Am J Surg Pathol, 1997 Jun, 21(6), 706 - 10 Can ischemic colitis be differentiated from C difficile colitis in biopsy specimens? Dignan CR, Greenson JK. Pseudomembranous colitis is often caused by Clostridium difficile; however, it may also be due to ischemia . To determine if any histologic features could be used to differentiate C difficile from ischemia, 49 biopsies of pseudomembranous colitis (25 from patients with C difficile colitis and 24 from patients with ischemic colitis) were coded, randomized, and evaluated for the presence of numerous variables, including the amount and distribution of mucosal necrosis, lamina propria hyalinization, and atrophic "micro-crypts." Hyalinization of the lamina propria was seen in 19 cases of ischemia but not in C difficile colitis (p < 0.0001) . Atrophic-appearing micro-crypts were seen in 18 ischemic cases and 6 C difficile cases (p < 0.0006) . Lamina propria hemorrhage, full-thickness mucosal necrosis, and a diffuse microscopic distribution of pseudomembranes were significantly more common in ischemia than C difficile . Endoscopic examination identified pseudomembranes significantly more often with C difficile than ischemia, while the endoscopic appearance of masses or polyps was seen exclusively in cases of ischemia . The presence of a hyalinized lamina propria appeared to be a specific and sensitive marker for ischemia in colon biopsies with pseudomembranes . The presence of atrophic micro-crypts, lamina propria hemorrhage, full-thickness mucosal necrosis, diffuse involvement of all the surface of all biopsies by pseudomembranes, and the endoscopic impression of a localized process, polyp, or mass were also markers of ischemia, while the endoscopic identification of diffuse pseudomembranes favored the diagnosis of C difficile. Gastroenterology, 1997 Jun, 112(6), 1887 - 94 Early functional effects of Clostridium difficile toxin A on human colonocytes; Branka JE et al.; BACKGROUND & AIMS: Previous in vitro studies have shown that Clostridium difficile toxin A is able to directly affect the intestinal epithelial barrier function . The aim of this study was to examine the early effects of toxin A on mucin exocytosis and determine whether this toxin can induce the production of the chemokine interleukin 8 (IL-8) from human colonic epithelial cells . METHODS: Two model systems were used: the HT29-CI.16E colonic goblet cell line and primary cultures of human normal colonocytes . RESULTS: Toxin A exerted a rapid and dose-related inhibition of stimulated mucin exocytosis without altering baseline (constitutive) mucin exocytosis from HT29-CI.16E cells . Toxin A was also able to induce the secretion of IL-8 from both HT29-CI.16E cells and primary cultures of human normal colonocytes, as early as 2-3 hours of incubation . CONCLUSIONS: The results show that while toxin A is able to down-regulate stimulated mucin exocytosis, it is able to up-regulate the secretion of an important chemoattractant chemokine, IL-8 . These modifications illustrate the ability of colonocytes to recruit inflammatory and immune cells that will eventually bring about major mucosal damage. J Bacteriol, 1997 Jun, 179(11), 3736 - 45 Evidence for the bacterial origin of genes encoding fermentation enzymes of the amitochondriate protozoan parasite Entamoeba histolytica; Rosenthal B et al.; Entamoeba histolytica is an amitochondriate protozoan parasite with numerous bacterium-like fermentation enzymes including the pyruvate:ferredoxin oxidoreductase (POR), ferredoxin (FD), and alcohol dehydrogenase E (ADHE) . The goal of this study was to determine whether the genes encoding these cytosolic E . histolytica fermentation enzymes might derive from a bacterium by horizontal transfer, as has previously been suggested for E . histolytica genes encoding heat shock protein 60, nicotinamide nucleotide transhydrogenase, and superoxide dismutase . In this study, the E . histolytica por gene and the adhE gene of a second amitochondriate protozoan parasite, Giardia lamblia, were sequenced, and their phylogenetic positions were estimated in relation to POR, ADHE, and FD cloned from eukaryotic and eubacterial organisms . The E . histolytica por gene encodes a 1,620-amino-acid peptide that contained conserved iron-sulfur- and thiamine pyrophosphate-binding sites . The predicted E . histolytica POR showed fewer positional identities to the POR of G . lamblia (34%) than to the POR of the enterobacterium Klebsiella pneumoniae (49%), the cyanobacterium Anabaena sp . (44%), and the protozoan Trichomonas vaginalis (46%), which targets its POR to anaerobic organelles called hydrogenosomes . Maximum-likelihood, neighbor-joining, and parsimony analyses also suggested as less likely E . histolytica POR sharing more recent common ancestry with G . lamblia POR than with POR of bacteria and the T . vaginalis hydrogenosome . The G . lamblia adhE encodes an 888-amino-acid fusion peptide with an aldehyde dehydrogenase at its amino half and an iron-dependent (class 3) ADH at its carboxy half . The predicted G . lamblia ADHE showed extensive positional identities to ADHE of Escherichia coli (49%), Clostridium acetobutylicum (44%), and E . histolytica (43%) and lesser identities to the class 3 ADH of eubacteria and yeast (19 to 36%) . Phylogenetic analyses inferred a closer relationship of the E . histolytica ADHE to bacterial ADHE than to the G . lamblia ADHE . The 6-kDa FD of E . histolytica and G . lamblia were most similar to those of the archaebacterium Methanosarcina barkeri and the delta-purple bacterium Desulfovibrio desulfuricans, respectively, while the 12-kDa FD of the T . vaginalis hydrogenosome was most similar to the 12-kDa FD of gamma-purple bacterium Pseudomonas putida . E . histolytica genes (and probably G . lamblia genes) encoding fermentation enzymes therefore likely derive from bacteria by horizontal transfer, although it is not clear from which bacteria these amebic genes derive . These are the first nonorganellar fermentation enzymes of eukaryotes implicated to have derived from bacteria. J Bacteriol, 1997 Jun, 179(11), 3488 - 93 The Helicobacter pylori ureC gene codes for a phosphoglucosamine mutase; De Reuse H et al.; The function of UreC, the product of a 1,335-bp-long open reading frame upstream from the urease structural genes (ureAB) of Helicobacter pylori, was investigated . We present data showing that the ureC gene product is a phosphoglucosamine mutase . D . Mengin-Lecreulx and J . van Heijenoort (J . Biol . Chem . 271:32-39, 1996) observed that UreC is similar (43% identity) to the GlmM protein of Escherichia coli . Those authors showed that GlmM is a phosphoglucosamine mutase catalyzing interconversion of glucosamine-6-phosphate into glucosamine-1-phosphate, which is subsequently transformed into UDP-N-acetylglucosamine . The latter product is one of the main cytoplasmic precursors of cell wall peptidoglycan and outer membrane lipopolysaccharides . The present paper reports that, like its E . coli homolog glmM, the H . pylori ureC gene is essential for cell growth . It was known that growth of a lethal conditional glmM mutant of E . coli at a nonpermissive temperature can be restored in the presence of the ureC gene . We showed that complete complementation of the glmM mutant can be obtained with a plasmid overproducing UreC . The peptidoglycan content and the specific phosphoglucosamine mutase activity of such a complemented strain were measured; these results demonstrated that the ureC gene product functions as a phosphoglucosamine mutase . Homologs of the UreC and GlmM proteins were identified in Haemophilus influenzae, Mycobacterium leprae, Clostridium perfringens, Synechocystis sp . strain PCC6803, and Methanococcus jannaschii . Significant conservation of the amino acid sequence of these proteins in such diverse organisms suggests a very ancient common ancestor for the genes and defines a consensus motif for the phosphoglucosamine mutase active site . We propose renaming the H . pylori ureC gene the glmM gene. J Am Vet Med Assoc, 1997 Jun 1, 210(11), 1610 - 4 Organisms isolated from dogs and cats with anaerobic infections and susceptibility to selected antimicrobial agents; Jang SS et al.; OBJECTIVE: To determine the prevalence of obligate anaerobic bacteria in bacterial infections in dogs and cats and susceptibility to selected antimicrobial agents . DESIGN: Case series . SAMPLE POPULATION: Specimens from 1,267 dogs and 243 cats . PROCEDURE: Standard anaerobic and aerobic bacterial culture methods were used . Anaerobic isolates were tested for susceptibility to selected antimicrobial agents . RESULTS: Obligate anaerobic bacteria were isolated from 199 (15.7%) and 69 (28.4%) specimens obtained from dogs and cats, respectively . More than half of the specimens that contained obligate anaerobic bacteria were from draining tracts (exclusively dogs), pleural fluid, abscesses, bones, the respiratory tract, or the abdominal cavity . The most commonly isolated obligate anaerobic bacteria (approx 70% of all isolates) were Bacteroides spp, Peptostreptococcus spp, Fusobacterium spp, and Porphyromonas spp . Eighty percent of the specimens that contained obligate anaerobic bacteria also contained facultative anaerobic or aerobic organisms . The organisms most commonly isolated in association with obligate anaerobic bacteria were members of the family Enterobacteriaceae (Escherichia coli was the most common), Pasteurella spp, and Staphylococcus intermedius . Ninety-seven obligate anaerobic isolates were tested for susceptibility to ampicillin, amoxicillin-clavulanic acid, chloramphenicol, clindamycin, and metronidazole . All were susceptible to amoxicillin-clavulanic acid and chloramphenicol, and most were susceptible to metronidazole . Only 71% of the Bacteroides isolates were susceptible to ampicillin, and only 83% were susceptible to clindamycin . Only 80% of the Clostridium isolates were susceptible to clindamycin, but all were susceptible to ampicillin . CLINICAL IMPLICATIONS: Data on sites and conditions from which anaerobic bacteria are commonly isolated, along with results of susceptibility testing, may be useful in designing antimicrobial treatment regimens. Infect Immun, 1997 Jun, 65(6), 2313 - 20 Clostridium perfringens urease genes are plasmid borne; Dupuy B et al.; Although many bacteria are ureolytic, and in some cases urease acts as a virulence factor, the urease phenotype has not been analyzed in the anaerobic pathogen Clostridium perfringens . In this study, approximately 2% of C . perfringens strains, representing the principal biotypes, were found to harbor the urease structural genes, ureABC, and these were localized on large plasmids that often encode, in addition, the lethal epsilon or iota toxins or the enterotoxin . This represents the first report of a plasmid-encoded urease in a gram-positive bacterium . The C . perfringens enzyme was highly similar to the ureases of other bacteria and cross-reacted with antibodies raised against the urease purified from Helicobacter pylori . Urease production was inhibited by urea and induced under growth conditions where the availability of nitrogen sources was limiting . To date, this form of regulation has been observed only for chromosomal ureABC genes. Presse Med, 1997 May 17, 26(16), 748 - 51 {Prospective study of pathogenic agents isolated from feces of patients with HIV infections}; Meynard JL et al.; OBJECTIVE: Determine the frequency of enteropathogenic agents isolated in diarrheic feces of patients with HIV infection and to compare findings with a control group (HIV + without diarrhea) in order to identify risk factors . PATIENTS AND METHODS: All HIV seropositive inpatients and outpatients seropositive for HIV, with or without diarrhea, seen between 1 November 1994 and 30 April 1995 were included . Samples of feces were obtained for culture, virology examination, parasite examination and search for Clostridium difficile . The same samples were obtained in case of diarrhea during the course of hospitalization . RESULTS: There were 113 samples . Analyses demonstrated a pathogenic agent in 73.6% of the samples in patients with diarrhea and in 31.6% of those without diarrhea . Clostridium difficile and parasites were the most frequently identified agents . An infectious agent was identified in one-fourth of the patients without clinical signs of diarrhea, and in one-fourth of those with diarrhea no pathogen could be demonstrated . No factor of risk for finding a particular microorganism in feces of patients with diarrhea could be identified . DISCUSSION: The exact pathogenic roles of Pseudomonas aeuriginosa, yeast, and adenovirus remain to be determined . It is hypothesized that the HIV has a direct effect on the host digestive tract. FEMS Microbiol Lett, 1997 May 15, 150(2), 311 - 6 Single-step polymerase chain reaction for combined gene detection and epidemiological typing in three bacterial models; Saulnier P et al.; We describe a new polymerase chain reaction (PCR) for combined gene detection and epidemiological typing (COGEDET), which allows bacterial typing and gene detection in a one-step assay . This assay, in which target gene-specific primers are used under low-stringency annealing conditions, was evaluated on 32 Staphylococcus aureus strains using toxic shock syndrome toxin 1 (tst) primers, 30 Clostridium difficile strains using toxin A (toxA) primers, and 30 Escherichia coli strains using cytotoxic necrotizing factor (cnf) primers . Typing performances with COGEDET were compared to those of conventional random amplification polymorphic DNA (RAPD), and gene detection performances, to those of conventional PCR followed by Southern blot hybridization . Concordances between conventional PCR/Southern blot and COGEDET were 96.9, 100 and 96.7% for the detection of the tst, toxA and cnf genes, respectively . Discriminatory indexes for the conventional RAPD and COGEDET techniques were similar in the three bacterial species tested . These results show that the COGEDET assay can replace two separate assays for typing and genes detection respectively, thus saving both technicians' time and reagents. FEMS Microbiol Lett, 1997 May 15, 150(2), 203 - 8 Cleavage of the synaptobrevin/vesicle-associated membrane protein (VAMP) of the mouse brain by the recombinant light chain of Clostridium botulinum type B toxin; Rhee SD et al.; The light chain of Clostridium botulinum type B toxin was expressed in Escherichia coli using the expression vector pEt-3a containing phage T7 promoter . The expressed protein was then purified by DEAE-cellulose and phosphocellulose chromatography and the proteolytic activity of the purified light chain was studied . The purified recombinant light chain cleaved synaptobrevin when mixed with the mouse brain microsome and the proteolytic activity of the light chain was inhibited if a metal chelating agent such as EDTA or 2,2'-dipyridyl was added . The recombinant light chain cleaved synaptobrevin more effectively than the native type B toxin . When the native toxin was trypinized and was reduced with DTT, its proteolytic activity was similar to that of the recombinant light chain. Rev Esp Cardiol, 1997 May, 50(5), 360 - 2 {Prosthetic endocarditis and splenic abscess caused by Clostridium clostridiformis}; Robles P et al.; We report the first case of Clostridium clostridiformis endocarditis in a 71 year old man with an aortic prosthetic valve . He was febrile with left upper quadrant pain and left lower lobe infiltrate in chest X ray . The diagnosis was made by gram-positive bacilli grown from three blood cultures . Transthoracic and transesophageal echocardiogram showed a paraaortic abscess . A computed tomographic scan of the abdomen revealed a large splenic abscess . He received penicillin G 4 million units every 4 hours intravenously . A successful percutaneous drainage guided computed tomographic scan was performed . The patient remained febrile and a new computed tomographic scan of the abdomen revealed residual splenic abscess . A splenectomy was performed . The patient defervesced on the second day of surgery and remained afebrile during the remainder of his hospitalization . He has returned for medical follow-up and two years later the patient is asymptomatic. Toxicon, 1997 May, 35(5), 743 - 52 Role of tumor necrosis factor and nitric oxide in the cytotoxic effects of Clostridium difficile toxin A and toxin B on macrophages; Melo Filho AA et al.; Clostridium difficile, the bacterium involved in antibiotic-associated colitis, produces two exotoxins, toxin A (TxA) and toxin B (TxB) . Although these toxins are well recognized as being cytotoxic to several mammalian cell types, the mechanisms involved are not fully understood . The aim of the present investigation was to examine the cytotoxicity of TxA and TxB to peritoneal macrophages in culture and to investigate whether tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) are involved in the process . As a control, the effect of E . coli LPS was also investigated . TxA, TxB and LPS were dose-dependently cytotoxic to macrophage monolayers, with TxB being the most potent . All of the toxins stimulated the release of TNF-alpha from macrophages . TxB was again the most active in inducing this response . The TNF-alpha released appears to be involved in the action of LPS and TxA, but not of TxB, since a mAb against TNF-alpha inhibited the cytotoxicity of the former two but had no effect on the latter . NO is not involved in the effects of TxA and TxB since these toxins did not induce the production of this mediator in macrophages, even in the presence of IFN-gamma . In addition, L-imino-ethyl-L-ornithine (L-NIO), a NO synthase inhibitor, did not modify the macrophage death caused by TxA or TxB . Although LPS was able to induce the production of high amounts of NO, NO did not mediate the LPS cytotoxicity since L-NIO did not influence the degree of macrophage death caused by LPS . TxA and TxB therefore appear to exert cytotoxic effects on cultured macrophages by different mechanisms . TNF-alpha is involved in TxA and LPS-mediated cytotoxicity but not in the toxicity caused by TxB . NO is not involved in the killing action of any of these toxins. Infection, 1997 May-Jun, 25(3), 171 - 4 Central nervous system infection due to Clostridium septicum: a case report and review of the literature; Cheng YT et al.; A patient with end stage breast cancer was admitted to hospital due to fever, chills, multiply eroded discharging wounds, and sudden onset of left hemiparesis . Clostridium septicum bacteremia and brain abscess were diagnosed . The patient was treated successfully with intravenous penicillin and clindamycin and stereotactic aspiration of the abscess . Eleven cases of C . septicum central nervous system infection are reviewed . They showed an extremely fulminant course and high fatality . Nevertheless, some relationship seems to exist between outcome and type of brain lesion . Hemolytic-uremic syndrome associated with central nervous system infection is also discussed, because all these cases in the literature were due to this organism . Early diagnosis and aggressive treatment, including surgical drainage and appropriate antibiotics, are the key to improving the prognosis . A long-term prophylactic oral antimicrobial agent is suggested for patients who survive this infection. Steroids, 1997 May, 62(5), 437 - 43 Preparation of 3-ketodesogestrel metabolites by microbial transformation and chemical synthesis; Groh H et al.; Specific microbial reactions were used for the preparation of metabolites of 3-ketodesogestrel (13-ethyl-17 beta-hydroxy-11-methylene-18,19-dinor-17 alpha-pregn-4-en-20-yn-3-one, the active from of the progestagen desogestrel . Clostridium paraputrificum transformed 3-ketodesogestrel (KDG) to the 5 beta-dihydro and tetrahydro metabolites 13-ethyl-17 beta-hydroxy-11-methylene-18,19-dinor-5 beta, 17 alpha-pregnan-20-yn-3-one and 13-ethyl-11-methylene-18,19-dinor-5 beta, 17 alpha-pregnan-20-yne-3 alpha, 17 beta-diol, respectively . The epimeric compound 13-ethyl-11-methylene-18,19-dinor-5 beta, 17 alpha-pregnan-20-yne-3 beta, 17 beta-diol was obtained by chemical reduction of the 3-oxo compound . Mycobacterium smegmatis converted KDG to metabolites of the 5 alpha H-series: 13-ethyl-17 beta-hydroxy-11-methylene-18,19-dinor-5 alpha, 17 alpha-pregnan-20-yn-3-one, 13-ethyl-11-methylene-18,19-dinor-5 alpha, 17 alpha-pregnan-20-yne-3 alpha, 17 beta-diol and 13-ethyl-11-methylene-18,19-dinor-5 alpha, 17 alpha-pregnan-20-yne-3 beta, 17 beta-diol . The ring A-aromatized analog of KDG 13-ethyl-11-methylene-18,19-dinor-17 alpha-pregna-1,3,5(10)-trien-20-yne-3,17 beta-diol was obtained by microbial 1-dehydrogenation with Rhodococcus rhodochrous . Additionally, chemical syntheses of the microbially obtained KDG metabolites listed above were carried out . These included Birch reduction, reduction of KDG with sodium borohydride in aqueous pyridine and in methanol, reduction of KDG with potassium selectride in tetrahydrofuran, and dehydrogenation of KDG with cupric-II bromide in acetonitrile . The problems encountered in chemical syntheses favor the microbial procedures . The compounds were characterized by mass spectra (MS), IR, and circular dichroism (CD) . Complete assignments of 1H and 13C chemical shifts were made using homo- and heteronuclear 2-DN-NMR spectroscopy . Chromatographic {gas-liquid chromatography (GLC), high-performance liquid chromatography (HPLC), thin-layer chromatography (TLC)} data of all the prepared KDG metabolites are presented. Lett Appl Microbiol, 1997 May, 24(5), 393 - 6 A small heat shock protein from Leuconostoc oenos induced by multiple stresses and during stationary growth phase; Guzzo J et al.; In Leuconostoc oenos, a malolactic bacterium, the synthesis of a stress protein called LO18 with an apparent molecular mass of 18 kDa was greatly induced after heat (42 degrees C), acid (pH 3) or ethanolic (12% (v/v)) shocks . Moreover, the LO18 protein synthesis was induced in stationary growth phase and was detected for a long time (30 h) during this growth phase . Significant identity was found between the N-terminal parts of the LO18 protein and the Hsp18 from Clostridium acetobutylicum suggesting that LO18 protein belongs to the family of small heat shock proteins conserved in prokaryotic and eukaryotic cells. J Appl Microbiol, 1997 May, 82(5), 619 - 24 Monoclonal antibody detection of Clostridium microcolonies directly on membrane used for milk filtration; Batina P et al.; The normal procedure for bacterial colony detection requires a nitrocellulose transfer step after membrane filtration and culture to prevent the development of a high background during the immunodetection . In this paper, we describe a modification of the basic protocol that omits the transfer step and reduces the risk of background . Previous observations indicated that interactions between milk components (principally cream) and membrane are responsible for the high non-specific staining observed . Experiments were performed to remove lipid components or to block the membrane binding sites before milk filtration . Samples of milks of different origin (collected at different times of the year) and different membranes were tested . The results obtained showed that removing lipids did not significantly improve the test but, on the contrary, led to an antigen diffusion . Incubation of the membrane in 0.1% (w/v) of Tween 20 in phosphate-buffered saline before milk filtration prevented non-specific binding, and allowed performance of the detection without any noticeable background. EMBO J, 1997 May 1, 16(9), 2397 - 407 The GTPase Rho has a critical regulatory role in thymus development; Henning SW et al.; The present study employs a genetic approach to explore the role of Rho GTPases in murine thymic development . Inactivation of Rho function in the thymus was achieved by thymic targeting of a transgene encoding C3 transferase from Clostridium botulinum which selectively ADP-ribosylates Rho within its effector domain and thereby abolishes its biological function . Thymi lacking functional Rho isolated from C3 transgenic mice were strikingly smaller and showed a marked (90%) decrease in cellularity compared with their normal litter mates . We also observed a similar decrease in levels of peripheral T cells in C3 transgenic mice . Analysis of the maturation status of thymocytes indicated that differentiation of progenitor cells to mature T cells can occur in the absence of Rho function, and both positive and negative selection of T cells appear to be intact . However, transgenic mice that lack Rho function in the thymus show maturational, proliferative and cell survival defects during T-cell development that severely impair the generation of normal numbers of thymocytes and mature peripheral T cells . The present study thus identifies a role for Rho-dependent signalling pathways in thymocyte development . The data show that the function of Rho GTPases is critical for the proliferative expansion of thymocytes . This defines a selective role for the GTPase Rho in early thymic development as a critical integrator of proliferation and cell survival signals. Pharmacotherapy, 1997 May-Jun, 17(3), 606 - 11 Recurrent Clostridium difficile diarrhea associated with mitoxantrone and etoposide: a case report and review; Jarvis B et al.; Clostridium difficile colitis most commonly occurs in association with antibiotic administration and infrequently with antineoplastic agents . Our patient experienced recurrent C . difficile diarrhea associated with mitoxantrone and etoposide . He received antibiotics during the 6 months, but each episode of diarrhea was preceded by at least a 6-week antibiotic-free period . In addition, antineoplastic therapy preceded each episode by 8 or 9 days . Clinicians should be aware that antineoplastic drugs may precipitate overgrowth of C . difficile in the bowel. Microb Pathog, 1997 May, 22(5), 275 - 84 Isolation of alpha-toxin, theta-toxin and kappa-toxin mutants of Clostridium perfringens by Tn916 mutagenesis; Awad MM et al.; Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and mediates infection and disease by producing numerous extracellular toxins, including alpha-toxin, theta-toxin and kappa-toxin . Tn916-mutagenesis was used to isolate mutants defective in their ability to produce either alpha-toxin or theta-toxin . Nine independently derived mutants were isolated . In four of these mutants Tn916 had inserted at sites located 193 bp or 198 bp upstream of the theta-toxin structural gene, pfoA . Four mutants contained large deletions, three in regions which encompassed the theta-toxin structural and regulatory genes pfoA and pfoR, respectively, and the kappa-toxin structural gene, colA, and one in a region encompassing the alpha-toxin structural gene, plc . These mutants should prove to be invaluable for further genetic studies aimed at determining the role of these toxins in virulence. Infect Control Hosp Epidemiol, 1997 May, 18(5), 342 - 4 Vancomycin-resistant enterococci in stool specimens submitted for Clostridium difficile cytotoxin assay; Rafferty ME et al.; The prevalence of, and clinical risk factors associated with, vancomycin-resistant enterococcal colonization were investigated in patients suspected of having Clostridium difficile infection . Stools submitted for C difficile cytotoxin testing were screened for vancomycin-resistant enterococci (VRE) . Isolates were speciated and characterized further by antibiotic susceptibility testing, DNA fingerprinting, and DNA:DNA hybridization for detection of specific vancomycin resistance genes . Of the 79 evaluable patients identified during a 3-month period, 16.5% were VRE-positive . The VRE isolates were genetically heterogeneous, although all carried the vanA gene . DNA fingerprinting data suggest that patient-to-patient transmission occurred, implicating colonized patients as potential reservoirs for VRE transmission . A positive C difficile cytotoxin assay and diabetes mellitus were the only identifiable risk factors associated with VRE colonization . Patients at risk for C difficile infection therefore may serve as reservoirs for VRE. Neurology, 1997 May, 48(5), 1253 - 60 Myasthenic crisis: clinical features, mortality, complications, and risk factors for prolonged intubation; Thomas CE et al.; We retrospectively reviewed the hospital records of 53 patients admitted for 73 episodes of myasthenic crisis at Columbia-Presbyterian Medical Center over a period of 12 years, from 1983 to 1994 . Median age at the onset of first crisis was 55 (range, 20 to 82), the ratio of women to men was 2:1, and the median interval from onset of symptoms to first crisis was 8 months . Infection (usually pneumonia or upper respiratory infection) was the most common precipitating factor (38%), followed by no obvious cause (30%) and aspiration (10%) . Twenty-five percent of patients were extubated at 7 days, 50% at 13 days, and 75% at 31 days; the longest crisis exceeded 5 months . Using survival analysis and backward stepwise Cox regression, we identified three independent predictors of prolonged intubation: (1) pre-intubation serum bicarbonate > or = 30 mg/dl (p = 0.0004, relative hazard 4.5), (2) peak vital capacity day 1 to 6 post-intubation < 25 ml/kg (p = 0.001, relative hazard 2.9), and (3) age > 50 (p = 0.01, relative hazard 2.4) . The proportion of patients intubated longer than 2 weeks was 0% among those with no risk factors, 21% with one risk factor, 46% with two risk factors, and 88% with three risk factors (p = 0.0004) . Complications independently associated with prolonged intubation included atelectasis (p = 0.002), anemia treated with transfusion (p = 0.03), Clostridium difficile infection (p = 0.01), and congestive heart failure (p = 0.03) . Three episodes of crisis were fatal, for a mortality rate of 4% (3/73); four additional patients died after extubation . All seven deaths were due to overwhelming medical comorbidity . Over half of those who survived were functionally dependent (home or institutionalized) at discharge . In addition to prospective controlled studies of immunotherapies, the prevention and treatment of medical complications offers the best opportunity for further improving the outcome of myasthenic crisis. Dis Colon Rectum, 1997 May, 40(5), 622 - 4 Pseudomembranous colitis with associated fulminant ileitis in the defunctionalized limb of a jejunal-ileal bypass . Report of a case; Kralovich KA et al.; Presented is what is believed to be the first reported case of a defunctionalized limb of small intestine serving as a reservoir for Clostridium difficile . Because of the altered intestinal continuity, the ensuing enteritis and colitis failed to respond to nonoperative management . Current treatment strategies are reviewed . Surgical intervention, including restoration of normal gastrointestinal continuity, should be considered early in the hospital course of this patient population. Naunyn Schmiedebergs Arch Pharmacol, 1997 May, 355(5), 566 - 70 Involvement of 5-hydroxytryptamine and bradykinin in the hyperalgesia induced in rats by collagenase from Clostridium histolyticum; Damas J et al.; The involvement of bradykinin, 5-hydroxytryptamine, substance P and prostanoids in the hyperalgesia elicited by collagenase in rat paw was investigated . Collagenase (100 micrograms) induced a slight hyperalgesia in kininogen deficient rats in comparison with the behavioural response obtained in normal rats . Lisinopril (10(-5) M), and angiotensin-converting enzyme inhibitor, increased the duration of the hyperalgesia elicited in normal rats . Ondansetron (0.5 to 5 mumol/kg), a 5-HT3 antagonist, suppressed the hyperalgesia as did methysergide (1.1 to 11 mumol/kg), a mixed 5-HT1 and 5-HT2 receptor antagonist . However, the hyperalgesia was not modified by RP 67580 (1.8 to 18 mumol/kg), a NK1 receptor antagonist, and was only slightly delayed by indomethacin (2 mg/kg), a cyclo-oxygenase inhibitor . The oedema-promoting effect of 5-HT (6 nmol) was inhibited by methysergide but not by ondansetron . The swelling induced by collagenase in rat paw was reduced by methysergide but not by ondansetron . We conclude that the behavioural response induced by collagenase depends on an interactions between bradykinin and 5-HT . Prostanoids play a minor role in the beginning of the reaction whereas substance P is not significantly involved in this hyperalgesia. J Bacteriol, 1997 May, 179(10), 3181 - 7 Molecular characterization of a germination-specific muramidase from Clostridium perfringens S40 spores and nucleotide sequence of the corresponding gene; Chen Y et al.; The exudate of fully germinated spores of Clostridium perfringens S40 in 0.15 M KCI-50 mM potassium phosphate (pH 7.0) was found to contain another spore-lytic enzyme in addition to the germination-specific amidase previously characterized (S . Miyata, R . Moriyama, N . Miyahara, and S . Makino, Microbiology 141:2643-2650, 1995) . The lytic enzyme was purified to homogeneity by anion-exchange chromatography and shown to be a muramidase which requires divalent cations (Ca2+, Mg2+, or Mn2+) for its activity . The enzyme was inactivated by sulfhydryl reagents, and sodium thioglycolate reversed the inactivation by Hg2+ . The muramidase hydrolyzed isolated spore cortical fragments from a variety of wild-type organisms but had minimal activity on decoated spores and isolated cell walls . However, the enzyme was not capable of digesting isolated cortical fragments from spores of Bacillus subtilis ADD1, which lacks muramic acid delta-lactam in its cortical peptidoglycan . This indicates that the enzyme recognizes the delta-lactam residue peculiar to spore peptidoglycan, suggesting an involvement of the enzyme in spore germination . Immunochemical studies indicated that the muramidase in its mature form is localized on the exterior of the cortex layer in the dormant spore . A gene encoding the muramidase, sleM, was cloned into Escherichia coli, and the nucleotide sequence was determined . The gene encoded a protein of 321 amino acids with a deduced molecular weight of 36,358 . The deduced amino acid sequence of the sleM gene indicated that the enzyme is produced in a mature form . It was suggested that the muramidase belongs to a separate group within the lysozyme family typified by the fungus Chalaropsis lysozyme . A possible mechanism for cortex degradation in C . perfringens S40 spores is discussed. Am J Gastroenterol, 1997 May, 92(5), 739 - 50 Guidelines for the diagnosis and management of Clostridium difficile-associated diarrhea and colitis . American College of Gastroenterology, Practice Parameters Committee; Fekety R; Guidelines for clinical practice are intended to suggest preferable approaches to particular medical problems as established by interpretation and collation of scientifically valid research, derived from extensive review of published literature . When data are not available that will withstand objective scrutiny, a recommendation may be made based on a consensus of experts . Guidelines are intended to apply to the clinical situation for all physicians without regard to specialty . Guidelines are intended to be flexible, not necessarily indicating the only acceptable approach, and should be distinguished from standards of care that are inflexible and rarely violated . Given the wide range of choices in any health care problem, the physician should select the course best suited to the individual patient and the clinical situation presented . These guidelines are developed under the auspices of the American College of Gastroenterology and its Practice Parameters Committee . These guidelines are also approved by the governing boards of American College of Gastroenterology and Practice Parameters Committee . Expert opinion is solicited from the outset for the document . Guidelines are reviewed in depth by the committee, with participation from experienced clinicians and others in related fields . The final recommendations are based on the data available at the time of the production of the document and may be updated with pertinent scientific developments at a later time . The following guidelines are intended for adults and not for pediatric patients. Antimicrob Agents Chemother, 1997 May, 41(5), 1037 - 41 Antianaerobic activity of the ketolide RU 64004 compared to activities of four macrolides, five beta-lactams, clindamycin, and metronidazole; Ednie LM et al.; Agar dilution methodology (with added Oxyrase in the case of the macrolide group to allow incubation without added CO2) was used to compare the activity of RU 64004, a new ketolide, with the activities of erythromycin, azithromycin, clarithromycin, roxithromycin, clindamycin, amoxicillin with and without clavulanate, piperacillin with and without tazobactam, metronidazole, and imipenem against 379 anaerobes . Overall, RU 64004 yielded an MIC at which 50% of the isolates are inhibited (MIC50) of 1.0 microg/ml and an MIC90 of 16.0 microg/ml . In comparison, MIC50s and MIC90s of erythromycin, azithromycin, clarithromycin, and roxithromycin were 2.0 to 8.0 and >64.0 microg/ml, respectively . MICs of macrolides, including RU 64004, were higher for Bacteroides ovatus, Fusobacterium varium, Fusobacterium mortiferum, and Clostridium difficile than for the other species . RU 64004 was more active against gram-positive rods and cocci, Prevotella and Porphyromonas spp., and fusobacteria other than F . mortiferum and F . varium than against the Bacteroides fragilis group . Overall MIC50s and MIC90s (in micrograms per milliliter), respectively, of other compounds were as follows: clindamycin, 1.0 and 16.0; amoxicillin, 4.0 and 64.0; amoxicillin-clavulanate, 0.5 and 4.0; piperacillin, 8.0 and >64.0; piperacillin-tazobactam, 1.0 and 16.0; metronidazole, 1.0 and 4.0; and imipenem, 0.25 and 1.0. Appl Environ Microbiol, 1997 May, 63(5), 1732 - 8 Isolation and characterization of two new homoacetogenic hydrogen-utilizing bacteria from the human intestinal tract that are closely related to Clostridium coccoides; Kamlage B et al.; Two gram-positive, strictly anoxic, coccoid- to rod-shaped strains of bacteria, Clostridium coccoides 1410 and C . coccoides 3110, were isolated from human feces on the typical homoacetogenic substrates formate plus H2 plus CO2 (strain 1410) and vanillate plus H2 plus CO2 (strain 3110) in the presence of 2-bromoethanesulfonate to inhibit methanogenesis . On the basis of 16S rRNA sequencing, DNA-DNA hybridization, and physiological and morphological parameters, both isolates are closely related to C . coccoides DSM 935T . The G+C contents of the DNA were 46.1 and 46.2 mol% for C . coccoides 1410 and C . coccoides 3110, respectively . Cytochromes could not be detected . Formate was degraded exclusively to acetate, whereas vanillate was O-demethylated, resulting in acetate and 3,4-dihydroxybenzoate, the latter being further decarboxylated to catechol . In the presence of organic substrates, H2 was cometabolized to acetate, but both strains failed to grow autotrophically . Lactose, lactulose, sorbitol, glucose, and various other carbohydrates supported growth as well . Untypical of homoacetogens, glucose and sorbitol were fermented not exclusively to acetate; instead, considerable amounts of succinate and D-lactate were produced . H2 was evolved from carbohydrates only in negligible traces . Acetogenesis from formate plus H2 plus CO2 or vanillate plus H2 plus CO2 was constitutive, whereas utilization of carbohydrates was inducible . Hydrogenase, CO dehydrogenase, formate dehydrogenase, and all of the tetrahydrofolic acid-dependent, C1 compound-converting enzymes of the acetyl-coenzyme A pathway of homoacetogenesis were present in cell extracts. Clin Infect Dis, 1997 May, 24(5), 920 - 4 Prevalence of and risk factors for Clostridium difficile colonization at admission to an infectious diseases ward; Hutin Y et al.; A study of 240 consecutive admissions to a single hospital ward over a 6-month period was conducted to determine the prevalence of and risk factors for Clostridium difficile colonization at admission . The prevalence rate of C . difficile colonization at admission was 13.3% . Seventy-four percent of the patients admitted to the ward were infected with human immunodeficiency virus (HIV) . Multivariate analysis identified three risk factors for C . difficile colonization: clindamycin use (adjusted odds ratio {OR}, 9.4; P < .001), penicillin use (adjusted OR, 3.9; P = .018), and a history of cytomegalovirus infection (adjusted OR, 4.2; P = .02) . C . difficile colonization at admission to our infectious diseases ward was common . Antibiotic treatments received before admission were the main risk factors for C . difficile colonization . HIV infection per se was not associated with C . difficile colonization . It is interesting that there was an association between C . difficile colonization and a history of cytomegalovirus infection. Clin Infect Dis, 1997 May, 24(5), 889 - 93 Isolation of various genotypes of Clostridium difficile from patients and the environment in an oncology ward; Cohen SH et al.; The epidemiology of Clostridium difficile-associated diarrhea (CDAD) is not well defined in nonepidemic situations because precise biotyping techniques have only recently become available . Arbitrarily primed polymerase chain reaction (AP-PCR) was used to determine strain identity of C . difficile isolates recovered on our oncology ward, at an incidence rate of 0.84% . Twenty-one strains of C . difficile, which were grouped into 18 different AP-PCR types, were isolated from patients' specimens . Forty-two C . difficile isolates recovered from the environment (33 toxigenic and 9 nontoxigenic) represented 9 different AP-PCR types . The most commonly found type, a toxigenic strain accounting for 29% of the environmental isolates, was widespread throughout the ward . None of the environmental types were found among the isolates from patients . Three patients' isolates were of the same AP-PCR type, and two of these patients had occupied neighboring rooms at the same time . The diversity of C . difficile isotypes suggests that endemic nosocomial CDAD is not necessarily clonally spread. J Bacteriol, 1997 May, 179(9), 2810 - 6 Role of scaffolding protein CipC of Clostridium cellulolyticum in cellulose degradation; Pages S et al.; The role of a miniscaffolding protein, miniCipC1, forming part of Clostridium cellulolyticum scaffolding protein CipC in insoluble cellulose degradation was investigated . The parameters of the binding of miniCipC1, which contains a family III cellulose-binding domain (CBD), a hydrophilic domain, and a cohesin domain, to four insoluble celluloses were determined . At saturating concentrations, about 8.2 micromol of protein was bound per g of bacterial microcrystalline cellulose, while Avicel, colloidal Avicel, and phosphoric acid-swollen cellulose bound 0.28, 0.38, and 0.55 micromol of miniCipC1 per g, respectively . The dissociation constants measured varied between 1.3 x 10(-7) and 1.5 x 10(-8) M . These results are discussed with regard to the properties of the various substrates . The synergistic action of miniCipC1 and two forms of endoglucanase CelA (with and without the dockerin domain {CelA2 and CelA3, respectively}) in cellulose degradation was also studied . Although only CelA2 interacted with miniCipC1 (K(d), 7 x 10(-9) M), nonhydrolytic miniCipC1 enhanced the activities of endoglucanases CelA2 and CelA3 with all of the insoluble substrates tested . This finding shows that miniCipC1 plays two roles: it increases the enzyme concentration on the cellulose surface and enhances the accessibility of the enzyme to the substrate by modifying the structure of the cellulose, leading to an increased available cellulose surface area . In addition, the data obtained with a hybrid protein, CelA3-CBD(CipC), which was more active towards all of the insoluble substrates tested confirm that the CBD of the scaffolding protein plays an essential role in cellulose degradation. J Clin Microbiol, 1997 May, 35(5), 1258 - 9 Use of cycloserine-cefoxitin-fructose agar and L-proline-aminopeptidase (PRO Discs) in the rapid identification of Clostridium difficile; Fedorko DP et al.; The PRO Disc (Carr-Scarborough Microbiologicals, Inc., Decatur, Ga.) can be used to screen for L-proline-aminopeptidase produced by Clostridium difficile grown on cycloserine-cefoxitin-fructose agar (CCFA) . Fifty stored isolates of C . difficile (48 toxin-positive and 2 toxin-negative isolates) and 47 fresh C . difficile isolates (39 toxin-positive and 8 toxin-negative isolates) were all PRO Disc positive . Other Clostridium species that were PRO Disc positive could be differentiated from C . difficile by failure to grow on CCFA, different colonial morphology on CCFA, or morphology upon Gram staining. Int J Food Microbiol, 1997 Apr 29, 36(1), 49 - 60 Time-to-turbidity model for non-protective type B Clostridium botulinum; Whiting RC et al.; A model to predict the time for growth to turbidity from spores of non-proteolytic type B strains of Clostridium botulinum was developed in broth media with varying temperatures (4-28 degrees C), pH values (5-7), NaCl additions (0-4%) and total spores (10(1)-10(5)) . The model estimates the probability that a sample will have growth on a given day for up to 90 days of storage . The parameters of the model include the probability of growth after 90 days (Pmax) and the mean time of growth (tau) for those that showed growth . The 95% confidence interval (CI95%) for tau was also determined . The tau decreased with increasing temperature and pH, but NaCl levels below 3% had little effect . Decreasing the number of spores in a sample increased both tau and the confidence intervals about tau, reflecting the increasing uncertainty about the estimation of growth times for low spore numbers. Proc Natl Acad Sci U S A, 1997 Apr 29, 94(9), 4788 - 93 Increased substance P responses in dorsal root ganglia and intestinal macrophages during Clostridium difficile toxin A enteritis in rats; Castagliuolo I et al.; Previously we reported that pretreatment of rats with the substance P (SP) antagonist CP-96,345 inhibits the enterotoxic responses following administration of toxin A from Clostridium difficile into ileal loops, indicating that SP participates in the intestinal responses to this toxin . We now report that injection of toxin A into rat ileum causes a rapid increase in SP content in lumbar dorsal root ganglia (DRG) and mucosal scrapings 30-60 min after toxin A administration . Toxin A-mediated fluid secretion, mannitol permeability, and ileal histologic damage is significantly increased only after 2 hr . Toxin A also causes an increase in the abundance of SP mRNA in lumbar DRG and ileal mucosa as measured by reverse transcription-PCR . Lamina propria macrophages (LPMs) obtained from toxin A-injected loops release greater amounts of tumor necrosis factor alpha (TNFalpha) and SP as compared with LPMs isolated from buffer-injected loops (P < 0.01) . Pretreatment of rats with the SP antagonist CP-96,345 inhibits toxin A-mediated TNFalpha release from isolated LPMs, whereas an inactive enantiomer (CP-96,344) of the SP antagonist has no effect . LPMs obtained from toxin A-injected ileal loops incubated in vitro with SP (10(-8) to 10(-9) M) show enhanced TNFalpha secretion, whereas LPMs isolated from buffer-injected loops do not respond to SP . In addition, LPMs obtained from toxin A-injected ileal loops incubated in vitro with CP-96,345 showed a diminished TNFalpha release . Our results indicate that activated LPMs secrete SP during toxin A enteritis that can lead to secretion of cytokines, suggesting an autocrine/paracrine regulation of cytokine secretion by SP from LPMs during intestinal inflammation. J Biol Chem, 1997 Apr 25, 272(17), 11074 - 8 Localization of the glucosyltransferase activity of Clostridium difficile toxin B to the N-terminal part of the holotoxin; Hofmann F et al.; Clostridium difficile toxin B that is one of the largest cytotoxins (270 kDa) known acts on Rho subfamily proteins by monoglucosylation (Just, I., Selzer, J., Wilm, M., von Eichel-Streiber, C., Mann, M., and Aktories, K . (1995) Nature 375, 500-503) . By deletion analysis we identified the enzyme and cytotoxic activity of the toxin to be located at the N terminus of the holotoxin . A 63-kDa fragment of toxin B covering the first 546 amino acid residues glucosylated Rho, Rac, and Cdc42, but not Ras, by using UDP-glucose as a cosubstrate . As known for the holotoxin, glucosylation by the toxin fragment was favored with the GDP-bound form of the low molecular mass GTPases . Microinjection of the toxin fragment into NIH-3T3 cells induced rounding up of cells and redistribution of the actin cytoskeleton . In contrast, a toxin fragment encompassing the first 516 amino acid residues was at least 1000-fold less active than toxin fragment 1-546 and cytotoxically inactive . The data give direct evidence for location of the enzyme activity of C . difficile toxin B at the N-terminal 546 amino acids residues and indicate a functionally and/or structurally important role of the region from amino acid residues 516 through 546 for enzyme and cytotoxic activities. Carbohydr Res, 1997 Apr 21, 299(3), 119 - 28 Structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5 as determined by NMR spectroscopy; Kalelkar S et al.; The complete primary structure of the capsular polysaccharide of Clostridium perfringens Hobbs 5, an anaerobic bacterium implicated in food poisoning, was determined . The polysaccharide was isolated from C . perfringens Hobbs 5 cells, after deproteination, by ethanol precipitation and by ion-exchange chromatography . The polysaccharide was comprised of glucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine, and glucuronic acid, in equimolar ratios . Sequence and linkage assignments of the glycosyl residues were obtained by NMR spectroscopy, specifically by the combination of two-dimensional homonuclear TOCSY and NOESY experiments and heteronuclear (1H, 13C) multiple-quantum coherence (HMQC, HMQC-COSY, HMQC-TOCSY and HMBC) experiments . Thus, the envelope polysaccharide of C . perfringens Hobbs 5 was found to be a polymer composed of a hexasaccharide repeating unit with the following structure: {formula: see text} This structure is novel among bacterial cell-surface polysaccharides, and it is the first of many serotypically distinct capsular polysaccharides of C . perfringens to be described. FEMS Microbiol Lett, 1997 Apr 15, 149(2), 213 - 9 Dockerin-like sequences in cellulases and xylanases from the rumen cellulolytic bacterium Ruminococcus flavefaciens; Kirby J et al.; Recent analysis of the endA cellulase gene from Ruminococcus flavefaciens 17 has revealed that it encodes a product of 759 amino acids that provides the first example of a multidomain cellulase from a Ruminococcus sp . Following the family 5 catalytic domain in the predicted EndA enzyme is a 282 amino acid domain of unknown function for which no close relationship was found to other protein sequences . However, the C-terminal sequences of EndA contain a 34 amino acid threonine-rich linker connected to an 81 amino acid region, both of which show strong similarities to sequences present in two xylanases from R . flavefaciens 17 . A distant relationship is evident between regions of the 80 amino acid sequences of EndA, XynD and XynB and the duplicated 23 amino acid dockerin sequences found in cellulolytic Clostridium sp., suggesting that as in Clostridium sp . these sequences could mediate the binding of enzymatic polypeptides to another component in the cell surface enzyme complex of R . flavefaciens. Arch Intern Med, 1997 Apr 14, 157(7), 786 - 90 The stethoscope . A potential source of nosocomial infection? Marinella MA, Pierson C, Chenoweth C. BACKGROUND: Stethoscope diaphragms have been shown to harbor potentially pathogenic bacteria . OBJECTIVES: To assess bacterial contamination on the diaphragm and under the plastic rim that secures the diaphragm of stethoscopes of physicians, nurses, medical students, and house staff in an intensive care unit and a general medical ward of a large university hospital . Also to compare the effectiveness of various cleaning agents and assess the transmissibility of bacteria from contaminated stethoscopes to human skin . METHODS: Aerobic and anaerobic bacterial cultures were performed on 40 randomly selected stethoscopes . We compared the effects of isopropyl alcohol, sodium hypochlorite (bleach), and benzalkonium chloride swabs, as well as soap and water, on reducing bacterial contamination on the stethoscope diaphragm and under the rim . The transmissibility of Micrococcus luteus inoculated onto a stethoscope diaphragm to clean human skin was also determined . RESULTS: Eleven genera and species of bacteria were isolated, with coagulase-negative staphylococcus present on 100% of stethoscopes and Staphylococcus aureus on 38% . Clostridium difficile was not isolated . The mean (+/-SE) number of total colony-forming units was 158 +/- 33 per diaphragm and 289 +/- 54 per rim . Physicians' stethoscope diaphragms had significantly more colony-forming units of coagulase-negative staphylococci than those of nurses: 163 +/- 44 vs 50 +/- 12, respectively (P = .02) . The most effective cleaning agent was isopropyl alcohol after cleaning the diaphragm surface, the stethoscope diaphragms contained 0.2 +/- 0.2 colony-forming units and the rims contained 2.2 +/- 1.5 colony-forming units (P = .01) . In addition, M luteus was transferred from inoculated stethoscopes to human skin . CONCLUSIONS: Most stethoscopes harbor potential pathogens but are not a source of C difficile . Physicians' stethoscopes generally had a higher bacterial load than nurses' stethoscopes . Isopropyl alcohol is an effective cleaning agent when applied to the stethoscope diaphragm . Stethoscopes transfer M luteus to human skin, making it likely that other bacteria can be transferred as well. J Mol Biol, 1997 Apr 11, 267(4), 916 - 32 Crystal structure of glutamate dehydrogenase from the hyperthermophilic eubacterium Thermotoga maritima at 3.0 A resolution; Knapp S et al.; The extremely thermostable glutamate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima has been crystallized and the three-dimensional structure has been determined by X-ray diffraction methods . Crystals up to a maximum size of 1.2 mm have been grown in 3% polyethylene glycol, 120 mM ammonium acetate and 50 mM bis-tris propane (pH 6.5) . The enzyme crystallized in the trigonal space group P3(1)21 with the cell dimensions a = b = 147.3 A, c = 273.6 A . The diffraction limit of these crystals is 3.0 A . Measured diffraction data have a completeness of 94% up to a resolution of 3.0 A and contain 75% of all possible data in the last resolution shell between 3.1 and 3.0 A . The crystal structure of T . maritima glutamate dehydrogenase has been solved by Patterson search methods using the hexameric Pyrococcus furiosus glutamate dehydrogenase as a search model . The crystallographic refinement has been carried out to a maximum resolution of 3.1 A and an crystallographic R-value of 22.5% (Rfree = 29.5%) . The three-dimensional structure of the T . maritima enzyme shows typical features of hexameric glutamate dehydrogenases: six subunits are arranged in 32 symmetry . Each subunit consists of two domains connected by a flexible hinge region . Secondary structure elements as well as residues important for the catalytic activity of the enzyme are highly conserved . A structural comparison of the two glutamate dehydrogenases from the hyperthermophiles T . maritima and P . furiosus with the enzyme from the mesophilic bacterium Clostridium symbiosum has revealed that common as well as distinct mechanisms contribute to the thermal stability of these enzymes . The number of intrasubunit ion pairs is increased and the volume of intrasubunit cavities decreased in both thermostable enzymes, whereas striking differences have been observed in the subunit interfaces . In P . furiosus glutamate dehydrogenase the subunit interactions are dominated by ionic interactions realized by large saltbridge networks . However, in T . maritima glutamate dehydrogenase the number of intersubunit ion pairs is reduced and the hydrophobic interactions are increased. Oncogene, 1997 Apr 10, 14(14), 1705 - 13 Rho regulates association of both the ERM family and vinculin with the plasma membrane in MDCK cells; Kotani H et al.; Rho small G protein regulates various actin-dependent cell functions . As to the functioning sites of Rho, Rho regulates formation of stress fibers and focal adhesions in many types of cultured cells, whereas we have shown that the association sites of actin filaments with the plasma membrane controlled by the ERM (Ezrin, Radixin, Moesin) family are the functioning sites of Rho in MDCK cells stably expressing myc-RhoA . We have investigated here the effect of microinjection of Rho GDI, a negative regulator of Rho which inhibits activation of Rho, C3, an exoenzyme of Clostridium botulinum which ADP-ribosylates Rho and inhibits its functions, or guanosine 5'-(3-O-thio) triphosphate-bound active form of Rho on the intracellular localization of both the ERM family and vinculin, which is one of the structural proteins of focal adhesions, in wild type MDCK cells . The ERM family was preferentially localized at peripheral bundles of actin filaments which are localized at the outer edge of colonies of the cells, microvilli and low Ca2+-induced cortical bundles of actin filaments in wild type MDCK cells . Microinjection of Rho GDI or C3 inhibited the localization of the ERM family at both the peripheral bundles and the low Ca2+-induced cortical bundles . On the other hand, vinculin was localized at both focal adhesions and basal edges of the colonies of the cells, and microinjection of Rho GDI or C3 inhibited the localization of vinculin at both of these sites . These results indicate that activation of Rho is necessary for the association of both the ERM family and vinculin with the plasma membrane in wild type MDCK cells . Microinjection of the guanosine 5'-(3-O-thio) triphosphate-bound form of Rho induced an increase in the localization of vinculin at focal adhesions, but did not induce an increase in the localization of the ERM family at the plasma membrane, indicating that activation of Rho itself is sufficient only for the association of vinculin with the plasma membrane at focal adhesions. Anal Biochem, 1997 Apr 5, 247(1), 89 - 95 Homogeneous liposome immunoassay for insulin using phospholipase C from Clostridium perfringens; Lim SJ et al.; A new homogeneous liposome immunoassay system was developed in which analyte-phospholipase C conjugates are used instead of complement or melittin . This system was applied for the determination of insulin . Liposomes incorporated with calcein as a marker were prepared by the reverse-phase evaporation method . The lysis of liposomes was determined by measuring the fluorescence intensity of calcein released from liposomes and it was increased with increasing concentration of phospholipase C . Insulin was conjugated to phospholipase C by a three-step procedure with hetero-bifunctional crosslinking reagents, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and 3-(2-pyridyldithio)propionic acid N-hydroxysuccinimide ester . The lytic activity of phospholipase C was not affected by the reaction for conjugation . Both p-nitrophenylphosphatidylcholine hydrolytic activity and liposome lytic activity of insulin-phospholipase C conjugate were inhibited in the presence of insulin antiserum . However, lower concentration of conjugate and shorter incubation time were required when liposomes were used in the inhibition study . The antibody inhibition of conjugate-induced lysis could be reversed by addition of a competing amount of free insulin . The standard calibration curve was obtained in the range between 4 and 130 microIU/ml (r = 0.994) . The detection limit (8 microIU/ml) was comparable with those of conventional heterogeneous enzyme immunoassays . This new assay approach offers a simple, sensitive, and inexpensive testing procedure for determining ultratrace amounts of bioactive substances. J Biol Chem, 1997 Apr 4, 272(14), 9587 - 96 Cytotoxic necrotizing factor 1 from Escherichia coli and dermonecrotic toxin from Bordetella bronchiseptica induce p21(rho)-dependent tyrosine phosphorylation of focal adhesion kinase and paxillin in Swiss 3T3 cells; Lacerda HM et al.; Treatment of Swiss 3T3 cells with cytotoxic necrotizing factor 1 (CNF1) from Escherichia coli and dermonecrotic toxin (DNT) from Bordetella bronchiseptica, which directly target and activate p21(rho), stimulated tyrosine phosphorylation of focal adhesion kinase (p125(fak)) and paxillin . Tyrosine phosphorylation induced by CNF1 and DNT occurred after a pronounced lag period (2 h), and was blocked by either lysosomotrophic agents or incubation at 22 degrees C . CNF1 and DNT stimulated tyrosine phosphorylation of p125(fak) and paxillin, actin stress fiber formation, and focal adhesion assembly with similar kinetics . Cytochalasin D and high concentrations of platelet-derived growth factor disrupted the actin cytoskeleton and completely inhibited CNF1 and DNT induced tyrosine phosphorylation . Microinjection of Clostridium botulinum C3 exoenzyme which ADP-ribosylates and inactivates p21(rho) function, prevented tyrosine phosphorylation of focal adhesion proteins in response to either CNF1 or DNT . In addition, our results demonstrated that CNF1 and DNT do not induce protein kinase C activation, inositol phosphate formation, and Ca2+ mobilization . Moreover, CNF1 and DNT stimulated DNA synthesis without activation of p42(mapk) and p44(mapk) providing additional evidence for a novel p21(rho)-dependent signaling pathway that leads to entry into the S phase of the cell cycle in Swiss 3T3. Nippon Ronen Igakkai Zasshi, 1997 Apr, 34(4), 298 - 304 {Effects of administration of Clostridium butyricum to patients receiving long-term tube feeding}; Ito I et al.; In patients who require total parenteral or enteral nutrition the intestinal lining may atrophy and the ability to absorb nutrients may be lost . To prevent atrophy of the small intestine, we administered a suspension of Clostridium butyricum to elderly patients receiving tube feeding, and then measured the activation of serum diamine oxidase and the number, form, water content, and bacteria content of stools, indicators of intestinal structure . We found a significant increase in diamine oxidase activity and an improvement in stool condition: the number of stools per day decreased, form improved, and water content and the number of aerobic bacteria decreased significantly . These results indicate that in patients receiving long-term tube feeding, administration of Clostridium butyricum can restore condition to a near-normal state. Home Care Provid, 1997 Apr, 2(2), 92 - 3 Clostridium difficile: a microbial enigma; Sharbaugh RJ; Nearly 150 years ago, Louis Pasteur introduced the world to the science of microbiology and to the fact that our environment contains microbes capable of causing disease . Subsequent to these discoveries, a pandemic of health care-related staphylococcal infections nearly a century later led to the recognition of hospital-associated (nosocomial) infection . Clearly such infections (nosohusial) now also afflict nursing home residents and patients who receive home health care. Arch Pediatr, 1997 Apr, 4(4), 347 - 9 {Clostridium perfringens meningitis with fatal outcome in a 3-week old infant}; Hiffler L et al.; BACKGROUND: Clostridium perfringens meningitis is a rare condition usually associated to skull injuries, surgery or gastro-intestinal disorders . CASE REPORT: A 21 day-old infant was admitted for the sudden worsening of a neonatal post-hemorrhagic hydrocephalus . Eighteen hours after admission, she developed a septic status with acute meningitis . A CT scan revealed the presence of intracerebral gas suggesting the responsibility of an anaerobic bacterium . The infant died within 24 hours from multiorgan failure, hemodynamic disorders, acute anemia with red urines . Clostridium perfringens was isolated both from the blood and CSF . CONCLUSIONS: The tissue alterations following meningeal hemorrhage may favor anaerobic growth . The presence of intracerebral gas is highly suggestive of the diagnosis . The prognosis is very poor. Nutr Clin Pract, 1997 Apr, 12(2), 72 - 5 Banana flakes control diarrhea in enterally fed patients; Emery EA et al.; Diarrhea occurs frequently in the critically ill tube-fed population and may result from a multitude of causes . Despite the availability of antidiarrheal medications, diarrhea associated with enteral feedings remains a problem for clinicians and for the patients affected by it . We tested the hypothesis that administration of banana flakes would control diarrhea in critically ill patients receiving enteral feedings . Thirty-one patients with diarrhea and receiving enteral feedings were randomized to receive either banana flakes or medical treatment for diarrhea . Medical treatments included the use of pharmacological agents according to the discretion of the patient's physician or reducing feeding rates . Both banana flakes and medical treatments reduced the severity of diarrhea in critically ill tube-fed patients . Over the course of treatment, mean diarrhea scores were 21.64 +/- 7.81 for the banana flake group and 25.41 +/- 9.76 for the medical group . These differences were not statistically significant . Both groups achieved similar levels of nutrition support . The banana flake group had less diarrhea clinically, with 57% of the subjects diarrhea free on their last study day as opposed to 24% of the medically treated subjects . This occurred despite a threefold increase in the number of patients testing positive for Clostridium difficile toxin in the banana flake group . We conclude that banana flakes can be used as a safe, cost-effective treatment for diarrhea in critically ill tube-fed patients . Banana flakes can be given concurrently with a workup for C . difficile colitis, thereby expediting treatment of diarrhea. J Antimicrob Chemother, 1997 Apr, 39(4), 537 - 41 Health care resource utilization and antimicrobial use in elderly patients with community-acquired lower respiratory tract infection who develop Clostridium difficile-associated diarrhoea; MacGowan AP et al.; We conducted a prospective observational study on the medical management and health service resource utilization associated with the hospital care of patients with community-acquired lower respiratory tract infection . Between January 1994 and June 1995, 28 such patients developed Clostridium difficile-associated diarrhoea; these 28 patients were matched with 56 age-matched patients, who were used as a control group in a comparative study . Progress during the first week after admission was similar as measured by fever days and pathology or radiology use . The use of iv cephalosporins (g/day) during the first week was greater in the group who developed C . difficile-associated diarrhoea than in controls . The length of hospital stay was 36.4 +/- 21.6 days in patients with C . difficile-associated diarrhoea compared with 19.8 +/- 13.3 days in controls . Cases also required more pathological and radiological tests and greater use of antimicrobials and other drugs; however, if pathology and radiology use was calculated per day of patient stay there was no difference between the two groups . When antimicrobial use was compared, controlling for the time taken until found to be C . difficile toxin positive, patients with C . difficile infection received more iv cefuroxime as well as more total cephalosporins, beta-lactams and macrolides measured in g/day . Interestingly, in this study we could not show an increased mortality associated with C . difficile diarrhoea despite obvious evidence of morbidity . The development of C . difficile-associated diarrhoea substantially increases health care resource utilization for individual patients who are admitted to hospital with lower respiratory tract infection. Biosci Biotechnol Biochem, 1997 Apr, 61(4), 599 - 603 Purification and properties of beta-fructofuranosidase from Clostridium perfringens; Ishimoto M et al.; beta-Fructofuranosidase {EC 3.2.1.26} in Clostridium perfringens was induced in the presence of sucrose and suppressed in the presence of glucose or maltose . The enzyme seems to be present in protoplasm in a soluble state . The beta-fructofuranosidase from C . perfringens cells grown on sucrose was purified by ammonium sulfate precipitation . DEAE-cellulose chromatography, Sephadex G-150 gel filtration, and hydroxylapatite chromatography to a homogeneous state . The molecular weight was 37,000 by gel filtration using Sephadex G-150 and by SDS-polyacrylamide gel electrophoresis . The amino acid composition is not much different from those of other microorganisms, but the Glx content was a little higher . The enzyme was inhibited by heavy metals, such as Hg2+, Cu2+, and Ag+, as well as pCMB; the activity was restored by incubating with mercaptoethanol . Fructose and amines including Tris and aniline had inhibitory effects. Trends Microbiol, 1997 Apr, 5(4), 156 - 61 Bacterial phospholipases and their role in virulence; Songer JG; Virulence of many bacterial pathogens is based, at least in part, on the action of phospholipases . The consequences may be immediate and direct, as in the action of Clostridium perfringens alpha toxin on red cells or platelets, or subtle, as with phosphatidylinositol-specific phospholipases of Listeria monocytogenes and other bacteria. Int Immunol, 1997 Apr, 9(4), 523 - 31 In vivo induction of type 1 and 2 immune responses against protein antigens; Comoy EE et al.; Polarization of the immune response towards Th1 or Th2 profiles is under the control of several, not yet well known, mechanisms . The present study was undertaken to investigate whether immune responses generated against major protein antigens, of parasitic (Schistosoma mansoni) and bacterial (Clostridium tetani) origin, present characteristic Th profiles . Mice were immunized with a single dose of S . mansoni 28 kDa glutathione-S-transferase (Sm28-GST) or tetanus toxin fragment c (TTc) in combination with different adjuvants, or Salmonelia typhimurium expressing these antigens as a fusion protein . Antigen-specific IgG isotypes and cytokine mRNA expression in vivo, as well as cytokine secretion after in vitro antigen stimulation were studied . Immunizations with either protein in aluminum hydroxide induced a strong Th2-associated antibody (IgG1) and cytokine (IL-4) response . In contrast, the recombinant S . typhimurium, expressing the TTc/Sm28-GST fusion protein, induced a Th1-like response, associated with the production of IFN-gamma and IgG2a antibodies against both antigens . When complete Freund's adjuvant was used, a non-polarized profile was observed, characterized by expression of both IL-4 and IFN-gamma, as well as strong specific IgG1 and IgG2a antibody responses . These results indicated that some protein antigens play a weak role in polarizing the immune response and that contrasting cytokine profiles could be induced against the same antigen, depending on the adjuvant employed. J Med Microbiol, 1997 Apr, 46(4), 270 - 5 Characteristics of toxicity and haemorrhagic toxin produced by Clostridium sporogenes in various animals and cultured cells; Hara-Kudo Y et al.; The toxic effects of the haemorrhagic toxin of Clostridium sporogenes were studied in mice, rats, guinea-pigs and rabbits, and in various cultured cells . In rabbits, but not in the other animals, intradermal injection with crude toxin and its injection into a ligated intestinal loop caused haemorrhage in both the skin and intestinal wall . Intraperitoneal (i.p.) injection of crude toxin similarly caused death only of rabbits, with marked haemorrhage in the serous surface of kidney, intestines, liver, spleen, mesentery and diaphragm . Histological examination of the rabbits killed after i.p . inoculation revealed leakage of blood into a space beneath the serous membranes of parenchymatous organs in the peritoneal cavity and within the loose connective tissues in the mesentery and diaphragm . Cytotoxicity of partially purified haemorrhagic toxin in vitro was noted with rabbit aorta endothelial cells, human skin capillary vein endothelial cells and bovine pulmonary artery endothelial cells, but not with Chinese hamster ovary cells, Vero cells, human epitheloid carcinoma cells, human colon carcinoma cells (T84) and human colon adenocarcinoma cells (Caco 2) . The results suggest that the haemorrhagic toxin of C . sporogenes exerts its effects in rabbits but not in mice, rats or guinea-pigs, through direct action on endothelial cells. Infect Immun, 1997 Apr, 65(4), 1402 - 7 Production of a complete binary toxin (actin-specific ADP-ribosyltransferase) by Clostridium difficile CD196; Perelle S et al.; A Clostridium difficile isolate was found to produce an actin-specific ADP-ribosyltransferase (CDT) homologous to the enzymatic components of Clostridium perfringens iota toxin and Clostridium spiroforme toxin (M . R . Popoff, E . J . Rubin, D . M . Gill, and P . Boquet, Infect . Immun . 56:2299-2306, 1988) . The CDT locus from C . difficile CD196 was cloned and sequenced . It contained two genes (cdtA and cdtB) which display organizations and sequences similar to those of the iota toxin gene . The deduced enzymatic (CDTa) and binding (CDTb) components have 81 and 84% identity, respectively, with the corresponding components of iota toxin . CDTa and CDTb induced actin cytoskeleton alterations similar to those caused by other clostridial binary toxins . The lower level of production of binary toxin by CD196 than of iota toxin by C . perfringens was related to a lower transcript level, possibly due to a promoter region different from that of iota toxin genes . The cdtA and cdtB genes have been detected in 3 of 24 clinical isolates examined, and cdtB alone was found in 2 additional strains . One strain (in addition to CD196) was shown by Western blotting to produce CDTa and CDTb . These results indicate that some C . difficile strains synthesize a binary toxin that could be an additional virulence factor. Int J Syst Bacteriol, 1997 Apr, 47(2), 569 - 72 Reclassification of Paenibacillus durum (formerly Clostridium durum Smith and Cato 1974) Collins et al . 1994 as a member of the species P . azotofixans (formerly Bacillus azotofixans Seldin et al . 1984) Ash et al . 1994; Rosado AS et al.; Phenotypic studies, as well as the reaction of Paenibacillus durum genomic DNA with a 16S ribosomal DNA (sequence of variable regions V1 to V4)-based Paenibacillus azotofixans-specific PCR system and oligonucleotide probe, the presence of sequences homologous to Klebsiella pneumoniae nifKDH in both P . durum and P . azotofixans, and the results of DNA-DNA hybridization experiments performed with the P . durum and P.azotofixans type strains and one additional P . durum strain, showed that these two species form a homogeneous group . In addition, evidence was found for the presence of nif genes in P . durum, and P . durum was shown to fix atmospheric nitrogen . Therefore, the names P . durum and P . azotofixans should be considered synonyms . As P . durum was capable of fixing nitrogen and fixation without inhibition by nitrate is a major characteristic of the group, we propose that P . durum be included in the species P . azotofixans. Int J Syst Bacteriol, 1997 Apr, 47(2), 420 - 4 Cultures of "Clostridium acetobutylicum" from various collections comprise Clostridium acetobutylicum, Clostridium beijerinckii, and two other distinct types based on DNA-DNA reassociation; Johnson JL et al.; The best-known acetone-butanol (solvent)-producing bacterium is the Weizmann organism, Clostridium acetobutylicum, which was used for starch-based industrial fermentation . In the past two decades, cultures of "C . acetobutylicum" from various culture collections have included organisms that were isolated for sugar (molasses)-based industrial solvent production . Recent biochemical and genetic studies have revealed significant differences among some of these "C . acetobutylicum" strains . We used DNA-DNA reassociation to analyze 39 cultures of "C . acetobutylicum" and phenotypically similar organisms from major collections . The results of this study clearly identified four groups intergroup reassociation values of less than 30% . All of the intragroup values except the value for one strain were 68% or more, which supported species status for each group . The C . acetobutylicum group (with ATCC 824 as the type strain) consisted of 17 cultures and had average reassociation values of 10% with the other three groups . All strains of C . acetobutylicum produced riboflavin in milk, and the cultures were bright yellow, which is useful for differentiating this species from the other three groups . The Clostridium beijerinckii group (with VPI 5481 {= ATCC 25752} as the type strain) consisted of 16 cultures and included strains NCIMB 8052 and NCP 270 . Strains NCP 262 and NRRL B643 constituted the third group, whereas strain N1-4 ("Clostridium saccharoperbutylacetonicum") and its derivative, strain N1-4081, formed the fourth group . At present, the last two groups are each represented by only one independent strain; definitive descriptions of these two groups as two new or revived species will require further phenotypic characterization, as well as identification of additional strains . C . beijerinckii NCP 270, Clostridium sp . strain NRRL B643, and "C . saccharoperbutylacetonicum" were used in industrial solvent production from molasses, which confirms that the new organisms used for the sugar-based processes are distinct from C . acetobutylicum. Int J Syst Bacteriol, 1997 Apr, 47(2), 352 - 8 Sporomusa silvacetica sp, nov., an acetogenic bacterium isolated from aggregated forest soil; Kuhner CH et al.; Sporomusa silvacetica sp . nov . DG-1T (= DSMZ 10669T) (T = type strain) was isolated from well-drained, aggregated forest soil (pH 6.0) in east-central Germany . The cells were obligately anaerobic, slightly curved rods and were motile by means of laterally inserted flagella on the concave side of each cell . Typical cells were approximately 3.5 by 0.7 micron . Cells stained weakly gram positive, but thin sections revealed a complex multilayer cell wall . Spores were spherical and distended the sporangia . Growth and substrate utilization occurred with ferulate, vanillate, fructose, betaine, fumarate, 2,3-butanediol, pyruvate, lactate, glycerol, ethanol, methanol, formate, and H2-CO2 . With most substrates, acetate was the primary reduced end product and was produced in stoichiometries indicative of an acetyl-coenzyme A pathway-dependent metabolism . Fumarate was dismutated to succinate and acetate . Methoxyl and acrylate groups of various aromatic compounds were O-demethylated and reduced, respectively . Yeast extract was not required for growth . Cells grew optimally at approximately 30 degrees C and pH 6.8; under these conditions and with fructose as the substrate, the doubling time was approximately 14 h . The lowest temperature that supported growth was between 5 and 10 degrees C . The carbon monoxide dehydrogenase and hydrogenase activities were approximately 9 and 102 mumol min-1 mg of protein-1, respectively . A type b cytochrome was detected in the membrane . The G + C content was approximately 43 mol% . Phylogenetic analysis of the 16S ribosomal DNA indicated that DG-1T was most closely related to members of the genus Sporomusa in the Clostridium subphylum of the gram-positive bacteria. J Bacteriol, 1997 Apr, 179(8), 2519 - 23 Characterization and subcellular localization of the Clostridium thermocellum scaffoldin dockerin binding protein SdbA; Leibovitz E et al.; This article reports the characterization of the Clostridium thermocellum SdbA protein thought to anchor the cellulosome to the bacterial cell surface . The NH2-terminal region of SdbA consists of a cohesin domain which specifically binds the dockerin domain of the cellulosomal scaffolding protein CipA . The COOH-terminal region consists of a triplicated segment, termed SLH repeats, which is present in the sequence of many bacterial cell surface polypeptides . The binding parameters of the interaction between the dockerin domain of CipA and the cohesin domain of SdbA were studied by using, as a probe, the chimeric polypeptide CelC-DSCipA, which carries the dockerin domain of CipA fused to endoglucanase CelC . In the presence of Ca2+, CelC-DSCipA bound to SdbA with an affinity constant of 1.26 x 10(7) M(-1) . Binding of CelC-DSCipA to SdbA as a function of Ca2+ concentration was sigmoidal, corresponding to a Hill coefficient of 2 and an affinity constant for Ca2+ of 4 x 10(6) M(-2) . This suggested the presence of two cooperatively bound Ca2+ ions in the cohesin-dockerin complex . Immunoblotting of C . thermocellum subcellular fractions and electron microscopy of immunocytochemically labeled cells indicated that SdbA is located on the cell surface and is a component of the cellulosome . Together, the data confirm that SdbA could mediate anchoring of the cellulosome to the surface of C . thermocellum cells by interacting with the dockerin domain of CipA. Appl Environ Microbiol, 1997 Apr, 63(4), 1598 - 601 Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectant and by free chlorine; Venczel LV et al.; Cryptosporidium parvum oocysts and Clostridium perfringens spores are very resistant to chlorine and other drinking-water disinfectants . Clostridium perfringens spores have been suggested as a surrogate indicator of disinfectant activity against Cryptosporidium parvum and other hardy pathogens in water . In this study, an alternative disinfectant system consisting of an electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.) was evaluated for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores . The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine on the basis of equal weight per volume concentrations of total oxidants . Batch inactivation experiments were done on purified oocysts and spores in buffered, oxidant demand-free water at pH 7 an 25 degrees C by using a disinfectant dose of 5 mg/liter and contact times of up to 24 h . The mixed-oxidant solution was considerably more effective than free chlorine in activating both microorganisms . A 5-mg/liter dose of mixed oxidants produced a > 3-log10-unit (> 99.9%) inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores in 4 h . Free chlorine produce no measurable inactivation of Cryptosporidium parvum oocysts by 4 or 24 h, although Clostridium perfringens spores were inactivated by 1.4 log10 units after 4 h . The on-site generation of mixed oxidants may be a practical and cost-effective system of drinking water disinfection protecting against even the most resistant pathogens, including Cryptosporidium oocysts. Appl Environ Microbiol, 1997 Apr, 63(4), 1505 - 14 Bacterial diversity of a Carolina bay as determined by 16S rRNA gene analysis: confirmation of novel taxa; Wise MG et al.; Carolina bays are naturally occurring shallow elliptical depressions largely fed by rain and shallow ground water . To identify members of the domain Bacteria which inhibit such an environment, we used PCR to construct a library of 16S rRNA genes (16S rDNAs) cloned from DNA extracted from the sediments of Rainbow bay, located on the Savannah River Site, near Aiken, S.C . Oligonucleotides complementary to conserved regions of 16S rDNA were used as primers for PCR, and gel-purified PCR products were cloned into vector pGEM-T . Partial sequencing of the cloned 16S rDNAs revealed an extensive amount of phylogenetic diversity within this system . Of the 35 clones sequenced, 32 were affiliated with five bacterial groups: 11 clustered with the Proteobacteria division (including members of the alpha, beta, and delta subdivisions), 8 clustered with the Acidobacterium subdivision of the Fibrobacter division (as categorized by the Ribosomal Database Project's taxonomic scheme, version 5.0), 7 clustered with the Verrucomicrobium subdivision of the Planctomyces division, 3 clustered with the gram-positive bacteria (Clostridium and relatives subdivision), and 3 clustered with the green nonsulfur bacteria . One sequence branched very deeply from the Bacteria and was found not to be associated with any of the major divisions when phylogenetic trees were constructed . Two clones did not consistently cluster with specific groups and may be chimeric sequences . None of the clones exhibited an exact match to any of the 16S rDNA sequences deposited in the databases, suggesting that most of the bacteria in Rainbow Bay are novel species . In particular, the clones related to the Acidobacterium subdivision and the Verrucomicrobium subdivision confirm the presence of novel taxa discovered previously in other molecular surveys of this type. Appl Environ Microbiol, 1997 Apr, 63(4), 1214 - 8 Epitope regions in the heavy chain of Clostridium botulinum type E neurotoxin recognized by monoclonal antibodies; Kubota T et al.; Seventeen monoclonal antibodies (MAbs) were previously established against the heavy chain (Hc) of botulinum type E neurotoxin in BALB/c mice immunized with the type E toxoid . Five MAbs (LE15-5, LE34-6, EK19-7, EK21-4, and AE27-9) showed toxin-neutralizing activity in mice . Two of the five MAbs, EK19-7 and EK21-4, recognized the regions located at amino acid positions 731 to 787 and 811 to 897, respectively . One of the remaining three antibodies (LE34-6) reacted with the amino acid sequence VIKAIN, at amino acid positions 663 to 668, closed by the ion channel-forming domain . It is suggested that the ion channel-forming domain may also be associated with the blocking of acetylcholine release . Furthermore, the amino acid sequence YLTHMRD within 30 residues of the C-terminal region of the Hc component seemed to be recognized by LE15-5 . It has been reported that the binding domain of the type E toxin is located on the C-terminal half of the Hc component . Therefore, the neutralizing activity of LE15-5 antibody may be attributed to its ability to block the binding of neurotoxin to the receptor of target cells. Vet Hum Toxicol, 1997 Apr, 39(2), 89 - 92 Botulism outbreak associated with poultry litter consumption in three Brazilian cattle herds; Ortolani EL et al.; One hundred fifty-five of 201 cattle from 3 different farms showed clinical signs and died of botulism after eating the same batch of poultry litter contaminated with poultry and rodent carcasses . The cattle had access to poultry litter for only 1 d; afterwards it was removed from the diet . Death occurred over a period of 17 d after the poultry litter intake . The peak mortality was on day 4; 20 animals died within 10 d of the ingestion . The greater the intake of poultry litter, the higher the cattle mortality . Three steers which died on the first day had peracute effects while the remaining cattle showed classical signs . Twenty-five of the 46 surviving cattle had mild clinical signs, but recovered in a few days . Type C Clostridium botulinum toxin was found in extracts of the poultry litter, carcasses and cattle intestinal contents . Nutrient composition of the poultry litter was normal but pH was lower (6.9) than usual (7.5 to 9.3). J Cell Biol, 1997 Mar 24, 136(6), 1239 - 47 Molecular cloning and functional characterization of the receptor for Clostridium perfringens enterotoxin; Katahira J et al.; A cDNA encoding the Clostridium perfringens enterotoxin receptor gene (CPE-R) was cloned from an expression library of enterotoxin-sensitive Vero cells . The nucleotide sequence of CPE-R showed that the enterotoxin receptor consists of 209 amino acids with a calculated molecular mass of 22,029 D . This receptor is highly hydrophobic, contains four putative transmembrane segments, and has significant similarity to the rat androgen withdrawal apoptosis protein RVP1 and the mouse oligodendrocyte specific protein, the functions of which are unknown . The expression of CPE-R was detected in the enterotoxin-sensitive Vero, Hep3B, and Intestine 407 cell lines, but not in the enterotoxin-insensitive K562 and JY cell lines . The CPE-R gene product expressed in enterotoxin-resistant L929 cells bound to enterotoxin specifically and directly and with high affinity and rendered the cells sensitive to the toxin, indicating that the cloned receptor is functional . Results showed that enterotoxin could not assemble into a complex with a defined structure unless it interacted with the receptor . From these results, it is proposed that the enterotoxin receptor is required for both target cell recognition and pore formation in the cell membrane. Eur J Biochem, 1997 Mar 15, 244(3), 735 - 42 Transcription analysis of the genes tcdA-E of the pathogenicity locus of Clostridium difficile; Hundsberger T et al.; To analyse the transcription pattern of the five tcdA-E genes of the pathogenicity locus (PaLoc) of Clostridium difficile a protocol was established to purify RNA from strain VPI10463 . Transcription analysis of the five tcdA-E genes showed that they were all transcribed . In the early exponential phase, a high level of tcdC and low levels of tcdA,B,D,E transcripts were detectable; this was inverted in the stationary phase, suggesting that TcdC might have a negative influence on transcription of the other genes . Three transcription initiation sites, one for tcdA and two for tcdB were determined by primer extension analysis . Readthrough transcripts from outside the locus were not obtainable, so that parts of the transcription of tcdD, tcdB, tcdA and tcdC must occur by monocistronic transcription . Within the locus all possible intergenic readthrough transcripts were detectable except that between tcdC and tcdA, a stretch of DNA interrupted by a functional transcription terminator . Thus we found mono- and polycistronic transcription of tcdA and tcdB to occur which should lead to production of a surplus of tcdA over tcdB transcripts . This would explain the surplus of TcdA over TcdB expression observed in vitro . Due to its basic nature and similarity to BcnA of Clostridium perfringens and to Orf-22 of Clostridium botulinum, TcdD is most probably a regulatory protein with DNA-binding properties . On the basis of the presented study we discuss a model for the growth-phase-related, coordinate regulation of toxin expression wherein tcdC has a negative and tcdD a positive regulatory function on transcription of the tcdD,B,E and tcdA genes. FEMS Microbiol Lett, 1997 Mar 15, 148(2), 197 - 202 Characterization of polymorphisms in the toxin A and B genes of Clostridium difficile; Rupnik M et al.; We have used six independent polymerase chain reactions (A1-A3 and B1-B3) for amplification of the entire sequence of the two toxin genes tcdA and tcdB of several Clostridium difficile strains . With this approach we have detected (1) restriction site polymorphisms which are distributed all over the genes, and (2) deletions that could be found only in tcdA . Characteristic differences between strains were mainly focused to the 5' third of tcdB (B1 fragment) and/or the 3' third of tcdA (A3 fragment) . The possible use of our approach for typing of C . difficile toxin genes is discussed. Structure, 1997 Mar 15, 5(3), 381 - 90 A cohesin domain from Clostridium thermocellum: the crystal structure provides new insights into cellulosome assembly; Shimon LJ et al.; BACKGROUND: The scaffoldin component of the cellulolytic bacterium Clostridium thermocellum is a non-hydrolytic protein which organizes the hydrolytic enzymes in a large complex, called the cellulosome . Scaffoldin comprises a series of functional domains, amongst which is a single cellulose-binding domain and nine cohesin domains which are responsible for integrating the individual enzymatic subunits into the complex . The cohesin domains are highly conserved in their primary amino acid sequences . These domains interact with a complementary domain, termed the dockerin domain, one of which is located on each enzymatic subunit . The cohesin-dockerin interaction is the crucial interaction for complex formation in the cellulosome . The determination of structural information about the cohesin domain will provide insights into cellulosome assembly and activity . RESULTS: We have determined the three-dimensional crystal structure of one of the cohesin domains from C . thermocellum (cohesin 2) at 2.15 A resolution . The domain forms a nine-stranded beta sandwich with a jelly-roll topology, somewhat similar to the fold displayed by its neighboring cellulose-binding domain . CONCLUSIONS: The compact nature of the cohesin structure and its lack of a defined binding pocket suggests that binding between the cohesin and dockerin domains is characterized by interactions between exposed surface residues . As the cohesin-dockerin interaction appears to be rather nonselective, the binding face would presumably be characterized by surface residues which exhibit both intraspecies conservation and interspecies dissimilarity . Within the same species, unconserved surface residues may reflect the position of a given cohesin domain within the scaffoldin subunit, its orientation and interactions with neighboring domains. MMWR Morb Mortal Wkly Rep, 1997 Mar 7, 46(9), 202 - 4 Methemoglobinemia attributable to nitrite contamination of potable water through boiler fluid additives--New Jersey, 1992 and 1996; Assimilation of sulfur from alkyl- and arylsulfonates by Clostridium spp; Fakultat fur Biologie, Universitat Konstanz, Postfach 5560, D-78434 Konstanz, Germany Organisms able to utilize one of several alkyl- and arylsulfonates as sole source of sulfur under anoxic conditions were enriched . Three fermenting bacteria, all putative Clostridium spp., were isolated in pure culture . All three organisms had wide substrate ranges for alkylsulfonates, taurine and arylsulfonates, presumably due to three different enzyme systems . One organism, strain KNNDS (DSM 10612) was selected for further characterization . The organism was possibly a new Clostridium sp., with Clostidium intestinalis as its nearest neighbor (97.6% similarity of rDNA) . Strain KNNDS catalyzed complete sulfonate utilization concomitant with growth . Growth yields of approximtely 3 kg protein/mol sulfur were observed, independent of the sulfur source {e.g . sulfate, sulfide, 4-(phenyl)butyl-1-sulfonate, 2,6-naphthyldisulfonate or 4-nitrocatechol sulfate} . We failed to detect significant amounts of either an arylsulfonatase or an arylsulfatase, and we hypothesize different arylsulfatases {EC 3.1.6.1} in aerobes and in Clostridium spp. Int J Food Microbiol, 1997 Mar 3, 34(3), 293 - 305 The effect of ethylenediaminetetraacetic acid on heat resistance and recovery of Clostridium sporogenes PA 3679 spores treated in HTST conditions; Silla Santos MH et al.; The effect of ethylenediaminetetraacetic acid (EDTA) on the heat resistance of Clostridium sporogenes PA 3679 spores was studied . EDTA was added to heating substrates and recovery media in order to establish which stage of the heat treatment registered the greatest EDTA activity . The heating substrates assayed were phosphate buffer (pH 7.0) and white asparagus puree, at natural pH (5.8) and acidified with citric acid and glucono-delta-lactone (GDL) to pH 5.5, 5.0 and 4.5 . Recovery of survivors was carried out in MPA3679A medium in various conditions of acidification with citric and GDL (250 and 500 ppm), at pH 7.5 6.5 and 6.0 . The results show greater activity of EDTA on spores when it was applied in recovery of heat injured spores, than during heating . The strongest influence of EDTA during heating was found in phosphate buffer (pH 7.0), with the effect being most evident at 121 and 126 degrees C, and in asparagus puree, at 121 degrees C and pH 5.8 rather than acidified . In recovery, the inhibiting activity of EDTA was more evident in spores subjected to more severe heat treatment, either by increasing the exposure time or by raising the temperature to 130 or 135 degrees C . The pH level of the recovery medium also affected the antimicrobial activity of EDTA, which had a greater inhibiting effect at pH 7.5 than at lower pH levels (6.5, 6.0). Z Arztl Fortbild Qualitatssich, 1997 Mar, 91(2), 165 - 70 {Anaerobic bacteria as the cause of endogenous infections}; Briedigkeit H et al.; Most mucocutaneous surfaces of humans harbor a rich indigenous microbial flora with predominance of anaerobes . Anaerobic infections are usually endogenous indicating that they originate from the host's own flora . Important exceptions are botulism, tetanus, food poisoning by Clostridium perfringens, some cases of gas gangrene and cases of hospital-acquired C . difficile-induced diarrhea . Endogenous anaerobic infections often occur in adjacent to the mucosal surfaces . Other organs are infected by penetration or hematogenous spread . A predisposing condition to anaerobic infections is a low redox potential resulting from tissue destruction, foreign bodies, malignancy or vascular insufficiency . A mixed anaerobic-aerobic infection is often found in abscesses or tissue necrosis . Antimicrobial therapy must take into account that anaerobic infections are often associated with aerobic bacteria. Anaesthesist, 1997 Mar, 46(3), 207 - 10 {Generalized gas gangrene infection with rhabdomyloysis following cholecystectomy}; Haerty W et al.; We report a rare case of spontaneously developing generalised gas gangrene with massive rhabdomyolysis after a cholecystectomy and drainage of a hepatic abscess . On preoperative physical examination the patient appeared severely ill and was icteric and oliguric . Laboratory evaluation showed signs of systemic inflammation, elevated lactate levels, evidence of disseminated intravascular coagulation (DIC), and increased levels of serum creatine kinase (CK) activity . Abdominal ultrasound and endoscopic retrograde cholangiography showed a gallbladder perforation and a hepatic abscess . Cholecystectomy and drainage of the abscess was performed immediately and without technical problems . After postoperative admission to the intensive care unit, the patient showed evidence of generalised myonecrosis with subcutaneous gas formation and acute renal failure . Initially, there were few other signs of systemic toxicity; the patient was not hypotensive and the pulmonary gas exchange was normal . Within hours diffuse swelling of his right leg developed with cutaneous gangrene and a compartment syndrome . After fasciectomy and extensive surgical debridement, uncontrollable bleeding due to DIC developed from the fasciectomy site, which finally required exarticulation of the leg at the hip joint . At this point, multiple organ failure including severe adult respiratory distress syndrome was present . Two days after cholecystectomy, the patient died from hypoxic cardiocirculatory failure . Clostridium perfringens was repeatedly isolated from the wounds . Besides gas gangrene, the differential diagnosis of such infections includes localised clostridial cellulitis, nonclostridial anaerobic cellulitis caused by mixed aerobes and anaerobes, and type I or type II necrotising fasciitis . Patients with systemic necrotising infections should be treated with broad-spectrum antimicrobial regimens (penicillin G, 3rd generation cephalosporins, clindamycin, and aminoglycosides) . An otherwise unexplained elevation of serum CK activity in the presence of acute cholecystitis may suggest haematologic spread of an aggressive myolytic agent and the beginning of myonecrosis . This should prompt immediate surgical exploration after establishing broad-spectrum antibiotic coverage . The role of hyperbaric oxygen treatment in this situation remains to be established . If hyperbaric oxygen is to be employed, it should neither delay surgical exploration nor jeopardize the patient with the hazards of an interhospital transport. J Clin Pathol, 1997 Mar, 50(3), 259 - 60 A fatal postpartum Clostridium sordellii associated toxic shock syndrome; Bitti A et al.; Clostridium sordellii is an infrequent human pathogen . It has been demonstrated to be occasionally responsible for myonecrosis or gas gangrene . Interestingly, in the obstetric literature, some cases of postpartum maternal deaths have been associated with C sordellii infection causing a rapidly lethal toxin mediated syndrome . This is the first reported case of postpartum death in a 29 year old woman, in which a toxigenic C sordellii was isolated from the patient's blood antemortem during the fatal toxic shock, strongly indicating its role in this rare syndrome. J Infect, 1997 Mar, 34(2), 127 - 32 Preparation for emergency relief after biological warfare; Steffen R et al.; Upon invitation by the World Health Organization during the Gulf War, a task force "Scorpio" independent from the nations involved in the armed conflict was formed whose task was to determine whether, which and to what extent biological warfare agents had been used . risk assessment concluded that anthrax and Clostridium botulinum toxin were the major risks . The 21 civilian experts had rapidly to decide on the doctrine of operation, to assemble material which could be used and to be immunized or protected otherwise against the potential risks . Biological warfare agents may be used anywhere any time, be it by terrorists or during open or clandestine hostilities . The general population cannot rely on the military to take care of civilian relief, thus international and national organizations may wish to establish similar task forces basing on the "Scorpio" model on a national or regional basis. Enzyme Microb Technol, 1997 Mar, 20(4), 277 - 85 Small neuraminidase gene of Clostridium perfringens ATCC 10543: cloning, nucleotide sequence, and production; Chien CH et al.; The small nanH gene encoding a neuraminidase from Clostridium perfringens ATCC 10543 was cloned in JM109 using pUC19 as a vector . Sequence analysis revealed an ORF, nt 310-1455, encoding 382 amino acids that was proceeded by a typical Shine-Dalgarno sequence, GGACGAGA . The nt sequence in the 15-402 region had in vivo promoter activity in an Escherichia coli promoter probe plasmid pKK232-8, which suggested that the small nanH promoter is functional in E . coli . Four regions of amino acids demonstrated great similarity to the "Asp boxes" (-Ser-X-Asp-X-Gly-X-Thr-Trp-) of other bacterial nanH proteins . The small nanH expressing clone, pCPN-1, which was cultured under aerobic conditions resulted in NanH activity which was 203-fold in culture and 211-fold in intracellular fraction compared to that of C . perfringens which has to be cultured under anaerobic conditions . Production of small NanH was also induced by adding sialyllactose to the culture medium of JM109 {pCPN-1} . The enzyme activity could be detected in the periplasmic fraction and the culture medium of JM109 {pCPN-1} after culturing to the stationary phase . The molecular weight, K(m), and optimum pH and pI of the cloned enzyme are identical to those of the parent strain . The cloned, small nanH could be used to study the structure-functional relationship of nanH, while the pCPN-1 clone could be used in the aerobic production of neuraminidase. Am J Emerg Med, 1997 Mar, 15(2), 152 - 4 Clostridium perfringens septicemia with massive hemolysis in a patient with Hodgkin's lymphoma; Singer AJ et al.; A 55-year-old woman with stage IV-B nodular sclerosing Hodgkin's lymphoma presented to the emergency department with fever and lethargy of 12 hours' duration . The patient developed massive intravascular hemolysis secondary to Clostridium perfringens sepsis and cardiac arrest unresponsive to transfusions and cardiac pulmonary resuscitation, and died within 4 hours of presentation . The differential diagnosis of massive intravascular hemolysis, as well as the pathogenesis and treatment of C perfringens-induced hemolysis, are discussed. Clin Infect Dis, 1997 Mar, 24(3), 324 - 33 Recurrent Clostridium difficile diarrhea: characteristics of and risk factors for patients enrolled in a prospective, randomized, double-blinded trial; Fekety R et al.; Recurrent Clostridium difficile diarrhea (RCDD) occurs in 20% of patients after they have received standard antibiotic treatment with vancomycin or metronidazole, but the reasons for the recurrences are largely unknown . Patients receiving vancomycin or metronidazole for active C . difficile diarrhea (CDD) were referred to our study centers for treatment and a 2-month follow-up as part of a randomized placebo-controlled trial . Sixty patients had RCDD (median number of episodes, 3.0; range, 2-9 episodes) and 64 were having their first episode of CDD . Patients with RCDD had more-severe abdominal pain and were more likely to have fever but initially responded well to antibiotic therapy . Data on sequential episodes showed no progression in disease severity . Five factors were associated with a higher risk of RCDD: the number of previous CDD episodes, onset of the initial disease in the spring, exposure to additional antibiotics for treatment of other infections, infection with immunoblot type 1 or 2 strains of C . difficile, and female gender . These factors may help to identify patients who are more likely to develop RCDD and require careful medical supervision. Ann Hematol, 1997 Mar, 74(3), 143 - 7 Clostridium septicum sepsis and neutropenic enterocolitis in a patient treated with intensive chemotherapy for acute myeloid leukemia; Pouwels MJ et al.; We report a case of neutropenic enterocolitis complicated by Clostridium septicum sepsis following intensive chemotherapy to induce remission of acute myeloid leukemia . With the trend towards more intensive chemotherapy, particularly using regimens that induce gastrointestinal toxicity, it is important to recognize the circumstances under which sepsis due to C . septicum is likely to occur, so that appropriate treatment can be instituted promptly. Vet Microbiol, 1997 Mar, 54(3-4), 301 - 8 Clostridial population and the intestinal lesions in chickens infected with Clostridium perfringens and Eimeria necatrix; Baba E et al.; Chickens infected with Clostridium perfringens and Eimeria necatrix were examined bacteriologically and pathologically . When chickens were inoculated with 1.0 x 10(8) C . perfringens and/or 2 x 10(4) E . necatrix sporulated oocysts, populations of C . perfringens in the intestinal contents were examined on 3, 5 and 7 days after E . necatrix inoculation . In both groups infected with E . necatrix, the mean clostridial counts were significantly higher than those of uninfected controls . The concurrent infection had no enhancing effects on increasing the clostridial population more than E . necatrix-alone . Mortality of 4-day-old chickens inoculated on 5 consecutive days with C.perfringens after receiving E . necatrix was higher than those of chickens inoculated with the both organisms . However, intestinal lesions of the concurrently infected group were not different from E . necatrix-alone-infected group on 5 and 7 days after the coccidial infection . When chickens received a large dose (1.5 x 10(9)) of C . perfringens after the inoculation with E . necatrix, edema in the duodenum through jejunum were observed early after the bacterial broth inoculation . These results suggest that the concurrent infection with E . necatrix and C . perfringens increases clostridial population in the intestine of the chickens and has synergic effects on mortality and edema in the upper intestine. Eur J Gastroenterol Hepatol, 1997 Mar, 9(3), 303 - 5 Splenic abscess with Clostridium novyi bacteraemia and sepsis; Vleminckx WG et al.; Splenic abscess is an uncommon entity and usually results in the death of the patient when left undiagnosed . A case is presented where bacteraemia with an anaerobic Gram-positive bacillus was associated with splenic abscess . Despite treatment with splenectomy and antibiotics the patient developed a multiple organ dysfunction syndrome (MODS) and died . Of particular interest was the isolation of Clostridium novyi type A from the blood in a patient without gas gangrene but with splenic suppuration. Biosci Biotechnol Biochem, 1997 Mar, 61(3), 427 - 31 Purification and characterization of the family J catalytic domain derived from the Clostridium thermocellum endoglucanase CelJ; Ahsan M et al.; The Clostridium thermocellum endoglucanase CelJ contains two different catalytic domains in a polypeptide, i.e., a subfamily E1 catalytic domain and a family J catalytic domain {J . Bacteriol., 178, 5732-5740 (1996)} . The family J catalytic domain (CDJ-CelJ) was produced by a recombinant Escherichia coli and purified . The purified CDJ-CelJ gave a single band on SDS-polyacrylamide gel electrophoresis and the molecular weight of this enzyme (60,000) was consistent with the value (60,333) calculated from the DNA sequence . CDJ-CelJ hydrolyzed various cellulosic substrates, xylan, and lichenan but not p-nitrophenyl (PNP)-cellobioside, PNP-glucoside, or PNP-xyloside at all . CDJ-CelJ was active on Avicel, a microcrystalline cellulose, and the specific activity of CDJ-CelJ on Avicel (0.0078 U/mg protein) was comparable to that of CelS, which is recognized as the most important catalytic subunit of the C . thermocellum, cellulosome, suggesting that CelJ is also an important catalytic subunit in the cellulosome of this bacterium, in addition to CelS. J Pediatr Surg, 1997 Mar, 32(3), 430 - 3 Effects of epidermal growth factor and Clostridium difficile toxin B in a model of mucosal injury; Lawrence JP et al.; Numerous factors have been advocated as being paramount to the development of necrotizing enterocolitis (NEC) including hypoxia, abnormal bacterial flora, and by products of enteral feedings . In an effort to better understand mechanisms involved at the level of the intestinal mucosal barrier the authors have chosen the CACO-2 cell line to model the neonatal intestinal epithelium . By growing CACO-2 cells in transwell inserts, the authors have investigated the ability of Clostridium difficile toxin B, epidermal growth factor (EGF), and a model of mechanical injury to alter transepithelial resistance of CACO-2 monolayers . The findings show that toxin B diminishes resistance in this setting, and EGF can alter that resistance drop. Naunyn Schmiedebergs Arch Pharmacol, 1997 Mar, 355(3), 328 - 34 Effects of Clostridium difficile toxin B on activation of rat peritoneal mast cells; Wex CB et al.; Clostridium difficile toxin B that inactivates Rho subfamily proteins by glucosylation, inhibited dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) and phorbol 12-myristate 13-acetate (PMA)-induced mast cell activation by 80 to 90% in a concentration- and time dependent manner with a delay of abou