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, intrav