Clostridia

Clin Infect Dis, 2005 Jan 15, 40(2), 265 - 72 Epub 2004 Dec 15.
A hospital outbreak of Clostridium difficile disease associated with isolates carrying binary toxin genes; McEllistrem MC et al.; INTRODUCTION: The binary toxin genes cdt and cdtB have been detected in approximately 5% of Clostridium difficile strains . Severe C . difficile disease (CDD) may be associated with strains that carry the binary toxin genes . METHODS: From April 2001 through March 2002, 8 severe and 41 nonsevere cases of nosocomial CDD were studied . Severe cases of CDD were defined by the presence of >or=2 of the following criteria: (1) abdominal pain, (2) a white blood cell count of >20,000 or <1500 cells/mm(3), and (3) ileus or bowel wall thickening with ascites . Underlying disease was assessed by 2 methods: a modified Horn score and the presence of comorbid conditions . The presence of cdtA, cdtB, and the toxin A and toxin B genes was determined, and molecular subtyping was performed . RESULTS: All strains were positive for the toxin A and B genes, and 65.3% of the strains carried the cdtA and cdtB genes . Strains that carried the binary toxin genes accounted for 87.5% of the cases of severe CDD and 61.0% of the nonsevere cases (P=.23) . Severity of CDD was not associated with either severe underlying disease or comorbid conditions . The strains that caused severe CDD belonged to 4 protein profile groups and >or=3 restriction endonuclease analysis (REA) groups . All (i.e., 5 of 5) strains in REA group BI, compared with none (i.e., 0 of 7) of the strains in REA group J carried the binary toxin genes (P=.001) . Strains that belonged to REA groups BK and BR also carried the binary toxin genes . CONCLUSIONS: The binary toxin genes were present in nearly two-thirds of the C . difficile strains, and they were correlated with the REA group . Severity of CDD was not closely associated with a specific clone or underlying disease, but it may be associated with the presence of the binary toxin genes . Larger studies are needed to discern whether a true association exists and whether the binary toxin alters the pathogenicity of the C . difficile strain.

FEBS J, 2005 Jan, 272(2), 550 - 61
2-Hydroxyisocaproyl-CoA dehydratase and its activator from Clostridium difficile; Kim J et al.; The hadBC and hadI genes from Clostridium difficile were functionally expressed in Escherichia coli and shown to encode the novel 2-hydroxyisocaproyl-CoA dehydratase HadBC and its activator HadI . The activated enzyme catalyses the dehydration of (R)-2-hydroxyisocaproyl-CoA to isocaprenoyl-CoA in the pathway of leucine fermentation . The extremely oxygen-sensitive homodimeric activator as well as the heterodimeric dehydratase, contain iron and inorganic sulfur; besides varying amounts of zinc, other metal ions, particularly molybdenum, were not detected in the dehydratase . The reduced activator transfers one electron to the dehydratase concomitant with hydrolysis of ATP, a process similar to that observed with the unrelated nitrogenase . The thus activated dehydratase was separated from the activator and ATP; it catalyzed about 10(4) dehydration turnovers until the enzyme became inactive . Adding activator, ATP, MgCl(2), dithionite and dithioerythritol reactivated the enzyme . This is the first demonstration with a 2-hydroxyacyl-CoA dehydratase that the catalytic electron is recycled after each turnover . In agreement with this observation, only substoichiometric amounts of activator (dehydratase/activator = 10 mol/mol) were required to generate full activity.

Haematologica . 2005 Jan;90(1):ECR01.
Cytomegalovirus and Clostridium Difficile co-infection in severeulcero-hemorrhagic colitis during induction chemotherapy for acute lymphoblastic leukemia; Riva G et al.; Here we describe the first case of a biopsy-proven Cytomegalovirus ulcero-hemorrhagic colitis, associated with Clostridium Difficile co-infection, occurring during standard induction chemotherapy for common B cell acute lymphoblastic leukemia . We discuss the case and focalize clinical management and diagnostic issues arising from it.

J Wildl Dis, 2004 Oct, 40(4), 749 - 53
Detection of Clostridium botulinum Type C Cells in the Gastrointestinal Tracts of Mozambique Tilapia (Oreochromis mossambicus) by Polymerase Chain Reaction; Nol P et al.; We established a method of directly detecting Clostridium botulinum type C cells, while minimizing spore detection, in the intestinal contents of Mozambique tilapia (Oreochromis mossambicus) . This technique involved extraction of predominantly cellular DNA from tilapia intestinal tracts and used a polymerase chain reaction assay to detect presence of type C(1) toxin gene . We consistently detected C . botulinum type C cells in tilapia gastrointestinal contents at a level of 7.5x10(4) cells per 0.25 g material or 1.9x10(3) cells . This technique is useful for determining prevalence of the potentially active organisms within a given population of fish and may be adapted to other types of C . botulinum and vertebrate populations as well.

J Microbiol Methods, 2005 Mar, 60(3), 403 - 11
Evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with Clostridium botulinum spores; Johnston MD et al.; The aim of this study was to evaluate the efficacy of hydrogen peroxide vapour (HPV) against spores of Clostridium botulinum, for use as a method for decontaminating environments where this pathogen has been handled . Spores were dried onto stainless steel slides and exposed to HPV in a sealed glovebox enclosure, transferred to a quenching agent at timed intervals during the exposure period, before survivors were cultured and enumerated . D-values were calculated from graphs of log(10) survivors plotted against time and were found to range from 1.41 to 4.38 min . HPV was found to be effective at deactivating spores of toxigenic Cl . botulinum, non-toxigenic Clostridium spp . and Geobacillus stearothermophilus dried onto stainless steel surfaces . HPV could be used to decontaminate cabinets and rooms where Cl . botulinum has been handled . The cycle parameters should be based on studies carried out with relevant spores of this organism, rather than based on inactivation data for G . stearothermophilus spores, which have been used in the past as a standard biological challenge for disinfection and sterilisation procedures . HPV could provide an attractive alternative to other decontamination methods, as it was rapid, residue-free and did not give rise to the health and safety concerns associated with other gaseous decontamination systems.

Biochem Soc Symp, 2005, (72), 199 - 209
Lipids, Rafts and Traffic: Chapter 19 - Translocation of the cell-penetrating Tat peptide across artificial bilayers and into living cells; Curnow P et al.; The ability of a short, charged peptide to penetrate synthetic DOPC (1,2-dioleoyl-sn-3-glycerophosphocholine) liposomes was investigated by fluorescence confocal microscopy . The peptide, termed Tat (trans-activating transcription factor), was a 14-mer derived from the region of the HIV-1 Tat protein responsible for cellular internalization . This Tat peptide was labelled at a C-terminal cysteine residue with the fluorescent probes IAF (5-iodoacetamidofluorescein) or A568 (Alexa Fluor 568) . The Tat-IAF conjugate was directly observed entering liposomes at room temperature (approx . 258C) in the absence of pH gradient, ATP or other energy source . The uptake of the Tat-A568 conjugate in unfixed, live HeLa cells was found to be via endocytosis, as expected . In contrast, when the peptide was attached to an IAF-labelled 25kDa protein corresponding to the catalytic domain of Clostridium botulinum C3 exotoxin, this larger, Tat-C3-IAF construct was not able to enter liposomes, although it localized similarly to Tat-A568 in live cells . The data suggest that Tat peptide can cross synthetic bilayers spontaneously in vitro, but that size and type of cargo may limit this behaviour.

J Neurosci, 2005 Jan 12, 25(2), 299 - 307
3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors attenuate beta-amyloid-induced microglial inflammatory responses; Cordle A et al.; Alzheimer's disease (AD) is characterized by extracellular deposits of fibrillar beta-amyloid (Abeta) in the brain, a fulminant microglial-mediated inflammatory reaction, and neuronal death . The use of 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors (statins) is associated with a reduced risk of AD, which has been attributed to the cholesterol-lowering actions of these drugs . Statins have been reported recently to have anti-inflammatory actions in addition to their classic lipid-lowering effects . We report that statins robustly inhibited the Abeta-stimulated expression of interleukin-1beta and inducible nitric oxide synthase and the production of nitric oxide by microglia and monocytes . Statin treatment also blocked the rac1-dependent activation of NADPH oxidase and superoxide production . The anti-inflammatory actions of the statins were attributable to their ability to reduce the levels of isoprenyl intermediates in the cholesterol biosynthetic pathway . The effect of statins could not be reversed by exogenous cholesterol supplementation, indicating that the anti-inflammatory actions are distinct from their cholesterol-lowering actions . The addition of the isoprenyl precursors, mevalonic acid, and geranylgeranyl pyrophosphate (GGpp) attenuated the statin-mediated downregulation of inflammatory markers . Prevention of protein isoprenylation by the GGpp transferase inhibitor (GGTI-286) or inhibition of Rho-family function with Clostridium difficile Toxin A blocked the inflammatory response similar to the effect of statin treatment . We argue that the statin-mediated decrease in AD risk arises from their pleiotropic actions, effecting a reduction in neuronal Abeta production and microglia-directed inflammation.

Jt Comm J Qual Saf, 2004 Dec, 30(12), 676 - 80
"Keeping each patient safe": quality safety teaching/learning packets; Benezo C et al.; BACKGROUND: University of Pittsburgh Medical Center (UPMC) McKeesport developed a tool, the UPMC McKeesport Quality Safety Teaching/Learning Packet, to provide physicians, nurses, and therapists with a common language to address complex safety issues . Teaching/learning packets were developed to "keep each patient safe": by calling for help early; from falls and confusion; and from hospital-acquired infections . TEACHING/LEARNING PACKETS: In July 2002, the concept of calling for help early became a requirement at UPMC McKeesport . The code team was to be called for any significant change in status and for traditional code arrests . In 2004, a teaching/learning packet addressed the concepts of fall risk and acute (delirium) and chronic (dementia) confusion . Strategies were implemented to reduce the rate of falls through risk screening and interventions for falls and delirium . In April 2004, a teaching/learning packet was introduced to reduce hospital-acquired infections, and professionals were positioned to better address isolation, hand hygiene, central-line-associated bacteremia, Clostridium difficile, and appropriate antibiotic usage . SUMMARY AND CONCLUSIONS: Three quality safety teaching/learning packets, which provided the professionals in the organization with the common language (culture) to advance patient safety, accomplished rapid change and were well accepted by staff and physicians.

Can J Microbiol, 2004 Oct, 50(10), 845 - 51
Cloning and expression of the Clostridium thermocellum L-lactate dehydrogenase gene in Escherichia coli and enzyme characterization; Ozkan M et al.; The structural gene for L-lactate dehydrogenase (LDH) (EC.1.1.1.27) from Clostridium thermocellum 27405 was cloned in Escherichia coli by screening the Lambda Zap II phage library of C . thermocellum genomic DNA . In one positive clone, an open reading frame of 948 base pairs corresponded to C . thermocellum ldh gene encoding for the predicted 315-residue protein . The ldh gene was successfully expressed in E . coli FMJ39 (ldh mutant) under the lac promoter . The recombinant enzyme was partially purified from E . coli cell extracts and its kinetic properties were determined . Clostridium thermocellum LDH was shown to catalyze a highly reversible reaction and to be an allosteric enzyme that is activated by fructose-1,6-diphosphate (FDP) . For pyruvate, partially purified LDH had Km and Vmax values of 7.3 mmol/L and 87 micromol/min, respectively, and in the presence of FDP, a 24-fold decrease in Km and a 5.7-fold increase in Vmax were recorded . The enzyme exhibited no marked catalytic activity for lactate in the absence of FDP, whereas Km and Vmax values were 59.5 mmol/L and 52 micromol/min, respectively, in its presence . The enzyme did not lose activity when incubated at 65 degrees C for 5 min.

J Vet Med Sci, 2004 Dec, 66(12), 1613 - 5
An outbreak of necrotic enteritis in the ostrich farm in Korea; Kwon YK et al.; An acute disease with high mortality occurred in the ostrich farm and characterized by depression, severe diarrhea and sternal recumbency . Four dead ostriches of the farm were submitted to the National Veterinary Research & Quarantine Service, and diagnosed as necrotic enteritis . In the gross and histopathological examination, extensive diffuse fibrinonecrotic enteritis was found in the small intestine, especially jejunum . Clostridium perfringens was isolated from a pure culture from the duodenum and jejunum of these birds . Based on our current knowledge, this is the first report of an outbreak of necrotic enteritis in the ostrich in Korea.

Breast Cancer Res, 2005, 7(1), R60 - 70 Epub 2004 Nov 08.
Prenylation inhibitors stimulate both estrogen receptor alpha transcriptional activity through AF-1 and AF-2 and estrogen receptor beta transcriptional activity; Cestac P et al.; INTRODUCTION: We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation . However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex . The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta . METHODS: The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine . Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor) . In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha . The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells . Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity . RESULTS: FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells . They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol . The roles of both AF-1 and AF-2 are significant in this effect . Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol . By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol . The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established . CONCLUSIONS: Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

Appl Environ Microbiol, 2005 Jan, 71(1), 530 - 7
Intracellular Butyryl Phosphate and Acetyl Phosphate Concentrations in Clostridium acetobutylicum and Their Implications for Solvent Formation; Zhao Y et al.; It has been suggested (L . H . Harris, R . P . Desai, N . E . Welker, and E . T . Papoutsakis, Biotechnol . Bioeng . 67:1-11, 2000) that butyryl phosphate (BuP) is a regulator of solventogenesis in Clostridium acetobutylicum . Here, we determined BuP and acetyl phosphate (AcP) levels in fermentations of C . acetobutylicum wild type (WT), degenerate strain M5, a butyrate kinase (buk) mutant, and a phosphotransacetylase (pta) mutant . A sensitive method was developed to measure BuP and AcP in the same sample . Compared to the WT, the buk mutant had higher levels of BuP and AcP; the BuP levels were high during the early exponential phase, and there was a peak corresponding to solvent production . Consistent with this, solvent formation was initiated significantly earlier and was much stronger in the buk mutant than in all other strains . For all strains, initiation of butanol formation corresponded to a BuP peak concentration that was more than 60 to 70 pmol/g (dry weight), and higher and sustained levels corresponded to higher butanol formation fluxes . The BuP levels never exceeded 40 to 50 pmol/g (dry weight) in strain M5, which produces no solvents . The BuP profiles were bimodal, and there was a second peak midway through solventogenesis that corresponded to carboxylic acid reutilization . AcP showed a delayed single peak during late solventogenesis corresponding to acetate reutilization . As expected, in the pta mutant the AcP levels were very low, yet this strain exhibited strong butanol production . These data suggest that BuP is a regulatory molecule that may act as a phosphodonor of transcriptional factors . DNA array-based transcriptional analysis of the buk and M5 mutants demonstrated that high BuP levels corresponded to downregulation of flagellar genes and upregulation of solvent formation and stress genes.

Appl Environ Microbiol, 2005 Jan, 71(1), 29 - 38
Development and Validation of PCR Primers To Assess the Diversity of Clostridium spp . in Cheese by Temporal Temperature Gradient Gel Electrophoresis; Le Bourhis AG et al.; A nested-PCR temporal temperature gradient gel electrophoresis (TTGE) approach was developed for the detection of bacteria belonging to phylogenetic cluster I of the genus Clostridium (the largest clostridial group, which represents 25% of the currently cultured clostridial species) in cheese suspected of late blowing . Primers were designed based on the 16S rRNA gene sequence, and the specificity was confirmed in PCRs performed with DNAs from cluster I and non-cluster I species as the templates . TTGE profiles of the PCR products, comprising the V5-V6 region of the 16S rRNA gene, allowed us to distinguish the majority of cluster I species . PCR-TTGE was applied to analyze commercial cheeses with defects . All cheeses gave a signal after nested PCR, and on the basis of band comigration with TTGE profiles of reference strains, all the bands could be assigned to a clostridial species . The direct identification of Clostridium spp . was confirmed by sequencing of excised bands . C . tyrobutyricum and C . beijerinckii contaminated 15 and 14 of the 20 cheese samples tested, respectively, and C . butyricum and C . sporogenes were detected in one cheese sample . Most-probable-number counts and volatile fatty acid were determined for comparison purposes . Results obtained were in agreement, but only two species, C . tyrobutyricum and C . sporogenes, could be isolated by the plating method . In all cheeses with a high amount of butyric acid (>100 mg/100 g), the presence of C . tyrobutyricum DNA was confirmed by PCR-TTGE, suggesting the involvement of this species in butyric acid fermentation . These results demonstrated the efficacy of the PCR-TTGE method to identify Clostridium in cheeses . The sensitivity of the method was estimated to be 100 CFU/g.

J Biotechnol, 2005 Feb 9, 115(3), 261 - 9
Enhanced biosynthesis of dihydrodaidzein and dihydrogenistein by a newly isolated bovine rumen anaerobic bacterium; Wang XL et al.; A rod-shaped and Gram-positive anaerobic bacterium, named Niu-O16, which was isolated from bovine rumen contents, was found to be capable of anaerobically converting isoflavones daidzein and genistein to dihydrodaidzein (DHD) and dihydrogenistein (DHG), respectively . The metabolites DHD and DHG were identified using EI-MS and NMR spectrometric analyses . Stereoisomeric metabolites, which were separated on chiral stationary phase HPLC, were formed in equal amounts by the strain Niu-O16 . Tautomerization reaction occurred on the B-ring of DHD and DHG seems to be attributed to the equal production of stereoisomeric metabolites . For the synthesis of DHD, the strain Niu-O16 showed an optimal pH range from 6.0 to 7.0 and completely reduced up to 800muM of daidzein to DHD with the initial OD(600nm)=1.0 and pH 7.0 for 3 days incubation . The strain Niu-O16, showed relatively faster reduction activity toward daidzein to produce DHD than the previously isolated human intestinal bacterium Clostridium sp . HGH6.

Respir Res . 2005 Jan 8;6(1):4 {Epub ahead of print}
Involvement of RhoA-mediated Ca2+ sensitization in antigen-induced bronchial smooth muscle hyperresponsiveness in mice; Chiba Y et al.; BACKGROUND: It has recently been suggested that RhoA plays an important role in the enhancement of the Ca2+ sensitization of smooth muscle contraction . In the present study, a participation of RhoA-mediated Ca2+ sensitization in the augmented bronchial smooth muscle (BSM) contraction in a murine model of allergic asthma was examined . METHODS: Ovalbumin (OA)-sensitized BALB/c mice were repeatedly challenged with aerosolized OA and sacrificed 24 hours after the last antigen challenge . The contractility and RhoA protein expression of BSMs were measured by organ-bath technique and immunoblotting, respectively . RESULTS: Repeated OA challenge to sensitized mice caused a BSM hyperresponsiveness to acetylcholine (ACh), but not to high K+-depolarization . In a-toxin-permeabilized BSMs, ACh induced a Ca2+ sensitization of contraction, which is sensitive to Clostridium botulinum C3 exoenzyme, indicating that RhoA is implicated in this Ca2+ sensitization . Interestingly, the ACh-induced, RhoA-mediated Ca2+ sensitization was significantly augmented in permeabilized BSMs of OA-challenged mice . Moreover, protein expression of RhoA was significantly increased in the hyperresponsive BSMs . CONCLUSION: These findings suggest that the augmentation of Ca2+ sensitizing effect, probably via an up-regulation of RhoA protein, might be involved in the enhanced BSM contraction in antigen-induced airway hyperresponsiveness.

J Clin Microbiol, 2005 Jan, 43(1), 511 - 3
Infant botulism acquired from household dust presenting as sudden infant death syndrome; Nevas M et al.; Clostridium botulinum type B was detected by multiplex PCR in the intestinal contents of a suddenly deceased 11-week-old infant and in vacuum cleaner dust from the patient's household . C . botulinum was also isolated from the deceased infant's intestinal contents and from the household dust . The genetic similarity of the two isolates was demonstrated by pulsed-field gel electrophoresis and randomly amplified polymorphic DNA analysis, thereby confirming that dust may act as a vehicle for infant botulism that results in sudden death.

J Clin Microbiol, 2005 Jan, 43(1), 472 - 5
Clonal spread of a Clostridium difficile strain with a complete set of toxin A, toxin B, and binary toxin genes among Polish patients with Clostridium difficile-associated diarrhea; Pituch H et al.; Clinically relevant Clostridium difficile strains usually produce toxins A and B . Some C . difficile strains can produce an additional binary toxin . We report clonality among five strains carrying all toxin genes from Polish patients with C . difficile-associated diarrhea . In another strain, possible recombination between binary toxin genes is documented.

J Food Prot, 2004 Dec, 67(12), 2682 - 7
Competitive inhibition between different Clostridium botulinum types and strains; Eklund MW et al.; Mixtures of proteolytic and nonproteolytic strains of toxigenic Clostridium botulinum types A, B, and F; nonproteolytic types B, E, and F; Clostridium sporogenes; and nontoxic E-like organisms resembling nonproteolytic C . botulinum were tested against each other for the purpose of selecting a mixture of compatible C . botulinum strains for inoculated pack studies on the basis of their sensitivity to bacteriophages and bacteriocin-like agents . All of the proteolytic strains produced bacteriocin-like agents that were inhibitory to three or more of the other proteolytic types and C . sporogenes . When selected strains of proteolytic types A and B were grown together, type A cultures produced neurotoxin, but type B toxin production was inhibited . Nonproteolytic strains of C . botulinum also produced bacteriocin-like agents against each other . Of these, type E strain EF4 produced bacteriocin-like agents against both proteolytic and nonproteolytic types of C . botulinum and C . sporogenes . EF4, however, was not inhibitory to the nontoxigenic E-like strains . When EF4 was grown with type A strain 62A, it had an inhibitory effect on type A toxin production . Strain 62A inactivated the type E toxin of EF4 after 7 to 21 days at 30 degrees C . On the basis of the production of these bacteriocin-like agents by different strains of C . botulinum and their potential effect on neurotoxin production, it is very important that compatible strains are used in mixtures for inoculated pack studies to determine the safety of a food process or product.

Microbiology, 2005 Jan, 151(Pt 1), 199 - 208
Characterization of the cleavage site and function of resulting cleavage fragments after limited proteolysis of Clostridium difficile toxin B (TcdB) by host cells; Rupnik M et al.; Clostridium difficile toxin B (TcdB) is a single-stranded protein consisting of a C-terminal domain responsible for binding to the host cell membrane, a middle part involved in internalization, and the N-terminal catalytic (toxic) part . This study shows that TcdB is processed by a single proteolytic step which cleaves TcdB(10463) between Leu(543) and Gly(544) and the naturally occurring variant TcdB(8864) between Leu(544) and Gly(545) . The cleavage occurs at neutral pH and is catalysed by a pepstatin-sensitive protease localized in the cytoplasm and on the cytoplasmic face of intracellular membranes . The smaller N-terminal cleavage products {63 121 Da (TcdB(10463)) and 62 761 Da (TcdB(8864))} harbour the cytotoxic and glucosyltransferase activities of the toxins . When microinjected into cultured Chinese hamster lung fibroblasts, the N-terminal cleavage fragment shows full cytotoxic activity shortly after injection whereas the holotoxin initially exhibits a very low activity which, however, increases with time . Twenty minutes after the start of internalization of TcdB, the larger cleavage products {206 609 Da (TcdB(10463)) and 206 245 Da (TcdB(8864))} are found exclusively in a membrane fraction, whereas the N-terminal cleavage products appear mainly in the cytosol and associated with the membrane . This is in line with a proposed model according to which the longer, C-terminal, part of these toxins forms a channel allowing for the translocation of the toxic N-terminal part, which is subsequently cleaved off at the cytoplasmic face of an intracellular compartment, most likely endosomes.

J Biochem (Tokyo), 2004 Nov, 136(5), 569 - 74
Clostridium perfringens Alpha-Toxin: Characterization and Mode of Action; Sakurai J et al.; Clostridium perfringens type A strains that produce alpha-toxin cause gas gangrene, which is a life-threatening infection with fever, pain, edema, myonecrosis and gas production . Intramuscular injection of the toxin or Bacillus subtilis carrying the alpha-toxin gene causes myonecrosis and produces histopathological features of the disease . Immunization of mice with alpha-toxin or fragments of the toxin prevents gas gangrene caused by C . perfringens . The toxin possesses phospholipase C (PLC), sphingomyelinase (SMase) and biological activities causing hemolysis, lethality and dermonecrosis . These biological activities are closely related to PLC and/or SMase activities . However, there is yet some uncertainty about the biological activities induced by the PLC and SMase activities of alpha-toxin . Based on the isolation and characterization of the gene for alpha-toxin and a comparison of the toxin with enzymes of the PLC family, significant progress has been made in determining the function-structure of alpha-toxin and the mode of action of the toxin . To provide a better understanding of the role of alpha-toxin in tissue damage in gas gangrene, this article summarizes current knowledge of the characteristics and mode of action of alpha-toxin.

Biochem Biophys Res Commun, 2005 Feb 4, 327(1), 361 - 70
Peptide antibiotic and actin-binding protein as mixed-type inhibitors of Clostridium difficile CDT toxin activities; Angeles DC et al.; CDT from Clostridium difficile is an ADP-ribosyltransferase that causes rapid actin disaggregation and cell death . For efficient catalysis, CDT required specific divalent cations and binding by NAD which can be substituted by ATP but not ADP . Increasing isolation of CDT-producing strains prompted our search for antagonists like the anti-C . difficile agents bacitracin and vancomycin which were effective CDT inhibitors . Other CDT transferase and glycohydrolase inhibitors with consistently low IC(50) values were heterocyclic peptide antibiotics containing modified amino acids such as polymyxin B and beta-lactam cephalosporins . The strongest inhibitors were actin-binding proteins which possess extensive interfaces with G-actin, adjoining the CDT-ADP-ribose(+) acceptor site and nucleotide cleft . Analysis of the extent and mode of inhibition and actin interaction sites provided fresh evidences on the designation of actin interface domains with actin-binding proteins . Our results uphold ADP-ribosylation as an innate physiologic process in cellular cytoskeletal reorganization regulated by endogenous metabolites.

Inflamm Bowel Dis, 2004 Nov, 10(6), 824 - 33
Ribosomal DNA sequence analysis of mucosa-associated bacteria in Crohn's disease; Prindiville T et al.; BACKGROUND: Enteric bacteria are implicated in the pathogenesis of Crohn's disease (CD); however, no specific causative organisms have been identified . AIMS: This study was undertaken to correlate disease activity with changes in intestinal biota in patients with CD . SUBJECTS: Ribosomal DNA analysis was used to explore the composition of the intestinal biota in patients with (1) CD undergoing colonoscopy, (2) CD undergoing surgical resection, and (3) no inflammatory bowel disease . METHODS: Primers targeting bacterial 16S ribosomal DNA (rDNA) were used to amplify bacterial DNA associated with active CD lesions, comparable normal tissue from patients with CD, and normal control tissue . Each amplicon was cloned . Seven hundred thirty-nine rDNA clones were sequenced from 16 biopsies from CD patients, 15 surgical samples, and 10 biopsies from normal control patients . RESULTS: Known extracellular or intracellular pathogens were not found . No rDNA sequence, phylogenetic group, or subgroup was consistently associated with CD lesions compared with normal tissues from the same patients . Colonic biopsies from CD-afflicted patients compared with biopsies from normal control subjects had an increase in facultative bacteria; in small bowel, CD patients had an increase in the Ruminococcus gnavus subgroup with a decrease in the Clostridium leptum and Prevotella nigrescens subgroups . However, differences in small bowel may have reflected individual variation rather than disease association . Surgical samples showed differences when compared with biopsy-derived samples . CONCLUSIONS: These findings suggest that CD is not caused by invasive pathogens associated specifically with the sites of lesions but that dysbiosis exists in this condition.

Am J Respir Cell Mol Biol . 2004 Dec 30; {Epub ahead of print}
Thromboxane prostanoid receptor signals through Gi protein to rapidly activate ERK in human airways; Citro S et al.; We showed previously that activation of the thromboxane prostanoid (TP) receptor causes human airway smooth muscle (HASM) cells to proliferate, suggesting a role in airway remodeling . This study aimed at determining the molecular mechanisms underlying this mitogenic action . We found that the MEK inhibitor PD98059 significantly affected agonist-induced DNA synthesis of HASM cells, which suggests that extracellular-regulated kinases (ERK) are involved . ERK activation by the agonist U46619 was rapid, sensitive to pertussis toxin, and significantly abrogated by the tyrosine kinase inhibitors genistein and PP1 . Stimulation of the TP receptor was also found to translocate phosphorylated ERK into the nucleus . TP receptor was found to activate Ras, as demonstrated by inhibition of ERK activation and DNA synthesis by Clostridium sordellii lethal toxin, and by the ability of U46619 to increase RasGTP . Finally, {(3)H}thymidine incorporation and ERK phosphorylation were also affected by prior treatment with protein kinase C (PKC) inhibitor GF109203X, although to different extents . In conclusion, in HASM cells TP receptor, predominantly coupled to Gi/o proteins, activates the Ras/ERK pathway to induce mitogenesis, probably with the involvement of non-receptor tyrosine kinases and PKC.

Br Poult Sci, 2004 Oct, 45(5), 648 - 56
A family 6 carbohydrate-binding module potentiates the efficiency of a recombinant xylanase used to supplement cereal-based diets for poultry; Fontes CM et al.; (1) Cellulases and xylanases display a modular architecture that comprises a catalytic module linked to one or more non-catalytic carbohydrate-binding modules (CBMs) . On the basis of primary structure similarity, CBMs have been classified into more than 30 different families . These non-catalytic modules mediate a prolonged and intimate contact of the enzyme with the target substrate, eliciting efficient hydrolysis of the insoluble polysaccharides . (2) Xylanases are very effective in improving the nutritive value of wheat- or rye-based diets for broiler chicks although the role of non-catalytic CBMs in the function of exogenous modular xylanases in vivo remains to be determined . (3) A study was undertaken to investigate the importance of a family 6 CBM in the function of recombinant derivatives of xylanase 11A (Xyn11A) of Clostridium thermocellum used to supplement cereal-based diets for poultry . (4) The data show that birds fed on a wheat-based diet supplemented with the modular xylanase display an increased final body weight when compared with birds receiving Xyn11A catalytic module or birds receiving the enzyme mixture Roxazyme G . (5) Interestingly, the modular xylanase was truncated and transformed into its single domain counterpart on the duodenum of birds fed on the wheat-based diets, most possibly due to the action of pancreatic proteases . (6) Together the data point to the importance of CBMs in the function of feed xylanases and suggest, that in chicken fed on wheat-based diets, the main sites for exogenous enzymes action might be the gastrointestinal (GI) compartments preceding the duodenum, most probably the crop.

Can Nurse, 2004 Nov, 100(9), 16 - 20
Nurses and the control of infectious disease . Understanding epidemiology and disease transmission is vital to nursing care; Stirling B et al.; Epidemiology examines the distribution and source of a disease in a population . Understanding epidemiology and disease transmission is vital to nursing care . Infectious disease transmission requires three components: an agent (virus, bacterium, parasite or other microbe), a vulnerable host and a conducive environment . Disease spread can occur through direct contact or via indirect methods (airborne droplets, vectors, fomites, water or food) . Intervention can occur by attacking the agent (e.g., using microbicides), changing the environment (e.g., providing negative pressure rooms) or strengthening the host (e.g., vaccination) . Three epidemiologically relevant microbes are the SARS (severe acute respiratory syndrome)-associated coronavirus, methicillin-resistant Staphylococcus aureus (MRSA) and Clostridium difficile (C . difficile) . The first is an emerging pathogen, and the latter two are existing agents that have mutated such that they are resistant to their standard treatments . For SARS, control measures include screening for possible cases and appropriate triage, respiratory and barrier precautions within the healthcare facility, and voluntary isolation in the community for contacts or healthcare workers who exhibit symptoms . Control measures for MRSA include the screening of patient lesions, isolating or cohorting patients who are already infected, covering wounds with impermeable dressings, treating staff and patient carriers with antibiotics, and improved hygiene . Control measures for C . difficile Control measures include paying close attention to the hygiene of the clinical setting, disinfecting using bleach and the isolation of infected patients.

FEMS Microbiol Lett, 2005 Jan 15, 242(2), 281 - 5
Identification and characterization of a cell-wall anchored DNase gene in Clostridium perfringens; Okumura K et al.; Completion of the whole genome sequence of Clostridium perfringens strain 13 revealed the presence of an extracellular nuclease gene, cadA . Transcriptional analysis showed that the cadA gene is negatively regulated by the two-component VirR/VirS system and its secondary regulator VR-RNA . The CadA protein possesses an N-terminal signal sequence and a Gram-positive cell wall anchoring motif consisting of a sorting signal (LPXTG motif), a hydrophobic domain, and positively charged residues at the end of C-terminus . By comparing the DNase production between the wild type and the cadA mutant, and DNase activity assay with the recombinant truncated CadA protein, we confirmed that the cadA gene product is one of the DNases produced by C . perfringens.

Med Mal Infect, 2004 Feb, 34(2), 57 - 61
{Risk factors for nosocomial Clostridium difficile diarrhoea in an infectious and tropical diseases department}; Henoun Loukili N et al.; FOREWORD: Clostridium difficile associated diarrhea (CDAD) accounts for 25% of all cases of diarrhea occurring in hospital . Infectious diseases departments are considered as presenting with an important risk of CDAD because of the large quantity of antibiotics used . OBJECTIVES AND METHOD: The authors made a prospective study in the first 6 months of 2001, in order to identify the risk factors of CDAD in their department . One hundred and fifty-two patients hospitalized for at least 6 days were included in this study . The studied factors were: age, mean number of days of hospitalization (MDH), antibiotic therapy, WHO scale of reduced mobility of patients, recent hospitalization (less than 3 months before) . RESULTS: MDH was 36 (IC95%: 23-48) . Beta-lactam antibiotics were found as significant risk factors, as reported in the literature . However, age and a recent hospitalization were not related to the CDAD as described in the literature . A reduced mobility of patients was identified as a significant risk factor for developing a CDAD in our department.

Microb Pathog, 2004 Dec, 37(6), 279 - 86 Epub 2004 Nov 13.
Cytotoxicity of Clostridium septicum alpha-toxin: its oligomerization in detergent resistant membranes of mammalian cells; Hang'ombe MB et al.; Alpha-toxin is an important agent of the virulence of Clostridium septicum . We examined cytotoxicity for alpha-toxin to various mammalian cells with recombinant toxin fused with a histidine-tag at the amino-terminal . The recombinant toxin retained the activity indistinguishable from the native form . Mammalian nucleated cells examined in this study are more sensitive to the protoxin than to the trypsinized toxin, except RAW 264.7 and P3U1 cells of myeloid lineage . Cellular proteins of various molecular sizes interacted with the toxin . The size and SDS-PAGE pattern of the proteins were different among cell lines but they were liberated from the cells by the treatment with phosphatidylinositol-specific phospholipase C . The toxin appeared to target and utilize detergent resistant membranes (DRMs) for binding and subsequent oligomerization . In discontinuous sucrose density gradient, we demonstrated by immunoblotting that the toxin bound to DRMs contained in L929 cells and caused the oligomer formation . Furthermore, cholesterol depletion with cholesterol-interacting agents reduced toxin oligomerization and lowered cytotoxicity of the toxin towards cells . These results suggest that alpha-toxin preferentially exploits DRMs for oligomerization.

J Biol Chem . 2004 Dec 22; {Epub ahead of print}
A clostridial endo-beta-galactosidase that cleaves blood group A and B glycotopes: The first member of a new glycoside hydrolase family GH98; Anderson KM et al.; We have isolated an endo-beta-galactosidase (E-ABase) from Clostridium perfringens ATCC 10543 capable of liberating both the A- trisaccharide (GalNAc-alpha-1,3(Fuc-alpha-1,2)Gal) and B-trisaccharide (Gal-alpha-1,3(Fuc-alpha-1,2)Gal) from glycoconjugates containing blood group A- and B- glycotope, respectively . We have subsequently cloned the gene (eabC) that encodes E-ABase from this organism . This gene was found to be identical to the gene CPE0329 of C . perfringens strain 13 whose product was labeled as a hypothetical protein (Shimizu, T., Ohtani, K., Hirakawa, H., Ohshima, K., Yamashita, A., Shiba, T., Ogasawara, N., Hattori, M., Kuhara, S., and Hayashi, H . (2002) Proc . Natl . Acad . Sci . U.S.A . 99, 996-1001) . Since the amino acid sequence of E-ABase does not bear detectable similarity to any of the existing 97 families of glycoside hydrolases (GH), we have proposed to assign this unusual enzyme to a new family, GH98 . We have also expressed eabC in Escherichia coli BL21(DE3) and obtained 27 mg of fully active recombinant E-ABase from 1 liter of culture . The recombinant E-ABase not only destroyed the blood group A- and B-antigenicity of human type A- and B-erythrocytes but also released A- and B-trisaccharide from blood group A+- and B+-glycoconjugates . The structures of A- and B-trisaccharide liberated from A+-porcine gastric mucin and B+-human ovarian cyst glycoprotein were established by NMR spectroscopy . The unique specificity of E-ABase should make it useful for studying the structure and function of blood group A- and B-containing glycoconjugates as well as for identifying other glycosidases belonging to the new GH98 family.

Infect Immun, 2005 Jan, 73(1), 652 - 6
Atypical cpb2 genes, encoding beta2-toxin in Clostridium perfringens isolates of nonporcine origin; Jost BH et al.; Beta2-toxin, encoded by cpb2, is implicated in the pathogenesis of Clostridium perfringens enteritis . However, cpb2 genes from nonporcine C . perfringens isolates were not always expressed, at least in vitro . Nucleotide sequencing identified atypical cpb2 genes with 70.2 to 70.7% DNA identity to previously identified (consensus) cpb2 . Atypical beta2-toxin displayed 62.3% identity and 80.4% similarity to consensus beta2-toxin . No porcine type C isolates (n = 16) and only 3.3% of porcine type A isolates (n = 60) carried atypical cpb2 genes . However, 88.5% of nonporcine isolates carried atypical cpb2 (n = 78), but beta2-toxin was not expressed . Almost half of the nonporcine consensus cpb2 genes (44.4%) carried a frameshift mutation (n = 9), resulting in an absence of beta2-toxin expression . These findings strengthen the role of beta2-toxin in the pathogenesis of enteritis in neonatal pigs . However, the identification of apparently nonexpressed, atypical cpb2 genes raises the question of whether this protein plays the same role in enteritis in other animal species.

Cell Microbiol, 2005 Jan, 7(1), 129 - 46
The importance of calcium influx, calpain and calmodulin for the activation of CaCo-2 cell death pathways by Clostridium perfringens enterotoxin; Chakrabarti G et al.; Summary CaCo-2 cells exhibit apoptosis when treated with low doses of Clostridium perfringens enterotoxin (CPE), but develop oncosis when treated with high CPE doses . This study reports that the presence of extracellular Ca(2+) in treatment buffers is important for normal activation of both those cell death pathways in CPE-treated CaCo-2 cells . Normal development of CPE-induced cell death pathway effects, such as morphologic damage, DNA fragmentation, caspase activation, mitochondrial membrane depolarization and cytochrome c release, was strongly inhibited when CaCo-2 cells were CPE-treated in Ca(2+)-free buffers . When treatment buffers contained Ca(2+), CPE caused a rapid increase in CaCo-2 cell Ca(2+) levels, apparently because of increased Ca(2+) influx through a CPE pore . High CPE doses caused massive changes in cellular Ca(2+) levels that appear responsible for activating oncosis, whereas low CPE doses caused less perturbations in cellular Ca(2+) levels that appear responsible for activating apoptosis . Both CPE-induced apoptosis and oncosis were found to be calmodulin- and calpain-dependent processes . As Ca(2+) levels present in the intestinal lumen resemble those of Ca(2+)-containing treatment buffers used in this study, perturbations in cellular Ca(2+) levels and calpain/calmodulin-dependent processes are also probably important for inducing enterocyte cell death during CPE-mediated gastrointestinal disease.

Antimicrob Agents Chemother, 2005 Jan, 49(1), 431 - 3
Interaction of bismuth subsalicylate with fruit juices, ascorbic acid, and thiol-containing substrates to produce soluble bismuth products active against Clostridium difficile; Mahony DE et al.; Bismuth subsalicylate (BSS), the active ingredient of Pepto-Bismol, has been used for many years to treat various disorders of the gastrointestinal tract . Using mass spectrometry and the agar dilution method, we determined that insoluble BSS interacts with certain dietary components and organic substrates to produce water-soluble products with activity against Clostridium difficile.

Antimicrob Agents Chemother, 2005 Jan, 49(1), 408 - 13
Comparative in vitro activities of XRP 2868, pristinamycin, quinupristin-dalfopristin, vancomycin, daptomycin, linezolid, clarithromycin, telithromycin, clindamycin, and ampicillin against anaerobic gram-positive species, actinomycetes, and lactobacilli; Goldstein EJ et al.; A comparative study of the in vitro activities of XRP 2868, a new oral streptogramin, against 266 anaerobic gram-positive clinical isolates using the agar dilution method showed that the XRP 2868 MICs for 95% (254 of 266) of isolates were < or =0.5 microg/ml . XRP 2868 MICs for only two strains, one being Clostridium clostridioforme (MIC, 16 microg/ml) and the other being Clostridium difficile (MIC, 32 microg/ml), were >2 microg/ml . Depending on its pharmacokinetics and pharmacodynamics, XRP 2868 has potential for use against infections with gram-positive anaerobes and deserves further clinical evaluation.

Poult Sci, 2004 Dec, 83(12), 1948 - 52
Evaluation of immunosuppressants and dietary mechanisms in an experimental disease model for necrotic enteritis; McReynolds JL et al.; Clostridium perfringens (CP) is the etiologic agent of necrotic enteritis (NE) . Clinical signs of this disease include depression, decreased appetite, diarrhea, and severe necrosis of the intestinal tract . Understanding the disease progression of NE has been difficult due to its complexity and the involvement of multiple factors (dietary components, immunosuppression, and mechanical irritation of the gut) that appear to contribute to this syndrome . In the present investigation, day-of-hatch broilers were fed a 55% wheat diet and randomly assigned to 1 of 8 groups . Treatments included positive control (CP challenge only), commercial coccidia vaccine (CCV), commercial bursal disease vaccine (CBDV), or the combination of CCV and CBDV, and an appropriate negative control for each (vaccinated and not challenged) . Challenged treatment groups received 10(7) cfu of CP twice daily . When compared with controls, broilers in each treatment group had increased (P < or = 0.05) lesion scores, with mean scores of 1.05 and 2.05 in the CP and CBDV + CP treatments, respectively . When compared with controls, the incidence of CP increased (P < or = 0.05) in all treatment groups (73 and 100% in the CCV + CP and CBDV + CP treatment groups, respectively) . Compared with controls, percentage mortality increased (P < or = 0.05) from 2% to 26 and 34% in the CP and CBDV + CP treatment groups, respectively . Results of this study indicate that the methodology used provides a good model for studying NE.

J Virol, 2005 Jan, 79(2), 1191 - 206
Kaposi's sarcoma-associated herpesvirus modulates microtubule dynamics via RhoA-GTP-diaphanous 2 signaling and utilizes the dynein motors to deliver its DNA to the nucleus; Naranatt PP et al.; Human herpesvirus 8 (HHV-8; also called Kaposi's sarcoma-associated herpesvirus), which is implicated in the pathogenesis of Kaposi's sarcoma (KS) and lymphoproliferative disorders, infects a variety of target cells both in vivo and in vitro . HHV-8 binds to several in vitro target cells via cell surface heparan sulfate and utilizes the alpha3beta1 integrin as one of its entry receptors . Interactions with cell surface molecules induce the activation of host cell signaling cascades and cytoskeletal changes (P . P . Naranatt, S . M . Akula, C . A . Zien, H . H . Krishnan, and B . Chandran, J . Virol . 77:1524-1539, 2003) . However, the mechanism by which the HHV-8-induced signaling pathway facilitates the complex events associated with the internalization and nuclear trafficking of internalized viral DNA is as yet undefined . Here we examined the role of HHV-8-induced cytoskeletal dynamics in the infectious process and their interlinkage with signaling pathways . The depolymerization of microtubules did not affect HHV-8 binding and internalization, but it inhibited the nuclear delivery of viral DNA and infection . In contrast, the depolymerization of actin microfilaments did not have any effect on virus binding, entry, nuclear delivery, or infection . Early during infection, HHV-8 induced the acetylation of microtubules and the activation of the RhoA and Rac1 GTPases . The inactivation of Rho GTPases by Clostridium difficile toxin B significantly reduced microtubular acetylation and the delivery of viral DNA to the nucleus . In contrast, the activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor significantly augmented the nuclear delivery of viral DNA . Among the Rho GTPase-induced downstream effector molecules known to stabilize the microtubules, the activation of RhoA-GTP-dependent diaphanous 2 was observed, with no significant activation in the Rac- and Cdc42-dependent PAK1/2 and stathmin molecules . The nuclear delivery of viral DNA increased in cells expressing a constitutively active RhoA mutant and decreased in cells expressing a dominant-negative mutant of RhoA . HHV-8 capsids colocalized with the microtubules, as observed by confocal microscopic examination, and the colocalization was abolished by the destabilization of microtubules with nocodazole and by the phosphatidylinositol 3-kinase inhibitor affecting the Rho GTPases . These results suggest that HHV-8 induces Rho GTPases, and in doing so, modulates microtubules and promotes the trafficking of viral capsids and the establishment of infection . This is the first demonstration of virus-induced host cell signaling pathways in the modulation of microtubule dynamics and in the trafficking of viral DNA to the infected cell nucleus . These results further support our hypothesis that HHV-8 manipulates the host cell signaling pathway to create an appropriate intracellular environment that is conducive to the establishment of a successful infection.

Lett Appl Microbiol, 2005, 40(1), 81 - 6
Inhibitory effects of various micro-organisms on the growth of Helicobacter pylori; Krausse R et al.; Abstract r . krausse, k . piening and u . ullmann . 2004.Aims: To examine the in vitro influence of various bacteria species on Helicobacter pylori (Hp) growth . Methods and Results: The effects of 29 micro-organisms on 31 Hp strains were determined using two modified 'cross streak' methods . Staphylococcus epidermidis, Staphylococcus aureus, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Morganella morganii, Serratia marcescens, Bacteroides fragilis, Fusobacterium nucleatum and Clostridium difficile showed the strongest inhibition . The inhibitory effects varied, depending on the bacteria spp . and Hp strains, and were method dependent . The cagA status of Hp strains did not correlate with the extent of inhibition . Conclusions: Helicobacter pylori is inhibited by a significant number of commensal bacteria species as well as opportunistic human pathogens . The success and progress of Hp infection may be influenced by the bacterial flora present, while the difficulty in cultivating Hp from the oral mucosa and faeces may be the result of antagonistic bacterial interaction . Significance and Impact of the Study: This study provides valuable data on the sensitivity of Hp to a variety of intestinal and oral commensals as well as opportunistic human pathogens . Hp's varying pathogenicity and the specific localization of infection may be the result of these sensitivities . These results can also serve as a basis for further studies to identify the inhibitory substances and make them available for therapeutic use.

Mol Microbiol, 2005 Jan, 55(1), 235 - 49
BotR/A and TetR are alternative RNA polymerase sigma factors controlling the expression of the neurotoxin and associated protein genes in Clostridium botulinum type A and Clostridium tetani; Raffestin S et al.; Summary Clostridium botulinum and Clostridium tetani, respectively, produce potent toxins, botulinum neurotoxin (BoNT) and tetanus neurotoxin (TeTx), which are responsible for severe diseases, botulism and tetanus . Neurotoxin synthesis is a regulated process in Clostridium . The genes botR/A in C . botulinum A and tetR in C . tetani positively regulate expression of BoNT/A and associated non-toxic proteins (ANTPs), as well as TeTx respectively . The botR/A gene lies in close vicinity of the two operons which contain bont/A and antps genes in C . botulinum A, and tetR immediately precedes the tetX gene in C . tetani . We show that BotR/A and TetR function as specific alternative sigma factors rather than positive regulators based on the following results: (i) BotR/A and TetR associated with target DNAs only in the presence of the RNA polymerase core enzyme (Core), (ii) BotR/A and TetR directly bound with the core enzyme, (iii) BotR/A-Core recognized -35 and -10 regions of ntnh-bont/A promoter and (iv) BotR/A and TetR triggered in vitro transcription from the target promoters . In C . botulinum A, bont/A and antps genes are transcribed as bi- and tricistronic operons controlled by BotR/A . BotR/A and TetR are seemingly related to a new subgroup of the sigma(70) family that includes TcdR and UviA, which, respectively, regulate production of toxins A and B in C . difficile and bacteriocin in C . perfringens . Sequences of -35 region are highly conserved in the promoter of target toxin genes in C . botulinum, C . tetani, C . difficile and C . perfringens . Overall, a common regulation mechanism probably controls toxin gene expression in these four toxigenic clostridial species.

Microbiol Immunol, 2004, 48(12), 917 - 29
Effects of Two Probiotic Lactobacillus Strains on Jejunal and Cecal Microbiota of Broiler Chicken under Acute Heat Stress Condition as Revealed by Molecular Analysis of 16S rRNA Genes; Lan PT et al.; We examined the effects of probiotic Lactobacillus strains of Lactobacillus agilis JCM 1048 and Lactobacillus salivarius subsp . salicinius JCM 1230 on jejunal and cecal microbiota of broiler chicken under heat stress condition using terminal restriction fragment length polymorphism (T-RFLP) analysis . The jejunal bacterial community was limited to a few bacterial groups, mostly Lactobacillus spp . A relatively abundant and higher prevalence of Lactobacillus spp . were observed in the jejunal and cecal microbiota of the probiotic chickens compared with those of the control chickens under heat stress condition . In general, the probiotic strains did not significantly affect the abundance of L . agilis and L . salivarius in chicken intestine but clearly contributed to increasing their prevalence in the probiotic chickens . The probiotic Lactobacillus strains enriched the diversity of Lactobacillus flora in chicken jejunum and cecum by increasing the abundance and prevalence of Lactobacillus spp . inhabiting the intestine . The richness of Lactobacillus species tended to be similar among the jejunal and cecal microbiota . The bacterial community of cecum was complex and age-dependent . The major components of the cecal microbiota were clostridia and lactobacilli . The Clostridium subcluster XIVa was the most predominant group in chicken cecum . Probiotic Lactobacillus strains restored the microbial balance and maintained the natural stability of indigenous bacterial microbiota following heat stress-induced changes.

J Biotechnol, 2005 Jan 26, 115(2), 179 - 87
Continuous butanol fermentation and feed starch retrogradation: butanol fermentation sustainability using Clostridium beijerinckii BA101; Ezeji TC et al.; Use of starch solution as feed for butanol bioconversion processes employing Clostridium beijerinckii BA101 may have added economic advantage over the use of glucose . Acetone butanol ethanol (ABE) was produced from 30gL(-1) starch solution using a continuous process . The bioreactor was fed at a dilution rate of 0.02h(-1) and starch solution/feed volume (3L) was replaced every 72h . The continuous reactor fed with cornstarch solution (feed temperature 19 degrees C) produced approximately 6.0gL(-1) total ABE . Increasing the feed storage temperature to 37 degrees C improved ABE production to 7.2gL(-1) suggesting that retrogradation was occurring more rapidly at 19 degrees C . In both these cases the fermentation drifted toward acid production after approximately 260h, consistent with the retrogradation of starch overtime . The use of soluble starch, which is less prone to retrogradation, resulted in the production of 9.9gL(-1) ABE at 37 degrees C feed storage temperature, as compared to 7.2gL(-1) ABE when cornstarch was used . It should be noted that gelatinized starch retrogradation takes place after sterilization and prior to use of the feed medium, and does not occur during long-term storage of the raw corn material in the months leading up to processing . The degree of hydrolysis of gelatinized starch decreased from 68.8 to 56.2% in 3 days when stored at 37 degrees C . Soluble starch which does not retrograde demonstrated no change in the degree of hydrolysis.

J Vet Med B Infect Dis Vet Public Health, 2004 Dec, 51(10), 423 - 6
Significance of beta2-Toxigenic Clostridium perfringens Infections in Animals and Their Predisposing Factors - A Review; Schotte U et al.; Summary The novel beta2-toxin of Clostridium perfringens has recently been described as the cause of enteric diseases in animals . The biological activity of beta2-toxin is similar to that of the beta1-toxin with a possibly weaker cytotoxic activity . However, the production of beta2-toxin in vitro is not seen in all beta2-toxin-gene (cpb2)-positive C . perfringens strains, and to deduce a clinical importance solely from the detection of cpb2 is difficult . Detection of cpb2-positive C . perfringens from various animal species with and without enteric diseases demonstrates the wide distribution of cpb2 in nature, and the presence of cpb2 gene is therefore not considered a risk by itself . Predisposing factors like low trypsin activity in the intestinal tract, antibiotic and/or antiphlogistic treatment or changes in diet can result in the selection of beta2-toxigenic C . perfringens which may lead to enteritis or enterotoxaemia.

J Intern Med, 2005 Jan, 257(1), 78 - 92
Probiotics and gastrointestinal diseases; Sullivan A et al.; Abstract . Sullivan A, Nord CE (Karolinska University Hospital Huddinge, Karolinska Institutet, Stockholm, Sweden) . Probiotics and gastrointestinal diseases (Review article) . J Intern Med 2005; 257: 78-92.There is increasing evidence indicating health benefits by consumption of foods containing microorganisms, i.e . probiotics . A number of clinical trials have been performed to evaluate the effects in the prevention and treatment of gastrointestinal diseases caused by pathogenic microorganisms or by disturbances in the normal microflora . Gastrointestinal infections caused by Helicobacter pylori, traveller's diarrhoea, rotavirus diarrhoea, antibiotic-associated diarrhoea (AAD) and Clostridium difficile-induced diarrhoea are conditions that have been studied . There are also studies performed on the preventive effect of probiotics on radiation-induced diarrhoea and diarrhoea in tube-fed patients . Inflammatory bowel disease and irritable bowel syndrome, two idiopathic conditions where alterations in the normal microflora have been implicated as responsible for initiation, are two further areas where the use of probiotics has been regarded as promising . The results from clinical studies have not been conclusive in that the effects of probiotics have been strain-dependent and different study designs have been used . Treatment of acute diarrhoea in children and prevention of AAD are the two most justified areas for the application of probiotics.

Acta Neurochir (Wien) . 2004 Dec 21; {Epub ahead of print}
Distal ventriculoperitoneal shunt failure secondary to Clostridium difficile colitis; Gottfried ON et al.; Distal ventriculoperitoneal shunt obstruction is typically associated with cerebrospinal fluid (CSF) infection, fluid pseudocysts, bowel obstruction, bowel perforation, or improper shunt placement in the abdomen . We describe a unique etiology for distal shunt obstruction secondary to Clostridium difficile pancolitis that occurred because of inflammation and ascites, which led to incomplete drainage and absorption of CSF . This case illustrates the importance of considering distal shunt obstruction in a patient with signs of abdominal pathology in the setting of mental status changes, and the effective treatment of this patient initially with distal catheter externalization followed by internalization of a new distal catheter after resolution of the pancolitis.

J Infect, 2005 Jan, 50(1), 76 - 80
Fatal Clostridium tertium septicemia in a nonneutropenic patient; Tappe D et al.; Clostridium tertium septicemia is a rare condition that predominantly occurs in neutropenic patients with concomitant abdominal disease . We report the fatal case of a nonneutropenic, 51-year-old patient with mechanical ileus and post-operative C . tertium septicemia, resulting in widespread pathology with multi-organ failure . As C . tertium is aerotolerant, often gram-variable and mostly resistant to broad-spectrum cephalosporins, differentiation is difficult and empirical therapeutic strategies may fail.

Mol Pharmacol . 2004 Dec 15; {Epub ahead of print}
A novel strategy for the enhancement of drug absorption using a claudin modulator; Kondoh M et al.; Claudin, a tight junction integral membrane protein and a family of proteins, forms the actual sealing element of the tight junction . There are more than 20 members of the claudin family with different tissue-specific expression and barrier function . Thus, a family of claudin may be a target for modifying the absorption of drugs . Here, we examined whether modulation of claudin could be used to enhance drug absorption . In the current studies, we used a C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE) as a modulator of claudin-4 . The absorption of dextran was assessed in an in situ loop assay in rats to evaluate the absorption-enhancing effects of C-CPE . Treatment with C-CPE dose-dependently enhanced the absorption of dextran (MW 4,000) . These effects were not accompanied by injury of the intestinal mucosa as assessed by leakage of lactose dehydrogenase and histological observation . C-CPE was over 400-fold more potent at enhancing dextran absorption than capric acid, a clinically used enhancer of absorption . C-CPE interacted directly with claudin-4, and C-CPE lacking a part the C-terminus neither bound claudin-4 nor enhanced absorption in the rat jejunum . These results suggest that C-CPE enhances the absorption of dextran in rat jejunum, apparently through interactions with claudin-4, and this effect may represent an effective novel strategy for enhancing the absorption of drugs.

J Bacteriol, 2005 Jan, 187(1), 99 - 106
Regulation of cellulase synthesis in batch and continuous cultures of Clostridium thermocellum; Zhang YH et al.; Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram {dry weight} of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal . Chem . 75:219-227, 2003) . The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures . In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate . The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis . Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter . Several factors suggest that cellulase synthesis in C . thermocellum is regulated by catabolite repression . These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C . thermocellum genome.

J Clin Endocrinol Metab . 2004 Dec 14; {Epub ahead of print}
Hypoxia up-regulates HIF-1{alpha} expression through RhoA activation in trophoblast cells; Hayashi M et al.; During early pregnancy, trophoblast cells are exposed to relative low oxygen tension . Recently, the rho GTPase family has been shown to play a key role in hypoxia-inducible factor-1 (HIF-1) alpha induction in renal cell carcinoma . The present study was designed to investigate the effect of low oxygen conditions on RhoA expression in trophoblast cells isolated from early stages of human placenta and in trophoblast-derived BeWo cells and JAR cells . Immunoblot and RT-PCR analyses showed that low oxygen conditions (1% O2 or 250 microM CoCl2) stimulated expression of RhoA protein and mRNA . Pull-down assays demonstrated that these low oxygen conditions increased RhoA activity . Preincubation of BeWo cells with Clostridium botulinum C3 exoenzyme, a specific inhibitor of rho, inhibited hypoxia-induced HIF-1alpha expression . Under 1% O2 or 250 microM CoCl2, BeWo cells transfected with a dominant-negative RhoA exhibited decreased levels of HIF-1alpha protein and mRNA compared with the control vector transfectants . BeWo cells expressing constitutively active RhoA showed enhanced protein levels of not only HIF-1alpha but also vascular endothelial growth factor (VEGF) and glucose transporter (GLUT) 1, which are target gene products of HIF-1alpha . These findings suggest that up-regulation of RhoA induced by low oxygen conditions may play an important role in regulation of HIF-1alpha expression in trophoblast cells.

Can Fam Physician, 2004 Nov, 50, 1536 - 40, 1543-5
Clostridium difficile-associated colitis; Hull MW et al.; OBJECTIVE: To review the basic microbiology, pathogenesis of disease, and diagnosis of the nosocomial pathogen Clostridium difficile and to examine therapies recommended by the Canadian Task Force on Preventive Health Care . QUALITY OF EVIDENCE MEDLINE: was searched using MeSH headings . Controlled trials for therapy were sought, but case-control studies and observational reviews were included . MAIN MESSAGE: Clostridium difficile causes approximately 20% of cases of diarrhea associated with antibiotics, including clindamycin and the second- and third-generation cephalosporins . Diarrhea is usually mild, but can be severe; extreme cases develop toxic megacolon . Diagnosis is dependent on demonstrating presence of clostridial toxin in stool specimens or of pseudomembranes through sigmoidoscopy . First-line therapy for C . difficile diarrhea is restricted to metronidazole . Second-line therapy for treatment failure is vancomycin . For relapse, a second course of metronidazole is recommended; tapering courses of vancomycin and probiotics are used for multiple recurrences . CONCLUSION: Clostridium difficile is an important nosocomial pathogen requiring prudent use of antibiotics and strict infection-control policies to prevent large health care costs.

Acta Clin Belg, 2004 Jul-Aug, 59(4), 223 - 4
Saccharomyces cerevisiae fungemia in an elderly patient with Clostridium difficile colitis; Cherifi S et al.; Saccharomyces boulardii is widely used as a probiotic compound and is generally thought to be safe . We report one case of fungemia caused by Saccharomyces cerevisiae occurring in an elderly patient treated orally with S . boulardii in association with vancomycin for Clostridium difficile colitis . We do not recommend administering this viable yeast particularly in debilited patient with active colitis.

Protein Eng Des Sel . 2004 Dec 13; {Epub ahead of print}
Interactions between immunoglobulin-like and catalytic modules in Clostridium thermocellum cellulosomal cellobiohydrolase CbhA; Kataeva IA et al.; Cellobiohydrolase CbhA from Clostridium thermocellum cellulosome is a multi-modular protein composed starting from the N-terminus, of a carbohydrate-binding module (CBM) of family 4, an immunoglobulin(Ig)-like module, a catalytic module of family 9 glycoside hydrolases (GH9), X11 and X12 modules, a CBM of family 3, and a dockerin module . Deletion of the Ig-like module from the Ig-GH9 construct results in complete inactivation of the GH9 module . The crystal structure of the Ig-GH9 module pair reveals the existence of an extensive module interface composed of over forty amino acid residues of both modules and maintained through a large number of hydrophilic and hydrophobic interactions . To investigate the importance of these interactions between the two modules, we compared the secondary and tertiary structure and thermostabilities of the individual Ig-like and GH9 modules and the Ig-GH9 module pair using both circular dichroism (CD) spectroscopy and differential scanning calorimetry (DSC) . Thr230, Asp262 and Asp264 of the Ig-like module are located in the module interface of the Ig-GH9 module pair and are suggested to be important in "communication" between the modules . These residues were mutated to alanyl residues . The structure, stability and catalytic properties of the native Ig-GH9 and its D264A and T230A/D262A mutants were compared . The results indicate that despite being able to fold relatively independently, the Ig-like and GH9 modules interact and these interactions affect the final fold and stability of each module . Mutations of one or two amino acid residues lead to destabilization, and change of the mechanism of thermal unfolding of the polypeptides . The enzymatic properties of native Ig-GH9, D264A and T230A/D262A mutants are similar . The results indicate that inactivation of the GH9 module occurs as a result of multiple structural disturbances finally affecting the topology of the catalytic center.

Theriogenology, 2005 Jan 1, 63(1), 17 - 23
A new periparturient disease in Eastern Europe, Clostridium difficile causes postparturient sow losses; Kiss D et al.; Postparturient sow losses caused by Clostridium difficile have not been reported in the veterinary literature . Recently in Croatia, in a large outdoor production unit with suboptimal environmental conditions, a sudden increase in postparturient sow mortality was diagnosed . After postpartal application of enrofloxacine to postparturient mastitis metritis agalactia (MMA) suffering sows, diarrhea, respiratory distress, and mortality of these sows were recorded . While 13% of MMA suffering and treated sows died, only 0.4% of the non-treated (no MMA suffering) sows died postpartum . Gross pathology revealed mesocolonic edema, hydrothorax, and ascites . Microscopic examination showed scattered foci of suppuration in the colonic lamina propria and accumulation of neutrophils and fibrin on colonic mucosa . Anaerobic cultures of the colon yielded heavy growth of C . difficile . Enzyme immunoassay revealed C . difficile toxins A and B . C . difficile infections of postparturient MMA suffering sows may be associated with environmental stress, the application of antibiotics, or both . C . difficile infections are an impending danger in Eastern Europe and does not only raise animal welfare issues, but seriously inflict the economical well being of outdoor production units.

Biochem Pharmacol, 2005 Jan 1, 69(1), 87 - 95
Inhibitory effects of mevastatin and a geranylgeranyl transferase I inhibitor (GGTI-2166) on mononuclear osteoclast formation induced by receptor activator of NFkappaB ligand (RANKL) or tumor necrosis factor-alpha (TNF-alpha); Woo JT et al.; We have previously reported that the statin mevastatin (compactin) reversibly inhibits the fusion of TRAP-positive mononuclear preosteoclasts (pOCs) into multinucleated osteoclasts and disrupts the actin ring in mature osteoclasts through the inhibition of protein prenylation . Protein geranylgeranylation, specifically, is known to be required for pOC fusion and for the function and survival of mature osteoclasts . However, it has not been determined whether protein geranylgeranylation is involved in early differentiation of osteoclasts (pOC formation) . The current study shows that statins and the geranylgeranyl transferase I inhibitor GGTI-2166 inhibit the pOC formation induced by RANKL or TNF-alpha in cultures of both mouse marrow-derived macrophage-colony-stimulating factor (M-CSF) dependent monocytes (MD cells) and the mouse monocyte cell line RAW 264.7 (RAW cells) . Mevastatin, 0.1-0.6muM, inhibited the formation of pOCs induced by receptor activator of nuclear factor-kappaB ligand (RANKL) or tumor necrosis factor (TNF-alpha) in both cell cultures . The inhibitory effects of mevastatin were overcome by the addition of mevalonate, farnesyl pyrophosphate or geranylgeranyl pyrophosphate . GGTI-2166 inhibited TRAP activity induced by RANKL or TNF-alpha in both cell cultures and prevented the incorporation of {(3)H}all-trans geranylgeraniol into prenylated proteins in RAW cells . However, the farnesyl transferase inhibitor FTI-2153 did not inhibit TRAP activity although FTI prevented the incorporation of {(14)C}mevalonate into farnesylated proteins in RAW cells . Clostridium difficile cytotoxin B (toxin B) inhibited pOC formation induced by RANKL or TNF-alpha in both cell cultures . The inhibitory effects of statins and GGTI-2166 on pOC formation may result from the inhibition of the geranylgeranylation of G-proteins, such as Rho or Rac, suggesting that the geranylgeranylation of these proteins is involved in the early differentiation of progenitor cells into pOCs.

J Vet Diagn Invest, 2004 Nov, 16(6), 515 - 21
A retrospective study of mortality in Pennsylvania captive white-tailed deer (Odocoileus virginianus): 20000--2003; Hattel AL et al.; The postmortem records of 160 white-tailed deer (Odocoileus virginianus) submitted for necropsy examination from 59 separate Pennsylvania captive deer farms over a 3.5-year period were reviewed to determine the primary cause of death of each animal . The most common causes of death were bronchopneumonia (39 cases), enterocolitis (30 cases), malnutrition (13 cases), and trauma (11 cases) . Other causes of mortality included severe gastrointestinal parasitism (6 cases), cellulitis with septicemia (5 cases), degenerative myopathy (4 cases), ruminal acidosis (4 cases), and nephritis (4 cases) . The cause of death was undetermined in 13 of the 160 animals . Arcanobacterium pyogenes (19 cases), Fusobacterium necrophorum (10 cases), Escherichia coli (7 cases), and Mannheimia haemolytica (4 cases) were the most commonly isolated bacteria from the pneumonic lungs . Bacterial agents associated with enterocolitis included Clostridium perfringens (15 cases), E . coli (12 cases), and Mycobacterium avium subsp . paratuberculosis (2 cases) . The majority (52.2%) of the death loss in white-tailed deer of known ages occurred in animals 1 year of age or less, with 46.2% of the bronchopneumonia cases and 50.0% of the enterocolitis cases occurring during this time period . Cases of degenerative myopathy, myocardial degeneration, hepatic necrosis, meningoencephalitis, peritonitis, and urolithiasis considered severe enough to be the primary cause of death appeared early in life, affecting deer 6 months of age or less in all cases . In conclusion, bronchopneumonia, enterocolitis, malnutrition, and trauma were considered the most common causes of death in confined white-tailed deer in this study.

J Clin Microbiol, 2004 Dec, 42(12), 5947 - 9
Bacteremia due to Clostridium hathewayi in a patient with acute appendicitis; Woo PC et al.; Clostridium hathewayi is a newly described Clostridium species isolated from the feces of healthy human individuals, but its clinical significance is not known . We describe a case of human infection associated with C . hathewayi . The bacterium (strain HKU18) was isolated from the blood culture of a 39-year-old patient with acute gangrenous appendicitis complicated by septic shock . The cells were strictly anaerobic, nonmotile rods that stained gram negative . Conventional phenotypic tests and commercial identification systems failed to identify HKU18 to the species level . 16S rRNA gene analysis showed 1.4% nucleotide difference between the sequence of HKU18 and that of C . hathewayi, indicating that HKU18 was a strain of C . hathewayi . The patient responded to appendectomy and antibiotic treatment . 16S rRNA gene sequencing would be useful in further characterizing the clinical disease spectrum of C . hathewayi.

J Clin Microbiol, 2004 Dec, 42(12), 5710 - 4
Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile; Lemee L et al.; A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates . Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A-B+) variant strains . The reliability of the multiplex PCR was established by using a panel of 72 C . difficile strains including A+B+, A-B-, and A-B+ toxigenic types and 11 other Clostridium species type strains . The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C . difficile in 1,343 consecutive human and animal stool samples . Overall, 111 (15.4%) of 721 human samples were positive for C . difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A-B+ variant strains . Fifty (8%) of 622 animal samples contained C . difficile strains, which were toxigenic in 27 (54%) cases, including 1 A-B+ variant isolate . Eighty of the 721 human stool samples (37 positive and 43 negative for C . difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C . difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively . The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C . difficile intestinal infections.

Water Sci Technol, 2004, 50(9), 223 - 8
Production of hydrogen and methane from wastewater sludge using anaerobic fermentation; Ting CH et al.; The hydrogen and methane were produced from wastewater sludge using a Clostridium strain . The original sludge and the pre-treated (acidified, sterilized, freeze/thawed, and sonicated) sludges were tested . Some pre-treatment could enhance hydrogen yield, and the other tests could enhance methane yield . Hydrogen yield followed freeze/thawed>acidified>sterilized>original sludge>sonicated; while methane yield followed sonicated>freeze/thawed>sterilized>acidified>original sludge . The production and consumption of acetate correlated closely with the trends in both yields.

Childs Nerv Syst . 2004 Dec 4; {Epub ahead of print}
Secondary infection of intracranial hydatid cyst with Clostridium ramosum; Turkoglu OF et al.; INTRODUCTION . Brain involvement in hydatid disease occurs in 1-2% of all Echinococcus granulosus infections . Secondary infection of intracranial hydatid cysts is extremely rare . CASE REPORT AND DISCUSSION . In this case report, we present a secondary infection of an intracranial hydatid cyst due to Clostridium ramosum, which is an extremely rare infectious pathogen in neurosurgical practice, and a potential pitfall in neuroradiological investigations.

Surg Endosc . 2004 Dec 9; {Epub ahead of print}
The laparoscopic repair of suprapubic ventral hernias; Carbonell AM et al.; BACKGROUND . The complexity of dissection and the close proximity of the hernia to bony, vascular, nerve, and urinary structures make the laparoscopic repair of suprapubic hernias (LRSPH) a formidable operation . We performed a prospective evaluation of the outcomes of patients undergoing LRSPH.METHODS . The study population comprised 36 patients undergoing LRSPH from July 1996 to January 2004 . Patient demographics, hernia sizes, mesh types and sizes, perioperative outcomes, and recurrences were documented . After our early experience with this operation, the repair evolved to include transabdominal suture fixation to the pubic bone, Cooper's ligament, and above the iliopubic tract.RESULTS . There were 26 women and 10 men . They had a mean age of 55.9 years (range, 33-76) and a mean body mass index (BMI) of 31.0 kg/m(2) (range, 22-67) . Twenty-two (61%) of the repairs were for recurrent hernias, with an average of 2.3 previously failed open repairs each (range, 1-11) . The mean hernia size was 191.4 cm(2) (range, 20-768), and the average mesh size was 481.4 cm(2) (range, 193-1,428) . All repairs were performed with expanded polytetrafluoroethylene (ePTFE) mesh . Mean operating time was 178.7 min (range, 95-290) . Mean blood loss was 40 cc (range, 20-100) . One patient undergoing her fifth repair required conversion due to adhesions to a polypropylene mesh . Hospital stay averaged 2.4 days (range, 1-7) . Mean follow-up was 21.1 months (range, 1-70) . Complications (16.6%) included deep venous thrombosis ( n = 1), prolonged pain for >6 weeks ( n = 1), trocar site cellulitis ( n = 1), ileus ( n = 1), prolonged seroma ( n = 1), and Clostridium difficile colitis ( n = 1) . Hernias recurred in two of our first nine patients, for an overall recurrence rate of 5.5% . Since we began using the technique of applying multiple sutures directly to the pubis and Cooper's ligament (in the subsequent 27 patients), no recurrences have been documented.CONCLUSIONS . Although technically demanding and time-consuming, the LRSPH is safe and technically feasible . Moreover, it results in a low recurrence rate and is applicable to large or multiply recurrent hernias . Transabdominal suture fixation to the bony and ligamentous structures produces a more durable hernia repair.

Aesthetic Plast Surg . 2004 Dec 2; {Epub ahead of print}
Complications with the Use of Botulinum Toxin Type A in Facial Rejuvenation: Report of 8 Cases; Ferreira MC et al.; The botulinum toxin A is produced by Clostridium botulinum and causes a reversible, selective muscle relaxation that leads to a temporary flattening of the mechanical component of wrinkling without the stigmata of invasive surgery . Since the end of the 1980s, this neurotoxin has been used to treat mimic facial lines with good results . Although this is considered a safe therapy, with adverse effects typically self-limited, more severe complications have been observed when it is used by nonskilled physicians or in improper dosages . This article reports eight patients treated with botulinum toxin A for aesthetic purposes who developed different complications . Treatment of the complication included the use of electrical stimulation, lymphatic drainage, antiinflammatory therapy, dipivefrine cloridrate drops, and other approaches . With specific treatment for each patient, the lengths of these complications seemed to be reduced.

Rev Gastroenterol Disord, 2004 Fall, 4(4), 186 - 95
Clostridium difficile-associated diarrhea: risk factors, diagnostic methods, and treatment; Oldfield EC 3rd; Clostridium difficile-associated diarrhea (CDAD) has become the most common cause of infectious diarrhea acquired in the hospital, with an estimated 3 million annual cases and an annual cost of $1 billion . Risk factors for CDAD include antibiotic use (especially ampicillin, clindamycin, and cephalosporins), advanced age, and gastrointestinal surgery . Specific diagnosis of CDAD is made with an enzyme immunoassay to detect toxins A and B . Metronidazole remains the initial treatment of choice, with a 95% success rate . Vancomycin is reserved for failures . Despite the high initial success rates, recurrence of CDAD remains a significant problem in 20% to 30% of cases, with increased cost and substantial morbidity . Efforts to prevent CDAD will need to be strengthened, including education and better compliance with isolation, use of gloves, and hand washing.

Neurourol Urodyn, 2005, 24(1), 2 - 12
Botulinum toxin for the treatment of lower urinary tract symptoms: a review; Sahai A et al.; AIMS: To review the available literature on the application of botulinum toxin in the urinary tract, with particular reference to its use in treating detrusor overactivity (DO) . METHODS: Botulinum toxin, overactive bladder (OAB), detrusor instability, DO, detrusor sphincter dyssynergia (DSD), and lower urinary tract dysfunction were used on Medline Services as a source of articles for the review process . RESULTS: DO poses a significant burden on patients and their quality of life . Traditionally patients have been treated with anti-cholinergic drugs if symptomatic, however, a significant number find this treatment either ineffective or intolerable due to side effects . Recent developments in this field have instigated new treatment options, including botulinum toxin, for patients' refractory to first line medication . Botulinum toxin, one of the most poisonous substances known to man, is a neurotoxin produced by the bacterium Clostridium botulinum . Botulinum toxin injections into the external urethral sphincter to treat detrusor sphincter dyssynergia has been successfully used for some years but recently its use has expanded to include voiding dysfunction . Intradetrusal injections of botulinum toxin into patients with detrusor overactivity and symptoms of the overactive bladder have resulted in significant increases in mean maximum cystometric capacity and detrusor compliance with a reduction in mean maximum detrusor pressures . Subjective and objective assessments in these patients has shown significant improvements that last for 9-12 months . Repeated injections have had the same sustained benefits . CONCLUSIONS: Application of botulinum toxin in the lower urinary tract has produced promising results in treating lower urinary tract dysfunction, which needs further evaluation with randomised, placebo-controlled trials.

J Bacteriol, 2004 Dec, 186(24), 8347 - 55
Isolation and expression of the xynB gene and its product, XynB, a consistent component of the Clostridium cellulovorans cellulosome; Han SO et al.; The nucleotide sequence of the Clostridium cellulovorans xynB gene, which encodes the XynB xylanase, consists of 1,821 bp and encodes a protein of 607 amino acids with a molecular weight of 65,976 . XynB contains a typical N-terminal signal peptide of 29 amino acid residues, followed by a 147-amino-acid sequence that is homologous to the family 4-9 (subfamily 9 in family 4) carbohydrate-binding domain . Downstream of this domain is a family 10 catalytic domain of glycosyl hydrolase . The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly . The XynB sequence from mass spectrometry and N-terminal amino acid sequence analyses agreed with that deduced from the nucleotide sequence . XynB was highly active toward xylan, but not active toward carboxymethyl cellulose . The enzyme was optimally active at 40 degrees C and pH 5.0 . Northern hybridizations revealed that xynB is transcribed as a monocistronic 1.9-kb mRNA . RNA ligase-mediated rapid amplification of 5' cDNA ends by PCR (RLM-5'RACE PCR) analysis of C . cellulovorans RNA identified a single transcriptional start site of xynB located 47 bp upstream from the first nucleotide of the translation initiation codon . Alignment of the xynB promoter region provided evidence for highly conserved sequences that exhibited strong similarity to the sigmaA consensus promoter sequences of gram-positive bacteria . Expression of xynB mRNA increased from early to middle exponential phase and decreased during the early stationary phase when the cells were grown on cellobiose . No alternative promoter was observed by RLM-5'RACE PCR and reverse transcriptase PCR analyses during expression . The analysis of the products from xylan hydrolysis by thin-layer chromatography indicated its endoxylanase activity . The results suggest that XynB is a consistent and major cellulosomal enzyme during growth on cellulose or xylan.

Appl Environ Microbiol, 2004 Dec, 70(12), 7497 - 510
Complete nucleotide sequence of the conjugative tetracycline resistance plasmid pFBAOT6, a member of a group of IncU plasmids with global ubiquity; Rhodes G et al.; This study presents the first complete sequence of an IncU plasmid, pFBAOT6 . This plasmid was originally isolated from a strain of Aeromonas caviae from hospital effluent (Westmorland General Hospital, Kendal, United Kingdom) in September 1997 (G . Rhodes, G . Huys, J . Swings, P . McGann, M . Hiney, P . Smith, and R . W . Pickup, Appl . Environ . Microbiol . 66:3883-3890, 2000) and belongs to a group of related plasmids with global ubiquity . pFBAOT6 is 84,748 bp long and has 94 predicted coding sequences, only 12 of which do not have a possible function that has been attributed . Putative replication, maintenance, and transfer functions have been identified and are located in a region in the first 31 kb of the plasmid . The replication region is poorly understood but exhibits some identity at the protein level with replication proteins from the gram-positive bacteria Bacillus and Clostridium . The mating pair formation system is a virB homologue, type IV secretory pathway that is similar in its structural organization to the mating pair formation systems of the related broad-host-range (BHR) environmental plasmids pIPO2, pXF51, and pSB102 from plant-associated bacteria . Partitioning and maintenance genes are homologues of genes in IncP plasmids . The DNA transfer genes and the putative oriT site also exhibit high levels of similarity with those of plasmids pIPO2, pXF51, and pSB102 . The genetic load region encompasses 54 kb, comprises the resistance genes, and includes a class I integron, an IS630 relative, and other transposable elements in a 43-kb region that may be a novel Tn1721-flanked composite transposon . This region also contains 24 genes that exhibit the highest levels of identity to chromosomal genes of several plant-associated bacteria . The features of the backbone of pFBAOT6 that are shared with this newly defined group of environmental BHR plasmids suggest that pFBAOT6 may be a relative of this group, but a relative that was isolated from a clinical bacterial environment rather than a plant-associated bacterial environment.

Appl Environ Microbiol, 2004 Dec, 70(12), 7192 - 9
Differentiation of the gene clusters encoding botulinum neurotoxin type A complexes in Clostridium botulinum type A, Ab, and A(B) strains; Franciosa G et al.; We describe a strategy to identify the clusters of genes encoding components of the botulinum toxin type A (boNT/A) complexes in 57 strains of Clostridium botulinum types A, Ab, and A(B) isolated in Italy and in the United States from different sources . Specifically, we combined the results of PCR for detecting the ha33 and/or p47 genes with those of boNT/A PCR-restriction fragment length polymorphism analysis . Three different type A toxin gene clusters were revealed; type A1 was predominant among the strains from the United States, whereas type A2 predominated among the Italian strains, suggesting a geographic distinction between strains . By contrast, no relationship between the toxin gene clusters and the clinical or food source of strains was evident . In two C . botulinum type A isolates from the United States, we recognized a third type A toxin gene cluster (designated type A3) which was similar to that previously described only for C . botulinum type A(B) and Ab strains . Total genomic DNA from the strains was subjected to pulsed-filed gel electrophoresis and randomly amplified polymorphic DNA analyses, and the results were consistent with the boNT/A gene clusters obtained.

Appl Environ Microbiol, 2004 Dec, 70(12), 6984 - 91
Design and production in Aspergillus niger of a chimeric protein associating a fungal feruloyl esterase and a clostridial dockerin domain; Levasseur A et al.; A chimeric enzyme associating feruloyl esterase A (FAEA) from Aspergillus niger and dockerin from Clostridium thermocellum was produced in A . niger . A completely truncated form was produced when the dockerin domain was located downstream of the FAEA (FAEA-Doc), whereas no chimeric protein was produced when the bacterial dockerin domain was located upstream of the FAEA (Doc-FAEA) . Northern blot analysis showed similar transcript levels for the two constructs, indicating a posttranscriptional bottleneck for Doc-FAEA production . The sequence encoding the first 514 amino acids from A . niger glucoamylase and a dibasic proteolytic processing site (kex-2) were fused upstream of the Doc-FAEA sequence . By using this fusion strategy, the esterase activity found in the extracellular medium was 20-fold-higher than that of the wild-type reference strain, and the production yield was estimated to be about 100 mg of chimeric protein/liter . Intracellular and extracellular production was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, dockerin-cohesin interaction assays, and Western blotting . Labeled cohesins detected an intact extracellular Doc-FAEA of about 43 kDa and a cleaved-off dockerin domain of about 8 kDa . In addition, an intracellular 120-kDa protein was recognized by using labeled cohesins and antibodies raised against FAEA . This protein corresponded to the unprocessed Doc-FAEA form fused to glucoamylase . In conclusion, these results indicated that translational fusion to glucoamylase improved the secretion efficiency of a chimeric Doc-FAEA protein and allowed production of the first functional fungal enzyme joined to a bacterial dockerin.

Pediatrics, 2004 Dec, 114(6), e757 - 60
Clostridium septicum myonecrosis in congenital neutropenia; Barnes C et al.; Severe congenital neutropenia (SCN) and Clostridium septicum myonecrosis is an uncommon and life-threatening association requiring urgent combined aggressive medical and surgical management . We report 2 cases of SCN (1 with known Kostmann's syndrome and 1 not known at presentation to have a congenital neutropenic disorder but subsequently received a diagnosis of cyclic neutropenia) who presented with spontaneous C septicum myonecrosis . The cases highlight the importance of response to recombinant human granulocyte colony-stimulating factor in obtaining a satisfactory outcome for these patients . Early, empirical use of recombinant human granulocyte colony-stimulating factor in patients who are suspected of having a congenital neutropenia and who present with life-threatening sepsis is recommended.

Obstet Gynecol, 2004 Dec, 104(6), 1244 - 7
Fulminant sepsis after invasive prenatal diagnosis; Plachouras N et al.; BACKGROUND: Sepsis is extremely rare after invasive prenatal diagnosis . CASE: A patient, who had undergone amniocentesis at 15 weeks, cordocentesis at 20 weeks, and repeat cordocentesis 24 hours before presentation, was admitted at 21 weeks gestation with vaginal bleeding, rupture of membranes, and intrauterine demise . Although clinical and laboratory findings were unremarkable at presentation, she rapidly developed septic syndrome with disseminated intravascular coagulation and eventually multiple organ failure . The fetus was disintegrated and the uterus had to be removed . She was discharged from the intensive care unit after 34 days . Cultures of the uterine content grew Clostridium perfringens . Review of the literature revealed 10 more cases of sepsis after transabdominal prenatal diagnosis . CONCLUSION: Sepsis after prenatal diagnosis can be devastating, unless promptly diagnosed and treated.

Crit Care Nurs Clin North Am, 2004 Dec, 16(4), 547 - 51
Clostridium difficile: causes and interventions; Posani T; Human infection with Clostridium difficile can take many forms . It can exist in many patients who are relatively well or who have symptoms similar to irritable bowel syndrome . It can also infect the patient in the acute care facility . These patients typically have received antibiotics for more than 3 days and begin to experience foul-smelling, watery stools within a few days of initiation of antibiotic coverage . Good hand washing and environmental cleanliness remain the primary ways of preventing the spread of this infection from patient to patient . The possibility of using probiotics to replace the beneficial bacteria should be pursued.

Antimicrob Agents Chemother, 2004 Dec, 48(12), 4898 - 902
In vitro activities of OPT-80 and comparator drugs against intestinal bacteria; Finegold SM et al.; The activities of OPT-80 against 453 intestinal bacteria were compared with those of seven other drugs . OPT-80 showed good activity against most clostridia, staphylococci, and enterococci, but streptococci, aerobic and facultative gram-negative rods, anaerobic gram-negative rods, and Clostridium ramosum were resistant . Poor activity against anaerobic gram-negative rods may maintain colonization resistance.

Kansenshogaku Zasshi, 2004 Oct, 78(10), 905 - 9
{A case of pseudomembranous enterocolitis caused by methicillin-resistant Staphylococcus aureus}; Fujita K et al.; A 47-year-old woman was hospitalized because of urinary-tract infection . She was treated with antibiotics for 6 days . However, severe watery diarrhea and pyrexia developed 6 days after stopping administration of antibiotics . Stool, throat and blood cultures were positive for methicillin-resistant Staphylococcus aureus (MRSA) and negative for Clostridium difficile DI toxin . In spite of administration of VCM, she died of septic shock . At autopsy, macroscopic observation revealed a pseudomembrane in the ileum . MRSA enterocolitis can occur in patients with antibiotic-related diarrhea, and physicians should be aware of its rapid clinical course and possible lethal outcome.

Rev Argent Microbiol, 2004 Jul-Sep, 36(3), 130 - 5
{"In vitro" activity of ten antimicrobial agents against anaerobic bacteria . A collaborative study, 1999-2002}; Litterio M et al.; The antimicrobial activity of ampicillin, ampicillin-sulbactam, cefoxitin, ceftriaxone, imipenem, piperacillin, piperacillin-tazobactam, clindamycin, metronidazole, and azitromycin was assesed against 166 strains of anaerobic bacteria recovered from eight hospitals in Buenos Aires . The strains studied were Bacteroides fragilis group (65), Fusobacterium spp . (26), Prevotella spp . (21), Porphyromonas spp . (10), Clostridium difficile (10), other clostridia (12), and gram-positive cocci (22) . The MICs were determined by the agar dilution method according to NCCLS document M11-A5 . Metronidazole and piperacillin-tazobactam were the most active antimicrobial agents tested and exhibited MIC90 values of < or = 2 microg/ml and < or = 4 microg/ml against gram-negative organisms, and < or = 2 microg/ml, and < or = 8 microg/ml against gram-positive organisms, respectively . Among beta-lactams the activity against gram-negative rods was in the following order: imipenem > piperacillin > cefoxitin > ceftriaxone > ampicillin . Among the gram-positive bacteria the decreased activity was: piperacillin > imipenem > cefoxitin > ceftriaxone > ampicillin . The majority of the species studied showed different degrees of resistance to clindamycin and azitromycin . Nevertheless, 90% of Fusobacterium nucleatum and Porphyromonas spp . isolates were inhibited by 0.125 mg/ml of clindamycin and azitromycin, respectively.

J Enzyme Inhib Med Chem, 2004 Aug, 19(4), 339 - 42
The inhibition of Clostridium chauvoei (jakari strain) neuraminidase activity by methanolic extracts of the stem barks of Tamarindus indicus and Combretum fragrans; Useh NM et al.; The inhibition of neuraminidase from Clostridium chauvoei (jakari strain) with partially purified methanolic extracts of some plants used in Ethnopharmacological practice was evaluated . Extracts of two medicinal plants, Tamarindus indicus and Combretum fragrans at 100-1000 microg/ml, both significantly reduced the activity of the enzyme in a dose-dependent fashion (P < 0.001) . The estimated IC50 values for Tamarindus indicus and Combretum fragrans were 100 and 150 microg/ml respectively . Initial velocity studies conducted, using fetuin as substrate revealed a non-competitive inhibition with the Vmax significantly altered from 500 micromole min(-1) mg(-1) to 240 micromole min(-1) mg(-1) and 340 micromole min(-1) mg(-1) in the presence of Tamarindus indicus and Combretum fragrans respectively . The KM remained unchanged at 0.42 mM . The computed Index of physiological efficiency was reduced from 1.19min(-1) to 0.57min(-1) and 0.75min(-1) with Tamarindus indicus and Combretum fragrans as inhibitors respectively.

Appl Microbiol Biotechnol, 2004 Dec, 66(2), 166 - 73 Epub 2004 Dec.
H2-producing bacterial communities from a heat-treated soil inoculum; Iyer P et al.; Hydrogen gas (approximately 60% H(2)) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l(-1)) . The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H(2) by methanogens . The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30 degrees C and 37 degrees C) . At 30-h HRT, the H(2) production rate was 80 ml h(-1) and yield was 0.91 mol H(2)/mol glucose . At 10-h HRT, the H(2) production rate was more than 5 times higher at 436 ml h(-1), and yield was 1.61 mol H(2)/mol glucose . Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA) . Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae . At 10-h HRT, only Clostridiaceae were detected . When the temperature of the 10-h HRT reactor was increased from 30 degrees C to 37 degrees C, the steady-state H(2) production rate increased slightly to 463 ml h(-1) and yield was 1.8 mol H(2)/mol glucose . Compared to 30 degrees C, RISA fingerprints at 37 degrees C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).

Infect Immun, 2004 Dec, 72(12), 6914 - 23
Fine mapping of the N-terminal cytotoxicity region of Clostridium perfringens enterotoxin by site-directed mutagenesis; Smedley JG 3rd et al.; Clostridium perfringens enterotoxin (CPE) has a unique mechanism of action that results in the formation of large, sodium dodecyl sulfate-resistant complexes involving tight junction proteins; those complexes then induce plasma membrane permeability alterations in host intestinal epithelial cells, leading to cell death and epithelial desquamation . Previous deletion and point mutational studies mapped CPE receptor binding activity to the toxin's extreme C terminus . Those earlier analyses also determined that an N-terminal CPE region between residues D45 and G53 is required for large complex formation and cytotoxicity . To more finely map this N-terminal cytotoxicity region, site-directed mutagenesis was performed with recombinant CPE (rCPE) . Alanine-scanning mutagenesis produced one rCPE variant, D48A, that failed to form large complexes or induce cytotoxicity, despite having normal ability to bind and form the small complex . Two saturation variants, D48E and D48N, also had a phenotype resembling that of the D48A variant, indicating that both size and charge are important at CPE residue 48 . Another alanine substitution rCPE variant, I51A, was highly attenuated for large complex formation and cytotoxicity, but rCPE saturation variants I51L and I51V displayed a normal large complex formation and cytotoxicity phenotype . Collectively, these mutagenesis results identify a core CPE sequence extending from residues G47 to I51 that directly participates in large complex formation and cytotoxicity.

Protein Expr Purif, 2004 Dec, 38(2), 258 - 63
Expression, purification and structural characterization of the scaffoldin hydrophilic X-module from the cellulosome of Clostridium thermocellum; Adams JJ et al.; The cellulosome is a membrane-bound, extracellular multi-subunit complex responsible for the degradation of crystalline cellulose by a number of organisms including anaerobic bacteria and fungi . The hydrophilic X-module (CipA-X) from the modular scaffoldin subunit of Clostridium thermocellum cellulosome has been proposed to play various roles in cellulosomal function, including thermal and structural stability . Towards elucidating the function of CipA-X using structural and biophysical studies, the region comprising residues 1692-1785 from the C . thermocellum CipA cDNA encoding CipA-X was cloned into a pET21b expression vector . When expressed in Escherichia coli, the C-terminal His-tagged protein accumulated in the insoluble fraction . Cell fractionation experiments showed that the recombinant protein was localized to inclusion bodies . Refolding and purification involved denaturation of the whole cell lysate by addition of urea, followed by a nickel-Sepharose chromatography step and dialysis into native conditions (25 mM Tris-HCl, pH 7.4, 50 mM NaCl, and 10 mM EDTA) . A final gel filtration step purified the protein to homogeneity, yielding 40 mg/L . The two-dimensional 1H-15N correlation spectrum of uniformly 15N-labelled CipA-X showed the characteristics of a well-folded protein comprising significant beta-structure, which is in agreement with the circular dichroism data.

Int J Antimicrob Agents, 2004 Dec, 24(6), 619 - 21
Resistance determinants in strains of Clostridium difficile from two geographically distinct populations; Bendle JS et al.; Ninety-three clinical isolates of Clostridium difficile, comprising 65 from Royal Gwent Hospital, Newport and 28 from Southmead Hospital, Bristol were examined to determine the prevalence of genes coding for macrolide resistance and to explore differences in susceptibility patterns . Antibiogram testing produced similar results for both sets of strains with respect to amoxicillin, tetracycline, erythromycin and cefotaxime . Results differed for rifampicin, where 53% of the Bristol isolates were resistant, compared with 3% of the Newport isolates . Clindamycin disc susceptibility testing produced similar resistance rates . However, clindamycin MIC determinations revealed that 53% of the Bristol strains exhibited high-level resistance (MIC > 256 mg/L), whereas strains from Newport had clindamycin MICs ranging from 0.25 to 3mg/L . erm (B) was present in 15 of the strains from Bristol and in none of the Newport strains . erm (F) and erm (Q) were not detected in either population . The two geographically distinct populations of C . difficile differed considerably in their susceptibility to antibiotics . The possibility that C . difficile may serve as a conservator for resistant determinants subsequent to exposure to antimicrobial agents, has important implications for infection control.

Int J Antimicrob Agents, 2004 Dec, 24(6), 562 - 6
Risk factors and mortality associated with Clostridium difficile-associated diarrhoea at a VA hospital; Changela U et al.; The objective of this study was to evaluate the risk of certain patient co-morbidities and antibiotics in the development of Clostridium difficile-associated diarrhoea (CDAD) . Hospitalized patients developing CDAD during a specified period were compared with a cohort of patients, matched by age, without a diagnosis of CDAD, who were hospitalized during the same time period . Data collection included demographics, hospital ward, co-morbid conditions, antibiotics received, and mortality . Gender and age were similar in both groups . Co-morbid conditions significantly associated with the case group included cancer and COPD . The most commonly prescribed antibiotics in the case versus control group included levofloxacin, intravenous vancomycin, clindamycin, and piperacillin/tazobactam . The case group was associated with a higher mortality rate.

Poult Sci, 2004 Nov, 83(11), 1868 - 75
Effects of copper-bearing montmorillonite on growth performance, digestive enzyme activities, and intestinal microflora and morphology of male broilers; Xia MS et al.; Avian commercial male broiler chicks (n = 240), 1 d of age, were used to investigate the effects of copper-bearing montmorillonite (Cu-MMT) on growth performance, digestive enzyme activities, and intestinal microflora and morphology . The chicks were allocated to 4 treatments, each of which had 5 pens of 12 chicks per pen . The 4 treatments were basal diet only (control group), basal diet + 1.5 g/kg montmorillonite (MMT), basal diet + 36.75 mg/kg Cu, in the form of CuSO4, and basal diet + 1.5 g/kg Cu-MMT . The results showed that supplementation with Cu-MMT significantly improved growth performance compared with the control diet, and that chicks fed with Cu-MMT had higher average daily gain (ADG) than those fed with MMT or CuSO4 . Supplementation with Cu-MMT significantly reduced the total viable counts of Escherichia coli and Clostridium in the small intestine and cecum . Supplementation with MMT or CuSO4 had no influence on intestinal microflora . Chicks fed with Cu-MMT had lower viable counts of E . coli in cecal contents than those fed with MMT or CuSO4 . The addition of either MMT or Cu-MMT to the diet improved the activities of total protease, amylase, and lipase in the small intestinal contents but had no effect on those in the pancreas . Morphological measurements of the small intestinal mucosa of chicks indicated that dietary addition of MMT or Cu-MMT improved intestinal mucosal morphology.

J Food Prot, 2004 Nov, 67(11), 2530 - 7
Comparison of pressure and heat resistance of Clostridium botulinum and other endospores in mashed carrots; Margosch D et al.; Inactivation of bacterial endospores in food requires a combination of pressure and moderate heat . Endospore resistance of seven Clostridium botulinum strains was compared with those of Bacillus spp . (B . cereus, B . subtilis, B . licheniformis, B . smithii, B . amyloliquefaciens) and Thermoanaerobacterium thermosaccharolyticum with respect to pressure (600 to 800 MPa) and temperature (80 to 116 degrees C) treatments in mashed carrots . A large variation was observed in the pressure resistance of C . botulinum spores . Their reduction after treatments with 600 MPa at 80 degrees C for 1 s ranged from more than 5.5 log units to no reduction . Spores of the proteolytic C . botulinum TMW 2.357 exhibited a greater resistance to pressure than spores from all other bacteria examined, with the exception of B . amyloliquefaciens . Heat resistance of spores did not correlate with pressure resistance, either within strains of C . botulinum or when C . botulinum spores were compared with spores of T . thermosaccharolyticum . A quantitative release of dipicolinic acid was observed from C . botulinum spores on combined pressure and temperature treatments only after inactivation of more than 99.999% of the spores . Thus, dipicolinic acid is released by a physicochemical rather than a physiological process . The resistance of spores to combined pressure and temperature treatments correlated with their ability to retain dipicolinic acid . B . amyloliquefaciens, a mesophilic organism that forms highly pressure-resistant spores is proposed as a nonpathogenic target organism for high-pressure process development.

Emerg Infect Dis, 2004 Nov, 10(11), 1950 - 2
Human Infection Caused by Clostridium hathewayi; Elsayed S; We describe a 27-year-old man with acute cholecystitis, hepatic abscess, and bacteremia caused by Clostridium hathewayi, a newly described gram-negative, endospore-forming, rod-shaped bacterium . This report is the first of human infection caused by this microorganism.

Rev Physiol Biochem Pharmacol, 2004, 152, 49 - 63 Epub 2004.
CNF and DNT; Hoffmann C et al.; The actin cytoskeleton of mammalian cells is involved in many processes that affect the growth and colonization of bacteria, such as migration of immune cells, phagocytosis by macrophages, secretion of cytokines, maintenance of epithelial barrier functions and others . With respect to these functions, it is not surprising that many bacterial protein toxins, which are important virulence factors and causative agents of human and/or animal diseases, target the actin cytoskeleton of the host . Some toxins target actin directly, such as the C2 toxin produced by Clostridium botulinum . Moreover, bacterial toxins target the cytoskeleton indirectly by modifying actin regulators such as the low-molecular-mass guanosine triphosphate (GTP)-binding proteins of the Rho family . Remarkably, toxins affect these GTPases in a bidirectional manner . Some toxins inhibit and some activate the GTPases . Here we review the Rho-activating toxins CNF1 and CNF2 (cytotoxic necrotizing factors) from Escherichia coli, the Yersinia CNF(Y) and the dermonecrotic toxin (DNT) from Bordetella species . We describe and compare their uptake into mammalian cells, mode of action, structure-function relationship, substrate specificity and role in diseases.

J Appl Microbiol, 2004, 97(6), 1185 - 91
Growth of enterotoxigenic Staphylococcus aureus in povi masima, a traditional Pacific island food; Wong TL et al.; AIMS: To obtain preliminary data on the microbiology and hurdles to pathogen growth in the traditional Pacific Island food, povi masima, which is essentially beef brisket cured in brine . METHODS AND RESULTS: Six containers of povi masima were prepared and two were inoculated with five enterotoxigenic strains of Staphyloccocus aureus . The povi masima were divided into two lots each containing two uninoculated control and an inoculated container . Lot 1 was incubated at room temperature (20 degrees C) and lot 2 under refrigeration (4-5 degrees C) for up to 98 days . During storage, samples were removed and tested for aerobic plate count, coagulase-producing Staphylococci, Clostridium perfringens, staphylococcal enterotoxin and various chemical parameters of the food . Coagulase-producing Staphylococci and aerobic plate counts grew to high levels in both the inoculated and uninoculated lots stored at room temperature, but enterotoxin was only detected at one time point in these lots and this may represent a false positive result . The concentration of NaCl in the meat increased with time as concentrations equilibrated, and nitrite was rapidly lost in those lots stored at room temperature . Storage at 4-5 degrees C prevented proliferation of coagulase-producing Staphylococci . CONCLUSIONS: For safe curing and storage, this food should be kept under refrigeration as this prevented growth of staphylococci . Optimum storage would also be achieved with improved attempts to ensure equal distribution of NaCl prior to storage . SIGNIFICANCE AND IMPACT OF THE STUDY: Under conditions traditionally used to cure and store this food, enterotoxigenic staphylococci can grow to numbers where toxigenesis might occur, especially during the early stages of curing where the salt has not diffused from the brine into the meat.

Int J Syst Evol Microbiol, 2004 Nov, 54(Pt 6), 2257 - 62
Acetanaerobacterium elongatum gen . nov., sp . nov., from paper mill waste water; Chen S et al.; Two mesophilic anaerobic bacterial strains (Z7(T) and Z1) were isolated from waste water sludge of the Xinanzhang paper mill, Beijing, China . The strains were Gram-positive, non-spore-forming and motile . Cells were thin rods (0.2-0.4x4.0-8.0 microm) . Growth of the strains was observed at 20-42 degrees C and pH 5.0-7.5 . Both strains hydrolysed gelatin and aesculin and fermented several kinds of mono-, di- and oligosaccharides . The fermentation end products formed from glucose were acetate, ethanol, hydrogen and carbon dioxide . The predominant cellular fatty acids were the branched-chain fatty acids isoC(15 : 0) (42.83 %) and isoC(14 : 0) (32.11 %) . The DNA G+C contents of strains Z7(T) and Z1 were 50.4 and 48.6 mol%, respectively . Phylogenetic analysis based on 16S rRNA gene sequences revealed that the isolates represent a new phyletic sublineage within the Clostridium leptum rRNA cluster, with <91 % 16S rRNA gene sequence similarity to currently described species . On the basis of polyphasic evidence from this study, Acetanaerobacterium elongatum gen . nov., sp . nov., a novel genus and species, is proposed, with strain Z7(T) (=JCM 12359(T)=AS 1.5012(T)) as the type strain.

Med Dosw Mikrobiol, 2004, 56(2), 155 - 9
{Comparative study of thermoresistance spores of Clostridium difficle strains belonging to the different toxigenicity groups}; Wultanska D et al.; The thermoresistance of spores of Clostridium difficile strains belonging to the different toxigenicity groups was compared in the study . Among spores of toxigenicity C . difficile strains (26 C . difficile strains produced toxins A and B (TcdA+TcdB+) and 32 C . difficile strains produced only toxin B (TcdA-TcdB+) were high thermoresistant . Between spores of non-toxigenic C . difficile strains much lower thermoresistance was observed . In conclusion, more studies are needed to clarify the importance of spores transmission in growing up quantity of AAD cases in Poland.

Chem Rec, 2004, 4(5), 305 - 14
Butanol fermentation research: upstream and downstream manipulations; Ezeji TC et al.; An overview of advances in acetone-butanol fermentation research is presented with specific reference to the history of acetone-butanol fermentation, genetic manipulation of the butanol-producing Clostridium beijerinckii NCIMB 8052, as well as upstream and downstream processing . Specific reference is made to the development of the hyperamylolytic, hyper-"butanolagenic" C . beijerinckii BA101 strain . Amylolytic enzyme production by C . beijerinckii BA101 was 1.8- and 2.5-fold greater than that of the C . beijerinckii NCIMB 8052 strain grown in starch and glucose, respectively . We confirmed the presence of a phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) associated with cell extracts of C . beijerinckii BA101 by glucose phosphorylation by PEP and ATP-dependent glucose phosphorylation . It was found that C . beijerinckii BA101 was defective in PTS activity and that it compensates for this defect with enhanced glucokinase activity, resulting in an ability to transport and utilize glucose during the solventogenic stage . The principal problem associated with acetone-butanol fermentation by C . beijerinckii or C . acetobutylicum is butanol toxicity/inhibition to the culture . To solve this problem, we have attempted various alternative in situ/online techniques of butanol removal including membrane-based systems such as pervaporation, liquid-liquid extraction, and gas stripping . We found that gas stripping and pervaporation appear to be the most promising of the in situ acetone-butanol fermentation and recovery techniques but, in terms of cost-effective industrial applications, gas stripping appears to be the most promising . (c) 2004 The Japan Chemical Journal Forum and Wiley Periodicals, Inc.

J Biol Chem . 2004 Nov 12; {Epub ahead of print}
Identification of the structural and functional domains of the large serine recombinase TnpX from clostridium perfringens; Lucet IS et al.; Members of the large serine resolvase family of site-specific recombinases are responsible for the movement of several mobile genetic elements, however, little is known about the structure or function of these proteins . TnpX is a serine recombinase that is responsible for the movement of the chloramphenicol resistance elements of the Tn4451/3 family . We have shown that TnpX binds differentially to its transposon and target sites, suggesting that resolvase-like excision and insertion were two distinct processes . To analyse the structural and functional domains of TnpX and, more specifically, to define the domains involved in protein-DNA and protein-protein interactions, we conducted limited proteolysis studies on the wild-type dimeric TnpX1-707 protein and its functional truncation mutant, TnpX1-597 . The results showed that TnpX was organised into three major domains: Domain I (amino acids (aa) 1-170), which included the resolvase catalytic domain; domain II (aa 170-266); and domain III (aa 267-707), which contained the dimerization region and two separate regions involved in binding to the DNA target . A small polypeptide (aa 533-587) was shown to bind specifically to the TnpX binding sites providing further evidence that it was the primary DNA binding region . In addition, a previously unidentified DNA binding site was shown to be located between residues 583 and 707 . Finally, the DNA binding and multerimization, but not the catalytic, functions of TnpX could be reconstituted by recombining separate polypeptides that contain the N-terminal and C-terminal regions of the protein . These data provide evidence that TnpX has separate catalytic, DNA binding and multimerization domains.

Crit Rev Food Sci Nutr, 2004, 44(5), 353 - 60
Target selection in designing pasteurization processes for shelf-stable high-acid fruit products; Silva FV et al.; This study is focused on the search for targets and criteria for the design of pasteurization processes for high-acid shelf-stable fruit products, such as juices, nectars, pastes, purees, concentrates, jams, jellies, etc . First, an overview of pasteurization is presented and then, frequently used targets for pasteurization processes are reviewed Enzymes naturally present in fruits, in decreasing order of heat resistance, were pectinesterase, peroxidase, and polyphenoloxidase, and they may be used as pasteurization targets . The heat resistance of each enzyme is strongly dependent on its fruit origin . The most heat resistant micro-organisms capable of spoiling high acid fruit products include ascospores of Neosartorya fischeri, Byssochlamys nivea, Talaromyces flavus, Eupenicillium javanicum, and Byssochlamys fulva moulds, as well as bacterial spores of Clostridium butyricum, Bacillus coagulans, and Bacillus megaterium . These micro-organisms, spores, and enzymes were, in general, less heat resistant than the spores of a particular spoilage micro-organism named Alicyclobacillus acidoterrestris, which has been causing problems in the fruit industry . Therefore, the use of Alicyclobacillus acidoterrestris spores as a reference micro-organism in the design of pasteurization processes for high-acid shelf-stable fruit products is suggested.

Dis Colon Rectum, 2004 Oct, 47(10), 1620 - 6
Outcome after colectomy for Clostridium difficile colitis; Longo WE et al.; PURPOSE: Clostridium difficile colitis is a relatively common entity, yet large series of patients with fulminant C . difficile colitis are infrequently reported . This study was designed to identify risk factors, clinical characteristics, and outcome of patients who required colectomy for fulminant C . difficile colitis . METHODS: A population-based study on all patients in 159 hospitals of the Department of Veterans Affairs from 1997 to 2001 was performed . Data were compiled from several national computerized Department of Veterans Affairs data sets . Supplementary information including demographic information, discharge summaries, operative reports, and pathology reports were obtained from local medical records . Patient variables were entered into a computerized database and analyzed using the Pearson chi-squared and Fisher's exact tests . Statistical significance was designated as P < 0.05 . RESULTS: Sixty-seven patients (mean age, 69 (range, 40-86) years; 99 percent males) were identified . All 67 patients had C . difficile verified in the colectomy specimens . Thirty-six of 67 patients (54 percent) developed C . difficile colitis during a hospitalization for an unrelated illness, and 30 of 36 patients (87 percent) after a surgical procedure . Thirty-one of 67 (46 percent) developed C . difficile colitis at home . There was no history of diarrhea in 25 of 67 patients (37 percent) . Thirty of 67 patients (45 percent) presented in shock (blood pressure, <90 mmHg) . Forty-three of 67 patients (64 percent) presented with an acute surgical abdomen . Mean white blood cell count was 27.2; mean percent bands was 12 . Twelve of 67 patients (18 percent) had a negative C difficile colitis stool assay . Abdominal computed tomography correctly diagnosed 45 of 46 patients (98 percent) who were imaged . Twenty-six of 67 patients (39 percent) underwent colonoscopy; all 26 were found to have severe inflammation or pseudomembranes . Fifty-three of 67 patients (80 percent) underwent total colectomy; 14 of 67 underwent segmental colonic resection . Perforation and infarction were found in 59 of 67 patients (58 percent) at surgery . Overall mortality was 48 percent (32/67) . Mean hospitalization was 36 (range, 2-297) days . CONCLUSIONS: Patients with fulminant C . difficile colitis often present with an unexplained abdominal illness with a marked leukocytosis that rapidly progresses to shock and peritonitis . Although frequently developed during a hospitalization and often after a surgical procedure, it may develop outside of a hospital setting . Diarrhea may be absent and stool cytology may be negative for C . difficile toxin . Perforation and infarction are frequently found at surgery . In those patients who survive, a prolonged hospitalization is common . Mortality from fulminant C . difficile colitis remains high despite surgical intervention.

Curr Infect Dis Rep, 2004 Dec, 6(6), 420 - 425
The Role of the Intestinal Tract As a Source for Transmission of Nosocomial Pathogens; Stiefel U et al.; The intestinal tract provides an important source for transmission of many nosocomial pathogens, including Enterococcus species, Clostridium difficile, Candida species, Enterobacteriaceae, and other gram-negative bacilli . Recent data suggest that the intestinal tracts of hospitalized patients may also be an important reservoir of Staphylococcus aureus . Although the clinical manifestations of these pathogens are diverse, a common pathogenesis is involved in their colonization of and dissemination from the intestinal tract . Of particular importance is the role that antibiotic selective pressure plays in promotion of colonization by antibiotic-resistant pathogens . Strategies to limit the spread of these pathogens must include efforts to improve adherence to standard infection control practices and promotion of good antimicrobial stewardship . New strategies that include application of novel technologies to the problem of pathogen transmission are needed, and additional research is needed to clarify the potential utility of selective decontamination of the digestive tract.

Int J Med Microbiol, 2004 Oct, 294(4), 261 - 5
PCR identification of the plasmid-borne enterotoxin gene (cpe) in Clostridium perfringens strains isolated from food poisoning outbreaks; Nakamura M et al.; It is generally accepted that Clostridium perfringens strains associated with food poisoning carry their enterotoxin gene, cpe, on the chromosome, while C . perfringens strains isolated from non-food-borne diseases, such as antibiotic-associated diarrhea and sporadic diarrhea, carry cpe on the plasmid . However, we recently encountered a food poisoning outbreak caused by C . perfringens bearing a plasmid cpe . We therefore investigated a total of 31 clinical and non-clinical C . perfringens strains to locate the cpe gene by PCR . The cpe of nine heat-sensitive (100 degrees C for 10min) strains isolated from three outbreaks of food poisoning were located on the plasmid, while those of six heat-resistant strains from other food poisoning outbreaks were located on the chromosome . Moreover, the cpe of 5 heat-sensitive strains isolated from healthy human feces and those of 11 heat-sensitive soil strains were also located on the plasmid . These findings indicate that heat-sensitive, cpe-plasmid-borne C . perfringens strains should not be disregarded as causative agents of food poisoning.

Indian J Pediatr, 2004 Oct, 71(10), 883 - 5
Nosocomial diarrhea; Uppal B et al.; OBJECTIVE: To study the occurrence of nosocomial diarrhea in pediatric wards and the role of infections in its causation . METHODS: 75 cases conforming to the CDC definition of nosocomial diarrhea were selected for the study . Stool samples were subjected to direct microscopic examination, bacteriological culture and direct antigen detection for Clostridium difficile (C . difficile) and Rota virus . RESULTS: All the cases were sporadic in origin . Rota virus was detected in 16.67% cases and Enteropathogenic Escherichia coli (E.P.E.C.) was detected in 12% cases . Cases in which no pathogen was detected (82.7%) outnumbered those in which a pathogen was identified (17.3%) . CONCLUSION: Rota virus and E.P.E.C were the most common etiological agents . C . difficile was detected in one case only . Most of the sporadic cases of nosocomial diarrhea in pediatric patients can be managed on fluids and electrolytes.

J Biol Chem, 2005 Jan 14, 280(2), 1499 - 1505 Epub 2004 Nov 5.
Clostridium difficile Toxin A Induces Expression of the Stress-induced Early Gene Product RhoB; Gerhard R et al.; Clostridium difficile toxin A monoglucosylates the Rho family GTPases Rho, Rac, and Cdc42 . Glucosylation leads to the functional inactivation of Rho GTPases and causes disruption of the actin cytoskeleton . A cDNA microarray revealed the immediate early gene rhoB as the gene that was predominantly up-regulated in colonic CaCo-2 cells after treatment with toxin A . This toxin A effect was also detectable in epithelial cells such as HT29 and Madin-Darby canine kidney cells, as well as NIH 3T3 fibroblasts . The expression of RhoB was time-dependent and correlated with the morphological changes of cells . The up-regulation of RhoB was approximately 15-fold and was based on the de novo synthesis of the GTPase because cycloheximide completely inhibited the toxin A effect . After 8 h, a steady state was reached, with no further increase in RhoB . The p38 MAPK inhibitor SB202190 reduced the expression of RhoB, indicating a participation of the p38 MAPK in this stress response . Surprisingly, newly formed RhoB protein was only partially glucosylated by toxin A, sparing a pool of potentially active RhoB, as checked by sequential C3(bot)-catalyzed ADP-ribosylation . A pull-down assay in fact revealed a significant amount of active RhoB in toxin A-treated cells that was not present in control cells . We demonstrate for the first time that toxin A has not only the property to inactivate the GTPases RhoA, Rac1, and Cdc42 by glucosylation, but it also has the property to generate active RhoB that likely contributes to the overall picture of toxin treatment.

Vaccine, 2004 Dec 2, 23(3), 343 - 52
Differential antibody responses to Plasmodium falciparum-derived B-cell epitopes induced by diepitope multiple antigen peptides (MAP) containing different T-cell epitopes; Vasconcelos NM et al.; Epitopes of universal character are needed when designing subunit vaccines against infectious diseases such as malaria . We have compared the immunogenicity of B-cell epitopes from the Plasmodium falciparum antigen repeats DPNANPNV (PfCS protein) and VTEEI (Pf332) when assembled with four different universal T-cell epitopes in diepitope multiple antigen peptides (MAP) . T-epitopes employed were from P . falciparum antigens (CS.T3, {T(*)}4 and EBP3) or from the Clostridium tetani toxin (P2) . In association with either of the T-epitopes, the genetic unresponsiveness to the B-epitopes was successfully bypassed . Our results show that the immunogenicity of a T-epitope alone does not necessarily predict the ability of the T-epitope to provide T-cell help when combined with other epitopes in an immunogen . Further, the nature of the immune responses in terms of total IgG antibodies and their subclass distribution, T-cell proliferation and IFN-gamma production, varied with the T-epitope and mouse strain, which may indicate the need for inclusion of a combination of different universal T-epitopes in a future malaria subunit vaccine.

Avian Dis, 2004 Sep, 48(3), 675 - 80
High mortality in egg layers as a result of necrotic enteritis; Dhillon AS et al.; A new facility was designed to hold 1.8 million birds in 10 houses; chickens were placed in five of the houses, and the remaining five houses were under construction when this outbreak occurred . An increase in mortality was reported in five houses; however, mortality in house 7 was quite high . Well-fleshed birds were suddenly found dead without a significant drop in egg production . The middle and distal intestines were distended with gas, congested, thin walled, atonic, and bluish or pale in color with sloughed mucosa in some places . Necrotic enteritis was diagnosed as the cause of increased mortality . The ingesta in the crop occasionally contained flies . The 4-wk mortality in house 7 was 6.55% with a loss of 10,898 chickens . The 4-wk mortality rate in the other houses ranged from 0.54% to 1.98% . The houses affected with necrotic enteritis were treated for coccidiosis with amprolium because low numbers of the oocysts were present in the intestinal specimens of some of the chickens . Household bleach was added to the water at a dilution of one part bleach to 1040 parts water to control bacterial contamination . The fly (Musca domestica) population was out of control . Clostridium perfringens was isolated from the alcohol-washed macerated flies caught from houses 4 and 7 . Dead flies were often seen in the feed troughs . The chickens may possibly have had C . perfringens infection as a result of consumption of dead flies or their secretions/excretions . The alcohol-washed, macerated, clarified fly extract from the affected houses caused death in 11 inoculated mice and paralysis in one mouse . Similarly, illness and mortality were present in four mice inoculated with clarified intestinal contents . The bacterium isolated on anaerobic culture was identified as C . perfringens by polymerase chain reaction . The disease was brought under control after straw was added and mixed in with the litter . As a result, the litter temperature increased, causing a decrease in the fly population . This study suggests that flies in the poultry houses acted as mechanical transmitters of C . perfringens and that the development of necrotic enteritis was by ingestion of bacteria present in the flies and their secretions/excretions.

Int J Clin Pract, 2004 Sep, 58(9), 892 - 3
Severe sepsis following wound infection by an unusual organism--Clostridium novyi; Majumdar S et al.; We present a case of post-operative wound infection with Clostridium novyi in a non-intravenous drug user . Clinical features included progressive cellulitis despite being on antibiotics, accompanied by hypotension, marked leucocytosis and oedema but minimal fever . While established infection with this organism is associated with high mortality, our patient survived . The administration of clindamycin and intravenous immunoglobulin in addition to early surgical assessment and aggressive debridement of affected tissue may have contributed to this successful outcome . To our knowledge, this is the only reported post-operative wound infection due to this pathogen.

J Clin Microbiol, 2004 Nov, 42(11), 5406 - 8
Natural Clostridium botulinum type C toxicosis in a group of cats; Elad D et al.; Clinical signs of botulism were observed in a group of eight cats, four of which died, after being fed pelican carrion . Clostridium botulinum type C was isolated from one cat . The microorganism and its toxin were found in the pelican . This is apparently the first report of natural botulism in cats.

J Clin Microbiol, 2004 Nov, 42(11), 5184 - 8
Effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants; Jaureguy F et al.; Early-onset group B streptococcal (GBS) infections remain a leading cause of morbidity and mortality in infants . To prevent the vertical transmission of GBS and neonatal GBS infection, guidelines recommend intrapartum penicillin or amoxicillin prophylaxis . This intrapartum antibiotic prophylaxis (IAP) is suspected to favor colonization by antibiotic-resistant bacteria . However, the effects of this prophylaxis on the patterns of acquisition of gastrointestinal bacterial flora in infants have never been studied . We collected stool samples from 3-day-old infants born to mothers who received intrapartum amoxicillin (antibiotic-exposed group; n = 25) and to untreated mothers (non-antibiotic-exposed group; n = 25) . The groups were matched for factors known to affect intestinal microbial colonization: gestational age, type of delivery, and type of feeding . Qualitative and quantitative differential analyses of the bacterial flora in stool samples were performed . Similar numbers of infants in the non-antibiotic-exposed and antibiotic-exposed groups were colonized by aerobic bacteria and amoxicillin-resistant enterobacteria (75 and 77%, respectively) (P = 0.79) . In contrast, significantly fewer infants in the antibiotic-exposed group than in the non-antibiotic-exposed group were colonized by anaerobic bacteria, especially Clostridium (12 and 40%, respectively) (P < 0.05) . Regarding intestinal bacterial colonization, the differences between antibiotic-exposed and non-antibiotic-exposed infants were remarkably few . The only statistically significant effect was the reduced initial bacterial colonization by Clostridium in the antibiotic-exposed group . In our study, the use of IAP did not favor colonization by beta-lactam-resistant bacteria . However, further evaluations are required to highlight the potential risks of the widespread use of antibiotics to prevent early-onset GBS infection.

Appl Environ Microbiol, 2004 Nov, 70(11), 6459 - 65
Real-time PCR quantitation of clostridia in feces of autistic children; Song Y et al.; Based on the hypothesis that intestinal clostridia play a role in late-onset autism, we have been characterizing clostridia from stools of autistic and control children . We applied the TaqMan real-time PCR procedure to detect and quantitate three Clostridium clusters and one Clostridium species, C . bolteae, in stool specimens . Group- and species-specific primers targeting the 16S rRNA genes were designed, and specificity of the primers was confirmed with DNA from related bacterial strains . In this procedure, a linear relationship exists between the threshold cycle (CT) fluorescence value and the number of bacterial cells (CFU) . The assay showed high sensitivity: as few as 2 cells of members of cluster I, 6 cells of cluster XI, 4 cells of cluster XIVab, and 0.6 cell of C . bolteae could be detected per PCR . Analysis of the real-time PCR data indicated that the cell count differences between autistic and control children for C . bolteae and the following Clostridium groups were statistically significant: mean counts of C . bolteae and clusters I and XI in autistic children were 46-fold (P = 0.01), 9.0-fold (P = 0.014), and 3.5-fold (P = 0.004) greater than those in control children, respectively, but not for cluster XIVab (2.6 x 10(8) CFU/g in autistic children and 4.8 x 10(8) CFU/g in controls; respectively) . More subjects need to be studied . The assay is a rapid and reliable method, and it should have great potential for quantitation of other bacteria in the intestinal tract.

Int J Food Microbiol, 2004 Dec 1, 97(1), 71 - 80
Behavior of Clostridium perfringens at low temperatures; de Jong AE et al.; Refrigerated storage is an important step in the preparation of foods and inadequate storage is one of the main causes of food poisoning outbreaks of Clostridium perfringens . Therefore, growth and germination characteristics of C . perfringens in a temperature range of 3-42 degrees C were determined in fluid thioglycollate broth (FTG) and Dutch pea soup . To study the effect of adaptation, cells were either inoculated from a 37 degrees C pre-culture or from a temperature-adapted pre-culture . Membrane fatty acid patterns were determined at all temperatures to examine the effect of temperature on membrane composition . Spores were either inoculated with and without heat treatment . Adaptation of cells did not influence growth rate nor lag phase . Growth in pea soup, however, was slower and lag phases tended to be more extended compared to FTG . No growth was observed at temperatures < or =10 degrees C and death rates in pea soup were higher than those in FTG at these low temperatures . Cells preserved the membrane fluidity by reducing the arachidic acid content and increasing the lauric acid content when the temperature dropped . This resulted in a net reduction in chain length . Microscopic analysis of cells grown at 15 degrees C revealed a morphological change: cells were elongated compared to those grown at 37 degrees C . These data demonstrate the ability of C . perfringens to adapt to lower temperatures . However, this did not influence growth characteristics compared to non-adapted cells . Spores of C . perfringens did germinate at all temperatures with and without heat-activation . Combining this fact with the extended survival at low temperatures emphasizes the need for adequate heating of refrigerated foods before consumption to eliminate health risks due to C . perfringens.

FEBS Lett, 2004 Nov 5, 577(1-2), 81 - 6
Glycogen synthase kinase-3beta negatively regulates group IIA phospholipase A2 expression in human aortic smooth muscle and HepG2 hepatoma cells; Menschikowski M et al.; The present study shows that the IFN-gamma-mediated upregulation of secretory phospholipase A2 of group IIA (sPLA2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3beta (GSK-3beta) by indirubin-3'-monoxime, 5-iodo or AR-A014418 . The effect of GSK-3beta inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase . Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3beta inhibition on sPLA2-IIA expression depends on Janus kinase-2 and NF-kappaB-signaling.

J Biol Chem, 2005 Jan 14, 280(2), 1582 - 93 Epub 2004 Nov 03.
Angiotensin II and Epidermal Growth Factor Induce Cyclooxygenase-2 Expression in Intestinal Epithelial Cells through Small GTPases Using Distinct Signaling Pathways; Slice LW et al.; Colorectal carcinogenesis is a multistep process involving genetic mutations and alterations in rigorously controlled signaling pathways and gene expression that control intestinal epithelial cell proliferation, differentiation, and apoptosis . Cyclooxygenase-2 (COX-2) is aberrantly expressed in premalignant adenomatous polyps and colorectal carcinomas and is associated with increased epithelial cell proliferation, decreased apoptosis, and increased cell invasiveness . Currently, knowledge of the regulation of expression of COX-2 by endogenous cell-surface receptors is inadequate . Recently, in a non-transformed rat intestinal epithelial cell line (IEC-18), we showed induction of cell proliferation and DNA synthesis by angiotensin II (Ang II) via the endogenous Ang II type 1 receptor (Chiu, T., Santiskulvong, C., and Rozengurt, E . (2003) Am . J . Physiol . 285, G1-G11) . We report that Ang II potently stimulated expression of COX-2 mRNA and protein as an immediate-early gene response through the Ang II type 1 receptor, correlating with an increase in prostaglandin I(2) production . Ang II induced Cdc42 activation and filopodial formation . COX-2 expression was induced by epidermal growth factor (EGF), which activated Rac with lamellipodial formation . Inhibition of small GTPases by Clostridium difficile toxin B blocked COX-2 expression by Ang II and EGF . Inhibition of ERK activation by U0126 or PD98059 significantly decreased EGF-dependent COX-2 expression, but did not affect Ang II-dependent COX-2 expression . Conversely, inhibition of p38(MAPK) by SB202190 or PD169316 inhibited COX-2 expression by Ang II, but did not block COX-2 induction by EGF . Ang II caused Ca(2+) mobilization . Inhibition of Ca(2+) signaling by 2-aminobiphenyl borate blocked Ang II-dependent COX-2 expression . EGF did not induce Ca(2+) mobilization, and 2-aminobiphenyl borate did not inhibit EGF-dependent COX-2 expression . Inhibition of COX-2 expression correlated with inhibition of prostaglandin I(2) production . Luciferase promoter assays showed that Ang II-dependent transcriptional activation of the COX-2 promoter was dependent on activation of small GTPases and p38(MAPK) and on Ca(2+) signaling via the cAMP-responsive element/activating transcription factor cis-acting element.

Biochemistry (Mosc), 2004 Sep, 69(9), 949 - 62
Catalase and superoxide dismutase: distribution, properties, and physiological role in cells of strict anaerobes; Brioukhanov AL et al.; This review considers the distribution of the main enzymes of antioxidative defense, superoxide dismutase (SOD) and catalase, in various groups of strictly anaerobic microorganisms: bacteria of the genus Clostridium, Bacteroides, sulfate-reducing and acetogenic bacteria, methanogenic archaea, etc . Molecular and biochemical properties of purified Fe-containing SODs, cambialistic SODs, and heme catalases are presented . The physiological role and origin of the enzymes of antioxidative defense in strict anaerobes are discussed . Physiological responses (induction of SOD and catalase) to factors provoking oxidative stress in the cells of strict anaerobes able to maintain viability under aerobic conditions are also considered.

Vet Clin North Am Equine Pract, 2004 Dec, 20(3), 615 - 30
Clostridium difficile diarrhea: infection control in horses; Baverud V; C difficile has emerged as an important cause of diarrheic disease in horses . C difficile diarrhea is usually diagnosed in mature horses, mostly when they are treated with antimicrobials and hospitalized . It is important for clinicians at veterinary hospitals to have knowledge about the organism and the infection . To prevent C difficile diarrhea, judicious use of antimicrobials is important, as is minimizing different stress factors at the animal hospital or clinic . Infected horses must be isolated . Routine examination for C difficile and toxin A or B is recommended in horses with antibiotic-associated diarrhea . When treating foals for R equi pneumonia, it is important to avoid accidental ingestion of erythromycin by the dams . To reduce the number of environmental spores, thorough cleaning and surface disinfection of the animal hospital and clinic are important . Routine handwashing should be performed by all staff.

Virology, 2004 Nov 24, 329(2), 226 - 33
Use of a Clostridium perfringens vector to express high levels of SIV p27 protein for the development of an oral SIV vaccine; Chen Y et al.; Clostridium perfringens is a normal bacterial flora of the small and large intestines of humans and other animals . The current study investigates the potential use of a noncytotoxic C . perfringens as an oral vaccine vehicle for expression and intestinal delivery of a large amount of SIV antigens . Here we report the construction of a recombinant C . perfringens vaccine vector expressing high levels of SIV p27 during sporulation . Following oral administration of this recombinant C . perfringens vaccine vector to mice, large amounts of intact p27 protein were detected in the terminal ileum where the majority of Peyer's Patches (PPs) are located . Furthermore, dendritic cells (DCs) beneath the mucosal surface in the PPs were able to capture SIV p27 antigen, when PPs were exposed to C . perfringens expressing SIV p27 antigen . In addition, uptake of C . perfringens was able to induce maturation of mouse DCs . These results support the potential use of C . perfringens as an oral SIV/HIV vaccine vector.

Protein J, 2004 Aug, 23(6), 371 - 8
Characterization of toxin complex produced by a unique strain of Clostridium botulinum serotype D 4947; Hasegawa K et al.; A unique strain of Clostridium botulinum, serotype D 4947 (D-4947), produces a considerable amount of a 650 kDa toxin complex (L-TC) and a small amount of a 280 kDa M-TC, a 540 kDa TC, and a 610 kDa TC . The complexes are composed of only un-nicked components, including neurotoxin (NT), nontoxic nonhemagglutinin (NTNHA) and hemagglutinin subcomponents (HA-70, HA-33 and HA-17) . Unlike other NTs from all serotype strains, separation of D-4947 NT from L-TC, except for M-TC, during chromatography required highly alkaline conditions around pH 8.8 . The separated NT and NTNHA/HAs complex can be reconstituted to L-TC that is indistinguishable from the parent L-TC with respect to toxicity, hemagglutination activity and gel filtration profile . The isoelectric points of NT and NTNHA/HAs were close together depending on the number of HA-33/17 molecules . We have established a new method to separate the unique D-4947 NT from the complex, which will yield valuable information on structure of botulinum toxin.

Rev Physiol Biochem Pharmacol, 2004, 152, 183 - 204 Epub 2004.
The enteric toxins of Clostridium perfringens; Smedley JG 3rd et al.; The Gram-positive pathogen Clostridium perfringens is a major cause of human and veterinary enteric disease largely because this bacterium can produce several toxins when present inside the gastrointestinal tract . The enteric toxins of C . perfringens share two common features: (1) they are all single polypeptides of modest (approximately 25-35 kDa) size, although lacking in sequence homology, and (2) they generally act by forming pores or channels in plasma membranes of host cells . These enteric toxins include C . perfringens enterotoxin (CPE), which is responsible for the symptoms of a common human food poisoning and acts by forming pores after interacting with intestinal tight junction proteins . Two other C . perfringens enteric toxins, epsilon-toxin (a bioterrorism select agent) and beta-toxin, cause veterinary enterotoxemias when absorbed from the intestines; beta- and epsilon-toxins then apparently act by forming oligomeric pores in intestinal or extra-intestinal target tissues . The action of a newly discovered C . perfringens enteric toxin, beta2 toxin, has not yet been defined but precedent suggests it might also be a pore-former . Experience with other clostridial toxins certainly warrants continued research on these C . perfringens enteric toxins to develop their potential as therapeutic agents and tools for cellular biology.

Appl Microbiol Biotechnol . 2004 Oct 23; {Epub ahead of print}
Wood adhesives prepared from lucerne fiber fermentation residues of Ruminococcus albus and Clostridium thermocellum; Weimer PJ et al.; Fermentation residues (consisting of incompletely fermented fiber, adherent bacterial cells, and a glycocalyx material that enhanced bacterial adherence) were obtained by growing the anaerobic cellulolytic bacteria Ruminococcus albus 7 or Clostridium thermocellum ATCC 27405 on a fibrous fraction derived from lucerne ( Medicago sativa L.) . The dried residue was able to serve as an effective co-adhesive for phenol-formaldehyde (PF) bonding of aspen veneer sheets to one another . Testing of the resulting plywood panels revealed that the adhesive, formulated to contain 30% of its total dry weight as fermentation residue, displayed shear strength and wood failure values under both wet and dry conditions that were comparable with those of industry standards for PF that contained much smaller amounts of fillers or extenders . By contrast, PF adhesives prepared with 30% of dry weight as either unfermented lucerne fiber or conventional fillers or extenders rather than as fermentation residues, displayed poor performance, particularly under wet conditions.

Obstet Gynecol, 2004 Nov, 104(5 Pt 2), 1142 - 4
A fatal case of Clostridium sordellii septic shock syndrome associated with medical abortion; Wiebe E et al.; BACKGROUND: Clostridia bacteria are infrequent human pathogens . In the obstetric and gynecologic literature, Clostridium sordellii infections have been very rarely reported . This is a case of infection following medical termination of early pregnancy with mifepristone and misoprostol . CASE: A 27-year-old woman presented for termination of pregnancy at 5.5 weeks from her last menstrual period . She received mifepristone 200 mg orally followed by 800 microg vaginal misoprostol . Three days after administration of misoprostol, she complained of dizziness, pelvic pain, and bleeding . The next day, she experienced worsening of symptoms and was hospitalized . She developed pulmonary edema, ascites, and heart failure . Despite supportive measures, antibiotics, and hysterectomy, she died 3 days later . The post mortem examinations indicated that death was caused by shock secondary to C sordellii infection . CONCLUSION: The frequency of infection following medical abortion is low . The rapid and fatal course of this infection is similar to other obstetric and gynecologic cases reported in the literature . Although providers should remain vigilant to the possibility of infection following medical abortion, the overall proven safety of medical abortion remains the same.

Clin Transplant, 2004 Dec, 18(6), 726 - 8
Clostridial infection in a liver transplant recipient; Solis DR et al.; Clostridium perfringens infection in a liver transplant recipient is a rare complication . We report a case of a liver allograft gas gangrene . The case illustrates the fulminant and rapidly devastating course of this complication.

J Vet Intern Med, 2004 Sep-Oct, 18(5), 734 - 8
Experimental Clostridium difficile enterocolitis in foals; Arroyo LG et al.; Despite empirical clinical association of infection with Clostridium difficile with colitis in horses, a causal link has not been confirmed . The objective of this study was to develop a model of C . difficile-associated diarrhea in foals with normal transfer of passive immunity . Nine 1-day-old pony foals were inoculated intragastrically with spores or vegetative cells of C . difficile . Five foals were challenged with spores, with 2 receiving 10(5) colony-forming units (CFUs) and concurrently 3 receiving 10(7) CFUs once daily for 3 days . Clindamycin was administered orally to disrupt gastrointestinal flora . A further 4 foals were challenged by orogastric administration of 10(10) CFUs of vegetative cells once daily for 3 days or until diarrhea developed . This group did not receive clindamycin . Spore and vegetative cell preparations were negative for toxins of C . difficile and common enteropathogens . Clinical signs varied from mild abdominal discomfort and pasty feces to colic and watery diarrhea in 8 of 9 foals . Four of 5 foals challenged with spores developed mild diarrhea, whereas all foals challenged with vegetative cells developed moderate to severe diarrhea . C . difficile was isolated from feces of all foals between 24 and 72 hours after inoculation and toxins A or B or both were detected in the feces of all foals by an enzyme-linked immunosorbent assay . We concluded that spores and vegetative cells of C . difficile are capable of colonizing the gastrointestinal tract, producing toxins, and inducing clinical signs similar to those encountered in naturally occurring cases . This study fulfilled Koch's postulates for C . difficile-associated diarrhea in foals and provides a model for consistent reproduction of the disease for future studies.

Glycoconj J, 2004, 21(6), 287 - 93
Glycosphingolipids-sweets for botulinum neurotoxin; Yowler BC et al.; A number of viruses, bacteria, and bacterial toxins can only act on cells that express the appropriate glycosphingolipids (GSLs) on the outer surface of their plasma membranes . An example of this dependency is provided by botulinum neurotoxin (BoNT) which is synthesized by Clostridium botulinum and inhibits neurotransmission at the neuromuscular junction by catalyzing hydrolysis of a SNARE protein, thereby inducing a flaccid paralysis . Haemagglutinin components of progenitor forms of BoNT mediate its adherence to glycosphingolipids (GSLs) on intestinal epithelial cells while the cellular activity of most isolated serotypes requires the presence of certain gangliosides, especially those of the Gg1b family . This review discusses available information about the identity and the roles of GSLs in the activity of BoNT . Observations that serotypes A-F of BoNT require gangliosides for optimum activity (serotype G apparently does not), permits the hypothesis that it should be possible to develop an antagonist of this interaction thereby inhibiting/reducing its effect.

Proc Natl Acad Sci U S A, 2004 Nov 9, 101(45), 15870 - 5 Epub 2004 Oct 28.
A locking mechanism preventing radical damage in the absence of substrate, as revealed by the x-ray structure of lysine 5,6-aminomutase; Berkovitch F et al.; Lysine 5,6-aminomutase is an adenosylcobalamin and pyridoxal-5'-phosphate-dependent enzyme that catalyzes a 1,2 rearrangement of the terminal amino group of dl-lysine and of l-beta-lysine . We have solved the x-ray structure of a substrate-free form of lysine-5,6-aminomutase from Clostridium sticklandii . In this structure, a Rossmann domain covalently binds pyridoxal-5'-phosphate by means of lysine 144 and positions it into the putative active site of a neighboring triosephosphate isomerase barrel domain, while simultaneously positioning the other cofactor, adenosylcobalamin, approximately 25 A from the active site . In this mode of pyridoxal-5'-phosphate binding, the cofactor acts as an anchor, tethering the separate polypeptide chain of the Rossmann domain to the triosephosphate isomerase barrel domain . Upon substrate binding and transaldimination of the lysine-144 linkage, the Rossmann domain would be free to rotate and bring adenosylcobalamin, pyridoxal-5'-phosphate, and substrate into proximity . Thus, the structure embodies a locking mechanism to keep the adenosylcobalamin out of the active site and prevent radical generation in the absence of substrate.

Eur J Biochem, 2004 Nov, 271(21), 4293 - 7
Coexpression, purification and characterization of the E and S subunits of coenzyme B(12) and B(6) dependent Clostridium sticklandii D-ornithine aminomutase in Escherichia coli; Chen HP et al.; D-Ornithine aminomutase from Clostridium sticklandii comprises two strongly associating subunits, OraS and OraE, with molecular masses of 12,800 and 82,900 Da . Previous studies have shown that in Escherichia coli the recombinant OraS protein is synthesized in the soluble form and OraE as inclusion bodies . Refolding experiments also indicate that the interactions between OraS and OraE and the binding of either pyridoxal phosphate (PLP) or adenosylcobalamin (AdoCbl) play important roles in the refolding process . In this study, the DNA fragment containing both genes was cloned into the same expression vector and coexpression of the oraE and oraS genes was carried out in E . coli . The solubility of the coexpressed OraS and OraE increases with decreasing isopropyl thio-beta-D-galactoside induction temperature . Among substrate analogues tested, only 2,4-diamino-n-butyric acid displays competitive inhibition of the enzyme with a K(i) of 96 +/- 14 microm . Lys629 is responsible for the binding of PLP . The apparent K(d) for coenzyme B(6) binding to d-ornithine aminomutase is 224 +/- 41 nm as measured by equilibrium dialysis . The mutant protein, OraSE-K629M, is successfully expressed . It is catalytically inactive and unable to bind PLP . Because no coenzyme is involved in protein folding during in vivo translation of OraSE-K629M in E . coli, in vitro refolding of the enzyme employs a different folding mechanism . In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation.

Wien Klin Wochenschr, 2001, 113 Suppl 4, 20 - 4
{Botulinum toxin treatment of hip adductor spasticity in multiple sclerosis}; Wissel J et al.; Spasticity results in a resistance to passive movement and decrease of passive mobility of the involved joints and is defined as a state of hypertonicity with exaggeration of tendon reflexes mediated by a loss of inhibitory control of upper motor neurons . In patients with severe stages of multiple sclerosis (MS) spasticity of the lower limbs often leeds to a spastic pattern with hip adduction resulting in decreased range-of-motion (ROM), increased pain, spasms, and functional disability (disturbed gait and sitting position) as well as difficulties with perineal hygiene . Local botulinum toxin type A (Btx-A) injections in spastic muscles offer a new treatment approach for managing spasticity and associated problems . Up to now Btx-A is approved for the treatment of blepharospasm and cervical dystonia and the treatment of equinous gait in children with cerebral palsy in Austria and Germany . Up to now only in Switzerland Botox is licensed for the treatment of focal spasticity . Btx-A is a neurotoxin derived from Clostridium botulinum . In most european countries Btx-A is available as Dysport (vial = 500 units) and Botox (vial = 100 units) . In prospective studies a ratio of 1 unit Botox to 3-4 units Dysport was found . Following intramuscular injection Btx-A blocks the release of acetylcholine at the neuromuscular junctions, thereby inhibiting muscle contraction, and decreases spastic muscle tone and muscle spindles afferent information to the spinal cord . The spectrum of side effects includes local weakening of the injected and adjacent muscles as well as pain and haematoma at the injection site . At therapeutic doses side effects are local and transient . According to a double blind, placebo controlled, dose ranging study published by Hyman et al . (2000, Dysport in a dose of 500, 1000 and 1500 units reduced the degree of hip adductor spasticity associated with MS, and this benefit was evident despite concomitant use of oral antispasticity medication . According to the results of the study there was a clear trend towards greater efficacy and duration of effects with higher doses of Dysport . Taking efficacy and adverse events into account (incidence of muscle weakness was higher for the 1500 units group than for placebo) the optimal dose for hip adductor spasticity seems to be 1000 units Dysport divided between the adductor magnus, longus and brevis muscles and between both legs . To increase Btx-A effects following injection of hip adductors additional physiotherapy and casting or orthosis to increase passive hip-abduction is recommended . According to the literature anatomical localisation of the adductor muscles for injection and aspiration following insertion of the needle, to avoid injection of the toxin into a vessel, should be performed . A maximum dose of 1500 units Dysport (400 units Botox) per treatment session and 250 units Dysport (50 units Botox) per injection site is recommended . See table for dose-range of Dysport, and Botox in the treatment of adult patients with hip-adductor spasticity . For evaluation of treatment effects in hip adductor spasticity clinical examination with specific scales and measurements (see Appendix) at baseline, 4 and 12 weeks following BtxA injection is recommended:--Global rating of severity (0-4; patient's self assessment and physician's rating) --Global rating of response (-4 - +4; patient's self assessment and physician's rating)--Visual Analogue Scale (patient's self assessment of pain)--Active and passive ROM (manual goniometer)--Distance between the medial femur condyles in thigh extension (distance in cm)--Modified Ashworth scale (0-4)--Ten meter walking time (seconds)--Functional Ambulation Categories (0-5)--Score of perineal hygiene (0-5).

Antimicrob Agents Chemother, 2004 Nov, 48(11), 4430 - 4
Activity of OPT-80, a novel macrocycle, compared with those of eight other agents against selected anaerobic species; Credito KL et al.; Agar dilution MIC was used to compare activities of OPT-80, linezolid, vancomycin, teicoplanin, quinupristin/dalfopristin, amoxicillin/clavulanate, imipenem, clindamycin, and metronidazole against 350 gram-positive and -negative anaerobes . OPT-80 was active against gram-positive strains only, especially Clostridium spp . (85 strains tested, including 21 strains of C . difficile), with MICs ranging between </=0.016 and 0.25 microg/ml.

Acta Crystallogr D Biol Crystallogr, 2004 Nov, 60(Pt 11), 2063 - 6 Epub 2004 Oct 20.
Crystallization and atomic resolution X-ray diffraction of the catalytic domain of the large sialidase, nanI, from Clostridium perfringens; Newstead S et al.; Sialidases catalyse the removal of terminal sialic acids from a range of glycoproteins, glycolipids and oligosaccharides . They have been found in bacteria, viruses and parasites, where they play important roles in pathogenesis and/or microbial nutrition, and in mammalian cells, where they modulate cell-surface glycosylation associated with a range of cellular activities . Clostridium perfringens, a causative agent of gas gangrene and peritonitis in humans, possesses three sialidases: nanH, nanI and nanJ, with molecular weights of 42, 77 and 129 kDa, respectively . The two larger enzymes are secreted by the bacterium and are involved in the pathogenesis and nutrition of Clostridium . As part of a study to examine the structures of all three enzymes, crystallization of the 77 kDa nanI isoenzyme was attempted . The expressed full-length protein was found to degrade easily; a stable 50 kDa catalytic domain was therefore subcloned . This domain was overexpressed in Escherichia coli and produced crystals belonging to space group P2(1)2(1)2(1), with unit-cell parameters a = 96.98, b = 69.41, c = 72.69 A and one monomer per asymmetric unit . The crystals diffract to at least 0.92 A . A molecular-replacement solution was obtained using the catalytic domain of the sialidase from the leech Macrobdella decora.

J Biol Chem, 2004 Dec 31, 279(53), 55985 - 94 Epub 2004 Oct 23.
Structural insights into the mechanism of formation of cellulosomes probed by small angle X-ray scattering; Hammel M et al.; Exploring the mechanism by which the multiprotein complexes of cellulolytic organisms, the cellulosomes, attain their exceptional synergy is a challenge for biologists . We have studied the solution structures of the Clostridium cellulolyticum cellulosomal enzyme Cel48F in the free and complexed states with cohesins from Clostridium thermocellum and Clostridium cellulolyticum by small angle x-ray scattering in order to investigate the conformational events likely to occur upon complexation . The solution structure of the free cellulase indicates that the dockerin module is folded, whereas the linker connecting the catalytic module to the dockerin is extended and flexible . Remarkably, the docking of the different cohesins onto Cel48F leads to a pleating of the linker . The global structure determined here allowed modeling of the atomic structure of the C . cellulolyticum dockerin-cohesin interface, highlighting the local differences between both organisms responsible for the species specificity.

J Enzyme Inhib Med Chem, 2004 Jun, 19(3), 237 - 48
Quantum theoretic QSAR of benzene derivatives: some enzyme inhibitors; Supuran CT et al.; Our previously developed approach to the development of QSAR equations for benzene derivatives, originally for phenylalkylamine hallucinogens, has been applied to four new systems: sulfonamide inhibitors of the enzymes carbonic anhydrase, thrombin, trypsin, and Clostridium histolyticum collagenase . The novel features involve the energies and nodal orientations of pi-like orbitals, and an allowance for the symmetry of the benzene nucleus . The resulting equations give better fits, better predictivity and are more easily interpretable than those resulting from traditional QSAR methods.

J Am Diet Assoc, 2004 Nov, 104(11), 1708 - 17
Food safety: emerging trends in foodborne illness surveillance and prevention; McCabe-Sellers BJ et al.; Between 250 and 350 million Americans are estimated to suffer acute gastroenteritis annually, with 25% to 30% thought to be caused by foodborne illnesses . Most vulnerable to foodborne diseases are elderly people, pregnant women, immune-compromised people, and children . While bacterial causes such as Salmonella are widely recognized and monitored as foodborne infections, other important bacterial causes such as Clostridium perfringens , Bacillus cereus , and Staphylococcus aureus are less well known . While the majority of cases of foodborne diseases are of unknown cause, bacteria and viruses are the most likely causative agents . Caliciviridae (Norwalk-like) virus cases are more difficult to identify, but represent the most common cause of known and probably unknown cases . Fresh produce has to be added to the traditional list of foods requiring careful selection and handling to prevent foodborne disease . To assess the disease burden in the United States, morbidity and mortality surveillance activities are done by several networks and systems with collaboration among federal agencies and health departments . Not all important causes are being equally monitored . Critical behaviors by food processors, food retailers, foodservice personnel, and consumers can reduce the risk of foodborne illness episodes . Dietetics professionals can more readily monitor new developments and update knowledge and practice through online resources.

Emerg Infect Dis, 2004 Sep, 10(9), 1606 - 11
Foodborne botulism in the United States, 1990-2000; Sobel J et al.; Foodborne botulism, a potentially lethal neuroparalytic disease, is caused by ingesting preformed Clostridium botulinum neurotoxin . We reviewed surveillance data and reports from 1990 to 2000 . Of 263 cases from 160 foodborne botulism events (episode of one or more related cases) in the United States, 103 (39%) cases and 58 events occurred in Alaska . Patients' median age was 48 years; 154 (59%) were female; the case-fatality rate was 4% . The median number of cases per event was 1 (range 1-17) . Toxin type A caused 51% of all cases; toxin type E caused 90% of Alaska cases . A particular food was implicated in 126 (79%) events . In the lower 49 states, a noncommercial food item was implicated in 70 (91%) events, most commonly home-canned vegetables (44%) . Two restaurant-associated outbreaks affected 25 persons . All Alaska cases were attributable to traditional Alaska Native foods . Botulism prevention efforts should be focused on those who preserve food at home, Alaska Natives, and restaurant workers.

Proc Natl Acad Sci U S A, 2004 Nov 2, 101(44), 15645 - 9 Epub 2004 Oct 20.
Crystal structure of 4-hydroxybutyryl-CoA dehydratase: radical catalysis involving a {4Fe-4S} cluster and flavin; Martins BM et al.; Dehydratases catalyze the breakage of a carbon-oxygen bond leading to unsaturated products via the elimination of water . The 1.6-A resolution crystal structure of 4-hydroxybutyryl-CoA dehydratase from the gamma-aminobutyrate-fermenting Clostridium aminobutyricum represents a new class of dehydratases with an unprecedented active site architecture . A {4Fe-4S}(2+) cluster, coordinated by three cysteine and one histidine residues, is located 7 A from the Re-side of a flavin adenine dinucleotide (FAD) moiety . The structure provides insight into the function of these ubiquitous prosthetic groups in the chemically nonfacile, radical-mediated dehydration of 4-hydroxybutyryl-CoA . The substrate can be bound between the {4Fe-4S}(2+) cluster and the FAD with both cofactors contributing to its radical activation and catalytic conversion . Our results raise interesting questions regarding the mechanism of acyl-CoA dehydrogenases, which are involved in fatty acid oxidation, and address the divergent evolution of the ancestral common gene.

J Med Microbiol, 2004 Nov, 53(Pt 11), 1129 - 36
Comparative analysis of Clostridium difficile clinical isolates belonging to different genetic lineages and time periods; Spigaglia P et al.; Recent studies have shown that Clostridium difficile strains with variant toxins and those with resistance to macrolide-lincosamide-streptogramin B (MLSB) are increasingly causing severe disease and outbreaks in hospital settings . Here, the pathogenicity locus (PaLoc), the acquisition of binary toxin, and the genotypic and phenotypic characteristics of antibiotic resistance of 74 C . difficile clinical strains isolated from symptomatic patients in Italy during different time periods were studied . These strains were found to belong to two different lineages, and those isolated before 1991 were genetically unrelated to the more recent strains . The majority of recent C . difficile strains showed variations in toxin genes and in the toxin negative regulator (tcdC) and had the binary toxin . In 62 % of them, variations in tcdC and the presence of the binary toxin were associated . Five classes of susceptibility/resistance pattern (EC-a to -e) for erythromycin and clindamycin were identified in all strains studied . Most of the recent isolates belonged to EC-d and EC-e and, although erythromycin-resistant in vitro, did not harbour the commonly associated ermB determinant . Interestingly, two strains of the EC-d class were resistant to clindamycin only after induction with subinhibitory concentrations of the antibiotic . A decrease in tetracycline and chloramphenicol MIC values was also observed in the recently isolated strains, associated with less frequent detection of the catD and tetM genes . Two tetM-positive strains were resistant in vitro only after induction with subinhibitory concentrations of the antibiotic . The acquisition of the binary toxin, the possible increase in toxin production due to a mutated negative regulator and a decrease in the fitness cost as a result of lower levels of antibiotic resistance or other mechanisms may have led to the successful establishment of these new phenotypes, with potentially serious clinical implications.

J Biol Chem, 2004 Dec 24, 279(52), 54896 - 904 Epub 2004 Oct 18.
G alpha 13 signals via p115RhoGEF cascades regulating JNK1 and primitive endoderm formation; Lee YN et al.; The heterotrimeric G-protein G(13) mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase . The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G alpha(13)-mediated pathway, whereas that between G alpha(13) and MEKKs is not known . The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members . Constitutively active G alpha(13) was found to activate RhoA as well as Cdc42 and Rac1 in these cells . Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G alpha(13) . Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities . Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G alpha(13) . Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G alpha(13)-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway . The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1 . Thus, the signal propagated from G alpha(13) to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1 . In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.

Vasc Endovascular Surg, 2004 Sep-Oct, 38(5), 431 - 8
Factor XIII contrasts the effects of metalloproteinases in human dermal fibroblast cultured cells; Zamboni P et al.; Matrix metalloproteinases (MMPs) are overexpressed in venous leg ulcers, determining a breakdown of the main extracellular matrix (ECM) components owing mainly to collagenase activities, and so playing a crucial role in ulcer pathogenesis . The authors studied the effects of coagulation factor XIII (FXIII), which cross-links collagen and other ECM components, in human fibroblast cultured cells in the presence and in the absence of matrix metalloproteinases from Clostridium histolyticum collagenase . Clostridium collagenase at concentrations of 2.0, 1.0, and 0.5 mg/mL was added to normal human dermal fibroblasts cultured in the presence of 0.0, 1.0, and 5.0 U/mL of FXIII concentrate (Fibrogammin P, Aventis Behring) . Cell counting and metabolically active fibroblast evaluation in the cultures were monitored for 72 hours, by means of trypan-blue dye and MTT test, respectively . The MTT test showed that at the highest collagenase concentration (2.0 mg/mL), the cell number decreased more than 95% in 72 hours of treatment and no significant differences were observed regardless of the FXIII concentrations utilized . At lower collagenase concentration (1.0 mg/mL), in absence or in presence of FXIII (1.0 U/mL), the cell number decreased by about 80% in 72 hours . In contrast, in the presence of higher FXIII levels (5.0 U/mL), cells suffered globally significantly less collagenase effects (p = 0.011) and the gain was appreciable at each time tested . Finally, at 0.5 mg/mL of collagenase concentration, in the absence of FXIII, the cell number decreased by about 60% in 72 hours, whereas in presence of FXIII 1.0 U/mL and 5.0 U/mL, cells decreased significantly less, by about 35% and 20%, respectively (p < 0.025 and p < 0.01, respectively) . These data were also confirmed by direct cell counting utilizing the trypan-blue test . Factor XIII contrasts effectively the detrimental action of Clostridium collagenases in human fibroblast cultured cells . These results support several in vivo reports about the effectiveness of its topical application in order to enhance the venous ulcer healing processes.

Mol Cell Probes, 2004 Dec, 18(6), 373 - 7
Detection of Staphylococcus aureus enterotoxin A and B genes with PCR-EIA and a hand-held electrochemical sensor; Aitichou M et al.; Two electrochemical assays for detecting Staphylococcus aureus enterotoxin A and B genes were developed . The assays are based on PCR amplification with biotinylated primers, hybridization to a fluorescein-labeled probe, and detection with horseradish peroxidase-conjugated anti-fluorescein antibody using a hand-held electrochemical detector . The limit of detection (LOD) for both assays was approximately 16 copies of the sea and seb genes . The assays were evaluated in blinded studies, each with 81 samples that included genomic and cloned S . aureus DNA, and genomic DNA from Alcaligens, Bacillus, Bacteroides, Bordetella, Borkholderia, Clostridium, Comanonas, Enterobacter, Enterococcus, Escherichia, Francisella, Haemophilus, Klebsiella, Listeria, Moraxella, Neisseria, Proteus, Pseudomonas, Salmonella, Serratia, Shigella, Streptococcus, Vibrio and Yersinia species . Both assays showed 100% sensitivity . The specificity was 96% for the SEA assay and 98% for the SEB assay . These results demonstrate the feasibility of performing probe-based detection of PCR products with a low-cost, hand-held, electrochemical detection device as a viable alternative to colorimetric enzyme-linked assays of PCR products.

Mol Cell Probes, 2004 Dec, 18(6), 359 - 67
Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization; Al-Khaldi SF et al.; A microarray-based method for characterization of six Clostridium perfringens toxin genes: iA (iota toxin), cpa (alpha toxin), cpe (enterotoxin E), etxD (epsilon toxin), cpb1 (beta toxin 1),and cpb2 (beta toxin 2) was developed and evaluated using 17 C . perfringens isolates . Three individual oligonucleotide probes (oligoprobes), complementary to the unique sequences of each toxin gene, were designed and immobilized on a surface of aldehyde-coated glass slides . Multiplex PCR was used to simultaneously amplify DNA target regions of all six genes . Single-stranded DNA (ssDNA) samples for microarray analysis were prepared by following a primer extension of amplicons in the presence of one primer . Fluorescent moieties (Cy3) were incorporated into the ssDNA by chemical modification of guanine bases . The presence of toxin genes in C . perfringens was established by hybridization of the fluorescently labeled ssDNA representing different samples to the microarray gene-specific oligoprobes . Results of the study showed sensitivity and specificity of genotyping C . perfringens using multiple microarray-based assays.

Biochem J, 2005 Jan 15, 385(Pt 2), 479 - 84
The interaction of carbohydrate-binding modules with insoluble non-crystalline cellulose is enthalpically driven; Boraston AB; Natural cellulose exists as a composite of cellulose forms, which can be broadly characterized as crystalline or non-crystalline . The recognition of both of these forms of cellulose by the CBMs (carbohydrate-binding modules) of microbial glycoside hydrolases is important for the efficient natural and biotechnological conversion of cellulosic biomass . The category of CBM that binds insoluble non-crystalline cellulose does so with an affinity approx . 10-20-fold greater than their affinity for cello-oligosaccharides and/or soluble polysaccharides . This phenomenon has been assumed to originate from the effects of changes in configurational entropy upon binding . The loss of configurational entropy is thought to be less profound upon binding to conformationally restrained insoluble non-crystalline cellulose, resulting in larger free energies of binding . However, using isothermal titration calorimetry, it is shown that this is not the case for the high-affinity interactions of CcCBM17 (the family 17 CBM from EngF of Clostridium cellulovorans) and BspCBM28 (the family 28 CBM from Cel5A of Bacillus species 1139) with regenerated cellulose, an insoluble preparation of primarily non-crystalline cellulose . The enhanced free energy of binding of non-crystalline cellulose relative to cello-oligosaccharides is by virtue of improved enthalpy, not entropy.

J Pediatr Surg, 2004 Oct, 39(10), 1590 - 2
Severe ciprofloxacin-associated pseudomembranous colitis in an eight-year-old child; Angel CA et al.; Clostridium difficile is the principal cause of antibiotic-associated diarrhea and pseudomembranous enterocolitis in children . A case of severe pseudomembranous colitis developing in an 8-year-old child who had received oral ciprofloxacin therapy as part of an investigational protocol is presented . The safety and efficacy of fluoroquinolones in children has not yet been established . Use of these antibiotics in children outside investigational protocols ("off-label" use) as oral antipseudomonas agents is discouraged.

Clin Infect Dis, 2004 Oct 15, 39(8), 1182 - 9 Epub 2004 Sep 27.
Contamination, disinfection, and cross-colonization: are hospital surfaces reservoirs for nosocomial infection?
Hota B.
Despite documentation that the inanimate hospital environment (e.g., surfaces and medical equipment) becomes contaminated with nosocomial pathogens, the data that suggest that contaminated fomites lead to nosocomial infections do so indirectly . Pathogens for which there is more-compelling evidence of survival in environmental reservoirs include Clostridium difficile, vancomycin-resistant enterococci, and methicillin-resistant Staphylococcus aureus, and pathogens for which there is evidence of probable survival in environmental reservoirs include norovirus, influenza virus, severe acute respiratory syndrome-associated coronavirus, and Candida species . Strategies to reduce the rates of nosocomial infection with these pathogens should conform to established guidelines, with an emphasis on thorough environmental cleaning and use of Environmental Protection Agency-approved detergent-disinfectants.

Clin Infect Dis, 2004 Oct 15, 39(8), 1161 - 9 Epub 2004 Sep 24.
Bloodstream infections: a trial of the impact of different methods of reporting positive blood culture results; Bouza E et al.; BACKGROUND: The impact of how positive blood culture results are reported on the evolution bloodstream infections (BSIs) has not been assessed . METHODS: We randomly assigned patients with BSIs into 3 groups: group A (for which physicians received a conventional report), group B (for which physicians received a conventional report and a written alert on the chart with clinical advice), and group C (for which physicians received the above plus oral clinical advice) . The adequacy of therapy before and after receipt of the different types of information was assessed . RESULTS: Overall, 297 episodes (109 in group A, 99 in group B, and 89 in group C) were studied . Patients who received inadequate treatment before receiving microbiological information had a longer mean (+/-SD) hospital stay (27.2+/-32.4 vs . 19.4+/-15.8 days; P=.017), a higher mean risk of Clostridium difficile-associated diarrhea (8.3% vs . 1.9%; P=.013), a higher mean overall mortality rate (30.8% vs . 19.4%; P=.025), and a higher mean risk of infection-related mortality (23.3% vs . 13.6%; P=.031) . After receipt of microbiological reports, recommendations for changes in therapy were issued for patients in groups B (52.3%) and C (53.1%) . For groups A, B, and C, the proportions of days on which adequate treatment was received were 66.3%, 92.1%, and 91.2% (P<.001); the mean numbers of defined daily doses of appropriate antibiotic therapy were 16.4, 22.2, and 20.7 (P=.003); the mean durations of hospital stay were 19.8, 23.6, and 24.1 days (P=.761); and the mortality rates during the late period were 12.9%, 15.6%, and 11% (P=.670), respectively . The mean costs of antimicrobials per episode in groups A, B, and C were 580.63, 537.98, and 434.53 (US707.85 dollars, US699.73 dollars, and US529.73 dollars, respectively) . CONCLUSIONS: Written- or oral-alert reports with clinical advice should complement traditional microbiological reports for patients with BSIs.

Dis Colon Rectum, 2004 Sep, 47(9), 1515 - 8 Epub 2004 Jul 08.
Small-bowel infarction from disseminated aspergillosis; Tresallet C et al.; INTRODUCTION: Despite the use of new, effective drugs, the disseminated invasive aspergillosis often remains lethal in neutropenic patients . Diagnosis is difficult because early symptoms are nonspecific . New tools could help in diagnosis and lead to early surgery when needed . METHODS: A neutropenic patient developed an acute abdomen . CT findings were a diffuse, small-bowel distention with a thickened, distal, ileum wall . Emergency surgery was performed with resection and immediate anastomosis of the distal ileum . Pathology of the small bowel showed a wall necrosis and invasion by Aspergillus fumigatus . RESULTS: The postoperative course was uneventful except for persisting diarrhea secondary to a coexistent infection with Clostridium difficile . Aspergillus antigene in serum was positive, whereas neither pulmonary nor central nervous system aspergillosis was observed on CT scan . CONCLUSIONS: This diagnosis should be considered when neutropenic patients show abdominal pain and distention with fever . Repetition of Aspergillus antigenemia, search for others aspergillosis localizations, CT scan, and colonoscopy with biopsies should be performed until diagnosis allows the administration of early antifungal therapy.

J Am Vet Med Assoc, 2004 Sep 15, 225(6), 897 - 902
Underlying cause, pathophysiologic abnormalities, and response to treatment in cats with septic peritonitis: 51 cases (1990-2001); Costello MF et al.; OBJECTIVE: To determine the underlying cause, pathophysiologic abnormalities, and response to treatment in cats with septic peritonitis and identify differences between cats that survived following treatment and cats that did not survive despite treatment . DESIGN: Retrospective study . ANIMALS: 51 cats with septic peritonitis . PROCEDURE: Medical records were reviewed for clinical findings; results of clinicopathologic testing, microbial culture, and radiography; diagnosis; treatment; and outcome . RESULTS: Signs of pain during palpation of the abdomen were reported for only 29 of 47 (62%) cats . Eight (16%) cats had relative bradycardia (heart rate < 140 beats/min) . The most commonly isolated organisms included Escherichia coli, Enterococcus spp, and Clostridium spp . The most common cause of peritonitis was gastrointestinal tract leakage (24 cats) . No definitive source could be identified in 7 cats . Treatment, including exploratory surgery, was pursued in 23 cats, of which 16 (70%) survived and were discharged . There were no significant differences between survivors and nonsurvivors in regard to heart rate, age, rectal temperature, serum lactate concentration, WBC count, PCV, blood glucose concentration, or serum albumin concentration . CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that clinicopathologic abnormalities and outcome in cats with septic peritonitis are similar to those reported for dogs . However, certain features may be unique, including an absence of signs of pain during abdominal palpation, relative bradycardia, and apparent spontaneous peritonitis in some cats.

Rev Gastroenterol Peru, 2004 Jul-Sep, 24(3), 263 - 9
Basic and clinical aspects of Clostridium difficile colitis; Morishita T et al.; Clostridium difficile, a gram-positive anaerobic bacillus dubbed as the difficult clostridium because it resisted early attempts of isolation and culture . After some decades in the darkness, it became famous, when in 1978, a cytotoxin of the C . difficile was found the responsible of the pseudomembranous colitis . We review in this paper aspects of the epidemiology of the C . difficile in health and disease . Also the importance of C . difficile as a cause of nosocomial infections . We review the characteristics of the toxins A and B produced by the pathogenic strains of C . difficile . Finally, clinical aspects of infection with C . difficile in special in the pseudomembranous colitis . The diagnosis, medical therapy, complications and surgical indications are briefly described.

Extremophiles, 2004 Oct, 8(5), 421 - 9 Epub 2004 Jul 28.
A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures; Grant S et al.; Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000 . Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis . Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga-Flexibacter-Bacteroides group . Similar clones were identified in the SS, but Bacillus spp . predominated . Many of the clones were most closely related to organisms already identified in alkaline or saline environments . Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample . A novel cellulase activity was identified and characterized in each . The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris . The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.

Int Orthop, 2004 Oct, 28(5), 315 - 8 Epub 2004 Jul 23.
Cadaveric allograft microbiology; Ibrahim T et al.; This study aims to determine the contamination rate of cadaveric bone allograft and blood cultures retrieved from 119 donors within Leicester between 1990 and 2003 . A contamination rate of 27% was present, with 120 of 437 bone allografts culturing positive at the time of retrieval . Similarly, a contamination rate of 37% was present, with 40 of 107 blood samples culturing positive . The time interval between death and procurement did not influence blood contamination . Coagulase-negative Staphylococcus was the commonest organism isolated in both blood and bone cultures . One donor had Clostridium grown in their blood culture . The available evidence confirms similar contamination rates with other studies . The majority of organisms isolated were skin commensals with a low rate of contamination of highly pathogenic organisms such as Clostridium.

Pol J Vet Sci, 2004, 7(3), 207 - 9
Microbiological quality of petfood in Poland; Wojdat E et al.; From epidemiological and safety point of view, petfoods produced from edible and non-edible animal by-products should be safe for slaughter animals, owners of these pets and environment . Domestic animals are recognized as highly important reservoir of pathogenic microorganisms for man . The close contact of people and especially children with pets may constitute a serious hazard for their health . Taking these aspects into account, the study was undertaken in which 2271 dried and 18 canned petfood samples were examined . Moreover, 14, 90, 23 and 22 samples of petfoods were examined for presence or number of Clostridium sp., Enterobacteriaceae, total plate count, and yeast and moulds, respectively . Salmonella was isolated from 22 (1%) out of 2271 dried petfood samples examined . Number of Enterobacteriaceae was higher than 300 cfu/g, i.e . the allowed maximum level was found in 9 (10%) samples examined . The rest of microbiological quality parameters did not exceed the allowed values.

J Spinal Cord Med, 2004, 27(3), 266 - 8
Fulminant Clostridium difficile colitis in a patient with spinal cord injury: case report; Bahadursingh AN et al.; BACKGROUND: In certain patients with Clostridium difficile colitis (CDC), a life-threatening systemic toxicity may develop despite appropriate and timely medical therapy . DESIGN: Literature search and case report . FINDINGS: A 39-year-old man with T10 paraplegia presented with a distended, quiet abdomen following recent treatment with antibiotics for pneumonia . Diarrhea was not present . Complete blood counts demonstrated a marked leukocytosis . A CT scan of the abdomen demonstrated a state of diffuse pancolonic inflammation with peritoneal fluid . The patient was taken to the operating room and underwent total abdominal colectomy with oversewing of the rectal stump and end ileostomy for treatment of the fulminant CDC . CONCLUSION: Patients with spinal cord injury (SCI) often receive antibiotics for infections of the aerodigestive tree and urinary tract and for problems with skin integrity . A heightened awareness of the development of fulminant CDC remains essential in the care of patients with SCI . Any unexplained abdominal illness after recent antibiotic administration should alert the physician to CDC and its potential as a fulminant, potentially fatal illness.

J Appl Toxicol, 2004 Sep-Oct, 24(5), 363 - 9
Responses of fish chromatophore-based cytosensor to a broad range of biological agents; Dierksen KP et al.; A cytosensor based on living chromatophores from Betta splendens Siamese fighting fish was used to test several classes of biologically active agents . Tested agents include neurotransmitters, adenyl cyclase activators, cytoskeleton effectors, cell membrane effectors and protein synthesis inhibitors . Characteristic cell responses were analyzed, and potential cytosensor applications were considered . Streptococcus pyogenes toxins streptolysin S and streptolysin O, Clostridium tetani tetanolysin, Staphylococcus aureus alpha-toxin and Vibrio parahemolyticus hemolysin, all bacterial toxins that act on cell membranes, elicited a strong response from chromatophores . A comparison of purified toxin to actual bacterial culture from Vibrio parahemolyticus demonstrated a nearly identical chromatophore cell response pattern . This suggests that the cytosensor response is reflective of bacterial toxin production . Copyright (c) 2004 John Wiley & Sons, Ltd.

J Bone Miner Res, 2004 Nov, 19(11), 1882 - 91 Epub 2004 Aug 16.
Rho and Rho kinase are involved in parathyroid hormone-stimulated protein kinase C alpha translocation and IL-6 promoter activity in osteoblastic cells; Radeff JM et al.; The role of small G-proteins in PTH-stimulated PKC translocation and IL-6 promoter expression in UMR-106 cells was determined . The effects of PTH(1-34) and PTH(3-34) in stimulating PKCalpha translocation and IL-6 were inhibited by agents that interfere with the activity of small G-proteins of the Rho family and with the downstream kinase Rho kinase . INTRODUCTION: Activation of protein kinase C (PKC) is a signaling mechanism by which parathyroid hormone (PTH) modulates interleukin-6 (IL-6) in osteoblasts, leading to osteoclastogenesis and bone resorption . PKCalpha and PKCbetaI are translocated after treatment with PTH in UMR-106 osteoblastic cells; however, the pathway leading to PKC isozyme translocation is not established . Diacylglycerol (DAG) generation from phospholipase D (PLD) is one pathway of PKC activation, and PTH-mediated PLD activity is dependent on small G-proteins of the Rho family . This study investigated whether Rho proteins modulate the PKCalpha translocation and IL-6 promoter activity stimulated by PTH in UMR-106 cells . MATERIALS AND METHODS: UMR-106 cells were treated with PTH(1-34) or PTH(3-34) . PKC translocation was determined by immunofluorescence, Rho A activation by Rhotekin assay and by translocation assessed by Western blotting in membrane and cytosol fractions, and IL-6 promoter expression by luciferase assay . RESULTS AND CONCLUSIONS: Inhibition of Rho proteins with Clostridium difficile toxin B or inhibition of Rho prenylation with GGTI attenuated PTH(1-34)- and PTH(3-34)-stimulated translocation of endogenous PKCalpha and IL-6 promoter activity . Expression of a constitutively active RhoA (RhoA63L) mimicked the effect of PTH(1-34) or PTH(3-34) to promote membrane localization of PKCalpha, whereas cells expressing a dominant negative RhoA (RhoA19N) did not respond to PTH(1-34) or PTH(3-34) . The Rho kinase inhibitor Y27632 attenuated PTH(1-34)- and PTH(3-34)-stimulated PKCalpha translocation and IL-6 promoter activation . Rho seemed to be acting at a step before production of diacylglycerol (DAG), because the stimulation of PKCalpha translocation by the DAG mimetic phorbol 12,13 dibutyrate (PDBu) was unaffected by C . difficile toxin B or Y27632 . These results indicate that Rho proteins are an important component of PTH signaling in osteoblastic cells and provide further demonstration of convergence between PKC and small G-protein signaling pathways.

Phytother Res, 2004 Aug, 18(8), 615 - 8
Identification of the antibacterial component of an ethanolic extract of the Australian medicinal plant, Eremophila duttonii; Shah A et al.; Activity-guided fractionation was used to determine the antibacterial component of an ethanolic extract of the leaves of an Australian native medicinal plant, Eremophila duttonii F . Muell . (Myoporaceae) . The extract, previously shown to have activity against Gram positive bacteria, was shown to have activity against additional Gram positive bacteria, including Clostridium perfringens, C . sporogenes and Listeria monocytogenes . Thin layer chromatography (TLC) was used to separate the extract into seven coloured fractions in visible light, one of which was shown by bioautography to contain antibacterial activity . Recovery of the component from the TLC plate and testing for antibacterial activity using a plate-hole diffusion assay supported this result . The purity of the component was verified by high-performance liquid chromatography and a time-kill experiment indicated that the purified component showed identical bactericidal activity to the whole extract . TLC spray reagents indicated that the component was a sterol, terpene or sugar but not a flavonoid, while the pigmented nature suggested a carotenoid . Copyright (c) 2004 John Wiley & Sons, Ltd.

Biochem Biophys Res Commun, 2004 Nov 12, 324(2), 753 - 60
Generation and characterization of Clostridium septicum alpha toxin mutants and their use in diagnosing paroxysmal nocturnal hemoglobinuria; Shin DJ et al.; Glycosylphosphatidylinositol (GPI) anchors various proteins to the membrane of eukaryotic cells . Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disorder that is primarily due to the lack of GPI-anchored proteins on the surface of blood cells . To detect the GPI-deficient cells in PNH patients, we modified alpha toxin, a pore-forming toxin of the Gram-positive bacterium Clostridium septicum . We first showed that aerolysin, a homologous toxin from Aeromonas hydrophila, bound to both of Chinese hamster ovary cells deficient of N-glycan maturation as well as GPI biosynthesis at a significant level . However, alpha toxin bound to the mutant cells of N-glycosylation, but not to GPI-deficient cells . It suggested that alpha toxin could be used as a specific probe to differentiate only GPI-deficient cells . As a diagnostic probe, alpha toxin must be the least cytotoxic while maintaining its affinity for GPI . Thus, we constructed several mutants . Of these, the mutants carrying the Y155G or S189C/S238C substitutions bound to GPI as well as the wild-type toxin . These mutants also efficiently underwent proteolytic activation and aggregated into oligomers on the cell surface, which are events that precede the formation of a pore in the host cell membrane, leading to cell death . Nevertheless, these mutants almost completely failed to kill host cells . It was revealed that the substitutions affect the events that follow oligomerization . The S189C/S238C mutant toxin differentiated GPI-deficient granulocyte and PMN, but not red blood cells, of a PNH patient from GPI-positive cells at least as sensitively as the commercial monoclonal antibodies that recognize the CD59 or CD55 GPI proteins on blood cells . Thus, this modified bacterial toxin can be employed instead of costly monoclonal antibodies to diagnose PNH patients.

J Clin Microbiol, 2004 Oct, 42(10), 4863 - 5
Performance of the TechLab C . DIFF CHEK-60 enzyme immunoassay (EIA) in combination with the C . difficile Tox A/B II EIA kit, the Triage C . difficile panel immunoassay, and a cytotoxin assay for diagnosis of Clostridium difficile-associated diarrhea; Snell H et al.; We compared a recently marketed enzyme immunoassay for glutamate dehydrogenase (GDH), TechLab's C . DIFF CHEK-60 (TL-GDH), in combination with the C . difficile Tox A/B II enzyme immunoassay (Tox-A/B) with (i) the Triage C . difficile test, which detects both GDH (TR-GDH) and toxin A (TR-Tox-A); (ii) an in-house cytotoxin assay (C-Tox); and (iii) stool cultures for C . difficile . All C . difficile isolates were tested for the presence of the toxin genes by PCR . If a toxin gene-positive strain of Clostridium difficile was recovered and a toxin was detected by any method, the result was considered to be truly positive . Eighty-seven of 93 and 79 of 93 C . difficile culture-positive samples were also TL-GDH and TR-GDH positive, respectively . No test was able to detect toxin in all samples with true-positive results . Tox-A/B and TR-Tox-A in combination with the GDH detection tests and C-Tox were able to identify 52 and 50 samples with true-positive results . Tox-A/B and TR-Tox-A would have missed 15 and 31% of cases of C . difficile-associated diarrhea, respectively, if used alone.

J Clin Microbiol, 2004 Oct, 42(10), 4718 - 25
Type C botulism due to toxic feed affecting 52,000 farmed foxes and minks in Finland; Lindstrom M et al.; The largest reported outbreak of type C botulism in fur production animals is described . Epidemiological investigation of 117 out of 157 (response rate, 74.5%) farms revealed that 44,130 animals died or were euthanized, while 8,033 animals with milder symptoms recovered . The overall death rate in all animals at risk was 21.7% . The death rates were significantly higher in blue and shadow foxes (24.2 and 27.8%, respectively) than in silver and blue silver foxes and minks (below 4%) . All minks had been immunized against botulinum toxin type C . Deaths were associated with feed manufactured by a local processor, 83 of whose customer farms (70.9%) reported dead or sick animals . Five feedlots out of 19 delivered to the farms on the day preceding the onset of the outbreak (day 2) were associated with a death rate higher than 40% . These feedlots consisted of fresh feed processed on day 2 and feed processed 1 day earlier (day 1) . In laboratory analysis, the day 2 feed contained botulinum toxin type C (>600 minimum lethal doses/g), while the day 1 feed did not contain toxin . Toxin was not detected in feed raw-material samples . Clostridium botulinum type C was detected by PCR in some feed components and in feed . However, as the feed temperature was continuously 8 degrees C or below and the pH was continuously 5.6 or below according to the manufacturer, it seems unlikely that spore germination and toxin formation occurred during overnight storage . Hence, the events leading to toxin formation were not determined.

Science, 2004 Nov 26, 306(5701), 1550 - 3 Epub 2004 Nov 26.
Femtomolar sensitivity of a NO sensor from Clostridium botulinum; Nioche P et al.; Nitric oxide (NO) is extremely toxic to Clostridium botulinum, but its molecular targets are unknown . Here, we identify a heme protein sensor (SONO) that displays femtomolar affinity for NO . The crystal structure of the SONO heme domain reveals a previously undescribed fold and a strategically placed tyrosine residue that modulates heme-nitrosyl coordination . Furthermore, the domain architecture of a SONO ortholog cloned from Chlamydomonas reinhardtii indicates that NO signaling through cyclic guanosine monophosphate arose before the origin of multicellular eukaryotes . Our findings have broad implications for understanding bacterial responses to NO, as well as for the activation of mammalian NO-sensitive guanylyl cyclase.

Proc Natl Acad Sci U S A, 2004 Oct 19, 101(42), 15172 - 7 Epub 2004 Oct 07.
Bacteriolytic therapy can generate a potent immune response against experimental tumors; Agrawal N et al.; When spores of the anaerobic bacterium Clostridium novyi-NT are systemically injected into animals, they germinate exclusively within the hypoxic regions of cancers . The germinated bacteria destroy adjacent tumor cells but spare a rim of well oxygenated tumor cells that subsequently expand . Surprisingly, we found that approximately 30% of mice treated with such spores were cured of their cancers despite the viable tumor rim initially remaining after spore germination . The mechanism underlying this effect was shown to be immune-mediated, because cured animals rejected a subsequent challenge of the same tumor . Similar effects were observed in rabbits with intrahepatic tumors . It was particularly notable that the induced immune response, when combined with the bacteriolytic effects of C . novyi-NT, could eradicate large established tumors.

J UOEH, 2004 Sep 1, 26(3), 349 - 67
{Evaluation of bacterial flora in contaminated soil as a countermeasure against H2S gas production}; Taniguchi H et al.; Gas productions in illegal dumping sites and waste landfills have caused serious problems . The gas production was induced by bacterial flora inhabited soils . In order to construct a culture independent evaluation system of the soil bacteria, bacterial communities were analyzed quantitatively and qualitatively, about 16 soil samples at 4 sites, both using culture and culture-independent methods . The real time PCR method was developed for counting total bacterial number . Sequencing analysis of 16S rDNA amplified by a direct PCR method revealed that non-spore forming sulfate reducing bacteria and sulfur-oxidizing bacteria were detected at a similar frequency at an illegal dumping site near mountains . On the other hand, spore-forming sulfate reducing bacteria and Clostridium sp . were mainly detected in deep samples at reclaimed landfills from the sea, whereas sulfur-oxidizing bacteria was hardly detected . The result regarding sulfate-reducing bacteria was confirmed also by an anaerobic culture method . Culture-independent molecular analyses of soil bacteria would give us useful information for prediction of gas production and for the evaluation of soil equilibrium.

J Wildl Dis, 2004 Jul, 40(3), 414 - 9
Prevalence of neurotoxic Clostridium botulinum type C in the gastrointestinal tracts of tilapia (Oreochromis mossambicus) in the Salton Sea; Nol P et al.; Tilapia (Oreochromis mossambicus) have been implicated as the source of type C toxin in avian botulism outbreaks in pelicans (Pelecanus erythrorhynchos, Pelecanus occidentalis californicus) at the Salton Sea in southern California (USA) . We collected sick, dead, and healthy fish from various sites throughout the Sea during the summers of 1999 through 2001 and tested them for the presence of Clostridium botulinum type C cells by polymerase chain reaction targeting the C(1) neurotoxin gene . Four of 96 (4%), 57 of 664 (9%), and five of 355 (1%) tilapia tested were positive for C . botulinum type C toxin gene in 1999, 2000, and 2001, respectively . The total number of positive fish was significantly greater in 2000 than in 2001 (P<0.0001) . No difference in numbers of positives was detected between sick and dead fish compared with live fish . In 2000, no significant relationships were revealed among the variables studied, such as location and date of collection.

Pathol Biol (Paris), 2004 Oct, 52(8), 444 - 9
{Characterization of an extracellular protease from Clostridium difficile}; Janoir C et al.; Clostridium difficile is an intestinal pathogen, which produces two main virulence factors, the exotoxins A and B . Other bacterial structures have been implicated in the colonization of the gastrointestinal tract, which is the first step of the pathogenic process . C . difficile expresses adherence factors and also, displays some surface-associated proteolytic activity, which could play a role in the physiopathology of this bacterium . The aim of this work was to study the protein named Cwp84 which displays significant homologies with many cysteine proteases . The coding catalytic domain of this protein has been cloned in the expression system pGEX-6P-1, as an in-frame fusion with the gluthatione S-transferase, and subsequently purified . The purified fraction showed proteolytic activity on gelatine and BAPNA, but not on azocoll, suggesting a highly selective substrate specificity . The results obtained from inhibition experiments confirmed that Cwp84 belongs to the cysteine protease family . Cwp84 could play a role in degrading some specific host proteins or in the maturation of surface-associated bacterial proteins.

Parasitol Today, 1993 May, 9(5), 183 - 6
Metronidazole and drug resistance; Johnson PJ; In recent years, the basis of metronidozole resistance has been examined in anaerobic protozoa, such as Trichomonas and Giardia, as well as anaerobic bacteria, such as Bacteroides and Clostridium . In this review, Patricia Johnson looks at a variety of mechanisms that lead to reduced susceptibility of these pathogens to the drug . The frequent correlation between metronidozole resistance and inefficient drug activation suggests that this is the level at which drug resistance operates.

Parasitol Today, 1987 Jul, 3(7), 214 - 6
Antibacterial action of Myiasis-causing flies; Erdmann GR; Some species of calliphorid blowflies lay their eggs in wounds; their larvae develop by feeding on the tissue, and the infection is known as myiasis or fly-strike . But wounds, from whatever cause, are frequently contaminated with bacteria - many o f which can spread in the bloodstream causing septicaemia and/or toxaemia . For example, wound contamination with Clostridium welchii - leading to 'gas gangrene' - was a frequent cause of death amongst battlefield casualties . It is from such situations that early observations were made on the beneficial effect of some blowfly larvae in limiting the bacterial infection of wounds . Indeed, some military surgeons would deliberately infest wounds with blowfly maggots in order to prevent bacterial complications . Now, a century or two later, the search for new antibiotics had led researchers back to these early observations, and in this article, Gory Erdmann describes progress in understanding the antibacterial action of blowfly maggots.

Commun Dis Intell, 2004, 28(2), 211 - 24
Foodborne disease outbreaks in Australia, 1995 to 2000; Dalton CB et al.; Health agencies are increasingly conducting systematic reviews of foodborne disease outbreak investigations to develop strategies to prevent future outbreaks . We surveyed state and territory health departments to summarise the epidemiology of foodborne disease outbreaks in Australia from 1995 to 2000 . From 1995 through 2000, 293 outbreaks were identified, with 214 being of foodborne origin . One hundred and seventy-four (81%) had a known aetiology, and accounted for 80 per cent (6,472/8,124) of illnesses . There were 20 deaths attributed to foodborne illness . Of the 214 outbreaks, bacterial disease was responsible for 61 per cent of outbreaks, 64 per cent of cases and 95 per cent of deaths . The most frequent aetiology of outbreaks was Salmonella in 75 (35%) outbreaks, Clostridium perfringens in 30 (14%), ciguatera toxin in 23 (11%), scombrotoxin in 7 (3%) and norovirus in 6 (3%) . Salmonellosis was responsible for eight of the 20 (40%) deaths, as was Listeria monocytogenes . Restaurants and commercial caterers were associated with the highest number of outbreak reports and cases . Outbreaks in hospitals and aged care facilities were responsible for 35 per cent of deaths . The most frequently implicated vehicles in the 173 outbreaks with known vehicles were meats 64 (30%), fish 34 (16%), seafood 13 (6%), salad 12 (6%), sandwiches 11 (5%) and eggs 9 (4%) . Chicken, the most frequently implicated meat, was associated with 27 (13%) outbreaks . This summary demonstrates the serious nature of foodborne disease and supports the move to risk-based food safety interventions focusing on mass catering and hospital and aged care facilities.

J Vet Diagn Invest, 2004 Sep, 16(5), 403 - 11
The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep; Uzal FA et al.; The pathological findings in sheep with peracute experimental Clostridium perfringens type D enterotoxemia are described . Of 16 animals inoculated intraduodenally with a whole culture of this microorganism and a starch solution in the abomasum, 12 developed clinical signs including increased respiratory efforts, recumbency, paddling, bleating, convulsions, blindness, and opisthotonus . Diarrhea was not observed in any of the animals . The time lapse between the beginning of intraduodenal infusion and onset of clinical signs varied between 30 minutes and 26 hours, and the clinical course varied between 1 and 9 hours . Gross postmortem changes were observed in these 12 animals and included pulmonary edema; excess pericardial, peritoneal, or pleural fluid with or without strands of fibrin; liquid small intestinal contents; leptomeningeal edema; cerebellar coning; and subcapsular petechiae on kidneys . Histological changes consisted of severe edema of pleura and interlobular septa and around blood vessels and airways and acidophilic, homogeneous, proteinaceous perivascular edema in the brain . Five of 12 animals (42%) with clinical signs consistent with enterotoxemia lacked specific histological lesions in the brain . None of the intoxicated or control animals developed nephrosis . Glucose was detected in the urine of 3 of 6 animals that were tested for this analyte . These results stress the importance of the use of histological examination of the brain, coupled with epsilon toxin detection, for a definitive diagnosis of C . perfringens type D enterotoxemia in sheep.

Postepy Hig Med Dosw (Online), 2004 Sep 20, 58, 349 - 51
{Anaerobic intestinal microflora in pathogenesis of autism?}; Martirosian G; The present study provides information on differences in the gastrointestinal microflora of children with autism compared with a control group . Special attention was paid to the microbiological characteristic of the new obligate anaerobic microorganisms: Clostridium bolteae sp.nov . and Cetobacterium somerae sp.nov . observed in stools of children with autism . In light of recent publications, the hypothesis of interactions between intestinal microflora - and the brain based on possible alterations in bacterial toxins and other metabolites in the pathogenesis of autism is discussed.

Mol Microbiol, 2004 Oct, 54(1), 89 - 98
Identification of SpyA, a novel ADP-ribosyltransferase of Streptococcus pyogenes; Coye LH et al.; Streptococcus pyogenes, the aetiological agent of both respiratory and skin infections, produces numerous exotoxins to establish infection . This report identifies a new exotoxin produced by this organism, termed SpyA, for S . pyogenesADP-ribosylating toxin . SpyA, MW 24.9, has amino acid identity with the ADP-riboslytransferases (ADPRTs) Staphylococcus aureus EDIN and Clostridium botulinum C3 . Recombinant SpyA was able to hydrolyse beta-NAD(+), and this activity was dependent on a glutamate at position 187 . SpyA has a putative biglutamate active site, and similar to most biglutamate ADPRTs, was able to ADP-ribosylate poly-l-arginine . SpyA modified numerous proteins in both CHO and HeLa cell lysates . Two-dimesional gel analysis and MALDI-TOF MS analysis of modified proteins indicated that vimentin, tropomyosin and actin, all cytoskeletal proteins, are targets . Expression of spyA in HeLa cells resulted in loss of actin microfilaments . We hypothesize that SpyA is produced by S . pyogenes to disrupt cytoskeletal structures and promote colonization of the host.

Arch Microbiol, 2004 Dec, 182(6), 482 - 6 Epub 2004 Dec.
Propanol as an end product of threonine fermentation; Janssen PH; Clostridium sp . strain 17cr1 was able to ferment L: -threonine to propionate and propanol . Electrons arising in the oxidation of 2-oxobutyrate to propionyl-CoA were apparently used in reductive pathway leading to propanol formation . Part of the propionyl-CoA was used to form propionate in an ATP-forming pathway via a propionate kinase, so that the final ATP yield was 0.5 mol per mol of L: -threonine metabolised . Other growth substrates were fermented mainly to acetate and butyrate, and the reductive formation of butyrate, from 2 mol of acetyl-CoA or from crotonate or 3-hydroxybutyrate, was the main route for recycling reduced electron carriers arising during oxidative pathways for most substrates.

J Food Prot, 2004 Sep, 67(9), 1840 - 7
Effect of spices and organic acids on the growth of Clostridium perfringens during cooling of cooked ground beef; Sabah JR et al.; This study evaluated the effect of organic acids and spices, alone or combined, on Clostridium perfringens growth in cooked ground beef during alternative cooling procedures . Ground beef was inoculated with a three-strain cocktail of C . perfringens (ATCC 10388, NCTC 8238, and NCTC 8239) at 2 log spores per g and prepared following an industrial recipe (10% water, 1.5% sodium chloride, and 0.5% sodium triphosphate {wt/wt}) . Treatments consisted of the base meat plus combinations of commercial solutions of sodium lactate or sodium citrate (0 or 2%, wt/wt) with chili, garlic and herbs, curry, oregano, or clove in commercial powder form (0 or 1%, wt/wt) . Untreated meat was used as a control . Vacuum-packaged samples of each treatment were cooked (75 degrees C for 20 min) and cooled from 54.4 to 7.2 degrees C in 15, 18, or 21 h . Spore counts were estimated after inoculation, cooking, and cooling . All treatments containing sodium citrate reduced the population of C . perfringens about 0.38 to 1.14 log units during each of the three cooling procedures . No sodium citrate and spice treatment combinations showed antagonisms or synergisms . Regardless of the cooling time, the control ground beef or treatments with any of the five spices alone supported C . perfringens growth above the U.S . Department of Agriculture stabilization guidelines of 1 log unit . Except for the 21-h cooling period, addition of sodium lactate prevented C . perfringens growth over 1 log unit . Depending on the cooling time and spice, some combinations of sodium lactate and spice kept C . perfringens growth below 1 log unit.

FEBS Lett, 1990 Sep 3, 269(2), 368 - 72
Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS); Fischer R et al.; Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1 . When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M . barkeri or from Clostridium pasteurianum . The apparent Km for ferredoxin from M . barkeri was determined to be 2.5 M . A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate . Reduction of methyl-coenzyme M with H2 did not require ferredoxin . These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate . Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.

Rev Physiol Biochem Pharmacol, 2004, 152, 23 - 47 Epub 2004.
Large clostridial cytotoxins; Just I et al.; The large clostridial cytotoxins are a family of structurally and functionally related exotoxins from Clostridium difficile (toxins A and B), C . sordellii (lethal and hemorrhagic toxin) and C . novyi (alpha-toxin) . The exotoxins are major pathogenicity factors which in addition to their in vivo effects are cytotoxic to cultured cell lines causing reorganization of the cytoskeleton accompanied by morphological changes . The exotoxins are single-chain protein toxins, which are constructed of three domains: receptor-binding, translocation and catalytic domain . These domains reflect the self-mediated cell entry via receptor-mediated endocytosis, translocation into the cytoplasm, and execution of their cytotoxic activity by an inherent enzyme activity . Enzymatically, the toxins catalyze the transfer of a glucosyl moiety from UDP-glucose to the intracellular target proteins which are the Rho and Ras GTPases . The covalent attachment of the glucose moiety to a conserved threonine within the effector region of the GTPases renders the Rho-GTPases functionally inactive . Whereas the molecular mode of cytotoxic effects is fully understood, the mechanisms leading to inflammatory processes in the context of disease (e.g., antibiotic-associated pseudomembranous colitis caused by Clostridium difficile) are less clear.

Int J Syst Evol Microbiol, 2004 Sep, 54(Pt 5), 1465 - 8
Clostridium jejuense sp . nov., isolated from soil; Jeong H et al.; A strictly anaerobic, mesophilic, endospore-forming bacterium, designated strain HY-35-12T, was isolated from a soil sample in Jeju, Korea . Cells of this isolate were Gram-positive, motile rods that formed oval to spherical terminal spores . Strain HY-35-12T grew optimally at 30 degrees C, pH 7.0 and 0-0.5 % (w/v) NaCl . The isolate produced pyruvate, lactate, acetate, formate and hydrogen as fermentation end products from glucose . The G + C content of DNA of the isolate was 41 mol% . Phylogenetic analysis based on 16S rRNA gene sequences revealed that the organism formed a monophyletic clade with Clostridium xylanovorans and Clostridium aminovalericum in cluster XIVa of the genus Clostridium . The closest phylogenetic neighbour was C . xylanovorans, with 96.65 % 16S rRNA gene sequence similarity . Several physiological and chemotaxonomic properties were identified that enable strain HY-35-12T to be distinguished from phylogenetically related clostridia . On the basis of polyphasic characteristics, it is proposed that strain HY-35-12T (= IMSNU 40003T = KCTC 5026T = DSM 15929T) represents a novel species, Clostridium jejuense sp . nov.

Antimicrob Agents Chemother, 2004 Oct, 48(10), 3975 - 9
Rifalazil treats and prevents relapse of clostridium difficile-associated diarrhea in hamsters; Anton PM et al.; Although vancomycin and metronidazole effectively treat Clostridium difficile-associated diarrhea and colitis (CDAD), their use is associated with a high incidence of relapsing C . difficile infection . Rifalazil is a new benzoxazinorifamycin that possesses activity against Mycobacterium tuberculosis and gram-positive bacteria . Here we compared rifalazil and vancomycin for effectiveness in preventing or treating clindamycin-induced cecitis in a hamster model of CDAD . Golden Syrian hamsters were injected subcutaneously with clindamycin phosphate (10 mg/kg), followed 24 h later by C . difficile gavage . Hamsters received by gavage for 5 days vehicle, vancomycin (50 mg/kg), or rifalazil (20 mg/kg) either simultaneously with (prophylactic protocol) or 24 h after C . difficile administration (treatment protocol) . While all vehicle-administered animals became moribund within 48 h of C . difficile administration, no rifalazil- or vancomycin-treated animals in either protocol showed signs of morbidity after 7 days . Ceca of rifalazil-treated animals showed absence of epithelial cell damage, significantly reduced congestion and edema, and less, but not statistically significantly less, neutrophil infiltration compared to those of vehicle-treated animals . In contrast, vancomycin-treated animals demonstrated severe epithelial cell damage and mildly reduced congestion and edema . Moreover, hamsters relapsed and tested C . difficile toxin positive (by enzyme-linked immunosorbent assay) 10 to 15 days after discontinuation of vancomycin treatment . None of the rifalazil-treated hamsters showed signs of disease or presence of toxins in their feces 30 days after discontinuation of treatment . Our results indicate that once daily rifalazil may be superior to vancomycin for curative treatment of CDAD.

J Emerg Med, 2004 Oct, 27(3), 257 - 60
Case report of tetanus in an immunized, healthy adult and no point of entry; Hahn BJ et al.; We report the case of a 58-year-old man born in the United States with a history of complete childhood immunizations who presented to the Emergency Department with trismus . Past medical history was significant only for Elephantiasis . After an exhaustive workup the patient was found to have Tetanus, with no identifiable portal of entry . The patient was successfully treated for Tetanus with complete recovery . Tetanus is caused by the organism Clostridium Tetani, which usually requires an open lesion to cause infection . Our patient was unique in that he was previously immunized with no obvious lesion . Tetanus should be suspected and treated empirically in any patient presenting with typical signs and symptoms even without an apparent entry site.

J Biol Chem, 2004 Nov 26, 279(48), 49876 - 82 Epub 2004 Nov 26.
A phosphatidylserine-binding site in the cytosolic fragment of Clostridium sordellii lethal toxin facilitates glucosylation of membrane-bound Rac and is required for cytotoxicity; Mesmin B et al.; Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators . We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT(cyt); amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids . The binding of LT(cyt) to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP . Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT(cyt) . Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT(cyt) on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT(cyt) microinjection into various cells, but it does not affect the intrinsic activity of LT(cyt) on non-geranyl-geranylated Rac-GDP in solution . We conclude that the avidity of LT(cyt) for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Microb Drug Resist, 2004 Fall, 10(3), 191 - 6
CdeA of Clostridium difficile, a new multidrug efflux transporter of the MATE family; Dridi L et al.; The cdeA gene, cloned from Clostridium difficile clinical strain 714 under the control of its natural promoter made Escherichia coli and Clostridium perfringens resistant to ethidium bromide and acriflavin but had no effect on the susceptibility of the hosts to the following antibiotics: norfloxacin, ciprofloxacin, gentamicin, erythromycin, tetracyclin, and chloramphenicol . However, it was responsible for fluoroquinolone resistance in E . coli when it was cloned under the control of the Plac promoter . Quantitative reverse transcriptase (RT)-PCR showed that growth of C . difficile clinical strain 253 in the presence of subinhibitory concentrations of ethidium bromide significantly increased the transcription of cdeA, but this was not observed with ciprofloxacin . The deduced protein was homologous to the protein sequences of known efflux pumps from the third cluster (the so-called DinF branch) of the multidrug and toxic compound extrusion (MATE) family . CdeA caused ethidium bromide energy-dependent efflux in whole cells of E . coli . Efflux activity was stimulated by addition of Na+ ions, suggesting that CdeA, like other pumps of the MATE family, is a Na+-coupled efflux pump . CdeA is the first multidrug efflux transporter identified in C . difficile . Copyright Mary Ann Liebert, Inc.

Proteins, 2004 Nov 15, 57(3), 618 - 25
Crystallographic studies of V44 mutants of Clostridium pasteurianum rubredoxin: effects of side-chain size on reduction potential; Park IY et al.; Understanding the structural origins of differences in reduction potentials is crucial to understanding how various electron transfer proteins modulate their reduction potentials and how they evolve for diverse functional roles . Here, the high-resolution structures of several Clostridium pasteurianum rubredoxin (Cp Rd) variants with changes in the vicinity of the redox site are reported in order to increase this understanding . Our crystal structures of {V44L} (at 1.8 A resolution), {V44A} (1.6 A), {V44G} (2.0 A) and {V44A, G45P} (1.5 A) Rd (all in their oxidized states) show that there is a gradual decrease in the distance between Fe and the amide nitrogen of residue 44 upon reduction in the size of the side chain of residue 44; the decrease occurs from leucine to valine, alanine or glycine and is accompanied by a gradual increase in their reduction potentials . Mutation of Cp Rd at position 44 also changes the hydrogen-bond distance between the amide nitrogen of residue 44 and the sulfur of cysteine 42 in a size-dependent manner . Our results suggest that residue 44 is an important determinant of Rd reduction potential in a manner dictated by side-chain size . Along with the electric dipole moment of the 43-44 peptide bond and the 44-42 NH--S type hydrogen bond, a modulation mechanism for solvent accessibility through residue 41 might regulate the redox reaction of the Rds.

Euro Surveill . 2004 Sep 1;9(9) {Epub ahead of print}
Outbreak of Clostridium histolyticum infections in injecting drug users in England and Scotland; Brazier JS et al.; Clostridial infections in injecting drug users in the United Kingdom are a relatively new phenomenon that came to light in 2000 when cases of serious illness and deaths due to Clostridium novyi were recorded . In the period December 2003 to April 2004, the Anaerobe Reference Laboratory received twelve referrals of an extremely rare isolate, Clostridium histolyticum, from cases of infection in injecting drug users submitted from nine different hospitals in England and Scotland . Molecular typing of these isolates by two different methods of pulsed-field gel electrophoresis and PCR ribotyping revealed they are all indistinguishable, indicating a common source of the infections, most probably a batch of heroin that was recently distributed across the UK.

Phytochemistry, 2004 Sep, 65(17), 2485 - 97
Variation in antimicrobial action of proanthocyanidins from Dorycnium rectum against rumen bacteria; Sivakumaran S et al.; The proanthocyanidin polymer fractions of the leaves of the forage legume Dorycnium rectum were analysed by acid catalysis with benzyl mercaptan, NMR and ES-MS . The results showed that D . rectum differs from other temperate proanthocyanidin-containing forage legumes in that the range of polymers extends up to very high degrees of polymerisation . Three fractions were characterised as low, medium, and high molecular weight proanthocyanidin fractions with mean degree of polymerisations of 10.3, 41 and 127, respectively . Epigallocatechin was the most abundant extension unit and the terminating flavan-3-ols comprised largely catechin and gallocatechin units in equal proportions . Formation of thiolyated dimer products showed the interflavan-linkages of the lower molecular weight proanthocyanidins to be predominantly C4-->C8 with a small amount of C4-->C6 . ES-MS spectra distinguished lower from higher polymeric proanthocyanidins from M2- to M8(2)- . The antibacterial activity of proanthocyanidin fractions against pure cultures of microbes selected from the ruminal population to represent fibre degrading, proteolytic and hyper ammonia producing bacteria in broth culture was evaluated . The activity of proanthocyanidin fractions against Clostridium aminophilum, Butyrivibrio fibrisolvens and Clostridium proteoclasticum was significantly dependent on their structure but not so against Ruminococcus albus and Peptostreptococcus anaerobius . The latter observation was unique in that they were sensitive to all proanthocyanidin fractions evaluated, even at the lowest concentration (100 microg/ml) . The results suggest the effects of the extractable proanthocyanidins on rumen microbes should be considered when evaluating an alternative proanthocyanidin-containing forage source for ruminants, such as D . rectum .

Arch Gerontol Geriatr, 2004 Nov-Dec, 39(3), 277 - 82
Diarrhea in elderly patients due to Clostridium difficile associated with Salmonella and Shigella infection; Grinblat J et al.; Clostridium difficile is the main cause of nosocomial diarrhea mainly in elderly hospitalized patients and in nursing homes, but less common in the community . We report three elderly patients living in the community, one frail and two in reasonable good health who recently developed diarrhea due to C . difficile associated with Salmonella group C in two cases and Shigella flexneri in the other . The association with Shigella has not previously been reported and there have only been a few cases with Salmonella . These findings may be important due to the possibility that diarrhea caused by C . difficile associated with other enteric bacteria may be more prevalent than expected in the aging population, thus necessitating awareness and consequently appropriate treatment and follow-up.

Br J Plast Surg, 2004 Oct, 57(7), 673 - 5
Fatal multifocal metastasis of Clostridium septicum: a case report; Tehrani H et al.; Clostridium septicum infection produces rapidly spreading tissue necrosis, often, but not exclusively, associated with trauma or large bowel malignancy . We present a unique case of atraumatic infection leading to multifocal metastatic spread in a neutropenic patient, emphasising the devastating potential of this disease.

Cancer, 2004 Oct 1, 101(7), 1508 - 13
Colitis in patients with breast carcinoma treated with taxane-based chemotherapy; Li Z et al.; BACKGROUND: Colitis is a rare but serious gastrointestinal complication associated with taxane-based chemotherapy in patients with cancer . The incidence, clinical presentation, and outcome of colitis in patients with breast carcinoma treated with taxane-based chemotherapy is not known . METHODS: The authors searched their electronic database and identified patients with breast carcinoma who were treated with taxane-based chemotherapy between January 1997 and December 1999 . Patients diagnosed with colitis were identified and their presentation and clinical outcomes were analyzed . RESULTS: During the study period, 1350 patients received taxane-based chemotherapy . Sixty-four patients were admitted to The University of Texas M . D . Anderson Cancer Center (Houston, TX) (73 admissions) for gastrointestinal complications . Neutropenia and/or fever accounted for 56 of these admissions . Fourteen patients (16 admissions) were diagnosed with colitis . All had abdominal pain at median Day 6 of their chemotherapy cycle (range, 3-8 days), with or without other symptoms . Computed tomography scans of the abdomen and pelvis were abnormal for the 10 patients tested, whereas only 3 of the 9 patients who underwent radiographic abdominal series had abnormal findings . Two patients had a colonoscopy that confirmed the diagnosis of colitis . Blood cultures were positive in only 3 (20%) of 15 colitis events . All eight patients tested for Clostridium difficile toxin were negative . One patient died of sepsis . An autopsy subsequently revealed cecal ulceration . Two patients had bowel perforations requiring hemicolectomy . Colitis recurred in two patients after they resumed taxane-based chemotherapy without dose reduction . Dose reductions prevented the recurrence of colitis in seven patients . Treatment discontinuation of taxane-based chemotherapy also prevented disease recurrence . CONCLUSIONS: Acute abdominal pain in patients with breast carcinoma treated with taxane-based chemotherapy signaled a potentially fatal colitis, and warrants aggressive supportive care . Dose reduction or discontinuation of taxane-based chemotherapy may prevent the recurrence of colitis . (c) 2004 American Cancer Society.

J Ind Microbiol Biotechnol, 2004 Oct, 31(9), 442 - 6 Epub 2004 Oct.
Production of 1,3-propanediol by Clostridium butyricum VPI 3266 using a synthetic medium and raw glycerol; Gonzalez-Pajuelo M et al.; Growth inhibition of Clostridium butyricum VPI 3266 by raw glycerol, obtained from the biodiesel production process, was evaluated . C . butyricum presents the same tolerance to raw and to commercial glycerol, when both are of similar grade, i.e . above 87% (w/v) . A 39% increase of growth inhibition was observed in the presence of 100 g l(-1) of a lower grade raw glycerol (65% w/v) . Furthermore, 1,3-propanediol production from two raw glycerol types (65% w/v and 92% w/v), without any prior purification, was observed in batch and continuous cultures, on a synthetic medium . No significant differences were found in C . butyricum fermentation patterns on raw and commercial glycerol as the sole carbon source . In every case, 1,3-propanediol yield was around 0.60 mol/mol glycerol consumed.

Liver Transpl, 2004 Oct, 10(10), 1315 - 9
Fulminant and fatal gas gangrene of the stomach in a healthy live liver donor; Miller C et al.; A 57-year-old male with a history of hypercholesterolemia and anxiety but otherwise in good health volunteered to donate the right lobe of his liver to his brother . The operation was performed uneventfully, without transfusion . Postoperatively he did well, until he developed tachycardia, profound hypotension, and coffee ground emesis on postoperative day 3 . Despite resuscitative measures, he arrested and expired . Autopsy demonstrated gas gangrene of the stomach as the underlying cause of the hemorrhage and numerous colonies of Gram-positive bacilli were identified . Subsequent polymerase chain reaction (PCR) analysis identified these bacteria to be Clostridium perfringens (C . perfringens) type D . This patient's death was devastating, both to his family and his medical team . The impact of his death has transcended that of an individual occurrence . In conclusion, herein we present the facts and discuss this extraordinary example of florid clostridial infection and toxin-mediated shock . It was completely unexpected and probably unpreventable, and its cause was almost inconceivable.

J Bacteriol, 2004 Oct, 186(19), 6575 - 85
Genome-wide molecular clock and horizontal gene transfer in bacterial evolution; Novichkov PS et al.; We describe a simple theoretical framework for identifying orthologous sets of genes that deviate from a clock-like model of evolution . The approach used is based on comparing the evolutionary distances within a set of orthologs to a standard intergenomic distance, which was defined as the median of the distribution of the distances between all one-to-one orthologs . Under the clock-like model, the points on a plot of intergenic distances versus intergenomic distances are expected to fit a straight line . A statistical technique to identify significant deviations from the clock-like behavior is described . For several hundred analyzed orthologous sets representing three well-defined bacterial lineages, the alpha-Proteobacteria, the gamma-Proteobacteria, and the Bacillus-Clostridium group, the clock-like null hypothesis could not be rejected for approximately 70% of the sets, whereas the rest showed substantial anomalies . Subsequent detailed phylogenetic analysis of the genes with the strongest deviations indicated that over one-half of these genes probably underwent a distinct form of horizontal gene transfer, xenologous gene displacement, in which a gene is displaced by an ortholog from a different lineage . The remaining deviations from the clock-like model could be explained by lineage-specific acceleration of evolution . The results indicate that although xenologous gene displacement is a major force in bacterial evolution, a significant majority of orthologous gene sets in three major bacterial lineages evolved in accordance with the clock-like model . The approach described here allows rapid detection of deviations from this mode of evolution on the genome scale.

J Bacteriol, 2004 Oct, 186(19), 6544 - 52
Towards designer cellulosomes in Clostridia: mannanase enrichment of the cellulosomes produced by Clostridium cellulolyticum; Perret S et al.; The man5K gene of Clostridium cellulolyticum was cloned and overexpressed in Escherichia coli . This gene encodes a 424-amino-acid preprotein composed of an N-terminal leader peptide, followed by a dockerin module and a C-terminal catalytic module belonging to family 5 of the glycosyl hydrolases . Mature Man5K displays 62% identity with ManA from Clostridium cellulovorans . Two forms of the protein were purified from E . coli; one form corresponds to the full-length enzyme (45 kDa), and a truncated form (39 kDa) lacks the N-terminal dockerin module . Both forms exhibit the same typical family 5 mannanase substrate preference; they are very active with the galactomannan locust bean gum, and the more galacto-substituted guar gum molecules are degraded less . The truncated form, however, displays fourfold-higher activity with galactomannans than the full-length enzyme . Man5K was successfully overproduced in C . cellulolyticum by using expression vectors . The trans-produced protein was found to be incorporated into the cellulosomes and became one of the major enzymatic components . Modified cellulosomes displayed 20-fold-higher specific activities than control fractions on galactomannan substrates, whereas the specific activity on crystalline cellulose was reduced by 20% . This work clearly showed that the composition of the cellulosomes is obviously regulated by the relative amounts of the enzymes produced and that this composition can be engineered in clostridia by structural gene cloning.

J Bacteriol, 2004 Oct, 186(19), 6351 - 9
Hydrophilic domains of scaffolding protein CbpA promote glycosyl hydrolase activity and localization of cellulosomes to the cell surface of Clostridium cellulovorans; Kosugi A et al.; CbpA, the scaffolding protein of Clostridium cellulovorans cellulosomes, possesses one family 3 cellulose binding domain, nine cohesin domains, and four hydrophilic domains (HLDs) . Among the three types of domains, the function of the HLDs is still unknown . We proposed previously that the HLDs of CbpA play a role in attaching the cellulosome to the cell surface, since they showed some homology to the surface layer homology domains of EngE . Several recombinant proteins with HLDs (rHLDs) and recombinant EngE (rEngE) were examined to determine their binding to the C . cellulovorans cell wall fraction . Tandemly linked rHLDs showed higher affinity for the cell wall than individual rHLDs showed . EngE was shown to have a higher affinity for cell walls than rHLDs have . C . cellulovorans native cellulosomes were found to have higher affinity for cell walls than rHLDs have . When immunoblot analysis was carried out with the native cellulosome fraction bound to cell wall fragments, the presence of EngE was also confirmed, suggesting that the mechanism anchoring CbpA to the C . cellulovorans cell surface was mediated through EngE and that the HLDs play a secondary role in the attachment of the cellulosome to the cell surface . During a study of the role of HLDs on cellulose degradation, the mini-cellulosome complexes with HLDs degraded cellulose more efficiently than complexes without HLDs degraded cellulose . The rHLDs also showed binding affinity for crystalline cellulose and carboxymethyl cellulose . These results suggest that the CbpA HLDs play a major role and a minor role in C . cellulovorans cellulosomes . The primary role increases cellulose degradation activity by binding the cellulosome complex to the cellulose substrate; secondarily, HLDs aid the binding of the CbpA/cellulosome to the C . cellulovorans cell surface.

FEMS Microbiol Rev, 2004 Oct, 28(4), 455 - 68
Dehydration of (R)-2-hydroxyacyl-CoA to enoyl-CoA in the fermentation of alpha-amino acids by anaerobic bacteria; Kim J et al.; Several clostridia and fusobacteria ferment alpha-amino acids via (R)-2-hydroxyacyl-CoA, which is dehydrated to enoyl-CoA by syn-elimination . This reaction is of great mechanistic interest, since the beta-hydrogen, to be eliminated as proton, is not activated (pK 40-50) . A mechanism has been proposed, in which one high-energy electron acts as cofactor and transiently reduces the electrophilic thiol ester carbonyl to a nucleophilic ketyl radical anion . The 2-hydroxyacyl-CoA dehydratases are two-component systems composed of an extremely oxygen-sensitive component A, an activator, and component D, the actual dehydratase . Component A, a homodimer with one {4Fe-4S}cluster, transfers an electron to component D, a heterodimer with 1-2 {4Fe-4S}clusters and FMN, concomitant with hydrolysis of two ATP . From component D the electron is further transferred to the substrate, where it facilitates elimination of the hydroxyl group . In the resulting enoxyradical the beta-hydrogen is activated (pK14) . After elimination the electron is handed-over to the next incoming substrate without further hydrolysis of ATP . The helix-cluster-helix architecture of component A forms an angle of 105 degrees, which probably opens to 180 degrees upon binding of ATP resembling an archer shooting arrows . Therefore we designated component A as 'Archerase' . Here, we describe 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans, Clostridium symbiosum and Fusobacterium nucleatum, 2-phenyllactate dehydratase from Clostridium sporogenes, 2-hydroxyisocaproyl-CoA dehydratase from Clostridium difficile, and lactyl-CoA dehydratase from Clostridium propionicum . A relative of the 2-hydroxyacyl-CoA dehydratases is benzoyl-CoA reductase from Thauera aromatica . Analogous but unrelated archerases are the iron proteins of nitrogenase and bacterial protochlorophyllide reductase . In anaerobic organisms, which do not oxidize 2-oxo acids, a second energy-driven electron transfer from NADH to ferredoxin, the electron donor of component A, has been established . The transfer is catalysed by a membrane-bound NADH-ferredoxin oxidoreductase driven by an electrochemical Na(+)-gradient . This enzyme is related to the Rnf proteins involved in Rhodobacter capsulatus nitrogen fixation.

Arch Gerontol Geriatr, 1991 Nov-Dec, 13(3), 255 - 61
Clostridium difficile infection in a geriatric ward; Monsieur I et al.; In a prospective, longitudinal study we searched for the presence of Clostridium difficile in the stools of 100 consecutively hospitalized elderly patients (mean age: 82; SD: 9.5 years) . C . difficile was found on admission in 6 patients, 3 of whom were asymptomatic carriers . Ten patients acquired C . difficile during hospitalization . Four different types of C . difficile were isolated . The various types were clustered in time, indicating that the infection was acquired from the environment.

J Agric Food Chem, 2004 Sep 8, 52(18), 5740 - 6
Glycopeptide derived from hen egg ovomucin has the ability to bind enterohemorrhagic Escherichia coli O157:H7; Kobayashi K et al.; Ovomucin glycopeptide (OGP) was prepared by size exclusion chromatography after Pronase digestion of hen egg ovomucin, and the binding of OGP to foodborne pathogens (Bacillus cereus,Clostridium perfringens, Escherichia coli O157:H7, Listeria monocytogenes, Salmonella enteritidis, Salmonella typhimurium, and Staphylococcus aureus) was investigaed . Binding assays with biotinylated bacteria as probes in microtiter plates showed that OGP bound to only E . coli O157:H7 among these foodborne pathogens . Periodate treatment markedly reduced the binding ability, indicating that E . coli O157:H7 bound to carbohydrate moieties of OGP . Lectin blot analysis with Maackia amurensis (MAA) and Sambucus nigra (SNA), which are specific for oligosaccharides containing sialic acid, revealed their binding sites in OGP were similar to the E . coli O157:H7 binding sites that were probed with biotinylated E . coli O157:H7 after Western blotting of OGP . Sialydase treatment of OGP abolished its ability to bind E . coli O157:H7, demonstrating that sialic acid played an important role in the binding . These results suggest that OGP has E . coli O157:H7-specific binding sites that consist of sialic acid . On the basis of these properties, OGP has the potential to be an ingredient with a protective effect against E . coli O157:H7 infection and to be a novel probe for the detection of E . coli O157:H7 in the food hygiene field.

Scand J Infect Dis, 2004, 36(9), 689 - 92
Endophthalmitis caused by non-perfringens Clostridium species; Bradley JC et al.; The role of other non-perfringens Clostridium species in endophthalmitis infections has not been analyzed . We describe a case of non-perfringens Clostridium endophthalmitis and review prior cases in the literature . Fulminant clinical presentations and poor visual outcomes are the rule, but a combined aggressive medical and surgical approach may conserve vision.

Leuk Lymphoma, 2004 Oct, 45(10), 2157 - 9
Massive intravascular hemolysis: a fatal complication of clostridium perfringens septicemia in a patient with acute myeloid leukemia; Vaiopoulos G et al.; We describe a fatal case of a 74-year old man with acute myeloid leukemia complicated by massive intravascular hemolysis due to Clostridium perfringens sepsis.

Eur J Immunol, 2004 Oct, 34(10), 2708 - 19
Early cytoskeletal rearrangement during dendritic cell maturation enhances synapse formation and Ca(2+) signaling in CD8(+) T cells; Averbeck M et al.; The interplay between dendritic cells (DC) and T cells is a dynamic process critically depending on DC maturation . Ca(2+) influx is one of the initial events occurring during DC/T cell contacts . To determine how DC maturation influences DC/T cell contacts, time-lapse video microscopy was established using TCR-transgenic CD8(+) T cells from P14 mice . DC maturation shifted DC/T cell contacts from short-lived interactions with transient Ca(2+) influx in T cells to long-lasting interactions and sustained Ca(2+) influx of 30 min and more . Follow-up of DC/T cell interactions after 2 h using confocal microscopy revealed that long-lasting Ca(2+) responses in T cells were preferentially associated with the formation of an immunological synapse involving CD54 and H2-K(b) at the DC/T cell interface . Such synapse formation preceded MHC or B7 up-regulation, since DC developed into potent Ca(2+) stimulators 7 h after initiation of maturation . Instead, the enhanced capacity of 7 h-matured DC to induce sustained Ca(2+) responses in CD8(+) T cells is critically dependent on the polarization and rearrangement of the cytoskeleton, as shown by Clostridium difficile toxin B inhibitor experiments . These data indicate that already very early after receiving a maturation stimulus, DC display enhanced cytoskeletal activity resulting in the rapid formation of immunological synapses and effective CD8(+) T cell stimulation.

J Clin Microbiol, 2004 Sep, 42(9), 4390 - 2
Bacteremia caused by Clostridium symbiosum; Elsayed S et al.; We describe a fatal case of Clostridium symbiosum bacteremia in a 70-year-old man with metastatic colon cancer . Our report is the first, in the world literature, of human infection caused by this microorganism.

J Clin Microbiol, 2004 Sep, 42(9), 4316 - 8
Community-acquired Clostridium difficile diarrhea caused by binary toxin, toxin A, and toxin B gene-positive isolates in Hungary; Terhes G et al.; The aim of this work was to study the toxin types of Clostridium difficile isolates originating from different parts of Hungary . A PCR method was used for amplification of the two major toxin genes and the binary toxin gene and to detect the deletion or insertion in the 3' end of the toxin A gene . The findings were compared with the results of cytotoxicity assays on the HeLa cell line . One hundred twelve isolates were tested; the toxin A and toxin B genes were detected in 79 strains by the PCR method . All of the isolates that were positive by the PCR method were also positive by the cytotoxicity assay . All of the other strains (n = 33) were negative for the toxin A and toxin B genes; in these cases, cytopathic effects on the cell line were not observed . No tcdA-negative and tcdB-positive isolates were found by the PCR method . In two cases, the presence of a binary toxin gene was observed by PCR; both isolates that were isolated from diarrheal feces carried the tcdA and tcdB genes . No prior hospitalization had occurred in either case.

Int J Food Microbiol, 2004 Nov 1, 96(2), 115 - 31
Prevalence of Clostridium species and behaviour of Clostridium botulinum in gnocchi, a REPFED of italian origin; Del Torre M et al.; Sales and consumption of refrigerated processed foods of extended durability (REPFEDs) have increased many-fold in Europe over the last 10 years . The safety and quality of these convenient ready-to-eat foods relies on a combination of mild heat treatment and refrigerated storage, sometimes in combination with other hurdles such as mild preservative factors . The major hazard to the microbiological safety of these foods is Clostridium botulinum . This paper reports on the prevalence and behaviour of proteolytic C . botulinum and non-proteolytic C . botulinum in gnocchi, a potato-based REPFED of Italian origin . Attempts to isolate proteolytic C . botulinum and non-proteolytic C . botulinum from gnocchi and its ingredients were unsuccessful . Based on assessment of the adequacy of the methods used, it was estimated that for proteolytic C . botulinum there was < 25 spores/kg of gnocchi and < 70 spores/kg of ingredients . The total anaerobic microbial load of gnocchi and its ingredients was low, with an estimated 1 MPN/g in processed gnocchi . Most of the anaerobic flora was facultatively anaerobic . A few obligately anaerobic bacteria were isolated from gnocchi and its ingredients and belonged to different Clostridium species . The protection factor, number of decimal reductions in the probability of toxigenesis from a single spore, was determined for eight different gnocchi formulations by challenge test studies . For all gnocchi stored at 8 degrees C (as recommended by the manufacturer) or 12 degrees C (mild temperature abuse), growth and toxin production were not detected in 75 days . The protection factor was >4.2 for proteolytic C . botulinum, and >6.2 for non-proteolytic C . botulinum . When inoculated packs were stored at 20 degrees C (severe temperature abuse), toxin production in 75 days was prevented by the inclusion of 0.09% (w/w) sorbic acid (protection factors as above), however in the absence of sorbic acid the packs became toxic before the end of the intended shelf-life and the protection factors were lower . Providing sorbic acid (0.09% w/w) is included in the gnocchi, the safety margin would seem to be very large with respect to the foodborne botulism hazard.

Vaccine, 2004 Sep 28, 22(29-30), 3942 - 6
Efficacy of DNA vaccines expressing the type F botulinum toxin Hc fragment using different promoters; Jathoul AP et al.; DNA vaccines which expressed the Hc fragment of the Clostridium botulinum type F neurotoxin (BoNT/F Hc) fused to a signal peptide downstream of four different eukaryotic promoters were prepared . Subsequently, the immunogenicity of the DNA vaccines and protection afforded in mice against challenge with 10(4) MLD of type F botulinum toxin was evaluated . The DNA vaccine containing the human ubiquitin gene (UbC) promoter induced the highest BoNT/F Hc-specific antibody concentration following two intramuscular immunisations and afforded 90% protection against challenge . The results from this study indicate that the selection of promoter used in DNA vaccination studies may be of importance in designing optimised vaccines.

Clin Infect Dis, 2004 Sep 1, 39(5), 747 - 50 Epub 2004 Aug 13.
Staphylococcus aureus and Clostridium difficile cause distinct pseudomembranous intestinal diseases; Froberg MK et al.; We report simultaneous infections with Clostridium difficile and methicillin-resistant Staphylococcus aureus (MRSA) in a patient with discrete colonic pseudomembranes typical of C . difficile infection, as well as confluent, loosely adherent pseudomembranes in the small bowel . Identification of MRSA in the small bowel pseudomembrane by polymerase chain reaction supports S . aureus as an enteric pathogen.

Lett Appl Microbiol, 2004, 39(4), 363 - 8
Response surface methodology for optimizing the fermentation medium of Clostridium butyricum; He GQ et al.; AIMS: Strains of Clostridium butyricum have been increasingly used as probiotics for both animals and humans . The aim of this study was to develop a growth medium for cultivating C . butyricum ZJUCB using a statistical methodology . METHODS AND RESULTS: Response surface methodology (RSM) was used to evaluate the effects of variables, namely the concentrations of the glucose, pectin, soyabean cake extract, casein, corn steep flour, ammonium sulphate, sodium bicarbonate and the medium initial pH . A fractional factorial design was applied to study the main factors that affected the growth of a probiotic strain of C . butyricum currently preserved in our lab and the central composite experimental design was adopted to derive a statistical model for optimizing the composition of the fermentation medium . The experimental results showed that the optimum fermentation medium for the growth of C . butyricum was composed of 2% glucose (w/v), 0.5% pectin (w/v), 0.2% casein (w/v), 3.98% soyabean cake extract, 0.1% (NH4)2SO4 (w/v), 0.124% NaHCO3 (w/v), 0.37% corn steep flour (w/v), 0.02% MnSO4 H2O (w/v), 0.02% MgSO4 7H2O (w/v) and 0.002% CaCl2 (w/v) at pH 7.5 . CONCLUSIONS: After incubating 24 h in the optimum fermentation medium, the populations of the viable organisms were estimated to be 10(9) CFU ml(-1) . In the present study, we report the optimization of a growth medium that produced increased yields using statistical approach . SIGNIFICANCE AND IMPACT OF THE STUDY: The use of bacteria as a probiotic is showing increasing potential . The development of a growth medium that has a high yield is an obvious need, and the approach to optimizing a growth medium is innovative.

Microbiol Mol Biol Rev, 2004 Sep, 68(3), 560 - 602, table of contents
Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion; Brussow H et al.; Comparative genomics demonstrated that the chromosomes from bacteria and their viruses (bacteriophages) are coevolving . This process is most evident for bacterial pathogens where the majority contain prophages or phage remnants integrated into the bacterial DNA . Many prophages from bacterial pathogens encode virulence factors . Two situations can be distinguished: Vibrio cholerae, Shiga toxin-producing Escherichia coli, Corynebacterium diphtheriae, and Clostridium botulinum depend on a specific prophage-encoded toxin for causing a specific disease, whereas Staphylococcus aureus, Streptococcus pyogenes, and Salmonella enterica serovar Typhimurium harbor a multitude of prophages and each phage-encoded virulence or fitness factor makes an incremental contribution to the fitness of the lysogen . These prophages behave like "swarms" of related prophages . Prophage diversification seems to be fueled by the frequent transfer of phage material by recombination with superinfecting phages, resident prophages, or occasional acquisition of other mobile DNA elements or bacterial chromosomal genes . Prophages also contribute to the diversification of the bacterial genome architecture . In many cases, they actually represent a large fraction of the strain-specific DNA sequences . In addition, they can serve as anchoring points for genome inversions . The current review presents the available genomics and biological data on prophages from bacterial pathogens in an evolutionary framework.

Microbiol Mol Biol Rev, 2004 Sep, 68(3), 373 - 402, table of contents
Binary bacterial toxins: biochemistry, biology, and applications of common Clostridium and Bacillus proteins; Barth H et al.; Certain pathogenic species of Bacillus and Clostridium have developed unique methods for intoxicating cells that employ the classic enzymatic "A-B" paradigm for protein toxins . The binary toxins produced by B . anthracis, B . cereus, C . botulinum, C . difficile, C . perfringens, and C . spiroforme consist of components not physically associated in solution that are linked to various diseases in humans, animals, or insects . The "B" components are synthesized as precursors that are subsequently activated by serine-type proteases on the targeted cell surface and/or in solution . Following release of a 20-kDa N-terminal peptide, the activated "B" components form homoheptameric rings that subsequently dock with an "A" component(s) on the cell surface . By following an acidified endosomal route and translocation into the cytosol, "A" molecules disable a cell (and host organism) via disruption of the actin cytoskeleton, increasing intracellular levels of cyclic AMP, or inactivation of signaling pathways linked to mitogen-activated protein kinase kinases . Recently, B . anthracis has gleaned much notoriety as a biowarfare/bioterrorism agent, and of primary interest has been the edema and lethal toxins, their role in anthrax, as well as the development of efficacious vaccines and therapeutics targeting these virulence factors and ultimately B . anthracis . This review comprehensively surveys the literature and discusses the similarities, as well as distinct differences, between each Clostridium and Bacillus binary toxin in terms of their biochemistry, biology, genetics, structure, and applications in science and medicine . The information may foster future studies that aid novel vaccine and drug development, as well as a better understanding of a conserved intoxication process utilized by various gram-positive, spore-forming bacteria.

Arch Oral Biol, 2004 Nov, 49(11), 931 - 5
Quantitative analysis of the impact of the organic matrix on the fluoride effect on erosion progression in human dentine using longitudinal microradiography; Ganss C et al.; After an initial demineralisation, an intensive fluoridation is capable of inhibiting the erosive mineral loss in dentine completely, which might be related to the presence of the exposed organic dentine matrix . Aim of the present study was, therefore, to evaluate whether fluoride is also effective when the organic material is removed . The study was a cyclic de- and remineralisation model over 5 days in vitro . Samples from human coronal dentine were demineralised with citric acid (pH 2.3; 6 x 10 min per day) and intermittently stored in a remineralisation solution . Groups (n = 25 each) were defined as follows: Group 1: erosion only, no fluoridation; Group 2: erosion, alternately fluoridation with toothpaste (NaF; 0.15% F-; 3 x 5 min per day), mouthrinse (Olaflur/SnF2; 0.025 F-; 3 x 5 min per day) and gel (Olaflur/NaF; 1.25% F-; at Days 1 and 3 instead of one toothpaste application); Group 3: erosion and fluoridation as Group 2, organic material was continuously removed with collagenase (from Clostridium Histolyticum type VII, 100 U/ml) added to the remineralisation solution . Mineral content was monitored daily using longitudinal microradiogaphy . After fluoridation in the presence of collagenase, a linear increase in mineral loss (73.3 +/- 17.6 microm at Day 5) was observed, which significantly (P < or = 0.001) exceeded that of the control group (45.9 +/- 14.3 microm at Day 5) . After fluoridation in the absence of collagenase, mineral loss ceased after the 2nd day (12.2 +/- 10.2 microm at Day 5) and was significantly lower compared to Groups 1 and 3 (P < or = 0.001) . The results indicate that the dentine matrix is essential for the effectiveness of fluoride in dental erosion.

Surg Infect (Larchmt), 2004 Summer, 5(2), 205 - 9
Fulminant liver failure following infection by Clostridium perfringens; Bergert H et al.; BACKGROUND: Since the first description of gas gangrene of an internal organ by Fraenkel in 1889, few cases of acute organ failure following Clostridium perfringens infection have been described in the medical literature . Isolated Clostridium perfringens infection with subsequent sepsis syndrome is an extremely rare clinical syndrome . A consecutive pattern of multiple organ failure generally has a very high mortality rate . METHODS: Individual case report and literature review . RESULTS: A 58-year-old male patient developed fulminant necrotic liver failure following a Clostridium perfringens infection . Despite all intensive care measures, including computed tomography-guided drainage, the condition of the patient deteriorated rapidly and the patient died . In this case report, we characterize the symptoms of gas gangrene isolated to the liver and compare the treatment measures instituted with the medical literature . CONCLUSIONS: In our presented case, primary malignant disease of the papilla of Vater and resection by a Whipple procedure with a hepatico-jejunostomy were a decisive cause of the gas gangrene in the liver . The origin is probably ascension up the common hepatic duct of gut-derived bacteria.

Microbiology, 2004 Sep, 150(Pt 9), 2921 - 30
Metabolic properties of Eubacterium pyruvativorans, a ruminal 'hyper-ammonia-producing' anaerobe with metabolic properties analogous to those of Clostridium kluyveri; Wallace RJ et al.; Eubacterium pyruvativorans I-6(T) is a non-saccharolytic, amino-acid-fermenting anaerobe from the rumen, isolated by its ability to grow on pancreatic casein hydrolysate (PCH) as sole C source . This study investigated its metabolic properties and its likely ecological niche . Additional growth was supported by pyruvate, vinyl acetate, and, to a lesser extent, lactate and crotonate, and also by a mixture of amino acids (alanine, glycine, serine and threonine) predicted to be catabolized to pyruvate . No single amino acid supported growth, and peptides were required for growth on amino acids . Alanine, followed by leucine, serine and proline, were used most extensively during growth, but only alanine and asparate were extensively modified before incorporation . Growth on PCH, but not on pyruvate, was increased by the addition of acetate, propionate and butyrate . l-Lactate was fermented incompletely, mainly to acetate, but no lactate-C was incorporated . Propionate and butyrate were utilized during growth, forming valerate and caproate, respectively . Labelling experiments suggested a metabolic pattern where two C atoms of butyrate, valerate and caproate were derived from amino acids, with the others being formed from acetate, propionate and butyrate . The metabolic strategy of E . pyruvativorans therefore resembles that of Clostridium kluyveri, which ferments ethanol only when the reaction is coupled to acetate, propionate or butyrate utilization . The fermentative niche of E . pyruvativorans appears to be to scavenge amino acids, lactate and possibly other metabolites in order to generate ATP via acetate formation, using volatile fatty acid elongation with C(2) units derived from other substrates to dispose of reducing equivalents.

Appl Environ Microbiol, 2004 Sep, 70(9), 5704 - 7
Production of minicellulosomes from Clostridium cellulovorans in Bacillus subtilis WB800; Cho HY et al.; Two genes encoding EngB endoglucanase and mini-CbpA1 scaffolding protein of Clostridium cellulovorans were constructed and coexpressed in Bacillus subtilis WB800 . The resulting minicellulosomes were isolated by gel filtration chromatography and characterized . Biochemical and immunological evidence indicated that fraction II contained minicellulosomes consisting of mini-CbpA1 and EngB . The in vivo synthesis of minicellulosomes suggests that it will be possible in the future to insert into B . subtilis cellulosomal genes that will allow growth on cellulosic materials and the production of various designer cellulosomes with specific functions.

Appl Environ Microbiol, 2004 Sep, 70(9), 5238 - 43
Substrate-induced production and secretion of cellulases by Clostridium acetobutylicum; Lopez-Contreras AM et al.; Clostridium acetobutylicum ATCC 824 is a solventogenic bacterium that grows heterotrophically on a variety of carbohydrates, including glucose, cellobiose, xylose, and lichenan, a linear polymer of beta-1,3- and beta-1,4-linked beta-D-glucose units . C . acetobutylicum does not degrade cellulose, although its genome sequence contains several cellulase-encoding genes and a complete cellulosome cluster of cellulosome genes . In the present study, we demonstrate that a low but significant level of induction of cellulase activity occurs during growth on xylose or lichenan . The celF gene, located in the cellulosome-like gene cluster and coding for a unique cellulase that belongs to glycoside hydrolase family 48, was cloned in Escherichia coli, and antibodies were raised against the overproduced CelF protein . A Western blot analysis suggested a possible catabolite repression by glucose or cellobiose and an up-regulation by lichenan or xylose of the extracellular production of CelF by C . acetobutylicum . Possible reasons for the apparent inability of C . acetobutylicum to degrade cellulose are discussed.

Appl Environ Microbiol, 2004 Sep, 70(9), 5208 - 13
Acidified litter benefits the intestinal flora balance of broiler chickens; Garrido MN et al.; The alterations in the balance of the normal intestinal bacterial flora of chickens exposed to acidified wood-derived litter were analyzed and compared to those of a control group exposed to nonacidified litter . A total of 1,728 broilers were divided into two groups, with six replicates in each . One group was exposed to dry wood-derived litter, and the other was exposed to dry wood-derived litter sprayed with a mixture of sodium lignosulfonate, formic acid, and propionic acid . At five different times, five chickens from each pen were killed and the intestinal contents from ileum and caeca were collected . The samples were diluted and plated onto selective media to identify coliforms, Lactobacillus spp., Clostridium perfringens, and Enterococcus spp . Covariance analysis of bacterial counts showed significantly lower counts for C . perfringens in the caeca and the ileum and for Enterococcus spp . and Lactobacillus spp . in the ileum in chickens exposed to the acidified litter . Lactobacillus spp . showed significantly higher counts in the caeca in chickens exposed to acidified litter . There was no difference between the two litters with regard to coliforms in the ileum and the caeca or to Enterococcus spp . in the caeca . The study shows that exposing the chickens to acidified litter lowers the intestinal bacterial number, especially in the ileum, without negative consequences for the chicken's health or performance . Of special interest are the lower counts of C . perfringens and Enterococcus spp . that might reduce the risk of developing clinical or subclinical necrotic enteritis and growth depression.

Eur J Pediatr Surg, 2004 Aug, 14(4), 265 - 73
Intra-abdominal, retroperitoneal, and visceral abscesses in children; Brook I; This review describes the microbiology, diagnosis, and management of intra-abdominal abscesses (including subphrenic, hepatic, splenic, and retroperitoneal abscesses) in children . They often occur as a complication of local or generalized peritonitis, commonly secondary to appendicitis, necrotizing enterocolitis, pelvic inflammatory disease, and tubo-ovarian infection, surgery or trauma . The original infection generally occurs because of the entry of enteric microorganisms into the peritoneal cavity through a defect in the wall of the intestine or other viscus as a result of obstruction, infarction, or direct trauma . Mixed aerobic and anaerobic flora can be recovered from most abscesses . The predominant aerobic isolates are Escherichia coli, Staphylococcus aureus, and Enterococcus spp . and the main anaerobic bacteria are Bacteroides fragilis group Peptostreptococcus spp., Fusobacterium spp., and Clostridium spp . The treatment of intraabdominal abscesses includes drainage, surgical correction of pathology, and administration of antimicrobials effective against both aerobic and anaerobic microorganisms.

Arch Microbiol, 2004 Oct, 182(2-3), 259 - 63 Epub 2004 Aug 31.
Characterization of a heme oxygenase of Clostridium tetani and its possible role in oxygen tolerance; Bruggemann H et al.; In order to colonize mammalian wounds, the anaerobic bacterium Clostridium tetani must presumably cope with temporary oxic conditions . Therefore, the recently decoded genome sequence was searched for genes which could confer oxygen tolerance . A few identified systems such as superoxide dismutases and peroxidases are probably responsible for this protection against toxic oxygen species . Another system was detected, a heme oxygenase which could have a role in establishing or maintaining an anoxic microenvironment in the process of wound colonization . The hemT gene encoding the heme oxygenase is expressed in C . tetani, as shown by reverse transcription-PCR . When overexpressed in Escherichia coli, the enzyme converts heme to biliverdin under strict oxic conditions.

Naunyn Schmiedebergs Arch Pharmacol, 2004 Sep, 370(3), 211 - 22 Epub 2004 Aug 26.
The small GTPase Rac is involved in clustering of hippocampal neurons and fasciculation of their neurites; Leemhuis J et al.; In hippocampal neurons cultured from brains of newborn rats, the glutamate receptor agonist N-methyl-D-aspartate induced the clustering of neuronal perikarya and the fasciculation of neurites . In addition, N-methyl-D-aspartate activated the small GTPase Rac1 . Other stimuli of Rac activity, such as the Rho kinase inhibitors Y-27632, H-1152, and H89, as well as the cytotoxic necrotizing factor-1 from Escherichia coli, also caused neuronal clustering and neurite bundling . In neurons transiently transfected with dominant negative Rac1N17 neither N-methyl-D-aspartate nor Y-27632 induced clustering and fasciculation . In addition, the PI3-kinase inhibitors wortmannin and LY-294002 prevented these effects, as did a dominant negative form of p110PI3-Kgamma . Time-lapse microscopy showed that lethal toxin from Clostridium sordellii, which inhibits Rac, and wortmannin blocked the neuronal migration induced by Y-27632 . In contrast, only lethal toxin reversed the clustering and fasciculation induced by pre-treatment with Y-27632 . This effect of the toxin may be due to inactivation of Ras, since FTI-277, which prevents the farnesylation of Ras and thereby inactivates the GTPase, also dissolved the preformed clusters . We suggest that active Rac and a PI3-kinase synergistically induce neuronal migration, whereas a Ras isoform is responsible for the lasting attachment of neurons necessary for clustering and neurite fasciculation.

CMAJ, 2004 Aug 31, 171(5), 466 - 72
Clostridium difficile-associated diarrhea in a region of Quebec from 1991 to 2003: a changing pattern of disease severity; Pepin J et al.; BACKGROUND: Recent reports suggest that Clostridium difficile colitis may be evolving into a more severe disease . During the second half of 2002 we noted an increase in the number of patients with severe C . difficile-associated diarrhea (CDAD) in our institution . We describe cases of CDAD at our institution over a 13-year period and investigate changes in illness severity . METHODS: We undertook a retrospective chart review of all cases of CDAD diagnosed at the Centre hospitalier universitaire de Sherbrooke from Jan . 1, 1991, to Dec . 31, 2003 . Because the hospital serves a well-defined population of Quebec, we were also able to calculate population-based incidence during this period . We abstracted data on individual patients from patient charts and from hospital and pharmacy computer databases . We defined cases of CDAD as having a positive C . difficile cytotoxicity assay result, or endoscopic or histopathological evidence of pseudomembranous colitis . A case was considered complicated if one or more of the following was observed: megacolon, perforation, colectomy, shock requiring vasopressor therapy, or death within 30 days after diagnosis . RESULTS: A total of 1721 cases of CDAD were diagnosed during the study period . The incidence increased from 35.6 per 100,000 population in 1991 to 156.3 per 100,000 in 2003; among patients aged 65 years or more, it increased from 102.0 to 866.5 per 100,000 . The proportion of cases that were complicated increased from 7.1% (12/169) in 1991-1992 to 18.2% (71/390) in 2003 (p < 0.001), and the proportion of patients who died within 30 days after diagnosis increased from 4.7% (8/169) in 1991-1992 to 13.8% (54/390) in 2003 (p < 0.001) . A high leukocyte count (20.0 small ha, Cyrillic 10(9)/L or greater) and an elevated creatinine level (200 micromol/L or greater) were strongly associated with adverse outcomes: in 2003, 45 (40.9%) of 110 patients with a high leukocyte count or creatinine level, or both, had complicated CDAD and 28 (25.5%) died within 30 days after diagnosis . After adjustment for age and other confounding factors, patients initially given oral vancomycin therapy had a risk of progression to complicated CDAD that was 79% lower than the risk among patients initially treated with metronidazole (adjusted odds ratio 0.2, 95% confidence interval 0.06-0.8, p = 0.02) . INTERPRETATION: An epidemic of CDAD with an increased case-fatality rate has had important consequences on the elderly population of our region . Our observational data suggest that the equivalence of vancomycin and metronidazole in the treatment of CDAD needs to be questioned.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 249 - 54
A rubrerythrin-like oxidative stress protein of Clostridium acetobutylicum is encoded by a duplicated gene and identical to the heat shock protein Hsp21; May A et al.; Comparison of the N-terminus of the heat shock protein Hsp21 of Clostridium acetobutylicum with proteins predicted to be encoded by the genome of this bacterium revealed that this stress protein is encoded by two almost identical open reading frames CAC3597 and CAC3598 . These genes encode a rubrerythrin-like protein with the rubredoxin-like FeS4 domain at the N-terminus and the ferritin-like diiron domain (rubrerythrin domain) at the C-terminus . Thus, the order of the two putative functional domains is reversed compared to "normal" rubrerythrins . This protein is proposed to be involved in the oxidative stress response of strict anaerobic bacteria . Northern blot analysis indicated that hsp21 is induced by heat and oxidative stress (air, H2O2) . Hsp21 of C . acetobutylicum can be considered as a "reverse" rubrerythrin and a role of this stress protein, which is conserved among clostridia and other strict anaerobic bacteria, in the heat and oxidative stress response is proposed.

FEMS Microbiol Lett, 2004 Sep 1, 238(1), 71 - 8
The N-terminal family 22 carbohydrate-binding module of xylanase 10B of Clostridium themocellum is not a thermostabilizing domain; Dias FM et al.; Xylanase Xyn10B from Clostridium thermocellum is a modular enzyme that contains two family 22 carbohydrate binding modules N- (CBM22-1) and C- (CBM22-2) terminal of the family 10 glycoside hydrolase catalytic domain (GH10) . In a previous study, we showed that removal of CBM22-1 reduces the resistance to thermoinactivation of the enzyme suggesting that this module is a thermostabilizing domain . Here, we show that it is the module border on the N-terminal side of GH10 that confers resistance to thermoinactivation and to proteolysis . Therefore, CBM22-1 does not function as a thermostabilizing domain and the role for this apparently non-functional CBM remains elusive.

J Clin Pathol, 2004 Sep, 57(9), 973 - 9
Colonic IgA producing cells and macrophages are reduced in recurrent and non-recurrent Clostridium difficile associated diarrhoea; Johal SS et al.; BACKGROUND: In Clostridium difficile associated diarrhoea (CDAD), histological changes in the colonic mucosa range from minimal inflammation to pseudomembranous colitis (PMC) . The disease also recurs in a considerable proportion of patients . AIM: To investigate mucosal immune system cells in colonic biopsies of patients with CDAD . METHODS: Colonic biopsies were obtained from 12 control patients with diarrhoea, six patients with CDAD and minimal inflammation, and 10 patients with CDAD with pseudomembranous colitis (samples obtained from areas with and without inflammatory exudate) . Immunohistochemical studies were performed using antibodies to T cells (CD3), macrophages (CD68), B/plasma cells (CD79alpha), and to IgA, IgM, and IgG . Labelled cells in lamina propria were quantified . RESULTS: In contrast to T cells, there were significant reductions in B/plasma cell and macrophage counts in all biopsies from patients with CDAD compared with controls (p<0.001) . Studies using anti-immunoglobulin antibodies showed significant reductions in IgA producing cells in CDAD biopsies (p<0.05), with the greatest reduction in samples from patients with PMC . In contrast, there was a significant increase (p<0.05) in IgG producing cells in CDAD biopsies . Only patients with PMC relapsed . In these patients, B/plasma cell and IgA producing cell counts (in biopsies with and without inflammatory exudates) were significantly lower (p<0.01) in mucosal samples from those who subsequently relapsed (five) than those who did not . CONCLUSIONS: A selective reduction in mucosal IgA producing cells and macrophages is associated with colonic disease in C difficile infected patients . Severe reduction in colonic IgA producing cells may predispose to recurrence of CDAD.

East Mediterr Health J, 2001 Jul-Sep, 7(4-5), 750 - 5
Prevalence of Clostridium difficile-associated diarrhoea among hospitalized Jordanian patients; Shehabi AA et al.; We investigated stool specimens of 400 patients at Jordan University Hospital (300 patients with clinical diarrhoea and 100 controls without diarrhoea) for the presence of Clostridium difficile or its toxin . We found a 9.7% prevalence rate of C . difficile or its toxin in stools of patients with diarrhoea . The prevalence of other potential enteric pathogens, such as Salmonella spp . (2.3%), Shigella spp . (1.0%) and Entamoeba histolytica (2.7%), was significantly less . Prevalence of C . difficile or its toxin in controls was 3.0% . Toxin A was detected in 93.1% of C . difficile-associated diarrhoea cases using an enzyme immunoassay . Our study indicates that C . difficile-associated diarrhoea is mostly observed among hospitalized patients aged > or = 50 years, in association with antimicrobial treatment.

Am J Physiol Gastrointest Liver Physiol, 2004 Sep, 287(3), G715 - 25
Role of small GTP binding proteins in the growth-promoting and antiapoptotic actions of gastrin; Stepan V et al.; G17 has growth promoting and antiapoptotic effects on the AR4-2J pancreatic acinar cell line . We previously reported that whereas MAPK regulates G17-stimulation of AR4-2J cell proliferation, Akt mediates the antiapoptotic action of G17 . We examined the signal-transduction pathways mediating G17 stimulation of AR4-2J cell growth and survival . G17 activated the small GTP binding proteins Ras, Rac, Rho, and Cdc42 . Transduction of the cells with adenoviral vectors expressing dominant negative Akt, Ras, Rho, and Cdc42 but not dominant negative Rac inhibited AR4-2J cell proliferation and survival . Both exoenzyme C3 from Clostridium botulinum (C3), a toxin known to inactivate Rho, and PD98059, a MAPK inhibitor, reversed G17 inhibition of AR4-2J cell apoptosis . G17 induction of Akt activation was reduced by >60% by both dominant negative Ras and Rho and by 30% by dominant negative Cdc42 . In contrast, G17-stimulated MAPK activation was blocked by >80% by dominant negative Ras but not by dominant negative Rho and Cdc42 . Similar results were observed in the presence of C3 . Dominant negative Rac failed to affect G17 induction of both Akt and MAPK, whereas it inhibited sorbitol by almost 50% but not G17-stimulated activation of p38 kinase . Thus G17 promotes AR4-2J cell growth and survival through the activation of multiple GTP binding proteins, which, in turn, regulate different protein kinase cascades . Whereas Ras activates Akt and MAPK, Rho and Cdc42 appear to regulate Akt and possibly other as yet unidentified kinases mediating the growth-stimulatory actions of G17.

J Food Prot, 2004 Aug, 67(8), 1765 - 9
Antibotulinal activity of process cheese ingredients; Glass KA et al.; Ingredients used in the manufacture of reduced-fat process cheese products were screened for their ability to inhibit growth of Clostridium botulinum serotypes A and B in media . Reinforced clostridial medium (RCM) supplemented with 0, 0.5, 1, 2, 3, 5, or 10% (wt/vol) of various ingredients, including a carbohydrate-based fat replacer, an enzyme-modified cheese (EMC) derived from a Blue cheese, sweet whey, modified whey protein, or whey protein concentrate, did not inhibit botulinal growth and toxin production when stored at 30 degrees C for 1 week . In contrast, RCM supplemented with 10% soy-based flavor enhancer, 10% Parmesan EMC, or 5 or 10% Cheddar EMC inhibited botulinal toxin production in media for at least 6 weeks of storage at 30 degrees C . Subsequent trials revealed that the antibotulinal effect varied significantly among 13 lots of EMC and that the antimicrobial effect was not correlated with the pH or water activity of the EMC.

Vet Rec, 2004 Jul 24, 155(4), 111 - 5
Association between Key-Gaskell syndrome and infection by Clostridium botulinum type C/D; Nunn F et al.; There is growing evidence that equine dysautonomia is a toxicoinfection with Clostridium botulinum type C . The possibility that feline dysautonomia has the same aetiology was investigated by attempting to detect botulinum type C neurotoxin in the food, faeces and the contents of the ileum of affected cats, and by serology . The toxin was detected directly in four of eight affected cats and after enrichment in seven of them, and in their dried food . No toxin was detected in healthy control cats or in their tinned food . Recent exposure to the organism was assessed by the detection of immunoglobulin A (IgA) in the faeces of healthy control cats and affected cats . The levels of IgA antibodies to the toxin and to surface antigens of C . botulinum type C in the faeces of the affected cats 14 weeks after the outbreak were significantly higher than in the faeces of the control cats.

Proteins, 2004 Oct 1, 57(1), 118 - 27
Absence of kinetic thermal stabilization in a hyperthermophile rubredoxin indicated by 40 microsecond folding in the presence of irreversible denaturation; LeMaster DM et al.; The striking kinetic stability of many proteins derived from hyperthermophilic organisms has led to the proposal that such stability may result from a heightened activation barrier for unfolding independent of a corresponding increase in the thermodynamic stability . This in turn implies a corresponding retardation of the folding reaction . A commonly cited model for kinetic thermal stabilization is the rubredoxin from Pyrococcus furiosus (Pf), which exhibits an irreversible denaturation lifetime at 100 degrees C of nearly a week . Utilizing protein resonances shifted well outside of the random coil chemical shift envelope, nuclear magnetic resonance (NMR) chemical exchange measurements on Pf rubredoxin as well as on the mesophile Clostridium pasteurianum (Cp) rubredoxin demonstrate reversible thermal transition temperatures of 144 degrees C (137 degrees C for the N-terminal modified A2K variant) and 104 degrees C, respectively, with similar (un)folding rates of approximately 25,000 s(-1), only modestly slower than the diffusion controlled rate . The absence of a substantial activation barrier to rubredoxin folding as well as the similar folding kinetics of the mesophile protein indicate that kinetic stabilization has not been utilized by the hyperthermophile rubredoxin in achieving its extreme thermal stability . The two-state folding kinetics observed for Pf rubredoxin contradict a previous assertion of multiphasic folding based on hydrogen exchange data extrapolated to an estimated midpoint of transition temperature (T(m)) of nearly 200 degrees C . This discrepancy is resolved by the observation that the base-catalyzed hydrogen exchange of the model dipeptide (N-acetyl-L-cysteine-N-methylamide)4-Cd2+ is 23-fold slower than that of the free cysteine model dipeptide used to normalize the Pf rubredoxin hydrogen exchange data .

Anesthesiol Clin North America, 2004 Sep, 22(3), 509 - 32, vii
Medical aspects of biologic toxins; Marks JD; Biologic toxins are molecules produced by living organisms that are poisonous to other species, such as humans . Some biologic toxins are so potent and relatively easy to produce that they have been classified as biothreat agents . These include the botulinum neurotoxins, ricin, staphylococcal enterotoxin B, and Clostridium perfringens epsilon toxin . This article focuses on these four biothreat toxins and their medical aspects . The majority of the article is spent on the botulinum neurotoxins, because these are the most poisonous substances known and are the only toxins classified as Category A threat agents-the highest level of threat agent . The remainder of the article is devoted to sections on the other three biothreat toxins: ricin, staphylococcal enterotoxin B, and C perfringens epsilon toxin.

FEMS Immunol Med Microbiol, 2004 Sep 1, 42(1), 76 - 84
Cot mattresses as reservoirs of potentially harmful bacteria and the sudden infant death syndrome; Sherburn RE et al.; Cot mattress materials were investigated as potential reservoirs of bacteria in relation to the sudden infant death syndrome (SIDS) . The sleeping position of the infant significantly influenced bacterial population density of cot mattress polyurethane foams (p<0.0000001) and their covers (p<0.004) . Staphylococcus aureus was isolated at significantly higher frequency (p<0.03) from the infant's head region of cot mattress materials . Significantly higher bacterial population densities (p<0.001) were associated with polyurethane foams from non-integral mattresses (exposed polyurethane foam), when compared to those from mattresses completely covered by polyvinyl chloride (integral type mattress) . The frequency of isolation of S . aureus from polyurethane foams from non-integral mattresses was also significantly higher (p=0.03) than from foams from the integral type . The following factors were significantly associated with increased frequency of isolation of S . aureus: from the polyurethane foam, previous use of non-integral mattresses by another child (p=0.03 for all sample sites, p=0.01 for torso region); from the covers, sleeping in the prone position (p=0.003 head region, p=0.001 torso region) . Prone sleeping was also significantly associated with increased bacterial population levels (p=0.01) and increased frequency of isolation of Escherichia coli (p=0.02) from the torso region of cot mattress covers . These findings could explain some recently identified risk factors for SIDS associated with type and previous use of cot mattresses . Clostridium perfringens was isolated at very low frequency and Streptococcus pyogenes was not isolated from any cot mattress materials tested.

Infect Immun, 2004 Sep, 72(9), 5204 - 15
Effects of Clostridium perfringens alpha-toxin (PLC) and perfringolysin O (PFO) on cytotoxicity to macrophages, on escape from the phagosomes of macrophages, and on persistence of C . perfringens in host tissues; O'Brien DK et al.; Clostridium perfringens is the most common cause of clostridial myonecrosis (gas gangrene) . Polymorphonuclear cells (PMNs) appear to play only a minor role in preventing the onset of myonecrosis in a mouse animal model of the disease (unpublished results) . However, the importance of macrophages in the host defense against C . perfringens infections is still unknown . Two membrane-active toxins produced by the anaerobic C . perfringens, alpha-toxin (PLC) and perfringolysin O (PFO), are thought to be important in the pathogenesis of gas gangrene and the lack of phagocytic cells at the site of infection . Therefore, C . perfringens mutants lacking PFO and PLC were examined for their relative cytotoxic effects on macrophages, their ability to escape the phagosome of macrophages, and their persistence in mouse tissues . C . perfringens survival in the presence of mouse peritoneal macrophages was dependent on both PFO and PLC . PFO was shown to be the primary mediator of C . perfringens-dependent cytotoxicity to macrophages . Escape of C . perfringens cells from phagosomes of macrophage-like J774-33 cells and mouse peritoneal macrophages was mediated by either PFO or PLC, although PFO seemed to play a more important role in escape from the phagosome in peritoneal macrophages . At lethal doses (10(9)) of bacteria only PLC was necessary for the onset of myonecrosis, while at sublethal doses (10(6)) both PFO and PLC were necessary for survival of C . perfringens in mouse muscle tissue . These results suggest PFO-mediated cytotoxicity toward macrophages and the ability to escape macrophage phagosomes may be important factors in the ability of C . perfringens to survive in host tissues when bacterial numbers are low relative to those of phagocytic cells, e.g., early in an infection.

Nurs Times, 2004 Jun 29-Jul 6, 100(26), 56 - 7, 59
The prevention of Clostridium difficile associated diarrhoea in hospital; Jenkins L; The number of health care-associated infections has increased over the years and generated a lot of interest and concern . The attention tends to be focused on methicillin-resistant Staphylococcus aureus (MRSA), but the less publicised Clostridium difficile is a growing problem . It increases length of hospital stay, causes significant morbidity in patients, affects nurses' workloads, adds to the cost of cleaning, laundry and disposables, and can lead to ward closures . All NHS trusts in England are now required to report C . difficile-associated diarrhoea (CDAD) in all patients over 65 years of age (National Clostridium Difficile Standards Group, 2003).

Water Sci Technol, 2004, 50(1), 141 - 6
Inactivation of enteric microbes in water by electro-chemical oxidant from brine (NaCl) and free chlorine; Venczel LV et al.; Oxidant solutions of mostly free chlorine can be electrochemically produced on-site from brine (NaCl) solution and used to disinfect water at the household or community level . In this study electrochemical oxidant (ECO) from brine and free chlorine were evaluated under laboratory conditions for inactivation of test microbes . Purified suspensions of Escherichia coli, the rugose strain of Vibrio cholerae, Clostridium perfringens spores, MS2 coliphage and Cryptosporidium parvum oocysts were treated with 2 mg/L or 5 mg/L solutions of ECO or free chlorine at 5 degrees C and 25 degrees C and pH 6, 8, and 10 (pH 7 and 25 degrees C only for C . parvum oocysts) for contact times <60 min . Under nearly all conditions, inactivation kinetics were more rapid for E . coli, V . cholerae, C . perfringens spores and MS2 coliphage with ECO than with free chlorine . ECO reduced E . coli, V . cholerae and MS2 by >4 log10 within 30 min and C . perfringens spores by >2 log10 within 10 min at pH 8 and 25 degrees C . Contrary to previous results, however, C . parvum oocysts were not inactivated by ECO, and the reasons for this difference are uncertain . The on-site electrolytic generation of oxidants from brine provided a convenient and inexpensive disinfectant containing free chlorine that was effective against many enteric microbes, for the treatment of household and community drinking-water supplies worldwide . However, the effectiveness of such oxidants for inactivating C . parvum oocysts was variable and sometimes ineffective.

Water Sci Technol, 2004, 50(1), 7 - 12
Bioaerosols from the land application of biosolids in the desert southwest USA; Brooks JP et al.; This study evaluated bioaerosol emissions during land application of Class B biosolids in and around Tucson, Arizona, to aid in developing models of the fate and transport of bioaerosols generated from the land application of biosolids . Samples were collected for 20 min at distances between 2 m and 20 m downwind of point sources, using an SKC BioSampler impinger . A total of six samples were collected per sampling event, which consisted of a biosolid spray applicator applying liquid biosolids to a cotton field . Each application represented one exposure . Samples were collected in deionised water amended with peptone and antifoam agent . Ambient weather conditions were also monitored every 10 min following initiation of sampling . Concurrently with downwind samples, background (ambient) air samples were collected to compensate for any ambient airborne microorganisms . In addition, biosolids samples were collected for analysis of target indicator and pathogenic organisms . Soil samples were also collected and analysed . Significant numbers of heterotrophic plate count (HPC) bacteria were found in air samples collected during the biosolid application process . These could have arisen from soil particles being aerosolised during the land application process . Aerosolised soil may contribute significantly to the amount of aerosolised microorganisms . Soil particles may be able to more readily aerosolise, due to their low density, small particle size and low mass . Aerosolised HPC bacteria found during biosolids land application were similar to those found during normal tractor operation on non-biosolids applied fields . Coliforms and coliphages were not routinely detected even though they were found to be present in the biosolids at relatively high concentrations, 10(6) and 10(4)/g (dry weight) of biosolids respectively . This could be due to the die-off rate of aerosolised Gram-negative bacteria or sorption to the solid portion of the biosolids . Low numbers of aerosolised coliphages may likewise be due to sorption phenomena . We theorise that only organisms in the aqueous phase of the biosolids were available to desorb and be aerosolised . Animal viruses, which were not detected in the biosolids, were likewise not detected in the aerosol samples . Clostridium perfringens was detected in only a small percent of aerosol samples although it was detected during all weather conditions; other microorganisms were detected during more favourable environmental conditions (relative humidity >10%) . Despite the fact that many of these organisms were present in the biosolids at significant concentrations, their presence in bioaerosols generated during the land application of biosolids was limited to only a small percentage of samples . Bacteria as well as viruses may sorb to biosolids, which contain a high percentage of organic matter, and desorption during land application of biosolids may not readily take place; therefore, these microorganisms may not be readily aerosolised.

Ann Pharm Fr, 2004 Sep, 62(5), 304 - 9
{Clostridium difficile, nosocomial enteropathogen: phylogeny and virulence}; Pons JL; Clostridium difficile is recognized as a potentially nosocomial enteric pathogen . It induces diarrhea or pseudomembranous colitis in patients whose digestive flora has been altered by antibiotic treatment and thus allows the colonization with a strain producing toxin A (enterotoxin) and toxin B (cytotoxin) (A+B+, sometimes A-B+ strains) . We studied the phylogeny of C . difficile by developing MultiLocus Sequence Typing (MLST) analysis, which reports allelic polymorphism of housekeeping genes through DNA sequencing . C . difficile exhibits genomic stability, with mutational clonal evolution and individualization of phylogenetic lineages . These lineages are not correlated with human or animal hosts . Strains involved in pseudomembranous colitis or in diarrhea do not define distinct lineages, and bi-toxinogenic strains do not segregate from non toxinogenic strains . Conversely, A-B+ strains define a unique clone, highly divergent from the population studied . Allelic sequence data may be available from an centralized internet site, allowing phylogenetic and macro-epidemiologic analyses.

J Med Microbiol, 2004 Sep, 53(Pt 9), 887 - 94
Distribution of Clostridium difficile variant toxinotypes and strains with binary toxin genes among clinical isolates in an American hospital; Geric B et al.; Genetic variants of Clostridium difficile have been reported with increasing frequency, but their true incidence is unknown . C . difficile strains have been classified into variant toxinotypes according to variations in the pathogenicity locus encoding the major virulence factors, toxins A and B . Some strains produce an additional toxin, binary toxin CDT . This survey of clinical isolates (153) from patients in a single hospital set out to ascertain the distribution of variant toxinotypes and strains possessing binary toxin genes . A PCR-RFLP-based method of toxinotyping identified 123 (80.4 %) isolates as toxinotype 0, 13 (8.5 %) strains as non-toxigenic and 17 (11.1 %) as belonging to variant toxinotypes . Binary toxin genes were amplified by PCR in nine strains (5.8 %), all of which were variant toxinotypes . Toxin variants of C . difficile are pathogenic and commonly isolated and need to be considered when evaluating new diagnostic testing strategies for C . difficile disease.

World J Gastroenterol, 2004 Sep 15, 10(18), 2756 - 8
Simplified purification method for Clostridium difficile toxin A; Fu SW et al.; AIM: To establish the purification method for Clostridium difficile (C . difficile) toxin A . METHODS: C . difficile VPI 10463 filtrate was cultured anaerobically by the dialysis bag methods . And then the toxin A was purified by precipitation with 500 g/L (NH4)2SO4 and acid precipitation at pH 5.5, followed by ion-exchange chromatography on DEAE-Toyopearl . RESULTS: Purified toxin A exhibited only one band on native polyacrylamide gel electrophoresis (native-PAGE) and Ouchterlony double immunodiffusion . The molecular weight of toxin A was estimated to be 550,000 . The purified toxin A had a protein concentration of 0.881 mg/mL . The minimum lethal dose was 1X10(6) MLD/mL (i.p.mice) . The cytotoxic titer was 10(7) CU/mg . The haemagglutinate activity was at a concentration of 1.72 microg/mL . The ratio of fluid volume (mL) accumulated to the length (cm) of the loop was 2.46 . CONCLUSION: The modified method for purification of toxin A of C . difficile was simple and convenient . It may be even more suitable for purification of toxin A on large scales.

World J Gastroenterol, 2004 Sep 15, 10(18), 2747 - 9
Development of an ELISA kit using monoclonal antibody to Clostridium difficile toxin A; Fu SW et al.; AIM: To establish an ELISA kit using monoclonal antibodies against Clostridium difficile (C . difficile) toxin A . METHODS: An indirect sandwich ELISA was described using the purified rabbit monospecific antiserum as capturing antibody . After the polystyrene microtitre plates with 96 flat-bottomed wells were coated with rabbit antiserum, the wells were blocked with 100 g/L BSA in PBS-T . C . difficile toxin A or culture filtrates were added to each well and then monoclonal antibodies IgG-horseradish peroxidase conjugate was added as detecting antibody, tetramethylbenzidine was used as substrate and A450 of the stopped reacting product was recorded in an automated plate reader . RESULTS: The tested specimens included culture filtrates of 2 strains of toxigenic C . difficile, 2 strains of non-toxigenic C . difficile, 26 strains of E . coli, 2 strains of S . dysenteriae, 1 strain of Bif . infantis, 5 strains of V . cholera, 2 strains of S . typhi, 7 strains of C . botulinum, 1 strain of toxigenic C . sordllii, and 1 strain of C . butyricum . A total of 47 strains of culture filtrates were all negative except for 2 strains of toxigenic C . difficile . The detective limitation of toxin A was 0.1 ng/mL . CONCLUSION: An ELISA kit with high specificity and excellent sensitivity for the rapid detection of C . difficile toxin A was established . It will be a useful tool for diagnostic test of C . difficile toxin A.

Cytogenet Genome Res, 2004, 107(1-2), 38 - 48
Genome architecture studied by nanoscale imaging: analyses among bacterial phyla and their implication to eukaryotic genome folding; Takeyasu K et al.; The proper function of the genome largely depends on the higher order architecture of the chromosome . Our previous application of nanotechnology to the questions regarding the structural basis for such macromolecular dynamics has shown that the higher order architecture of the Escherichia coli genome (nucleoid) is achieved via several steps of DNA folding (Kim et al., 2004) . In this study, the hierarchy of genome organization was compared among E . coli, Staphylococcus aureus and Clostridium perfringens . A one-molecule-imaging technique, atomic force microscopy (AFM), was applied to the E . coli cells on a cover glass that were successively treated with a detergent, and demonstrated that the nucleoids consist of a fundamental fibrous structure with a diameter of 80 nm that was further dissected into a 40-nm fiber . An application of this on-substrate procedure to the S . aureus and the C . perfringens nucleoids revealed that they also possessed the 40- and 80-nm fibers that were sustainable in the mild detergent solution . The E . coli nucleoid dynamically changed its structure during cell growth; the 80-nm fibers releasable from the cell could be transformed into a tightly packed state depending upon the expression of Dps . However, the S . aureus and the C . perfringens nucleoids never underwent such tight compaction when they reached stationary phase . Bioinformatic analysis suggested that this was possibly due to the lack of a nucleoid protein, Dps, in both species . AFM analysis revealed that both the mitotic chromosome and the interphase chromatin of human cells were also composed of 80-nm fibers . Taking all together, we propose a structural model of the bacterial nucleoid in which a fundamental mechanism of chromosome packing is common in both prokaryotes and eukaryotes .

Appl Biochem Biotechnol, 2004 Jun, 117(3), 133 - 41
Production of ethanol from cellulosic biomass by Clostridium thermocellum SS19 in submerged fermentation: screening of nutrients using Plackett-Burman design; Balusu R et al.; Plackett-Burman design, a statistical methodology, was used to screen 23 nutrients belonging to three categories--carbon, nitrogen, and salt/mineral sources--for the production of ethanol from cellulosic biomass by Clostridium thermocellum SS19 in anaerobic submerged fermentation . In this design, just n number of experiments is required for screening n-1 variables . The experimental data were subjected to statistical analysis for calculating the regression coefficients and t-values . Filter paper, Solka Floc, corn steep liquor (CSL), cysteine HCl, magnesium chloride, and ferrous sulfate showed relatively higher regression coefficients on ethanol production and growth . Among the 23 nutrients screened, based on their performance in terms of product-promoting ability, availability, and cost, filter paper, CSL, cysteine HCl, magnesium chloride, and ferrous sulfate were identified as the most effective and, therefore, selected for inclusion in further optimization studies . Copryright 2004 Humana Press Inc.

FEBS Lett, 2004 Aug 13, 572(1-3), 195 - 200
Cohesin-dockerin interaction in cellulosome assembly: a single Asp-to-Asn mutation disrupts high-affinity cohesin-dockerin binding; Handelsman T et al.; The cohesive cellulosome complex is sustained by the high-affinity cohesin-dockerin interaction . In previous work, we demonstrated that a single Thr-to-Leu replacement in the Clostridium thermocellum dockerin component differentiates between non-recognition and high-affinity recognition by the interspecies rival cohesin from C . cellulolyticum . In this report, we show that a single Asp-to-Asn substitution on the cohesin counterpart also disrupts normal recognition of the dockerin . The Asp34 carboxyl group of the cohesin appears to play a central role in the resultant hydrogen-bonding network as an acceptor of two crucial hydrogen bonds from Ser45 of the dockerin domain . The results underscore the fragile nature of the intermolecular contact interactions that maintain this very high-affinity protein--protein interaction.

J Biol Chem, 2004 Oct 22, 279(43), 45266 - 78 Epub 2004 Aug 09.
Hyperosmotic stress induces rapid focal adhesion kinase phosphorylation at tyrosines 397 and 577 . Role of Src family kinases and Rho family GTPases; Lunn JA et al.; Hyperosmotic stress induced by treatment of Swiss 3T3 cells with the non-permeant solutes sucrose or sorbitol, rapidly and robustly stimulated endogenous focal adhesion kinase (FAK) phosphorylation at Tyr-397, the major autophosphorylation site, and at Tyr-577, within the kinase activation loop . Hyperosmotic stress-stimulated FAK phosphorylation at Tyr-397 occurred via a Src-independent pathway, whereas Tyr-577 phosphorylation was completely blocked by exposure to the Src family kinase inhibitor PP-2 . Inhibition of p38 MAP kinase or phosphatidylinositol 3-kinases did not prevent FAK phosphorylation stimulated by hyperosmotic stress . Overexpression of N17 RhoA did not reduce hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts and treatment with the Rho-associated kinase inhibitor Y-27632 did not prevent FAK translocation and tyrosine phosphorylation in response to hyperosmotic stress . Overexpression of N17 Rac only slightly altered the hyperosmotic stress-mediated localization of phosphorylated FAK to focal contacts . In contrast, overexpression of the N17 mutant of Cdc42 disrupted hyperosmotic stress-stimulated FAK Tyr-397 localization to focal contacts . Additionally, treatment of cells with Clostridium difficile toxin B potently inhibited hyperosmotic stress-induced FAK tyrosine phosphorylation . Furthermore, FAK null fibroblasts compared with their FAK containing controls show markedly increased sensitivity, manifest by subsequent apoptosis, to sustained hyperosmotic stress . Our results indicate that FAK plays a fundamental role in protecting cells from hyperosmotic stress, and that the pathway(s) that mediates FAK autophosphorylation at Tyr-397 in response to osmotic stress can be distinguished from the pathways utilized by many other stimuli, including neuropeptides and bioactive lipids (Rho- and Rho-associated kinase-dependent), tyrosine kinase receptor agonists (phosphatidylinositol 3-kinase-dependent), and integrins (Src-dependent).

Anal Biochem, 2004 Sep 1, 332(1), 32 - 7
Measurement of bile acid in serum and bile with arylamine-glass-bound 3alpha-hydroxysteroid dehydrogenase and diaphorase; Rani K et al.; 3Alpha-hydroxysteroid dehydrogenase (3-HSD) from Pseudomonas testosteroni and diaphorase (lipoyl dehydrogenase) from Clostridium spp . have been immobilized individually onto arylamine glass beads through diazotization . A cost-effective enzymic colorimetric method for determination of bile acid in serum and bile employing a mixture of these immobilized enzymes was developed . The method is based on measurement of reduced nicotinamide adenine dinucleotide generated from bile acid in serum/bile by immobilized 3alpha-HSD with a color reagent consisting of nitro blue tetrazolium chloride salt, oxidized nicotinamide adenine dinucleotide, and immobilized lipoyl dehydrogenase in 0.065 M sodium phosphate buffer, pH 7.0 . Analytical recovery of added bile acid (50 and 200 micromol/L) was 95.57 and 85.46% in serum and 97.6 and 91.6% in bile, respectively . Within- and between-batch coefficients of variation (CV) for bile acid determination were <1.2 and <0.2% in serum and >0.1 and <0.1% in bile, respectively . Good correlations for bile acid in serum (r1=0.92) and in bile (r2=0.97) were obtained by use of a standard chemical method and the present method . The mixture of immobilized 3alpha-HSD dehydrogenase and lipoyl dehydrogenase lost 50% of its initial activity after 6 months of regular use . The cost of bile acid determination in 100 serum and bile samples by the present method has been compared with that of the Sigma kit method.

Acta Crystallogr D Biol Crystallogr, 1997 Jan, 53(Pt 1), 114 - 5
Crystallization and preliminary X-ray analysis of a cohesin domain of the cellulosome from Clostridium thermocellum; Shimon LJ; Recombinant cohesin-2, a unique type of protein-recognition domain from the cellulosome of Clostridium thermocellum, has been crystallized by the hanging-drop vapor-diffusion method . The crystals are monoclinic, space group C2 with unit-cell dimensions a = 79.91, b = 47.86, c = 51.13 A, beta = 126.77 degrees . There is most likely to be one molecule per asymmetric unit, corresponding to a packing density of 2.16 A(3) Da(-1) . The crystals diffract to beyond 2.3 A on a conventional laboratory rotating-anode source.

Acta Crystallogr D Biol Crystallogr, 1995 Nov, 51(Pt 6), 1092 - 3
Preliminary X-ray crystallographic study of methyltetrahydrofolate: corrinoid/iron sulfur protein methyltransferase from Clostridium thermoaceticum; Doukov TI; Methyltetrahydrofolate:corrinoid/iron sulfur protein methyltransferase from Clostridium thermoaceticum has been crystallized in two polymorphic forms and characterized by X-ray diffraction measurements . Form I displayed orthorhombic symmetry with a = 63.9, b = 53.8, c = 164.0 A . Form II also displayed orthorhombic symmetry with a = 63.5, b = 87.1, c = 117.9 A . Crystals of form I diffract to approximately . 3 A resolution; those of form II diffract to approximately 2.7 A.

Acta Crystallogr D Biol Crystallogr, 1994 Nov, 50(Pt 6), 913 - 4
Crystallization and preliminary X-ray diffraction study of the flavoenzyme 2,4-pentadienoyl-CoA reductase from Clostridium aminovalericum; Eikmanns U; The tetrameric flavoenzyme 2,4-pentadienoyl-CoA reductase has been crystallized from solutions containing polyethylene glycol as precipitant . The crystals grow in the monoclinic space group C2 with unit-cell dimensions a = 160.2, b = 120.2, c = 95.3 A, beta = 99.0 degrees . The packing parameter V(M) is 2.3 A(3) Da(-1) (Matthews parameter) for four monomers per asymmetric unit . Complete data sets to about 2.9 A resolution have been collected.

Acta Crystallogr D Biol Crystallogr, 1995 Mar, 51(Pt 2), 155 - 9
Structure of the ferredoxin from Clostridium acidurici: model at 1.8 A resolution; TranQui D; Ferredoxins (Fd) are electron-carrier proteins, the active sites of which are organized around clusters made of iron and inorganic sulfur . The Fd from Clostridium acidurici is 55 amino acids long and contains two {4Fe-4S} clusters . Crystals have been obtained in the space group P4(3)2(1)2, a = b = 34.441 (5), c = 74.778 (9) A . The structure was solved by molecular replacement using the Fd from Peptostreptcoccus asaccharolyticus as a search model, these two ferredoxins having 37 residues in common . Refinement using molecular-dynamics techniques was then initiated . Successive rounds of model building and refinement gave a structure that includes 45 water molecules with R = 15% . At this stage, the electron-density map clearly revealed discrepancies in the position of two amino acids in the published primary sequence . Refinement based on these modifications led to R = 14.3% for 3921 reflections up to 1.8 A, resolution . The geometry of the two clusters has been found to be in good agreement with that previously obtained at a lower resolution . Interactions of polypeptide chain with the {4Fe-4S} clusters, the cluster geometry as well as the hydrogen bonds involving S, Sgamma, N and water molecules are reported.

Curr Microbiol, 2004 Aug, 49(2), 99 - 107
Identification and functional analysis of dTDP-glucose-4,6-dehydratase gene and its linked gene cluster in an aminoglycoside antibiotics producer of Streptomyces tenebrarius H6; Du Y et al.; Streptomyces tenebrarius H6 produces a variety of aminoglycoside antibiotics, such as apramycin, tobramycin, and kanamycin B . Primers were designed according to the highly conserved sequences of the dTDP-glucose-4,6-dehydratase genes, and a 0.6-kb PCR product was obtained from S . tenebrarius H6 genomic DNA . With the 0.6-kb PCR product as a probe, a BamHI 7.0-kb fragment was isolated . DNA sequence analysis of the 7.0-kb fragment revealed four ORFs and an incomplete ORF . In search of databases, the deduced product of one ORF (orfE) showed 62% identity to the dTDP-glucose-4,6-dehydratase, StrE of S . griseus . Three other ORFs (orfG1, orfG2, and orfGM) showed 55%, 62%, and 42% similarities, respectively, to glycosyltransferase from Clostridium acetobutylicum and mannosyltransferase from Xanthomonas axonopodis pv . citri str . 306 and glycosyltransferase from Pseudomonas putida KT2440 . Upstream of the orfE was an incomplete ORF, and the deduced product showed 56% similarity to dTDP-4-dehydrorhamnose, StrL from S . griseus . The function of the orfE gene was studied by targeted gene disruption . The resulting mutant failed to produce tobramycin and kanamycin B, but still produced apramycin, suggesting that the orfE gene and linked gene cluster are essential for the biosynthesis of tobramycin and kanamycin B in S . tenebrarius H6.

J Clin Microbiol, 2004 Aug, 42(8), 3635 - 43
Molecular epidemiology of hospital-associated and community-acquired Clostridium difficile infection in a Swedish county; Noren T et al.; All episodes of Clostridium difficile associated diarrhea (CDAD) diagnosed in a defined population of 274,000 including one tertiary and two primary hospitals and their catchment areas were studied during 12 months . The annual CDAD incidence in the county was 97 primary episodes per 100,000, and 78% of all episodes were classified as hospital associated with a mean incidence of 5.3 (range, 1.4 to 6.5) primary episodes per 1,000 admissions . The incidence among hospitalized individuals was 1,300-fold higher than that in the community (33,700 versus 25 primary episodes per 100,000 persons per year), reflecting a 37-fold difference in antibiotic consumption (477 versus 13 defined daily doses {DDD}/1,000 persons/day) and other risk factors . Three tertiary hospital wards with the highest incidence (13 to 36 per 1,000) had CDAD patients of high age (median age of 80 years versus 70 years for other wards, P < 0.001), long hospital stay (up to 25 days versus 4 days), or a high antibiotic consumption rate (up to 2,427 versus 421 DDD/1,000 bed days) . PCR ribotyping of C . difficile isolates available from 330 of 372 CDAD episodes indicated nosocomial acquisition of the strain in 17 to 27% of hospital-associated cases, depending on the time interval between index and secondary cases allowed (2 months or up to 12 months), and only 10% of recurrences were due to a new strain of C . difficile (apparent reinfection) . In other words, most primary and recurring episodes were apparently caused by the patient's endogenous strain rather than by one of hospital origin . Typing also indicated that a majority of C . difficile strains belonged to international serotypes, and the distribution of types was similar within and outside hospitals and in primary and relapsing CDAD . However, type SE17 was an exception, comprising 22% of hospital isolates compared to 6% of community isolates (P = 0.008) and causing many minor clusters and a silent nosocomial outbreak including 36 to 44% of the CDAD episodes in the three high-incidence wards.

Bioelectrochemistry, 2004 Sep, 64(2), 157 - 63
A comparison between the use of a redox mediator in solution and of surface modified electrodes in the electrocatalytic oxidation of nicotinamide adenine dinucleotide; Antiochia R et al.; Cyclic voltammetry was successfully applied to study the oxidation of nicotinamide adenine dinucleotide (NADH) both in homogeneous and heterogeneous phase . The first case was realized with a solution containing p-methylamino-phenolsulphate (MAP) as redox mediator and the diaphorase (DI) from Clostridium kluveri as enzyme while the second one by using both a glassy carbon (GC) and a carbon nanotube paste (CNTP) electrode modified with electrodeposited films derived from 3,4-dihydroxybenzaldehyde (3,4-DHB) . Such systems were successively coupled with glucose dehydrogenase (GDH) reaction to realize the redox chain present in glucose biosensors . A critical comparison of the two systems was also reported.

Am J Physiol Cell Physiol, 2004 Dec, 287(6), C1657 - 66 Epub 2004 Aug 04.
Lysophospholipids increase ICAM-1 expression in HUVEC through a Gi- and NF-kappaB-dependent mechanism; Lee H et al.; Lysophosphatidic acid (LPA) and sphingosine 1-phosphate (S-1-P) are both low molecular weight lysophospholipid (LPL) ligands that are recognized by the Edg family of G protein-coupled receptors . In endothelial cells, these two ligands activate Edg receptors, resulting in cell proliferation and cell migration . The intercellular adhesion molecule-1 (ICAM-1, CD54) is one of many cell adhesion molecules belonging to the immunoglobulin superfamily . This study showed that LPA and S-1-P enhance ICAM-1 expression at both the mRNA and protein levels in human umbilical cord vein endothelial cells (HUVECs) . This enhanced ICAM-1 expression in HUVECs was first observed at 2 h postligand treatment . Maximal expression appeared at 8 h postligand treatment, as detected by flow cytometry and Western blotting . Furthermore, the effects of S-1-P on ICAM-1 expression were shown to be concentration dependent . Prior treatment of HUVECs with pertussis toxin, a specific inhibitor of G(i), ammonium pyrrolidinedithiocarbamate and BAY 11-7082, inhibitors of the nuclear factor (NF)-kappaB pathway, or Clostridium difficile toxin B, an inhibitor of Rac, prevented the enhanced effect of LPL-induced ICAM-1 expression . However, pretreatment of HUVECs with exoC3, an inhibitor of Rho, had no effect on S-1-P-enhanced ICAM-1 expression . In a static cell-cell adhesion assay system, pretreatment of LPL enhanced the adhesion between HUVECs and U-937 cells, a human mononucleated cell line . The enhanced adhesion effect could be prevented by preincubation with a functional blocking antibody against human ICAM-1 . These results suggest that LPLs released by activated platelets might enhance interactions of leukocytes with the endothelium through a G(i)-, NF-kappaB-, and possibly Rac-dependent mechanism, thus facilitating wound healing and inflammation processes.

Protein Expr Purif, 2004 Sep, 37(1), 187 - 95
The C-terminus of botulinum neurotoxin type A light chain contributes to solubility, catalysis, and stability; Baldwin MR et al.; Botulinum neurotoxin type A (BoNT/A) is the etiological agent responsible for botulism, a disease characterized by peripheral neuromuscular blockade . BoNT/A is produced by Clostridium botulinum as a single chain protein that is activated by proteolytic cleavage to form a 50 kDa light chain (LC, 448 amino acids) and a disulfide bond-linked 100 kDa heavy chain (HC, 847 amino acids) . Whilst HC comprises the receptor binding and translocation domains, LC is a Zn2+-endopeptidase that cleaves at a single glutaminyl-arginine bond corresponding to residues 197 and 198 at the C-terminus of SNAP25 . Cleavage of SNAP25 uncouples the neural exocytosis docking/fusion machinery . LC/A (LC 1-448) and several C-terminal deletion proteins of LC/A were engineered and expressed as His-tagged fusion proteins in Escherichia coli . LC 1-448 was purified, but precipitated upon storage . Approximately 40% of LC 1-448 was a covalent dimer due to the formation of inter-chain disulfide bond formation at Cys430 . Conversion of Cys430 to Ser abolished dimer formation of LC 1-448, but did not improve solubility . Three C-terminal deletion peptides were engineered; LC 1-425 and LC 1-418 were expressed and could be purified as soluble and stable proteins, whilst LC 1-398 was soluble, but not stable to storage . Kinetic studies showed that LC 1-448 and LC 1-425 efficiently cleaved GST-SNAP25 and the fluorescent substrate SNAPtide, while LC 1-418 catalyzed the cleavage of both the SNAP25 and the fluorescent substrate SNAPtide with a similar Km, but at a 10-fold slower kcat . Thus, regions within the C-terminus of LC/A contribute to solubility, stability, and catalysis.

Mikrobiyol Bul, 2004 Jan-Apr, 38(1-2), 45 - 50
{Six years evaluation of Clostridium difficile associated diarrhea}; Ercis S et al.; This study was aimed to detect the presence of Clostridium difficile toxin in the stool samples of patients with antibiotic-associated diarrhea or pseudomembranous colitis, and to relate its presence with the clinical findings of the patients . Between January 1997-April 2003, a total of 726 stool samples were investigated for C . difficile toxin A and/or B by enzyme immunoassay . Of them, 68 (9.4%) were found positive for C . difficile toxin (62 were toxin A, 6 were toxin B) . C . difficile associated diarrhea were found to be related mostly with the use of beta-lactam/beta-lactamase inhibitor combinations (32/68), followed by aminoglycosides (12/68), and cephalosporins (8/68) . The ages of the patients were between 1-86 years old (mean: 43.3 years), and 36 (52.9%) of them had an underlying conditions . Chronic obstructive pulmonary disease and chronic renal failure were the underlying disease in 18, malignancy in 11, and others (diabetes, hepatitis, transplantation, multiple sclerosis) in 7 of the patients . In conclusion, toxin detection and knowledge of the risk factors are the beneficial guidelines for the diagnosis of C . difficile associated diarrhea in the routine setting.

J Bacteriol, 2004 Aug, 186(16), 5221 - 9
The CcpA protein is necessary for efficient sporulation and enterotoxin gene (cpe) regulation in Clostridium perfringens; Varga J et al.; Clostridium perfringens is the cause of several human diseases, including gas gangrene (clostridial myonecrosis), enteritis necroticans, antibiotic-associated diarrhea, and acute food poisoning . The symptoms of antibiotic-associated diarrhea and acute food poisoning are due to sporulation-dependent production of C . perfringens enterotoxin encoded by the cpe gene . Glucose is a catabolite repressor of sporulation by C . perfringens . In order to identify the mechanism of catabolite repression by glucose, a mutation was introduced into the ccpA gene of C . perfringens by conjugational transfer of a nonreplicating plasmid into C . perfringens, which led to inactivation of the ccpA gene by homologous recombination . CcpA is a transcriptional regulator known to mediate catabolite repression in a number of low-G+C-content gram-positive bacteria, of which C . perfringens is a member . The ccpA mutant strain sporulated at a 60-fold lower efficiency than the wild-type strain in the absence of glucose . In the presence of 5 mM glucose, sporulation was repressed about 2,000-fold in the wild-type strain and 800-fold in the ccpA mutant strain compared to sporulation levels for the same strains grown in the absence of glucose . Therefore, while CcpA is necessary for efficient sporulation in C . perfringens, glucose-mediated catabolite repression of sporulation is not due to the activity of CcpA . Transcription of the cpe gene was measured in the wild-type and ccpA mutant strains grown in sporulation medium by using a cpe-gusA fusion (gusA is an Escherichia coli gene encoding the enzyme beta-glucuronidase) . In the exponential growth phase, cpe transcription was two times higher in the ccpA mutant strain than in the wild-type strain . Transcription of cpe was highly induced during the entry into stationary phase in wild-type cells but was not induced in the ccpA mutant strain . Glucose repressed cpe transcription in both the wild-type and ccpA mutant strain . Therefore, CcpA appears to act as a repressor of cpe transcription in exponential growth but is required for efficient sporulation and cpe transcription upon entry into stationary phase . CcpA was also required for maximum synthesis of collagenase (kappa toxin) and acted as a repressor of polysaccharide capsule synthesis in the presence of glucose, but it did not regulate synthesis of the phospholipase PLC (alpha toxin).

Clin Gastroenterol Hepatol, 2004 Aug, 2(8), 669 - 74
Clinical application of polymerase chain reaction to diagnose Clostridium difficile in hospitalized patients with diarrhea; Morelli MS et al.; BACKGROUND & AIMS: Clostridium difficile is a common cause of diarrhea in hospitalized patients and is associated with significant morbidity and cost . The current diagnostic standard, enzyme immunoassay (EIA), has low sensitivity, leading to duplicate testing and empiric treatment . We sought to show the usefulness and potential cost effectiveness of polymerase chain reaction (PCR) amplification of toxin B gene for diagnosis of C . difficile-induced diarrhea . METHODS: A total of 148 stool samples from academic and community-based hospitals were sent for EIA testing and were evaluated prospectively for the presence of toxin B gene by PCR . Results were compared with EIA regarding sensitivity, specificity, and predictive values . Medical charts were reviewed to determine the following: (1) number of EIAs sent per admission, (2) number sent within a 24-hour time period, and (3) how caregivers practiced based on EIA results . RESULTS: The mean age of 130 patients was 55 years . EIA and PCR were positive in 6.8% and 13.6% of patients, respectively . EIA sensitivity was 40%, specificity was 98%, and positive and negative predictive values were 80% and 91%, respectively . The cost of the PCR was $22/sample . Empiric treatment for C . difficile was given unnecessarily in 42% of EIA-negative results . Thirty percent of patients had 3 or more EIAs sent during their hospital admission . Of patients with multiple samples sent, 57% had more than 1 sample sent in a 24-hour period . CONCLUSIONS: Many physicians do not conform to practice guidelines regarding recommended diagnosis and empiric treatment of C . difficile . Toxin B gene PCR represents a more sensitive and potentially cost-effective method to diagnose C . difficile-induced diarrhea than EIA and should be considered for use as an alternative diagnostic standard.

Extremophiles . 2004 Jul 28; {Epub ahead of print}
A phylogenetic analysis of Wadi el Natrun soda lake cellulase enrichment cultures and identification of cellulase genes from these cultures; Grant S et al.; Samples of sediments and surrounding soda soils (SS) from the extremely saline and alkaline lakes of the Wadi el Natrun in the Libyan Desert, Egypt, were obtained in October 2000 . Anaerobic enrichment cultures were grown from these samples, DNA isolated, and the bacterial diversity assessed by 16S rRNA gene clone analysis . Clones derived from lake sediments (LS) most closely matched Clostridium spp., Natronoincola histidinovorans, Halocella cellulolytica, Bacillus spp., and the Cytophaga- Flexibacter- Bacteroides group . Similar clones were identified in the SS, but Bacillus spp . predominated . Many of the clones were most closely related to organisms already identified in alkaline or saline environments . Two genomic DNA libraries were made from the pooled LS enrichments and a single SS-enrichment sample . A novel cellulase activity was identified and characterized in each . The lake cellulase ORF encoded a protein of 1,118 amino acids; BLASTP analysis showed it was most closely related to an endoglucanase from Xanthomonas campestris . The soil-cellulase ORF encoded a protein of 634 amino acids that was most closely related to an endoglucanase from Fibrobacter succinogenes.

Appl Microbiol Biotechnol . 2004 Jul 29; {Epub ahead of print}
H(2)-Producing bacterial communities from a heat-treated soil inoculum; Iyer P et al.; Hydrogen gas ( approximately 60% H(2)) was produced in a continuous flow bioreactor inoculated with heat-treated soil, and fed synthetic wastewater containing glucose (9.5 g l(-1)) . The pH in the bioreactor was maintained at 5.5 to inhibit consumption of H(2) by methanogens . The objective of this study was to characterize bacterial communities in the reactor operated under two different hydraulic retention times (HRTs of 30-h and 10-h) and temperatures (30show $132# degrees show $132#C and 37show $132# degrees show $132#C) . At 30-h HRT, the H(2) production rate was 80 ml h(-1) and yield was 0.91 mol H(2)/mol glucose . At 10-h HRT, the H(2) production rate was more than 5 times higher at 436 ml h(-1), and yield was 1.61 mol H(2)/mol glucose . Samples were removed from the reactor under steady-state conditions for PCR-based detection of bacterial populations by ribosomal intergenic spacer analysis (RISA) . Populations detected at 30-h HRT were more diverse than at 10-h HRT and included representatives of Bacillaceae, Clostridiaceae, and Enterobacteriaceae . At 10-h HRT, only Clostridiaceae were detected . When the temperature of the 10-h HRT reactor was increased from 30show $132# degrees show $132#C to 37show $132# degrees show $132#C, the steady-state H(2) production rate increased slightly to 463 ml h(-1) and yield was 1.8 mol H(2)/mol glucose . Compared to 30show $132# degrees show $132#C, RISA fingerprints at 37show $132# degrees show $132#C from the 10-h HRT bioreactor exhibited a clear shift from populations related to Clostridium acidisoli (subcluster Ic) to populations related to Clostridium acetobutylicum (subcluster Ib).

J Pediatr Hematol Oncol, 2004 Aug, 26(8), 515 - 7
Probiotics in relapsing and chronic diarrhea; Benchimol EI et al.; Diarrhea is common in oncology patients; if it becomes chronic and relapsing, it can be debilitating, hinder planned management, and be difficult to treat . The authors describe two patients, one with leukemia who developed recurrent Clostridium difficile colitis and another who developed chronic diarrhea after bone marrow transplantation . In both patients, administration of antibiotics was suspected as the cause . In one patient, relapsing diarrhea resolved after probiotics were given with a 2-day course of metronidazole, and in the other patient, chronic diarrhea resolved after probiotics were given; resolution was maintained after the probiotics were stopped . Probiotics may offer a way to bring about resolution in antibiotic-associated chronic diarrhea.

FEBS Lett, 2004 Jul 30, 571(1-3), 21 - 5
Identification of O2-induced peptides in an obligatory anaerobe, Clostridium acetobutylicum; Kawasaki S et al.; Clostridium acetobutylicum DSM792 (=ATCC824), a solvent producing obligate anaerobe, grew well after a shift in growth conditions from anoxic to microoxic at the mid exponential phase . In two-dimensional gel electrophoresis, a spot migrating at 45 kDa and three spots at 23 kDa accumulated after 30 min of flushing with 5% O(2)/95% N(2) . Based on peptide mass fingerprints, the 45 kDa polypeptide was determined to be NP_347663 (A-type flavoprotein homologue) and the 23 kDa polypeptides were determined to be NP_350180 or NP_350181 (novel type rubrerythrin homologue) . Northern blot analysis indicated that the expressions of these peptide transcripts were upregulated within 10 min after flushing with 5% O(2)/95% N(2).

Rev Physiol Biochem Pharmacol . 2004 Jul 27; {Epub ahead of print}
CNF and DNT; Hoffmann C et al.; The actin cytoskeleton of mammalian cells is involved in many processes that affect the growth and colonization of bacteria, such as migration of immune cells, phagocytosis by macrophages, secretion of cytokines, maintenance of epithelial barrier functions and others . With respect to these functions, it is not surprising that many bacterial protein toxins, which are important virulence factors and causative agents of human and/or animal diseases, target the actin cytoskeleton of the host . Some toxins target actin directly, such as the C2 toxin produced by Clostridium botulinum . Moreover, bacterial toxins target the cytoskeleton indirectly by modifying actin regulators such as the low-molecular-mass guanosine triphosphate (GTP)-binding proteins of the Rho family . Remarkably, toxins affect these GTPases in a bidirectional manner . Some toxins inhibit and some activate the GTPases . Here we review the Rho-activating toxins CNF1 and CNF2 (cytotoxic necrotizing factors) from Escherichia coli, the Yersinia CNF(Y) and the dermonecrotic toxin (DNT) from Bordetella species . We describe and compare their uptake into mammalian cells, mode of action, structure-function relationship, substrate specificity and role in diseases.

Int Orthop . 2004 Jul 23; {Epub ahead of print}
Cadaveric allograft microbiology; Ibrahim T et al.; This study aims to determine the contamination rate of cadaveric bone allograft and blood cultures retrieved from 119 donors within Leicester between 1990 and 2003 . A contamination rate of 27% was present, with 120 of 437 bone allografts culturing positive at the time of retrieval . Similarly, a contamination rate of 37% was present, with 40 of 107 blood samples culturing positive . The time interval between death and procurement did not influence blood contamination . Coagulase-negative Staphylococcus was the commonest organism isolated in both blood and bone cultures . One donor had Clostridium grown in their blood culture . The available evidence confirms similar contamination rates with other studies . The majority of organisms isolated were skin commensals with a low rate of contamination of highly pathogenic organisms such as Clostridium.

Parasitol Today, 1996 May, 12(5), 175 - 9
Mosquitocidal toxins, genes and bacteria: The hit squad; Porter AG; Certain entomopathogenic species of bacilli and Clostridium produce one or more toxins that kill mosquito larvae even at concentrations in the picomolar range . Altogether, 19 distinct genes are known that encode mosquitocidal toxins, which vary in their potency, species specificity and mode of action . Unlike chemical insecticides, mosquitocidal bacilli used as larvicides are safe for animals and the environment, and do not affect non-pest insects . Mosquitocidal bacteria are effective to varying degrees against Culex, Anopheles and Aedes mosquito larvae, but their rapid sedimentation from the larval feeding zone, UV-light sensitivity and narrow host range have hampered their development . New genetic engineering approaches are being investigated that could overcome these limitations and allow stable expression of broad host range combinations of toxins in UV-resistant, buoyant recombinant bacteria, as discussed here by Alan Porter.

J Environ Sci (China), 2004, 16(3), 525 - 8
Degradation of phenol in an upflow anaerobic sludge blanket (UASB) reactor at ambient temperature; Ke SZ et al.; A synthetic wastewater containing phenol as sole substrate was treated in a 2.8 L upflow anaerobic sludge blanket (UASB) reactor at ambient temperature . The operation conditions and phenol removal efficiency were discussed, microbial population in the UASB sludge was identified based on DNA cloning, and pathway of anaerobic phenol degradation was proposed . Phenol in wastewater was degraded in an UASB reactor at loading rate up to 18 gCOD/(L x d), with a 1:1 recycle ratio, at 26 +/- 1 degrees C, pH 7.0-7.5 . An UASB reactor was able to remove 99% of phenol up to 1226 mg/L in wastewater with 24 h of hydraulic retention time (HRT) . For HRT below 24 h, phenol degradation efficiency decreased with HRT, from 95.4% at 16 h to 93.8% at 12 h . It further deteriorated to 88.5% when HRT reached 8 h . When the concentration of influent phenol of the reactor was 1260 mg/L (corresponding COD 3000 mg/L), with the HRT decreasing (from 40 h to 4 h, corresponding COD loading increasing), the biomass yields tended to increase from 0.265 to 3.08 g/(L x d) . While at 12 h of HRT, the biomass yield was lower . When HRT was 12 h, the methane yield was 0.308 L/(gCOD removed), which was the highest . Throughout the study, phenol was the sole organic substrate . The effluent contained only residual phenol without any detectable intermediates, such as benzoate, 4-hydrobenzoate or volatile fatty acids (VFAs) . Based on DNA cloning analysis, the sludge was composed of five groups of microorganisms . Desulfotomaculum and Clostridium were likely responsible for the conversion of phenol to benzoate, which was further degraded by Syntrophus to acetate and H2/CO2 . Methanogens lastly converted acetate and H2/CO2 to methane . The role of epsilon-Proteobacteria was, however, unsure.

J Infect Dis, 2004 Aug 15, 190(4), 767 - 73 Epub 2004 Jul 19.
Immunization with the C-Domain of alpha -Toxin prevents lethal infection, localizes tissue injury, and promotes host response to challenge with Clostridium perfringens; Stevens DL et al.; Clostridium perfringens gas gangrene is characterized by rapid tissue destruction, impaired host response, and, often, death . Phospholipase C (alpha -toxin) is the virulence factor most responsible for these pathologies . The present study investigated the efficacy of active immunization with the C-terminal domain of alpha -toxin (Cpa247-370) in a murine model of gas gangrene . Primary end points of the study were survival, progression of infection, and tissue perfusion . Secondary end points, which were based on findings of histologic evaluation of tissues, included the extent of tissue destruction and microvascular thrombosis, as well as the magnitude of the tissue inflammatory response . Survival among C-domain-immunized animals was significantly greater than that among sham-immunized control animals . Furthermore, immunization with the C-domain localized the infection and prevented ischemia of the feet . Histopathologic findings demonstrated limited muscle necrosis, reduced microvascular thrombosis, and enhanced granulocytic influx in C-domain-immunized mice . We conclude that immunization with the C-domain of phospholipase C is a viable strategy for the prevention of morbidity and mortality associated with C . perfringens gas gangrene.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1502 - 5 Epub 2004 Jul 21.
C3 exoenzyme from Clostridium botulinum: structure of a tetragonal crystal form and a reassessment of NAD-induced flexure; Evans HR et al.; C3 exoenzyme from Clostridium botulinum (C3bot1) ADP-ribosylates and thereby inactivates Rho A, B and C GTPases in mammalian cells . The structure of a tetragonal crystal form has been determined by molecular replacement and refined to 1.89 A resolution . It is very similar to the apo structures determined previously from two different monoclinic crystal forms . An objective reassessment of available apo and nucleotide-bound C3bot1 structures indicates that, contrary to a previous report, the protein possesses a rigid core formed largely of beta-strands and that the general flexure that accompanies NAD binding is concentrated in two peripheral lobes . Tetragonal crystals disintegrate in the presence of NAD, most likely because of disruption of essential crystal contacts.

Acta Crystallogr D Biol Crystallogr, 2004 Aug, 60(Pt 8), 1364 - 73 Epub 2004 Jul 21.
A neutron crystallographic analysis of a rubredoxin mutant at 1.6 A resolution; Chatake T et al.; A neutron diffraction study has been carried out at 1.6 A resolution on a mutant rubredoxin from Pyrococcus furiosus using the BIX-3 single-crystal diffractometer at the JRR-3 reactor of the Japan Atomic Energy Research Institute . In order to study the unusual thermostability of rubredoxin from P . furiosus (an organism that grows optimally at 373 K), the hydrogen-bonding patterns were compared between the wild-type protein and a 'triple-mutant' variant . In this mutant protein, three residues were changed (Trp3-->Tyr3, Ile23-->Val23, Leu32-->Ile32) so that they are identical to those in a mesophilic rubredoxin from Clostridium pasteurianum . In the present study, some minor changes were found between the wild-type and mutant proteins in the hydrogen-bonding patterns of the Trp3/Tyr3 region . In this investigation, the H/D-exchange ratios in the protein were also studied . Because the target protein was soaked in D2O during the crystallization procedure, most of the N-H and O-H bonds have become deuterated, while essentially all of the C-H bonds have not . In particular, the H/D-exchange pattern of the N-H amide bonds of the protein backbone is of interest because it may contain some indirect information about the mechanism of unfolding of this small protein . The results are in broad agreement with those from solution NMR studies, which suggest that the backbone amide bonds near the four Cys residues of the FeS4 redox center are most resistant to H/D exchange . Finally, the detailed geometries of the water molecules of hydration around the rubredoxin molecule are also reported . The 1.6 A resolution of the present neutron structure determination has revealed a more detailed picture than previously available of some portions of the water structure, including ordered and disordered O-D bonds . Crystallographic details: space group P2(1)2(1)2(1) (orthorhombic), unit-cell parameters a = 34.48, b = 35.70, c = 43.16 A; final agreement factors R = 0.196 and Rfree = 0.230 for 19,384 observed and 6548 unique neutron reflections collected at room temperature; crystal size 4 mm3; a total of 423 non-H atoms, 290 H atoms and 88 D atoms were located in this study.

Am J Physiol Heart Circ Physiol, 2004 Dec, 287(6), H2427 - 33 Epub 2004 Dec.
cAMP protects endothelial barrier functions by preventing Rac-1 inhibition; Waschke J et al.; cAMP enhances endothelial barrier properties and is protective against various inflammatory mediators both in vivo and in vitro . However, the mechanisms whereby cAMP stabilizes the endothelial barrier are largely unknown . Recently we demonstrated that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in vivo and in vitro . Therefore, in the present study we investigated the effect of forskolin (5 microM)- and rolipram (10 microM)-induced cAMP increase on reduction of barrier functions in response to Rac-1 inhibition by Clostridium sordellii lethal toxin (LT) . Forskolin and rolipram treatment blocked LT (200 ng/ml)-induced hydraulic conductivity (Lp) increase in mesenteric microvessels in vivo . Likewise, LT-induced intercellular gap formation in monolayers of cultured microvascular myocardial endothelial (MyEnd) cells and LT-induced loss of adhesion of vascular endothelial cadherin-coated microbeads were abolished . Inhibition of PKA by myristoylated inhibitor peptide (14-22) of PKA (100 microM) reduced the protective effect of cAMP on LT-induced Lp increase in vivo and gap formation in vitro, indicating that the effect of cAMP on Rac-1 inhibition was PKA dependent . Glucosylation assays demonstrated that cAMP prevents inhibitory Rac-1 glucosylation by LT, indicating that one way that cAMP enhances endothelial barrier functions may be by regulating Rac-1 signaling . Our study suggests that cAMP may provide its well-established protective effects at least in part by regulation of Rho proteins.

Mol Cell Probes, 2004 Aug, 18(4), 271 - 4
Cloning and expression of Clostridium difficile toxin A gene (tcdA) by PCR amplification and use of an expression vector; Ackermann G et al.; Toxigenic Clostridium difficile isolates harbor a 19 kb pathogenicity locus that encodes the genes for toxins A and B . Toxins A and B are among the largest known bacterial toxins expressing potent cytotoxicity and enterotoxicity, and thus the major virulence factors in C . difficile associated diarrhea . Cloning and sequencing of toxin genes is of interest for studies of molecular pathogenesis . We report the amplification and cloning of the complete toxin A gene into an Escherichia coli expression vector . Ten clones analyzed contained the complete toxin A gene . Four of these clones showed cytotoxic activity in cell culture, and were positive for toxin A as determined by ELISA . Toxin A expression was confirmed by Western immunoblot analysis . The presence of cytotoxic activity in cell culture suggests that toxin A activity is independent of other genes in the pathogenicity locus.

J Food Prot, 2004 Jul, 67(7), 1537 - 47
Growth potential of Clostridium perfringens during cooling of cooked meats; Taormina PJ et al.; Many meat-based food products are cooked to temperatures sufficient to inactivate vegetative cells of Clostridium perfringens, but spores of this bacterium can survive, germinate, and grow in these products if sufficient time, temperature, and other variables exist . Because ingestion of large numbers of vegetative cells can lead to concomitant sporulation, enterotoxin release in the gastrointestinal tract, and diarrhea-like illness, a necessary food safety objective is to ensure that not more than acceptable levels of C . perfringens are in finished products . As cooked meat items cool they will pass through the growth temperature range of C . perfringens (50 to 15 degrees C) . Therefore, an important step in determining the likely level of C . perfringens in the final product is the estimation of growth of the pathogen during cooling of the cooked product . Numerous studies exist dealing with just such estimations, yet consensual methodologies, results, and conclusions are lacking . There is a need to consider the bulk of C . perfringens work relating to cooling of cooked meat-based products and attempt to move toward a better understanding of the true growth potential of the organism . This review attempts to summarize observations made by researchers and highlight variations in experimental approach as possible explanations for different outcomes . An attempt is also made here to identify and justify optimal procedures for conducting C . perfringens growth estimation in meat-based cooked food products during cooling.

AJR Am J Roentgenol, 2004 Aug, 183(2), 409 - 13
Relationship between diffuse esophageal spasm and lower esophageal sphincter dysfunction on barium studies and manometry in 14 patients; Prabhakar A et al.; OBJECTIVE: We sought to reassess the function and clinical characteristics of the lower esophageal sphincter in a series of patients with radiographically defined diffuse esophageal spasm . MATERIALS AND METHODS: In reviewing records in the radiology database at our hospital, we identified 14 patients with diffuse esophageal spasm confirmed on barium studies who also underwent esophageal manometry . The radiographic findings were reviewed and correlated with the manometric findings . Medical records were also reviewed to determine the clinical presentation, treatment, and patient course . RESULTS: All 14 patients were symptomatic, presenting with dysphagia, chest pain, or both . All the barium studies revealed intermittently absent or weakened peristalsis, with nonperistaltic contractions that were moderate in six patients (43%) and marked in eight patients (57%) (contractions nearly obliterating the lumen in six and completely obliterating the lumen in two) . Nine patients (64%) had impaired opening of the lower esophageal sphincter, manifested by beaklike narrowing of the distal esophagus, and five (36%) had normal opening of the lower esophageal sphincter . Manometry revealed abnormal peristalsis in all 14 patients, with repetitive simultaneous contractions in eight (57%) and lower esophageal sphincter dysfunction in 12 (86%) . All eight patients with lower esophageal sphincter dysfunction or incomplete relaxation of the lower esophageal sphincter on barium studies or manometry who were treated with the Clostridium botulinum toxin or endoscopic balloon dilatation had a positive response . CONCLUSION: Our preliminary data show that diffuse esophageal spasm is characterized on barium studies by frequent lower esophageal sphincter dysfunction rather than a classic corkscrew appearance . Barium and manometric studies may have complementary roles in the evaluation of patients with diffuse esophageal spasm.

FEMS Microbiol Lett, 2004 Aug 1, 237(1), 65 - 72
Metabolism of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine by Clostridium bifermentans strain HAW-1 and several other H2-producing fermentative anaerobic bacteria; Zhao JS et al.; Several H2-producing fermentative anaerobic bacteria including Clostridium, Klebsiella and Fusobacteria degraded octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) (36 microM) to formaldehyde (HCHO) and nitrous oxide (N2O) with rates ranging from 5 to 190 nmol h(-1)g {dry weight} of cells(-1) . Among these strains, C . bifermentans strain HAW-1 grew and transformed HMX rapidly with the detection of the two key intermediates the mononitroso product and methylenedinitramine . Its cellular extract alone did not seem to degrade HMX appreciably, but degraded much faster in the presence of H2, NADH or NADPH . The disappearance of HMX was concurrent with the release of nitrite without the formation of the nitroso derivative(s) . Results suggest that two types of enzymes were involved in HMX metabolism: one for denitration and the second for reduction to the nitroso derivative(s).

J Bacteriol, 2004 Aug, 186(15), 5129 - 37
The phosphoenolpyruvate carboxylase from Methanothermobacter thermautotrophicus has a novel structure; Patel HM et al.; In Methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential CO(2)-fixation reaction . This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase . The phosphoenolpyruvate carboxylase from this organism was purified to homogeneity . The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (PPCs) . The NH(2)-terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence . A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved . This is the first report of a new type of phosphoenolpyruvate carboxylase that we call PpcA ("A" for "archaeal") . Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (Clostridium perfringens, Oenococcus oeni, and Leuconostoc mesenteroides) and no eukaryotic genomes . PpcA was the only recognizable oxaloacetate-producing enzyme in Methanopyrus kandleri, a hydrothermal vent organism . Each PpcA-containing organism lacked a PPC homolog . The activity of M . thermautotrophicus PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the Escherichia coli PPC . The catalytic core (including His(138), Arg(587), and Gly(883)) of the E . coli PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys(773), Arg(832), and Asn(881)) were not . PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme . The reverse is also equally possible.

Nat Struct Mol Biol, 2004 Aug, 11(8), 797 - 8 Epub 2004 Jul 18.
Clostridium perfringens epsilon-toxin shows structural similarity to the pore-forming toxin aerolysin; Cole AR et al.; Epsilon-toxin from Clostridium perfringens is a lethal toxin . Recent studies suggest that the toxin acts via an unusually potent pore-forming mechanism . Here we report the crystal structure of epsilon-toxin, which reveals structural similarity to aerolysin from Aeromonas hydrophila . Pore-forming toxins can change conformation between soluble and transmembrane states . By comparing the two toxins, we have identified regions important for this transformation.

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