end . The modified primer, 16S-Sal, was designed with one or more mismatched bases near the 3' end of the primer annealing to the corresponding sequences of non-Salmonella strains . Such modification eliminates interference from Citrobacter freundii and Enterobacter cloacae as occurs with the 16SFI primer . When 16S-Sal and a degenerate primer, 16S-CCR, were used as a primer pair, detection specificity of Salmonella serovars was achieved . Because this primer pair was used for PCR detection of the salmonellae in food samples, such as whole milk and chicken meat, as low as 1 to 9 CFU/g (ml) of the food sample could be detected when a 8-h preculture step was performed prior to the PCR . For chicken meat, the endogenous microflora did not interfere with the PCR results.

J Basic Microbiol, 2004, 44(4), 320 - 4
Isolation of Citrobacter sp . mutants defective in decolorizing malachite green; Jang MS et al.; To identify genes involved in the decolorization of malachite green, random mutants generated by transposon insertion in the malachite green-decolorizing bacterium, Citrobacter sp . were isolated . The resulting mutant bank yielded 24 mutants with complete defects in their abilities to decolorize malachite green . Southern hybridization with a Tn5 fragment as a probe showed a single hybridized band in 7 mutants, which appeared to have insertions at different sites of the chromosome . The Tn5-inserted genes were isolated and the DNA sequence flanking Tn5 was determined . Based on a sequence database, the putative protein products encoded by the mg genes were identified as follows . mg3, an ABC transporter homolog; mg6, a LysR-type regulatory protein; m11, an oxidoreductase; mg17, a MalG protein in the maltose transport system; and mg21, a sugar kinase . The deduced sequences from two mg genes (mg7 and mg18) showed no significant similarity to any protein with a known function, suggesting that these two mg genes encode unidentified proteins that are responsible for the decolorization of malachite green .

J Immunol, 2004 Aug 1, 173(3), 2109 - 17
Protective role of arginase in a mouse model of colitis; Gobert AP et al.; Arginase is the endogenous inhibitor of inducible NO synthase (iNOS), because both enzymes use the same substrate, l-arginine (Arg) . Importantly, arginase synthesizes ornithine, which is metabolized by the enzyme ornithine decarboxylase (ODC) to produce polyamines . We investigated the role of these enzymes in the Citrobacter rodentium model of colitis . Arginase I, iNOS, and ODC were induced in the colon during the infection, while arginase II was not up-regulated . l-Arg supplementation of wild-type mice or iNOS deletion significantly improved colitis, and l-Arg treatment of iNOS(-/-) mice led to an additive improvement . There was a significant induction of IFN-gamma, IL-1, and TNF-alpha mRNA expression in colitis tissues that was markedly attenuated with l-Arg treatment or iNOS deletion . Treatment with the arginase inhibitor S-(2-boronoethyl)-l-cysteine worsened colitis in both wild-type and iNOS(-/-) mice . Polyamine levels were increased in colitis tissues, and were further increased by l-Arg . In addition, in vivo inhibition of ODC with alpha-difluoromethylornithine also exacerbated the colitis . Taken together, these data indicate that arginase is protective in C . rodentium colitis by enhancing the generation of polyamines in addition to competitive inhibition of iNOS . Modulation of the balance of iNOS and arginase, and of the arginase-ODC metabolic pathway may represent a new strategy for regulating intestinal inflammation.

Chem Commun (Camb), 2004 Jul 21, (14), 1634 - 5 Epub 2004 Jun 08.
Multienzyme system for dihydroxyacetone phosphate-dependent aldolase catalyzed C-C bond formation from dihydroxyacetone; Sanchez-Moreno I et al.; A multienzyme system composed by recombinant dihydroxyacetone kinase from Citrobacter freundii, fuculose-1-phosphate aldolase and acetate kinase, allows a practical one-pot C-C bond formation catalysed by dihydroxyacetone phosphate-dependent aldolases from dihydroxyacetone and an aldehyde.

Cell Tissue Bank, 2003, 4(2-4), 95 - 100
Properties of Air Dried Radiation Processed Amniotic Membranes under Different Storage Conditions; Singh R et al.; Amniotic membranes collected from the placentae of screened donors were processed, air dried and sterilized by gamma irradiation at 25 kGy . Effect of storage under different temperature and humidity conditions (10 degrees C, RH 80-90%; 10 degrees C, RH 40-50%; 40 degrees C, RH 50-60% and 40 degrees C, RH 10-20%) on the properties of the membrane were examined . Infrared (IR) spectral scanning was carried out to examine degradation or change if any in the tissue under different storage conditions . The degradation of amnion on irradiation with gamma rays or during storage after irradiation would tend to produce the relative variation in IR absorption troughs . This kind of addendum was absent in all the samples indicating no qualitative change in the material property of amnion . Water absorption and water vapour transmission rate (WVTR) of the membrane remained unchanged even after 6 months . No effect on the microbial permeability of membrane was observed during storage . The amniotic membranes were found to be impermeable to different strains of bacteria - Bacillus, Escherichia coli, Pseudomonas, Citrobacter, Flavimonas and Staphylococcus . The results indicate that amniotic membranes processed by air-drying are stable and can be stored under different environmental conditions without compromise to their clinical performance.

Ann Clin Microbiol Antimicrob . 2004 Jul 15;3(1):13.
Kirby-Bauer disc approximation to detect inducible third-generation cephalosporin resistance in Enterobacteriaceae; Qin X et al.; Resistance to beta-lactam antibiotics in enteric Gram-negative bacilli may be difficult to detect using standard methods of either Kirby-Bauer disc diffusion (KBDD) or broth dilution for minimal inhibitory concentration (MIC) . This difficulty is due to genetic differences in resistance determinants, differences in levels of gene expression, and variation in spectra of enzymatic activity against the substrate beta-lactams used for susceptibility testing . We have examined 95 clinical isolates reportedly susceptible to ceftazidime and ceftriaxone, as originally determined by either KBDD or MIC methods . The organisms studied here were isolated in 2002 from two pediatric hospital centers (Seattle, USA and Shanghai, China) . They belong to the inducible beta-lactamase producing Gram-negative bacilli, such as Enterobacter spp., Citrobacter spp., Serratia spp., Morganella spp., Providencia spp., and Proteus vulgaris . A Kirby-Bauer disc approximation (KBDA) method identified inducible phenotypes of third-generation cephalosporin resistance in 76% of isolates, which would otherwise be considered susceptible by standard KBDD methods.

J Antimicrob Chemother, 2004 Aug, 54(2), 410 - 7 Epub 2004 Jul 14.
In vitro activity of AVE1330A, an innovative broad-spectrum non-beta-lactam beta-lactamase inhibitor; Bonnefoy A et al.; OBJECTIVES: Production of beta-lactamases is the main mechanism of beta-lactam resistance in Gram-negative bacteria . Despite the current use of clavulanic acid, sulbactam and tazobactam, the prevalence of class A and class C enzymes is increasing worldwide, demanding new beta-lactamase inhibitors . Here we report the antimicrobial properties of AVE1330A, a representative of a novel class of bridged bicyclico{3.2.1}diazabicyclo-octanones in combination with ceftazidime . Materials and methods: IC(50) and kinetic parameters of the hydrolysis reaction were used to characterize beta-lactamase inhibition by AVE1330A . MICs for >600 strains were determined with the combination ceftazidime/AVE1330A at a fixed ratio of 4:1 . RESULTS: IC(50)s of AVE1330A for TEM-1 and P99 enzymes were 0.0023 mg/L (8 nM) and 0.023 mg/L (80 nM), compared with 0.027 mg/L (130 nM) and 205.1 mg/L (1 x 10(6) nM) of clavulanic acid and 0.013 mg/L (40 nM) and 1.6 mg/L (5000 nM) of tazobactam . A highly stable covalent complex led to a low turnover of AVE1330A . MICs of ceftazidime/AVE1330A for Enterobacteriaceae were at least eight-fold lower than those of ceftazidime alone . All of the Escherichia coli, Klebsiella pneumoniae, Citrobacter and Proteus mirabilis strains, including ceftazidime-resistant isolates, were inhibited at 4-8 mg/L . Only 2 mg/L were required to inhibit other Proteeae, Enterobacter, Salmonella and Serratia . CONCLUSION: The combination of ceftazidime with AVE1330A exhibited broad-spectrum activity against Ambler class A- and class C-producing Enterobacteriaceae.

Transfusion, 2004 Jul, 44(7), 1041 - 6
Neonatal neutropenia and bacterial sepsis associated with placental transfer of maternal neutrophil-specific autoantibodies; Davoren A et al.; BACKGROUND: Passively acquired neonatal neutropenia is an infrequently reported complication of maternal autoimmune neutropenia (AIN) . Two affected siblings are described . The firstborn developed Citrobacter meningitis and was permanently disabled . The second was success-fully managed with pre- and postnatal injections of recombinant human granulocyte colony-stimulating factor (rHuG-CSF) . STUDY DESIGN AND METHODS: Neutrophil-specific antibodies were evaluated by flow cytometry (FC), monoclonal antibody immobilization of granulocyte antigens, and granulocyte agglutination assays . RESULTS: A neutrophil-reactive antibody was detected by FC in samples of the mother's serum spanning a 4-year time frame . This antibody reacted with neutrophils from the mother, father, and their first infant and with 18 of 20 target neutrophils tested . In serologic studies, it was shown that the antibody was not specific for the commonly recognized neutrophil-specific alloantigens HNA-1a (NA1), HNA-1b (NA2), HNA-1c (SH), HNA-2a (NB1), or HNA-3a (5b) . CONCLUSION: Severe neonatal neutropenia in the two siblings appears to have been caused by placental transfer of a maternal neutrophil-reactive autoantibody of undetermined specificity . Neutrophil counts should be evaluated in infants born to mothers with chronic neutropenia of possible autoimmune origin so that neutropenic infants can be carefully monitored and antibiotics and/or rHuG-CSF administered if indicated.

Pediatr Nephrol, 2004 Sep, 19(9), 982 - 6 Epub 2004 Jun 18.
Antibiotic resistance of urinary tract pathogens and rationale for empirical intravenous therapy; Haller M et al.; Empirical antibiotic treatment in urinary tract infection (UTI) in children must rely on surveillance data on the epidemiology and resistance patterns of common uropathogens . A retrospective analysis of bacteria isolated from children with UTI irrespective of underlying disease or pre-treatment was performed at the University Hospital of Freiburg, Germany, in 1997, and from 1999 to 2001 . In the first study period, 261 positive urine samples and in the second period 684 positive samples were analyzed . Escherichia coli (57.2%) was the leading uropathogen followed by Enterococcus spp . (13.7%), Pseudomonas aeruginosa (7.0%), Proteus spp . (5.9%), Klebsiella spp . (4.7%), and Enterobacter/Citrobacter spp . (4.3%) . Almost 50% of the E . coli isolates were resistant to ampicillin, but effectively no resistance against cephalosporins, aminogylcosides, ciprofloxacin, nitrofurantoin, and imipenem was observed . In Enterococcus spp . the resistance to ampicillin was about 15% and 40% to netilmicin, while none of the latter showed high-level aminoglycoside resistance . In P . aeruginosa, there was no resistance to aminoglycosides . No difference in resistance patterns between the two study periods was observed . We conclude that an empirical combination treatment of ampicillin and gentamicin, netilmicin, or tobramycin is appropriate in children with UTI independent of pre-treatment or underlying disease . This therapy should be clinically efficacious, well tolerated, and cost effective, and should prevent unnecessary development of antimicrobial resistance.

Pediatr Crit Care Med, 2004 Jul, 5(4), 393 - 5
Pneumocephalus in neonatal meningitis: diffuse, necrotizing meningo-encephalitis in Citrobacter meningitis presenting with pneumatosis oculi and pneumocephalus; Pooboni SK et al.; OBJECTIVE/PATIENT: Gas-containing encephalitis is rarely associated with neonatal meningitis . We report a case of a 19-day-old baby who presented with a rapid onset of septic shock complicated by progressively increasing gas accumulation within the brain and anterior chamber of the eye . We describe the evolution of the clinical picture and the management . INTERVENTIONS: Ventilatory support, fluid resuscitation, and continuous venovenous hemofiltration were provided in view of multiple system failure . Despite effective antibiotic therapy and supportive management, the patient died with worsening accumulation of gas within the brain, resulting in brainstem death . RESULTS: Computed tomographic images were characteristic of diffuse necrotizing meningo-encephalitis . Postmortem examination showed friable brain tissue with venous infarction and extensive gas accumulation . Citrobacter koseri was identified from the blood and cerebrospinal fluid cultures . CONCLUSION: This case re-emphasises the importance of C . koseri as both a community-acquired and nosocomial neonatal pathogen . Radiologic evidence suggestive of diffuse necrotizing meningo-encephalitis in combination with pneumocephalus and pneumatosis oculi in Citrobacter infections has never been described before . Diagnostic imaging with computed tomographic scanning of the brain and initiation of broad-spectrum antibiotics with good penetration into cerebrospinal fluid are indicated as soon as infection with Citrobacter species is suspected clinically, with appearance of pneumatosis oculi as a rare, late finding.

Immunol Res, 2004, 29(1-3), 241 - 52
Initiation and resolution of mucosal inflammation; Sherman MA et al.; Antigens entering the body through the mucosal surface are screened by a highly developed immune system comprised not only of traditional lymphoid cells but also epithelial cells, fibroblasts, and antigen-presenting cells (APCs) . For example, in the intestinal tract, gut-associated lymphoid tissue (GALT) is tolerant to the approx 400 separate commensal strains residing mainly in the colon, but also retains the capacity to detect and remove virulent bacteria before they infect systemically . This review summarizes recent work characterizing the molecular mechanisms involved in acute and chronic intestinal inflammation . We will also describe a natural murine pathogen, Citrobacter rodentium, which is being used to explore the host response to enteric pathogens and the resulting immunopathology.

Pediatrics, 2004 Jun, 113(6), 1765 - 70
Intracerebral abscess in children: historical trends at Children's Hospital Boston; Goodkin HP et al.; OBJECTIVES: A previous study performed at Children's Hospital Boston describing the natural history of intracerebral abscess between 1945 and 1980 demonstrated a decline in mortality after 1970 . This current study examines the occurrence of intracerebral abscess at Children's Hospital Boston between 1981 and 2000, inclusive, and compares the results with the previous study . Our objectives were to determine whether there had been a change in the predisposing factors, whether there were changes in the microbiology of intracerebral abscesses, and whether mortality rate had continued to decline . METHODS: To ensure that all occurrences of intracerebral abscess treated at Children's Hospital Boston between 1981 and 2000 were identified, we searched 4 separately maintained databases for the keywords "brain" or "abscess" or the International Classification of Diseases, Ninth Revision code 324.x . This search yielded the names of 386 patients . Of these 386 patients, a solitary intracerebral abscess or multiple noncontiguous intracerebral abscesses could be confirmed in 54 patients on the basis of cranial imaging (computed tomography or magnetic resonance imaging) or autopsy reports . The complete retrospective review of the medical records of these 54 patients constitutes the basis for this study . RESULTS: Congenital heart disease was the most common predisposing factor during both eras . Compared with the previous era, important historical trends identified include a reduction in the number of abscesses that occurred in the settings of sinus or otitic infection (11% during 1981-2000 vs 26% during 1945-1980), an increase in number of intracranial abscesses in infants (18% vs 7%) and in the setting of immunosuppression (16% vs 1%), an increase in the number of children who were treated with antibiotics alone (22% vs 1%), a stable overall mortality rate (24% vs 27%), and the identification of Citrobacter and fungus as causes of intracranial abscess not observed during the previous era of 1945-1980 . Citrobacter was observed only during the neonatal period . Fungi were the causative organisms predominantly in the setting of immunosuppression . CONCLUSIONS: Intracerebral abscess in children continues to be associated with high rates of neurologic impairment and death . Because earlier detection may reduce morbidity and mortality, intracranial abscess should be considered when evaluating children with new-onset neurologic signs or symptoms, especially in children who have acute immunosuppression and disseminated fungal disease or fungemia.

J Biol Chem, 2004 Aug 6, 279(32), 33751 - 8 Epub 2004 Jun 01.
Intimin types alpha, beta, and gamma bind to nucleolin with equivalent affinity but lower avidity than to the translocated intimin receptor; Sinclair JF et al.; The outer membrane adhesins of enteropathogenic Escherichia coli, Citrobacter rodentium, and enterohemorrhagic E . coli (EHEC) O157:H7 that mediate attach and efface intestinal lesions are classified as intimin alpha, beta, and gamma, respectively . Each of these intimin types binds to its cognate, bacterially encoded receptor (called Tir for translocated intimin receptor) to promote tight adherence of the organism to the host-cell plasma membrane . We previously reported that gamma intimin of EHEC O157:H7 also bound to a eucaryotic receptor that we determined was nucleolin . The objective of this study was to investigate in vitro and in vivo the interactions of intimins alpha, beta, and gamma with nucleolin in the presence of Tir from EHEC O157:H7 . Protein binding experiments demonstrated that intimin of types alpha, beta, and gamma bound nucleolin with similar affinity . Moreover, all three intimin types co-localized with regions of nucleolin expressed on the surface of HEp-2 cells . When intimin alpha, beta, or gamma bound to Tir in vitro, the intimin interaction with nucleolin was blocked . Both Tir and nucleolin accumulated beneath intimin-presenting bacteria that had attached to the surface of HEp-2 cells . Taken together, these findings suggest that nucleolin is involved in bacterial adherence promoted by all intimin types and that Tir and nucleolin compete for intimin during adherence.

Infect Immun, 2004 Jun, 72(6), 3315 - 24
Clearance of Citrobacter rodentium requires B cells but not secretory immunoglobulin A (IgA) or IgM antibodies; Maaser C et al.; Citrobacter rodentium, a murine model pathogen for human enteropathogenic Escherichia coli, predominantly colonizes the lumen and mucosal surface of the colon and cecum and causes crypt hyperplasia and mucosal inflammation . Mice infected with C . rodentium develop a secretory immunoglobulin A (IgA) response, but the role of B cells or secretory antibodies in host defense is unknown . To address this question, we conducted oral C . rodentium infections in mice lacking B cells, IgA, secreted IgM, polymeric Ig receptor (pIgR), or J chain . Normal mice showed peak bacterial numbers in colon and feces at 1 week and bacterial eradication after 3 to 4 weeks . B-cell-deficient mice were equally susceptible initially but could not control infection subsequently . Tissue responses showed marked differences, as infection of normal mice was accompanied by transient crypt hyperplasia and mucosal inflammation in the colon and cecum at 2 but not 6 weeks, whereas B-cell-deficient mice had few mucosal changes at 2 weeks but severe epithelial hyperplasia with ulcerations and mucosal inflammation at 6 weeks . The functions of B cells were not mediated by secretory antibodies, since mice lacking IgA or secreted IgM or proteins required for their transport into the lumen, pIgR or J chain, cleared C . rodentium normally . Nonetheless, systemic administration of immune sera reduced bacterial numbers significantly in normal and pIgR-deficient mice, and depletion of IgG abrogated this effect . These results indicate that host defense against C . rodentium depends on B cells and IgG antibodies but does not require production or transepithelial transport of IgA or secreted IgM.

J Antimicrob Chemother, 2004 Jun, 53(6), 964 - 70 Epub 2004 May 12.
Plasmid-encoded functions compensate for the biological cost of AmpC overexpression in a clinical isolate of Salmonella typhimurium; Hossain A et al.; OBJECTIVES: In a previous study with a Salmonella typhimurium strain containing cloned ampC-ampR from Enterobacter cloacae, it was suggested that ampC expression must be kept at low levels by AmpR to maintain normal growth and virulent phenotype . The purpose of this study was to determine whether findings obtained with a laboratory model can be extended to a virulent clinical isolate of S . typhimurium expressing the plasmid-encoded bla(CMY-7) . METHODS: Disc induction assays were carried out to investigate inducibility of bla(CMY-7) . Primer extension and sequence analyses were carried out to map the transcriptional start site of bla(CMY-7) and determine the relative expression . Growth and invasion potential of Salmonella strains were monitored by optical density, viable counts and cell invasion assays . RESULTS: Sequence analysis confirmed the absence of ampR upstream of bla(CMY-7) therefore confirming the negative results observed using the disc induction assay . Primer extension analysis mapped the start site of bla(CMY-7) transcription within an ISEcp1-like element . The relative expression of bla(CMY-7) was approximately 965-fold higher than the expression of a wild-type Citrobacter freundii chromosomal ampC and approximately 4.1-fold higher than ampC expression from a derepressed mutant of C . freundii . Growth and the capacity to invade mammalian cells were not compromised for either the clinical isolate or the S . typhimurium transconjugant containing bla(CMY-7) . However, a Salmonella transformant containing bla(CMY-7) exhibited a compromised phenotype with respect to growth and invasion of mammalian cells . CONCLUSION: These findings indicate that the biological cost of high-level AmpC production can be compensated by plasmid-encoded factors and not by regulating ampC expression.

Eur Spine J . 2004 May 8; {Epub ahead of print}
Detection of bacterial DNA in painful degenerated spinal discs in patients without signs of clinical infection; Fritzell P et al.; A local inflammatory and potentially painful response, of which the ultimate cause is unknown, has been described in nervous tissues in contact with degenerated disc material in patients with low back and leg pain . With the rationale that a possible cause of such inflammation could be bacterial infection, we utilized PCR (polymerase chain reaction) amplification of the 16S rRNA (ribosomal RNA) gene followed by gene sequencing, to investigate whether bacterial DNA might be detected in the degenerative discs of 10 patients operated for disc herniation or post-discectomy syndrome . One patient with disc hernia harbored DNA homologous to Bacillus cereus, and in one patient suffering from post-discectomy syndrome, Citrobacter braaki/freundii DNA was detected . The finding demonstrates that 16S rRNA PCR can be a useful tool in search of bacterial DNA in degenerated discs, which in turn may be indicative of low-grade infection, manifesting itself only as pain rather than as clinical infection.

J Antimicrob Chemother, 2004 Jun, 53(6), 1076 - 80 Epub 2004 May 05.
Citrobacter koseri and Citrobacter amalonaticus isolates carry highly divergent beta-lactamase genes despite having high levels of biochemical similarity and 16S rRNA sequence homology; Underwood S et al.; OBJECTIVES: Isolates previously identified as Citrobacter diversus are now known as Citrobacter koseri . We measured sequence variation at the beta-lactamase structural gene among a group of clinical isolates originally identified as C . diversus by API 20E profiling . METHODS: beta-Lactamase and 16S rRNA genes were amplified by PCR and sequenced by standard methods . beta-Lactamase induction was attempted in liquid-grown cultures using cefoxitin . Nitrocefin hydrolysis assays were performed using a spectrophotometer . RESULTS: Analysis of 16S rRNA gene sequences showed that Citrobacter spp . isolates with an inducible beta-lactamase gene, cdiA, closely related to 'C . koseri ' NF85 and ULA27 are actually Citrobacter amalonaticus . C . koseri isolates, whose identities were confirmed by 16S rRNA sequencing, produce a class A beta-lactamase, Cko, constitutively at low levels . The cko and cdiA beta-lactamase genes share <45% identity . CONCLUSIONS: We have confirmed that cko is a beta-lactamase gene carried by C . koseri, and that isolates previously identified as 'C . koseri ', but carrying the cdiA beta-lactamase gene are C . amalonaticus . Thus, beta-lactamase-gene-specific PCR may provide a valuable tool to differentiate these biochemically homogeneous Citrobacter species.

Int J Infect Dis, 2004 May, 8(3), 147 - 54
Antimicrobial resistance of Gram-negative bacilli isolates from inpatients and outpatients at Yaounde Central Hospital, Cameroon; Pieboji JG et al.; OBJECTIVE: To determine and compare antimicrobial susceptibility patterns of pathogenic bacteria from inpatients and outpatients at a university teaching hospital in Yaounde, Cameroon . METHODS: Gram-negative bacilli isolates (n = 522), obtained from a wide range of clinical specimens (urine, pus and blood) from inpatients and outpatients at Yaounde Central Hospital between March 1995 and April 1998, were evaluated for resistance to antibiotics (amoxicillin, amoxicillin/clavulanate, piperacillin, cefazolin, cefoxitin, cefotaxime, ceftazidime, aztreonam, imipenem, gentamicin, tobramicin, ofloxacin and trimethoprim/sulfamethoxazole) . RESULTS: Of the 522 isolates recorded, 80.3% were Enterobacteriaceae . A high incidence of resistance to amoxicillin (85%), piperacillin (75%) and trimethoprim/sulfamethoxazole (71%) was observed . The proportion of antimicrobial-resistant isolates from inpatients was significantly higher than that from outpatients (P < 0.05), except for piperacillin, tobramicin and trimethoprim/sulfamethoxazole . The combinations of antimicrobial and organism showed that the percentage of ceftazidime-resistant Pseudomonas aeruginosa and ceftazidime-resistant Enterobacter cloacae were 26.8% and 24% respectively . The rate of antimicrobial resistance in isolates from inpatients was not significantly higher than that in isolates from outpatients for all the antimicrobial/organism combinations, except for ceftazidime-resistant Escherichia coli, which was exclusively found in isolates from inpatients . Among Enterobacteriaceae, high and low level penicillinase (mostly in E . coli (13.6% and 11% respectively) and Klebsiella spp . (9% and 8% respectively) were the most important beta-lactam resistance phenotypes (31.2% and 23.6%, respectively) . Wild type (exclusively observed in E . coli, Proteus mirabilis and Salmonella spp.) and low level penicillinase were higher in outpatient than inpatient isolates (wild type--17.9% vs 10.8% and low level penicillinase--29.4% vs 20.5%, respectively; P < 0.05) . However, extended spectrum beta-lactamase strains (Klebsiella spp . (3.5%), E . coli (2.6%), Citrobacter spp . (0.7%), Enterobacter spp . (0.4%) and P . mirabilis (0.2%)) were exclusively recovered from inpatients . Penicillinase and high level cephalosporinase resistance phenotypes were frequently observed in non-fermenter Gram-negative bacilli (46.6% and 29.1% respectively) . However, there were no significant differences in penicillinase and cephalosporinase resistance between inpatient and outpatient isolates . CONCLUSION: As the incidence of antimicrobial resistance is substantially higher in isolates from inpatient than outpatient pathogens, more resources should be allocated within the hospital to encourage good antibiotic practices and good hospital hygiene.

Am J Trop Med Hyg, 2004 Apr, 70(4), 412 - 9
Responses of small intestinal architecture and function over time to environmental factors in a tropical population; Kelly P et al.; To determine the response of the small intestinal mucosa to environmental conditions, we studied changes in mucosal architecture and function in a longitudinal cohort study in African adults . Over three consecutive years, 238 adults submitted monthly stool samples for parasitologic and bacteriologic analysis and underwent an annual endoscopic jejunal biopsy for mucosal morphometry . Absorption and permeability assays were performed on the same day as the enteroscopy . Variation in mucosal architecture and function was correlated with environmental factors and stool microbiology . The whole cohort had structural and functional evidence of tropical enteropathy, but structure and function were only weakly correlated . There were marked changes over time, and seasonal variation was observed in villous height (16%), xylose recovery (16%), and permeability (28%) . Asymptomatic intestinal infections were common . Enteropathy was more severe in participants with Citrobacter rodentium or hookworm ova in the stool sample taken one month before the investigations were performed.

J Am Acad Dermatol, 2004 May, 50(5 Suppl), S114 - 7
Nonpseudomonal ecthyma gangrenosum; Reich HL et al.; Ecthyma gangrenosum is a cutaneous infection associated most commonly with pseudomonal sepsis in the patient who is immunocompromised . We describe an 8-month-old girl with acute myelocytic leukemia who developed perineal ecthyma gangrenosum caused by Citrobacter freundii, a gram-negative pathogen that has been rarely associated with cutaneous disease . We also review the literature to categorize the range of pseudomonal and nonpseudomonal pathogens associated with ecthyma gangrenosum.

Antimicrob Agents Chemother, 2004 Apr, 48(4), 1151 - 8
CFE-1, a novel plasmid-encoded AmpC beta-lactamase with an ampR gene originating from Citrobacter freundii; Nakano R et al.; A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded beta-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins . Resistance arising from production of a beta-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E . coli ML4947 . The substrate and inhibition profiles of this enzyme resembled those of the AmpC beta-lactamase . The resistance gene of pKU601, which was cloned and expressed in E . coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3 . DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C . freundii GC3 . In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C . freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a beta-lactamase-associated gene region from the chromosome to a plasmid . Finally, we describe a novel plasmid-encoded AmpC beta-lactamase, CFE-1, with an ampR gene derived from C . freundii.

Berl Munch Tierarztl Wochenschr, 2004 Mar-Apr, 117(3-4), 116 - 29
{Plasticity of bacterial genomes: pathogenicity islands and the locus of enterocyte effacement (LEE)}; Kirsch P et al.; Many bacterial virulence attributes, like toxins, adhesins, invasins, iron uptake systems, are encoded within specific regions of the bacterial genome . These in size varying regions are termed pathogenicity islands (PAIs) since they confer pathogenic properties to the respective micro-organism . Per definition PAIs are exclusively found in pathogenic strains and are often inserted near transfer-RNA genes . Nevertheless, non-pathogenic bacteria also possess foreign DNA elements that confer advantageous features, leading to improved fitness . These additional DNA elements as well as PAIs are termed genomic islands and were acquired during bacterial evolution . Significant G+C content deviation in pathogenicity islands with respect to the rest of the genome, the presence of direct repeat sequences at the flanking regions, the presence of integrase gene determinants as other mobility features,the particular insertion site (tRNA gene) as well as the observed genetic instability suggests that pathogenicity islands were acquired by horizontal gene transfer . PAIs are the fascinating proof of the plasticity of bacterial genomes . PAIs were originally described in human pathogenic Escherichia (E.) coli strains . In the meantime PAIs have been found in various pathogenic bacteria of humans, animals and even plants . The Locus of Enterocyte Effacement (LEE) is one particular widely distributed PAI of E coli . In addition, it also confers pathogenicity to the related species Citrobacter (C.) rodentium and Escherichia (E.) alvei . The LEE is an important virulence feature of several animal pathogens . It is an obligate PAI of all animal and human enteropathogenic E . coli (EPEC), and most enterohaemorrhegic E . coli (EHEC) also harbor the LEE . The LEE encodes a type III secretion system, an adhesion (intimin) that mediates the intimate contact between the bacterium and the epithelial cell, as well as various proteins which are secreted via the type III secretion system . The LEE encoded virulence features are responsible for the formation of so called attaching and effacing (AE) lesions in the intestinal epithelium . Due to its wide distribution in animal pathogens, LEE encoded antigens are suitable vaccine antigens . Acquisition and structure of the LEE pathogenicity island is the crucial point of numerous investigations . However, the evolution of the LEE, its origin and further spread in E . coli, are far from being resolved.

Syst Appl Microbiol, 2004 Mar, 27(2), 219 - 28
DNA based classification of food associated Enterobacteriaceae previously identified by Biolog GN Microplates; Olsson C et al.; Enterobacteriaceae are frequently isolated from food products and it is essential to have methods for correct identification for both food hygiene and epidemiology reasons . Phenotypic methods are not always sufficient and have to be supplemented by DNA based methods . In the present study, 70 strains of Enterobacteriaceae derived from milk, fish and meat that had previously been identified by Biolog GN Microplates were genomically classified together with 15 representative type strains of species of Enterobacteriaceae . The field strains were dominated by Hafnia alvei, Serratia liquefaciens and Rahnella aquatilis . All strains were subjected to temporal temperature gel electrophoresis (TTGE) analysis using amplicons encompassing the V3, V4 and V9 variable regions of the 16S rRNA gene . Selected strains were analysed by ribotyping and partial 16S rDNA sequencing . The type strains were differentiated into 10 different TTGE groups . Two of the groups contained two type strains . Enterobacter aerogenes and Klebsiella planticola were not distinguished due to their identical sequences and Yersinia ruckeri and Citrobacter freundii showed the same migration pattern . The 70 food strains could be differentiated into 14 TTGE groups where 33 strains (47.1%) could be assigned to TTGE groups including type or reference strains . Rahnella strains were dispersed into three TTGE groups of which one group corresponded to Rahnella genomospecies 1 and one to genomospecies 3 . The grouping of Rahnella strains was supported by ribotyping and phylogenetic analysis . TTGE can be a useful additional tool for identification on the species level of food related Enterobacteriaceae.

Infect Immun, 2004 Apr, 72(4), 2288 - 302
Identification of a novel Citrobacter rodentium type III secreted protein, EspI, and roles of this and other secreted proteins in infection; Mundy R et al.; Citrobacter rodentium is a member of a group of pathogens that colonize the lumen of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation . C . rodentium, which causes transmissible colonic hyperplasia in mice, is used as an in vivo model system for the clinically significant A/E pathogens enterohemorrhagic and enteropathogenic Escherichia coli . These bacteria all contain a pathogenicity island called the locus of enterocyte effacement (LEE), which encodes a type III secretion system that is designed to deliver effector proteins into eukaryotic host cells . These effectors are involved in the subversion of host eukaryotic cell functions to the benefit of the bacterium . In this study we used mutant strains to determine the effects of the C . rodentium LEE-encoded effectors EspF, EspG, EspH, and Map on virulence in the mouse model . In addition, we identified a novel secreted protein, EspI encoded outside the LEE, whose secretion is also dependent on a functional type III secretion system . Mutant strains with each of the effectors investigated were found to be outcompeted by wild-type bacteria in mixed-infection experiments in vivo, although the effects of EspF and EspH were only subtle . In single-infection experiments, we found that EspF, EspG, and EspH are not required for efficient colonization of the mouse colon or for the production of hyperplasia . In contrast, strains producing EspI and Map had significant colonization defects and resulted in dramatically reduced levels of hyperplasia, and they exhibited very different growth dynamics in mice than the wild-type strain exhibited.

Int J Antimicrob Agents, 2004 Mar, 23 Suppl 1, S2 - 5
Etiology and susceptibility of urinary tract isolates in Kosova; Raka L et al.; Urinary tract infections are amongst the most common pathogenic infections with an increasing resistance to antimicrobials . The objective of this study was to determine the etiology and antimicrobial susceptibility patterns of urinary tract infection pathogens isolated in Kosovo . A retrospective study was carried from urine samples of both inpatients and outpatients that were received in our laboratory throughout 2001 . During the study period, 16500 urine samples were analysed, of which 4260 (25.8%) had significant bacteriuria obtained from 1420 patients . Of this, 1059 (74.6%) were collected from females and 361 (25.4%) from males . Urine samples processed from outpatients were 72.5% (1029), whereas 27.5% (391) were from hospitalised patients . Escherichia coli was the most common aetiologic agent isolated (80.5%), followed by Proteus spp . (6.1%), Klebsiella spp . (5.9%), Citrobacter (5.1%) and Mycobacterium tuberculosis (0.8%) . Gram-positive bacteria accounted for only 0.3% . Pseudomonas aeruginosa was only isolated from inpatients and was responsible for 0.6% of infections . Amoxicillin, ampicillin and trimethoprim-sulphamethoxazole resistance rates were 48.7, 46.5 and 32.1%, respectively . Nitrofurantoin, cefalexin and ciprofloxacin expressed the highest susceptibility among these isolates . E . coli isolates from inpatients and outpatients showed more than 25% resistance to trimethoprim-sulphamethoxazole . Of all isolates, 16% (225) were resistant to three or more agents and considered multi-drug resistant . Current data on the prevalence of multidrug resistance among urinary tract isolates should be a consideration to change the current empiric treatment of urinary tract infections.

Przegl Epidemiol, 2003, 57(4), 655 - 62
{Characteristics of bacteria isolated from body surface of German cockroaches caught in hospitals}; Czajka E et al.; The objective of the study was to identify bacterial flora from external parts of German cockroaches caught in hospitals . The susceptibility of the bacteria to the most important groups of antimicrobial agents was also examined . 80 strains of bacteria were isolated, among them 34 strains of Gram-positive cocci and 31 strains of Gram-negative rods . One of isolated strains of Citrobacter freundii and two strains of Serratia liquefaciens showed ESBL mechanism of resistance and extended level of AmpC--type beta-lactamases . Two Staphylococcus strains (S . epidermidis and S . equorum) were resistant to erythromycin and clindamycin (MLSB mechanism of resistance) . Such strains, resistant to antibiotics and chemiotherapeutics may be reservoirs of resistance genes which can be transmitted into other bacteria . Presence of such pathogens on the body surface of German cockroaches, very mobile insects, might create conditions for easy dissemination of them in hospital environment.

J Antimicrob Chemother, 2004 Apr, 53(4), 584 - 91 Epub 2004 Mar 10.
Analysis of AmpC beta-lactamase expression and sequence in biochemically atypical ceftazidime-resistant Enterobacteriaceae from paediatric patients; Avison MB et al.; OBJECTIVES: To analyse the variation of ampC beta-lactamase gene sequence and expression in biochemically atypical Enterobacteriaceae isolates, and to identify them definitively . METHODS: beta-Lactamase gene-containing recombinant plasmids transformed into Escherichia coli were selected using ampicillin . PCR analysis was used to locate specific ampC and 16S rRNA genes, and the amplicons were sequenced . Random amplified polymorphic DNA PCR was used to group isolates and API 20E biochemical profiling was used to identify them putatively . RESULTS: Of 50 ceftazidime-resistant clinical Enterobacteriaceae isolates, 36 were identified (>95% confidence)-using API 20E test strips-as being organisms known to express inducible class C beta-lactamases (Citrobacter freundii, Enterobacter cloacae, Morganella morganii or Hafnia alvei) . The rest were biochemically atypical . Of these, isolate I113, putatively identified as E . coli, possesses a chromosomally encoded ampC which differs by 15% from C . freundii OS60 ampC and by >30% from E . coli ampC . A related ampC gene was found in another seven of the atypical isolates . The use of various identification methods, including ampC sequence analysis, revealed that these I113-like ampC-positive isolates represent Citrobacter murliniae and Citrobacter youngae . CONCLUSIONS: We report sequences for two new Citrobacter spp . ampC genes, and provide evidence that ampC sequencing is a discriminatory method for identifying atypical Citrobacter spp . isolates.

Proc Natl Acad Sci U S A, 2004 Mar 9, 101(10), 3597 - 602 Epub 2004 Feb 26.
Dissecting virulence: systematic and functional analyses of a pathogenicity island; Deng W et al.; Bacterial pathogenicity islands (PAI) often encode both effector molecules responsible for disease and secretion systems that deliver these effectors to host cells . Human enterohemorrhagic Escherichia coli (EHEC), enteropathogenic E . coli, and the mouse pathogen Citrobacter rodentium (CR) possess the locus of enterocyte effacement (LEE) PAI . We systematically mutagenized all 41 CR LEE genes and functionally characterized these mutants in vitro and in a murine infection model . We identified 33 virulence factors, including two virulence regulators and a hierarchical switch for type III secretion . In addition, 7 potential type III effectors encoded outside the LEE were identified by using a proteomics approach . These non-LEE effectors are encoded by three uncharacterized PAIs in EHEC O157, suggesting that these PAIs act cooperatively with the LEE in pathogenesis . Our findings provide significant insights into bacterial virulence mechanisms and disease.

Mol Microbiol, 2004 Mar, 51(5), 1233 - 49
Identification and characterization of NleA, a non-LEE-encoded type III translocated virulence factor of enterohaemorrhagic Escherichia coli O157:H7; Gruenheid S et al.; Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 uses a specialized protein translocation apparatus, the type III secretion system (TTSS), to deliver bacterial effector proteins into host cells . These effectors interfere with host cytoskeletal pathways and signalling cascades to facilitate bacterial survival and replication and promote disease . The genes encoding the TTSS and all known type III secreted effectors in EHEC are localized in a single pathogenicity island on the bacterial chromosome known as the locus for enterocyte effacement (LEE) . In this study, we performed a proteomic analysis of proteins secreted by the LEE-encoded TTSS of EHEC . In addition to known LEE-encoded type III secreted proteins, such as EspA, EspB and Tir, a novel protein, NleA (non-LEE-encoded effector A), was identified . NleA is encoded in a prophage-associated pathogenicity island within the EHEC genome, distinct from the LEE . The LEE-encoded TTSS directs translocation of NleA into host cells, where it localizes to the Golgi apparatus . In a panel of strains examined by Southern blot and database analyses, nleA was found to be present in all other LEE-containing pathogens examined, including enteropathogenic E . coli and Citrobacter rodentium, and was absent from non-pathogenic strains of E . coli and non-LEE-containing pathogens . NleA was determined to play a key role in virulence of C . rodentium in a mouse infection model.

Carbohydr Res, 2004 Mar 15, 339(4), 881 - 4
Structures of two O-polysaccharides of the lipopolysaccharide of Citrobacter youngae PCM 1538 (serogroup O9); Ovchinnikova OG et al.; Mild acid degradation of the lipopolysaccharide of Citrobacter youngae O9, strain PCM 1538 released a homopolysaccharide of 4-acetamido-4,6-dideoxy-D-mannose (D-Rha4NAc, N-acetyl-D-perosamine) . Studies by methylation analysis and (1)H and (13)C NMR spectroscopy, using two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and H-detected (1)H,(13)C HSQC experiments showed the presence of two structurally different polysaccharides consisting of the following units: -->)-alpha-D-Rhap4NAc-(1 --> and --> 3)-alpha-D-Rhap4NAc-(1 --> 3)-beta-D-Rhap4NAc-(1 -->.

Salud Publica Mex, 2003, 45 Supp 5, S694 - 7
{Etiology of cervical vaginal infection among patients of the Juárez Hospital of Mexico}; Flores-Paz R et al.; OBJECTIVE: To identify the etiologic agents of cervicovaginal infection in order to establish an accurate diagnosis and proper treatment . MATERIAL AND METHODS: From January 1995 to December 1999, bacteriological studies were done in cervical discharge specimens from 6,811 patients aged 13 to 65 years, seen at Hospital Juarez in Mexico City . All patients had leucorrhea, pruritus, hyperemia, and abdominal pain . Statistical significance was assessed using the chi-squared test . RESULTS: The frequencies of infectious agents were as follows: G . vaginalis, 22.65%, Candida spp, 19.13%, C, albicans, 7.8%, T . vaginalis, 1.5%, Streptococcus group D, 11.78%, Streptococcus beta hemolytic, 4.59%, E . coli, 13.46%, and Klebsiella spp, 2.0% . Less frequent enterobacteria were: Citrobacter spp, Enterobacter spp, Pseudomonas spp, M . morganii, and P . mirabilis . Almost 3% of patients presented anaerobic species, which were always associated with G . vaginalis . Neisseria spp and N . weaveri were isolated in 0.15% each; N . gonorrhoeae was not isolated in any of the patients . Comparative data showed that Streptococcus beta hemolytic and E . coli increased markedly in the past two years (p < 0.001 for the latter) . CONCLUSIONS: The diversity of etiologic agents requires performing bacteriological cultures of cervical and vaginal discharge to all symptomatic patients . The English version of this paper is available at:http://www.insp.mx/salud/index.html.

Org Biomol Chem, 2004 Feb 21, 2(4), 554 - 61 Epub 2004 Jan 16.
Stereoselective reductase-catalysed deoxygenation of sulfoxides in aerobic and anaerobic bacteria; Boyd DR et al.; Direct and indirect evidence, of unexpected stereoselective reductase-catalysed deoxygenations of sulfoxides, was found . The deoxygenations proceeded simultaneously, with the expected dioxygenase-catalysed asymmetric sulfoxidation of sulfides, during some biotransformations with the aerobic bacterium Pseudomonas putida UV4 . Stereoselective reductase-catalysed asymmetric deoxygenation of racemic alkylaryl, dialkyl and phenolic sulfoxides was observed, without evidence of the reverse sulfoxidation reaction, using anaerobic bacterial strains . A purified dimethyl sulfoxide reductase, obtained from the intact cells of the anaerobic bacterium Citrobacter braakii DMSO 11, yielded, from the corresponding racemates, enantiopure alkylaryl sulfoxide and thiosulfinate samples.

Indian J Med Res, 2003 Jul, 118, 29 - 32
Occurrence & detection of AmpC beta-lactamases at a referral hospital in Karnataka; Ratna AK et al.; BACKGROUND & OBJECTIVES: AmpC beta-lactamases confer resistance to a wide variety of beta-lactam drugs except for cefepime, cefpirome and carbapenems . They are known to be responsible for nosocomial outbreaks, therapeutic failures and multidrug resistance . Although reported with increasing frequency the true rate of occurrence of these beta-lactamases in Enterobacteriaceae is not known . Hence the present study was undertaken to determine the occurrence of AmpC enzymes among clinical isolates . METHODS: A total of 520 consecutive, non-repeat clinical isolates were included in the present study . Twenty eight strains resistant to cefoxitin were tested for AmpC beta-lactamases by the modified 3-dimensional extract method . Isolates harbouring AmpC beta-lactamases were tested for inducible beta-lactamases by disc diffusion . RESULTS: Sixteen (3.3%) isolates were positive for AmpC beta-lactamases . Based on the species 9 (3.3%) Escherichia coli, 4 (2.2%) Klebsiella pneumoniae, 2 (5%) Citrobacter freundii and 1 (5.5%) isolate of Enterobacter aerogenes harboured AmpC enzymes . Nine (56.3%) of AmpC harbouring strains, were urinary isolates . All the isolates were sensitive to imipenem and variably sensitive to aminoglycosides and co-trimoxazole . INTERPRETATION & CONCLUSION: Our findings document the presence of AmpC enzymes in this region . Hence AmpC beta-lactamase detection should be undertaken in clinical isolates showing resistance to broad-spectrum cephalosporins.

FEMS Immunol Med Microbiol, 2004 Jan 15, 40(1), 51 - 5
Yersiniabactin and other siderophores produced by clinical isolates of Enterobacter spp . and Citrobacter spp; Mokracka J et al.; We analyzed the ability of extraintestinal strains of Enterobacter spp . and Citrobacter spp . to employ different siderophore-mediated strategies of iron acquisition . All strains produced iron-chelating compounds . Cross-feeding assays indicated that most isolates of both Enterobacter spp . and Citrobacter spp . excreted catecholate siderophore enterobactin, less produced aerobactin, and single strains excreted hydroxamates different from aerobactin . Besides, we analyzed if the strains had the ability to produce the siderophore yersiniabactin coded by the Yersinia high-pathogenicity island (HPI) . The presence of HPI genes was observed in single isolates of three species: E . cloaceae, E . aerogenes and C . koseri . A detailed polymerase chain reaction analysis revealed differences in the genetic organization of the HPIs; however, in a cross-feeding test we proved that yersiniabactin was produced and the island was functional.

Arch Microbiol, 2004 Feb, 181(2), 163 - 70 Epub 2004 Jan 10.
Utilization of aminoaromatic acids by a methanogenic enrichment culture and by a novel Citrobacter freundii strain; Savelieva O et al.; Following incubation of mesophilic methanogenic floccular sludge from a lab-scale upflow anaerobic sludge bed reactor used to treat cattle manure wastewater, a stable 5-aminosalicylate-degrading enrichment culture was obtained . Subsequently, a Citrobacter freundii strain, WA1, was isolated from the 5-aminosalicylate-degrading methanogenic consortium . The methanogenic enrichment culture degraded 5-aminosalicylate completely to CH4, CO2 and NH4+, while C . freundii strain WA1 reduced 5-aminosalicylate with simultaneous deamination to 2-hydroxybenzyl alcohol during anaerobic growth with electron donors such as pyruvate, glucose or serine . When grown on pyruvate, C . freundii WA1 converted 3-aminobenzoate to benzyl alcohol and also reduced benzaldehyde to benzyl alcohol . Pyruvate was fermented to acetate, CO2, H2 and small amounts of lactate, succinate and formate . Less lactate (30%) was produced from pyruvate when C . freundii WA1 grew with 5-aminosalicylate as co-substrate.

Cell Prolif, 2003 Dec, 36(6), 361 - 75
Dietary pectin and calcium inhibit colonic proliferation in vivo by differing mechanisms; Umar S et al.; Diet plays an important role in promoting and/or preventing colon cancer; however, the effects of specific nutrients remain uncertain because of the difficulties in correlating epidemiological and basic observations . Transmissible murine colonic hyperplasia (TMCH) induced by Citrobacter rodentium, causes significant hyperproliferation and hyperplasia in the mouse distal colon and increases the risk of subsequent neoplasia . We have recently shown that TMCH is associated with an increased abundance of cellular beta-catenin and its nuclear translocation coupled with up-regulation of its downstream targets, c-myc and cyclin D1 . In this study, we examined the effects of two putatively protective nutrients, calcium and soluble fibre pectin, on molecular events linked to proliferation in the colonic epithelium during TMCH . Dietary intervention incorporating changes in calcium {high (1.0%) and low (0.1%)} and alterations in fibre content (6% pectin and fibre-free) were compared with the standard AIN-93 diet (0.5% calcium, 5% cellulose), followed by histomorphometry and immunochemical assessment of potential oncogenes . Dietary interventions did not alter the time course of Citrobacter infection . Both 1.0% calcium and 6% pectin diet inhibited increases in proliferation and crypt length typically seen in TMCH . Neither the low calcium nor fibre-free diets had significant effect . Pectin diet blocked increases in cellular beta-catenin, cyclin D1 and c-myc levels associated with TMCH by 70%, whereas neither high nor low calcium diet had significant effect on these molecules . Diets supplemented with either calcium or pectin therefore, exert anti-proliferative effects in mouse distal colon involving different molecular pathways . TMCH is thus a diet-sensitive model for examining the effect of specific nutrients on molecular characteristics of the pre-neoplastic colonic epithelium.

Carbohydr Res, 2004 Jan 22, 339(2), 321 - 5
Structure of the O-polysaccharide of Citrobacter youngae O1 containing an alpha-D-ribofuranosyl group; Kocharova NA et al.; The lipopolysaccharide of Citrobacter youngae O1, strain PCM 1492 was degraded with acid or alkali under mild conditions, and the resultant polysaccharide was isolated by GPC and studied by sugar and methylation analyses and 1H and 13C NMR spectroscopies, including 2D COSY, TOCSY, NOESY and 1H, 13C HSQC experiments . The following structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established: {structure: see text} where substitution with the alpha-D-Ribf group is nonstoichiometric . This group occurs rarely in bacterial polysaccharides and is easily cleaved under mild acidic conditions . Studies with polyclonal rabbit antisera against whole cells of C . youngae PCM 1492 and PCM 1506 showed the serological identity of the lipopolysaccharides of C . youngae PCM 1492, PCM 1493 and PCM 1506, which are classified in serogroup O1.

J Immunol, 2004 Jan 1, 172(1), 433 - 41
Critical role of T cell-dependent serum antibody, but not the gut-associated lymphoid tissue, for surviving acute mucosal infection with Citrobacter rodentium, an attaching and effacing pathogen; Bry L et al.; Citrobacter rodentium uses virulence factors similar to the enteropathogenic Escherichia coli to produce attaching and effacing lesions in the distal colon of mice . We used this infection model to determine components of adaptive immunity needed to survive infection . During acute infection, wild-type mice develop breaks across infected epithelial surfaces but resolve infection . Surprisingly, mice markedly deficient in mucosal lymphocyte populations from beta(7) integrin deficiency resolve infection, as do CD8alpha-/- or TCR-delta-/- mice . In contrast, CD4-/- or TCR-beta-/- mice develop polymicrobial sepsis and end-organ damage, and succumb during acute infection, despite epithelial damage similar to wild-type mice . B cell-deficient (MuMT-/-) or B and T cell-deficient (recombinase-activating gene 2-/-) mice develop severe pathology in colon and internal organs, and deteriorate rapidly during acute infection . Surviving mice develop robust Citrobacter-specific serum IgM responses during acute infection, whereas mice that succumb do not . However, CD4-/- mice receiving serum Igs from infected wild-type mice survive and clear the infection . Our data show that survival of apparently self-limited and luminal mucosal infections requires a systemic T cell-dependent Ab response against bacteria that enter through damaged mucosa . These findings have implications for understanding host defense against mucosal infections, including the pathogenesis of these diseases in immunocompromised populations.

Acta Crystallogr D Biol Crystallogr, 2004 Jan, 60(Pt 1), 125 - 8 Epub 2003 Dec 18.
Crystallization and preliminary X-ray diffraction study of the class A beta-lactamase SED-1 and its mutant SED-G238C from Citrobacter sedlakii; Petrella S et al.; SED-1, a class A beta-lactamase from Citrobacter sedlakii, is a CTX-M-type extended-spectrum beta-lactamase that has the ability to hydrolyze expanded-spectrum cephalosporins such as cefotaxime . SED-1 and a SED mutant in which Gly238 has been replaced by a cysteine, forming a disulfide bridge with the other Cys residue located at position 69 (SED-G238C), have been crystallized . The crystals belong to the monoclinic space group C2, with unit-cell parameters a = 188.09, b = 73.65, c = 105.41 A, beta = 121.67 degrees for SED-1 and a = 187.64, b = 73.2, c = 103.89 A, beta = 121.89 degrees for the SED-G238C mutant . X-ray diffraction data were collected to maximum resolutions of 2.4 A for SED-1 and 2.0 A for SED-G238C.

Ann Chim, 2003 Sep-Oct, 93(9-10), 729 - 37
Bioremediation of soil contaminated with organic compounds with special reference to acrylonitrile; Deshkar A et al.; Enrichment of acrylonitrile (AN) degrading bacterial culture from contaminated soil resulted in the isolation of two cultures which were identified as gram negative small rods (C1) and gram positive cocci (C2) . One of the cultures (C1) was identified as Citrobacter fruendii on the basis of biochemical and physiological tests . Both the cultures (C1 and C2) were able to utilize acrylonitrile up to a concentration of 2000 mg/l as the sole source of carbon and nitrogen . The studies also confirmed that the acrylonitrile contaminated soil when ploughed with well mixed AN degrading culture, diammonium phosphate and farmyard manure, could be completely remediated within two months from the date of soil amendment.

Medicine (Baltimore), 2003 Nov, 82(6), 385 - 91
Etiologic diagnosis of 204 pericardial effusions; Levy PY et al.; The etiologic evaluation of pericardial effusion is frequently unsuccessful when noninvasive methods are used . To determine the cause of the current episode, all patients with echographically identified pericardial effusion from May 1998 to December 2002 underwent noninvasive diagnostic testing of blood, throat, and stool samples . Patients with postpericardiotomy syndrome were excluded . To analyze the value of our tests, we tested randomly selected blood donors as negative controls . Among 204 included patients, 107 (52.4%) had a final etiologic diagnosis: the etiology of 52 was highly suspected at first examination and later confirmed (thyroid deficiency, 5 cases; systemic lupus erythematous, 7; rheumatoid arthritis, 7; scleroderma, 3; cancer, 25; and renal insufficiency, 5) . A definite etiologic diagnosis was made in 11 patients from pericardial fluid analysis (cancer, 5 cases; tuberculosis, 3; Streptococcus pneumoniae, Citrobacter freundii, and Actinomyces, 1 case each) . Among 141 patients considered to have idiopathic pericarditis, 44 (32.1%) gained an etiologic diagnosis by our systematic testing strategy . This included serologic evaluation of serum (Coxiella burnetii, 10 cases; Bartonella quintana, 1; Legionella pneumophila, 1; Mycoplasma pneumoniae, 4; influenza virus, 1), viral culture of throat swabs (enterovirus, 8 cases; and adenovirus, 1), high-level antinuclear antibodies (>1/400, 3 cases), and thyroid-stimulating hormone (15 abnormal results) . Antibodies to Toxoplasma and cytomegalovirus, enterovirus recovered from rectal swabs, and low-level antinuclear antibodies were seen with equal frequency in patients and controls.Using our evaluation strategy, the number of pericardial effusions classified as idiopathic was less than in other series . Systematic testing for Q fever, Mycoplasma pneumoniae, thyroid abnormalities, and antinuclear antibodies, accompanied by viral throat cultures, frequently enabled us to diagnose diseases not initially suspected in patients with pericardial effusion.

J Biol Chem, 2004 Mar 5, 279(10), 9344 - 52 Epub 2003 Dec 03.
Hydrolysis of third-generation cephalosporins by class C beta-lactamases . Structures of a transition state analog of cefotoxamine in wild-type and extended spectrum enzymes; Nukaga M et al.; Bacterial resistance to the third-generation cephalosporins is an issue of great concern in current antibiotic therapeutics . An important source of this resistance is from production of extended-spectrum (ES) beta-lactamases by bacteria . The Enterobacter cloacae GC1 enzyme is an example of a class C ES beta-lactamase . Unlike wild-type (WT) forms, such as the E . cloacae P99 and Citrobacter freundii enzymes, the ES GC1 beta-lactamase is able to rapidly hydrolyze third-generation cephalosporins such as cefotaxime and ceftazidime . To understand the basis for this ES activity, m-nitrophenyl 2-(2-aminothiazol-4-yl)-2-{(Z)-methoxyimino}acetylaminomethyl phosphonate has been synthesized and characterized . This phosphonate was designed to generate a transition state analog for turnover of cefotaxime . The crystal structures of complexes of the phosphonate with both ES GC1 and WT C . freundii GN346 beta-lactamases have been determined to high resolution (1.4-1.5 Angstroms) . The serine-bound analog of the tetrahedral transition state for deacylation exhibits a very different binding geometry in each enzyme . In the WT beta-lactamase the cefotaxime-like side chain is crowded against the Omega loop and must protrude from the binding site with its methyloxime branch exposed . In the ES enzyme, a mutated Omega loop adopts an alternate conformation allowing the side chain to be much more buried . During the binding and turnover of the cefotaxime substrate by this ES enzyme, it is proposed that ligand-protein contacts and intra-ligand contacts are considerably relieved relative to WT, facilitating positioning and activation of the hydrolytic water molecule . The ES beta-lactamase is thus able to efficiently inactivate third-generation cephalosporins.

Biochem Biophys Res Commun, 2003 Dec 26, 312(4), 914 - 21
Mutagenesis of SugE, a small multidrug resistance protein; Son MS et al.; The small multidrug resistance protein family has two subclasses . In this study we used a mutation approach to see what is necessary to convert a SUG subgroup member into a quaternary ammonium compound (QAC) transporter . We chose four key residues (H24, M39, I43, and A44) conserved within SUGs but conserved differently within the QAC transporters . Altogether, seven mutants were generated in Citrobacter freundii SugE . Surprisingly, the mutated SugE demonstrated an increased sensitivity to representative QACs . Additionally, ethidium uptake is found to be more prominent in the hypersensitive mutants . We conducted orientation studies using topology reporter gene fusions which indicated that SugE and the QAC transporter EmrE both have their N- and C-termini in the cytoplasm as predicted . The results imply that SugE can be converted to a QAC transporter with only a single mutation . However, because hypersensitivity was observed, the SugE mutant proteins are behaving as importers rather than as exporters.

J Evol Biol, 2003 Nov, 16(6), 1236 - 48
A molecular phylogeny of enteric bacteria and implications for a bacterial species concept; Wertz JE et al.; A molecular phylogeny for seven taxa of enteric bacteria (Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca, Klebsiella pneumoniae, and Serratia plymuthica) was made from multiple isolates per taxa taken from a collection of environmental enteric bacteria . Sequences from five housekeeping genes (gapA, groEL, gyrA, ompA, and pgi) and the 16S rRNA gene were used to infer individual gene trees and were concatenated to infer a composite molecular phylogeny for the species . The isolates from each taxa formed tight species clusters in the individual gene trees, suggesting the existence of 'genotypic' clusters that correspond to traditional species designations . These sequence data and the resulting gene trees and consensus tree provide the first data set with which to assess the utility of the recently proposed core genome hypothesis (CGH) . The CGH provides a genetically based approach to applying the biological species concept to bacteria.

FEMS Microbiol Lett, 2003 Nov 21, 228(2), 175 - 9
Antimicrobial resistance in Gram-negative bacilli isolated from infant formulas; Carneiro LA et al.; A total of 90 samples of infant formula (IF) were collected from the lactary of a teaching hospital, during a 4-month period from July to August 1999 . The sanitary conditions of the formulas were analyzed, and a physiological characterization of Gram-negative bacillus isolates and antimicrobial susceptibility testing were performed . Colony counts were considered to be unacceptable for the majority of the IF samples and the contamination rates were related to inadequate handling . Coliforms (35 degrees C and 45 degrees C growth) were detected in most of the IF tested . Klebsiella pneumoniae, Citrobacter freundii, Cedacea davisae, Klebsiella planticola and Enterobacter cloacae were the isolates most commonly identified . Antimicrobial susceptibility testing showed significant resistance rates, particularly to amoxicillin/clavulanic acid, cefoxitin, cephalotin or ampicillin . One extended-spectrum beta-lactamase-producing K . pneumoniae strain was also recovered.

Braz J Infect Dis, 2003 Dec, 7(6), 429 - 32 Epub 2004 Mar 01.
Fulminant citrobacter meningitis with multiple periventricular abscesses in a three-month-old infant; Anoop P et al.; Citrobacter, a Gram-negative enteric bacillus, is a rare cause of septicemia and meningitis, seldom reported beyond the neonatal period . It is characterized by a fulminant clinical course and a high incidence of complications, including brain abscesses . We studied a three-month-old infant with Citrobacter meningitis, who developed acute communicating hydrocephalus and multiple periventricular brain abscesses while on treatment . The patient died, despite intensive antibiotic treatment directed towards the causative organism, C . diversus.

Braz J Infect Dis, 2003 Dec, 7(6), 360 - 9 Epub 2004 Mar 01.
The use of molecular typing to evaluate the dissemination of antimicrobial resistance among Gram-negative rods in Brazilian hospitals; Tosin I et al.; Antimicrobial resistance has increased rapidly in Brazil and worldwide during the past few years, giving rise to a growing necessity for antimicrobial resistance surveillance programs . These programs have been instituted in order to monitor bacterial resistance in various regions, and to guide empirical antimicrobial therapy . We evaluated the use of molecular typing in multicenter surveillance programs . We also studied the dissemination modes of selected resistance profiles . Antimicrobial susceptibility to various antimicrobial agents was evaluated by the reference broth microdilution method . Bacterial isolates with selected susceptibility patterns were characterized by pulsed field-gel electrophoresis (PFGE) . A total of 119 Gram-negative bacteria were molecularly typed, including 22 imipenem-resistant Pseudomonas aeruginosa, 26 ESBL-producing Escherichia coli, 27 cefoxitin-resistant-ESBL-producing Klebsiella pneumoniae, 33 Enterobacter spp., 8 Citrobacter spp., and 3 S . marcescens isolates resistant to ceftazidime . The isolates were from clinically apparent bacteremia of patients hospitalized in medical centers located in 13 cities of 11 Brazilian states . Our molecular typing results revealed a great genetic diversity among isolates of the same species . However, some major PFGE patterns were found in more than one isolate . All repeated PFGE patterns were detected in only 2 isolates, which were isolated within the same institutions or in different medical centers . We conclude that the ability to characterize organisms phenotypically and genotypically is a powerful epidemiologic tool and it provides unique information that is very important for multicenter surveillance programs.

Lik Sprava, 2003 Jul-Aug, (5-6), 46 - 8
{Plasmid profile of common neonatal sepsis causative agents}; Madaminov MS et al.; The plasmid profile of 25 hospital strains of Gram-negative bacteria that are known to be originators of neonatal sepsis was studied by the authors . Of these, E . coli, Enterobacter spp., and Citrobacter spp . proved to be the most frequently seen causative agents in the above health problem, with 13, 5, and 7 strains respectively having been recovered in the studied material . There have been isolated 26 plasmids from the studied 25 bacterial strains . Of these, fifty percent were classified as Citrobacter spp . The molecular mass of the plasmids recovered has been found to vary among 30 and 120.

J Antimicrob Chemother, 2003 Dec, 52(6), 1015 - 7 Epub 2003 Nov 12.
SHV-34: an extended-spectrum beta-lactamase encoded by an epidemic plasmid; Heritage J et al.; OBJECTIVES: To elucidate the causes for treatment failure in children given extended-spectrum cephalosporins . METHODS: During April 1998-March 2000, 18 isolates of members of the family Enterobacteriaceae, fulfilling microbiological criteria for carriage of extended-spectrum beta-lactamases (ESBLs) and carrying blaSHV, were isolated from paediatric inpatients . The collection was subjected to a retrospective molecular analysis . RESULTS: Three species were represented in the collection: Citrobacter koseri (one isolate), Escherichia coli (one isolate) and Klebsiella pneumoniae (16 isolates) . A common plasmid was found in these bacteria, as judged by restriction endonuclease digestion . This was able to transfer an ESBL phenotype from donors to a laboratory strain of E . coli . Nucleotide sequence analysis revealed that this phenotype was associated with a new variant in blaSHV encoding SHV-34 . CONCLUSIONS: Analysis reveals the presence of an epidemic plasmid in this collection of bacteria . This carries a gene encoding the SHV-34 ESBL, described for the first time in this report . Nucleotide sequence analysis shows that there is a mutation from A-->G affecting the codon at amino acid position 64 (GAA-->GGA), changing the glutamic acid typically seen in this position to glycine.

Scand J Infect Dis, 2003, 35(10), 765 - 8
Citrobacter bacteremia in a tertiary care hospital; Gupta N et al.; From January 1998 to December 2001, 176 cases of Citrobacter bacteremia occurred of which repeat isolation was possible in 48 cases . Of 48 isolates, 79.1% were C . diversus and 20.9% C . freundii . Citrobacter bacteremia was polymicrobial in 46.1% cases, and maximum number of cases (54.1%) occurred in the age group less than 10 years . Portal of entry was unknown (42.3%), respiratory tract (20.9%), gastrointestinal tract (15.3%) and urinary tract 15.3% . C . freundii isolates were relatively more resistant than C . diversus against 10 tested antimicrobial agents, while 79.1% isolates were multiresistant . Sensitivity based on MIC was highest for ceftizoxime, ciprofloxacin and cefotaxime . Overall mortality of Citrobacter bacteremia was seen in 56% of cases . Therefore greater caution is required in selection of antibiotic therapy in order to avoid selection of strains and treatment failure.

Mikrobiyol Bul, 2003 Apr-Jun, 37(2-3), 157 - 62
{Activity of frequently used disinfectants and antiseptics against nosocomial bacterial types}; Ozkurt Z et al.; In this study, bactericidal activity of widely used disinfectants and antiseptics (glutaraldehyde, laurylbispropylidentriamin 5 g and benzalkoniumchlorid 20 g, polyvinylpyrolidon iodine, benzalkonium chloride and sodium hypochloride) against some nosocomial bacterial isolates were investigated by qualitative and quantitative suspension test methods . One methicillin-resistant Staphylococcus aureus (MRSA), 6 multi drug-resistant Gram-negative hospital isolates (Escherichia coli, Pseudomonas aeruginosa, Enterobacter cloaca, Klebsiella pneumoniae, Citrobacter diversus, Serratia marcescens) and 3 standard strains of American Type Culture Collection (ATCC) bacteria (S . aureus, P . aeruginosa, E . coli) were tested against three different concentrations of disinfectants of which, manufacturer's recommended use-dilution, 1/2 and 1/4 of those recommended dilutions . All tested disinfectants were found effective in all three concentrations against nosocomial isolates in five minutes, by using both qualitative and quantitative suspension methods . When the test was repeated with albumine, bactericidal activities of disinfectants were found the same . Our findings showed that these disinfectants can be still used in safe for the sterilization in hospital, and routine disinfection protocols do not need to be alerted.

Acta Otorhinolaryngol Belg, 2003, 57(3), 205 - 8
Bacteriology of chronic suppurative otitis media in congolese children; Nyembue DT et al.; AIM: The study intended to identify bacteria active in the chronic suppurative otitis media and to determine their sensitivity to current antibiotics . METHODS: After clinical evaluation, middle-ear secretions were taken for bacteriological examination from 78 children meeting the inclusion criteria . All children with cholesteatoma and those with tumors occluding the ear canal were excluded . RESULTS: The most frequent isolated germs, in descending order frequency, were as follows: Proteus mirabilis (23%), Pseudomonas aeruginosa (22%), Citrobacter (20%) and Salmonella (5%) . There were no cases of mixed flora . Ofloxacin was susceptible on all isolates . Neomycin, gentamicin and polymyxin B were susceptible on 96%, 83% and 67% of the isolates respectively . All isolates were resistant to amoxycillin . CONCLUSION: Peudomonas, Proteus and Citrobacter are the most common causes of chronic otitis media among children in our community of congolese children . Ofloxacin and neomycin are the most highly effective against most of the isolated germs, and are therefore recommended as the first line local treatment . Amoxycillin and chloramphenicol should be avoided.

Infection, 2003 Aug, 31(4), 202 - 7
Resistance to extended-spectrum cephalosporins and mortality in patients with Citrobacter freundii bacteremia; Kim BN et al.; BACKGROUND: This study was performed to characterize the clinical features and to identify the risk factors for resistance to extended-spectrum cephalosporins (ESCs) and for mortality in patients with Citrobacter freundii bacteremia . PATIENTS AND METHODS: 105 patients (aged > or = 15 years) with C . freundii bacteremia in 1991-2000 were retrospectively analyzed . RESULTS: Nosocomial acquisition was identified in 78.1% of the patients . Hepatic, biliary and pancreatic disease was the most common underlying disease (65.7%) and the biliary tract was the most common site of infection (50.5%) . The overall resistance rate to ESCs was 59.0% and was significantly associated with hepatic, biliary and pancreatic disease, recent surgery and procedure, biliary drainage catheter and previous antibiotic therapy in univariate analysis . However, only previous antibiotic therapy with ESCs (OR = 5.0, 95% CI 1.6-15.7, p = 0.006) and recent surgery or procedure (OR = 3.1, 95% CI 1.1-8.4, p = 0.03) were strong, independent risk factors in multivariate analysis . Mortality directly related to C . freundii bacteremia was 21.9% and there was no difference between cases with resistance and susceptibility to ESCs (19.4% vs 25.6%; p = 0.45) . Mortality was significantly associated with rapidly fatal or ultimately fatal underlying disease, a solid tumor, septic shock and polymicrobial bacteremia in univariate analysis . Among patients who had therapeutic surgical procedures, mortality was lower (4.5%, p = 0.04) . Multivariate analysis revealed rapidly or ultimately fatal disease, septic shock and polymicrobial bacteremia as independent prognostic factors . CONCLUSION: Biliary infection was the leading cause of C . freundii bacteremia . Previous antibiotic therapy, especially with ESCs, frequently predisposed for resistance to these antibiotics . However, resistance to ESCs was not associated with increased mortality.

Carbohydr Res, 2003 Oct 10, 338(21), 2169 - 75
A practical synthesis of alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-{alpha-D-Glcp-(1-->3)}-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp, an O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c; Chen L et al.; An O-specific heterohexasaccharide fragment of Citrobacter braakii O7a, 3b, 1c, alpha-D-Manp-(1-->3)-alpha-D-Manp-(1-->2)-{alpha-D-Glcp-(1-->3)}-alpha-D-Manp-(1-->2)-alpha-D-Manp-(1-->2)-alpha-D-Manp was synthesized as its methyl glycoside . Acetylation of allyl 4,6-O-benzylidene-alpha-D-mannopyranoside, followed by debenzylidenization and benzoylation gave allyl 2,3-di-O-acetyl-4,6-di-O-benzoyl-alpha-D-mannopyranoside (3), and subsequent deacetylation of 3 with CH(3)COCl-MeOH gave the monosaccharide acceptor 4 . Condensation of isopropyl 2,3,4,6-tetra-O-benzyl-1-thio-beta-D-glucopyranoside (6) with 4 selectively afforded the alpha-(1-->3)-linked disaccharide 7 . Condensation of 7 with the (1-->3)-linked disaccharide donor 9, followed by deallylation and trichloroacetimidation, afforded the tetrasaccharide donor 12 . Coupling of 12 with disaccharide acceptor 13, followed by debenzylation and deacylation, furnished the target heterohexasaccharide 16.

Biotechnol Adv, 1993, 11(3), 481 - 93
Tempe fermentation: some aspects of formation of gamma-linolenic acid, proteases and vitamins; Bisping B et al.; During a tempe fermentation the concentrations of linoleic, and alpha-linolenic acids (ALA) decreased while the concentration of oleic acid increased . During fatty acid synthesis Rhizopus sp . produced only gamma-linolenic acid (GLA) instead of ALA . The amount of GLA in tempe were influenced by varying external parameters.The proteolytic capacity of 36 strains of the genus Rhizopus isolated from Indonesian tempe or tempe inocula was examined . There was a distinct increase in the amount of free amino acids during tempe fermentation . Fermentations with mixed populations of bacteria and Rhizopus yielded a lower level of free amino acids, but an increase in total amount of amino acids . In comparison to intracellular, and extracellular proteases the proteases of the cell wall fraction are most responsible for proteolytic capacity of the different Rhizopus strains.Two isolated strains of Citrobacter freundii were found to be the best vitamin B(12) producers during the soaking of soybeans . In the solid substrate fermentation the Rhizopus molds formed vitamin B(6), riboflavin, and nicotinic acid . The addition of bacteria to the solid substrate fermentation resulted in a strong increase of active vitamin B(12) in tempe . In the presence of the Rhizopus mold, the vitamin B(12) formation by C . freundii was three times higher than that of a fermentation without the mold.

Int J Antimicrob Agents, 2003 Oct, 22(4), 406 - 19
Epidemiology and antibiotic susceptibility of bacteria causing skin and soft tissue infections in the USA and Europe: a guide to appropriate antimicrobial therapy; Jones ME et al.; Susceptibility data for all organisms associated with a range of skin and soft tissue infections (SSTI) in hospitalised patients were studied . Data were reported by clinical laboratories in the USA, France, Germany, Italy and Spain during 2001 which participate in The Surveillance Network (TSN) . Staphylococcus aureus, Enterococcus spp . and coagulase-negative staphylococci (CNS), Escherichia coli and Pseudomonas aeruginosa were the most prevalent pathogens in all countries . MRSA was detected in 44.4, 34.7, 12.4, 41.8 and 32 . 4% of S . aureus in each country, respectively . The majority of MRSA were cross resistant to other compound classes tested except for vancomycin (100% susceptible) trimethoprim-sulphamethoxazole with range 1.7% (France) to 15.9% (Italy) resistant, and gentamicin with range 12.2% (France) to 87.0% (Italy) resistant . More than 99.0% of MSSA tested susceptible to ceftriaxone and >94.9% to trimethoprim-sulphamethoxazole . 87.2% (France) to 94.6% of MSSA (Germany) were ciprofloxacin susceptible; 73.2% (USA) to 86.6% (Spain) were erythromycin susceptible; 85.4% (Italy) to 99.2% (France) were gentamicin susceptible . MSSA were more frequently found and generally more antibiotic susceptible from out patients . Overall, 100% of Streptococcus agalactiae and Streptococcus pyogenes were susceptible to penicillin, ceftriaxone and cefotaxime . Macrolide resistance was common among S . agalactiae (20.7%, Germany to 10%, Italy and Spain), S . pyogenes (19.2%, France to 11.1%, USA) and viridans streptococci (25.7%, France to 14.1%, Germany) . Vancomycin-resistant Enterococcus spp . were uncommon outside the USA (17.5%) and Italy (7.4%) . For all countries susceptibility of E . coli was 100% to imipenem, >98.7% to amikacin, >96.0% to ceftriaxone and cefotaxime . Susceptibility of E . coli isolates to ciprofloxacin was 77.6% in Spain to 94.3% in Germany . Klebsiella spp., Proteus spp., Citrobacter spp . and Enterobacter spp . displayed varying susceptibilities between countries to drugs tested . Putative extended spectrum beta-lactamase expression in E . coli remained rare comprising 4-5% of isolates in USA, Italy and Spain and in France and Germany <2% . For P . aeruginosa piperacillin-tazobactam, amikacin, imipenem and ceftazidime were the most active compounds tested irrespective of region . Surveillance data should be considered when selecting empirical therapy for treating SSTI.

South Med J, 2003 Aug, 96(8), 796 - 8
Sepsis in a renal transplant recipient due to Citrobacter braakii; Gupta R et al.; Cellulitis is usually caused by organisms such as beta-hemolytic streptococci and Staphylococcus aureus . Citrobacter are gram-negative bacilli that can cause opportunistic infections in immunocompromised hosts . They are rarely implicated in skin or soft tissue infections . The genus Citrobacter has been respeciated according to genetic relatedness . Citrobacter braakii refers to the genomospecies 6 of the Citrobacter freundii complex . There are no detailed studies of infections caused by the newly formed specific genetic species . We report a case of C . braakii infection in a renal transplant patient receiving immunosuppressive therapy . The patient's lower extremity cellulitis did not respond to conventional antibiotic therapy . Blood cultures grew C . braakii . Sensitivity studies and treatment with appropriate antibiotics resulted in prompt recovery . Immunosuppressive therapy in renal transplant recipients predisposes to infection by unusual pathogens, and this should be suspected when lack of a clinical response to conventional antibiotics is observed . We believe this is the first reported case of C . braakii cellulitis and bacteremia in a renal transplant recipient.

Biotechnol Lett, 2003 Aug, 25(15), 1231 - 4
Isolation and characterization of a phytase with improved properties from Citrobacter braakii; Kim HW et al.; Citrobacter braakii YH-15 produced an intracellular phytase which was purified 12800 fold to homogeneity with the specific activity of 3457 units mg(-1), which is 1.9 times higher than E . coli phytase previously recorded as having the highest specific activity . Its molecular weight was 47 kDa by SDS-PAGE gel . Enzyme activity was optimal at pH 4 and at 50 degrees C . The Km value for sodium phytate was 0.46 mM with a Vmax 6027 U mg(-1) . The phytase was resistant to proteases such as trypsin, pepsin, papain, pancreatin, and elastase.

Biochem J, 2004 Jan 1, 377(Pt 1), 111 - 20
Mutational analysis of the catalytic centre of the Citrobacter freundii AmpD N-acetylmuramyl-L-alanine amidase; Genereux C et al.; Citrobacter freundii AmpD is an intracellular 1,6-anhydro-N-acetylmuramyl-L-alanine amidase involved in both peptidoglycan recycling and beta-lactamase induction . AmpD exhibits a strict specificity for 1,6-anhydromuropeptides and requires zinc for enzymic activity . The AmpD three-dimensional structure exhibits a fold similar to that of another Zn2+ N-acetylmuramyl-L-alanine amidase, the T7 lysozyme, and these two enzymes define a new family of Zn-amidases which can be related to the eukaryotic PGRP (peptidoglycan-recognition protein) domains . In an attempt to assign the different zinc ligands and to probe the catalytic mechanism of AmpD amidase, molecular modelling based on the NMR structure and site-directed mutagenesis were performed . Mutation of the two residues presumed to act as zinc ligands into alanine (H34A and D164A) yielded inactive proteins which had also lost their ability to bind zinc . By contrast, the active H154N mutant retained the capacity to bind the metal ion . Three other residues which could be involved in the AmpD catalytic mechanism have been mutated (Y63F, E116A, K162H and K162Q) . The E116A mutant was inactive, but on the basis of the molecular modelling this residue is not directly involved in the catalytic mechanism, but rather in the binding of the zinc by contributing to the correct orientation of His-34 . The K162H and K162Q mutants retained very low activity (0.7 and 0.2% of the wild-type activity respectively), whereas the Y63F mutant showed 16% of the wild-type activity . These three latter mutants exhibited a good affinity for Zn ions and the substituted residues are probably involved in the binding of the substrate . We also describe a new method for generating the N-acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-tripeptide AmpD substrate from purified peptidoglycan by the combined action of two hydrolytic enzymes.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3260 - 9
In vitro antibacterial potency and spectrum of ABT-492, a new fluoroquinolone; Nilius AM et al.; ABT-492 demonstrated potent antibacterial activity against most quinolone-susceptible pathogens . The rank order of potency was ABT-492 > trovafloxacin > levofloxacin > ciprofloxacin against quinolone-susceptible staphylococci, streptococci, and enterococci . ABT-492 had activity comparable to those of trovafloxacin, levofloxacin, and ciprofloxacin against seven species of quinolone-susceptible members of the family Enterobacteriaceae, although it was less active than the comparators against Citrobacter freundii and Serratia marcescens . The activity of ABT-492 was greater than those of the comparators against fastidious gram-negative species, including Haemophilus influenzae, Moraxella catarrhalis, Neisseria gonorrhoeae, and Legionella spp . and against Pseudomonas aeruginosa and Helicobacter pylori . ABT-492 was as active as trovafloxacin against Chlamydia trachomatis, indicating good intracellular penetration and antibacterial activity . In particular, ABT-492 was more active than trovafloxacin and levofloxacin against multidrug-resistant Streptococcus pneumoniae, including strains resistant to penicillin and macrolides, and H . influenzae, including beta-lactam-resistant strains . It retained greater in vitro activity than the comparators against S . pneumoniae and H . influenzae strains resistant to other quinolones due to amino acid alterations in the quinolone resistance-determining regions of the target topoisomerases . ABT-492 was a potent inhibitor of bacterial topoisomerases, and unlike the comparators, DNA gyrase and topoisomerase IV from either Staphylococcus aureus or Escherichia coli were almost equally sensitive to ABT-492 . The profile of ABT-492 suggested that it may be a useful agent for the treatment of community-acquired respiratory tract infections, as well as infections of the urinary tract, bloodstream, and skin and skin structure and nosocomial lung infections.

Infect Immun, 2003 Oct, 71(10), 5871 - 80
Citrobacter koseri brain abscess in the neonatal rat: survival and replication within human and rat macrophages; Townsend SM et al.; A unique feature of Citrobacter koseri is the extremely high propensity to initiate brain abscesses during neonatal meningitis . Previous clinical reports and studies on infant rats have documented many Citrobacter-filled macrophages within the ventricles and brain abscesses . It has been hypothesized that intracellular survival and replication within macrophages may be a mechanism by which C . koseri subverts the host response and elicits chronic infection, resulting in brain abscess formation . In this study, we showed that C . koseri causes meningitis and brain abscesses in the neonatal rat model, and we utilized histology and magnetic resonance imaging technology to visualize brain abscess formation . Histology and electron microscopy (EM) revealed that macrophages (and not fibroblasts, astrocytes, oligodendrocytes, or neurons) were the primary target for long-term C . koseri infection . To better understand C . koseri pathogenesis, we have characterized the interactions of C . koseri with human macrophages . We found that C . koseri survives and replicates within macrophages in vitro and that uptake of C . koseri increases in the presence of human pooled serum in a dose-dependent manner . EM studies lend support to the hypothesis that C . koseri uses morphologically different methods of uptake to enter macrophages . FcgammaRI blocking experiments show that this receptor primarily facilitates the entry of opsonized C . koseri into macrophages . Further, confocal fluorescence microscopy demonstrates that C . koseri survives phagolysosomal fusion and that more than 90% of intracellular C . koseri organisms are colocalized within phagolysosomes . The ability of C . koseri to survive phagolysosome fusion and replicate within macrophages may contribute to the establishment of chronic central nervous system infection including brain abscesses.

J Microbiol Methods, 2003 Oct, 55(1), 35 - 40
Development of a rapid 1-h fluorescence-based cytotoxicity assay for Listeria species; Shroyer ML et al.; Listeria monocytogenes is cytotoxic to the lymphocyte-origin hybridoma Ped-2E9 cell line . The relative cytotoxicity can be calculated by assaying the release of alkaline phosphatase (ALP) from the infected cell line . In this study, a fluorogenic substrate (4-methylumbelliferyl phosphate, MUP) was used to quantify the ALP activity . The assay is 3.5-fold more sensitive than the colorimetric-based assay and requires only 1 h to differentiate virulent from avirulent strains . In addition to various Listeria species, 27 different common foodborne or clinical microorganisms were tested with the fluorescence-based cytotoxicity assay and only six cultures (Bacillus cereus, Citrobacter freundii, Serratia marcescens, Pseudomonas putida, Corynebacterium glutamicum and Micrococcus luteus) showed cytotoxic effects similar to L . monocytogenes . To use this assay as a confirmatory test for virulent L . monocytogenes suspect strains, pure cultures must be isolated from the sample prior to testing.

Int J Antimicrob Agents, 2003 Sep, 22(3), 223 - 7
Enhancement of plasmid curing by 9-aminoacridine and two phenothiazines in the presence of proton pump inhibitor 1-(2-benzoxazolyl)-3,3,3-trifluoro-2-propanone; Spengler G et al.; Plasmid-containing bacteria often cause serious therapeutic failure during the treatment of infectious diseases . The selection of resistant-mutant strains and the transfer of mobile genetic determinants (such as plasmids and transposons) of resistance promote increased antibiotic resistance . In the last 30 years the antiplasmid effect of acridine dyes, ethidium bromide, sodium dodecyl sulphate and phenothiazines was described . The main aim of this study was to test the mechanism of the antiplasmid effect of promethazine and 9-aminoacridine on doxycycline-resistant enteric bacteria . The antiplasmid effects of promethazine and 9-aminoacridine were studied on plasmid elimination of native plasmid DNA and plasmid DNA isolated from drug-treated cells of plasmid-containing Escherichia coli, Citrobacter freundii and Enterobacter cloacae . The effects of some phenothiazines on plasmid profiles of bacterial strains isolated from urinary tract infections were analysed by agarose gel electrophoresis . Various complex of plasmid DNA were identified in the presence of promethazine, trifluoperazine and 9-aminoacridine in the agarose gel electrophoresis . Doxycycline resistance of tested enteric bacteria was the target of "curing" in the presence of promethazine and trifluoperazine . The frequency of elimination of tetracycline resistance was low despite the formation of antiplasmid compounds complex with isolated plasmid DNA . Tetracycline resistance plasmid was isolated and re-transformed . The plasmid curing effects of promethazine, trifluoperazine and 9-aminoacridine were increased in the presence of a trifluoroketone proton pump inhibitor on E . coli K12 LE140 strain in a model experiment . We propose that the inefficient penetration of antiplasmid compounds could be responsible for the weak plasmid-curing effect in some clinical isolates and that membrane active, calmodulin- and proton pump inhibitors may be combined for plasmid curing in antibiotic-resistant bacteria.

J Biol Chem, 2003 Nov 28, 278(48), 48236 - 44 Epub 2003 Sep 09.
Crystal structure of the Citrobacter freundii dihydroxyacetone kinase reveals an eight-stranded alpha-helical barrel ATP-binding domain; Siebold C et al.; Dihydroxyacetone kinases are a sequence-conserved family of enzymes, which utilize two different phosphoryldonors, ATP in animals, plants and some bacteria, and a multiphosphoprotein of the phosphoenolpyruvate carbohydrate phosphotransferase system in bacteria . Here we report the 2.5-A crystal structure of the homodimeric Citrobacter freundii dihydroxyacetone kinase complex with an ATP analogue and dihydroxyacetone . The N-terminal domain consists of two alpha/beta-folds with a molecule of dihydroxyacetone covalently bound in hemiaminal linkage to the N epsilon 2 of His-220 . The C-terminal domain consists of a regular eight-helix alpha-barrel . The eight helices form a deep pocket, which includes a tightly bound phospholipid . Only the lipid headgroup protrudes from the surface . The nucleotide is bound on the top of the barrel across from the entrance to the lipid pocket . The phosphate groups are coordinated by two Mg2+ ions to gamma-carboxyl groups of aspartyl residues . The ATP binding site does not contain positively charged or aromatic groups . Paralogues of dihydroxyacetone kinase also occur in association with transcription regulators and proteins of unknown function pointing to biological roles beyond triose metabolism.

Appl Environ Microbiol, 2003 Sep, 69(9), 5336 - 42
Similarity of tetracycline resistance genes isolated from fish farm bacteria to those from clinical isolates; Furushita M et al.; Tetracycline-resistant (Tet(r)) bacteria were isolated from fishes collected at three different fish farms in the southern part of Japan in August and September 2000 . Of the 66 Tet(r) gram-negative strains, 29 were identified as carrying tetB only . Four carried tetY, and another four carried tetD . Three strains carried tetC, two strains carried tetB and tetY, and one strain carried tetC and tetG . Sequence analyses indicated the identity in Tet(r) genes between the fish farm bacteria and clinical bacteria: 99.3 to 99.9% for tetB, 98.2 to 100% for tetC, 99.7 to 100% for tetD, 92.0 to 96.2% for tetG, and 97.1 to 100% for tetY . Eleven of the Tet(r) strains transferred Tet(r) genes by conjugation to Escherichia coli HB-101 . All transconjugants were resistant to tetracycline, oxycycline, doxycycline, and minocycline . The donors included strains of Photobacterium, Vibrio, Pseudomonas, Alteromonas, Citrobacter, and Salmonella spp., and they transferred tetB, tetY, or tetD to the recipients . Because NaCl enhanced their growth, these Tet(r) strains, except for the Pseudomonas, Citrobacter, and Salmonella strains, were recognized as marine bacteria . Our results suggest that tet genes from fish farm bacteria have the same origins as those from clinical strains.

Antimicrob Agents Chemother, 2003 Sep, 47(9), 2882 - 7
Epidemiological risk factors for isolation of ceftriaxone-resistant versus -susceptible citrobacter freundii in hospitalized patients; Kim PW et al.; Antimicrobial resistance is an emerging problem among nosocomial bacteria . Risk factors for the recovery of ceftriaxone-resistant (CRCF) or -susceptible (CSCF) Citrobacter freundii in clinical cultures from hospitalized patients were determined by using a case-case-control study design . CRCF was isolated from 43 patients (case group 1) and CSCF was isolated from 87 patients (case group 2) over a 3-year period . Risk factors for CRCF were exposure to imipenem (odds ratio {OR}, 7.5; 95% confidence interval {CI}, 1.2 to 45.4), broad-spectrum cephalosporins (OR, 6.9; 95% CI, 1.8 to 26.7), vancomycin (OR, 3.0; 95% CI, 1.2 to 7.4), or piperacillin-tazobactam (OR, 2.6; 95% CI, 1.1 to 6.2), as well as hospital length of stay >or=1 week (OR, 3.6; 95% CI, 1.3 to 10.2) and intensive care unit (ICU) stay (OR, 2.6; 95% CI, 1.1 to 6.2) . Risk factors for CSCF were peripheral vascular disease (OR, 23.2; 95% CI, 4.3 to 124.6), AIDS (OR, 9.5; 95% CI, 1.6 to 55.5), cerebrovascular disease (OR, 4.2; 95% CI, 1.6 to 10.8), and ICU stay (OR, 3.1; 95% CI, 1.8 to 5.4).

Infect Immun, 2003 Sep, 71(9), 5077 - 86
Central role for B lymphocytes and CD4+ T cells in immunity to infection by the attaching and effacing pathogen Citrobacter rodentium; Simmons CP et al.; Citrobacter rodentium, an attaching-effacing bacterial pathogen, establishes an acute infection of the murine colonic epithelium and induces a mild colitis in immunocompetent mice . This study describes the role of T-cell subsets and B lymphocytes in immunity to C . rodentium . C57Bl/6 mice orally infected with C . rodentium resolved infection within 3 to 4 weeks . Conversely, systemic and colonic tissues of RAG1(-/-) mice orally infected with C . rodentium contained high and sustained pathogen loads, and in the colon this resulted in a severe colitis . C57Bl/6 mice depleted of CD4(+) T cells, but not CD8(+) T cells, were highly susceptible to infection and also developed severe colitis . Mice depleted of CD4(+) T cells also had diminished immunoglobulin G (IgG) and IgA antibody responses to two C . rodentium virulence-associated determinants, i.e., EspA and intimin, despite having a massively increased pathogen burden . Mice with an intact T-cell compartment, but lacking B cells ( micro MT mice), were highly susceptible to C . rodentium infection . Systemic immunity, but not mucosal immunity, could be restored by adoptive transfer of convalescent immune sera to infected micro MT mice . Adoptive transfer of immune B cells, but not naive B cells, provided highly variable immunity to recipient micro MT mice . The results suggest that B-cell-mediated immune responses are central to resolution of a C . rodentium infection but that the mechanism through which this occurs requires further investigation . These data are relevant to understanding immunity to enteric attaching and effacing bacterial pathogens of humans.

J Food Prot, 2003 Aug, 66(8), 1385 - 92
Detection of Morganella morganii, a prolific histamine former, by the polymerase chain reaction assay with 16S rDNA-targeted primers; Kim SH et al.; A polymerase chain reaction (PCR) assay for the rapid and sensitive detection of the most prolific histamine former, Morganella morganii, was developed . 16S rDNA targeted PCR primers were designed, and the primer specificity and sensitivity of the PCR assay were evaluated . The 16S rDNA sequence (1,503 bp) for M . morganii showed 95% identity to those for enteric bacteria, i.e., Enterobacter spp., Klebsiella spp., Citrobacter spp., Hafnia alvei, Proteus spp., and Providencia spp . The unique primers for M . morganii were designed on the basis of the variable regions in the 16S rDNA sequence . The primers showed positive reactions with all M . morganii strains tested . However, PCR amplification was not detected when the primers were tested with other enteric or marine bacteria . When the sensitivity of the assay was evaluated, M . morganii was detected at levels ranging from 10(6) to 10(8) CFU/ml in albacore homogenate after the PCR amplification . The sensitivity of the assay was greatly improved with the enrichment of samples, and 9 CFU of M . morganii per ml of albacore homogenate was detected after 6 h of enrichment at 37 degrees C.

Int J Antimicrob Agents, 2003 Aug, 22(2), 106 - 11
Outcome of antibiotic therapy for third-generation cephalosporin-resistant Gram-negative bacteraemia: an analysis of 249 cases caused by Citrobacter, Enterobacter and Serratia species; Kim BN et al.; Two hundred and forty-nine patients with monomicrobial bacteraemia due to third-generation cephalosporin (TGC)-resistant Citrobacter freundii (42), E . aerogenes (23), E . cloacae (143), and Serratia marcescens (41) were analyzed for antibiotic therapy used and outcome . For isolates with resistance to any of the TGCs, all beta-lactams, except imipenem, were considered ineffective . Of 152 patients given appropriate treatment, the mortality rates were 10.9% for 128 patients given monotherapy and 25.0% for 24 patients given combination therapy (P=0.09) . Of patients given monotherapy, there were no significant differences in mortality between imipenem, aminoglycoside, and ciprofloxacin treatment groups (P=0.57) . Only shock was associated with mortality in multivariate analysis . In conclusion, for patients with TGC-resistant Gram-negative bacteraemia, no significant difference in outcome was observed between single antibiotic therapy groups or monotherapy and combination therapy groups.

Int J Food Microbiol, 2003 Oct 15, 87(1-2), 29 - 34
Evaluation of increased incubation temperature and cefixime-tellurite treatment for the isolation of Escherichia coli O157:H7 from minced beef; Dogan HB et al.; To improve enrichment and isolation of Escherichia coli O157:H7, this study evaluated increased incubation temperature and cefixime-tellurite (CT) on five strains of each of the following bacteria, E . coli, Hafnia alvei, Enterobacter spp., Citrobacter freundii and E . coli O157:H7, and two strains of E . coli O157:nH7 . These were grown in pure culture in LST broth with varying cefixime-tellurite concentrations . A range of incubation temperatures from 37 to 46 degrees C was investigated for the inhibition of cohabitant microorganisms . Minced beef, spiked with E . coli O157:H7 and cohabitant microorganisms was investigated . Increased incubation temperature (42 degrees C) and treatment with half of the prescribed amount of cefixime-tellurite by BAM for SMAC agar in enrichment step were the most effective in selectively growing E . coli O157:H7 . The results show that E . coli O157:H7 is more resistant to these two conditions than the other cohabitant bacteria.

J Clin Microbiol, 2003 Aug, 41(8), 3933 - 5
Modification of the double-disk test for detection of enterobacteriaceae producing extended-spectrum and AmpC beta-lactamases; Pitout JD et al.; Detection of extended-spectrum beta-lactamases (ESBLs) in AmpC-producing Enterobacteriaceae is problematic . A modification of the double-disk test (MDDT) has been developed for successful detection of ESBLs in gram-negative bacilli producing well-characterized beta-lactamases as well as 212 clinical isolates of Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens, and Citrobacter freundii . MDDT accurately differentiated between ESBL producers and derepressed chromosomal AmpC mutants . MDDT provides a cost-effective alternative approach for clinical microbiology laboratories for routine susceptibility testing with simultaneous detection of ESBLs in enterobacteriaceae.

Antimicrob Agents Chemother, 2003 Aug, 47(8), 2669 - 73
Molecular and biochemical characterization of a novel class A beta-lactamase (HER-1) from Escherichia hermannii; Beauchef-Havard A et al.; Escherichia hermannii showed a low level of resistance to amoxicillin and ticarcillin, reversed by clavulanate, and a moderate susceptibility to piperacillin but was susceptible to all cephalosporins . A bla gene was cloned and encoded a typical class A beta-lactamase (HER-1, pI 7.5), which shares 45, 44, 41, and 40% amino acid identity with other beta-lactamases, AER-1 from Aeromonas hydrophila, MAL-1/Cko-1 from Citrobacter koseri, and TEM-1 and LEN-1, respectively . No ampR gene was detected . Only penicillins were efficiently hydrolyzed, and no hydrolysis was observed for cefuroxime and broad-spectrum cephalosporins . Sequencing of the bla gene in 12 other strains showed 98 to 100% identity with bla(HER-1).

Presse Med, 2003 Jun 14, 32(21), 985 - 7
{Purple urine bag syndrome}; Fain-Ghironi N et al.; INTRODUCTION: The report of purple discoloration in a urinary drainage system, known as Purple Urine Bag Syndrome (P.U.B.S.) is rarely described in the literature . OBSERVATION: In an 85 year-old woman, with permanent indwelling urinary catheter, the appearance of purple coloration in the urine collecting bag, without change in the colour of the urine, was observed four times in one year . During these different episodes, a Gram negative lower urinary infection diagnoses . The germs identified were Providencia stuartii and Citrobacter koseri . Symptoms resolved completely after treatment with ceftriaxone . DISCUSSION: The clinical and biological symptoms usually described in cases of P.U.B.S . are observed in the medical history of this elderly woman: indwelling catheter with delay before onset of coloration greater than 15 days following catheterization, alkaline urinary pH, Gram negative lower urinary tract infection . However, during one of the episodes of PUBS in our patient, Citrobacter koseri was identified, germ not mentioned, as far as we know, in the literature . Moreover, in the published cases, Proteus species was identified as potentially associated with P.U.B.S., but a Proteus mirabilis urinary infection with was diagnosed in our patient, without any purple coloration of the urine in the collection bag.

Eur J Biochem, 2003 Jul, 270(13), 2732 - 8
Structural and serological studies on a new 4-deoxy-d-arabino-hexose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter braakii PCM 1531 (serogroup O6); Katzenellenbogen E et al.; The O-specific polysaccharide of Citrobacter braakii PCM 1531 (serogroup O6) was isolated by mild acid hydrolysis of the lipopolysaccharide (LPS) and found to contain d-fucose, l-rhamnose, 4-deoxy-d-arabino-hexose and O-acetyl groups in molar ratios 2 : 1 : 1 : 1 . On the basis of methylation analysis and 1H and 13C NMR spectroscopy data, the structure of the branched tetrasaccharide repeating unit of the O-specific polysaccharide was established . Using various serological assays, it was demonstrated that the LPS of strain PCM 1531 is not related serologically to other known 4-deoxy-d-arabino-hexose-containing LPS from Citrobacter PCM 1487 (serogroup O5) or C . youngae PCM 1488 (serogroup O36) . Two other strains of Citrobacter, PCM 1504 and PCM 1505, which, together with strain PCM 1531, have been classified in serogroup O6, were shown to be serologically distinct from strain PCM 1531 and should be reclassified into another serogroup.

Phytomedicine, 2003 May, 10(4), 344 - 7
Bioactivity of secoiridoid glycosides from Centaurium erythraea; Kumarasamy Y et al.; As part of our on-going search for bioactive compounds from Scottish plants, two secoiridoid glycosides, swertiamarin and sweroside, have been isolated from the aerial parts of Centaurium erythraea Rafn (Family: Gentianaceae) by reversed-phase preparative HPLC coupled with a photo-diode-array detector . The structures of these compounds were elucidated unambiguously by UV, FABMS and extensive 1D and 2D NMR spectroscopic analyses and also by comparing experimental data with literature data . Antibacterial, free radical scavenging activities and general toxicity of these glycosides have been assessed . Both compounds inhibited the growth of Bacillus cereus, Bacillus subtilis, Citrobacter freundii and Escherichia coli . While swertiamarin was also active against Proteus mirabilis and Serratia marcescens, sweroside inhibited the growth of Staphylococcus epidermidis . Swertiamarin and sweroside exhibited significant general toxicity in brine shrimp lethality bioassay and the LD50 values were 8.0 microg/ml and 34 microg/ml, respectively, whereas that of the positive control podophyllotoxin, a well known cytotoxic lignan, was 2.79 microg/ml . Chemotaxonomic implications of these compounds in the family Gentianaceae have also been discussed briefly.

West Indian Med J, 2003 Mar, 52(1), 31 - 3
Extended-spectrum beta-lactamase producing enterobacteriaceae in a tertiary care hospital in Trinidad and Tobago; Cherian BP et al.; Extended-spectrum beta-lactamase (ESBL) mediated resistance to third generation cephalosporins, amongst the family Enterobacteriaceae, is emerging worldwide . This is the Caribbean's first survey on ESBL production, and was conducted during two six-month periods in 1998 and 2001, in a tertiary health institution in Trinidad and Tobago . Consecutive ampicillin resistant isolates of the family Enterobacteriaceae from in-patients were screened for resistance to third generation cephalosporins, and for ESBL production . The proportion of isolates found to be ESBL producers was similar in both samples (40 of 560 and 23 of 361) . Overall, ESBL production was more frequent in enterobacter, citrobacter and proteus (and related organisms) than in Klebsiella and Escherichia (11.2% and 4.6%, respectively, p < 0.001) . In the 1998 sample, this proportion (9.8% versus 5.8%) was significant (p < 0.05), but the difference was more marked in the 2001 sample (13.6% versus 2.9%, p < 0.001) . Continued distribution of these resistant bacterial strains is of concern . In the Caribbean region, more laboratory surveillance and increased infection control vigilance are recommended, with focus on specific genera in the family.

Carbohydr Res, 2003 Jun 23, 338(13), 1389 - 95
Structure of the O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii O11, strain PCM 1540; Katzenellenbogen E et al.; The O-specific polysaccharide of the lipopolysaccharide of Citrobacter gillenii PCM 1540 (serogroup O11) consists of D-Glc, D-Man, D-GalNAc, D-GlcNAc, 2-acetamido-2,6-dideoxy-D-galactose (D-FucNAc) and O-acetyl groups in the ratios 2:1:1:1:1:1 . On the basis of sugar and methylation analyses and Smith-degradation along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched hexasaccharide repeating unit was established: {structure: see text} . Citrobacter werkmanii PCM 1541 belonging to the same serogroup O11 was found to have an R-form lipopolysaccharide devoid of the O-specific polysaccharide.

J Natl Med Assoc, 2003 May, 95(5), 352 - 62
Antimicrobial susceptibility patterns of urinary pathogens in Trinidad, 1996-1999; Orrett FA; The prevalence of antimicrobial resistance among urinary pathogens has been increasing worldwide . Laboratory diagnosed urinary tract infections were retrospectively evaluated for the years 1996 through 1999, to document the common pathogens and their changing antimicrobial profiles . From 14,853 hospital specimens, an isolation rate of 6.1% was found; and from 5330 community specimens, the isolation rate was 27.9% . E . coli was the predominant cause of urinary tract infections in both hospital and community practices . The rate of isolation of the other pathogens was relatively stable except for citrobacter species, which increased from 1.3% in 1996 to 20.1% in 1999 (p < 0.001) among community isolates . Significant changes in the susceptibility patterns of uropathogens also were observed . E . coli strains from hospitals were significantly more resistant to cefuroxime than community strains (p < 0.001), while resistance to ampicillin and nalidixic acid was high in both practices . No substantial changes in susceptibility to gentamicin and tetracycline were noticed during the four-year period compared to the 99% resistance to tetracycline in 1995 . In relation to nitrofurantoin, no significant changes were noted in both practices where resistant rates remained low, but susceptibility to augmentin showed much improvement among all isolates, including E . coli . Urinary isolates were more commonly recovered from the paediatric age group (1-10 years) and those older than 50 years of age, and males were the predominant gender in both age groups . The study showed that the antibiotics useful in the treatment of UTI in decreasing order of effectiveness in community practice were gentamicin, norfloxacin, nitrofurantoin and cefuroxime . For nosocomial UTI, the drugs most effective include norfloxacin, nitrofurantoin, gentamicin, co-trimoxazole and amoxicillin-clavulanic acid.

Appl Environ Microbiol, 2003 Jun, 69(6), 3048 - 60
Identification and characterization of coenzyme B12-dependent glycerol dehydratase- and diol dehydratase-encoding genes from metagenomic DNA libraries derived from enrichment cultures; Knietsch A et al.; To isolate genes encoding coenzyme B(12)-dependent glycerol and diol dehydratases, metagenomic libraries from three different environmental samples were constructed after allowing growth of the dehydratase-containing microorganisms present for 48 h with glycerol under anaerobic conditions . The libraries were searched for the targeted genes by an activity screen, which was based on complementation of a constructed dehydratase-negative Escherichia coli strain . In this way, two positive E . coli clones out of 560,000 tested clones were obtained . In addition, screening was performed by colony hybridization with dehydratase-specific DNA fragments as probes . The screening of 158,000 E . coli clones by this method yielded five positive clones . Two of the plasmids (pAK6 and pAK8) recovered from the seven positive clones contained genes identical to those encoding the glycerol dehydratase of Citrobacter freundii and were not studied further . The remaining five plasmids (pAK2 to -5 and pAK7) contained two complete and three incomplete dehydratase-encoding gene regions, which were similar to the corresponding regions of enteric bacteria . Three (pAK2, -3, and -7) coded for glycerol dehydratases and two (pAK4 and -5) coded for diol dehydratases . We were able to perform high-level production and purification of three of these dehydratases . The glycerol dehydratases purified from E . coli Bl21/pAK2.1 and E . coli Bl21/pAK7.1 and the complemented hybrid diol dehydratase purified from E . coli Bl21/pAK5.1 were subject to suicide inactivation by glycerol and were cross-reactivated by the reactivation factor (DhaFG) for the glycerol dehydratase of C . freundii . The activities of the three environmentally derived dehydratases and that of glycerol dehydratase of C . freundii with glycerol or 1,2-propanediol as the substrate were inhibited in the presence of the glycerol fermentation product 1,3-propanediol . Taking the catalytic efficiency, stability against inactivation by glycerol, and inhibition by 1,3-propanediol into account, the hybrid diol dehydratase produced by E . coli Bl21/pAK5.1 exhibited the best properties of all tested enzymes for application in the biotechnological production of 1,3-propanediol.

Infect Immun, 2003 Jun, 71(6), 3443 - 53
Host susceptibility to the attaching and effacing bacterial pathogen Citrobacter rodentium; Vallance BA et al.; Many studies have shown that genetic susceptibility plays a key role in determining whether bacterial pathogens successfully infect and cause disease in potential hosts . Surprisingly, whether host genetics influence the pathogenesis of attaching and effacing (A/E) bacteria such as enteropathogenic and enterohemorrhagic Escherichia coli has not been examined . To address this issue, we infected various mouse strains with Citrobacter rodentium, a member of the A/E pathogen family . Of the strains tested, the lipopolysaccharide (LPS) nonresponder C3H/HeJ mouse strain experienced more rapid and extensive bacterial colonization than did other strains . Moreover, the high bacterial load in these mice was associated with accelerated crypt hyperplasia, mucosal ulceration, and bleeding, together with very high mortality rates . Interestingly, the basis for the increased susceptibility was not due to LPS hyporesponsiveness, as the genetically related but LPS-responsive C3H/HeOuJ and C3H/HeN mouse strains were also susceptible to infection . Analysis of the intestinal pathology in these susceptible strains revealed significant crypt epithelial cell apoptosis (terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end label staining) as well as bacterial translocation to the mesenteric lymph nodes . Further studies with infection of SCID (T- and B-lymphocyte-deficient) C3H/HeJ mice demonstrated that loss of lymphocytes had no effect on bacterial numbers but did reduce crypt cell apoptosis and delayed mortality . These studies thus identify the adaptive immune system, crypt cell apoptosis, and bacterial translocation but not LPS responsiveness as contributing to the tissue pathology and mortality seen during C . rodentium infection of highly susceptible mouse strains . Determining the basis for these strains' susceptibility to intestinal colonization by an A/E pathogen will be the focus of future studies.

Int J Med Microbiol, 2003 Apr, 293(1), 87 - 93
Host defences to Citrobacter rodentium; MacDonald TT et al.; Citrobacter rodentium is a natural non-invasive bacterial pathogen which infects the distal colon of mice . It uses the same molecular mechanisms of type III secretion as human enteropathogenic and enterohemorrhagic Escherichia coli to colonise the epithelial cells of the gut and is therefore an ideal model to study host-bacterial pathogen interactions in vivo . Infection elicits mucosal inflammation with similarities to inflammatory bowel disease, and so it is a readily accessible model to investigate the relationship between inflammation and anti-bacterial immunity in the gut.

Scand J Infect Dis, 2003, 35(3), 202 - 4
Citrobacter koseri (diversus) meningitis in an otherwise healthy adolescent; Prais D et al.; Citrobacter infection is commonly reported in neonates and immunocompromised patients . Citrobacter koseri (diversus) is an important cause of neonatal meningitis and brain abscess formation . It adults, however, Citrobacter infection with central nervous system involvement is rare, and is usually associated with an underlying disorder . This report describes a 12-y-old previously healthy girl with Citrobacter koseri meningitis . Intravenous treatment with ceftriaxone for 10 d led to complete recovery . Head computed tomography and brainstem-evoked response audiometry were normal . On follow-up, the patient was completely healthy . Previously reported cases of C . koseri meningitis in the adult population were associated with underlying predisposing factors . In this case a normal, healthy adolescent was treated with intravenous ceftriaxone without any of the known neurological complications observed in the neonatal cases.

Genetics, 2003 May, 164(1), 23 - 9
Experimental prediction of the evolution of cefepime resistance from the CMY-2 AmpC beta-lactamase; Barlow M et al.; Understanding of the evolutionary histories of many genes has not yet allowed us to predict the evolutionary potential of those genes . Intuition suggests that current biochemical activity of gene products should be a good predictor of the potential to evolve related activities; however, we have little evidence to support that intuition . Here we use our in vitro evolution method to evaluate biochemical activity as a predictor of future evolutionary potential . Neither the class C Citrobacter freundii CMY-2 AmpC beta-lactamase nor the class A TEM-1 beta-lactamase confer resistance to the beta-lactam antibiotic cefepime, nor do any of the naturally occurring alleles descended from them . However, the CMY-2 AmpC enzyme and some alleles descended from TEM-1 confer high-level resistance to the structurally similar ceftazidime . On the basis of the comparison of TEM-1 and CMY-2, we asked whether biochemical activity is a good predictor of the evolutionary potential of an enzyme . If it is, then CMY-2 should be more able than the TEMs to evolve the ability to confer higher levels of cefepime resistance . Although we generated CMY-2 evolvants that conferred increased cefepime resistance, we did not recover any CMY-2 evolvants that conferred resistance levels as high as the best cefepime-resistant TEM alleles.

J Vet Diagn Invest, 2003 May, 15(3), 297 - 9
Citrobacter freundii septicemia in two dogs; Galarneau JR et al.; A 4-month-old Maltese puppy and a 7.5-year-old Collie were diagnosed with septicemia associated with Citrobacter freundii . The puppy died soon, after developing weakness and mucohemorragic diarrhea . The Collie had immune-mediated hemolytic anemia and thrombocytopenia and was treated with immunosuppressive drugs before being euthanized . Gross examination of the puppy revealed mucohemorrhagic intestinal contents . Focal necrotic hepatitis, fibrinous peritonitis, interstitial pneumonia, and hemorrhagic gastrointestinal contents were observed in the older dog . Histologically, there was a diffuse, moderate, histiocytic meningitis in the puppy and a focal fibrinonecrotic hepatitis in the adult dog . Lesions in both dogs contained numerous gram-negative rods . Citrobacter freudii is a potential cause of monomicrobic bacteraemia-septicemia in puppies or immunocompromized adult dogs.The gastrointestinal tract is probably the main site of entry.

Appl Environ Microbiol, 2003 May, 69(5), 2568 - 79
Cloning and sequencing of the histidine decarboxylase genes of gram-negative, histamine-producing bacteria and their application in detection and identification of these organisms in fish; Takahashi H et al.; The use of molecular tools for early and rapid detection of gram-negative histamine-producing bacteria is important for preventing the accumulation of histamine in fish products . To date, no molecular detection or identification system for gram-negative histamine-producing bacteria has been developed . A molecular method that allows the rapid detection of gram-negative histamine producers by PCR and simultaneous differentiation by single-strand conformation polymorphism (SSCP) analysis using the amplification product of the histidine decarboxylase genes (hdc) was developed . A collection of 37 strains of histamine-producing bacteria (8 reference strains from culture collections and 29 isolates from fish) and 470 strains of non-histamine-producing bacteria isolated from fish were tested . Histamine production of bacteria was determined by paper chromatography and confirmed by high-performance liquid chromatography . Among 37 strains of histamine-producing bacteria, all histidine-decarboxylating gram-negative bacteria produced a PCR product, except for a strain of Citrobacter braakii . In contrast, none of the non-histamine-producing strains (470 strains) produced an amplification product . Specificity of the amplification was further confirmed by sequencing the 0.7-kbp amplification product . A phylogenetic tree of the isolates constructed using newly determined sequences of partial hdc was similar to the phylogenetic tree generated from 16S ribosomal DNA sequences . Histamine accumulation occurred when PCR amplification of hdc was positive in all of fish samples tested and the presence of powerful histamine producers was confirmed by subsequent SSCP identification . The potential application of the PCR-SSCP method as a rapid monitoring tool is discussed.

Chirurgia (Bucur), 2001 Nov-Dec, 96(6), 547 - 52
{Cefepime (maxipime), large spectrum 4th generation cephalosporin, resistant to beta-lactamases}; Angelescu M et al.; As a result of the appearance of bacterial strains resistant to 3rd generation cephlosporin, since 1993 cephalosporins of 4th generation have been developed and introduced in therapy; among them are: cefepime and cefpirome . Cefepime is the most active 4th generation cephalosporin possessing the following advantages over the 3rd generation cephalosporins: high intrinsic potency due to rapid penetration into the periplasmic space; an extended spectrum of activity that includes many Gram-positive and Gram-negative organisms; Activity against multi-resistant Gram-negative bacteria, including Enterobacter and Klebsiella species; Low potential for beta-lactamase induction, especially Bush group 1 beta-lactamases, even ar low periplasmic concentrations; Minimal selection of resistant mutant strains . Its spectrum is very large being very active against Gram-negative bacilii: Enterobacter, Pseudomonas aeruginosa, Klebsiella pneumoniae, Serratia, Citrobacter, Proteus mirabilis and less active against Bacillus fragillis . Cefepime is also very active against Gram-positive cocci: Staphylococcus aureus (methicillin-susceptible strains only), Streptococcus pneumoniae, Streptococcus pyogenes . Some of the methicillin-resistant strains of staphylococcus are susceptible to cefepime; Enterococcus is resistant . Due to its high resistance against beta-lactamases Cefepime (Maxipime) is the best choice in life threatening nosocomial infections occuring in patients in the intensive care units . Cefepime can be synergically associated with aminoglycosides and fluoroquinolones.

Chirurgia (Bucur), 2002 Nov-Dec, 97(6), 571 - 5
{Cefepime (Maxipime) treatment efficacy in surgical patients }; Simion L et al.; Between September and December 2001 in the "Caritas" Surgical Clinic of Bucharest has been conducted a clinical study for the efficiency of Cefepime (Maxipime) treatment in surgical patients . Introduced in therapy in the last decade of the XXth Century, Cefepime (Maxipime) is the most active 4th generation cephalosporin, due to its extended spectrum of activity and its high resistance against beta-lactamases . Cefepime (Maxipime) has a very large spectrum, including the majority of the microorganisms implicated in surgical infections: Enterobacter, Klebsiella pneumoniae/speciae, Proteus mirabilis, Bacillus fragillis, Pseudomonas aeruginosa, Serratia, Citrobacter and other Gram-negative bacilii, Gram-positive cocci (Staphylococcus aureus, Streptococcus pneumoniae, Streptococcus pyogenes) . The clinical study included 30 surgical patients, the selection criteria being the severity of the present infection or the potential risk after major (abdominal) surgery . We introduced Cefepime (Maxipime) as first choice of monotheraphy, except: severe, life threatening nosocomial infections, when we associated Cefepime (Maxipime) with aminoglycosides; failure of another antibiotheraphy schema, when we associated Cefepime (Maxipime) with aminoglycosides; suspicion of anaerobe contamination, when we associated Cefepime (Maxipime) with metronidazole . The results of our study support the utilization of Cefepime (Maxipime) as the best choice antibiotic in severe surgical infections, especially in the intensive care and surgical units . Cefepime (Maxipime) can be synergically associated with aminoglycosides and imidazoles (metronidazole).

Diagn Microbiol Infect Dis, 2003 Apr, 45(4), 295 - 301
Susceptibility patterns of orally administered antimicrobials among urinary tract infection pathogens from hospitalized patients in North America: comparison report to Europe and Latin America . Results from the SENTRY Antimicrobial Surveillance Program (2000); Gordon KA et al.; Urinary tract infections (UTIs) remain a worldwide nosocomial infection problem . Geographic variations in pathogen occurrence and susceptibility profiles require monitoring to provide information to guide new (garenoxacin {BMS284756}) therapeutic options . Two thousand seven hundred-eighty UTI isolates from Europe (n = 783), Latin America (531), and North America (1,466) were tested and compared against 44 agents by reference methods in the SENTRY Antimicrobial Surveillance Program . The top seven pathogens accounted for 90% of all isolates and the rank order for all regions was: Escherichia coli (1,316; 47%), Enterococcus spp . (351; 13%), Klebsiella spp . (306; 11%), Pseudomonas aeruginosa (210; 8%), Proteus mirabilis (145; 5%), Enterobacter spp . (97; 4%), and Citrobacter spp . (78; 3%) . The pathogen rank order was similar among regions except for the rarer occurrence of Enterococcus spp . (Rank #6, 4%) in Latin America . E . coli ampicillin resistance was highest in Europe and Latin America (51-55%) . Ampicillin (37%), ciprofloxacin or garenoxacin (4%), and trimethoprim/sulfamethoxazole (23%) resistance remained lowest in North America . Nitrofurantoin susceptibility in E . coli was still at acceptable levels and ranged from 91 to 96% across regions . The regional ciprofloxacin-resistant rank order for P . aeruginosa by region was: Latin America (55%) > Europe (41%) > North America (29%) . Vancomycin-resistant enterococci (VRE) were only detected in North America (7%) . Garenoxacin possessed a 34 to 44% wider spectrum compared to ciprofloxacin against enterococci UTI isolates . Extended spectrum beta-lactamase rates for E . coli and Klebsiella spp . were 4 and 19%, respectively . These results emphasized the need to assess the often striking differences in pathogen occurrence and resistance rates among the commonly encountered UTI pathogens.

Epidemiol Infect, 2003 Apr, 130(2), 179 - 86
Environmental isolates of Citrobacter braakii that agglutinate with Escherichia coli O157 antiserum but do not possess the genes responsible for the biosynthesis of O157 somatic antigen; Khan A et al.; While searching for Escherichia coli O157 in the aquatic environment of Calcutta using an immunodetection procedure, we fortuitously detected five strains of Citrobacter braakii, which cross-reacted with the commercially available O157 polyvalent antiserum . The five C . braakii isolates gave positive results when a sensitive dot-ELISA was performed with E . coli O157 monoclonal antibody . Further, the O157 monoclonal antibody recognized the bands of proteinase K treated whole cells of lipopolysaccharide of all the C . braakii isolates . Apart from weak reactions with two or three of the DNA probes, all the C . braakii strains did not hybridize with the other probes spanning the minimum region required for O157 O-antigen biosynthesis . These strains did not possess any of the virulence genes that are commonly found in the Shiga toxin-producing E . coli (STEC) specially the serotype O157: H7 . Therefore, it appears that the serological cross-reaction between C . braakii and E . coli O157 antiserum is based on structural mimicry between the O-polysaccharide of C . braakii and E . coli O157.

FEMS Immunol Med Microbiol, 2003 May 15, 36(1-2), 71 - 6
The identity of the O-specific polysaccharide structure of Citrobacter strains from serogroups O2, O20 and O25 and immunochemical characterisation of C . youngae PCM 1507 (O2a,1b) and related strains; Mieszala M et al.; Serological studies using SDS-PAGE and immunoblotting revealed that from five strains that are ascribed to Citrobacter serogroup O2, four strains, PCM 1494, PCM 1495, PCM 1496 and PCM 1507, are reactive with specific anti-Citrobacter O2 serum . In contrast, strain PCM 1573 did not react with anti-Citrobacter O2 serum and, hence, does not belong to serogroup O2 . The LPS of Citrobacter youngae O2a,1b (strain PCM 1507) was degraded under mild acidic conditions and the O-specific polysaccharide (OPS) released was isolated by gel chromatography . Sugar and methylation analyses along with (1)H- and (13)C-NMR spectroscopy, including two-dimensional (1)H,(1)H COSY, TOCSY, NOESY and (1)H,(13)C HSQC experiments, showed that the repeating unit of the OPS has the following structure: {structure: see text} . NMR spectroscopic studies demonstrated that Citrobacter werkmanii O20 and C . youngae O25 have the same OPS structure as C . youngae O2 . Sugar and methylation analyses of the core oligosaccharide fractions demonstrated structural differences in the lipopolysaccharide core regions of these strains, which may substantiate their classification in different serogroups.

J Clin Microbiol, 2003 Apr, 41(4), 1463 - 8
Characterization of clinical isolates of Enterobacteriaceae from Italy by the BD Phoenix extended-spectrum beta-lactamase detection method; Sanguinetti M et al.; Production of extended-spectrum beta-lactamases (ESBLs) is an important mechanism of beta-lactam resistance in Enterobacteriaceae: Identification of ESBLs based on phenotypic tests is the strategy most commonly used in clinical microbiology laboratories . The Phoenix ESBL test (BD Diagnostic Systems, Sparks, Md.) is a recently developed automated system for detection of ESBL-producing gram-negative bacteria . An algorithm based on phenotypic responses to a panel of cephalosporins (ceftazidime plus clavulanic acid, ceftazidime, cefotaxime plus clavulanic acid, cefpodoxime, and ceftriaxone plus clavulanic acid) was used to test 510 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Proteus mirabilis, Providencia stuartii, Morganella morganii, Enterobacter aerogenes, Enterobacter cloacae, Serratia marcescens, Citrobacter freundii, and Citrobacter koseri . Of these isolates, 319 were identified as ESBL producers, and the remaining 191 were identified as non-ESBL producers based on the results of current phenotypic tests . Combined use of isoelectric focusing, PCR, and/or DNA sequencing demonstrated that 288 isolates possessed bla(TEM-1)- and/or bla(SHV-1)-derived genes, and 28 had a bla(CTX-M) gene . Among the 191 non-ESBL-producing isolates, 77 isolates produced an AmpC-type enzyme, 110 isolates possessed TEM-1, TEM-2, or SHV-1 beta-lactamases, and the remaining four isolates (all K . oxytoca strains) hyperproduced K1 chromosomal beta-lactamase . The Phoenix ESBL test system gave positive results for all the 319 ESBL-producing isolates and also for two of the four K1-hyperproducing isolates of K . oxytoca . Compared with the phenotypic tests and molecular analyses, the Phoenix system displayed 100% sensitivity and 98.9% specificity . These findings suggest that the Phoenix ESBL test can be a rapid and reliable method for laboratory detection of ESBL resistance in gram-negative bacteria.

Mol Microbiol, 2003 May, 48(3), 795 - 809
Identification of a novel type IV pilus gene cluster required for gastrointestinal colonization of Citrobacter rodentium; Mundy R et al.; Citrobacter rodentium is used as an in vivo model system for clinically significant enteric pathogens such as enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E . coli (EPEC) . These pathogens all colonize the lumen side of the host gastrointestinal tract via attaching and effacing (A/E) lesion formation . In order to identify genes required for the colonization of A/E-forming pathogens, a library of signature-tagged transposon mutants of C . rodentium was constructed and screened in mice . Of the 576 mutants tested, 14 were attenuated in their ability to colonize the descending colon . Of these, eight mapped to the locus of enterocyte effacement (LEE), which is required for the formation of A/E lesions, underlying the importance of this mechanism for pathogenesis . Another mutant, P5H2, was found to have a transposon insertion in an open reading frame that has strong similarity to type IV pilus nucleotide-binding proteins . The region flanking the transposon insertion was sequenced, identifying a cluster of 12 genes that encode the first described pilus of C . rodentium (named colonization factor Citrobacter, CFC) . The proteins encoded by cfc genes have identity to proteins of the type IV COF pilus of enterotoxigenic E . coli (ETEC), the toxin co-regulated pilus of Vibrio cholerae and the bundle-forming pilus of EPEC . A non-polar mutation in cfcI, complementation of this strain with wild-type cfcI and complementation of strain P5H2 with wild-type cfcH confirmed that these genes are required for colonization of the gastrointestinal tract by C . rodentium . Thus, CFC provides a convenient model to study type IV pilus-mediated pathogen-host interactions under physiological conditions in the natural colonic environment.

Biochim Biophys Acta, 2003 Apr 11, 1647(1-2), 167 - 72
Structure and mechanism of tryptophan indole-lyase and tyrosine phenol-lyase; Phillips RS et al.; Tyrosine phenol-lyase (TPL) and tryptophan indole-lyase (Trpase) catalyse the reversible hydrolytic cleavage of L-tyrosine or L-tryptophan to phenol or indole, respectively, and ammonium pyruvate . These enzymes are very similar in sequence and structure, but show strict specificity for their respective physiological substrates . We have mutated the active site residues of TPL (Thr(124), Arg(381), and Phe(448)) to those of Trpase and evaluated the effects of the mutations . Tyr(71) in Citrobacter freundii TPL, and Tyr(74) in E . coli Trpase, are essential for activity with both substrates . Mutation of Arg(381) of TPL to Ala, Ile, or Val (the corresponding residues in the active site of Trpase) results in a dramatic decrease in L-Tyr beta-elimination activity, with little effect on the activity of other substrates . Arg(381) may be the catalytic base with pK(a) of 8 seen in pH-dependent kinetic studies . T124D TPL has no measureable activity with L-Tyr or 3-F-L-Tyr as substrate, despite having high activity with SOPC . T124A TPL has very low but detectable activity, which is about 500-fold less than wild-type TPL, with L-Tyr and 3-F-L-Tyr . F448H TPL also has very low activity with L-Tyr . None of the mutant TPLs has any detectable activity with L-Trp as substrate . H463F Trpase also exhibits low activity with L-Trp, but retains high activity with other substrates . Thus, additional residues remote from the active site may be needed for substrate specificity . Both Trpase and TPL may react by a rare S(E)2-type mechanism.

Biotechnol Prog, 2003 Mar-Apr, 19(2), 263 - 72
Comparative genomic analysis of dha regulon and related genes for anaerobic glycerol metabolism in bacteria; Sun J et al.; The dihydroxyacetone (dha) regulon of bacteria encodes genes for the anaerobic metabolism of glycerol . In this work, genomic data are used to analyze and compare the dha regulon and related genes in different organisms in silico with respect to gene organization, sequence similarity, and possible functions . Database searches showed that among the organisms, the genomes of which have been sequenced so far, only two, i.e., Klebsiella pneumoniae MGH 78578 and Clostridium perfringens contain a complete dha regulon bearing all known enzymes . The components and their organization in the dha regulon of these two organisms differ considerably from each other and also from the previously partially sequenced dha regulons in Citrobacter freundii, Clostridium pasteurianum, and Clostridium butyricum . Unlike all of the other organisms, genes for the oxidative and reductive pathways of anaerobic glycerol metabolism in C . perfringens are located in two separate organization units on the chromosome . Comparisons of deduced protein sequences of genes with similar functions showed that the dha regulon components in K . pneumoniae and C . freundii have high similarities (80-95%) but lower similarities to those of the Clostridium species (30-80%) . Interestingly, the protein sequence similarities among the dha genes of the Clostridium species are in many cases even lower than those between the Clostridium species and K . pneumoniae or C . freundii, suggesting two different types of dha regulon in the Clostridium species studied . The in silico reconstruction and comparison of dha regulons revealed several new genes in the microorganisms studied . In particular, a novel dha kinase that is phosphoenolpyruvate-dependent is identified and experimentally confirmed for K . pneumoniae in addition to the known ATP-dependent dha kinase . This finding gives new insights into the regulation of glycerol metabolism in K . pneumoniae and explains some hitherto not well understood experimental observations.

Indian J Med Res, 2002 Oct, 116, 145 - 9
Extended spectrum beta-lactamase mediated resistance in urinary tract isolates of family Enterobacteriaceae; Khurana S et al.; BACKGROUND & OBJECTIVES: Extended spectrum beta-lactamases (ESBL) hydrolyse expanded spectrum cephalosporins like ceftazidime, cefotaxime and monobactam aztreonam . ESBL producing bacteria may not be detectable in the routine disk diffusion susceptibility tests leading to inappropriate use of antibiotics and treatment failures . As information on ESBL producers causing urinary tract infection (UTI) is not available from our country, the occurrence of ESBL producing strains causing UTI was studied as also the differences between the antimicrobial susceptibility patterns of ESBL and non-ESBL producers . METHODS: Urinary isolates (233) were tested for antimicrobial susceptibility by Stoke's method and ESBL production by double disk diffusion method (ceftazidime and clavulanic acid) . The clinical and demographic profile of the patients was noted . RESULTS: Sixty two of the 233 isolates tested (26.6%) were ESBL producers . Approximately 38.5 per cent of Klebsiellae, 24.7 per cent of Escherichia coli, 24 per cent of Enterobacter aerogenes, 33.3 per cent of Proteus sp . and the only Citrobacter strain produced ESBL . Recent surgery was found to be a significant (P < 0.01) risk factor for acquisition of ESBLs . Approximately 30 per cent of the ESBL producers appeared falsely sensitive or moderately sensitive to cefotaxime/ceftazidime in routine susceptibility testing . There was no difference between the ESBL producers and non-producers in the susceptibility to non-beta lactam agents except for gentamicin . INTERPRETATION & CONCLUSION: A high percentage of urinary tract isolates produced ESBL . Since they are likely to be missed in routine disk diffusion susceptibility tests, all microbiology laboratories should look for ESBL production routinely to avoid treatment failures.

Acad Emerg Med, 2003 Apr, 10(4), 347 - 51
Antibiotic resistance patterns of uropathogens in pediatric emergency department patients; McLoughlin TG Jr et al.; OBJECTIVES: To evaluate the prevalence of resistance of the various urinary tract infection (UTI) pathogens obtained from patients in an urban pediatric emergency department (PED), and to identify risk factors for infection with resistant strains . METHODS: The data were collected retrospectively in an urban, academic PED in northeastern Florida . The microbiology-computerized database was used to identify all positive urine cultures from October 1999 through June 2000 . All patients aged 17 years or less, whose urine specimen was collected in the ED and grew cultures with greater than 10,000 colony forming units (CFU) per milliliter of a single organism on Maconkey or blood agar, were included . The medical records of the patients were reviewed and selective demographic and clinical data were collected . Patients were excluded if their charts were unavailable for review or if the pathogen that grew in culture was a suspected contaminant . All patients lacking clinical symptoms of UTI (frequency, dysuria, abdominal pain, fever, or urgency) and whose urine was collected by clean-catch were excluded if their culture grew between 10,000 and 100,000 CFU . Resistance to trimethoprim-sulfamethoxazole (T-S) was estimated for the subset of gram-negative pathogens . Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated to compare rates of resistance among patients with and without the following risk factors: age greater than 4 years; current or recent antibiotic use; day care attendance; and previous UTI . RESULTS: A total of 126 urine cultures were identified for inclusion . Of these, 45 patients were excluded, leaving 81 who met the study criteria . The majority of isolated organisms were Escherichia coli, accounting for 89% of the patients (n = 72) . Other organisms identified were Klebsiella 3.7%, Proteus 1.2%, Citrobacter 1.2%, Staphylococcus 1.2%, and Enterococcus 3.7% (all in children < 4 years old) . The resistance to T-S was 6.5% (95% CI = 0.9% to 12.1%) for gram-negative pathogens . Overall, 48% of gram-negative isolates were resistant to one or more antibiotics, any resistance (95% CI = 36.5% to 59.5%) . T-S resistance was nominally higher for older children and for those with a history of antibiotic use, although not to a significant degree . Children less than age 4 were more likely to have any resistance (OR 2.6; 95% CI = 1.0 to 6.7) . CONCLUSIONS: The resistance to T-S in this study was 6.7% for gram-negative pathogens . These rates are lower than rates reported in adult populations, international pediatric studies, and the authors' hospital antibiograms, demonstrating the importance of local, population-specific data in selecting antibiotics . This study did not identify any statistically significant risk factors for resistance to T-S, but suggests that those with a recent history of antibiotic use may be at highest risk . While children less than 4 years old with gram-negative pathogens have nominally lower rates of T-S resistance, they are at higher risk for resistance to one or more antibiotics (any resistance) and are at risk for UTI caused by enterococcus (uniformly nonsusceptible to T-S) . Prospective studies are needed to validate these results and to identify predisposing factors for urinary pathogens with antibiotic resistance.

Mol Microbiol, 2003 Apr, 48(1), 95 - 115
Citrobacter rodentium translocated intimin receptor (Tir) is an essential virulence factor needed for actin condensation, intestinal colonization and colonic hyperplasia in mice; Deng W et al.; Citrobacter rodentium infection of mice serves as a relevant small animal model to study enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E . coli (EPEC) infections in man . Enteropathogenic E . coli and EHEC translocate Tir into the host cytoplasmic membrane, where it serves as the receptor for the bacterial adhesin intimin and plays a central role in actin condensation beneath the adherent bacterium . In this report, we examined the function of C . rodentium Tir both in vitro and in vivo . Similar to EPEC, C . rodentium Tir is tyrosine phosphorylated and is essential for actin condensation . Citrobacter Tir and EPEC Tir are functionally interchangeable and both require tyrosine phosphorylation to mediate actin rearrangements . In contrast, Citrobacter Tir supports actin nucleation in EHEC independent of tyrosine phosphorylation, while EHEC Tir cannot replace Citrobacter Tir for this function . This indicates that C . rodentium and EPEC use an actin nucleating mechanism different from EHEC . We also found that Tir is expressed and translocated into mouse enterocytes in vivo by C . rodentium during infections . This represents the first direct demonstration of a type III effector translocated in vivo into a natural host by any pathogen . In addition, we showed that Tir, but not its tyrosine phosphorylation, is essential for C . rodentium to colonize the large bowel and induce attaching/effacing (A/E) lesions and colonic hyperplasia in mice, and that both EPEC Tir and EHEC Tir can substitute for Citrobacter Tir for these activities in vivo . These results thus demonstrate that Tir is an essential virulence factor in this infection model . The data also show that the function of Tir tyrosine phosphorylation and its subsequent actin nucleating activity are not essential for C . rodentium colonization of the mouse gut nor for inducing A/E lesions and colonic hyperplasia, thereby uncoupling colonization and disease from actin condensation for this A/E pathogen.

Infect Immun, 2003 Apr, 71(4), 2130 - 41
CesD2 of enteropathogenic Escherichia coli is a second chaperone for the type III secretion translocator protein EspD; Neves BC et al.; Enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli are extracellular pathogens that employ a type III secretion system to export translocator and effector proteins, proteins which facilitates colonization of the mucosal surface of the intestine via formation of attaching and effacing (A/E) lesions . The genes encoding the proteins for A/E lesion formation are located on a pathogenicity island, termed the locus of enterocyte effacement (LEE), which contains eae encoding intimin as well as the type III secretion system and effector genes . Many type III secreted proteins are stabilized and maintained in a secretion-competent conformation in the bacterial cytosol by specific chaperone proteins . Three type III chaperones have been described thus far within the EPEC LEE region: CesD, for the translocator proteins EspB and EspD; CesT, for the effector proteins Tir and Map; and CesF, for EspF . In this study we report the characterization of CesD2 (previously Orf27), a second LEE-encoded chaperone for EspD . We show specific CesD2-EspD protein interaction which appears to be necessary for proper EspD secretion in vitro and pathogenesis in vivo as demonstrated in the A/E-lesion-forming mouse pathogen Citrobacter rodentium.

J Antimicrob Chemother, 2003 Apr, 51(4), 791 - 802 Epub 2003 Feb 25.
Analyses of ampC gene expression in Serratia marcescens reveal new regulatory properties; Mahlen SD et al.; Serratia marcescens encodes an inducible, chromosomal beta-lactamase, ampC . Studies addressing the regulation of inducible ampC genes have focused primarily on Enterobacter cloacae and Citrobacter freundii . The purpose of this study was to clone and sequence the ampC, ampR and intergenic region of S . marcescens and examine both inducible and basal level ampC expression . Sequence analysis of the S . marcescens ampC gene identified an extended 5' untranslated region (UTR) of 126 nucleotides, which formed a prominent stem-loop structure . Induction of ampC expression required AmpR, and the start of transcription was determined using primer extension analysis . In vivo half-life analysis revealed that the half-life of the S . marcescens ampC transcript was 7 min . Confirmation of the in vivo half-life and the role of the stem-loop structure in the 5' UTR was demonstrated by comparing transcript half-life and luciferase expression between a wild-type (WT) and a 5' UTR stem-loop deletion mutant . These data demonstrated that the stem-loop structure was involved in transcript stability . Taken together, these findings indicate that constitutive expression of S . marcescens ampC is regulated by both transcriptional initiation and post-transcriptional events.

J Mol Biol, 2003 Apr 4, 327(4), 833 - 42
NMR structure of Citrobacter freundii AmpD, comparison with bacteriophage T7 lysozyme and homology with PGRP domains; Liepinsh E et al.; AmpD is a bacterial amidase involved in the recycling of cell-wall fragments in Gram-negative bacteria . Inactivation of AmpD leads to derepression of beta-lactamase expression, presenting a major pathway for the acquisition of constitutive antibiotic resistance . Here, we report the NMR structure of AmpD from Citrobacter freundii (PDB accession code 1J3G) . A deep substrate-binding pocket explains the observed specificity for low molecular mass substrates . The fold is related to that of bacteriophage T7 lysozyme . Both proteins bind zinc at a conserved site and require zinc for amidase activity, although the enzymatic mechanism seems to differ in detail . The structure-based sequence alignment identifies conserved features that are also conserved in the eukaryotic peptidoglycan recognition protein (PGRP) domains, including the zinc-coordination site in several of them . PGRP domains thus belong to the same fold family and, where zinc-binding residues are conserved, may have amidase activity . This hypothesis is supported by the observation that human serum N-acetylmuramyl-L-alanine amidase seems to be identical with a soluble form of human PGRP-L .

Br J Dermatol, 2003 Mar, 148(3), 456 - 66
Anaerobic cocci populating the deep tissues of chronic wounds impair cellular wound healing responses in vitro; Stephens P et al.; BACKGROUND: Anaerobic cocci are estimated to be present in the deep tissues of over 50% of chronic skin wounds . While the part they play in the chronicity of these wounds is uninvestigated, anaerobic cocci have previously been shown to be involved in other chronic inflammatory human conditions . METHODS: In this study the anaerobic microflora of the deep tissues of 18 patients with refractory chronic venous leg ulcers (mean age 80.3 years; mean duration > 24 months) was characterized using strict anaerobic culture conditions . The effect of the anaerobic organisms isolated from these tissues on extracellular matrix (ECM) proteolysis and cellular wound healing responses was studied using in vitro models . RESULTS: Anaerobic organisms were present in the deep tissues of 14 of 18 wounds and were principally Peptostreptococcus spp . The effects of three Peptostreptococcus spp . isolated from these wounds (P . magnus, P . vaginalis and P . asaccharolyticus) on cellular wound healing responses were compared with those of two pathogenic organisms also isolated from these wounds (Pseudomonas aeruginosa and Citrobacter diversus) . While the direct ECM proteolytic activity exhibited by the Peptostreptococcus spp . was limited, they did significantly inhibit both fibroblast and keratinocyte proliferation, but only at high concentrations . However, at lower concentrations peptostreptococcal supernatants profoundly inhibited keratinocyte wound repopulation and endothelial tubule formation . The magnitude of these effects varied between strains and they were distinct from those demonstrated by Pseudomonas aeruginosa and Citrobacter diversus . CONCLUSIONS: These studies confirm the importance of anaerobic organisms in chronic wounds and demonstrate an indirect, strain-specific mechanism by which these microorganisms may play a part in mediating the chronicity of these wounds.

J Small Anim Pract, 2003 Mar, 44(3), 117 - 20
Septic fibrinous pericarditis in a cocker spaniel; Stafford Johnson JM et al.; A four-year-old cocker spaniel presented with cardiac tamponade due to a pericardial effusion, in addition to pyrexia and peripheral neutrophilia and a recent history of chest trauma . Cytological examination of the pericardial effusion revealed a predominant neutrophilia . The echocardiographic findings were of numerous hyperechoic densities in the pericardial space, due to fibrin, with concurrent thickening and distortion of the pericardium . Postmortem examination, including microbiology, revealed the presence of organising septic fibrinous pericarditis associated with a mixed infection of Streptococcus canis, Citrobacter species, Pseudomonas species and alpha-haemolytic streptococci.

J Food Prot, 2003 Mar, 66(3), 457 - 65
Prophylactic feeding of Lactobacillus acidophilus NCFM to mice attenuates overt colonic hyperplasia; Varcoe JJ et al.; The objective of this project was to determine if the probiotic Lactobacillus acidophilus NCFM would protect mice from developing transmissible murine colonic hyperplasia (TMCH) caused by Citrobacter rodentium . Our hypothesis was that the oral administration of L . acidophilus NCFM to mice would mitigate colonic hyperplasia and modulate the host immune response . A concurrent administration (CA) study was performed by feeding mice phosphate-buffered saline (PBS), C . rodentium only, L . acidophilus NCFM only, or C . rodentium and NCFM concurrently on the same day . The mice in the CA study were not protected by the probiotic, since their mean colon sample weights (0.109 g) were significantly higher than those of the PBS controls (0.0774 g), and the hematoxylin and eosin-stained samples showed histological changes typically associated with TMCH . A prophylactic feeding (PF) study was performed by orally feeding mice PBS or NCFM once daily for 20 consecutive days; in addition, on day 7, mice were challenged with either PBS or C . rodentium . Mice in the PF study were protected when they consumed the probiotic prior to the pathogen challenge, since their mean colon sample weights (0.0812 g) were not significantly higher than those of the controls (0.0753 g) . The hematoxylin and eosin-stained samples appeared similar to the control samples, and the intestinal interleukin (IL)-15 and gamma interferon (IFN-gamma) mRNA levels were reduced . L . acidophilus NCFM did attenuate overt colonic hyperplasia when fed to mice prior to challenge with C . rodentium . The mouse model used in this study enabled us to investigate the efficacy of the L . acidophilus NCFM in preventing gastrointestinal disease and is a valid model for future probiotic research.

Indian J Exp Biol, 2002 Feb, 40(2), 227 - 9
Reversibility of oxygen induced inactivation of nitrogenase in some enterobacteria; Kannan V et al.; Aerobic and microaerobic diazotrophs possess numerous oxygen restriction strategies to protect nitrogenase from inactivation by oxygen without interfering with energy generation through oxidative phosphorylation . Protection by conformational change in nitrogenase was first detected and described in Azotobacter . This strategy once considerd unique for Azotobacter has been shown in this study to occur in Citrobacterfreundii (Braak) Werkman and Gillen and Klebsiella pneumoniae subspecies rhinoscleromatis (Trevisan) Migula also . However, in these enteric bacteria the entire enzyme is not protected probably due to the absence of any respiratory protection similar to that found in the aerobe, Azotobacter.

Jpn J Antibiot, 2002 Dec, 55(6), 827 - 43
{Antibacterial activity of cefpodoxime against clinical isolates in 2000 and 2001}; Abe T et al.; As the post-marketing surveillance of cefpodoxime proxetil (Banan), MICs of cefpodoxime (CPDX, an active form of Banan) against 1090 clinical isolates of 22 species from 15 medical institutions all over Japan from June 2000 to March 2001 were measured using the broth microdilution method approved by the Japanese Society of Chemotherapy and compared with those of oral cephem antibacterials, cefaclor, cefdinir, cefditoren, and cefcapene . In this study, remarkable change in the activity of CPDX was observed in Streptococcus pneumoniae and Haemophilus influenzae compared with the susceptibility in the studies before Banan was launched . This cause is considered to be the increase in the incidence of the following resistant strains: penicillin-intermediate S . pneumoniae (47.3%), penicillin-resistant S . pneumoniae (PRSP, 15.1%), and beta-lactamase-negative ampicillin-resistant (BLNAR) H . influenzae (24.0%), which were scarcely isolated in 1989 when Banan was launched . Other tested drugs also exhibited low activity against these resistant strains . However, CPDX showed comparatively good activity with MIC90 of 2 micrograms/mL against PRSP . Against methicillin-susceptible Staphylococcus spp., Streptococcus pyogenes, Streptococcus agalactiae, and Moraxella catarrhalis, CPDX also showed comparatively good activity with MIC90 of < or = 4 micrograms/mL, which was almost equal to that in the studies before its marketing . Against quinolones-resistant Neisseria gonorrhoeae, CPDX showed excellent activity with MIC90 of 0.5 microgram/mL . Against members of the family Enterobacteriaceae except for Citrobacter freundii, Enterobacter spp., Proteus vulgaris, and Morganella morganii, CPDX showed good activity . However, in Escherichia coli, Klebsiella spp . Proteus spp., and Providencia spp., there are some high-resistant strains to all tested drugs including CPDX . Against Peptostreptococcus spp., MIC90 of CPDX was 8 micrograms/mL and its MIC range was widely distributed from 0.03 to 32 micrograms/mL, which were similar to those in the studies before its marketing . In this study, CPDX showed the decrease in the activity against several species as did other drugs tested, but against most of species tested, CPDX maintained good activity . Furthermore, it is necessary to pay much attention to the trend of resistant strains.

Biochemistry, 2003 Feb 25, 42(7), 1950 - 7
A dynamic structure for the acyl-enzyme species of the antibiotic aztreonam with the Citrobacter freundii beta-lactamase revealed by infrared spectroscopy and molecular dynamics simulations; Wilkinson AS et al.; Infrared difference spectra show that at least 4 conformations coexist for the ester carbonyl group of the stable acyl-enzyme species formed between the antibiotic aztreonam and the class C beta-lactamase from Citrobacter freundii . A novel method for the assignment of the bands that arise from the ester carbonyl group has been employed . This has made use of the finding that the infrared absorption intensity of aliphatic esters is surprisingly constant, so a direct comparison with simple model esters has been possible . This has allowed a clear distinction to be made between ester and amide (protein) absorptions . The polarity of the conformer environment varies from hexane-like to strongly hydrogen-bonded . We assume that the conformer with the lowest frequency (1,690 cm(-)(1)) and hence the strongest hydrogen-bonding is the singular conformer observed in the X-ray crystallographic structure, since a good interaction via two hydrogen bonds with the oxyanion hole is seen . Molecular dynamics simulation by the method of locally enhanced sampling revealed that the motion of the ester carbonyl of the acyl-enzyme species in and out of the oxyanion hole is facile . The simulation revealed two pathways for this motion that would go through intermediates that first break one or the other of the two hydrogen bonds to the oxyanion hole, prior to departure of the carbonyl moiety out of the active site . It is likely that such motion for the acyl-enzyme species might also occur with more typical beta-lactam substrates for beta-lactamases, but their detection in the more rapid time scale may prove a challenge.

Arch Immunol Ther Exp (Warsz), 2002, 50(6), 379 - 91
Structures and serology of the O-specific polysaccharides of bacteria of the genus Citrobacter; Knirel YA et al.; The review presents the structures of the O-specific polysaccharides (O-antigens) of the lipopolysaccharides isolated from over 25 Citrobacter strains, which represent different species and serogroups . The correlation between O-antigen structure and immunospecificity as well as numerous cross-reactions between Citrobacter and other enterobacterial species are discussed.

Antimicrob Agents Chemother, 2003 Feb, 47(2), 790 - 3
Clinical isolates of Enterobacteriaceae producing extended-spectrum beta-lactamases: prevalence of CTX-M-3 at a hospital in China; Wang H et al.; The prevalence of extended-spectrum beta-lactamase-producing strains was demonstrated in 5 of 44 (11.4%) Escherichia coli, 17 of 43 (39.5%) Klebsiella pneumoniae, 3 of 50 (6.0%) Enterobacter cloacae, and 2 of 25 (8.0%) Citrobacter freundii strains at a teaching hospital in China . Nineteen of these 27 strains expressed CTX-M-3 beta-lactamase (pI 8.6) . A subset of the clinical isolates expressing the CTX-M-3 enzyme, tested by pulsed-field gel electrophoresis, revealed multiple clones . Five isolates expressed a novel enzyme, SHV-43 (pI 8.0), which had two substitutions (Leu113Phe and Thr149Ser) compared with SHV-1.

Jpn J Antibiot, 2002 Oct, 55(5), 514 - 23
{Antibacterial activities and PK/PD parameters of aminoglycosides against recent clinical isolates of gram-negative rods}; Okubo T et al.; We examined antibacterial activities and PK/PD parameters of six kinds of aminoglycosides against seven bacterial species of clinical isolates in 2001 . Aminoglycoseides examined were gentamicin (GM), dibekacin (DKB), tobramycin (TOB), amikacin (AMK), netilmicin (NTL), and isepamicin (ISP), and bacterial isolates used were each 50 strains of Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacae, Citrobacter freundii, Proteus spp., Serratia marcescens and Pseudomonas aeruginosa . All aminoglycosides showed good activities with low MICs against 6 species of Enterobacteriacea except S . marcescens . Eight strains (3.2%) among them were resistant to one or more aminoglycosides . Resistance to multiple aminoglycosides were detected in 16 strains (32%) of S . marcescens, among which 13 strains were resistant to AMK but susceptible to ISP . Three (6%) strains of P . aeruginosa were resistant to multiple drugs, one of which was resistant to all six aminoglycosides, and others were moderately susceptible to AMK and ISP, and susceptible to GM, AMK and ISP . Using a ratio of peak serum concentration to MIC90 (Cmax/MIC90) or a ratio of area under the curve to MIC90 (AUC/MIC90) as a pharmacokinetic and pharmacodynamic (PK/PD) parameter, we estimated the efficacy of the drug . An excellent effect of ISP, which was injected intramuscularly or intravenously at a dose of 400 mg, was expected for strains of Enterobacteriacea except S . marcescens . The Cmax/MIC90 ratios for S . marcescens were comparably higher in GM and ISP and that for P . aeruginosa were rather high in TOB when compared to other aminoglycosides . Another PK/PD parameter, AUC/MIC90 ratio, was high enough in NTL and ISP for Enterobacteriacea, suggesting good efficacy of these drugs . The (AUC/MIC90) ratios for S . marcescens were comparably high in GM and ISP, and that for P . aeruginosa were high in TOB, DKB, and ISP.

J Infect Chemother, 2002 Dec, 8(4), 353 - 7
Evaluation of antibacterial efficacy of drugs for urinary tract infections by genotyping based on pulsed-field gel electrophoresis (PFGE); Osawa K et al.; We attempted to determine whether the same bacterium isolated from patients with urinary tract infections (UTI) before and after treatment with antimicrobial agents was of the same strain ("persisted") or of a different strain ("changed") . Furthermore, to verify the effectiveness of antimicrobial agents for UTI, we investigated whether bacterial strains could be classified as persisted or changed based on their electrophoretic patterns in pulsed-field gel electrophoresis (PFGE) . We examined eight species of bacteria ( Enterococcus avium, Staphylococcus epidermidis, Pseudomonas fluorescens, Serratia marcescens, Citrobacter freundii, Enterobacter cloacae, Enterobacter aerogenes, and Escherichia coli), consisting of 41 strains (19 pairs isolated before and after treatment with antimicrobial agents) isolated from patients with complicated UTI . It was concluded that all bacteria were unchanged, based on the clinical effect on bacteriuria . The chromosomal DNA of these bacteria was digested with restriction enzymes and classified based on their electrophoretic pattern in PFGE . A comparison of the patterns of the fragments revealed that 14 pairs were indistinguishable before and after treatment, 3 pairs were closely related, 1 pair was possibly related, and 1 pair was different . These results demonstrated that 18 pairs of isolates (indistinguishable, closely related, and possibly related) were highly likely to be continued infections by the same strain, and that 1 pair (different) had been replaced by another strain . Based on these results, we believe that bacterium genotyping by PFGE is a more effective method for evaluating the antibacterial efficacy of antimicrobial agents.

J Clin Microbiol, 2003 Jan, 41(1), 155 - 8
Occurrence and phenotypic characteristics of extended-spectrum beta-lactamases among members of the family Enterobacteriaceae at the Tel-Aviv Medical Center (Israel) and evaluation of diagnostic tests; Navon-Venezia S et al.; We assessed the prevalence and phenotypic characteristics of extended-spectrum beta-lactamase (ESBL) producers among cefuroxime-resistant (CXM-R) (MIC > or = 32 micro g/ml) members of the family Enterobacteriaceae in our institution . The 438 CXM-R clinical isolates obtained from nonurine sources among inpatients were screened . ESBL production was confirmed by disk diffusion assay using cefpodoxime (CPD), cefotaxime (CTX), and ceftazidime (CTZ) with and without clavulanate (CLAV) . A difference of > or =5 mm in the size of the zone of inhibition in the presence of CLAV for at least one of the agents was considered representative of the ESBL phenotype: 186 isolates (42.5%) were confirmed as ESBL producers . The isolates tested and the rates of ESBL producers were as follows: Klebsiella spp . (n = 81), 79%; Proteus spp . (n = 58), 62%; Escherichia coli (n = 64), 53%; Enterobacter spp . (n = 69), 42%; Serratia spp . (n = 70), 14%; Citrobacter spp . (n = 25), 24%; Providencia spp . (n = 21), 24%; Morganella spp . (n = 41), 5%; and Kluyvera (n = 3), 0% . The overall sensitivity of isolated ESBL confirmatory tests was 79% for CPD-CLAV, 66% for CTZ-CLAV, and 91% for CTX-CLAV . Sensitivities of CTZ-CLAV confirmatory tests for Klebsiella spp., Proteus spp., E . coli, and Enterobacter spp . were 84, 22, 76, and 62%, respectively, and those for CTX-CLAV were 95, 97, 94, and 83%, respectively . They were 90% for CPD-CLAV and CTZ-CLAV, 95% for CPD-CLAV and CTX-CLAV, and 100% for CTZ-CLAV and CTX-CLAV . ESBL production was highly prevalent among Enterobacteriaceae . Using resistance to CXM as an ESBL screening criterion is a suitable option in high-incidence areas where Klebsiella spp . are not the dominant ESBL producers . This screening criterion may simplify the screening test and improve its sensitivity, although at the price of testing more isolates . The CTX-CLAV combination confirmed ESBL producers better than the CTZ-CLAV combination, with sensitivity varying between species . Combined CTZ-CLAV and CTX-CLAV testing detected all these strains; CPD-CLAV provided no additional benefit.

Biochem J, 2003 Apr 1, 371(Pt 1), 175 - 81
pKa measurements from nuclear magnetic resonance of tyrosine-150 in class C beta-lactamase; Kato-Toma Y et al.; 13C-NMR spectroscopy was used to estimate the p K a values for the Tyr(150) (Y150) residue in wild-type and mutant class C beta-lactamases . The tyrosine residues of the wild-type and mutant lactamases were replaced with (13)C-labelled L-tyrosine ({ phenol -4-(13)C}tyrosine) in order to observe the tyrosine residues selectively . Spectra of the wild-type and K67C mutant (Lys(67)-->Cys) enzyme were compared with the Y150C mutant lactamase spectra to identify the signal originating from Tyr(150) . Titration experiments showed that the chemical shift of the Tyr(150) resonance in the wild-type enzyme is almost invariant in a range of 0.1 p.p.m . up to pH 11 and showed that the p K (a) of this residue is well above 11 in the substrate-free form . According to solvent accessibility calculations on X-ray-derived structures, the phenolic oxygen of Tyr(150), which is near the amino groups of Lys(315) and Lys(67), appears to have low solvent accessibility . These results suggest that, in the native enzyme, Tyr(150) in class C beta-lactamase of Citrobacter freundii GN346 is protonated and that when Tyr(150) loses a proton, a proton from Lys(67) would replace it . Consequently, Tyr(150) would be protonated during the entire titration.

Zh Mikrobiol Epidemiol Immunobiol, 2002 Nov-Dec, (6), 5 - 9
{Pathogenicity islands in opportunistic Enterobacteria}; Mavziutov AR et al.; The presence of fragments of genomes hlyA, hlyB, papAH, papC, sfaG, sfaA and kps MT, associated with the pathogenicity islands of Escherichia coli, in clinical strains of other genera of the family Enterobacteriaceae, has been experimentally evaluated with the use of PCR . The presence of DNA fragments specific to the known genes of the pathogenicity clusters of E . coli in representatives of the genera Citrobacter, Enterobacter, Hafnia, Klebsiella, Morganella, Proteus, Providencia, Serratia and Yersinia of rarely occurring groups has been established . In Enterobacteriaceae cultures isolated from the intestine amplicons homologous to hlyB were detected significantly less frequently than among strains of nonintestinal origin . In Enterobacteriaceae strains isolated in respiratory pathology amplicons of the pili gene (sfaG) were detected significantly more frequently than in collection cultures . The total evaluation of the detection rate of the genes of pathogenicity islands among Enterobacteriaceae clinical strains under study in comparison with E . coli showed that they occurred significantly less frequently . Klebsiella spp . were found to differ most essentially from E . coli as regards the occurrence of fragments of the genes of pathogenicity islands . The conclusion was made on the high probability of genetic exchange in DNA fragments between different species of bacteria with corresponding changes in their pathogenicity.

Antimicrob Agents Chemother, 2003 Jan, 47(1), 395 - 7
Escherichia coli with a self-transferable, multiresistant plasmid coding for metallo-beta-lactamase VIM-1; Miriagou V et al.; An Escherichia coli strain exhibiting decreased susceptibility to carbapenems was isolated from a hospitalized patient in Greece . The strain carried a self-transferable plasmid coding for metallo-beta-lactamase VIM-1 . bla(VIM-1), along with aacA7, dhfrI, and aadA, was included as a gene cassette in a novel class 1 integron . A Citrobacter freundii ampC-derived gene, not associated with the integron, was also located in the same plasmid.

Microbes Infect, 2002 Nov, 4(14), 1389 - 99
Tyrosine residues at the immunoglobulin-C-type lectin inter-domain boundary of intimin are not involved in Tir-binding but implicated in colonisation of the host; Reece S et al.; Intimin is an outer membrane adhesion molecule involved in bacterial adhesion to intestinal epithelium by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium . Intimin binds to the translocated intimin receptor, Tir, which is delivered to the plasma membrane of the host cell by a type III protein translocation system . Intimin is also implicated in binding to a host cell-encoded intimin receptor (Hir) . The receptor-binding activity of intimin resides within the carboxy terminus 280 amino acids (Int280) of the polypeptide . Structural analysis of this region revealed two immunoglobulin-like domains, the second of which forms a number of contacts with the distal C-type lectin-like module . Specific orientation differences at this inter-domain boundary, which consists of several tyrosine residues, were detected between the crystal and solution structures . In this study, we determined the influence of site-directed mutagenesis of each of four tyrosine residues on intimin-Tir interactions and on intimin-mediated intimate attachment . The mutant intimins were also studied using a variety of in vitro and in vivo infection models . The results show that three of the four Tyr, although not essential for A/E lesion formation in vitro, are required for efficient colonisation of the mouse host following oral challenge.

Clin Microbiol Infect, 2002 Nov, 8(11), 725 - 33
Infectious complications in patients undergoing unrelated donor bone marrow transplantation: experience from a single institution; Saavedra S et al.; OBJECTIVE: To analyze the incidence and characteristics of documented infections in patients with hematologic malignancies undergoing unrelated donor bone marrow transplantation (UD-BMT) . METHODS: We studied the occurrence of infections in 22 patients with hematologic malignancies or severe aplastic anemia who underwent UD-BMT from April 1990 to December 2000 . The median age was 26 years (range 13-46) . Acyclovir-ganciclovir, co-trimoxazole, fluconazole-nystatin and ciprofloxacin were administered for anti-infectious prophylaxis . RESULTS: We registered 61 infectious episodes . During the early post-transplant period, there were eight clinically documented infections (CDIs), four cases of fever of unknown origin (FUO), seven cases of bacteremia, two cases of cytomegalovirus (CMV) antigenemia, and one case of CMV disease . In the intermediate period (days 30-100 after BMT), there were nine cases of CMV antigenemia, three bacterial infections, two fungal infections, one case of disseminated toxoplasmosis, and one case of FUO . In the late period (day 100 and later), we documented 13 viral infections, eight bacterial infections, one CDI, and one case of invasive aspergillosis . Infections contributed to death in 10 of 17 patients . Citrobacter bacteremia and sepsis of unknown origin were the main causes of infectious mortality in the early period . Infection was the main cause of death in six of seven patients in the late period . CONCLUSION: A high incidence of life-threatening infections and infection-related mortality was observed . A high rate of CMV infection in the early period, and death caused by multiresistant Gram-negative microorganisms in the late period, were the main findings in this series.

Infect Immun, 2002 Dec, 70(12), 7153 - 5
Cloning and characterization of genes encoding homologues of the B subunit of cholera toxin and the Escherichia coli heat-labile enterotoxin from clinical isolates of Citrobacter freundii and E . coli; Karasawa T et al.; We identified and characterized a gene encoding a homologue of the B subunits of cholera toxin (CTB) and heat-labile enterotoxin (LTB) of Escherichia coli from a clinical isolate of Citrobacter freundii that was found to produce a factor in the culture supernatant that cross-reacted with antibodies to CTB and LTB when assayed by enzyme-linked immunosorbent assay (ELISA) . The gene encoding the ELISA-positive factor, cfxB, consisted of 375 nucleotides and was located downstream of an 852-nucleotide open reading frame, cfxA, with a 56-nucleotide intergenic space . The cfxB gene was predicted to encode a 125-amino-acid polypeptide, which had 73.8 and 72.8% identities with the amino acid sequences of LTB and CTB, respectively . However, the amino acid sequence of the deduced polypeptide CFXA had no homologies to those of the A subunits of CT or LT . DNA probes developed from the sequences of cfxA and cfxB were used to screen 67 C . freundii isolates and 152 E . coli isolates from diarrheal patients by colony blot hybridization . Two strains, C . freundii 48 and E . coli 176, reacted with both DNA probes under conditions of high stringency . We cloned homologues of the cfxA and cfxB genes from E . coli 176 and designated them ecxA and ecxB, respectively . The ecxA gene and the ecxB gene comprise 855 and 375 nucleotides, respectively, with a 50-nucleotide intergenic space, and encode a 285- and a 125-amino-acid residue polypeptides, respectively . The results of the present study may provide important clues to the origin and evolution of immunologically related factors sharing a common enterotoxin-like A and B subunit structures.

Res Microbiol, 2002 Sep, 153(7), 455 - 9
Host/pathogen interactions at mucosal surfaces: immune consequences; Clare S et al.; The mucosal immune system has evolved to protect the host against the establishment of infections at or through the mucosal surfaces of the body . Protective immunity must be activated to specific pathogenic agents or their products but inappropriate immune responses to food/environmental antigens must be avoided . Thus, the mucosal immune system is under tight regulation . Pathogenic bacteria and their products can be exploited as specific probes of mucosal immune responses . Bacterial enterotoxins such as cholera toxin are potent mucosal immunogens and adjuvants that activate both mucosal and systemic immune responses . Infection models involving microorganisms such as Citrobacter rodentium can also be used to investigate the consequences of mucosal colonisation that lead to immune disfunction.

Microbiol Res, 2002, 157(3), 233 - 8
Characterisation of local isolates of Enterobacteriaceae from Turkey; Icgen B et al.; 20 local isolates of enterics belonging to the genera Salmonella, Enterobacter, Proteus, Citrobacter from human, chicken and/or egg were characterised for their antibiotic resistance patterns, plasmid profiles, phage types, outer membrane proteins, and lipopolysaccharide patterns . Relatedness of these characteristics for epidemiological analysis was assessed . 18 (90%) strains were resistant to at least one antibiotic and those (multi-drug resistant ones) resisting to two or more antibiotics constituted 50% of all isolates . A common 54 kb plasmid was harboured by 55% of the isolates . 14 isolates showed smooth type lipopolysaccharide . 60% of the 20 isolates contained outer membrane proteins in a molecular weight range of 34.6 to 30.6 kDa . The data reveal the lack of correlation between the characteristics investigated.

FEMS Microbiol Lett, 2002 Sep 24, 215(1), 81 - 7
Cloning and sequencing of the beta-lactamase gene and surrounding DNA sequences of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii, Escherichia fergusonii and Enterobacter cancerogenus; Naas T et al.; To further identify the origins of plasmid-mediated cephalosporinases that are currently spreading worldwide, the chromosomal beta-lactamase genes of Citrobacter braakii, Citrobacter murliniae, Citrobacter werkmanii reference strains and of Escherichia fergusonii and Enterobacter cancerogenus clinical isolates were cloned and expressed into Escherichia coli and sequenced . These beta-lactamases had all a single pI value >8 and conferred a typical AmpC-type resistance pattern in E . coli recombinant strains . The cloned inserts obtained from genomic DNAs of each strain encoded Ambler class C beta-lactamases . The AmpC-type enzymes of C . murliniae, C . braakii and C . werkmanii shared 99%, 96% and 95% amino acid sequence identity, respectively, with chromosomal AmpC beta-lactamases from Citrobacter freundii . The AmpC-type enzyme of E . cancerogenus shared 85% amino acid sequence identity with the chromosomal AmpC beta-lactamase of Enterobacter cloacae OUDhyp and the AmpC-type enzyme of E . fergusonii shared 96% amino acid sequence identity with that of E . coli K12 . The ampC genes, except for E . fergusonii, were associated with genes homologous to regulatory ampR genes of other chromosomal class C beta-lactamases that explain inducibility of beta-lactamase expression in these strains . This work provides further evidence of the molecular heterogeneity of class C beta-lactamases.

East Afr Med J, 2002 Mar, 79(3), 140 - 2
Antibiotic sensitivities of common bacterial pathogens in urinary tract infections at Gondar Hospital, Ethiopia; Moges AF et al.; OBJECTIVE: To determine the prevalence and sensitivity trends of urinary bacterial isolates . DESIGN: A cross-sectional study . SETTING: Gondar College of Medical Sciences (GCMS) Teaching and Referral Hospital, north west Ethiopia . SUBJECTS AND METHODS: Four hundred and twenty urine specimens from 70 in-patient and 350 out-patient cases were studied by quantitative culture method and anti-microbial sensitivity test was done by disc diffusion technique . RESULTS: One hundred and seventy two pathogenic organisms were isolated from 166 patients; the isolation rate was 39.5 % . Among the isolates E . coli, S . aureus, Klebsiella species, coagulase negative Staphylococcus species and Citrobacter species were common accounting for 46.0%, 18.0%, 10.0%, 8.0% and 6.0%, respectively . Of the total isolates 71.5% were Gram negatives . Sensitivity tested against ten antibiotics showed that resistance was common, and the effectiveness of tetracycline, ampicillin, co-trimoxazole, chloramphenicol and penicillin was under 50.0% . The resistance rate was 71.5%, 62.2%, and 62.2%, 54.7% and 40.8%, respectively . Polymixin B, cefoxitin, gentamycin and erythromycin controlled over 76.0% of the common infective agents . Ciprofloxacin did control 98.3% of the organisms . CONCLUSION: Resistance was found to be very high to the commonly used antibiotics . The sensitivity rate for the recently introduced ciprofloxacin was above 98% . Therefore, this antibiotic may be used for empirical therapy of urinary tract infection (UTI) when culture and sensitivity testing is impossible . Strict control on the use of antibiotics and appropriate measures against over the counter availability and self-medication is recommended.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3555 - 60
Low-virulence Citrobacter species encode resistance to multiple antimicrobials; Pepperell C et al.; Citrobacter spp . are gram-negative commensal bacteria that infrequently cause serious nosocomial infections in compromised hosts . They are often resistant to cephalosporins due to overexpression of their chromosomal beta-lactamase . During a recent study of multidrug-resistant Enterobacteriaceae (MDRE) in solid-organ transplant patients, we found that almost half of patients colonized with MDRE carried one or more cefpodoxime-resistant Citrobacter freundii, Citrobacter braakii, or Citrobacter amalonaticus strains . Pulsed-field gel electrophoresis showed that 36 unique strains of Citrobacter were present among 32 patients . Genetic and phenotypic analysis of the resistance mechanisms of these bacteria showed that the extended-spectrum beta-lactamase (ESBL) SHV-5 or SHV-12 was encoded by 8 strains (26%) and expressed by 7 strains (19%) . A number of strains were resistant to other drug classes, including aminoglycosides (28%), trimethoprim-sulfamethoxazole (31%), and fluoroquinolones (8%) . PCR and DNA analysis of these multiresistant strains revealed the presence of class I integrons, including the first integrons reported for C . braakii and C . amalonaticus . The integrons encoded aminoglycoside resistance, trimethoprim resistance, or both . Despite the prevalence of MDR Citrobacter spp . in our solid-organ transplant patients, only a single infection with a colonizing strain was recorded over 18 months . Low-virulence Citrobacter spp., which can persist in the host for long periods, could influence pathogen evolution by accumulation of genes encoding resistance to multiple antimicrobial classes.

Antimicrob Agents Chemother, 2002 Nov, 46(11), 3401 - 5
Identification of a chromosome-borne expanded-spectrum class a beta-lactamase from Erwinia persicina; Vimont S et al.; From whole-cell DNA of an enterobacterial Erwinia persicina reference strain that displayed a penicillinase-related antibiotic-resistant phenotype, a beta-lactamase gene was cloned and expressed in Escherichia coli . It encoded a clavulanic-acid-inhibited Ambler class A beta-lactamase, ERP-1, with a pI value of 8.1 and a relative molecular mass of ca . 28 kDa . ERP-1 shared 45 to 50% amino acid identity with the most closely related enzymes, the chromosomally encoded enzymes from Citrobacter koseri, Kluyvera ascorbata, Kluyvera cryocrescens, Klebsiella oxytoca, Proteus vulgaris, Proteus penneri, Rahnella aquatilis, Serratia fonticola, Yersinia enterocolitica, and the plasmid-mediated enzymes CTX-M-8 and CTX-M-9 . The substrate profile of the noninducible ERP-1 was similar to that of these beta-lactamases . ERP-1 is the first extended-spectrum beta-lactamase from an enterobacterial species that is plant associated and plant pathogenic.

J Food Prot, 2002 Oct, 65(10), 1656 - 9
Comparison between VIDAS automatic enzyme-linked fluorescent immunoassay and culture method for Salmonella recovery from pork carcass sponge samples; Yeh KS et al.; VIDAS Salmonella (VIDAS-SLM) is an automated system that uses the enzyme-linked fluorescent assay method to detect Salmonella species . This study evaluated the efficacy of the VIDAS-SLM method in detecting Salmonella species in pork carcass sponge samples gathered from 10 slaughter plants in Taiwan . Two hundred fifty-seven pork carcass sponge samples were screened by the VIDAS-SLM method and by the culture method in parallel . While 18 sponge samples were found to test positive by both methods, the VIDAS-SLM method detected four additional positive samples for which the culture method failed to recover Salmonella . The specificity of the VIDAS-SLM method was found to be 0.98, and its sensitivity was 1.0, since no false-negative results occurred . Artificially inoculated Salmonella at concentrations as low as 5.0 x 10(0) CFU/ml was detected in the heat-inactivated sponge sample in the presence or absence of 5.0 x 10(4) CFU of Citrobacter freundii per ml . Thus, the VIDAS-SLM method is a rapid screening method and a potential alternative to the time- and labor-intensive culture method.

Infect Immun, 2002 Nov, 70(11), 6424 - 35
Modulation of inducible nitric oxide synthase expression by the attaching and effacing bacterial pathogen citrobacter rodentium in infected mice; Vallance BA et al.; Citrobacter rodentium belongs to the attaching and effacing family of enteric bacterial pathogens that includes both enteropathogenic and enterohemorrhagic Escherichia coli . These bacteria infect their hosts by colonizing the intestinal mucosal surface and intimately attaching to underlying epithelial cells . The abilities of these pathogens to exploit the cytoskeleton and signaling pathways of host cells are well documented, but their interactions with the host's antimicrobial defenses, such as inducible nitric oxide synthase (iNOS), are poorly understood . To address this issue, we infected mice with C . rodentium and found that iNOS mRNA expression in the colon significantly increased during infection . Immunostaining identified epithelial cells as the major source for immunoreactive iNOS . Finding that nitric oxide (NO) donors were bacteriostatic for C . rodentium in vitro, we examined whether iNOS expression contributed to host defense by infecting iNOS-deficient mice . Loss of iNOS expression caused a small but significant delay in bacterial clearance without affecting tissue pathology . Finally, immunofluorescence staining was used to determine if iNOS expression was localized to infected cells by staining for the C . rodentium virulence factor, translocated intimin receptor (Tir), as well as iNOS . Interestingly, while more than 85% of uninfected epithelial cells expressed iNOS, fewer than 15% of infected (Tir-positive) cells expressed detectable iNOS . These results demonstrate that both iNOS and intestinal epithelial cells play an active role in host defense during C . rodentium infection . However, the selective expression of iNOS by uninfected but not infected cells suggests that this pathogen has developed mechanisms to locally limit its exposure to host-derived NO.

J Infect Chemother, 2002 Sep, 8(3), 207 - 10
Cefcapene inactivates chromosome-encoded class C beta-lactamases; Alba J et al.; The stability of cefcapene and cefpodoxime, oral antibacterial cephalosporins, toward different classes of beta-lactamases was evaluated . For the class A beta-lactamases, TEM-1, SHV-1, and NMC-A, only the steady-state kinetic parameter ( k(cat)/ Km) values were calculated (3100 - 1.1 x 10(7) M(-1) x s(-1)), because these enzymes have very high Km values for cefpodoxime and cefotaxime . As for class B beta-lactamases L1, IMP-1, and CcrA, in general, similar k(cat)/ Km values were obtained . However, regarding class C beta-lactamases from Enterobacter cloacae, Escherichia coli, Pseudomonas aeruginosa, and Citrobacter freundii, we found major differences in stability between the two compounds . Cefpodoxime acted as a good substrate for the class C beta-lactamases, except for the enzyme from E . cloacae; its k(cat) and Km values were successfully calculated ( k(cat)/ Km, 1.8 x 10(5) - 1.2 x 10(7) M(-1) x s(-1)) . On the other hand, cefcapene acted as a poor substrate or an inactivator for class C beta-lactamases; its k(2)/ K value was successfully calculated (8.7 x 10(5) - 7.0 x 10(6) M(-1) x s(-1)) . In addition, k(3) values were determined for beta-lactamases from P . aeruginosa (2.3 x 10(-2) x s(-1)) and C . freundii (2.1 x 10(-1) x s(-1)) . Even though these values could be calculated, transient inactivation as an enzyme reactivation reaction for all these enzymes was observed . These findings suggest the potential of cephem compounds as inhibitors of class C beta-lactamases.

J Antimicrob Chemother, 2002 Oct, 50(4), 503 - 11
Metallo-beta-lactamase-producing Enterobacteriaceae isolates in a university hospital in Taiwan: prevalence of IMP-8 in Enterobacter cloacae and first identification of VIM-2 in Citrobacter freundii; Yan JJ et al.; A total of 9082 clinical isolates of Enterobacteriaceae other than Klebsiella spp . collected in 1999 and 2000 at a university hospital in Taiwan were investigated for the production of metallo- beta-lactamases (MBLs) . Thirty-six (2.9%) of the 1261 Enterobacter cloacae isolates and one (0.3%) of the 340 Citrobacter freundii isolates were found to carry bla(IMP-8) and bla(VIM-2), respectively, by colony hybridization, PCR and sequence analysis . The IMP-8 producers were recovered from 20 patients and four of them had recently transferred from other hospitals, implying spread of IMP-8-producing E . cloacae among different healthcare settings . Of the 20 non-repetitive IMP-8 producers, 17 (85%) isolates also harboured bla(SHV-12), which was on the same transferable plasmids with bla(IMP-8) . The bla(VIM-2)-positive isolate and all non-repetitive bla(IMP-8)-positive isolates appeared susceptible to imipenem (MICs < 8 mg/L) and meropenem (MICs < 4 mg/L), indicating the difficulty in detection of MBLs in Enterobacteriaceae by routine susceptibility testing . Ribotyping of the IMP-8-producing E . cloacae isolates indicated that the dissemination of bla(IMP-8) was due largely to the spread of an epidemic clone, but horizontal transfer among unrelated strains also occurred.

Carbohydr Res, 2002 Sep 27, 337(17), 1541 - 6
Structure of a new 2-deoxy-2-{(R)-3-hydroxybutyramido}-D-glucose-containing O-specific polysaccharide from the lipopolysaccharide of Citrobacter gillenii PCM 1542; Katzenellenbogen E et al.; The O-specific polysaccharide of Citrobacter gillenii PCM 1542 from serotype O-12a,12 b is composed of one residue each of D-glucose, D-GlcNAc, 2-deoxy-2-{(R)-3-hydroxybutyramido}-D-glucose (D-GlcNAcyl) and two GalNAc residues . On the basis of sugar and methylation analyses of the intact and Smith degraded polysaccharides, along with 1D and 2D 1H and 13C NMR spectroscopy, the following structure of the branched pentasaccharide repeating unit of the O-specific polysaccharide was established:This structure differs significantly from that of the O-specific polysaccharide of C . gillenii PCM 1544 from the same serotype O-12a,12 b, which has been established earlier (Kubler-Kielz.shtsls;b, J . et al . Carbohydr . Res . 2001, 331, 331-336) . Serological studies confirmed that the two O-antigens are not related and suggested that strains PCM 1542 and 1544 should be classified into different O-serogroups.

Appl Environ Microbiol, 2002 Oct, 68(10), 4788 - 94
Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer; Reissbrodt R et al.; Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium . Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time . The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine . Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect . Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating . Patterns of resuscitation of S . enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation . Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.

J Vet Diagn Invest, 2002 Sep, 14(5), 431 - 3
Bacterial pathogens isolated from cultured bullfrogs (Rana castesbeiana); Mauel MJ et al.; A commercial bullfrog (Rana castesbeiana) operation in south Georgia had multiple epizootics of systemic bacterial infections over a 3-year period, 1998-2000 . A number of potential pathogens (Aeromonas hydrophila, Chryseobacterium (Flavobacterium) meningosepticum, Chryseobacterium (Flavobacterium) indolgenes, Edwardsiella tarda, Citrobacterfreundii, Pseudomonas spp., and (Streptococcus iniae) were isolated from various tissues . Clinically, frogs demonstrated acute onset of torticolis, stupor, and indifference to stimuli . Cutaneous hyperemia, subcutaneous and muscular hemorrhage, and peripheral edema were consistent gross findings . Histologically, clusters of lymphocytes, monocytes, and occasional acidophiles with scattered granulomas occurred in liver, kidney, and spleen . This is the first report of S . inae and C . meningosepticum as potential disease agents in R . castesbeiana . These findings suggest that a variety of bacteria may be associated with redleg and that culture results must be obtained for accurate diagnosis.

Vet Microbiol, 2002 Sep 24, 88(4), 339 - 49
Development of an ELISA using a recombinant 41 kDa partial protein (P45N') for the detection of Riemerella anatipestifer infections in ducks; Huang B et al.; Riemerella anatipestifer, a gram-negative bacillus, is the causative agent of duck septicemia, a disease which could incur much economic loss in the duck industry . An indirect enzyme-linked immunosorbent assay (ELISA) has been developed to facilitate early detection of R . anatipestifer infection in ducks . The antigen used was a recombinant 41 kDa N-terminal fragment (rP45N') of a newly characterized R . anatipestifer potential surface protein, P45, which was expressed in Escherichia coli as an N-terminal GST fusion protein . The rP45N'-based ELISA successfully detected P45 antibodies in the sera of 20 ducks immunized with bacterin preparations of R . anatipestifer serotypes 1, 10 15, 19 and the ATCC11845 strain . Antibodies to P45 were also detected in the sera of 25% (75/296) of White Pekin ducks which were imported into Singapore from three different farms . Successful discrimination was obtained between sera from infected ducks and that of specific-pathogen free ducks (p<0.01) . The rP45N'-GST antigen did not cross-react with antibodies in sera from guinea pigs which were infected with other gram-negative and gram-positive bacterial pathogens, including Aeromonas hydrophila, Citrobacter freundii, E . coli, Klebsiella pneumoniae, Pastuerella multocida, Proteus mirabilis, Salmonella spp., Serratia maccescens, Shigella sonnei and Yersinia enterocolitica . In addition, the DNA sequence encoding P45 was detected in R . anatipestifer serotypes 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 14, 15, 16, 17, 18, 19 and the ATCC11845 strain, suggesting that P45 is probably also universally expressed in these R . anatipestifer serotypes . Thus, the ELISA described is applicable to the detection of R . anatipestifer infection in ducks.

East Afr Med J, 2001 Dec, 78(12), 646 - 9
Antimicrobial resistance of bacterial organisms isolated from rats; Gakuya FM et al.; OBJECTIVE: To determine if antimicrobial resistance occurs in various bacterial species isolated from rats . METHOD: Two hundred and fifteen rats were trapped from areas in and around Nairobi, Kenya . They were sacrificed and their intestinal, liver and spleen specimens obtained . Various bacterial species were isolated from these specimens . The species were analysed for antimicrobial susceptibility to 12 commonly used antimicrobials using the disc diffusion technique . RESULTS: The bacterial species isolated included pathogenic and potentially pathogenic ones such as Escherichia coli 137, Salmonella typhimurium 1, Klebsiella pneumoniae 2, Enterobacter cloacae 4, Enterobacter sakazakii 2, Citrobacter freundii 3, Morganella morganii (2), Pseudomonas aeruginosa 2 and Burkhoddria cepacia 6 . Depending on the species, the resistance to the various antimicrobials were: 0-100% for cefotaxime, nalidixic acid, cefuroxime, tetracycline, chloramphenicol, co-amoxyclav, sulfamethoxazole, ampicillin, trimethoprim and cephradine, 0-66.6% for gentamicin and 0-25% for apramycin . CONCLUSION: The results showed that, rats from the study area harboured bacterial species with antimicrobial resistance . These micro-organisms may form an important reservoir for antibiotic resistance which could pose a public health hazard . Control of rat populations, better management of sewer systems and waste dumping sites are recommended in order to reduce occurrence of these drug resistance reservoirs.

Med Pregl, 2002 May-Jun, 55(5-6), 225 - 8
{Bacteriologic examination of gallbladder contents}; Petakovic G et al.; INTRODUCTION: Acute calculous (obstructive) cholecystitis develops as a consequence of cystic obstruction and obstruction of bile flow into choledochus . Most often it is a result of impacted gallstones in Hartman's pouch or the cystic duct . Their direct pressure on gallbladder mucosa causes ischemia, necrosis and ulceration with consequential wall edema and obstructed venous flow . This mechanism is further increasing and spreading the inflammatory process . Ulcerations may be that extensive, that mucosa is highly recognizable on the microscopic preparation . Leukocyte infiltration of all segments occurs . Results of necrosis are as follows: perforation with pericholecystic abscess formation, fistulization or biliary peritonitis . AIM: The aim of this investigation was to use microbial sensitivity tests in order to establish possibilities of antibiotic therapy in patients with acute cholecystitis . MATERIAL AND METHODS: Using random sampling a total of 240 patients with acute cholecystitis were included in the investigation . They were all treated at the Clinic of Abdominal and Endocrine Surgery of the Clinical Center Novi Sad in the period 1997-1999 . All patients underwent bacteriological examination and were coherent in regard to sex and age . Microbial sensitivity tests analyzed two groups of bacteria: Group I: Escherichia coli, Klebsiella and Staphylococcus and Group II: other isolated bacteria (Citrobacter, Enterobacter, Enterococcus, Proteus, Pseudomonas, Serratia and Streptococcus) . RESULTS: In our material Escherichia coli was isolated in most patients--32 (55.17%), Klebsiella and Staphylococcus in 6 (10.34%) patients and Streptococcus in 4 (6.90%), whereas other bacteria were infrequent (Citrobacter and Serratia in 3.45%, Enterobacter, Proteus and Pseudomonas in 1.75%) . Thus, E . coli, Klebsiella and Staphylococcus were established in 75.85% of bacteriologic findings, and all the rest in 24.15% . Assessment regarding premedication with antibiotics started with an assumption that cholecystitis was caused by one of the bacteria isolated in 75% of cases . That is why antibiotics should be given prior to surgery, primarily those to which these bacteria are susceptible in more than 95% of cases . CONCLUSIONS: E . coli, Klebsiella and Staphylococcus participate with 75.85% of all bacteriologic findings, whereas all the others make 24.15%; Amikacin, Cefalexin, Ceftriaxone, Ofloxacin and Pefloxacin are recommended in premedication; considering the fact that new generation antibiotics have not been tested yet, they were not taken into consideration for this study.

Environ Technol, 2002 Jul, 23(7), 731 - 45
Reduction of Cr(VI) and bioaccumulation of chromium by gram positive and gram negative microorganisms not previously exposed to Cr-stress; Pattanapipitpaisal P et al.; Resistance to Cr(VI) is usually associated with its cellular exclusion, precluding enrichment techniques for the isolation of organisms accumulating Cr(VI) via bioreduction to insoluble Cr(III) . A technique was developed to screen for potential Cr(VI) reduction in approx . 2000 isolates from a coastal environment, based on the non-specific reduction of selenite and tellurite to Se0 and Te0, and reduction of tetrazolium blue to insoluble blue formazan . The most promising strains were further screened in liquid culture, giving three, which were identified by 16S rRNA sequence analysis as Bacillus pumilus, Exiguobacterium aurantiacum and Pseudomonas synxantha, all of which reduced 100 microM Cr(VI) anaerobically, without growth . The respective removal of Cr(VI) was 90% and 80% by B . pumilus and E . aurantiacum after 48 h and 80% and by P . synxantha after 192 h . With the gram positive strains Cr(VI) promoted loss of flagella and, in the case of B . pumilus, lysis of some cells, but Cr was deposited as an exocellular precipitate which was identified as containing Cr and P using energy dispersive X-ray microanalysis (EDAX) . This prompted the testing of Citrobacter sp . N14 (subsequently re-assigned by 16S rRNA sequence analysis and biochemical studies as a strain of Serratia) which bioprecipitates metal cation phosphates via enzymatically-liberated phosphate . This strain reduced Cr(VI) at a rate comparable to that of P . synxantha but Cr(III) was not bioprecipitated where La(III) was removed as LaPO4, even though a similar amount of phosphate was produced in the presence of Cr(III) . Since B . pumilus removed most of the Cr(VI), with the formation of cell-bound CrPO4 implicated, this suggests that this strain could have future bioprocess potential.

Can J Vet Res, 2002 Jul, 66(3), 137 - 44
Occurrence and characterization of resistance to extended-spectrum cephalosporins mediated by beta-lactamase CMY-2 in Salmonella isolated from food-producing animals in Canada; Allen KJ et al.; Resistance of Salmonella to extended-spectrum cephalosporins (ESCs) is being reported with increasing frequency . In humans, infections with Salmonella resistant to ESCs threaten the efficacy of ceftriaxone, the drug of choice for treating salmonellosis in children . To determine the occurrence of resistance to ESCs, we examined 8426 strains isolated from food-producing animals in Canada in 1994-99 for reduced susceptibility or resistance to ceftriaxone . Of the 8 such strains identified (7 from turkeys and 1 from cattle), 5 had reduced susceptibility, and 3 were resistant; 2 were isolated in 1995, 1 was isolated in each of 1996 and 1997, and 4 were isolated in 1999 . Isoelectric focusing showed that all 8 isolates produced a beta-lactamase with a pI > or = 9 . The strains were resistant to cefoxitin and not inhibited by clavulanic acid . Primers specific for the Citrobacter freundii blaAmpC gene produced the expected product in the polymerase chain reaction . DNA sequencing showed that all isolates possessed the blaCMY-2 gene . Plasmid DNA from all 8 isolates transformed Escherichia coli DH10B, whereas only 1 isolate transferred blaCMY-2 conjugally . All transformants and the transconjugant were resistant to ampicillin, cefoxitin, ceftiofur, cephalothin, streptomycin, sulfisoxazole, and tetracycline . Southern blots of plasmids from the isolates, the transformants, and the transconjugant showed that blaCMY-2 was located on similar-sized plasmids (60 or 90 MDa) in the transformants and the transconjugant . In the S . Typhimurium DT104 and S . Ohio isolates, the floSt gene was found on the same plasmid . Class 1 integrons with the aadB gene cassette were detected in the S . Bredeney isolates but not in their transformants or the transconjugant . Pulsed-field gel electrophoresis and plasmid profiles indicated that both clonal dispersion and horizontal transfer of blaCMY-2 may have caused dissemination of the resistance determinant.

Anal Chem, 2002 Jul 1, 74(13), 3174 - 82
Mass spectrometric methods for generation of protein mass database used for bacterial identification; Wang Z et al.; The availability of a suitable database is critical in a proteomic approach for bacterial identification by mass spectrometry (MS) . The major limitation of the present public proteome database is the lack of extensive low-mass bacterial protein entries with masses experimentally verified for most bacteria . Here, we present a method based on mass spectrometry to create protein mass tables specifically tailored for bacterial identification . Several issues related to the detection of bacterial proteins for the purpose of database creation are addressed . Three species of bacteria, namely, Escherichia coli, Bacillus megaterium, and Citrobacter freundii, which can be found in the ambient environment, were chosen for this study . Direct matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis of each bacterial extract reveals 20-29 protein components in the mass range from 2000 to 20,000 Da . HPLC fractionation of bacterial extracts followed by off-line MALDI-TOF analysis of individual fractions detects 156-423 components . Analysis of the extracts by HPLC/electrospray ionization MS shows the number of detectable proteins in the range of 46-59 . Although a number of components were common to the three detection schemes employed, some unique components were found using each of these techniques . In addition, for E . coli where a large proteome database exists in the public domain, a number of masses detected by the mass spectrometric methods do not match with the proteome database . Compared to the public proteome database, the mass tables generated in this work are demonstrated to be more useful for bacterial identification in an application where the bacteria of interest have limited protein entries in the public database . The implication of this work for future development of a comprehensive mass database is discussed.

FEMS Microbiol Lett, 2002 Jul 16, 213(1), 103 - 11
BUT-1: a new member in the chromosomal inducible class C beta-lactamases family from a clinical isolate of Buttiauxella sp; Fihman V et al.; An atypical Enterobacteriaceae strain with a beta-lactam susceptibility pattern of inducible cephalosporinase was isolated in Tenon Hospital (Paris, France) from a patient's skull wound infection . Identifications by the API-50CHE biochemical system and 16S rRNA gene sequencing concluded that it was a member of the Buttiauxella genus . The bla gene was cloned and sequenced . The deduced translated product was a 383-amino acid protein (BUT-1) with 75-78% identity with the chromosomal AmpC beta-lactamases of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae and Escherichia coli . The isoelectric point of 9.0 and the kinetic constants of BUT-1 were comparable with results described for other Ambler class C enzymes . bla(BUT-1) and the associated ampR transcriptional regulator gene were divergently transcribed from a common intercistronic region, a genetic organization already described for other inducible class C beta-lactamases . The deduced amino acid sequence of AmpR shared 85% and 81% identity with AmpR from E . cloacae and C . freundii respectively.

J Clin Pathol, 2002 Jul, 55(7), 524 - 9
A comparison of the performance of cystine lactose electrolyte deficient (CLED) agar with Oxoid chromogenic urinary tract infection (CUTI) medium for the isolation and presumptive identification of organisms from urine; Fallon D et al.; AIMS: As part of the UK antimicrobial resistance strategy and action plan, the Public Health Laboratory Service (PHLS) is required to collect antibiotic susceptibility data so that resistance trends and patterns can be monitored . Most laboratories report urine Gram negative isolates, as "coliforms" according to morphological appearance, but without an acceptable identification system the antimicrobial surveillance data will be meaningless . Commercially available identification systems tend to be expensive and time consuming . Chromogenic agars, which claim to improve the detection of mixed cultures and identification of organisms from urine, have now become available and may provide a cost effective alternative . The primary aim of this study was to compare the performance of cystine lactose electrolyte deficient (CLED) agar with a chromogenic agar (Oxoid urinary tract infection medium; CUTI) in terms of isolation rates and ability to detect mixed cultures . Secondary aims were to evaluate the correlation of "presumptive" identification of isolates from chromogenic media with that of two commercial identification systems and to appraise the sensitivity of the semiquantitative loop and filter paper strip culture techniques . METHOD: One thousand, four hundred and sixty six urine samples were examined in four laboratories using the semiquantitative culture methods of 1 microl loop and filter paper strip . The degree of accuracy of organism identification was measured by comparing the presumptive identification using colony colour supplemented with simple bench tests, with identification obtained from two more complex commercial systems . RESULTS: There was no significant difference between the performance of the loop and filter paper strip methods on the CLED agar, but the CUTI agar performed significantly better than the CLED agar for the detection of significant isolates and mixed cultures . This difference was greater using the loop method . Identification of the organisms using the commercial systems gave > 99% agreement and was therefore considered suitable as a standard against which to compare the presumptive CUTI identification . Using the manufacturer's colony colour criteria in combination with a bench indole test, the CUTI medium was 99% specific for Escherichia coli, although this was reduced to 97% if the indole test was omitted . Citrobacter spp were the most commonly misidentified organisms, giving false presumptive identification as E coli . By testing oxidase activity to differentiate Pseudomonas spp and the absence of indole production to support the identification of Proteus mirabilis, the CUTI medium provided a suitable identification for 86.8% of Gram negative isolates . The remaining 13.2% would require further identification . CONCLUSION: CUTI medium improves the detection of mixed cultures, thereby improving the reliability of reporting of significant isolates when compared with CLED agar . When supplemented with simple bench tests it provides an identification system capable of speciating 86.8% of Gram negative isolates and providing a valuable cost effective mechanism for antimicrobial resistance surveillance.

J Microbiol Immunol Infect, 2002 Jun, 35(2), 109 - 14
Clinical features and antimicrobial susceptibility trends in Citrobacter freundii bacteremia; Chen YS et al.; This retrospective study investigated the clinical characteristics and antimicrobial susceptibilities of 36 cases of Citrobacter freundii bacteremia treated at the Taipei Veterans General Hospital from 1996 through 1999 . The results showed that the predominant infection site was the intraabdominal region and the mortality was 22% . The resistance of C . freundii to most third-generation cephalosporins and broad-spectrum penicillins increased in both nosocomial and community-acquired C . freundii bacteremia during the study period, and the strategy of using a combination of antimicrobial agents to treat C . freundii infection was effective . This study also demonstrated the importance of appropriate antimicrobial therapy to the successful outcome of C . freundii bacteremia . The guidelines of the National Nosocomial Infections Surveillance System were followed to determine trends of resistance of C . freundii to various antimicrobial agents . The resistance of C . freundii to antibiotics in 1999 (n = 10), compared with the period from 1996 through 1998 (n = 26), increased 66% for ciprofloxacin, 36% for ticarcillin/clavulanate, 70% for piperacillin/tazobactam, and 62.8% for piperacillin, but remained uniformly susceptible to imipenem/cilastatin and the new fluoroquinolone (levofloxacin) . This increase in resistance was attributable to the use of third-generation cephalosporin instead of first-generation cephalosporins . These findings indicate the need for new measures to facilitate the appropriate choice of antimicrobial agents for patients with C . freundii bacteremia.

Wilderness Environ Med, 2002 Summer, 13(2), 113 - 8
An analysis of human pathogens found in horse/mule manure along the John Muir Trail in Kings Canyon and Sequoia and Yosemite National Parks; Derlet RW et al.; OBJECTIVE: To determine the prevalence of microorganisms that are potentially pathogenic for humans in horse/mule manure along the John Muir Trail (JMT) . METHODS: Random samples of horse/mule manure were collected along sections of the JMT in Yosemite, Kings Canyon, and Sequoia national parks (NP), as well as in portions of the Pacific Crest Trail (PCT) and selected JMT/PCT access trails . Convenience samples of wild animal scat found within I mile of trails were also collected . The fresh specimens were individually preserved both in 0.9% saline and polyvinyl alcohol (PVA)-containing tubes and stored at 4 degrees C until time of analysis . Bacteriological analysis was performed using standard microbiology laboratory procedures . PVA samples were stained with trichrome and were then examined by a parasitologist . RESULTS: Collection: A total of 186 trail miles were sampled, including 113 on the JMT (Yosemite 37, Kings 53, and Sequoia 23) . The PCT samplings included 24 miles, and NP and wilderness area access trails added an additional 49 miles . A total of 102 samples were collected, which included 81 samples from pack animals and 21 identified as having come from wild animals . Pack Animal Bacteria: All plated specimens grew large numbers of commensal gut flora . Potential pathogenic bacteria were found in only 12 samples and included Hafnia alvei (4), Serratia odorifera (1), Citrobacter freundii (1), Escherichia vulneris (1), Clostridium clostridioforme (1), Yersinia enterocolitica (1), Sherwinella putraformus (1), and Enterobacter spp (4) . No Escherichia coli O157, Salmonella, or Aeromonas were found . Microscopic examination for protozoal organisms revealed occasional commensal ciliates and I Giardia . Wild Animal Pathogens: One specimen grew Y enterocolitica, and another grew Enterobacter amnigenus . CONCLUSIONS: We found a low prevalence of human pathogens in pack animal manure on the JMT.

Trends Microbiol, 2002 Jun, 10(6), 293 - 9
How bacteria could cause cancer: one step at a time; Lax AJ et al.; Helicobacter pylori highlighted the potential for bacteria to cause cancer . It is becoming clear that chronic infection with other bacteria, notably Salmonella typhi, can also facilitate tumour development . Infections caused by several bacteria (e.g . Bartonella spp., Lawsonia intracellularis and Citrobacter rodentium) can induce cellular proliferation that can be reversed by antibiotic treatment . Other chronic bacterial infections have the effect of blocking apoptosis . However, the underlying cellular mechanisms are far from clear . Conversely, several bacterial toxins interfere with cellular signalling mechanisms in a way that is characteristic of tumour promoters . These include Pasteurella multocida toxin, which uniquely acts as a mitogen, and Escherichia coli cytotoxic necrotizing factor, which activates Rho family signalling . This leads to activation of COX2, which is involved in several stages of tumour development, including inhibition of apoptosis . Such toxins could provide valuable models for bacterial involvement in cancer, but more significantly they could play a direct role in cancer causation and progression.

Eur J Clin Microbiol Infect Dis, 2002 Apr, 21(4), 283 - 9 Epub 2002 Apr 10.
Comparative evaluation of two commercial chromogenic media for detection and presumptive identification of urinary tract pathogens; Scarparo C et al.; The performance of two commercial chromogenic media for the isolation and presumptive identification of urinary tract pathogens, the CPS ID2 (bioMerieux, France) and the CHROMagar Orientation (BBL Becton Dickinson, USA), was evaluated and compared with that of cystine-lactose-electrolyte-deficient agar and tryptic soy agar with 5% sheep blood . The detection, determination of bacterial counts, and presumptive identification of bacteria causing urinary tract infections were evaluated in 3,000 urine specimens . The two chromogenic media showed excellent correlation with the standard media for the detection and the bacterial count of urinary pathogens . The Escherichia coli strains produced the expected colour on the CHROMagar Orientation and the CPS ID2 media in 99% and 90% of the cases, respectively . The Klebsiella-Enterobacter-Citrobacter and the Proteus-Morganella-Providencia groups were easily identified on both chromogenic media, but further biochemical tests were needed to differentiate them to a species level . Both media enabled the differentiation, with varying degrees of difficulty, of Pseudomonas spp . strains from members of the family Enterobacteriaceae . All isolates of Enterococcus spp . were correctly identified and were easily distinguished from the Streptococcus agalactiae isolates . Staphylococcus saprophyticus isolates were easy to identify only on the CHROMagar Orientation medium . No substantial difference was observed when comparing the results of the susceptibility tests, which were performed according to the standardized disk diffusion method as described by the National Committee for Clinical Laboratory Standards, for colonies recovered from the blood agar versus those recovered from the chromogenic media . In conclusion, the CPS ID2 and CHROMagar Orientation media enabled excellent detection, count determination, and presumptive identification of urinary pathogens, both in pure and mixed cultures, and reliable and accurate antimicrobial susceptibility testing directly from primary isolates . Moreover, these media allowed a remarkable reduction in the workload and a significant savings of time . On the basis of their performance, these media can replace the standard primary plating media used in the routine diagnosis of urinary tract infections.

Diagn Microbiol Infect Dis, 2002 May, 43(1), 49 - 60
Potency and antimicrobial spectrum update for piperacillin/tazobactam (2000): emphasis on its activity against resistant organism populations and generally untested species causing community-acquired respiratory tract infections; Johnson DM et al.; The in vitro activity of piperacillin/tazobactam and several comparison broad-spectrum compounds was assessed against recent clinical isolates of Gram-positive and -negative bacteria from geographically diverse medical centers in Europe, North and Latin America participating in various surveillance programs in 2000 . Several organisms were characterized for phenotypic expression of various resistant determinants such as extended-spectrum beta-lactamase (ESBL) or amp C cephalosporinase hyperproduction, and vancomycin resistance in enterococci (VRE) . Piperacillin/tazobactam retained activity (MIC50) against oxacillin-susceptible Staphylococcus spp . (0.12-0.5 microg/ml), Bacillus spp . (0.5 microg/ml), vancomycin-susceptible enterococci (>4 microg/ml), and Corynebacterium spp . (2 microg/ml; not including C . jeikeium) with susceptibility rates of 100.0, 91.7, 85.7 and 81.8%, respectively . Piperacillin/tazobactam inhibited all Streptococcus spp . strains at < or = 16 microg/ml, including penicillin-resistant strains many of which were co-resistant to erythromycin (90%) and other beta-lactams . A specific breakpoint for these streptococci when testing piperacillin/tazobactam appears needed to prevent false-resistant reports using penicillin as a class representative . The carbapenems among beta-lactams were the most active agents against the ESBL-producing species of Escherichia coli and Klebsiella pneumoniae and those strains which hyper-express amp C enzymes including Citrobacter spp . and Enterobacter spp . Piperacillin/tazobactam only exhibited modest activity against the "amp C resistance group" strains (68.8% susceptible or intermediate, MIC < or = 64 microg/ml) . Piperacillin/tazobactam (MIC50, 8 microg/ml; 79.5% susceptible) was the most active agent tested against multi-drug resistant isolates of Pseudomonas aeruginosa . Against sampled Haemophilus influenzae (39.2% ampicillin-resistant), piperacillin/tazobactam (MIC(90,) < or = 0.06 microg/ml), ceftriaxone and ceftazidime inhibited 100.0% of the isolates at < or = 0.25 microg/ml . These in vitro surveillance results from the year 2000 on three continents, demonstrated a sustained potent activity of piperacillin/tazobactam against selected problematic nosocomial and community-acquired pathogens . The potential importance of these findings is that this beta-lactamase inhibitor combination can be used an empiric treatment of serious infections in hospital environments where resistance has emerged, as well as covering nearly all isolates of fastidious respiratory tract pathogens acquired in the community setting.

Cutis, 2002 May, 69(5), 393 - 4
Citrobacter koseri in scalp folliculitis; Garcia-Bustinduy M et al.; Gram-negative folliculitis, an uncommon condition, is most often seen in older patients who have acne and who either have received prolonged courses of antibiotic therapy or have used antibacterial cleansers that selectively inhibit gram-positive organisms . Citrobacter infections are uncommon, and dermatologists seldom encounter them . In the past, these infections occurred in hospitals, particularly in neonatal intensive care units . Bacteremias also occur in elderly or immunocompromised patients . In this article, we present a case of Citrobacter koseri scalp folliculitis in an otherwise healthy patient.

Southeast Asian J Trop Med Public Health, 2001, 32 Suppl 2, 240 - 4
Parasitic and bacterial contamination in collards using effluent from treated domestic wastewater in Chiang Mai, Thailand; Keawvichit R et al.; Thailand often has inadequate water supply for agriculture during the dry season . The reuse of treated wastewater treatment plants could solve this problem . Treatment of domestic wastewater of Chiang Mai municipality by the aerated lagoon system (AL) releases more than 25,000 m3 of treated water everyday . The reuse of wastewater in agriculture is an efficient use of water, especially in tropical countries or in drought zones . The objective of this study is to demonstrate the possibility of using treated wastewater in growing edible vegetables, ie collards (kale), without pathogenic parasite and bacterial contamination . Collards (Brassica oleracea var acephala) were grown using either the treated wastewater from the aerated lagoon system (AL) or ground water (GW) . Three cropping times were scheduled in February, May and July, 2000 . Samples of water from AL system and GW were taken two times per month (the consecutive weeks) from February to July and examined for bacteria and parasites . Irrigation water (IW) that was normally used in agriculture was also collected, at the same time of the AL and GW collection, for bacteria and parasite investigation . A soil sample was taken before and after each crop for parasite examination . Collards were also collected at the end of the crop for parasite investigation . The results showed that GW seems to be a clean water since no pathogenic bacteria were found although small amount of Escherichia coli was noted in May . For AL and IW, similar number and types of bacteria were found . They were Aeromonas sobria, A . hydrophila, E . coli, Citrobacter freundii, Pseudomonas aeruginosa, non-pathogenic type of Vibrio cholerae . The small number of Salmonella enteritidis gr E was found in AL in April . After investigating 12 samples in 6 months of each kind of water, ie GW, Al, and IW, no parasite was found . Only unidentified free living nematodes were found in IW but those parasites are non pathogenic . A small number of unidentified free living nematodes (UFLN), a natural parasite, were found in soil after cropping . After each cropping time, similar number of hookworm was found in the plots which used either GW or AL . Collards grown by using either GW or AL showed no harmful parasite contamination . We conclude that the effluent from wastewater treatment, using aerated lagoon system, of Chiang Mai municipality could be safely used for growing collards.

J Clin Microbiol, 2002 Jun, 40(6), 1947 - 57
Development of a sensitive and specific enzyme-linked immunosorbent assay for detecting and quantifying CMY-2 and SHV beta-lactamases; Hujer AM et al.; Polyclonal rabbit antibodies against SHV-1 and CMY-2 beta-lactamases were produced and characterized, and enzyme-linked immunosorbent assays (ELISAs) were developed . Immunoblots revealed that the anti-SHV-1 antibody recognized SHV-1 but did not recognize TEM-1, K-1, OXA-1, or any AmpC beta-lactamase tested . The anti-CMY-2 antibody detected Escherichia coli CMY-2, Enterobacter cloacae P99, Klebsiella pneumoniae ACT-1, and the AmpC beta-lactamases of Enterobacter aerogenes, Morganella morganii, and Citrobacter freundii . No cross-reactivity of the anti-CMY-2 antibody was seen against laboratory strains of E . coli possessing TEM-1, SHV-1, K-1, or OXA-1 beta-lactamases . Operating conditions for performing ELISAs were optimized . Both anti-CMY-2 and anti-SHV-1 antibodies detected picogram quantities of purified protein in ELISAs . The reactivity of the anti-CMY-2 antibody was tested against a number of AmpC beta-lactamases by assaying known quantities of purified enzymes in ELISAs (AmpC beta-lactamases of M . morganii, C . freundii, E . coli, and E . cloacae) . As the homology to CMY-2 beta-lactamase decreased, the minimum level needed for detection increased (e.g., 94% homology recognized at 1 ng/ml and 71% homology recognized at 10 ng/ml) . The ELISAs were used to assay unknown clinical isolates for AmpC and SHV beta-lactamases, and the results were confirmed with PCR amplification of bla(AmpC) and bla(SHV) genes . Overall, we found that our ELISAs were at least 95% sensitive and specific for detecting SHV and AmpC beta-lactamases . The ELISA format can facilitate the identification of AmpC and SHV beta-lactamases and can be used to quantify relative amounts of beta-lactamase enzymes in clinical and laboratory isolates.

J Biol Chem, 2002 Aug 2, 277(31), 27725 - 32 Epub 2002 May 16.
Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization; Hawkes DB et al.; Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes . Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms . We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy . This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined . By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA) . Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin . This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner . Sequencing also identified the unprecedented substitution of a highly conserved, catalytically important active site threonine with an asparagine residue . The P450 gene was subcloned and heterologously expressed in Escherichia coli at approximately 2000 nmol/liter of original culture, and purification was achieved by standard protocols . Postulating the native E . coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450(cin), it was expressed in E . coli in the presence of cineole 1 . A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2 . Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K(D) = 0.7 microm), and a large spin state change of the heme iron associated with binding of cineole 1 . These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation . This constitutes the first characterization of an enzyme involved in this pathway.

Vet Pathol, 2002 May, 39(3), 393 - 5
Myocarditis in sibling boxer puppies associated with Citrobacter koseri infection; Cassidy JP et al.; Two sibling Boxer puppies presented with severe suppurative myocarditis in the absence of additional disseminated suppurative foci . The identification of gram-negative bacteria within areas of myocarditis in both puppies and the pure growth of large numbers of Citrobacter koseri from the myocardial lesions in one of the dogs were consistent with a bacterial etiology . The fact that C . koseri is an opportunist pathogen suggested intercurrent immunosuppression . The finding of a concomitant bacterial myocarditis in two canine siblings is novel . The case is also unusual in that syncope could be related to the myocardial injury.

J Antibiot (Tokyo), 2002 Mar, 55(3), 288 - 95
Identification of compounds that inhibit late steps of peptidoglycan synthesis in bacteria; DeCenzo M et al.; A screening system is described that can detect and confirm inhibitors of the late steps of cell wall biosynthesis . The primary high through-put screen monitors induction of beta-lactamase following exposure to samples, in an Escherichia coli envA- strain that carries the beta-lactamase gene from Citrobacter freundii on a plasmid . Positive samples were detected from compound libraries, from natural products libraries, and from fractions of natural products crude preparations . These samples were then subjected to in vitro assays that could detect the incorporation of soluble cell wall precursor into Lipid I, Lipid II, and polymerized cell wall, using a TLC system that was very accurate and unambiguous in detecting known cell wall inhibitors . One partially purified sample containing a novel antibacterial agent derived from natural products was found to inhibit the formation of Lipid I (50% inhibition at < or = 62.5 ng/ml), whereas another partially purified sample also derived from natural products inhibited transglycosylation into cell wall polymer (50% inhibition at < or = 10 microg/ml) . This screening system proved to be especially useful because it was sufficiently sensitive and robust to detect inhibitors among samples of crude preparations or varying states of purity.

J Antibiot (Tokyo), 2002 Mar, 55(3), 279 - 87
A pathway-specific cell based screening system to detect bacterial cell wall inhibitors; Sun D et al.; A pathway-specific cell-based screen is described to detect compounds that inhibit the biosynthesis of the cell wall of bacteria . The basis for detection is the discovery that the beta-lactamase gene from Citrobacterfreundii, cloned into Escherichia coli, is induced when cells are exposed to known cell wall inhibitors, and not just beta-lactam-based antibiotics . In a wild type host, cell wall inhibitors such as moenomycin, vancomycin, and ramoplanin, which are excluded by the outer membrane, only induce at high concentrations . However, these compounds, as well as fosfomycin, cycloserine, and cefoxitin, induce at concentrations at or below the MIC of a host carrying the envA-mutation, which causes a defect in the outer membrane . As additional proof that induction of beta-lactamase is the direct result of cell wall inhibition, a host strain carrying a temperature-sensitive mutation in the murG gene, whose product converts the cell wall intermediate Lipid I, to Lipid II, also induced beta-lactamase at the restrictive temperature . A protocol is described for screening samples in high-throughput mode.

Int J Antimicrob Agents, 2002 May, 19(5), 383 - 8
Antimicrobial susceptibility of inducible AmpC beta-lactamase-producing Enterobacteriaceae from the Meropenem Yearly Susceptibility Test Information Collection (MYSTIC) Programme, Europe 1997-2000; Pfaller MA et al.; Among 11345 clinical isolates from 31 European centres in the MYSTIC (Meropenem Yearly Susceptibility Test Information Collection) Programme (1997-2000), the potential AmpC-producing pathogens were Enterobacter spp . (904 cases), Citrobacter spp . (144) and Serratia marcescens (288) . Resistance to ceftazidime or cefotaxime (11-34%) and piperacillin/tazobactam (12%) was observed, indicating stably derepressed expression of AmpC cephalosporinases . Meropenem (MIC(90), 0.25 mg/l; >99% susceptible) and imipenem (MIC(90), 2 mg/l; 98% susceptible) were active, even against these stably derepressed AmpC-producers: Enterobacter spp . (305 strains), Citrobacter spp . (29) and S . marcescens (35) . The overall rank order of spectrum (% susceptible) versus the stably derepressed subset of isolates was: meropenem=imipenem (98%)>cefepime (89%)>gentamicin (73%)>ciprofloxacin (62%)>tobramycin (60%) >piperacillin/tazobactam (21%) . We suggest increased attention in Europe to: (1) the recognition of these resistant phenotypes, (2) infection control practices, and (3) limiting the overuse of certain selecting extended-spectrum beta-lactam agents (e.g . ceftazidime).

J Biomol Screen, 2002 Apr, 7(2), 127 - 34
A luminescent Escherichia coli biosensor for the high throughput detection of beta-lactams; Valtonen SJ et al.; A group-specific bioluminescent Escherichia coli strain for studying the action of beta-lactam antibiotics is described . The strain contains a plasmid, pBlaLux1, in which the luciferase genes from Photorhabdus luminescens are inserted under the control of the beta-lactam-responsive element ampR/ampC from Citrobacter freundii . In the presence of beta-lactams, the bacterial cells are induced to express the luciferase enzyme and three additional enzymes generating the substrate for the luciferase reaction . This biosensor for beta-lactams does not need any substrate or cofactor additions, and the bioluminescence can be measured very sensitively in real time by using a luminometer . Basic parameters affecting the light production and induction in the gram-negative model organism E . coli SNO301/pBlaLux1 by various beta-lactams were studied . The dose-response curves were bell shaped, indicating toxic effects for the sensor strain at high concentrations of beta-lactams . Various beta-lactams had fairly different assay ranges: ampicillin, 0.05-1.0 microg/ml; piperacillin, 0.0025-25 microg/ml; imipenem, 0.0025-0.25 microg/ml; cephapirin, 0.025-2.5 microg/ml; cefoxitin, 0.0025-1.5 microg/ml; and oxacillin, 25-500 microg/ml . Also, the induction coefficients (signal over background noninduced control) varied considerably from 3 to 158 in a 2-hour assay . Different non-beta-lactam antibiotics did not cause induction . Because the assay can be automated using microplate technologies, the approach may be suitable for higher throughput analysis of beta-lactam action.

Microb Drug Resist, 2002 Spring, 8(1), 35 - 7
CTX-m-3 beta-lactamase-producing Escherichia coli from Greece; Mavroidi A et al.; An Escherichia coli clinical strain resistant to all beta-lactams except carbapenems was isolated in a Greek hospital . Analysis of beta-lactamase content by isoelectric focusing, PCR assays specific for various bla genes, and DNA sequencing showed that the strain produced TEM-1, a Citrobacter freundii AmpC-related cephalosporinase, and CTX-M-3 . The blaCTX.M-3 gene was carried by a 120-kb plasmid that was readily transferable to a susceptible E . coli host.

J Am Vet Med Assoc, 2002 May 1, 220(9), 1321 - 4
Bacterial colonization of intravenous catheters in young dogs suspected to have parvoviral enteritis; Lobetti RG et al.; OBJECTIVE: To determine the prevalence of bacterial colonization of IV catheters among young dogs suspected to have parvoviral enteritis, to identify the organisms responsible for catheter colonization, and to determine the antimicrobial susceptibility of organisms that were obtained . DESIGN: Case series . ANIMALS: 100 dogs . PROCEDURE: Catheters were aseptically removed when fluid therapy was discontinued, the catheter was replaced, or the dog died . The distal tip of the catheter was cut off, split open, and vortexed with sterile saline (0.9% NaCl) solution . The saline solution was plated on culture plates, which were then incubated and examined for bacterial growth every 24 hours for 72 hours . All bacteria cultured were identified, and antimicrobial susceptibility was determined . RESULTS: Bacteria were isolated from 22 catheters . Most bacteria that were isolated were of gastrointestinal tract or environmental origin (Serratia odorifera, S . liquefaciens, S . marcescens, Acinobacter anitratus, Citrobacter freundii, Klebsiella pneumoniae, K . oxytoca, Escherichia coli, Enterobacter spp) . Only 2 gram-positive organisms were isolated (Staphylococcus intermedius and Streptococcus spp) . High percentages of organisms were resistant to penicillin, lincomycin, cloxacillin, erythromycin, and cephalexin . Percentages of organisms resistant to amikacin, enrofloxacin, chloramphenicol, potentiated sulfonamides, and amoxicillin-clavulanic acid were low . CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that IV catheters may be colonized with bacteria in 22% of young dogs suspected to have parvovirus infection.

Trop Doct, 2002 Jan, 32(1), 20 - 2
Urinary pathogens' resistance to common antibiotics: a retrospective analysis; Navaneeth BV et al.; The susceptibility of urinary pathogens to common antibiotics was investigated and the results analysed retrospectively using the WHONET computer program . Of 1776 urine samples (44 catheterized) processed, 510 (28.7%) urinary pathogens were isolated . Of these 510 positive cultures, 455 (89.2%) were gram-negative bacilli, 45 (8.8%) Candida species and 10 (1.9%) gram-positive cocci . Of the 44 catheterized samples, 32 (72.7%) yielded significant bacteriuria and these were mainly gram-negative bacteria (24/32) . The commonest pathogen isolated was Escherichia coli (47.3%) followed by Klebsiella species (10.3%), non-fermenters other than Pseudomonas species (9%), Candida species (8.8%), Providentia species (7%), Pseudomonas species (5.6%), Citrobacter species (3.7%), Enterobacter species (3.3%) and Proteus species (2.5%) . The isolation of gram-negative bacteria among inpatients and outpatients was 71.6% and 28.3%, respectively . The critical care unit, nephrourology, obstetric and gynaecology, medical and surgical wards were found to be high-risk areas constituting 58.7% of the major isolates . The highest and lowest mean resistance among gram-negative bacteria to common antibiotics was 93.5% to ampicillin and to 61% gentamicin . The mean resistance to norfloxacin, amoxy-clavulanic acid, nitrofurantoin, trimethoprim-sulfamethoxazole and cefazolin was 65%, 67%, 75.5%, 76% and 77.5%, respectively . The most resistant pathogen to common antibiotics was found to be Proteus species (resistance 80% and above) . Overall susceptibility testing demonstrated decreased usefulness of common antibiotics and demonstrates a need for a re-evaluation of common antibiotics used in the therapy for urinary tract infection.

J Ind Microbiol Biotechnol, 2002 Apr, 28(4), 225 - 31
Potential microbiological hazards in the production of refined paper products for food applications; Raaska L et al.; This study sought to investigate the significance of raw materials (starch-based glues, raw material papers) at different microbiologically critical stages in the manufacturing process of refined paper products . The study examined the occurrence of microorganisms in the process and in end-product samples . Microbiological surveys verified that the production and use of pasteurized starch-based glue was the most important factor threatening the process hygiene and product safety . Subsequently, the production and use of starch-based glue was changed, and a follow-up programme targeting the microbiological quality of glue was developed as part of a hygiene and safety management system . A total of 33 spore-forming bacterial and 15 enterobacterial isolates were ribotyped, and 22 and 10 different ribogroups (ribotypes), respectively, were generated . These isolates from starch-based glue, raw material paper and end products were atypical and, thus, in many cases physiological, chemotaxonomic (FAME) and molecular (partial 16S rDNA) results did not correspond . The most common spore-forming bacteria (55% of the isolates) were Paenibacillus sp . and within this genus several new species were also proposed . The most common enterobacteria (87%) were Enterobacter cloacae and Citrobacter freundii belonging to bacteria in hazard group 2, or species closely related to them . It was demonstrated that the same spore-forming bacteria (ribotypes) were present in both the glue samples and the end products (45% of isolates) . All RiboPrint patterns were saved at the VTT identification library for future use.

Biochem J, 2002 May 1, 363(Pt 3), 745 - 52
Threonine-124 and phenylalanine-448 in Citrobacter freundii tyrosine phenol-lyase are necessary for activity with L-tyrosine; Demidkina TV et al.; Thr-124 and Phe-448 are located in the active site of Citrobacter freundii tyrosine phenol-lyase (TPL) near the phenol ring of a bound substrate analogue, 3-(4'-hydroxyphenyl)propionic acid {Sundararaju, Antson, Phillips, Demidkina, Barbolina, Gollnick, Dodson and Wilson (1997) Biochemistry 36, 6502-6510} . Thr-124 is replaced by Asp and Phe-448 is replaced by His in the crystal structure of a structurally similar enzyme, Proteus vulgaris tryptophan indole-lyase, which has 50% identical residues . Hence, Thr-124 and Phe-448 in TPL were mutated to Ala or Asp, and His, respectively, in order to probe the role of these residues in the reaction specificity for L-Tyr . These mutant enzymes have little or no beta-elimination activity with L-Tyr or 3-fluoro-L-Tyr as a substrate, but retain significant elimination activity with S-(o-nitrophenyl)-L-cysteine, S-alkyl-L-cysteines and beta-chloroalanine . Furthermore, the binding of L-Tyr and other non-substrate amino acids is not significantly affected by the mutations . The mutant TPLs form intermediates in rapid-scanning stopped-flow experiments with L-Phe, L-Tyr and L-Trp, similar to those seen with wild-type TPL . These results demonstrate that Thr-124 and Phe-448 are necessary for the reaction specificity of TPL for L-Tyr, and probably play a role in the elimination stage of the reaction mechanism . Thr-124 is within hydrogen-bonding distance of the phenolic group of the bound substrate, and may help to orientate the ring for beta-elimination to occur . Phe-448 may be important to allow the formation of the closed conformation during the reaction.

Prikl Biokhim Mikrobiol, 2002 Mar-Apr, 38(2), 145 - 8
{Anaerobic degradation of biphenyl by the facultative anaerobic strain Citrobacter freundi BS2211}; Grishchenkov VG et al.; Using a synthetic medium supplemented with biphenyl (a polycyclic aromatic hydrocarbon), a new bacterial strain of Citrobacter freundii was isolated from enrichment cultures containing soil and industrial wastewater samples of the Serpukhov Condenser Factory . This strain was found to be capable of degrading biphenyl under anaerobic conditions in the course of nitrate reduction . When the initial concentration of biphenyl in culture medium equaled 150 mg/l, the culture with a titer of 10(9) cells/ml degraded up to 26-28% of biphenyl in 3 days (28 degrees C) . At 250 mg/l, the culture with a titer of 10(7) cells/ml degraded 15% of biphenyl in 21 days . Approximately 10% of the substrate consumed was utilized completely, whereas the remainder underwent transformation.

Antimicrob Agents Chemother, 2002 May, 46(5), 1190 - 8
Origin and evolution of the AmpC beta-lactamases of Citrobacter freundii; Barlow M et al.; To determine whether the widespread clinical use of beta-lactams has been selective for Citrobacter freundii-derived alleles of plasmid ampC genes, we generated a Bayesian consensus phylogeny of the published ampC sequences and compared the MICs of 16 beta-lactam antibiotics for Escherichia coli strains containing cloned copies of the C . freundii ampC alleles . We found that for the majority of compounds investigated, there has been essentially no increase in beta-lactam resistance conferred by those alleles . We also found that ampC alleles from the chromosomes of two beta-lactam-sensitive C . freundii strains isolated in the 1920s, before the clinical use of antibiotics, were as effective at providing beta-lactam resistance in E . coli as were the plasmid-borne alleles from beta-lactam-resistant clinical isolates . These results suggest that selection for increased resistance to beta-lactam antibiotics has not been a significant force directing the evolution of the C . freundii ampC alleles found in beta-lactam-resistant clinical isolates.

J Biol Chem, 2002 Jun 14, 277(24), 21592 - 7 Epub 2002 Apr 04.
Crystals of tryptophan indole-lyase and tyrosine phenol-lyase form stable quinonoid complexes; Phillips RS et al.; The binding of substrates and inhibitors to wild-type Proteus vulgaris tryptophan indole-lyase and to wild type and Y71F Citrobacter freundii tyrosine phenol-lyase was investigated in the crystalline state by polarized absorption microspectrophotometry . Oxindolyl-lalanine binds to tryptophan indole-lyase crystals to accumulate predominantly a stable quinonoid intermediate absorbing at 502 nm with a dissociation constant of 35 microm, approximately 10-fold higher than that in solution . l-Trp or l-Ser react with tryptophan indole-lyase crystals to give, as in solution, a mixture of external aldimine and quinonoid intermediates and gem-diamine and external aldimine intermediates, respectively . Different from previous solution studies (Phillips, R . S., Sundararju, B., & Faleev, N . G . (2000) J . Am . Chem . Soc . 122, 1008-1114), the reaction of benzimidazole and l-Trp or l-Ser with tryptophan indole-lyase crystals does not result in the formation of an alpha-aminoacrylate intermediate, suggesting that the crystal lattice might prevent a ligand-induced conformational change associated with this catalytic step . Wild-type tyrosine phenol-lyase crystals bind l-Met and l-Phe to form mixtures of external aldimine and quinonoid intermediates as in solution . A stable quinonoid intermediate with lambda(max) at 502 nm is accumulated in the reaction of crystals of Y71F tyrosine phenol-lyase, an inactive mutant, with 3-F-l-Tyr with a dissociation constant of 1 mm, approximately 10-fold higher than that in solution . The stability exhibited by the quinonoid intermediates formed both by wild-type tryptophan indole-lyase and by wild type and Y71F tyrosine phenol-lyase crystals demonstrates that they are suitable for structural determination by x-ray crystallography, thus allowing the elucidation of a key species of pyridoxal 5'-phosphate-dependent enzyme catalysis.

Microbiology, 2002 Apr, 148(Pt 4), 943 - 50
Development of a P1 phagemid system for the delivery of DNA into Gram-negative bacteria; Westwater C et al.; The inability to transform many clinically important Gram-negative bacteria has hampered genetic studies addressing the mechanism of bacterial pathogenesis . This report describes the development and construction of a delivery system utilizing the broad-host-range transducing bacteriophage P1 . The phagemids used in this system contain a P1 pac initiation site to package the vector, a P1 lytic replicon to generate concatemeric DNA, a broad-host-range origin of replication and an antibiotic-resistance determinant to select bacterial clones containing the recircularized phagemid . Phagemid DNA was successfully introduced by infection and stably maintained in members of the families Enterobacteriaceae (Escherichia coli, Shigella flexneri, Shigella dysenteriae, Klebsiella pneumoniae and Citrobacter freundii) and Pseudomonadaceae (Pseudomonas aeruginosa) . In addition to laboratory strains, these virions were used successfully to deliver phagemids to a number of strains isolated from patients . This ability to deliver genetic information to wild-type strains raises the potential for use in antimicrobial therapies and DNA vaccine development.

Int J Syst Evol Microbiol, 2002 Mar, 52(Pt 2), 531 - 47
Evolution of the gyrB gene and the molecular phylogeny of Enterobacteriaceae: a model molecule for molecular systematic studies; Dauga C; Phylogenetic trees showing the evolutionary relatedness of Enterobacteriaceae based upon gyrB and 16S rRNA genes were compared . Congruence among trees of these molecules indicates that the genomes of these species are not completely mosaic and that molecular systematic studies can be carried out . Phylogenetic trees based on gyrB sequences appeared to be more reliable at determining relationships among Serratia species than trees based on 16S rRNA gene sequences . gyrB sequences from Serratia species formed a monophyletic group validated by significant bootstrap values . Serratia fonticola had the most deeply branching gyrB sequence in the Serratia monophyletic group, which was consistent with its atypical phenotypic characteristics . Klebsiella and Enterobacter genera seemed to be polyphyletic, but the branching patterns of gyrB and 16S rRNA gene trees were not congruent . Enterobacter aerogenes was grouped with Klebsiella pneumoniae on the gyrB phylogenetic tree, which supports that this species could be transferred to the Klebsiella genus . Unfortunately, 16S rRNA and gyrB phylogenetic trees gave conflicting evolutionary relationships for Citrobacter freundii because of its unusual gyrB evolutionary process . gyrB lateral gene transfer was suspected for Hafnia alvei . Saturation of gyrB genes was observed by the pairwise comparison of Proteus spp., Providencia alcalifaciens and Morganella morganii sequences . Depending on their level of variability, 16S rRNA gene sequences were useful for describing phylogenetic relationships between distantly related Enterobacteriaceae, whereas gyrB sequence comparison was useful for inferring intra- and some intergeneric relationships.

Infect Immun, 2002 Apr, 70(4), 2070 - 81
Mice lacking T and B lymphocytes develop transient colitis and crypt hyperplasia yet suffer impaired bacterial clearance during Citrobacter rodentium infection; Vallance BA et al.; The bacterial pathogen Citrobacter rodentium belongs to a family of gastrointestinal pathogens that includes enteropathogenic and enterohemorrhagic Escherichia coli and is the causative agent of transmissible colonic hyperplasia in mice . The molecular mechanisms used by these pathogens to colonize host epithelial surfaces and form attaching and effacing (A/E) lesions have undergone intense study . In contrast, little is known about the host's immune response to these infections and its importance in tissue pathology and bacterial clearance . To address these issues, wild-type mice and mice lacking T and B lymphocytes (RAG1 knockout {KO}) were infected with C . rodentium . By day 10 postinfection (p.i.), both wild-type and RAG1 KO mice developed colitis and crypt hyperplasia, and these responses became more exaggerated in wild-type mice over the next 2 weeks, as they cleared the infection . By day 24 p.i., bacterial clearance was complete, and the colitis had subsided; however, crypt heights remained increased . In contrast, inflammatory and crypt hyperplastic responses in the RAG1 KO mice were transient, subsiding after 2 weeks . By day 24 p.i., RAG1 KO mice showed no signs of bacterial clearance and infection was often fatal . Surprisingly, despite remaining heavily infected, tissues from RAG1 KO mice surviving the acute colitis showed few signs of disease . These results thus emphasize the important contribution of the host immune response during infection by A/E bacterial pathogens . While T and/or B lymphocytes are essential for host defense against C . rodentium, they also mediate much of the tissue pathology and disease symptoms that occur during infection.

Microbiology, 2002 Mar, 148(Pt 3), 657 - 65
Mutagenesis of conserved tryptophan residues within the receptor-binding domain of intimin: influence on binding activity and virulence; Reece S et al.; Intimate bacterial adhesion to intestinal epithelium is a pathogenic mechanism shared by several human and animal enteric pathogens, including enteropathogenic and enterohaemorrhagic Escherichia coli and Citrobacter rodentium . The proteins directly involved in this process are the outer-membrane adhesion molecule intimin and the translocated intimin receptor, Tir . The receptor-binding activity of intimin resides within the carboxy terminus 280 aa (Int280) of the polypeptide . Four tryptophan residues, W117/776, W136/795, W222/881 and W240/899, are conserved within different Int280 molecules that otherwise show considerable sequence variation . In this study the influence of site-directed mutagenesis of each of the four tryptophan residues on intimin-Tir interactions and on intimin-mediated intimate attachment was determined . The mutant intimins were also studied using a variety of in vitro and in vivo infection models . The results show that all the substitutions modulated intimin activity, although some mutations had more profound effects than others.

Clin Microbiol Infect, 1997 Feb, 3 Suppl 4, S10 - S19
beta-Lactamases: quantity and resistance; Livermore DM; Rates of enzyme-mediated catalysis are proportional to enzyme quantity, so increasing the amount of beta-lactamase should increase resistance . Kinetic considerations support this argument for both Gram-positive and Gram-negative bacteria . Direct relationships between resistance and enzyme quantity are most obvious with constitutive beta-lactamases, e.g . the TEM types from Gram-negative bacteria . As the level of TEM enzyme rises, so do the MICs of substrates and the concentrations of inhibitors required to potentiate these substrates . The position is more complex for inducible beta-lactamases, e.g . the AmpC types of Enterobacter spp., Citrobacter freundii, Serratia spp . and Pseudomonas aeruginosa . Third-generation cephalosporins are labile to these enzymes, but are only weak inducers, so the beta-lactamase-inducible strains appear susceptible . Once, however, the enzyme is derepressed, resistance is apparent . The behavior of inhibitor combinations against inducible beta-lactamases is complicated by the propensity of some inhibitors to induce further enzyme synthesis . As the inoculum is raised in laboratory tests, the amount of beta-lactamase and MICs also rise . This is notorious for staphylococci but is seen also for some Gram-negative organisms, notably klebsiellae with extended-spectrum beta-lactamases . A beta-lactamase-related inoculum effect generally predicts clinical failure.

Infect Control Hosp Epidemiol, 2002 Jan, 23(1), 27 - 31
The direct costs of nosocomial catheter-associated urinary tract infection in the era of managed care; Tambyah PA et al.; OBJECTIVE: To determine the additional direct costs of hospitalization attributable to catheter-associated urinary tract infection (CAUTI) in 1,497 newly catheterized patients . DESIGN: Prospective observational and laboratory study . SETTING: University hospital . METHODS: Data were collected on risk factors for CAUTI (defined as > 10(3) colony-forming units {CFU}/mL), severity of illness, and diagnostic and therapeutic interventions in consenting newly catheterized patients . Daily urine cultures were obtained from each newly catheterized patient, but the results of these cultures were not revealed to his or her physician . During the study, one of the investigators (DGM) reviewed each patient's record and made a judgment as to which of the diagnostic tests and treatments ordered and what incremental length of stay could reasonably be ascribed to his or her CAUTI . The total hospital costs for each patient were also obtained . RESULTS: Overall, 235 patients acquired CAUTIs during the study; most of the CAUTIs were completely asymptomatic, and only 52% were diagnosed by the patients' physicians using the hospital laboratory . Only 1 patient with a CAUTI had a secondary bloodstream infection . Thirty-three (13%) of the CAUTIs were caused by Escherichia coli; 63 (25%) by Klebsiella, Enterobacter, Citrobacter, Pseudomonas aeruginosa, or other antibiotic-resistant, gram-negative bacilli; 87 (35%) by enterococci or staphylococci; and 67 (27%) by Candida species . The 123 CAUTIs diagnosed by the hospital laboratory were judged to have been responsible for an additional $20,662 in extra costs of diagnostic tests and $35,872 in extra medication costs, a mean of $589 (median, $356) per CAUTI . CAUTIs caused by E . coli cost considerably less than infections caused by other gram-negative bacilli ($363.3 +/- $228.2 vs $690.4 +/- $783.7; P = .02) or yeasts ($821.2 +/- $2,169.9) . There were less striking differences in the costs per CAUTI caused by staphylococci or enterococci ($387.1 +/- $434.8) . CONCLUSIONS: The extra direct costs associated with nosocomial CAUTI found in this prospective study, which was done in the era of managed care during the late 1990s, are substantially lower than those reported in the largest comparable studies done more than 15 years ago, most of which were retrospective, reflecting the powerful impact of cost-containment measures that are now implemented in managed care.

Clin Microbiol Infect, 1997 Jun, 3(3), 335 - 344
CQ-397 and CQ-414: antimicrobial activity and spectrum of two fluoroquinolone---cephalosporin, dual-action compounds with carboxamido bonds; Johnson DM et al.; OBJECTIVE: To evaluate the potential spectrum of activity of two novel dual-action compounds with carboxamido bonds (CQ-397 and CQ-414; Laboratorios Aranda, San Rafael, Mexico) against human pathogens . METHODS: Approximately 800 Gram-positive and Gram-negative aerobic clinical bacteria were tested in vitro using the Mueller-Hinton broth microdilution method of the National Committee of Clinical Laboratory Standards . RESULTS: CQ-397 (cefamandole+enrofloxacin) and CQ-414 (cefamandole+norfloxacin) were equally potent against Enterobacteriaceae (MIC90 range, 0.06--0.5 microg/mL and 0.06--1 microg/mL, respectively) . Citrobacter freundii (MIC90, 4 microg/mL) and Providencia spp . (MIC90, >32 microg/mL) exhibited elevated study drug MICs . Enterobacteriaceae resistant to fluoroquinolones generally remained resistant . CQ-397 and CQ-414 were active against Stenotrophomonas maltophilia (MIC90, 4 microg/mL) and oxacillin-susceptible staphylococci (MIC90, 0.25 microg/mL), but not oxacillin-resistant Staphylococcus aureus (MIC90, >32 microg/mL), Staphylococcus epidermidis (MIC90, 8 microg/mL), and enterococci (MIC90s, 8 to >32 microg/mL) . There was no difference in the dual-action drug activity (MIC90, 2 microg/mL) between penicillin-susceptible and -resistant pneumococci . Haemophilus influenzae and Moraxella catarrhalis were very susceptible (MIC range, less-than-or-equal0.015--0.06 microg/mL) to both compounds . CONCLUSIONS: The activity of these novel dual-action compounds, formed from the bonding of older antimicrobials, warrants further investigation for potential human and/or animal health use, including toxicology and pharmacokinetics.

Clin Microbiol Infect, 1997 Feb, 3(1), 53 - 57
Differentiation and susceptibility of Citrobacter isolates from patients in a university hospital; Arens S et al.; OBJECTIVE: Recently a publication of Brenner et al . introduced 11 genetically distinct species within the genus Citrobacter . These newly recognized Citrobacter species can be classified by means of their biochemical characteristics . The aim of this study was to examine the distribution and susceptibility of Citrobacter isolates in our patient population . METHODS: A total of 126 samples---containing a Citrobacter species---was collected from 116 hospitalized patients during a 6-month period . Organisms were identified according to standard procedures . Antimicrobial susceptibility testing was performed by agar dilution on Mueller-Hinton agar, and interpretation was based on NCCLS criteria . RESULTS: C . freundii was the most common organism isolated (n=59), followed by C . braakii (n=25) and C . koseri (n=23) . The urinary tract and the respiratory tract were found to be the predominant sites of colonization or infection, accounting for 45% and 32% of all isolates respectively . It appeared that young children (<12 months old) and the elderly were most at risk of acquiring Citrobacter . Two-thirds of all specimens contained other organisms in addition to Citrobacter . Most Citrobacter isolates were related with a predisposing factor . Species-related differences were found in the susceptibility pattern . CONCLUSIONS: These findings suggest that citrobacteria are important opportunistic pathogens contributing to colonization or infection in our hospital population.

Cell Microbiol, 2002 Jan, 4(1), 29 - 42
Species-specific cell adhesion of enteropathogenic Escherichia coli is mediated by type IV bundle-forming pili; Tobe T et al.; Enteropathogenic Escherichia coli (EPEC) is a causative agent of diarrhoea in humans . Localized adherence of EPEC onto intestinal mucosa was reproduced in an in vitro adherence assay with cultured human epithelial cells . We found that the efficiency of EPEC adherence to a mouse-derived colonic epithelial cell line, CMT-93, was remarkably lower than its adherence to human-derived intestinal cell lines, such as Intestine-407 or Caco-2 . Although EPEC did adhere to some cell lines derived from non-human species, fixing the cells with formalin to inactivate one or more formalin-sensitive factors allowed us to observe species-specific differences in EPEC adherence . In contrast to these results, an EPEC mutant that is defective in bundle-forming pili (BFP) production adhered as efficiently to CMT-93 cells as to Caco-2 cells . Furthermore, Citrobacter rodentium expressing BFP adhered to Caco-2 cells much more efficiently than to CMT-93 cells . Finally, a purified BfpA-His6 fusion protein showed higher affinity for Caco-2 cells than for CMT-93 cells, and inhibited EPEC adherence . Following BFP-mediated adherence, secretion of EspB from adherent bacteria and reorganization of F-actin in the host cells was observed . EPEC adhering to CMT-93 cells induced far less secretion of EspB, or reorganization of F-actin in the host CMT-93 cells, than did EPEC adhering to Caco-2 cells . These results indicated that BFP plays an important role in the cell-type-dependent adherence of EPEC and in the progression to the later steps in EPEC adherence.

Structure (Camb), 2002 Jan, 10(1), 105 - 13
Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase; Levy CW et al.; Methylaspartate ammonia lyase (MAL) catalyzes the magnesium-dependent reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid . The 1.3 A MAD crystal structure of the dimeric Citrobacter amalonaticus MAL shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel . Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton alpha to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate . This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.

Folia Microbiol (Praha), 2001, 46(4), 339 - 44
Occurrence and transferability of beta-lactam resistance in Enterobacteriaceae isolated in Children's University Hospital in Bratislava; Bujdakova H et al.; Occurrence and transferability of beta-lactam resistance in 30 multi-resistant Escherichia coli, Klebsiella spp., Enterobacter spp., Pantoea agglomerans, Citrobacter freundii and Serratia marcescens strains isolated from children between 0 and 3 years of age is presented . The strains were resistant to ampicillin (30), cefoxitin (22), cefotaxime (30), ceftriaxone (30), ceftazidime (30) and aztreonam (28), but susceptible to cefepime (30) and imipenem (26) . Twenty-eight of 30 isolates possessed a transferable resistance confirmed by conjugation and isolation of 79-89-kb plasmids . The beta-lactam resistance was due to production of beta-lactamases and ceftazidime proved to be stronger beta-lactamase inductor than ceftriaxone . Twenty-five clinical isolates expressed transferable extended spectrum beta-lactamases, and chromosomally encoded AmpC beta-lactamase.

J Immunol, 2002 Feb 15, 168(4), 1804 - 12
Impaired resistance and enhanced pathology during infection with a noninvasive, attaching-effacing enteric bacterial pathogen, Citrobacter rodentium, in mice lacking IL-12 or IFN-gamma; Simmons CP et al.; Mice infected with Citrobacter rodentium represent an excellent model in which to examine immune defenses against an attaching-effacing enteric bacterial pathogen . Colonic tissue from mice infected with C . rodentium harbors increased transcripts for IL-12 and IFN-gamma and displays mucosal pathology compared with uninfected controls . In this study, the role of IL-12 and IFN-gamma in host defense and mucosal injury during C . rodentium infection was examined using gene knockout mice . IL-12p40(-/-) and IFN-gamma(-/-) mice were significantly more susceptible to mucosal and gut-derived systemic C . rodentium infection . In particular, a proportion of IL-12p40(-/-) mice died during infection . Analysis of the gut mucosa of IL-12p40(-/-) mice revealed an influx of CD4(+) T cells and a local IFN-gamma response . Infected IL-12p40(-/-) and IFN-gamma(-/-) mice also mounted anti-Citrobacter serum and gut-associated IgA responses and strongly expressed inducible NO synthase (iNOS) in mucosal tissue, despite diminished serum nitrite/nitrate levels . However, iNOS does not detectably contribute to host defense against C . rodentium, as iNOS(-/-) mice were not more susceptible to infection . However, C57BL/6 mice infected with C . rodentium up-regulated expression of the mouse beta-defensin (mBD)-1 and mBD-3 in colonic tissue . In contrast, expression of mBD-3, but not mBD-1, was significantly attenuated during infection of IL-12- and IFN-gamma-deficient mice, suggesting mBD-3 may contribute to host defense . These studies are among the first to examine mechanisms of host resistance to an attaching-effacing pathogen and show an important role for IL-12 and IFN-gamma in limiting bacterial infection of the colonic epithelium.

Vet Microbiol, 2002 Feb 26, 85(1), 69 - 82
Immunochemical analyses of serum antibodies from pig herds in a Salmonella non-endemic region; Wiuff C et al.; In a large comparative survey of Danish and Swedish slaughter pig herds performed prior to this work, it was unexpectedly found that some Swedish herds harbored seropositive pigs . Serum samples from the Swedish herds had moderate responses in the Salmonella mix-ELISA (detecting serogroup B and C1 infections) compared to the Danish herds classifying some of them as seropositive using a cut-off value at 40 OD% . In Sweden, extensive Salmonella control is carried out by bacteriological screening of feces and lymph nodes, and the overall prevalence has been proven to be below 0.1% . The serological positive results were therefore unexpected; hence the reactivities of the Swedish sera were studied by a number of immunochemical analyses (Western blot, indirect ELISA, inhibition ELISA, avidity ELISA) and compared to sera from Danish pig herds with verified Salmonella infections ("the reference sera").In Western blot, the Swedish sera had high binding reactivities against Salmonella Typhimurium LPS of different molecular weights, and gave binding patterns similar to that of the reference sera . Pre-incubation with free S . Typhimurium LPS or PS (the polysaccharide part of LPS) was able to inhibit the reactivity of the Swedish sera in the mix-ELISA . Reactivities against other related bacterial LPS such as Citrobacter freundii LPS and Yersinia enterocolitica O:3 LPS were observed in the Swedish sera, but these LPS antigens were unable to inhibit the reactivities in the mix-ELISA as efficiently as S . Typhimurium LPS . Furthermore, the Swedish sera did not bind Salmonella LPS of another serogroup (S . Meleagridis LPS, serogroup E1) or rough Salmonella LPS, both lacking the specific O-antigenic parts of S . Typhimurium LPS . The avidity of the Swedish sera was much lower than the avidity of the reference sera, which could indicate the presence of transient low-dose infections or stimulation by inactivated bacteria in feed . The results obtained in this investigation strongly indicate that the Swedish sera contain antibodies directed against the O-antigenic part of LPS from S . Typhimurium or possibly on as yet unknown bacterium.

Eur J Biochem, 2002 Jan, 269(1), 93 - 9
Structures of two O-chain polysaccharides of Citrobacter gillenii O9a,9b lipopolysaccharide . A new homopolymer of 4-amino-4,6-dideoxy-D-mannose (perosamine); Lipinski T et al.; Mild acid degradation of the lipopolysaccharide of Citro- bacter gillenii O9a,9b released a polysaccharide (PS), which was found to consist of a single monosaccharide, 4- acetamido-4,6-dideoxy-d-mannose (d-Rha4NAc, N-acetyl-d-perosamine) . PS was studied by methylation analysis and (1)H-NMR and (13)C-NMR spectroscopy, using two-dimensional (1)H,(1)H COSY, TOCSY, NOESY, and H-detected (1)H,(13)C heteronuclear correlation experiments . It was found that PS includes two structurally different polysaccharides: an alpha1-->2-linked homopolymer of N-acetyl-d-perosamine {-->2)-alpha-d-Rhap4NAc-(1-->, PS2} and a polysaccharide composed of tetrasaccharide repeating units (PS1) with the following structure: -->3)-alpha-d-Rhap4NAc-(1-->2)-alpha-d-Rhap4NAc-(1-->2)-alpha-d-Rhap4NAc-(1-->3)-alpha-d-Rhap4 N Ac2Ac-(1--> where the degree of O-acetylation of a 3-substituted Rha4NAc residue at position 2 is approximately 70% . PS could be fractionated into PS1 and PS2 by gel-permeation chromatography on TSK HW-50S . Matrix-assisted laser desorption ionization MS data indicate sequential chain elongation of both PS1 and PS2 by a single sugar unit, with O-acetylation in PS1 beginning at a certain chain length . Anti-(C . gillenii O9a,9b) serum reacted with PS1 in double immunodiffusion and immunoblotting, whereas neither PS2 nor the lipopolysaccharide of Vibrio cholerae O1 with a structurally related O-chain polysaccharide were reactive.

J Clin Microbiol, 2002 Jan, 40(1), 123 - 7
BetalasEN: microdilution panel for identifying beta-lactamases present in isolates of Enterobacteriaceae; Sanders CC et al.; A dried investigational use-only microdilution panel named betalasEN (a short named derived from the panel's purpose, to identify beta-lactamases in Enterobacteriaceae) containing 10 beta-lactam drugs with and without beta-lactamase inhibitors was developed to identify beta-lactamases among clinical isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter koseri, Citrobacter freundii group, Enterobacter spp., and Serratia marcescens . The MICs obtained with a collection of 383 organisms containing well-characterized beta-lactamases were used to develop numeric codes and logic pathways for computerized analysis of results . The resultant logic pathways and betalasEN panel were then used to test and identify beta-lactamases among 885 isolates of Enterobacteriaceae recovered in cultures obtained at six different hospital laboratories across the United States . beta-Lactamases present in 801 (90.5%) of the 885 isolates were identified by betalasEN by using the existing logic pathways and codes or after minor modifications were made to the existing codes . The 84 strains that gave codes that betalasEN could not identify were collected, reidentified, and retested by using betalasEN . Three strains had been misidentified, 54 strains gave different codes upon repeat testing that could be identified by betalasEN, and 27 strains repeated new codes . The beta-lactamases in these strains were identified, and the new codes were added to the betalasEN logic pathways . These results indicate that betalasEN can identify clinically important beta-lactamases among most isolates of Enterobacteriaceae . The results also show that good quality control and attention to proper performance of the tests are essential to the correct performance of betalasEN.

Int J Food Microbiol, 2001 Nov 8, 70(3), 255 - 65
Evaluation of culture media for enrichment and isolation of Shigella sonnei and S . flexneri; Uyttendaele M et al.; The performance of Gram-negative (GN) broth with (10 microg/ml) and without novobiocin, Shigella broth (SB) with 0.5 and 3.0 microg/ml novobiocin, all incubated at 37 degrees C (SB with 3.0 microg/ml novobiocin also at 42 degrees C) and buffered brilliant green bile glucose (EE) broth with 1.0 microg/ml novobiocin incubated at 37 and 42 degrees C were evaluated for resuscitation and growth of Shigella sonnei and S . flexneri (eight strains; unstressed, chill-stressed and acid-stressed) and non-shigellae (11 strains) . GN broth with or without novobiocin supported significantly less growth of Shigella sp . No significant differences in growth of shigellae were obtained between the other culture media . Performance depended more on the Shigella strain used . None of the tested media were significantly superior for suppressing the competitive flora . Electivity and selectivity of MacConkey agar (MAC), tergitol-7 agar (T7), desoxycholate citrate agar (DCA), xylose lysine desoxycholate agar (XLD), Salmonella Shigella agar and Hektoen enteric agar (HEA) were determined by ecometric testing . HEA confirmed to be a high selective medium for both non-shigellae and stressed Shigella sp . Klebsiella sp., Enterobacter sp., Citrobacter sp. . Salmonella sp . and the Escherichia strains can mask the presence of shigellae . In vitro competition experiments and experiments with artificially contaminated foods showed higher resistance of S . sonnei than S . flexneri towards the stress imposed by the food matrix and its indigenous flora . Reliable detection, however, of shigellae in foods with the current enrichment and isolation media was not achieved.

Antimicrob Agents Chemother, 2002 Jan, 46(1), 151 - 9
Countrywide spread of CTX-M-3 extended-spectrum beta-lactamase-producing microorganisms of the family Enterobacteriaceae in Poland; Baraniak A et al.; Eighty-four clinical isolates of the family Enterobacteriaceae, recovered from 1998 to 2000 in 15 hospitals in 10 Polish cities, were analyzed . All the isolates produced beta-lactamases with pIs of 8.4 and 5.4, and the pI 8.4 enzymes were demonstrated to hydrolyze cefotaxime but not ceftazidime in the in vitro bioassay . PCR analysis and DNA sequencing have revealed that in all cases the pI 8.4 beta-lactamase was probably the CTX-M-3 extended-spectrum beta-lactamase (ESBL) variant, which was originally identified in 1996 in Praski Hospital in Warsaw . In the majority of isolates, bla(CTX-M-3) genes resided within large conjugative plasmids with similar fingerprints, which, in the context of the high degree of diversity of the randomly amplified polymorphic DNA types of the isolates, suggested that horizontal transfer of plasmids was likely the main mechanism of CTX-M-3 spread . The dissemination of plasmids was probably preceded by the center-to-center transmission of several strains, as indicated by the identification by pulsed-field gel electrophoresis of closely related or possibly related Klebsiella pneumoniae, Escherichia coli, and Citrobacter freundii isolates in five different hospitals . CTX-M-3-producing organisms revealed a very high degree of diversity in beta-lactam resistance levels and patterns . This was attributed to several factors, such as the production of other beta-lactamases including additional ESBLs, possible quantitative variations in CTX-M-3 expression, segregation of AmpC derepressed mutants, and permeability alterations.

J Antimicrob Chemother, 2001 Dec, 48(6), 839 - 52
SHV-12, SHV-5, SHV-2a and VEB-1 extended-spectrum beta-lactamases in Gram-negative bacteria isolated in a university hospital in Thailand; Chanawong A et al.; Sixty-one extended-spectrum beta-lactamase (ESBL)-producing isolates were collected from Srinagarind Hospital, Thailand . These included 43 Enterobacteriaceae and 18 Pseudomonadaceae . The 43 Enterobacteriaceae were found to produce the following ESBLs: 26 (60.5%) SHV-12, 13 (30.2%) SHV-5, two (4.7%) SHV-2a, one (2.3%) VEB-1 and one (2.3%) unidentified . Twenty-four isolates (55.8%) also carried bla(TEM-1B), as well as bla(SHV) or bla(VEB-1) . Plasmid DNA from transconjugants carrying the bla(SHV-12) gene showed various restriction patterns, indicating the distribution of the bla(SHV-12) gene among different antibiotic resistance plasmids . In contrast, bla(SHV-5) in 13 isolates was found on a single plasmid of c . 130 kb . Pulsed-field gel electrophoresis (PFGE) analysis of genomic DNA from these isolates revealed that nine of 11 Klebsiella pneumoniae gave the same pattern, indicating clonal spread of the strain within the hospital, together with the occasional spread of the plasmid to other strains . Among the pseudomonad isolates, 16 Pseudomonas aeruginosa and one Pseudomonas putida had bla(VEB-like) and one P . aeruginosa had bla(SHV-12) . Nine of the 16 isolates carrying bla(VEB-like) (56.3%) had identical PFGE patterns, suggesting the dissemination of this gene, also by clonal spread . At least six different bla(VEB-like-)containing integrons were found among the 18 isolates . This is the first report of bacteria producing SHV-12 and SHV-2a in Thailand and the first report of SHV-12 in P . aeruginosa, of VEB-1 in Citrobacter freundii and a VEB-1-like beta-lactamase in P . putida . These findings indicate that ESBL genes in the Far East are part of a gene pool capable of broad horizontal gene transfer, in that these genes can transfer between different families of Gram-negative bacilli.

Biochemistry, 2001 Dec 11, 40(49), 14862 - 8
Inhibition of tyrosine phenol-lyase from Citrobacter freundii by 2-azatyrosine and 3-azatyrosine; Watkins EB et al.; The interactions of 2-azatyrosine and 3-azatyrosine with tyrosine phenol-lyase (TPL) from Citrobacter freundii have been examined . 2-Aza-DL-tyrosine and 3-aza-DL-tyrosine were synthesized by standard methods of amino acid synthesis, while the L-isomers were prepared from 3-hydroxypyridine and 2-hydroxypyridine, respectively, with TPL (Watkins, E . B., and Phillips, R . S . (2001) Bioorg . Med . Chem . Lett . 11, 2099-2100) . 3-Azatyrosine was examined as a potential transition state analogue inhibitor of TPL . Both compounds were found to be competitive inhibitors of TPL, with K(i) values of 3.4 mM and 135 microM for 3- and 2-aza-L-tyrosine, respectively . Thus, 3-azatyrosine does not act as a transition state analogue, possibly due to the lack of tetrahedral geometry at C-1 . However, 2-aza-L-tyrosine is the most potent competitive inhibitor of TPL found to date . The K(i) value of 2-aza-L-tyrosine is half that of 2-aza-DL-tyrosine, indicating that the D-enantiomer is inactive as an inhibitor . Neither azatyrosine isomer was shown to be a substrate for beta-elimination, based on coupled assays with lactate dehydrogenase and on HPLC measurements . Both isomers of azatyrosine form equilibrium mixtures of external aldimine and quinonoid intermediates when they bind to TPL . However, 2-azatyrosine reacts about 10-fold faster to form a quinonoid intermediate than does 3-azatyrosine . Since 2-azatyrosine is in the zwitterion or phenolate ion form at all the pH values examined, the strong binding of this compound suggests that L-tyrosine may be bound to the active site of TPL as the phenolate anion.

Mol Ecol, 2001 Oct, 10(10), 2499 - 513
Host and geographical factors influence the thermal niche of enteric bacteria isolated from native Australian mammals; Okada S et al.; The thermal profiles of 118 bacterial strains, representing six species of the family Enterobacteriaceae, isolated from a variety of native Australian mammals were determined under in vitro conditions . Each of the bacterial species had a unique thermal profile and differed in their minimum or maximum temperature for growth and in their response to changing temperatures . The taxonomic classification of the host from which the bacterial strains were isolated explained a significant amount of the variation in thermal profile among strains of a species . Host effects were detected at all taxonomic levels: order, family, genus, and species . The locality (State or Territory) or climate zone from which the strain was collected explained a significant amount of the variation in the thermal profile of Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae strains . Genetically similar strains, as determined by allozyme profiles, had similar thermal profiles for the bacterial species Hafnia alvei and Escherichia coli . The results of this study indicate that there are potentially many aspects of host biology that may determine the thermal profile of these bacteria.

Biotechnol Prog, 2001 Nov-Dec, 17(6), 1008 - 13
Influence of sulfobetaines on the stability of the Citrobacterdiversus ULA-27 beta-lactamase; Spreti N et al.; The activity and stability of beta-lactamase from Citrobacter diversus ULA-27 have been investigated in the presence of different ionic and zwitterionic surfactants . All the sulfobetaine surfactants tested allow the enzyme to retain its full activity, but the best stabilizing effect is greatly dependent on their structure . Very little variations on the monomer headgroup can significantly reduce enzyme deactivation or speed up the loss of activity with respect to buffer alone . The whole hydrophobic/hydrophilic balance on the headgroup seems to have a determining role in preserving beta-lactamase activity and structure . The presence of zwitterionic surfactants stabilizes the protein conformation toward denaturation by urea and low-temperature inactivation . Similar experiments were performed in the presence of other two zwitterionic surfactants, an amine oxide, dimethylmyristylamine oxide (DMMAO) and a carboxybetaine, cetyldimethylammonium methanecarboxylate (CB1-16) . The former stabilizes the enzyme even better than the sulfobetaines, the latter quickly deactivates it . Therefore, the factors responsible for beta-lactamase stabilization are dependent not only on the zwitterionic nature of the surfactant headgroup but also specific interactions between the surfactant and the protein may be important.

Eur J Biochem, 2001 Nov, 268(22), 5740 - 6
Structural studies on the O-polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459); MacLean LL et al.; Citrobacter rodentium is the etiologic agent of transmissible murine colonic hyperplasia (TMCH) and is the only Citrobacter species known to possess virulence factors homologous to human enteropathogenic and enterohemorrhagic Escherichia coli . Members of this species are considered clonal and represent the only known attaching and effacing bacterial pathogen of mice and thus provides a useful animal model for studying the molecular basis of attaching and effacing pathology . The lipopolysaccharide (LPS) produced by C . rodentium has not been previously studied or its possible role as a virulence factor determined . The structure of the LPS has been undertaken as a first step in an investigation of its possible role in pathogenesis . The structure of C . rodentium (ATCC 51459, prototype TMCH isolate, original biotype 4280, previously designated DBS 100) LPS was determined from composition and methylation analyses, mass spectrometry, and two-dimensional nuclear magnetic resonance spectroscopy . The antigenic O-polysaccharide was found to be a high molecular mass branched polymer of repeating pentasaccharide units composed of 2-acetamido-2-deoxy-D-glucose (D-GlcNAc), d-glucose (D-Glc), and L-rhamnose (L-Rha) in the molar ratio 2 : 2 : 1 linked through phosphate, and has the structure: {structure: see text}

Acta Crystallogr D Biol Crystallogr, 2001 Dec, 57(Pt 12), 1922 - 4 Epub 2001 Nov 21.
Crystallization and preliminary X-ray analysis of Citrobacter amalonaticus methylaspartate ammonia lyase; Levy CW et al.; Methylaspartate ammonia lyase (MAL) catalyses the reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid . Crystals of Citrobacter amalonaticus MAL have been obtained by the hanging-drop method of vapour diffusion using ammonium sulfate as the precipitant . Three crystal forms were obtained from identical crystallization conditions, two of which (forms A and B) diffract to high resolution, whilst the third form diffracted poorly . Crystals of form A diffract to beyond 2.1 A and have been characterized as belonging to one of the enantiomorphic space groups P4(1)22 or P4(3)22, with unit-cell parameters a = b = 66.0, c = 233.1 A, alpha = beta = gamma = 90 degrees and a monomer in the asymmetric unit . Crystals of form B diffract to beyond 1.5 A and belong to space group C222, with unit-cell parameters a = 128.3, b = 237.4, c = 65.8 A, alpha = beta = gamma = 90 degrees and a dimer in the asymmetric unit . Determination of the structure of MAL will be an important step in resolving current conflicts concerning the enzyme mechanism which differ between one which places MAL as a member of the superfamily of ammonia lyases whose catalytic activity requires a cofactor formed by post-translational modification of the enzyme and another which links MAL to the enolase superfamily.

Antimicrob Agents Chemother, 2001 Dec, 45(12), 3548 - 54
Cefepime, piperacillin-tazobactam, and the inoculum effect in tests with extended-spectrum beta-lactamase-producing Enterobacteriaceae; Thomson KS et al.; There is little information about the clinical effectiveness of cefepime and piperacillin-tazobactam in the treatment of infections caused by extended-spectrum beta-lactamase (ESBL)-producing pathogens . Some inferences have been drawn from laboratory studies, which have usually involved only one or a few strains of ESBL-producing Klebsiella pneumoniae or Escherichia coli that produced only a limited range of ESBLs . Such studies are indirect, sometimes conflicting, indicators of efficacy . To extend previous laboratory findings, a study was designed to investigate organism-drug interactions by determining the in vitro activities of eight parenteral beta-lactam agents against 82 clinical and laboratory strains of Klebsiella, Escherichia, Enterobacter, Citrobacter, Serratia, Morganella, and Proteus species that produced 22 different ESBLs, alone or in combination with other beta-lactamases . Activities were determined in broth microdilution MIC tests using standard and 100-fold-higher inocula . An inoculum effect, defined as an eightfold or greater MIC increase on testing with the higher inoculum, was most consistently detected with cefepime, cefotaxime, and ceftriaxone and least frequently detected with meropenem and cefoteten . Piperacillin-tazobactam was intermediate between these two groups of agents . Although the inoculum effect is an in vitro laboratory phenomenon, if it has any predictive value in identifying increased risk of therapeutic failure in serious infections, these results support suggestions that cefepime may be a less-than-reliable agent for therapy of infections caused by ESBL-producing strains.

Infection, 2001 Oct, 29(5), 280 - 2
Long-Term outcome of neonatal Citrobacter koseri (diversus) meningitis treated with imipenem/meropenem and surgical drainage; Straussberg R et al.; Neonatal Citrobacter koseri (diversus) meningitis is often complicated by the formation of brain abscesses and has a poor neurological outcome with seizures, mental retardation and paresis as sequelae in 50% of the cases . As there is emerging resistance to ampicillin, gentamicin and third-generation cephalosporins, we attempted to treat this infection with carbapenems . Carbapenems in combination with cefotaxime and surgical drainage may play an important role in treating C . koseri meningitis.

Rev Soc Bras Med Trop, 2001 Sep-Oct, 34(5), 407 - 11
{Identification of Vibrio spp bacteria on skin lesions of fisherman in the county of Raposa-MA}; Rodrigues SM et al.; The study was undertaken aiming at identifying bacteria from the county of Raposa in the state of Maranhao . The clinical sample was collected by using a swab and held in a Cary-Blair transport medium . Enrichment in alkaline peptone water, isolation in TCBS selective indicator medium and biochemical coding of species were used for laboratory processing . Fifty fisherman with age varying from 12-65 years took part on the study . Vibrio bacteria isolated in 21 subjects had been identified . There was a predominance of V . alginolyticus (66.6%) followed by V . parahaemolyticus (42.8%), and V . cholerae non-O1 (9.5%) . Lesions predominated on lower limbs, presenting hyperhemia, swelling, secretion, and pain . Some species of gram-negative bacteria of the Serratia, Proteus, Escherichia, Citrobacter, Enterobacter associated to the vibrios were isolated, as well as other non-fermenting bacteria (30.9) and gram-positive bacteria of the genos Staphylococcus.

Infect Immun, 2001 Nov, 69(11), 6651 - 9
Critical role for tumor necrosis factor alpha in controlling the number of lumenal pathogenic bacteria and immunopathology in infectious colitis; Goncalves NS et al.; Infection of mice with the intestinal bacterial pathogen Citrobacter rodentium results in colonic mucosal hyperplasia and a local Th1 inflammatory response similar to that seen in mouse models of inflammatory bowel disease . In these latter models, and in patients with Crohn's disease, neutralization of tumor necrosis factor alpha (TNF-alpha) is of therapeutic benefit . Since there is no information on the role of TNF-alpha in either immunity to noninvasive bacterial pathogens or on the role of TNF-alpha in the immunopathology of infectious colitis, we investigated C . rodentium infection in TNFRp55(-/-) mice . In TNFRp55(-/-) mice, there were higher colonic bacterial burdens, but the organisms were cleared at the same rate as C57BL/6 mice, showing that TNF-alpha is not needed for protective antibacterial immunity . The most striking feature of infection in TNFRp55(-/-) mice, however, was the markedly enhanced pathology, with increased mucosal weight and thickness, increased T-cell infiltrate, and a markedly greater mucosal Th1 response . Interleukin-12 p40 transcripts were markedly elevated in C . rodentium-infected TNFRp55(-/-) mice, and this was associated with enhanced mucosal STAT4 phosphorylation . TNF-alpha is not obligatory for protective immunity to C . rodentium in mice; however, it appears to play some role in downregulating mucosal pathology and Th1 immune responses.

Antimicrob Agents Chemother, 2001 Oct, 45(10), 2831 - 7
Activity of ertapenem (MK-0826) versus Enterobacteriaceae with potent beta-lactamases; Livermore DM et al.; Ertapenem (MK-0826; L-749,345), a new carbapenem with a long serum half-life, was tested, in vitro, against beta-lactamase-producing bacteria . The new compound had a MIC at which 90% of the isolates were inhibited of 0.06 microg/ml for extended-spectrum beta-lactamase (ESBL)-producing klebsiellas, compared with 0.5 microg/ml for imipenem, 16 microg/ml for cefepime, and >128 microg/ml for ceftazidime and piperacillin-tazobactam . MICs of ertapenem for AmpC-derepressed mutant Enterobacteriaceae were 0.015 to 0.5 microg/ml, whereas imipenem MICs were 0.25 to 1 microg/ml and those of cefepime were 0.5 to 4 microg/ml, and resistance to ceftazidime and piperacillin-tazobactam was generalized . Despite this good activity, the MICs of ertapenem for ESBL-positive klebsiellas mostly were two- to fourfold above those for ESBL-negative strains, and the MICs for AmpC-hyperproducing Enterobacter cloacae and Citrobacter freundii mutants exceeded those for the corresponding AmpC-basal mutants . These differentials did not increase when the inoculum was raised from 10(4) to 10(6) CFU/spot, contraindicating significant lability . Carbapenemase producers were also tested . The IMP-1 metallo-beta-lactamase conferred substantial ertapenem resistance (MIC, 128 microg/ml) in a porin-deficient Klebsiella pneumoniae strain, whereas a MIC of 6 microg/ml was recorded for its porin-expressing revertant . SME-1 carbapenemase was associated with an ertapenem MIC of 2 microg/ml for Serratia marcescens S6, compared with <0.03 microg/ml for Serratia strains lacking this enzyme . In summary, ertapenem had good activity against strains with potent beta-lactamases, except for those with known carbapenemases.

Infect Immun, 2001 Oct, 69(10), 6323 - 35
Locus of enterocyte effacement from Citrobacter rodentium: sequence analysis and evidence for horizontal transfer among attaching and effacing pathogens; Deng W et al.; The family of attaching and effacing (A/E) bacterial pathogens, which includes diarrheagenic enteropathogenic Escherichia coli (EPEC) and enterohemorrhagic E . coli (EHEC), remains a significant threat to human and animal health . These bacteria intimately attach to host intestinal cells, causing the effacement of brush border microvilli . The genes responsible for this phenotype are encoded in a pathogenicity island called the locus of enterocyte effacement (LEE) . Citrobacter rodentium is the only known murine A/E pathogen and serves as a small animal model for EPEC and EHEC infections . Here we report the full DNA sequence of C . rodentium LEE and provide a comparative analysis with the published LEEs from EPEC, EHEC, and the rabbit diarrheagenic E . coli strain RDEC-1 . Although C . rodentium LEE shows high similarities throughout the entire sequence and shares all 41 open reading frames with the LEE from EPEC, EHEC, and RDEC-1, it is unique in its location of the rorf1 and rorf2/espG genes and the presence of several insertion sequences (IS) and IS remnants . The LEE of EPEC and EHEC is inserted into the selC tRNA gene . In contrast, the Citrobacter LEE is flanked on one side by an operon encoding an ABC transport system, and an IS element and sequences homologous to Shigella plasmid R100 and EHEC pO157 flank the other . The presence of plasmid sequences next to C . rodentium LEE suggests that the prototype LEE resided on a horizontally transferable plasmid . Additional sequence analysis reveals that the 3-kb plasmid in C . rodentium is nearly identical to p9705 in EHEC O157:H7, suggesting that horizontal plasmid transfer among A/E pathogens has occurred . Our results indicate that the LEE has been acquired by C . rodentium and A/E E . coli strains independently during evolution.

Infect Immun, 2001 Oct, 69(10), 6217 - 24
Enteropathogenic Escherichia coli infection induces expression of the early growth response factor by activating mitogen-activated protein kinase cascades in epithelial cells; de Grado M et al.; Enteropathogenic Escherichia coli (EPEC) is an extracellular bacterial pathogen that infects the human intestinal epithelium and is a major cause of infantile diarrhea in developing countries . EPEC belongs to the group of attaching and effacing (A/E) pathogens . It uses a type III secretion system to deliver proteins into the host cell that mediate signal transduction events in host cells . We used gene array technology to study epithelial cell responses to EPEC infection at the level of gene expression . We found that EPEC induces the expression of several genes in infected HeLa cells by a lipopolysaccharide (LPS)-independent mechanism, including cytokines and early growth response factor 1 (Egr-1) . The transcription factor Egr-1 is an immediate-early-induced gene that is activated in most cell types in response to stress . EPEC-induced upregulation of egr-1 is mediated by the activation of the MEK/extracellular signal-regulated kinase signal transduction pathway and is dependent on the type III secretion system . egr-1 is also induced during infection of mice by the A/E pathogen Citrobacter rodentium, suggesting that both Egr-1 and the activation of this mitogen-activated protein kinase signal transduction pathway may play a role in disease.

J Bacteriol, 2001 Oct, 183(19), 5782 - 7
Intervening sequences in rrl genes and fragmentation of 23S rRNA in genera of the family Enterobacteriaceae; Pronk LM et al.; Intervening sequences (IVSs) in the rrl genes for 23S rRNA are transcribed but later removed by RNase III without religation during RNA processing, leading to fragmented rRNA . We examined about 240 strains of the family Enterobacteriaceae for presence of IVSs using PCR . No IVSs were detected in strains belonging to Escherichia, Shigella, Enterobacter, Erwinia, Ewingella, Hafnia, Kluyvera, Morganella, Pantoea, or Serratia . Previously unreported IVSs were detected in Klebsiella oxytoca, Citrobacter amalonaticus, and Providencia stuartii; previously reported IVSs are in species of Salmonella, Proteus, Providencia, and Yersinia . The sporadic distribution of IVSs indicates lateral genetic transfer of IVSs.

Clin Infect Dis, 2001 Oct 1, 33(7), 947 - 53 Epub 2001 Sep 05.
Evolution, incidence, and susceptibility of bacterial bloodstream isolates from 519 bone marrow transplant patients; Collin BA et al.; Bacteria remain an important cause of infection in bone marrow transplants . To examine shifts in the etiology and susceptibility of bacterial isolates from transplants, we reviewed the incidence and susceptibility of blood isolates during a 7-year period . The infection rate fell dramatically during this time . Gram-positive organisms were isolated more often than gram-negative organisms, but the trend is reversing . Streptococci surpassed staphylococci for 5 years as the leading pathogen . Increasing resistance to penicillin, ciprofloxacin, and imipenem was noted in Streptococcus species . With the exception of type 1 beta-lactamase-producing bacteria and Pseudomonas aeruginosa, gram-negative isolates remained overall susceptible to ceftazidime . Increased antibiotic prophylaxis coincided with the reduction in percentage of infected patients and increase in resistance to beta-lactam antibiotics . Mortality attributed to bacteremia was low except for infections caused by P . aeruginosa and the Enterobacter, Serratia, Citrobacter group . There was no mortality attributable to gram-positive organisms such as Staphylococcus aureus and viridans streptococci.

J Clin Microbiol, 2001 Sep, 39(9), 3147 - 55
Group G beta-hemolytic streptococcal bacteremia characterized by 16S ribosomal RNA gene sequencing; Woo PC et al.; Little is known about the relative importance of the four species of Lancefield group G beta-hemolytic streptococci in causing bacteremia and the factors that determine the outcome for patients with group G beta-hemolytic streptococcal bacteremia . From 1997 to 2000, 75 group G beta-hemolytic streptococcal strains were isolated from the blood cultures of 66 patients . Sequencing of the 16S rRNA genes of the group G beta-hemolytic streptococci showed that all 75 isolates were Streptococcus dysgalactiae subspecies equisimilis . The API system (20 STREP) and Vitek system (GPI) successfully identified 65 (98.5%) and 62 (93.9%) isolates, respectively, as S . dysgalactiae subspecies equisimilis with >95% confidence, whereas the ATB Expression system (ID32 STREP) only successfully identified 49 isolates (74.2%) as S . dysgalactiae subspecies equisimilis with >95% confidence . The median age of the patients was 76 years (range, 33 to 99 years) . Fifty-six patients (85%) were over 60 years old . All patients had underlying diseases . No source of the bacteremia was identified (primary bacteremia) in 34 patients (52%), whereas 17 (26%) had cellulitis and 8 (12%) had bed sore or wound infections . Fifty-eight patients (88%) had community-acquired group G streptococcal bacteremia . Sixty-two patients (94%) had group G Streptococcus recovered in one blood culture, whereas 4 patients (6%) had it recovered in multiple blood cultures . Fifty-nine patients (89%) had group G Streptococcus as the only bacterium recovered in their blood cultures, whereas in 7 patients other bacteria were recovered concomitantly with the group G Streptococcus in the blood cultures (Staphylococcus aureus in 3, Clostridium perfringens in 2, Citrobacter freundii in 1, and Bacteroides fragilis in 1) . Overall, 10 patients (15%) died . Male sex, diagnosis other than cellulitis, hospital-acquired bacteremia, and multiple positive blood cultures were associated with mortality {P < 0.005 (relative risk {RR} = 7.6), P < 0.05 (RR = 3.7), P < 0.005 (RR = 5.6), and P < 0.05 (RR = 5.6), respectively} . Unlike group C beta-hemolytic streptococcal bacteremia, group G beta-hemolytic streptococcal bacteremia is not a zoonotic infection in Hong Kong.

Int J Antimicrob Agents, 2001 Aug, 18(2), 141 - 5
Comparative antimicrobial spectrum and activity of BMS284756 (T-3811, a desfluoroquinolone) tested against 656 Enterobacteriaceae, including preliminary in vitro susceptibility test comparisons and development; Fix AM et al.; BMS284756 (T-3811), a novel des-F(6)-quinolone, was evaluated using isolates of Enterobacteriaceae from the SENTRY Antimicrobial Surveillance Program tested by Etest (AB BIODISK, Solna, Sweden), reference broth microdilution and disk diffusion (5-microg) methods . Ciprofloxacin, levofloxacin, gemifloxacin and gatifloxacin were also tested by broth microdilution as comparator antimicrobial agents within the same drug class . The 656 isolate collection included species from the genera Citrobacter, Enterobacter, Escherichia, Hafnia, Klebsiella, Morganella, Pantoea, Proteus, Providencia, Salmonella, and Serratia . BMS284756 was slightly less active than comparison fluoroquinolones against these isolates (MIC(90), 4 mg/l versus 0.06-2 mg/l) . However, at a proposed susceptible breakpoint of < or =4 mg/l, 90.7% of the isolates processed were susceptible to BMS284756, demonstrating an equivalent spectrum of activity to all other agents except gemifloxacin (86.6%) . In general, isolates requiring >4 mg/l of BMS284756 for inhibition of growth were also less susceptible to the comparators suggesting cross-resistance is common between des-F(6)- and fluoro-quinolones . Excellent correlation was observed between broth microdilution MIC results and 5-microg disk zone diameters (r=0.94), and between broth microdilution dilution and Etest MIC values (r=0.96) . In conclusion, BMS284756 has an activity and spectrum similar to contemporary fluoroquinolones and in vitro test methods (NCCLS, Etest) appear accurate and reproducible

Bioorg Med Chem Lett, 2001 Aug 20, 11(16), 2099 - 100
Enzymatic synthesis of aza-l-tyrosines; Watkins EB et al.; Tyrosine phenol-lyase from Citrobacter freundii synthesizes 2-aza-L-tyrosine and 3-aza-L-tyrosine from 3-hydroxypyridine and 2-hydroxypyridine, respectively, and ammonium pyruvate.

Infect Immun, 2001 Sep, 69(9), 5597 - 605
Intimin-specific immune responses prevent bacterial colonization by the attaching-effacing pathogen Citrobacter rodentium; Ghaem-Maghami M et al.; The formation of attaching and effacing (A/E) lesions on gut enterocytes is central to the pathogenesis of enterohemorrhagic (EHEC) Escherichia coli, enteropathogenic E . coli (EPEC), and the rodent pathogen Citrobacter rodentium . Genes encoding A/E lesion formation map to a chromosomal pathogenicity island termed the locus of enterocyte effacement (LEE) . Here we show that the LEE-encoded proteins EspA, EspB, Tir, and intimin are the targets of long-lived humoral immune responses in C . rodentium-infected mice . Mice infected with C . rodentium developed robust acquired immunity and were resistant to reinfection with wild-type C . rodentium or a C . rodentium derivative, DBS255(pCVD438), which expressed intimin derived from EPEC strain E2348/69 . The receptor-binding domain of intimin polypeptides is located within the carboxy-terminal 280 amino acids (Int280) . Mucosal and systemic vaccination regimens using enterotoxin-based adjuvants were employed to elicit immune responses to recombinant Int280alpha from EPEC strain E2348/69 . Mice vaccinated subcutaneously with Int280alpha, in the absence of adjuvant, were significantly more resistant to oral challenge with DBS255(pCVD438) but not with wild-type C . rodentium . This type-specific immunity could not be overcome by employing an exposed, highly conserved domain of intimin (Int388-667) as a vaccine . These results show that anti-intimin immune responses can modulate the outcome of a C . rodentium infection and support the use of intimin as a component of a type-specific EPEC or EHEC vaccine.

Arch Microbiol, 2001 Jun, 175(6), 395 - 404
Further studies on RpoS in enterobacteria: identification of rpoS in Enterobacter cloacae and Kluyvera cryocrescens; Martinez-Garcia E et al.; RpoS, the alternative sigma factor sigma(s), is important for bacterial survival under extreme conditions . Many enterobacteria are opportunistic human pathogens and their ability to survive in a changing environment could be an essential step for their virulence . To determine the presence of this gene in enteric bacteria, an Escherichia coli rpoS probe was constructed and used to detect the presence of this gene in different species . A gene homologous to rpoS was found in Citrobacter amalonaticus, Enterobacter cloacae, Klebsiella planticola, Kluyvera cryocrescens, Serratia rubidaea, Shigella sonnei, and Yersinia ruckeri . Providencia stuartii and Proteus vulgaris were the only tested enterobacteria that did not show any signal with the E . coli rpoS probe or that did not lead to amplification of an rpoS fragment using specific primers . The rpoS gene from E . cloacae and from K . cryocrescens was cloned and sequenced and a mutant allele was constructed in E . cloacae . Survival rates under different harsh conditions were followed in order to determine the effect of rpoS inactivation in exponential- and stationary-phase cells of both strains . E . cloacae rpoS mutants were more sensitive to extreme pH, high osmolarity, and high temperature than the wild-type.

Anal Chem, 2001 Jul 15, 73(14), 3432 - 40
UV Raman spectral intensities of E . coli and other bacteria excited at 228.9, 244.0, and 248.2 nm; Wu Q et al.; Resonance Raman spectral intensities per average bacterial cell have been measured quantitatively for Gram-negative Escherichia coli, Citrobacter freundii, and Enterobacter aerogenes, as well as Gram-positive Bacillus subtilis and Staphylococcus epidermidis . Spectra have been obtained from cultures in the lag, log, and stationary growth phases excited in turn by 228.9, 244.0, and 248.2 nm light . Although Raman spectral peak positions (cm(-1)) excited by a given wavelength are very similar for all five bacterial species, the organisms are characterized by significantly different spectral intensity values . Intensity changes are associated with growth phase changes in all of the species as well . A comparison of measured with estimated average intensities has been made for spectra of log-phase E . coli . It is possible to compare measured intensities with intensities estimated for log-phase E . coli on the basis of the knowledge of its known average cellular molecular composition . A significant degree of hypochromism is observed in E . coli nucleic acid spectra . In contrast, strong average hyperchromism characterizes all aromatic amino acid peaks belonging to the same E . coli cells . Results suggest that knowledge of spectral intensity values will enhance significantly the capability to identify bacteria by means of their UV resonance Raman spectra.

J Food Prot, 2001 Jul, 64(7), 1035 - 44
Source and identification of histamine-producing bacteria from fresh and temperature-abused albacore; Kim SH et al.; Histamine-producing bacteria were isolated from fresh and temperature-abused albacore using two different isolation procedures . Typically, the bacterial isolates on Niven's or modified Niven's medium produced negligible or low levels of histamine (<300 ppm) in histamine enumeration broth . The most frequently found species using this approach was Hafnia alvei . By prescreening on selective media (eosin methylene blue {EMB} agar for enteric bacteria; deMan Rogosa Sharpe agar for lactic acid bacteria: KF streptococcus agar for streptococci; pseudomonas isolation {PI} agar for pseudomonads; and staphylococcus medium 110 agar for staphylococci) prior to plating on histidine decarboxylase differential media, detection rate of true histamine formers increased . Prolific histamine producers capable of forming >1,000 ppm histamine in culture broth were isolated when PI and EMB agars were used for prescreening . Among the selective media tested, EMB agar was most effective in selecting high histamine producers, as demonstrated by the highest rate of true positives based on histamine analysis . Histamine-producing isolates were mostly enteric bacteria, including Morganella morganii, H . alvei, Klebsiella spp., Citrobacter freundii, Enterobacter spp., and Serratia spp . M . morganii isolated on PI agar from temperature-abused albacore muscle was found to be the highest histamine former . This species was not isolated from fresh albacore . while other enteric bacteria were frequently detected on the gills . However, only a few species isolated from both fresh and temperature-abused muscles were identified as high histamine formers.

Carbohydr Res, 2001 Jul 19, 333(4), 335 - 8
Structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c; Kocharova NA et al.; The following structure of the O-specific polysaccharide of Citrobacter braakii O7a,3b,1c was established using sugar and methylation analyses and NMR spectroscopy, including 2D COSY, TOCSY, NOESY, and 1H, 13C heteronuclear single-quantum coherence (HSQC) experiments: (struture: see text) . The main D-mannan chain of the polysaccharide studied has the same structure as the O-specific polysaccharide of Escherichia coli O9, Klebsiella pneumoniae O3, and Hafnia alvei PCM 1223.

Antimicrob Agents Chemother, 2001 Aug, 45(8), 2287 - 98
Novel class A beta-lactamase Sed-1 from Citrobacter sedlakii: genetic diversity of beta-lactamases within the Citrobacter genus; Petrella S et al.; Citrobacter sedlakii 2596, a clinical strain resistant to aminopenicillins, carboxypenicillins, and early cephalosporins such as cephalothin, but remaining susceptible to acylureidopenicillins, carbapenems, and later cephalosporins such as cefotaxime, was isolated from the bile of a patient treated with beta-lactam and quinolone antibiotics . The isolate produced an inducible class A beta-lactamase of pI 8.6, named Sed-1, which was purified . Characterized by a molecular mass of 30 kDa, Sed-1 preferentially hydrolyzed benzylpenicillin, cephalothin, and cloxacillin . The corresponding gene, bla(Sed-1), was cloned and sequenced . Its deduced amino acid sequence shared more than 60% identity with the chromosome-encoded beta-lactamases from Citrobacter koseri (formerly C . diversus) (84%), Klebsiella oxytoca (74%), Serratia fonticola (67%), and Proteus vulgaris (63%) and 71% identity with the plasmid-mediated enzyme MEN-1 . A gene coding for a LysR transcriptional regulator was found upstream from bla(Sed-1) . This regulator, named SedR, displayed 90% identity with the AmpR sequence of the chromosomal beta-lactamase from C . koseri and 63 and 50% identity with the AmpR sequences of P . vulgaris and Enterobacter cloacae, respectively . By using DNA-DNA hybridization, a bla(Sed-1)-like gene was identified in two reference strains, C . sedlakii (CIP-105037) and Citrobacter rodentium (CIP-104675), but not in the 18 strains of C . koseri studied . Two DNA fragments were amplified and sequenced from the reference strains of C . sedlakii CIP-105037 and C . rodentium CIP-104675 using two primers specific for bla(Sed-1) . They shared 98 and 80% identity with bla(Sed-1), respectively, confirming the diversity of the chromosomally encoded class A beta-lactamases found in Citrobacter.

J Med Microbiol, 2001 Jul, 50(7), 636 - 41
Clonal origin of aminoglycoside-resistant Citrobacter freundii isolates in a Danish county; Norskov-Lauritsen N et al.; During 1997, attention was drawn to an increased frequency of aminoglycoside-resistant Citrobacterfreundii in a Danish county, when a total of 24 resistant C . freundii isolates was detected . In this study, 15 such isolates were typed by pulsed-field gel electrophoresis, riboprinting and partial sequencing of the gene encoding translation initiation factor 2 . Fourteen of the 15 isolates were identical, as evaluated by their antibiograms and by all these typing methods . This epidemic strain harboured the aminoglycoside resistance genes aac(3)-II and ant(3")-I, with the latter located in tandem with a dihydrofolate reductase gene in a class I integron . The source of the strain remains unresolved . Representative isolates were obtained from various specimens from hospitals and general practice throughout the county, with no evidence of patient-to-patient transmission.

Can J Urol, 2001 Jun, 8 Suppl 1, 13 - 7
Complicated urinary tract infections in patients with voiding dysfunction; MacMillan RD; Complicated urinary tract infections (UTIs) occur in patients who have structurally or functionally abnormal urinary tracts -- in contrast to simple cystitis, which usually occurs in young, healthy females who have normal anatomy . Because of their abnormal anatomy, patients with complicated UTIs can be exceedingly difficult to treat . Successful treatment usually requires correction of the underlying anatomical problem, but this may not possible, so relapse of the infection is the general rule . Although most infections are still caused by Escherichia coli, many are caused by more exotic and highly resistant coliforms, such as citrobacter and pseudomonas . As well, gram-positive bacteria such as staphylococcus and enterococcus can be found . In patients with spinal cord injuries, high bladder pressure due to obstruction at the sphincter level can cause obstruction and/or reflux . In these patients, as well as in patients with benign prostatic hypertrophy, and in geriatric women, high residual bacteria levels prevent washout of bacteria . Other possibly significant risk factors include poor fluid intake, poor hygiene, and poor nutrition that results in decreased immunity . Finally, the unavoidable use of catheters actively introduces bacteria into the urinary tracts of these patients . Prevention by modifying as many of these risk factors as possible is essential to reduce attack rates of UTIs . Treatment must be guided by the results of urine cultures . Broad-spectrum coverage including anti-pseudomonal activity is preferable, and the fluoroquinolones, eg . ciprofloxacin or levofloxacin, seem to fit this bill perfectly . If possible, physicians should start by prescribing oral antibiotics for a minimum of 10 days and maybe for a lot longer, as indicated . There is no role for short-course therapy . Other options include oral, second-generation cephalosporins, or intravenous therapy with aminoglycosides and piperacillin . Physicians must be prepared to treat aggressively for prolonged periods . Unfortunately, reinvestigation, reculturing, and retreatment are usually required.

Microb Drug Resist, 2001 Summer, 7(2), 171 - 5
Detection and typing of extended-spectrum beta-lactamases in clinical isolates of the family Enterobacteriaceae in a medical center in Turkey; Durmaz R et al.; To determine and type the extended-spectrum beta-lactamases (ESBLs) among the family Enterobacteriaceae in a medical center, a total of 668 clinical isolates were screened . Of the 668 isolates, the 80 strains were presumptively defined as ESBL producers according to the result of disk method using ESBL marker antibiotics (aztreonam, ceftazidime, and cefoxitin) . These 80 strains were retested with the double-disk synergy test (DDST), the E-test ESBL strip, a 5-microg ceftazidime disk, and agar dilution MICs of ceftazidime with and without clavulonic acid . Isoelectric focusing was performed to confirm ESBL production and type the beta-lactamases . By evaluation of the results of all tests used for ESBL detection together with isoelectric focusing, 33 (4.9%) of the 668 isolates were described as ESBL producer . The positive results of the agar dilution test, DDST, the E-test strip, and 5-microg ceftazidime disk were 32, 26, 27, and 26 of the 33 strains, respectively . ESBL positivity was 48.8% in Klebsiella species, 15.4% in Citrobacter species, 4.9% in Enterobacter species and 1.1% in Escherichia coli strains . The ESBL enzymes frequently determined were SHV-2/6-like (pI 7.6), SHV-5-like (pI 8.2), SHV-4-like (pI 7.8), and SHV-3-like (pI 7) . SHV-derived enzymes were commonly observed in Klebsiella spp whereas TEM-related enzymes were seen in E . coli strains . The results of this study indicated that SHV-2/6-derived (pI 7.6) ESBL expression among the isolates of the family Enterobacteriaceae is an important problem in our medical center.

J Infect Dis, 2001 Jul 15, 184(2), 227 - 30 Epub 2001 Jun 18.
Bacterial infection promotes colon tumorigenesis in Apc(Min/+) mice; Newman JV et al.; The Min mouse, which has a germ line mutation in 1 allele of the Apc tumor suppressor gene, is a model for the early steps in human colorectal cancer . Helicobacter pylori infection, a known risk factor for gastric cancer in humans, causes chronic inflammation and increased epithelial cell proliferation in the stomach . Infection with the bacterium Citrobacter rodentium is known to increase epithelial cell proliferation and to promote chemically initiated tumors in the colon of mice . Min mice infected with C . rodentium at 1 month of age were found to have a 4-fold increase in the number of colonic adenomas at 6 months of age, compared with uninfected Min mice . Most of the colonic adenomas in the infected Min mice were in the distal colon, where C . rodentium-induced hyperplasia occurs . These data demonstrate that bacterial infection promotes colon tumor formation in genetically susceptible mice.

J Antimicrob Chemother, 2001 Jul, 48 Suppl 1, 59 - 64
Detection of beta-lactamase-mediated resistance; Livermore DM et al.; beta-Lactams are the most widely used antibiotics, and beta-lactamases are the greatest source of resistance to them . An understanding of beta-lactamase detection and identification is therefore valuable . Colorimetric, acidimetric and iodometric tests of beta-lactamase production are good, rapid indicators of penicillin and ampicillin resistance in Haemophilus, Moraxella and Neisseria spp . These methods can also be applied to Gram-negative aerobic bacilli but are less useful, since the usual question is not whether a beta-lactamase is produced by these organisms, but which beta-lactamase? Accurate identification of the beta-lactamases of Enterobacteriaceae demands gene or protein sequencing, but the broad type of enzyme produced by an isolate can often be inferred from antibiotic susceptibility data . Resistance to ceftazidime or cefpodoxime implies extended-spectrum beta-lactamase (ESBL) production in Escherichia coli and Klebsiella spp., especially if susceptibility to cefoxitin is retained . ESBL production can be confirmed with double disc tests or with various commercial kits . Derepression of AmpC beta-lactamases in Enterobacter spp . and Citrobacter freundii is another important mechanism and can be inferred from cross-resistance to beta-lactamase inhibitor combinations and to all cephalosporins except fourth-generation agents . Antagonism between cefoxitin and cefotaxime can be used to infer the presence of inducible AmpC enzymes in these species, indicating the risk of segregation of derepressed mutants, but in general this risk is better predicted from accurate speciation.

Microbes Infect, 2001 Jun, 3(7), 561 - 9
The Yersinia high-pathogenicity island: an iron-uptake island; Carniel E; Highly pathogenic Yersinia carry a pathogenicity island termed high-pathogenicity island (HPI) . The Yersinia HPI comprises genes involved in the synthesis of the siderophore yersiniabactin and can thus be regarded as an iron-uptake island . A unique characteristic of the HPI is its wide distribution among different enterobacteria such as Escherichia coli, Klebsiella, Citrobacter and Salmonella . Other types of iron-uptake systems are also carried by different pathogenicity islands in enterobacteria.

Southeast Asian J Trop Med Public Health, 2000, 31 Suppl 1, 157 - 61
Mechanical carrier of bacterial enteric pathogens by Chrysomya megacephala (Diptera: Calliphoridae) in Chiang Mai, Thailand; Sukontason K et al.; Chrysomya megacephala was studied regarding its mechanically bacterial carrier in urban areas of Chiang Mai, northern Thailand . Fifty-six adult flies were randomly collected using sweep insect net during April-May, 1999 from 3 fresh food markets and examined for bacteriological isolation . Among them, 49 flies (87.5%) were bacterial carriers . The total 22 bacterial species and 8 groups were isolated . Three species previously reported as the bacterial enteric pathogens causing diarrheal disease were isolated from 5 flies, ie Aeromonas hydrophila, Edwardsiella tarda and Vibrio cholerae non-01, with their prevalence rates in flies being 3.579, 1.79% and 3.57%, respectively . Five possible bacteria enteric pathogens, ie Aermononas sobria, Citrobacter freundii, Escherichia coli, Providencia alcalifaciens and Pseudomonas aeruginosa, were isolated from 21 flies with the prevalence rates in flies being 5.34%, 3.57%, 26.79%, 7.14% and 1.79%, respectively . The bacterial load isolated from all 3 pathogenic species was entirely found more than 10 colony per fly, indicating the high chance for disease transmission via this fly species . C . megacephala may play the possible and/or important role of bacterial enteric pathogens transmission, thereby promoting the public health personnel for sanitation improvement in fresh food markets and fly control management in these particular areas.

J Food Prot, 2001 Jun, 64(6), 833 - 7
Primers and a specific DNA probe for detecting lactic acid bacteria producing 3-hydroxypropionaldehyde from glycerol in spoiled ciders; Claisse O et al.; Of the 40 strains isolated from several spoiled ciders where glycerol was degraded, 36 were identified as Lactobacillus collinoides, three were Lactobacillus hilgardii, and one was Lactobacillus mali . However, only 30 L . collinoides and two L . hilgardii could degrade glycerol . The glycerol dehydratase activity was shown . The main product of the transformation was 1.3 propanediol . Two DNA primers GD1 and GD2 were chosen in the region encoding one of the subunits of glycerol dehydratase of Citrobacter freundii, Klebsiella pneumoniae, Klebsiella oxytoca, Salmonella Typhimurium, and Clostridium pasteurianum . A 279-bp amplicon in polymerase chain reaction amplification was obtained with the genomic L . collinoides IOEB 9527 DNA as template . The amino acid sequence deduced from the amplicon DNA sequence showed a very high similarity and identity with the gene of gram-negative and C . pasteurianum species . After labeling, the amplicon was used as DNA probe in dot-blot hybridization with the genomic DNA of all the tested strains . Only strains that could degrade glycerol hybridized . Moreover, polymerase chain reactions using GDI and GD2 revealed only glycerol dehydratase genes of positive L . collinoides and L . hilgardii strains . The primers and the amplicon proved to be suitable and reliable tools to detect the lactic acid bacteria involved in the deterioration of cider.

Microbiol Immunol, 2001, 45(4), 277 - 83
Characterization of an extended-spectrum class C beta-lactamase of Citrobacter freundii; Haruta S et al.; Citrobacter freundii GC3 is a clinical isolate which showed moderate resistance to oxyimino beta-lactams such as ceftazidime and aztreonam . This drug resistance was due to an extended-spectrum class C beta-lactamase encoded by chromosomal gene(s) . The GC3 beta-lactamase showed high amino acid sequence homology to a known C . freundii beta-lactamase, i.e., 346 of 361 amino acids were identical with those of C . freundii GN346 beta-lactamase (Tsukamoto, K . et al, Eur . J . Biochem . 188, 15-22, 1990) . Asp198 was the only dissimilar amino acid found in the omega loop region, known as the hot spot for extended-spectrum resistance in class C beta-lactamases (Haruta, S . et al, Microbiol . Immunol . 42, 165-169, 1998) . However, Asp198 was eliminated as a cause of the extended-spectrum resistance by the substitution of Asn for Asp198 . Subsequent investigation suggested that the moderate resistance to oxyimino beta-lactams is attributable to the replacement of amino acids on the enzyme's surface area, far from the active-site . Some or all of the replacements are assumed to delicately modify the active-site configuration . The GC3 beta-lactamase is the first example of an extended-spectrum class C beta-lactamase in which mutations are independent of the omega loop.

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