Citrobacter

Carbohydr Res, 2001 Apr 12, 331(3), 331 - 6
Structure of the O-specific polysaccharide isolated from the lipopolysaccharide of Citrobacter gillenii serotype O12a, 12b strain PCM 1544; Kubler-Kielb J et al.; A neutral O-specific polysaccharide was isolated from the lipopolysaccharide of Citrobacter gillenii strain PCM 1544, representing serotype O12a,12b . The polysaccharide was studied by sugar and methylation analyses and Smith degradation along with 1H and 13C NMR spectroscopy, including a ROESY experiment . The following structure of the tetrasaccharide repeating unit was established, in which substitution with terminal GlcNAc is approximately 60% . {structure: see text}

Exp Anim, 2001 Apr, 50(2), 183 - 6
Comparison of bacteriological, genetic and pathological characters between Escherichia coli O115a,c:K(B) and Citrobacter rodentium; Okutani A et al.; Murine pathogenic Escherichia coli O115a,c:K(B) (MPEC) is the causative agent of mouse megaenteron, the pathology of which resembles that of transmissible murine colonic hyperplasia caused by Citrobacter rodentium . We compared their genetic and pathological features to reveal the relationship between these two bacteria . To evaluate the genetic distances, 16S rDNA genes were sequenced and biochemical reactions were tested . Mouse strain susceptibility tests, using CF1 MPEC-susceptible germfree mice and BALB/cA(Jic) resistant mice were performed . MPEC strains and C . rodentium showed more than 99.6% identity by comparison of 16S rDNA gene sequences . All results from biochemical reactions and the mouse strain susceptibility tests were identical . It is proposed that MPEC should be reclassified as C . rodentium.

Biotechniques, 2001 May, 30(5), 1044 - 8, 1050-1
Use of an ALFexpress DNA sequencer to analyze protein-nucleic acid interactions by band shift assay; Filee P et al.; Gel retardation analysis, or band shift assay, is technically the simplest method to investigate protein-nucleic acid interactions . In this report, we describe a nonradioactive band shift assay using a fluorescent DNA target and an ALFexpress automatic DNA sequencer in place of the current method that utilizes radioactively end-labeled DNA target and a standard electrophoresis unit . In our study, the dsDNA targets were obtained by annealing two synthetic oligonucleotides or by PCR . In both cases, a molecule of indodicarbocyanine (CY5) was attached at the 5' OH end of one of the two synthetic oligonucleotides, with a ratio of one molecule of fluorescent dye per molecule of dsDNA . To demonstrate the feasibility of this new band shift assay method, the DNA-binding proteins selected as models were the BlaI and AmpR repressors, which are involved in the induction of the Bacillus licheniformis 749/I and Citrobacter freundii beta-lactamases, respectively . The results show that the use of an automatic DNA sequencer allows easy gel retardation analysis and provides a fast, sensitive, and quantitative method . The ALFexpress DNA sequencer has the same limit of detection as a laser fluorescence scanner and can be used instead of a FluorImager or a Molecular Imager.

J Bacteriol, 2001 Jun, 183(11), 3417 - 27
Identification and functional characterization of arylamine N-acetyltransferases in eubacteria: evidence for highly selective acetylation of 5-aminosalicylic acid; Delomenie C et al.; Arylamine N-acetyltransferase activity has been described in various bacterial species . Bacterial N-acetyltransferases, including those from bacteria of the gut flora, may be involved in the metabolism of xenobiotics, thereby exerting physiopathological effects . We characterized these enzymes further by steady-state kinetics, time-dependent inhibition, and DNA hybridization in 40 species, mostly from the human intestinal microflora . We report for the first time N-acetyltransferase activity in 11 species of Proteobacteriaceae from seven genera: Citrobacter amalonaticus, Citrobacter farmeri, Citrobacter freundii, Klebsiella ozaenae, Klebsiella oxytoca, Klebsiella rhinoscleromatis, Morganella morganii, Serratia marcescens, Shigella flexneri, Plesiomonas shigelloides, and Vibrio cholerae . We estimated apparent kinetic parameters and found that 5-aminosalicylic acid, a compound efficient in the treatment of inflammatory bowel diseases, was acetylated with a catalytic efficiency 27 to 645 times higher than that for its isomer, 4-aminosalicylic acid . In contrast, para-aminobenzoic acid, a folate precursor in bacteria, was poorly acetylated . Of the wild-type strains studied, Pseudomonas aeruginosa was the best acetylator in terms of both substrate spectrum and catalytic efficiency . DNA hybridization with a Salmonella enterica serovar Typhimurium-derived probe suggested the presence of this enzyme in eight proteobacterial and four gram-positive species . Molecular aspects together with the kinetic data suggest distinct functional features for this class of microbial enzymes.

Microbes Infect, 2001 Apr, 3(4), 333 - 40
Molecular pathogenesis of Citrobacter rodentium and transmissible murine colonic hyperplasia; Luperchio SA et al.; Here we review the history, clinical significance, pathology and molecular pathogenesis of Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia . C . rodentium serves as an important model pathogen for investigating the mechanisms controlling attaching and effacing pathology, epithelial hyperproliferation, and tumor promotion in the distal colon of the mouse.

J Appl Microbiol, 2001 Apr, 90(4), 543 - 9
Development of a monoclonal sandwich ELISA for the detection of animal and human Escherichia coli O157 strains; Kerr P et al.; AIMS: Production of a monoclonal antibody (MAb) to Escherichia coli O157 to develop a rapid test using a sandwich ELISA (sELISA) format . METHODS AND RESULTS: A MAb (7A6) was developed to the long-chain lipopolysaccharide of E . coli O157 . A sELISA developed with the MAb reacted with 28 bovine and seven human enterohaemorrhagic E . coli (EHEC) O157 strains and also with two enterotoxigenic E . coli O157 strains . Cross-reaction to a rabbit diarrhoeal E.coli O15, Citrobacter freundii, Salmonella urbana and Vibrio cholerae O1 Inaba was detected . CONCLUSION: A MAb-based sELISA to detect E . coli O157 was produced . Its application to field samples is required to fully determine its prospective use for the detection of EHEC O157, to evaluate the non-specific interference of the cross-reacting strains . SIGNIFICANCE AND IMPACT OF THE STUDY: The assay produced is not wholly specific to EHEC O157, but has the potential to be used as a rapid method for screening large numbers of samples for E . coli O157.

Pediatr Infect Dis J, 2001 Mar, 20(3), 331 - 6
Cefepime microbiologic profile and update; Kessler RE; BACKGROUND: The evolution of the cephalosporin class of antibiotics through modifications of the basic cephem structure has resulted in a new generation with improved antibacterial activity . Cefepime is a prototypic agent of this new class of fourth generation cephalosporins . OBJECTIVE: To review the microbiologic profile of cefepime . RESULTS: Cefepime, which is a zwitterion, has a net neutral charge that allows it to penetrate the outer membrane of Gram-negative bacteria faster than third generation cephalosporins . It is more stable against beta-lactamases because of the lower affinity of the enzymes for cefepime when compared with third generation cephalosporins . As a result of these structural attributes, cefepime has in vitro activity against pathogens that are prevalent in pediatric infections . This agent offers the advantage of Gram-positive coverage similar to that of cefotaxime and ceftriaxone, as well as good activity against Pseudomonas aeruginosa and many enteric bacilli that are resistant to third generation cephalosporins, including clinical isolates of Enterobacter spp . and Citrobacter freundii . CONCLUSIONS: Based on its spectrum of activity cefepime is an option for the treatment of pediatric infections caused by susceptible pathogens.

Eur J Biochem, 2001 Apr, 268(8), 2369 - 78
Identification and expression of the genes and purification and characterization of the gene products involved in reactivation of coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii; Seifert C et al.; The coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii is subject to suicide inactivation by the natural substrate glycerol during catalysis . We identified dhaF and dhaG as the genes responsible for reactivation of inactivated dehydratase . Northern blot analyses revealed that both genes were expressed during glycerol fermentation . The dhaF gene is transcribed together with the three structural genes coding for glycerol dehydratase (dhaBCE), whereas dhaG is coexpressed with the dhaT gene encoding 1,3-propanediol dehydrogenase . The dhaF and dhaG gene products were copurified to homogeneity from cell-free extracts of a recombinant E . coli strain producing both His6-tagged proteins . Both proteins formed a tight complex with an apparent molecular mass of 150 000 Da . The subunit structure of the native complex is probably alpha2beta2 . The factor rapidly reactivated glycerol- or O2-inactivated hologlycerol dehydratase and activated the enzyme-cyanocobalamin complex in the presence of coenzyme B12, ATP, and Mg2+ . The DhaF-DhaG complex and DhaF exhibited ATP-hydrolyzing activity, which was not directly linked to the reactivation of dehydratase . The purified DhaF-DhaG complex of C . freundii efficiently cross-activated the enzyme-cyanocobalamin complex and the glycerol-inactivated glycerol dehydratase of Klebsiella pneumoniae . It was not effective with respect to the glycerol dehydratase of Clostridium pasteurianum and to diol dehydratases of enteric bacteria.

Int J Antimicrob Agents, 2001 Apr, 17(4), 253 - 8
Secretion of cytokines by uroepithelial cells stimulated by Escherichia coli and Citrobacter spp; Funfstuck R et al.; Urinary tract epithelial cells (T 24/83) are able to express interleukin (IL)-6, IL-8, platelet-derived growth factor (PDGF) and tumour necrosis factor-alpha, but not IL-1 beta, IL-2, IL-4 and IL-10 in response to an infection with uropathogenic bacteria . The process of cytokine secretion is time dependent, with a significant increase in the cytokine activity after 60 min . The expression of virulence factors of the bacteria does not seem to play a role . The interaction between bacterial products (e.g . lipopolysaccharide) and/or bacterial adhesion mediated by adhesins and specific receptor molecules of cell surfaces may be responsible for the activity of mediator protein expression in the epithelial cells . The release of PDGF and IL-8 was found to be higher when due to Escherichia coli HB 101 (rough form) than that caused by other bacterial strains . Citrobacter CB 3009 provoked the highest level of IL-6 . The PDGF level correlated significantly with IL-6 and IL-8 values (P<0.001) . There was a significant correlation between the time-dependent release of IL-6 and IL-8 (P<0.05) . In epithelial cytokine response to bacterial infection, the reaction of the epithelial cells may modify themselves (e.g . internalization of bacteria) and the immuno-regulatory processes that are caused by infection and responsible for parenchymal injury.

Surg Endosc . 2000 Aug;14(8):767 . Epub 2000 Jul 12.
Liver hematoma following endoscopic retrograde cholangiopancreatography (ERCP); Ortega Deballon P et al.; We report the case of an 81-year-old man who presented with abdominal pain following endoscopic retrograde cholangiopancreatography (ERCP) for choledocholithiasis . A diagnosis of infected hematoma was made . A CT-guided puncture produced bloody matter that grew Citrobacter freundii . A catheter was left in place for 3 weeks before the patient could be discharged from hospital . We hypothesize that the hepatic parenchyma had been torn by the guide used during the ERCP . This case represents the first report of this type of iatrogenic injury.

Appl Environ Microbiol, 2001 Apr, 67(4), 1558 - 64
Identification and characterization of integron-mediated antibiotic resistance among Shiga toxin-producing Escherichia coli isolates; Zhao S et al.; A total of 50 isolates of Shiga toxin-producing Escherichia coli (STEC), including 29 O157:H7 and 21 non-O157 STEC strains, were analyzed for antimicrobial susceptibilities and the presence of class 1 integrons . Seventy-eight (n = 39) percent of the isolates exhibited resistance to two or more antimicrobial classes . Multiple resistance to streptomycin, sulfamethoxazole, and tetracycline was most often observed . Class 1 integrons were identified among nine STEC isolates, including serotypes O157:H7, O111:H11, O111:H8, O111:NM, O103:H2, O45:H2, O26:H11, and O5:NM . The majority of the amplified integron fragments were 1 kb in size with the exception of one E . coli O111:H8 isolate which possessed a 2-kb amplicon . DNA sequence analysis revealed that the integrons identified within the O111:H11, O111:NM, O45:H2, and O26:H11 isolates contained the aadA gene encoding resistance to streptomycin and spectinomycin . Integrons identified among the O157:H7 and O103:H2 isolates also possessed a similar aadA gene . However, DNA sequencing revealed only 86 and 88% homology, respectively . The 2-kb integron of the E . coli O111:H8 isolate contained three genes, dfrXII, aadA2, and a gene of unknown function, orfF, which were 86, 100, and 100% homologous, respectively, to previously reported gene cassettes identified in integrons found in Citrobacter freundii and Klebsiella pneumoniae . Furthermore, integrons identified among the O157:H7 and O111:NM strains were transferable via conjugation to another strain of E . coli O157:H7 and to several strains of Hafnia alvei . To our knowledge, this is the first report of integrons and antibiotic resistance gene cassettes in STEC, in particular E . coli O157:H7.

J Laryngol Otol, 2001 Apr, 115(4), 327 - 9
Acute tonsillitis complicated by retropharyngeal and thyroid abscess infected with de-repressed beta lactamase Citrobacter mutans; Maini S et al.; An unusual presentation of acute tonsillitis complicated by retropharyngeal and thyroid abscess is reported . Spontaneous rupture of retropharyngeal abscess resulted in necrotic fistulae between the pharyngeal wall and the retropharyngeal space.

J Biochem (Tokyo), 2001 Apr, 129(4), 607 - 13
A cryptic melibiose transporter gene possessing a frameshift from Citrobacter freundii; Shimamoto T et al.; Wild-type Citrobacter freundii cannot grow on melibiose as a sole source of carbon . The melibiose transporter gene melB was cloned from a C . freundii mutant M4 that could utilize melibiose as a sole carbon source . Although the cloned melB gene is closely similar to the melB genes of other bacteria, it is cryptic because of a frameshift mutation . Site-directed mutagenesis was used to construct a functional melB gene by deleting one nucleotide, resulting in the production of an active melibiose transporter . The active MelB transporter could utilize Na(+) and H(+) as coupling cations to melibiose transport . The amino acid sequence of the C . freundii MelB was found to be most similar to those of Salmonella typhimurium and Escherichia coli MelB . These facts are consistent with the phylogenetic relationship of bacteria and the cation coupling properties of the melibiose transporters.

J Microbiol Immunol Infect, 2000 Dec, 33(4), 258 - 62
Comparison of antimicrobial susceptibility of Citrobacter freundii isolates in two different time periods; Wang JT et al.; Citrobacter freundii was first identified in 1932, since then it has been reported to cause a variety of infections in aged, immunocompromised, and debilitated patients . With the use of broad-spectrum antibiotics, C . freundii has become increasingly resistant to antimicrobial agents . In order to determine the chronological changes in susceptibility and current susceptibility status of C . freundii, we compared the antimicrobial susceptibility of C . freundii in two different time periods, from 1987 to 1988 and from 1997 to 1998 . In both time periods, 61 isolates of C . freundii were randomly selected for study from all clinical isolates at National Taiwan University Hospital . The minimum inhibitory concentrations and susceptible rates of 15 antimicrobial agents were compared, and it was found that most C . freundii isolates were resistant to anti-pseudomonal penicillins, first, second, and third generation cephalosporins, gentamicin, tobramycin, and aztreonam . The results indicate that the susceptible rates of C . freundii to aminoglycosides and ciprofloxacin decreased markedly during the period from 1987 to 1998 . Cefepime, cefpirome, imipenem, and meropenem remained the most active agents against C . freundii.

Enferm Infecc Microbiol Clin, 2001 Jan, 19(1), 11 - 4
{Characterization and distribution of Citrobacter species in a university hospital}; Manganello S et al.; OBJETIVE: a) To identify Citrobacter strains following the conventional biochemical reaction of Brenner and col; b) to evaluate the sensitivity and specificity of the O'Hara's method compared with Brenner's method, and c) to determine the rate and distribution of the strains in the clinical isolates . MATERIAL AND METHODS: One hundred and twenty two clinical isolates, characterized as Citrobacter spp . were collected between May of 1994 and August of 1997 . Clinical isolates included inpatients and outpatients from Hospital de Clinicas . Strains were identified following the methods of Brenner and O'Hara . RESULTS: Methods of Brenner identified 111 of 122 strains: C . freundii 59 of 111; C . koseri 18 of 111; C . werkmanii 15 of 111; C . braakii 9 of 111; C . youngae 6 of 111 and C . amalonaticus 4 of 111 . O'Hara's methods identified 104 of 111 strains (94%) . C . freundii was recovered most frequently from urine and feces (p Fisher < 0.026 and 0.039 respectively), while C . koseri was isolated from urine principally (p Fisher < 0.0372) . CONCLUSIONS: The genus Citrobacter is an important opportunistic pathogen that can be identified in clinical microbiology laboratories using O'Hara's method.

Jpn J Antibiot, 2000 Sep, 53(9), 593 - 608
{In vitro antibacterial activity of prulifloxacin, a new oral quinolone, and comparative susceptibility rate at clinical breakpoint MIC}; Inoue M et al.; In vitro drug sensitivity of clinically isolated bacteria against prulifloxacin (PUFX), which is a new quinolone, was investigated, and the antibacterial activity and susceptibility rate at clinical breakpoint were compared with those of norfloxacin, ofloxacin (OFLX), ciprofloxacin, tosufloxacin, fleroxacin, sparfloxacin and levofloxacin (LVFX) . The following results were obtained . 1) PUFX showed a broad-spectrum antibacterial activity against both Gram-positive and Gram-negative bacteria . 2) MIC80 of PUFX was 0.25 and 1 microgram/ml, against methicillin susceptible Staphylococcus aureus and Streptococcus pneumoniae, respectively and below 0.125 microgram/ml against Gram-negative Enterobacteriaceae . MIC90 of PUFX against Pseudomonas aeruginosa, which has MIC not exceeding 4 micrograms/ml to OFLX, was 0.5 microgram/ml . 3) PUFX was judged as active against the bacteria under the criteria proposed presented by "the Sensitivity Determination Committee for Antibiotics, Japan Society of Chemotherapy: Break Point for Respiratory Infectious Diseases and Sepsis" . It is suggested that the sensitivity of each bacterial species to PUFX was high . 4) From the correlation analysis of MIC, PUFX was shown to have two to eight times higher antibacterial acitivity than LVFX for Citrobacter freundii, Serratia marcescens and Pseudomonas aeruginosa . 5) PUFX showed potent short-time bactericidal activity against S . aureus and P . aeruginosa.

Zh Mikrobiol Epidemiol Immunobiol, 2000 Nov-Dec, (6), 15 - 8
{Diagnostic value of a novel nutrient medium for isolation and cultivation of pathogens causing enteric yersiniosis and pseudotuberculosis}; Saiapina LV et al.; A new nutrient medium for isolation and cultivation of the causative agents of enteric yersiniosis and pseudotuberculosis was found to have advantages over Endo medium in its differentiating and inhibiting properties . This medium permitted the easy differentiation of Yersinia pseudotuberculosis from Y . enterocolitica, as well as from Escherichia coli, Shigella flexneri, Klebsiella pneumoniae, K . rhinoscleromatis, Hafnia, Enterobacter and Citrobacter by color; from Proteus inconstans by swarming . In addition, weakly swarming of P . vulgaris differed by their light bluish color and Pseudomonas aeruginosa, by the brilliance and size of colonies . Endo medium could be used only for differentiation of E . coli from lactose-negative Yersinia colonies, Klebsiella (by mucous growth) and, to a certain extent, all Proteus species (by swarming) . The medium under test and the control medium inhibited the growth of Staphylococcus aureus . In contrast to Endo medium, the medium under test partially inhibited the growth of K . rhinoscleromatis and the swarming of P . inconstans . The new medium is now introduced into practice.

Am Surg, 2001 Jan, 67(1), 80 - 5
Splenic abscess: report of six cases and review of the literature; Green BT; Splenic abscesses are rare but appear to be increasing in frequency . Recent advances in radiologic techniques have affected the diagnosis and management . The purpose of this study was to evaluate these effects . The medical records of one institution were retrospectively reviewed and six cases of splenic abscesses seen between 1989 and 1999 were identified . All patients had predisposing conditions with metastatic hematogenous infection in three and one each with trauma, immunodeficiency, and a contiguous site of infection . Fever was present in all patients with chills and vomiting in five and three patients, respectively . Left upper quadrant tenderness appeared in four patients and leukocytosis was found in every patient except one with the acquired immunodeficiency syndrome . Chest roentgenograms were abnormal in five patients with a left pleural effusion most common . Ultrasound revealed the defect in both patients it was utilized in and computed tomography was diagnostic in all cases . The causative organisms were anaerobes in two cases and Candida albicans, Streptococcus viridans, Escherichia coli, and Citrobacter freundii each present in one case . Radiology guided percutaneous drainage was attempted in four patients but was only successful in one . Splenectomy with antibiotics was curative in the remainder with a 100 per cent survival . These data suggest that percutaneous drainage may be appropriate for certain patients initially, but the high failure rate demonstrates that splenectomy remains the standard treatment.

Curr Microbiol, 2001 Apr, 42(4), 290 - 4
Isolation and identification of bacteria associated with adult laboratory Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae); Kuzina LV et al.; From the guts of new and old colonies (female and male) of Mexican fruit flies, Anastrepha ludens (Diptera: Tephritidae), we identified a total of 18 different bacterial species belonging to the family Enterobacteriaceae, Pseudomonadaceae, Vibrionaceae, Micrococcaceae, Deinococcacea, Bacillaceae, and the genus Listeria . Enterobacter, Providencia, Serratia, and Staphylococcus spp . were the most frequently isolated genera, with Citrobacter, Streptococcus, Aerococcus, and Listeria found less frequently . We found Bacillus cereus, Enterobacter sakazakii, Providencia stuartii, and Pseudomonas aeruginosa only in the new colony, Aeromonas hydrophila and Klebsiella pneumoniae spp . pneumoniae only in the old colony . We also studied resistance/sensitivity to 12 antibiotics for six bacterial isolates such as Enterobacter cloacae, E . sakazakii, K . pneumoniae spp., Providencia rettgeri, P . aeruginosa, and Bacillus cereus . Isolates on the whole were resistant to penicillin and ampicillin (five of six isolates) and sensitive to rifampin and streptomycin (six of six isolates) . Antibiotic resistance profiles might be useful characteristics for distinguishing among species and strains of these bacteria, probably having ecological significance with respect to intra- and inter-specific competition within host cadavers, and could have implications for the utility of these organisms for biological control, including the alternative control strategy, paratransgenesis.

Microb Pathog, 2001 Jan, 30(1), 19 - 28
Bacterial invasion and transcytosis in transfected human brain microvascular endothelial cells; Stins MF et al.; Most cases of neonatal bacterial meningitis develop as a result of a hematogenous spread, but it is not clear how circulating bacteria cross the blood-brain barrier . Attempts to answer these questions have been hampered by the lack of a reliable model of the human blood-brain barrier . Human brain microvascular endothelial cells (HBMEC) were isolated and transfected with a pBR322 based plasmid containing simian virus 40 large T antigen (SV40-LT) . The transfected HBMEC exhibited similar brain endothelial cell characteristics as the primary HBMEC, i.e . gamma glutamyl transpeptidase and a high transendothelial electrical resistance . Escherischia coli and Citrobacter spp, two important Gram-negative bacilli causing neonatal meningitis, were found to transcytose across primary and transfected HBMEC, without affecting the integrity of the monolayer . In addition, E . coli and C . freundii invaded transfected HBMEC as shown previously with primary HBMEC . We conclude that E . coli and C . freundii are able to invade and transcytose HBMEC and these bacterial-HBMEC interactions are similar between primary and transfected HBMEC . Therefore, our transfected HBMEC should be useful for studying pathogenesis of CNS infections .

Can J Microbiol, 1993 Sep, 39(9), 821 - 5
Use of 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside for the isolation of beta-galactosidase-positive bacteria from municipal water supplies; Ley A et al.; A new medium, mX-Gal, has been developed for the membrane filter enumeration of beta-galactosidase-positive bacteria in municipal water supplies . mX-Gal medium contains the chromogenic beta-galactosidase substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) . All Aeromonas, Citrobacter, and Enterobacter strains isolated from raw water on mX-Gal medium were beta-galactosidase positive . In contrast, only 10 to 20% of these strains produced a red colony with a metallic sheen on m-Endo agar LES medium . Of 674 chlorinated water samples analyzed for total coliforms on m-Endo agar LES medium and for beta-galactosidase-positive bacteria on mX-Gal medium, 18 that were negative for coliforms on m-Endo agar LES showed beta-galactosidase-positive bacteria on mX-Gal . Of a total of 50 beta-galactosidase-positive bacteria isolated from these samples, 76% were identified as Aeromonas hydrophila.

Urology . 1999 Dec;54(6):1097.
Citrobacter diversus urosepsis and cerebral abscess in a child with antenatal hydronephrosis; Ferrer FA et al.; One percent of all pregnancies are found to have an antenatal abnormality; of these, 20% involve the genitourinary system . Today, controversy still exists regarding the postnatal management of some antenatal abnormalities detected by ultrasound . We present a case in which antenatal hydronephrosis initially detected by ultrasound appeared to resolve in utero . Postnatally, the child developed Citrobacter diversus urosepsis, meningitis, and cerebral abscess . Voiding cystourethrogram obtained after resolution of sepsis revealed grade IV reflux . This case underscores the importance of a full postnatal evaluation for all children with antenatal hydronephrosis and alerts clinicians to a virulent pathogen not commonly associated with urinary tract infection.

Am J Med Sci, 2000 Dec, 320(6), 408 - 10
Citrobacter diversus endocarditis; Tellez I et al.; Citrobacter species are motile Gram-negative bacilli that cause disease in humans, such as urinary tract infection, pneumonia, superficial and deep wound infections, gastroenteritis, meningitis, bacteremia, and rarely endocarditis . In those cases of endocarditis, intravenous drug use has been associated with Citrobacter species . Gram-negative organisms are present in less than 10% of cases of endocarditis in intravenous drug users . We present a case of tricuspid valve endocarditis in an intravenous drug user caused by Citrobacter diversus alone.

Nucleic Acids Res, 2001 Jan 15, 29(2), 380 - 6
Conserved regulatory elements of the promoter sequence of the gene rpoH of enteric bacteria; Ramirez-Santos J et al.; The rpoH regulatory region of different members of the enteric bacteria family was sequenced or downloaded from GenBank and compared . In addition, the transcriptional start sites of rpoH of Yersinia frederiksenii and Proteus mirabilis, two distant members of this family, were determined . Sequences similar to the sigma(70) promoters P1, P4 and P5, to the sigma(E) promoter P3 and to boxes DnaA1, DnaA2, cAMP receptor protein (CRP) boxes CRP1, CRP2 and box CytR present in Escherichia coli K12, were identified in sequences of closely related bacteria such as: E.coli, Shigella flexneri, Salmonella enterica serovar Typhimurium, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae . In more distant bacteria, Y.frederiksenii and P.mirabilis, the rpoH regulatory region has a distal P1-like sigma(70) promoter and two proximal promoters: a heat-induced sigma(E)-like promoter and a sigma(70) promoter . Sequences similar to the regulatory boxes were not identified in these bacteria . This study suggests that the general pattern of transcription of the rpoH gene in enteric bacteria includes a distal sigma(70) promoter, >200 nt upstream of the initiation codon, and two proximal promoters: a heat-induced sigma(E)-like promoter and a sigma(70) promoter . A second proximal sigma(70) promoter under catabolite-regulation is probably present only in bacteria closely related to E.coli.

JAMA, 2000 Dec 27, 284(24), 3151 - 6
Emergence of domestically acquired ceftriaxone-resistant Salmonella infections associated with AmpC beta-lactamase; Dunne EF et al.; CONTEXT: Ceftriaxone, an expanded-spectrum cephalosporin, is an antimicrobial agent commonly used to treat severe Salmonella infections, especially in children . Ceftriaxone-resistant Salmonella infections have recently been reported in the United States, but the extent of the problem is unknown . OBJECTIVES: To summarize national surveillance data for ceftriaxone-resistant Salmonella infections in the United States and to describe mechanisms of resistance . DESIGN AND SETTING: Case series and laboratory evaluation of human isolates submitted to the Centers for Disease Control and Prevention from 17 state and community health departments participating in the National Antimicrobial Resistance Monitoring System (NARMS) for enteric bacteria between 1996 and 1998 . PATIENTS: Patients with ceftriaxone-resistant Salmonella infections between 1996 and 1998 were interviewed and isolates with decreased ceftriaxone susceptibility were further characterized . MAIN OUTCOME MEASURES: Exposures and illness outcomes, mechanisms of resistance . RESULTS: The prevalence of ceftriaxone-resistant Salmonella was 0.1% (1 of 1326) in 1996, 0.4% (5 of 1301) in 1997, and 0.5% (7 of 1466) in 1998 . Ten (77%) of the 13 patients with ceftriaxone-resistant infections were aged 18 years or younger . The patients lived in 8 states (California, Colorado, Kansas, Massachusetts, Maryland, Minnesota, New York, and Oregon) . Nine (82%) of 11 patients interviewed did not take antimicrobial agents and 10 (91%) did not travel outside the United States before illness onset . Twelve of the 15 Salmonella isolates with ceftriaxone minimum inhibitory concentrations of 16 microg/mL or higher were serotype Typhimurium but these isolates had different pulsed-field gel electrophoresis patterns . Thirteen of these 15 isolates collected between 1996 and 1998 were positive for a 631-base pair polymerase chain reaction product obtained by using primers specific for the ampC gene of Citrobacter freundii . CONCLUSIONS: Domestically acquired ceftriaxone-resistant Salmonella has emerged in the United States . Most ceftriaxone-resistant Salmonella isolates had similar AmpC plasmid-mediated resistance.

Int Microbiol, 1999 Sep, 2(3), 161 - 7
The Yersinia high-pathogenicity island; Carniel E; A pathogenicity island present only in highly pathogenic strains of Yersinia (Y . enterocolitica 1B, Y . pseudotuberculosis I and Y . pestis) has been identified on the chromosome of Yersinia spp . and has been designated High-Pathogenicity Island (HPI) . The Yersinia HPI carries a cluster of genes involved in the biosynthesis, transport and regulation of the siderophore yersiniabactin . The major function of this island is thus to acquire iron molecules essential for in vivo bacterial growth and dissemination . The presence of an integrase gene and att sites homologous to those of phage P4, together with a G + C content much higher than the chromosomal background, suggests that the HPI is of foreign origin and has been acquired by chromosomal integration of a phage . The HPI can excise from the chromosome of Y . pseudotuberculosis and is found inserted into any of the three copies of the asn tRNA loci present in this species . A unique characteristic of the HPI is its wide distribution in various enterobacteria . Although first identified in Yersinia spp., it has subsequently been detected in other genera such as E . coli, Klebsiella and Citrobacter.

J Clin Microbiol, 2000 Dec, 38(12), 4586 - 92
Cost-effective and rapid presumptive identification of gram-negative bacilli in routine urine, pus, and stool cultures: evaluation of the use of CHROMagar orientation medium in conjunction with simple biochemical tests; Ohkusu K; The algorithm for a new identification system was designed on the basis of colony color and morphology on CHROMagar Orientation medium in conjunction with simple biochemical tests such as indole (IND), lysine decarboxylase (LDC), and ornithine decarboxylase (ODC) utilization tests with gram-negative bacilli isolated from urine samples as well as pus, stool, and other clinical specimens by the following colony characteristics, biochemical reactions, and serological results: pinkish to red, IND positive (IND(+)), Escherichia coli; metallic blue, IND(+), LDC(+), and ODC negative (ODC(-)), Klebsiella oxytoca; IND(+), LDC(-), and ODC(+), Citrobacter diversus; IND(+) or IND(-), LDC(-), and ODC(-), Citrobacter freundii; IND(-), LDC(+), and ODC(+), Enterobacter aerogenes; IND(-), LDC(-), and ODC(+), Enterobacter cloacae; IND(-), LDC(+), and ODC(-), Klebsiella pneumoniae; diffuse brown and IND(+), Morganella morganii; IND(-), Proteus mirabilis; aqua blue, Serratia marcescens; bluish green and IND(+), Proteus vulgaris; transparent yellow-green, serology positive, Pseudomonas aeruginosa; clear and serology positive, Salmonella sp.; other colors and reactions, the organism was identified by the full identification methods . The accuracy and cost-effectiveness of this new system were prospectively evaluated . During an 8-month period, a total of 345 specimens yielded one or more gram-negative bacilli . A total of 472 gram-negative bacillus isolates were detected on CHROMagar Orientation medium . For 466 of the isolates (98.7%), no discrepancies in the results were obtained on the basis of the identification algorithm . The cost of identification of gram-negative bacilli during this period was reduced by about 70% . The results of this trial for the differentiation of the most commonly encountered gram-negative pathogens in clinical specimens with the new algorithm were favourable in that it permitted reliable detection and presumptive identification . In addition, this rapid identification system not only significantly reduced costs but it also improved the daily work flow within the clinical microbiology laboratory.

J Clin Microbiol, 2000 Dec, 38(12), 4343 - 50
Citrobacter rodentium, the causative agent of transmissible murine colonic hyperplasia, exhibits clonality: synonymy of C . rodentium and mouse-pathogenic Escherichia coli; Luperchio SA et al.; Citrobacter rodentium (formerly Citrobacter freundii biotype 4280 and Citrobacter genomospecies 9) was described on the basis of biochemical characterization and DNA-DNA hybridization data and is the only Citrobacter species known to possess virulence factors homologous to those of the human pathogens enteropathogenic Escherichia coli and enterohemorrhagic E . coli . These virulence factors are encoded on the locus of enterocyte effacement (LEE), a pathogenicity island required for the characteristic attaching and effacing (AE) pathology seen in infection with these three pathogens . C . rodentium, which apparently infects only mice, provides a useful animal model for studying the molecular basis of AE pathology . No work has been done to assess differences in pathogenicity between C . rodentium isolates from diverse sources . Here, we report the examination of 15 C . rodentium isolates using a battery of genetic and biochemical approaches . No differences were observed between the isolates by repetitive-element sequence-based PCR analysis, biochemical analysis, and possession of LEE-specific virulence factors . These data suggest that members of the species are clonal . We further characterized an atypical E . coli strain from Japan called mouse-pathogenic E . coli (MPEC) that, in our hands, caused the same disease as C . rodentium . Applying the same battery of tests, we found that MPEC possesses LEE-encoded virulence factors and is indistinguishable from the previously characterized C . rodentium isolate DBS100 . These results demonstrate that MPEC is a misclassified C . rodentium isolate and that members of this species are clonal and represent the only known attaching and effacing bacterial pathogen of mice.

Antimicrob Agents Chemother, 2000 Dec, 44(12), 3478 - 80
Levofloxacin pharmacokinetics and serum bactericidal activities against five enterobacterial species; Geerdes-Fenge HF et al.; After oral administration of 500 mg of levofloxacin to 12 volunteers, we investigated the pharmacokinetics and serum bactericidal activities (SBAs) against five strains of members of the family Enterobacteriaceae . Pharmacokinetic data were as follows: maximum concentration in serum, 6.36 +/- 0.57 mg/liter; area under the concentration-time curve, 43.6 +/- 6.23 mg . h/liter; elimination half-life 4.23 +/- 0.87 h . SBAs were present for 24 h against Escherichia coli and Citrobacter freundii . The SBAs at 1, 12, and 24 h after administration against E . coli were 1:108, 1:29, and 1:7, respectively, and those against Citrobacter freundii were 1:74, 1:25, and 1:7, respectively . The SBAs were present for 12 h against the other three organisms tested . The SBAs against Serratia marcescens were 1:28 and 1:9 at 1 and 12 h, respectively; the SBAs against Klebsiella pneumoniae were 1:25 and 1:7 at 1 and 12 h, respectively; and the SBAs against Enterobacter cloacae were 1:24 and 1:10 at 1 and 12 h, respectively.

Expert Opin Investig Drugs, 2000 Aug, 9(8), 1877 - 95
Gatifloxacin: a new fluoroquinolone; Blondeau JM; Gatifloxacin is a new 8-methoxy-fluoroquinoline antimicrobial agent . It has enhanced activity against Gram-positive and atypical agents, while retaining broad-spectrum antiGram-negative activity . For example, the MIC(90) values for respiratory tract pathogens are < or = 0.5 microg/ml for organisms such as Streptococcus pneumoniae (regardless of penicillin susceptibility), Haemophilus influenzae (beta-lactamase positive or negative), Moraxella catarrhalis (beta-lactamase positive or negative), Legionella species, Mycoplasma pneumoniae, methicillin-sensitive Staphylococcus aureus, beta-haemolytic Streptococci (macrolide sensitive or resistant), Neisseria species, most Enterobacteriaceae, Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella species, Vibrio species and Yersinia enterocolitica . For methicillin-resistant S . aureus, ciprofloxacin-resistant S . aureus, Citrobacter freundii, Providencia species, Serratia species, Pseudomonas aeruginosa and other non-fermentative Gram-negative bacilli, the MIC(90) are elevated . Gatifloxacin is bactericidal and exhibits a post-antibiotic effect against Gram-positive and -negative bacteria . The standard dose is 400 mg once daily and is available in both oral and iv . formulation . Gatifloxacin appears to have a low propensity for the selection of resistant mutants . Clinical trial data supports the use of gatifloxacin for treatment of patients with respiratory tract, urinary tract, skin and soft tissue infections . The side effect profile for gatifloxacin is similar to that with other agents.

J Clin Microbiol, 2000 Nov, 38(11), 3946 - 52
Outbreak of nosocomial infections due to extended-spectrum beta-lactamase-producing strains of enteric group 137, a new member of the family Enterobacteriaceae closely related to Citrobacter farmeri and Citrobacter amalonaticus; Warren JR et al.; A member of the Enterobacteriaceae initially identified as Kluyvera cryocrescens by the MicroScan Gram-Negative Combo 13 panel caused an outbreak of nosocomial infections in four patients (pneumonia, n = 2; urinary tract infection, n = 1; wound infection, n = 1) and urinary tract colonization in one patient . When the strains were tested by the Enteric Reference Laboratory of the Centers for Disease Control and Prevention, biochemical results were most compatible with Yersinia intermedia, Kluyvera cryocrescens, and Citrobacter farmeri but identification scores were low and test results were discrepant . However, when the biochemical test profile was placed in the computer database as a new organism, all strains were identified as the organism with high identification scores (0 . 999968 to 0.999997) and no discrepant test results . By 16S rRNA sequence analysis the organism clustered most closely with, but was distinct from, Citrobacter farmeri and Citrobacter amalonaticus . Based on its unique biochemical profile and rRNA sequence, this organism is designated Enteric Group 137 . Restriction endonuclease analysis and taxonomic antibiograms of strains causing the outbreak demonstrated a single clone of Enteric Group 137, and antibiotic susceptibility testing revealed the presence of extended-spectrum beta-lactamase (ESBL) resistance . Enteric Group 137 appears to be a new opportunistic pathogen that can serve as a source of ESBL resistance in the hospital.

J Endotoxin Res, 2000, 6(3), 205 - 14
Enteric bacteria, lipopolysaccharides and related cytokines in inflammatory bowel disease: biological and clinical significance; Caradonna L et al.; Ulcerative colitis (UC) and Crohn's disease (CD) {inflammatory bowel disease (IBD)} are both characterized by an exaggerated immune response at the gut associated lymphoreticular tissue level . Such an abnormal and dysregulated immune response may be directed against luminal and/or enteric bacterial antigens, as also supported by murine models of inflammatory bowel disease (IBD) caused by organisms such as Citrobacter rodentium and Helicobacter hepaticus . Bacterial endotoxins or lipopolysaccharides (LPS) have been detected in the plasma of IBD patients and an abnormal microflora and/or an increased permeability of the intestinal mucosa have been invoked as cofactors responsible for endotoxemia . At the same time, the evidence that phagocytosis and killing exerted by polymorphonuclear cells and monocytes and the T-cell dependent antibacterial activity are decreased in IBD patients may also explain the origin of LPS in these diseases . In IBD, pro-inflammatory cytokines and chemokines have been detected in elevated amounts in mucosal tissue and/or in peripheral blood, thus suggesting a monocyte/macrophage stimulation by enteric bacteria and/or their constituents (e.g . LPS) . On these grounds, in experimental models and in human IBD, anti-cytokine monoclonal antibodies and interleukin receptor antagonists are under investigation for their capacity to neutralize the noxious effects of immune mediators . Finally, the administration of lactobacilli is beneficial in human IBD and, in murine colitis, this treatment leads to a normalization of intestinal flora, reducing the number of colonic mucosal adherent and translocated bacteria.

FEMS Microbiol Ecol, 2000 Oct 1, 34(1), 81 - 90
Detection and distribution of insertion sequence 1 (IS1)-containing bacteria in the freshwater environment(1); Rhodes G et al.; The distribution of insertion sequence 1 (IS1)-containing bacteria was investigated in Windermere (Cumbria, UK), a freshwater body impacted by treated sewage discharge and run-off from the surrounding catchment . Culturable IS1-containing bacteria were recovered from the water column at three depths in Windermere North Basin (WNB) and South Basin (WSB), and from sediment at both sites (at the sediment surface in WSB and to a depth of 12-13 cm in WNB) . Polymerase chain reaction amplification of IS1 and the Escherichia coli/Shigella sp . specific gene uidA, from community DNA from shallow sediments, extended the detection limit beyond that of culture at both sites . This detection was extended further into deep sediment extracted from WNB as IS1 and uidA were detected in sub-samples to a depth of 4.7 and 2.3 m, respectively . Analysis of a representative subset of 90 IS1-carrying isolates recovered from water and sediment at both sites demonstrated 21 heterogeneous IS1 profiles with estimated copy numbers ranging from 1 to 16 . Identification of the host bacteria showed that the element was confined mainly to Enterobacter spp . However, this study showed IS1 to be present in Citrobacter freundii for the first time . Plasmids were carried by 75.3% of enterobacterial isolates and four plasmids (2.6%) carried IS1 . DNA sequence analysis of five IS1 clones demonstrated that IS1 isoforms from this study were similar (>89% nucleotide identity) to known IS1 isoforms . Two isoforms of IS1 from a single Enterobacter cloacae isolate differed by 6.7% at the nucleotide level suggesting that they had been acquired independently.

Antimicrob Agents Chemother, 2000 Nov, 44(11), 3137 - 43
Biological cost of AmpC production for Salmonella enterica serotype Typhimurium; Morosini MI et al.; Chromosomally mediated AmpC-type beta-lactamases are frequently found among Enterobacteriaceae . Hyperproduction of AmpC beta-lactamase results in high-level resistance to beta-lactam antibiotics . One striking feature of Salmonella is the absence of the structural ampC gene, encoding AmpC beta-lactamase, in contrast with other members in the Enterobacteriaceae family, such as Escherichia, Citrobacter, or Enterobacter . The horizontal acquisition of ampC genes is one of the causes of the increased resistance to extended-spectrum cephalosporins and beta-lactamase inhibitors among gram-negative rods . Nevertheless, despite the high number of beta-lactam-resistant Salmonella isolates so far described, only two strains expressing resistance to cephalosporin and beta-lactamase inhibitors which is mediated by AmpC-type enzymes have been found . In this work, data are provided which support the possibility that the maintenance and expression of the ampC gene may represent an unbearable cost for Salmonella in terms of reduction of some of its lifestyle attributes, such as growth rate and invasiveness . The deleterious AmpC burden can be eliminated by decreasing the production of AmpC when both the regulatory gene, ampR, and ampC are present in Salmonella . Thus, it is suggested that the two genes have to be acquired together by Salmonella, leading to an inducible beta-lactam resistance phenotype . AmpC synthesis did not produce major variations in the peptidoglycan composition of Salmonella.

Microbes Infect, 2000 Aug, 2(10), 1237 - 44
Bacterial penetration across the blood-brain barrier during the development of neonatal meningitis; Huang SH et al.; Bacterial pathogens may breach the blood-brain barrier (BBB) and invade the central nervous system through paracellular and/or transcellular mechanisms . Transcellular penetration, e.g., transcytosis across the BBB has been demonstrated for Escherichia coli K1, group B streptococcus, Listeria monocytogenes, Citrobacter freundii and Streptococcus pneumonia strains . Genes contributing to invasion of brain microvascular endothelial cells include E . coli K1 genes ompA, ibeA, ibeB, and yijP . Understanding the mechanisms of bacterial penetration across the BBB may help develop novel approaches to preventing bacterial meningitis.

Nucleic Acids Res, 2000 Oct 1, 28(19), 3817 - 22
DNA methylation at the CfrBI site is involved in expression control in the CfrBI restriction-modification system; Beletskaya IV et al.; We have previously found that genes of the CFR:BI restriction-modification (R-M) system from Citrobacter freundii are oriented divergently and that their promoter regions overlap . The overlapping promoters suggest regulation of gene expression at the transcriptional level . In this study the transcription regulation of CFR:BI R-M genes was analyzed in vivo and in vitro in Escherichia coli . It was shown that in the presence of CFR:BI methyltransferase (M.CFR:BI), cell galactokinase activity decreases 10-fold when the galactokinase gene (galK) is under the control of the cfrBIM promoter and increases 20-fold when galK is under the control of the cfrBIR promoter . The CFR:BI site, proven to be unique for the entire CFR:BI R-M gene sequence, is located in the -35 cfrBIM promoter region and is in close vicinity of the -10 cfrBIR promoter region . A comparison of the cfrBIM and the cfrBIR promoter activities in the in vitro transcription system using methylated and unmethylated DNA fragments as templates demonstrated that the efficiency of CFR:BI R-M gene transcription is regulated by enzymatic modification at the N-4-position of cytosine bases of the CFR:BI site by M.CFR:BI . From the results of the in vivo and in vitro experiments we suggest a new model of gene expression regulation in type II R-M systems.

J Food Prot, 2000 Sep, 63(9), 1273 - 6
Microbial evaluation of Spanish potato omelette and cooked meat samples in University restaurants; Soriano JM et al.; The focus of this study was to evaluate the microbial quality of Spanish potato omelette and cooked meat samples including pork loin, chicken croquettes, long pork sausage, chicken breast, and meatballs from University restaurants . Microbiological analyses of Spanish potato omelette and cooked meat samples resulted in aerobic plate counts from <1.00 to 2.90 and from <1.00 to 6.04 log10 CFU g(-1), respectively . Total coliforms ranged from <3 to 43 most probable number (MPN) g(-1) and from <3 to >2,400 MPN g(-1) for Spanish potato omelette and meat products, respectively . Escherichia coli, coagulase-positive staphylococci, and Lancefield group-D streptococci were detected in 1.7%, 3.5%, and 12.9% of Spanish potato omelette samples, respectively . For cooked meat samples, 8.8%, 7.6%, and 24.6% contained E . coli, coagulase-positive staphylococci, and Lancefield group-D streptococci, respectively . E . coli O157:H7, Salmonella spp., and Shigella spp . were not detected . Klebsiella pneumoniae, Klebsiella oxytoca, Citrobacter freundii, Enterobacter cloacae, and Serratia spp . were isolated from Spanish potato omelette samples . For cooked meat samples, C . freundii, E . cloacae, and Aeromonas hydrophila were detected . The results suggest that some handling practices should require more attention, and as a consequence, a hazard analysis and critical control point program should be developed and implemented.

FEMS Microbiol Lett, 2000 Sep 1, 190(1), 157 - 61
Serological cross-reaction between the lipopolysaccharide O-polysaccharaide antigens of Escherichia coli O157:H7 and strains of Citrobcter freundii and Citrobacter sedlakii; Vinogradov E et al.; A strain of Citrobacter sedlakii showing serological cross-reaction with Escherichia coli O157 antisera was demonstrated to produce a lipopolysaccharide O-antigen having an identical structure with that of the E . coli O157 O-antigen . A strain of Citrobacter freunndii showing similar cross-reaction with E . coli O157 specific monoclonal antibody was shown to produce a lipopolysaccharide O-antigen composed of a trisaccharide repeating unit having the structure { 2)-alpha-D Rhap-(1-3)-beta-D-Rhap-(1-4)-beta-D-Glcp-(1-} . This O-antigen differs from that of the E . coli O157 O-antigen and also lacks a component 2-substituted 4-amino-4,6-dideoxy-alpha-D-mannopyranosyl residue implicated as the common epitope in the lipopolysaccharide O-antigens of previously investigated bacterial species showing serological cross-reactivity with E . coli O157 antisera . The C freundii O-antigen presents an interesting example of structural mimicry within a bacterial polysaccharide antigen.

J Infect Dis, 2000 Oct, 182(4), 1268 - 71 Epub 2000 Sep 05.
High-pathogenicity island of Yersinia pestis in enterobacteriaceae isolated from blood cultures and urine samples: prevalence and functional expression; Schubert S et al.; Production of the siderophore yersiniabactin is associated with virulence in Yersinia species . The genes for biosynthesis and uptake of yersiniabactin are located on a high-pathogenicity island (HPI) . The distribution and functioning of the Yersinia HPI were assessed in different Enterobacteriaceae strains isolated from blood cultures and urine samples . In total, 550 clinical isolates from 10 Enterobacteriaceae species were investigated by polymerase chain reaction and DNA hybridization . The Yersinia HPI was most prevalent in Escherichia coli (overall prevalence, 72.3%) and, to a lesser extent, in Klebsiella oxytoca (58.3%), Citrobacter species (25%), Klebsiella pneumonia (17.7%), and Enterobacter species (12.2%) . The production of the siderophore yersiniabactin was also demonstrated in these HPI-positive strains by use of a reporter gene bioassay . These findings indicate that the HPI of Yersinia is distributed and functions in clinical specimens of different Enterobacteriaceae species.

Arq Neuropsiquiatr, 2000 Sep, 58(3A), 736 - 40
Brain abscess by citrobacter diversus in infancy: case report; Feferbaum R et al.; Citrobacter diversus is closely related to brain abscess in newborn infants . We describe a case of brain abscess by this bacteria in a newborn infant and his clinical and cranial computed tomographic evaluation until the fourth month of life and discuss therapeutic management of this patient.

Lett Appl Microbiol, 2000 Aug, 31(2), 169 - 73
The capacity of Enterobacteriaceae species to produce biogenic amines in cheese; Marino M et al.; The amino acid decarboxylating activity and production of biogenic amines by 104 cheese-associated Enterobacteriaceae species (58 Enterobacter, 18 Serratia, eight Escherichia, seven Hafnia, six Arizona, four Citrobacter and three Klebsiella) were investigated . All strains could decarboxylate at least two amino acids in Moller's broth and in Niven's medium, and the decarboxylase activity was strain specific . In a laboratory medium containing all free amino acids, all strains could produce more than 100 ppm cadaverine, putrescine was produced by 96% of strains . Tyramine and histamine were produced in the lowest concentrations . A positive correlation existed between cadaverine concentration and Enterobacteriaceae counts in cheese, that may have caused the increase in decarboxylase content . This study suggests that it is possible to limit the presence of cadaverine in cheese, thereby controlling the Enterobacteriaceae counts, a sign of contamination during cheese making and/or storage.

Appl Environ Microbiol, 2000 Sep, 66(9), 4131 - 5
Rapid 5' nuclease (TaqMan) assay for detection of virulent strains of Yersinia enterocolitica; Vishnubhatla A et al.; We have developed a rapid procedure for the detection of virulent Yersinia enterocolitica in ground pork by combining a previously described PCR with fluorescent dye technologies . The detection method, known as the fluorogenic 5' nuclease assay (TaqMan), produces results by measuring the fluorescence produced during PCR amplification, requiring no post-PCR processing . The specificity of the chromosomal yst gene-based assay was tested with 28 bacterial isolates that included 7 pathogenic and 7 nonpathogenic serotypes of Y . enterocolitica, other species of Yersinia (Y . aldovae, Y . pseudotuberculosis, Y . mollaretti, Y . intermedia, Y . bercovieri, Y . ruckeri, Y . frederiksenii, and Y . kristensenii), and other enteric bacteria (Escherichia, Salmonella, Citrobacter, and Flavobacterium) . The assay was 100% specific in identifying the pathogenic strains of Y . enterocolitica . The sensitivity of the assay was found to be >/=10(2) CFU/ml in pure cultures and >/=10(3) CFU/g in spiked ground pork samples . Results of the assay with food enrichments prespiked with Y . enterocolitica serotypes O:3 and O:9 were comparable to standard culture results . Of the 100 field samples (ground pork) tested, 35 were positive for virulent Y . enterocolitica with both 5' nuclease assay and conventional virulence tests . After overnight enrichment the entire assay, including DNA extraction, amplification, and detection, could be completed within 5 h.

Infection, 2000 Jul-Aug, 28(4), 243 - 5
CSF interleukin-6 in neonatal Citrobacter ventriculitis after meningitis; Baumeister FA et al.; An infant with neonatal severe Citrobacter koseri (formerly Citrobacter diversus) meningoencephalitis developed necrosis with multicystic regression of both hemispheres . The ventriculitis persisted over months in spite of antibiotic therapy.The treatment succeeded with cefotaxime in a high dose (300 mg/kg/day) without surgical intervention.The infant had been previously treated with cefotaxime (200 mg/kg/day) over 5 weeks . High levels of CSF interleukin-6 (IL-6) permitted to attribute persisting CSF pleocytosis in spite of sterile CSF cultures to chronic infection and not to reminiscence of brain necrosis . This report reveals two main points . On the one hand, the importance of therapy monitoring with IL-6 in CSF for the consequent treatment of Citrobacter meningitis and on the other hand, high-dose cefotaxime (300 mg/kg/day) treatment of Citrobacter ventriculitis, which succeeded without surgical intervention.

Pathol Biol (Paris), 2000 Jun, 48(5), 485 - 9
{Antibiotic sensitivity of enterobacteria in intensive care units}; Pina P et al.; Antibiotic therapy of intensive care patients is usually undocumented . The treatment is chosen according to epidemiologic and susceptibility data from microbiological laboratories . The aim of our study is to determine antibiotic susceptibility of enterobacteria isolated from intensive care patients during a five-month multicenter study in 18 French hospitals . Numerous (n = 1,113) strains were studied: 447 enterobacteria isolated from urine (n = 229), blood cultures (n = 106), respiratory tract specimens (n = 72), peritoneal fluids (n = 22), pus (n = 15) and catheters (n = 2) . MICs of group 2 and group 3 enterobacteria were determined using the dilution agar method and were interpreted according to the CASFM (Comite de l'antibiogramme de la societe francaise de microbiology) recommendations . Group 1 enterobacteria were most frequently isolated (67%) . Only one Escherichia coli strain produced ESBL (0.3%) . Among group 2 enterobacteria, one Citrobacter koseri strain produced ESBL . We did not isolate Klebsiella pneumoniae ESBL . Isolation of group 3 enterobacteria was frequent (24%) . Thirty-five percent of group 3 enterobacteria were resistant to cefotaxime, 26% to ceftazidime and 16% to cefepime and cefpirome . Fourteen strains of this group produced ESBL: 13 Enterobacter aerogenes and one E . amnigenus.

Infect Immun, 2000 Sep, 68(9), 5120 - 5
Enhancement of neonatal innate defense: effects of adding an N-terminal recombinant fragment of bactericidal/permeability-increasing protein on growth and tumor necrosis factor-inducing activity of gram-negative bacteria tested in neonatal cord blood ex vivo; Levy O et al.; Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides . Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria . We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O . Levy et al., Pediatrics 104:1327-1333, 1999) . The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense . Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI(21)) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates . Bacteria were tested in citrated newborn cord blood or adult peripheral blood . Bacterial viability was assessed by plating assay, and TNF-alpha release was measured by enzyme-linked immunosorbent assay . Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri . Bacteria varied in their susceptibility to rBPI(21)'s bactericidal action: E . coli K1/r was relatively susceptible (50% inhibitory concentration {IC(50)}, approximately 10 nM), C . koseri was intermediate (IC(50), approximately 1,000 nM), Klebsiella pneumoniae was resistant (IC(50), approximately 10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC(50), >10,000 nM) . All isolates were potent inducers of TNF-alpha activity in both adult and newborn cord blood . In contrast to its variable antibacterial activity, rBPI(21) consistently inhibited the TNF-inducing activity of all strains tested (IC(50), 1 to 1,000 nM) . The antibacterial effects of rBPI(21) were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin) . Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI(21) either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine . Thus, supplementing newborn cord blood with rBPI(21) potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity . These results suggest that administration of rBPI(21) may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.

Enzyme Microb Technol, 2000 Sep 1, 27(6), 399 - 405
Purification and characterization of the 1-3-propanediol dehydrogenase of Clostridium butyricum E5; Malaoui H et al.; 1-3 PPD dehydrogenase (EC 1.1.1.202) was purified to homogeneity from Clostridium butyricum E5 grown anaerobically on glycerol in continuous culture . The native enzyme was estimated by gel filtration to have a molecular weight of 384 200 +/- 31 100 Da; it is predicted to exist as an octamer or a decamer of identical molecular weight subunits . When tested as a dehydrogenase, the enzyme was most active with 1-3 propane diol . In the physiological direction, 3-hydroxypropionaldehyde was the preferred substrate . The apparent K(m) values of the enzyme for 3-hydroxypropionaldehyde and NADH were 0.17 mM and 0.06 mM, respectively . The enzyme requires only Mn(2+) for full activity . The enzyme was found to have properties similar to those reported for Klebsellia pneumoniae, Citrobacter freundii, and Clostridium pasteurianum.

J Mol Microbiol Biotechnol, 2000 Jul, 2(3), 277 - 81
Prediction of two- and three-amino-acid sequences of Citrobacter Freundii beta-lactamase from its amino acid composition; Wu G et al.; The repeated amino-acid sequences in Citrobacter Freundii beta-lactamase may be indispensable for its function, because such repetitions cannot be simply attributed to a chance . In order to fully explore the functional units in Citrobacter Freundii beta-lactamase, it may need to analyse all the amino acid pairs, triplets, etc . along Citrobacter Freundii beta-lactamase from one terminal to the other terminal, to count their frequencies and calculate their probabilities . The amino-acid sequence of Citrobacter Freundii beta-lactamase was counted according to two-, three- and four-amino-acid sequences . The counted frequency and probability were compared with the predicted frequency and probability . The amino acid sequences, which appear in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately evolved and conserved . By contrast, the amino acid sequences, which appear in Citrobacter Freundii beta-lactamase but cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately evolved and conversed . Accordingly 99 (26.053%) and 33 (8.684%) of 380 two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism . Some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and can be predicted from its amino acid composition according to a purely random mechanism, should not be deliberately excluded from Citrobacter Freundii beta-lactamase . By contrast, some kinds of amino acid sequences, which absent in Citrobacter Freundii beta-lactamase and cannot be predicted from its amino acid composition according to a purely random mechanism, should be deliberately excluded from Citrobacter Freundii beta-lactamase . Accordingly 89 (48.370%) and 41 (22.283%) of 184 kinds of absent two-amino-acid sequences can be predicted by the frequency and probability according to a purely random mechanism, and 7236 (99.848%) of 7247 kinds of absent three-amino-acid sequences can be predicted by the frequency according to a purely random mechanism . The amino acids, whose probabilities in following certain preceding amino acids can be predicted from Citrobacter Freundii beta-lactamase amino acid composition according to a purely random mechanism, should not be deliberately evolved and conversed, accordingly 2 (0.526%) of 380 counted first order Markov transition probabilities for the second amino acid in two-amino-acid sequences match the predicted conditional probabilities.

Microbiology, 2000 Aug, 146 ( Pt 8), 1855 - 67
Enzymically mediated bioprecipitation of uranium by a Citrobacter sp . : a concerted role for exocellular lipopolysaccharide and associated phosphatase in biomineral formation; Macaskie LE et al.; A Citrobacter sp . accumulated uranyl ion (UO2(2+)) via precipitation with phosphate ligand liberated by phosphatase activity . The onset and rate of uranyl phosphate deposition were promoted by NH4(+), forming NH(4)UO(2)PO(4), which has a lower solubility product than NaUO(2)PO(4) . This acceleration decoupled the rate-limiting chemical crystallization process from the biochemical phosphate ligand generation . This provided a novel approach to monitor the cell-surface-associated changes using atomic-force microscopy in conjunction with transmission electron microscopy and electron-probe X-ray microanalysis, to visualize deposition of uranyl phosphate at the cell surface . Analysis of extracted surface materials by (31)P NMR spectroscopy showed phosphorus resonances at chemical shifts of 0.3 and 2.0 p.p.m., consistent with monophosphate groups of the lipid A backbone of the lipopolysaccharide (LPS) . Addition of fUO2(2+) to the extract gave a yellow precipitate which contained uranyl phosphate, while addition of Cd(2+) gave a chemical shift of both resonances to a single new resonance at 3 p.p.m . Acid-phosphatase-mediated crystal growth exocellularly was suggested by the presence of acid phosphatase, localized by immunogold labelling, on the outer membrane and on material exuded from the cells . Metal deposition is proposed to occur via an initial nucleation with phosphate groups localized within the LPS, shown by other workers to be produced exocellularly in association with phosphatase . The crystals are further consolidated with additional, enzymically generated phosphate in close juxtaposition, giving high loads of LPS-bound uranyl phosphate without loss of activity and distinguishing this from simple biosorption, or periplasmic or cellular metal accumulation mechanisms . Accumulation of 'tethered' metal phosphate within the LPS is suggested to prevent fouling of the cell surface by the accumulated precipitate and localization of phosphatase exocellularly is consistent with its possible functions in homeostatis and metal resistance.

Int J Antimicrob Agents, 2000 Aug, 15(3), 213 - 9
In vitro activity of 19 antimicrobial agents against 3513 nosocomial pathogens collected from 48 Canadian medical centres . The Canadian Antimicrobial Study Group; Blondeau JM et al.; Antimicrobial resistance is a global concern . Differentiation between susceptibility rates for nosocomial versus community pathogens is important epidemiologically because it impacts on the appropriate empirical selection of antimicrobial therapy for infected patients . We studied resistance rates for 3513 nosocomial pathogens from 48 Canadian medical centres tested against 19 antimicrobial agents . The following are percent susceptibility for ceftazidime, ceftriaxone, ciprofloxacin, imipenem, netilmicin, and ticarcillin/clavulanic acid, respectively: Enterobacteriaceae 95, 95, 97, 99 98, 89; Escherichia coli, all 99 except ticarcillin/clavulanic acid (91); Enterobacter spp . 78, 78, 96, 99, 99, 71; Citrobacter spp . 79, 80, 89, 100, 94, 73; Proteus spp . 99, 88, 99, 88, 99, 98; Pseudomonas aeruginosa 88, 20, 82, 88, 81, 36; Staphylococcus aureus, all > 95; Enterococcus spp . 4, 9, 62, 95, 43, 38 . Susceptibility rates for other species of microorganisms and agents tested varied considerably . Some institutions had higher than average resistance rates for some pathogens (i.e . P . aeruginosa) and some agents . Detection and continued surveillance of antimicrobial resistance amongst nosocomial pathogens is vital to patient care and health care resources . The control of antimicrobial resistance can help maintain antibiotic usage and costs associated with the use of ever more potent drugs and the treatment of increasingly resistant infections.

Cesk Patol, 2000 May, 36(2), 65 - 70
{Diagnosis of malacoplakia in a transplanted kidney by needle biopsy}; Husek K et al.; Malakoplakia is an uncommon inflammatory condition rarely involving parenchyma of transplanted kidney . We report a 44-year-old female recipient of a cadaveric renal allograft treated with cyclosporin A and prednisone . After transplantation, E . coli and Citrobacter bacteruria occurred and three years later decreased graft function developed . Percutaneous needle biopsy was performed and diagnosis of malakoplakia was established . Histologically, interstitial sheets of plasmocytes and histiocytes with periodic acid-Shiff positive cytoplasm containing Michaelis-Gutmann bodies were present . Ultrastructurally, phagolysosomes containing membrane fragments and various developmental stages of inclusions to fully developed Michaelis-Gutmann bodies were found . The patient was treated with co-piperacillin and subsequently pefloxacin and renal functions improved after six months follow-up . Our case suggests that malakoplakia represents an abnormal defective histiocytic response to the infection in the setting of immunosuppressive therapy.

Biochemistry, 2000 Jul 25, 39(29), 8546 - 55
The role of glutamic acid-69 in the activation of Citrobacter freundii tyrosine phenol-lyase by monovalent cations; Sundararaju B et al.; Tyrosine phenol-lyase (TPL) from Citrobacter freundii is activated about 30-fold by monovalent cations, the most effective being K(+), NH(4)(+), and Rb(+) . Previous X-ray crystal structure analysis has demonstrated that the monovalent cation binding site is located at the interface between subunits, with ligands contributed by the carbonyl oxygens of Gly52 and Asn262 from one chain and monodentate ligation by one of the epsilon-oxygens of Glu69 from another chain {Antson, A . A., Demidkina, T . V., Gollnick, P., Dauter, Z., Von Tersch, R . L., Long, J., Berezhnoy, S . N., Phillips, R . S., Harutyunyan, E . H., and Wilson, K . S . (1993) Biochemistry 32, 4195} . We have studied the effect of mutation of Glu69 to glutamine (E69Q) and aspartate (E69D) to determine the role of Glu69 in the activation of TPL . E69Q TPL is activated by K(+), NH(4)(+), and Rb(+), with K(D) values similar to wild-type TPL, indicating that the negative charge on Glu69 is not necessary for cation binding and activation . In contrast, E69D TPL exhibits very low basal activity and only weak activation by monovalent cations, even though monovalent cations are capable of binding, indicating that the geometry of the monovalent cation binding site is critical for activation . Rapid-scanning stopped-flow kinetic studies of wild-type TPL show that the activating effect of the cation is seen in an acceleration of rates of quinonoid intermediate formation (30-50-fold) and of phenol elimination . Similar rapid-scanning stopped-flow results were obtained with E69Q TPL; however, E69D TPL shows only a 4-fold increase in the rate of quinonoid intermediate formation with K(+) . Preincubation of TPL with monovalent cations is necessary to observe the rate acceleration in stopped flow kinetic experiments, suggesting that the activation of TPL by monovalent cations is a slow process . In agreement with this conclusion, a slow increase (k < 0.5 s(-)(1)) in fluorescence intensity (lambda(ex) = 420 nm, lambda(em) = 505 nm) is observed when wild-type and E69Q TPL are mixed with K(+), Rb(+), and NH(4)(+) but not Li(+) or Na(+) . E69D TPL shows no change in fluorescence under these conditions . High concentrations (>100 mM) of all monovalent cations result in inhibition of wild-type TPL . This inhibition is probably due to cation binding to the ES complex to form a complex that releases pyruvate slowly.

Infect Immun, 2000 Aug, 68(8), 4637 - 46
Expression of intimin gamma from enterohemorrhagic Escherichia coli in Citrobacter rodentium; Hartland EL et al.; The carboxy-terminal 280 amino acids (Int280) of the bacterial adhesion molecule intimin include the receptor-binding domain . At least five different types of Int280, designated alpha, beta, gamma, delta, and epsilon, have been described based on sequence variation in this region . Importantly, the intimin types are associated with different evolutionary branches and contribute to distinct tissue tropism of intimin-positive bacterial pathogens . In this study we engineered a strain of Citrobacter rodentium, which normally displays intimin beta, to express intimin gamma from enterohemorrhagic Escherichia coli . We show that intimin gamma binds to the translocated intimin receptor (Tir) from C . rodentium and has the ability to produce attaching and effacing lesions on HEp-2 cells . However, C . rodentium expressing intimin gamma could not colonize orally infected mice or induce mouse colonic hyperplasia . These results suggest that intimin may contribute to host specificity, possibly through its interaction with a receptor on the host cell surface.

Int J Food Microbiol, 2000 Jun 30, 58(1-2), 123 - 8
Assessment of the microbiological quality and wash treatments of lettuce served in University restaurants; Soriano JM et al.; One hundred and forty-four samples of lettuce from 16 University restaurants were analyzed . The mesophilic aerobic counts of all samples ranged from 3.01 to 7.81 log10 CFU g(-1) . Results of total coliforms ranged from < 0.47 to > 3.38 log10 most probable number (MPN) g(-1) . Of the lettuce samples, 25.7% harbored Escherichia coli, 22.9% Staphylococcus aureus and 84% group D streptococci . Similarly, 10.4% of the samples harbored Aeromonas hydrophila, 2.8% Pseudomonas aeruginosa, and coliforms such as 14.6% Citrobacter freundii, 8.3% Klebsiella pneumoniae, 4.2% Enterobacter cloacae and 1.4% Providencia spp . Salmonella, Shigella and E . coli O157:H7 were not detected . When sodium hypochlorite or potassium permanganate solutions were used in washing procedures, the aerobic microorganisms were reduced by more than two log units, and total coliforms by at least one log.

J Perinatol, 2000 Jun, 20(4), 265 - 6
Citrobacter sepsis and severe newborn respiratory failure supported with extracorporeal membrane oxygenation; Rais-Bahrami K et al.; An infant with fulminant Citrobacter sepsis and respiratory failure is presented . The severity of respiratory failure and the need for systemic heparinization on extracorporeal membrane oxygenation delayed the opportunity of initial lumbar puncture to rule out meningitis . The infant was successfully treated with extracorporeal membrane oxygenation and long-term antibiotics . Repeated cranial computed tomography scans remained negative for intracerebral abscesses, and the infant is within normal limits for growth, neurologic status, and developmental status.

Int J Food Microbiol, 2000 Jun 15, 57(3), 211 - 8
Modification of the bile salts-Irgasan-brilliant green agar for enumeration of Aeromonas species from food; Neyts K et al.; The present study evaluated the productivity of BIBG medium for the isolation of Aeromonas spp . from food and describes a modification of the BIBG medium (mBIBG) (increased pH (8.7), replacement of xylose by soluble starch as a carbon source, decreased concentration of bile salts) to increase its selectivity and electivity . Using the mBIBG medium, growth of the majority of the Enterobacteriaceae (9/10) was suppressed except for Citrobacter freundii . The mBIBG medium supported growth of Pseudomonas species but a clear distinction between Aeromonas and Pseudomonas colonies could be made . Interpretation of the mBIBG medium should be performed after 24 h of incubation . It was noted that three of the 27 Aeromonas strains tested did not develop on the mBIBG medium . The ability or inability to grow on a selective medium is strain-dependent . Enumeration of Aeromonas species (A . hydrophila LMG 3771, A . caviae LMG 3775, A . veronii biovar veronii LMG 9075, A . veronii biovar sobria LMG 13071) from artificially contaminated foods (shrimp, minced meat (beef/pork), precut leek, and shredded carrots) confirmed that the mBIBG medium is suitable for quantitative recovery of aeromonads (ca . 10(2)-10(7) cfu/g) in the presence of a high background flora (10(5)-10(6) cfu/g) . Screening of naturally contaminated foods (vegetables, seafood, meat) for the presence of Aeromonas resulted in three out of 14 food samples showing presumptive Aeromonas colonies on mBIBG.

Chemotherapy, 2000 Jul-Aug, 46(4), 253 - 66
In vitro activity of rifaximin, metronidazole and vancomycin against Clostridium difficile and the rate of selection of spontaneously resistant mutants against representative anaerobic and aerobic bacteria, including ammonia-producing species; Marchese A et al.; BACKGROUND: Rifaximin is a rifamycin derivative characterized by a wide antibacterial activity . This drug is neither absorbed by the gastrointestinal tract nor inactivated by gastric juices, and exerts its action entirely within the intestinal lumen . METHODS: In this study, the activity of this antibiotic was compared with that of metronidazole and vancomycin against 93 Clostridium difficile isolates . The rate of emergence of bacteria spontaneously resistant to the new compound was also evaluated in relation to representative gram-positive and gram-negative strains . In terms of MIC(50) values, rifaximin showed an intrinsic activity superior to that of the other agents . The emergence of spontaneously resistant strains was assessed with 46 aerobic (staphylococci, enterococci, Proteus spp., Citrobacter freundii, Providencia rettgeri, enteropathogenic, enteroinvasive, enterotoxigenic and entero- hemorrhagic Escherichia coli, and Salmonella enteritidis) and anaerobic (Clostridium spp., Bacteroides spp., Fusobacterium nucleatum and Peptococcus spp.) pathogens, most of them also ammonium producers . Two different methods, broth and agar dilution, were employed . RESULTS: When liquid medium was employed, bacteria capable of sustained growth in 100 microg/ml of rifaximin were obtained after 2-5 transfers with gram-positive aerobic cocci, 2-3 transfers with gram-negative aerobic strains and 2-5 transfers with anaerobic species . At the highest dose used with the agar dilution method (8 x MIC), the frequency of emergence of spontaneously resistant mutants ranged from <1 x 10(-9) to 1.6 x 10(-8) with gram-positive aerobic and anaerobic cocci, while with aerobic and anaerobic gram-negative bacteria, this value ranged from <1 x 10(-9) to 1.7 x 10(-7) . C . difficile showed a particularly low incidence of spontaneously resistant mutants (<1 x 10(-9)) . The low incidence of resistant subpopulations selected by levels of 8 x MIC of rifaximin suggests that the high levels of the drug which were reached in the gastrointestinal lumen may further prevent the selection of mutants . CONCLUSION: The low toxicity, broad antibacterial activity and very poor absorption from the gastrointestinal tract of rifaximin suggest a potential therapeutic use for this drug in gastrointestinal diseases, as well as in the management of patients with cirrhosis and chronic portal-systemic encephalopathy .

Antimicrob Agents Chemother, 2000 Jul, 44(7), 1936 - 42
A novel CTX-M beta-lactamase (CTX-M-8) in cefotaxime-resistant Enterobacteriaceae isolated in Brazil; Bonnet R et al.; To estimate the diversity of extended-spectrum beta-lactamases in Brazil, 18 strains from different species of the family Enterobacteriaceae exhibiting a positive double-disk synergy test were collected by a clinical laboratory from several hospitals in Rio de Janeiro, Brazil, in 1996 and 1997 . Four strains (Proteus mirabilis, Enterobacter cloacae, Enterobacter aerogenes, and Citrobacter amalonaticus) hybridized with a 550-bp CTX-M probe . The P . mirabilis strain produced a CTX-M-2 enzyme . The E . cloacae, E . aerogenes, and C . amalonaticus isolates harbored a bla gene which was identified by cloning and sequencing as a bla(CTX-M) gene . E . coli HB101 transconjugants and the E . coli DH5alpha transformant harboring a recombinant plasmid produced a CTX-M beta-lactamase with an isoelectric point of 7.6 conferring a resistance phenotype characterized by a higher level of resistance to cefotaxime than to ceftazidime, as observed with the other CTX-M enzymes . The deduced protein sequence showed a novel Ambler class A CTX-M enzyme, named CTX-M-8, which had 83 to 88% identity with the previously described CTX-M enzymes . The phylogenic study of the CTX-M family including CTX-M-8 revealed four CTX-M types, CTX-M-8 being the first member of a new phylum of CTX-M enzymes . The evolutionary distances between the four types of CTX-M were large, suggesting that the four clusters branched off early from a distant unknown enzyme and that intermediate enzymes probably existed.

Int J Antimicrob Agents, 2000 Jul, 15(2), 111 - 8
In vitro efficacy of six cephalosporins tested against Enterobacteriaceae isolated at 38 North American medical centres participating in the SENTRY Antimicrobial Surveillance Program, 1997-1998; Jones RN et al.; The SENTRY Antimicrobial Surveillance Program is an ongoing international collaboration that monitors the predominant bacterial and fungal pathogens and antimicrobial susceptibility patterns associated with community-acquired and nosocomial infections . SENTRY data on the current cephalosporin susceptibility patterns (1997-98) of North American isolates of clinically important Enterobacteriaceae were analyzed . Susceptibility to a selection of cephalosporins was assessed at a central laboratory using reference broth microdilution methods and interpretive criteria specified by the National Committee for Clinical Laboratory Standards . The third- and fourth-generation cephalosporins tested demonstrated excellent activity against Escherichia coli and Klebsiella pneumoniae, whereas some of the older agents maintained good efficacy . Extended spectrum beta-lactamases were detected in all regions of the United States and Canada (1.8-10.7%) . Cefepime was the most active agent tested against pathogens with the potential for enzyme-mediated resistance due to Amp C . The third-generation agents maintained acceptable efficacy against Serratia marcescens, but were less effective against Citrobacter and Enterobacter species . The older cephalosporins were generally inadequate against these pathogens, in contrast to cefepime, which was the widest spectrum cephalosporin overall . Some significant regional variations in spectrum were detected.

Trop Gastroenterol, 2000 Jan-Mar, 21(1), 35 - 6
Enteropathogenic Escherichia coli isolates in paediatric diarrhoea; Vaishnavi C et al.; Enteropathogenic Escherichia coli (EPEC) are known to cause infantile enteritis . We studied the prevalence of EPEC in paediatric patients with acute and persistent diarrhoea . A total of 56 stool samples from paediatric patients were studied . There were 28 significant bacterial isolates . Of these 21 were untypable E . coli, 5 were typable E . coli, four of which belonged to members considered to be enteropathogenic . Non E . coli isolates grown in pure culture were one each of Pseudomonas aeruginosa and Citrobacter freundi . The study reveals the definitive role of EPEC in childhood diarrhoea at all age groups and emphasizes the need for characterisation of all significant E . coli isolates in this age group.

J Food Prot, 2000 May, 63(5), 655 - 8
Prevalence of Salmonella spp . in poultry broilers and shell eggs in Korea; Chang YH; This study was conducted to determine the presence of Salmonella spp . in raw broilers and shell eggs in Korea . In total, 135 dozen shell eggs and 27 raw broilers were tested . None of the egg yolks were found to contain Salmonella organisms but Escherichia coli, Escherichia hermanii, and Citrobacter freundii were isolated from egg shells . Salmonella spp . were detected in 25.9% of raw broilers, and Salmonella serotypes isolated from raw broilers were Salmonella Enteritidis, Salmonella Virchow, and Salmonella Virginia . D-values and antibiotic resistance of Salmonella isolates were also investigated . D-values of Salmonella enteritidis, Salmonella Virginia, and Salmonella Virchow in tryptic soy broth at 55 degrees C were 2.36, 2.13, and 0.70 min and 0.53, 0.37, and 0.20 min at 60 degrees C, respectively . All Salmonella isolates showed multiple antibiotic resistance patterns and were resistant to penicillin and vancomycin . One strain of Salmonella Enteritidis showed resistance to 12 antibiotics used in this study.

Anal Biochem, 2000 Apr 10, 280(1), 166 - 72
Molecular beacons: a real-time polymerase chain reaction assay for detecting Salmonella; Chen W et al.; Molecular beacons are oligonucleotide probes that become fluorescent upon hybridization . We developed a real-time PCR assay to detect the presence of Salmonella species using these fluorogenic reporter molecules . A 122-base-pair section of the himA was used as the amplification target . Molecular beacons were designed to recognize a 16-base-pair region on the amplicon . As few as 2 colony-forming unit (CFU) per PCR reaction could be detected . We also demonstrated the ability of the molecular beacons to discriminate between amplicons obtained from similar species such as Escherichia coli and Citrobacter freundii in real-time PCR assays . These assays could be carried out entirely in sealed PCR tubes, enabling fast and direct detection of Salmonella in a semiautomated format.

Subcell Biochem, 2000, 33, 47 - 59
E . coli invasion of brain microvascular endothelial cells as a pathogenetic basis of meningitis; Kim KS; A major limitation to advances in prevention and therapy of bacterial meningitis is our incomplete understanding of the pathogenesis of this disease . Successful isolation and cultivation of BMEC, which constitute the blood brain barrier, and the development of experimental hematogenous meningitis animal model, which mimics closely the pathogenesis of human meningitis, enabled us to dissect the pathogenetic mechanisms of bacterial meningitis . We have shown for the first time using E . coli as a paradigm the mechanisms of bacterial crossing of the blood-brain barrier into the central nervous system . We have shown that invasion of BMEC is a requirement for E . coli K1 crossing of the blood-brain barrier in vivo (Prasadarao et al., 1996b; Huang et al., 1995) . We have identified several novel E . coli proteins (i.e., Ibe10, Ibe7, and Ibe23) contributing to invasion of BMEC . We have also established a novel phenotype, i.e., invasion of BMEC, of a well known major E . coli protein, OmpA . In addition, we have shown that some of these E . coli proteins (i.e., OmpA, Ibe10) interact with novel endothelial receptors present on BMEC, not on systemic vascular endothelial cells . Further understanding and characterization of these E . coli-BMEC interactions should allow us to develop novel strategies to prevent this serious infection . In addition, the in vitro and in vivo models of the blood-brain barrier and the information derived from our study should be beneficial to investigating the pathogenesis of meningitis due to other organisms such as group B streptococci, Listeria monocytogenes, Streptococcus pneumoniae and Citrobacter.

Med Dosw Mikrobiol, 1999, 51(3-4), 311 - 21
{Differentiation of Citrobacter strains using chromosomal DNA restriction patterns}; Cieslik A et al.; The aim of this study was checking of the usefulness of chromosomal DNA restriction patterns in differentiation of Citrobacter strains . Molecular characterization of total 56 isolates of Citrobacter from Poland and Czech Republic, was performed by pulsed-field gel electrophoresis after digestion of chromosomal DNA with restriction endonuclease Xba I (5'-TCTAGA-3') . Chromosomal DNA of all tested Citrobacter strains gave after electrophoresis 12 to 21 bands and patterns consisting of 12 to 21 fragments ranging in size from 790 kb to 48.5 kb and smaller, which where not distinguishable . Pulsed-field gel electrophoresis patterns were useful for comparing Citrobacter strains . Identical restriction patterns generated by PFGE were observed in the case of selected strains e.g . strains C . sedlakii studied in this study, coming from an outbreak, having the some phenotype . In addition, PFGE patterns can be used to evaluate the clonal relatedness among bacterial isolates . PFGE can be helpful for assessing genetic relatedness among strains epidemiologicaly unrelated e.g . C . werkmanii strains tested in this study . The sum of DNA fragments after Xba I digestion indicates the genome size of Citrobacter strains . This suggests that PFGE should be useful for epidemiological investigations of Citrobacter strains.

Med Dosw Mikrobiol, 1999, 51(3-4), 301 - 9
{Differentiation of Citrobacter strains using electrophoretic protein patterns}; Cieslik A; The aim of this study was checking of the usefulness of electrophoretic protein patterns in differentiation of Citrobacter strains . For analysis of whole-cell proteins 181 Citrobacter strains were prepared . Electrophoresis was performed in Mini Protean Duall Slab Cell (Bio-Rad) apparatus . Electrophoresis was carried out in 10% polyacrylamide gel according to the SDS-PAGE method of Laemmli . Whole-cell proteins of all tested Citrobacter strains gave after electrophoresis 12 to 20 bands . Patterns consisting of 12 to 20 fragments ranging in size from 70,000 to 14,000 and smaller, were not distinguishable . There were no significant differences between electrophorograms of Citrobacter strains belonging to the different species, useful for species differentiation . Identical protein band patterns were observed in the case of selected strains e.g . strains C . sedlakii studied in this study, coming from an outbreak, having the some phenotype.

Med Dosw Mikrobiol, 1999, 51(3-4), 289 - 99
{Taxonomy of Citrobacter rods found in human specimens}; Cieslik A; The aim of this study was the identification of 181 Citrobacter strains on the basis of the recently proposed taxonomic changes of Brenner . All strains were isolated from diarrhoeic patients; 124 strains were originally sent for identification to Laboratory of Enterobacteriaceae DB NIH, 57 strains was isolated in Czech Republic . Citrobacter isolates were initially identified as C . koseri (3 strains), C . amalonaticus (1 strain) or as members of the C . freundii complex (177 strains) . Additionally some biochemical tests were performed . The ability to grow in medium containing KCN, lysine decarboxylase production, lactose fermentation and PYR test were examined . Strains belonging to the C . freundii complex were identified to the species level by biochemical methods on the basis of the results of Brenner, who found some tests to be useful in separating Citrobacter species . These test included citrate and acetate utilization, arginine dihydrolase and ornithine decarboxylase activities, motility, urease production, esculin hydrolysis, and acid production from sucrose, dulcitol, melibiose, raffinose and salicin . On the basis of the criteria described above, 96.6% of the strains tested could be assigned to one of the recently named species of C . freundii complex . Using biochemical tests suggested by Brenner we were able to identify Citrobacter strains members of newly recognised species . A five-test system is proposed to identify the most frequently encountered species currently residing in the C . freundii complex.

FEMS Immunol Med Microbiol, 2000 Jun, 28(2), 163 - 71
Structure and serologic properties of O-specific polysaccharide from Citrobacter freundii possessing cross-reactivity with Escherichia coli O157:H7; Nishiuchi Y et al.; Citrobacter freundii OCU158 is a serologically cross-reactive strain with Escherichia coli O157:H7 . To explore the close relationship between two strains, we have analyzed the chemical structures of O-specific polysaccharides and antigenic properties of lipopolysaccharides (LPSs) of both strains . The structure of O-specific polysaccharides from both strains was found to be identical by chemical and nuclear magnetic resonance analyses, in which D-PerNAc was 4-acetamido-4,6-dideoxy-D-mannose: {-->4)-beta-D-Glc-(1-->3)-alpha-D-PerNAc-(1-->4)-alpha-D-GalNAc-(1 --> 3)-alpha-L-Fuc-(1-->}(n) . The enzyme immunoassay using LPS derived either from E . coli O157 or from C . freundii could equally detect high levels of serum antibodies against LPS in patients with enterohemorrhagic E . coli (EHEC) O157 infection . Absorption of antibodies in EHEC patient serum by LPS from E . coli O157 or C . freundii, however, showed a difference in the epitopes . This difference was attributable to the epitope specificity of the core region and/or lipid A structure in LPS.

East Mediterr Health J, 1999 Jan, 5(1), 130 - 5
Isolation of Yersinia enterocolitica from cases of acute appendicitis and ice-cream; el-Sherbini M et al.; Seventy (70) appendiceal specimens and 80 ice-cream samples were analysed to detect Yersinia enterocolitica using three different media . Both Y . enterocolitica and Citrobacter freundii were recovered in appendiceal specimens (17.1% and 8.6%) and ice-cream (26.25% and 18.75%) respectively . Thioglycollate medium was more selective and productive in isolating Yersinia . Y . enterocolitica was the major causative agent of acute appendicitis (11/25, 44%) . It was sensitive to chloramphenicol, gentamicin, tetracycline and trimethoprim- sulfamethoxazole.

Kansenshogaku Zasshi, 2000 Mar, 74(3), 250 - 8
{Detection of extended spectrum beta-lactamases producing Enterobacteriaceae in feces}; Komatsu M et al.; A study was made of 366 feces for detection of extended spectrum beta-lactamases producing Enterobacteriaceae from feces . The selective agar was used for modified drigalski agar (Eiken Chemical Co., LTD) with 2 micrograms/ml cefotaxime (ESBL screen agar) . 92 strains of Enterobacteriaceae, 41 Escherichia coli, 15 Citrobacter freundii, 13 Enterobacter cloacae, 11 Klebsiella pneumoniae, and other 12, were isolated from ESBL screen agar . And, R-plasmid that were selected by 2 micrograms/ml cefotaxime were transferred by conjugation from two of the 92 strains . These strain were E . coli TH9809927 and Proteus mirabilis TH9808262 that were amplified by "Toho-1 type" primer . The clude enzyme from two strains (donor) and transconjugants were especially hydrolysed cepharoridine and cefotaxime . Accordingly, two strains (0.5%) were detected as ESBL producers . We think that the result of our survey suggests the increase of ESBLs producing bacterial infection in Japan, and believe that there is a trend of infection of its by surveilance of the feces.

J Clin Gastroenterol, 2000 Apr, 30(3), 317 - 20
Ascending cholangitis as a cause of pyogenic liver abscesses complicated by a gastric submucosal abscess and fistula; Yamada T et al.; Ruptures of nonamebic (pyogenic) liver abscesses into the thorax and peritoneum are very uncommon; but, hepatoduodenal and hepatocolonic fistulas are ever more rare . We report a case where ascending cholangitis was associated with pyogenic liver abscess formation and a gastric fistula . Drainage into the stomach was demonstrated by gastroduodenal endoscopy for gastric bleeding . After fistula formation, we could successfully treat the inflammation caused by infection of Citrobacter freundii and Candida albicans with intravenous infusion of both antibiotic and antifungal agents.

Protein Eng, 2000 Mar, 13(3), 207 - 15
Citrobacter freundii tyrosine phenol-lyase: the role of asparagine 185 in modulating enzyme function through stabilization of a quinonoid intermediate; Barbolina MV et al.; Asn185 is an invariant residue in all known sequences of TPL and of closely related tryptophanase and it may be aligned with the Asn194 in aspartate aminotransferase . According to X-ray data, in the holoenzyme and in the Michaelis complex Asn185 does not interact with the cofactor pyridoxal 5'-phosphate, but in the external aldimine a conformational change occurs which is accompanied by formation of a hydrogen bond between Asn185 and the oxygen atom in position 3 of the cofactor . The substitution of Asn185 in TPL by alanine results in a mutant N185A TPL of moderate residual activity (2%) with respect to adequate substrates, L-tyrosine and 3-fluoro-L-tyrosine . The affinities of the mutant enzyme for various amino acid substrates and inhibitors, studied by both steady-state and rapid kinetic techniques, were lower than for the wild-type TPL . This effect mainly results from destabilization of the quinonoid intermediate, and it is therefore concluded that the hydrogen bond between Asn185 and the oxygen at the C-3 position of the cofactor is maintained in the quinonoid intermediate . The relative destabilization of the quinonoid intermediate and external aldimine leads to the formation of large amounts of gem-diamine in reactions of N185A TPL with 3-fluoro-L-tyrosine and L-phenylalanine . For the reaction with 3-fluoro-L-tyrosine it was first possible to determine kinetic parameters of gem-diamine formation by the stopped-flow method . For the reactions of N185A TPL with substrates bearing good leaving groups the observed values of k(cat) could be accounted for by taking into consideration two effects: the decrease in the quinonoid content under steady-state conditions and the increase in the quinonoid reactivity in a beta-elimination reaction . Both effects are due to destabilization of the quinonoid and they counterbalance each other . Multiple kinetic isotope effect studies on the reactions of N185A TPL with suitable substrates, L-tyrosine and 3-fluoro-L-tyrosine, show that the principal mechanism of catalysis, suggested previously for the wild-type enzyme, does not change . In the framework of this mechanism the observed considerable decrease in k(cat) values for reactions of N185A TPL with L-tyrosine and 3-fluoro-L-tyrosine may be ascribed to participation of Asn185 in additional stabilization of the keto quinonoid intermediate.

J Antimicrob Chemother, 2000 Apr, 45(4), 521 - 4
In vitro selected fluoroquinolone-resistant mutants of Citrobacter freundii: analysis of the quinolone resistance acquisition; Tavio M et al.; Ten quinolone-resistant mutants of Citrobacter freundii, which were selected in vitro with fluoroquinolones from two clinical isolates, were studied . The parent isolates were susceptible to quinolones in spite of showing a single substitution in the GyrB (His-417 --> Leu) . No change was observed in the outer membrane proteins or in the lipopolysaccharide in any of the ten mutants studied with respect to their parent isolates . The development of quinolone resistance in selected mutants was associated with the appearance of a substitution in the GyrA (Thr-83 --> Ile) in nine of the ten mutants plus enhanced active efflux in all of them.

Avian Dis, 2000 Jan-Mar, 44(1), 85 - 98
Minimum inhibitory concentration determinations for various antimicrobial agents against 1570 bacterial isolates from turkey poults; Salmon SA et al.; Minimum inhibitory concentrations (MICs) were determined for 1570 bacteria from eight geographic locations (1204 Escherichia coli, 231 other enteric gram-negative bacilli {including Citrobacter spp., Enterobacter spp., Klebsiella spp., Proteus spp., and Salmonella spp.}, 31 Pseudomonas spp., 18 coagulase-positive staphylococci, 26 coagulase-negative staphylococci, and 55 streptococci and enterococci) by the National Committee for Clinical Laboratory Standards broth microdilution procedure . Antimicrobial agents tested included ampicillin, ceftiofur, enrofloxacin, erythromycin, florfenicol, gentamicin, neomycin, spectinomycin, sulfamethazine, tetracycline, and trimethoprim/sulfadiazine . Against the E . coli strains tested, ceftiofur, enrofloxacin, gentamicin, and trimethoprim/sulfadiazine were the most active compounds with MIC at which 50% of the strains are at or below (MIC50) = 0.5, < or = 0.03, 0.5, and 0.13 microg/ml, respectively, and MIC at which 90% of the strains are at or below (MIC90) = 1.0, 0.13, 32.0, and 2.0 microg/ml, respectively . Ampicillin, florfenicol, neomycin, and spectinomycin were the next most active compounds against the E . coli strains, with MIC50 = 4.0, 4.0, 16.0, and 16.0 microg/ml, respectively . MIC90 values for these compounds against E . coli strains were > 32.0, 8.0, 512.0, and > 128.0 microg/ml, respectively . The remaining compounds exhibited limited, strain-dependent activity against the E . coli strains tested . As with the E . coli, enrofloxacin, ceftiofur, and trimethoprim/sulfadiazine were also the most active compounds against the 231 other enteric organisms tested, with MIC50 < or = 1.0 microg/ml for all of these genera . The remaining compounds exhibited limited activity against these genera . Against the gram-positive cocci tested, ampicillin, enrofloxacin, ceftiofur, and trimethoprim/sulfadiazine were most active, whereas the remaining compounds exhibited strain-dependent activity . When MIC data for E . coli were summarized separately, differences were observed between the geographic locations for the various antimicrobial agents . In conclusion, ceftiofur, enrofloxacin, and trimethoprim/sulfadiazine were the most active of the compounds tested against all of the bacterial strains.

APMIS, 2000 Feb, 108(2), 145 - 52
The prevalence of gentamicin resistance among clinical isolates of enterobacteria in a Danish region; Schonheyder HC et al.; The susceptibility of enterobacteria to gentamicin was studied in the County of North Jutland during 1993-1998 . A total of 378 patients had a first-time gentamicin-resistant isolate . The annual number rose from 34 patients in 1993 to 89 in 1998 . The prevalence of resistance per 1000 patients with isolates examined was 13.5 for Escherichia coli, 15.8 for Klebsiella pneumoniae, 26.1 for Klebsiella oxytoca, and 150.4 for Citrobacter freundii . E . coli accounted for 67% of gentamicin-resistant isolates . K . oxytoca was probably associated with a nosocomial epidemic, whereas a source for C . freundii remained unresolved . An analysis confined to E . coli identified specimens other than urine as risk factors for gentamicin resistance . Likewise, resistance to sulfonamides and to fluoroquinolones were strong risk factors for gentamicin resistance among urinary isolates of E . coli . Thus, it is likely that aminoglycoside resistance may be promoted by other antibiotic groups due to co-resistance . The therapeutic and prophylactic uses of aminoglycosides have as yet not been undermined, but continuous population-based surveillance and vigilance are recommended.

Eur J Biochem, 2000 Mar, 267(6), 1830 - 6
Pyridoxal 5'-phoshate schiff base in Citrobacter freundii tyrosinephenol-lyase . Ionic and tautomeric equilibria; Bazhulina NP et al.; Spectral properties of the internal Schiff base in tyrosine phenol-lyase have been investigated in the presence of an activating cation K+ and a cation-inhibitor Na+ . The holoenzyme absorption spectra in the pH range 6.5-8.7 were recorded in the presence of K+ . No apparent pKa value of the coenzyme chromophore was found in this pH range, indicating that the internal Schiff base does not change its ionic form on going from pH 6.5 to 8.7 . To determine the ionic state and tautomeric composition of the Schiff base in tyrosine phenol-lyase, the absorption and circular dichroism spectra were analyzed using lognormal distribution curves . The predominant form of the internal Schiff base is that with protonated pyridinium and aldimine nitrogen atoms and deprotonated 3'-hydroxy group, i.e . the ketoenamine . This form is in prototropic equilibrium with its enolimine tautomer . The internal aldimine ionic form is changed upon replacement of K+ with Na+ . This replacement leads to a significant decrease in the pKa value of pyridinium nitrogen of the pyridoxal-P.

Jpn J Antibiot, 2000 Jan, 53(1), 46 - 59
{The resistance of recent clinical isolates against isepamycin, other aminoglycosides and injectable beta-lactams}; O'Hara K et al.; Clinical isolates collected from clinical facilities across Japan in 1998 were tested against five aminoglycosides and three beta-lactams . The resistance of 50 strains each of methicillin sensitive Staphylococcus aureus, methicillin resistant Staphylococcus aureus (MRSA), Staphylococcus epidermidis, Escherichia coli, Citrobacter freundii, Klebsiella pneumoniae, Enterobacter sp., Serratia sp., Pseudomonas aeruginosa and Proteus sp . (P . mirabilis 25 strains and P . vulgaris 25 strains) to the aminoglycosides isepamicin (ISP), amikacin (AMK), gentamicin, tobramycin and dibekacin, and to the beta-lactams imipenem, ceftazidime and piperacillin (all three known to be effective against P . aeruginosa) were investigated using a micro liquid dilution method with the following results: 1 . ISP was effective against all strains except for 14% of MRSA, 2% of Proteus sp., and 4% of P . aeruginosa . 2 . Six strains of MRSA were resistant to all eight drugs; however, in these cases ISP exhibited a relatively low minimum inhibitory concentration (MIC) compared to the other compounds . 3 . Four strains of MRSA were resistant to all drugs except ISP . MRSA was the only isolate to demonstrate a resistance to seven or more drugs . 4 . Twenty-one strains of MRSA and 1 strain of P . aeruginosa were resistant to six drugs; however, all of these were susceptible to both ISP and AMK . 5 . Against all strains tested, ISP generally exhibited a lower MIC compared to AMK . These results suggest that, even ten years after its entering the market, ISP is still an aminoglycoside having a high anti-bacterial activity against a wide range of clinical isolates.

Cytobios, 2000, 101(396), 15 - 21
Occurrence and antibiotic sensitivity of Enterobacteriaceae isolated from a group of Jordanian patients with community acquired urinary tract infections; Abu Shaqra Q; The type and antibiotic sensitivity of urinary tract pathogens may differ in various communities . Of 207 isolates recovered from midstream urine specimens collected from a group of patients with community acquired urinary tract infections (UTI), 86% were species of Enterobacteriaceae . The most frequently recovered pathogens were Escherichia coli (82%), Klebsiella spp . (7.3%), Proteus spp . (6.2%), Enterobacter spp . (3.4%) and Citrobacter spp . (1.1%) . High rates of resistance were found against ampicillin (95%), tetracycline (86%), carbenicillin (84%), trimethoprim/sulphamethoxazole (48%), and amoxycillin/clavulanic acid (45%) . For the antibiotics tobramycin, aztreonam, ceftriaxone and gentamicin 7% of the isolates were resistant, while resistance varied from 9-18% for amikacin, ciprofloxacin, norfloxacin, nalidixic acid and cefuroxime . The incidence of UTI caused by Enterobacteriaceae was three times higher in females than in males, particularly in young and middle age groups (< or = 19 and 20-39 years).

J Food Prot, 2000 Feb, 63(2), 264 - 7
Specificity of a conductance assay for enumeration of Escherichia coli from broiler carcass rinse samples containing genetically similar species; Edmiston AL et al.; Experiments were conducted to evaluate the specificity of a rapid method for enumeration of Escherichia coli from fresh broiler chicken carcasses . In three separate trials, E . coli, Citrobacter freundii, Salmonella Enteritidis, and Shigella sonnei were serially diluted and then inoculated into identical broiler chicken carcass rinses . Inoculated rinses were mixed with double-strength Coliform Medium supplemented with 2% dextrose . This mixture was placed in a Bactometer module in duplicate, and conductance was measured at 44 degrees C . Results indicated that C . freundii did not grow to an appreciable degree in the selective medium at 44 degrees C . Salmonella Enteritidis grew similarly to E . coli; however, an initial level of 10(6) Salmonella in the food product would be required for Salmonella to interfere with enumeration of E . coli using this method . S . sonnei grew at a more rapid rate than E . coli; however, there was an interaction between the regression lines formed when serial dilutions (log10 CFU/ml) were compared to E . coli detection times for these two species of bacteria . Therefore, high levels of S . sonnei in a food sample may interfere with the enumeration of E . coli . In general, Salmonella and Shigella are not found at high enough levels on poultry products to interfere with enumeration of E . coli using this method and, if found at high levels, would be detected and rejected using this procedure . Hence, the presence of organisms that are genetically and phylogenetically similar to E . coli would not preclude enumeration of E . coli using conductance under these conditions.

Changgeng Yi Xue Za Zhi, 1999 Dec, 22(4), 649 - 53
Adult Citrobacter freundii meningitis: case report; Chuang YC et al.; Citrobacter is a distinct group of Gram-negative bacilli belonging to the Enterobacteriaceae family . Central nervous system (CNS) infections due to Citrobacter are uncommon, though they occur more frequently in neonates and young children . In adults, Citrobacter meningitis is extremely unusual with only 6 cases reported in the literature before 1998 . This rare CNS infection has been seen in patients with head trauma, following neurosurgical procedures, and in those who are immunocompromised . Of the patients in the 6 reported cases, only one developed multi-antibiotic resistant Citrobacter CNS infection . Adding to this small number of reported cases, we report an adult case of post-neurosurgical meningitis and subdural empyema caused by multi-antibiotic resistant Citrobacter freundii and also review the literature related to this infection . Antimicrobial therapy with imipenem and third-generation cephalosporins failed to result in cerebrospinal fluid sterilization in our patient . Because of the use of broad-spectrum antibiotics, multi-antibiotic resistant Citrobacter species have developed in this nosocomial CNS infection and now present a therapeutic challenge . Therefore, further clinical studies are needed to determine updated therapeutic modalities for treating this life-threatening infection.

FEMS Microbiol Lett, 2000 Feb 15, 183(2), 289 - 94
The Yersinia high-pathogenicity island is present in different members of the family Enterobacteriaceae; Bach S et al.; A pathogenicity island termed high-pathogenicity island (HPI) is present in pathogenic Yersinia . This 35 to 45 kb island carries genes involved in synthesis, regulation and transport of the siderophore yersiniabactin . Recently, the HPI was also detected in various strains of Escherichia coli . In this study, the distribution of the HPI in the family Enterobacteriaceae was investigated . Among the 67 isolates pertaining to 18 genera and 52 species tested, nine (13.4%) harbored the island . These isolates were three E . coli, one Citrobacter diversus and five Klebsiella of various species (Klebsiella pneumoniae, Klebsiella rhinoscleromatis, Klebsiella ozaenae, Klebsiella planticola, and Klebsiella oxytoca) . As in Yersinia sp., all nine isolates synthesized the HPI-encoded iron-repressible proteins HMWP1 and HMWP2 . In the K . oxytoca strain, the right-end portion of the HPI was deleted, whereas the entire core region of the island was present in the eight other enterobacteria strains analyzed . In most of these isolates, the HPI was bordered by an asn tRNA locus, as in Yersinia sp . This report thus demonstrates the spread of the HPI among various members of the family Enterobacteriaceae.

FEMS Microbiol Lett, 2000 Feb 1, 183(1), 95 - 8
Association of qacE and qacEDelta1 with multiple resistance to antibiotics and antiseptics in clinical isolates of Gram-negative bacteria; Kucken D et al.; Clinical isolates of Enterobacter cloacae, Citrobacter freundii, Pseudomonas aeruginosa, and Stenotrophomonas maltophilia were tested for resistance to antibiotics and to the antiseptics benzalkonium chloride and cetyltrimethylammonium bromide . Furthermore, they were examined for the presence of the resistance genes qacE and qacEDelta1 . qacEDelta1 was detected by PCR in 10% of all (n=103) and in 81% of multiply antibiotic-resistant strains (n=15) . qacE was found in only one of 37 P . aeruginosa strains . The minimum inhibitory concentrations of benzalkonium chloride, cetyltrimethylammonium bromide, and ethidium bromide were not significantly different for qacEDelta1/qacE-positive or -negative strains . Our data indicate that multiply antibiotic-resistant Gram-negative bacteria are not necessarily more resistant to quaternary ammonium compounds than antibiotic-sensitive strains even though qacE or qacEDelta1 is present.

J Bacteriol, 2000 Feb, 182(4), 1109 - 17
Fragmentation of 23S rRNA in strains of Proteus and Providencia results from intervening sequences in the rrn (rRNA) genes; Miller WL et al.; Intervening sequences (IVSs) were originally identified in the rrl genes for 23S rRNA (rrl genes, for large ribosomal subunit, part of rrn operon encoding rRNA) of Salmonella enterica serovars Typhimurium LT2 and Arizonae . These sequences are transcribed but later removed during RNase III processing of the rRNA, resulting in fragmentation of the 23S species; IVSs are uncommon, but have been reported in at least 10 bacterial genera . Through PCR amplification of IVS-containing regions of the rrl genes we showed that most Proteus and Providencia strains contain IVSs similar to those of serovar Typhimurium in distribution and location in rrl genes . By extraction and Northern blotting of rRNA, we also found that these IVSs result in rRNA fragmentation . We report the first finding of two very different sizes of IVS (113 bp and 183 to 187 bp) in different rrl genes in the same strain, in helix 25 of Proteus and Providencia spp.; IVSs from helix 45 are 113 to 123 bp in size . Analysis of IVS sequence and postulated secondary structure reveals striking similarities of Proteus and Providencia IVSs to those of serovar Typhimurium, with the stems of the smaller IVSs from helix 25 being similar to those of Salmonella helix 25 IVSs and with both the stem and the central loop domain of helix 45 IVSs being similar . Thus, IVSs of related sequences are widely distributed throughout the Enterobacteriaceae, in Salmonella, Yersinia, Proteus, and Providencia spp., but we did not find them in Escherichia coli, Citrobacter, Enterobacter, Klebsiella, or Morganella spp.; the sporadic distribution of IVSs of related sequence indicates that lateral genetic transfer has occurred.

Microb Drug Resist, 1999 Winter, 5(4), 235 - 40
Decreased production of AmpC-type beta-lactamases associated with the development of resistance to quinolones in Citrobacter freundii strains; Tavio Perez MM et al.; The effect of fluoroquinolones in Citrobacter freundii strains that results in a decreased expression of cephalosporin-hydrolysing beta-lactamases was studied . Resistance to broad-spectrum cephalosporins and penicillins in two C . freundii clinical isolates was associated with moderate production of chromosomal AmpC-type-beta-lactamase in addition to changes in the outer membrane proteins profile with respect to wild-type C . freundii strains . Ten quinolone-resistant mutants were derived from the two clinical isolates using increasing fluoroquinolone concentrations . The level of susceptibility to cephalosporins and meropenem of these 10 mutants was increased and was associated with a 3.6-32% diminution in the hydrolyzing activity of their periplasmic extracts containing beta-lactamases on cephaloridine as compared with those from their parent strains . Susceptibility to cephalosporins and meropenem, as well as the expression of chromosomal AmpC-type-beta-lactamase in C . freundii strains, was influenced by the exposure to quinolones.

J Endourol, 1999 Dec, 13(10), 735 - 8
Predictive value of urinary cultures in assessment of microbial colonization of ureteral stents; Lifshitz DA et al.; BACKGROUND AND PURPOSE: Bacterial colonization of indwelling ureteral stents may serve as a nidus for bacteriuria in operations where stents are manipulated . The predictive value of urine cultures in the assessment of stent colonization was examined in 65 patients with indwelling ureteral stents . PATIENTS AND METHODS: Prophylactic antibiotic treatment was administered prior to stent insertion . All patients were ambulatory at the time of investigation and were examined in the outpatient clinic . Urine cultures were taken prior to stent removal after 8 to 150 (mean 64) days . The stents were removed under aseptic conditions, and the proximal and distal ends were cut off and placed in a culture medium for evaluation . None of the patients was treated for urinary tract infection prior to stent removal . RESULTS: Bacteriuria was found in 15% of the patients . In 35 patients (54%), urine and stent cultures were sterile . In 20 patients (31%), the urine culture was sterile but the stent was colonized (Enterococcus 9, E . coli 5, Staphylococcus aureus 2, S . epidermidis 2, Candida 1, Citrobacter diversus 1) . One patient had a sterile stent culture with bacteriuria . In 9 patients (13.5%), urine and stent cultures were identical (E . coli 4, Pseudomonas 4, Candida 1) . The incidence of stent colonization did not correlate with stent dwelling time . The sensitivity of urine cultures for the detection of stent colonization was poor, being 31% only . In a specific patient with negative urine culture, the probability of stent colonization was 36% . CONCLUSION: A sterile urine culture does not rule out the stent itself being colonized . Therefore, patients with indwelling ureteral stents and a sterile urine culture may benefit from prophylactic antibiotic treatment prior to endourologic procedures . The prophylactic regimen must provide coverage for common gram-negative uropathogens as well as gram-positive bacteria, including enterococci.

J Chemother, 1999 Oct, 11(5), 391 - 5
Intestinal protozoa in HIV-infected patients: effect of rifaximin in Cryptosporidium parvum and Blastocystis hominis infections; Amenta M et al.; In HIV-1 infected patients severe enteritis and chronic diarrhea are often documented as a consequence of multiple opportunistic infections . We analyzed 48 HIV-1 positive patients for the presence of intestinal pathogenic protozoa . Patients with CD4 > or = 200/mm3 showed a higher prevalence of a single pathogenic protozoa than patients with CD4 < or =200/mm3, who showed the presence of multiple protozoal infections . Patients who proved positive for only a single protozoa, Cryptosporidium or Blastocystis, were also positive, by stool culture, for the presence of Proteus mirabilis (3 samples), Citrobacter freundii (3 samples), Escherichia coli (one sample) or Enterobacter cloacae (one sample) . Treatment with rifaximin (600 mg, 3 times a day, for 14 days) was efficacious in resolving the clinical symptoms and clearing protozoan infections in HIV-1 infected patients with CD4 > or = 200/mm3, who presented enteric and systemic symptoms due to Criptosporidium or Blastocystis associated with enteropathogenic bacteria.

Antibiot Khimioter, 1999, 44(11), 7 - 16
{The results of a multicenter study of the comparative activity of cefepime and other antibiotics against the causative agents of severe hospital infections (the Micromax program)}; Sidorenko SV et al.; A multicentre trial was performed on the activity of cefepime in comparison with ceftazidime, ceftriaxone, piperacillin/tazobactam, imipenem and ciprofloxacin against severe hospital infection pathogens in intensive care units . The isolates of Escherichia coli and Proteus spp . from the majority of the centres were highly susceptible to the antibiotics (90 to 100 per cent of the isolates) . In some centres up to 40 per cent of the isolates produced ESBL . The isolates of Klebsiella spp . were characterized by lower susceptibility, in some centres the frequency of the strains producing ESBL exceeded 90 per cent, by the MIC geometric mean cefepime was superior to the third generation cephalosporins, the frequency of resistance to ciprofloxacin ranged from 0 to 31 per cent, no resistance to imipenem was recorded . The frequency of resistance to the third generation cephalosporins and piperacillin/tazobactam in Enterobacter spp., Serratia spp., Citrobacter spp., Morganella spp., and Providencia spp . ranged from 10 to 52 per cent, the resistance to cefepime equaled 0-11 per cent, 0 to 17 per cent of the isolates were resistant to ciprofloxacin, some isolates were resistant to imipenem . As for the nonfermenting microorganisms their resistance to all the antibiotics tested was comparatively high and markedly differed in various centres . As a whole, 7 per cent of all the isolates of the nonfermenting organisms was resistant to cefepime, 10 per cent was resistant to imipenem, 17 per cent was resistant to ceftazidime, 21 per cent was resistant to piperacillin/tazobactam and 36 per cent was resistant to ciprofloxacin.

J Food Prot, 2000 Dec, 63(12), 1643 - 7
Improvement of mannitol lysine crystal violet brilliant green agar for the selective isolation of H2S-positive Salmonella; Kodaka H et al.; Mannitol lysine crystal violet brilliant green agar (MLCB) is widely used in Japan for Salmonella isolation because the medium has been commercially available . Colonies of Salmonella on MLCB appear colorless with black centers due to H2S gas production; however, most Citrobacter freundii also produce H2S gas . In order to distinguish H2S-positive Salmonella from C . freundii we have improved MLCB . To MLCB was added 1% lactose (L-MLCB) . The relation for pH and black center colony formation was examined . The pH of MLCB and L-MLCB inoculated with Salmonella species was slightly acid after 7 h, but the pH of L-MLCB inoculated with C . freundii did not become acid for 24 h . The colony of C . freundii did not have a black center because the production of acid from lactose lowers the pH below 10 where it is needed for H2S to react with iron to produce black pigments . Of 99 Salmonella strains including 13 serotypes tested, all strains had the same colony morphologies on MLCB and L-MLCB . When MLCB and L-MLCB were evaluated with 36 C . freundii strains isolated from foods, only colonies on MLCB had black centers . We conclude that L-MLCB is useful for detection of nonlactose-fermenting, H2S-positive Salmonella in food samples.

Nucleic Acids Res, 2000 Dec 15, 28(24), 4974 - 86
Comparison of the Escherichia coli K-12 genome with sampled genomes of a Klebsiella pneumoniae and three salmonella enterica serovars, Typhimurium, Typhi and Paratyphi; McClelland M et al.; The Escherichia coli K-12 genome (ECO) was compared with the sampled genomes of the sibling species Salmonella enterica serovars Typhimurium, Typhi and Paratyphi A (collectively referred to as SAL) and the genome of the close outgroup Klebsiella pneumoniae (KPN) . There are at least 160 locations where sequences of >400 bp are absent from ECO but present in the genomes of all three SAL and 394 locations where sequences are present in ECO but close homologs are absent in all SAL genomes . The 394 sequences in ECO that do not occur in SAL contain 1350 (30.6%) of the 4405 ECO genes . Of these, 1165 are missing from both SAL and KPN . Most of the 1165 genes are concentrated within 28 regions of 10-40 kb, which consist almost exclusively of such genes . Among these regions were six that included previously identified cryptic phage . A hypothetical ancestral state of genomic regions that differ between ECO and SAL can be inferred in some cases by reference to the genome structure in KPN and the more distant relative Yersinia pestis . However, many changes between ECO and SAL are concentrated in regions where all four genera have a different structure . The rate of gene insertion and deletion is sufficiently high in these regions that the ancestral state of the ECO/SAL lineage cannot be inferred from the present data . The sequencing of other closely related genomes, such as S.bongori or Citrobacter, may help in this regard.

Pharmacotherapy, 1999 Dec, 19(12), 1392 - 9
Comparison of five beta-lactam antibiotics against common nosocomial pathogens using the time above MIC at different creatinine clearances; Kays MB; STUDY OBJECTIVE: To compare the time above the minimum inhibitory concentration (T>MIC) for five parenteral beta-lactam antibiotics against common nosocomial bacterial pathogens at different creatinine clearances (Clcr) . INTERVENTIONS: Serum concentration-time profiles were simulated for cefepime, ceftazidime, piperacillin, piperacillin-tazobactam, and imipenem at Clcr ranging from 120-30 ml/minute . The MIC data for 90% of organisms (MIC90) were collected for Escherichia coli, Klebsiella pneumoniae, Serratia marcescens, Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, Pseudomonas aeruginosa, and oxacillin-susceptible Staphylococcus aureus, and a weighted geometric mean MIC90 was calculated . The T>MIC was calculated as percentage of the dosing interval in which free concentrations exceeded the weighted geometric mean MIC90 . A T>MIC of 70% or greater was considered desirable for all organisms except S . aureus (> or = 50%) . MEASUREMENTS AND MAIN RESULTS: Cefepime 2 g every 12 hours (Clcr > or = 70 ml/min) and every 24 hours (Clcr < or = 60 ml/min) achieved desirable T>MIC for all Enterobacteriaceae and S . aureus at every Clcr . Imipenem 0.5 g achieved desirable T>MIC for E . coli, K . pneumoniae, C . freundii, and S . aureus at every Clcr . However, imipenem T>MIC was less than 70% for the following regimens and organisms: S . marcescens 0.5 g every 6 hours (Clcr > or = 90 ml/min), E . aerogenes 0.5 g every 6 hours (Clcr > or = 80 ml/min), E . cloacae 0.5 g every 6 hours (Clcr > or = 100 ml/min), S . marcescens 0.5 g every 8 hours (Clcr 60-70 ml/min), E . cloacae 0.5 g every 8 hours (Clcr 60-70 ml/min), and E . aerogenes 0.5 g every 8 hours (Clcr 50-70 ml/min) . Ceftazidime 2 g every 8 hours (Clcr 60-100 ml/min) and every 12 hours (Clcr 40-50 ml/min) achieved desirable T>MIC for E . coli, K . pneumoniae, S . marcescens, and S . aureus only . At every dose and Clcr, piperacillintazobactam achieved desirable T>MIC for S . aureus but not for any Enterobacteriaceae at Clcr > 50 ml/minute . Piperacillin did not achieve desirable T>MIC for any organism, and none of the beta-lactams attained a T>MIC of 70% or above for P . aeruginosa at any Clcr . CONCLUSION: At every Clcr, cefepime achieved a desirable T>MIC for more nosocomial pathogens than any other beta-lactam evaluated . Based on pharmacodynamic data, cefepime is an appropriate empiric choice for treatment of nosocomial infections . However, when P . aeruginosa is a potential pathogen, empiric combination therapy should be considered.

Curr Pharm Des, 1999 Nov, 5(11), 881 - 94
Regulation of inducible AmpC beta-lactamase expression among Enterobacteriaceae; Hanson ND et al.; AmpC ss-lactamases are active-site serine enzymes that are primarily cephalosporinases . In many gram negative organisms, including Enterobacter spp.,Citrobacter freundii, Serratia marcescens, Morganella morganii and Pseudomonas aeruginosa, the expression of chromosomal ampC genes is low but inducible in response to ss-lactams and other stimuli . The current working model for AmpC induction requires exposure of bacterial cells to ss-lactam drugs or other stimuli and is linked to the cell wall recycling pathway . Induction of ampC appears to involve several gene products associated with this pathway . These gene products include AmpR, AmpD, and AmpG . In addition, anhydro forms of cell wall precursor muropeptides are believed to act as cofactors for AmpC induction . These cofactors bind to the DNA binding protein, AmpR, and define the role of AmpR as activator . Recent debate has ensued in the literature as to the identification of the precursor muropeptide involved in the activation process . Two candidate muropeptides include 1,6-anhydro-N-acetylmuramic acid L-Ala-D-Glu-meso-diaminopimelic acid (anhydro-MurNAc-tripeptide) and anhydro-MurNAc-L-Ala-D-Glu-meso-diaminopimelic acid- D-Ala-D-Ala (pentapeptide) . The intent of this review is to address the general mechanism involved in AmpC induction . In doing so, the genes and gene products required for the process of AmpC induction are described . In addition, we review the data addressing cell wall recycling as it relates to AmpC induction.

J Antimicrob Chemother, 1999 Dec, 44(6), 743 - 8
Analysis of the mechanisms of quinolone resistance in clinical isolates of Citrobacter freundii; Navia MM et al.; The presence of gyrA, gyrB and/or parC mutations, quinolone uptake, outer membrane protein profiles and epidemiological relationship were studied in 12 clinical isolates of Citrobacter freundii . No alterations were observed in the gyrB gene of any of the strains, or gyrA or parC of the four quinolone-susceptible strains (nalidixic acid MIC of 2-4 mg/L, and a ciprofloxacin MIC of 0.006-0.06 mg/L) . The quinolone-resistant strains were classified into two groups: one group (group A) composed of strains resistant to nalidixic acid but not to ciprofloxacin and another (group B) including those resistant to both antibiotics with a mutation at codon 83 of the gyrA gene (Thr-->Ile), but no alteration in either parC or gyrB genes . In group B, three of the four resistant isolates, with a nalidixic acid MIC > 1024 mg/L and ciprofloxacin MIC of 8-32 mg/L, showed concomitant mutations at codons 83 and 87 of the gyrA gene (Thr-->Ile and Asp-->Tyr, respectively) as well as a single mutation in codon 80 of the parC gene (Ser-->Ile) . The fourth isolate did not possess the mutation at codon 87 of gyrA . Two strains belong to the same clone and, although they had the same type of mutations in the gyrA and parC genes, showed different MICs of ciprofloxacin . This difference was related to an efflux pump mechanism . Mutations in the gyrA and parC genes play the main role in quinolone resistance development in Citrobacter freundii, although other factors such as overexpression of efflux pumps can play a complementary role and thus modulate the final quinolone MIC.

J Antimicrob Chemother, 1999 Oct, 44(4), 489 - 99
Concurrent outbreaks of extended-spectrum beta-lactamase-producing organisms of the family Enterobacteriaceae in a Warsaw hospital; Palucha A et al.; The increasing use of broader-spectrum cephalosporins in the first half of the 1990s has become one of the major factors responsible for the high rate of selection of extended-spectrum beta-lactamase (ESBL)-producing microorganisms in Polish hospitals . Thirty-five isolates of seven different species of the family Enterobacteriaceae were identified as ESBL producers, over a 4 month period, in one of Warsaw's hospitals between the end of 1996 and the beginning of 1997 . Sixteen per cent of all Klebsiella pneumoniae isolates, 16% of Citrobacter freundii isolates and 32% of Serratia marcescens isolates collected by the hospital microbiology laboratory at that time were expressing these enzymes . The majority of these (27 isolates) were found to express CTX-M-type ESBLs (pI 8.4) . This outbreak was due to both plasmid dissemination among unrelated strains and clonal spread of some strains in several wards of the hospital . The remaining isolates produced ESBLs (pI 8.2) belonging to the SHV family of beta-lactamases and demonstrated a high degree of genetic diversity.

Antimicrob Agents Chemother, 1999 Dec, 43(12), 2990 - 5
Activities of a nitrofurazone-containing urinary catheter and a silver hydrogel catheter against multidrug-resistant bacteria characteristic of catheter-associated urinary tract infection; Johnson JR et al.; The in vitro inhibitory activity of a nitrofurazone-coated urinary catheter (NFC) against 86 recently obtained susceptible and multidrug-resistant (MDR) clinical isolates of Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Staphylococcus aureus, coagulase-negative staphylococci, and Enterococcus faecium, which are species implicated in catheter-associated urinary tract infection and which traditionally have been susceptible to nitrofuran derivatives, was determined using an agar diffusion assay . In a subset of these strains, the activity of the NFC was compared with that of a silver hydrogel urinary catheter (SHC), and the durability of each catheter's inhibitory activity was assessed during serial daily transfers of catheter segments to fresh culture plates . Except for vancomycin-resistant E . faecium, the NFC was active against all isolates tested and showed comparable inhibition zones with susceptible and MDR strains of each species . In contrast, the SHC inhibited only certain staphylococci (P < 0.01 versus the NFC), and among these strains, the SHC produced smaller inhibition zones than did the NFC (P < 0.01) . Inhibition was evident for up to 5 days with the NFC, but for only 1 day (if at all) with the SHC (P < 0.01) . These data document that, for most genera which traditionally have been susceptible to nitrofuran derivatives, the NFC remains active against contemporary MDR isolates . They also demonstrate that the in vitro antibacterial activity of the NFC is markedly superior to that of the SHC in several respects . Thus, the NFC shows promise for clinical use in the current era of MDR bacteria.

J Microbiol Methods, 2000 Jan, 39(2), 145 - 8
Resolution of high-molecular-weight components in lipopolysaccharides of Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus strains with sodium dodecyl sulfate polyacrylamide gels; Tavio MM et al.; The use of 0.5% sodium dodecyl sulfate in polyacrylamide separation gels allowed the resolution in several bands of high-molecular-mass components in smooth lipopolysaccharide of bacterial outer membrane from Escherichia coli, Morganella morganii, Citrobacter freundii and Citrobacter diversus . With or without 0.1% SDS, however, such a result was not possible.

Vet Rec, 1999 Oct 2, 145(14), 400 - 3
Epidemic infection caused by Citrobacter rodentium in a gerbil colony; de la Puente-Redondo VA et al.; Non-motile, Gram-negative rods, isolated from the intestinal tract and kidney of several dead animals in a gerbil colony, were identified as Citrobacter rodentium (formerly included in C . freundii species) on the basis of 31 biochemical tests . The isolates were tested against 40 antimicrobial agents and were all susceptible to ticarcillin plus clavulanate, ceftazidime and most of the quinolones studied, but were all resistant to most of the penicillins and aminoglycosides tested, and to fosfomycin, metronidazole and tiamulin . This bacterial species has been primarily associated with transmissible murine colonic hyperplasia, and this appears to be the first report of an epidemic infection in a gerbil colony with a fatal outcome in most of the animals affected.

J Clin Microbiol, 1999 Dec, 37(12), 4065 - 70
Detection and reporting of organisms producing extended-spectrum beta-lactamases: survey of laboratories in Connecticut; Tenover FC et al.; Extended-spectrum beta-lactamases (ESBLs) are enzymes produced in some gram-negative bacilli that mediate resistance to extended-spectrum cephalosporins and aztreonam . They are most common in Klebsiella spp . and Escherichia coli but are present in a variety of Enterobacteriaceae . Resistance mediated by these enzymes can be difficult to detect depending on the antimicrobial agents tested . AmpC beta-lactamases are related to the chromosomal enzymes of Enterobacter and Citrobacter spp . and also mediate resistance to extended-spectrum cephalosporins and aztreonam in addition to cephamycins, such as cefoxitin . Unlike ESBLs, however, AmpC beta-lactamases are not inhibited by clavulanic acid or other similar compounds . To assess the abilities of various antimicrobial susceptibility testing methods to detect ESBLs, we sent three ESBL-producing organisms, one AmpC-producing organism, and a control strain that was susceptible to extended-spectrum cephalosporins to 38 laboratories in Connecticut for testing . Eight (21.0%) of 38 labs failed to detect extended-spectrum cephalosporin or aztreonam resistance in any of the ESBL- or AmpC-producing isolates . Errors were encountered with both automated and disk diffusion methods . Conversely, seven (18.4%) labs categorized at least some of the four resistant isolates as potential ESBL producers and reported the results with the extended-spectrum cephalosporins and aztreonam as resistant as suggested by current National Committee for Clinical Laboratory Standards (NCCLS) guidelines . The percentage of laboratories that failed to detect resistance in the ESBL or AmpC isolates ranged from 23.7 to 31.6% depending on the type of enzyme present in the test organism . This survey suggests that many laboratories have difficulty detecting resistance in ESBL and AmpC-producing organisms and may be unaware of the NCCLS guidelines on modifying susceptibility testing reports for ESBL-producing strains.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1433 - 8
The phylogenetic position of Serratia, Buttiauxella and some other genera of the family Enterobacteriaceae; Sproer C et al.; The phylogenetic relationships of the type strains of 38 species from 15 genera of the family Enterobacteriaceae were investigated by comparative 16S rDNA analysis . Several sequences of strains from the genera Citrobacter, Erwinia, Pantoea, Proteus, Rahnella and Serratia, analysed in this study, have been analysed previously . However, as the sequences of this study differ slightly from the published ones, they were included in the analysis . Of the 23 enterobacterial genera included in an overview dendrogram of relatedness, members of the genera Xenorhabdus, Photorhabdus, Proteus and Plesiomonas were used as a root . The other genera formed two groups which could be separated, although not exclusively, by signature nucleotides at positions 590-649 and 600-638 . Group A contains species of Brenneria, Buttiauxella, Citrobacter, Escherichia, Erwinia, Klebsiella, Pantoea, Pectobacterium and Salmonella . All seven type strains of Buttiauxella share 16S rDNA similarities greater than 99% . Group B embraces two phylogenetically separate Serratia clusters, a lineage containing Yersinia species, Rahnella aquatica, Ewingella americana, and also the highly related pair Hafnia alvei and Obesumbacterium proteus.

Microbiology, 1999 Oct, 145 ( Pt 10), 2673 - 82
The genetic structure of enteric bacteria from Australian mammals; Gordon DM et al.; A total of 246 isolates representing five species of the family Enterobacteriaceae, taken from a variety of Australian mammal species, were characterized using multi-locus enzyme electrophoresis . Genome diversity estimates varied significantly among species, with the Klebsiella pneumoniae sample exhibiting the lowest diversity and the Citrobacter freundii sample the highest . Multi-locus linkage disequilibrium estimates revealed that alleles were non-randomly associated in all five species samples, but the magnitude of the estimates differed significantly among species . Escherichia coli had the lowest linkage disequilibrium estimate and Klebisella oxytoca the largest . Molecular analyis of variance was used to determine the extent to which population structure explained the observed genetic variation in a species . Two population levels were defined: the taxonomic family of the host from which the isolate was collected and the geographical locality where the host was collected . The amount of explained variation varied from 0% for K . oxytoca to 22% for K . pneumoniae . Host locality explained a significant amount of the genetic variation in the C . freundii (12%), E . coli (5%), Hafnia alvei (17%) and K . pneumoniae (22%) samples . Host family explained a significant fraction of the variation in E . coli (6%) H . alvei (7%) and K . pneumoniae (20%) . Estimates of effective population size for all five species, based on the probability that two randomly chosen isolates will be identical, failed to reveal any relationship between the effective population size and the genetic diversity of a species.

Microbiology, 1999 Oct, 145 ( Pt 10), 2663 - 71
The distribution of enteric bacteria from Australian mammals: host and geographical effects; Gordon DM et al.; Bacteria of the family Enterobacteriaceae were isolated from 642 mammalian hosts, representing 16 families and 79 species, collected from throughout Australia . Escherichia coli was the most common of the 24 enteric species recovered and represented almost half of the isolates . Association analysis revealed that most other species of bacteria were less likely to be recovered from hosts in which E . coli was present . The composition of the enteric community of a host was found to be determined by both the taxonomic family to which the host belonged and the geographical area from which the host was collected . Hosts collected from the northern areas of Queensland and the Northern Territory had more diverse enteric communities than hosts collected from New South Wales or Western Australia . Hosts of the families Petauridae and Vespertilionidae had more diverse enteric communities than did members of the Macropodidae or Phalangeridae . The probability of occurrence of Citrobacter freundii, Enterobacter cloacae, Escherichia coli, Hafnia alvei, Klebsiella oxytoca and K . pneumoniae in a host was found to vary with respect to host family and/or host locality . The non-random distribution of these species demonstrates the presence of extensive population structure and may suggest the existence of adaptations specific to both the primary and secondary habitats of these enteric bacteria.

Infect Immun, 1999 Nov, 67(11), 6019 - 25
Citrobacter rodentium espB is necessary for signal transduction and for infection of laboratory mice; Newman JV et al.; Citrobacter rodentium is the causative agent of transmissible murine colonic hyperplasia and contains a locus of enterocyte effacement (LEE) similar to that found in enteropathogenic Escherichia coli (EPEC) . EPEC espB is necessary for intimate attachment and signal transduction between EPEC and cultured cell monolayers . Mice challenged with wild-type C . rodentium develop a mucosal immunoglobulin A response to EspB . In this study, C . rodentium espB has been cloned and its nucleotide sequence has been determined . C . rodentium espB was found to have 90% identity to EPEC espB . A nonpolar insertion mutation in C . rodentium espB was constructed and used to replace the chromosomal wild-type allele . The C . rodentium espB mutant exhibited reduced cell association and had no detectable fluorescent actin staining activity on cultured cell monolayers . The C . rodentium espB mutant also failed to colonize laboratory mice following experimental inoculation . The espB mutation could be complemented with a plasmid-encoded copy of the gene, which restored both cell association and fluorescent actin staining activity, as well as the ability to colonize laboratory mice . These studies indicate that espB is necessary for signal transduction and for colonization of laboratory mice by C . rodentium.

Pediatr Infect Dis J, 1999 Oct, 18(10), 889 - 92
Citrobacter urinary tract infections in children; Gill MA et al.; BACKGROUND: Citrobacter species have been described as the etiologic agents in cases of bacteremia, meningitis, diarrhea and brain abscess, but little is known of their role as a cause of urinary tract infections in children . The purpose of this study was to define the role of Citrobacter species in pediatric urinary tract infections . METHODS: The project consisted of a retrospective chart review of microbiologic and medical records of patients younger than 18 years of age with urine cultures positive for Citrobacter species during a 3-year period . RESULTS: Thirty-four patients with 37 infections were included in the review . The average patient age was 6.9 years (range, 1 month to 18 years) and 71% were female . Fifty-six percent of the patients had urinary tract/renal anomalies or neurologic impairment and 26% represented nosocomial infections . Thirty-seven percent of patients were asymptomatic at the time of diagnosis, whereas 63% complained of at least one of the following findings: gastrointestinal symptoms; dysuria; fever; incontinence; penile/vaginal discharge; frequency; flank pain; and hematuria . Twenty-six of the isolates were Citrobacter freundii and 11 were Citrobacter koseri . Blood cultures were obtained in 9 patients and all were negative for Citrobacter isolates . CONCLUSIONS: Although it is uncommon Citrobacter can cause urinary tract infections in the pediatric population, which occur more frequently in children with underlying medical conditions . It appears that treatment similar to that of other gram-negative enteric organisms is the most prudent approach to these children until more information can be gathered.

Diagn Microbiol Infect Dis, 1999 Sep, 35(1), 65 - 73
Multicenter evaluation of the antimicrobial activity for seven broad-spectrum beta-lactams in Turkey using the Etest method . Turkish Antimicrobial Resistance Study Group; Pfaller MA et al.; From March through July 1997, a nine laboratory surveillance project was initiated in Turkey to monitor the potency and spectrum of seven broad-spectrum antimicrobial agents (cefepime, ceftazidime, cefotaxime, imipenem, aztreonam, cefoperazone/sulbactam, and ticarcillin/clavulanate) tested against approximately 100 organisms (average 82; range 70 to 95 isolates) per participant center (736 strains) . Eleven groups of organisms were tested by the Etest method (AB BIODISK, Solna, Sweden) with results validated by concurrent quality control strain analysis . Results from all centers were tabulated and 91.1% of quality assurance tests were within ranges recommended by the National Committee for Clinical Laboratory Standards . Among the seven beta-lactam-class drugs tested, imipenem and cefepime were the most active beta-lactams tested against all isolates . Overall, the rank order of susceptibility of the seven agents was imipenem > cefepime > cefoperazone/sulbactam > ceftazidime > cefotaxime > aztreonam > ticarcillin/clavulanate . Both cefepime and imipenem were active against ceftazidime-resistant strains of Enterobacteriaceae as well as against Streptococcus spp . and oxacillin-susceptible Staphylococcus aureus . Resistance phenotypes consistent with extended spectrum beta-lactamases were documented among Escherichia coli and Klebsiella spp., and profiles consistent with stably derepressed Bush-Jacoby-Mederios group 1 (Amp C) cephalosporinases were common among Enterobacter spp., Citrobacter spp., and Serratia spp . These data should be used to guide empiric therapy with beta-lactams in Turkey, and additionally will provide a reference statistical baseline to which future national studies of drugs in this class can be compared.

APMIS, 1999 Sep, 107(9), 859 - 62
The use of CHROMagar Orientation as a primary isolation medium with presumptive identification for the routine screening of urine specimens; Houang ET et al.; The aim of the study was to compare the use of a novel differential culture medium CHROMagar, for both primary isolation and presumptive identificaton, with the method currently used in our laboratory for screening mid-stream-urine samples (MSU) . Routine methods (RM) included blotting paper imprinting of all specimens and additional quantitative culture on cysteine lactose electrolyte-deficient agar (CLED) for selected samples together with Microbact 12E for further identification . The CHROMagar method (CH) relied on the use of blotting paper imprints, colonial colour and morphology on CHROMagar only . With respect to the 3390 MSU specimens examined, both methods yielded similar results in 3240, including > or = 87% of Escherichia coli, Pseudomonas spp., Staphylococcus spp., Proteus mirabilis/Morganella morganii and Enterobacter/Serratia/Klebsiella/Citrobacter spp . Of the 52 discordant identifications, yeasts were reported as staphylococci on CHROMagar in 10 . The overall cost of materials per specimen was US$ 0.30 by RM and $ 0.24 by CH . It took about 3 min to perform each Microbact test . Thus, CHROMagar plus Gram stain and other simple bench tests gave results similar to those using our current method, but had the advantage of saving time and materials.

J Bacteriol, 1999 Oct, 181(19), 6124 - 32
Phylogenetic analysis of L4-mediated autogenous control of the S10 ribosomal protein operon; Allen T et al.; We investigated the regulation of the S10 ribosomal protein (r-protein) operon among members of the gamma subdivision of the proteobacteria, which includes Escherichia coli . In E . coli, this 11-gene operon is autogenously controlled by r-protein L4 . This regulation requires specific determinants within the untranslated leader of the mRNA . Secondary structure analysis of the S10 leaders of five enterobacteria (Salmonella typhimurium, Citrobacter freundii, Yersinia enterocolitica, Serratia marcescens, and Morganella morganii) and two nonenteric members of the gamma subdivision (Haemophilus influenzae and Vibrio cholerae) shows that these foreign leaders share significant structural homology with the E . coli leader, particularly in the region which is critical for L4-mediated autogenous control in E . coli . Moreover, these heterologous leaders produce a regulatory response to L4 oversynthesis in E . coli . Our results suggest that an E . coli-like L4-mediated regulatory mechanism may operate in all of these species . However, the mechanism is not universally conserved among the gamma subdivision members, since at least one, Pseudomonas aeruginosa, does not contain the required S10 leader features, and its leader cannot provide the signals for regulation by L4 in E . coli . We speculate that L4-mediated autogenous control developed during the evolution of the gamma branch of proteobacteria.

An Med Interna, 1999 Jul, 16(7), 363 - 4
{Citrobacter freundii endocarditis}; Clemente Gonzalez C et al.; Infection by Citrobacter appears in man only in certain circumstances, since it usually acts as contaminant or colonizer . Bacteraemia by this bacillus can affect immunodeficient people, elderly people or those patients who have undergone invasive hospital processes . Although incidence of bacteraemia is low (0.3-0.9%), the death rate is very high, about 48% . This bacillus is seldom the cause of endocarditis . That is why we describe a case of endocarditis by Citrobacter freundii, in an aged person with previous valvulopathy.

Jpn J Antibiot, 1999 May, 52(5), 439 - 47
{Bacteriological and clinical break points: evaluation of the usefulness of cefozopran based on MIC}; Ishigo S et al.; Drug sensitivity of clinically isolated bacteria to cefozopran (CZOP), which is a new cephem antibiotic, was investigated, and the usefulness of the drug was evaluated from the viewpoint of bacteriological and clinical (pneumonia) break points . The following results were obtained . 1 . According to bacteriological break points, methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus spp . showed low sensitivity to cefozopran (CZOP) . However, the sensitivity of methicillin-sensitive Staphylococcus aureus (MSSA), E . coli, and Klebsiella spp., which are often isolated as pathogens of common infections, was 100%, that of Enterobacter spp., Serratia sp., and Citrobacter sp . was 90% or higher, and that of Pseudomonas aeruginosa was 80% or higher; the values were comparable to or better than those for ceftazidime (CAZ) . These results suggest a expanded antibacterial spectrum and enhanced antibacterial potency of CZOP . 2 . The estimated response rate of pneumonia to CZOP was 87.5% in outpatients and 51.9% in inpatients . Therefore, CZOP is considered to be one of the first choices especially in outpatient empiric therapy.

Mol Cell Probes, 1999 Aug, 13(4), 261 - 8
Use of PNA oligonucleotides for the in situ detection of Escherichia coli in water; Prescott AM et al.; A species-specific Peptide Nucleic Acid (PNA) oligonucleotide probe directed against the V(1)region of the 16S rRNA molecule was synthesized for the detection of Escherichia coli in water . The specificity of the probe was tested in dot blot hybridizations against a number of environmental isolates including those from the genera Escherichia, Klebsiella, Enterobacter and Citrobacter . In situ hybridization experiments were performed with biotinylated PNA oligonucleotide probes with subsequent detection of the biotin label using a combination of Streptavidin-Horseradish Peroxidase and a tyramide signal amplification system . The results obtained enabled the specific detection of E . coli in under 3 h . Hybridizations were also performed on cells which were treated with chlorine (1.5 mg l(-1)) for up to 30 min . Escherichia coli cells were still detected after storage for 14 days at room temperature . No cells were detected by conventional plate count or the <<Colilert>> assay, a method currently used for the routine detection of E . coli and coliforms in the water industry . Cell viability was assessed by the ability of cells to reduce 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) to highly fluorescent formazan crystals through bacterial respiration . Only cells that had not been chlorinated were detected . These results confirm that ribosomal RNA exists within the cell long after cell death has occurred and that rRNA cannot be used to assess the viability of individual cells . However rRNA probes in combination with viability markers should enable the specific detection of viable cells in situ . Hybridization experiments were also performed successfully on seeded tap water samples . The number of fluorescent cells detected correlated well with those obtained by plate count analysis . This represents the first reported use of PNA oligonucleotides for in situ detection of micro-organisms and offers a fast efficient alternative to conventional DNA approaches .

Antimicrob Agents Chemother, 1999 Aug, 43(8), 1895 - 900
In vitro and in vivo activities of Syn2190, a novel beta-lactamase inhibitor; Nishida K et al.; Syn2190, a monobactam derivative containing 1,5-dihydroxy-4-pyridone as the C-3 side chain, is a potent inhibitor of group 1 beta-lactamase . The concentrations of inhibitor needed to reduce the initial rate of hydrolysis of substrate by 50% for Syn2190 against these enzymes were in the range of 0.002 to 0.01 microM . These values were 220- to 850-fold lower than those of tazobactam . Syn2190 showed in vitro synergy with ceftazidime and cefpirome . This synergy was dependent on the concentration of the inhibitor against group 1 beta-lactamase-producing strains, such as Pseudomonas aeruginosa, Enterobacter cloacae, Citrobacter freundii, and Morganella morganii . However, against beta-lactamase-derepressed mutants of P . aeruginosa, the MICs of ceftazidime plus Syn2190 were not affected by the amount of beta-lactamase, and the values were the same for the parent strains . The MICs at which 50% of isolates are inhibited (MIC(50)s) of ceftazidime plus Syn2190 were 2- to 16-fold lower than those of ceftazidime alone for ceftazidime-resistant, clinically isolated gram-negative bacteria . Similarly, the MIC(50)s of cefpirome plus Syn2190 were two- to eightfold lower for cefpirome-resistant clinical isolates . The synergies of Syn2190 plus ceftazidime or cefpirome observed in vitro were also reflected in vivo . Syn2190 improved the efficacies of both cephalosporins in both a murine systemic infection model with cephalosporin-resistant rods and urinary tract infection models with cephalosporin-resistant P . aeruginosa.

J Basic Microbiol, 1999, 39(3), 161 - 8
Biodegradation of tannic acid by Citrobacter freundii isolated from a tannery effluent; Kumar RA et al.; A bacterial strain capable of utilizing tannic acid as sole carbon source was isolated from the effluent of a tannery and was identified as Citrobacter freundii . This organism could grow at concentrations as high as 5% (w/v) of tannic acid and produced extracellular tannase to hydrolyze the same . When grown in minimal medium containing 1% tannic acid (w/v) at 30 degrees C, this strain produced 1.87 U/ml of tannase at 6 h . At that time, tannic acid degradation products, namely glucose and gallic acid, were detectable in the culture filtrate; the other intermediate metabolites formed were pyrogallol (extracellular) and pyruvate (intracellular) . 2-hydroxymuconic acid is presumed to form as a result of ortho-cleavage of pyrogallol . The proposed biochemical pathway for the degradation of tannic acid by Citrobacter freundii is: Tannic acid-->{Glucose + Gallic acid}-->Pyrogallol -->2-hydroxymuconic acid -->{?}-->Pyruvate.

Lik Sprava, 1999 Jan-Feb, (1), 101 - 3
{The clinico-morphological characteristics of salmonellosis typhimurium in children}; Nezhoda II; Clinical and morphological features were studied of the course of gastrointestinal and generalized forms of salmonellosis . The grave gastrointestinal form runs its course against the unfavourable premorbid background getting associated with other flora: Proteus, Citrobacter, Klebsiella, Staphylococcus aureus . A distinguishing feature of the generalized form is its variegated clinical symptomatology . Noted in some instances is an acute onset of the illness with a clinical picture of purulent meningitis, but it happens that it runs a little-by-little, and a wave-like course presenting with poorly manifest phenomena of gastroenteritis and subsequent development of meningitis . Morphological changes in Salmonella enterocolitis are characterized by macrophagal infiltration of the mucosal lamina proper and by disordered hemomicrocirculation in postcapillaries and venules . The generalized form is associated with profound irreversible processes in the brain, liver, kidneys, and myocardium.

Science, 1999 Jul 23, 285(5427), 588 - 91
Role of bacterial intimin in colonic hyperplasia and inflammation; Higgins LM et al.; Enteropathogenic Escherichia coli (EPEC) cells adhere to gut epithelial cells through intimin alpha: the ligand for a bacterially derived epithelial transmembrane protein called the translocated intimin receptor . Citrobacter rodentium colonizes the mouse colon in a similar fashion and uses a different intimin: intimin beta . Intimin alpha was found to costimulate submitogenic signals through the T cell receptor . Dead intimin beta+ C . rodentium, intimin alpha-transfected C . rodentium or E . coli strain K12, and EPEC induced mucosal hyperplasia identical to that caused by C . rodentium live infection, as well as a massive T helper cell-type 1 immune response in the colonic mucosa . Mutation of cysteine-937 of intimin to alanine reduced costimulatory activity in vitro and prevented immunopathology in vivo . The mucosal changes elicited by C . rodentium were interferon-gamma-dependent . Immunopathology induced by intimin enables the bacteria to promote conditions that are favorable for increased microbial colonization.

Infect Immun, 1999 Aug, 67(8), 4208 - 15
Citrobacter freundii invades and replicates in human brain microvascular endothelial cells; Badger JL et al.; Neonatal bacterial meningitis remains a disease with unacceptable rates of morbidity and mortality despite the availability of effective antimicrobial therapy . Citrobacter spp . cause neonatal meningitis but are unique in their frequent association with brain abscess formation . The pathogenesis of Citrobacter spp . causing meningitis and brain abscess is not well characterized; however, as with other meningitis-causing bacteria (e.g., Escherichia coli K1 and group B streptococci), penetration of the blood-brain barrier must occur . In an effort to understand the pathogenesis of Citrobacter spp . causing meningitis, we have used the in vitro blood-brain barrier model of human brain microvascular endothelial cells (HBMEC) to study the interaction between C . freundii and HBMEC . In this study, we show that C . freundii is capable of invading and trancytosing HBMEC in vitro . Invasion of HBMEC by C . freundii was determined to be dependent on microfilaments, microtubules, endosome acidification, and de novo protein synthesis . Immunofluorescence microscopy studies revealed that microtubules aggregated after HBMEC came in contact with C . freundii; furthermore, the microtubule aggregation was time dependent and seen with C . freundii but not with noninvasive E . coli HB101 and meningitic E . coli K1 . Also in contrast to other meningitis-causing bacteria, C . freundii is able to replicate within HBMEC . This is the first demonstration of a meningitis-causing bacterium capable of intracellular replication within BMEC . The important determinants of the pathogenesis of C . freundii causing meningitis and brain abscess may relate to invasion of and intracellular replication in HBMEC.

J Clin Microbiol, 1999 Aug, 37(8), 2619 - 24
Biochemical identification of Citrobacter species defined by DNA hybridization and description of Citrobacter gillenii sp . nov . (formerly Citrobacter genomospecies 10) and Citrobacter murliniae sp . nov . (formerly Citrobacter genomospecies 11); Brenner DJ et al.; Recent work describing six named species and two unnamed genomospecies within Citrobacter has enlarged the genus to 11 species . DNA relatedness and phenotypic tests were used to determine how well these species can be identified . One hundred thirty-six strains were identified to species level by DNA relatedness and then identified phenotypically in a blinded fashion . By using conventional tests, 119 of the 136 strains (88%) were correctly identified to species level . Three additional strains (2%) were identified as citrobacteria but were not identified to species level, and 14 strains (10%) were misidentified as other Citrobacter species . Carbon source utilization tests were used to identify 86 of the strains . Eighty-four strains (98%) were correctly identified, and two strains (2%) were misidentified as other Citrobacter species . Additional strains of Citrobacter genomospecies 10 and Citrobacter genomospecies 11 were identified, allowing these species to be formally named as Citrobacter gillenii sp . nov . and Citrobacter murliniae sp . nov., respectively.

J Trop Pediatr, 1999 Jun, 45(3), 146 - 51
The bacteriology of neonatal septicaemia in Ile-Ife, Nigeria; Ako-Nai AK et al.; The incidence of septicaemia among neonates categorized as being at high risk was 55 per cent in Ile-Ife, Nigeria . Gram-positive organisms, specifically Staphylococcus aureus, were predominant (33.8 per cent) among bacteria cultured from proven cases of septicaemia . Other coagulase-negative staphylococci also contributed 21 per cent, with Staphylococcus epidermidis occurring in 5 per cent of the isolates . Listeria monocytogenes was cultured from 8.4 per cent of septic neonates . Pseudomonas aeruginosa was cultured from 3 per cent, Klebsiella pneumoniae from 14 per cent, and Escherichia coli from 7 per cent . Other Gram-negative bacilli cultured were Enterobacter aerogenes (5 per cent), Citrobacter freundii, Salmonella sp., and Proteus sp . (2 per cent each) . The bacterial isolates were relatively resistant to antibiotics traditionally employed to treat cases of septicaemia . The study shows a high prevalence of neonatal bacterial sepsis at the centre and the emerging role of Listeria monocytogenes in the aetiology of neonatal sepsis . It highlights the preponderance of multiple antibiotic resistant organisms among these neonates early in life which is of epidemiological importance in the control of the infectious agents.

Jpn J Antibiot, 1999 Apr, 52(4), 333 - 52
{A consideration on the results of nationwide surveillance of antimicrobial susceptibilities--gram-negative bacilli}; Kuroyama M et al.; The results of the semi-annual nationwide surveillance of antimicrobial susceptibilities, conducted by the Japanese Ministry of Health and Welfare during the period of January 1993 to July 1995, were analyzed for typical Gram-negative bacilli in the purpose of provision of an index for antimicrobial selection . During these 3 years, Escherichia coli, Citrobacter freundii, Enterobacter aerogenes and Proteus mirabilis showed slightly increasing tendency in susceptibility to fosfomycin (FOM) and Citrobacter freundii . Klebsiella pneumoniae and Enterobacter aerogenes showed slightly increasing tendency to minocycline (MINO) . While Haemophilus influenzae and Haemophilus parainfluenzae showed slightly decreasing tendency to cefmetazole (CMZ) . However, these annual changes were almost negligible . Generally, these microorganisms showed relatively good susceptibilities, every year, to the principal antimicrobial agents being approved for use against Gram-negative bacilli . However, Enterobacter cloacae, Enterobacter aerogenes, Serratia marcescens and Pseudomonas aeruginosa showed tendencies of decreased susceptibility to some of the antimicrobial agents . On the other hand, sulfamethoxazole-trimethoprim (ST), CMZ, latamoxef (LMOX), gentamicin (GM) and amikacin (AMK) showed good activities against some of the Gram-negative bacilli to which no indications are approved . In conclusion, bedside the identification of the causative microorganisms and the performance of antimicrobial susceptibility testing, such analyses (graphics of susceptibility tendency of clinical isolates to variety of antimicrobial agents) could be used as an index for selection of antimicrobial agents, when emergent and urgent selection of antimicrobial agents is necessary.

Antimicrob Agents Chemother, 1999 Jun, 43(6), 1393 - 400
Use of microdilution panels with and without beta-lactamase inhibitors as a phenotypic test for beta-lactamase production among Escherichia coli, Klebsiella spp., Enterobacter spp., Citrobacter freundii, and Serratia marcescens; Thomson KS et al.; Over the past decade, a number of new beta-lactamases have appeared in clinical isolates of Enterobacteriaceae that, unlike their predecessors, do not confer beta-lactam resistance that is readily detected in routine antibiotic susceptibility tests . Because optimal methodologies are needed to detect these important new beta-lactamases, a study was designed to evaluate the ability of a panel of various beta-lactam antibiotics tested alone and in combination with beta-lactamase inhibitors to discriminate between the production of extended-spectrum beta-lactamases, AmpC beta-lactamases, high levels of K1 beta-lactamase, and other beta-lactamases in 141 isolates of Escherichia coli, Klebsiella pneumoniae, Klebsiella oxytoca, Enterobacter cloacae, Enterobacter aerogenes, Citrobacter freundii, and Serratia marcescens possessing well-characterized beta-lactamases . The microdilution panels studied contained aztreonam, cefpodoxime, ceftazidime, cefotaxime, and ceftriaxone, with and without 1, 2, and 4 microg of clavulanate per ml or 8 microg of sulbactam per ml and cefoxitin and cefotetan with and without 8 microg of sulbactam per ml . The results indicated that a minimum panel of five tests would provide maximum separation of extended-spectrum beta-lactamase high AmpC, high K1, and other beta-lactamase production in Enterobacteriaceae . These included cefpodoxime, cefpodoxime plus 4 microg of clavulanate per ml, ceftazidime, ceftriaxone, and ceftriaxone plus 8 microg of sulbactam per ml . Ceftriaxone plus 2 microg of clavulanate per ml could be substituted for cefpodoxime plus 4 microg of clavulanate per ml without altering the accuracy of the tests . This study indicated that tests with key beta-lactam drugs, alone and in combination with beta-lactamase inhibitors, could provide a convenient approach to the detection of a variety of beta-lactamases in members of the family Enterobacteriaceae.

Antimicrob Agents Chemother, 1999 Jun, 43(6), 1350 - 7
Characterization and nucleotide sequence of a Klebsiella oxytoca cryptic plasmid encoding a CMY-type beta-lactamase: confirmation that the plasmid-mediated cephamycinase originated from the Citrobacter freundii AmpC beta-lactamase; Wu SW et al.; Plasmid pTKH11, originally obtained by electroporation of a Klebsiella oxytoca plasmid preparation into Escherichia coli XAC, expressed a high level of an AmpC-like beta-lactamase . The enzyme, designated CMY-5, conferred resistance to extended-spectrum beta-lactams in E . coli; nevertheless, the phenotype was cryptic in the K . oxytoca donor . Determination of the complete nucleotide sequence of pTKH11 revealed that the 8,193-bp plasmid encoded seven open reading frames, including that for the CMY-5 beta-lactamase (blaCMY-5) . The blaCMY-5 product was similar to the plasmidic CMY-2 beta-lactamase of K . pneumoniae and the chromosomal AmpC of Citrobacter freundii, with 99.7 and 97.0% identities, respectively; there was a substitution of phenylalanine in CMY-5 for isoleucine 105 in CMY-2 . blaCMY-5 was followed by the Blc and SugE genes of C . freundii, and this cluster exhibited a genetic organization identical to that of the ampC region on the chromosome of C . freundii; these results confirmed that C . freundii AmpC was the evolutionary origin of the plasmidic cephamycinases . In the K . oxytoca host, the copy number of pTKH11 was very low and the plasmid coexisted with plasmid pNBL63 . Analysis of the replication regions of the two plasmids revealed 97% sequence similarity in the RNA I transcripts; this result implied that the two plasmids might be incompatible . Incompatibility of the two plasmids might explain the cryptic phenotype of blaCMY-5 in K . oxytoca through an exclusion effect on pTKH11 by resident plasmid pNBL63.

J Appl Microbiol, 1999 May, 86(5), 731 - 40
The serodiagnosis of infections caused by Verocytotoxin-producing Escherichia coli; Chart H et al.; Patients with haemolytic uraemic syndrome (HUS) and haemorrhagic colitis (HC) produce serum antibodies to the lipopolysaccharides (LPS) of Escherichia coli O157 and certain other E . coli serogroups . Patients may also make salivary antibodies to the LPS of E . coli O157 . Serological tests based on these antibodies can be used to provide evidence of infection in the absence of culturable VTEC or the toxins they produce . Serum antibodies to LPS persist for several months following onset of disease, enabling both current and retrospective serological testing . The LPS of E . coli O157 shares epitopes with strains of Brucella abortus, Yersinia enterocolitica O9, Vibrio cholerae O1 Inaba, group N Salmonella and certain strains of Citrobacter freundii and E . hermanni . Serological tests for serum antibodies to E . coli O157 should be evaluated in the light of these cross-reactions . Serological tests to supply evidence of infection with E . coli O157 have been shown to provide a valuable adjunct to bacteriological procedures for detecting culturable VTEC and VT . The use of well characterized LPS antigens in association with the techniques of ELISA and immunoblotting provide valuable procedures for detecting evidence of infection with E . coli O157 and possibly other VTEC.

FEMS Microbiol Lett, 1999 May 15, 174(2), 295 - 301
Metabolism of L(-)-carnitine by Enterobacteriaceae under aerobic conditions; Elssner T et al.; Different Enterobacteriaceae, such as Escherichia coli, Proteus vulgaris and Proteus mirabilis, are able to convert L(-)-carnitine, via crotonobetaine, into gamma-butyrobetaine in the presence of carbon and nitrogen sources under aerobic conditions . Intermediates of L(-)-carnitine metabolism (crotonobetaine, gamma-butyrobetaine) could be detected by thin-layer chromatography . In parallel, L(-)-carnitine dehydratase, carnitine racemasing system and crotonobetaine reductase activities were determined enzymatically . Monoclonal antibodies against purified CaiB and CaiA from E . coli O44K74 were used to screen cell-free extracts of different Enterobacteriaceae (E . coli ATCC 25922, P . vulgaris, P . mirabilis, Citrobacter freundii, Enterobacter cloacae and Klebsiella pneumoniae) grown under aerobic conditions in the presence of L(-)-carnitine.

Infect Immun, 1999 Jun, 67(6), 3031 - 9
Citrobacter rodentium infection in mice elicits a mucosal Th1 cytokine response and lesions similar to those in murine inflammatory bowel disease; Higgins LM et al.; Citrobacter rodentium is a classically noninvasive pathogen of mice that is similar to enteropathogenic Escherichia coli (EPEC) in man . Following oral infection of young mice, the organism colonizes the distal colon, and within 1 week the colonic mucosa doubles in thickness and there is massive epithelial cell hyperplasia . Since T-cell responses in mouse models of inflammatory bowel disease (IBD) also cause epithelial hyperplasia, we have investigated the possibility that C . rodentium promotes similar T-cell responses in the mucosa, thereby increasing epithelial shedding, transmission, and replication of the organism . Beginning 6 days after infection, bacteria were observed to be in close association with the epithelial surface and were also visible scattered throughout the lamina propria and in the submucosa . There was a CD3(+)-cell infiltrate into the colonic lamina propria and epithelium as well as mucosal thickening and crypt hyperplasia . The majority of CD3(+) cells were CD4(+) and were not gammadelta+ . Reverse transcription-PCR analysis of cytokines also revealed a highly polarized Th1 response (interleukin-12, gamma interferon, and tumor necrosis factor alpha) in the mucosa and a large increase in the epithelial cell mitogen keratinocyte growth factor . None of the changes were seen in mice inoculated with bacteria lacking intimin (which is necessary for colonization), but they were seen in mice inoculated with C . rodentium complemented with intimin from EPEC . This is the first example of a classically noninvasive bacterial pathogen which elicits a strong mucosal Th1 response and which produces pathology similar to that seen in mouse models of IBD, which is also characterized by a strong Th1 response . These results also suggest that the colonic mucosa responds in a stereotypic way to Th1 responses.

Anticancer Res, 1999 Jan-Feb, 19(1A), 133 - 9
The effects of plant phenolics, caffeic acid, chlorogenic acid and ferulic acid on arylamine N-acetyltransferase activities in human gastrointestinal microflora; Lo HH et al.; The possible effects of naturally occurring plant phenolics, caffeic acid (CA), chlorogenic acid (CGA) and ferulic acid (FA) on arylamine N-acetyltransferase (NAT) activities on human gastrointestinal microflora, Escherichia coli, Klebsiella pneumoniae, Enterobacter aerogenes, Citrobacter koseri and Pseudomonas aeruginosa, were examined . The bacterial NAT activities were determined by HPLC measuring the acetylation of 2-aminofluorene (2-AF) . Among all examined bacteria, P . aeruginosa exerted the highest NAT activity while C . koseri possessed the lowest NAT activity . CA, CGA and FA could suppress the bacterial NAT activities dose-dependently both in the intact cell and cytosolic fraction analysis . According to the analysis of kinetic parameters in E . coli and P . aeruginosa, CA, CGA and FA were shown to be potent noncompetitive inhibitors of bacterial NAT activities . For the time course experiment, 4 mM of CA and FA could inhibit bacterial NAT activities for at least 4 hour but 4 mM of CGA could only significantly suppress NAT activity in E . coli for the same reaction time . These results strongly demonstrated that CA, CGA and FA inhibited NAT activities in human gastrointestinal bacteria.

Ethiop Med J, 1998 Jul, 36(3), 185 - 92
Asymptomatic bacteriuria in pregnancy: epidemiological, clinical and microbiological approach; Gebre-Selassie S; Asymptomatic urinary tract infection is a risk factor for fetal and maternal morbidity including development of pyelonephritis, premature labor and impaired intra-uterine development . In this study, 326 pregnant and 100 non-pregnant control women were screened for significant asymptomatic bacteriuria from April 8 to July 25, 1997 to gain insight into the prevalence rate, clinical characteristics of the disease and microbiological assessments of the causative agents . All the subjects were clinically identified to have no signs and symptoms of urinary tract infection (UTI) . The age ranges of the study and control groups were between 15-40 years for both groups with mean of 25.1 and 25.3 years, respectively . Bacteriological screening of mid-stream urine (MSU) revealed that 24/326 (7%) and 3/100 (3%) were positive for asymptomatic bacteriuria in the study group and controls, respectively (P < 0.05) . Further biochemical species identification showed that Escherichia coli was found in 11/24 (46%) followed by coagulase-negative staphylococci (CNS) in 8/24 (33%) and Citrobacter freundii in 2/24 (8%) . Others found in smaller number included Staphylococcus aureus, Enterobacter cloacae and Proteus rettgeri in 1/24 (4%) each . Antimicrobial susceptibility test revealed that 10/11 (91%) of the E . coli isolates were resistant to ampicillin and amoxicillin and 10/11 (91%) of them sensitive to nitrofurantoin.

Lett Appl Microbiol, 1999 Mar, 28(3), 175 - 8
Use of pyrrolidonyl peptidase to distinguish Citrobacter from Salmonella; Bennett AR et al.; In the routine testing of foods for Salmonella, Citrobacter and other members of the Enterobacteriaceae often produce colonies which are almost indistinguishable from Salmonella on commonly used selective agars . Biochemical confirmation of such colonies can be expensive and time-consuming . It has been suggested that the enzyme pyrrolidonyl peptidase (PYRase) could be used as a rapid test to distinguish Citrobacter colonies (PYRase-positive) from Salmonella (PYRase-negative) . Pure cultures of Salmonella, Citrobacter and other Enterobacteriaceae were tested for PYRase activity; all strains of Salmonella tested were PYRase-negative, and all Citrobacter tested were PYRase-positive . Inoculated and naturally contaminated food samples were tested for the presence of Salmonella by a standard cultural method . A PYR test was used to test Salmonella-like colonies isolated on selective agar and potentially, eliminate PYR-positive isolates from further biochemical testing . The test was able to screen out 6% of colonies selected from samples inoculated with Salmonella, and 43% of colonies selected from uninoculated samples.

Biotechnol Prog, 1999 Mar-Apr, 15(2), 228 - 37
Biosorption of lead, cadmium, and zinc by Citrobacter strain MCM B-181: characterization studies; Puranik PR et al.; The biosorption process for removal of lead, cadmium, and zinc by Citrobacter strain MCM B-181, a laboratory isolate, was characterized . Effects of environmental factors and growth conditions on metal uptake capacity were studied . Pretreatment of biomass with chemical agents increased cadmium sorption efficiency; however, there was no significant enhancement in lead and zinc sorption capacity . Metal sorption by Citrobacter strain MCM B-181 was found to be influenced by the pH of the solution, initial metal concentration, biomass concentration, and type of growth medium . The metal sorption process was not affected by the age of the culture or change in temperature . Equilibrium metal sorption was found to fit the Langmuir adsorption model . Kinetic studies showed that metal uptake by Citrobacter strain MCM B-181 was a fast process, requiring <20 min to achieve >90% adsorption efficiency . The presence of cations reduced lead, zinc, and cadmium sorption to the extent of 11 . 8%, 84.3%, and 33.4%, respectively . When biomass was exposed to multimetal solutions, metals were adsorbed in the order Co2+ < Ni2+ < Cd2+ < Cu2+ < Zn2+ < Pb2+ . Among various anions tested, only phosphate and citrate were found to hamper metal sorption capacity of cells . Biosorbent beads prepared by immobilizing the Citrobacter biomass in polysulfone matrix exhibited high metal loading capacities . A new mathematical model used for batch kinetic studies was found to be highly useful in prediction of experimentally obtained metal concentration profiles as a function of time . Metal desorption studies indicated that Citrobacter beads could, in principle, be regenerated and reused in adsorption-desorption cycles . In an expanded scale trial, biosorbent beads were found to be useful in removal/recovery of metals such as lead from industrial wastewaters.

Biochemistry, 1999 Mar 30, 38(13), 3851 - 6
Multiple conformations of the acylenzyme formed in the hydrolysis of methicillin by Citrobacter freundii beta-lactamase: a time-resolved FTIR spectroscopic study; Wilkinson AS et al.; Time-resolved infrared difference spectroscopy has been used to show that the carbonyl group of the acylenzyme reaction intermediate in the Citrobacter freundii beta-lactamase-catalyzed hydrolysis of methicillin can assume at least four conformations . A single-turnover experiment shows that all four conformations decline during deacylation with essentially the same rate constant . The conformers are thus in exchange on the reaction time scale, assuming that deacylation takes place only from the conformation which is most strongly hydrogen bonded or from a more minor species not visible in these experiments . All conformers have the same (10 cm-1) narrow bandwidth compared with a model ethyl ester in deuterium oxide (37 cm-1) which shows that all conformers are well ordered relative to free solution . The polarity of the carbonyl group environment in the conformers varies from 'ether-like' to strongly hydrogen bonding (20 kJ/mol), presumably in the oxyanion hole of the enzyme . From the absorption intensities, it is estimated that the conformers are populated approximately proportional to the hydrogen bonding strength at the carbonyl oxygen . A change in the difference spectrum at 1628 cm-1 consistent with a perturbation (relaxation) of protein beta-sheet occurs slightly faster than deacylation . Consideration of chemical model reactions strongly suggests that neither enamine nor imine formation in the acyl group is a plausible explanation of the change seen at 1628 cm-1 . A turnover reaction supports the above conclusions and shows that the conformational relaxation occurs as the substrate is exhausted and the acylenzymes decline . The observation of multiple conformers is discussed in relation to the poor specificity of methicillin as a substrate of this beta-lactamase and in terms of X-ray crystallographic structures of acylenzymes where multiple forms are not apparently observed (or modeled) . Infrared spectroscopy has shown itself to be a useful method for assessment of the uniqueness of enzyme-substrate interactions in physiological turnover conditions as well as for determination of ordering, hydrogen bonding, and protein perturbation.

Biotechnol Bioeng, 1998 Apr 20-May 5, 58(2-3), 339 - 43
Production of L-DOPA(3,4-dihydroxyphenyl-L-alanine) from benzene by using a hybrid pathway; Park HS et al.; As an alternative approach to the production of L-DOPA from a cheap raw material, we constructed a hybrid pathway consisting of toluene dioxygenase, toluene cis-glycol dehydrogenase, and tyrosine phenol-lyase . In this pathway, catechol is formed from benzene through the sequential action of toluene dioxygenase and toluene cis-glycol dehydrogenase, and L-DOPA is synthesized from the resulting catechol in the presence of pyruvate and ammonia by tyrosine phenol-lyase cloned from Citrobacter freundii . When the hybrid pathway was expressed in E . coli, production of L-DOPA was as low as 3 mM in 4 h due to the toxic effect of benzene on the cells . In order to reduce lysis of cells, Pseudomonas aeruginosa was employed as an alternative, which resulted in accumulation of about 14 mM L-DOPA in 9 h, showing a stronger resistance to benzene .

Antimicrob Agents Chemother, 1999 Apr, 43(4), 769 - 76
Cloning, sequence analyses, expression, and distribution of ampC-ampR from Morganella morganii clinical isolates; Poirel L et al.; Shotgun cloning experiments with restriction enzyme-digested genomic DNA from Morganella morganii 1, which expresses high levels of cephalosporinase, into the pBKCMV cloning vector gave a recombinant plasmid, pPON-1, which encoded four entire genes: ampC, ampR, an hybF family gene, and orf-1 of unknown function . The deduced AmpC beta-lactamase of pI 7.6 shared structural and functional homologies with AmpC from Citrobacter freundii, Escherichia coli, Yersinia enterocolitica, Enterobacter cloacae, and Serratia marcescens . The overlapping promoter organization of ampC and ampR, although much shorter in M . morganii than in the other enterobacterial species, suggested similar AmpR regulatory properties . The MICs of beta-lactams for E . coli MC4100 (ampC mutant) harboring recombinant plasmid pACYC184 containing either ampC and ampR (pAC-1) or ampC (pAC-2) and induction experiments showed that the ampC gene of M . morganii 1 was repressed in the presence of ampR and was activated when a beta-lactam inducer was added . Moreover, transformation of M . morganii 1 or of E . coli JRG582 (delta ampDE) harboring ampC and ampR with a recombinant plasmid containing ampD from E . cloacae resulted in a decrease in the beta-lactam MICs and an inducible phenotype for M . morganii 1, thus underlining the role of an AmpD-like protein in the regulation of the M . morganii cephalosporinase . Fifteen other M . morganii clinical isolates with phenotypes of either low-level inducible cephalosporinase expression or high-level constitutive cephalosporinase expression harbored the same ampC-ampR organization, with the hybF and orf-1 genes surrounding them; the organization of these genes thus differed from those of ampC-ampR genes in C . freundii and E . cloacae, which are located downstream from the fumarate operon . Finally, an identical AmpC beta-lactamase (DHA-1) was recently identified as being plasmid encoded in Salmonella enteritidis, and this is confirmatory evidence of a chromosomal origin of the plasmid-mediated cephalosporinases.

Yonsei Med J, 1998 Dec, 39(6), 520 - 5
Plasmid-encoded AmpC beta-lactamases: how far have we gone 10 years after the discovery?
Bauernfeind A, Chong Y, Lee K.
The dogma that ampC genes are located exclusively on the chromosome was dominant until about 10 years ago . Since 1989 over 15 different plasmid-encoded AmpC beta-lactamases have been reported from several countries . Most of these enzymes evolved in two clusters . The major cluster includes several enzymes with a high similarity to CMY-2, which is the closest related chromosomal AmpC enzyme of Citrobacter freundii . A second cluster centers around CMY-1 . It is less homogeneous and not closely related chromosomal AmpC enzymes . Molecular diversification by amino acid substitutions does not usually translate into a change in the resistance phenotype . At this time, CMY-2 appears to be the most prevalent and widely distributed . Further global increase of prevalence and diversity of plasmidic AmpC beta-lactamases have to be anticipated in the next millenium.

Appl Microbiol Biotechnol, 1999 Feb, 51(2), 193 - 200
Cloning and overexpression in Escherichia coli of the gene encoding dihydroxyacetone kinase isoenzyme I from Schizosaccharomyces pombe, and its application to dihydroxyacetone phosphate production; Itoh N et al.; The gene dak1 encoding a dihydroxyacetone kinase (DHAK) isoenzyme I, one of two isoenzymes in the Schizosaccharomyces pombe IFO 0354 strain, was cloned and sequenced . The dak1 gene comprises 1743 bp and encodes a protein of 62,245 Da . The deduced amino acid sequence showed a similarity to a putative DHAK of Saccharomyces cerevisiae and DHAK of Citrobacter freundii . The dak1 gene was expressed at a high level in Escherichia coli, and the recombinant enzyme was purified to homogeneity and characterized . The acetone powder of recombinant E . coli cells was used to produce dihydroxyacetone phosphate.

Lab Anim Sci, 1998 Apr, 48(2), 145 - 55
Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: possible role of Citrobacter rodentium; Maggio-Price L et al.; Citrobacter rodentium from an undetermined source was detected in a breeding colony of T-cell receptor transgenic mice housed in a conventional mouse facility in which murine hepatitis virus had been endemic and Helicobacter spp . had been detected . Citrobacter rodentium, isolated from blood, spleen, and colon, correlated with a significant increase in mortality and morbidity in this breeding colony . Transgenic mice of all ages were affected by chronic debilitation, loss in reproductive efficiency, rectal prolapse, and acute death, resulting in the near loss of these noncommercially available strains . Several alterations in immunologic parameters were observed, including outgrowth of an unusual population of cells in the spleen and blood, reduction in ascites production, loss of the capacity of peritoneal exudate cells to serve as feeders for the cloning of long-term T-cell lines, and inhibition of antigen-specific cytotoxic T-cell activity . These altered immune functions also were apparent in commercially-derived nontransgenic mice cohoused with the infected colony and in overtly healthy transgenic and nontransgenic littermates . Citrobacter rodentium and murine hepatitis virus were eliminated ultimately on rederivation of the affected strains by embryo transfer . However, the rapid decrease in the health of the colony necessitated more immediate action . To reduce mortality and allow breeding to continue during rederivation of the transgenic lines, animals were treated with enrofloxacin and moved to a barrier facility . Antibiotic therapy significantly reduced morbidity and mortality, markedly increased litter size and frequency, and resulted in the normalization of many of the immunologic assays . The involvement of C . rodentium in altering viability of the colony and perturbing immunologic assays is suggested by correlation of the onset of the syndrome with the appearance of Citrobacter sp . and its resolution with the elimination of Citrobacter sp . from the colony . Whether infection with Citrobacter alone is causative or whether superinfection of murine hepatitis virus- and Helicobacter-infected mice is required remains to be determined.

Arch Pharm (Weinheim), 1999 Jan, 332(1), 7 - 12
Synthesis and evaluation of 2 beta-oxyimino and alkenylpenicillanic acid sulfone derivatives as beta-lactamase inhibitors; Cho YS et al.; The synthesis and in vitro synergies of 2 beta-alkenyl and oxyiminopenam sulfone derivatives are described . Most of the compounds synthesized exhibited good inhibitory activities and synergistic antibacterial activities with piperacillin and ceftriaxone, respectively, against several beta-lactamase producing strains . Particularly the 2 beta-alkenylpenam sulfone derivatives . 1e and 1g, showed good synergistic activity with ceftriaxone against Citrobacter freundi NIH 10018-68 and Proteus vulgaris 20 . Also the compounds 2a, 2c, and 2f, 2 beta-oxyiminopenam sulfone derivatives, exhibited improved synergistic activity with piperacillin against Citrobacter freundi NIH 10018-68.

J Basic Microbiol, 1999, 39(1), 29 - 35
Cloning and expression in Escherichia coli of an azoreductase gene from Clostridium perfringens and comparison with azoreductase genes from other bacteria; Rafii F et al.; A genomic library of Clostridium perfringens ATCC 3626 was constructed in phage lambda gt11 and screened with an antibody against the C . perfringens azoreductase, which catalyzes the reduction of azo dyes to aromatic amines . A positive recombinant phage, containing a 3.8 kb DNA fragment insert was selected and purified . Lytic and lysogenic Escherichia coli cultures infected with the recombinant phage had higher azoreductase activity than cultures infected only with the vector lambda gt11 . The 3.8 kb DNA fragment was amplified by PCR and found to hybridize with one band from C . perfringens DNA digested with EcoR1, indicating the presence of a single copy of the azoreductase gene . The fragment also hybridized with DNA from other azoreductase-producing Clostridium species, a Eubacterium sp., Enterobacter cloacae, Citrobacter amalonaticus and E . coli, but not with DNA from some other species of anaerobic bacteria capable of reducing azo dyes . The data indicate that the sequence of the azoreducatse gene of C . perfringens is conserved in some anaerobes and facultative anaerobes, but not in others, and that different types of azoreductase genes must be found in other anaerobic bacteria.

J Zoo Wildl Med, 1998 Dec, 29(4), 413 - 8
Abdominal ascites in electric eels (Electrophorus electricus) associated with hepatic hemosiderosis and elevated water pH; Marselas GA et al.; Six electric eels (Electrophorus electricus) from various centers that house aquatic organisms presented clinically with abdominal distension following prolonged exposure to elevated environmental pH . Postmortem examination revealed marked ascites . Culture of the abdominal fluid from three of the eels yielded either Aeromonas hydrophila or Citrobacter freundii, which were most likely secondary invaders . Histopathology showed marked iron accumulation in both hepatocytes and hepatic macrophage aggregates.

Clin Infect Dis, 1999 Feb, 28(2), 384 - 94
The role of Citrobacter in clinical disease of children: review; Doran TI; Various species of Citrobacter may cause infections in neonates and immunocompromised hosts . Citrobacter koseri (formerly Citrobacter diversus) is best known as the cause of sepsis and meningitis leading to central nervous system (CNS) abscesses in neonates and young infants . Early onset and late-onset infections occur as for other neonatal bacterial infections . The majority of cases are sporadic, with no clear source of infection . A few have been confirmed to be vertically transmitted, and nosocomial outbreaks have occurred in neonatal care units . The pathophysiology is not well understood, but a surface protein has been identified as a possible virulence factor among strains that cause citrobacter brain abscesses in neonates . Despite improvements in diagnostic imaging techniques, surgery, and antibiotic therapy, approximately one-third of infants with abscesses die, and one-half sustain CNS damage . In this article, the taxonomy, epidemiology, pathogenesis, diagnosis, treatment, and outcome of citrobacter disease in children are reviewed.

Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 1998, 9(2), 55 - 8
Study of rapidity and quality for the VITEK susceptibility cards with 45 wells; Miyamoto H et al.; VITEK 45 wells Card was evaluated . Recent clinical isolates of gram-positive cocci (107) and gram-negative bacilli (179) were antimicrobial susceptibility testing by VITEK system in comparison with MIC-2000 system . The antimicrobial susceptibility results were available within of 8 h for gram-positive cocci without a part of MRSA . Enterobac-teriaceae were available within of 8 h and Pseudomonas aeruginosa were from 8 to 11 h . The antimicrobial susceptibility data had disagreement of 60% with EM, 46% with CLDM for Enterococcus faecium, 50% with CMZ for Citrobacter freundii, and 71% with CTM for Morganella morganii, whereas it was satisfactory agreement of 97.5% for gram-positive cocci and 98.2% for gram-negative bacilli . In using this procedure it was possible to provide accurate and rapid results of anti-microbial susceptibility tests for all organisms.

Appl Environ Microbiol, 1999 Feb, 65(2), 807 - 12
A novel chromogenic ester agar medium for detection of Salmonellae; Cooke VM et al.; A novel agar medium, chromogenic Salmonella esterase (CSE) agar, for the differentiation of salmonellae is described . The agar contains peptones and nutrient extracts together with the following (grams per liter unless otherwise specified): 4-{2-(4-octanoyloxy-3, 5-dimethoxyphenyl)-vinyl}-quinolinium-1-(propan-3-yl carboxylic acid) bromide (SLPA-octanoate; bromide form), 0.3223; lactose, 14 . 65; trisodium citrate dihydrate, 0.5; Tween 20, 3.0; ethyl 4-dimethylaminobenzoate, 0.035% (wt/vol), novobiocin, 70 mg liter-1 . The key component of the medium is SLPA-octanoate, a newly synthesized ester formed from a C8 fatty acid and a phenolic chromophore . In CSE agar, the ester is hydrolyzed by Salmonella spp . to yield a brightly colored phenol which remains tightly bound within colonies . After 24 h of incubation at 37 or 42 degreesC, colonies of typical Salmonella spp . were burgundy colored on a transparent yellow background, whereas non-Salmonella spp . were white, cream, yellow or transparent . CSE agar was evaluated by using a panel of strains including a high proportion of Salmonella and non-Salmonella strains giving atypical reactions on other differential agars . The sensitivity (93.1%) of CSE agar for non-typhi salmonellae compared favorably with those of Rambach (82 . 8%), xylose-lysine-deoxycholate (XLD; 91.4%), Hektoen-enteric (89.7%), and SM ID (91.4%) agars . The specificity (93.9%) was also comparable to those of other Salmonella media (SM ID agar, 95.9%; Rambach agar, 91.8%; XLD agar, 91.8%; Hektoen-enteric agar, 87.8%) . Strains of Citrobacter freundii and Proteus spp . giving false-positive reactions with other media gave a negative color reaction on CSE agar . CSE agar enabled the detection of >30 Salmonella serotypes, including agona, anatum, enteritidis, hadar, heidelberg, infantis, montevideo, thompson, typhimurium, and virchow, which accounted for 91.8% of the salmonella isolates recorded by the Public Health Laboratory Service (Colindale, London, England) for 1997.

Antimicrob Agents Chemother, 1999 Feb, 43(2), 307 - 13
Characterization of SFO-1, a plasmid-mediated inducible class A beta-lactamase from Enterobacter cloacae; Matsumoto Y et al.; Enterobacter cloacae 8009 produced an inducible class A beta-lactamase which hydrolyzed cefotaxime efficiently . It also hydrolyzed other beta-lactams except cephamycins and carbapenems . The activity was inhibited by clavulanic acid and imipenem . The bla gene was transferable to Escherichia coli by electroporation of plasmid DNA . The molecular mass of the beta-lactamase was 29 kDa and its pI was 7.3 . All of these phenotypic characteristics of the enzyme except for inducible production resemble those of some extended-spectrum class A beta-lactamases like FEC-1 . The gene encoding this beta-lactamase was cloned and sequenced . The deduced amino acid sequence of the beta-lactamase was homologous to the AmpA sequences of the Serratia fonticola chromosomal enzyme (96%), MEN-1 (78%), Klebsiella oxytoca chromosomal enzymes (77%), TOHO-1 (75%), and FEC-1 (72%) . The conserved sequences of class A beta-lactamases, including the S-X(T)-X(S)-K motif, in the active site were all conserved in this enzyme . On the basis of the high degree of homology to the beta-lactamase of S . fonticola, the enzyme was named SFO-1 . The ampR gene was located upstream of the ampA gene, and the AmpR sequence of SFO-1 had homology with the AmpR sequences of the chromosomal beta-lactamases from Citrobacter diversus (80%), Proteus vulgaris (68%), and Pseudomonas aeruginosa (60%) . SFO-1 was also inducible in E . coli . However, a transformant harboring plasmid without intact ampR produced a small amount of beta-lactamase constitutively, suggesting that AmpR works as an activator of ampA of SFO-1 . This is the first report from Japan describing an inducible plasmid-mediated class A beta-lactamase in gram-negative bacteria.

J Vet Med Sci, 1998 Dec, 60(12), 1315 - 9
Analysis of enterohemorrhagic Escherichia coli serotype O157:H7 by flow cytometry using monoclonal antibodies; Kusunoki H et al.; To develop a rapid and specific method to detect and/or identify enterohemorrhagic Escherichia coli O157:H7, two mouse monoclonal antibodies (MAbs) were prepared . Specificities of these two MAbs (1D9 and 3E8) were determined by flow cytometry method (FCM) . MAbs 3E8 and 1D9 were found to react with E . coli O157:H7, Citrobacter freundii and Salmonella group N (O:30), but not with Escherichia hermannii . With a mixture containing strains of E . coli O157:H7 and E . coli O6:H1, two different peaks appeared in FCM with MAbs, whereas a single peak appeared with polyclonal rabbit antiserum . From these findings, FCM with MAb is suggested to be a rapid, specific, and useful method to detect and identify strain(s) of E . coli O157:H7 in food ingredients.

Arch Pharm Res, 1998 Oct, 21(5), 527 - 30
Comparative activities of novel beta-lactamase inhibitors, 6-exomethylene penamsulfones (CH1240, CH2140) in experimental mouse infection model; Park KW et al.; The antibacterial activity of novel beta-lactamase inhibitors, 6-exomethylene penamsulfones (CH1240, CH2140), has been compared in vivo with that of sulbactam and clavulanic acid against beta-lactamase producing strains . In vivo microbiological assessment was used as experimental mouse infection model by gram negative strains . Against Pseudomonas aeruginosa F0013, cefoperazone/CH1240 was slightly less active than sulbactam . Ampicillin/CH1240 was more active than sulbactam against Citrobacter diversus species . That of ampicillin/CH2140 was less effective than sulbactam against Escheriachia coli 3457 . Especially against Citrobacter diversus 2046E, amoxicillin/CH2140 was the most potent and amoxicillin/CH1240 was slightly more active than clavulanic acid . Consequently the difference in efficacy between the drug combinations appears to be related to the degree of protection afforded the animals by the beta-lactamase inhibitors . CH1240 and CH2140 are promising new agents and should undergo further investigations.

FEMS Microbiol Lett, 1998 Dec 15, 169(2), 235 - 40
Characterisation of CMY-4, an AmpC-type plasmid-mediated beta-lactamase in a Tunisian clinical isolate of Proteus mirabilis; Verdet C et al.; A strain of Proteus mirabilis resistant to beta-lactams, including cefoxitin, was isolated from the urine of a woman from Tunisia . Its antibiotic susceptibility pattern and that of the Escherichia coli transconjugant suggested the presence of an AmpC-type beta-lactamase . Two bands of beta-lactamase activity (pI 5.4 and 9.2) were detected by isoelectric focusing . The nucleotide sequence of the gene encoding the AmpC-type enzyme was determined . The deduced amino acid sequence was 98-99% identical to CMY-3 and to those of the plasmid-mediated AmpC-type beta-lactamases originated from Citrobacter freundii and 97% identical to the chromosome-encoded beta-lactamase of a Tunisian clinical isolate of C . freundii . This enzyme differs from CMY-2 by one substitution (Arg for Trp at position 221) and from CMY-3 by two substitutions (Glu for Gly at position 42 and Ser for Asn at position 363) and we propose the denomination CMY-4.

Eur J Clin Microbiol Infect Dis, 1998 Oct, 17(10), 727 - 30
Utility of serial rectal swab cultures for detection of ceftazidime- and imipenem-resistant gram-negative bacilli from patients in the intensive care unit; Arbo MD et al.; Forty-four patients receiving intensive care were studied prospectively to assess the utility of serial rectal swab cultures and clinical correlates of resistance for Pseudomonas aeruginosa, Enterobacter spp., Citrobacter spp., Morganella morganii, and Serratia marcescens strains resistant to ceftazidime or imipenem . Strains of Pseudomonas aeruginosa, Enterobacter spp., Citrobacter spp., or Morganella morganii were found in 26 of 44 (59%) patients: 17 (65%) in clinical sites (11 with concomitant rectal isolates) and nine (35%) in a rectal site only . Of 49 total isolates, 13 (26.5%) were resistant: 10 (20.4%) to ceftazidime and three (6.1%) to imipenem . Surveillance rectal swabs from 27 patients without a clinical isolate identified two patients with resistant organisms (15% of all resistant isolates) . The majority of resistance to ceftazidime or imipenem among Pseudomonas or Enterobacter can be detected by the use of clinical specimens alone.

Appl Microbiol Biotechnol, 1998 Oct, 50(4), 468 - 74
Cloning, nucleotide sequencing, and expression of the 3-methylaspartate ammonia-lyase gene from Citrobacter amalonaticus strain YG-1002; Kato Y et al.; The gene coding for 3-methylaspartate ammonia-lyase (3-methylaspartase, MAL, EC 4.3.1.2) from Citrobacter amalonaticus strain YG-1002 (TPU 6323) was cloned onto plasmid pBluescript II KS(+), and the nucleotide sequence of the 1239-bp open reading frame (ORF), consisting of 413 codons, was identified as the mal gene coding for MAL . The predicted polypeptide has 62.5% identity with MAL from the obligate anaerobe, Clostridium tetanomorphum NCIMB 11547 . ORF1, which showed 58.6% and 58.8% identities with subunit E of the glutamate mutases of C . tetanomorphum and Clostridium cochlearium respectively, was found in the upstream region of the mal gene . An expression plasmid pMALCA3 (5.4 kb), in which the mal gene was expressed under control of the lac promoter on the vector, was constructed . With feeding of 1 mM isopropyl beta-D-thiogalactopyranoside, the amount of the enzyme in a cell-free extract of the transformant, E . coli JM109/pMALCA3, was elevated to 51,800 units/l culture, which is about 50-fold that of C . amalonaticus strain YG-1002 . It was calculated that the enzyme comprised over 40% of the total extractable cellular proteins . The enzyme produced by the E . coli transformant was purified in a crystalline form and shown to be identical to that of the wild-type strain with respect to specific activity, molecular mass, subunit structure, enzymological properties, and N-terminal amino acid sequences.

J Bacteriol, 1998 Dec, 180(23), 6173 - 86
Integration host factor and cyclic AMP receptor protein are required for TyrR-mediated activation of tpl in Citrobacter freundii; Bai Q et al.; The tpl gene of Citrobacter freundii encodes an enzyme that catalyzes the conversion of L-tyrosine to phenol, pyruvate, and ammonia . This gene is known to be positively regulated by TyrR . The amplitude of regulation attributable to this transcription factor is at least 20-fold . Three TyrR binding sites, designated boxes A, B, and C, centered at coordinates -272.5, -158.5, and -49.5, respectively, were identified in the upstream region of the tpl promoter . The results of mutational experiments suggest that TyrR binds in cooperative fashion to these sites . The nonavailability of any TyrR site impairs transcription . Full TyrR-mediated activation of tpl required integration host factor (IHF) and the cAMP receptor protein (CRP) . By DNase I footprinting, it was shown that the IHF binding site is centered at coordinate -85 and that there are CRP binding sites centered at coordinates -220 and -250 . Mutational alteration of the IHF binding site reduced the efficiency of the tpl promoter by at least eightfold . The proposed roles of CRP and IHF are to introduce bends into tpl promoter DNA between boxes A and B or B and C . Multimeric TyrR dimers were demonstrated by a chemical cross-linking method . The formation of hexameric TyrR increased when tpl DNA was present . The participation of both IHF and CRP in the activation of the tpl promoter suggests that molecular mechanisms quite different from those that affect other TyrR-activated promoters apply to this system . A model wherein TyrR, IHF, and CRP collaborate to regulate the expression of the tpl promoter is presented.

J Food Prot, 1998 Nov, 61(11), 1480 - 3
Aspects of microbiological and chemical quality of turmus, lupin seeds debittered by soaking in water; Yamani MI et al.; Eleven species of spherical lactic acid bacteria (LAB) belonging to the genera Leuconostoc, Lactococcus, Enterococcus and Pediococcus were the predominant microorganisms in 40 samples of turmus, ready-to-eat lupin seeds debittered by boiling and soaking in water . The average counts of the LAB in the 20 winter samples and the 20 summer samples were 7.4 and 8.7 log CFU/g, respectively . The averages of the Enterobacteriaceae counts were 5.1 and 6.6 log CFU/g, respectively, and the 11 species isolated belonged to the genera Enterobacter, Citrobacter, Escherichia and Klebsiella . The average yeast counts in winter and summer samples were 3 and 3.2 log CFU/g, respectively, and the 5 species isolated were in the genera Saccharomyces, Cryptococcus, Rhodotorula and Candida . Although Salmonella was not isolated from any sample and the Staphylococcus aureus count in all samples was < 1 log CFU/g, microbial hazards could be associated with the high Enterobacteriaceae counts and the presence of Escherichia coli . Total alkaloid concentration in 30% of the samples examined was higher than 0.02%, thus making the seeds a potential chemical hazard . Boiling the turmus directly before consumption and discarding the seeds with a bitter taste may help in avoiding some of the microbial and chemical hazards which could be associated with turmus consumption.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Sep-Oct, (5), 36 - 9
{Conservative regions of the gene of thermostable enterotoxin in Enterobacteriaceae--the principal possibility of their practical use}; Mavziutov AR et al.; The nucleotide sequences connected with the production of thermostable enterotoxins (ST) by the representatives of Enterobacteriaceae were analyzed . The conservative area sized up to 30 pairs of nucleotides at the 3'-end of all ST-genes, present in the bank, and responsible for the enterotoxicity of ST-toxin molecules was detected . On its basis 3 oligonucleotides were synthesized; 2 of them were used as primers in experiments on the amplification of the DNA of enterotoxigenic strains of Citrobacter spp., Escherichia coli and Yersinia spp . and the third one was used as a probe in experiments on dot-blot hybridization with the DNA of the above-mentioned cultures . The universal diagnostic test system making it possible to detect the ST-enterotoxin of opportunistic enterobacteria irrespective of their species was proposed.

Diagn Microbiol Infect Dis, 1998 Oct, 32(2), 85 - 94
Activity of a broad-spectrum cephalosporin (Ro 48-8391) alone and in combination with two novel beta-lactamase inhibitors (Ro 48-5545 and Ro 48-8724); Jones RN et al.; The susceptibility of a group of beta-lactamase-producing and drug-resistant Gram-positive and Gram-negative organisms was tested against a novel cephalosporin (Ro 48-8391) alone and in combination with two bridged carbacephem beta-lactamase inhibitors (Ro 48-5545 or Ro 48-8724) and compared with that of ceftriaxone, ceftazidime, and cefepime (representative "third- and fourth-generation" cephalosporins), imipenem, and a combination of piperacillin and tazobactam . Five hundred and one selected clinical isolates were tested using the reference broth microdilution method (National Committee for Clinical Laboratory Standards) . Ro 48-8391 has a spectrum of activity and potency most similar to ceftriaxone but with improved activity against Gram-positive species . The two beta-lactamase inhibitors, Ro 48-5545 and Ro 48-8724, have modest antimicrobial activity . When combined with Ro 48-8391, the beta-lactamase inhibitor Ro 48-8724 was superior to the combination of Ro 48-8391 and Ro 48-5545 in spectrum and enzyme inhibition against extended spectrum beta-lactamase enzyme-producing Escherichia coli and Klebsiella pneumoniae, and against Enterobacteriaceae with "stably derepressed" Bush-Jacoby-Medeiros gr 1 enzymes (ceftazidime-resistant Enterobacter and Citrobacter) . Ro 48-5545 and Ro 48-8724 appear to be promising beta-lactamase inhibitors with potential application against chromosomal- and plasmid-mediated enzymes . Ro 48-8391, although superior to some currently available "third-generation" cephems, was not a well-matched active codrug because of limited activity against several commonly isolated species of clinically important bacteria . Further efforts are necessary to find a penicillin or cephem with activity more complementary to that of the tested beta-lactamase inhibitors and the Ro 48-8391 compound could be focused for therapeutic use in serious streptococcal infections.

Biochemistry, 1998 Nov 17, 37(46), 16082 - 92
Three-dimensional structure of AmpC beta-lactamase from Escherichia coli bound to a transition-state analogue: possible implications for the oxyanion hypothesis and for inhibitor design; Usher KC et al.; The structures of AmpC beta-lactamase from Escherichia coli, alone and in complex with a transition-state analogue, have been determined by X-ray crystallography . The native enzyme was determined to 2.0 A resolution, and the structure with the transition-state analogue m-aminophenylboronic acid was determined to 2.3 A resolution . The structure of AmpC from E . coli resembles those previously determined for the class C enzymes from Enterobacter cloacae and Citrobacter freundii . The transition-state analogue, m-aminophenylboronic acid, makes several interactions with AmpC that were unexpected . Perhaps most surprisingly, the putative "oxyanion" of the boronic acid forms what appears to be a hydrogen bond with the backbone carbonyl oxygen of Ala318, suggesting that this atom is protonated . Although this interaction has not previously been discussed, a carbonyl oxygen contact with the putative oxyanion or ligand carbonyl oxygen appears in most complexes involving a beta-lactam recognizing enzyme . These observations may suggest that the high-energy intermediate for amide hydrolysis by beta-lactamases and related enzymes involves a hydroxyl and not an oxyanion, although the oxyanion form certainly cannot be discounted . The involvement of the main-chain carbonyl in ligand and transition-state recognition is a distinguishing feature between serine beta-lactamases and serine proteases, to which they are often compared . AmpC may use the interaction between the carbonyl of Ala318 and the carbonyl of the acylated enzyme to destabilize the ground-state intermediate, this destabilization energy might be relieved in the transition state by a hydroxyl hydrogen bond . The structure of the m-aminophenylboronic acid adduct also suggests several ways to improve the affinity of this class of inhibitor and points to the existence of several unusual binding-site-like features in the region of the AmpC catalytic site.

J Antimicrob Chemother, 1998 Oct, 42(4), 419 - 25
Transferable class C beta-lactamases in Escherichia coli strains isolated in Greek hospitals and characterization of two enzyme variants (LAT-3 and LAT-4) closely related to Citrobacter freundii AmpC beta-lactamase; Gazouli M et al.; Among 2133 isolates of Escherichia coli obtained during 1996 from 10 Greek hospitals, 63 (3%) were resistant to cefoxitin . Typing by ERIC2-PCR indicated that the cefoxitin-resistant (FOXr) isolates were distinct . beta-Lactamase studies and hybridization experiments showed that most strains produced beta-lactamases related to the AmpC chromosomal cephalosporinase of Citrobacter freundii . The enzymes were encoded by similar non-self-transmissible plasmids . The bla genes encoding two beta-lactamases (LAT-3 and LAT-4) with isoelectric points 8.9 and 9.4, respectively, were cloned and sequenced . The deduced amino acid sequences displayed a high degree of homology (>95%) with the AmpC beta-lactamase of C . freundii . The patterns of resistance to beta-lactams of the FOXr E . coli depended on the quantity of class C enzymes and the simultaneous expression of other beta-lactamases . In a few isolates a 36 kDa outer-membrane protein, presumably a porin, was not expressed at detectable quantities . These isolates were resistant to cefoxitin, and their susceptibility to the other beta-lactams tested was not significantly decreased.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Jul-Aug, (4), 61 - 3
{The use of random amplification of polymorphous bacterial DNA for the typing of bacteria in the genus Citrobacter}; Akhunov ED et al.; To obtain the profiles of randomly amplified DNA, isolated from bacteria of the genus Citrobacter, the method of polymerase chain reaction was used . Nine oligonucleotides were evaluated for the possibility of their use as primers for the amplification of random polymorphous sequences of DNA; of these, 2 oligonucleotides which generated profiles, sufficiently reproducible and typical for different C . freundii and C . diversus strains, were selected . The possibility of using the above oligonucleotides in pair for amplification of species-specific fragments of polymorphous bacterial DNA for typing was shown.

Zh Mikrobiol Epidemiol Immunobiol, 1998 Jul-Aug, (4), 17 - 9
{An additional dehydrogenase or oxidation-reduction test in the classification of enterobacteria}; Bril'man IaE; The dehydrogenase (DHG) or oxidation-reduction test is proposed for use together with the determination of such enzymes as hydrolases, cytochrome oxidase, dehydrocarboxylase, urease, etc . 200 Citrobacter freundii cultures and 76 strains of enteropathogenic Escherichia (EPE) were studied with the determination of their DHG activity in semiliquid mannitol and in Kligler's medium . The study revealed that this test, characterized by the reduction of the indicator, similarly to that in salmonellae and shigellae, was constantly negative in semiliquid mannitol in C . freundii and in 97.3% of cases in EPE . In 17.5% of C . freundii lactose-positive cultures the DHG test in Kligler's medium was positive, which made it possible to regard them as a separate biovar . Taking into account the results of this investigation, the subdivision of C . freundii into 3 biovars is proposed.

Syst Appl Microbiol, 1998 Aug, 21(3), 384 - 97
Phylogenetic position of phytopathogens within the Enterobacteriaceae; Hauben L et al.; The almost complete 16S rDNA sequences of twenty nine plant-associated strains, representing species of the genera Erwinia, Pantoea and Enterobacter were determined and compared with those of other members of the Enterobacteriaceae . The species of the genus Erwinia may be divided into three phylogenetic groups . Cluster I represents the true erwinias and comprises E . amylovora, E . mallotivora, E . persicinus, E . psidii, E . rhapontici and E . tracheiphila . We propose to unite the species of cluster II, E . carotovora subsp . atroseptica, E . carotovora subsp . betavasculorum, E . carotovora subsp . carotovora, E . carotovora subsp . odorifera, E . carotovora subsp . wasabiae, E . cacticida, E . chrysanthemi and E . cypripedii in the genus Pectobacterium respectively as P . carotovorum subsp . atrosepticum comb . nov., P . carotovorum subsp . betavasculorum comb . nov., P . carotovorum subsp . carotovorum comb . nov., P . carotovorum subsp . odoriferum comb . nov., P . carotovorum subsp . wasabiae comb . nov., P . cacticidum comb . nov., P . chrysanthemi and P . cypripedii . The species E . alni, E . nigrifluens, E . paradisiaca, E . quercina, E . rubrifaciens and E . salicis, comprising cluster III, are being classified into a new genus Brenneria gen . nov . respectively as B . alni comb . nov., B . nigrifluens comb . nov., B . paradisiaca comb . nov., B . quercina comb . nov., B . rubrifaciens comb . nov . and B . salicis comb . nov . The species of the genus Pantoea, included in this study, form a monophyletic unit (cluster IV), closely related with Erwinia, whereas the three phytopathogenic species of the genus Enterobacter are scattered among the genera Citrobacter and Klebsiella.

J Bacteriol, 1998 Oct, 180(20), 5478 - 83
Gram-negative bacteria produce membrane vesicles which are capable of killing other bacteria; Li Z et al.; Naturally produced membrane vesicles (MVs), isolated from 15 strains of gram-negative bacteria (Citrobacter, Enterobacter, Escherichia, Klebsiella, Morganella, Proteus, Salmonella, and Shigella strains), lysed many gram-positive (including Mycobacterium) and gram-negative cultures . Peptidoglycan zymograms suggested that MVs contained peptidoglycan hydrolases, and electron microscopy revealed that the murein sacculi were digested, confirming a previous modus operandi (J . L . Kadurugamuwa and T . J . Beveridge, J . Bacteriol . 174:2767-2774, 1996) . MV-sensitive bacteria possessed A1alpha, A4alpha, A1gamma, A2alpha, and A4gamma peptidoglycan chemotypes, whereas A3alpha, A3beta, A3gamma, A4beta, B1alpha, and B1beta chemotypes were not affected . Pseudomonas aeruginosa PAO1 vesicles possessed the most lytic activity.

Appl Microbiol Biotechnol, 1998 Aug, 50(2), 266 - 72
The use of Escherichia coli bearing a phoN gene for the removal of uranium and nickel from aqueous flows; Basnakova G et al.; A Citrobacter sp . originally isolated from metal-polluted soil accumulates heavy metals via metalphosphate deposition utilizing inorganic phosphate liberated via PhoN phosphatase activity . Further strain development was limited by the non-transformability of this environmental isolate . Recombinant Escherichia coli DH5 alpha bearing cloned phoN or the related phoC acquired metal-accumulating ability, which was compared with that of the Citrobacter sp . with respect to removal of uranyl ion (UO2(2+)) from dilute aqueous flows and its deposition in the form of polycrystalline hydrogen uranyl phosphate (HUO2PO4) . Subsequently, HUO2PO4-laden cells removed Ni2+ from dilute aqueous flows via intercalation of Ni2+ into the HUO2PO4 lattice . Despite comparable acid phosphatase activity in all three strains, the E . coli DH5 alpha (phoN) construct was superior to Citrobacter N14 in both uranyl and nickel accumulation, while the E . coli DH5 alpha (phoC) construct was greatly inferior in both respects . Expression of phosphatase activity alone is not the only factor that permits efficient and prolonged metal phosphate accumulation, and the data highlight possible differences in the PhoN and PhoC phosphatases, which are otherwise considered to be related in many respects.

Arch Pediatr, 1998, 5 Suppl 3, 266S - 268S
{Antibiotic sensitivity to isolated bacteria in pediatric urinary tract infections}; Vu-Thien H; Of the childhood urinary tract infections, more than 50% are caused by Escherichia coli (E Coli), followed by Proteus mirabilis (P mirabilis), Klebsiella sp, other enterobacteria, enterococci, Pseudomonas aeruginosa, and staphylococci . Of E coli isolates, 50 to 60% are resistant to ampicillin (ampi-R), 10% being susceptible to amoxicillin + clavulanic acid (AMC) . For P mirabilis, ampi-R isolates are less frequent and more often susceptible to AMC . Klebsiella sp is resistant to ampicillin, 75% of isolates being susceptible to AMC . In these three species, the susceptibility of isolates to third generation cephalosporins, aminogly-cosides, and ciprofloxacin is still high (> 90%), but 15 to 35% are resistant to cotrimoxazole . In the other enterobacteria (Enterobacter cloacae, Morganella morganii, P vulgaris, Citrobacter freundii and Serratia marcescens) the resistance to cefalotin (hence to ampicillin and AMC) is permanent, with an exception: the susceptibility of P vulgaris to AMC . Enterococci are mostly susceptible to ampicillin, and P aeruginosa to ceftazidime, but in both species, the percentage of resistant strains in hospitalised patients is greater than in outpatients . For Staphylococcus aureus, the community-acquired isolates are susceptible to oxacillin and other anti-staphylococcal agents . All the coagulase negative staphylococci isolates are susceptible to vancomycin, but 70% of those from hospitalised patients are resistant to oxacillin, aminoglycosides and cotrimoxazole.

Antimicrob Agents Chemother, 1998 Oct, 42(10), 2661 - 7
gyrA mutations associated with fluoroquinolone resistance in eight species of Enterobacteriaceae; Weigel LM et al.; Fluoroquinolone resistance (FQ-R) in clinical isolates of Enterobacteriaceae species has been reported with increasing frequency in recent years . Two mechanisms of FQ-R have been identified in gram-negative organisms: mutations in DNA gyrase and reduced intracellular drug accumulation . A single point mutation in gyrA has been shown to reduce susceptibility to fluoroquinolones . To determine the extent of gyrA mutations associated with FQ-R in enteric bacteria, one set of oligonucleotide primers was selected from conserved sequences in the flanking regions of the quinolone resistance-determining regions (QRDR) of Escherichia coli and Klebsiella pneumoniae . This set of primers was used to amplify and sequence the QRDRs from 8 Enterobacteriaceae type strains and 60 fluoroquinolone-resistant clinical isolates of Citrobacter freundii, Enterobacter aerogenes, Enterobacter cloacae, E . coli, K . pneumoniae, Klebsiella oxytoca, Providencia stuartii, and Serratia marcescens . Although similarity of the nucleotide sequences of seven species ranged from 80.8 to 93.3%, when compared with that of E . coli, the amino acid sequences of the gyrA QRDR were highly conserved . Conservative amino acid substitutions were detected in the QRDRs of the susceptible type strains of C . freundii, E . aerogenes, K . oxytoca (Ser-83 to Thr), and P . stuartii (Asp-87 to Glu) . Strains with ciprofloxacin MICs of >2 microg/ml expressed amino acid substitutions primarily at the Gly-81, Ser-83, or Asp-87 position . Fluoroquinolone MICs varied significantly for strains exhibiting identical gyrA mutations, indicating that alterations outside gyrA contribute to resistance . The type and position of amino acid alterations also differed among these six genera . High-level FQ-R frequently was associated with single gyrA mutations in all species of Enterobacteriaceae in this study except E . coli.

J Hosp Infect, 1998 Aug, 39(4), 291 - 300
A hospital outbreak of extended-spectrum beta-lactamase-producing Klebsiella pneumoniae investigated by RAPD typing and analysis of the genetics and mechanisms of resistance; Shannon K et al.; Between July and September 1997 a ceftazidime- and aminoglycoside-resistant strain of Klebsiella pneumoniae infected or colonized seven patients on three paediatric wards at Guy's Hospital in London . The patients were mostly neonates or infants recovering from cardiac surgery for congenital defects . The organism was probably introduced by an asymptomatic patient from Greece and the subsequent outbreak could largely be explained by person-to-person spread on individual wards and frequent transfers of patients between wards . The outbreak was controlled by patient isolation and attention to handwashing, and there were no fatalities . The organisms were non-typeable by serology but had a characteristic RAPD profile . They produced the extended spectrum beta-lactamase SHV-5 and the aminoglycoside-modifying enzymes AAC(6') + probably AAC(3)II, encoded on a conjugative plasmid of approximately 160 kb . Two other patients had multi-resistant klebsiellas, one of them an SHV-5 producer and one a TEM-5 producer, but these could be distinguished from each other and from the outbreak strain by serological and RAPD typing and by the genetics and mechanisms of their resistances . Three other multi-resistant enterobacteria were isolated during the outbreak: an Escherichia coli that had acquired the 160 kb resistance plasmid from the epidemic klebsiella, a Citrobacter isolated from one of the patients with the klebsiella but which did not produce SHV-5, and a TEM-5-producing Enterobacter . This outbreak illustrates the importance of screening patients from high-risk areas for multiply-resistant organisms on admission, and the value of bacterial typing and analysis of resistance mechanisms to define the epidemiology of hospital infection.

Skin Pharmacol Appl Skin Physiol, 1998 May-Jun, 11(3), 140 - 51
Silver nitrate: antimicrobial activity related to cytotoxicity in cultured human fibroblasts; Hidalgo E et al.; The aims of this study were to ascertain whether silver nitrate (AgNO3) concentrations below those used in clinical practice inhibit bacterial growth, and in parallel study the cytotoxic effects on human fibroblasts . The cytoprotective effects of fetal calf serum (FCS) were also evaluated . The cytotoxic effects of eight different silver nitrate concentrations were determined by assessing mitochondrial activity of viable cells capable of cleaving tetrazolium salts . Antimicrobial activity of AgNO3, range: 7-550 x 10(-5)%, was tested against Staphylococcus aureus, Citrobacter freundii, and Pseudomonas aeruginosa . Silver nitrate concentrations exerting antimicrobial effects were: S . aureus, >70 x 10(-5)%; P . aeruginosa, >/=270 x 10(-5)%, and C . freundii, >/=550 x 10(-5)% . With 2% FCS, the lowest AgNO3 concentration studied (7 x 10(-5)%) showed cytotoxic effects (cell survival 71 +/- 19%) at only 2 h of incubation . Under these conditions AgNO3 cytotoxicity was time- and concentration-dependent in all exposure periods . Cytotoxicity was greatly enhanced causing 76% fibroblast growth inhibition at concentrations of 14 x 10(-5)% and contact time of 2 h . The AgNO3 concentration of 7 x 10(-5)% was also cytotoxic with 5% FCS in the media compared with controls, although cell survival was higher than with 2% FCS . The cytoprotective action of FCS was clearly shown at the concentration of 10% at which AgNO3 cytotoxicity of 7 x 10(-5)% to 28 x 10(-5)% was partially or completely inhibited . Our results show that AgNO3 at concentrations 100-700 times more diluted than that normally used in clinical practice retained effective inhibitory activity against some of the above-mentioned microorganisms . However, even these concentrations are cytotoxic for cultured fibroblasts . Thus, silver nitrate concentrations up to 100 times more diluted can be used, since they possess bacterial growth-inhibiting power, are less cytotoxic and therefore favour wound healing.

Kansenshogaku Zasshi, 1998 Jul, 72(7), 738 - 41
{Identification of Escherichia coli O157 antigen by polymerase chain reaction}; Tsukamoto T et al.; An assay was developed for the specific detection of Escherichia coli O157 using PCR, because O serological cross-reactivities have been reported between E . coli O157 and some E . coli, other bacterial species . PCR amplification of E . coli O157 rfbE (Ec O157:H7) gene that is necessary for the expression of the O157 antigen, was performed for the identification of E . coli O157 . All Shiga toxin-producing Escherichia coli (STEC) O157:H7 and O157:H, non-STEC O157 strains were positive, and other non-O157 E . coli strains were negative by PCR . All tested strains of other bacterial species, like Salmonella O30 and Citrobacter freundii which gave positive results with O157 detection kits, were negative by PCR . It is recommended that PCR amplification of O157 rfbE gene is one of the most specific method for E . coli O157 identification.

J Appl Microbiol, 1998 Mar, 84(3), 399 - 403
Development of a new culture medium for the rapid detection of Salmonella by indirect conductance measurements; Blivet D et al.; The main difficulties in conductance medium development are to allow Salmonella to grow and produce a conductance signal while impeding growth of related species such as Escherichia coli and Citrobacter freundii . Various selective agents were screened for these capacities and a new medium was derived, named KIMAN (Whitley Impedance Broth basal medium supplemented with three selective components: novobiocin, malachite green and potassium iodide) . This medium supported the growth of Salmonella serotypes and inhibited non-salmonella strains in pure cultures.

Mol Microbiol, 1998 Jul, 29(2), 559 - 70
Generation of Escherichia coli intimin derivatives with differing biological activities using site-directed mutagenesis of the intimin C-terminus domain; Frankel G et al.; Intimins, encoded by eae genes, are outer membrane proteins involved in attaching-effacing (A/E) lesion formation and host cell invasion by pathogenic bacteria, including enteropathogenic Escherichia coli (EPEC) and Citrobacter rodentium . A series of intimins, harbouring specific mutations close to the C-terminus, were constructed using pCVD438, which encodes the eae gene from EPEC strain E2348/69 . These mutant plasmids were introduced into EPEC strain CVD206 and C . rodentium strain DBS255, which both contain deletion mutations in their eae genes . CVD206, CVD206(pCVD438) and CVD206(pCVD438) derivatives were assessed for their ability to promote A/E lesion formation or invasion of HEp-2 cells and to induce A/E lesions on fresh human intestinal in vitro organ cultures (IVOC) . The pathogenicity of C . rodentium DBS255 harbouring these plasmid derivatives was also studied in mice . Here, we report that intimin-mediated A/E lesion formation can be segregated from intimin-mediated HEp-2 cell invasion . Moreover, adherence to IVOC, EPEC-induced microvillus elongation and colonization of the murine intestine by C . rodentium were also modulated by the modified intimins.

Int J Antimicrob Agents, 1998 May, 10(2), 135 - 41
Study of beta-lactam resistance in ceftazidime-resistant clinical isolates of Enterobacteriaceae; Bujdakova H et al.; Mechanisms and transferability of beta-lactam resistance in 50 ceftazidime resistant strains of Enterobacteriaceae was studied . These strains were selected from 1991 E . coli, 1035 Enterobacter spp., 168 Citrobacter spp . and 1371 Klebsiella spp., isolated from patients hospitalized in ICUs and in the pediatric and urology departments of six hospitals in Bratislava during the years 1994-1996 . The selected strains expressed the resistance not only to ceftazidime (50/50) but also to ampicillin (50/50), ceftriaxone (50/50), cefotaxime (49/50) and cefoxitin (45/50) . The mechanism of resistance in all 50 strains was the production of beta-lactamases by conjugation, using either ceftazidime or cefotaxime for the selection of transconjugants and by isolation of R-plasmids ranging from to 55-87 kb from donor strains and from transconjugants . A total of 21 isolates possessed chromosomally encoded resistance and 25 clinical isolates and their transconjugants expressed ESBL sensitive to clavulanate . Selected E . coli and Klebsiella pneumoniae isolates expressed the presence of TEM and SHV enzymes determined by isoelectric focusing . The possible trends in the development of antimicrobial resistance in Slovakia in the future are indicated.

Infect Immun, 1998 Sep, 66(9), 4305 - 12
Comparison of sample sequences of the Salmonella typhi genome to the sequence of the complete Escherichia coli K-12 genome; McClelland M et al.; Raw sequence data representing the majority of a bacterial genome can be obtained at a tiny fraction of the cost of a completed sequence . To demonstrate the utility of such a resource, 870 single-stranded M13 clones were sequenced from a shotgun library of the Salmonella typhi Ty2 genome . The sequence reads averaged over 400 bases and sampled the genome with an average spacing of once every 5,000 bases . A total of 339,243 bases of unique sequence was generated (approximately 7% representation) . The sample of 870 sequences was compared to the complete Escherichia coli K-12 genome and to the rest of the GenBank database, which can also be considered a collection of sampled sequences . Despite the incomplete S . typhi data set, interesting categories could easily be discerned . Sixteen percent of the sequences determined from S . typhi had close homologs among known Salmonella sequences (P < 1e-40 in BlastX or BlastN), reflecting the proportion of these genomes that have been sequenced previously; 277 sequences (32%) had no apparent orthologs in the complete E . coli K-12 genome (P > 1e-20), of which 155 sequences (18%) had no close similarities to any sequence in the database (P > 1e-5) . Eight of the 277 sequences had similarities to genes in other strains of E . coli or plasmids, and six sequences showed evidence of novel phage lysogens or sequence remnants of phage integrations, including a member of the lambda family (P < 1e-15) . Twenty-three sample sequences had a significantly closer similarity a sequence in the database from organisms other than the E . coli/Salmonella clade (which includes Shigella and Citrobacter) . These sequences are new candidate lateral transfer events to the S . typhi lineage or deletions on the E . coli K-12 lineage . Eleven putative junctions of insertion/deletion events greater than 100 bp were observed in the sample, indicating that well over 150 such events may distinguish S . typhi from E . coli K-12 . The need for automatic methods to more effectively exploit sample sequences is discussed.

Carbohydr Res, 1998 Jan, 306(1-2), 331 - 3
Structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c; Kocharova NA et al.; The following structure of the O-specific polysaccharide of Citrobacter freundii O3a,3b,1c containing D-mannose and D-rhamnose was established using sugar analysis and NMR spectroscopy, including computer-assisted analysis of the 13C NMR spectrum, 2D COSY, H,H-relayed COSY, heteronuclear 13C, 1H correlation (HETCOR), and rotating-frame NOE spectroscopy (ROESY):-->4)-alpha-D-Manp-(1-->3)-beta-D-Rhap-(1-->4) -beta-D-Rhap-(1-->.

J Antimicrob Chemother, 1998 Jun, 41 Suppl D, 25 - 41
Beta-lactamase-mediated resistance and opportunities for its control; Livermore DM; Clinical use of beta-lactams has selected for beta-lactamase-producing organisms . Numerous beta-lactamases are known, and sequencing allows them to be divided into four Classes, A to D, with Classes A and C being the most important . Pharmaceutical chemists have responded to the spread of beta-lactamase-producing organisms by developing stable agents and inhibitors . Stability in penicillins and cephalosporins is achieved by attaching a bulky substituent to the amino group of 6-aminopenicillanic acid or 7-aminocephalosporanic acid, or by replacing the hydrogen on carbon 6 (penicillins) or 7 (cephalosporins) with an alpha-methoxy group . In carbapenems, stability is achieved by incorporation of a simple trans-6-hydroxyethyl group . Beta-lactamase-inhibitory activity occurs in many beta-lactam classes but only clavams and penicillanic acid sulphones have been developed specifically as beta-lactamase inhibitors . These inhibit most Class A and some Class D enzymes but act poorly against Class B and C enzymes . Their success is affected by the amount of enzyme, the permeability of the bacterial cell wall, the partner beta-lactam and the pH . Piperacillin/tazobactam, which combines a good inhibitor of Class A enzymes with a broad-spectrum, easily-protected penicillin, has wide activity against common pathogens, the major exceptions being strains of Enterobacter, Serratia and Citrobacter freundii that produce large amounts of Class C enzymes, and Gram-positive cocci with modified penicillin-binding proteins . Beta-lactamase-stable beta-lactams and inhibitor combinations overcome many existing resistance mechanisms but are themselves selecting new resistances . Few new beta-lactams able to overcome these resistances are advanced in development and consequently the opportunities for control lie mostly in the more prudent use of compounds already available.

Antimicrob Agents Chemother, 1998 Aug, 42(8), 2006 - 11
Rapid detection and evaluation of clinical characteristics of emerging multiple-drug-resistant gram-negative rods carrying the metallo-beta-lactamase gene blaIMP; Hirakata Y et al.; Gram-negative rods (GNR) carrying the transferable carbapenem resistance gene blaIMP, including Pseudomonas aeruginosa and Serratia marcescens, have been isolated from more than 20 hospitals in Japan . Although the emergence of such multiple-drug-resistant bacteria is of utmost clinical concern, little information in regard to the distribution of blaIMP-positive GNR in hospitals and the clinical characteristics of infected patients is available . To address this, a system for the rapid detection of the blaIMP gene with a simple DNA preparation and by enzymatic detection of PCR products was developed . A total of 933 ceftazidime-resistant strains of GNR isolated between 1991 and 1996 at Nagasaki University Hospital, Nagasaki, Japan, were screened for the blaIMP gene; 80 isolates were positive, including 53 P . aeruginosa isolates, 13 other glucose-nonfermenting bacteria, 13 S . marcescens isolates, and 1 Citrobacter freundii isolate . Most of the patients from whom blaIMP-positive organisms were isolated had malignant diseases (53 . 8%) . The organisms caused urinary tract infections, pneumonia, or other infections in 46.3% of the patients, while they were just colonizing the other patients evaluated . It was possible that blaIMP-positive P . aeruginosa strains contributed to the death of four patients, while the other infections caused by GNR carrying blaIMP were not lethal . DNA fingerprinting analysis by pulsed-field gel electrophoresis suggested the cross transmission of strains within the hospital . The isolates were ceftazidime resistant and were frequently resistant to other antibiotics . Although no particular means of pathogenesis of blaIMP-positive GNR is evident at present, the rapid detection of such strains is necessary to help with infection control practices for the prevention of their dissemination and the transmission of the resistance gene to other pathogenic bacteria.

J Mol Biol, 1998 Jul 24, 280(4), 583 - 96
The entericidin locus of Escherichia coli and its implications for programmed bacterial cell death; Bishop RE et al.; Antidote/toxin gene pairs known as "addiction modules" can maintain plasmids in bacterial populations by means of post-segregational killing . However, several chromosome-encoded addiction modules may provide an entirely distinct function in the programmed cell death of moribund subpopulations under starvation conditions . We now report a novel chromosomal bacteriolytic module of Escherichia coli called the entericidin locus, which is activated in stationary phase under high osmolarity conditions by sigmaS and simultaneously repressed by the osmoregulatory EnvZ/OmpR signal transduction pathway . The entericidin locus encodes tandem paralogous genes (ecnAB) and directs the synthesis of two small cell-envelope lipoproteins . An attenuator precedes ecnA and an ompR-sensitive sigmaS promoter governs expression of ecnB . The entericidin A lipoprotein is an antidote to the bacteriolytic lipoprotein entericidin B . The entericidins are predicted to adopt amphipathic alpha-helical structures and to reciprocally modulate membrane stability . The entericidin locus is not present on any known plasmids, but is conserved in the homologous region of the Citrobacter freundii chromosome . Although the cloned C . freundii entericidin locus is expressed in E . coli independently of ompR, it carries an additional ompR-like gene called ecnR . The organization of the entericidin locus as a chromosomal antidote/toxin gene pair, which is regulated by both positive and negative osmotic signals during starvation, is consistent with an emerging paradigm of programmed bacterial cell death .

Aust Vet J, 1998 Jun, 76(6), 415 - 7
Gram-negative bacterial infections and cardiovascular parasitism in green sea turtles (Chelonia mydas); Raidal SR et al.; OBJECTIVE: To investigate causes of ill health and mortality in juvenile wild green sea turtles (Chelonia mydas) found along the mid-north west coast of Western Australia between June and October of 1997 . PROCEDURE: Department of Conservation and Land Management rangers submitted four dead or dying green sea turtles from separate incidents for veterinary examination, necropsy, and bacteriological, parasitological and histopathological examination . RESULTS: Numerous different species of trematodes belonging to the families Pronocephalidae, Microscaphidiidae and Paramphistomidae were detected in the intestines of two turtles examined, and in all turtles there was severe spirorchid fluke infection including Haemoxenicon sp, Amphiorchis sp and Hapalotrema sp . Histopathological examination demonstrated severe multifocal to diffuse granulomatous vasculitis, aggregations of spirorchid fluke eggs and microabscesses throughout various tissues including intestines, kidney, liver, lung and brain . Cultures and or histopathological examination demonstrated disseminated Gram-negative bacterial infections including salmonella, E coli, Citrobacter freundii and Moraxella sp . CONCLUSION: Infections caused by salmonellae, E coli and other Gram-negative bacteria should be considered as causes of systemic illness and death in wild green sea turtles infected with spirorchid cardiovascular flukes and other internal parasites.

J Clin Microbiol, 1998 Aug, 36(8), 2326 - 30
Direct detection of eae-positive bacteria in human and veterinary colorectal specimens by PCR; Hubbard AL et al.; A PCR test based on the amplification of an eae-specific sequence was designed and evaluated for its ability to directly detect homologous sequences in enteropathogenic Escherichia coli and Citrobacter spp . (amplification of eae open reading frame, 178 bp) in sections of the intestines of humans and animals with colonic lesions . Positive PCR results were observed with eae-positive reference strains of E . coli and Citrobacter rodentium (Citrobacter freundii biotype 4280) . Known eae-negative reference strains of E . coli and other laboratory strains of enteric bacteria were negative by the amplification test . The sensitivity of the PCR for detection of eae-positive E . coli and C . rodentium was between 1 and 2 CFU . To detect these sequences directly from sections of fixed colon from human and veterinary sources, PCR conditions were modified by the addition of 0.1 mM 8-methoxypsoralen to eliminate extraneous bacterial DNA from the PCR amplification cocktail without added template . Sections of colon from three pigs experimentally affected with colon lesions due to enteropathogenic (attaching and effacing) E . coli were PCR positive for bacterial eae genome . Sections from control animals were negative . Sections of colon from one of 18 biopsies from confirmed AIDS patients and from 22 of 35 colorectal cancer patients were PCR positive for bacterial eae genome . The PCR test was a simple and quick method of detecting bacterial eae genome in human and veterinary clinical specimens . This method may remove the need for initial culture and detection of the gene by DNA probing from potential associated lesions . The clear relationship of bacteria containing the eae gene with colonic lesions in the pigs and mice indicates that a similar relationship is possible for human patients having similar lesions.

J Bacteriol, 1998 Jul, 180(13), 3480 - 2
Mutants of Citrobacter freundii that transport and utilize melibiose; Okazaki N et al.; We have isolated mutants of Citrobacter freundii that can grow on melibiose . Inducible alpha-galactosidase activity and melibiose transport activity were detected in the mutant cells but not in the wild-type cells . We detected a DNA region which hybridized with melB (the gene for the melibiose transporter) DNA of Escherichia coli in the chromosomal DNA of wild-type C . freundii . Protons, but not sodium ions, were found to be the coupling cations for melibiose (and methyl-beta-D-thiogalactoside) transport in the mutant cells.

Infect Immun, 1998 Jul, 66(7), 3120 - 7
Inhibition of murine splenic and mucosal lymphocyte function by enteric bacterial products; Malstrom C et al.; Previously we showed that lysates of enteropathogenic Escherichia coli (EPEC) inhibit lymphokine production by mitogen-activated human peripheral blood and lamina propria mononuclear cells . The aims of the present study were to determine whether EPEC-inhibitory factors have similar effects on murine lymphoid populations in order to further delineate the mechanisms of alteration of cytokine production . Preexposure to EPEC lysates inhibited mitogen-stimulated interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) production by murine spleen cells, but IL-10 production was increased . The inhibition was not due to increased apoptosis and was not blocked by neutralizating antibodies against IL-10 or transforming growth factor beta (TGF-beta) . EPEC lysates also inhibited mitogen-stimulated IL-2 and IFN-gamma production by CD11b-depleted spleen cells, IL-2 and IL-4 production by intraepithelial and Peyer's patch lymphocytes, IL-2 production by the human T-cell line Jurkat, and antigen-stimulated IL-2 production by murine spleen cells . Lysates obtained from Shiga-like toxin-producing E . coli, E . coli RDEC-1, Citrobacter rodentium, and an EPEC espB insertion mutant all inhibited IL-2 and IL-4 production by mitogen-stimulated lymphoid cells . In conclusion, lysates of EPEC and related bacteria directly inhibit cytokine production by lymphoid cells from multiple sites by a mechanism that does not increase apoptosis or result from secondary effects of IL-10 or TGF-beta.

Diagn Microbiol Infect Dis, 1998 Jun, 31(2), 379 - 88
Bacterial resistance: a worldwide problem; Jones RN et al.; The therapeutic crisis produced by emerging antimicrobial resistances has compromised the chemotherapy of hospitalized patients with serious infections . For the most prevalent resistance problems, meropenem, a new carbapenem, appears to provide a potency and spectrum for: 1) extended-spectrum beta-lactamase-producing Enterobacteriaceae; 2) Bush-Jacoby-Merdeiros group 1 enzyme-producing ceftazidime-resistant Enterobacter spp., Citrobacter freundii, and some Serratia spp.; 3) ceftazidime- and imipenem-resistant Pseudomonas aeruginosa; and 4) some Streptococcus spp . with elevated penicillin MICs . Documented in vitro study results using 1997 gram-negative blood stream infection isolates indicate a wider spectrum and a two- to fourfold greater potency for meropenem compared with imipenem . This was especially true for P . aeruginosa where 93.4% of strains were susceptible to meropenem (84.1% for imipenem) . Also among over 30,000 reported in vitro meropenem results from the United States and Europe, 90.6% of gram-positive cocci and 99.1% of anaerobes were inhibited at < or = 4 microg/ml . Over 90% of ceftazidime-resistant blood stream infection strains were meropenem susceptible, a rate greater than those of imipenem, ciprofloxacin, and gentamicin . As the clinical utility of many contemporary antimicrobial agents is challenged by emerging resistance, the carbapenems (meropenem, imipenem) appear positioned for a greater role in the treatment of infections in hospitalized patients.

Diagn Microbiol Infect Dis, 1998 Jul, 31(3), 461 - 6
Important and emerging beta-lactamase-mediated resistances in hospital-based pathogens: the Amp C enzymes; Jones RN; Resistance to third-generation cephalosporins mediated by beta-lactamases is an increasing problem for clinical therapeutics . A wide range of Enterobacteriaceae produce these AmpC enzymes (Bush-Jacoby-Medeiros group 1), including Enterobacter spp., Citrobacter freundii, Morganella morganii, Providencia spp., and Serratia marcescens . Resistance via this mechanism has been shown to be statistically correlated with the use of some third-generation cephalosporins, and the infections caused by these stably derepressed enzyme-producing species seem to occur most frequently in the seriously ill . More recently the genes encoding this enzyme have been documented on plasmids capable of transfer into other species such as Klebsiella pneumoniae . Fourth-generation cephalosporins, with stability and low affinity for the Amp C beta-lactamases and the ability to penetrate rapidly into the periplasmic space of Gram-negative organisms, offer a viable alternative in the treatment of these infections or as empiric regimens . Furthermore, these compounds (example: cefpirome) possess greater potency against the frequently occurring Gram-positive cocci such as oxacillin-susceptible staphylococci and the streptococci (including some penicillin-resistant strains) as compared to previously used anti-pseudomonal cephalosporias, ceftazidime.

Nat Biotechnol, 1996 May, 14(5), 635 - 8
Bioaccumulation of nickel by intercalation into polycrystalline hydrogen uranyl phosphate deposited via an enzymatic mechanism; Bonthrone KM et al.; A Citrobacter sp . accumulates uranyl ion (UO2(2+)) as crystalline HUO2PO4.4H2O (HUP), using enzymatically generated inorganic phosphate . Ni was not removed by this mechanism, but cells already loaded with HUP removed Ni2+ by intercalative ion-exchange, forming Ni(UO2PO4)2.7H2O, as concluded by x-ray diffraction (XRD) and proton induced x-ray emission (PIXE) analyses . The loaded biomass became saturated with Ni rapidly, with a molar ratio of Ni:U in the cellbound deposit of approx . 1:6; Ni penetration was probably surface-localized . Cochallenge of the cells with Ni2+ and UO2(2+), and glycerol 2-phosphate (phosphate donor for phosphate release and metal bioprecipitation) gave sustained removal of both metals in a flow through bioreactor, with more extensively accumulated Ni . We propose 'Microbially Enhanced Chemisorption of Heavy Metals' (MECHM) to describe this hybrid mechanism of metal bioaccumulation via intercalation into preformed, biogenic crystals, and note also that MECHM can promote the removal of the transuranic radionuclide neptunium, which is difficult to achieve by conventional methods.

Microbiol Immunol, 1998, 42(4), 321 - 4
Inhibitory effect of bacterial attachment on candidal growth due to adherence with mannose-sensitive pili; Seki K et al.; A bacterial strain with affinity to Candida albicans was successfully obtained from a natural environment . An uncovered Petri dish containing a suspension of heat-killed C . albicans cells was allowed to stand in a laboratory for several days . Some bacteria which had adhered to the candidal cells were tested for their ability to agglutinate the cells . A bacterial strain, designated later as CAB-1, was found to agglutinate candidal cells through bridging by mannose-sensitive pili . CAB-1 showed similar bacteriological characteristics to those of Citrobacter freundii by ID test . The adherence of CAB-1 to candidal cell was precisely presented by scanning electron microscopy . The inhibitory effect of CAB-1 attachment to candidal cells on the growth of Candida was also preliminarily confirmed.

Antimicrob Agents Chemother, 1998 May, 42(5), 1110 - 4
Chromosomally encoded ampC-type beta-lactamase in a clinical isolate of Proteus mirabilis; Bret L et al.; A clinical strain of Proteus mirabilis (CF09) isolated from urine specimens of a patient displayed resistance to amoxicillin (MIC >4,096 microg/ml), ticarcillin (4,096 microg/ml), cefoxitin (64 microg/ml), cefotaxime (256 microg/ml), and ceftazidime (128 microg/ml) and required an elevated MIC of aztreonam (4 microg/ml) . Clavulanic acid did not act synergistically with cephalosporins . Two beta-lactamases with apparent pIs of 5.6 and 9.0 were identified by isoelectric focusing on a gel . Substrate and inhibition profiles were characteristic of an AmpC-type beta-lactamase with a pI of 9.0 . Amplification by PCR with primers for ampC genes (Escherichia coli, Enterobacter cloacae, and Citrobacter freundii) of a 756-bp DNA fragment from strain CF09 was obtained only with C . freundii-specific primers . Hybridization results showed that the ampC gene is only chromosomally located while the TEM gene is plasmid located . After cloning of the gene, analysis of the complete nucleotide sequence (1,146 bp) showed that this ampC gene is close to blaCMY-2, from which it differs by three point mutations leading to amino acid substitutions Glu --> Gly at position 22, Trp --> Arg at position 201, and Ser --> Asn at position 343 . AmpC beta-lactamases derived from that of C . freundii (LAT-1, LAT-2, BIL-1, and CMY-2) have been found in Klebsiella pneumoniae, E . coli, and Enterobacter aerogenes and have been reported to be plasmid borne . This is the first example of a chromosomally encoded AmpC-type beta-lactamase observed in P . mirabilis . We suggest that it be designated CMY-3.

Am J Med Sci, 1998 May, 315(5), 314 - 6
Citrobacter freundii empyema in a patient with occult pulmonary histoplasmosis; Chuang AW et al.; The genus Citrobacter includes three species of organisms that are uncommonly associated with human infection . When they are pathogenic, there are usually one or more associated respiratory, urinary, skin-soft tissue, and central nervous system infections and neonatal sepsis . These infections occur in the wake of significant systemic illness or complicate antibiotic usage . Rarely, infection has been associated with active tuberculosis . The authors report a case of Citrobacter freundii empyema in a patient with occult pulmonary histoplasmosis.

Ethiop Med J, 1997 Apr, 35(2), 93 - 102
Bacterial isolates from indigenous weaning foods in rural Ethiopian setting, Jimma Zone, south west Ethiopia; Tenssay ZW et al.; A community based bacteriological study of weaning foods was conducted from November 1994 to August 1995 in six peasant associations, Jimma Zone . The households in the study community were found to be in poor sanitary conditions with cattle and pets living in the same room with humans, and the community gets water from unprotected sources . The predominant weaning foods in the study community were cereals . These and other foods given to weaning age children were found to be grossly contaminated, aerobic mesophilic bacterial counts being 10(5) cfu/ml of sample . The most frequent bacterial isolates were Enterobacter sp, Gram positive cocci including Staphylococcus aureus, Klebsiella sp., Citrobacter sp., and Escherichia coli in that order . Various factors such as unsafe water, unhygienic handling of food, storage of food at ambient temperature for a long time, poor domestic and personal hygiene may have contributed for the gross contamination of weaning foods in the study community . This calls for educating the community on the relationship between contamination of weaning foods and diarrhoeal diseases, and promoting measures such as reheating of weaning foods which have been kept at ambient temperature for a long time before serving infants and children.

J Clin Microbiol, 1998 May, 36(5), 1408 - 9
Isolation of a nonpathogenic strain of Citrobacter sedlakii which expresses Escherichia coli O157 antigen; Park CH et al.; A nonpathogenic strain of Citrobacter sedlakii which expresses the Escherichia coli O157 antigen is described . The discovery of this strain emphasizes the necessity of additional biochemical and/or toxigenicity testing when isolates react with E . coli O157 latex reagents.

Diagn Microbiol Infect Dis, 1998 Mar, 30(3), 215 - 28
Antimicrobial activity and spectrum investigation of eight broad-spectrum beta-lactam drugs: a 1997 surveillance trial in 102 medical centers in the United States . Cefepime Study Group; Jones RN et al.; Because antimicrobial agents become less effective after the emergence of resistance mechanisms in clinically prevalent pathogens, physicians must utilize local, regional, and national antimicrobial susceptibility surveillance data to assist in choices of appropriate agents . An investigation of the spectrum and potency of eight broad-spectrum beta-lactam drugs (cefepime, cefotaxime, ceftazidime, ceftriaxone, imipenem, piperacillin with or without tazobactam, and ticarcillin/clavulanic acid) was performed using a common protocol and method (Etest; AB BIODISK, Solna, Sweden) in 102 clinical microbiology laboratories in the United States . A total of 9777 strains of Gram-negative bacilli were tested from late 1996 through April 1997 . Quality assurance measures using three control strains observed quality control failures in 13 laboratories (usually ticarcillin/clavulanic acid or piperacillin), but only 2% of results required deletion . A total of 33.4% of Enterobacter spp . (1977 strains) were either resistant or intermediately susceptible to ceftazidime . Only imipenem (99.6% susceptible) and cefepime (99.1%) remained highly active against strains of Enterobacter, as well as Citrobacter freundii, indole-positive Proteae, and Serratia spp . Ceftazidime-resistant Escherichia coli and Klebsiella pneumoniae were detected at rates of 10.3% and 23.8%, respectively . Although these were participant-selected strains, only imipenem and cefepime had broad-spectrum coverage (> or = 97.1%) against these extended-spectrum beta-lactamase phenotypes . A dominant number of these extended-spectrum beta-lactamase phenotypes were reported from medical centers in the Northeast, but a nationwide distribution was observed . Among the nonenteric Gram-negative bacilli (4057 strains), the rank order of susceptibility (percent inhibited at published breakpoint concentrations) was: imipenem (86.1%) > piperacillin/tazobactam (80.1%) > cefepime (77.1%) > ceftazidime = piperacillin (74.9%) > ticarcillin/clavulanic acid (61.6%) > cefotaxime (18.2%) > ceftriaxone (12.9%) . The cephalosporins, cefepime and ceftazidime, had rates of resistance for the 3005 Pseudomonas aeruginosa isolates of 10.1% and 14.4%, respectively . For all Gram-negative strains tested, only two contemporary beta-lactam antimicrobials exhibited > 90% inhibition of strains, imipenem at 93.6% and cefepime at 90.2% . These drugs were superior to the other tested compounds (48.8-84.3%) . Ticarcillin/clavulanic acid had the narrowest spectrum of activity (48.8% of isolates susceptible) . These results indicate that carbapenems and a new fourth-generation cephalosporin, cefepime, possess usable in vitro potencies against current clinical strains of Gram-negative bacilli, many of which harbored resistance to other antimicrobial agents.

Scand J Infect Dis, 1997, 29(6), 615 - 21
In vitro activity of cefpirome against microorganisms isolated in haematology, oncology and intensive care units in Switzerland; Liassine N et al.; The in vitro activity of cefpirome, a new parenteral fourth-generation cephalosporin, was investigated in the 5 university hospitals of Switzerland, and compared to 9 other antibiotics mainly used in hospitals, such as ceftazidime, ceftriaxone, cefotaxime, piperacillin, imipenem, gentamicin, vancomycin, ciprofloxacin and ofloxacin . A total number of 992 strains collected only from intensive care units and haematology-oncology units were tested by microdilution according to NCCLS . Cefpirome showed an excellent activity against all Enterobacteriaceae (MIC90 = 4 mg/l), methicillin-susceptible staphylococci (MIC90 = 1 mg/l), Streptococcus pneumoniae (MIC90 = 0.25 mg/l) and Haemophilus influenzae (MIC90 = 0.12 mg/l) isolates . Its activity was superior to that of third-generation cephalosporins against cephalosporinase-depressed mutants of Enterobacter cloacae and Citrobacter freundii isolates (MIC90 > 32 mg/l for third-generation cephalosporins vs 4 mg/l for cefpirome) . The MICs of cefpirome of 3 strains of Klebsiella spp . with an extended-spectrum-beta-lactamase were lower (MIC90 = 2 mg/l) than those of third-generation cephalosporins (MICs90 > 32 mg/l) . Against Pseudomonas aeruginosa cefpirome was as active as ceftazidime . The activity of cefpirome was poor against methicillin-resistant staphylococci, enterococci and nosocomial Gram-negative bacteria such as Stenotrophomonas maltophilia.

Microbiol Immunol, 1998, 42(3), 165 - 9
Resistance to oxyimino beta-lactams due to a mutation of chromosomal beta-lactamase in Citrobacter freundii; Haruta S et al.; The duplicative mutation of an Ala-Val-Arg sequence at positions 208 to 210 in the loop structure of Enterobacter cloacae class C beta-lactamase caused substrate specificity extension to oxyimino beta-lactam antibiotics and this chromosomal mutation provided bacterial cells with high resistance to the beta-lactams (M . Nukaga et al, 1995, J . Biol . Chem . 270, 5729-5735) . In order to confirm the universality of this phenomenon among other class C beta-lactamases, the duplicative mutation was applied to a class C beta-lactamase of Citrobacter freundii, which has 74% homology to the E . cloacae beta-lactamase amino acid sequence . The counterpart sequence to the Ala-Val-Arg of the E . cloacae enzyme in C . freundii beta-lactamase was identified to be Pro-Val-His . A Pro-Val-His sequence was inserted just after the native Pro-Val-His sequence at positions 208 to 210 in the C . freundii beta-lactamase . The resulting mutant of C . freundii beta-lactamase obtained a striking characteristic that we expected, showing substrate specificity extension to oxyimino beta-lactams . Nearly the same result was obtained with the insertion of an Ala-Val-Arg sequence after the native Pro-Val-His sequence . These results indicate that structural modification of this locus commonly induces modification of the substrate specificity to unfavorable substrates for many chromosomal class C beta-lactamases produced by gram-negative bacteria.

J Paediatr Child Health, 1998 Feb, 34(1), 90 - 1
Osteomyelitis and septic arthritis due to Citrobacter freundii and Haemophilus influenzae type b; Stricker T et al.; This report describes dual infection with Citrobacter freundii and Haemophilus influenzae type b causing septic arthritis and osteomyelitis of the elbow in a previously healthy 5-year-old boy . The patient was treated successfully with intravenous fosfomycin for 4 weeks . Infections with Citrobacter beyond the neonatal period are rare in paediatric patients . When Citrobacter spp . is isolated, coinfection with other bacterial pathogens should be considered.

Int J Food Microbiol, 1998 Jan 6, 39(1-2), 11 - 7
Enterobacteriaceae found in high numbers in fish, minced meat and pasteurised milk or cream and the presence of toxin encoding genes; Lindberg AM et al.; Enterobacteriaceae were found in high numbers after storage at 7 degrees C in 6% of consumers packs of pasteurised milk or cream, in 31% of retailed fish and in 100% of retail packs of minced meat . Seventy two fresh-water fishes, 40 packs of minced meat and 430 milk packs were sampled . One hundred and eighty four isolates were randomly picked from Tryptone glucose extract (TGE) agar (30 degrees C for 3d) or Violet red bile glucose (VRBG) agar (37 degrees C for 1d) . In minced meat, Serratia liquefaciens, Hafnia alvei, Rahnella aquatilis were frequently encountered . On fish, the most frequently found species were R . aquatilis, and in milk, the dominating species were S . liquefaciens, H . alvei and R . aquatilis . One to three isolates of Citrobacter freundii were found in all three food categories . Using a polymerase chain reaction (PCR) technique, the gene of Escherichia coli heat-labile toxin (lt) was indicated in one fish isolate of R . aquatilis whereas heat-stable toxin genes (s.t.) were indicated in four H . alvei isolates, two originating from fish and two from minced meat . Positive PCR-reaction for vero cytotoxin genes were found in one H . alvei strain originating from fish (vt1), in two S . liquefaciens strains from minced meat (vt2), and in a C . freundii reference strain . One of the st-positive H . alvei strains from meat harboured the eaeA gene involved in the attaching phenotype of enteropathogenic E . coli.

FEMS Microbiol Lett, 1998 Apr 1, 161(1), 135 - 8
Phosphatase activity and lead resistance in Citrobacter freundii and Staphylococcus aureus; Levinson HS et al.; It has been previously reported that strains of Citrobacter freundii and of Staphylococcus aureus accumulated lead as Pb-phosphate when grown on media supplemented with high levels of lead salts . Phosphatase activity, which has been postulated to be involved in lead accumulation, was unrelated to lead resistance, resistant and sensitive cells displaying similar levels and patterns of enzyme activity.

FEMS Microbiol Lett, 1998 Apr 1, 161(1), 89 - 96
Phylogenetic relationships of Salmonella based on DNA sequence comparison of atpD encoding the beta subunit of ATP synthase; Christensen H et al.; DNA sequences covering 57% of atpD encoding the beta subunit of ATP synthase were determined for 16 strains of Salmonella enterica, two strains of S . bongori, and one strain each of Citrobacter freundii and Yersinia enterocolitica, and comparison was made with the published Escherichia coli and Enterobacter aerogenes sequences . The phylogenetic tree based on maximum-likelihood analysis showed separation of the subspecies of S . enterica except for two serotypes of subspecies II which were unsupported by a common node . The two serotypes of S . bongori were separated from S . enterica and related to the serotypes of subspecies II . A tight relationship was found between S . enterica subspecies IIIa consisting of monophasic serotypes and subspecies IIIb consisting of diphasic serotypes . This is in conflict with results obtained for most other housekeeping genes and the 23S rRNA gene separating mono- from diphasic subspecies.

Antimicrob Agents Chemother, 1998 Apr, 42(4), 827 - 32
Cefotaxime-resistant Enterobacteriaceae isolates from a hospital in Warsaw, Poland: identification of a new CTX-M-3 cefotaxime-hydrolyzing beta-lactamase that is closely related to the CTX-M-1/MEN-1 enzyme; Gniadkowski M et al.; A group of cefotaxime-resistant Citrobacter freundii and Escherichia coli isolates were collected by a clinical laboratory in a hospital in Warsaw, Poland, in July 1996 . Detailed analysis has shown that all of these produced a beta-lactamase (pI, 8.4) belonging to the CTX-M family, one of the minor extended-spectrum beta-lactamase families with a strong cefotaxime-hydrolyzing activity . Sequencing has revealed that C . freundii isolates produced a new CTX-M-3 enzyme which is very closely related to the CTX-M-1/MEN-1 beta-lactamase, sporadically identified in Europe over a period of 6 years . Amino acid sequences of these two beta-lactamases differ at four positions: Val77Ala, Asp114Asn, Ser140Ala, and Asn288Asp (the first amino acid of each pair refers to CTX-M-1/MEN-1 and second refers to CTX-M-3) . The partial sequence of the E . coli CTX-M gene was identical to the corresponding region of bla(CTX-M-3), but a transconjugant of the E . coli isolate expressed higher levels of resistance to beta-lactams than did C . freundii transconjugants . These resistance differences correlated with differences in plasmid DNA restriction patterns . Our results suggest that CTX-M genes have been spread among different species of the family Enterobacteriaceae in the hospital and that the CTX-M-3-expressing C . freundii strain causing routine urinary tract infections has been maintained for a relatively long time in the hospital environment.

Cutis, 1998 Mar, 61(3), 158 - 9
Cellulitis caused by Citrobacter diversus in a patient with multiple myeloma; Bishara J et al.; The most common cause of cellulitis is streptococci . Coagulase-negative staphylococci and gram-negative bacteria, such as Serratia spp., Proteus spp., and other Enterobacteriaceae may produce cellulitis in the immunocompromised patient . We report a case of Citrobacter diversus-induced cellulitis, resembling streptococcal infection, in a patient with multiple myeloma.

Pathol Biol (Paris), 1997 Nov, 45(9), 716 - 20
Prevalence and sensitivity to antibiotics of Enterobacteriaceae isolated from urinary cultures in some microbiology laboratories of a city in west Greece; Papapetropoulou M et al.; The prevalence of Enterobacteriaceae from midstream urine samples from patients with community acquired urinary tract infections (UTI) of a town in SW Greece during one year period and their susceptibility to antibiotics were studied . The most frequently recovered pathogens were E . coli (77%), Proteus mirabilis (10%), Klebsiella spp (8.7%), Enterobacter spp (2.5%) and Citrobacter freundii (1.8%) . E . coli were found more resistant to carbenicillin, the combination of amoxicillin/clavulanic acid and cotrimoxazole . Half of the strains were found resistant to more than one antibiotics . All strains were found sensitive to aminoglucosides, 2nd generation cephalosporines (except cefoxitin), 3rd generation cephalosporines, aztreonam and imipeneme . According to our results a statistically significant increase of the resistance to antibiotics at individuals over 45 years of age was noticed . The positivity of the samples was not correlated to prior antibiotic consumption and to the occupation of the participants or their residence.

Antimicrob Agents Chemother, 1998 Feb, 42(2), 209 - 15
Aminoglycoside 6'-N-acetyltransferase variants of the Ib type with altered substrate profile in clinical isolates of Enterobacter cloacae and Citrobacter freundii; Casin I et al.; Three clinical isolates, Enterobacter cloacae EC1562 and EC1563 and Citrobacter freundii CFr564, displayed an aminoglycoside resistance profile evocative of low-level 6'-N acetyltransferase type II {AAC(6')-II} production, which conferred reduced susceptibility to gentamicin but not to amikacin or isepamicin . Aminoglycoside acetyltransferase assays suggested the synthesis in the three strains of an AAC(6') which acetylated amikacin practically as well as it acetylated gentamicin in vitro . Both compounds, however, as well as isepamicin, retained good bactericidal activity against the three strains . The aac genes were borne by conjugative plasmids (pLMM562 and pLMM564 of ca . 100 kb and pLMM563 of ca . 20 kb) . By PCR mapping and nucleotide sequence analysis, an aac(6')-Ib gene was found in each strain upstream of an ant(3")-I gene in a sulI-type integron . The size of the AAC(6')-Ib variant encoded by pLMM562 and pLMM564, AAC(6')-Ib7, was deduced to be 184 (or 177) amino acids long, whereas in pLMM563 a 21-bp duplication allowing the recruitment of a start codon resulted in the translation of a variant, AAC(6')-Ib8, of 196 amino acids, in agreement with size estimates obtained by Western blot analysis . Both variants had at position 119 a serine instead of the leucine typical for the AAC(6')-Ib variants conferring resistance to amikacin . By using methods that predict the secondary structure, these two amino acids appear to condition an alpha-helical structure within a putative aminoglycoside binding domain of AAC(6')-Ib variants.

Arch Microbiol, 1998 Feb, 169(2), 166 - 73
Purification and characterization of acid-type phosphatases from a heavy-metal-accumulating Citrobacter sp; Jeong BC et al.; An acid phosphatase from a heavy-metal-accumulating strain of a Citrobacter sp . was resolved into two forms on the basis of their nonbinding (phosphatase I) or binding (phosphatase II) behaviour on the cation-exchange resin SP-Sephadex C50 . Both holoenzymes had a molecular mass of 103-108 kDa as determined by Superose Q-6 column chromatography in the presence of 150 mM KCl and a subunit molecular mass of 27 kDa as determined by SDS-PAGE; the enzyme was tetrameric . Both enzymes had a pI approximately 9.0 and were immunologically cross-reactive . There were minor differences in amino acid composition and in peptide maps following tryptic digest . The pH optimum for phosphatases I and II was 5.5 and 6.25, respectively; phosphatase II alone retained activity at pH values up to 9.0 . Phosphatase I was more resistant to mechanical shear, gamma-irradiation, high temperature, and toxins (F- and formaldehyde) . Glycerol increased the thermostability of both enzymes, particularly the more thermosensitive phosphatase II . Phosphatase II had a lower Km and a lower Vmax for glycerol 2-phosphate hydrolysis . The production of enzyme isoforms is a phenomenon similar to that described previously for the alkaline phosphatase of Escherichia coli, where the isoforms relate to precursive and final processed forms of the enzyme . Acid phosphatase is physiologically distinct, with a role that is still obscure but that may relate to cellular stress responses.

J Clin Microbiol, 1998 Mar, 36(3), 662 - 8
Detection of intimins alpha, beta, gamma, and delta, four intimin derivatives expressed by attaching and effacing microbial pathogens; Adu-Bobie J et al.; Intimins are outer membrane proteins expressed by enteric bacterial pathogens capable of inducing intestinal attachment-and-effacement lesions . A eukaryotic cell-binding domain is located within a 280-amino-acid (Int280) carboxy terminus of intimin polypeptides . Polyclonal antiserum was raised against Int280 from enteropathogenic Escherichia coli (EPEC) serotypes O127:H6 and O114:H2 (anti-Int280-H6 and anti-Int280-H2, respectively), and Western blot analysis was used to explore the immunological relationship between the intimin polypeptides expressed by different clinical EPEC and enterohemorrhagic E . coli (EHEC) isolates, a rabbit diarrheagenic E . coli strain (RDEC-1), and Citrobacter rodentium . Anti-Int280-H6 serum reacted strongly with some EPEC serotypes, whereas anti-Int280-H2 serum reacted strongly with strains belonging to different EPEC and EHEC serotypes, RDEC-1, and C . rodentium . These observations were confirmed by using purified Int280 in an enzyme-linked immunosorbent assay and by immunogold and immunofluorescence labelling of whole bacterial cells . Some bacterial strains were recognized poorly by either antiserum (e.g., EPEC O86:H34 and EHEC O157:H7) . By using PCR primers designed on the basis of the intimin-encoding eae gene sequences of serotype O127:H6, O114:H2, and O86:H34 EPEC and serotype O157:H7 EHEC, we could distinguish between different eae gene derivatives . Accordingly, the different intimin types were designated alpha, beta, delta, and gamma, respectively.

Infect Control Hosp Epidemiol, 1998 Jan, 19(1), 38 - 40
Stool colonization of healthcare workers with selected resistant bacteria; Carmeli Y et al.; We examined the carriage of selected resistant bacteria in the stools of healthcare workers who provided direct patient care . Neither vancomycin-resistant enterococci, methicillin-resistant Staphylococcus aureus, nor Clostridium difficile was recovered from the 55 stool specimens collected . A ceftazidime-resistant Citrobacter freundii was isolated from one specimen . We conclude that the stool of healthcare workers is colonized infrequently with these resistant organisms.

Arch Microbiol, 1997 Dec, 168(6), 457 - 63
3-Methylaspartate ammonia-lyase as a marker enzyme of the mesaconate pathway for (S)-glutamate fermentation in Enterobacteriaceae; Kato Y et al.; The enzyme 3-methylaspartase (3-methylaspartate ammonia-lyase, EC 4 . 3.1.2) was found in the cells of enteric bacteria, especially in the genera Citrobacter and Morganella, that were grown under anoxic and oxygen-limited conditions . The enzymes were purified to homogeneity from the cell-free extracts of 18 active strains and had similar enzymological properties such as action on columns, specific activity, molecular weight, subunit structure, and N-terminal amino acid sequence similarity . The production of the enzyme was dependent on the limitation of oxygen during growth and was arrested by aeration . The addition of external electron acceptors such as dimethylsulfoxide could support cell growth and production of the enzyme . Activities of glutamate mutase (EC 5.4.99.1) and (S)-citramalate hydrolyase (EC 4.2.1.34), key enzymes of the mesaconate pathway of (S)-glutamate fermentation in the genus Clostridium, were detected in the cells of the active strains grown under oxygen-limited conditions . Based on the results, the mesaconate pathway is proposed to explain the (S)-glutamate fermentation process observed in Enterobacteriaceae, and 3-methylaspartase could be a marker enzyme for this pathway.

Acta Microbiol Immunol Hung, 1997, 44(3), 241 - 7
Studies on antimicrobial effect of the antihistaminic phenothiazine trimeprazine tartrate; Dastidar SG et al.; The antibacterial and bactericidal activities of the antihistamine trimeprazine were studied against 243 strains of bacteria which included both Gram positive and Gram negative types . The susceptibility of these bacterial strains to trimeprazine was assessed by determining their minimum inhibitory concentration (MIC) which was found to be between 10 and 100 micrograms/ml . Nineteen strains of Staphylococcus spp . and Salmonella spp . were trimeprazine . Most of the strains belonging to Bacillus spp . and Salmonella spp . were inhibited by less than 100 micrograms/ml . Trimeprazine could also inhibit strains of Shigella spp . Vibrio cholerae and V . parahaemolyticus at 10-100 micrograms/ml . Strains of klebsiella, proteus, pseudomonas and citrobacter were moderately sensitive to trimeprazine . In in vivo studies it was seen that when trimeprazine was used at a concentration of 0.75 and 0.4 micrograms/gm body weight of the mouse both levels offered significant protection to Swiss mice challenged with 50LD50 of virulent strain of S . typhimurium 74 . Statistical analysis of the data was found to be significant, p < 0.001 according to chi 2 test.

Folia Microbiol (Praha), 1997, 42(4), 353 - 6
Degradation of commercial detergent products by microbial populations of the Lagos lagoon; Amund OO et al.; The biodegradability potentials of three detergent products with the trade names Omo, Teepol and sodium dodecyl sulfate (SDS) by the native bacteria of the Lagos lagoon was carried out using the lagoon die-away method . Physicochemical parameters of the water samples showed that the lagoon in Apapa was more polluted than at the University of Lagos . In 12 days, approximately 30, 60 and 97% of Omo, Teepol and SDS respectively were degraded . SDS with an alkyl sulfate moiety as surfactant supported the highest growth of the detergent-utilizing organisms, indicating that the components of Omo and Teepol are more resistant to microbial attack . The detergent-utilizing bacteria identified were mainly Gram-negative and of the following genera: Vibrio, Klebsiella, Flavobacterium, Pseudomonas, Escherichia, Enterobacter, Proteus, Shigella and Citrobacter . Vibrio was the most frequently encountered organism while Proteus was the rarest . Results of this investigation had shown that detergents made in Nigeria may still contain components that are recalcitrant to biodegradation.

J Ind Microbiol Biotechnol, 1997 Oct, 19(4), 286 - 93
Detection and speciation of bacteria through PCR using universal major cold-shock protein primer oligomers; Francis KP et al.; The detection of bacteria using PCR is a well-established diagnostic technique . However, conventional PCR requires the use of DNA primer oligomers that are specific to the target organism and, as a consequence, a sample can only be tested for the presence of that specific target . A significant advantage would be to probe a sample for the presence of any bacteria, followed by identification . To achieve this it is necessary to identify a DNA sequence common to all bacteria . Here we demonstrate that such a sequence may be that encoding the major cold-shock proteins . Using two universal PCR primer oligomers from conserved regions of these gene homologues, we have amplified a 200 base-pair DNA sequence from more than 30 diverse Gram-positive and Gram-negative bacteria, including representatives from the genera Aeromonas, Bacillus, Citrobacter, Enterobacter, Enterococcus, Escherichia, Klebsiella, Lactobacillus, Lactococcus, Listeria, Pediococcus, Photobacterium, Proteus, Salmonella, Shigella, Staphylococcus, Streptococcus, and Yersinia . Sequence analysis of the amplified products confirmed a high level of DNA homology . Significantly, however, there are sufficient nucleotide variations to allow the unique allocation of each amplified sequence to its parental bacterium.

Antimicrob Agents Chemother, 1997 Dec, 41(12), 2652 - 63
In vitro and in vivo antibacterial activities of CS-834, a novel oral carbapenem; Fukuoka T et al.; CS-834 is a novel oral carbapenem antibiotic . This compound is an ester-type prodrug of the active metabolite R-95867 . The antibacterial activity of R-95867 was tested against 1,323 clinical isolates of 35 species and was compared with those of oral cephems, i.e., cefteram, cefpodoxime, cefdinir, and cefditoren, and that of a parenteral carbapenem, imipenem . R-95867 exhibited a broad spectrum of activity covering both gram-positive and -negative aerobes and anaerobes . Its activity was superior to those of the other compounds tested against most of the bacterial species tested . R-95867 showed potent antibacterial activity against clinically significant pathogens: methicillin-susceptible Staphylococcus aureus including ofloxacin-resistant strains, Streptococcus pneumoniae including penicillin-resistant strains, Clostridium perfringens, Neisseria spp., Moraxella catarrhalis, most members of the family Enterobacteriaceae, and Haemophilus influenzae (MIC at which 90% of strains are inhibited, < or =0.006 to 0.78 microg/ml) . R-95867 was quite stable to hydrolysis by most of the beta-lactamases tested except the metallo-beta-lactamases from Stenotrophomonas maltophilia and Bacteroides fragilis . R-95867 showed potent bactericidal activity against S . aureus and Escherichia coli . Penicillin-binding proteins 1 and 4 of S . aureus and 1Bs, 2, 3, and 4 of E . coli had high affinities for R-95867 . The in vivo efficacy of CS-834 was evaluated in murine systemic infections caused by 16 strains of gram-positive and -negative pathogens . The efficacy of CS-834 was in many cases superior to those of cefteram pivoxil, cefpodoxime proxetil, cefdinir, and cefditoren pivoxil, especially against infections caused by S . aureus, penicillin-resistant S . pneumoniae, E . coli, Citrobacter freundii, and Proteus vulgaris . Among the drugs tested, CS-834 showed the highest efficacy against experimental pneumonia in mice caused by penicillin-resistant S . pneumoniae.

FEMS Microbiol Lett, 1997 Dec 1, 157(1), 55 - 7
Comparison of ViaB regions of Vi-positive organisms; Hashimoto Y et al.; We cloned the vipR genes from Salmonella paratyphi C, S . dublin, and Citrobacter freundii strains and compared them with the S . typhi sequence to clarify the genetic relationship of the ViaB regions of Vi-positive organisms . ViaB regions were divided into two groups based on their sequences, the Salmonella and C . freundii groups . The vipR coding sequences of the Salmonella group were identical . Southern blot hybridization results using the full-length ViaB region as a probe support these findings.

Microbios, 1997, 90(364-365), 209 - 18
Production of Escherichia coli group I-like heat-labile enterotoxin by Enterobacteriaceae isolated from environmental water; Jolivet-Gougeon A et al.; Strains of Klebsiella oxytoca (2), Citrobacter freundii (2), Enterobacter cloacae (1) and Escherichia coli (1) isolated from environmental water were identified as heat-labile enterotoxin (LT) producing strains by immunological methods and polymerase chain amplification . A 322 bp amplified fragment was obtained with specific primers LTR and LTL, and hybridized to a digoxigenin-labelled LTB probe only under low stringency conditions, and not with a cholera toxin probe . These results suggest that Enterobacteriaceae may produce a LT-like toxin antigenically and genetically related to the LT enterotoxin of E . coli.

J Clin Microbiol, 1997 Dec, 35(12), 3353 - 4
Citrobacter farmeri bacteremia in a child with short-bowel syndrome; Bruckner DA et al.; A case of sepsis in a pediatric patient due to the newly described Citrobacter species C . farmeri is described . Factors predisposing this child to infection included short-bowel syndrome requiring total parenteral nutrition.

APMIS, 1997 Nov, 105(11), 854 - 60
Antibiotic susceptibility of blood culture isolates of Enterobacteriaceae from six Norwegian hospitals 1991-1992; Digranes A et al.; From August 1991 to February 1992, each of the six largest hospitals throughout Norway collected 84 to 107 consecutive blood culture isolates of Enterobacteriaceae, altogether 571 isolates . The distribution of various species and genera at the different hospitals was uniform; Escherichia coli being most prevalent (57-67%), followed by Klebsiella spp . (12-18%) and Proteus mirabilis (7-11%) . Twenty-one and 4% of E . coli isolates were resistant to ampicillin and cefuroxime, respectively, and 11% of Klebsiella isolates were cefuroxime resistant . Five Enterobacter isolates and one Citrobacter isolate were resistant to ceftazidime, and one Salmonella isolate was resistant to imipenem . All isolates were susceptible to ciprofloxacin and tobramycin . These results were compared with the antibiotic consumption in each hospital region . Although hospitals in the regions with the highest consumption of ampicillin tended to have a higher percentage of isolates resistant to this agent, no significant differences were found . There was no significant difference between hospitals regarding prevalence of cefuroxime-resistant isolates.

Antimicrob Agents Chemother, 1997 Nov, 41(11), 2439 - 47
Cloning and characterization of an aminoglycoside 6'-N-acetyltransferase gene from Citrobacter freundii which confers an altered resistance profile; Wu HY et al.; A novel gene encoding a 6'-N-aminoglycoside acetyltransferase, aac(6')-In, has been cloned and sequenced from Citrobacter freundii 13996-19, a clinical isolate from Venezuela . This gene mediates resistance to amikacin, 2'-N-ethylnetilmicin, isepamicin, kanamycin, netilmicin, and tobramycin . The aac(6')-In gene is 573 nucleotides in length and encodes a putative protein of 190 amino acids . AAC(6')-In is most closely related to AAC(6')-Im and AAC(6')-Ie, demonstrating 64.4% and 62.3% similarity, respectively, at the protein level, suggesting these proteins share a common ancestor . The aac(6')-In flanking sequences demonstrated homology to integron- and transposon-related elements which are often found associated with resistance determinants . Hybridization studies performed with an intragenic probe specific for aac(6')-In indicate that this gene is prevalent within Venezuela but has not been observed outside of the country . Furthermore, the aac(6)-In gene was found in 10 different species of gram-negative bacteria.

Antimicrob Agents Chemother, 1997 Nov, 41(11), 2352 - 4
Carbapenem resistance in a clinical isolate of Citrobacter freundii; Mainardi JL et al.; Carbapenem resistance was studied in two sets of Citrobacter freundii strains: (i) strain CFr950, resistant to imipenem (MIC, 16 microg/ml) and isolated in vivo during imipenem therapy, and strain CFr950-Rev, the spontaneous, imipenem-susceptible revertant of CFr950 selected in vitro, and (ii) strains CFr801 and CFr802, two imipenem-resistant mutants selected in vitro from the susceptible clinical isolate CFr800 . In all strains, whether they were imipenem-susceptible or -resistant strains, production of the cephalosporinase was derepressed and their Km values for cephaloridine were in the range of 128 to 199 microM . No carbapenemase activity was detected in vitro . The role of cephalosporinase overproduction in the resistance was demonstrated after introduction of the ampD gene which decreased the level of production of cephalosporinase at least 250-fold and resulted in an 8- to 64-fold decrease in the MICs of the carbapenems . The role of reduced permeability in the resistance was suggested by the absence, in CFr950 and CFr802, of two outer membrane proteins (the 42- and 40-kDa putative porins whose levels were considerably decreased in CFr801) and the reappearance of the 42-kDa protein in imipenem-susceptible strain CFr950-Rev . This role was confirmed after introduction of the ompF gene of Escherichia coli into the CFr strains, which resulted in 8- to 16-fold decreases in the MICs of carbapenems for CFr802 and CFr950 . We infer from these results that the association of reduced, porin-mediated permeability with high-level cephalosporinase production, observed previously in other gram-negative bacteria, may also confer carbapenem resistance on C . freundii.

Pediatr Pathol Lab Med, 1997 Nov-Dec, 17(6), 977 - 82
Neonatal meningitis and multiple brain abscesses due to Citrobacter diversus; Tse G et al.; We report a fatal sporadic case of neonatal Citrobacter diversus meningitis with rapid clinical progression . At autopsy, multiple brain abscesses and abscesses in the spinal cord had complicated purulent meningitis . Septic omphalitis was identified as the most likely portal of entry.

J Bacteriol, 1997 Nov, 179(21), 6633 - 9
Propanediol utilization genes (pdu) of Salmonella typhimurium: three genes for the propanediol dehydratase; Bobik TA et al.; The propanediol utilization (pdu) operon of Salmonella typhimurium encodes proteins required for the catabolism of propanediol, including a coenzyme B12-dependent propanediol dehydratase . A clone that expresses propanediol dehydratase activity was isolated from a Salmonella genomic library . DNA sequence analysis showed that the clone included part of the pduF gene, the pduABCDE genes, and a long partial open reading frame (ORF1) . The clone included 3.9 kbp of pdu DNA which had not been previously sequenced . Complementation and expression studies with subclones constructed via PCR showed that three genes (pduCDE) are necessary and sufficient for propanediol dehydratase activity . The function of ORF1 was not determined . Analyses showed that the S . typhimurium propanediol dehydratase was related to coenzyme B12-dependent glycerol dehydratases from Citrobacter freundii and Klebsiella pneumoniae . Unexpectedly, the S . typhimurium propanediol dehydratase was found to be 98% identical in amino acid sequence to the Klebsiella oxytoca propanediol dehydratase; this is a much higher identity than expected, given the relationship between these organisms . DNA sequence analyses also supported previous studies indicating that the pdu operon was inherited along with the adjacent cobalamin biosynthesis operon by a single horizontal gene transfer.

Diagn Microbiol Infect Dis, 1997 Aug, 28(4), 211 - 9
Inducible amp C beta-lactamase producing gram-negative bacilli from blood stream infections: frequency, antimicrobial susceptibility, and molecular epidemiology in a national surveillance program (SCOPE); Pfaller MA et al.; A surveillance study of nosocomial blood stream infections {Surveillance and Control of Pathogens of Epidemiologic Importance (SCOPE)} was conducted during a 14-month period in 1995 to 1996 in approximately 50 American medical centers . Among the 4725 blood stream infections, the etiologic agent was Enterobacter spp . in 230, Citrobacter freundii in 24, and Serratia marcescens in 65 . The vast majority of these isolates (89%) had been sent to the University of Iowa including 198 Enterobacter spp . (46 Enterobacter aerogenes, 141 Enterobacter cloacae, 11 other Enterobacter spp.), 23 C . freundii, and 62 S . marcescens . Because these species are capable of producing Amp C beta-lactamase, we examined their susceptibility to 12 broad-spectrum antimicrobial agents . The frequency of resistance to ceftazidime and the molecular epidemiology of ceftazidime-resistant strains was also examined . Among the Enterobacter spp . and C . freundii isolates, resistance to third generation cephalosporins (ceftazidime, ceftriaxone) and broad-spectrum semisynthetic penicillins (piperacillin), with or without an enzyme inhibitor (piperacillin/tazobactam), was high, e.g., 35 to 50% . The S . marcescens isolates were quite susceptible to all agents tested . Both imipenem and cefepime were active against virtually all isolates tested including 84 stably derepressed Amp C-producing ceftazidime-resistant strains of Enterobacter spp . and C . freundii . The overall rank order of activity for the six best agents against these Amp C-producing strains was: imipenem (100% susceptible) > amikacin = cefepime (98.6%) > ciprofloxacin = gentamicin = ofloxacin (93.6 to 94.0%) . Molecular typing studies of ceftazidime-resistant E . cloacae using an automated ribotyping system, as well as pulsed-field gel electrophoresis, indicated that although clonal spread of a single strain occurred in some of the medical centers, most of the episodes of bacteremia were caused by patient-unique strains . Control of these resistant organisms will require attention to microbiologic recognition of phenotypes, to infection control practices, and to limiting the overuse of certain extended spectrum beta-lactams.

Antimicrob Agents Chemother, 1997 Oct, 41(10), 2177 - 83
Mechanism of suppression of piperacillin resistance in enterobacteria by tazobactam; Kadima TA et al.; Resistance to piperacillin in several isolates of Citrobacter freundii and Enterobacter cloacae was investigated and confirmed to occur at a frequency of 10(-7) to 10(-6) . Development of resistance to piperacillin was significantly suppressed by tazobactam but not by clavulanic acid . To elucidate the mechanism by which resistance suppression occurs, the effect of piperacillin plus tazobactam on the induction of AmpC beta-lactamase was analyzed by monitoring the beta-galactosidase activity of an inducible ampC-lacZ gene fusion in Escherichia coli . The combination exerted no inhibitory effect on AmpC beta-lactamase induction . Tazobactam also had no effect on the accumulation of a key intermediate in the AmpC beta-lactamase induction pathway, 1,6-anhydromurotripeptide, in an ampD mutant strain of E . coli . However, the addition of tazobactam to liquid cultures of E . cloacae 40001 in the presence of piperacillin at four times the MIC caused a delay in the recovery of the culture to piperacillin-induced stress . At 16 times the MIC, a complete suppression of regrowth occurred . Analysis of culture viability on piperacillin plates showed that the culture recovery was due to growth by moderately resistant mutants preexisting in the cell population, which at 16 times the MIC became susceptible to the combination . Evidence from the kinetics of inhibition of the E . cloacae 40001 AmpC beta-lactamase by clavulanic acid, sulbactam, and tazobactam and from the effects of these drugs on the frequency of resistance to piperacillin suggests that the suppressive effect of tazobactam on the appearance of resistance is primarily mediated by the beta-lactamase inhibitory activity.

J Clin Microbiol, 1997 Oct, 35(10), 2686 - 8
Citrobacter sedlakii meningitis and brain abscess in a premature infant; Dyer J et al.; Citrobacter sedlakii was isolated from blood and cerebrospinal fluid cultures of a 5-day-old premature infant with sepsis, meningitis, and brain abscess . This newly described organism was difficult to identify due to discrepancies between the Vitek and API 20E identification systems . To our knowledge, this is the first report of the isolation of C . sedlakii from cerebrospinal fluid.

J Clin Microbiol, 1997 Oct, 35(10), 2593 - 7
Occurrence and detection of extended-spectrum beta-lactamases in members of the family Enterobacteriaceae at a veterans medical center: seek and you may find; Coudron PE et al.; A total of 907 consecutive isolates of members of the family Enterobacteriaceae recovered during a 20-week period were tested for production of extended-spectrum beta-lactamases (ESBLs) by the double-disk (DD) potentiation method . Of 84 DD-positive isolates, 83 (9.2%) produced ESBLs based on isoelectric focusing . SHV-derived ESBLs and several TEM-derived ESBLs were present in nine species, including the first isolate of Citrobacter koserii and Morganella morganii known to harbor an SHV-derived ESBL . Results of testing 58 nonrepeat isolates for ESBL production by several recommended methods were as follows (percent detected in parentheses): DD method with aztreonam (95), ceftazidime (79), ceftriaxone (88), or cefpodoxime (90); broth microdilution method with ceftazidime (86) or cefotaxime (91) alone or in combination with clavulanate; and the standard disk diffusion method with new breakpoints and standard concentrations of aztreonam (78), ceftazidime (79), ceftriaxone (83), or cefpodoxime (98) or a novel concentration (5 microg) of ceftazidime (88) . In three instances during an extended part of the study, an ESBL-producing isolate and a non-ESBL-producing isolate of the same species were recovered from a single blood culture bottle . These data indicate that ESBLs occur in several species of Enterobacteriaceae and at a relatively high incidence at our institution and that the standard disk diffusion method with cefpodoxime and the DD method with several beta-lactams are practical and cost-effective methods for detecting ESBL-producing isolates of Enterobacteriaceae.

J Clin Microbiol, 1997 Oct, 35(10), 2561 - 7
Molecular epidemiology of a nosocomial outbreak due to SHV-4-producing strains of Citrobacter diversus; El Harrif-Heraud Z et al.; Over a 6-month period, eight strains of Citrobacter diversus (Citrobacter koseri) resistant to extended-spectrum cephalosporins and monobactams were isolated from seven colonized and/or infected patients from the same intensive care unit . All strains harbored a single large conjugative plasmid which mediated an extended-spectrum beta-lactamase of the SHV-4 type (ceftazidimase phenotype; enzyme pI, 7.8; plasmid DNA hybridization with a blaSHV-specific probe) . All strains were characterized by antibiotic resistance pattern analysis, beta-lactamase content analysis, plasmid profiling, ribotyping with EcoRI, and arbitrarily primed (AP)-PCR with primers O8 and O12 . Among the eight C . diversus strains, strains Cd5 to Cd12, six isolates (isolates Cd6 to Cd11) were identical by all markers; one strain (strain Cd5) differed by two markers (antibiotype and AP-PCR pattern with primer O8), and the remaining strain (strain Cd12) differed by two other markers (ribotype and AP-PCR pattern with primer O12) . Our results suggest that six of the eight SHV-4-producing C . diversus strains studied (strains Cd6 to Cd11) were a single epidemic strain . Strain Cd5 could be related to the epidemic strain; the origin of strain Cd12 remains uncertain.

FEBS Lett, 1997 Sep 8, 414(2), 397 - 401
Distribution of a sub-class of bacterial ABC polar amino acid transporter and identification of an N-terminal region involved in solute specificity; Walshaw DL et al.; A new sub-class of binding protein-dependent transporter with specificity for a broad range of polar amino acids has been identified by sequence comparison, in Rhizobium leguminosarum, Rhodobacter capsulatus, Escherichia coli and Pseudomonas fluorescens . Southern blotting and PCR analysis has shown that transporters from this new sub-class are widely distributed in Gram-negative bacteria, including, in addition to the above, Citrobacter freundii, Erwinia carotovorum and Rhizobium meliloti . ABC transporters of polar amino acids can be divided into two groups: those with narrow solute specificity and the newly identified sub-class with broad solute specificity . The binding and inner membrane proteins from transporters with a broad solute specificity are larger by approximately 30% than those with a narrow solute specificity . Multiple alignment of the inner membrane proteins from all sequenced polar amino acid transporters indicates there is an N-terminal conserved region that may be involved in solute specificity . A conserved arginine or lysine at residue 30 of this region is changed to glutamate in arginine transporters . Residue 53 also has a strong correlation with the charge on the transported solute, with basic amino acid transporters replacing an aliphatic amino acid at this position with a negatively charged amino acid . The general amino acid permease from R . leguminosarum, which will transport aliphatic as well as basic and acidic amino acids, juxtaposes two prolines at residues 52 and 53 of the N-terminal conserved region.

Am J Perinatol, 1997 Sep, 14(8), 465 - 7
Vertical transmission of a Citrobacter infection; Mastrobattista JM et al.; Citrobacter species have rarely been described as-etiological factors of intraamniotic infections . Citrobacter is not a normal inhabitant of the female genital tract . Vertical transmission of Citrobacter from mother to fetus has rarely been reported . A 21-year-old primigravida presented to labor and delivery at 40 6/7 weeks' gestation complaining of ruptured membranes, painful uterine contractions, and fever . An intraamniotic infection was diagnosed and antibiotics begun . She was subsequently delivered of a live male infant . Mother and infant had positive cultures for Citrobacter and overwhelming sepsis . Citrobacter species are rarely described as etiological factors of intraamniotic infections, and vertical transmission has rarely been reported . This pathogen should be considered in cases of chorioamnionitis or maternal sepsis as overwhelming maternal and fetal infection are possible sequelae.

FEMS Microbiol Lett, 1997 Sep 15, 154(2), 409 - 14
Mutations in the gyrA and parC genes associated with fluoroquinolone resistance in clinical isolates of Citrobacter freundii; Nishino Y et al.; We determined partial sequences of the gyrA and parC genes of Citrobacter freundii type strain, and then examined 38 C . freundii clinical strains isolated from patients with urinary tract infections for the association of alterations in GyrA and ParC with susceptibility to fluoroquinolones . Our results suggest that in C . freundii DNA gyrase may be a primary target of quinolones, that an amino acid change at Thr-83 or Asp-87 in GyrA is sufficient to decrease susceptibility to fluoroquinolones, and that accumulation of changes in GyrA with the simultaneous presence of an alteration at Ser-80 or Glu-84 in ParC may be associated with the development of high-level fluoroquinolone resistance in C . freundii clinical isolates.

FEMS Microbiol Lett, 1997 Sep 15, 154(2), 337 - 45
Glycerol conversion to 1,3-propanediol by Clostridium pasteurianum: cloning and expression of the gene encoding 1,3-propanediol dehydrogenase; Luers F et al.; When grown on glycerol as sole carbon and energy source, cell extracts of Clostridium pasteurianum exhibited activities of glycerol dehydrogenase, dihydroxyacetone kinase, glycerol dehydratase and 1,3-propanediol dehydrogenase . The genes encoding the latter two enzymes were cloned by colony hybridization using the dhaT gene of Citrobacter freundii as a heterologous DNA probe and expressed in Escherichia coli . The native molecular mass of 1,3-propanediol dehydrogenase (DhaT) is 440,000 Da . The dhaT gene of C . pasteurianum was subcloned and its nucleotide sequence (1158 bp) was determined . The deduced gene product (41,776 Da) revealed high similarity to DhaT of C . freundii (80.5% identity; 89.8% similarity).

Biochim Biophys Acta, 1997 Aug 15, 1341(1), 58 - 70
Characterization and amino acid sequence analysis of a new oxyimino cephalosporin-hydrolyzing class A beta-lactamase from Serratia fonticola CUV; Peduzzi J et al.; Serratia fonticola CUV produces two isoenzymes (forms I and II) with beta-lactamase activity which were purified by a five-step procedure . The isoenzymes had identical kinetic parameters and isoelectric point (pI = 8.12) . They were characterized by a specific activity towards benzylpenicillin of 1650 U/mg . The beta-lactamase hydrolyzed benzylpenicillin, amoxycillin, ureidopenicillins, first- and second-generation cephalosporins . Carboxypenicillins and isoxazolylpenicillins were hydrolyzed to a lesser extent . Towards cefotaxime and ceftriaxone (third-generation cephalosporins), the S . fonticola enzyme exhibited catalytic efficiencies much higher than those of MEN-1 and extended-spectrum TEM derivative beta-lactamases . The beta-lactamase from S . fonticola was markedly inhibited by beta-lactamase inhibitors such as clavulanic acid, sulbactam and tazobactam . The purified isoenzymes were digested by trypsin, endoproteinase Asp-N and chymotrypsin . Amino acid sequence determinations of the resulting peptides allowed the alignment of 267 amino acid residues (Swiss-Prot, accession number P 80545) for form I beta-lactamase . Form II is five residues shorter than form I at its N-terminus . From amino acid sequence comparisons, S . fonticola CUV beta-lactamase was found to share more than 69.3% identity with the chromosomally encoded beta-lactamases of Klebsiella oxytoca, Proteus vulgaris, Citrobacter diversus and the plasmid-mediated enzymes MEN-1 and Toho-1 . Therefore, the oxyimino cephalosporin-hydrolyzing beta-lactamase of S . fonticola belongs to Ambler's class A . Contribution of the serine at ABL 237 in the broad-spectrum activity of these beta-lactamases is discussed.

J Bacteriol, 1997 Sep, 179(18), 5914 - 21
The tpl promoter of Citrobacter freundii is activated by the TyrR protein; Smith HQ et al.; The ability of microorganisms to degrade L-tyrosine to phenol, pyruvate, and ammonia is catalyzed by the inducible enzyme L-tyrosine phenol lyase (EC 4.1.99.2) . To investigate possible mechanisms for how the synthesis of this enzyme is regulated, a variety of biochemical and genetic procedures was used to analyze transcription from the tpl promoter of Citrobacter freundii ATCC 29063 (C . braakii) . By computer analysis of the region upstream of the tpl structural gene, two segments of DNA bearing strong homology to the known operator targets of the TyrR protein of Escherichia coli were detected . A DNA fragment of 509 bp carrying these operator targets plus the presumptive tpl promoter was synthesized by PCR and used to construct a single-copy tpl-lacZ reporter system . The formation of beta-galactosidase in strains carrying this reporter system, which was measured in E . coli strains of various genotypes, was strongly dependent on the presence of a functional TyrR protein . In strains bearing deletions of the tyrR gene, the formation of beta-galactosidase was reduced by a factor of 10 . Several mutationally altered forms of TyrR were deficient in their abilities to activate the tpl promoter . The pattern of loss of activation function was exactly parallel to the effects of the same tyrR mutations on the mtr promoter, which is known to be activated by the TyrR protein . When cells carrying the tpl-lacZ reporter system were grown on glycerol, the levels of beta-galactosidase were 10- to 20-fold higher than those observed in glucose-grown cells . The effect was the same whether or not TyrR-mediated stimulation of the tpl promoter was in effect . By deleting the cya gene, it was shown that the glycerol effect was attributable to stimulation of the tpl promoter by the cyclic AMP (cAMP)-cAMP reporter protein system . A presumptive binding site for this transcription factor was detected just upstream of the -35 recognition hexamer of the tpl promoter . The transcriptional start point of the tpl promoter was determined by chemical procedures . The precise locations of the TyrR binding sites, which were established by DNase I footprinting, agreed with the computer-predicted positions of these regulatory sites . The two TyrR operators, which were centered at coordinates -272.5 and -158.5 with respect to the transcriptional start point, were independently disabled by site-directed mutagenesis . When the upstream operator was altered, activation was completely abolished . When the downstream operator was altered, there was a fourfold reduction in reporter enzyme levels . The tpl system presents a number of intriguing features not previously encountered in TyrR-activated promoters . First among these is the question of how the TyrR protein, bound to widely separated operators, activates the tpl promoter which is also widely separated from the operators.

Arch Microbiol, 1997 Aug, 168(2), 160 - 3
Sensitivity to pH, product inhibition, and inhibition by NAD+ of 1,3-propanediol dehydrogenase purified from Enterobacter agglomerans CNCM 1210; Barbirato F et al.; Because of its key role in the metabolism of glycerol during fermentation, 1,3-propanediol dehydrogenase (EC 1.1.1.202) of Enterobacter agglomerans CNCM 1210 was purified to homogeneity and studied with respect to its sensitivity to pH and to nucleotide and 1,3-propanediol concentrations . Enzyme activity was optimal at pH 7.8 . The enzyme was competitively inhibited by NAD+ (Ki of 0.29 mM), and 1,3-propanediol exerted a strong inhibitory effect according to a mixed-type inhibition with a Ki of 13.7 mM and an a-factor of 9.0 . It is proposed that these dehydrogenase properties be extended to the dehydrogenases of Citrobacter freundii and Klebsiella pneumoniae, which exhibited numerous similar physical properties.

Eur J Clin Microbiol Infect Dis, 1997 Jul, 16(7), 523 - 9
Molecular epidemiology of an outbreak due to extended-spectrum beta-lactamase-producing enterobacteria in a French hospital; Bermudes H et al.; Between June 1992 and June 1993, 128 extended-spectrum beta-lactamase-producing enterobacteria (123 Klebsiella pneumoniae, 3 Escherichia coli, 1 Enterobacter aerogenes, and 1 Citrobacter diversus) were collected in a French university hospital . These isolates were recovered mainly from patients hospitalized in intensive care and neurosurgery units . The 128 strains were divided into 14 antibiotypes (ATBs; ATB1 to ATB14); 102 of 103 nonredundant isolates were shown to produce an SHV-4-related extended-spectrum beta-lactamase (pl 7.8, hybridization with a blaSHV probe); the remaining strain (Kp 2108) produced a TEM-3-related extended-spectrum beta-lactamase (pl 6.3, hybridization with a blaTEM probe) . For representative isolates, five plasmid profiles (Pl to Pll-4), eight ribotypes (E1 to E8), and seven arbitrarily primed polymerase chain reaction profiles (A1 to A7) were obtained . The results suggest the spread of an epidemic strain of Klebsiella pneumoniae (E1, A1, Pl, various ATBs) from an intensive care unit throughout the hospital . Another epidemic strain (E2, A2, pl, ATB4) was confined to the neurosurgery unit . Other extended-spectrum beta-lactamase-producing Klebsiella pneumoniae of ribotypes distinct from E1 or E2 might result from the spread of an epidemic plasmid, such as reported for isolates of other enterobacterial species . Conversely, they might represent imported cases, such as the strain Kp 2108, which produced a TEM-3-related beta-lactamase.

Microbiology, 1997 Jul, 143 ( Pt 7), 2497 - 507
Localization of enzymically enhanced heavy metal accumulation by Citrobacter sp . and metal accumulation in vitro by liposomes containing entrapped enzyme; Jeong BC et al.; A heavy-metal-accumulating Citrobacter sp . has been used for the treatment of metal-laden industrial wastes . Metal uptake is mediated via a cell-bound phosphatase that liberates inorganic phosphate which precipitates with heavy metals as cell-bound metal phosphate . A phosphatase-efficient mutant accumulated little UO(2)2+, while a phosphatase-overproducing mutant accumulated correspondingly more metal, with a uranium loading equivalent to the bacterial dry weight achieved after 6 h exposure of resting cells to uranyl ion in the presence of phosphatase substrate (glycerol 2-phosphate) . The phosphatase, visualized by immunogold labelling in the parent and overproducing strains, but not seen in the deficient mutant, was held within the periplasmic space with, in some cells, a higher concentration at the polar regions . Enzyme was also associated with the outer membrane and found extracellularly . Accumulated uranyl phosphate was visible as cell-surface- and polar-localized deposits, identified by energy-dispersive X-ray analysis (EDAX), proton-induced X-ray emission analysis (PIXE) and X-ray diffraction analysis (XRD) as polycrystalline HUO2PO4.4H2O . Nucleation sites for initiation of biocrystallization were identified at the cytoplasmic and outer membranes, prompting consideration of an in vitro biocatalytic system for metal waste remediation . Phosphatidylcholine-based liposomes with entrapped phosphatase released phosphate comparably to whole cells, as shown by 31P NMR spectroscopy in the presence of 'NMR-silent' 112Cd2+ . Application of liposome-immobilized enzyme to the decontamination of uranyl solutions was, however, limited by rapid fouling of the biocatalyst by deposited uranyl phosphate . It is suggested that the architecture of the bacterial cell surface provides a means of access of uranyl ion to the inner and outer membranes and enzymically liberated phosphate in a way that minimizes fouling in whole cells.

Protein Sci, 1997 Jul, 6(7), 1503 - 10
Binding of monoclonal antibody 4B1 to homologs of the lactose permease of Escherichia coli; Sun J et al.; The conformationally sensitive epitope for monoclonal antibody (mAb) 4B1, which uncouples lactose from H+ translocation in the lactose permease of Escherichia coli, is localized in the periplasmic loop between helices VII and VIII (loop VII/VIII) on one face of a short helical segment (Sun J, et al., 1996, Biochemistry 35;990-998) . Comparison of sequences in the region corresponding to loop VII/VIII in members of Cluster 5 of the Major Facilitator Superfamily (MFS), which includes five homologous oligosaccharide/H+ symporters, reveals interesting variations . 4B1 binds to the Citrobacter freundii lactose permease or E . coli raffinose permease with resultant inhibition of transport activity . Because E . coli raffinose permease contains a Pro residue at position 254 rather than Gly, it is unlikely that the mAb recognizes the peptide backbone at this position . Consistently, E . coli lactose permease with Pro in place of Gly254 also binds 4B1 . In contrast, 4B1 binding is not observed with either Klebsiella pneumoniae lactose permease or E . coli sucrose permease . When the epitope is transferred from E . coli lactose permease (residues 245-259) to the sucrose permease, the modified protein binds 4B1, but the mAb has no significant effect on sucrose transport . The studies provide further evidence that the 4B1 epitope is restricted to loop VII/VIII, and that 4B1 binding induces a highly specific conformational change that uncouples substrate and H+ translocation.

Acta Paediatr Jpn, 1997 Jun, 39(3), 390 - 1
Citrobacter koseri osteomyelitis in an infant; Hayani KC; A 3-week-old infant developed left shoulder swelling and was found to have septic arthritis and osteomyelitis of the humerus caused by Citrobacter koseri (formerly C . diversus) . Citrobacter species are Gram-negative rods that are best known for their propensity to cause neonatal meningitis, ventriculitis and concomitant brain abscess . Non-central nervous system infections are rare . The present case illustrates that neonatal osteomyelitis caused by unusual organisms can present to pediatricians in an inner-city setting, and can respond favorably to surgical and medical management.

Biochemistry, 1997 May 27, 36(21), 6502 - 10
The crystal structure of Citrobacter freundii tyrosine phenol-lyase complexed with 3-(4'-hydroxyphenyl)propionic acid, together with site-directed mutagenesis and kinetic analysis, demonstrates that arginine 381 is required for substrate specificity; Sundararaju B et al.; The X-ray structure of tyrosine phenol-lyase (TPL) complexed with a substrate analog, 3-(4'-hydroxyphenyl)propionic acid, shows that Arg 381 is located in the substrate binding site, with the side-chain NH1 4.1 A from the 4'-OH of the analog . The structure has been deduced at 2.5 A resolution using crystals that belong to the P2(1)2(1)2 space group with a = 135.07 A, b = 143.91 A, and c = 59.80 A . To evaluate the role of Arg 381 in TPL catalysis, we prepared mutant proteins replacing arginine with alanine (R381A), with isoleucine (R381I), and with valine (R381V) . The beta-elimination activity of R381A TPL has been reduced by 10(-4)-fold compared to wild type, whereas R381I and R381V TPL exhibit no detectable beta-elimination activity with L-tyrosine as substrate . However, R381A, R381I, and R381V TPL react with S-(o-nitrophenyl)-L-cysteine, beta-chloro-L-alanine, O-benzoyl-L-serine, and S-methyl-L-cysteine and exhibit k(cat) and k(cat)/Km values comparable to those of wild-type TPL . Furthermore, the Ki values for competitive inhibition by L-tryptophan and L-phenylalanine are similar for wild-type, R381A, and R381I TPL . Rapid-scanning-stopped flow spectroscopic analyses also show that wild-type and mutant proteins can bind L-tyrosine and form quinonoid complexes with similar rate constants . The binding of 3-(4'-hydroxyphenyl)propionic acid to wild-type TPL decreases at high pH values with a pKa of 8.4 and is thus dependent on an acidic group, possibly Arg404, which forms an ion pair with the analog carboxylate, or the pyridoxal 5'-phosphate Schiff base . R381A TPL shows only a small decrease in k(cat)/Km for tyrosine at lower pH, in contrast to wild-type TPL, which shows two basic pKas with an average value of about 7.8 . Thus, it is possible that Arg 381 is one of the catalytic bases previously observed in the pH dependence of k(cat)/Km of TPL with L-tyrosine {Kiick, D . M., & Phillips . R . S . (1988) Biochemistry 27, 7333-7338}, and hence Arg 381 is at least partially responsible for the substrate specificity of TPL.

Gig Sanit, 1997 May-Jun, (3), 24 - 6
{Microbiological contamination of fish in the delta of Volga}; Lartseva LV et al.; Examining 213 bacteriological samples from 57 pike perch has revealed the high contamination of the fish with Enterobacteria in the branchia and intestine, which spread into the viscera and tissues . In the kidney there prevalent Escherichia coli (27.3%) . Enterobacteria (20.), and Edvardsiella (16.6) . The liver most commonly displayed yeasts and fungi (18.2%) . Citrobacteria (14.3) . Acinobacteria (14.3%) . Many microbial strains had proteolytic and hemolytic activities and a considerable resistance to sodium chloride (7.5%) . Acinobacteria, Providencia, and Proteus remain viable in its 10% solution . The findings suggest that the foods from the pike perch should undergo thermal treatment and a thorough bacteriological monitoring from its raw material to finished products to be made.

Drugs, 1997 Apr, 53(4), 637 - 56
Fosfomycin tromethamine . A review of its antibacterial activity, pharmacokinetic properties and therapeutic efficacy as a single-dose oral treatment for acute uncomplicated lower urinary tract infections; Patel SS et al.; Fosfomycin tromethamine is a phosphonic acid bactericidal agent with in vitro activity against most urinary tract pathogens . It is particularly active against Escherichia coli, and Citrobacter, Enterobacter, Klebsiella, Serratia and Enterococcus spp . There appears to be little cross-resistance between fosfomycin and other antibacterial agents, possibly because it differs from other agents in its general chemical structure and site of action . In its new formulation as the oral tromethamine salt, fosfomycin has 34 to 41% oral bioavailability, has a mean elimination half-life of 5.7 hours, and is primarily excreted unchanged in the urine . Following a single 3 g oral dose, peak urinary concentrations occur within 4 hours and remain high (> 128 mg/L) for 24 to 48 hours, which is sufficient to inhibit most urinary tract pathogens . In clinical trials in patients with acute uncomplicated lower urinary tract infection, single-dose fosfomycin tromethamine therapy was effective, and comparable with several other antibacterial agents given either as single-dose or multiple-dose treatments {e.g . beta-lactam and fluoroquinolone agents, cotrimoxazole (trimethoprim-sulfamethoxazole), nitrofurantoin and pipemidic acid} . Bacteriological eradication rates of 75 to 90% were achieved 5 to 11 days after therapy, with eradication rates of 62 to 93% 4 to 6 weeks after therapy . In 3 large double-blind comparisons with ciprofloxacin, cotrimoxazole and nitrofurantoin, 99% of fosfomycin tromethamine recipients and 100% of patients receiving comparator agents were considered clinically cured or improved after therapy . Fosfomycin tromethamine is well tolerated, with a low incidence of adverse events . These comprise mainly gastrointestinal symptoms that are transient, mild and self-limiting . Thus, fosfomycin tromethamine achieves high clinical and bacteriological cure rates in patients with acute uncomplicated lower urinary tract infection and is well tolerated . The single-dose administration regimen and favourable US pregnancy category rating of fosfomycin tromethamine should also encourage its use in this indication.

FEMS Microbiol Lett, 1997 Mar 15, 148(2), 239 - 45
Cloning and characterization of the eae gene from a dog attaching and effacing Escherichia coli strain 4221; An H et al.; We have cloned and determined the nucleotide sequence of the eae gene from a dog attaching and effacing (A/E) Escherichia coli (DEPEC) strain 4221 . When comparing the predicted amino acid sequence of the eaeDEPEC to that of the Eae proteins from enteropathogenic E . coli (EPEC), enterohaemorrhagic E . coli O157:H7 (EHEC), Citrobacter freundii biotype 4280, and a swine A/E E . coli strain O45 (PEPEC), the overall sequence identity was 84, 81, 83 and 83%, respectively, with the greatest divergence at the C-terminal end, the putative receptor-binding portion . Interestingly, the DEPEC Eae shares the greatest identity at the C-terminal region with the Citrobacter freundii Eae protein . We have constructed and purified a maltose-binding fusion protein (MBP) containing the product of the entire eae gene of the DEPEC strain 4221 . Binding of MBP-EaeDEPEC fusion protein to HEp-2 cells was demonstrated by immunofluorescence microscopy . In addition, the Eae protein of DEPEC (4221) demonstrated a strong serological relationship with that of EPEC (E2348/69) as observed using a polyclonal antiserum against MBP-EaeDEPEC fusion protein.

Zh Mikrobiol Epidemiol Immunobiol, 1997 Mar-Apr, (2), 29 - 33
{New data on the specificity and genetic control of the capsular antigen (F1) in Yersinia pestis}; Kosse LV et al.; The specific features of the synthesis of antigenic fractions F1, obtained from the strains of different species of enterobacteria and their hybrids inheriting Y . pestis plasmid pYT, were determined at different cultivation temperatures and the preparations of these antigenic fractions were characterized . Preparations containing the diagnostic antigenic determinant F1 were obtained from disintegrated bacteria of Y . pestis plasmid-free strain and microbial cells of Klebsiella pneumoniae, Citrobacter freundii and Escherichia coli initial recipient cultures by Baker's method . These data were indicative of the chromosomal control of the synthesis of antigen F1, responsible for the manifestation of protective activity . The antigen synthesized in the absence of pYT was practically, not secreted on the cell-wall surface and could not be determined by tests made with the use of whole bacterial cells . The antigen thus detected did not possess the properties of the preparation of antigen F1, obtained from Y . pestis pYT-containing strain, though it was capable of inducing the formation of F1-specific antibodies.

J Indian Med Assoc, 1997 Mar, 95(3), 72 - 4, 77
Reservoirs of nosocomial pathogens in neonatal intensive care unit; Chandrashekar MR et al.; A total of 256 swabs taken from different areas of neonatal intensive care units (ICU) in KCG Hospital and AMC Hospital, Bangalore were bacteriologically investigated for prevalence, source and spread of nosocomial bacteria . Culture studies revealed growth in 217 (84.8%) swab samples indicating considerable contamination of different areas of the units and sources of infection . Klebsiella pneumoniae (27.3%) was the predominant organism followed by Esch coli (16.8%), Staph aureus (11.7%), Staph epidermidis and Pseudomonas aeruginosa (10.2%), enterococcus and proteus (4.7%), Citrobacter freundi (3.5%) and Clostridium tetani (2.4%) isolated from the equipment, cradles, other inanimate objects and environmental surfaces . Out of 312 isolates, monobacterial prevalence was 43.6% in contrast to polybacterial prevalence of 56.4% . Klebsiella pneumoniae (74.3%) was the predominant monobacterial isolate . The indoor air of the units was found to carry common nosocomial bacteria of 4 or more different bacterial species at dangerous levels as observed by colony counts of 15 to 30 on exposed blood agar plates . Almost all sources in ICU revealed the presence of Klebsiella Pneumoniae, Esch coli, pseudomonas and staphylococcus thus forming the potential reservoirs of nosocomial infections to babies and this could be attributed to overcrowding, poor ventilation system and failure to follow basic principles of strict protective barrier nursing.

East Afr Med J, 1997 Mar, 74(3), 147 - 50
Rational use of antibiotics in neonatal infections; Musoke RN; Review of the management of neonatal infections is done with the aim of guiding the clinician on appropriate therapy . Minimum investigations should include a white blood cell count including the L:T ratio and a blood culture . The bulk of infections at Kenyatta National Hospital newborn unit are caused by Klebsiela, Citrobacter and Staphylococcus aureus . During the 1990's considerable resistance to gentamicin has developed . Currently, cephalosporins chloramphenicol have the best sensitivity pattern . The diagnosis must be carefully verified at different stages of treatment to ensure that only those requiring antimicrobial therapy get it . Indiscriminate use is thus avoided . This in turn minimises development of antibiotic resistant organisms . Failure of response to antimicrobials sometimes means a non infectious cause of illness or poor supportive management . Continuous surveillance is recommended with emphasis on primary prevention of infection as well as cross infections.

Vet Med (Praha), 1997 Mar, 42(3), 87 - 91
{Identification of Citrobacter species and their occurrence in raw products and foods}; Urbanova E et al.; Fifty-nine strains of bacteria were tested in study that were isolated from raw materials and foods (raw milk, farm milking parlors, sausage meat, raw potatoes, cheese, frozen oven-ready foods, confectionery products, cold dishes), and they were included in the genus Citrobacter using a commercial diagnostic kit ENTEROtest 16 (Lachema a.s., Brno), numeric identification program TNW (Czech Collection of Microorganisms, Faculty of Science of Masaryk University at Brno) and identification key (O'Hara et al., 1995) . The results of the ENTEROtest itself, including OXI and ONPtests, did not provide satisfactory discrimination of detected strains to the level of species since 64.4% were identified by the genus by TNW program or designated as intermediary strains . Correct species identification was obtained in 33.9% only (Tab . I) . Five next conventional tests (dulcitol, alpha-methyl-glucoside, raffinose, melibiose, arginine dihydrolase) were used and their results successfully specified 94.9% tested strains to the species level (Tab . I) . A dichotomic identification key (O'Hara et al., 1995) enabled to classify the strains on the basis of the results of ENTEROtest 16 and two conventional tests (dulcitol, melibiosis) relatively easily, and it can be recommended for routine identification of typical Citrobacters from foods . Only ten strains were classified in a wrong way (16.9%) due to false results in ENTEROtest (Tab . II) . Tab . III shows the strains classified wrongly by both identifications systems . The tested set contained Citrobacter braakii in a majority of cases (30 strains), followed by C . freundii (17 strains), C . youngae (6 strains), 2 strains of genomospecies 10 and one strain of C . koseri, C . amalonaticus and C farmeri . Tab . IV shows the presence of Citrobacter species in the particular raw materials and foods . The most frequently present species in raw materials: C . braakii (26 strains, 68.4%), C . freundii (3 strains, 7.9%) and C . youngae (3 strains, 7.9%) . Foods contained these three species only: C . freundii (14 strains, 66.7%), C . braakii (4 strains, 19.0%) and C . youngae (3 strains, 14.3%) . The percentage of the three most frequently present species in raw materials is substantially different from their percentage in foods (Fig . 1) . The higher percentage of C . freundii presence in foods can be ascribed to secondary contamination.

Vet Med (Praha), 1997 Mar, 42(3), 81 - 5
{Changes in the microbial picture during the production of poultry salami}; Pipova M et al.; Changes in microbiological parameters during the production of fine poultry salami was monitored in a private poultry-processing plant in Kosice within a period of six months . This product consists of meat paste or mechanically deboned poultry meat (MDPM), which is produced with the help of a Protecon MPB 30 E deboning machine, pork trimmings, water (in the form of crushed ice), salt, spices, and garlic paste . The main raw food material (hand-boned meat and skin from poultry carcasses intended for mechanical deboning), MDPM immediately after production, salted MDPM after its 24-hour-storage and ripening in the chiller, pork trimmings added to MDPM at an amount of 25%, prepared salami emulsion, and the final product (fine poultry salami) were examined for the presence of pathogenic and potentially pathogenic microorganisms . The total plate count, the counts of indicatory microorganisms (Coliforms and Enterococci), Staphylococci, psychrotrophic, proteolytic and lipolytic bacteria were also determined according to the Slovak government regulations . The results of the quantitative microbiological examination are shown in Figs . 1 and 2 . It follows from them that the microbial load of poultry skin is considerably higher than that of hand-boned meat . During the deboning process an increase in all microbial parameters (on average by 1-3 radices) was noticed as compared with the raw food material (defrosted poultry carcasses before the mechanical deboning) . MDPM contained 10(4)-10(7) CFU/g; the count of coliforms ranged between 10(3) and 10(6)/g; the count of proteolytic and lipolytic bacteria between 10(4) and 10(6)/g; the count of Staphylococci and Enterococci between 10(2) and 10(4)/g . Staphylococcus aureus was discovered in about 50% samples of MDPM in an average concentration of 10(2)/g . In addition to the groups of bacteria listed above, a wide range of potentially pathogenic microorganisms belonging to the family Enterobacteriaceae, especially those of the genus Proteus, Escherichia coli, Citrobacter and Providencia, was also found in MDPM . In four cases Salmonella enteritidis was also detected in the samples of both the skin of chicken carcasses and the MDPM . The nitrate salting mixture added at a concentration of 2% did not significantly reduce the counts of bacteria present in the MDPM . The prepared salami emulsion was very high in all groups of microorganisms, which was also caused by the addition of pork trimmings . This component seems to be a very important source of microbial contamination, showing the following average bacterial counts: total plate count 4.8 x 10(6)/g, the count of psychrotrophic bacteria 1.0 x 10(6)/g, the count of coliforms 7.0 x 10(5)/g, the count of Enterococci 9.2 x 10(3)/g, the count of Staphylococci 3.0 x 10(4)/g, the count of proteolytic microorganisms 2.7 x 10(5)/g, and the count of lipolytic microorganisms 4.2 x 10(5)/g . However, heat treatment of the final product reliably and almost completely devitalized all the bacteria found in the salami emulsion before heat processing . No pathogenic or potentially pathogenic bacteria were detected in any sample of fine poultry salami . Quite a high number of bacteria in the salami emulsion was reduced by heat treatment to a final average value of 10(2)/g.

FEMS Microbiol Lett, 1997 Mar 1, 148(1), 15 - 20
Cloning and sequencing of the gene encoding the AmpC beta-lactamase of Morganella morganii; Barnaud G et al.; The chromosomal beta-lactamase gene of a clinical isolate of Morganella morganii was cloned in Escherichia coli and sequenced . The beta-lactamase had a pI of 7.4 and conferred a typical AmpC susceptibility pattern . The insert obtained was found to encode a protein of 379 amino acids . Its deduced amino acid sequence revealed it to be a class C beta-lactamase: 39-56% identity with chromosomal AmpC beta-lactamases of Serratia marcescens, Yersinia enterocolitica, Citrobacter freundii, Enterobacter cloacae and Escherichia coli; and 37-56% identity with plasmid-mediated beta-lactamases (MOX-1, CMY-1, FOX-1, ACT-1, LAT-1, BIL-1 and CMY-2) . The ampC gene was linked to a gene only part of which (450 bp) was cloned homologous to the regulatory ampR genes of chromosomal class C beta-lactamases.

Anal Biochem, 1997 Feb 15, 245(2), 207 - 12
An homogeneous fluorescence polymerase chain reaction assay to identify Salmonella; Tseng SY et al.; We have developed a semiquantitative homogeneous fluorescence assay that combines polymerase chain reaction (PCR) amplification with direct fluorescence detection (HF-PCR) . The assay eliminates the need to perform gel electrophoresis on test samples . Using a set of Salmonella-specific primers, this system was used to verify suspect colonies from culture plates as Salmonella . The fluorescence signal is generated by a nucleic acid dye, YO-PRO-1, that is included in the amplification reaction . This homogeneous PCR assay was used to test 84 Salmonella strains picked from selective culture plates . All data indicated positive results when compared with 17 non-Salmonella strains (in general, Citrobacter, Hafnia, Proteus, and Escherichia) . The HF-PCR assay is a sensitive, simple, accurate, and reproducible method that correlates well with size-exclusion high-performance liquid chromatography and gel electrophoresis techniques as a means to monitor PCR-mediated DNA amplification . This assay can confirm suspect colonies within 2.5 h.

Zentralbl Bakteriol, 1997 Feb, 285(3), 389 - 96
Detection and identification of Citrobacter sedlakii in the Czech Republic; Aldova E et al.; The biochemical characters of eight strains identified as Citrobacter sedlakii were investigated with the aid of six methods (tube tests, API 50 CH, API 20 E, MICROSCAN, BIOLOG, and CRYSTAL) . All the strains were well defined on the basis of biochemical properties investigated with the aid of laboratory-prepared tube tests . The results obtained by the identification kits could not be correctly interpreted . Commercial diagnostic kits should include the reference data necessary for the identification of new Citrobacter species.

Antimicrob Agents Chemother, 1997 Feb, 41(2), 314 - 8
Characterization of the 6'-N-aminoglycoside acetyltransferase gene aac(6')-Im {corrected} associated with a sulI-type integron; Hannecart-Pokorni E et al.; The amikacin resistance gene aac(6')-Im {corrected} from Citrobacter freundii Cf155 encoding an aminoglycoside 6'-N-acetyltransferase was characterized . The gene was identified as a coding sequence of 521 bp located down-stream from the 5' conserved segment of an integron . The sequence of this aac(6')-Im {corrected} gene corresponded to a protein of 173 amino acids which possessed 64.2% identity in a 165-amino-acid overlap with the aac(6')-Ia gene product (F.C . Tenover, D . Filpula, K.L . Phillips, and J . J . Plorde, J . Bacteriol . 170:471-473, 1988) . By using PCR, the aac(6')-Im {corrected} gene could be detected in 8 of 86 gram-negative clinical isolates from two Belgian hospitals, including isolates of Citrobacter, Klebsiella spp., and Escherichia coli . PCR mapping of the aac(6')-Im {corrected} gene environment in these isolates indicated that the gene was located within a sulI-type integron; the insert region is 1,700 bases long and includes two genes cassettes, the second being ant (3")-Ib.

Clin Oncol (R Coll Radiol), 1997, 9(3), 172 - 5
Citrobacter freundii and fatal neutropenic enterocolitis following adjuvant chemotherapy for breast cancer; Clemons MJ et al.; Neutropenic enterocolitis is increasingly being recognized as a life-threatening complication of chemotherapy, mainly for haematological and lymphoproliferative malignancies . It is under-recognized clinically, with the diagnosis often being made on post-mortem examination . Although active medical management is generally preferred, surgical intervention may be indicated . We report a case of fatal neutropenic enterocolitis, secondary to Citrobacter freundii, following adjuvant chemotherapy for breast cancer . We also review the literature, examining the aetiology, diagnosis and management of this often fatal entity.

Plasmid, 1997, 37(1), 2 - 14
Small cryptic plasmids of multiplasmid, clinical Escherichia coli; Burian J et al.; Clinical isolates of Escherichia coli were found to host a multiplicity of plasmids . These were resolved from plasmid gel profiles, from the properties of various transconjugants and transformants of E . coli DH1, by the topoisomerase I relaxation of covalently closed circle plasmid DNA, by electron microscopy, and by the determination of their compatibilities . The majority of these were unusually small, cryptic plasmids (SCPs) . From one strain, KL4, 13 electrophoretic bands were resolved to five plasmids, three of which were SCPs . SCPs were phenotypically barren, and the smallest of these, pKL1, contained barely enough information for self-replication . A derivative of pKL1, pKL1Km, in which the transposon was restricted to a small 350-bp region, was stably maintained in Shigella, Salmonella, Serratia, and Citrobacter species and its replication was polA independent . pKL1 encoded only a single protein, RepA (Mr 17960), which specifically bound to pKL1 DNA . No apparent homologies with other RepA protein sequences could be detected . Thus the SCP, pKL1, is a novel minimal plasmid replicon encoding only enough information to ensure perpetuation . A hypothesis is presented describing SCPs as a class of selfish DNA that persists simply due to its ability to replicate and to its stability based on high copy number.

Med Vet Entomol, 1997 Jan, 11(1), 58 - 64
The responses of Lucilia cuprina to odours from sheep, offal and bacterial cultures; Morris MC et al.; The responses of gravid female Lucilia cuprina to odours from sheep urine, faeces and gut mucus, and to odours from liver/sodium sulphide mixtures was tested using a bioassay which measured the movement and probing response of walking flies . The same bioassay was used to test the response to odours from cultures of bacteria isolated from liver/sodium sulphide and liver/water mixtures . A significant movement towards odours from faeces, gut mucus and urine was observed . Odours from cultures of the bacteria Proteus mirabilis, Dermatophilus congolensis and Serratia marcescens also elicited significant movement . A probing response was elicited by odours from gut mucus, fresh urine, liver/sodium sulphide mixtures and cultures of P.mirabilis, D.congolensis and gram-positive species . Odours from cultures of Pseudomonas aeruginosa, Enterobacter aerogenes and Citrobacter freundii did not elicit significant movement or probing . The movement and probing responses are discussed with reference to the possible uses of the substances tested as a bait for attracting L.cuprina.

J Antimicrob Chemother, 1997 Jan, 39(1), 31 - 4
Antibacterial activity of T-5575, a novel 2-carboxypenam, and its stability to beta-lactamase; Matsumura N et al.; The in-vitro activity of T-5575, 2-carboxypenam, a new parenteral antibiotic and its stability to beta-lactamases were compared with those of ceftazidime and imipenem . The activity of T-5575 was equal or superior to that of ceftazidime or imipenem against Gram-negative bacteria that produced penicillinase with the exception of the enzyme OXA-1 . Against strains that produced Cephalosporinase and zinc-dependent beta-lactamase, the activity of T-5575 was superior to that of ceftazidime or imipenem . T-5575 was a poor substrate and had low affinity for beta-lactamases produced by Citrobacter freundii, Enterobacter cloacae and Pseudomonas aeruginosa . The activity of T-5575 was less influenced by the derepressed production of chromosomal enzymes than that of ceftazidime . Overall, T-5575 had excellent activity against Gram-negative pathogens that produced various types of beta-lactamases.

Jpn J Antibiot, 1996 Dec, 49(12), 1073 - 84
{Antimicrobial activities of cefetamet against clinical isolates from urinary tract infection}; Ishii Y et al.; In order to evaluate antimicrobial activity of cefetamet (CEMT), minimum inhibitory concentrations (MICs) of CEMT and control drugs were determined against Gram-negative rods mainly from complicated urinary tract infections examined in our laboratory from April to September of 1994 . The results are summarized as follows; 1 . The obtained strains were Citrobacter diversus 20, Citrobacter freundii 30, Enterobacter aerogenes 20, Enterobacter cloacae 30, Serratia marcescens 30, Proteus mirabilis 30, Proteus vulgaris 20 and Morganella morganii 30 strains, a total of 210 strains . 2 . Excluding some resistant strains, the MIC-distribution showed showed that CEMT had strong antimicrobial activities against those strains from the MIC-distribution of this investigation . Compared to reports on CEMT in 1989, the MIC80 of CEMT in this investigation against clinical isolates were similar . The MIC50's of CEMT against E . aerogenes, S . marcescens, P . mirabilis, P . vulgaris and M . morganii in the previous examination were equal to or similar to the current results, but the MIC50's against C . freundii and E . cloacae were lower than the value of this report . The detection frequency of highly resistant strains of C . freundii and E . cloacae to cefteram and cefixime were similar to that of CEMT-resistant strains . Multiple drug resistant strains, among these bacterial species seemed to be increasing . 3 . Compared to oral antibacterial agents of oxime cephems that were used in the past, CEMT showed higher peak values of urinary excretion concentration and higher blood levels were sustained for a longer period of time . CEMT-PI will be effective against urinary tract infections.

Infect Immun, 1996 Dec, 64(12), 5315 - 25
Intimin from enteropathogenic Escherichia coli restores murine virulence to a Citrobacter rodentium eaeA mutant: induction of an immunoglobulin A response to intimin and EspB; Frankel G et al.; The formation of attaching and effacing (A/E) lesions is central to the pathogenesis of enteropathogenic Escherichia coli (EPEC)-mediated disease in humans and Citrobacter rodentium (formerly C . freundii biotype 4280)-mediated transmissible colonic hyperplasia in mice . Closely related outer membrane proteins, known as intimins, are required for formation of the A/E lesion by both EPEC (Int(EPEC)) and C . rodentium (Int(CR)) . A secreted protein, EspB (formally EaeB), is also necessary for A/E-lesion formation . Here we report that expression of a cloned Int(EPEC), encoded by plasmid pCVD438, restores murine virulence to an intimin-deficient mutant of C . rodentium DBS255 . Replacement of Cys937 with Ala abolished the ability of the cloned EPEC intimin to complement the deletion mutation in DBS255 . Ultrastructural examination of tissues from wild-type C . rodentium and DBS255(pCVD438)-infected mice revealed multiple A/E lesion on infected cells and loss of contact between enterocytes and basement membrane . Histological investigation showed that although both wild-type C . rodentium and DBS255(pCVD438) colonized the descending colon and induced colonic hyperplasia in orally infected 21-day-old mice, the latter strain adhered to epithelial cells located deeper within crypts . Nonetheless, infection with the wild-type strain was consistently more virulent, as indicated by a higher mortality rate . All the surviving mice, challenged with either wild-type C . rodentium or DBS255(pCVD438), developed a mucosal immunoglobulin A response to intimin and EspB . These results show that C . rodentium infection provides a relevant, simple, and economic model to investigate the role of EPEC proteins in the formation of A/E lesions in vivo and in intestinal disease.

J Bacteriol, 1996 Nov, 178(22), 6546 - 54
Characterization of EspC, a 110-kilodalton protein secreted by enteropathogenic Escherichia coli which is homologous to members of the immunoglobulin A protease-like family of secreted proteins; Stein M et al.; Enteropathogenic Escherichia coli (EPEC) secretes at least five proteins . Two of these proteins, EspA and EspB (previously called EaeB), activate signal transduction pathways in host epithelial cells . While the role of the other three proteins (39, 40, and 110 kDa) remains undetermined, secretion of all five proteins is under the control of perA, a known positive regulator of several EPEC virulence factors . On the basis of amino-terminal protein sequence data, we cloned and sequenced the gene which encodes the 110-kDa secreted protein and examined its possible role in EPEC signaling and interaction with epithelial cells . In accordance with the terminology used for espA and espB, we called this gene espC, for EPEC-secreted protein C . We found significant homology between the predicted EspC protein sequence and a family of immunoglobulin A (IgA) protease-like proteins which are widespread among pathogenic bacteria . Members of this protein family are found in avian pathogenic Escherichia coli (Tsh), Haemophilus influenzae (Hap), and Shigella flexneri (SepA) . Although these proteins and EspC do not encode IgA protease activity, they have considerable homology with IgA protease from Neisseria gonorrhoeae and H . influenzae and appear to use a export system for secretion . We found that genes homologous to espC also exist in other pathogenic bacteria which cause attaching and effacing lesions, including Hafnia alvei biotype 19982, Citrobacter freundii biotype 4280, and rabbit diarrheagenic E . coli (RDEC-1) . Although these strains secrete various proteins similar in molecular size to the proteins secreted by EPEC, we did not detect secretion of a 110-kDa protein by these strains . To examine the possible role of EspC in EPEC interactions with epithelial cells, we constructed a deletion mutant in espC by allelic exchange and characterized the mutant by standard tissue culture assays . We found that EspC is not necessary for mediating EPEC-induced signal transduction in HeLa epithelial cells and does not play a role in adherence or invasion of tissue culture cells.

FEMS Microbiol Lett, 1996 Nov 1, 144(2-3), 249 - 58
Characterization of the eaeA gene from rabbit enteropathogenic Escherichia coli strain RDEC-1 and comparison to other eaeA genes from bacteria that cause attaching-effacing lesions; Agin TS et al.; A number of enteric pathogens, including enteropathogenic (EPEC) and enterohemorrhagic (EHEC) Escherichia coli, Hafnia alvei, a strain of Citrobacter freundii, and rabbit EPEC strain RDEC-1 cause attaching-effacing (AE) lesions in the gut mucosa . These bacteria have a pathogenicity cassette (locus of enterocyte effacement or LEE) containing the eaeA gene . This gene encodes intimin, an outer membrane protein required for production of AE lesions . RDEC-1, a non-invasive enteropathogen in young rabbits, produces AE lesions morphologically indistinguishable from lesions caused by human AE bacterial strains . The RDEC-1 example of E . coli diarrhea in rabbits is an important model for studying the pathogenesis of AE bacteria in a natural infection and for analyzing specific roles of the components of LEE . In order to better understand the role of intimin in the development of AE lesions, a portion of DNA within RDEC-1 LEE, containing the eaeA gene and an upstream open reading frame (ORF), was sequenced . The RDEC-1 eaeA gene shared 87%, 92%, and 93% DNA sequence identity and > 80% amino acid sequence identity with the eaeA genes of C . freundii biotype 4280, EHEC O157:H7, and EPEC O127:116, respectively . The carboxy-terminal 280 amino acid residues of intimin has 80%, 56%, and 54% identity with C . freundii, EHEC O157:H7, and EPEC O127:H6 intimins, respectively . The predicted protein encoded by the upstream ORF (156 amino acids) shares 95%, 97%, and 99% amino acid identity with predicted proteins from C . freundii . EHEC O157:H7, and EPEC O127:H6, respectively . The high degree of sequence homology of the ORF and the eueA gene of RDEC-1 with those of other AE bacteria suggests an evolutionary relationship of LEE and supports and facilitates the use of the RDEC-1 model for studying the role of LEE in pathogenesis.

Jpn J Antibiot, 1996 Oct, 49(10), 947 - 65
{Antimicrobial activity of cefodizime against clinical isolates}; Suzuki Y et al.; In order to evaluate antimicrobial activity of cefodizime (CDZM), minimum inhibitory concentrations (MICs) of CDZM and control drugs were determined against clinical isolates collected from nation-wide medical institutions and in our laboratory from September to December of 1992 and from September to December of 1995 . The results are summarized as follows: 1 . Bacterial species with no or few strains resistant to CDZM included Streptococcus pyogenes, Haemophilus influenzae, Citrobacter koseri, Proteus mirabilis and Neisseria gonorrhoeae . The range of MIC values of CDZM against Klebsiella pneumoniae was spread . Other strains, Streptococcus pneumoniae, Moraxella subgenus Branhamella catarrhalis, Escherichia coli, Citrobacter freundii, Enterobacter spp., Serratia marcescens, Proteus vulgaris, Morganella morganii, Providencia spp., Peptostreptococcus spp . and Bacteroides fragilis group were resistant to cephems including CDZM . 2 . The MIC90's of CDZM were 0.05 approximately 3.13 micrograms/ml against Streptococcus spp., H . influenzae, M . (B.) catarrhalis, E . coli, Klebsiella spp., P . mirabilis, N . gonorrhoeae and Peptostreptococcus spp . obtained in 1995 that were frequently found in daily treatment of infections . It appears that the effectiveness of CDZM was still relatively high against community-acquired infections . 3 . Among H . influenzae isolates included imipenem (IPM)-resistant and norfloxacin (NFLX)-resistant strains . The MIC-range of CDZM against strains collected in 1995 including IPM-resistant and NFLX-resistant strains was < or = 0.025 approximately 0.1 microgram/ml, and MIC90 against these strains was 0.05 microgram/ml . CDZM showed strong antimicrobial activities against H . influenzae strains resistant to carbapenems and new-quinolones.

Diagn Microbiol Infect Dis, 1996 Oct, 26(2), 63 - 7
Transmission of Citrobacter koseri from mother to infant documented by ribotyping and pulsed-field gel electrophoresis; Papasian CJ et al.; We describe a case in which Citrobacter koseri (formerly C . diversus) was transmitted from a pregnant mother with chorioamnionitis and bacteremia to her infant who was bacteremic at birth and in apparent septic shock . Two highly discriminating molecular methods, ribotyping and pulsed field gel electrophoresis, were used to examine restriction fragment length polymorphisms within the genomic DNA of maternal and infant isolates . Both techniques identified the maternal and infant isolates as the same strain, distinct from epidemiologically unrelated controls, thus confirming their common origin.

J Bacteriol, 1996 Oct, 178(19), 5793 - 6
Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii; Seyfried M et al.; The genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli . The B12-free enzyme was purified to homogeneity . It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively . The enzyme complex has the composition alpha2beta2gamma2 . Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T . Tobimatsu, T . Hara, M . Sakaguchi, Y . Kishimoto, Y . Wada, M . Isoda, T . Sakai, and T . Toraya, J . Biol . Chem . 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.

J Bacteriol, 1996 Oct, 178(19), 5592 - 601
Characterization and expression of the plasmid-borne bedD gene from Pseudomonas putida ML2, which codes for a NAD+-dependent cis-benzene dihydrodiol dehydrogenase; Fong KP et al.; The catabolic plasmid pHMT112 in Pseudomonas putida ML2 contains the bed gene cluster encoding benzene dioxygenase (bedC1C2BA) and a NAD+-dependent dehydrogenase (bedD) required to convert benzene into catechol . Analysis of the nucleotide sequence upstream of the benzene dioxygenase gene cluster (bedC1C2BA) revealed a 1,098-bp open reading frame (bedD) flanked by two 42-bp direct repeats, each containing a 14-bp sequence identical to the inverted repeat of IS26 . In vitro translation analysis showed bedD to code for a polypeptide of ca . 39 kDa . Both the nucleotide and the deduced amino acid sequences show significant identity to sequences of glycerol dehydrogenases from Escherichia coli, Citrobacter freundii, and Bacillus stearothermophilus . A bedD mutant of P . putida ML2 in which the gene was disrupted by a kanamycin resistance cassette was unable to utilize benzene for growth . The bedD gene product was found to complement the todD mutation in P . putida 39/D, the latter defective in the analogous cis-toluene dihydrodiol dehydrogenase . The dehydrogenase encoded by bedD) was overexpressed in Escherichia coli and purified . It was found to utilize NAD+ as an electron acceptor and exhibited higher substrate specificity for cis-benzene dihydrodiol and 1,2-propanediol compared with glycerol . Such a medium-chain dehydrogenase is the first to be reported for a Pseudomonas species, and its association with an aromatic ring-hydroxylating dioxygenase is unique among bacterial species capable of metabolizing aromatic hydrocarbons.

J Med Chem, 1996 Sep 13, 39(19), 3712 - 22
Design, synthesis, and evaluation of 2 beta-alkenyl penam sulfone acids as inhibitors of beta-lactamases; Richter HG et al.; A general method for synthesis of 2 beta-alkenyl penam sulfones has been developed . The new compounds inhibited most of the common types of beta-lactamase . The level of activity depended very strongly on the nature of the substituent in the 2 beta-alkenyl group . The inhibited species formed with the beta-lactamase from Citrobacter freundii 1205 was sufficiently stable for X-ray crystallographic studies . These, together with UV absorption spectroscopy and studies of chemical degradation, suggested a novel reaction mechanism for the new inhibitors that might account for their broad spectrum of action . The (Z)-2 beta-acrylonitrile penam sulfone Ro 48-1220 was the most active inhibitor from this class of compound . The inhibitor enhanced the action of, for example, ceftriaxone against a broad selection of organisms producing beta-lactamases . The organisms included strains of Enterobacteriaceae that produce cephalosporinases, which is an exceptional activity for penam sulfones.

Biochem Biophys Res Commun, 1996 Sep 13, 226(2), 481 - 7
Mutational analysis of the CitA citrate transporter from Salmonella typhimurium: altered substrate specificity; Shimamoto T et al.; The CitA citrate transporter in Salmonella typhimurium is encoded by the citA gene and consists of 434 amino acid residues that probably include 12 membrane-spanning segments {Shimamoto . T., et al . (1991) J . Biochem . 110, 22-28} . CitA mutants with altered substrate specificities were isolated by in vitro mutagenesis using nitrous acid . The mutants could grow on isocitrate as a sole carbon source which normally cannot be transported well by the CitA transporter of S . typhimurium . The mutation sites in the citA gene of the nine mutants were determined to involve single residues at seven sites (one mutation per mutant) . The original amino acid residues at these sites (Arg-19, Ala-38, Glu-51, Gly-132, Ala-169, Pro-262 and Leu-271) were identified to be responsible for the altered substrate specificity . All these amino acid residues were conserved in four other homologous citrate transporters from Escherichia coli, Citrobacter amalonaticus and Klebsiella pneumoniae and are suggested to be involved in substrate recognition by the CitA transporter.

Vet Med (Praha), 1996 Sep, 41(9), 283 - 8
{Use of semi-solid Diasalm medium for isolation of Salmonella enteritidis}; Ruzickova V et al.; The Diagnostic Semi-solid Salmonella Agar (DIASALM) was compared with two commercial semi-solid Rappaport-Vasiliadis media (MSRV by Oxoid and SRVA, basis by HiMedia) using 52 strains of Salmonella and 10 strains of interfering Gram-negative bacteria . The diagnostic potency for salmonellae was higher in DIASALM than in MSRV or SRVA . Unlike the Rappaport-Vasiliadis media, DIASALM contains a diagnostic system consisting of lactose, saccharose and bromocresol purple, allowing the differentiation of salmonellae from non-pathogenic Gram-negative sugar-fermenting bacteria (Tab . I) . The semi-solid media were supplemented with 0.0015% nitrofurantoine to recover specifically Salmonella enteritidis . Eighty percent of strains of the latter serovar were resistant to this chemotherapeutic agent, while all the other serovars were sensitive to it . The use of discs soaked with the monovalent Salmonella antiserum H:g,m increased the recovery rate in 95 percent of S . enteritidis strains . Compared with the cultures from peptone water, the diameters of the migration zones formed by the positive cultures grown in M-broth were larger by 3 to 5 mm . Pure cultures of salmonellae were isolated in 98% of cases from the borders of the migration zones when mixed cultures of salmonellae (a S . enteritidis isolate from a patient, density 10(3) to 1 or 2 cells per drop) and Citrobacter koseri or Edwardsiella tarda, or Proteus mirabilis, or Psedomonas aeruginosa (densities > or = 10(3) cells per drop) were inoculated onto DIASALM (Tab III).

Rinsho Byori, 1996 Sep, 44(9), 895 - 8
{Evaluation of a new disk containing indoxyl-beta-D-glucuronide for rapid identification of Escherichia coli}; Fujita S et al.; To establish a simple identification procedure for Escherichia coli, we developed a disk containing indoxyl-beta-D-glucuronide, the chromogenic substrate of beta-D-glucuronidase . Of 188 isolates of Enterobacteriaceae, 101 (97%) of 104 strains of E . coli and two (67%) of three strains of Shigella species were positive for beta-glucuronidase . We also tested 495 strains (466 strains of E . coli and 29 of Citrobacter freundii) that might be considered lactose-fermenting E . coli under routine conditions, and 458 strains of E . coli showed beta-glucuronidase activity: the sensitivity of the disk method was 92%, specificity was 100%, and the negative predictive value was 44% . Our results suggest that for routine identification of E . coli on primary isolation plates, the disk method is sufficiently rapid (within 3h), simpler, and far less costly than identification methods using commercially available kits.

J Hosp Infect, 1996 Sep, 34(1), 11 - 22
Risk factors for nosocomial infections caused by gram-negative bacilli . The Hellenic Antibiotic Resistance Study Group; Vatopoulos AC et al.; Two hundred and ninety-nine Gram-negative hospital-acquired infections from 257 patients, consecutively identified during one month (November 1992) in five hospitals in the greater Athens area, were divided into four groups on the basis of the bacterium isolated: Group 1 (Escherichia coli group) included infections owing to E . coli, Group 2 (Proteus group) consisted of infections owing to Proteus spp . and Providencia spp., Group 3 (Kiebsiella/Enterobacter group) involved infections owing to Kiebsiella spp., Enterobacter spp., Citrobacter spp . and Serratia spp . Infections owing to Pseudomonas spp . and other non-fermenters were allocated into Group 4 (non-fermenters group) . The four groups were studied in relation to risk factors including the duration of hospitalization, type of ward, underlying disease, history of operation, medical procedures/devices and antimicrobial therapy . A stepwise multiple logistic regression technique (SPSS Inc) was used to analyse the data, and the three groups (the Proteus group, the Klebsiella/Enterobacter group and the non-fermenters group) were analysed separately against the E . coli group . Infections with the Kiebsiella/Enterobacter group were associated with: (a) length of hospital stay before the infection, (b) treatment with newer antibiotics, and (c) hospitalization in an intensivecare unit (ICU) . Infections with non-fermenters were associated with: (a) length of hospital stay before infection, (b) a urinary catheter, (c) type of disease (chronic infection being negatively associated), (d) treatment with newer antibiotics and (e) hospitalization in an ICU . Proteus group infections were associated with (a) length of hospital stay before infection, (b) treatment with newer antibiotics and (c) operation during present hospitalization (negative association) . Interestingly, no specific hospitals were identified as risk factors . Identification of patients at risk for acquiring an infection owing to a nosocomial pathogen is vital in the development of a preventive strategy for hospital-acquired infections.

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