Clostridia

Res Microbiol, 1999 Sep, 150(7), 465 - 73
Induction of lactate production associated with a decrease in NADH cell content enables growth resumption of Clostridium cellulolyticum in batch cultures on cellobiose; Payot S et al.; When grown in batch cultures in fermentors with 23.4 mM cellobiose, Clostridium cellulolyticum displayed biphasic growth kinetics not associated with sequential substrate consumption and which led to a twofold higher production of biomass than previously reported . In the first growth phase, acetate was the major product of cellobiose metabolism, since lactate and ethanol productions remained low . Furthermore, an accumulation of intracellular NADH was observed . The transition towards the second growth phase was accompanied by an induction of lactate production, in such a way that lactate became the major product of C . cellulolyticum metabolism . In addition, a decrease in NADH concentration was measured, concomitant with this induction of lactate production and with the growth resumption . During both growth phases, the NADH-ferredoxin reductase-hydrogenase system played a major function in NADH regeneration, since H2 production was 1.4- to 1.5-fold higher than that of CO2 . Thus, we found that lactate production serves as an additional catabolic pathway enabling C . cellulolyticum to cope with excesses of carbon and NADH produced . Growth experiments on C . cellulolyticum under an atmosphere of carbon monoxide mimicked this phenomenon and confirmed that a high intracellular level of NADH can provide a barrier to bacterial growth.

J Cell Sci, 1999 Dec, 112 ( Pt 24), 4763 - 71
Involvement of Rho GTPases in calcium-regulated exocytosis from adrenal chromaffin cells; Gasman S et al.; The Rho GTPase family, including Rho, Rac and Cdc42 proteins, is implicated in various cell functions requiring the reorganization of actin-based structures . In secretory cells, cytoskeletal rearrangements are a prerequisite for exocytosis . We previously described that, in chromaffin cells, the trimeric granule-bound Go protein controls peripheral actin and prevents exocytosis in resting cells through the regulation of RhoA . To provide further insight into the function of Rho proteins in exocytosis, we focus here on their intracellular distribution in chromaffin cells . By confocal immunofluorescence analysis, we found that Rac1 and Cdc42 are exclusively localized in the subplasmalemmal region in both resting and nicotine-stimulated cells . In contrast, RhoA is associated with the membrane of secretory granules . We then investigated the effects of clostridial toxins, which differentially impair the function of Rho GTPases, on the subplasmalemmal actin network and catecholamine secretion . Clostridium difficile toxin B, which inactivates Rho, Rac and Cdc42, markedly altered the distribution of peripheral actin filaments . Neither Clostridium botulinum C3 toxin, which selectively ADP-ribosylates Rho, nor Clostridium sordellii lethal toxin, which inactivates Rac, affected cortical actin, suggesting that Cdc42 plays a specific role in the organization of subplasmalemmal actin . Indeed, toxin B strongly reduced secretagogue-evoked catecholamine release . This effect on secretion was not observed in cells having their actin cytoskeleton depolymerized by cytochalasin E or Clostridium botulinum C2 toxin, suggesting that the inhibition of secretion by toxin B is entirely linked to the disorganization of actin . C . sordellii lethal toxin also inhibited catecholamine secretion, but this effect was not related to the actin cytoskeleton as seen in cells pretreated with cytochalasin E or C2 toxin . In contrast, C3 exoenzyme did not affect secretion . We propose that Cdc42 plays an active role in exocytosis by coupling the actin cytoskeleton to the sequential steps underlying membrane trafficking at the site of exocytosis.

Int J Food Microbiol, 1999 Nov 1, 52(1-2), 57 - 65
PCR-based 16S ribosomal DNA detection technique for Clostridium estertheticum causing spoilage in vacuum-packed chill-stored beef; Helps CR et al.; Chill stored vacuum-packaged meat is sometimes spoiled by psychrotrophic or psychrophilic clostridia . Clostridium estertheticum was first isolated from vacuum-packed beef from southern Africa, but has recently been found in beef originating from northern Europe . This organism is difficult to isolate using conventional methods, and two PCR-based methods have been devised for use in measures to control the bacterium in the abattoir and to study its ecology . In the first method, primers were designed having a high annealing temperature of 65 degrees C to increase specificity, producing a PCR product of 567 bp from the 16S rDNA . Two species of Enterobacteriaceae found in meat cross-reacted in this test, and so it was necessary to use a second step, digesting the PCR product with two restriction enzymes . Subsequently a further set of primers was designed, producing a PCR product of 641 bp, and using an annealing temperature of 60 degrees C . The second procedure was more specific and did not require subsequent restriction analysis of the PCR product . The two sets of primers appeared to have similar sensitivity, detecting 10-100 cells of C . estertheticum in broth, meat or meat purge (drip) . A semiquantitative method is described for estimating numbers of the target bacterium.

N Engl J Med, 1999 Nov 25, 341(22), 1645 - 51
Epidemics of diarrhea caused by a clindamycin-resistant strain of Clostridium difficile in four hospitals; Johnson S et al.; BACKGROUND: Large outbreaks of diarrhea caused by a newly recognized strain of Clostridium difficile occurred in four hospitals located in different parts of the United States between 1989 and 1992 . Since frequent use of clindamycin was associated with the outbreak in one of the hospitals, we examined the resistance genes of the epidemic-strain isolates and studied the role of clindamycin use in these outbreaks . METHODS: Case-control studies were performed at three of the four hospitals to assess the relation of the use of clindamycin to C . difficile-associated diarrhea . All isolates of the epidemic strain and representative isolates of other strains identified during each outbreak were tested for susceptibility to clindamycin . Chromosomal DNA from these representative isolates was also analyzed by dot blot hybridization and amplification with the polymerase chain reaction (PCR) with the use of probes and primers from a previously described determinant of erythromycin resistance - the erythromycin ribosomal methylase B (ermB) gene - found in C . perfringens and C . difficile . RESULTS: In a stratified analysis of the case-control studies with pooling of the results according to the Mantel-Haenszel method, we found that the use of clindamycin was significantly increased among patients with diarrhea due to the epidemic strain of C . difficile, as compared with patients whose diarrhea was due to nonepidemic strains (pooled odds ratio, 4.35; 95 percent confidence interval, 2.02 to 9.38; P<0.001) . Exposure to other types of antibiotics or hospitalization in a surgical ward was not significantly associated with the risk of C . difficile-associated diarrhea due to the epidemic strain . All epidemic-strain isolates were highly resistant to clindamycin (minimal inhibitory concentration, >256 microg per milliliter) . DNA hybridization and PCR analysis showed that all these isolates had an ermB gene, which encodes a 23S ribosomal RNA methylase that mediates resistance to macrolide, lincosamide, and streptogramin antibiotics . Only 15 percent of the nonepidemic strains were resistant to clindamycin . CONCLUSIONS: A strain of C . difficile that is highly resistant to clindamycin was responsible for large outbreaks of diarrhea in four hospitals in different states . The use of clindamycin is a specific risk factor for diarrhea due to this strain . Resistance to clindamycin further increases the risk of C . difficile-associated diarrhea, an established complication of antimicrobial use.

Appl Microbiol Biotechnol, 1999 Nov, 52(5), 670 - 4
Effect of dilution rate, cellobiose and ammonium availabilities on Clostridium cellulolyticum sporulation; Payot S et al.; The nutritional and physiological factors affecting sporulation of Clostridium cellulolyticum were studied using steady-state continuous cultures grown in both complex and synthetic media . Under cellobiose limitation, the probability that cells will sporulate appears to be directly related to the growth rate . In complex medium, the highest percentage of sporulation was 20% at a dilution rate of 0.015 h-1 whereas in synthetic medium it was 10% at 0.035 h-1 . In both media, when the dilution rate was either higher or lower the percentage of sporulation decreased by between 2% and 4% . At low dilution rates, endospore formation was repressed under cellobiose-sufficient concentrations, suggesting catabolite repression by cellobiose . Furthermore, the concentration of ammonium was important in determining the percentage of sporulation, as ammonium limitation induced extensive sporulation at low growth rates even in an excess of cellobiose . The sporulation process is not triggered when cells are cellobiose-exhausted both in complex and synthetic media . These data suggest that, in C . cellulolyticum, an exogenous supply of carbon is required throughout the sporulation process . In the experimental conditions used in this work, no relationship between glycogen accumulation or glycogen mobilization and endospore formation was detected in C . cellulolyticum.

Am J Physiol, 1999 Nov, 277(5 Pt 1), C955 - 64
RhoA inactivation enhances endothelial barrier function; Carbajal JM et al.; The modulation of endothelial barrier function is thought to be a function of contractile tension mediated by the cell cytoskeleton, which consists of actomyosin stress fibers (SF) linked to focal adhesions (FA) . We tested this hypothesis by dissociating SF/FA with Clostridium botulinum exoenzyme C3 transferase (C3), an inhibitor of the small GTP-binding protein RhoA . Bovine pulmonary artery endothelial cell (EC) monolayers given C3, C3 + thrombin, thrombin, or no treatment were examined using a size-selective permeability assay and quantitative digital imaging measurements of SF/FA . C3 treatment disassembled SF/FA, stimulated diffuse myosin II immunostaining, and reduced the phosphotyrosine (PY) content of paxillin and 130- to 140-kDa proteins that included p125(FAK) . C3-treated monolayers displayed a 60-85% decline in F-actin content and a 170-300% increase in EC surface area with enhanced endothelial barrier function . This activity correlated with reorganization of F-actin and PY protein(s) to beta-catenin-containing cell-cell junctions . Because C3 prevented the thrombin-induced formation of myosin ribbons, SF/FA, and the increased PY content of proteins, these characteristics were Rho dependent . Our data show that C3 inhibition of Rho proteins leads to cAMP-like characteristics of reduced SF/FA and enhanced endothelial barrier function.

J Immunol, 1999 Nov 15, 163(10), 5183 - 91
Roles of intracellular calcium and NF-kappa B in the Clostridium difficile toxin A-induced up-regulation and secretion of IL-8 from human monocytes; Jefferson KK et al.; Clostridium difficile causes an intense inflammatory colitis through the actions of two large exotoxins, toxin A and toxin B . IL-8 is believed to play an important role in the pathophysiology of C . difficile-mediated colitis, although the mechanism whereby the toxins up-regulate the release of IL-8 from target cells is not well understood . In this study, we investigated the mechanisms through which toxin A induces IL-8 secretion in human monocytes . We found that cellular uptake of toxin A is required for the up-regulation of IL-8, an effect that is not duplicated by a recombinant toxin fragment comprising the cell-binding domain alone . Toxin A induced IL-8 expression at the level of gene transcription and this effect occurred through a mechanism requiring intracellular calcium and calmodulin activation . Additionally, the effects of toxin A were inhibited by the protein tyrosine kinase inhibitor genistein, but were unaffected by inhibitors of protein kinase C and phosphatidylinositol-3 kinase . We determined that toxin A activates nuclear translocation of the transcription factors NF-kappa B and AP-1, but not NF-IL-6 . NF-kappa B inhibitors blocked the ability of toxin A to induce IL-8 secretion, and supershift analysis indicated that the major isoform of NF-kappa B activated by the toxin is a p50-p65 heterodimer . This study is the first to identify intracellular signaling pathways and transcription factors involved in the C . difficile toxin-mediated up-regulation of IL-8 synthesis and release by target cells . This information should increase our understanding of the pathogenesis of C . difficile colitis and the nature of IL-8 gene regulation as well.

Vet Pathol, 1999 Nov, 36(6), 613 - 5
Enterocolitis associated with dual infection by Clostridium piliforme and feline panleukopenia virus in three kittens; Ikegami T et al.; Dual infection by Clostridium piliforme and feline panleukopenia virus (FPLV) was found in three kittens . In all cases, we found focal necrosis and desquamation of epithelial cells with occasional neutrophil infiltration in the large intestine . Large filamentous bacilli and spores were observed in the epithelium by using the Warthin-Starry method . Electron microscopy revealed the vegetative forms with characteristic peritrichous flagella and spore forms . Immunohistochemically, these bacilli showed a positive reaction with mouse antisera against the RT and MSK C . piliforme strains . Polymerase chain reaction (PCR) using cecum specimens demonstrated the 196-bp band specific to C . piliforme 16S rRNA . All three kittens were also diagnosed as FPLV-infected on the basis of the characteristic mucosal lesions, including intranuclear inclusions and PCR study for the FPLV genomic DNA . The PCR techniques are useful for confirming the C . piliforme and FPLV infection in spontaneous cases.

Dev Biol Stand, 1999, 101, 261 - 6
Veterinary vaccines: In-VITRO--International Veterinary Industry Test Replacement Organisation; Redhead K et al.; The International Veterinary Industry Test Replacement Organisation (In-VITRO) was established in 1995 with the aim of developing, validating and harmonising in vitro alternatives to replace in vivo methods for in-process and potency testing of veterinary clostridial vaccines . The emphasis has been on the reduction of animal usage in the Clostridium chauvoei potency assay and its eventual replacement by an in vitro assay . Replacement of the toxin neutralisation assay for Cl . tetani by an internationally validated indirect ELISA has already started . A validation programme involving a collaboration organised through EDQM which could ultimately lead to the standardisation of in vitro tests for all clostridial vaccines is in progress . In addition In-VITRO is now considering the setting up of a programme for Erysipelas vaccines . The collaboration between manufacturers of veterinary vaccines in the development and validation of in vitro tests is a major step towards the reduction and replacement of animal tests.

Dev Biol Stand, 1999, 101, 85 - 94
Development of monoclonal antibodies suitable for use in antigen quantification potency tests for clostridial veterinary vaccines; Hauer PJ et al.; The quality control testing of clostridial veterinary vaccines currently requires large numbers of animals . Alternative in vitro test methods are being investigated by researchers in industry and by regulatory authorities in many countries . Monoclonal antibodies that neutralize Clostridium perfringens alpha toxin, C . perfringens beta toxin, C . perfringens epsilon toxin, and C . sordellii lethal toxin as well as a monoclonal antibody directed against C . chauvoei flagellar antigen have been developed by the Center for Veterinary Biologics-Laboratory for use in antigen quantification assays . A proposal to create an international standard collection of clostridial-specific monoclonal antibodies is made.

Am J Gastroenterol, 1999 Nov, 94(11), 3263 - 6
Clostridium difficile-associated diarrhea in the elderly; Brandt LJ et al.; OBJECTIVE: It is widely believed that Clostridium difficile (C . difficile)-associated diarrhea is a more severe disease in the elderly than in the young, associated with increased morbidity and mortality . These beliefs are largely anecdotal, and there are few data supporting them . METHODS: We conducted an evaluation in an urban, tertiary care hospital of 89 inpatients in whom C . difficile-associated diarrhea was identified . These patients were evaluated prospectively, and the group was divided by age into those < 60 yr of age (younger) and those > or = 60 yr (elderly) . RESULTS: There was no difference in mortality or morbidity in elderly individuals with C . difficile-associated diarrhea when compared with younger persons similarly infected . The response to standard treatment was similar in both groups . Older patients were more likely to have an elevated white blood cell count in association with C . difficile-associated diarrhea (60% vs 26%, p < 0.05), and were more likely to have acquired their infection in the hospital (89% vs 50%, p < 0.0001) . CONCLUSIONS: In the elderly, C . difficile-associated diarrhea is almost always acquired in institutions, and may not be obvious among patients' other problems . The elderly do not seem to have an increase in C . difficile diarrhea-associated morbidity or mortality . There is no evidence that C . difficile-associated diarrhea is more severe in the elderly than it is in the young.

Dis Colon Rectum, 1999 Nov, 42(11), 1502 - 4
Can quinolones cause hemorrhagic colitis of late onset? Report of three cases; Koga H et al.; PURPOSE: This study was undertaken to demonstrate that quinolones may cause acute colitis resembling penicillin-induced hemorrhagic colitis . METHODS: We reviewed the medical records of patients with acute colitis in our institutes . Twenty-eight patients with acute hemorrhagic colitis in which no pathogenic microorganisms were identified were the subjects of this study . Pseudomembranous colitis caused by Clostridium difficile was excluded . Ulcerative colitis, Crohn's disease, and radiation proctocolitis were also excluded . RESULTS: Among these patients, 25 had a history of recent administration of penicillin derivatives . The remaining three patients had never been given any penicillin derivatives, but had ingested quinolones approximately four weeks before the developing colitis had been identified . Klebsiella oxytoca was also isolated in these three patients . CONCLUSIONS: Quinolones may cause acute hemorrhagic colitis . The time interval from antibiotic ingestion to onset of the condition may be much longer in quinolones than in penicillin derivatives.

Biochim Biophys Acta, 1999 Nov 16, 1472(3), 550 - 4
Clostridium innocuum: a glucoseureide-splitting inhabitant of the human intestinal tract; Mohr C et al.; Glycosylureides were recently described as non-invasive markers of intestinal transit time . The underlying principle is an enzymatic splitting of (13)C-labelled ureides by intestinal bacteria . The (13)CO(2) released from the urea moiety of the glycosylureides can be measured in breath samples when the ingested tracer substrate reaches the caecum that is colonised with microbes . To date, the microbes that degrade glycosylureides are unknown . In order to identify the glucoseureide (GU)-splitting bacteria, 174 different strains of intestinal microbes obtained from five healthy adults were checked for their ability to degrade GU . The results of the microbial cultures and thin layer chromatography revealed that GU was exclusively degraded by Clostridium innocuum, belonging to the normal human intestinal microflora . C . innocuum probably synthesises a yet unknown enzyme that splits the glucose-urea bond . We suggest that the term glucoseureidehydrolase is the appropriate designation for this enzyme.

J Bacteriol, 1999 Nov, 181(22), 7136 - 9
Transcriptional analysis of the rubrerythrin and superoxide dismutase genes of Clostridium perfringens; Geissmann TA et al.; We cloned and sequenced a 2.7-kb fragment of chromosomal DNA from Clostridium perfringens containing the superoxide dismutase-encoding gene, sod . Previously, rubrerythrin from C . perfringens had been isolated and its gene (rbr) had been cloned (Y . Lehmann, L . Meile, and M . Teuber, J . Bacteriol . 178:7152-7158, 1996) . Northern blot experiments revealed a length of approximately 800 bases for each transcript of rbr and sod of C . perfringens . Thus, rbr and sod each represent a monocistronic operon . Their transcription start points were located by primer extension analyses . sod transcription was shown to depend on the growth phase, and it reached a maximum during the transition from log phase to stationary phase . Neither sod nor rbr transcription was influenced by oxidative stress.

Biochemistry, 1999 Nov 9, 38(45), 14803 - 9
Modulation of the redox potential of the {Fe(SCys)(4)} site in rubredoxin by the orientation of a peptide dipole; Eidsness MK et al.; Rubredoxins (Rds) may be separated into two classes based upon the correlation of their reduction potentials with the identity of residue 44; those with Ala44 have reduction potentials that are approximately 50 mV higher than those with Val44 . The smaller side chain volume occupied by Ala44 relative to that occupied by Val44 has been proposed to explain the increase in the reduction potential, based upon changes in the Gly43-Ala44 peptide bond orientation and the distance to the {Fe(SCys)(4)} center in the Pyrococcus furiosus (Pf) Rd crystal structure compared to those of Gly43-Val44 in the Clostridium pasteurianum (Cp) Rd crystal structure . As an experimental test of this hypothesis, single-site Val44 <--> Ala44 exchange mutants, {V44A}Cp and {A44V}Pf Rds, have been cloned and expressed . Reduction potentials of these residue 44 variants and pertinent features of the X-ray crystal structure of {V44A}Cp Rd are reported . Relative to those of wild-type Cp and Pf Rds, the V44A mutation in Cp Rd results in an 86 mV increase in midpoint reduction potential and the {A44V} mutation in Pf Rd results in a 95 mV decrease in midpoint reduction potential, respectively . In the crystal structure of {V44A}Cp Rd, the peptide bond between residues 43 and 44 is approximately 0.3 A closer to the Fe center and the hydrogen bond distance between the residue 44 peptide nitrogen and the Cys42 gamma-sulfur decreases by 0.32 A compared to the analogous distances in the wild-type Cp Rd crystal structure . The results described herein support the prediction that the identity of residue 44 alone determines whether a Rd reduction potential of about -50 or 0 mV is observed.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 241 - 8
Sequence analysis of a new open reading frame located in the pathogenicity locus of Clostridium difficile strain 8864; Song KP et al.; Strain 8864 is a natural isolate of Clostridium difficile that is toxin B-positive and toxin A-negative . Recent work showed that there is a genetic rearrangement occurring at the pathogenicity locus (PaLoc) of the bacteria . Our investigation in the PaLoc region revealed an open reading frame (tcdF) of 543 bp DNA not reported before . This tcdF could encode a putative polypeptide of 22 kDa . Although no peptide homology was found with other known proteins, we postulate that it could be a novel protein because not only highly consensus ribosome-binding and promoter sequences were found upstream of tcdF, transcript was also identified at the region occupied by tcdF.

FEMS Microbiol Lett, 1999 Nov 15, 180(2), 197 - 203
Identification of the syntrophic partners in a coculture coupling anaerobic methanol oxidation to Fe(III) reduction; Daniel R et al.; From enrichments with methanol and ferric pyrophosphate a coculture was isolated which coupled methanol oxidation to carbon dioxide with the reduction of Fe(III) to Fe(II) . 16S rRNA gene analysis of the isolated syntrophic partners revealed 99.5% similarity to Clostridium sphenoides and 98.5% to Shewanella putrefaciens . Formation of Fe(II) coupled to methanol oxidation was confirmed by using strains of culture collections (C . sphenoides DSM 632 and S . putrefaciens DSM 9461) . The importance of this process is discussed, also with respect to the anaerobic oxidation of methane.

J Immunol Methods, 1999 Aug 31, 228(1-2), 151 - 62
Phage display of a cellulose binding domain from Clostridium thermocellum and its application as a tool for antibody engineering; Berdichevsky Y et al.; Phage display of antibody fragments has proved to be a powerful tool for the isolation and in vitro evolution of these biologically important molecules . However, the general usefulness of this technology is still limited by some technical difficulties . One of the most debilitating obstacles to the widespread application of the technology is the accumulation of "insert loss" clones in the libraries; phagemid clones from which the DNA encoding part or all of the cloned antibody fragment had been deleted . Another difficulty arises when phage technology is applied for cloning hybridoma-derived antibody genes, where myeloma derived light chains, irrelevant to the hybridoma's antibody specificity may be fortuitously cloned . Here, we report the construction of a novel phage-display system designed to address these problems . In our system a single-chain Fv (scFv) is expressed as an in-frame fusion protein with a cellulose-binding domain (CBD) derived from the Clostridium thermocellum cellulosome . The CBD domain serves as an affinity tag allowing rapid phage capture and concentration from crude culture supernatants, and immunological detection of both displaying phage and soluble scFv produced thereof . We demonstrate the utility of our system in solving the technical difficulties described above, and in speeding up the process of scFv isolation from combinatorial antibody repertoires.

Arch Surg, 1999 Nov, 134(11), 1235 - 41; discussion 1241-2
Clostridium difficile toxins may augment bacterial penetration of intestinal epithelium; Feltis BA et al.; BACKGROUND: Clostridium difficile can be recovered from many high-risk hospitalized patients receiving broad-spectrum antibiotic therapy . Clostridium difficile toxins A and B have been associated with increased intestinal permeability in vitro and there is growing evidence that increased intestinal permeability may be a common mechanism whereby enteric bacteria penetrate the intestinal epithelium . HYPOTHESIS: Clostridium difficile-induced alterations in the intestinal barrier facilitate microbial penetration of the intestinal epithelium, which in turn facilitates the translocation of intestinal bacteria . DESIGN: Mature Caco-2 enterocytes were pretreated with varying concentrations of toxin A or toxin B followed by 1 hour of incubation with pure cultures of either Salmonella typhimurium, Escherichia coli, or Proteus mirabilis . The effects of toxins A and B on enterocyte viability, cytoskeletal actin, and ultrastructural topography were assessed using vital dyes, fluorescein-labeled phalloidin, and scanning electron microscopy, respectively . The toxins' effects on bacterial adherence and bacterial internalization by cultured enterocytes were assessed using enzyme-linked immunosorbent assay and quantitative culture, respectively . Epithelial permeability was assessed by changes in transepithelial electrical resistance and by quantifying paracellular bacterial movement through Caco-2 enterocytes cultivated on permeable supports . RESULTS: Neither toxin A nor toxin B had a measurable effect on the numbers of enteric bacteria internalized by Caco-2 enterocytes; however, both toxins were associated with alterations in enterocyte actin, decreased transepithelial electrical resistance, and increased bacterial adherence and paracellular transmigration . CONCLUSION: Clostridium difficile toxins A or B may facilitate bacterial adherence and penetration of the intestinal epithelial barrier.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1539 - 50
Clostridium frigidicarnis sp . nov., a psychrotolerant bacterium associated with 'blown pack' spoilage of vacuum-packed meats; Broda DM et al.; Two strains of a psychrotolerant Clostridium, isolated from vacuum-packed, temperature-abused beef, were characterized using a multiphasic approach . The strains were Gram-positive motile rods producing elliptical subterminal spores during early stationary growth phase . The strains were psychrotolerant . At pH 7.0, they grew between 3.8 and 40.5 degrees C; their optimum growth temperature was 30.0-38.5 degrees C . At 30 degrees C, the pH range for growth was between 4.7 and 9.5; the optimum pH for growth was 6.4-7.2 . The organisms were proteolytic and saccharolytic, lecithinase-positive and hydrolysed gelatin . The fermentation products formed in peptone/yeast extract/glucose/starch broth were acetate, ethanol, butyrate, isovalerate, butanol, isobutyrate, oxalacetate, lactate, hydrogen and carbon dioxide . The DNA G + C compositions of the two meat strains were 27.3 and 28.4 mol% . Phylogenetic analyses indicated that the strains belong to Cluster I of the genus Clostridium (sensu Collins et al., 1994) . The new strains differed from phylogenetically related clostridia in terms of cellular fatty acid composition, soluble protein profiles and phenotypic properties . On the basis of phenotypic and genotypic characterization data, the strains were assigned to a new species for which the name Clostridium frigidicarnis is proposed; strain SPL77AT (= DSM 12271T) is the type strain.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1387 - 94
A protein-based phylogenetic tree for gram-positive bacteria derived from hrcA, a unique heat-shock regulatory gene; Ahmad S et al.; The dnaK operon from Bacillus subtilis and other Gram-positive bacteria with low G + C DNA content contains additional heat-shock genes, including hrcA . The hrcA gene encodes a transcription factor that negatively regulates heat-shock genes and is uniformly present in all Gram-positive bacteria studied to date . An hrcA homologue is also present in Synechocystis species, Leptospira interrogans, Chlamydia trachomatis, Caulobacter crescentus and Methanococcus jannaschii, organisms that diverged early on from the common ancestor of all Gram-positive bacteria and Proteobacteria, according to 16S rRNA phylogeny . A partial, protein-based phylogenetic tree, derived using amino acid sequence homology of hrcA proteins from Gram-positive bacteria, is presented here, and the results are compared with the phylogenetic trees generated from 16S rRNA, dnaK and dnaJ sequences . The location of the hrcA gene and the genome organization of the dnaK operon support the division of all Gram-positive bacteria into three major groups: one group contains high-G + C Gram-positive bacteria, and two others contain low-G + C Gram-positive bacteria . Among the Gram-positive bacteria with low G + C DNA content, the results indicate that there is a close phylogenetic relationship between Bacillus species and Clostridium species on the one hand and between Lactococcus lactis and Streptococcus mutans on the other . Streptomyces and Mycobacterium species also exhibited a close relationship . A hierarchical arrangement of Gram-positive bacteria based on HrcA sequences is proposed as an additional refinement of the phylogenetic relationships within this important bacterial group.

Int J Syst Bacteriol, 1999 Oct, 49 Pt 4, 1375 - 9
Phylogenetic analysis of Fusobacterium alocis and Fusobacterium sulci based on 16S rRNA gene sequences: proposal of Filifactor alocis (Cato, Moore and Moore) comb . nov . and Eubacterium sulci (Cato, Moore and Moore) comb . nov; Jalava J et al.; Genes encoding the 16S rRNA of Fusobacterium alocis ATCC 35896T and Fusobacterium sulci ATCC 35585T were sequenced . These sequences did not have any affinity with the 16S rRNA gene sequences of members of the genus Fusobacterium . Fusobacterium alocis ATCC 35896T and Fusobacterium sulci ATCC 35585T belonged to Clostridium cluster XI; the species most closely related to these strains were Filifactor villosus NCTC 11220T and Eubacterium infirmum W1471, respectively . Two new combinations are proposed: Filifactor alocis (Cato, Moore and Moore) comb . nov . (type strain ATCC 35896T) and Eubacterium sulci (Cato, Moore and Moore) comb . nov . (type strain ATCC 35585T).

Cell, 1999 Oct 29, 99(3), 293 - 9
The mechanism of membrane insertion for a cholesterol-dependent cytolysin: a novel paradigm for pore-forming toxins; Shatursky O et al.; Perfringolysin O (PFO), a water-soluble monomeric cytolysin secreted by pathogenic Clostridium perfringens, oligomerizes and forms large pores upon encountering cholesterol-containing membranes . Whereas all pore-forming bacterial toxins examined previously have been shown to penetrate the membrane using a single amphipathic beta hairpin per polypeptide, cysteine-scanning mutagenesis and multiple independent fluorescence techniques here reveal that each PFO monomer contains a second domain involved in pore formation, and that each of the two amphipathic beta hairpins completely spans the membrane . In the soluble monomer, these transmembrane segments are folded into six alpha helices . The insertion of two transmembrane hairpins per toxin monomer and the major change in secondary structure are striking and define a novel paradigm for the mechanism of membrane insertion by a cytolytic toxin.

Am Surg, 1999 Nov, 65(11), 1049 - 53
Microbiology of subphrenic abscesses: a 14-year experience; Brook I et al.; The objective of the review was to study the aerobic and anaerobic microbiology of subphrenic abscesses in relation with predisposing conditions . A retrospective review of clinical and laboratory data of 52 patients treated between 1974 and 1988 was conducted . Forty-three (83%) patients developed the abscesses after an operative procedure . These included 11 patients after colonic, 9 patients after gastric or duodenal, 7 patients after abdominal trauma, 7 patients after biliary, and 6 patients after appendix surgery . A total of 194 organisms (3.7 isolates/specimen), 83 aerobic (1.6/specimen), and 111 anaerobes (2.1/specimen) were recovered . Aerobic bacteria only were recovered in 7 (13%) abscesses, anaerobic bacteria only in 11 (21%), and mixed aerobic and anaerobic bacteria in 34 (65%) . Polymicrobial infection was present in 47 (90%) . The predominant aerobic isolates were Escherichia coli (28 isolates), Enterococcus group D(9), and Staphylococcus aureus (9) . The predominant anaerobes were Peptostreptococcus species (33 isolates), Bacteroides fragilis group (25), Clostridium species (13), and Prevotella species (6) . The number of isolates/site varied . The number of anaerobic bacteria/site outnumbered or was equal to the number of aerobic or facultatives in all instances, except in abscesses after biliary surgery . Their number/site was especially high in abscesses after appendectomy, and the number of aerobic and anaerobic bacteria was the lowest after gastric or duodenal surgery . S . aureus predominated after gastric, duodenal and posttrauma surgery; B . fragilis predominated after colonic, appendix, and postabdominal trauma surgery; Enterococcus group D predominated after biliary surgery; Fusobacterium and Prevotella species predominated after gastric or duodenal surgery; and Clostridium species predominated after colonic or appendix surgery . These data highlight the polymicrobial aerobic-anaerobic nature of subphrenic abscesses and their correlation with predisposing surgery.

Drugs, 1999 Oct, 58(4), 683 - 96; discussion 697-8
Gatifloxacin; Perry CM et al.; Gatifloxacin is a novel extended-spectrum fluoroquinolone with improved gram-positive and anaerobe coverage compared with older agents such as ciprofloxacin . It has good activity (but is slightly less active than ciprofloxacin) against Enterobacteriaceae . Gatifloxacin is generally 2- to 4-fold more active than ciprofloxacin against staphylococci, streptococci and enterococci and 4- to 16-fold more active than ciprofloxacin against anaerobes, including Clostridium and Bacteroides spp . In comparative clinical trials that included patients with lower respiratory tract, urinary tract, skin and soft tissue or gonococcal infections, clinical cure rates of > or = 89% were achieved with oral gatifloxacin 400 mg/day for 7 to 14 days . Data from a subset of North American patients included in a multinational trial showed that oral gatifloxacin 400 mg/day produced a significantly higher clinical cure rate than cefuroxime axetil 250 mg twice daily (89 vs 77%; p = 0.01) in patients with acute exacerbations of chronic bronchitis . The clinical efficacy of gatifloxacin was similar to that of clarithromycin or levofloxacin or ceftriaxone (with or without erythromycin) in the treatment of patients with community-acquired pneumonia . Oral gatifloxacin 400 mg/day showed clinical and bacteriological efficacy similar to that of levofloxacin in patients with skin and soft tissue infections . In patients with urinary tract infections, clinical cure and bacterial eradication rates achieved with a single 400 g oral dose of gatifloxacin were similar to those produced with ciprofloxacin . In a pooled analysis of tolerability data from trials that included 3021 patients treated with oral gatifloxacin 400 mg/day, the most commonly reported adverse events were nausea (8%), diarrhoea (4%), headache (4%) and dizziness (3%) . The drug was reported to be well tolerated . Gatifloxacin does not appear to cause phototoxic effects.

Dig Dis Sci, 1999 Oct, 44(10), 2119 - 23
Probiotic bacteria stimulate gut epithelial cell proliferation in rat; Ichikawa H et al.; Probiotics are used for various intestinal diseases . However, their effects on gut epithelial cell proliferation have not been investigated . We administered 10(7) colony-forming units of Lactobacillus casei or Clostridium butyricum, or no probiotics (control) by gastric intubation once a day for seven days to rats fed an elemental diet . We estimated the crypt cell production rate of the jejunum, ileum, cecum, and distal colon . We also quantified cecal bacteria . Both probiotics increased the crypt cell production rate of the jejunum and ileum by 25-40%, of the cecum by 70%, and of the distal colon by more than 200% compared with control . Only minor variance in the cecal bacterial composition existed among the three groups . Probiotics enhanced gut epithelial cell proliferation in rats fed an elemental diet.

Protein Expr Purif, 1999 Nov, 17(2), 249 - 59
Matrix-assisted refolding of single-chain Fv- cellulose binding domain fusion proteins; Berdichevsky Y et al.; We describe a method for the isolation of recombinant single-chain antibodies in a biologically active form . The single-chain antibodies are fused to a cellulose binding domain as a single-chain protein that accumulates as insoluble inclusion bodies upon expression in Escherichia coli . The inclusion bodies are then solubilized and denatured by an appropriate chaotropic solvent, then reversibly immobilized onto a cellulose matrix via specific interaction of the matrix with the cellulose binding domain (CBD) moiety . The efficient immobilization that minimizes the contact between folding protein molecules, thus preventing their aggregation, is facilitated by the robustness of the Clostridium thermocellum CBD we use . This CBD is unique in retaining its specific cellulose binding capability when solubilized in up to 6 M urea, while the proteins fused to it are fully denatured . Refolding of the fusion proteins is induced by reducing with time the concentration of the denaturing solvent while in contact with the cellulose matrix . The refolded single-chain antibodies in their native state are then recovered by releasing them from the cellulose matrix in high yield of 60% or better, which is threefold or higher than the yield obtained by using published refolding protocols to recover the same scFvs . The described method should have general applicability for the production of many protein-CBD fusions in which the fusion partner is insoluble upon expression .

Appl Environ Microbiol, 1999 Nov, 65(11), 4973 - 80
The ald gene, encoding a coenzyme A-acylating aldehyde dehydrogenase, distinguishes Clostridium beijerinckii and two other solvent-producing clostridia from Clostridium acetobutylicum; Toth J et al.; The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia . It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol . The ALDH of Clostridium beijerinckii NRRL B593 was purified . It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde . The N-terminal amino acid sequence of the purified ALDH was determined . The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C . beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH . The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M(r) of 51, 353 . The position of the ald gene in C . beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792 . In Southern analyses, a probe derived from the C . acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C . beijerinckii and two other species of solvent-producing clostridia . In contrast, a probe derived from the C . beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C . acetobutylicum, indicating a key difference among the solvent-producing clostridia . The amino acid sequence of the ALDH of C . beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C . acetobutylicum, E . coli, Lactococcus lactis, and amitochondriate protozoa . The predicted secondary structure of the C . beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD(+) binding . A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.

J Bacteriol, 1999 Nov, 181(21), 6720 - 9
A novel cellulosomal scaffoldin from Acetivibrio cellulolyticus that contains a family 9 glycosyl hydrolase; Ding SY et al.; A novel cellulosomal scaffoldin gene, termed cipV, was identified and sequenced from the mesophilic cellulolytic anaerobe Acetivibrio cellulolyticus . Initial identification of the protein was based on a combination of properties, including its high molecular weight, cellulose-binding activity, glycoprotein nature, and immuno-cross-reactivity with the cellulosomal scaffoldin of Clostridium thermocellum . The cipV gene is 5,748 bp in length and encodes a 1,915-residue polypeptide with a calculated molecular weight of 199,496 . CipV contains an N-terminal signal peptide, seven type I cohesin domains, an internal family III cellulose-binding domain (CBD), and an X2 module of unknown function in tandem with a type II dockerin domain at the C terminus . Surprisingly, CipV also possesses at its N terminus a catalytic module that belongs to the family 9 glycosyl hydrolases . Sequence analysis indicated the following . (i) The repeating cohesin domains are very similar to each other, ranging between 70 and 90% identity, and they also have about 30 to 40% homology with each of the other known type I scaffoldin cohesins . (ii) The internal CBD belongs to family III but differs from other known scaffoldin CBDs by the omission of a 9-residue stretch that constitutes a characteristic loop previously associated with the scaffoldins . (iii) The C-terminal type II dockerin domain is only the second such domain to have been discovered; its predicted "recognition codes" differ from those proposed for the other known dockerins . The putative calcium-binding loop includes an unusual insert, lacking in all the known type I and type II dockerins . (iv) The X2 module has about 60% sequence homology with that of C . thermocellum and appears at the same position in the scaffoldin . (v) Unlike the other known family 9 catalytic modules of bacterial origin, the CipV catalytic module is not accompanied by a flanking helper module, e.g., an adjacent family IIIc CBD or an immunoglobulin-like domain . Comparative sequence analysis of the CipV functional modules with those of the previously sequenced scaffoldins provides new insight into the structural arrangement and phylogeny of this intriguing family of microbial proteins . The modular organization of CipV is reminiscent of that of the CipA scaffoldin from C . thermocellum as opposed to the known scaffoldins from the mesophilic clostridia . The phylogenetic relationship of the different functional modules appears to indicate that the evolution of the scaffoldins reflects a collection of independent events and mechanisms whereby individual modules and other constituents are incorporated into the scaffoldin gene from different microbial sources.

Antimicrob Agents Chemother, 1999 Nov, 43(11), 2607 - 11
Antimicrobial susceptibilities and serogroups of clinical strains of Clostridium difficile isolated in France in 1991 and 1997; Barbut F et al.; Glycopeptides (vancomycin and teicoplanin) and metronidazole are the drugs of choice for the treatment of Clostridium difficile infections, but trends in susceptibility patterns have not been assessed in the past few years . The objective was to study the MICs of glycopeptides and metronidazole for unrelated C . difficile strains isolated in 1991 (n = 100) and in 1997 (n = 98) by the agar macrodilution, the E-test, and the disk diffusion methods . Strain susceptibilities to erythromycin, clindamycin, tetracycline, rifampin, and chloramphenicol were also determined by the ATB ANA gallery (bioMerieux, La Balme-les-Grottes, France) . The MICs at which 50% of isolates are inhibited (MIC(50)s) and MIC(90)s of glycopeptides and metronidazole remained stable between 1991 and 1997 . All the strains were inhibited by concentrations that did not exceed 2 microgram/ml for vancomycin and 1 microg/ml for teicoplanin . Comparison of MICs determined by the agar dilution method recommended by the National Committee for Clinical Laboratory Standards and the E test showed correlations (+/-2 dilutions) of 86 . 6, 95.9, and 99% for metronidazole, vancomycin, and teicoplanin, respectively . The E test always underestimated the MICs . Strains with decreased susceptibility to metronidazole (MICs, >/=8 microgram/ml) were isolated from six patients (n = 4 in 1991 and n = 2 in 1997) . These strains were also detected by the disk diffusion method (zone inhibition diameter, </=21 mm); they belonged to nontoxigenic serogroup D (n = 5) and toxigenic serogroup H (n = 1) . Decreased susceptibility to erythromycin (MICs, >/=1 microgram/ml), clindamycin (MICs, >/=2 microgram/ml), tetracycline (MICs, >/=8 microgram/ml), rifampin (MICs, >/=4 microgram/ml), and chloramphenicol (MICs, >/=16 microgram/ml) was observed in 64.2, 80.3, 23.7, 22.7, and 14.6% of strains, respectively . Strains isolated in 1997 were more susceptible than those isolated in 1991, and this trend was correlated to a major change in serogroup distribution . Periodic studies are needed in order to detect changes in serogroups and the emergence of strains with decreased susceptibility to therapeutic drugs.

Microbiology, 1999 Oct, 145 ( Pt 10), 2947 - 55
Identification of structural and functional domains of the tetracycline efflux protein TetA(P) from Clostridium perfringens; Bannam TL et al.; The Clostridium perfringens tetracycline-resistance protein, TetA(P), is an integral inner-membrane protein that mediates the active efflux of tetracycline from the cell . TetA(P) acts as an antiporter, presumably transporting a divalent cation-tetracycline complex in exchange for a proton, and is predicted to have 12 transmembrane domains (TMDs) . Two glutamate residues that are located in predicted TMD 2 were previously shown to be required for the active efflux of tetracycline by TetA(P) . To identify additional residues that are required for the structure or function of TetA(P), a random mutagenesis approach was used . Of the 61 tetracycline-susceptible mutants that were obtained in Escherichia coli, 31 different derivatives were shown to contain a single amino acid change that resulted in reduced tetracycline resistance . The stability of the mutant TetA(P) proteins was examined by immunoblotting and 19 of these strains were found to produce a detectable TetA(P) protein . The MIC of these derivatives ranged from 2 to 15 microg tetracycline ml(-1), compared to 30 microg tetracycline ml(-1) for the wild-type . The majority of these mutants clustered into three potential loop regions of the TetA(P) protein, namely the cytoplasmic loops 2-3 and 4-5, and loop 7-8, which is predicted to be located in the periplasm in E . coli . It is concluded that these regions are of functional significance in the TetA(P)-mediated efflux of tetracycline from the bacterial cell.

Infect Control Hosp Epidemiol, 1999 Oct, 20(10), 664 - 70
Enteric carriage of vancomycin-resistant Enterococcus faecium in patients tested for Clostridium difficile; Garbutt JM et al.; OBJECTIVE: To identify independent risk factors for enteric carriage of vancomycin-resistant Enterococcus faecium (VREF) in hospitalized patients tested for Clostridium difficile toxin . DESIGN: Retrospective case-cohort study . SETTING: Tertiary-care teaching hospital . PATIENTS: Convenience sample of 215 adult inpatients who had stool tested for C difficile between January 29 and February 25, 1996 . RESULTS: 41 (19%) of 215 patients had enteric carriage of VREE Five independent risk factors for enteric VREF were identified: history of prior C difficile (odds ratio {OR}, 15.21; 95% confidence interval {CI95}, 3.30-70.10; P < .001), parenteral treatment with vancomycin for > or = 5 days (OR, 4.06; CI95, 1.54-10.73; P = .005), treatment with antimicrobials effective against gram-negative organisms (OR, 3.44; CI95, 1.20-9.87; P = .021), admission from another institution (OR, 2.95; CI95, 1.21-7.18; P =.017), and age > 60 years (OR 2.57; CI95, 1.13-5.82; P = .024) . These risk factors for enteric VREF were independent of the patient's current C difficile status . CONCLUSIONS: Antimicrobial exposures are the most important modifiable independent risk factors for enteric carriage of VREF in hospitalized patients tested for C difficile.

Infect Immun, 1999 Nov, 67(11), 5634 - 41
Identification of a Clostridium perfringens enterotoxin region required for large complex formation and cytotoxicity by random mutagenesis; Kokai-Kun JF et al.; Clostridium perfringens enterotoxin (CPE), a single polypeptide of 319 amino acids, has a unique multistep mechanism of action . In the first step, CPE binds to claudin proteins and/or a 50-kDa eukaryotic membrane protein receptor, forming a small ( approximately 90-kDa) complex . This small complex apparently then associates with a 70-kDa eukaryotic membrane protein, resulting in formation of a large complex that induces the onset of membrane permeability alterations . To better define the boundaries of CPE functional regions and to identify specific amino acid residues involved in various steps of CPE action, in this study we subjected the cloned cpe gene to random mutagenesis in XL-1 Red strains of Escherichia coli . Seven CPE random mutants with reduced cytotoxicity for Vero cells were phenotypically characterized for the ability to complete each step in CPE action . Five of these seven recombinant CPE (rCPE) random mutants (G49D, S59L, R116S, R137G, and S167P) exhibited binding characteristics similar to those of rCPE or native CPE, while the Y310C and W226Stop mutants showed reduced binding and no binding, respectively, to brush border membranes . Interestingly, two completely nontoxic mutants (G49D and S59L) were able to bind and form small complex but they did not mediate any detectable large complex formation . Another strongly attenuated mutant, R116S, formed reduced amounts of an anomalously migrating large complex . Collectively, these results provide further support for large complex formation being an essential step in CPE action and also identify the CPE region ranging from residues approximately 45 to 116 as important for large complex formation . Finally, we also report that limited removal of extreme N-terminal CPE sequences, which may occur in vivo during disease, stimulates cytotoxic activity by enhancing large complex formation.

Biochemistry, 1999 Oct 5, 38(40), 12969 - 73
Binding of exogenously added carbon monoxide at the active site of the iron-only hydrogenase (CpI) from Clostridium pasteurianum; Lemon BJ et al.; A site for the binding of exogenously added carbon monoxide has been identified at the active site of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum . The binding and inhibition of carbon monoxide have been exploited in biochemical and spectroscopic studies to gain mechanistic insights . In the present study, we have taken advantage of the ability to generate an irreversibly carbon monoxide bound state of CpI . The crystallization and structural characterization of CpI inhibited in the presence of carbon monoxide indicates the addition of a single molecule of carbon monoxide . The ability to generate crystals of the carbon monoxide bound state of the hydrogenase that are isomorphous to those of the native enzyme has allowed for a direct comparison of the crystallographic data and an unambiguous identification of the site of carbon monoxide binding at the active site of CpI . Carbon monoxide binds to an Fe atom of the 2Fe subcluster at the site of a terminally bound water molecule in the as crystallized native state of CpI that has been previously suggested to be a potential site of reversible hydrogen oxidation . Binding of carbon monoxide at this site results in an active site that is coordinately saturated with strong ligands (S, CO, and CN), providing a rational potential mechanism for inhibition of reversible hydrogen oxidation at the active site of CpI.

Int J Antimicrob Agents, 1999 Aug, 12 Suppl 2, S11 - 5
Fusidic acid in other infections; Golledge C; Fusidic acid, both systemic and topical, has been used for a wide variety of less common infections . Efficacy for oral fusidic acid has been demonstrated in the treatment of Clostridium difficile colitis and in staphylococcal infections in patients with cystic fibrosis . Topical fusidic acid gel is also effective in bacterial conjunctivitis and other minor external eye infections, and may be effective in reducing bacterial flora in the conjunctival sac prior to eye surgery . Studies suggest a potential role for fusidic acid in neurosurgical prophylaxis, as adjunctive therapy in bacterial endophthalmitis and Legionella pneumonia, and in leprosy . Topical fusidic acid has no effect in the treatment of chlamydial conjunctivitis or the prevention of staphylococcal infections in patients on continuous ambulatory peritoneal dialysis.

J Food Prot, 1999 Oct, 62(10), 1157 - 61
Effect of pH and CO2 on growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres; Daifas DP et al.; The effect of pH and CO2 on both growth of and toxin production by Clostridium botulinum in English-style crumpets, packaged under modified atmospheres was investigated using a 2 x 2 factorial experiment . English-style crumpets (water activity, 0.990; pH 6.5 and 8.3) were inoculated with C . botulinum spores types A and proteolytic B (500 spores/g), packaged in either 60% CO2 (balance N2) or 100% CO2, stored at ambient temperature (25 degrees C), and monitored daily for toxicity . Toxin was detected after 4 days in crumpets packaged in 60% CO2, irrespective of initial product pH . Toxin production was delayed 1.5 to 3 days in crumpets packaged under 100% CO2 . Analysis of variance indicated a significant interaction effect of pH and %CO2 on time of earliest toxin detection . Delay of toxin production was greatest for high pH (8.3) crumpets . All products were organoleptically acceptable at the time of toxigenesis, and therefore, high moisture-high pH bakery products, if contaminated with spores of C . botulinum, could become hazardous if packaged in atmospheres containing CO2.

J Calif Dent Assoc, 1999 May, 27(5), 405 - 9, 411-3
Clostridium difficile-associated diarrhea and colitis; Pallasch TJ; Clostridium difficile-induced diarrhea (CDAD) and colitis (CDAC) are important nosocomial (hospital)-acquired infections resulting almost exclusively from antibiotic therapy and certain host factors . The severity of these disorders may range from simple diarrhea that can be resolved easily with antibiotic cessation to fulminant pseudomembranous colitis with fever, severe dehydration, abdominal pain and distention, and plaque formation over part or all of the colon . Community-acquired CDAD and CDAC are far less problematic but nevertheless may affect 20,000 or more people in the United States every year . Knowledge of the risk factors for CDAD and CDAC, including certain antibiotics, and recognition of the entire spectrum of signs and symptoms of this disorder are imperative for good dental practice . Likewise the prevention of recurrence of CDAD by judicious use of antibiotics in its immediate posttreatment period is an important consideration.

Rev Neurol, 1999 Jul 16-31, 29(2), 157 - 62
{Indications and management of botulinum toxin}; Singer C; INTRODUCTION: Botulinus toxin (BTX) is the most potent biological toxin yet known . It is produced by Clostridium botulinium, a Gram positive bacteria . DEVELOPMENT: Type A Botulinus toxin is the most widely used in human drug trials . It has become the treatment of choice for blepharospasm, hemifacial spasm, cervical dystonia and laryngeal dystonia . It may also be used in the treatment of patients with oromandibular dystonia and limb dystonia, especially writer's cramp, and has been used successfully in the treatment of spasticity and cerebral paralysis . There are many benefits from this treatment, including improved walking, improved posture of wheelchair patients, improvement of patients with spasms and easier extension of their arms and knees . The toxin also alleviates pain and may be used in therapeutic trials for prediction of the response to surgical elongation.

Biochim Biophys Acta, 1999 Oct 12, 1434(2), 365 - 71
The NAD-dependent glutamate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum: cloning, sequencing, and expression of the enzyme gene(1); Kujo C et al.; The NAD-dependent glutamate dehydrogenase (GluDH) gene from the hyperthermophilic archaeon Pyrobaculum islandicum was cloned and expressed in Escherichia coli . Analysis of the nucleotide sequence revealed an open reading frame of 1266 bp encoding a protein of 421 amino acids with a molecular weight of 46,905 . In the alignment of the amino acid sequence with those of mesophilic Clostridium symbiosum NAD-dependent GluDH and hyperthermophilic NADP-dependent enzymes from Thermococcus profundus and Pyrococcus furiosus, substitutions in the residues involved in dinucleotide binding were observed . On the other hand, the residues involved in glutamate binding were well conserved . This is the first description of the primary structure of NAD-dependent GluDH in hyperthermophilic archaea.

Microbiol Immunol, 1999, 43(8), 737 - 42
Detection of Clostridium difficile toxin A by reversed passive latex agglutination; Toma C et al.; A reversed passive latex agglutination (RPLA) assay for detecting Clostridium difficile toxin A is presented . Purified monoclonal antibody (mAb 37B5) was used for latex sensitization . The culture supernatants of 93 strains of C . difficile were tested by RPLA assay and the results compared with those of a commercially available latex agglutination test, PCR and cytotoxin assay with Vero cells . There was agreement between RPLA, cytotoxicity and PCR assays, but 29 strains were positive in the RPLA assay while 35 were positive in the cytotoxicity test and PCR using primer pair NK3-NK2 directed to the nonrepeating portion of the C . difficile toxin A gene . The 6 cytotoxic but RPLA-negative strains were demonstrated to be toxin A-negative/toxin B-positive strains in the PCR assay by using primer pair NK11-NK9 directed to the repeating portion of the C . difficile toxin A gene . There were no cross-reactions with culture supernatants of the other clostridial strains except for two strains of C . sordelli that produced hemorrhagic toxin (which is immunologically related to C . difficile toxin A).

Nurs Times, 1999 Jul 14-20, 95(28), 49 - 50, 53
Infection control . Dazed and confused; Prieto J et al.; This research project aims to uncover the practical concerns of health care staff on a hospital ward while attempting to implement isolation precaution guidelines for patients with Clostridium difficile-associated diarrhoea (CDAD) and methicillin-resistant Staphylococcus aureus (MRSA) . The project is still in progress so this article describes the research methods used and some preliminary findings.

J Clin Microbiol, 1999 Nov, 37(11), 3778 - 9
Septicemia in neutropenic patients infected with Clostridium tertium resistant to cefepime and other expanded-spectrum cephalosporins; Steyaert S et al.; Clostridium tertium was isolated from two immunocompromised patients with septicemia, fever, and gastrointestinal symptoms . The strains were resistant to ceftazidime, cefepime, and clindamycin; intermediately resistant to penicillin; and susceptible to metronidazole, quinolones, and vancomycin.

J Biol Chem, 1999 Oct 22, 274(43), 30361 - 4
Mildly oxidized low density lipoprotein induces contraction of human endothelial cells through activation of Rho/Rho kinase and inhibition of myosin light chain phosphatase; Essler M et al.; Mildly oxidized low density lipoprotein (mox-LDL) is critically involved in the early atherogenic responses of the endothelium and increases endothelial permeability through an unknown signal pathway . Here we show that (i) exposure of confluent human endothelial cells (HUVEC) to mox-LDL but not to native LDL induces the formation of actin stress fibers and intercellular gaps within minutes, leading to an increase in endothelial permeability; (ii) mox-LDL induces a transient decrease in myosin light chain (MLC) phosphatase that is paralleled by an increase in MLC phosphorylation; (iii) phosphorylated MLC stimulated by mox-LDL is incorporated into stress fibers; (iv) cytoskeletal rearrangements and MLC phosphorylation are inhibited by C3 transferase from Clostridium botulinum, a specific Rho inhibitor, and Y-27632, an inhibitor of Rho kinase; and (v) mox-LDL does not increase intracellular Ca(2+) concentration . Our data indicate that mox-LDL induces endothelial cell contraction through activation of Rho and its effector Rho kinase which inhibits MLC phosphatase and phosphorylates MLC . We suggest that inhibition of this novel cell signaling pathway of mox-LDL could be relevant for the prevention of atherosclerosis.

J Microbiol Methods, 1999 Oct, 38(1-2), 53 - 62
Amplification of fluorescently labelled DNA within gram-positive and acid-fast bacteria; Vaid A et al.; Representative organisms from a variety of Gram-positive genera were subjected to varying regimes in order to optimise the intracellular amplification of DNA . The bacteria were subjected to treatments with paraformaldehyde, muramidases and mild acid hydrolysis to discover which regime made each organism permeable to the amplification reagents yet allowed retention of the fluorescein-labelled amplified products within the cell . Scanning electron micrographs were used to corroborate the effectiveness of the treatments, as seen by fluorescent photomicrographs, with the damage caused to the bacterial walls . A combination of mutanolysin and lysozyme was found most effective for Bacillus cereus, whereas permeabilisation of Streptomyces coelicolor, Lactococcus lactis and Clostridium sporogenes was most effective when exposed to lysozyme only . Surprisingly, direct amplification with no pre-treatment gave the brightest fluorescence in Mycobacterium phlei . Comparing the techniques of whole cell PCR, primed in situ labelling (PRINS), and cycle PRINS showed that under the conditions used the strongest intensity of fluorescence was obtained with in situ PCR; only L . lactis and M . phlei produced signals with cycle PRINS, fluorescence was not seen for any of the organisms with PRINS.

Am J Kidney Dis . 1999 Oct;34(4):e16.
Association of IgA nephropathy with Clostridium difficile colitis; Gaughan WJ et al.; Immunoglobulin A (IgA) nephropathy, the most common cause of glomerulonephritis worldwide, is usually idiopathic in origin and renal limited . Secondary IgA nephropathy has been associated with systemic disease, including such gastrointestinal tract disturbances as celiac sprue and inflammatory bowel disease . We describe gross hematuria and reversible acute renal failure from IgA nephropathy in a patient with cephalosporin-induced Clostridium difficile colitis . In addition to mesangial IgA and C3 deposition, renal histological examination showed glomerular bleeding, intratubular red blood cell casts, and acute tubular necrosis . To the best of our knowledge, this is the first report of an association between IgA nephropathy and C difficile colitis.

Microb Comp Genomics, 1999, 4(1), 47 - 58
Molecular cloning of the dnaK gene region from Bacillus sphaericus in the context of genomic comparisons; Ahmad S et al.; The dnaK gene region of Bacillus sphaericus was cloned as a 3.8 kb HindIII fragment and an overlapping 1.7 kb EcoRI fragment by using an internal B . sphaericus specific dnaK gene probe generated by polymerase chain reaction (PCR) . Complete DNA sequencing of the two fragments revealed three complete open reading frames (ORFs) . These ORFs exhibited a high degree of identity to the grpE dnaK, and dnaJ heat shock genes from other gram-positive bacteria . The order of the genes was found to be grpE-dnaK-dnaJ . Additionally, the 5'-end and 3'-end contained amino acid sequences that were homologous to the C-terminal sequence of the hrcA gene and the N-terminal sequence of ORF35 (yqeT), respectively, from Bacillus subtilis . The entire hrcA gene from B . sphaericus was then isolated by high-fidelity PCR and completely sequenced . A transcription stop site is located between the dnaK and dnaJ genes but not after the dnaJ gene . Consistent with this observation, the dnaJ gene is immediately followed by an ORF that shows a high degree of identity to ORF35 from B . subtilis, Staphylococcus aureus, and Clostridium acetobutylicum . The presence of ORF35 is not indicated in other genera representing the gram-positive bacteria . The amino acid sequence of ORF35 exhibited nearly 30% identity with the methyltransferase for large subunit ribosomal protein L11 from gram-negative Proteobacteria and the related protein from cyanobacteria, other gram-negative bacteria, and Archaea, suggesting the presence of the gene for this protein in the common ancestor of Bacteria and Archaea . The absence of the ORF35 gene in Mycobacterium tuberculosis and other gram-positive bacteria indicates that the loss of this gene must have occurred in an ancestor of other gram-positive bacteria following their divergence from the ancestor of Bacillus/Clostridium/staphylococcus lineage.

Microbiology, 1999 Sep, 145 ( Pt 9), 2533 - 42
Characterization of haemagglutinin activity of Clostridium botulinum type C and D 16S toxins, and one subcomponent of haemagglutinin (HA1); Inoue K et al.; The 16S toxin and one subcomponent of haemagglutinin (HA), designated HA1, were purified from a type D culture of Clostridium botulinum by a newly established procedure, and their HA activities as well as that of purified type C 16S toxin were characterized . SDS-PAGE analysis indicated that the free HA1 forms a polymer with a molecular mass of approximately 200 kDa . Type C and D 16S toxins agglutinated human erythrocytes in the same manner . Their HA titres were dramatically reduced by employing erythrocytes that had been previously treated with neuraminidase, papain or proteinase K, and were inhibited by the addition of N-acetylneuraminic acid to the reaction mixtures . In a direct-binding test to glycolipids such as SPG (NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc beta1-Cer) and GM3 (NeuAc alpha2-3Gal beta1-4Glc beta1-Cer), and glycoproteins such as glycophorin A and/or B prepared from the erythrocytes, both toxins bound to sialylglycolipids and sialoglycoproteins, but bound to neither neutral glycolipids nor asialoglycoproteins . On the basis of these results, it was concluded that type C and D 165 toxins bind to erythrocytes through N-acetylneuraminic acid . HA1 showed no haemagglutination activity, although it did bind to sialylglycolipids . We therefore speculate that binding to glycoproteins rather than to glycolipids may be important in causing haemagglutination by type C and D 16S toxins.

Biopolymers, 1999 Nov, 50(6), 656 - 66
Dependence of the Raman signature of genomic B-DNA on nucleotide base sequence; Deng H et al.; The vibrational spectra of four genomic and two synthetic DNAs, encompassing a wide range in base composition {poly(dA-dT) . poly(dA-dT), 0% G + C; Clostridium perfringens DNA, 27% G + C; calf thymus DNA, 42% G + C; Escherichia coli DNA, 50% G + C; Micrococcus luteus DNA, 72% G + C; poly(dG-dC).poly(dG-dC), 100% G + C} (dA: deoxyadenosine; dG: deoxyguanosine; dC: deoxycytidine; dT: thymidine), have been analyzed using Raman difference methods of high sensitivity . The results show that the Raman signature of B DNA depends in detail upon both genomic base composition and sequence . Raman bands assigned to vibrational modes of the deoxyribose-phosphate backbone are among the most sensitive to base sequence, indicating that within the B family of conformations major differences occur in the backbone geometry of AT- and GC-rich domains . Raman bands assigned to in-plane vibrations of the purine and pyrimidine bases-particularly of A and T-exhibit large deviations from the patterns expected for random base distributions, establishing that Raman hypochromic effects in genomic DNA are also highly sequence dependent . The present study provides a basis for future use of Raman spectroscopy to analyze sequence-specific DNA-ligand interactions . The demonstration of sequence dependency in the Raman spectrum of genomic B DNA also implies the capability to distinguish genomic DNAs by means of their characteristic Raman signatures .

J Cell Biol, 1999 Oct 4, 147(1), 195 - 204
Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier; Sonoda N et al.; Claudins, comprising a multigene family, constitute tight junction (TJ) strands . Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half . We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4 . C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2 . In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface . In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation . At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased . In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner . These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

Appl Environ Microbiol, 1999 Oct, 65(10), 4295 - 300
Stable Escherichia coli-Clostridium acetobutylicum shuttle vector for secretion of murine tumor necrosis factor alpha; Theys J et al.; Recombinant plasmids were constructed to secrete mouse tumor necrosis factor alpha (mTNF-alpha) from Clostridium acetobutylicum . The shuttle plasmids contained the clostridial endo-beta1, 4-glucanase (eglA) promoter and signal sequence that was fused in frame to the mTNF-alpha cDNA . The construction was first tested in Escherichia coli and then introduced in C . acetobutylicum DSM792 by electroporation . Controls confirmed the presence and stability of the recombinant plasmids in this organism . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an in vitro cytotoxic assay were used to monitor expression and secretion of mTNF-alpha during growth . Significant levels of biologically active mTNF-alpha were measured in both lysates and supernatants . The present report deals with investigations on the elaboration of a gene transfer system for cancer treatment using anaerobic bacteria.

J Biol Chem, 1999 Oct 8, 274(41), 29050 - 6
Monoglucosylation of RhoA at threonine 37 blocks cytosol-membrane cycling; Genth H et al.; The small GTPases Rho, Rac, and Cdc42 are monoglucosylated at effector domain amino acid threonine 37/35 by Clostridium difficile toxins A and B . Glucosylation renders the Rho proteins inactive by inhibiting effector coupling . To understand the functional consequences, effects of glucosylation on subcellular distribution and cycling of Rho GTPases between cytosol and membranes were analyzed . In intact cells and in cell lysates, glucosylation leads to a translocation of the majority of RhoA GTPase to the membranes whereas a minor fraction is monomeric in the cytosol without being complexed with the guanine nucleotide dissociation inhibitor (GDI-1) . Rho complexed with GDI-1 is not substrate for glucosylation, and modified Rho does not bind to GDI-1 . However, a membranous factor inducing release of Rho from the GDI complex makes cytosolic Rho available as a substrate for glucosylation . The binding of glucosylated RhoA to the plasma membranes is saturable, competable with unmodified Rho-GTPgammaS guanosine 5'-O-(3-thiotriphosphate), and takes place at a membrane protein with a molecular mass of about 70 kDa . Membrane-bound glucosylated Rho is not extractable by GDI-1 as unmodified Rho is, leading to accumulation of modified Rho at membranous binding sites . Thus, in addition to effector coupling inhibition, glucosylation also inhibits Rho cycling between cytosol and membranes, a prerequisite for Rho activation.

Biochemistry, 1999 Sep 28, 38(39), 12822 - 32
Cellulosome from Clostridium cellulolyticum: molecular study of the Dockerin/Cohesin interaction; Fierobe HP et al.; Clostridium cellulolyticum produces cellulolytic complexes (cellulosomes) made of 10-13 cell wall degrading enzymes tightly bound to a scaffolding protein (CipC) by means of their dockerin domain . It has previously been shown that the receptor domains in CipC are the cohesin domains and that the cohesin/dockerin interaction is calcium-dependent . In the present study, surface plasmon resonance was used to demonstrate that the free cohesin1 from CipC and dockerin from CelA have the same K(D) (2.5 x 10(-)(10) M) as that of the entire CelA and a larger fragment of CipC, the latter of which contains, in addition to cohesin1, a cellulose binding domain and a hydrophilic domain of unknown function . This demonstrates that neither the catalytic domain of CelA nor the noncohesin domains of CipC have any influence on the interaction . Dockerin domains are composed of two conserved segments of 22 residues: removal of the second segment abolishes the affinity for cohesin1, whereas modified dockerins having twice the first segment, twice the second, or both segments but in a reverse order have K(D) values for cohesin1 in the same range as that observed for wild-type dockerin . These data indicate that if two segments are required for the complexation with the cohesin, segments 1 and 2 are similar enough to replace each other . Calcium overlay experiments revealed that the dockerin domain has one calcium binding site per conserved segment . Circular dichroism performed on wild-type and mutant dockerins indicates that this domain is well structured and that removal of calcium only weakly affects the secondary structure, which remains 40-45% helical.

Pathology, 1999 Aug, 31(3), 261 - 3
Clostridium beijerinckii endophthalmitis secondary to penetrating ocular injury; Newton PJ et al.; Endophthalmitis occurs in five to 10% of injuries involving intraocular foreign bodies . A 52 year old abattoir worker sustained such penetrating ocular trauma and developed fulminant endophthalmitis . Clostridium beijerinckii was isolated from the vitreous humor . Intravitreal vancomycin and amikacin and intravenous penicillin and clindamycin were given . Despite therapeutic vancomycin and amikacin levels in the vitreous, vision was lost and enucleation was ultimately required.

FEMS Microbiol Lett, 1999 Sep 15, 178(2), 355 - 61
The 105-kDa protein component of Bacillus cereus non-haemolytic enterotoxin (Nhe) is a metalloprotease with gelatinolytic and collagenolytic activity; Lund T et al.; A sequence of 91 amino acids residues, probably starting from the N-terminal of the mature protein, was determined for the 105-kDa protein of the non-haemolytic enterotoxin of Bacillus cereus . The last part of this sequence was similar to parts of the N-terminal portions of two collagenases of Clostridium histolyticum and Clostridium perfringens . Zymography, with intact collagen fibril and gelatin as substrates, showed that the 105-kDa protein had collagenolytic and gelatinolytic activity . The 105-kDa protein also showed activity against a typical collagenase substrate, azocoll, and was inhibited by EDTA and 1,10-phenanthroline . We conclude that the 105-kDa protein is a collagenase.

Appl Microbiol Biotechnol, 1999 Aug, 52(2), 170 - 3
Acetate enhances solvent production and prevents degeneration in Clostridium beijerinckii BA101
Chen CK, Blaschek HP.
Addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production by Clostridium beijerinckii BA101, a solvent-hyperproducing mutant derived from C . beijerinckii NCIMB 8052 . C . beijerinckii BA101 demonstrated a greater increase in solvent production than C . beijerinckii NCIMB 8052 when sodium acetate was added to MP2 medium . In 1-l batch fermentations, C . beijerinckii BA101 produced 32.6 g/l total solvents, with butanol at 20.9 g/l, when grown in MP2 medium containing 60 mM sodium acetate and 8% glucose . To our knowledge, these values represent the highest solvent and butanol concentrations produced by a solventogenic Clostridium strain when grown in batch culture.

J Biol Chem, 1999 Oct 1, 274(40), 28087 - 95
Protein kinase B stimulates the translocation of GLUT4 but not GLUT1 or transferrin receptors in 3T3-L1 adipocytes by a pathway involving SNAP-23, synaptobrevin-2, and/or cellubrevin; Foran PG et al.; An interaction of SNAP-23 and syntaxin 4 on the plasma membrane with vesicle-associated synaptobrevin-2 and/or cellubrevin, known as SNAP (soluble N-ethyl-maleimide-sensitive factor attachment protein) receptors or SNAREs, has been proposed to provide the targeting and/or fusion apparatus for insulin-stimulated translocation of the GLUT4 isoform of glucose transporter to the plasma membrane . By microinjecting 3T3-L1 adipocytes with the Clostridium botulinum toxin B or E, which proteolyzed synaptobrevin-2/cellubrevin and SNAP-23, respectively, we investigated the role of these SNAREs in GLUT4, GLUT1, and transferrin receptor trafficking . As expected, insulin stimulated the translocation of GLUT4, GLUT1, and transferrin receptors to the plasma membrane . By contrast, a constitutively active protein kinase B (PKB-DD) only stimulated a translocation of GLUT4 and not GLUT1 or the transferrin receptor . The GLUT4 response to PKB-DD was abolished by toxins B or E, whereas the insulin-evoked translocation of GLUT4 was inhibited by approximately 65% . These toxins had no significant effect on insulin-stimulated transferrin receptor appearance at the cell surface . Thus, insulin appears to induce GLUT4 translocation via two distinct routes, only one of which involves SNAP-23 and synaptobrevin-2/cellubrevin, and can be mobilized by PKB-DD . The PKB-, SNAP-23-, and synaptobrevin-2/cellubrevin-independent GLUT4 translocation pathway may involve movement through recycling endosomes, together with GLUT1 and transferrin receptors.

Infect Immun, 1999 Oct, 67(10), 5124 - 32
Local and systemic neutralizing antibody responses induced by intranasal immunization with the nontoxic binding domain of toxin A from Clostridium difficile; Ward SJ et al.; Fourteen of the 38 C-terminal repeats from Clostridium difficile toxin A (14CDTA) were cloned and expressed either with an N-terminal polyhistidine tag (14CDTA-HIS) or fused to the nontoxic binding domain from tetanus toxin (14CDTA-TETC) . The recombinant proteins were successfully purified by bovine thyroglobulin affinity chromatography . Both C . difficile toxin A fusion proteins bound to known toxin A ligands present on the surface of rabbit erythrocytes . Intranasal immunization of BALB/c mice with three separate 10-microg doses of 14CDTA-HIS or -TETC generated significant levels of anti-toxin A serum antibodies compared to control animals . The coadministration of the mucosal adjuvant heat labile toxin (LT) from Escherichia coli (1 microg) significantly increased the anti-toxin A response in the serum and at the mucosal surface . Importantly, the local and systemic antibodies generated neutralized toxin A cytotoxicity . Impressive systemic and mucosal anti-toxin A responses were also seen following coadministration of 14CDTA-TETC with LTR72, an LT derivative with reduced toxicity which shows potential as a mucosal adjuvant for humans.

Infect Immun, 1999 Oct, 67(10), 5083 - 90
Clostridium botulinum C2 toxin delays entry into mitosis and activation of p34cdc2 kinase and cdc25-C phosphatase in HeLa cells; Barth H et al.; The Clostridium botulinum C2 toxin ADP-ribosylates monomeric actin, thereby inducing disassembly of actin filaments, alteration of focal adhesions, and rounding of cells . After treatment with C2 toxin, cells stop to proliferate but remain viable for about 2 days . In view of reported correlations between the structure of the actin cytoskeleton and cell cycle transition, the effects of C2 toxin on the G(2)/M phase transition of the cell division cycle were studied . Since C2 toxin delayed entry into mitosis in HeLa cells, those enzymes which control entry into mitosis, the cyclin-dependent protein kinase mitosis-promoting factor (MPF) and the phosphatase cdc25-C were examined after treatment of synchronized cells with C2 toxin . MPF is composed of the regulatory cyclin B and the enzymatic p34cdc2 kinase subunits . For its activation at the G2/M border, p34cdc2 needs to be associated with cyclin B and additionally dephosphorylated at Tyr-15 by the specific phosphatase cdc25-C . Treatment of synchronized cells in S or G2 phase with C . botulinum C2 toxin prevented p34cdc2 protein kinase activation by inhibiting its tyrosine dephosphorylation at the G2/M border . Furthermore, the activity of cdc25-C phosphatase was decreased after treatment of cells with C2 toxin . Our results suggest that the prevented activation of the mitotic inducers p34cdc2 kinase and cdc25-C phosphatase represents the final downstream events in the action of C2 toxin resulting in a G(2) phase cell cycle delay in synchronized HeLa cells.

Avian Dis, 1999 Jul-Sep, 43(3), 484 - 90
Cecal carriage of Clostridium perfringens in broiler chickens given Mucosal Starter Culture; Craven SE et al.; Day-of-hatch broiler chicks housed in isolation units were each given, by oral gavage, 0.1 ml of Mucosal Starter Culture (MSC) or saline control . Each of the treated and control chicks was subsequently given a composite culture of three strains of bacitracin-resistant Clostridium perfringens (Cp) previously isolated from chickens with symptoms of necrotic enteritis . Some chicks were maintained on a corn-based diet provided ad libitum . Others were given the feed supplemented with 50% rye (a predisposing factor for necrotic enteritis) . At 7, 14, and 21 days after receiving Cp, chicks were euthanatized, and cecal contents were diluted and plated on selective agar containing bacitracin . For chicks on corn feed, Cp numbers were similar in control birds and birds given MSC in three of four trials . In two of the trials that demonstrated no effect of MSC on Cp numbers, enterotoxin presence was determined . The number of birds with detectable Cp enterotoxin in their small intestine and the mean toxin levels were lower in the MSC-treated birds . In a fourth trial with birds on corn-based feed, mean Cp numbers and the number of Cp-positive birds were lower in the MSC-treated birds . For the two trials involving chickens on rye-supplemented feed, Cp numbers and the percentage of Cp-positive birds were significantly reduced in MSC-treated birds compared with control birds . Enterotoxin in birds receiving the 50% rye diet was at low levels or not detected in control and MSC-treated birds . Results suggest that MSC may reduce intestinal proliferation of Cp, a causative agent of necrotic enteritis in poultry and of foodborne disease in humans.

Biochemistry, 1999 Sep 21, 38(38), 12377 - 86
Role of glutamate-59 hydrogen bonded to N(3)H of the flavin mononucleotide cofactor in the modulation of the redox potentials of the Clostridium beijerinckii flavodoxin . Glutamate-59 is not responsible for the pH dependency but contributes to the stabilization of the flavin semiquinone; Bradley LH et al.; The midpoint potentials for both redox couples of the noncovalently bound flavin mononucleotide (FMN) cofactor in the flavodoxin are known to be pH dependent . While the pH dependency for the oxidized-semiquinone (ox/sq) couple is consistent with the formation of the blue neutral form of the flavin semiquinone, that of the semiquinone-hydroquinone (sq/hq) couple is more enigmatic . The apparent pK(a) of 6.7 for this couple in the flavodoxin from Clostridium beijerinckii has been attributed to the ionization of the FMN(HQ); however, nuclear magnetic resonance data strongly suggest the FMN(HQ) remains anionic over the entire pH range testable . As an alternative explanation, a specific glutamate residue (Glu59 in this flavodoxin), which is hydrogen-bonded to N(3)H of the FMN, has been postulated to be the primary redox-linked proton acceptor responsible for the pH effect in some flavodoxins . This model was directly tested in this study by permanently neutralizing Glu59 by its replacement with glutamine . This conservative substitution resulted in an increase of 86 mV (at pH 7) in midpoint potential of the sq/hq couple; however, the pH dependency of this couple was not altered . Thus, the redox-linked protonation of Glu59 clearly cannot be responsible for this effect as proposed . The pH dependency of the ox/sq couple was also similar to wild type, but the midpoint potential has decreased by 65 mV (pH 7) . The K(d) values for the oxidized, semiquinone, and hydroquinone complexes increased by 43-, 590-, and 20-fold, respectively, relative to the wild type . Thus, the Glu59 to glutamine substitution substantially effects the stability of the semiquinone but, on a relative basis, slightly favors the formation of the hydroquinone . On the basis of (1)H-(15)N HSQC nuclear magnetic resonance spectroscopic studies, the increased temperature coefficients for the protons on N(3) and N(5) of the reduced FMN in E59Q suggest that the hydrogen-bonding interactions at these positions are significantly weakened in this mutant . The increase for N(5)H correlates with the reduced stability of the FMN(SQ) and the more negative midpoint potential for the ox/sq couple . On the basis of the X-ray structure, an "anchoring" role is proposed for the side chain carboxylate of Glu59 that stabilizes the structure of the 50's loop in such a way so as to promote the crucial hydrogen-bonding interaction that stabilizes the flavin semiquinone, contributing to the low potential of this flavodoxin.

J Rheumatol, 1999 Sep, 26(9), 1934 - 8
Specificity of antibodies to bacterial DNA in the sera of healthy human subjects and patients with systemic lupus erythematosus; Pisetsky D et al.; OBJECTIVE: To elucidate the epitope structure to DNA by identifying antigenic determinants on bacterial DNA bound by anti-DNA antibodies from normal human subjects (NHS) and patients with systemic lupus erythematosus (SLE) . METHODS: Sera from NHS and patients with SLE were tested by ELISA for the presence of antibodies to single stranded DNA from calf thymus, Micrococcus lysodeikticus, Staphylococcus epidermidis, Clostridium perfringens, and Klebsiella pneumoniae . To assess binding to conserved and nonconserved determinants, sera were absorbed on DNA-cellulose affinity columns bearing each of the bacterial DNA and then tested for binding to the other DNA antigens . RESULTS: Absorption of SLE sera with any of the bacterial DNA caused a loss of binding to all other bacterial DNA as well as calf thymus DNA . In contrast, absorption of NHS sera with bacterial DNA caused a loss of binding to the DNA on the affinity column with much less effect on binding to the other DNA antigens . CONCLUSION: These results indicate a marked difference in the specificity of antibodies to bacterial DNA in NHS and patients with SLE . The binding of SLE anti-DNA to predominantly conserved determinants suggests that a shift in patterns of anti-DNA specificity may be associated with the autoimmune state.

Ann Surg Oncol, 1999 Sep, 6(6), 582 - 90
Treatment of primary peritoneal mesothelioma by continuous hyperthermic peritoneal perfusion (CHPP); Park BJ et al.; BACKGROUND: Primary peritoneal mesothelioma is a locally aggressive disease that is difficult to treat or even palliate . Continuous hyperthermic peritoneal perfusion (CHPP) with cisplatin (CDDP) allows uniform, high regional delivery of chemotherapeutics and hyperthermia to the peritoneal surface for the treatment of peritoneal tumors . This article summarizes the results of 18 patients with peritoneal mesothelioma treated with CHPP . METHODS: From June 1993 through April 1998, 18 patients with primary peritoneal mesothelioma (13 male, 5 female; median age, 51 years) underwent surgical exploration and tumor debulking followed by a 90-minute CHPP with CDDP and hyperthermia as part of three consecutive phase I trials conducted at the National Cancer Institute . Seventeen of 18 patients had malignant peritoneal mesothelioma, 13 with associated ascites . One patient had a symptomatic, multiply recurrent, benign, cystic peritoneal mesothelioma . Three patients who had a recurrence after a prolonged progression-free interval (>6 months) after CHPP underwent re-treatment . CHPP parameters included median cisplatin dose of 530 mg (range, 187-816), perfusate volume 6.0 liter (range, 4-9), flow 1.5 liter/min (range, 1-2), intraperitoneal temperature 41 degrees C (range, 38.7-43.2), and central temperature 38.6 degrees C (range, 36.8-39.7) . RESULTS: Median follow-up after CHPP is 19 months (range, 2-56) with no operative or treatment-related mortality . Overall operative morbidity was 24% and included two patients with superficial wound infection and one patient each with atrial fibrillation, pancreatitis, fascial dehiscence, ileus, line sepsis, and clostridium difficile colitis . The major treatment-related toxicity was systemic renal toxicity at doses above what was defined as the maximum tolerated dose of cisplatin . Nine of 10 patients had resolution of their ascites postoperatively . Three patients who developed recurrent ascites (27, 22, and 10 months after initial treatment) were re-treated and had resolution of their ascites with ongoing responses at 24, 6, and 4 months after the second perfusion . The median progression-free survival was 26 months, and the overall 2-year survival was 80% . The median overall survival has not been reached . CONCLUSIONS: CHPP with cisplatin can be performed safely with no mortality and minimal morbidity . In selected patients, successful palliation in the abdomen and long-term survival, compared with historical controls, can be achieved with aggressive surgical debulking and CHPP . Re-treatment after initial response can result in a second long-term response.

Vet Res Commun, 1999 Aug, 23(5), 275 - 84
Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro; Uzal FA et al.; Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin . Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control . No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin . MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin . All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin . The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro.

Br J Neurosurg, 1999 Feb, 13(1), 85 - 6
Localized pneumocephalus caused by Clostridium perfringens meningitis; Penrose-Stevens A et al.; Clostridium meningitis is a rare complication of elective surgery, but the presence of pneumocephalus on CT in the absence of penetrating injuries, should raise the possibility of anaerobic infections . We report a case of fatal Clostridium perfringens meningitis which occurred 4 months after a craniotomy for glioblastoma multiforme . The diagnosis was suspected based on the CT findings . The literature of this rare condition is reviewed.

J Food Prot, 1999 Sep, 62(9), 1041 - 4
Comparison of five anaerobic incubation methods for enumeration of Clostridium perfringens from foods; Riley A et al.; This study compared the cost, speed, convenience, and sensitivity of five anaerobic systems . Fung's double-tube (FDT) method, the minitube method (MT), the sandwiched microtiter plate (SMP) method, and the Mitsubishi AnaeroPack System were compared with the Brewer anaerobic jar for total anaerobic bacterial counts in foods . Incubation was at 37 degrees C for 4, 8, 12, and 24 h . The results indicated that FDT, MT, SMP, and the Mitsubishi AnaeroPack System were as sensitive as the Brewer anaerobic jar for the detection and enumeration of Clostridium perfringens from food products . The FDT, MT, and SMP methods recovered higher numbers of C . perfringens compared to the Brewer anaerobic jar (P < 0.05) after 12 and 24 h incubation . The Brewer anaerobic jar was the most expensive method.

Eur J Biochem, 1999 Oct 1, 265(1), 404 - 14
2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum; Hans M et al.; Component D (HgdAB) of 2-hydroxyglutaryl-CoA dehydratase from Clostridium symbiosum was purified to homogeneity . It is able to use component A from Acidaminococcus fermentans (HgdC) to initiate catalysis together with ATP, Mg2+ and a strong reducing agent such as Ti(III)citrate . Component D from C . symbiosum has a 6 x higher specific activity compared with that from A . fermentans and contains a second {4Fe-4S} cluster but the same amount of riboflavin 5'-phosphate (1.0 per heterodimeric enzyme, m = 100 kDa) . Mossbauer spectroscopy revealed symmetric cube-type structures of the two {4Fe-4S}2+ clusters . EPR spectroscopy showed the resistance of the clusters to reducing agents, but detected a sharp signal at g = 2 . 004 probably due to a stabilized flavin semiquinone . Three genes from C . symbiosum coding for components D (hgdA and hgdB) and A (hgdC) were cloned and sequenced . Primer extension experiments indicated that the genes are transcribed in the order hgdCAB from an operon only half the size of that from A . fermentans . Sequence comparisons detected a close relationship to the dehydratase system from A . fermentans and HgdA from Fusobacterium nucleatum, as well as to putative proteins of unknown function from Archaeoglobus fulgidus . Lower, but significant, identities were found with putative enzymes from several methanogenic Archaea and Escherichia coli, as well as with the mechanistically related benzoyl-CoA reductases from the Proteobacteria Rhodopseudomonas palustris and Thauera aromatica.

J Immunol, 1999 Oct 1, 163(7), 3819 - 25
ADP-ribosylation of rho by C3 ribosyltransferase inhibits IL-2 production and sustained calcium influx in activated T cells; Angkachatchai V et al.; Activation of the T lymphocyte induces dramatic cytoskeletal changes, and there is increasing evidence that disruption of the cytoskeleton inhibits early and late events of T cell signal transduction . However, relatively little is known about the signaling molecules involved in activation-induced cytoskeletal rearrangement . The rho family of small GTP-binding proteins, which include rho, rac, and cdc42, regulates the cytoskeleton and coordinates various cellular functions via their many effector targets . In prior studies, the Clostridium botulinum toxin C3 exoenzyme has been used to ADP-ribosylate and inactivate rho . In this study, we demonstrate that treatment of T cells with C3 exoenzyme inhibits IL-2 transcription following ligation of the TCR . Inhibition of IL-2 expression correlated with loss of sustained increase in {Ca+2}i and mitogen activated protein kinase (MAPK/Erk) activity, but not with activation of the tyrosine kinase, lck . These findings are the first to show that ADP-ribosylation of rho by C3 ribosyltransferase (exoenzyme) inhibits IL-2 production due, in part, to the requirement for sustained calcium influx and MAPK activation after Ag receptor ligation.

Int J Food Microbiol, 1999 Aug 15, 49(3), 139 - 49
Brining time effect on physicochemical and microbiological parameters in Idiazábal cheese; Perez Elortondo FJ et al.; Physicochemical and microbiological parameters were compared for three brining times (12, 24 and 36 h) for fresh, young, semihard and hard Idiazabal cheese . Longer brining time produced higher salt, dry matter and salt-moisture ratio and lower water activity values for all types of cheese according to ripening time, while non-significant changes were observed for pH . In fresh cheese (1-15 days ripening), non-significant differences for microbiological counts in relation to brining time were observed, except for moulds . In young and hard cheeses, Lactobacillus and Leuconostoc showed lower counts with longer brining times . In contrast, Micrococaceae, yeast and moulds were stimulated by higher salt content in matured cheeses . In addition . this work has proved that there are lower water activity values and lower microbiological counts in longer-matured Idiazabal cheeses . For the different brining and ripening times, positive correlations were observed among most of the microbial groups studied, but a different behavior was established for Enterococcus, Clostridium tyrobutyricum, yeast and moulds.

Singapore Med J, 1999 Jun, 40(6), 405 - 9
A review of 5 years' experience in the use of botulinium toxin A in the treatment of sixth cranial nerve palsy at the Singapore National Eye Centre; Quah BL et al.; INTRODUCTION: This retrospective study reports our experience on the use of botulinum toxin A (BTXA) in the treatment of sixth cranial nerve palsy at the Singapore National Eye Centre . BTXA is derived from clostridium botulinum; it causes temporary paralysis of the extraocular muscle (medial rectus) into which it is injected, thus preventing its contracture and allows the antagonist lateral rectus muscle to take up the slack and reduce or correct the ocular misalignment . METHODS: Nineteen patients had BTXA injection for estropia due to sixth cranial nerve palsy during the period September 1992 to August 1997 . The sixth cranial nerve palsy was related to nasopharyngeal carcinoma in 76.7% of cases . Follow-up after the last injection ranged from zero (defaulted) to 21 months (mean 8, median 6 months) . RESULTS: A total of 25 injections were given to 19 patients . Seven patients (36.8%) had final ocular alignment within 10 prism dioptres of orthotropia of which six achieved fusion at primary gaze position . There was no correlation between the number of injections per patient and the size of strabismus or grade of lateral rectus muscle function . The incidence of ptosis was 48%, subconjunctival haemorrhage 16% and hypertropia 16% . DISCUSSION: Our results suggest that those patients with smaller strabismus and a shorter time interval between onset of strabismus and botulinum injection tend to achieve better outcome in terms of fusion or ocular alignment within 10 prism dioptres of orthotropia . The treatment of strabismus with BTXA is an acceptable approach in selected patients . The procedure is simple, safe, cheap, effective, and avoids the risks of general anaesthesia . It can substitute for or eliminate the need for strabismus surgery in some cases of sixth nerve palsy.

J Biol Chem, 1999 Sep 24, 274(39), 27562 - 6
Galpha(13) stimulates Rho-dependent activation of the cyclooxygenase-2 promoter; Slice LW et al.; Cyclooxygenase-2 (COX-2) gene expression is rapidly increased by cytokines, tumor promoters, and growth factors and is markedly enhanced in various cancer cells . Here, we examine the regulation of COX-2 promoter activity by alpha subunits of heterotrimeric G proteins in NIH 3T3 cells . Using a transient transfection assay with a reporter vector in which the murine COX-2 promoter drives the production of luciferase and expression vectors encoding for alpha subunits of G-proteins, we show that overexpression of wild type and constitutively active Galpha(13) and Galpha(q) induced transcription from the COX-2 promoter . The highest level of induced luciferase activity (5.8-fold) occurred in cells expressing the constitutively active Galpha(13)(Q226L) . We also show that expression of a constitutively active mutant of Rho (RhoQ63L) also induced transcription from the COX-2 promoter . Co-expression of Clostridium botulinum C3 toxin specifically blocked induction of the COX-2 promoter by either Galpha(13)Q226L or RhoQ63L but did not prevent the activation of this promoter by Ras, Rac, v-src, or forskolin . We conclude that Galpha(13) signals through a Rho-dependent pathway leading to activation of the COX-2 promoter . This pathway is not inhibited by either cytochalasin D, which disrupts actin filament organization, or genistein, a broad spectrum tyrosine kinase inhibitor, indicating a bifurcation of the signaling pathway used by Galpha(13)/Rho to induce COX-2 expression from that used to induce stress fiber formation and tyrosine phosphorylation of focal adhesion proteins.

J Biol Chem, 1999 Sep 24, 274(39), 27407 - 14
Neosynthesis and activation of Rho by Escherichia coli cytotoxic necrotizing factor (CNF1) reverse cytopathic effects of ADP-ribosylated Rho; Barth H et al.; Clostridium botulinum exoenzyme C3 inactivates the small GTPase Rho by ADP-ribosylation . We used a C3 fusion toxin (C2IN-C3) with high cell accessibility to study the kinetics of Rho inactivation by ADP-ribosylation . In primary cultures of rat astroglial cells and Chinese hamster ovary cells, C2IN-C3 induced the complete ADP-ribosylation of RhoA and concomitantly the disassembly of stress fibers within 3 h . Removal of C2IN-C3 from the medium caused the recovery of stress fibers and normal cell morphology within 4 h . The regeneration was preceded by the appearance of non-ADP-ribosylated RhoA . Recovery of cell morphology was blocked by the proteasome inhibitor lactacystin and by the translation inhibitors cycloheximide and puromycin, indicating that intracellular degradation of the C3 fusion toxin and the neosynthesis of Rho were required for reversal of cell morphology . Escherichia coli cytotoxic necrotizing factor CNF1, which activates Rho by deamidation of Gln(63), caused reconstitution of stress fibers and cell morphology in C2IN-C3-treated cells within 30-60 min . The effect of CNF1 was independent of RhoA neosynthesis and occurred in the presence of completely ADP-ribosylated RhoA . The data show three novel findings; 1) the cytopathic effects of ADP-ribosylation of Rho are rapidly reversed by neosynthesis of Rho, 2) CNF1-induced deamidation activates ADP-ribosylated Rho, and 3) inhibition of Rho activation but not inhibition of Rho-effector interaction is a major mechanism underlying inhibition of cellular functions of Rho by ADP-ribosylation.

Muscle Nerve, 1999 Oct, 22(10), 1388 - 92
Neurophysiological assessment in the diagnosis of botulism: usefulness of single-fiber EMG; Padua L et al.; We report the clinical, serological, and neurophysiological findings in seven patients with foodborne botulism caused by ingestion of black olives in water . The clinical picture was characterized by mild symptoms with a long latency of onset and by involvement of cranial and upper limb muscles; only one patient, a child, developed respiratory failure . Spores of Clostridium botulinum were found in stools in some but not all cases . Conventional neurophysiological tests had low sensitivity; abnormal findings were present only in the patient with severe clinical involvement, in whom compound muscle action potentials (CMAPs) appeared reduced . Repetitive nerve stimulation at a high rate showed pseudofacilitation and not true posttetanic facilitation, but single-fiber electromyography (SFEMG) showed abnormalities of neuromuscular transmission in every case . Neurophysiological evaluation, particularly SFEMG, is important because it allows rapid identification of abnormal neuromuscular transmission while bioassay studies are in progress .

Cleve Clin J Med, 1999 Sep, 66(8), 503 - 7
Clostridium difficile diarrhea and colitis: a clinical overview; Taege AJ et al.; Infection with toxin-producing strains of Clostridium difficile is common and potentially life-threatening . It occurs mostly in patients in the hospital or nursing home who are taking or have recently taken antibiotics . Two toxins, A and B, damage the colonic mucosa, resulting in symptoms ranging from mild diarrhea to bloody diarrhea with fever and abdominal pain, colitis, or even pseudomembranous colitis . Severe cases may involve dehydration, toxic megacolon, or colonic perforation . This article reviews the microbiology, epidemiology, clinical manifestations, diagnosis, treatment, and prevention of this disease.

Mol Biol Evol, 1999 Sep, 16(9), 1280 - 91
A single eubacterial origin of eukaryotic pyruvate: ferredoxin oxidoreductase genes: implications for the evolution of anaerobic eukaryotes; Horner DS et al.; The iron sulfur protein pyruvate: ferredoxin oxidoreductase (PFO) is central to energy metabolism in amitochondriate eukaryotes, including those with hydrogenosomes . Thus, revealing the evolutionary history of PFO is critical to understanding the origin(s) of eukaryote anaerobic energy metabolism . We determined a complete PFO sequence for Spironucleus barkhanus, a large fragment of a PFO sequence from Clostridium pasteurianum, and a fragment of a new PFO from Giardia lamblia . Phylogenetic analyses of eubacterial and eukaryotic PFO genes suggest a complex history for PFO, including possible gene duplications and horizontal transfers among eubacteria . Our analyses favor a common origin for eukaryotic cytosolic and hydrogenosomal PFOs from a single eubacterial source, rather than from separate horizontal transfers as previously suggested . However, with the present sampling of genes and species, we were unable to infer a specific eubacterial sister group for eukaryotic PFO . Thus, we find no direct support for the published hypothesis that the donor of eukaryote PFO was the common alpha-proteobacterial ancestor of mitochondria and hydrogenosomes . We also report that several fungi and protists encode proteins with PFO domains that are likely monophyletic with PFOs from anaerobic protists . In Saccharomyces cerevisiae, PFO domains combine with fragments of other redox proteins to form fusion proteins which participate in methionine biosynthesis . Our results are consistent with the view that PFO, an enzyme previously considered to be specific to energy metabolism in amitochondriate protists, was present in the common ancestor of contemporary eukaryotes and was retained, wholly or in part, during the evolution of oxygen-dependent and mitochondrion-bearing lineages.

J Comp Pathol, 1999 Oct, 121(3), 217 - 25
Duodenal adenomas in Balb/-c mice monoinfected with Clostridium perfringens; Rozengurt N et al.; An outbreak of disease in multiparous females occurred in one isolator in a colony of Balb/-c germ-free mice . The affected isolator had been accidentally contaminated with Copyright Clostridium perfringens . Gross and histological examination of the diseased mice revealed lesions in the lungs, heart and intestinal tract . Lesions in the valvular endocardium and vascular walls were closely associated with bacterial colonies and septic thrombi containing Gram-positive rods . C perfringens type B was recovered in pure culture from the faeces, intestinal contents and atrial thrombi of the sick mice . Intestinal lesions varied, depending on the region of the intestine . The ileum showed shortened villi and ulceration of the mucosa . The duodenum of all the affected mice showed microscopic foci of polypoid adenomatous growth of the crypt epithelium . The significance of these unusual neoplastic lesions is discussed in the context of the growing evidence of an association between cell growth and bacterial cell products . 1999 Harcourt Publishers Ltd

Curr Microbiol, 1999 Oct, 39(4), 226 - 30
The ability of "low G + C gram-positive" ruminal bacteria to resist monensin and counteract potassium depletion; Callaway TR et al.; Gram-negative ruminal bacteria with an outer membrane are generally more resistant to the feed additive, monensin, than Gram-positive species, but some bacteria can adapt and increase their resistance . 16S rRNA sequencing indicates that a variety of ruminal bacteria are found in the "low G + C Gram-positive group," but some of these bacteria are monensin resistant and were previously described as Gram-negative species (e.g., Selenomonas ruminantium and Megasphaera elsdenii) . The activity of monensin can be assayed by its ability to cause potassium loss, and results indicated that the amount of monensin needed to catalyze half maximal potassium depletion (K(d)) from low G + C gram-positive ruminal bacteria varied by as much as 130-fold . The K(d) values for Butyrivibrio fibrisolvens 49, Streptococcus bovis JB1, Clostridium aminophilum F, S . ruminantium HD4, and M . elsdenii B159 were 10, 65, 100, 1020, and 1330 nM monensin, respectively . B . fibrisolvens was very sensitive to monensin, and it did not adapt . S . bovis and C . aminophilum cultures that were transferred repeatedly with sub-lethal doses of monensin had higher K(d) values than unadapted cultures, but the K(d) was always less than 800 nM . S . ruminantium and M . elsdenii cells were highly resistant (K(d) > 1000 nM), and this resistance could be explained by the ability of these low G + C Gram-positive bacteria to synthesize outer membranes.

Clin Transplant, 1999 Aug, 13(4), 318 - 23
Clostridium difficile colitis after kidney and kidney-pancreas transplantation; West M et al.; OBJECTIVE: To determine the timing and risk factors involved in the development of Clostridium difficile (CD) colitis in kidney and kidney-pancreas transplant recipients . BACKGROUND DATA: The incidence of CD colitis after kidney and kidney-pancreas transplantation has not been studied in detail . The question of whether the immunosuppressed transplant recipient is more prone to CD colitis and its complications (i.e., megacolon, perforations) and the risk factors involved have not been determined . METHODS: We retrospectively reviewed our experience in kidney and kidney-pancreas recipients who received transplants between January 1, 1985 and December 31, 1994 . We divided these recipients into three groups: pediatric kidney recipients, adult kidney recipients, and kidney-pancreas recipients . For each group, we assessed the timing of infection, primary disease, colitis treatment, and any concurrent complications or risk factors . RESULTS: Of 1932 transplants, 159 recipients developed post-transplant CD colitis . 132 charts were available for review . Forty-three pediatric kidney recipients developed CD colitis . Their mean age was 3.2 yr; 74% (n = 37) of them developed their colitis during their initial hospital stay, with the mean timing of infection being 33 d . Forty-one (95%) had undergone intra-abdominal placement of the graft, with renal artery anastomoses to the aorta . Fifty adult kidney recipients developed CD colitis . Thirteen (26%) developed colitis during their initial hospital stay, with the mean timing of infection (for all adult kidney recipients) being 15 months . Thirty-nine kidney-pancreas recipients developed CD colitis . Mean timing of infection was 6 months . The overall incidence of CD colitis was 8%, with 16% in the pediatric kidney group, 15.5% in the kidney-pancreas group, and 3.5% in the adult kidney group . The difference in mean timing of infection was significant between the three groups (p < 0.001 for pediatric versus adult kidney recipients, p = 0.002 for pediatric kidney versus kidney-pancreas recipients, and p = 0.2846 for adult kidney versus kidney-pancreas recipients) . CONCLUSION: The incidence of CD colitis is increased in pediatric kidney and kidney-pancreas recipients . Young recipient age ( < 5 yr), female gender, treatment of rejection with monoclonal antibodies, antibiotic use, and intra-abdominal graft placement have been shown to increase the incidence of this disease . Further studies concerning prevention in the high-risk groups are needed.

J Zoo Wildl Med, 1999 Jun, 30(2), 293 - 6
Bacillary hemoglobinuria in a free-ranging elk calf; Bender LC et al.; A dead elk (Cervus elaphus roosevelti) calf was diagnosed with bacillary hemoglobinuria, a toxemia caused by the bacterium Clostridium haemolyticum . The mortality occurred in southwest Washington, USA (46 degrees 13'N, 123 degrees 22'W), in an area in which several previous mortalities, suspected but not conclusively diagnosed to be either bacillary hemoglobinuria, enterotoxemia, or leptospirosis, occurred . This is the first reported incidence of mortality attributable to bacillary hemoglobinuria in free-ranging elk . Similar deaths of young elk in the area suggest that mortality from this disease may be common locally.

FEMS Microbiol Lett, 1999 Sep 1, 178(1), 163 - 8
A nonsense mutation abrogates production of a functional enterotoxin A in Clostridium difficile toxinotype VIII strains of serogroups F and X; von Eichel-Streiber C et al.; Clostridium difficile strains of toxinotype VIII from serogroups F and X are described as toxin B-positive, toxin A-negative (TcdB+ A-), although they harbour almost the entire tcdA gene . To identify the reason for the lack of TcdA detection, we analyzed catalytic and ligand domains of TcdA-1470 of the type strain of serogroup F, strain 1470 . Using recombinant fragments, the C-terminal immunodominant ligand domain TcdA3-1470, spanning amino acid residues 1694-2711 (corresponding to VPI 10463 sequence), was detected in Western blots . Similar experiments using the recombinant N-terminal catalytic fragment TcdAc1-2-1470 (amino acid positions 1-544) failed . In addition, this fragment showed no glucosylation activity . We determined the size and the position of alterations in the ligand domain tcdA3-1470 by DNA sequencing . Within the N-terminal fragment tcdAc1-2-1470, a nonsense mutation was identified introducing a stop codon at amino acid position 47 . Identical mutations were found in the two serogroup X strains 17663 and 10355 . The mutation might explain the lack of TcdA production observed in strains of serotypes F and X.

J Biotechnol, 1999 May 28, 71(1-3), 191 - 205
Stoichiometric modeling of Clostridium acetobutylicum fermentations with non-linear constraints; Desai RP et al.; A stoichiometric model of Clostridium acetobutylicum and related strains has been previously derived . The stoichiometric matrix of the model contains a singularity which has prevented the calculation of a unique set of fluxes which describe the primary metabolic activity . To resolve the singularity, we have developed a non-linear constraint relating the acetate and butyrate uptake fluxes . Subsequently, we developed a software package utilizing a model independent heuristic global optimization approach to solve the resultant non-linear problem . We have validated the use of the non-linear constraint by correlating calculated butyrate production pathway flux profiles with measured intracellular pH profiles . Finally, we examined a controlled batch fermentation to determine that the acid formation pathways play critical roles throughout solventogenesis . The broader usefulness of reformulating the stoichiometric model as a constrained minimization problem is discussed.

Cell, 1999 Aug 20, 98(4), 475 - 85
Activation of store-operated Ca2+ current in Xenopus oocytes requires SNAP-25 but not a diffusible messenger; Yao Y et al.; Depletion of Ca2+ stores in Xenopus oocytes activated entry of Ca2+ across the plasma membrane, which was measured as a current I(soc) in subsequently formed cell-attached patches . I(soc) survived excision into inside-out configuration . If cell-attached patches were formed before store depletion, I(soc) was activated outside but not inside the patches . I(soc) was potentiated by microinjection of Clostridium C3 transferase, which inhibits Rho GTPase, whereas I(soc) was inhibited by expression of wild-type or constitutively active Rho . Activation of I(soc) was also inhibited by botulinum neurotoxin A and dominant-negative mutants of SNAP-25 but was unaffected by brefeldin A . These results suggest that oocyte I(soc) is dependent not on aqueous diffusible messengers but on SNAP-25, probably via exocytosis of membrane channels or regulatory molecules.

J Biol Chem, 1999 Sep 17, 274(38), 27274 - 80
Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors; Gordon VM et al.; The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism . Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors . Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin . FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor . The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1 . We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis . Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin . On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor . We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors . Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.

Biol Pharm Bull, 1999 Aug, 22(8), 880 - 2
Conversion of dieldrin to aldrin by intestinal bacteria in rats; Kitamura S et al.; The present study provides the evidence that dieldrin is reductively metabolized to aldrin by intestinal bacteria in rats . When dieldrin was incubated with the cecal contents of rats, aldrin, a reduced metabolite of the epoxide, was isolated from the incubation mixture . The metabolite was identified unequivocally by UV and mass spectral comparison with an authentic sample, and on the basis of its TLC and HPLC behavior . The cecal contents of rats exhibited epoxide reductase activity toward dieldrin under anaerobic conditions . However, only marginal activity was observed under aerobic conditions . Four pure strains of intestinal bacteria exhibited epoxide reductase activities to varying degrees under anaerobic conditions . The highest activity was observed in Clostridium sporogenes . Cell-free extracts of the intestinal bacteria in rat cecal contents showed reductase activity when supplemented with both NAD(P)H and FMN under anaerobic conditions.

Int J Food Microbiol, 1999 Aug 1, 49(1-2), 75 - 83
Bacterial populations associated with a sorghum-based fermented weaning cereal; Kunene NF et al.; Microbiological surveys, to determine the quality and safety, were conducted on 45 sorghum samples comprising dry powders (n = 15) and corresponding fermented (n = 15) and cooked fermented porridge (n = 15) samples collected from households in an informal settlement of the Gauteng Province of South Africa . Mean aerobic plate counts, Gram-negative counts and bacterial spore counts of sorghum powder samples decreased in fermented and cooked fermented porridge samples . However, mean lactic acid bacteria counts increased in fermented porridge samples, but decreased slightly in cooked fermented porridge samples . The mean pH value of sorghum powder samples decreased in fermented and cooked fermented porridge, respectively . Bacillus (B.) cereus was detected in all 15 sorghum powder samples, while Escherichia (E.) coli was detected in 53%, Clostridium perfringens in 27%, Listeria monocytogenes in 13% and Aeromonas spp., Salmonella spp., Staphylococcus aureus, Shigella spp . and Yersinia spp., each in 7% of sorghum powder samples . Of the fermented porridge samples, 40% contained B . cereus and 7% contained E . coli . None of the pathogens tested for were detected in cooked fermented porridge samples . B . cereus (53%), B . subtilis (21%), B . thuringiensis (13%), B . licheniformis (10%) and B . coagulans (3%) were identified from 120 isolates randomly selected from spore count plates of the highest dilution showing growth.

Int J Food Microbiol, 1999 Aug 1, 49(1-2), 57 - 62
Modelling the overall effect of pH on the apparent heat resistance of Bacillus cereus spores; Couvert O et al.; A simple overall model is proposed to describe the effect of both the pH of the heating menstruum and the pH of the recovery medium on the apparent spore heat resistance of Bacillus cereus . Applied to foods making up both heating and recovery media, the model can be reduced to only two parameters . Its goodness of fit and its robustness enable it to be applied to the optimisation of heat treatments . However . further experiments should be undertaken to validate the model for other species and to determine the parameters related to reference species such as Clostridium botulinum.

Clin Infect Dis, 1999 Aug, 29(2), 367 - 74
Clostridium difficile colitis associated with infant botulism: near-fatal case analogous to Hirschsprung's enterocolitis; Schechter R et al.; We present the first five reported cases of Clostridium difficile-associated diarrhea (CDAD) in children with infant botulism caused by Clostridium botulinum . We compare two fulminant cases of colitis in children with colonic stasis, the first caused by infant botulism and the second caused by Hirschsprung's disease . In both children, colitis was accompanied by hypovolemia, hypotension, profuse ascites, pulmonary effusion, restrictive pulmonary disease, and femoral-caval thrombosis . Laboratory findings included pronounced leukocytosis, hypoalbuminemia, hyponatremia, coagulopathy, and, when examined in the child with infant botulism, detection of C . difficile toxin in ascites . CDAD recurred in both children, even though difficile cytotoxin was undetectable in stool after prolonged initial therapy . Four children who had both infant botulism and milder CDAD also are described . Colonic stasis, whether acquired, as in infant botulism, or congenital, as in Hirschsprung's disease, may contribute to the susceptibility to and the severity of CDAD.

Mol Microbiol, 1999 Sep, 33(5), 946 - 58
Inactivation of the gene (cpe) encoding Clostridium perfringens enterotoxin eliminates the ability of two cpe-positive C . perfringens type A human gastrointestinal disease isolates to affect rabbit ileal loops; Sarker MR et al.; Previous epidemiological studies have implicated Clostridium perfringens enterotoxin (CPE) as a virulence factor in the pathogenesis of several gastrointestinal (GI) illnesses caused by C . perfringens type A isolates, including C . perfringens type A food poisoning and non-food-borne GI illnesses, such as antibiotic-associated diarrhoea and sporadic diarrhoea . To further evaluate the importance of CPE in the pathogenesis of these GI diseases, allelic exchange was used to construct cpe knock-out mutants in both SM101 (a derivative of a C . perfringens type A food poisoning isolate carrying a chromosomal cpe gene) and F4969 (a C . perfringens type A non-food-borne GI disease isolate carrying a plasmid-borne cpe gene) . Western blot analyses confirmed that neither cpe knock-out mutant could express CPE during either sporulation or vegetative growth, and that this lack of CPE expression could be complemented by transforming these mutants with a recombinant plasmid carrying the wild-type cpe gene . When the virulence of the wild-type, mutant and complementing strains were compared in a rabbit ileal loop model, sporulating (but not vegetative) culture lysates of the wild-type isolates induced significant ileal loop fluid accumulation and intestinal histopathological damage, but neither sporulating nor vegetative culture lysates of the cpe knock-out mutants induced these intestinal effects . However, full sporulation-associated virulence could be restored by complementing these cpe knock-out mutants with a recombinant plasmid carrying the wild-type cpe gene, which confirms that the observed loss of virulence for the cpe knock-out mutants results from the specific inactivation of the cpe gene and the resultant loss of CPE expression . Therefore, in vivo analysis of our isogenic cpe mutants indicates that CPE expression is necessary for these two cpe-positive C . perfringens type A human disease isolates to cause GI effects in the culture lysate:ileal loop model system, a finding that supports CPE as an important virulence factor in GI diseases involving cpe-positive C . perfringens type A isolates.

Eur J Gynaecol Oncol, 1999, 20(4), 277 - 80
Primary intravenous paclitaxel and platinum chemotherapy for high-risk Stage I epithelial ovarian carcinoma; Chi DS et al.; OBJECTIVE: To evaluate the efficacy of intravenous (i.v.) paclitaxel and platinum chemotherapy in patients with high-risk Stage I epithelial ovarian carcinoma . METHODS: We performed a retrospective chart review of all patients with Stage I ovarian cancer treated at our institution between March 1993 and June 1995 . RESULTS: Twenty patients received adjuvant paclitaxel-containing chemotherapy for Stage I ovarian carcinoma after comprehensive surgical staging . Five patients (25%) had Stage IA disease and 15 patients (75%) had Stage IC disease . Tumor grades were: 1, five patients (25%); 2, nine patients (45%); and 3, six patients (30%) . Histologic cell types were: clear-cell, ten (50%); endometrioid, five (25%); mucinous, three (15%); and serous, two (10%) . Nineteen patients (95%) were treated with i.v . paclitaxel and platinum chemotherapy . One patient (5%) received i.v . paclitaxel alone . Eighteen patients (90%) had five cycles of chemotherapy, while two patients (10%) had three . The 96 total cycles were associated with nine episodes (9%) of significant toxicity: fever, four (4%); severe nausea and vomiting, two (2%); Clostridium difficile enteritis, one (1%); congestive heart failure, one (1%); and anemia, requiring blood transfusion, one (1%) . With a median follow-up of 36 months (range 24-50 mos), all 20 patients are alive, and 19 (95%) are disease-free . The one patient (5%) treated with i.v.++paclitaxel alone developed an abdominal recurrence 22 months after diagnosis . CONCLUSION: Primary i.v.++paclitaxel and platinum chemotherapy in patients with high-risk Stage I epithelial ovarian carcinoma is reasonably well tolerated and may improve survival . Larger studies with long-term follow-up are needed.

Appl Environ Microbiol, 1999 Sep, 65(9), 4234 - 8
Clostridium difficile cell attachment is modified by environmental factors; Waligora AJ et al.; Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation . The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains . Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa.

Appl Environ Microbiol, 1999 Sep, 65(9), 3990 - 5
Characterization of an acetyl xylan esterase from the anaerobic fungus Orpinomyces sp . strain PC-2; Blum DL et al.; A 1,067-bp cDNA, designated axeA, coding for an acetyl xylan esterase (AxeA) was cloned from the anaerobic rumen fungus Orpinomyces sp . strain PC-2 . The gene had an open reading frame of 939 bp encoding a polypeptide of 313 amino acid residues with a calculated mass of 34,845 Da . An active esterase using the original start codon of the cDNA was synthesized in Escherichia coli . Two active forms of the esterase were purified from recombinant E . coli cultures . The size difference of 8 amino acids was a result of cleavages at two different sites within the signal peptide . The enzyme released acetate from several acetylated substrates, including acetylated xylan . The activity toward acetylated xylan was tripled in the presence of recombinant xylanase A from the same fungus . Using p-nitrophenyl acetate as a substrate, the enzyme had a K(m) of 0.9 mM and a V(max) of 785 micromol min(-1) mg(-1) . It had temperature and pH optima of 30 degrees C and 9.0, respectively . AxeA had 56% amino acid identity with BnaA, an acetyl xylan esterase of Neocallimastix patriciarum, but the Orpinomyces AxeA was devoid of a noncatalytic repeated peptide domain (NCRPD) found at the carboxy terminus of the Neocallimastix BnaA . The NCRPD found in many glycosyl hydrolases and esterases of anaerobic fungi has been postulated to function as a docking domain for cellulase-hemicellulase complexes, similar to the dockerin of the cellulosome of Clostridium thermocellum . The difference in domain structures indicated that the two highly similar esterases of Orpinomyces and Neocallimastix may be differently located, the former being a free enzyme and the latter being a component of a cellulase-hemicellulase complex . Sequence data indicate that AxeA and BnaA might represent a new family of hydrolases.

Appl Environ Microbiol, 1999 Sep, 65(9), 3793 - 9
Development and characterization of a gene expression reporter system for Clostridium acetobutylicum ATCC 824; Tummala SB et al.; A gene expression reporter system (pHT3) for Clostridium acetobutylicum ATCC 824 was developed by using the lacZ gene from Thermoanaerobacterium thermosulfurogenes EM1 as the reporter gene . In order to test the reporter system, promoters of three key metabolic pathway genes, ptb (coding for phosphotransbutyrylase), thl (coding for thiolase), and adc (coding for acetoacetate decarboxylase), were cloned upstream of the reporter gene in pHT3 in order to construct vectors pHT4, pHT5, and pHTA, respectively . Detection of beta-galactosidase activity in time course studies performed with strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA) demonstrated that the reporter gene produced a functional beta-galactosidase in C . acetobutylicum . In addition, time course studies revealed differences in the beta-galactosidase specific activity profiles of strains ATCC 824(pHT4), ATCC 824(pHT5), and ATCC 824(pHTA), suggesting that the reporter system developed in this study is able to effectively distinguish between different promoters . The stability of the beta-galactosidase produced by the reporter gene was also examined with strains ATCC 824(pHT4) and ATCC 824(pHT5) by using chloramphenicol treatment to inhibit protein synthesis . The data indicated that the beta-galactosidase produced by the lacZ gene from T . thermosulfurogenes EM1 was stable in the exponential phase of growth . In pH-controlled fermentations of ATCC 824(pHT4), the kinetics of beta-galactosidase formation from the ptb promoter and phosphotransbutyrylase formation from its own autologous promoter were found to be similar.

Appl Environ Microbiol, 1999 Sep, 65(9), 3787 - 92
Development of an in vitro bioassay for Clostridium botulinum type B neurotoxin in foods that is more sensitive than the mouse bioassay; Wictome M et al.; A novel, in vitro bioassay for detection of the botulinum type B neurotoxin in a range of media was developed . The assay is amplified by the enzymic activity of the neurotoxin's light chain and includes the following three stages: first, a small, monoclonal antibody-based immunoaffinity column captures the toxin; second, a peptide substrate is cleaved by using the endopeptidase activity of the type B neurotoxin; and finally, a modified enzyme-linked immunoassay system detects the peptide cleavage products . The assay is highly specific for type B neurotoxin and is capable of detecting type B toxin at a concentration of 5 pg ml(-1) (0.5 mouse 50% lethal dose ml(-1)) in approximately 5 h . The format of the test was found to be suitable for detecting botulinum type B toxin in a range of foodstuffs with a sensitivity that exceeds the sensitivity of the mouse assay . Using highly specific monoclonal antibodies as the capture phase, we found that the endopeptidase assay was capable of differentiating between the type B neurotoxins produced by proteolytic and nonproteolytic strains of Clostridium botulinum type B.

Antimicrob Agents Chemother, 1999 Sep, 43(9), 2231 - 5
In vitro activity of gemifloxacin (SB 265805) against anaerobes; Goldstein EJ et al.; Gemifloxacin mesylate (SB 265805), a new fluoronaphthyridone, was tested against 359 recent clinical anaerobic isolates by the National Committee for Clinical Laboratory Standards reference agar dilution method with supplemented brucella blood agar and an inoculum of 10(5) CFU/spot . Comparative antimicrobials tested included trovafloxacin, levofloxacin, grepafloxacin, sparfloxacin, sitafloxacin (DU-6859a), penicillin G, amoxicillin clavulanate, imipenem, cefoxitin, clindamycin, and metronidazole . The MIC(50) and MIC(90) (MICs at which 50 and 90% of the isolates were inhibited) of gemifloxacin against various organisms (with the number of strains tested in parentheses) were as follows (in micrograms per milliliter): for Bacteroides fragilis (28), 0.5 and 2; for Bacteroides thetaiotaomicron (24), 1 and 16; for Bacteroides caccae (12), 1 and 16; for Bacteroides distasonis (12), 8 and >16; for Bacteroides ovatus (12), 4 and >16; for Bacteroides stercoris (12), 0.5 and 0.5; for Bacteroides uniformis (12), 1 and 4; for Bacteroides vulgatus (11), 4 and 4; for Clostridium clostridioforme (15), 0.5 and 0.5; for Clostridium difficile (15), 1 and >16; for Clostridium innocuum (13), 0.125 and 2; for Clostridium perfringens (13), 0.06 and 0.06; for Clostridium ramosum (14), 0.25 and 8; for Fusobacterium nucleatum (12), 0.125 and 0.25; for Fusobacterium necrophorum (11), 0.25 and 0.5; for Fusobacterium varium (13), 0.5 and 1; for Fusobacterium spp . (12), 1 and 2; for Peptostreptococcus anaerobius (13), 0.06 and 0.06; for Peptostreptococcus asaccharolyticus (13), 0.125 and 0.125; for Peptostreptococcus magnus (14), 0.03 and 0.03; for Peptostreptococcus micros (12), 0.06 and 0.06; for Peptostreptococcus prevotii (14), 0.06 and 0.25; for Porphyromonas asaccharolytica (11), 0.125 and 0.125; for Prevotella bivia (10), 8 and 16; for Prevotella buccae (10), 2 and 2; for Prevotella intermedia (10), 0.5 and 0.5; and for Prevotella melaninogenica (11), 1 and 1 . Gemifloxacin mesylate (SB 265805) was 1 to 4 dilutions more active than trovafloxacin against fusobacteria and peptostreptococci, and the two drugs were equivalent against clostridia and P . asaccharolytica . Gemifloxacin was equivalent to sitafloxacin (DU 6859a) against peptostreptococci, C . perfringens, and C . ramosum, and sitafloxacin was 2 to 3 dilutions more active against fusobacteria . Sparfloxacin, grepafloxacin, and levofloxacin were generally less active than gemifloxacin against all anaerobes.

Clin Rheumatol, 1999, 18(4), 337 - 8
Reactive arthritis induced by Clostridium difficile enteritis as a complication of Helicobacter pylori eradication; Soderlin MK et al.; Clostridium difficile has recently been established as a cause of reactive arthritis (ReA) . We present a case of Clostridium difficile-induced ReA as a complication of Helicobacter pylori eradication, which, to the best of our knowledge, is the first such case reported.

Structure Fold Des, 1999 Aug 15, 7(8), 891 - 902
Glutamate mutase from Clostridium cochlearium: the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights; Reitzer R et al.; BACKGROUND: Glutamate mutase (Glm) equilibrates (S)-glutamate with (2S,3S)-3-methylaspartate . Catalysis proceeds with the homolytic cleavage of the organometallic bond of the cofactor to yield a 5'-desoxyadenosyl radical . This radical then abstracts a hydrogen atom from the protein-bound substrate to initiate the rearrangement reaction . Glm from Clostridium cochlearium is a heterotetrameric molecule consisting of two sigma and two epsilon polypeptide chains . RESULTS: We have determined the crystal structures of inactive recombinant Glm reconstituted with either cyanocobalamin or methylcobalamin . The molecule shows close similarity to the structure of methylmalonyl CoA mutase (MCM), despite poor sequence similarity between its catalytic epsilon subunit and the corresponding TIM-barrel domain of MCM . Each of the two independent B12 cofactor molecules is associated with a substrate-binding site, which was found to be occupied by a (2S,3S)-tartrate ion . A 1:1 mixture of cofactors with cobalt in oxidation states II and III was observed in both crystal structures of inactive Glm . CONCLUSIONS: The long axial cobalt-nitrogen bond first observed in the structure of MCM appears to result from a contribution of the species without upper ligand . The tight binding of the tartrate ion conforms to the requirements of tight control of the reactive intermediates and suggests how the enzyme might use the substrate-binding energy to initiate cleavage of the cobalt-carbon bond . The cofactor does not appear to have a participating role during the radical rearrangement reaction.

Radiographics, 1999 Jul-Aug, 19(4), 887 - 97
Pseudomembranous colitis: spectrum of imaging findings with clinical and pathologic correlation; Kawamoto S et al.; Pseudomembranous colitis (PMC) is a potentially life-threatening acute infectious colitis caused by one or more toxins produced by an unopposed proliferation of Clostridium difficile bacteria . PMC is characterized by the presence of elevated, yellow-white plaques forming pseudomembranes on the colonic mucosa . These plaques can be visualized at both pathologic analysis and endoscopy . Plain radiography, contrast enema studies, and computed tomography (CT) are useful in the evaluation of PMC . Plain radiography of the abdomen can demonstrate polypoid mucosal thickening, "thumbprinting" (wide transverse bands associated with haustral fold thickening), or gaseous distention of the colon . A toxic megacolon with distention and occasionally pneumoperitoneum may be seen in the most severe cases of PMC involving perforation . At contrast enema studies, the primary finding in mild cases of PMC is small nodular filling defects representing the mucosal plaques . With more extensive colonic involvement, the plaques are larger and coalesce to form an irregular bowel wall margin . Mural thickening and wide haustral folds caused by intramural edema may also be seen . A contrast enema study is contraindicated in patients with severe PMC due to the danger of perforation . Common CT findings include wall thickening, low-attenuation mural thickening corresponding to mucosal and submucosal edema, the "accordion sign," the "target sign" ("double halo sign"), pericolonic stranding, and ascites . Familiarity with these imaging characteristics may allow early diagnosis and treatment and prevent progression to more serious pathologic conditions.

Trop Gastroenterol, 1999 Jan-Mar, 20(1), 33 - 5
Detection of Clostridium difficile toxin by an indigenously developed latex agglutination assay; Vaishnavi C et al.; An indigenously developed latex agglutination assay using C . sordelli antitoxin was used to screen 211 stool samples received from hospitalized patients . Of 126 samples from patients receiving single to multiple antibiotics for various ailments, 38 (30%) were positive by the toxin assay, whereas only 6/85 (7%) of samples of patients not receiving antibiotics were also positive . Thus, of 211 samples a total of 44 (20.8%) were positive by our toxin assay, giving titers ranging from 1 in 5 to 1 in 320 . The test developed by us is simple, rapid, easy and reliable and can be easily adapted to all microbiology laboratories.

J Bacteriol, 1999 Sep, 181(17), 5288 - 95
Cloning and sequence analysis of a new cellulase gene encoding CelK, a major cellulosome component of Clostridium thermocellum: evidence for gene duplication and recombination; Kataeva I et al.; The cellulolytic and hemicellulolytic complex of Clostridium thermocellum, termed cellulosome, consists of up to 26 polypeptides, of which at least 17 have been sequenced . They include 12 cellulases, 3 xylanases, 1 lichenase, and CipA, a scaffolding polypeptide . We report here a new cellulase gene, celK, coding for CelK, a 98-kDa major component of the cellulosome . The gene has an open reading frame (ORF) of 2,685 nucleotides coding for a polypeptide of 895 amino acid residues with a calculated mass of 100,552 Da . A signal peptide of 27 amino acid residues is cut off during secretion, resulting in a mature enzyme of 97,572 Da . The nucleotide sequence is highly similar to that of cbhA (V . V . Zverlov et al., J . Bacteriol . 180:3091-3099, 1998), having an ORF of 3,690 bp coding for the 1,230-amino-acid-residue CbhA of the same bacterium . Homologous regions of the two genes are 86.5 and 84.3% identical without deletion or insertion on the nucleotide and amino acid levels, respectively . Both have domain structures consisting of a signal peptide, a family IV cellulose binding domain (CBD), a family 9 glycosyl hydrolase domain, and a dockerin domain . A striking distinction between the two polypeptides is that there is a 330-amino-acid insertion in CbhA between the catalytic domain and the dockerin domain containing a fibronectin type 3-like domain and family III CBD . This insertion, missing in CelK, is responsible for the size difference between CelK and CbhA . Upstream and downstream flanking sequences of the two genes show no homology . The data indicate that celK and cbhA in the genome of C . thermocellum have evolved through gene duplication and recombination of domain coding sequences . celK without a dockerin domain was expressed in Escherichia coli and purified . The enzyme had pH and temperature optima at 6.0 and 65 degrees C, respectively . It hydrolyzed p-nitrophenyl-beta-D-cellobioside with a Km and a Vmax of 1.67 microM and 15.1 U/mg, respectively . Cellobiose was a strong inhibitor of CelK activity, with a Ki of 0.29 mM . The enzyme was thermostable, after 200 h of incubation at 60 degrees C, 97% of the original activity remained . Properties of the enzyme indicated that it is a cellobiohydrolase.

J Food Prot, 1996 Mar, 59(3), 299 - 305
Quality characteristics of fresh blue crab meat held at 0 and 4 degrees C in tamper-evident containers; Gates KW et al.; There has been a regulatory movement toward the required use of tamper-evident containers for fresh blue crab meat . North Carolina passed tamper-evident regulations in 1993 . Blue crab processors had little information on possible changes in head-space gases, microbial growth, chemical decomposition, sensory quality, or shelf life caused by the new containers . Chemical, microbiological, physical, and sensory changes in fresh crab meat were monitored during 18 days of storage in ice and 13 days of storage refrigerated at 4 degrees C . "Special" blue crab meat, chosen for the study, is the least expensive commercial form of white crab meat . The crab meat was packaged in four retail containers: copolymer polyethylene cups with polyethylene snap-on lids, copolymer polyethylene cups with snap-on polyethylene lids fastened to the cup with heat-shrink low-density polypropylene seals, copolymer polyethylene cans with aluminum easy-open ends, and copolymer polypropylene cups with a tamper-evident pull-tab on the lid . Control samples packaged in industry standard copolymer polyethylene cups maintained higher oxygen levels than meat stored in tamper-evident containers . No consistent differences in quality or shelf life were detected among the containers . Market shelf life was limited to 6 days for meat held at 4 degrees C and 15 days for meat held at 0 degrees C . Sensory quality deteriorated 6 days earlier for crab meat held at 4 degrees C than meat held at 0 degrees C . Collateral work showed that toxin production by Clostridium botulinum neither occurred following 18 days of storage at 4 degrees C nor after 15 days of storage at 10 degrees C . Definite spoilage occurred before any toxin production . The study suggests that blue crab processors can safely use the new tamper-evident packaging, which has little or no effect on product quality or shelf life . Processors may choose appropriate packaging options using price, packaging quality, market appearance, and ease of production as the deciding criteria.

J Food Prot, 1996 Mar, 59(3), 257 - 60
Risk of Clostridium botulinum type E toxin production in blue crab meat packaged in four commercial-type containers; Harrison MA et al.; The aim of this investigation was to determine if a risk of Clostridium botulinum growth and toxin production existed in four different packaged crabmeat products . Freshly picked blue crab meat was inoculated with 10(3) to 10(4) spores per g of a mixed pool of four strains of C . botulinum type E (Beluga, Minnesota, G21-5, and 070) . The lump crabmeat was packaged in four different packaging containers: (i) 12-oz copolymer polyethylene cups currently used by most crab processors; (ii) 12-oz copolymer polyethylene cups with heat-shrink, tamper-evident low-density polypropylene seals; (iii) 8-oz copolymer polyethylene cups with easy-open aluminum ends: and (iv) 8-oz copolymer polypropylene cups with integral tamper-evident pull-tabs . The packages were stored at either 4 degrees C for 21 days or 10 degrees C for 15 days . Storage at 10 degrees C was used to simulate temperature abuse . The mouse bioassay was used to detect the presence of C . botulinum toxin . Psychotrophic and anaerobic populations were enumerated and were found to increase with time regardless of packaging type . No botulinum toxin was detected in any of the four packaging types stored at 4 degrees C or 10 degrees C throughout the entire storage period.

Microbiology, 1999 Aug, 145 ( Pt 8), 1839 - 47
The glyceraldehyde-3-phosphate dehydrogenase of Clostridium acetobutylicum: isolation and purification of the enzyme, and sequencing and localization of the gap gene within a cluster of other glycolytic genes; Schreiber W et al.; Glyceraldehyde-3-phosphate dehydrogenase was purified from Clostridium acetobutylicum by sequential ammonium sulfate precipitation, gel filtration and anion-exchange chromatography (to a specific activity of 27 U mg-1) . The enzyme had a molecular mass of 40 kDa as determined by SDS-PAGE and a native molecular mass of 160 kDa as determined by nondenaturing PAGE, indicating that it has a homotetrameric composition . Its pH optimum was between 8.5 and 9.3 . The corresponding gene (gap) was cloned and sequenced from C . acetobutylicum DSM 792 and found to cluster with other genes of enzymes from the glycolytic pathway (pgk, phosphoglycerate kinase; tpi, triosephosphate isomerase; pgm(i), 2,3-bisphosphoglycerate-independent phosphoglycerate mutase) . No sequences resembling rho-independent transcription terminators were found in the intergenic regions . A plasmid carrying the clostridial gap gene complemented an Escherichia coli gap mutant.

Microbiology, 1999 Aug, 145 ( Pt 8), 1831 - 8
Growth inhibition of Clostridium cellulolyticum by an inefficiently regulated carbon flow; Guedon E et al.; Carbon flow in Clostridium cellulolyticum was investigated either in batch or continuous culture using a synthetic medium with cellobiose as the sole source of carbon and energy . Previous experiments carried out using a complex growth medium led to the conclusion that the carbon flow was stopped by intracellular NADH . In this study, results showed that cells cultured in a synthetic medium were better able to control electron flow since the NADH/NAD+ ratios were in the range 0.3-0.7, whereas a ratio as high as 57 was previously found in cells cultured on a complex medium . Furthermore, a specific rate of cellobiose consumption of 2.13 mmol (g cells)-1 h-1 was observed on synthetic medium whereas the highest value obtained on complex medium was 0.68 mmol (g cells)-1 h-1 . When C . cellulolyticum was grown in continuous culture and cellobiose in the feed medium was increased from 5.84 to 17.57 mM in stepwise fashion, there was an increase in cellobiose utilization without growth inhibition . In contrast, when the reactor was fed directly with 14.62 mM cellobiose, residual cellobiose was observed (4.24 mM) and growth was limited . These data indicate that C . cellulolyticum is not able to optimize its growth and carbon flow in response to a sudden increase in the concentration of growth substrate cellobiose . This interpretation was confirmed (i) by the study of cellobiose batch fermentation where it was demonstrated that growth inhibition was not due to nutritional limitation or inhibition by fermentation products but was associated with carbon excess and (ii) by the growth of C . cellulolyticum in dialysis culture where no growth inhibition was observed due to the limitation of carbon flow by the low rate of cellobiose diffusion through the dialysis tubing.

J Hosp Infect, 1999 Sep, 43(1), 25 - 32
Detection of glycopeptide-resistant enterococci in routine diagnostic faeces specimens; Taylor ME et al.; Faeces received in a diagnostic laboratory were screened for glycopeptide-resistant enterococci (GRE) on modified Lewisham medium, with and without enrichment in Enterococcosel broth . Colonization by GRE was detected in 102/838 patients (12.2%) . In 74 (73%) of colonized patients GRE were detected by both methods and in 28 (27%) they were detected only after enrichment . The carriage rate in hospitalized patients was 32% (93/289) compared with 2.3% (11/425) in the community (GP patients and food-handlers) . Carriage of GRE increased with age . Clostridium difficile isolation was associated with GRE colonization, odds ratio 6.76 (P<0.001) . Fifty-nine percent (60/102) of the GRE had the VanA phenotype and 41% (42/102) had the VanB phenotype . In the community VanA predominated (91%), whereas 64% (57/89) of the isolates from hospitalised patients were of the VanB phenotype .

Arch Microbiol, 1999 Sep, 172(3), 139 - 49
Isolation and characterization of a Clostridium sp . with cinnamoyl esterase activity and unusual cell envelope ultrastructure; McSweeney CS et al.; Microorganisms that hydrolyse the ester linkages between phenolic acids and polysaccharides in plant cell walls are potential sources of enzymes for the degradation of lignocellulosic waste . An anaerobic, mesophilic, spore-forming, xylanolytic bacterium with high hydroxy cinnamic acid esterase activity was isolated from the gut of the grass-eating termite Tumilitermes pastinator . The bacterium was motile and rod-shaped, stained gram-positive, had an eight-layered cell envelope, and formed endospores . Phylogenetic analysis based on 16S rRNA indicated that the bacterium is closely related to Clostridium xylanolyticum and is grouped with polysaccharolytic strains of clostridia . A wide range of carbohydrates were fermented, and growth was stimulated by either xylan or cellobiose as substrates . The bacterium hydrolysed and then hydrogenated the hydroxy cinnamic acids (ferulic and p-coumaric acids), which are esterified to arabinoxylan in plant cell walls . Three cytoplasmic enzymes with hydroxy cinnamic acid esterase activity were identified using non-denaturing gel electrophoresis . This bacterium possesses an unusual multilayered cell envelope in which both leaflets of the cytoplasmic membrane, the peptidoglycan layer and the S layer are clearly discernible . The fate of all these components was easily followed throughout the endospore formation process . The peptidoglycan component persisted during the entire morphogenesis . It was seen to enter the septum and to pass with the engulfing membranes to surround the prespore . It eventually expanded to form the cortex, verification for the peptidoglycan origin of the cortex . Sporogenic vesicles, which are derived from the cell wall peptidoglycan, were associated with the engulfment process . Spore coat fragments appeared early, in stage II, though spore coat formation was not complete until after cortex formation.

Infect Immun, 1999 Sep, 67(9), 4902 - 7
Use of genetically manipulated strains of Clostridium perfringens reveals that both alpha-toxin and theta-toxin are required for vascular leukostasis to occur in experimental gas gangrene; Ellemor DM et al.; A hallmark of gas gangrene (clostridial myonecrosis) pathology is a paucity of leukocytes infiltrating the necrotic tissue . The cause of this paucity most likely relates to the observation of leukocyte aggregates at the border of the area of tissue necrosis, often within the microvasculature itself . Infecting mice with genetically manipulated strains of Clostridium perfringens type A (deficient in either alpha-toxin or theta-toxin production) resulted in significantly reduced leukocyte aggregation when alpha-toxin was absent and complete abrogation of leukocyte aggregation when theta-toxin was absent . Thus, both alpha-toxin and theta-toxin are necessary for the characteristic vascular leukostasis observed in clostridial myonecrosis.

Infect Immun, 1999 Sep, 67(9), 4708 - 12
Pure botulinum neurotoxin is absorbed from the stomach and small intestine and produces peripheral neuromuscular blockade; Maksymowych AB et al.; Clostridium botulinum serotype A produces a neurotoxin composed of a 100-kDa heavy chain and a 50-kDa light chain linked by a disulfide bond . This neurotoxin is part of a ca . 900-kDa complex, formed by noncovalent association with a single nontoxin, nonhemagglutinin subunit and a family of hemagglutinating proteins . Previous work has suggested, although never conclusively demonstrated, that neurotoxin alone cannot survive passage through the stomach and/or cannot be absorbed from the gut without the involvement of auxiliary proteins in the complex . Therefore, this study compared the relative absorption and toxicity of three preparations of neurotoxin in an in vivo mouse model . Equimolar amounts of serotype A complex with hemagglutinins, complex without hemagglutinins, and purified neurotoxin were surgically introduced into the stomach or into the small intestine . In some experiments, movement of neurotoxin from the site of administration was restricted by ligation of the pylorus . Comparison of relative toxicities demonstrated that at adequate doses, complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin can be absorbed from the stomach . The potency of neurotoxin in complex was greater than that of pure neurotoxin, but the magnitude of this difference diminished as the dosage of neurotoxin increased . Qualitatively similar results were obtained when complex with hemagglutinins, complex without hemagglutinins, and pure neurotoxin were placed directly into the intestine . This work establishes that pure botulinum neurotoxin serotype A is toxic when administered orally . This means that pure neurotoxin does not require hemagglutinins or other auxiliary proteins for absorption from the gastrointestinal system into the general circulation.

J Food Prot, 1999 Aug, 62(8), 948 - 52
Evaluation of botulinal toxin production in packaged fresh-cut cantaloupe and honeydew melons; Larson AE et al.; The ability of Clostridium botulinum to produce toxin on cubed, packaged melons was investigated relative to microbial spoilage at various incubation temperatures and in different packaging systems . Freshly cut cubes (approximately 2.5 cm3) of cantaloupe and honeydew melons were surface inoculated with a 10 strain mixture of proteolytic and nonproteolytic spores of C . botulinum (10 to 15 cubes per package; approximately 100 total spores per package) . To initially evaluate toxin production and spoilage in a passively modified atmosphere, melon cubes were loosely packaged in air in polyethylene pouches, sealed, and incubated at 7 or 15 degrees C for up to 21 days . At various sampling intervals, samples were tested for headspace oxygen and carbon dioxide levels, pH, presence of botulinal toxin, aerobic and anaerobic plate counts, and counts of yeasts and molds . During incubation, headspace oxygen levels decreased, headspace carbon dioxide levels increased, aerobic and anaerobic plate counts increased, and the pH remained constant or decreased slightly . Botulinal toxin was not detected in any cantaloupe samples or in honeydew samples incubated at 7 degrees C . Botulinal toxin was detected in some honeydew samples at 15 degrees C after 9 days of incubation, but the toxic honeydews were severely spoiled and considered organoleptically unacceptable . A similar second experiment was performed in which half of the melon cubes were treated with UV light to inactivate vegetative organisms before packaging, and these were incubated at 7, 15, or 27 degrees C . In this second experiment, toxin production occurred in the UV-treated samples at 15 degrees C with gross spoilage and at 27 degrees C with only marginal spoilage . These data indicate that inhibition of spoilage organisms with UV light could result in botulinal toxin formation in packaged melons before overt spoilage.

J Food Prot, 1999 Aug, 62(8), 899 - 904
Identification and characterization of two bacteriocin-producing bacteria isolated from garlic and ginger root; Janes ME et al.; Two bacteriocin-producing bacterial strains were isolated from garlic and ginger root by the agar overlay method . The bacteria were identified by 16S rRNA sequence analyses and fermentation patterns as Leuconostoc mesenteroides (garlic isolate) and Lactococcus lactis (ginger isolate) . The bacteriocins were assigned the names leucocin BC2 and lactocin GI3, respectively . Physiochemical properties and antimicrobial spectra of the bacteriocins were determined by the spot-on-lawn method . Both bacteriocins were inhibited by proteolytic enzymes . Leucocin BC2 exhibited a narrow antimicrobial spectrum, inhibiting only Bacillus, Enterococcus, and Listeria species . Lactocin GI3 had a broader spectrum, inhibiting Bacillus, Clostridium, Listeria, Enterococcus, Leuconostoc, Pediococcus, and Staphylococcus species . Both bacteriocins remained active when heated at 90 degrees C for 15 min or 120 degrees C for 20 min . Leucocin BC2 assayed at 37 degrees C showed an inhibitory activity of 1,600 AU/ml, whereas at 8 degrees C the activity was 12,800 AU/ml . Conversely, lactocin GI3 activity was the same at both assay temperatures . Both bacteriocins remained active over a pH range of 2.0 to 9.0 and in various organic solvents . The activity of leucocin BC2 was increased when treated with 0.5% acetic acid and 0.5% lactic acid, whereas lactocin GI3 activity was decreased with either acid . The molecular mass values were 3.7 kDa for leucocin BC2 and 3.9 kDa for lactocin GI3 . These results show that the inhibitory substances produced by the bacteria isolated from garlic and ginger are bacteriocins that appear to be different in some characteristics from previously reported bacteriocins.

J Food Prot, 1999 Aug, 62(8), 872 - 6
Toxin production by Clostridium botulinum in pasteurized milk treated with carbon dioxide; Glass KA et al.; The addition of carbon dioxide to milk at levels of <20 mM inhibits the growth of selected spoilage organisms and extends refrigerated shelf life . Our objective was to determine if the addition of CO2 influenced the risk of botulism from milk . Carbon dioxide was added to pasteurized 2% fat milk at approximately 0, 9.1, or 18.2 mM using a commercial gas-injection system . The milk was inoculated with a 10-strain mixture of proteolytic and nonproteolytic Clostridium botulinum spore strains to yield 10(1) to 10(2) spores/ml . Milk was stored at 6.1 or 21 degrees C for 60 or 6 days, respectively, in sealed glass jars or high-density polyethylene plastic bottles . Milk stored at 21 degrees C curdled and exhibited a yogurt-like odor at 2 days and was putrid at 4 days . Botulinal toxin was detected in 9.1 mM CO2 milk at 4 days and in all treatments after 6 days of storage at 21 degrees C . All toxic samples were grossly spoiled based on sensory evaluation at the time toxin was detected . Although botulinal toxin appeared earlier in milk treated with 9.1 mM CO2 compared to both the 18.2 mM and untreated milk, gross spoilage would act as a deterrent to consumption of toxic milk . No botulinal toxin was detected in any treatment stored at 6.1 degrees C for 60 days . At 6.1 degrees C, the standard plate counts (SPCs) were generally lower in the CO2-treated samples than in controls, with 18.2 mM CO2 milk having the lowest SPC . These data indicate that the low-level addition of CO2 retards spoilage of pasteurized milk at refrigeration temperatures and does not increase the risk of botulism from treated milk stored at refrigeration or abuse temperatures.

J Food Prot, 1999 Aug, 62(8), 867 - 71
Clostridium botulinum spores and toxin in mascarpone cheese and other milk products; Franciosa G et al.; A total of 1,017 mascarpone cheese samples, collected at retail, were analyzed for Clostridium botulinum spores and toxin, aerobic mesophilic spore counts, as well as pH, a(w) (water activity), and Eh (oxidation-reduction potential) . In addition 260 samples from other dairy products were also analyzed for spores and botulinum toxin . Experiments were carried out on naturally and artificially contaminated mascarpone to investigate the influence of different temperature conditions on toxin production by C . botulinum . Three hundred and thirty-one samples (32.5%) of mascarpone were positive for botulinal spores, and 7 (0.8%) of the 878 samples produced at the plant involved in an outbreak of foodborne botulism also contained toxin type A . The chemical-physical parameters (pH, a(w), Eh) of all samples were compatible with C . botulinum growth and toxinogenesis . Of the other milk products, 2.7% were positive for C . botulinum spores . Growth and toxin formation occurred in naturally and experimentally contaminated mascarpone samples after 3 and 4 days of incubation at 28 degrees C, respectively.

Br J Pharmacol, 1999 Jul, 127(5), 1060 - 3
Rundown of somatodendritic N-methyl-D-aspartate (NMDA) receptor channels in rat hippocampal neurones: evidence for a role of the small GTPase RhoA; Norenberg W et al.; Actin filament (F-actin) depolymerization leads to the use-dependent rundown of N-methyl-D-aspartate (NMDA) receptor activity in rat hippocampal neurones . Depolymerization is promoted by Ca2+ which enters the cells via NMDA receptor channels . The ras homologue (Rho) GTPases (RhoA, Rac1 and Cdc42) promote actin polymerization and thus control the actin cytoskeleton . We have investigated, by means of the whole-cell patch clamp technique, whether the actin fibres which interact with NMDA receptors are controlled by Rho GTPases . In the presence of intracellular ATP which attenuates rundown, the C3 toxin from Clostridium (C.) botulinum was used to inactivate RhoA . Indeed, it enhanced the use-dependent rundown of NMDA-evoked inward currents to a level similar to that obtained in the absence of ATP . Lethal toxin from Clostridium sordellii which inactivates Rac1 and Cdc42 lacked this effect . We suggest that the function of somatodendritic NMDA receptor channels in rat hippocampal neurones can be modulated by RhoA via its action on F-actin.

Surg Today, 1999, 29(7), 626 - 8
Tetanus after a resection for a gangrenous perforated small intestine: report of a case; Furui J et al.; We report herein the case of a 75-year-old man who developed severe tetanus 24 h after the resection of a gangrenous perforated small intestine . It seemed that the tetanus was caused by a spillage of the intestinal contents harboring Clostridium tetani; however, this was not identified by a culture . The diagnosis of tetanus was made only when opisthotonus in this patient became evident and normal tetanus treatment proved to be successful.

J Clin Microbiol, 1999 Sep, 37(9), 3044 - 7
Evaluation of two rapid assays for detection of Clostridium difficile toxin A in stool specimens; Fedorko DP et al.; Rapid laboratory diagnosis of Clostridium difficile-associated diarrhea (CDAD) is highly desirable in the setting of hospital cost containment . We tested 654 stool specimens to compare the performance of two assays for rapid detection of toxin A, the Immunocard Toxin A test (Meridian Diagnostics, Inc.) and the Culturette Brand Toxin CD enzyme immunoassay (EIA) (Becton Dickinson Microbiology Systems), with a cytotoxin assay (Cytotoxi Test; Advanced Clinical Diagnostics) and culture on cycloserine-cefoxitin-fructose agar followed by determination of the production of toxins A and B . A chart review was performed for patients whose stool specimens provided positive results on one to three of the assays . With the "gold standard" of all four assays positive or chart review evidence of CDAD, 97 (14.8%) stool specimens were positive by one or more assays and 557 (85.2%) were negative by all methods . Total agreement for all assays was 90.5% (592 of 654) . The sensitivity, specificity, positive predictive value, and negative predictive value for toxigenic culture were 94.7, 98.6, 87.1, and 99.5%, respectively, for toxigenic culture; 87.7, 98.6, 86.2, and 98.8%, respectively, for the cytotoxin assay; 71.9, 99.3, 91.1, and 97.3%, respectively, for the Immunocard; and 68.4, 99.1, 88.6, and 96.9%, respectively, for the Culturette EIA . While easy to perform and highly specific, these rapid assays do not appear to be sufficient for accurate diagnosis of CDAD.

Chirurg, 1999 Jul, 70(7), 813 - 7
{Limb preservation after clostridial myonecrosis following internal fixation of a closed femoral fracture}; Keel M et al.; A 24-year-old healthy man developed Clostridium perfringens myonecrosis with severe sepsis after plating of a closed femoral fracture . The leg could be preserved by complete resection of the fascia from all muscle compartments of the leg, including the pelvitrochanteric and the iliopsoas muscles, radical removal of necrotic muscle tissue, dissection of the para-aortal infrarenal lymphatics, daily debridements over 2 weeks, and systemic antibiotic therapy . The plate was removed because of a second septic episode followed by temporary stabilization with external fixation . After soft-tissue healing, plate fixation was carried out . The patient developed significant deficit of knee flexion (0 degree/0/20 degrees) due to heterotopic ossification of the quadriceps femoris muscle that could be improved by partial resection of heterotopic bone formation . In the same operation the bony defect of the femur was filled with autologous bone graft . The fracture healed 10 months after the accident . The patient can work full time in his previous profession as a mechanic, but again needs operative mobilization of the knee joint, including open arthrolysis and quadriceps plasty.

Int J Food Microbiol, 1999 Jun 1, 48(3), 179 - 89
Characterisation of Clostridium botulinum groups I and II by randomly amplified polymorphic DNA analysis and repetitive element sequence-based PCR; Hyytia E et al.; Random amplified polymorphic DNA analysis (RAPD) and repetitive element sequence-based PCR (rep-PCR) were evaluated with respect to their applicability to characterise Clostridium botulinum group I and II strains, the species causing human botulism . Fifteen group I and 21 group II strains of various geographical and temporal origins were characterised with four single arbitrary RAPD primers at low stringency amplification conditions and with a degenerate REP primer pair at moderately stringent conditions . Ready-To-Go RAPD Analysis Beads and Ready-To-Go PCR Beads were used for PCR reactions with RAPD and rep-PCR, respectively . Arbitrary primer OPJ 6 yielded the most discriminating patterns, and distinguished group II C . botulinum serotypes at the strain level . Group I strains were mainly discriminated at the serotype level . The discriminatory power of rep-PCR was found to be inferior to that of RAPD . The REP1R-Dt and REP2R-Dt primer pair generated group I- and II-specific fragments and arbitrary primer OPJ 13 produced a serotype E-specific fragment . The use of pre-dispensed and pre-optimised beads attributed to highly reproducible results . As compared to more time-consuming typing methods, such as pulsed-field gel electrophoresis (PFGE), both RAPD and rep-PCR were characterised by rapid performance and a typeability of 100%.

FEMS Immunol Med Microbiol, 1999 Aug 1, 25(1-2), 175 - 82
The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): III . Detection of IgA antibodies in human milk that bind to bacterial toxins implicated in SIDS; Gordon AE et al.; Two toxin-producing bacteria implicated in sudden infant death syndrome (SIDS) are Staphylococcus aureus and Clostridium perfringens . Epidemiological studies have shown that breast feeding reduces an infant's risk of SIDS . This protective effect could be due partly to IgA antibodies to these toxins in human milk . The aim of this work was to use a quantitative ELISA to determine levels of IgA antibodies that bound to toxic shock syndrome toxin (TSST-1), staphylococcal enterotoxin C (SEC) and C . perfringens enterotoxin A (CEA) in individual samples of human milk . All samples of milk tested contained IgA antibodies that bound to the bacterial toxins . For individual samples, IgA bound to TSST-1, SEC and CEA were in the range of 900-3100 ng ml(-1), 1000-3600 ng ml(-1) and 1000-4300 ng ml(-1) respectively . Isolation of S . aureus from mothers donating breast milk samples was used to determine if the presence of bacteria affected IgA levels which bound TSST-1 and SEC . For 3/5 samples with levels above the upper limit of the standard deviation (2375 ng ml(-1)) for IgA bound to TSST-1, S . aureus was isolated from the mother whilst 4/5 samples found to contain levels above the upper limit of the standard deviation (2627 ng ml(-1)) for IgA bound to SEC, had S . aureus isolated from the mother . In conclusion, if bacterial toxins do play a role in precipitating a SIDS death, the presence of IgA antibodies to toxins in breast milk, but not in infant formula, might contribute to the protective effect of breast feeding in relation to SIDS.

FEMS Immunol Med Microbiol, 1999 Aug 1, 25(1-2), 167 - 73
The protective effect of breast feeding in relation to sudden infant death syndrome (SIDS): II . The effect of human milk and infant formula preparations on binding of Clostridium perfringens to epithelial cells; Gordon AE et al.; Breast feeding is known to protect an infant against gastrointestinal pathogens and epidemiological studies indicate that compared to breast fed infants, formula fed infants are at a greater risk of dying from sudden infant death syndrome (SIDS) . Many SIDS infants have symptoms of gastrointestinal infections prior to death and one gastrointestinal pathogen associated with SIDS is Clostridium perfringens . Studies have found that a significantly higher number of formula fed SIDS infants have C perfringens and its enterotoxin in their faeces compared to breast fed infants . The aim of the study was to compare the effects of human milk and infant formula on binding of C perfringens to epithelial cells . Two protocols were used to assess the effect of human milk and infant formula to inhibit binding of C perfringens to epithelial cells . Binding was assessed by flow cytometry . For the in vivo protocol which more closely represents interactions on the mucosal surface, breast milk enhanced bacterial binding but infant formula caused inhibition of binding; however for the in vitro method, both human milk and infant formula resulted in consistent enhancement of binding . Flow cytometry studies indicated that enhancement of binding was due to the formation of bacterial aggregates . Lewis(a) and Lewis(b) antigens, found in both breast milk and infant formula, inhibited C . perfringens binding in a dose dependent manner . The Lewis(a) and Lewis(b) antigens in human milk and infant formula can inhibit C . perfringens binding to epithelial cells . While infant formula reduced binding of C . perfringens to epithelial cells in the experiments carried out with the in vivo protocol, the protective effects of breast feeding in relation to colonisation with C . perfringens are more likely to be due to formation of bacterial aggregates . These findings have implications for improving infant formula preparations.

Biochem Biophys Res Commun, 1999 Aug 11, 261(3), 885 - 9
A {2Fe-2S} protein from the hyperthermophilic bacterium Aquifex aeolicus; Chatelet C et al.; Overexpression in Escherichia coli of the fdx4 gene from Aquifex aeolicus has allowed isolation and characterization of the first hyperthermophilic {2Fe-2S}(Scys)(4) protein, a homodimer of M = 2 x 12.4 kDa with one {2Fe-2S} cluster per subunit . This protein is undamaged by heating to 100 degrees C for at least three hours . The primary structure, in particular the characteristic distribution of the four cysteine ligands of the metal site, and the spectroscopic properties of the A . aeolicus protein relate it to well characterized {2Fe-2S} proteins from Clostridium pasteurianum and Azotobacter vinelandii . These proteins are also homologous to subunits or domains of hydrogenases and NADH-ubiquinone oxidoreductase (Complex I) of respiratory chains . The A . aeolicus {2Fe-2S} protein is thus representative of a presumably novel protein fold involved in a variety of functions in very diverse cellular backgrounds .

Biotechnol Prog, 1999 Jul-Aug, 15(4), 594 - 602
Production of acetone butanol ethanol (ABE) by a hyper-producing mutant strain of Clostridium beijerinckii BA101 and recovery by pervaporation; Qureshi N et al.; A silicone membrane was used to study butanol separation from model butanol solutions and fermentation broth . Depending upon the butanol feed concentration in the model solution and pervaporation conditions, butanol selectivities of 20.88-68.32 and flux values of 158.7-215.4 g m(-)(2) h(-)(1) were achieved . Higher flux values (400 g m(-)(2) h(-)(1)) were obtained at higher butanol concentrations using air as sweep gas . In an integrated process of butanol fermentation-recovery, solvent productivities were improved to 200% of the control batch fermentation productivities . In a batch reactor the hyper-butanol-producing mutant strain C . beijerinckii BA101 utilized 57.3 g/L glucose and produced 24.2 g/L total solvents, while in the integrated process it produced 51.5 g/L (culture volume) total solvents . Concentrated glucose medium was also fermented . The C . beijerinckii BA101 mutant strain was not negatively affected by the pervaporative conditions . In the integrated experiment, acids were not produced . With the active fermentation broth, butanol selectivity was reduced by a factor of 2-3 . However, the membrane flux was not affected by the active fermentation broth . The butanol permeate concentration ranged from 26.4 to 95.4 g/L, depending upon butanol concentration in the fermentation broth . Since the permeate of most membranes contains acetone, butanol, and ethanol (and small concentrations of acids), it is suggested that distillation be used for further purification.

J Bacteriol, 1999 Aug, 181(16), 5119 - 22
Cloning, characterization, and expression of a novel gene encoding a reversible 4-hydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum; Huang J et al.; A novel gene, designated ohb1, which encodes the oxygen-sensitive and biotin-, ATP-, thiamin-, pyridoxal phosphate-, and metal-ion-independent, reversible 4-hydroxybenzoate decarboxylase (4-HOB-DC) from the obligate anaerobe Clostridium hydroxybenzoicum JW/Z-1(T) was sequenced (GenBank accession no . AF128880) and expressed . The 1,440-bp open reading frame (ORF) (ohb1) encodes 480 amino acids . Major properties of the heterologous enzyme (Ohb1) expressed in Escherichia coli DH5alpha were the same as those described for the native 4-HOB-DC (Z . He and J . Wiegel, J . Bacteriol . 178:3539-3543, 1996) . The deduced amino acid sequence shows up to 57% identity and up to 74% similarity to hypothetical proteins deduced from ORFs in genomes from bacteria and archaea, suggesting a possible novel gene family.

Scand J Gastroenterol, 1999 Jun, 34(6), 587 - 90
Prognosis of adult-onset idiopathic bile acid malabsorption; Rossel P et al.; BACKGROUND: From 1986 to 1993, 150 patients were investigated with the 75Se-homocholic acid taurine (SeHCAT) test as a late step in the investigation of chronic diarrhoea . On basis of low SeHCAT values and response to cholestyramine treatment, 33 patients were initially classified as having idiopathic bile acid malabsorption (IBAM) . The aim was to describe the long-term clinical course of the disease and to assess the reliability of the SeHCAT test in diagnosing IBAM . METHODS: The methods included 1) clinical follow-up with patient interview combined with information from medical records and 2) repeated SeHCAT test . RESULTS: The diagnosis of IBAM had to be revised in three cases (inflammatory bowel disease in two patients, Clostridium difficile infection in one) . Six patients were lost to follow-up and a further four patients were excluded from re-examination either because of old age (>80 years) or bowel resection, leaving 20 patients for re-examination, of which 16 completed both clinical follow-up and a new SeHCAT test . The median duration of symptoms before initial SeHCAT test was 2.5 (1-30) years . In 13 of 16 patients symptoms persisted, and SeHCAT values remained low and almost identical to the initial value after a median observation time of 88 (51-113) months . Despite initial response to treatment with cholestyramine, six patients had to discontinue treatment because of adverse effects or other compliance problems . In three patients the SeHCAT value showed a considerable increase, and bowel function had correspondingly normalized in these cases . CONCLUSION: The study confirms the reliability of the SeHCAT test in diagnosing IBAM . Despite adult onset of symptoms, only a few patients improve after several years' observation . Treatment with cholestyramine is generally effective but not always tolerated.

Microbiology, 1999 Jul, 145 ( Pt 7), 1683 - 93
Suppression of toxin production in Clostridium difficile VPI 10463 by amino acids; Karlsson S et al.; The impact of various growth conditions on the expression of toxins and other proteins by Clostridium difficile VPI 10463 was studied . During non-starved conditions, the rate of toxin synthesis paralleled that of total protein during both exponential growth and stationary phase, and in both defined and complex media . Biotin limitation reduced growth rate and bulk protein synthesis, whereas toxin expression continued, leading to a 50- to 200-fold increase in intracellular toxin levels . Concomitantly, several 22 kDa proteins were up-regulated as revealed by two-dimensional PAGE analysis . The toxin yield was 30-fold higher in peptone yeast extract (PY) than in PY containing glucose (PYG) . By contrast, glucose limitation reduced toxin yields by 20- to 100-fold in defined media . By elevating the buffering capacity and bicarbonate concentration, toxin yields were increased by 10-fold in PY and PYG . The high toxin production by C . difficile during growth in PY was lowered 100-fold by adding a blend of nine amino acids and several 60-100 kDa proteins were concomitantly down-regulated . It was concluded that toxin expression in C . difficile VPI 10463 was not affected by growth rate, growth phase, catabolite repression or the stringent response . Instead the co-expression of toxins and a few specific additional proteins appeared to be influenced by metabolic pathways involving CO2 assimilation, carboxylation reactions and metabolism of certain amino acids.

J Biol Inorg Chem, 1999 Jun, 4(3), 311 - 7
The {2Fe-2S} protein I (Shetna protein I) from Azotobacter vinelandii is homologous to the {2Fe-2S} ferredoxin from Clostridium pasteurianum; Chatelet C et al.; The {2Fe-2S} protein from Azotobacter vinelandii that was previously known as iron-sulfur protein I, or Shethna protein I, has been shown to be encoded by a gene belonging to the major nif gene cluster . Overexpression of this gene in Escherichia coli yielded a dimeric protein of which each subunit comprises 106 residues and contains one {2Fe-2S} cluster . The sequence of this protein is very similar to that of the {2Fe-2S} ferredoxin from Clostridium pasteurianum (2FeCpFd), and the four cysteine ligands of the {2Fe-2S} cluster occur in the same positions . The A . vinelandii protein differs from the C . pasteurianum one by the absence of the N-terminal methionine, the presence of a five-residue C-terminal extension, and a lesser number of acidic and polar residues . The UV-visible absorption and EPR spectra, as well as the redox potentials of the two proteins, are nearly identical . These data show that the A . vinelandii FeS protein I, which is therefore proposed to be designated 2FeAvFdI, is the counterpart of the {2Fe-2S} ferredoxin from C . pasteurianum . The occurrence of the 2FeAvFdI-encoding gene in the nif gene cluster, together with the previous demonstration of a specific interaction between the 2FeCpFd and the nitrogenase MoFe protein, suggest that both proteins might be involved in nitrogen fixation, with possibly similar roles.

J Chemother, 1999 Jun, 11(3), 215 - 9
Successful treatment of cytomegalovirus colitis with ganciclovir in a patient with adult T cell leukemia lymphoma: case report; Oshima Y et al.; An 84-year-old patient with adult T cell leukemia lymphoma (ATLL) developed diarrhea on day 5 of chemotherapy and was diagnosed with cytomegalovirus (CMV) colitis . Sigmoidoscopy revealed multiple superficial erosions surrounded by a flare . Computed tomography (CT) and ultrasonogram of the abdomen revealed marked thickening of the colonic mucosa . There were 186 CMV antigen-positive leukocytes per 31,000 white blood cells (WBC) . A colonic biopsy specimen showed typical CMV nuclear inclusions . Immunohistological study of the specimen was positive for CMV antigen . Administration of ganciclovir (DHPG) 500 mg/day for 14 days improved the diarrhea and other symptoms . On day 30 of the chemotherapy, the patient developed diarrhea again but was diagnosed with pseudomembranous colitis instead of CMV colitis . At that time, CMV antigenemia and a histologic study for CMV were negative . The stool was positive for Clostridium difficile toxin antigen . ATLL patients are believed to be immunocompromised hosts and often develop opportunistic infections such as CMV infection . Most suffer from CMV pneumonia at the end of their course of therapy . Few gastrointestinal (GI) CMV infections are seen in ATLL patients and details of CMV colitis have never been reported . When an ATLL patient develops diarrhea that barely responds to conventional therapy, CMV colitis and pseudomembranous colitis should be listed in the differential diagnosis.

Yakugaku Zasshi, 1999 Jul, 119(7), 472 - 94
{Development from actions of bacterial phospholipases C on eucaryotic plasma membranes to molecular biology of GPI-anchored proteins}; Ikezawa H; Bacterial phospholipases C are known to act on biomembranes, since they can cleave the phosphodiester linkage between the polar head and the hydrophobic moiety of each phospholipid in these membranes . These enzymes have been classified into three groups; phosphatidylcholine (PC)-, sphingomyelin (SM)- and phosphatidylinositol (PI)-degrading phospholipases C . Enzymatic properties and toxicities of these phospholipases C are reviewed, in relation to author's research . Studies on the hemolytic phospholipases of Clostridium sp., Bacillus cereus etc., revealed that hydrolysis of choline-containing phospholipids such as PC and SM was responsible for the hemolysis of mammalian erythrocytes by these enzymes in the presence of Ca2+ and/or Mg2+ . Also, the studies on a structure-activity correlation of SM-hydrolyzing phospholipase C from B . cereus disclosed the similarity of active sites between this enzyme and bovine pancreatic DNase I . By action of PI-degrading phospholipases C, several membrane proteins such as alkaline phosphatase, 5'-nucleotidase, VSG (protozoal surface glycoprotein) etc., were shown to be released from the plasma membranes of eucaryotic cells . From structural analysis, these proteins have been revealed to be glycosylphosphatidylinositol (GPI)-anchored proteins bound to the plasma membranes with carboxyl terminal-attached glycolipid . Biochemistry and molecular biology of GPI-anchored proteins, including the structures and biosynthetic routes of GPI glycolipids as well as the process of GPI attachment to proteins, requirements of C-terminal signal peptide for the protein modification by GPI, and distribution of GPI-anchored proteins in living world, are described in relation to our studies.

Ugeskr Laeger, 1999 May 3, 161(18), 2678 - 80
{Acute hemolytic anemia in Clostridium perfringens infection}; Duun EH et al.; Haemolytic anaemia caused by Clostridium perfringens is a rare complication in patients with neoplastic diseases . According to the literature, our patient seems to be the first patient with underlying malignancy (multiple myeloma) who has survived C . perfringens septicaemia complicated with acute haemolysis and acute anuria . It is concluded that treatment with penicillin should be considered in case of acute haemolytic anaemia, if the patient is febrile and no other obvious cause of the haemolytic condition can be found.

Biochemistry, 1999 Aug 3, 38(31), 9956 - 63
Imaging real-time proteolysis of single collagen I molecules with an atomic force microscope; Lin H et al.; The dynamic process of synthesis and degradation of extracellular matrix molecules, including various collagens, is important in normal physiological functions and pathological conditions . Existing models of collagen enzymatic degradation reactions are derived from bulk biochemical assays . In this study, we have imaged in real-time individual collagen I molecules and their proteolysis by Clostridium histolyticum collagenases in phosphate-buffered saline (PBS) with atomic force microscopy (AFM) . We have also imaged the likely binding and unbinding of collagenase molecules to single triple-helical collagen I molecules and subsequent proteolysis of subsets of the collagen molecules . The proteolysis of collagen molecules was inhibited by reduced calcium and acidification . Results from AFM study of collagen proteolysis are consistent with SDS-PAGE biochemical assays . The real-time proteolysis of single collagen I molecules followed simple Michaelis-Menton kinetics previously derived from bulk biochemical assays . This is the first report of imaging real-time proteolysis of single macromolecules and its inhibition on a molecular scale . A strong correspondence between the kinetics of proteolysis of single collagen molecules and the kinetics of proteolysis derived from bulk biochemical assays will have a wide applicability in examining real-time enzymatic reactions and their regulation at single molecule structural level . Such real-time study of single molecule proteolysis could provide a better understanding of the interactions between proteases and target proteins as well as proteases and protease inhibitors.

Lett Appl Microbiol, 1999 Jul, 29(1), 15 - 9
PCR detection of Clostridium perfringens type D in formalin-fixed, paraffin-embedded tissues of goats and sheep; Warren AL et al.; A polymerase chain reaction (PCR) was used to identify the genes encoding the alpha, epsilon and beta toxins of Clostridium perfringens in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep . When pure cultures of Cl . perfringens types B and D were used as control templates in the PCR, products of the following sizes were observed on the agarose gel: 247 bp (alpha primers), 1025 bp (beta primers) and 403 bp (epsilon primers) . When used to identify Cl . perfringens type D in formalin-fixed, paraffin-embedded intestinal tissues of goats and sheep, the PCR technique resulted in the detection of this micro-organism in 11 out of 13 samples known to be infected with Cl . perfringens . No false positive results were obtained when 13 culturally negative samples were analysed by the PCR technique.

Biochem J, 1999 Aug 15, 342 ( Pt 1), 105 - 10
Homologous xylanases from Clostridium thermocellum: evidence for bi-functional activity, synergism between xylanase catalytic modules and the presence of xylan-binding domains in enzyme complexes; Fernandes AC et al.; Clostridium thermocellum produces a consortium of plant-cell-wall hydrolases that form a cell-bound multi-enzyme complex called the cellulosome . In the present study two similar xylanase genes, xynU and xynV, were cloned from C . thermocellum strain YS and sequenced . The deduced primary structures of both xylanases, xylanase U (XylU) and xylanase V (XylV), were homologous with the previously characterized xylanases from C . thermocellum strain F1 . Truncated derivatives of XylV were produced and their biochemical properties were characterized . The xylanases were shown to be remarkably thermostable and resistant to proteolytic inactivation . The catalytic domains hydrolysed xylan by a typical endo-mode of action . The type VI cellulose-binding domain (CBD) homologue of XylV bound xylan and, to a smaller extent, Avicel and acid-swollen cellulose . Deletion of the CBD from XylV abolished the capacity of the enzymes to bind polysaccharides . The polysaccharide-binding domain was shown to have a key role in the hydrolysis of insoluble substrates by XylV . The C-terminal domain of XylV, which is absent from XylU, removed acetyl groups from acetylated xylan and acted in synergy with the glycosyl hydrolase catalytic domain of the enzyme to elicit the hydrolysis of acetylated xylan.

Proc Natl Acad Sci U S A, 1999 Aug 3, 96(16), 9190 - 5
A chimeric prokaryotic ancestry of mitochondria and primitive eukaryotes; Karlin S et al.; We provide data and analysis to support the hypothesis that the ancestor of animal mitochondria (Mt) and many primitive amitochondrial (a-Mt) eukaryotes was a fusion microbe composed of a Clostridium-like eubacterium and a Sulfolobus-like archaebacterium . The analysis is based on several observations: (i) The genome signatures (dinucleotide relative abundance values) of Clostridium and Sulfolobus are compatible (sufficiently similar) and each has significantly more similarity in genome signatures with animal Mt sequences than do all other available prokaryotes . That stable fusions may require compatibility in genome signatures is suggested by the compatibility of plasmids and hosts . (ii) The expanded energy metabolism of the fusion organism was strongly selective for cementing such a fusion . (iii) The molecular apparatus of endospore formation in Clostridium serves as raw material for the development of the nucleus and cytoplasm of the eukaryotic cell.

Am J Prev Med, 1999 Jul, 17(1), 48 - 54
Seafood-associated disease outbreaks in New York, 1980-1994; Wallace BJ et al.; BACKGROUND: Seafood-associated disease outbreaks in New York were examined to describe their epidemiology and to identify areas for prevention and control efforts . METHODS: We reviewed reports submitted to the New York State Department of Health (NYSDOH) of seafood-associated outbreaks occurring from January 1, 1980, through December 31, 1994 . RESULTS: During 1980-1994, 339 seafood-associated outbreaks were reported, resulting in 3959 illnesses, 76 hospitalizations, and 4 deaths . During this period, seafood-associated outbreaks accounted for 19% of all reported foodborne outbreaks and 10% of foodborne illnesses . Shellfish, the most frequently implicated seafood item, accounted for 64% of seafood outbreaks, followed by finfish (31% of outbreaks) . Of the 148 seafood-associated outbreaks with a confirmed etiologic agent, Norwalk virus and scombrotoxin were the most frequently identified agents: Norwalk virus accounted for 42% of outbreaks and 42% of illnesses, and scombrotoxin accounted for 44% of outbreaks and 19% of illnesses . Three of the 4 seafood-associated deaths were caused by Clostridium botulinum; the remaining death was caused by Vibrio vulnificus . CONCLUSIONS: Reducing the number of seafood outbreaks will require continued and coordinated efforts by many different agencies, including those involved with water quality; disease surveillance; consumer education; and seafood harvesting, processing, and marketing . New York's foodborne disease surveillance data highlight potential areas on which to focus prevention efforts, including: (1) commodities and associated pathogens causing the largest number of seafood-associated outbreaks and illnesses, namely shellfish-associated viral gastroenteritis and finfish-associated scombroid fish poisoning, and (2) venues at which seafood were most frequently consumed in reported outbreaks, such as commercial food establishments and catered events.

Eur J Biochem, 1999 Jul, 263(1), 178 - 88
Structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium cochlearium; Hoffmann B et al.; Glutamate mutase (Glm) is an adenosylcobamide-dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S, 3S)-3-methylaspartate . The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an alpha2beta2 tetramer, whose assembly is mediated by coenzyme B12 . The smaller of the protein components, GlmS, has been suggested to be the B12-binding subunit . Here we report the solution structure of GlmS, determined from a heteronuclear NMR-study, and the analysis of important dynamical aspects of this apoenzyme subunit . The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C . tetanomorphum, which has previously been investigated using NMR-spectroscopy . Both solution structures of the two Glm B12-binding subunits share striking similarities with that determined by crystallography for the B12-binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii, which is B12 bound . In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide . However, in GlmS and MutS the sequence motif, Asp-x-His-x-x-Gly, which includes the cobalt-coordinating histidine residue, and a predicted alpha-helical region following the motif, are present as an unstructured and highly mobile loop . In the absence of coenzyme, the B12-binding site apparently is only partially formed . By comparing the crystal structure of Mcm with the solution structures of B12-free GlmS and MutS, a consistent picture on the mechanism of B12 binding has been obtained . Important elements of the binding site only become structured upon binding B12; these include the cobalt-coordinating histidine residue, and an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the coenzyme.

Appl Environ Microbiol, 1999 Aug, 65(8), 3449 - 57
A predictive model that describes the effect of prolonged heating at 70 to 90 degrees C and subsequent incubation at refrigeration temperatures on growth from spores and toxigenesis by nonproteolytic Clostridium botulinum in the presence of lysozyme; Fernandez PS et al.; Refrigerated processed foods of extended durability such as cook-chill and sous-vide foods rely on a minimal heat treatment at 70 to 95 degrees C and then storage at a refrigeration temperature for safety and preservation . These foods are not sterile and are intended to have an extended shelf life, often up to 42 days . The principal microbiological hazard in foods of this type is growth of and toxin production by nonproteolytic Clostridium botulinum . Lysozyme has been shown to increase the measured heat resistance of nonproteolytic C . botulinum spores . However, the heat treatment guidelines for prevention of risk of botulism in these products have not taken into consideration the effect of lysozyme, which can be present in many foods . In order to assess the botulism hazard, the effect of heat treatments at 70, 75, 80, 85, and 90 degrees C combined with refrigerated storage for up to 90 days on growth from 10(6) spores of nonproteolytic C . botulinum (types B, E, and F) in an anaerobic meat medium containing 2,400 U of lysozyme per ml (50 microg per ml) was studied . Provided that the storage temperature was no higher than 8 degrees C, the following heat treatments each prevented growth and toxin production during 90 days; 70 degrees C for >/=2,545 min, 75 degrees C for >/=463 min, 80 degrees C for >/=230 min, 85 degrees C for >/=84 min, and 90 degrees C for >/=33.5 min . A factorial experimental design allowed development of a predictive model that described the incubation time required before the first sample showed growth, as a function of heating temperature (70 to 90 degrees C), period of heat treatment (up to 2,545 min), and incubation temperature (5 to 25 degrees C) . Predictions from the model provided a valid description of the data used to generate the model and agreed with observations made previously.

Appl Environ Microbiol, 1999 Aug, 65(8), 3287 - 92
Phylogeny of the defined murine microbiota: altered Schaedler flora; Dewhirst FE et al.; The "altered Schaedler flora" (ASF) was developed for colonizing germfree rodents with a standardized microbiota . The purpose of this study was to identify each of the eight ASF strains by 16S rRNA sequence analysis . Three strains were previously identified as Lactobacillus acidophilus (strain ASF 360), Lactobacillus salivarius (strain ASF 361), and Bacteroides distasonis (strain ASF 519) based on phenotypic criteria . 16S rRNA analysis indicated that each of the strains differed from its presumptive identity . The 16S rRNA sequence of strain ASF 361 is essentially identical to the 16S rRNA sequences of the type strains of Lactobacillus murinis and Lactobacillus animalis (both isolated from mice), and all of these strains probably belong to a single species . Strain ASF 360 is a novel lactobacillus that clusters with L . acidophilus and Lactobacillus lactis . Strain ASF 519 falls into an unnamed genus containing {Bacteroides} distasonis, {Bacteroides} merdae, {Bacteroides} forsythus, and CDC group DF-3 . This unnamed genus is in the Cytophaga-Flavobacterium-Bacteroides phylum and is most closely related to the genus Porphyromonas . The spiral-shaped strain, strain ASF 457, is in the Flexistipes phylum and exhibits sequence identity with rodent isolates of Robertson . The remaining four ASF strains, which are extremely oxygen-sensitive fusiform bacteria, group phylogenetically with the low-G+C-content gram-positive bacteria (Firmicutes, Bacillus-Clostridium group) . ASF 356, ASF 492, and ASF 502 fall into Clostridium cluster XIV of Collins et al . Morphologically, ASF 492 resembles members of this cluster, Roseburia cecicola, and Eubacterium plexicaudatum . The 16S rRNA sequence of ASF 492 is identical to that of E . plexicaudatum . Since the type strain and other viable original isolates of E . plexicaudatum have been lost, strain ASF 492 is a candidate for a neotype strain . Strain ASF 500 branches deeply in the low-G+C-content gram-positive phylogenetic tree but is not closely related to any organisms whose 16S rRNA sequences are currently in the GenBank database . The 16S rRNA sequence information determined in the present study should allow rapid identification of ASF strains and should permit detailed analysis of the interactions of ASF organisms during development of intestinal disease in mice that are coinfected with a variety of pathogenic microorganisms.

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1201 - 9
Clostridium methoxybenzovorans sp . nov., a new aromatic o-demethylating homoacetogen from an olive mill wastewater treatment digester; Mechichi T et al.; A strictly anaerobic, spore-forming bacterium (3.0-5.0 x 0.4-0.8 microns), designated strain SR3T (T = type strain), which stained Gram-positive and possessed a Gram-positive type cell wall was isolated from a methanogenic pilot-scale digester fed with olive mill wastewater (Sfax, Tunisia) . It utilized a number of carbohydrates (glucose, fructose, sorbose, galactose, myo-inositol, sucrose, lactose, cellobiose), organic compounds (lactate, betaine, sarcosine, dimethylglycine, methanethiol, dimethylsulfide), alcohol (methanol) and all methoxylated aromatic compounds only in the presence of yeast extract (0.1%) . The end products from carbohydrate fermentation were H2, CO2, formate, acetate and ethanol, that from lactate was methanol, those from methoxylated aromatics were acetate and butyrate, and that from betaine, sarcosine, dimethylglycine, methanethiol and dimethylsulfide was only acetate . Strain SR3T was non-motile, had a G+C content of 44 mol% and grew optimally at 37 degrees C and pH 7.4 on a glucose-containing medium . Phylogenetically, the closest relatives of strain SR3T were the non-methoxylated aromatic-degrading Clostridium xylanolyticum, Clostridium aerotolerans, Clostridium sphenoides and Clostridium celerecrescens (mean similarity of 98%) . On the basis of the phenotypic, genotypic and phylogenetic characteristics of the isolate, it is proposed to designate strain SR3T as Clostridium methoxybenzovorans sp . nov . The type strain is SR3T (= DSM 12182T).

Int J Syst Bacteriol, 1999 Jul, 49 Pt 3, 1119 - 24
Phylogenetic status of Anaerobacter polyendosporus, an anaerobic, polysporogenic bacterium; Siunov AV et al.; The almost complete sequence of the 16S rRNA gene of the Gram-positive polysporogenic bacterium Anaerobacter polyendosporus was determined . This allowed phylogenetic analysis of A . polyendosporus by comparing sequences of the 16S rRNA gene of this bacterium to similar genes of other Gram-positive bacteria . It was shown that this polysporogenic bacterium belongs to the Clostridium cluster I, subcluster A . Phylogenetically, A . polyendosporus is distantly related to another polysporogenic, but non-cultivatable, bacterium, 'Metabacterium polyspora' and can be satisfactorily clustered within the saccharolytic clostridia with a low DNA G+C content grouped in subcluster A . A . polyendosporus was most closely related to Clostridium intestinale (94.8% identity of 16S rRNA genes) and Clostridium fallax (93.1%) . Like other members of the Clostridium cluster I, subcluster A, A . polyendosporus possesses such common phenotypic features as a Gram-positive cell wall structure, anaerobiosis, derivation of energy from carbohydrate fermentation yielding butyric acid among other organic acids and the capacity for endogenous spore-formation . However, the scale of evolutionary change in the 16S rRNA gene between A . polyendosporus and phylogenetically related Clostridium species does not correspond to the profound changes in the phenotype of A . polyendosporus . Distinctive phenotypic features of the latter are large cell size, polysporogenesis (up to seven spores per cell), alternative modes of development and an unusual membrane ultrastructure.

Structure Fold Des, 1999 Jul 15, 7(7), 769 - 82
The structure of bovine glutamate dehydrogenase provides insights into the mechanism of allostery; Peterson PE et al.; BACKGROUND: Bovine glutamate dehydrogenase (boGDH) is a homohexameric, mitochondrial enzyme that reversibly catalyzes the oxidative deamination of L-glutamate to 2-oxoglutarate using either NADP(H) or NAD(H) with comparable efficacy . GDH represents a key enzymatic link between catabolic and biosynthetic pathways, and is therefore ubiquitous in both higher and lower organisms . Only mammalian GDH exhibits strong negative cooperativity with respect to the coenzyme, however, and is regulated by a large number of allosteric effectors . RESULTS: The atomic structure of boGDH in complex with NADH, glutamate, and the allosteric inhibitor GTP has been determined to 2.8 A resolution . The major difference between the bacterial and bovine GDH structures is the presence of an additional 'antenna' in boGDH that protrudes from each trimer, twisting counterclockwise along the threefold axis . NADH and glutamate are clearly observed in the active site, but the contacts differ slightly from those observed in Clostridium symbiosum GDH . A second, inhibitory NADH molecule lies buried in the core of the hexamer . Finally, two GTP molecules bind near the hinge region connecting the NAD(+)- and glutamate-binding domains . CONCLUSIONS: We propose that the antenna serves as an intersubunit communication conduit during negative cooperativity and allosteric regulation . GTP and NADH inhibit GDH by keeping the catalytic cleft in a closed conformation . In contrast, ADP probably binds to the back of the NAD(+)-binding domain and activates the enzyme by keeping the catalytic cleft open . Extensive contacts between antennae within the crystal lattice may represent hexamer interactions in solution and, perhaps, with other enzymes within the mitochondrial matrix.

Crit Rev Immunol, 1999, 19(3), 219 - 60
Structure, activity, and immune (T and B cell) recognition of botulinum neurotoxins; Atassi MZ et al.; Botulism, which was first reported over a century ago, is caused by botulinum neurotoxins produced by Clostridium botulinum in seven immunological serotypes (A through G) . The primary structures of a number of these BoNTs have been determined and are reviewed here, together with their gene structure and synthesis . The biological actions of BoNTs, which result in their ability to block neurotransmitter release have been the subject of intensive study, and in this review we discuss the binding of BoNTs to the cell surface as well as the mechanism of their intercellular action . The ability of BoNTs to block neurotransmitter release has been exploited in therapeutic applications to reduce muscle hyperactivity for the treatment of a variety of clinical conditions associated with involuntary muscle spasm and contractions . The advantages, limitations, and risks of these applications are discussed . Certain compounds provide some limited protection against BoNT . However, more effective protection has been obtained immunologically either by passive immunity (i.e., by administration of anti-BoNT Abs) or by immunization with inactivated toxin . More recently, excellent protection has been obtained by immunization with the receptor-binding region comprising the C-terminal (residues 860 to 1296) fragment (Hc) of the heavy chain of BoNT/A . Here we review the mapping of the epitopes on the Hc region of BoNT/A that are recognized by anti-BoNT/A Abs raised in horse, human, and mouse . The epitopes on the Hc that are recognized by anti-Hc Abs and by Hc-primed T lymphocytes were mapped in two mouse strains {BALB/c (H-2d) and SJL (H-2s)} . The peptides, which contain Ab or T cell epitopes (or both) on the Hc, were used as immunogens in BALB/c and SJL mice and we identified those peptides whose Ab and/or T-cell response cross-react with Hc . Identification of these peptides is an important first step in the intricate requirements for the design of a synthetic vaccine.

Bacteriol Virusol Parazitol Epidemiol, 1998 Oct-Dec, 43(4), 241 - 5
{Acute infectious diarrheal disease in Romania: 1993-1998}; Popa MI et al.; The decrease of morbidity-mortality caused by gastroenteritis is in relation to the factors largely responsible for the fall in infant mortality and mortality from communicable diseases in developing countries . Nevertheless, diarrhea is still a considerable public health problem in these countries, especially among children under 5 years old . 98% of all deaths in children younger than 15 years are in the developing world . Five of the ten leading killers are communicable, perinatal, and nutritional disorders largely affecting children . The knowledge of the etiology and epidemiology of childhood diarrhea in a given area is needed to plan any measure designed to prevent or ameliorate diarrheal illness and to develop practical guidelines for the most appropriate examination procedures . In Romania, although the real data of morbidity by acute diarrhea are not known, the reports show a significant decrease in the past 10 years . In 1993, 420.2 cases at 100,000 inhabitants were reported, the most commonly affected being the children age 0-4 years . The incidence decreased to 338.5 cases at 100,000 inhabitants in 1997 (and a quite similar incidence for the first 11 month of 1998) . Between 1993-1998, 527,977 cases were reported (58.1% in urban area), with a higher frequency in spring-autumn season . Antibiotics are not required in case of acute diarrhea with little or no fever . Antibiotics could be discussed for cholera-like diarrhea and are required in case of invasive bacterial diarrhea, shigellosis, cholera, and Clostridium difficile as well as diarrhea with fever and sanguinolent stools in infants or salmonella-induced diarrhea with signs of extradigestive complications . Importance of oral rehydration solution in the treatment of diarrhoeal diseases is well known . It can be applied to all types of diarrhoea, practically, without any side effects, complications, such aas hypernatraemia is avoidable . It has proved to be effective for dehydration caused by diarrhoea and for diarrhoea, too . There is a need for effective infection control policies, which include appropriate training of staff; simple surveillance systems and readily available expert advice to ensure that outbreaks are rapidly controlled . Approaches to prevention include education about risk factors, which often fails to lead to modification of risky behavior . Further regional epidemiological studies are necessary to develop more appropriate management guidelines.

Appl Microbiol Biotechnol, 1999 Jun, 51(6), 852 - 9
Duplicated Clostridium thermocellum cellobiohydrolase gene encoding cellulosomal subunits S3 and S5; Zverlov VV et al.; The upstream region of the cellobiohydrolase gene cbhA of Clostridium thermocellum F7 was sequenced . It was found that this region contains the previously sequenced gene celK encoding an enzyme closely related to CbhA (cellulosomal subunit S3) . The presence of a putative transcription terminator in the 524-bp intergenic region indicates that celK and cbhA are not cotranscribed as an operon . Sequence comparison between the two cellobiohydrolases revealed high sequence conservation in the catalytic domain and in the N-terminal cellulose-binding domain (CBD) homologous to CBD family IV, which binds specifically to amorphous cellulose and soluble cellooligosaccharides . In contrast to CbhA, CelK lacks a family III CBD capable of binding to crystalline cellulose . By partial amino acid sequence determination CelK was shown to be identical to cellulosomal subunit S5 . CelK and CbhA were found to be members of subfamily E1 of cellulase family E (glycosylhydrolase family 9) . Sequence comparison of catalytic domains of family E1 cellulases with C . thermocellum CelD, a family E1 endoglucanase of known three-dimensional structure, revealed a significant variation in the lengths of substrate-binding loops connecting the helices of the (alpha/alpha)6 barrel fold . The extended loops of CelK and CbhA might form an active-site tunnel, as found in the catalytic domains of fungal cellobiohydrolases.

J Bacteriol, 1999 Aug, 181(15), 4526 - 32
Cloning, sequence, and transcriptional regulation of the operon encoding a putative N-acetylmannosamine-6-phosphate epimerase (nanE) and sialic acid lyase (nanA) in Clostridium perfringens; Walters DM et al.; Clostridium perfringens can obtain sialic acid from host tissues by the activity of sialidase enzymes on sialoglycoconjugates . After sialic acid is transported into the cell, sialic acid lyase (NanA) then catalyzes the hydrolysis of sialic acid into pyruvate and N-acetylmannosamine . The latter is converted for use as a biosynthetic intermediate or carbohydrate source in a pathway including an epimerase (NanE) that converts N-acetylmannosamine-6-phosphate to N-acetylglucosamine-6-phosphate . A 4.0-kb DNA fragment from C . perfringens NCTC 8798 that contains the nanE and nanA genes has been cloned . The identification of the nanA gene product as sialic acid lyase was confirmed by overexpressing the gene and measuring sialic acid lyase activity in a nanA Escherichia coli strain, EV78 . The nanA gene product was also shown to restore growth to EV78 in minimal medium with sialic acid as the sole carbon source . By using Northern blot experiments, it was demonstrated that the nanE and nanA genes comprise an operon and that transcription of the operon in C . perfringens is inducible by the addition of sialic acid to the growth medium . The Northern blot experiments also showed that there is no catabolite repression of nanE-nanA transcription by glucose . With a plasmid construct containing a promoterless cpe-gusA gene fusion, in which beta-glucuronidase activity indicated that the gusA gene acted as a reporter for transcription, a promoter was localized to the region upstream of the nanE gene . Primer extension experiments then allowed us to identify a sialic acid-inducible promoter located 30 bp upstream of the nanE coding sequence.

J Biol Chem, 1999 Jul 30, 274(31), 21926 - 31
3-Hydroxy-3-methylglutaryl-CoA reductase inhibitors attenuate vascular smooth muscle proliferation by preventing rho GTPase-induced down-regulation of p27(Kip1); Laufs U et al.; The mechanism by which platelet-derived growth factor (PDGF) regulates vascular smooth muscle cell (SMC) DNA synthesis is unknown, but may involve isoprenoid intermediates of the cholesterol biosynthetic pathway . Inhibition of isoprenoid synthesis with the 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor, simvastatin (Sim, 1-10 microM), inhibited PDGF-induced SMC DNA synthesis by >95%, retinoblastoma gene product hyperphosphorylation by 90%, and cyclin-dependent kinases (cdk)-2, -4, and -6 activity by 80 +/- 5, 50 +/- 3, and 48 +/- 3%, respectively . This correlated with a 20-fold increase in p27(Kip1) without changes in p16, p21(Waf1), or p53 levels compared with PDGF alone . Since Ras and Rho require isoprenoid modification for membrane localization and are implicated in cell cycle regulation, we investigated the effects of Sim on Ras and Rho . Up-regulation of p27(Kip1) and inhibition of Rho but not Ras membrane translocation by Sim were reversed by geranylgeranylpyrophosphate, but not farnesylpyrophosphate . Indeed, inhibition of Rho by Clostridium botulinum C3 transferase or overexpression of dominant-negative N19RhoA mutant increased p27(Kip1) and inhibited retinoblastoma hyperphosphorylation . In contrast, activation of Rho by Escherichia coli cytotoxic necrotizing factor-1 decreased p27(Kip1) and increased SMC DNA synthesis . These findings indicate that the down-regulation of p27(Kip1) by Rho GTPase mediates PDGF-induced SMC DNA synthesis and suggest a novel direct effect of 3-hydroxy-3-methylglutaryl-CoA reductase inhibitors on the vascular wall.

J Food Prot, 1999 Jul, 62(7), 766 - 72
Psychrotrophic clostridia causing spoilage in cooked meat and poultry products; Kalinowski RM et al.; Certain types of commercially produced noncured turkey breast and roast beef are precooked in situ, stored at 4 degrees C or below, and typically given use by dates of greater than 50 days . While of rare, sporadic occurrence, an unpleasant spoilage characterized by strong H2S odor and gas production has been observed in these products . This spoilage is due to the growth of psychrotrophic anaerobic sporeformers . Isolates from roast beef resemble Clostridium laramie while isolates from uncured turkey have been designated C . ctm for cooked turkey meat . The turkey breast isolates were characterized by temperature growth ranges, carbohydrate fermentations, and other biochemical reactions . Growth of all isolates was inhibited in broth media by 3.0% NaCl, 100 ppm nitrite, 2.0% sodium lactate, or 0.2% sodium diacetate . Inoculated studies were performed with three isolates in cooked turkey product . All three isolates grew and spoiled product at 10 and 3.3 degrees C, and one isolate grew at 0.5 and -3 degrees C . Some differences in growth were observed with the lactate and diacetate treatments in turkey meat among the three isolates . One isolate appeared to utilize the lactate, two were inhibited . Overall, 0.1% diacetate consistently delayed growth, although to different degrees, for all isolates.

J Food Prot, 1999 Apr, 62(4), 349 - 55
Growth and toxin production by Clostridium botulinum in English-style crumpets packaged under modified atmospheres; Daifas DP et al.; To determine the safety of a high moisture bakery product, packaged under modified atmospheres, challenge studies were done on English-style crumpets (water activity {a(w)} 0.990, pH 6.5) inoculated postbaking with Clostridium botulinum types A and proteolytic B spores (5 X 10(2) spores/g) . Products were packaged either in air, in air with an Ageless FX200 oxygen absorbent, or in a CO2/N2 (60:40) gas mixture, stored at ambient temperature (25 degrees C), and monitored for toxicity daily . All inoculated crumpets were toxic within 4 to 6 days and were organoleptically acceptable at the time of toxigenesis . Counts of C . botulinum increased to approximately 10(5) CFU/g at the time of toxicity . To determine the effect of baking on product safety, subsequent challenge studies were done on crumpets inoculated with 5 x 10(2) spores/g (baked weight basis) prior to baking . All crumpets were toxic after only 6 days, irrespective of packaging conditions, and toxigenesis again preceded spoilage . Temperature profile studies showed that the maximum internal temperature reached during baking was 97 degrees C, and the total baking process was equivalent to 0.03 min at 121 degrees C . The actual time to toxin production in both studies (4 to 6 days) correlated well with the predicted time (3.4 days) using the U.S . Department of Agriculture Pathogen Modeling Program (version 5.1) for proteolytic strains of C . botulinum . These studies confirm that high moisture bakery products, if contaminated with C . botulinum spores either pre- or postbaking, could pose a public health hazard, if packaged in air (in a high gas barrier package where O2 was depleted and CO2 was generated during storage) or under modified atmosphere packaging conditions and stored at ambient temperature.

Vet Microbiol, 1999 Jun 30, 67(3), 231 - 7
Characterization of flagellin from Clostridium chauvoei; Kojima A et al.; Differential centrifugation and cesium chloride-equilibrium centrifugation were used to purify the flagella from the strain Okinawa of the formalin-fixed Clostridium chauvoei . SDS-PAGE profile of purified flagella showed that a major protein band with a molecular mass of 46 kDa, corresponding to the flagellin monomer, and at least two minor protein bands with molecular masses of approximately 73 and 100 kDa were found . The amino acid composition of C . chauvoei flagellin was similar to the flagellin of Salmonella typhimurium and Bacillus subtilis . In addition, C . chauvoei flagellin monomer shared limited sequence homology with the N-terminal amino acid sequence reported for other bacterial flagellins . N-terminal sequences of two minor bands corresponded to the flagellin monomer, indicating that higher molecular mass bands were polymeric forms of the flagellin monomer.

Acta Vet Scand, 1999, 40(1), 11 - 21
Comparison between a live, attenuated anticoccidial vaccine and an anticoccidial ionophore, on performance of broilers raised with or without a growth promoter, in an initially Eimeria-free environment; Waldenstedt L et al.; An experiment was carried out to study the effects of vaccination with Paracox, a live, attenuated vaccine against avian coccidiosis, on broilers isolated from extraneous Eimeria parasites . The study involved 3200 broiler chickens raised in floor pens similar to commercial conditions, but in an initially Eimeria-free environment . Forty percent of the chickens were vaccinated at 3 days of age and given either a basal unmedicated feed or a feed supplemented with the feed antibiotic virginiamycin . Unvaccinated birds were given either the basal feed or feed supplemented either with virginiamycin or the anticoccidial ionophore narasin . At slaughter at 36 days of age vaccinated birds had a lower live weight than non-vaccinated birds . The difference was 4.6% in unmedicated, and 6.0% in virginiamycin medicated chickens . Feed conversion ratio at slaughter was 2.5% higher for unmedicated vaccinated birds, and 1.3% higher for virginiamycin medicated vaccinated birds, compared to respective non-vaccinated groups . There was no significant difference in overall performance of unvaccinated birds given narasin as compared to virginiamycin . At 10 days post vaccination vaccinated birds had a higher number of Clostridium perfringens in the caeca, but there was no difference thereafter . Throughout the experiment, caecal clostridial counts were considerably higher in vaccinated unmedicated birds than in unvaccinated birds given narasin . The number of oocysts shed in the vaccinated groups was very low, but during a subsequent challenge with E . maxima and E . tenella the birds' immunity was found to be satisfactory.

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 131 - 8
Dissimilatory Fe(III) reduction by Clostridium beijerinckii isolated from freshwater sediment using Fe(III) maltol enrichment; Dobbin PS et al.; A microorganism which reduces Fe(III) during the fermentation of glucose was isolated from freshwater sediment . The Fe(III) was supplied to enrichment cultures as a soluble complex with the bidentate ligand maltol (3-hydroxy-2-methyl-4-pyrone) . Advantages that were afforded by the use of Fe(III)(maltol)3 over previously published methods included negation of the requirement for assays of Fe(II) formation . Because Fe(III)(maltol)3 has a characteristic deep red colour, Fe(III) reduction could be quantified spectrophotometrically by monitoring the disappearance of the complex in liquid cultures . Furthermore, Fe(III) reduction on agar plates containing the complex was apparent by zones of decolourisation around the bacterial colonies . 16S rRNA gene sequencing indicated the isolate to be a strain of Clostridium beijerinckii . Growth experiments were performed on the isolate in batch cultures with varying concentrations of Fe(III) citrate and 50 mM glucose . Increasing the level of Fe(III) citrate present was found to alter the fermentation balance, with less acidic products being formed . The presence of Fe(III) led to increases in the growth rate and growth yield, which were both approximately doubled when the supply of the cation reached 25 mM . A NAD(P)H-dependent Fe(III) reductase activity was localised to the bacterial membrane and found not to be sensitive to respiratory inhibitors . Taken together, these data suggest that dissimilatory Fe(III) reduction by the isolate provides a means of utilising the cation as an electron sink, thus facilitating pyridine nucleotide to be recycled during fermentative metabolism.

FEMS Microbiol Lett, 1999 Jul 1, 176(1), 117 - 24
The actin-based motility of intracellular Listeria monocytogenes is not controlled by small GTP-binding proteins of the Rho- and Ras-subfamilies; Ebel F et al.; In this study, we analyzed whether the actin-based motility of intracellular Listeria monocytogenes is controlled by the small GTP-binding proteins of the Rho- and Ras-subfamilies . These signalling proteins are key regulatory elements in the control of actin dynamics and their activity is essential for the maintenance of most cellular microfilament structures . We used the Clostridium difficile toxins TcdB-10463 and TcdB-1470 to specifically inactivate these GTP-binding proteins . Treatment of eukaryotic cells with either of these toxins led to a dramatic breakdown of the normal actin cytoskeleton, but did not abrogate the invasion of epithelial cells by L . monocytogenes and had no effect on the actin-based motility of this bacterial parasite . Our data indicate that intracellular Listeria reorganize the actin cytoskeleton in a way that circumvents the control mechanisms mediated by the members of the Rho- and Ras-subfamilies that can be inactivated by the TcdB-10463 and TcdB-1470 toxins.

Pathol Biol (Paris), 1999 May, 47(5), 515 - 8
{Susceptibility of Clostridium difficile to metronidazole using the E-test: effect of the culture medium}; Poilane I et al.; The treatment of intestinal Clostridium difficile infections rests on administration of either a glycopeptide or metronidazole . Given the current shifts in resistance patterns of anaerobes to antimicrobials, a study of the susceptibility of C . difficile to metronidazole was timely . The objective of this study was to evaluate the influence of the culture medium on the Minimal Inhibitory Concentration (MIC) of metronidazole as determined using the E-test . Thirty-one strains were grown on three different media supplemented with 5% horse blood, namely Columbia agar, Wilkens Chalgren agar, and Brucella agar . Results were compared to those obtained using the reference agar dilution method (ADM) . As recommended by the French Society for Microbiology, susceptibility was defined as an MIC < or = 4 mg/L . When used on strains susceptible by the ADM, the E-test yielded lower values than the ADM with all three media . Furthermore, findings suggest that E-test results obtained with strains whose MIC is in the 4 to 8 mg/L range by the ADM should be interpreted with caution and, in some cases, tested using the ADM.

Pathol Biol (Paris), 1999 May, 47(5), 415 - 21
{"Second look" at cytotoxin B of Clostridium difficile in the course of diarrhea associated with antibiotic therapy}; Sednaoui P et al.; Clostridium difficile is a sporulated obligate anaerobe responsible for most cases of antibiotic-associated colitis, for 15 to 25% of cases of antibiotic-related diarrhea, and for a substantial proportion of nosocomial infections . The most important laboratory test for the diagnosis of C . difficile infection is examination of the stool for C . difficile toxins A and/or B . Detection of cytotoxin B using the direct cytotoxicity assay (D-CA) is the gold standard test . Whether routine isolation of the organism from stool is warranted remains controversial . OBJECTIVES: To evaluate second-look CA done on C . difficile culture-positive filtrates from stool samples negative by the D-CA . METHODS: 300 consecutive stool samples sent to the Alfred Fournier Institute from April through October 1998 for a CA were routinely cultured on modified Cefoxitin Cycloserine Fructose Agar medium (CCFA) . All CA-negative samples that grew C . difficile were examined by second-look CA . RESULTS: 245 stool specimens (81.7%) were negative by both CA and culture . The remaining 55 specimens all yielded C . difficile by culture; 32 (58.2%) had a positive D-CA and nine (16.4%) a negative D-CA with a positive second-look CA done on culture filtrates . CONCLUSION: Our data suggest that stool specimens sent for a direct CA should be routinely cultured to provide material for a second-look CA on culture-positive filtrates if the first CA prove negative . Culturing also allows to study antimicrobial drug resistance phenotypes and epidemiological markers.

Protein Sci, 1999 Jun, 8(6), 1241 - 9
Oligomeric integrity--the structural key to thermal stability in bacterial alcohol dehydrogenases; Korkhin Y et al.; Principles of protein thermostability have been studied by comparing structures of thermostable proteins with mesophilic counterparts that have a high degree of sequence identity . Two tetrameric NADP(H)-dependent alcohol dehydrogenases, one from Clostridium beijerinckii (CBADH) and the other from Thermoanaerobacter brockii (TBADH), having exceptionally high (75%) sequence identity, differ by 30 degrees in their melting temperatures . The crystal structures of CBADH and TBADH in their holo-enzyme form have been determined at a resolution of 2.05 and 2.5 A, respectively . Comparison of these two very similar structures (RMS difference in Calpha = 0.8 A) revealed several features that can account for the higher thermal stability of TBADH . These include additional ion pairs, "charged-neutral" hydrogen bonds, and prolines as well as improved stability of alpha-helices and tighter molecular packing . However, a deeper structural insight, based on the location of stabilizing elements, suggests that enhanced thermal stability of TBADH is due mainly to the strategic placement of structural determinants at positions that strengthen the interface between its subunits . This is also supported by mutational analysis of structural elements at critical locations . Thus, it is the reinforcement of the quaternary structure that is most likely to be a primary factor in preserving enzymatic activity of this oligomeric bacterial ADH at elevated temperatures.

Pharmacotherapy, 1999 Jul, 19(7), 885 - 90
Generalized tetanus in a patient with a diabetic foot infection; Panning CA et al.; Tetanus is a preventable disease that continues to affect people in the United States due to poor immunization practices in our health care system . A 57-year-old man with type 2 diabetes mellitus, hypertension, and end-stage renal disease with many hospital admissions came to the hospital emergency department because of a blackened great toe . He denied pain in the toe or knowledge of foot injury . The patient also complained of temporomandibular tenderness accompanied by inability to open his mouth completely . The man's problems progressed to generalized tetanus and required a long hospitalization . Clostridium tetani can flourish in the anaerobic environment of a diabetic foot infection . Practitioners should be aware of tetanus as a rare but potentially serious complication of diabetic foot infections.

Am J Forensic Med Pathol, 1999 Jun, 20(2), 158 - 62
Nontraumatic clostridial myonecrosis; Burke MP et al.; We describe three cases of nontraumatic clostridial myonecrosis seen at the Victorian Institute of Forensic Medicine . Nontraumatic clostridial myonecrosis is an uncommon and often fatal condition that requires immediate institution of appropriate medical and surgical therapy . It is most commonly caused by Clostridium perfringens and Clostridium septicum and is associated with gastrointestinal and hematologic malignancies, diabetes mellitus, and peripheral vascular disease . The clinical features include a rapidly evolving acute illness with severe pain, marked tachycardia, and brawny discoloration of the skin with bullae formation and crepitus, followed by hypotension and acute renal failure . Features at autopsy include reddish brown skin discoloration with bullae formation and necrotic skeletal muscle . Radiographs may be of use prior to the postmortem in detecting gas within the soft tissues . Gram stain and microbiologic culture are important in establishing a definitive diagnosis; although the major factors in suggesting the diagnosis are the recognition of the typical clinical history and macroscopic autopsy findings.

Ugeskr Laeger, 1999 May 10, 161(19), 2815 - 6
{Botulism caused by a commercially produced product}; Poulstrup A et al.; Insufficient conserved home-made products are the main cause of botulism in Denmark . We present a case of botulism caused by a commercially produced vegetable pie conserved by traditional bottling without addition of preservatives . Clostridium botulinum was grown from faeces of the index case indicating an intestinal infection . An action plan set up by the medical and veterinarian authorities functioned well and further spread of the disease was avoided . More cases of botulism may be seen in the future, if procedures ensuring proper conservation in food production are not adhered to.

Eur J Biochem, 1999 Jun, 262(3), 879 - 89
Phosphoinositide-dependent activation of Rho A involves partial opening of the RhoA/Rho-GDI complex; Faure J et al.; Rho GTPases have two interconvertible forms and two cellular localizations . In their GTP-bound conformation, they bind to the cell membrane and are activated . In the inactive GDP-bound conformation, they associate with a cytosolic protein called GDP dissociation inhibitor (GDI) . We previously reported that the RhoA component of the RhoA/Rho-GDI complex was not accessible to the Clostridium botulinum C3 ADP-ribosyl transferase, unless the complex had been incubated with phosphoinositides . We show here that PtdIns, PtdIns4P, PtdIns3,4P2, PtdIns4,5P2 and PtdInsP3 enhance not only the C3-dependent ADP-ribosylation, but also the GDP/GTP exchange in the RhoA component of the prenylated RhoA/Rho-GDI complex . In contrast, in the nonprenylated RhoA/Rho-GDI complex, the levels of ADP-ribosylation and GDP/GTP exchange are of the same order as those measured on free RhoA and are not modified by phosphoinositides . In both cases, phosphoinositides partially opened, but did not fully dissociate the complex . Upon treatment of the prenylated RhoA/Rho-GDI complex with phosphoinositides, a GTP-dependent transfer to neutrophil membranes was evidenced . Using an overlay assay with the prenylated RhoA/Rho-GDI complex pretreated with PtdIns4P and labeled with {alpha32P}GTP, three membrane proteins with molecular masses between 26 and 32 kDa were radiolabeled . We conclude that in the presence of phosphoinositides, the prenylated RhoA/Rho-GDI complex partially opens, which allows RhoA to exchange GDP for GTP . The opened GTP-RhoA/Rho-GDI complex acquires the capacity to target specific membrane proteins.

Microbiology, 1999 Jun, 145 ( Pt 6), 1461 - 72
The genes controlling sucrose utilization in Clostridium beijerinckii NCIMB 8052 constitute an operon; Reid SJ et al.; The sucrose operon of Clostridium beijerinckii NCIMB 8052 comprises four genes, which encode a sucrose-specific enzyme IIBC(Scr) protein of the phosphotransferase system (ScrA), a transcriptional repressor (ScrR), a sucrose hydrolase (ScrB) and an ATP-dependent fructokinase (ScrK) . The scrARBK operon was cloned in Escherichia coli in three stages . Initial isolation was achieved by screening a C . beijerinckii genomic library in E . coli for clones able to utilize sucrose, while the remainder of the operon was isolated by inverse PCR and by plasmid rescue of flanking regions from a scrB mutant constructed by targeted gene disruption . Substrate specificity assays confirmed that the sucrose hydrolase was a beta-fructofuranosidase, able to hydrolyse sucrose and raffinose but not inulin or levans, and that the scrK gene encoded an ATP/Mg2+-dependent fructokinase . Both enzyme activities were induced by sucrose in C . beijerinckii . Disruption of the scr operon of C . beijerinckii by targeted plasmid integration into either the scrR or the scrB gene resulted in strains unable to utilize sucrose, indicating that this was the only inducible sucrose catabolic pathway in this organism . RNA analysis confirmed that the genes of the scr operon were co-transcribed on a 5 kb mRNA transcript and that transcription was induced by sucrose, but not by glucose, fructose, maltose or xylose . Primer extension experiments identified the transcriptional start site as lying 44 bp upstream of the scrA ATG start codon, immediately adjacent to the imperfect pelindrome sequence proposed to be a repressor binding site . Disruption of the scrR gene resulted in constitutive transcription of the upstream scrA gene, suggesting that ScrR encodes a transcriptional repressor which acts at the scrA operator sequence . The scrR gene is therefore itself negatively autoregulated as part of the polycistronic scrARBK mRNA

Clin Exp Metastasis, 1999 Mar, 17(2), 141 - 8
Involvement of small GTPases Rho and Rac in the invasion of rat ascites hepatoma cells; Imamura F et al.; Lysophosphatidic acid (LPA) triggers the invasion of a mesothelial cell monolayer by rat ascites hepatoma (MM1) cells . LPA also induces rapid morphological changes of MM1 cells, cell surface blebbing and pseudopodia formation . Pseudopodia formation is tightly correlated with cellular invasiveness . Clostridium Botulinum C3 exoenzyme and genistein abrogated the formation of blebs and pseudopodia together with the inhibition of invasion, indicating that GTPase Rho and certain tyrosine kinases are involved in both processes . MM1 cells expressing constitutively active Rho exhibited the invasion and the formation of blebs and pseudopodia in the absence of LPA . In contrast, MM1 cells expressing constitutively active Rac were not invasive in the absence of LPA, but were invasive in the presence of LPA . Their morphological response to LPA was almost the same as that of parental MM1 cells . Expression of dominant negative Rac suppressed the invasiveness to approximately 3% of that of parental MM1 cells, together with the inhibition of pseudopodia formation . Thus, Rho and Rac are cooperatively involved in both the invasion and the related morphological changes of MM1 cells . Rho activation is sufficient both for the induction of invasion and the morphological changes leading to the invasion, whereas Rac activation is necessary but not sufficient by itself . We propose that Rho activation is not mediated by Rac but the cooperation of both GTPases is essential to trigger the invasive behavior of MM1 cells.

Can J Microbiol, 1999 Mar, 45(3), 242 - 9
Regulation of cellulose-inducible structures of Clostridium cellulovorans; Blair BG et al.; Scanning electron microscopy was used to detect ultrastructural protuberances on the cellulolytic anaerobe Clostridium cellulovorans . Numerous ultrastructural protuberances were observed on cellulose-grown cells, but few were detected on glucose-, fructose-, cellobiose-, or carboxymethylcellulose (CMC)-grown cells . Formation of these protuberances was detected within 2 h of incubation in cellulose medium, but 4 h incubation was required before numerous structures were observed on the cells . When a soluble carbohydrate or CMC was mixed with cellulose-grown cells, the ultrastructural protuberances could no longer be detected . In fact, no protuberances were observed within 5 min following the addition of glucose, cellobiose, or methylglucose to cellulose-grown cells . The presence of these protuberances corresponded with the binding of the Bandeiraea simplicifolia BSI-B4 isolectin to the cell . Cellulose-grown cells had a greater level of observable lectin binding than cellobiose-grown cells, and lectin binding was not detected on glucose- or fructose-grown cells . In addition, lectin binding ability was lost by cellulose-grown cells following the addition of glucose, fructose, or methylglucose to the cellulose medium . A cellulose-affinity protein fraction expressing cellulase activity was also detected in cell extracts of cellobiose- or cellulose-grown cultures . However, this protein fraction was not detected in extracts of glucose-grown cultures, and was rapidly lost (within 5 min) following the addition of glucose to cellulose-grown cultures . The ability of C . cellulovorans to adhere to cellulose was also affected by the energy substrate, but not in the same manner as the protuberance formation or the cellulase-containing protein fraction . Rather, cellobiose-, cellulose-, and CMC-grown cultures adhered to cellulose, but this adherence was not affected by addition of glucose to the medium . This is the first report that soluble carbohydrates caused the rapid loss of some cellulose-inducible systems of C . cellulovorans.

Am J Gastroenterol, 1999 Jul, 94(7), 1969 - 70
Clostridium difficile-associated diarrhea after short term vaginal administration of clindamycin; Vikenes K et al.; A 32-yr-old woman developed frequent watery diarrhea with occult blood after 3 days treatment with clindamycin vaginal cream . Clostridium difficile toxin was demonstrated in stool samples and was considered the cause of an antibiotic-associated diarrhea . No other antibiotic was used at least 3 months before the start of diarrhea . To our knowledge, antibiotic-associated diarrhea after vaginal application has previously been reported only once.

J Biotechnol, 1999 Jun 11, 72(1-2), 95 - 101
Bacterial xylanase expression in mammalian cells and transgenic mice; Fontes CM et al.; The energy which simple-stomached livestock can derive from dietary plant material is limited by the lack of plant polysaccharide degrading enzymes in their gastro-intestinal (GI) tract and the inefficient microbial fermentation of such material in their hind-gut . In poultry the non-starch polysaccharides found in cereal grains can also impair normal digestive function as they form viscous gels in the GI tract inhibiting the breakdown and absorption of nutrients . The nutrition of such livestock could, therefore, be improved by the introduction of enzymes able to degrade plant polysaccharides in the small intestine . We describe the expression of a xylanase, XYLY', from the bacterium Clostridium thermocellum in mammalian cells and the exocrine pancreas of transgenic mice . The enzyme is synthesised, secreted and functionally active in the eukaryote system . This work demonstrates the feasibility of generating animals with the endogenous capacity to depolymerise the xylan component of hemi-cellulose.

J Comp Pathol, 1999 Aug, 121(2), 127 - 38
Comparison of the effects of Clostridium perfringens type D culture supernates in ligated intestinal loops of goats and sheep; Uzal FA et al.; The effects of Clostridium perfringens type D culture supernates were compared in ligated loops of the small intestine (ileum) and colon of four goat kids and four lambs, the loops being examined histopathologically and electron microscopically 7 h after inoculation . No lesions were observed in the small intestine of any animal, or in control colonic loops . In the caprine and ovine colonic loops treated with culture supernates, most goblet cells were empty and the lumina contained a layer of mucus, polymorphonuclear leucocytes, bacteria and sloughed epithelial cells . The apical cytoplasm of the superficial epithelial cells was lost . Moderate oedema was observed in the submucosa and muscular layer . The colonic lesions were more severe in kids than in lambs . No changes were seen in vascular endothelial cells in any loop . 1999 W.B . Saunders and Company Ltd.

FEBS Lett, 1999 Jun 18, 453(1-2), 124 - 8
N-acetylcysteine protects epithelial cells against the oxidative imbalance due to Clostridium difficile toxins; Fiorentini C et al.; Toxins A and B from the anaerobic bacterium Clostridium difficile are the causative agents of the antibiotic-associated pseudomembraneous colitis . At the subcellular level, they inhibit the Rho family GTPases, thus causing alterations of the actin cytoskeleton . The cytoskeletal integrity is also controlled by the redox state of cells . Therefore, we have evaluated whether an oxidative imbalance could be involved in the toxin-induced cytopathic effects . Our results indicate that both toxins induce oxidative stress with a significant depletion of protein SH-groups . These responses and the cytoskeleton-dependent cell retraction and rounding are significantly counteracted by N-acetylcysteine but not by alpha-tocopherol . Our study provides the first evidence that the thiol supplier N-acetylcysteine impairs the cellular intoxication by acting on the cytoskeleton integrity . This also suggests a possible beneficial role for this drug during therapeutic intervention.

Vet Res Commun, 1999 May, 23(3), 143 - 50
Antibody response in goats vaccinated with liposome-adjuvanted Clostridium perfringens type D epsilon toxoid; Uzal FA et al.; A trial was performed using 20 goats to evaluate the antibody responses to a liposome-adjuvanted Clostridium perfringens epsilon toxoid vaccine (LIPV) . The antibody response was compared with those produced by epsilon toxoid vaccines prepared using aluminium hydroxide (ALV) and incomplete Freud's adjuvant (FAV) . The animals were allocated to four groups at the beginning of the trial . The animals in group 1 were vaccinated with ALV, while the animals in group 2 received FAV and those in groups 3 and 4 were vaccinated with LIPV . The animals in groups 1 to 3 received three doses of the corresponding vaccine at intervals of three weeks, while those in group 4 received only 1 dose of vaccine at the beginning of the trial . A blood sample was obtained from all the goats at the beginning of the trial and then weekly for 8 weeks . The samples were analysed for epsilon toxoid antibodies by an indirect ELISA technique . No major clinical abnormalities were observed in the animals after vaccination, with the exception of those that received the FAV, which experienced transient lameness . The highest antibody response was observed in the animals vaccinated with FAV, but they presented moderate to severe inflammatory tissue reactions at the injection site . Moderately high antibody responses were obtained with the ALV, with which only minor local reactions were observed . No significant antibody responses were obtained with the LIPV, nor were local reactions observed.

Br J Pharmacol, 1999 Jun, 127(3), 597 - 600
Augmented acetylcholine-induced, Rho-mediated Ca2+ sensitization of bronchial smooth muscle contraction in antigen-induced airway hyperresponsive rats; Chiba Y et al.; Treatment with acetylcholine (ACh) of a beta-escin-permeabilized intrapulmonary bronchial smooth muscle of the rat induced force when the Ca2+ concentration was clamped at 1 microM . The ACh-induced Ca2+ sensitization of myofilaments was significantly greater in antigen-induced airway hyperresponsive rats than in control rats . The ACh-induced Ca2+ sensitization was completely blocked by treatment with Clostridium botulinum C3 exoenzyme, an inactivator of Rho family of proteins . Moreover, the protein level of RhoA in the intrapulmonary bronchi was significantly increased in the airway hyperresponsive rats . Thus, increased airway smooth muscle contractility observed in asthmatics may be related to augmented agonist-induced, Rho-mediated Ca2+ sensitization of myofilaments.

Eur Cytokine Netw, 1999 Jun, 10(2), 227 - 36
Neutrophil F-actin and myosin but not microtubules functionally regulate transepithelial migration induced by interleukin 8 across a cultured intestinal epithelial monolayer; Hofman P et al.; The role of the polymorphonuclear leukocyte (PMN) cytoskeleton during the transmigration across colonic epithelial cells is not very well understood . In order to study the role of different components of the PMN cytoskeleton during transepithelial migration across a colonic epithelial cell monolayer (T84), PMN were preincubated with drugs affecting either the actin cytoskeleton (cytochalasin B, iota toxin of Clostridium perfringens, and phalloidin) or the microtubules (colchicine and taxol) . The role of PMN myosin during transepithelial migration was investigated using the inhibitor 2,3-butanedione monoxime (BDM) and DC3B toxin . PMN intracellular Ca2+, during neutrophil adhesion and translocation across the epithelium, was assessed by the Ca2+ chelator 1, 2bis-(2-aminophenoxy)-ethane-N,N,N', N'-tetra-acetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM) . Transmigration of PMN was initiated by applying either interleukin-8 or formyl-met-leu-phe (fMLP) . While colchicine and taxol preexposure did not influence PMN transepithelial migration, treatment with cytochalasin B, iota toxin, phalloidin, BDM, DC3B toxin and BAPTA-AM greatly diminished migration of PMN across T84 monolayers . Similarly, cell-cell contacts established between PMN and epithelial cells during the transmigration were diminished after treatment of PMN with iota toxin or cytochalasin B . These data show that the neutrophil actin cytokeleton and myosin, but not the microtubules, evoke a Ca2+ -dependent motility that facilitates migration across the colonic epithelial barrier.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 379 - 82
Study of the presence of the spores of Clostridium botulinum in honey in Brazil; Schocken-Iturrino RP et al.; The isolation of Clostridium botulinum from honey samples is described . Botulism is characterized as an intoxication provoked by ingestion of contaminated foods with this toxin . Infant botulism happens by the ingestion of spores of C . botulinum together with food that in special conditions of the intestinal tract, such as those present in babies of less than 1 year old, will allow the germination and colonization of the intestine with production and absorption of botulinic toxin . The samples were subjected to dilution and to a thermal shock and cultivated in modified CMM (Difco) . Cultures were subjected to Gram smears and toxicity tests in mice . The toxic cultures were purified in RFCA (Oxoid) plates and incubated in anaerobic jars . Positive samples were typed using the mouse assay neutralization test . From the 85 honey samples analyzed, six were positive for C . botulinum (7.06%), and identified as producers of type A, B, and D toxins.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 373 - 7
Detection of the etx gene (epsilon-toxin inducer) in plasmids of high molecular weight in Clostridium perfringens type D; Bentancor AB et al.; The purpose of this work is to correlate the production of epsilon-toxin in a set of strains of Clostridium perfringens type D with the presence of the etx gene, either genomic or in plasmids . Total DNA obtained from strains with a different level of toxin production was explored by PCR and all the strains showed the amplification signal . Different methods were used to obtain plasmid profiles and all of the bands were assayed by PCR . The detection of the etx gene was only shown in several high molecular plasmids . These results were confirmed by a Southern blot . We suggest that the localization of the etx gene in different plasmids could be associated with the epsilon-toxin production level.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 369 - 72
In vitro evaluation methods for Clostridium botulinum type C and D vaccines; Ellis CE et al.; Reagents were prepared for use in ELISAs to determine the concentration of the antigenic components of Clostridium botulinum type C and D . The results obtained were compared with the L+dose assay and a good correlation was found between the two assays for measurement of the C and D neurotoxin concentration . These ELISAs were also used to determine the concentration of the neurotoxins in toxoid form . The relationship between the C neurotoxin dose, in toxoid form, and the immune response in guinea pigs could be deduced from the data obtained . The relationship for the D neurotoxin was not that clear, as the same concentration of the antigen resulted in variable potency values . However, these ELISAs can be used to formulate the concentration of the C and D components in the final bivalent vaccine . Replacement of the preliminary potency assay on the monovalent components after production with the in vitro assays will shorten the total production time of the vaccine by about 60 days . The economical and ethical implications are the reduction in the use of animals to evaluate the vaccine.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 361 - 7
Production and purification of Clostridium botulinum type C and D neurotoxin; Gessler F et al.; Neurotoxins of Clostridium botulinum are needed in basic neurologic research, but as therapeutic agent for certain neuromuscular disorders like strabism as well . A method for the production and purification of botulinum neurotoxins C and D is reported using a two-step hollow-fiber cross flow filtration and a newly developed chromatographic purification procedure . Hollow-fiber filtration proved to be a rapid and safe concentration and pre-purification step, which can easily be scaled up . The chromatographic purification included hydrophobic interaction, anion exchange and size exclusion chromatography runs . Botulinum neurotoxins C and D could be recovered with an overall yield of 12.6% and 10.6%, respectively . A specific toxicity of 1.86 x 10(7) minimal lethal dose mg(-1) (type C) and 5.26 x 10(7) minimal lethal dose mg(-1) (type D) was determined in the mouse bioassay.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 353 - 9
Detection of neutralizing antibodies against alpha-toxin of different Clostridium septicum strains in cell culture; Roth F et al.; Clostridium septicum, a ubiquitious organism, is the pathogen which causes the classical malignant edema after injuries . Because of its strong cytotoxic alpha-toxin, infections are often lethal . To prevent losses in animals, vaccination with alpha-toxoid vaccines is carried out . Quality control of the vaccines is done by a neutralization test in mice . A cytotoxin test and as an alternative method to detect neutralizing antibodies, a cytotoxin inhibition test was standardized . In the studies, alpha-toxin of the C . septicum reference strain (NC 547) from the National Collection of Type Cultures was compared with alpha-toxin of a field strain from an outbreak in Germany . Sera from five heterologous polyvalent and three monovalent vaccines from eight rabbit groups were available . Each vaccination had been carried out according to the procedure of the German Pharmacopoeia . In three out of the five sera of the groups vaccinated with the heterologous polyvalent vaccine, cytotoxin neutralizing antibodies were detected . High antibody titers were observed in sera of rabbits vaccinated with a vaccine of strain NC 547, lower titers in the sera of rabbits vaccinated with a vaccine of the field strain . No cytotoxin neutralizing antibodies could be found in the sera of rabbits vaccinated with the monovalent C . chauvoei vaccine . The toxins of all strains showed the same ranking of the vaccines . Vaccines which caused high antibody titers in the animals were detected by all toxins as such, as well as vaccines which had medium or low antibody inducing capacity . The results were independent of the C . septicum strain used for the production of alpha-toxin.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 345 - 51
Liver lesions seen at slaughter as an indicator of necrotic enteritis in broiler flocks; Lovland A et al.; Historical data of necrotic enteritis incidence and carcass condemnations owing to liver lesions in south-eastern Norway during 1978-1998 revealed covariance in the occurrence of the two variables . In histological examination during the first half of 1998, 33 of 45 sampled carcasses condemned owing to liver lesions showed histopathological changes consistent with earlier described Clostridium perfringens-associated hepatitis . The results suggest that the occurrence of necrotic enteritis in broiler flocks may be monitored by using meat inspection data on liver lesions.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 337 - 43
Necrotic enteritis challenge models with broiler chickens raised on litter: evaluation of preconditions, Clostridium perfringens strains and outcome variables; Kaldhusdal M et al.; The effect of Clostridium perfringens challenge, number of challenge days, and pre-challenge antibiotic treatment on the induction of necrotic enteritis in broiler chickens raised on litter was studied, and the relationship between bacterial counts and frequency of gut lesions was evaluated . Specific intestinal lesions in randomly selected birds were present despite a lack of disease-specific mortality . Challenge, number of challenge days and frequency of lesions were associated with median counts of C . perfringens . The effect of pre-challenge C . perfringens counts and antibiotics cannot be evaluated unless procedures for the control of pre-challenge infection and methods for the differentiation between wild-type and challenge strains are established.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 333 - 6
The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine; Springer S et al.; Necrotic enteritis in sucking piglets constitutes a serious problem in piglet rearing units because of the high morbidity and mortality associated with the disease . The primary causal agent is Clostridium perfringens type C . The beta-toxin plays a decisive role in the pathogenesis of this disease . A toxoid vaccine for use in sows has been developed and studied in field trials . The European Pharmacopoeia Monograph on vaccines for use in animals lays down a method of the efficacy testing based on the immunization of rabbits, the collection of pooled sera and the subsequent assay of anti-toxin antibodies in mice using an appropriate test toxin . The vaccine is regarded as effective if it induces a minimum of 10 IU of beta-anti-toxin per ml of rabbit serum . We have established a range of 17.14-98.23 IU beta-anti-toxin per ml rabbit serum induced by a sample of C . perfringens toxoid vaccine . The vaccine has been used under field conditions in different rearing units at the same time, mostly in the form of emergency vaccinations following the outbreak of disease . The outcome of vaccination was evaluated by recording the total numbers of piglets born alive and the piglet losses . Use of the vaccine, coupled with other measures, resulted in an approximately 30% reduction in the number of losses.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 325 - 32
Detection of the beta2 toxin gene of Clostridium perfringens in diarrhoeic piglets in The Netherlands and Switzerland; Klaasen HL et al.; The two studies presented here were done to determine the prevalence of the alpha, beta, epsilon and enterotoxin genes and the novel beta2 toxin gene of Clostridium perfringens in neonatal or pre-weaned piglets with diarrhoea or necrotic enteritis . All C . perfringens isolates were positive for the alpha and negative for the epsilon and enterotoxin gene, implying that only non-enterotoxigenic type A and C strains were detected . The most important findings were the relatively high prevalence of the beta2 toxin gene in isolates from diarrhoeic piglets in both studies, and, in one of the two studies, absence of strains with only the alpha and beta toxin gene . These data are supportive for the suggestion of a causal relationship of beta2 toxin-producing strains with digestive tract diseases in piglets.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 313 - 7
Quantitation of commercial equine tetanus antitoxin by competitive enzyme-linked immunosorbent assay; Kolbe DR et al.; In the USA, the potency of commercially prepared equine tetanus antitoxin is determined by the method outlined in the Code of Federal Regulations, Title 9, Part 113.451 . In the current test, commercial equine tetanus antitoxin is tested by a toxin neutralization test in guinea pigs . The in vivo test measures antitoxin content through effectiveness of protection of guinea pigs injected with diluted mixtures of antitoxin and a standard toxin . A competitive enzyme-linked immunosorbent assay, designed as an in vitro alternative to the in vivo test, measures antitoxin content based on a competitive reaction between standard or unknown serum and murine monoclonal antibody specific for tetanus toxin . The monoclonal antibody used in the assay delayed death in mouse passive protection studies and reacted with the C fragment of tetanus toxin . No cross-reaction was observed when the antibody was tested with the toxins of Clostridium chauvoei, C . novyi, C . perfringens, or C . sordellii . The in vitro test will measure the antitoxin content of serum samples containing 100-1500 units of antitoxin . Tetanus antitoxin titers obtained by the competitive enzyme-linked immunosorbent assay compared favorably with the toxin neutralization test conducted in guinea pigs . The in vitro assay serves as a feasible alternative to the in vivo test because it can be completed in less time, is reproducible, and eliminates the use of test animals.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 299 - 311
Development and prevalidation of two different ELISA systems for the potency testing of Clostridium perfringens beta and epsilon-toxoid containing veterinary vaccines; Ebert E et al.; The potency testing of Clostridium perfringens mono- and multicomponent veterinary vaccines is currently performed with the mouse neutralisation test (MNT) to estimate levels of C . perfringens beta- and epsilon-antitoxin levels in the sera of rabbits immunised with the vaccine . Two in vitro methods based on monoclonal antibodies (mAb) have been developed for the determination of specific antibodies against C . perfringens beta-toxin (capture ELISA) and epsilon-toxin (competitive ELISA) in these sera . Both test systems show high specificity and good reproducibility . These ELISA procedures were used in addition to the routine batch potency test in mice (MNT) to determine beta- and epsilon-antitoxin levels in 523 samples of rabbit serum . There was good agreement between the rank order of sera determined in vivo and the rank order determined in vitro . Linear regression analysis gave correlation coefficients of 0.88 for the capture ELISA and 0.41 for the competitive ELISA, with a significance level of P < 0.01 in both cases . Furthermore, a prevalidation study was carried out in four laboratories to evaluate the transferability of the ELISA procedures and the interlaboratory reproducibility of the results . Three coded serum samples were tested several times . The results indicated that both ELISA systems are suitable candidates to replace the MNT used for the potency testing of beta- and epsilon-toxoid in mono- and multicomponent veterinary vaccines . However, these assays still need to be validated in an international collaborative study.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 293 - 7
Development and evaluation of various enzyme-linked immunosorbent assays for the detection of Clostridium perfringens beta anti-toxins; Krt B; The aim of our work was to develop an enzyme-linked immunosorbent assay for the detection of antibodies against the Clostridium perfringens beta toxin . For this purpose, five different ways of performing an enzyme-linked immunosorbent assay were investigated . Positive and negative sera of different animals and partially purified beta toxin were used . In all enzyme-linked immunosorbent assay tests, microplates were first coated with monoclonal antibodies against the C . perfringens beta toxin . Actually, the first three ways of performing enzyme-linked immunosorbent assay proved to be an inhibition or a blocking enzyme-linked immunosorbent assay . In the first of these modifications, the examined serum was added on a microplate after the toxin . In the second two tests, they were added simultaneously after they were incubated together (60 min at room temperature or overnight at 4 degrees C, respectively) . An anti-toxin conjugate was used for the detection . It was also used in a competitive enzyme-linked immunosorbent assay, where it was added together with the examined serum on the microplate, to which the toxin was already bound . The fifth way of performing an enzyme-linked immunosorbent assay differed from others by the use of conjugated anti-species immunoglobulin for the detection . The biggest differences in absorbances between positive and negative sera were found in the blocking enzyme-linked immunosorbent assay, where the mixture of the toxin and the examined serum were previously incubated overnight at 4 degrees C . The smallest differences in absorbance were found when anti-species conjugates were used.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 287 - 92
Short protocol for pulsed field gel electrophoresis of a variety of Clostridia species; Sperner B et al.; While pulsed field gel electrophoresis has become an important tool for genotyping of bacteria, one of its drawbacks is that standard methods are rather time-consuming . In order to overcome this problem, shortened procedures for DNA preparation have been developed for some bacterial species . The aim of this study was to examine if a short procedure used for pulsed field gel electrophoresis of Clostridium botulinum could be applied to other Clostridia species . For this, the protocol was modified and used to prepare the DNA of 34 strains of 25 different Clostridia species . In contrast to a standard procedure, which takes at least 5 days from DNA extraction to completion of the electrophoresis, this protocol yielded results within 2 days . In order to directly compare the results of the short protocol with those of the standard, long procedure, parallel DNA preparations were performed using both methods and the two DNA samples thus obtained per strain were then run on the same gel . Briefly, the procedure was as follows . After embedding the bacterial cells in agarose, the agarose blocks were incubated for 1 h in lysis solution containing lysozyme, mutanolysin, lysostaphin and RNase . This was followed by a 1-h proteinase K treatment . Then, slices were cut from the agarose blocks and washed for 15 min in TE buffer, these washes were repeated four times with fresh TE . After a 2-h restriction with SmaI, electrophoresis was carried out overnight.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 275 - 80
Detection of Clostridium novyi type B alpha toxin by cell culture systems; Borrmann E et al.; Ten permanent cell lines were examined for their reaction to the Clostridium novyi alpha toxin . The action of the toxin was determined after 3 days by microscopic examination and the MTT assay . The alpha toxin exhibited the strongest effect on ESH-L cells rather than other cell lines . Vero and SFT-R cells reacted in a comparable way, but less sensitively . We were able to show that the cytopathic effect on the three types of cells was neutralised by the international standard for gas gangrene antitoxin (C . novyi) but in no case by heterologous antisera . Our results have shown that the three cell lines were specific indicators for the detection of the cytopathic effect of alpha toxin . The cytopathic effect can be measured reproducibly by the cell culture assay used . These results are suitable as the starting point for the development of the neutralisation test using cell cultures.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 267 - 74
Identification of Clostridium botulinum with API 20 A, Rapid ID 32 A and RapID ANA II; Lindstrom MK et al.; Three commercially available test systems for the identification of anaerobic bacteria were evaluated for the identification of 18 proteolytic group I and 69 non-proteolytic group II Clostridium botulinum, four Clostridium sporogenes and 18 non-toxigenic group II C . botulinum-like strains . All proteolytic C . botulinum strains were misidentified by the Rapid ID 32 A and RapID ANA II, while 14 strains and all C . sporogenes strains were identified as C . botulinum or C . sporogenes by the API 20 A . Reversely, all non-proteolytic C . botulinum strains were misidentified by the API 20 A while the Rapid ID 32 A recognized 67 and RapID ANA II 68 strains . All C . sporogenes strains were recognized by the RapID ANA II, while the Rapid ID 32 A recognized one strain . All non-proteolytic non-toxigenic strains were identified as C . botulinum group II by the Rapid ID 32 A, 17 strains by the RapID ANA II, and one strain by the API 20 A . The results show that these test systems do not provide a reliable method for identification of C . botulinum.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 259 - 66
Typing of sheep clinical isolates and identification of enterotoxigenic Clostridium perfringens strains by classical methods and by polymerase chain reaction (PCR); Kadra B et al.; A simple procedure was developed to identify toxitypes of Clostridium perfringens of different origins . Ninety strains of C . perfringens were identified by classical bacteriological methods, typing of the strains was done by a seroneutralisation test on mice . Production of enterotoxin was tested and all strains were analysed by PCR using gene of toxin alpha, gene of toxin E, gene of toxin beta and gene of enterotoxin . Simple amplification (amplifying one gene), and duplex and triplex amplification (amplifying two and three genes simultaneously) were performed . In the conditions of the experiment, the PCR method has proved efficacious . The specificity and sensitivity are excellent and superior to those of the classical methods . The prophylaxis of enterotoxaemia in animals is achieved by vaccination, the PCR technique can thus become a first-choice tool for the identification and typing of the C . perfringens strains which initiate these diseases . In turn, this would simplify the development of vaccines adapted to the epidemiological situation.

FEMS Immunol Med Microbiol, 1999 Jul, 24(3), 253 - 8
Phylogenetic basis for a taxonomic dissection of the genus Clostridium; Stackebrandt E et al.; The 16S rDNA-based phylogenetic analysis of the genus Clostridium has been completed by determination of the phylogenetic position of the type strains of 15 species and two non-validated species . These strains are members of phylogenetic clusters I, III, IV, V, IX, XIVa and XVIII as defined previously by Collins et al . {Int . J . Syst . Bacteriol . 44 (1994) 812-826} . Members of the genus Clostridium span a large evolutionary distance and the genus is not a phylogenetically coherent taxon but is intermixed with members of different genera, exhibiting a combination of Clostridium- and non-Clostridium-type properties . Anaerobacter polyendosporus, Syntrophococcus sucromutans and Acetivibrio multivorans also cluster within the radiation of Clostridium species . Although several taxa have been described for former Clostridium species with distinct phenotypic properties, the majority of Clostridium species, which are not members of the core cluster I, can at present not be reclassified as long as taxon-specific, phenotypic properties are not available.

Appl Environ Microbiol, 1999 Jul, 65(7), 3258 - 60
Influence of 1-{(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl}pyrrolidine-2-carboxylic acid and diphenyliodonium chloride on ruminal protein metabolism and ruminal microorganisms; Floret F et al.; The effects of 1-{(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl}pyrrolidine-2-carboxy lic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet . Both compounds decreased NH3 production by inhibiting deamination of amino acids . LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii.

Appl Environ Microbiol, 1999 Jul, 65(7), 3244 - 7
Characterization of methylglyoxal synthase from Clostridium acetobutylicum ATCC 824 and its use in the formation of 1, 2-propanediol; Huang K et al.; A gene encoding a putative 150-amino-acid methylglyoxal synthase was identified in Clostridium acetobutylicum ATCC 824 . The enzyme was overexpressed in Escherichia coli and purified . Methylglyoxal synthase has a native molecular mass of 60 kDa and an optimum pH of 7.5 . The Km and Vmax values for the substrate dihydroxyacetone phosphate were 0.53 mM and 1.56 mmol min(-1) microgram(-1), respectively . When E . coli glycerol dehydrogenase was coexpressed with methylglyoxal synthase in E . coli BL21(DE3), 3.9 mM 1,2-propanediol was produced.

Appl Environ Microbiol, 1999 Jul, 65(7), 3240 - 3
In situ detection of the Clostridium botulinum type C1 toxin gene in wetland sediments with a nested PCR assay; Williamson JL et al.; A nested PCR was developed for detection of the Clostridium botulinum type C1 toxin gene in sediments collected from wetlands where avian botulism outbreaks had or had not occurred . The C1 toxin gene was detected in 16 of 18 sites, demonstrating both the ubiquitous distribution of C . botulinum type C in wetland sediments and the sensitivity of the detection assay.

Appl Environ Microbiol, 1999 Jul, 65(7), 3075 - 83
Isolation and characterization of proteolytic ruminal bacteria from sheep and goats fed the tannin-containing shrub legume Calliandra calothyrsus; McSweeney CS et al.; Tannins in forages complex with protein and reduce the availability of nitrogen to ruminants . Ruminal bacteria that ferment protein or peptides in the presence of tannins may benefit digestion of these diets . Bacteria from the rumina of sheep and goats fed Calliandra calothyrsus (3.6% N and 6% condensed tannin) were isolated on proteinaceous agar medium overlaid with either condensed (calliandra tannin) or hydrolyzable (tannic acid) tannin . Fifteen genotypes were identified, based on 16S ribosomal DNA-restriction fragment length polymorphism analysis, and all were proteolytic and fermented peptides to ammonia . Ten of the isolates grew to high optical density (OD) on carbohydrates (glucose, cellobiose, xylose, xylan, starch, and maltose), while the other isolates did not utilize or had low growth on these substrates . In pure culture, representative isolates were unable to ferment protein that was present in calliandra or had been complexed with tannin . One isolate, Lp1284, had high protease activity (80 U), a high specific growth rate (0.28), and a high rate of ammonia production (734 nmol/min/ml/OD unit) on Casamino Acids and Trypticase Peptone . Phylogenetic analysis of the 16S ribosomal DNA sequence showed that Lp1284 was related (97 . 6%) to Clostridium botulinum NCTC 7273 . Purified plant protein and casein also supported growth of Lp1284 and were fermented to ammonia . This is the first report of a proteolytic, ammonia-hyperproducing bacterium from the rumen . In conclusion, a diverse group of proteolytic and peptidolytic bacteria were present in the rumen, but the isolates could not digest protein that was complexed with condensed tannin.

J Struct Biol, 1999 Jun 15, 126(2), 175 - 7
Crystallization and preliminary X-ray studies of the Ia component of Clostridium perfringens iota toxin complexed with NADPH; Tsuge H et al.; A recombinant Ia component of Clostridium perfringens iota toxin, which ADP-ribosylates actin, was crystallized by the hanging drop vapor diffusion method using PEG4000 as a precipitating agent . The crystals were obtained in the presence of NADPH, which is similar to a real substrate, NADH, and belongs to the space group P1 (a = 47.9 A, b = 54.5 A, c = 103.1 A, alpha = 99.0 degrees, beta = 93.3 degrees, and gamma = 107.2 degrees ) . The Matthews coefficient of native crystal was 2.7, assuming 2 mol/asymmetric unit . Native data were collected at 2.4-A resolution . The results from a heavy-atom search showed that lanthanide ions (samarium, holmium) altered the molecular packing, judging from the unit-cell difference . The crystals also belonged to the space group P1 (a = 47.7 A, b = 53.9 A, c = 54.6 A, alpha = 68.9 degrees, beta = 78.3 degrees, and gamma = 73.7 degrees ), which is consistent with only one molecule per asymmetric unit .

Curr Microbiol, 1999 Jul, 39(1), 43 - 8
Characterization, production, and purification of leucocin H, a two-peptide bacteriocin from Leuconostoc MF215B; Blom H et al.; Leuconostoc MF215B was found to produce a two-peptide bacteriocin referred to as leucocin H . The two peptides were termed leucocin Halpha and leucocin Hbeta . When acting together, they inhibit, among others, Listeria monocytogenes, Bacillus cereus, and Clostridium perfringens . Production of leucocin H in growth medium takes place at temperatures down to 6 degrees C and at pH below 7 . The highest activity of leucocin H in growth medium was demonstrated in the late exponential growth phase . The bacteriocin was purified by precipitation with ammonium sulfate, ion-exchange (SP Sepharose) and reverse phase chromatography . Upon purification, specific activity increased 10(5)-fold, and the final specific activity was 2 x 10(7) BU/OD280 . Amino acid composition analyses of leucocin Halpha and leucocin Hbeta indicated that both peptides consisted of around 40 amino acid residues . Their N-termini were blocked for Edman degradation, and the methionin residues of leucocin Hbeta did not respond to Cyanogen Bromide (CNBr) cleavage . Absorbance at 280 nm indicated the presence of tryptophan residues and tryptophan-fracturing opened for partial sequencing by Edman degradation . From leucocin Halpha, the sequence of 20 amino acids was obtained; from leucocin Hbeta the sequence of 28 amino acid residues was obtained . No sequence homology to other known bacteriocins could be demonstrated . It also appeared that the two peptides themselves shared little or no sequence homology . The presence of soy oil did not affect the activity of leucocin H in agar.

Biochemistry, 1999 Jun 15, 38(24), 7874 - 80
Redox properties of mesophilic and hyperthermophilic rubredoxins as a function of pressure and temperature; Gilles de Pelichy LD et al.; The formal equilibrium reduction potentials of recombinant electron transport protein, rubredoxin (MW = 7500 Da), from both the mesophilic Clostridium pasteurianum (Topt = 37 degrees C) and hyperthermophilic Pyrococcus furiosus (Topt = 95 degrees C) were recorded as a function of pressure and temperature . Measurements were made utilizing a specially designed stainless steel electrochemical cell that easily maintains pressures between 1 and 600 atm and a temperature-controlled cell that maintains temperatures between 4 and 100 degrees C . The reduction potential of P . furiosus rubredoxin was determined to be 31 mV at 25 degrees C and 1 atm, -93 mV at 95 degrees C and 1 atm, and 44 mV at 25 degrees C and 400 atm . Thus, the reduction potential of P . furiosus rubredoxin obtained under standard conditions is likely to be dramatically different from the reduction potential obtained under its normal operating conditions . Thermodynamic parameters associated with electron transfer were determined for both rubredoxins (for C . pasteurianum, DeltaV degrees = -27 mL/mol, DeltaS degrees = -36 cal K-1 mol-1, and DeltaH degrees = -10 kcal/mol, and for P . furiosus, DeltaV degrees = -31 mL/mol, DeltaS degrees = -41 cal K-1 mol-1, and DeltaH degrees = -13 kcal/mol) from its pressure- and temperature-reduction potential profiles . The thermodynamic parameters for electron transfer (DeltaV degrees, DeltaS degrees, and DeltaH degrees ) for both proteins were very similar, which is not surprising considering their structural similarities and sequence homology . Despite the fact that these two proteins exhibit dramatic differences in thermostability, it appears that structural changes that confer dramatic differences in thermostability do not significantly alter electron transfer reactivity . The experimental changes in reduction potential as a function of pressure and temperature were simulated using a continuum dielectric electrostatic model (DELPHI) . A reasonable estimate of the protein dielectric constant (epsilonprotein) of 6 for both rubredoxins was determined from these simulations . A discussion is presented regarding the analysis of electrostatic interaction energies of biomolecules through pressure- and temperature-controlled electrochemical studies.

FEMS Microbiol Lett, 1999 Jun 15, 175(2), 261 - 6
Development of a new PCR-ribotyping method for Clostridium difficile based on ribosomal RNA gene sequencing; Bidet P et al.; PCR-ribotying, a typing method based on polymorphism in the 16S-23S intergenic spacer region, has been recently used to investigate outbreaks due to Clostridium difficile . However, this method generates bands of high and close molecular masses which are difficult to separate on agarose gel electrophoresis . To improve reading of banding patterns of PCR-ribotyping applied to C . difficile, a partial sequencing of the rRNA genes (16S and 23S) and intergenic spacer region has been performed, then a new set of primers located closer to the intergenic spacer region has been defined . The new PCR gave reproducible patterns of bands easy to separate on agarose gel electrophoresis . Each of the 10 serogroups and 11 subgroups of serogroup A produced a different pattern . This typing method has evidenced major qualities such as easiness, rapidity and reproducibility . However, its discriminatory power has to be evaluated to validate its importance as a typing tool for C . difficile.

FEMS Microbiol Lett, 1999 Jun 15, 175(2), 197 - 203
Deletions in the repeating sequences of the toxin A gene of toxin A-negative, toxin B-positive Clostridium difficile strains; Kato H et al.; The repeating sequences of the toxin A gene from toxin A-negative, toxin B-positive (toxin A-, toxin B+) strains of Clostridium difficile which were isolated in geographically separated facilities in Japan and Indonesia were determined . All six strains tested had identical repeating sequences with two deletions (1548 and 273 nucleotides in size) in the toxin A gene . A PCR method was designed to detect the deletions and the deletions were confirmed in all 50 toxin A-, toxin B+ strains examined by this method . Western immunoblot analysis revealed that polyclonal antiserum against native toxin A did not react with the concentrated culture filtrates of the toxin A-, toxin B+ strains . These results may suggest that toxin A-, toxin B+ strains have deletions of the two thirds of the repeating regions of the toxin A gene, which encodes the epitopes fully responsible for the reaction with the polyclonal antiserum.

Kansenshogaku Zasshi, 1999 May, 73(5), 467 - 72
{Detection of Clostridium difficile toxin A from stool specimens by an enzyme immunoassay kit}; Kato N et al.; Toxin detection from stool specimens is prerequisite for Clostridium difficile-associated diarrhea and colitis . However, in Japan only one toxin detection kit is commercially available, which requires computerized VIDAS fluorescence reader . In this study we evaluated ImmunoCard Toxin A, which is an enzyme immunoassay with a format of individual cassette and needs no special equipment to perform, by comparing with the VIDAS CDA kit . Of 61 stool specimens 12 were positive and 39 were negative by both assays, 7 were VIDAS positive-ImmunoCard negative, 2 were VIDAS invalid-ImmunoCard negative, and 1 was invalid by both assays . Stool specimens which gave inconsistent results between two assays were subjected to cell culture assay to detect toxin B and culture for C . difficile followed by testing of toxin producibility of isolates . Of 7 VIDAS positive-ImmunoCard negative specimens 5 were negative for cell culture assay and toxigenic C . difficile culture and 2 were positive for cell culture assay . By omitting 3 VIDAS invalid specimens and counting 5 specimens, which were cell culture negative but VIDAS positive, as negative and 2, which were cell culture positive and VIDAS positive, as positive, 12 of the 14 VIDAS positive specimens were ImmunoCard positive (sensitivity, 85.7%) and all 44 VIDAS negative were ImmunoCard negative (specificity, 100%) . These results indicate that although in comparison with VIDAS CDA, ImmunoCard Toxin A is slightly less sensitive, ImmunoCard is a rapid, simple, and reliable C . difficile toxin A detection kit, which has the advantage of requiring no special equipment to perform and no centrifuge step, as small as 25 muL of a specimen, and as short as approximately 15 min of time to complete the whole testing.

J Bacteriol, 1999 Jul, 181(13), 4035 - 40
The extracellular xylan degradative system in Clostridium cellulolyticum cultivated on xylan: evidence for cell-free cellulosome production; Mohand-Oussaid O et al.; In this study, we demonstrate that the cellulosome of Clostridium cellulolyticum grown on xylan is not associated with the bacterial cell . Indeed, the large majority of the activity (about 90%) is localized in the cell-free fraction when the bacterium is grown on xylan . Furthermore, about 70% of the detected xylanase activity is associated with cell-free high-molecular-weight complexes containing avicelase activity and the cellulosomal scaffolding protein CipC . The same repartition is observed with carboxymethyl cellulase activity . The cellulose adhesion of xylan-grown cells is sharply reduced in comparison with cellulose-grown cells . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan- and cellulose-grown cells have different compositions . In both cases, the scaffolding protein CipC is present, but the relative proportions of the other components is dramatically changed depending on the growth substrate . We propose that, depending on the growth substrate, C . cellulolyticum is able to regulate the cell association and cellulose adhesion of cellulosomes and regulate cellulosomal composition.

Infect Immun, 1999 Jul, 67(7), 3504 - 11
Differential roles of segmented filamentous bacteria and clostridia in development of the intestinal immune system; Umesaki Y et al.; The presence of microflora in the digestive tract promotes the development of the intestinal immune system . In this study, to evaluate the roles of two types of indigenous microbe, segmented filamentous bacteria (SFB) and clostridia, whose habitats are the small and large intestines, respectively, in this immunological development, we analyzed three kinds of gnotobiotic mice contaminated with SFB, clostridia, and both SFB and clostridia, respectively, in comparison with germfree (GF) or conventionalized (Cvd) mice associated with specific-pathogen-free flora . In the small intestine, the number of alpha beta T-cell receptor-bearing intraepithelial lymphocytes (alpha betaIEL) increased in SFB-associated mice (SFB-mice) but not in clostridium-associated mice (Clost-mice) . There was no great difference in Vbeta usage among GF mice, Cvd mice, and these gnotobiotic mice, although the association with SFB decreased the proportion of Vbeta6(+) cells in CD8beta- subsets to some extent, compared to that in GF mice . The expression of major histocompatibility complex class II molecules on the epithelial cells was observed in SFB-mice but not in Clost-mice . On the other hand, in the large intestine, the ratio of the number of CD4(-) CD8(+) cells to that of CD4(+) CD8(-) cells in alpha betaIEL increased in Clost-mice but not in SFB-mice . On association with both SFB and clostridia, the numbers and phenotypes of IEL in the small and large intestines changed to become similar to those in Cvd mice . In particular, the ratio of the number of CD8alpha beta+ cells to that of CD8alpha alpha+ cells in alpha betaIEL, unusually elevated in the small intestines of SFB-mice, decreased to the level in Cvd mice on contamination with both SFB and clostridia . The number of immunoglobulin A (IgA)-producing cells in the lamina propria was more elevated in SFB-mice than in Clost-mice, not only in the ileum but also in the colon . The number of IgA-producing cells in the colons of Clost-mice was a little increased compared to that in GF mice . Taken together, SFB and clostridia promoted the development of both IEL and IgA-producing cells in the small intestine and that of only IEL in the large intestine, respectively, suggesting the occurrence of compartmentalization of the immunological responses to the indigenous bacteria between the small and large intestines.

Infect Immun, 1999 Jul, 67(7), 3297 - 301
Differences in the carboxy-terminal (Putative phospholipid binding) domains of Clostridium perfringens and Clostridium bifermentans phospholipases C influence the hemolytic and lethal properties of these enzymes; Jepson M et al.; The phospholipases C of C . perfringens (alpha-toxin) and C . bifermentans (Cbp) show >50% amino acid homology but differ in their hemolytic and toxic properties . We report here the purification and characterisation of alpha-toxin and Cbp . The phospholipase C activity of alpha-toxin and Cbp was similar when tested with phosphatidylcholine in egg yolk or in liposomes . However, the hemolytic activity of alpha-toxin was more than 100-fold that of Cbp . To investigate whether differences in the carboxy-terminal domains of these proteins were responsible for differences in the hemolytic and toxic properties, a hybrid protein (NbiCalpha) was constructed comprising the N domain of Cbp and the C domain of alpha-toxin . The hemolytic activity of NbiCalpha was 10-fold that of Cbp, and the hybrid enzyme was toxic . These results confirm that the C-terminal domain of these proteins confers different properties on the enzymatically active N-terminal domain of these proteins.

Microbiology, 1999 May, 145 ( Pt 5), 1097 - 104
The small GTP-binding protein Rho is expressed differentially during spore germination of Phycomyces blakesleeanus; Ramirez-Ramirez N et al.; Evidence has been obtained for the presence of the small 22 kDa GTP-binding Rho protein in dormant spores of Phycomyces blakesleeanus . Immunoblotting with a polyclonal antibody against RhoA revealed a soluble and membrane-associated 22 kDa protein . When {32P}ADP-ribosylated by Clostridium botulinum C3 exotoxin the protein had a pI of 5.7, a value similar to that reported for other RhoA proteins . The 22 kDa protein was expressed at all stages of growth investigated, but radiolabelling of the {32P}ADP-ribosylated protein increased when tube-formation occurred and decreased as the hyphae branched . Localization of RhoA during spore germination studied by immunofluorescence microscopy revealed the presence of RhoA in the cell membrane of the spore . When the spore started to swell, RhoA was observed as patches in the cell membrane which become concentrated in the neck region of the site of the protuberation tube, but this protein was never observed at the point of growth of the hyphal tip . The above results suggest that RhoA associated with one or more membrane proteins could participate in the molecular mechanism involved in maintaining cell integrity during the extrusion of the germ-tube of P . blakesleeanus.

DNA Seq, 1999, 10(2), 93 - 6
Nucleotide and peptide sequences of the open reading frame encoding a truncated toxin A gene of Clostridium difficile strain CCUG 20309; Song KP et al.; The open reading frame encoding a putative truncated toxin A gene of Clostridium difficile in strain CCUG 20309 (ATCC 8864), a strain that produces toxin B but not toxin A, was sequenced by cycle sequencing method . The coding region contains 2097 base pairs and has a GC content of 26.4% . The deduced polypeptide is 50 kDa and is generally hydrophilic . Although strain CCUG 20309 of C . difficile was reported not to produce toxin A, it is enterotoxic, an inherent property of toxin A in pathogenic strains of C . difficile . It therefore remains to be determined whether a truncated form of toxin A could actually be expressed from the open reading frame by this strain of C . difficile.

Curr Biol, 1999 Jun 17, 9(12), 657 - 60
Inhibition of Rho at different stages of thymocyte development gives different perspectives on Rho function; Cleverley S et al.; Development of thymocytes can be staged according to the levels of expression of the cell-surface markers CD4, CD8, CD44, CD25 and CD2 . Thymocyte development is regulated by a complex signalling network {1}, one component of which is the GTPase Rho . The bacterial enzyme C3 transferase from Clostridium botulinum selectively ADP-ribosylates Rho in its effector-binding domain and thereby abolishes its biological function {2,3} . To explore the function of Rho in thymocyte development, we previously used the proximal promoter of the gene encoding the Src-family kinase p56lck to make transgenic mice that selectively express C3 transferase in the thymus {4,6} . In these mice, which lack Rho function from the earliest thymocyte stages, thymocyte numbers are reduced by approximately 50- to 100-fold . Here, we describe transgenic mice that express C3 transferase under the control of the locus control region (LCR) of the CD2 gene; this regulatory element drives expression at a later stage of thymocyte development than the lck proximal promoter {7} . In these mice, thymocyte numbers were also reduced by 50- to 100-fold, but unlike the lck-C3 mice, in which the reduction predominantly results from defects in cell survival of CD25(+) thymocyte progenitors, the CD2-C3 transgenic mice had a pre-T-cell differentiation block at the CD25(+) stage after rearrangement of the T-cell receptor (TCR) beta chains . Analysis of CD2-C3 mice demonstrated that Rho acts as an intracellular switch for TCR beta selection, the critical thymic-differentiation checkpoint . These results show that Rho-mediated survival signals for CD25(+) pre-T cells are generated by the extracellular signals that act on earlier thymocyte precursors and also that temporal cell-type-specific elimination of Rho can reveal different functions of this GTPase in vivo.

J Biol Chem, 1999 Jun 25, 274(26), 18605 - 12
Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells; Meacci E et al.; Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways . Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro . We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate . To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins . Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity . Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N . In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme . Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible . To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange . BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e . it did not alter a rate-limiting step in PLD activation . It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway . A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.

Bone Marrow Transplant, 1999 May, 23(10), 1039 - 42
Incidence and outcome of Clostridium difficile infection following autologous peripheral blood stem cell transplantation; Bilgrami S et al.; A retrospective evaluation of 200 consecutive recipients of autologous peripheral blood stem cell transplantation (PBSCT) was conducted to ascertain the incidence and outcome of infection with Clostridium difficile . The diagnosis was confirmed in 14 patients with diarrhea (15 episodes) at a median of 33 days after stem cell infusion . Five patients were neutropenic at the time of diagnosis . Every individual had adverse known risk factors such as recent or current use of antibiotic, corticosteroid and antiviral therapy, recent administration of myeloablative chemotherapy and numerous, prolonged periods of hospitalization . Diarrhea, frequently hemorrhagic, was the most common presenting feature along with fever, abdominal cramps and abdominal distention . Diagnosis was established by the stool-cytotoxin test . Response to standard treatment with oral vancomycin or metronidazole was prompt despite the presence of several adverse prognostic features in these patients . There was only one instance of relapse which was also treated successfully . Several transplant-related variables such as age, sex, underlying malignancy, myelo-ablative regimen, duration of neutropenia, and prophylactic use of oral ampicillin underwent statistical analysis but failed to be predictive of C . difficile infection in such a setting . Finally, C . difficile is not uncommon after autologous PBSCT and must be included in the differential diagnosis in any such patient with diarrhea.

EMBO J, 1999 Jun 15, 18(12), 3442 - 50
Promoter upstream bent DNA activates the transcription of the Clostridium perfringens phospholipase C gene in a low temperature-dependent manner; Katayama S et al.; The phospholipase C gene (plc) of Clostridium perfringens possesses three phased A-tracts forming bent DNA upstream of the promoter . An in vitro transcription assay involving C.perfringens RNA polymerase (RNAP) showed that the phased A-tracts have a stimulatory effect on the plc promoter, and that the effect is proportional to the number of A-tracts, and more prominent at lower temperature . A gel retardation assay and hydroxyl radical footprinting revealed that the phased A-tracts facilitate the formation of the RNAP-plc promoter complex through extension of the contact region . The upstream (UP) element of the Escherichia coli rrnB P1 promoter stimulated the downstream promoter activity temperature independently, differing from the phased A-tracts . When the UP element was placed upstream of the plc promoter, low temperature-dependent stimulation was observed, although this effect was less prominent than that of the phased A-tracts . These results suggest that both the phased A-tracts and UP element cause low temperature-dependent activation of the plc promoter through a similar mechanism, and that the more efficient low temperature-dependent activation by the phased A-tracts may be due to an increase in the bending angle at a lower temperature.

J Bacteriol, 1999 Jun, 181(12), 3837 - 41
Biochemical and molecular characterization of the Bacillus subtilis acetoin catabolic pathway; Huang M et al.; A recent study indicated that Bacillus subtilis catabolizes acetoin by enzymes encoded by the acu gene cluster (F . J . Grundy, D . A . Waters, T . Y . Takova, and T . M . Henkin, Mol . Microbiol . 10:259-271, 1993) that are completely different from those in the multicomponent acetoin dehydrogenase enzyme system (AoDH ES) encoded by aco gene clusters found before in all other bacteria capable of utilizing acetoin as the sole carbon source for growth . By hybridization with a DNA probe covering acoA and acoB of the AoDH ES from Clostridium magnum, genomic fragments from B . subtilis harboring acoA, acoB, acoC, acoL, and acoR homologous genes were identified, and some of them were functionally expressed in E . coli . Furthermore, acoA was inactivated in B . subtilis by disruptive mutagenesis; these mutants were impaired to express PPi-dependent AoDH E1 activity to remove acetoin from the medium and to grow with acetoin as the carbon source . Therefore, acetoin is catabolized in B . subtilis by the same mechanism as all other bacteria investigated so far, leaving the function of the previously described acu genes obscure.

Am Surg, 1999 Jun, 65(6), 507 - 11; discussion 511-2
Clinical characteristics and antibiotic utilization in surgical patients with Clostridium difficile-associated diarrhea; Crabtree TD et al.; Clostridium difficile-associated diarrhea (CDAD) remains a significant problem in surgical patients . To address this, we prospectively studied all episodes of treated CDAD in surgical inpatients at the University of Virginia hospital from December 1996 through March 1998 . CDAD accounted for 3.2 per cent (32) of 1000 total infections . Compared with a randomly selected control group with other nosocomial infections, patients with CDAD had a longer period from the time of admission to diagnosis of infection (19 +/- 4 versus 9 +/- 1; P = 0.01), were more likely to be female (66% versus 37%; P = 0.009), and had a higher overall crude mortality (31% versus 11%; P = 0.01), although there were no deaths directly attributable to CDAD . Ciprofloxacin (19%) and cefoxitin (16%) were the most common individual antibiotics prescribed before the diagnosis of CDAD . The average time from completion of antibiotic therapy to diagnosis of CDAD was 7 +/- 2 days (range, 0-58) . Sixteen per cent (5 of 32) developed CDAD after administration of prophylactic perioperative antibiotics only . The high crude mortality rate associated with CDAD suggests that this may be a significant predictor of poor outcome among infected surgical patients . Antibiotics used commonly but not classically associated with CDAD frequently precipitate this infection . Finally, the use of prophylactic antibiotics is not without risk, as demonstrated by the significant percentage of CDAD occurring after routine administration of these agents.

J Clin Microbiol, 1999 Jul, 37(7), 2209 - 14
Molecular subtyping of Clostridium perfringens by pulsed-field gel electrophoresis to facilitate food-borne-disease outbreak investigations; Maslanka SE et al.; Clostridium perfringens is a common cause of food-borne illness . The illness is characterized by profuse diarrhea and acute abdominal pain . Since the illness is usually self-limiting, many cases are undiagnosed and/or not reported . Investigations are often pursued after an outbreak involving large numbers of people in institutions, at restaurants, or at catered meals . Serotyping has been used in the past to assist epidemiologic investigations of C . perfringens outbreaks . However, serotyping reagents are not widely available, and many isolates are often untypeable with existing reagents . We developed a pulsed-field gel electrophoresis (PFGE) method for molecular subtyping of C . perfringens isolates to aid in epidemiologic investigations of food-borne outbreaks . Six restriction endonucleases (SmaI, ApaI, FspI, MluI, KspI, and XbaI) were evaluated with a select panel of C . perfringens strains . SmaI was chosen for further studies because it produced 11 to 13 well-distributed bands of 40 to approximately 1,100 kb which provided good discrimination between isolates . Seventeen distinct patterns were obtained with 62 isolates from seven outbreak investigations or control strains . In general, multiple isolates from a single individual had indistinguishable PFGE patterns . Epidemiologically unrelated isolates (outbreak or control strains) had unique patterns; isolates from different individuals within an outbreak had similar, if not identical, patterns . PFGE identifies clonal relationships of isolates which will assist epidemiologic investigations of food-borne-disease outbreaks caused by C . perfringens.

J Biol Chem, 1999 Jun 18, 274(25), 17593 - 8
Evidence that MgATP accelerates primary electron transfer in a Clostridium pasteurianum Fe protein-Azotobacter vinelandii MoFe protein nitrogenase tight complex; Chan JM et al.; The nitrogenase catalytic cycle involves binding of the iron (Fe) protein to the molybdenum-iron (MoFe) protein, transfer of a single electron from the Fe protein to the MoFe protein concomitant with the hydrolysis of at least two MgATP molecules, followed by dissociation of the two proteins . Earlier studies found that combining the Fe protein isolated from the bacterium Clostridium pasteurianum with the MoFe protein isolated from the bacterium Azotobacter vinelandii resulted in an inactive, nondissociating Fe protein-MoFe protein complex . In the present work, it is demonstrated that primary electron transfer occurs within this nitrogenase tight complex in the absence of MgATP (apparent first-order rate constant k = 0.007 s-1) and that MgATP accelerates this electron transfer reaction by more than 10,000-fold to rates comparable to those observed within homologous nitrogenase complexes (k = 100 s-1) . Electron transfer reactions were confirmed by EPR spectroscopy . Finally, the midpoint potentials (Em) for the Fe protein {4Fe-4S}2+/+ cluster and the MoFe protein P2+/N cluster were determined for both the uncomplexed and complexed proteins and with or without MgADP . Calculations from electron transfer theory indicate that the measured changes in Em are not likely to be sufficient to account for the observed nucleotide-dependent rate accelerations for electron transfer.

Biochem J, 1999 Jun 15, 340 ( Pt 3), 829 - 35
Digestion of crystalline cellulose substrates by the clostridium thermocellum cellulosome: structural and morphological aspects; Boisset C et al.; The action of cellulosomes from Clostridium thermocellum on model cellulose microfibrils from Acetobacter xylinum and cellulose microcrystals from Valonia ventricosa was investigated . The biodegradation of these substrates was followed by transmission electron microscopy, Fourier-transform IR spectroscopy and X-ray diffraction analysis, as a function of the extent of degradation . The cellulosomes were very effective in catalysing the complete digestion of bacterial cellulose, but the total degradation of Valonia microcrystals was achieved more slowly . Ultrastructural observations during the digestion process suggested that the rapid degradation of bacterial cellulose was the result of a very efficient synergistic action of the various enzymic components that are attached to the scaffolding protein of the cellulosomes . The degraded Valonia sample assumed various shapes, ranging from thinned-down microcrystals to crystals where one end was pointed and the other intact . This complexity may be correlated with the multi-enzyme content of the cellulosomes and possibly to a diversity of the cellulosome composition within a given batch . Another aspect of the digestion of model celluloses by cellulosomes is the relative invariability of their crystallinity, together with their Ialpha/Ibeta composition throughout the degradation process . Comparison of the action of cellulosomes with that of fungal enzymes indicated that the degradation of cellulose crystals by cellulosomes occurred with only limited levels of processivity, in contrast with the observations reported for fungal enzymes . The findings were consistent with a mechanism whereby initial attack by a cellulosome of an individual cellulose crystal results in its 'commitment' towards complete degradation.

Int J Food Microbiol, 1999 Mar 15, 47(3), 161 - 9
Predicted and observed growth and toxigenesis by Clostridium botulinum type E in vacuum-packaged fishery product challenge tests; Hyytia E et al.; The observed growth and toxigenesis by Clostridium botulinum type E in vacuum-packaged unprocessed, raw pickled and cold-smoked rainbow trout stored at slightly abusive temperatures were compared to predictions generated by two currently available predictive microbiological programs, Food MicroModel and Pathogen Modelling Program . In unprocessed fish there was only a 2 log increase in type E cell count at the time the toxicity first occurred after 2 weeks storage at 8 degrees C . Neither growth or toxin production was observed in raw pickled fish with a NaCl concentration of 6.7% (w/v) during 6 weeks storage at 6 degrees C . In cold-smoked fish with a NaCl level of 3.2% (w/v) toxic samples were detected after 3 and 4 weeks storage at 8 degrees C and 4 degrees C, respectively, without any increase in type E count . Both models were hampered by limitations to controlling environmental factors set by the programs which also had an adverse effect on the reliability of predictions . Most predictions generated by the models were inconsistent with the results observed in the challenge studies . In certain situations, the models seemed to be 'fail-safe', in that, the growth rate predicted from the model was faster or a predicted time to toxicity shorter than that which actually occurred in the food . In other situations, the predictions showed the product to be safe when it was not . The results demonstrate the need for further development and rigorous validation of the models before they are accepted for wider use by inspecting officials and the food industry.

Int J Food Microbiol, 1999 Mar 1, 47(1-2), 121 - 31
Ribotyping as an identification tool for Clostridium botulinum strains causing human botulism; Hielm S et al.; Ribotyping was used for characterisation of 68 Clostridium botulinum strains and five related Clostridium species to determine the applicability of this method for identification of species causing human botulism . Thirteen restriction enzymes were initially tested for suitability for ribotyping of C . botulinum, of which EcoRI and HindIII were selected . Both enzymes clearly differentiated between proteolytic (group I) and a nonproteolytic (group II) strains of C . botulinum, and can be recommended for Group/species identification . Using a commercial software package (GelCompar), a numerical analysis of the discriminatory abilities of EcoRI and HindIII ribotyping within and between the two C . botulinum groups was performed . EcoRI had the higher discriminatory index (0.982), but the ribopatterns generated with group II strains were partly muddled and difficult to interpret . All HindIII ribopatterns were easy to analyse and the discriminatory index for all strains was almost equally high (0.954), whereas this enzyme did not discriminate well between group I isolates . The Clostridium strains diverged at 35+/-13% (mean+/-standard deviation) Dice similarity in dendrograms based on cluster analysis of the ribotyping results . These findings are in good agreement with taxonomical ribotyping studies with other bacterial genera, indicating that ribotyping is a highly suitable method for C . botulinum species identification.

Int J Food Microbiol, 1999 Mar 1, 47(1-2), 51 - 7
Risk analysis of the thermal sterilization process . Analysis of factors affecting the thermal resistance of microorganisms; Akterian SG et al.; A risk analysis was applied to experimental heat resistance data . This analysis is an approach for processing experimental thermobacteriological data in order to study the variability of D and z values of target microorganisms depending on the deviations range of environmental factors, to determine the critical factors and to specify their critical tolerance . This analysis is based on sets of sensitivity functions applied to a specific case of experimental data related to the thermoresistance of Clostridium sporogenes and Bacillus stearothermophilus spores . The effect of the following factors was analyzed: the type of target microorganism; nature of the heating substrate; pH, temperature; type of acid employed and NaCl concentration . The type of target microorganism to be inactivated, the nature of the substrate (reference or real food) and the heating temperature were identified as critical factors, determining about 90% of the alteration of the microbiological risk . The effect of the type of acid used for the acidification of products and the concentration of NaCl can be assumed to be negligible factors for the purposes of engineering calculations . The critical non-uniformity in temperature during thermobacteriological studies was set as 0.5% and the critical tolerances of pH value and NaCl concentration were 5% . These results are related to a specific case study, for that reason their direct generalization is not correct.

Curr Microbiol, 1999 May, 38(5), 264 - 7
Distribution of the rubredoxin gene among the Clostridium butyricum species; Gerard P et al.; With PCR methods, the rubredoxin gene was systematically identified among 11 strains of Clostridium butyricum; this ubiquity means major functions in the metabolism of the Clostridia . The 11 PCR products allowed deduction of a sequence of 26 amino acids corresponding to positions 11-36 of the rubredoxin . They all contained the tyrosines at positions 11 and 13 and the phenylalanine at position 30 characteristic of the rubredoxin, but differed at positions 14-17, 20, 25, 29, and 31, allowing determination of three types of rubredoxins among these 11 strains of C . butyricum.

Biochim Biophys Acta, 1999 May 31, 1454(1), 97 - 105
Clostridium perfringens beta-toxin is sensitive to thiol-group modification but does not require a thiol group for lethal activity; Nagahama M et al.; The beta-toxin gene isolated from Clostridium perfringens type B was expressed as a glutathione S-transferase (GST) fusion gene in Escherichia coli . The purified GST-beta-toxin fusion protein from the E . coli transformant cells was not lethal . The N-terminal amino acid sequence of the recombinant beta-toxin (r toxin) isolated by thrombin cleavage of the fusion protein was G-S-N-D-I-G-K-T-T-T . Biological activities and molecular mass of r toxin were indistinguishable from those of native beta-toxin (n toxin) purified from C . perfringens type C . Replacement of Cys-265 with alanine or serine by site-directed mutagenesis resulted in little loss of the activity . Treatment of C265A with N-ethylmaleimide (NEM), which inactivated lethal activity of r toxin and n toxin, led to no loss of the activity . The substitution of tyrosine or histidine for Cys-265 significantly diminished lethal activity . In addition, treatment of C265H with ethoxyformic anhydride which specifically modifies histidyl residue resulted in significant decrease in lethal activity, but that of r toxin with the agent did not . These results showed that replacement of the cysteine residue at position 265 with amino acids with large size of side chain or introduction of functional groups in the position resulted in loss of lethal activity of the toxin . Replacement of Tyr-266, Leu-268 or Trp-275 resulted in complete loss of lethal activity . Simultaneous administration of r toxin and W275A led to a decrease in lethal activity of beta-toxin . These observations suggest that the site essential for the activity is close to the cysteine residue.

Biochemistry, 1999 Jun 1, 38(22), 7168 - 76
The midpoint potentials for the oxidized-semiquinone couple for Gly57 mutants of the Clostridium beijerinckii flavodoxin correlate with changes in the hydrogen-bonding interaction with the proton on N(5) of the reduced flavin mononucleotide cofactor as measured by NMR chemical shift temperature dependencies; Chang FC et al.; In the Clostridium beijerinckii flavodoxin, the reduction of the flavin mononucleotide (FMN) cofactor is accompanied by a local conformation change in which the Gly57-Asp58 peptide bond "flips" from primarily the unusual cis O-down conformation in the oxidized state to the trans O-up conformation such that a new hydrogen bond can be formed between the carbonyl group of Gly57 and the proton on N(5) of the neutral FMN semiquinone radical {Ludwig, M . L., Pattridge, K . A., Metzger, A . L., Dixon, M . M., Eren, M., Feng, Y., and Swenson, R . P . (1997) Biochemistry 36, 1259-1280} . This interaction is thought to contribute to the relative stabilization of the flavin semiquinone and may be at least partially responsible for the substantial separation of the midpoint potentials of the two one-electron reduction steps . Through a series of amino acid substitutions, the above cited study demonstrated the critical role of the often conserved glycine residue in this process . However, it has not been directly established experimentally as to whether these substitutions brought about the changes in the midpoint potentials by altering the strength of this hydrogen-bonding interaction as proposed . In this study, the relative strengths of the FMN N(5)H.O57 hydrogen bond in wild type and the G57A, G57N, and G57T mutants were evaluated by measuring the temperature dependency of the chemical shift for the proton on N(5) of the fully reduced cofactor by 1H-15N HSQC nuclear magnetic resonance spectroscopy . Based on the established correlation between the temperature coefficient of amide protons and the strength of hydrogen bonding in small peptides, the apparent strength of the N(5)H.O57 interaction was found to depend on the properties of the side chain at position 57 . The glycine residue found in the wild-type flavodoxin appears to provide the strongest interaction while the beta-branched side chain in the G57T mutant provides the weakest . A good correlation was noted between the temperature coefficients of N(5)H and the one-electron reduction potential for the ox/sq couple as well as the binding free energy of the FMN semiquinone in this group of mutants . These results provide more direct quantitative evidence that support the previous hypothesis that this conformation change and the associated formation of the hydrogen bonding interaction with N(5)H of the reduced FMN represent an important means of stabilizing the neutral semiquinone and in modulating the oxidation-reduction potentials of the flavin cofactor in this and perhaps other flavodoxins.

Radiology, 1999 Jun, 211(3), 743 - 6
The accordion sign at CT: a nonspecific finding in patients with colonic edema; Macari M et al.; PURPOSE: To determine whether the "accordion sign" is a specific computed tomographic (CT) sign of Clostridium difficile colitis . MATERIALS AND METHODS: Fifty-seven patients with CT evidence of severe colitis, as judged by colonic wall thickening, an abnormal haustral pattern, the target sign, and stranding of the pericolic fat, were identified from a computerized CT database for 25 months . CT images were retrospectively evaluated for the presence of oral contrast material in the colon and for the accordion sign . The medical and laboratory records of all patients were reviewed and correlated with CT findings to establish the cause of colitis . RESULTS: Oral contrast material had reached the colon in 35 of 57 patients at the time of the CT examination . The images in 15 of these patients demonstrated the accordion sign, and those in 20 patients did not . C difficile colitis was documented in four of the 15 cases displaying the accordion sign . In the remaining 11 patients, a different cause was documented . Oral contrast material had not reached the colon in the remaining 22 patients . Within this group with findings similar to the accordion sign, five patients had documented C difficile colitis, and four had colitis from other causes . CONCLUSION: The accordion sign is indicative of severe colonic edema or inflammation, but it is not specific for C difficile colitis.

Biochemistry, 1999 May 25, 38(21), 6903 - 10
Enhancement of the endopeptidase activity of botulinum neurotoxin by its associated proteins and dithiothreitol; Cai S et al.; Botulinum neurotoxins type A (BoNT/A), the most toxic substance known to man, is produced by Clostridium botulinum type A as a complex with a group of neurotoxin-associated proteins (NAPs), possibly through a polycistronic expression of a clustered group of genes . The botulinum neurotoxin complex is the only known example of a protein complex where a group of proteins (NAPs) protect another protein (BoNT) against acidity and proteases of the GI tract . We now report that NAPs also potentiate the Zn2+ endopeptidase activity of BoNT/A in both in vitro and in vivo assays against its known intracellular target protein, 25 kDa synaptosomal associated protein (SNAP-25) . While BoNT/A exhibited no protease activity prior to reduction with dithiothreitol (DTT), the BoNT/A complex exhibited a high protease activity even in its nonreduced form . Our results suggest that the bacterial production of NAPs along with BoNT is designed for the NAPs to play an accessory role in the neurotoxin function, in contrast to their previously known limited role in protecting the neurotoxin in the GI tract and in the external environment . Structural features of BoNT/A change considerably upon disulfide reduction, as revealed by near-UV circular dichroism spectroscopy . BoNT/A in the reduced form adopts a more flexible structure than in the unreduced form, as also indicated by large differences in DeltaH values (155 vs 248 kJ mol-1) of temperature-induced unfolding of BoNT/A.

Rapid Commun Mass Spectrom, 1999, 13(8), 695 - 703
Characterization of surface layer proteins from Clostridium difficile by liquid chromatography/electrospray ionization mass spectrometry; Mauri PL et al.; Surface layers (S-layers) are regularly ordered protein subunits found as the outermost cell envelope component of many bacteria . Most S-layers are composed of a single protein or glycoprotein species with a molecular weight varying between 40 and 200 kDa . Clostridium difficile is the most common cause of antibiotic associated diarrhea (AAD) and pseudomembranous colitis (PMC) in humans . Detection of the S-layer in some C . difficile strains, and preliminary characterization of two glycoproteins, P36 and P47, involved in the composition of the S-layer of one of these strains (C . difficile C253), led us to investigate the most appropriate conditions for purification and chemical characterization of these proteins . This work describes the results obtained when liquid chromatography (LC) coupled to mass spectrometry (MS) using electrospray ionization was applied to the analysis of C . difficile S-layer proteins (SLPs) . In this way the molecular weights of the two SLP components, P36 and P47, were detected to be 34,258 +/- 2 and 39,545 +/- 3 Da, respectively . These data deviate from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) results by 1.85 and 7.5 kDa . To confirm the LC-MS results, an alternative molecular weight analysis was performed: the two S-layer proteins were isolated by semipreparative high performance liquid chromatography (HPLC), concentrated, and analyzed by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) . The two SLP subunits were digested with protease V8, and the peptide maps were determined by LC-MS using a C18 column . Finally, preliminary results about peptide glycosylation were obtained.

Curr Microbiol, 1999 Jun, 38(6), 324 - 8
Detection and transcription of toxin DNA in a nontoxigenic strain of Clostridium difficile; Mathis JN et al.; Genomic DNA from three Clostridium difficile strains was analyzed by PCR for DNA sequences encoding toxin A (tcdA) and toxin B (tcdB) . Toxigenic control strain VPI 10463 possessed tcdA, tcdB, and an open reading frame (tcdE) between these two genes, whereas nontoxigenic control strain 85 lacked each of these genetic determinants . However, strain M90, also a nontoxigenic strain, was found to possess tcdA, tcdB, and tcdE . Normally the presence of toxin genes is associated with toxigenicity . Analysis of tcdA and tcdB mRNA revealed toxin gene transcription in strains VPI 10463, 23 (a mildly toxigenic strain), and M90, but not in strain 85 . However, for strain M90, tcdA and tcdB mRNA was at the lower limit of detection, whereas mRNAs encoding tcdA and tcdB were easily detected in strains VPI 10463 and 23 . Low levels of toxin gene transcription is the probable cause of M90's lack of toxigenicity.

J Infect, 1999 Mar, 38(2), 128 - 9
Fatal Clostridium sordellii ischio-rectal abscess with septicaemia complicating ultrasound-guided transrectal prostate biopsy; Borer A et al.; Clostridium sordellii is a Gram-positive spore-forming anaerobic bacillus rarely encountered in human infection . A case of C . sordellii ischio-rectal abscess with rapidly fatal septicaemia is described which complicated ultrasound-guided transrectal biopsy of the prostate, despite ciprofloxacin prophylaxis . Neither C . sordellii ischio-rectal abscess nor ischio-rectal abscess complicating transrectal biopsy have been reported previously . Judging from our experience and the reviewed literature, the addition of prophylactic anti-anaerobe drugs should be strongly considered until an optimal prophylactic regimen will be defined by randomized controlled trials.

J Cell Sci, 1999 Jun, 112 ( Pt 12), 2019 - 32
Teneurin-1, a vertebrate homologue of the Drosophila pair-rule gene ten-m, is a neuronal protein with a novel type of heparin-binding domain; Minet AD et al.; The Drosophila gene ten-m is the first pair-rule gene not encoding a transcription factor, but an extracellular protein . We have characterized a highly conserved chicken homologue that we call teneurin-1 . The C-terminal part harbors 26 repetitive sequence motifs termed YD-repeats . The YD-repeats are most similar to the core of the rhs elements of Escherichia coli . Related repeats in toxin A of Clostridium difficile are known to bind specific carbohydrates . We show that recombinantly expressed proteins containing the YD-repeats of teneurin-1 bind to heparin . Furthermore, heparin lyase treatment of extracts of cells expressing recombinant YD-repeat protein releases this protein from high molecular mass aggregates . In situ hybridization and immunostaining reveals teneurin-1 expression in neurons of the developing visual system of chicken and Drosophila . This phylogenetic conservation of neuronal expression from flies to birds implies fundamental roles for teneurin-1 in neurogenesis . This is supported by the neurite outgrowth occurring on substrates made of recombinant YD-repeat proteins, which can be inhibited by heparin . Database searches resulted in the identification of ESTs encoding at least three further members of the teneurin family of proteins . Furthermore, the human teneurin-1 gene could be identified on chromosome Xq24/25, a region implied in an X-linked mental retardation syndrome.

J Food Prot, 1999 May, 62(5), 499 - 508
Microbiological quality and production of botulinal toxin in film-packaged broccoli, carrots, and green beans; Hao YY et al.; The production of toxin by a 10-strain mixture of proteolytic Clostridium botulinum in fresh produce packaged in polyethylene films with different oxygen permeability was determined . Broccoli florets, shredded carrots, and green beans inoculated with approximately 10(2) C . botulinum spores per g were placed in bags (1.4 kg per bag) composed of four films with different oxygen transmission rates (OTRs) . Broccoli was packaged in bags with OTRs of 3 (7,000 cm3/m2/24 h) and 4 (16,000 cm3/m2/24 h), and green beans were packaged in bags with OTRs of 2 (6,000 cm3/m2/24 h) and 4 . Broccoli and green beans in bags were compressed and heat-sealed . Shredded carrots were packaged in bags with OTRs of 1 (3,000 cm3/m2/24 h) and 3 and vacuum-sealed . Produce was stored at 4, 13, and 21 degrees C for up to 27 (broccoli) or 28 (carrots and green bean) days and analyzed periodically . At each sampling time, gas composition within the bags, pH of the produce microbial population (total aerobic and anaerobic microorganisms, lactic acid bacteria, psychrotrophic bacteria, yeasts, and molds), and the presence or absence of botulinal toxin were determined . Packaging material affected the quality of vegetables, especially broccoli stored at 4 and 13 degrees C . For example, broccoli was scored as "good" after 22 days at 4 degrees C when it was packaged in film with higher gas permeability (OTR of 4), whereas broccoli appeared to be in "poor" condition when packaged in film with lower gas permeability (OTR of 3) . With the exception of lactic acid bacteria, packaging material did not noticeably influence the growth of microorganisms . Lactic acid bacteria grew better in broccoli packaged in bags with an OTR of 3 than in those with an OTR of 4 at all temperatures . Botulinal toxin was detected in broccoli packaged in bags with an OTR of 3 and stored at 13 degrees C for 21 days and in those with an OTR of 4 and 3 and stored at 21 degrees C for 10 days . All toxic samples were visibly spoiled . Toxin was not detected in produce packaged under any other test conditions.

J Am Vet Med Assoc, 1999 May 15, 214(10), 1519 - 22, 1496
Presumed clostridial and aerobic bacterial infections of the cornea in two horses; Rebhun WC et al.; Microscopic examination of Gram-stained tissue specimens collected from severe corneal ulcers in 2 horses revealed large gram-positive rods suggestive of Clostridium spp . Clostridium perfringens was isolated from specimens collected from horse 1; anaerobic organisms were not detected in specimens from horse 2 . Aerobic bacterial culture revealed Aeromonas hydrophila and Enterobacter cloacae in specimens collected from horses 1 and 2, respectively . An insect exoskeleton was presumed to be the underlying cause of ulceration in horse 1 . Cause of ulceration in horse 2 was not determined . Antibiotics used to treat the corneal infections included ticarcillin disodium-clavulanic acid injected one time subconjunctivally and chloramphenicol applied topically at frequent intervals . Horse 2 also received penicillin or trimethoprim-sulfadiazine . Small leukomas were the only lesion remaining between 2 and 7 months after initial evaluation . Chloramphenicol applied topically appears to be an effective treatment against clostridial corneal infections in horses.

Infect Immun, 1999 Jun, 67(6), 3002 - 8
Effects of cytotoxic necrotizing factor 1 and lethal toxin on actin cytoskeleton and VE-cadherin localization in human endothelial cell monolayers; Vouret-Craviari V et al.; Integrity of the vascular endothelium is largely dependent on endothelial cell shape and establishment of intercellular junctions . Certain pathogenic bacterial toxins alter the cytoskeletal architecture of intoxicated cells by modulating the GTPase activity of p21 Rho family proteins . In the present study we have analyzed the effect of Rho-directed toxins on the actin cytoskeleton and monolayer integrity of endothelial cells . We report here that Escherichia coli cytotoxic necrotizing factor 1 (CNF1) activates Rho in human umbilical vein endothelial cells (HUVEC) . In confluent monolayers, CNF1 treatment induces prominent stress fiber formation without significantly modifying peripheral localization of VE-cadherin, a specific marker of vascular endothelial cell adherens junctions . Further, Rho activation with CNF1 blocks thrombin-induced redistribution of VE-cadherin staining and gap formation in HUVEC monolayers . Inhibition of Rho by prolonged treatment of cells with C3 exoenzyme (Clostridium botulinum) eliminates actin stress fibers without disrupting the continuity of VE-cadherin staining, indicating that Rho-dependent stress fibers are not required for maintaining this adhesion receptor at sites of intercellular contact . Lethal toxin (Clostridium sordellii), an inhibitor of Rac as well as Ras and Rap, potently disrupts the actin microfilament system and monolayer integrity in HUVEC cultures.

FEBS Lett, 1999 Apr 23, 449(2-3), 165 - 8
The energy conserving methyltetrahydromethanopterin:coenzyme M methyltransferase complex from methanogenic archaea: function of the subunit MtrH; Hippler B et al.; In methanogenic archaea the transfer of the methyl group of N5-methyltetrahydromethanopterin to coenzyme M is coupled with energy conservation . The reaction is catalyzed by a membrane associated multienzyme complex composed of eight different subunits MtrA-H . The 23 kDa subunit MtrA harbors a corrinoid prosthetic group which is methylated and demethylated in the catalytic cycle . We report here that the 34 kDa subunit MtrH catalyzes the methylation reaction . MtrH was purified and shown to exhibit methyltetrahydromethanopterin:cob(I)alamin methyltransferase activity . Sequence comparison revealed similarity of MtrH with MetH from Escherichia coli and AcsE from Clostridium thermoaceticum: both enzymes exhibit methyltetrahydrofolate:cob(I)alamin methyltransferase activity.

J Med Microbiol, 1999 Mar, 48(3), 235 - 43
Cationic currents induced by Clostridium perfringens type A enterotoxin in human intestinal CaCO-2 cells; Hardy SP et al.; Clostridium perfringens type A produces an enterotoxin that induces diarrhoea experimentally in man and animals . The enterotoxin causes increased membrane permeability in susceptible cells which is thought to be due to pore formation in the host cell membrane . The effect of purified C . perfringens enterotoxin on intact intestinal CaCO-2 monolayers was examined in Ussing chambers and on single cells by whole-cell patch clamp . Mucosal application of C . perfringens enterotoxin resulted in prompt increases in short-circuit current coupled with a reduction in transepithelial resistance consistent with movement of sodium and other cations smaller than diethanolamine from mucosa to serosa . These changes were independent of extracellular calcium . Increases in short-circuit current were also observed in the apical membranes of CaCO-2 monolayers permeabilised across the basolateral membrane with nystatin . Currents were blocked by subsequent exposure to mucosal barium and zinc . Zinc also prevented the development of the current increases in apical membranes . Cationic currents were also observed following exposure of single CaCO-2 cells in whole-cell patch clamp recordings . These data indicate that C . perfringens enterotoxin is able to form cation permeant pores in the apical membrane of human intestinal CaCO-2 epithelia and the increases in short-circuit current can be prevented by pre-exposure to zinc ions.

Vet Pathol, 1999 May, 36(3), 253 - 5
Naturally occurring Tyzzer's disease in a calf; Ikegami T et al.; Naturally occurring Clostridium piliforme infection (Tyzzer's disease) was found in a calf . Light microscopic examination revealed multifocal coagulative necrosis in the liver, catarrhal gastroenteritis, tracheitis and pneumonia, and thymic atrophy . Warthin-Starry staining clearly showed large filamentous bacilli in bundles or criss-cross patterns within the hepatocytes and epithelium and smooth muscle cells of the ileum and cecum . Immunohistochemistry using anti-C . piliforme RT and MSK strain antisera showed positive reaction against the bacilli . Electron microscopic examination revealed bacilli within the hepatocytes that demonstrated a characteristic vegetative form, with peritrichous flagella, and spores . The polymerase chain reaction (PCR) study using the paraffin-embedded liver sections, the 196-bp DNA fragment specific to 16S ribosomal RNA of C . piliforme was amplified . The characteristics of these bacilli are consistent with those of of C . piliforme . The PCR technique using paraffin-embedded sections should be useful for confirming C . piliforme infection in spontaneous cases.

J Chemother, 1999 Apr, 11(2), 90 - 2
In vitro activity of LY 333328 against anaerobic gram-positive bacteria; Sillerstrom E et al.; LY 333328 is a new semisynthetic glycopeptide with reported activity against aerobic Gram-positive cocci such as enterococci, pneumococci, streptococci and staphylococci . The present investigation was undertaken to determine the in vitro activity of LY 333328 against 178 Gram-positive anaerobic bacteria recently isolated from human infections . The activity was compared with that of vancomycin, teicoplanin, cefoxitin, imipenem, clindamycin and metronidazole . Peptostreptococci (48 strains): LY 333328, range 0.016-1.0 mg/l; vancomycin, 0.125-2.0 mg/l; teicoplanin, 0.032-2.0 mg/l; cefoxitin, 0.064-8.0 mg/l; imipenem, 0.016-0.064 mg/l; clindamycin, 0.016-1.0 mg/l; metronidazole, 0.125-8.0 mg/l . Propionibacterium acnes (31 strains): LY 333328, range 0.032-0.125 mg/l; vancomycin, 0.25-0.5 mg/l; teicoplanin, 0.25-0.5 mg/l; cefoxitin, 0.125-1.0 mg/l; imipenem, 0.032-0.064 mg/l; clindamycin, 0.016-0.064 mg/l; metronidazole, 32-> or =64 mg/l . Clostridium difficile (50 strains): LY 333328, range 0.016-2.0 mg/l; vancomycin, 0.5-4.0 mg/l; teicoplanin, 0.064-0.5 mg/l; cefoxitin, 64-128 mg/l; imipenem, 8.0 mg/l; clindamycin, 0.25-128 mg/l; metronidazole, 0.125-0.25 mg/l . Clostridium perfringens (49 strains): LY 333328, range 0.016-2.0 mg/l; vancomycin, 0.025-4.0 mg/l; teicoplanin, 0.064-4.0 mg/l; cefoxitin, 0.5-1.0 mg/l; imipenem, 0.016-0.5 mg/l; clindamycin, 0.008-1.0 mg/l; metronidazole, 1.0-4.0 mg/l . LY 333328 had an excellent in vitro activity against anaerobic Gram-positive bacteria.

J Bacteriol, 1999 May, 181(10), 3270 - 6
Three surface layer homology domains at the N terminus of the Clostridium cellulovorans major cellulosomal subunit EngE; Tamaru Y et al.; The gene engE, coding for endoglucanase E, one of the three major subunits of the Clostridium cellulovorans cellulosome, has been isolated and sequenced . engE is comprised of an open reading frame (ORF) of 3,090 bp and encodes a protein of 1,030 amino acids with a molecular weight of 111,796 . The amino acid sequence derived from engE revealed a structure consisting of catalytic and noncatalytic domains . The N-terminal-half region of EngE consisted of a signal peptide of 31 amino acid residues and three repeated surface layer homology (SLH) domains, which were highly conserved and homologous to an S-layer protein from the gram-negative bacterium Caulobacter crescentus . The C-terminal-half region, which is necessary for the enzymatic function of EngE and for binding of EngE to the scaffolding protein CbpA, consisted of a catalytic domain homologous to that of family 5 of the glycosyl hydrolases, a domain of unknown function, and a duplicated sequence (DS or dockerin) at its C terminus . engE is located downstream of an ORF, ORF1, that is homologous to the Bacillus subtilis phosphomethylpyrimidine kinase (pmk) gene . The unique presence of three SLH domains and a DS suggests that EngE is capable of binding both to CbpA to form a CbpA-EngE cellulosome complex and to the surface layer of C . cellulovorans.

J Bacteriol, 1999 May, 181(10), 3262 - 9
Carbon and electron flow in Clostridium cellulolyticum grown in chemostat culture on synthetic medium; Guedon E et al.; Previous results indicated poor sugar consumption and early inhibition of metabolism and growth when Clostridium cellulolyticum was cultured on medium containing cellobiose and yeast extract . Changing from complex medium to a synthetic medium had a strong effect on (i) the specific cellobiose consumption, which was increased threefold; and (ii) the electron flow, since the NADH/NAD+ ratios ranged from 0.29 to 2.08 on synthetic medium whereas ratios as high as 42 to 57 on complex medium were observed . These data indicate a better control of the carbon flow on mineral salts medium than on complex medium . By continuous culture, it was shown that the electron flow from glycolysis was balanced by the production of hydrogen gas, ethanol, and lactate . At low levels of carbon flow, pyruvate was preferentially cleaved to acetate and ethanol, enabling the bacteria to maximize ATP formation . A high catabolic rate led to pyruvate overflow and to increased ethanol and lactate production . In vitro, glyceraldehyde-3-phosphate dehydrogenase, lactate dehydrogenase, and ethanol dehydrogenase levels were higher under conditions giving higher in vivo specific production rates . Redox balance is essentially maintained by NADH-ferredoxin reductase-hydrogenase at low levels of carbon flow and by ethanol dehydrogenase and lactate dehydrogenase at high levels of carbon flow . The same maximum growth rate (0.150 h-1) was found in both mineral salts and complex media, proving that the uptake of nutrients or the generation of biosynthetic precursors occurred faster than their utilization . On synthetic medium, cellobiose carbon was converted into cell mass and catabolized to produce ATP, while on complex medium, it served mainly as an energy supply and, if present in excess, led to an accumulation of intracellular metabolites as demonstrated for NADH . Cells grown on synthetic medium and at high levels of carbon flow were able to induce regulatory responses such as the production of ethanol and lactate dehydrogenase.

Surg Neurol, 1999 May, 51(5), 568 - 70
Postcraniotomy gas-containing brain abscess: a neurosurgical emergency . Case report; Cohen JE et al.; BACKGROUND: Gas-containing brain abscesses are very rare, and the majority are caused by Clostridium perfringens . We report a case of gas-containing brain abscess that required urgent surgery after a craniotomy for a brain tumor . METHODS AND RESULTS: The patient was a 53-year-old male who presented with a cerebral neoplasm . A temporal lobectomy was performed and the diagnosis of low grade glioma was confirmed . Although the surgery was uneventful the postoperative course was complicated; the patient became agitated and febrile and deteriorated to a deep coma . A computed tomography scan demonstrated gas in the temporal fossa at the lobectomy site, producing mass effect . Urgent surgical debridement and drainage was performed and C . perfringens and mixed flora were found . Antibiotics were started and the patient's condition markedly improved . He was awake and alert, followed commands adequately and was extubated; however, after a week he suffered massive gastrointestinal bleeding and died . CONCLUSIONS: Early recognition of a gas-containing brain abscess is of great interest to immediately start the appropriate treatment . Urgent surgical debridement and broad spectrum chemotherapy are major components in the management of this entity.

Biochim Biophys Acta, 1999 May 5, 1411(2-3), 475 - 88
Nitrite and nitrosyl compounds in food preservation; Cammack R et al.; Nitrite is consumed in the diet, through vegetables and drinking water . It is also added to meat products as a preservative . The potential risks of this practice are balanced against the unique protective effect against toxin-forming bacteria such as Clostridium botulinum . The chemistry of nitrite, and compounds derived from it, in food systems and bacterial cells are complex . It is known that the bactericidal species is not nitrite itself, but a compound or compounds derived from it during food preparation . Of a range of nitrosyl compounds tested, the anion of Roussin's black salt {Fe4S3(NO)7}- was the most inhibitory to C . sporogenes . This compound is active against both anaerobic and aerobic food-spoilage bacteria, while some other compounds are selective, indicating multiple sites of action . There are numerous possible targets for inhibition in the bacterial cells, including respiratory chains, iron-sulfur proteins and other metalloproteins, membranes and the genetic apparatus.

Neurogastroenterol Motil, 1999 Apr, 11(2), 79 - 92
The neurology and enterology of equine grass sickness: a review of basic mechanisms; Cottrell DF et al.; Autonomic dysfunction constitutes a prominent clinical feature of equine grass sickness (EGS) . Significant injury to the nervous control of the alimentary system is life threatening, partly because of dysphagia but also because of the failure of the unique regulatory mechanisms in equine digestion involving water and electrolyte balance . The neuropathology also indicates the presence of a somatic polyneuropathy . The morphological features of EGS are similar to those of excitotoxic neuronal degeneration, which resembles neuronal apoptosis . It is difficult to ascertain from published accounts the degree of damage to central neurones: the distribution is well documented and selective but the proportion of damage is poorly quantified . If lesions involve a significant number of regulatory neurones they should produce functional deficits . Any clinical assessment of horses, especially those with chronic EGS, should include a thorough neurological examination . Although this will not necessarily improve the outcome of the case, it may enable the rational selection of animals with a reasonable prognosis for recovery which is partly determined by the extent of CNS lesions . The evidence supports the following pathogenesis . There is an initial lesion in the enteric nervous system of susceptible horses . In the acute form of EGS, massive enteric neuronal damage occurs first functionally, then structurally leading to generalized alimentary smooth muscle atony, enhanced secretions and altered fluid fluxes . Severe distension of the stomach and small intestines rapidly develops, which augments the intestinal ileus by intersegmental inhibitory reflexes and causes colic and dehydration . In subacute cases, failure of intestinal bicarbonate buffer together with alimentary stasis rapidly reduces caecal-colonic fermentation . Thus the osmolality of large intestinal digesta reduces and water travels out of the bowel along osmotic gradients . Water returns to the circulation, but is eventually lost in the gastric and small intestinal secretions . The observation that pathological lesions may not be seen in the prevertebral ganglia within the first few days of acute cases supports the view that a functional deficit precedes structural lesions which may be secondary to a retrograde degeneration . It is therefore possible to resolve the observations that less damage may be seen in prevertebral ganglia and elsewhere in peracute and acute cases with the more common finding that greater neuronal damage is present in acute than in chronic cases . These different observations are probably time dependent . Chronic EGS occurs when there is less initial enteric nerve damage which may lead to less secondary prevertebral ganglionic pathology, and more time for functional and structural compensatory mechanisms to develop . Denervation hypersensitivity develops at target sites both in the gut and in peripheral somatic nerves which may account, in part, for the clinical signs of patchy sweating and muscle tremors . Raised circulating adrenaline levels may also account for generalized sweating, may contribute to gastrointestinal atony and may affect pacemakers at the pelvic flexure . Many of the features of EGS make worthwhile the re-investigation of Clostridium botulinum Group III toxins, which are known to prevent vesicular exocytosis, stimulate neurosecretion, produce neuronal chromatolysis and inhibit neutrophil migration . Also, evidence from other species suggests that increased nitrergic neuronal activity can account for many of the clinical signs of EGS, namely dysphagia, generalized ileus, gastric dilatation, sweating, peripheral vasodilatation, tachycardia, salivary hypersecretion, muscle wastage and cachexia.

J Biol Chem, 1999 May 14, 274(20), 14021 - 31
G-protein-stimulated phospholipase D activity is inhibited by lethal toxin from Clostridium sordellii in HL-60 cells; El Hadj NB et al.; Lethal toxin (LT) from Clostridium sordellii has been shown in HeLa cells to glucosylate and inactivate Ras and Rac and, hence, to disorganize the actin cytoskeleton . In the present work, we demonstrate that LT treatment provokes the same effects in HL-60 cells . We show that guanosine 5'-O-(3-thiotriphosphate)-stimulated phospholipase D (PLD) activity is inhibited in a time- and dose-dependent manner after an overnight treatment with LT . A similar dose response to the toxin was found when PLD activity was stimulated by phorbol 12-myristate 13-acetate via the protein kinase C pathway . The toxin effect on actin organization seemed unlikely to account directly for PLD inhibition as cytochalasin D and iota toxin from Clostridium perfringens E disorganize the actin cytoskeleton without modifying PLD activity . However, the enzyme inhibition and actin cytoskeleton disorganization could both be related to a major decrease observed in phosphatidylinositol 4,5-bisphosphate (PtdIns(4, 5)P2) . Likely in a relationship with this decrease, recombinant ADP-ribosylation factor, RhoA, Rac, and RalA were not able to reconstitute PLD activity in LT-treated cells permeabilized and depleted of cytosol . Studies of phosphoinositide kinase activities did not allow us to attribute the decrease in PtdIns(4,5)P2 to inactivation of PtdIns4P 5-kinase . LT was also found to provoke a major inhibition in phosphatidylinositol 3-kinase that could not account for the inhibition of PLD activity because wortmannin, at doses that fully inhibit phosphatidylinositol 3-kinase, had no effect on the phospholipase activity . Among the three small G-proteins, Ras, Rac, and RalA, inactivated by LT and involved in PLD regulation, inactivation of Ral proteins appeared to be responsible for PLD inhibition as LT toxin (strain 9048) unable to glucosylate Ral proteins did not modify PLD activity . In HL-60 cells, LT treatment appeared also to modify cytosol components in relationship with PLD inhibition as a cytosol prepared from LT-treated cells was less efficient than one from control HL-60 cells in stimulating PLD activity . Phosphatidylinositol transfer proteins involved in the regulation of polyphosphoinositides and ADP-ribosylation factor, a major cytosolic PLD activator in HL-60 cells, were unchanged, whereas the level of cytosolic protein kinase Calpha was decreased after LT treatment . We conclude that in HL-60 cells, lethal toxin from C . sordellii, in inactivating small G-proteins involved in PLD regulation, provokes major modifications at the membrane and the cytosol levels that participate in the inhibition of PLD activity . Although Ral appeared to play an essential role in PLD activity, we discuss the role of other small G-proteins inactivated by LT in the different modifications observed in HL-60 cells.

Am J Gastroenterol, 1999 May, 94(5), 1327 - 31
Bacterial populations contaminating the upper gut in patients with small intestinal bacterial overgrowth syndrome; Bouhnik Y et al.; OBJECTIVE: Small intestinal bacterial overgrowth syndrome (SIBOS) is characterized by an abnormally high bacterial population level in the upper gut, exceeding 10(5) organisms/ml (5 log colony-forming unit (CFU)/ml) . To understand its origin and select an appropriate antibiotic treatment, we have analyzed the bacterial populations contaminating the upper gut in SIBOS patients . METHODS: Jejunal samples of 63 consecutive patients with diarrhea or malabsorption and conditions predisposing to SIBOS were cultured and antibiotic sensitivities determined . RESULTS: Concentrations of total, microaerophilic, and anaerobic bacteria were confirmed in 55 patients with SIBOS (mean +/- SE) 7.6 +/- 0.8, 7.4 +/- 0.9, and 6.1 +/- 0.7 log CFU/ml, respectively . Mean number of bacterial genera was 4.6 +/- 0.8 . The main bacteria recovered were (mean +/- SE log CFU/ml) Streptococcus (71%; 6.4 +/- 0.8), Escherichia coli (69%; 7.2 +/- 0.9), Staphylococcus (25%; 6.2 +/- 0.6), Micrococcus (22%; 6.0 +/- 0.7), Klebsiella (20%; 7.1 +/- 0.8), Proteus (11%; 6.1 +/- 0.8) for microaerophilic bacteria, and Lactobacillus (75%; 6.1 +/- 1.1), Bacteroides (29%; 6.9 +/- 1.3), Clostridium (25%; 5.5 +/- 1.0), Veillonella (25%; 5.3 +/- 0.7), Fusobacterium (13%; 4.8 +/- 0.5), and Peptostreptococcus (13%; 6.1 +/- 0.7) for anaerobic bacteria . Amoxicillin-clavulanic acid and cefoxitin were efficient on >90% of strains . CONCLUSIONS: Contaminating flora isolated in SIBOS include commonly identified oropharyngeal and colonic flora, but these occur in SIBOS at different levels from those usually found in their original location . These data may hopefully serve as a starting point to further therapeutic controlled studies.

Biochemistry, 1999 May 4, 38(18), 5736 - 45
Binding of (6R,S)-methyltetrahydrofolate to methyltransferase from Clostridium thermoaceticum: role of protonation of methyltetrahydrofolate in the mechanism of methyl transfer; Seravalli J et al.; The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoacetium catalyzes transfer of the N5-methyl group of (6S)-methyltetrahydrofolate (CH3-H4folate) to the cob(I)amide center of a corrinoid/iron-sulfur protein (CFeSP), forming H4folate and methylcob(III)amide . We have investigated binding of 13C-enriched (6R,S)-CH3-H4folate and (6R)-CH3-H4folate to MeTr by 13C NMR, equilibrium dialysis, fluorescence quenching, and proton uptake experiments . The results described here and in the accompanying paper {Seravalli, J., Shoemaker, R . K., Sudbeck, M . J., and Ragsdale, S . W . (1999) Biochemistry 38, 5728-5735} constitute the first evidence for protonation of the pterin ring of CH3-H4folate . The pH dependence of the chemical shift in the 13C NMR spectrum for the N5-methyl resonance indicates that MeTr decreases the acidity of the N5 tertiary amine of CH3-H4folate by 1 pK unit in both water and deuterium oxide . Binding of (6R,S)-CH3H4folate is accompanied by the uptake of one proton . These results are consistent with a mechanism of activation of CH3-H4folate by protonation to make the methyl group more electrophilic and the product H4folate a better leaving group toward nucleophilic attack by cob(I)amide . When MeTr is present in excess over (6R,S)-13CH3-H4folate, the 13C NMR signal is split into two broad signals that reflect the bound states of the two diastereomers . This unexpected ability of MeTr to bind both isomers was confirmed by the observation of MeTr-bound (6R)-13CH3-H4folate by NMR and by the measurement of similar dissociation constants for (6R)- and (6S)-CH3-H4folate diastereomers by fluorescence quenching experiments . The transversal relaxation time (T2) of 13CH3-H4folate bound to MeTr is pH independent between pH 5.50 and 7.0, indicating that neither changes in the protonation state of bound CH3-H4folate nor the previously observed pH-dependent MeTr conformational change contribute to broadening of the 13C resonance signal . The dissociation constant for (6R,S)-CH3-H4folate is also pH independent, indicating that the role of the pH-dependent conformational change is to stabilize the transition state for methyl transfer, and not to favor the binding of CH3-H4folate.

Biochemistry, 1999 May 4, 38(18), 5728 - 35
Mechanism of transfer of the methyl group from (6S)-methyltetrahydrofolate to the corrinoid/iron-sulfur protein catalyzed by the methyltransferase from Clostridium thermoaceticum: a key step in the Wood-Ljungdahl pathway of acetyl-CoA synthesis; Seravalli J et al.; The methyltetrahydrofolate:corrinoid/iron-sulfur protein methyltransferase (MeTr) from Clostridium thermoaceticum catalyzes transfer of the N5-methyl group from (6S)-methyltetrahydrofolate (CH3-H4folate) to the cobalt center of a corrinoid/iron-sulfur protein (CFeSP), forming methylcob(III)amide and H4folate . This reaction initiates the unusual biological organometallic reaction sequence that constitutes the Wood-Ljungdahl or reductive acetyl-CoA pathway . The present paper describes the use of steady-state, product inhibition, single-turnover, and kinetic simulation experiments to elucidate the mechanism of the MeTr-catalyzed reaction . These experiments complement those presented in the companion paper in which binding and protonation of CH3-H4folate are studied by spectroscopic methods {Seravalli, J., Shoemaker, R . K., Sudbeck, M . J., and Ragsdale, S . W . (1999) Biochemistry 38, 5736-5745} . Our results indicate that a pH-dependent conformational change is required for methyl transfer in the forward and reverse directions; however, this step is not rate-limiting . CH3-H4folate and the CFeSP {in the cob(I)amide state} bind randomly and independently to form a ternary complex . Kinetic simulation studies indicate that CH3-H4folate binds to MeTr in the unprotonated form and then undergoes rapid protonation . This protonation enhances the electrophilicity of the methyl group, in agreement with a 10-fold increase in the pKa at N5 of CH3-H4folate . Next, the Co(I)-CFeSP attacks the methyl group in a rate-limiting SN2 reaction to form methylcob(III)amide . Finally, the products randomly dissociate . The following steady-state constants were obtained: kcat = 14.7 +/- 1.7 s-1, Km of the CFeSP = 12 +/- 4 microM, and Km of (6S)-CH3-H4folate = 2.0 +/- 0.3 microM . We assigned the rate constants for the elementary reaction steps by performing steady-state and pre-steady-state kinetic studies at different pH values and by kinetic simulations.

Eur J Ophthalmol, 1999 Jan-Mar, 9(1), 21 - 31
Post-traumatic endophthalmitis: causative organisms and visual outcome; Abu el-Asrar AM et al.; PURPOSE: Post-traumatic endophthalmitis makes up a distinct subset of intraocular infections . The purpose of the present study was to identify the causative organisms and record the visual outcome after infectious endophthalmitis in eyes with penetrating trauma . METHODS: We reviewed 18 consecutive cases of culture-positive endophthalmitis that developed after penetrating ocular trauma . All cases were treated with pars plana vitrectomy and intravenous and intraocular antibiotics . RESULTS: The 15 males and 3 females ranged in age from 4 to 43 years (mean 25.1 +/- 11 years) . Nine (50%) had intraocular foreign bodies . A single species was isolated in 16 cases, and multiple organisms in two . Staphylococcus epidermidis and gram-negative organisms were the most frequent and were cultured either alone or in association with other organisms in respectively five (27.7%) and four cases (22.2%) . Clostridium perfringens was isolated in three cases (16.6%) . Bacillus was not found as a cause of endophthalmitis . Final visual acuity was better than 20/400 in eight cases (44%) . In five cases (27.7%), the eye was saved but visual acuity was counting fingers . Two eyes (11%) had no light perception . The remaining three eyes (16.6%) were enucleated or eviscerated . Clostridium perfringens was isolated from two eyes and Aspergillus niger from one . Postoperative retinal detachment developed in four eyes, which were successfully operated . CONCLUSIONS: Organisms isolated in this series were similar to those in previous reports of post-traumatic endophthalmitis from other parts of the world, except that the frequency of Clostridium perfringens isolation was high and no Bacillus species were cultured . In view of its devastating outcome, post-traumatic endophthalmitis must be treated promptly with vitrectomy and intravitreal antibiotics.

Hepatogastroenterology, 1999 Jan-Feb, 46(25), 343 - 8
The diagnosis and treatment of Clostridium difficile in antibiotic-associated diarrhea; Gorenek L et al.; BACKGROUND/AIMS: This study was initiated to evaluate the role of C . difficile in diarrhea associated with the use of antibiotics, to determine which antibiotics are most often responsible, to characterize the response to several different treatment regimens, and to define the relapse rate as seen in a large teaching hospital in Turkey . METHODOLOGY: Three different patient groups were studied . The first group consisted of 154 individuals with antibiotic-associated diarrhea . The stools of all 154 cases were cultured on cycloserine-cefoxitin-fructose agar (CCFA) . If any bacteria grew out, they were identified specifically as C . difficile using a commercially available latex agglutination kit specific for bacterial antigens of C . difficile (MicroScreen C . difficile Latex Slide Test; Merica Diagnostic Limited, Guilford, England) . The presence of toxin-A (CDTA) was determined using a MicroScreen CDTA Enzyme Immunoassay kit . RESULTS: The stools of 31 of these patients grew out enteric pathogens . Twenty-eight of these 31 were CCFA positive . Three different drug regimens (Ornidazole, Ornidazole + Cholestyramine, and Vancomycin) were used to treat these 28 C . difficile positive cases . The second group consisted of 37 hospitalized patients who had been in hospital for more than 30 days without any gastrointestinal symptoms . This group was used to identify the in-hospital carrier rate for C . difficile . Stools from these 37 cases were cultured on CCFA and were analyzed for the presence of CDTA by EIA . Colonization with C . difficile was detected in 4 cases . The third group consisted of 40 healthy subjects who served as a population-based control group . The stools obtained from these 40 cases were cultured on CCFA and analyzed for CDTA as were the stools for the other 2 groups . None were CDTA positive . One case was positive for the presence of non-toxigenic C . difficile . CONCLUSIONS: It can be concluded from these data that, in Turkey, C . difficile is responsible for 20% of antibiotic-associated diarrheas . Lincomycin, Azithromycin and Ampicillin were most often associated with the development of antibiotic-associated diarrhea . Ornidazole and Vancomycin were effective agents for C . difficile-associated diarrhea with the latter agent being associated with no relapses.

FEMS Microbiol Lett, 1999 Apr 15, 173(2), 467 - 73
Germination-specific cortex-lytic enzymes from Clostridium perfringens S40 spores: time of synthesis, precursor structure and regulation of enzymatic activity; Urakami K et al.; Germination-specific enzymes, an amidase and a muramidase, of Clostridium perfringens S40 were synthesized at the time of forespore formation during sporulation . The amidase had a unique precursor structure consisting of four domains: the N-terminal pre-sequence, the N-terminal pro-sequence, mature enzyme and the C-terminal pro-sequence . The N-terminal pre-sequence and the C-terminal pro-sequence were sequentially processed at the time of development of phase-bright spores, and the resulting inactive pro-enzyme was activated by cleavage of the N-terminal pro-sequence with a specific protease during germination . A possible mechanism for the regulation of activity of muramidase, which is produced as a mature form and does not need processing for activation, is presented.

Infect Immun, 1999 May, 67(5), 2145 - 52
Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A; Ward SJ et al.; The C-terminal repeat domain of Clostridium difficile toxin A harbors toxin-neutralizing epitopes and is considered to be a candidate component of a vaccine against C . difficile-associated disease (CDAD) . Fourteen of the 38 C-terminal toxin A repeats (14CDTA) were cloned into pTECH-1 in frame with the immunogenic fragment C of tetanus toxin (TETC) to generate plasmid p56TETC . Expression of the TETC-14CDTA fusion protein was driven from the anaerobically inducible nirB promoter within attenuated Salmonella typhimurium BRD509 (aroA aroD) . The TETC-14CDTA fusion protein was purified and shown to bind to known toxin A receptors found on the surface of rabbit erythrocytes . Intranasal (i.n.) and intragastric (i.g.) immunization with 10(7) and 10(10) CFU, respectively, of BRD509(p56TETC) generated significant (P < 0.05) anti-toxin A serum responses after a single dose . Antibody titers were elevated following a boosting dose with either live vaccine or a subcutaneous injection of 0.5 microgram of purified 14CDTA protein . Importantly, serum from mice immunized with BRD509(p56TETC) neutralized toxin A cytotoxicity . Both i.n . and i.g . immunizations also generated toxin A-specific immunoglobulin A on the pulmonary and intestinal mucosa, respectively . Intranasal vaccination induced consistently higher serum and mucosal anti-toxin A antibody responses . Significant anti-tetanus toxoid serum and mucosal antibodies were also generated by both immunization routes . The availability of live attenuated Salmonella typhi for human use may allow the development of a multivalent mucosal vaccine against CDAD, tetanus, and typhoid.

Appl Environ Microbiol, 1999 May, 65(5), 2269 - 71
Examination of physiological and molecular factors involved in enhanced solvent production by clostridium beijerinckii BA101
Chen CK, Blaschek HP.
The specific activities and the mRNA expression levels associated with coenzyme A transferase, acetoacetate decarboxylase, and butyraldehyde dehydrogenase were elevated in hyper-solvent-producing Clostridium beijerinckii BA101 during the exponential growth phase . The increase in expression of the sol operon and associated enzyme activities may be responsible for enhanced solvent production by C . beijerinckii BA101.

Appl Environ Microbiol, 1999 May, 65(5), 2136 - 42
Growth from spores of nonproteolytic Clostridium botulinum in heat-treated vegetable juice; Stringer SC et al.; Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media . With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80 degrees C . The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media . Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80 degrees C for 100 min . We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C . botulinum that can lead to colony formation.

Appl Environ Microbiol, 1999 May, 65(5), 2057 - 64
Biodiversity of Clostridium botulinum type E strains isolated from fish and fishery products; Hyytia E et al.; The genetic biodiversity of Clostridium botulinum type E strains was studied by pulsed-field gel electrophoresis (PFGE) with two macrorestriction enzymes (SmaI-XmaI and XhoI) and by randomly amplified polymorphic DNA (RAPD) analysis with two primers (OPJ 6 and OPJ 13) to characterize 67 Finnish isolates from fresh fish and fishery products, 15 German isolates from farmed fish, and 10 isolates of North American or North Atlantic origin derived mainly from different types of seafood . The effects of fish species, processing, and geographical origin on the epidemiology of the isolates were evaluated . Cluster analysis based on macrorestriction profiles was performed to study the genetic relationships of the isolates . PFGE and RAPD analyses were combined and resulted in the identification of 62 different subtypes among the 92 type E isolates analyzed . High genetic biodiversity among the isolates was observed regardless of their source . Finnish and North American or North Atlantic isolates did not form distinctly discernible clusters, in contrast with the genetically homogeneous group of German isolates . On the other hand, indistinguishable or closely related genetic profiles among epidemiologically unrelated samples were detected . It was concluded that the high genetic variation was probably a result of a lack of strong selection factors that would influence the evolution of type E . The wide genetic biodiversity observed among type E isolates indicates the value of DNA-based typing methods as a tool in contamination studies in the food industry and in investigations of botulism outbreaks.

Antimicrob Agents Chemother, 1999 May, 43(5), 1105 - 10
Heterogeneity in the vanB gene cluster of genomically diverse clinical strains of vancomycin-resistant enterococci; Dahl KH et al.; Molecular analysis of 17 genomically unrelated clinical VanB-type vancomycin-resistant enterococcus isolates from hospital patients in Germany, Norway, Sweden, the United Kingdom and the United States revealed three subtypes of the vanB gene cluster-vanB1, vanB2, and vanB3-which was in accordance with previous subtyping of the ligase gene sequence . There was no correlation between vanB subtype and levels of vancomycin resistance . All strains studied carried a structurally conserved vanB gene cluster as shown by long-range PCR (long PCR) covering 5,959 bp of the published sequence in vanB1 strain V583 . Restriction analysis of long PCR amplicons displayed one unique vanB1 pattern and a second vanB2- and vanB3-specific pattern . The vanSB-vanYB intergenic sequences with flanking coding regions were identical within each vanB subtype with one exception . A U.S . vanB2 isolate had a 789-bp enlargement of this region containing a putative open reading frame (ORF) with substantial homology to an ORF in the Clostridium perfringens IS1469 insertion element . The molecular heterogeneity within the vanB gene cluster has implications for the selection of PCR primers, as the primers must ensure detection of all vanB subtypes, and is of importance when considering reservoirs and dissemination of vanB resistance . The molecular identity within the vanB1 and the vanB2 subtype indicates horizontal transmission of both gene clusters between isolates in different geographical areas . Restriction analysis of long PCR vanB amplicons may reveal specific varieties that can be used as epidemiological markers for mobile determinants conferring VanB-type resistance . The finding of three distinct vanB gene clusters should encourage a search for different environmental reservoirs of vanB resistance determinants.

Antimicrob Agents Chemother, 1999 May, 43(5), 1077 - 84
In vitro and in vivo antimicrobial activities of T-3811ME, a novel des-F(6)-quinolone; Takahata M et al.; The in vitro and in vivo activities of T-3811ME, a novel des-F(6)-quinolone, were evaluated in comparison with those of some fluoroquinolones, including a newly developed one, trovafloxacin . T-3811, a free base of T-3811ME, showed a wide range of antimicrobial spectra, including activities against Chlamydia trachomatis, Mycoplasma pneumoniae, and Mycobacterium tuberculosis . In particular, T-3811 exhibited potent activity against various gram-positive cocci, with MICs at which 90% of the isolates are inhibited (MIC90s) of 0.025 to 6.25 microgram/ml . T-3811 was the most active agent against methicillin-resistant Staphylococcus aureus and streptococci, including penicillin-resistant Streptococcus pneumoniae (PRSP) . T-3811 also showed potent activity against quinolone-resistant gram-positive cocci with GyrA and ParC (GrlA) mutations . The activity of T-3811 against members of the family Enterobacteriaceae and nonfermentative gram-negative rods was comparable to that of trovafloxacin . In common with other fluoroquinolones, T-3811 was highly active against Haemophilus influenzae, Moraxella catarrhalis, and Legionella sp., with MIC90s of 0.0125 to 0.1 microgram/ml . T-3811 showed a potent activity against anaerobic bacteria, such as Bacteroides fragilis and Clostridium difficile . T-3811 was the most active agent against C . trachomatis (MIC, 0.008 microgram/ml) and M . pneumoniae (MIC90, 0.0313 microgram/ml) . The activity of T-3811 against M . tuberculosis (MIC90, 0.0625 microgram/ml) was potent and superior to that of trovafloxacin . In experimental systemic infection with a GrlA mutant of S . aureus and experimental pneumonia with PRSP in mice, T-3811ME showed excellent therapeutic efficacy in oral and subcutaneous administrations.

Appl Microbiol Biotechnol, 1999 Mar, 51(3), 348 - 57
Nucleotide sequences of two contiguous and highly homologous xylanase genes xynA and xynB and characterization of XynA from Clostridium thermocellum; Hayashi H et al.; A 5.7-kbp region of the Clostridium thermocellum F1 DNA was sequenced and found to contain two contiguous and highly homologous xylanase genes, xynA and xynB . The xynA gene encoding the xylanase XynA consists of 2049 bp and encodes a protein of 683 amino acids with a molecular mass of 74,511 Da, and the xynB gene encoding the xylanase XynB consists of 1371 bp and encodes a protein of 457 amino acids with a molecular mass of 49,883 Da . XynA is a modular enzyme composed of a typical N-terminal signal peptide and four domains in the following order: a family-II xylanase domain, a family-VI cellulose-binding domain, a dockerin domain, and a NodB domain . XynB exhibited extremely high overall sequence homology with XynA (identity 96.9%), while lacking the NodB domain present in the latter . These facts suggested that the xynA and xynB genes originated from a common ancestral gene through gene duplication . XynA was purified from a recombinant Escherichia coli strain and characterized . The purified enzyme was highly active toward xylan; the specific activity on oat-spelt xylan was 689 units/mg protein . Immunological and zymogram analyses suggested that XynA and XynB are components of the C . thermocellum F1 cellulosome.

Appl Microbiol Biotechnol, 1999 Mar, 51(3), 340 - 7
Sequencing, expression, and transcription analysis of the Clostridium paraputrificum chiA gene encoding chitinase ChiA; Morimoto K et al.; Immediately (17 bp) upstream of the Clostridium paraputrificum chiB gene {J . Bacteriol . 179: 7306-7314 (1997)}, we found another chitinase gene chiA encoding chitinase A (ChiA) . The chiA gene consists of an open reading frame of 2496 nucleotides and encodes 832 amino acids with a deduced molecular mass of 92,585 Da . The mature ChiA is a modular enzyme composed of a family-18 catalytic domain responsible for chitinase activity, two cadherin-like domains, and a chitin-binding domain . The domain organization of ChiA is fundamentally identical to that of ChiB and the overall sequence identity between them is 35.4% . ChiA was purified from the periplasm fraction of Escherichia coli harboring the chiA gene . The molecular mass of purified ChiA (89,000 Da), determined by sodium dodecyl sulfate/polyacrylamide gel electrophoresis analysis, was in good agreement with the value (89,119 Da) calculated from the deduced amino acid sequence, excluding the signal peptide . Immunological and N-terminal amino acid sequence analyses revealed that ChiA and ChiB are major chitinases of C . paraputrificum and their production is inducible by ball-milled chitin . Northern blot analysis indicated that the chiA and chiB genes constitute a polycistronic operon . Primer-extension analysis confirmed that the transcription of this operon starts upstream of chiA.

Med Pediatr Oncol, 1999 May, 32(5), 336 - 43
Trends in infection morbidity in a pediatric oncology ward, 1986-1995; Wehl G et al.; BACKGROUND AND PROCEDURE: We retrospectively studied the type, severity, frequency, and outcome of febrile infectious complications in 217 cancer patients receiving cytotoxic chemotherapy (603 episodes) over a 10-year period in a single pediatric institution . RESULTS: A total of 48.8% of the episodes occurred in severely leukopenic patients (WBC < 1.0 x 10(9)/l, absolute neutrophil count < 500 x 10(6)/l) . In the second half of the study period febrile episodes occurred at increased frequency . The number of patients with gram-positive isolates in blood cultures increased over the years, most frequently coagulase-negative staphylococci were found . Remarkably, gram-negative bacteria increasingly resistant to the administered first-line antibiotic regimen emerged, necessitating modifications of the antimicrobial strategy every 3 years . Furthermore, Clostridium difficile-associated enterocolitis posed a clinical problem at increasing frequency since 1993 . As expected, the speed of leukocyte recovery within 5 days from the onset of a febrile complication had an influence on the outcome of these episodes . CONCLUSIONS: Rapid recovery of the WBC was associated with an excellent prognosis whereas persisting neutropenia was found to be a negative factor associated with fatal outcomes . The fatality rate of all febrile episodes (2.3%) remained the same throughout the study period despite the availability and wider use of recombinant hematopoietic growth factors since 1991.

Microbiology, 1999 Apr, 145 ( Pt 4), 819 - 26
Proline biosynthesis from L-ornithine in Clostridium sticklandii: purification of delta1-pyrroline-5-carboxylate reductase, and sequence and expression of the encoding gene, proC; Kenklies J et al.; Clostridium sticklandii utilizes combinations of amino acids for growth by Stickland reactions . Proline is an efficient electron acceptor in these reactions and is reduced to 5-aminovalerate . Proline can be partly synthesized from ornithine by the action of ornithine aminotransferase and delta1-pyrroline-5-carboxylate (PCA) reductase . Both enzymes were present in crude extracts of C . sticklandii in sufficient activity of 0.93 nkat (mg protein)(-1) and 4.3 nkat (mg protein)(-1), respectively, whereas enzymes involved in proline biosynthesis from glutamate were not detected . PCA reductase was purified to homogeneity in a three-step procedure involving ammonium sulfate precipitation, affinity chromatography with Procion Red and gel filtration on Sephadex GF200 . The homogeneous enzyme was most likely an octamer of 230 kDa with a subunit size of 25 kDa as obtained by SDS-PAGE and 28.9 kDa as calculated from the sequence . Apparent Km values for PCA and NADH were 0.19 mM and 0.025 mM, respectively . The enzyme also catalysed in vitro the reverse reaction, the oxidation of proline, at alkaline pH values above 8 and higher substrate concentrations (apparent Km values: 1.55 mM for proline and 10.5 mM for NAD at pH 10.0) . Studies with growing cells of C . sticklandii and {15N}proline revealed that proline is not oxidized in vivo because 15N was solely detected by HPLC-MS in 5-aminovalerate as the product of proline reduction . The proC gene encoding PCA reductase of C . sticklandii was cloned, sequenced and heterologously expressed in Escherichia coli . The enzyme exhibited high homologies to PCA reductases from different sources . Thus, C . sticklandii is able to synthesize the electron acceptor proline from ornithine (a degradation product of arginine) by action of ornithine aminotransferase and PCA reductase.

J Bacteriol, 1999 May, 181(9), 2816 - 22
Identification of metal ligands in the Clostridium histolyticum ColH collagenase; Jung CM et al.; A Clostridium histolyticum 116-kDa collagenase has an H415EXXH motif but not the third zinc ligand, as found in already characterized zinc metalloproteinases . To identify its catalytic site, we mutated the codons corresponding to the three conserved residues in the motif to other amino acid residues . The mutation affecting His415 or His419 abolished catalytic activity and zinc binding, while that affecting Glu416 did the former but not the latter . These results suggest that the motif forms the catalytic site . We also mutated the codons corresponding to other amino acid residues that are likely zinc ligands . The mutation affecting Glu447 decreased markedly both the enzymatic activity and the zinc content, while that affecting Glu446 or Glu451 had smaller effects on activity and zinc binding . These mutations caused a decrease in kcat but no significant change in Km . These results are consistent with the hypothesis that Glu447 is the third zinc ligand . The spacing of the three zinc ligands is the same in all known clostridial collagenases but not in other known gluzincins, indicating that they form a new gluzincin subfamily . The effects of mutations affecting Glu446 and Glu451 suggest that the two residues are also involved in catalysis, possibly through an interaction with the two zinc-binding histidine residues.

Acta Crystallogr D Biol Crystallogr, 1999 May, 55(5), 962 - 8
Rubredoxin from Clostridium pasteurianum . Structures of G10A, G43A and G10VG43A mutant proteins . Mutation of conserved glycine 10 to valine causes the 9-10 peptide link to invert; Maher MJ et al.; The four cysteine ligands which coordinate the Fe atom in the electron-transfer protein rubredoxin lie on loops of the polypeptide which form approximate local twofold symmetry . The cysteine ligands in the protein from Clostridium pasteurianum lie at positions 6, 9, 39 and 42 . Two glycine residues adjacent to the cysteine ligands at positions 10 and 43 are conserved in all rubredoxins, consistent with the proposal that a beta-carbon substituent at these positions would eclipse adjacent peptide carbonyl groups {Adman et al . (1975) . Proc . Natl Acad . Sci . USA, 72, 4854-4858} . X-ray crystal structures of the three mutant proteins G10A, G43A and G10VG43A are reported . The crystal structures of the single-site mutations are isomorphous with the native protein, space group R3; unit-cell parameters are a = 64.3, c = 32.9 A for G10A and a = 64.4, c = 32.8 A for G43A . The crystals of the double mutant, G10VG43A, were in space group P43212, unit-cell parameters a = 61.9, c = 80.5 A, with two molecules per asymmetric unit . The observed structural perturbations support the hypothesis that mutation of the conserved glycine residues would introduce strain into the polypeptide . In particular, in the G10VG43A protein substitution of valine at Gly10 causes the 9-10 peptide link to invert, relieving steric interaction between Cys9 O and Val10 Cbeta . This dramatic change in conformation is accompanied by the loss of the 10N-HcO6 hydrogen bond, part of the chelate loop Thr5-Tyr11 . The new conformation allows retention of the 11N-HcS9 hydrogen bond, but converts it from a type II to a type I hydrogen bond . This occurs at the cost of a less tightly packed structure . The structural insights allow rationalization of 1H NMR data reported previously for the 113CdII-substituted proteins and of the negative shifts observed in the FeIII/FeII mid-point potentials upon mutation.

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