Clostridia

Annu Rev Pharmacol Toxicol, 2004, 44, 167 - 93
Identification of the major steps in botulinum toxin action; Simpson LL; Botulinum toxin is a uniquely potent substance synthesized by the organisms Clostridium botulinum, Clostridium baratii, and Clostridium butyricum . This toxin, which acts preferentially on peripheral cholinergic nerve endings to block acetylcholine release, is both an agent that causes disease (i.e., botulism) as well as an agent that can be used to treat disease (e.g., dystonia) . The ability of botulinum toxin to produce its effects is largely dependent on its ability to penetrate cellular and intracellular membranes . Thus, toxin that is ingested or inhaled can bind to epithelial cells and be transported to the general circulation . Toxin that reaches peripheral nerve endings binds to the cell surface then penetrates the plasma membrane by receptor-mediated endocytosis and the endosome membrane by pH-induced translocation . Internalized toxin acts in the cytosol as a metalloendoprotease to cleave polypeptides that are essential for exocytosis . This review seeks to identify and characterize all major steps in toxin action, from initial absorption to eventual paralysis of cholinergic transmission.

Int J Syst Evol Microbiol, 2004 Jan, 54(Pt 1), 241 - 5
Hespellia stercorisuis gen . nov., sp . nov . and Hespellia porcina sp . nov., isolated from swine manure storage pits; Whitehead TR et al.; Four Gram-positive-staining, strictly anaerobic, non-spore-forming, rod-shaped organisms were isolated from a pig manure storage pit . Comparative 16S rRNA gene sequence analysis revealed that the isolates belonged to two related but distinct groups . Sequence analysis showed that the two groups of isolates were highly related to each other (approx . 97% 16S rRNA gene sequence similarity), forming a distinct cluster within the Clostridium coccoides suprageneric rDNA grouping . Biochemical and physiological studies confirmed the division of the isolates into two related, albeit distinct, groups . Based on both phenotypic and phylogenetic evidence, it is proposed that the unidentified rod-shaped isolates from pig manure should be classified in a novel genus, Hespellia gen . nov., as Hespellia stercorisuis sp . nov . and Hespellia porcina sp . nov . The type species of the novel genus is H . stercorisuis (type strain, PC18T=NRRL B-23456T=CCUG 46279T=ATCC BAA-677T) and the type strain of H . porcina is PC80T (=NRRL B-23458T=ATCC BAA-674T).

Biomaterials, 2004 May, 25(10), 1841 - 9
Engineering a bifunctional starch-cellulose cross-bridge protein; Levy I et al.; Biodegradable starch- and cellulose-based polymers have a range of properties which make them suitable for use in a wide array of biomedical applications ranging from bone replacement to engineering of tissue scaffolds and drug delivery systems . A novel polysaccharide cross-bridging protein was designed which was comprised of a cellulose-binding domain from Clostridium cellulovorans (CBD(clos)) and a starch-binding domain from Aspergillus niger B1 (SBD(Asp)) . The two genes were fused in-frame via a synthetic elastin gene to construct a Cellulose/Starch Cross bridging Protein (CSCP) . Recombinant CSCP was expressed in Escherichia coli, and successfully refolded from inclusion bodies . CSCP demonstrated cross-bridging ability in different model systems composed of insoluble or soluble starch and cellulose . The aspect that different carbohydrate-binding module maintain their binding capacity over a wide range of conditions, without the need for chemical reactions, makes them attractive domains for designing new classes of chimeric polysaccharide-binding domains which demonstrate potential for use in a wide range of biomaterials.

J Am Assoc Gynecol Laparosc, 2003 Nov, 10(4), 481 - 3
Outpatient total laparoscopic hysterectomy; Thiel J et al.; STUDY OBJECTIVE: To assess the safety of and patient satisfaction with total laparoscopic hysterectomy as an outpatient procedure . DESIGN: Retrospective case study (Canadian Task Force classification III) . SETTING: Tertiary care hospital . PATIENTS: Sixty-six consecutive women . INTERVENTION: Outpatient total laparoscopic hysterectomy . MEASUREMENTS AND MAIN RESULTS: Of 66 procedures completed, 6 patients (7.5%) required overnight hospitalization, with 4 of them discharged the next day . Seven (11%) minor postoperative complications occurred . One patient required hospitalization to receive intravenous antibiotics and one for drainage of a cuff hematoma . There were three (4.5%) minor intraoperative complications . One woman developed Clostridium difficile diarrhea as well as deep vein thrombosis and pulmonary embolus . Sixty-three women (95%) were satisfied with the procedure and would recommend it to others . CONCLUSION: Outpatient total laparoscopic hysterectomy is well tolerated, safe, and cost effective.

Internist (Berl), 2004 Jan, 45(1), 85 - 9
{Generalized pain syndrome, fever and somnolence in an 81-year-old patient}; Fuchs KF et al.; We present a case of an 81-year-old diabetic man with anaerobic sepsis due to acalculous cholecystitis . The patient was admitted to our hospital with a seven-day history of severe abdominal pain accompanied with fever and somnolence . Blood cultures taken during the initial procedure developed Clostridium perfringens . The patient was immediately treated with parenteral penicillin . The ultrasonography pointed out the case: the gall bladder was found to be distended and slightly thickened . This result was interpreted as an acute non-emphysematous cholecystitis . The material obtained by needle aspiration and therapeutical emptying of the gall bladder revealed large gram positive rods, that also proved to be Clostridium perfringens . The patients course afterwards was uneventful . Antibiotics were continued and he was discharged after 13 days in a stable condition.

J Cell Biol, 2004 Jan 19, 164(2), 279 - 90
Atypical protein kinase C (PKCzeta/lambda) is a convergent downstream target of the insulin-stimulated phosphatidylinositol 3-kinase and TC10 signaling pathways; Kanzaki M et al.; Insulin stimulation of adipocytes resulted in the recruitment of atypical PKC (PKCzeta/lambda) to plasma membrane lipid raft microdomains . This redistribution of PKCzeta/lambda was prevented by Clostridium difficile toxin B and by cholesterol depletion, but was unaffected by inhibition of phosphatidylinositol (PI) 3-kinase activity . Expression of the constitutively active GTP-bound form of TC10 (TC10Q/75L), but not the inactive GDP-bound mutant (TC10/T31N), targeted PKCzeta/lambda to the plasma membrane through an indirect association with the Par6-Par3 protein complex . In parallel, insulin stimulation as well as TC10/Q75L resulted in the activation loop phosphorylation of PKCzeta . Although PI 3-kinase activation also resulted in PKCzeta/lambda phosphorylation, it was not recruited to the plasma membrane . Furthermore, insulin-induced GSK-3beta phosphorylation was mediated by both PI 3-kinase-PKB and the TC10-Par6-atypical PKC signaling pathways . Together, these data demonstrate that PKCzeta/lambda can serve as a convergent downstream target for both the PI 3-kinase and TC10 signaling pathways, but only the TC10 pathway induces a spatially restricted targeting to the plasma membrane.

Biochim Biophys Acta, 2004 Jan 5, 1676(1), 71 - 82
GltX from Clostridium saccharobutylicum NCP262: glutamate synthase or oxidoreductase?
Stutz HE, Reid SJ.
A full-length gene encoding a homologue of the small subunit of the glutamate synthase (GOGAT) enzyme was isolated from the anaerobic bacterium, Clostridium saccharobutylicum NCP262, which has been used extensively for the commercial production of solvents . Using a screening system designed to isolate genes involved in electron transport, plasmid pMET13C1 was isolated . Analysis of this plasmid identified a gene (1245 bp) with a predicted approximately 46-kDa product, which was associated with reductive activation of the pro-drug metronidazole . The deduced 414-amino-acid sequence was not typical of electron transport proteins, but rather shared striking homology to the small (beta) subunit of the GOGAT enzyme and other beta subunit-like polypeptides, and was thus designated gltX . Although all the functional domains typical of GOGAT beta subunits were conserved in this GltX protein, certain sequence features were not conserved . Furthermore, it was independently transcribed, did not lie adjacent to a GOGAT large subunit (alpha) domain, and its expression was not regulated by nitrogen conditions . These results provide additional support for current theories on the evolutionary relationships of GOGAT beta subunit domains in bacteria, and suggest that GltX belongs to a more general family of oxidoreductases, which is used in a context other than glutamate biosynthesis to transfer electrons to a currently unknown protein domain.

Trends Cell Biol, 1995 Dec, 5(12), 441 - 3
Monoglucosylation of low-molecular-mass GTP-binding Rho proteins by clostridial cytotoxins; Aktories K et al.; Rho proteins, which are involved in receptor-mediated regulation of the actin cytoskeleton, are substrates for ADP-ribosylation by Clostridium botulinum C3 toxins . Recently, it was shown that Rho and other members of the Rho subfamily of low-molecular-mass GTP-binding proteins are glucosylated by C . difficile toxins A and B . Glucosylation occurs at threonine-37, which is a crucial amino acid residue for the regulatory functions of the small GTP-binding proteins . These toxins should prove useful as tools for studying the functions of Rho proteins.

J Med Microbiol, 2004 Feb, 53(Pt 2), 167 - 72
Clostridium difficile colonization in healthy adults: transient colonization and correlation with enterococcal colonization; Ozaki E et al.; The aim of the present study was to investigate the colonization status of Clostridium difficile in healthy individuals . In total, 139 healthy adults from two study groups were examined at intervals of 3 months . Among the 18 positive subjects, the number of subjects from whom C . difficile was isolated once, twice, three times or four times was 10 (55.6%), three (16.7%), two (11.1%) and three (16.7%), respectively . In the student group, different subjects were colonized by different PCR ribotype/PFGE types . However, the same PCR ribotype/PFGE types of C . difficile were isolated from different subjects in the employee group, indicating that cross-transmission may have occurred in this group . Continuous colonization by the same PCR ribotype/PFGE type was only observed in three subjects . C . difficile-positive subjects were significantly more densely colonized by enterococci (P<0.05) than C . difficile-negative subjects: subjects that were found to be C . difficile-positive three or four times appeared to have higher concentrations of enterococci . The present results demonstrate that, although colonization by a C . difficile strain is transient in many cases, there are healthy individuals that are colonized persistently by C . difficile . They also suggest that dense colonization of the intestine by enterococci may be associated with C . difficile colonization.

Scand J Infect Dis, 2003, 35(11-12), 883 - 6
Clostridium perfringens pleuropulmonary infection and septic shock: case report and population-based laboratory surveillance study; Jackson S et al.; We report a rare case of necrotizing pneumonia, empyema, and septic shock caused by Clostridium perfringens . Population-based laboratory surveillance was subsequently conducted that identified an annual incidence of invasive C . perfringens disease of 0.83 per 100,000 population with a striking age-related increased risk for acquisition.

Am J Respir Med, 2003, 2(5), 395 - 411
Prevention and treatment of gastrointestinal complications in patients on mechanical ventilation; Mutlu GM et al.; There exists a complex, dynamic interaction between mechanical ventilation and the splanchnic vasculature that contributes to a myriad of gastrointestinal tract complications that arise during critical illness . Positive pressure-induced splanchnic hypoperfusion appears to play a pivotal role in the pathogenesis of these complications, the most prevalent of which are stress-related mucosal damage, gastrointestinal hypomotility and diarrhea . Furthermore, characteristics of the splanchnic vasculature make the gastrointestinal tract vulnerable to adverse effects related to positive pressure ventilation . While most of these complications seen in mechanically ventilated patients are reflections of altered gastrointestinal physiology, some may be attributed to medical interventions instituted to treat critical illness.Since maintenance of normal hemodynamics cannot always be achieved, pharmacologic prophylactic therapy has become a mainstay in the prevention of gastrointestinal complications in the intensive care unit . Improved understanding of the systemic effects of mechanical ventilation and greater application of lung-protective ventilatory strategies may potentially minimize positive pressure-induced reductions in splanchnic perfusion, systemic cytokine release and, consequently, reduce the incidence of gastrointestinal complications associated with mechanical ventilation . Herein, we discuss the pathophysiology of gastrointestinal complications associated with mechanical ventilation, summarize the most prevalent complications and focus on preventive strategies and available treatment options for these complications.The most common causes of gastrointestinal hemorrhage in mechanically ventilated patients are bleeding from stress-related mucosal damage and erosive esophagitis . In general, histamine H(2) receptor antagonists and proton pump inhibitors prevent stress-related mucosal disease by raising the gastric fluid pH . Proton pump inhibitors tend to provide more consistent pH control than histamine H(2) receptor antagonists . There is no consensus on the drug of choice for stress ulcer prophylaxis with several meta-analyses providing conflicting results on the superiority of any medication . Prevention of erosive esophagitis include careful use of nasogastric tubes and institution of strategies that improve gastric emptying . Many mechanically ventilated patients have gastrointestinal hypomotility and diarrhea . Treatment options for gastrointestinal motility are limited, thus, preventive measures such as correction of electrolyte abnormalities and avoidance of medications that impair gastrointestinal motility are crucial . Treatment of diarrhea depends on the underlying cause . When associated with Clostridium difficile infection antibacterial therapy should be discontinued, if possible, and treatment with oral metronidazole should be initiated.More studies are warranted to better understand the systemic effects of mechanical ventilation on the gastrointestinal tract and to investigate the impact of lung protective ventilatory strategies on gastrointestinal complications.

Am J Vet Res, 2004 Jan, 65(1), 26 - 30
Effects of injection of botulinum toxin type B into the external anal sphincter on anal pressure of horses; Adam-Castrillo D et al.; OBJECTIVE: To determine effects on anal pressure of horses after local injection of the external anal sphincter with Clostridium botulinum toxin type B . ANIMALS: 11 healthy adult horses . PROCEDURE: Peak and resting anal sphincter pressures were measured with a custom-made rectal probe that was connected to a pressure transducer . Pressures were measured before treatment and after injection with botulinum toxin type B (BTB) or saline (0.9% NaCl) solution . Dose titration with 500, 1,000, 1,500, and 2,500 U of BTB was completed . Physical changes, behavior, and anal pressure were recorded for each horse . RESULTS: Injection of 1,000 U of BTB caused a significant reduction in peak anal pressure from days 2 to 84, compared with pressure in control horses . Maximal effect of the toxin was observed within the first 15 days after injection, followed by a slow return to baseline during the 168-day period . Injection of 2,500 U of BTB in the anal sphincter in 1 horse resulted in lethargy, generalized weakness, and dysphagia for 14 days . Adverse clinical effects were not observed in horses after injections with 500, 1,000, or 1,500 U of BTB . CONCLUSIONS AND CLINICAL RELEVANCE: The effect of focal intramuscular injection of BTB in horses is similar to that reported for other species . However, horses appear to be more sensitive to BTB, compared with other species, and clinical signs of botulism may develop at doses exceeding 1,500 U . Injections of BTB in the external anal sphincter of mares may be useful to reduce incisional dehiscence after repair of perineal lacerations.

J Food Prot, 2004 Jan, 67(1), 199 - 202
Antibacterial efficiency of Finnish spice essential oils against pathogenic and spoilage bacteria; Nevas M et al.; The antibacterial properties of 13 essential oils, derived from spices grown in Finland, were examined with an agar diffusion method against 12 bacterial strains . The organisms tested included both spoilage and pathogenic bacteria . The gram-positive bacteria appeared to be more sensitive than the gram-negative organisms, Clostridium botulinum and Clostridium perfringens being the most sensitive . Oregano, savory, and thyme showed the broadest antibacterial activity by distinctly inhibiting the growth of all the organisms tested . By gas chromatography-mass spectrometry analysis, differences were noted in the composition of oregano and thyme oils in comparison to previous reports.

J Food Prot, 2004 Jan, 67(1), 83 - 9
Growth of heat-treated enterotoxin-positive Clostridium perfringens and the implications for safe cooling rates; Andersen KG et al.; Clostridium perfringens 790-94 and 44071.C05 carrying a chromosomal and a plasmid cpe gene, respectively, were used to determine differences in heat resistance and growth characteristics between the genotypes . Heat inactivation experiments were conducted using an immersed coil apparatus . Spore germination, outgrowth, and lag phase, together named GOL time, as well as generation times were determined during constant temperatures in fluid thioglycollate (FTG) medium as well as in vacuum-packed, heat-treated minced turkey . GOL time and growth were also monitored during cooling scenarios from 65 to 10 degrees C for 3, 4, 5, 6, and 7 h in vacuum-packed, heat-treated minced turkey . Spores of strain 790-94 were approximately 10-fold more heat resistant at 85 degrees C than those of strain 44071.C05, and strain 790-94 also had a higher temperature growth range in FTG . The higher growth range for a chromosomal enterotoxin-producing CPE+ strain was confirmed using two other strains carrying a chromosomal (NCTC8239) and plasmid (945P) cpe gene . Moreover, strain 790-94 had shorter GOL times at 50 degrees C in turkey and approximately half the generation time compared with strain 44071.C05 at temperatures > or = 45 degrees C in both FTG and turkey . Strain 790-94 increased with 0.3, 1.0, 1.7, and 2.0 logs, respectively, during cooling from 65 to 10 degrees C in 4, 5, 6, and 7 h, which was significantly higher than for strain 44071.C05 . A maximum acceptable cooling time of 5 h between 65 and 10 degrees C is suggested.

J Clin Microbiol, 2004 Jan, 42(1), 484 - 6
Osteosynthesis-associated bone infection caused by a nonproteolytic, nontoxigenic Clostridium botulinum-like strain; Carlier JP et al.; A nonproteolytic, nontoxigenic Clostridium botulinum strain identified by conventional and molecular techniques as type B-, E-, or F-like (BEF-like) was isolated from a human postsurgical wound . All previous reports of such strains have been from environmental sources . Since toxin production is the main taxonomic denominator for C . botulinum, a new name is needed for nonproteolytic, nontoxigenic BEF-like clinical isolates.

J Biol Chem, 2004 Apr 2, 279(14), 14315 - 22 Epub 2004 Jan 10.
The identification and structure of the membrane-spanning domain of the Clostridium septicum alpha toxin; Melton JA et al.; Alpha toxin (AT) is a pore-forming toxin produced by Clostridium septicum that belongs to the unique aerolysin-like family of pore-forming toxins . The location and structure of the transmembrane domains of these toxins have remained elusive . Using deletion mutagenesis, cysteine-scanning mutagenesis and multiple spectrofluorimetric methods a membrane-spanning amphipathic beta-hairpin of AT has been identified . Spectrofluorimetric analysis of cysteine-substituted residues modified with an environmentally sensitive fluorescent probe via the cysteine sulfydryl showed that the side chains of residues 203-232 alternated between the aqueous milieu and the membrane core when the AT oligomer was inserted into membranes, consistent with the formation of an amphipathic transmembrane beta-hairpin . AT derivatives that contained deletions that removed up to 90% of the beta-hairpin did not form a pore but were similar to native toxin in all other aspects of the mechanism . Furthermore, a mutant of AT that contained an engineered disulfide, predicted to restrict the movement of the beta-hairpin, functioned similarly to native toxin except that it did not form a pore unless the disulfide bond was reduced . Together these studies revealed the location and structure of the membrane-spanning domain of AT.

Pain, 2004 Jan, 107(1-2), 125 - 33
Subcutaneous administration of botulinum toxin A reduces formalin-induced pain; Cui M et al.; Botulinum toxin type A (BoNT-A) produced by the bacterium Clostridium botulinum is a potent inhibitor of acetylcholine release in the neuromuscular junction and has been used to treat many disorders related to excessive muscle contraction . However, BoNT-A has recently been used in pain therapy to treat myofascial pain, low back pain and various types of headaches, including migraine . The purpose of this study is to investigate the antinociceptive effect of BoNT-A and its underlying mechanism in the rat formalin inflammatory pain model . BoNT-A (3.5, 7, 15 and 30 U/kg) or vehicle was administered to the plantar surface of the right hindpaw of male Sprague-Dawley rats . BoNT-A dose-dependently (P<0.05) inhibited formalin-induced nociceptive behavior during phase 2 but not during phase 1 when administered 5 h to 12 days before formalin challenge . The onset of the antinociceptive effect started at 5 h after pre-treatment and this effect lasted for at least 12 days . BoNT-A (7 U/kg) also reduced edema . Consistent with the lack of effect in the formalin phase 1, BoNT-A, at 15 U/kg, had no effect on acute thermal nociception; no local muscle weakness was observed at this dose . Pre-treatment of rats with BoNT-A (3.5, 7 or 15 U/kg) all significantly reduced formalin-evoked glutamate (Glu) release . These results demonstrate that local peripheral injection of BoNT-A significantly reduces formalin-induced nociceptive behaviors with the absence of obvious muscle weakness . Such an antinociceptive effect of BoNT-A is associated with the inhibition of formalin-induced release of Glu (and/or neuropeptides) from primary afferent terminals.

Vet Q, 2003 Dec, 25(4), 155 - 9
Pathogenesis and pathology of blackleg in ruminants: the role of toxins and neuraminidase . A short review; Useh NM et al.; There is no consensus on the pathogenesis of blackleg infection that occurs in ruminants, but toxins and neuraminidase produced by Clostridium chauvoei are believed to play a significant role in the pathogenesis of the disease . This paper provides an update on the role of toxins and neuraminidase in the pathogenesis and pathology of the disease . The use of neuraminidase inhibitors to manage clinical blackleg infections is therefore an alternative therapeutic protocol that should be thoroughly investigated . It is suggested that in vivo clinical trials should be carried out to determine the mechanism of action and clinical efficacy of neuraminidase inhibitors.

Surg Today, 2004, 34(1), 80 - 1
Primary gas gangrene of the pancreas: report of a case; Ikegami T et al.; Spontaneous gas gangrene of the pancreas, caused by an infection of Clostridium perfringens, is an extremely rare but severe form of acute pancreatitis . A 67-year-old man complaining of severe epigastric pain with diffuse guarding underwent an emergency laparotomy . During surgery, hemorrhagic pancreatic necrosis was observed with a large amount of peripancreatic gas . Cultures demonstrated C . perfringens . The identification of hemolysis and the accumulation of peripancreatic gas on computed tomography, which were both caused by an infection of C . perfringens, led us to make a diagnosis of clostridial infection of the pancreas.

Biotechnol Bioeng, 2003 Dec 30, 84(7), 842 - 54
Transcriptional analysis of product-concentration driven changes in cellular programs of recombinant Clostridium acetobutylicumstrains; Tummala SB et al.; Antisense RNA (asRNA) downregulation alters protein expression without changing the regulation of gene expression . Downregulation of primary metabolic enzymes possibly combined with overexpression of other metabolic enzymes may result in profound changes in product formation, and this may alter the large-scale transcriptional program of the cells . DNA-array based large-scale transcriptional analysis has the potential to elucidate factors that control cellular fluxes even in the absence of proteome data . These themes are explored in the study of large-scale transcriptional analysis programs and the in vivo primary-metabolism fluxes of several related recombinant C . acetobutylicum strains: C . acetobutylicum ATCC 824(pSOS95del) (plasmid control; produces high levels of butanol snd acetone), 824(pCTFB1AS) (expresses antisense RNA against CoA transferase (ctfb1-asRNA); produces very low levels of butanol and acetone), and 824(pAADB1) (expresses ctfb1-asRNA and the alcohol-aldehyde dahydrogenase gene (aad); produce high alcohol and low acetone levels) . DNA-array based transcriptional analysis revealed that the large changes in product concentrations (snd notably butanol concentration) due to ctfb1-asRNA expression alone and in combination with aad overexpression resulted in dramatic changes of the cellular transcriptome . Cluster analysis and gene expression patterns of established and putative operons involved in stress response, motility, sporulation, and fatty-acid biosynthesis indicate that these simple genetic changes dramatically alter the cellular programs of C . acetobutylicum . Comparison of gene expression and flux analysis data may point to possible flux-controling steps and suggest unknown regulatory mechanisms .

J Immunol, 2004 Jan 15, 172(2), 1118 - 24
Role of commensal bacteria in development of gut-associated lymphoid tissues and preimmune antibody repertoire; Rhee KJ et al.; Intestinal bacteria are required for development of gut-associated lymphoid tissues (GALT), which mediate a variety of host immune functions, such as mucosal immunity and oral tolerance . In rabbits, the intestinal microflora are also required for developing the preimmune Ab repertoire by promoting somatic diversification of Ig genes in B cells that have migrated to GALT . We studied the mechanism of bacteria-induced GALT development . Bacteria were introduced into rabbits in which the appendix had been rendered germfree by microsurgery (we refer to these rabbits as germfree-appendix rabbits) . We then identified specific members of the intestinal flora that promote GALT development . The combination of Bacteroides fragilis and Bacillus subtilis consistently promoted GALT development and led to development of the preimmune Ab repertoire, as shown by an increase in somatic diversification of VDJ-C micro genes in appendix B cells . Neither species alone consistently induced GALT development, nor did Clostridium subterminale, Escherichia coli, or Staphylococcus epidermidis . B . fragilis, which by itself is immunogenic, did not promote GALT development; hence, GALT development in rabbits does not appear to be the result of an Ag-specific immune response . To identify bacterial pathways required for GALT development, we introduced B . fragilis along with stress-response mutants of B . subtilis into germfree-appendix rabbits . We identified two Spo0A-controlled stress responses, sporulation and secretion of the protein YqxM, which are required for GALT development . We conclude that specific members of the commensal, intestinal flora drive GALT development through a specific subset of stress responses.

J Hosp Infect, 2004 Jan, 56(1), 10 - 5
How do we assess hospital cleaning? A proposal for microbiological standards for surface hygiene in hospitals; Dancer SJ; Increasing numbers of hospital-acquired infections have generated much attention over the last decade . The public has linked the so-called 'superbugs' with their experience of dirty hospitals, but the precise role of cleaning in the control of these organisms in unknown . Hence the importance of a clean environment is likely to remain speculative unless it becomes an evidence-based science . This proposal is a call for bacteriological standards with which to assess clinical surface hygiene in hospitals, based on those used by the food industry . The first standard concerns any finding of a specific 'indicator' organism, the presence of which suggests a requirement for increased cleaning . Indicators would include Staphylococcus aureus, including methicillin-resistant S . aureus, Clostridium difficile, vancomycin-resistant enterococci and various Gram-negative bacilli . The second standard concerns a quantitative aerobic colony count of <5 cfu/cm(2) on frequent hand touch surfaces in hospitals . The principle relates to modern risk management systems such as HACCP, and reflects the fact that pathogens of concern are widespread . Further work is required to evaluate and refine these standards and define the infection risk from the hospital environment.

Med Dosw Mikrobiol, 2003, 55(3), 253 - 8
{Assessment of susceptibility to metronidazole and vancomycin of Clostridium difficile strains isolated between 1998-2002}; Pituch H et al.; The drugs of choice used to treat C . diffcile associated diarrhoea (CDAD) are metronidazole and vancomycin . C . difficile strains isolated in most laboratories are susceptible to metronidazole and vancomycin . Communication about emergence of antimicrobial resistance among C . difficile strains in some countries to metronidazole and intermediate resistance to vancomycin are alarming . This study was performed to determine the susceptibility to metronidazole and vancomycin of 140 C . difficile strains isolated from patients with CDAD hospitalised in academic hospital between 1999-2002 . Resistance to metronidazole and vancomycin was not observed.

Microbiology, 2004 Jan, 150(Pt 1), 127 - 34
The Clostridium perfringens TetA(P) efflux protein contains a functional variant of the Motif A region found in major facilitator superfamily transport proteins; Bannam TL et al.; The Clostridium perfringens tetracycline resistance protein, TetA(P), is an inner-membrane protein that mediates the active efflux of tetracycline from the bacterial cell . This protein comprises 420 aa and is predicted to have 12 transmembrane domains (TMDs) . Comparison of the TetA(P) amino acid sequence to that of several members of the major facilitator superfamily (MFS) identified a variant copy of the conserved Motif A . This region consists of the sequence E59xPxxxxxDxxxRK72 and is located within the putative loop joining TMDs 2 and 3 in the predicted structural model of the TetA(P) protein . To study the functional importance of the conserved residues, site-directed mutagenesis was used to construct 17 point mutations that were then analysed for their effect on tetracycline resistance and their ability to produce an immunoreactive TetA(P) protein . Changes to the conserved Phe-58 residue were tolerated, whereas three independent substitutions of Pro-61 abolished tetracycline resistance . Examination of the basic residues showed that Arg-71 is required for function, whereas tetracycline resistance was retained when Lys-72 was substituted with arginine . These results confirm that the region encoding this motif is important for tetracycline resistance and represents a distant version of the Motif A region found in other efflux proteins and members of the MFS family . In addition, it was shown that Glu-117 of the TetA(P) protein, which is predicted to be located in TMD4, is important for resistance although a derivative with an aspartate residue at this position is also functional.

J Biol Chem, 2004 Mar 26, 279(13), 12181 - 9 Epub 2003 Dec 30.
Clostridium perfringens alpha-toxin activates the sphingomyelin metabolism system in sheep erythrocytes; Ochi S et al.; Clostridium perfringens alpha-toxin induces hemolysis of rabbit erythrocytes through the activation of glycerophospholipid metabolism . Sheep erythrocytes contain large amounts of sphingomyelin (SM) but not phosphatidylcholine . We investigated the relationship between the toxin-induced hemolysis and SM metabolic system in sheep erythrocytes . Alpha-toxin simultaneously induced hemolysis and a reduction in the levels of SM and formation of ceramide and sphingosine 1-phosphate (S1P) . N-Oleoylethanolamine, a ceramidase inhibitor, inhibited the toxin-induced hemolysis and caused ceramide to accumulate in the toxin-treated cells . Furthermore, dl-threo-dihydrosphingosine and B-5354c, isolated from a novel marine bacterium, both sphingosine kinase inhibitors, blocked the toxin-induced hemolysis and production of S1P and caused sphingosine to accumulate . These observations suggest that the toxin-induced activation of the SM metabolic system is closely related to hemolysis . S1P potentiated the toxin-induced hemolysis of saponin-permeabilized erythrocytes but had no effect on that of intact cells . Preincubation of lysated sheep erythrocytes with pertussis toxin blocked the alpha-toxin-induced formation of ceramide from SM . In addition, incubation of C . botulinum C3 exoenzyme-treated lysates of sheep erythrocytes with alpha-toxin caused an accumulation of sphingosine and inhibition of the formation of S1P . These observations suggest that the alpha-toxin-induced hemolysis of sheep erythrocytes is dependent on the activation of the SM metabolic system through GTP-binding proteins, especially the formation of S1P.

Ir J Med Sci, 2003 Jul-Sep, 172(3), 143 - 4
Botulism as a cause of respiratory failure in injecting drug users; FitzGerald S et al.; BACKGROUND: Wound botulism occurs as a consequence of inoculation of Clostridium botulinum spores into a wound . AIM: To describe such a case of wound botulism . RESULTS: A 23-year-old drug-injecting user presented with bulbar symptoms and progressive signs over a three-day period . The diagnosis of botulism was suspected and was treated with large doses of penicillin and botulinum antitoxin . The diagnosis was confirmed by the presence of serum botulinum toxin A . CONCLUSION: Physicians should be aware of the association of botulism in injecting drug users, particularly in Ireland.

Microbiol Immunol, 2003, 47(12), 1005 - 16
Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics; Goto T et al.; We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F . magna . The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage . Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid . The contigs were localized on the physical map of F . magna ATCC 29328 DNA . A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F . magna . The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F . magna, was sequenced and localized on the physical map . Homology analysis of 121 BAC-end sequences revealed that F . magna is most closely related to clostridia, particularly Clostridium tetani . This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis . The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.

Microbiol Immunol, 2003, 47(12), 951 - 8
Effects of high amylose maize starch and Clostridium butyricum on metabolism in colonic microbiota and formation of azoxymethane-induced aberrant crypt foci in the rat colon; Nakanishi S et al.; High amylose maize starch (HAS) is not digested in the small intestine and most of it reaches the large intestine . In the large intestine, HAS is fermented by intestinal bacteria, resulting in production of short-chain fatty acids (SCFA), particularly butyrate . Clostridium butyricum can utilize HAS and produce butyrate and acetate . It has been proposed that butyrate inhibits carcinogenesis in the colon . In this study, we examined the inhibitory effects of HAS and C . butyricum strain MIYAIRI588 (CBM588) on azoxymethane-induced aberrant crypt foci (ACF) formation in rats . In the group of rats administered only CBM588 spores, the concentration of butyrate in the cecum increased, but there was no decrease in the number of ACF . In the group of rats fed an HAS diet, a decrease in the number of ACF was observed, and in the group of rats administered HAS and CBM588, the number of ACF decreased significantly . In these two groups, the concentrations of acetate and propionate in intestinal contents significantly increased, but the concentration of butyrate did not change . It was found that the beta-glucuronidase activity level of colonic contents decreased significantly in the two groups of rats fed HAS . This study showed that HAS and CBM588 changed the metabolism of colonic microbiota and decreased the level of beta-glucuronidase activity, phenomena that may play a role in the inhibition of ACF formation in the rat colon.

J Mol Model (Online), 2004 Feb, 10(1), 69 - 75 Epub 2003 Dec 23.
Improvement of comparative modeling by the application of conserved motifs amongst distantly related proteins as additional restraints; Chakrabarti S et al.; Protein comparative modeling has useful applications in large-scale structural initiatives and in rational design of drug targets in medicinal chemistry . The reliability of a homology model is dependent on the sequence identity between the query and the structural homologue used as a template for modeling . Here, we present a method for the utilization and conservation of important structural features of template structures by providing additional spatial restraints in comparative modeling programs like MODELLER . We show that root mean square deviation at C(alpha) positions between the model and the corresponding experimental structure and the quality of the models can be significantly improved for distantly related systems by utilizing additional spatial restraints of the template structures . We demonstrate the influence of such approaches to homology modeling during distant relationships in understanding functional properties of protein such as ligand binding using cytochrome P450 as an example.FIGURE Example of a modeling triad . Structures of flavodoxin from Clostridium beijerinckii (PDB code, 5nul) and flavodoxin from Chondrus crispus (PDB code, 2fcr) that are equidistant in sequence identity (Seq . ID.) and structural similarity (RMSD) to the third flavodoxin member (from Desulfovibrio vulgaris, PDB code, 1akr)

J Infect Chemother, 2003 Dec, 9(4), 351 - 4
Pseudomembranous colitis caused by toxin A-negative/toxin B-positive variant strain of Clostridium difficile; Toyokawa M et al.; We report the first case of pseudomembranous colitis (PMC) due to a toxin A-negative, toxin B-positive strain (toxin A variant strain) of Clostridium difficile in Japan . The toxin A variant strain of C . difficile is capable of causing PMC and is undetectable in clinical laboratories that use only toxin A immunoassays for C . difficile testing . If C . difficile-associated diarrhea is clinically suspected and toxin A is not detected, then the possibility of a toxin A variant strain should be considered, and further diagnostic testing, such as polymerase chain reaction (PCR) analysis for the detection of C . difficile toxin genes should be performed.

Biochemistry, 2003 Dec 30, 42(51), 15284 - 91
Cellular uptake of Clostridium botulinum C2 toxin: membrane translocation of a fusion toxin requires unfolding of its dihydrofolate reductase domain; Haug G et al.; The Clostridium botulinum C2 toxin is the prototype of the family of binary actin-ADP-ribosylating toxins . C2 toxin is composed of two separated nonlinked proteins . The enzyme component C2I ADP-ribosylates actin in the cytosol of target cells . The binding/translocation component C2II mediates cell binding of the enzyme component and its translocation from acidic endosomes into the cytosol . After proteolytic activation, C2II forms heptameric pores in endosomal membranes, and most likely, C2I translocates through these pores into the cytosol . For this step, the cellular heat shock protein Hsp90 is essential . We analyzed the effect of methotrexate on the cellular uptake of a fusion toxin in which the enzyme dihydrofolate reductase (DHFR) was fused to the C-terminus of C2I . Here, we report that unfolding of C2I-DHFR is required for cellular uptake of the toxin via the C2IIa component . The C2I-DHFR fusion toxin catalyzed ADP-ribosylation of actin in vitro and was able to intoxicate cultured cells when applied together with C2IIa . Binding of the folate analogue methotrexate favors a stable three-dimensional structure of the dihydrofolate reductase domain . Pretreatment of C2I-DHFR with methotrexate prevented cleavage of C2I-DHFR by trypsin . In the presence of methotrexate, intoxication of cells with C2I-DHFR/C2II was inhibited . The presence of methotrexate diminished the translocation of the C2I-DHFR fusion toxin from endosomal compartments into the cytosol and the direct C2IIa-mediated translocation of C2I-DHFR across cell membranes . Methotrexate had no influence on the intoxication of cells with C2I/C2IIa and did not alter the C2IIa-mediated binding of C2I-DHFR to cells . The data indicate that methotrexate prevented unfolding of the C2I-DHFR fusion toxin, and thereby the translocation of methotrexate-bound C2I-DHFR from endosomes into the cytosol of target cells is inhibited.

Tohoku J Exp Med, 2003 Dec, 201(4), 239 - 49
The role of rho and rho-dependent kinase in serotonin-induced contraction observed in bovine middle cerebral artery; Nishikawa Y et al.; The current study was designed to characterize the role of Rho and Rho-dependent kinase (Rho-kinase) in isometric contractile responses induced by serotonin (5-HT) and a solution containing 40 mM K(+) (high K(+)) in ring preparations of the middle cerebral artery of bovine . Application of W-7, a Ca(2+)-calmodulin inhibitor, reversibly and equally attenuated the amplitudes of contractions produced by both 5-HT and high K(+) . Similar effects were observed with ML-7, an inhibitor of myosin light chain kinase . Surprisingly, the protein kinase C inhibitors, calphostin C and Ro-31-8220, had no effect on the 5-HT-induced contraction . Incubation of preparations with Clostridium difficile toxin A and B or with Clostridium botulinum C3 exoenzyme for 48 hours attenuated the 5-HT-induced response but not the high K(+)-induced response . Application of the Rho-kinase inhibitor, Y-27632, resulted in marked inhibition of the 5-HT-induced response but had negligible effect on the high K(+)-induced response . These results suggest that the activation of Rho and Rho-kinase may be involved in the generation of the contraction produced by 5-HT in the bovine middle cerebral artery, while protein kinase C plays, if any, an insignificant role on the contraction.

J Biol Chem, 2004 Mar 12, 279(11), 9867 - 74 Epub 2003 Dec 19.
Cohesin-dockerin interactions within and between Clostridium josui and Clostridium thermocellum: binding selectivity between cognate dockerin and cohesin domains and species specificity; Jindou S et al.; The cellulosome components are assembled into the cellulosome complex by the interaction between one of the repeated cohesin domains of a scaffolding protein and the dockerin domain of an enzyme component . We prepared five recombinant cohesin polypeptides of the Clostridium thermocellum scaffolding protein CipA, two dockerin polypeptides of C . thermocellum Xyn11A and Xyn10C, four cohesin polypeptides of Clostridium josui CipA, and two dockerin polypeptides of C . josui Aga27A and Cel8A, and qualitatively and quantitatively examined the cohesin-dockerin interactions within C . thermocellum and C . josui, respectively, and the species specificity of the cohesin-dockerin interactions between these two bacteria . Surface plasmon resonance (SPR) analysis indicated that there was a certain selectivity, with a maximal 34-fold difference in the K(D) values, in the cohesin-dockerin interactions within a combination of C . josui, although this was not detected by qualitative analysis . Affinity blotting analysis suggested that there was at least one exception to the species specificity in the cohesin-dockerin interactions, although species specificity was generally conserved among the cohesin and dockerin polypeptides from C . thermocellum and C . josui, i.e . the dockerin polypeptides of C . thermocellum Xyn11A exceptionally bound to the cohesin polypeptides from C . josui CipA . SPR analysis confirmed this exceptional binding . We discuss the relationship between the species specificity of the cohesin-dockerin binding and the conserved amino acid residues in the dockerin domains.

Neurosci Res, 2004 Jan, 48(1), 33 - 43
The small GTP-binding protein RhoA regulates serotonin-induced Na+-current response in the neurons of Aplysia; Kawasaki S et al.; Application of serotonin (5-HT) induces a slow inward current response in identified neurons of Aplysia ganglia under voltage clamp . The 5-HT-induced current response was depressed in Na+-free media, but augmented in Ca2+-free media, and unaffected by a change in external K+ . The 5-HT-induced response was markedly blocked by intracellular injection of guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS) . After the injection of guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), the responses to 5-HT gradually and significantly increased at the initial period, reached its plateau, and finally decreased . Intracellular injection of Clostridium difficile toxin B, a blocker of small G-protein Rho family members such as Rho (RhoA, RhoB and RhoC), Rac and Cdc42, markedly depressed the 5-HT-induced response . Intracellular injection of Clostridium botulinum C3 exoenzyme, a specific blocker of RhoA, RhoB, RhoC, exhibited a similar depressing effect observed with toxin B . In contrast, intracellular injection of recombinant L63RhoA, a constitutively active form of RhoA, significantly augmented the 5-HT-induced response without affecting the resting membrane . These results suggested that the 5-HT-induced Na+-current response might be facilitated by the activation of Aplysia Rho which is closely homologous to RhoA, RhoB or RhoC in mammalian neuron.

Hum Exp Toxicol, 2003 Nov, 22(11), 593 - 605
The development of tolerance to Clostridium perfringens type D epsilon-toxin in MDCK and G-402 cells; Beal DR et al.; The epithelial Madin Darby canine kidney (MDCK) cell line, Caucasian renal leiomyoblastoma (G-402) cells, human small airways epithelial (HSAE) cells, human bronchial epithelial (HBE) cells and human renal proximal tubule (HRPT) epithelial cells were examined for sensitivity to Clostridium perfringens biotype D epsilon-toxin . MDCK and G-402 cells were confirmed as being the only established cell lines that are sensitive to the toxin . HSAE, HBE and HRPT epithelial cells were only found to be sensitive to the toxin at concentrations of > 1 mg/ mL . Cultures of MDCK and G-402 cells, with increased resistance (tolerance) to the cytotoxic effects of epsilon-toxin, were developed by exposing these cultures to progressively higher concentrations of toxin . The greatest relative increase in tolerance to epsilon-toxin was developed in MDCK cells, in which the LC50 in control cultures was 2 microg/mL as determined by the MTS/PMS assay system; after selection for tolerance, this was raised to 100 microg/mL . This represents a 50-fold increase in tolerance as measured by this index . Using G-402 cells, it was possible to increase the LC50 by twofold from 290 to 590 microg/mL . Subsequent 2-D electrophoresis of membrane preparations from tolerant and control MDCK cells revealed that the expression of a discrete group of proteins found in control cells with a range of molecular weights from 32-36 kDa, all with acidic isoelectric points (IEPs), were either not expressed in epsilon-toxin tolerant cells or had undergone a shift in IEP to a more alkaline pH in tolerant cells . This suggests that epsilon-toxin lethality in MDCK cells may be mediated by membrane-located proteins . Their absence or alteration in toxin-resistant cells would, at least partly, explain the failure of most cell lines to demonstrate sensitivity to this toxin, despite being derived from tissues that are damaged by epsilon-toxin . This approach may have utility in the study of other toxin-cell interactions and could be used in the development of novel medical countermeasures by identifying cellular targets which mediate toxin lethality.

J Pediatr (Rio J), 1999 Nov-Dec, 75(6), 463 - 6
{Pseudomembranous colitis}; Ferreira CT et al.; OBJECTIVE: To alert about the pseudomembranous colitis in children, a consequence of the use of antibiotics . METHODS: This report is the result of the clinical follow-up of a patient with chronic diarrhea after the use of several antibiotics . The bibliography was obtained through Medline system . RESULTS: Case report of a girl two years and seven months old, previously healthy, with a clinical picture of chronic diarrhea with dysenteric characteristics after the use of antibiotics, associated with important weight lost and hypoproteinemia . The diagnosis of pseudomembranous was established clinically and was confirmed by colonoscopy and the presence of Clostridium difficile toxin A in the stools . CONCLUSIONS: It is necessary to consider the C . difficile infection in any child with diarrhea associated to the use of antibiotics . The antibiotics in pediatric patients should always be prescribed with caution and precise indications.

Avian Pathol, 2004 Feb, 33(1), 83 - 92
Maternal vaccination against subclinical necrotic enteritis in broilers; Lovland A et al.; The inclusion of antibacterial feed additives has until now been the major strategy for controlling Clostridium perfringens-associated necrotic enteritis in broilers . In the present study, the effect of maternal immunization against the disease was examined . Broiler breeder hens were injected intramuscularly with candidate vaccines based on C . perfringens type A and type C toxoids adjuvanted with aluminium hydroxide . Vaccination resulted in a strong serum immunoglobulin G response to C . perfringens alpha-toxin in parent hens, and specific antibodies were transferred to their progeny . Subclinical necrotic enteritis in broilers was induced under field conditions or in a disease model, and the occurrence of specific enteric and hepatic lesions was evaluated in randomly selected birds . In three experiments, estimates of odds ratio for developing such lesions were 0.23, 0.33 and 0.56 in maternally toxoid C-immunized broilers compared with non-immunized controls . In toxoid A-immunized birds, odds ratios were estimated at 0.41, 0.61 and 0.63 . From these results, immunoprophylaxis seems to be an interesting alternative for the control of necrotic enteritis in broilers.

Avian Pathol, 2004 Feb, 33(1), 19 - 24
Fatal necrotic enteritis associated with Clostridium perfringens in wild crows (Corvus macrorhynchos); Asaoka Y et al.; Sporadic outbreaks of fatal enteritis occurred among free-living wild crows ('large billed' or 'wok' crow; Corvus macrorhynchos) in an open-air park in Japan in 2002 . Eight crows were found dead during February, followed by two more in September, and five of the eight were examined histopathologically . At necropsy, all cases showed a markedly dilated small intestine, especially the jejunum and ileum, with large amounts of gas, and dark red to greenish-brown soft content . The necrotic intestinal wall was markedly thickened with multifocal haemorrhages . All cases had multifocal white foci in the liver, and four cases showed marked splenomegaly . Histologically, there was severe necrotic enteritis characterized by extensive mucosal necrosis and multifocal haemorrhages, as well as inflammatory cell infiltrations . A prominent pseudo-membrane formation was noted in the affected intestine . Severe adhesive peritonitis was also observed in three cases . Gram-positive bacilli were present in large numbers in the lumen, and in and around necrotic lesions in the affected intestine . The bacilli were positive for Clostridium perfringens enterotoxin type A by immunohistochemistry, and were also positive for C . perfringens type A using the immunofluorescence method . C . perfringens was isolated by anaerobic culture from the intestinal contents . The present enteritis was thought to be induced by proliferated C . perfringens in the intestine, and to be the cause of death.

Avian Pathol, 2004 Feb, 33(1), 3 - 7
Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials; Martel A et al.; Clostridium perfringens strains isolated in 2002 from the intestines of broiler chickens from 31 different farms located in Belgium were tested for susceptibility to 12 antibiotics used for therapy, growth promotion or prevention of coccidiosis . All strains were uniformly sensitive to the ionophore antibiotics monensin, lasalocid, salinomycin, maduramycin and narasin . All were sensitive to avilamycin, tylosin and amoxicillin, while flavomycin (bambermycin) showed low or no activity . Chlortetracycline and oxytetracycline were active at very low concentrations, but low-level acquired resistance was detected in 66% of the strains investigated . Fifty percent of these strains carried the tetP(B) resistance gene, while the tet(Q) gene was detected in only one strain . One strain with high-level resistance against tetracyclines carried the tet(M) gene . Sixty-three percent of the strains showed low-level resistance to lincomycin . The lnu(A) and lnu(B) genes were each only found in one strain . Compared with a similar investigation carried out in 1980, an increase was seen in resistance percentages with lincomycin (63% against 49%) and a slight decrease with tetracycline (66% against 74%).

FEMS Microbiol Lett, 2003 Dec 12, 229(2), 265 - 70
The insecticidal toxin makes caterpillars floppy 2 (Mcf2) shows similarity to HrmA, an avirulence protein from a plant pathogen; Waterfield NR et al.; The Photorhabdus luminescens W14 toxin encoding gene makes caterpillars floppy (mcf) was discovered due to its ability to kill caterpillars when expressed in Escherichia coli . Here we describe a homologue of mcf (renamed as mcf1), termed mcf2, discovered in the same genome . The mcf2 gene predicts another large toxin whose central domain, like Mcf1, also shows limited homology to Clostridium cytotoxin B . However, the N-terminus of Mcf2 shows significant similarity to the type-III secreted effector HrmA from the plant pathogen Pseudomonas syringae and no similarity to the N-terminus of Mcf1 . HrmA is a plant avirulence gene whose transient expression in tobacco cells results in cell death . Here we show that E . coli expressing Mcf2 can, like E . coli expressing Mcf1, kill insects . Further, expression of the c-Myc tagged N-terminus of Mcf2, the region showing similarity to HrmA, results in nuclear localisation of the fusion protein and subsequent destruction of transfected mammalian cells . The Mcf1 and Mcf2 toxins therefore belong to a family of high molecular mass toxins, differing at their N-termini, which encode different effector domains.

J Bacteriol, 2004 Jan, 186(1), 253 - 7
Production of heterologous and chimeric scaffoldins by Clostridium acetobutylicum ATCC 824; Perret S et al.; Clostridium acetobutylicum ATCC 824 converts sugars and various polysaccharides into acids and solvents . This bacterium, however, is unable to utilize cellulosic substrates, since it is able to secrete very small amounts of cellulosomes . To promote the utilization of crystalline cellulose, the strategy we chose aims at producing heterologous minicellulosomes, containing two different cellulases bound to a miniscaffoldin, in C . acetobutylicum . A first step toward this goal describes the production of miniCipC1, a truncated form of CipC from Clostridium cellulolyticum, and the hybrid scaffoldin Scaf 3, which bears an additional cohesin domain derived from CipA from Clostridium thermocellum . Both proteins were correctly matured and secreted in the medium, and their various domains were found to be functional.

J Bacteriol, 2004 Jan, 186(1), 136 - 45
Ruminococcus albus 8 mutants defective in cellulose degradation are deficient in two processive endocellulases, Cel48A and Cel9B, both of which possess a novel modular architecture; Devillard E et al.; The cellulolytic bacterium Ruminococcus albus 8 adheres tightly to cellulose, but the molecular biology underpinning this process is not well characterized . Subtractive enrichment procedures were used to isolate mutants of R . albus 8 that are defective in adhesion to cellulose . Adhesion of the mutant strains was reduced 50% compared to that observed with the wild-type strain, and cellulose solubilization was also shown to be slower in these mutant strains, suggesting that bacterial adhesion and cellulose solubilization are inextricably linked . Two-dimensional polyacrylamide gel electrophoresis showed that all three mutants studied were impaired in the production of two high-molecular-mass, cell-bound polypeptides when they were cultured with either cellobiose or cellulose . The identities of these proteins were determined by a combination of mass spectrometry methods and genome sequence data for R . albus 8 . One of the polypeptides is a family 9 glycoside hydrolase (Cel9B), and the other is a family 48 glycoside hydrolase (Cel48A) . Both Cel9B and Cel48A possess a modular architecture, Cel9B possesses features characteristic of the B(2) (or theme D) group of family 9 glycoside hydrolases, and Cel48A is structurally similar to the processive endocellulases CelF and CelS from Clostridium cellulolyticum and Clostridium thermocellum, respectively . Both Cel9B and Cel48A could be recovered by cellulose affinity procedures, but neither Cel9B nor Cel48A contains a dockerin, suggesting that these polypeptides are retained on the bacterial cell surface, and recovery by cellulose affinity procedures did not involve a clostridium-like cellulosome complex . Instead, both proteins possess a single copy of a novel X module with an unknown function at the C terminus . Such X modules are also present in several other R . albus glycoside hydrolases and are phylogentically distinct from the fibronectin III-like and X modules identified so far in other cellulolytic bacteria.

J Toxicol Clin Toxicol, 2003, 41(6), 841 - 7
Polar poisons: did Botulism doom the Franklin expedition?
Horowitz BZ.
In 1845 the Franklin expedition left London with 2 ships and 134 men on board in an attempt to find the route through the Northwest Passage . The ships were built with state-of-the-art technology for their day, but provisioned with supplies from the lowest bidder . After taking on fresh provisions in the Whalefish Islands, off the coast of Greenland, the entire crew was never heard from again . Graves found on remote Beechey Island indicate that three able-bodied seamen died during the first winter . A note written on a ship's log, later found in a cairn, indicate that the expedition's leader, Sir John Franklin, died during the second winter entrapped on the ice, by which time 24 men had also perished . The remaining crew failed in their attempt to walk out of the Arctic by an overland route . In 1981 Owen Beattie, from the University of Alberta, exhumed the remains of the sailors from the three graves on Beechey Island . Elevated lead levels were found in all three sailors . While lead poisoning has been a leading theory of the cause of the crew's deaths, blamed on the crudely tinned provisions the ships carried with them from England, chronic lead exposure may only have weakened the crew, not necessarily killed them . One of three exhumed sailors also had in his intestine the spores of an unspecified Clostridium species . The theory put forth by this article is that Botulism, type E, which is endemic in the Arctic, may have been responsible for their deaths.

J Infect Dis, 2003 Dec 15, 188(12), 1922 - 7 Epub 2003 Dec 09.
Prevention of fatal Clostridium difficile-associated disease during continuous administration of clindamycin in hamsters; Merrigan MM et al.; Clostridium difficile-associated disease (CDAD) due to toxigenic strains is prevented in hamsters by colonization by nontoxigenic C . difficile after administration of clindamycin (Cm) . To prevent CDAD during treatment with antibiotics, we gave a Cm-resistant nontoxigenic C . difficile strain, M13 (minimal inhibitory concentration {MIC}, >256 microg/mL), and a Cm-susceptible strain, M3 (MIC, 0.5 microg/mL), to hamsters receiving Cm daily for days 1-5 . Either M13 or M3 was given orogastrically (1 x 10(6) spores/day) to each hamster in 3 groups of 5 each, on either day 3, days 3-5, or days 3-7 . M13 colonized at a higher rate and faster than did M3 (P<.001) . When hamsters were challenged by toxigenic strain B1 on days 5, 7, or 9, M13 prevented CDAD in 100% of the hamsters . M3 protected no hamsters challenged by B1 on day 5, 20% on day 7, and 100% on day 9 . M13 contains the erm(B) resistance gene but not the mobilizable element Tn5398 . The benefits of use of Cm-resistant nontoxigenic C . difficile to prevent CDAD must be balanced against the risk that resistance might be transferred to other enteric bacteria.

Int J Food Microbiol, 2004 Jan 1, 90(1), 83 - 91
The late blowing in cheese: a new molecular approach based on PCR and DGGE to study the microbial ecology of the alteration process; Cocolin L et al.; A molecular biology method based on polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) was developed to detect Clostridium spp . in cheese samples suspected of late blowing . Strains of Clostridium spp . and different Lactic Acid Bacteria species, obtained from international collections, were used to determine the experimental conditions for the PCR amplification and DGGE differentiation . DNA extracted directly from cheeses with late blowing symptoms was subjected to PCR and DGGE analysis and traditional agar plating was performed for samples pasteurized and enriched overnight . Moreover, volatile fatty acids were determined for comparison purposes . The PCR-DGGE results were in agreement with the plating performed, and only samples presenting DGGE bands migrating at the same position as Clostridium spp . bands, showed the presence of Clostridium colonies on Reinforced Clostridial Medium plates . Butyric acid contents were high (>100 mg/kg) in the cases of positive DGGE results, underlining the suitability of the protocol for the study of cheese spoilage . The sensitivity of the method is estimated to be 10(4) CFU/g.

J Food Prot, 2003 Dec, 66(12), 2332 - 5
Microbiology of charcoal-broiled European river lampreys (Lampetra fluviatilis) stored at 3 and 22 degrees C; Merivirta LO et al.; The microbiological quality of 30 production lots of charcoal-broiled river lampreys was studied at three lamprey processing plants (plants A, B, and C) . Samples were taken directly after charcoal broiling and stored at 22 and 3 degrees C . Lampreys were examined on the day of manufacture, and those kept at 22 degrees C were examined every second day for 6 days . Samples kept at 3 degrees C were examined every fourth day for up to 24 days . On the production day, the mean aerobic plate counts (APCs) for broiled lampreys from plants A, B, and C were 2.29 log CFU/g, 1.88 log CFU/g, and undetectable (1.67 log CFU/g), respectively . At 22 degrees C, the mean APCs for samples from plants A, B, and C increased markedly within 4 days, and after 6 days the counts for samples from these plants were 8.56, 5.04, and 6.23 log CFU/g, respectively . Chilling and storage at 3 degrees C remarkably improved the shelf life of the product . The levels of bacteria in charcoal-broiled river lampreys from plant A were higher than those in lampreys from plants B and C . No significant increases in APCs were observed during storage at 3 degrees C for 24 days; mean APCs did not exceed 2.80 log CFU/g for samples from any plant . Staphylococcus aureus was found in two samples . No lactic acid bacteria, thermotolerant coliforms, enterococci, Clostridium perfringens, or Listeria monocytogenes was detected . Microbiological data from this study will be used for the development of a hazard analysis for the determination of critical control points.

Anal Chem, 2003 Dec 15, 75(24), 6886 - 93
Bacillus spore identification via proteolytic peptide mapping with a miniaturized MALDI TOF mass spectrometer; English RD et al.; An approach is tested here as a rapid screening method for Bacillus spore species employing bacterial peptide analysis with a miniaturized MALDI TOF mass spectrometer . A limited set of tryptic peptides was generated in situ following selective solubilization of the small, acid-soluble protein family (SASP) from spore samples on the MALDI sample holder . To facilitate species identification, a compact database was created comprising masses of the tryptic cleavage products generated in silico from all Bacillus and Clostridium SASPs whose sequences are available in public databases . Experimental measurements were matched against the custom-made database, and a published statistical model was then used to evaluate the probability of false identifications.

Curr Microbiol, 2003 Nov, 47(5), 425 - 30
Metabolic changes in Clostridium absonum ATCC 27555 accompanying induction of epimerization of a primary bile acid; Warchol M et al.; Some parameters of fermentation have been determined for Clostridium absonum in a chemostat by using a chemically defined medium with glucose as the sole source of carbon and energy . Steady-state continuous cultures were achieved at two dilution rates (D) . Trends of the carbon flow were determined by comparison of ratios between the specific rates of formation of the three products of metabolism (lactate, acetate, butyrate) . Chenodeoxycholate induced the 7alpha- and 7beta-hydroxysteroid dehydrogenases of C . absonum . In the presence of this inducer, the growth yield and the carbon recovery decreased, the carbon flow distribution was altered favoring acetate production, and a deficit in the reoxydation of nucleotidic cofactors was observed . In the presence of chenodeoxycholate, C . absonum would favor the production of energy at the expense of the reoxidation of nucleotidic cofactors so as to ensure its growth, and the epimerization of chenodeoxycholate to ursodeoxycholate.

Biochem Pharmacol, 2004 Jan 1, 67(1), 129 - 34
Adenosine A3 receptor-mediated regulation of p38 and extracellular-regulated kinase ERK1/2 via phosphatidylinositol-3'-kinase; Hammarberg C et al.; The adenosine A(3) receptor generally couples to the G(i) class of heterotrimeric G proteins, thereby decreasing cAMP levels and also mediating signaling via release of betagamma subunits . Here we describe the central role of phosphatidylinositol-3'-kinase (PI3K) for adenosine A(3) receptor-induced intracellular signaling to the stress-activated protein kinase p38 and the extracellular signal-regulated protein kinases ERK1/2 . We used Chinese hamster ovary cells expressing the human adenosine A(3) receptor, phospho-specific antibodies and different pharmacological tools to dissect the signaling pathways involving PI3K . The adenosine receptor agonist 5'N-ethylcarboxamidoadenosine induced a time- and dose-dependent increase in p38 and ERK1/2 phosphorylation, two signaling pathways that appeared also to be activated in the immortalized microglia cell line N13, which expressed endogenous adenosine A(3) receptors . The 5'N-ethylcarboxamidoadenosine-induced effects on p38 and ERK1/2 in CHO cells were blocked by pertussis toxin pretreatment and were sensitive to pharmacological inhibition of PI3K . In addition, inhibition of Rac/Cdc42, small GTPases of the Rho family, by clostridium toxin B, diminished p38 phosphorylation but did not affect ERK1/2 . Furthermore, we identified the serine 727 site of signal transducer and activator of transcription STAT3 as a probable downstream target of ERK1/2, and thereby provide evidence that adenosine A(3) receptor mediated ERK1/2 activation has functional consequences.

J Vet Diagn Invest, 2003 Nov, 15(6), 523 - 6
Determination of the median toxic dose of type C botulinum toxin in lactating dairy cows; Moeller RB Jr et al.; Because of the difficulty in identifying botulinum toxin in cattle, it is hypothesized that cattle are sensitive to levels of toxin below the detection limits of current diagnostic techniques (the mouse protection bioassay and the immunostick enzyme-linked immunosorbent assay {ELISA} for type C botulinum toxin) . Using an up-down method for toxicologic testing, the median toxic dose (MTD50) for cattle was determined . Four lactating Holstein cows were dosed at 0.125 or 0.25 ng/kg with Clostridium botulinum type C toxin and failed to develop clinical signs of botulism during the 7-day observation period . Three cows given 0.50 ng/kg of toxin developed clinical signs of botulism . From these results, the MTD50 was calculated at 0.388 ng/kg (3.88 mouse lethal doses/kg) using the trim-logit method . These results suggest that cattle are 12.88 times more sensitive to type C botulinum toxin than a mouse on a per kilogram weight basis . The mouse protection bioassay and the immunostick ELISA for type C botulinum toxin failed to identify the presence of the toxin in the serum, blood, and milk samples taken from all 7 animals.

J Vet Med Sci, 2003 Nov, 65(11), 1249 - 51
Necrotic hepatitis due to Clostridium perfringens infection in newly hatched broiler chicks; Sasaki J et al.; Multiple necrotic hepatitis lesions of 5 newly hatched broiler chicks in three flocks derived from two hatcheries were examined pathologically . The livers were brittle, and multiple yellowish or green foci were scattered on the surface and cut surface . The main histological finding was well demarcated multi-focal necrosis in the liver . Many Gram-positive large bacilli that reacted positively with polyclonal anti-Clostridium perfringens serum were observed in necrotic areas.

J Vet Med Sci, 2003 Nov, 65(11), 1245 - 7
Clostridium perfringens type A myonecrosis in a horse in Korea; Choi YK et al.; Acute hemorrhagic myonecrosis accompanied by severe inter- and intrafascicular edema and hemorrhage of the right gluteal area was diagnosed in a 13-year-old male thoroughbred horse . Once the muscular and fascicular changes were subsided, the horse then developed acute respiratory problem . Histologically, the lung had diffuse severe hemorrhage with mild neutrophilic infiltration . The cause of death was acute respiratory failure that is believed to occur secondary to toxaemic event . Alpha and beta2 toxin secreting Clostiridum perfringens type A was isolated from the muscle and lung . The diagnosis was based on the light microscopic examination, bacterial toxinotyping and toxin genotyping from the muscular and pulmonary lesion . Also, susceptibility of the isolates to antimicrobial agents was determined.

Proc Natl Acad Sci U S A, 2003 Dec 23, 100(26), 15422 - 7 Epub 2003 Dec 08.
Trans-editing of mischarged tRNAs; Ahel I et al.; Aminoacyl-tRNA synthetases (aaRSs) are multidomain proteins that specifically attach amino acids to their cognate tRNAs . Their most conserved, and presumably evolutionarily oldest, domains are the catalytic cores, which activate amino acids and transfer them to the 3' ends of tRNAs . Additional domains appended to or inserted in the body of aaRSs increase efficiency and specificity of the aminoacylation process, either by providing additional tRNA contacts, or by hydrolyzing noncognate amino acid products (cis-editing) . Here, we report specific tRNA-dependent trans-editing by aaRS-like proteins that reciprocate the editing domains of aaRSs, but not the remainder of the corresponding enzyme . A freestanding homologue of the prolyl-tRNA synthetase-editing domain, the PrdX protein from Clostridium sticklandii, efficiently and specifically hydrolyzes Ala-tRNAPro . Similarly, autonomous alanyl-tRNA synthetase-editing domain homologues (AlaX proteins) from Methanosarcina barkeri and Sulfolobus solfataricus hydrolyze Ser-tRNAAla and Gly-tRNAAla substrates . The discovery of autonomous editing proteins efficient in hydrolyzing misacylated products provides a direct link between ancestral aaRSs consisting solely of the catalytic core and extant enzymes to which functionally independent modules are appended.

Microbiology, 2003 Dec, 149(Pt 12), 3361 - 70
Structural analysis by X-ray crystallography and calorimetry of a haemagglutinin component (HA1) of the progenitor toxin from Clostridium botulinum; Inoue K et al.; Botulism food poisoning is caused primarily by ingestion of the Clostridium botulinum neurotoxin (BoNT) . The 1300 amino acid BoNT forms a progenitor toxin (PTX) that, when associated with a number of other proteins, increases its oral toxicity by protecting it from the low pH of the stomach and from intestinal proteases . One of these associated proteins, HA1, has also been suggested to be involved with internalization of the toxin into the bloodstream by binding to oligosaccharides lining the intestine . Here is reported the crystal structure of HA1 from type C Clostridium botulinum at a resolution of 1.7 Angstrom . The protein consists of two beta-trefoil domains and bears structural similarities to the lectin B-chain from the deadly plant toxin ricin . Based on structural comparison to the ricin B-chain lactose-binding sites, residues of type A HA1 were selected and mutated . The D263A and N285A mutants lost the ability to bind carbohydrates containing galactose moieties, implicating these residues in carbohydrate binding.

J Biol Chem, 2004 Feb 20, 279(8), 7208 - 12 Epub 2003 Dec 01.
A Rho exchange factor mediates fMet-Leu-Phe-induced NF-kappaB activation in human peripheral blood monocytes; Chen LY et al.; We reported previously that fMLP stimulates NF-kappaB activation, and this function of fMLP requires small GTPase RhoA in human peripheral blood monocytes (Huang, S., Chen, L.-Y., Zuraw, B . L., Ye, R . D., and Pan, Z . K . (2001) J . Biol . Chem . 276, 40977-40981) . Here we present evidence that RhoA associates specifically with the guanine nucleotide exchange factor Lbc in human peripheral blood monocytes stimulated with fMLP and that Lbc specifically catalyzes the guanine nucleotide exchange activity of RhoA in human peripheral blood monocytes . Cotransfection of the monocytic THP1 cells with lbc with a kappaB promoter reporter plasmid results in a marked increase in NF-kappaB-mediated reporter gene expression . Finally, Lbc-enhanced NF-kappaB activation is inhibited by a RhoA inhibitor, C3 transferase from Clostridium botulinum . A dominant-negative form of RhoA (T19N) also inhibited Lbc-enhanced reporter gene expression in a kappaB-dependent manner . These results indicate that guanine nucleotide exchange factor Lbc is a novel signal transducer for RhoA-mediated NF-kappaB activation in human peripheral blood monocytes stimulated with bacterial products.

Appl Environ Microbiol, 2003 Dec, 69(12), 6969 - 78
Cloning, expression, and cell surface localization of Paenibacillus sp . strain W-61 xylanase 5, a multidomain xylanase; Ito Y et al.; We have shown that a xylan-degrading bacterium, W-61, excretes multiple xylanases, including xylanase 5 with a molecular mass of 140 kDa . Here, we emend the previously used classification of the bacterium (i.e., Aeromonas caviae W-61) to Paenibacillus sp . strain W-61 on the basis of the nucleotide sequence of the 16S rRNA gene, and we clone and express the xyn5 gene encoding xylanase 5 (Xyn5) in Escherichia coli and study the subcellular localization of Xyn5 . xyn5 encodes 1,326 amino acid residues, including a 27-amino-acid signal sequence . Sequence analysis indicated that Xyn5 comprises two family 22 carbohydrate-binding modules (CBM), a family 10 catalytic domain of glycosyl hydrolases, a family 9 CBM, a domain similar to the lysine-rich region of Clostridium thermocellum SdbA, and three S-layer-homologous (SLH) domains . Recombinant Xyn5 bound to a crystalline cellulose, Avicel PH-101, while an N-terminal 90-kDa fragment of Xyn5, which lacks the C-terminal half of the family 9 CBM, did not bind to Avicel PH-101 . Xyn5 was cell bound, and the cell-bound protein was digested by exogenous trypsin to produce immunoreactive and xylanolytic fragments with molecular masses of 80 and 60 kDa . Xyn5 was exclusively distributed in the cell envelope fraction consisting of a peptidoglycan-containing layer and an associated S layer . Thus, Paenibacillus sp . strain W-61 Xyn5 is a cell surface-anchored modular xylanase possessing a functional cellulose-binding module and SLH domains . Possible cooperative action of multiple xylanases produced by strain W-61 is discussed on the basis of the modular structure of Xyn5.

FEMS Microbiol Lett, 2003 Dec 5, 229(1), 103 - 10
Gene transfer into Clostridium difficile CD630 and characterisation of its methylase genes; Herbert M et al.; Ignorance of pathogenesis in Clostridium difficile may be attributable to a lack of effective genetic tools . We have now shown that oriT-based shuttle vectors may be conjugated from Escherichia coli donors to the C . difficile strain CD630, at frequencies of around 10(-6) transconjugants per donor cell . Transfer is unaffected by either sequences present on the vector or its methylation status . Whilst the genome of this strain carries five methylase genes, there is no in silico or experimental evidence for cognate restriction enzymes . It would seem that the identified methylases do not participate in restriction-modification, and must, therefore, fulfil another role . A similar situation most likely applies to other clostridia.

Proc Natl Acad Sci U S A, 2003 Dec 9, 100(25), 15083 - 8 Epub 2003 Dec 01.
Overcoming the hypoxic barrier to radiation therapy with anaerobic bacteria; Bettegowda C et al.; The low level of oxygenation within tumors is a major cause of radiation treatment failures . We theorized that anaerobic bacteria that can selectively destroy the hypoxic regions of tumors would enhance the effects of radiation . To test this hypothesis, we used spores of Clostridium novyi-NT to treat transplanted tumors in mice . The bacteria were found to markedly improve the efficacy of radiotherapy in several of the mouse models tested . Enhancement was noted with external beam radiation derived from a Cs-137 source, systemic radioimmunotherapy with an I-131-conjugated monoclonal antibody, and a previously undescribed form of experimental brachytherapy using plaques loaded with I-125 seeds . C . novyi-NT spores added little toxicity to the radiotherapeutic regimens, and the combination resulted in long-term remissions in a significant fraction of animals.

MMWR Morb Mortal Wkly Rep, 2003 Dec 5, 52(48), 1176 - 9
Clostridial endophthalmitis after cornea transplantation--Florida, 2003; Centers for Disease Control and Prevention (CDC); Endophthalmitis is a severe condition caused by inflammation of the ocular cavity that often is associated with infection of the internal structures of the eye . The source of infection can include bacteria disseminated through the bloodstream and contamination of the cornea at the time of ocular surgery or trauma . Complications include rapid, reversible vision loss that can progress quickly to panophthalmitis, requiring surgical removal of the eye . Clostridium perfringens, an anaerobic gram-positive bacillus found in soil and bowel flora, is an infrequent cause of endophthalmitis . Although the majority of cases are caused by penetrating injury with soil-contaminated foreign bodies, C . perfringens endophthalmitis has been reported in patients after cataract surgery . This report describes two cases of C . perfringens endophthalmitis that occurred within 24 hours after transplant of contaminated corneas . These cases demonstrate the potential for transmission of Clostridium infection from donor to recipient . Clinicians should be aware of potential infection risks associated with transplantation of corneal tissues and report any infections to the appropriate eye bank.

Eur J Biochem, 2003 Dec, 270(24), 4962 - 72
A 49 kDa microtubule cross-linking protein from Artemia franciscana is a coenzyme A-transferase; Oulton MM et al.; Embryos and larvae of the brine shrimp, Artemia franciscana, were shown previously to possess a protein, now termed p49, which cross-links microtubules in vitro . Molecular characteristics of p49 were described, but the protein's identity and its role in the cell were not determined . Degenerate oligonucleotide primers designed on the basis of peptide sequence obtained by Edman degradation during this study were used to generate p49 cDNAs by RT-PCR and these were cloned and sequenced . Comparison with archived sequences revealed that the deduced amino acid sequence of p49 resembled the Drosophila gene product CG7920, as well as related proteins encoded in the genomes of Anopheles and Caenorhabditis . Similar proteins exist in several bacteria but no evident homologues were found in vertebrates and plants, and only very distant homologues resided in yeast . When evolutionary relationships were compared, p49 and the homologues from Drosophila, Anopheles and Caenorhabditis formed a distinct subcluster within phylogenetic trees . Additionally, the predicted secondary structures of p49, 4-hydroxybutyrate CoA-transferase from Clostridium aminobutyricum and glutaconate CoA-transferase from Acidaminococcus fermentans were similar and the enzymes may possess related catalytic mechanisms . The purified Artemia protein exhibited 4-hydroxybutyrate CoA-transferase activity, thereby establishing p49 as the first crustacean CoA-transferase to be characterized . Probing of Western blots with an antibody against p49 revealed a cross-reactive protein in Drosophila that associated with microtubules, but to a lesser extent than did p49 from Artemia.

Eur Arch Otorhinolaryngol, 2004 Sep, 261(8), 456 - 8 Epub 2003 Dec 03.
Thyroid abscess--an acute emergency; Kale SU et al.; An abscess in a long-standing goitre is usually life threatening . A patient with a large goitre was lost to follow-up for about 10 years and presented with a large thyroid abscess causing acute respiratory obstruction . The abscess caused erosion of the tracheal wall and perforation of the esophagus . Clostridium septicum was the causative organism, which is almost always associated with cancer of the colon . This led to the detection of colonic malignancy in this patient on further investigations . We describe the complications of a thyroid abscess and discuss the pathophysiology and treatment.

Dtsch Med Wochenschr, 2003 Nov 28, 128(48), 2531 - 3
{Fungemia after oral treatment with Saccharomyces boulardii in a patient with multiple comorbidities}; Lestin F et al.; HISTORY AND CLINICAL FINDINGS: A 48-year-old diabetic with multiple co-morbidities presented with generalized micro- and macroangiopathy including peripheral artery disease stage IV with necroses in several digits of both feet . He was admitted to the department of surgery for the insertion of femoropopliteal bypasses . INVESTIGATIONS: Infectious parameters were elevated (CRP 66.1 mg/l, sedimentation rate 90/96), accompanied by anemia (Hb 7.1 mmol/l), leukocytosis (14.8 Gpt/l) and thrombocytosis (514 Gpt/l) . Body temperature was normal (36.8 degrees C) . With insulin treatment the patient became nearly normoglycemic (HbA1c 6.8 %) . TREATMENT AND FOLLOW UP: After receiving different broad-spectrum antibiotics over seven weeks the patient developed Clostridium difficile toxin-positive diarrhea that resolved after administration of oral metronidazole and Saccharomyces boulardii (Perenterol ((R))) . Three days after bypass insertion, both legs had to be amputated due to infection and beginning sepsis . The condition of the patient improved . However, eight days after bypass-insertion the patient developed a toxic megacolon and sepsis . Blood cultures yielded the growth of Saccharomyces cerevisae . Despite of intensive care treatment the patient died five days later from to multi-organ failure . CONCLUSION: S . boulardii (synonym: S . cerevisiae) is considered an non-pathogenic probiotic yeast, and live yeast cells are used for supportive therapy of diarrhea . The present case and a review of the literature demonstrate that fungemia and sepsis are rare complications of the administration of S . boulardii in immunocompromised patients . For this reason the therapeutic usage of probiotics should be carefully considered regarding its risk-benefit potential.

J Pediatr (Rio J), 2000 Sep-Oct, 76(5), 391 - 4
{Neonatal tetanus}; Guardiola A et al.; OBJECTIVE: Neonatal tetanus is an infection with high mortality, constituting a problem in underdeveloped countries, where there is faulty prenatal attendance . In spite of this and of the decreasing incidence of the disease in the state of Rio Grande do Sul, we report a case of neonatal tetanus identified in 1997 . METHOD: We interviewed the infantacute;s mother and verified her vaccination history . We conducted a clinical examination of the newborn, anaerobic culture of umbilical stump and review of medical records . We also reviewed the literature and verified the epidemic profile of neonatal tetanus in Rio Grande do Sul.RESULTS: Healthy mother, with complete outline of tetanus vaccination 4 years ago, prenatal uncomplicated and hospital delivery . Family of good socioeconomic level, differing from the other cases notified in the State, in that most did not have prenatal care nor hospital delivery . The patient is a boy . In the 24th day of life he presented fever, cyanosis, lower and upper limbs muscle hypertonia and opisthotonos episodes . An anaerobic culture of the umbilical stump grew Clostridium tetani . He was treated with penicillin G, gentamicin, sedation and tetanus immunoglobulin . He left hospital in good conditions.CONCLUSIONS: Faulty prenatal care in underdeveloped countries is the largest risk factor for high incidence of this disease in these areas . However, in pregnant women with good socioeconomic level and prenatal care, as in the case described here, this diagnosis should not be excluded if there are suggestive signs of it.

J Med Assoc Thai, 2003 Oct, 86(10), 970 - 5
Detection of Clostridium difficile toxin A and B genes from stool samples of Thai diarrheal patients by polymerase chain reaction technique; Wongwanich S et al.; The prevalence of Clostridium difficile isolated from stools of Thai adult patients with suspected antibiotic-associated diarrhea (AAD) was 18.64 per cent . The recovery rate of toxin genes (tcdA and tcdB) by polymerase chain reaction (PCR) from stool samples yielded almost the same compared to the recovery rate of the toxin detection by enzyme immunoassay (EIA), which were 44.9 per cent and 46.7 per cent, respectively . Correlation of toxin gene detection by PCR and toxin detection by EIA was 90.6 per cent . All but one stool sample, the tcdA gene was detected together with the tcdB gene . Both genes were always detected together from tox gene-positive strains . Although, there were some discrepancy results for certain samples, the direct PCR-based-detection of C . difficile tox genes in stool samples seems to be the appropriate method for the diagnosis of C . difficile diarrhea . The PCR assay should be a recommended technique to be used routinely in laboratories . Further optimization of the technique to increase the sensitivity of the PCR assays is still needed . However, a quantitative isolation of the organism from stools of suspected antibiotic-associated diarrhea (AAD) or antibiotic-associated colitis (AAC) patients may give some evidence for clinicians in hospitals who cannot perform PCR-based or EIA-based techniques, since 48.6 per cent of the isolates were demonstrated as toxigenic strains.

Equine Vet J, 2003 Nov, 35(7), 638 - 41
Evaluation of in vitro properties of di-tri-octahedral smectite on clostridial toxins and growth; Weese JS et al.; REASONS FOR PERFORMING STUDY: Clostridial colitis and endotoxaemia of intestinal origin are significant causes of morbidity and mortality in horses . Intestinal adsorbents are available for treatment of these conditions; however, little information exists supporting their use . OBJECTIVES: To evaluate the ability of di-tri-octahedral smectite to bind to Clostridium difficile toxins A and B, C . perfringens enterotoxin and endotoxin, inhibit clostridial growth and the actions of metronidazole in vitro . METHODS: Clostridium difficile toxins, C . perfringens enterotoxin and endotoxin were mixed with serial dilutions of di-tri-octahedral smectite, then tested for the presence of clostridial toxins or endotoxin using commercial tests . Serial dilutions of smectite were tested for the ability to inhibit growth of C . perfringens in culture broth, and to interfere with the effect of metronidazole on growth of C . perfringens in culture broth . RESULTS: Clostridium difficile toxins A and B, and C . perfringens enterotoxin were completely bound at dilutions of 1:2 to 1:16 . Partial binding of C . difficile toxins occurred at dilutions up to 1:256 while partial binding of C . perfringens enterotoxin occurred up to a dilution of 1:128 . Greater than 99% binding of endotoxin occurred with dilutions 1:2 to 1:32 . No inhibition of growth of C . difficile or C . perfringens was present at any dilution, and there was no effect on the action of metronidazole . CONCLUSIONS: Di-tri-octahedral smectite possesses the ability to bind C . difficile toxins A and B, C . perfringens enterotoxin and endotoxin in vivo while having no effect on bacterial growth or the action of metronidazole . POTENTIAL RELEVANCE: In vivo studies are required to determine whether di-tri-octahedral smectite might be a useful adjunctive treatment of clostridial colitis and endotoxaemia in horses.

Vet Ophthalmol, 2003 Dec, 6(4), 279 - 84
Normal ocular features, conjunctival microflora and intraocular pressure in the Canadian beaver (Castor canadensis); Cullen CL; OBJECTIVE: The aim of the study was to assess the ocular features, normal conjunctival bacterial and fungal flora, and intraocular pressure (IOP) in the Canadian beaver (Castor canadensis) . SAMPLE POPULATION: Sixteen, apparently healthy beavers with no evidence of ocular disease, and live-trapped in regions throughout Prince Edward Island . PROCEDURES: The beavers were sedated with intramuscular ketamine (12-15 mg/kg) . Two culture specimens were obtained from the ventral conjunctival sac of both eyes of 10/16 beavers for aerobic and anaerobic bacterial and fungal identifications . The anterior ocular structures of all beavers were evaluated using a transilluminator and slit lamp biomicroscope . Palpebral fissure length (11/16 beavers), and horizontal and vertical corneal diameters (10/16 beavers) were measured . IOPs were measured in both eyes of 11/16 beavers using applanation tonometry . Both eyes of 3/16 beavers and one eye of 1/16 beavers were dilated using topical tropicamide prior to sedation to effect timely maximal dilation . Culture specimens and IOPs were not evaluated in these four animals . Indirect ophthalmoscopy was performed on 7/8 eyes of these four beavers . RESULTS: Conjunctival specimens from all eyes cultured positively for one or more isolates of aerobic bacteria . The most common isolate was Micrococcus spp . (five beavers; 9/20 eyes) . Other isolates included a Gram-positive coccobacilli-like organism (four beavers; 7/20 eyes), Aeromonas hydrophila (three beavers; 4/20 eyes), Staphylococcus spp . (three beavers; 4/20 eyes), Gram positive bacilli (one beaver; 2/20 eyes), Enterobacter spp . (two beavers; 2/20 eyes), Streptococcus spp . (two beavers; 2/20 eyes), aerobic diphtheroids (one beaver; 1/20 eyes), and Pseudomonas spp . (one beaver; 1/20 eyes) . Clostridium sordellii (one beaver; 1/20 eyes) and Peptostreptococcus spp . (one beaver; 1/20 eyes) were the sole anaerobic bacteria isolated . All conjunctival specimens were negative for growth of fungi . Ophthalmic examinations revealed the normal beaver eye and ocular adnexa included dorsal and ventral puncta, a vestigial third eyelid, and a circular pupil . Average palpebral fissure length was 9.36 mm (SD = 1.00) for both eyes . Mean horizontal and vertical corneal diameters of both eyes were 9.05 mm (SD = 0.64) and 8.45 mm (SD = 0.69), respectively . Mean IOP for the right and left eyes were 17.11 mmHg (SD = 6.39) and 18.79 mmHg (SD = 5.63), respectively . Indirect ophthalmoscopic examinations revealed normal anangiotic retinas . CONCLUSIONS: Gram-positive aerobes were most commonly cultured from the conjunctival sac of normal beavers, with Micrococcus spp . predominating . The overall mean IOP in ketamine-sedated beavers was 17.95 mmHg . The beaver, an amphibious rodent, has an anangiotic retina.

Environ Microbiol, 2003 Nov, 5(11), 1212 - 20
Phylogenetic analysis of the microbial populations in the wild herbivore gastrointestinal tract: insights into an unexplored niche; Nelson KE et al.; At present, there is little information on the phylogenetic diversity of microbial species that inhabit the gastrointestinal tracts of wildlife . To increase understanding in this area, we initiated a characterization of the bacterial diversity in the digestive tracts of three wild African ruminant species namely eland (Taurotragus oryx), Thompson's gazelle (Gazella rufifrons) and Grant's gazelle (Gazella granti), together with a domesticated ruminant species, zebu cattle (Bos indicus), and a non-ruminant species, zebra (Equus quagga) . Bacterial diversity was analysed by PCR amplification, sequencing and phylogenetic analysis of 16S ribosomal DNA (rDNA) sequences . A total of 252 full-length 16S rDNA sequences averaging 1,500 base pairs (bp) in length, and an additional 27 partial sequences were obtained and subject to phylogenetic analysis . Using a 98% criterion for similarity, all except for one of the sequences were derived from distinct phylotypes . At least 24 distinct operational taxonomic units (OTU's) could be identified, with the majority of these sequences representing hitherto uncharacterized species and genera . The sequences were generally affiliated with four major bacterial phyla, the majority being members of the Firmicutes (low G+C Gram-positives) related to the genera Clostridium and Ruminococcus . By contrast, with earlier studies using 16S rDNA sequences to assess biodiversity in Bos taurus dairy cattle, Gram-negative bacteria in the Bacteroidales (Prevotella-Bacteroides group) were poorly represented . The lack of redundancy in the 16S rDNA dataset from the five African ungulate species, and the presence of novel sequences not previously described from the gastrointestinal tract of any animal species, highlights the level of diversity that exists in these ecosystems and raises the question as to the functional role of these species in the gastrointestinal tract.

Cell Microbiol, 2003 Dec, 5(12), 957 - 71
Activation of NF-kappaB and IL-8 by Yersinia enterocolitica invasin protein is conferred by engagement of Rac1 and MAP kinase cascades; Grassl GA et al.; Yersinia enterocolitica triggers activation of the nuclear factor (NF)-kappaB and production of the proinflammatory chemokine interleukin (IL)-8 in intestinal epithelial cells . This activation is due to adhesion of the bacteria via their outer membrane protein invasin to the host cells . Using Clostridium difficile toxins that specifically inactivate small GTPases, and transfection of inhibitory proteins of the Rho-GTPases, we demonstrate that Rac1, but not Cdc42 or Rho, is required for activation of NF-kappaB by invasin . Invasin activated the mitogen activated protein kinases (MAPK) p38 and c-Jun N-terminal protein kinase (JNK) but not extracellular signal regulated kinase (ERK) . The functional relevance of these pathways for invasin-mediated IL-8 expression was assessed by protein kinase inhibitors and dominant-negative kinase mutants . While NF-kappaB and JNK contribute to IL-8 transcription, p38 MAPK also acts through stabilization of IL-8 mRNA, as confirmed by quantitative RT-PCR and electrophoretic mobility shift assays . Transfection experiments with I-kappaB kinase (IKK)1 and IKK2 mutants indicate that the release of NF-kappaB from its cytoplasmic inhibitor I-kappaB and its translocation into the nucleus is mediated by these kinases . Our data identify Rac1 as a key intermediate in invasin-triggered IL-8 synthesis and demonstrate that maximum IL-8 induction involves several MAP kinase cascades.

Nucleic Acids Res, 2003 Dec 1, 31(23), 6748 - 57
Regulation of lysine biosynthesis and transport genes in bacteria: yet another RNA riboswitch?
Rodionov DA, Vitreschak AG, Mironov AA, Gelfand MS.
Comparative analysis of genes, operons and regulatory elements was applied to the lysine biosynthetic pathway in available bacterial genomes . We report identification of a lysine-specific RNA element, named the LYS element, in the regulatory regions of bacterial genes involved in biosynthesis and transport of lysine . Similarly to the previously described RNA regulatory elements for three vitamins (riboflavin, thiamin and cobalamin), purine and methionine regulons, this regulatory RNA structure is highly conserved on the sequence and structural levels . The LYS element includes regions of lysine-constitutive mutations previously identified in Escherichia coli and Bacillus subtilis . A possible mechanism of the lysine-specific riboswitch is similar to the previously defined mechanisms for the other metabolite-specific riboswitches and involves either transcriptional or translational attenuation in various groups of bacteria . Identification of LYS elements in Gram-negative gamma-proteobacteria, Gram-positive bacteria from the Bacillus/Clostridium group, and Thermotogales resulted in description of the previously uncharacterized lysine regulon in these bacterial species . Positional analysis of LYS elements led to identification of a number of new candidate lysine transporters, namely LysW, YvsH and LysXY . Finally, the most likely candidates for genes of lysine biosynthesis missing in Gram- positive bacteria were identified using the genome context analysis.

J Food Prot, 2003 Nov, 66(11), 2171 - 5
Jerusalem artichokes stimulate growth of broiler chickens and protect them against endotoxins and potential cecal pathogens; Kleessen B et al.; Control of intestinal pathogens during the earliest phases of broiler production may be the best strategy for the reduction of human pathogens on processed broiler carcasses . The recent ban on antibiotics in poultry feed has served to focus much attention on alternative methods of controlling the gastrointestinal microflora . A field trial was conducted to evaluate the effect of the fructan-rich Jerusalem artichoke, or topinambur (administered as 0.5% topinambur syrup in drinking water), on cultural numbers of selected cecal bacteria (total aerobes, Enterobacteriaceae, Bdellovibrio spp., and Clostridium perfringens) and levels of bacterial endotoxins as well as on body weights and relative weights of organs (the pancreas and the bursa of Fabricius) of chickens in the first 35 days of life (with weekly investigations being conducted) . One-day-old broiler chickens (Ross 308) were randomly assigned to experimental (with topinambur) and control (without topinambur) groups . They were allowed free access to a standard broiler diet without growth-promoting antibiotics . Topinambur treatment resulted in a significant increase (P < 0.01) in cecal counts of B . bacteriovorus, which parasitizes susceptible gram-negative pathogens . Topinambur led to significantly smaller numbers of total aerobes, Enterobacteriaceae, and C . perfringens as well as to reduced levels of endotoxins in the blood compared with those for control birds . Increased body weights resulting from topinambur consumption were observed on day 35 of the trial period (P < 0.05) . The relative weights of the pancreas and the bursa of Fabricius, however, were higher (P < 0.05) for topinambur-treated broilers than for control birds at the ages of 14, 21, 28, and 35 days . These results indicate that a small amount of topinambur in broilers' drinking water has a beneficial effect on growth performance, reduces bacterial endotoxin levels, and suppresses potential pathogens in broilers' ceca.

Ann Transplant, 2003, 8(2), 34 - 42
Peracetic acid-ethanol treatment of allogeneic avital bone tissue transplants--a reliable sterilization method; Pruss A et al.; OBJECTIVES: Based on the European Standard EN 1040, the validation guidelines of the German Federal Institute for Drugs and Medical Devices and CPMP guidelines we tested the antimicrobial effectiveness of a peracetic acid-ethanol sterilization procedure (PES) in allogenic avital bone transplants . STUDY DESIGN: Delipidated human bone spongiosa cubes (15 x 15 x 15 mm) served as tissue . Three enveloped viruses (human immunodeficiency virus type 2, pseudorabies virus, bovine virus diarrhoea virus) and three non-enveloped viruses (hepatitis A virus, poliovirus, porcine parvovirus) were used . The reduction of virus infectivity was measured as TCID50/ml in neutralized supernatants and bone homogenates . Staphylococcus aureus . Enterococcus faecium, Pseudomonas aeruginosa . Bacillus subtilis . Clostridium sporogenes, Mycobacterium terrae . Candida albicans, Aspergillus niger as well as spores of Bacillus subtilis were tested additionally . PES led to a reduction of virus titres by more than 4 log10 . Only HAV showed a reduction below 4 log10 (2.87) with residual infectivity . After including a delipidating step for HAV-infected cells, a reduction of over 7 log10 HAV titre was found . For viable bacteria, fungi and spores a titre reduction below the detection level (5 log10) was achieved after an incubation time of 2 hours . CONCLUSIONS: The peracetic acid-ethanol procedure proved to be a reliable method for the sterilization of human bone transplants (layer thickness < or = 15 mm) . However, additional safety measures (anamnestic informations, infectious serology, HIV-/HBV-/HCV-PCR in case of multiorgan donors) should be taken.

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

Biologicals, 2003 Dec, 31(4), 287 - 94
Validation of the 'Marburg bone bank system' for thermodisinfection of allogenic femoral head transplants using selected bacteria, fungi, and spores; Pruss A et al.; The Marburg Bone Bank System 'Lobator sd-2' is widely used to process human femoral heads removed during aseptic surgery by thermal disinfection . The inactivating capacity of the thermodisinfection system was validated in compliance with current standards using a newly developed femoral head model . The following micro-organisms, bacteria and fungi, taken from the American Type Culture Collection were investigated: Staphylococcus aureus, Staphyloccus epidermidis, Enterococcus faecium, Pseudomonas aeruginosa, Bacillus subtilis including spores, Clostridium sporogenes, Mycobacterium terrae, Candida albicans and Aspergillus niger spores . Highly enriched suspensions of these micro-organisms were applied to the centre of the femoral heads . The reduction in the number of micro-organisms was determined by counting the colony-forming units (cfu) before and after processing the spiked test device in the 'Lobator sd-2' system.Vegetative bacteria, fungi and fungal spores were completely inactivated (reduction factor >/=6 log(10)) . The numbers of B . subtilis and C . sporogenes spores, both known to be heat-resistant, were reduced by one to two orders of magnitude . These bacteria serve as a model for spore forming pathogens which are not relevant in femoral heads from living donors . By processing human femoral heads from living donors by thermal disinfection using the Marburg Bone Banking system, a high level of safety is achieved regarding clinically relevant pathogens . To further increase the safety of the thermally treated femoral heads, we recommend that the medical history and present state of the donor, as well as the necessary serological tests should be taken into account.

Arch Microbiol, 2004 Jan, 181(1), 45 - 51 Epub 2003 Nov 18.
Identification and characterization of the cytoplasmic tungstate/molybdate-binding protein (Mop) from Eubacterium acidaminophilum; Makdessi K et al.; The mop gene, encoding the molybdate-binding protein from Eubacterium acidaminophilum, was cloned using Clostridium pasteurianum mopI as a probe for heterologous hybridization . mop encodes a 69-amino-acid protein ( M(r) 7,328) with high sequence similarities to members of the molbindin protein family, which have been implicated in molybdenum storage and homeostasis . Northern blot analysis showed three mRNA transcripts (1.0, 1.6, and 2.6 kb) for mop . This result was obtained independent of the availability of tungstate in the growth medium . mop was overexpressed in Escherichia coli as a C-terminal Strep-tag fusion protein . On the basis of gel filtration, the native protein was a homohexamer of 48 kDa . The specificity of oxyanion binding was examined by protein mobility shift assay . Molybdate, tungstate, and chromate strongly changed the mobility of the protein in a native polyacrylamide gel, indicating the binding of these oxyanions to Mop . Other oxyanions, such as sulfate and phosphate, had no effect on Mop mobility . Mutational analysis revealed that the positive charge of the Arg-6, located in the conserved SARN region of Mop, was not directly involved in oxyanion binding.

Proc Natl Acad Sci U S A, 2003 Nov 25, 100(24), 13809 - 14 Epub 2003 Nov 17.
Cellulosome assembly revealed by the crystal structure of the cohesin-dockerin complex; Carvalho AL et al.; The utilization of organized supramolecular assemblies to exploit the synergistic interactions afforded by close proximity, both for enzymatic synthesis and for the degradation of recalcitrant substrates, is an emerging theme in cellular biology . Anaerobic bacteria harness a multiprotein complex, termed the "cellulosome," for efficient degradation of the plant cell wall . This megadalton catalytic machine organizes an enzymatic consortium on a multifaceted molecular scaffold whose "cohesin" domains interact with corresponding "dockerin" domains of the enzymes . Here we report the structure of the cohesin-dockerin complex from Clostridium thermocellum at 2.2-A resolution . The data show that the beta-sheet cohesin domain interacts predominantly with one of the helices of the dockerin . Whereas the structure of the cohesin remains essentially unchanged, the loop-helix-helix-loop-helix motif of the dockerin undergoes conformational change and ordering compared with its solution structure, although the classical 12-residue EF-hand coordination to two calcium ions is maintained . Significantly, internal sequence duplication within the dockerin is manifested in near-perfect internal twofold symmetry, suggesting that both "halves" of the dockerin may interact with cohesins in a similar manner, thus providing a higher level of structure to the cellulosome and possibly explaining the presence of "polycellulosomes." The structure provides an explanation for the lack of cross-species recognition between cohesin-dockerin pairs and thus provides a blueprint for the rational design, construction, and exploitation of these catalytic assemblies.

Int J Food Microbiol, 2003 Dec 31, 89(2-3), 251 - 63
Combined effects of ionizing-irradiation and different environments on Clostridium botulinum type E spores; Lim YH et al.; We examined the combined effects of gamma-radiation (24 degrees C) on spores of Clostridium botulinum-type Eklund strain suspended in different gas-saturated Na-phosphate buffer in absence or presence of protectors or sensitizers . Response surface methodology (RSM) was also used to ascertain the effects of radiation on the recovery of spores using a medium containing various levels of NaCl or Na-thioglycollate . The former (< 0.5%) decreased viable spore counts, but the latter (0.15%) did not . Irradiation inactivation of Eklund spores was most effective in air-saturated buffers compared to N2O and N2 gas . The Na2-EDTA (0.01 M) was the most efficient radioprotector of spores due to its reactivity toward hydroxy radicals, followed by t-butanol (0.1 M) in NO2 or N(2)-saturated buffers, respectively . Catalase (10.0 mg ml(-1)) and DL-cysteine (0.1 mM) sensitized the spores during irradiated N2O or N(2)-saturated buffers, and NaCl (0.01 M) only sensitized spores in N2 environment . Spores frozen at -75 degrees C for 30 days and thawed prior to use were more sensitive to radiation damage compared to freshly prepared spores . Glycerol (15%), in Na-phosphate buffer (pH 7.0, 0.06 M), protected Eklund spores and increased the number of spores from 10(6) to 10(11) colony forming unit (CFU) ml(-1), and enhanced their radiosensitivities . Seven strains of C . botulinum type E were screened for plasmids and strain BL764 had two plasmids (15.8 and 46.8 mDa), BL4028 also had two (4.4 and 13.2 mDa), BL4850 contained only one (4.9 mDa), whereas EQA, BL211, Eklund, and Beluga had none . Gamma-Radiation (10 kGy, absorbed dose) cured the 15.8-mDa plasmid in strain BL764, but its absence yielded no changes in toxigenicity.

Biochem Biophys Res Commun, 2003 Nov 28, 311(4), 1067 - 71
SDF-1alpha-induced intracellular calcium transient involves Rho GTPase signalling and is required for migration of hematopoietic progenitor cells; Henschler R et al.; Signalling through the chemokine stromal derived factor (SDF)-1alpha and its receptor CXCR4 has been recognized as a key event in the migratory response of hematopoietic stem and progenitor cells (HPC) . Small GTPases of the Rho/Rac family might be involved in SDF-1alpha signalling at several different levels . In the present study we report that two toxins from Clostridium species which inhibit the small GTPase Rho suppressed SDF-1alpha-induced generation of intracellular calcium transients in HPC . Chelation of intracellular Ca(2+) with BAPTA or depletion of intracellular Ca(2+) stores with thapsigargin demonstrated that calcium transients are essential for SDF-1alpha-induced chemotactic migration of HPC . Furthermore, transplantation of HPC pretreated with Ca(2+) flux inhibitors into mice revealed a suppression of HPC homing to the bone marrow and increased levels of cells remaining in the bloodstream or circulating to the spleen . Our data indicate that the small GTPase Rho is required for the induction of Ca(2+) transients in HPC, which in turn are necessary for the coordinated migratory response of HPC both in vitro and in vivo.

Eur J Biochem, 2003 Dec, 270(23), 4628 - 34
Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology-based modelling; Seah SY et al.; Through comparison with the high-resolution structure of Clostridium symbiosum glutamate dehydrogenase, the different substrate specificities of the homologous enzymes phenylalanine dehydrogenase and leucine dehydrogenase were attributed to two residues, glycine 124 and leucine 307, in Bacillus sphaericus phenylalanine dehydrogenase, which are replaced with alanine and valine in leucine dehydrogenases . As predicted, making these substitutions in phenylalanine dehydrogenase decreased the specific activity towards aromatic substrates and enhanced the activity towards some aliphatic amino acids in standard assays with fixed concentrations of both substrates . This study did not, however, distinguish effects on affinity from those on maximum catalytic rate . A fuller kinetic characterization of the single- and double-mutant enzymes now reveals that the extent of the shift in specificity was underestimated in the earlier study . The maximum catalytic rates for aromatic substrates are reduced for all the mutants, but, in addition, the apparent Km values are higher for the single-mutant G124A and double-mutant G124A/L307V compared with the wild-type enzyme . Conversely, specificity constants (kcat/Km) for the nonpolar aliphatic amino acids and the corresponding 2-oxoacids for the mutants are all markedly higher than for the wild type, with up to a 40-fold increase for l-norvaline and a 100-fold increase for its 2-oxoacid in the double mutant . In some cases a favourable change in Km was found to outweigh a smaller negative change in kcat . These results emphasize the risk of misjudging the outcome of protein engineering experiments through too superficial an analysis . Overall, however, the success of the predictions from molecular modelling indicates the usefulness of this strategy for engineering new specificities, even in advance of more detailed 3D structural information.

J Neurochem, 2003 Dec, 87(5), 1272 - 83
Cyclic phosphatidic acid elicits neurotrophin-like actions in embryonic hippocampal neurons; Fujiwara Y et al.; Cyclic phosphatidic acid (cPA; 1-acyl-sn-glycerol-2,3-cyclic phosphate) is an analog of the growth factor-like phospholipid mediator lysophosphatidic acid (LPA) . As brain tissue is the richest source of cPA we tested its effects on hippocampal neurons from day 16/17 embryonic rat cultured in a serum-free medium . Nanomolar concentrations of cPA elicited a neurotrophic effect and promoted neurite outgrowth that exceeded that of 50 ng/mL nerve growth factor (NGF) . Pertussis toxin, the LPA1/LPA3 receptor-selective antagonist dioctylglycerol pyrophosphate, the myristoylated inhibitory pseudosubstrate peptide of protein kinase A (PKI), Wortmannin and PD98059 abolished the neurite-promoting effect . cPA elicited a sustained activation of extracellular signal-related kinases (ERK) 1/2 and Akt . Clostridium difficile toxin B, an inhibitor of the Rho family of GTPases, reduced cPA-induced enhancement of neurite outgrowth . In B5P cells, a clonal cell line of PC12 cells overexpressing tyrosine kinase NGF receptor (TrkA), cPA elicited transphosphorylation of TrkA . cPA-elicited ERK activation was blocked by K252a and PKI . These results suggest that cPA mimics the effects of, and activates signaling pathways similar to, the neurotrophin NGF in cultured embryonic hippocampal neurons and B5P cells.

Water Sci Technol, 2003, 48(5), 111 - 7
Polish experience with sewage sludge dewatering in reed systems; Obarska-Pempkowiak H et al.; Macrophyte plants e.g . reed, cattails, bulrush, can be applied to sewage sludge utilisation . One of the first facilities of this type in the Gdansk region (Northern Poland) was established in Darzlubie in 1995 and primary sludge has been utilised in reed beds there . The objective of the undertaken research was evaluating the influence of sewage sludge storage on its chemical and biological properties . A total of 5.5 m thick layer of primary, anaerobically stabilised sludge (moisture 90-96%) was loaded to the reed bed during 6 years of operation . As a consequence of dewatering and biochemical transformation, the sludge layer decreased to 30 cm . Moisture, organic matter, total nitrogen and total phosphorus contents, as well as coli index, Clostridium perfringens index and the number of Ascaris lumbricoides ova were measured . Also the contents of heavy metals (Cu, Pb, Zn, Ni, Cr and Cd) were determined in sludge samples . The study showed that sludge storage in reed beds results in dewatering and partial stabilisation . In the reed lagoons in Swarzewo and Zambrow, dewatering of secondary sludges was investigated . The results of measurements of the quality of sludge and effluent from the reed lagoon in Zambrow are also presented.

Pharmacotherapy, 2003 Nov, 23(11), 1517 - 9
Moxifloxacin-induced Clostridium difficile-associated diarrhea; Carroll DN; A 22-year-old woman was admitted to the hospital with pneumonia, urinary tract infection, anemia, thrombocytopenia, and leukocytosis . After receiving moxifloxacin for 5 days, she experienced diarrhea with cramping and abdominal pain . She was diagnosed with Clostridium difficile-associated diarrhea (CDAD) after C . difficile toxin was identified in a stool specimen . Metronidazole was begun, and the CDAD resolved with continued moxifloxacin administration . Virtually any antibiotic can lead to development of CDAD through disruption of the normal colonic flora, allowing for overgrowth of C . difficile . Although moxifloxacin is generally well tolerated, clinicians should be aware of its potential for inducing CDAD.

Clin Microbiol Infect, 2003 Oct, 9(10), 989 - 96
A European survey of diagnostic methods and testing protocols for Clostridium difficile; Barbut F et al.; OBJECTIVE: To conduct a survey of the methods used in clinical microbiology laboratories in Europe to diagnose infection with Clostridium difficile . METHODS: A questionnaire was devised and sent to a co-ordinating member of the Study Group in each of eight European countries . This co-ordinator was in charge of forwarding the questionnaire to hospital laboratories arbitrarily selected . The number of laboratories in each country was determined on the basis of one laboratory for 10,000 beds of hospitalization . This questionnaire covered different aspects pertaining to Clostridium difficile associated to diarrhea (CDAD) diagnosis such as circumstances of request, criteria used for undertaking C . difficile investigations, methods used for the diagnosis, etc . RESULTS: A total of 212 questionnaires were completed and submitted for analysis: 87.7% of laboratories reported routinely performing C . difficile diagnostic tests . Methods used included toxin detection (93%), culture (55%), and glutamate dehydrogenase (GDH) detection (5.9%) . Among the laboratories detecting toxins, different enzyme immunoassays (EIA) and cytotoxicity assays were used in 79% and 17.3% of cases, respectively . Among the different strategies reported, 4.8% were considered suboptimal for the diagnosis of C . difficile infections, but marked discrepancies could be observed between countries . The overall incidence (median) of CDAD was estimated at 1.1 for 1,000 patient admissions . CONCLUSION: The results of this study suggest marked discrepancies between laboratories and also between countries regarding the criteria by which C . difficile is investigated for, and the methods and the strategies that are used for the diagnosis of C . difficile . These discrepancies could be explained by the lack of clear guidelines for C . difficile diagnosis in each country, and by the importance that physicians attach to C . difficile . Precise guidelines for C . difficile diagnosis would be the first step to make possible accurate comparison of the incidence and the epidemiology of CDAD from one hospital to another or from one country to another.

APMIS, 2003 Oct, 111(10), 969 - 77
Cholera toxin protects against action by Clostridium difficile toxin A . The role of antisecretory factor in intestinal secretion and inflammation in rat; Lonnroth I et al.; The protein antisecretory factor (AF) inhibits intestinal fluid secretion induced by the cholera toxin (CT) and Clostridium difficile toxin A (CDA) . The present work investigated whether CT-induced AF protects against the enterotoxin action by CDA . Rats were pretreated perorally with CT or buffer as control, whereafter CDA-induced fluid secretion and cytotoxicity was tested in vivo in ligated intestinal loops; the mucosal level of AF was estimated using the Western blot technique . Rats given repeated peroral doses of CT became tolerant to CDA, the inhibition of fluid secretion and of cytotoxicity being 79% in eight out of nine animals . The repeated CT-treatment also induced long-lasting rise of AF in the mucosal epithelium . Recombinant AF given either perorally or intravenously inhibited both fluid secretion and cytotoxicity by CDA; similar results were obtained with a truncated 16-mer AF peptide . In conclusion: peroral CT-treatment induced tolerance to CDA in rat small intestine . The tolerance was probably mediated by AF induced via action of cholera toxin on the enteric nervous and immune system.

Clin Infect Dis, 2003 Dec 1, 37(11), 1490 - 5 Epub 2003 Oct 17.
An outbreak of foodborne botulism associated with food sold at a salvage store in Texas; Kalluri P et al.; Foodborne botulism is caused by potent neurotoxins of Clostridium botulinum . We investigated a large outbreak of foodborne botulism among church supper attendees in Texas . We conducted a cohort study of attendees and investigated the salvage store that sold the implicated foods . We identified 15 cases of botulism (40%) among 38 church supper attendees . Nine patients (60%) had botulinum toxin type A detected in stool specimens . The diagnosis was delayed in 3 cases . Fifteen (63%) of 24 attendees who ate a chili dish developed botulism (relative risk, undefined; P<.001) . The chili dish was prepared with "brand X" or "brand Y" frozen chili, "brand Z" canned chili, and hot dogs . An unopened container of brand X chili yielded type A toxin . Brand X chili was purchased at a salvage store where perishable foods were inadequately refrigerated . Our investigation highlights the need to improve clinicians' awareness of botulism . More rigorous and more unannounced inspections may be necessary to detect food mishandling at salvage stores.

J Med Microbiol, 2003 Dec, 52(Pt 12), 1033 - 8
Effects of sub-MIC concentrations of antibiotics on growth of and toxin production by Clostridium difficile; Drummond LJ et al.; Effects on growth and toxin A production of sub-MIC concentrations of six different antibiotics were investigated in three strains of Clostridium difficile: reference strain NCTC 11223, a fully sequenced strain (630) and a locally endemic isolate (strain 338a) . The antibiotics chosen for investigation were the agents used to treat C . difficile-associated disease (CDAD), i.e . vancomycin and metronidazole, and four antibiotics that are commonly involved in precipitating CDAD (amoxycillin, clindamycin, cefoxitin and ceftriaxone) . Strains were cultured in sublethal concentrations of antibiotics (1/2, 1/4 and 1/8 MIC) over 104 h and growth and toxin A production were measured three times a day . Effects varied between strain and antibiotic . The most common effect on growth of the strains was to increase the initial lag period by approximately 4 h, compared with antibiotic-free controls; however, strain NCTC 11223, which has high-level clindamycin resistance (> or = 512 microg ml(-1)), showed no lag whatsoever in comparison with the controls when grown in this antibiotic . The most common effect on production of toxin A was in the onset of toxin elaboration . Normally, toxins began to appear at low levels in the early stationary phase, before accumulating to high levels by the start of decline . In the presence of sub-MIC antibiotics, this onset appeared before that of the antibiotic-free controls . This effect was seen with metronidazole, amoxycillin and clindamycin, rarely with vancomycin and never with cefoxitin . These results suggest a very complex, strain-dependent relationship between the effects of growth and toxin production.

Vet Pathol, 2003 Nov, 40(6), 710 - 3
Necrotizing enterotyphlocolitis in dogs treated with a potent antimuscarinic; Rudmann DG et al.; To assess the safety of a potent muscarinic receptor antagonist (antimuscarinic) for human clinical testing, repeat-dose oral toxicity studies were conducted in the Beagle dog . Treatment resulted in unexpected gastrointestinal system effects that were likely mediated by the exaggerated pharmacologic effects of the antimuscarinic on intestinal motility . Dogs developed profound anorexia followed by severe bloody diarrhea, and at necropsy, the intestinal tract was distended, filled with red-tinged fluid, and the mucosa was reddened . Histologically, intestinal lesions consisted of severe epithelial necrosis and mucosal atrophy localized predominantly to the ileum and large intestine . Feces from dogs with diarrhea were culture-positive for Clostridium perfringens (CP), suggesting that CP might be the etiologic agent; however, the intestinal lesions were not consistent with histologic findings reported in dogs with hemorrhagic canine enteritis.

J Microbiol Methods, 2003 Dec, 55(3), 617 - 24
Development of an integrative vector for the expression of antisense RNA in Clostridium difficile; Roberts AP et al.; A method was developed to use the conjugative transposon Tn916 as a vector for introducing recombinant DNA into Clostridium difficile . This was used to introduce antisense RNA for the adhesin encoding gene cwp66 into C . difficile 79-685 . RT-PCR demonstrated that cwp66 specific antisense RNA was produced . However, there was no statistically significant difference in the protein expression or in the adherence of recombinant C . difficile strains . This may be due to the amount of transcripts of the wild-type (sense) cwp66 outnumbering the antisense transcripts or secondary structures present within the cwp66 mRNA . Unlike in other strains of C . difficile, where Tn916 inserts into the genome at highly preferred sites, in C . difficile 79-685, it integrates into multiple sites opening up the possibility of using Tn916 as a mutagen in this strain.

Cells Tissues Organs, 2003, 175(1), 1 - 8
Human uterine epithelial RL95-2 cells reorganize their cytoplasmic architecture with respect to Rho protein and F-actin in response to trophoblast binding; Heneweer C et al.; Embryo implantation is initiated by interaction of trophoblast with uterine epithelium via the apical cell poles of both partners . Using spheroids of human trophoblastoid JAR cells and monolayers of human uterine epithelial RL95-2 cells to simulate this initial interaction, we previously demonstrated that formation of stable cell-to-cell bonds depends on actin cytoskeleton (F-actin) and small GTPases of the Rho family, most likely RhoA . In this study, we determined the apical as well as the basal distribution of these proteins by fluorescence confocal microscopy before and after binding of JAR spheroids . We focussed on changes in cytoplasmic organization with respect to apicobasal polarity of RL95-2 cells . Before binding of spheroids, significantly higher fluorescence signals of RhoA {37 +/- 6 grey scale values (gsv)} and of F-actin (41 +/- 3 gsv) were found in the basal region of RL95-2 cells as compared to the apical pole (RhoA: 24 +/- 3 gsv, F-actin: 28 +/- 2 gsv) . After binding of JAR spheroids, this apicobasal asymmetry was inverted (RhoA: 55 +/- 10 gsv apical vs . 25 +/- 3 gsv basal; F-actin: 108 +/- 17 gsv apical vs . 57 +/- 7 gsv basal) . Inactivation of Rho GTPases in RL95-2 cells by Clostridium difficile toxin A leads to a loss of their apical adhesion competence, as previously published . Here, we observed a uniform distribution of RhoA and F-actin between apical and basal region rather than an asymmetric one in toxin A-treated cells . These data suggest that activation of Rho GTPases and coordinated rearrangement of F-actin within uterine epithelial cells in response to trophoblast binding are part of a generalized structural and functional reorganization of the cytoplasm . This involves not only the immediate contact zone (apical) but also the opposite (basal) cell pole and may be a critical element of uterine epithelial reactions during transition between trophoblast adhesion and transmigration .

J Clin Microbiol, 2003 Nov, 41(11), 5227 - 32
Frequency of binary toxin genes among Clostridium difficile strains that do not produce large clostridial toxins; Geric B et al.; Pathogenic strains of Clostridium difficile commonly produce two large clostridial toxins (LCTs), A and B, virulence factors responsible for C . difficile disease . Some strains have been reported to produce an additional toxin, a binary toxin designated CDT . Binary toxin has cytotoxic effects on cells in culture, but its role in human disease is not yet defined . In this study we examined the frequency of binary toxin genes (cdtB and cdtA) among C . difficile isolates that do not produce LCTs (A(-) B(-)) from a large United States-based collection organized by restriction endonuclease analysis (REA) typing . Of 58 strains tested, 9 (15.5%) were cdtB and cdtA positive, including 4 of 46 (8.7%) non-LCT-producing REA groups, with an estimated prevalence of at least 2% of all non-LCT-producing isolates within the collection . Five of the binary toxin-positive strains belonged to toxinotype XI, which does not produce LCTs but has minor parts of the LCT coding region or pathogenicity locus (PaLoc) . We describe two new binary toxin-positive variants, one without any remnant of the LCT genes . This previously unknown variation was found in three isolates that were unrelated by REA typing . LCT-negative, binary toxin-positive strains were isolated from symptomatic and asymptomatic patients and from the hospital environment.

Appl Environ Microbiol, 2003 Nov, 69(11), 6455 - 63
A new Bacteroides conjugative transposon that carries an ermB gene; Gupta A et al.; The erythromycin resistance gene ermB has been found in a variety of gram-positive bacteria . This gene has also been found in Bacteroides species but only in six recently isolated strains; thus, the gene seems to have entered this genus only recently . One of the six Bacteroides ermB-containing isolates, WH207, could transfer ermB to Bacteroides thetaiotaomicron strain BT4001 by conjugation . WH207 was identified as a Bacteroides uniformis strain based on the sequence of its 16S rRNA gene . Results of pulsed-field gel electrophoresis experiments demonstrated that the transferring element was normally integrated into the Bacteroides chromosome . The element was estimated from pulsed-field gel data to be about 100 kb in size . Since the element appeared to be a conjugative transposon (CTn), it was designated CTnBST . CTnBST was able to mobilize coresident plasmids and the circular form of the mobilizable transposon NBU1 to Bacteroides and Escherichia coli recipients . A 13-kb segment that contained ermB was cloned and sequenced . Most of the open reading frames in this region had little similarity at the amino acid sequence level to any proteins in the sequence databases, but a 1,723-bp DNA segment that included a 950-bp segment downstream of ermB had a DNA sequence that was virtually identical to that of a segment of DNA found previously in a Clostridium perfringens strain . This finding, together with the finding that ermB is located on a CTn, supports the hypothesis that CTnBST could have entered Bacteroides from some other genus, possibly from gram-positive bacteria . Moreover, this finding supports the hypothesis that many transmissible antibiotic resistance genes in Bacteroides are carried on CTns.

Microbiology, 2003 Nov, 149(Pt 11), 3083 - 91
A novel type of DNA curvature present in a Clostridium perfringens ferredoxin gene: characterization and role in gene expression; Kaji M et al.; This study has revealed that a Clostridium perfringens ferredoxin gene (per-fdx) possesses a novel type of DNA curvature, which is formed by five phased A-tracts extending from upstream to downstream of the -35 region . The three A-tracts upstream of the promoter and the two within the promoter are located at the positions corresponding to A-tracts present in a C . perfringens phospholipase C gene (plc) and a Clostridium pasteurianum ferredoxin gene (pas-fdx), respectively . DNA fragments of the per-fdx, pas-fdx and plc genes (nucleotide positions -69 to +1 relative to the transcription initiation site) were fused to a chloramphenicol acetyltransferase reporter gene on a plasmid, pPSV, and their in vivo promoter activities were examined by assaying the chloramphenicol acetyltransferase activity of each C . perfringens transformant . Comparison of the three constructs showed that the order of promoter activity is, in descending order, per-fdx, pas-fdx and plc . Deletion of the three upstream A-tracts of the per-fdx gene drastically decreased the promoter activity, as demonstrated previously for the plc promoter . Substitution of the most downstream A-tract decreased the promoter activities of the per-fdx and pas-fdx genes . These results indicate that not only the phased A-tracts upstream of the promoter but also those within the promoter stimulate the promoter activity, and suggest that the high activity of the per-fdx promoter is due to the combined effects of these two types of A-tracts.

Int J Food Microbiol, 2003 Dec 1, 88(2-3), 215 - 22
Functionality of enterococci in dairy products; Giraffa G; Enterococci have important implications in the dairy industry . They occur as nonstarter lactic acid bacteria (NSLAB) in a variety of cheeses, especially artisan cheeses produced in southern Europe from raw or pasteurised milk, and in natural milk or whey starter cultures . They play an acknowledged role in the development of sensory characteristics during ripening of many cheeses and have been also used as components of cheese starter cultures . The positive influence of enterococci on cheese seems due to specific biochemical traits such as lipolytic activity, citrate utilisation, and production of aromatic volatile compounds . Some enterococci of dairy origin have also been reported to produce bacteriocins (enterocins) inhibitory against food spoilage or pathogenic bacteria, such as Listeria monocytogenes, Staphylococcus aureus, Vibrio cholerae, Clostridium spp., and Bacillus spp . The technological application of enterocins, shown to be produced during cheese manufacture, led to propose enterococci as adjunct starter or protective cultures in cheeses . There is evidence that enterococci, either added as adjunct starters or present as nonstarter NSLAB, could find potential application in the processing of some fermented dairy products . Literature suggest that the complex biochemical and ecological phenomena explaining the technological functionality of the enterococci in dairy products, are still to be fully understood . Clearly, the clinical research on enterococci underlines also that the safety of dairy products containing enterococci is an issue that the industry must carefully address before proceeding to their application.

Phytother Res, 2003 Nov, 17(9), 1005 - 10
Antimicrobial activity of essential oil and methanol extracts of Achillea sintenisii Hub . Mor . (Asteraceae); Sokmen A et al.; The essential oil, obtained by Clevenger distillation, and water-soluble and water-insoluble parts of the methanol extracts of Achillea sintenisii Hub . Mor . were individually assayed for their antimicrobial activities against 12 bacteria and two yeasts, Candida albicans and C . krusei . No activity was exhibited by the water-soluble subfraction, whereas both the water-insoluble subfraction of the methanol extracts and the essential oil were found to be active against some test microorganisms studied . Since the essential oil possessed stronger activity than the other extracts tested, it was further fractionated and the fractions were evaluated for their antimicrobial activity, followed by GC-MS analysis, resulting in the identification of 32 compounds which constituted 90.2% of the total oil . The GC-MS analysis of the oil and its fractions revealed that the main components of the oil, e.g . camphor and eucalyptol, possessed appreciable activity against C . albicans and Clostridium perfringens . The fi ndings presented here also suggest that the other constituents of the oil, e.g . borneol and piperitone can also be taken into account for the activity observed .

Mol Biol Cell, 2004 Feb, 15(2), 411 - 9 Epub 2003 Oct 31.
Epithelial cell polarity alters Rho-GTPase responses to Pseudomonas aeruginosa; Kazmierczak BI et al.; Pseudomonas aeruginosa is an opportunistic human pathogen that preferentially infects damaged epithelial tissues . Previous studies have failed to distinguish whether the increased susceptibility of injured epithelium results from the loss of cell polarity or increased access to the basolateral surface . We have used confluent monolayers of Madin-Darby canine kidney (MDCK) cells cultured on porous filter supports for 1-3 d as a model system to investigate whether the differentiation state of a polarized model epithelium affected the response of epithelial cells to this pathogen . Confluent incompletely polarized MDCK cell monolayers (day 1) efficiently internalized apically applied P . aeruginosa via a pathway that required actin polymerization and activation of Rho-family GTPases and was accompanied by an increase in the amount of activated RhoA . In contrast, P . aeruginosa entry into highly polarized MDCK monolayers (day 3) was 10- to 100-fold less efficient and was insensitive to inhibitors of actin polymerization or of Rho-family GTPase activation . There was no activation of RhoA; instead, Cdc42-GTP levels increased significantly . Basolateral infection of highly polarized MDCK monolayers was less efficient and insensitive to Clostridium difficile Toxin B, whereas basolateral infection of incompletely polarized MDCK monolayers was more efficient and required activation of Rho-family GTPases . Together, our findings suggest that as epithelial barrier differentiates and becomes highly polarized, it becomes resistant to P . aeruginosa infection . Nevertheless, polarized epithelial cells still sense the presence of apically infecting P . aeruginosa, but they may do so through a different group of surface proteins and/or downstream signaling pathways than do incompletely polarized cells.

Int J Environ Health Res, 2003 Dec, 13(4), 349 - 59
Wessex shopping basket survey - a structured approach to local food sampling; Willis C et al.; The shopping basket approach to food sampling, in which ready-to-eat food items from a shopping list were sampled at premises chosen at random, was adopted by 15 Environmental Health Departments in the Wessex region . A total of 2037 samples were analysed over a 30-month period . The microbiological quality varied considerably between food categories, with gravy and stock samples giving the highest proportion of satisfactory results . Sliced meats, cooked rice and sandwiches gave the poorest overall results . Whilst the majority of unsatisfactory results were due to elevated levels of indicator organisms (Aerobic Colony Count, Enterobacteriaceae and Escherichia coli), unsatisfactory or potentially hazardous levels of pathogens (Staphylococcus aureus, Bacillus cereus, Clostridium perfringens or Listeria monocytogenes) were detected in 1% of samples . Salmonella was not detected in any sample . The shopping basket survey was considered to be a useful way of structuring local sampling, and the random selection of premises was found to be a considerable advantage over previous sampling plans . In addition, the survey drew attention to a number of microbiological problems with specific food types, leading to more detailed investigations.

J Mol Microbiol Biotechnol, 2003, 6(1), 6 - 11
Analysis of the elements of catabolite repression in Clostridium acetobutylicum ATCC 824; Tangney M et al.; The PTSH gene, encoding the phosphotransferase protein HPr, from Clostridium acetobutylicum ATCC 824 was identified from the genome sequence, cloned and shown to complement a PTSH mutant of Escherichia coli . The deduced protein sequence shares significant homology with HPr proteins from other low-GC gram-positive bacteria, although the highly conserved sequence surrounding the Ser-46 phosphorylation site is not well preserved in the clostridial protein . Nevertheless, the HPr was phosphorylated in an ATP-dependent manner in cell-free extracts of C . Acetobutylicum . Furthermore, purified His-tagged HPr from Bacillus Subtilis was also a substrate for the clostridial HPr kinase/phosphorylase . This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B . Subtilis and other low-GC gram-positive bacteria . Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C . Acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism .

Bioresour Technol, 2004 Jan, 91(2), 189 - 93
Optimization of initial substrate and pH levels for germination of sporing hydrogen-producing anaerobes in cow dung compost; Fan Y et al.; Biohydrogen production by anaerobic microbes enriched from heat-shocked cow dung compost was studied by using an artificial medium containing sucrose . Initial pH and substrate levels were selected as target factors in this study . Our experimental results demonstrated that optimal substrate concentration and pH for the composts generating hydrogen gas were 4.0+/-0.5 g sucrose/l and 5.4+/-0.2, respectively . Supplementary experiments confirmed that chemical oxygen demand reduction efficiency (69%) obtained from the conditions of sucrose=4.0 g/l and pH=5.5 was significantly greater than that (37%) from sucrose=5.0 g/l and pH=5.0 . Experimental results of metabolites analysis led us to the conclusion that Clostridium sp . predominated in the anaerobic composts, suggesting that inocula used to seed the batch experiment can be obtained from a common natural source.

FEMS Microbiol Lett, 2003 Oct 24, 227(2), 175 - 81
A comparative study between an endoglucanase IV and its fused protein complex Cel5-CBM6; Bae HJ et al.; The recombinant endoglucanase IV (Cel5; encoded by egIV) of Ruminococcus albus was compared with protein Cel5-CBM6 comprised of Cel5 fused at the C-terminus with the single-cellulose binding domain II (CBM6) of Clostridium stercorarium xylanase A, in order to improve its binding ability . Previous analyses using ball-milled cellulose had suggested that a cellulose binding domain of xylanase A could enhance cellulase activity, especially with insoluble substrates . Comparison of the catalytic activities of Cel5 and Cel5-CBM6 were determined using carboxymethylcellulose, Avicel, and filter paper as substrates . This study confirmed previous findings, and provided further evidence suggesting that Cel5-CBM6 exhibits enhanced activity with insoluble cellulose compared to native Cel5 . However, its hydrolytic activity with soluble substrates such as carboxymethylcellulose was comparable to Cel5 . For both cellulases, central linkages of cellulooligosaccharides (up to six glucose residues) were found to be the preferred points of cleavage . The rates of hydrolysis with both cellulases increased with cellulooligosaccharide chain length, and at least three consecutive glycosyl residues seemed to be necessary for hydrolysis to occur . Cel5-CBM6 showed a higher affinity for cellulose substrates than did Cel5, as demonstrated by transmission electron microscopy . Taken together, these results suggest that CBM6 increases the affinity of Cel5 for insoluble substrates, and this increased binding capacity seems to result in increased catalytic activity.

Infect Control Hosp Epidemiol, 2003 Oct, 24(10), 765 - 8
Activity of three disinfectants and acidified nitrite against Clostridium difficile spores; Wullt M et al.; OBJECTIVE: To identify environmentally safe, rapidly acting agents for killing spores of Clostridium difficile in the hospital environment . DESIGN: Three classic disinfectants (2% glutaraldehyde, 1.6% peracetyl ions, and 70% isopropanol) and acidified nitrite were compared for activity against C . difficile spores . Four strains of C . difficile belonging to different serogroups were tested using a dilution-neutralization method according to preliminary European Standard prEN 14347 . For peracetyl ions and acidified nitrite, the subjective cleaning effect and the sporicidal activity was also tested in the presence of organic load . RESULTS: Peracetyl ions were highly sporicidal and yielded a minimum 4 log10 reduction of germinating spores already at short exposure times, independent of organic load conditions . Isopropanol 70% showed low or no inactivation at all exposure times, whereas glutaraldehyde and acidified nitrite each resulted in an increasing inactivation factor (IF) over time, from an IF greater than 1.4 at 5 minutes of exposure time to greater than 4.1 at 30 minutes . Soiling conditions did not influence the effect of acidified nitrite . There was no difference in the IF among the 4 strains tested for any of the investigated agents . Acidified nitrite demonstrated a good subjective cleaning effect and peracetyl ions demonstrated a satisfactory effect . CONCLUSIONS: Cidal activity was shown against C . difficile spores by glutaraldehyde, peracetyl ions, and acidified nitrite . As acidified nitrite and peracetyl ions are considered to be environmentally safe chemicals, these agents seem well suited for the disinfection of C . difficile spores in the hospital environment.

Med Pregl, 2003 Jul-Aug, 56(7-8), 381 - 3
{Pseudomembranous colitis during antibiotic therapy}; Canovic P et al.; INTRODUCTION: The use of antibiotics is commonly accompanied by diarrhea: idiopathic diarrhea with a benign process and diarrhea caused by Clostridium difficile and pseudomembranous colitis . Clostridium difficile colonizes the gastrointestinal tract and produces a toxin in cases when normal flora is suppressed by antibiotics . Pseudomembranous colitis most frequently appears after application of clindamycin, lincomycin, ampicillin, cephalosporins and other antibiotics . Diagnosis is established after rectoscopic findings of adherent pseudomembrane and pathohistological verification . The diagnosis is confirmed if there is evidence of Cl . difficile toxin in feces . CASE REPORT: We report about the clinical course of two patients with antibiotic-associated colitis . The diagnosis were made by clinical examinations, rectoscopy and pathohistologic verification of biopsy specimen of the intestinal mucosa . Neutralization test was not done due to technical reasons . Patients were treated with metronidazole . Unwanted side-effects of metronidazole therapy were not observed . DISCUSSION: Both our patients confirmed that they previously used different antibiotics . In the first case, diarrhea appeared during the antibiotic therapy, and in the second case, after finishing it . After antibiotic use, diarrhea appears in 5.30% cases, but fortunately pseudomembranous colitis is rare . However, taking into consideration that pseudomembranous colitis has a severe course and requires urgent treatment, one has to consider the possibility of pseudomembranous colitis when diarrhea appears during and after antibiotic use in order to initiate adequate therapy.

Biosci Biotechnol Biochem, 2003 Oct, 67(10), 2160 - 6
Cloning, sequencing, and expression of the gene encoding the Clostridium stercorarium alpha-galactosidase Aga36A in Escherichia coli; Suryani et al.; The alpha-galactosidase gene aga36A of Clostridium stercorarium F-9 was cloned, sequenced, and expressed in Escherichia coli . The aga36A gene consists of 2,208 nucleotides encoding a protein of 736 amino acids with a predicted molecular weight of 84,786 . Aga36A is an enzyme classified in family 36 of the glycoside hydrolases and showed sequence similarity with some enzymes of family 36 such as Geobacillus (formerly Bacillus) stearothermophilus GalA (57%) and AgaN (52%) . The enzyme purified from a recombinant E . coli is optimally active at 70 degrees C and pH 6.0 . The enzyme hydrolyzed raffinose and guar gum with specific activities of 3.0 U/mg and 0.46 U/mg for the respective substrates.

Microb Ecol, 2003 Jul, 46(1), 1 - 11
Competitive and interactions affecting a fermentative spirochete in anaerobic chemostats; Dollhopf SL et al.; Terminal restriction fragment length polymorphism and fluorescent in situ hybridization revealed that spirochete-related populations dominated two glucose-fed methanogenic bioreactor communities at dilution rates of 0.06, 0.13, and 0.17 day(-1) . At dilution rates of 0.25 and 0.50 day(-1), spirochete-related populations decreased while Clostridium-related populations increased . Isolates representing both dominant populations were obtained (Treponema R8 and Clostridium S9) and competed against each other in continuous culture . Treponema R8 out-competed Clostridium S9 at all dilution rates applied (0.17 to 1.0 day(-1)) when sufficient pantothenate was supplied in the medium . Without sufficient pantothenate, the population size of Treponema R8 was limited to 40% of the total cells . Coculture of Treponema R8 with Methanobacterium bryantii increased the cell yield of Treponema R8 and relieved the pantothenate requirement . Triculture of Treponema R8, Clostridium S9, and M . bryantii in pantothenate-deficient medium allowed Treponema R8 to outcompete Clostridium S9 in continuous culture upto a dilution rate of 0.50 day(-1) . These experiments demonstrate that cofactor and vitamin requirements can affect the competitive success of a microbial species.

Biophys J, 2003 Nov, 85(5), 2818 - 29
Prediction of reduction potential changes in rubredoxin: a molecular mechanics approach; Ergenekan CE et al.; Predicting the effects of mutation on the reduction potential of proteins is crucial in understanding how reduction potentials are modulated by the protein environment . Previously, we proposed that an alanine vs . a valine at residue 44 leads to a 50-mV difference in reduction potential found in homologous rubredoxins because of a shift in the polar backbone relative to the iron site due to the different side-chain sizes . Here, the aim is to determine the effects of mutations to glycine, isoleucine, and leucine at residue 44 on the structure and reduction potential of rubredoxin, and if the effects are proportional to side-chain size . Crystal structure analysis, molecular mechanics simulations, and experimental reduction potentials of wild-type and mutant Clostridium pasteurianum rubredoxin, along with sequence analysis of homologous rubredoxins, indicate that the backbone position relative to the redox site as well as solvent penetration near the redox site are both structural determinants of the reduction potential, although not proportionally to side-chain size . Thus, protein interactions are too complex to be predicted by simple relationships, indicating the utility of molecular mechanics methods in understanding them.

Astrobiology, 2003 Summer, 3(2), 331 - 41
Supercooled water brines within permafrost-an unknown ecological niche for microorganisms: a model for astrobiology; Gilichinsky D et al.; This study describes brine lenses (cryopegs) found in Siberian permafrost derived from ancient marine sediment layers of the Arctic Ocean . The cryopegs were formed and isolated from sediment ~100,000-120,000 years ago . They remain liquid at the in situ temperature of -10 degrees C as a result of their high salt content (170-300 g/L) . {(14)C} Glucose is taken up by the cryopeg biomass at -15 degrees C, indicating microbial metabolism at low temperatures in this habitat . Furthermore, aerobic, anaerobic heterotrophs, sulfate reducers, acetogens, and methanogens were detected by most probable number analysis . Two psychrophilic microbes were isolated from the cryopegs, a Clostridium and a Psychrobacter . The closest relatives of each were previously isolated from Antarctica . The cryopeg econiche might serve as a model for extraterrestrial life, and hence is of particular interest to astrobiology.

J Food Prot, 2003 Oct, 66(10), 1822 - 31
Bactericidal effects of konjac fluid on several food-poisoning bacteria; Kitamoto N et al.; In this study, the bactericidal effects of Japanese alkaline foods on food-poisoning bacteria were evaluated . Konjac is an alkaline food soaked in calcinated calcium (the pH of konjac fluid ranges from 11.42 to 12.53) . Konjac fluids completely inactivated Escherichia coli, enterohemorrhagic E . coli O157:H7 and E . coil O26:H9, Salmonella Enteritidis, Vibrio parahemolyticus . and Staphylococcus aureus . The initial level of 6 log CFU/ml dramatically decreased after incubation with konjac fluid, and no viable gram-negative bacterium cells could be detected within 1 to 2 days and no viable S . aureus cells could be detected within 3 to 5 days . On the other hand, treatment with konjac fluid was also effective in reducing levels of spore-forming bacteria (Bacillus subtilis, Bacillus cereus, Clostridium perfringens, and Clostridium botulinum type E and type A) . At least a 4-log reduction of spore-forming bacteria was obtained in konjac fluid within 7 to 14 days . Vegetative cells were more susceptible to konjac fluid than spores were . When the initial cell count was 6 log CFU/ml, a few surviving spores remained for 60 to 90 days, but no spores could be detected after 120 days . When the initial count of spore-forming bacteria was 3 to 4 log CFU/ml, the cells considered vegetative were completely inactivated within I to 3 days . Repeated treatment with konjac fluid caused complete inactivation of spores in less than 1 to 3 days . Our studies indicate that konjac fluid, which has a long history of use in food, will control food-poisoning bacterial contamination during the production or preservation of konjac and other foods and has a preventive effect on bacteria that can cause severe disease at uniquely low levels.

J Biol Chem, 2004 Jan 9, 279(2), 1553 - 61 Epub 2003 Oct 21.
Genes malh and pagl of Clostridium acetobutylicum ATCC 824 encode NAD+- and Mn2+-dependent phospho-alpha-glucosidase(s); Thompson J et al.; The genome of Clostridium acetobutylicum 824 contains two genes encoding NAD+, Mn2+, and dithiothreitol-dependent phospho-alpha-glucosidases that can be assigned to family 4 of the glycosylhydrolase superfamily . The two genes, designated malh (maltose 6-phosphate hydrolase) and pagl (phospho-alpha-glucosidase), respectively, reside in separate operons that also encode proteins of the phosphoenolpyruvate-dependent:sugar phosphotransferase system . C . acetobutylicum grows on a variety of alpha-linked glucosides, including maltose, methyl-alpha-d-glucoside, and the five isomers of sucrose . In the presence of the requisite cofactors, extracts of these cells readily hydrolyzed the chromogenic substrate p-nitrophenyl-alpha-d-glucopyranoside 6-phosphate, but whether hydrolysis reflected expression of enzymes encoded by the malh or pagl genes was not discernible by spectrophotometric analysis or polyacrylamide gel electrophoresis . Resolution of this question required the cloning of the malh and pagl genes, and subsequent high expression, purification, and characterization of maltose-6'-phosphate hydrolase (MalH) and phospho-alpha-glucosidase (PagL), respectively . MalH and PagL exhibit 50% residue identity, and in solution are tetramers comprising similar sized ( approximately 50 kDa) subunits . The two proteins cross-react with polyclonal rabbit antibody against phospho-alpha-glucosidase from Fusobacterium mortiferum . Purified MalH and PagL cleaved p-nitrophenyl-alpha-d-glucopyranoside 6-phosphate with comparable efficiency, but only MalH catalyzed the hydrolysis of disaccharide 6'-phosphates formed via the phosphoenolpyruvate-dependent:sugar phosphotransferase system . Importantly, analysis of the proteome of C . acetobutylicum 824 by electrospray ionization-mass spectrometry confirmed expression of MalH during growth on many alpha-glucosides tested . Site-directed changes C169S and D170N yielded full-length, but catalytically inactive MalH . Of the two putative operons, our findings suggest that only proteins encoded by the mal operon participate in the dissimilation of maltose and related O-alpha-linked glucosides by C . acetobutylicum 824.

J Mol Biol, 2003 Oct 31, 333(4), 759 - 69
Clostridium absonum alpha-toxin: new insights into clostridial phospholipase C substrate binding and specificity; Clark GC et al.; Clostridium absonum phospholipase C (Caa) is a 42.7 kDa protein, which shows 60% amino acid sequence identity with the Clostridium perfringens phospholipase C, or alpha-toxin (Cpa), and has been isolated from patients suffering from gas gangrene . We report the cloning and sequencing, purification, characterisation and crystal structure of the Caa enzyme . Caa had twice the phospholipid-hydrolysing (lecithinase) activity, 1.5 times the haemolytic activity and over seven times the activity towards phosphatidylcholine-based liposomes when compared with Cpa . However, the Caa enzyme had a lower activity than Cpa to the free (i.e . not in lipid bilayer) substrate para-nitrophenylphosphorylcholine, towards sphingomyelin-based liposomes and showed half the cytotoxicity . The lethal dose (LD(50)) of Caa in mice was approximately twice that of Cpa . The crystal structure of Caa shows that the 72-93 residue loop is in a conformation different from those of previously determined open-form alpha-toxin structures . This conformational change suggests a role for W84 in membrane binding and a possible route of entry into the active site along a hydrophobic channel created by the re-arrangement of this loop . Overall, the properties of Caa are compatible with a role as a virulence-determinant in gas gangrene caused by C.absonum.

FEMS Microbiol Lett, 2003 Oct 10, 227(1), 1 - 7
Phylogenetic reconstruction of Gram-positive organisms based on comparative sequence analysis of molecular chaperones from the ruminal microorganism Ruminococcus flavefaciens FD-1; Antonopoulos DA et al.; Primers designed on the basis of nucleotide sequences conserved in DnaK and GroEL from Gram-positive organisms were used to PCR amplify internal regions of the cognate genes from the anaerobic ruminal cellulolytic bacterium Ruminococcus flavefaciens FD-1 . Genome walking was then utilized to elucidate the remainder of the sequences in addition to upstream and downstream regions . The full sequence of the gene encoding the GroES protein (groES) was found directly upstream from groEL . The deduced amino acid sequence of the groEL gene showed the highest homology with the amino acid sequence of the Clostridium thermocellum GroEL protein (72% amino acid identity) . Similarly, translation of the groES nucleotide sequence showed highest homology to the C . thermocellum GroES protein (61% amino acid identity) . Analysis of the upstream region of this chaperonin operon revealed a CIRCE regulatory element 45 bp upstream from the putative start of the groES ORF . The deduced amino acid sequence of the putative dnaK gene showed the highest homology with the amino acid sequence of the Clostridium acetobutylicum DnaK protein (68% amino acid identity) . Phylogenetic analyses based on the translated sequences reiterate this relationship between R . flavefaciens and the Clostridia . However, when the nucleotide sequences of Gram-positive organisms are analyzed, a different topology occurs of the relationship between high- and low-G+C Gram-positive organisms to the 16S rRNA interpretation.

Arch Pathol Lab Med, 2003 Nov, 127(11), 1465 - 70
Autopsy findings in an outbreak of severe systemic illness in heroin users following injection site inflammation: an effect of Clostridium novyi exotoxin?
Finn SP, Leen E, English L, O'Briain DS.
CONTEXT: An epidemic of unexplained illness among injecting drug users characterized by injection site inflammation and severe systemic toxicity occurred in Ireland and the United Kingdom from April to August 2000 . One hundred eight persons became ill, and 43 persons died . In Dublin, 8 of 22 patients died . Six of the 8 fatal cases were epidemiologically linked to a source of heroin . Most had experienced local injection site lesions for 7 to 14 days before developing a rapidly fatal systemic illness characterized by hypotension, thirst, pulmonary edema, pericardial and pleural effusions, and leukocytosis . OBJECTIVE: To document the clinical course and autopsy findings of the fatal cases in Dublin . DESIGN: To study the clinical, autopsy, microbiologic, and toxicologic findings from the 8 fatal cases in Dublin . RESULTS: In Dublin, there were 6 men and 2 women who were fatally involved in the epidemic, with the mean age being 34 years (range, 22-51 years) . The injection site inflammations involved the buttock (n = 4), leg, iliac region, arm, and a Portacath site . At autopsy, the local lesions were ulcerated, swollen, and indurated but were inconspicuous in 2 patients . All the deceased had pulmonary edema . There were pleural effusions in 7, 2 of whom had pericardial effusions . Five had prominent left ventricular subendocardial hemorrhages . Five had splenomegaly . Microscopy showed pulmonary edema and a granulocytic reaction mainly in the spleen, marrow, and myocardium . Toxicology showed a range of narcotic drugs in the toxic or fatal range . Clostridium novyi type A, a fastidious toxin-producing anaerobe, was identified in 2 cases . CONCLUSION: The clinicopathologic findings of a local inflammatory lesion followed 7 to 14 days later by a rapidly fatal systemic illness are consistent with the effect of exotoxin produced by organisms growing in the local inflammatory site . Clostridium novyi-derived exotoxin is the likely cause of such a syndrome, although the fastidious organism was isolated from only 2 of 8 cases (from none of the 14 surviving patients and from only 13 of 60 cases in Scotland) . In the setting of an epidemic, the toxic and fatal range blood levels of narcotics are unlikely to explain these events, and no other candidate organism could be isolated . The heroin is likely to have come from Afghanistan, but local contamination at a putative distribution site in the United Kingdom is more likely than international terrorism to be the initiating factor.

J Neurosurg, 2003 Oct, 99(4), 693 - 700
Fusobacterial brain abscess: a review of five cases and an analysis of possible pathogenesis; Han XY et al.; OBJECT: The cases of five patients with fusobacterial brain abscess are presented . The authors discuss their attempt to determine the pathogenesis . METHODS: The clinical and microbiological features of five cases of fusobacterial brain abscess are reviewed . Isolates of 2031 Fusobacterium spp . and other anaerobes collected (1989-2002) at our institution were analyzed and compared for incidences and isolation sources . The findings were correlated with extensive literature on the subject . The five patients were men between 45 and 74 years of age . All experienced an insidious onset of the disease and probable hematogenous seeding of the organism(s) . One patient had a monomicrobic Fusobacterium necrophorum abscess, whereas the others had polymicrobic F . nucleatum abscesses . Despite surgery and a regimen of antibiotic medications and dexamethasone, three patients experienced a paradoxical deterioration 3 days postoperatively that necessitated reevacuation of the lesion . The evacuants observed at that time contained numerous leukocytes but no microorganisms, suggesting intensified inflammation as the likely cause of deterioration . This explanation is supported by literature that fusobacteria strongly activate neutrophils . An analysis of the 2031 anaerobes from blood, wounds, and abscesses showed the considerable virulence of Fusobacterium spp., which were able to enter and/or sustain themselves in the blood circulation . This pattern was similar to that of Clostridium spp., but different from those of Peptostreptococcus spp., Bacteroides spp., and Prevotella spp., which were less invasive but more abundant . CONCLUSIONS: Some fusobacterial brain abscesses may be associated with a paradoxical postoperative deterioration, which is probably due to intensified inflammation following treatment . The blood-borne dissemination and invasive behavior of fusobacteria likely initiate such a brain abscess, and further seeding of other synergic bacteria leads to a polymicrobic abscess.

Clin Auton Res, 2003 Oct, 13(5), 337 - 8
Botulism type B presenting as pure autonomic dysfunction; Merz B et al.; Botulism nowadays is a rare mostly food-borne disease caused by the toxin of Clostridium botulinum . On the other hand, botulinum toxin blocking cholinergic transmission has become a most powerful treatment option for many focal movement disorders and is increasingly used to treat autonomic disorders {2, 7, 8} . Although muscle weakness is the hallmark of botulism, autonomic dysfunction may be the leading clinical symptom in rare cases and should be included in the differential diagnosis of pure dysautonomia . Here, we describe a patient with autonomic dysfunction as the leading symptom of botulism type B.

J Antimicrob Chemother, 2003 Nov, 52(5), 856 - 9 Epub 2003 Oct 16.
Chloramphenicol-resistant Neisseria meningitidis containing catP isolated in Australia; Shultz TR et al.; Earlier workers have described chloramphenicol resistance in meningococci isolated from cerebrospinal fluid sampled in patients in Vietnam (11 cases) and France (one case) during 1987-1996 . Here we describe two distinct serogroup B strains isolated in Australia in 1994 and 1997, and found among approximately 1400 invasive meningococcal isolates examined in Australia over a 9 year period . Both were phenotypically chloramphenicol resistant on disc, Etest and agar inclusion MIC and acetylated chloramphenicol examination . DNA amplification and sequencing confirmed the presence of catP and the 3' end of tnpV from Tn4451, a mobilizable element from Clostridium perfringens, although other sequences were not present . Tn4451 has inserted into a gene designated TIGR locus NMB1350 in both isolates with no loss of DNA and no apparent interruption of virulence genes . This second report of chloramphenicol-resistant meningococci is in a setting with a very low volume of systemic chloramphenicol use, but where the high topical use may contribute to recombination events in vivo . Methods for screening for chloramphenicol resistance in meningococci and the in vitro parameters that define this resistance are ill defined.

J Biol Chem, 2003 Dec 26, 278(52), 53055 - 62 Epub 2003 Oct 16.
Involvement of Cdc42 signaling in apoA-I-induced cholesterol efflux; Nofer JR et al.; Cholesterol efflux, an important mechanism by which high density lipoproteins (HDL) protect against atherosclerosis, is initiated by docking of apolipoprotein A-I (apoA-I), a major HDL protein, to specific binding sites followed by activation of ATP-binding cassette transporter A1 (ABCA1) and translocation of cholesterol from intracellular compartments to the exofacial monolayer of the plasma membrane where it is accessible to HDL . In this report, we investigated potential signal transduction pathways that may link apoA-I binding to cholesterol translocation to the plasma membrane and cholesterol efflux . By using pull-down assays we found that apoA-I substantially increased the amount of activated Cdc42, Rac1, and Rho in human fibroblasts . Moreover, apoA-I induced actin polymerization, which is known to be controlled by Rho family G proteins . Inhibition of Cdc42 and Rac1 with Clostridium difficile toxin B inhibited apoA-I-induced cholesterol efflux, whereas inhibition of Rho with Clostridium botulinum C3-exoenzyme exerted opposite effects . Adenoviral expression of a Cdc42(T17N) dominant negative mutant substantially reduced apoA-I-induced cholesterol efflux, whereas dominant negative Rac1(T17N) had no effect . We further found that two downstream effectors of Cdc42/Rac1 signaling, c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (p38 MAPK), are activated by apoA-I . Pharmacological inhibition of JNK but not p38 MAPK decreased apoA-I-induced cholesterol efflux, whereas anisomycin and hydrogen peroxide, two direct JNK activators, could partially substitute for apoA-I in its ability to induce cholesterol efflux . These results for the first time demonstrate activation of Rho family G proteins and stress kinases by apoA-I and implicate the involvement of Cdc42 and JNK in the apoA-I-induced cholesterol efflux.

Avian Dis, 2003 Jul-Sep, 47(3), 759 - 63
Beneficial effect of beta-mannanase feed enzyme on performance of chicks challenged with Eimerla sp . and Clostridium perfringens; Jackson ME et al.; Two experiments were conducted to determine the effect of a beta-mannanase feed enzyme on the performance of broiler chicks subject to a necrotic enteritis disease challenge model involving oral inoculation of Eimeria sp . and Clostridium pefringens . Beta-mannanase is known to improve productive performance when added to poultry and swine diets . In both experiments, disease challenge in the absence of feed additives demonstrated significant reductions in performance as measured by weight gain, feed conversion, and the incidence of coccidial lesion scores . Significant mortality was also observed in challenged groups in Experiment 1 . The disease challenge model was therefore judged as highly effective . Additions of a commonly used antibiotic, bacitracin methylene disalicilate (BMD), and coccidiostat, salinomycin, were highly effective in partially counteracting negative effects of the disease challenge . In both experiments, addition of beta-mannanase significantly improved performance and reduced lesion scores in disease-challenged groups . The degree of improvement was somewhat less than that afforded by a combination of BMD and salinomycin in Experiment 1 but was not different from that afforded by BMD alone in Experiment 2 . We conclude that the beta-mannanase enzyme can play a role in circumstances where the use of antibiotics is not desired.

Avian Dis, 2003 Jul-Sep, 47(3), 707 - 11
Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation; Craven SE et al.; Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations . In a previous study, ribotypes of certain strains of C . perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining trays used to transport commercial chicks from the hatchery to the grow-out facility on the farm . These results suggest that C . perfringens contaminating the processed product could originate from facilities in the integrated poultry operation prior to grow out . In this study, samples were collected from the breeder farm, hatchery, previous grow-out flock, during grow out and after processing . In the first trial, C . perfringens was recovered from the breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, from processed carcasses, and from the breeder farm after processing in 4%, 30%, 4%, 0%, 2% and 16%, and 4% of the samples, respectively . In the second trial, the incidence of C . perfringens in samples collected from breeder farms, the hatchery, previous grow-out flock, grow-out flock at 3 weeks of age, grow-out flock at 5 weeks of age, and fromprocessed carcasses was 38%, 30%, 32%, 8%, 4%, and 8%, respectively . The genetic relatedness of the isolated strains as determined by ribotyping suggests that C . perfringens may be transmitted between facilities within the integrated broiler chicken operation.

J Vasc Surg, 2003 Oct, 38(4), 847 - 51
Mycotic aneurysm of the infrarenal abdominal aorta infected by Clostridium septicum: a case report of surgical management and review of the literature; Takano H et al.; We report a surgical case of mycotic aneurysm of the infrarenal abdominal aorta infected by Clostridium septicum . The patient was first treated with an in situ prosthetic graft replacement . When the infection recurred 5 weeks after the aortic surgery, the patient was successfully treated by transposition of rectus abdominis muscle flap around the graft . Only 19 cases of mycotic aneurysm or aortic dissection caused by Clostridium septicum have been reported . Ten of 12 patients who underwent vascular surgery survived, whereas all 7 patients who did not undergo surgery died . Surgical treatment should be undertaken since the surgical results seem satisfactory.

Gene, 2003 Oct 2, 315, 21 - 32
Complete DNA sequences of the botulinum neurotoxin complex of Clostridium botulinum type A-Hall (Allergan) strain; Zhang L et al.; BOTOX is manufactured with the purified native 900-kDa type A neurotoxin complex from Clostridium botulinum type A-Hall (Allergan) strain . This complex is composed of the botulinum neurotoxin (BoNT) and several toxin associated proteins known as the hemagglutinins (HAs) and the non-toxic non-hemagglutinin protein (NTNH) . We describe here the complete gene sequences of the BoNT complex of type A-Hall (Allergan) strain . Using a polymerase chain reaction-based approach, we sequenced six open reading frames (ORFs) encoding BoNT (1296 amino acids), the toxin-associated proteins: HA70, 625 aa; HA17, 147 aa; HA34, 291 aa; NTNH, 1193 aa; and the regulatory component botR/OrfX, 178 aa . Comparative alignments of the amino acid sequence of BoNT/A shows a 98-100% sequence identity among different strains of the type A, except for the Kyoto-F strain (90%), whereas the sequence identity between BoNT/A and other toxin serotypes is only 30.4-39.1% . Similar to the neurotoxin, the toxin-associated proteins and botR from type A-Hall strain also share more than 95% identity to the homologous proteins found in type A-NCTC2916 strain . Among all the toxin associated proteins, NTNHs and HA70s are the most conserved with 65-87% identity across different serotypes . On the other hand, HA34s, present only in serotypes A-D, show greater diversity than all other toxin-associated proteins; HA34/A has 90% identity to HA34/B and only approximately 35% identity to HA34/C and HA34/D . Relatively higher sequence identity ( approximately 60%) is seen in HA17 and botR of Hall A when compared to their counterparts in serotypes C or D . Of all proteins within the toxin complex, NTNH and HA70 have the highest degree of conservation across serotypes and this may underscore a critical role for these proteins in the formation of the complexes . Physiologically, different duration of action in different serotypes may be due to different modifications of toxins by neuronal enzymes, which lead to different compartmentalization of different toxins . Computer-assisted motif analysis reveals that toxins contain several potential sites for phosphorylation by casein kinase II, protein kinase C, tyrosine kinases, glycogen synthase kinase 3, cGMP dependent protein kinase (PKG) that are well conserved . The reported sequence information for type A-Hall strain will potentially facilitate elucidation of the toxin interactions with the nontoxin proteins in the complex.

J Mol Biol, 2003 Oct 24, 333(3), 527 - 40
Mechanism of C2-toxin inhibition by fluphenazine and related compounds: investigation of their binding kinetics to the C2II-channel using the current noise analysis; Bachmeyer C et al.; The binding component C2II of the binary actin ADP-ribosylating C2-toxin from Clostridium botulinum is essential for intoxication of target cells . Activation by a protease leads to channel formation and this is presumably required for the transport of the toxic C2I component into cells . The C2II-channel is cation selective and contains a binding site for fluphenazine and structurally related compounds . Ion transport through C2II and in vivo intoxication is blocked when the sites are occupied by the ligands . C2II was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels . The binding constant of chloroquine, primaquine, quinacrine, chloropromazine and fluphenazine to the C2II-channel was determined using titration experiments, which resulted in its block . The ligand-induced current noise of the C2II-channels was investigated using fast Fourier transformation . The noise of the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz . Upon addition of ligands to the aqueous phase the current through C2II decreased in a dose-dependent manner . Simultaneously, the spectral density of the current noise increased drastically and its frequency dependence was of Lorentzian type, which was caused by the on and off-reactions of the ligand-mediated channel block . The ligand-induced current noise of C2II was used for the evaluation of the binding kinetics for different ligands to the channel . The on-rate constant of ligand binding was between 10(7) and 10(9) M(-1) s(-1) and was dependent on the ionic strength of the aqueous phase . The off-rate varied between about 10 s(-1) and 3900 s(-1) and depended on the structure of the ligand . The role of structural requirements for the effective block of C2II by the different ligands is discussed.

Biochemistry, 2003 Oct 21, 42(41), 11951 - 9
Glucosylation of Ras by Clostridium sordellii lethal toxin: consequences for effector loop conformations observed by NMR spectroscopy; Geyer M et al.; The lethal toxin (LT) from Clostridium sordellii, which belongs to the family of large clostridial cytotoxins, acts as a monoglucosyltransferase for the Rho subfamily GTPase Rac and also modifies Ras . In the present study we investigated structural changes of H-Ras in its di- and triphosphate form that occur upon glucosylation of the effector domain amino acid threonine-35 by LT . (31)P NMR experiments recorded during the enzymatic glucosylation process, using UDP-glucose as a cosubstrate, show that the modification of the threonine side chain influences the chemical shifts of the phosphate groups of the bound nucleotides . In the diphosphate-bound form (Ras.GDP) glucosylation of Thr35 induces only small changes in the chemical environment of the active center . In the triphosphate form with the GTP analogue GppNHp bound (Ras.GppNHp) Ras shows at least two different conformations in the active center that exchange on a medium-range time scale (10 to 0.1 ms) . Glucosylation selectively stabilizes one distinct conformation of the effector loop (state 1) with tyrosine-32 probably apart from the nucleotide and threonine-35 not involved in magnesium ion coordination . This conformation is known to have a low affinity to effector proteins such as Raf-1, AF-6, or Byr2 and thus prevents the transduction of the activation signal in the Ras-mediated pathway . NMR correlation spectra of Ras(T35glc).GDP and denaturation experiments with urea indicate that the glucose is bound in the alpha-anomeric form to the hydroxyl group of the threonine-35 side chain . Inhibition of the glucosylation reaction by 1,5-gluconolactone suggests a stereospecific reaction mechanism with a glucosyl oxonium ion transition state for the enzymatic activity of LT.

Environ Toxicol Chem, 2003 Oct, 22(10), 2293 - 7
Mutagenicity of nitroaromatic degradation compounds; Padda RS et al.; The mutagenicity of 2,4-dinitrotoluene (24DNT), and 2,6-dinitrotoluene (26DNT), and their related transformation products such as hydroxylamine and amine derivatives, which are formed by Clostridium acetobutylicum, were tested in crude cell extracts using Salmonella typhimurium TA100 . A previous publication already reported the mutagenic activities of 2,4,6-trinitrotoluene (TNT) and its related hydroxylamine derivatives in this test system . A time course of the mutagenicity during the anaerobic transformation of TNT, 24DNT, and 26DNT was also investigated under the same conditions to compare with the results from the pure compounds . The monohydroxylamino intermediates 2-hydroxylamino-4-nitrotoluene (2HA4NT), 4-hydroxylamino-2-nitrotoluene (4HA2NT) and 2-hydroxylamino-6-nitrotoluene (2HA6NT) formed during anaerobic transformation of dinitrotoluenes were proven to be mutagenic in the Ames test using Salmonella typhimurium TA100 . This study reports that 4HA2NT is the most stable derivative, whereas 2HA4NT and 2HA6NT are less stable and these intermediates are mutagenic in the Ames test . Both 24DNT and 26DNT and their final metabolites 2,4-diaminotoluene (24DAT) and 2,6-aminotoluene (26DAT) appeared nonmutagenic . In a time-course study of TNT degradation, the temporal sample containing 85% of 2,4-dihydroxylamino-6-nitrotoluene (24HA6NT) is most mutagenic . These observations suggest that the bioremediation approach for treatment of 24DNT and 26DNT should be carried past the hydroxylamino intermediate.

Protein Expr Purif, 2003 Oct, 31(2), 276 - 85
Molecular cloning, overexpression in Escherichia coli, and purification of 6x his-tagged C-terminal domain of Clostridium difficile toxins A and B; Letourneur O et al.; Genomic DNA from ribotype-01 and -17 Clostridium difficile strains was used for amplification of the sequences encoding the carboxy-terminal domain of toxins A (TcdA) and B (TcdB) . The deduced C-terminal TcdB ribotype-01 and -17 domains share 99.5% amino acid sequence identity while TcdA ribotype-17 comprises a 607 amino acid deletion compared to TcdA-01 . When compared to previously sequenced C . difficile toxins, 99.3% amino acid identity was found between TcdA-01 and TcdA from strain VPI10643 and 98.8% identity between TcdA-17 and TcdA from strain F-1470 . The obtained sequences were fused in 3' to a sequence encoding a hexahistidine tag and cloned into an Escherichia coli expression vector . The recombinant proteins were expressed in E . coli and purified using single-step metal-chelate chromatography . The recombinant carboxy-terminal domain of TcdA-01 was purified from the soluble E . coli lysate fraction whereas TcdA-17 and TcdB-17 carboxy-terminal domains were purified from inclusion bodies . At least 40 mg of each protein was purified per liter of bacterial culture . The recombinant toxin domains were detected specifically by Western blot and ELISA with antibodies against native C . difficile toxins . This study demonstrated that the carboxy-terminal domains of TcdA and TcdB can be produced using an E . coli expression system and easily purified . These recombinant, stable, and non-toxic proteins provide a convenient source for use in the diagnosis of C . difficile infections, instead of native toxins, as controls and calibrators in immunoassay kits and to obtain specific monoclonal antibodies.

Biochem Biophys Res Commun, 2003 Oct 24, 310(3), 685 - 90
A Rho GTPase controls the rate of protein synthesis in the sea urchin egg; Manzo S et al.; Fertilization of the sea urchin egg triggers a Ca(2+)-dependent cortical granule exocytosis and cytoskeletal reorganization, both of which are accompanied by an accelerated protein synthesis . The signaling mechanisms leading to these events are not completely understood . The possible role of Rho GTPases in sea urchin egg activation was studied using the Clostridium botulinum C3 exotoxin, which specifically ADP-ribosylates Rho proteins and inactivates them . We observed that incubation of eggs with C3 resulted in in situ ADP-ribosylation of Rho . Following fertilization, C3-treated eggs were capable of performing cortical granule exocytosis but not the first cytokinesis . C3 caused in both unfertilized eggs and early embryos alterations in the state of actin polymerization and inhibition of the spindle formation . Moreover, C3 diminished markedly the rate of protein synthesis . These findings suggested that Rho is involved in regulating the acceleration of protein synthesis that accompanies the egg activation by sperm.

Biotechnol Adv, 1984, 2(2), 161 - 81
The enzymatic hydrolysis and fermentation of pretreated wood substrates; Saddler JN et al.; Aspenwood chips were pretreated by steam explosion . The various wood fractions obtained were assayed for their ability to act as substrates for growth and cellulase production of different Trichoderma and Clostridium thermocellum species . Steam exploded aspenwood was as efficiently utilized as solka floc and correspondingly high cellulase activities were detected in the various culture filtrates . When T . harzianum E58 was grown on increasing concentrations of solka floc, highest cellulase and xylanase activities were detected at 1% substrate concentrations while high substrate concentrations (10-20%) inhibited growth and enzyme production . When the cellulosic substrates were supplemented with increasing amounts of glucose, cellulase and xylanase production were inhibited when the glucose concentration exceeded 0.1% . Highest xylanase activities were detected after growth of T . reesei C30 and T . harianum E58 on xylan and solka floc respectively . All of the steam exploded fractions were at least partially hydrolyzed by the T . harzianum E58 cellulase system . The extent of the pretreatment also influenced the ability of Zymomonas mobilis and Saccharomyces cerevisiae to ferment the liberated sugars to ethanol . About 85% of the theoretical yield of ethanol from cellulose could be obtained from the combined hydrolysis and fermentation of pretreated aspenwood.

Biotechnol Adv, 1985, 3(1), 13 - 28
Acetone-butanol fermentation and its variants; Haggstrom L; Recent intensive research on the acetone-butanol-ethanol and the isopropanol-butanol-ethanol fermentation has increased the basic understanding of these processes substantially . Metabolic investigations on Clostridium acetobutylicum, and Clostridium beijerinkii show that enzyme activities necessary for solvent production are induced only in solvent-producing cells . Although produced, or added, acetic and butyric acid have significant effects on the metabolic activities, the transition from acid to solvent production cannot as yet be fully explained . Based on studies in continuous cultures, the kinetics of product formation can be described . Knowledge of the mechanism of butanol toxicity is accumulating but no dramatic increase in butanol tolerance has so far been obtained . Successful results, approaching the limitations determined by biological and technological possibilities, have been obtained in batch and continuous cultures, and in continuous processes based on immobilized cells . Continuous processes are superior to batch cultures in respect of their productivity.

J Vet Med B Infect Dis Vet Public Health, 2003 Sep, 50(7), 360 - 2
Genotyping of Clostridium perfringens isolated from domestic and exotic ruminants and swine; Sipos W et al.; Clostridium perfringens types A, B, C, D and E are known to cause severe enteritis/enterotoxaemia and diseases (especially caused by type A) belonging to the gas oedema complex in many species . Samples from the small intestine as well as faeces of domestic and exotic animals suffering from enterotoxaemic signs or having died within days after first occurance of toxaemia were submitted for typing C . perfringens toxovars by multiplex PCR . The following species have been investigated: domestic sheep (Ovis ammon; n = 10), domestic goat (Capra aegagrus hircus; n = 26), Japanese serow (Capricornis sumatraensis; n = 4), lechwe waterbuck (Hydrotragus leche; n = 1), blackbuck (Antilope cervicapra; n = 1), European reindeer (Rangifer tarandus tarandus; n = 4), domestic swine (Sus scrofa; n = 52), and collared peccary (Tayassu albirostris; n = 1) . Interestingly, the predominant C . perfringens toxovar in domestic sheep was type A . This toxovar could also be diagnosed in all reindeer, in three Japanese serows, one lechwe waterbuck and most pigs (n = 47), the majority of those being at suckling age . Type D was the most prevalent toxovar (n = 18) in domestic goats, but also types A and E could be identified as pathogens in this species . Type C could only be found in domestic swine (n = 5) and in one case of clostridiosis in a Japanese serow . Two cases of enterotoxaemia in goats, one case in reindeer, and a single case in blackbuck and collared peccary were caused by C . perfringens type E . Genotyping of C . perfringens is recommended before starting vaccination programmes as it could be shown, that the importance of specific toxovars has been underestimated in specific species and/or age groups.

Endocr Res, 2003 Aug, 29(3), 363 - 76
Novel roles for the rho subfamily of GTP-binding proteins in succinate-induced insulin secretion from betaTC3 cells: further evidence in support of the succinate mechanism of insulin release; Kowluru A et al.; We have previously demonstrated regulatory roles for Rho subfamily of G-proteins in glucose- and calcium-induced insulin secretion . Herein, we examined regulation by these proteins of insulin secretion from betaTC3 cells elicited by mitochondrial fuels, such as the succinic acid methyl ester (SAME) . Preincubation of these cells with Clostridium difficile toxin-B (200 ng/mL), which monoglucosylates and inactivates Cdc42 and Rac1, markedly decreased (> 70%) SAME-induced insulin secretion . Furthermore, exposure of betaTC3 cells to GGTI-2147 (20 microM), a selective inhibitor of the requisite prenylation of Rac1 and Cdc42, significantly reduced (> 80%) SAME-induced insulin release, suggesting that post-translational prenylation of these proteins is necessary for SAME-induced insulin release . Western blot analysis indicated localization of Cdc42, Rac1, and Ras in the beta cell mitochondrial fraction . Confocal microscopy revealed a modest, but inconsistent, increase in the association of either Rac1 or Cdc42 with Mitotracker, a mitochondrial marker, following exposure to SAME . These data suggest that activation of preexisting intramitochondrial Rac1 and Cdc42 may be sufficient to regulate SAME-induced insulin secretion . Together, our findings support a role for G-proteins in insulin secretion at a step dependent on mitochondrial metabolism . They also identify mevalonate-derived, isoprenoid modified Rho G-proteins as specific signaling molecules in recently proposed succinate mechanism of insulin release.

Electromyogr Clin Neurophysiol, 2003 Sep, 43(6), 373 - 6
Food-borne botulism cases in Van region in eastern Turkey: importance of electromyography in the diagnosis; Anlar O et al.; OBJECTIVES: Food-borne botulism is an acute form of poisoning that results from ingestion of a toxin produced by Clostridium botulinum . Botulism toxin causes its major effect by blocking neuromuscular transmission in autonomic and motor nerve terminals . METHODS: In this study, we present the features of eleven cases of food-borne botulism admitted to our hospital in 2001 . All of the cases were caused by home-prepared foods; green beans . In these cases, the main symptoms and signs were generalized muscular weakness, dry mouth, dysphagia, disponea and diplopia . Electrophysiological studies were performed on four patients . RESULTS: Motor conduction studies showed that compound muscle action potentials were decreased with normal latencies and conduction velocities . The needle electromyography showed signs of denervation potentials like fibrillation and positive waves in four patients . Repetitive nerve stimulation with high frequency (20 Hz) induced an increment close to 100% in the amplitudes in 2 of 4 patients . CONCLUSION: Although toxin could not be detected in the patients, the electromyographic findings supported our diagnosis . We concluded that electromyography has an important role in diagnosis of botulism, especially in the condition that serologic tests are negative or cannot be performed.

Plast Surg Nurs, 2003 Summer, 23(2), 64 - 9
BOTOX: a review; Mendez-Eastman SK; BOTOX cosmetic is proving to be an affordable alternative for thousands of people looking to enhance their appearance without the cost, risks and downtime associated with surgical procedures . Botulinum toxin is a neuromuscular blocking agent produced by Clostridium Botulinum, an anaerobic bacterium . When first discovered BOTOX was pinpointed as the cause of severe paralysis acquired through the ingestion of contaminated food . Paralysis related to BOTOX is caused by chemodenervation, which is a result of blockage of the presynaptic release of acetylcholine at the neuromuscular junction . The corrugator (medial eyebrows), crow's feet, forehead, platysmal neckbands, and the jowl are frequently targeted areas for BOTOX injections . There are no current guidelines for physical limitations following the administration of BOTOX . Side effects associated with BOTOX infections appear to be either local, due to the paralysis of adjacent muscles, or mild flu-like symptoms lasting a few days in a small number of patients.

Appl Environ Microbiol, 2003 Oct, 69(10), 6321 - 6
Formate-dependent growth and homoacetogenic fermentation by a bacterium from human feces: description of Bryantella formatexigens gen . nov., sp . nov; Wolin MJ et al.; Formate stimulates growth of a new bacterium from human feces . With high formate, it ferments glucose to acetate via the Wood-Ljungdahl pathway . The original isolate fermented vegetable cellulose and carboxymethylcellulose, but it lost this ability after storage at -76 degrees C . 16S rRNA gene sequencing identifies it as a distinct line within the Clostridium coccoides supra-generic rRNA grouping . We propose naming it Bryantella formatexigens gen . nov., sp . nov.

Appl Environ Microbiol, 2003 Oct, 69(10), 6073 - 81
Molecular diversity of sulfate-reducing bacteria from two different continental margin habitats; Liu X et al.; This study examined the natural diversity and distributions of sulfate-reducing bacteria along a natural carbon gradient extending down the shelf-slope transition zone of the eastern Pacific continental margin . Dissimilatory (bi)sulfite reductase gene sequences (dsrAB) were PCR amplified and cloned from five different sampling sites, each at a discrete depth, from two different margin systems, one off the Pacific coast of Mexico and another off the coast of Washington State . A total of 1,762 clones were recovered and evaluated by restriction fragment length polymorphism (RFLP) analysis . The majority of the gene sequences recovered showed site and depth restricted distributions; however, a limited number of gene sequences were widely distributed within and between the margin systems . Cluster analysis identified 175 unique RFLP patterns, and nucleotide sequences were determined for corresponding clones . Several different continental margin DsrA sequences clustered with those from formally characterized taxa belonging to the delta subdivision of the class Proteobacteria (Desulfobulbus propionicus, Desulfosarcina variabilis) and the Bacillus-Clostridium (Desulfotomaculum putei) divisions, although the majority of the recovered sequences were phylogenetically divergent relative to all of the other DsrA sequences available for comparison . This study revealed extensive new genetic diversity among sulfate-reducing bacteria in continental margin sedimentary habitats, which appears to be tightly coupled to slope depth, specifically carbon bioavailability.

Appl Environ Microbiol, 2003 Oct, 69(10), 5957 - 67
Cloning, characterization, and functional expression of the Klebsiella oxytoca xylodextrin utilization operon (xynTB) in Escherichia coli; Qian Y et al.; Escherichia coli is being developed as a biocatalyst for bulk chemical production from inexpensive carbohydrates derived from lignocellulose . Potential substrates include the soluble xylodextrins (xyloside, xylooligosaccharide) and xylobiose that are produced by treatments designed to expose cellulose for subsequent enzymatic hydrolysis . Adjacent genes encoding xylobiose uptake and hydrolysis were cloned from Klebsiella oxytoca M5A1 and are functionally expressed in ethanologenic E . coli . The xylosidase encoded by xynB contains the COG3507 domain characteristic of glycosyl hydrolase family 43 . The xynT gene encodes a membrane protein containing the MelB domain (COG2211) found in Na(+)/melibiose symporters and related proteins . These two genes form a bicistronic operon that appears to be regulated by xylose (XylR) and by catabolite repression in both K . oxytoca and recombinant E . coli . Homologs of this operon were found in Klebsiella pneumoniae, Lactobacillus lactis, E . coli, Clostridium acetobutylicum, and Bacillus subtilis based on sequence comparisons . Based on similarities in protein sequence, the xynTB genes in K . oxytoca appear to have originated from a gram-positive ancestor related to L . lactis . Functional expression of xynB allowed ethanologenic E . coli to metabolize xylodextrins (xylosides) containing up to six xylose residues without the addition of enzyme supplements . 4-O-methylglucuronic acid substitutions at the nonreducing termini of soluble xylodextrins blocked further degradation by the XynB xylosidase . The rate of xylodextrin utilization by recombinant E . coli was increased when a full-length xynT gene was included with xynB, consistent with xynT functioning as a symport . Hydrolysis rates were inversely related to xylodextrin chain length, with xylobiose as the preferred substrate . Xylodextrins were utilized more rapidly by recombinant E . coli than K . oxytoca M5A1 (the source of xynT and xynB) . XynB exhibited weak arabinosidase activity, 3% that of xylosidase.

Cell Microbiol, 2003 Nov, 5(11), 761 - 71
Lethal toxin from Clostridium sordellii induces apoptotic cell death by disruption of mitochondrial homeostasis in HL-60 cells; Petit P et al.; Lethal toxin (LT) from Clostridium sordellii (strain IP82) inactivates in glucosylating the small GTPases Ras, Rap, Ral and Rac . In the present study we show that LT-IP82 induces cell death via an intrinsic apoptotic pathway in the myeloid cell-line HL-60 . LT-IP82 was found to disrupt mitochondrial homeostasis as characterized by a decrease in mitochondrial transmembrane potential and cardiolipin alterations, associated with the release of cytochrome c in the cytosol . Time-course studies of caspase activation revealed that caspase-9 and caspase-3 were activated before caspase-8 . Moreover, although LT-IP82-induced cell death was abrogated by caspase-inhibitors, these inhibitors did not suppress mitochondrial alterations, indicating that caspase activation occurs downstream of mitochondria . Protection of mitochondria by Bcl-2 overexpression prevented mitochondrial changes as well as apoptosis induction . Furthermore, evidence is provided that LT-IP82-induced apoptosis is not a consequence of cortical actin disorganization, suggesting that Rac inactivation does not initiate the apoptotic process . Cell exposure to LT-IP82 leads to a co-localization of the toxin with a mitochondrial marker within 2 h . Therefore, we suggest that LT-IP82 could act at the mitochondrion level independently of its enzymatic effect on small GTPases.

Int Ophthalmol, 2001, 24(5), 269 - 77
Ocular infections due to anaerobic bacteria; Brook I; The increased recovery of anaerobic bacteria in clinical infection has led to greater appreciation of these organisms in ocular infections . In studies that employed adequate method for recovery of anaerobes they were isolated from about a third of patients with conjunctivitis, half of the time in pure culture . The predominant recovered anaerobes were Clostridium spp., gram-negative anaerobic bacilli, and Peptostreptococcus spp . Anaerobic bacteria were also recovered from patients who wore contact lenses and developed conjunctivitis . Anaerobic bacteria were also reported in cases of keratitis . The most frequently recovered anaerobes were Propionibacterium spp., Peptostreptococcus spp., Clostridium spp., Prevotella spp., and Fusobacterium spp . The most frequently recovered anaerobes from dacryocystitis were Peptostreptococcus spp., Propionibacterium spp., Prevotella spp., and Fusobacterium spp . This review describes the microbiology, diagnosis and management of ocular infections due to anaerobic bacteria in children.

J Hosp Infect, 2003 Oct, 55(2), 124 - 30
Comparison of AP-PCR typing and PCR-ribotyping for estimation of nosocomial transmission of Clostridium difficile; Wullt M et al.; We recently attempted to clarify an increased incidence of Clostridium difficile-associated diarrhoea (CDAD) in our hospital by arbitrarily primed polymerase chain reaction (AP-PCR) typing of isolates from 147 consecutive patients collected during a 12 month period (Wullt et al . J Hosp Infect 1999;43:265-273) . In the present study we compared the results based on previous AP-PCR data with those based on recent PCR ribotyping of the same isolates and re-analysis of a subset of isolates by AP-PCR typing . The pattern of PCR ribotypes was similar among inpatients and outpatients . A cluster of three closely related PCR ribotypes, related to those of the serogroup H and A8 type strains, dominated and comprised 31% of inpatient and 28% of outpatient C . difficile isolates . The apparent nosocomial transmission rate among inpatients with CDAD was only 9% by AP-PCR typing compared with 18 or 36% by PCR ribotyping depending on the definition used (proportion of patients sharing C . difficile type and ward within two or 12 months) . Corresponding rates for all CDAD patients were 5% by AP-PCR and 11 or 21% by PCR ribotyping . Thus, most CDAD patients apparently became ill due to their endogenous strain of C . difficile . Because of the low concordance between the two typing methods the proportion of patients fulfilling the criteria for nosocomial transmission by both methods was only 1% . Re-examination of isolates from patients with recurrences revealed a reproducibility problem with AP-PCR typing . We conclude, that of these two PCR-based options for typing of C . difficile PCR ribotyping offers a superior experimental robustness compared with AP-PCR typing.

Int J Food Microbiol, 2003 Nov 1, 87(3), 217 - 27
Dynamic computer simulation of Clostridium perfringens growth in cooked ground beef; Huang L; The objective of this study was to develop a computer simulation algorithm to dynamically estimate and predict the growth of Clostridium perfringens in cooked ground beef . The computational algorithm was based on the implicit form of the Gompertz model, the growth kinetics of C . perfringens in cooked ground beef, and the fourth-order Runge-Kutta numerical method . This algorithm was validated using a cocktail of three strains of C . perfringens spores grown under isothermal, square-waved, linear cooling, and exponential cooling temperature profiles . In general, the results of computer simulation matched closely with the experimental data with the absolute errors less than 0.5 log(10) CFU/g . This method may be a useful tool for the food industry, regulatory agencies, distributors, and retailers to predict the effect of temperature abuse on the microbial safety of C . perfringens and other foodborne pathogens in processed meat products.

Wiad Lek, 2003, 56(5-6), 278 - 82
{Treatment of infections associated with Clostridium difficile}; Szczesny A et al.; Cases of antibiotic-associated diarrhoea (AAD) associated with toxigenic C . difficile strains more frequently are described recently . In Polish studies diagnosis of AAD or PMC (pseudomembranous colitis) was confirmed by isolation of C . difficile strains in 52% of suspected cases . Strains of C . difficile were isolated also from samples taken from hospital environment (10.5% positive samples) . Problem of C . difficile-associated infection is more frequently observed in Poland also because of uncontrolled using of wide spectrum antibiotics . These antibiotics destroy intestinal microflora--"colonization resistance factor" . Cases of AAD or PMC were described even after treatment of patients by vancomycin or metronidazol--antibiotics of choice for treatment of C . difficile-associated diseases . Other risk factors as: age, long term hospitalization, previous surgery, colonoscopy are also taken into account, when C . difficile-associated infections are suspected . Different treatment methods of C . difficile-associated infections are reviewed here and discussed in light of recent publications.

J Bacteriol, 2003 Oct, 185(20), 6205 - 8
The SKHR motif is required for biological function of the VirR response regulator from Clostridium perfringens; McGowan S et al.; The response regulator VirR and its cognate sensor histidine kinase, VirS, are responsible for toxin gene regulation in the human pathogen Clostridium perfringens . The C-terminal domain of VirR (VirRc) contains the functional FxRxHrS motif, which is involved in DNA binding and is conserved in many regulatory proteins . VirRc was cloned, purified, and shown by in vivo and in vitro studies to comprise an independent DNA binding domain . Random and site-directed mutagenesis was used to identify further amino acids that were required for the functional integrity of the protein . Random mutagenesis identified a unique residue, Met-172, that was required for biological function . Site-directed mutagenesis of the SKHR motif (amino acids 216 to 219) revealed that these residues were also required for biological activity . Analysis of the mutated proteins indicated that they were unable to bind to the DNA target with the same efficiency as the wild-type protein.

J Bacteriol, 2003 Oct, 185(20), 6067 - 75
Regulation of expression of cellulosomal cellulase and hemicellulase genes in Clostridium cellulovorans; Han SO et al.; The regulation of expression of the genes encoding the cellulases and hemicellulases of Clostridium cellulovorans was studied at the mRNA level with cells grown under various culture conditions . A basic pattern of gene expression and of relative expression levels was obtained from cells grown in media containing poly-, di- or monomeric sugars . The cellulase (cbpA and engE) and hemicellulase (xynA) genes were coordinately expressed in medium containing cellobiose or cellulose . Growth in the presence of cellulose, xylan, and pectin gave rise to abundant expression of most genes (cbpA-exgS, engH, hbpA, manA, engM, engE, xynA, and/or pelA) studied . Moderate expression of cbpA, engH, manA, engE, and xynA was observed when cellobiose or fructose was used as the carbon source . Low levels of mRNA from cbpA, manA, engE, and xynA were observed with cells grown in lactose, mannose, and locust bean gum, and very little or no expression of cbpA, engH, manA, engE, and xynA was detected in glucose-, galactose-, maltose-, and sucrose-grown cells . The cbpA-exgS and engE genes were most frequently expressed under all conditions studied, whereas expression of xynA and pelA was more specifically induced at higher levels in xylan- or pectin-containing medium, respectively . Expression of the genes (cbpA, hbpA, manA, engM, and engE) was not observed in the presence of most soluble di- or monosaccharides such as glucose . These results support the hypotheses that there is coordinate expression of some cellulases and hemicellulases, that a catabolite repression type of mechanism regulates cellulase expression in rapidly growing cells, and that the presence of hemicelluloses has an effect on cellulose utilization by the cell.

Microbiology, 2003 Oct, 149(Pt 10), 2779 - 87
Identification and characterization of a fibronectin-binding protein from Clostridium difficile; Hennequin C et al.; A 68 kDa fibronectin-binding protein (Fbp68) from Clostridium difficile displaying significant homology to several established or putative Fbps from other bacteria was identified . The one-copy gene is highly conserved in C . difficile isolates . Fbp68 was expressed in Escherichia coli in fusion with glutathione S-transferase; the fusion protein and the native Fbp68 were purified . Immunoblot analysis and cell fractionation experiments revealed that Fbp68 is present on the surface of the bacteria . Far-immuno dot-blotting demonstrated that Fbp68 was capable of fixing fibronectin . Indirect immunofluorescence and ELISA were employed to demonstrate that C . difficile could bind both soluble and immobilized fibronectin . With competitive adherence inhibition assays it was shown that antibodies raised against Fbp68 partially inhibited attachment of C . difficile to fibronectin and Vero cells . Furthermore, Vero cells could fix purified membrane-immobilized Fbp68 . Thus Fbp68 appears to be one of the several adhesins identified to date in C . difficile.

Avian Pathol, 2003 Oct, 32(5), 527 - 34
Diagnosing Clostridium perfringens-associated necrotic enteritis in broiler flocks by an immunoglobulin G anti-alpha-toxin enzyme-linked immunosorbent assay; Lovland A et al.; The tools available for monitoring necrotic enteritis caused by Clostridium perfringens in broiler chickens have been limited, particularly for identifying subclinical disease . In this study, a modified enzyme-linked immunosorbent assay was used to quantify levels of specific immunoglobulin G to C . perfringens alpha-toxin in serum from broilers . We found significantly higher antibody levels in broilers with a history of subclinical necrotic enteritis compared with a zinc-bacitracin-treated group with a low level of gut lesions . Furthermore, in 4.5-week-old commercial broiler flocks, there was an association between the occurrence of C . perfringens-associated hepatitis at slaughter and the immune response to alpha-toxin . Practical solutions for defining cut-off levels for positive serum samples at individual and flock levels are proposed, and were found to be useful on a set of samples available from flocks with different histories regarding the occurrence of C . perfringens-associated disease . This serological approach seems promising as a diagnostic tool in research and disease monitoring regarding C . perfringens-associated disease.

Biochem Biophys Res Commun, 2003 Oct 17, 310(2), 280 - 5
Distribution of potential type II restriction sites (palindromes) in prokaryotes; Fuglsang A; Restriction-modification systems are used as a defensive mechanism against inappropriate invasion of foreign DNA . The recognition sequences for the common type II restriction enzymes and their corresponding methylases are usually palindromes . In this study, we identified the most over- and underrepresented words in DNA of four bacteria: Escherichia coli, Bacillus subtilis, Clostridium perfringens, and Pseudomonas aeruginosa . Using maximum order Markov chain analysis, we found that palindromic words were most often more underrepresented than their non-palindromic counterparts . No strict rule for the intragenic palindrome content could be derived, but for three of the bacteria there was a weak correlation between codon usage bias and palindrome content . A clear drop in palindrome counts was observed in the Shine-Dalgarno region for B . subtilis and C . perfringens, but not in E . coli or P . aeruginosa . It was also shown that palindromes in eubacteria and archaebacteria seem to occur slightly more infrequently than expected on the basis of the genomic GC-content, but some exceptions to this principle exist.

Gastrointest Endosc, 2003 Oct, 58(4), 505 - 9
Do pancreatic duct stents cause or prevent pancreatic sepsis?
Kozarek R, Hovde O, Attia F, France R.
BACKGROUND: Pancreatic sepsis can occur after contrast injection into an obstructed or disrupted pancreatic duct . Whether stents cause or prevent pancreatic sepsis is unknown . Accordingly, the pancreatic duct bacteriology in patients with pancreatic duct stents was retrospectively reviewed and contrasted with biliary cultures taken from patients at the time of bile duct stent retrieval and/or exchange . METHODS: Of 61 patients (29 men, 32 women; 72 stents; mean age 51 {16} years, range 14-88 years), 36 with pancreatic duct stents had pancreatic duct cultures obtained at the time of stent exchange and/or retrieval . The results of these cultures were compared with bile duct cultures taken from 36 patients at the time of biliary stent exchange/retrieval . Eleven of the 36 patients with pancreatic duct stents also had bile duct stents . Data collected included stent patency, clinical sepsis at initial stent placement or retrieval, administration of antibiotics before the procedure, indication for stent placement, stent duration, and culture results . RESULTS: At stent retrieval and/or exchange, all 61 patients with pancreatic and/or biliary stents had contamination of the respective ducts with multiple enteric bacteria (mean 3.4 organisms in patients with pancreatic duct stents vs . 3.3 in those with bile duct stents) . Clostridium perfringens was found in 17% and 0% of patients with, respectively, bile duct and pancreatic duct stents . Among the most common indications for pancreatic duct stent placement were stricture (28), sphincterotomy (9), leak (7), stones (3), and dilated pancreatic duct (1) . Indications for a biliary stent included benign stricture (29), malignancy (6), stones (2), cholangitis (1), chronic pancreatitis (1), and dilated common bile duct (1) . Pancreatic cultures were taken at a median of 85 days (interquartile range 60-126; range 13-273) and biliary cultures at a median of 87 days (interquartile range 45-149; range 19-927) after stent placement . Eleven patients, 6 with a bile duct stent, 4 with a pancreatic duct stent, and one with dual stents, developed pre-exchange/retrieval clinical sepsis; 3 had pancreatic sepsis . All had received antibiotics at initial placement . In the 11 patients with sepsis (12 stents), 8 stents were completely occluded at exchange/retrieval, 3 were partially occluded, and one was patent . In 50 patients (60 stents), no clinical sepsis developed; 7 stents were patent, 31 partially occluded, and 22 completely obstructed . CONCLUSIONS: (1) Comparable to patients with biliary stents, all patients with pancreatic stents had contamination of the pancreatic ductal system by enteric flora . (2) In contrast to the 17% of patients with bile duct stents who had intraductal Clostridium perfringens, there were no instances of contamination with this organism in patients with pancreatic stent (p = 0.025), although, after adjusting for multiple comparisons, statistical significance was lost . (3) There was a tendency for stent occlusion to predispose to pancreatic sepsis, but occlusion by itself was insufficient (p = 0.106) . (4) Further investigation is required to define the additional variables that are associated with the development of pancreatic sepsis.

Prostaglandins Other Lipid Mediat, 2003 Jul, 71(3-4), 235 - 51
Involvement of prenylated proteins in calcium signaling induced by LTD4 in differentiated U937 cells; Capra V et al.; We investigated signal transduction pathways for LTD4 in the human promonocytic cell line U937 known, upon differentiation, to express CysLT1 receptors . We confirmed the presence of high-affinity binding sites for 3H-LTD4, which, in functional studies, displayed the features of CysLT1 receptor . In fact, three potent and selective CysLT1 receptor antagonists were able to completely inhibit LTD4-induced response . In turn, cytosolic Ca2+ ({Ca2+}i) increase (EC50 = 3.4 nM +/- 27% CV) was only partially sensitive to pertussis toxin (PTx) as well as to the prenylation inhibitor fluvastatin and to the specific geranylgeranylation and farnesylation inhibitors BAL 9504 and FPT II . Finally, Clostridium sordellii lethal toxin, inhibitor of the Ras family of GTPases, and FTS, a potent methyltransferase inhibitor, were both able to partially inhibit LTD4-induced {Ca2+} increase, suggesting a role for a Ras family member in {Ca2+}i regulation . In conclusion, in dU937 LTD4 signal transduction involves: (a) at least two pathways, one sensitive and one insensitive to PTx; (b) isoprenylated proteins, such as betagamma subunits and, possibly, a small G protein of the Ras family.

Vnitr Lek, 2003 Aug, 49(8), 645 - 9
{Clostridium difficile infection associated with pseudomembranous colitis in district hospitals}; Zemanova E et al.; At a regional hospital serving 150,000 inhabitants, the authors were tracking the occurrence of antibiotic-associated diarrhea caused by a nosocomial infection of the Clostridium difficile, especially its most serious form--pseudomembranous colitis . Six cases of the disease were found in a retrospective study in 2001, another 20 cases were diagnosed while actively searching for the disease from January till September of 2002 . The patients suffered from many complications, were of an average age of 69.24 years, their hospitalization averaged 34 days and 15 (52%) of them underwent surgery during their hospitalization . It could be demonstrably proved that 25 cases of nosocomial infection occurred during hospitalization and 3 patients were admitted to hospital with the disease . However, 2 of them had been released from hospital less than 20 days before being admitted again . Twenty-five patients (96%) had been treated with antibiotics, often in combined therapy . Most often this involved penicillin with betalactamas activity (50%), clindamycin (42%) and cefalosporins (42%) . We used methods for detecting enterotoxin A in the stool, rectoscopy and anaerobic cultivation of the stool in the diagnostic process . Rectoscopy discovered pseudomembranous colitis in 14 of 17 patients examined this way . The sensitivity for proving enterotoxin A in the stool using EIA, for patients with proven pseudomembranous colitis via rectoscopy, was 75% . Anaerobic cultivation of the stool was done in 12 patients and all the results were negative . The mortality rate of 38% for our group of patients testifies to the seriousness of this disease, which we consider to be the results of antibiotic therapy . In conclusion, nosocomial infection caused by Clostridium difficile is quite often a nosocomial disease, a prognosis that especially worsens for seriously ill patients.

J Bone Joint Surg Br, 2003 Sep, 85(7), 1051 - 4
Incidence of clostridial contamination in donors' musculoskeletal tissue; Malinin TI et al.; Reports of infection by Clostridium sordellii associated with allograft transplantation have generated considerable interest . We report our experience in recognising clostridial contamination in cadaver donors of musculoskeletal tissue . Tissues obtained from 795 consecutive donors were excised using standard surgical techniques . Samples of blood and bone marrow were also obtained . Donors with clostridia recovered from any site were matched with the preceding donor without clostridia as a procedural and environmental control . The histories of the donors were analysed to determine which variables had a relationship to contamination by running a contingency table and chi-squared test on the variables against the event of a donor being contaminated . Sixty-four donors (8.1%) had clostridia, most commonly C . sordellii . Clostridia were grown from the blood, marrow and tissue samples of 52, 37 and 30 donors, respectively . In eight cases, they were cultured from the tissue samples alone . There was no significant difference in age or gender between the contaminated donors and the control group . Open wounds were more common in control than in contaminated subjects, but only death by drowning in the contaminated group was statistically significant (p = 0.02) . The time between death and the excision of tissue which was contaminated (16 hrs 10 mins) compared with control (11 hrs 10 mins) donors was also significant (p < 10(-6)) . We conclude that there is clostridial contamination in a significant number of tissue donors, particularly with increasing time between death and tissue excision . Among the most commonly encountered species is C . sordellii . Multiple microbiological cultures, including blood, are necessary in order to identify clostridial contamination.

Am J Physiol Heart Circ Physiol, 2004 Jan, 286(1), H394 - 401 Epub 2003 Sep 25.
Requirement of Rac activity for maintenance of capillary endothelial barrier properties; Waschke J et al.; Our previous experiments indicated that GTPases, other than RhoA, are important for the maintenance of endothelial barrier integrity in both intact microvessels of rats and mice and cultured mouse myocardial endothelial (MyEnd) cell monolayers . In the present study, we inhibited the endothelial GTPase Rac by Clostridium sordellii lethal toxin (LT) and investigated the relation between the degree of inhibition of Rac by glucosylation and increased endothelial barrier permeability . In rat venular microvessels, LT (200 ng/ml) increased hydraulic conductivity from a control value of 2.5 +/- 0.6 to 100.8 +/- 18.7 x 10-7 cm x s(-1) x cm H2O(-1) after 80 min . In cultured MyEnd cells exposed to LT (200 ng/ml), up to 60% of cellular Rac was glucosylated after 90 min, resulting in depolymerization of F-actin and interruptions of junctional distribution of vascular endothelial cadherin (VE-cadherin) and beta-catenin as well as the formation of intercellular gaps . To understand the mechanism by which inhibition of Rac caused disassembly of adherens junctions, we used laser tweezers to quantify VE-cadherin-mediated adhesion . LT and cytochalasin D, an actin depolymerizing agent, both reduced adhesion of VE-cadherin-coated microbeads to the endothelial cell surface, whereas the inhibitor of Rho kinase Y-27632 did not . Stabilization of actin filaments by jasplakinolide completely blocked the effect of cytochalasin D but not of LT on bead adhesion . We conclude that Rac regulates endothelial barrier properties in vivo and in vitro by 1) modulation of actin filament polymerization and 2) acting directly on the tether between VE-cadherin and the cytoskeleton.

Biochem Biophys Res Commun, 2003 Oct 10, 310(1), 78 - 83
Molecular modelling of S1 and S2 subunits of SARS coronavirus spike glycoprotein; Spiga O et al.; The S1 and S2 subunits of the spike glycoprotein of the coronavirus which is responsible for the severe acute respiratory syndrome (SARS) have been modelled, even though the corresponding amino acid sequences were not suitable for tertiary structure predictions with conventional homology and/or threading procedures . An indirect search for a protein structure to be used as a template for 3D modelling has been performed on the basis of the genomic organisation similarity generally exhibited by coronaviruses . The crystal structure of Clostridium botulinum neurotoxin B appeared to be structurally adaptable to human and canine coronavirus spike protein sequences and it was successfully used to model the two subunits of SARS coronavirus spike glycoprotein . The overall shape and the surface hydrophobicity of the two subunits in the obtained models suggest the localisation of the most relevant regions for their activity.

Infect Control Hosp Epidemiol, 2003 Sep, 24(9), 699 - 706
Favorable impact of a multidisciplinary antibiotic management program conducted during 7 years; Carling P et al.; OBJECTIVE: To evaluate the impact of an interventional multidisciplinary antibiotic management program on expenditures for antibiotics and on the incidence of nosocomial infections caused by Clostridium difficile and antibiotic-resistant pathogens during 7 years . DESIGN: Prospective study with comparison with preintervention trends . SETTING: University-affiliated teaching hospital . PATIENTS: All adult inpatients . INTERVENTION: A multidisciplinary antibiotic management program to minimize the inappropriate use of third-generation cephalosporins was implemented in 1991 . Its impact was evaluated prospectively . The incidence of nosocomial C . difficile and resistant Enterobacteriaceae infections as well as the rate of vancomycin-resistant enterococci (VRE) and methicillin-resistant Staphylococcus aureus (MRSA) were compared with those of National Nosocomial Infections Surveillance System hospitals of similar size . RESULTS: Following implementation of the program, there was a 22% decrease in the use of parenteral broad-spectrum antibiotics (P < .0001) despite a 15% increase in acuity of patient care during the following 7 years . Concomitantly, there was a significant (P = .002) decrease in nosocomial infections caused by C . difficile and a significant (P = .02) decrease in nosocomial infections caused by resistant Enterobacteriaceae . The program also appeared to have a favorable impact on VRE rates without a sustained impact on MRSA rates . CONCLUSION: These results suggest that an ongoing multidisciplinary antibiotic management program may have a sustained beneficial impact on both expenditures for antibiotics and the incidence of nosocomial infection by C . difficile and resistant bacterial pathogens.

Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 2003, 13(2), 137 - 45
{Present state and disadvantage of rapid diagnosis method in clinical microbiology--analysis of questionnaire survey}; Nishiyama H; To investigate the present state and the disadvantages of rapid diagnosis methods in clinical microbiology in Japan, We have conducted a questionnaire survey of 360 medical facilities accredited by the Japanese Association for Rapid Method and Automation in Microbiology (JARMAM) . Major rapid diagnosis methods being used in clinical microbiology are, in the order of its use rate, influenza virus, adenovirus, Mycobacterium tuberculosis, Clostridium difficile toxin A, Rotavirus, hepatitis B virus, group A Streptococcus, RS virus, hepatitis C virus antibody . The fact found by this survey is that there is some gap between two groups, as one group views that the result of rapid diagnosis method can be considered as the final, another views that it is a supplementary diagnosis under conventional methods such as culturing . Some problems related to rapid diagnosis methods are also pointed out; how to interpret and report a test result obtained by the rapid diagnosis method when it is different from that of culture method, it can not perform antibiotic sensitivity tests, a problem of non-specific reaction, comparatively higher cost of rapid diagnosis kits . This survey finds that rapid diagnosis methods have greatly contributed to early medical treatment and appropriate therapy with its quickness, such as the use of rapid diagnosis kits for bacterial meningitis or viral infectious diseases requiring clinical urgency is highly necessitated even though some problems mentioned above exist.

Am J Surg Pathol, 2003 Oct, 27(10), 1375 - 9
Pseudomembranous collagenous colitis; Yuan S et al.; The classic clinical and histologic features of collagenous colitis are well characterized; however, the acute or neutrophilic inflammatory changes that may accompany this entity are less well established . In this report of 10 patients, we describe the first series of pseudomembranous collagenous colitis . Because superimposed Clostridium difficile infection was only demonstrated in one patient and no other causes of pseudomembranous colitis were evident in the remaining nine patients, we conclude that pseudomembranes are part of the spectrum of collagenous colitis itself . This case series illustrates the importance of searching for collagenous colitis in the evaluation of pseudomembranous colitis . At the same time, superimposed infectious or ischemic etiologies need to be excluded clinically in any patient with superimposed pseudomembranes . The existence of pseudomembranes in collagenous colitis also lends support to the hypothesis that toxin- and/or ischemia-mediated injury may be involved in the pathogenesis of collagenous colitis.

Biophys J, 2003 Oct, 85(4), 2351 - 62
Diacylglycerol-rich domain formation in giant stearoyl-oleoyl phosphatidylcholine vesicles driven by phospholipase C activity; Riske KA et al.; We have studied the effect of phospholipase C from Bacillus cereus and Clostridium perfringens (alpha-toxin) on giant stearoyl-oleoyl phosphatidylcholine (SOPC) vesicles . Enzyme activity leads to a binary mixture of SOPC and the diacylglycerol SOG, which phase separates into a SOPC-rich bilayer phase and a SOG-rich isotropic bulk-like domain embedded within the membrane, as seen directly by phase contrast microscopy . After prolonged enzymatic attack, all bilayer membranes are transformed into an isotropic pure SOG phase as characterized by fluorescence microscopy, differential scanning calorimetry, fluorescence anisotropy measurements, and small angle x-ray scattering . These domains may have biological relevance, serving as storage compartments for hydrophobic molecules and/or catalyzing cellular signaling events at their boundaries . Furthermore, in the early stages of asymmetric enzymatic attack to the external monolayer of giant vesicles, we observe a transient coupling of the second-messenger diacylglycerol to membrane spontaneous curvature, which decreases due to enzyme activity, before domain formation and final vesicle collapse occurs.

Best Pract Res Clin Gastroenterol, 2003 Oct, 17(5), 755 - 73
Probiotics to enhance anti-infective defences in the gastrointestinal tract; Gill HS; Several clinical studies have demonstrated the therapeutic and/or prophylactic efficacy of specific probiotics against acute viral gastroenteritis and antibiotic-associated diarrhoea (including Clostridium difficile infection) . Emerging evidence also suggests beneficial effects against Helicobacter pylori infection . The evidence of efficacy against traveller's diarrhoea remains, however, inconclusive . The precise mechanisms by which probiotics potentiate host gastrointestinal defences and mediate protection are not fully known . There is evidence to suggest, however, that probiotics might contribute to host defence by reinforcing non-immunological defences and stimulating both specific and non-specific host immune responses . Little is known about the relative importance of the probiotic-stimulated mechanisms in host protection . This review summarises the evidence for the anti-infective effects of probiotics and discusses the effect of orally delivered probiotics on non-immunological and immunological defence mechanisms in the host, especially in the gastrointestinal tract.

J Enzyme Inhib Med Chem, 2003 Jun, 18(3), 233 - 42
Protease inhibitors: synthesis of bacterial collagenase and matrix metalloproteinase inhibitors incorporating succinyl hydroxamate and iminodiacetic acid hydroxamate moieties; Santos MA et al.; A series of succinyl hydroxamates/bishydroxamates as well as a new structural type of matrix metalloproteinase (MMP)/bacterial protease (BP) inhibitors, incorporating iminodiacetic (IDA) hydroxamate/bishydroxamate moieties, has been synthesized and tested for interaction with four vertebrate proteases, MMP-1, MMP-2, MMP-8 and MMP-9, and a BP, the collagenase isolated from Clostridium histolyticum (ChC) . The new derivatives generally showed inhibition constants in the range of 8-62 nM against the five proteases mentioned above.

Antimicrob Agents Chemother, 2003 Oct, 47(10), 3311 - 7
Effects of tylosin on bacterial mucolysis, Clostridium perfringens colonization, and intestinal barrier function in a chick model of necrotic enteritis; Collier CT et al.; Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens . Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats . Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics . However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C . perfringens as well as the pathophysiological consequences of its growth . We report a study with a chick model of NE, which used molecular (16S rRNA gene {16S rDNA}) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C . perfringens . The effects of tylosin and pectin on mucolytic activity of the microbiota and C . perfringens colonization and their relationship to pathological indices of NE were of particular interest . The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C . perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function . The presence of pectin did not significantly affect the variables measured . Thus, it appears that tylosin can control NE through its modulation of C . perfringens colonization and the mucolytic activity of the intestinal microbiota.

Plast Reconstr Surg, 2003 Oct, 112(5 Suppl), 177S - 88S; quiz 188S, 192S; discussion 189S-191S
The cosmetic use of botulinum toxin; Rohrich RJ et al.; The approach to facial rejuvenation continues to evolve . For decades, the primary focus on rejuvenation has centered on modalities such as skin care, skin resurfacing, soft-tissue augmentation, and surgical intervention . A better understanding of the physiologic changes that occur with facial aging lends itself to new approaches and techniques that are mindful of the causes . As animation has shown to be a significant contributor to both the appearance of facial lines and soft-tissue malposition, there has been recent interest in chemodenervating agents and their applications in the field of facial rejuvenation . These agents, by and large, efface rhytides by selective and precise focal paralysis of the underlying facial musculature and, therefore, reduce or eliminate the prominence of the overlying rhytides . In addition, chemodenervation can serve as an adjunct for facial rejuvenation because of its influence on facial soft-tissue position and shape . Botulinum toxin, derived from Clostridium botulinum, is the most widely used agent; therefore, this new modality, its applications in cosmetic plastic surgery, and its applications to other areas will be discussed.

Biochemistry, 2003 Sep 30, 42(38), 11382 - 90
Cofactor determination and spectroscopic characterization of the selenium-dependent purine hydroxylase from Clostridium purinolyticum; Self WT et al.; Purine hydroxylase (PH) from Clostridium purinolyticum contains a labile selenium cofactor and belongs to a class of enzymes known as the selenium-dependent molybdenum hydroxylases . The presence of approximately 1.1 mol of molybdenum, 0.87 mol of selenium, and 3.3 mol of iron per mol of PH was determined by atomic absorption spectroscopy . Enzyme preparations with lower than stoichiometric amounts of selenium exhibited correspondingly lower hydroxylase activities . Bound FAD, 1 mol per mol enzyme, was confirmed by UV-vis and fluorescence spectroscopy . CMP, released by acid hydrolysis, indicated the presence of a molybdopterin cytosine dinucleotide cofactor . The fully active PH utilized NADP(+) as an electron acceptor, and kinetic analysis revealed an optimal k(cat) of 412 s(-1) using hypoxanthine as the hydroxylase substrate . Xanthine, NAD(+), and NADPH had no significant effect on this reaction rate . A selenium-independent NADPH oxidase activity was exhibited by native PH . Electron paramagnetic resonance spectroscopy revealed the presence of a Mo(V) desulfo signal, FAD radical, and 2Fe-2S centers in hypoxanthine-reduced PH . No hyperfine coupling of selenium, using (77)Se isotope-enriched PH, was observed in any of the EPR active signals studied . The appearance of the desulfo signal suggests that the ligands of Mo in selenium-dependent molybdenum hydroxylases are different from the well-studied mammalian xanthine oxidoreductases (XOR) and aldehyde oxidoreductases (AOR) and suggests a unique role for Se in catalysis.

J Food Prot, 2003 Sep, 66(9), 1599 - 603
Influence of NaCl content and cooling rate on outgrowth of Clostridium perfringens spores in cooked ham and beef; Zaika LL; The effect of NaCl concentration and cooling rate on the ability of Clostridium perfringens to grow from spore inocula was studied with the use of a process that simulates the industrial cooking and cooling of smoked boneless ham and beef roasts . NaCl was added to ground cooked hams A and B (which were commercially obtained) to obtain levels of 2.4, 3.1, 3.6, and 4.1% (wt/wt) and 2.8, 3.3, 3.8, and 4.3% (wt/wt), respectively, and to raw ground beef to obtain levels of 0, 1, 2, 3, and 4% (wt/wt) . Ham C, a specially formulated, commercially prepared product, was supplemented with NaCl to obtain levels of 2.0, 2.5, 3.0, and 3.5% . The samples were inoculated with a three-strain mixture of C . perfringens spores to obtain concentrations of ca . 3 log10 CFU/g . Portions of meat (5 g each) were spread into thin layers (1 to 2 mm) in plastic bags, vacuum packaged, and stored at -40 degrees C . Thawed samples were heated at 75 degrees C for 20 min and subsequently cooled in a programmed water bath from 54.4 to < or = 8.5 degrees C in 15, 18, or 21 h . For the enumeration of C . perfringens, samples were plated on tryptose-sulfite-cycloserine agar and incubated in an anaerobic chamber at 37 degrees C for 48 h . Population densities for cooked ham and beef increased as cooling time increased, and NaCl exerted a strong inhibitory effect on the germination and outgrowth of C . perfringens . For beef, while 3% NaCl completely arrested growth, pathogen numbers increased by > or = 3, 5, and 5 log10 CFU/g in 15, 18, and 21 h, respectively, when the NaCl level was <2% . C . perfringens did not grow during cooling for 15, 18, or 21 h in ham samples containing > or = 3.1% NaCl . Results obtained in this study suggest that a 15-h cooling time for cooked ham, which is normally formulated to contain >2% NaCl, would yield an acceptable product (with an increase of <1 log10 CFU/g in the C . perfringens count); however, for beef containing <2% NaCl, C . perfringens populations may reach levels high enough to cause illness.

Cell Biol Int, 2003, 27(10), 831 - 6
Structural organization of the intact bacterial cellulosome as revealed by electron microscopy; Madkour M et al.; The architecture of the intact cellulosome of Clostridium thermocellum, a huge extracellular multi-polypetide bacterial enzyme complex engaged in degradation of cellulose, was investigated by electron microscopy . This was done because former electron microscopic studies aimed at elucidation of the structure of polycellulosomes and cellulosomes were restricted by the fact that data on macromolecular details could only be derived from deformed or disrupted enzyme complexes, or by application of cryo preparation and imaging techniques yielding insufficient resolution . The shape of well-preserved cellulosomes was more or less spherical, often similar to that of an olive fruit with a cavity . Therein, multiple fibrillar structures could be visualized, interpreted to be the proximal stretches of copies of the fibrillar protein Cip A ('scaffoldin'), the nonenzymatic scaffolding protein known to function as attachment site for the enzymatic subunits, as well as fibrillar parts of anchoring proteins . The enzymatic subunits were depicted to be attached, in a repetitive fashion, to the distal stretches of the Cip A proteins . The enzymatic subunits were seen, in the intact cellulosome, to form a shell-like complex substructure surrounding the cavity . Obviously, this kind of architecture makes sure that the catalytic domains of the enzymatic subunits are exposed to the environment, and, hence, to the substrate, the cellulose fibrils . Attempts were made to demonstrate the alternating occurrence of coiled domains and fibrillar stretches along the elongated protein Cip A previously characterized by sequencing, X-ray, and NMR studies . To this end, Cip A molecules, with adhering enzymatic subunits, were partially removed from their native location within the cellulosome, "stretched" by hydromechanical forces directly on the electron microscopic support film, negatively stained, and depicted by electron microscopy . The alternating occurrence of presumed coiled domains and fibrillar stretches along Cip A could be visualized, together with detached enzymatic subunits found on the support film.

Med Clin (Barc), 2003 Sep 20, 121(9), 331 - 3
{Diarrhea associated with Clostridium difficile: experience at a secondary hospital}; de Isusi AM et al.; BACKGROUND AND OBJECTIVE: Our purpose was to know the incidence, clinical profile, evolution and mortality of Clostridium difficile-associated diarrhea . PATIENTS AND METHOD: Retrospective study . Inpatients were included who had a positive toxin on stools or a diagnostic colonoscopy (pseudomembranous colitis) after ruling out other causes (n=113) . RESULTS: We observed a growing annual incidence (1.05, 1.7, and 2.6 per 1000 admissions in 1999, 2000 and 2001, respectively) . Hyponatremia (OR=5.16) and vomiting (OR=3.09) were found to be worse-prognosis factors . Hyponatremia (OR=2.72) and chronic renal disease (OR=4.45) increased the mortality risk . CONCLUSIONS: The incidence of C . difficile-associated diarrhea is growing, probably in relation to increased clinical suspicion, wide antibiotics utilization, and old age of inpatients . Hyponatremia could be a predictive factor of both illness and worse-prognosis.

Eur J Clin Microbiol Infect Dis, 2003 Oct, 22(10), 615 - 8 Epub 2003 Sep 12.
Clostridium difficile-associated diarrhoea after internal fixation of intertrochanteric femoral fractures; Sharma P et al.; The aim of the study presented here was to record the incidence and outcome of Clostridium difficile-associated diarrhoea in patients undergoing surgery for intertrochanteric femoral fractures . Between January 2000 and June 2001, a total of 239 patients who underwent surgery after sustaining an intertrochanteric femoral fracture were included in the study . Seventeen patients developed Clostridium difficile-associated diarrhoea (incidence risk, 7.1%), and six patients died after developing the condition (case fatality rate, 35%) . Thus, Clostridium difficile-associated diarrhoea was found to cause significant morbidity and mortality after surgery for intertrochanteric femoral fractures, and antibiotics should be used judiciously in this group of patients.

J Child Neurol, 2003 Sep, 18 Suppl 1, S50 - 66
Injectable neuromuscular blockade in the treatment of spasticity and movement disorders; Tilton AH; Neuromuscular blockade via injection of alcohol, phenol, or botulinum toxin reduces the tone of overactive muscles in order to restore the appropriate balance between agonists and antagonists . Such a restoration allows improved stretch and increased resting length and can reduce the likelihood of contracture . Alcohol or phenol, injected onto the motor nerve, denatures proteins and promotes axonal degeneration . The onset of action is within hours, whereas the duration of action is variable, ranging from 2 weeks to 6 months and beyond . The advantages of alcohol or phenol chemodenervation lie in their low cost and lack of antigenicity . The disadvantages include the technical difficulty of the injections and significant risk for pain as a result of treatment . Botulinum toxins, purified forms of Clostridium botulinum exotoxins, are injected directly into muscle, where they cleave one or more vesicle fusion proteins, thus blocking release of acetylcholine at the neuromuscular junction . Three commercial products--two of serotype A and one of B--are available . Each differs in its unit potency, side effects, and duration of action . On average, botulinum toxin has a clinical onset of action approximately 12 to 72 hours after injection, with a peak effect at 1 to 3 weeks . Effects then plateau for 1 to 2 months, with patients often requiring reinjection approximately every 3 months . Side effects may include local discomfort at the site of the injection and excessive weakness of the injected or nearby muscles, although more distant effects may occur . Antibody formation is a significant clinical concern and eventually obviates treatment benefit in approximately 5% of patients . Switching serotypes may be effective, at least temporarily . Consensus dosing guidelines have been developed and are presented within . Numerous studies have suggested that botulinum toxin has a role in the care of children with spasticity or dystonia related to cerebral palsy, and may improve equinus, gait, upper extremity use, comfort, and care . Evidence of functional improvement remains equivocal in the severely impaired child; however, there is evidence for improvement in less impaired children . The optimal candidate for injectable neuromuscular blockade is one who has a limited number of muscles that need treatment, who does not have fixed contracture, and who retains selective motor control . The ultimate goal of treatment for the hypertonic child is to maximize function, comfort, and independence . Hypertonia is only one aspect of the upper motoneuron syndrome, which includes both positive and negative symptoms . The treatment program, in which chemodenervation is only one tool, requires a multidisciplinary evaluation and individualized plan to address the whole patient.

Am J Sports Med, 2003 Sep-Oct, 31(5), 791 - 7
The safe and effective use of allograft tissue--an update; Barbour SA et al.; The use of allograft tissue in orthopaedic surgery has increased tremendously over the last several years . Tissue availability, reduced surgical times, and lack of donor site morbidity are attractive characteristics to surgeons and patients alike . Although complications, such as disease transmission, are relatively uncommon when using allograft tissue, they do occur . This article will review the literature concerning the safe and effective use of allograft tissue, as well as present four case reports of Clostridium septicum infection caused by implantation of contaminated allograft tissue.

Biochemistry, 2003 Sep 23, 42(37), 10991 - 7
Complete subunit structure of the Clostridium botulinum type D toxin complex via intermediate assembly with nontoxic components; Mutoh S et al.; Clostridium botulinum serotype D strains usually produce two types of stable toxin complex (TC), namely, the 300 kDa M (M-TC) and the 660 kDa L (L-TC) toxin complexes . We previously proposed assembly pathways for both TCs {Kouguchi, H., et al . (2002) J . Biol . Chem . 277, 2650-2656}: M-TC is composed by association of neurotoxin (NT) and nontoxic nonhemagglutinin (NTNHA); conjugation of M-TC with three auxiliary types of hemagglutinin subcomponents (HA-33, HA-17, and HA-70) leads to the formation of L-TC . In this study, we found three TC species, 410, 540, and 610 kDa TC species, in the culture supernatant of type D strain 4947 . The 540 and 610 kDa TC species displayed banding patterns on SDS-PAGE similar to that of L-TC but with less staining intensity of the HA-33 and HA-17 bands than those of L-TC, indicating that these are intermediate species in the pathway to L-TC assembly . In contrast, the 410 kDa TC species consisted of M-TC and two molecules of HA-70 . All of the TC species, except L-TC, demonstrated no hemagglutination activity . When the intermediate TC species were mixed with an isolated HA-33/17 complex, every TC species converted to 650 kDa L-TC with full hemagglutination activity and had the same molecular composition of L-TC . On the basis of titration analysis with the HA-33/17 complex, the stoichiometry of the HA-33/17 complex molecules in the L-TC, 610 kDa, and 540 kDa TC species was estimated as 4, 3, and 2, respectively . In conclusion, the complete subunit composition of mature L-TC is deduced to be a dodecamer assembled by a single NT, a single NTNHA, two HA-70, four HA-33, and four HA-17 molecules.

Lett Appl Microbiol, 2003, 37(4), 304 - 8
Fermentation of cellulose to acetic acid by Clostridium lentocellum SG6: induction of sporulation and effect of buffering agent on acetic acid production; Tammali R et al.; AIMS: To determine the growth, correlation between sporulation and acetic acid production and effect of buffering agent at high substrate cellulose concentrations of the strain Clostiridium lentocellum SG6 . METHODS AND RESULTS: The strain SG6 was grown in cellulose mineral salt medium containing cellulose (Whatman No . 1 filter paper, Whatmore International Ltd., Maidstone, UK) or cellobiose . The strain fermented cellulose even after several transfers on cellobiose medium . The formation of endospores on third day onwards indicated the lowering of pH in the medium because of the formation of acetic acid . Maintaining the pH 7.2 at higher substrate concentrations resulted in increase of biomass, cellulose fermentation, acetic acid production, etc . CONCLUSIONS: The strain SG6, with its high fermentation yields and sporulating character can become a potential strain for acetic acid production and also as a probiotic strain in animal nutrition . SIGNIFICANCE AND IMPACT OF THE STUDY: The direct conversion of cellulosic biomass to acetic acid can eliminate expensive three-step saccharification, fermentation processes . The strain SG6 can ferment cellulose at high substrate concentrations.

Exp Biol Med (Maywood), 2003 Sep, 228(8), 972 - 81
Involvement of rho and rho-associated kinase in sphincteric smooth muscle contraction by angiotensin II; Rattan S et al.; The tonic smooth muscles of lower esophageal sphincter (LES) and internal anal sphincter (IAS) are subject to modulation by the neurohumoral agents . We report that angiotensin (Ang) II-induced contraction of rat IAS and LES smooth muscle cells (SMC) was inhibited by Clostridium botulinum C3 exozyme, HA 1077 and Y 27632, suggesting a role for Rho kinase and a Rho-associated kinase (ROK) . Ang II-induced contraction of the SMC was also attenuated by genistein, antibodies to the pp60(c-src), p(190) RhoGTPase-activating protein (p190 RhoGAP), carboxyl terminus of Galpha13, carboxyl terminus peptide, and ADP ribosylation factor (ARF) antibody . Ang II-induced increase in p(190) RhoGAP tyrosine phosphorylation was attenuated by genistein . Furthermore, Ang II-induced increase in smooth muscle tone and phosphorylation of myosin light chain (MLC; 20 kDa; MLC20-P) were attenuated by Y 27632 and genistein . The results suggest an important role for Galpha13 and pp60(c-src) in the intracellular events responsible for the activation of RhoA/ROK in Ang II-induced contraction of LES and IAS SMC.

Mol Gen Mikrobiol Virusol, 2003, (3), 29 - 33
{Production of recombinant fragments of the Clostridium tetani neurotoxin for the development of new immune-prophylaxis preparations against tetanus}; Varfolomeeva NA et al.; Tetanus belongs to dangerous infection diseases, whose effective prevention can be ensured by vaccines . The acting substance of tetanus vaccines, presently in use, is a partially purified and deprived-of-lethal-action Clostridium tetani neurotoxin . The construction of a subunit preparation on the basis of toxin fragments obtained through gene engineering could be a method aimed at promoting the quality of the used tetanus vaccines . With this goal in mind, we built, within the present case study, the expressing genetic constructions and obtained, in the pure form, an extensive tetanus-vaccine chain with its C-terminal (Hc) fragment, hydride peptides, containing the Hc-fragment and C-terminal fragment of toxin B C . difficile, as well as Hc-fragment and S3 collagen-binding domain of collagenase C . histolyticum . The thus obtained proteins can be used in testing their immunogenic and protective properties, while the conducted study could be a basis for further research of a new-generation vaccine against tetanus and other human infection diseases.

Zh Mikrobiol Epidemiol Immunobiol, 2003 Jul-Aug, (4), 74 - 8
{Effect of a polyoxydonium immunoregulator on the biological properties of microorganisms}; Kirillov DA et al.; The effect of the synthetic immunomodulator polyoxydonium (PO) on some biological properties of pathogenic bacteria (Shigella flexneri, Salmonella enteritidis), opportunistic bacteria (Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacteroides fragilis, Peptostreptococcus anaerobius, Prevotella melaninogenica, Propionibacterium propionicum, Clostridium difficile) and fungi (Candida albicans), isolated during enteric infections, enteric dysbiosis, pyoinflammatory diseases, was evaluated in a number of in vitro experiments . The study revealed that the recommended therapeutic concentration of PO decreased antilysozyme activity (ALA) and the anticomplement activity in Klebsiella, Shigella, Propionibacterium, Clostridium, bacteroids, fungi of the genus Candida, but increased ALA in nonhemolytic Escherichia . Under the action of PO an increased sensitivity of the microorganisms under study to definite antibiotics of the lincosamide, fluoroquinolon, carbapenem and cephalosporin groups was noted . The data obtained in this study reveal one of the possible mechanisms of the corrective action of PO on the microbiocenosis of the intestine in dysbiosis, enteric infections and pyoinflammatory diseases.

Microbiol Mol Biol Rev, 2003 Sep, 67(3), 429 - 53, table of contents
Surviving the acid test: responses of gram-positive bacteria to low pH; Cotter PD et al.; Gram-positive bacteria possess a myriad of acid resistance systems that can help them to overcome the challenge posed by different acidic environments . In this review the most common mechanisms are described: i.e., the use of proton pumps, the protection or repair of macromolecules, cell membrane changes, production of alkali, induction of pathways by transcriptional regulators, alteration of metabolism, and the role of cell density and cell signaling . We also discuss the responses of Listeria monocytogenes, Rhodococcus, Mycobacterium, Clostridium perfringens, Staphylococcus aureus, Bacillus cereus, oral streptococci, and lactic acid bacteria to acidic environments and outline ways in which this knowledge has been or may be used to either aid or prevent bacterial survival in low-pH environments.

Lancet Oncol, 2003 Sep, 4(9), 548 - 56
Bacteria as tumour-targeting vectors; Pawelek JM et al.; Live bacteria were first actively used in the treatment of cancer nearly 150 years ago, work that ultimately led to the study of immunomodulation . Today, with the discovery of bacterial strains that specifically target tumours, and aided by genomic sequencing and genetic engineering, there is new interest in the use of bacteria as tumour vectors . Bifodobacterium, Clostridium, and Salmonella have all been shown to preferentially replicate within solid tumours when injected from a distal site, and all three types of bacteria have been used to transport and amplify genes encoding factors such as prodrug-converting enzymes, toxins, angiogenesis inhibitors, and cytokines . In this review we provide a historical discussion of this area, and describe the development of the bacteria, which are currently being prepared for use in clinical trials in patients with cancer.

Int J Mol Med, 2003 Oct, 12(4), 571 - 5
The long-term oral administration of a product derived from a probiotic, Clostridium butyricum induced no pathological effects in rats; Araki Y et al.; Recent studies have suggested that short chain fatty acids (SCFAs) exert a therapeutic effect on some human and experimental animal diseases . In our previous study, we showed that Clostridium butyricum produces high levels of SCFAs in the culture system used . In addition, an additive based on yogurt was effective in eliminating and masking the odor derived from these SCFAs in the product . Recently, we reported that the oral administration of a high concentration (50% w/w) of this product derived from Clostridium butyricum for 17 days caused no pathological abnormalities in rats . The aim of the present study was to investigate the effects of the prolonged oral administration of this product in rats . Male and female Wistar Hannover GALAS rats, 5 weeks old, were given a mixture of a standard diet plus the product derived from Clostridium butyricum (5% w/w) with 0.1% additive for 16 months (n=6) . The control rats were allowed the same standard diet plus tap water (5% w/w) with 0.1% additive (n=6) . After 16 months, a laparotomy was performed . A hemocyte count, and biochemical and electrolyte analyses were subsequently carried out . The esophagus, stomach, small intestine, large intestine and pancreas were investigated macroscopically and microscopically . The results showed that the rats grew normally for the duration of the experimental period . The body weights of the product-fed rats were comparable with those of the control-fed rats . There were no significant differences in the organ weight between the product- and control-fed rats, except for a significantly increased weight of the large intestine in the product-fed male rats . No pathological abnormalities were found in the hemocyte count, the biochemical and electrolyte analyses, or the macroscopic and microscopic findings.

J Infect, 2003 Oct, 47(3), 248 - 50
Fatal Clostridium perfringens infection of a liver cyst; Quigley M et al.; We report a case of Clostridium perfringens infection of a pre-existing hepatic cyst leading to a fatal outcome.

Endocrinology, 2003 Oct, 144(10), 4508 - 18 Epub 2003 Jun 19.
Mastoparan-induced insulin secretion from insulin-secreting betaTC3 and INS-1 cells: evidence for its regulation by Rho subfamily of G proteins; Amin RH et al.; Mastoparan, a tetradecapeptide from wasp venom, stimulates insulin secretion from the islet beta-cells, presumably via activation of trimeric G proteins . Herein, we used Clostridial toxins, which selectively modify and inactivate the Rho subfamily of G proteins, to examine whether mastoparan-induced insulin secretion also involves activation of these signaling proteins . Mastoparan, but not mastoparan 17 (an inactive analog of mastoparan), significantly stimulated insulin secretion from betaTC3 and INS-1 cells . Preincubation of betaTC3 cells with either Clostridium difficille toxin B, which inactivates Rho, Cdc42, and Rac, or Clostridium sordellii toxin, which inactivates Ras, Rap, and Rac, markedly attenuated the mastoparan-induced insulin secretion, implicating Rac in this phenomenon . Mastoparan-stimulated insulin secretion was resistant to GGTI-2147, a specific inhibitor of geranylgeranylation of Rho G proteins (e.g . Rac), suggesting that mastoparan induces direct activation of Rac via GTP/GDP exchange . This was confirmed by a pull-down assay that quantifies the binding of activated (i.e . GTP-bound) Rac to p21-activated kinase . However, glucose-induced insulin secretion from these cells was abolished by toxin B or GGTI-2147, suggesting that the geranylgeranylation step is critical for glucose-stimulated secretion . Mastoparan significantly increased the translocation of cytosolic Rac and Cdc42 to the membrane fraction . Confocal light microscopy revealed a substantial degree of colocalization of Rac (and, to a lesser degree, Cdc42) with insulin in beta-cells exposed to mastoparan . Further, stable expression of a dominant negative (N17Rac) form of Rac into INS-1 cells resulted in a significant reduction in mastoparan-stimulated insulin secretion from these cells . Taken together, our findings implicate Rho G proteins, specifically Rac, in mastoparan-induced insulin release.

Pneumonol Alergol Pol, 2003, 71(1-2), 95 - 8
{A rare case of gaseous gangrenous mediastinitis}; Buczynski K et al.; We report a case of a cervical phlegmone and mediastinitis descending from peridental abscess in 32-year old student . Emergency left thoracotomy was performed after CT evaluation in which multiple fluid levels and gas spaces were found . After 72 hours bacterial strains showed Clostridium perfringens in fluid taken from left pleural cavity and patient was sent to Hiberbaric Center in Gdynia, where he underwent hyperbaric oxygen therapy . After 10 days the patient was brought back in good condition to our Department . After 3 days he died because of sudden massive bleeding caused by necrosis of aorta wall.

J Clin Microbiol, 2003 Sep, 41(9), 4184 - 7
Recent emergence of an epidemic clindamycin-resistant clone of Clostridium difficile among Polish patients with C . difficile-associated diarrhea; Pituch H et al.; Analysis of both the antibiotic resistance and the virulence characteristics of anaerobic human microbial pathogens is important in order to improve our understanding of a number of clinically significant infectious diseases, including Clostridium difficile-associated diarrhea (CDAD) . We determined the presence of the clindamycin resistance-associated gene ermB and the ribotype of 33 C . difficile strains isolated from Polish patients suffering from CDAD . While all strains produced cytotoxin B (TcdB), enterotoxin A (TcdA) was produced by a subset of 15 strains only . The results showed that a single ermB-positive, TcdA(-)B(+) C . difficile strain with ribotype A has disseminated widely in the two Warsaw hospitals under investigation . Although different strains with the same phenotype were detected, the genotype A strain appeared to be the only one with a clear epidemic character . Apparently, enhanced local spread of CDAD-causing C . difficile may be restricted to a limited number of bacterial genotypes only.

Scand J Infect Dis, 2003, 35(6-7), 365 - 7
Lactobacillus plantarum 299v for the treatment of recurrent Clostridium difficile-associated diarrhoea: a double-blind, placebo-controlled trial; Wullt M et al.; A double-blind, placebo-controlled trial was performed to analyse the ability of Lactobacillus plantarum 299v to prevent further recurrent episodes of Clostridium difficile-associated diarrhoea (RCDAD) . Recurrence of clinical symptoms (main outcome) was seen in 4 of 11 patients who received metronidazole in combination with L . plantarum 299v and in 6 of 9 treated with metronidazole in combination with placebo . The lactobacilli treatment had no side-effects . Although the small sample size does not allow any conclusion to be drawn concerning the efficacy of L . plantarum in patients with RCDAD, these results may contribute to the ongoing discussion about the benefits of probiotics in patients with RCDAD and encourage the performance of larger multicentre studies.

J Antimicrob Chemother, 2003 Oct, 52(4), 623 - 8 Epub 2003 Sep 01.
The susceptibility of ionophore-resistant Clostridium aminophilum F to other antibiotics; Houlihan AJ et al.; OBJECTIVE: To determine if ionophore-resistant ruminal bacteria are cross-resistant to other classes of antibiotics . Clostridium aminophilum was used as a model organism because this Gram-positive ruminal bacterium can adapt to ionophores (monensin and lasalocid) . Non-adapted cultures lagged for at least 12 h with 1 microM monensin or lasalocid, but initiated no growth if the concentration was 10 microM . Adapted cultures did not lag with 1 microM monensin or lasalocid, grew well even if the ionophore concentration was 10 microM and contained cells at least 100,000-fold more resistant than those in non-adapted cultures . METHODS: Ionophore-adapted and non-adapted cultures were assayed for their susceptibility to other classes of antibiotics (penicillin G, ampicillin, cephalosporin C, vancomycin, carbenicillin, tetracycline, chloramphenicol, erythromycin, streptomycin, linocomycin, rifampicin, trimethoprim, novobiocin, polymyxin B and bacitracin) using a broth microdilution method . RESULTS: Adapted cultures retained their resistance phenotype for at least 28 generations even if ionophore was no longer present . Monensin-adapted cultures were as resistant to lasalocid as those adapted to lasalocid, but lasalocid-adapted cultures lagged with 1 microM monensin . Monensin- and lasalocid-resistant C . aminophilum F cultures were as susceptible to most antibiotics as non-adapted cultures . The only antibiotic that seemed to have a common mechanism of resistance was bacitracin, and the ionophore-adapted cultures had a 32-fold greater MIC . CONCLUSION: The use of ionophores in cattle feed and the selection of ionophore-resistant ruminal bacteria does not necessarily lead to other types of antibiotic resistance.

FEMS Microbiol Lett, 2003 Aug 29, 225(2), 325 - 9
Construction of a fusion protein carrying antigenic determinants of enteric clostridial toxins; Belyi IF et al.; Clostridium difficile and Clostridium perfringens type A are infectious agents of enteric diseases . The main virulence factors of these microorganisms include toxins A and B of C . difficile (ToxA and ToxB) and enterotoxin of C . perfringens (Cpe) . In this study genetic constructions have been created for the expression of ToxA, ToxB and Cpe fragments either as individual components or as a hybrid multidomain (ToxA-ToxB-Cpe) protein . Rabbit monospecific sera raised against individual peptides reacted with the chimeric product indicating that the corresponding antigenic determinants were correctly expressed on the hybrid molecule . Furthermore, mice immunized with the fusion protein produced antibodies specific to each of the three separate components . These data suggest that the constructed three-domain molecule could be used in future studies for development of a vaccine against enteric clostridial diseases.

J Bacteriol, 2003 Sep, 185(18), 5527 - 35
Clostridium pasteurianum F1Fo ATP synthase: operon, composition, and some properties; Das A et al.; The atp operon encoding F1Fo ATP synthase in the fermentative obligate anaerobic bacterium Clostridium pasteurianum was sequenced . It consisted of nine genes arranged in the order atpI(i), atpB(a), atpE(c), atpF(b), atpH(delta), atpA(alpha), atpG(gamma), atpD(beta), and atpC(epsilon), which was identical to that found in many bacteria . Reverse transcription-PCR confirmed the presence of the transcripts of all nine genes . The amount of ATPase activity in the membranes of C . pasteurianum was low compared to what has been found in many other bacteria . The F1Fo complexes solubilized from membranes of C . pasteurianum and Escherichia coli had similar masses, suggesting similar compositions for the F1Fo complexes from the two bacteria . Western blotting experiments with antibodies raised against the purified subunits of F1Fo detected the presence of eight subunits, alpha, beta, gamma, delta, epsilon, a, b, and c, in the F1Fo complex from C . pasteurianum . The F1Fo complex from C . pasteurianum was activated by thiocyanate, cyanate, or sulfhydryl compounds; inhibited by sulfite, bisulfite, or bicarbonate; and had tolerance to inhibition by dicyclohexylcarbodiimide . The target of thiol activation of the F1Fo complex from C . pasteurianum was F1 . Thiocyanate and sulfite were noncompetitive with respect to substrate Mg ATP but competitive with respect to each other . The F1 and Fo parts of the F1Fo complexes from C . pasteurianum and E . coli bound to each other, but the hybrid F1Fo complexes were not functionally active.

Ann Chir, 2003 Jul, 128(6), 391 - 3
{Fulminant spontaneous Clostridium septicum gas gangrene}; Carron P et al.; Clostridium septicum gas gangrene is a rare fulminant infectious disease for which signs and symptoms like extreme pain, subcutaneous crepitus and violaceous necrotic bullae must be recognised . Early diagnosis followed by large surgical debridement are the only ways to ameliorate the bad prognosis . Here we describe a case of cervico-thoracic spontaneous gas gangrene.

J Biol Chem, 2003 Nov 7, 278(45), 44535 - 41 Epub 2003 Aug 26.
Cellular uptake of Clostridium difficile toxin B . Translocation of the N-terminal catalytic domain into the cytosol of eukaryotic cells; Pfeifer G et al.; Clostridium difficile toxin B (269 kDa) is one of the causative agents of antibiotic-associated diarrhea and pseudomembranous colitis . Toxin B acts in the cytosol of eukaryotic target cells where it inactivates Rho GTPases by monoglucosylation . The catalytic domain of toxin B is located at the N terminus (amino acid residues 1-546) . The C-terminal and the middle region of the toxin seem to be involved in receptor binding and translocation . Here we studied whether the full-length toxin or only a part of the holotoxin is translocated into the cytosol . Vero cells were treated with recombinant glutathione S-transferase-toxin B, and thereafter, toxin B fragments were isolated by affinity precipitation of the glutathione S-transferase-tagged protein from the cytosolic fraction of intoxicated cells . The toxin fragment (approximately 65 kDa) was recognized by an antibody against the N terminus of toxin B and was identified by matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis as the catalytic domain of toxin B . The toxin fragment located in the cytosol possessed glucosyltransferase activity that could modify RhoA in vitro, but it was not able to intoxicate intact cells . After treatment of Vero cells with a radiolabeled fragment of toxin B (amino acid residues 547-2366), radioactivity was identified in the membrane fraction of Vero cells but not in the cytosolic fraction of Vero cells . Furthermore, analysis of cells by fluorescence microscopy revealed that the C terminus of toxin B was located in endosomes, whereas the N terminus was detected in the cytosol . Protease inhibitors, which were added to the cell medium, delayed intoxication of cells by toxin B and pH-dependent translocation of the toxin from the cell surface across the cell membrane . The data indicate that toxin B is proteolytically processed during its cellular uptake process.

J Environ Sci Health A Tox Hazard Subst Environ Eng, 2003 Sep, 38(9), 1867 - 75
Hydrogen production from wastewater sludge using a Clostridium strain; Wang CC et al.; Limited data in literature revealed a relatively low hydrogen yield from wastewater sludge, ca . 0.16 mg/g-dried solids, using anaerobic fermentation . We demonstrated in this work a much higher hydrogen yield, around 1.1 mg-H2/g-dried solids using a clostridium strain isolated from the sludge sample . The formed hydrogen would be consumed after passing the peak value at around 30-36 h of fermentation . We examined the effects of employing five different pre-treatments on substrate sludge, but noted no appreciable enhancement in hydrogen yield as commonly expected for methane production . Since a vast amount of organic matters had been released to water after hydrogen fermentation, we externally dosed methanogenic bacteria to the fermented liquor to produce methane . The fermented liquor could produce more methane than the non-fermented sample, indicating that the dosed methanogenic bacteria readily utilized the organic matters derived from the fermentation test.

Eur J Clin Microbiol Infect Dis, 2003 Sep, 22(9), 525 - 9 Epub 2003 Aug 21.
High frequency of antibiotic-associated diarrhea due to toxin A-negative, toxin B-positive Clostridium difficile in a hospital in Japan and risk factors for infection; Komatsu M et al.; Patients hospitalized in a hospital with a high incidence of antibiotic-associated diarrhea due to toxin A-negative, toxin B-positive (A-/B+) Clostridium difficile were retrospectively investigated to determine the clinical manifestations and risk factors for infection . Of 77 Clostridium difficile isolates obtained from 77 patients during the 1-year investigation period, 30 were A-/B+ and 47 were toxin A-positive, toxin B-positive (A+/B+) . By pulsed-field gel electrophoresis analysis, 23 of the 30 A-/B+ strains were outbreak-related, suggesting nosocomial spread of a single type of bacterium, which mainly affected patients in the wards of respiratory medicine, hematology and neurology . Using regression analysis, three factors were found to be associated with infection by A-/B+ isolates: (i) exposure to antineoplastic agents ( P=0.01, odds ratio {OR}=5.1), (ii) the use of nasal feeding tubes ( P=0.008, OR=5.2), and (iii) assignment to a certain internal medicine ward ( P=0.05, OR=3.0) . Between patients with Clostridium difficile-associated diarrhea caused by A-/B+ strains and those with A+/B+ strains, no statistically significant difference was found in body temperature, serum concentration of C-reactive protein, leukocyte count in whole blood, frequency of diarrhea, or type of underlying disease . These results indicate that A-/B+ strains of Clostridium difficile can cause intestinal infection in humans and they spread nosocomially in the same manner as A+/B+ strains.

J Ind Microbiol Biotechnol, 2003 Jul, 30(7), 427 - 32 Epub 2003 Jul 09.
Heterologous expression of the Saccharomyces cerevisiae alcohol acetyltransferase genes in Clostridium acetobutylicum and Escherichia coli for the production of isoamyl acetate; Horton CE et al.; Esters are formed by the condensation of acids with alcohols . The esters isoamyl acetate and butyl butyrate are used for food and beverage flavorings . Alcohol acetyltransferase is one enzyme responsible for the production of esters from acetyl-CoA and different alcohol substrates . The genes ATF1 and ATF2, encoding alcohol acetyltransferases from the yeast Saccharomyces cerevisiae have been sequenced and characterized . The production of acids and alcohols in mass quantities by the industrially important Clostridium acetobutylicum makes it a potential organism for exploitation of alcohol acetyltransferase activity . This report focuses on the heterologous expression of the alcohol acetyltransferases in Escherichia coli and C . acetobutylicum . ATF1 and ATF2 were cloned and expressed in E . coli and ATF2 was expressed in C . acetobutylicum . Isoamyl acetate production from the substrate isoamyl alcohol in E . coli and C . acetobutylicum cultures was determined by head-space gas analysis . Alcohol acetyltransferase I produced more than twice as much isoamyl acetate as alcohol acetyltransferase II when expressed from a high-copy expression vector . The effect of substrate levels on ester production was explored in the two bacterial hosts to demonstrate the efficacy of utilizing ATF1 and ATF2 in bacteria for ester production.

Leg Med (Tokyo), 2002 Dec, 4(4), 246 - 50
Pseudomembranous colitis with fatal outcome in a 43-year-old man; Turk EE et al.; Pseudomembranous colitis is a life-threatening complication of broad spectrum antibiotic therapy caused by Clostridium difficile . Untreated, the disease can lead to severe and in many cases fatal complications such as peritonitis due to colonic wall perforation, shock as a consequence of volume depletion, toxic megacolon and massive lower gastrointestinal haemorrhage . Fatal complications mostly occur in elderly people with a high degree of comorbidity . We report the case of a 43-year-old patient with AIDS who was admitted to hospital with abdominal pain of unknown origin and died before the correct diagnosis could be established . Autopsy and postmortem stool cultures revealed severe pseudomembranous colitis due to C . difficile with toxic cardiac and circulatory failure as cause of death.

Infect Immun, 2003 Sep, 71(9), 5371 - 5
Accumulation of Clostridium perfringens epsilon-toxin in the mouse kidney and its possible biological significance; Tamai E et al.; In this paper we show that Clostridium perfringens epsilon-toxin accumulates predominantly in the mouse kidney, where it is distributed mainly in glomeruli, capillaries, and collecting ducts . Although some pycnotic and exfoliated epithelial cells were observed in distal tubuli and collecting ducts, there were no findings indicative of severe renal injury . Bilateral nephrectomy increased the mouse lethality of the toxin, suggesting that the kidney contributes to the host defense against the lethal toxicity of epsilon-toxin.

J Biol Chem, 2003 Nov 14, 278(46), 45924 - 30 Epub 2003 Aug 21.
The crystal structure of C3stau2 from Staphylococcus aureus and its complex with NAD; Evans HR et al.; The C3stau2 exoenzyme from Staphylococcus aureus is a C3-like ADP-ribosyltransferase that ADP-ribosylates not only RhoA-C but also RhoE/Rnd3 . In this study we have crystallized and determined the structure of C3stau2 in both its native form and in complex with NAD at 1.68- and 2.02-A resolutions, respectively . The topology of C3stau2 is similar to that of C3bot1 from Clostridium botulinum (with which it shares 35% amino acid sequence identity) with the addition of two extra helices after strand beta1 . The native structure also features a novel orientation of the catalytic ARTT loop, which approximates the conformation seen for the "NAD bound" form of C3bot1 . C3stau2 orients NAD similarly to C3bot1, and on binding NAD, C3stau2 undergoes a clasping motion and a rearrangement of the phosphate-nicotinamide binding loop, enclosing the NAD in the binding site . Comparison of these structures with those of C3bot1 and related toxins reveals a degree of divergence in the interactions with the adenine moiety among the ADP-ribosylating toxins that contrasts with the more conserved interactions with the nicotinamide . Comparison with C3bot1 gives some insight into the different protein substrate specificities of these enzymes.

J Bone Miner Res, 2003 Aug, 18(8), 1453 - 60
Regulation of parathyroid hormone-stimulated phospholipase D in UMR-106 cells by calcium, MAP kinase, and small G proteins; Singh AT et al.; Signaling intermediates for PTH and phorbol activation of PLD in UMR-106 cells were determined . Calcium was required, and the effects of PTH, phorbol, and calcium were dependent on p42/44 MAP kinase and small G proteins, specifically RhoA, acting through Rho kinase . INTRODUCTION: Phospholipase D (PLD) plays a key signaling role in numerous cellular processes . PLD-stimulated hydrolysis of phosphatidylcholine (PC) generates phosphatidic acid, a source of diacylglycerol (DAG) . We previously reported that parathyroid hormone (PTH) stimulates PLD activity in UMR-106 osteoblastic cells by a protein kinase C (PKC)-independent mechanism . The current study investigated the roles of calcium, MAP kinase, and small G proteins in PTH- and phorbol-12,13-dibutyrate (PDBu)-stimulated transphosphatidylation of ethanol, a reaction catalyzed by PLD . METHODS: UMR-106 cells were labeled with 3H-palmitic and treated in the presence of ethanol . Phosphatidylethanol was separated by thin-layer chromatography and detected by autoradiography, and the bands were scraped and counted . Statistical significance of the responses from three to nine replicates was determined by ANOVA and Tukey's post-test . RESULTS AND CONCLUSIONS: PTH and PDBu effects were attenuated by EGTA, BAPTA, nifedipine, and dantrolene, whereas ionomycin or 2X calcium increased basal PLD activity . PTH activated p42/p44 MAP kinase, and the effects of PTH, PDBu, and ionomycin on PLD, but not on calcium influx, were prevented by the MEK inhibitors PD98059 and U0126 . Small G proteins were shown to be involved in the effects of PTH, PDBu, and ionomycin on PLD . Inhibition of ARF by brefeldin prevented the PLD activation by all three agonists . A nonselective Rho/Rac/cdc-42 inhibitor, Clostridium difficile toxin B, also inhibited the effects of all three agonists on PLD . More selective inhibition of RhoA with a dominant negative RhoA construct or by inhibiting geranylgeranyltransferase I antagonized the effects of PTH, PDBu, and ionomycin, as did inhibiting the downstream kinase, Rho kinase . The current results reveal the importance of calcium, MAP kinase, and small G proteins in PTH and PDBu stimulation of PLD activity in UMR-106 cells.

J Food Prot, 2003 Aug, 66(8), 1474 - 8
Inhibition of nonproteolytic, psychrotrophic clostridia and anaerobic sporeformers by sodium diacetate and sodium lactate in cook-in-bag turkey breast; Meyer JD et al.; A nonproteolytic, psychrotrophic Clostridium isolate, designated strain OMFRI1, was recovered from cook-in-bag turkey breasts (CIBTB) that displayed an intense pink discoloration and an off-odor following extended refrigerated storage . The viability of strain OMFRI1 in CIBTB containing sodium diacetate (at 0, 0.25, and 0.5%) and/or sodium lactate (at 0, 1.25, and 2.5%) was subsequently evaluated . Raw CIBTB batter was inoculated with 9 to 30 spores of strain OMFRI1 per g, vacuum packaged, cooked to an instantaneous internal temperature of 71.1 degrees C, chilled, and incubated at 4 degrees C for up to 22 weeks . In the absence of food-grade antimicrobial agents, spoilage (i.e., an off-odor) occurred within 6 weeks, and anaerobic plate counts reached 6.6 log10 CFU/g . The CIBTB containing sodium diacetate (0.25%) and that containing sodium lactate (1.25%) required 12 weeks for spoilage to occur and for anaerobic plate counts to reach 7.0 and 6.0 log10 CFU/g, respectively . When sodium diacetate (0.25%) and sodium lactate (1.25%) were used in combination, no off-odor was detected and anaerobic plate counts did not exceed 2.3 log10 CFU/g over 22 weeks of storage at 4 degrees C . In related experiments, sodium diacetate (at 0, 0.25, and 0.5%), sodium lactate (at 0, 1.25, and 2.5%), and combinations of both ingredients were evaluated in uninoculated CIBTB incubated at 25 degrees C for up to 22 days . In the absence of antimicrobial agents and in CIBTB containing sodium diacetate (0.5%), spoilage occurred within 8 days and anaerobic plate counts reached 6.8 and 6.6 log10 CFU/g, respectively . Samples of CIBTB containing sodium lactate (2.5%) showed signs of spoilage within 22 days, and anaerobic plate counts for these samples ranged from < or = 1.0 to 6.3 log10 CFU/g . In CIBTB containing both sodium lactate (2.5%) and sodium diacetate (0.25%), spoilage was not evident and anaerobic plate counts were < or = 1.0 log10 CFU/g within 22 days . These data validate the efficacy of sodium lactate and sodium diacetate in extending the shelf life of CIBTB.

J Food Prot, 2003 Aug, 66(8), 1408 - 12
Use of organic acids for the control of Clostridium perfringens in cooked vacuum-packaged restructured roast beef during an alternative cooling procedure; Sabah JR et al.; This study was conducted to determine how well Clostridium perfringens spores germinate and grow in restructured roast beef treated with different commercial organic salts during an alternative chilling procedure . The meat was prepared according to an industrial recipe (10% water, 1.5% sodium chloride, and 0.5% sodium triphosphate) . The base meat was treated with sodium citrate at 2 or 4.8% (wt/wt), buffered to a pH of 5.6, 5.0, or 4.4 (six treatments); a 60% (wt/wt) solution of sodium lactate at 2 or 4.8% (wt/wt); sodium acetate at 0.25% (wt/wt); or sodium diacetate at 0.25% (wt/wt) . Untreated meat was used as a control . Meat samples were inoculated with a three-strain cocktail of C . perfringens spores (strains ATCC 10388, NCTC 8238, and NCTC 8239) . Meat was vacuum packaged in bags and cooked in a stirred water bath to an internal temperature of 75 degrees C for 20 min, and then the bags were cooled from 54.4 to 4.4 degrees C within 18 h . Samples were taken after inoculation, after cooking, and after chilling . Spore and vegetative cell counts were obtained after incubation at 37 degrees C for 8 to 10 h in Fung's Double Tubes containing tryptose sulfite agar without egg yolk enrichment . Cooking was not sufficient to eliminate C . perfringens spores . Over the 18-h cooling period, sodium citrate, sodium lactate, and sodium diacetate reduced the growth of C . perfringens to < 1 log unit, a growth level that meets U.S . Department of Agriculture performance standards . The use of sodium citrate or sodium lactate at a concentration of > or = 2% (wt/wt) inhibited C . perfringens growth over the 18-h cooling period.

J Food Prot, 2003 Aug, 66(8), 1402 - 7
Inactivation of Clostridium botulinum type A spores by high-pressure processing at elevated temperatures; Reddy NR et al.; The effects of high-pressure treatments at various temperature-time combinations on the inactivation of spores of Clostridium botulinum type A strains 62-A and BS-A in phosphate buffer (0.067 M, pH 7.0) and in a crabmeat blend were investigated . The log unit reduction of strain 62-A spores increased significantly as the processing pressure increased from 417 to 827 MPa (from 60,000 to 120,000 lb/in2) at 75 degrees C . The reduction of BS-A and 62-A spores in either medium increased as processing temperatures increased from 60 to 75 degrees C and processing times increased from 5 to 15 or 20 min at a maximum pressure of 827 MPa . Approximately 2- and 3-log reductions of BS-A and 62-A spores, respectively, in phosphate buffer were obtained at the maximum pressure-maximum temperature combination of 827 MPa and 75 degrees C for a processing time of 20 min . Processing for 15 min at the maximum pressure-maximum temperature combination resulted in maximum reductions of 3.2 and 2.7 log units for BS-A and 62-A spores, respectively, in the crabmeat blend . Results obtained in this study indicate that the crabmeat blend did not protect BS-A and 62-A spores against inactivation by high-pressure processing.

J Immunol, 2003 Sep 1, 171(5), 2625 - 30
Inhibition of Rho family GTPases results in increased TNF-alpha production after lipopolysaccharide exposure; Monick MM et al.; These studies demonstrate that treatment of macrophages with lovastatin, a cholesterol-lowering drug that blocks farnesylation and geranylgeranylation of target proteins, increases LPS-induced TNF-alpha production . This is reversed by the addition of mevalonate, which bypasses the lovastatin block . Examination of membrane localization of RhoA, Cdc42, Rac1, and Ras demonstrated decreased membrane localization of the geranylgeranylated Rho family members (RhoA, Cdc42, and Rac1) with no change in the membrane localization of farnesylated Ras . LPS-induced TNF-alpha production in the presence of the Rho family-specific blocker (toxin B from Clostridium difficile) was significantly enhanced consistent with the lovastatin data . One intracellular signaling pathway that is required for TNF-alpha production by LPS is the extracellular signal-regulated kinase (ERK) . Significantly, we found prolonged ERK activation after LPS stimulation of lovastatin-treated macrophages . When we inhibited ERK, we blocked the lovastatin-induced increase in TNF-alpha production . As a composite, these studies demonstrate a negative role for one or more Rho family GTPases in LPS-induced TNF-alpha production.

Ann Readapt Med Phys, 2003 Jul, 46(6), 386 - 8
{Botulism toxin in practice}; Durand A et al.; Botulinum toxins (A and B) are neurotoxins derived from Clostridium botulinum . Clostridium are anaerobic bacteria . C . botulinum produces exotoxins (A to G) with distinct antigenicities . The neurotoxins inhibit the release of the neurotransmitter acetylcholine from the axon terminals of motor neurons . Botulinum toxin is officially used in clinic for the treatment of muscular hyperactivity (strabismus, blepharospam, cervical dystonia) . Botulinum toxins are also used in non recognized clinical applications: neurogenic incontinence, palmar and axillary hyperhidrosis, chronic anal fissure . The respective formulations of Botox, Dysport and Neurobloc are described . Special considerations for administration are introduced.

Ann Readapt Med Phys, 2003 Jul, 46(6), 265 - 75
{Mode of action of botulinum neurotoxin: pathological, cellular and molecular aspect}; Poulain B et al.; Several bacteria of the Clostridium genus (C . botulinum) produce 150 kDa di-chainal protein toxins referred as botulinum neurotoxins or BoNTs . They associate with non-toxic companion proteins and form a complex termed botulinum toxin or BoTx . The latter is used in clinic for therapeutic purpose . BoNTs affect cholinergic nerve terminals in periphery where they block acetylcholine release, thereby causing dysautonomia and motorparalysis (i.e . botulism) . The cellular action of BoNTs can be depicted according to a three steps model: binding, internalisation and intraneuronal action . The toxins heavy chain mediates binding to specific receptors followed by endocytotic internalisation of BoNT/receptor complex . BoNT receptors may comprise gangliosides and synaptic vesicle-associated proteins as synaptotagmins . Vesicle recycling induces BoNT internalisation . Upon acidification of vesicles, the light chain of the neurotoxin is translocated into the cytosol . Here, this zinc-endopeptidase cleaves one or two among three synaptic proteins (VAMP-synaptobrevin, SNAP25, and syntaxin) . As the three protein targets of BoNT play major role in fusion of synaptic vesicles at the release sites, their cleavage is followed by blockage of neurotransmitter exocytosis . The duration of the paralytic effect of the BoNTs is determined by 1) the turnover of their protein target; 2) the time-life of the toxin light chain in the cytosol, and 3) the sprouting of new nerve-endings that are retracted when the poisoned nerve terminal had recovered its full functionality.

Am J Clin Dermatol, 2003, 4(9), 609 - 16
Botulinum toxin A: its expanding role in dermatology and esthetics; Said S et al.; The use of botulinum toxin A in cosmetic dermatology has increased in popularity due to the efficacy and relative safety of the treatment . Botulinum toxin A is one of eight exotoxins produced by Clostridium botulinum, a Gram-positive, spore-forming anaerobe . Flaccid paralysis results from the denervation of muscle fibers at the neuromuscular junction after botulinum toxin A administration . While treating blepharospasm, the Carruthers incidentally found that botulinum toxin A improved glabellar frown lines . Dynamic rhytides occur in areas of dynamic motion . These types of lines may be improved with botulinum toxin A . There are two types of botulinum toxin A commercially available (BOTOX and Dysport); only BOTOX is currently available in the US.The efficacy and tolerability of BOTOX was best demonstrated with a multicenter, double-blind, randomized, placebo-controlled study of the efficacy and safety of botulinum toxin type A in the treatment of glabellar lines in 264 patients . There was a significantly greater reduction in glabellar line severity with BOTOX . The effect was maintained for the duration of the study (120 days) . There was low occurrence (5.4%) of mostly mild blepharoptosis in the BOTOX group.In another prospective study, it was found that about 1% of BOTOX patients reported severe headache . Botulinum toxin A can provide an alternative treatment of palmar and axillary hyperhidrosis when options such as topical agents (aluminum chloride) and iontophoresis have failed.

Ugeskr Laeger, 2003 Jul 21, 165(30), 2962 - 3
{Botulism an a 38-year-old man after ingestion of garlic in chilli oil}; Lohse N et al.; Botulism is a rare but potentially fatal disease caused by toxins produced by Clostridium botulinum . We report a case of botulism in a 38-year-old man after eating canned "garlic in chilli-oil" . The patient was treated with antiserum . The diagnosis was confirmed by detection of botulinum B toxin by a bio-assay and growth of Clostridium botulinum from the food left-overs.

Eur J Gastroenterol Hepatol, 2003 Sep, 15(9), 1051 - 3
Association of common variable immunodeficiency with atypical collagenous colitis; Byrne MF et al.; Collagenous colitis is a condition characterized by chronic, watery diarrhoea, which is diagnosed histologically as most cases reveal a normal colonoscopic appearance . The aetiology is poorly understood, but nonsteroidal anti-inflammatory drugs or infections may act as triggers for an immune-mediated process . In this report, an unusual case of collagenous colitis associated with pseudomembrane formation is described . Stool assay was negative for Clostridium difficile cytotoxin B . There are only three reports of pseudomembranes in collagenous colitis in the absence of C . difficile infection . In addition, the patient had a deficiency in immunoglobulin production, which may suggest an infective trigger to collagenous colitis . This is the first report of an association between an immunoglobulin deficiency and this unusual variant of collagenous colitis . The implications of these findings are discussed.

RNA, 2003 Sep, 9(9), 1084 - 97
Regulation of the vitamin B12 metabolism and transport in bacteria by a conserved RNA structural element; Vitreschak AG et al.; Cobalamin in the form of adenosylcobalamin (Ado-CBL) is known to repress expression of genes for vitamin B(12) biosynthesis and be transported by a posttranscriptional regulatory mechanism, which involves direct binding of Ado-CBL to 5'untranslated gene regions (5'UTR) . Using comparative analysis of genes and regulatory regions, we identified a highly conserved RNA structure, the B12-element, which is widely distributed in 5'UTRs of vitamin B(12)-related genes in eubacteria . Multiple alignment of approximately 200 B12-elements from 66 bacterial genomes reveals their common secondary structure and several extended regions of sequence conservation, including the previously known B12-box motif . In analogy to the model of regulation of the riboflavin and thiamin biosynthesis, we suggest Ado-CBL-mediated regulation based on formation of alternative RNA structures including the B12-element . In Gram-negative proteobacteria, as well as in cyanobacteria, actinobacteria, and the CFB group, the cobalamin biosynthesis and vitamin B(12) transport genes are predicted to be regulated by inhibition of translation initiation, whereas in the Bacillus/Clostridium group of Gram-positive bacteria, these genes seem to be regulated by transcriptional antitermination . Phylogenetic analysis of the B12-elements reveals a large number of likely duplications of B12-elements in several bacterial genomes . These lineage-specific duplications of RNA regulatory elements seem to be a major evolutionary mechanism for expansion of the vitamin B(12) regulon.

J Bacteriol, 2003 Sep, 185(17), 5109 - 16
Regulation of expression of scaffoldin-related genes in Clostridium thermocellum; Dror TW et al.; Clostridium thermocellum produces an extracellular multienzyme complex, termed the cellulosome, that allows efficient solubilization of crystalline cellulose . The complex is organized around a large noncatalytic protein subunit, termed CipA or scaffoldin, and is found either free in the supernatant or cell bound . The binding of the complex to the cell is mediated by three cell surface anchoring proteins, OlpB, Orf2p, and SdbA, that interact with the CipA scaffoldin . The transcriptional level of the olpB, orf2, sdbA, and cipA genes was determined quantitatively by RNase protection assays in batch and continuous cultures, under carbon and nitrogen limitation . The mRNA level of olpB, orf2, and cipA varied with growth rate, reaching 40 to 60 transcripts per cell under carbon limitation at a low growth rate of 0.04 h(-1) and 2 to 10 transcripts per cell at a growth rate of 0.35 h(-1) in batch culture . The mRNA level of sdbA was about three transcripts per cell and was not influenced by growth rate . Primer extension analysis revealed two major transcriptional start sites, at -81 and -50 bp, upstream of the translational start site of the cipA gene . The potential promoters exhibited homology to the known sigma factors sigma(A) and sigma(L) (sigma(54)) of Bacillus subtilis . Transcription from the sigma(L)-like promoter was found under all growth conditions, whereas transcription from the sigma(A)-like promoter was significant only under carbon limitation . The overall expression level obtained in the primer extension analysis was in good agreement with the results of the RNase-protection assays.

Dev Biol, 2003 Aug 15, 260(2), 522 - 35
Involvement of Rho family G protein in the cell signaling for sperm incorporation during fertilization of mouse eggs: inhibition by Clostridium difficile toxin B; Kumakiri J et al.; Sperm-egg interaction was investigated in mouse eggs freed from the zona pellucida and injected with Clostridium difficile toxin B, the inhibitor of Rho family small G proteins . Toxin B reduced in a dose-dependent manner the percentage of eggs associated with sperm fusion on the surface or sperm nucleus decondensation in the ooplasm, examined by injection of a DNA-staining dye into the egg and transfer of the dye to the fused sperm head after recording intracellular Ca(2+) responses for 100 min postinsemination . The mean number of decondensed sperm nuclei per egg was remarkably decreased by approximately 1 microg/ml toxin B in the ooplasm . This was because spermatozoa were arrested at the fusion state without developing to sperm incorporation and tended to lose cytoplasmic continuity to the egg . The fusion-arrested spermatozoa caused transient small Ca(2+) oscillations in most of eggs, while an injected spermatozoon produced repetitive large Ca(2+) spikes unaffected by toxin B . A decrease in the rate of fused spermatozoa and decondensed sperm nuclei was also caused by 20-40 microM cytochalasin D, the inhibitor of actin polymerization . Immunostaining of Rho proteins showed that Rac1 and RhoB are present in the cortical ooplasm, but Cdc42 is absent . Actin filaments in the cortex appeared to be reduced in toxin B-injected eggs . This study suggests that Rho protein(s) regulating actin-based cytoskeletal reorganization is involved in the process leading to sperm incorporation.

Environ Microbiol, 2003 Sep, 5(9), 730 - 6
Characterization of bacterial pectinolytic strains involved in the water retting process; Tamburini E et al.; Pectinolytic microorganisms involved in the water retting process were characterized . Cultivable mesophilic anaerobic and aerobic bacteria were isolated from unretted and water-retted material . A total of 104 anaerobic and 23 aerobic pectinolytic strains were identified . Polygalacturonase activity was measured in the supernatant of cell cultures; 24 anaerobic and nine aerobic isolates showed an enzymatic activity higher than the reference strains Clostridium felsineum and Bacillus subtilis respectively . We performed the first genotypic characterization of the retting microflora by a 16S amplified ribosomal DNA restriction analysis (ARDRA) . Anaerobic isolates were divided into five different groups, and the aerobic isolates were clustered into three groups . 84.6% of the anaerobic and 82.6% of the aerobic isolates consisted of two main haplotypes . Partial 16S rRNA gene sequences were determined for 12 strains, representative of each haplotype . All anaerobic strains were assigned to the Clostridium genus, whereas the aerobic isolates were assigned to either the Bacillus or the Paenibacillus genus . Anaerobic isolates with high polygalacturonase (PG) activity belong to two clearly distinct phylogenetic clusters related to C . acetobutylicum-C . felsineum and C . saccharobutylicum species . Aerobic isolates with high PG activity belong to two clearly distinct phylogenetic clusters related to B . subtilisT and B . pumilusT.

J Antimicrob Chemother, 2003 Sep, 52(3), 375 - 81 Epub 2003 Aug 13.
Antimicrobial activity of SMAP-29 against the Bacteroides fragilis group and clostridia; Arzese A et al.; OBJECTIVES: The cathelicidin-derived peptide SMAP-29 exerts rapid and broad-spectrum antimicrobial activity against aerobic bacteria and fungi . In this study, the effects of the peptide against the Bacteroides fragilis group, including antibiotic-resistant isolates, Clostridium perfringens and Clostridium difficile reference and clinical isolates, were investigated . METHODS: The microbicidal activity of SMAP-29 against eight reference and 100 clinical anaerobic strains from a national collection was assessed using a microdilution susceptibility assay, and by determining the killing kinetics on selected strains . The killing mechanism was investigated further by means of a two-colour fluorescent permeabilization assay, and by scanning electron microscopy (SEM) . RESULTS: The Bacteroides fragilis group, Clostridium reference strains and most clinical isolates were inhibited in vitro by 1-2 microM (3.2-6.4 mg/L) SMAP-29, and killed by 1.5- to 2-fold higher peptide concentrations . The anaerobic bacterial cells were 90%-100% permeabilized within 2 h of exposure to bactericidal concentrations of the peptide . The SEM images of bacteria exposed to SMAP-29 provide morphological evidence that the envelope is an important target of the bactericidal activity of this peptide . These results are consistent with earlier studies indicating that SMAP-29 kills aerobic bacteria with a membranolytic mechanism, and suggest that both aerobic and anaerobic bacteria share surface features that are targeted by this peptide . CONCLUSIONS: These studies demonstrate that the spectrum of antibacterial activity of SMAP-29 includes the B . fragilis group and Clostridium species, and encourage further investigations of the therapeutic potential of this peptide.

J Vet Med B Infect Dis Vet Public Health, 2003 May, 50(4), 178 - 82
Two cases of equine grass sickness with evidence for soil-borne origin involving botulinum neurotoxin; Bohnel H et al.; Botulism is caused by different types of Clostridium botulinum, a soil bacterium . Equine grass sickness (equine dysautonomia) is suspected of being a clinical form of this disease . On a stud where this disease occurred twice within 8 months, grass and soil samples and necropsy specimens of one horse were tested for the presence of bacterial forms and toxin of C . botulinum . Different types and type mixtures (A-E) of C . botulinum and botulinum neurotoxin were found . For the first time, it has been shown that green grass blades contain botulinum toxin . The results support the hypothesis that equine grass sickness is a clinical form of botulism, a soil-borne disease.

Int J Food Microbiol, 2003 Sep 15, 86(3), 239 - 47
An ultrastructural comparison of spores from various strains of Clostridium perfringens and correlations with heat resistance parameters; Novak JS et al.; It has been shown that Clostridium perfringens isolates associated with food poisoning carry a chromosomal cpe gene, whereas nonfood-borne human gastrointestinal disease isolates carry a plasmid cpe gene . In addition, the chromosomal cpe gene isolates exhibit greater heat resistance as compared with the plasmid cpe strains . Therefore, the current study conducted ultrastructural measurements of spores from several plasmid and chromosomal cpe-positive C . perfringens isolates . In support of the dehydration mechanism of spore heat resistance, the C . perfringens spore core average size was found to show a negative correlation with D-values for spores obtained at 100 degrees C . Dipicolinic acid (DPA) concentrations assayed for the spores did not correlate well with C . perfringens spore core averages nor with D(10)-values at 100 degrees C . Spore core thickness might be a distinguishing phenotypic characteristic used to identify heat resistance and survival potential of C . perfringens in improperly cooked foods.

J Appl Microbiol, 2003, 95(3), 508 - 20
Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis; Wang X et al.; AIM: The aim of this study was to characterize the bacterial community adhering to the mucosa of the terminal ileum, and proximal and distal colon of the human digestive tract . METHODS AND RESULTS: Pinch samples of the terminal ileum, proximal and distal colon were taken from a healthy 35-year-old, and a 68-year-old subject with mild diverticulosis . The 16S rDNA genes were amplified using a low number of PCR cycles, cloned, and sequenced . In total, 361 sequences were obtained comprising 70 operational taxonomic units (OTU), with a calculated coverage of 82.6% . Twenty-three per cent of OTU were common to the terminal ileum, proximal colon and distal colon, but 14% OTU were only found in the terminal ileum, and 43% were only associated with the proximal or distal colon . The most frequently represented clones were from the Clostridium group XIVa (24.7%), and the Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides) cluster (27.7%) . CONCLUSION: Comparison of 16S rDNA clone libraries of the hindgut across mammalian species confirms that the distribution of phylogenetic groups is similar irrespective of the host species . Lesser site-related differences within groups or clusters of organisms, are probable . SIGNIFICANCE AND IMPACT: This study provides further evidence of the distribution of the bacteria on the mucosal surfaces of the human hindgut . Data contribute to the benchmarking of the microbial composition of the human digestive tract.

Biotechnol Appl Biochem, 2003 Dec, 38(Pt 3), 241 - 51
The use of microcalorimetry to characterize tetanus neurotoxin, pertussis toxin and filamentous haemagglutinin; Krell T et al.; Tetanus neurotoxin (TeNT), pertussis toxin (PT) and pertussis filamentous haemagglutinin (FHA) are major virulence factors of Clostridium tetani and Bordetella pertussis, which are the causative agents of tetanus and whooping cough respectively . Inactivated forms of these virulence factors are the protein components of vaccines against these diseases . Here we report microcalorimetric studies to characterize these proteins . The microcalorimetric titration curves of TeNT with micelles of gangliosides GD1b, GT1b and GQ1b were biphasic . For these gangliosides a high-affinity binding site (KD 45-277 nM) can be distinguished from a lower-affinity binding event (KD 666-1190 nM) . This is direct evidence for multiple binding sites for gangliosides of the 1b series at TeNT as proposed by Emsley et al . {Emsley, Fotinou, Black, Fairweather, Charles, Watts, Hewitt and Isaacs (2000) J . Biol . Chem . 275, 8889-8894} . In agreement with previous reports, no binding was observed for gangliosides GM1, GM2, GM3 and GD2 . The thermal denaturation of TeNT was characterized by two unfolding transitions centred around 57.4 and 62.4 degrees C . The conversion of TeNT into the toxoid form by formaldehyde treatment was accompanied by a large increase in Tm (the midpoint of protein unfolding transition, that is, the temperature at which half the protein is denatured and the other half is still present in its native form) . Fetuin and asialofetuin bound to PT with similar affinities (KD 420 and 335 nM respectively) . Binding was largely enthalpy-driven and counterbalanced by an unfavourable entropy change, indicating a loss of conformational flexibility . The latter could account for the observed inhibition of ATP binding after binding to fetuin . Furthermore, the molecular limits of mature PT subunit S5 were defined by MS and N-terminal peptide sequencing . The differential-scanning-calorimetry thermogram of FHA shows four well-resolved unfolding transitions, a finding consistent with the sequential denaturation of four structural domains.

Biochemistry, 2003 Aug 19, 42(32), 9694 - 702
Rho-specific Bacillus cereus ADP-ribosyltransferase C3cer cloning and characterization; Wilde C et al.; C3-like ADP-ribosyltransferases represent an expanding family of related exoenzymes, which are produced by Clostridia and various Staphylococcus aureus strains . Here we report on the cloning and biochemical characterization of an ADP-ribosyltransferase from Bacillus cereus strain 2339 . The transferase encompasses 219 amino acids; it has a predicted mass of 25.2 kDa and a theoretical isoelectric point of 9.3 . To indicate the relationship to the family of C3-like ADP-ribosyltransferases, we termed the enzyme C3cer . The amino acid sequence of C3cer is 30 to 40% identical to other C3-like exoenzymes . By site-directed mutagenesis, Arg(59), Arg(97), Tyr(151), Arg(155), Thr(178), Tyr(180), Gln(183), and Glu(185) of recombinant C3cer were identified as pivotal residues of enzyme activity and/or protein substrate recognition . Precipitation experiments with immobilized RhoA revealed that C3cerTyr(180), which is located in the so-called "ADP-ribosylating toxin turn-turn" (ARTT) motif, plays a major role in the recognition of RhoA . Like other C3-like exoenzymes, C3cer ADP-ribosylates preferentially RhoA and RhoB and to a much lesser extent RhoC . Because the cellular accessibility of recombinant C3cer is low, a fusion toxin (C2IN-C3cer), consisting of the N-terminal 225 amino acid residues of the enzyme component of C2 toxin from Clostridium botulinum and C3cer was used to study the cytotoxic effects of the transferase . This fusion toxin caused rounding up of Vero cells comparable to the effects of Rho-inactivating toxins.

Int Microbiol, 2003 Dec, 6(4), 259 - 61 Epub 2003 Aug 09.
Stress proteins of Clostridium perfringens type A immunoreact with antiserum from rabbits infected with gas gangrene; Heredia N et al.; Various stressors were used to induce stress proteins in Clostridium perfringens . Cultures of C . perfringens FD-1041 were subjected to cold shock (28 degrees C for 1 h), acid shock (pH 4.5 for 30 min), or heat shock (50 degrees C for 30 min) . Cells were lysed and protein samples were analyzed by immunoblotting with antiserum derived from rabbits suffering from gas gangrene . Eight cold shock proteins (approximate Mr 101, 82, 70, 37, 22, 12, 10 and 6 kDa) and also eight heat shock proteins (approximate Mr 101, 82, 70, 27, 22, 16, 12 and 10 kDa) were immunoreactive with the serum . No immunoreactive proteins were detected in samples subjected to acid shock proteins and purified DnaK protein was also non-immunoreactive with the serum . These immunogenic stress proteins may be important in regulating diseases caused by C . perfringens . Such proteins could be involved in cell survival mechanisms, serve as targets during infection, or play a role in recognition of the bacteria by the host.

Appl Microbiol Biotechnol, 2004 Feb, 63(6), 653 - 8 Epub 2003 Aug 09.
Acetone butanol ethanol (ABE) production from concentrated substrate: reduction in substrate inhibition by fed-batch technique and product inhibition by gas stripping; Ezeji TC et al.; Acetone butanol ethanol (ABE) was produced in an integrated fed-batch fermentation-gas stripping product-recovery system using Clostridium beijerinckii BA101, with H(2) and CO(2) as the carrier gases . This technique was applied in order to eliminate the substrate and product inhibition that normally restricts ABE production and sugar utilization to less than 20 g l(-1) and 60 g l(-1), respectively . In the integrated fed-batch fermentation and product recovery system, solvent productivities were improved to 400% of the control batch fermentation productivities . In a control batch reactor, the culture used 45.4 g glucose l(-1) and produced 17.6 g total solvents l(-1) (yield 0.39 g g(-1), productivity 0.29 g l(-1) h(-1)) . Using the integrated fermentation-gas stripping product-recovery system with CO(2) and H(2) as carrier gases, we carried out fed-batch fermentation experiments and measured various characteristics of the fermentation, including ABE production, selectivity, yield and productivity . The fed-batch reactor was operated for 201 h . At the end of the fermentation, an unusually high concentration of total acids (8.5 g l(-1)) was observed . A total of 500 g glucose was used to produce 232.8 g solvents (77.7 g acetone, 151.7 g butanol, 3.4 g ethanol) in 1 l culture broth . The average solvent yield and productivity were 0.47 g g(-1) and 1.16 g l(-1) h(-1), respectively.

Resuscitation, 2003 Aug, 58(2), 219 - 25
Gas gangrene pyaemia with myocardial abscess formation--fatal outcome from a rare infection nowadays; Keese M et al.; We report a case of sudden death after gas gangrene . A 67-year-old male patient with diabetes mellitus and chronic renal failure (on haemodialysis three times a week) presented in the surgical emergency department with a severe swelling and crepitation in the right groin . No signs of trauma were present-except for a well-healed, 1-year-old scar after femoro-popliteal bypass surgery . Two days earlier, he had presented to the internal medicine department with epigastric pain and had left against medical advice . On readmission the patient was initially conscious and in a stable cardiopulmonary condition but developed sudden cardiocirculatory failure and underwent resuscitation . Despite all resuscitation measures, including the administration of high doses of catecholamines and the treatment of hyperkalemia, the patient died . Autopsy revealed septicaemia with rod-shaped gram-positive bacteria, typical of Clostridium perfringens, evidenced by multiple areas of myonecrosis . Abscess formation was found in the myocardium . Clostridial gas gangrene is a rare clinical condition . Unless immediate diagnosis and adequate therapy measures are taken, the outcome and chances for survival are poor as demonstrated by this case.

Med Dosw Mikrobiol, 2003, 55(1), 53 - 60
{Comparison of AP-PCR methods, ribotyping (PCR-ribotyping) and pulsed-field electrophoresis (PFGE) for strains of Clostridium difficile producing toxin B and not producing toxin A}; Pituch H et al.; In this study were used AP-PCR, PCR-ribotyping and pulsed-field elecrophoresis (PFGE) for comparative study of toxin A-negative/toxin B-posi-tive Clostridium difficile strains with deletion in toxin A gen . We investigated nine unrelated clinical strains, isolated from different units and different time from patients suffering to antibiotic associated diarrhea (AAD) . We found that toxin A-negative/toxin B-positive C . difficile strains isolated in Poland belonging to a single genotype A, are being similar to the Japanese strains.

Expert Opin Drug Saf, 2003 Jul, 2(4), 421 - 9
Gastrointestinal side effects of drugs; Makins R et al.; Drugs can have adverse effects on any part of the gastrointestinal (GI) tract from mouth to colon . It is essential that a detailed and accurate drug history is taken in patients presenting with GI complaints . Many drug-induced effects will regress or heal on cessation of treatment . NSAIDs are usually associated with gastric and duodenal ulcers but are also recognised to cause lichen planus in the mouth, oesophageal inflammation and strictures, and small bowel and colonic ulcers and strictures . A newer class of anti-inflammatory drugs, the cyclooxygenase-2 (COX-2)-selective inhibitors, have been developed and have a more favourable GI safety profile than standard NSAIDs . Acute diarrhoea, relapse of inflammatory bowel disease (IBD), microscopic colitis and acute pancreatitis are also induced by ingestion of standard NSAIDs . The calcium antagonists, phenytoin and cyclosporin, induce gum hyperplasia, particularly in patients with poor oral hygiene . Alendronate, a bisphosphonate, has been associated with development of oesophageal ulcers, and specific recommendations are now given to reduce this complication . Of the many different forms of colitis associated with drug ingestion, the most frequent is pseudomembranous colitis . This is a complication of antibiotics and is caused by the toxin produced by Clostridium difficile . Many drugs have been associated with the development of acute pancreatitis, although a definite cause and effect relationship has been shown for only a few drugs . These include didanosine, furosomide, corticosteroids, azathioprine and sodium valproate.

Appl Environ Microbiol, 2003 Aug, 69(8), 4985 - 8
Development of a sensitive gene expression reporter system and an inducible promoter-repressor system for Clostridium acetobutylicum; Girbal L et al.; A sensitive gene expression reporter system was developed for Clostridium acetobutylicum ATCC 824 by using a customized gusA expression cassette . In discontinuous cultures, time course profiles of beta-glucuronidase specific activity reflected adequately in vivo dynamic up- and down-regulation of acidogenesis- and/or solventogenesis-associated promoter expression in C . acetobutylicum . Furthermore, a new inducible gene expression system was developed in C . acetobutylicum, based on the Staphylococcus xylosus xylose operon promoter-repressor regulatory system.

Appl Environ Microbiol, 2003 Aug, 69(8), 4951 - 65
Overexpression of groESL in Clostridium acetobutylicum results in increased solvent production and tolerance, prolonged metabolism, and changes in the cell's transcriptional program; Tomas CA et al.; DNA array and Western analyses were used to examine the effects of groESL overexpression and host-plasmid interactions on solvent production in Clostridium acetobutylicum ATCC 824 . Strain 824(pGROE1) was created to overexpress the groESL operon genes from a clostridial thiolase promoter . The growth of 824(pGROE1) was inhibited up to 85% less by a butanol challenge than that of the control strain, 824(pSOS95del) . Overexpression of groESL resulted in increased final solvent titers 40% and 33% higher than those of the wild type and plasmid control strains, respectively . Active metabolism lasted two and one half times longer in 824(pGROE1) than in the wild type . Transcriptional analysis of 824(pGROE1) revealed increased expression of motility and chemotaxis genes and a decrease in the expression of the other major stress response genes . Decreased expression of the dnaKJ operon upon overexpression of groESL suggests that groESL functions as a modulator of the CIRCE regulon, which is shown here to include the hsp90 gene . Analysis of the plasmid control strain 824(pSOS95del) revealed complex host-plasmid interactions relative to the wild-type strain, resulting in prolonged biphasic growth and metabolism . Decreased expression of four DNA gyrases resulted in differential expression of many key primary metabolism genes . The ftsA and ftsZ genes were expressed at higher levels in 824(pSOS95del), revealing an altered cell division and sporulation pattern . Both transcriptional and Western analyses revealed elevated stress protein expression in the plasmid-carrying strain.

Expert Rev Vaccines, 2002 Dec, 1(4), 495 - 505
Live bacteria as the basis for immunotherapies against cancer; Chabalgoity JA et al.; For more than a century, bacteria and bacterial products have been used for the treatment of cancer . Starting from the practical observation of tumor regression in individuals with concomitant bacterial infection, the field has evolved into some standard clinical practices, such as the use of BCG for the treatment of superficial bladder cancer . However, in the last few years, new applications have started to emerge that may profoundly change the perspective of the field . BCG can be engineered to express cytokines to improve its efficacy . Bacteria such as Salmonella and Listeria can be attenuated by genetically-defined mutations and provide effective vehicles for DNA vaccines encoding tumor-associated antigens . Salmonella and nonpathogenic strains of Clostridium can selectively accumulate in tumors in vivo, providing attractive delivery systems to target immunomodulatory molecules and therapeutic agents to the tumor site . Many of these new developments have been attempted for prophylactic or therapeutic vaccination in several different experimental models of cancer and in many cases, results from clinical trials are now emerging . There is still some way to go before achieving products that could be in routine use, but the field has great promise for the development of more effective immunotherapies for several different cancers . In this paper, we will review the current state of such applications and highlight some of the directions that the field may take.

J Biol Chem, 2003 Oct 17, 278(42), 40455 - 63 Epub 2003 Aug 04.
Molecular cloning, expression, and characterization of novel hemolytic lectins from the mushroom Laetiporus sulphureus, which show homology to bacterial toxins; Tateno H et al.; We describe herein the cDNA cloning, expression, and characterization of a hemolytic lectin and its related species from the parasitic mushroom Laetiporus sulphureus . The lectin designated LSL (L . sulphureus lectin), is a tetramer composed of subunits of approximately 35 kDa associated by non-covalent bonds . From a cDNA library, three similar full-length cDNAs, termed LSLa, LSLb, and LSLc, were generated, each of which had an open reading frame of 945 bp encoding 315 amino acid residues . These proteins share 80-90% sequence identity and showed structural similarity to bacterial toxins: mosquitocidal toxin (MTX2) from Bacillus sphaericus and alpha toxin from Clostridium septicum . Native and recombinant forms of LSL showed hemagglutination and hemolytic activity and both activities were inhibited by N-acetyllactosamine, whereas a C-terminal deletion mutant of LSLa (LSLa-D1) retained hemagglutination, but not hemolytic activity, indicating the N-terminal domain is a carbohydrate recognition domain and the C-terminal domain functions as an oligomerization domain . The LSL-mediated hemolysis was protected osmotically by polyethylene glycol 4000 and maltohexaose . Inhibition studies showed that lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc) was the best inhibitor for LSL . These results indicate that LSL is a novel pore-forming lectin homologous to bacterial toxins.

QJM, 2003 Aug, 96(8), 579 - 82
Reducing delays in the diagnosis and treatment of Clostridium difficile diarrhoea; Frenz MB et al.; BACKGROUND: The diagnosis of Clostridium difficile diarrhoea is often delayed . AIM: To assess and reduce delays in diagnosis and treatment . DESIGN: Two-part study: retrospective audit then prospective observational . METHODS: The retrospective study audited cases positive for C . difficile culture or toxin A between June 2000 and January 2001 . Cases were reviewed regarding demographic characteristics and the timing from onset of symptoms to testing and treatment (n = 27) . In the prospective study, efforts were made to increase the awareness of medical staff about C . difficile diarrhoea, and testing for C . difficile toxin A assay was substituted for stool culture and external toxin analysis . Data were prospectively analysed for demographic characteristics and time from onset of symptoms to testing and treatment (n = 24) . RESULTS: There were no significant differences in age (76 vs . 78 years), male to female ratio (1:2 vs . 1:4), reason for admission, specialty responsible, or mortality rate (40% vs . 37%) during the presenting admission . Preceding antibiotic use, the combination of antibiotics used and subsequent treatment also did not differ between groups . The time from onset of diarrhoea to sampling was reduced from 4.7 days (range 3-30) to 0.8 days (range 0-5; p </= 0.05) . The time from sampling to treatment was reduced from 7.7 days (5-34) to 2.8 days (2-9; p </= 0.05) . DISCUSSION: The combination of increased awareness of C . difficile diarrhoea and the change to in-house toxin testing significantly reduced the time from onset of symptoms to sampling and treatment.

J Bacteriol, 2003 Aug, 185(16), 4727 - 33
A rhamnogalacturonan lyase in the Clostridium cellulolyticum cellulosome; Pages S et al.; Clostridium cellulolyticum secretes large multienzymatic complexes with plant cell wall-degrading activities named cellulosomes . Most of the genes encoding cellulosomal components are located in a large gene cluster: cipC-cel48F-cel8C-cel9G-cel9E-orfX-cel9H-cel9J-man5K-cel9M . Downstream of the cel9M gene, a new open reading frame was discovered and named rgl11Y . Amino acid sequence analysis indicates that this gene encodes a multidomain pectinase, Rgl11Y, containing an N-terminal signal sequence, a catalytic domain belonging to family 11 of the polysaccharide lyases, and a C-terminal dockerin domain . The present report describes the biochemical characterization of a recombinant form of Rgl11Y . Rgl11Y cleaves the alpha-L-Rhap-(1-->4)-alpha-D-GalpA glycosidic bond in the backbone of rhamnogalacturonan I (RGI) via a beta-elimination mechanism . Its specific activity on potato pectic galactan and rhamnogalacturonan was found to be 28 and 3.6 IU/mg, respectively, indicating that Rgl11Y requires galactan decoration of the RGI backbone . The optimal pH of Rgl11Y is 8.5 and calcium is required for its activity . Rgl11Y was shown to be incorporated in the C . cellulolyticum cellulosome through a typical cohesin-dockerin interaction . Rgl11Y from C . cellulolyticum is the first cellulosomal rhamnogalacturonase characterized.

Berl Munch Tierarztl Wochenschr, 2003 Jul-Aug, 116(7-8), 269 - 73
{Diagnosis of botulism since 1995 . Report of test results}; Bohnel H et al.; Since 1970 our laboratory is specialized in diagnosis of clostridial diseases, including Clostridium botulinum and botulism . Since 1995, samples from more than 900 suspected botulinal cases were received, mainly in cattle, horses and men . 524 outbreaks were diagnosed as clearly positive by toxin neutralisation; 83 cases remained inconclusive with the toxin neutralisation . The geographical distribution of the positive cases in Germany is demonstrated for cattle and horses . Dispatch and treatment of specimens and interpretation of results are discussed.

Biochem Biophys Res Commun, 2003 Aug 1, 307(3), 584 - 8
Expression of recombinant Clostridium difficile toxin A using the Bacillus megaterium system; Burger S et al.; Pathogenic Clostridium difficile produces two major protein toxins, toxin A and toxin B . We used the Bacillus megaterium expression system for expression of recombinant toxin A . The construct for the toxin A gene was obtained by the following cloning strategy: the gene for toxin A was generated in three parts, each of them ligated into a cloning vector . The three parts were sequentially fused to the complete gene . The holotoxin gene was ligated into the expression vector pWH1520 . This vector was modified to generate a toxin with a C-terminally located His-tag . Gene expression in the B . megaterium system resulted in an approximate 300 kDa protein, which was identified by specific antibody as toxin A . Recombinant, His-tagged toxin A was purified by Ni(2+) as well as thyroglobulin affinity chromatography . Characterization of the recombinant toxin A showed identical cytotoxicity and in vitro-glucosyltransferase activity as the native toxin A from C . difficile.

FEMS Microbiol Lett, 2003 Jul 29, 224(2), 191 - 5
New integration vector using a cellulase gene as a screening marker for Lactobacillus; Jang SJ et al.; The new integration vector for Lactobacillus, pJC4, was developed using the extracellular endoglucanase A gene (celA) of Clostridium thermocellum as a screening marker . pJC4 was transformed into four Lactobacillus species, Lb . johnsonii, Lb . gasseri, Lb . bulgaricus, and Lb . plantarum . In each species, the pJC4 integrants were easily and accurately detected by the appearance of a clear halo on a cellulase screening plate without any false transformants . Polymerase chain reaction and Southern hybridization indicated that all transformants with clear halos contained pJC4 in their chromosomal DNAs . The celA gene could be a useful screening marker for other lactic acid bacteria.

Int J Syst Evol Microbiol, 2003 Jul, 53(Pt 4), 1019 - 29
Characterization of novel psychrophilic clostridia from an Antarctic microbial mat: description of Clostridium frigoris sp . nov., Clostridium lacusfryxellense sp . nov., Clostridium bowmanii sp . nov . and Clostridium psychrophilum sp . nov . and reclassification of Clostridium laramiense as Clostridium estertheticum subsp . laramiense subsp . nov; Spring S et al.; Taxonomic studies were performed on four strains (D-1/D-an/IIT, C/C-an/B1T, A-1/C-an/C1T and A-1/C-an/IT) of anaerobic, gram-positive, spore-forming bacteria originally isolated from a mat sample retrieved from a shallow, moated area around Lake Fryxell, an Antarctic freshwater lake . Phylogenetic analyses based on 16S rRNA gene sequence data indicated that these strains are affiliated with cluster I clostridia and form a coherent group with Clostridium estertheticum and Clostridium laramiense . Similarity values among 16S rRNA gene sequences within this assemblage ranged between 96.7 and 99.8% . Despite the close phylogenetic relationship, several distinguishing phenotypic traits were found among the novel strains using a polyphasic approach . All strains were psychrophilic, but the temperature optimum for growth differed markedly, ranging from 4 to 16 degrees C . In addition, substrate utilization patterns, fermentation end products, cellular fatty acid profiles and morphological traits enabled a clear differentiation between the strains . DNA-DNA hybridization experiments revealed that each of the four novel strains represents a distinct species, with DNA-DNA similarity values to related strains in the range 16-62% . In contrast, the type strains of C . estertheticum and C . laramiense shared 79% DNA-DNA similarity, indicating a close relationship at the species level . On the basis of genetic and phenotypic properties, it is proposed to designate four novel species of the genus Clostridium to harbour the newly isolated strains: Clostridium frigoris sp . nov . (type strain D-1/D-an/IIT=DSM 14204T=ATCC BMAA-579T), Clostridium lacusfryxellense sp . nov . (type strain C/C-an/B1T=DSM 14205T=ATCC BAA-580T), Clostridium bowmaniisp . nov . (type strain A-1/C-an/C1T=DSM 14206T=ATCC BAA-581T) and Clostridium psychrophilum sp . nov . (type strain A-1/C-an/IT=DSM 14207T=ATCC BAA-582T) . It is also proposed to unite C . laramiense and C . estertheticum under C . estertheticum . The subspecies C . estertheticum subsp . laramiense subsp . nov . is established, represented by strain ATCC 51254T (=DSM 14884T) . The type strain of C . estertheticum subsp . estertheticum remains NCIMB 12511T (=DSM 8809T).

Gastroenterology, 2003 Aug, 125(2), 413 - 20
Clostridium difficile toxin B is an inflammatory enterotoxin in human intestine; Savidge TC et al.; BACKGROUND & AIMS: Clostridium difficile causes antibiotic-associated diarrhea and pseudomembranous colitis, diseases afflicting millions of people each year . Although C . difficile releases 2 structurally similar exotoxins, toxin A and toxin B, animal experiments suggest that only toxin A mediates diarrhea and enterocolitis . However, toxin A-negative/toxin B-positive strains of C . difficile recently were isolated from patients with antibiotic-associated diarrhea and colitis, indicating that toxin B also may be pathogenic in humans . METHODS: Here we used subcutaneously transplanted human intestinal xenografts in immunodeficient mice to generate a chimeric animal model for C . difficile toxin-induced pathology of human intestine . RESULTS: We found that intraluminal toxin B, like equivalent concentrations of toxin A, induced intestinal epithelial cell damage, increased mucosal permeability, stimulated interleukin (IL)-8 synthesis, and caused an acute inflammatory response characterized by neutrophil recruitment and tissue damage . Laser capture microdissection and real-time quantitative reverse-transcription polymerase chain reaction (RT-PCR) showed that intestinal epithelial cell-specific IL-8 gene expression also was increased significantly after luminal exposure to C . difficile toxins in vivo . CONCLUSIONS: We conclude that C . difficile toxin B, like toxin A, is a potent inflammatory enterotoxin for human intestine . Future therapeutic or vaccine strategies for C . difficile infection therefore need to target both toxins.

Oncol Rep, 2003 Sep-Oct, 10(5), 1351 - 6
Rho regulates the hepatocyte growth factor/scatter factor-stimulated cell motility of human oral squamous cell carcinoma cells; Kitajo H et al.; To investigate the effects of hepatocyte growth factor/scatter factor (HGF/SF) on the invasion and metastasis of human oral squamous cell carcinoma (SCC) cells, we examined cell motility and intercellular signal transduction of a human oral SCC cell line (SAS) obtained from the primary lesion of a tongue carcinoma . HGF/SF stimulation significantly enhanced the motility of SAS cells in a dose-dependent manner . Clostridium botulinum C3 exoenzyme (C3), which is known to selectively impair the function of Ras-related small G-protein p21rho (Rho), significantly reduced the motility of SAS cells . HGF/SF stimulation also enhanced the tyrosine phosphorylation of HGF receptors (c-Met) and focal adhesion kinase (FAK) on SAS cells, but C3 completely inhibited the phosphorylation of FAK . Furthermore, it was observed that Rho A protein, normally located around the nuclear area, was translocated to the membrane and levels in the cytolysate increased following HGF/SF stimulation with no change in Rho A mRNA . These results suggest that the activation of FAK caused by phosphorylation of c-Met may mediate the HGF/SF-induced motility of human oral SCC cells, and that Rho protein regulates the tyrosine phosphorylation of FAK through translocation from the nucleus to the membrane.

Ann Emerg Med, 2003 Aug, 42(2), 226 - 9
Foodborne botulism in a six-month-old infant caused by home-canned baby food; Armada M et al.; Previously reported cases of botulism in infants have been diagnosed as infant botulism; that is, botulism caused by intestinal colonization by Clostridium botulinum with intraluminal production and absorption of toxin . Foodborne botulism is caused by ingestion of preformed toxin . We describe an unusual case of foodborne botulism in a 6-month-old infant caused by the ingestion of improperly prepared home-canned baby food . This represents the youngest age of onset for foodborne botulism in the United States of which we are aware and illustrates the need to rule out foodborne botulism, which represents a public health emergency, regardless of the patient's age . The diagnosis could have been readily missed or delayed in this case because the weakness was rapidly progressive rather than insidious, as is typical of infant botulism.

Biotechnol Lett, 2003 Jan, 25(2), 121 - 5
Continuous cultures of Clostridium acetobutylicum: culture stability and low-grade glycerol utilisation; Andrade JC et al.; Continuous cultures of two strains of Clostridium acetobutylicum were stable for over 70 d when grown on glucose/glycerol mixtures . Butanol was the major fermentation end-product, accounting for 43 to 62% (w/w) of total products . Low-grade glycerol {65% (w/v) purity} could replace commercial glycerol {87% (w/v) purity}, leading to a similar fermentation pattern: a butanol yield of 0.34 (mol/mol), a butanol productivity of 0.42 g l(-1) h(-1) and a 84% (w/w) glycerol consumption were attained when cultures were grown at pH 6 and D = 0.05 h(-1); butanol accounted for 94% (w/w) of total solvents . These values are among the highest reported in literature for C . acetobutylicum simple chemostats.

Presse Med, 2003 Jun 7, 32(20), 935 - 41
{Diarrhea during enteral feeding}; Schneider SM et al.; PREVALENCE: Diarrhea occurs in 2 to 70% of tube-fed patients, depending on their disease (with an increased risk in critically ill patients) and on the definition of diarrhea used . CONSEQUENCES: Diarrhea increases morbidity, particularly since the nutritional goals are harder to reach . CAUSES: Relevant causes today are related to the nutrition (irregular and too high output, jejunal site, low sodium and fiber contents), to the patient (malnutrition, stress, underlying diseases), and predominantly to concomitant treatments (antibiotics, with an increased risk of Clostridium difficile infection, laxative-containing drugs) . TREATMENT: Treatment of the cause is only valid when it has been identified . Symptomatic treatment principally combines compensation for water and electrolyte loss and drugs that slow down the transit . Prevention associates the regulation of the administration rate and the fight against Clostridium difficile . Restoration of the colonic microflora appears the key to success and some prebiotics and probiotics have demonstrated preventive effects . Further research in this direction is warranted.

Equine Vet J, 2003 Jul, 35(5), 465 - 71
Clostridium difficile: prevalence in horses and environment, and antimicrobial susceptibility; Baverud V et al.; REASONS FOR PERFORMING STUDY: Clostridium difficile has been associated with acute colitis in mature horses . OBJECTIVES: To survey C . difficile colonisation of the alimentary tract with age, occurrence of diarrhoea and history of antibiotic therapy; and to study the occurrence and survival of C . difficile in the environment and antimicrobial susceptibility of isolated strains . METHODS: A total of 777 horses of different breeds, age and sex were studied . Further, 598 soil samples and 434 indoor surface samples were examined . Antimicrobial susceptibility of 52 strains was investigated by Etest for 10 antibiotics . RESULTS: In horses that developed acute colitis during antibiotic treatment, 18 of 43 (42%) were positive to C . difficile culture and 12 of these (28%) were positive in the cytotoxin B test . Furthermore, C . difficile was isolated from a small number of diarrhoeic mature horses (4 of 72 {6%}) with no history of antibiotic treatment, but not from 273 healthy mature horses examined or 65 horses with colic . An interesting new finding was that, in normal healthy foals age < 14 days, C . difficile was isolated from 1/3 of foals (16 of 56 {29%}) . All older foals (170) except one were negative . Seven of 16 (44%) nondiarrhoeic foals treated with erythromycin or gentamicin in combination with rifampicin were also excretors of C . difficile . On studfarms, 14 of 132 (11%) outdoor soil samples were positive for C . difficile in culture, whereas only 2 of 220 (1%) soil samples from farms with mature horses were positive for C . difficile (P = < 0.001) . By PCR, it was demonstrated that strains from the environment and healthy foals can serve as a potential reservoir of toxigenic C . difficile . The experimental study conducted here found that C . difficile survived in nature and indoors for at least 4 years in inoculated equine faeces . The susceptibility of 52 strains was investigated for 10 antibiotics and all were susceptible to metronidazole (MIC < or = 4 mg/l) and vancomycin (MIC < or = 2 mg/l) . CONCLUSIONS: C . difficile is associated with acute colitis in mature horses, following antibiotic treatment . Furthermore, C . difficile was isolated from 1 in 3 normal healthy foals age < 14 days . POTENTIAL RELEVANCE: Strains from healthy foals and the environment can serve as a potential reservoir of toxigenic C . difficile.

Infect Immun, 2003 Aug, 71(8), 4260 - 70
Death pathways activated in CaCo-2 cells by Clostridium perfringens enterotoxin; Chakrabarti G et al.; Clostridium perfringens enterotoxin (CPE), a 35-kDa polypeptide, induces cytotoxic effects in the enterocyte-like CaCo-2 cell culture model . To identify the mammalian cell death pathway(s) mediating CPE-induced cell death, CaCo-2 cultures were treated with either 1 or 10 micro g of CPE per ml . Both CPE doses were found to induce morphological damage and DNA cleavage in CaCo-2 cells . The oncosis inhibitor glycine, but not a broad-spectrum caspase inhibitor, was able to transiently block both of those pathological effects in CaCo-2 cells treated with the higher, but not the lower, CPE dose . Conversely, a caspase 3/7 inhibitor (but not glycine or a caspase 1 inhibitor) blocked morphological damage and DNA cleavage in CaCo-2 cells treated with the lower, but not the higher, CPE dose . Collectively, these results indicate that lower CPE doses cause caspase 3/7-dependent apoptosis, while higher CPE doses induce oncosis . Apoptosis caused by the lower CPE dose was shown to proceed via a classical pathway involving mitochondrial membrane depolarization and cytochrome c release . As the CPE concentrations used in this study for demonstrating apoptosis and oncosis have pathophysiologic relevance, these results suggest that both oncosis and apoptosis may occur in the intestines during CPE-associated gastrointestinal disease.

Am J Gastroenterol, 2003 Jul, 98(7), 1569 - 72
Clinical correlation of toxin and common antigen enzyme immunoassay testing in patients with Clostridium difficile disease; Lee SD et al.; OBJECTIVE: The aim of the present study was to assess the correlation of Triage Micro Clostridium difficile Panel and toxin B cytotoxicity assay with the clinical diagnosis of C . difficile diarrhea . METHODS: The subjects evaluated were 98 patients with diarrhea for whom stool was submitted for testing for C . difficile . Clinical symptoms prompting evaluation, laboratory values, comorbid illness, and treatment outcomes that provided clinical insight into the etiology of the diarrhea were recorded . These data were then reviewed by two experienced clinical gastroenterologists who were blinded to the results of the Triage enzyme immunoassay and cytotoxin B assay . The final diagnosis of C . difficile diarrhea was based on the patient's clinical evaluation and symptoms, treatment, and subsequent outcome . RESULTS: Of 98 patients evaluated, 33 were diagnosed with C . difficile diarrhea by clinical criteria . The toxin B assay displayed 88% sensitivity and 100% specificity and positive predictive value . The toxin A component of the Triage Panel displayed 45% sensitivity but 98% specificity and 94% positive predictive value . The common antigen had 97% sensitivity and 95% negative predictive value . Among the 45 patients with only a common antigen detected, the most common diagnoses for diarrhea were chemotherapy-related, antibiotic-related diarrhea, and graft versus host disease . CONCLUSIONS: Our data show that both the Triage Micro C . difficile Panel and cytotoxin B for C . difficile have a high positive predictive value and negative predictive value for C . difficile diarrhea . The Triage Micro C . difficile Panel provides a reasonable alternative to the cytotoxin B assay in the assessment of clinically relevant C . difficile . The Triage Micro C . difficile Panel is less labor intensive and less expensive than cytotoxin B assay . The panel approach improves on the individual assay performances by increasing sensitivity and negative predictive value . When both common antigen and toxin A are positive, the likelihood of C . difficile diarrhea is high; conversely, when both results are negative, the likelihood of C . difficile diarrhea is low.

J Biol Chem, 2003 Oct 10, 278(41), 40020 - 5 Epub 2003 Jul 18.
Increased vesicle recycling in response to osmotic cell swelling . Cause and consequence of hypotonicity-provoked ATP release; van der Wijk T et al.; Osmotic swelling of Intestine 407 cells leads to an immediate increase in cell surface membrane area as determined using the fluorescent membrane dye FM 1-43 . In addition, as measured by tetramethylrhodamine isothiocyanate (TRITC)-dextran uptake, a robust (>100-fold) increase in the rate of endocytosis was observed, starting after a discrete lag time of 2-3 min and lasting for approximately 10-15 min . The hypotonicity-induced increase in membrane surface area, like the cell swelling-induced release of ATP (Van der Wijk, T., De Jonge, H . R., and Tilly, B . C . (1999) Biochem . J . 343, 579-586), was diminished after 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester loading or cytochalasin B treatment . Uptake of TRITC-dextrans, however, was not affected . Treatment of the cells with the vesicle-soluble N-ethylmaleimide-sensitive factor attachment protein receptor-specific protease Clostridium botulinum toxin F not only nearly eliminated the hypotonicity-induced increase in membrane surface area but also strongly diminished the release of ATP, indicating the involvement of regulated exocytosis . Both the ATP hydrolase apyrase and the MEK inhibitor PD098059 diminished the osmotic swelling-induced increase in membrane surface area as well as the subsequent uptake of TRITC-dextrans . Taken together, the results indicate that extracellular ATP is required for the hypotonicity-induced vesicle recycling and suggest that a positive feedback loop, involving purinergic activation of the Erk-1/2 pathway, may contribute to the release of ATP from hypo-osmotically stimulated cells.

J Food Prot, 2003 Jul, 66(7), 1304 - 9
A review of recent taxonomic changes in seven genera of bacteria commonly found in foods; Jay JM; The purpose of this review is to call attention to the new approaches now in use that classify bacteria primarily on the basis of phylogenetic criteria . The primary laboratory methods for ascertaining phylogenetic relationships entail the use of 16S rRNA and DNA sequence comparisons . These methods have been applied to some long-established genera of bacteria of importance in foods, and seven genera (Bacillus, Clostridium, Flavobacterium, Micrococcus, Pseudomonas, Staphylococcus, and Xanthomonas) are summarized in this review . Some 121 species and strains from these seven genera were recently transferred to 36 newly created genera . The new genera consist of general environmental contaminants and some food spoilage species . To date, significant taxonomic changes have not been reported for foodborne pathogens . These taxonomic shifts need to be examined with respect to the utility of widely used microscopic, cultural, and serologic methods for future use in food microbiology . Phylogeny-based detection and identification methods for foodborne bacteria must be further developed for larger numbers of foodborne genera in order to keep up with the current trends in taxonomy.

J Food Prot, 2003 Jul, 66(7), 1227 - 32
Impact of cooking, cooling, and subsequent refrigeration on the growth or survival of Clostridium perfringens in cooked meat and poultry products; Kalinowski RM et al.; In January 1999, the Food Safety and Inspection Service (FSIS) finalized performance standards for the cooking and chilling of meat and poultry products in federally inspected establishments . More restrictive chilling (stabilization) requirements were adopted despite the lack of strong evidence of a public health risk posed by industry practices employing the original May 1988 guidelines (U.S . Department of Agriculture FSIS Directive 7110.3) . Baseline data led the FSIS to estimate a "worst case" of 10(4) Clostridium perfringens cells per g in raw meat products . The rationale for the FSIS performance standards was based on this estimate and the assumption that the numbers detected in the baseline study were spores that could survive cooking . The assumptions underlying the regulation stimulated work in our laboratory to help address why there have been so few documented outbreaks of C . perfringens illness associated with the consumption of commercially processed cooked meat and poultry products . Our research took into account the numbers of C . perfringens spores in both raw and cooked products . One hundred ninety-seven raw comminuted meat samples were cooked to 73.9 degrees C and analyzed for C . perfringens levels . All but two samples had undetectable levels (<3 spores per g) . Two ground pork samples contained 3.3 and 66 spores per g . Research was also conducted to determine the effect of chilling on the outgrowth of C . perfringens spores in cured and uncured turkey . Raw meat blends inoculated with C . perfringens spores, cooked to 73.9 degrees C, and chilled according to current guidelines or under abuse conditions yielded increases of 2.25 and 2.44 log10 CFU/g for uncured turkey chilled for 6 h and an increase of 3.07 log10 CFU/g for cured turkey chilled for 24 h . No growth occurred in cured turkey during a 6-h cooling period . Furthermore, the fate of C . perfringens in cooked cured and uncured turkey held at refrigeration temperatures was investigated . C . perfringens levels decreased by 2.52, 2.54, and 2.75 log10 CFU/g in cured turkey held at 0.6, 4.4, and 10 degrees C, respectively, for 7 days . Finally, 48 production lots of ready-to-eat meat products that had deviated from FSIS guidelines were analyzed for C . perfringens levels . To date, 456 samples have been tested, and all but 25 (ranging from 100 to 710 CFU/g) of the samples contained C . perfringens at levels of <100 CFU/g . These results further support historical food safety data that suggest a very low public health risk associated with C . perfringens in commercially processed ready-to-eat meat and poultry products.

Rev Med Chil, 2003 Apr, 131(4), 397 - 403
{High incidence and complications of Clostridium difficile diarrhea among patients with renal diseases}; Herrera P et al.; BACKGROUND: Clostridium difficile is the main agent causing antimicrobial associated nosocomial diarrhea . Chronic renal failure is a risk factor for this type of diarrhea . AIM: To study the incidence and complications of Clostridium difficile diarrhea in a university hospital and among patients with renal diseases . PATIENTS AND METHODS: Retrospective review of all cases of Clostridium difficile diarrhea that occurred in a university hospital, between June 2000 and May 2001 . RESULTS: In the Nephrology Unit, 48 episodes of Clostridium difficile diarrhea occurred in 35 patients (7 cases per 100 discharges/year) . This figure is higher than the global incidence in the hospital (0.53 cases per 100 discharges/year, p < 0.001) . The mean age of the 33 patients with renal diseases was 63 years old and 17 of them were female . Their main diagnoses were chronic renal failure in hemodialysis in 48%, uremic syndrome in 36% and renal transplant in 6% . Seventy nine percent had a history of antimicrobial use (42% quinolones and 36% cephalosporins) . In 3 patients, the only risk factor was chronic renal failure . Seventy five percent responded to metronidazole and in 27%, diarrhea recidivated, compared with a 6% recurrence rate in other units, p < 0.02) . Eight patients died during hospital stay . CONCLUSIONS: Among patients with renal diseases, Clostridium difficile is frequent and associated with a high recurrence rate and mortality . Chronic renal failure may be a risk factor for its development.

J Biol Chem, 2003 Sep 26, 278(39), 37360 - 7 Epub 2003 Jul 17.
Clostridium botulinum C2 toxin: low pH-induced pore formation is required for translocation of the enzyme component C2I into the cytosol of host cells; Blocker D et al.; The binary Clostridium botulinum C2 toxin consists of two individual proteins, the transport component C2II (80 kDa) and the enzyme component C2I, which ADP-ribosylates G-actin in the cytosol of cells . Trypsin-activated C2II (C2IIa) forms heptamers that bind to the cell receptor and mediate translocation of C2I from acidic endosomes into the cytosol of target cells . Here, we report that translocation of C2I across cell membranes is accompanied by pore formation of C2IIa . We used a radioactive rubidium release assay to detect C2IIa pores in the membranes of Chinese hamster ovary cells . Pore formation by C2IIa was dependent on the cellular C2 toxin receptor and an acidic pulse . Pores were formed when C2IIa was bound to cells at neutral pH and when cells were subsequently shifted to acidic medium (pH < 5.5), but no pores were detected when C2IIa was added to cells directly in acidic medium . Most likely, acidification induces a change from "pre-pore" to "pore" conformation of C2IIa, and formation of the pore conformation before membrane binding precludes insertion into membranes . When C2I was present during binding of C2IIa to cells prior to the acidification step, C2IIa-mediated rubidium release was decreased, suggesting that C2I interacted with the lumen of the C2IIa pore . A decrease of rubidium efflux was also detected when C2I was added to C2IIa-treated cells after the acidification step, suggesting that C2I interacted with C2IIa in its pore conformation . Moreover, C2I also interacted with C2IIa channels in artificial lipid membranes and blocked them partially . C2I was only translocated across the cell membrane when C2IIa plus C2I were bound to cells at neutral pH and subsequently shifted to acidic pH . When cell-bound C2IIa was exposed to acidic pH prior to C2I addition, only residual intoxication of cells was observed at high toxin concentrations, and binding of C2I to C2IIa was slightly decreased . Overall, C2IIa pores were essential but not sufficient for translocation of C2I . Intoxication of target cells with C2 toxin requires a strictly coordinated pH-dependent sequence of binding, pore formation by C2IIa, and translocation of C2I.

J Med Microbiol, 2003 Aug, 52(Pt 8), 705 - 9
Prevalent PCR ribotypes of clinical and environmental strains of Clostridium difficile isolated from intensive-therapy unit patients in Kuwait; Rotimi VO et al.; Ninety-five isolates of Clostridium difficile from symptomatic and asymptomatic patients and 18 from their environment in the intensive-therapy units (ITUs) of four teaching hospitals in Kuwait were typed by PCR amplification of rRNA intergenic spacer regions (PCR ribotyping) . A total of 32 different ribotypes was detected among the clinical isolates . The predominant ribotypes from the clinical isolates were types 097 and 078, which accounted for approximately 40 % of all isolates in the ITUs in Kuwait . Ribotypes 097 (toxigenic), 078 (toxigenic) and 039 (non-toxigenic) were three distinct clones that were circulating in all four hospitals . Ribotypes 097, 078 and 076 (i.e . 50 % of isolates from symptomatic patients) were the predominant isolates associated with C . difficile-associated disease (CDAD) . The environmental isolates belonged to a diverse range of ribotypes, with no particular types common to all the hospitals . Ribotype 078 was found only in the patient environment in Mubarak hospital, while ribotype 097 was restricted to Amiri hospital . The hospital environment occupied by symptomatic as well as symptom-free patients was contaminated with C . difficile . Eight new strains that did not match any in the PCR ribotype library established at the PHLS Anaerobe Reference Unit, Cardiff, UK, were assigned ribotypes 105, 125, 128, 129, 131, 134, 140 and 141 . These findings show that the isolates associated with CDAD in Kuwait are different from those found in the UK and some other European countries.

J Bacteriol, 2003 Aug, 185(15), 4539 - 47
DNA array-based transcriptional analysis of asporogenous, nonsolventogenic Clostridium acetobutylicum strains SKO1 and M5; Tomas CA et al.; The large-scale transcriptional program of two Clostridium acetobutylicum strains (SKO1 and M5) relative to that of the parent strain (wild type {WT}) was examined by using DNA microarrays . Glass DNA arrays containing a selected set of 1,019 genes (including all 178 pSOL1 genes) covering more than 25% of the whole genome were designed, constructed, and validated for data reliability . Strain SKO1, with an inactivated spo0A gene, displays an asporogenous, filamentous, and largely deficient solventogenic phenotype . SKO1 displays downregulation of all solvent formation genes, sigF, and carbohydrate metabolism genes (similar to genes expressed as part of the stationary-phase response in Bacillus subtilis) but also several electron transport genes . A major cluster of genes upregulated in SKO1 includes abrB, the genes from the major chemotaxis and motility operons, and glycosylation genes . Strain M5 displays an asporogenous and nonsolventogenic phenotype due to loss of the megaplasmid pSOL1, which contains all genes necessary for solvent formation . Therefore, M5 displays downregulation of all pSOL1 genes expressed in the WT . Notable among other genes expressed more highly in WT than in M5 were sigF, several two-component histidine kinases, spo0A, cheA, cheC, many stress response genes, fts family genes, DNA topoisomerase genes, and central-carbon metabolism genes . Genes expressed more highly in M5 include electron transport genes (but different from those downregulated in SKO1) and several motility and chemotaxis genes . Most of these expression patterns were consistent with phenotypic characteristics . Several of these expression patterns are new or different from what is known in B . subtilis and can be used to test a number of functional-genomic hypotheses.

J Bacteriol, 2003 Aug, 185(15), 4461 - 70
Transcription and analysis of polymorphism in a cluster of genes encoding surface-associated proteins of Clostridium difficile; Savariau-Lacomme MP et al.; Recent investigations of the Clostridium difficile genome have revealed the presence of a cluster of 17 genes, 11 of which encode proteins with similar two-domain structures, likely to be surface-anchored proteins . Two of these genes have been proven to encode proteins involved in cell adherence: slpA encodes the precursor of the two proteins of the S-layer, P36 and P47, whereas cwp66 encodes the Cwp66 adhesin . To gain further insight into the function of this cluster, we further focused on slpA, cwp66, and cwp84, the latter of which encodes a putative surface-associated protein with homology to numerous cysteine proteases . It displayed nonspecific proteolytic activity when expressed as a recombinant protein in Escherichia coli . Polymorphism of cwp66 and cwp84 genes was analyzed in 28 strains, and transcriptional organization of the three genes was explored by Northern blots . The slpA gene is strongly transcribed during the entire growth phase as a bicistronic transcript; cwp66 is transcribed only in the early exponential growth phase as a polycistronic transcript encompassing the two contiguous genes upstream . The putative proteins encoded by the cotranscribed genes have no significant homology with known proteins but may have a role in adherence . No correlation could be established between sequence patterns of Cwp66 and Cwp84 and virulence of the strains . The cwp84 gene is strongly transcribed as a monocistronic message . This feature, together with the highly conserved sequence pattern of cwp84, suggests a significant role in the physiopathology of C . difficile for the Cwp84 protease, potentially in the maturation of surface-associated adhesins encoded by the gene cluster.

Ai Zheng, 2003 Jul, 22(7), 719 - 24
Comparative study on apoptosis induction of SMMC7721 and Vero cells by Clostridium difficile toxin A; Zhao J et al.; BACKGROUND & OBJECTIVE: Clostridium difficile is recognized as a frequent cause of antibiotic-induced diarrhea . This study was designed to investigate whether Clostridium difficile toxin A might induce apoptosis on human hepatoma cell line SMMC7721 and African green monkey kidney Vero cells . METHODS: Highly purified toxin A was obtained by bovine thyroglobulin affinity purification followed by ion exchange chromatography on Q sepharose . The apoptosis induction of toxin A was examined on SMMC7721 cells with Vero cells as a control . Inhibition of proliferation was measured by MTT assay . Morphological assessment of apoptosis was performed with fluorescence and electronic microscopy . DNA fragmentation was observed by agarose gel electrophoresis . Cell cycle distribution was analyzed by flow cytometry . RESULTS: Toxin A (0.293-4.690 mg x L(-1) ) inhibited proliferation of SMMC7721 and Vero cells in a time- and concentration-dependent manner . Morphological changes of typical apoptosis showed that SMMC7721 had a higher percentage of apoptosis than Vero cells . Agarose gel electrophoresis of DNA from SMMC7721 treated with toxin A for 48 hours revealed a "ladder" pattern . CONCLUSION: Clostridium difficile toxin A induced apoptosis of SMMC7721 and Vero cells . The apoptosis induction on SMMC7721 cells was more effective than that on Vero cells.

J Appl Microbiol, 2003, 95(2), 294 - 300
Characteristics and identification of enterocins produced by Enterococcus faecium JCM 5804T; Park SH et al.; AIMS: To screen bacteriocin-producing lactic acid bacteria (LAB) in 52 type and reference strains, which have not previously been studied, with respect to bacteriocins, and to characterize the presence of bacteriocins . METHODS AND RESULTS: Only Enterococcus faecium JCM 5804T showed bacteriocin-like activity . It inhibited the growth of Lactobacillus spp., Enterococcus spp., Clostridium spp., Listeria monocytogenes, and vancomycin resistant Enterococcus (VRE) . However, it was not effective against Gram-negative strains, Weisella spp., Leuconostoc spp., Lactococcus spp., or methicillin resistant Staphylococcus aureus (MRSA) . The inhibitory activity of Ent . faecium JCM 5804T was inactivated by proteinase K, trypsin, alpha-chymotrypsin, and papain, but not by lysozyme, lipase, catalase, or beta-glucosidase . The inhibitory activity was stable at 100 degrees C for 30 min, and had a pH range from 2 to 10 . The molecular weight of the partially purified bacteriocin(s) was approx . 4.5 kDa, according to tricine-sodium dodecyl sulphate-polyacrylamide gel electrophoresis . Polymerase chain reaction and direct sequencing methods identified three different types of bacteriocins produced by Ent . faecium JCM 5804T, enterocin A, enterocin B, and enterocin P-like bacteriocin . CONCLUSION: Enterococcus faecium JCM 5804T produced three different types of bacteriocins, and they inhibited LAB and pathogens . SIGNIFICANCE AND IMPACT OF STUDY: This is the first report of enterocin A, enterocin B, and enterocin P-like bacteriocin, detected in Ent . faecium JCM 5804T among LAB type and reference strains.

Clin Microbiol Rev, 2003 Jul, 16(3), 451 - 62
Biology and pathogenesis of thrombosis and procoagulant activity in invasive infections caused by group A streptococci and Clostridium perfringens; Bryant AE; Group A streptococcal necrotizing fasciitis/myonecrosis and Clostridium perfringens gas gangrene are two of the most fulminant gram-positive infections in humans . Tissue destruction associated with these infections progresses rapidly to involve an entire extremity . Multiple-organ failure is common, and morbidity and mortality remain high . Systemic activation of coagulation and dysregulation of the anticoagulation pathways contribute to the pathogenesis of many diverse disease entities of infectious etiology, and it has been our hypothesis that microvascular thrombosis contributes to reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis and organ failure in these invasive gram-positive infections . This article reviews the coagulation, anticoagulation, and fibrinolytic systems from cellular players to cytokines to novel antithrombotic therapies and discusses the mechanisms contributing to occlusive microvascular thrombosis and tissue destruction in invasive group A streptococcal and C . perfringens infections . A thorough understanding of these mechanisms may suggest novel therapeutic targets for patients with these devastating infections.

J Hosp Infect, 2003 Jul, 54(3), 243 - 5
Proton pump inhibitors as a risk factor for Clostridium difficile diarrhoea; Cunningham R et al.; Clostridium difficile is the main infectious cause of colitis in hospital inpatients . The incidence is increasing, and it is associated with significant mortality, morbidity, and increased length of stay . The main risk factor is use of broad-spectrum antibiotics, and antibiotic restriction is the most effective control measure . We carried out a retrospective case-control study to investigate whether use of proton pump inhibitors (PPI) was an additional risk factor . PPI use within the preceding eight weeks was associated with an increased risk of C . difficile diarrhoea (odds ratio 2.5, 95% CI 1.5-4.2) . Reduction of unnecessary PPI use may be an additional strategy to reduce the incidence of this infection.

J Hosp Infect, 2003 Jul, 54(3), 202 - 6
Clostridium difficile infections related to antibiotic use and infection control facilities in two university hospitals; Berild D et al.; We investigated whether a reduction in antibiotic use at the Aker University Hospital (Aker) led to a reduction in Clostridium difficile-associated diarrhoea (CDAD) . We compared the incidence of CDAD in Aker and Tromsoe University Hospitals (Tromsoe) and related it to antibiotic use and facilities for infection control between 1993-2001 . For this purpose we also performed point prevalence studies . Total antibiotic use was the same in the two hospitals . In spite of a reduction in the use of broad-spectrum antibiotics in Aker the incidence of CDAD increased during 1993-1999 . In Tromsoe the use of broad-spectrum antibiotics and clindamycin was two to three times higher than in Aker, but until 1999 the incidence of CDAD remained constant and only half that of Aker . After 1999 the incidence of CDAD was halved in Aker, and increased three-fold in Tromsoe . Point prevalence studies in 2001 revealed an equal prevalence of antibiotic-associated diarrhoea . The facilities for infection control were better in Tromsoe . The percentage of single rooms were 8% in Aker and 14% in Tromsoe, and the percentage of single rooms with a WC was 6% in Aker and 12% in Tromsoe . The bed occupancy was much higher in Aker than in Tromsoe . Lack of facilities for infection control and higher bed occupancy could have contributed to the higher incidence of CDAD in Aker in spite of decreased use of broad-spectrum antibiotics and clindamycin . To limit CDAD in hospitals the focus must be on both rational antibiotic use and infection control.

Clin Diagn Lab Immunol, 2003 Jul, 10(4), 587 - 95
Recombinant single-chain variable fragment antibodies directed against Clostridium difficile toxin B produced by use of an optimized phage display system; Deng XK et al.; Recombinant antibody cloning and phage display technologies were used to produce single-chain antibodies (scFv) against Clostridium difficile toxin B . The starting material was the mouse B cell hybridoma line 5A8, which generates a monoclonal antibody against the toxin . The integrated cloning, screening, and phage display system of Krebber et al . (J . Immunol . Methods 201:35-55, 1997) allowed us to rapidly obtain toxin B-binding scFv sequences derived from the hybridoma cell line . The best candidate scFv sequences, based on preliminary enzyme-linked immunosorbent assay (ELISA) screening data were then subcloned into the compatible expression vector . Recombinant single-chain antibodies were expressed in Escherichia coli . A 29-kDa band was observed on polyacrylamide gel electrophoresis as predicted . The expressed product was characterized by immunoblotting and detection with an anti-FLAG antibody . The toxin B-binding function of the single-chain antibody was shown by a sandwich ELISA . The antibody was highly specific for toxin B and did not cross-react with material isolated from a toxin B-negative C . difficile strain . The sensitivity of the soluble single-chain antibody is significantly higher than the original monoclonal antibody based on ELISA data and could detect a minimum of 10 ng of toxin B/well . Competitive ELISAs established that the affinity of the 5A8 parent antibody and the best representative (clone 10) of the single-chain antibodies were similar and in the range of 10(-8) M . We propose that recombinant antibody technology is a rapid and effective approach to the development of the next generation of immunodiagnostic reagents.

Am J Physiol Cell Physiol, 2003 Nov, 285(5), C1197 - 206 Epub 2003 Jul 09.
CCK-A receptor activates RhoA through G alpha 12/13 in NIH3T3 cells; Le Page SL et al.; Cholecystokinin (CCK) is a major regulator of pancreatic acinar cells and was shown previously to be capable of inducing cytoskeletal changes in these cells . In the present study, using NIH3T3 cells stably transfected with CCK-A receptors as a model cell, we demonstrate that CCK can induce actin stress fibers through a G13- and RhoA-dependent mechanism . CCK induced stress fibers within minutes similar to those induced by lysophosphatidic acid (LPA), the active component of serum . The effects of CCK were mimicked by active RhoV14 and blocked by dominant-negative RhoN19, Clostridium botulinum C3 transferase, and the Rho-kinase inhibitor Y-27632 . CCK rapidly induced active Rho in cells as shown with a pull-down assay using the Rho binding domain of rhotekin and by a serum response element (SRE)-luciferase reporter assay . To evaluate the G protein mediating the action of CCK, cells were transfected with active alpha-subunits; Galpha13 and Galpha12 but not Galphaq induced stress fibers and in some cases cell rounding . A p115 Rho guanine nucleotide exchange factor (GEF) regulator of G protein signaling (RGS) domain known to interact with G12/13 inhibited active alpha12/13-and CCK-induced stress fibers, whereas RGS2 and RGS4, which are known to inhibit Gq, had no effect . Cotransfection with plasmids coding for the G protein alpha-subunit carboxy-terminal peptide from alpha13 and, to a lesser extent alpha12, also inhibited the effect of CCK, whereas the peptide from alphaq did not . These results show that in NIH3T3 cells bearing CCK-A receptors, CCK activates Rho primarily through G13, leading to rearrangement of the actin cytoskeleton.

J Biol Chem, 2003 Sep 19, 278(38), 36934 - 41 Epub 2003 Jul 08.
Biological activities and pore formation of Clostridium perfringens beta toxin in HL 60 cells; Nagahama M et al.; Clostridium perfringens beta toxin is an important agent of necrotic enteritis . Of the 10 cell lines tested, only the HL 60 cell line was susceptible to beta toxin . The toxin induced swelling and lysis of the cell . Treatment of the cells with the toxin resulted in K+ efflux from the cells and Ca2+, Na+, and Cl- influxes . These events reached a maximum just before the cells were lysed by the toxin . Incubation of the cells with the toxin showed the formation of toxin complexes of about 191 and 228 kDa, which were localized in the domains that fulfilled the criteria of lipid rafts . The complex of 228 kDa was observed until 30 min after incubation, and only the complex of 191 kDa was remained after 60 min . Treatment of the cells with methyl-beta-cyclodextrin or cholesterol oxidase blocked binding of the toxin to the rafts and the toxin-induced K+ efflux and swelling . The toxin-induced Ca2+ influx and morphological changes were inhibited by an increase in the hydrodynamic diameter of polyethylene glycols from 200 to 400 and markedly or completely inhibited by polyethylene glycol 600 and 1000 . However, these polyethylene glycols had no effect on the toxin-induced K+ efflux . The toxin induced carboxyfluorescein release from phosphatidyl-choline-cholesterol liposomes containing carboxyfluorescein and formed an oligomer with 228 kDa in a dose-dependent manner but did not form an oligomer with the 191-kDa complex . We conclude that the toxin acts on HL 60 cells by binding to lipid rafts and forming a functional oligomer with 228 kDa.

Age Ageing, 2003 Jul, 32(4), 388 - 93
A comparison of two feeding methods in the alleviation of diarrhoea in older tube-fed patients: a randomised controlled trial; Lee JS et al.; OBJECTIVE: to compare the effect of two feeding methods on older tube-fed patients suffering from diarrhoea . DESIGN: randomised controlled study . SETTING: geriatric wards in a regional hospital . SUBJECTS: 105 patients over the age of 60 who developed diarrhoea while on intermittent bolus tube feeding . METHODS: subjects were randomised either to switch to continuous tube feeding or to continue with intermittent bolus tube feeding for 3 days, with some extended to 5 days . Incontinence pads were inspected 6 times per day by independent observers . Each motion was evaluated using a validated score according to its volume and consistency . Stools were sent for pathogen culture and Clostridium difficile cytotoxin assay . Those with positive assays were excluded from analysis . Gastric aspirations were performed at regular intervals and significant residual volumes recorded . RESULTS: 86 patients completed the study . Twelve patients (14%) were excluded due to positive CD stool assays . Seventy-four patients were entered into analysis . The median diarrhoea scores of the continuous infusion group and the intermittent bolus group were: day 0 (12 versus 10, P=0.18), day 3 (4 versus 6, P=0.23) and day 5 (5 versus 4, P=0.83) respectively . There was no statistical difference between the two groups . Two patients in the continuous infusion group (n=37) and 4 patients in the intermittent bolus group (n=37) had significant gastric residual volumes . The incidence did not differ significantly . CONCLUSION: contrary to common belief and recommendation, we could not find a significant difference between the continuous feeding method and the intermittent feeding method in the alleviation of diarrhoea in tube-fed patients . We do not recommend routine switch to continuous pump feeding in the management of tube-fed patients suffering from diarrhoea.

Clin Microbiol Infect, 2003 Jun, 9(6), 560 - 2
A parent as a vector of Salmonella brandenburg nosocomial infection in a neonatal intensive care unit; Cartolano GL et al.; A newborn baby was admitted to the Neonatal Intensive Care Unit (NICU) of St Germain en Laye Hospital (France) because of premature birth . On day 12, he contracted gastroenteritis due to Salmonella brandenbourg . The salmonellosis led to a septic shock syndrome with a brief cardiopulmonary arrest . He was treated with intravenous ceftriaxone and gentamicin, and the evolution was favorable . Microbiological investigations revealed that the mother was the vector for this nosocomial infection . S . brandenbourg was isolated from the feces of the baby, despite recent recommendations on managing stool specimens from patients hospitalized for more than three days: according to these recommendations, these stools should be processed for viruses and Clostridium difficile toxin only.

Clin Microbiol Infect, 2003 Jun, 9(6), 526 - 30
Antecedent use of fluoroquinolones is associated with resistance to moxifloxacin in Clostridium difficile; Ackermann G et al.; OBJECTIVE: Moxifloxacin is characterized by high activity against Gram-positive cocci and some Gram-positive and -negative anaerobes, including Clostridium difficile . This study investigates the role of prior quinolone use in relation to patterns of susceptibility of C . difficile to moxifloxacin . METHODS: Sixty-three clinical isolates of C . difficile were investigated for toxigenicity, susceptibility to moxifloxacin, and mutations in the DNA gyrase gene . The medical histories for 50 of these patients were available and used to identify previous fluoroquinolone use . RESULTS: Thirty-three (52.4%) strains showed resistance to moxifloxacin (MICs > or = 16 mg/L) . All moxifloxacin-resistant strains harbored a mutation at amino acid codon Ser-83 of gyrA . Forty-five isolates (71.4%) were toxigenic; all moxifloxacin-resistant strains were in this group . Resistance to moxifloxacin was associated with prior use of fluoroquinolones (P-value 0.009, chi-square) . CONCLUSIONS: Although the use of moxifloxacin to treat C . difficile-associated diarrhea is not likely to be common, these data show a relationship between antecedent fluoroquinolone use and resistance to moxifloxacin in C . difficile isolates, and raise questions regarding selection pressure for resistance placed on colonizing bacteria exposed to fluoroquinolones . Mutations in gyrA are involved in moxifloxacin resistance.

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