Clostridia

J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 307 - 13
Orf5/SolR: a transcriptional repressor of the sol operon of Clostridium acetobutylicum?
Thormann K, Durre P.
The gene of Orf5 (SolR) of Clostridium acetobutylicum DSM 792 was subcloned and overexpressed in Escherichia coli . The protein was purified with Ni-NTA agarose and used for DNA binding assays . No DNA binding of Orf5 to regions upstream of the sol operon from C . acetobutylicum was observed . Overexpression of Orf5 in C . acetobutylicum led to a change in the organism's pattern of glycosylated exoproteins . The Orf5 protein was localized in the cell membrane fraction and to a small extent in the supernatant medium . Based on these results Orf5 (SolR) appears not to act as a transcriptional repressor in C . acetobutylicum, but instead may be an enzyme involved in glycosylation or deglycosylation.

J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 298 - 306
Characterization of a maltose transport system in Clostridium acetobutylicum ATCC 824; Tangney M et al.; The utilization of maltose by Clostridium acetobutylicum ATCC 824 was investigated . Glucose was used preferentially to maltose, when both substrates were present in the medium . Maltose phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity was detected in extracts prepared from cultures grown on maltose, but not glucose or sucrose, as the sole carbon source . Extract fractionation and PTS reconstitution experiments revealed that the specificity for maltose is contained entirely within the membrane in this organism . A putative gene system for the maltose PTS was identified (from the C . acetobutylicum ATCC 824 genome sequence), encoding an enzyme II(Mal) and a maltose 6-phosphate hydrolase.

J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 292 - 7
ABE production from corn: a recent economic evaluation; Qureshi N et al.; This article details an economic assessment of butanol production from corn using the newly developed hyper-butanol-producing strain of Clostridium beijerinckii BA101 . Butanol is produced in batch reactors and recovered by distillation . For a plant with 153,000 metric tons of acetone, butanol, and ethanol (ABE) production capacity, the production equipment cost and total working capital cost is US$33.47x10(6) and US$110.46x10(6), respectively . Based on a corn price (C(p)) of US$79.23 x ton(-1) (US$2.01 x bushel(-1)), an ABE yield of 0.42 (g ABE/g glucose) butanol price is projected to be US$0.34 x kg(-1) . An improved yield of 0.50 will reduce this price to US$0.29 x kg(-1) . Assumptions, such as by-product credit for gases and complete conversion of corn steep liquor (CSL) to fermentation by-products, have been taken into consideration . An increased price of corn to US$197.10 x ton(-1) would result in a butanol price of US$0.47 x kg(-1) . A grass-rooted plant would result in a butanol price of US$0.73 x kg(-1) (C(p) US$79.23 x ton(-1)) . In a worst case scenario, the price of butanol would increase to US$1.07 x kg(-1) (C(p) 197.10 x ton(-1) for a grass-rooted plant and assuming no credit for gases) . This is based on the assumption that corn price would not increase to more than US$197.10 x ton(-1).

J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 287 - 91
Recent advances in ABE fermentation: hyper-butanol producing Clostridium beijerinckii BA101; Qureshi N et al.; This is an overview of the mutant strain Clostridium beijerinckii BA101 which produces solvents (acetone-butanol-ethanol, ABE) at elevated levels . This organism expresses high levels of amylases when grown on starch . C . beijerinckii BA101 hydrolyzes starch effectively and produces solvent in the concentration range of 27-29 g x l(-1) . C . beijerinckii BA101 has been characterized for both substrate and butanol inhibition . Supplementing the fermentation medium (MP2) with sodium acetate enhances solvent production to 33 g x l(-1) . The results of studies utilizing commercial fermentation medium and pilot plant-scale reactors are consistent with the results using small-scale reactors . Pervaporation, a technique to recover solvents, has been applied to fed-batch reactors containing C . beijerinckii BA101, and solvent production as high as 165 g x l(-1) has been achieved . Immobilization of C . beijerinckii BA101 by adsorption and use in a continuous reactor resulted in reactor productivity of 15.8 g x l(-1) x h(-1) . Recent economic studies employing C . beijerinckii BA101 suggested that butanol can be produced at US$0.20-0.25 x lb(-1) by employing batch fermentation and distillative recovery . Application of new technologies such as pervaporation, fed-batch culture, and immobilized cell reactors is expected to further reduce these prices.

J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 281 - 6
Nitrogen-fixation genes and nitrogenase activity in Clostridium acetobutylicum and Clostridium beijerinckii; Chen JS et al.; Several solvent-producing clostridia, including Clostridium acetobutylicum and C . beijerinckii, were previously shown to be nitrogen-fixing organisms based on the incorporation of 15N2 into cellular material . The key nitrogen-fixation (nif) genes, including nifH, nifD, and nifK for nitrogenase component proteins as well as nifE, nifN, nifB and nifV for synthesis of the iron-molybdenum cofactor (FeMoco) of nitrogenase, have now been identified in C . acetobutylicum or C . beijerinckii or both . The organization of these genes is similar to the distinctive pattern that was first observed in Clostridium pasteurianum, with the nifN and nifB genes fused into the nifN-B gene and with the nifV gene split into the nifVomega and nifValpha genes . The corresponding nif genes of these three clostridial species are highly related to each other . However, in the two solvent-producing clostridia, the nifH and nifD genes are interspersed by two glnB-like genes, which are absent in the corresponding region in C . pasteurianum . However, the nifN-B and nifVomega genes of C . pasteurianum are interspersed by the putative modA and modB genes (for molybdate transport), which are absent in the corresponding region in C . acetobutylicum . C . acetobutylicum and C . beijerinckii grew well under nitrogen-fixing conditions, and the acetylene-reducing activity of nitrogenase was measured in the two species . Acetone, butanol, and isopropanol production occurred in nitrogen-fixing cultures, but the peak of nitrogen-fixing activity preceded the active solventogenic phase.

J Ind Microbiol Biotechnol, 2001 Nov, 27(5), 271 - 4
Electrotransformation studies in Clostridium cellulolyticum; Tardif C et al.; Electropermeabilization of Clostridium cellulolyticum was optimized using ATP leakage assays . Electrotransformation was then performed under optimized conditions (6 to 7.5 kV x cm(-1) field strength applied during 5 ms to a mixture containing methylated plasmids and late exponential phase cell suspensions (10 molecules:1 cell) in a sucrose-containing buffer) . Transformants were only obtained when 7 or 7.5 kV x cm(-1) pulses were applied . Transformation efficiencies evaluated from the growth curves of transformed cells were between 10(5) and 10(7) transformants per microgram of plasmid DNA for five different replicon-based plasmids . Restriction nuclease digestion patterns of pJIR418 purified from transformed Clostridia and Escherichia coli were indistinguishable, indicating that heterologous DNA was structurally stable in the Clostridium strain . Copy numbers of 130, 70 and 10 were estimated from purification yield for pCTC1, pKNT19 and pJIR418, respectively.

Appl Microbiol Biotechnol, 2001 Dec, 57(5-6), 660 - 6
Sequence of celQ and properties of celQ, a component of the Clostridium thermocellum cellulosome; Arai T et al.; The nucleotide sequence of the Clostridium thermocellum F1 celQ gene, which codes for the endoglucanase CelQ, consists of 2,130 bp encoding 710 amino acids . The precursor form of CelQ has a molecular weight of 79,809 and is composed of a signal peptide, a family 9 cellulase domain, a family IIIc carbohydrate-binding module (CBM), and a dockerin domain . Truncated derivatives of CelQ were constructed: CelQdeltadoc consisted of the catalytic domain and the CBM; CelQcat consisted of the catalytic domain only . CelQdeltadoc showed strong activity toward carboxymethylcellulose (CMC) and barley beta-glucan and low activity toward Avicel, acid-swollen cellulose, lichenan, and xylan . The Vmax and Km values were 235 micromol/min/mg and 3.3 mg/ml, respectively, for CMC . By contrast, CelQcat, which was devoid of the CBM, showed negligible activity toward CMC, i.e., about 1/1,000 of the activity of CelQdeltadoc, supporting the previously proposed idea that family IIIc CBMs participate in the catalytic function of the enzyme . Immunological analysis using an antiserum raised against CelQdeltadoc confirmed that CelQ is a component of the C . thermocellum cellulosome.

Presse Med, 2001 Dec 8, 30(37), 1825 - 6
{Clostridium difficile bacteremia}; Duthilly A et al.; BACKGROUND: Extra-digestive manifestations of Clostridium difficile infection are very uncommon . Exceptional cases of C . difficile bacteremia or severe sepsis have been described in intensive care patients, demonstrating the capacity of this agent to generate generalized infection . CASE REPORT: C . difficile bacteremia occurred in a 66-year-old immunodepressed patient treated for acute myeloblastic leukemia . Bacteremia was associated with a abscess of the anal margin . Outcome was favorable after treatment with metronidosole . DISCUSSION: Clostridium difficile is generally selected by prior antibiotic treatment . It is the principal agent of nosocomial diarrhea . In immunodepressed patients, systemic dissemination is a rare but possible development.

Chin Med J (Engl), 2000 Sep, 113(9), 858 - 61
Anaerobic bacteria and intrahepatic stones: detections of Clostridium sp . and Bacteroides fragilis; Liu Y et al.; OBJECTIVE: To detect anaerobic bacteria Clostridium sp . and Bacteroides fragilis in intrahepatic stones by molecular genetic method . METHODS: DNA was extracted from 59 stone samples and subjected to polymerase chain reaction (PCR) amplification targeting the 16S rRNA gene of Clostridium sp . and the glutamine synthetase gene of Bacteroides fragilis . Single-strand conformational polymorphism (SSCP) analysis was performed to identify the Clostridium sp . RESULTS: 16S rRNA gene sequences for Clostridium sp . were identified in 49 stones (83%, 49/59) . The two most common groups were detected in 19 (41%) and 17 (37%) of the 46 samples using SSPC analysis, and 25/59 (42%) stones were tested positive for Bacteroides fragilis . CONCLUSIONS: Anaerobes such as Clostridium sp . and Bacteroides fragilis present in intrahepatic stones and may play a role in stone formation . PCR is a useful technique to detect fastidious pathogens, which are difficult to culture . SSCP of PCR products is a rapid method in differentiating bacterial species.

Clin Infect Dis, 2002 Feb 1, 34(3), 346 - 53 Epub 2001 Dec 17.
Health care costs and mortality associated with nosocomial diarrhea due to Clostridium difficile; Kyne L et al.; A total of 271 patients were prospectively followed up to determine whether patients whose hospital stay is complicated by diarrhea due to Clostridium difficile experience differences in cost and length of stay and survival rates when compared with patients whose stay is not complicated by C . difficile-associated diarrhea . Forty patients (15%) developed nosocomial C . difficile-associated diarrhea . These patients incurred adjusted hospital costs of $3669--that is, 54% (95% confidence interval {CI}, 17%-103%)--higher than patients whose course was not complicated by C . difficile-associated diarrhea . The extra length of stay attributable to C . difficile-associated diarrhea was 3.6 days (95% CI, 1.5-6.2) . C . difficile-associated diarrhea was not associated with excess 3-month or 1-year mortality after adjustment for age, comorbidity, and disease severity . On the basis of the findings of this study, a conservative estimate of the cost of this disease in the United States exceeds $1.1 billion per year.

Obstet Gynecol Surv, 2002 Jan, 57(1), 53 - 7
Diagnosis and management of clostridium perfringens sepsis and uterine gas gangrene; Halpin TF et al.; The progression of Clostridium perfringens endomyometritis to gas gangrene is a rare, but greatly feared complication in the obstetrical patient . While endometritis following cesarean delivery is a common complication, recognition of C . perfringens as the pathogen as well as its progression to gas formation in the myometrium is essential to the survival of the patient . We present a patient that we recently cared for, and review the bacteriology, clinical diagnosis, and management.

J Clin Microbiol, 2002 Jan, 40(1), 101 - 4
Molecular fingerprinting of Clostridium difficile isolates: pulsed-field gel electrophoresis versus amplified fragment length polymorphism; Klaassen CH et al.; Two molecular fingerprinting techniques, pulsed-field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP), were used to investigate the epidemiological relatedness among Clostridium difficile isolates from suspected outbreaks in three general hospitals . Analysis by PFGE yielded inconclusive data as a result of extensive DNA degradation . Although this degradation could be prevented to a certain extent by the inclusion of thiourea in the electrophoresis buffer, the weak DNA banding patterns obtained in this way were still far from optimal . AFLP data were obtained by using fluorescently labeled PCR primers and analysis on an ABI PRISM automated DNA analysis platform . AFLP analysis yielded high resolution and highly reproducible DNA fingerprinting patterns from which the epidemiological relatedness among the isolates could easily be determined . AFLP results could be readily obtained within 24 h, whereas 3 to 4 days were routinely required to complete the lengthy PFGE protocol . AFLP clearly proved to be a much more fail-safe fingerprinting method for C . difficile isolates, especially for those isolates for which a standard PFGE procedure yielded inconclusive results due to DNA degradation.

Appl Environ Microbiol, 2002 Jan, 68(1), 53 - 8
Improvement of cellulolytic properties of Clostridium cellulolyticum by metabolic engineering; Guedon E et al.; Cellulolytic clostridia have evolved to catabolize lignocellulosic materials at a seasonal biorhythm, so their biotechnological exploitation requires genetic improvements . As high carbon flux leads to pyruvate accumulation, which is responsible for the cessation of growth of Clostridium cellulolyticum, this accumulation is decreased by heterologous expression of pyruvate decarboxylase and alcohol dehydrogenase from Zymomonas mobilis . In comparison with that of the wild strain, growth of the recombinant strain at the same specific rate but for 145 h instead of 80 h led to a 150% increase in cellulose consumption and a 180% increase in cell dry weight . The fermentation pattern was shifted significantly: lactate production decreased by 48%, whereas the concentrations of acetate and ethanol increased by 93 and 53%, respectively . This study demonstrates that the fermentation of cellulose, the most abundant and renewable polymer on earth, can be greatly improved by using genetically engineered C . cellulolyticum.

J Endod, 2001 Dec, 27(12), 730 - 3
Antibacterial activity of fifth-generation dentin bonding systems; Atac AS et al.; Past concepts that the pulp does not become infected until an actual carious exposure takes place have been challenged . The antibacterial effects of the dentin bonding systems Single Bond, Prime&Bond NT, and Excite were evaluated using the bacteria Streptococcus mutans ATCC 25175, Streptococcus intermedius, Lactobacillus acidophilus, Prevotella oris, Prevotella bivia, Prevotella denticola, Porphyromonas gingivalis, Porphyromonas endodontalis, and Clostridium ramosum with a disk diffusion method . Chlorhexidine (0.2%) was used as a positive control . After incubation zones of inhibited bacterial growth were measured . Prime&Bond NT showed growth inhibition for all bacterial strains . Lactobacillus acidophilus and Streptococcus mutans were remarkably resistant to Single Bond, whereas EX produced no inhibitory effect on Porphyromonas endodontalis, although the adhesive produced the maximum halo inhibition to Streptococcus mutans (15+/-1 mm), showing an antibacterial effect closest to chlorhexidine . The variety of results obtained in this study suggest that antibacterial properties of current dentin adhesives may depend on components that are originally incorporated to promote adhesion.

J Food Prot, 2001 Dec, 64(12), 1956 - 60
Occurrence of Clostridium perfringens in the broiler chicken processing plant as determined by recovery in iron milk medium; Craven SE; Over 30 years ago, Clostridium perfringens was reported as a contaminant of the processing plant and processed carcasses of broiler chickens . Poultry processing procedures and methods for detecting C . perfringens have changed since that time . Therefore, a study was conducted to determine the incidence and numbers of C . perfringens in the water of the scald tank, the water of the chill tank, and the rinse water of the processed carcasses from modern broiler chicken processing plants . In trial 1, collected samples were inoculated into iron milk medium (IMM) and incubated at 46 degrees C for 18 h (the traditional method) or at 37 degrees C for 3 h followed by incubation at 46 degrees C for 15 h (an injury recovery method) . Each of three preselected broiler chicken flocks from two integrators were the first processed for that processing shift . The overall incidence of confirmed C . perfringens in samples associated with the three flocks was 40% of postprocessing scald water samples, 13% of preprocessing chill water samples, 13% of postprocessing chill water samples, and 19% of carcass rinses . The incidence of C . perfringens in samples incubated in IMM using the injury recovery procedure was significantly higher than in samples incubated in IMM by the traditional method, but only when all samples associated with the three flocks were pooled . In trial 2, water samples from each tank of a three-tank counterflow scalder, water samples from the prechill and chill tank, and samples of carcass rinses were collected in the middle of a processing shift during multiple visits to a processing plant . Samples were inoculated into IMM with neomycin and polymyxin B sulfate (IMMA) and incubated using the traditional and injury recovery procedures . The incidence of C . perfringens in water samples was 100% from scald tank 1, 100% from scald tank 2, 100% from scald tank 3, 88% from the prechill tank, and 63% from the chill tank . The incidence in carcass rinse samples was 67% . The mean most probably number (MPN) of C . perfringens for contaminated samples decreased from log10 5.07/100 ml of water in scald tank 1 to log10 1.26/100 ml of water in the chill tank . The mean MPN in carcass rinse samples was log10 1.20 C . perfringens per 100 ml . The incidence and mean MPN of C . perfringens in these samples after heat shock at 75 degrees C for 20 min was somewhat less, but high enough to indicate that much of the contamination arises from heat-resistant spores of this organism . In trial 2, there were no differences in incidence and MPN of C . perfringens in samples incubated in IMMA with the traditional method or the injury recovery method.

DNA Seq, 2001, 12(2), 115 - 20
ADP-ribosylating binary toxin genes of Clostridium difficile strain CCUG 20309; Chang SY et al.; The cdt genes that encode a binary ADP-ribosylating toxin in Clostridium difficile were first characterized from a toxigenic C . difficile strain CD196 in 1997 . We report here C . difficile strain CCUG 20309 (ATCC 8864), a strain that produces toxin B but not toxin A, also carry a complete set of cdtA and cdtB genes . These genes were sequenced by cycle sequencing method . The 2 ORFs and the intergenic sequences of these 2 strains have a homology of 99.6% . Interestingly, 9 extra bases were found within the cdtA gene of strain CCUG 20309 which do not affect the downstream region of the ORF . Using the same homologous primers, the highly toxigenic reference strain VPI 10463 was found to carry only parts of the 2 ORFs while a nontoxigenic strain ATCC 8884 does not carry any of the cdt genes . Though it remains to be determined whether these genes are expressed, it is significant that strain CCUG 20309 contains the complete set of cdt genes . We speculate that the putative expressed proteins may contribute to pathogenesis, for example, enterotoxicity, of this unique strain of bacteria.

Vet Rec, 2001 Nov 17, 149(20), 618 - 20
Clostridium perfringens beta2-toxin in an African elephant (Loxodonta africana) with ulcerative enteritis; Bacciarini LN et al.; A 22-year-old female African elephant (Loxodonta africana) developed diarrhoea of unknown cause which lasted for two days . The animal was euthanased after it remained recumbent and refused to get up . Gross pathological changes were present mainly in the gastrointestinal tract . The intestinal contents were watery and dark brown . Several areas of the mucosa of the small intestine were covered minimally to moderately with fibrin and had a few 0.1 x 10 to 15 cm linear ulcerations . Microscopical lesions consisted of discrete areas of necrosis of the surface and crypt epithelium without overt inflammatory infiltrates . Culture of the small intestinal contents resulted in a moderate growth of Clostridium perfringens . No salmonella were found in the small or large intestine . PCR of the isolate of C . perfringens revealed the presence of the beta2-toxin gene cpb2 and the alpha-toxin gene cpa but no other known toxin genes . The expression of the beta2-toxin gene in vivo was demonstrated by the immunohistochemical localisation of the beta2-toxin to the microscopical lesions in the small intestine.

J Med Microbiol, 2001 Dec, 50(12), 1082 - 6
PCR ribotyping of clinically important Clostridium difficile strains from Hungary; Urban E et al.; Isolates of Clostridium difficile from different hospital wards at the University Hospital of Szeged in Hungary were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) . A total of 15 different ribotypes was detected among the 65 isolates tested . The predominant type, PCR ribotype 087, accounted for 39% of all isolates, in contrast with an international typing study where ribotype 001 was the most common . Two non-toxigenic C . difficile strains were found to exhibit the same pattern, which was distinct from those of all the ribotypes described previously, suggesting that this is a new type.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2105 - 11
Metabolism of cinnamic acids by some Clostridiales and emendation of the descriptions of Clostridium aerotolerans, Clostridium celerecrescens and Clostridium xylanolyticum; Chamkha M et al.; The ability of Clostridium aerotolerans DSM 5434T, Clostridium celerecrescens DSM 5628T, Clostridium methoxybenzovorans DSM 12182T, Clostridium stercorarium ATCC 35414T, Clostridium subterminale DSM 2636, Clostridium termitidis DSM 5398T, Clostridium thermolacticum DSM 2910T, Clostridium thermopalmarium DSM 5974T and Clostridium xylanolyticum DSM 6555T to metabolize cinnamic acid and various derivatives, with or without glucose supplementation, was examined . Only C aerotolerans DSM 5434T and C . xylanolyticum DSM 6555T, closely related species, transformed cinnamic acid to 3-phenylpropionic acid . Both species also reduced a wide range of cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic, isoferulic and 3,4,5-trimethoxycinnamic acids to their corresponding 3-phenylpropionic acid derivatives . C . aerotolerans DSM 5434T, however, also decarboxylated p-coumaric acid into 4-vinylphenol, which was then reduced to 4-ethylphenol . C . celerecrescens was grouped with C . aerotolerans and C . xylanolyticum in subcluster XIVa of the Clostridiales . C . celerecrescens DSM 5628T only metabolized m- and p-methoxycinnamic and p-methylcinnamic acids to their corresponding 3-phenylpropionic acid derivatives, reducing the double bond in the C3 aliphatic side chain . Addition of glucose markedly increased the yield of the biotransformations by these three species . An emendation of the descriptions of C . aerotolerans, C . celerecrescens and C . xylanolyticum is proposed, based on these observations.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2095 - 103
Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp . nov . and Clostridium saccharobutylicum sp . nov; Keis S et al.; On the basis of 16S rRNA gene sequencing and DNA-DNA reassociation, industrial solvent-producing clostridia have been assigned to four species . In this study, the phenotypic characteristics of Clostridium acetobutylicum, Clostridium beijerinckii, 'Clostridium saccharoperbutylacetonicum', and an unnamed Clostridium sp . represented by the strains NCP 262T and NRRL B643 are compared . In addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, DNA fingerprinting and 16S rRNA gene sequencing . These included 14 C . beijerinckii strains, two strains currently designated as 'Clostridium kaneboi' and 'Clostridium butanologenum', and 24 production strains used in the commercial acetone-butanol fermentation . All of the C . beijerinckii strains were confirmed to have been classified correctly . The 'C . kaneboi' and 'C . butanologenum' strains require reclassification as C . acetobutylicum and C . beijerinckii, respectively . The commercial production strains were found to belong either to C . beijerinckii or to the unnamed Clostridium sp . For the comparative phenotypic studies of the four species, representative strains were selected from each of the DNA-fingerprint subgroups within each species . These strains were analysed for their ability to utilize different carbohydrates, hydrolyse gelatin or aesculin, and produce indole, and were tested for the presence of catalase and urease . On the basis of these results, several phenotypic traits were found to be useful for differentiating between the four species . The descriptions of C . acetobutylicum and C . beijerinckii have been emended . The names Clostridium saccharoperbutylacetonicum sp . nov . {type strain = N1-4 (HMT) = ATCC 27021T} and Clostridium saccharobutylicum sp . nov . (type strain = DSM 13864T = ATCC BAA-117T) are proposed for the two new species.

Int J Syst Evol Microbiol, 2001 Nov, 51(Pt 6), 2049 - 54
Isolation of a cinnamic acid-metabolizing Clostridium glycolicum strain from oil mill wastewaters and emendation of the species description; Chamkha M et al.; A strictly anaerobic, gram-positive, motile, sporulated bacterium, designated strain CIN5, was isolated from olive mill wastewaters after enrichment on cinnamic acid . The rod-shaped cells were slightly curved (0.4-1.1 x 2.0-15 microm) and occurred singly or in pairs . Strain CIN5 utilized a limited number of carbohydrates (glucose, fructose, maltose, sorbitol), grew optimally at 37 degrees C and at pH 7.3-7.5 and had a DNA G+C content of 29.1+/-0.3 mol% . Strain CIN5 was very closely related to Clostridium glycolicum DSM 1288T . Both strain CIN5 and the type strain of C . glycolicum transformed cinnamic acid to hydrocinnamic acid and a wide range of other cinnamic acid derivatives, including o-, m- and p-coumaric, o-, m- and p-methoxycinnamic, p-methylcinnamic, caffeic, ferulic and isoferulic acids, to their corresponding 3-phenylpropionic acids by reducing the double bond of the side chain . Glucose supplementation increased the rate of conversion markedly . The emendation of the description of C . glycolicum is proposed to include these new characteristics.

Pharmacoepidemiol Drug Saf, 2001 Jun-Jul, 10(4), 303 - 8
Risk factors for the development of Clostridium difficile toxin-associated diarrhoea: a pilot study; Aziz EE et al.; This study was a pilot investigation of risk factors for the development of Clostridium difficile toxin-associated diarrhoea and in particular the differential influence of antimicrobial agents . The study was a retrospective case-control design conducted at Freeman Hospital, Newcastle upon Tyne . Cases were inpatients with stool positive C . difficile toxin diarrhoea and two controls were drawn for each case matched for age (+/- 5 years) and type of admission (emergency or elective) . Using conditional logistic regression analysis, cephalosporins and erythromycin were found to be statistically significantly associated with Clostridium difficile toxin associated-diarrhoea . The results form the basis for designing a larger, prospective study.

Trends Microbiol, 2002 Jan, 10(1), 5 - 7
C3stau, a new member of the family of C3-like ADP-ribosyltransferases; Wilde C et al.; C3-like ADP-ribosyltransferases, which are produced by Clostridium botulinum, Clostridium limosum, Bacillus cereus and Staphylococcus aureus, are exoenzymes lacking a translocation unit . These enzymes specifically inactivate Rho GTPases in host target cells . Recently, a novel C3-like transferase from S . aureus with new properties was identified, raising questions regarding its function . As Rho GTPases are master regulators of several eukaryotic signal processes and S . aureus can invade eukaryotic cells, C3 might play a role as a virulence factor.

Nucleic Acids Res, 2002 Jan 1, 30(1), 179 - 82
The tmRNA Website: invasion by an intron; Williams KP; tmRNA (also known as 10Sa RNA or SsrA) plays a central role in an unusual mode of translation, whereby a stalled ribosome switches from a problematic mRNA to a short reading frame within tmRNA during translation of a single polypeptide chain . Research on the mechanism, structure and biology of tmRNA is served by the tmRNA Website, a collection of sequences for tmRNA and the encoded proteolysis-inducing peptide tags, alignments, careful documentation and other information; the URL is Four pseudoknots are usually present in each tmRNA, so the database is rich with information on pseudoknot variability . Since last year it has doubled (227 tmRNA sequences as of September 2001), a sequence alignment for the tmRNA cofactor SmpB has been included, and genomic data for Clostridium botulinum has revealed a group I (subgroup IA3) intron interrupting the tmRNA T-loop.

J Bacteriol, 2002 Jan, 184(2), 600 - 4
alpha-Galactosidase Aga27A, an enzymatic component of the Clostridium josui cellulosome; Jindou S et al.; The Clostridium josui aga27A gene encodes the cellulosomal alpha-galactosidase Aga27A, which comprises a catalytic domain of family 27 of glycoside hydrolases and a dockerin domain responsible for cellulosome assembly . The catalytic domain is highly homologous to those of various alpha-galactosidases of family 27 of glycoside hydrolases from eukaryotic organisms, especially plants . The recombinant Aga27A alpha-galactosidase devoid of the dockerin domain preferred highly polymeric galactomannan as a substrate to small saccharides such as melibiose and raffinose.

FEMS Microbiol Lett, 2001 Dec 18, 205(2), 305 - 14
Transcriptional regulation of pentose utilisation systems in the Bacillus/Clostridium group of bacteria; Rodionov DA et al.; In Bacillus subtilis, utilisation of xylose, arabinose and ribose is controlled by the transcriptional factors XylR, AraR and RbsR, respectively . Here we apply the comparative approach to the analysis of these regulons in the Bacillus/Clostridium group . Evolutionary variability of operon structures is demonstrated and operator sites for the main transcription factors are predicted . The consensus sequences for the XylR and RbsR binding sites vary in different subgroups of genomes . The functional coupling of gene clusters and the conservation of regulatory sites allow for detection of non-orthologous gene displacement of ribulose kinase in Enterococcus faecium and Clostridium acetobutylicum . Moreover, candidate catabolite responsive elements found upstream of most pentose-utilising genes suggest CcpA-mediated catabolite repression.

J Korean Med Sci, 2001 Dec, 16(6), 742 - 4
Pseudomonas aeruginosa as a potential cause of antibiotic-associated diarrhea; Kim SW et al.; Although Pseudomonas aeruginosa is not generally considered as a cause of antibiotic-associated diarrhea, several cases of diarrhea caused by P . aeruginosa have been reported . We experienced seven cases of nosocomial diarrhea presumably caused by P . aeruginosa, which was the predominant organism isolated from stool cultures . Clostridium difficile toxin was also positive in one patient . No other potential or recognized enteropathogens were identified from stools . All patients had underlying diseases and had been receiving antibiotics before the diarrheal onset . All of the seven P . aeruginosa isolates were resistant to previously given antibiotics . Diarrhea stopped three days after withdrawal of probable offending antibiotics without specific treatment in two patients . The other five patients having continuous diarrhea despite withdrawal of probable offending antibiotics, were successfully treated with antipseudomonal agents . The median duration of diarrhea after the initiation of treatment was 6.3 days . These data suggest that P . aeruginosa can be a potential cause of antibiotic-associated diarrhea . Further investigations are warranted to evaluate the possible etiologic role of P . aeruginosa in antibiotic-associated diarrhea.

J Biol Chem, 2002 Mar 8, 277(10), 8306 - 11 Epub 2001 Dec 17.
The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step; Asuncion M et al.; Methylaspartate ammonia-lyase (3-methylaspartase, MAL; EC ) catalyzes the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid . This reaction lies on the main catabolic pathway for glutamate in Clostridium tetanomorphum . MAL requires monovalent and divalent cation cofactors for full catalytic activity . The enzyme has attracted interest because of its potential use as a biocatalyst . The structure of C . tetanomorphum MAL has been solved to 1.9-A resolution by the single-wavelength anomalous diffraction method . A divalent metal ion complex of the protein has also been determined . MAL is a homodimer with each monomer consisting of two domains . One is an alpha/beta-barrel, and the other smaller domain is mainly beta-strands . The smaller domain partially occludes the C terminus of the barrel and forms a large cleft . The structure identifies MAL as belonging to the enolase superfamily of enzymes . The metal ion site is located in a large cleft between the domains . Potential active site residues have been identified based on a combination of their proximity to a metal ion site, molecular modeling, and sequence homology . In common with all members of the enolase superfamily, the carboxylic acid of the substrate is co-ordinated by the metal ions, and a proton adjacent to a carboxylic acid group of the substrate is abstracted by a base . In MAL, it appears that Lys(331) removes the alpha-proton of methylaspartic acid . This motif is the defining mechanistic characteristic of the enolase superfamily of which all have a common fold . The degree of structural conservation is remarkable given only four residues are absolutely conserved.

Cell Signal, 2002 Jan, 14(1), 75 - 81
Phospholipase D stimulation is required for sphingosine-1-phosphate activation of actin stress fibre assembly in human airway epithelial cells; Porcelli AM et al.; In human airway epithelial cells, sphingosine-1-phosphate (SPP) and lysophosphatidic acid (LPA) stimulated the production of phosphatidic acid (PA), which was inhibited by the primary alcohol butan-1-ol, but not by the inactive butan-2-ol, clearly indicating phospholipase D (PLD) involvement . Both SPP and LPA stimulated actin stress fibre formation, which was also butan-2-ol-insensitive and inhibited by butan-1-ol . SPP-induced PLD activation and cytoskeletal remodelling were insensitive to brefeldin A and toxin B from Clostridium difficile, which conversely blocked the effect of LPA, suggesting that the monomeric GTPases ADP ribosylation factor (ARF) and Rho are involved in LPA, but not in SPP responses . Pertussis toxin inhibited SPP- but not LPA-induced effects . PLD activation and stress fibre formation by both lysolipids were abolished by the tyrosine kinase inhibitor genistein . Addition of PA to cells caused a massive stress fibre assembly . In conclusion, PLD is one of the signalling components linking SPP-receptor activation to assembly of actin stress fibres.

Phytother Res, 2001 Nov, 15(7), 586 - 8
Antimicrobial activity of some Pacific Northwest woods against anaerobic bacteria and yeast; Johnston WH et al.; Extracts of woods commonly used for animal bedding were tested for antimicrobial activity . Essential oils from Alaska cedar (Chamaecyparis nootkatensis), western juniper (Juniperus occidentalis) and old growth Douglas fir (Pseudotsuga menziesii) as well as methanol extracts of wood from these trees plus western red cedar (Thuja plicata) and ponderosa pine (Pinus ponderosa) were tested for antimicrobial activity against anaerobic bacteria and yeast . The test microbes included Fusobacterium necrophorum, Clostridium perfringens, Actinomyces bovis and Candida albicans which are common to foot diseases and other infections in animals . The essential oils and methanol extracts were tested using a standardized broth assay . Only extracts of Alaska cedar and western juniper showed significant antimicrobial activity against each of the microbes tested . The essential oil of Douglas fir did show antimicrobial activity against A . bovis at the concentrations tested . The methanol extracts of the heartwood of Douglas fir and the sapwood of ponderosa pine showed no antimicrobial activity . The major chemical components of western juniper (cedrol and alpha- and beta-cedrene) and Alaska cedar (nootkatin) were also tested . In western juniper, alpha- and beta-cedrene were found to be active components . Nootkatin showed activity only against C . albicans . The inhibitory activity in Alaska cedar oil was high enough to justify further efforts to define the other chemical components responsible for the antimicrobial activity .

Int J Mol Med, 2002 Jan, 9(1), 53 - 7
Oral administration of a product derived from Clostridium butyricum in rats; Araki Y et al.; Recent studies have suggested that short chain fatty acids (SCFAs) exert a therapeutic effect on some human and experimental animal diseases . Clostridium butyricum produces high levels of SCFAs in the gut lumen . The aim of the present study was to analyze the product derived from Clostridium butyricum in a culture system, and to develop methods to eliminate the odor derived from SCFAs in the product . Clostridium butyricum was incubated in CS medium for 24 h and subsequently in CS broth for 24 h . The suspension of Clostridium butyricum in the broth was centrifugated and the supernatant was analyzed . The results showed this product contained high levels of SCFAs, especially acetic acid and n-butyric acid . Many food materials were tested in order to eliminate the odor derived from SCFAs in the product . Of the food materials tested, yogurt was shown to most effectively eliminate the odor . Using a yogurt base, we prepared a special food additive . Use of the additive completely eliminated the odor of the product derived from Clostridium butyricum . Finally, we administered the product with the additive to Sprague-Dawley rats for 14 days . The rats grew normally for the duration of the experimental period . It is possible that this novel product with the additive exerts therapeutic effects on some gastointestinal disorders.

Dis Colon Rectum, 2001 Dec, 44(12), 1871 - 2
Intracolonic use of vancomycin for treatment of clostridium difficile colitis in a patient with a diverted colon: report of a case; Nathanson DR et al.; Clostridium difficile-associated pseudomembranous colitis (PMC) is a common affliction of postoperative patients . Risk factors include antibiotic therapy, recent surgery, and hospitalization (1,2,3) . We present a case of PMC in a diverted colon and its treatment using vancomycin enemas.

J Biol Chem, 2002 Feb 22, 277(8), 6143 - 52 Epub 2001 Dec 10.
Interaction of Clostridium perfringens iota-toxin with lipid bilayer membranes . Demonstration of channel formation by the activated binding component Ib and channel block by the enzyme component Ia; Knapp O et al.; The interaction between model lipid membranes and the binding component (Ib) of the ADP-ribosylating iota-toxin of Clostridium perfringens was studied in detail . Ib had to be activated by trypsin to result in channel formation in artificial lipid bilayers . The channels formed readily by Ib had a small single-channel conductance of about 85 picosiemens in 1 m KCl . Channel function was blocked in single-channel and multichannel experiments by the enzymatic component Ia in a pH-dependent manner . The strong Ia-mediated channel block of Ib occurred only when the pH was at least lowered to pH 5.6 . The single-channel conductance showed a linear dependence on the bulk aqueous KCl concentration, which indicated that the channel properties were more general than specific . Zero current membrane potential measurements suggested the Ib channel has an approximately 6-fold higher permeability for potassium ions than for chloride . The selectivity ratio changed for salts composed of cations and anions of different mobility in the aqueous phase, again suggesting that Ib formed a water-filled general diffusion pore . Asymmetric addition of activated Ib to lipid bilayer membranes resulted in an asymmetric voltage dependence, indicating its full orientation within the membrane . Titration experiments with chloroquine and different tetraalkylammonium ions suggested that the Ib channel was blocked by these compounds but had only a weak affinity to them . In vivo measurements using Vero cells demonstrate that chloroquine and related molecules also did not efficiently block intoxication of the cells by iota-toxin . The possible role of Ib in the translocation of iota-toxin across the target cell membrane is discussed.

J Biol Chem, 2002 Feb 15, 277(7), 5074 - 81 Epub 2001 Dec 06.
The binary Clostridium botulinum C2 toxin as a protein delivery system: identification of the minimal protein region necessary for interaction of toxin components; Barth H et al.; The binary Clostridium botulinum C2 toxin is composed of the enzyme component C2I and the binding component C2II, which are individual and non-linked proteins . Activated C2IIa mediates cell binding and translocation of C2I into the cytoplasm . C2I ADP-ribosylates G-actin at Arg-177 to depolymerize actin filaments . A fusion toxin containing the N-terminal domain of C2I (residues 1-225) transports C3 ADP-ribosyltransferase from Clostridium limosum into cells (Barth, H., Hofmann, F., Olenik, C., Just, I., and Aktories, K . (1998) Infect . Immun . 66, 1364-1369) . We characterized the adaptor function of C2I and its interaction with C2IIa . The fusion toxin GST-C2I(1-225)-C3 was efficiently transported by C2IIa, indicating that C2IIa translocates proteins into the cytosol even when the C2I(1-225) adaptor was positioned in the middle of a fusion protein . Amino acid residues 1-87 of C2I were sufficient for interaction with C2IIa and for translocation of C2I fusion toxins into HeLa cells . Residues 1-87 were the minimal part of C2I to bind to C2IIa on the cell surface, as detected by fluorescence-activated cytometry . An excess of C2I(1-87) (but not of further truncated C2I fragments) competed with Alexa488-labeled C2I for binding to C2IIa . Also, the fragment C2I(30-431) and the fusion toxin C2I(30-225)-C3 competed with C2I-Alexa488 for binding to C2IIa . C2I(30-225)-C3 did not induce cytotoxic effects on cells when applied together with C2IIa, indicating that amino acid residues 1-29 are involved in translocation of C2I but are not absolutely essential for binding to C2IIa.

J Bacteriol, 2002 Jan, 184(1), 76 - 81
Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes; Murashima K et al.; In cellulosomes produced by Clostridium spp., the high-affinity interaction between the dockerin domain and the cohesin domain is responsible for the assembly of enzymatic subunits into the complex . Thus, heterologous expression of full-length enzymatic subunits containing the dockerin domains and of the scaffolding unit is essential for the in vitro assembly of a "designer" cellulosome, or a recombinant cellulosome with a specific function . We report the preparation of Clostridium cellulovorans recombinant cellulosomes containing the enzymatic subunit EngB and the scaffolding unit, mini-CbpA, containing a cellulose binding domain, a putative cell wall binding domain, and two cohesin units . The full-length EngB containing the dockerin domain was expressed by Bacillus subtilis WB800, which is deficient in eight extracellular proteases, to prevent the proteolytic cleavage of the enzymatic subunit between the catalytic and dockerin domains that was observed in previous attempts to express EngB with Escherichia coli . The assembly of recombinant EngB with the mini-CbpA was confirmed by immunostaining, a cellulose binding experiment, and native polyacrylamide gel electrophoresis analysis.

FEBS Lett, 2001 Dec 7, 509(2), 235 - 8
Phased A-tracts bind to the alpha subunit of RNA polymerase with increased affinity at low temperature; Katayama S et al.; Previously we showed that the expression of a Clostridium perfringens phospholipase C gene (plc) is activated by promoter upstream phased A-tracts in a low temperature-dependent manner . In this paper we characterize the interaction between the alpha subunit of C . perfringens RNA polymerase and the phased A-tracts . Hydroxyl radical footprinting and fluorescence polarization assaying revealed that the alpha subunit binds to the minor grooves of the phased A-tracts through its C-terminal domain with increased affinity at low temperature . The result provides a molecular mechanism underlying the activation of the plc promoter by the phased A-tracts.

J Pediatr Gastroenterol Nutr, 2001 Nov, 33(5), 592 - 601
Bacterial translocation in neonatal rats: the relation between intestinal flora, translocated bacteria, and influence of milk; Yajima M et al.; BACKGROUND: A high incidence of bacterial translocation in neonates results not only from immaturity of host-defense functions, but also from the dominant colonization of aerobic bacteria in the intestine . Bacterial colonization develops differently among breast-fed, formula-fed, premature, and full-term infants . The purpose of this study was to examine the incidence of bacterial translocation and to identify the translocated bacterial species, relating these findings to the intestinal microflora and to the type of feeding in neonatal rats . METHODS: Animals were divided into three groups: breast-fed normal pups (MR group), formula-fed pups fed via an intragastric cannula implanted esophageally (AR group), and breast-fed pups after the removal of the cannula (Sham group) . Artificial rearing was achieved using a machine feeding system . Culture and identification of the bacteria in the intestine, mesenteric lymph nodes, liver, portal blood, and lungs were made using a simplified version of Mitsuoka's method . RESULTS: At 14 days of age, the dominant bacteria in the feces of the MR and Sham Groups were Enterobacteriaceae, Lactobacillus, and Enterococcus, but Enterobacteriaceae and Clostridium were significantly more common in the AR group than in the MR group . The dominant bacteria in the mesenteric lymph nodes were Enterobacteriaceae, Lactobacillus, and Staphylococcus . The extent of systemic bacterial translocation decreased earlier in the Sham group than in the AR group . CONCLUSIONS: The frequency with which species of bacteria were cultured from mesenteric lymph nodes and other peripheral sites did not mirror the composition of the intestinal flora . Among the translocated bacteria, Staphylococcus may be especially hard to recognize and difficult for the host-defense systems to destroy . Breast-feeding inhibited systemic bacterial translocation in the suckling period of the rat.

Toxicon, 2002 Apr, 40(4), 409 - 18
Isolation and identification of Clostridium perfringens in the venom and fangs of Loxosceles intermedia (brown spider): enhancement of the dermonecrotic lesion in loxoscelism; Monteiro CL et al.; Loxoscelism or the envenoming by the brown spiders (Loxosceles genus spiders), may produce extensive dermonecrosis and hemorrhage at the bite site and, eventually, systemic reactions that may be lethal . Isolation and identification of many different bacteria, among them Clostridium perfringens, of great medical importance due to its involvement in dermonecrotizing and systemic conditions, was carried out from the venomous apparatus (fangs and venom) of spiders obtained directly from nature, through microbiological cultures in aerobic and anaerobic conditions . Working with Loxosceles intermedia venom (alone) and with the venom conjugated with Clostridium perfringens using rabbits as experimental models for dermonecrosis, allowed for the observation that venom and anaerobic bacteria conjugated resulted in a striking increase of the dermonecrotic picture when compared to venom alone, suggesting a role for Clostridium perfringens in the severe dermonecrotic picture of these patients and opening the possibility for the association of antibiotic therapy in treating loxoscelism.

Structure (Camb), 2001 Dec, 9(12), 1183 - 90
The structure of the feruloyl esterase module of xylanase 10B from Clostridium thermocellum provides insights into substrate recognition; Prates JA et al.; BACKGROUND: Degradation of the plant cell wall requires the synergistic action of a consortium of predominantly modular enzymes . In Clostridiae, these biocatalysts are organized into a supramolecular assembly termed a "cellulosome." This multienzyme complex possesses, in addition to its well-described cellulolytic activity, an apparatus specific for xylan degradation . Cinnamic acid esterases hydrolyze the ferulate groups involved in the crosslinking of arabinoxylans to lignin and thus play a key role in the degradation of the plant cell wall in addition to having promising industrial and medical applications . RESULTS: We have cloned and overexpressed the feruloyl esterase module from a 5 domain xylanase, Xyn10B from Clostridium thermocellum . The native structure at 1.6 A resolution has been solved with selenomethionine multiple wavelength anomalous dispersion and refined to a final R(free) of 17.8% . The structure of a hydrolytically inactive mutant, S954A, in complex with the reaction product ferulic acid has been refined at a resolution of 1.4 A with an R(free) of 16.0% . CONCLUSIONS: The C . thermocellum Xyn10B ferulic acid esterase displays the alpha/beta-hydrolase fold and possesses a classical Ser-His-Asp catalytic triad . Ferulate esterases are characterized by their specificity, and the active center reveals the binding site for ferulic acid and related compounds . Ferulate binds in a small surface depression that possesses specificity determinants for both the methoxy and hydroxyl ring substituents of the substrate . There appears to be a lack of specificity for the xylan backbone, which may reflect the intrinsic chemical heterogeneity of the natural substrate.

Eur J Biochem, 2001 Dec, 268(24), 6473 - 86
The sialate-pyruvate lyase from pig kidney . Elucidation of the primary structure and expression of recombinant enzyme activity; Traving C et al.; The first complete primary structure of a mammalian sialate-pyruvate lyase, namely of the enzyme from porcine kidney, was elucidated by a combination of different PCR techniques followed by sequencing of the resulting fragments . The primers used were either deduced from four porcine lyase peptides or from an alignment of human and mouse expressed sequence tags (ESTs), which were found to be homologous to already known microbial lyase sequences, and cDNA alone or after ligation with a plasmid vector served as a template . The lyase primary structure consists of 319 amino acids with a calculated protein molecular mass of approximately 35 kDa, which fits well to the value determined for the native enzyme . The porcine lyase sequence made it possible to assemble several ESTs from mouse and man in order to obtain the complete putative lyase genes . The three mammalian sequences reveal a high degree of homology both on the nucleotide (83% of the nucleotides are identical between all three sequences) and on the amino-acid level (72% of the amino acids are identical between all three sequences), and thus form a tightly related group . In contrast, the identity between the lyase primary structures from pig kidney and the microbial enzyme from Clostridium perfringens is much less pronounced (25%) . Thirty-one amino acids were found to be absolutely conserved in all lyase sequences . Among them are two amino acids (lysine 173 and tyrosine 143 in the porcine lyase) that are most important for the catalytic reaction . After expression cloning, recombinant enzyme activity was expressed in Escherichia coli BL21(DE3)pLysS, which confirms the identity of the cloned sequence and verifies one of the putative human and murine sequences . After SDS/PAGE of a cell extract of the expression clone, a band of 35kDa was stained on the gel.

ANZ J Surg, 2001 Nov, 71(11), 647 - 9
Clostridium septicum and malignancy; Chew SS et al.; BACKGROUND: Clostridium septicum is known to be associated with malignancy or immunosuppression . It has a variable clinical presentation and is associated with a high mortality . The aim of the present study was to review the experience at St George Hospital, Sydney, over a 10-year period, with particular reference to the association of this condition with colorectal cancer . METHODS: The records of five patients with blood culture-proven Clostridium septicum infection, among a larger group of 31 patients with clostridial infections, presenting to St George Hospital between 1990 and 2000 were reviewed . RESULTS: Associated malignancy was found in four (80%) of the patients with Clostridium septicum infection . Two infections were related to colorectal cancer, two to haematological malignancies and one to radiation-induced recto-urethral fistula . Those patients who had colorectal cancer presented with septicaemia and vague abdominal symptoms . CONCLUSIONS: Clostridium septicum infections have a strong association with malignancy . When this infection occurs without an obvious underlying aetiology there should be a high index of suspicion about associated malignancy . In the absence of haematological malignancy a colonoscopy is warranted . Early diagnosis and aggressive treatment is essential in order to improve prognosis.

Biochem J, 2001 Dec 15, 360(Pt 3), 717 - 26
Influence of electrochemical properties in determining the sensitivity of {4Fe-4S} clusters in proteins to oxidative damage; Tilley GJ et al.; Interconversion between {4Fe-4S} cubane and {3Fe-4S} cuboidal states represents one of the simplest structural changes an iron-sulphur cluster can undertake . This reaction is implicated in oxidative damage and in modulation of the activity and regulation of certain enzymes, and it is therefore important to understand the factors governing cluster stability and the processes that activate cluster conversion . In the present study, protein film voltammetry has been used to induce and monitor the oxidative conversion of {4Fe-4S} into {3Fe-4S} clusters in different variants of Azotobacter vinelandii ferredoxin I (AvFdI; the 8Fe form of the native protein), and DeltaThr(14)/DeltaAsp(15), Thr(14)-->Cys (T14C) and C42D mutants . The electrochemical results have been correlated with the differing oxygen sensitivities of {4Fe-4S} clusters, and comparisons have been drawn with other ferredoxins (Desulfovibrio africanus FdIII, Clostridium pasteurianum Fd, Thauera aromatica Fd and Pyrococcus furiosus Fd) . In contrast with high-potential iron-sulphur proteins (HiPIPs) for which the oxidized species {4Fe-4S}(3+) is inert to degradation and can be isolated, the hypervalent state in these ferredoxins (most obviously the 3+ level) is very labile, and the reduction potential at which this is formed is a key factor in determining the cluster's resistance to oxidative damage.

Biochem J, 2001 Dec 15, 360(Pt 3), 651 - 6
Allosteric behaviour of 1:5 hybrids of mutant subunits of Clostridium symbiosum glutamate dehydrogenase differing in their amino acid specificity; Goyal A et al.; Hybrid hexamers were made by refolding mixtures of two mutant forms of clostridial glutamate dehydrogenase . Mutant Cys320Ser (C320S) has a similar activity to the wild-type enzyme, but is unreactive with Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoate) (DTNB) . The triple mutant Lys89Leu/Ala163Gly/Ser380Ala (K89L/A163G/S380A), active with norleucine but not glutamate, is inactivated by DTNB, since the amino acid residue at position 320 is a cysteine residue . The chosen ratio favoured 1:5 hybrids of the triple mutant and C320S . The renatured mixture was treated with DTNB and separated on an NAD(+)-agarose column to which only C320S subunits bind tightly . Fractions were monitored for glutamate and norleucine activity and for releasable thionitrobenzoate to establish subunit stoichiometry . A fraction highly enriched in the 1:5 hybrid was identified . Homohexamers (C320S with 40 mM glutamate and 1 mM NAD(+) at pH 8.8, or K89L/A163G/S380A with 70 mM norleucine and 1 mM NAD(+) at pH 8.5) showed allosteric activation; succinate activated C320S approx . 50-fold (EC(50)=70 mM, h=2.4), and glutarate gave approx . 30-fold activation (EC(50)=35 mM, h=2.3) . For the triple mutant, corresponding values were 80 mM and 2.2 for succinate, and 75 mM and 1.7 for glutarate, but maximal activation was only about 2-fold . In the 1:5 hybrid, with only one norleucine-active subunit per hexamer, responses to glutarate and succinate were still co-operative, and activation was more extensive than in the triple mutant homohexamer . A single norleucine-active subunit can thus respond co-operatively to a substrate analogue at the other five inactive sites . On the other hand, similar hyperbolic dependence on the norleucine concentration for the hybrid and the triple mutant homohexamer reflected the inability of C320S subunits to bind norleucine . With glutamate at pH 8.8, an h value of 3.6 was obtained for the 1:5 hybrid, in contrast with an h value of 5.2 for the C320S homohexamer . The "foreign" subunit evidently impedes inter-subunit communication to some extent.

Biochemistry, 2001 Dec 18, 40(50), 15327 - 33
Role of the disulfide cleavage induced molten globule state of type a botulinum neurotoxin in its endopeptidase activity; Cai S et al.; Botulinum neurotoxins are produced by anaerobic Clostridium botulinum in an inactive form . The endopeptidase activity of type A botulinum neurotoxin (BoNT/A) is triggered by reduction of its disulfide bond between its heavy chain and light chain . By using circular dichroism spectroscopy, we show that, upon reduction of BoNT/A and under physiological temperature (37 degrees C), the BoNT/A loses most of its native tertiary structure, while retaining most of its secondary structure . This type of structure is characterized as a molten globule type conformation, which was further confirmed for BoNT/A by the characteristic binding of 1-anilinonaphthalene-8-sulfonic acid . Under nonreducing conditions where the interchain disulfide bond is intact, the enzymatically inactive BoNT/A did not show a molten globule type of structure . A temperature profile of the structure and enzyme activity of BoNT/A revealed that, under reducing conditions, there was a strong correlation in the existence of the molten globule structure and optimum endopeptidase activity at about 37 degrees C.

Plasmid, 2001 Nov, 46(3), 229 - 32
Induction of pCW3-encoded tetracycline resistance in Clostridium perfringens involves a host-encoded factor; Johanesen PA et al.; The tetracycline resistance determinant Tet P, which is encoded by the conjugative plasmid pCW3 from Clostridium perfringens, is induced by subinhibitory concentrations of tetracycline . In this study we have shown that the inducible phenotype is strain dependent . When pCW3 is present in derivatives of the wild-type strains CW234 and CW362 resistance is inducible . However, transfer to derivatives of strain 13 leads to a constitutive phenotype that is only observed in this strain background . Based on these results it is proposed that induction of the pCW3-encoded tet(P) genes in C . perfringens requires a host-encoded factor that is either absent or nonfunctional in strain 13 derivatives .

Mol Cell Probes, 2001 Oct, 15(5), 301 - 6
Electroporation of DNA sequences from the pathogenicity locus (PaLoc) of toxigenic Clostridium difficile into a non-toxigenic strain; Ackermann G et al.; Toxigenic Clostridium difficile is the etiologic agent of C . difficile-associated diarrhoea (CDAD), the most common cause of hospital-acquired infectious diarrhoea . The genes tcdA and tcdB, which encode for the toxin A and B proteins, are part of the pathogenicity locus (PaLoc) of toxigenic C . difficile . Genetic and virulence studies at the molecular level in C . difficile have been hindered by the lack of techniques for DNA manipulation in this species . We describe the electroporation of DNA fragments from a toxigenic isolate into a non-toxigenic strain of C . difficile . Using previously described methods of electroporation into Clostridium spp., the complete toxin B gene and polymerase chain reaction (PCR) fragments of the PaLoc were cloned and electroporated into a non-toxigenic strain of C . difficile . The resulting transformed clones were screened for the introduced gene fragments by PCR, which confirmed their presence . This is the first description of introduction of DNA into C . difficile by electroporation .

J Mol Biol, 2001 Dec 7, 314(4), 797 - 806
Recognition of cello-oligosaccharides by a family 17 carbohydrate-binding module: an X-ray crystallographic, thermodynamic and mutagenic study; Notenboom V et al.; The crystal structure of the Clostridium cellulovorans carbohydrate-binding module (CBM) belonging to family 17 has been solved to 1.7 A resolution by multiple anomalous dispersion methods . CBM17 binds to non-crystalline cellulose and soluble beta-1,4-glucans, with a minimal binding requirement of cellotriose and optimal affinity for cellohexaose . The crystal structure of CBM17 complexed with cellotetraose solved at 2.0 A resolution revealed that binding occurs in a cleft on the surface of the molecule involving two tryptophan residues and several charged amino acids . Thermodynamic binding studies and alanine scanning mutagenesis in combination with the cellotetraose complex structure allowed the mapping of the CBM17 binding cleft . In contrast to the binding groove characteristic of family 4 CBMs, family 17 CBMs appear to have a very shallow binding cleft that may be more accessible to cellulose chains in non-crystalline cellulose than the deeper binding clefts of family 4 CBMs . The structural differences in these two modules may reflect non-overlapping binding niches on cellulose surfaces .

Infect Control Hosp Epidemiol, 2001 Sep, 22(9), 572 - 5
Quinolone use as a risk factor for nosocomial Clostridium difficile-associated diarrhea; Yip C et al.; OBJECTIVE: To determine modifiable risk factors for nosocomial Clostridium difficile-associated diarrhea (CDAD) . DESIGN: Case-control study . SETTING: 300-bed tertiary-care hospital . PARTICIPANTS: Hospital inpatients present during the 3-month study period . METHODS: Case-patients identified with nosocomial CDAD over the study period were compared to two sets of control patients: inpatients matched by age, gender, and date of admission; and inpatients matched by duration of hospital stay . Variables including demographic data, comorbid illnesses, antibiotic exposure, and use of gastrointestinal medications were assessed for case- and control-patients . Conditional logistic regression was performed to identify risk factors for nosocomial CDAD . RESULTS: 27 case-patients were identified and were compared to the two sets of controls (1:1 match for each comparison set) . For the first set of controls, use of ciprofloxacin (odds ratio {OR}, 5.5; 95% confidence interval {CI 95}, 1.2-24.8; P=.03) was the only variable that remained significant in the multivariable model . For the second set of controls, prior exposure to cephalosporins (OR, 6.7; CI 95, 1.3-33.7; P=.02) and to ciprofloxacin (OR, 9.5; CI 95, 1.01-88.4; P=.05) were kept in the final model . CONCLUSIONS: Along with cephalosporins, prior quinolone use predisposed hospitalized patients to nosocomial CDAD . Quinolones should be used judiciously in acute-care hospitals, particularly in those where CDAD is endemic.

Biotechniques . 2001 Nov;31(5):1064, 1066, 1068.
New E . coli cloning vector using a cellulase gene (celA) as a screening marker; Jang SJ et al.; The extracellular endoglucanase A gene of Clostridium thermocellum (celA) was used as a screening marker for E . coli cloning vector A 1.4-kb EcoRI fragment containing celA from pTvec/celA was isolated and cloned into a pUC18 deleting beta-galactosidase gene fragment . The constructed vectors, pCEL1, pCEL10, pCEL11, and pCEL20, have different multiple cloning sites within celA . If the cellulase, CelA, is inactivated by insertion of a foreign DNA fragment into multiple cloning sites, the recombinant transformants show no clear halos on an agar plate containing cellulose . This process overcomes the ambiguity of color screening in the X-gal/beta-galactosidase system, and over 90% of the recombinant transformants with no halos have foreign DNA inserts . Several E . coli strains were transformed successfully with pCEL series vectors regardless of mutation for alpha-complementation . Because E . coli strains do not have a cellulase gene, a vector using a cellulase gene screening marker can be used in any E . coli strain without limit . The new cloning system is very efficient, convenient, and cost effective.

J Biol Chem, 2002 Feb 8, 277(6), 4247 - 54 Epub 2001 Nov 29.
Protein kinase C signaling regulates ZO-1 translocation and increased paracellular flux of T84 colonocytes exposed to Clostridium difficile toxin A; Chen ML et al.; Clostridium difficile toxin A increases paracellular permeability in colonic epithelial T84 cells by mechanisms involving RhoA glucosylation and actin depolymerization . However, we previously observed that toxin A-mediated decline in transepithelial electrical resistance preceded changes in cell morphology and tight junction ultrastructure (Hecht, G., Pothoulakis, C., LaMont, J . T., and Madara, J . L . (1988) J . Clin . Invest . 82, 1516-1524) . Recent studies also showed that C . difficile toxins induce early cellular responses, including activation of mitogen-activated protein kinases, generation of reactive oxygen metabolites, and calcium influx . The aim of this study was to investigate whether toxin A-induced early cellular responses contribute to the permeability changes . We found that toxin A stimulated the activities of membrane and cytosolic protein kinase Calpha (PKCalpha) and cytosolic PKCbeta . A specific PKCalpha/beta antagonist (myristoylated PKCalpha/beta peptide) blocked toxin A-mediated RhoA glucosylation . Furthermore, decreased transepithelial electrical resistance and increased translocation of ZO-1 from tight junction occurred within 2-3 h of toxin A exposure and were also inhibited by PKCalpha/beta antagonist . During this time period, toxin exposure did not induce translocation of ZO-2, dephosphorylation or translocation of occludin, or cell rounding . Our data indicate that PKC signaling regulates toxin A-mediated paracellular permeability changes and ZO-1 translocation.

Biochem Pharmacol, 2001 Dec 1, 62(11), 1459 - 68
Positive modulation by Ras of interleukin-1beta-mediated nitric oxide generation in insulin-secreting clonal beta (HIT-T15) cells; Tannous M et al.; In the present study, we have shown that exposure of insulin-secreting clonal beta (HIT-T15) cells to interleukin-1beta (IL-1beta) results in a time- and concentration-dependent increase in nitric oxide (NO) release . These effects by IL-1beta on NO release were mediated by induction of inducible nitric oxide synthase (iNOS) from the cells . Preincubation of HIT cells with Clostridium sordellii lethal toxin-82, which irreversibly glucosylates and inactivates small G-proteins, such as Ras, Rap, Ral, and Rac, but not Cdc42, completely abolished IL-1beta-induced NO release . Pre-exposure of HIT cells to C . sordellii lethal toxin-9048, which monoglucosylates and inhibits Ras, Cdc42, Rac, and Rap, but not Ral, also attenuated IL-1beta-mediated NO release . These data indicate that activation of Ras and/or Rac may be necessary for IL-1beta-mediated NO release . Preincubation of HIT cells with C . difficile toxin-B, which monoglucosylates Rac, Cdc42, and Rho, had no demonstrable effects on IL-mediated NO release, ruling out the possibility that Rac may be involved in this signaling step . Further, two structurally dissimilar inhibitors of Ras function, namely manumycin A and damnacanthal, inhibited, in a concentration-dependent manner, the IL-1beta-mediated NO release from these cells . Together, our data provide evidence, for the first time, that Ras activation is an obligatory step in IL-1beta-mediated NO release and, presumably, the subsequent dysfunction of the pancreatic beta cell . Our data also provide a basis for future investigations to understand the mechanism of cytokine-induced beta cell death leading to the onset of insulin-dependent diabetes mellitus.

Scand J Infect Dis, 2001, 33(10), 731 - 3
The role of Clostridium difficile in childhood nosocomial diarrhea; Oguz F et al.; The role of Clostridium difficile was investigated in 100 children with nosocomial diarrhea . An etiologic agent was identified in 69 cases, 8 of whom had dual infection . C . difficile-associated diarrhea (Cdad) was defined in 16 children (16%) . The mean age of the patients with Cdad was 5.4 y (range 2 months to 13 y) and the male:female ratio was 1.2 . All cases with Cdad were on antibiotic therapy . Cdad occurred more frequently in the cases given combined antibiotic treatment than in those given single antibiotic treatment (p < 0.05) . One case with neutropenic sepsis died . C . difficile was also investigated in the stool samples of 50 hospitalized children treated with antibiotics who did not develop diarrhea . C . difficile toxins A and B were found in 5 children aged < 2 y in the control group . This study shows that C . difficile is an important cause of nosocomial diarrhea in our hospital population.

J Biomol NMR, 2001 Oct, 21(2), 107 - 16
Simultaneous measurement of intra- and intermolecular NOEs in differentially labeled protein-ligand complexes; Eichmuller C et al.; A new NOE strategy is presented that allows the simultaneous observation of intermolecular and intramolecular NOEs between an unlabeled ligand and a 13C,15N-labeled protein . The method uses an adiabatic 13C inversion pulse optimized to an empirically observed relationship between 1 J(CH) and carbon chemical shift to selectively invert the protein protons (attached to 13C) . Two NOESY data sets are recorded where the intermolecular and intramolecular NOESY cross peaks have either equal or opposite signs, respectively . Addition and subtraction yield two NOESY spectra which contain either NOEs within the labeled protein (or unlabeled ligand) or along the binding interface . The method is demonstrated with an application to the B12-binding subunit of Glutamate Mutase from Clostridium tetanomorphum complexed with the B12-nucleotide loop moiety of the natural cofactor adenosylcobalamin (Coenzyme B12).

Proc Natl Acad Sci U S A, 2001 Dec 18, 98(26), 15155 - 60 Epub 2001 Nov 27.
Combination bacteriolytic therapy for the treatment of experimental tumors; Dang LH et al.; Current chemotherapeutic approaches for cancer are in part limited by the inability of drugs to destroy neoplastic cells within poorly vascularized compartments of tumors . We have here systematically assessed anaerobic bacteria for their capacity to grow expansively within avascular compartments of transplanted tumors . Among 26 different strains tested, one (Clostridium novyi) appeared particularly promising . We created a strain of C . novyi devoid of its lethal toxin (C . novyi-NT) and showed that intravenously injected C . novyi-NT spores germinated within the avascular regions of tumors in mice and destroyed surrounding viable tumor cells . When C . novyi-NT spores were administered together with conventional chemotherapeutic drugs, extensive hemorrhagic necrosis of tumors often developed within 24 h, resulting in significant and prolonged antitumor effects . This strategy, called combination bacteriolytic therapy (COBALT), has the potential to add a new dimension to the treatment of cancer.

J Biol Chem, 2002 Mar 8, 277(10), 8366 - 71 Epub 2001 Nov 27.
Rapid determination of substrate specificity of Clostridium histolyticum beta-collagenase using an immobilized peptide library; Hu Y et al.; The molecular basis of the substrate specificity of Clostridium histolyticum beta-collagenase was investigated using a combinatorial method . An immobilized positional peptide library, which contains 24,000 sequences, was constructed with a 7-hydroxycoumarin-4-propanoyl (Cop) fluorescent group attached at the N terminus of each sequence . This immobilized peptide library was incubated with C . histolyticum beta-collagenase, releasing fluorogenic fragments in the solution phase . The relative substrate specificity (k(cat)/K(m)) for each member of the library was determined by measuring fluorescence intensity in the solution phase . Edman sequencing was used to assign structure to subsites of active substrate mixtures . Collectively, the substrate preference for subsites (P(3)-P(4)') of C . histolyticum beta-collagenase was determined . The last position on the C-terminal side in which the identity of the amino acids affects the activity of the enzyme is P(4)', and an aromatic side chain is preferred in this position . The optimal P(1)'-P(3)' extended substrate sequence is P(1)'-Gly/Ala, P(2)'-Pro/Xaa, and P(3)'-Lys/Arg/Pro/Thr/Ser . The Cop group in either the P(2) or P(3) position is required for a high substrate activity with C . histolyticum beta-collagenase . S(2) and S(3) sites of the protease play a dominant role in fixing the substrate specificity . The immobilized peptide library proved to be a powerful approach for assessing the substrate specificity of C . histolyticum beta-collagenase, so it may be applied to the study of other proteases of interest.

Biochemistry, 2001 Dec 4, 40(48), 14384 - 91
Reduced temperature dependence of collective conformational opening in a hyperthermophile rubredoxin; Hernandez G et al.; Spatially localized differences in the conformational dynamics of the rubredoxins from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophile Clostridium pasteurianum (Cp) are monitored via amide exchange measurements . As shown previously for the hyperthermophile protein, nearly all backbone amides of the Cp rubredoxin exhibit EX(2) hydrogen exchange kinetics with conformational opening rates of >1 s(-)(1) . Significantly slower amide exchange is observed for Pf rubredoxin in the region surrounding the metal site and the proximal end of the three-stranded beta-sheet, while for the rest of the structure, the exchange rates at 23 degrees C are similar for both proteins . For the multiple-turn region comprising residues 14-32 in both rubredoxins, the uniformity of both the exchange rate constants and the values of the activation energy at the slowly exchanging sites is consistent with a model of solvent exposure via a subglobal cooperative conformational opening . In contrast to the common expectation of increased rigidity in the hyperthermophile proteins, below room temperature Pf rubredoxin exhibits a larger apparent flexibility in this multiple-turn region . The smaller enthalpy for the conformational opening process of this region in Pf rubredoxin reflects the much weaker temperature dependence of the underlying conformational equilibrium in the hyperthermophile protein.

Appl Environ Microbiol, 2001 Dec, 67(12), 5656 - 67
Percent G+C profiling accurately reveals diet-related differences in the gastrointestinal microbial community of broiler chickens; Apajalahti JH et al.; Broiler chickens from eight commercial farms in Southern Finland were analyzed for the structure of their gastrointestinal microbial community by a nonselective DNA-based method, percent G+C-based profiling . The bacteriological impact of the feed source and in-farm whole-wheat amendment of the diet was assessed by percent G+C profiling . Also, a phylogenetic 16S rRNA gene (rDNA)-based study was carried out to aid in interpretation of the percent G+C profiles . This survey showed that most of the 16S rDNA sequences found could not be assigned to any previously known bacterial genus or they represented an unknown species of one of the taxonomically heterogeneous genera, such as Ruminococcus or Clostridium . The data from bacterial community profiling were analyzed by t-test, multiple linear regression, and principal-component statistical approaches . The percent G+C profiling method with appropriate statistical analyses detected microbial community differences smaller than 10% within each 5% increment of the percent G+C profiles . Diet turned out to be the strongest determinant of the cecal bacterial community structure . Both the source of feed and local feed amendment changed the bacteriological profile significantly, whereas profiles of individual farms with identical feed regimens hardly differed from each other . This suggests that the management of typical Finnish farms is relatively uniform or that hygiene on the farm, in fact, has little impact on the structure of the cecal bacterial community . Therefore, feed compounders should have a significant role in the modulation of gut microflora and consequently in prevention of gastrointestinal disorders in farm animals.

J Appl Microbiol, 2001 Nov, 91(5), 814 - 21
Preliminary microbiological investigation of the preparation of two traditional Maori foods (Kina and Tiroi); Hudson JA et al.; AIMS: Little information exists regarding the microbiology of two traditional Maori food preparation processes which may involve fermentations . Preliminary microbiological and chemical analyses were carried out on these two foods in order to identify the fermentations involved (if any) . METHODS AND RESULTS: Testing was carried out on freshly-prepared foods and on those that had been processed and stored . Kina (sea urchins, Evechinus chloroticus) are harvested and then stored either under fresh water or buried underground . The most frequently-occurring process appeared to be an alkaline fermentation . Large numbers of Clostridium perfringens were detected in one set of samples prepared outside of the traditional season, but this was the only pathogen detected . In Kina stored in buried plastic bottles during the traditionally-accepted time of the year, bacterial numbers decreased . Tiroi is prepared from mussels and Puha (sow thistle, Sonchos asper) that have been cooked to some degree, combined and stored . Of three methods used to prepare and store Tiroi, the results for one indicated the possible involvement of a lactic acid fermentation, but the other two methods were effectively only cooking and bottling processes . CONCLUSIONS: In the case of Kina, the use of an alkaline fermentation to prepare a seafood for consumption is unusual . One method of Tiroi production is a lactic acid fermentation . SIGNIFICANCE AND IMPACT OF THE STUDY: If these foods are produced as described and are not either eaten immediately or cooked before consumption, then growth, and intoxication by, Clostridium botulinum might occur.

J Anim Sci, 2001 Oct, 79(10), 2558 - 64
Clostridial antibody response from injection-site lesions in beef cattle, long-term response to single or multiple doses, and response in newborn beef calves; Troxel TR et al.; Experiments were conducted to compare clostridial antibody response of beef heifers that do and do not develop injection-site lesions, evaluate long-term antibody response of a single- and multiple-dose toxoid, and evaluate the ability of a clostridial toxoid to elicit an active antibody response in newborn calves . In Exp . 1, 37 weaned heifers were vaccinated (d 0) with a clostridial vaccine (Alpha-7, 2 mL, s.c.) . Serum samples were collected on d 0, 28, 56, 84, and 112 to determine clostridial antibody titers . On d 28, heifers were visually inspected and palpated for injection-site lesions . The percentage of heifers that developed lesions was 64.9% . Lesioned heifers had elevated antibody titers for Clostridium chauvoei (CC) on d 28 (P < 0.08) and 84 (P < 0.07) compared with non-lesioned heifers . Clostridium sordellii (CS) and perfringens type D (CPD) antibody titers were greater in lesioned heifers than in non-lesioned heifers on d 28 and 56 . In Exp . 2, long-term antibody response of Alpha-7 (A7) and Ultrabac 7 (UB7) was investigated in stocker heifers . The A7 heifers (n = 15) received one 2-mL vaccination (d 0), and the UB7 heifers (n = 15) received a 5-mL vaccination on d 0 and 28 . Blood samples were collected on d 0, 28, 56, 84, 112, 140, and 180 . Clostridium chauvoei, CPD, and Cl . novyi (CN) antibody titers from the A7 heifers were greater than those from the UB7 heifers on d 28 . Due to the second UB7 injection, CC, CS, CN, and Cl . perfringens type C (CPC) antibody titers were greater in UB7 heifers than in A7 heifers on d 56 . By d 112, titers were not different, and by d 140 all antibody titers were below detectable levels . In Exp . 3, 58 pregnant, mature, crossbred cows were vaccinated with A7 before calving . At birth, calves were carefully observed to ensure consumption of colostrum . Calves were blocked according to parturition date, and calves in each block were randomly allocated to receive A7 (s.c . at 3 +/- 3 d of age) or remain unvaccinated controls . Calves were bled at the time of vaccination (d 0) and on d 28, 56, 84, and 112 . Antibody titers for CC, CPC, and CPD were elevated on d 0 and decreased throughout the experimental period (P < 0.01), but no titer differences (P > 0.10) were detected between treatment groups on any of the days sampled . These data indicated that antibody titers against clostridial diseases are enhanced when injection-site lesions develop . One injection of Alpha-7 seemed to provide the same length of protection as two injections of Ultrabac 7.

Equine Vet J, 2001 Nov, 33(6), 547 - 53
Systemic antibodies to Clostridium botulinum type C: do they protect horses from grass sickness (dysautonomia)?
Hunter LC, Poxton IR.
The aetiology of equine grass sickness (EGS) is still unknown . There is increasing evidence that toxicoinfection with Clostridium botulinum type C is involved . Epidemiological evidence shows that resistance to EGS can occur in older horses and those that have been on a particular pasture for longer or have been in prior contact with the disease . This resistance may be in the form of an immune response to the aetiological agent . Levels of systemic antibodies to the surface antigens of C . botulinum type C (using the closely related and safe C . novyi type A as a phenotypic marker) and to the botulinum type C neurotoxin (BoNT/C) were investigated in horses with and without EGS . Horses with grass sickness were found to have significantly lower levels of systemic IgG to both surface antigens and BoNT/C . Horses with low levels of systemic immunity to these antigens may be more susceptible to developing EGS . There were no significant differences in antibody levels between the different categories of EGS, suggesting systemic immunity to C . botulinum type C does not play a significant role in influencing the severity of the disease . However, horses that had been in contact with EGS or that were grazing land where it had occurred frequently in the past had significantly higher antibody levels to these antigens . These horses may have been exposed to subclinical doses of C . botulinum type C and BoNT/C, resulting in the production of a protective immune response against the putative aetiological agent . This finding is of potential significance for the prospect of prevention of EGS by vaccination against C . botulinum type C.

FEBS Lett, 2001 Nov 16, 508(2), 253 - 8
Protein-binding partners of the tobacco syntaxin NtSyr1; Kargul J et al.; Syntaxins and other SNARE (soluble NSF-attachment protein receptor) complex proteins play a key role in the cellular processes of vesicle trafficking, vesicle fusion and secretion . Intriguingly, the SNARE NtSyr1 (=NtSyp121) from Nicotiana tabacum also appears to have a role in signalling evoked by the plant stress hormone abscisic acid . However, partner proteins contributing to its function(s) remain unknown . We used an affinity chromatography approach to identify proteins from tobacco leaf microsomes that directly interact with the hydrophilic (cytosolic) domains of NtSyr1 and report several interacting proteins with sensitivities to the endopeptidase activity of Clostridium botulinum neurotoxins, including one protein that was recognised by alphaAtSNAP33 antiserum, raised against the Arabidopsis SNAP25 homologue . Treatment of microsomal membrane fractions indicated a protein near 55 kDa was sensitive to proteolysis by BotN/A and BotN/E, yielding degradation products of approximately 34 and 23 kDa . Expressed and purified AtSNAP33 also bound directly to the cytosolic domain of NtSyr1 and was sensitive to proteolysis by these toxins, suggesting that NtSyr1, a tobacco homologue of AtSNAP33, and coordinate SNAREs are likely to associate as partners for function in vivo.

J Bacteriol, 2001 Dec, 183(24), 7110 - 9
Transcriptional analysis of the tet(P) operon from Clostridium perfringens; Johanesen PA et al.; The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms . Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon . Deletion of P3 or alteration of the spacing between the -35 and -10 regions significantly reduced the level of transcription in a reporter construct . Induction was shown to be mediated at the level of transcription . Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the tetA(P) gene . Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct . It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.

J Bacteriol, 2001 Dec, 183(24), 7037 - 43
Characterization of xylanolytic enzymes in Clostridium cellulovorans: expression of xylanase activity dependent on growth substrates; Kosugi A et al.; Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized . Most of the activity was secreted into the growth medium when the bacterium was grown on xylan . Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions . Each of these fractions contained at least two major and three minor xylanase activities . In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis . The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000 . High alpha-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions . Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan . These results strongly indicated that C . cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate.

J Hosp Infect, 2001 Nov, 49(3), 204 - 9
Prospective evaluation of environmental contamination by Clostridium difficile in isolation side rooms; Verity P et al.; We determined prospectively the frequency, persistence and molecular epidemiology of Clostridium difficile environmental contamination after detergent-based cleaning in side rooms used to isolate patients with C . difficile diarrhoea . Approximately one-quarter of all environmental sites in side rooms sampled over four-week periods were contaminated with C . difficile . The overall side room prevalence of environmental C . difficile declined from 35% initially, to 24% in week 2, 18% in week 3, and 16% in week 4 . The bed frame was the most common site from which C . difficile was recovered, although the floor was the most contaminated site in terms of total numbers of colonies . C . difficile was recovered significantly more frequently from swabs plated directly on to C . difficile selective media containing lysozyme than from enrichment broth (P< 0.001), emphasizing the benefit of lysozyme supplementation . The great majority of C . difficile isolates (87% of all isolates, 84% of patient isolates) was indistinguishable from the UK epidemic strain (PCR ribotype 1) . It thus could not be determined whether environmental contamination was a cause or a consequence of diarrhoea . Our findings highlight the need for improved approaches to hospital environmental hygiene, and call into question current UK guidelines that recommend detergent-based cleaning to remove environmental C . difficile . In particular, improved cleaning of frequently touched sites in the immediate bed space area is required .

Biochem Biophys Res Commun, 2001 Nov 30, 289(2), 623 - 9
High sensitivity of mouse neuronal cells to tetanus toxin requires a GPI-anchored protein; Munro P et al.; Tetanus neurotoxin (TeNT) produced by Clostridium tetani specifically cleaves VAMP/synaptobrevin (VAMP) in central neurons, thereby causing inhibition of neurotransmitter release and ensuing spastic paralysis . Although polysialogangliosides act as components of the neurotoxin binding sites on neurons, evidence has accumulated indicating that a protein moiety is implicated as a receptor of TeNT . We have observed that treatment of cultured mouse neuronal cells with the phosphatidylinositol-specific phospholipase C (PIPLC) inhibited TeNT-induced cleavage of VAMP . Also, we have shown that the blocking effects of TeNT on neuroexocytosis can be prevented by incubation of Purkinje cell preparation with PIPLC . In addition, treatment of cultured mouse neuronal cells with cholesterol sequestrating agents such as nystatin and filipin, which disrupt clustering of GPI-anchored proteins in lipid rafts, prevented intraneuronal VAMP cleavage by TeNT . Our results demonstrate that high sensitivity of neurons to TeNT requires rafts and one or more GPI-anchored protein(s) which act(s) as a pivotal receptor for the neurotoxin.

J Biol Chem, 2002 Jan 25, 277(4), 2650 - 6 Epub 2001 Nov 16.
In vitro reconstitution of the Clostridium botulinum type D progenitor toxin; Kouguchi H et al.; Clostridium botulinum type D strain 4947 produces two different sizes of progenitor toxins (M and L) as intact forms without proteolytic processing . The M toxin is composed of neurotoxin (NT) and nontoxic-nonhemagglutinin (NTNHA), whereas the L toxin is composed of the M toxin and hemagglutinin (HA) subcomponents (HA-70, HA-17, and HA-33) . The HA-70 subcomponent and the HA-33/17 complex were isolated from the L toxin to near homogeneity by chromatography in the presence of denaturing agents . We were able to demonstrate, for the first time, in vitro reconstitution of the L toxin formed by mixing purified M toxin, HA-70, and HA-33/17 . The properties of reconstituted and native L toxins are indistinguishable with respect to their gel filtration profiles, native-PAGE profiles, hemagglutination activity, binding activity to erythrocytes, and oral toxicity to mice . M toxin, which contained nicked NTNHA prepared by treatment with trypsin, could no longer be reconstituted to the L toxin with HA subcomponents, whereas the L toxin treated with proteases was not degraded into M toxin and HA subcomponents . We conclude that the M toxin forms first by assembly of NT with NTNHA and is subsequently converted to the L toxin by assembly with HA-70 and HA-33/17.

Microb Pathog, 2001 Nov, 31(5), 255 - 60
Analysis of expression of GroEL (Hsp60) of Clostridium difficile in response to stress; Hennequin C et al.; Our laboratory has previously shown that adherence of Clostridium difficile to tissue culture cells is augmented by various stresses and that GroEL, a heat shock protein, serves an adhesive function in this bacterium . In this communication, RT-PCR, SDS-PAGE and immunoblotting were used to study the stress response in C . difficile following heat, acid or osmotic shock, iron deprivation or presence of a subinhibitory concentration of ampicillin in the culture medium . All these stresses increased transcription of groEL and production of GroEL to various degrees . Furthermore, the protein was found in membrane fractions and in the extracellular space after heat stress .

FEBS Lett, 2001 Nov 9, 508(1), 95 - 8
Sialic acids in gastropods; Burgmayr S et al.; The occurrence of N-acetylneuraminic acid and N-glycolylneuraminic acid residues in preparations of the slug Arion lusitanicus (Gastropoda) was determined by sodium dodecyl sulphate electrophoresis of the proteins followed by lectin blots stained with the sialic acid specific lectin from Maackia amurensis, by the sensitivity of this binding to sialidase from Clostridium perfringens, by specific fluorescent labelling of sialic acids with 1,2-diamino-4,5-methylenedioxybenzene, by the determination of the sensitivity to sialate-pyruvate-lyase, by co-migration with standards on high performance anion exchange chromatography with pulsed amperometric detection and by identification of the typical masses in the fragmentation patterns of the trimethylsilyl derivatives after gas chromatography . It is the first time sialic acids are identified in gastropods.

Infect Immun, 2001 Dec, 69(12), 7937 - 40
Role of FliC and FliD flagellar proteins of Clostridium difficile in adherence and gut colonization; Tasteyre A et al.; In vitro and in vivo adhesive properties of flagella and recombinant flagellin FliC and flagellar cap FliD proteins of Clostridium difficile were analyzed . FliC, FliD, and crude flagella adhered in vitro to axenic mouse cecal mucus . Radiolabeled cultured cells bound to a high degree to FliD and weakly to flagella deposited on a membrane . The tissue association in the mouse cecum of a nonflagellated strain was 10-fold lower than that of a flagellated strain belonging to the same serogroup, confirming the role of flagella in adherence.

Infect Immun, 2001 Dec, 69(12), 7904 - 10
Synergistic effects of alpha-toxin and perfringolysin O in Clostridium perfringens-mediated gas gangrene; Awad MM et al.; To examine the synergistic effects of alpha-toxin and perfringolysin O in clostridial myonecrosis, homologous recombination was used to construct an alpha-toxin deficient derivative of a perfringolysin O mutant of Clostridium perfringens . The subsequent strain was complemented with separate plasmids that carried the alpha-toxin structural gene (plc), the perfringolysin O gene (pfoA), or both toxin genes, and the resultant isogenic strains were examined in a mouse myonecrosis model . Synergistic effects were clearly observed in these experiments . Infection with the control strain, which did not produce either toxin, resulted in very minimal gross pathological changes, whereas the isogenic strain that was reconstituted for both toxins produced a pathology that was clearly more severe than when alpha-toxin alone was reconstituted . These changes were most apparent in the rapid spread of the disease, the gross pathology of the footpad and in the rate at which the mice had to be euthanatized for ethical reasons . Elimination of both alpha-toxin and perfringolysin O production removed most of the histopathological features typical of clostridial myonecrosis . These effects were restored when the mutant was complemented with the alpha-toxin structural gene, but reconstituting only perfringolysin O activity produced vastly different results, with regions of coagulative necrosis, apparently enhanced by vascular disruption, being observed . Reconstitution of both alpha-toxin and perfringolysin O activity produced histopathology most similar to that observed with the alpha-toxin reconstituted strain . The spreading of myonecrosis was very rapid in these tissues, and coagulative necrosis appeared to be restricted to the lumen of the blood vessels . The results of these virulence experiments clearly support the hypothesis that alpha-toxin and perfringolysin O have a synergistic effect in the pathology of gas gangrene.

Am J Physiol Renal Physiol, 2001 Dec, 281(6), F1092 - 101
Rho inhibits cAMP-induced translocation of aquaporin-2 into the apical membrane of renal cells; Tamma G et al.; First published August 8, 2001; 10.1152/ajprenal.00091.2001.-We have recently demonstrated that actin depolymerization is a prerequisite for cAMP-dependent translocation of the water channel aquaporin-2 (AQP2) into the apical membrane in AQP2-transfected renal CD8 cells (29) . The Rho family of small GTPases, including Cdc42, Rac, and Rho, regulates the actin cytoskeleton . In AQP2-transfected CD8 cells, inhibition of Rho GTPases with Clostridium difficile toxin B or with C . limosum C3 fusion toxin, as well as incubation with the Rho kinase inhibitor, Y-27632, caused actin depolymerization and translocation of AQP2 in the absence of the cAMP-elevating agent forskolin . Both forskolin and C3 fusion toxin-induced AQP2 translocation were associated with a similar increase in the osmotic water permeability coefficient . Expression of constitutively active RhoA induced formation of stress fibers and abolished AQP2 translocation in response to forskolin . Cytochalasin D induced both depolymerization of F-actin and AQP2 translocation, suggesting that depolymerization of F-actin is sufficient to induce AQP2 translocation . Together, these data indicate that Rho inhibits cAMP-dependent translocation of AQP2 into the apical membrane of renal principal cells by controlling the organization of the actin cytoskeleton.

Vox Sang, 2001 Oct, 81(3), 154 - 60
Evaluation of the BacT/Alert automated blood culture system for detecting bacteria and measuring their growth kinetics in leucodepleted and non-leucodepleted platelet concentrates; McDonald CP et al.; BACKGROUND AND OBJECTIVE: To evaluate the BacT/Alert automated blood culture system for the detection of bacteria in platelet concentrates, and to determine bacterial growth kinetics in leucodepleted and non-leucodepleted units . MATERIALS AND METHODS: Apheresis (Cobe Leucocyte Reduction System {LRS}) and pooled buffy coat-derived (Optipress) platelet concentrates (PCs) were tested . Six organisms were used for spiking the PCs: Clostridium perfringens, Bacillus cereus, Group B Streptococcus, Staphylococcus epidermidis, Staphylococcus aureus and Escherichia coli . Units were inoculated to give a final concentration of approximately equal to 1 and 50 colony-forming units (CFU)/ml . On days 0, 2 and 5, BacT/Alert standard aerobic and anaerobic bottles were inoculated with a 5-ml fill volume and bacteria were enumerated . RESULTS: The BacT/Alert Automated blood culture system gave rapid determination times of spiked units, with all positives detected within 48 h and 98.1% detected within 24 h . In general, as the inoculum concentration increased, the detection time decreased . Rapid growth was obtained with all organisms tested except for B . cereus, which failed to grow on four occasions . Bacterial numbers on day 2 ranged from 10(5) to 10(11) CFU/ml and on day 5 ranged from 10(4) to 10(12) CFU/ml . Growth was not significantly greater in leucodepleted units . CONCLUSIONS: The study confirmed that PCs are an excellent growth medium for bacteria . Rapid and substantial growth was obtained with all organisms under test . Leucodepletion does not appear to enhance bacterial proliferation . The BacT/Alert automated blood culture system could rapidly detect contamination of units . Bacterial screening using an automated blood culture system is therefore a potential option.

Folia Microbiol (Praha), 2001, 46(3), 197 - 204
Anaerobic fermentation of gelatinized sago starch-derived sugars to acetone-1-butanol-ethanol solvent by Clostridium acetobutylicum; Madihah MS et al.; A study of the kinetics and performance of solvent-yielding batch fermentation of individual sugars and their mixture derived from enzymic hydrolysis of sago starch by Clostridium acetobutylicum showed that the use of 30 g/L gelatinized sago starch as the sole carbon source produced 11.2 g/L total solvent, i.e . 1.5-2 times more than with pure maltose or glucose used as carbon sources . Enzymic pretreatment of gelatinized sago starch yielding maltose and glucose hydrolyzates prior to the fermentation did not improve solvent production as compared to direct fermentation of gelatinized sago starch . The solvent yield of direct gelatinized sago starch fermentation depended on the activity and stability of amylolytic enzymes produced during the fermentation . The pH optima for alpha-amylase and glucoamylase were found to be at 5.3 and 4.0-4.4, respectively . alpha-Amylase showed a broad pH stability profile, retaining more than 80% of its maximum activity at pH 3.0-8.0 after a 1-d incubation at 37 degrees C . Since C . acetobutylicum alpha-amylase has a high activity and stability at low pH, this strain can potentially be employed in a one-step direct solvent-yielding fermentation of sago starch . However, the C . acetobutylicum glucoamylase was only stable at pH 4-5, maintaining more than 90% of its maximum activity after a 1-d incubation at 37 degrees C.

Eur J Clin Microbiol Infect Dis, 2000 Jan, 19(1), 9 - 15
Factors associated with nosocomial diarrhea and Clostridium difficile-associated disease on the adult wards of an urban tertiary care hospital; Schwaber MJ et al.; A prospective survey of the adult inpatient population of an urban tertiary care hospital was conducted to determine factors associated with the development of nosocomial diarrhea and the acquisition of Clostridium difficile-associated disease . During the 3-month survey, 98 patients with nosocomial diarrhea were enrolled, and 38 controls were recruited . The controls were patients without diarrhea lying in beds adjacent to the affected patients . Factors significantly associated with nosocomial diarrhea were the administration of a special diet (P=0.02) and receipt of a greater number of different antibiotics (P=0.02) . Among the 98 patients with diarrhea, Clostridium difficile toxin B was identified in the stool of 13 . Factors found to be associated with the presence of toxin B as compared to other causes of nosocomial diarrhea were a greater number of individual antibiotics used during hospitalization (P=0.02) and receipt of a cephalosporin (P=0.03) or, more specifically, a third-generation cephalosporin (P=0.02) . Among patients with nosocomial diarrhea, those who had toxin in their stool had a significantly higher total antibiotic burden (expressed as antibiotic days) than those with diarrhea due to other causes (P=0.01).

Am J Physiol Cell Physiol, 2001 Dec, 281(6), C2010 - 9
Enhancement of survival by LPA via Erk1/Erk2 and PI 3-kinase/Akt pathways in a murine hepatocyte cell line; Sautin YY et al.; First published September 5, 2001; 10.1152/ajpcell.00077.2001.-Protective mechanisms for lysophosphatidic acid (LPA) against cell death caused by Clostridium difficile toxin, or tumor necrosis factor-alpha (TNF-alpha) plus D-galactosamine, were investigated in a murine hepatocyte cell line AML12 expressing Edg2 LPA receptor . In these models of hepatocellular injury, LPA prevented hepatocyte damage, suppressed apoptosis, and enhanced cell survival in a dose-dependent fashion . The protective effects of LPA were abolished by wortmannin and LY-294002, specific inhibitors of phosphatidylinositol 3-phosphate kinase (PI 3-kinase), and by PD-98059 and U-0126, inhibitors of MEK1/MEK2 . In nontreated hepatocytes, LPA elicited a gradual and sustained increase in phosphorylation of Erk1/Erk2 over 180 min of stimulation and downstream phosphorylation of p90RSK and transcription factor Elk-1 . In C . difficile toxin-treated cells, LPA-induced phosphorylation of Erk1/Erk2 was rapid but transient, while p90RSK and Elk-1 phosphorylation did not change significantly . LPA stimulated phosphorylation of Akt in a time-dependent manner in both intact and toxin-treated AML12 hepatocytes . Wortmannin and LY-294002 abolished phosphorylation of Akt, further supporting activation of PI 3-kinase/Akt as a signaling pathway, which mediates hepatocyte protection by LPA . Taken together, these results demonstrate that LPA prevents cell apoptosis induced by C . difficile toxin and TNF-alpha/D-galactosamine in the AML12 murine hepatocyte cell line . Cell protection by LPA involves activation of the mitogen-activated protein kinase Erk1/Erk2 cascade and PI 3-kinase-dependent phosphorylation of Akt.

Clin Exp Dermatol, 2001 Oct, 26(7), 619 - 30
Botulinum toxin A in the therapy of mimic facial lines; Becker-Wegerich P et al.; In aesthetic medicine, many different methods of skin rejuvenation are available . At the end of the 1980s, the neurotoxin Botulinum toxin A (BT-A) led to a revolution in aesthetic-corrective dermatology for the treatment of mimic facial wrinkles . The toxin is produced by Clostridium botulinum and causes a reversible, selective muscle relaxation that leads to a temporary flattening of the mechanical part of wrinkling without the stigmata of invasive surgery . After two decades of experience in different medical disciplines, there has been remarkable clinical development and progress in research, the identification of new botulinum toxin serotypes, and also innovation in indications and combined modalities . These lead to new and interesting questions . BT-A offers the experienced, critical dermatologist a time-saving, effective, cosmetically satisfactory, non-invasive treatment for mimic facial wrinkles and neck and decollete lines, with only minor side effects . Dermatologists should have a profound anatomical knowledge and should be able to perform all injection techniques to meet the needs of ever more demanding patients and to ensure optimization of patient satisfaction . The following review summarizes the historical development and the mechanism of action of both frequently and rarely used injection techniques with BT-A for the treatment of wrinkles and lines of the upper face, neck and decollete, and gives an update of different experiences encountered.

Biochemistry, 2001 Nov 13, 40(45), 13548 - 55
Alanine-scanning of the 50's loop in the Clostridium beijerinckii flavodoxin: evaluation of additivity and the importance of interactions provided by the main chain in the modulation of the oxidation-reduction potentials; Kasim M et al.; The four-residue reverse turn -Met56-Gly-Asp-Glu59- in the Clostridium beijerinckii flavodoxin provides the majority of the critical interactions with the isoalloxazine ring of the flavin mononucleotide (FMN) cofactor that contribute to the binding and the differential stabilization of its three redox states . Direct side chain contacts include the sulfur-ring interaction of Met56, which primarily influences the oxidized and hydroquinone states, and the hydrogen bond by Glu59 with the N3H, which directly (and indirectly through its "anchoring" function) influences all three states to various extents . Involving a novel redox-dependent conformational change, the hydrogen bond formed between the carbonyl group of Gly57 and the N5H of the reduced cofactor strongly influences the stability of the semiquinone state . In this study, the sequential elimination of all side chain interactions in various combinations through a systematic alanine-scanning mutagenesis approach was conducted to more completely understand the functional inter-relationships as well as any synergistic interactions that might occur within the loop . In general, additive effects for each side chain on the midpoint potentials for both couples were observed except for the hydroquinone state where some degree of nonadditivity was noted in multiple mutants involving Glu59 . The study concluded with the generation of the triple mutant -Ala56-Gly-Ala-Ala59- in which all side chain interactions are removed . Gly57 was left unchanged because of its critical conformational contribution . Remarkably, this mutant retained the ability to bind the FMN and to thermodynamically stabilize the semiquinone state despite the absence of all side chain interactions . Collectively, these observations emphasize the overriding importance of the main chain interactions with the N5H of the FMN and the associated redox-dependent conformational change in this loop and leaves little doubt as to its role in the thermodynamic stabilization of the neutral semiquinone state of the FMN cofactor.

Biochemistry, 2001 Nov 13, 40(45), 13466 - 73
Investigation of the role of the domain linkers in separate site catalysis by Clostridium symbiosum pyruvate phosphate dikinase; Wei M et al.; Pyruvate phosphate dikinase (PPDK) catalyzes the reversible reaction: ATP + P(i) + pyruvate <--> AMP + PP(i) + PEP using Mg2+ and NH4+ ions as cofactors . The reaction takes place in three steps, each mediated by a carrier histidine residue located on the surface of the central domain of this three-domain enzyme: (1) E-His + ATP <--> E-His-PP.AMP, (2) E-His-PP.AMP + P(i) <--> E-His-P + AMP + PP(i), (3) E-His-P + pyruvate <--> E-His + PEP . The first two partial reactions are catalyzed at an active site located on the N-terminal domain, and the third partial reaction is catalyzed at an active site located on the C-terminal domain . For catalytic turnover, the central domain travels from one terminal domain to the other . The goal of this work is to determine whether the two connecting linkers direct the movement of the central domain between active sites during catalytic turnover . The X-ray crystal structure of the enzyme suggests interaction between the two linkers that may result in their coordinated movement . Mutations were made at the linkers for the purpose of disrupting the linker-linker interaction and, hence, synchronized linker movement . Five linker mutants were analyzed . Two of these contain 4-Ala insertions within the solvated region of the linker, and three have 3-residue deletions in this region . The efficiencies of the mutants for catalysis of the complete reaction as well as the E-His + ATP <--> E-His-PP.AMP partial reaction at the N-terminal domain and the E-His + PEP <--> E-His-P + pyruvate reaction at the C-terminal domain were measured to assess linker function . Three linker mutants are highly active catalysts at both active sites, and the fourth is highly active at one site but not the other . These results are interpreted as evidence against coordinated linker movement, and suggest instead that the linkers move independently as the central domain travels between active sites . It is hypothesized that while the linkers play a passive role in central domain-terminal domain docking, their structural design minimizes the conformational space searched in the diffusion process.

Appl Microbiol Biotechnol, 2001 Oct, 57(1-2), 138 - 45
Cloning of the cel9A gene and characterization of its gene product from marine bacterium Pseudomonas sp . SK38; Ryu SK et al.; The yellow-pigmented bacterial strain causing green spot rot and death of layer was isolated from Porphyra dentata . This strain has been identified as Pseudomonas sp., harboring agarase, xylanase, and protease activity, as well as carboxymethyl-cellulase (CMCase) . Using genomic DNA from the Pseudomonas sp . SK38 digested with Sau3AI and ligated into pBluescript II KS+, we isolated a cel gene encoding a CMCase in Pseudomonas sp . SK38 . A 4.5-kb fragment was subcloned into pKR400 . The structure of the cel9A gene consists of an open reading frame of 1,521 bp starting with a GTG start codon and ending with a TAG stop codon . It thus encodes 506 amino acid residues of a protein with a calculated molecular weight of 52,636 daltons plus a signal peptide of 22 amino acids . The deduced amino acid sequence of the cel9A protein is similar to the same protein of Clostridium thermocellum . It contains, in particular, the two conserved regions of the glycoside hydrolase family 9 . The apparent molecular mass of the Cel9A protein is 52 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The enzyme is most active at pH 6-7 and an optimal temperature of around 30 degrees C.

Khirurgiia (Sofiia), 2000, 56(5-6), 25 - 7
{Retroperitoneal postoperative necrotizing fasciitis}; Fichev G et al.; This is a report on clinical experience had with 17 patients presenting necrotizing fasciitis--a complication ever more frequently encountered . The case material is distributed in two group differing by origin and clinical course of the complication . In group one (n = 11) it is a matter of postoperative development of postoperative complication, consistent with the classical "per continuitatem" and "per contiguitatem" mechanisms, while in group two (n = 6) the process originates, evolves and speads within the retroperitoneal space proper . Comprehensive microbiological examinations performed in 13 cases show that in either group different microorganisms are identified . In group one aerobic-anaerobic mixed infection is documented in all patients, with predominance of Enterobacteroidaceae among aerobic ones . In group two, anaerobic bacterial species, mainly Clostridium sp, prevail in all the isolates . The clinical study points to a substantial difference in the time of septic complication occurrence, as well as between the clinical picture of the two species . Accordingly, the final results are radically different--in group one survivorship amounts to 62.6%, whereas in group two--to 16.6% only.

Rev Esp Enferm Dig, 2001 Aug, 93(8), 535 - 43
Antibiotic-associated diarrhea and Clostridium difficile colitis: an update; Giannella RA; C . difficile colitis ranges from mild diarrhea to life-threatening "toxic" illness with fever, severe diarrhea, and abdominal pain . A colitis, frequently with a pseudomembrone, is the characteristic finding on sigmoidoscopy and is caused by one of more toxins elaborated by the organism Clostridium difficile . The clinical syndrome is not specific and can be mimicked by other colonic infections, inflammatory bowel disease, radiation colitis, or ischemic colitis . The diagnosis should be suspected in any patient who develops diarrhea during antibiotic therapy or within 6-8 weeks of treatment . Diagnosis should be confirmed by the detection of C . difficile toxin in stool along with sigmoidoscopy or colonoscopy for special situations . Most patients will respond promptly to discontinuation of the antibiotic especially early . However, if the diarrhea persists or is severe of the patient appears to be ill, then specific antimicrobial therapy should be employed . Antimicrobial agents that have been shown to be effective in this syndrome include metronidazole and vancomycin . In some patients who do not respond to this therapy or have complications, subtotal colectomy may be required.

Eur J Gastroenterol Hepatol, 2001 Nov, 13(11), 1371 - 3
Treatment of proctalgia fugax with botulinum A toxin; Katsinelos P et al.; Two recent studies described a temporal association between a high-amplitude and high-frequency myoelectrical activity of the anal sphincter and the occurrence of proctalgia, which suggest that paroxysmal hyperkinesis of the anus may cause proctalgia fugax . We describe a single case of proctalgia fugax responding to anal sphincter injection of Clostridium botulinum type A toxin . The presumed aetiology of proctalgia fugax is discussed and the possible mechanism of action of botulinum toxin (BTX) in this condition is outlined . Botulinum A toxin seems to be a promising treatment for patients with proctalgia fugax, and further trials appear to be worthwhile for this condition, which has been described as incurable.

Cancer Res, 2001 Nov 1, 61(21), 7878 - 81
Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium; Long H et al.; The mRNA for Rvp.1 (rat ventral prostate) increases in abundance before gland involution after androgen deprivation . Rvp.1 is homologous to CPE-R, the high-affinity intestinal epithelial receptor for Clostridium perfringens enterotoxin (CPE), and is sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis . Rvp.1 (claudin-3) and CPE-R (claudin-4) are members of a larger family of transmembrane tissue-specific claudin proteins that are essential components of intercellular tight junction structures regulating paracellular ion flux . However, claudin-3 and claudin-4 are the only family members capable of mediating CPE binding and cytolysis . The present study was designed to study the expression of claudin-3 and claudin-4 in human prostate tissue as potential targets for CPE toxin-mediated therapy for prostate cancer . On human multiple-tissue Northern blot analysis, mRNAs for both claudin-3 and claudin-4 were expressed at high levels in prostate tissue . In normal prostate tissue, expression of claudin-3 was localized exclusively within acinar epithelial cells by in situ mRNA hybridization . Compared with expression within prostate epithelial cells in surrounding normal glandular tissue, expression of claudin-3 mRNA remained high in the epithelium of prostate adenocarcinoma (10 of 10) and prostatic intraepithelial neoplasia (five of five) . Prostate adenocarcinoma cells metastatic to bone were obtained from a patient with disease progression during antiandrogen therapy . These metastatic cells were prostate-specific antigen-positive by immunohistochemical staining and also expressed functional CPE receptors as measured by sensitivity to CPE-induced cell lysis . The persistent high level of claudin-3 expression in prostate adenocarcinoma and functional cytotoxicity of CPE in metastatic androgen-independent prostate adenocarcinoma suggests a new potential therapeutic strategy for prostate cancer.

J Virol, 2001 Dec, 75(23), 11474 - 82
Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines; Shi Z et al.; The effectiveness of vaccination programs would be enhanced greatly through the availability of vaccines that can be administered simply and, preferably, painlessly without the need for timed booster injections . Tetanus is a prime example of a disease that is readily preventable by vaccination but remains a major threat to public health due to the problems associated with administration of the present vaccine . Here we show that a protective immune response against live Clostridium tetani infection in mice can be elicited by an adenovirus vector encoding the tetanus toxin C fragment when administered as a nasal or epicutaneous vaccine . The results suggest that these vaccination modalities would be effective needle-free alternatives . This is the first demonstration that absorption of a small number of vectored vaccines into the skin following topical application of a patch can provide protection against live bacteria in a disease setting.

Clin Microbiol Infect, 2001, 7 Suppl 4, 53 - 65
Limitations of presently available glycopeptides in the treatment of Gram-positive infection; Ziglam HM et al.; The glycopeptide antibacterial drugs vancomycin and teicoplanin are widely used in hospitals for therapy of severe or multiresistant Gram-positive infections, notably staphylococcal, enterococcal and rarely pneumococcal . Vancomycin has also been used in the management of Clostridium difficile enteropathy . The incidence and potential for resistance differ between agents . The in vitro activity, pharmacokinetics and clinical use of glycopeptide, as well as epidemiology of glycopeptide resistance are discussed . There are limited comparative studies indicating the need for further investigation . Therapeutic drug monitoring has been widely used for vancomycin and less commonly for teicoplanin, but remains controversial . Advances in our understanding of their pharmacodynamics and clinical studies are helping clarify the situation . This paper reviews the current literature and highlights limitations of glycopeptides in treating Gram-positive infection.

Clin Microbiol Infect, 2001, 7 Suppl 4, 16 - 33
Human infections caused by glycopeptide-resistant Enterococcus spp: are they a zoonosis?
Sundsfjord A, Simonsen GS, Courvalin P.
Following the detection of glycopeptide-resistant enterococci (GRE) in 1986 and their subsequent global dissemination during the 1990s, many studies have attempted to identify the reservoirs and lines of resistance transmission as a basis for intervention . The eradication of reservoirs and the prevention of GRE spread is of major importance for two reasons: (i) the emergence of high-level glycopeptide resistance in invasive enterococcal clinical isolates that are already multiresistant, has left clinicians with therapeutic options that are only at the experimental stage; and (ii) the resistance genes may spread to more virulent bacterial species such as Staphylococcus aureus, Streptococcus pneumoniae and Clostridium difficile . VanA-type strains, resistant to high levels of both vancomycin and teicoplanin, are the most commonly encountered enterococci with acquired glycopeptide resistance in humans . A widespread VanA-type GRE reservoir was detected early in farm animals that were exposed to the glycopeptide growth-promoter avoparcin . Numerous studies have provided indirect evidence for the transfer of VanA-type GRE and their resistance determinants from animal reservoirs to humans . The data collected have expanded our understanding of the promiscuous nature of antibiotic resistance, and have provided the groundwork for logical decision-making with the objective of deterring the dissemination of resistant bacteria and of their resistance genes.

Presse Med, 2001 Oct 6, 30(28), 1399 - 400
{Recurrent fatal pseudomembranous colitis}; Auvray L et al.; BACKGROUND: Clostridium difficile pseudomembranous colitis may trace a fulminent course and require surgery . CASE REPORTS: Outcome was fatal despite subtotal colectomy in the reported case of recurrent Clostridium difficile pseudomembranous colitis . Infrequent localization, low serum albumin and an unfavorable clinical course were observed . DISCUSSION: Rapid surgical treatment with large resection is mandatory in such cases, particularly in patients of rectal and sigmoid remnant involvement.

J Ind Microbiol Biotechnol, 2001 Oct, 27(4), 220 - 7
Expression of the Klebsiella pneumoniae CG21 acetoin reductase gene in Clostridium acetobutylicum ATCC 824; Wardwell SA et al.; Acetoin reductase catalyzes the production of 2,3-butanediol from acetoin . The gene encoding the acetoin reductase of Klebsiella pneumoniae CG21 was cloned and expressed in Escherichia coli and Clostridium acetobutylicum ATCC 824 . The nucleotide sequence of the gene encoding the enzyme was determined to be 768 bp long . Expression of the K . pneumoniae acetoin reductase gene in E . coli revealed that the enzyme has a molecular mass of about 31,000 Da based on sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis . The K . pneumoniae acetoin reductase gene was cloned into a clostridial/E . coli shuttle vector, and expression of the gene resulted in detectable levels of acetoin reductase activity in both E . coli and C . acetobutylicum . While acetoin, the natural substrate of acetoin reductase, is a typical product of fermentation by C . acetobutylicum, 2,3-butanediol is not . Analysis of culture supernatants by gas chromatography revealed that introduction of the K . pneumoniae acetoin reductase gene into C . acetobutylicum was not sufficient for 2,3-butanediol production even though the cultures were producing acetoin . 2,3-Butanediol was produced by cultures of C . acetobutylicum containing the gene only when commercial acetoin was added.

Eur J Pediatr, 2001 Oct, 160(10), 623 - 8
Is there a link between infant botulism and sudden infant death? Bacteriological results obtained in central Germany; Bohnel H et al.; Despite the fact that botulism was described in Germany for the first time by Kerner in 1820, the disease is almost forgotten in this country . Only about 10-20 cases of classical botulism (intoxication) are recorded every year, including 1-2 cases of clinical infant botulism . As we assumed a high incidence of botulism to be connected with cases of sudden infant death (SID), we undertook the research work presented here . From every case of unexpected infant death up to 12 months of age, standardised specimens (blood, liver and intestine) were taken at autopsy . They were tested for the presence of botulinum neurotoxin (BoNT) and/or bacterial forms of Clostridium botulinum with subsequent BoNT neutralisation tests by the international standard mouse bioassay . Age, sex, pathological findings and season were recorded . Over a 5-year period, 75 samples including 57 SID cases were tested . Free toxin was found in nine and bacterial forms were detected in six samples . Toxin neutralisation revealed the definite presence of BoNT/BoNT producing bacteria (mainly type E), whereas another 11 toxin tests were inconclusive . According to international literature, these 15 cases are to be interpreted as infant botulism . Conclusion: the results show a remarkable incidence of infant botulism without any known previous medical history, partly hidden as sudden infant death . We propose to systematically search for botulism in connection with sudden infant death.

Curr Microbiol, 2001 Dec, 43(6), 434 - 9
Both pH and carbon flux influence the level of rubredoxin in Clostridium butyricum; Guedon E et al.; The rubredoxin expression level in Clostridium butyricum DSM 5431 grown in continuous culture was monitored using primer extension analyses of the rub gene and a specific enzymatic assay of the iron-sulfur protein . In this way, we showed that variations in rubredoxin content and in rub mRNA level were influenced by the pH of the culture and were directly dependent on the carbon flux . The maximum rubredoxin level reached 1227.3 pmol (mg of proteins)(-1) (i.e . 0.7% of the total protein content) under strictly anaerobic conditions when cells grew at pH 6.5 with an excess of glucose . In addition, primer extension analyses established that the control for all the variations observed operates at the level of gene transcription . Altogether, these results suggested a main function of rubredoxin in Clostridium butyricum independent of the protection against oxygen as has already been reported for Desulfovibrio gigas and Pyrococcus furiosus.

Curr Microbiol, 2001 Oct, 43(4), 238 - 43
Effects of acetate and butyrate during glycerol fermentation by Clostridium butyricum; Colin T et al.; The effects of acetate and butyrate during glycerol fermentation to 1,3-propanediol at pH 7.0 by Clostridium butyricum CNCM 1211 were studied . At pH 7.0, the calculated quantities of undissociated acetic and butyric acids were insufficient to inhibit bacterial growth . The initial addition of acetate or butyrate at concentrations of 2.5 to 15 gL(-1) had distinct effects on the metabolism and growth of Clostridium butyricum . Acetate increased the biomass and butyrate production, reducing the lag time and 1,3-propanediol production . In contrast, the addition of butyrate induced an increase in 1,3-propanediol production (yield: 0.75 mol/mol glycerol, versus 0.68 mol/mol in the butyrate-free culture), and reduced the biomass and butyrate production . It was calculated that reduction of butyrate production could provide sufficient NADH to increase 1,3-propanediol production . The effects of acetate and butyrate highlight the metabolic flexibility of Cl . butyricum CNCM 1211 during glycerol fermentation.

Eur J Clin Microbiol Infect Dis, 2001 Aug, 20(8), 528 - 34
Nosocomial outbreak of Clostridium difficile-associated diarrhoea due to a clindamycin-resistant enterotoxin A-negative strain; Kuijper EJ et al.; A clindamycin-resistant toxin A-negative, toxin B-positive Clostridium difficile strain caused an outbreak among 24 hospitalized patients at the Department of Surgery, the Intensive Care unit, and the Department of Internal Medicine of an 800-bed academic hospital . Nineteen patients had undergone a surgical intervention and all 24 patients received at least one dose of antibiotics prior to the development of Clostridium difficile-associated diarrhoea . Twenty-seven episodes of Clostridium difficile-associated diarrhoea in 24 patients were categorized as mild (n=19), severe (n=7), or fatal (n=1) . Relapses occurred in three patients . Nineteen of the 27 episodes required anti-Clostridium difficile treatment . Molecular typing performed by arbitrary primer polymerase chain reaction (PCR) and PCR amplification of rRNA intergenic spacer regions revealed that the outbreak strains recovered from culture were identical . The outbreak strain belonged to serogroup F and was resistant to erythromycin, clindamycin, and tetracycline, whereas susceptibility to chloramphenicol varied . No phenotypic activity of enterotoxin A was detected . A deletion of approximately 1.7 kb was found in the toxin A gene . Cytotoxin B had an unusual effect on cell culture assays that, at first, was not recognized as Clostridium difficile specific but could be neutralized with anti-Clostridium difficile B cytotoxin.

Int J Med Microbiol, 2001 Sep, 291(4), 243 - 50
Bacterial protein toxins inhibiting low-molecular-mass GTP-binding proteins; Just I et al.; The Rho GTPases, which belong to the Ras superfamily of low-molecular-mass GTP-binding proteins, are the preferred intracellular targets of bacterial protein toxins . The Rho GTPases RhoA/B/C, Rac1/2 and Cdc42 are the master regulators of the actin cytoskeleton . Clostridium difficile toxins A and B, the causative agents of the antibiotic-associated pseudomembranous colitis, are intracellularly acting cytotoxins which mono-glucosylate the Rho GTPases . Clostridium botulinum C3 toxin, which is not related to the clostridial neurotoxins, catalyses ADP-ribosylation of RhoA/B/C but not of other Rho GTPases . Glucosylation as well as ADP-ribosylation result in functional inactivation of Rho causing disassembly of the actin cytoskeleton.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1743 - 6 Epub 2001 Oct 25.
Crystallographic evidence for doxorubicin binding to the receptor-binding site in Clostridium botulinum neurotoxin B; Eswaramoorthy S et al.; The neurotoxins of Clostridium botulinum and tetanus bind to gangliosides as a first step of their toxin activity . Identifying suitable receptors that compete with gangliosides could prevent toxin binding to the neuronal cells . A possible ganglioside-binding site of the botulinum neurotoxin B (BoNT/B) has already been proposed and evidence is now presented for a drug binding to botulinum neurotoxin B from structural studies . Doxorubicin, a well known DNA intercalator, binds to the neurotoxin at the receptor-binding site proposed earlier . The structure of the BoNT/B-doxorubicin complex reveals that doxorubicin has interactions with the neurotoxin similar to those of sialyllactose . The aglycone moiety of the doxorubicin stacks with tryptophan 1261 and interacts with histidine 1240 of the binding domain . Here, the possibility is presented of designing a potential antagonist for these neurotoxins from crystallographic analysis of the neurotoxin-doxorubicin complex, which will be an excellent lead compound.

Acta Crystallogr D Biol Crystallogr, 2001 Nov, 57(Pt 11), 1715 - 7 Epub 2001 Oct 25.
Crystallization and X-ray diffraction measurements on recombinant molbindin, MopII, from Clostridium pasteurianum; Harrison JA et al.; Clostridium pasteurianum carries three genes termed mopI, II and III encoding three molbindin isoforms, one of which has been cloned, the gene product expressed in high yield and crystallized using the hanging-drop vapour-diffusion method . Well ordered monoclinic crystals in two different crystal forms, both with space group C2, were obtained in the presence and absence of Na(2)MoO(4) and Na(2)WO(4) . Ligand-bound MopII crystallized with polyethylene glycol (PEG) 400 as a precipitant, whereas apo MopII required PEG 6000 . High-resolution diffraction data were collected for ligand-bound MopII structures using synchrotron radiation to 1.8 and 1.6 A resolution for the molybdate and tungstate complexes, respectively . Data were collected on apoMopII crystals to a resolution of 1.8 A in-house.

Appl Environ Microbiol, 2001 Nov, 67(11), 5127 - 33
Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced lichenan utilization and solvent production; Lopez-Contreras AM et al.; Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed . On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium . On lichenan growth and solvent production occurred, but this polymer was only partially utilized . To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C . beijerinckii . To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene . Stable C . beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD) . The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red . In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD) . Although C . beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain.

Appl Environ Microbiol, 2001 Nov, 67(11), 5025 - 31
Glucose uptake in Clostridium beijerinckii NCIMB 8052 and the solvent-hyperproducing mutant BA101; Lee J et al.; Glucose uptake and accumulation by Clostridium beijerinckii BA101, a butanol hyperproducing mutant, were examined during various stages of growth . Glucose uptake in C . beijerinckii BA101 was repressed 20% by 2-deoxyglucose and 25% by mannose, while glucose uptake in C . beijerinckii 8052 was repressed 52 and 28% by these sugars, respectively . We confirmed the presence of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) associated with cell extracts of C . beijerinckii BA101 by glucose phosphorylation by PEP . The PTS activity associated with C . beijerinckii BA101 was 50% of that observed for C . beijerinckii 8052 . C . beijerinckii BA101 also demonstrated lower PTS activity for fructose and glucitol . Glucose phosphorylation by cell extracts derived from both C . beijerinckii BA101 and 8052 was also dependent on the presence of ATP, a finding consistent with the presence of glucokinase activity in C . beijerinckii extracts . ATP-dependent glucose phosphorylation was predominant during the solventogenic stage, when PEP-dependent glucose phosphorylation was dramatically repressed . A nearly twofold-greater ATP-dependent phosphorylation rate was observed for solventogenic stage C . beijerinckii BA101 than for solventogenic stage C . beijerinckii 8052 . These results suggest that C . beijerinckii BA101 is defective in PTS activity and that C . beijerinckii BA101 compensates for this defect with enhanced glucokinase activity, resulting in an ability to transport and utilize glucose during the solventogenic stage.

Biochem Biophys Res Commun, 2001 Nov 2, 288(3), 650 - 7
Role of C-terminal region of HA-33 component of botulinum toxin in hemagglutination; Sagane Y et al.; Using SDS-PAGE, we found that one subcomponent, hemagglutinin (HA-33), from the Clostridium botulinum progenitor toxin of type D strain 1873 and type C strain Yoichi had slightly smaller molecular sizes than those of type C and D reference strains, but other components did not . Based on N- and C-terminal sequence analyses of HA-33, a deletion of 31 amino acid residues from the C-terminus at a specific site was observed in the HA-33 proteins of both strains . The progenitor toxins from both strains showed poor hemagglutination activities, titers of 2(1) or less, which were much lower than titers from the reference strains (2(6)), and did not bind to erythrocytes . These results suggest strongly that the short C-terminal region of the HA-33 plays an essential role in the hemagglutination activity of the botulinum progenitor toxin . Additionally, a sequence motif search predicted that the C-terminal region of HA-33 has a carbohydrate-recognition subdomain .

J Biol Chem, 2001 Dec 21, 276(51), 48580 - 7 Epub 2001 Oct 22.
The location of the ligand-binding site of carbohydrate-binding modules that have evolved from a common sequence is not conserved; Czjzek M et al.; Polysaccharide-degrading enzymes are generally modular proteins that contain non-catalytic carbohydrate-binding modules (CBMs), which potentiate the activity of the catalytic module . CBMs have been grouped into sequence-based families, and three-dimensional structural data are available for half of these families . Clostridium thermocellum xylanase 11A is a modular enzyme that contains a CBM from family 6 (CBM6), for which no structural data are available . We have determined the crystal structure of this module to a resolution of 2.1 A . The protein is a beta-sandwich that contains two potential ligand-binding clefts designated cleft A and B . The CBM interacts primarily with xylan, and NMR spectroscopy coupled with site-directed mutagenesis identified cleft A, containing Trp-92, Tyr-34, and Asn-120, as the ligand-binding site . The overall fold of CBM6 is similar to proteins in CBM families 4 and 22, although surprisingly the ligand-binding site in CBM4 and CBM22 is equivalent to cleft B in CBM6 . These structural data define a superfamily of CBMs, comprising CBM4, CBM6, and CBM22, and demonstrate that, although CBMs have evolved from a relatively small number of ancestors, the structural elements involved in ligand recognition have been assembled at different locations on the ancestral scaffold.

Inorg Chem, 1996 Jan 17, 35(2), 434 - 438
Electron Paramagnetic Resonance Spectroscopy of the Iron-Molybdenum Cofactor of Clostridium pasteurianum Nitrogenase; George GN et al.; We report a computer simulation study of the electron paramagnetic resonance (EPR) spectral line shape of the iron-molybdenum cofactor of nitrogenase . The unusually broad and asymmetric line shape of the EPR spectrum can be interpreted in terms of a distribution of zero-field splitting parameters called D-strain . The best fit simulations were computed using D = 2.5 cm(-1) and E = 0.317 cm(-1) and distributions in D and E approximated by Gaussians of half-widths 0.446 cm(-1) and 0.108 cm(-1), respectively . The value of D estimated in the present work is smaller than previous estimates by others but consistent with the temperature dependence of the EPR spectrum . The large D-strain is most likely caused by an ensemble of nearly isoenergetic conformational states and should not be considered as being indicative of chemical inhomogeneity.

Pediatr Surg Int, 2001 Sep, 17(7), 515 - 20
The effects of mesenteric ischemia on ileal colonization, intestinal integrity, and bacterial translocation in newborn piglets; Meddah AT et al.; The effects of mesenteric ischemia on ileal colonization, intestinal integrity, and bacterial translocation (BT) in newborn piglets were investigated in 362-day-old Pietrain piglets . Group I, controls were not operated upon; group II underwent a sham laparotomy; and group III underwent ligation of the mesenteric vessels in the distal ileum . After 3 days, the kidneys, spleens, livers, and ileal segments were harvested for microbial and histologic analyses . Two piglets in the ischemic group died; microscopic examination showed severe histologic lesions of the ischemic area . Escherichia coli counts were increased in the ischemic segment compared to the upper loop (P < 0.05) . Ischemia favoured staphylococcal colonization, whereas in the sham group a drastic reduction of these organisms was observed (P < 0.005) . BT to the kidneys, spleen, and liver occurred normally in the control group . Ischemia significantly increased the total microflora in the spleen and liver (P < 0.05) and furthered dissemination of Clostridium perfringens in the kidneys (P < 0.05); 50% of ischemic animals had proteolytic clostridia in this organ (P < 0.05) . Moreover, the incidence of E . coli in the kidneys, spleen, and liver was significantly higher in the sham and ischemic groups than in the controls (P < 0.05) . Ileal ischemia thus induced significant histologic lesions, and surgery rather than gut microflora controls translocation.

J Hist Neurosci, 1999 Apr, 8(1), 43 - 50
On the discovery of Clostridium botulinum; Devriese PP; A description is given of a food intoxication in 1895 at Ellezelles, a village in Belgium . As a result 3 persons died within a few days and others became seriously ill . A thorough investigation by E . van Ermengem led to the discovery of Clostridium botulinum and botulinum toxin . About 75 years later a subtype of the toxin proved to be highly effective in the treatment of dystonias and is now widely used.

Int J Food Microbiol, 2001 Sep 28, 69(3), 247 - 53
Selection of primers for specific detection of Clostridium botulinum types B and E neurotoxin genes using PCR method; Alsallami AA et al.; Improved oligonucleotide primers were designed to flank 370- and 307-bp fragments of the bont genes encoding botulinum neurotoxins types B and E, respectively . Primer specificity was confirmed for reference strains of Clostridium botulinum types B and E for strains representing bacterial species common in food, and for the DNA mixtures of C . botulinum types B and E in the presence of background DNA isolated from cold smoked salmon and ham . The detection limit of template DNAs of C . botulinum types B and E from the DNA mixtures increased from 1 to 0.1 ng by raising annealing temperature from 50 degrees C to 62 degrees C.

Tidsskr Nor Laegeforen . 2001 Aug 30;121(20):2381.
{Clostridium septicum infection and cancer}; Bernardshaw SV et al.; BACKGROUND: Unusual infections may occasionally be the first sign of cancer . MATERIAL, METHODS AND RESULTS: We present a patient admitted with septicaemia caused by Clostridium septicum . After successful treatment of the infection, cancer of the transverse colon was revealed by further examination . INTERPRETATION: In cases of unusual infections, underlying malignant disease should be suspected.

Biochemistry, 2001 Oct 23, 40(42), 12524 - 32
Structural basis for the substrate specificity of the feruloyl esterase domain of the cellulosomal xylanase Z from Clostridium thermocellum; Schubot FD et al.; Feruloyl esterases function in the cleavage of ferulic acid's bonds to arabinoxylan and pectin where the ferulic acid moieties cross-link the layers of polysaccharide chains within hemicellulose . This work presents the crystal structure of FAE_XynZ, the domain of Clostridium thermocellum's cellulosomal xylanase Z that displays feruloyl esterase activity . The structure was obtained via multiple isomorphous replacement with anomalous scattering (MIRAS) using three heavy atom derivatives and refined against X-ray diffraction data of up to 1.75 A resolution . The R-value of the final model was 0.187 (R(free) = 0.21) . FAE_XynZ displays an eight-stranded alpha/beta-fold with the characteristic "catalytic triad" at the heart of the active site . To define the substrate specificity determinants of the enzyme, the crystal structures of FAE_XynZ and the inactive FAE_XynZ(S172A) mutant were determined in complexes with the feruloyl-arabinoxylans FAXX and FAX(3), respectively . In the complex crystals, the ferulic acid moieties are clearly recognizable and allowed identification of the hydrophobic binding pocket . The carbohydrate part of both substrates is not visible in either structure . The location of the putative carbohydrate binding-pocket was inferred based on the location and orientation of the adjacent ferulic acid molecule . Five of the six residues lining the pocket were found to be conserved in FAE A from Orpinomyces sp., which further supports the proposed role of these amino acids.

J Food Prot, 2001 Oct, 64(10), 1527 - 34
Heat treatment adaptations in Clostridium perfringens vegetative cells; Novak JS et al.; Vegetative cells of Clostridium perfringens enterotoxigenic strains NCTC 8679, NCTC 8238 . and H6 were grown at 37 degrees C followed by a 60-min exposure to 28 degrees C or 46 degrees C . D10-values, as a measure of thermal resistance at 60 degrees C, were significantly lower for 28 degrees C exposures as compared with cultures given 37 and 46 degrees C exposures . Following refrigeration at 4 degrees C for 24 h, D10-values for the 37 and 46 degrees C samples could not be differentiated from 28 degrees C samples . Western immunoblot analyses of lysates from heat-adapted cells also detected the increased expression of proteins reacting with antiserum directed against the molecular chaperonins from Escherichia coli; GroEL, DnaJ, and the small acid soluble protein from Bacillus subtilis, SspC . Differential scanning calorimetry (DSC) identified thermal transitions corresponding to ribosomal protein denaturations at 72.1 +/- 0.5 degrees C . Any cellular heat adaptations in the DSC profiles were lost following refrigeration for several days to simulate minimally processed food storage conditions . Further analyses of high-speed pellets from crude cell extract fractions using two-dimensional gel electrophoresis detected the differential gene expression of at least four major proteins in heat-adapted vegetative cells of C . perfringens . N-terminal amino acid analyses identified two of the proteins as glyceraldehyde 3-phosphate dehydrogenase and rubrerythrin . Both appear to have roles in this anaerobe under stressful conditions.

Bioresour Technol, 2001 Dec, 80(3), 171 - 7
Clostridium lentocellum SG6--a potential organism for fermentation of cellulose to acetic acid; Ravinder T et al.; A cellulolytic, acetic acid producing anaerobic bacterial isolate, Gram negative, rod-shaped, motile, terminal oval shaped endospore forming bacterium identified as Clostridium lentocellum SG6 based on physiological and biochemical characteristics . It produced acetic acid as a major end product from cellulose fermentation at 37 degrees C and pH 7.2 . Acetic acid production was 0.67 g/g cellulose substrate utilized in cellulose mineral salt (CMS) medium . Yeast extract (0.4%) was the best nitrogen source among the various nitrogenous nutrients tested in production medium containing 0.8% cellulose as substrate . No additional vitamins or trace elemental solution were required for acetic acid fermentation . This is the highest acetic acid fermentation yield in monoculture fermentation for direct conversion of cellulose to acetic acid.

J AOAC Int, 2001 Sep-Oct, 84(5), 1460 - 4
Detection of preformed type A botulinal toxin in hash brown potatoes by using the mouse bioasssay and a modified ELISA test; Ferreira JL et al.; A foodborne illness caused by type A toxin-producing Clostridium botulinum was investigated by using the standard mouse bioassay and a rapid invitro test for toxin detection . The patient, who consumed improperly stored hash brown potatoes that contained the preformed toxin, was diagnosed with type A botulism . C . botulinum type A toxin was detected in the hash brown potatoes as well as in the tryptone-peptone-glucose-yeast extract (TPGY) medium subcultures of this food using the mouse bioassay and an amplified ELISA technique . The mouse bioassay revealed preformed toxin at 10,000 minimum lethal dose (MLD)/g uncooked product and the amplified ELISA an equivalent 50,000 MLD/g . The cultural toxin from the uncooked product killed mice at the 10(6) dilution and a modification of the ELISA procedure was positive at the 10(3) dilution . Cooked food obtained from the consumer's waste can contained 100 MLD/g and the ELISA was also positive at the same dilution of the product . The culture of the cooked product obtained from the waste can was lethal for mice at the 10(7) dilution and positive using the modified ELISA at the 10(4) dilution . The unmodified amplified ELISA method indicated a toxin level of approximately 1 ng/mL (equivalent to 5 x 10(5) MLD/mL) in diluted culture fluid from the uncooked food and the culture of cooked food obtained from the waste can . The hash brown potatoes were negative for types B, E, and F preformed and cultural botulinal toxins using both assays.

Antimicrob Agents Chemother, 2001 Nov, 45(11), 3246 - 9
Novel tetracycline resistance gene, tet(32), in the Clostridium-related human colonic anaerobe K10 and its transmission in vitro to the rumen anaerobe Butyrivibrio fibrisolvens; Melville CM et al.; A novel tetracycline resistance gene, designated tet(32), which confers a high level of tetracycline resistance, was identified in the Clostridium-related human colonic anaerobe K10, which also carries tet(W) . tet(32) was transmissible in vitro to the rumen anaerobe Butyrivibrio fibrisolvens 2221(R) . The predicted gene product of tet(32) has 76% amino acid identity with Tet(O) . PCR amplification indicated that tet(32) is widely distributed in the ovine rumen and in porcine feces.

Antimicrob Agents Chemother, 2001 Nov, 45(11), 3113 - 21
Antibacterial activities and pharmacokinetics of E-4767 and E-5065, two new 8-chlorofluoroquinolones with a 7-azetidin ring substituent; Gargallo-Viola D et al.; E-4767 {(-)-7-{3-(R)-amino-2-(S)-methyl-1-azetidinyl}-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid} and E-5065 {(-)-7-(3-amino-1-azetidinyl)-8-chloro-1-cyclopropyl-1,4-dihydro-6-fluoro-4-oxo-3-quinolinecarboxylic acid} are two new chlorofluoroquinolones with an azetidine moiety at position 7 . Their in vitro activities were evaluated in comparison with those of ciprofloxacin, ofloxacin, fleroxacin, and tosufloxacin, while ciprofloxacin was used as a reference for in vivo studies . Against gram-positive organisms, E-4767 and E-5065 were, in general, eight- and fourfold more active than tosufloxacin, which is the most potent of the reference compounds . E-4767 and E-5065 were also more potent than the reference compounds against all species of enteric bacteria tested . The MICs of E-4767 and E-5065 at which 90% of the isolates tested were inhibited (MIC(90)s) were 0.007 to 0.5 microg/ml and 0.03 to 2 microg/ml, respectively, for gram-positive organisms and <or=0.003 to 0.06 microg/ml and 0.007 to 0.12 microg/ml, respectively, for members of the family Enterobacteriaceae except Serratia marcescens and Providencia spp . (MIC(90)s of E-4767 and E-5065 for these species were <or=0.5 microg/ml and <or=2 microg/ml, respectively) . For Pseudomonas aeruginosa both compounds had a MIC(90) of 0.5 microg/ml . E-4767 and E-5065 were 356- and 32-fold more potent than ciprofloxacin against Bacteroides spp., and their MIC(90)s for Clostridium spp . were 0.25 and 0.5 microg/ml, respectively . Both products showed a remarkable reduction of activity when the pH was below 4.8 and, in general, were less active in the presence of 5 or 10 mM Mg(2+) . The presence of horse serum or human urine (pH 7.2) decreased the activity of E-4767 and E-5065 only two- to fourfold more than the activity observed in broth . After an oral dose of 50 mg/kg of body weight, the maximum levels in serum (the maximum concentration of drug in serum was reached 30 min postadministration) of E-4767 and E-5065 were approximately threefold higher than that of ciprofloxacin . The area under the concentration-time curve from 0 to 4 h for ciprofloxacin was about two- and fourfold lower than that for E-4767 and E-5065, respectively . These two new chlorofluoroquinolones were as effective as or more effective than ciprofloxacin against all experimental infections evaluated, not only against gram-negative bacteria, such as Escherichia coli or P . aeruginosa, but also against gram-positive pathogens, such as Staphylococcus aureus or Streptococcus pneumoniae . E-4767 was the most effective compound, with a 50% effective dose (ED(50)) of <or=17 mg/kg for all strains tested except ciprofloxacin-resistant S . aureus strains . The ED(50) of E-4767 for these strains was <or=47.5 mg/kg . Against gram-positive experimental infections, the ED(50) values of E-4767 were 3- to 14-fold lower than those of E-5065 and up to 25 times lower than those of ciprofloxacin.

Poult Sci, 2001 Oct, 80(10), 1451 - 4
Efficacy of in-feed tylosin phosphate for the treatment of necrotic enteritis in broiler chickens; Brennan J et al.; The efficacy of tylosin phosphate for the treatment of necrotic enteritis (NE) was investigated in a floor pen study of 2,000 broiler chickens . A model in which Clostridium perfringens was administered in the feed on Days 14 to 16 was used to initiate an outbreak of NE . Treatments, allocated at the pen level in a randomized complete block design, consisted of five levels of tylosin phosphate (0, 50, 100, 200, or 300 ppm) administered in the feed on Days 15 to 22, following the identification of an outbreak of NE on Day 15 . Mortality due to NE was significantly reduced (P < 0.05) for medicated birds at all dose levels of tylosin phosphate compared to unmedicated birds . Mean NE lesion scores on Day 17 were significantly reduced (P < 0.05) by all levels of tylosin treatment compared to those of unmedicated birds, decreasing linearly from 2.66 at 0 ppm to 0.38 at 100 ppm and 0 at higher doses . Tylosin at all levels provided improvement in Day 29 body weight, average daily gain, feed to gain ratio, and average daily feed intake compared to unmedicated birds . The results of this study provide evidence that tylosin phosphate, when administered in feed, is effective in the treatment of clinical outbreaks of NE in broiler chickens and suggest that the optimal dose for this purpose is 100 ppm.

J Ind Microbiol Biotechnol, 2001 Jul, 27(1), 18 - 26
Fermentation of glycerol by Clostridium pasteurianum--batch and continuous culture studies; Biebl H; The fermentation of glycerol by Clostridium pasteurianum was studied with respect to product formation as influenced by the culture conditions . In the majority of batch cultures, butanol was the main fermentation product, but a varying fraction of glycerol was also converted to 1,3-propanediol, butyric and acetic acids and ethanol . More than 60 g/l glycerol was utilized, and up to 17 g/l butanol was produced . Fed-batch cultures did not offer an advantage . When molecular nitrogen was used as a nitrogen source, the fermentation time was prolonged by a factor of 1.5 . Fermentations at constant pH values between 4.5 and 7.5 did not reveal significant differences in product formation except for an increase in the ethanol content starting at pH 6.5 . Chemostat cultures also yielded predominantly n-butanol, but in some fermentations, the 1,3-propanediol fraction was relatively high . The pH auxostat cultures, which were operated at a glycerol excess, contained 1,3-propanediol as the main product . As a whole, the fermentations were characterized by a certain variability in product formation under seemingly equal or slightly varied conditions . It appears that the regulation of the numerous fermentation pathways occurring in this organism is not very strict.

Infect Immun, 2001 Nov, 69(11), 7194 - 6
Naturally occurring Clostridium perfringens nontoxic alpha-toxin variant as a potential vaccine candidate against alpha-toxin-associated diseases; Schoepe H et al.; Clostridium perfringens mutant strain 121A/91 shows neither enzymatic (phospholipase C) nor hemolytic activity . Nevertheless, the cpa gene and the corresponding alpha-toxin variant are detectable . Vaccination with this genetically constructed alpha-toxin variant, rAT121/91, induces antibodies capable of significantly reducing activities induced by wild-type toxin . Thus, rAT121/91 could be a useful vaccine candidate.

Biochim Biophys Acta, 2001 Nov 1, 1515(1), 38 - 43
Clostridium perfringens type A enterotoxin forms mepacrine-sensitive pores in pure phospholipid bilayers in the absence of putative receptor proteins; Hardy SP et al.; Clostridium perfringens enterotoxin (CPE) is an important cause of food poisoning with no significant homology to other enterotoxins and its mechanism of action remains uncertain . Although CPE has recently been shown to complex with tight junction proteins, we have previously demonstrated that CPE increases ionic permeability in single Caco-2 cells using the whole-cell patch-clamp technique, thereby excluding any paracellular permeability . In this paper we demonstrate that CPE forms pores in synthetic phospholipid membranes in the absence of receptor proteins . The properties of the pores are consistent with CPE-induced permeability changes in Caco-2 cells suggesting that CPE has innate pore-forming ability.

Toxicon, 2001 Nov, 39(11), 1781 - 91
The complex interactions between Clostridium perfringens enterotoxin and epithelial tight junctions; McClane BA; Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal symptoms of C . perfringens type A food poisoning and antibiotic-associated diarrhea . The CPE protein consists of a single 35 kDa polypeptide with a C-terminal receptor-binding region and an N-terminal toxicity domain . Under appropriate conditions, CPE can interact with structural components of the epithelial tight junctions, including certain claudins and occludin . Those interactions can affect tight junction structure and function, thereby altering paracellular permeability and (possibly) contributing to CPE-induced diarrhea . However, the tight junction effects of CPE require cellular damage as a prerequisite . CPE induces cellular damage via its cytotoxic activity, which results from plasma membrane permeability alterations caused by formation of a approximately 155 kDa CPE-containing complex that may correspond to a pore . Thus, CPE appears to be a bifunctional toxin that first induces plasma membrane permeability alterations; using the resultant cell damage, CPE then gains access to tight junction proteins and affects tight junction structure and function.

Toxicon, 2001 Nov, 39(11), 1769 - 80
Clostridial hydrolytic enzymes degrading extracellular components; Matsushita O et al.; Bacteria belonging to the genus Clostridium, both glycolytic and proteolytic, and both pathogenic and non-pathogenic, produce a battery of hydrolytic enzymes to obtain nutrients from various biopolymers . The clostridial hydrolytic enzymes are diverse, and are used or are potentially useful for fundamental and applied research purposes . Among them, enzymes degrading the major components in the extracellular matrix or on the cell surface in vertebrates are herein reviewed with special emphasis on recent knowledge gained through molecular biology of clostridial collagenases, sialidases and hyaluronidases . This paper also reviews some literature on the biotechnological approach to the designing of new molecular tools and drug delivery systems involving clostridial hydrolytic enzymes.

Toxicon, 2001 Nov, 39(11), 1703 - 22
Clostridium botulinum and its neurotoxins: a metabolic and cellular perspective; Johnson EA et al.; Clostridium botulinum comprises a diverse assemblage of clostridia that have the common property of producing a distinctive protein neurotoxin (BoNT) of similar pharmacological activity and extraordinary potency . BoNTs are produced in culture as molecular complexes consisting of BoNT, hemagglutinin (HA) and associated subcomponent proteins, nontoxic nonhemagglutinin (NTNH), and RNA . The genes encoding the protein components reside as a cluster on the chromosome, on bacteriophages, or on plasmids depending on the C . botulinum serotype . A gene BotR coding for a regulatory protein has been detected in toxin gene clusters from certain strains, as well as ORFs coding for uncharacterized components . The gene encoding TeNT is located on a large plasmid, and expression of the structural gene is controlled by the regulatory gene, TetR, located immediately upstream of the TeNT structural gene . TeNT is not known to be assembled into a protein/nucleic acid complex in culture . Cellular synthesis of BoNT and TeNT have been demonstrated to be positively regulated by the homologous proteins, BotR/A and TetR . Evidence suggests that negative regulatory factors and general control cascades such as those involved in nitrogen regulation and carbon catabolite repression also regulate synthesis of BoNTs . Neurotoxigenic clostridia have attracted considerable attention from scientists and clinicians during the past decade, and many excellent reviews are available on various aspects of these organisms and their neurotoxins . However, certain areas have not been well-studied, including metabolic regulation of toxin formation and genetic tools to study neurotoxigenic clostridia . These topics are the focus of this review.

Toxicon, 2001 Nov, 39(11), 1681 - 9
The family of thiol-activated, cholesterol-binding cytolysins; Palmer M; Several species of both pathogenic and non-pathogenic grampositive bacteria within the genera Streptococcus, Clostridium and Bacillus secrete cytolytic proteins that belong to a single, highly homologous family . The most widely known members of this family are streptolysin O, listeriolysin, perfringolysin, and pneumolysin . These toxins specifically require membrane cholesterol but, apparently, do not depend on any other specific cell surface receptor, so that they are able to lyse the cytoplasmic membranes of virtually any animal cell . Upon binding as monomers, they oligomerize to form large pores with up to 30 nm internal diameter . These are the largest pores known, permitting permeation not only of ions and small metabolites but also of macromolecules . The latter property renders these toxins useful tools in cell biology.While several of these cytolysins have been shown to be determinants of bacterial pathogenicity, their biological roles may vary, as do the lifestyles of the bacteria secreting them . A unique function is surely fulfilled by listeriolysin O, which helps the intracellular pathogen Listeria monocytogenes escape from phagolysosomes and then spread to adjacent host cells.

Toxicon, 2001 Nov, 39(11), 1661 - 72
Mode of action of beta-barrel pore-forming toxins of the staphylococcal alpha-hemolysin family; Menestrina G et al.; Staphylococcal alpha-hemolysin is the prototype of a family of bacterial exotoxins with membrane-damaging function, which share sequence and structure homology . These toxins are secreted in a soluble form which finally converts into a transmembrane pore by assembling an oligomeric beta-barrel, with hydrophobic residues facing the lipids and hydrophilic residues facing the lumen of the channel . Besides alpha-hemolysin the family includes other single chain toxins forming homo-oligomers, e.g . beta-toxin of Clostridium perfringens, hemolysin II and cytotoxin K of Bacillus cereus, but also the staphylococcal bi-component toxins, like gamma-hemolysins and leucocidins, which are only active as the combination of two similar proteins which form hetero-oligomers . The molecular basis of membrane insertion has become clearer after the determination of the crystal structure of both the oligomeric pore and the soluble monomer . Studies on this family of beta-barrel pore-forming toxins are important for many aspects: (i) they are involved in serious pathologies of humans and farmed animals, (ii) they are a good model system to investigate protein-membrane interaction and (iii) they are the basic elements for the construction of nanopores with biotechnological applications in various fields.

Surg Endosc, 2001 Jul, 15(7), 653 - 9 Epub 2001 May 07.
Decompressive colonoscopy with intracolonic vancomycin administration for the treatment of severe pseudomembranous colitis; Shetler K et al.; BACKGROUND: We explored the potential of early decompressive colonoscopy with intracolonic vancomycin administration as an adjunctive therapy for severe pseudomembranous Clostridium difficile colitis with ileus and toxic megacolon . METHODS: We reviewed the symptoms, signs, laboratory tests, radiographic findings, and outcomes from the medical records of seven patients who experienced eight episodes of severe pseudomembranous colitis with ileus and toxic megacolon . All seven patients underwent decompressive colonoscopy with intracolonic perfusion of vancomycin . RESULTS: Fever, abdominal pain, diarrhea, abdominal distention, and tenderness were present in all patients . Five of seven patients were comatose, obtunded, or confused, and six of the seven required ventilatory support . The white blood cell count was greater than 16,000 in seven cases (six patients) . Colonoscopy showed left-side pseudomembranous colitis in one patient, right-side colitis in one patient, and diffuse pseudomembranous pancolitis in five patients . Two patients were discharged with improvement . Five patients had numerous medical problems leading to their death . Complete resolution of pseudomembranous colitis occurred in four patients . One patient had a partial response, and two patients failed therapy . CONCLUSION: Colonoscopic decompression and intracolonic vancomycin administration in the management of severe, acute, pseudomembranous colitis associated with ileus and toxic megacolon is feasible, safe, and effective in approximately 57% to 71% of cases.

J Biol Chem, 2001 Dec 14, 276(50), 46849 - 55 Epub 2001 Oct 08.
Modulation of COX-2 expression by statins in human aortic smooth muscle cells . Involvement of geranylgeranylated proteins; Degraeve F et al.; Cyclooxygenase (COX)-2 and COX-1 play an important role in prostacyclin production in vessels and participate in maintaining vascular homeostasis . Statins are inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase, which is crucial in cholesterol biosynthesis . Recently, cholesterol-independent effects of statins have been described . In this study, we evaluated the effect of two inhibitors of HMG CoA reductase, mevastatin and lovastatin, on the production of prostacyclin and the expression of COX in human aortic smooth muscle cells . Treatment of cells with 25 microm mevastatin or lovastatin resulted in the induction of COX-2 and increase in prostacyclin production . Mevalonate, the direct metabolite of HMG CoA reductase, and geranylgeranyl-pyrophosphate reversed this effect . GGTI-286, a selective inhibitor of geranylgeranyltransferases, increased COX-2 expression and prostacyclin formation, thus indicating the involvement of geranylgeranylated proteins in the down-regulation of COX-2 . Furthermore, Clostridium difficile toxin B, an inhibitor of the Rho GTP-binding protein family, the Rho selective inhibitor C3 transferase, and Y-27632, a selective inhibitor of the Rho-associated kinases, targets of Rho A, increased COX-2 expression whereas the activator of the Rho GTPase, the cytotoxic necrotizing factor 1, blocked interlukin-1alpha-dependent COX-2 induction . These results demonstrate that statins up-regulate COX-2 expression and subsequent prostacyclin formation in human aortic smooth muscle cells in part through inhibition of Rho.

Clin Microbiol Infect, 2001 Aug, 7(8), 453 - 7
Extra-intestinal infections caused by Clostridium difficile; Garcia-Lechuz JM et al.; The objective of this paper was to investigate the incidence of extra-intestinal infections caused by Clostridium difficile . During a 10-year period, the microbiology laboratory of our institution isolated 2034 isolates of C . difficile . Of the 2034 isolates, 21 (1.08%) were obtained from extra-intestinal sources . This represents an incidence of extra-intestinal isolation of four cases per 100 000 admissions . We were able to review the records of 17 patients for our study . The isolates in 12 patients were obtained from structures or fluids anatomically close to the colon and included the following infections: peritonitis in five cases (three primary and two secondary), intra-abdominal abscesses in three patients and abdominal wound infections in four cases . The infections in the other five patients were not in the anatomic vicinity of the colon . They included one case with a brain abscess, two episodes of bacteremia and two cases of foot infections (one chronic osteomyelitis) . In all but one case, C . difficile isolation was obtained as part of a polymicrobial flora . The isolates were frequently non-toxigenic and the extra-intestinal infections occurred without concomitant diarrhea or prior anti-microbial therapy . Out of the 17 patients, eight died and nine survived . Death could not be directly attributed to C . difficile in any of the cases . The isolation of C . difficile outside the intestinal tract is very uncommon . Its clinical significance should be interpreted with caution.

Clin Microbiol Infect, 2001 Aug, 7(8), 447 - 50
Clostridium difficile cytotoxin B in adults with diarrhea: a comparison of patients treated or not treated with antibiotics prior to infection; Svenungsson B et al.; OBJECTIVE: To study the detection rate of Clostridium difficile cytotoxin B in stool specimens from adults with diarrhea as related to previous antimicrobial treatment . METHODS: Stool specimens from 802 adult patients with diarrhea and 203 healthy controls were tested for C . difficile cytotoxin B using a cell cytotoxicity assay . Antibiotic susceptibility testing of C . difficile was performed with the E test . RESULTS: Of 173 patients treated with antimicrobial medication within 5 weeks of onset of diarrhea, 60 (35%) were positive for C . difficile cytotoxin B (group A) compared to only 41 (7%) of 629 untreated patients (group B) and two of the 203 (1%) healthy controls . Compared to patients in group A, patients in group B possessed characteristics not usually connected with C . difficile disease . They were generally younger (median age 40 years vs . 73 years), had been hospitalized less frequently (10% vs . 67%), had more often travelled abroad within the previous 2 weeks (46% vs . 1%), and more often had multiple enteropathogens (41% vs . 3%) . Minimal inhibitory concentrations for vancomycin, metronidazole and fucidic acid to C . difficile isolates ranged from 0.5 to 4 mg/L, from 0.125 to 256 mg/L and 0.25 to 4 mg/L, respectively . CONCLUSIONS: The detection rate of C . difficile cytotoxin B in patients with diarrhea, not associated with antibiotic treatment, is comparable to that in healthy control subjects . It probably merely reflects a carrier state without clinical significance.

Clin Microbiol Infect, 2001 Aug, 7(8), 432 - 7
Mathematical modeling of Clostridium difficile infection; Starr JM et al.; Clostridium difficile diarrhea is a major cause of morbidity and mortality in hospitals . However, the number of cases in an outbreak is usually relatively small . This precludes many traditional statistical methods of modeling epidemics . Stochastic models are designed to deal with small numbers and are promising methods of understanding C . difficile epidemiology . This is illustrated by a reversible jump Markov chain Monte Carlo model based on the herd immunity hypothesis of C . difficile outbreaks.

Clin Microbiol Infect, 2001 Aug, 7(8), 428 - 31
Typing of Clostridium difficile; Brazier JS; Clostridium difficile is primarily recognised as a nosocomially acquired pathogen manifesting in gastrointestinal disease subsequent to the patient receiving broad-spectrum antibiotics . Infection can be sporadic, but outbreaks commonly occur within a ward or hospital as a result of cross-infection . Since the 1980s, the epidemiology of C . difficile disease has been studied by the application of many different typing or fingerprinting methods; these, and the lessons learned, are reviewed herein.

Clin Microbiol Infect, 2001 Aug, 7(8), 421 - 7
The pathogenicity of Clostridium difficile; Poxton IR et al.; It is now well established that the major virulence factors of C . difficile are the two toxins A and B . However, the organism possesses an array of other putative virulence factors that may be important for localisation within the colon, and in evasion of the immune system . It has been observed that certain types of C . difficile are more commonly found causing disease than others, and this seems to be independent of toxin production . Is this simply a reflection of their abundance in the hospital environment, or is it due to their virulence determinants? This review covers our current knowledge of the modes of action of toxins A and B at the cellular and molecular level . Many unanswered questions are posed that require answers before we can fully understand the pathogenic mechanisms of the organism and be in a position to manage better the spectrum of diseases it causes.

Clin Microbiol Infect, 2001 Aug, 7(8), 417 - 20
How to detect Clostridium difficile variant strains in a routine laboratory; Rupnik M; Toxin A-negative, toxin B-positive strains (A-/B+) are the best studied examples of Clostridium difficile variant strains . In addition, there are some other groups of variant C . difficile strains that produce both toxins (A+/B+) or are non-cytotoxic (A-/B-) but differ from the reference strain VPI 10463 in their toxin genes . Here we describe two simple methods (amplification of the tcdA gene and amplification of the binary toxin gene cdtA) which can be used in rapid screening for variant C . difficile strains in collections or in routine laboratories.

Clin Microbiol Infect, 2001 Aug, 7(8), 405 - 10
Epidemiology of Clostridium difficile-associated infections; Barbut F et al.; Clostridium difficile is responsible for 15-25% of cases of antibiotic-associated diarrhea (AAD) and for virtually all cases of antibiotic-associated pseudomembranous colitis (PMC) . This anaerobic bacterium has been identified as the leading cause of nosocomial infectious diarrhea in adults and can be responsible for large outbreaks . Nosocomial C . difficile infection results in an increased length of stay in hospital ranging from 8 to 21 days . Risk factors for C . difficile-associated diarrhea include antimicrobial therapy, older age (>65 years), antineoplastic chemotherapy and length of hospital stay . Other interventions with high risk associations are enemas, nasogastric tubes, gastrointestinal surgery and antiperistaltic drugs . Prospective studies have shown that nosocomial transmission of C . difficile is frequent but often remains asymptomatic . Patients can be contaminated from environmental surfaces, shared instrumentation, hospital personnel hands and infected roommates . Once an outbreak starts, C . difficile may be spread rapidly throughout the hospital environment where spores may persist for months . Measures that are effective in reducing incidence of C . difficile infections and cross-infection include: (i) an accurate and rapid diagnosis, (ii) appropriate treatment, (iii) implementation of enteric precautions for symptomatic patients, (iv) reinforcement of hand-washing, (v) daily environmental disinfection, and (vi) a restrictive antibiotic policy . C . difficile is a common cause of infectious diarrhea and should be therefore systematically investigated in patients with nosocomial diarrhea.

Eur J Biochem, 2001 Oct, 268(19), 5081 - 91
Molecular characterization of a phosphatidylcholine-hydrolyzing phospholipase C; Preuss I et al.; While searching for a phospholipase C (PLC) specific for phosphatidylcholine in mammalian tissues, we came across such an activity originating from a contamination of Pseudomonas fluorescens . This psychrophilic bacterium was found to contaminate placental extracts upon processing in the cold . The secreted phosphatidylcholine-hydrolyzing PLC was purified by a combination of chromatographic procedures . As substrates, the enzyme preferred dipalmitoyl-phosphatidylcholine and 1-palmitoyl-2-arachidonoyl-phosphatidylcholine over phosphatidylinositol . The active enzyme is a monomer of approximately 40 kDa . As for other bacterial PLCs, the enzyme requires Ca2+ and Zn2+ for activity; dithiothreitol affected the activity due to its chelation of Zn2+, but this inhibition could be compensated for by addition of ZnCl2 . The compound D609, described to selectively inhibit phosphatidylcholine-specific PLCs, caused half-inhibition of the P . fluorescens enzyme at approximately 420 microM, while 50-fold lower concentrations similarly affected PLCs from Bacillus cereus and Clostridium perfringens . Partial peptide sequences obtained from the pure P . fluorescens enzyme after tryptic cleavage were used to clone a DNA fragment of 3.5 kb from a P . fluorescens gene library prepared from our laboratory isolate . It contains an ORF of 1155 nucleotides encoding the PLC . There is no significant sequence homology to other PLCs, suggesting that the P . fluorescens enzyme represents a distinct subclass of bacterial PLCs . The protein lacks cysteine residues and consequently contains no disulfide bonds . Interestingly, P . fluorescens reference strain DSMZ 50090 is devoid of the PLC activity described here as well as of the relevant coding sequence.

Glycobiology, 2001 Oct, 11(10), 831 - 41
The major gangliosides of human peripheral blood monocytes/macrophages: absence of ganglio series structures; Yohe HC et al.; Sialoglycosphingolipids (gangliosides) are membrane components of eukaryotic cells that modulate cell signal transduction events . Discrepancies exist in the published descriptions of the gangliosides present in the human peripheral monocyte/macrophage . Macrophages were isolated from healthy human volunteers by two different methods . Their ganglioside fractions were isolated and examined by 2D thin-layer mobility, enzymatic susceptibility, and mass spectral-collision induced dissociation-mass spectral analyses . Thin-layer ganglioside chromatographic patterns displayed four major doublets and were similar for monocytes/macrophages isolated by either apheresis/elutriation or density gradient centrifugation . All gangliosides were resistant to beta-galactosidase but sensitive to Clostridium perfringens sialidase, indicating the absence of terminal galactose residues and sialidase-resistant sialic acid moieties . Mass spectra indicated only three major sets of glycolipid components with mass heterogeneity in the ceramide portion of each set . In all the gangliosides, the ceramide moiety contained only C18 sphingosine with the heterogeneity produced by the presence of C16 or C24 fatty acid . One doublet was resistant to Newcastle disease virus sialidase, indicating the presence of an alpha(2-6)-linked sialic acid residue with the same mass as another doublet . All data was consistent with the following structures as the major gangliosides of human peripheral monocyte/macrophages: II(3)NeuAcLacCer (sialolactosyl ceramide, GM3), IV(3)- and IV(6)NeuAcnLcOse(4)Cer (sialoparagloboside, nLM1), and IV(3)NeuAcnLcOse(6)Cer (a sialohexosylceramide).

Gastroenterol Clin North Am, 2001 Sep, 30(3), 753 - 77, ix-x
Clostridium difficile; Kyne L et al.; Clostridium difficile is a major cause of antibiotic-associated diarrhea and colitis . The incidence of infection with this organism is increasing in hospitals worldwide, consequent to the widespread use of broad-spectrum antibiotics . Pathogenic strains of C . difficile produce two protein exotoxins, toxin A and toxin B, that cause colonic mucosal injury and inflammation . Many patients who are colonized are asymptomatic, and recent evidence indicates that diarrhea and colitis occur in those individuals who lack a protective antitoxin immune response . In patients who do develop symptoms, the spectrum of C . difficile disease ranges from mild diarrhea to fulminant pseudomembranous colitis . Prevention of nosocomial C . difficile infection involves judicious use of antibiotics and multidisciplinary infection control measures to reduce environmental contamination and patient cross-infection . Ultimately, active or passive immunization against C . difficile may be an effective means of controlling the growing problem of nosocomial C . difficile diarrhea and colitis.

Vasc Surg, 2001 Jul-Aug, 35(4), 303 - 10
Clostridial mycotic aneurysm of the thoracoabdominal aorta--a case report; Morrison RC Jr et al.; Clostridial infection of the aorta is a rare and life-threatening condition . The management of a mycotic aneurysm involving the thoracoabdominal aorta due to Clostridium septicum infection is presented . Successful surgical management of the aortic infection involved arterial resection, wide debridement of the surrounding tissues, and in situ graft replacement . Sixteen additional cases of clostridial infection of the aortoiliac segment reported in the literature are also summarized . In ten of these 17 cases, an associated colonic adenocarcinoma was documented.

Vet Res Commun, 2001 Oct, 25(7), 555 - 63
Clostridium infection (jisizheng) in yaks in Qinghai, China; Changqing Q et al.; Since the mid-1980s, outbreaks of a disease characterized by a sudden onset, acute deaths and extensive haemorrhages in the viscera and digestive tract of yaks have been prevalent in Qilian, Qinghai, China . The disease is known as jisiheng by local people . Virulent Clostridium perfringens type A and Clostridium haemolytica were isolated from yaks that had died of jisizheng . In 1996 and 1997, yaks were immunized with a polyvalent inactivated vaccine against C . perfringens and with an inactivated vaccine against C . haemolyticum, and this prevented the occurrence of jisizheng.

J Antimicrob Chemother, 2001 Oct, 48(4), 463 - 78
The problem with cephalosporins; Dancer SJ; The cephalosporin antibiotics have become a major part of the antibiotic formulary for hospitals in affluent countries . They are prescribed for a wide variety of infections every day . Their undoubted popularity relies upon lesser allergenic and toxicity risks as well as a broad spectrum of activity . It is the latter feature, however, that encourages the selection of microorganisms that are resistant to these agents . There are long-term implications for the treatment and control of this heterogeneous group of superinfections . When clinicians evaluate a septic patient, it is understandable that they choose empirical therapy with a cephalosporin whilst awaiting microbiology and other tests, since bacterial identification and antimicrobial testing still usually require 24-48 h . The broad-spectrum capability of these drugs, however, encourages rapid overgrowth of some microorganisms that are neither eliminated nor inhibited by therapy . These organisms not only have pathogenic potential, they may also be multiply resistant to antibiotics . This review discusses the evidence that cephalosporin usage is the most important factor in the selection and propagation of microorganisms such as Clostridium difficile, methicillin-resistant Staphylococcus aureus, penicillin-resistant pneumococci, multiply resistant coliforms and vancomycin-resistant enterococci, the continuing increase of which threatens the future of antimicrobial therapy.

Biologicals, 2001 Jun, 29(2), 59 - 66
Validation of the sterilization procedure of allogeneic avital bone transplants using peracetic acid-ethanol; Pruss A et al.; Different procedures are available to inactivate bacteria and fungi, including their spores, as well as viruses in human bone transplants . The most efficient methods are considered to be gamma irradiation and thermal inactivation as well as chemical sterilization methods like the peracetic acid-ethanol treatment (PES) . Following national and international standards or draft standards, the antimicrobial effectiveness of this procedure was evaluated . Due to the standardizable size as well as the clinical relevance, defatted human spongiosa cuboids (15x15x15 mm) served as model system . After treatment with PES for 2 and 4 hours, respectively, the titre of living micro-organisms was determined in the supernatant and the cuboid . A reduction in the titre of viable micro-organisms below the detection level (reduction factor >5 log10) was already achieved after an incubation time of 2 hours (Staphylococcus aureus, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis, Clostridium sporogenes, Mycobacterium terrae, Candida albicans as well as spores of Bacillus subtilis) . No viable micro-organisms could be detected in any of the PES-treated test cuboids . Spores of Aspergillus niger were also completely inactivated . The PES procedure proved to be a reliable method for the sterilization of human bone transplants derived from spongiosa .

Int J Gynecol Cancer, 1993 Jul, 3(4), 231 - 238
Radical hysterectomy and pelvic lymphadenectomy for early invasive cancer of the cervix - 14-year experience; Sivanesaratnam V et al.; During a 14-year period, 397 radical hysterectomies and pelvic lymphadenectomies were performed for early invasive carcinoma of the cervix . Twenty-one patients were in stage IA2 with lymphatic/vascular channel permeation (5.2%), 340 in stage IB (85.6%) and 34 in early stage 2A disease (8.5%) . Eighteen patients (4.5%) were pregnant . Adenocarcinoma comprised 26.9% of cases . The mean operative time was 4.14 h; the intraoperative blood loss was less than 1.51 in 77.3% patients . There was no operative mortality; one patient died 3 weeks after surgery from clostridium difficile enterocilitis . Eleven patients (2.7%) developed venous thrombosis; severe lymphedema occurred in four (1%) . The incidence of uretero-vaginal fistula was 0.2% and that of vesico-vaginal fistula 0.5% . Ovarian metastases were noted in 4.3% of cases with adenocarcinoma . Sixty-six patients had positive nodes (16.6%) . Five-year survival in patients with more than 2 positive nodes was 68% . The use of adjuvant chemotherapy in patients with 'high risk' factors resulted in survival rates approaching those without risk factors . Neo-adjuvant chemotherapy was used in 10 patients with large bulky tumors; the results were favorable . Recurrences occurred in 47 patients (11.8%); 36 patients have died (9.1%) . Age did not appear to influence survival . The overall 5-year survival was 92.2%.

J Gastroenterol, 2001 Sep, 36(9), 629 - 32
Pseudomembranous colitis without diarrhea presenting clinically as acute intestinal pseudo-obstruction; Sheikh RA et al.; Pseudomembranous colitis usually presents with diarrhea in a clinical setting of recent antibiotic use . It is uncommon to see it as a cause of obstipation and colonic pseudo-obstruction . We report an unusual case of an elderly woman with hypertension, congestive heart failure, chronic obstructive pulmonary disease, chronic renal insufficiency, and diabetes mellitus, who was admitted with fever, abdominal pain, and distension without diarrhea . She presented with decreased stool frequency and obstipation . She did not respond to conservative management . Colonoscopy revealed a picture of pseudomembranous colitis, and Clostridium difficile toxin was positive . She responded well to metronidazole therapy.

Neurotoxicology, 2001 Aug, 22(4), 447 - 53
Clostridium botulinum neurotoxins act with a wide range of potencies on SH-SY5Y human neuroblastoma cells; Purkiss JR et al.; We have described, in undifferentiated SH-SY5Y human neuroblastoma cells, the relative potency of Clostridium botulinum neurotoxin (BoNT) serotypes A-F Sensitivity of stimulated {3H}-noradrenaline ({3H}-NA) release to the toxins had a rank order of potency of: C > D > A > B > F after 3 days exposure . The difference between the most potent (BoNT/C: IC50 0.54 nM) and the least (BoNT/F: IC50 > 300 nM) was approximately 1,000-fold . Though fluid phase endocytosis may have been the mechanism of entry for low potency toxins the far higher potency of BoNT/C would suggest receptor-driven entry . Potency was not a reflection of the dependence of the release mechanism on a particular SNARE since the substrate specificities were mixed throughout the potency order . This indicated that the toxins differed in their efficiency of binding/endocytosis or enzymatic activity inside the cell . The serotypes that cleaved vesicle-associated membrane protein (VAMP) isoforms (BoNT/B, D and F) did not fully inhibit {3H}-NA release . Cleavage of the appropriate substrate proteins was observed for all serotypes . SNAP-25 cleavage by BoNT/A was shown to be a dose-dependent and correlated closely with reduction of release, supporting proteolysis as the mechanism by which toxin inhibited secretion . Comparison of the SH-SY5Y cell line sensitivity to BoNT/A with glycine releasing rat primary spinal cord neuron cultures, revealed a massive difference in potency; the primary cultures being approximately 200,000-fold more sensitive . The demonstration, using BoNTs, of the crucial role of SNAP-25, VAMP and syntaxin in SH-SY5Y cells suggests the use of this neuroblastoma as a model in the study of these proteins in neurotransmitter release.

Microbiology, 2001 Oct, 147(Pt 10), 2717 - 28
Genomic analysis of the erythromycin resistance element Tn5398 from Clostridium difficile; Farrow KA et al.; Clostridium difficile is a nosocomial pathogen that causes a range of chronic intestinal diseases, usually as a result of antimicrobial therapy . Macrolide-lincosamide-streptogramin B (MLS) resistance in C . difficile is encoded by the Erm B resistance determinant, which is thought to be located on a conjugative transposon, Tn5398 . The 9630 bp Tn5398 element has been cloned and completely sequenced and its insertion site determined . Analysis of the resultant data reveals that Tn5398 is not a classical conjugative transposon but appears to be a mobilizable non-conjugative element . It does not carry any transposase or site-specific recombinase genes, nor any genes likely to be involved in conjugation . Furthermore, using PCR analysis it has been shown that isolates of C . difficile obtained from different geographical locations exhibit heterogeneity in the genetic arrangement of both Tn5398 and their Erm B determinants . These results indicate that genetic exchange and recombination between these determinants occurs in the clinical and natural environment.

Microbiology, 2001 Oct, 147(Pt 10), 2679 - 88
Functional assembly of two membrane-binding domains in listeriolysin O, the cytolysin of Listeria monocytogenes; Dubail I et al.; Listeriolysin O (LLO) is a major virulence factor secreted by the pathogenic Listeria monocytogenes and acts as pore-forming cytolysin . Based on sequence similarities between LLO and perfringolysin (PFO), the cytolysin from Clostridium perfringens of known crystallographic structure, two truncated LLO proteins were produced: LLO-d123, comprising the first three predicted domains, and LLO-d4, the last C-terminal domain . The two proteins were efficiently secreted into the culture supernatant of L . monocytogenes and were able to bind to cell membranes . Strikingly, when expressed simultaneously, the two secreted domains LLO-d123 and LLO-d4 reassembled into a haemolytically active form . Two in-frame linker insertions were generated in the hinge region between the d123 and d4 domains . In both cases, the insertion created a major cleavage site for proteolytic degradation and abolished cytolytic activity, which might suggest that the region connecting d123 and d4 participates in the interaction between the two portions of the monomer.

J Biol Chem, 2001 Nov 30, 276(48), 44744 - 50 Epub 2001 Sep 27.
Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-ornithine aminomutase from Clostridium sticklandii; Chen HP et al.; D-Ornithine aminomutase from Clostridium sticklandii catalyzes the reversible rearrangement of d-ornithine to (2R,4S)-2,4-diaminopentanoic acid . The two genes encoding d-ornithine aminomutase have been cloned, sequenced, and expressed in Escherichia coli . The oraS gene, which encodes a protein of 121 amino acid residues with M(r) 12,800, is situated upstream of the oraE gene, which encodes a protein of 753 amino acid residues with M(r) 82,900 . The holoenzyme appears to comprise a alpha(2)beta(2)-heterotetramer . OraS shows no significant homology to other proteins in the Swiss-Prot data base . The deduced amino acid sequence of OraE includes a conserved base-off/histidine-on cobalamin-binding motif, DXHXXG . OraE was expressed in E . coli as inclusion bodies . Refolding experiments on OraE indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate or adenosylcobalamin play important roles in refolding process . The K(m) values for d-ornithine, 5'-deoxyadenosylcobalamin (AdoCbl), and pyridoxal 5'-phosphate (PLP) are 44.5 +/- 2.8, 0.43 +/- 0.04, and 1.5 +/- 0.1 microm, respectively; the k(cat) is 6.3 +/- 0.1 s(-1) . The reaction was absolutely dependent upon OraE, OraS, AdoCbl, PLP, and D-ornithine being present in the assay; no other cofactors were required . A red-shift in UV-visible absorption spectrum is observed when free adenosylcobinamide is bound by recombinant D-ornithine aminomutase and no significant change in spectrum when free adenosylcobinamide is bound by mutant OraE-H618G, demonstrating that the enzyme binds adenosylcobalamin in base-off/histidine-on mode.

J Appl Microbiol, 2001 Oct, 91(4), 677 - 85
Subcellular distribution of glycanases and related components in Ruminococcus albus SY3 and their role in cell adhesion to cellulose; Miron J et al.; AIMS: To compare the subcellular distribution of glycanase-related components between wild-type Ruminococcus albus SY3 and an adhesion-defective mutant, to identify their possible contribution to the adhesion process, and to determine their association with cellulosome-like complexes . METHODS AND RESULTS: Cell fractionation revealed that most of the cellulases and xylanases were associated with capsular and cell-wall fractions . SDS-PAGE and gel filtration indicated that most of the bacterial enzyme activity was not integrated into cellulosome-like complexes . The adhesion-defective mutant produced significantly less (5- to 10-fold) overall glycanase activity, and the 'true cellulase activity' appeared to be entirely confined to the cell membrane fractions . Antibodies specific for the cellulosomal scaffoldin of Clostridium thermocellum recognized a single 240 kDa band in R . albus SY3 . CONCLUSIONS: The adhesion-defective mutant appeared to be blocked in exocellular transport of enzymes involved in true cellulase activity . A potential cellulosomal scaffoldin candidate was identified in R . albus SY3 . SIGNIFICANCE AND IMPACT OF THE STUDY: Several glycanase-related proteins and more than one mechanism appear to be involved in the adhesion of R . albus SY3 to cellulose.

Can J Gastroenterol, 2001 Sep, 15(9), 586 - 90
Colonic disorders in adult cystic fibrosis; Chaun H; By 1996, the median survival of patients with cystic fibrosis (CF) in North America had increased to 31 years . With the markedly improved life expectancy, many CF patients are now adults . There is an associated increased risk of certain colonic disorders, and the emergence of other previously unrecognized disorders, in adult CF patients . The distal intestinal obstruction syndrome (DIOS), which is more common in older patients, is a frequent cause of abdominal pain . Intussusception may complicate DIOS; other differential diagnoses include appendiceal disease, volvolus, Crohn's disease, fibrosing colonopathy and colonic carcinoma . The diagnosis of acute appendicitis, although uncommon in patients with CF, is often delayed, and appendiceal abscess is a frequent complication . The prevalence of Crohn's disease in CF has been shown to be 17 times that of the general population . Right-sided microscopic colitis is a recently recognized entity in CF of uncertain clinical significance . Fibrosing colonopathy has been confined mostly to children with CF, attributed to the use of high strength pancreatic enzyme supplements, but it has been reported in three adults . Nine cases of carcinoma of the large intestine have been reported worldwide, associated with an apparent excess risk of digestive tract cancers in CF . Despite high carrier rates of Clostridium difficile in patients with CF, pseudomembranous colitis is distinctly rare, but severe cases complicated by toxic megacolon have been reported . In these patients, watery diarrhea is often absent . Adult CF patients with refractory or unexplained intestinal symptoms merit thorough investigations.

Appl Environ Microbiol, 2001 Oct, 67(10), 4734 - 41
Physiological ecology of Clostridium glycolicum RD-1, an aerotolerant acetogen isolated from sea grass roots; Kusel K et al.; An anaerobic, H(2)-utilizing bacterium, strain RD-1, was isolated from the highest growth-positive dilution series of a root homogenate prepared from the sea grass Halodule wrightii . Cells of RD-1 were gram-positive, spore-forming, motile rods that were linked by connecting filaments . Acetate was produced in stoichiometries indicative of an acetyl coenzyme A (acetyl-CoA) pathway-dependent metabolism when RD-1 utilized H(2)-CO(2), formate, lactate, or pyruvate . Growth on sugars or ethylene glycol yielded acetate and ethanol as end products . RD-1 grew at the expense of glucose in the presence of low initial concentrations (up to 6% {vol/vol}) of O(2) in the headspace of static, horizontally incubated culture tubes; the concentration of O(2) decreased during growth in such cultures . Peroxidase, NADH oxidase, and superoxide dismutase activities were detected in the cytoplasmic fraction of cells grown in the presence of O(2) . In comparison to cultures incubated under strictly anoxic conditions, acetate production decreased, higher amounts of ethanol were produced, and lactate and H(2) became significant end products when RD-1 was grown on glucose in the presence of O(2) . Similarly, when RD-1 was grown on fructose in the presence of elevated salt concentrations, lower amounts of acetate and higher amounts of ethanol and H(2) were produced . When the concentration of O(2) in the headspace exceeded 1% (vol/vol), supplemental H(2) was not utilized . The 16S rRNA gene of RD-1 had a 99.7% sequence similarity to that of Clostridium glycolicum DSM 1288(T), an organism characterized as a fermentative anaerobe . Comparative experiments with C . glycolicum DSM 1288(T) demonstrated that it had negligible H(2)- and formate-utilizing capacities . However, carbon monoxide dehydrogenase was detected in both RD-1 and C . glycolicum DSM 1288(T) . A 91.4% DNA-DNA hybridization between the genomic DNA of RD-1 and that of C . glycolicum DSM 1288(T) confirmed that RD-1 was a strain of C . glycolicum . These results indicate that (i) RD-1 metabolizes certain substrates via the acetyl-CoA pathway, (ii) RD-1 can tolerate and consume limited amounts of O(2), (iii) oxic conditions favor the production of ethanol, lactate, and H(2) by RD-1, and (iv) the ability of RD-1 to cope with limited amounts of O(2) might contribute to its survival in a habitat subject to daily gradients of photosynthesis-derived O(2).

Appl Environ Microbiol, 2001 Oct, 67(10), 4464 - 70
Insertion or deletion of the Cheo box modifies radiation inducibility of Clostridium promoters; Nuyts S et al.; Radiation-inducible promoters are being used in many viral vector systems to obtain spatial and temporal control of gene expression . It was previously proven that radiation-induced gene expression can also be obtained in a bacterial vector system using anaerobic apathogenic clostridia . The effect of radiation inducibility was detected using mouse tumor necrosis factor alpha (mTNF-alpha) as a model protein under regulation of the radiation-inducible recA promoter . In this report, experiments are described in which this recA promoter was modified in order to increase radiation responsiveness . Incorporation of an extra Cheo box in the recA promoter region resulted in an increase in mTNF-alpha secretion from 44% for the wild-type promoter to 412% for the promoter with an extra Cheo box after a single irradiation dose of 2 Gy . Deletion of the Cheo box in the promoter region eliminated radiation inducibility . These results prove that the Cheo box in the recA promoter is indeed the radiation-responsive element . We also tested whether we could induce the constitutive endo-beta-1,4-glucanase promoter (eglA) via ionizing irradiation by introducing a Cheo box in the promoter region . While the use of the constitutive promoter did not lead to an increase in mTNF-alpha secretion after irradiation, the introduction of a Cheo box resulted in a 242% increase in mTNF-alpha secretion . Reverse transcriptase PCR of RNA samples isolated from irradiated and nonirradiated bacterial cultures demonstrated that the increase in secretion was the result of enhanced transcription of the mTNF-alpha gene.

Berl Munch Tierarztl Wochenschr, 2001 Sep-Oct, 114(9-10), 327 - 30
The Hazard Analysis and Critical Control Points (HACCP) generic model for the production of Thai fermented pork sausage (Nham); Paukatong KV et al.; Nham is a traditional Thai fermented pork sausage . The major ingredients of Nham are ground pork meat and shredded pork rind . Nham has been reported to be contaminated with Salmonella spp., Staphylococcus aureus, and Listeria monocytogenes . Therefore, it is a potential cause of foodborne diseases for consumers . A Hazard Analysis and Critical Control Points (HACCP) generic model has been developed for the Nham process . Nham processing plants were observed and a generic flow diagram of Nham processes was constructed . Hazard analysis was then conducted . Other than microbial hazards, the pathogens previously found in Nham, sodium nitrite and metal were identified as chemical and physical hazards in this product, respectively . Four steps in the Nham process have been identified as critical control points . These steps are the weighing of the nitrite compound, stuffing, fermentation, and labeling . The chemical hazard of nitrite must be controlled during the weighing step . The critical limit of nitrite levels in the Nham mixture has been set at 100-200 ppm . This level is high enough to control Clostridium botulinum but does not cause chemical hazards to the consumer . The physical hazard from metal clips could be prevented by visual inspection of every Nham product during stuffing . The microbiological hazard in Nham could be reduced in the fermentation process . The critical limit of the pH of Nham was set at lower than 4.6 . Since this product is not cooked during processing, finally, educating the consumer, by providing information on the label such as "safe if cooked before consumption", could be an alternative way to prevent the microbiological hazards of this product.

Avian Dis, 2001 Jul-Sep, 45(3), 760 - 3
Enteritis as a cause of mortality in the western bluebird (Sialia mexicana); Bildfell RJ et al.; Increased mortalities in adult western bluebirds utilizing nestboxes were noted in western Oregon during 1998 and 1999 . A necrohemorrhagic enteritis was found in 8 of 10 birds submitted for necropsy . Acanthocephalan parasites were present in four of eight birds with enteritis . Microscopic changes consistent with necrotic or ulcerative enteritis were commonly present . Anaerobic culture of the intestine yielded Clostridium perfringens in three of three birds . Genotype analysis of two of these isolates revealed them to be C . perfringens type A . Bacterial enteritis is believed to be the cause of the increased mortality rate, but further investigation is required to prove a definitive link to a clostridial agent.

Avian Dis, 2001 Jul-Sep, 45(3), 741 - 4
Sudden death of a bearded vulture (Gypaetus barbatus) possibly caused by Newcastle disease virus; Lublin A et al.; An adult female bearded vulture (Gypaetus barbatus) in the Tel Aviv University Research Zoo was found dead without previous clinical signs . The predominant pathologic changes were considerable bloody content in the intestines and enlargement of the liver, which had a rubbery consistency with color changes . Microscopic lesions consisted of multifocal histiocytic infiltration in the liver . Newcastle disease virus (NDV) was isolated from a cloacal swab and from the lungs and liver . Intracerebral pathogenicity index of the virus, as estimated in 1-day-old chicks, was repeated three times and had an average value of 1.68, indicating a velogenic strain . Numerous Clostridium septicum bacteria were found on the intestinal surface, but bioassays in which they were orally administered into chickens and mice revealed that, even though they were heavily multiplied in the intestines, they were nonpathogenic . It seems that NDV, documented for the first time in a bearded vulture in Israel, was the likely cause of sudden death.

Avian Dis, 2001 Jul-Sep, 45(3), 724 - 32
A field study of naturally occurring specific antibodies against Clostridium perfringens alpha toxin in Norwegian broiler flocks; Heier BT et al.; Necrotic enteritis (NE), a disease associated with high numbers of the intestinal bacterium Clostridium perfringens, is common in intensive broiler production . Antimicrobial feed additives may control the disease, but their use is now being questioned in many countries . A field study was undertaken at the end of 1997 to study the level of naturally occurring specific humoral immunity against phospholipase C (PLC; C perfringens alpha toxin) in Norwegian broiler flocks . Blood samples were collected at hatch from 61 study flocks, and the sampling was repeated for 56 of the same flocks at processing . The level of specific antibodies against PLC was analyzed in an enzyme-linked immunosorbent assay (ELISA) test . Data on production performance and weekly mortality were recorded . The relationship between the age of the hens and the level of specific maternal antibodies in the progenies was studied . The association between the level of the maternal antibodies and the production performance, including mortality, was analyzed . The level of specific antibodies against PLC in day-old broiler flocks was relatively high and varied considerably compared with the levels in the broilers at processing . The progenies from the oldest hens had significantly higher levels of specific antibodies than the chicks from younger hens . No outbreak of NE occurred during the study period, making it impossible to analyze the association between naturally occurring specific immunity against PLC and the occurrence of the disease . However, the results showed that the flocks with high titers of specific maternal antibodies against PLC had lower mortality during the production period than flocks with low titers.

Avian Dis, 2001 Jul-Sep, 45(3), 659 - 62
Differences in the pathogenicity of various bacterial isolates used in an induction model for gangrenous dermatitis in broiler chickens; Wilder TD et al.; A gangrenous dermatitis model was developed in broiler chickens, in which birds previously vaccinated at 14 days of age with a bursal disease virus vaccine were challenged at 4 wk of age with various bacterial combinations with the combination of subcutaneous and intramuscular injection . Gangrenous dermatitis lesions were not produced in birds injected with one of the Staphylococcus aureus isolates, either alone or in combination with various Clostridium septicum isolates . Other S . aureus isolates produced significant levels of gangrenous dermatitis either alone or in combination with the same C . septicum isolates . These same C . septicum isolates when given alone did not produce gangrenous lesions . Data from this experiment show the highest level of mortality occurred in birds challenged with a mixture of C . septicum and S . aureus isolates, whereas lower or no mortality was associated with the same isolates given separately . The data clearly demonstrate that the pathogenicity of isolates responsible for gangrenous dermatitis varies widely, indicating that the frequency and severity of lesion production, as well as the occurrence of mortality, are largely dependent upon the specific isolate or isolates with which the birds are challenged.

Am J Gastroenterol, 2001 Sep, 96(9), 2688 - 90
Failure of single-toxin assays to detect clostridium difficile infection in pediatric inflammatory bowel disease; Markowitz JE et al.; OBJECTIVES: The aims of this retrospective study were 1) to determine the ability of single-toxin assays for Clostridium difficile to detect infection among pediatric patients with inflammatory bowel disease (IBD) and 2) to determine the toxin assays routinely used by pediatric tertiary care hospitals in the United States . METHODS: Stool specimens from patients with IBD (submitted from January, 1996, to August, 1999) were evaluated for the presence of C . difficile toxin A and toxin B . Toxin profile (toxin A alone, toxin B alone, toxin A and B together) was compared in positive specimens . A phone interview was conducted with representatives from laboratories in 22 pediatric hospitals to investigate which toxin assays were routinely used . RESULTS: A total of 697 specimens were submitted from 284 IBD patients . In all, 81 IBD patients (28.5%) had at least one documented infection . Toxin A assay failed to identify 41.5% of C . difficile infections . Toxin B assay failed to detect 34.9% of C . difficile infections . Toxin profile changed in 55% of patients with multiple infections . Of the hospitals surveyed, 59% did not test for both toxins . CONCLUSIONS: Single-toxin assays for C . difficile fail to detect a significant percentage of infections . The toxins identified during one infection are not predictive of the toxins identified in subsequent infections . Despite this, many pediatric hospitals do not routinely use both toxin assays to diagnose C . difficile infection . When infection is suspected, assays for C . difficile toxin A and toxin B should be requested.

Vaccine, 2001 Oct 12, 20(1-2), 31 - 41
The WHO Vaccine Trial Registry; Robertson SE et al.; The WHO Vaccine Trial Registry prospectively registers clinical vaccine studies supported by WHO . Through December 1999, the registry includes 103 studies from 43 countries, with nearly 80% in developing countries . The registry documents an expanding research capacity, with an average of 3.9 new studies per year during 1987-1993, rising to 10.7 per year during 1994-2000 . The studies concern a broad spectrum of infectious organisms, including: Clostridium tetani (tetanus), dengue virus, enterotoxigenic Escherichia coli (ETEC), Haemophilus influenzae type b (Hib), hepatitis B virus, measles virus, Mycobacterium tuberculosis, Neisseria meningitidis (meningococcus), poliovirus, respiratory syncytial virus (RSV), rotavirus, Salmonella typhi, Shigella, Streptococcus pneumoniae (pneumococcus), and Vibrio cholerae.

Biochim Biophys Acta, 2001 Sep 10, 1549(1), 32 - 6
Mapping the interaction of the {2Fe-2S} Clostridium pasteurianum ferredoxin with nitrogenase MoFe protein; Chatelet C et al.; The {2Fe-2S} ferredoxin from Clostridium pasteurianum had previously been shown to interact specifically with the nitrogenase MoFe protein, and electrostatic forces were found to be important contributors to the interaction . This phenomenon has now been analyzed in detail by using ferredoxin variants in which charge inversions or cancellations were introduced on all charged residues . The mutated forms of the ferredoxin were covalently cross-linked to the MoFe protein . The reaction products were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and their nitrogenase activity was measured . The latter displayed a consistent inverse correlation with the amount of cross-linked MoFe protein . The data allowed an unambiguous identification of the ferredoxin residues (glutamates 31, 34, 38, 39, 84, 85) that are involved in the interaction with the MoFe protein . Furthermore, whereas the wild-type ferredoxin yielded approximately equal amounts of cross-linked products with the alpha and beta subunits of the MoFe protein, some of its molecular variants displayed a differential decrease of reactivity towards one or the other of these subunits . The positions on the ferredoxin molecule of the residues interacting with the MoFe protein were determined using the recently elucidated crystal structure of the homologous {2Fe-2S} ferredoxin from Aquifex aeolicus.

Clin Infect Dis, 2001 Oct 15, 33(8), 1429 - 31; discussion 1432 Epub 2001 Sep 20.
Clostridium difficile small bowel enteritis occurring after total colectomy; Freiler JF et al.; Clostridium difficile infection is usually associated with antibiotic therapy and is almost always limited to the colonic mucosa . Small bowel enteritis is rare: only 9 cases have been previously cited in the literature . This report describes a case of C . difficile small bowel enteritis that occurred in a patient after total colectomy and reviews the 9 previously reported cases of C . difficile enteritis.

J Immunol, 2001 Oct 1, 167(7), 3585 - 91
A pivotal role of Rho GTPase in the regulation of morphology and function of dendritic cells; Kobayashi M et al.; Dendritic cell (DC) is the most potent activator of CD4+ T cells and has unique dendrites and veils . To explore the function of Rho in DC, exoenzyme C3 from Clostridium botulinum was used as a specific inhibitor of Rho . Treatment of DC with C3 (DC/C3) resulted in profound morphological changes by losing dendrites and emerging of shrunk membrane processes that were in parallel with marked reduction of polymerized actin in the marginal area . Inactivation of Rho-associated coiled coil-containing kinase (p160ROCK) by a specific ROCK inhibitor Y-27632 also led to disappearance of dendrites of DC with retaining large membrane expansions . In scanning electron microscopy, untreated DCs interacted with CD4+ T cells more efficiently than DC/C3 . Conjugate formation assay showed that the number of DCs associated with CD4+ T cells was 2-fold higher in untreated DCs than that of DC/C3 . Alloantigen-presenting capacity of DC/C3 was significantly suppressed in a dose-dependent manner . Because C3 treatment did not affect the surface expression of HLA, costimulatory, and adhesion molecules of DC, we examined cytokine production of DC and naive CD4+ T cells to further elucidate the inhibitory mechanism of MLR . Unexpectedly, DC/C3 increased IL-12 production after LPS stimulation . Naive CD4+ T cells cocultured with DC/C3 produced the increased percentage of IFN-gamma-producing cells, whereas the percentage of IL-2-producing T cells was decreased . These results demonstrate that Rho GTPase in DC controls both characteristic shape and immunogenic capacity.

Biochemistry (Mosc), 2001 Jul, 66(7), 808 - 13
Exploring the properties of thermostable Clostridium thermocellum cellulase CelE for the purpose of its expression in plants; Abdeev RM et al.; The main properties (pH and temperature range, stability, substrate specificity) of the modified cellulase CelE (endo-beta-1,4-glucanase) from Clostridium thermocellum have been analyzed with the goal of its expression in plants . The modified enzyme is similar to plant cellulases . Deletions in the N-terminus of the enzyme do not affect its biochemical properties . Based on the present investigation, we conclude that the modified beta-1,4-glucanase CelEM1, when expressed in plants, will be a good model to study the role of cellulases in plants.

J Food Prot, 2001 Sep, 64(9), 1388 - 91
Monitoring of microbial hazards at farms, slaughterhouses, and processing lines of swine in Korea; Rho MJ et al.; This study was executed to investigate microbiological hazards at swine farms, slaughterhouses, dressing operations, and local markets for the application of the hazard analysis critical control point system in Korea by analyzing total aerobic plate count (APC) and presence of pathogens . Six integrated pig farms and meat packers were selected from six different provinces, and samples were collected from pig carcasses by swabbing and excision methods at the slaughterhouses, processing rooms, and local markets, respectively . APCs of water in water tanks were relatively low, 1.9 to 3.1 log10 CFU/ml; however, they were increased to 4.6 to 6.9 log10 CFU/ml when sampled from water nipples in the pigpen . APCs of feeds in the feed bins and in the pigpens were 4.4 to 5.4 and 5.2 to 6.7 log10 CFU/g, respectively . Salmonella spp., Staphylococcus aureus, and Clostridium perfringens were detected from water and feed sampled in pigpens and pigpen floors . S . aureus was the most frequently detected pathogenic bacteria in slaughterhouses and processing rooms . Listeria monocytogenes and Yersinia enterocolitica were also detected from the processing rooms of the Kyonggi, Kyongsang, and Cheju provinces . Even though APCs were maintained at the low level of 3.0 log10 CFU/g during slaughtering and processing steps, those of final pork products produced by the same companies showed relatively high numbers when purchased from the local market . These results indicated that the cold chain system for transporting and merchandising of pork products was deficient in Korea . Water supply and feed bins in swine farms and individual operations can be identified as critical control points to reduce microbiological hazards in swine farms, slaughterhouses, and processing plants.

J Am Anim Hosp Assoc, 2001 Sep-Oct, 37(5), 453 - 60
Dog-bite wounds: bacteriology and treatment outcome in 37 cases; Griffin GM et al.; Bite wounds in 37 dogs were prospectively evaluated . Ninety-five percent of animals presented within 12 hours of injury . The most common wound locations were neck, limbs, head, chest, shoulder region, and abdomen . Eighty-six percent had wounds to multiple locations . Fifty-seven percent of wounds were Class 4 (i.e., most severe) . Based on results of all samples, 65% had positive aerobic cultures, 15% had positive anaerobic cultures, and 33% had negative cultures . The most commonly isolated aerobic bacteria were Staphylococcus intermedius, Enterococcus spp., Staphylococcus coagulase negative, and Escherichia coli . Most common anaerobic isolates were Bacillus spp., Clostridium spp., and Corynebacterium spp . Severe bite wounds had a high rate of bacterial contamination at presentation . No single antibiotic or antibiotic combination was effective against all bacteria that were cultured.

CMAJ, 2001 Sep 4, 165(5), 609 - 11
Unexplained deaths among injection drug users: a case of probable Clostridium myonecrosis; Williamson N et al.; A series of unexplained deaths associated with soft-tissue inflammation and severe systemic sepsis was reported among injection drug users (IDUs) in the United Kingdom and the Republic of Ireland in 2000 . Health Canada has identified one reported fatality in an IDU that matched the case definition . Although the cause of the epidemic in the UK and Ireland is not fully understood, contributing factors include injecting into muscle or beneath the skin, rather than directly into a vein, and the use of acid to dissolve the heroin . This single Canadian case is considered to be a sporadic event that occurs at a low background rate among IDUs . These cases serve to remind primary health care providers to be vigilant in cases of soft-tissue infection among IDUs and not to underestimate the potential severity of the situation.

J Microbiol Immunol Infect, 1999 Jun, 32(2), 116 - 20
Antimicrobial susceptibility of Clostridium difficile by E test; Cheng SH et al.; The in vitro inhibitory activity of 11 antimicrobials against 44 clinical isolates of Clostridium difficile was investigated . Minimum inhibitory concentrations (MICs) were determined using E test . Metronidazole (MIC90 0.38 microg/mL), teicoplanin (MIC90 0.75 microg/mL) and vancomycin (MIC90 1.0 microg/mL) were very active against the isolates examined, whereas, resistance to imipenem, cefoxitin, clindamycin and ciprofloxacin was found in most of the tested strains . We concluded that teicoplanin warrants clinical trials to determine its adequate dosage to treat C . difficile infection . The commonly used regimens to treat intra-abdominal and/or anaerobic infections (eg . imipenem, cefoxitin, clindamycin or ciprofloxacin) need special attention, while considering the side effects of C . difficile-associated diarrhea.

Ann Intern Med, 2001 Sep 18, 135(6), 434 - 8
Fatal pseudomembranous colitis associated with a variant clostridium difficile strain not detected by toxin A immunoassay; Johnson S et al.; BACKGROUND: Many clinical laboratories use toxin A immunoassays to test for Clostridium difficile . OBJECTIVE: To describe the clinical course of a patient infected with a toxin variant strain of C . difficile that was not detected by toxin A immunoassay; to genetically characterize this strain; and to estimate the number of laboratories that use only toxin A immunoassays . DESIGN: Case report, molecular investigation, and laboratory survey . SETTING: Tertiary care hospital in Chicago, Illinois . PATIENT: An 86-year-old man . MEASUREMENTS: Restriction endonuclease analysis, polymerase chain reaction, and survey of regional clinical laboratories . RESULTS: An elderly hospitalized man died of advanced pseudomembranous colitis . Four stool specimens submitted over a 2-month period had tested negative on toxin A immunoassay, but a strain of C . difficile with a 1.8-kb deletion of the toxin A gene was recovered from each specimen . This strain, identified as restriction endonuclease analysis type CF4, is closely related to a widely disseminated variant, toxinotype VIII . Toxin A immunoassay was the only test being performed for detection of C . difficile at 31 of 67 (46%) regional clinical laboratories . CONCLUSIONS: Toxin A variant strains of C . difficile cause serious disease and are undetectable in clinical laboratories that use only toxin A immunoassays for C . difficile testing.

Mayo Clin Proc, 2001 Sep, 76(9), 883 - 9
Lack of effect of Lactobacillus GG on antibiotic-associated diarrhea: a randomized, placebo-controlled trial; Thomas MR et al.; OBJECTIVES: To assess the efficacy of Lactobacillus GG in preventing antibiotic-associated diarrhea (AAD) in adults and, secondarily, to assess the effect of coadministered Lactobacillus GG on the number of tests performed to determine the cause of diarrhea . PATIENTS AND METHODS: In this prospective, randomized, double-blind, placebo-controlled trial conducted from July 1998 to October 1999, 302 hospitalized patients receiving antibiotics were randomized to receive Lactobacillus GG, 20 x 10(9) CFU/d, or placebo for 14 days . Subjects recorded the number of stools and their consistency daily for 21 days . The primary outcome was the proportion of patients who developed diarrhea in the first 21 days after enrollment . Weekly telephone follow-up was also performed . Results were analyzed in an intention-to-treat fashion . RESULTS: Diarrhea developed in 39 (29.3%) of 133 patients randomized to receive Lactobacillus GG and in 40 (29.9%) of 134 patients randomized to receive placebo (P=.93) . No additional difference in the rate of occurrence of diarrhea was found between treatment and placebo patients in a subgroup analysis of those treated with beta-lactam vs non-beta-lactam antibiotics . Too few patients had stool cultures, additional laboratory tests for diarrhea, or a positive diagnosis of Clostridium difficile infection to assess between-group differences . CONCLUSION: Lactobacillus GG in a dose of 20 x 10(9) CFU/d did not reduce the rate of occurrence of diarrhea in this sample of 267 adult patients taking antibiotics initially administered in the hospital setting.

J Vet Med Sci, 2001 Aug, 63(8), 879 - 83
Protective effects of clostridium sordellii LT and HT toxoids against challenge with spores in guinea pigs; Amimoto K et al.; The protective effects of Clostridium sordellii lethal toxin (LT) and hemorrhagic toxin (HT) toxoids against challenge with spores in guinea pigs were investigated . Purified LT and partially purified HT were obtained from the culture supernatant of C . sordellii strain 3703, and then were treated with formalin to make toxoids . LT . HT and combined LT and HT (LT/HT) toxoid vaccines were prepared by mixing each toxoid with an aluminum phosphate gel as adjuvant . Guinea pigs immunized twice with the respective toxoid vaccines were challenged with spores of strains 3703 or KZ1047 . The latter strain does not produce HT . LT toxoid vaccine conferred protection against challenge with strain KZ1047, but not strain 3703, in guinea pigs . All guinea pigs immunized with HT toxoid vaccine died after challenge with spores of either strain . LT/HT toxoid vaccine gave complete protection against challenge with spores of strains 3703 and KZ1047 to guinea pigs . These results suggest that not only LT toxoid, but also HT toxoid, are essential protective antigens of C . sordellii.

Am J Physiol Lung Cell Mol Physiol, 2001 Oct, 281(4), L816 - 23
Signal transduction pathways of IL-1beta-mediated iNOS in pulmonary vascular smooth muscle cells; Finder JD et al.; Interleukin (IL)-1beta is an important early mediator of inflammation in pulmonary artery smooth muscle cells . We previously reported that a geranylgeranyltransferase inhibitor elevated basal levels of inducible nitric oxide synthase (iNOS) and enhanced IL-1beta-mediated induction, suggesting that Rac or Rho small G proteins are candidates for antagonism of such induction . In this study, overexpression of constitutively active Rac1 or its dominant negative mutant did not affect IL-1beta induction of iNOS . Alternatively, treatment with Clostridium botulinum C3 exoenzyme, which ADP-ribosylates Rho, was associated with superinduction of iNOS, suggesting an inhibitory role for Rho . IL-1beta activated the three mitogen-activated protein kinase (extracellular signal-regulated kinases 1 and 2, c-Jun NH2-terminal kinase/stress-activated protein kinase, and p38) and the Janus kinase (JAK)-signal transducer and activator of transcription pathways . The former two pathways were not associated with IL-1beta-mediated iNOS induction, whereas the latter two appeared to have inhibitory roles in iNOS expression . These data suggest that a broad intracellular signaling response to IL-1beta in rat pulmonary artery smooth muscle cells results in elevated levels of iNOS that is opposed by the geranylgeranylated small G protein Rho as well as the p38 and JAK2 pathways.

FEBS Lett, 2001 Sep 7, 505(1), 125 - 8
Novel EPR signals associated with FeMoco centres of MoFe protein in MgADP-inhibited turnover of nitrogenase; Maritano S et al.; Two novel electron paramagnetic resonance (EPR) signals arising from the {1Mo-7Fe-9S-homocitrate} (FeMoco) centres of MoFe protein of Klebsiella pneumoniae nitrogenase (Kp1) were observed following turnover under MgATP-limited conditions . The combination of the nitrogenase Fe protein of Clostridium pasteurianum showed similar signals . The accumulation of MgADP under these conditions causes the normal EPR signal of dithionite-reduced Kp1 (with g=4.3, 3.6, 2.01) to be slowly converted to novel signals with g=4.74, 3.32, 2.00 and g=4.58, 3.50, 1.99 . These signals do not form in incubation of protein mixtures containing only MgADP, thus they may be associated with trapped intermediates of the catalytic cycle.

J Vet Med A Physiol Pathol Clin Med, 2001 Aug, 48(6), 373 - 83
Visceral botulism--a new form of bovine Clostridium botulinum toxication; Bohnel H et al.; There are reports of a hitherto unknown bovine disease in Germany . The symptoms are, in general, indigestion (constipation alternating with diarrhoea), non-infectious chronic laminitis, engorged veins, oedemas, retracted abdomen, emaciation and apathy . Most cases occur during the peripartal period and often result in unexpected death . In addition, there are findings of delayed growth and wasting in heifers, as well as decreasing milk yield . Clinical and standard laboratory examinations leave the origin undisclosed . Bioassays for Clostridium botulinum, its spores and toxins in animals of affected farms revealed the presence of free botulinum toxin in the contents of the lower sections of the intestine . In two control farms without signs of the disease, the tests remained negative . This seems to support our hypothesis that long-lasting absorption of low quantities of botulinum toxin may interfere with the neurological control of intestinal physiology . The authors propose to name this disease complex 'visceral botulism'.

Infect Immun, 2001 Oct, 69(10), 6004 - 11
Characterization of the enzymatic component of the ADP-ribosyltransferase toxin CDTa from Clostridium difficile; Gulke I et al.; Certain strains of Clostridium difficile produce the ADP-ribosyltransferase CDT, which is a binary actin ADP-ribosylating toxin . The toxin consists of the binding component CDTb, which mediates receptor binding and cellular uptake, and the enzyme component CDTa . Here we studied the enzyme component (CDTa) of the toxin using the binding component of Clostridium perfringens iota toxin (Ib), which is interchangeable with CDTb as a transport component . Ib was used because CDTb was not expressed as a recombinant protein in Escherichia coli . Similar to iota toxin, CDTa ADP-ribosylates nonmuscle and skeletal muscle actin . The N-terminal part of CDTa (CDTa1-240) competes with full-length CDTa for binding to the iota toxin binding component . The C-terminal part (CDTa244-263) harbors the enzyme activity but was much less active than the full-length CDTa . Changes of Glu428 and Glu430 to glutamine, Ser388 to alanine, and Arg345 to lysine blocked ADP-ribosyltransferase activity . Comparison of CDTa with C . perfringens iota toxin and Clostridium botulinum C2 toxin revealed full enzyme activity of the fragment Ia208-413 but loss of activity of several N-terminally deleted C2I proteins including C2I103-431, C2I190-431, and C2I30-431 . The data indicate that CDTa belongs to the iota toxin subfamily of binary actin ADP-ribosylating toxins with respect to interaction with the binding component and substrate specificity . It shares typical conserved amino acid residues with iota toxin and C2 toxin that are suggested to be involved in NAD-binding and/or catalytic activity . The enzyme components of CDT, iota toxin, and C2 toxin differ with respect to the minimal structural requirement for full enzyme activity.

Biochemistry, 2001 Sep 18, 40(37), 11140 - 8
Involvement of coenzyme A esters and two new enzymes, an enoyl-CoA hydratase and a CoA-transferase, in the hydration of crotonobetaine to L-carnitine by Escherichia coli; Elssner T et al.; Two proteins (CaiB and CaiD) were found to catalyze the reversible biotransformation of crotonobetaine to L-carnitine in Escherichia coli in the presence of a cosubstrate (e.g., gamma-butyrobetainyl-CoA or crotonobetainyl-CoA) . CaiB (45 kDa) and CaiD (27 kDa) were purified in two steps to electrophoretic homogeneity from overexpression strains . CaiB was identified as crotonobetainyl-CoA:carnitine CoA-transferase by MALDI-TOF mass spectrometry and enzymatic assays . The enzyme exhibits high cosubstrate specificity to CoA derivatives of trimethylammonium compounds . In particular, the N-terminus of CaiB shows significant identity with other CoA-transferases (e.g., FldA from Clostridium sporogenes, Frc from Oxalobacter formigenes, and BbsE from Thauera aromatica) and CoA-hydrolases (e.g., BaiF from Eubacterium sp.) . CaiD was shown to be a crotonobetainyl-CoA hydratase using MALDI-TOF mass spectrometry and enzymatic assays . Besides crotonobetainyl-CoA CaiD is also able to hydrate crotonyl-CoA with a significantly lower Vmax (factor of 10(3)) but not crotonobetaine . The substrate specificity of CaiD and its homology to the crotonase confirm this enzyme as a new member of the crotonase superfamily . Concluding these results, it was verified that hydration of crotonobetaine to L-carnitine proceeds at the CoA level in two steps: the CaiD catalyzed hydration of crotonobetainyl-CoA to L-carnitinyl-CoA, followed by a CoA transfer from L-carnitinyl-CoA to crotonobetaine, catalyzed by CaiB . When gamma-butyrobetainyl-CoA was used as a cosubstrate (CoA donor), the first reaction is the CoA transfer . The optimal ratios of CaiB and CaiD during this hydration reaction, determined to be 4:1 when crotonobetainyl-CoA was used as cosubstrate and 5:1 when gamma-butyrobetainyl-CoA was used as cosubstrate, are different from that found for in vivo conditions (1:3).

Chemotherapy, 2001, 47 Suppl 3, 15 - 23; discussion 44-8
A comparison of side effects of levofloxacin to other agents concerning the ecological and microbiological effects on normal human flora; Acar JF; The safety of levofloxacin was compared to that of non-fluoroquinolone alternatives used for respiratory tract infections . Results from five randomised controlled trials revealed that the incidence of any adverse events possibly associated with levofloxacin ranged from 5.8% to 22.7%, whereas that of comparators (ceftriaxone, cefuroxime axetil, clarithromycin and amoxicillin-clavulanic acid) ranged from 8.5% to 39.3% . The rate of adverse drug reactions (ADRs) was lower for levofloxacin in all trials . The most common adverse events for all agents tended to be gastrointestinal in nature . Levofloxacin was associated with a mild effect on the normal microflora, reaching a maximum at four days of therapy, with complete recovery being achieved by seven days post-therapy . No colonisation with resistant strains was observed during the period of levofloxacin therapy . Amoxicillin-clavulanic acid administration selected for resistant strains of Enterobacteriaceae, and ampicillin administration was associated with both resistant strains of Enterobacteriacae as well as Candida spp . Ceftriaxone selected resistant strains of Clostridium difficile and Candida spp . Thus, microflora effects favoured levofloxacin over all of the agents tested, including macrolides and tetracyclines . These results confirm that the ecological impact of levofloxacin is markedly less than that associated with non-fluoroquinolone comparators .

FEMS Immunol Med Microbiol, 2001 Aug, 31(2), 131 - 5
Variation in the surface layer proteins of Clostridium difficile; McCoubrey J et al.; Surface layers (S-layers) form regular crystalline structures on the outermost surface of many bacteria . Clostridium difficile possesses such an S-layer consisting of two protein subunits . Treatment of whole cells of C . difficile with 5 M guanidine hydrochloride revealed two major proteins of different molecular masses characteristic of the S-layer on SDS-PAGE . In this study 25 isolates were investigated . A high degree of variability in the molecular mass of the two S-layer proteins was evident . Molecular masses ranged from 48 to 56 kDa for the heavier protein and from 37 to 45 kDa for the lighter protein . A further protein component of 70 kDa was detectable in all isolates . No cross-reaction was seen between the two major proteins from isolates that produced different S-layer patterns, and most S-layer proteins from isolates with the same or similar banding patterns did not cross-react . The S-layer proteins, when detected by a combination of Coomassie blue staining and immunoblotting, are a useful marker for phenotyping.

FEMS Immunol Med Microbiol, 2001 Aug, 31(2), 85 - 92
Effect of Clostridium perfringens epsilon toxin on MDCK cells; Borrmann E et al.; Epsilon toxin is one of the major lethal toxins produced by Clostridium perfringens type D and B . It is responsible for a rapidly fatal disease in sheep and other farm animals . Many facts have been published about the physical properties and the biological activities of the toxin, but the molecular mechanism of the action inside the cells remains unclear . We have found that the C . perfringens epsilon toxin caused a significant decrease of the cell numbers and a significant enlargement of the mean cell volume of MDCK cells . The flow cytometric analysis of DNA content revealed the elongation of the S phase and to a smaller extent of the G2+M phase of toxin-treated MDCK cells in comparison to untreated MDCK cells . The results of ultrastructural studies showed that the mitosis is disturbed and blocked at a very early stage, and confirmed the toxin influence on the cell cycle of MDCK cells.

Can J Microbiol, 2001 Jul, 47(7), 626 - 33
The effect of condensed tannins from Lotus pedunculatus and Lotus corniculatus on the growth of proteolytic rumen bacteria in vitro and their possible mode of action; Molan AL et al.; Five strains of proteolytic rumen bacteria were treated with condensed tannins (CT) purified from Lotus pedunculatus and Lotus corniculatus to investigate their effect on the growth of these bacteria in vitro . Streptococcus bovis NCFB 2476, Eubacterium sp . C124b, Prevotella bryantii B14, Butyrivibrio fibrisolvens H17c, and Clostridium proteoclasticum B316T were tested against 200, 400, and 600 microg CT x mL(-1) extracted from L . pedunculatus and L . corniculatus . In the absence of CT, all bacterial strains showed typical growth and reached maximum optical density (OD) after 6-8 h of incubation in a plant protein medium . Growth of Eubacterium sp., P . bryantii, and B . fibrisolvens was inhibited (P < 0.01-0.001) more by the CT from L . pedunculatus than by the CT from L . corniculatus . All strains continued to grow in the presence of 200 microg x mL(-1) of the CT from L . pedunculatus, but attained significantly (P < 0.05-0.01) lower maximum OD600 values than (minus CT) controls, except for S . bovis . At 400 and 600 microg x mL(-1), the addition of CT from L . pedunculatus inhibited (P < 0.05-0.001) the growth of all bacterial strains tested compared with controls . The growth of Eubacterium sp . and P . bryantii was stimulated for the first 4-6 h of incubation (P < 0.001) by 200 microg x mL(-1) of CT from L . corniculatus, but then declined leading to a significant difference in OD values compared with the controls . At 400 microg x mL(-1), the CT from L . corniculatus reduced (P < 0.05-0.01) the growth of all strains except S . bovis, while 600 microg x mL(-1) inhibited (P < 0.01-0.001) the growth of all strains . To study the mechanism of CT action, the degradation of the large subunit (LSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco; Fraction 1 Leaf protein) was followed after bacterial cells or Rubisco were preincubated with CT extracted from L . corniculatus and L . pedunculatus . Both preincubations decreased LSU degradation, but they differed in their response to polyethylene glycol (PEG) addition . Addition of PEG to CT-Rubisco preincubations negated the effects of CT, while PEG addition to CT-bacteria preincubations did not . This implies that the CT-bacterial interaction is stronger than the CT-Rubisco interaction or the interaction is of a different type . Also, L . pedunculatus CT reduced the degradation of the LSU to a greater extent than the CT from L . corniculatus when preincubated with bacteria.

J Biol Chem, 2001 Nov 9, 276(45), 42436 - 44 Epub 2001 Sep 06.
Insulin-stimulated GLUT4 translocation in adipocytes is dependent upon cortical actin remodeling; Kanzaki M et al.; Rhodamine-labeled phalloidin staining of morphologically differentiated 3T3L1 adipocytes demonstrated that F-actin predominantly exists juxtaposed to and lining the inner face of the plasma membrane (cortical actin) with a smaller amount of stress fiber and/or ruffling actin confined to the cell bottom in contact with the substratum . The extent of cortical actin disruption with various doses of either latrunculin B or Clostridium difficile toxin B (a Rho family small GTP-binding protein toxin) directly correlated with the inhibition of insulin-stimulated glucose uptake and GLUT4 translocation . The dissolution of the cortical actin network had no significant effect on proximal insulin receptor signaling events including insulin receptor autophosphorylation, tyrosine phosphorylation of insulin receptor substrate and Cbl, or serine/threonine phosphorylation of Akt . Surprisingly, however, stabilization of F-actin with jasplakinolide also resulted in a dose-dependent inhibition of insulin-stimulated glucose uptake and GLUT4 translocation . In vivo time-lapse confocal fluorescent microscopy of actin-yellow fluorescent protein demonstrated that insulin stimulation initially results in cortical actin remodeling followed by an increase in polymerized actin in the peri-nuclear region . Importantly, the insulin stimulation of cortical actin rearrangements was completely blocked by treatment of the cells with latrunculin B, C . difficile toxin B, and jasplakinolide . Furthermore, expression of the dominant-interfering TC10/T31N mutant completely disrupted cortical actin and prevents any insulin-stimulated actin remodeling . Together, these data demonstrate that cortical actin, but not stress fibers, lamellipodia, or filopodia, plays an important regulatory role in insulin-stimulated GLUT4 translocation . In addition, cortical F-actin does not function in a static manner (e.g . barrier or scaffold), but insulin-stimulated dynamic cortical actin remodeling is necessary for the GLUT4 translocation process.

J Mol Microbiol Biotechnol, 2001 Oct, 3(4), 585 - 600
Comparative genomics and evolution of genes encoding bacterial (p)ppGpp synthetases/hydrolases (the Rel, RelA and SpoT proteins); Mittenhuber G; In the gram-negative model organism Escherichia coli, the effector molecule of the stringent response, (p)ppGpp, is synthesized by two different enzymes, RelA and SpoT, whereas in the gram-positive model organism Bacillus subtilis only one enzyme named Rel is responsible for this activity . Rel and SpoT also possess (p)ppGpp hydrolase activity . BLAST searches were used to identify orthologous genes in databases . The construction and bootstrapping of phylogenetic trees allowed classification of these orthologs . Four groups could be distinguished: With the exception of Neisseria and Bordetella (beta subdivision), the RelA and SpoT groups are exclusively found in the gamma subdivision of proteobacteria . Two Rel groups representing the actinobacterial and the Bacillus/Clostridium group were also identified . The SpoT proteins are related to the gram positive Rel proteins . RelA proteins carry substitutions in the HD domain (Aravind and Koonin, 1998, TIBS 23: 469-472) responsible for ppGpp degradation . A theory for the evolution of the specialized, paralogous relA and spoT genes is presented: After gene duplication of an ancestral rellike gene, the spoT and relA genes evolved from the duplicated genes . The distribution pattern of the paralogous RelA and SpoT proteins supports a new model of linear bacterial evolution (Gupta, 2000, FEMS Microbiol . Rev . 24: 367-402) . This model postulates that the gamma subdivision of proteobacteria represents the most recently evolved bacterial lineage . However, two paralogous, closely related genes of Porphyromonas gingivalis (Cytophaga-Flavobacterium-Bacteroides phylum) encoding proteins with functions probably identical to the RelA and SpoT proteins do not fit in this model . Completely sequenced genomes of several obligately parasitic organisms (Treponema pallidum, Chlamydia species, Rickettsia prowazekii) and the obligate aphid symbiont Buchnera sp . APS as well as archaea do not contain rel-like genes but they are present in the Arabidopsis genome . In crosslinking experiments using different analogs of ppGpp as crosslinking reagents and RNA polymerase preparations of Escherichia coli, binding of ppGpp to distinct regions at the C-terminus of the beta subunit (the RpoB gene product) and/or at the N-terminus of the beta subunit (the RpoC gene product) was observed previously . RpoB and RpoC sequences of the species which do not possess a rel like gene do not exhibit specific insertions or deletions in the ppGpp binding regions.

Int J Food Microbiol, 2001 Aug 15, 68(1-2), 149 - 53
Evaluation of microbiological quality of medicinal plants used in natural infusions; Martins HM et al.; Consumption of preparations of medicinal plants has been increasing during the last decades in occidental societies . However, there are no effective sanitary controls of these products . To evaluate the nature and content of microbiological contamination, 62 samples of seven medicinal plants (chamomile, leaves of orange tree, flowers of linden, corn silk, marine alga, pennyroyal mint and garden sage) were studied, using conventional microbiological methods . Practically all samples (96.8%) were contaminated with Bacillus cereus; 19.2% of them with levels higher than 10(3) spores/g . The highest levels were found in corn silk samples (> 10(7) spores/g) . Spores of Clostridium perfringens were detected in 83.9% of samples, but only 19.2% had levels greater than 10(3)/g . The mean level of fungal population was 10(5.5) cfu/g . Corn silk samples were the most contaminated, with levels above 10(6) cfu/g . Fusarium spp., Penicillium spp., Aspergillus flavus and Asp . niger were predominant in all samples with the exception of garden sage samples . Many yeasts were found in chamomile, flowers of linden, corn silk, pennyroyal mint and garden sage . Predominant species were Cryptococcus laurentii (28.1%) and Rhodotorula mucilaginosa (22.8%) . The mean level of Crypt . laurentii contamination in corn silk was greater than 10(4) cfu/g.

Int J Food Microbiol, 2001 Aug 15, 68(1-2), 105 - 13
Prevalence of Staphylococcus aureus and staphylococcal enterotoxins in raw pork and uncooked smoked ham--a comparison of classical culturing detection and RFLP-PCR; Atanassova V et al.; In many countries Staphylococcus aureus is considered to be the second or third most common pathogen causing outbreaks of food poisoning, only outnumbered by Salmonella spp . and in competition with Clostridium perfringens . Often the consumption of ham or meat containing staphylococcal enterotoxins (SE) is identified as cause of the illness . Thus, to gain an insight into the prevalence of S . aureus and its emetic enterotoxins in raw pork and uncooked smoked ham and to investigate how the prevalence of the pathogen is influenced during the fabrication process, a total of 135 samples of raw pork, salted meat and ready-for-sale uncooked smoked ham were examined for the prevalence of S . aureus and staphylococcal enterotoxins A to D (SEA-SED) . To this means classical cultural methods were employed as well as molecular biological techniques (PCR) and the results were compared . In 25.9% of all samples S . aureus was detected by culture whereas 51.1% of the samples showed a positive result when PCR was used for the detection of the pathogen . Fresh meat was contaminated most often . By PCR, 62.2% were identified as being S . aureus positive compared to 57.7% positive samples using the cultural technique . The detection rate during the fabrication process declined significantly . The pathogen was cultivated from 8.9% of the salted meat samples . Here, 55.6% of the samples reacted positively in the PCR, and finally, in approximately a third of the ready-for-sale smoked hams, S . aureus genes were found . From 11.1% of these samples, the pathogen could be isolated by culture . From these results, we conclude that the PCR used in this study is more sensitive than the classical cultural method . By PCR, one or more staphylococcal enterotoxin genes were found in 24 of the 135 examined samples . This means that 34.8% of the staphylococcal strains identified using the PCR technique were enterotoxigenic . Using the SET-RPLA, a percentage of 28.6% enterotoxigenic isolates was ascertained . No staphylococcal enterotoxin formation was detected by the SET-RPLA in ready-for-sale ham, although SE-genes were found by PCR . The detection of SE-genes by PCR is faster and easier to perform than the SET-RPLA.

Acta Astronaut, 1993 Aug, 29(8), 629 - 32
Planetary quarantine in the solar system . Survival rates of some terrestrial organisms under simulated space conditions by proton irradiation; Koike J et al.; We have been studying the survival rates of some species of terrestrial unicellular and multicellular organism (viruses, bacteria, yeasts, fungi, algae, etc.) under simulated interstellar conditions, in connection with planetary quarantine . The interstellar environment in the solar system has been simulated by low temperature, high vacuum (77 K, 4 x 10(-8) torr), and proton irradiation from a Van de Graaff generator . After exposure to a barrage of protons corresponding to about 250 years of irradiation in solar space, tobacco mosaic virus, Bacillus subtilis spores, Staphylococcus aureus, Micrococcus flavus, Aspergillus niger spores, and Clostridium mangenoti spores showed survival rates of 82, 45, 74, 13, 28, and 25%, respectively.

Adv Space Res, 1995 Mar, 15(3), 211 - 4
Studies in the search for life on Mars; Koike J et al.; The ability of living organisms to survive extraterrestrial conditions has implications for the origins of life in the solar system . We have therefore studied the survival of viruses, bacteria, yeast, and fungi under simulated Martian conditions . The environment on Mars was simulated by low temperature, proton irradiation, ultraviolet irradiation, and simulated Martian atmosphere (CO2 95.46%, N2 2.7%, water vapor 0.03%) in a special cryostat . After exposure to these conditions, tobacco mosaic virus and spores of Bacillus, Aspergillus, Clostridium, and some species of coccus showed significant survival.

Int J Syst Bacteriol, 1993 Apr, 43(2), 278 - 86
Assignment of fatty acid-beta-oxidizing syntrophic bacteria to Syntrophomonadaceae fam . nov . on the basis of 16S rRNA sequence analyses; Zhao H et al.; After enrichment from Chinese rural anaerobic digestor sludge, anaerobic, sporing and nonsporing, saturated fatty acid-beta-oxidizing syntrophic bacteria were isolated as cocultures with H2- and formate-utilizing Methanospirillum hungatei or Desulfovibrio sp . strain G-11 . The syntrophs degraded C4 to C8 saturated fatty acids, including isobutyrate and 2-methylbutyrate . They were adapted to grow on crotonate and were isolated as pure cultures . The crotonate-grown pure cultures alone did not grow on butyrate in either the presence or the absence of some common electron acceptors . However, when they were reconstituted with M . hungatei, growth on butyrate again occurred . In contrast, crotonate-grown Clostridium kluyveri and Clostridium sticklandii, as well as Clostridium sporogenes, failed to grow on butyrate when these organisms were cocultured with M . hungatei . The crotonate-grown pure subcultures of the syntrophs described above were subjected to 16S rRNA sequence analysis . Several previously documented fatty acid-beta-oxidizing syntrophs grown in pure cultures with crotonate were also subjected to comparative sequence analyses . The sequence analyses revealed that the new sporing and nonsporing isolates and other syntrophs that we sequenced, which had either gram-negative or gram-positive cell wall ultrastructure, all belonged to the phylogenetically gram-positive phylum . They were not closely related to any of the previously known subdivisions in the gram-positive phylum with which they were compared, but were closely related to each other, forming a new subdivision in the phylum . We recommend that this group be designated Syntrophomonadaceae fam . nov.; a description is given.

Dis Colon Rectum, 2001 Aug, 44(8), 1176 - 80
Intravenous metronidazole for the treatment of Clostridium difficile colitis; Friedenberg F et al.; PURPOSE: Severe Clostridium difficile colitis may produce abdominal distention and ileus, precluding oral antibiotic therapy . Stimulated by several case reports in which intravenous metronidazole was used, we reviewed our experience . METHODS: Using pharmacy and microbiology laboratory records, we retrospectively identified patients with C . difficile colitis who received intravenous metronidazole as initial monotherapy . To be included, patients had to fulfill the following criteria: 1) at least six doses (equivalent to two days of therapy) of intravenous metronidazole were administered, 2) no other potential cause for colitis was found, and 3) the diagnosis of C . difficile colitis was firmly established . For eligible patients, five clinical parameters were assessed before and after intravenous metronidazole . RESULTS: Our patient group (n = 10) received an average of 13.7 (range, 6-24) doses of intravenous metronidazole as initial therapy for C . difficile colitis . All received a dose of 500 mg three times daily . The majority of patients with vomiting, fever, and/or abdominal pain present at the beginning of therapy had resolution with intravenous metronidazole . Only one patient developed a symptom (vomiting) while on therapy; however, this eventually resolved when oral metronidazole was instituted . No patient required colectomy for refractory colitis or developed toxic megacolon . No patient, including those on prolonged courses, developed toxicity related to intravenous metronidazole such as peripheral neuropathy . CONCLUSIONS: Intravenous metronidazole may be effective therapy in patients with C . difficile colitis . A randomized, prospective study appears warranted.

Nahrung, 2001 Aug, 45(4), 286 - 92
Nutritional and bacteriological properties of some game duck carcasses; Khalifa AH et al.; The aim of this work was to evaluate the body composition, bacteriological quality, proximate composition, amino acids content, total lipids fractionation, as well as fatty acids profile in breast and thigh meat (with skin) of males and females of two species of game ducks namely: Pintail (Anas acuta) and garganey (Anas querquedula) . The obtained results are as follows . The live weight of pentail and garganey females constituted 59.0 and 86.0% of male's weight in pintail and garganey, respectively . The bacteriological quality revealed that the mean values of psychrotrophs, enterobacteriaceae, pseudomonas, coliforms, streptococci and Staph . aureus were 4.1, 2.8, 1.7, 2.0, 2.5 and 3.1 log 10 n/g of pintail breast muscle . The corresponding values in garganey breast muscle was 3.8, 3.2, 2.0, 3.0, 2.9 and 3.1, respectively . In the thigh of pintail and garganey, the results were more or less different . Neither salmonella nor Clostridium perfringens could be isolated from examined game duck carcasses . Protein content ranged from 19.0 to 23.8%, fat 4.8 to 23.2%, ash 1.0 to 1.4% and energy value 580 to 1191 kJ/100 g in pintail meat against 20.8 to 23.3% protein, 9.3 to 16.1% fat, 1.3 to 1.4% ash and 741 to 952 kJ/100 g in garganey meat . Breast meat of pintail recorded high content of iron (5.12 and 6.19 mg/100 g wet basis) in males and females, respectively, against 4.22 and 6.14 mg/100 g in garganey meat . The essential amino acids content ranged from 34.3 to 38.6 g/100 g protein in pintail meat against 36.3 to 38.1 g/100 g protein in garganey meat . The total lipids of pintail and garganey were fractionated to seven fractions . The major fatty acids in pintail and garganey lipids were oleic, palmitic and stearic . Besides, garganey lipids had more unsaturated fatty acids content compared with pintail.

J Biol Chem, 2001 Nov 2, 276(44), 40977 - 81 Epub 2001 Aug 31.
Chemoattractant-stimulated NF-kappaB activation is dependent on the low molecular weight GTPase RhoA; Huang S et al.; Chemoattractants bind to seven transmembrane-spanning, G-protein-coupled receptors on monocytes and neutrophils and induce a variety of functional responses, including activation of the transcription factor NF-kappaB . The signaling mechanisms utilized by chemoattractants to activate NF-kappaB in human peripheral blood monocytes are poorly defined . We previously demonstrated that fMet-Leu-Phe (fMLP) stimulates NF-kappaB activation, and this function of fMLP requires phosphatidylinositol 3-kinase (PI3K) . Here we present evidence that fMLP activates RhoA and that fMLP-induced NF-kappaB activation requires this small GTPase . Stimulation of monocytes with fMLP rapidly activated RhoA as well as NF-kappaB, and their activation was markedly reduced by pertussis toxin treatment . Pretreatment of monocyte with a RhoA inhibitor, C3 transferase from Clostridium botulinum, effectively blocked fMLP-induced NF-kappaB activation as well as interleukin-1beta gene expression . A dominant negative form of RhoA (T19N) also inhibited fMLP-stimulated reporter gene expression in a kappaB-dependent manner . Cotransfection of the monocytic THP1 cells with a constitutively active form of RhoA (Q63L) with the promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression . Furthermore, the PI3K inhibitors wortmannin and LY294002 block RhoA activation induced by fMLP . These results demonstrate that low molecular weight GTPase RhoA is a novel signal transducer for fMLP-induced NF-kappaB activation and Galpha(i) or Galpha(o) class of heterotrimeric G proteins likely mediate RhoA activation via PI3K in human peripheral blood monocytes.

Korean J Ophthalmol, 2001 Jun, 15(1), 54 - 7
A case of anaerobic abscessed hydroxyapatite orbital implants; Park KS et al.; The purpose of this report is to document an unusual case of implant infection in a patient who had undergone enucleation and hydroxyapatite orbital implant surgery . A 32-year-old woman presented with chronic orbital discomfort and discharge following a history of hydroxyapatite orbital implant surgery at another hospital 4 years previous . She exhibited profuse discharge with a yellow, creamy color and marked conjunctival chemosis . Granulation tissue was noted on the central conjunctival surface . Following the removal of the conjunctival granulation tissue, a central 3x5 mm conjunctival dehiscence was present with exposure of the hydroxyapatite implant . A culture of purulent drainage emanating from the exposed implant showed a growth of Clostridium acetobutylicum . Removal of the orbital implant was done . The implant was noted to be filled with pus . This case suggests that anaerobic infection may be suspected when the granulation tissue is observed and a discharge with a foul odor is found.

Microbiol Mol Biol Rev, 2001 Sep, 65(3), 335 - 52, table of contents
Biological degradation of 2,4,6-trinitrotoluene; Esteve-Nunez A et al.; Nitroaromatic compounds are xenobiotics that have found multiple applications in the synthesis of foams, pharmaceuticals, pesticides, and explosives . These compounds are toxic and recalcitrant and are degraded relatively slowly in the environment by microorganisms . 2,4,6-Trinitrotoluene (TNT) is the most widely used nitroaromatic compound . Certain strains of Pseudomonas and fungi can use TNT as a nitrogen source through the removal of nitrogen as nitrite from TNT under aerobic conditions and the further reduction of the released nitrite to ammonium, which is incorporated into carbon skeletons . Phanerochaete chrysosporium and other fungi mineralize TNT under ligninolytic conditions by converting it into reduced TNT intermediates, which are excreted to the external milieu, where they are substrates for ligninolytic enzymes . Most if not all aerobic microorganisms reduce TNT to the corresponding amino derivatives via the formation of nitroso and hydroxylamine intermediates . Condensation of the latter compounds yields highly recalcitrant azoxytetranitrotoluenes . Anaerobic microorganisms can also degrade TNT through different pathways . One pathway, found in Desulfovibrio and Clostridium, involves reduction of TNT to triaminotoluene; subsequent steps are still not known . Some Clostridium species may reduce TNT to hydroxylaminodinitrotoluenes, which are then further metabolized . Another pathway has been described in Pseudomonas sp . strain JLR11 and involves nitrite release and further reduction to ammonium, with almost 85% of the N-TNT incorporated as organic N in the cells . It was recently reported that in this strain TNT can serve as a final electron acceptor in respiratory chains and that the reduction of TNT is coupled to ATP synthesis . In this review we also discuss a number of biotechnological applications of bacteria and fungi, including slurry reactors, composting, and land farming, to remove TNT from polluted soils . These treatments have been designed to achieve mineralization or reduction of TNT and immobilization of its amino derivatives on humic material . These approaches are highly efficient in removing TNT, and increasing amounts of research into the potential usefulness of phytoremediation, rhizophytoremediation, and transgenic plants with bacterial genes for TNT removal are being done.

Appl Environ Microbiol, 2001 Sep, 67(9), 4382 - 4
Rapid confirmation of Clostridium perfringens by using chromogenic and fluorogenic substrates; Adcock PW et al.; The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-beta-D-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated . A liquid assay containing both MUP and ONPG was a highly specific alternative method for C . perfringens confirmation, reducing incubation time from 48 to only 4 h . The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life.

Appl Environ Microbiol, 2001 Sep, 67(9), 3846 - 51
Flux analysis of the metabolism of Clostridium cellulolyticum grown in cellulose-fed continuous culture on a chemically defined medium under ammonium-limited conditions; Desvaux M et al.; An investigation of cellulose degradation by the nonruminal, cellulolytic, mesophilic bacterium Clostridium cellulolyticum was performed in cellulose-fed chemostat cultures with ammonium as the growth-limiting nutrient . At any dilution rate (D), acetate was always the main product of the catabolism, with a yield of product from substrate ranging between 37.7 and 51.5 g per mol of hexose equivalent fermented and an acetate/ethanol ratio always higher than 1 . As D rose, the acetyl coenzyme A was rerouted in favor of ethanol pathways, and ethanol production could represent up to 17.7% of the carbon consumed . Lactate was significantly produced, but with increasing D, the specific lactate production rate declined, as did the specific rate of production of extracellular pyruvate . The proportion of the original carbon directed towards phosphoglucomutase remained constant, and the carbon surplus was balanced mainly by exopolysaccharide and glycogen biosyntheses at high D values, while cellodextrin excretion occurred mainly at lower ones . With increasing D, the specific rate of carbon flowing down catabolites increased as well, but when expressed as a percentage of carbon it declined, while the percentage of carbon directed through biosynthesis pathways was enhanced . The maximum growth and energetic yields were lower than those obtained in cellulose-limited chemostats and were related to an uncoupling between catabolism and anabolism leading to an excess of energy . Compared to growth on cellobiose in ammonium-limited chemostats (E . Guedon, M . Desvaux, and H . Petitdemange, J . Bacteriol . 182:2010-2017, 2000), (i) a specific consumption rate of carbon of as high as 26.72 mmol of hexose equivalent g of cells(-1) x h(-1) could not be reached and (ii) the proportions of carbon directed towards cellodextrin, glycogen, and exopolysaccharide pathways were not as high as first determined on cellobiose . While the use of cellobiose allows highlighting of metabolic limitation and regulation of C . cellulolyticum under ammonium-limited conditions, some of these events should then rather be interpreted as distortions of the metabolism . Growth of cellulolytic bacteria on easily available carbon and nitrogen sources represents conditions far different from those of the natural lignocellulosic compounds.

Appl Environ Microbiol, 2001 Sep, 67(9), 3837 - 45
Kinetics and metabolism of cellulose degradation at high substrate concentrations in steady-state continuous cultures of Clostridium cellulolyticum on a chemically defined medium; Desvaux M et al.; The hydrolysis and fermentation of insoluble cellulose were investigated using continuous cultures of Clostridium cellulolyticum with increasing amounts of carbon substrate . At a dilution rate (D) of 0.048 h(-1), biomass formation increased proportionately to the cellulose concentration provided by the feed reservoir, but at and above 7.6 g of cellulose x liter(-1) the cell density at steady state leveled off . The percentage of cellulose degradation declined from 32.3 to 8.3 with 1.9 and 27.0 g of cellulose x liter(-1), respectively, while cellodextrin accumulation rose and represented up to 4.0% of the original carbon consumed . The shift from cellulose-limited to cellulose-sufficient conditions was accompanied by an increase of both the acetate/ethanol ratio and lactate biosynthesis . A kinetics study of C . cellulolyticum metabolism in cellulose saturation was performed by varying D with 18.1 g of cellulose x liter(-1) . Compared to cellulose limitation (M . Desvaux, E . Guedon, and H . Petitdemange, J . Bacteriol . 183:119-130, 2001), in cellulose-sufficient continuous culture (i) the ATP/ADP, NADH/NAD+, and q(NADH produced)/q(NADH used) ratios were higher and were related to a more active catabolism, (ii) the acetate/ethanol ratio increased while the lactate production decreased as D rose, and (iii) the maximum growth yield (Y(max)X/S) (40.6 g of biomass per mol of hexose equivalent) and the maximum energetic yield (Y(max)ATP) (19.4 g of biomass per mol of ATP) were lowered . C . cellulolyticum was then able to regulate and optimize carbon metabolism under cellulose-saturated conditions . However, the facts that some catabolized hexose and hence ATP were no longer associated with biomass production with a cellulose excess and that concomitantly lactate production and pyruvate leakage rose suggest the accumulation of an intracellular inhibitory compound(s), which could further explain the establishment of steady-state continuous cultures under conditions of excesses of all nutrients . The following differences were found between growth on cellulose in this study and growth under cellobiose-sufficient conditions (E . Guedon, S . Payot, M . Desvaux, and H . Petitdemange, Biotechnol . Bioeng . 67:327-335, 2000): (i) while with cellobiose, a carbon flow into the cell of as high as 5.14 mmol of hexose equivalent g of cells(-1) x h(-1) could be reached, the maximum entering carbon flow obtained here on cellulose was 2.91 mmol of hexose equivalent g of cells(-1) x h(-1); (ii) while the NADH/NAD+ ratio could reach 1.51 on cellobiose, it was always lower than 1 on cellulose; and (iii) while a high proportion of cellobiose was directed towards exopolysaccharide, extracellular protein, and free amino acid excretions, these overflows were more limited under cellulose-excess conditions . Such differences were related to the carbon consumption rate, which was higher on cellobiose than on cellulose.

Surv Ophthalmol, 2001 Jul-Aug, 46(1), 25 - 34
Clostridium botulinum and the ophthalmologist: a review of botulism, including biological warfare ramifications of botulinum toxin; Caya JG; The anaerobic bacterium Clostridium botulinum causes disease by elaborating an extremely potent neurotoxin that inhibits release of acetylcholine at presynaptic nerve endings, thereby resulting in a descending flaccid paralysis and autonomic nervous system dysfunction . Possible ophthalmological effects of this neurotoxin are many and typically constitute the earliest manifestations of botulism . This review summarizes the medical literature on botulism with regard to historical perspective, epidemiology, clinical manifestations, and treatment . Ophthalmological findings of botulism are tabulated and their frequencies are provided . Finally, the bioterrorism/biologic warfare ramifications of botulinum toxin are briefly discussed.

Vet Microbiol, 2001 Oct 22, 83(1), 61 - 9
Tetracycline-resistance genes of Clostridium perfringens, Clostridium septicum and Clostridium sordellii isolated from cattle affected with malignant edema; Sasaki Y et al.; The minimal inhibitory concentrations (MICs) of 10 antimicrobial agents against a total of 33 isolates of Clostridium perfringens, Clostridium septicum and Clostridium sordellii from cattle affected with malignant edema in Japan was determined . The low MIC activities of benzylpenicillin confirm the place of benzylpenicillin as the antibiotics of choice for treatment of malignant edema . Five (22%) of 23 C . septicum strains, five (71%) of seven C . perfringens strains and all strains of C . sordellii showed resistance to oxytetracycline . These oxytetracycline-resistant strains carried tetracycline-resistance genes {tetA(P), tetA408(P), tetB(P) and tetM} . The sequences of the tetracycline-resistance genes of some C . septicum strains were completely or nearly completely identical to those of strains belonging to other clostridiual species . This is the first report of resistance of C . septicum to tetracycline.

Gastroenterology, 2001 Sep, 121(3), 678 - 84
Claudin-4: a new target for pancreatic cancer treatment using Clostridium perfringens enterotoxin; Michl P et al.; BACKGROUND & AIMS: Recently, several members of the claudin family have been identified as integral constituents of tight junctions . Using expression profiling, we previously found claudin-4 to be overexpressed in pancreatic cancer . Because claudin-4 has been described as a receptor for the cytotoxic Clostridium perfringens enterotoxin (CPE), we investigated the effect of CPE on pancreatic cancer cells . METHODS: Expression of claudin-4 was analyzed by Northern blots . In vitro toxicity of CPE was determined by trypan blue exclusion and the (86)Rb-release assay . The in vivo effect of CPE was studied in claudin-4-expressing nude mouse xenografts of the Panc-1 cell line . RESULTS: Expression analyses showed that claudin-4 was overexpressed in most pancreatic cancer tissues and cell lines and several other gastrointestinal tumors . CPE led to an acute dose-dependent cytotoxic effect, restricted to claudin-4-expressing cells and dependent on claudin-4 expression levels . Furthermore, transforming growth factor beta was identified as a negative modulator of both claudin-4 expression and susceptibility to CPE . In vivo, intratumoral injections of CPE in Panc-1 xenografts led to large areas of tumor cell necrosis and significant reduction of tumor growth . CONCLUSIONS: Our findings suggest that targeting claudin-4-expressing tumors with CPE represents a promising new treatment modality for pancreatic cancer and other solid tumors.

J Biomol NMR, 2001 Jul, 20(3), 195 - 202
Mapping the ligand binding site at protein side-chains in protein-ligand complexes through NOE difference spectroscopy; Eichmuller C et al.; This report describes a novel NMR approach for mapping the interaction surface between an unlabeled ligand and a 13C,15N-labeled protein . The method relies on the spin inversion properties of the dipolar relaxation pathways and records the differential relaxation of two spin modes, where ligand and protein 1H magnetizations are aligned either in a parallel or anti-parallel manner . Selective inversion of protein protons is achieved in a straightforward manner by exploiting the one-bond heteronuclear scalar couplings (1J(CH), 1J(NH)) . Suppression of indirect relaxation pathways mediated by bulk water or rapidly exchanging protons is achieved by selective inversion of the water signal in the middle of the NOESY mixing period . The method does not require deuteration of the protein or well separated spectral regions for the protein and the ligand, respectively . Additionally, in contrast to previous methods, the new experiment identifies side-chain enzyme ligand interactions along the intermolecular binding interface . The method is demonstrated with an application to the B12-binding subunit of glutamate mutase from Clostridium tetanomorphum for which NMR chemical shift changes upon B12-nucleotide loop binding and a high-resolution solution structure are available.

Eur Radiol, 2001, 11(8), 1433 - 4
The accordion sign at CT: report of a case of Crohn's disease with diffuse colonic involvement; Mountanos GI et al.; The accordion sign is a finding that could be seen on CT scans of the abdomen in patients who have received oral contrast material . Initially, it was described as a sign specific of Clostridium difficile colitis, but it is also reported to represent a sign of diffuse colonic edema of several other etiologies . We report a case of a patient with Crohn's pancolitis whose abdominal CT scan presented the accordion sign throughout the entire large bowel together with signs of Crohn's disease of the small bowel.

Facial Plast Surg, 2001 Feb, 17(1), 11 - 20
Botulinum A exotoxin for rejuvenation of the upper third of the face; Carucci JA et al.; Botulinum A exotoxin, derived from the gram-positive anaerobe Clostridium botulinum, has proven to be safe and effective in the temporary treatment of facial rhytides . In order to obtain reproducible results and avoid complications, it is necessary to understand the relevant physiology and anatomic relationships . Technical considerations including injection technique, dilution, storage, and potential complications will be discussed.

J Am Soc Nephrol, 2001 Sep, 12(9), 1853 - 61
Expression of connective tissue growth factor in human renal fibroblasts: regulatory roles of RhoA and cAMP; Heusinger-Ribeiro J et al.; The induction of connective tissue growth factor (CTGF) was investigated in a human renal fibroblast cell line that exhibited many characteristics of primary human renal fibroblasts . Induction of CTGF mRNA was observed after treatment of the cells with transforming growth factor-beta (TGF-beta) or, even more prominently, lysophosphatidic acid (LPA) . LPA induced a rapid transient increase in CTGF mRNA expression, with maximal levels being observed after 1 to 2 h . This increase was accompanied by CTGF protein synthesis . Induction of CTGF was insensitive to pertussis toxin and was not dependent on the activation of p42/44 mitogen-activated protein kinases . Inhibition of the proteins of the Rho family with toxin B from Clostridium difficile abrogated basal and LPA-mediated induction of CTGF . Specific targeting of RhoA with C3 exotoxin or of the Rho kinases with the inhibitor Y-27632 similarly prevented induction of CTGF, implicating RhoA as a signaling module downstream of LPA . Inhibition of RhoA depolymerized the actin cytoskeleton, as did treatment with cytochalasin D . Preincubation of the human renal fibroblasts with cytochalasin D prevented induction of CTGF by LPA, indicating a strong contribution of an intact cytoskeleton . Interference with RhoA signaling similarly inhibited the induction of CTGF by TGF-beta . Elevation of intracellular levels of cAMP and thus activation of protein kinase A prevented induction of CTGF expression . The cytoskeletal effects of cAMP, however, were reversed by LPA . These data indicate complex interactions involving LPA-mediated activation of RhoA- and protein kinase A-dependent signaling pathways . The data thus demonstrate the regulatory functions of the small GTPase RhoA and of an intact cytoskeleton in the expression of CTGF after stimulation with LPA or TGF-beta . Analogous signal transduction pathways were previously demonstrated in rat mesangial cells, suggesting a more general role for RhoA in the regulation of CTGF expression.

Am J Physiol Gastrointest Liver Physiol, 2001 Sep, 281(3), G635 - 44
Persistent epithelial dysfunction and bacterial translocation after resolution of intestinal inflammation; Asfaha S et al.; Epithelial secretion may play an important role in reducing bacterial colonization and translocation in intestine . If so, secretory dysfunction could result in increased susceptibility to infection and inflammation . We investigated whether long-term colonic secretory dysfunction occurs after a bout of colitis and if this is accompanied by an increase in bacterial colonization and translocation . Rats were studied 6 wk after induction of colitis with trinitrobenzene sulfonic acid when inflammation had completely resolved, and epithelial permeability was normal . Intestinal loops were stimulated with either Clostridium difficile toxin A or a phosphodiesterase inhibitor . In vitro, colonic tissue from previously sensitized rats was exposed to antigen (ovalbumin) . Secretory responses to all three stimuli were suppressed in rats that had previously had colitis . These rats exhibited increased (16-fold) numbers of colonic aerobic bacteria and increased (>3-fold) bacterial translocation, similar to results in rats studied after resolution of enteritis . Postcolitis bacterial translocation was prevented by daily treatment with an inhibitor of inducible nitric oxide synthase . This study demonstrates that intestinal inflammation results in prolonged impairment of colonic epithelial secretion, which may contribute to increases in bacterial load and bacterial translocation . Epithelial dysfunction of this type could underlie an increased propensity for further bouts of inflammation, a hallmark of diseases such as inflammatory bowel disease.

Int J Food Microbiol, 2001 Aug 5, 67(3), 247 - 52
Microbiological and biochemical studies on Gergoush fermentation; Sherfi SA et al.; According to the results obtained, three steps in Gergoush fermentation were identified . Step one is the primary starter preparation and comprises a 12-15 h propagation of the natural thermotolerant bacterial flora of the legume ingredient of Gergoush using the legume and boiled milk as a propagation medium . This primary starter is then used in step two to inoculate a wheat flour dough to produce the adapted starter in a 1-2-h fermentation time . The adapted starter is finally used in step three to raise the main Gergoush dough . In all of the three steps of Gergoush fermentation, three genera of bacteria dominated . They were tentatively identified as lactic acid bacteria, Bacillus spp . and Clostridium spp . Their counts reached a maximum in the primary starter stage of 2.2 x 10(7), 2.8 x 10(8) and 7.3 x 10(7) CFU/g, respectively . These bacteria produced lactic, acetic and butyric acids . The concentrations of the acids were maximum in the primary starter and reached values of 0.6%, 0.4% and 0.5%, respectively, and the pH decreased from 6.1 to 4.1 . Baked Gergoush has a pH of about 5 and contains about 59% starch, 16% protein, 18% fat, 6.5% water and 0.5% ash.

Syst Appl Microbiol, 2001 Jul, 24(2), 149 - 56
Sequence heterogeneity of the ten rRNA operons in Clostridium perfringens; Shimizu T et al.; We have cloned and sequenced rRNA operons of Clostridium perfringens strain 13 and analyzed the sequence structure in view of the phylogenesis . The organism had ten copies of rRNA operons all of that comprised of 16S, 23S and 5S rDNAs except for one operon . The operons clustered around the origin of replication, ranging within one-third of the whole genome sequence as it is arranged in a circle . Seven operons were transcribed in clockwise direction, and the remaining three were transcribed in counter clockwise direction assuming that the gyrA was transcribed in clockwise direction . Two of the counter clockwise operons contained tRNA(Ile) genes between the 16S and 23S rDNAs, and the other had a tRNA(Ile) genes between the 16S and 23S rDNAs and a tRNA(Asn) gene in the place of the 5S rDNA . Microheterogeneity was found within the rRNA structural genes and spacer regions . The length of each 16S, 23S and 5S rDNA were almost identical among the ten operons, however, the intergenic spacer region of 16S-23S and 23S-5S were variable in the length depending on loci of the rRNA operons on the chromosome . Nucleotide sequences of the helix 19, helix 19a, helix 20 and helix 21 of 23S rDNA were divergent and the diversity appeared to be correlated with the loci of the rRNA operons on the chromosome.

J Bacteriol, 2001 Sep, 183(18), 5431 - 5
Cohesin-dockerin interactions of cellulosomal subunits of Clostridium cellulovorans; Park JS et al.; The cellulosome of Clostridium cellulovorans consists of three major subunits: CbpA, EngE, and ExgS . The C . cellulovorans scaffolding protein (CbpA) contains nine hydrophobic repeated domains (cohesins) for the binding of enzymatic subunits . Cohesin domains are quite homologous, but there are some questions regarding their binding specificity because some of the domains have regions of low-level sequence similarity . Two cohesins which exhibit 60% sequence similarity were investigated for their ability to bind cellulosomal enzymes . Cohesin 1 (Coh1) was found to contain amino acid residues corresponding to amino acids 312 to 453 of CbpA, which contains a total of 1,848 amino acid residues . Coh6 was determined to contain amino acid residues corresponding to residues 1113 to 1254 of CbpA . By genetic construction, these two cohesins were each fused to MalE, producing MalE-Coh1 and MalE-Coh6 . The abilities of two fusion proteins to bind to EngE, ExgS, and CbpA were compared . Although MalE-Coh6 could bind EngE and ExgS, little or no binding of the enzymatic subunits was observed with MalE-Coh1 . Significantly, the abilities of the two fusion proteins to bind CbpA were similar . The binding of dockerin-containing enzymes to cohesin-containing proteins was suggested as a model for assembly of cellulosomes . In our examination of the role of dockerins, it was also shown that the binding of endoglucanase B (EngB) to CbpA was dependent on the presence of EngB's dockerin . These results suggest that different cohesins may function with differing efficiency and specificity, that cohesins may play some role in the formation of polycellulosomes through Coh-CbpA interactions, and that dockerins play an important role during the interaction of cellulosomal enzymes and cohesins present in CbpA.

Peptides, 2001 Sep, 22(9), 1439 - 46
The capsaicin VR1 receptor mediates substance P release in toxin A-induced enteritis in rats; McVey DC et al.; The mechanism by which Clostridium difficile toxin A causes substance P (SP) release and subsequent inflammation in the rat ileum is unknown . Pretreatment with the vanilloid receptor subtype 1 (VR1) antagonist, capsazepine, before toxin A administration significantly inhibited toxin A-induced SP release and intestinal inflammation . Intraluminal administration of the VR1 agonist capsaicin caused intestinal inflammation similar to the effects of toxin A . Pretreatment with capsazepine before capsaicin administration also significantly inhibited capsaicin-induced intestinal inflammation . These results suggest that intraluminal toxin A causes SP release from primary sensory neurons via stimulation of VR1 receptors resulting in intestinal inflammation.

Singapore Med J, 2001 May, 42(5), 214 - 6
Fatal chemotherapy associated Clostridium difficile infection--a case report; Wong AS et al.; Clostridium difficile associated diarrhoea or Pseudomembranous colitis occasionally occurs without prior antibiotic usage.While the association of chemotherapy and Clostridium difficile infection has previously been well recorded, the true incidence is unknown . We report a case of Clostridium difficile associated diarrhoea after chemotherapy for lung cancer.The fatal outcome in this case and the increasing use of chemotherapy in this country highlights the need to have a high index of suspicion in any case of unexplained diarrhoea post chemotherapy . A review of the literature is presented.

Clin Infect Dis, 2001 Sep 15, 33(6), 786 - 91 Epub 2001 Aug 10.
Clostridium difficile infection in patients with neutropenia; Gorschluter M et al.; Clostridium difficile is the most important cause of nosocomial infectious diarrhea . The importance of C . difficile-associated diarrhea (CDAD) has been poorly investigated in patients with neutropenia who have hematologic malignancies . A retrospective chart review of all patients treated in the leukemia ward of a university medical center during 1991-2000 determined that 875 courses of myelosuppressive chemotherapy were administered . CDAD occurred in 7.0% of all cycles . In 8.2% of the patients, severe enterocolitis developed . Two patients died while they had diarrhea . However, in no patient was C . difficile infection clinically considered to be the primary cause of death . The response rate to oral metronidazole was 90.9% . These data indicate that C . difficile infection is not rare and should be suspected whenever a hospitalized patient with neutropenia develops diarrhea . Oral metronidazole can be recommended as initial drug of choice for treatment of patients with neutropenia who have hematologic malignancies and CDAD.

Pflugers Arch, 2001 Aug, 442(5), 675 - 87
Role of actin filaments in endothelial cell-cell adhesion and membrane stability under fluid shear stress; Schnittler HJ et al.; Clostridium botulinum C2 toxin (C2 toxin) and purified ADP-ribosylated-alpha-actin (ADP-r-alpha-actin) cause specific actin depolymerisation in living cells . This effect was used to investigate the actin microfilament system with particular emphasis on cell-cell adhesion and plasma membrane integrity in endothelial cells . C2 toxin caused time- and dose-dependent (15-100 ng/ml) changes in endothelial surface morphology (investigated by atomic force microscopy), intercellular gap formation and cell detachment under shear stress . Low concentrations of C2 toxin (1.5 ng/ml), however, did not induce cell detachment but inhibited shear stress-dependent cell alignment . Gap formation as well as cell loss under shear stress was also observed in cells microinjected with purified ADP-r-alpha-actin . Intercellular gap formation was mediated by increased alpha-catenin solubility (40%) due to actin filament depolymerisation . Disintegration of plasma membranes (measured by LDH release) and cell fragmentation during simultaneous exposure to shear stress and C2 toxin were due to a loss of more than 50% of membrane-associated actin . These data show that small disturbances in actin dynamics inhibit shear stress-dependent cell alignment; that depolymerisation of actin filaments increases the solubility of alpha-catenin, thus resulting in cell dissociation and that actin filaments of the membrane cytoskeleton are required to protect the cells from haemodynamic injury such as shear stress . Together, the study shows a heterogeneous regulation of actin filament dynamics at subcellular locations . Junction-associated actin filaments displayed the highest sensitivity whereas stress fibres were far more stable.

J Food Prot, 2001 Aug, 64(8), 1252 - 4
Occurrence of anaerobic bacterial, clostridial, and Clostridium perfringens spores in raw goose livers from a poultry processing plant in Hungary; Turcsan J et al.; Anaerobic bacterial, clostridial, and Clostridium perfringens spores were enumerated in raw goose liver samples taken after evisceration of the birds (EB) in the slaughterhouse and after removal of blood vessels from the liver (RBVL) in the cannery . The samples taken after RBVL had significantly higher (P < 0.05) spore counts than did those taken after EB, indicating contamination of livers during processing . The number of C . perfringens spores was one log cycle higher in the samples taken after RBVL than in those taken after EB (P < 0.05) . The confirmation of C . perfringens according to the profiles of Rapid ID 32 A tests was carried out by means of the ATB Plus computer program . With an identification percentage of 99.9 and a T-value of 0.65, the suspect colonies proved to be C . perfringens . Therefore, the importance of an appropriate cleaning and sanitation program and of personnel hygiene should be emphasized in the industry.

Rev Med Chil, 2001 Jun, 129(6), 620 - 5
{Diagnosis of Clostridium difficile diarrhea: in search of a more efficient clinical focus}; Alvarez M et al.; BACKGROUND: The clinical parameters for the suspicion of Clostridium difficile infections, namely the use of antimicrobials and diarrhea, have a low predictive value for the diagnosis . AIM: To search other clinical variables and determine a clinical prediction model for (Clostridium difficile diarrhea . PATIENTS AND METHODS: All patients to whom a Clostridium difficile study was requested, were prospectively studied during 5 months . Clinical variables of these patients were registered . The diagnosis of Clostridium difficile was done using the cytotoxicity test in fibroblast cultures . RESULTS: Ninety two patients were analyzed and in 26, the diagnosis of Clostridium difficile was confirmed . A logistic regression model disclosed an age over 60 years old, the presence of mucus in the stools and a temperature over 37.8 degrees C in the previous 24 h, as significant predictors of the infection . The correlation of the model, between the predicted probability and the observed condition, was 81.5% . CONCLUSIONS: The presence of the clinical variables identified in this study are associated with a high probability of an infection by Clostridium difficile in patients with diarrhea and the recent use of antimicrobials.

J Biol Chem, 2001 Oct 19, 276(42), 39123 - 31 Epub 2001 Aug 16.
An essential role for Rac/Cdc42 GTPases in cerebellar granule neuron survival; Linseman DA et al.; Rho family GTPases are critical molecular switches that regulate the actin cytoskeleton and cell function . In the current study, we investigated the involvement of Rho GTPases in regulating neuronal survival using primary cerebellar granule neurons . Clostridium difficile toxin B, a specific inhibitor of Rho, Rac, and Cdc42, induced apoptosis of granule neurons characterized by c-Jun phosphorylation, caspase-3 activation, and nuclear condensation . Serum and depolarization-dependent survival signals could not compensate for the loss of GTPase function . Unlike trophic factor withdrawal, toxin B did not affect the antiapoptotic kinase Akt or its target glycogen synthase kinase-3beta . The proapoptotic effects of toxin B were mimicked by Clostridium sordellii lethal toxin, a selective inhibitor of Rac/Cdc42 . Although Rac/Cdc42 GTPase inhibition led to F-actin disruption, direct cytoskeletal disassembly with Clostridium botulinum C2 toxin was insufficient to induce c-Jun phosphorylation or apoptosis . Granule neurons expressed high basal JNK and low p38 mitogen-activated protein kinase activities that were unaffected by toxin B . However, pyridyl imidazole inhibitors of JNK/p38 attenuated c-Jun phosphorylation . Moreover, both pyridyl imidazoles and adenoviral dominant-negative c-Jun attenuated apoptosis, suggesting that JNK/c-Jun signaling was required for cell death . The results indicate that Rac/Cdc42 GTPases, in addition to trophic factors, are critical for survival of cerebellar granule neurons.

J Biol Chem, 2001 Oct 19, 276(42), 38619 - 27 Epub 2001 Aug 15.
Activation of protein kinase D by signaling through Rho and the alpha subunit of the heterotrimeric G protein G13; Yuan J et al.; Protein kinase D (PKD/PKCmu) immunoprecipitated from COS-7 cells transiently transfected with either a constitutively active mutant of Rho (RhoQ63L) or the Rho-specific guanine nucleotide exchange factor pOnco-Lbc (Lbc) exhibited a marked increase in basal activity . Addition of aluminum fluoride to cells co-transfected with PKD and wild type Galpha(13) also induced PKD activation . Co-transfection of Clostridium botulinum C3 toxin blocked activation of PKD by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13) . Treatment with the protein kinase C inhibitors GF I or Ro 31-8220 prevented the increase in PKD activity induced by RhoQ63L, Lbc, or aluminum fluoride-stimulated Galpha(13) . PKD activation in response to Galpha(13) signaling was also completely prevented by mutation of Ser-744 and Ser-748 to Ala in the kinase activation loop of PKD . Co-expression of C . botulinum C3 toxin and a COOH-terminal fragment of Galpha(q) that acts in a dominant-negative fashion blocked PKD activation in response to agonist stimulation of bombesin receptor . Expression of the COOH-terminal region of Galpha(13) also attenuated PKD activation in response to bombesin receptor stimulation . Our results show that Galpha(13) contributes to PKD activation through a Rho- and protein kinase C-dependent signaling pathway and indicate that PKD activation is mediated by both Galpha(q) and Galpha(13) in response to bombesin receptor stimulation.

Drug Resist Updat, 1999 Oct, 2(5), 289 - 294
Molecular basis of metronidazole resistance in pathogenic bacteria and protozoa; Land KM et al.; The molecular basis of metronidazole resistance has been examined in anaerobic bacteria, such as Bacteroides, Clostridium, and Helicobacter, and anaerobic parasitic protists such as Giardia, Entamoeba, and trichomonads . A variety of enzymatic and cellular alterations have been shown to correlate with metronidazole susceptibility in these pathogens; however, a common theme has been revealed . Resistant cells are typically deficient in drug activation . The frequent correlation between metronidazole resistance and ineffective drug activation suggests that drug resistance is the result of modification of proteins involved in drug activation .

Diagn Microbiol Infect Dis, 2001 Jul, 40(3), 103 - 6
Isolation of Clostridium innocuum from cases of recurrent diarrhea in patients with prior Clostridium difficile associated diarrhea; Ackermann G et al.; Clostridium innocuum isolates resistant to vancomycin (MIC values of 16-24 microg/mL) were isolated from three patients with recurrent Clostridium difficile -associated diarrhea (CDAD) . We discuss the clinical significance and problems associated with the identification and differentiation of these two clostridial species, which may result in misdiagnosis of patients.

Res Microbiol, 2001 Jul-Aug, 152(6), 563 - 7
Recombinant Listeria strains producing the nontoxic L-chain of botulinum neurotoxin A in a soluble form; Vertiev YV et al.; Fragments of Clostridium botulinum neurotoxin A (BoNT/A) gene (botA) were expressed in Listeria monocytogenes ATCC10527 to produce the L-chain of the toxin in a soluble form . A shuttle vector pAT19 (EmR) was used to make plasmid pAT-RL containing a botA gene fragment placed under C . botulinum ntnH-gene promoter control . The plasmid also contained a C . botulinum botR/A gene, a positive transcriptional regulator of botA . The cytoplasmic fraction of the L . monocytogenes (pAT-RL) strain was found to contain up to 3 mg/L of a soluble protein of expected size and immunologically positive towards BoNT antibodies . This is the first evidence of heterologous botA gene expression producing a soluble safe derivative of botulinum neurotoxin A needed as a molecular tool for exploratory research in neurosciences as well as a basis for raising protective immunity in humans.

Folia Microbiol (Praha), 2001, 46(1), 76 - 8
Chitinolytic bacteria of the mammal digestive tract; Simunek J et al.; Chitinolytic bacteria were isolated from the digestive tract of different mammals and characterized . All isolates were facultatively anaerobic, long Gram-positive, straight rods resembling Clostridium sp . Only one isolate consisted of Gram-positive ovoid cells . All cultures grew on glucose, N-acetylglucosamine, glucosamine, galactose, starch, hemicellulose and xylan . Fermentation products were mainly formate, acetate, butyrate and lactate . The isolates were identified as Clostridium sartagoforme (2 species), C . aminovalericum, C . bifermentans and Enterococcus durans (1 isolate of each species) . Exocellular fractions of all strains exhibited higher activities of all enzymes than cellular ones . Inductive effects of hemicelluloses, pectin and laminarine on chitinases were demonstrated . High exocellular endochitinase activity was found in cultures grown on chitin . N-Acetylglucosaminidase activity was low with the exception of exocellular fractions of two strains of C . sartagoforme.

Infect Immun, 2001 Sep, 69(9), 5487 - 93
pH-enhanced cytopathic effects of Clostridium sordellii lethal toxin; Qa'Dan M et al.; Clostridium sordellii lethal toxin (TcsL) is a large clostridial toxin (LCT) that glucosylates Ras, Rac, and Ral . TcsL differs from other LCTs because it modifies Ras, which does not cycle from cytosol to membrane . By using a suite of inhibitors, steps in cell entry by TcsL were dissected, and entry appears to be dependent on endosomal acidification . However, in contrast to TcdB, TcsL was substantially slower in its time course of entry . TcsL cytopathic effects (CPE) were blocked by bafilomycin A1 and neutralized by antiserum up to 2 h following treatment of cells with the toxin . The slow time course of intoxication and relatively high cytopathic dose were alleviated by exposing TcsL to acid pH, resulting in a time course similar to that of TcdB . The optimal pH range for activation was 4.0 to 5.0, which increased the rate of intoxication over 5-fold, lowered the minimal intoxicating dose by over 100-fold, and allowed complete substrate modification within 2 h, as shown by differential glucosylation . Fluorescence analysis of TcsL with 2-(p-toluidinyl) naphthalene-6-sulfonic acid as a probe suggested the acid pH stimulated a hydrophobic transition in the protein, a likely prelude to membrane insertion . Finally, acid entry by TcsL caused TcdB-like morphological changes in CHO cells, which suggesting that acid activation may impact substrate recognition profiles for TcsL.

J Ind Microbiol Biotechnol, 2001 May, 26(5), 290 - 5
Soy molasses as fermentation substrate for production of butanol using Clostridium beijerinckii BA101; Qureshi N et al.; Spray-dried soy molasses (SDSM) contains the sugars dextrose, sucrose, fructose, pinitol, raffinose, verbascose, melibiose, and stachyose . Of the 746 g kg(-1) total sugars in SDSM, 434 g kg(-1) is fermentable using Clostridium beijerinckii BA101 . SDSM was used to produce acetone, butanol, and ethanol (ABE) by C . beijerinckii BA101 in batch cultures . Using 80 g l(-1) SDSM, 10.7 g l(-1) ABE was produced in P2 medium . Higher concentrations of SDSM resulted in poor solvent production due to the presence of excessive salt and inhibitory components . C . beijerinckii BA101 in SDSM at 80 g l(-1) concentration produced 22.8 g l(-1) ABE when supplemented with 25.3 g l(-1) glucose . SDSM contains 57.4 g kg(-1) mineral ash and 2% tri-calcium phosphate . Tri-calcium phosphate up to 43.1 g l(-1) was not inhibitory and at a tri-calcium phosphate concentration of 28.8 g l(-1), the culture produced more solvents (30.1 g l(-1)) than the control experiment (23.8 g l(-1)) . In contrast, sodium chloride was a strong inhibitor of C . beijerinckii BA101 cell growth . At a concentration of 10 g l(-1) sodium chloride, a maximum cell concentration of 0.6 g l(-1) was achieved compared to 1.7 g l(-1) in the control experiment . The effects of two salts on specific growth rate constant (mu) and specific rate of ABE production (nu) for C . beijerinckii BA101 were examined.

J Biol Chem, 2001 Oct 5, 276(40), 37141 - 8 Epub 2001 Jul 30.
alpha 2,3-Sialylation of terminal GalNAc beta 1-3Gal determinants by ST3Gal II reveals the multifunctionality of the enzyme . The resulting Neu5Ac alpha 2-3GalNAc linkage is resistant to sialidases from Newcastle disease virus and Streptococcus pneumoniae; Toivonen S et al.; Enzymatic alpha 2,3-sialylation of GalNAc has not been described previously, although some glycoconjugates containing alpha 2,3-sialylated GalNAc residues have been reported . In the present experiments, recombinant soluble alpha 2,3-sialyltransferase ST3Gal II efficiently sialylated the X(2) pentasaccharide GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, globo-N-tetraose GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and the disaccharide GalNAc beta 1-3Gal in vitro . The purified products were identified as Neu5Ac alpha 2-3GalNAc beta 1-3Gal beta 1-4GlcNAc beta 1-3Gal beta 1-4Glc, Neu5Ac alpha 2-3GalNAc beta 1-3Gal alpha 1-4Gal beta 1-4Glc, and Neu5Ac alpha 2-3GalNAc beta 1-3Gal, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, enzymatic degradations, and one- and two-dimensional NMR-spectroscopy . In particular, the presence of the Neu5Ac alpha 2-3GalNAc linkage was firmly established in all three products by a long range correlation between Neu5Ac C2 and GalNAc H3 in heteronuclear multiple bond correlation spectra . Collectively, the data describe the first successful sialyltransfer reactions to the 3-position of GalNAc in any acceptor . Previously, ST3Gal II has been shown to transfer to the Gal beta 1-3GalNAc determinant . Consequently, the present data show that the enzyme is multifunctional, and could be renamed ST3Gal(NAc) II . In contrast to ST3Gal II, ST3Gal III did not transfer to the X(2) pentasaccharide . The Neu5Ac alpha 2-3GalNAc linkage of sialyl X(2) was cleaved by sialidases from Arthrobacter ureafaciens and Clostridium perfringens, but resisted the action of sialidases from Newcastle disease virus and Streptococcus pneumoniae . Therefore, the latter two enzymes cannot be used to differentiate between Neu5Ac alpha 2-3GalNAc and Neu5Ac alpha 2-6GalNAc linkages, as has been assumed previously.

Toxicon, 2001 Oct, 39(10), 1515 - 31
Botulinum neurotoxin types B and E: purification, limited proteolysis by endoproteinase Glu-C and pepsin, and comparison of their identified cleaved sites relative to the three-dimensional structure of type A neurotoxin; Prabakaran S et al.; Botulinum neurotoxin (NT) serotypes B and E are approximately 150 kDa proteins . Isolated from the liquid culture of Clostridium botulinum the NT type E is a single chain protein while the NT type B, from the proteolytic strain of the bacteria, is a mixture of dichain (nicked within a disulfide loop located about one-third the way from the N-terminus to the C-terminus) protein and its precursor single-chain protein . Endoproteinase Glu-C (EC 3.4.21.19) and pepsin (EC 3.4.23.1) were used for controlled digestion of NT types B and E; the amino acid residues flanking many of the cleavable peptide bonds were identified and the corresponding proteolytic fragments partially characterized . Chemical identification of 82 and 108 residues of types B and E NT, respectively, revealed that the residue 738 and 1098 in type E NT, identified as Leu and Asn, respectively, differ from those deduced from nucleotide sequences . Several fragments overlapped spanning various segments of the NT's functional domains; they appear to have potential for structure-function studies of the NT . The cleavage sites were compared with the previously determined proteolyzed sites on NT types A and E . The cleavage sites of the NT types A, B and E, all exposed on the protein surface, were scrutinized in the context of the three-dimensional structure of crystallized NT type A {Lacy, D.B., Stevens, R.C., 1999 . J . Mol . Biol . 291, 1091-1104} . Detailed procedures for isolation of pure NT types B and E in large quantities (average yield 92 and 62 mg, respectively) suitable for crystallization are reported.

Biochemistry, 2001 Aug 7, 40(31), 9167 - 76
Clostridium thermocellum Xyn10B carbohydrate-binding module 22-2: the role of conserved amino acids in ligand binding; Xie H et al.; The majority of plant cell wall hydrolases are modular enzymes which, in addition to a catalytic module, possess one or more carbohydrate-binding modules (CBMs) . These carbohydrate-active enzymes and their constituent modules have been classified into a number of families based upon amino acid sequence similarity . The Clostridium thermocellum xylanase, Xyn10B, contains two CBMs that belong to family 22 (CBM22) . The crystal structure of the C-terminal CBM22 (CBM22-2) was determined in a previous study {Charnock, S . J., et al . (2000) Biochemistry 39, 5013--5021} and revealed a surface cleft which presents several conserved residues that are implicated in ligand binding . These amino acids have been substituted and the structure and biochemical properties of the mutants analyzed . The data show that R25A, W53A, Y103A, Y136A, and E138A exhibit greatly reduced affinity for xylotetraose relative to that of the wild-type protein . Conversely, mutations Y103F and Y136F have little effect on ligand binding . Using thermodynamic, X-ray, and NMR measurements on the mutants, we show that the cleft of CBM22-2 does indeed form the ligand-binding site . Trp 53 and Tyr 103 most likely participate in hydrophobic stacking interactions with the ligand, while Glu 138 makes one or more important hydrogen bonds with the tetrasaccharide . Although Arg 25 and Tyr 136 are likely to form hydrogen bonds with the ligand, they are also shown to play a critical role in maintaining the structural integrity of the binding cleft.

Transplantation, 2001 Jul 27, 72(2), 338 - 40
Bile leak from duct of Luschka after liver transplantation; Albishri SH et al.; BACKGROUND: We report a case of bile leak from an accessory duct of Luschka during cholecystectomy during liver transplantation . METHODS: Radiological findings suggested that the collection was septated . An intra-operative cholangiogram was obtained by cannulation of the accessory hepatic duct . RESULTS: An infected biloma with Clostridium perfringens was drained surgically . The bile leak that emanated from the gall bladder fossa was found to communicate with an accessory right hepatic duct draining a segmental duct in the right liver lobe . The bile leak resolved completely after direct suture of the accessory duct . CONCLUSIONS: Excessive use of electrocautery to the liver bed during donor cholecystectomy may injure subcapsular ducts in the gallbladder fossa . In liver transplantation, dissection should be kept close to the serosal lining of the gall bladder, preserving the areolar tissue in the gall bladder bed, to avoid injury to the duct of Luschka.

J Biol Chem, 2001 Oct 5, 276(40), 37649 - 58 Epub 2001 Jul 26.
The uptake and degradation of matrix-bound lipoproteins by macrophages require an intact actin Cytoskeleton, Rho family GTPases, and myosin ATPase activity; Sakr SW et al.; A key cellular event in atherogenesis is the interaction of macrophages with lipoproteins in the subendothelium . In vivo, these lipoproteins are bound to matrix and often aggregated, yet most cell-culture experiments explore these events using soluble monomeric lipoproteins . We hypothesized that the internalization and degradation of matrix-retained and aggregated low density lipoprotein (LDL) by macrophages may involve the actin-myosin cytoskeleton in a manner that distinguishes this process from the endocytosis of soluble LDL . To explore these ideas, we plated macrophages on sphingomyelinase-aggregated LDL bound to smooth muscle cell-derived matrix in the presence of lipoprotein lipase . The macrophages internalized and degraded the LDL, which was mediated partially by the LDL receptor-related protein . Cytochalasin D and latrunculin A, which block actin polymerization, markedly inhibited the uptake and degradation of matrix-retained LDL but not soluble LDL . Inhibition of Rho family GTPases by Clostridium difficile toxin B blocked the degradation of matrix-retained and aggregated LDL by >90% without any inhibition of soluble LDL degradation . However, specific inhibition of Rho had no effect, suggesting the importance of Rac1 and Cdc42 . Degradation of matrix-retained, but not soluble, LDL was also blocked by inhibitors of tyrosine kinase, phosphatidylinositol 3-kinase, and myosin ATPase . These findings define fundamental cytoskeletal pathways that may be involved in macrophage foam cell formation in vivo but have been missed by the use of previous cell culture models.

Semin Cutan Med Surg, 2001 Jun, 20(2), 127 - 36
Botulinum toxin type B: an overview of its biochemistry and preclinical pharmacology; Callaway JE et al.; Produced by Clostridium botulinum, botulinum toxins are high molecular weight protein complexes consisting of the neurotoxin and additional nontoxic proteins that function to protect the toxin molecule . The neurotoxin acts to inhibit the release of acetylcholine at the neuromuscular junction, causing muscle paralysis . Purified toxin complexes have found a niche in the treatment of clinical disorders involving muscle hyperactivity . The different serotypes are structurally and functionally similar; however, specific differences in neuronal acceptor binding sites, intracellular enzymatic sites, and species sensitivities suggest that each serotype is its own unique pharmacologic entity . Recently, botulinum toxin type B has been developed as a liquid formulation to avoid the lyophilization (vacuum-drying) and reconstitution processes associated with decreasing the potency and stability of current type A toxin preparations . Biochemical tests were conducted to evaluate the quality of toxin in this formulation . In 3 consecutive manufacturing lots, the botulinum toxin type B complex was found to be highly purified, intact, uniform, and consistent from lot to lot . Also, it showed long-term stability at refrigerator and room temperatures (2 to 25 degrees C) . Electrophysiologic studies in cynomolgus monkeys showed that botulinum toxin type B is effective in paralyzing injected muscle groups, with minimal spread to relatively distant noninjected muscles.

J Infect Dis, 2001 Sep 1, 184(5), 648 - 52 Epub 2001 Jul 26.
Role of inducible cyclooxygenase and prostaglandins in Clostridium difficile toxin A-induced secretion and inflammation in an animal model; Alcantara C et al.; Cyclooxygenase (Cox)-2 expression and inhibition were investigated in a rabbit ileal loop model of Clostridium difficile colitis and diarrhea . Intestinal tissue stimulated with C . difficile toxin A showed up-regulation of Cox-2 expression in lamina propria macrophages and elevated prostaglandin levels . Toxin A-stimulated loops exhibited severe inflammation and increased secretory volume . Celecoxib, a specific Cox-2 inhibitor, significantly reduced toxin A-induced prostaglandin production . Furthermore, celecoxib (> or =0.02 mg/mL) blocked both histologic damage (mean histologic grade, 1.25 vs . 3.44 in rabbits receiving toxin A alone; P<.0005) and secretion (volume:length ratio, 0.18 vs . 0.72 in those receiving toxin A alone; P=.002) in toxin A-stimulated loops in a dose-related manner . Thus, toxin A induced expression of Cox-2 in the host, and prostaglandins produced through Cox-2 were involved in the mediation of the increased secretion of electrolytes and water and the inflammatory response induced by toxin A.

J Clin Microbiol, 2001 Aug, 39(8), 2846 - 9
Evaluation of methods for detection of toxins in specimens of feces submitted for diagnosis of Clostridium difficile-associated diarrhea; O'Connor D et al.; Clostridium difficile is the principal pathogen associated with hospital-acquired acute diarrheal disease . We have evaluated the performances of six approaches for diagnosis of C . difficile-associated diarrhea (CDAD) . Consecutive stool specimens (n = 200) from 133 patients were examined by cytotoxin assay, by culture of C . difficile on cycloserine-cefoxitin-fructose agar, and by toxin detection using four rapid immunoassay systems (Oxoid Toxin A test, ImmunoCard Toxin A test, TechLab Tox A/B II test, and Premier Toxins A&B test) . A diagnosis of CDAD was established for 35 (27%) patients (representing 29% of specimens) . The adjusted sensitivity and specificity of the methods were, respectively, 98 and 99% for the cytotoxin assay, 54 and 99% for ImmunoCard, 50 and 98% for Oxoid, 79 and 98% for TechLab, 80 and 98% for Premier, and 57 and 100% for culture . The TechLab and Premier assays are acceptable tests for diagnosis of CDAD but are not equivalent to the cytotoxin assay.

J Appl Microbiol, 2001 Aug, 91(2), 364 - 72
Analysis of the role of bacterial endospore cortex structure in resistance properties and demonstration of its conservation amongst species; Atrih A et al.; AIMS: The aim of this work was to compare the chemical structure of the spore cortex of a range of species, and to determine any correlation between cortex structure and spore resistance properties . METHODS AND RESULTS: The fine chemical structure of the cortex of Bacillus subtilis, Bacillus megaterium, Bacillus cereus and Clostridium botulinum was examined by muropeptide analysis using reverse phase HPLC . There is a conserved basic structure between peptidoglycan of these species, with the only difference being the level of de-N-acetylation of an amino sugar . In order to determine if an alteration in cortex structure correlates with heat resistance properties, the peptidoglycan structure and properties of B . subtilis spores prepared under different conditions were compared . Peptidoglycan from spores prepared in Nutrient Broth (NB) showed reduction in single L-alanine substituted muramic acid to only 13.9% compared with 20.6% in CCY-grown spores . NB-prepared spores are also unstable, with 161-fold less heat resistance (60 min, 85 degrees C) and 43 times less Mn(2+) content than CCY-grown spores . Addition of MnCl(2) to NB led to a peptidoglycan profile similar to CCY-grown spores, sevenfold more heat resistance (60 min, 85 degrees C) and an 86-fold increase in Mn(2+) content . Addition of CCY salts to NB led all parameters to be comparable with CCY-grown spore levels . CONCLUSION: It has been shown that peptidoglycan structure is conserved in four spore-forming bacteria . Also, spore heat resistance is multifactorial and cannot be accounted for by any single parameter . SIGNIFICANCE AND IMPACT OF THE STUDY: Endospores made by diverse species most likely have common mechanisms of heat resistance . However, the molecular basis for their resistance remains elusive.

J Biol Inorg Chem, 2001 Jun, 6(5-6), 590 - 600
Long-range interactions between the Fe protein binding sites of the MoFe protein of nitrogenase; Maritano S et al.; We report the properties and reactivity of the catalytically active heterologous nitrogenase formed between the Fe protein from Clostridium pasteurianum (Cp2) and the MoFe protein from Klebsiella pneumoniae (Kp1) . Under turnover conditions, in the presence of MgATP, a stable 2:1 (Cp2)2Kp1 electron transfer complex is formed, in which the {4Fe-4S}+ centre of Cp2 is protected from chelation by alpha,alpha'-bipyridyl . However, the two Fe protein-binding sites on Kp1 are not equivalent, since a 1:1 Cp2.Kp1 complex was isolated by gel filtration . The non-equivalence of the Fe protein binding sites was also indicated by the inhibition pattern of Klebsiella nitrogenase by Cp2 . The EPR spectrum of the isolated 1:1 Cp2.Kp1 complex showed an S=1/2 signal characteristic of dithionite-reduced Cp2 and signals with g values of 4.27, 3.73, 2.01 and 4.32, 3.63, 2.00 characteristic of the high- and low-pH forms of the FeMoco centre of Kp1, respectively . The unoccupied binding site of Kp1 of the isolated 1:1 Cp2Kp1 complex was shown to be catalytically fully functional in combination with Kp2 . In contrast to homologous nitrogenases, which require MgATP for detectable rates of electron transfer from the Fe protein, stopped-flow kinetic studies revealed that electron transfer from Cp2 to Kp1 occurred in the absence of MgATP with a rate constant of 0.065 s(-1) . Subsequently, a slower transient decrease and restoration of absorption in the electronic spectrum in the 500-700 nm region was observed . These changes corresponded with those in the intensity of the S=3/2 EPR signal of the FeMoco centres of Kp1 and were consistent with the transient reduction of the FeMoco centre of Kp1 to an EPR-silent form, followed by restoration of the signal at longer reaction times . These changes were not associated with catalysis since no evolution of H2 was detectable.

Curr Treat Options Gastroenterol, 2001 Apr, 4(2), 139 - 146
Bacterial Cholangitis; Lum DF et al.; The treatment of acute bacterial cholangitis requires broad-spectrum antibiotics to cover against gram-negative aerobic enteric organisms (Escherichia coli, Klebsiella species, and Enterobacter species), gram-positive Enterococcus and anaerobic bacteria (Bacteroides fragilis and Clostridium perfringens) . Approximately 20% of patients with acute cholangitis fail to respond to conservative treatment with antibiotic therapy and require urgent biliary decompression, which is the mainstay of therapy . This is best accomplished by endoscopic retrograde cholangiopancreatography (ERCP) and placement of a nasobiliary drainage tube or a large bore (10 F or larger) indwelling plastic stent . Alternative therapy includes percutaneous transhepatic biliary drainage or surgical biliary decompression, but these carry a significantly higher morbidity and mortality . Supportive care includes intravenous fluid hydration to prevent renal failure and close monitoring of vital signs for determination of potential septicemia.

J Clin Gastroenterol, 2001 Aug, 33(2), 159 - 60
Ischemic colitis associated with paclitaxel; Daniele B et al.; Systemic chemotherapy can be complicated by colonic toxicity, which usually determines the onset of pseudomembranous colitis and, rarely, of ischemic colitis in patients with cancer . This report describes the case of a 49-year-old woman with liver metastases from a neuroendocrine tumor of unknown origin who developed mild ischemic colitis after chemotherapy with carboplatin and paclitaxel . The patient developed symptoms of gastrointestinal toxicity with abdominal pain and bloody diarrhea, which resolved in about 10 days . She had a normal white blood cell count throughout her illness; the assay of stool specimens for Clostridium difficile toxins and the stool cultures were both negative . A sigmoidoscopy showed a mild, transient ischemic colitis, which was confirmed by pathologic examination of the biopsy specimens . Although carboplatin is not related to severe colonic cytotoxicity, it has been previously reported that paclitaxel induces necrosis of the gastrointestinal mucosa and inhibits angiogenesis . Pseudomembranous colitis is the most frequent complication in patients with cancer who undergo paclitaxel-based chemotherapy and develop gastrointestinal toxicity . Once C . difficile infection has been excluded, a diagnosis of ischemic colitis should be considered, especially in patients with cancer who have normal white blood cell counts.

J Biol Chem, 2001 Oct 5, 276(40), 37630 - 9 Epub 2001 Jul 23.
Investigation of the catalytic site within the ATP-grasp domain of Clostridium symbiosum pyruvate phosphate dikinase; Ye D et al.; Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, P(i), and pyruvate with AMP, PP(i), and phosphoenolpyruvate (PEP) in three partial reactions as follows: 1) E-His + ATP --> E-His-PP.AMP; 2) E-His-PP.AMP + P(i) --> E-His-P.AMP.PP(i); and 3) E-His-P + pyruvate --> E.PEP using His-455 as the carrier of the transferred phosphoryl groups . The crystal structure of the Clostridium symbiosum PPDK (in the unbound state) reveals a three-domain structure consisting of consecutive N-terminal, central His-455, and C-terminal domains . The N-terminal and central His-455 domains catalyze partial reactions 1 and 2, whereas the C-terminal and central His-455 domains catalyze partial reaction 3 . Attempts to obtain a crystal structure of the enzyme with substrate ligands bound at the nucleotide binding domain have been unsuccessful . The object of the present study is to demonstrate Mg(II) activation of catalysis at the ATP/P(i) active site, to identify the residues at the ATP/P(i) active site that contribute to catalysis, and to identify roles for these residues based on their positions within the active site scaffold . First, Mg(II) activation studies of catalysis of E + ATP + P(i) --> E-P + AMP + PP(i) partial reaction were carried out using a truncation mutant (Tem533) in which the C-terminal domain is absent . The kinetics show that a minimum of 2 Mg(II) per active site is required for the reaction . The active site residues used for substrate/cofactor binding/activation were identified by site-directed mutagenesis . Lys-22, Arg-92, Asp-321, Glu-323, and Gln-335 mutants were found to be inactive; Arg-337, Glu-279, Asp-280, and Arg-135 mutants were partially active; and Thr-253 and Gln-240 mutants were almost fully active . The participation of the nucleotide ribose 2'-OH and alpha-P in enzyme binding is indicated by the loss of productive binding seen with substrate analogs modified at these positions . The ATP, P(i), and Mg(II) ions were docked into the PPDK N-terminal domain crevice, in an orientation consistent with substrate/cofactor binding modes observed for other members of the ATP-Grasp fold enzyme superfamily and consistent with the structure-function data . On the basis of this docking model, the ATP polyphosphate moiety is oriented/activated for pyrophosphoryl transfer through interaction with Lys-22 (gamma-P), Arg-92 (alpha-P), and the Gly-101 to Met-103 loop (gamma-P) as well as with the Mg(II) cofactors . The P(i) is oriented/activated for partial reaction 2 through interaction with Arg-337 and a Mg(II) cofactor . The Mg(II) ions are bound through interaction with Asp-321, Glu-323, and Gln-335 and substrate . Residues Glu-279, Asp-280, and Arg-135 are suggested to function in the closure of an active site loop, over the nucleotide ribose-binding site.

Biochemistry, 2001 Jul 31, 40(30), 8686 - 95
Role of hydrogen bonding interactions to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin; Bradley LH et al.; The role of the hydrogen bonding interaction with the N(3)H of the flavin cofactor in the modulation of the redox properties of flavoproteins has not been extensively investigated . In the flavodoxin from Clostridium beijerinckii, the gamma-carboxylate group of glutamate-59 serves as a dual hydrogen bond acceptor with the N(3)H of flavin mononucleotide (FMN) cofactor and the amide hydrogen of the adjacent polypeptide backbone in all three oxidation states . This "bridging" interaction serves to anchor the FMN in the binding site, which, based on the E59Q mutant, indirectly affects the stability of the neutral flavin semiquinone by facilitating a strong and critical interaction at the FMN N(5)H {Bradley, L . H., and Swenson, R . P . (1999) Biochemistry 38, 12377-12386} . In this study, the specific role of the N(3)H interaction itself was investigated through the systematic replacement of Glu59 by aspartate, asparagine, and alanine in an effort to weaken, disrupt, and/or eliminate this interaction, respectively . Just as for the E59Q mutant, each replacement significantly weakened the binding of the cofactor, particularly for the semiquinone state, affecting the midpoint potentials of each one-electron couple in opposite directions . (1)H-(15)N HSQC nuclear magnetic resonance (NMR) spectroscopic studies revealed that not only was the N(3)H interaction weakened as anticipated, but so also was the hydrogen bonding interaction with the N(5)H . Using the temperature coefficients of the N(5)H to quantify and correct for changes in this interaction, the contribution of the N(3)H hydrogen bond to the binding of each redox state of the FMN was isolated and estimated . Based on this analysis, the N(3)H hydrogen bonding interaction appears to contribute primarily to the stability of the oxidized state (by as much as 2 kcal/mol) and to a lesser extent the reduced states . It is concluded that this interaction contributes only modestly (<45 mV) to the modulation of the midpoint potential for each redox couple in the flavodoxin . These conclusions are generally consistent with ab initio calculations and model studies on the non-protein-bound cofactor.

Epidemiol Infect, 2001 Jun, 126(3), 343 - 50
Molecular epidemiology of endemic Clostridium difficile infection; Fawley WN et al.; This is the first study to provide a comprehensive insight into the molecular epidemiology of endemic Clostridium difficile and particularly that associated with a recently recognized epidemic strain . We DNA fingerprinted all C . difficile isolates from the stools of patients with symptomatic antibiotic-associated diarrhoea and from repeated samples of the inanimate ward environment on two elderly medicine hospital wards over a 22-month period . Notably, C . difficile was not recoverable from either ward immediately before opening, but was found on both wards within 1-3 weeks of opening, and the level of environmental contamination rose markedly during the first 6 months of the study period . C . difficile infection (CDI) incidence data correlated significantly with the prevalence of environmental C . difficile on ward B (r = 0.76, P < 0.05) but not on ward A (r = 0.26, P > 0.05) . We found that RAPD and RS-PCR typing had similar discriminatory power, although, despite fingerprinting over 200 C . difficile isolates, we identified only six distinct types . Only two distinct C . difficile strains were identified as causing both patient infection and ward contamination . Attempts to determine whether infected patients or contaminated environments are the prime source for cross-infection by C . difficile had limited success, as over 90% of C . difficile isolates were the UK epidemic clone . However, a non-epidemic strain caused a cluster of six cases of CDI, but was only isolated from the environment after the sixth patient became symptomatic . The initial absence of this strain from the environment implies patient-to-patient and/or staff-to-patient spread . In general, routine cleaning with detergent was unsuccessful at removing C . difficile from the environment . Understanding the epidemiology and virulence of prevalent strains is important if CDI is to be successfully controlled.

J Bacteriol, 2001 Aug, 183(16), 4823 - 38
Genome sequence and comparative analysis of the solvent-producing bacterium Clostridium acetobutylicum; Nolling J et al.; The genome sequence of the solvent-producing bacterium Clostridium acetobutylicum ATCC 824 has been determined by the shotgun approach . The genome consists of a 3.94-Mb chromosome and a 192-kb megaplasmid that contains the majority of genes responsible for solvent production . Comparison of C . acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases closer, phylogenetic proximity . This conservation allows the prediction of many previously undetected operons in both bacteria . However, the C . acetobutylicum genome also contains a significant number of predicted operons that are shared with distantly related bacteria and archaea but not with B . subtilis . Phylogenetic analysis is compatible with the dissemination of such operons by horizontal transfer . The enzymes of the solventogenesis pathway and of the cellulosome of C . acetobutylicum comprise a new set of metabolic capacities not previously represented in the collection of complete genomes . These enzymes show a complex pattern of evolutionary affinities, emphasizing the role of lateral gene exchange in the evolution of the unique metabolic profile of the bacterium . Many of the sporulation genes identified in B . subtilis are missing in C . acetobutylicum, which suggests major differences in the sporulation process . Thus, comparative analysis reveals both significant conservation of the genome organization and pronounced differences in many systems that reflect unique adaptive strategies of the two gram-positive bacteria.

Eur J Immunol, 2001 May, 31(5), 1610 - 9
Regulation by rho family GTPases of IL-1 receptor induced signaling: C3-like chimeric toxin and Clostridium difficile toxin B inhibit signaling pathways involved in IL-2 gene expression; Dreikhausen U et al.; In this study the participation of Rho family GTPases in the regulation of IL-1-activated protein kinase cascades controlling IL-2 synthesis was investigated in murine EL-4 thymoma cells . The recombinant C3-like chimeric toxin, which consists of the C3 toxin of Clostridium limosum and the N-terminal part of Clostridium botulinum C2 toxin (C2IN-C3) interacting with the C2II binding subunit to facilitate uptake into cells, and selectively inactivates Rho A by ADP-ribosylation, prevented IL-1-stimulated activation of Jun-NH2-terminal-kinases (JNK) and p38 mitogen-activated-protein kinases (MAPK) . UDP-monoglucosylation and concomitant inactivation of Rho A and of Rac-2 by Clostridium difficile toxin B also inhibited IL-1-induced activation of JNK and p38 MAPK, but additionally inhibited activation of the extracellular-regulated-kinase pathway and DNA binding of the transcription factor NFkappaB . Accordingly, pre-treatment of cells with C21N-C3 fusion toxin only decreased IL-1-stimulated IL-2 synthesis by 50%, while in C . difficile toxin B-treated cells IL-1-induced IL-2 secretion was reduced by 90% . These results imply that together with Rho A an additional member of the Rho family G proteins, i.e . Rac-2, is critically involved as an upstream regulator in IL-1-induced activation of different MAPK, stress-activated protein kinases, and in NFkappaB activation controlling IL-2 gene expression in response to IL-1, acting in close proximity to the IL-1-receptor complex.

Water Sci Technol, 2001, 43(12), 201 - 4
Evaluation of fluorogenic TSC agar for recovering Clostridium perfringens in groundwater samples; Araujo M et al.; Clostridium perfringens is widely recognised as a reliable water pollution indicator . Since several media can be employed for the membrane filtration enumeration of this microorganism, the main aim of this work was to investigate the ability of fluorocult-supplemented TSC-agar (Merck) for recovering Cl . perfringens from public springs used for direct human consumption . Cl . perfringens recovery was also performed on mCP agar (Cultimed) according to Directive 98/83 as well as on TSC-Agar (Merck), TSN-Agar (Merck) and SPS-Agar (BBL) media . Variance analysis of data obtained showed no statistically significant differences in the counts obtained among all media employed in this work . However, the Cl . perfringens recovery efficiencies with TSC and fluorogenic TSC agars were significantly greater (P = < 0.05) than the corresponding values of mCP and TSN media . On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for Cl . perfringens recovery in groundwater samples (85.3% of typical colonies and 82.8% of atypical colonies confirmed) . In summary, the membrane filtration technique with fluorogenic TSC agar showed the best performance characteristics of all the media tested as judged by their recovery efficiency and specificity in these water samples.

Otolaryngol Head Neck Surg, 2001 Jul, 125(1), 101 - 2
Sphenoid sinusitis caused by Clostridium perfringens; Rosenblum BN et al.; Anaerobic bacterial infections in chronic sinusitis are well described in literature . We present what is believed to be the first reported case of Clostridium perfringens presenting as the causative pathogen in paranasal sinusitis . This patient presented with severe headaches and, with CT and MRI findings of unilateral sphenoid sinus opacification, with bone demineralization and intrasinus calcification . This patient responded to endoscopic debridement and long-term antibiotics without sequelae.

Facial Plast Surg Clin North Am, 2001 Aug, 9(3), 395 - 404
Current practices in the use of botulinum toxin A in the management of facial lines and wrinkles; Blitzer A et al.; Botulinum toxin A (BTX-A) is a potent neurotoxin produced by the bacterium Clostridium botulinum . There are eight antigenically distinct serotypes, and they share a similar structure--a light chain with an associated molecule of zinc and a heavy chain linked by a disulfide bond . Each serotype has a separate site of action within the nerve ending . Only serotype A (Botox, Allergan, Irvine, CA) is available for clinical use in the United States.

J Am Chem Soc, 2001 May 23, 123(20), 4697 - 703
Kinetic mechanism of acetyl-CoA synthase: steady-state synthesis at variable Co/Co2 pressures; Maynard EL et al.; Steady-state initial rates of acetyl-CoA synthesis (upsilon/{E(tot)}) catalyzed by acetyl-CoA synthase from Clostridium thermoaceticum (ACS) were determined at various partial pressures of CO and CO2 . When {CO} was varied from 0 to 100 microM in a balance of Ar, rates increased sharply from 0.3 to 100 min(-1) . At {CO} > 100 microM, rates declined sharply and eventually stabilized at 10 min(-1) at 980 microM CO . Equivalent experiments carried out in CO2 revealed similar inhibitory behavior and residual activity under saturating {CO} . Plots of upsilon/{E(tot)} vs {CO2} at different fixed inhibitory {CO} revealed that Vmax/{E(tot)} (kcat) decreased with increasing {CO} . Plots of upsilon/{E(tot)} vs {CO2} at different fixed noninhibitory {CO} showed that Vmax/{E(tot)} was insensitive to changes in {CO} . Of eleven candidate mechanisms, the simplest one that fit the data best had the following key features: (a) either CO or CO2 (at a designated reductant level and pH) activate the enzyme (E' + CO right arrow over left arrow E, E' + CO2/2e-/2H+ right arrow over left arrow E); (b) CO and CO2 are both substrates that compete for the same enzyme form (E + CO right arrow over left arrow ECO, E + CO2/2e-/2H+ right arrow over left arrow ECO, and ECO --> E + P); (c) between 3 and 5 molecules of CO bind cooperatively to an enzyme form different from that to which CO2 and substrate CO bind (nCO + ECO right arrow over left arrow (CO)nECO), and this inhibits catalysis; and (d) the residual activity arises from either the (CO)nECO state or a heterogeneous form of the enzyme . Implications of these results, focusing on the roles of CO and CO2 in catalysis, are discussed.

J Microbiol Immunol Infect, 2001 Jun, 34(2), 113 - 8
Clostridium bacteremia: emphasis on the poor prognosis in cirrhotic patients; Chen YM et al.; Bacteremic episodes caused by anaerobes are unusual and the clinical importance of Clostridium bacteremia remains unclear . This retrospective case study examined the risk factors among a group of patients who developed Clostridium bacteremia . Medical records from 73 episodes of clostridial bacteremia in 73 patients treated in a medical center during an 11-year period were reviewed . Of all episodes, 96% were community-acquired . Twelve percent of patients had polymicrobial bacteremia, with Escherichia coli being the most common accompanying bacterium . Diabetes mellitus (26%) and liver cirrhosis (25%) were the most common underlying diseases . The most common etiological organisms were Clostridium perfringens (77%), Clostridium bifermentans (9%), and Clostridium septicum (4%) . Only one patient with C . septicum bacteremia had a histocytotoxic infection, which was a fatal gas gangrene . Univariate analysis of data from patients with monomicrobial Clostridium bacteremia revealed that younger age (age < 65 years), underlying liver cirrhosis, and presence of septic shock at initial presentation were associated with fatality; but only the latter two variables were independently associated with fatality in multivariate logistic regression analysis . Appropriate antimicrobial therapy for monomicrobial Clostridium bacteremia did not significantly affect clinical outcomes, which might suggest that Clostridium species in the bloodstream can be regarded as merely contaminants or transient bacteremia . This suggestion was not supported by the finding that seven of 13 cirrhotic patients with monomicrobial Clostridium bacteremia died of sepsis, of whom six had not receive appropriate antimicrobial therapy . Therefore, the clinical importance of Clostridium bacteremia should be interpreted with caution because of its high risk of mortality in susceptible hosts, particularly cirrhotic patients, who do not receive appropriate therapy timely.

Eur J Biochem, 2001 Jul, 268(14), 4019 - 26
Characterization and reconstitution of functional hemagglutinin of the Clostridium botulinum type C progenitor toxin; Kouguchi H et al.; The purified progenitor toxin of Clostridium botulinum type C strain 6814 (C-6814) forms a large complex composed of 150-kDa neurotoxin (NT), 130-kDa nontoxic-nonhemagglutinin (NTNHA), and hemagglutinin (HA) components . The HA component consisted of a mixture of several subcomponents with molecular masses of 70, 55, 33, 26-21 and 17 kDa . We isolated the HA subcomponents from the progenitor toxin by chromatography in the presence of denaturants . The isolated HA subcomponents, designated as i-HA-33, i-HA-55, i-HA-70 and i-HA-33/17, were nearly homogeneous on SDS/PAGE, but the HA-17 and HA-26-21 components were not purified . Some HA subcomponents, designated as f-HA-33 and f-HA-33/17 complex, existed free of the progenitor toxin in the culture medium and they were separately purified . Every HA subcomponent so far isolated shows binding activity to erythrocytes . The hemagglutination activities of each HA subcomponent had a titer of 25 for the f-HA-33/17 complex, and below 23 for the other f- and i-HA subcomponents, while the parent progenitor L toxin was 28 . The reconstitution of various combinations of f- and i-HA subcomponents was attempted via mixing and tested for hemagglutination activity . When the i-HA-33/17 complex and i-HA-55 were mixed, the hemagglutination activity was recovered to a titer of 29, which was slightly higher than that of the parent toxin . These data imply that a combination of at least HA-33, -17 and -55 subcomponents is required for full hemagglutination activity of the botulinum progenitor toxin, but each single HA subcomponent shows weak or no aggregation of erythrocytes.

Pharmacol Toxicol, 2001 Jun, 88(6), 313 - 8
The effect of tumour necrosis factor (TNF) inhibitors in Clostridium difficile toxin-induced paw oedema and neutrophil migration; Carneiro-Filho BA et al.; Clostridium difficile produces a potent enterotoxin and a cytotoxin, toxin A and toxin B, respectively . These toxins are associated with pseudomembranous colitis and antibiotic-associated diarrhoea . In the present study, we investigated the oedematogenic activity of both toxins, characterizing the time-course and dose-response of this pro-inflammatory event . We also explored the effects of two inhibitors of tumour necrosis factor (TNF) production, thalidomide and pentoxifylline, in neutrophil recruitment and the oedematogenic activity of these toxins . Subplantar injection of toxin A induced paw oedema with a maximal response at 1 microg, reaching a maximal value 9 hr after toxin A challenge (toxin A 1 microg:1.39+/-0.09 ml) . Toxin B also showed a dose-dependent oedematogenic activity with a late peak at 24 hr and a maximal response at a dose of 0.1 microg (toxin B 0.1 microg:1.74+/-0.12 ml) . Pentoxifylline, but not thalidomide, significantly reduced the oedema induced by Toxin A (pentoxifylline 135 mg/kg:60% of inhibition) and Toxin B (pentoxifylline 135 mg/kg:33.6% of inhibition) . Both thalidomide and pentoxifylline were able to significantly reduce neutrophil influx into the peritoneal cavities of rats evoked with Toxin A (thalidomide 45 mg/kg: 53.1% of inhibition; pentoxifylline 45 mg/kg:47.1% of inhibition) and Toxin B (thalidomide 45 mg/kg:46.8% of inhibition; pentoxifylline 45 mg/kg:63.1% of inhibition) . This study demonstrates the oedematogenic activities of both toxins with distinct potencies and time-courses . These data also show an inhibitory effect of pentoxifylline in toxin A and B-induced paw oedema . Furthermore, both pentoxifylline and thalidomide significantly inhibited the Clostridium difficile toxins-induced neutrophil migration.

Antimicrob Agents Chemother, 2001 Aug, 45(8), 2348 - 53
Resistance to moxifloxacin in toxigenic Clostridium difficile isolates is associated with mutations in gyrA; Ackermann G et al.; Clostridium difficile is the etiological agent of antibiotic-associated colitis and the most common cause of hospital-acquired infectious diarrhea . Fluoroquinolones such as ciprofloxacin are associated with lower risks of C . difficile-associated diarrhea . In this study, we have analyzed 72 C . difficile isolates obtained from patients with different clinical courses of disease, such as toxic megacolon and relapses; the hospital environment; public places; and horses . They were investigated for their susceptibilities to moxifloxacin (MXF), metronidazole (MEO), and vancomycin (VAN) . Mutants highly resistant to fluoroquinolones were selected in vitro by stepwise exposure to increasing concentrations of MXF . The resulting mutants were analyzed for the presence of mutations in the quinolone resistance-determining regions of DNA gyrase (gyrA), the production of toxins A and B, and the epidemiological relationship of these isolates . These factors were also investigated using PCR-based methods . All strains tested were susceptible to MEO and VAN . Twenty-six percent of the clinical isolates (19 of 72) were highly resistant to MXF (MIC > or = 16 microg/ml) . Fourteen of these 19 strains contained nucleotide changes resulting in amino acid substitutions at position 83 in the gyrA protein . Resistant strains selected in vitro did not contain mutations at that position . These findings indicate that resistance to MXF in a majority of cases may be due to amino acid substitution in the gyrA gene.

Antimicrob Agents Chemother, 2001 Aug, 45(8), 2340 - 7
GT160-246, a toxin binding polymer for treatment of Clostridium difficile colitis; Kurtz CB et al.; GT160-246, a high-molecular-weight soluble anionic polymer, was tested in vitro and in vivo for neutralization of Clostridium difficile toxin A and B activities . Five milligrams of GT160-246 per ml neutralized toxin-mediated inhibition of protein synthesis in Vero cells induced by 5 ng of toxin A per ml or 1.25 ng of toxin B per ml . In ligated rat ileal loops, 1 mg of GT160-246 neutralized fluid accumulation caused by 5 microg of toxin A . At doses as high as 80 mg/loop, cholestyramine provided incomplete neutralization of fluid accumulation caused by 5 microg of toxin A . GT160-246 protected 80% of the hamsters from mortality caused by infection with C . difficile, whereas cholestyramine protected only 10% of animals . Treatment of C . difficile-infected hamsters with metronidazole initially protected 100% of the hamsters from mortality, but upon removal of treatment, 80% of the hamsters had relapses and died . In contrast, removal of GT160-246 treatment did not result in disease relapse in the hamsters . GT160-246 showed no antimicrobial activity in tests with a panel of 16 aerobic bacteria and yeast and 22 anaerobic bacteria and did not interfere with the in vitro activities of most antibiotics . GT160-246 offers a novel, nonantimicrobial treatment of C . difficile disease in humans.

Acta Neurochir Suppl, 2000, 76, 231 - 6
Toxin-induced vasogenic cerebral oedema in a rat model; Ghabriel MN et al.; Vasogenic cerebral oedema (VCO) was induced in Hooded Wistar rats by intraperitoneal injection of Clostridium perfringens type D epsilon prototoxin . Animals were killed, 1 h to 14 d postinjection, by perfusion fixation under general anaesthesia . VCO was detected by the presence of endogenous albumin in the brain, visualised by immunocytochemistry . As early as 1 h postinjection, albumin was detected in the walls of cerebral microvessels . Maximal diffuse leakage within the neural parenchyma was seen at 24 and 48 h and immunoreactivity was still present at 4 d . At 7 d only few foci were seen, and at 14 d albumin distribution was similar to that in controls . Ultrastructural assessment of the microvessels showed swelling of many astrocytic processes and abnormalities of the endothelial cells varying from swelling with loss of cytoplasmic organelles to cells showing increased electron density . Immunostaining for the endothelial barrier antigen (EBA) showed strongly immunoreactive vessels throughout normal brains . Experimental animals showed partial reduction in EBA expression, most evident at 24 and 48 h, with gradual recovery to normal by 14 d . The exact role that EBA plays in the intact BBB remains obscure.

Am J Physiol Gastrointest Liver Physiol, 2001 Aug, 281(2), G544 - 51
Deletion of neutral endopeptidase exacerbates intestinal inflammation induced by Clostridium difficile toxin A; Kirkwood KS et al.; Toxin A (TxA) of Clostridium difficile induces acute inflammation of the intestine initiated by release of substance P (SP) and activation of the neurokinin-1 receptor . However, the mechanisms that terminate this response are unknown . We determined whether the SP-degrading enzyme neutral endopeptidase (NEP, EC 3.4.24.11) terminates TxA-induced enteritis . We used both genetic deletion and pharmacological inhibition of NEP to test this hypothesis . In wild-type mice, instillation of TxA (0.5-5 microg) into ileal loops for 3 h dose dependently increased ileal fluid secretion, stimulated granulocyte transmigration determined by myeloperoxidase activity, and caused histological damage characterized by depletion of enterocytes, edema, and neutrophil accumulation . Deletion of NEP reduced the threshold secretory and inflammatory dose of TxA and exacerbated the inflammatory responses by more than twofold . This exacerbated inflammation was prevented by pretreatment with recombinant NEP . Conversely, pretreatment of wild-type mice with the NEP inhibitor phosphoramidon exacerbated enteritis . Thus NEP terminates enteritis induced by C . difficile TxA, underlying the importance of SP degradation in limiting neurogenic inflammation.

J Biol Chem, 2001 Sep 7, 276(36), 33402 - 12 Epub 2001 Jul 09.
Comparative biochemical and immunocytochemical studies reveal differences in the effects of Clostridium perfringens enterotoxin on polarized CaCo-2 cells versus Vero cells; Singh U et al.; Since most in vitro studies exploring the action of Clostridium perfringens enterotoxin (CPE) utilize either Vero or CaCo-2 cells, the current study directly compared the CPE responsiveness of those two cell lines . When CPE-treated in suspension, both CaCo-2 and Vero cells formed SDS-resistant, CPE-containing complexes of approximately 135, approximately 155, and approximately 200 kDa . However, confluent Transwell cultures of either cell line CPE-treated for 20 min formed only the approximately 155-kDa complex . Since those Transwell cultures also exhibited significant (86)Rb release, approximately 155-kDa complex formation is sufficient for CPE-induced cytotoxicity . Several differences in CPE responsiveness between the two cell lines were also detected . (i) CaCo-2 cells were more sensitive when CPE-treated on their basal surface, whereas Vero cells were more sensitive when CPE-treated on their apical surface; those sensitivity differences correlated with CPE binding the apical versus basolateral surfaces of these two cell lines . (ii) CPE-treated Vero cells released (86)Rb into both Transwell chambers, whereas CaCo-2 cells released (86)Rb only into the CPE-containing Transwell chamber . (iii) Vero cells express the tight junction (TJ) protein occludin but (unlike CaCo-2 cells) cannot form TJs . The ability of TJs to affect CPE responsiveness is supported by the similar effects of CPE on Transwell cultures of CaCo-2 cells and Madin-Darby canine kidney cells, another polarized cell forming TJs . Confluent CaCo-2 Transwell cultures CPE-treated for >1 h formed the approximately 200-kDa CPE complex (which also contains occludin), exhibited morphologic damage, and had occludin removed from their TJs . Collectively, these results identify CPE as a bifunctional toxin that, in confluent polarized cells, first exerts a cytotoxic effect mediated by the approximately 155-kDa complex . Resultant damage then provides CPE access to TJs, leading to approximately 200-kDa complex formation, internalization of some TJ proteins, and TJ damage that may increase paracellular permeability and thereby contribute to the diarrhea of CPE-induced gastrointestinal disease.

Biochem Biophys Res Commun, 2001 Jul 13, 285(2), 496 - 502
Identification of the gene encoding NADH-rubredoxin oxidoreductase in Clostridium acetobutylicum; Guedon E et al.; NADH-rubredoxin oxidoreductase (NROR), a flavoprotein from the obligately anaerobe Clostridium acetobutylicum is encoded by an ORF (nror) of 1140 nucleotides . Whereas primary structure analysis reveals that NROR has amino acid sequence patterns homologous with those involved in FAD and NAD-binding, the enzyme is distantly related to other flavoproteins in the databank . NROR is highly active for reducing clostridial rubredoxin (Rd) especially against C . acetobutylicum Rd with an efficiency (k(cat)/K(m)) of 400,000 mM(-1)s(-1) . These results suggest that Rd from C . acetobutylicum, C . pasteurianum, C . butyricum, and C . cellulolyticum can be interchanged with each other . Since C . acetobutylicum is the sole Clostridium strain that possesses such an enzyme, possible functions are discussed with regard to Desulfovibrio gigas and Pyrococcus furiosus, the only two other anaerobic systems for which a similar activity was reported, but no gene isolated .

J Med Microbiol, 2001 Jul, 50(7), 613 - 9
Evidence for holin function of tcdE gene in the pathogenicity of Clostridium difficile; Tan KS et al.; Toxigenic strains of Clostridium difficile produce two large bacterial toxins called toxins A (TcdA) and B (TcdB) . tcdA and tcdB genes are located on the pathogenicity locus of C . difficile, a unique characteristic of toxigenic strains of this species . Intergenic to the two toxin genes is tcdE, a small 501-bp open reading frame of unknown function . Expression of the tcdE gene in Escherichia coli caused bacterial cell death . Computational analysis of the amino acid sequence of TcdE revealed structural features that are strikingly similar to a class of bacteriophage proteins called holins . Holins are cytolytic proteins that cause lysis of bacterial hosts to effect the release of progeny phages . Further analysis of the recombinant clone expressing TcdE by transmission electron microscopy confirmed that the site of action of TcdE is on the bacterial cell membrane . The results provide evidence that TcdE is structurally and functionally similar to holin proteins . TcdE may function as a lytic protein to facilitate the release of TcdA and TcdB to the extracellular environment, as these toxins lack signal peptide.

Mayo Clin Proc, 2001 Jul, 76(7), 725 - 30
Clostridium difficile-associated diarrhea and colitis; Yassin SF et al.; Clostridium difficile is a spore-forming toxigenic bacterium that causes diarrhea and colitis, typically after the use of broad-spectrum antibiotics . The clinical presentation ranges from self-limited diarrhea to fulminant colitis and toxic megacolon . The incidence of this disease is increasing, resulting in major medical and economic consequences . Although most cases respond quickly to medical treatment, C difficile colitis may be serious, especially if diagnosis and treatment are delayed . Recurrent disease represents a particularly challenging problem . Prevention is best accomplished by limiting the use of broad-spectrum antibiotics and following good hygienic techniques and universal precautions to limit the transmission of bacteria . A high index of suspicion results in early diagnosis and treatment and potentially reduces the incidence of complications.

Poult Sci, 2001 Jun, 80(6), 813 - 6
Cooling rate effect on outgrowth of Clostridium perfringens in cooked, ready-to-eat turkey breast roasts; Steele FM et al.; The potential for Clostridium perfringens spores to germinate and grow in cooked, ready-to-eat turkey products was evaluated to determine a safe cooling rate within the critical temperatures of 48.9 C (120 F) through 12.8 C (55 F) . Raw turkey deli breast roasts were inoculated with a cocktail of C . perfringens spores (NCTC 8238, NCTC 8239, and NCTC 10388) and cooked in a steam oven to an internal temperature of 72 C . The sample roasts were then cooled through the critical cooling range at rates yielding cooling times of 6, 8, and 10 h . Turkey roasts were analyzed for spore growth and multiplication using tryptose-sulfite-cycloserine agar and anaerobic incubation at 37 C for 48 h . Cooling times of 6 and 8 h showed no proliferation of C . perfringens that would violate the USDA/Food Safety Inspection Service safe cooling standard criteria, which would allow no more than a 1 log10 multiplication between 48.9 and 12.8 C . A 9.6-h cooling period between the designated temperatures at a 95% confidence interval was determined to be adequate for nonproliferation of C . perfringens . On the other hand, a 95% tolerance interval would be more stringent in that it suggests no more than an 8.9-h cooling period . Tolerance intervals required that 95% of all our observations did not exceed the limit of 1 log10 increase in C . perfringens . This study indicated that in cooked, ready-to-eat turkey deli breasts, a cooling period between 48.9 C (120 F) and 12.8 C (55 F) of no greater than 8.9 h should be utilized to prevent possible C . perfringens foodborne outbreaks.

J Hosp Infect, 2001 Jul, 48(3), 238 - 41
Bacterial contamination of uniforms; Perry C et al.; Microbiological sampling of nurses' uniforms was undertaken using a Casella slit sampler . Staphylococcus aureus, Clostridium difficile and vancomycin-resistant enterococci were detected on uniforms both before and after a span of duty . Recommendations for provision and changing of nurses' uniforms are made .

J Hosp Infect, 2001 Jul, 48(3), 233 - 7
"Second-look" cytotoxicity: an evaluation of culture plus cytotoxin assay of Clostridium difficile isolates in the laboratory diagnosis of CDAD; Bouza E et al.; Clostridium difficile is one of the most frequent causes of hospital-acquired diarrhoea . Our objective was to prove that some stool samples with a direct negative cytotoxicity assay may indeed harbour toxigenic C . difficile and that this can be demonstrated by performing a "second-look" cytotoxicity assay using the isolated C . difficile strains . Over an eight-year period (1992-1999), the 8241 stool samples submitted for direct cell culture from patients with suspected C . difficile-associated diarrhoea (CDAD) were simultaneously plated on cycloserine cefoxitin fructose agar . C . difficile strains isolated from samples with a negative direct cell culture assay were re-tested for toxin production "second-look" cell culture assay) . Using both methods 6423 samples (78%) were negative . Of the remaining 1818 samples, 127 (7%) yielded C . difficile isolates which were confirmed as non-producers of toxin by both methods, 1437 (85%) were positive in direct cell culture assay, and 254 were positive only after the "second-look" cell culture assay . Thus, our approach allowed us to detect an extra 15% of toxin-producing strains that could have gone undetected otherwise.The combination of direct-cell culture assay, culture for toxigenic C . difficile and "second-look" cell culture assay enhances the potential for diagnosis of CDAD and enables us to be more efficient with our patient care resources .

Biomed Chromatogr, 2001 Jun, 15(4), 257 - 62
Ascertainment of D-amino acids in germ-free, gnotobiotic and normal laboratory rats; Bruckner H et al.; Free D-amino acids were ascertained in the blood serum, urine and aqueous ethanolic extracts of feces of germ-free laboratory rats and a rat made gnotobiotic (tetra-associated) with species of Streptococcus, Lactobacillus and Clostridium . D-Amino acids were also determined in the brains of two germ-free rats . For comparison, D-amino acids were also measured in the blood serum of normal rats and the blood plasma, urine and feces of normal white mice . D-Enantiomers of most protein L-amino acids were detected in all physiological samples of animals . Quantities of free D-amino acids were determined as N(O)-pentafluoropropionyl-(2)-propyl esters by enantioselective gas chromatography and mass spectrometry . Stereoisomers of the bacterial marker 2,6-diaminopimelic acid, analyzed as N-trifluoroacetyl-(2)-propyl esters, were detected in feces of the gnotobiotic but not of the germ-free rat .

Clin Infect Dis, 2001 Aug 1, 33(3), 349 - 53 Epub 2001 Jun 22.
Clinical features of clostridial bacteremia: a review from a rural area; Rechner PM et al.; Blood samples, which were obtained from patients who lived in a rural area with approximately 500 acute-care hospital beds, were cultured from 1990 through 1997 . We retrospectively reviewed the blood cultures that yielded Clostridium species (74 {0.12%} of 63,296 cultures) . These were obtained from 46 different hospitalized patients (incidents per hospital, 0.03%) . The source of the Clostridium species was a gastrointestinal site in 24 patients (52.2%) . The most frequently identified Clostridium species was Clostridium perfringens (in 10 {21.7%} of patients), followed by Clostridium septicum (in 9 {19.6%}) . Thirty-one patients (67.4%) were aged > or =65 years, 13 patients (28.3%) had diabetes mellitus, and underlying malignancy was present in 22 patients (47.8%) . The mortality rate of patients whose condition had been managed surgically was 33%; for those patients whose conditions required medical management, the mortality rate was 58% . Clostridium bacteremia in these patients usually had a gastrointestinal source, it often occurred in patients with serious underlying medical conditions, and it rarely was the result of traumatic farm accidents.

Clin Infect Dis, 2001 Aug 1, 33(3), 338 - 43 Epub 2001 Jul 05.
Risk factors for anaerobic bloodstream infections in bone marrow transplant recipients; Lark RL et al.; The incidence of anaerobic bloodstream infections (BSI) in patients who underwent bone marrow transplantation (BMT) recently increased at our institution . A retrospective case-control study of patients undergoing BMT from January 1995 through December 1998 was performed to determine the microbiological characteristics, epidemiology, and outcome of anaerobic BSI and to identify independent risk factors for infection . Anaerobic BSI occurred in 23 patients, for a rate of 4 BSIs per 100 BMT procedures, and it accounted for 17% of all BSIs that occurred during the study period . Infection occurred at a mean (+/- standard deviation) of 7+/-4 days after BMT and 7+/-5 days after the onset of neutropenia . Fusobacterium nucleatum was the most frequently isolated pathogen (in 17 patients), followed by Leptotrichia buccalis (in 4), Clostridium septicum (in 1), and Clostridium tertium (in 1) . Two case patients (9%) died . Severity of mucositis was an independent predictor of anaerobic BSI (odds ratio, 4.4; P=.01) . Controlling mucositis is critical for the prevention of anaerobic BSI in this patient population.

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