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