Clostridia

Extremophiles, 2004 Dec, 8(6), 475 - 88 Epub 2004 Jul 14.
Diversity and cold-active hydrolytic enzymes of culturable bacteria associated with Arctic sea ice, Spitzbergen; Groudieva T et al.; The diversity of culturable bacteria associated with sea ice from four permanently cold fjords of Spitzbergen, Arctic Ocean, was investigated . A total of 116 psychrophilic and psychrotolerant strains were isolated under aerobic conditions at 4 degrees C . The isolates were grouped using amplified rDNA restriction analysis fingerprinting and identified by partial sequencing of 16S rRNA gene . The bacterial isolates fell in five phylogenetic groups: subclasses alpha and gamma of Proteobacteria, the Bacillus-Clostridium group, the order Actinomycetales, and the Cytophaga-Flexibacter-Bacteroides (CFB) phylum . Over 70% of the isolates were affiliated with the Proteobacteria gamma subclass . Based on phylogenetic analysis (<98% sequence similarity), over 40% of Arctic isolates represent potentially novel species or genera . Most of the isolates were psychrotolerant and grew optimally between 20 and 25 degrees C . Only a few strains were psychrophilic, with an optimal growth at 10-15 degrees C . The majority of the bacterial strains were able to secrete a broad range of cold-active hydrolytic enzymes into the medium at a cultivation temperature of 4 degrees C . The isolates that are able to degrade proteins (skim milk, casein), lipids (olive oil), and polysaccharides (starch, pectin) account for, respectively, 56, 31, and 21% of sea-ice and seawater strains . The temperature dependences for enzyme production during growth and enzymatic activity were determined for two selected enzymes, alpha-amylase and beta-galactosidase . Interestingly, high levels of enzyme productions were measured at growth temperatures between 4 and 10 degrees C, and almost no production was detected at higher temperatures (20-30 degrees C) . Catalytic activity was detected even below the freezing point of water (at -5 degrees C), demonstrating the unique properties of these enzymes.

J Ind Microbiol Biotechnol, 2004 Jun, 31(5), 229 - 34 Epub 2004 Jun 08.
Thermostable xylanase10B from Clostridium acetobutylicum ATCC824; Ali MK et al.; The Clostridium acetobutylicum xylanase gene xyn10B (CAP0116) was cloned from the type strain ATCC 824, whose genome was recently sequenced . The nucleotide sequence of C . acetobutylicum xyn10B encodes a 318-amino acid protein . Xyn10B consists of a single catalytic domain that belongs to family 10 of glycosyl hydrolases . The enzyme was purified from recombinant Escherichia coli . The Xyn10B enzyme was highly active toward birchwood xylan, oat-spelt xylan, and moderately active toward avicel, carboxymethyl cellulose, polygalacturonic acid, lichenan, laminarin, barley-beta-glucan and various p-nitrophenyl monosaccharides . Xyn10B hydrolyzed xylan and xylooligosaccharides to produce xylobiose and xylotriose . The pH optimum of Xyn10B was 5.0, and the optimal temperature was 70 degrees C . The enzyme was stable at 60 degrees C at pH 5.0-6.5 for 1 h without substrate . This is one of a number of xylan-related activities encoded on the large plasmid in C . acetobutylicum ATCC 824.

Schweiz Arch Tierheilkd, 2004 Jun, 146(6), 295 - 302
{Tetanus in cats: 3 case descriptions}; Tomek A et al.; Three cats with spasticity on one leg or on all four limbs were presented between 1996 and 1998 at the Department of clinical veterinary medicine, Section of neurology, Vetsuisse-Faculty of Bern . The presumptive diagnosis was tetanus . A focal form was present in two cases and generalised tetanus in one cat . All cats had a history of injury at the affected legs respectively at the neck . The first clinical signs were seen between two days and three weeks after injury . The bacteriologic examination of serous fluid from the site of injury revealed an infection with Clostridium . EMG in one cat during anaesthesia showed motor united potentials (MUPs) on the spastic leg . All patients received antibiotics (Penicillin, respectively Amoxicillin/Clavulanic acid and Metronidazol) . Supportive aid were initially sedation, wound revision and in one cat nutrition through oesophageal sonde . In a second phase physiotherapy was performed . All three animals were significantly better after a couple of weeks, two cats were without symptoms after eight and five weeks respectively.

J Biol Chem, 2004 Oct 15, 279(42), 43547 - 54 Epub 2004 Jul 06.
Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells; Zhao D et al.; Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation . We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion . Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation . We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation . Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation . Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases . Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion . This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling . Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

Int J Food Microbiol, 2004 Aug 1, 94(3), 313 - 22
Effects of mastic resin and its essential oil on the growth of proteolytic Clostridium botulinum; Daifas DP et al.; Studies were done to determine the effect of mastic resin and its essential oil, alone and in conjunction with ethanol, on the growth of proteolytic strains of Clostridium botulinum in media, and on neurotoxin production in challenge studies with English-style crumpets . Preliminary studies, using a spot-on-the-lawn method, indicated that high levels of mastic resin in ethanol ( approximately 8% w/w) were required for complete inhibition of all strains of C . botulinum tested, but mastic resin in ethanol had a greater anti-botulinal effect than ethanol alone . However, only low levels of mastic oil ( approximately 0.3% v/v) were required for inhibition of proteolytic strains of C . botulinum . Both studies showed a strain specific inhibition, with C . botulinum type A strains being more sensitive to mastic resin and its essential oil than type B strains . However, mastic resin in ethanol proved to be more effective when used as a vapor phase inhibitor applied to cotton pads and placed inside inoculated plates than when added directly to media . While both mastic resin and its essential oil inhibited the growth of proteolytic strains of C . botulinum in vitro, they failed to inhibit neurotoxin production in challenge studies with C . botulinum in English-style crumpets.

Clin Med, 2004 May-Jun, 4(3), 258 - 61
The story of Clostridium botulinum: from food poisoning to Botox; Ting PT et al.; In the last fifty years, Clostridium botulinum has become notorious for its ability to produce the deadly botulinum neurotoxins . While botulinum toxin A, better known as Botox, is universally recognised by the public as a cosmetic enhancement tool, the botulinum neurotoxins are commonly used off-label for many medical conditions in ophthalmology, neurology and dermatology . The versatility of these botulinum toxins has made Clostridium botulinum one of the most widely known bacterial pathogens in medical history . This article outlines the discovery of botulinum toxins through to their present day applications in medicine.

J Appl Microbiol, 1998 Jan, 84(1), 133 - 7
A high prevalence of Clostridium botulinum type E in Finnish freshwater and Baltic Sea sediment samples; Hielm S et al.; The distribution of Clostridium botulinum serotypes A, B, E and F in aquatic environments of the Baltic Sea and Finnish mainland was examined . A total of 110 samples were tested with a neurotoxin-specific PCR assay . Clostridium botulinum type E was found in 81% of sea and 61% of freshwater samples . No other toxinotypes were found . Spore counts were quantified by the most probable number method, Cl . botulinum type E kg(-1) averaging 940 in sea and 370 in freshwater samples . The overall prevalence and spore counts of Cl . botulinum type E in aquatic sediments correlated significantly with offshore bottom oxygen content, depth, and bioturbation activity, whereas there was no correlation with bottom water temperature . These findings indicate the possibility of Cl . botulinum type E multiplication or at least, suitable conditions for spore survival, in anoxic sediments.

J Appl Microbiol, 1998 Jan, 84(1), 5 - 17
Phylogeny and taxonomy of the food-borne pathogen Clostridium botulinum and its neurotoxins; Collins MD et al.; Until recently, all clostridia producing neurotoxins able to cause paralysis symptomatic of botulism were deemed to be Clostridium botulinum . Defining Cl . botulinum on the basis of this single phenotypic trait has resulted in the species encompassing metabolically very diverse organisms, and four distinct phenotypic groups are recognized within this taxon (designated groups I-IV) . Nucleic acid hybridization and 16S ribosomal RNA sequencing studies have revealed the presence of four phylogenetically distinct lineages within the species, which correlate with these phenotypic divisions . In addition to marked phenotypic and genotypic heterogeneity between groups, the taxonomy of the species is further complicated by the existence of strains which are closely related, if not genetically identifiable, to members of each Cl . botulinum group, but are non-toxigenic . Furthermore, strains of species other than Cl . botulinum (viz . Cl . baratii, Cl . butyricum) have been found which express botulinum neurotoxin (BoNT) . Great advances have been made in recent years in elucidating the nucleotide sequences of genes encoding the various BoNT antigenic types (A through to G) . Genealogical trees derived from BoNTs show marked discordance with those depicting 'natural' relationships inferred from 16S rRNA and phenotypic clusters, and strong evidence exists for BoNT gene transfer between some groups of Cl . botulinum (e.g . groups I and II), and with non-botulinum species . Botulinum neurotoxin is produced by Cl . botulinum as a non-covalently bound progenitor toxin complex of two or more protein components . Information on the evolutionary histories of the various non-toxic progenitor proteins is currently limited, although there is evidence of gene recombination . In particular, chimera-like or mosaic non-toxic-non-haemagglutinins (NTNH) genes in group I Cl . botulinum have been described, and it is now apparent that the phylogeny of the NTNHs is not going to 'mirror' that of botulinal neurotoxins, although their genes are physically contiguous . In this article, the current state of knowledge of the phylogenetics of the species Cl . botulinum and its neurotoxins is reviewed, and a view is presented that a nomenclature based rigidly on BoNT production is no longer tenable.

Exp Cell Res, 2004 Aug 1, 298(1), 74 - 82
Newcastle disease virus neuraminidase primes neutrophils for stimulation by galectin-3 and formyl-Met-Leu-Phe; Almkvist J et al.; Human neutrophils are activated by the beta-galactoside-binding lectin galectin-3, provided that the cells are primed by in vivo extravasation or by in vitro preactivation with, for example, LPS . Removal of terminal sialic acid can change neutrophil functionality and responsiveness due to exposure of underlying glycoconjugate receptors or change in surface charge . Here, we investigated whether such alteration of the cell surface carbohydrate composition can alter the responsiveness of the cells to galectin-3 . Neutrophils were treated with neuraminidases (NA) of different origins: Clostridium perfringens (CP), Salmonella typhimurium, Vibrio cholerae, and Newcastle disease virus (NDV) . In the presence of NDV-NA, but no other NA, the otherwise non-responding neutrophils responded readily to galectin-3 by activation of the NADPH-oxidase . The galectin-3 priming effect was inhibited by the sialidase inhibitor 2,3-dehydro-2-deoxy-N-acetyl-neuraminic acid . Earlier studies have shown that priming of the neutrophil response to galectin-3 with, for example, LPS is paralleled by degranulation of intracellular vesicles and granules and upregulation of potential galectin-3 receptors . Also, NDV-NA (but not CP-NA) treatment induced degranulation, shown as an upregulation of complement receptor 3 . Since not only the galectin response but also the response to the chemoattractant fMLF was primed, NDV-NA appears to induce a general priming phenomenon, possibly due to receptor upregulation by degranulation.

Curr Opin Infect Dis, 2004 Aug, 17(4), 323 - 7
Nosocomial diarrhoea due to Clostridium difficile; Riley TV; PURPOSE OF REVIEW: The purpose of this review is to summarize recent developments in the diagnosis, epidemiology, treatment and prevention of nosocomial diarrhoea due to Clostridium difficile . RECENT FINDINGS: Twenty-five years after its discovery, the diagnosis of C . difficile-associated diarrhoea is still problematic with laboratories trying to reconcile the time and expense of the diagnostic process . Newer molecular techniques may offer hope . With the introduction of new antibiotics into clinical practice, confusion has arisen about the risk they pose for C . difficile-associated diarrhoea . Strains of C . difficile that fail to produce an active toxin A are an emerging problem and good molecular epidemiology is required to determine whether highly infectious clones exist . Little progress has been made in the treatment of recurrent C . difficile-associated diarrhoea; however, the development of a vaccine is imminent . More effort is being made to rid the hospital environment of C . difficile through infection-control procedures or changes in antibiotic-prescribing policies . SUMMARY: C . difficile continues to be a major nosocomial infection in many health-care institutions throughout the world . Strategies that reduce exposure to the organism or to antibiotics will have an impact on rates of C . difficile-associated diarrhoea.

Biophys J, 2004 Jul, 87(1), 688 - 95
Controlled pseudopod extension of human neutrophils stimulated with different chemoattractants; Zhelev DV et al.; The formation of pseudopods and lamellae after ligation of chemoattractant sensitive G-protein coupled receptors (GPCRs) is essential for chemotaxis . Here, pseudopod extension was stimulated with chemoattractant delivered from a micropipet . The chemoattractant diffusion and convection mass transport were considered, and it is shown that when the delivery of chemoattractant was limited by diffusion there was a strong chemoattractant gradient along the cell surface . The diffusion-limited delivery of chemoattractant from a micropipet allowed for maintaining an almost constant chemoattractant concentration at the leading edge of single pseudopods during their growth . In these conditions, the rate of pseudopod extension was dependent on the concentration of chemoattractant in the pipet delivering chemoattractant . The pseudopod extension induced using micropipets was oscillatory even in the presence of a constant delivery of chemoattractant . This oscillatory pseudopod extension was controlled by activated RhoA and its downstream effector kinase ROCK and was abolished after the inhibition of RhoA activation with Clostridium botulinium C3 exoenzyme (C3) or the blocking of ROCK activation with Y-27632 . The ability of the micropipet assay to establish a well-defined chemoattractant distribution around the activated cell over a wide range of molecular weights of the used chemoattractants allowed for comparison of the effect of chemoattractant stimulation on the dynamics of pseudopod growth . Pseudopod growth was stimulated using N-formylated peptide (N-formyl-methionyl-leucyl-phenylalanine (fMLP)), platelet activating factor (PAF), leukotriene B4 (LTB(4)), C5a anaphylotoxin (C5a), and interleukin-8 (IL-8), which represent the typical ligands for G-protein coupled chemotactic receptors . The dependence of the rate of pseudopod extension on the concentration of these chemoattractants and their equimolar mixture was measured and shown to be similar for all chemoattractants . The inhibition of the activity of phosphoinositide-3 kinase (PI3K) with wortmannin showed that 72%-80% of the rate of pseudopod extension induced with N-formyl-methionyl-leucyl-phenylalanine, platelet activating factor, and leukotriene B4 was phosphoinositide-3 kinase-dependent, in contrast to 55% of the rate of pseudopod extension induced with interleukin-8 . The dependence of the rate of pseudopod extension on the concentration of individual chemoattractants and their equimolar mixture suggests that there is a common rate-limiting mechanism for the polymerization of cytoskeletal F-actin in the pseudopod region induced by G-protein coupled chemoattractant receptors.

Biophys J, 2004 Jul, 87(1), 540 - 52
The solution structure and oligomerization behavior of two bacterial toxins: pneumolysin and perfringolysin O; Solovyova AS et al.; Pneumolysin (PLY), an important protein virulence factor of the human bacterial pathogen Streptococcus pneumoniae, could be a candidate for inclusion in a new anti-streptococcal vaccine . PLY solution species from monomer via multimeric intermediates to ring-shaped oligomers were studied with time-dependent sedimentation velocity in the analytical ultracentrifuge (AUC) . Hydrodynamic bead modeling was used to interpret the data obtained . PLY remained mostly monomeric in solution; intermediate PLY multimers were detected in small quantities . Current understanding of PLY molecular mechanism is guided by a model built on the basis of its homology with perfringolysin O (PFO) for which there is an atomic structure . PFO, a virulence factor of the organism Clostridium perfringens, has almost the same molecular mass as PLY and shares 48% sequence identity and 60% sequence similarity with PLY . We report a comparative low-resolution structural study of PLY and PFO using AUC and small-angle x-ray scattering (SAXS) . AUC data demonstrate that both proteins in solution are mostly monodisperse but PLY is a monomer whereas PFO is mostly dimeric . Ab initio dummy atom and dummy residue models for PFO and PLY were restored from the distance distribution function derived from experimental small-angle x-ray scattering curves . In solution, PLY is elongated, consistent with the shape predicted by its high-resolution homology model . The PFO dimer is also an elongated particle whose shape and volume are consistent with a staggered antiparallel dimer.

Biophys J, 2004 Jul, 87(1), 534 - 9
Toxin binding of tolevamer, a polyanionic drug that protects against antibiotic-associated diarrhea; Braunlin W et al.; Tolevamer, (GT160-246), is a sodium salt of styrene sulfonate polymer that is under development for the treatment of diarrhea caused by infection with Clostridium difficile . Pulsed ultrafiltration binding experiments in phosphate buffer containing 0.15 M Na(+) provide per polymer chain dissociation constants of 133 nM and 8.7 microM for the binding of tolevamer to C . difficile toxins A and B, respectively . At 0.05 M Na(+), the binding of toxin A to tolevamer is irreversible, whereas the dissociation constant to toxin B under these conditions is 120 nM . Binding constants obtained from fluorescence polarization data for toxin A binding to tolevamer at 0.15 M Na(+) agree substantially with those obtained by pulsed ultrafiltration . The binding activity of tolevamer reported here correlates well with previously reported results for the inhibition of the biological activity of C . difficile toxins A and B . From the fluorescence polarization data, it is estimated that one toxin A molecule interacts with between 600 to 1000 monomer units on tolevamer at 0.15 M Na(+) . Thus, the data suggest a very large interaction surface between polymer and toxin A.

Appl Environ Microbiol, 2004 Jul, 70(7), 4267 - 75
Identification of bacterial populations in dairy wastewaters by use of 16S rRNA gene sequences and other genetic markers; McGarvey JA et al.; Hydraulic flush waste removal systems coupled to solid/liquid separators and circulated treatment lagoons are commonly utilized to manage the large amounts of animal waste produced on high-intensity dairy farms . Although these systems are common, little is known about the microbial populations that inhabit them or how they change as they traverse the system . Using culture-based and non-culture-based methods, we characterized the microbial community structure of manure, water from the separator pit, and water from the circulated treatment lagoon from a large dairy in the San Joaquin Valley of California . Our results show that both total bacterial numbers and bacterial diversity are highest in manure, followed by the separator pit water and the lagoon water . The most prevalent phylum in all locations was the Firmicutes (low-G+C, gram-positive bacteria) . The most commonly occurring operational taxonomic unit (OTU) had a 16S rRNA gene (rDNA) sequence 96 to 99% similar to that of Clostridium lituseburense and represented approximately 6% of the manure derived sequences, 14% of the separator pit-derived sequences and 20% of the lagoon-derived sequences . Also highly prevalent was an OTU with a 16S rDNA sequence 97 to 100% similar to that of Eubacterium tenue, comprising approximately 3% of the manure-derived sequences, 6% of the separator pit-derived sequences and 9% of the lagoon-derived sequences . Taken together, these sequences represent approximately one-third of the total organisms in the lagoon waters, suggesting that they are well adapted to this environment.

Appl Environ Microbiol, 2004 Jul, 70(7), 4170 - 6
Identification of type A, B, E, and F botulinum neurotoxin genes and of botulinum neurotoxigenic clostridia by denaturing high-performance liquid chromatography; Franciosa G et al.; Denaturing high-performance liquid chromatography (DHPLC) is a recently developed technique for rapid screening of nucleotide polymorphisms in PCR products . We used this technique for the identification of type A, B, E, and F botulinum neurotoxin genes . PCR products amplified from a conserved region of the type A, B, E, and F botulinum toxin genes from Clostridium botulinum, neurotoxigenic C . butyricum type E, and C . baratii type F strains were subjected to both DHPLC analysis and sequencing . Unique DHPLC peak profiles were obtained with each different type of botulinum toxin gene fragment, consistent with nucleotide differences observed in the related sequences . We then evaluated the ability of this technique to identify botulinal neurotoxigenic organisms at the genus and species level . A specific short region of the 16S rRNA gene which contains genus-specific and in some cases species-specific heterogeneity was amplified from botulinum neurotoxigenic clostridia and from different food-borne pathogens and subjected to DHPLC analysis . Different peak profiles were obtained for each genus and species, demonstrating that the technique could be a reliable alternative to sequencing for the rapid identification of food-borne pathogens, specifically of botulinal neurotoxigenic clostridia most frequently implicated in human botulism.

CMAJ, 2004 Jul 6, 171(1), 51 - 8
Clostridium difficile-associated diarrhea in adults; Poutanen SM et al.; Clostridium difficile is the most important cause of nosocomial diarrhea in adults . Illness may range from mild watery diarrhea to life-threatening colitis . An antecedent disruption of the normal colonic flora followed by exposure to a toxigenic strain of C . difficile are necessary first steps in the pathogenesis of disease . Diagnosis is based primarily on the detection of C . difficile toxin A or toxin B . First-line treatment is with oral metronidazole therapy . Treatment with oral vancomycin therapy should be reserved for patients who have contraindications or intolerance to metronidazole or who fail to respond to first-line therapy.

CMAJ, 2004 Jul 6, 171(1), 33 - 8
Risk of Clostridium difficile diarrhea among hospital inpatients prescribed proton pump inhibitors: cohort and case-control studies; Dial S et al.; BACKGROUND: Antibiotic disruption of the normal intestinal flora is a well-known risk factor for Clostridium difficile-associated diarrhea . Reduced gastric acidity has been suggested as a risk factor, and we hypothesized that proton pump inhibitors, because of their potency, may be an independent risk factor for this problem . METHODS: For the cohort study we identified from a pharmacy database 1187 inpatients at a Montreal teaching hospital who received antibiotics over a 9-month period beginning in August 2002 . We compared patients in this group who had also received a proton pump inhibitor or an H(2) blocker with patients who had not received acid suppressive therapy . Hospital laboratory reports of positive assay results for C . difficile toxin were used to ascertain cases in the cohort . To assess the possibility that proton pump inhibitors were prescribed to patients who were sicker and had other risk factors for C . difficile infection, we did a case-control study at a second Montreal teaching hospital . Cases were defined as patients who were positive for C . difficile toxin and who had a history of diarrhea (n = 94) . Control subjects were selected from among patients who had received an antibiotic and were matched to cases by ward, age within 5 years and class of antibiotics (n = 94) . RESULTS: In the cohort study, C . difficile diarrhea developed in 81 (6.8%) of the 1187 patients who received antibiotics while in hospital . In a multivariate analysis, C . difficile diarrhea was significantly associated with use of proton pump inhibitors (adjusted odds ratio {OR} 2.1, 95% confidence interval {CI} 1.2- 3.5), receipt of 3 or more antibiotics (OR 2.1, 95% CI 1.3- 3.4) and admission to a medical ward (OR 4.1, 95% CI 2.3- 7.3) . In the case-control study C . difficile diarrhea was associated with female sex (adjusted OR 2.1, 95% CI 1.1-4.0), prior renal failure (adjusted OR 4.3, 95% CI 1.5-11.9), hospital admission in the 3 months before the index admission (adjusted OR 2.6, 95% CI 1.4-5.2) and use of proton pump inhibitors (adjusted OR 2.7, 95% CI 1.4-5.2) . INTERPRETATION: Patients in hospital who received proton pump inhibitors were at increased risk of C . difficile diarrhea.

Biochim Biophys Acta, 2004 Jul 6, 1673(1-2), 66 - 74
Large clostridial cytotoxins: cellular biology of Rho/Ras-glucosylating toxins; Schirmer J et al.; Mono-O-glycosylation of eukaryotic target proteins is the molecular mechanism of bacterial protein toxins of the family of large clostridial cytotoxins . This toxin family encompasses several high molecular mass proteins (>250 kDa) of various Clostridia species that are implicated in severe human diseases . Toxin A and toxin B from Clostridium difficile are the causative agents of pseudomembranous colitis and antibiotic-associated diarrhea . Lethal toxin and hemorrhagic toxin from Clostridium sordellii as well as alpha-toxin from Clostridium novyi are involved in the gas gangrene syndrome . The common mode of action of large clostridial cytotoxins is elicited by specific glycosylation of small GTP-binding proteins in the cytosol of target cells using activated nucleotide sugars as cosubstrates . Specific modification at a single threonine residue in the small GTPases renders these important key players of various signaling pathways inactive . This minireview intends to give an overview on structure-function analysis and mode of action of the large clostridial cytotoxins, as well as on the resulting functional consequences of glycosylation of target proteins.

Pediatr Emerg Care, 2004 Jul, 20(7), 457 - 9
Gas gangrene secondary to Clostridium perfringens in pediatric oncology patients; Temple AM et al.; OBJECTIVE: To report 2 cases of severe gas gangrene secondary to Clostridium perfringens in pediatric oncology patients . METHODS: We describe 2 children with acute presentations of gas gangrene secondary to C . perfringens . Both children were initially seen and treated in a community hospital emergency department and subsequently were cared for in a pediatric intensive care unit in a tertiary care, university-based children's hospital . RESULTS: Both children demonstrated severe and unrelenting decompensation and required operative intervention within the first hospital day, which included amputation of the infected limb . One child survived and one child expired despite heroic measures . CONCLUSIONS: Gas gangrene secondary to C . perfringens is an uncommon but life-threatening and limb-threatening condition in pediatric cancer patients . A high index of suspicion in a immunocompromised child with cancer who presents with extremity pain in combination with neutropenia is the key to early diagnosis and may lead to improved survival . This disease requires prompt recognition and aggressive treatment to allow any hope of recovery . Emergency medicine physicians who treat these children should be aware of this severe and potentially fatal infectious process and should not delay treatment or prompt orthopedic surgery consultation.

Folia Microbiol (Praha), 2004, 49(2), 194 - 8
Chitinolytic enzymes from Clostridium aminovalericum: activity screening and purification; Simunek J et al.; A strain isolated from the feces of takin was identified as Clostridium aminovalericum . In response to various types of chitin used as growth substrates, the bacterium produced a complete array of chitinolytic enzymes: chitinase ('endochitinase'), exochitinase, beta-N-acetylglucosaminidase, chitosanase and chitin deacetylase . The highest activities of chitinase (536 pkat/mL) and exochitinase (747 pkat/mL) were induced by colloidal chitin . Fungal chitin also induced high levels of these enzymes (463 pkat/mL and 502 pkat/mL, respectively) . Crab shell chitin was the best inducer of chitosanase activity (232 pkat/mL) . The chitinolytic enzymes of this strain were separated from culture filtrate by ion-exchange chromatography on the carboxylic sorbent Polygran 27 . At pH 4.5, some isoforms of the chitinolytic enzymes (30% of total enzyme activity) did not bind to Polygran 27 . The enzymes were eluted under a stepwise pH gradient (pH 5-8) in 0.1 mol/L phosphate buffer . At merely acidic pH (4.5-5.5), the adsorbed enzymes were co-eluted . However, at pH close to neutral values, the peaks of highly purified isoforms of exochitinases and chitinases were isolated . The protein and enzyme recovery reached 90%.

Toxicon, 2004 Jul, 44(1), 19 - 25
Isolation and characterization of a neutralizing antibody specific to internalization domain of Clostridium botulinum neurotoxin type B; Yang GH et al.; Botulinum neurotoxins (BoNTs), the causative agents for life-threatening human disease botulism, have been recognized as biological warfare agents . In this study, a neutralizing mouse monoclonal antibody against botulinum neurotoxin serotype B (BoNT/B), named BTBH-N1, was developed from mice immunized with BoNT/B toxoid without non-toxic components, which are generally associated with the toxin . Western blot analysis, using recombinant toxin fragments containing light (L), N-terminal half of heavy (HN) and C-terminal half of heavy chains, indicated that BTBH-N1 recognizes linear epitopes located on the HN domain . An in vivo neutralization assay with mice, was conducted to characterize the neutralization capacity of the BTBH-N1 . Only 10 microg of BTBH-N1 completely neutralized 20 units (1 unit = one 50% lethal dose) of BoNT/B . Even though the Mab (up to 100 microg) failed to protect mice challenged with 100 units, it significantly prolonged the time to death in a dose dependent manner . BTBH-N1, the first neutralizing antibody against BoNT/B, could be further developed as effective biological therapeutics for preventing and treating botulism, as well as other diseases caused by BoNT/B.

Transpl Infect Dis, 2004 Mar, 6(1), 10 - 4
Clostridium difficile colitis in patients after kidney and pancreas-kidney transplantation; Keven K et al.; Limited data exist about Clostridium difficile colitis (CDC) in solid organ transplant patients . Between 1/1/99 and 12/31/02, 600 kidney and 102 pancreas-kidney allograft recipients were transplanted . Thirty-nine (5.5%) of these patients had CDC on the basis of clinical and laboratory findings . Of these 39 patients, 35 have information available for review . CDC developed at a median of 30 days after transplantation, and the patients undergoing pancreas-kidney transplantation had a slightly higher incidence of CDC than recipients of kidney alone (7.8% vs . 4.5%, P>0.05) . All but one patient presented with diarrhea . Twenty-four patients (64.9%) were diagnosed in the hospital, and CDC occurred during first hospitalization in 14 patients (40%) . Treatment was with oral metronidazole (M) in 33 patients (94%) and M+oral vancomycin (M+V) in 2 patients . Eight patients had recurrent CDC, which occurred at a median of 30 days (range 15-314) after the first episode . Two patients (5.7%) developed fulminant CDC, presented with toxic megacolon, and underwent colectomy . One of them died; the other patient survived after colectomy . CDC should be considered as a diagnosis in transplant patients with history of diarrhea after antibiotic use, and should be treated aggressively before the infection becomes complicated.

J Mol Biol, 2004 Jul 16, 340(4), 869 - 79
Binding sub-site dissection of a carbohydrate-binding module reveals the contribution of entropy to oligosaccharide recognition at "non-primary" binding subsites; Lammerts van Bueren A et al.; The optimal ligands for many carbohydrate-binding proteins are often oligosaccharides comprising two, three, or more monosaccharide units . The binding affinity for these sugars is increased incrementally by contributions from binding subsites on the protein that accommodate the individual monosaccharide residues of the oligosaccharide . Here, we use CsCBM6-1, a xylan-specific type B carbohydrate-binding module (CBM) from Clostridium stercorarium falling into amino acid sequence family CBM6, as a model system to investigate the structural and thermodynamic contributions of binding subsites in this protein to carbohydrate recognition . The three-dimensional structures of uncomplexed CsCBM6-1 (at 1.8 A resolution) and bound to the oligosaccharides xylobiose, xylotriose, and xylotetraose (at 1.70 A, 1.89 A, and 1.69 A resolution, respectively) revealed the sequential occupation of four subsites within the binding site in the order of subsites 2, 3, 4 then 1 . Overall, binding to all of the xylooligosaccharides tested was enthalpically favourable and entropically unfavourable, like most protein-carbohydrate interactions, with the primary subsites 2 and 3 providing the bulk of the free energy and enthalpy of binding . In contrast, the contributions to the changes in entropy of the non-primary subsites 1 and 4 to xylotriose and xylotetraose binding, respectively, were positive . This observation is remarkable, in that it shows that the 10-20-fold improvement in association constants for oligosaccharides longer than a disaccharide is facilitated by favourable entropic contributions from the non-primary binding subsites.

J Food Prot, 2004 Jun, 67(6), 1133 - 7
Evaluation of a predictive model for Clostridium perfringens growth during cooling; Smith S et al.; Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production . The U.S . Department of Agriculture Food Safety and Inspection Service offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimes result in no more than a 1-log CFU/g increase of C . perfringens and no growth of Clostridium botulinum . The Juneja 1999 model for C . perfringens growth during cooling may be helpful in determining whether the C . perfringens performance standard has been achieved, but this model has not been extensively validated . The objective of this study was to validate the Juneja 1999 model under a variety of temperature situations . The Juneja 1999 model for C . perfringens growth during cooling is fail safe when low (<1 log CFU/ml) or high (>3 log CFU/ml) observed increases occur during exponential cooling . The Juneja 1999 model consistently underpredicted growth at intermediate observed increases (1 to 3 log CFU/ml) . The Juneja 1999 model also underpredicted growth whenever exponential cooling took place at two different rates in the first and second portions of the cooling process . This error may be due to faster than predicted growth of C . perfringens cells during cooling or to an inaccuracy in the Juneja 1999 model.

J Food Prot, 2004 Jun, 67(6), 1128 - 32
Influence of several methodological factors on the growth of Clostridium perfringens in cooling rate challenge studies; Smith S et al.; Proper temperature control is essential in preventing Clostridium perfringens food poisoning . The U.S . Department of Agriculture Food Safety and Inspection Service cooling guidelines offer two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log CFU/g increase of C . perfringens and no growth of Clostridium botulinum . The latter option requires laboratory challenge studies to validate the efficacy of a given cooling process . Accordingly, the objective of this study was to investigate the role of several methodological variables that might be encountered during typical C . perfringens challenge studies . Variables studied included plastic bag type (Whirlpak or Spiral Biotech), sealing method (Multivac or FoodSaver), initial spore inoculum size (1 to approximately 3 log CFU/g), and growth environment (ground beef or Trypticase-peptone-glucose-yeast extract {TPGY} broth) . The major factors that affected growth were sample bag type and growth environment . Samples incubated in Whirlpak bags showed significantly less growth than those incubated in Spiral Biotech bags, which was likely due to the former bag's greater oxygen permeability . C . perfringens spores showed shorter germination, outgrowth, and lag times and C . perfringens cells showed faster growth rates in ground beef compared with TPGY broth . No significant difference was observed between two different sealing methods . Initial spore inoculum levels in the range studied had no significant effect on final C . perfringens cell concentration.

Cornea, 2004 Jul, 23(5), 522 - 3
Clostridium sordellii endophthalmitis after suture removal from a corneal transplant; Zink JM et al.; PURPOSE: To report a case of endophthalmitis caused by Clostridium sordellii . METHODS: A 33-year-old man sustained a penetrating injury of the right eye that resulted in several ocular surgical procedures including pars plana vitrectomy, scleral buckling, scleral-sutured posterior chamber intraocular lens, and penetrating keratoplasty . More than 4 years after the penetrating injury he presented for examination with pain, photophobia, redness, decreased vision, and floaters in the right eye . Vitreous culture grew Clostridium sordellii . RESULTS: Following intravitreal injection of antibiotics, the patient's vision improved from 3/200 to 20/80 (baseline visual acuity) within 2 days . All signs of inflammation resolved without recurrence . CONCLUSIONS: Clostridium sordellii endophthalmitis may have a more benign course than the fulminant endophthalmitis typically seen with other Clostridium species.

J Clin Gastroenterol, 2004 Jul, 38(6 Suppl), S86 - 90
Bacillus clausii probiotic strains: antimicrobial and immunomodulatory activities; Urdaci MC et al.; The clinical benefits observed with probiotic use are mainly attributed to the antimicrobial substances produced by probiotic strains and to their immunomodulatory effects . Currently, the best-documented probiotic bacteria used in human therapy are lactic acid bacteria . In contrast, studies aiming to characterize the mechanisms responsible for the probiotic beneficial effects of Bacillus are rare . The current work seeks to contribute to such characterization by evaluating the antimicrobial and immunomodulatory activities of probiotic B . clausii strains . B . clausii strains release antimicrobial substances in the medium . Moreover, the release of these antimicrobial substances was observed during stationary growth phase and coincided with sporulation . These substances were active against Gram-positive bacteria, in particular against Staphylococcus aureus, Enterococcus faecium, and Clostridium difficile . The antimicrobial activity was resistant to subtilisin, proteinase K, and chymotrypsin treatment, whereas it was sensitive to pronase treatment . The evaluation of the immunomodulatory properties of probiotic B . clausii strains was performed in vitro on Swiss and C57 Bl/6j murine cells . The authors demonstrate that these strains, in their vegetative forms, are able to induce NOS II synthetase activity, IFN-gamma production, and CD4 T-cell proliferation.

Public Health Rep, 2004 Jul-Aug, 119(4), 427 - 34
A review of outbreaks of foodborne disease associated with passenger ships: evidence for risk management; Rooney RM et al.; OBJECTIVE: Foodborne disease outbreaks on ships are of concern because of their potentially serious health consequences for passengers and crew and high costs to the industry . The authors conducted a review of outbreaks of foodborne diseases associated with passenger ships in the framework of a World Health Organization project on setting guidelines for ship sanitation . METHODS: The authors reviewed data on 50 outbreaks of foodborne disease associated with passenger ships . For each outbreak, data on pathogens/toxins, type of ship, factors contributing to outbreaks, mortality and morbidity, and food vehicles were collected . RESULTS: The findings of this review show that the majority of reported outbreaks were associated with cruise ships and that almost 10,000 people were affected . Salmonella spp were most frequently associated with outbreaks . Foodborne outbreaks due to enterotoxigenic E . coli spp, Shigella spp, noroviruses (formally called Norwalk-like viruses), Vibrio spp, Staphylococcus aureus, Clostridium perfringens, Cyclospora sp, and Trichinella sp also occurred on ships . Factors associated with the outbreaks reviewed include inadequate temperature control, infected food handlers, contaminated raw ingredients, cross-contamination, inadequate heat treatment, and onshore excursions . Seafood was the most common food vehicle implicated in outbreaks . CONCLUSIONS: Many ship-associated outbreaks could have been prevented if measures had been taken to ensure adequate temperature control, avoidance of cross-contamination, reliable food sources, adequate heat treatment, and exclusion of infected food handlers from work.

J Chemother, 2004 Apr, 16(2), 119 - 21
In-vitro activity of nisin against clinical isolates of Clostridium difficile; Bartoloni A et al.; Nisin is a cationic peptide produced by Lactococcus lactis . Its activity against clinical isolates of Clostridium difficile was compared to that of vancomycin and metronidazole by minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and time-kill studies . Nisin was more active than the other agents, with a MIC90 of 0.256 mg/L and strong bactericidal activity . Nisin may be a promising agent for the management of C . difficile associated diarrhea.

Nucleic Acids Res, 2004 Jun 23, 32(11), 3340 - 53 Print 2004.
Comparative genomics of the methionine metabolism in Gram-positive bacteria: a variety of regulatory systems; Rodionov DA et al.; Regulation of the methionine biosynthesis and transport genes in bacteria is rather diverse and involves two RNA-level regulatory systems and at least three DNA-level systems . In particular, the methionine metabolism in Gram-positive bacteria was known to be controlled by the S-box and T-box mechanisms, both acting on the level of premature termination of transcription . Using comparative analysis of genes, operons and regulatory elements, we described the methionine metabolic pathway and the methionine regulons in available genomes of Gram-positive bacteria . A large number of methionine-specific RNA elements were identified . S-boxes were shown to be widely distributed in Bacillales and Clostridia, whereas methionine-specific T-boxes occurred mostly in Lactobacillales . A candidate binding signal (MET-box) for a hypothetical methionine regulator, possibly MtaR, was identified in Streptococcaceae, the only family in the Bacillus/Clostridium group of Gram-positive bacteria having neither S-boxes, nor methionine-specific T-boxes . Positional analysis of methionine-specific regulatory sites complemented by genome context analysis lead to identification of new members of the methionine regulon, both enzymes and transporters, and reconstruction of the methionine metabolism in various bacterial genomes . In particular, we found candidate transporters for methionine (MetT) and methylthioribose (MtnABC), as well as new enzymes forming the S-adenosylmethionine recycling pathway . Methionine biosynthetic enzymes in various bacterial species are quite variable . In particular, Oceanobacillus iheyensis possibly uses a homolog of the betaine-homocysteine methyltransferase bhmT gene from vertebrates to substitute missing bacterial-type methionine synthases.

Lett Appl Microbiol, 2004, 38(4), 301 - 5
Comparative analysis of Cryptosporidium, Giardia and indicator bacteria during sewage sludge hygienization in various composting processes; Rimhanen-Finne R et al.; AIMS: To evaluate the suitability of Clostridium perfringens, Escherichia coli and enterococci as indicator organisms for Cryptosporidium and Giardia in treated sludge . METHODS AND RESULTS: Occurrence of Cryptosporidium oocysts and Giardia cysts, detected and enumerated by direct immunofluorescence microscopy, were compared with counts of indicator bacteria during six different sewage sludge hygienization processes, including closed reactor and open windrow composting, and sludge sanitation by quicklime or peat addition . No statistical correlation existed between the counts of indicator bacteria, Cl . perfringens, E . coli, and enterococci and occurrence of Cryptosporidium or Giardia . In sludge end-products, Giardia cysts were detected more frequently than Cryptosporidium oocysts . SIGNIFICANCE OF THE STUDY: Direct analysis is the best method to confirm the presence of (oo)cysts in sludge.

Biotechnol Bioeng, 2004 Jul 5, 87(1), 119 - 27
Biological hydrogen production using a membrane bioreactor; Oh SE et al.; A cross-flow membrane was coupled to a chemostat to create an anaerobic membrane bioreactor (MBR) for biological hydrogen production . The reactor was fed glucose (10,000 mg/L) and inoculated with a soil inoculum heat-treated to kill non-spore-forming methanogens . Hydrogen gas was consistently produced at a concentration of 57-60% in the headspace under all conditions . When operated in chemostat mode (no flow through the membrane) at a hydraulic retention time (HRT) of 3.3 h, 90% of the glucose was removed, producing 2200 mg/L of cells and 500 mL/h of biogas . When operated in MBR mode, the solids retention time (SRT) was increased to SRT = 12 h producing a solids concentration in the reactor of 5800 mg/L . This SRT increased the overall glucose utilization (98%), the biogas production rate (640 mL/h), and the conversion efficiency of glucose-to-hydrogen from 22% (no MBR) to 25% (based on a maximum of 4 mol-H(2)/mol-glucose) . When the SRT was increased from 5 h to 48 h, glucose utilization (99%) and biomass concentrations (8,800 +/- 600 mg/L) both increased . However, the biogas production decreased (310 +/- 40 mL/h) and the glucose-to-hydrogen conversion efficiency decreased from 37 +/- 4% to 18 +/- 3% . Sustained permeate flows through the membrane were in the range of 57 to 60 L/m(2) h for three different membrane pore sizes (0.3, 0.5, and 0.8 microm) . Most (93.7% to 99.3%) of the membrane resistance was due to internal fouling and the reversible cake resistance, and not the membrane itself . Regular backpulsing was essential for maintaining permeate flux through the membrane . Analysis of DNA sequences using ribosomal intergenic spacer analysis indicated bacteria were most closely related to members of Clostridiaceae and Flexibacteraceae, including Clostridium acidisoli CAC237756 (97%), Linmingia china AF481148 (97%), and Cytophaga sp . MDA2507 AF238333 (99%) . No PCR amplification of 16s rRNA genes was obtained when archaea-specific primers were used .

QJM, 2004 Jul, 97(7), 423 - 9
Antibiotic prescribing policy and Clostridium difficile diarrhoea; O'Connor KA et al.; BACKGROUND: Broad-spectrum antibiotics, particularly intravenous cephalosporins, are associated with Clostridium difficile diarrhoea . Diarrhoea due to C . difficile is a growing problem in hospitals, especially among elderly patients . AIM: To establish whether changing an antibiotic policy with the aim of reducing the use of injectable cephalosporins leads to a reduction in the incidence of C . difficile diarrhoea in elderly patients . DESIGN: Retrospective analysis . METHODS: A group of patients who were subject to the new antibiotic policy from the period following July 2000, were compared with patients who were admitted prior to July 2000 and were not subject to the new policy . Infections, antibiotic prescriptions and mortality rates were determined from case notes, and C . difficle diarrhoea rates from microbiological data . RESULTS: Intravenous cephalosporin use fell from 210 to 28 defined daily doses (p < 0.001) following the change in antibiotic policy, with a corresponding increase in piperacillin-tazobactam (p < 0.001) and moxifloxacin (p < 0.001) use . The new policy led to a significant reduction in C . difficile diarrhoea cases . The relative risk of developing C . difficile infection with the old policy compared to the new policy was 3.24 (95%CI 1.07-9.84, p = 0.03) . DISCUSSION: The antibiotic policy was successfully introduced into an elderly care service . It reduced both intravenous cephalosporin use and C . difficile diarrhoea.

J Histochem Cytochem, 2004 Jul, 52(7), 931 - 42
Effect of epsilon toxin-GFP on MDCK cells and renal tubules in vivo; Soler-Jover A et al.; Epsilon toxin (epsilon-toxin), produced by Clostridium perfringens types B and D, causes fatal enterotoxemia, also known as pulpy kidney disease, in livestock . Recombinant epsilon-toxin-green fluorescence protein (epsilon-toxin-GFP) and epsilon-prototoxin-GFP were successfully expressed in Escherichia coli . MTT assays on MDCK cells confirmed that recombinant epsilon-toxin-GFP retained the cytotoxicity of the native toxin . Direct fluorescence analysis of MDCK cells revealed a homogeneous peripheral pattern that was temperature sensitive and susceptible to detergent . epsilon-Toxin-GFP and epsilon-prototoxin-GFP bound to endothelia in various organs of injected mice, especially the brain . However, fluorescence mainly accumulated in kidneys . Mice injected with epsilon-toxin-GFP showed severe kidney alterations, including hemorrhagic medullae and selective degeneration of distal tubules . Moreover, experiments on kidney cryoslices demonstrated specific binding to distal tubule cells of a range of species . We demonstrate with new recombinant fluorescence tools that epsilon-toxin binds in vivo to endothelial cells and renal tubules, where it has a strong cytotoxic effect . Our binding experiments indicate that an epsilon-toxin receptor is expressed on renal distal tubules of mammalian species, including human.

Biochem Biophys Res Commun, 2004 Jul 16, 320(1), 256 - 61
Inhibition effects of (+)-catechin-aldehyde polycondensates on proteinases causing proteolytic degradation of extracellular matrix; Kim YJ et al.; Inhibition effects of (+)-catechin-aldehyde polycondensates against the activity of proteinases, Clostridium histolyticum collagenase (ChC) and human neutrophil elastase (HNE) causing proteolytic degradation of extracellular matrix (ECM), have been investigated . In normal tissues, a balance is reached between the formation and destruction of ECM, leading to a state of homeostasis . However, uncontrolled destruction of ECM contributes to tumor invasion and metastasis . In the measurement of the inhibition activity on ChC and HNE, the polycondensates exhibited superior effects compared to the catechin monomer . Kinetic assays of ChC and HNE inhibition by the polycondensate clearly showed a mixed-type inhibition . These data demonstrate that the polycondensates are a new class of proteinase inhibitors useful for a potent therapeutic agent.

Poult Sci, 2004 Jun, 83(6), 925 - 38
Influence of whole wheat and xylanase on broiler performance and microbial composition and activity in the digestive tract; Engberg RM et al.; An experiment was carried out to study the effect of different forms of wheat (airtight silo stored whole wheat, conventionally stored whole wheat, and ground wheat included in pellets) and dietary xylanase addition on production results and gastrointestinal characteristics of broiler chickens . Ileal viscosity, pancreatic digestive enzyme activities, and the composition and activity of the intestinal microflora were considered as response parameters . Differences between the 2 types of whole wheat with respect to the various measured parameters were marginal, whereas distinct differences were found between pellet-fed birds and birds receiving whole wheat . Whole wheat feeding improved feed conversion ratio and reduced water consumption (P < 0.001) . Compared with pellets, whole wheat increased the relative weight of pancreas and gizzard and the dry matter concentration of gizzard content (P < 0.001) . Whole wheat feeding reduced the pH in the gizzard contents (P < 0.01) and increased ileal viscosity . The addition of xylanase reduced ileal viscosity in birds receiving whole wheat to the same level as in pellet-fed birds . Whole wheat feeding resulted in lower activities of amylase in pancreatic tissue (P = 0.054), whereas xylanase addition increased chymotrypsin (P = 0.030) and lipase activities (P = 0.052) . Whole wheat feeding resulted in lower intestinal numbers of lactose-negative enterobacteria (P < 0.05) and tended to reduce the ileal and cecal numbers of Clostridium perfringens (P < or = 0.08) . It is concluded that whole wheat feeding stimulates gizzard function, which in turn prevents potentially pathogenic bacteria from entering the intestinal tract.

J Bacteriol, 2004 Jul, 186(13), 4218 - 27
Regulation of expression of cellulosomes and noncellulosomal (hemi)cellulolytic enzymes in Clostridium cellulovorans during growth on different carbon sources; Han SO et al.; Cellulosomes and noncellulosomal (hemi)cellulolytic enzymes are produced by Clostridium cellulovorans to degrade plant cell walls . To understand their synergistic relationship, changes in mRNA and protein expression in cellulosomes and noncellulosomal (hemi)cellulolytic enzymes (hereafter called noncellulosomal enzymes) of cultures grown on cellobiose, cellulose, pectin, xylan, and corn fiber or mixtures thereof were examined . Cellulase expression, favored particularly by the presence of Avicel, was found with all substrates . Comparison of cellulosome and noncellulosomal enzymes showed that expression profiles were strongly affected by the carbon source . High xylanase or pectate lyase expression was observed when C . cellulovorans was grown on xylan or pectin, respectively . Mixed carbon substrates (cellulose-pectin-xylan mixture or corn fiber) induced a wider variety of enzymes than a single carbon source, such as cellobiose, pectin, or xylan . Cellulosomal proteome profiles were more affected by the carbon source than the noncellulosomal enzymes . Transcription and protein analyses revealed that cellulosomes and noncellulosomal enzymes were expressed simultaneously on mixed carbon sources, but their degree of inducibility varied when the substrate was either cellulose or cellobiose . Cellulosomes and noncellulosomal enzymes had synergistic activity on various carbon substrates . These results indicated that expression of plant cell wall-degrading enzymes is highly influenced by the available carbon source and that synergy between cellulosomes and noncellulosomal enzymes contribute to plant cell wall degradation.

Vet Clin North Am Food Anim Pract, 2004 Jul, 20(2), 379 - 91, vii-viii
Clostridial disease associated with neurologic signs: tetanus, botulism, and enterotoxemia; Rings DM; Clostridial infections are found worldwide in almost all species of animals and may involve a variety of body systems and present with a diversity of clinical signs . Most damage done through clostridial infections is due to the action of toxins released from the bacteria.Thus, disease caused by Clostridium spp should more correctly be called intoxication . Two prominent clostridial infections are associated with neurologic signs: Clostridium botulinum and C tetani . In both infections, the mechanism that is responsible for causing the problem is similar, despite the remarkable difference in clinical presentation . In addition, neurologic signs are described with C perfringens types C and D but are not the dominant feature of these diseases.

N Engl J Med, 2004 Jun 17, 350(25), 2564 - 71
Clostridium infections associated with musculoskeletal-tissue allografts; Kainer MA et al.; BACKGROUND: Allografts are commonly used in orthopedic reconstructive surgery . In 2001, approximately 875,000 musculoskeletal allografts were distributed by U.S . tissue banks . After the death from Clostridium sordellii sepsis of a 23-year-old man who had received a contaminated allograft from a tissue bank (Tissue Bank A), the Centers for Disease Control and Prevention initiated an investigation, including enhanced case finding, of the methods used for the recovery, processing, and testing of tissue . METHODS: A case of allograft-associated clostridium infection was defined as a culture-proven infection of a surgical site within one year after allograft implantation, from January 1998 to March 2002 . We traced tissues to tissue banks that recovered and processed these tissues . We also estimated the rates of and risk ratios for clostridium infections for tissues processed by the implicated tissue bank and reviewed processing and testing methods used by various tissue banks . RESULTS: Fourteen patients were identified, all of whom had received allografts processed by Tissue Bank A . The rates of clostridium infection were 0.12 percent among patients who received sports-medicine tissues (i.e., tendons, femoral condyles, menisci) from Tissue Bank A and 0.36 percent among those who received femoral condyles in particular . The risk-ratio estimates for clostridium infections from tissues processed by Tissue Bank A, as compared with those from other tissue banks, were infinite (P<0.001) for musculoskeletal allografts, sports-medicine tissues, or tendons . Because Tissue Bank A cultured tissues only after treating them with a nonsporicidal antimicrobial solution, some test results were probably false negatives . Tissues from implicated donors were released despite the isolation of clostridium or bowel flora from other anatomical sites or reports of infections in other recipients . CONCLUSIONS: Clostridium infections were traced to allograft implantation . We provide interim recommendations to enhance tissue-transplantation safety . Tissue banks should validate processes and culture methods . Sterilization methods that do not adversely affect the functioning of transplanted tissue are needed to prevent allograft-related infections .

Pediatr Res, 2004 Sep, 56(3), 366 - 70 Epub 2004 Jun 16.
Early intestinal bacterial colonization and necrotizing enterocolitis in premature infants: the putative role of Clostridium; de la Cochetiere MF et al.; Necrotizing enterocolitis (NEC) is among the most severe conditions that can affect preterm infants . Although the etiology of NEC remains unknown, initial bacterial colonization could play a pivotal role in the development of NEC . To further explore the putative relationship between pathogen microorganisms and NEC, we conducted a prospective case-control study in 12 preterm infants with a new approach based on molecular techniques . Over an inclusion period of 24 mo, 12 neonates of <34 wk gestational age admitted to the neonatal unit were enrolled . The group included three cases of NEC, and nine control infants without evidence of NEC who were matched for gestational age and birth weight . Stool samples were collected at weekly intervals from all infants . PCR and temporal temperature gradient gel electrophoresis of 16S ribosomal DNA were used to detect the establishment of bacterial communities in the digestive tract . A salient feature of the bacteriological pattern was observed only in the three infants who later developed NEC: A band corresponding to the Clostridium perfringens subgroup could be detected in early samples, before diagnosis . There was no evidence for this specific band in any of the nine controls . To our knowledge, the current report is the first to demonstrate that the use of molecular techniques based on the study of bacterial 16S rRNA genes allowed the recognition of C . perfringens species in the first 2 wk of life of three infants who later displayed symptoms of NEC . A significant temporal relationship was thus established between early colonization by Clostridium and the later development of NEC . Compared with conventional bacteriological culturing methods, the use of this new molecular approach to analyze the gastrointestinal ecosystem should therefore allow a more complete and rapid assessment of intestinal flora . Although the current data do not constitute definitive proof that the identified bacterial species was a causative agent in the development of NEC, they outline the promise of this new technique based on molecular biology, and suggest that large-scale studies on a much wider population at high risk for NEC may be warranted.

Glycobiology, 2004 Oct, 14(10), 923 - 9 Epub 2004 Jun 16.
Crystal structures of Erythrina cristagalli lectin with bound N-linked oligosaccharide and lactose; Turton K et al.; Erythrina cristagalli lectin (ECL) is a galactose-specific legume lectin . Although its biological function in the legume is unknown, ECL exhibits hemagglutinating activity in vitro and is mitogenic for T lymphocytes . In addition, it has been recently shown that ECL forms a novel conjugate when coupled to a catalytically active derivative of the type A neurotoxin from Clostridium botulinum, thus providing a therapeutic potential . ECL is biologically active as a dimer in which each protomer contains a functional carbohydrate-combining site . The crystal structure of native ECL was recently reported in complex with lactose and 2'-fucosyllactose . ECL protomers adopt the legume lectin fold but form non-canonical dimers via the handshake motif as was previously observed for Erythrina corallodendron lectin . Here we report the crystal structures of native and recombinant forms of the lectin in three new crystal forms, both unliganded and in complex with lactose . For the first time, the detailed structure of the glycosylated hexasaccharide for native ECL has been elucidated . The structure also shows that in the crystal lattice the glycosylation site and the carbohydrate binding site are involved in intermolecular contacts through water-mediated interactions.

Pathol Res Pract, 2004, 200(3), 241 - 5
Hemophagocytic syndrome associated with clostridial infection in a pancreatic carcinoma patient; Chinen K et al.; This report describes the autopsy case of a 71-year-old man presenting with clostridial infection and hemophagocytic syndrome (HS) . The patient underwent pancreatoduodenectomy for a pancreatic tumor, and a histological examination revealed an invasive ductal adenocarcinoma . Multiple peritoneal metastases were noted when laparotomy was performed because of postoperative ileus 2 months after the initial operation . Then, acutely progressive anemia associated with fever developed in the patient before death . The autopsy revealed advanced cancer dissemination and HS . In addition, systemic spread of clostridium, confirmed by the polymerase chain reaction method, had resulted in generalized bleb formation . The clostridial infection appeared to be responsible for the HS . This case indicates that HS may occur as a result of clostridial infection.

J Neurochem, 2004 Jul, 90(1), 9 - 18
Differential effects of Rho GTPases on axonal and dendritic development in hippocampal neurones; Ahnert-Hilger G et al.; Formation of neurites and their differentiation into axons and dendrites requires precisely controlled changes in the cytoskeleton . While small GTPases of the Rho family appear to be involved in this regulation, it is still unclear how Rho function affects axonal and dendritic growth during development . Using hippocampal neurones at defined states of differentiation, we have dissected the function of RhoA in axonal and dendritic growth . Expression of a dominant negative RhoA variant inhibited axonal growth, whereas dendritic growth was promoted . The opposite phenotype was observed when a constitutively active RhoA variant was expressed . Inactivation of Rho by C3-catalysed ADP-ribosylation using C3 isoforms (Clostridium limosum, C3(lim) or Staphylococcus aureus, C3(stau2)), diminished axonal branching . By contrast, extracellularly applied nanomolar concentrations of C3 from C . botulinum (C3(bot)) or enzymatically dead C3(bot) significantly increased axon growth and axon branching . Taken together, axonal development requires activation of RhoA, whereas dendritic development benefits from its inactivation . However, extracellular application of enzymatically active or dead C3(bot) exclusively promotes axonal growth and branching suggesting a novel neurotrophic function of C3 that is independent from its enzymatic activity.

FEMS Immunol Med Microbiol, 2004 Jul 1, 41(3), 237 - 42
Human antibody response to surface layer proteins in Clostridium difficile infection; Drudy D et al.; Clostridium difficile is a major cause of infectious diarrhoea in hospitalised patients . Surface layer proteins (SLPs) are the most abundant surface localised proteins expressed by C . difficile . The aim of this study was to examine the humoral immune response to C . difficile SLPs and its potential role in protection from C . difficile associated diarrhoea (CDAD) . Serum antibodies to SLPs from C . difficile were measured by ELISA in a cohort of 146 patients (55 patients with CDAD, 34 asymptomatic carriers, and 57 controls) . No significant difference was detected in serum IgM, IgA or IgG antibody levels between cases, carriers or control groups at any of the time points tested . However, patients with recurrent episodes of C . difficile diarrhoea had significantly lower IgM-anti-SLP levels than patients with a single episode on days 1, 3, 6 and 9 (p = 0.05, p = 0.009, p = 0.02, p = 0.049) . The adjusted odds ratio for recurrent diarrhoea associated with a low day 3 serum IgM anti-SLP antibody level was 24.5 (95% confidence interval; 1.6-376.3) . Further studies which examine the specific anti-SLP antibody responses to the colonising strain are warranted to determine if immune responses to C . difficile SLPs play a role in protection from CDAD.

Microb Pathog, 2004 Jul, 37(1), 47 - 54
Intracellular signalling and cytoskeletal rearrangement involved in Yersinia pestis plasminogen activator (Pla) mediated HeLa cell invasion; Benedek O et al.; Yersinia pestis, the etiologic agent of plague is a highly invasive organism being able to invade non-phagocytic epithelial cells . Its plasminogen activator (Pla), encoded by the pPCP1 plasmid plays a pivotal role in internalisation of bacteria by HeLa cells . The aim of this study was to analyse the intracellular signalling processes and cytoskeletal rearrangement events associated with invasion . Wortmannin caused a 50% decrease of invasiveness at 50nM concentration pointing to the involvement of phosphatidyl-inosinol-4 kinase (PtINs4) . Pre-treatment with staurosporin, a potent inhibitor of protein kinases (PKs) and with genistein, a specific tyrosine kinase inhibitor decreased the number of internalised bacteria about seven-fold and two-fold, respectively, indicating the involvement of PKs including tyrosine kinases in Pla-mediated internalisation . Cytochalasin D, an actin polymerisation inhibitor, C3 exoenzyme of Clostridium botulinum, a specific inhibitor of small GTPase Rho, and NDGA, a 5-lipoxygenase inhibitor also involved in Rho activation strongly reduced the number of internalised bacteria revealing the role of cytoskeletal events in the invasion process . All the tested inhibitors changed the invasion but not the adhesion pattern of the Pla producing recombinant strain . Actin rearrangement could also be visualised also with rhodamin-phalloidin staining.

Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi, 2004 May, 20(3), 376 - 8
{Preparation and characterization of monoclonal antibody against Clostridium difficile toxin A}; Fu SW et al.; AIM: To prepare monoclonal antibodies (mAbs) against Clostridium difficile toxin A and identify their properties . METHODS: BALB/c mice were immunized with C.difficile toxin A . The splenocytes from immunized mice were fused with myeloma cells Sp2/0 . The hybridoma cells were screened by indirect ELISA and limiting dilution method . The titer and relative affinity of ascitic mAbs were determined by ELISA . Specificity of mAbs was analyzed by Western blot . RESULTS: Six hybridoma cell lines (2H7, 3E9, 4B5, 5C10, 6G8 and 8A1) secreting mAbs against C.difficile toxin A were obtained . The Ig classes and subclasses of mAbs 2H7, 3E9 and 6G8 were IgM, mAbs 4B5 and 8A1 were IgG1, and mAb 5C10 was IgG2a . All 6 mAbs had no neutralization activity . Epitope recognized by 5 mAbs(2H7, 4B5, 5C10, 6G8 and 8A1) differed from that by mAb 3E9 . Relative affinities of mAbs 8A1 and 4B5 were all above 10(5), and those of other 4 mAbs were 10(4) . Western blot analysis no-denatured PAGE showed that all 6 mAbs reacted to C.difficile toxin A with M(r) being 55 x 10(4), and under the condition of denatured SDS-PAGE, Western blot analysis showed that all 6 mAbs reacted to subunits of C.difficile toxin A with M(r) being 5 x 10(4)-24 x 10(4) . CONCLUSION: Six mAbs against C.difficile toxin A with high titers were obtained successfully with satisfactory specificity and relative affinity, which will be useful for detection of C.difficile toxin A.

J Biol Chem, 2004 Aug 13, 279(33), 34785 - 93 Epub 2004 Jun 10.
The family 11 carbohydrate-binding module of Clostridium thermocellum Lic26A-Cel5E accommodates beta-1,4- and beta-1,3-1,4-mixed linked glucans at a single binding site; Carvalho AL et al.; Modular glycoside hydrolases that attack recalcitrant polymers generally contain noncatalytic carbohydrate-binding modules (CBMs), which play a critical role in the action of these enzymes by localizing the appended catalytic domains onto the surface of insoluble polysaccharide substrates . Type B CBMs, which recognize single polysaccharide chains, display ligand specificities that are consistent with the substrates hydrolyzed by the associated catalytic domains . In enzymes that contain multiple catalytic domains with distinct substrate specificities, it is unclear how these different activities influence the evolution of the ligand recognition profile of the appended CBM . To address this issue, we have characterized the properties of a family 11 CBM (CtCBM11) in Clostridium thermocellum Lic26A-Cel5E, an enzyme that contains GH5 and GH26 catalytic domains that display beta-1,4- and beta-1,3-1,4-mixed linked endoglucanase activity, respectively . Here we show that CtCBM11 binds to both beta-1,4- and beta-1,3-1,4-mixed linked glucans, displaying K(a) values of 1.9 x 10(5), 4.4 x 10(4), and 2 x 10(3) m(-1) for Glc-beta1,4-Glc-beta1,4-Glc-beta1,3-Glc, Glc-beta1,4-Glc-beta1,4-Glc-beta1,4-Glc, and Glc-beta1,3-Glc-beta1,4-Glc-beta1,3-Glc, respectively, demonstrating that CBMs can display a preference for mixed linked glucans . To determine whether these ligands are accommodated in the same or diverse sites in CtCBM11, the crystal structure of the protein was solved to a resolution of 1.98 A . The protein displays a beta-sandwich with a concave side that forms a potential binding cleft . Site-directed mutagenesis revealed that Tyr(22), Tyr(53), and Tyr(129), located in the putative binding cleft, play a central role in the recognition of all the ligands recognized by the protein . We propose, therefore, that CtCBM11 contains a single ligand-binding site that displays affinity for both beta-1,4- and beta-1,3-1,4-mixed linked glucans.

Zhonghua Er Ke Za Zhi, 2004 May, 42(5), 367 - 70
{Detection of four human herpesviruses DNA and virus-specific IgM antibody in blood specimens of infants}; Dong GP et al.; OBJECTIVE: To establish a restriction endonuclease pattern which could detect and differentiate four major human herpesviruses by polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), DNA cloning and sequence analysis . METHODS: A pair of primer, which was designed according to sequences in well-conserved regions of the DNA polymerase gene in human herpesviruses, was designed to amplify herpes simplex virus type 1 and 2 (HSVI/II), Epstein-Barr virus (EBV) and cytomegalovirus (CMV) . Sequences of the primers are as follows: P(1) (5'-CGACTTTGCCAGCCTGACC-3') and P(2) (5'-AGTCCGTGTCCCCGTAGATG-3') . DNA of four strains of standard herpesviruses were amplified by PCR, and further studied by DNA cloning, sequence analysis and RFLP . At last, the authors established the PCR-RFLP technique to differentiate the four different herpesviruses . Meanwhile, 75 clinical blood specimens from infants with suspected viral infection and 38 blood specimens from healthy children were evaluated for herpesviruses DNA or virus-specific IgM antibody by PCR-RFLP or by ELISA . RESULTS: The PCR amplified products of four human herpesviruses were from 510 bp to 592 bp in length and were analyzed for herpesvirus types with restriction endonuclease technique . The specificity and sensitivity of this PCR-RFLP were examined . There was no cross-reaction with Escherichia coli, Staphylococcus aureus, hepatitis B virus (HBV), Clostridium neoformans and human-genomic DNA and the lowest detection level was 0.1 fg DNA . Among 75 specimens, 23 were positive by PCR and the positive rate was 30.7%, including 13 for CMV, four for EBV, five for HSVII and one for HSVI after restriction enzyme digestion with BamHI and BstUI, while 10 were positive by ELISA and positive rate was 13.3% . All ELISA-positive specimens were likewise positive by PCR . Thirteen of 65 specimens that were ELISA-negative were tested positive by PCR . An infant with CMV infection was determined with viral DNA and virus-specific IgM antibody in blood at 3, 4 and 6 months after birth, respectively . The result showed that she was still CMV DNA-positive in blood whereas IgM antibody was positive only at month 3 after birth . None of the 38 control blood specimens was positive for herpesvirus by this PCR-RFLP or by ELISA . CONCLUSIONS: This PCR-RFLP technique was specific, sensitive, rapid and accurate in diagnosing herpesviruses infection in infants, and it could detect herpesviruses DNA in specimens which were negative for IgM antibody by ELISA.

Infect Control Hosp Epidemiol, 2004 May, 25(5), 413 - 7
Failure to develop vancomycin-resistant Enterococcus with oral vancomycin treatment of Clostridium difficile; Salgado CD et al.; OBJECTIVE: Oral vancomycin therapy has been a risk factor for turning culture positive for vancomycin-resistant Enterococcus (VRE) . VRE colonization status was reviewed for all patients who received oral vancomycin and underwent prospective cultures . METHODS: Data were extracted from the medical records of all patients receiving oral vancomycin between August 1995 and February 2001 regarding history, hospital course, and perirectal VRE cultures . Hospital policy required contact isolation for patients receiving oral vancomycin until colonization with VRE was excluded . RESULTS: Twenty-six courses of oral vancomycin were given to 22 patients . VRE colonization status after completion of therapy was evaluated for 23 courses in 20 (91%) of these patients . None of these patients became VRE culture positive during a median follow-up of 18 days (range, 9 to 39 days), with a median duration of treatment of 10 days (range, 3 to 58 days), and with a median total dose of 6,500 mg (range, 1,250 to 29,000 mg) . All patients received other antibiotics within 30 days prior to therapy with oral vancomycin, during therapy with oral vancomycin, or both; 95% had received anti-anaerobic therapy and 35% had received parenteral vancomycin . CONCLUSIONS: Even when other risk factors were present, no patient receiving oral vancomycin at our facility subsequently became culture positive for VRE . This suggests that oral vancomycin therapy or other antibiotic use, including anti-anaerobic therapy, may not be a significant independent risk factor for turning culture positive for VRE among patients not previously exposed to the microbe.

Infect Control Hosp Epidemiol, 2004 May, 25(5), 395 - 401
Do infection control measures work for methicillin-resistant Staphylococcus aureus?
Boyce JM, Havill NL, Kohan C, Dumigan DG, Ligi CE.
OBJECTIVE: To review evidence regarding the effectiveness of control measures in reducing transmission of methicillin-resistant Staphylococcus aureus (MRSA) in hospitals . DESIGN: Literature review and surveillance cultures of hospitalized patients at high risk for MRSA colonization or infection . SETTING: A 500-bed, university-affiliated, community teaching hospital . RESULTS: The percentage of nosocomial S . aureus infections caused by MRSA increased significantly between 1982 and 2002, despite the use of various isolation and barrier precaution policies . The apparent ineffectiveness of control measures may be due to several factors including the failure to identify patients colonized with MRSA . For example, cultures of stool specimens submitted for Clostridium difficile toxin assays at one hospital found that 12% of patients had MRSA in their stool, and 41% of patients with unrecognized colonization were cared for without using barrier precautions . Other factors include the use of barrier precaution strategies that do not account for multiple reservoirs of MRSA, poor adherence of healthcare workers (HCWs) to recommended barrier precautions and handwashing, failure to identify and treat HCWs responsible for transmitting MRSA, and importation of MRSA by patients admitted from other facilities . Control programs that include active surveillance cultures (ASCs) of high-risk patients and use of barrier precautions have reduced MRSA prevalence rates and have been cost-effective . Using a staged approach to implementing ASCs can minimize logistic problems . CONCLUSION: MRSA control programs are effective if they include ASCs of high-risk patients, use of barrier precautions when caring for colonized or infected patients, hand hygiene, and treating HCWs implicated in MRSA transmission.

Behav Pharmacol, 2004 May, 15(3), 233 - 40
Central injection of botulinum neurotoxins: behavioural effects in mice; Luvisetto S et al.; Strains of Clostridium botulinum produce seven antigenically distinct botulinum neurotoxins (BoNTs) designated as serotypes A-G . All serotypes interfere with neural transmission by blocking the release of acetylcholine in cholinergic neurons . They cleave specific sites on proteins of the SNARE {soluble n-ethylmaleimide-sensitive factor (NSF) attachment protein receptor} complex, which play a key role in neuroexocytosis . This study assessed the behavioural effects due to central administration of BoNTs in mice . CD1 mice were injected intracerebroventricularly (icv) with sub-lethal doses of BoNT/A or /B and their behavioural responses in conditioning of active avoidance, object recognition test and pharmacologically induced locomotor activity were tested . Compared to control mice, BoNT-treated mice showed: (1) a reduced capacity to discriminate a novel object within a familiar environment; (2) an enhanced stimulant effect by scopolamine and a depressant effect by oxotremorine on locomotor activity . In contrast, central injection of BoNTs did not alter active avoidance acquisition . These results suggest an in vivo functional alteration due to the action of BoNTs directly administered into the central nervous system . The present data demonstrate that BoNTs may represent an analytical tool for studying the functional role of cholinergic neurons.

Microbiology, 2004 Jun, 150(Pt 6), 1649 - 59
Oxidative stress response in Clostridium perfringens; Jean D et al.; Clostridium perfringens, a strictly anaerobic bacterium, is able to survive when exposed to oxygen for short periods of time and exhibits a complex adaptive response to reactive oxygen species, both under aerobic and anaerobic conditions . However, this adaptive response is not completely understood . C . perfringens possesses specialized genes that might be involved in this adaptive process, such as those encoding superoxide dismutase (SOD), superoxide reductase and alkyl hydroperoxide reductase, but their contribution to the oxidative stress response and their control mechanisms are unknown . By a combination of functional complementation of Escherichia coli strains impaired in either SOD, alkyl hydroperoxide reductase (AhpC) or catalase activity (Cat), transcription analysis and characterization of mutants impaired in regulatory genes, it was concluded that: (i) the product of the sod gene is certainly essential to scavenge superoxide radicals, (ii) the ahpC gene, which is fully induced in all oxidative stress conditions, is probably involved in the scavenging of all intracellular peroxides, (iii) the three rubrerythrin (rbr) genes of C . perfringens do not encode proteins with in vivo H(2)O(2) reductase activity, and (iv) the two rubredoxin (rub) genes do not contribute to the hypothetical superoxide reductase activity, but are likely to belong to an electron transfer chain involved in energy metabolism.

J Clin Microbiol, 2004 Jun, 42(6), 2609 - 17
Multilocus sequence typing analysis of human and animal Clostridium difficile isolates of various toxigenic types; Lemee L et al.; A multilocus sequence typing (MLST) scheme was developed to study the genetic relationships and population structure of 72 Clostridium difficile isolates from various hosts, geographic sources, PCR ribotypes, and toxigenic types (determined by PCR targeting tcdA and tcdB genes) . MLST was performed by DNA sequence analysis of seven housekeeping genes (aroE, ddl, dutA, tpi, recA, gmk, and sodA) . The number of alleles ranged from five (dutA and ddl) to eleven (recA) . Allelic profiles allowed the definition of 34 different sequence types (STs) . These STs lacked correlation with geographic source but were well correlated to toxigenic type . The dendrogram generated from a matrix of pairwise genetic distances showed that animal isolates did not constitute a distinct lineage from human isolates and that there was no hypervirulent lineage within the population of toxigenic human isolates (isolates recovered from pseudomembranous colitis and antibiotic-associated diarrhea did not cluster in distinct lineages) . However, A(-) B(+) variant isolates shared the same ST that appeared as a divergent lineage in the population studied, indicating a single evolutionary origin . The population structure was further examined by analysis of allelic polymorphism . The dendrogram generated from composite sequence-based analysis revealed a homogeneous population associated with three divergent lineages, one of which was restricted to A(-) B(+) variant isolates . C . difficile exhibited a clonal population structure, as revealed by the estimation of linkage disequilibrium (Ia) between loci . The analysis of alleles within clonal complexes estimated that point mutation generated new alleles at a frequency eightfold higher than recombinational exchange, and the congruence of the dendrograms generated from separate housekeeping loci confirmed the mutational evolution of this species.

J Biol Chem, 2004 Sep 3, 279(36), 37544 - 50 Epub 2004 Jun 07.
Stabilization of exocytosis by dynamic F-actin coating of zymogen granules in pancreatic acini; Nemoto T et al.; Reorganization of F-actin in the apical region of mouse pancreatic acinar cells during Ca(2+)-dependent exocytosis of zymogen granules was investigated by two-photon excitation microscopy with intact acini . Granules were rapidly coated with F-actin in response to either agonist stimulation or photolysis of a caged-Ca(2+) compound . Such F-actin coating occurred exclusively at the surface of granules undergoing exocytosis and was prevented either by latrunculin-A, which inhibits actin polymerization, or by Clostridium botulinum exoenzyme C3, which inhibits the small GTPase Rho . Latrunculin-A or exoenzyme C3 also triggered the formation of vacuoles in acinar cells, a characteristic of acute pancreatitis . Stimulation of acini with high concentrations of cholecystokinin, which cause acute pancreatitis in mice, also impaired the F-actin coating of granules and induced vacuole formation . Latrunculin-A reduced the latency to exocytosis but did not affect the total number of exocytic events, suggesting that F-actin slows and further stabilizes exocytosis by facilitating F-actin coating . Rho-dependent F-actin coating of granule membranes thus stabilizes exocytic structures and is necessary for physiological progression of sequetial compound exocytosis in the exocrine pancreas and for prevention of acute pancreatitis.

FEMS Microbiol Lett, 2004 Jun 15, 235(2), 289 - 95
Organization and transcriptional regulation of myo-inositol operon in Clostridium perfringens; Kawsar HI et al.; myo-Inositol operon of Clostridium perfringens strain 13 consists of 13 genes with an upstream divergent regulator, iolR . Transcriptional analysis showed three separate transcripts for the operon of 15.6, 4.6 and 2.0 kb in length . iolR mutation studies showed that IolR is a negative regulator of the operon at transcriptional level . All the transcripts were induced by myo-Inositol in dose- and time-dependent manner . Glucose repressed the expression of all the transcripts of myo-Inositol operon . We also found that the operon was positively regulated by the two-component VirR/VirS system both in the presence and absence of myo-Inositol . This study shows that the global regulatory VirR/VirS system controls the expression of genes related to energy production (e.g . myo-Inositol operon) in addition to the virulence genes of C . perfringens strain 13.

J Hosp Infect, 2004 Jun, 57(2), 144 - 8
The effect of Perasafe and sodium dichloroisocyanurate (NaDCC) against spores of Clostridium difficile and Bacillus atrophaeus on stainless steel and polyvinyl chloride surfaces; Block C; Clostridium difficile is an important cause of nosocomial diarrhoea . The aim of this study was to evaluate the potential for Perasafe, a recently introduced biocide, to contribute to control of C . difficile spores in the patient environment, in comparison with the chlorine-releasing agent sodium dichloroisocyanurate (NaDCC) . These agents were evaluated against a water control, in a surface test on stainless steel and polyvinyl chloride (PVC) floor covering, materials commonly found in the hospital environment . The organisms studied were a toxigenic clinical isolate of C . difficile, and Bacillus atrophaeus (formerly B . subtilis var niger) . The data indicate that in our in vitro system, Perasafe was significantly more active than NaDCC (1000 ppm available chlorine) against C . difficile spores dried on stainless steel surfaces, and against B . atrophaeus on PVC floor covering material, achieving mean log10 reduction factors in viable counts of 6 and 5.5, respectively, at 10 min exposures . Perasafe appeared to be less lethal in 10 min exposures to C . difficile spores fixed on PVC floor covering material . In general, 1000 ppm chlorine generated from NaDCC showed lower log10 reduction factors in viable counts at 10 min, ranging from 0.7 to 1.5, than Perasafe which ranged from 2.7 to 6.0 . The potential efficacy of Perasafe in reducing the density of C . difficile spores in the patient environment in hospitals, nursing homes or other long-stay facilities should be evaluated in field studies .

J Immunol Methods, 2004 May, 288(1-2), 55 - 60
Novel application of an in vitro technique to the detection and quantification of botulinum neurotoxin antibodies; Hall YH et al.; Detection of Clostridium botulinum neurotoxin (BoNT) neutralising antibodies is currently achieved using the mouse lethality assay (MLA) . This technique has provided the majority of the data for vaccine development and, with the increasing use of BoNT as a therapeutic agent, the MLA is the assay of choice to evaluate 'non-responder' antisera . However, the MLA is semi-quantitative and has an animal consumption rate that raises ethical concerns . The development of an alternative is therefore desirable . Here, we describe an in vitro neuronal release assay that may represent such an alternative in terms of both its sensitivity and ability to produce quantitative data . Initially recognised in the course of assessing a novel vaccine candidate, the suitability of this assay has been further explored using an International standard . The results support the conclusion that the detection of neutralising antibodies in human sera should be attempted using this method .

Int Microbiol, 2004 Mar, 7(1), 41 - 52
Signature sequences in diverse proteins provide evidence for the late divergence of the Order Aquificales; Griffiths E et al.; The Aquificales species are presently believed to be the earliest branching lineage within Bacteria . However, the branching order of this group in different phylogenetic trees is highly variable and not resolved . In the present work, the phylogenetic placement of Aquificales was examined by means of a cladistic approach based on the shared presence or absence of definite signature sequences (consisting of conserved inserts or deletions) in many highly conserved and important proteins, e.g . RNA polymerase beta (RpoB), RNA polymerase beta (RpoC), alanyl-tRNA synthetase (AlaRS), CTP synthase, inorganic pyrophosphatase (PPase), Hsp70 and Hsp60 . For this purpose, fragments of the above genes that contained the signature regions were cloned from different Aquificales species (Calderobacterium hydrogenophilum, Hydrogenobacter marinus, and Thermocrinis ruber) and the sequence data were compared with those available from all other species . The presence in Aquificales species of distinctive inserts in Hsp70 and Hsp60 that are not found in any Firmicutes, Actinobacteria, or Thermotoga-Clostridium species excluded them from these groups of Bacteria . The shared presence of prominent indels in the RpoB (>100 amino acids), RpoC (>100 amino acids) and AlaRS (4 amino acids) proteins, which are only found in the various Aquificales species, the Chlamydiae, the CFBG (Cytophaga-Flavobacteria-Bacteroides-green sulfur bacteria) group, and Proteobacteria, strongly suggests their placement within these groups of Bacteria . A specific relationship between Proteobacteria and Aquificales is suggested by the presence in inorganic pyrophosphatase of a 2-amino-acid insert that is uniquely found in these phyla . However, the Aquificales species lacked a number of other protein signatures (e.g . indels in CTP synthase and Hsp70) that are characteristic of Proteobacteria, indicating that they constitute a distinct phylum related to Proteobacteria . These results provide strong and consistent evidence that the Aquificales diverged after the branching of Firmicutes, Actinobacteria, Thermotoga, Deinococcus-Thermus, green nonsulfur bacteria, Cyanobacteria, Spirochetes, Chlamydiae, and CFBG group, but before the emergence of the Proteobacteria.

Int Microbiol, 2004 Mar, 7(1), 3 - 12
IS200: an old and still bacterial transposon; Beuzon CR et al.; IS200 is a mobile element found in a variety of eubacterial genera, such as Salmonella, Escherichia, Shigella, Vibrio, Enterococcus, Clostridium, Helicobacter, and Actinobacillus . In addition, IS200-like elements are found in archaea . IS200 elements are very small (707-711 bp) and contain a single gene . Cladograms constructed with IS200 DNA sequences suggest that IS200 has not spread among eubacteria by horizontal transfer; thus it may be an ancestral component of the bacterial genome . Self-restraint may have favored this evolutionary endurance; in fact, unlike typical mobile elements, IS200 transposes rarely . Tight repression of transposase synthesis is achieved by a combination of mechanisms: inefficient transcription, protection from impinging transcription by a transcriptional terminator, and repression of translation by a stem-loop mRNA structure . A consequence of IS200 self-restraint is that the number and distribution of IS200 elements remain fairly constant in natural populations of bacteria . This stability makes IS200 a suitable molecular marker for epidemiological and ecological studies, especially when the number of IS200 copies is high . In Salmonella enterica, IS200 fingerprinting is extensively used for strain discrimination.

Biochem Biophys Res Commun, 2004 Jun 25, 319(2), 327 - 33
The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells; Nishikawa A et al.; Orally ingested botulinum toxin enters the circulatory system and eventually reaches the peripheral nerves, where it elicits a response of neurological dysfunction . In this study, we report the important findings concerning the mechanism of Clostridium botulinum type C progenitor toxin (C16S) endocytic mechanism . C16S toxin bound to high molecular weight proteins on the surface of human colon carcinoma HT-29 cells and was internalized, but not if the cells were pretreated with neuraminidase . Benzyl-GalNAc which inhibited O-glycosylation of glycoproteins also interfered in the toxin's ability to bind the cell surface . On the other hand, the toxin was internalized in spite of pretreatment of the cells with PPMP, an inhibitor of ganglioside synthesis . These results suggest that the glycoproteins, like mucin, fulfill the important roles of receptor and transporter of C16S toxin.

Protein Expr Purif, 2004 Jul, 36(1), 70 - 5
High-level expression of clostridial sialidase using a ferredoxin gene promoter-based plasmid; Takamizawa A et al.; A "large" sialidase isozyme (NanI) from Clostridium perfringens is a representative microbial sialidase with broad substrate specificity, being used for the analysis of sialoglycoconjugates . It is also a possible virulence factor . However, purification of the native enzyme in a large quantity is not practical due to its low productivity . To obtain the enzyme in a satisfactory yield, a gene encoding the NanI was transcriptionally fused to the fdx gene promoter (P(fdx)) in a shuttle-vector, pFF, and transformed into C . perfringens 13 . The resultant strain released the enzyme into the culture medium, as the original strain does . The enzyme activity increased during the first 6 h of culture and thereafter remained at maximal levels . The maximal activity was approximately 3000-fold compared with that of the original strain, and 15-fold compared with that of recombinant Escherichia coli, which possesses extra copies of the tRNA gene for selected rare codons . This suggests the usefulness of a P(fdx)-based plasmid for expressing AT-rich genes in C . perfringens . The enzyme was successfully purified by two-step procedure with a specific activity of 2860 U/mg using 2'-(4-methylumbelliferyl)-alpha-D-N-acetylneuraminic acid and a yield of 1.69 mg of NanI per 100 ml of culture . The method described here can facilitate purification of NanI in enough quality and quantity to analyze the role of sialoglycoconjugates in cells and the pathogenic importance of NanI sialidase.

Kansenshogaku Zasshi, 2004 Apr, 78(4), 312 - 9
{A nosocomial outbreak of diarrhea caused by toxin A-negative, toxin B-positive Clostridium difficile in a cancer center hospital}; Sato H et al.; Between February and July 2001, 15 patients were diagnosed as Clostridium difficile-associated diarrhea in a ward of hematological neoplasm and lung cancer in a cancer center hospital . Of these 15 patients, 10 had malignant lymphoma, and 12 and 11 had exposure to antimicrobial agents and cancer chemotherapy, respectively, before the onset of diarrhea . Toxin A-positive, toxin B-positive (A+ B+) C . difficile was recovered from five patients and the remaining 10 patients suffered from diarrhea caused by toxin A-negative, toxin B-positive (A- B+) strains . All of the 10A- B+ isolates represented an identical banding pattern by PCR ribotyping and classified into one type (two subtypes) by pulsed field gel electrophoresis typing, indicating that a nosocomial outbreak of diarrhea caused by A- B+ C . difficile occurred among the patients hospitalized on this ward . Detection of toxin A in stool specimens by a toxin A detection kit was performed on 14 patients . Although two patients who carried A+ B+ strains were positive for toxin A assay, toxin A detection test was negative in 12 patients including 10 patients with A- B+ C . difficile infection . Diagnosis of C . difficile-associated diarrhea by combination of toxin A assay in feces and culture of C . difficile could successfully lead to recognition of an outbreak caused by A- B+ C . difficile in a cancer center hospital.

J Bacteriol, 2004 Jun, 186(12), 3922 - 7
Spreading factors of Mycoplasma alligatoris, a flesh-eating mycoplasma; Brown DR et al.; Mycoplasma alligatoris causes lethal invasive disease of alligators and caimans . A homolog of the nagH gene, encoding a hyaluronidase secreted by Clostridium perfringens, and a C . perfringens hyaluronidase nagI or nagK pseudogene were discovered in the M . alligatoris genome . The nagH gene was detected by PCR in the closest relative of M . alligatoris, Mycoplasma crocodyli, but not in 40 other species representing the Mycoplasma hominis, Mycoplasma pneumoniae, and Spiroplasma phylogenetic clusters . The hyaluronidase activity in the cellular fraction of M . alligatoris and M . crocodyli SP4 broth cultures was equivalent to 10(-16) U of Streptomyces hyalurolyticus hyaluronidase CFU(-1) . Negligible activity was present in the cell-free supernatant fraction . No chondroitinase activity was detected . There is also a novel homolog of the nanI gene, which encodes a sialidase secreted by C . perfringens, in the M . alligatoris genome . The signature YRIP and SXDXGXTW motifs and catalytic residues of the clostridial sialidase are conserved in the mycoplasmal gene, but the leader sequence necessary for its secretion by C . perfringens is absent . The gene was not detected by PCR in any other mycoplasma . Potent cell-associated sialidase activity was present in M . alligatoris colonies on agar but not in the cell-free supernatants of broth cultures or in M . crocodyli . The presence of hyaluronidase and sialidase in M . alligatoris is consistent with the rapid invasiveness and necrotizing effects of this organism, and the lack of sialidase in M . crocodyli is consistent with its comparatively attenuated virulence . This genetic and biochemical evidence suggests that the spreading factors hyaluronidase and sialidase, a combination unprecedented in mycoplasmas, are the basis of the virulence of M . alligatoris.

J Nutr, 2004 Jun, 134(6), 1487 - 92
Dietary antibiotic growth promoters enhance the bioavailability of alpha-tocopheryl acetate in broilers by altering lipid absorption; Knarreborg A et al.; The influence of intestinal microbial bile salt deconjugation on absorption of fatty acids and alpha- and gamma-tocopherol was investigated in a trial with Ross 208 broilers . Birds (n = 1600) were assigned to 4 dietary treatments: no supplementation or supplementation of antibiotics (salinomycin, 40 mg/kg feed and avilamycin, 10 mg/kg feed), and inclusion of either animal fat (10 g/100 g feed) or soybean oil (10 g/100 g feed) in the diet . At d 7, 14, 21, and 35 of age, the intestinal number of the bile salt hydrolase-active bacteria Clostridium perfringens, the concentration of conjugated and unconjugated bile salts, the ileal absorption of fatty acids and tocopherols, and the blood plasma concentrations of tocopherols were measured . All variables were significantly influenced by bird age . C . perfringens counts were lower and bile salt concentrations were greater in birds fed soybean oil . The supplementation of antibiotics reduced the numbers of C . perfringens in the small intestine and reduced the concentration of unconjugated bile salts . The ileal absorption of fatty acids and alpha-tocopherol, as well as the plasma concentration of alpha-tocopherol, was greater in birds fed antibiotics . The absorption and plasma concentration of gamma-tocopherol were not influenced by antibiotics . Unlike gamma-tocopherol, which is present solely as the free alcohol, the major proportion of dietary alpha-tocopherol is present as alpha-tocopheryl acetate, which requires a bile salt-dependent enzymatic hydrolysis before absorption . In conclusion, proper digestion of lipid-soluble compounds is highly dependent on an adequate concentration of bile salts in the small intestine to provide proper lipid emulsification and activation of lipolytic enzymes.

J Biochem (Tokyo), 2004 May, 135(5), 631 - 7
Functional role of RhoA in growth regulation of primary hepatocytes; Dohda T et al.; The expression, activation and involvement in growth regulation of a small GTPase, RhoA, were examined in rat primary hepatocyte cultures . Hepatocytes freshly isolated from liver expressed RhoA protein at high levels . The total level of RhoA protein in the cells decreased markedly within a day in monolayer cultures . Thereafter, RhoA expression recovered as cell-cell attachment occurred during the culture . On the other hand, the level of the active form of RhoA decreased as the culture proceeded . Ca(2+) depletion in the medium to disrupt cadherin engagement triggered RhoA activation without de novo protein synthesis, indicating cadherin engagement regulates RhoA activation in hepatocytes . Hepatocyte growth stimulation by HGF was enhanced by Ca(2+) depletion or introduction of a constitutively active form of RhoA . The Clostridium botulinum C3 enzyme inhibited hepatocyte growth with stimulation by HGF . These results suggest that RhoA has a crucial role in hepatocyte growth control.

Biochemistry (Mosc), 2004 Apr, 69(4), 427 - 8
Comparative characterization of extracellular and intracellular hydrocarbons of Clostridium pasteurianum; Bagaeva TV et al.; Extracellular and intracellular hydrocarbons produced by Clostridium pasteurianum VKM 1774 during cultivation on glucose-containing media in an argon atmosphere or in the presence of carbon dioxide and molecular hydrogen were analyzed by gas-liquid chromatography . Intracellular hydrocarbons were 50-55% (C25-C35) n-alkanes . Carbon dioxide and molecular hydrogen stimulated synthesis of extracellular hydrocarbons, which comprised 90-95% (C11-C24) n-alkanes.

Am J Perinatol, 2004 May, 21(4), 173 - 82
Tetanus in pregnancy; Sheffield JS et al.; Tetanus remains a leading cause of maternal and neonatal morbidity and mortality in developing countries . It is caused by the release of two toxins produced by Clostridium tetani, a noninvasive gram-positive anaerobic bacillus . Tetanospasmin is taken up by the neuronal end plates and prevents neurotransmitter release at the synaptic junction . This leads to spasms and is irreversible . Recovery requires the formation of new neurons and may take months . Generalized muscle spasm, respiratory compromise, and autonomic dysfunction are all common clinical manifestations . Diagnosis is based mainly on history and clinical examination . The management of the pregnant woman is similar to the nonpregnant individual . The main objectives are prompt prevention of further toxin absorption, wound debridement, antibiotic therapy, and aggressive supportive care . Primary and secondary prevention protocols are important worldwide because tetanus is a preventable disease . The tetanus toxoid vaccine can be given in pregnancy.

New Microbiol, 2004 Apr, 27(2), 187 - 9
Immunity to tetanus of a rural population in a Greek county; Mendrinou E et al.; Tetanus is still a common problem in countries with poor health conditions . On the contrary, where there is a systematic program of vaccination in children it is very rare . The aim of this study was to check the immunity level of a representative sample of rural people from villages of Achaia County in Southern Greece . Samples were taken from 140 locals during a six-month period (January till July 2002) . In each sample, we estimated IgG antibodies against Clostridium tetani toxin . A protection level of 0.1 IU/ml was set . 15.7% of the people had sufficient immunization cover while a statistically significant superiority of immunized men was found . The results of this study revealed low immunity percentage of the examined inhabitants and a vaccination program against the disease has been proposed to the local health authorities.

J Antimicrob Chemother, 2004 Jul, 54(1), 168 - 72 Epub 2004 May 26.
Long-term surveillance of cefotaxime and piperacillin-tazobactam prescribing and incidence of Clostridium difficile diarrhoea; Wilcox MH et al.; OBJECTIVES: We followed the effects of changes to a new antibiotic policy favouring a ureidopenicillin as opposed to a third-generation cephalosporin on the long-term incidence of Clostridium difficile diarrhoea (CDD) and antibiotic utilization in a large Elderly Medicine Unit . PATIENTS AND METHODS: In 1999, piperacillin-tazobactam was added to the formulary in Elderly Medicine and its use promoted in preference to cefotaxime . Following review and feedback to clinicians of surveillance data, cefotaxime prescribing was actively restricted during 2000-2001 . An audit of prescriber adherence to antibiotic policy was carried out by reviewing the records of 159 patients during February-April 2001 . In December 2001, due to manufacturer production problems, supply of piperacillin-tazobactam was stopped . We performed standardized period prevalence surveillance (February-April) allowing comparisons of antibiotic utilization and CDD incidence during the 5 year study period (1998-2002) . RESULTS: CDD incidence did not change significantly (P>0.1) during 1998-1999 despite a marked increase in piperacillin-tazobactam prescribing . However, when cefotaxime prescribing was curtailed in 2001, CDD rates decreased (in four of five wards) and overall by 52% (P=0.008) . When piperacillin-tazobactam became unavailable in 2002, despite advice to the contrary cefotaxime prescribing rose five-fold, and CDD rates increased in four of five wards and by 232% (P<0.01) overall . Adherence to antibiotic policy introduced in 2000 was good (81% accordance); 94%, 88% and 73% of patients with cellulitis, urinary tract and respiratory tract infection, respectively, received appropriate antibiotics . CONCLUSIONS: Long-term prescribing of piperacillin-tazobactam in Elderly Medicine in preference to cefotaxime is associated with reduced rates of CDD . However, unless cephalosporin prescribing is curtailed, the beneficial effects on CDD rates may be missed . This is one of few studies to document adverse effects due to loss of antibiotic supply.

J Antimicrob Chemother, 2004 Jul, 54(1), 211 - 6 Epub 2004 May 26.
A double-blind randomized controlled trial of fusidic acid and metronidazole for treatment of an initial episode of Clostridium difficile-associated diarrhoea; Wullt M et al.; OBJECTIVES: Few treatment options are currently available to treat patients suffering from an initial episode of Clostridium difficile-associated diarrhoea (CDAD) . PATIENTS AND METHODS: A prospective, randomized controlled, double-blind trial was conducted to compare the efficacy of fusidic acid and metronidazole for treatment of patients experiencing a first episode of CDAD . The primary outcomes were clinical cure and clearance of C . difficile toxin determined on days 8-13, and secondary outcomes were clinical recurrence and reappearance of C . difficile toxin evaluated on days 35-40 . RESULTS: Of the patients in the fusidic acid group, 83% were clinically cured in comparison to 93% in the metronidazole group (P=0.116) at the first follow-up visit . Clearance of C . difficile toxin did not differ between the two groups at that time . Clinical recurrence and reappearance of C . difficile toxin were noted in 27% and in 13% of the patients receiving fusidic acid, respectively and in 29% and 10% of those given metronidazole at the second follow-up on days 35-40 . CONCLUSION: Since three of the four primary and secondary outcomes were almost identical for the two groups, the results indicate that fusidic acid is as effective as metronidazole in curing an initial episode of CDAD and can therefore be considered as an adequate alternative for treatment of this disease.

Otolaryngol Clin North Am, 2004 Jun, 37(3), 559 - 66
Management of pharyngoesophageal spasm with Botox; Chao SS et al.; Pharyngoesophageal spasm following laryngectomy can result in failure of tracheoesophageal (TE) speech and dysphagia . Chemical denervation with Clostridium botulinum toxin (Botox) is effective in relieving pharyngeal constrictor spasm, thereby facilitating TE speech production . This article reviews the technique, results,and complications regarding the use of Botox in the management of TE speech failure associated with pharyngoesophageal spasm.

Arch Pathol Lab Med, 2004 Jun, 128(6), 653 - 62
Clostridium botulinum and the clinical laboratorian: a detailed review of botulism, including biological warfare ramifications of botulinum toxin; Caya JG et al.; OBJECTIVE: This review article is designed to thoroughly familiarize all health care professionals with the history, classification, epidemiology, clinical characteristics, differential diagnosis, diagnostic evaluation (including laboratory-based testing), treatment, and prognosis of botulism . It is especially targeted toward clinical laboratorians and includes a detailed enumeration of the important clinical laboratory contributions to the diagnosis, treatment, and monitoring of patients with botulism . Finally, the bioterrorism potential for botulism is discussed, with an emphasis on the clinical laboratory ramifications of this possibility . DATA SOURCES: Included medical periodicals and textbooks accessioned from computerized and manual medical literature searches . More than 1000 medical works published from the 1800s through 2003 were retrieved and reviewed in this process . DATA SYNTHESIS: Pertinent data are presented in textual and tabular formats, the latter including 6 tables presenting detailed information regarding the clinical parameters, differential diagnosis, diagnostic studies, laboratory testing, and therapeutic approaches to botulism . CONCLUSIONS: Because botulism is such a rare disease, a keen awareness of its manifestations and prompt diagnosis are absolutely crucial for its successful treatment . The bioterrorism potential of botulism adds further urgency to the need for all health care professionals to be familiar with this disease, its proper evaluation, and timely treatment; the need for such urgency clearly includes the clinical laboratory.

Shanghai Kou Qiang Yi Xue, 1997 Mar, 6(1), 23 - 5
{The influences of lanthanum,cerium and fluorine ions to the activity of collagenase degrading cemental collagens}; Li J et al.; The influences of trace elements lanthanum,cerium and fluorine to the activity of clostridium histolyticum collagenase(CHC) during the period of CHC degrading the cemental root collagen in vitro is studied.The results reveal that La(3+) can significantly inhibit the activity of the CHC than Ce(3+) and F(-) . Therefore it suggests that lanthanum may be used as a new kind of element in preventing root caries.The mechanisms of above functions are also discussed.

Acta Crystallogr D Biol Crystallogr, 2004 Jun, 60(Pt 6), 1139 - 41 Epub 2004 May 21.
Crystallization and preliminary crystallographic analysis of a novel haemolytic lectin from the mushroom Laetiporus sulphureus; Mancheno JM et al.; The novel haemolytic lectin from the parasitic mushroom Laetiporus sulphureus (LSL) is a homotetramer (approximately 140 kDa) composed of subunits associated by non-covalent bonds . It exhibits haemagglutination and haemolytic activities, both of which are inhibited by N-acetyllactosamine . The structural similarity found between LSL and the bacterial pore-forming toxins mosquitocidal toxin (MTX2) from Bacillus sphaericus and alpha-toxin from Clostridium septicum points to a mechanism of biological action involving the formation of pores in the target membranes . LSL has been crystallized using the hanging-drop vapour-diffusion method at 291 K . Diffraction-quality hexagonal crystals have unit-cell parameters a = b = 101.8, c = 193.9 angstroms and belong to space group P6(3)22 . A 2.7 angstroms native data set was collected with an Rmerge of 9.2% .

Proc Natl Acad Sci U S A, 2004 Jun 1, 101(22), 8503 - 8 Epub 2004 May 24.
Corticotropin-releasing hormone (CRH) requirement in Clostridium difficile toxin A-mediated intestinal inflammation; Anton PM et al.; Clostridium difficile, the causative agent of antibiotic-associated colitis, mediates inflammatory diarrhea by releasing toxin A, a potent 308-kDa enterotoxin . Toxin A-induced inflammatory diarrhea involves many steps, including mucosal release of substance P (SP) corticotropin-releasing hormone (CRH) and neutrophil transmigration . Here we demonstrate that, compared with wild type, mice genetically deficient in CRH (Crh(-/-)) have dramatically reduced ileal fluid secretion, epithelial cell damage, and neutrophil transmigration 4 h after intraluminal toxin A administration . This response is associated with diminished mucosal activity of the neutrophil enzyme myeloperoxidase compared with that of wildtype mice . In wild-type mice, toxin A stimulates an increase in intestinal SP content compared with buffer administration . In contrast, toxin A administration in Crh(-/-) mice fails to result in an increased SP content . Moreover, immunohistochemical experiments showed that CRH and SP are colocalized in some enteric nerves of wild-type mice, and this colocalization is more evident after toxin A administration . These results provide direct evidence for a major proinflammatory role for CRH in the pathophysiology of enterotoxin-mediated inflammatory diarrhea and indicate a SP-linked pathway.

FEMS Microbiol Lett, 2004 Jun 1, 235(1), 139 - 46
Maturation of the murine cecal microbiota as revealed by terminal restriction fragment length polymorphism and 16S rRNA gene clone libraries; Kibe R et al.; The maturation of murine cecal microbiota was determined by terminal restriction fragment polymorphism (T-RFLP) and 16S rRNA gene clone libraries . Cecal microbiota in specific pathogen free (SPF) mice aged four to 10 weeks were collected . The cluster of samples in 4-week-old mice was different from those of other ages based on T-RFLP profiles . The majority of clones obtained in this study belonged to the Clostridium coccoides (C . coccoides) group, the Bacteroides group or the Lactobacillus group . Phylogenetic analysis showed characteristic clusters composed of new operational taxonomic unit (OTU) of the C . coccoides and Bacteroides groups . The existence of a large number of yet unidentified bacteria inhabiting the murine cecum was demonstrated by 16S rRNA gene clone libraries . T-RFLP analysis data were more complex and more sensitive than the patterns generated by computer simulation of 16S rRNA gene clone library analysis data . T-RFLP revealed development with maturation of cecal microbiota including unidentified bacteria of SPF mice.

FEMS Microbiol Lett, 2004 Jun 1, 235(1), 65 - 72
Epimerization of chenodeoxycholic acid to ursodeoxycholic acid by Clostridium baratii isolated from human feces; Lepercq P et al.; Ursodeoxycholic acid-producing bacteria are of clinical and industrial interest due to the multiple beneficial effects of this bile acid on human health . This work reports the first isolation of 7-epimerizing bacteria from feces of a healthy volunteer, on the basis of their capacity to epimerize the primary bile acid, chenodeoxycholic acid, to ursodeoxycholic acid . Five isolates were found to be active starting from unconjugated chenodeoxycholic acid and its tauro-conjugated homologue, but none of these strains could epimerize the glyco-conjugated form . Biochemical testing and 16S ribosomal DNA sequencing converged to show that all five isolates were closely related to Clostridium baratii (99% sequence similarity), suggesting that this bacterial species could be responsible at least partially, for this bioconversion in the human gut.

FEMS Microbiol Lett, 2004 Jun 1, 235(1), 9 - 16
Nucleotide sequence and transcriptional analysis of the type A2 neurotoxin gene cluster in Clostridium botulinum; Dineen SS et al.; The nucleotide sequences of the upstream regions of the botulinum neurotoxin type A1 (BoNT/A1) cluster of Clostridium botulinum strain NCTC 2916 and the BoNT/A2 cluster of strain Kyoto-F were determined . A novel gene, designated orfx3, was identified following the orfx2 gene in both clusters . ORF-X2 and ORF-X3 exhibit similarity to the BoNT cluster associated P-47 protein . The BoNT/A1 and BoNT/A2 clusters share a similar gene arrangement, but exhibit differences in the spacing between certain genes . Sequences with similarity to transposases were identified in these intergenic regions, suggesting that these differences arose from an ancestral insertion event . Transcriptional analysis of the BoNT/A2 cluster revealed that the genes of the cluster are primarily synthesized as three polycistronic transcripts . Two divergent polycistronic transcripts, one encoding the orfx1, orfx2, and orfx3 genes, the second encoding the p47, ntnh, and bont/a2 genes, are transcribed from conserved BoNT cluster promoters . The third polycistronic transcript, expressed at low levels, encodes the positive regulatory botR gene and the orfx genes . This is the first complete analysis of a botulinum toxin A2 cluster.

Biochemistry, 2004 Jun 1, 43(21), 6637 - 44
Structural analysis of botulinum neurotoxin type E catalytic domain and its mutant Glu212-->Gln reveals the pivotal role of the Glu212 carboxylate in the catalytic pathway; Agarwal R et al.; The seven serotypes of botulinum neurotoxins (A-G) produced by Clostridium botulinum share significant sequence homology and structural similarity . The functions of their individual domains and the modes of action are also similar . However, the substrate specificity and the peptide bond cleavage selectivity of their catalytic domains are different . The reason for this unique specificity of botulinum neurotoxins is still baffling . If an inhibitor leading to a therapeutic drug common to all serotypes is to be developed, it is essential to understand the differences in their three-dimensional structures that empower them with this unique characteristic . Accordingly, high-resolution structures of all serotypes are required, and toward achieving this goal the crystal structure of the catalytic domain of C . botulinum neurotoxin type E has been determined to 2.1 A resolution . The crystal structure of the inactive mutant Glu212-->Gln of this protein has also been determined . While the overall conformation is unaltered in the active site, the position of the nucleophilic water changes in the mutant, thereby causing it to lose its ability to activate the catalytic reaction . The structure explains the importance of the nucleophilic water and the charge on Glu212 . The structural differences responsible for the loss of activity of the mutant provide a common model for the catalytic pathway of Clostridium neurotoxins since Glu212 is conserved and has a similar role in all serotypes . This or a more nonconservative mutant (e.g., Glu212-->Ala) could provide a novel, genetically modified protein vaccine for botulinum.

Methods Mol Biol, 2004, 268, 331 - 6
Purification of bacteriocins produced by lactic acid bacteria; Saavedra L et al.; Bacteriocins are antibacterial substances of a proteinaceous nature that are produced by different bacterial species . Lactic acid bacteria (LAB) produce biologically active peptides or protein complexes that display a bactericidal mode of action almost exclusively toward Gram-positive bacteria and particularly toward closely related species . Generally they are active against food spoilage and foodborne pathogenic microorganisms including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, and Listeria monocytogenes.There is an increased tendency to use natural occurring metabolites to prevent the growth of undesirable flora in foodstuffs . These metabolites could replace the use of chemical additives such as sorbic acid, sulfur dioxide, nitrite, nitrate, and others . For instance, bacteriocins produced by LAB may be promising for use as bio-preservaties.Bacteriocins of lactic acid bacteria are typically cationic, hydrophobic peptides and differ widely in many characteristics including molecular weight, presence of particular groups of amino acids, pI, net positive charge, and post-translational modifications of certain amino acids . This heterogeneity within the LAB bacteriocins may explain the different procedures for isolation and purification developed so far . The methods most frequently used for isolation, concentration, and purification involve salt precipitation of bacteriocins from culture supernatants, followed by various combinations of gel filtration, ion-exchange chromatography, and reverse-phase high-performance liquid chromatography (RP-HPLC).In this chapter, a protocol is described that combines several methods used in our laboratory for the purification of two cationic bacteriocins, Lactocin 705AL and Enterocin CRL10, produced by Lactobacillus casei CRL705 and Enterococcus mundtii CRL10, respectively.

Methods Mol Biol, 2004, 268, 311 - 6
Spectrophotometric determination of histamine in fisheries using an enzyme immunoassay method; Pessatti TL et al.; The biogenic amines are low-weight organic bases that exhibit variable biological activity . Approximately 30 vasoactive and psycoactive amines have been found in foodstuffs . Histamine, tyramine, phenylethylamine, tryptamine, cadaverine, putrescine, spermine, and spermidine are examples of biogenic amines detectable in meat and its derivatives . These amines occur naturally in animals, plants, and microorganisms owing to their natural metabolic processes, and are usually formed by decarboxylation of amino acids.Histamine, 5-imidazol-ethylamine (MW 111), is produced by oxidative decarboxylation of histidine, and decarboxylase-positive microorganisms are able to produce it as a byproduct of their action on host tissues . This process, associated with enterobacteria such as Escherichia, Salmonella, Clostridium, and Bacillus, can be fast if the meat storage conditions are inadequate.Additionally, because of its nonvolatile behavior, histamine may bestow toxicity on the product even before it is considered decayed or organoleptically unacceptable . Secondary amines such as piperidine and pyrrolidine can be produced in the catabolism of histamine, and they, in turn, are precursors of nitrosamines, substances with known carcinogenic activity . Some cases of histamine toxicity have been reported.

Infect Immun, 2004 Jun, 72(6), 3366 - 72
Clostridium sordellii lethal toxin is maintained in a multimeric protein complex; Voth DE et al.; Clostridium sordellii lethal toxin (TcsL) is distinct among large clostridial toxins (LCTs), as it is markedly reduced in its rate of intoxication at pH 8.0 yet is cytotoxic at pH 4.0 . Results from the present study suggest that TcsL's slow rate of intoxication at pH 8.0 is linked to formation of a high-molecular-weight complex containing dissociable pH 4.0-sensitive polypeptides . The cytosolic delivery of TcsL's enzymatic domain by using a surrogate cell entry system resulted in cytopathic effect rates similar to those of other LCTs at pH 8.0, further indicating that rate-limiting steps occurred at the point of cell entry . Since these rate-limiting steps could be overcome at pH 4.0, TcsL was examined across a range of pH values and was found to dissociate into distinct 45- to 55-kDa polypeptides between pH 4.0 and pH 5.0 . The polypeptides reassociated when shifted back to pH 8.0 . At pH 8.0, this complex was resistant to sodium dodecyl sulfate (SDS) and multiple proteases; however, following dissociation, the polypeptides became protease sensitive . Dissociation of TcsL, and cytotoxicity, could be blocked by preincubation with ethylene glycol bis(sulfosuccinimidylsuccinate), resulting in cross-linking of the polypeptides . TcsL was also examined at pH 8.0 by using SDS-agarose gel electrophoresis and transmission electron microscopy and was found to exist in a higher-molecular-weight complex which resolved at a size exceeding 750 kDa and also dissociated at pH 4.0 . However, this complex did not reassemble following a shift back to pH 8.0 . Collectively, these data suggest that TcsL is maintained in a protease-resistant, high-molecular-weight complex, which dissociates at pH 4.0, leading to cytotoxicity.

Infect Immun, 2004 Jun, 72(6), 3267 - 75
Binding and internalization of Clostridium perfringens iota-toxin in lipid rafts; Nagahama M et al.; Clostridium perfringens iota-toxin is a binary toxin composed of an enzymatic component (Ia) and a binding component (Ib) . The oligomer of Ib formed in membranes induces endocytosis . We examined the binding and internalization of Ib by using Cy3-labeled Ib . Labeled Ib was retained at the membranes of MDCK cells for 60 min of incubation at 37 degrees C, and later it was detected in cytoplasmic vesicles . To determine whether Ib associates with lipid rafts, we incubated MDCK cells with Ib at 4 or 37 degrees C and fractionated the Triton-insoluble membranes . An Ib complex of 500 kDa was localized at 37 degrees C to the insoluble fractions that fulfilled the criteria of lipid rafts, but it did not form at 4 degrees C . The amount of complex in the raft fraction reached a maximum after 60 min of incubation at 37 degrees C . When the cells that were preincubated with Ib at 4 degrees C were incubated at 37 degrees C, the complex was detected in the raft fraction . The treatment of MDCK cells with methyl-beta-cyclodextrin reduced the localization of the Ib complex to the rafts and the rounding of the cells induced by Ia plus Ib . When 125I-labeled Ia was incubated with the cells in the presence of Ib at 37 degrees C, it was localized in the raft fraction . Surface plasmon resonance analysis revealed that Ia binds to the oligomer of Ib . We conclude that Ib binds to a receptor in membranes and then moves to rafts and that Ia bound to the oligomer of Ib formed in the rafts is internalized.

Infect Immun, 2004 Jun, 72(6), 3120 - 8
Degeneration and regeneration of murine skeletal neuromuscular junctions after intramuscular injection with a sublethal dose of Clostridium sordellii lethal toxin; Barbier J et al.; Clostridium sordellii lethal toxin (LT), a 250-kDa protein which is the bacteria's major virulence factor, belongs to a family of large clostridial cytotoxins which glucosylate small GTP-binding proteins . Here, we report the results of our ex vivo analysis of the structure and function of skeletal neuromuscular tissue obtained from mice at various times after intramuscular injection of a sublethal dose of LT (0.25 ng/g of body wt) . The toxin caused, within 24 h, pronounced localized edema, inflammation, myofibril disassembly, and degeneration of skeletal muscle fibers in the injected area, and it glucosylated the muscle tissue's small GTPases . Regeneration of the damaged fibers was evident 6 to 9 days postinjury and was completed by 60 days . The expression of dystrophin, laminin, and fast and neonatal myosin in regenerating fibers, detected by immunofluorescence microscopy, confirmed that LT does not impair the high regenerative capacity of murine skeletal muscle fibers . Functional studies revealed that LT affects muscle contractility and neuromuscular transmission . However, partial recovery of nerve-evoked muscle twitches and tetanic contractions was observed by day 15 postinjection, and extensive remodeling of the neuromuscular junction's nerve terminals and clusters of muscle acetylcholine receptors was still evident 30 days postinjection . In conclusion, to the best of our knowledge, this is the first report to characterize the degeneration and regeneration of skeletal neuromuscular tissue after in vivo exposure to a large clostridial cytotoxin . In addition, our data may provide an explanation for the severe neuromuscular alterations accompanying wound infections caused by C . sordellii.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2280 - 2
In vitro activity of OPT-80 against Clostridium difficile; Ackermann G et al.; Clostridium difficile remains the major cause of nosocomial diarrhea . Reports on impaired susceptibility of C . difficile to metronidazole and vancomycin and frequent relapses of patients after therapy necessitate the search for new substances . With this study, the activity of OPT-80, a new macrocycle, against 207 C . difficile strains and against other obligately anaerobic bacteria was tested . OPT-80 showed high in vitro activity against all C . difficile strains tested.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2149 - 52
In vitro activities of the new semisynthetic glycopeptide telavancin (TD-6424), vancomycin, daptomycin, linezolid, and four comparator agents against anaerobic gram-positive species and Corynebacterium spp; Goldstein EJ et al.; Telavancin is a new semisynthetic glycopeptide anti-infective with multiple mechanisms of action, including inhibition of bacterial membrane phospholipid synthesis and inhibition of bacterial cell wall synthesis . We determined the in vitro activities of telavancin, vancomycin, daptomycin, linezolid, quinupristin-dalfopristin, imipenem, piperacillin-tazobactam, and ampicillin against 268 clinical isolates of anaerobic gram-positive organisms and 31 Corynebacterium strains using agar dilution methods according to National Committee for Clinical Laboratory Standards procedures . Plates with daptomycin were supplemented with Ca(2+) to 50 mg/liter . The MICs at which 90% of isolates tested were inhibited (MIC(90)s) for telavancin and vancomycin were as follows: Actinomyces spp . (n = 45), 0.25 and 1 microg/ml, respectively; Clostridium difficile (n = 14), 0.25 and 1 microg/ml, respectively; Clostridium ramosum (n = 16), 1 and 4 microg/ml, respectively; Clostridium innocuum (n = 15), 4 and 16 microg/ml, respectively; Clostridium clostridioforme (n = 15), 8 and 1 microg/ml, respectively; Eubacterium group (n = 33), 0.25 and 2 microg/ml, respectively; Lactobacillus spp . (n = 26), 0.5 and 4 microg/ml, respectively; Propionibacterium spp . (n = 34), 0.125 and 0.5 microg/ml, respectively; Peptostreptococcus spp . (n = 52), 0.125 and 0.5 microg/ml, respectively; and Corynebacterium spp . (n = 31), 0.03 and 0.5 microg/ml, respectively . The activity of TD-6424 was similar to that of quinupristin-dalfopristin for most strains except C . clostridioforme and Lactobacillus casei, where quinupristin-dalfopristin was three- to fivefold more active . Daptomycin had decreased activity (MIC > 4 microg/ml) against 14 strains of Actinomyces spp . and all C . ramosum, Eubacterium lentum, and Lactobacillus plantarum strains . Linezolid showed decreased activity (MIC > 4 microg/ml) against C . ramosum, two strains of C . difficile, and 15 strains of Lactobacillus spp . Imipenem and piperacillin-tazobactam were active against >98% of strains . The MICs of ampicillin for eight Clostridium spp . and three strains of L . casei were >1 microg/ml . The MIC(90) of TD-6424 for all strains tested was </=2 microg/ml . TD-6424 has potential for use against infections with gram-positive anaerobes and deserves further clinical evaluation.

Antimicrob Agents Chemother, 2004 Jun, 48(6), 2144 - 8
Efficacy of oral ramoplanin for inhibition of intestinal colonization by vancomycin-resistant enterococci in mice; Stiefel U et al.; Ramoplanin is a glycolipodepsipeptide antibiotic with activity against gram-positive bacteria that is in clinical trials for prevention of vancomycin-resistant Enterococcus (VRE) bloodstream infections and treatment of Clostridium difficile diarrhea . Orally administered ramoplanin suppresses VRE intestinal colonization, but recurrences after discontinuation of treatment have frequently been observed . We used a mouse model to examine the efficacy of ramoplanin for inhibition of VRE colonization and evaluated the etiology of recurrences of colonization . Eight days of treatment with ramoplanin (100 microg/ml) in drinking water suppressed VRE to undetectable levels, but 100% of mice developed recurrent colonization; a higher dose of 500 microg/ml in water was associated with recurrent colonization in 50% of mice . Two of eight (25%) mice treated with the 100-microg/ml dose of ramoplanin had low levels of VRE in their cecal tissues on day 8 despite undetectable levels in stool and cecal contents . Mice that received prior ramoplanin treatment did not develop VRE overgrowth when challenged with 10(7) CFU of oral VRE 1, 2, or 4 days later . In communal cages, rapid cross-transmission and overgrowth of VRE was observed among clindamycin-treated mice; ramoplanin treatment effectively suppressed VRE overgrowth in such communal cages . Ramoplanin treatment promoted increased density of indigenous Enterobacteriaceae and overgrowth of an exogenously administered Klebsiella pneumoniae isolate . These results demonstrate the efficacy of ramoplanin for inhibition of VRE colonization and suggest that some recurrences occur due to reexpansion of organisms that persist within the lining of the colon . Ramoplanin treatment may be associated with overgrowth of gram-negative bacilli.

Prev Vet Med, 2004 Jan 30, 62(1), 59 - 72
What has happened in norway after the ban of avoparcin? Consumption of antimicrobials by poultry; Grave K et al.; When avoparcin was prohibited for use as feed additive in poultry in Norway on 31 May 1995, an increased incidence of Clostridium perfringens-associated necrotic enteritis (NE) and an increase in the use of antibacterial (AB) drug therapy in meat-type poultry was expected . The consumption of AB drugs for use against NE in poultry in the period 1990-2001 was investigated by use of sales statistics at the drug-wholesaler level . Defined daily dose (DDD) per kg live weight poultry was the unit of measurement for drug use (to correct for differences in the dosages) . Sales figures of the AB drugs were converted to number of DDDpoultry sold for the numbers of broilers at risk (broilers were 97% of the slaughter poultry) . Estimated annual percentages of the broilers treated against NE increased abruptly after the avoparcin ban--but in 1996, this figure declined to the same level as before the ban and has remained at that low level since then . In November 1995, narasin was approved temporarily as an ionophore feed additive (IFA) in broilers . The usage patterns of IFAs in broilers were measured as the weight of feed to which an IFA was added per broiler chicken produced . In 1996-2001, the IFAs used in broilers were predominantly narasin . We note that the temporary increase in NE after the avoparcin ban coincide with the period before narasin became available . The increase in the consumption of AB drugs for the treatment of NE in poultry following the avoparcin ban has been negligible.

Eur J Biochem, 2004 Jun, 271(11), 2225 - 30
Subunit composition of the glycyl radical enzyme p-hydroxyphenylacetate decarboxylase . A small subunit, HpdC, is essential for catalytic activity; Andrei PI et al.; p-Hydroxyphenylacetate decarboxylase from Clostridium difficile catalyses the decarboxylation of p-hydroxyphenylacetate to yield the cytotoxic compound p-cresol . The three genes encoding two subunits of the glycyl-radical enzyme and the activating enzyme have been cloned and expressed in Escherichia coli . The recombinant enzymes were used to reconstitute a catalytically functional system in vitro . In contrast with the decarboxylase purified from C . difficile, which was an almost inactive homo-dimeric protein (beta(2)), the recombinant enzyme was a hetero-octameric (beta(4)gamma(4)), catalytically competent complex, which was activated using endogenous activating enzyme from C . difficile or recombinant activating enzyme to a specific activity of 7 U.mg(-1) . Preliminary results suggest that phosphorylation of the small subunit is responsible for the change of the oligomeric state . These data point to an essential function of the small subunit of the decarboxylase and may indicate unique regulatory properties of the system.

J Food Prot, 2004 May, 67(5), 1031 - 5
Characterization of Bacillus cereus isolates from raw soybean sprouts; Kim HJ et al.; Raw soybean sprouts, which are used as ingredients in cook-chilled products, were analyzed to evaluate the incidence of mesophilic aerobic microorganisms, psychrotrophic microorganisms, anaerobic microorganisms, coliforms, and spore-forming microorganisms Bacillus cereus, Clostridium botulinum, and Clostridium perfringens . Mesophilic microorganisms on raw soybean sprouts were present in large populations, 5.5 x 10(6) to 1.4 x 10(8) CFU/g, and psychrotrophic microorganisms were found to be more numerous than the other groups . Coliforms accounted for 15% of mesophilic microorganism counts on average, and the average for spore-forming microorganisms was 5.2 x 10(2) CFU/g . B . cereus was isolated from 12 of 17 soybean sprout samples, whereas C . botulinum and C . perfringens were not isolated . B . cereus was isolated in greater numbers at 30 degrees C compared with other temperatures and was not isolated at 4 degrees C . Of the 55 strains isolated from soybean sprouts, 52 were positive for starch hydrolysis, and only 3 strains did not hydrolyze starch . Among the 55 strains of B . cereus isolates, 53 showed the ability to produce diarrheal enterotoxin by CRET-RPLA.

J Med Microbiol, 2004 Jun, 53(Pt 6), 581 - 3
Clostridium fallax associated with sudden death in a 16-year-old boy; Hausmann R et al.; Clostridial myonecrosis or gas gangrene occurs most frequently in contaminated wounds following trauma or surgery . It is caused by a wide variety of Clostridium species, the most common being Clostridium perfringens . Spontaneous, non-traumatic clostridial myonecrosis is uncommon and is usually associated with gastrointestinal and haematological malignancy, diabetes mellitus and peripheral vascular disease . The case of a previously healthy 16-year-old boy with acute onset of gastrointestinal symptoms, who died of bacterial sepsis without apparent preceding trauma, is presented here . Clostridium fallax was identified as the most probable causative agent . As far as is known, this is the first report of fatal sepsis in humans due to C . fallax, which has been described only rarely as a cause of gas oedema in animals.

J Med Microbiol, 2004 Jun, 53(Pt 6), 551 - 4
Inhibition of Clostridium difficile strains by intestinal Lactobacillus species; Naaber P et al.; Indigenous intestinal microflora (including lactobacilli) has an important role in protection against Clostridium difficile infection . To assess in vitro interaction between lactobacilli and C . difficile, antagonistic activity of 50 intestinal Lactobacillus spp . strains against 23 pathogenic C . difficile strains was determined . Phenotypic properties of C . difficile strains {production of short-chain fatty acids (SCFAs) and toxin A, and antimicrobial susceptibility} and lactobacilli (production of SCFAs and H(2)O(2)) were investigated . Five lactobacilli (Lactobacillus paracasei and Lactobacillus plantarum species) were antagonistic to all C . difficile strains, 18 were antagonistic to some C . difficile strains and 27 showed no antagonistic activity . This antagonistic activity was strain-specific and seemed to correlate with H(2)O(2) and lactic acid production . C . difficile strains that were more sensitive to lactobacilli (n = 9) usually produced higher toxin levels and more SCFAs, and were more resistant to antibiotics, than strains that were resistant to lactobacilli (n = 14) . Compatibility of C . difficile strain properties (resistance to lactobacilli or antibiotics) with intestinal microecological conditions (e.g . presence of antagonistic lactobacilli, concentration of antibiotics) may determine expression of disease.

J Biol Chem, 2004 Jul 30, 279(31), 32001 - 7 Epub 2004 May 18.
Protein splicing and auto-cleavage of bacterial intein-like domains lacking a C'-flanking nucleophilic residue; Dassa B et al.; Bacterial intein-like (BIL) domains are newly identified homologs of intein protein-splicing domains . The two known types of BIL domains together with inteins and hedgehog (Hog) auto-processing domains form the Hog/intein (HINT) superfamily . BIL domains are distinct from inteins and Hogs in sequence, phylogenetic distribution, and host protein type, but little is known about their biochemical activity . Here we experimentally study the auto-processing activity of four BIL domains . An A-type BIL domain from Clostridium thermocellum showed both protein-splicing and auto-cleavage activities . The splicing is notable, because this domain has a native Ala C'-flanking residue rather than a nucleophilic residue, which is absolutely necessary for intein protein splicing . B-type BIL domains from Rhodobacter sphaeroides and Rhodobacter capsulatus cleaved their N' or C' ends . We propose an alternative protein-splicing mechanism for the A-type BIL domains . After an initial N-S acyl shift, creating a thioester bond at the N' end of the domain, the C' end of the domain is cleaved by Asn cyclization . The resulting amino end of the C'-flank attacks the thioester bond next at the N' end of the domain . This aminolysis step splices the two flanks of the domain . The B-type BIL domain cleavage activity is explained in the context of the canonical intein protein-splicing mechanism . Our results suggest that the different HINT domains have related biochemical activities of proteolytic cleavages, ligation and splicing . Yet the predominant reactions diverged in each HINT type according to their specific biological roles . We suggest that the BIL domain cleavage and splicing reactions are mechanisms for post-translationally generating protein variability, particularly in extracellular bacterial proteins.

J Bacteriol, 2004 Jun, 186(11), 3321 - 30
The spatial organization of the VirR boxes is critical for VirR-mediated expression of the perfringolysin O gene, pfoA, from Clostridium perfringens; Cheung JK et al.; The transcriptional regulation of toxin production in the gram-positive anaerobe Clostridium perfringens involves a two-component signal transduction system that comprises the VirS sensor histidine kinase and its cognate response regulator, VirR . Previous studies showed that VirR binds independently to a pair of imperfect direct repeats, now designated VirR box 1 and VirR box 2, located immediately upstream of the promoter of the pfoA gene, which encodes the cholesterol-dependent cytolysin, perfringolysin O . For this study, we introduced mutated VirR boxes into a C . perfringens pfoA mutant and found that both VirR boxes are essential for transcriptional activation . Furthermore, the spacing between the VirR boxes and the distance between the VirR boxes and the -35 region are shown to be critical for perfringolysin O production . Other VirR boxes that were previously identified from the strain 13 genome sequence were also analyzed, with perfringolysin O production used as a reporter system . The results showed that placement of the different VirR boxes at the same position upstream of the pfoA promoter yields different levels of perfringolysin O activity . In all of these constructs, VirR was still capable of binding to the target DNA, indicating that DNA binding alone is not sufficient for transcriptional activation . Finally, we show that the C . perfringens RNA polymerase binds more efficiently to the pfoA promoter in the presence of VirR, indicating that interactions must occur between these proteins . We propose that these interactions are required for VirR-mediated transcriptional activation.

Curr Treat Options Gastroenterol, 2004 Jun, 7(3), 235 - 239
Treatment of Clostridium difficile Infection; Stroehlein JR; With the introduction of broad-spectrum antibiotics into clinical practice, Clostridium difficile infection has become the most common cause of infectious diarrhea in hospitalized patients . Although mild cases may resolve by discontinuing antibiotics, thus allowing re-establishment of colonic microflora, oral metronidazole or vancomycin is indicated if the process is more severe . Metronidazole may be given intravenously, with intracolonic therapeutic levels achieved by excretion of drug into bile and exudation across inflamed tissue . Vancomycin is preferred treatment of severe cases . Bacitracin given orally is a therapeutic alternative and cholestyramine is a useful adjunct . Most patients with diarrhea or colitis caused by C . difficile respond to initial therapy; however, up to 20% experience relapse when treatment is discontinued . Repeating initial therapy for 10 to 14 days is indicated for first relapse . Multiple relapses require prolonged treatment with vancomycin, which may be supplemented with cholestyramine . Saccharomyces boulardii alone or in combination with vancomycin has been reported to be an effective therapeutic alternative for recurrent infection . Intravenous immunoglobulin can be effective in patients with severe recurrent Clostridium difficile colitis and immune deficiency or low pretreatment levels of serum antitoxin . Surgery is indicated only if recurrent infections are severe and associated with serious complications.

Int J Med Microbiol, 2004 Apr, 293(7-8), 557 - 64
Clostridium botulinum C2 toxin--new insights into the cellular up-take of the actin-ADP-ribosylating toxin; Aktories K et al.; Clostridium botulinum C2 toxin is a member of the family of binary actin-ADP-ribosylating toxins . It consists of the enzyme component C2I, and the separated binding/translocation component C2II . Proteolytically activated C2II forms heptamers and binds to a carbohydrate cell surface receptor . After attachment of C2I, the toxin complex is endocytosed to reach early endosomes . At low pH of endosomes, C2II-heptamers insert into the membrane, form pores and deliver C2I into the cytosol . Here, C2I ADP-ribosylates actin at Arg177 to block actin polymerization and to induce depolymerization of actin filaments . The mini-review describes main properties of C2 toxin and discusses new findings on the involvement of chaperones in the up-take process of the toxin.

Anal Chem, 2004 May 15, 76(10), 2938 - 50
2,5-Dihydroxybenzoic acid butylamine and other ionic liquid matrixes for enhanced MALDI-MS analysis of biomolecules; Mank M et al.; The performance of the new ionic liquid MALDI-MS matrix 2,5-dihydroxybenzoic acid butylamine (DHBB) was assessed and compared to results obtained with the ionic liquid MALDI-MS matrixes alpha-cyano-4-hydroxycinnamic acid butylamine (CHCAB), 3,5-dimethoxycinnamic acid triethylamine (SinTri), and the frequently used solid MALDI matrixes 2,5-dihydroxybenzoic acid (DHB) and alpha-cyano-4-hydroxycinnamic acid (CHCA) . The vacuum-stable, liquid consistency of ionic liquid matrix sample preparations considerably enhanced MALDI-MS analysis in terms of shot-to-shot reproducibility . Consequently, relative standard deviations serving as a measure for reproducibility of intensity-values acquired from 90 different spots on one MALDI-MS preparation were approximately one-half as high when solid DHB was replaced by the ionic liquid DHBB and eight times lower after exchange of solid CHCA by ionic liquid CHCAB . Interestingly, the ionic liquid MALDI matrix DHBB conserved the broad applicability of its solid analogue DHB, reduced MALDI induced fragmentation of monosialylated glycans and gangliosides, and was the superior ionic liquid matrix for MALDI-MS analysis of oligosaccharides and polymers, such as poly(ethylene glycol) . It also worked well with glycoconjugates, peptides, and proteins; however, the tendency of DHBB to form multiple alkali adduct ions with peptides and proteins made CHCAB the ionic liquid matrix of choice for peptides . SinTri was the best ionic liquid matrix for proteins of high molecular weight, such as IgG . Furthermore, it was demonstrated for the first time that solvent properties and MALDI matrix properties of ionic liquids, such as DHBB, can be combined to enable fast, direct screening of an enzymatic reaction . This was proven by the desialylation of sialylactose with sialidase from Clostridium perfringens in the presence of diluted aqueous DHBB and subsequent direct MALDI-MS analysis of the reaction mixture.

Wien Klin Wochenschr, 2004 Apr 30, 116(7-8), 264 - 7
Gas gangrene due to Clostridium perfringens in two injecting drug users in Vienna, Austria; Assadian O et al.; We describe two cases of severe myonecrotic infections caused by Clostridium perfringens in injecting drug users (IDUs) in Vienna, Austria . Clostridial myonecrosis, or gas gangrene, is a clostridial infection primarily of muscle tissue . C . perfringens is isolated in 90% of these infections . Other clostridial species isolated are C . novyi, C . septicum, C . histolyticum, C . fallax, and C . bifermentans . Classically, clostridial myonecrosis has an acute presentation and a fulminant clinical course . It is diagnosed mainly on a clinical basis . The infection may be so rapidly progressive that any delay in recognition or treatment may be fatal . The onset is sudden, often within 4 to 6 hours after an injury . An early clinical finding is sudden severe pain in the area of infection . Swelling and edema in the area of infection is pronounced . At surgery, the infected muscle is dark-red to black, is noncontractile, and does not bleed when cut . Crepitus, although not prominent, is sometimes detected . We were able to demonstrate spores that were morphologically indistinguishable from spores of C . perfringens in a drug sample obtained from case 2 . General practitioners and accident and emergency staff should be aware of the possibility of C . perfringens infection in IDUs, especially if injection into soft tissue is suspected.

J Infect Dis, 2004 Jun 1, 189(11), 2110 - 9 Epub 2004 Apr 30.
Differential effects of varying concentrations of clostridium difficile toxin A on epithelial barrier function and expression of cytokines; Johal SS et al.; BACKGROUND: Presentation after Clostridium difficile infection may depend on the level of epithelial exposure to toxins . We investigated epithelial barrier function and expression of interleukin (IL)-8 and transforming growth factor (TGF)-beta in response to varying concentrations of C . difficile toxin A . METHODS: T84 cells were either preexposed or continuously exposed to C . difficile toxin A (0.01-1000 ng/mL) . Barrier function was assessed by measurements of transepithelial electrical resistance . RESULTS: Preexposure to < or =10 ng/mL toxin A led to an increase in the release of TGF-beta 1, but there was no change in the expression of IL-8 . In contrast, after preexposure to >10 ng/mL toxin A, there was enhanced expression of IL-8, but release of TGF-beta 1 was similar to that in control monolayers . After preexposure to >10 ng/mL toxin A, there was complete and irreversible loss of electrical resistance . At lower concentrations, loss of resistance across monolayers was followed by recovery, which was enhanced by all 3 recombinant isoforms of TGF-beta . Pretreatment with recombinant isoforms of TGF-beta or coculture with TGF-beta 3-expressing colonic subepithelial myofibroblasts was also protective . CONCLUSIONS: In C . difficile infection, the development and severity of colonic inflammation may depend on the exposure of intestinal epithelial cells to toxins and the expression of proinflammatory (IL-8) and protective (TGF-beta) factors.

Int J Syst Evol Microbiol, 2004 May, 54(Pt 3), 947 - 9
Clostridium hastiforme is a later synonym of Tissierella praeacuta; Bae JW et al.; The previously proposed species Clostridium hastiforme and Tissierella praeacuta appear to be similar from their published descriptions . Accordingly, the aim of the current study was to perform phenotypic and genetic analyses of the type strains of both species, in order to clarify their taxonomic positions . The type strains of C . hastiforme (DSM 5675(T)) and T . praeacuta (NCTC 11158(T)) exhibited identical biochemical profiles and their 16S rRNA gene sequences displayed 99.9 % similarity . DNA-DNA hybridization was also estimated to be 96.5 % . Thus, it was concluded that C . hastiforme and T . praeacuta are synonyms, where T . praeacuta has priority . An emended description of the genus Tissierella is also given.

Bioorg Med Chem, 2004 Jun 1, 12(11), 3055 - 62
Small tripeptide surrogates with low nanomolar affinity as potent inhibitors of the botulinum neurotoxin B metallo-proteolytic activity; Blommaert A et al.; Botulinum neurotoxin type B is a high-weight (150 kDa) protein produced by the anaerobic bacillus Clostridium botulinum . This metallo-protease neurotoxin cleaves synaptobrevin, a protein, which is crucial to neurotransmission, resulting in the muscle paralysis, which characterizes botulism . Inhibition of the metallo-peptidase activity is a possible approach to obtain specific therapeutics to treat botulism . We have previously reported a successful attempt to block the proteolytic activity of this neurotoxin with new, selective amino-thiol inhibitors endowed with Ki values in the 15-20 nanomolar range . With the aim of increasing the affinity and bioavailability of this first series of inhibitors we have optimized the residue that fits the P(1) subsite of the enzyme by comparing a series of ligands that contain subtle but significant variants of the parent structure . In addition, this strategy provided a simplification of the synthesis of BoNT/B inhibitors by reducing the possible number of stereoisomers . As such we were able to enhance the inhibitory potency whilst reducing the size as compared to the initial privileged structure yielding the first pseudo-tripeptide inhibitors with Ki values in the low nanomolar range.

J Pharmacol Exp Ther, 2004 Sep, 310(3), 881 - 9 Epub 2004 May 12.
Botulinum toxin type A targets RhoB to inhibit lysophosphatidic acid-stimulated actin reorganization and acetylcholine release in nerve growth factor-treated PC12 cells; Ishida H et al.; Botulinum toxin type A (BoNT/A) produced by Clostridium botulinum inhibits Ca2+-dependent acetylcholine (ACh) release (neuroexocytosis) at peripheral neuromuscular junctions, sometimes causing neuromuscular paralysis . This inhibitory effect is attributed to its metalloprotease activity to cleave the 25-kDa synaptosomal-associated protein, which is essential for the exocytotic machinery . However, deletion of this protein does not result in a complete block of neuroexocytosis, suggesting that botulinum-mediated inhibition may occur via another mechanism . Rho GTPases, a class of small GTP-binding proteins (G proteins), control actin cytoskeletal organization, thereby regulating a variety of cellular functions in various cells, including neuronal cells . We have shown that the G protein activator lysophosphatidic acid (LPA) triggered actin reorganization followed by Ca2+-dependent ACh release in nerve growth factor-treated PC12 cells and that BoNT/A blocked both events through degradation of RhoB by the proteasome . Overexpression of wild-type RhoB caused actin reorganization and enhanced the release of ACh by LPA to overcome toxin's inhibitory effect on actin reorganization/exocytosis stimulated by LPA, whereas overexpression of a dominant negative RhoB inhibited ACh release, regardless of LPA and/or toxin treatment . Finally, a knockdown of the RhoB gene via sequence-specific, post-transcriptional gene silencing reduced RhoB expression in PC12 cells, resulting in total inhibition of both actin reorganization and ACh release induced by LPA . We conclude that the RhoB signaling pathway regulates ACh release via actin cytoskeletal reorganization and that botulinum toxin inhibits neuroexocytosis by targeting RhoB pathway.

Microb Drug Resist, 2004 Spring, 10(1), 57 - 63
Antimicrobial susceptibility of equine and environmental isolates of Clostridium difficile; Baverud V et al.; The antimicrobial susceptibility of 50 Clostridium difficile isolates, 36 of them from horse feces and 14 from environmental sites, was determined by broth microdilution . The antimicrobial agents tested were avilamycin, cephalothin, chloramphenicol, clindamycin, erythromycin, gentamicin, neomycin, oxacillin, oxytetracycline, penicillin, spiramycin, streptomycin, trimethoprim/sulfamethoxazole, vancomycin, and virginiamycin . All isolates were susceptible to vancomycin (MIC </=1 microg/ml) . The MICs of erythromycin, oxytetracycline, spiramycin, and virginiamycin showed a bimodal distribution . Compared with the majority of isolates, the MICs of erythromycin (MIC > 16 microg/ml), oxytetracycline (MIC >/=32 microg/ml), spiramycin (MIC > 16 microg/ml), and virginiamycin (MIC 8-16 microg/ml) were higher for 18 isolates . Those were mainly isolated from horses at animal hospitals and further from environmental sites at a stud farm . In contrast, all isolates, except one, from healthy foals had low MICs of erythromycin, spiramycin, virginiamycin, and oxytetracycline . The isolates from soil in public parks had also low MICs of these antimicrobial agents . Broth microdilution appeared both reliable and reproducible for susceptibility testing of C . difficile . The method was also readily performed and the MIC endpoints were easily read.

J Appl Microbiol, 2004, 96(6), 1333 - 41
Screening of bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis, focussed on Bacillus and related endospore-forming genera; De Clerck E et al.; AIMS: To screen for bacterial contamination during gelatine production by means of denaturing gradient gel electrophoresis (DGGE) . As members of Bacillus and related genera were found to persist in the final product, this study focussed on these taxa . METHODS AND RESULTS: Template DNA was extracted from gelatine samples at five crucial points of a gelatine production process . A primer specific for Bacillus and related genera was designed and used in a selective PCR, followed by a nested DGGE-PCR targeting the V9 region of the 16S rDNA . DGGE analysis of the resulting amplicons, and sequence analysis of selected bands, showed high sequence similarities of these bands with Bacillus fumarioli, B . licheniformis, B . coagulans and Clostridium perfringens . When the selective PCR was omitted, primarily Lactobacillus bands were retrieved . CONCLUSIONS: PCR-DGGE analysis of gelatine extracts can be used for tracing and screening of bacterial contamination during gelatine production . A selective PCR, nested with DGGE-PCR, gave much more accurate information about endospore-forming contaminants than did the direct DGGE procedure alone . Significance AND IMPACT OF THE STUDY: Use of this nested DGGE-PCR protocol may provide important information about possible hazards to the final microbiological quality and/or safety of gelatine, so allowing production parameters and/or remediation procedures may be adjusted on-line.

Clin Diagn Lab Immunol, 2004 May, 11(3), 496 - 502
Vaccination with recombinant whole heavy chain fragments of Clostridium botulinum Type C and D neurotoxins; Arimitsu H et al.; Mice and ducks were subcutaneously immunized with recombinant whole heavy (H) chains of Clostridium botulinum type C and D neurotoxins, which were expressed as glutathione S-transferase fusion proteins . In the case of mice, it was confirmed that two immunizations with type C- and D-H chains, 10 microg each time, significantly increased the specific antibodies against 100-kDa H chains of type C and D neurotoxins in an immunoblot analysis and an enzyme-linked immunosorbent assay, respectively . The mice immunized with type C- and D-H chains showed no symptoms of botulism when they were challenged with C- and D-16 S toxins at doses, given intraperitoneally, of up to 10(5) and 10(6) minmum lethal doses (MLD), respectively, per mouse . Ducks were immunized with a total of 100 microg of type C-H chain . The ducks also developed specific antibodies to the type C-H chain and showed significant protection against a challenge with 10(3) duck MLD of C-16 S toxin given intravenously . These results indicate that recombinant whole H chains can be used as an effective and safe vaccine for type C and D botulism in domestic animals.

Int J Food Microbiol, 2004 Jun 1, 93(2), 155 - 63
Inhibitory effects of organic acid salts on growth of Clostridium perfringens from spore inocula during chilling of marinated ground turkey breast; Juneja VK et al.; Inhibition of Clostridium perfringens germination and outgrowth by salts of organic acids such as sodium lactate, sodium acetate, buffered sodium citrate and buffered sodium citrate supplemented with sodium diacetate was evaluated during continuous chilling of ground turkey . Turkey breast meat was injected with a brine-containing NaCl, potato starch and potassium tetra pyrophosphate to yield final in-product concentrations of 0.85%, 0.25% and 0.20%, respectively . The meat was ground, mixed with either sodium lactate (1%, 2%, 3% or 4%), sodium acetate (1% or 2%), buffered sodium citrate (Ional, 1%) or buffered sodium citrate supplemented with sodium diacetate (Ional Plus trade mark, 1%), in addition to a control that did not contain added antimicrobials . Each product was mixed with a three-strain C . perfringens spore cocktail to obtain final spore concentrations of ca . 2.8 log10 spores/g . Inoculated products (10 g) were packaged into cook-in-bags (2 x 3 in.), vacuum sealed, cooked at 60 degrees C for 1 h, and subsequently chilled from 54.4 to 7.2 degrees C in 15, 18 and 21 h following exponential chilling rates . Products were sampled immediately after cooking and then after chilling . Chilling of cooked turkey following 15, 18 and 21 h chill rates resulted in germination and outgrowth of C . perfringens spores to 6.6, 7.58 and 7.95 log10 CFU/g populations, respectively, from initial spore populations of ca . 2.80 log10 CFU/g . Incorporation of sodium lactate (1%), sodium acetate (1%), Ional or Ional Plus (1%) substantially inhibited germination and outgrowth of C . perfringens spores compared to controls . Final C . perfringens total populations of 3.12, 3.10, 2.38 and 2.92 log10 CFU/g, respectively, were observed following a 15-h exponential chill rate . Similar inhibitory effects were observed for 18 and 21 chill rates with the antimicrobials at 1% concentrations . While sodium lactate and sodium acetate concentrations of 1% were sufficient to control C . perfringens germination and outgrowth (<1.0 log10 CFU/g growth) following 15 h chill rates, higher concentrations were required for 18 and 21 h chill rates . Ional at 1% concentration was effective in inhibiting germination and outgrowth to <1.0 log10 CFU/g of C . perfringens for all three chill rates (15, 18 and 21 h) tested . Use of sodium salts of organic acids in formulation of ready-to-eat meat products can reduce the risk of C . perfringens spore germination and outgrowth during chilling .

FEMS Microbiol Lett, 2004 May 15, 234(2), 357 - 70
Attenuation regulation of amino acid biosynthetic operons in proteobacteria: comparative genomics analysis; Vitreschak AG et al.; Candidate attenuators were identified that regulate operons responsible for biosynthesis of branched amino acids, histidine, threonine, tryptophan, and phenylalanine in gamma- and alpha-proteobacteria, and in some cases in low-GC Gram-positive bacteria, Thermotogales and Bacteroidetes/Chlorobi . This allowed us not only to describe the evolutionary dynamics of regulation by attenuation of transcription, but also to annotate a number of hypothetical genes . In particular, orthologs of ygeA of Escherichia coli were assigned the branched chain amino acid racemase function . Three new families of histidine transporters were predicted, orthologs of yuiF and yvsH of Bacillus subtilis, and lysQ of Lactococcus lactis . In Pasteurellales, the single bifunctional aspartate kinase/homoserine dehydrogenase gene thrA was predicted to be regulated not only by threonine and isoleucine, as in E . coli, but also by methionine . In alpha-proteobacteria, the single acetolactate synthase operon ilvIH was predicted to be regulated by branched amino acids-dependent attenuators . Histidine biosynthetic operons his were predicted to be regulated by histidine-dependent attenuators in Bacillus cereus and Clostridium difficile, and by histidine T-boxes in L . lactis and Streptococcus mutans .

Vet Microbiol, 2004 May 20, 100(1-2), 11 - 6
Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates; Baums CG et al.; In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR . This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions . The efficiency of the protocol was demonstrated by typing C . perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

Microbiology, 2004 May, 150(Pt 5), 1529 - 38
Molecular characterization of binding subcomponents of Clostridium botulinum type C progenitor toxin for intestinal epithelial cells and erythrocytes; Fujinaga Y et al.; Clostridium botulinum type C 16S progenitor toxin consists of a neurotoxin (NTX), a non-toxic non-HA (NTNH), and a haemagglutinin (HA) . The HA acts as an adhesin, allowing the 16S toxin to bind to intestinal epithelial cells and erythrocytes . In type C, these bindings are dependent on sialic acid . The HA consists of four distinct subcomponents designated HA1, HA2, HA3a and HA3b . To identify the binding subcomponent(s) of HA of type C 16S toxin, all of the HA-subcomponents and some of their precursor forms were produced as recombinant proteins fused to glutathione S-transferase (GST) . These proteins were evaluated for their capacity to adhere to intestinal epithelial cells of guinea pig and human erythrocytes . GST-HA1, GST-HA3b and GST-HA3 (a precursor form of HA3a and HA3b) bound intestinal epithelial cells and erythrocytes, whereas GST alone, GST-HA2 and GST-HA3a did not . GST-HA3b and GST-HA3 showed neuraminidase-sensitive binding to the intestinal epithelial cells and erythrocytes, whereas GST-HA1 showed neuraminidase-insensitive binding . TLC binding assay revealed that GST-HA3b and GST-HA3 recognized sialosylparagloboside (SPG) and GM3 in the ganglioside fraction of the erythrocytes, like native type C 16S toxin {Inoue, K . et al . (1999) . Microbiology 145, 2533-2542} . On the other hand, GST-HA1 recognized paragloboside (PG; an asialo- derivative of SPG) in addition to SPG and GM3 . Deletion mutant analyses of GST-HA3b showed that the C-terminal region of HA3b is important for its binding activity . Based on these data, it is concluded that the HA component contains two distinct carbohydrate-binding subcomponents, HA1 and HA3b, which recognize carbohydrates in different specificities.

J Clin Microbiol, 2004 May, 42(5), 1933 - 9
Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile; Goncalves C et al.; In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT . We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C . difficile-associated diarrhea or colitis . Twenty-two strains (a prevalence of 6%) harbored both genes . When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C . perfringens (anti-Ia and anti-Ib) . Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains . Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis . All binary toxin-positive strains also produced TcdB and/or TcdA . However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII . We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.

Appl Environ Microbiol, 2004 May, 70(5), 2928 - 34
Quantitative interaction effects of carbon dioxide, sodium chloride, and sodium nitrite on neurotoxin gene expression in nonproteolytic Clostridium botulinum type B; Lovenklev M et al.; The effects of carbon dioxide, sodium chloride, and sodium nitrite on type B botulinum neurotoxin (BoNT/B) gene (cntB) expression in nonproteolytic Clostridium botulinum were investigated in a tryptone-peptone-yeast extract (TPY) medium . Various concentrations of these selected food preservatives were studied by using a complete factorial design in order to quantitatively study interaction effects, as well as main effects, on the following responses: lag phase duration (LPD), growth rate, relative cntB expression, and extracellular BoNT/B production . Multiple linear regression was used to set up six statistical models to quantify and predict these responses . All combinations of NaCl and NaNO(2) in the growth medium resulted in a prolonged lag phase duration and in a reduction in the specific growth rate . In contrast, the relative BoNT/B gene expression was unchanged, as determined by the cntB-specific quantitative reverse transcription-PCR method . This was confirmed when we measured the extracellular BoNT/B concentration by an enzyme-linked immunosorbent assay . CO(2) was found to have a major effect on gene expression when the cntB mRNA levels were monitored in the mid-exponential, late exponential, and late stationary growth phases . The expression of cntB relative to the expression of the 16S rRNA gene was stimulated by an elevated CO(2) concentration; the cntB mRNA level was fivefold greater in a 70% CO(2) atmosphere than in a 10% CO(2) atmosphere . These findings were also confirmed when we analyzed the extracellular BoNT/B concentration; we found that the concentrations were 27 ng x ml(-1) . unit of optical density(-1) in the 10% CO(2) atmosphere and 126 ng x ml(-1) . unit of optical density(-1) in the 70% CO(2) atmosphere.

Appl Environ Microbiol, 2004 May, 70(5), 2919 - 27
Relative neurotoxin gene expression in clostridium botulinum type B, determined using quantitative reverse transcription-PCR; Lovenklev M et al.; A quantitative reverse transcription-PCR (qRT-PCR) method was developed to monitor the relative expression of the type B botulinum neurotoxin (BoNT/B) gene (cntB) in Clostridium botulinum . The levels of cntB mRNA in five type B strains were accurately monitored by using primers specific for cntB and for the reference gene encoding the 16S rRNA . The patterns and relative expression of cntB were different in the different strains . Except for one of the strains investigated, an increase in cntB expression was observed when the bacteria entered the early stationary growth phase . In the proteolytic strain C . botulinum ATCC 7949, the level of cntB mRNA was four- to fivefold higher than the corresponding levels in the other strains . This was confirmed when we quantified the production of extracellular BoNT/B by an enzyme-linked immunosorbent assay and measured the toxicity of BoNT/B by a mouse bioassay . When the effect of exposure to air on cntB expression was investigated, no decline in the relative expression was observed in spite of an 83% reduction in the viable count based on the initial cell number . Instead, the level of cntB mRNA remained the same . When there was an increase in the sodium nitrite concentration, the bacteria needed a longer adjustment time in the medium before exponential growth occurred . In addition, there was a reduction in the expression of cntB compared to the expression of the 16S rRNA gene at higher sodium nitrite concentrations . This was most obvious in the late exponential growth phase, but at the highest sodium nitrite concentration investigated, 45 ppm, a one- to threefold decline in the cntB mRNA level was observed in all growth phases.

Appl Environ Microbiol, 2004 May, 70(5), 2728 - 33
Evaluation of a Clostridium perfringens predictive model, developed under isothermal conditions in broth, to predict growth in ground beef during cooling; Smith S et al.; Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production . The U.S . Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimens result in no more than a 1-log(10) CFU/g increase of C . perfringens and no growth of Clostridium botulinum . A mathematical model developed by Juneja et al . (Food Microbiol . 16:335-349, 1999) may be helpful in determining if the C . perfringens performance standard has been achieved, but this model has not been extensively validated . The objective of this study was to validate the Juneja 1999 model in ground beef under a variety of changing temperature and temperature abuse situations . The Juneja 1999 model consistently underpredicted growth of C . perfringens during exponential cooling of ground beef . The model also underpredicted growth of C . perfringens in ground beef cooled at two different rates . The results presented here show generally good agreement with published data on the growth of C . perfringens in similar products . The model error may be due to faster-than-expected exponential growth rates in ground beef during cooling or an error in the mathematical formulation of the model.

Appl Environ Microbiol, 2004 May, 70(5), 2685 - 91
Detection of enterotoxigenic Clostridium perfringens type A isolates in American retail foods; Wen Q et al.; Currently there is only limited understanding of the reservoirs for Clostridium perfringens type A food poisoning . A recent survey (Y.-T . Lin and R . Labbe, Appl . Environ . Microbiol . 69:1642-1646, 2003) of non-outbreak American retail foods did not identify the presence of a single C . perfringens isolate carrying the enterotoxin gene (cpe) necessary for causing food poisoning . The present study revisited this issue, using revised methodology and food sampling strategies . In our survey, cpe-positive C . perfringens isolates were detected in approximately 1.4% of approximately 900 surveyed non-outbreak American retail foods . Interestingly, those enterotoxigenic isolates in non-outbreak foods appear indistinguishable from C . perfringens isolates known to cause food poisoning outbreaks: i.e., the enterotoxigenic retail food isolates all carry a chromosomal cpe gene, are classified as type A, and exhibit exceptional heat resistance . Collectively, these findings indicate that some American foods are contaminated, at the time of retail purchase, with C . perfringens isolates having full potential to cause food poisoning . Furthermore, demonstrating that type A isolates carrying a chromosomal cpe gene are the enterotoxigenic isolates most commonly present in foods helps to explain why these isolates (rather than type A isolates carrying a plasmid cpe gene or cpe-positive type C or D isolates) are strongly associated with food poisoning outbreaks . Finally, since type A chromosomal cpe isolates present in the surveyed raw foods exhibited strong heat resistance, it appears that exceptional heat resistance is not a survivor trait selected for by cooking but is instead an intrinsic trait possessed by many type A chromosomal cpe isolates.

Nurs Manage, 2000 Jun, 31(6), 32 - 7; quiz 37-8
Minimizing the threat of C . difficile; Sheff B; Learn the signs and symptoms of Clostridium difficile-associated diarrhea (CDAD), outline infection control measures to help stop its spread and review treatment regimens.

Clin Infect Dis, 2004 May 1, 38(9), e87 - 91 Epub 2004 Apr 14.
Outbreak of necrotizing fasciitis due to Clostridium sordellii among black-tar heroin users; Kimura AC et al.; In California, black tar heroin (BTH) use among injection drug users (IDUs) has resulted in an increased number of cases of wound botulism due to Clostridium botulinum, tetanus due to Clostridium tetani, and necrotizing soft-tissue infections due to a variety of clostridia . From December 1999 to April 2000, nine IDUs in Ventura County, California, developed necrotizing fasciitis; 4 died . Cultures of wound specimens from 6 case patients yielded Clostridium sordellii . Some of the patients appeared to have the toxic shock syndrome previously reported to be characteristic of toxin-mediated C . sordellii infection, which is characterized by hypotension, marked leukocytosis, and hemoconcentration . The suspected source of this outbreak was contaminated BTH that was injected subcutaneously or intramuscularly ("skin popped") . This outbreak of C . sordellii infection serves as another example of how BTH can potentially serve as a vehicle for transmitting severe and often deadly clostridial infections, and reinforces the need to educate IDUs and clinicians about the risks associated with skin popping of BTH.

Am J Ophthalmol, 2004 May, 137(5), 942 - 4
Panophthalmitis due to clostridium septicum; Schickner DC et al.; PURPOSE: To describe patient survival in a rare case of endogenous Clostridium septicum sepsis with panophthalmitis . DESIGN: Observational case report . METHODS: Both eyes of a female patient were examined in a hospital setting . RESULTS: A 68-year-old woman had right orbital pain, proptosis, panophthalmitis, mental confusion and fever for 2 days . Blood cultures were significant for Clostridium septicum . The patient did not improve after treatment with intravenous broad-spectrum antibiotics and the right eye was enucleated . The patient survived the acute infection and extensive systemic evaluation revealed an undiagnosed colon carcinoma that may have been responsible for colonization and vascular dissemination of Clostridium septicum . CONCLUSIONS: Clostridium septicum panophthalmitis and sepsis can be the presenting sign in patients with unsuspected malignancies, particularly colon cancer . Patients can survive the infection with aggressive therapy with systemic antibiotics combined with removal of the infected tissue.

J Biol Chem, 2004 Jul 16, 279(29), 30622 - 33 Epub 2004 Apr 28.
Phosphatidylinositol 4,5-bisphosphate regulates adipocyte actin dynamics and GLUT4 vesicle recycling; Kanzaki M et al.; To investigate the potential role of phosphatidylinositol 4, 5-bisphosphate (PI(4,5)P2) in the regulation of actin polymerization and GLUT4 translocation, the type I phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) were expressed in 3T3L1 adipocytes . In preadipocytes (fibroblasts) PIP5K expression promoted actin polymerization on membrane-bound vesicles to form motile actin comets . In contrast, expression of PIP5K in differentiated 3T3L1 adipocytes resulted in the formation of enlarged vacuole-like structures coated with F-actin, cortactin, dynamin, and N-WASP . Treatment with either latrunculin B (an inhibitor for actin polymerization) or Clostridium difficile toxin B (a general Rho family inhibitor) resulted in a relatively slower disappearance of coated F-actin from these vacuoles, but the vacuoles themselves remained unaffected . Functionally, the increased PI(4,5)P2 levels resulted in an inhibition of transferrin receptor and GLUT4 endocytosis and a slow accumulation of these proteins in the PI(4,5)P2-enriched vacuoles along with the non-clathrin-derived endosome marker (caveolin) and the AP-2 adaptor complex . However, these structures were devoid of early endosome markers (EEA1, clathrin) and the biosynthetic membrane secretory machinery markers p115 (Golgi) and syntaxin 6 (trans-Golgi Network) . Taken together, these data demonstrate that PI(4,5)P2 has distinct morphologic and functional properties depending upon specific cell context . In adipocytes, altered PI(4,5)P2 metabolism has marked effects on GLUT4 endocytosis and intracellular vesicle trafficking due to the derangement of actin dynamics.

Best Pract Res Clin Gastroenterol, 2004 Apr, 18(2), 387 - 404
Microbial-gut interactions in health and disease . Mucosal immune responses; Acheson DW et al.; The host gastrointestinal tract is exposed to countless numbers of foreign antigens and has embedded a unique and complex network of immunological and non-immunological mechanisms, often termed the gastrointestinal 'mucosal barrier', to protect the host from potentially harmful pathogens while at the same time 'tolerating' other resident microbes to allow absorption and utilization of nutrients . Of the many important roles of this barrier, it is the distinct responsibility of the mucosal immune system to sample and discriminate between harmful and beneficial antigens and to prevent entry of food-borne pathogens through the gastrointestinal (GI) tract . This system comprises an immunological network termed the gut-associated lymphoid tissue (GALT) that consists of unique arrangements of B cells, T cells and phagocytes which sample luminal antigens through specialized epithelia termed the follicle associated epithelia (FAE) and orchestrate co-ordinated molecular responses between immune cells and other components of the mucosal barrier . Certain pathogens have developed ways to bypass and/or withstand defence by the mucosal immune system to establish disease in the host . Some 'opportunistic' pathogens (such as Clostridium difficile) take advantage of host or other factors (diet, stress, antibiotic use) which may alter or weaken the response of the immune system . Other pathogens have developed mechanisms for invading gastrointestinal epithelium and evading phagocytosis/destruction by immune system defences . Once cellular invasion occurs, host responses are activated to limit local mucosal damage and repel the foreign influence . Some pathogens (Shigella spp, parasites and viruses) primarily establish localized disease while others (Salmonella, Yersinia, Listeria) use the lymphatic system to enter organs or the bloodstream and cause more systemic illness . In some cases, pathogens (Helicobacter pylori and Salmonella typhi) colonize the GI tract or associated lymphoid structures for extended periods of time and these persistent pathogens may also be potential triggers for other chronic or inflammatory diseases, including inflammatory bowel disease and malignancies . The ability of certain pathogens to avoid or withstand the host's immune assault and/or utilize these host responses to their own advantage (i.e . enhance further colonization) will dictate the pathogen's success in promoting illness and furthering its own survival.

Best Pract Res Clin Gastroenterol, 2004 Apr, 18(2), 337 - 52
Microbial-gut interactions in health and disease . Antibiotic-associated diarrhoea; Beaugerie L et al.; Most cases of antibiotic-associated diarrhoea (AAD) are directly or indirectly due to the alteration of gut microflora by antibiotics . 'Functional' diarrhoea, usually limited to a mild and brief change in stool frequency, is considered as the most frequent pattern of AAD . Reduced carbohydrate fermentation and impaired metabolism of bile acids have been claimed as the potential causes of this transient digestive discomfort but a critical analysis of the data supporting these theories is necessary . Alternatively, changes in the gut flora ecosystem allow pathogens to proliferate . Clostridium difficile is responsible for approximately 10% of cases of AAD and almost all cases of antibiotic-associated pseudomembranous colitis . The level of evidence which supports the potential responsibility of other candidate pathogens (Klebsiella oxytoca, enterotoxin-producing Clostridium perfringens and Staphylococcus aureus, Candida) needs to be appreciated according to the updated postulates of causality relationships between a bacterium and a disease.

J Biotechnol, 2004 May 27, 110(2), 143 - 57
Effect of pH on metabolic pathway shift in fermentation of xylose by Clostridium tyrobutyricum; Zhu Y et al.; The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by Clostridium tyrobutyricum was studied . At pH 6.3, the fermentation gave a high butyrate production of 57.9 g l(-1) with a yield of 0.38-0.59 g g(-1) xylose and a reactor productivity up to 3.19 g l(-1)h(-1) . However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid . The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes . The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3 . In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate . Also, PTA was very sensitive to the inhibition by butyric acid . Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.

J Gastrointest Surg, 2004 May-Jun, 8(4), 489 - 92
Pneumoperitoneum from gas gangrene of the pancreas: three unusual findings in a single case; Stockinger ZT et al.; A 62-year-old man was first seen with acute pancreatitis with diffuse intrapancreatic gas and pneumoperitoneum . An immediate exploratory operation revealed diffuse pancreatic necrosis but no perforated viscus; postoperatively, the patient rapidly died . This case represents a constellation of extremely rare findings: Clostridium perfringens infection of the pancreas, pancreatic emphysema or "gas gangrene," and pneumoperitoneum without a perforated viscus.

Biosci Biotechnol Biochem, 2004 Apr, 68(4), 924 - 6
Interaction between a type-II dockerin domain and a type-II cohesin domain from Clostridium thermocellum cellulosome; Jindou S et al.; The interaction between the type-II dockerin domain of the scaffoldin protein CipA and the type-II cohesin domain of the outer layer protein SdbA is the fundamental mechanism for anchoring the cellulosome to the cell surface of Clostridium thermocellum . We constructed and purified a dockerin polypeptide and a cohesin polypeptide, and determined affinity constants of the interaction between them by the surface plasmon resonance method . The dissociation constant (K(D)) value was 1.8 x 10(-9) M, which is a little larger than that for the combination of a type-I dockerin and a type-I cohesin.

J Chromatogr A, 2004 Apr 30, 1035(1), 97 - 114
Characterisation of botulinum toxins type C, D, E, and F by matrix-assisted laser desorption ionisation and electrospray mass spectrometry; van Baar BL et al.; In a follow-up of the earlier characterisation of botulinum toxins type A and B (BTxA and BTxB) by mass spectrometry (MS), types C, D, E, and F (BTxC, BTxD, BTxE, BTxF) were now investigated . Botulinum toxins are extremely neurotoxic bacterial toxins, likely to be used as biological warfare agent . Biologically active BTxC, BTxD, BTxE, and BTxF are comprised of a protein complex of the respective neurotoxins with non-toxic non-haemagglutinin (NTNH) and, sometimes, specific haemagglutinins (HA) . These protein complexes were observed in mass spectrometric identification . The BTxC complex, from Clostridium botulinum strain 003-9, consisted of a 'type C1 and D mosaic' toxin similar to that of type C strain 6813, a non-toxic non-hemagglutinating and a 33 kDa hemagglutinating (HA-33) component similar to those of strain C-Stockholm, and an exoenzyme C3 of which the sequence was in full agreement with the known genetic sequence of strain 003-9 . The BTxD complex, from C . botulinum strain CB-16, consisted of a neurotoxin with the observed sequence identical with that of type D strain BVD/-3 and of an NTNH with the observed sequence identical with that of type C strain C-Yoichi . Remarkably, the observed protein sequence of CB-16 NTNH differed by one amino acid from the known gene sequence: L859 instead of F859 . The BTxE complex, from a C . botulinum isolated from herring sprats, consisted of the neurotoxin with an observed sequence identical with that from strain NCTC 11219 and an NTNH similar to that from type E strain Mashike (1 amino acid difference with observed sequence) . BTxF, from C . botulinum strain Langeland (NCTC 10281), consisted of the neurotoxin and an NTNH; observed sequences from both proteins were in agreement with the gene sequence known from strain Langeland . As with BTxA and BTxB, matrix-assisted laser desorption/ionisation (MALDI) MS provided provisional identification from trypsin digest peptide maps and liquid chromatography-electrospray (tandem) mass spectrometry (LC-ES MS) afforded unequivocal identification from amino acid sequence information of digest peptides obtained in trypsin digestion.

J Infect Dis, 2004 May 1, 189(9), 1585 - 9 Epub 2004 Apr 20.
Secular trends in hospital-acquired Clostridium difficile disease in the United States, 1987-2001; Archibald LK et al.; We reviewed Clostridium difficile-associated disease (CDAD) data from the intensive care unit (ICU) and hospital-wide surveillance components of the National Nosocomial Infections Surveillance System hospitals during 1987-2001 . ICU CDAD rates increased significantly only in hospitals with >500 beds (P<.01) and correlated with the duration of ICU stay (r=0.82; P<.05) . Hospital-wide (non-ICU) rates increased only in hospitals with <250 beds (P<.01) and in general medicine patients versus surgery patients (P<.0001) . CDAD predominated in general hospitals versus other facility types, and rates were significantly higher during winter versus nonwinter months (P<.01) . Thus, prevention efforts should be targeted to high-risk groups in these settings.

Isr Med Assoc J, 2004 Apr, 6(4), 201 - 4
Clinical manifestations and outcome of Pseudomembranous colitis in an elderly population in Israel; Moshkowitz M et al.; BACKGROUND: Pseudomembranous colitis is a well-recognized cause of diarrhea in patients receiving antibiotics and has significant consequences in terms of morbidity, mortality and cost . Clostridium difficile infection is the single most important infectious cause of PMC . PMC is frequently nosocomial, with an increased risk of spread among institutionalized patients, both in hospitals and nursing homes . OBJECTIVE: To investigate the demographic, clinical and laboratory characteristics of PMC patients in an Israeli elderly population . METHODS: We studied 72 hospitalized patients with endoscopically proven PMC . The medical records of all patients including clinical history and laboratory data were reviewed, such as: age, pre-hospitalization status (dependency or not, in the community as compared to the nursing home), background medical history, presenting symptoms, antibiotic history, physical examination on admission, hematologic and biochemical parameters, treatment, duration of hospitalization, complications, mortality, and recurrence of disease . RESULTS: Of the 72 patients (34 males and 38 females, mean age 77 years) 47% were nursing home residents . Pre-hospitalization antibiotic treatment was given to 91.4% for infections of the upper respiratory tract (45%) and urinary tract (45%) . The most common antibiotics were cephalosporin (64%), penicillins (42%) and quinolones (28%) . Sixty-four percent of the patients were treated with more than one antibiotic, 26% of patients received anti-acid therapy and 36% had been fed with a nasogastric tube . On admission, leukocytosis was found in 79% of patients, > 20,000/mm3 in half of them; 60% were anemic, 60% had elevated erythrocyte sedimentation rate, and 78% had hypoalbuminemia . Treatment consisted of metronidazole (41%) or a combination of metronidazole and vancomycin (56%) . Overall, 31% of patients recovered without complications, 29% died within 30 days of hospitalization, and 24% were re-hospitalized due to recurrence of PMC . CONCLUSION: The most common antibiotics implicated in PMC are cephalosporin, penicillins and quinolones . The disease is associated with high mortality and recurrence rates.

Am J Pathol, 2004 May, 164(5), 1627 - 33
Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4; Kominsky SL et al.; Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4 . In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy . CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively . Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4 . Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis . Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour . Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

J Hosp Infect, 2004 Apr, 56 Suppl 2, S10 - 2
Provision of alcohol hand rub at the hospital bedside: a case study; King S; A pilot study was performed on a 28-bed acute hospital ward, promoting hand disinfection by providing Sterillium alcohol hand rub at the bedside . Bottles of Sterillium with pump dispensers were attached to the end of each patient's bed, next to the observation charts . Posters promoting the use of the product were prominently displayed and several ward staff attended a presentation on the effectiveness of Sterillium . Although this was a small study, Alert Organism rates during the three-month trial indicated a reduction in methicillin-resistant Staphylococcus aureus (MRSA) incidence and an increased Clostridium difficile incidence . Several other factors that may have influenced the study outcomes will be discussed . There were no health and safety problems during the trial, but other issues were identified which have implications for introducing the bedside rub on a hospital wide basis, including the type of holder used, logistical arrangements and the staff and patient information requirements . The strategies adopted to address these issues will be discussed.

FEMS Microbiol Lett, 2004 May 1, 234(1), 9 - 17
Analysis of orthologous hrcA genes in Escherichia coli and Bacillus subtilis; Wiegert T et al.; The hrcA gene codes for a transcriptional repressor protein interacting with the CIRCE operator thereby reducing expression of the groE operon of more than 120 bacterial species . At least in Bacillus subtilis, the activity of the HrcA protein is modulated by the GroE chaperonin system . We amplified the hrcA gene from five different bacterial species and analyzed its activity in Escherichia coli and Bacillus subtilis . While those from Clostridium acetobutylicum and Staphylococcus aureus turned out to be active, those of Helicobacter pylori, Lactococcus lactis and Thermotoga maritima were inactive in E . coli, but that of T . maritima turned out to repress expression of the reporter gene in B . subtilis . All these results strongly suggest to us a specific recognition of HrcA by the GroE chaperonin system.

Trans R Soc Trop Med Hyg, 2004 May, 98(5), 290 - 5
Fatal type A botulism in South Africa, 2002; Frean J et al.; Although wildfowl and domestic livestock botulism has been recognized as a problem in southern Africa, very few human cases have ever been described in the region . In late February 2002, two siblings aged eight and 12 years developed acute flaccid paralysis and died . Mouse bioassays revealed the presence of type A botulinum toxin in the serum of both children, and in the retrieved remains of the implicated food . The implicated vehicle of the toxin was tinned fish in tomato sauce, commercially produced in South Africa . Type A Clostridium botulinum was cultured from the food . The most likely scenario was that corrosion damage had allowed entry of environmental organisms, including Clostridium botulinum, to the tinned food . This is the first outbreak of human type A botulism in southern Africa to be documented, and the first fatal outbreak described; previous human cases in this region have involved type B botulinum toxin, which tends to produce milder disease . A few other outbreaks elsewhere in Africa have been published, the most extensive being a type E epidemic in Egypt . Commercially tinned products were not involved in any of those outbreaks.

Biochem J, 2004 Jul 15, 381(Pt 2), 397 - 403
Control of expression of the lectin-like protein Reg-1 by gastrin: role of the Rho family GTPase RhoA and a C-rich promoter element; Ashcroft FJ et al.; The expression of members of the Reg family of secreted lectin-like proteins is increased in response to stress, inflammation and damage in many tissues . In the stomach, Reg is located in enterochromaffin-like cells, where its expression is stimulated by the gastric hormone gastrin . We have examined the mechanisms by which gastrin stimulates expression of Reg-1 . Deletional mutations of 2.1 to 0.1 kb of the rat Reg-1 promoter in a luciferase reporter vector were transiently transfected into gastric cancer AGS-G(R) cells . All promoter fragments tested showed similar relative increases in luciferase expression in response to gastrin (1 nM) . The response to gastrin of the smallest (104 bp) construct was 4.2+/-0.4-fold over basal . These responses were reduced by Ro-32-0432, a protein kinase C inhibitor, by C3-transferase, a Clostridium botulinum toxin and a selective inhibitor of the Rho family GTPase RhoA, and by co-transfection with a dominant negative form of RhoA . Co-transfection with a constitutively active form of RhoA stimulated expression 11.6+/-1.7-fold over basal . Mutations through the 104 bp construct identified a C-rich element (C-79CCCTCCC-72) required for responses to gastrin, PKC (protein kinase C) and L63RhoA (the constitutively active form of human RhoA protein containing a glutamine-to-leucine substitution at position 63) . EMSAs (electrophoretic-mobility-shift assays) using nuclear extracts of control and gastrin-stimulated AGS-G(R) cells and a probe spanning -86 to -64 bp revealed multiple binding proteins . There was no effect of gastrin on the pattern of binding . Supershift assays indicated that transcription factors Sp1 and Sp3 bound the C-rich sequence . We conclude that gastrin stimulates Reg expression via activation of PKC and RhoA, that a C-rich region (-79 to -72) is critical for the response and that Sp-family transcription factors bind to this region of the promoter.

Poult Sci, 2004 Apr, 83(4), 669 - 75
The effect of two different blends of essential oil components on the proliferation of Clostridium perfringens in the intestines of broiler chickens; Mitsch P et al.; The effect of 2 different blends of essential oils on Clostridium perfringens (Cp) in the intestine and feces of broiler chickens was tested in 6 field trials for each blend . One hundred parts per million of the blends were mixed in a commercial corn-based diet throughout the entire growing period for experimental flocks . Samples from the jejunum, cecum, cloaca, and feces were taken on d 14, 21, and 30 from experimental and control flocks and tested quantitatively for Cp via blood agar plate, litmus milk medium, and ELISA . Blend A reduced (P < or = 0.05) the average Cp concentration in the feces on all sampling days, in the jejunum and cecum on d 14 and 21, and in the cloaca on d 14 . Blend B effected a significant reduction of Cp concentration in the jejunum on d 14 and 30 and in the cloaca on d 14 . The percentages of specimens from the control group that tested positive for Cp were 83.3% for feces, 88.0% for jejunum and cloaca, and 82.6% for cecum . Specimens from the feces and 3 sections of the intestine were Cp positive in groups treated with blend A (60.8, 64.6, 47.9, and 70.8%) and with blend B (65.9, 63.6, 63.6, and 72.7%) . Our results indicate that specific blends of essential oil components can control Cp colonization and proliferation in the gut of broilers and therefore may be of help to prevent problems with Cp and necrotic enteritis.

South Med J, 2004 Apr, 97(4), 388 - 92
Leukemoid reaction due to Clostridium dificile infection in acquired immunodeficiency syndrome: two case reports and a review of the literature; De Toledo FG et al.; The clinical presentation of colitis associated with Clostridium difficile infection in immunosuppressed patients with acquired immunodeficiency syndrome (AIDS) has not been completely characterized . Previous reports suggest that these patients present with low blood leukocyte counts, consistent with the impaired myelopoiesis that can occur with human immunodeficiency virus (HIV) infection . In contrast, we describe the cases of two patients with colitis associated with C difficile infection who developed intense leukemoid reactions despite being in advanced stages of AIDS . To the best of our knowledge, these are the first described cases of leukemoid reaction associated with C difficile or other bacterial infection in AIDS patients . We review the literature on C difficile colitis in patients infected with HIV and suggest that severe C difficile infection should be considered in such patients presenting with leukemoid reaction and diarrhea.

Proc Natl Acad Sci U S A, 2004 May 4, 101(18), 6888 - 93 Epub 2004 Apr 23.
Crystal structure of Clostridium botulinum neurotoxin protease in a product-bound state: Evidence for noncanonical zinc protease activity; Segelke B et al.; Clostridium botulinum neurotoxins (BoNTs), the most potent toxins known, disrupt neurotransmission through proteolysis of proteins involved in neuroexocytosis . The light chains of BoNTs are unique zinc proteases that have stringent substrate specificity and require exceptionally long substrates . We have determined the crystal structure of the protease domain from BoNT serotype A (BoNT/A) . The structure reveals a homodimer in a product-bound state, with loop F242-V257 from each monomer deeply buried in its partner's catalytic site . The loop, which acts as a substrate, is oriented in reverse of the canonical direction for other zinc proteases . The Y249-Y250 peptide bond of the substrate loop is hydrolyzed, leaving the Y249 product carboxylate coordinated to the catalytic zinc . From the crystal structure of the BoNT/A protease, detailed models of noncanonical binding and proteolysis can be derived which we propose are also consistent with BoNT/A binding and proteolysis of natural substrate synaptosome-associated protein of 25 kDa (SNAP-25) . The proposed BoNT/A substrate-binding mode and catalytic mechanism are markedly different from those previously proposed for the BoNT serotype B.

Kansenshogaku Zasshi, 2004 Jan, 78(1), 32 - 9
{An outbreak of diarrheal disease caused by enterotoxigenic Clostridium perfringens following exposure to a contaminated environment in a nursing home}; Fukao T et al.; We herein report an outbreak of non-food-borne diarrhea which occurred in a nursing home due to enterotoxigenic Clostridium perfringens . The regional public health center in Gifu, Japan, recognized 7 patients with diarrhea in a nursing home, suspecting a food-borne illness . Bacteriological and epidemiological studies indicated that enterotoxigenic C . perfringens was the causative agent . However, suspected foods, the kitchen and the cooks carried no enteropathogenic bacteria, indicating that this outbreak was a non-food-borne diarrhea . The swab specimens obtained from the residential area of the nursing home were found to have enterotoxigenic C . perfringens . Isolates from the stool specimens of patients and environment were all serotype TW47, showing susceptibilities to ampicillin, levofloxacin, and clindamycin very similar to each other, and had banding patterns identical to each other by pulsed-field gel electrophoresis . These results strongly supported the existence of monoclonal spread of an enterotoxigenic C . perfringens among the environment of the nursing home and the residents . During 3 weeks 14 residents were involved in this outbreak . The extensive effort of keeping the residential area clean led to a prompt cease of this outbreak.

Infect Immun, 2004 May, 72(5), 3066 - 8
The host cell chaperone Hsp90 is necessary for cytotoxic action of the binary iota-like toxins; Haug G et al.; The heat shock protein Hsp90 is essential for uptake of the binary actin ADP-ribosylating toxins Clostridium perfringens iota-toxin and Clostridium difficile transferase into eukaryotic cells . Inhibition of Hsp90 by its specific inhibitor radicicol delayed intoxication of Vero cells by these toxins . A common Hsp90-dependent mechanism for their translocation is discussed.

Infect Immun, 2004 May, 72(5), 2827 - 36
Clostridium difficile toxin A carboxyl-terminus peptide lacking ADP-ribosyltransferase activity acts as a mucosal adjuvant; Castagliuolo I et al.; The receptor binding domains of the most potent mucosal adjuvants, bacterial toxins and plant lectins, are organized in repeat units to recognize specific sugar residues . The lectin-like structure of the C-terminal region of Clostridium difficile toxin A prompted us to investigate the mucosal adjuvant properties of a nontoxigenic peptide corresponding to amino acids 2394 to 2706 (TxA(C314)) . We compared TxA(C314) adjuvant activity to those of cholera toxin (CT) and Escherichia coli heat-labile enterotoxin subunit B (EtxB) coadministered orally or nasotracheally with poor peptide antigens (keyhole limpet hemocyanin {KLH} and hen egg lysozyme {HEL}) . Levels of anti-KLH-specific serum immunoglobulin G (IgG) and IgA as well as that of mucosal IgA were significantly higher in animals immunized orally with TxA(C314) plus KLH than with KLH alone, CT plus KLH, or EtxB plus KLH . Following intranasal immunization with TxA(C314) plus HEL, levels of serum- and mucosa-specific antibodies were comparable to those induced by coadministering HEL with CT or EtxB . The TxA(C314) adjuvant effect following oral, but not intranasal, immunization was dose dependent . The analysis of the subclasses of anti-KLH-specific IgG isotypes and the cytokines released from splenocytes of immunized mice challenged in vitro with KLH indicates the induction of a mixed Th1/Th2-type immune response, with prevalence of the Th1 branch . We conclude that TxA(C314) enhances immune responses against mucosa-coadministered foreign antigens and represents a promising mucosal adjuvant, especially because its ability to stimulate mixed Th1/Th2 responses with a strong a Th1 component is extremely worthwhile against intracellular pathogens.

Pain Med . 2001 Sep;2(3):239.
(203) Manufacturing Process for a Highly Purified, Stable Liquid Formulation of Botulinum Toxin Type B (Myobloc trade mark ); Grethlein A et al.; Botulinum toxin type B (BoNT-B; Myobloc trade mark ) is a new botulinum toxin with demonstrated efficacy in patients with cervical dystonia, and has been found to decrease neck pain associated with this disorder . Developmental work has led to the commercial-scale production of a uniform, highly purified toxin complex in a liquid formulation . Production of BoNT-B involves fermentation, recovery and purification from Clostridium botulinum . The reliability and robustness of the process were tested by altering critical process parameters (eg, pH, temperature) and showing that a high-quality product resulted even in conditions detrimental to C botulinum fermentation . Consistency and key quality attributes (purity, complex integrity, percent nicking, specific activity) of the toxin were assessed using a series of biochemical tests, which were validated as precise and accurate and are now routinely used in quality control analysis . Results confirmed production of an intact, uniform toxin complex with consistent purity, percentage nicking (a measure of toxin activation) of over 70%, and specific activity over 90 U/ng in three manufacturing runs . BoNT-B is manufactured as a slightly acidic liquid formulation that maintains complex integrity, reducing the potential for generating neutralizing antibodies . The potency of drug substance, dilute bulk solution, and finished product was shown to be reliable using the validated mouse intraperitoneal LD50 potency assay . The liquid formulation of BoNT-B was found to be stable for at least 9 months at 25 degrees C and at least 3 years at 2-8 degrees C . BoNT-B has a long shelf-life and may be produced on a commercial scale reliably and reproducibly, making it readily available and convenient to store and use . Support of Elan Pharmaceuticals is gratefully acknowledged.

J Clin Gastroenterol, 2004 May-Jun, 38(5), 414 - 8
Factors associated with failure of metronidazole in Clostridium difficile-associated disease; Fernandez A et al.; GOAL: To identify patients likely to fail metronidazole as initial treatment of C . difficile infection . BACKGROUND: For moderate to severe Clostridium difficile-associated diarrhea, metronidazole is the drug of choice for treatment . Oral vancomycin is given to patients who fail metronidazole or have intolerable side effects . STUDY: Retrospective review identified all patients treated for C . difficile-associated diarrhea during hospitalization from January 2000 to September 2001 . C difficile was documented by a positive toxin assay or pseudomembranes on colonoscopy . Metronidazole failure was defined as persistent symptoms of C . difficile-associated diarrhea after 5 days of uninterrupted therapy . Response was defined as improvement in symptoms at day 5 of therapy including reduction of diarrhea to <or=2 bowel movements per day . RESULTS: 119 C . difficile-associated diarrhea patients were identified, and 99 met inclusion criteria . There were 61 (62%) metronidazole responders and 38 (38%) treatment failures . Albumin <2.5g/l and intensive care unit stay at/prior to diagnosis were the only variables associated with treatment failure . The odds ratios for treatment failure were 11.7 (95% confidence interval: 4.0-31.6) and 4.1 (95% confidence interval: 1.3-12.2), respectively . When considering these 2 variables together (low albumin, intensive care unit care), the area under the receiver operating characteristic curve was 0.80 for predicting treatment failure . CONCLUSIONS: Albumin level <2.5g/l and intensive care unit stay were predictors of failure of metronidazole therapy for C . difficile-associated diarrhea . These patients may benefit from oral vancomycin therapy at outset.

Prog Urol, 2004 Feb, 14(1), 87 - 9
{Emphysematous cystitis complicated by bladder perforation: diagnosis and treatment of a rare case}; Bracq A et al.; Emphysematous cystitis is a rare disease mainly encountered in poorly controlled diabetics, immunodepressed patients or patients with infravesical obstruction . The pathophysiology of this disease is characterized by the formation of carbon dioxide (CO2) present in the lumen and/or bladder wall, derived from bacterial fermentation of carbohydrates . The bacteria most frequently responsible are facultative aerobes-anaerobes (Escherichia coli) and more rarely strict anaerobes (Clostridium perfringens) . The authors report a case of emphysematous cystitis in a context of pelvic trauma and fracture of the pelvis, complicated by secondary bladder rupture . The diagnostic and therapeutic aspects are discussed.

Biochemistry, 2004 Apr 27, 43(16), 4791 - 8
Enhancement of the endopeptidase activity of purified botulinum neurotoxins A and E by an isolated component of the native neurotoxin associated proteins; Sharma SK et al.; In botulism disease, neurotransmitter release is blocked by a group of structurally related neurotoxin proteins produced by Clostridium botulinum . Botulinum neurotoxins (BoNT, A-G) enter nerve terminals and irreversibly inhibit exocytosis via their endopeptidase activities against synaptic proteins SNAP-25, VAMP, and Syntaxin . Type A C . botulinum secretes the neurotoxin along with 5 other proteins called neurotoxin associated proteins (NAPs) . Here, we report that hemagglutinin-33 (Hn-33), one of the NAP components, enhances the endopeptidase activity of not only BoNT/A but also that of BoNT/E, both under in vitro conditions and in rat synaptosomes . BoNT/A endopeptidase activity in vitro is about twice as high as that of BoNT/E under disulfide-reduced conditions . Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E (which otherwise have only residual endopeptidase activity) enhanced their in vitro endopeptidase activity by 21- and 25-fold, respectively . Cleavage of rat-brain synaptosome SNAP-25 by BoNTs was used to assay endopeptidase activity under nerve-cell conditions . Reduced BoNT/A and BoNT/E cleaved synaptosomal SNAP-25 by 20% and 15%, respectively . Addition of Hn-33 separately to nonreduced BoNT/A and BoNT/E enhanced their endopeptidase activities by 13-fold for the cleavage of SNAP-25 in synaptosomes, suggesting a possible functional role of Hn-33 in association with BoNTs . We believe that Hn-33 could be used as an activator in the formulation of the neurotoxin for therapeutic use.

Biochemistry, 2004 Apr 27, 43(16), 4635 - 45
Insight into the mechanism of the B12-independent glycerol dehydratase from Clostridium butyricum: preliminary biochemical and structural characterization; O'Brien JR et al.; The molecular characterization of a B12-independent glycerol dehydratase from Clostridium butyricum has recently been reported {Raynaud, C., et al . (2003) Proc . Natl . Acad . Sci . U.S.A . 100, 5010-5015} . In this work, we have further characterized this system by biochemical and crystallographic methods . Both the glycerol dehydratase (GD) and the GD-activating enzyme (GD-AE) could be purified to homogeneity under aerobic conditions . In this form, both the GD and GD-AE were inactive . A reconstitution procedure, similar to what has been reported for pyruvate formate lyase activating enzyme (PFL-AE), was employed to reconstitute the activity of the GD-AE . Subsequently, the reconstituted GD-AE could be used to reactivate the GD under strictly anaerobic conditions . We also report here the crystal structure of the inactive GD in the native (2.5 A resolution, Rcryst = 17%, Rfree = 20%), glycerol-bound (1.8 A resolution, Rcryst = 21%, Rfree = 24%), and 1,2-propanediol-bound (2.4 A resolution, Rcryst = 20%, Rfree = 24%) forms . The overall fold of the GD monomer was similar to what has been observed for pyruvate formate lyase (PFL) and anaerobic ribonucleotide reductase (ARNR), consisting of a 10-stranded beta/alpha barrel motif . Clear density was observed for both substrates, and a mechanism for the dehydration reaction is presented . This mechanism clearly supports a concerted pathway for migration of the OH group through a cyclic transition state that is stabilized by partial protonation of the migrating OH group . Finally, despite poor alignment (rmsd approximately 6.8 A) of the 10 core strands that comprise the barrel structure of the GD and PFL, the C-terminal domains of both proteins align well (rmsd approximately 0.7 A) and have structural properties consistent with this being the docking site for the activating enzyme . A single point mutation within this domain, at a strictly conserved arginine residue (R782K) in the GD, resulted in formation of a tight protein-protein complex between the GD and the GD-AE in vivo, thereby supporting this hypothesis.

Microb Ecol, 2004 Jul, 48(1), 66 - 77 Epub 2004 Apr 19.
Microbial population dynamics in the sediments of a eutrophic lake (Aydat, France) and characterization of some heterotrophic bacterial isolates; Mallet C et al.; The bacterial populations of anoxic sediments in a eutrophic lake (Aydat, Puy-de-Dome-France) were studied by phospholipid fatty acid analysis (PLFA) and also by culturing heterotrophic bacteria under strictly anaerobic conditions . The mean PLFA concentrations of prokaryotes and microeukaryotes were 5.7 +/- 2.9 mgC g(-1) DS and 9.6 +/- 6.7 mgC g(-1) DS, respectively . The analysis of bacterial PLFA markers was used to determine the dynamics of the Gram-positive and Gram-negative species of anaerobic bacteria, Clostridiae, and sulfate-reducing bacteria . Throughout the sampling period the concentrations of i15:0 (from 20 nmol g(-1) DS to 130 nmol g(-1) DS), markers of Gram-positive bacteria, were higher than those for Gram-negative bacteria . The dynamics of Clostridiae (Cy15:0) paralleled those of sulfate-reducing bacteria that were marked by i17:1omega7 . Partial 16S rDNA sequencing and the physiological study of the various fermenting strains, whose abundance in the superficial sediment layer was 1.1 +/- 0.4 x 10(6) cells mL(-1), showed that all the isolates belonged to the Clostridiae and related taxa ( Lactosphaera pasteurii, Clostridium vincentii, C . butyricum, C . algidixylanolyticum, C . puniceum, C . lituseburense, and C . gasigenes) . All the isolates were capable of metabolizing a wide range of organic substrates.

Gut, 2004 May, 53(5), 673 - 7
Clostridium difficile associated diarrhoea in hospitalised patients: onset in the community and hospital and role of flexible sigmoidoscopy; Johal SS et al.; OBJECTIVES: Clostridium difficile associated diarrhoea (CDAD) is a hospital acquired infection in which optimal methods for diagnosis and the scale of the problem in the community remain to be determined . In hospitalised patients with CDAD, we aimed to (i) study patients in whom the onset of diarrhoea was in the community and (ii) investigate the role of bedside flexible sigmoidoscopy in diagnosis . METHODS: Patients with CDAD (onset in hospital or community) were studied prospectively . In those with diarrhoea of unknown aetiology, flexible sigmoidoscopy was compared with stool assay for C difficile cytotoxin . RESULTS: Of 136 patients with CDAD (which was associated with antibiotic exposure in 96%), diarrhoea started in the community in 38 (28%; majority in own home) and while an inpatient in 98 (72%) . The majority with CDAD onset in the community had been hospitalised over the preceding 12 months (86.8% v 57.1% in the hospital onset group; p<0.001) . In 56 patients with pseudomembranous colitis at sigmoidoscopy, the stool C difficile cytotoxin test was negative in 29 (52%) but toxigenic C difficile was isolated from all of nine stool samples cultured . Of patients with pseudomembranous colitis, 30.4% relapsed over the subsequent 57.7(4.2) days . CONCLUSIONS: In a significant proportion of hospitalised patients with CDAD, diarrhoea started in the community . However, the majority of these had been hospital inpatients previously when they may have acquired C difficile, with the subsequent onset of diarrhoea in the community following exposure to antibiotics . Flexible sigmoidoscopy is superior to the stool C difficile cytotoxin test in a subgroup of patients with pseudomembranous colitis . Sigmoidoscopy should therefore be considered in all hospitalised patients with diarrhoea in whom the stool test for C difficile cytotoxin and enteric pathogens is negative.

Bioorg Med Chem, 2004 May 1, 12(9), 2035 - 41
Homology modeling and molecular dynamics studies of a novel C3-like ADP-ribosyltransferase; Xiao JF et al.; The novel C3-like ADP-ribosyltransferase is produced by a Staphylococcus aureus strain that especially ADP-ribosylates RhoE/Rnd3 subtype proteins, and its three-dimensional (3D) structure has not known . In order to understand the catalytic mechanism, the 3D structure of the protein is built by using homology modeling based on the known crystal structure of exoenzyme C3 from Clostridium botulinum (1G24) . Then the model structure is further refined by energy minimization and molecular dynamics methods . The putative nicotinamide adenine dinucleotide (NAD(+))-binding pocket of exoenzyme C3(Stau) is determined by Binding-Site Search module . The NAD(+)-enzyme complex is developed by molecular dynamics simulation and the key residues involved in the combination of enzyme binding to the ligand-NAD(+) are determined, which is helpful to guide the experimental realization and the new mutant designs as well . Our results indicated that the key binding-site residues of Arg48, Glu180, Ser138, Asn134, Arg85, and Gln179 play an important role in the catalysis of exoenzyme C3(Stau), which is in consistent with experimental observation.

Aust Vet J, 2003 Apr, 81(4), 219 - 21
Pathogenesis of brain damage produced in sheep by Clostridium perfringens type D epsilon toxin: a review; Finnie JW; Microvascular endothelial damage by the epsilon toxin of Clostridium perfringens type D appears to be the fundamental cause of cerebral parenchymal injury and lesions occur in a seemingly dose- and time-dependent manner . Large doses of circulating toxin produce a severe, generalised, vasogenic cerebral oedema and an acute or peracute clinical course to death . With lower doses of toxin, or in partially immune sheep, focal necrosis, often bilaterally symmetrical, occurs in certain selectively vulnerable brain regions, which appear to become fewer as the toxin dose is reduced . These cases follow a more protracted clinical course, but death is the usual outcome . The precise pathogenesis of the focal brain damage found in subacutely intoxicated sheep is unresolved, but several possible mechanisms are discussed.

J Gen Physiol, 2004 May, 123(5), 491 - 504 Epub 2004 Apr 12.
Inhibition and redistribution of NHE3, the apical Na+/H+ exchanger, by Clostridium difficile toxin B; Hayashi H et al.; NHE3, the apical isoform of the Na(+)/H(+) exchanger, is central to the absorption of salt and water across the intestinal epithelium . We report that treatment of epithelial cells with toxin B of Clostridium difficile, a diarrheal pathogen, causes a pronounced inhibition of NHE3 activity, with little effect on the basolateral NHE1 isoform . Depression of NHE3 activity is accompanied by the translocation of apical exchangers to a subapical endomembrane compartment . Treatment of cells with toxin B increased the fraction of exchangers that were solubilized by nonionic detergents and induced dephosphorylation and extensive redistribution of ezrin . The Rho-kinase inhibitor, Y-27632, also altered the distribution and activity of NHE3 . We suggest that inactivation of Rho-family GTPases by clostridial toxin B alters the interaction between NHE3 and the microvillar cytoskeleton, possibly by impairing the ability of ezrin to bridge the exchangers to filamentous actin . Detachment of NHE3 from the actin skeleton would facilitate its internalization, resulting in net disappearance from the apical surface . The consequent inhibition of transport is likely to contribute to the diarrheal effects of C . difficile.

Crit Rev Food Sci Nutr, 2004, 44(1), 19 - 55
Shelf life and safety concerns of bakery products--a review; Smith JP et al.; Bakery products are an important part of a balanced diet and, today, a wide variety of such products can be found on supermarket shelves . This includes unsweetened goods (bread, rolls, buns, crumpets, muffins and bagels), sweet goods (pancakes, doughnuts, waffles and cookies) and filled goods (fruit and meat pies, sausage rolls, pastries, sandwiches, cream cakes, pizza and quiche) . However, bakery products, like many processed foods, are subject to physical, chemical and microbiological spoilage . While physical and chemical spoilage limits the shelf life of low and intermediate moisture bakery products, microbiological spoilage by bacteria, yeast and molds is the concern in high moisture products i.e., products with a water activity (a(w)) > 0.85 . Furthermore, several bakery products also have been implicated infoodborne illnesses involving Salmonella spp., Listeria monoctyogenes and Bacillus cereus, while Clostridium botulinum is a concern in high moisture bakery products packaged under modified atmospheres . This extensive review is divided into two parts . Part I focuses on the spoilage concerns of low, intermediate and high moisture bakery products while Part II focuses on the safety concerns of high moisture bakery products only . In both parts, traditional and novel methods of food preservation that can be used by the bakery industry to extend the shelf life and enhance the safety of products are discussed in detail.

J Antimicrob Chemother, 2004 May, 53(5), 882 - 4 Epub 2004 Apr 08.
Descriptive study of intravenous immunoglobulin for the treatment of recurrent Clostridium difficile diarrhoea; Wilcox MH; OBJECTIVES: Clostridium difficile diarrhoea (CDD) cases treated with intravenous immunoglobulin during a 2 year period were reviewed to determine disease severity and response to treatment . PATIENTS AND METHODS: Of 580 CD cytotoxin-positive patients, five received intravenous immunoglobulin because of protracted and/or recurrent CDD (median duration 50 days, range 45-64); two had biopsy- proven pseudomembranous colitis . The five patients received a median three non-CDD antibiotic courses (range 2-8) . Indices of CDD severity included hypoalbuminaemia (n = 5, median 27 g/L, range 11-29), marked hypokalaemia (n = 3, range 1.9-2.7 mM), markedly raised peripheral white cell count (n = 3, 18-34 x 10(9) cells/L), abdominal signs (n = 3) and pyrexia (n = 1) . The five cases received metronidazole for median 17 days (range 0-63) plus vancomycin for median 14 days (range 10-42) before intravenous immunoglobulin . One also received rifampicin plus vancomycin and one was given Saccharomyces boulardii . RESULTS: Intravenous immunoglobulin was given at a dosage of 300-500 mg/kg (most commonly 400 mg/kg) for one dose (two patients), two doses (two patients) and in one case for six doses . The latter patient died of intractable CDD, three had a good therapeutic response to intravenous immunoglobulin and CDD recurred within 6 weeks in one case . In the three successfully treated cases, CDD resolved within 11 days . CONCLUSIONS: Intravenous immunoglobulin is useful for the treatment of intractable and severe CDD . Controlled studies are needed to assess the true value of this and other forms of passive immunotherapy.

Can Vet J, 2004 Mar, 45(3), 251 - 3
Clostridium perfringens type A and beta2 toxin associated with enterotoxemia in a 5-week-old goat; Dray T; Postmortem examination of a Boer buck kid that died peracutely revealed diffusely congested, edematous bowel . Clostridium perfringens Type A was isolated . Some isolates carried the gene for beta2 toxin, suggesting a role for beta2 toxin in caprine enterotoxemia, a common cause of death in growing kids.

J Clin Microbiol, 2004 Apr, 42(4), 1713 - 5
Dual toxin-producing strain of Clostridium botulinum type Bf isolated from a California patient with infant botulism; Barash JR et al.; A retrospective study of Clostridium botulinum strains isolated from patients from California with infant botulism identified the fourth known C . botulinum strain that produces both type B and type F botulinum toxins . This unique strain represented 0.12% of the California infant botulism case isolates from 1976 to 2003 . The relative concentrations of type B and F toxins produced were temperature dependent.

J Clin Microbiol, 2004 Apr, 42(4), 1552 - 8
Multiplex PCR genotyping assay that distinguishes between isolates of Clostridium perfringens type A carrying a chromosomal enterotoxin gene (cpe) locus, a plasmid cpe locus with an IS1470-like sequence, or a plasmid cpe locus with an IS1151 sequence; Miyamoto K et al.; Clostridium perfringens type A isolates carrying the enterotoxin (cpe) gene are important causes of both food poisoning and non-food-borne diarrheas in humans . In North America and Europe, food poisoning isolates were previously shown to carry a chromosomal cpe gene, while non-food-borne gastrointestinal (GI) disease isolates from those two geographic locations were found to have a plasmid cpe gene . In this report, we describe the development of an economical multiplex PCR cpe genotyping assay that works with culture lysates to distinguish among type A isolates carrying a chromosomal cpe gene, a plasmid cpe gene with a downstream IS1470-like sequence, or a plasmid cpe gene with a downstream IS1151 sequence . When this multiplex PCR assay was applied in molecular epidemiologic studies, it was found that (i) all 57 examined type A isolates with a plasmid cpe gene have either IS1470-like or IS1151 sequences downstream of the plasmid cpe gene; (ii) an IS1470-like sequence, rather than an IS1151 sequence, is more commonly present downstream of the plasmid cpe gene (particularly in North American non-food-borne human GI disease isolates); and (iii) as previously shown in the United States and Europe, isolates carrying the chromosomal cpe gene also appear to be the major cause of C . perfringens food poisoning in Japan . The superiority of this new multiplex PCR assay over existing cpe genotyping approaches should facilitate further molecular epidemiologic investigations of C . perfringens enterotoxin-associated GI illnesses and their associated cpe-positive type A isolates.

Proc Natl Acad Sci U S A, 2004 Apr 20, 101(16), 6182 - 7 Epub 2004 Apr 06.
Comparative analysis of gene expression among low G+C gram-positive genomes; Karlin S et al.; We present a comparative analysis of predicted highly expressed (PHX) genes in the low G+C Gram-positive genomes of Bacillus subtilis, Bacillus halodurans, Listeria monocytogenes, Listeria innocua, Lactococcus lactis, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Clostridium acetobutylicum, and Clostridium perfringens . Most enzymes acting in glycolysis and fermentation pathways are PHX in these genomes, but not those involved in the TCA cycle and respiration, suggesting that these organisms have predominantly adapted to grow rapidly in an anaerobic environment . Only B . subtilis and B . halodurans have several TCA cycle PHX genes, whereas the TCA pathway is entirely missing from the metabolic repertoire of the two Streptococcus species and is incomplete in Listeria, Lactococcus, and Clostridium . Pyruvate-formate lyase, an enzyme critical in mixed acid fermentation, is among the highest PHX genes in all these genomes except for C . acetobutylicum (not PHX), and B . subtilis, and B . halodurans (missing) . Pyruvate-formate lyase is also prominently PHX in enteric gamma-proteobacteria, but not in other prokaryotes . Phosphotransferase system genes are generally PHX with selection of different substrates in different genomes . The various substrate specificities among phosphotransferase systems in different genomes apparently reflect on differences in habitat, lifestyle, and nutrient sources.

J Biol Inorg Chem, 2004 Jun, 9(4), 423 - 8 Epub 2004 Apr 06.
The unique hydrogen bonded water in the reduced form of Clostridium pasteurianum rubredoxin and its possible role in electron transfer; Park IY et al.; Rubredoxin is a small iron-sulfur (FeS4) protein involved in oxidation-reduction reactions . The side chain of Leu41 near the iron-sulfur center has two conformations, which we suggested previously serve as a gate for a water molecule during the electron transfer process . To establish the role of residue 41 in electron transfer, an {L41A} mutant of Clostridium pasteurianum rubredoxin was constructed and crystallized in both oxidation states . Despite the lack of the gating side chain in this protein, the structure of the reduced {L41A} rubredoxin reveals a specific water molecule in the same position as observed in the reduced wild-type rubredoxin . In contrast, both the wild-type and {L41A} rubredoxins in the oxidized state do not have water molecules in this location . The reduction potential of the {L41A} variant was approximately 50 mV more positive than wild-type . Based on these observations, it is proposed that the site around the Sgamma of Cys9 serves as a port for an electron acceptor . Lastly, the Fe-S distances of the reduced rubredoxin are expanded, while the hydrogen bonds between Sgamma of the cysteines and the backbone amide nitrogens are shortened compared to its oxidized counterpart . This small structural perturbation in the Fe(II)/Fe(III) transition is closely related to the small energy difference which is important in an effective electron transfer agent .

Appl Environ Microbiol, 2004 Apr, 70(4), 2414 - 9
Identification, detection, and spatial resolution of Clostridium populations responsible for cellulose degradation in a methanogenic landfill leachate bioreactor; Burrell PC et al.; An anaerobic landfill leachate bioreactor was operated with crystalline cellulose and sterile landfill leachate until a steady state was reached . Cellulose hydrolysis, acidogenesis, and methanogenesis were measured . Microorganisms attached to the cellulose surfaces were hypothesized to be the cellulose hydrolyzers . 16S rRNA gene clone libraries were prepared from this attached fraction and also from the mixed fraction (biomass associated with cellulose particles and in the planktonic phase) . Both clone libraries were dominated by Firmicutes phylum sequences (100% of the attached library and 90% of the mixed library), and the majority fell into one of five lineages of the clostridia . Clone group 1 (most closely related to Clostridium stercorarium), clone group 2 (most closely related to Clostridium thermocellum), and clone group 5 (most closely related to Bacteroides cellulosolvens) comprised sequences in Clostridium group III . Clone group 3 sequences were in Clostridium group XIVa (most closely related to Clostridium sp . strain XB90) . Clone group 4 sequences were affiliated with a deeply branching clostridial lineage peripherally associated with Clostridium group VI . This monophyletic group comprises a new Clostridium cluster, designated cluster VIa . Specific fluorescence in situ hybridization (FISH) probes for the five groups were designed and synthesized, and it was demonstrated in FISH experiments that bacteria targeted by the probes for clone groups 1, 2, 4, and 5 were very abundant on the surfaces of the cellulose particles and likely the key cellulolytic microorganisms in the landfill bioreactor . The FISH probe for clone group 3 targeted cells in the planktonic phase, and these organisms were hypothesized to be glucose fermenters.

Vet Microbiol, 2004 Apr 19, 99(3-4), 251 - 7
Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes; Johansson A et al.; This study was undertaken to determine the in vitro susceptibility of Clostridium perfringens, isolated from poultry to antimicrobials used in poultry production . The minimal inhibitory concentration (MIC) of eight antimicrobials, including the ionophoric coccidiostat narasin, was determined for 102 C . perfringens isolates, 58 from Sweden, 24 from Norway and 20 from Denmark . Susceptibility to each antimicrobial compound was determined by broth microdilution . The isolates were obtained from broilers (89), laying hens (9) and turkeys (4), affected by necrotic enteritis (NE) or by C . perfringens associated hepatitis (CPH), and from healthy broilers . All strains, regardless of origin, proved inherently susceptible to ampicillin, narasin, avilamycin, erythromycin and vancomycin . A low frequency of resistance to virginiamycin and bacitracin was also found . Resistance to tetracycline was found in strains isolated in all three countries; Sweden (76%), Denmark (10%) and Norway (29%) . In 80% of the tetracycline-resistant isolates, the two resistance genes tetA(P) and tetB(P) were amplified by PCR whereas in 20% only the tetA(P) gene was detected . No tetM gene amplicon was obtained from any of the tetracycline-resistant isolates . The uniform susceptibility to narasin revealed in this study shows that the substance can still be used to control clostridiosis . In this study, C . perfringens also showed a low degree of resistance to most other antimicrobials tested . Despite the small amounts of tetracycline used in poultry, a considerable degree of resistance to tetracycline was found in C . perfringens isolates from Swedish broilers.

Extremophiles, 2004 Feb, 8(1), 63 - 71 Epub 2003 Oct 29.
Diversity of Kenyan soda lake alkaliphiles assessed by molecular methods; Rees HC et al.; DNA was extracted from water and sediment samples taken from soda lakes of the Kenyan-Tanzanian Rift Valley . DNA was also extracted from microbial enrichment cultures of sediment samples . 16S rRNA genes were amplified by the polymerase chain reaction and microbial diversity was studied using denaturing gradient gel electrophoresis (DGGE) of 16S rDNA amplicons . Cloning and sequencing of single DGGE bands showed that they usually contained mixed amplicons . Several of the amplicon sequences had high identities, up to 99%, with 16S rRNA genes of organisms previously isolated from soda lakes, while others were only distantly related, with identities as low as 82% . Phylogenetic analysis of the sequenced amplicons indicated that sequences were related to the haloarchaeal, Bacillus/Clostridium, Rhodobacterium/ Thioalcalovibrio/ Methylobacter, and Cytophaga/Flavobacterium/Bacteroides (CFB) groups and the enterobacteria/ Aeromonas/Vibrio part of the gamma3 subdivision of the Proteobacteria.

Extremophiles, 2004 Apr, 8(2), 169 - 73 Epub 2003 Nov 19.
Pressure effects on Clostridium strains isolated from a cold deep-sea environment; Lauro FM et al.; Three Clostridium strains were isolated from deep-sea sediments collected at a depth of 6.3-7.3 km in the Japan Trench . Physiological characterization and 16S rDNA analysis revealed that the three isolates were all closely related to Clostridium bifermentans . The spores of all three isolates were resistant to inactivation at high pressure and low temperature . However, despite the fact that the vegetative cells were halotolerant and eurythermal they did not appear to be adapted for growth or viability under the conditions prevailing in the deep-sea sediments from which they were obtained . The results suggest that the isolates had survived as spores in the deep-sea sediments and that the marine benthos could be a source of clostridia originating in other environments.

Biochem Biophys Res Commun, 2004 Apr 30, 317(2), 384 - 9
The Rac/Cdc42 guanine nucleotide exchange factor beta1Pix enhances mastoparan-activated Gi-dependent pathway in mast cells; Chahdi A et al.; Carbachol stimulates granule exocytosis, phospholipase C (PLC), and phospholipase D (PLD) in RBL-2H3hm1 mast cells by a mechanism that involves Galphaq . However, mastoparan stimulates the same responses through Gi protein . Both Gi and Galphaq pathways are suppressed by Clostridium difficile toxin B, suggesting that Rac and Cdc42 small GTPases are also involved . Over-expression of beta1Pix, a guanine nucleotide exchange factor for Rac and Cdc42, enhances mastoparan-but not carbachol-induced hexosaminidase secretion and PLC and PLD activation . Furthermore, cells expressing beta1Pix exhibit elevated levels of mastoparan-stimulated IP3 production . Taken together, these findings implicate beta1Pix in regulating hexoasaminidase secretion and IP3 production in early stage upon mastoparan stimulation.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 325 - 31
Cel6B of Thermobifidus fusca and a Cel5-CBM6 of Ruminococcus albus containing a cellulose binding site show synergistic effect on hydrolysis of native plant cellulose; Bae HJ et al.; Hydrolysis of cellulose requires two different types of cellulases: exo- and endocellulase . Here, we investigated for the hydrolysis of cellulose by two types of cellulases, an endoglucanase (Cel5) from Ruminococcus albus fused with the xylanase A cellulose binding domain II (CBM6) of Clostridium stercorarium and Thermobifidus fusca E3, an exoglucanase (Cel6B) . Cel5-CBM6 or Cel6B showed a linear relationship between the production of soluble sugars and the incubation time when native alfalfa cellulose was used as a substrate . Cel5-CBM6 produces more soluble sugars than Cel6B and the hydrolysis of cellulose by a mixture of the two enzymes produces substantially more (22%) soluble sugars than the total amount produced by these enzymes individually . Although Cel5-CBM6 solubilized high quantities of sugars from alfalfa cellulose, it did not significantly decrease its crystallinity, while Cel6B decreased the crystallinity of cellulose by 34% . When the two cellulases were combined, a decrease of more than 50% in the content of crystalline cellulose was observed . The enzyme-gold labeling experiments revealed that both enzymes showed a high affinity for all substrates . Furthermore, simultaneous visualization of the enzyme-binding sites revealed the preferred substrates in native lignocellulosic material . When plant cellulose was pre-incubated with Cel5-CBM6, density of the gold labeling greatly increased suggesting that preliminary exposure of lignocellulosic material to Cel5-CBM6 may have enhanced the accessibility of the substrate to Cel5-CBM6 and Cel6B . This result provides a plausible explanation for the observed endo/exo cellulase synergism during hydrolysis.

FEMS Microbiol Lett, 2004 Apr 15, 233(2), 233 - 40
Disruption of the gene (spo0A) encoding sporulation transcription factor blocks endospore formation and enterotoxin production in enterotoxigenic Clostridium perfringens type A; Huang IH et al.; This study identified a functional spo0A ORF in enterotoxigenic Clostridium perfringens type A . To evaluate the function of spo0A, an isogenic spo0A knock-out mutant was constructed . The spo0A mutant was unable to form endospores and produce enterotoxin, however, these defects could be restored by complementing the mutant with a recombinant plasmid carrying the wild-type spo0A gene . These results provide evidence that spo0A expression is essential for sporulation and enterotoxin production in C . perfringens.

J Microbiol Methods, 2004 May, 57(2), 175 - 80
Enumeration of Clostridium perfringens spores in groundwater samples: comparison of six culture media; Araujo M et al.; In order to investigate the ability of Fluorocult-supplemented TSC agar (TSCF (Fluorocult supplemented TSC-agar): prepared from Tryptose Sulfite Cycloserine Agar Base (Merck), D-cycloserine (Fluka Chemika, USA), and fluorocult TSC-Agar supplement (Merck)) for detecting spores of Clostridium perfringens in water, we analyzed groundwater samples, pretreated by heating to 80 degrees C/5 min, using this fluorogenic medium together with five other media: mCP agar (Panreac; Cultimed), TSC agar (Merck, Germany), TSN agar (Merck), and SPS agar (BBL, USA) by the membrane filtration technique, and Wilson-Blair agar (WB) following the still-in-force Spanish official method . Variance analysis of the data obtained shows statistically significant differences in the counts obtained between media employed in this work . The C . perfringens spore counts on mCP agar were significantly lower (P<0.05) than the corresponding values of TSC, TSCF, SPS, and WB media . No statistically significant differences were found between C . perfringens spore counts on TSCF compared with those of other methods used . On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for C . perfringens spore recovery in groundwater samples . Additionally, the results obtained indicate that mCP agar, which is the reference method in the European Union, is not suitable medium for recovering C . perfringens spores from groundwater samples.

Facial Plast Surg Clin North Am, 2003 Nov, 11(4), 431 - 8
The basic science of botulinum toxin; Lam SM; Understanding the basic science of botulinum toxin should serve as a fundamental first step for clinical therapy . This article endeavors to cover many aspects of basic research that also have clinical import . The two principal toxins of the clostridial family, Clostridium tetani and C botulinum, are described in detail . The five clinical manifestations of botulism poisoning are also outlined, and structural aspects and the mechanism of action of botulinum toxin are then presented . Finally, the immunologic and pharmacologic principles that define the various serotypes of botulinum toxin are set forth.

Nucleic Acids Res, 2004 Apr 01, 32(6), 1973 - 81 Print 2004.
Transcriptional organization of the Clostridium acetobutylicum genome; Paredes CJ et al.; Prokaryotic genes are frequently organized in multicistronic operons (or transcriptional units, TUs), and usually the regulatory motifs for the whole TU are located upstream of the first TU gene . Although the number of sequenced genomes has increased dramatically, experimental information on TU organization is extremely limited . Even for organisms as extensively studied as Escherichia coli and Bacillus subtilis, TU annotation is far from complete . It therefore becomes imperative to rely on computational approaches to complement experimental information . Here we present a TU map for the obligate anaerobe Clostridium acetobutylicum ATCC 824 . This map is largely based on the distance between pairs of consecutive genes but enhanced and refined by predictions of several types of promoters (sigmaA, sigmaE and sigmaF/G) and rho-independent terminator structures . Based on the set of known C.acetobutylicum TUs, the presented TU map offers an 88% prediction accuracy.

J Bacteriol, 2004 Apr, 186(8), 2508 - 10
Clostridium difficile IStron CdISt1: discovery of a variant encoding two complete transposase-like proteins; Hasselmayer O et al.; Screening a Clostridium difficile strain collection for the chimeric element CdISt1, we identified two additional variants, designated CdISt1-0 and CdISt1-III . In in vitro assays, we could prove the self-splicing ribozyme activity of these variants . Structural comparison of all known CdISt1 variants led us to define four types of IStrons that we designated CdISt1-0 through CdISt1-III . Since CdISt1-0 encodes two complete transposase-like proteins (TlpA and TlpB), we suggest that it represents the original genetic element, hypothesized before to have originated by fusion of a group I intron and an insertion sequence element.

J Bacteriol, 2004 Apr, 186(8), 2495 - 8
Analysis of an engineered Salmonella flagellar fusion protein, FliR-FlhB; Van Arnam JS et al.; Salmonella FliR and FlhB are membrane proteins necessary for flagellar export . In Clostridium a fliR-flhB fusion gene exists . We constructed a similar Salmonella fusion gene which is able to complement fliR, flhB, and fliR flhB null strains . Western blotting revealed that the FliR-FlhB fusion protein retains the FlhB protein's cleavage properties . We conclude that the FliR and FlhB proteins are physically associated in the wild-type Salmonella basal body, probably in a 1:1 ratio.

ALTEX, 2004, 21 Suppl 3, 65 - 9
Quality assurance of C . perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test; Rosskopf-Streicher U et al.; Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium . Disease caused by C . perfringens infection is called enterotoxaemia . C . perfringens strains are classified on the basis of the lethal exotoxins formed by the bacteria . Epsilon toxin is one of the major lethal toxins and is formed by C . perfringens types B and D . C . perfringens is an ubiquitous bacterium . Infection occurs via food, water, animal litter or soil . Affected animals include mainly sheep, pigs and cattle . C . perfringens infection manifests as pulpy kidney disease and diarrhoea in suckling lambs . Enterotoxaemia development is peracute in most cases . Animals die suddenly while grazing on the pasture, without any prior signs of disease . Therefore, treatment is possible only in very rare cases . Suitable immunoprophylactic measures are the treatment of choice to combat the disease: Vaccines and immunosera have therefore been used extensively for a long time . The requirements for quality, efficacy and safety testing of the inactivated vaccines are laid down in the Ph . Eur . in the monograph: Clostridium perfringens vaccines for veterinary use . After a marketing authorisation is attained, the product batches must be tested in laboratory animal models for their potency against all vaccine components (Pharmeuropa, 1997) . For potency testing (batch control) of C . perfringens types B and D, the induction of specific antibodies against epsilon toxin in rabbits must be verified . For this purpose, 10 rabbits are immunised twice with the product to be tested . Their blood is taken 14 days after the last immunisation and the serum is pooled . The pooled serum is then tested for its protective effect . This is done by means of the toxin neutralisation test in mice (optionally also in guinea pigs) in comparison with an international reference serum . The evaluation criterion is the death rate of the mice in the test and reference groups after administration of lethal doses of epsilon toxin . The exact efficacy of the test serum is given in International Units (IU) . The tested serum must show a minimum content of 5 IU . This in vivo method requires a very high number of experimental animals . Approximately 400 mice (or 50 guinea pigs) are used per vaccine batch . The monograph for C . perfringens vaccines, which has recently been revised, expressly indicates that a validated serological method may be used for batch testing . In addition, a reference serum known as clostridium multicomponent serum has been available since 2000 . The objective is to test vaccine batches against this reference and by means of a competitive ELISA developed in the precursor project, using a monoclonal antibody for direct determination of specific antitoxins in rabbit sera . This ELISA method was subjected to an international validation to verify whether the protocol and the precision can be transferred within and between the participating laboratories.

Biosci Biotechnol Biochem, 2004 Mar, 68(3), 609 - 14
Sequencing and expression of the gene encoding the Clostridium stercorarium beta-xylosidase Xyl43B in Escherichia coli; Suryani et al.; The Clostridium stercorarium F-9 xyl43B gene encoding the beta-xylosidase Xyl43B consists of an open reading frame of 1,491 nucleotides that encodes a putative protein, classified in family 43, of 497 amino acids with a predicted molecular weight of 56,355 . The deduced amino acid sequence of Xyl43B has sequence similarity with beta-xylosidases from Bacteriodes thetaiotaomicron (57% sequence identity), Prevotella ruminicola (45%), Streptomyces coelicolor (40%), and Clostridium acetobutylicum (36%), all of which have been classified in family 43 of the glycoside hydrolases . Xyl43B was purified from a recombinant Escherichia coli and characterized . The optimum pH of the purified enzyme was 3.5 and it was stable over pH from 3.0 to 8.0 . Its optimum temperature was 80 degrees C and it showed thermostability in the temperature range from 50 to 70 degrees C . Xyl43B had a K(m) of 6.2 mM and a V(max) of 15 micromol min(-1) mg(-1) for p-nitrophenyl-beta-D-xylopyranoside.

Am Surg, 2004 Mar, 70(3), 268 - 71
Pneumoretroperitoneum in two patients with Clostridium perfringens necrotizing pancreatitis; Anderson CM et al.; Pancreatic gas gangrene is an uncommon and often fatal complication of acute pancreatitis, due to the sporulating anaerobe Clostridium perfringens . C . perfringens is a normal constituent of colonic flora, but infects the pancreas by either transmural spread from the colon or via the biliary tree . Only three reported cases in the world literature describe acute pancreatitis with pneumoretroperitoneum and clostridial infection . Two separate cases, at the same institution, of acute pancreatitis complicated by C . perfringens were analyzed . The records of patients were reviewed for admission history, laboratory and radiology results, intensive care support, surgical intervention, and outcome . Retroperitoneal air was visualized early in the clinical course of both patients by computed tomography . Early surgical debridement, drainage, parental antibiotics, and reexploration resulted in an uncomplicated recovery . Early computed tomography in patients with suspected necrotizing pancreatitis contributes to early intervention and may advantageously enhance survival.

Appl Biochem Biotechnol, 2004 Spring, 113-116, 887 - 98
Continuous production of butanol by Clostridium acetobutylicum immobilized in a fibrous bed bioreactor; Huang WC et al.; We explored the influence of dilution rate and pH in continuous cultures of Clostridium acetobutylicum . A 200-mL fibrous bed bioreactor was used to produce high cell density and butyrate concentrations at pH 5.4 and 35degreesC . By feeding glucose and butyrate as a cosubstrate, the fermentation was maintained in the solventogenesis phase, and the optimal butanol productivity of 4.6 g/(L h) and a yield of 0.42 g/g were obtained at a dilution rate of 0.9 h-1 and pH 4.3 . Compared to the conventional acetone-butanol-ethanol fermentation, the new fermentation process greatly improved butanol yield, making butanol production from corn an attractive alternative to ethanol fermentation.

Brain Res, 2004 Mar 12, 1000(1-2), 85 - 91
A role for monomeric G-proteins in synaptic plasticity in the rat dentate gyrus in vitro; Murray HJ et al.; Recent studies have implicated Ras signalling in synaptic plasticity . In this study we have investigated a role for the low molecular weight G proteins Ras, Rap, Ra1 and Rac in long-term potentiation and depression using Clostridium Sordelli Lethal Toxin-82 (LT-82), which inactivates Ras, Rap, Ra1 and Rac, and manumycin A, a Ras inhibitor . Perfusion of hippocampal slices with LT-82 (200 ng/ml) attenuated LTP (83+/-10%, n=5, P<0.01, compared with controls of 160+/-11% at 60 min post HFS, n=5) . LT-82 had no effect on LTD (63+/-1% at 100 ng/ml, n=5 and 66+/-1% at 200 ng/ml, n=4, compared to controls of 56+/-6%, n=6) . Manumycin A (2 microM) had no effect on LTP (162+/-2%, n=5, compared to controls of 167+/-13%, n=5), but significantly attenuated LTD (88+/-6%, n=5, P<0.01, compared to controls of 63+/-9%, n=7) . LT-82 (200 ng/ml) significantly increased the amplitude of the isolated NMDA-EPSP at 60 min post-drug application (240+/-40%, n=5, P<0.01, compared with controls of 100+/-4%, n=5) . However, manumycin A, had no significant effect on NMDAR-EPSP amplitude (92+/-2%, n=5, compared with controls) . These results demonstrate an important role for Ras in LTD and a role for Rap, Ra1 and Rac in LTP.

Transplant Proc, 2004 Mar, 36(2), 379 - 80
Infectious enteritis after intestinal transplantation: incidence, timing, and outcome; Ziring D et al.; AIM: To review the incidence, timing, and outcome of infectious enteritis after intestinal transplantation (IT) . METHOD: A retrospective review of all patients undergoing IT at a single institution between 1991 and 2003 was analyze with standard statistical tools . RESULTS: Among 33 IT recipients, 13 (39%) developed 20 culture- or biopsy-proven episodes of infectious enteritis . The recipient demographics were 77% men and median age 2.6 years . Infections were diagnosed at a median of 76 days (32 to 1800) after IT . There were 14 viral (CMV one, rotavirus eight, adenovirus four, EBV one, three bacterial (Clostridium difficile), and three other infections (Giardia lamblia one, cryptosporidium two) . Complete resolution was achieved in 17 (94%) infectious after appropriate antimicrobial or conservative therapy . Interestingly, there were six rejection episodes following infectious enteritis . Grafts were lost to rejection after rotaviral enteritis (n = 1) and adenoviral enteritis misdiagnosed as rejection (n = 1) . Patient and graft survival were not adversely affected by infections . CONCLUSIONS: Infectious enteritis occurs frequently after IT . Viral agents are the cause in two-thirds of cases . With supportive care and appropriate treatment, resolution is possible in the majority of cases . Differentiating rejection and infection by histopathology can be difficult.

Poult Sci, 2004 Mar, 83(3), 414 - 20
Effects of dietary protein source and level on intestinal populations of Clostridium perfringens in broiler chickens; Drew MD et al.; Two experiments were conducted to examine the effect of the level of dietary crude protein and protein source on intestinal populations of Clostridium perfringens in broilers . In experiment 1, 6 groups of 12 birds were fed diets containing 230,315 or 400 g/kg crude protein with soy protein concentrate (SPC) or low-temperature-dried fishmeal as the major protein sources in a 2 x 3 factorial arrangement of treatments . A significant interaction between protein source and level was observed where the number of C . perfringens present in the ileum and cecum increased as the level of crude protein in the diets increased from 230 to 400 g/kg in the birds fed fishmeal-based diets (P < 0.05) but not in the birds fed SPC-based diets . In experiment 2, the dietary treatments used were arranged in a 2 x 2 factorial arrangement with 2 levels of crude protein (230 and 400 g/kg) and 2 protein sources (SPC or fishmeal) . The main effects of protein source and protein level significantly (P < 0.05) affected numbers of C . perfringens without interaction . Amino acid analysis of the diets showed that the glycine and methionine contents of the fishmeal diets were elevated compared with the SPC diets . This suggests that the level of crude protein, protein source, and amino acid content of diets affect the growth of C . perfringens in the lower intestinal tract of the broiler chicken and might be predisposing factors to outbreaks of clinical necrotic enteritis.

Can J Microbiol, 2001 Jan, 47(1), 96 - 101
Phospholipid analogue profiles of Peptostreptococcus, Micromonas, and Finegoldia species analysed by fast atom bombardment mass spectrometry; Radcliffe CE et al.; Species of Peptostreptococcus cause a variety of infections, primarily abscesses of soft tissues, joints, and mucous membranes . The aim of this study was to compare the phospholipid analogue profiles of Peptostreptococcus species, represented by P . anaerobius, P . asaccharolyticus, P . indolicus, P . lacrimalis, and P . prevotii; Micromonas micros (P . micros) and Finegoldia magna (P . magnus) . After anaerobic growth on blood-FAA, lipids extracted by chloroform-methanol (2:1 v/v) were purified, then analysed by fast atom bombardment mass spectrometry (FAB-MS) in negative ion mode . The major peaks with mass to charge (m/z) 719, 721, and 749, corresponded to phosphatidylglycerol analogues, namely PG (32:1), PG (32:0), and PG (34:0), which have been found previously in Lactobacillus spp., Clostridium difficile, and Staphylococcus spp . Other major peaks observed, with m/z 619, 647, 665, 675, 677, 687, 691, 693, 701, 703, 707, 733, and 746 have also been reported in one or more of these three species . However, other major peaks found here in Peptostreptococcus, Micromonas, and Finegoldia have not been described elsewhere; these are 501, 514, 515, 618, 659, 673, 676, 688, 690, 692, 694, 700, 706, 715, 718, 722, and 750 . We conclude that Peptostreptococcus, Micromonas, and Finegoldia isolates are chemically unique.

J Biol Chem, 2004 Jun 4, 279(23), 24714 - 23 Epub 2004 Mar 23.
Alpha C protein of group B Streptococcus binds host cell surface glycosaminoglycan and enters cells by an actin-dependent mechanism; Baron MJ et al.; Group B Streptococcus (GBS) colonizes mucosal surfaces of the human gastrointestinal and gynecological tracts and causes disease in a wide range of patients . Invasive illness occurs after organisms traverse an epithelial boundary and enter deeper tissues . Previously we have reported that the alpha C protein (ACP) on the surface of GBS mediates GBS entry into ME180 cervical epithelial cells and GBS translocation across layers of these cells . We now demonstrate that ACP interacts with host cell glycosaminoglycan (GAG); the interaction of ACP with ME180 cells is inhibited if cells are pretreated with sodium chlorate, an inhibitor of sulfate incorporation, or with heparitinases . The interaction is also inhibited in the presence of soluble heparin or heparan sulfate or host cell-derived GAG . In addition, ACP binds soluble heparin specifically in inhibition and dot blot assays . After interaction with host GAG, soluble ACP enters ME180 cells and fractionates to the eukaryotic cell cytosol . These events are inhibited in cells pretreated with cytochalasin D or with Clostridium difficile toxin B . These data indicate that full-length ACP interacts with ME180 cell GAG and enters the eukaryotic cell cytosol by a mechanism that involves Rho GTPase-dependent actin rearrangements . We suggest that these molecular interactions drive ACP-mediated translocation of GBS across epithelial barriers, thereby facilitating invasive GBS infection.

Am J Physiol Heart Circ Physiol, 2004 Aug, 287(2), H704 - 11 Epub 2004 Mar 25.
Role of adhesion and contraction in Rac 1-regulated endothelial barrier function in vivo and in vitro; Waschke J et al.; We demonstrated previously that inhibition of the small GTPase Rac-1 by Clostridium sordellii lethal toxin (LT) increased the hydraulic conductivity (L(p)) of rat venular microvessels and induced gap formation in cultured myocardial endothelial cells (MyEnd) . In MyEnd cells, we also demonstrated that both LT and cytochalasin D reduced cellular adhesion of vascular endothelial (VE)-cadherin-coated beads . Here we further evaluate the contribution of actin depolymerization, myosin-based contraction, and VE-cadherin linkage to the actin cytoskeleton to LT-induced permeability . The actin-depolymerizing agent cytochalasin D increased L(p) in single rat mesenteric microvessels to the same extent as LT over 80 min . However, whereas the actin-stabilizing agent jasplakinolide blunted the L(p) increase due to cytochalasin D by 78%, it had no effect on the LT response . This conforms to the hypothesis that the predominant mechanism whereby Rac-1 stabilizes the endothelial barrier in intact microvessels is separate from actin polymerization and likely at the level of the VE-cadherin linkage to the actin cytoskeleton . In intact vessels, neither inhibition of contraction (butanedione monoxime, an inhibitor of myosin ATPase) nor inhibition of Rho kinase (Y-27632) modified the response to LT, even though both inhibitors lowered resting L(p) . In contrast butanedione monoxime and inhibition of myosin light chain kinase completely inhibited LT-induced intercellular gap formation and largely reduced the LT-induced permeability increase in MyEnd monolayers . These results support the hypothesis that the contractile mechanisms that contribute to the formation of large gaps between cultured endothelial cells exposed to inflammatory conditions do not significantly contribute to increased permeability in intact microvessels.

FEMS Microbiol Lett, 2004 Apr 1, 233(1), 65 - 8
Phospholipids of Clostridium perfringens: a reexamination; Johnston NC et al.; We have identified phosphatidylethanolamine as one of the major phospholipids of Clostridium perfringens by two dimensional thin layer chromatography of the intact lipids and of their deacylation products and by liquid chromatography followed by mass spectrometry of the intact neutral phospholipid fraction . The principal fatty acids of phosphatidylethanolamine are myristic acid (14:0), lauric acid (12:0), and palmitic acid (16:0) and the major molecular species are 14:0,14:0 (26.3%); 12:0,14:0 (19.0%); 14:0,16:0 (22.4%) and 16:0,16:0 (17.6%) . A similar distribution of molecular species was found in the other major phospholipid, O-alanyl phosphatidylglycerol.

Biophys J, 2004 Apr, 86(4), 2030 - 6
Protein control of electron transfer rates via polarization: molecular dynamics studies of rubredoxin; Dolan EA et al.; The protein matrix of an electron transfer protein creates an electrostatic environment for its redox site, which influences its electron transfer properties . Our studies of Fe-S proteins indicate that the protein is highly polarized around the redox site . Here, measures of deviations of the environmental electrostatic potential from a simple linear dielectric polarization response to the magnitude of the charge are proposed . In addition, a decomposition of the potential is proposed here to describe the apparent deviations from linearity, in which it is divided into a "permanent" component that is independent of the redox site charge and a dielectric component that linearly responds or polarizes to the charge . The nonlinearity measures and the decomposition were calculated for Clostridium pasteurianum rubredoxin from molecular dynamics simulations . The potential in rubredoxin is greater than expected from linear response theory, which implies it is a better electron acceptor than a redox site analog in a solvent with a dielectric constant equivalent to that of the protein . In addition, the potential in rubredoxin is described well by a permanent potential plus a linear response component . This permanent potential allows the protein matrix to create a favorable driving force with a low activation barrier for accepting electrons . The results here also suggest that the reduction potential of rubredoxin is determined mainly by the backbone and not the side chains, and that the redox site charge of rubredoxin may help to direct its folding.

Biochem Pharmacol, 2004 Apr 15, 67(8), 1569 - 77
CysLT1 signal transduction in differentiated U937 cells involves the activation of the small GTP-binding protein Ras; Capra V et al.; We investigated the signal transduction pathway(s) of leukotriene D(4) (LTD(4)) in the human promonocytic U937 cells, a cell line known to constitutively express CysLT(1) receptors . Herein, we demonstrate that LTD(4) specifically acts on a CysLT(1) receptor to dose-dependently increase (three to five-fold over basal) RasGTP through a G(i/o) protein . In fact, while cytosolic Ca(2+) ({Ca(2+)}(i)) increase was only partially sensitive to pertussis toxin (PTx), Ras activation was almost completely inhibited by the same toxin . Furthermore, the phospholipase C (PLC) inhibitor U73122 completely inhibited both {Ca(2+)}(i) and RasGTP increase, suggesting that in these cells PLC is the point of convergence for both PTx insensitive and sensitive pathways leading to {Ca(2+)}(i) release and Ras activation . Indeed, chelating intracellular Ca(2+) strongly (>70%) prevented LTD(4)-induced Ras activation, indicating that this ion plays an essential role for CysLT(1)-induced downstream signaling in differentiated U937 (dU937) cells . In addition, while Src did not appear to be substantially involved in CysLT(1)-induced signaling, genistein was able to partially inhibit LTD(4)-induced {Ca(2+)}(i) transient ( approximately 34%) and almost completely prevented Ras activation (>90%), suggesting a potential role for other Ca(2+)-dependent tyrosine kinases in LTD(4)-induced signaling . Finally, agonist-induced CysLT(1) stimulation was followed by a specific extracellular regulated kinase (ERK) 1/2 phosphorylation, an event with a pharmacological profile similar to that of Ras activation, partially ( approximately 40%) sensitive to Clostridium sordellii lethal toxin and totally blocked by PTx . In conclusion, LTD(4)-induced CysLT(1) receptor activation in dU937 cells leads to Ras activation and ERK phosphorylation mostly through a PTx-sensitive G(i/o) protein, PLC, and Ca(2+)-dependent tyrosine kinase(s).

Vaccine, 2004 Feb 17, 22(7), 848 - 56
Active and passive immunization against Clostridium difficile diarrhea and colitis; Giannasca PJ et al.; Clostridium difficile, a gram-positive bacterium, is the major cause of hospital-acquired infectious diarrhea and colitis in industrialized nations . C . difficile colonization results from antibiotic administration and subsequent loss of protection provided by intestinal flora . C . difficile induced-colitis is caused by the release of two exotoxins, toxin A and B . Host factors including advanced age, pre-existing severe illness and weakened immune defenses predispose individuals to symptomatic infection . The generation of antibody responses to toxin A through natural exposure is associated with protection from disease . In addition, an inability to acquire immunity to toxin A puts individuals at risk for recurrent and/or severe disease . Immunological approaches for the management of this disease are being developed which could reduce the reliance on antibiotics for treatment and allow for re-establishment of the natural barrier provided by an intact commensal flora . An active vaccine and various immunotherapeutic strategies under evaluation may prove to be effective against severe or relapsing C . difficile infection.

Infect Immun, 2004 Apr, 72(4), 2186 - 93
Detergent-resistant membrane microdomains facilitate Ib oligomer formation and biological activity of Clostridium perfringens iota-toxin; Hale ML et al.; Clostridium perfringens iota-toxin consists of two separate proteins identified as a cell binding protein, iota b (Ib), which forms high-molecular-weight complexes on cells generating Na(+)/K(+)-permeable pores through which iota a (Ia), an ADP-ribosyltransferase, presumably enters the cytosol . Identity of the cell receptor and membrane domains involved in Ib binding, oligomer formation, and internalization is currently unknown . In this study, Vero (toxin-sensitive) and MRC-5 (toxin-resistant) cells were incubated with Ib, after which detergent-resistant membrane microdomains (DRMs) were extracted with cold Triton X-100 . Western blotting revealed that Ib oligomers localized in DRMs extracted from Vero, but not MRC-5, cells while monomeric Ib was detected in the detergent-soluble fractions of both cell types . The Ib protoxin, previously shown to bind Vero cells but not form oligomers or induce cytotoxicity, was detected only in the soluble fractions . Vero cells pretreated with phosphatidylinositol-specific phospholipase C before addition of Ib indicated that glycosylphosphatidyl inositol-anchored proteins were minimally involved in Ib binding or oligomer formation . While pretreatment of Vero cells with filipin (which sequesters cholesterol) had no effect, methyl-beta-cyclodextrin (which extracts cholesterol) reduced Ib binding and oligomer formation and delayed iota-toxin cytotoxicity . These studies showed that iota-toxin exploits DRMs for oligomer formation to intoxicate cells.

Equine Vet J, 2004 Mar, 36(2), 123 - 9
Equine grass sickness is associated with low antibody levels to Clostridium botulinum: a matched case-control study; McCarthy HE et al.; REASONS FOR PERFORMING STUDY: Equine grass sickness is a high mortality disease which, despite many years of investigation, is of unknown aetiology . Recent findings indicating that the disease is associated with Clostridium botulinum require support from an epidemiological study that recognises and controls for potential confounders, e.g . age, time of year and premises . HYPOTHESIS: EGS is associated with low antibody levels to C . botulinum antigens . METHODS: A matched case-control study was conducted . Data were collected from 66 histologically confirmed cases of EGS and 132 premises-matched control horses . The probability of EGS in horses was modelled using conditional logistic regression . RESULTS: EGS was significantly associated (age-adjusted P < 0.005) with low antibody levels to each of 3 clostridial antigens; C . botulinum type C and C . novyi type A surface antigens and a C . botulinum type C toxin complex toxoid . These serological risk factors for EGS remained highly significant when entered into multivariable models . This study also identified new horse-level risk factors for EGS; feeding hay or haylage was associated with a decreased risk of disease, change of feed type or quantity during the 14 days prior to disease was associated with increased risk, and the use of an ivermectin anthelmintic at both the ultimate and penultimate treatments was also associated with a significantly increased risk of EGS . CONCLUSIONS: This study provides strong support for the role of C . botulinum in the aetiology of EGS and identifies managemental risk factors for the disease . POTENTIAL RELEVANCE: Increasing anticlostridial antibody levels by vaccination and appropriate managemental interventions may decrease the risk of EGS occurring.

Equine Vet J, 2004 Mar, 36(2), 105 - 12
An epidemiological study of risk factors associated with the recurrence of equine grass sickness (dysautonomia) on previously affected premises; Newton JR et al.; REASONS FOR PERFORMING STUDY: The reasons why equine grass sickness (EGS) recurs on premises are unknown and, consequently, practical methods for reducing the risk of recurrence are not available . OBJECTIVES: To identify risk factors associated with recurrence of EGS on premises and to gain possible insights into the pathogenesis of the disease . METHODS: Data on disease history and risk factors were collected by postal questionnaire from premises with EGS cases between 1st January 1997 and 31st December 2001 . Data on variation in rates of recurrence of EGS for different risk factors were analysed using Poisson regression analysis . RESULTS: Of 509 premises contacted, 305 (60%) returned useable questionnaires and 100 of these (33%) were classified as 'recurrent' premises . An overall median incidence rate for EGS of 2.1 EGS incidents/100 horses/premises/year was recorded . There was an increased rate of recurrence with higher numbers of horses, presence of younger animals, stud farms and livery/riding establishments, loam and sand soils, rearing of domestic birds and mechanical droppings removal . The rate of recurrence decreased with chalk soil, cograzing ruminants, grass cutting on pastures and removal of droppings by hand . Several statistically significant interactions were identified . CONCLUSIONS: Many of the findings are consistent with the theory that EGS is a toxico-infectious form of botulism . Several of the significant factors identified may directly or indirectly relate to soil disturbance and consequent soil contamination of grass, thereby increasing the rate of exposure of grazing horses to Clostridium botulinum, which resides in soil . Potential relevance: Identification of potentially modifiable risk factors may, ideally following validation in appropriately designed, controlled and randomised intervention studies, lead to practical measures to reduce the incidence of EGS on previously affected premises.

Toxicon, 2004 Jan, 43(1), 27 - 34
A highly sensitive immuno-polymerase chain reaction assay for Clostridium botulinum neurotoxin type A; Chao HY et al.; Our goal was to develop a sensitive method for detecting Clostridium botulinum neurotoxin type A (BoNT/A) . We were able to detect BoNT/A in the femtogram (10(-15)g) range using an indirect immuno-polymerase chain reaction (immuno-PCR) assay and an indirect sandwich immuno-PCR assay . For the indirect immuno-PCR assay, enzyme-linked immunosorbent assay (ELISA) plates were coated with BoNT/A that was recognized by anti-BoNT/A monoclonal antibody . For the indirect sandwich immuno-PCR assay, the monoclonal antibody was immobilized on ELISA plates for detecting BoNT/A that was recognized by its polyclonal antibodies . Reporter DNA was prepared by PCR amplification using biotinylated 5'-primers, and it was coupled with biotinylated antibodies through streptavidin . In order to increase sensitivity and reduce background noise, the amounts of reporter DNA (ranging from 50 fg to 50 ng) and streptavidin (ranging from 0.125 ng to 8 ng) were optimized . Using the optimized concentration of reporter DNA and streptavidin, both indirect and indirect sandwich immuno-PCR assays detected BoNT/A as low as 50 fg . These results are a 10(5)-fold improvement over conventional indirect ELISA and indirect sandwich ELISA methods . The assays we developed are currently the most sensitive methods for detecting BoNT/A.

Biochem Biophys Res Commun, 2004 Apr 9, 316(3), 816 - 21
Chemotaxis-mediated biodegradation of cyclic nitramine explosives RDX, HMX, and CL-20 by Clostridium sp . EDB2; Bhushan B et al.; Cyclic nitramine explosives, RDX, HMX, and CL-20 are hydrophobic pollutants with very little aqueous solubility . In sediment and soil environments, they are often attached to solid surfaces and/or trapped in pores and distribute heterogeneously in aqueous environments . For efficient bioremediation of these explosives, the microorganism(s) must access them by chemotaxis ability . In the present study, we isolated an obligate anaerobic bacterium Clostridium sp . strain EDB2 from a marine sediment . Strain EDB2, motile with numerous peritrichous flagella, demonstrated chemotactic response towards RDX, HMX, CL-20, and NO(2)(-) . The three explosives were biotransformed by strain EDB2 via N-denitration with concomitant release of NO(2)(-) . Biotransformation rates of RDX, HMX, and CL-20 by the resting cells of strain EDB2 were 1.8+/-0.2, 1.1+/-0.1, and 2.6+/-0.2nmol h(-1)mgwet biomass(-1) (mean+/-SD; n=3), respectively . We found that commonly seen RDX metabolites such as TNX, methylenedinitramine, and 4-nitro-2,4-diazabutanal neither produced NO(2)(-) during reaction with strain EDB2 nor they elicited chemotaxis response in strain EDB2 . The above data suggested that NO(2)(-) released from explosives during their biotransformation might have elicited chemotaxis response in the bacterium . Biodegradation and chemotactic ability of strain EDB2 renders it useful in accelerating the bioremediation of explosives under in situ conditions.

FEMS Microbiol Lett, 2004 Mar 19, 232(2), 139 - 44
Quantification of toxin-encoding mRNA from Clostridium botulinum type E in media containing sorbic acid or sodium nitrite by competitive RT-PCR; Sharkey FH et al.; Competitive reverse transcription polymerase chain reaction (cRT-PCR) was used to quantify the toxin-encoding mRNA production of a Clostridium botulinum type E strain in media containing either sorbic acid or sodium nitrite . A 10-fold reduction in toxin mRNA production and a 25-fold reduction in the proportion of toxin mRNA to total RNA, was estimated when either 1 mg ml(-1) sorbic acid or 100 microg ml(-1) sodium nitrite were added to the medium at pH 7.0.

J Bacteriol, 2004 Apr, 186(7), 2006 - 18
Transcriptional analysis of butanol stress and tolerance in Clostridium acetobutylicum; Tomas CA et al.; The effects of challenges with low (0.25%, vol/vol) and high (0.75%) concentrations of butanol on the growth, glucose metabolism, product formation, and transcriptional program of the solvent-tolerant Clostridium acetobutylicum strain 824(pGROE1) and the plasmid control strain 824(pSOS95del) were used to study solvent tolerance and stress response . Strain 824(pGROE1) was generated by groESL overexpression . The growth of 824(pGROE1) was less inhibited than that of 824(pSOS95del), and 824(pGROE1) was able to metabolize glucose over the entire course of the culture (60 h postchallenge) while glucose metabolism in 824(pSOS95del) lasted 24 h . A comparison of their respective DNA array-based transcriptional profiles identified genes with similar expression patterns (these genes are likely to be part of a general butanol stress response) and genes with opposite expression patterns (these genes are likely to be associated with increased tolerance to butanol) . Both strains exhibited a butanol dose-dependent increase in expression of all major stress protein genes, including groES, dnaKJ, hsp18, and hsp90; all major solvent formation genes, including aad, ctfA and -B, adc, and bdhA and -B (an unexpected and counterintuitive finding); the butyrate formation genes (ptb and buk); the butyryl coenzyme A biosynthesis operon genes; fructose bisphosphate aldolase; and a gene with homology to Bacillus subtilis kinA . A dose-dependent decrease in expression was observed for the genes of the major fatty acid synthesis operon (also an unexpected and counterintuitive finding), several glycolytic genes, and a few sporulation genes . Genes with opposite expression kinetics included rlpA, artP, and a gene encoding a hemin permease . Taken together, these data suggest that stress, even when it derives from the solvent product itself, triggers the induction of the solvent formation genes.

J Bacteriol, 2004 Apr, 186(7), 1959 - 71
Transcriptional analysis of spo0A overexpression in Clostridium acetobutylicum and its effect on the cell's response to butanol stress; Alsaker KV et al.; Spo0A is the regulator of stationary-phase events and is required for transcription of solvent formation genes in Clostridium acetobutylicum . In order to elucidate the role of spo0A in differentiation, we performed transcriptional analysis of 824(pMSPOA) (a spo0A-overexpressing C . acetobutylicum strain with enhanced sporulation) against a plasmid control strain . DNA microarray data were contrasted to data from a spo0A knockout strain (SKO1) that neither sporulates nor produces solvents . Transcripts of fatty acid metabolism genes, motility and chemotaxis genes, heat shock protein genes, and genes encoding the Fts family of cell division proteins were differentially expressed in the two strains, suggesting that these genes play roles in sporulation and the solvent stress response . 824(pMSPOA) alone showed significant downregulation of many glycolytic genes in stationary phase, which is consistent with metabolic flux analysis data . Surprisingly, spo0A overexpression resulted in only nominal transcriptional changes of regulatory genes (abrB and sigF) whose expression was significantly altered in SKO1 . Overexpression of spo0A imparted increased tolerance and prolonged metabolism in response to butanol stress . While most of the differentially expressed genes appear to be part of a general stress response (similar to patterns in two plasmid control strains and a groESL-overexpressing strain), several genes were expressed at higher levels at early time points after butanol challenge only in 824(pMSPOA) . Most of these genes were related to butyryl coenzyme A and butyrate formation and/or assimilation, but they also included the cell division gene ftsX, the gyrase subunit-encoding genes gyrB and gyrA, DNA synthesis and repair genes, and fatty acid synthesis genes, all of which might play a role in the immediate butanol stress response, and thus in enhanced butanol tolerance.

Mov Disord, 2004 Mar, 19 Suppl 8, S42 - 7
Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain; Chaddock JA et al.; Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types . Previously reported data have demonstrated that the catalytically active LH(N) endopeptidase fragment of botulinum neurotoxin type A (termed LH(N)/A) can be retargeted to a range of cell types in vitro to lead to inhibition of secretion of a range of transmitters . Here, we report the synthesis of endopeptidase conjugates with in vitro selectivity for nociceptive afferents compared to spinal neurons . Chemical conjugates prepared between Erythrina cristagalli lectin and LH(N)/A are assessed in vitro and in in vivo models of pain . Chemical conjugates prepared between E . cristagalli lectin and either natively sourced LH(N)/A, or recombinant LH(N)/A purified from Escherichia coli are assessed, and equivalence of the recombinant material is demonstrated . The duration of action of inhibition of neurotransmitter release by the conjugate in vitro is also assessed and is comparable to that observed with Clostridium botulinum neurotoxin . Selectivity of targeting and therapeutic potential have been confirmed by in vivo electrophysiology studies . Furthermore, the analgesic properties of the conjugate have been assessed in in vivo models of pain and extended duration effects observed . These data provide proof of principle for the concept of retargeted clostridial endopeptidases as novel analgesics .

Mov Disord, 2004 Mar, 19 Suppl 8, S2 - 6
Historical notes on botulism, Clostridium botulinum, botulinum toxin, and the idea of the therapeutic use of the toxin; Erbguth FJ; Food-borne botulism probably has accompanied mankind since its beginning . However, we have only few historical sources and documents on food poisoning before the 19th century . Some ancient dietary laws and taboos may reflect some knowledge about the life-threatening consumption of poisoned food . One example of such a dietary taboo is the 10th century edict of Emperor Leo VI of Byzantium in which manufacturing of blood sausages was forbidden . Some ancient case reports on intoxications with Atropa belladonna probably described patients with food-borne botulism, because the combination of dilated pupils and fatal muscle paralysis cannot be attributed to an atropine intoxication . At the end of the 18th century, some well-documented outbreaks of "sausage poisoning" in Southern Germany, especially in Wurttemberg, prompted early systematic botulinum toxin research . The German poet and district medical officer Justinus Kerner (1786-1862) published the first accurate and complete descriptions of the symptoms of food-borne botulism between 1817 and 1822 . Kerner did not succeed in defining the suspected "biological poison" which he called "sausage poison" or "fatty poison." However, he developed the idea of a possible therapeutic use of the toxin . Eighty years after Kerner's work, in 1895, a botulism outbreak after a funeral dinner with smoked ham in the small Belgian village of Ellezelles led to the discovery of the pathogen Clostridium botulinum by Emile Pierre van Ermengem, Professor of bacteriology at the University of Ghent . The bacterium was so called because of its pathological association with the sausages (Latin word for sausage = "botulus") and not-as it was suggested-because of its shape . Modern botulinum toxin treatment was pioneered by Alan B . Scott and Edward J . Schantz .

Water Res, 2004 Apr, 38(7), 1822 - 30
Stimulation of microbial sulphate reduction in a constructed wetland: microbiological and geochemical analysis; Lloyd JR et al.; Microbial sulphate reduction was stimulated successfully in enclosures installed in a constructed wetland . When sucrose (2.4mM) and NH(4)Cl (600 microM) were added to water in the test enclosures, the indigenous microbial community was able to remove over 90% of the sulphate, present as a contaminant from nearby mining activity at a concentration of 384 mg x l(-1) (4mM), over 50 days . Over 90% of the sucrose was also removed . Sulphate was not reduced in control enclosures containing no added sucrose or NH(4)Cl . Fermentation of sucrose by obligate anaerobes including Clostridium sp . and Bacteriodes sp . preceded sulphate reduction in the test enclosures . Sulphate reduction was biphasic, with maximum rates noted between 2-5 and 23-27 days after the addition of the growth substrates . Relatively unbiased 16S rDNA analysis suggested that nitrogen-fixing bacteria were important constituents of the microbial community in the test enclosures at day 23, suggesting that soluble nitrogen was limiting in the amended test enclosures during the experiment.

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 439 - 43
Tepidibacter formicigenes sp . nov., a novel spore-forming bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent; Urios L et al.; A novel anaerobic, Gram-positive, sporulating and strictly chemoorganoheterotrophic bacterium, designated strain DV1184(T), was isolated from a deep-sea hydrothermal vent sample from the Mid-Atlantic Ridge . The cells were short, straight rods (4 micro m long and 0.8 micro m wide) and were motile with peritrichous flagella . They grew between 35 and 55 degrees C (optimum, 45 degrees C), between pH 5.0 and 8.0 (optimum, 6.0) and at 20-60 g sea salts l(-1) (optimum sea salts concentration, 30 g l(-1)) . Strain DV1184(T) was able to ferment yeast extract, tryptone, peptone, glucose, sucrose, maltose and pyruvate . The main fermentation products from glucose were (in decreasing order) formate, acetate and ethanol . The genomic DNA G+C content was 29 mol% . Phylogenetic analysis of the 16S rRNA gene located the strain within cluster XI of the lineage that encompasses the genus Clostridium and related genera in the bacterial domain . On the basis of 16S rDNA sequence comparison and physiological and biochemical characteristics, it is proposed that the isolate should be described as a novel species, Tepidibacter formicigenes sp . nov . The type strain is DV1184(T) (=CIP 107893(T)=DSM 15518(T)).

Int J Syst Evol Microbiol, 2004 Mar, 54(Pt 2), 413 - 7
Anaerotruncus colihominis gen . nov., sp . nov., from human faeces; Lawson PA et al.; Phenotypic and phylogenetic studies were performed on two isolates of an unidentified Gram-positive, anaerobic, non-spore-forming, rod-shaped bacterium that was isolated from human faeces . The organisms were catalase-negative, produced acetic and butyric acids as end products of metabolism and possessed a DNA G+C content of approximately 54 mol% . Comparative 16S rRNA gene sequencing demonstrated that the two isolates were related closely to each other and formed a hitherto unknown sublineage within the Clostridium leptum rRNA cluster of organisms . Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium should be classified in a novel genus as Anaerotruncus colihominis gen . nov., sp . nov . The type strain of Anaerotruncus colihominis is WAL 14565(T)=CCUG 45055(T)=CIP 107754(T).

Toxicon, 2003 Dec 15, 42(8), 979 - 86
Role of Clostridium perfringens phospholipase C in the pathogenesis of gas gangrene; Flores-Diaz M et al.; Gas gangrene is an acute and devastating infection most frequently caused by Clostridium perfringens and characterized by severe myonecrosis, intravascular leukocyte accumulation, and significant thrombosis . Several lines of evidence indicate that C . perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is the major virulence factor in this disease . This toxin is a Zn2+ metalloenzyme with lecithinase and sphingomyelinase activities . Its three dimensional structure shows two domains, an N-terminal domain which contains the active site, and a C-terminal domain required for the Ca2+dependent interaction with membranes . Cp-PLC displays several biological activities: it increases capillary permeability, induces platelet aggregation, hemolysis, myonecrosis, decreases cardiac contractility, and is lethal . Experiments with genetically engineered Cp-PLC variants have revealed that the sphingomyelinase activity and the C-terminal domain are required for toxicity . The myotoxicity of Cp-PLC is largely dependent on its membrane damaging effect . In addition, it has been suggested that the alterations in the blood flow induced by this toxin also contribute to muscle damage . In gas gangrene, Cp-PLC dysregulates transduction pathways in endothelial cells, platelets and neutrophils leading to the uncontrolled production of several intercellular mediators and adhesion molecules . Thus, Cp-PLC alters the traffic of neutrophils to the infected tissue and promotes thrombotic events, enhancing the conditions for anaerobic growth.

Pharm World Sci, 2004 Feb, 26(1), 8 - 9
Clostridium difficile colitis associated with valaciclovir; De Andres S et al.; OBJECTIVE: To report a case of Clostridium difficile colitis associated with valaciclovir treatment . CASE SUMMARY: A 73-year-old man with lumbar herpes-zoster started valaciclovir 1 g tid . After three days he began vomiting and developed diarrhea, three to four stools per day . Symptoms worsened over the following days and he was admitted . Valaciclovir was stopped and fluid and electrolyte replacement was started . He continued 6 days later with diarrhea of 7 to 13 stools per day and a stool test for diagnosis of C . difficile infection was performed with a positive result . The patient received oral metronidazole (500 mg/t.i.d . for 10 days) and rapid improvement and eventual resolution of his diarrhea was observed after 3 days of therapy . DISCUSSION: Although no conclusive reports of this reaction exist, we think this is a case of C difficile colitis that appeared three days after valaciclovir was initiated . Colitis improved with metronidazole . Other causes of diarrhea were excluded, such as diabetes mellitus, renal failure, intestinal surgery and intestinal obstruction . Infection was confirmed by a positive test for C . difficile . The application of Naranjo's algorithm asserts the reaction as 'probable' . CONCLUSIONS: Valaciclovir-associated C . difficile colitis, although rare, can have severe consequences for the patient's health . It should be included as a possible adverse effect of valaciclovir treatment by health professionals.

Clin Gastroenterol Hepatol, 2003 Sep, 1(5), 370 - 6
Klebsiella oxytoca as an agent of antibiotic-associated hemorrhagic colitis; Beaugerie L et al.; BACKGROUND & AIMS: Klebsiella oxytoca has been isolated from stools and colonic biopsy specimens of patients with Clostridium difficile-negative antibiotic-associated hemorrhagic colitis (AAHC), but the pathogenic role of the germ has not been established . The purpose of this study was to investigate the presence of K . oxytoca in patients with AAHC from a prospective cohort of patients with acute colitis, and to test the cytotoxicity on HEp-2 cells of K . oxytoca strains from patients with AAHC and healthy carriers . METHODS: Colonic biopsy specimens and a sample of colonic fluid from 93 consecutive patients with acute colitis were cultured on selective media for 7 established pathogens and K . oxytoca . The 2 K . oxytoca strains isolated in the 4 patients with C . difficile-negative AAHC of this cohort and 105 additional K . oxytoca strains from patients with C . difficile-negative AAHC (n = 15) and healthy carriers (n = 90) were tested for cytotoxicity using a HEp-2 cell culture assay . RESULTS: K . oxytoca was isolated in 50% (2 of 4) of the patients of the prospective cohort with C . difficile-negative AAHC compared with 2% (1 of 41) of the patients with acute colitis caused by established pathogens (P = 0.02) . The rate of cytotoxic strains of K . oxytoca was higher in patients with AAHC (82%) than in healthy carriers (42%, P = 0.003) . CONCLUSIONS: We conclude that K . oxytoca is isolated with a significant high rate in patients with C . difficile-negative AAHC, and that K . oxytoca strains from patients with AAHC are cytotoxic more frequently on HEp-2 cells than strains from healthy carriers . These results strengthen the hypothesis of a causative role of K . oxytoca in some of the patients with AAHC.

Arch Microbiol, 2004 Apr, 181(4), 324 - 30 Epub 2004 Mar 11.
Purification and characterization of an H2O-forming NADH oxidase from Clostridium aminovalericum: existence of an oxygen-detoxifying enzyme in an obligate anaerobic bacteria; Kawasaki S et al.; Clostridium aminovalericum, an obligate anaerobe, is unable to form colonies on PYD agar plates in the presence of 1% O(2) . When grown anaerobically in PYD liquid medium, the strain can continue normal growth after the shift from anoxic (sparged with O(2)-free N(2) carrier-gas) to microoxic (sparged with 3% O(2)/97% N(2) mixed carrier-gas) growth conditions in the mid exponential phase (OD(660)=1.0) . When the strain grew under 3% O(2)/97% N(2), the medium remains anoxic . Thirty minutes after beginning aeration with 3% O(2), the activity of NADH oxidase in cell-free extracts increased more than five-fold from the level before aeration . We purified NADH oxidase to determine the characteristics of this enzyme in an obligate anaerobe . The purified NADH oxidase dominated the NADH oxidase activity detected in cell-free extracts . The enzyme is a homotetramer composed of a subunit with a molecular mass of 45 kDa . The enzyme shows a spectrum typical of a flavoprotein, and flavin adenine dinucleotide (FAD) was identified as a cofactor . The final product of NADH oxidation was H(2)O, and the estimated K(m) for oxygen was 61.9 microM . These data demonstrate that an O(2)-response enzyme that is capable of detoxifying oxygen to water exists in C . aminovalericum.

FEBS Lett, 2004 Mar 12, 561(1-3), 155 - 8
Essential role of the family-22 carbohydrate-binding modules for beta-1,3-1,4-glucanase activity of Clostridium stercorarium Xyn10B; Araki R et al.; Clostridium stercorarium Xyn10B is a modular enzyme comprising two family-22 carbohydrate-binding modules (CBMs), a family-10 catalytic module of glycoside hydrolases, a family-9 CBM, and two S-layer homologous modules consecutively from the N-terminus . To investigate the role of the family-22 CBMs, truncated proteins were constructed: a recombinant catalytic module polypeptide (rCD), a CBM polypeptide composed of two family-22 CBMs (rCBM) and a polypeptide composed of the family-22 CBMs and the catalytic module (rCBM-CD) . We found that rCBM-CD was highly active toward beta-1,3-1,4-glucan; however, rCD was negligibly active toward the same substrate . The V(max)/K(m) value of rCBM-CD for beta-1,3-1,4-glucan was 7.8 times larger than that for oat-spelt xylan, indicating that rCBM-CD should be specified as a beta-1,3-1,4-glucanase rather than a xylanase despite the fact that family-10 catalytic modules are well-known xylanase modules . These results indicate that the family-22 CBMs in rCBM-CD are essential for hydrolysis of beta-1,3-1,4-glucan.

Urology, 2004 Mar, 63(3 Suppl 1), 65 - 73
Mechanisms involved in new therapies for overactive bladder; Cruz F; During the last few years, vanilloid substances and botulinum-A toxin were extensively investigated as new therapies for overactive bladder . Intravesical administration of capsaicin or resiniferatoxin--2 members of the vanilloid family--has been shown to increase bladder capacity and decrease urge incontinence in patients with neurogenic, as well as nonneurogenic, forms of detrusor overactivity . In addition, vanilloids have been shown also to reduce bladder pain in patients with hypersensitive disorders . Vanilloids are exogenous ligands of vanilloid receptor type 1 (VR1), an ion channel present in the membrane of type C primary afferent nerve fibers . This receptor, which plays a key role in pain perception and control of the micturition reflex, may be upregulated by nerve growth factor (NGF), a neurotrophic molecule detected in high concentrations in overactive detrusor tissue . Vanilloids, by reducing uptake of NGF through sensory neurons, may counteract VR1 upregulation . Intravesical injections of botulinum-A toxin, a neurotoxin produced by Clostridium botulinum, were shown to increase bladder capacity and to decrease urge incontinence episodes in patients with neurogenic detrusor overactivity . Botulinum-A toxin impedes the release of acetylcholine from cholinergic nerve endings at the neuromuscular junction, leading to paralysis of the detrusor smooth muscle.

Clin Lab Sci, 2004 Winter, 17(1), 30 - 4
Botulin toxin: a weapon in terrorism; Josko D; Clostridium botulinum, the causative agent of botulism is an anaerobic, spore forming gram-positive bacillus . C . botulinum causes three types of botulism; foodborne botulism, wound botulism, and infant botulism . Most strains of the bacterium produce a potent, muscle-paralyzing neurotoxin . Respiratory failure secondary to paralysis of the respiratory muscles can lead to death unless appropriate therapy is promptly initiated . Due to the severity and potency of this neurotoxin, its importance as a biological weapon is of major concern to public health officials.

Int J Mol Med, 2004 Apr, 13(4), 577 - 80
Clostridium butyricum, a probiotic derivative, suppresses dextran sulfate sodium-induced experimental colitis in rats; Araki Y et al.; Recent studies have suggested that probiotics or short chain fatty acids (SCFAs) exert a therapeutic effect on inflammatory bowel disease (IBD) patients . In a previous study, we demonstrated that Clostridium butyricum produces high levels of SCFAs in culture . In addition, a yogurt-based additive effectively masked, completely eliminating the unpleasant odor derived from the SCFAs . We recently reported that the oral administration of both high and low dose diets (50% w/w for 17 days and 5% w/w for 16 months, respectively) of the Clostridium butyricum derivative did not cause pathological abnormalities in rats . In the present study, we evaluated the effects of this product against dextran sulfate sodium (DSS)-induced experimental colitis in rats . Five-week-old male Wistar Hannover GALAS rats were given a mixture of a standard diet containing 3% (w/w) of DSS for 8 days . In the derivative-fed group, Clostridium butyricum derivative (20% w/w) with 0.1% (w/w) additive was also added to their diet . The control-fed group was given tap water (20% w/w) with 0.1% (w/w) additive . After 8 days, a laparotomy was performed, and macroscopic and microscopic inflammation scoring was determined . The Clostridium butyricum derivative effectively prevented bloody diarrhea . In addition, mucosal damage to the derivative-fed group was significantly reduced macroscopically compared to that of the control-fed group . The potential clinical efficacy of the Clostridium butyricum derivative in IBD patients is also discussed.

Mol Microbiol, 2004 Mar, 51(6), 1787 - 800
The large resolvase TnpX is the only transposon-encoded protein required for transposition of the Tn4451/3 family of integrative mobilizable elements; Lyras D et al.; Chloramphenicol resistance in Clostridium perfringens and Clostridium difficile is often encoded by catP genes located within the 6.3 kb integrative mobilizable elements Tn4451 and Tn4453 respectively . This family of transposons is capable of being mobilized into a recipient cell in the presence of another conjugative element . Transposition is mediated by the large resolvase TnpX, which excises the element to produce a circular molecule that is the integrative intermediate . In this study, in vivo deletion analysis of the transposon-encoded tnpV and tnpY genes showed that they are not essential for excision or integration of this group of elements . Similar studies on tnpW suggested either that this gene is not essential for these functions or that TnpW does not function when provided in trans . Development and use of an in vivo insertion assay showed that TnpX is the only transposon-encoded protein required for the integration reaction . Subsequently, a TnpXLEH6 protein was purified and shown to catalyse excision in vitro in the absence of any other protein and preferentially to excise a supercoiled DNA substrate . In summary, these studies have shown that TnpX is the only transposon protein required in vivo and in vitro for the excision process and that, like excision, integration also occurs by a serine recombinase-mediated site-specific recombination mechanism.

Int J Urol, 2004 Mar, 11(3), 133 - 41
Urinary tract and genito-urinary suppurative infections due to anaerobic bacteria; Brook I; Anaerobes have been involved in many different types of urinary tract infection . This review describes the microbiology, diagnosis and management of urinary tract and genito-urinary suppurative infections caused by anaerobic bacteria . The types of infections of the urinary tract in which anaerobes have been involved include para- or periurethral cellulitis or abscess, acute and chronic urethritis, cystitis, acute and chronic prostatitis, prostatic and scrotal abscesses, periprostatic phlegmon, ureteritis, periureteritis, pyelitis, pyelonephritis, renal abscess, scrotal gangrene, metastatic renal infection pyonephrosis, perinephric abscess, retroperitoneal abscess and other infections . The anaerobes recovered in these studies were Gram-negative bacilli (including Bacteroides fragilis and pigmented Prevotella and Porphyromonas sp.), Clostridium sp., anaerobic Gram-positive cocci and Actinomyces sp . In many cases, they were recovered mixed with coliforms or streptococci . The recovery of anaerobes requires the administration of antimicrobial therapy that is effective against these organisms . These antimicrobials include metronidazole, chloramphenicol, clindamycin, a carbapenem, cefoxitin and the combination of a penicillin and a beta-lactamase inhibitor . Percutaneous drainage, open surgical drainage or nephectomy might be indicated for abscesses.

Eur J Biochem, 2004 Mar, 271(5), 983 - 92
The structure-function relationship in the clostripain family of peptidases; Labrou NE et al.; In this study we investigate the active-site structure and the catalytic mechanism of clostripain by using a combination of three separate techniques: affinity labelling, site-directed mutagenesis and molecular modelling . A benzamidinyl-diazo dichlorotriazine dye (BDD) was shown to act as an efficient active site-directed affinity label for Clostridium histolyticum clostripain . The enzyme, upon incubation with BDD in 0.1 m Hepes/NaOH buffer pH 7.6, exhibits a time-dependent loss of activity . The rate of inactivation exhibits a nonlinear dependence on the BDD concentration, which can be described by reversible binding of dye to the enzyme prior to the irreversible reaction . The dissociation constant of the reversible formation of an enzyme-BDD complex is KD = 74.6 +/- 2.1 micro m and the maximal rate constant of inactivation is k3 = 0.21 x min(-1) . Effective protection against inactivation by BDD is provided by the substrate N-benzoyl-L-arginine ethyl ester (BAEE) . Cleavage of BDD-modified enzyme with trypsin and subsequent separation of peptides by reverse-phase HPLC gave only one modified peptide . Amino acid sequencing of the modified tryptic peptide revealed the target site of BDD reaction to be His176 . Site-directed mutagenesis was used to study further the functional role of His176 . The mutant His176Ala enzyme exhibited zero activity against BAEE . Together with previous data, these results confirm that a catalytic dyad of His176 and Cys231 is responsible for cysteine peptidase activity in the C11 peptidase family . A molecular model of the catalytic domain of clostripain was constructed using a manually extended fold recognition-derived alignment with caspases . A rigorous iterative modelling scheme resulted in an objectively sound model which points to Asp229 as responsible for defining the strong substrate specificity for Arg at the P1 position . Two possible binding sites for the calcium required for auto-activation could be located . Database searches show that clostripain homologues are not confined to bacterial lineages and reveal an intriguing variety of domain architectures.

Appl Environ Microbiol, 2004 Mar, 70(3), 1563 - 9
Kinetics and relative importance of phosphorolytic and hydrolytic cleavage of cellodextrins and cellobiose in cell extracts of Clostridium thermocellum; Zhang YH et al.; Rates of phosphorolytic cleavage of beta-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage . Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)- and cellobiose-grown cultures, with more limited data also obtained for cellotetraose . To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60 degrees C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35 degrees C . Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by > or = 20-fold for both cellobiose and cellopentaose over a 10-fold range of beta-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM) . Rates of phosphorolytic cleavage of beta-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures . Comparisons of V(max) values indicated that cellobiose- and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control . The apparent K(m) for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel- and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

J Clin Microbiol, 2004 Mar, 42(3), 1345 - 7
Highly conserved alpha-toxin sequences of avian isolates of Clostridium perfringens; Sheedy SA et al.; Clostridium perfringens causes necrotic enteritis in chickens, and alpha-toxin has been suggested to be a key virulence determinant . Analysis of the alpha-toxin of 25 chicken-derived C . perfringens strains demonstrated high homology to mammal-derived strains rather than to the only avian-derived C . perfringens alpha-toxin sequence reported previously.

J Clin Microbiol, 2004 Mar, 42(3), 1035 - 41
Characterization of toxin A-negative, toxin B-positive Clostridium difficile isolates from outbreaks in different countries by amplified fragment length polymorphism and PCR ribotyping; van den Berg RJ et al.; Clinical Clostridium difficile isolates of patients with diarrhea or pseudomembranous colitis usually produce both toxin A and toxin B, but an increasing number of reports mention infections due to toxin A-negative, toxin B-positive (A(-)/B(+)) strains . Thirty-nine clinical toxin A(-)/B(+) isolates, and 12 other unrelated isolates were obtained from Canada, the United States, Poland, the United Kingdom, France, Japan, and The Netherlands . The isolates were investigated by high-resolution genetic fingerprinting by use of amplified fragment length polymorphism (AFLP) and two well-described PCR ribotyping methods . Furthermore, the toxin profile and clindamycin resistance were determined . Reference strains of C . difficile representing 30 known serogroups were also included in the analysis . AFLP discriminated 29 types among the reference strains, whereas the two PCR ribotyping methods distinguished 25 and 26 types . The discriminatory power of AFLP and PCR ribotyping among 12 different unrelated isolates was similar . Typing of 39 toxin A(-)/B(+) isolates revealed 2 AFLP types and 2 and 3 PCR ribotypes . Of 39 toxin A(-)/B(+) isolates, 37 had PCR ribotype 017/20 and AFLP type 20 (95%) . A deletion of 1.8 kb was seen in 38 isolates, and 1 isolate had a deletion of approximately 1.7 kb in the tcdA gene, which encodes toxin A . Clindamycin resistance encoded by the erm(B) gene was found in 33 of 39 toxin A(-)/B(+) isolates, and in 2 of the 12 unrelated isolates (P < 0.001, chi-square test) . We conclude that clindamycin-resistant C . difficile toxin A(-)/B(+) strain (PCR ribotype 017/20, AFLP type 20, serogroup F) has a clonal worldwide spread.

Vaccine, 2004 Mar 12, 22(9-10), 1177 - 87
Display of heterologous antigens on the Bacillus subtilis spore coat using CotC as a fusion partner; Mauriello EM et al.; We report the use of CotC, a major component of the Bacillus subtilis spore coat, as a fusion partner for the expression of two heterologous antigens on the spore coat . Recombinant spores expressing tetanus toxin fragment C (TTFC) of Clostridium tetani or the B subunit of the heat-labile toxin of Escherichia coli (LTB) were used for oral dosing and shown to generate specific systemic and mucosal immune responses in a murine model . This report, expanding the previously described expression of TTFC on the spore surface by fusion to CotB {J Bacteriol 183 (2001) 6294} and its use for oral vaccination {Infect Immun 71 (2003) 2810} shows that different antigens can be successfully presented on the spore coat and supports the use of the spore as an efficient vehicle for mucosal immunisation.

Surg Today, 2004, 34(3), 261 - 4
Primary abscess of the omentum: report of a case; Otagiri N et al.; We report a case of a primary abscess of the omentum without any obvious etiology . A 62-year-old man was referred to our clinic with lower abdominal pain, and computed tomography showed an intra-abdominal abscess in the left pelvic area . Laparotomy revealed that the abscess adhered to the urinary bladder and abdominal wall, but no perforation of the alimentary tract was identified and there was no foreign body in the abscess cavity . A culture of the abscess fluid grew Clostridium perfringens . The patient was discharged on the 16th hospital day after an uneventful postoperative course without any complications.

Int J Food Microbiol, 2004 Mar 1, 91(2), 141 - 5
Prevalence of Clostridium botulinum in food raw materials used in REPFEDs manufactured in France; Carlin F et al.; Food raw materials used in refrigerated processed foods of extended durability (REPFEDs) manufactured in France were surveyed for Clostridium botulinum types A, B and E using PCR-Enzyme-linked Immunosorbent assay (PCR-ELISA) and mouse bioassay for detection respectively of cells and toxins in enrichment broth . Portions of 25 to 50 g of food were analysed . A total of 8 out of the 102 samples of fish and shellfish, 12 out of the 143 samples of meat and poultry, 1 out of the 62 samples of aroma, sauce and gravy, 4 out of the 25 samples of thickening agents, 3 out of the 26 samples of dehydrated dairy ingredients, and none of the 65 samples of spices, herbs and dehydrated mushroom were positive for C . botulinum in PCR-ELISA, i.e., 6.6% of all the samples tested . The 28 positive samples comprised 10 type A, 10 type B, 4 with both types A and B, and 4 undetermined by PCR typing . No sample positive for type E was detected . Of the 28 samples positive in PCR-ELISA, 15 were also positive in the mouse bioassay . The MPN count was between 1 and 3 C . botulinum/kg of food, which is similar to or in the lower range of values reported in the literature.

Acta Crystallogr D Biol Crystallogr, 2004 Mar, 60(Pt 3), 537 - 8 Epub 2004 Feb 25.
Crystallization and preliminary X-ray analysis of GlcNAc alpha 1,4Gal-releasing endo-beta-galactosidase from Clostridium perfringens; Deng L et al.; The unique clostridial endo-beta-galactosidase (Endo-beta-Gal(GnGa)) capable of releasing the disaccharide GlcNAc alpha 1,4Gal from O-glycans expressed in the gastric gland mucous cell-type mucin has been crystallized . The crystal belongs to space group P6(3), with unit-cell parameters a = 160.4, c = 86.1 A . Under cryocooled conditions and using a synchrotron X-ray source, the crystals diffract to 1.82 A resolution . The asymmetric unit contains two or three molecules.

World J Gastroenterol, 2004 Mar 1, 10(5), 765 - 6
Successful treatment with rifampin for fulminant antibiotics-associated colitis in a patient with non-Hodgkin's lymphoma; Nomura K et al.; A 74-year-old man was admitted to the hospital because of chemotherapy for relapsed non-Hodgkin's lymphoma (NHL) . The patient became febrile and experienced diarrhea after chemotherapy . Although ceftazidime and amikacin sulfate were administered as empiric therapy, diarrhea was continued . After several days, stool cytotoxin assay for clostridium difficile (C . difficile) was positive and he was diagnosed as having antibiotics-associated colitis (AAC) . Although antibiotics were discontinued and both oral vancomycin and metronidazole were administrated, disease was not improved . To rule out the presence of an additional cause of diarrhea, colon fibroscopic examination was performed . It revealed multiple deep ulcerative lesions at right side colon, surface erosive and minute erosive lesions in all continuous colon . Pseudomembranes were not seen . These findings are compatible with AAC without pseudomembranes . There are no reports that the rifampin is effective on refractory AAC . However, we administered oral rifampin for the current patient . The reasons are 1) conventional antibiotics were not effective, 2) rifampin has excellent in vitro activity against C . difficile, and 3) the efficacy of rifampin on relapsing colitis due to C . difficile is established . After administration of rifampin, fever alleviated and diarrhea was improved . Because AAC may result in significant mortality, patients with refractory or fulminant AAC should be treated with oral rifampin from outset.

J Clin Oncol, 2004 Mar 1, 22(5), 829 - 37
Phase I and pharmacokinetic study of topotecan administered orally once daily for 5 days for 2 consecutive weeks to pediatric patients with refractory solid tumors; Daw NC et al.; PURPOSE: We conducted a phase I trial of the injectable formulation of topotecan given orally once daily for 5 days for 2 consecutive weeks (qd x 5 x 2) in pediatric patients with refractory solid tumors . PATIENTS AND METHODS: Cohorts of two to six patients received oral topotecan at 0.8, 1.1, 1.4, 1.8, and 2.3 mg/m(2)/d every 28 days for a maximum of six courses . Twenty patients (median age, 10.6 years) received a total of 51 courses . Eight patients received topotecan capsules during course 2 only . RESULTS: Dose-limiting toxicity occurred at 2.3 mg/m(2)/d and consisted of prolonged grade 4 neutropenia (n = 2), grade 3 stomatitis as a result of radiation recall (n = 1), grade 3 hemorrhage (epistaxis) in the presence of grade 4 thrombocytopenia (n = 1), and grade 3 diarrhea in the presence of Clostridium difficile infection (n = 1) . Dose-limiting, prolonged grade 4 neutropenia and thrombocytopenia occurred in one patient at 1.4 mg/m(2)/d . Infrequent toxicities were mild nausea, vomiting, elevated liver ALT or AST, and rash . The maximum-tolerated dosage was 1.8 mg/m(2)/d; the mean (+/- standard deviation) area under the plasma concentration-time curve for topotecan lactone at this dosage was 20.9 +/- 8.4 ng/mL . h . The population mean (+/- standard error) oral bioavailability of the injectable formulation was 0.27 +/- 0.03; that of capsules was 0.36 +/- 0.06 (P =.16) . Disease stabilized in nine of 19 assessable patients for 1.5 to 6 months . CONCLUSION: Oral topotecan (1.8 mg/m(2)/d) on a qd x 5 x 2 schedule is well tolerated and warrants additional testing in pediatric patients.

Carcinogenesis, 2004 Aug, 25(8), 1335 - 44 Epub 2004 Feb 26.
Ionizing radiation-induced E-selectin gene expression and tumor cell adhesion is inhibited by lovastatin and all-trans retinoic acid; Nubel T et al.; E-selectin mediated tumor cell adhesion plays an important role in metastasis . Here we show that ionizing radiation (IR) induces E-selectin gene and protein expression in human endothelial cells at therapeutically relevant dose level . E-selectin expression is accompanied by an increase in the adhesion of human colon carcinoma cells to primary human umbilical vein endothelial cells (HUVEC) . The HMG-CoA reductase inhibitor lovastatin impairs IR-stimulated E-selectin expression as analyzed at the level of the protein, mRNA and promoter . Inactivation of Rho GTPases either by use of Clostridium difficile toxin A or by co-expression of dominant-negative Rho blocked IR-induced E-selectin gene induction, indicating Rho GTPases to be essential . Radiation-induced expression of E-selectin was also blocked by all-trans retinoic acid (at-RA), whereas 9-cis retinoic acid was ineffective . Abrogation of IR-stimulated E-selectin expression by lovastatin and at-RA reduced tumor cell adhesion in a dose-dependent manner . Combined treatment with lovastatin and at-RA exerted additive inhibitory effects on radiation-induced E-selectin expression and tumor cell adhesion . Therefore, application of statins and at-RA might have clinical impact in protecting against E-selectin-promoted metastasis, which might arise as an unwanted side effect from radiation treatment.

FEMS Microbiol Lett, 2004 Feb 16, 231(2), 159 - 64
Characterization of six type A strains of Clostridium botulinum that contain type B toxin gene sequences; Kirma N et al.; Six Clostridium botulinum isolates exhibiting type A toxicity as measured by the mouse bioassay were found to contain both type A and type B neurotoxin DNA sequences . The six strains were divided into three groups based on the DNA sequence of the type B neurotoxin gene . Members of each group exhibited 100% sequence identity over the 3876 bp type B toxin open reading frame . The type B toxin sequence of all groups differed at more than 60 positions when compared to the BGB control strain.

Transpl Infect Dis, 2003 Dec, 5(4), 199 - 202
Clostridium innocuum bacteremia secondary to infected hematoma with gas formation in a kidney transplant recipient; Castiglioni B et al.; Clostridium innocuum is a relatively antimicrobial resistant, frequently misidentified anaerobe that has only rarely been associated with bacteremia . A 38-year-old female with chronic hepatitis C underwent a second kidney transplant operation . Two weeks after surgery a computed tomography scan of the abdomen showed a heterogeneous hematoma with pockets of gas adjacent to the allograft, which extended into the pelvis and left abdominal wall, associated with low-grade fever . An anaerobic blood culture grew a Clostridium initially identified as C . subterminale and later re-identified as C . innocuum . At abdominal exploration liquefied blood was evacuated, and the patient completed a course of antibiotics and recovered . C . innocuum should be considered as a cause of gas-producing anaerobic infection in transplant patients . Because C . innocuum is frequently misidentified by the use of commercial anaerobic identification kits, its true incidence in serious infections is likely underestimated.

Clin Infect Dis, 2004 Mar 1, 38(5), 640 - 5 Epub 2004 Feb 11.
Outbreak of Clostridium difficile infection in a long-term care facility: association with gatifloxacin use; Gaynes R et al.; To determine the cause of an increase in the rate of Clostridium difficile-associated diarrhea (CDAD) in a long-term care facility (LTCF), we analyzed CDAD cases among LTCF patients from October 2001 through June 2002 . CDAD cases were identified from review of all enzyme immunoassays positive for C . difficile toxin A . The increase coincided with a formulary change from levofloxacin to gatifloxacin . We performed a case-control study in which we randomly selected control subjects from 612 LTCF admissions during this period . Although we examined a variety of risk factors, logistic regression analysis only demonstrated associations between CDAD and use of clindamycin (P=.005) and gatifloxacin, the latter being associated with an increasing risk of CDAD with increasing duration of gatifloxacin therapy (P<.0001) . We concluded that an outbreak of CDAD in an LTCF was associated with a formulary change from levofloxacin to gatifloxacin . The rate of CDAD in the LTCF decreased after a change back to levofloxacin.

Int J Food Microbiol, 2004 Mar 15, 91(3), 289 - 96
Identification and characterization of Clostridium perfringens using single target DNA microarray chip; Al-Khaldi SF et al.; A DNA microarray method was developed to identify the presence of toxin genes: encoding beta toxin (cpb), epsilon toxin (etx), enterotoxin (cpe), alpha toxin (cpa), and iota toxin (iA) in Clostridium perfringens . To build the DNA chip, each gene sequence was represented by one approximately 22-bp amino-modified oligonucleotide printed twice on aldehyde-coated slides . Multiplex PCR with Cy3 and Cy5-dCTP derivatized fluorescent nucleotides was used to label five genes and fluorescent probes were prepared . The PCR probes were denatured and single-strand-labeled DNAs were separated and purified using magnetic beads . The presence of toxin genes in C . perfringens was detected by hybridization of amplified ssDNA probes to oligonucleotides on the chip representing one target sequence of each toxin gene . The DNA chip was able to identify eight strains of C . perfringens.

South Med J, 2004 Feb, 97(2), 172 - 4
Isolated gastrointestinal histoplasmosis: case report and review of the literature; Jain S et al.; The usual manifestation of histoplasmosis is in the form of respiratory illness . We report the case of a 67-year-old man who presented with chronic diarrhea and did not respond to the conventional treatment, including that for Clostridium difficile . He was found to have isolated colonic histoplasmosis infection, which was treated with itraconazole . There was no evidence of any disseminated disease . His only immunocompromised state was end-stage renal disease, for which he was on chronic hemodialysis . Although it is well documented as a part of disseminated histoplasmosis, our extensive review of the literature did not reveal any reported case of isolated colonic histoplasmosis in a patient on hemodialysis.

Biochemistry, 2004 Mar 2, 43(8), 2209 - 16
Role of metals in the biological activity of Clostridium botulinum neurotoxins; Eswaramoorthy S et al.; Clostridium botulinum neurotoxins are the most potent toxins to humans and cause paralysis by blocking neurotransmitter release at the presynaptic nerve terminals . The toxicity involves four steps, viz., binding to neuronal cells, internalization, translocation, and catalytic activity . While the catalytic activity is a zinc endopeptidase activity on the SNARE complex proteins, the translocation is believed to be a pH-dependent process allowing the translocation domain to change its conformation to penetrate the endosomal membrane . Here, we report the crystal structures of botulinum neurotoxin type B at various pHs and of an apo form of the neurotoxin, and discuss the role of metal ions and the effect of pH variation in the biological activity . Except for the perturbation of a few side chains, the conformation of the catalytic domain is unchanged in the zinc-depleted apotoxin, suggesting that zinc's role is catalytic . We have also identified two calcium ions in the molecule and present biochemical evidence to show that they play a role in the translocation of the light chain through the membrane.

Water Sci Technol, 2004, 49(1), 95 - 102
Anaerobic degradation of phenol in wastewater at ambient temperature; Fang HH et al.; Treating a synthetic wastewater containing phenol as the sole substrate at 26 degrees C, an upflow anaerobic sludge blanket reactor was able to remove over 98% of phenol up to 1,260 mg/l in wastewater with 12 h of hydraulic retention time, corresponding to 6.0 g-COD/(l x day) . Results showed that benzoate was the key intermediate of phenol degradation . Conversion of benzoate to methane was suppressed by the presence of phenol . Based on DNA cloning analysis, the sludge was composed of five groups of microorganisms . Desulfotomaculum and Clostridium were likely responsible for the conversion of phenol to benzoate, which was further degraded by Syntrophus to acetate and H2/CO2 . Methanogens lastly converted acetate and H2/CO2 to methane . The role of epsilon-Proteobacteria was, however, unclear.

J Immunol, 2004 Mar 1, 172(5), 3018 - 25
Essential involvement of IFN-gamma in Clostridium difficile toxin A-induced enteritis; Ishida Y et al.; Clostridium difficile has emerged as the important causative agent of antibiotics-associated pseudomembranous colitis; especially its toxin A is presumed to be responsible for the colitis . We examined the pathophysiological roles of IFN-gamma in toxin A-induced enteritis using IFN-gamma knockout (KO) mice . When toxin A of C . difficile was injected into the ileal loops of BALB/c wild-type (WT) mice, massive fluid secretion, disruption of intestinal epithelial structure, and massive neutrophil infiltration developed within 4 h after the injection . IFN-gamma protein was faintly detected in some CD3-positive lymphocytes in the lamina propria and submucosa of the ileum of untreated WT mice . On the contrary, at 2 and 4 h after toxin A injection, IFN-gamma protein was detected in infiltrating neutrophils and to a lesser degree in CD3-positive lymphocytes . In the ileum of WT mice, toxin A treatment markedly enhanced the gene expression of TNF-alpha, macrophage inflammatory protein-1alpha and -2, KC, and ICAM-1 >2 h after treatment . In contrast, the histopathological changes were marginal, without enhanced fluid secretion in the ileum of toxin A-treated IFN-gamma KO mice . Moreover, toxin A-induced gene expression of TNF-alpha, neutrophil chemotactic chemokines, and ICMA-1 was remarkably attenuated in IFN-gamma KO mice . Furthermore, pretreatment of WT mice with a neutralizing anti-IFN-gamma Ab prevented toxin A-induced enteritis . These observations indicate that IFN-gamma is the crucial mediator of toxin A-induced acute enteritis and suggest that IFN-gamma is an important molecular target for the control of C . difficile-associated pseudomembranous colitis.

J Med Microbiol, 2004 Mar, 53(Pt 3), 197 - 205
Immunological detection and cytotoxic properties of toxins from toxin A-positive, toxin B-positive Clostridium difficile variants; Blake JE et al.; Clostridium difficile is a major nosocomial pathogen and a causative agent of antibiotic-associated diarrhoea and pseudomembranous colitis . PCR analysis of the toxin A and B genes of this bacterium has revealed 20 variant types (toxinotypes I-XX), many of which can cause human disease . Strains comprising the 15 toxin A-positive, toxin B-positive toxinotypes are not usually differentiated from non-variant strains by routine laboratories that do not utilize PCR tests . Consequently, the toxins from these variant strains have not been investigated thoroughly . The present studies revealed that toxin A-positive (A+B+) strains representing 12 variant toxinotypes all express considerably lower levels of toxin A and are less cytotoxic in vitro than non-variant strain VPI 10463 . Truncated forms of toxin A were detected by immunoblotting in toxinotype VI and VII strains and these toxins were differentiated from each other and from toxin A of the non-variant strain . A further novel finding was the ability of toxin A-positive (A+B+) strains of toxinotypes IX, XIV and XV to exhibit an alternative Clostridium sordellii-like cytopathic effect on Vero cells, characterized by marked cell clumping . A rapid and simple method for toxin A removal from culture filtrates was developed . This enabled confirmation that the abnormal cytotoxicity observed for these strains is due to an altered toxin B, as has been found in toxin A-negative (A-B+) strains . These findings indicate the potential for differentiation of certain toxin A-positive (A+B+) toxinotypes without the need for PCR techniques.

Hepatobiliary Pancreat Dis Int, 2004 Feb, 3(1), 133 - 5
Expression of the bacterial gene in gallbladder carcinoma tissue and bile; Lu Y et al.; BACKGROUND: The major causive factors of gallbladder carcinoma are very complex . Cholecystitis with gallstone was reported one of the most important factors . Many research revealed that cholecystitis or gallstone can give rise to epithelial hyperplasia of gallbladder mucusa or canceration secondarily . In this study, 46 patients were detected in order to find the relationship between infection of different bacteria and formation of gallbladder carcinoma . METHODS: Using the common gene primer of bacteria 16S ribosomal RNA (rRNA), we detected bacterial gene fragments of gallbladder carcinoma tissues in 46 patients by polymerase chain reaction (PCR) . Relative bile was also detected by PCR in 18 patients who underwent operations, including U-tube drainage (1), right or left biliary tube drainage (4), radical cholecystectomy (9), and cholecystorrhaphy (4) . The tissue fragments of gallbladder carcinoma from the remaining 28 patients were paraffin slices . RESULTS: The positive rate of bacterial DNA in gallbladder carcinoma tissue was 78.3% (36/46) . The sequence of 16S ribosomal RNA gene fragments amplified by PCR was approximately 371 base pairs (bp) . Multiple kinds of standard bacterial gene fragments obtained from 36 patients included Colibacillus, B.fragilis, Klebsiella, C.perfringens and Clostridium, with a positive rate of 78.3% (36/46) . Among the 36 patients, 14 patients with gallbladder carcinoma received operation and their relative bile at operation was detected bacterial gene fragments with a positive rate of 77.8% (14/18) . This result was close to that in gallbladder carcinoma tissues . CONCLUSIONS: Our results suggested that there might be a relationship between occurrence of gallbladder carcinoma and infection of different kinds of bacteria, especially anaerobic bacteria C.perfringens . This reminds us that the gallbladder mucosa stimulated by anaerobic and aerobic bacteria might be the principal cause for the development of carcinoma.

J Food Prot, 2004 Feb, 67(2), 352 - 6
Effect of cooling on Clostridium perfringens in pea soup; de Jong AE et al.; Foods associated with Clostridium perfringens outbreaks are usually abused after cooking . Because of their short generation times, C . perfringens spores and cells can grow out to high levels during improper cooling . Therefore, the potential of C . perfringens to multiply in Dutch pea soup during different cooling times was investigated . Tubes of preheated pea soup (50 degrees C) were inoculated with cocktails of cells or heat-activated spores of this pathogen . The tubes were linearly cooled to 15 degrees C in time spans of 3, 5, 7.5, and 10 h and were subsequently stored in a refrigerator at 3 or 7 degrees C for up to 84 h . Cell numbers increased by 1-log cycle during the 3-h cooling period and reached their maximum after 10 h of cooling . Subsequent refrigeration hardly reduced cell numbers . Cooling of 3.75 liters of pea soup in an open pan showed that this amount of pea soup cooled from 50 to 15 degrees C in 5 h, which will allow a more than 10-fold increase in cell numbers . These findings emphasize the need of good hygienic practices and quick cooling of heated foods after preparation.

J Food Prot, 2004 Feb, 67(2), 342 - 6
Increased inactivation of ozone-treated Clostridium perfringens vegetative cells and spores on fabricated beef surfaces using mild heat; Novak JS et al.; Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75 degrees C against enterotoxin-producing strains of Clostridium perfringens . Vegetative cells on beef surfaces at an initial concentration of 5.59 +/- 0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09 +/- 0.72 log CFU/g and 3.50 +/- 0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55 degrees C, respectively . Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94 +/- 0.37 log spores per g to 2.07 +/- 0.38 log spores per g and 1.70 +/- 0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75 degrees C, respectively . Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury . This study provides evidence for the decreased resistance of both vegetative cells and spores of C . perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.

Protein Expr Purif, 2003 Dec, 32(2), 309 - 16
Purification and characterization of a recombinant alpha-N-acetylgalactosaminidase from Clostridium perfringens; Hsieh HY et al.; Clostridium perfringens alpha-N-acetylgalactosaminidase (alphaNAG) hydrolyzed the terminal N-acetyl-alpha-d-galactosamine from the blood type A(2) antigen producing H antigen, blood type O . Blood type O is universally compatible in the ABO system . Purification of the native enzyme is difficult with very low yields . To obtain the enzyme in satisfactory yield, the gene encoding the clostridial enzyme was cloned in an Escherichia coli T7 expression system . A highly purified preparation of recombinant alphaNAG was obtained from cell lysates by ion-exchange chromatography and high-pressure liquid chromatography . The final preparation was homogeneous by SDS-PAGE with a molecular mass of 71.96kDa and the native molecular weight of 72.42kDa . The enzyme was highly selective for terminal N-acetylgalactosamine residues . No other significant exoglycosidase activities, particularly neuraminidase, were detected . The pH optimum of the enzyme was between 6.5 and 7.0 and activity was relatively unaffected by ionic strength . ELISA experiments demonstrated activity against blood type A(2) epitope . These characteristics were similar to those of native alphaNAG from C . perfringens . With adequate expression in E . coli, sufficient recombinant alphaNAG enzyme mass can be obtained for potential use in enzymatic conversion of human blood type A(2) red blood cells to universally transfusable type O red blood cells.

Lett Appl Microbiol, 2004, 38(3), 197 - 205
In vivo characterization of Lactobacillus johnsonii FI9785 for use as a defined competitive exclusion agent against bacterial pathogens in poultry; La Ragione RM et al.; AIMS: To test the efficacy of Lactobacillus johnsonii FI9785 in reducing the colonization and shedding of Salmonella enterica serotype Enteritidis, Escherichia coli O78:K80 and Clostridium perfringens in poultry . METHODS AND RESULTS: Specific pathogen-free chicks (1 day old) were dosed with a single oral inoculum of 1x10(9) CFU . Lactobacillus johnsonii FI9785 and 24 h later were challenged in separate experiments with S . Enteritidis (S1400, nalr) and E . coli O78:K80 (EC34195, nalr) . There were no significant effects against S . Enteritidis whereas colonization of the small intestine by E . coli O78:K80 was reduced significantly . Both S . Enteritidis and E . coli colonized the caeca and colon to levels equivalent to control birds and there was no reduction in shedding as assessed by a semi-quantitative cloacal swabbing technique . Specific pathogen-free chicks (20 day old) were dosed with a single oral inoculum of 1x10(9) CFU L . johnsonii FI9785 and 24 h later were challenged with C . perfringens . A single oral dose of L . johnsonii FI9785 was sufficient to suppress all aspects of colonization and persistence of C . perfringens . CONCLUSIONS: Lactobacillus johnsonii FI9785 may be given to poultry for use as a competitive exclusion agent to control C . perfringens . SIGNIFICANCE AND IMPACT OF THE STUDY: Lactobacillus johnsonii FI9785 may be a valuable tool to control the endemic disease of necrotic enteritis, thereby reducing economic losses associated with reduced use of antimicrobials in the poultry industry.

Pharmeuropa Bio, 2004 Jan, 2003(2), 91 - 6
Control of Clostridium perfringens vaccines using an indirect competitive ELISA for the epsilon toxin component - examination of the assay by a collaborative study; Rosskopf-Streicher U et al.; Investigations on the replacement of the mouse neutralisation test for proving vaccine batches of Clostridium (C.) perfringens toxoid vaccines were performed since several years . The European Pharmacopoeia (Ph . Eur.) monograph Clostridium perfringens vaccines for veterinary use (0363) is prescribing a potency test by immunisation of rabbits and checking the induction of specific antibodies against the toxins in a mouse neutralisation test . Since the monograph was revised, immunochemical methods are favoured to detect directly specific antibodies in the rabbit sera . An indirect competitive ELISA using a monoclonal antibody was established at the Paul-Ehrlich-Institut for the detection of antibodies against the epsilon toxin component of C . perfringens . It was revised using the Clostridia rabbit antiserum Ph . Eur . Biological Reference Preparation (BRP) Batch 1 as reference serum . With a defined content of 11 International Units (IU) of C . perfringens epsilon antitoxin this reference serum enables the calculation of the potency of rabbit sera under test . For the collaborative study vaccine products of different composition licensed for the German and European markets were used . Seven international laboratories were included . Aim was to make a prediction on the transferability and precision of the test method . The results showing a satisfactory intermediate precision and transferability of the test confirmed the applicability of the ELISA method for the batch control of C . perfringens vaccines . Therefore a replacement of the mouse neutralisation test is available.

Childs Nerv Syst . 2004 Feb 10; {Epub ahead of print}
Clostridium infection resulting in paralysis in a child; Tubbs RS et al.; CASE REPORT . We report an 11-year-old boy who fell from an All Terrain Vehicle and sustained multiple minor soft tissue contusions and a small midthoracic laceration . Irrigation and closure of the small wound was performed at another hospital . There was no history of a penetrating wound . Within 48 h of injury, the patient developed profound dysesthesia and paralysis of the lower extremities and was transferred to our hospital . MRI disclosed a paraspinal abnormality without bony involvement . At exploration a portion of a tree branch was removed . Wound cultures were positive for Clostridium botulinum, tetani, and perfringens . CONCLUSIONS . To our knowledge, this is the first case of direct Clostridium intoxication of the spinal cord in man . Moreover, this report demonstrates the invasive manner in which Clostridium toxins may breach both the intact ligamentum flavum and the dura mater to deliver their toxicity to the intradural contents . Although the patient's dysesthesia resolved and paraplegia improved to ambulation he is still left with a significant motor deficit.

J Biol Inorg Chem, 2004 Apr, 9(3), 297 - 306 Epub 2004 Feb 10.
Contribution of the {FeII(SCys)4} site to the thermostability of rubredoxins; Bonomi F et al.; The thermostabilities of Fe(2+) ligation in rubredoxins (Rds) from the hyperthermophile Pyrococcus furiosus (Pf) and the mesophiles Clostridium pasteurianum (Cp) and Desulfovibrio vulgaris (Dv) were compared . Residue 44 forms an NH.S(Cys) hydrogen bond to one of the cysteine ligands to the {Fe(SCys)(4)} site, and substitutions at this location affect the redox properties of the {Fe(SCys)(4)} site . Both Pf Rd and Dv Rd have an alanine residue at position 44, whereas Cp Fd has a valine residue . Wild-type proteins were examined along with V44A and A44V "exchange" mutants of Cp and Pf Rds, respectively, in order to assess the effects of the residue at position 44 on the stability of the {Fe(SCys)(4)} site . Stability of iron ligation was measured by temperature-ramp and fixed-temperature time course experiments, monitoring iron release in both the absence and presence of more thiophilic metals (Zn(2+), Cd(2+)) and over a range of pH values . The thermostability of the polypeptide fold was concomitantly measured by fluorescence, circular dichroism, and (1)H NMR spectroscopies . The A44V mutation strongly lowered the stability of the {Fe(II)(SCys)(4)} site in Pf Rd, whereas the converse V44A mutation of Cp Rd significantly raised the stability of the {Fe(II)(SCys)(4)} site, but not to the levels measured for wild-type Dv Rd . The region around residue 44 is thus a significant contributor to stability of iron coordination in reduced Rds . This region, however, made only a minor contribution to the thermostability of the protein folding, which was found to be higher for hyperthermophilic versus mesophilic Rds, and largely independent of the residue at position 44 . These results, together with our previous studies, show that localized charge density, solvent accessibility, and iron site/backbone interactions control the thermostability of the {Fe(SCys)(4)} site . The iron site thermostability does make a minor contribution to the overall Rd thermostability . From a mechanistic standpoint, we also found that attack of displacing ions (H(+), Cd(2+)) on the Cys42 sulfur ligand at the {Fe(SCys)(4)} site occurs through the V8 side and not the V44 side of the iron site.

Am J Med Sci, 2004 Feb, 327(2), 91 - 3
Coinfection with Giardia lamblia and Clostridium difficile after use of ranitidine; Khatami SS et al.; A 49-year-old man presented with a 3-week history of vomiting and diarrhea . He reported foamy stools but no blood or melena and had crampy epigastric pain . He denied usage of antibiotics . He had been taking ranitidine for intermittent epigastric pain for the last few months and noted an 11-pound weight loss during the 3 weeks before admission . Stool was positive for Clostridium difficile toxin and Giardia lamblia antigen . Cultures and occult blood tests were negative . Oral metronidazole, 500 mg 3 times a day, was administered, and the patient was hydrated . The diarrhea resolved, and patient was discharged on the fourth hospital day . Prior antibiotic therapy is the most common risk factor for C difficile colitis . This patient developed concomitant infection with C difficile and G lamblia while he used ranitidine . He had no other risk factors for these infections . Hence, we propose that ranitidine-induced hypochlorhydria predisposed this patient to the enteric infections.

Joint Bone Spine, 2004 Jan, 71(1), 60 - 2
Clostridium difficile-associated reactive arthritis in two children; Loffler HA et al.; In adults, reactive arthritis (ReA) following Clostridium difficile-enterocolitis has been documented . In children, only one case of C . difficile-associated ReA has been reported . We now describe two other cases of ReA associated with C . difficile in children . The characteristics of ReA due to C . difficile appear to be similar in adults and children . Both children show polyarthritis after an episode of diarrhoea with positive stool cultures for C . difficile . Arthritis is asymmetrical with a self-limiting course . Nonsteroidal antiinflammatory drug (NSAID) therapy is sufficient . One case is remarkable because of its prolonged course of ReA despite NSAID therapy, and its association with the presence of HLA-B27 antigen.

Mikrobiologiia, 2003 Nov-Dec, 72(6), 752 - 8
{Analysis of the anaerobic microbial community capable of degrading p-toluene sulphonate}; Shcherbakova VA et al.; Three strains of Clostridium sp., 14 (VKM B-2201), 42 (VKM B-2202), and 21 (VKM B-2279), two methanogens, Methanobacterium formicicum MH (VKM B-2198) and Methanosarcina mazei MM (VKM B-2199), and one sulfate-reducing bacterium, Desulfovibrio sp . SR1 (VKM B-2200), were isolated in pure cultures from an anaerobic microbial community capable of degrading p-toluene sulfonate . Strain 14 was able to degrade p-toluene sulfonate in the presence of yeast extract and bactotryptone and, like strain 42, to utilize p-toluene sulfonate as the sole sulfur source with the production of toluene . p-Toluene sulfonate stimulated the growth of Ms . mazei MM on acetate . The sulfate-reducing strain Desulfovibrio sp . SR1 utilized p-toluene sulfonate as an electron acceptor . The putative scheme of p-toluene sulfonate degradation by the anaerobic microbial community is discussed.

Appl Environ Microbiol, 2004 Feb, 70(2), 1081 - 7
Development and validation of experimental protocols for use of cardinal models for prediction of microorganism growth in food products; Pinon A et al.; An experimental protocol to validate secondary-model application to foods was suggested . Escherichia coli, Listeria monocytogenes, Bacillus cereus, Clostridium perfringens, and Salmonella were observed in various food categories, such as meat, dairy, egg, or seafood products . The secondary model validated in this study was based on the gamma concept, in which the environmental factors temperature, pH, and water activity (aw) were introduced as individual terms with microbe-dependent parameters, and the effect of foodstuffs on the growth rates of these species was described with a food- and microbe-dependent parameter . This food-oriented approach was carried out by challenge testing, generally at 15 and 10 degrees C for L . monocytogenes, E . coli, B . cereus, and Salmonella and at 25 and 20 degrees C for C . perfringens . About 222 kinetics in foods were generated . The results were compared to simulations generated by existing software, such as PMP . The bias factor was also calculated . The methodology to obtain a food-dependent parameter (fitting step) and therefore to compare results given by models with new independent data (validation step) is discussed in regard to its food safety application . The proposed methods were used within the French national program of predictive microbiology, Sym'Previus, to include challenge test results in the database and to obtain predictive models designed for microbial growth in food products.

Appl Environ Microbiol, 2004 Feb, 70(2), 883 - 90
Electrotransformation of Clostridium thermocellum; Tyurin MV et al.; Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin . A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube . Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C . thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli . Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA . Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium . The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072 . Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse . Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency . The effect of isoniacin was also strain specific . The results reported here provide for the first time a gene transfer method functional in C . thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign gene products or knockout of native genes.

Appl Environ Microbiol, 2004 Feb, 70(2), 798 - 803
Characterization and development of two reporter gene systems for Clostridium acetobutylicum; Feustel L et al.; The use of lacZ from Thermoanaerobacterium thermosulfurigenes (encoding beta-galactosidase) and lucB from Photinus pyralis (encoding luciferase) as reporter genes in Clostridium acetobutylicum was analyzed with promoters of genes required for solventogenesis and acidogenesis . Both systems proved to be well suited and allowed the detection of differences in promoter strength at least up to 100-fold . The luciferase assay could be performed much faster and comes close to online measurement . Resequencing of lacZ revealed a sequence error in the original database entry, which resulted in beta-galactosidase with an additional 31 amino acids . Cutting off part of the gene encoding this C terminus resulted in decreased enzyme activity . The lacZ reporter data showed that bdhA (encoding butanol dehydrogenase A) is expressed during the early growth phase, followed by sol (encoding butyraldehyde/butanol dehydrogenase E and coenzyme A transferase) and bdhB (encoding butanol dehydrogenase B) expression . adc (encoding acetoacetate decarboxylase) was also induced early . There is about a 100-fold difference in expression between adc and bdhB (higher) and bdhA and the sol operon (lower) . The lucB reporter activity could be increased 10-fold by the addition of ATP to the assay . Washing of the cells proved to be important in order to prevent a red shift of bioluminescence in an acidic environment (for reliable data) . lucB reporter measurements confirmed the expression pattern of the sol and ptb-buk (encoding phosphotransbutyrylase and butyrate kinase) operons as determined by the lacZ reporter and showed that the expression level from the ptb promoter is 59-fold higher than that from the sol operon promoter.

Protein Expr Purif, 2004 Mar, 34(1), 95 - 102
Cloning, high level expression, purification, and crystallization of the full length Clostridium botulinum neurotoxin type E light chain; Agarwal R et al.; The catalytic activity of the highly potent botulinum neurotoxins are confined to their N-terminal light chains ( approximately 50kDa) . A full-length light chain for the type E neurotoxin with a C-terminal 6x His-tag, BoNT/E-LC, has been cloned in a pET-9c vector and over-expressed in BL21 (DE3) cells . BoNT/E-LC was purified to homogeneity by affinity chromatography on Ni-NTA agarose followed by exclusion chromatography using a Superdex-75 sizing column . The purified protein has very good solubility and can be stored stably at -20 degrees C; however, it seems to undergo auto-proteolysis when stored at temperature #10878;4-10 degrees C . BoNT/E-LC is active on its natural substrate, the synaptosomal associated 25kDa protein, SNAP-25, indicating that it retains a native-like conformation and therefore can be considered as a useful tool in studying the structure/function of the catalytic light chain . Recombinant BoNT/E-LC has been crystallized under five different conditions and at various pHs . Crystals diffract to better than 2.1A.

Toxicon, 2003 Dec, 42(7), 687 - 707
The role of bacterial and non-bacterial toxins in the induction of changes in membrane transport: implications for diarrhea; Laohachai KN et al.; Bacterial toxins induce changes in membrane transport which underlie the loss of electrolyte homeostasis associated with diarrhea . Bacterial- and their secreted toxin-types which have been linked with diarrhea include: (a) Vibrio cholerae (cholera toxin, E1 Tor hemolysin and accessory cholera enterotoxin); (b) Escherichia coli (heat stable enterotoxin, heat-labile enterotoxin and colicins); (c) Shigella dysenteriae (shiga-toxin); (d) Clostridium perfringens (C . perfringens enterotoxin, alpha-toxin, beta-toxin and theta-toxin); (e) Clostridium difficile (toxins A and B); (f) Staphylococcus aureus (alpha-haemolysin); (g) Bacillus cereus (cytotoxin K and haemolysin BL); and (h) Aeromonas hydrophila (aerolysin, heat labile cytotoxins and heat stable cytotoxins) . The mechanisms of toxin-induced diarrhea include: (a) direct effects on ion transport in intestinal epithelial cells, i.e . direct toxin interaction with intrinsic ion channels in the membrane and (b) indirect interaction with ion transport in intestinal epithelial cells mediated by toxin binding to a membrane receptor . These effects consequently cause the release of second messengers, e.g . the release of adenosine 3',5'-cyclic monophosphate/guanosine 3',5'-monophosphate, IP(3), Ca2+ and/or changes in second messengers that are the result of toxin-formed Ca2+ and K+ permeable channels, which increase Ca2+ flux and augment changes in Ca2+ homeostasis and cause depolarisation of the membrane potential . Consequently, many voltage-dependent ion transport systems, e.g . voltage-dependent Ca2+ influx, are affected . The toxin-formed ion channels may act as a pathway for loss of fluid and electrolytes . Although most of the diarrhea-causing toxins have been reported to act via cation and anion channel formation, the properties of these channels have not been well studied, and the available biophysical properties that are needed for the characterization of these channels are inadequate.

Mol Microbiol, 2004 Jan, 51(2), 599 - 607
Use of antisense RNA to modify the composition of cellulosomes produced by Clostridium cellulolyticum; Perret S et al.; The enzymatic composition of the cellulosomes produced by Clostridium cellulolyticum was modified by inhibiting the synthesis of Cel48F that is the major cellulase of the cellulosomes . The strain ATCC 35319 (pSOSasrF) was developed to over-produce a 469 nucleotide-long antisense-RNA (asRNA) directed against the ribosome-binding site region and the beginning of the coding region of the cel48F mRNAs . The cellulolytic system secreted by the asRNA-producing strain showed a markedly lower amount of Cel48F, compared to the control strain transformed with the empty plasmid (pSOSzero) . This was correlated with a 30% decrease of the specific activity of the cellulolytic system on Avicel cellulose, indicating that Cel48F plays an important role in the recalcitrant cellulose degradation . However, only minor effects were observed on the growth parameters on cellulose . In both transformant strains, cellulosome production was found to be reduced and two unknown proteins (P105 and P98) appeared as major components of their cellulolytic systems . These proteins did not contain any dockerin domain and were shown to be not included into the cellulosomes; they are expected to participate to the non-cellulosomal cellulolytic system of C . cellulolyticum.

Mol Microbiol, 2004 Jan, 51(2), 589 - 98
Cellulolysis is severely affected in Clostridium cellulolyticum strain cipCMut1; Maamar H et al.; Progress towards understanding the molecular basis of cellulolysis by Clostridium cellulolyticm was obtained through the study of the first cellulolysis defective mutant strain, namely cipCMut1 . In this mutant, a 2 659 bp insertion element, disrupts the cipC gene at the sequence encoding the seventh cohesin of the scaffoldin CipC . cipC is the first gene in a large 'cel' gene cluster, encoding several enzymatic subunits of the cellulosomes, including the processive cellulase Cel48F, which is the major component . Physiological and biochemical studies showed that the mutant strain was affected in cellulosome synthesis and severely impaired in its ability to degrade crystalline cellulose . It produced small amounts of a truncated CipC protein (P120), which had functional cohesin domains and assembled complexes which did not contain any of the enzymes encoded by genes of the 'cel' cluster . The mutant cellulolytic system was mainly composed of three proteins designated P98, P105 and P125 . Their N-termini did not match any of the known cellulase sequences from C . cellulolyticum . A large amount of entire CipC produced in the cipCMut1 strain by trans-complementation with plasmid pSOScipC did not restore the cellulolytic phenotype, in spite of the assembly of a larger amount of complexes . The complexes produced in the mutant and complemented strains contained at least 12 different dockerin-containing proteins encoded by genes located outside of the 'cel' cluster . The disturbances observed in the mutant and trans-complemented strains were the result of a strong polar effect resulting from the cipC gene disruption . In conclusion, this study provided genetic evidence that the cellulases encoded by the genes located in the 'cel' cluster are essential for the building of cellulosomes efficient in crystalline cellulose degradation.

Biochemistry, 2004 Feb 10, 43(5), 1163 - 70
Structural basis for the exocellulase activity of the cellobiohydrolase CbhA from Clostridium thermocellum; Schubot FD et al.; Numerous bacterial and fungal organisms have evolved elaborate sets of modular glycoside hydrolases and similar enzymes aimed at the degradation of polymeric carbohydrates . Presently, on the basis of sequence similarity catalytic modules of these enzymes have been classified into 90 families . Representatives of a particular family display similar fold and catalytic mechanisms . However, within families distinctions occur with regard to enzymatic properties and type of activity against carbohydrate chains . Cellobiohydrolase CbhA from Clostridium thermocellum is a large seven-modular enzyme with a catalytic module belonging to family 9 . In contrast to other representatives of that family possessing only endo- and, in few cases, endo/exo-cellulase activities, CbhA is exclusively an exocellulase . The crystal structures of the combination of the immunoglobulin-like module and the catalytic module of CbhA (Ig-GH9_CbhA) and that of an inactive mutant Ig-GH9_CbhA(E795Q) in complex with cellotetraose (CTT) are reported here . The detailed analysis of these structures reveals that, while key catalytic residues and overall fold are conserved in this enzyme and those of other family 9 glycoside hydrolases, the active site of GH9_CbhA is blocked off after the -2 subsite . This feature which is created by an extension and altered conformation of a single loop region explains the inability of the active site of CbhA to accommodate a long cellulose chain and to cut it internally . This altered loop region is responsible for the exocellulolytic activity of the enzyme.

Eur J Epidemiol, 2003, 18(12), 1153 - 4
Considering the antimicrobial sensitivity of the intestinal botulism agent Clostridium butyricum when treating concomitant infections; Fenicia L et al.; In Italy, neurotoxigenic Clostridium butyricum has been reported as a new agent of intestinal toxemia botulism, and most of the cases have been associated with enterocolitis . Although infections concomitant with botulism must be treated with antibiotics, this can increase the severity of botulism . We discuss the sensitivity of this agent to certain antibiotics, compared to findings on the sensitivity of C . botulinum.

Eur J Epidemiol, 2003, 18(12), 1147 - 52
Serological survey of immunity to tetanus in adult population of Northern Halkidiki, Greece; Symeonidis N et al.; BACKGROUND: Despite the implementation of the obligatory anti-tetanus vaccination, the tetanus cases in Greece are not eliminated . Because of the increased possibility of Clostridium tetani infection of the Northern Halkidiki population--like any other rural population the evaluation of the immunity to tetanus in the area is considered necessary . METHODS: The tetanus antitoxin levels were determined using the enzyme-linked immunosorbent assay (ELISA) in 405 healthy adult individuals attending the health center for routine laboratory tests . RESULTS: 64.4% of the studied population was found protected (tetanus antitoxin levels > or = 0.1 IU/ml) . The percentage of protected people decreased as age increased from 83.3% in the 21-30 to 51.2% in the > 60 age group . 82.1% of the tested males and 52.6% of the tested females had tetanus antitoxin levels > or = 0.1 IU/ml (p < 0.0001) . The percentages of immune men (100-66.2% in various age groups) were found higher than those of women (80.8-35.5% in the respective age groups) . The geometrical mean titres (GMTs) were 0.44 in all of 261 immune people, 0.53 in 133 immune men and 0.37 in 128 immune women (p = 0.0021) . CONCLUSION: The proportion of protection among men over 60 and women over 30 years old is inadequate, the levels of tetanus antitoxin decline with age and a significant difference was found between the proportion of protection of males and females.

Am J Med, 2004 Feb 1, 116(3), 198 - 200
Cases from the Osler Medical Service at Johns Hopkins University; Zetola-Burneo N et al.; A 47-year-old white woman with a history of stage III squamous cell carcinoma of the anus was transferred to Johns Hopkins Hospital for further evaluation of renal failure, hemolytic anemia, and thrombocytopenia.The patient was first diagnosed with squamous cell carcinoma of the anus 1 year before admission . She was treated with external beam radiation of the pelvis and two cycles of mitomycin C-based chemotherapy (a cumulative dose, 34 mg/m(2)) . Her clinical course was complicated by Clostridium difficile colitis and myositis successfully treated with prednisone.Three months before admission, the patient developed dysuria . Her creatinine increased from normal to 1.7 mg/dL, and microscopic hematuria was present . A renal ultrasound and an abdominal computed tomographic scan showed no abnormalities or obstruction . One month before admission, she underwent a cystoscopy, which showed only radiation-induced changes in the bladder . Two weeks before admission, the patient became delirious and was taken to a hospital, where she was found to be anemic, with a hematocrit level of 23.7%, and thrombocytopenic with a platelet count of 110,000/mm(3) . Her creatinine level was 5.9 mg/dL . Previous values of hematocrit, platelet count, and serum creatinine were normal.On admission at Johns Hopkins Hospital the patient had no complaints . She was afebrile on physical examination and had normal vital signs . Head, neck, chest, cardiovascular, and abdominal examinations were normal . There was skin pallor, but no echymoses or petechiae . She was alert and oriented with normal mental status . Her neurologic examination was normal . Laboratory data showed a white blood cell count of 6390/mm(3), a hematocrit level of 26.5%, and a platelet count of 26,000/mm(3) . Her blood urea nitrogen level was 57 mg/dL, creatinine level was 4.0 mg/dL, and lactate dehydrogenase was 550 U/L (reference, 115 to 275 U/L) . Urinalysis showed innumerable red blood cells and large protein . A peripheral blood smear showed fragmented red blood cells, schistocytes, no abnormal white blood cells, and few platelets . There was no radiographic or clinical evidence of relapse of her squamous cell carcinoma.What is the diagnosis?

Klin Padiatr, 2004 Jan-Feb, 216(1), 31 - 5
{Sudden death of twins: botulism because of contamination by pap vegetables}; Fischer D et al.; Botulism is caused by the blockage of the neural transmission in the cholinergic synapses by botulinum neurotoxin (BoNT) which is produced by Clostridium botulinum or other Clostridia . The classic form of botulism occurs after the ingestion of food contaminated by BoNT . The course of the infection can be asymptomatic, mild with subtle paralysis ("failure to thrive") oder severe with generalized paralysis ("floppy infant") . Infected infants can also die sudden and unexpectedly . These deaths often are attributed to Sudden Infant Death Syndrome (SIDS), unless a thorough postmortem examination reveals Botulism . The rate of fatal Botulism falsely attributed to SIDS is not known, because it is difficult in most cases to show the causal relationship between contamination, disease and death . We report the sudden and unexpected simultaneous death of twins of 22 months which could be attributed to Botulism . Contamination of food, colonization of the gut by Clostridia and infection with specific pathomorphological changes could be proven . The initial suspicion of infanticide could be excluded . lt could be shown, that Botulism is a potential cause of simultaneous unexpected deaths in twins.

Klin Padiatr, 2004 Jan-Feb, 216(1), 26 - 30
{Infant botulism and sudden infant death syndrome}; Bartram U et al.; Infant botulism represents a distinct entity of botulism . Ingestion of the ubiquitously present spores of Clostridium botulinum leads to germination of the organism and neurotoxin production in the infant intestine . Symptoms typically develop gradually in contrast to classical food botulism in which an acute onset of symptoms shortly after the ingestion of preformed toxin in a food is characteristic . Microbiologically, the diagnosis is established by identification of Clostridium botulinum organism and toxin in stool specimen . However, positive results in these tests provide only indirect evidence for the clinical relevance of the neurotoxin since asymptomatic carriers have been found . The toxin irreversibly blocks the release of acetylcholin from the motoric end plate which results in muscle weakness and paralysis . Depending on the amount of toxin produced, infant botulism exhibits a broad clinical spectrum ranging from oligosymptomatic forms to a fulminant course with acute respiratory failure within hours leading to sudden death . Unrecognized mild forms or beginning muscle weakness can be a co-factor for other risk factors of sudden infant death (SIDS) . In studies analyzing infants who died from SIDS, botulism bacteria or toxin were found in up to 20 % of cases . Infant botulism therefore represents an important differential diagnosis of unexplained and inconclusive muscular hypotonia in the first year of life.

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 347 - 9 Epub 2004 Jan 23.
Crystallization and preliminary X-ray analysis of the human-specific toxin intermedilysin; Polekhina G et al.; Intermedilysin is a human-specific toxin from Streptococcus intermedius, which is part of normal human oral flora . The bacterium is an opportunistic pathogen with a tendency for deep-seated infection in the brain and liver . Intermedilysin belongs to the cholesterol-dependent cytolysin (CDCs) family of toxins, which have been identified in several different bacteria including the serious human pathogens S . pneumoniae and Clostridium perfringens . Intermedilysin, however, is the only member that shows exclusive specificity for human cells . The toxin has a couple of non-conservative amino-acid substitutions in a tryptophan-rich region of the molecule (Cys to Ala and Trp to Pro), the most conserved region amongst the CDCs . Mutations in this region are known to render other CDCs inactive . In order to investigate the structure-function relationships of the unusual features of intermedilysin, which will help us to understand the molecular mechanism of the toxin family in general, recombinant intermedilysin has been crystallized . The crystals belong to an orthorhombic space group and contain two molecules per asymmetric unit . Diffraction data were collected to 2.3 A using synchrotron radiation.

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 342 - 3 Epub 2004 Jan 23.
Crystallization and preliminary X-ray analysis of xylanase B from Clostridium stercorarium; Nishimoto M et al.; Recombinant mature xylanase B from Clostridium stercorarium has been prepared and crystallized by the sitting-drop vapour-diffusion method using 4 mg ml(-1) purified enzyme, 10.3%(w/v) polyethylene glycol 1500, 8.6%(v/v) glycerol and 0.34 M non-detergent sulfobetaine 195 . A suitable crystal grew after incubation for ten weeks at 293 K . The crystal belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 64.76, b = 96.60, c = 138.44 A . X-ray diffraction data were collected to 1.80 A resolution.

Acta Crystallogr D Biol Crystallogr, 2004 Feb, 60(Pt 2), 298 - 303 Epub 2004 Jan 23.
Metal-substituted derivatives of the rubredoxin from Clostridium pasteurianum; Maher M et al.; Five different metal-substituted forms of Clostridium pasteurianum rubredoxin have been prepared and crystallized . The single Fe atom present in the Fe(S-Cys)(4) site of the native form of the protein was exchanged in turn for Co, Ni, Ga, Cd and Hg . All five forms of rubredoxin crystallized in space group R3 and were isomorphous with the native protein . The Co-, Ni- and Ga-substituted proteins exhibited metal sites with geometries similar to that of the Fe form (effective D(2d) local symmetry), as did the Cd and Hg proteins, but with a significant expansion of the metal-sulfur bond lengths . A knowledge of these structures contributes to a molecular understanding of the function of this simple iron-sulfur electron-transport protein.

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