Clin Microbiol Infect, 2003 Jun, 9(6), 512 - 7 Mechanism of quinolone resistance in anaerobic bacteria; Oh H et al.; Several recently developed quinolones have excellent activity against a broad range of aerobic and anaerobic bacteria and are thus potential drugs for the treatment of serious anaerobic and mixed infections . Resistance to quinolones is increasing worldwide, but is still relatively infrequent among anaerobes . Two main mechanisms, alteration of target enzymes (gyrase and topoisomerase IV) caused by chromosomal mutations in encoding genes, or reduced intracellular accumulation due to increased efflux of the drug, are associated with quinolone resistance . These mechanisms have also been found in anaerobic species . High-level resistance to the newer broad-spectrum quinolones often requires stepwise mutations in target genes . The increasing emergence of resistance among anaerobes may be a consequence of previous widespread use of quinolones, which may have enriched first-step mutants in the intestinal tract . Quinolone resistance in the Bacteroides fragilis group strains is strongly correlated with amino acid substitutions at positions 82 and 86 in GyrA (equivalent to positions 83 and 87 of Escherichia coli) . Several studies have indicated that B . fragilis group strains possess efflux pump systems that actively expel quinolones, leading to resistance . DNA gyrase seems also to be the primary target for quinolones in Clostridium difficile, since amino acid substitutions in GyrA and GyrB have been detected in resistant strains . To what extent other mechanisms, such as mutational events in other target genes or alterations in outer-membrane proteins, contribute to resistance among anaerobes needs to be further investigated. Drugs Today (Barc), 2000 Sep, 36(9), 631 - 9 Linezolid: a new antibiotic; Xiong YQ et al.; The U.S . Food and Drug Administration recently approved linezolid for the treatment of patients with methicillin-resistant staphylococcal and vancomycin-resistant enterococcal infections . This oxazolidinone antibacterial agent represents the first approved antibiotic of a new structural class in 35 years . Linezolid is a synthetic compound that acts by inhibiting the initiation complex formation in bacterial protein synthesis, a mechanism of action distinct from other commercially available antibiotics . Thus, cross-resistance between linezolid and other current antimicrobial agents has not been demonstrated to date . Linezolid has a wide spectrum of in vitro activity against Gram-positive organisms, including methicillin-resistant staphylococci, penicillin-resistant pneumococci and vancomycin-resistant enterococci . Some anaerobes, such as Clostridium spp., Peptostreptococcus spp . and Prevotella spp . are also susceptible to linezolid . In addition, linezolid has exhibited good efficacy in experimental animal models of acute otitis media, endocarditis and meningitis due to many common aerobic Gram-positive bacteria . In clinical trials involving hospitalized patients with skin/soft tissue infections, community-acquired pneumonia and serious Gram-positive bacterial infections, linezolid appeared to be an effective treatment option, comparable in efficacy to vancomycin. J Appl Physiol, 2003 Nov, 95(5), 1889 - 95 Epub 2003 Jul 03. Rho inhibition decreases TNF-induced endothelial MAPK activation and monolayer permeability; Nwariaku FE et al.; Our laboratory previously demonstrated that MAPK activation is an important signal during cytokine-induced endothelial permeability (Nwariaku FE, Liu Z, Terada L, Duffy S, Sarosi G, and Turnage R . Shock 18: 82-85, 2002) . Because GTP-binding proteins have been implicated in MAPK activation, we now hypothesize that the GTP-binding protein Rho is a mediator of TNF-induced MAPK activation and increased endothelial permeability . Transmonolayer permeability was assessed in human lung microvascular cells by measuring transmonolayer electrical resistance . MAPK activity was assessed by using a phospho-specific immunoprecipitation kinase assay and by comparing Western blots for phospho-MAPK with total MAPK . MAPK inhibitors used were SB-202190 and PD-098059, whereas Clostridium botulinum C3 transferase was used as a Rho inactivator . Rho-associated coiled-coil kinase was inhibited with Y-27632 . TNF increased pulmonary endothelial permeability in vitro and caused a rapid, sustained increase in endothelial p38 and extracellular signal-regulated kinase MAPK activity . Inhibition of p38 and extracellular signal-regulated kinase MAPK with SB-202190 and PD-098059, respectively, decreased TNF-induced endothelial permeability . C3 transferase attenuated TNF-induced MAPK activation and blocked TNF-induced endothelial permeability . Finally, inhibition of Rho-associated coiled-coil kinase with Y-27632 prevented both MAPK activation and TNF-induced decreases in transmonolayer resistance . Rho acts upstream of mitogen-activated protein kinases in mediating TNF-induced pulmonary endothelial leak. J Clin Microbiol, 2003 Jul, 41(7), 3413 - 4 Molecular analysis of Clostridium difficile strains isolated from 18 cases of recurrent clostridium difficile-associated diarrhea; Tang-Feldman Y et al.; Recurrence of Clostridium difficile-associated diarrhea (CDAD) occurs in 15 to 20% of patients after discontinuation of treatment . Arbitrarily primed PCR was used to investigate the epidemiology of recurrent CDAD in 18 patients . Reinfection with a new strain occurred in 6 of 18 patients (33.3%), while 12 patients relapsed with the original strain shortly after discontinuation of treatment . These data suggest that reinfection with exogenous C . difficile is a common problem and that not all recurrences are due to relapse. J Microbiol Methods, 2003 Sep, 54(3), 359 - 66 Comparison of media for enumeration of Clostridium perfringens from foods; de Jong AE et al.; Many media have been developed to enumerate Clostridium perfringens from foods . In this study, six media {iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin polymyxin sulfadiazine perfringens (OPSP) agar} were compared in a prestudy, of which four (IS, TSC, SCA, and DCA) were selected for an international collaborative trial . Recovery of 15 pure strains was tested in the prestudy and recovery of one strain from foodstuffs was tested in the collaborative trial . Results from the prestudy did reveal statistical difference of the media but recoveries on all media were within the microbiological limits (+/-30%) of IS, which was set as a reference medium . Recoveries on the media tested in the collaborative trial were statistically different as well, but these differences were of no microbiological-analytical relevance . Food matrices did not affect the recovery of C . perfringens in general . DCA and SCA, in particular, are labor-intensive to prepare and DCA frequently failed to produce black colonies; gray colonies were quite common . Since IS medium is nonselective, it was concluded that TSC was the most favorable medium for the enumeration of C . perfringens from foods. Appl Environ Microbiol, 2003 Jul, 69(7), 4029 - 36 Thermal inactivation of nonproteolytic Clostridium botulinum type E spores in model fish media and in vacuum-packaged hot-smoked fish products; Lindstrom M et al.; Thermal inactivation of nonproteolytic Clostridium botulinum type E spores was investigated in rainbow trout and whitefish media at 75 to 93 degrees C . Lysozyme was applied in the recovery of spores, yielding biphasic thermal destruction curves . Approximately 0.1% of the spores were permeable to lysozyme, showing an increased measured heat resistance . Decimal reduction times for the heat-resistant spore fraction in rainbow trout medium were 255, 98, and 4.2 min at 75, 85, and 93 degrees C, respectively, and those in whitefish medium were 55 and 7.1 min at 81 and 90 degrees C, respectively . The z values were 10.4 degrees C in trout medium and 10.1 degrees C in whitefish medium . Commercial hot-smoking processes employed in five Finnish fish-smoking companies provided reduction in the numbers of spores of nonproteolytic C . botulinum of less than 10(3) . An inoculated-pack study revealed that a time-temperature combination of 42 min at 85 degrees C (fish surface temperature) with >70% relative humidity (RH) prevented growth from 10(6) spores in vacuum-packaged hot-smoked rainbow trout fillets and whole whitefish stored for 5 weeks at 8 degrees C . In Finland it is recommended that hot-smoked fish be stored at or below 3 degrees C, further extending product safety . However, heating whitefish for 44 min at 85 degrees C with 10% RH resulted in growth and toxicity in 5 weeks at 8 degrees C . Moist heat thus enhanced spore thermal inactivation and is essential to an effective process . The sensory qualities of safely processed and more lightly processed whitefish were similar, while differences between the sensory qualities of safely processed and lightly processed rainbow trout were observed. Can Vet J, 2003 Jun, 44(6), 493 - 5 Clostridium botulinum type D intoxication in a dairy herd in Ontario; Martin S; Thirty-four Holstein cows died after exposure to Clostridium botulinum type D toxin, presumably from contaminated haylage . The presence of type D toxin in ruminal contents was confirmed by mouse inoculation . This is the first confirmation by direct toxin isolation of C . botulinum type D toxin in cattle in North America. J Bacteriol, 2003 Jul, 185(14), 4127 - 35 X-Ray crystal structure of the multidomain endoglucanase Cel9G from Clostridium cellulolyticum complexed with natural and synthetic cello-oligosaccharides; Mandelman D et al.; Complete cellulose degradation is the first step in the use of biomass as a source of renewable energy . To this end, the engineering of novel cellulase activity, the activity responsible for the hydrolysis of the beta-1,4-glycosidic bonds in cellulose, is a topic of great interest . The high-resolution X-ray crystal structure of a multidomain endoglucanase from Clostridium cellulolyticum has been determined at a 1.6-A resolution . The endoglucanase, Cel9G, is comprised of a family 9 catalytic domain attached to a family III(c) cellulose-binding domain . The two domains together form a flat platform onto which crystalline cellulose is suggested to bind and be fed into the active-site cleft for endolytic hydrolysis . To further dissect the structural basis of cellulose binding and hydrolysis, the structures of Cel9G in the presence of cellobiose, cellotriose, and a DP-10 thio-oligosaccharide inhibitor were resolved at resolutions of 1.7, 1.8, and 1.9 A, respectively. Rev Argent Microbiol, 2003 Jan-Mar, 35(1), 45 - 8 {Presence of botulinum-producing clostridia in the soils of Entre Ríos}; Luquez C et al.; Botulism, well known for centuries, continues to preoccupy the Public Health authorities, food and agricultural industries . In its oldest form it results from ingestion of food containing botulinum toxin . To correctly evaluate the incidence of botulism it is fundamental to know the distribution of botulinum toxin-producing clostridia (Clostridium botulinum, C . baratii, C . butyricum and C . argentinense) in nature, specially in the soil, its main reservoir . In our country, this study has been carried out in a partial way . With the aim to contribute to such knowledge, 240 soil samples from Entre Rios province were examined for C . botulinum and 35 (14.6%) samples resulted positive. Biochim Biophys Acta, 2003 Jun 27, 1613(1-2), 79 - 86 Clostridium perfringens alpha-toxin-induced hemolysis of horse erythrocytes is dependent on Ca2+ uptake; Ochi S et al.; Clostridium perfringens alpha-toxin is able to lyse various erythrocytes . Exposure of horse erythrocytes to alpha-toxin simultaneously induced hot-cold hemolysis and stimulated production of diacylglycerol and phosphorylcholine . When A23187-treated erythrocytes were treated with the toxin, these events were dependent on the concentration of extracellular Ca2+ . Incubation with the toxin of BAPTA-AM-treated horse erythrocytes caused no hemolysis or production of phosphorylcholine, but that of the BAPTA-treated erythrocytes did . When Quin 2-AM-treated erythrocytes were incubated with the toxin in the presence of 45Ca2+, the cells accumulated 45Ca2+ in a dose- and a time-dependent manner . These results suggest that the toxin-induced hemolysis and hydrolysis of phosphatidylcholine are closely related to the presence of Ca2+ in the cells . Flunarizine, a T-type Ca2+ channel blocker, and tetrandrine, an L- and T-type Ca2+ channel blocker, inhibited the toxin-induced hemolysis and Ca2+ uptake . However, L-type Ca2+ channel blockers, nifedipine, verpamil and diltiazem, an N-type blocker, omega-conotoxin SVIB, P-type blockers, omega-agatoxin TK and omega-agatoxin IVA, and a Q-type blocker, omega-conotoxin MVII C, had no such inhibitory effect . The observation suggests that Ca2+ taken up through T-type Ca2+ channels activated by the toxin plays an important role in hemolysis induced by the toxin. Arch Biochem Biophys, 2003 Jul 15, 415(2), 235 - 44 Molecular identification of an alpha-L-rhamnosidase from Bacillus sp strain GL1 as an enzyme involved in complete metabolism of gellan; Hashimoto W et al.; The genes (rhaA and rhaB) for two alpha-L-rhamnosidases of Bacillus sp . strain GL1, which assimilates a bacterial polysaccharide (gellan), were cloned from a genomic DNA library of the bacterium constructed in Escherichia coli, and the nucleotide sequences of the genes were determined . Gene rhaA (2661 bp) contained an open reading frame (ORF) encoding a protein (RhaA: 886 amino acids) with a molecular weight (MW) of 98280 . Gene rhaB (2871 bp) contained an ORF encoding a protein (RhaB: 956 amino acids) with a MW of 106049 . RhaA exhibited significant identity (41%) with alpha-L-rhamnosidase of Clostridium stercorarium, while RhaB showed slight homology with enzymes from other sources . An overexpression system for the two enzymes was constructed in E . coli, and the enzymes were purified and characterized . Both RhaA and RhaB were highly specific for rhamnosyl saccharides, including gellan disaccharide (rhamnosyl glucose) and naringin, and released rhamnose from substrates most efficiently at pH 6.5-7.0 and 40 degrees C . Bacillus sp . strain GL1 cells grown in a gellan medium produced only RhaB, indicating that RhaB plays a crucial role in the complete metabolism of gellan. Med Clin (Barc), 2003 Jun 14, 121(2), 58 - 60 {Foodborne outbreak by Clostridium perfringens in a school dining room}; Dominguez-Berjon MF et al.; BACKGROUND AND OBJECTIVE: The purpose was to investigate the etiology and epidemiological characteristics of a food-borne infection outbreak in a school dining room . SUBJECTS AND METHOD: Retrospective cohort study . Information about gastrointestinal symptoms and the use of the service of the school dining room was obtained . Coprocultures and detection of Clostridium perfringens enterotoxin in stool samples by means of reverse passive latex agglutination were carried out in 7 ill persons . Relative risks (RR) at 95% confidence intervals (95% CI) were calculated . RESULTS: The attack rate was 17.5% (48/275) and the probability of becoming sick was higher in students who ate at the second shift than in those eating at the first one (RR=13.8; 95% CI: 4.4-43.1) . C perfringens enterotoxin was detected in 6 stool samples from patients . A high recount of C perfringens was not observed in those food samples kept frozen after their elaboration . CONCLUSIONS: The determination of C perfringens enterotoxin in feces allowed to confirm the etiologic agent of the outbreak. Appl Microbiol Biotechnol, 2003 Dec, 63(2), 187 - 93 Epub 2003 Jun 24. Metabolism of hexahydro-1,3,5-trinitro-1,3,5-triazine through initial reduction to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine followed by denitration in Clostridium bifermentans HAW-1; Zhao JS et al.; A fast hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX)-degrading {28.1 micromol h(-1) g (dry weight) cells(-1); biomass, 0.16 g (dry weight) cells(-1)} and strictly anaerobic bacterial strain, HAW-1, was isolated and identified as Clostridium bifermentans using a 16S-rRNA-based method . Based on initial rates, strain HAW-1 transformed RDX to hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine (MNX), hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine (DNX), and hexahydro-1,3,5-trinitroso-1,3,5-triazine (TNX) with yields of 56, 7.3 and 0.2%, respectively . Complete removal of RDX and its nitroso metabolites produced (%, of total C or N) methanol (MeOH, 23%), formaldehyde (HCHO, 7.4%), carbon dioxide (CO2, 3.0%) and nitrous oxide (N2O, 29.5%) as end products . Under the same conditions, strain HAW-1 transformed MNX separately at a rate of 16.9 micromol h(-1) g (dry weight) cells(-1) and produced DNX (25%) and TNX (0.4%) as transient products . Final MNX transformation products were (%, of total C or N) MeOH (21%), HCHO (2.9%), and N2O (17%) . Likewise strain HAW-1 degraded TNX at a rate of 7.5 micromol h(-1) g (dry weight) cells(-1 )to MeOH and HCHO . Furthermore, removal of both RDX and MNX produced nitrite (NO2-) as a transient product, but the nitrite release rate from MNX was quicker than from RDX . Thus, the predominant pathway for RDX degradation is based on initial reduction to MNX followed by denitration and decomposition . The continued sequential reduction to DNX and TNX is only a minor route. Prim Care, 2003 Mar, 30(1), 63 - 80, vi Gastroenteritis and antibiotic-associated diarrhea; Jabbar A et al.; Acute gastroenteritis is a common cause of emergency and office visits . This article reviews causes, pathogenesis, diagnosis, prevention, and treatment . The incidence of antibiotic-associated colitis is increasing worldwide as a consequence of widespread use of broad-spectrum antibiotics for various illnesses . The pathogenic organism, Clostridium difficile, produces two enterotoxins, toxin A and toxin B, that cause colonic mucosal inflammation . C . difficile infection presents with a wide range of clinical manifestations, from asymptomatic carriers to life-threatening pseudomembranous colitis. Jpn J Infect Dis, 2003 Apr, 56(2), 73 - 4 A botulism case of a 12-year-old girl caused by intestinal colonization of Clostridium botulinum type Ab; Kobayashi H et al.; We encountered a 12-year-old girl, who had contracted food-borne botulism, and subsequently suffered from obstinate constipation for more than half a year . Even on hospital day 122, Clostridium botulinum and its toxin were detected in her stool specimens . The potency of the toxin of the blood serum sampled before treatment was 20 mouse minimum lethal dose per ml . The toxin in the blood had a molecular size equivalent to that of type A botulinum neurotoxin . On hospital day 250, the patient's serum detoxified type A neurotoxin . We confirmed that the patient had food-borne botulism caused by C . botulinum type Ab, followed by intestinal colonization-type botulism. Vet Pathol, 2003 Jul, 40(4), 376 - 81 Immunohistochemical localization of Clostridium perfringens beta2-toxin in the gastrointestinal tract of horses; Bacciarini LN et al.; Clostridia-associated intestinal disease in horses was generally reported to be due to infection with Clostridium perfringens type A, which harbors the cpa-encoded alpha-toxin . A recent study demonstrated a high incidence of beta2-toxigenic C . perfringens in horses suffering or dying from typhlocolitis, suggesting that this novel type of C . perfringens might play an important role in typhlocolitis and possibly other equine intestinal diseases . A retrospective study was conducted to assess the presence of the beta2-toxin in tissues of the equine gastrointestinal tract . Monospecific polyclonal antibodies against recombinant beta2-toxin were produced in rabbits and used to demonstrate the beta2-toxin in sections of the gastrointestinal tract by immunohistochemical methods . Sections from 69 horses were stained and beta2-toxin was observed immunohistochemically in 40 animals . Sections from the stomach, small intestine, and large intestine were positive . Immunopositivity for beta2-toxin was significantly associated with presence of beta2-toxigenic bacteria . This investigation demonstrates local production of beta2-toxin and suggests that immunohistochemistry using antitoxin antibodies represents a useful diagnostic method in those cases where isolation of bacteria and polymerase chain reaction typing is not feasible . Although the association between the presence of beta2-toxin and development of gastrointestinal disease in horses remains uncertain, the findings of this study indicate that the potential causal relationship warrants further investigation. Eur J Biochem, 2003 Jul, 270(13), 2750 - 8 Expression of two {Fe}-hydrogenases in Chlamydomonas reinhardtii under anaerobic conditions; Forestier M et al.; We have isolated and characterized a second {Fe}-hydrogenase gene from the green alga, Chlamydomonas reinhardtii . The HydA2 gene encodes a protein of 505 amino acids that is 74% similar and 68% identical to the known HydA1 hydrogenase from C . reinhardtii . HydA2 contains all the conserved residues and motifs found in the catalytic core of the family of {Fe}-hydrogenases . We demonstrate that both the HydA1 and the HydA2 transcripts are expressed upon anaerobic induction, achieved either by neutral gas purging or by sulfur deprivation of the cultures . Furthermore, the expression levels of both transcripts are regulated (in some cases differently) by incubation conditions, such as the length of anaerobiosis, the readdition of O2, the presence of acetate, and/or the absence of nutrients such as sulfate during growth . Antibodies specific for HydA2 recognized a protein of about 49 kDa in extracts from anaerobically induced C . reinhardtii cells, strongly suggesting that HydA2 encodes for an expressed protein . Homology-based 3D modeling of the HydA2 hydrogenase shows that its catalytic site models well to the known structure of Clostridium pasteurianum CpI, including the H2-gas channel . The major differences between HydA1, HydA2 and CpI are the absence of the N-terminal Fe-S centers and the existence of extra sequences in the algal enzymes . To our knowledge, this work represents the first systematic study of expression of two algal {Fe}-hydrogenases in the same organism. Acta Physiol Scand, 2003 Jul, 178(3), 241 - 50 Involvement of enteric nerves in permeability changes due to deoxycholic acid in rat jejunum in vivo; Fihn BM et al.; AIM: Stress and Clostridium difficile toxin A increase epithelial permeability in the small intestine via vagus and visceral afferents, in turn activating mucosal mast cells . Bile acids also increase epithelial permeability but it is not known if nerves or mast cells are involved in this effect in the small intestine . METHOD: In jejunum of anesthetized rats, the effects of hexamethonium and atropine on deoxycholic acid (DCA) induced fluid secretion and increase in epithelial permeability was therefore studied by determining the appearance and disappearance rates of 14C-mannitol and 51Cr-EDTA into and from a perfusion system containing 4 or 8 mm DCA and expressed as clearance . RESULTS: DCA increased net fluid transport and appearance and to a less extent disappearance rates of the probes . Hexamethonium but not atropine, chronic denervation or the NO synthase inhibitor L-NNA did significantly decrease the appearance rate and net fluid secretion . The levels of the mast cell protease II (RMCP II) in perfusate and plasma were not increased by DCA . The clearance ratio Cr-EDTA/mannitol indicates that the plasma clearance of the permeability probes is partly secondary to net fluid transport only at higher DCA concentrations . CONCLUSION: We conclude that the DCA effect on epithelial permeability is to a large part induced by intramural reflex(es) containing nicotinic receptors . The results also suggest that mast cell degranulation and NO release are not involved in the mechanism . This indicates that the nerve effect on intestinal paracellular permeability is not mediated by the mechanisms described for stress or Clostridium difficile toxin A. Bioorg Med Chem, 2003 Jul 17, 11(14), 3065 - 71 A quantitative structure-activity relationship study on Clostridium histolyticum collagenase inhibitors: roles of electrotopological state indices; Gupta SP et al.; A quantitative structure-activity relationship (QSAR) study has been made on eight different series of Clostridium histolyticum collegenase (ChC) inhibitors . These series are comprised of four different groups of sulfonylated amino acids and their corresponding hydroxamates . In each series, the inhibition potency of the compounds has been found to be significantly correlated with the electrotopological state (E-state) indices of nitrogen and sulfur atoms of the sulfonylated amino group in the molecules, showing the importance of the electronic characterstics of these atoms in controlling the inhibition potency of the compounds. J Hosp Infect, 2003 Jun, 54(2), 109 - 14 Comparison of the effect of detergent versus hypochlorite cleaning on environmental contamination and incidence of Clostridium difficile infection; Wilcox MH et al.; To determine how best to decontaminate the hospital environment of Clostridium difficile, we carried out a cross-over study on two elderly medicine wards to determine whether cleaning with a hypochlorite disinfectant was better than using neutral detergent in reducing the incidence of C . difficile infection (CDI) . We examined 1128 environmental samples in two years, 35% of which grew C . difficile . There was a significant decrease of CDI incidence on ward X, from 8.9 to 5.3 cases per 100 admissions (P<0.05) using hypochlorite, but there was no significant effect on ward Y . On ward X the incidence of CDI was significantly associated with the proportion of culture-positive environmental sites (P<0.05) . On ward Y the only significant correlation between CDI and C . difficile culture-positive environmental sites was in patient side-rooms (r=0.41, P<0.05) . The total daily defined doses of cefotaxime, cephradine and aminopenicillins were similar throughout the trial . These results provide some evidence that use of hypochlorite for environmental cleaning may significantly reduce incidence of CDI, but emphasize the potential for confounding factors. J Hosp Infect, 2003 Jun, 54(2), 104 - 8 Impact of changes in antibiotic policy on Clostridium difficile-associated diarrhoea (CDAD) over a five-year period in a district general hospital; Khan R et al.; The impact of changes in antibiotic policy on Clostridium difficile-associated diarrhoea (CDAD), over a five-year period between 1995 and 2000, were studied in the Preston Acute Hospitals Trust . In 1996 the policy was changed in the Preston Acute Hospitals Trust from cefotaxime to ceftriaxone for initial treatment of severe sepsis or pneumonia in medical patients . Over the next nine months the average number of patients with C . difficile toxin-positive stools per quarter increased from 16 to 39 . The predicted use of ceftriaxone exceeded by 65% an estimate based on prior use of cefotaxime . A policy of restricted duration of ceftriaxone was introduced, and although this reduced usage by over 50%, CDAD continued at an average of 9.2 cases per month, despite withdrawal of oral cephalosporins in December 1998 . In August 1999 levofloxacin was substituted for ceftriaxone in the policy . The incidence of CDAD fell progressively to five cases per month by 2000 . It would appear that a short (typically three dose) course of third-generation cephalosporin poses a similar risk for CDAD as a more prolonged course . The six-month delay in the decline of CDAD after virtual withdrawal of cephalosporins may reflect a slowly diminishing environmental reservoir. J Am Med Dir Assoc, 2000 Mar-Apr, 1(2), 58 - 61 Clostridium difficile colitis in older adults in long-term facilities and the community: do their outcomes differ? Dharmarajan TS, Patel B, Sipalay M, Norkus EP. OBJECTIVE: To determine if older adults from long-term care facilities (LTCF) have a greater risk of death than older people in the community after the development of Clostridium difficile (CD) colitis during hospitalization . DESIGN: A retrospective review of medical records from all older hospitalized patients with a confirmed diagnosis of CD colitis from February 1995 to February 1997 at Our Lady of Mercy Medical Center, Bronx, NY, a University hospital of the New York Medical College . METHODS: A total of 108 patients (aged 60-97 yrs.) with a positive diagnosis of CD colitis (EIA of CD cytotoxin A and B) were identified . Residence (nursing home vs . community), sex and age, length of hospital stay, laboratory values, the number, dose, and duration of all antibiotics used, and co-morbid medical conditions were examined as potential risk factors for adverse outcome (mortality) . RESULTS: Fifty-two nursing home and 56 older patients living in the community were compared . Outcome (survival vs . death) was equivalent between nursing home (13 of 52 died, 25% death rate) and community elderly (13 of 56 died, 23% death rate) . The patients in the nursing home were, on average, 3 years older than community those in the community, but age was not related to outcome in either group . Death occurred significantly more often in LTCF and community patients who received prolonged antibiotic therapy (P = 0.0056) or were prescribed four or more antibiotics (P = 0.036) during hospitalization . Low serum albumin level was found to be a strong predictor of death (P = 0.002) . However, nursing home and community elderly had similar mean serum albumin levels (P = 0.2797) . Death was also predicted by the use of clindamycin alone (P = 0.046) or penicillin-like antibiotics (excluding cephalosporins) and clindamycin (P = 0.021), or a history of cardiac disease (coronary artery disease or congestive heart failure) (P = 0.022) . CONCLUSIONS: Patients from LTCF do not have an increased risk of mortality compared with older people in the community after developing CD colitis during hospitalization . Factors such as low serum albumin, prolonged antibiotic therapy, the number of antibiotics used, use of specific antibiotics, and cardiac disease were significantly related to an increased risk of death in both LTCF and community older adults . Age did not influence outcome in either group of older adults. J Rehabil Med, 2003 May, (41 Suppl), 56 - 9 Botulinum toxin--mechanisms of action and clinical use in spasticity; Barnes M; Botulinum toxin is a potent neurotoxin produced by the bacterium Clostridium botulinum . There are seven serotypes, all of which block the release of acetylcholine from nerve endings, which gives the compound its theoretical base for reducing spasticity . Initial studies of the use of botulinum toxin in the management of spasticity were promising and now there are a number of well-designed, double-blind, placebo-controlled studies that confirm the place of botulinum toxin in our treatment armoury against focal spasticity . The studies have demonstrated both efficacy and safety . There is still more work to be done in terms of disability although early reports confirm functional improvements, particularly reduction of pain as well as improvements in nursing care, hygiene and carer burden . Further studies also need to be done to confirm the place of botulinum toxin in the overall context of other treatment possibilities in the management of spasticity. Vet Microbiol, 2003 Jul 17, 94(3), 257 - 66 Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens; Nauerby B et al.; The aim of this study was to analyse the genetic diversity among Clostridium perfringens isolates from Danish broiler chickens since both sick and presumably healthy animals were investigated . Isolates (n=279) collected from chickens from 25 farms were analysed by pulsed-field gel electrophoresis (PFGE) with the restriction enzyme SmaI . A high genetic diversity was found . Isolates with different PFGE types were toxin typed by PCR and all were found to be of type A . The results showed that healthy broiler chickens carried several different C . perfringens clones both within a flock and even within individual birds, whereas flocks suffering from necrotic enteritis (NE) or cholangio-hepatitis carried only one or two clones. Vet Microbiol, 2003 Jul 17, 94(3), 225 - 35 Molecular typing of isolates of Clostridium perfringens from healthy and diseased poultry; Engstrom BE et al.; The bacterium Clostridium perfringens can cause both clinical and subclinical disease in poultry . To study the pathogenesis and epidemiology of disease caused by C . perfringens, methods for typing its various strains need to be evaluated . C . perfringens isolates from healthy and diseased poultry from different parts of Sweden were analysed by polymerase chain reaction (PCR) in order to establish the presence of alpha-, beta-, beta2-, epsilon -, iota- and enterotoxin genes . In order to subtype C . perfringens isolates, the two methods amplified fragment length polymorphism (AFLP) and pulsed field gel electrophoresis (PFGE) were compared on 21 C . perfringens isolates from 10 different farms . In a second study, 32 isolates of C . perfringens type A from three broilers from a healthy flock reared without ionophorous anticoccidials were subtyped by PFGE . All 53 isolates analysed with PCR belonged to the toxin type A of C . perfringens, with the gene coding for alpha-toxin production . Two isolates possessed the beta2-gene as well, but none had the other toxin genes . Both AFLP and PFGE differentiated 21 strains into 10 different subtypes . This differentiation correlated closely with the origins of the isolates . Unique subtypes were isolated from seven farms . Only isolates from birds of one farm demonstrated more than one subtype of C . perfringens . The subtyping of the isolates from a healthy flock showed that each bird carried two to three different subtypes and two different subtypes were found in the same kind of tissue sample in four cases . Three of the four different subtypes found in this study were new, compared with the first study . AFLP and PFGE were found to be equally suitable for subtyping of C . perfringens isolates . The wide variation in subtypes in the healthy broilers could be the result of the antibiotic-free rearing of these birds. J Gend Specif Med, 2003, 6(2), 27 - 30 Prevalence of asymptomatic Clostridium difficile colonization in a nursing home population: a cross-sectional study; Rivera EV et al.; OBJECTIVE: We conducted a cross-sectional study to determine the prevalence of asymptomatic Clostridium difficile in a nursing home population . METHODS: Inclusion criteria for the study required that the subjects be residents of the facility for more than 1 month . Exclusion criteria included: (1) patients who developed C . difficile infection within 2 months prior to stool collection; (2) metronidazole or vancomycin therapy within 2 months prior to stool collection; (3) past surgical history of colectomy; and (4) possible signs and symptoms of active C . difficile infection . RESULTS: The prevalence of asymptomatic C . difficile infection in this population was 5% . There was no significantly higher prevalence of asymptomatic C . difficile based on age, race, length of stay, gender, diabetes, renal failure, history of cancer, recent antibiotic use, gastric tube, histamine2 blocker/proton pump inhibitor use, Foley catheter, or dementia . CONCLUSIONS: The prevalence of asymptomatic C . difficile in this population is similar to previously studied populations . Further research should follow a cohort of asymptomatic individuals with C . difficile to determine if colonization may be protective against symptomatic infection. Can J Gastroenterol, 2003 Jun, 17(6), 369 - 73 Clostridium difficile-associated diarrhea: predictors of severity in patients presenting to the emergency department; Andrews CN et al.; OBJECTIVE: Experiences with Clostridium difficile-associated diarrhea (CDAD) were reviewed to determine predictors of severity in patients presenting from the community . METHODS: All patients admitted to two hospitals over 4.5 years with a primary diagnosis of CDAD were reviewed . Patients requiring a hospital stay of greater than 14 days, colectomy, intensive care unit admission or who died were classified as 'severe CDAD' and compared with the remainder of the patients (termed 'mild CDAD') . RESULTS: One hundred fifty-three patients (mean age 63.4+/-20.5 years, range 21 to 93, 64.7% female) were reviewed . Forty-four per cent of the patients had community-acquired CDAD, and the remainder had hospital-acquired disease . There were 44/153 (28.8%) patients with severe CDAD, of which 10/153 (6.5%) died . The severe group had more patients over 70 years old (75% versus 43% in the mild group, OR 3.09, CI 1.81-8.63, P<0.001) and had more comorbid disease (median two major organ systems affected {range zero to five} versus one {range zero to four} in the mild group, OR 1.52, CI 1.27-2.65, P<0.05) . Patients with recurrent CDAD were more likely to have severe CDAD (12/44 versus 10/109 in the mild group, OR 4.10, CI 1.47-9.40, P<0.01) . CONCLUSION: Age over 70 years, comorbid illness and CDAD recurrence are significant risk factors for severe disease and a poor outcome in patients admitted to hospital for CDAD. Int J Food Microbiol, 2003 Aug 15, 85(1-2), 23 - 33 Inhibition of Clostridium tyrobutyricum in Vidiago cheese by Lactococcus lactis ssp . lactis IPLA 729, a nisin Z producer; Rilla N et al.; Lactococcus lactis ssp . lactis IPLA 729 is a nisin Z producer isolated from raw milk cheese able to grow and produce nisin Z in milk . The ability of this strain to inhibit the growth of Clostridium tyrobutyricum CECT 4011, a late blowing agent, in Vidiago cheese, a semi-hard farmhouse variety, manufactured in Asturias, Northern Spain, was investigated . For control purposes, cheeses were manufactured with the mesophilic mixed starter IPLA-001 . In experimental cheeses, the nisin-producing strain L . lactis IPLA 729 was combined with this starter . Nisin Z activity reached a concentration of 1600 AU/ml in 1-day cheeses and this level was maintained until 15 days of ripening . Furthermore, to compare the inhibitory activity of the nisin-producing strain to nitrate, cheeses were also manufactured with a commercial starter culture and potassium nitrate as anti-blowing agent was added in accordance with Vidiago's cheesemakers . The control, experimental and commercial cheeses were contaminated with C . tyrobutyricum CECT 4011 . The composition of the three different cheeses showed only slight differences with respect to total solids, protein and fat, although control and experimental cheeses showed a richer flavour-compound profile than commercial cheeses . The level of the spoilage strain C . tyrobutyricum CECT 4011 decreased from 1.2x10(6) to 1.3x10(3) cfu/g during ripening in presence of the nisin Z producer, while it increased to 1.99x10(9) cfu/g in control cheeses and to 3.5x10(7) cfu/g in commercial cheeses. J Med Microbiol, 2003 Jul, 52(Pt 7), 573 - 8 Clostridium difficile in a geriatric unit: a prospective epidemiological study employing a novel S-layer typing method; McCoubrey J et al.; Clostridium difficile is the major identifiable cause of antibiotic-associated diarrhoea in the UK . The aim of this study was to employ traditional culture, toxin detection and a novel typing method to determine the level of C . difficile colonization and disease in a population of elderly patients and to investigate the association between strains in the patients and their environment . Three hundred and ninety patients between 62 and 101 years of age admitted to a geriatric unit in the Royal Victoria Hospital (RVH), Edinburgh, were investigated for the presence of C . difficile . C . difficile was cultured from 100 (26 %) patients using pre-reduced cycloserine-cefoxitin egg yolk agar, and toxin(s) was detected in the faeces of 34 of these patients using the Techlab ELISA test kit for the detection of C . difficile toxins A and/or B . Toxin(s) was detected in a further 18 patients from whom no C . difficile was detected in culture . Of the patients in whom C . difficile was detected, 49 % had diarrhoea, with the highest proportion of patients with diarrhoea being both culture- and toxin-positive for C . difficile . Environmental sampling of the patient environment yielded C . difficile from 14 % of samples . The organism was most frequently isolated from floors, sluice-rooms and toilet areas . The variation in the molecular mass of the C . difficile S-layer proteins was exploited as the basis of a novel typing method for C . difficile . Isolates from patients in the RVH were given a four-digit 'S-type' number based on their S-layer protein profile . A total of seven S-types were identified, with one type, toxigenic S-type 5236, accounting for 73 % of all clinical isolates and 91 % of environmental isolates. Commun Dis Intell, 2003, 27 Suppl, S143 - 6 Non-antibiotic therapies for infectious diseases; Carson CF et al.; The emergence of multiple antibiotic resistant organisms in the general community is a potentially serious threat to public health . The emergence of antibiotic resistance has not yet prompted a radical revision of antibiotic utilisation . Instead it has prompted the development of additional antibiotics . Unfortunately, this does not relieve the underlying selection pressure that drives the development of resistance . A paradigm shift in the treatment of infectious disease is necessary to prevent antibiotics becoming obsolete and, where appropriate, alternatives to antibiotics ought to be considered . There are already several non-antibiotic approaches to the treatment and prevention of infection including probiotics, phages and phytomedicines . There is some evidence that probiotics such as Lactobacillus spp . or Saccharomyces boulardii are useful in the prevention and treatment of diarrhoea, including Clostridium difficile-associated diarrhoea that can be difficult to treat and recurs frequently . Bacteriophages have received renewed attention for the control of both staphylococcal and gastrointestinal infections . Phytomedicines that have been utilised in the treatment of infections include artesunate for malaria, tea tree oil for skin infections, honey for wound infections, mastic gum for Helicobacter pylori gastric ulcers and cranberry juice for urinary tract infections . Many infections may prove amenable to safe and effective treatment with non-antibiotics. Commun Dis Intell, 2003, 27 Suppl, S28 - 31 Restriction of third generation cephalosporin use reduces the incidence of Clostridium difficile-associated diarrhoea in hospitalised patients; Thomas C et al.; Third generation cephalosporin antibiotics (3GC) have become the antibiotics of choice in many hospitals in recent years for the treatment of infections such as community-acquired pneumonia . However, increased use of 3GCs has also been associated with a rise in the occurrence of antibiotic-associated diarrhoea due to Clostridium difficile, as well as an increase in the prevalence of antibiotic resistant organisms such as methicillin resistant Staphylococcus aureus, vancomycin resistant entrococci, and extended-spectrum beta-lactamase-producing gram negative bacilli . In Western Australia, greater use of 3GCs was shown to correlate with more Clostridium difficile-associated diarrhoea (CDAD) in a large acute care teaching hospital during the 1980s . During the 1990s, the use of 3GCs in this hospital remained high and, at the end of 1998, a policy was introduced to prevent the use of ceftriaxone (the only 3GC in use) without prior approval . This resulted in a decline in 3GC use and a 50 per cent reduction in the incidence of CDAD during 1999 and 2000 . To strengthen these observations, the impact of the 3GC policy on the occurrence of CDAD was analysed using time-series intervention analysis that showed a statistically significant decrease in the occurrence of CDAD during the post-intervention period after controlling for exogenous factors . Thus, changes in antibiotic prescribing practices can influence the incidence of CDAD and, potentially, antibiotic resistant pathogens. Int J Syst Evol Microbiol . 2003 May;53(Pt 3):931. Paenibacillus durus (Collins et al . 1994, formerly Clostridium durum Smith and Cato 1974) has priority over Paenibacillus azotofixans (Seldin et al . 1984) . Opinion 73; The host cell chaperone Hsp90 is essential for translocation of the binary Clostridium botulinum C2 toxin into the cytosol; Institut fur Experimentelle und Klinische Pharmakologie und Toxikologie der Albert-Ludwigs-Universitat Freiburg, Albertstrasse 25 (Otto-Krayer-Haus), D-79104 Freiburg, GermanyClostridium botulinum C2 toxin is the prototype of the binary actin-ADP-ribosylating toxins and consists of the binding component C2II and the enzyme component C2I . The activated binding component C2IIa forms heptamers, which bind to carbohydrates on the cell surface and interact with the enzyme component C2I . This toxin complex is taken up by receptor-mediated endocytosis . In acidic endosomes, heptameric C2IIa forms pores and mediates the translocation of C2I into the cytosol . We report that the heat shock protein (Hsp) 90-specific inhibitors, geldanamycin or radicicol, block intoxication of Vero cells, rat astrocytes, and HeLa cells by C2 toxin . ADP-ribosylation of actin in the cytosol of toxin-treated cells revealed that less active C2I was translocated into the cytosol after treatment with Hsp90 inhibitors . Under control conditions, C2I was localized in the cytosol of toxin-treated rat astrocytes, whereas geldanamycin blocked the cytosolic distribution of C2I . At low extracellular pH (pH 4.5), which allows the direct translocation of C2I via C2IIa heptamers across the cell membrane into the cytosol, Hsp90 inhibitors retarded intoxication by C2I . Geldanamycin did not affect toxin binding, endocytosis, and pore formation by C2IIa . The ADP-ribosyltransferase activity of C2I was not affected by Hsp90 inhibitors in vitro . The cytotoxic actions of the actin-ADP-ribosylating Clostridium perfringens iota toxin and the Rho-ADP-ribosylating C2-C3 fusion toxin was similarly blocked by Hsp90 inhibitors . In contrast, radicicol and geldanamycin had no effect on anthrax lethal toxin-induced cytotoxicity of J774-A1 macrophage-like cells or on cytotoxic effects of the glucosylating Clostridium difficile toxin B in Vero cells . The data indicate that Hsp90 is essential for the membrane translocation of ADP-ribosylating toxins delivered by C2II. Vaccine, 2003 Jul 4, 21(23), 3200 - 7 Cellulose beads bound to cellulose binding domain-fused recombinant proteins; an adjuvant system for parenteral vaccination of fish; Maurice S et al.; A recombinant form of the outer membrane protein (A-layer protein) associated with atypical Aeromonas salmonicida was expressed, fused to a cellulose binding domain (CBD) isolated from Clostridium cellulovorans . The resultant chimerical protein was bound to either Sigmacell 20((R)) or Orbicell cellulose particles . Common goldfish were injected intraperitoneally with the cellulose-protein complex and blood serum antibody levels produced against A-protein were examined weekly by means of ELISA . These titers were compared to those induced by immunization of goldfish with the same protein, with or without Freund's incomplete adjuvant, as well as to a standard bacterin-adjuvant system . Small Orbicell beads (1-10 microM) induced antibody levels that were equal to the titers produced by the adjuvanted protein and bacterin formulae . In comparison, the larger Sigmacell particles (10-20 microM) proved to be poor immunopotentiators . The long-term titer elicited from a single injection of A-protein bound to Orbicell beads was equivalent to that induced by two injections . All the vaccinated fish demonstrated memory to the A-layer protein after exposure to a pathogenic load of atypical A . salmonicida with Orbicell treated fish displaying the highest titer . No direct correlation was found between the presence of anti-A-protein antibodies and protection against infection . The paper describes a simple and safe method to increase the potential immunogenicity of soluble recombinant proteins by employing relatively inexpensive cellulose particles. Vaccine, 2003 Jul 4, 21(23), 3110 - 7 DNA vaccination protects against botulinum neurotoxin type F; Bennett AM et al.; A DNA vaccine was constructed which expressed the binding domain of Clostridium botulinum neurotoxin serotype F fused to a signal peptide . Three intra-muscular doses fully protected Balb/c mice against 10(4) MLD of serotype F toxin . Priming of the immune response by DNA vaccination followed by a single booster with type F binding domain protein resulted in high levels of antibody against the binding domain . This study demonstrates the utility of DNA vaccination for protection against botulinum neurotoxin type F and indicates that a prime-boost regimen could be an efficient method of generating antibody for passive immune therapy in cases of botulism involving serotype F toxin. J Hematother Stem Cell Res, 2003 Apr, 12(2), 155 - 60 Infectious complications in patients receiving mobilization chemotherapy for autologous peripheral blood stem cell collection; Jillella AP et al.; The purpose of this retrospective study was to evaluate infectious complications in patients receiving mobilization chemotherapy for stem cell collection prior to autologous peripheral blood stem cell transplantation . An additional goal was to evaluate risk factors associated with the development of infectious complications . At the Medical College of Georgia BMT center, 54 patients were administered mobilization chemotherapy for the purpose of collecting stem cells between June, 1997, and May, 2002 . All patients received Filgrastim in addition to chemotherapy, and 50 of 54 patients received prophylactic acyclovir, fluconazole, and ciprofloxacin until neutrophil recovery . The median duration to neutrophil recovery was 11 days . Fourteen of 54 (26%) patients developed fever/infections during the mobilization phase . One patient developed both a catheter-related infection and Clostridium difficile colitis, increasing the total number of infectious episodes to 15 . Twelve patients had a documented site of infection whereas 2 patients had neutropenic fever with no identifiable source . Eight of the 15 (55%) infections were Gram-positive catheter infections . All the patients were treated successfully with antibiotics . No systemic fungal infections were identified and none of the patients died from complications related to mobilization chemotherapy . Logistic regression was applied for univariate and multivariate analysis and showed that age, sex, diagnosis, neutrophil recovery, disease status, use of salvage chemotherapy, and mobilization regimen used did not affect the infection rate . In our series of 54 patients, 14 patients developed fever/infections during mobilization . Although there is a substantial risk of infectious complications among patients who receive mobilization chemotherapy, it is not clear that prophylactic antibiotics decrease infectious complications . Because the vast majority of infections are Gram-positive catheter infections, it appears reasonable to employ Gram-positive prophylaxis . Controlled studies should be conducted to define the optimum mobilization regimens as well as the optimum combination of prophylactic antibiotics. Verh K Acad Geneeskd Belg, 2003, 65(1), 29 - 46 Vascular targeting: a potential additional anti-cancer treatment; Lambin P et al.; Our preclinical in vivo investigations were aimed to evaluate the potential of selectively targeting the tumour vasculature as an additional anti-cancer strategy . Using a clinical angiography method and the tumour growth delay assay, the efficacy of the vascular targeting compound combretastatin A-4 phosphate was demonstrated in rat rhabdomyosarcomas: specifically, an inverse efficacy as compared to radio- or chemotherapy was measured when comparing small and large tumours . The combination of this vascular targeting compound with ionising radiation indicated, depending on the timing and the sequence, a potential benefit . Within the limits of our experiments, no significant increase in tumour growth delay was measured when TNP-470 anti-angiogenesis was given after the combretastatin A-4 phosphate treatment . The use of the vascular targeting agent did advance the in vivo application of a non-apathogenic anaerobe Clostridium transfer system of therapeutic proteins . A strong improvement of the selective expression of cytosine deaminase in the tumour microenvironment was observed, even with very small tumours . In summary, the present preclinical results demonstrate several advantages from the introduction of vascular targeting next to classical and novel anti-cancer therapies. Am J Physiol Gastrointest Liver Physiol, 2003 Nov, 285(5), G1049 - 55 Epub 2003 Jun 11. Toxin B of Clostridium difficile activates human VIP submucosal neurons, in part via an IL-1beta-dependent pathway; Neunlist M et al.; This study investigated whether toxin B of Clostridium difficile can activate human submucosal neurons and the involved pathways . Isolated segments of human colon were placed in organ culture for 3 h in the presence of toxin B or IL-1beta . Whole mounts of internal submucosal plexus were stained with antibodies against c-Fos, neuron-specific enolase (NSE), vasoactive intestinal polypeptide (VIP), and substance P (SP) . The membrane potential (Vm) response of submucosal neurons to local application of toxin B and IL-1beta was determined by a multisite optical recording technique . Toxin B (0.1 to 10 ng/ml) increased the proportion of c-Fos-positive neurons dose dependently compared with the control . In the presence of toxin B (10 ng/ml), most c-Fos-positive neurons were immunoreactive for VIP (79.8 +/- 22.5%) but only 19.4 +/- 14.0% for SP . Toxin B induced a rapid rise in IL-1beta mRNA level and a sixfold increase in IL-1beta protein in supernatant after 3 h of incubation . c-Fos expression induced by toxin B was reduced dose dependently by IL-1 receptor antagonist (0.1-10 ng/ml) . IL-1beta significantly increased c-Fos expression in submucosal neurons compared with the control (34.2 +/- 10.1 vs . 5.1 +/- 1.3% of NSE neurons) . Microejection of toxin B had no effect on the Vm of enteric neurons . Evidence of a direct excitatory effect of IL-1beta on Vm was detected in a minority of enteric neurons . Therefore, toxin B of C . difficile activates VIP-positive submucosal neurons, at least in part, via an indirect IL-1beta-dependent pathway. Mol Gen Mikrobiol Virusol, 2003, (2), 13 - 6 {Gene-engineering approach for the production of A and B toxin fragments for diagnostic and immunotherapeutic use in Clostridium difficile infection}; Varfolomeeva NA et al.; Clostridium difficle is a causative agent of severe and difficult-to-diagnose human infections . Toxins A and B, which modify the RAS-like proteins of eukaryotic cells, are the major factor in the pathogenicity of the discussed causative agent . These very toxins are considered as the key components of the developed diagnostic and therapeutic-and-preventive preparations . The C-terminal fragments of toxins A and B as well as hybrid products, consisting of fragments of both toxins, were cloned, within the present case study, by using the pET28 plasmid vector . The recombinant plasmids were transformed into strains Escherichia Coli BL21 (DE3), and they were used later to produce the appropriate proteins . The purified protein preparations were isolated from the ultrasound bacterial-cell lysate by using the method of metal-affinity chromatography to be used later in the production of hyper-immune rabbit sera. Clin Pediatr (Phila), 2003 May, 42(4), 347 - 52 Clostridium difficile-associated diarrhea in a pediatric hospital; Spivack JG et al.; This retrospective cohort analysis examined the risk factors, symptoms, and severity of disease associated with C . difficile in pediatric inpatients . Risk factors for a C . difficile-positive test were an oncologic diagnosis, diarrhea of more than 2 days' duration, and gastrointestinal symptoms, especially abdominal pain . Over a 3.5-year period, there was a total of 22 C . difficile-positive patients, and most had mild, self-limiting diarrheal illness . No cases of C . difficile diarrhea were identified . Seventy-eight percent of the C . difficile-positive patients were found to have alternate risk factors for diarrhea . Our data indicate that C . difficile rarely causes severe diarrhea in pediatric inpatients and that C . difficile testing should be limited to patients with severe prolonged diarrhea and abdominal pain. Exp Cell Res, 2003 Jul 1, 287(1), 106 - 15 Rho protein-mediated changes in the structure of the actin cytoskeleton regulate human inducible NO synthase gene expression; Witteck A et al.; Rho proteins (Rho, Rac, Cdc 42) are known to control the organization of the actin cytoskeleton as well as gene expression . Inhibition of Rho proteins by Clostridium difficile toxin B disrupted the F-actin cytoskeleton and enhanced cytokine-induced inducible nitric oxide synthase (iNOS) expression in human epithelial cells . Also specific inhibition by Y-27632 of p160ROCK, which mediates Rho effects on actin fibers, caused a disruption of the actin cytoskeleton and a superinduction of cytokine-induced iNOS expression . Accordingly, direct disruption of the actin cytoskeleton by cytochalasin D, latrunculin B, or jasplakinolide enhanced cytokine-induced iNOS expression . The transcription factor serum response factor (SRF) has been described as mediating actin cytoskeleton-dependent regulation of gene expression . Direct targets of SRF are activating protein 1 (AP1)-dependent genes . All compounds used inhibited SRF- and AP1-dependent reporter gene expression in DLD-1 cells . However, the enhancing effect of the actin cytoskeleton-disrupting compounds on human iNOS promoter activity was much less pronounced than the effect on iNOS mRNA expression . Therefore, besides transcriptional mechanisms, posttranscriptional effects seem to be involved in the regulation of iNOS expression by the above compounds . In conclusion, our data suggest that Rho protein-mediated changes of the actin cytoskeleton negatively modulate the expression of human iNOS. J Infect, 2003 May, 46(4), 253 - 5 Clostridium perfringens septicemia with thrombophlebitis of the portal vein; Shin DH et al.; We report a case of patient with Clostridium perfringens septicemia and thrombophlebitis of the portal vein (pylephlebitis), probably secondary to an initially unrecognized gastric ulcer . The extension of the thrombosis from the superior mesenteric vein to the main portal vein on a repeat CT scan and subsequent partial resolution of the thrombus with antibiotic therapy alone, suggested that Clostridium perfringens bacteremia may have enhanced the formation of thrombus . The coexistence of Clostridium perfringens septicemia and pylephlebitis should prompt a search for intra-abdominal processes as the portal of entry of infection. Inflammation, 2003 Apr, 27(2), 89 - 95 Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells; Schmeck B et al.; Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells . We made use of Clostridium difficile toxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC) . Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)-or tumor necrosis factor-alpha (TNFalpha)-related COX-2 expression but had no effect on COX-1 protein levels . TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-kappaB activation (NF-kappaB) . Rho inhibition did not affect COX-1 activity . Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F(1alpha), (6k-PGF(1alpha)) . In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF(1alpha) liberation when TcdB-10643 was added 10 h after LPS or TNFalpha stimulation of EC . Therefore, RhoA/Rac1/Cdc42 contribute to NF-kappaB-dependent LPS- and TNFalpha-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2. J Chemother, 2003 Apr, 15(2), 113 - 7 Comparative in vitro activity of PH-027 versus linezolid and other anti-anaerobic antimicrobials against clinical isolates of Clostridium difficile and other anaerobic bacteria; Phillips OA et al.; PH-027 is a new 5-triazole oxazolidinone synthesized in our laboratories, which shows strong activity against gram-positive aerobic bacteria including clinical isolates . The objective of this study was to investigate the in vitro activity of this compound in comparison with linezolid and other antibiotics against gram-positive and gram-negative anaerobes . The in vitro activity of PH-027 in comparison with those of linezolid and other antimicrobial agents was evaluated against 201 clinical isolates of gram-positive and gram-negative anaerobic bacteria by agar dilution and Etest methods . PH-027 showed excellent activity, with minimum inhibitory concentrations (MIC) in the range of 0.12-4.0 microg/ml against all isolates; MIC90s being 4.0, 1.0, 2.0, 2.0 and 2.0 microg/ml against Clostridium difficile, Peptostreptococcus spp., Bacteroides fragilis, Prevotella bivia and Fusobacterium spp . respectively . In comparison, linezolid had MIC in the range of 0.5-4.0 microg/ml against all isolates, with MIC90s of 2.0, 4.0, 4.0, 4.0 and 2.0 microg/ml against the same set of bacteria respectively . PH-027 demonstrated excellent in vitro activity that is superior to linezolid against Peptostreptococcus spp., B . fragilis and P . bivia . However, against C . difficile and Fusobacterium spp, PH-027 and linezolid showed comparable in vitro activity . Against all anaerobes, metronidazole, PH-027 and, to a lesser extent, linezolid had the most potent activity . From the results of in vitro susceptibility testing, both linezolid and PH-027 show promise in the treatment of anaerobic infections. J Clin Microbiol, 2003 Jun, 41(6), 2454 - 7 Evaluation of repetitive element sequence-based PCR as a molecular typing method for Clostridium difficile; Spigaglia P et al.; Repetitive element sequence-based PCR (rep-PCR) is a typing method that enables the generation of DNA fingerprinting that discriminates bacterial strains . In this study, we evaluated the applicability of rep-PCR in typing Clostridium difficile clinical isolates . The results obtained by rep-PCR were compared with those obtained by pulsed-field gel electrophoresis (PFGE) and PCR ribotyping . A high correspondence between pattern differentiations produced by rep-PCR and PFGE was observed, whereas PCR ribotyping showed a lower level of discriminatory power. Am J Physiol Cell Physiol, 2003 Oct, 285(4), C935 - 44 Epub 2003 Jun 04. Hypotonicity induces membrane protrusions and actin remodeling via activation of small GTPases Rac and Cdc42 in Rat-1 fibroblasts; Carton I et al.; An important consequence of cell swelling is the reorganization of the F-actin cytoskeleton in different cell types . We demonstrate in this study by means of rhodamine-phalloidin labeling and fluorescence microscopy that a drastic reorganization of F-actin occurs in swollen Rat-1 fibroblasts: stress fibers disappear and F-actin patches are formed in peripheral extensions at the cell border . Moreover, we demonstrate that activation of both Rac and Cdc42, members of the family of small Rho GTPases, forms the link between the hypotonic stimulation and F-actin reorganization . Indeed, inhibition of the small GTPases RhoA, Rac, and Cdc42 (by Clostridium difficile toxin B) prevents the hypotonicity-induced reorganization of the actin cytoskeleton, whereas inhibition of RhoA alone (by C . limosum C3 exoenzyme) does not preclude this rearrangement . Second, a direct activation and translocation toward the actin patches underneath the plasma membrane is observed for endogenous Rac and Cdc42 (but not for RhoA) during cell swelling . Finally, transfection of Rat-1 fibroblasts with constitutively active RhoA, dominant negative Rac, or dominant negative Cdc42 abolishes the swelling-induced actin reorganization . Interestingly, application of cRGD, a competitor peptide for fibronectin-integrin association, induces identical membrane protrusions and changes in the F-actin cytoskeleton that are also inhibited by C . difficile toxin B and dominant negative Rac or Cdc42 . Moreover, cRGD also induces a redistribution of endogenous Rac and Cdc42 to the newly formed submembranous F-actin patches . We therefore conclude that hypotonicity and cRGD remodel the F-actin cytoskeleton in Rat-1 fibroblasts in a Rac/Cdc42-dependent way. Knee, 2003 Jun, 10(2), 149 - 53 Clostridium infection in a knee extensor mechanism allograft: case report and review; Pearle AD et al.; An unusual case of Clostridium perfringens infection after an allograft reconstruction of an extensor mechanism in a multiply revised total knee arthroplasty is reported . The case occurred in an elderly patient with an underlying bleeding dyscrasia who had previously undergone two revision total knee arthroplasties . The patient was treated by open irrigation, debridement, removal of allograft and prosthesis, intravenous antibiotics, and implantation of an antibiotic impregnated cement spacer utilizing an intramedullary rod. Ital Heart J Suppl, 2003 Mar, 4(3), 244 - 7 {Type-A aortic dissection without chest pain in a patient with Clostridium fallax infection}; Zucchelli G et al.; We describe the case of a 64-year-old patient admitted to our hospital because of syncope and suspicion of cardiac tamponade . At admission he had temporary alteration of conscience with clinical evidence of sepsis without chest pain . There was a mild pericardial effusion in absence of clinical and echocardiographic signs of cardiac tamponade . About 36 hours later we found evidence of an aortic dissection and in the blood culture an isolation of Clostridium fallax that we consider the probable cause of this lesion. Eur J Pharmacol, 2003 May 23, 469(1-3), 125 - 34 S1P3 receptors mediate the potent constriction of cerebral arteries by sphingosine-1-phosphate; Salomone S et al.; We characterized the effect of Sphingosine-1-phosphate (S1P) on vascular tone . S1P selectively constricted isolated cerebral, but not peripheral arteries, despite ubiquitous expression of S1P(1), S1P(2), S1P(3) and S1P(5) receptor mRNA . Clostridium B and C3 toxins and the rho-kinase inhibitor Y27632 (trans-N-(4-pyridyl)-4-(l-aminoethyl)-cyclohexane carboxamide) reduced this vasoconstriction to S1P, indicating that the response was mediated through Rho . Pertussis toxin displayed only weak inhibition, suggesting minor involvement of G(i/o) protein . The S1P effect was specifically reduced by adenovirus bearing a s1p(3) but not s1p(2), antisense construct . Furthermore, suramin, which selectively blocks S1P(3) receptors, inhibited the vasoconstrictor effect of S1P, indicating that S1P(3) receptors account for at least part of S1P-mediated vasoconstriction in cerebral arteries . In vivo, intracarotid injection of S1P decreased cerebral blood flow, an effect prevented by suramin treatment . Because S1P constricts cerebral blood vessels and is released from platelets during clotting, the S1P/S1P(3) system constitutes a novel potential target for cerebrovascular disease therapy. Vet Microbiol, 2003 Jul 1, 94(2), 121 - 9 Prevalence of cpb2, encoding beta2 toxin, in Clostridium perfringens field isolates: correlation of genotype with phenotype; Bueschel DM et al.; Beta2 toxin, encoded by the cpb2 gene, has been implicated in the pathogenesis of porcine, equine and bovine enteritis by type A Clostridium perfringens . By incorporating primers to cpb2 into a multiplex genotyping PCR, we screened 3270 field isolates of C . perfringens . Of these, 37.2% were PCR positive for the cpb2 gene . The majority of isolates from cases of porcine enteritis were positive for cpb2 (>85%), and this was even more true for C . perfringens isolated from cases of porcine neonatal enteritis (91.8%) . In contrast, isolates from normal pigs only contained cpb2 in 11.1% of cases . The correlation between enteritis in other animal species and the presence of cpb2 was not so strong . cpb2 was found in 21.4% of C . perfringens isolates from cattle with enteritis, and in 47.3% of isolates from calves with enteritis or abomastitis . The prevalence of cpb2 varied with genotype, with type A isolates being positive for this gene in 35.1% of cases . Furthermore, enterotoxigenic type D or type E strains almost always carried cpb2 . We cloned a 6xHIS-tagged beta2 (HIS-beta2) and used this protein to raise antiserum against beta2 . Culture supernatants from 68 cpb2-positive and 13 cpb2-negative strains were tested for the presence of beta2 by Western blotting . In cpb2-positive isolates of porcine origin, beta2 was almost always detected (96.9%) . However, in cpb2-positive isolates from other animal species, only 50.0% expressed beta2 protein . The high rate of cpb2-positivity among strains from neonatal pigs with enteritis and the high correlation of genotype with phenotype, supports the contention that beta2 toxin plays a role in the pathogenesis of these infections . However, it may be important to consider the use of an additional method for the detection of beta2 toxin in non-porcine cpb2-positive isolates when making claims about the role of beta2 in enteritis in non-porcine species. Emerg Infect Dis, 2003 Jun, 9(6), 730 - 3 Fluoroquinolone use and Clostridium difficile-associated diarrhea; McCusker ME et al.; We performed a case-control study to evaluate the association between antibiotic use and Clostridium difficile-associated diarrhea (CDAD), matching for admission unit and time at risk for CDAD . A multivariable regression model showed that treatment with fluoroquinolones (odds ratio 12.7; 95% confidence interval 2.6 to 61.6) was the strongest risk factor for CDAD. Am J Kidney Dis . 2003 May;41(5):E14. Clostridium difficile colitis associated with hemolytic-uremic syndrome; Mbonu CC et al.; The authors report the case of a 46-year-old woman who presented with vomiting and profuse bloody diarrhea . Laboratory studies were significant for a hematocrit of 27% and lactate dehydrogenase of 5,394 U/L (5,394 U/L) . Her renal function deteriorated rapidly with a peak creatinine of 12.4 mg/dL (1,096.4 micromol/L), and platelet count dropped simultaneously to a nadir of 123,000/microL (123 x 10(9)/L} . Schistocytes were observed in peripheral blood smear . Stool was positive for Clostridium difficile toxin A by enzyme immunoassay (EIA) . Stool assay for Shiga-like toxin was negative by EIA, and stool cultures returned negative for Escherichia coli O157:H7 and other enteric pathogens . A diagnosis of C difficile colitis associated with hemolytic-uremic syndrome was made; the patient received plasmapheresis and recovered with no relapse after 10 months of follow-up . This is the second reported case of C difficile colitis associated with hemolytic-uremic syndrome in an adult. Microbiology, 2003 Jun, 149(Pt 6), 1377 - 86 Multiple effects on Clostridium perfringens binding, uptake and trafficking to lysosomes by inhibitors of macrophage phagocytosis receptors; O'Brien DK et al.; Clostridium perfringens is a Gram-positive, anaerobic bacterium that is the most common cause of gas gangrene (clostridial myonecrosis) in humans . C . perfringens produces a variety of extracellular toxins that are thought to be the major virulence factors of the organism . However, C . perfringens has recently been shown to have the ability to survive in a murine macrophage-like cell line, J774-33, even under aerobic conditions . In J774-33 cells, C . perfringens can escape the phagosome and gain access to the cytoplasm . Since the receptor that is used for phagocytosis can determine the fate of an intracellular bacterium, we used a variety of inhibitors of specific receptors to identify those used by J774-33 cells to phagocytose C . perfringens . It was found that the scavenger receptor and mannose receptor(s) were involved in the phagocytosis of C . perfringens . In the presence of complement, the complement receptor (CR3) was also involved in the binding and/or uptake of C . perfringens . Since the receptor inhibition studies indicated that the scavenger receptor played a major role in phagocytosis, C . perfringens binding studies were performed with a Chinese hamster ovary (CHO) cell line expressing the mouse SR-A receptor . The cell line expressing the SR-A receptor showed a significant increase in C . perfringens binding in comparison to the non-transfected CHO cells . In the absence of opsonizing antibodies, the Fc receptor was not used to phagocytose C . perfringens . Forcing the macrophages to use a specific receptor by using combinations of different receptor inhibitors led to only a slight increase in co-localization of intracellular C . perfringens with the late endosome-lysosome marker LAMP-1 . Carbohydrate analysis of C . perfringens strain 13 extracellular polysaccharide confirmed the presence of mannose and negatively charged residues of glucuronic acid, which may provide the moieties that promote binding to the mannose and scavenger receptors, respectively. Vet Res Commun, 2003 Apr, 27(3), 231 - 41 The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep; Fernandez Miyakawa ME et al.; Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals . The disease is caused by C . perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine . A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine . In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine . Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species . Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin . Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin . Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment: these changes were more marked in goats than in sheep . No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin . These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms. J Bacteriol, 2003 Jun, 185(12), 3644 - 53 Antisense RNA downregulation of coenzyme A transferase combined with alcohol-aldehyde dehydrogenase overexpression leads to predominantly alcohologenic Clostridium acetobutylicum fermentations; Tummala SB et al.; Plasmid pAADB1 for the overexpression of the alcohol-aldehyde dehydrogenase (aad) gene and downregulation of the coenzyme A transferase (CoAT) using antisense RNA (asRNA) against ctfB (the second CoAT gene on the polycistronic aad-ctfA-ctfB message) was used in order to increase the butanol/acetone ratio of Clostridium acetobutylicum ATCC 824 fermentations . Acetone and butanol levels were drastically reduced in 824(pCTFB1AS) (expresses only an asRNA against ctfB) compared to 824(pSOS95del) (plasmid control) . Compared to strain 824(pCTFB1AS), 824(pAADB1) fermentations exhibited two profound differences . First, butanol levels were ca . 2.8-fold higher in 824(pAADB1) and restored back to plasmid control levels, thus supporting the hypothesis that asRNA downregulation of ctfB leads to degradation of the whole aad-ctfA-ctfB transcript . Second, ethanol titers in 824(pAADB1) were ca . 23-fold higher and the highest (ca . 200 mM) ever reported in C . acetobutylicum . Western blot analysis confirmed that CoAT was downregulated in 824(pAADB1) at nearly the same levels as in strain 824(pCTFB1AS) . Butyrate depletion in 824(pAADB1) fermentations suggested that butyryl-CoA was limiting butanol production in 824(pAADB1) . This was confirmed by exogenously adding butyric acid to 824(pAADB1) fermentations to increase the butanol/ethanol ratio . DNA microarray analysis showed that aad overexpression profoundly affects the large-scale transcriptional program of the cells . Several classes of genes were differentially expressed {strain 824(pAADB1) versus strain 824(pCTFB1AS)}, including genes of the stress response, sporulation, and chemotaxis . The expression patterns of the CoAT genes (ctfA and ctfB) and aad were consistent with the overexpression of aad and asRNA downregulation of ctfB. Int J Colorectal Dis, 2003 Jul, 18(4), 361 - 4 Epub 2003 Jan 03. Current indications for blow-hole colostomy:ileostomy procedure . A single center experience; Remzi FH et al.; BACKGROUND AND AIMS: Because of improved medical care and surgical techniques blow-hole colostomy with loop ileostomy is now rarely performed to reduce operative risks in patients with toxic megacolon related to inflammatory bowel disease (IBD) . We reviewed patient charts to identify continuing indications for this procedure . PATIENTS AND METHODS: Seventeen patients underwent blow-hole colostomy procedure with ( n=15) or without ( n=2) ileostomy (8 men, 9 women; median age 51 years, range 21-79) during the past 18 years (1983-2001) . RESULTS: The indications for the procedure were: toxic megacolon related to IBD ( n=6), toxic megacolon related to IBD and associated with pregnancy ( n=2), Clostridium difficile colitis ( n=3), adult Hirschsprung's disease ( n=1), pancreatitis with obstructing pseudocyst ( n=1), and palliation for malignant bowel obstruction with metastases ( n=4) . Patients were discharged home after a median stay of 10 days (range 4-32 days) . The 4 patients who underwent a palliative blow-hole procedure had died secondary to their underlying disease by the time of follow-up . Of the remaining 13 patients 12 had their alimentary tract reconstituted, and one still awaits a definitive procedure . CONCLUSION: The blow-hole colostomy-ileostomy procedure is still indicated for select patients with toxic megacolon and large-bowel obstruction . The procedure acts as a bridge to definitive operation for toxic patients with benign disease and palliates those with malignant obstructions and metastasis. J Ind Microbiol Biotechnol, 2003 Jul, 30(7), 414 - 20 Epub 2003 May 28. Sequences affecting the regulation of solvent production in Clostridium acetobutylicum; Scotcher MC et al.; The high solvent phenotype of Clostridium acetobutylicum mutants B and H was complemented by the introduction of a plasmid that contains either an intact or partially-deleted copy of solR, restoring acetone and butanol production to wild-type levels . This demonstrates that the solR open reading frame on pSOLThi is not required to restore solvent levels . The promoter region upstream of alcohol dehydrogense E (adhE) was examined in efforts to identify sites that play major roles in the control of expression . A series of adhE promoter fragments was constructed and the expression of each in acid- and solvent-phases of growth was analyzed using a chloramphenicol acetyl-transferase reporter system . Our results show that a region beyond the 0A box is needed for full induction of the promoter . Additionally, we show that the presence of sequences around a possible processing site designated S2 may have a negative role in the regulation of adhE expression. FEMS Microbiol Lett, 2003 May 28, 222(2), 211 - 20 Regulation of biosynthesis and transport of aromatic amino acids in low-GC Gram-positive bacteria; Panina EM et al.; Computational comparative techniques were applied to analysis of the aromatic amino acid regulon in Gram-positive bacteria . A new candidate transcription regulation signal of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and shikimate kinase genes was identified in Streptococcus and Lactococcus species . New T-boxes were found upstream of aromatic amino acid biosynthesis and transport genes in the Bacillus/Clostridium group . The substrate specificity of proteins from the PabA/TrpG family was assigned based on metabolic reconstruction and analysis of regulatory signals and phylogenetic patterns . New candidate tryptophan transporters were identified; their specificity was predicted by analysis of T-box regulatory sites . Comparison of all available genomes shows that regulation of genes of the aromatic amino acid biosynthesis pathway is quite labile and involves at least four regulatory systems, two at the DNA level and two more involving competition of alternative RNA secondary structures for transcription and/or translation regulation at the RNA level. Support Care Cancer, 2003 Sep, 11(9), 581 - 6 Epub 2003 May 24. Pseudomembranous and neutropenic enterocolitis in pediatric oncology patients; van de Wetering MD et al.; Neutropenic enterocolitis in oncological patients represents a wide spectrum of clinicopathological pictures each with its own entity . Early diagnosis of enterocolitis can lead to improved supportive care and therefore better outcome . We present two cases--patient A, a child with pseudomembranous colitis caused by Clostridium difficile, and patient B, a child with neutropenic enterocolitis, where no organism was found . By allowing an insight into the pathology, immunology and culture results, we demonstrated that early diagnosis leads to improved management and therefore improved outcome. J Hosp Infect, 2003 May, 54(1), 74 - 7 Evidence to support the existence of subgroups within the UK epidemic Clostridium difficile strain (PCR ribotype 1); Fawley WN et al.; We used three different DNA fingerprinting techniques and clindamycin susceptibility testing to confirm that Clostridium difficile PCR ribotype I isolates can be divided into two subclones . This observation may permit a better understanding of the epidemiology and pathogenicity of C . difficile infection. Best Pract Res Clin Gastroenterol, 2003 Jun, 17(3), 475 - 93 Gastrointestinal disorders and the critically ill . Clostridium difficile infection and pseudomembranous colitis; Wilcox MH; Clostridium difficile causes a spectrum of diseases ranging from diarrhoea to pseudomembranous colitis, primarily in the hospitalized elderly, although community-acquired infection is probably under-documented . Host factors are increasingly recognized as critical determinants of disease expression . Exposure to antibiotics, particularly those adversely affecting anaerobic gut flora, appears to create a niche which is exploited by C . difficile . Several retrospective and intervention studies have indicated that third-generation cephalosporins have a high propensity to induce C . difficile diarrhoea . Conversely, some broad-spectrum antibiotics, including ureidopenicillins (e.g . piperacillin-tazobactam) and ciprofloxacin, are less likely to induce C . difficile infection . Effective control of C . difficile in the hospital requires both antibiotic control and prevention of environmental seeding and bacterial spread . Epidemic C . difficile strains are widely distributed in the hospital environment, both as a cause and result of nosocomial diarrhoea . Current treatment options are antibiotic-based, which is less than ideal . Although many biotherapeutic approaches have been tried few have shown real benefit. Curr Gene Ther, 2003 Jun, 3(3), 207 - 21 Tumor-specific gene delivery using genetically engineered bacteria; Theys J et al.; The loco-regional control of cancer remains a major contributor to the treatment outcome for many cancer patients prescribed conventional radiotherapy or chemotherapy . Failure of treatment coupled with the realisation that cancer is essentially a genetic disease has led to development of many clinical protocols based on gene therapy . In this review, we will describe an alternative gene delivery system based on the use of non-pathogenic bacteria . Tumor regressions have been reported long ago in patients with bacterially infected tumors, suggesting that bacteria could target tumors and have local anti-tumor effects . The basis of this phenomenon is attributable to the unique properties of the tumor micro-environment . The presence of hypoxic and/or necrotic areas provides a haven for a number of anaerobic bacteria and over the past 60 years, several strains of anaerobic bacteria have been shown to localise within and cause cell lysis of experimental animal tumors . One of the most important strains in that context is Clostridium . Other bacteria have also been implicated in experimental anti-cancer settings . Of these, attenuated Salmonella strains capable of both selective amplification within tumors and expression of effector genes encoding therapeutic proteins are probably the most promising . We will discuss the potential advantages and the pitfalls of this alternative delivery approach . We will emphasize the importance of hypoxia in solid tumors and discuss the potential of radiation-inducible promoters and combined treatment modalities, involving vascular targeting and radiotherapy . We believe that this approach will act in a complementary way to current radiotherapy and chemotherapy treatments of solid tumors. Infect Immun, 2003 Jun, 71(6), 3294 - 301 Mutational analysis of the enzymatic domain of Clostridium difficile toxin B reveals novel inhibitors of the wild-type toxin; Spyres LM et al.; Toxin B (TcdB), a major Clostridium difficile virulence factor, glucosylates and inactivates the small GTP-binding proteins Rho, Rac, and Cdc42 . In the present study we provide evidence that enzymatically inactive fragments of the TcdB enzymatic domain are effective intracellular inhibitors of native TcdB . Site-directed and deletion mutants of the TcdB enzymatic region (residues 1 to 556), lacking receptor binding and cell entry domains, were analyzed for attenuation of glucosyltransferase and glucosylhydrolase activity . Five of six derivatives from TcdB(1-556) were found to be devoid of enzymatic activity . In order to facilitate cell entry, mutants were genetically fused to lfn, which encodes the protective antigen binding region of anthrax toxin lethal factor and mediates the cell entry of heterologous proteins . In line with reduced enzymatic activity, the mutants also lacked cytotoxicity . Remarkably, pretreatment or cotreatment of cells with four of the mutants provided protection against the cytotoxic effects of native TcdB . Furthermore, a CHO cell line expressing enzymatically active TcdB(1-556) was also protected by the mutant-derived inhibitors, suggesting that inhibition occurred at an intracellular location . Protection also was afforded by the inhibitor to cells treated with Clostridium sordellii lethal toxin (TcsL), which uses the same cosubstrate as TcdB but shares Rac only as a common substrate target . Finally, the inhibitor did not provide protection against Clostridium novyi alpha-toxin (Tcnalpha), which shares similar substrates with TcdB yet uses a different cosubstrate . This is the first report to demonstrate that the potential exists to inhibit toxins at their intracellular site of action by using inactive mutants. Antimicrob Agents Chemother, 2003 Jun, 47(6), 1968 - 71 In vitro activities of dalbavancin and nine comparator agents against anaerobic gram-positive species and corynebacteria; Goldstein EJ et al.; Dalbavancin is a novel semisynthetic glycopeptide with enhanced activity against gram-positive species . Its comparative in vitro activities and those of nine comparator agents, including daptomycin, vancomycin, linezolid, and quinupristin-dalfopristin, against 290 recent gram-positive clinical isolates strains, as determined by the NCCLS agar dilution method, were studied . The MICs of dalbavancin at which 90% of various isolates tested were inhibited were as follows: Actinomyces spp., 0.5 microg/ml; Clostridium clostridioforme, 8 microg/ml; C . difficile, 0.25 microg/ml; C . innocuum, 0.25 microg/ml; C . perfringens, 0.125 microg/ml; C . ramosum, 1 microg/ml; Eubacterium spp., 1 microg/ml; Lactobacillus spp., >32 microg/ml, Propionibacterium spp., 0.5 microg/ml; and Peptostreptococcus spp., 0.25 microg/ml . Dalbavancin was 1 to 3 dilutions more active than vancomycin against most strains . Dalbavancin exhibited excellent activity against gram-positive strains tested and warrants clinical evaluation. FEMS Microbiol Lett, 2003 May 16, 222(1), 137 - 41 The VirR/VirS regulatory cascade affects transcription of plasmid-encoded putative virulence genes in Clostridium perfringens strain 13; Ohtani K et al.; We analyzed the transcriptional regulation of the putative virulence genes encoded on the plasmid pCP13 from Clostridium perfringens strain 13 . The transcription of the beta2-toxin (cpb2) and possible collagen adhesin (cna) genes were regulated in both a positive and negative manner, respectively, by the two-component VirR/VirS system . The secondary regulator of the VirR/VirS system, VR-RNA, also affects the expression of both of these genes in the same fashion as the VirR/VirS system . This indicates that the global regulatory cascade of the VirR/VirS system controls the expression of virulence genes located on the plasmid, as well as those found chromosomally in C . perfringens strain 13. Clin Oral Implants Res, 2003 Jun, 14(3), 263 - 8 Enzymatic degradation of collagen-guided tissue regeneration membranes by periodontal bacteria; Sela MN et al.; Bacterial infection in the vicinity of guided tissue regeneration barrier membranes was shown to have a negative effect on the clinical outcomes of this increasingly used technique . Several oral and specifically periodontal bacteria were shown to adhere to such membranes in vivo and in vitro with a higher affinity to membranes constructed from collagen . The present study examined the role of periodontal bacteria and their enzymes in the degradation of commercially used collagen membranes . Degradation of two collagen membranes {Biomend (Calcitek, Colla-Tec Inc., Plainsboro, NJ) and Bio-Gide (Geistlich Biomaterials, Wolhousen, Switzerland)} labeled by fluorescein isothiocyanate was examined by measuring soluble fluorescence . Porphyromonas gingivalis, Treponema denticola and Actinobacillus actinomycetemcomitans and their enzymes were evaluated . Collagenase from Clostridium hystolyticum was used as a positive control . While whole cells of P . gingivalis were able to degrade both types of membranes, T . denticola could degrade Bio-Gide membranes only and A . actinomycetemcomitans whole cells could degrade none of the membranes . Fractionation of P . gingivalis cells revealed that cell membrane associated proteases were responsible for the degradation of the two collagen membranes . In T . denticola, the purified major phenylalanine protease was found to be responsible for the degradation of Bio-Gide membranes . These results suggest that proteolytic bacterial enzymes may take part in the degradation of collagen barrier membranes used for guided tissue regeneration. J Enzyme Inhib Med Chem, 2003 Feb, 18(1), 7 - 13 A comparative QSAR study on carbonic anhydrase and matrix metalloproteinase inhibition by sulfonylated amino acid hydroxamates; Gupta SP et al.; A quantitative structure-activity relationship (QSAR) study is made on the inhibition of a few isozymes of carbonic anhydrase (CA) and some matrix metalloproteinases (MMPs), both zinc containing families of enzymes, by sulfonylated amino acid hydroxamates . For both enzymes, the inhibition potency of the hydroxamates is found to be well correlated with Kier's first-order valence molecular connectivity index 1chi(v) of the molecule and electrotopological state indices of some atoms . From the results, it is suggested that while hydroxamate-CA binding may involve mostly polar interactions, hydroxamate-MMP and hydroxamate-ChC (ChC: Clostridium histolyticum collagenase, another zinc enzyme related to MMPs) bindings may involve some hydrophobic interactions . Both MMPs and ChC also possess some electronic sites of exactly opposite nature to the corresponding sites in CAs . A group such as C6F5 present in the sulfonyl moiety is shown to be advantageous in both CA and MMP (also ChC) inhibitions, which is supposed to be due to the interaction of this group with Zn2+ ion present in the catalytic site of both families of enzymes. J Biol Chem, 2003 Aug 1, 278(31), 28523 - 7 Epub 2003 May 15. Entrapment of Rho ADP-ribosylated by Clostridium botulinum C3 exoenzyme in the Rho-guanine nucleotide dissociation inhibitor-1 complex; Genth H et al.; RhoA, -B, and -C are ADP-ribosylated by Clostridium botulinum exoenzyme C3 to induce redistribution of the actin filaments in intact cells, a finding that has led to the notion that the ADP-ribosylation blocks coupling of Rho to the downstream effectors . ADP-ribosylation, however, does not alter nucleotide binding, intrinsic, and GTPase-activating protein-stimulated GTPase activity . ADP-ribosylated Rho is even capable of activating the effector protein ROK in a recombinant system . Treatment of cells with a cell-permeable chimeric C3 toxin led to complete localization of modified Rho to the cytosolic fraction based on the complexation of ADP-ribosylated Rho with the guanine-nucleotide dissociation inhibitor-1 (GDI-1) . The modified complex turned out to be resistant to phosphatidylinositol 4,5-bisphosphate- and GTPgammaS-induced release of Rho from GDI-1 . Thus, ADP-ribosylation leads to entrapment of Rho in the GDI-1 complex . The increased stability of the GDI complex prevented binding of Rho to membrane-associated players of the GTPase cycle such as the activating guanine nucleotide exchange factors and effector proteins. Am J Physiol Cell Physiol, 2003 Sep, 285(3), C555 - 66 Epub 2003 May 14. Hyperosmotic stress activates Rho: differential involvement in Rho kinase-dependent MLC phosphorylation and NKCC activation; Di Ciano-Oliveira C et al.; Hyperosmotic stress initiates adaptive responses, including phosphorylation of myosin light chain (MLC) and concomitant activation of Na+-K+-Cl- cotransporter (NKCC) . Because the small GTPase Rho is a key regulator of MLC phosphorylation, we investigated 1) whether Rho is activated by hyperosmotic stress, and if so, what the triggering factors are, and 2) whether the Rho/Rho kinase (ROK) pathway is involved in MLC phosphorylation and NKCC activation . Rho activity was measured in tubular epithelial cells by affinity pulldown assay . Hyperosmolarity induced rapid (<1 min) and sustained (>20 min) Rho activation that was proportional to the osmotic concentration and reversed within minutes upon restoration of isotonicity . Both decreased cell volume at constant ionic strength and elevated total ionic strength at constant cell volume were capable of activating Rho . Changes in {Na+} and {K+} at normal total salinity failed to activate Rho, and Cl- depletion did not affect the hyperosmotic response . Thus alterations in cellular volume and ionic strength but not individual ion concentrations seem to be the critical triggering factors . Hyperosmolarity induced mono- and diphosphorylation of MLC, which was abrogated by the Rho-family blocker Clostridium toxin B . ROK inhibitor Y-27632 suppressed MLC phosphorylation under isotonic conditions and prevented its rise over isotonic levels in hypertonically stimulated cells . ML-7 had a smaller inhibitory effect . In contrast, it abolished the hypertonic activation of NKCC, whereas Y-27632 failed to inhibit this response . Thus hyperosmolarity activates Rho, and Rho/ROK pathway contributes to basal and hyperosmotic MLC phosphorylation . However, the hypertonic activation of NKCC is ROK independent, implying that the ROK-dependent component of MLC phosphorylation can be uncoupled from NKCC activation. J Food Prot, 2003 May, 66(5), 833 - 9 Growth and germination of proteolytic Clostridium botulinum in vegetable-based media; Braconnier A et al.; The growth of proteolytic Clostridium botulinum from spore inocula and changes in spore counts in mushroom, broccoli, and potato purees were monitored . Four strains of proteolytic C . botulinum types A and B were inoculated separately at approximately 10(4) spores per ml in nutrient broth and vegetable purees incubated at 15, 20, and 30 degrees C for up to 52 days . The times for the cell populations to increase 1,000-fold (T1,000) in the tested vegetables (1 to 5 days at 30 degrees C, 3 to 16 days at 20 degrees C, 7 to > 52 days at 15 degrees C) were similar to those for meat or fish . Only temperature significantly influenced growth rate . In contrast, the lag phase depended on the strains and media tested, in addition to temperature . Lag times and T1,000S for proteolytic C . botulinum were longer for potato and broccoli purees than for mushroom puree . These differences were not related to different pHs or redox potentials . The germination level, evaluated as the decrease in the spore count, was low . The addition of a germinant mixture (L-cysteine, L-alanine, and sodium lactate) to some strains inoculated in vegetable purees resulted in an increase in germination, suggesting a lack of germination-triggering agents in the vegetable purees. Syst Appl Microbiol, 2003 Mar, 26(1), 90 - 6 Genotypic differentiation of twelve Clostridium species by polymorphism analysis of the triosephosphate isomerase (tpi) gene; Dhalluin A et al.; Housekeeping genes encoding metabolic enzymes may provide alternative markers to 16S ribosomal DNA (rDNA) for genotypic and phylogenetic characterization of bacterial species . We have developed a PCR-restriction fragment length polymorphism (PCR-RFLP) assay, targeting the triosephosphate isomerase (tpi) gene, which allows the differentiation of twelve pathogenic Clostridium species . Degenerate primers constructed from alignments of tpi sequences of various gram-positive bacteria allowed the amplification of a 501 bp target region in the twelve Clostridium type strains . A phylogenetic tree constructed from the nucleotidic sequences of these tpi amplicons was well correlated with that inferred from analysis of 16S rDNA gene sequences . The analysis of tpi sequences revealed restriction sites of enzyme AluI that could be species-specific . Indeed, AluI digestion of amplicons from the twelve type strains provided distinct restriction patterns . A total of 127 strains (three to sixteen strains for each species) was further analyzed by PCR-RFLP of the tpi gene, and confirmed that each species could be characterized by one to three restriction types (RTs) . The differences between RTs within species could be explained by point mutations in AluI restriction sites of the tpi sequences . PCR-restriction analysis of the tpi gene offers an accurate tool for species identification within the genus Clostridium, and provides an alternative marker to 16S rDNA for phylogenetic analyses. Syst Appl Microbiol, 2003 Mar, 26(1), 84 - 9 Clostridium bolteae sp . nov., isolated from human sources; Song Y et al.; Seven obligately anaerobic, gram-positive, rod-shaped, spore-forming organisms isolated from human sources were characterized using phenotypic and molecular taxonomic methods . Comparative 16S rRNA gene sequencing showed that the strains were genetically highly related to each other (displaying >99% sequence similarity) and represent a previously unknown sub-line within the Clostridium coccoides rRNA group of organisms . Strains of the unidentified bacterium used carbohydrate as fermentable substrates, producing acetic acid and lactic acid as the major products of glucose metabolism . The closest described species to the novel bacterium corresponded to Clostridium clostridioforme, although a 16S rRNA sequence divergence of 3% demonstrated they represent different species . Genomic DNA-DNA pairing studies confirmed the separateness of the unknown species and Clostridium clostridioforme . Based on phenotypic and phylogenetic evidence, it is therefore proposed that the unknown bacterium, be classified as Clostridium bolteae sp . nov . The type strain of Clostridium bolteae is WAL 16351T (= ATCC(T) = BAA-613T, CCUG(T) = 46953T). J Antimicrob Chemother, 2003 Jun, 51(6), 1339 - 50 Epub 2003 May 13. Antibiotics and hospital-acquired Clostridium difficile-associated diarrhoea: a systematic review; Thomas C et al.; A systematic review of studies that investigated the association of antibiotics with hospital-acquired Clostridium difficile-associated diarrhoea (CDAD) was undertaken to summarize the strength of the evidence for this relationship . The results from the studies identified were considered after critically reviewing the design and conduct of each study . Although the majority of studies found an association with various antibiotics, antibiotic classes or components of antibiotic administration, most were limited in their ability to establish a causal relationship by the use of incorrect control groups, the presence of bias, inadequate control of confounding and small sample sizes . The limitations identified in this review prevented the pooling of results in a meta-analysis . Two studies of reasonable quality suggested an association between clindamycin, cephalosporins, penicillins and CDAD . Well-designed studies grounded in epidemiological principles are needed to identify true risk factors for CDAD and to provide reliable estimates of the strength of association. Mediators Inflamm, 2003 Feb, 12(1), 3 - 8 Human intestinal epithelial and smooth muscle cells are potent producers of IL-6; Ng EK et al.; BACKGROUND: Interleukin-6 (IL-6), a pluripotent cytokine, has traditionally been considered the product of proinflammatory cells . However, many other cell types have been shown to produce IL-6 . Since intestinal inflammation is commonly associated with a vigorous systemic inflammatory response, we hypothesized that intestinal epithelial and smooth muscle cells might contribute to that response by producing IL-6 . We therefore studied the capacity of differentiated human intestinal epithelial and smooth muscle cell lines to produce IL-6 in response to various proinflammatory stimuli . MATERIALS AND METHODS: CCL-241, a human intestinal epithelial cell line, and HISM, a human intestinal muscle cell line, were grown to confluency and then treated for 24 h with various concentrations of lipopolysaccharide, Clostridium difficile culture extract containing both toxin A and toxin B, recombinant human tumor necrosis factor-alpha (TNF-alpha), or recombinant human interleukin-1 beta (IL-1beta) . Supernatants were then collected for IL-6 determination using an enzyme-linked immunosorbent assay . Cell numbers were determined using a Coulter counter . For comparison, parallel studies were performed using phorbol ester-primed U-937 and THP-1 human macrophage cell lines . RESULTS: Both human intestinal epithelial and smooth muscle cells produced IL-6 under basal conditions . In HISM cells, but not in CCL-241 cells, IL-6 release was increased slightly by treatment with C . difficile culture extract containing both toxin A and toxin B and with lipopolysaccharide . In both cell lines, IL-6 production was profoundly stimulated by treatment with IL-1beta and less so with TNF-alpha . Combinations of high-dose TNF-alpha and IL-1beta may have a slightly additive, but not synergistic, effect on IL-6 release . The amount of IL-6 produced by IL-1-stimulated intestinal cell lines was 70-fold higher than that produced by stimulated macrophage cell lines . CONCLUSIONS; Both intestinal epithelial and smooth muscle cells demonstrate the ability to release significant amounts of IL-6 . The profound response to IL-1beta and TNF-alpha stimulation by both cell lines suggests that human intestinal parenchymal cells, influenced by paracrine mediators liberated from proinflammatory cells, might significantly contribute to the overall systemic inflammatory response by producing IL-6. Avian Pathol, 2003 Feb, 32(1), 57 - 62 Hepatitis associated with Clostridium difficile in an ostrich chick; Shivaprasad HL; A live 19-day-old male ostrich chick was euthanized and necropsied . It was one of 12 chicks in a group in which 8 had died with history of anorexia, diarrhoea and weight loss . The birds had been treated with amikacin, piperacillin and enrofloxacin . Necropsy of the ostrich revealed dehydration, mild ascites and serous atrophy of fat around the heart . The liver had numerous yellow tan foci on the capsular surface as well as on the cut surface . Caecal contents were watery . Microscopic examination of the liver revealed multifocal necrosis of hepatocytes with infiltration of heterophils mixed with fibrin, few lymphocytes, and multinucleated giant cells . A Gram stain of the liver revealed a few gram-positive bacilli scattered within the necrotic foci . Clostridium difficile was isolated from the liver, and toxin A was detected by ELISA . A retrospective examination of approximately 1000 ostriches submitted during a seven year period to the laboratory system revealed seven cases of hepatitis due to Clostridium perfringens, two additional cases due to C . difficile and two cases due to C . sordelli. J Emerg Med, 2003 May, 24(4), 401 - 5 Atraumatic Clostridial myonecrosis: case report and literature review; Abella BS et al.; Clostridial myonecrosis (CM) is a rare, life-threatening infection that is most often associated with recent surgery or skeletal muscle trauma . It usually affects patients with some degree of underlying immunocompromise or vascular insufficiency . Occasionally, CM can occur at remote sites, with seeding from a gastrointestinal source in the setting of malignancy . We report a case of a 75-year-old man who developed rapidly progressive myonecrosis in the right shoulder, without prior trauma, caused by Clostridium septicum . On autopsy, this patient was found to have previously undiagnosed radiation colitis with ulcerations and abscess formation, secondary to recent prostate cancer radiation therapy . Although several case reports discuss CM in the setting of bowel malignancy, our case illustrates that non-malignant bowel inflammation may be a sufficient source for the infection . Clinical features of this uncommon disease are discussed, and the relevant literature is reviewed with regard to Clostridium septicum as an etiologic agent. Parasitol Res, 2003 May, 90(1), 19 - 26 Epub 2003 Jan 28. A new method for the experimental production of necrotic enteritis and its use for studies on the relationships between necrotic enteritis, coccidiosis and anticoccidial vaccination of chickens; Williams RB et al.; A new method for the experimental production of necrotic enteritis in chickens is described . The main features are the use of a diet high in wheat and fish meal content; oral administration of a non-lethal inoculum of the coccidium Eimeria maxima followed 6 days later by the bacterium Clostridium perfringens type A per cloaca, so that the bacterial inoculum is deposited at the time and place when and where the intestinal coccidial lesions are maximal; grading of coccidial and clostridial lesions in individual birds sampled during the 14 days following the coccidial infection . The new method was used to examine the relationship between clostridial and coccidial infections . Frank coccidiosis, caused by virulent E . maxima, exacerbated the lesions of necrotic enteritis and other clinical effects due to a subsequent challenge with virulent C . perfringens type A . Immunization with a live, pentavalent, attenuated anticoccidial vaccine (Paracox-5) protected against a severe challenge with heterologous E . maxima . Furthermore, vaccination with Paracox-5, by virtue of its protection against clinical coccidiosis due to the E . maxima challenge, indirectly protected birds against a subsequent challenge with virulent C . perfringens . The results are reconciled with previous field observations on concomitant coccidiosis and necrotic enteritis in chicken flocks. Appl Microbiol Biotechnol, 2003 Sep, 62(4), 400 - 6 Epub 2003 May 13. Phylogenetic analysis of an anaerobic microbial consortium deiodinating 5-amino-2,4,6-triiodoisophthalic acid; Lecouturier D et al.; The dehalogenating performance of an anaerobic 5-amino-2,4,6-triiodoisophthalic acid (ATIA) fixed-bed reactor was evaluated . The reactor operating conditions were set for ATIA deiodination . A phylogenetic survey for a stable anaerobic ATIA-deiodinating microbial consortium was carried out using 16S rDNA restriction fragment length polymorphism, and unique clones were sequenced . Four phylotypes were identified . Two sequences were related to those of Desulfitobacterium frappieri species and another was closest to that of Desulfitobacterium hafniense, but may have represented a new Desulfitobacterium species . Desulfitobacteria were previously described as aryl-dechlorinating and debrominating bacteria . The new strains identified in this study were probably responsible for the ATIA deiodination . The fourth clone was related to the Clostridium-Flavobacterium-Bacteroides group. J Protein Chem, 2003 Jan, 22(1), 99 - 108 Molecular characterization of GroES and GroEL homologues from Clostridium botulinum; Sagane Y et al.; We report novel findings of significant amounts of 60- and 10-kDa proteins on SDS-PAGE in a culture supernatant of the Clostridium botulinum type D strain 4947 (D-4947) . The N-terminal amino acid sequences of the purified proteins were closely related to those of other bacterial GroEL and GroES proteins, and both positively cross-reacted with Escherichia coli GroEL and GroES antibodies . Native GroEL homologue as an oligomeric complex is a weak ATPase whose activity is inhibited by the presence of GroES homologue . The 2634-bp groESL operon of D-4947 was isolated by PCR and sequenced . The sequence included two complete open reading frames (282 and 1629 bp), which were homologous to the groES and groEL gene family of bacterial proteins . Southern and Northern blot analyses indicate that the groESL operon is encoded on the genomic DNA of D-4947 as a single copy, and not on that of its specific toxin-converting phage. J Biol Chem, 2003 May 2, 278(18), 15484 - 94 Biochemical characterization of proline racemases from the human protozoan parasite Trypanosoma cruzi and definition of putative protein signatures; Chamon