Clostridia

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 about 30 min . Activation of mast cells by compound 48/80 and calcium ionophore A23187 was maximally inhibited by about 50% . Inhibition by toxin B was observed with suspended, attached and permeabilised mast cells . C3 ADP-ribosyltransferase, which selectively inactivates RhoA,B,C subtype proteins inhibited antigen, compound 48/80, PMA, A23187 and GTP{S}-induced degranulation of permeabilised mast cells C3-induced inhibition of stimulated histamine release was smaller than that observed with toxin B and both inhibitory effects were not additive . These findings suggest the involvement of Rho subtype GTPases and additionally, of other members of the Rho subfamily GTPases in activation of rat peritoneal mast cells.

Naunyn Schmiedebergs Arch Pharmacol, 1997 Mar, 355(3), 319 - 27
Effects of Clostridium botulinum C2 toxin-induced depolymerisation of actin on degranulation of suspended and attached mast cells; Wex CB et al.; We studied the effects of C . botulinum C2 toxin, which ADP-ribosylates G-actin, on mast cell degranulation . C2 toxin inhibited degranulation of suspended rat peritoneal mast cells induced by compound 48/80 and dinitrophenyl-conjugated bovine serum albumin (DNP-BSA) maximally by about 50 and 90%, respectively . Inhibition by C2 toxin occurred in a time- and concentration-dependent manner . Half-maximal inhibition of DNP-BSA-induced degranulation by C2 toxin occurred at about 0.015 ng/ml, whereas stimulation of mast cells induced by compound 48/80 was half-maximally inhibited at 0.15 ng/ml C2 toxin . C2 toxin also inhibited stimulated {3H}serotonin release from suspended mast cells . Phorbol 12-myristate 13-acetate (PMA)-induced histamine release of suspended mast cells was inhibited by C2 toxin by about 80-90% . C2 toxin had no effect on calcium ionophore A23187-induced histamine release . Toxin treatment of mast cells caused ADP-ribosylation of actin and depolymerisation of F-actin . Attachment of mast cells, which largely increased the diameter of the subcortical actin network, reduced degranulation stimulated by compound 48/80, antigen and calcium ionophore but not by PMA . Opposite to its effect on suspended cells, in adherent mast cells C2 toxin stimulated degranulation by compound 48/80, antigen, and calcium ionophore but not by PMA . The data indicate that mast cell degranulation and responsiveness towards the actin-depolymerising C2 toxin depend largely on mast cell attachment.

Microbiology, 1997 Mar, 143 ( Pt 3), 891 - 8
Structure of the Clostridium stercorarium gene celY encoding the exo-1,4-beta-glucanase Avicelase II; Bronnenmeier K et al.; The nucleotide sequence of the celY gene coding for the thermostable exo-1,4-beta-glucanase Avicelase II of Clostridium stercorarium was determined . The gene consists of an ORF of 274Z bp which encodes a preprotein of 914 amino acids with a molecular mass of 103 kDa . The signal-peptide cleavage site was identified by comparison with the N-terminal amino acid sequence of Avicelase II purified from C stercorarium . The celY gene is located in close vicinity to the celZ gene coding for the endo-1,4-beta-glucanase Avicelase I . The CelY-encoding sequence was isolated from genomic DNA of C . stercorarium with the PCR technique . The recombinant enzyme produced in Escherichia coli as a LacZ'-CelY fusion protein could be purified using a simple two-step procedure . The properties of CelY proved to be consistent with those of Avicelase II purified from C . stercorarium . Sequence comparison revealed that CelY consists of an N-terminal catalytic domain flanked by a domain of 95 amino acids with unknown function joined to a type III cellulose-binding domain . The catalytic domain belongs to the recently proposed family L of cellulases (family 48 of glycosyl hydrolases).

Microbiology, 1997 Mar, 143 ( Pt 3), 885 - 90
The recA gene from Clostridium perfringens is induced by methyl methanesulphonate and contains an upstream Cheo box; Johnston JL et al.; The recA gene from Clostridium perfringens was cloned using degenerate oligonucleotide primers designed from conserved regions of RecA proteins from other bacteria . The 1089 bp gene encoded a putative RecA protein with 69% amino acid sequence similarity to the RecA protein from Bacillus subtilis . The C . perfringens recA gene was induced by exposure to methyl methanesulphonate and complemented a recA mutant of Escherichia coli . A cheo box was identified in the region upstream of the gene . Since this SOS-like operator site is conserved in many DNA-damage-inducible recA gene regions from Gram-positive bacteria, the results suggest that the regulation of the C . perfringens recA gene also involves the binding of a LexA-like protein to this site.

Arch Otolaryngol Head Neck Surg, 1997 Mar, 123(3), 321 - 6
Patient selection in the treatment of glabellar wrinkles with botulinum toxin type A injection; Pribitkin EA et al.; OBJECTIVES: To determine the dose-response characteristics and side-effects profile of Clostridium botulinum type A exotoxin (Botox) used to treat glabellar wrinkles and develop guidelines for patient selection based on the nature and severity of the treated wrinkles . DESIGN: Prospective, nonrandomized pilot and electromyogram (EMG)-guided studies . SETTING: Two ambulatory care clinics at university hospitals . PARTICIPANTS: For the pilot study, volunteer samples of 23 patients with glabellar wrinkles; for the EMG-guided study, volunteer samples of 57 patients with glabellar wrinkles . INTERVENTIONS: For the pilot study, 23 patients were serially injected with up to 10.0 mouse units (MU) of Botox into each corrugator muscle; for the EMG-guided study, 57 patients were injected under EMG guidance with an initial dose of 10.0 MU of Botox into each corrugator muscle . Eleven patients with persistent corrugator activity were reinjected with 10.0 MU of Botox . MAIN OUTCOME MEASURES: For the pilot study, slide photographs were obtained before and 2 weeks after injection; for the EMG-guided study, slide photographs were obtained before and at 2 weeks and at 2 months after injection . Patients were asked to evaluate results numerically . RESULTS: For the pilot study, injection of up to 10.0 MU of Botox into each corrugator muscle produced a satisfactory improvement in 12 patients; for the EMG-guided study, 43 patients were satisfied with improvement after full abolition of corrugator or accessory lateral brow muscle activity . Women were more likely to achieve satisfactory results than were men (80% {40/50} vs 43% {3/7}; P < or = .03) . Improvement was not age related . No significant side effects or complications were observed . CONCLUSIONS: Glabellar wrinkles may be satisfactorily treated with Botox injection into the corrugator supercilii muscles . Improvement is temporary, dose dependent, and may not be seen in some patients even with successful denervation of the treated muscles . Clinicians may begin treatment with a dose of 10.0 MU of Botox into each corrugator muscle, and may select candidates for injection by determining the type of wrinkle to be treated and its spreadability (glabellar spread test).

Microb Pathog, 1997 Mar, 22(3), 143 - 54
Transcriptional analysis of the toxigenic element of Clostridium difficile; Hammond GA et al.; A transcriptional analysis was undertaken for the toxigenic element of Clostridium difficile in five strains which differ greatly in toxigenicity . The toxigenic element has recently been described in C . difficile strain VPI 10463 and consists of three small open reading frames in addition to the toxin A and B genes . A large, polycistronic transcript (17.5 kb) was detected, in addition to processing intermediates, and individual transcripts for toxin A, toxin B, and two of the three small open reading frames . A transcription initiation site and a promoter for the toxigenic element were identified, as well as major extension products upstream of the toxin A and B genes . These data support models in which the toxin A and B genes are cotranscribed along with the open reading frames, in addition to being transcribed individually . Transcriptional analyses, using probes for the transcripts for toxin A and toxin B, revealed quantitative differences among strains which correlated with quantitative differences in toxin production among these strains . However, DNA sequencing of intergenic regions in these strains showed remarkable DNA sequence conservation in these intergenic regions.

Am J Gastroenterol, 1997 Mar, 92(3), 454 - 6
A prospective randomized controlled trial of oral ciprofloxacin in acute ulcerative colitis; Mantzaris GJ et al.; OBJECTIVES: The aim of this prospective, randomized, controlled trial was to evaluate the role of ciprofloxacin as an adjunct to corticosteroids in acute ulcerative colitis . METHODS: Seventy consecutive patients with mild (n = 37) or moderately active (n = 33) ulcerative colitis were randomized to receive oral ciprofloxacin (250 mg b.i.d., n = 34) or placebo (n = 36) for 14 days . In addition, they were given oral prednisolone (initial dose 20 or 40 mg for mild and moderately active ulcerative colitis, respectively) and rectal betamethasone enemas (2 g at night) for 7-9 weeks . All patients were receiving olsalazine (0.5 g twice daily) . At study entry, the groups were similar with respect to age, sex, extent, duration, and severity of disease, and previous treatments . Patients were assessed clinically, endoscopically, and histologically before, at the end of the trial (day 14), and on completion of steroid treatment, or at any time worsening of symptoms or a complication of ulcerative colitis occurred . RESULTS: At the end of the study, 24 patients (70.5%) in the ciprofloxacin group and 26 patients (72%) in the placebo group achieved remission (p > 0.1, Yates chi 2) . Ten patients in each group necessitated higher doses of oral (n = 12) or intravenous (n = 8) steroids . Of the latter patients, two underwent emergency colectomy without perioperative deaths . Clostridium difficile toxin A was not detected in nonresponders to ciprofloxacin treatment . CONCLUSIONS: A short course of oral ciprofloxacin treatment does not seem to increase the proportion of patients with active ulcerative colitis going into remission.

Appl Environ Microbiol, 1997 Mar, 63(3), 1148 - 50
Recovery of a strain of Clostridium botulinum producing both neurotoxin A and neurotoxin B from canned macrobiotic food; Franciosa G et al.; A rare strain of Clostridium botulinum subtype Ab was isolated from a canned macrobiotic food suspected of being linked to a fatal case of food-borne botulism . The strain was recovered and identified by conventional methods modified by the inclusion of a PCR assay (G . Franciosa, J.L . Ferreira, and C.L . Hatheway, J . Clin . Microbiol . 32:1911-1917, 1994) . The titers of neurotoxins produced by the strain were evaluated by a mouse bioassay.

Appl Environ Microbiol, 1997 Mar, 63(3), 903 - 9
Characterization of the cellulolytic complex (cellulosome) produced by Clostridium cellulolyticum; Gal L et al.; The cellulolytic complex was isolated from Clostridium cellulolyticum grown on cellulose . Upon gel filtration, the complex was found to consist mainly of 600-kDa units, along with a 16-MDa aggregate . Its ability to degrade various substrates and its capacity to bind to the crystalline cellulose were measured . The results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, N-terminal sequencing, and blotting analysis showed that all of the known cellulases of this organism are present in this complex . Three major components were observed: the first component, a noncatalytic, large (160-kDa) protein, was identified based on its ability to bind to the dockerin-containing cellulases as scaffolding protein CipC . The other two components, which had molecular masses of 94 and 80.6 kDa, were identified as CelE and CelF, respectively . The identified cellulases and some other components of the cellulosome were able to bind to a miniCipC1 construct . In addition to providing an extensive description of the system, the results of the present study confirm that the dockerin-cohesin domain interaction plays an essential role in the constitution of the cellulosome.

J Bacteriol, 1997 Mar, 179(5), 1714 - 20
Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum; Das A et al.; The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column . The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5 . It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa . These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no . U64318), demonstrating the close similarity of the F1F0 complexes from C . thermoaceticum and C . thermoautotrophicum . Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium . The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria . The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive . The F1F0 complex of C . thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities . Immunoblot analyses of the subunits of ATP synthases from C . thermoautotrophicum, C . thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E . coli.

J Clin Microbiol, 1997 Mar, 35(3), 578 - 83
Sensitive assay for measurement of antibodies to Clostridium botulinum neurotoxins A, B, and E: use of hapten-labeled-antibody elution to isolate specific complexes; Doellgast GJ et al.; The measurement of chicken and human antibodies to Clostridium botulinum neurotoxins A, B, and E was accomplished by affinity isolation of complexes containing these antibodies . By this approach, a mixture of toxin with the test antibody, fluoresceinated antibody, and enzyme (Russell's viper venom factor X activator)-labeled antibody is allowed to form a complex in solution phase . This complex is then bound to a matrix containing antifluorescein antibody . All components not bound to the matrix are washed off, and the complex is isolated intact by elution with fluorescein, which competes with the complex for binding to the antifluorescein matrix . The eluted complex is then bound to a matrix which specifically binds the test antibody (anti-chicken immunoglobulin Y {IgY} or anti-human IgG), and the bound complex is measured by using the enzyme label . Using this approach, we were able to measure as little as 1 ng of specific antibody per ml from affinity-isolated, monospecific chicken antibody preparations and to measure antibody specifically from IgY fractions of monospecific chicken antibody preparations . Human antibodies from subjects immunized with pentavalent toxoid preparations were detectable at dilutions as great as 24,300-fold, and undiluted serum from most control subjects showed no measurable antibody . Antibody was also measured in 65 subjects who were receiving preparations of neurotoxin A (BOTOX) for the treatment of spastic disorders . Eighteen of them had toxin-specific antibody reactive with toxin B, and two of them had toxin-specific antibody reactive with toxin A . The two patients having antibody to toxin A were refractory to treatment with this toxin . This approach of isolation of hapten-labeled immune complexes under nondenaturing conditions with hapten is broadly applicable to the specific measurement of antibodies present at very low concentrations in serum.

J Clin Microbiol, 1997 Mar, 35(3), 558 - 62
Evaluation of the AnaeroPack system for growth of clinically significant anaerobes; Delaney ML et al.; The AnaeroPack (Mitsubishi Gas Chemical America, Inc., New York, N.Y.) system was compared with the GasPak (Becton Dickinson Microbiology Systems, Cockeysville, Md.) system and a conventional anaerobe chamber to evaluate the ability of the AnaeroPack system to support the growth of clinically significant anaerobes . The AnaeroPack system requires no catalyst or water, produces no hydrogen, and is oxygen absorbing and carbon dioxide generating . It is simple to use and reduces preparation time to a minimum . One hundred forty clinical isolates obtained from various anatomic sites and 10 American Type Culture Collection type strains were evaluated . Isolates were plated on various media, and bacterial growth was examined after 24, 48, 72, and 168 h of incubation . Criteria for evaluation and comparison of systems included rate and quality of growth, colonial morphology, hemolytic reactions, and pigment production . Results indicate that the AnaeroPack system is highly effective in creating an anaerobic atmosphere . The AnaeroPack system never failed to reduce the methylene blue indicator, while the GasPak system failed 15% of the time . The rate or quality of growth achieved by the AnaeroPack system compared with that of established anaerobic culturing techniques was similar and significantly better for several genera including the Bacteroides fragilis group, Fusobacterium, Clostridium, and Peptostreptococcus . The AnaeroPack system appears to be an excellent alternative to established methods for generating an environment for anaerobic incubation.

J Infect Dis, 1997 Mar, 175(3), 633 - 7
Endogenous antibody production to botulinum toxin in an adult with intestinal colonization botulism and underlying Crohn's disease; Griffin PM et al.; A patient with obstruction of the terminal ileum from Crohn's disease developed complete paralysis in week 1 of hospitalization . Features initially suggested Guillain-Barre syndrome, but botulinum toxin was identified in serum and stool specimens from week 1 and type A toxin-producing Clostridium botulinum in stool specimens from weeks 3 to 19, confirming botulism due to intestinal colonization . In week 19, the inflamed small bowel was resected, and C . botulinum disappeared from the stool . In week 31, the patient was able to breath without assistance . Testing for an active immune response with neutralizing antibodies to C . botulinum at week 19 was positive; these antibodies remained at a protective level for >1 year . Intestinal colonization botulism, rare in adults, should be considered for patients with descending paralysis, especially those with a preceding alteration in small bowel function . An active immune response to botulinum toxin with production of protective antibodies has not been demonstrated previously in a patient with botulism and may have contributed to this patient's recovery.

Infect Immun, 1997 Mar, 65(3), 1105 - 8
Positive regulation of Clostridium difficile toxins; Moncrief JS et al.; The toxigenic element of Clostridium difficile VPI 10463 contains a small open reading frame (ORF) immediately upstream of the toxin B gene (G . A . Hammond and J . L . Johnson, Microb . Pathog . 19:203-213, 1995) . The deduced amino acid sequence of the ORF, which we have designated txeR, encodes a 22-kDa protein which contains a helix-turn-helix motif with sequence identity to DNA binding regulatory proteins . We used a DNA fragment containing the C . difficile toxin A repeating units (ARU) as a reporter gene to determine if txeR regulates expression from the toxin A and toxin B promoters in Escherichia coli . To test the affect of txeR on expression, we fused the ARU gene fragment in frame with the toxin promoters . The fusions expressed a 104-kDa protein that contained the epitopes for monoclonal antibody PCG-4, which we used to measure levels of recombinant ARU by enzyme-linked immunosorbent assay . When txeR was expressed in trans with the toxin B promoter-ARU fusion contained on separate low-copy-number plasmid, expression of ARU increased over 800-fold . Furthermore, when we tested the toxin A promoter fused to ARU, expression increased over 500-fold with txeR supplied in trans . Our results suggest that TxeR is a positive regulator that activates expression of the C . difficile toxins.

Infect Immun, 1997 Mar, 65(3), 1014 - 22
Deletion analysis of the Clostridium perfringens enterotoxin; Kokai-Kun JF et al.; To further our knowledge of the structure-function relationship and mechanism of action of the Clostridium perfringens enterotoxin (CPE), a series of recombinant CPE (rCPE) species containing N- and C-terminal CPE deletion fragments was constructed by recombinant DNA approaches . Each rCPE species was characterized for its ability to complete the first four early steps in the action of CPE, putatively ordered as specific binding, a postbinding physical change to bound CPE, large-complex formation, and induction of alterations in small-molecule membrane permeability . These studies demonstrated that (i) at least 44 amino acids can be removed from the N terminus of CPE without loss of cytotoxicity, (ii) removal of the first 53 amino acids from the N terminus of CPE produces a fragment that appears to be noncytotoxic because it cannot undergo the post-binding physical change step in CPE action, (iii) removal of as few as five amino acids from the C terminus of CPE produces a noncytotoxic fragment lacking receptor binding activity, and (iv) a fragment lacking the first 44 N-terminal amino acids of native CPE formed twice as much large complex and was twice as cytotoxic as native CPE . From these structure-function results, it appears that the minimum-size cytotoxic CPE fragment comprises approximately residues 45 to 319 of native CPE . Results from these deletion fragment studies have also contributed to our understanding of CPE action by (i) independently supporting previous suggestions that binding, the postbinding physical change step, and large-complex formation represent important steps in CPE cytotoxicity and (ii) providing independent evidence confirming the putative sequential order of these early events in CPE action.

Mol Cell Biol, 1997 Mar, 17(3), 1201 - 11
Rho family GTPases and neuronal growth cone remodelling: relationship between increased complexity induced by Cdc42Hs, Rac1, and acetylcholine and collapse induced by RhoA and lysophosphatidic acid; Kozma R et al.; Rho family GTPases have been assigned important roles in the formation of actin-based morphologies in nonneuronal cells . Here we show that microinjection of Cdc42Hs and Rac1 promoted formation of filopodia and lamellipodia in N1E-115 neuroblastoma growth cones and along neurites . These actin-containing structures were also induced by injection of Clostridium botulinum C3 exoenzyme, which abolishes RhoA-mediated functions such as neurite retraction . The C3 response was inhibited by coinjection with the dominant negative mutant Cdc42Hs(T17N), while the Cdc42Hs response could be competed by coinjection with RhoA . We also demonstrate that the neurotransmitter acetylcholine (ACh) can induce filopodia and lamellipodia on neuroblastoma growth cones via muscarinic ACh receptor activation, but only when applied in a concentration gradient . ACh-induced formation of filopodia and lamellipodia was inhibited by preinjection with the dominant negative mutants Cdc42Hs(T17N) and Rac1(T17N), respectively . Lysophosphatidic acid (LPA)-induced neurite retraction, which is mediated by RhoA, was inhibited by ACh, while C3 exoenzyme-mediated neurite outgrowth was inhibited by injection with Cdc42Hs(T17N) or Rac1(T17N) . Together these results suggest that there is competition between the ACh- and LPA-induced morphological pathways mediated by Cdc42Hs and/or Rac1 and by RhoA, leading to either neurite development or collapse.

Curr Microbiol, 1997 Mar, 34(3), 162 - 6
NAD-independent lactate and butyryl-CoA dehydrogenases of Clostridium acetobutylicum P262; Diez-Gonzalez F et al.; Clostridium acetobutylicum P262 cells that were growing on lactate and acetate had an NAD-independent lactate dehydrogenase(iLDH) activity of 200 nmol mg protein-1 min-1.Ammonium sulfate precipitation and DEAE cellulose caused a 35-fold purification . Gel filtration indicated that the iLDH had a molecular weight of approximately 55 kDa, but two bands were always observed . Phenyl sepharose could not separate the two proteins, and hydroxyapatite caused a complete loss of activity . The semi-purified iLDH had a Vmax of 13,000 nmol mg protein-1 min-1 and a Km value of 3.5 mM for D-lactate . The Vmax and Km values for L-lactate were 300 nmol mg protein-1 min-1 and 0.7 mM . The iLDH had a pH optimum of 7.5, was not activated by fructose-1,6-bisphosphate (FDP), and could be coupled to either 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) or dichlorophenol-indophenol (DCPIP), but not methyl viologen (MV) or benzyl viologen (BV) . The iLDH did not have strong absorbance between 500 and 300 nm, and trichloroacetic acid or acid ammonium sulfate extracts had virtually no fluorescence at 450 nm . The crude extracts also had MTT-linked butyryl-CoA dehydrogenase activity (60 nmol mg protein-1 min-1) . The NAD-independent butyryl-CoA dehydrogenase eluted from DEAE-cellulose as two fractions . The yellow fraction was extremely unstable, but the green fraction could be stored for short periods of time at 5 degrees C . The green-colored butyryl-CoA dehydrogenase had strong absorption at 450 nm, and gel filtration indicated that it had a molecular weight of 90 kDa . The NAD-independent butyryl-CoA dehydrogenase could be coupled to MTT, DCPIP, or MV, but not BV . Because the NAD-independent lactate and butyryl-CoA dehydrogenase could both be linked to low potential carriers, these two enzymes may function as oxidation-reduction system in vivo.

Mol Gen Genet, 1997 Feb 27, 253(6), 770 - 6
The heterocyst-specific fdxH gene product of the cyanobacterium Anabaena sp . PCC 7120 is important but not essential for nitrogen fixation; Masepohl B et al.; To clarify the role of the heterocyst-specific {2Fe-2S} ferredoxin in cyanobacterial nitrogen fixation, mutational analysis of the Anabaena 7120 fdxH gene region was carried out . First, the DNA sequence of the wild-type 3509-bp EcoRI fragment downstream of the fdxH gene was determined . Genes homologous to ORF3 from the fdxH gene regions of A . variabilis and Plectonema boryanum, the mop genes of Clostridium pasteurianum encoding molybdo-pterin binding proteins, and ORF3 from the A . variabilis hydrogenase gene cluster were identified within the sequenced region . For mutational analysis the Anabaena 7120 mutant strains LAK4, BMB92, and KSH10 were constructed . In LAK4 the fdxH coding region is disrupted by an interposon, whereas BMB92 is deleted for a 2799-bp NheI fragment encompassing fdxH, ORF3, mop, ORF4, and ORF5 . Mutant strain KSH10 is a derivative of BMB92, complemented for fdxH but not for the other genes located further downstream . Analysis of the Nif phenotype of these mutant strains showed that FdxH is necessary for maximum nitrogenase activity and optimal growth under nitrogen-fixing conditions, but not absolutely essential for diazotrophic growth . The role of alternative electron donors for nitrogenase, which might substitute for FdxH, is discussed . Iron concentrations (1 microM Fe) sufficient to induce synthesis of the vegetative cell flavodoxin did not stimulate diazotrophic growth of the fdxH mutant strains, suggesting that FdxH was not replaced by a NifJ-flavodoxin system . Comparison of LAK4 and BMB92 indicated that one of the genes located downstream of fdxH might also play a (minor) role in nitrogen fixation.

J Immunol, 1997 Feb 15, 158(4), 1516 - 22
IL-2 signaling controls actin organization through Rho-like protein family, phosphatidylinositol 3-kinase, and protein kinase C-zeta; Gomez J et al.; IL-2 and IL-4 induce proliferation of TS1 alpha beta cells . Activation of the zeta isoform of protein kinase C is an important step in IL-2-, but not IL-4-mediated proliferation . In addition, protein kinase C-zeta is implicated in IL-2-mediated actin organization . Given the established involvement of the Rho family of small guanine nucleotide-binding proteins in organization of actin structures, we analyze the possible relationships between Rho and protein kinase C-zeta . Using the Rho-like protein family-specific toxin B from Clostridium difficile, we report in this work that IL-2, but not IL-4, induces a Rho-dependent activation of protein kinase C-zeta . This signaling event is mediated by the activation of phosphatidylinositol 3-kinase . In contrast, IL-4 induces a Rho-independent, phosphatidylinositol 3-kinase-mediated activation of protein kinase C-zeta, but this pathway has no implications in cytoskeleton organization.

Biochemistry, 1997 Feb 11, 36(6), 1259 - 80
Control of oxidation-reduction potentials in flavodoxin from Clostridium beijerinckii: the role of conformation changes; Ludwig ML et al.; X-ray analyses of wild-type and mutant flavodoxins from Clostridium beijerinckii show that the conformation of the peptide Gly57-Asp58, in a bend near the isoalloxazine ring of FMN, is correlated with the oxidation state of the FMN prosthetic group . The Gly-Asp peptide may adopt any of three conformations: trans O-up, in which the carbonyl oxygen of Gly57 (O57) points toward the flavin ring; trans O-down, in which O57 points away from the flavin; and cis O-down . Interconversions among these conformers that are linked to oxidation-reduction of the flavin can modulate the redox potentials of bound FMN . In the semiquinone and reduced forms of the protein, the Gly57-Asp58 peptide adopts the trans O-up conformation and accepts a hydrogen bond from the flavin N5H {Smith, W . W., Burnett, R . M., Darling, G . D., & Ludwig, M . L . (1977) J . Mol . Biol . 117, 195-225; Ludwig, M . L., & Luschinsky, C . L . (1992) in Chemistry and Biochemistry of Flavoenzymes III (Muller, F., Ed.) pp 427-466, CRC Press, Boca Raton, FL} . Analyses reported in this paper confirm that, in crystals of wild-type oxidized C . beijerinckii flavodoxin, the Gly57-Asp58 peptide adopts the O-down orientation and isomerizes to the cis conformation . This cis form is preferentially stabilized in the crystals by intermolecular hydrogen bonding to Asn137 . Structures for the mutant Asn137Ala indicate that a mixture of all three conformers, mostly O-down, exists in oxidized C . beijerinckii flavodoxin in the absence of intermolecular hydrogen bonds . Redox potentials have been manipulated by substitutions that alter the conformational energies of the bend at 56M-G-D-E . The mutation Asp58Pro was constructed to study a case where energies for cis-trans conversion would be different from that of wild type . Intermolecular interactions with Asn137 are precluded in the crystal, yet Gly57-Pro58 is cis, and O-down, when the flavin is oxidized . Reduction of the flavin induces rearrangement to the trans O-up conformation . Redox potential shifts reflect the altered energies associated with the peptide rearrangement; E(ox/sq) decreases by approximately 60 mV (1.3 kcal/mol) . Further, the results of mutation of Gly57 agree with predictions that a side chain at residue 57 should make addition of the first electron more difficult, by raising the energy of the O-up conformer that forms when the flavin is reduced to its semiquinone state . The ox/sq potentials in the mutants Gly57Ala, Gly57Asn, and Gly57Asp are all decreased by approximately 60 mV (1.3 kcal/mol) . Introduction of the beta-branched threonine side chain at position 57 has much larger effects on the conformations and potentials . The Thr57-Asp58 peptide adopts a trans O-down conformation when the flavin is oxidized; upon reduction to the semiquinone, the 57-58 peptide rotates to a trans O-up conformation resembling that found in the wild-type protein . Changes in FMN-protein interactions and in conformational equilibria in G57T combine to decrease the redox potential for the ox/sq equilibrium by 180 mV (+4.0 kcal/mol) and to increase the sq/hq potential by 80 mV (-1.7 kcal/mol) . A thermodynamic scheme is introduced as a framework for rationalizing the properties of wild-type flavodoxin and the effects of the mutations.

Lancet, 1997 Feb 8, 349(9049), 426 - 8
Hospital-acquired Clostridium difficile diarrhoea and herd immunity; Starr JM et al.; Clostridium difficile diarrhoea represents a significant health-service burden . We recently experienced an outbreak of C difficile diarrhoea associated with increased use of cefotaxime . The question we pose in this paper is how did the introduction and withdrawal of a single antibiotic so greatly affect rates of C difficile diarrhoea? Other antibiotics had nearly as high a risk of causing diarrhoea as cefotaxime, and the majority of patients never received cefotaxime . We believe that such outbreaks of C difficile diarrhoea are best understood in terms of a population model, and that taking antibiotics like cefotaxime should be thought of as a population rather than an individual risk factor . We postulate a herd-immunity model of C difficile diarrhoea, and examine the implications of this hypothesis.

J Endod, 1997 Feb, 23(2), 110 - 4
Identification and antibiotic sensitivity of bacteria isolated from periapical lesions; Vigil GV et al.; Periradicular tissues from 28 refractory endodontic cases requiring surgical intervention were submitted for histological diagnosis and microbiological culture . Bacteria isolated from these lesions were identified and then tested for their antibiotic sensitivity to a panel of common antibiotics . The periapical tissue specimens of 22 out of 28 lesions (79%) contained microorganisms . Of the 22 cases showing positive growth cultures, 15 were polymicrobial and 7 were single species isolates . Fifty-three different species were recovered: 29 anaerobes, 19 facultative anaerobes, and 5 aerobes . Microbes were observed under light microscopy in only one case . The most common organisms isolated were Propionibacterium acnes, Staphylococcus epidermidis, Streptococcus intermedius, Wolinella recta, Fusobacterium species, and Clostridium species . Antibiotic susceptibility results showed no clear cut evidence of significant antibiotic resistance among the species tested . The results of this study seem to corroborate earlier studies regarding the microbial population of periapical lesions refractory to nonsurgical endodontics.

J Ind Microbiol Biotechnol, 1997 Feb-Mar, 18(2-3), 82 - 8
2,4,6-Trinitrotoluene (TNT) transformation by clostridia isolated from a munition-fed bioreactor: comparison with non-adapted bacteria; Ederer MM et al.; Several bacterial strains were examined for their ability to degrade the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) . The strains examined included various clostridial strains isolated from a 4-year-old munition enrichment, related clostridial strains obtained from a culture collection, two enteric bacteria, and three lactobacilli . All Clostridium species tested were able to reduce TNT rapidly in a complex medium . In cell suspension experiments, these strains were also able to reduce 2,4-diamino-6-nitrotoluene (DANT) to 2,4,6-triaminotoluene (TAT) and to produce a compound that is not yet identified; thus, they could not be distinguished from one another with regard to the pathway of transformation . The enteric strains and the lactobacilli were able to perform the initial reduction of TNT, but none was capable of reducing DANT in cell suspensions.

Arch Microbiol, 1997 Feb-Mar, 167(2-3), 177 - 81
Assimilation of sulfur from alkyl- and arylsulfonates by Clostridium spp; Denger K et al.; 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.

Zentralbl Bakteriol, 1997 Feb, 285(3), 397 - 402
Comparison of a new, bismuth-iron-sulfite-cycloserine agar for isolation of Clostridium perfringens with the tryptose-sulfite-cycloserine and blood agars; Gubash SM et al.; A new differential and selective, bismuth-iron-sulfite-cycloserine (BISC) medium, for isolation and enumeration of Clostridium perfringens from food and feces, was developed . The medium was compared with the widely-used tryptose-sulfite-cycloserine (TSC) medium and blood agar (BA) in recovering actively growing cells, cold- (refrigerated and frozen) stressed, and heat-stressed C . perfringens cells, and heat-activated spores from human feces . Both selective media were satisfactory in recovering actively growing cells and heat-activated spores of C . perfringens . Both were inferior to non-inhibitory blood agar in recovering heat or cold-stressed cells . The advantages of the new BISC medium over the TSC medium were: elimination of the need to prepare pour- or overlay-agar plates, which simplified inoculation of specimens on the medium and simplified the subcultures of colonies for confirmatory identification . All colonies of C . perfringens developed on BISC were black or dark gray . This was contrary to TSC medium, which gave, on average, 39.6% of white colonies when inoculated with the pure cultures of C . perfringens.

Lett Appl Microbiol, 1997 Feb, 24(2), 95 - 100
Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature; Graham AF et al.; The effect of combinations of temperature (2 degrees, 3 degrees, 4 degrees, 5 degrees, 8 degrees and 10 degrees C), pH (5.0-7.2) and NaCl (0.1-5.0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium . Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth . Growth and toxin production were detected at 3 degrees C in 5 weeks, at 4 degrees C in 3/4 weeks and at 5 degrees C in 2/3 weeks . The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time . This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl . botulinum in minimally processed chilled foods.

Gastroenterol Hepatol, 1997 Feb, 20(2), 55 - 8
{Gas gangrene due to Clostridium septicum in a patient with an occult colonic neoplasm . A case report and review of the literature}; Saez Castillo AI et al.; A case of gaseous gangrene by Clostridium septicum associated with colorectal cancer is presented . The patient evolved rapidly towards septic shock and death . Autopsy showed occult neoplasm and pelvic and retroperitoneal myonecrosis . An exceptional finding was that of myocarditis in which thick gram-positive bacilli were identified . A review of the literature was carried out regarding the pathogenesis and clinical manifestations of this disease . The association of colonic neoplasm and Clostridium septicum may be related with the sensitivity of the cells of this neoplasm to the toxins of the microorganisms . The usefulness of this cytotoxicity is being tested in the therapeutic reduction of tumoral mass . With respect to clinical attitude, all the authors agree on the need for clinical suspicion as to the possible existence of occult colon neoplasm in individuals with septic shock by gaseous gangrene with no obvious entry site . Diagnosis is performed by imaging techniques with barium enema and if this is normal colonoscopy is carried out . Emergency treatment consists in laparotomy with resection of the neoplasm and debridement of the area accompanied by hyperbaric oxygen and antibiotics.

J Vet Med Sci, 1997 Feb, 59(2), 89 - 92
Most probable number method combined with nested polymerase chain reaction for detection and enumeration of enterotoxigenic Clostridium perfringens in intestinal contents of cattle, pig and chicken; Miwa N et al.; The most probable number (MPN) method combined with a nested polymerase chain reaction (nested PCR) for the detection and enumeration of enterotoxigenic Clostridium perfringens in the intestinal contents of cattle, pig and chicken was examined . Ten-fold serial dilutions of samples were added to three tubes of enrichment medium, which were incubated at 37 degrees C for 20-24 hr, and the C . perfringens enterotoxin gene was detected by nested PCR from the enrichment culture without isolating the organism . The results obtained by this method with artificially contaminated intestinal contents were significantly correlated with those obtained by a plate count method . When the method was applied to the detection and enumeration of indigenous enterotoxigenic C . perfringens, the organism was found in two, two and three samples of 10 intestinal contents of cattle, pig and chicken, respectively . Most of the positive samples contained fewer than 10 MPN/g of enterotoxigenic C . perfringens, except one sample of chicken, which contained 1.5 x 10(2) MPN/g . The MPN method combined with nested PCR is easy to perform and may be a useful tool for the detection and enumeration of enterotoxigenic C . perfringens in intestinal contents.

Berl Munch Tierarztl Wochenschr, 1997 Feb, 110(2), 46 - 7
Clostridium perfringens toxins absorbed on filter paper--a simple method for sample conservation; Rohner E et al.; Clostridium perfringens type C and type D toxins were absorbed on filter paper, dried and stored at room temperature (18-20 degrees C), at 37 degrees C, at 4 degrees C and at -20 degrees C . Type specific toxin was correctly identified in the EIA for 74 days . Absorption on filter paper may offer a simple method for conservation and transport of post mortem samples from cases of suspected enterotoxaemia.

Vet Microbiol, 1997 Feb, 54(2), 195 - 200
In-vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin; Watkins KL et al.; The minimum inhibitory concentrations (MIC) of eight antibiotics and two anticoccidial agents were determined for Clostridium perfringens strains isolated from 26 commercial broiler farms and 22 commercial turkey farms . Isolates were obtained from the intestines of birds on the farm or as the processing plant using standard culture and identification techniques . The microbroth dilution test was used to determine the MIC for each compound . Most isolates from chickens had MICs in the range of 2-16 mg/L for tilmicosin, tylosin and virginiamycin, whereas the MICs for avilamycin, avoparcin, monensin, narasin and penicillin were < or = 1 mg/L . Most strains from chickens had high MICs (> or = 64 mg/L) and appeared to be resistant to bacitracin and lincomycin . Most turkey isolates had MICs in the range of 2-16 mg/L for bacitracin, tilmicosin, tylosin and virginiamycin, with strains exhibiting MICs < or = 1 mg/L for avilamycin, avoparcin, monensin, narasin and penicillin . Several turkey isolates had MICs > or = 64 mg/L to lincomycin . No attempt was made to associate farm usage of a particular antibiotic to the antibiograms.

Dig Dis Sci, 1997 Feb, 42(2), 359 - 65
Concordance of bacterial cultures with endotoxin and interleukin-6 in necrotizing enterocolitis; Duffy LC et al.; Concordance between gram-negative enteric and other toxin-producing bacteria in blood and stool culture, endotoxin (lipopolysaccharide), and interleukin-6 (IL-6) was measured in 60 preterm infants (600-1600 g) as a clinical index in neonatal necrotizing enterocolitis (NEC) . E . coli, Klebsiella, Enterobacter, and Clostridium spp., identified by routine bacteriology, were each strongly associated with elevated concentrations of endotoxin (P < 0.01) in stool filtrates, with Clostridium spp . most strongly associated with NEC disease . Stool filtrate endotoxin (EU/g) measured by a Limulus amebocyte lysate assay was age dependent . Samples from stage I NEC (61%) and infants with advanced disease (67%) had notably elevated levels of stool endotoxin (> 10 ln EU/g) compared to NEC-negative (47%) samples tested . Plasma and stool IL-6 generally tested at the low, nonmeasurable limit of the ELISA for NEC-negative (88%) and stage I NEC (93%), although a small proportion of samples (25%) from infants with stage II or III NEC had elevated stool concentrations of IL-6 . We conclude that identification of toxin-producing organisms and endotoxin elevations in stool filtrates are more useful than circulating levels of endotoxin in plasma in predicting mucosally limited disease in the gastrointestinal tract . The prognostic value of monitoring stool endotoxin in infants with overgrowth of gram-negative bacteria has implications for therapeutic strategies in patients with early and advanced stages of disease . Monitoring inflammatory cytokines (IL-6) in relation to endotoxin values in stool appears of limited clinical value in controlling this devastating disease in preterm neonates.

Br J Surg, 1997 Feb, 84(2), 150 - 9
Surgical aspects of Clostridium difficile colitis; Bradbury AW et al.; BACKGROUND: There has been a marked increase in the number of surgical patients developing Clostridium difficile colitis . The epidemiology, pathogenesis, diagnosis and management of C . difficile infection were reviewed from a surgical perspective . METHODS: A literature review was carried out based primarily on a Medline search of all English language publications containing the term C . difficile . RESULTS: The recent dramatic increase in diagnosis of C . difficile infection amongst surgical patients results from heightened awareness of the condition, better methods of diagnosis, more widespread use of antibiotics for treatment and prophylaxis, and the increasing numbers of elderly and immunocompromised patients with malignancy, sepsis, and (multiple) organ failure being cared for within intensive therapy and high-dependency units . In addition to morbidity and mortality, the economic burden of C . difficile infection in terms of delayed discharge and other hospital costs is considerable . CONCLUSION: Appropriate use of antibiotics, isolation of affected patients and meticulous hygiene measures on the part of staff are vital if the morbidity, mortality and economic consequences of this nosocomial infection are to be minimized.

J Appl Physiol, 1997 Feb, 82(2), 382 - 8
Ultrastructural changes of lung capillary endothelium in response to botulinum C2 toxin; Ermert L et al.; The role of the endothelial cytoskeleton for the structural integrity of the pulmonary gas exchange area was probed with the use of Clostridium botulinum C2 toxin . This agent causes selective loss of nonmuscle F-actin . In buffer-perfused rabbit lungs, vascular pressures were kept within physiological ranges . In different groups, low-dose {0.3 (C2,I)/0.6 (C2,II) ng/ml} and high-dose {10 (C2,I)/20 (C2,II) ng/ml} toxin were applicated into the buffer fluid; experiments were terminated after a total weight gain of either 1 or 7.5 g . Electron microscopy revealed extensive attenuations, undulations, and protrusions of the endothelial layer, suggestive of "remodeling" and "flowing" of the cell membrane in low C2 toxin-treated lungs accompanied by few disruptions of the endothelial layer and edema formation . In addition, endothelial cells displayed vesiculation and bleb formation . Lungs that were exposed to high-toxin doses displayed marked attenuations of the endothelial layer in addition to large endothelial cell disruptions, which did not include interendothelial junctions . Interestingly, type II epithelial cells displayed fusion of lamellar bodies . Collectively, these data suggest that the actin microfilament system is instrumental in supporting endothelial cell membrane configuration and integrity and maintains the intimal barrier function of the lung microvasculature.

Mol Microbiol, 1997 Feb, 23(3), 551 - 8
Generation of a membrane-bound, oligomerized pre-pore complex is necessary for pore formation by Clostridium septicum alpha toxin; Sellman BR et al.; Low-temperature inhibition of the cytolytic activity of alpha toxin has facilitated the identification of an important step in the cytolytic mechanism of this toxin . When alpha toxin-dependent haemolysis was measured on erythrocytes at various temperatures it was clear that at temperatures < or = 15 degrees C the haemolysis rate was significantly inhibited with little or no haemolysis occurring at 4 degrees C . Alpha toxin appeared to bind to and oligomerize on erythrocyte membranes with similar kinetics at 4 degrees C and 37 degrees C . The slight differences in these two processes at 4 degrees C and 37 degrees C could not account for the loss of cytolytic activity at low temperature . At 4 degrees C alpha toxin neither stimulated potassium release from erythrocytes nor formed pores in planar membranes . In contrast, at temperatures > or = 25 degrees C both processes proceeded rapidly . Pores that were opened in osmotically stabilized erythrocytes could not be closed by low temperature . Therefore, low temperature appeared to prevent the oligomerized complex from forming a pore in the membrane . These data support the hypothesis that alpha toxin oligomerizes into a membrane-bound, pre-pore complex prior to formation of a pore in a lipid bilayer.

Arch Pediatr Adolesc Med, 1997 Feb, 151(2), 142 - 5
Yield from stool testing of pediatric inpatients; Meropol SB et al.; OBJECTIVES: To quantify the yield from stool testing in pediatric inpatients and to identify criteria to test stool more deliberately without sacrificing diagnostic sensitivity . DESIGN: A retrospective review was performed of all stool cultures, ova and parasite examinations, and Clostridium {correction of Clostridia} difficile toxin assays performed on pediatric inpatients, aged 3 days to 18 years, at Thomas Jefferson University Hospital, Philadelphia, Pa, for 1 year . Medical records were reviewed for positive cases, each with 2 controls matched for age and test type . For this study, the term admission refers to the interval between the times each patient was admitted to and discharged from the hospital . Some patients had multiple stool tests sent to the laboratory during a single admission; some patients had more than 1 admission during the study period . Statistical analysis was performed using X2 analysis and the Student 2-tailed t test with a commercially available statistical software package (Statworks, Cricket Software, Philadelphia) . RESULTS: Of 250 patient admissions to the hospital for which stool was cultured, 7 cultures (2.8%) were positive . Of 63 patient admissions having ova and parasite testing, 1 (2%) had a positive result . Clostridium {correction of Clostridia} difficile toxin assays were performed on 40 patient admissions to the hospital, and 7 (18%) had a positive result . Only 18 (3.0%) of 598 of all test results reviewed were positive . Costs of negative test results totaled $26,084 . More patients (71%) with positive stool cultures than control patients (21%) had a temperature higher than or equal to 38 degrees C (X2, P < .05); however, relying on this sign missed 29% of the children with bacterial infection . A white blood cell band count of at least 0.10 was 100% sensitive and 79% specific in identifying patients with positive stool culture . There was no statistically significant relationship between stool culture results and age, total white blood cell count or white blood cell segmented neutrophil count, and no relationship between C . difficile toxin assay results and any of the above characteristics . Clostridium {correction of Clostridia} difficile was the most common pathogen identified (6 of 9) in patients developing gastrointestinal symptoms after admission; however, Salmonella enteritidis and Shigella sonnei were also significant causes (3 of 9) . CONCLUSIONS: There is low yield from stool testing of pediatric inpatients: C . difficile toxin assay has the highest yield . Clostridium {correction of Clostridia} difficile testing is most valuable for children with nosocomial gastrointestinal symptoms although other bacterial pathogens do cause nosocomial symptoms in children . More selective stool testing could help us be more efficient with our patient care resources.

Int Arch Allergy Immunol, 1997 Feb, 112(2), 115 - 24
Role of hemolytic and nonhemolytic phospholipase C from Pseudomonas aeruginosa for inflammatory mediator release from human granulocytes; Konig B et al.; BACKGROUND: Pseudomonas aeruginosa phospholipase C (PLC) is a critical component in the pathogenesis of severe P . aeruginosa infections . However, P . aeruginosa can produce a hemolytic (PLC-H) as well as a nonhemolytic (PLC-N) variant, both having a MW of about 77 kD . In the past, studies did not distinguish between both types of PLC with regard to the induction of inflammatory mediators from human cells . METHODS: We compared the ability of P . aeruginosa PLC-H and PLC-N to generate leukotriene B4 (by HPLC) and oxygen (O2-) metabolites (luminol-enhanced chemiluminescence), and to release beta-glucuronidase and histamine (fluorophotometry from human granulocytes . Therefore, human neutrophilic granulocytes (PMN; 1 x 10(7)) or human peripheral blood mononuclear cells (5 x 10(6)) were treated with purified P . aeruginosa PLC-H (up to 10 units) as well as culture supernatants (cutoff: MW > 50,000) of P . aeruginosa PAOl capable of producing both PLC-H and PLC-N, and PAOl mutant strains deficient in the production of either or both phospholipases . Controls were PLC-H from Clostridium perfringens and PLC-N from Bacillus cereus . RESULTS: PLC-H-containing P . aeruginosa culture supernatant, purified P . aeruginosa PLC-H as well as PLC-H from P . perfringens activated human leukocytes for a significant (p < 0.05) increase in inflammatory mediator release . In this regard, purified PLC-H (10 units) from P . aeruginosa activated human PMN for a significant increase in the generation of oxygen metabolites (30 +/- 5.4 x 10(3) cpm) and in leukotriene B4 (6.1 +/- 2.0 ng), in the release of beta-glucuronidase (15.8 +/- 1.1%) and of histamine (25.8 +/- 6.2%) as compared to the corresponding control values (3 +/- 1 x 10(3) cpm; 0.2 +/- 0.1 ng; 5.1 +/- 1.0%, 5.1 +/- 1.5%) . Culture supernatants containing no PLC or only PLC-N, as well as PLC-N from B . cereus, failed to activate or only slightly stimulated human granulocytes for inflammatory mediator release . CONCLUSION: The data thus provide evidence that P . aeruginosa PLC-H can be a potent inducer of inflammatory mediator release, at least in vitro . Our results therefore contribute to the understanding of the pathophysiological role of P . aeruginosa PLCs.

Appl Environ Microbiol, 1997 Feb, 63(2), 482 - 7
Monoclonal antibodies for use in detection of Bacillus and Clostridium spores; Quinlan JJ et al.; Five monoclonal antibodies against bacterial spores of Bacillus cereus T and Clostridium sporogenes PA3679 were developed . Two antibodies (B48 and B183) were selected for their reactivity with B . cereus T spores, two (C33 and C225) were selected for their reactivity with C . sporogenes spores, and one (D89) was selected for its reactivity with both B . cereus and C sporogenes spores . The isotypes of the antibodies were determined to be immunoglobulin G2a (IgG2a) (B48), IgG1 (B183), and IgM (C33, C225, and D89) . The antibodies reacted with spores of B . cereus T, Bacillus subtilis subsp . globigii, Bacillus megaterium, Bacillus stearothermophilus, C . sporogenes, Clostridium perfringens, and Desulfotomaculum nigrificans . Antibody D89 also reacted with vegetative cells of B . cereus and C . sporogenes . Analysis of B . cereus spore extracts showed that two of the antigens with which the anti-Bacillus antibodies reacted had molecular masses of 76 kDa and approximately 250 kDa . Immunocytochemical localization indicated that antigens with which B48, B183, and D89 react are on the exosporium of the B . cereus T spore . Antibody D89 reacted with the exosporium and outer cortex of C . sporogenes spores in immunocytochemical localization studies but did not react with extracts of C . sporogenes or B . cereus spores in Western blotting . Some C . sporogenes antigens were not stable during long-term storage at -20 degrees C . Antibodies B48, B183, and D89 should prove to be useful tools for developing immunological methods for the detection of bacterial spores.

Antimicrob Agents Chemother, 1997 Feb, 41(2), 484 - 7
Comparative in vitro activities of trovafloxacin (CP 99,219) and other antimicrobials against clinically significant anaerobes; Aldridge KE et al.; A total of 590 strains of clinically important anaerobes were tested to determine their susceptibility to trovafloxacin . Overall, trovafloxacin had a mode MIC of 0.25 micrograms/ml and a MIC at which 90% of the isolates were inhibited of 1 micrograms/ml and had activity comparable to that of metronidazole . Trovafloxacin was 8-, 8-, 16-, 32-, and 64-fold more active than ampicillin-sulbactam, clindamycin, ciprofloxacin, cefoxitin, and cefotetan, respectively . Of the Bacteroides fragilis group, 97% of the isolates were inhibited by trovafloxacin at 21 micrograms/ml, and trovafloxacin was more active than ciprofloxacin, cefoxitin, cefotetan, ampicillin-sulbactam, and clindamycin against Clostridium, Fusobacterium, Porphyromonas, and Prevotella strains.

J Bacteriol, 1997 Feb, 179(3), 643 - 9
Characterization of four outer membrane proteins that play a role in utilization of starch by Bacteroides thetaiotaomicron; Reeves AR et al.; Results of earlier work had suggested that utilization of polysaccharides by Bacteroides spp . did not proceed via breakdown by extracellular polysaccharide-degrading enzymes . Rather, it appeared that the polysaccharide was first bound to a putative outer membrane receptor complex and then translocated into the periplasm, where the degradative enzymes were located . In a recent article, we reported the cloning and sequencing of susC, a gene from Bacteroides thetaiotaomicron that encoded a 115-kDa outer membrane protein . SusC protein proved to be essential for utilization not only of starch but also of intermediate-sized maltooligosaccharides (maltose to maltoheptaose) . In this paper, we report the sequencing of a 7-kbp region of the B . thetaiotaomicron chromosome that lies immediately downstream of susC . We found four genes in this region (susD, susE, susF, and susG) . Transcription of these genes was maltose inducible, and the genes appeared to be part of the same operon as susC . Western blot (immunoblot) analysis using antisera raised against proteins encoded by each of the four genes showed that all four were outer membrane proteins . Protein database searches revealed that SusE had limited similarity to a glucanohydrolase from Clostridium acetobutylicum and SusG had high similarity to amylases from a variety of sources . SusD and SusF had no significant similarity to any proteins in the databases . Results of 14C-starch binding assays suggested that SusD makes a major contribution to binding . SusE and SusF also appear to contribute to binding but not to the same extent as SusD . SusG is essential for growth on starch but appears to contribute little to starch binding . Our results demonstrate that the binding of starch to the B . thetaiotaomicron surface involves at least four outer membrane proteins (SusC, SusD, SusE, and SusF), which may form a surface receptor complex . The role of SusG in binding is still unclear.

Endocrinology, 1997 Feb, 138(2), 607 - 12
Insulin promotes and cyclic adenosine 3',5'-monophosphate impairs functional insertion of insulin receptors in the plasma membrane of rat adipocytes: evidence for opposing effects of tyrosine and serine/threonine phosphorylation; Eriksson JW et al.; The aim of the present study was to elucidate events in the plasma membrane (PM) associated with the previously described effect of insulin to rapidly enhance the number of cell surface insulin binding sites in rat adipocytes . {125I}insulin was cross-linked to cell surface insulin receptors of intact cells that had been preincubated with or without insulin . Subsequently prepared PM displayed a approximately 3-fold increase in bound {125I}insulin when cells had been pretreated with 6 nM insulin for 20 min compared to membranes from control cells, and SDS-PAGE with autoradiography showed that this occurred at the insulin receptor alpha-subunit . The magnitude of the effect was similar to that found for insulin binding to intact cells that had been preincubated with insulin . In contrast, the insulin binding capacity in the PM was not affected by prior treatment of cells with insulin when assessed with the addition of {125I}insulin directly to solubilized PM; this suggests an unchanged total number of PM receptors . Thus, the enhancement of cell surface insulin binding capacity produced by insulin is not due to the translocation of receptors, but instead appears to be confined to receptors already present in the PM . The addition of phospholipase C (from Clostridium perfringens), which cleaves PM phospholipids, mimicked the effect of insulin to enhance cell surface binding in adipocytes, and this suggests a pool of cryptic PM receptors . Both the nonmetabolizable cAMP analog N6-monobutyryl cAMP (N6-mbcAMP) and the serine/threonine phosphatase inhibitor okadaic acid abolished the effect of concomitant insulin treatment to increase binding capacity . In contrast, the tyrosine phosphatase inhibitor vanadate increased insulin binding even in the presence of okadaic acid or N6-mbcAMP . The effect of N6-mbcAMP to impair cell surface insulin binding was also evident in the presence of a peptide derived from the major histocompatibility complex type I that effectively impairs receptor internalization, but the amount of PM receptors assessed by immunoblot was unaltered . Taken together, the data suggest that insulin exposure leads to the uncovering of cryptic receptors associated with the PM . It is also suggested that tyrosine phosphorylation promotes this process, whereas enhanced serine phosphorylation, e.g . produced by cAMP, impairs the functional insertion of the receptors, rendering them unable to bind insulin.

J Exp Med, 1997 Jan 20, 185(2), 281 - 92
rho, a small GTP-binding protein, is essential for Shigella invasion of epithelial cells; Watarai M et al.; Shigella, the causative agents of bacillary dysentery, are capable of invading mammalian cells that are not normally phagocytic . Uptake of bacteria by the mammalian cells is directed by bacterial factors named IpaB, IpaC, and IpaD invasins, in which Ipa invasins secreted into the bacterial environment can interact with alpha5beta1 integrin . We report here that Shigella invasion of epithelial cells requires rho activity, a ras-related GTP-binding protein . The invasive capacity of Shigella flexneri for Chinese hamister ovary (CHO) cells and other epithelial cells were greatly reduced when treated with Clostridium botulinum exoenzyme C3 transferase . Conversely, uptake of bacteria by CHO cells was promoted upon microinjection of an activated rho variant, Val14RhoA . Attachment of S . flexneri to CHO cells can elicit tyrosine phosphorylation of pp125FAK and paxillin, localized accumulation of F-actin, vinculin, and talin, and activation of protein kinase C, which were all blocked by the treatment with C3 transferase . Our results indicate that cellular signal transduction regulated by rho is essential for Shigella invasion of epithelial cells.

Presse Med, 1997 Jan 18-25, 26(1), 39 - 44
{Infections in pediatrics}; Aujard Y et al.; PROBABILITY-BASED ANTIBIOTIC THERAPY: In children, the risk of an unfavorable course of bacterial infections requires careful selection of the initial antibiotic prescription based on the disease state, bacterial epidemiology and the child's age . ACUTE COMMUNITY ACQUIRED PNEUMONIA: Before the age of 5 years, antibiotics active against Haemophilus influenzae such as amoxicillin or clavulanic acid can be given orally . In children over 5, amoxicillin or a macrolide are effective . SEVERE EAR, NOSE AND THROAT INFECTIONS: For sore throats, clinical and bacterial results of a 4-day antibiotic regimen using a second generation cephalosporin are equivalent (with better compliance) to a 10-day regimen of penicillin V . For acute middle ear infections, a combination of amoxicillin-clavulanic acid is usually prescribed as initial treatment . COMMUNITY ACQUIRED BACTERIAL MENINGITIS: The most recent consensus established the indication for cefotaxime or ceftriaxone . The increasing number of peni-R pneumococci and the major drop in the frequency of Haemophilus infections have led to new therapeutic propositions currently under investigation . ACUTE SKIN INFECTIONS: For impetigo, general antibiotics-oxacillin or a derivative-are required due to the risk of contagion . BONE AND JOINT INFECTIONS: For these urgent situations, in vitro sensitivity and antibiotic penetration into the infected tissue are the determining factors . BACTERIAL DIARRHEA: Antibiotics are not required in case of acute diaarhea with little or no fever . Antibiotics could be discussed for cholera-like diarrhea and are required in case of invasive bacterial diarrhea, shigelosis, cholera, and Clostridium difficile as well as diarrhea with fever and blood loss in infants or salmonella-induced diarrhea with signs of extradigestive complications . URINARY TRACT INFECTIONS: The choice of the antibiotic and the duration of treatment depend on the clinical presentation: lower tract infection, acute pyeloephritis, or prophylaxis . The causal germ must be identified for adapted antibiotic treatment.

J Biol Chem, 1997 Jan 17, 272(3), 1990 - 6
Increased activity of small GTP-binding protein-dependent phospholipase D during differentiation in human promyelocytic leukemic HL60 cells; Ohguchi K et al.; In response to dibutyryl cyclic AMP (dbcAMP) and all-trans retinoic acid, human promyelocytic leukemic HL60 cells differentiate into granulocyte-like cells . In cell lysate and in vitro reconstitution system, phospholipase D (PLD) activity in response to guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) was up-regulated by dbcAMP or all-trans retinoic acid treatment . In the present study, the mechanism(s) for increased PLD activity during differentiation was examined . Western blot analysis revealed that the contents of ADP-ribosylation factor, Rac2, and Cdc42Hs but not RhoA and Rac1 in the cytosolic fraction were elevated during differentiation . However, the cytosolic fraction from undifferentiated cells was almost equally potent as the cytosolic fraction from differentiated cells in the ability to stimulate membrane PLD activity . It was shown that the GTPgammaS-dependent PLD activity in membranes from differentiated cells was much higher than that in membranes from undifferentiated cells, suggesting that the increased PLD activity during differentiation was due to alterations in some membrane component(s) . Clostridium botulinum ADP-ribosyltransferase C3 and C . difficile toxin B, which are known as inhibitors of RhoA and Rho family proteins, respectively, effectively suppressed PLD activity in membranes from differentiated cells . In fact, the amount of membrane-associated RhoA was increased during differentiation . Furthermore, the extent of GTPgammaS-dependent PLD activity partially purified from membranes from differentiated cells was greater than that from membranes from undifferentiated cells in the presence of recombinant ADP-ribosylation factor 1 . The PLD (hPLD1) mRNA level was observed to be up-regulated during differentiation, as inferred by reverse transcription-polymerase chain reaction . Our results suggest the possibility that the increased Rho proteins in membranes and the changed level of PLD itself may be, at least in part, responsible for the increase in GTPgammaS-dependent PLD activity during granulocytic differentiation of HL60 cells.

J Biol Chem, 1997 Jan 17, 272(3), 1615 - 20
Role of Rho family proteins in phospholipase D activation by growth factors; Hess JA et al.; Treatment of fibroblasts with growth factors results in activation of phospholipase D (PLD) . In order to determine the role of the Rho family of small GTPases in growth factor-mediated PLD activation, we used cells transfected with wild type and mutant Rac1 . In response to epidermal growth factor (EGF), PLD activity was greatly increased in Rat1 fibroblasts expressing wild type Rac1 (wtRac1), and completely abrogated in cells expressing dominant negative N17Rac1, consistent with Rac1 mediating the action of this growth factor . In contrast, in cells treated with platelet-derived growth factor (PDGF) or phorbol ester, the wtRac1 cells showed little or no enhancement of PLD activity, and the response was not affected in the N17Rac1 cells, implying that Rac1 played a minimal role in the activation of PLD by PDGF or protein kinase C . Both growth factors produced an attenuated PLD response in cells expressing constitutively active V12Rac1, but these cells showed other changes, including altered morphology, increased basal PLD, and decreased growth factor receptor autophosphorylation . The effects of EGF and PDGF on phosphoinositide phospholipase C activity were not enhanced in cells expressing wtRac1 or inhibited in those expressing N17Rac1 . In cells expressing constitutively active V12Rac1, basal phosphoinositide phospholipase C was elevated, but there were no significant effects of EGF or PDGF . We used C3 transferase of Clostridium botulinum, which ADP-ribosylates and inactivates RhoA, to investigate the involvement of RhoA in the activation of PLD by PDGF . Cells expressing wtRac1 and N17Rac1 showed a decreased PLD in response to PDGF when treated with C3 transferase, indicating a role for RhoA . In summary, these data indicate a major role for Rac1 in the activation of PLD by EGF, but not PDGF or protein kinase C.

FEMS Microbiol Lett, 1997 Jan 15, 146(2), 199 - 204
13C-NMR study of glucose and pyruvate catabolism in four acetogenic species isolated from the human colon; Leclerc M et al.; Glucose fermentation by four acetogenic species (two Clostridium strains, one Streptococcus strain and Ruminococcus hydrogenotrophicus) isolated from the human colon was of a mixed-acid type, whereas pyruvate metabolism was characterised by homoacetogenesis . Acetate formation from {1-13C} and {2-13C}glucose was consistent with the formation of acetyl-SCoA from pyruvate generated by the Embden-Meyerhof-Parnas pathway . Labelling of lactate and ethanol demonstrated that these metabolites were formed by reduction of pyruvate and acetyl-SCoA, respectively . In contrast, the reductive pathway of acetate formation was the preferential means of re-oxidising cofactors formed during {1-13C}pyruvate catabolism.

Exp Cell Res, 1997 Jan 10, 230(1), 163 - 8
Bordetella bronchiseptica dermonecrotizing toxin, which activates a small GTP-binding protein rho, induces membrane organelle proliferation and caveolae formation; Senda T et al.; We recently demonstrated that Bordetella bronchiseptica dermonecrotizing toxin (DNT) modifies a small GTP-binding protein rho and causes assembly of actin stress fibers and focal adhesions in MC3T3-E1 cells, indicating that DNT activates the function of rho protein (Horiguchi et al., 1995, J . Cell Sci . 108, 3243-3251) . In this study, we examined by electron microscopy ultrastructural changes in DNT-treated MC3T3-E1 cells under conditions in which DNT elicited the above morphological changes . We found that DNT induced proliferation of cytoplasmic membrane organelles, such as Golgi apparatus, smooth and rough endoplasmic reticulum, and mitochondria, and formation of plasmalemmal caveolae . Therefore we examined also the effect of Clostridium botulinum C3 exoenzyme (C3), which has been known to ADP-ribosylate and inactivate rho protein, and found that C3 exoenzyme inhibited the development of membrane organelles in the MC3T3-E1 cells . These findings show that proliferation of membrane organelles and caveolae formation are controlled by the function of rho, which is the target of DNT action.

Schweiz Med Wochenschr Suppl, 1997, 89, 5S - 8S
{Clostridium difficile associated diarrhea in cephalosporin administration: experiences of the Swiss Adverse Drug Reaction Reporting System 1981-1995}; Werth B et al.; 20-25% of antibiotics-associated diarrhea cases are caused by infection with toxin-producing Clostridium difficile . Since the advent of broad-spectrum antibiotics Clostridium difficile-associated diarrhea has been observed both in ambulatory practice and as a nosocomial infection in medical and nursing institutions . Clindamycin, aminopenicillins, and cephalosporines are by far the most common triggers for this infection . We reviewed all cases of Clostridium difficile-associated diarrhea due to cephalosporines which were reported to the Swiss Drug Monitoring Center (SANZ) between 1981 and 1995 . 87 cases were reported (0.9% of 9720 spontaneous reports in this period), 69 (79%) of which were considered to be due to cephalosporines . In 74% of the cases the indication for the antibiotic treatment was an upper respiratory tract infection . 61 patients received cephalosporines by oral route and 9 patients by intravenous route . Two patients had to be hospitalized . There were no deaths . The pathogenesis, clinical picture, and therapy of Clostridium difficile-associated diarrhea is discussed . We conclude from these cases in the spontaneous reporting system of SANZ that Clostridium difficile infection due to cephalosporines is a frequent occurrence . Because the course can be severe, cephalosporines should be used restrictively.

World Health Stat Q, 1997, 50(1-2), 30 - 50
Epidemiology of foodborne diseases: a worldwide review; Todd EC; Acute foodborne disease infections and intoxications are much more of a concern to governments and the food industry today than a few decades ago . Some of the factors that have led to this include the identification of new agents that have caused life-threatening conditions; the finding that traditional agents are being associated with foods that were of no concern previously: an increasing number of large outbreaks being reported; the impact of foodborne disease on children, the aging population and the immunocompromised; migrant populations demanding their traditional foods in the countries of settlement; the ease of worldwide shipment of fresh and frozen food; and the development of new food industries, including aquaculture . However, to meaningfully monitor increases or decreases in foodborne disease requires an effective surveillance system at the local, national and international levels . To date, resources have been limited for most countries and regions to do this, and our current knowledge is based, for the most part, on passive reporting mechanisms . Laboratory isolation data and reports of notifiable diseases have some value in observing timely changes in case numbers of some enteric diseases, but they usually do not indicate the reasons for these trends . Special epidemiological studies are useful for the area covered, but it is often questionable whether they can be extrapolated to other areas or countries . Outbreak investigations tell us that a certain set of circumstances led to illness and that another outbreak may occur under similar but not necessarily identical conditions . Control programmes have often been triggered by the conclusions from investigations of specific outbreaks . Unfortunately, the agent/ food combination leading to illness in many of the reported incidents were not predicted from existing databases, and no doubt foodborne agents will continue to surprise food control agencies in the foreseeable future . Nevertheless, data from around the world do show some common elements . Salmonella is still the most important agent causing acute foodborne disease, with Salmonella enteritidis and S . typhimurium being of most concern . Foods of animal origin, particularly, meat and eggs, were most often implicated . Desserts, ice cream and confectionery items were products also mentioned, but some of these would have egg as a raw or incompletely cooked ingredient . Incidents most frequently occurred in homes or restaurants, and the main factors contributing to outbreaks were poor temperature control in preparing, cooking and storing food . Clostridium botulinum, Salmonella and VTEC are more frequently documented in industrialized than in developing countries . ETEC, EPEC, Shigella, Vibrio cholerae and parasites are the main scourges in developing countries, but it is uncertain how many cases are attributed to food, to water or to person-to-person transmission . The apparent decrease of S . aureus and C . perfringens outbreaks in industrialized countries may be related to improved temperature control in the kitchen . An increasing number of illnesses are international in scope, with contamination in a commercial product occurring in one country and affecting persons in several other countries, or tourists being infected abroad and possibly transmitting the pathogen to others at home . For Salmonella, a rapid alert and response coordination is being encouraged through Salm-Net and other international programs . However, unless such a network is worldwide, tracking clusters of illnesses is going to fall on the countries where the first cases occur, and some of these have very limited resources for investigation and control . It was heartening to see funds recently being allocated to foodborne disease surveillance and control in several industrialized countries, but the same commitment is required by the World Health Organization for the international community.

SAAS Bull Biochem Biotechnol, 1997, 10, 33 - 6
Genetic manipulation of anaerobic cellulolytic bacteria; Anderson KL; Transposon Tn1545 was introduced into the chromosome of the ruminal cellulolytic bacterium Eubacterium cellulosolvens . This was achieved by conjugal transfer of the transposon from Clostridium beijerinckii at a frequency of about 1 per 10(4) recipient cells . Transconjugants of E . cellulosolvens were resistant to both tetracycline and erythromycin . E . cellulosolvens could also serve as a donor for conjugal transfer of Tn1545 back into C . beijerinckii.

Microbiol Immunol, 1997, 41(7), 527 - 35
Lambda-toxin of Clostridium perfringens activates the precursor of epsilon-toxin by releasing its N- and C-terminal peptides; Minami J et al.; The effect of lambda-toxin, a thermolysin-like metalloprotease of Clostridium perfringens, on the inactive epsilon-prototoxin produced by the same organism was examined . When the purified epsilon-prototoxin was incubated with the purified lambda-toxin at 37 C for 2 hr, the 32.5-kDa epsilon-prototoxin was processed into a 30.5-kDa polypeptide, as determined by SDS-polyacrylamide gel electrophoresis . A mouse lethality test showed that the treatment activated the prototoxin: the 50% lethal doses (LD50) of the prototoxin with and without lambda-toxin treatment were 110 and 70,000 ng/kg of body weight, respectively . The lethal activity of the prototoxin activated by lambda-toxin was comparable to that with trypsin plus chymotrypsin and higher than that with trypsin alone: LD50 of the prototoxin treated with trypsin and trypsin plus chymotrypsin were 320 and 65 ng/kg of body weight, respectively . The epsilon-toxin gene was cloned and sequenced . Determination of the N-terminal amino acid sequence of each activated epsilon-prototoxin revealed that lambda-toxin cleaved between the 10th and 11th amino acid residues from the N-terminus of the prototoxin, while trypsin and trypsin plus chymotrypsin cleaved between the 13th and 14th amino acid residues . The molecular weight of each activated epsilon-prototoxin was also determined by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry . The C-terminus deduced from the molecular weight is located at the 23rd or 30th amino acid residue from the C-terminus of the prototoxin, suggesting that removal of not only N-terminal but also C-terminal peptide is responsible for activation of the prototoxin.

Clin Oncol (R Coll Radiol), 1997, 9(3), 184 - 5
Spontaneous gas gangrene: an unusual complication of colonic carcinoma; Hawkins C et al.; Clostridium septicum myonecrosis is an acutely painful and rapidly fatal infection occurring in the absence of trauma . Urgent surgery is essential both to maximize survival and to provide effective pain control . The majority of patients who develop this infection have an underlying malignancy and clinicians involved in the care of cancer patients should be aware of this condition . We present a case report and summarize the literature to date.

Arch Virol, 1997, 142(7), 1365 - 80
A novel synthetic reversible inhibitor of sialidase efficiently blocks secondary but not primary influenza virus infection of MDCK cells in culture; Barrere B et al.; The sodium salts of 2-difluoromethyl-phenyl-alpha-ketoside of N-acetyl-neuraminic acid (compound 1) and of 4-difluoromethyl-2-methoxy-phenyl-alpha-ketoside of N-acetylneuraminic acid (compound 2) were designed as potential mechanism-based inhibitors of sialidase . In vitro both of these compounds competitively inhibited the sialidases of Clostridium perfringens and of influenza virus A/HK/1/68 . Inhibition was irreversible with the sialidase of Clostridium perfringens whereas it was reversible with that of A/HK/1/68 . Compound 2 did not inhibit the hemagglutinin of the virus but exhibited significant anti-influenza activity when added to the medium of Madin-Darby canine kidney (MDCK) cells infected by influenza virus . In non-infected MDCK cells no inhibition of cellular sialidase was observed . Compound 2 did not block primary infection, but inhibited the release of progeny virus from infected cells . Even after 8 passages in its presence, no resistant strains were detected . Because of its high Ki (8 x 10(-5) M) compared to the low Ki (1' x 1(-10) M) of 4 guanidino-Neu 5 Ac 2en and its reversible inhibition of viral sialidase, its development as an anti-influenza agent is no longer envisaged . Nevertheless, as a mechanism-based irreversible inhibitor of the bacterial enzyme, it could at least be useful for investigating the intrinsic role of sialidase in infections caused by this strain.

Adv Exp Med Biol, 1997, 419, 349 - 53
ADP-ribosylating and glucosylating toxins as tools to study secretion in RBL cells; Prepens U et al.; The influence of different ADP-ribosylating and glucosylating cytotoxins on stimulated protein tyrosine phosphorylation and secretion in rat basophilic leukemia (RBL) cells was studied . Treatment of RBL cells with Clostridium botulinum C2 toxin, which specifically ADP-ribosylated monomeric G-actin and caused complete depolymerization of the actin cytoskeleton in intact cells, inhibited Fc epsilon RI receptor-mediated tyrosine phosphorylation of various proteins in a time- and concentration-dependent manner with maximal effects at 100 ng/ml C2I and 200 ng/ml C2I . C2 toxin (10 ng/ml C2I and 20 ng/ml C2II) increased antigen- or calcium ionophore (A23187)-stimulated {3H}serotonin release maximally by about 3 fold . Clostridium botulinum C3, which ADP-ribosylated Rho in intact RBL cells, had no effect on protein tyrosine phosphorylation and stimulated secretion . In contrast, the cytotoxic Clostridium difficile toxin B (ToxB), which glucosylated the Rho-subtype family members RhoA and Cdc42, blocked or reduced antigen- or calcium ionophore-mediated {3H}serotonin release, respectively, and decreased tyrosine phosphorylation of a 110 kDa protein . The data indicate that different actin pools control tyrosine phosphorylation and secretion in RBL cells and suggest that Rho subfamily proteins regulate secretion independently of the actin cytoskeleton.

Adv Exp Med Biol, 1997, 412, 225 - 32
Intracellular transport and processing of protein toxins produced by enteric bacteria; Sandvig K et al.; Bacterial toxins are associated with disease in humans and animals . Toxins can either be preformed in food or produced by bacteria in the intestine . There are two types of toxins: heat-labile protein toxins and heat stabile toxins . Heat labile toxins are produced by Bacillus cereus, Clostridium perfringens, Escherichia coli, and Vibrio cholerae, and heat-stabile enterotoxins consisting of relatively few amino acids are produced by Escherichia coli and acts by activation of guanylate cyclase . Similarly, heat-stabile entero-toxins are also produced by Staphylococcus aureus, a common cause of food poisoning in the United States, and Yersenia enterocolitica . Protein toxins produced by enteric bacteria can intoxicate intestinal cells and can also be taken up from the gut and reach other cells in the body . For example the Shiga-like toxins (vero-toxins) can intoxicate endothelial cells in the kidney and cause kidney failure . Intracellular transport and processing of a few of the protein toxins produced by enteric bacteria, namely Clostridium difficile toxin A and B, cholera toxin and the related heat-labile toxin produced by Escherichia coli, and Shiga toxin and Shiga-like toxins are presented.

Adv Exp Med Biol, 1997, 412, 63 - 75
Dynamics of Clostridium difficile infection . Control using diet; Ward PB et al.; Understanding the dynamics of the establishment of C . difficile within the gut is vital to effective prevention, control and therapy of disease due to this nosocomial pathogen . Factors affecting the establishment of C . difficile in the gut were investigated including the role of bacterial metabolic products (BMPs), the composition of colonic flora, diet, and properties of the infecting strain . Concentrations of 9/12 bacterial metabolic products (BMPs), both volatile and non-volatile were significantly higher in mice which eliminated oral challenge with 10(8) spores of C . difficile (E mice) than in mice harbouring the organism (H mice) . Growth of C . difficile in vitro was inhibited 10(4) fold at combinations of BMPs at concentrations found in stools of E mice but not in stools of H mice . The in situ production and concentrations of BMPs were increased by augmenting the amount of fermentable fibre in the diet . This resulted in elimination of C . difficile from 6/7 C . difficile colonized mice within 6 days of beginning a diet containing 20% fermentable fibre . Whereas mice fed diets containing 2% fermentable fibre or 20% non-fermentable fibre continued excreting the organism . Elimination of C . difficile was associated with increased concentrations of BMPs and changes in the numbers of organisms already present within the colonic flora . Properties of two microbial phenotypes (smooth (S), and rough (R)) of one strain of C . difficile were examined in vitro and the ID50s determined . The S phenotype survived, germinated and grew in media containing higher concentrations of BMPs, acquired iron when grown under iron restriction, utilized haem and bound Congo red more readily than the R phenotype . In mice fed the 2% fermentable fibre diet the ID50 for the S phenotype was 10(3) spores and 10(8) spores for the R phenotype, whereas for mice fed the 20% fermentable fibre diet it was > 10(6) spores for the S phenotype . The ability of this opportunistic pathogen to adapt to changing environmental conditions is an important factor in determining whether the organism will colonize and cause disease . Diets supplemented with fermentable fibre may be a valuable method of preventing and treating C . difficile related disease.

Eur Biophys J, 1997, 25(5-6), 417 - 22
Alteration of the quaternary structure of glutamate dehydrogenase from Clostridium symbiosum by a single mutation distant from the subunit interfaces; Dean JL et al.; X-ray crystallographic studies have previously shown that glutamate dehydrogenase from Clostridium symbiosum is a homohexamer . Mutation of the active-site aspartate-165 to histidine causes an alteration in the structural properties of the enzyme . The mutant enzyme, D165H exists predominantly as a single species of lower molecular mass than the wild-type enzyme as indicated by gel filtration and sedimentation velocity analysis . The latter technique gives an S20,w value for D165H of (6.07 +/- 0.01)S which compares with (11.08 +/- 0.01)S for the wild-type, indicative of alteration of the homohexameric quaternary structure of the native enzyme to a dimeric form, a result confirmed by sedimentation equilibrium experiments . Further support for this is provided by chemical modification by Ellman's reagent of cysteine-144 in the mutant, a residue which is buried at the dimer-dimer interface in the wild-type enzyme and is normally inaccessible to modification . The results suggest a possible structural route for communication between the active sites and subunit interfaces which may be important for relaying signals between subunits in allosteric regulation of the enzyme.

Biomed Pharmacother, 1997, 51(2), 63 - 7
Risk of diarrhoea, Clostridium difficile and cefotaxime in the elderly; Starr JM et al.; Clostridium difficile diarrhoea is an increasingly important nosocomial infection . Clostridium difficile infection is associated with antibiotic use . The elderly are at greatest risk . We reported an outbreak associated with the use of cefotaxime, a third-generation cephalosporin . We review the extent of this association, putative causal mechanisms and suggest an integrated approach to the control of C difficile infection which focuses on both limiting environmental contamination and reducing patient susceptibility . Future developments are also considered, especially the potential for vaccination.

Microbiol Immunol, 1997, 41(4), 295 - 9
Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens; Yamagishi T et al.; In order to avoid the use of experimental animals, the polymerase chain reaction (PCR) method was applied to differentiate Clostridium perfringens into five toxin types . Twenty-two out of 23 strains tested produced the toxin(s) corresponding to the toxin gene(s) identified by PCR, and vice versa . Consequently, the gene typing was consistent with conventional typing by animal tests . Twenty-five strains were identified as types different from original ones by the PCR method as well as a toxin neutralization test . These findings suggest that the PCR method, which is easy and timesaving, is applicable to identify the toxin types of C . perfringens as an alternative to animal tests, and that beta-, epsilon- and iota-toxin genes might be lost by long-term preservation . The reasons why the strains lost the genes are discussed.

Kansenshogaku Zasshi, 1997 Jan, 71(1), 83 - 6
{A case of pseudomembranous enterocolitis which diagnosis was delayed in the outpatient clinic}; Watanabe H et al.; A 71-year-old male visited our hospital because of diarrhea . At first we suspected infectious colitis and levofloxacin (300 mg/day) was administered for four days . But the diarrhea continued, so performed a barium enema after about 3 weeks from onset . Round polyposis from the rectum to the sigmoid colon were found . Colonoscopy was suggested to the patient, but was rejected . However diarrhea continued and fever appeared . Ceftriaxone (1 g/day) and sparfloxacin (200 mg/day) were administered, but the symptoms increased . He was admitted about 6 weeks from onset, and the colonoscopy showed multiple round yellow-white pseudomembranes . Pseudomembranous enterocolitis diagnosed because Clostridium difficile and its toxin were positive in the feces . After oral administration of vancomycin (1.5 g/day) was started, the symptoms alleviated rapidly and disappearance of pseudomembrane was confirmed by colonoscopy.

Life Sci, 1997, 60(13-14), 1093 - 100
Regulation of phospholipase C and D activities by small molecular weight G proteins and muscarinic receptors; Schmidt M et al.; The role of small molecular weight guanine nucleotide-binding proteins (G proteins) of the Rho family in muscarinic acetylcholine receptor (mAChR) signaling to phospholipase C (PLC) and phospholipase D (PLD) was studied in human embryonic kidney (HEK) cells, stably expressing the human m3 receptor subtype . Evidence for the involvement of Rho proteins in m3 mAChR signaling to both phospholipases is based on findings obtained with Clostridium (C.) difficile toxin B and C . botulinum C3 exoenzyme, both of which specifically, although by different mechanisms, inactivate Rho family G proteins . Toxin B potently inhibited both the mAChR-stimulated PLC and PLD activities in intact cells as well as the stimulation of both phospholipases by the stable GTP analog GTPgammaS in permeabilized cells, the latter effect being mimicked by C3 exoenzyme . In contrast, PLC and PLD activities, measured in the presence of exogenous phosphatidylinositol 4,5-bisphosphate {PtdIns(4,5)P2}, a substrate and cofactor for PLC and PLD, respectively, were not altered . These data suggested that the Rho-inactivating toxins inhibit stimulation of PLC and PLD by reducing the cellular level of PtdIns(4,5)P2, which was indeed found with both toxin B and C3 exoenzyme . In agreement with a crucial role of cellular PtdIns(4,5)P2 supply for PLC signaling, we observed that short-term agonist (carbachol) treatment of HEK cells caused a long-lasting increase in PtdIns(4,5)P2 level, accompanied by a potentiation of receptor- and G protein-stimulated inositol phosphate formation . Finally, studies with tyrosine kinase and tyrosine phosphatase inhibitors strongly suggest that PtdIns(4,5)P2 synthesis and mAChR-stimulated PLD activity in HEK cells apparently also involve a tyrosine phosphorylation-dependent mechanism(s) . Thus, m3 mAChR signaling to PLC and PLD in HEK cells requires the concerted action of various intracellular components, most notably the complex regulation of PtdIns(4,5)P2 synthesis.

J Infect, 1997 Jan, 34(1), 49 - 54
Outbreaks of infectious intestinal disease in residential institutions in England and Wales 1992-1994; Ryan MJ et al.; Data from the surveillance scheme of all general outbreaks of infectious intestinal disease in England and Wales reported to or otherwise identified, by the Public Health Laboratory Service Communicable Disease Surveillance Centre (CDSC) in 1992 and 1994 were used to describe the epidemiology of outbreaks of infectious intestinal disease in residential institutions . Outbreaks in residential institutions accounted for 22% (282/1275) of all outbreaks with most, 95% (268/282), occurring in homes for the elderly . The commonest pathogens in these 282 outbreaks were small round structured viruses 48% (132), salmonellas 17% (49) . Clostridium perfringens 8% (23), rotavirus 5% (15) and Shigella sonnei 2% (6) . The mode of transmission was described as mainly person to person in 71% (200 outbreaks) and mainly foodborne in 21% (58 outbreaks) . The mean duration of outbreaks was 9 days . Duration of outbreaks varied with both the mode of transmission and the pathogen involved . The mean attack rate was 37% . Illness was reported in 5872 people . One or more individuals were admitted to hospital in 22% of outbreaks . Twenty-six deaths were reported, of which 18 were attributed to salmonellosis . Outbreaks in residential institutions are common . Attack rates are high and outbreaks are often prolonged, with high morbidity and mortality . There is a need for effective infection control policies which include appropriate training of staff, simple surveillance systems and readily available expert advice to ensure outbreaks are rapidly controlled.

J Appl Microbiol, 1997 Jan, 82(1), 128 - 36
Combining heat treatment and subsequent incubation temperature to prevent growth from spores of non-proteolytic Clostridium botulinum; Stringer SC et al.; Refrigerated processed foods of extended durability rely on a mild heat treatment combined with refrigerated storage to ensure microbiological safety and quality . The principal microbiological safety risk in foods of this type is non-proteolytic Clostridium botulinum . In this article the combined effect of mild heat treatment and refrigerated storage on the time to growth and probability of growth from spores of non-proteolytic Cl . botulinum is described . Spores of non-proteolytic Cl . botulinum (two strains each of type B, E and F) were heated at 90 degrees C for between 0 and 60 min and subsequently incubated at 5 degrees, 10 degrees or 30 degrees C in PYGS broth in the presence or absence of lysozyme . The number of spores that resulted in turbidity depended on the combination of heat treatment, incubation time and incubation temperature they received . Heating at 90 degrees C for 1 or more min ensured a 10(6) reduction when spores were subsequently incubated at 5 degrees C for up to 23 weeks . Heating at 90 degrees C for 60 min ensured a 10(6) reduction over 23 weeks when subsequent incubation was at 10 degrees C in the presence of added lysozyme . The same treatment did not reduce the spore population by 10(6) when subsequent incubation was at 30 degrees C.

J Appl Microbiol, 1997 Jan, 82(1), 48 - 56
The effect of temperature on the germination of single spores of Clostridium botulinum 62A; Billon CM et al.; Phase-contrast microscopy coupled with image analysis has been used to study the germination of single spores of Clostridium botulinum and to investigate the variation of germination lag of individual spores in a population (biovariability) . The experiment was repeated at five different temperatures between 20 degrees C and 37 degrees C to look at the effect of temperature on the biovaribility of the spore germination . Data analysis shows that the germination lag distribution is skewed, with a tail, and that its shape is affected by the temperature . The origin of this biovariability is not exactly known, but could be due to a distribution of characteristics (e.g . permeabilities) or molecules (e.g . lytic enzymes) in the spore population . The method developed in this study will help us to describe and better understand the kinetics of spore germination and how this is influence by different environmental factors such as temperature and other factors that influence germination.

Arch Pharm (Weinheim), 1997 Jan-Feb, 330(1-2), 17 - 20
Carbohydrate natural products as a scaffolding for the preparation of potential neuraminidase inhibitors; Maione AM et al.; Compound 10b, 6-acetamido-6,8-dideoxy-D-erythro-beta-D- galacto-octopyranosyl-1-oxyacetic acid sodium salt, was synthesised by hydrazinolysis of Lincomycin, acetylation of methylthiolincosaminide (MTL) 9a, and by subsequent glycosylation of acetate 9b with methyl glycolate under mild conditions (NIS/TfOH) . The methyl ester 10a was hydrolysed by treatment with Amberlite Ira-4OO (OH-) resin and aqueous sodium hydroxide, followed by neutralisation with Dowex-50 W x 8 (H+) resin and lyophilisation to give 10b . This carboxylate may represent the first derivative in a novel series of sialidase inhibitors utilising carbohydrate natural products . The phosphonate 11c, prepared under the same experimental conditions with dibenzyl(hydroxymethyl)phosphonate as acceptor, also displays an inhibitory activity towards Clostridium perfringens sialidase (Ki in mM range as with Neu5Ac).

Immunol Lett, 1997 Jan, 55(1), 19 - 26
Hepatobiliary transport of IgA in the golden Syrian hamster (Mesocricetus auratus); Vaerman JP et al.; Do hamsters, like rats, rabbits and mice, possess an hepatocyte 'IgA pump' whereby circulating plasma polymeric IgA (pIgA) is actively transported into bile, against a concentration gradient, via the polymeric Ig receptor or secretory component (SC)? Precipitating antisera, raised against rat Igs and serum proteins, and crossreacting with their hamster homologues, detected hamster SC by immunoelectrophoresis in bile, but not serum . Gel filtration of hamster bile indicated that free SC eluted between IgG and albumin, as for other mammals . Hamster bile IgA was pIgA, and was true secretory IgA (SIgA) by its reaction with anti-SC antiserum and by SDS-PAGE with reduction . Hamster serum IgA comprised both pIgA and IgA monomers . Mean bile-to-serum concentration ratios (B/S) for IgA, IgG, transferrin and albumin, measured by radial immunodiffusion, were 2.65, 0.019, 0.024, and 0.016, respectively, demonstrating strongly selective enrichment of bile in IgA . Human 125I-labelled dimeric IgA was injected into the circulation of five hamsters with cannulated bile ducts; 20% of the {125I}IgA (> 95% precipitable by trichloroacetic acid) was recovered in bile within 5 h, a figure close to that for mice, but smaller than that for rats and rabbits . The data suggest that bile significantly contributes to hamster intestinal SIgA, as shown for rats, rabbits and mice . This could be relevant to studies where hamsters are used as an experimental model for infection by the human intestinal pathogen, Clostridium difficile.

Berl Munch Tierarztl Wochenschr, 1997 Jan, 110(1), 1 - 4
{Airborne microorganisms in animal stables . 1 . Anaerobic airborne bacteria in a calf stable with special regard to Clostridium perfringens}; Chai T et al.; The total number of airborne anaerobic bacteria with special regard to Clostridium perfringens (C . perfringens) as well as the total number of airborne aerobic bacteria was estimated indoor and outdoor of a calf stable . 65 indoor samples and 63 outdoor samples were collected by using a 6 stage Andersen sampler . The total number of airborne anaerobic bacteria of the indoor samples ranged from 3,216 to 24,222 CFU/m3 (Colony Forming Units) with a part of 4 to 1179 CFU/m3 of C . perfringens . 734 to 1588 CFU/m3 of airborne anaerobic bacteria were found in the outdoor samples . The number of C . perfringens CFU varied between 2 and 15 CFU/m3 . The total number of airborne aerobic bacteria indoor ranged from 9,166 to 53,932 CFU/m3, outdoor from 2,443 to 21,205 CFU/m3 . Furthermore the results suggested that C . perfringens is suitable for indicating the bacterial contamination of air by faeces of animals.

Microbiol Immunol, 1997, 41(2), 161 - 3
Nitrogen-gas bubbling during the cultivation of Clostridium tetani produces a higher yield of tetanus toxin for the preparation of its toxoid; De Luca MM et al.; We investigated the effect of exposing cultures of Clostridium tetani to nitrogen (N2) gas on the recovery of tetanus toxin to be processed for the preparation of its toxoid . N2 was bubbled through nine 10-liter cultures during the growth of the bacteria, while nine parallel control incubations were maintained without bubbling . We found that treatment of the C . tetani anaerobes with an inert gas in this manner during cultivation produced a highly significant increase in the yield of tetanus toxin from them in comparison with the standard procedure.

J Comp Pathol, 1997 Jan, 116(1), 63 - 71
Effects of the intravenous administration of Clostridium perfringens type D epsilon toxin on young goats and lambs; Uzal FA et al.; Young goats (n = 18) and lambs (n = 10) were compared in respect of the effects of Clostridium perfringens type D epsilon toxin . Toxin produced neurological signs within 0.5-3 h of intravenous injection in (1) all of six kids given doses of 250, 185 or 120 mouse lethal doses 50% (MLD50)/kg body weight, (2) two of the three kids given 60 MLD50/kg, and (3) all of five lambs given 250 or 120 MLD50/kg . Six kids and three lambs given 45, 30 or 15 MLD50/kg, one lamb given 60 MLD50/kg, and three kids and one lamb given saline (controls) all remained clinically normal . Gross post-mortem changes were observed only in the kids and lambs that showed clinical signs . In the kids these changes consisted of severe acute interstitial and alveolar oedema of the lungs . However, only two out of five lambs that presented clinical signs showed pulmonary oedema . No histological changes were observed in the brain of any of the kids inoculated with epsilon toxin . In the brain of four out of the five lambs given doses of 120 or 250 MLD50/kg, there were histological lesions consisting of perivascular proteinaceous oedema and haemorrhages . These results show that kids and lambs are equally susceptible to the intravenous injection of epsilon toxin, but that they differ in the histological response of the central nervous system to the toxin.

Glycoconj J, 1997 Jan, 14(1), 67 - 73
Arthrobacter ureafaciens sialidase isoenzymes, L, M1 and M2, cleave fucosyl GM1; Iwamori M et al.; Among bacterial, fungal and viral sialidases, the sialidase from Arthrobacter ureafaciens has the unique property of cleaving sialic acids linked to the internal galactose of gangliotetraose . In this study, we examined the ability to cleave the internal sialic acids of GM1 and fucosyl GM1 of sialidases from several bacterial and fungal origins, including Clostridium perfringens and Vibrio cholerae . We found that A . ureafaciens sialidase could liberate the sialic acid of GM1 at the highest rate, and was the only enzyme which could cleave fucosyl GM1 among the sialidases examined . The affinity-purified sialidase derived from the culture medium of A . ureafaciens was comprised of four isoenzymes with different molecular weights and isoelectric points, the isoenzymes that cleaved fucosyl GM1 being L (88 kDa, pl 5.0), M1 (66 kDa, pl 6.2) and M2 (66 kDa, pl 5.5), but not S (52 kDa, pl 6.2) which showed the highest specific activity toward colominic acid among the four isoenzymes.

Nippon Yakurigaku Zasshi, 1997 Jan, 109(1), 13 - 7
{Analysis of the cellular functions of the small GTP-binding protein rho p21 with Clostridium botulinum C3 exoenzyme}; Saito Y; rho p21 is a member of the ras superfamily of small GTPases . Clostridium botulinum C3 exoenzyme ADP-ribosylates rho p21 at the Asp41 residue located at an effector domain and inhibits its biological activity by interfering with the interaction with its downstream effectors . The amount of rho p21 in cells or tissues is determined by the in vitro ADP-ribosylation reaction with C3 exoenzyme and 32P NAD . The studies using C3 exoenzyme have revealed that rho p21 is involved in the regulation of stress fiber formation, cell adhesion, contractile ring formation during cytokinesis and serum response factor-mediated activation of immediate early genes . C3 exoenzyme is a valuable tool for elucidating the unidentified function of rho p21 because the exoenzyme specifically inhibits rho p21-mediated signal transduction pathways . A Glu173 substitution mutant of the C3 exoenzyme lacking ADP-ribosyltransferase activity is useful for a control experiment.

J Clin Pathol, 1997 Jan, 50(1), 70 - 1
Involvement of the appendix in pseudomembranous colitis; Coyne JD et al.; Pseudomembranous colitis (PMC) is an inflammatory disorder usually limited to the large intestine and is the consequence of antibiotic associated Clostridium difficile overgrowth with production of its toxin . It has a characteristic gross and microscopic appearance . PMC-like changes, usually associated with peri-operative hypotension and with more extensive gastrointestinal tract involvement, have also been described . In neither clinical setting has pseudomembranous appendicitis been recorded . A case of pseudomembranous appendicitis in a 76 year old woman is described.

FEMS Microbiol Lett, 1997 Jan 1, 146(1), 155 - 9
Collagenase gene (colA) is located in the 3'-flanking region of the perfringolysin O (pfoA) locus in Clostridium perfringens; Ohtani K et al.; The 3'-flanking region of the beta-galactosidase gene (pbg), which is located downstream of the perfringolysin O gene (pfoA), and the 5'-flanking region of the collagenase gene (colA) of Clostridium perfringens strains NCTC8237 and 13, respectively, were analyzed . Southern analysis revealed that the colA gene is located 6.5 kb downstream of the pbg gene in the chromosome of C . perfringens . Sequence analysis showed that between the pbg and colA genes were the arcABDC and ahrC genes, whose putative products were quite similar to enzymes of the arginine deiminase pathway of Pseudomonas aeruginosa and the arginine repressor/activator of Bacillus subtilis, respectively . It is concluded that the genomic structure of the pfoA-colA region consists of pfoR-pfoA-ORF54-pbg-arcABDC-ahrC-colA.

Protein Eng, 1997 Jan, 10(1), 69 - 75
Synthetic mutants of Clostridium pasteurianum ferredoxin: open iron sites and testing carboxylate coordination; Feinberg BA et al.; The entire polypeptide chains for two new Clostridium pasteurianum ferredoxin (Fd) mutants were prepared with the following site-specific substitutions: Cys11Asp and Cys11 alpha-aminobutyric acid (Cys11 alpha-Aba), the latter being a non-naturally occurring amino acid . Standard t-Boc procedures were used for the synthesis and the peptides . The two apoproteins were reconstituted to the 2{4Fe-4S} holoprotein and their spectroscopic, redox and thermal properties were compared with those of native C.pasteurianum Fds . The fully reconstituted Cys11Asp and Cys11 alpha-Aba mutants were initially found to have both clusters intact, i.e . they were 2{4Fe-4S} ferredoxins . The unconventional ligands of Asp and alpha-Aba led to holo-Fds that were not very stable and easily released an iron to form the {3Fe-4S} cluster, presumably through oxidation . The Cys11 alpha-Aba mutant was somewhat more thermally stable than Cys11Asp . In contrast, while both mutants were less stable than the native protein upon exposure to oxygen, the Cys11 alpha-Aba mutant was less stable than Cys11Asp . The Cys11Gly mutant was also prepared, but all attempts, despite repeated and varied experimental conditions, at reconstitution to the Cys11Gly holo 2{4Fe-4S} Fd were unsuccessful, probably because a Gly-Gly sequence is known to break structure . This work, when compared with molecular biological site-specific mutagenesis, shows some of the advantages of chemical/in vitro reconstitution: certain mutants which cannot be detected as holoproteins by site-specific mutagenesis can be formed after all in vitro . Nonetheless, it seems apparent that altering any of the Cys coordination sites of the Fd clusters results in fundamentally more unstable ferredoxins.

Am J Physiol, 1997 Jan, 272(1 Pt 1), L38 - 43
Glucosylation of small GTP-binding Rho proteins disrupts endothelial barrier function; Hippenstiel S et al.; The endothelial cytoskeleton is important for the regulation of endothelial barrier function . Small GTP-binding Rho proteins play a central role in the organization of the microfilament system . Clostridium difficile toxin B (TcdB) inactivates Rho proteins by glucosylation at Thr-37 . We used TcdB as a probe to study the role of Rho proteins in the regulation of endothelial barrier function . TcdB time (50-170 min) and dose (10-100 ng/ml) dependently increased the hydraulic conductivity of cultured porcine pulmonary artery endothelial cell monolayers approximately 10-fold . Simultaneously, the albumin reflection coefficient decreased substantially from 0.8 to 0.15 . Before endothelial hyperpermeability, TcdB reduced F-actin content in a dose-dependent manner, whereas G-actin content remained unchanged . Finally, we proved that TcdB caused dose (5-100 ng/ml)- and time-dependent glucosylation of Rho proteins in endothelial cells . Phalloidin, which stabilizes filamentous actin, prevented the effect of TcdB on endothelial permeability . In contrast to thrombin-, hydrogen peroxide-, or Escherichia coli hemolysin-induced hyperpermeability, the elevation of cyclic nucleotides did not block TcdB-related permeability . The data demonstrate a central role of small GTP-binding Rho proteins for the control of endothelial barrier function.

Poult Sci, 1997 Jan, 76(1), 24 - 8
The effect of added complex carbohydrates or added dietary fiber on necrotic enteritis lesions in broiler chickens; Branton SL et al.; Two trials utilizing two corn diets and four wheat diets were conducted . In Trial 2, all chicks were crop-infused at 9 d of age with Eimeria acervulina . In both trials, a broth culture of Clostridium perfringens was mixed with the diets for 3 consecutive d . Necrotic enteritis lesion scores were lowest in chickens consuming the corn diet with no C . perfringens and highest in chickens fed the wheat diets with C . perfringens . Chickens consuming a wheat diet with no added complex carbohydrates or added fiber exhibited the highest lesion score . Chickens on wheat diets with 4% new, ground, pine shavings had intestinal lesion scores intermediate to those of chickens that consumed the wheat or corn diets . Chickens consuming corn diets yielded the lowest lesion scores . Chickens provided diets containing either guar gum or pectin were not fully consumed and thus probably reduced the number of challenge organisms ingested.

J Vet Med Sci, 1997 Jan, 59(1), 5 - 8
Mitogenic activity of Clostridium perfringens enterotoxin in human peripheral lymphocytes; Nagata K et al.; Clostridium perfringens enterotoxin (CPE) was found to possess interferon (IFN)-producing and mitogenic activities to human peripheral blood mononuclear cells . Both activities were demonstrated only in the T lymphocyte-rich fraction from healthy volunteers . The IFN produced appeared to be gamma-type since the activity of the IFN was neutralized by antiserum against human IFN-gamma . With formalin-treated CPE, the IFN-producing and mitogenic activities were weakly found . Similar findings were also obtained in the mouse lethality and cytotoxicity to Vero (African green monkey) cells, suggesting that the biological activities of the CPE molecule may be existing on the similar (or the same) sites . From these findings, human peripheral T cells may be one of useful reagents to study the mode of action of CPE since CPE was found to be a T cell mitogen which is supposed to be a superantigen.

J Gen Intern Med, 1997 Jan, 12(1), 57 - 62
Predicting Clostridium difficile stool cytotoxin results in hospitalized patients with diarrhea; Katz DA et al.; OBJECTIVE: To validate a model for the prediction of Clostridium difficile cytotoxin assay results, and to identify a subgroup of patients with a very low likelihood of C . difficile-associated disease in whom the yield of routine cytotoxin testing is low . DESIGN: Prospective cohort study . Relevant clinical symptoms, signs, and antibiotic exposure were recorded before reporting of assay results . Each predictor was assigned a score based on regression coefficients, and patients were stratified according to their total score . SETTING: Two urban, tertiary care, university hospitals . PATIENTS: A total of 609 consecutive adult inpatients who received testing for C . difficile cytotoxin during a 3-month period in 1994 . MEASUREMENTS AND MAIN RESULTS: The prevalence of positive cytotoxin assays was 8% in the validation set, compared with 14% in the derivation set . Defining patients without both prior antibiotic use and at least one symptom predictor (significant diarrhea or abdominal pain) as a low-risk subgroup, the misclassification rate was 2.8% (5/177) for assay results; of the five misclassified cases patients, only one was judged to have C . difficile-associated disease . Use of this rule to identify low-risk patients could have potentially averted 29% of all cytotoxin assays . CONCLUSIONS: Patients without a history of antibiotic use and either significant diarrhea or abdominal pain are unlikely to have positive C . difficile cytotoxin assays and may not require cytotoxin testing.

Microb Pathog, 1997 Jan, 22(1), 31 - 8
Effect of hemorrhagic toxin produced by Clostridium sporogenes on rabbit ligated intestinal loop; Hara-Kudo Y et al.; In order to characterize the toxicity of the hemorrhagic toxin of Clostridium sporogenes isolated from the rabbit with antibiotic-associated hemorrhagic diarrhoea, we studied the toxin productivity of C . sporogenes cultured in various media and the toxic effect of the partially purified preparation of the culture supernatant in rabbit intestinal loops . The hemorrhagic activity, which was determined by rabbit skin test for assessment of the toxin production, of culture supernatant of C . sporogenes reached maximum in the early stationary phase of the bacterial growth in GAM or Trypticase soy broth that contained rich glucose, ammonia and peptide . The partially purified toxin prepared by hydroxyapatite, phenyl Toyopearl and Superdex 200 columns caused marked hemorrhage in rabbit ligated intestinal loops . Histological examination of the intestinal loops injected with the toxin revealed noticeable pathological alterations which seemed to be a characteristic of this toxin . Marked hemorrhage could be observed in the mucosa, submucosa, muscular layer and subserous spaces . There were various degrees of infiltration of inflammatory cells such as polymorphonuclear leukocytes and granulated leukocytes throughout the intestinal wall, but it was not associated with any necrotic changes of the tissue . These findings indicate that hemorrhagic toxin produced by C . sporogenes induces vascular changes in the intestine without any direct effects on the parenchymal cells.

J Anim Sci, 1997 Jan, 75(1), 19 - 25
Clostridial vaccination efficacy on stimulating and maintaining an immune response in beef cows and calves; Troxel TR et al.; Experiments were conducted to determine the efficacy in stimulating and maintaining an immune response in the presence of maternal antibodies, compare the extent of the anamnestic responses to revaccination, and compare the maternal antibody response of 2- or 5-mL clostridial vaccination . In Exp . 1, 118 nursing calves were randomly assigned to receive a 2-mL (Alpha-7, A7) or a 5-mL clostridial vaccine (Ultrabac 7; UB7) at 50.4 +/- 15.30 (X +/- SD) d of age (d 0 = date of calving) . Calves were revaccinated with the same treatment on d 170 . Blood samples were collected from 10 calves of each treatment group on d 50, 170, and 191 to determine antitoxin units for Clostridium perfringens type C (PC) and D (PD) and agglutination titers for Cl . chauvoei (CC) . The A7-treated calves tended (P < .10) to have higher PC units on d 170, an increased rate of change in PD units from d 170 to d 191 (P < .06), and a tendency (P < .10) for enhanced CC titers on d 191 compared with UB7-treated calves . In Exp . 2, 109 pregnant cows and 83 pregnant heifers were randomly assigned within a 2 x 2 x 2 factorial design . The main effects were dam age (cow or heifer), dam treatment (A7 or UB7), and calf treatment (A7 or UB7) . Dams were vaccinated with A7 or UB7 d 124 prepartum (d -124) and d 53 after birth . At d 53.4 +/- 12.88, calves were vaccinated with A7 or UB7 (d 53) . Calves were revaccinated with the same treatment on d 173 . Blood samples were collected from 10 dams per treatment group on d -124, 53, and 173 and from their calves on d 53, 173, and 194 . Cows had higher antitoxin levels for PC (P < .01) and PD (P < .05) than heifers . The A7-treated dams had higher (P < .01) PD units on d 53 and d 173 and CC on d 173 than did UB7-treated dams . Calves from A7-treated cows had higher (P < .03) PD units on d 53 than calves from UB7-treated cows . The A7-treated calves had higher titers for CC (P < .01) on d 173 than did UB7-treated calves, and this enhanced level seemed to continue to d 194 (P < .08) . In conclusion, titers for clostridial diseases in 50- to 53-d-old calves can be enhanced if dams are vaccinated approximately 4 mo before calving, and 120 d between clostridial vaccinations seems to be too long for adequate protection.

Lett Appl Microbiol, 1997 Jan, 24(1), 19 - 22
New media for detection and counting of Clostridia in foods; Dromigny E et al.; The growth of pure cultures of Clostridium perfringens (ATCC 12922) and Cl . sporogenes (PA 3679) in five non-selective media, fluid thioglycollate medium (FTM), rapid perfringens medium (RPM), Columbia broth Malthus (CBM), reinforced clostridial medium (RCM) and lactose sulphite (LS), was monitored using conductance measurements with a Malthus analyser . Only FTM and CBM gave useful results . The correlation of log10 plate counts on blood agar of the pure strain of Cl . sporogenes with detection times in FTM was highly significant (r = 0.96, n = 73), and with detection times in CBM less so (r = -0.909, n = 33) . The correlation of log10 counts on tryptose sulphite neomycin medium (TSN) of wild strains of Cl . sporogenes and Cl . perfringens with detection times with FTM in meat was also highly significant (r = 0.933, n = 54).

Heart Lung, 1997 Jan-Feb, 26(1), 83 - 4
Non-Clostridium difficile nosocomial diarrhea in the intensive care unit; Caines C et al.; It is assumed that most cases of nosocomial diarrhea are due to Clostridium difficile because of the widespread use of broad-spectrum antibiotic agents . Enteral tube feedings are another important cause of hospital-acquired diarrhea, especially in intensive care units (ICUs) . We report the results of a recent survey of patients in the ICU with nosocomial diarrhea and describe an illustrative case . We conclude on the basis of this and a previous larger study that C . difficile diarrhea is very uncommon in enterally fed patients in the ICU; nosocomial diarrhea in the ICU is most commonly caused by enteral tube feedings.

J Fam Pract, 1997 Jan, 44(1), 97 - 100
Clostridium difficile colitis presenting as an acute abdomen: case report and review of the literature; Muehlhausen VL et al.; Pseudomembranous colitis associated with Clostridium difficile rarely manifests as an acute abdomen and even more rarely as an acute abdomen without abnormal radiologic studies . The following is a case report of a 52-year-old white man who had an acute abdomen without abnormal radiologic studies, and was given a final diagnosis of C difficile colitis . Surgery was averted only by the ability to do an expeditious flexible sigmoidoscopy with the visualization of pseudomembranes . Diagnosis was later confirmed by a positive toxin assay and culture of C difficile . Treatment for C difficile colitis is usually medical, with oral vancomycin the preferred agent . Surgery may be needed when there is an acute abdomen with other systemic signs (fever or leukocytosis) or abnormal radiologic studies.

Neurology, 1997 Jan, 48(1), 265 - 7
Isolated meningeal vasculopathy associated with Clostridium septicum infection; Crowley RS et al.; A 28-year-old patient with acute lymphoblastic leukemia and neutropenia developed necrotizing enterocolitis and Clostridium septicum bacteremia, followed by rhabdomyolysis, skin rash, and acute neurologic changes . Numerous cortical leptomeningeal enhancements were present on head MRI . Meningeal and brain biopsy showed segmental, full-thickness lysis of smooth muscle cells of medium-sized meningeal vessels with overall preservation of the structure of the vessel wall.

Arch Microbiol, 1997 Jan, 167(1), 54 - 60
Clostridium vincentii sp . nov., a new obligately anaerobic, saccharolytic, psychrophilic bacterium isolated from low-salinity pond sediment of the McMurdo Ice Shelf, Antarctica; Mountfort DO et al.; A gram-positive, motile, rod-shaped, strictly anaerobic bacterium was isolated from an enrichment initiated with sediment taken from below the cyanobacterial mat of a low-salinity pond on the McMurdo Ice Shelf, Antarctica . The organism grew optimally at 12 degrees C, at pH 6.5, and at an NaCl concentration of< 0.5% (w/v) . It survived freeze-thawing at low salt concentrations,but not exposure to temperatures over 25 degrees C for more than 20 h or short-term exposure to temperatures> 50 degrees C . Out of a variety of polysaccharides tested as growth substrates, only xylan supported growth . The organism also grew on a variety of mono- and disaccharides including the cyanobacterial cell wall constituent, N-acetyl glucosamine . Fermentation products on a mol product per 100 mol of hexose monomer fermented basis were: acetate, 72; formate, 72; butyrate, 55; hydrogen, 114; and CO2, 100 . Not detectable in the culture medium(< 2 mol per 100 mol of monomer) were lactate, propionate, ethanol,n-propanol, n-butanol, and succinate . The G+C content of the DNA from the bacterium was 33 mol%, and a phylogenetic analysis indicated that it grouped closely with members of the RNA-DNA homology group 1 of the genus Clostridium . It differed from other species of this genus with regard to growth temperature optimum, substrate range, and fermentation pattern, and is therefore designated as a new species of Clostridium for which the name Clostridium vincentii is proposed . The type strain is lac-1 (DSM 10228).

Int J Syst Bacteriol, 1997 Jan, 47(1), 164 - 70
Clostridium pascui sp . nov., a new glutamate-fermenting sporeformer from a pasture in Pakistan; Wilde E et al.; Four strains of an obligately anaerobic spore-forming bacterium were isolated from soil samples from a donkey pasture in Pakistan . Comparative 16S rRNA sequence analysis demonstrated that the strains are members of phylogenetic cluster I of the genus Clostridium (Collins et al . 1994) . The strains are mesophilic, nonsaccharolytic, and nonproteolytic, utilize glutamate and histidine, and produce indole . Acetate, butyrate, ethanol, hydrogen, and carbon dioxide are the products of fermentation . Although the strains phenotypically resemble the classical glutamate-fermenting clostridia, such as Clostridium cochlearium, Clostridium tetanomorphum, Clostridium tetani, and especially Clostridium malenominatum, they differ from these organisms in sugar utilization, cellular fatty acid composition, and cellular protein pattern and by a 16S rRNA sequence divergence value of approximately 4 to 8% . Phylogenetically, the strains are more closely related to Clostridium estertheticum (sequence divergence, approximately 5%) and Clostridium subterminale (sequence divergence, approximately 5%) but are phenotypically readily distinguished from these species . On the basis of phenotypic and genotypic criteria, we conclude that the four strains are members of a new species of the genus Clostridium, for which the name Clostridium pascui is proposed . The type strain is strain Cm19 (= DSM 10365).

J Bacteriol, 1997 Jan, 179(1), 46 - 52
The processive endocellulase CelF, a major component of the Clostridium cellulolyticum cellulosome: purification and characterization of the recombinant form; Reverbel-Leroy C et al.; The recombinant form of the cellulase CelF of Clostridium cellulolyticum, tagged by a C-terminal histine tail, was overproduced in Escherichia coli . The fusion protein was purified by affinity chromatography on a Ni-nitrilotriacetic acid column . The intact form of CelF (Mr, 79,000) was rapidly degraded at the C terminus, giving a shorter stable form, called truncated CelF (Mr, 71,000) . Both the entire and the truncated purified forms degraded amorphous cellulose (kcat = 42 and 30 min(-1), respectively) and microcrystalline cellulose (kcat = 13 and 10 min(-1), respectively) . The high ratio of soluble reducing ends to insoluble reducing ends released by truncated CelF from amorphous cellulose showed that CelF is a processive enzyme . Nevertheless, the diversity of the cellodextrins released by truncated CelF from phosphoric acid-swollen cellulose at the beginning of the reaction indicated that the enzyme might randomly hydrolyze beta-1,4 bonds . This hypothesis was supported by viscosimetric measurements and by the finding that CelF and the endoglucanase CelA are able to degrade some of the same cellulose sites . CelF was therefore called a processive endocellulase . The results of immunoblotting analysis showed that CelF was associated with the cellulosome of C . cellulolyticum . It was identified as one of the three major components of cellulosomes . The ability of the entire form of CelF to interact with CipC, the cellulosome integrating protein, or mini-CipC1, a recombinant truncated form of CipC, was monitored by interaction Western blotting (immunoblotting) and by binding assays using a BIAcore biosensor-based analytical system.

Antimicrob Agents Chemother, 1997 Jan, 41(1), 101 - 6
In vitro activity of BAY 12-8039, a new fluoroquinolone; Woodcock JM et al.; The in vitro activity of BAY 12-8039, a new fluoroquinolone, was studied in comparison with those of ciprofloxacin, trovafloxacin (CP 99,219), cefpodoxime, and amoxicillin-clavulanate against gram-negative, gram-positive, and anaerobic bacteria . Its activity against mycobacteria and chlamydia was also investigated . BAY 12-8039 was active against members of the family Enterobacteriaceae (MIC at which 90% of strains tested were inhibited {MIC90S} < or = 1 microgram/ml, except for Serratia spp . MIC90 2 microgram/ml), Neisseria spp . (MIC90S, 0.015 microgram/ml), Haemophilus influenzae (MIC90, 0.03 microgram/ml), and Moraxella catarrhalis (MIC90, 0.12 micrgram/ml), and these results were comparable to those obtained for ciprofloxacin and trovafloxacin . Against Pseudomonas aeruginosa, the quinolones were more active than the beta-lactam agents but BAY 12-8039 was less active than ciprofloxacin . Strains of Stenotrophomonas maltophilia were fourfold more susceptible to BAY 12-8039 and trovafloxacin (MIC90S, 2 micrograms/ml) than to ciprofloxacin . BAY 12-8039 was as active as trovafloxacin but more active than ciprofloxacin against Streptococcus pneumoniae (MIC90, 0.25 microgram/ml) and methicillin-susceptible Staphylococcus auerus (MIC90S, 0.12 micrograms/ml) . The activity of BAY 12-8039 against methicillin-resistant S . aureus (MIC90, 2 micrograms/ml) was lower than that against methicillin-susceptible strains . BAY 12-8039 was active against anaerobes (MIC90S < or = 2 micrograms/ml), being three- to fourfold more active against Bacteroides fragilis, Prevotella spp., and Clostridium difficile than was ciprofloxacin . Against Mycobacterium tuberculosis, BAY 12-8039 exhibited activity comparable to that of rifampin (MICs < or = 0.5 micrograms/ml) . Against Chlamydia trachomatis and Chlamydia pneumoniae BAY 12-8039 was more active (MICs < or = 0.12 microgram/ml) than either ciprofloxacin or erythromycin and exhibited a greater lethal effect than either to these two agents . The protein binding of BAY 12-8039 was determined at 1 and 5 micrograms/ml as 30 and 26.4%, respectively . The presence of human serum (at 20 or 70%) had no marked effect on the in vitro activity of BAY 12-8039.

Rev Environ Contam Toxicol, 1997, 150, 75 - 94
Human disease associated with Clostridium perfringens enterotoxin; Meer RR et al.; Clostridium perfringens continues to be a common cause of food-borne disease . Characteristics of this organism that contribute to its ability to cause food-borne illness include the formation of heat-resistant spores that survive normal cooking/heating temperatures, a rapid growth rate in warm food, and the production of enterotoxin (CPE) in the human gut . Time and temperature abuse associated with food preparation contributes to the majority of outbreaks of C . perfringens food-borne disease . CPE-induced diarrhea has been reported in the absence of a defined food vehicle . These cases have been typically associated with the elderly and following a course of antibiotic therapy . The incidence of CPE-induced diarrhea may be expected to increase with the growing population of immunocompromised (disease-, treatment-, or age-induced) individuals . Clostridium perfringens has been implicated as a possible contributor to the development of SIDS in susceptible individuals . Specifically, it has been hypothesized that CPE acts as a triggering agent, initiating the events associated with the development of SIDS . Continued refinement of both immunoassays and molecular methods for toxin and gene detection, respectively, will facilitate their eventual availability as commercial kits, providing rapid and simplified methods for the detection of C . perfringens isolates that produce or have the capacity to produce CPE as well as other toxins associated with this organism.

J Clin Microbiol, 1997 Jan, 35(1), 228 - 32
Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR; Yoo HS et al.; Clostridium perfringens has been classified into five toxigenic types (A through E) on the basis of its capability to produce major lethal toxins (alpha, beta, epsilon, and iota toxins) . Seroneutralization with mice or guinea pigs has been used to type each toxin, but this conventional method has some disadvantages . Therefore, we used a molecular biological technique to type the bacterium in the present study . A multiplex PCR was developed for this purpose . This method has several advantages in comparison with seroneutralization with mice or guinea pigs . By this method, we also investigated the most prevalent type(s) of the organism in Korean calves, piglets, and chickens showing clinical symptoms such as diarrhea, enterotoxemia, and necrotic enteritis . Only type A was isolated from calves and chickens, while type C (2 of 14 isolates), in addition to type A, was isolated from piglets . These results suggested that seroneutralization could be replaced by our new method and that type A of C . perfringens is the most prevalent type in livestock in Korea.

Curr Microbiol, 1997 Jan, 34(1), 49 - 54
Identification and analysis of a collagenolytic activity in Streptococcus mutans; Jackson RJ et al.; Streptococcus mutans is an important pathogen in coronal caries and is implicated in dental root decay by its ability to bind collagen from various sources . In the present study, electron microscopic analysis demonstrated the ability of S . mutans to bind and to disrupt collagen fibrils of the amniotic membrane . The synthetic peptide FALGPA, which is similar in structure to collagen, was degraded by S . mutans, with a lower level of FALGPA hydrolytic activity observed in sucrose-grown cells compared with cells grown in the absence of sucrose . Inhibition studies of FALGPA hydrolytic activity showed a pattern characteristic of collagenase activity, with inhibition by 1,10-phenanthroline and EDTA, but not by phenylmethylsulfonyl fluoride (PMSF) . Additionally, immunological cross-reactivity was observed between proteins from disrupted cells of S . mutans and antiserum to collagenase from Clostridium histolyticum . Gelatinolytic activity was demonstrated by gelatin zymogram analysis . These findings suggest that collagenolytic activity by S . mutans may be an important virulence factor in dental root decay.

Am J Med Genet, 1996 Dec 30, 66(4), 413 - 22
Genetics, phenotype, and natural history of autosomal dominant cyclic hematopoiesis; Palmer SE et al.; Cyclic hematopoiesis, (CH, or cyclic neutropenia) is a rare disease manifested by transient severe neutropenia that recurs approximately every 21 days . The hematologic profile of families with the autosomal dominant form (ADCH) has not been well characterized, and it is unknown if the phenotype is distinct from the more common sporadic congenital or acquired forms of CH . We studied nine ADCH families whose children displayed typical CH blood patterns . Pedigrees confirmed dominant inheritance without evidence of heterogeneity or decreased penetrance; three pedigrees suggested new mutations . Families were Caucasian with exception of one with a Cherokee Native American founder: A wide spectrum of symptom severity, ranging from asymptomatic to life-threatening illness, was observed within families . The phenotype changed with age . Children displayed typical neutrophil cycles with symptoms of mucosal ulceration, lymphadenopathy, and infections . Adults often had fewer and milder chronic neutropenia without distinct cycles . While CH is commonly described as "benign", four children in three of the nine families died of Clostridium or E . coli colitis, documenting the need for urgent evaluation of abdominal pain . Misdiagnosis with other neutropenias was common but can be avoided by serial blood counts in index cases . Genetic counseling requires specific histories and complete blood counts in relatives at risk to assess status regardless of symptoms, especially to determine individuals with new mutations . We propose diagnostic criteria for ADCH in affected children and adults . Recombinant human granulocyte colony-stimulating factor treatment resulted in dramatic improvement of neutropenia and morbidity . The differential diagnosis from other forms of familial neutropenia is reviewed.

Biochemistry, 1996 Dec 24, 35(51), 16483 - 8
Mechanistic interpretation of tryptophan fluorescence quenching in the time courses of glutamate dehydrogenase catalyzed reactions; Saha SK et al.; We have related the ratios of the protein fluorescence quenching and nucleotide absorbance time courses for the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate to identify the occurrence and sequential location of a previously demonstrated charge-transfer intermediate . Static studies showed the major portion of the fluorescence quenching signal to be due to radiationless singlet energy transfer from tryptophan to reduced coenzyme chromophores and that conformational changes contribute little to this signal . The ratio approach applied to the transient time courses shows correspondingly that, over most of the time range, the fluorescence quenching signal provides a quantitative measure of the sum of all posthydride transfer species . However, it also indicates the very early occurrence of a species of anomalous optical properties for the reaction catalyzed by the Clostridium symbiosum enzyme as well as that from bovine liver . Transient-state kinetic isotope effect time courses of both the fluorescence and the absorbance signals confirm that this species must be the prehydride charge-transfer complex in both enzyme reactions . Kinetic analysis of alpha-deuterio- and alpha-protio-L-glutamate reaction time courses proves the kinetic competence of the assignments . These results also demonstrate that the intramolecular transfer of a proton from the alpha-amino group of the substrate to an immediately adjacent aspartate carboxylate group on the enzyme is an obligatory initial event in the reactions catalyzed by both enzyme species, even though the occurrence of protein release from a critical lysine residue to the solvent occurs at different phases in those two reactions . The abnormally low intrinsic KIE required to simulate both the alpha-deuterio-L-glutamate reaction and its protio counterpart implies that the transition state of the hydride transfer step must be highly asymmetric.

Ned Tijdschr Geneeskd, 1996 Dec 21, 140(51), 2547 - 50
{Gas gangrene of ischemic myocardial tissue caused by Clostridium septicum}; den Bakker MA et al.; A 86-year-old woman hospitalized for analysis of persistent abdominal discomfort died, apparently of myocardial infarction, shortly after admission . Autopsy revealed extensive myocarditis caused by infection with Clostridium septicum . As a portal of entry a carcinoma of the colon was found . The association of colon carcinoma and bacteraemia with C . septicum is well established while colon carcinoma can lead to cardiac hypoxia through blood loss and anaemia . However, myocarditis caused by this organism is extremely rare.

Biochem J, 1996 Dec 15, 320 ( Pt 3), 825 - 30
Adenosylcobalamin-dependent glutamate mutase: properties of a fusion protein in which the cobalamin-binding subunit is linked to the catalytic subunit; Holloway DE et al.; Adenosylcobalamin-dependent glutamate mutase (EC 5.4.99.1) from Clostridium tetanomorphum comprises two protein components, MutE and MutS . The formation of the holoenzyme is a kinetically complex process that involves the co-operative association of MutS, MutE and adenosylcobalamin . The MutS portion of the cobalamin-binding site is conserved within a group of adenosylcobalamin-dependent enzymes that catalyse similar isomerizations . However, in contrast with glutamate mutase, in these other enzymes the cobalamin-binding region represented by MutS is present as a C-terminal domain . We have investigated the effect on the structural and kinetic properties of glutamate mutase of linking MutS to the C-terminus of MutE . Kinetic analysis of this protein, MutES, showed, unexpectedly, that enzyme activity was still co-operatively dependent on protein concentration . The Km for L-glutamate was unchanged from the wild type, whereas Vmax was decreased to approx . one-thirtieth and the Km for coenzyme increased approx . 10-fold . Investigation of the quaternary structure of MutES by equilibrium ultra-centrifugation indicated that the protein existed in equilibrium between monomeric and dimeric forms . Thus linking MutE and MutS together seems to substantially weaken the contacts that are responsible for the dimerization of MutE . The two domains of the MutES monomer seem unable to communicate, so that active enzyme is formed by the intermolecular association of two MutES subunits in a co-operative manner.

Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 430 - 9
Clostridium botulinum C3 exoenzyme stimulates GLUT4-mediated glucose transport, but not glycogen synthesis, in 3T3-L1 adipocytes--a potential role of rho?
van den Berghe N, Barros LF, van Mackelenbergh MG, Krans HM.
The signal transduction pathway by which insulin stimulates glucose transport is largely unknown, but a role of PI-3-kinase and small GTP-binding proteins has been proposed . In previous studies we, among many others, excluded a role for the ras/MAP kinase pathway in insulin-mediated glucose transport . In this study we examined a possible role of the small GTP-binding protein rho in this process . Pretreatment of 3T3-L1 adipocytes with botulinum C3 exoenzyme (C3), which is known to ADP-ribosylate and inactivate rho, potently stimulated glucose uptake to a level similar to insulin . Interestingly, glycogen synthesis was not affected by C3 treatment . Insulin stimulates glucose uptake by triggering the translocation of GLUT4, the insulin-sensitive glucose transporter isotype, from an intracellular compartment to the plasma membrane . Similarly, C3-induced glucose uptake was paralleled by GLUT4 translocation . These data point to an important and novel role of the target of C3 (likely rho) in the regulation of GLUT4-mediated glucose transport . Our data suggest that insulin might stimulate glucose uptake through inactivation of rho.

Biochem Biophys Res Commun, 1996 Dec 13, 229(2), 370 - 4
Difference in protein substrate specificity between hemorrhagic toxin and lethal toxin from Clostridium sordellii; Genth H et al.; The hemorrhagic toxin (HT) from Clostridium sordellii is pharmacologically related to Clostridium difficile toxins A and B and Clostridium sordellii lethal toxin which have been recently identified as mono-glucosyl-transferases . Here we report that HT, which is coexpressed with lethal toxin, is also a glucosyltransferase . Whereas lethal toxin glucosylates the Rho subfamily proteins Rac and Cdc42 and the Ras subfamily proteins H-Ras and Rap, the substrate specificity of HT is strictly confined to the Rho subfamily proteins Rho, Rac and Cdc42 . Comparable to lethal toxin, transferase activity of HT is stimulated by Mn2+ . Acceptor amino acid in Rho was identified by mutagenesis as threonine-37 . C . sordellii HT is a novel member of the family of clostridial mono-glucosyl-transferases, a family which modifies the Rho and Ras GTPases.

Biochemistry, 1996 Dec 10, 35(49), 15814 - 21
Unleashing hydrogenase activity in carbon monoxide dehydrogenase/acetyl-CoA synthase and pyruvate:ferredoxin oxidoreductase; Menon S et al.; These results demonstrate that two well-studied metalloenzymes, carbon monoxide dehydrogenase/acetyl-CoA synthase (CODH/ACS) and pyruvate:ferredoxin oxidoreductase (PFOR), can reduce protons to H2 and, at much lower rates, oxidize H2 to protons and electrons . To our knowledge, this if the first time that PFOR has been shown to have hydrogenase activity . CODH/ACS and PFOR evolved H2 at maximum rates when CO and pyruvate were the electron donors, respectively, and when electron acceptors are absent; dithionite was a very poor substitute . PFOR, when purified to greater than 99% homogeneity, exhibited a specific activity for pyruvate-dependent H2 production of 135 nmol min-1 mg-1 . The H2 evolution activity divided by the H2 uptake activity was 282:1; the highest ratio previously reported (22:1) was with the membrane-bound hydrogenase from Rhodospirillum rubrum {Fox, J.D., Kerby, R . L., Roberts, G . P., & Ludden, P . W . (1996) J . Bacteriol . 178, 1515-1524} . Highly purified samples of CODH/ACS (> 99% homogeneity) exhibited a specific activity of CO-dependent H2 evolution in the absence of electron carrier of 590 nmol min-1 mg-1 . Equivalent rates of CO oxidation and H2 production were observed when determined in the absence of electron acceptor . This level of activity can account for the rate of H2 production that has been observed by growing cultures of Clostridium thermoaceticum and could solve the paradox that the highly CO-sensitive hydrogenases from acetogenic bacteria evolve H2 when grown on CO . The ratio of the rates of (H2 evolution):(H2 uptake) for purified CODH/ACS is between 20:1 and 30:1 . H2 evolution and uptake by CODH/ACS were strongly inhibited by cyanide (ki = 1 microM), indicating that these reactions are catalyzed by cluster C, the site of CO oxidation . Our results extend earlier findings that the CODHs from Methanosarcina barkeri {Bhatnagar, L., Krzycki, J . A., & Zeikus, J . G . (1987) FEMS Microbiol . Lett . 41, 337-343} and Oligotropha carboxydovorans {Santiago, B., & Meyer, O . (1996) FEMS Microbiol . Lett . 136, 157-162} exhibit hydrogenase activity . Mechanistic implications of hydrogenase activity are discussed . Several physiological roles for proton reduction by CODH/ACS and PFOR are discussed, including the prevention of radical formation from reduced metal clusters when electron carriers (ferredoxin, flavodoxin, etc.) are limiting.

J Biol Chem, 1996 Dec 6, 271(49), 31185 - 90
Rho is required for Galphaq and alpha1-adrenergic receptor signaling in cardiomyocytes . Dissociation of Ras and Rho pathways; Sah VP et al.; G protein-coupled receptor agonists initiate a cascade of signaling events in neonatal rat ventricular myocytes that culminates in changes in gene expression and cell growth characteristic of hypertrophy . These responses have been previously shown to be dependent on Gq and Ras . Rho, a member of the Ras superfamily of GTPases, regulates cytoskeletal rearrangement and transcriptional activation of the c-fos serum response element . Immunofluorescence staining of cardiomyocytes shows that Rho is present and predominantly cytosolic . We used two inhibitors of Rho function, dominant negative N19RhoA and Clostridium botulinum C3 transferase, to examine the possible requirement for Rho in alpha1-adrenergic receptor-mediated hypertrophy . Both inhibitors markedly attenuated atrial natriuretic factor (ANF) reporter gene expression induced by alpha1-adrenergic receptor stimulation with phenylephrine, and virtually abolished the increase in ANF reporter gene expression induced by GTPase-deficient Galphaq . These effects were reproduced with the myosin light chain-2 reporter gene . Notably, N19RhoA did not block the ability of activated Ras to induce ANF and myosin light chain-2 reporter gene expression . Furthermore, activation of the extracellular signal-regulated kinase by phenylephrine was not blocked by N19RhoA, nor was it stimulated by an activated mutant of RhoA . Since activated RhoA and Ras produce a large synergistic effect on ANF-luciferase gene expression, we conclude that Rho functions in a pathway separate from but complementary to Ras . Our results provide direct evidence that Rho is an effector of Galphaq signaling and suggest for the first time that a low molecular weight GTPase other than Ras is involved in regulating myocardial cell growth and gene expression in response to heterotrimeric G protein-linked receptor activation.

Gene, 1996 Dec 5, 182(1-2), 163 - 7
Characterization of engF, a gene for a non-cellulosomal Clostridium cellulovorans endoglucanase; Sheweita SA et al.; A new Clostridium cellulovorans (strain ATCC 35296) endoglucanase gene engF has been isolated and sequenced . The gene contains 1671 bp and codes for a protein containing 557 amino acids and a mass of 60.1 kDa . A putative signal peptide of 29 amino acids is present and the mature protein has a mass of 57.1 kDa . EngF does not have amino acid sequence homology to previously isolated EngB and EngD, but does show sequence homology to family 5 glycosyl hydrolases from Bacillus, Erwinia carotovora, and C . acetobutylicum species . EngF is not a component of the cellulosome and does not contain a duplicated sequence (DS) at its C-terminal region . EngF is capable of binding to cellulose and hydrolyzing carboxymethylcellulose but not xylan . The cellulose binding domain (CBD) differs from types I, II and III CBDs and no obvious homology has been found to other CBD types . The maximum activity of EngF occurs at pH 5.5 and at 47 degrees C . Its properties suggest that EngF plays an ancillary role in the degradation of cellulosic materials.

Antimicrob Agents Chemother, 1996 Dec, 40(12), 2737 - 42
Lymphocyte proliferative response and subset profiles during extended periods of chloroquine or primaquine prophylaxis; Fryauff DJ et al.; Immune suppression and disturbances of normal leukocyte populations are side effects attributed to many antimalarial drugs and were concerns during a recent year-long placebo-controlled trial that compared daily primaquine (0.5 mg of base per kg of body weight per day) with weekly chloroquine (300 mg of base one time per week) for malaria prophylaxis . The study took place in Irian Jaya, Indonesia, from July 1994 to August 1995 and enrolled 129 Javanese men with normal glucose-6-phosphate dehydrogenase function . Tests for lymphocyte function and subset composition were conducted blindly on a cross-section of subjects during weeks 10 (n = 42) and 48 (n = 72) of supervised prophylaxis . Lymphocyte function, measured as the proliferative response of peripheral blood mononuclear cells to a panel of mitogens (pokeweed mitogen, phytohemagglutinin, and concanavalin A) and antigens (purified protein derivative of Mycobacterium tuberculosis and Clostridium tetani toxoid) and expressed as a stimulation index, allowed for statistical comparison between groups and sampling times . The lymphocyte subset composition for each group and time point was based on flow cytometry profiling, and the results were expressed as the mean percentages of CD3 (total T cells), CD19 (total B cells), CD4+ (T-helper and inducer cells), and CD8+ (T suppressor and cytotoxic cells), CD3/CD16+ CD56 (natural killer cells), CD3/anti-HLA-DR (activated T cells) cells and the CD4+/CD8+ ratios . Lymphocyte stimulation indices were statistically comparable among the placebo, primaquine, and chloroquine groups at both time points, although the primaquine group was distinguished by having repeatedly greater proportions of subjects with high ( > 3.0) stimulation indices . The lymphocyte subset profiles of these groups at both time points were also similar and undistorted relative to those of healthy reference populations matched for age, sex, and ethnicity . The results provide quantitative support for the safety of daily primaquine prophylaxis.

Nippon Geka Gakkai Zasshi, 1996 Dec, 97(12), 1060 - 5
{Perioperative managements for postoperative severe infections in compromised host}; Yokoyama T et al.; The incidence of postoperative infections, especially due to multi-drug resistant strains such as Pseudomonas sp., Enterococcus sp., and Methicillin resistant Staphylococcus aureus (MRSA), is high in compromised hosts . Among them, respiratory infection, catheter sepsis, and drug-associated enteritis are frequently observed and respiratory infection is liable to fall into serious illness . These infections have characteristics in causative organisms . Pseudomonas aeruginosa or MRSA are frequently isolated in respiratory infections and Candida or coagulase-negative staphylococcus are frequently isolated in catheter sepsis . G-test in addition to blood culture is necessary for early diagnosis of Candida sepsis, vancomycin should be administered in early phase of antibiotic-associated enteritis, since this infection is usually caused by MRSA or Clostridium difficile and frequently falls into serious illness . The patients with protein-calorie malnutrition, liver cirrhosis, renal failure, diabetes melitis, administration of anticancer drugs and/or radiation therapy, serious injury, or severe operative stress are considered to be compromised hosts in surgical field, and the adequate perioperative managements according to these disorders should be carried out against postoperative infections.

Am J Surg, 1996 Dec, 172(6A), 33S - 37S
Nosocomial infections and nosocomial pneumonia; Hong J et al.; Nosocomial infections are a major source of revenue loss, morbidity, and even mortality to surgical patients . This review presents current issues regarding nosocomial infections and nosocomial pneumonias . This study is a literature review that presents material on nosocomial infections in general and details regarding Clostridium difficile and vancomycin-resistant enterococcus infections . Nosocomial infections, including pneumonias, are serious medical complications, and prevention by strict adherence to barrier precaution is the most important means of protecting the patient from hospital-acquired bacterial flora.

J Virol Methods, 1996 Dec, 62(2), 169 - 78
Isolation of viruses from clinical specimens in microtitre plates with cells inoculated in suspension; O'Neill HJ et al.; Virus isolation is essential for the provision of a full diagnostic virology service . Present methods are time consuming, expensive and relatively inflexible for routine use . Our objective was to audit our existing virus isolation system and to develop a sensitive, flexible virus isolation system which could be adapted for use in a busy routine laboratory which is required to provide a service for a wide range of clinical situations . We carried out a pilot study which compared conventional roller tube monolayer cultures to a microplate system using cells inoculated in suspension and showed that the microplate method using extra cell lines could provide a more sensitive system for virus isolation . This system was adapted for routine use using six cell lines inoculated in suspension and the results are presented for 2610 specimens for virus isolation and 972 for Clostridium difficile toxin (CDT) detection . There were 516 viruses isolated and 229 specimens positive for CDT using this system . Polioviruses (92), echoviruses (35), coxsackieviruses (15) and untyped enteroviruses (13) were isolated in RMK, E6-vero and RD cells . Adenoviruses (137) were isolated in HEp2 and E6-vero cells . Herpes simplex virus (HSV) was isolated from 149 specimens in E6-vero, FCL and HFF9 cells . Myxoviruses (38) and paramyxoviruses were isolated in RMK cells . HEp2 was the only cell line necessary to isolate the 33 respiratory syncytial viruses (RSV) . Cytomegaloviruses (CMV) (2) and varicella zoster (1) virus (VZV) were isolated only in the human fibroblast cell line HFF9 . Rubella virus was isolated from a baby with congenital rubella in RMK, E6-vero and additionally in BGM cells . In conclusion, the use of cells inoculated in suspension in microtitre plates for virus isolation was sensitive and convenient . It allowed the use of six cell lines for routine virus isolation without using additional laboratory staff time . It improved turnaround times . It was also safer microbiologically than conventional isolation in tube monolayers . The precise identification of virus isolates was simplified.

Jpn J Vet Res, 1996 Dec, 44(3), 175 - 8
Bovine clostridial infections in Zambia (1985-1994); Munang'andu HM et al.; Retrospective surveillance study of clostridial infections of cattle in Zambia, for the period 1985 to 1994, showed that out of the 318 cases observed, 62.8% and 24.2% were from Western and Southern provinces, respectively . Of the 6 clostridia species identified, Clostridium septicum (38.1%) followed by C . chauvoei (36.2%) and C . perfringens (13.2%) were dominant . Although the highest incidence for clostridial infections was in 1989 (75 cases) and 1990 (77 cases), the number of C . perfringens cases seemed to increase . More cases were found in the dry season until the onset of the rains, that is, the period August to December.

Naunyn Schmiedebergs Arch Pharmacol, 1996 Dec, 354(6), 693 - 7
ADP-ribosylation of actin by Clostridium perfringens iota toxin and turkey erythrocyte ADP-ribosyltransferase A: effects on profilin-regulated nucleotide exchange and ATPase activity; Sehr P et al.; Effects of ADP-ribosylation of skeletal muscle alpha-actin by Clostridium perfringens iota toxin and by turkey erythrocyte ADP-ribosyltransferase A on profilin-regulated nucleotide exchange and ATPase activity were compared . ADP-ribosylation of actin at Arg 177 by Clostridium perfringens iota toxin increased the nucleotide dissociation rate from 2.2 x 10(-3) s-1 to 4.5 x 10(-3) s-1 without affecting the profilin-induced stimulation of nucleotide exchange . In contrast, ADP-ribosylation of actin at Arg95/Arg372 induced by turkey erythrocyte transferase decreased the nucleotide dissociation rate to 1.5 x 10(3) s-1 and inhibited the profilin-induced stimulation of nucleotide exchange . Whereas toxin-induced ADP-ribosylation at Arg177 blocked actin ATPase, basal G-actin ATPase was not altered by ADP-ribosylation at Arg95/Arg372 but inhibited profilin effects on actin ATPase.

Aliment Pharmacol Ther, 1996 Dec, 10(6), 835 - 41
Review article: antibiotic-induced Clostridium difficile infection; Settle CD et al.; The great majority of cases of Clostridium difficile infection are hospital-acquired, and the reported incidence in England and Wales has increased sixfold between 1990 and 1993, with at least 17 patients dying in a recent large nosocomial outbreak . C . difficile infection accounts for an average 3-week increased length of stay in hospital . Acquisition of a toxigenic strain of Clostridium difficile may be followed by asymptomatic carriage, diarrhoea, colitis or pseudomembranous colitis . Antibiotic treatment and older age are major risk factors for the development of symptomatic disease, but less well-defined differences in strain virulence and host susceptibility are also probably important . Accurate data on the relative risks of different antibiotics to induce symptomatic C . difficile infection are scarce, but third-generation cephalosporins are frequently implicated . New kits are becoming available for the laboratory diagnosis of C . difficile infection but many of these lack sensitivity . Oral metronidazole or vancomycin are the main treatment options but avoidance of further antibiotics should also be encouraged where possible . The role of environmental C . difficile spores, which are highly resistant to conventional disinfectants, needs to be defined . Proven strategies for the prevention of C . difficile infection are required, in particular protocols to ensure that cross-infection does not occur.

FEMS Microbiol Lett, 1996 Dec 1, 145(2), 239 - 43
Production and purification of Clostridium perfringens alpha-toxin using a protein-hyperproducing strain, Bacillus brevis 47; Nagahama M et al.; Clostridium perfringens alpha-toxin was produced in a protein-hyperproducing strain, Bacillus brevis 47, by cloning the gene into the constructed expression-secretion vector which has the multiple promoters and the signal peptide coding region of an outer cell wall protein gene . The amount of alpha-toxin produced by the B . brevis 47 transformant carrying the gene was approximately 10 times greater than that produced by a B . subtilis transformant carrying the toxin gene . Biological activities and the N-terminal amino acid sequence of the toxin secreted by the B . brevis 47 transformant were identical to those of wild-type alpha-toxin.

Ann Thorac Surg, 1996 Dec, 62(6), 1835 - 7
Mycotic aneurysm of the thoracic aorta caused by Clostridium septicum; Murphy DP et al.; We describe a case-of a 78-year-old man who presented with a mycotic aneurysm of the thoracic aorta caused by Clostridium septicum and underwent successful resection . There are only 3 cases of mycotic aneurysms caused by Clostridium septicum reported in the literature . Clostridium septicum infections have been shown to have a high association with gastrointestinal and hematologic malignancies . All patients with Clostridium septicum infections, therefore, require a search for gastrointestinal lesions, as they may represent a source of persistent bacteremia . This patient had no malignant lesions but did have multiple benign sigmoid polyps.

Arch Surg, 1996 Dec, 131(12), 1333 - 7
Diarrhea and Clostridium difficile-associated diarrhea on a surgical service; McCarter MD et al.; OBJECTIVE: To identify the incidence, risk factors, and treatment of diarrhea and Clostridium difficile-associated diarrhea (CDAD) in surgery patients . DESIGN: Prospective and historical retrospective analysis . SETTING: Major urban tertiary care referral hospital . PATIENTS: Consecutive patients (N = 475) admitted to the vascular, trauma, and general surgical surgery services, prospectively evaluated during a 10-week period . A retrospective historical control of the same surgical services was used for comparison . INTERVENTION: None . MAIN OUTCOME MEASURES: Incidence of diarrhea and CDAD, use of bowel preparations, surgical procedure, use of C difficile toxin assay, white blood cell count, symptoms, treatment, and delay in hospital discharge . RESULTS: The incidence of diarrhea in surgery patients analyzed prospectively was 6.1%; the incidence of CDAD during the prospective and retrospective periods was 2% . Preoperative bowel preparations were associated with an increased risk of diarrhea (relative risk, 4.2; 95% confidence interval, 2.6-6.8; P < .001) and CDAD (relative risk, 3.2; 95% confidence interval, 1.5-7.2; P < .03) . Leukocytosis (white blood cell count > 11 x 10(9)/L) was significantly higher in the CDAD group compared with the diarrhea group only on the day of diagnosis (P < .05) . By subjective analysis, diarrhea was directly responsible for a delay in discharge in 7 of 29 patients for a mean (+/-SEM) of 4.0 +/- 1.0 days . CONCLUSIONS: Patients undergoing preoperative bowel preparations are at increased risk of experiencing diarrhea and CDAD . Among patients with diarrhea, an elevated white blood cell count may help identify those with C difficile . Early treatment of diarrhea with oral metronidazole while awaiting the results of the stool toxin assay is recommended for treating diarrhea in surgery patients . Prophylactic treatment of surgery patients undergoing bowel preparations should be considered.

J Bacteriol, 1996 Dec, 178(24), 7152 - 8
Rubrerythrin from Clostridium perfringens: cloning of the gene, purification of the protein, and characterization of its superoxide dismutase function; Lehmann Y et al.; The food-borne pathogen Clostridium perfringens, which is an obligate anaerobe, showed growth under conditions of oxidative stress . In protein extracts we looked for superoxide dismutase (SOD) activities which might scavenge highly toxic superoxide radicals evolving under such stress conditions . Using the classical assay to detect SOD activity on gels after electrophoresis of C . perfringens proteins, we obtained a pattern of three major bands indicating SOD activity . The protein representing the brightest band was purified by three chromatographic steps . On the basis of 20 amino acids determined from the N terminus of the protein, we designed a degenerate oligonucleotide probe to isolate the corresponding gene . We finally sequenced an open reading frame of 195 amino acids (molecular mass, 21,159 Da) with a strong homology to the Desulfovibrio vulgaris rubrerythrin; therefore, we assumed to have cloned a rubrerythrin gene from C . perfringens, and we named it rbr . The C-terminal region of the newly detected rubrerythrin from C . perfringens contains a characteristic non-heme, non-sulfur iron-binding site -Cys-X-X-Cys-(X)12-Cys-X-X-Cys- similar to that found in rubrerythrin from D . vulgaris . In addition, three -Glu-X-X-His- sequences could represent diiron binding domains . We observed SOD activity in extracts of Escherichia coli strains containing the recombinant rbr gene from C . perfringens . A biological function of rubrerythrin as SOD was confirmed with the functional complementation by the rbr gene of an E . coli mutant strain lacking SOD activity . We therefore suppose that rubrerythrin plays a role as a scavenger of oxygen radicals.

J Bacteriol, 1996 Dec, 178(23), 6983 - 90
Transcriptional organization and regulation of the dnaK and groE operons of Chlamydia trachomatis; Tan M et al.; The transcriptional organization and regulation of the dnaK and groE heat shock operons of Chlamydia trachomatis were studied and found to resemble those of the cognate operons of Bacillus subtilis and Clostridium acetobutylicum . The gene order is conserved (hrcA-grpE-dnaK), but no dnaJ homolog could be identified in this region . The dnaK operon was transcribed as a low-abundance polycistronic mRNA whose levels did not increase upon exposure to heat shock . In contrast, a more abundant 2.3-kb mRNA encoding only the dnaK sequence was detectable, and its steady-state level increased upon heat shock . The transcription initiation sites of the dnaK and groE operons were found to be preceded by sequences that resemble an Escherichia coli sigma70 consensus promoter . Upstream of each putative promoter is an inverted repeat sequence which resembles a similar element (CIRCE {controlling inverted repeat of chaperone expression}) found upstream of the dnaK and groE operons in at least 27 eubacterial species . In vitro transcription studies utilizing partially purified C . trachomatis RNA polymerase demonstrated that the regions containing the putative promoter elements of the dnaK and groE operons are functional, although heat shock-regulated expression could not be demonstrated.

Clin Infect Dis, 1996 Dec, 23 Suppl 1, S102 - 6
Increased incidence of Clostridium difficile-associated diarrhea following decreased restriction of antibiotic use; Ho M et al.; Removal of antimicrobial agents from formulary restriction status at our center was followed by an increase in the incidence of Clostridium difficile-associated diarrhea . The mean monthly incidence of C . difficile diarrhea for the 12-month period before institution of decreased restriction of antibiotic use was 3.4 infections per 1,000 admissions and rose to 6.2 infections per 1,000 admissions during the following 4-month period (P < .05) . Patients who developed disease before and after decreased restriction of antibiotics were similar in terms of the mean number of antimicrobial agents administered and mean duration of therapy . The most commonly administered agents whose use preceded the onset of disease were cefazolin, trimethoprim-sulfamethoxazole, ampicillin, ticarcillin/clavulanate, and gentamicin (the latter drug was always used in combination with other agents) . Immunoblot typing indicated that there was no association between C . difficile strains and administration of specific agents . There was no coincidental epidemic to account for the increased incidence of infection . The increased incidence of C . difficile disease is a potential problem that may occur following removal of extended-spectrum antimicrobial agents from formulary restriction status.

Clin Infect Dis, 1996 Dec, 23 Suppl 1, S73 - 7
Emerging resistance of anaerobic bacteria to antimicrobial agents in South Korea; Lee K et al.; In previous studies, Bacteroides fragilis group organisms isolated from Korean patients were more frequently resistant to various antimicrobial agents, including clindamycin, than were isolates in other countries . A recent report of increased resistance of Peptostreptococcus species prompted us to include such isolates in a study of antimicrobial susceptibility . anaerobes isolated in 1994 at a tertiary care hospital in Seoul were tested by agar dilution method . None of the B . fragilis group organisms were resistant to imipenem, cefoxitin, chloramphenicol, or metronidazole . However, 6.7% were resistant to ampicillin/sulbactam, 20.2% to cefotetan, 30.3% to piperacillin, 48.3% to cefotaxime, and 42.7% to clindamycin . Almost all of the Clostridium perfringens isolates were susceptible to all of the agents tested, except tetracycline . Peptostreptococcus isolates were susceptible to piperacillin, cefotaxime, and imipenem, while 7.4% were resistant to penicillin G, cefotetan, and metronidazole, and 25.9% were resistant to clindamycin . The isolates resistant to penicillin G, cefotetan, and metronidazole were identified as Peptostreptococcus anaerobius . In conclusion, besides the well-known high rate of resistance of B . fragilis group organisms to clindamycin, the emergence of resistance of Peptostreptococcus species isolates to beta-lactam drugs has become obvious in Korea.

Clin Infect Dis, 1996 Dec, 23 Suppl 1, S44 - 50
Comparison of activities of trovafloxacin (CP-99,219) and five other agents against 585 anaerobes with use of three media; Hecht DW et al.; Agar dilution testing was used to compare the in vitro activities of trovafloxacin and ofloxacin, ciprofloxacin, imipenem, metronidazole, and clindamycin against 585 anaerobes . The activity of trovafloxacin was superior to those of ofloxacin and ciprofloxacin, with 94%, 91%, 96%, 100%, 90%, and 100% inhibition of Bacteroides fragilis, non-fragilis Bacteroides species, Peptostreptococcus magnus/Peptostreptococcus micros, Clostridium perfringens, Prevotella bivia/Prevotella disiens, and Fusobacterium species, respectively, at a breakpoint of 2 micrograms/mL . Trovafloxacin was more active than clindamycin against most anaerobes and slightly less active than imipenem and metronidazole . Different testing media, which were compared side by side, did affect the in vitro activities of trovafloxacin, ofloxacin, and ciprofloxacin but did not affect those of the other antibiotics . Testing with supplemented brain-heart infusion agar demonstrated lower minimum inhibitory concentrations than did testing with Wilkins-Chalgren agar and supplemented brucella agar . The activity of trovafloxacin was twofold lower when the pH of the testing media was adjusted to 6.0 than when the pH of the testing media was adjusted to 7.0 or 7.5.

Clin Infect Dis, 1996 Dec, 23 Suppl 1, S15 - 8
In vitro activity of quinolones and other antimicrobial agents against anaerobic bacteria; Nord CE; The in vitro activities of ciprofloxacin, ofloxacin, sparfloxacin, and DU-6859a against peptostreptococci, Clostridium perfringens, Clostridium difficile, Bacteroides fragilis, Porphyromonas, Prevotella, and Fusobacterium were determined by an agar dilution method . These activities were compared with those of piperacillin/tazobactam, cefoxitin, imipenem, clindamycin, and metronidazole . Imipenem, metronidazole, and DU-6859a were the most active antimicrobial agents that were tested . The in vitro activity of DU-6859a was superior to those of ciprofloxacin, lomefloxacin, ofloxacin, and sparfloxacin.

Clin Infect Dis, 1996 Dec, 23 Suppl 1, S2 - 8
In vitro susceptibility of anaerobes to quinolones in the United States; Hecht DW et al.; The in vitro activity of early fluoroquinolone antibodies--including ciprofloxacin, ofloxacin, fleroxacin, pefloxacin, enoxacin, and lomefloxacin--against most anaerobes has been limited, a characteristic making them poor choices as antianaerobic agents . Newer fluoroquinolones, including levofloxacin, sparfloxacin, and grepafloxacin, have moderate activity against anaerobes, including the Bacteroides fragilis group as well as Clostridium, Peptostreptococcus, Prevotella, and Fusobacterium species . Fluoroquinolones that demonstrate the greatest activity against the B . fragilis group and other anaerobes include DU-6859a, clinafloxacin, and the related naphthyridone, trovafloxacin . There has been wide variation in the susceptibility results among different studies testing the same antibiotic; such variation may be due in part to the use of different methodologies, inoculum sizes, and testing media . In a direct comparison of susceptibility findings for ciprofloxacin, ofloxacin, and levofloxacin in three different media, we have determined that twofold dilution differences in minimum inhibitory concentration (MIC) values (MIC90, mode MIC, and geometric mean MIC) may occur in association with the choice of testing media . Thus, testing media should be considered when comparing results of different studies on the susceptibility of anaerobes to fluoroquinolones.

Infect Immun, 1996 Dec, 64(12), 5225 - 32
Saccharomyces boulardii protease inhibits Clostridium difficile toxin A effects in the rat ileum; Castagliuolo I et al.; Saccharomyces boulardii, a nonpathogenic yeast, is effective in treating some patients with Clostridium difficile diarrhea and colitis . We have previously reported that S . boulardii inhibits rat ileal secretion in response to C . difficile toxin A possibly by releasing a protease that digests the intestinal receptor for this toxin (C . Pothoulakis, C . P . Kelly, M . A . Joshi, N . Gao, C . J . O'Keane, I . Castagliuolo, and J . T . LaMont, Gastroenterology 104: 1108-1115, 1993) . The aim of this study was to purify and characterize this protease . S . boulardii protease was partially purified by gel filtration on Sephadex G-50 and octyl-Sepharose . The effect of S . boulardii protease on rat ileal secretion, epithelial permeability, and morphology in response to toxin A was examined in rat ileal loops in vivo . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified S . boulardii protease revealed a major band at 54 kDa . Pretreatment of rat ileal brush border (BB) membranes with partially purified protease reduced specific toxin A receptor binding (by 26%) . Partially purified protease digested the toxin A molecule and significantly reduced its binding to BB membranes in vitro (by 42%) . Preincubation of toxin A with S . boulardii protease inhibited ileal secretion (46% inhibition, P < 0.01), mannitol permeability (74% inhibition, P < 0.01), and histologic damage caused by toxin A . Thus, S . boulardii protease inhibits the intestinal effects of C . difficile toxin A by proteolysis of the toxin and inhibition of toxin A binding to its BB receptor . Our results may be relevant to the mechanism by which S . boulardii exerts its protective effects in C . difficile infection in humans.

Infect Immun, 1996 Dec, 64(12), 5029 - 34
Bacteroides fragilis toxin rapidly intoxicates human intestinal epithelial cells (HT29/C1) in vitro; Saidi RF et al.; Enterotoxigenic Bacteroides fragilis strains associated with childhood diarrhea produce a 20-kDa protein toxin (BFT) . Purified BFT causes striking morphologic changes in subconfluent human colonic epithelial cells (HT29/C1) . In a 3-h HT29/C1 cell assay, the estimated half-maximal effective concentration of BFT was 12.5 pM, and morphologic effects were detectable as early as 30 min and nearly complete by 1.5 h . Concentrations as low as 0.5 pM could also cause intoxication, but morphologic changes were detectable only when the assay was extended to 18 h . The onset of this intoxication was concentration dependent and rapid, occurring within minutes (<7 min at 0.25 nM, <2 min at 2.5 nM) . Notably, the onset of intoxication at 37 degrees C became irreversible to washing within 2 min after exposure to BFT . Morphologic changes were completely inhibited by treatment of HT29/C1 cells with BFT at 4 degrees C but could be demonstrated by subsequent warming to temperatures of 15 degrees C or higher after washing . The time required for the association of BFT with HT29/C1 cells at 4 degrees C was inversely correlated with concentration . Inhibitors of endosomal and Golgi trafficking (NH4Cl and brefeldin A) prevented the intoxication of HT29/C1 cells by Clostridium difficile toxin A and cholera toxin, respectively, but not by BFT . Agents altering microtubule structure did not affect the cellular activity of BFT . These data indicate that a purified toxin from B . fragilis strains associated with diarrhea rapidly and irreversibly intoxicates human intestinal epithelial cells (HT29/C1) in a concentration- and temperature-dependent manner and that the process of intoxication may not involve internalization mechanisms utilizing microtubules or sensitive to pH or brefeldin A.

Infect Immun, 1996 Dec, 64(12), 5022 - 8
Human intestinal epithelial cells swell and demonstrate actin rearrangement in response to the metalloprotease toxin of Bacteroides fragilis; Koshy SS et al.; Enterotoxigenic Bacteroides fragilis (ETBF) cells produce a 20-kDa heat-labile metalloprotease toxin which is potentially important in the pathogenesis of diarrhea associated with this infection . Previous studies indicate that subconfluent HT29/C1 cells treated with the B . fragilis toxin (BFT) develop morphologic changes with dissolution of tight clusters and apparent swelling . Such alterations suggest toxin-stimulated reorganization of the cellular cytoskeleton . The purpose of the current study was to evaluate the effect of BFT on actin microfilaments (F-actin) and cell volume . As assessed by fluorescent phallicidin staining which detects F-actin, BFT treatment of HT29/C1 cells resulted in redistribution of F-actin with loss of stress fibers, a floccular staining pattern, and cellular membrane blebbing without quantitative changes in F-actin fluorescence intensity . The F-actin redistribution was time and concentration dependent . In contrast to the cell shrinkage observed in response to the F-actin-depolymerizing agents cytochalasin D and Clostridium difficile toxin A, BFT stimulated an increase in HT29/C1 cell volume of 10 to 25% (compared with control cells) over a 24-h time course . Only 10 to 30 ng of BFT per ml was necessary to stimulate a maximal increase in HT29/C1 cell volume . The effect of BFT on cell volume was persistent and dependent on the proteolytic activity of BFT . In agreement with cell viability assays indicating that BFT did not injure HT29/C1 cells, intoxicated cells exhibited regulatory volume decrease, suggesting that toxin-treated cells remain physiologically dynamic . We conclude that BFT acts on the intestinal epithelial cell cytoskeleton to alter F-actin structure and to stimulate an increase in HT29/C1 cell volume . Although these two activities of BFT appear to be linked, the precise sequence of cellular events following intoxication of HT29/C1 cells with BFT remains unclear . We hypothesize that these F-actin and cell volume changes may lead to an alteration in tight junction function in the polarized intestinal epithelium, contributing to the pathogenesis of diarrhea in ETBF infections.

J Immunol, 1996 Dec 1, 157(11), 5070 - 5
Rho is a negative regulator of human monocyte spreading; Aepfelbacher M et al.; Monocyte spreading is a sequential series of events including cell flattening, formation of new actin structures and focal adhesions, as well as development of cytoplasmic projections . To investigate the involvement of the GTP-binding protein Rho in spreading, we treated human blood monocytes or PMA-stimulated U937 or THP1 cells with Clostridium botulinum C3-transferase (C3), which ADP-ribosylates and inactivates Rho in intact cells . The C3 treatment caused 1) a four- to fivefold increase in the number of THP1 cells that spread on fibronectin within 24 h of PMA stimulation, 2) a greater area covered by the spread cells, and 3) accelerated and enhanced development of macrophage-like filopodial and pseudopodial projections . Similar results were obtained with PMA-stimulated U937 cells and human blood monocytes . Furthermore, cell staining revealed disorganization of subcortical actin in C3-treated THP1 cells, whereas circular actin formations at the substrate-attached part of the cells and vinculin-containing focal complexes/adhesions were unaffected . Finally, we found a decrease in membrane-associated RhoA in normal spreading THP1 cells, which suggests endogenous inactivation of Rho and might provide an explanation for the acceleration of spreading caused by the C3-transferase . In conclusion, these results indicate that active Rho is an important, negative regulator of human monocyte spreading by maintaining cell tension and cortical actin organization.

Plast Reconstr Surg, 1996 Dec, 98(7), 1225 - 9
Microbial evaluation: 139 implants removed from symptomatic patients; Ahn CY et al.; Possible adverse effects of microbial organisms have been implicated in symptomatic silicone implant patients . In the literature, numerous authors have investigated the possible role of infection with respect to implant problems . To date, various bacterial species have been reported, including Staphylococcus aureus, Staphylococcus epidermidis, peptostreptococci, and Clostridium perfringens . Infections in polyurethane-coated prostheses also have been shown to prolong morbidity . Antibiotic use has been relatively empirical in this regard . The purpose of this study was, first, to determine the frequency, type, and clinical relevance of microbial colonization on implant surfaces removed from symptomatic patients and, second, to determine possible effects of microbial colonization on implant integrity (gel bleed, rupture) . A total of 139 implants from 72 symptomatic patients were entered into the prospective clinical study between February of 1993 and July of 1994 at the UCLA Medical Center . The implant shell types included smooth (79 percent), polyurethane (8 percent), textured (7 percent), and smooth and Dacron (6 percent) . The implant locations were subglandular (71 percent), submuscular (28 percent), and subcutaneous (1 percent) . Of the 139 implants removed, 69 percent were intact and 31 percent were ruptured . Forty-seven percent of 139 implants were culture-positive . Propionibacterium acnes was isolated most frequently (57.5 percent), followed by Staphylococcus epidermidis (41 percent), and then Escherichia coli (1.5 percent) . No fungal infections were identified . Culture positivity was not significantly associated with systemic symptoms . Sixty-seven percent of the positive culture implants were intact; 33 percent were ruptured . The frequency (47 percent) and types (P . acnes and S . epidermidis) of microbial colonization are determined in symptomatic silicone implant patients.

J Clin Microbiol, 1996 Dec, 34(12), 3049 - 55
Genotyping of outbreak-related and sporadic isolates of Clostridium difficile belonging to serogroup C; van Dijck P et al.; Serogroup C of Clostridium difficile is the serogroup most frequently related to outbreaks . Fifty-six toxigenic serogroup C isolates of C . difficile were genotyped by ribotyping PCR (ribo-PCR), random amplified polymorphic DNA (RAPD) assay, and pulsed-field gel electrophoresis (PFGE) . Thirty-five of the 56 isolates were recovered from four unrelated outbreaks (Belgium, 1987, 1992, and 1995; France, 1992 to 1993) 7 derived from a spatiotemporal cluster in Cotonou, Benin (1992), and 14 were sporadic isolates . The serogroup C reference strain, also isolated during an outbreak (Belgium, 1983), was genotyped too . Ribo-PCR, the RAPD assay, and PFGE generated 2, 5, and 11 major genotypes, respectively . Combination of the three methods finally yielded 13 general types, although ribo-PCR did not play any role in enhancing resolution . Three general types were recovered from all the isolates from the five outbreaks and the cluster, with two types being predominant . The 14 sporadic serogroup C isolates were divided into 11 overall genotypes . These results indicate that genotyping methods, and more particularly the combination of the RAPD assay and PFGE, can resolve genetic diversity within toxigenic, serogroup C C . difficile strains . Also, this study suggests that outbreak-related serogroup C strains are limited to a few genetically stable and apparently very widely (internationally and intercontinentally) distributed genotypes.

Gene, 1996 Nov 28, 181(1-2), 29 - 38
Definition of the single integration site of the pathogenicity locus in Clostridium difficile; Braun V et al.; We determined the nucleotide sequence 3.8 kb upstream and 5.2 kb downstream of the toxin genes A and B of Clostridium difficile . Nine ORFs were discovered . Based on PCR-directed approaches, two were attributed to the pathogenicity locus (PaLoc) . The other seven were found in every C . difficile isolate obtained from the human gastrointestinal tract, respectless of their toxinogenicity . The ORFs cdu1 and cdu2/2' upstream of the PaLoc displayed similarity to repressors of Gram-positive bacteria (cdu1), and to an Na+/H+ antiporter described for Enterococcus hirae (cdu2/2') . Downstream of the locus a putative ABC transporter (cdd2-4) was identified . With a set of three paired primers used in polymerase chain reactions we succeeded in delineating the PaLoc . Sequencing of the appropriate stretch of DNA in C . difficile VPI10463 and four additional toxinogenic strains proved a high conservation of the borders of the PaLoc in all these strains . Our data define the locus as a distinct genetic element . Comparing the sequences of five toxinogenic and five non-toxinogenic strains the integration site of the PaLoc was defined . This showed that a stretch of 115 bp found in non-toxinogenic strains is replaced by the 19-kb locus in toxinogenic strains . Analysis of the boundary sequences showed that the locus is obviously not a mobile genetic element by itself . Instead we propose that it is the independent pathogenic part of a more extended genetic element associated with virulence . The 115 bp of non-toxinogenic strains replaced by the locus in toxinogenic strains carry the putative transcription terminator of the cdu1, a predicted repressor protein . A possible polar effect of the loss of this terminator on transcription of the TcdABCDE genes is discussed . Such an effect would explain the unidirectional insertion of the PaLoc at a single site of the C . difficile genome and might give a rationale for the development of the disease which is induced after antibiotical treatment.

J Biol Chem, 1996 Nov 22, 271(47), 29780 - 4
Prostaglandin E receptor EP3 subtype induces neurite retraction via small GTPase Rho; Katoh H et al.; Prostaglandin E receptor EP3 subtype is widely distributed in the nervous system and is specifically localized to neurons, suggesting that the EP3 receptor plays important roles in the nervous system . We established a PC12 cell line that stably expresses the EP3B receptor isoform isolated from bovine adrenal chromaffin cells and examined the effect of agonist stimulation on the neuronal morphology of the PC12 cells . In the differentiated cells, M&B28767, an EP3 agonist, caused neurite retraction in a pertussis toxin-insensitive manner . 12-O-Tetradecanoylphorbol-13-acetate (TPA) also induced neurite retraction . However, when protein kinase C was down-regulated by long term exposure to TPA, TPA failed to induce neurite retraction, while the EP3B receptor-mediated retraction occurred normally . Clostridium botulinum C3 exoenzyme completely inhibited both EP3 agonist- and TPA-induced neurite retraction when microinjected into the cells, indicating that the morphological effect of the EP3B receptor is dependent on Rho activity . Thus, the activation of the EP3B receptor induced neurite retraction through a protein kinase C-independent Rho-activation pathway.

Gene, 1996 Nov 21, 180(1-2), 13 - 21
Molecular characterization of a family of choline-binding proteins of Clostridium beijerinckii NCIB 8052 . Evolution and gene redundancy in prokaryotic cell; Sanchez-Beato AR et al.; Three genes homologous to cspA, which encodes the major secretable protein of Clostridium beijerinckii NCIB 8052 have been cloned and sequenced . The Csp proteins showed the typical modular structure of cell-wall associated proteins and, that found in the choline-binding proteins of Streptococcus pneumoniae . The variable number of repeats that constitute the C-terminal choline-binding domain suggests that the csp genes have evolved by deletion-duplication events . Northern blot analysis indicated that under the culture conditions employed only two genes, cspA and cspC, are efficiently expressed and their products are detected in the culture medium . The csp genes are not contiguously located in the chromosome and appear to be expressed independently . Primer extension experiments located a transcription start site 29 bp upstream of the cspA initiation codon . The -10 and -35 promoter regions are closely related to the consensus sequence of Escherichia coli sigma 70 promoters . The cell wall binding capacity of the clostridial proteins, their abundance in the extracellular media, together with the existence of gene redundancy suggest that the Csp proteins should play an important role in the interaction of this microorganism with its surrounding environment.

FEBS Lett, 1996 Nov 18, 397(2-3), 290 - 2
Crystallization and preliminary X-ray analysis of a thiol-activated cytolysin; Feil SC et al.; We present the first reported crystallization of a member of the thiol-activated family of protein toxins . Perfringolysin O, a virulence factor of Clostridium perfringens, has been crystallized in two different forms by the hanging drop vapor diffusion method . In one form the toxin crystallizes with PEG 20000 in the orthorhombic space group C222(1) with cell dimensions of a = 47.8 A, b = 182.0 A and c = 175.5 A and the crystals diffract to beyond 2.5 A resolution . In the second form the toxin crystallizes in a large variety of organic solvents including malt whisky . This crystal form belongs to the orthorhombic space group P222(1) with unit cell dimensions a = 47.1 A, b = 166.1 A and c = 214.0 A and with diffraction observed to 2.4 A resolution.

J Biotechnol, 1996 Nov 15, 51(3), 243 - 9
Expression, purification and crystallization of a cohesin domain from the cellulosome of Clostridium thermocellum; Yaron S et al.; The cellulosome of the cellulolytic bacterium, Clostridium thermocellum, is a multi-enzyme complex in which the enzymatic (cellulolytic) subunits are attached to a unique nonhydrolytic subunit called scaffoldin . The attachment is mediated by two mutually interacting domains: namely multiple cohesin domains on the scaffoldin subunit and a dockerin domain on each of the enzymatic subunits . Knowledge of the three-dimensional structure of each of the interacting components would be critical to a better understanding of the cohesin-dockerin interaction at the molecular level . In this report, we describe the purification of one of the nine cohesin domains of the scaffoldin subunit from C . thermocellum . A DNA segment containing the cohesin 2 sequence was fused to a hexa-histidine tag, and the resultant construct was expressed in Escherichia coli . The expressed peptide was efficiently isolated by metal-chelate affinity chromatography . The purified recombinant form of the cohesin was crystallized pending determination of its structure.

Vet Med (Praha), 1996 Nov, 41(11), 335 - 8
Prevention of necrotic enteritis in piglets by vaccination of pregnant gilts with a Clostridium perfringens type C and D bacterin-toxoid; Kelneric Z et al.; On a large pig farm with a known history of necrotic enteritis 12 pregnant gilts were vaccinated s . c . 7 and 2 weeks before expected farrowing with a commercial bacterin-toxoid preparation of toxigenic strains C . perfringens type C and D (DizevakR-Pliva, Zagreb) . At the farrowing the titers of beta-antitoxins in serum samples from vaccinated gilts ranged from 9.0 to 26.0 IU/ml with a mean value of 14.16 IU/ml . Colostral titers varied from 12.0 IU/ml with a mean of 16.12 IU/ml . On the second day of life the mean serum titers between litters differed greatly from 4.75 to 24.0 IU/ml . By the age of 7 days the average serum titers were commonly lower and varied between the litters from 2.25 to 15.0 IU/ml, with a low of 1.5 to a high of 16.0 IU/ml in single animals . Ten (8.47%) out of a total of 118 piglets from vaccinated gilts died during the first 7 days of life but the losses were not caused by C . perfringens infection . In unvaccinated control animals 18 (15.9%) of 113 piglets died, eleven of them with clinical and pathoanatomical signs of necrotic enteritis . The affected piglets predominantly succumbed in the first 4 days of life . These data indicate that the investigated bacterin-toxoid can be successfully used in immunoprophylaxis of necrotic enteritis in piglets.

Toxicon, 1996 Nov-Dec, 34(11-12), 1335 - 43
An overview of Clostridium perfringens enterotoxin; McClane BA; Clostridium perfringens enterotoxin (CPE) is considered to be the virulence factor responsible for causing the symptoms of C . perfringens type A food poisoning and may also be involved in other human and veterinary illnesses . CPE has a unique four-step membrane action that apparently involves: (1) CPE binding to a 50,000 mol . wt mammalian protein receptor, forming a small complex of 90,000 mol . wt; (2) the development of a post-binding physical change to this small complex; this physical change could represent either the insertion of CPE into the membrane or a conformational change to small complex; (3) an interaction between this physically changed small complex and a 70,000 mol . wt mammalian protein, forming a large, 160,000 mol . wt complex in membranes; and (4) a breakdown in normal plasma membrane permeability properties for small (< 200,000 mol . wt) molecules . Structure-function analyses have identified a receptor binding region at the C-terminus of CPE and indicate that residues in the N-terminal half of CPE are required for the second step in CPE action to occur . Finally, cpe genetic studies are in their infancy but already indicate that cpe can be either chromosomal or plasmid-borne and that only a tiny minority of the global C . perfringens population is cpe positive . CPE expression appears to be transcriptionally regulated during sporulation, at least in part, by regulatory factors that are common to all C . perfringens isolates.

Vaccine, 1996 Nov, 14(16), 1538 - 44
Mapping of protective and cross-reactive domains of the type A neurotoxin of Clostridium botulinum; Dertzbaugh MT et al.; The purpose of this study was to identify the location of domains within the serotype A neurotoxin of Clostridium botulinum (BoNT/A) that conferred protection against botulism . The BoNT/A gene was subcloned into a series of 10 overlapping fragments that were expressed in Escherichia coli . The expressed proteins were partially purified and used to immunize mice . The resulting antisera were screened by immunoblotting analysis for the presence of BoNT/A-specific antibody . All fragments, except one, elicited antibody that recognized BoNT/A in an immunoblot . Serological screening identified several fragment-specific cross-reactive epitopes that were shared by heterologous serotypes of BoNT . Most of these epitopes immunoreactive by enzyme-linked immunosorbent assay, but not by immunoblot . Only two fragments were shown to confer protection against BoNT/A intoxication . Both of these proteins were derived from segments of the heavy chain and encoded amino acid residues H455-661 and H1150-1289 of BoNT/A.

Vaccine, 1996 Nov, 14(16), 1517 - 22
Tagging a Vibrio cholerae El Tor candidate vaccine strain by disruption of its hemagglutinin/protease gene using a novel reporter enzyme: Clostridium thermocellum endoglucanase A; Robert A et al.; The celA gene encoding Clostridium thermocellum endoglucanase A was expressed in Vibrio cholerae on its own promoter and used to tag a candidate El Tor biotype cholera vaccine strain . Colonies of the tagged strain could be unequivocally distinguished by overlaying them with CM-cellulose indicator agar and Congo Red staining . Expression of celA did not affect growth of V . cholerae in vitro and in vivo . The celA gene was inserted in the chromosomal hap locus encoding V . cholerae hemagglutinin/protease, a putative "detachase", to create a hap- mutant that could be identified and scored by its halo of cellulolytic activity . The inactivation of hap had a positive effect on colonization in the infant mice model . The above results indicate that celA is a suitable marker gene for V . cholerae and hap is an appropriate locus for insertion of foreign DNA in vaccine development . Inactivation of hap, by increasing the duration of adherence, might decrease excretion of the resulting vaccine vector strain and thus increase its immunogenicity.

Bioorg Med Chem, 1996 Nov, 4(11), 1849 - 55
Design and chemoenzymatic synthesis of thiooligosaccharide inhibitors of 1,3:1,4-beta-D-glucanases; Moreau V et al.; A successful chemoenzymatic synthesis of oligosaccharides with an interglucosidic sulfur atom as inhibitors of 1,3:1,4-D-glucanases is described . The key compound 3a was synthesized from acetylated 1-thio-beta-laminaribiose 4 and the methyl 4'-O-triflyl-lactoside 5 . After de-O-acylation, the tetrasaccharide 3b was used as an acceptor and glucose-1-P as a donor in a phosphorolytic elongation catalysed by cellodextrin phosphorylase from Clostridium thermocellum . The expected pentasaccharide 2a and hexasaccharide 1 were isolated in 56% and 13% yield, respectively . As expected, the thiooligosaccharides 1, 2a, and 3b were resistant to enzymatic cleavage by 1,3:1,4-beta-D-glucanase isolated from Bacillus licheniformis . Furthermore, they have been shown to act as competitive inhibitors of the hydrolysis of the chromophoric trisaccharide substrate 11 by this enzyme.

Lett Appl Microbiol, 1996 Nov, 23(5), 275 - 8
Production of chitinase and beta-N-acetylglucosaminidase by intestinal bacteria of Pinnipedian animals; Sugita H et al.; The chitinase- and beta-N-acetylglucosaminidase(GlcNAcase)-producing ability of intestinal bacteria from Pinnipedian animals was determined using fluorogenic 4-methylumbelliferone glycosides of N-acetylglucosamine oligosaccharides . Intestinal microflora of a single Cape fur seal, three California sea lions and three South American sea lions were characterized by a predominance of isolates of the Bacteroidaceae and Enterobacteriaceae families and the genus Clostridium . Of the 711 isolates tested 26.0, 10.0 and 8.7% could hydrolyse 4-MU(GlcNAc)1, 4-MU(GlcNAc)2 and 4-MU(GlcNAc)3, respectively . This result suggests that beta-GlcNAcase producers occur at a higher density than do chitinase producers . Moreover, beta-GlcNAcase, and to a lesser degree, chitinase seem to be efficiently produced by facultative anaerobes in the Cape fur seal and the California sea lion, and by both facultative and obligated anaerobes in the South American sea lion . To our knowledge, this is the first paper to report that isolates of the family Bacteroidaceae and the genus Streptococcus produce chitinase and/or beta-GlcNAcase.

Electrophoresis, 1996 Nov, 17(11), 1776 - 80
ADP-ribosylation of actins in fibroblasts and myofibroblasts by botulinum C2 toxin: influence on microfilament morphology and migratory behavior; Ronnov-Jessen L et al.; Actins comprise six isoforms of which the nonmuscle isoforms beta-/gamma-actins are expressed by all eukaryotic cells . The expression pattern of one of the muscle actin isoforms, alpha-sm actin, previously believed to be restricted to smooth muscle, has been broadened to encompass activated fibroblasts (myofibroblasts) as well . The significance of this molecular conversion has remained largely unknown . We have recently shown that a reduction in filamentous alpha-sm actin by electroinjected specific antibodies or antisense oligodeoxynucleotides leads to increased motility in breast myofibroblasts (Ronnov-Jessen, L., Petersen, O . W . J . Cell Biol . 1996, 134, 67-80) . In the present study we have expanded on the functional significance of actin isotypes in fibroblasts from the opposite point of view, namely filamentous nonmuscle actin . Nonmuscle actins in fibroblasts and myofibroblasts were ADP-ribosylated by Clostridium botulinum C2 toxin . The substrate for C2 toxin is globular actin, which upon ribosylation cannot incorporate into microfilaments . The pattern of actin ADP-ribosylation in (myo)fibroblasts in the presence of {32P}NAD was analyzed by isoelectric focusing, fluorography and immunoblotting . The influence of C2 toxin on microfilaments in intact cells was further assessed by immunofluorescence, and motility was measured in a mass migration assay and by computerized video time-lapse microscopy . We show here that C2 toxin specifically ribosylates beta- and gamma-actin in both fibroblasts and myofibroblasts . Whereas fibroblasts rapidly round up and stop migrating when filamentous beta-/gamma-actin is reduced by short-term ADP-ribosylation, myofibroblasts maintain their flattened morphology and a basic low motility.

J Pharm Pharmacol, 1996 Nov, 48(11), 1206 - 9
The effect of gamma-irradiation on the antibacterial activity of honey; Molan PC et al.; There is increasing usage of honey as a dressing on infected wounds, burns and ulcers, but there is some concern that there may be a risk of wound botulism from the clostridial spores sometimes found in honey . It is well-established that the antibacterial activity is heat-labile so would be destroyed if honey were sterilized by autoclaving, but the effect of gamma-irradiation on the antibacterial activity of honey is not known . Therefore an investigation was carried out to assess the effect on the antibacterial activity of honey when the honey was subjected to a commercial sterilization procedure using gamma-irradiation (25 kGy) . Two honeys with antibacterial activity due to enzymically-generated hydrogen peroxide and three manuka honeys with non-peroxide antibacterial activity were investigated . The honeys were tested against Staphylococcus aureus in an agar well diffusion assay . There was no significant change found in either type of antibacterial activity resulting from this form of sterilization of honey, even when the radiation was doubled (to 50 kGy) . Testing of honey seeded with spores of Clostridium perfringens and C . tetani (10000 and 1000 spores g-1 of honey, respectively) showed that 25 kGy of gamma-irradiation was sufficient to achieve sterility.

Chemotherapy, 1996 Nov-Dec, 42(6), 410 - 25
In vitro activity of BAY 12-8039, a new 8-methoxyquinolone; Dalhoff A et al.; BAY 12-8039 is a new 8-methoxyquinolone with antibacterial activity against gram-positive bacteria which is significantly better than those of sparfloxacin or ciprofloxacin . The minimal inhibitory concentrations (MICs) for 90% of methicillin-susceptible Staphylococcus aureus and Staphylococcus epidermidis were 0.062 and 2 mg/l, respectively . The MIC90s for ciprofloxacin-resistant, methicillin-susceptible and methicillin-resistant S . aureus were 8 mg/l . Against the staphylococcal strains tested sparfloxacin was 2-fold and ciprofloxacin > or = 10-fold less active . MIC90s for Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus agalactiae were 0.125-0.5 mg/l, irrespective of whether strains with diminished ciprofloxacin susceptibility or ciprofloxacin-susceptible strains were tested . Against the streptococci sparfloxacin was 2- to 4-fold less active . Against gram-negative bacteria BAY 12-8039 is almost as active as ciprofloxacin, except for Pseudomonas aeruginosa . Against Bacteroides fragilis, Bacteroides spp . and Clostridium spp . BAY 12-8039 was as active as metronidazole . The bactericidal activity against S . aureus and S . pneumoniae was in contrast to that of the other quinolones tested, penicillin G, amoxicillin+/-clavulanate, cefuroxime and clarithromycin, concentration-dependent . As compared to ciprofloxacin, development of resistance was less pronounced . The spontaneous mutation frequency towards BAY 12-8039 resistance was 2.8 x 10(-8) in Escherichia coli, 7.06 x 10(-8) in S . aureus and < 1.4 x 10(-9) in S . pneumoniae.

Eur J Biochem, 1996 Nov 1, 241(3), 941 - 7
Contribution of individual tryptophan residues to the structure and activity of theta-toxin (perfringolysin O), a cholesterol-binding cytolysin; Sekino-Suzuki N et al.; theta-Toxin (perfringolysin O), secreted by Clostridium perfringens, shares with other known thiol-activated toxins a conserved undecapeptide, ECTGLAWEWWR, located in the C-terminal region of the protein and containing the unique cysteine of the molecule . Single and double amino acid substitutions were created in the theta-toxin molecule to investigate the role of individual tryptophan residues in the lytic activity of theta-toxin . Wild-type and mutant theta-toxins were overproduced in Escherichia coli by means of a T7 RNA polymerase/promoter system and purified . The relative hemolytic activities of four mutant toxins, each with a Trp to Phe substitution outside the common Cys-containing region, were more than 60% that of wild-type theta-toxin . In contrast, mutant toxins with Phe replacements within the Cys-containing region (at Trp436, Trp438 or Trp439) showed significantly reduced hemolytic and erythrocyte-membrane-binding activities . The largest reduction in binding affinity, more than 100-fold, was observed for Trp438 mutant toxins . However, the mutants retain binding specificity for cholesterol and the ability to form arc-shaped and ring-shaped structures on membranes . These results indicate that the low hemolytic activities of these mutant toxins can be ascribed, at least in part, to reduced binding activities . With respect to protease susceptibility and far-ultraviolet circular-dichroism spectra, only the W436-->F mutant toxin, showed any considerable difference from wild-type toxin in secondary or higher-order structures, indicating that Trp436 is essential for maintenance of toxin structure.

Eur J Gastroenterol Hepatol, 1996 Nov, 8(11), 1054 - 61
Management of Clostridium difficile infection and other antibiotic-associated diarrhoeas; Bartlett JG; Most cases of antibiotic-associated diarrhoea are due to Clostridium difficile or are enigmatic . Patients with C . difficile-associated disease are more likely to have colitis, severe disease and nosocomial acquisition . The preferred diagnostic test is a C . difficile toxin assay using a tissue culture assay or enzyme immunoassay . The usual treatment is withdrawal of the inducing agent, sometimes supplemented with oral vancomycin or metronidazole . Response rates approach 100%; the major complication is relapse . Major complications include toxic megacolon, devastating diarrhoea, pseudomembranous colitis and hypoalbuminemia . Antibiotic-associated diarrhoea with negative tests for C . difficile toxin is usually dose-related and responds to antibiotic withdrawal.

Eur J Gastroenterol Hepatol, 1996 Nov, 8(11), 1048 - 53
Immune response to Clostridium difficile infection; Kelly CP; Clostridium difficile produces two toxins (A and B) which cause antibiotic-associated diarrhoea and pseudomembranous colitis . One of the most puzzling aspects of C . difficile infection is the wide spectrum of clinical presentation which ranges from asymptomatic carriage to fulminant, life-threatening colitis . This review examines the hypothesis that immune responses to C . difficile underlie these dramatic variations in disease presentation and course . Animals can be protected from C . difficile colitis by passive or active immunization against toxins A and B . Human antibody responses to these toxins are evident in approximately 60% of the general population . A number of clinical studies indicate that antitoxin responses in both serum and intestinal secretions may be protective whereas an inadequate immune response predisposes to severe or recurrent C . difficile diarrhoea . Thus there is now considerable interest in developing methods for passive and active immunization to combat this prevalent nosocomial intestinal pathogen.

Eur J Gastroenterol Hepatol, 1996 Nov, 8(11), 1041 - 7
Pathogenesis of Clostridium difficile-associated diarrhoea; Pothoulakis C; Clostridium difficile is now regarded as a major enteric pathogen in hospitals and nursing-home facilities . The pathophysiology of this pathogen involves alterations of the indigenous colonic flora by antibiotics, ingestion of spores and colonization by C . difficile, followed by release of its toxins . Although most of the research on the intestinal effects of C . difficile had been focused on its enterotoxin or toxin A, recent results indicate that toxin B, the cytotoxin of C . difficile, is also active in human colon . The cloning and sequencing of the toxin A and toxin B gene and the identification of the GTP-binding protein Rho as their intracellular target represent major advances in our understanding of the mode of action of these toxins . An important characteristic of C . difficile infection is the dramatic inflammation seen in pseudomembranous colitis . Recent studies indicate that an interplay between lamina propria neuroimmune cells and intestinal epithelial cells may be central in pathogenesis of this toxin-mediated inflammatory response.

Eur J Gastroenterol Hepatol, 1996 Nov, 8(11), 1035 - 40
Epidemiology and typing of Clostridium difficile; Jumaa P et al.; Clostridium difficile is now established as the most common nosocomial enteric pathogen causing pseudomembranous colitis, antibiotic-associated colitis and antibiotic-associated diarrhoea . Antibiotic therapy is the most important risk factor in colonization and infection with C . difficile . However, other factors are involved such as age and underlying illness . The introduction of reliable typing and fingerprinting methods has demonstrated hospital acquisition and cross-infection with C . difficile and has been important in improving our understanding of the epidemiology and pathogenicity of C . difficile.

Hum Exp Toxicol, 1996 Nov, 15(11), 904 - 8
Assessment of aspects of the toxicity of Clostridium perfringens epsilon-toxin using the MDCK cell line; Lindsay CD; 1 . The epithelial Madin Darby Canine Kidney (MDCK) cell line was used to study the toxicity of epsilon-toxin from Clostridium perfringens . The epithelial MDCK cell line is known to be sensitive to epsilon-toxin of Clostridium perfrigens and to investigate its mechanism of action, the neutral red assay has been used to dermine the viability of cultures of this cell line . 2 . Comparison of the LC50s obtained at 34 degrees C and 0 degree C showed that the lethality of epsilon-toxin was reduced by 18-fold at the lower temperature . The effect of temperature on epsilon-toxin lethality is unlikely to be due to reductions in membrane fluidity for the addition of Ca2+ or Mg2+ (2 mM) to buffer containing toxin was without effect . Varying the pH of the toxin-containing buffer from 6.9 to 8.7 did not increase the lethality of the toxin, though the most acidic pH used (5.8) was found to potentiate its action on MDCK cells . 3 . The effect of inhibiting endocytosis on the lethality of epsilon-toxin was also investigated by incubating cultures of MDCK cells with and without sodium azide over a range of concentrations of toxin . The co-administration of sodium azide did not reduce the toxicity of epsilon-toxin, suggesting that energy-dependent uptake processes such as endocytosis were unlikely to be involved in its mechanism of action . The results are, however, consistent with known receptor-based mechanisms of uptake and with other mechanisms of internalisation across the plasma membrane . epsilon-toxin thus interacts with cell surfaces by a temperature sensitive mechanism potentiated by low pH.

Biochem J, 1996 Nov 1, 319 ( Pt 3), 843 - 9
Microinjection of ADP-ribosylated actin inhibits actin synthesis in hepatocyte-hepatoma hybrid cells; Reuner KH et al.; Treatment of hepatocyte-hepatoma hybrid cells with Clostridium botulinum C2 toxin led to a 167% increase in monomeric globular actin (G-actin) and to a 57% decrease in filamentous actin (F-actin) within 2 h . Simultaneously, the level of actin mRNA was specifically decreased to 49% and actin synthesis was significantly diminished . In contrast, treatment of hybrid cells with phalloidin led to a decrease in G-actin to 55% and to a reciprocal increase in actin mRNA to 244% and an increase in actin synthesis . These alterations of actin synthesis depending on the G-actin/F-actin ratio corresponded to the autoregulation of actin synthesis observed in primary cultures of rat hepatocytes . Microinjection of C2 toxin or of phalloidin into hepatocyte-hepatoma hybrid cells had the same effects on actin synthesis as incubation with either toxin in the culture medium . Microinjection of nonpolymerizable ADP-ribosylated G-actin into hepatocyte-hepatoma hybrid cells specifically decreased the incorporation of {35S}methionine into newly synthesized actin within 1 h . This decrease continued for at least 19 h . Microinjection of ADP-ribosylated actin led to rounding of cells and obvious disaggregation of actin filaments, which might be due to capping of actin filaments by the ADP-ribosylated actin . Because stabilization of actin filaments by phalloidin before microinjection of ADP-ribosylated actin also resulted in decreased actin synthesis, the concentration of monomeric G-actin seems to be responsible for the regulation of actin synthesis in hepatocyte-hepatoma hybrid cells, which can be regarded as immortalized hepatocytes.

EMBO J, 1996 Nov 1, 15(21), 5739 - 51
Crystal structure of a bacterial family-III cellulose-binding domain: a general mechanism for attachment to cellulose; Tormo J et al.; The crystal structure of a family-III cellulose-binding domain (CBD) from the cellulosomal scaffoldin subunit of Clostridium thermocellum has been determined at 1.75 A resolution . The protein forms a nine-stranded beta sandwich with a jelly roll topology and binds a calcium ion . conserved, surface-exposed residues map into two defined surfaces located on opposite sides of the molecule . One of these faces is dominated by a planar linear strip of aromatic and polar residues which are proposed to interact with crystalline cellulose . The other conserved residues are contained in a shallow groove, the function of which is currently unknown, and which has not been observed previously in other families of CBDs . On the basis of modeling studies combined with comparisons of recently determined NMR structures for other CBDs, a general model for the binding of CBDs to cellulose is presented . Although the proposed binding of the CBD to cellulose is essentially a surface interaction, specific types and combinations of amino acids appear to interact selectively with glucose moieties positioned on three adjacent chains of the cellulose surface . The major interaction is characterized by the planar strip of aromatic residues, which align along one of the chains . In addition, polar amino acid residues are proposed to anchor the CBD molecule to two other adjacent chains of crystalline cellulose.

Clin Diagn Lab Immunol, 1996 Nov, 3(6), 782 - 5
Macrofragment localization of the toxin A and toxin B genes of Clostridium difficile; Norwood DA Jr et al.; We report the physical mapping of the toxin A and B genes to the bacterial chromosome of Clostridium difficile ATCC 43594 by pulsed-field gel electrophoresis . Single and double digestions with restriction endonucleases NruI and SacII allowed localization of the toxin genes to a specific 577-kb fragment and estimation of genome size to be approximately 3.8 megabases . This effort represents the initial step in the construction of a physical map of the whole genome.

Antimicrob Agents Chemother, 1996 Nov, 40(11), 2500 - 4
Genetic organization and distribution of tetracycline resistance determinants in Clostridium perfringens; Lyras D et al.; The Tet P determinant from the conjugative Clostridium perfringens R plasmid pCW3 two functional overlapping tetracycline resistance genes, tetA(P) and tetB(P) . The tetA(P) gene encodes a putative 46-kDa transmembrane protein which mediates active efflux of tetracycline from the cell, while tetB(P) encodes a putative 72.6-kDa protein which has significant similarity to Tet M-like tetracycline resistance proteins (J . Sloan, L.M . McMurry, D . Lyras, S . B . Levy, and J . I . Rood, Mol . Microbiol . 11:403-415, 1994) . In the present study, hybridization and PCR analysis of 81 tetracycline-resistant isolates of C . perfringens showed that they all carried the tetA(P) gene . Most of these isolates (93%) carried a second tetracycline resistance gene, with 53% carrying tetB(P) and 40% carrying a tet(M)-like gene . Despite the wide distribution of the tetB(P) and tet(M) genes, no isolate which carried both of these determinants was detected . In isolates that carried both tetA(P) and tetB(P) these genes overlapped, as in pCW3 . Isolates carrying this combination of genes originated from diverse geographical locations and environmental sources . The single Clostridium paraputrificum isolate examined carried tetA(P), indicating that this gene is not confined to C.perfringens . However, neither tetA(P) nor tetB(P) was detected in the nine Clostridium difficile isolates tested . Nucleotide sequence analysis of isolates lacking tetB(P) revealed that they contained the tetA408(P) gene, which lacked the codons for the 12 carboxy-terminal amino acids of the TetA(P) protein.

Arch Surg, 1996 Nov, 131(11), 1193 - 201
Management of intra-abdominal infections . The case for intraoperative cultures and comprehensive broad-spectrum antibiotic coverage . The Canadian Intra-abdominal Infection Study Group; Christou NV et al.; OBJECTIVE: To test the hypothesis that comprehensive broad-spectrum empirical antimicrobial therapy is superior to limited-spectrum empirical antimicrobial therapy in intra-abdominal infections . DESIGN: Prospective, randomized, double-blinded study . SETTING: University-affiliated hospitals in Canada . PATIENTS: Two hundred thirteen patients with intra-abdominal infections and planned operative or percutaneous drainage . INTERVENTION: Limited-spectrum empirical antimicrobial therapy consisted of cefoxitin sodium, 2 g, intravenously, every 6 hours (n = 109) . Comprehensive broad-spectrum empirical antimicrobial therapy consisted of a combination of imipenem and cilastatin sodium, 500 mg, intravenously, every 6 hours (n = 104) . MAIN OUTCOME MEASURES: Failure to cure the intra-abdominal infection (persistence of infection or death) . RESULTS: Of initial isolates, 98% were sensitive to imipenem plus cilastin sodium compared with 72% for cefoxitin . No difference was found in the failure rate between treatment groups . Among various reasons for failure (including technical), 12 of 80 patients in the limited-spectrum empirical antimicrobial therapy group had resistant organisms at a second intervention compared with 1 of 74 in the comprehensive broad-spectrum empirical antimicrobial therapy group (P < .003, chi 2) . One death in the limited-spectrum empirical antimicrobial therapy group was due to autopsy-proved disseminated Pseudomonas aeruginosa (blood, peritoneum, lung, and pleural fluid) that was resistant to cefoxitin, and the other was associated with peritonitis due to cefoxitin-resistant Enterobacter cloacae . One death in the comprehensive broad-spectrum empirical antimicrobial therapy group was associated with peritonitis from Clostridium perfringens that was sensitive to imipenem plus cilastin sodium, and the other was associated with peritonitis from Pseudomonas aeruginosa that was resistant to imipenem plus cilastin sodium . CONCLUSION: Treatment failure of intra-abdominal infection may be due, in part, to the presence of resistant pathogens at the site of infection . Therefore, routine culture of these sites seems worthwhile and empirical therapy should be as comprehensive as possible and should cover all potential pathogens.

Am J Med Sci, 1996 Nov, 312(5), 242 - 5
Case report: empyema with hydropneumothorax and bacteremia caused by Clostridium sporogenes; Corbett CE et al.; Clostridia species are rare causes of pleuropulmonary infections . This report describes an immunocompromised patient who had a renal transplant, had multiple risk factors for anaerobic pleuropulmonary infection, and developed an acute empyema with hydropneumothorax that was associated with Clostridium sporogenes bacteremia . Therapy included antibiotics and surgical drainage of the empyema . Species identification of clostridia can usually be limited to whether the species is perfringens or nonperfringens because the majority of clinically significant clostridial infections are caused by Clostridium perfringens . Increased cost and consumption of time limits the usefulness of species identification of nonperfringens species . However, the identification of clostridia species that are known to be associated with specific underlying diseases or known to have variable and unpredictable antibiotic susceptibilities may affect patient management . The role of the laboratory in identifying such anaerobic isolates is discussed.

Gastroenterology, 1996 Nov, 111(5), 1272 - 80
Substance P activation of enteric neurons in response to intraluminal Clostridium difficile toxin A in the rat ileum; Mantyh CR et al.; BACKGROUND & AIMS: Nerves have been suggested to mediate the effects of bacterial toxins in intestinal diseases . However, the mechanisms involved are unknown . This study examined endogenous substance P (SP) activation of the substance P receptor (SPR) on enteric neurons in the rat ileum after exposure to intraluminal Clostridium difficile toxin A . METHODS: After intraluminal injection of toxin A in ileal loops, tissue was examined for pathological changes by histology and for SPR activation by immunocytochemical analysis of SP-induced SPR endocytosis . RESULTS: After toxin A administration, > 70% of enteric neurons showed SPR endocytosis and became swollen with thickened dendrites . In contrast, SPRs in control rats were largely confined to the plasma membrane . Rats denervated of primary afferent fibers with neonatal capsaicin injection and animals pretreated with a nonpeptide SPR antagonist showed few endosomal SPRs, and the pathological inflammatory effects of toxin A were ablated . CONCLUSIONS: Intraluminal toxin A causes the release of SP from primary afferent neurons: this endogenous SP then acts on enteric neurons in the submucosal and myenteric plexuses . SP is the primary mediator of an axon reflex mediating neurogenic inflammation in the intestine . SPR blockade may prove to be a novel therapy used to prevent intestinal inflammation.

J Clin Microbiol, 1996 Nov, 34(11), 2718 - 21
Multicenter evaluation of four methods for Clostridium difficile detection: ImmunoCard C . difficile, cytotoxin assay, culture, and latex agglutination; Staneck JL et al.; A three-center study was undertaken to compare several test methods for the detection of Clostridium difficile, associated toxin, or related markers by using 927 stool specimens . Methods included direct assay of cytotoxin in stool by tissue culture, C . difficile bacterial culture followed by cytotoxin assay, bacterial culture alone, latex agglutination assay, and the ImmunoCard C . difficile test (Meridian Diagnostics, Inc.) . The sensitivities, as determined against direct cytotoxin assay results, of the ImmunoCard C . difficile and latex agglutination assays were 84 and 67%, respectively (92 and 77%, respectively, when adjusted for bacterial culture outcomes) . Evaluation for C . difficile-associated disease (CDAD) among 864 patients was based on clinical criteria for antibiotic-associated diarrhea combined with laboratory evidence of toxin or toxin-producing C . difficile in stool specimens . The sensitivity of each test method for screening of CDAD was as follows: bacterial culture, 95%; culture with cytotoxin assay of isolates, 90%; ImmunoCard C . difficile test, 83%; cytotoxin assay 82%; and latex agglutination assay, 67% (P < or = 0.05 versus all other methods) . The standard deviations of the test sensitivity statistics between study sites were ranked as follows: cytotoxin assay (+/- 3.1%) < ImmunoCard C . difficile test (+/- 5.7%) < latex agglutination assay (+/- 12.3%) < culture (+/- 24.7%) < culture with cytotoxin assay (+/- 28.0%) . The data support the use of the ImmunoCard C . difficile test as an adjunct for the diagnosis of CDAD.

Infect Immun, 1996 Nov, 64(11), 4433 - 7
Effects of Clostridium difficile toxin A and toxin B on phospholipase D activation in human promyelocytic leukemic HL60 cells; Ohguchi K et al.; The possible involvement of Rho family GTP-binding proteins in the regulation of phospholipase D (PLD) activity has recently been demonstrated . In the present study, to further examine the role of Rho family proteins in PLD activation of human promyelocytic leukemic HL60 cells, we used toxin A and toxin B from the anaerobic bacterium Clostridium difficile, which was shown to glucosylate Rho family proteins and inhibit their interaction with effectors . Pretreatment of {3H}oleic acid-labeled HL60 cell lysates with either one of the toxins resulted in a remarkable inhibition of membrane PLD activity stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) . The magnitude of inhibition of PLD activity was correlated well with the extent of toxin A- or B-induced glucosylation of 22-kDa RhoA in HL60 cells, toxin B being more effective than toxin A . GTPgammaS-stimulated PLD activation measured with the exogenous substrate containing phosphatidylinositol 4,5-bisphosphate was also inhibited by toxin B . Toxin B had no effect on GTP-gammaS-induced translocation of RhoA from cytosol to membranes . Furthermore, the toxin B pretreatment also suppressed PLD activation induced by 4beta-phorbol 12-myristate 13-acetate in HL60 cell lysates . Thus, it was indicated that Rho family proteins play a key role in GTPgammaS- and 40-phorbol 12-myristate 13-acetate-induced PLD activity in HL60 cells . In addition, the results obtained here indicate that C . difficile toxins are a useful tool for researching the regulation of the Rho family protein-mediated PLD activation and also provide a clue toward understanding the pathogenic background of pseudomembranous colitis from the viewpoint of signal transduction.

Curr Microbiol, 1996 Nov, 33(5), 281 - 6
Clostripain linker deletion variants yield active enzyme in Escherichia coli: a possible function of the linker peptide as intramolecular inhibitor of clostripain automaturation; Witte V et al.; The clostripain core protein is composed of the light and heavy chain subunits linked by a nonapeptide into a single polypeptide chain {Mol . Gen . Genet . 240: 140, 1993} . Linker removal is due to autocatalytic processing yielding active heterodimeric enzyme . We have expressed mutationally altered core protein variants in the heterologous host Escherichia coli to gain further insight into the process of clostripain automaturation . In a mutationally created Cys231 --> Ser variant, heterodimer formation was largely impaired, providing molecular evidence that the capacity for automaturation is attributed to the active site cysteine, Cys231, of the native enzyme . Artificially generated deletions of the linker peptide did not prevent the formation of active enzyme . One variant gave rise to a single-chain molecule devoid of the authentic processing sites while retaining enzymatic activity . Experiments performed with linker substitution variants suggested that the efficacy of automaturation depends on a proper configuration of the linker region . According to computerized predictions, the formation of a turn-structured protein loop or hinge with hydrophilic characteristics in the linker region is probably a prerequisite for the interaction of the active site cysteine with the processing sites, Arg181 and Arg190 . We propose that the clostripain linker nonapeptide serves as an important transient intramolecular inhibitor in the cellular self-defense program evolved by the natural host Clostridium histolyticum.

Biochemistry, 1996 Oct 29, 35(43), 13772 - 9
Temperature dependence of the redox potential of rubredoxin from Pyrococcus furiosus: a molecular dynamics study; Swartz PD et al.; Molecular dynamics simulations are used to evaluate the temperature dependent differences in structure, solvation, and energies for the iron-sulfur protein rubredoxin from the hyperthermophilic archebacterium Pyrococcus furiosus to understand the unusual temperature dependence of its redox potential {Adams, M . W . W . (1992) Adv . Inorg . Chem . 38, 341-396} . Simulations of both redox states performed at 295 and 363 K reveal that almost no backbone structure alteration occurs at the higher temperature and that the radius of gyration of the protein is temperature and redox state independent . The most striking change is that the penetration of the redox site by solvent molecules in the reduced from at 295 K, which was also seen in simulations of the reduced form of the mesophilic Clostridium pasteurianum rubredoxin at 295 K (Yelle, R . B., et al . (1995) Proteins 22, 154-167}, is no longer seen to a significant extent in either redox state at 363 K . Comparing 295 to 363 K, the calculated change in the electrostatic potential of about -300 mV and in the negative of the potential energy of about -550 meV is consistent with the observed change in redox potential of -160 mV . Moreover, the calculated change is in the wrong direction if the penetrating water is excluded . These results show that changing solvent accessibility may be responsible for the temperature dependence of the redox potential of P . furiosus rubredoxin.

MMWR CDC Surveill Summ, 1996 Oct 25, 45(5), 1 - 66
Surveillance for foodborne-disease outbreaks--United States, 1988-1992; Bean NH et al.; PROBLEM/CONDITION: Since 1973, CDC has maintained a collaborative surveillance program for collection and periodic reporting of data concerning the occurrence and causes of foodborne-disease outbreaks (FBDOs) . REPORTING PERIOD COVERED: This summary reviews data from January 1988 through December 1992 . DESCRIPTION OF SYSTEM: The surveillance system reviews data concerning FBDOs--defined as the occurrence of two or more cases of a similar illness resulting from the ingestion of a common food . Before 1992, only one case of intoxication by chemical, marine toxin, or Clostridium botulinum toxin as a result of the ingestion of food was required to constitute an FBDO . Since 1992, two or more cases have been required . State and local public health departments have primary responsibility for the identifying and investigating FBDOs . State and territorial health departments report these outbreaks to CDC on a standard form . RESULTS: During 1988-1992, a total of 2,423 outbreaks of foodborne disease were reported (451 in 1988, 505 in 1989, 532 in 1990, 528 in 1991, and 407 in 1992) . These outbreaks caused a reported 77,373 persons to become ill . Among outbreaks for which the etiology was determined, bacterial pathogens caused the largest percentage of outbreaks (79%) and the largest percentage of cases (90%) . Salmonella serotype Enteritidis accounted for the largest number of outbreaks, cases, and deaths; most of these outbreaks were attributed to eating undercooked, infected eggs . Chemical agents caused 14% of outbreaks and 2% of cases; parasites, 2% of outbreaks and 1% of cases; and viruses, 4% of outbreaks and 6% of cases . INTERPRETATION: The number of FBDOs reported per year did not change substantially during the first 4 years but declined in 1992 as a result of the revised definition of an outbreak . During this reporting period, S . Enteritidis continued to be a major cause of morbidity and mortality . In addition, multistate outbreaks caused by contaminated produce and outbreaks caused by Escherichia coli O157:H7 became more prominent . ACTIONS TAKEN: State and local public health departments investigate FBDOs . At the regional and national level, surveillance data provide an indication of the etiologic agents, vehicles of transmission, and contributing factors associated with FBDOs and help direct public health actions.

FEBS Lett, 1996 Oct 21, 395(2-3), 191 - 4
Evidence that Arg-295, Glu-378, and Glu-380 are active-site residues of the ADP-ribosyltransferase activity of iota toxin; Perelle S et al.; The active site of the enzymatic component (Ia) of the Clostridium perfringens iota toxin has been studied by site-directed mutagenesis . Sequence alignment showed that Ia and C3 enzymes display a segment in their C-terminal part which is homologous to that forming the active domain of pertussis toxin, cholera toxin, and Escherichia coli thermolabile toxins . This structure consists of a beta-strand and an alpha-helix which forms the NAD-binding cavity and which is flanked by two catalytic spatially conserved residues involved in catalysis {Domenighini et al . (1994) Mol . Microbiol . 14, 41-50} . Substitutions (Arg-295-Lys, Glu-378-Ala, Glu-380-Asp, and Glu-380-Ala) induced a drastic decrease in ADP-ribosylation and cytotoxic activities, while substitution of the adjacent Arg (Arg-296-Lys) only partially affected the enzymatic activity and cytotoxicity . These results indicate that Arg-295, Glu-378 and Glu-380 of Ia are involved in the ADP-ribosylation activity which is essential for the morphological changes of cells treated with iota toxin.

Biochim Biophys Acta, 1996 Oct 17, 1297(2), 149 - 58
Construction of a dimeric form of glutamate dehydrogenase from Clostridium symbiosum by site-directed mutagenesis; Pasquo A et al.; By using site-directed mutagenesis, Phe-187, one of the amino-acid residues involved in hydrophobic interaction between the three identical dimers comprising the hexamer of Clostridium symbiosum glutamate dehydrogenase (GDH), has been replaced by an aspartic acid residue . Over-expression in Escherichia coli led to production of large amounts of a soluble protein which, though devoid of GDH activity, showed the expected subunit M(r) on SDS-PAGE, and cross-reacted with an anti-GDH antibody preparation in Western blots . The antibody was used to monitor purification of the inactive protein . F187D GDH showed altered mobility on non-denaturing electrophoresis, consistent with changed size and/or surface charge . Gel filtration on a calibrated column indicated an M(r) of 87000 +/- 3000 . The mutant enzyme did not bind to the dye column routinely used in preparing wild-type GDH . Nevertheless suspicions of major misfolding were allayed by the results of chemical modification studies: as with wild-type GDH, NAD+ completely protected one-SH group against modification by DTNB, implying normal coenzyme binding . A significant difference, however, is that in the mutant enzyme both cysteine groups were modified by DTNB, rather than C320 only . The CD spectrum in the far-UV region indicated no major change in secondary structure in the mutant protein . The near-UV CD spectrum, however, was less intense and showed a pronounced Phe contribution, possibly reflecting the changed environment of Phe-199, which would be buried in the hexamer . Sedimentation velocity experiments gave corrected coefficients S20,W of 11.08 S and 5.29 S for the wild-type and mutant proteins . Sedimentation equilibrium gave weight average molar masses M(r,app) of 280000 +/- 5000 g/mol . consistent with the hexameric structure for the wild-type protein and 135000 +/- 3000 g/mol for F187D . The value for the mutant is intermediate between the values expected for a dimer (98000) and a trimer (147000) . To investigate the basis of this, sedimentation equilibrium experiments were performed over a range of protein concentrations . M(r,app) showed a linear dependence on concentration and a value of 108 118 g/mol at infinite dilution . This indicates a rapid equilibrium between dimeric and hexameric forms of the mutant protein with an equilibrium constant of 0.13 l/g . An independent analysis of the radial absorption scans with Microcal Origin software indicated a threefold association constant of 0.11 l/g . Introduction of the F187D mutation thus appears to have been successful in producing a dimeric GDH species . Since this protein is inactive it is possible that activity requires subunit interaction around the 3-fold symmetry axis . On the other hand this mutation may disrupt the structure in a way that cannot be extrapolated to other dimers . This issue can only be resolved by making alternative dimeric mutants.

J Physiol, 1996 Oct 15, 496 ( Pt 2), 317 - 29
Role of Rho proteins in carbachol-induced contractions in intact and permeabilized guinea-pig intestinal smooth muscle; Otto B et al.; 1 . The aim of this study was to determine whether the low molecular mass GTPase RhoA or related proteins are involved in carbachol- and high-K(+)-induced contractions in intact intestinal smooth muscle as well as the carbachol-induced increase in Ca2+ sensitivity of the myofilaments in permeabilized preparations . 2 . The carbachol-induced increase in the Ca2+ sensitivity of force production in beta-escin-permeabilized intestinal smooth muscle was enhanced in preparations that were loaded with the constitutively active mutant of RhoA, Val14RhoA, and was inhibited by exoenzyme C3 from Clostridium botulinum, which ADP-ribosylates and inactivates small GTPases of the Rho family . The effect of C3 on Ca2+ sensitivity in the absence of the agonist was negligible, while the maximal Ca(2+)-activated force was inhibited by about 20% . 3 . Inhibition of carbachol-induced force was associated with an increase in ADP-ribosylation of a protein band with a molecular mass of approximately 22 kDa, corresponding to Rho, and was partially reversed in the presence of Ile41RhoA, which is not a substrate for C3 . Val14RhoA did not restore carbachol-induced Ca2+ sensitization in C3-treated smooth muscle . 4 . In intact intestinal smooth muscle, toxin B from Clostridium difficile, which monoglucosylates members of the Rho family, inhibited high-K(+)-induced contractions and the initial phasic response to carbachol by about 30% . The delayed contractile response to carbachol was completely inhibited . 5 . In smooth muscle preparations that were permeabilized with beta-escin after treatment with toxin B, carbachol-and GTP gamma S-induced Ca2+ sensitization was significantly inhibited . 6 . These findings are consistent with a role for Rho or Rho-like proteins in agonist-induced increase in Ca2+ sensitivity of force production in intact and permeabilized intestinal smooth muscle.

Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 413 - 8
Purification and some properties of Clostridium sporogenes hemorrhagic toxin; Hara-Kudo Y et al.; Clostridium sporogenes was isolated from rabbits with antibiotic-associated hemorrhagic diarrhea, then its hemorrhagic toxin was purified from the supernatant by means of hydrophobic interaction chromatography, hydroxyapatite chromatography, and gel filtration . The purified toxin's hemorrhagic activity was completely inhibited by EDTA, but not by PMSF or ovomucoid; and fully restored by the addition of such divalent cations as Zn2+, Ca2+ and Mg2+ . The purified toxin did not hydrolyze azocasein, or type I or II collagens . But the purified toxin did hydrolyze type III and IV collagens, and also gelatins prepared from type I, II, III and IV collagens . It appears, therefore that C . sporogenes's toxin is a collagenase that hydrolyzes type III and IV collagens, which are major constituents of the tunica intima and media of blood vessels . And that fact suggests that hemorrhage caused by the toxin depend on its collagenase activity.

J Biol Chem, 1996 Oct 11, 271(41), 25173 - 7
Clostridium novyi alpha-toxin-catalyzed incorporation of GlcNAc into Rho subfamily proteins; Selzer J et al.; The lethal and edema-inducing alpha-toxin from Clostridium novyi causes rounding up of cultured cell lines by redistribution of the actin cytoskeleton . alpha-Toxin belongs to the family of large clostridial cytotoxins that encompasses Clostridium difficile toxin A and B and the lethal toxin from Clostridium sordellii . Toxin A and toxin B have been recently identified as monoglucosyltransferases to modify the low molecular mass GTPases of the Rho subfamily (Just, I., Selzer, J., Wilm, M., Von Eichel-Streiber, C., Mann, M., and Aktories, K . (1995) Nature 375, 500-503 and Just, I., Wilm, M., Selzer, J., Rex, G., Von Eichel-Streiber, C., Mann, M., and Aktories, K . (1995) J . Biol . Chem . 270, 13932-13936) . We report here the identification of the alpha-toxin-catalyzed modification of Rho . Using electrospray mass spectrometry, the mass of the modification was determined as 203 Da, consistent with a N-acetyl-hexosamine moiety . UDP-N-acetyl-glucosamine selectively served as cosubstrate for alpha-toxin-catalyzed modification into the Rho subfamily proteins Rho, Rac, Cdc42, and RhoG . The acceptor amino acid of N-acetyl-glucosaminylation was identified by mutagenesis as Thr-37 in Rho (equivalent to Thr-35 in Rac/Cdc42), which is located in the effector domain of the GTPases . C . novyi alpha-toxin seems to mediate its cytotoxic effects on cells by mimicking endogenous post-translational modification of cellular proteins.

Br J Hosp Med, 1996 Oct 16-Nov 5, 56(8), 398 - 400
Clostridium difficile; Settle CD; The current impact of Clostridium difficile will have been noticed by many clinicians, particularly those managing elderly patients . Infection with this bacterium can give rise to a wide range of symptoms, from diarrhoea to fulminating colitis and toxic megacolon . Patients may also be asymptomatically colonized by C . difficile . In this article the epidemiology and aetiology of C.difficile infection will be discussed, followed by an explanation of how diagnosis of cases is best achieved, how the disease is optimally treated and how cross-infection can be minimized.

J Mol Biol, 1996 Oct 4, 262(4), 413 - 20
Domain structure of the prokaryotic selenocysteine-specific elongation factor SelB; Kromayer M et al.; Incorporation of the non-canonical amino acid selenocysteine into proteins requires the activity of the elongation factor SelB which substitutes for the function of EF-Tu in contrast to EF-Tu, SelB binds selenocystylated tRNASec and an mRNA secondary structure adjacent to the UGA selenocysteine codon . To gain information on the domain structure of this specialized translation factor, the selB genes from two bacteria unrelated to Escherichia coli (Clostridium thermoaceticum and Desulfomicrobium baculatum) were cloned and sequenced . The derived amino acid residue sequences were compared to those of SelB from E . coli and Haemophilus influenzae and to EF-Tu sequences . The alignment revealed that SelB contains all three domains characterized for EF-Tu . A fourth, C-terminally located domain shows only limited sequence conservation within the four SelB proteins . To elucidate the function of this C-terminal part a structure-function analysis of SelB from E . coli was performed . It showed that a C-terminal 17 kDa subdomain of the translation factor, when expressed separately, specifically binds the mRNA secondary structure . The recognition motif itself could be reduced to a 17 nucleotide minihelix without loss of binding affinity and specificity . A truncated SelB lacking the mRNA binding domain was still able to interact with selenocysteyl-tRNASec . Expression of the mRNA binding domain alone suppressed selenocysteine insertion in vivo by competing with SelB for its binding site at the mRNA . The results indicate that SelB can be considered as an EF-Tu homolog hooked to the mRNA via its C-terminal domain.

Biochem Biophys Res Commun, 1996 Oct 3, 227(1), 77 - 81
The ras-related protein Ral is monoglucosylated by Clostridium sordellii lethal toxin; Hofmann F et al.; Clostridium sordellii lethal toxin (LT), a cytotoxin which causes preferential destruction of the actin cytoskeleton, has been recently identified as glucosyltransferase to modify the low molecular mass GTPases Rac, Ras and Rap . We report here on LT produced by C . sordellii strain 6018 which glucosylates in addition to Rac, Ras and Rap the Ral protein . LT from strain VPI9048 however does not glucosylate Ral . Besides recombinant Ral, cellular Ral is also substrate . In the GDP-bound form, Ral is a superior substrate to the GTP form . Acceptor amino acid for glucose is threonine-46 which is equivalent to threonine-35 in H-Ras located in the effector region . The Ral-glucosylating toxin is a novel isoform of Ras-modifying clostridial cytotoxins.

Proc Natl Sci Counc Repub China B, 1996 Oct, 20(4), 101 - 9
Immunopotentiating activity of Clostridium butyricum in mice; Wang GR et al.; Bacterial vaccine, as generated by heat-inactivated Clostridium butyricum cells, displayed antitumor activity against sarcoma 180 in DDY mice and antimetastatic activity against B16-F10 melanoma in BDF1 mice . According to our results, the vaccine has no direct growth inhibitory effect toward the tumor cell lines tested in this study . The vaccine increased gamma-interferon production, elicited delayed type hypersensitivity reaction, and enhanced IgM antibody formation and mitogenicity . The phagocytic activity of macrophage and killing activity of NK cells from mice were enhanced in a dose-dependent manner by stimulating with the heat-inactivated vaccine . Among those responses in the mice treated with CB, elevated NK cell activity may play a prominent role in manifesting antitumor activity in the B16-F10 metastasis experiment.

Lett Appl Microbiol, 1996 Oct, 23(4), 218 - 22
16S rDNA analysis of Butyrivibrio fibrisolvens: phylogenetic position and relation to butyrate-producing anaerobic bacteria from the rumen of white-tailed deer; Forster RJ et al.; Complete 16S rDNA sequences of six strains of Butyrivibrio fibrisolvens, including the type strain (ATCC 19171), were determined . The type strain was found to have less than 89% sequence similarity to the other isolates that were examined . The five plasmid-bearing strains formed a closely related cluster and three of these strains (OB156, OB157 and OB192) were very highly related (> 99%), indicating that they are isolates of the same genomic species . The phylogenetic position of Butyrivibrio was found to be within the subphylum Clostridium, of Gram-positive bacteria . The closest relatives to the type strain were Eubacterium cellulosolvens and Cl . xylanolyticum and the closest relatives to the separately clustered strains were Roseburia ceciola, Lachnospira pectinoschiza and Eubacterium rectale.

Lett Appl Microbiol, 1996 Oct, 23(4), 208 - 12
Studies of Clostridium cellulolyticum ATCC 35319 under dialysis and co-culture conditions; Gehin A et al.; The degradation of cellulose by Clostridium cellulolyticum has been studied in several ways: (1) in batch fermentation in 50-ml sealed-cap flasks, referred to as the control; (2) in batch fermentation with pH at 7.2; (3) fermentation in dialysis which permits elimination of all the products of metabolism; (4) fermentation in dialysis with a constant bubbling of nitrogen; (5) in co-culture with Clostridium A22 in batch with and without pH regulation and with dialysis . H2, CO2, acetate, ethanol and lactate were the major end-products of cellobiose and cellulose fermentation . Compared to batch culture, growth of Cl . cellulolyticum on cellobiose increased by a factor of 10 in dialysed culture . The end products from the dialysed culture were detected in a small range compared to the concentration for the batch culture . Related to the biomass, CMCase activities were of the same level, showing a direct relation between the biomass formation and the cellulase production . The percentage of cellulose degradation (50%) by Cl . cellulolyticum was greater when dialysis of end products with a constant bubbling of nitrogen took place during the course of fermentation (6 d) in comparison with cultures in 50-ml sealed-cap flasks (23%), in a fermentor (36%) or using dialysis without N2 bubbling (40%) . The presence of two micro-organisms produced no further enzyme activities and hence the percentage of cellulose degradation was quite similar in mono- and co-culture . No synergistic action was found between two cellulolytic strains.

J Protein Chem, 1996 Oct, 15(7), 691 - 700
Mapping of the antibody-binding regions on botulinum neurotoxin H-chain domain 855-1296 with antitoxin antibodies from three host species; Atassi MZ et al.; Botulism due to food poisoning is caused mainly by protein toxins, botulinum neurotoxins (BoNTs), produced by Clostridium botluinum in seven known immunological serotypes . These are the most potent toxins and poisons known . BoNT effects blockade of neuromuscular transmission by preventing neurotransmitter release . Human botulism is most frequently caused by types A, B, and E . Recent studies have shown that immunization with a 43-kDa C-terminal fragment (Hc, residues 860-1296) of BoNT/A affords excellent protection against BoNT/A poisoning . We raised antibodies (Abs) against BoNT/A in horse, and against pentavalent toxoid (BoNTs A, B, C, D, E) in human volunteers and outbred mice . Thirty-one 19-residue peptides that started at residue 855, overlapped consecutively by 5 residues, and encompassed the entire length of the Hc of BoNT/A were synthesized and used for mapping the Ab-binding regions recognized by the anti-BoNT/A antisera . Horse Abs against BoBT/A were bound by peptides 855-873, 939-957, 1079-1097/1093-1111 overlap, 1191-1209/1205-1223 overlap, 1261-1279 and 1275-1296 . In addition, peptides 883-901, 911-929, 995-1013, 1023-1041/1037-1055 overlap, 1121-1139, and 1149-1167 gave low, but significant and reproducible, binding . With human antisera, high amounts of Abs were bound by peptides 869-887, 925-943, 981-999, 995-1013, 1051-1069, and 1177-1195 . In addition, lower amounts of Abs were bound by peptides 911-929, 939-957, 967-985, and the overlaps 1121-1139/1135-1153 and 1247-1265/1261-1279/1275-1296 . With outbred mouse antisera, high amounts of Abs were bound by peptides 869-887, 1051-1069, and 1177-1195, while peptides 939-957, 995-1013, 1093-1111, and 1275-1296 bound lower amounts of Abs . The results indicate that horse antiserum against BoNT/A or human and mouse (outbred) antisera against the toxoid recognized similar regions on BoNT/A, but exhibited some boundary frame shifts and differences in immunodominance of these regions among the antisera . Selected synthetic epitopes will be used as immunogens to stimulate active or passive (by Ab transfer) immunity against toxin poisoning.

Schweiz Rundsch Med Prax, 1996 Oct 1, 85(40), 1249 - 52
{Infectious diarrhea}; Krause M; Infectious diarrhea is a very common, usually self-limited disease . Among travellers to developing countries, diarrhea is by far the most common medical problem . The intake of sufficient glucose-electrolyte solutions is the most important step to prevent dehydration . Loperamide may be prescribed as a valuable antimotility agent: however, this drug should not be used in patients with high fevers, bloody diarrhea and severe abdominal cramps . Stool cultures are recommended in cases without improvement, when clostridium difficile is suspected and when multiple cases occur . Antibiotics are indicated for treatment of certain microorganisms, for patients with immunosuppression and in dysenteric syndromes . They are not recommended for prophylaxis on a routine basis.

Acta Paediatr Jpn, 1996 Oct, 38(5), 541 - 3
The first case of type B infant botulism in Japan; Kakinuma H et al.; A six-month-old girl with a 5 consecutive day history of constipation and poor feeding developed generalized weakness, poor head control, difficulties in sucking and swallowing, and cranial nerve dysfunction within a few days . These characteristic manifestations and clinical course prompted examination of the possibility of infant botulism, although no history of eating honey was obtained . Mouse bioassay performed with enema effluent demonstrated type B botulinum toxin . Culture of the effluent was positive for Clostridium botulinum type B . This is the first case of type B infant botulism in Japan.

Cardiovasc Res, 1996 Oct, 32(4), 699 - 708
Leukocyte-endothelial cell interactions evoked by mast cells; Kubes P et al.; In this review we have summarized some of the evidence to support the view that mast cells play a critical role in leukocyte recruitment to sites of inflammation . Initially, data using a pharmacological tool, compound 48/80, which directly activates mast cells, is reviewed, demonstrating that this reagent can induce the multi-step recruitment of leukocytes (rolling, adhesion and emigration) to sites of inflammation . The adhesive mechanisms and pro-inflammatory mediators implicated in mast cell-induced leukocyte recruitment are discussed . Additionally, data are presented to implicate mast cells in delayed-type hypersensitivity reactions as they pertain to leukocyte recruitment . There is a growing body of evidence to suggest that mast cells also recruit leukocytes in IgE-independent leukocyte recruitment . Ischemia/reperfusion- and bacterial toxin- (Helicobacter pylori and Clostridium difficile) induced leukocyte recruitment is at least in part mast cell dependent . Future directions including preliminary work highlighting the role of nitric oxide as a modulator of mast cell function and subsequent leukocyte recruitment is also discussed.

Lab Anim Sci, 1996 Oct, 46(5), 493 - 6
Detection of Clostridium piliforme by enzymatic assay of amplified cDNA segment in microtitration plates; Goto K et al.; We developed a simple enzymatic assay system using reverse transcription-polymerase chain reaction products in a microtitration plate to detect Clostridium piliforme in tissue specimens . The reverse transcription-polymerase chain reaction was performed with a biotin-labeled primer directed in a highly conserved area of the C . piliforme 16S rRNA gene . The amplified cDNA was hybridized to an alkaline phosphatase-labeled DNA probe in a microtube, and the mixture was applied to a microtitration well precoated with streptavidin . Then hybridization signals in the microtitration plate were visualized by reaction with substrate for the alkaline phosphatase . When an optical density > 0.200 at 405 nm was considered a positive result, we found that the system could detect the RNA from as few as 10 organisms . The system was also capable of detecting organisms from the liver, mesenteric lymph nodes, and ileum of experimentally infected mice . In the ileum the C . piliforme gene was detected from 1 to 21 days after infection . This result suggests that the intestinal epithelium is one of the sites of persistent infection . We conclude that this enzymatic assay system of reverse transcription-polymerase chain reaction products is simple, rapid, and convenient for the detection of C . piliforme.

J Exp Zool, 1996 Oct 1, 276(2), 125 - 31
Light-sensitive response in melanophores of Xenopus laevis: II.Rho is involved in light-induced melanin aggregation; Miyashita Y et al.; Melanophores of the isolated tail fin of the Xenopus tadpole aggregate melanin granules in response to light . This aggregation was found to be inhibited by subcutaneous injection of exoenzyme C3 of Clostridium botulinum . A 26 kDa protein in homogenate obtained from the Xenopus tail fin was ADP-ribosylated by exoenzyme C3 . This reaction was inhibited effectively by a monoclonal antibody, anti-Rho mab A5 . raised against the small GTP-binding protein Rho . The extent of ADP-ribosylation depended on light and guanine nucleotide . Incubation under illumination partly reduced ADP-ribosylation and the reduction was restored by addition of guanine nucleotide during incubation . These findings suggest that Rho is involved in the photo-sensitive melanophore response as a signal transducer linking photo-stimuli to melanin granule translocation with Xenopus melanophores.

Trends Microbiol, 1996 Oct, 4(10), 375 - 82
Large clostridial cytotoxins--a family of glycosyltransferases modifying small GTP-binding proteins; von Eichel-Streiber C et al.; Some Clostridium species produce ABX-type protein cytotoxins of high molecular weight . These toxins constitute the group of large clostridial cytotoxins (LCTs), which have homologous protein sequences, exert glycosyltransferase activity and modify GTP-binding proteins of the Ras-superfamily . These characteristics render the LCTs valuable tools for developmental and cell biologists.

South Med J, 1996 Oct, 89(10), 1014 - 7
Ascites associated with antibiotic-associated pseudomembranous colitis; Jafri SF et al.; We report the case of an elderly patient who had ascites due to pseudomembranous colitis and associated hypoalbuminemia . Computed tomography showed diffuse colonic wall thickening . An indium-111 scan to localize the site of infection showed abnormal localization of 111In throughout the colon . Despite treatment, the patient died . Autopsy disclosed no other cause for the ascites, except for possible sepsis . To study the cause of ascites in patients with pseudomembranous colitis, we reviewed our institutions' experience with ascites in association with Clostridium difficile colitis, identifying 16 cases over a 1-year period (which included our case) . In most of the other cases, the ascites could be attributed primarily to another mechanism, including portal hypertension, congestive heart failure, and sepsis (intra-abdominal and systemic) . We also reviewed the literature regarding the association of ascites with C difficile colitis.

Mol Pharmacol, 1996 Oct, 50(4), 864 - 9
Inhibition by toxin B of inositol phosphate formation induced by G protein-coupled and tyrosine kinase receptors in N1E-115 neuroblastoma cells: involvement of Rho proteins; Zhang C et al.; G protein-coupled receptors activate phospholipase C (PLC)-beta isoforms by the alpha or beta gamma subunits of G proteins, whereas growth-factor receptors activate PLC-gamma isoforms by phosphorylating tyrosine residues of the enzyme . As a common substrate for PLC enzymes, phosphatidylinositol 4,5-bisphosphate {Ptdins(4,5)P2} may play a pivotal role in the regulation of cellular PLC activity . Because small-molecular-weight G proteins have been implicated in the synthesis of Ptdins(4,5)P2, we studied the effect of Clostridium difficile toxin B, which glucosylates and thereby inactivates small G proteins of the Rho family, on receptor-stimulated PLC activity . We report here that in N1E-115 neuroblastoma cells, stimulation of inositol phosphate formation by the G protein-coupled receptor agonists bradykinin and lysophosphatidic acid and by the tyrosine kinase receptor agonist platelet-derived growth factor is largely attenuated by toxin B treatment . Furthermore, inositol phosphate production stimulated by the stable GTP analog guanosine 5'-O-(3-thio)-triphosphate in permeabilized N1E-115 cells was inhibited by C3 exoenzyme, which specifically inactivates Rho proteins . The inhibition by toxin B was apparently not caused by its effect on the cytoskeleton . In addition, the level of platelet-derived growth factor receptors, which was studied with immunoblotting, was unaffected by toxin B . Using exogenous Ptdlns(4,5)P2 as PLC substrate, it was found that the intrinsic enzymatic activity of PLC activated either by Ca2+ or by guanosine 5'-O-(3-thio)triphosphate was not altered by toxin B . However, toxin B decreased strongly, by up to 80%, the cellular level of Ptdins(4,5)P2 in a concentration-dependent manner, without changing those of phosphatidylinositol and phosphatidylinositol 4-phosphate . These results, together with the recent finding that Rho family proteins can regulate phosphatidylinositol 4-phosphate 5-kinase activity, demonstrate that Rho proteins are presumably important regulators of Ptdins(4,5)P2 synthesis and, thereby, play an integral role in the regulation of cellular signaling by PLC enzymes.

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