Postgrad Med, 1991 Oct, 90(5), 157 - 60 Clostridium septicum infection . Beware of associated cancer; Kirchner JT; For reasons that are not fully understood, there is an association between Clostridium septicum infection and carcinoma . In this article, Dr Kirchner describes the examination and treatment of a patient with C septicum infection in whom metastatic cancer was found on laparotomy. J Parasitol, 1991 Oct, 77(5), 742 - 8 Lectin-binding properties of the surfaces of in vitro-transformed Schistosoma mansoni and Echinostoma paraensei sporocysts; Uchikawa R et al.; As carbohydrates on the surfaces of sporocysts of digenetic trematodes may be targets of attack by the molluscan internal defense system, the lectin-binding patterns of living, in vitro-transformed sporocysts of Schistosoma mansoni and Echinostoma paraensei were characterized . Schistosoma mansoni sporocysts specifically bound 8 and E . paraensei 6 of 11 lectins examined . Sporocysts of the 2 species responded differently to 7 of the 11 lectins . Lectins inhibitable by mannose, galactose, and N-acetylgalactosamine were bound by both species . Lectins inhibited by fucose and N-acetylglucosamine bound uniquely to S . mansoni, and an N-acetylneuraminic acid (NeuNAc)-inhibitable lectin bound only to E . paraensei . Preincubation of sporocysts of either species in the plasma of the host snail Biomphalaria glabrata for as long as 24 hr only marginally altered the subsequent binding of lectins . Pretreatment of S . mansoni sporocysts with pronase E and trypsin substantially altered subsequent lectin binding, but similar treatment of E . paraensei sporocysts had little effect . A neuraminidase enzyme derived from Clostridium perfringens diminished binding of the NeuNAc-inhibitable lectin to E . paraensei sporocysts . This study indicates that lectin-binding monosaccharides are expressed abundantly on sporocyst surfaces, they vary considerably between 2 species parasitizing the same host, and they are not obscured readily or altered by exposure to host plasma. J Cell Biol, 1991 Oct, 115(2), 309 - 19 The small GTP-binding protein Rho1p is localized on the Golgi apparatus and post-Golgi vesicles in Saccharomyces cerevisiae; McCaffrey M et al.; In Saccharomyces cerevisiae the ras-related protein Rho1p is essentially the only target for ADP-ribosylation by exoenzyme C3 of Clostridium botulinum . Using C3 to detect Rho1p in subcellular fractions, Rho1p was found primarily in the 10,000 g pellet (P2) containing large organelles; small amounts also were detected in the 100,000 g pellet (P3), and cytosol . When P2 organelles were separated in sucrose density gradients Rho1p comigrated with the Kex-2 activity, a late Golgi marker . Rho1p distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles . Rho1p comigrated with post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells . Vesicles containing Rho1p were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane . Immunofluorescence microscopy detected Rho1p as a punctate pattern, with signal concentrated towards the cell periphery and in the bud . Thus, in S . cerevisiae Rho1p resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles. J Bacteriol, 1991 Oct, 173(19), 6025 - 9 Purification and characterization of ADP-ribosyltransferases (exoenzyme C3) of Clostridium botulinum type C and D strains; Moriishi K et al.; By cation-exchange column chromatography followed by gel filtration or hydroxylapatite column chromatography, ADP-ribosyltransferases (exoenzyme C3) were isolated from culture supernatants of Clostridium botulinum type C strains Stockholm (CST) and 6813 (C6813) and from type D strains South African (DSA) and 1873 (D1873), and their molecular properties were compared . The purified C3 enzymes were homogeneous in polyacrylamide gel electrophoresis . The C3 enzymes existed as single-chain polypeptides with molecular masses of 25.0 to 25.5 kDa and transferred ADP-riboses to the same substrates in rat brain membrane extract . The C3 enzymes could be roughly classified into two groups with respect to amino acid composition, amino-terminal sequence, and antigenicity . One group contains the C3 enzymes of strains C6813 and DSA, and the other contains those of strains CST and D1873 . The specific activity of the C3 enzyme of strain C6813 was about 15 times higher than that of the C3 enzyme of strain CST . These results indicate that the classification of the C3 molecules differs from that of the neurotoxin molecules. J Bacteriol, 1991 Oct, 173(19), 5983 - 91 Interaction of selenoprotein PA and the thioredoxin system, components of the NADPH-dependent reduction of glycine in Eubacterium acidaminophilum and Clostridium litorale {corrected}; Dietrichs D et al.; Purification of protein PA of the glycine reductase complex from Eubacterium acidaminophilum and Clostridium litorale {corrected} was monitored by a new spectrophotometric assay . The procedure depended on a specific two- to threefold stimulation of a dihydrolipoamide dehydrogenase activity that is elicited by the interaction of a thioredoxin reductase-like flavoprotein and thioredoxin from both organisms . Protein PA isolated from E . acidaminophilum by 75Se labeling and monitoring of the dithioerythritol-dependent glycine reductase activity was identical in its biochemical, structural, and immunological properties to the protein isolated by using the stimulation assay . Proteins PA from both organisms were glycoproteins of Mr about 18,500 and exhibited very similar N-terminal amino acid sequences . Depletion of thioredoxin from crude extracts of E . acidaminophilum totally diminished the NADPH-dependent but not the dithioerythritol-dependent glycine reduction . The former activity could be fully restored by adding thioredoxin . Antibodies raised against the thioredoxin reductase-like flavoprotein or thioredoxin inhibited to a high extent NADPH-dependent but not dithioerythritol-dependent glycine reductase activity . These results indicate the involvement of the thioredoxin system in the electron flow from reduced pyridine nucleotides to glycine reductase. Infect Immun, 1991 Oct, 59(10), 3673 - 9 Characterization of the C3 gene of Clostridium botulinum types C and D and its expression in Escherichia coli; Popoff MR et al.; Clostridium botulinum type C and D strains produce exoenzyme C3, which ADP-ribosylates the Rho protein, a 21-kDa regulatory GTP-binding protein . In a previous work, we demonstrated that the C3 gene is encoded by bacteriophages C and D of C . botulinum by using DNA-DNA hybridizations with oligonucleotides deduced from the C3 protein N-terminal sequence . The C3 coding gene was cloned and sequenced, but its upstream DNA region could not be studied because of its instability in Escherichia coli . In this work, the upstream DNA region of the C3 gene was directly amplified by the polymerase chain reaction and sequenced . The C3 gene encodes a polypeptide of 251 amino acids (27,823 Da) consisting of a 40-amino-acid signal peptide and a mature protein of 211 amino acids (23,546 Da) . The C3 mature protein was expressed in E . coli under the control of the trc promoter . The recombinant polypeptide obtained was recognized by C3 antibodies and ADP-ribosylated the Rho protein . The C3 gene nucleotide sequence is identical on C and D phage DNAs . At the amino acid sequence level, no similarity was found among C3, other ADP-ribosylating toxins, or tetanus or botulinal A, C1, and D neurotoxins. Infect Immun, 1991 Oct, 59(10), 3659 - 66 Cytokine response by human monocytes to Clostridium difficile toxin A and toxin B; Flegel WA et al.; Clostridium difficile toxins A and B isolated from strain VPI 10463 were tested for induction of cytokine release by human monocytes . Toxin B at 10(-12) M activated human monocytes as measured by release of interleukin-1 (IL-1), tumor necrosis factor (TNF), or IL-6 . These effects of toxin B were heat labile (51 degrees C, 30 min) . Toxin B was as effective as bacterial lipopolysaccharides in inducing IL-1 beta but less effective in inducing TNF or IL-6 . Toxin B and lipopolysaccharides were synergistic in induction of IL-1 beta, TNF, and IL-6 . The toxin A preparation used was 1,000-fold less active than toxin B . Apart from the difference in activity, the two toxins showed identical patterns of reaction and there was no synergism between them . A short pulse with toxin B was sufficient to trigger IL-1 release . Toxin B was also extremely toxic for monocytes . The toxicity and the induced proinflammatory monokines (IL-1 and TNF) may contribute to the pathogenic mechanisms of C . difficile infection and pseudomembranous colitis. G E N, 1991 Oct-Dec, 45(4), 273 - 80 {Bacterial translocation in a model of intestinal obstruction . II . Bacteriological study and role of cellular immunity}; Zapata-Sirvent RL et al.; Bacterial translocation (Bt) from the gastrointestinal (GI) tract to systemic organs creates the possibility of Infection and sepsis in a great number of pathologic entities . In a mouse model of Intestinal Obstruction (IO), we evaluated the type of micro-organisms and the organs that bacteria frequent translocated . At 24 hours post-10, positive cultures where obtained at the MLN, portal, systemic circulation and peritoneal cavity, establishing that the translocation is bi-directional . The more frequent bacteria isolated were the Streptococcus group D, Proteus mirabilis, Escherichia coli, Pseudomonas sp., an clostridium . BT occurs at 24 hour post-OI and was due to increased intestinal permeability, at 48 hrs BT increased and related to the physical disruption of the mucosal barrier in the intestinal mucosa . Cell mediated immunity (CMI) response in this model was not altered, although a progressive decrease was observed at 48 hrs it was not significant, suggesting that the CMI play no role in the pathogenesis of BT . In the Control-Laparotomy group, CMI response was increased significantly at 48 hours, suggesting that a simple laparotomy boost the immune defense response. Biologicals, 1991 Oct, 19(4), 293 - 8 Development of an enzyme immunoassay for the detection of Clostridium novyi type B alpha toxin; Pietrzykowski E et al.; Clostridium novyi type B alpha toxin was purified to homogeneity and shown to have a molecular weight of 200 kD by SDS polyacrylamide gel electrophoresis . The toxin was toxoided and used to produce a pair of non-interfering monoclonal antibodies . Their specificity was confirmed by immunoblotting and bioassay . The monoclonal antibodies were used to develop an enzyme immunoassay which was more sensitive than bioassay, and permitted less than 1 ng/ml toxin to be detected in a rapid 10 min assay format . Use of the assay can eliminate the requirement for in vivo testing of novyi toxin and toxoid, provided measurements of biological activity are not required . Because of its speed and sensitivity, the assay can be used to monitor toxin production during fermentation and as an alternative to bioassay to measure antigen content during toxoiding and vaccine formulation. Biologicals, 1991 Oct, 19(4), 281 - 6 An alternative to the toxin neutralization assay in mice for the potency testing of the Clostridium tetani, Clostridium septicum, Clostridium novyi type B and Clostridium perfringens type D epsilon components of multivalent sheep vaccines; Wood KR; Potency testing of veterinary vaccines containing clostridial antigens currently requires the vaccination of laboratory rabbits followed by the determination of specific antitoxin concentration in the rabbit sera by toxin neutralization test in mice . ELISAs are described as an alternative method to toxin neutralization for the determination of Clostridium tetani, Clostridium septicum, Clostridium novyi Type B and Clostridium perfringens Type D epsilon antitoxins . The assays were found to be rapid, specific and economical and showed good correlation with the toxin neutralization test. Pathology, 1991 Oct, 23(4), 346 - 9 Diarrheal disease due to Clostridium difficile in general practice; Riley TV et al.; A total of 288 stool samples from patients attending their general practitioners was examined for the presence of Clostridium difficile . C . difficile or its cytotoxin was found in 16 patients (5.5%) and was the most common enteric pathogen detected . Most patients had only mild to moderate diarrhea but in the majority of patients the diarrhea was protracted . Eleven of the 16 patients had received antimicrobial agents in the 3 mths preceding onset of diarrhea and there was good circumstantial evidence that 2 other patients had also been exposed . None of the patients had a history of any inflammatory bowel disease or possible occupational exposure . The prescribing habits of general practitioners with regard to antimicrobial agents were monitored for a 1 yr period . Tetracyclines and amoxycillin accounted for approximately 25% each of all prescriptions dispensed . Ten of the 16 patients were treated with antimicrobials (mainly metronidazole) and in most cases the diarrhea resolved . We conclude that C . difficile may be a significant cause of community-acquired diarrhea. Biokhimiia, 1991 Oct, 56(10), 1787 - 97 {Cellobiohydrolase from Clostridium thermocellum, synthesized by a recombinant E . coli strain}; Mel'nik MS et al.; Clostridium thermocellum cellobiohydrolase was isolated in preparative amounts from the recombinant strain of E . coli K12 C600 carrying plasmid pCU 304 with a C . thermocellum chromosomal DNA insertion . The isolation procedure included chromatography on Ultrogel AcA 44, ion-exchange chromatography on DEAE-Sepharose CL-6B, rechromatography on Ultrogel and FPLC on Mono Q resulting in a 17.6% yield and 1530-fold purification . According to data from sodium dodecylsulfate polyacrylamide gel electrophoresis performed under nondenaturing conditions and analytical gel isoelectrofocusing, the enzyme preparation contains only one active protein band with Mr 56.2 +/- 1.0 kDa and pI 4.15 . The enzyme does not reduce the viscosity of the CM-cellulose solution but forms reducing sugars from this soluble substrate . Cellobiose (93-97%) is the major component produced by the enzyme from crystalline and amorphous cellulose (specific activity 2.3 x 10(-3) and 2.8 x 10(-2) U/mg, respectively) . The activity optimum of the enzyme is at pH 5.6, 60 degrees C . The half-inactivation time at 60 degrees C and 65 degrees C is 450 and 15.5 min, respectively . The action pattern of the enzyme on the low molecular fluorogenic cellooligosaccharides suggests that the enzyme pertains to typical cellobiohydrolases. Eur J Cell Biol, 1991 Oct, 56(1), 68 - 78 Phosphorylation of cellular proteins in response to treatment with Clostridium difficile toxin B and Clostridium sordellii toxin L; Ciesielski-Treska J et al.; Toxin B from Clostridium difficile induces typical morphological changes of cultured cells consisting of rounding up and arborization, which are associated with a dramatic disruption of microfilaments . In this study, we show that toxin L, a cytotoxin produced by bacterial strain Clostridium sordellii, has similar effects on cultured cells including the redistribution of F-actin and of the adhesion plaque protein vinculin . It has been assumed that the mechanisms involved in cytopathic effects of toxin B are related to the function of an unidentified component that regulates the organization of the actin cytoskeleton . We demonstrate that the treatment of cultured astrocytes with toxin B or toxin L alters the incorporation of inorganic phosphate into several proteins . Immunoblot analysis revealed that one of these proteins is tropomyosin . Since tropomyosin stabilizes microfilaments and inhibits the severing activity of gelsolin, the toxin-induced phosphorylation may counteract this inhibition resulting in severing of microfilaments and capping of short filaments . A decrease in the radioactivity associated with intermediate filament protein vimentin was also detected using a monoclonal antibody which specifically recognizes a phosphorylated epitope of vimentin . Since vimentin is an in vivo substrate for various protein kinases, these data are in favor of broad effects of these toxins . Direct measurement of protein kinase C in cells exposed to toxin B or to toxin L did not reveal a significant change in protein kinase C activity . Furthermore, treatments with toxins do not increase cAMP levels, suggesting that toxins do not activate protein kinase A . Although further studies are required to determine the primary target site for the clostridial cytotoxin B and L, our results show that they provoke the alteration in the phosphorylation of cellular proteins. Microbiologica, 1991 Oct, 14(4), 295 - 300 Capsule-like structures in Clostridium difficile strains; Baldassarri L et al.; Fourteen strains of Clostridium difficile, previously characterized by SDS-PAGE, were examined for the presence of surface structures . None of the strains were fimbriated but, when grown in the presence of glucose, all exhibited a thin ruthenium red-positive layer . Two strains, belonging to different electrophoretic groups, were also observed after treatment with homologous and heterologous antisera . The strain belonging to the electrophoretic group 2, usually associated with the disease, showed a 30-80nm thick capsule in ultrathin sections . The strains belonging to group 5, mainly observed in isolates from healthy children, exhibited a thinner polysaccharide layer (10-20 nm) . No stabilization was observed when these strains were treated with heterologous antisera . This capsule-like material did not seem to be associated with adhesive properties of C . difficile strains. J Gen Virol, 1991 Oct, 72 ( Pt 10), 2467 - 74 Gangliosides as binding sites in SA-11 rotavirus infection of LLC-MK2 cells; Superti F et al.; The chemical nature of receptors involved in the attachment of simian rotavirus (SA-11) to a monkey kidney cell line (LLC-MK2) was investigated . Enzymic treatment of cells before virus infection indicated that membrane proteins and phospholipids are not involved in virus attachment, whereas sialic acid and galactose participate in the receptor structure to differing extents . Incubation of SA-11 with bovine brain gangliosides before infection strongly reduced its ability to bind to cell membranes . Similar experiments with individual purified gangliosides from bovine brain showed that virus infection was prevented by preincubation with GM1 . Moreover, desialylated cells regained susceptibility to virus infection when coated with whole gangliosides or GM1 immediately after Clostridium perfringens neuraminidase treatment . The binding of SA-11 to whole gangliosides or GM1 was quantified by an ELISA procedure . The results suggest that gangliosides, mainly GM1, are part of the receptor structure for SA-11 of susceptible LLC-MK2 cells. Gene, 1991 Sep 30, 106(1), 13 - 9 Cloning and analysis of the beta-galactosidase-encoding gene from Clostridium thermosulfurogenes EM1; Burchhardt G et al.; Clostridium thermosulfurogenes EM1 produced a thermostable (up to 70 degrees C) beta-galactosidase (beta Gal) with a pH optimum of 7 during growth on lactose . The gene (lacZ) encoding this enzyme was cloned and expressed in Escherichia coli using pUC18 as a vector . The nucleotide sequence of a 2.7-kb PstI fragment carrying the lacZ gene was determined . The open reading frame for lacZ, which encoded a protein of 716 amino acids with a calculated Mr of 83,728, was confirmed by the identity of its deduced aa sequence with the chemically determined N-terminal aa sequence of the purified beta Gal of C . thermosulfurogenes EM1 . The structural gene was preceded by a possible promoter sequence, 5'-TTGTAG (-35), 5'-TAATAT (-10); and a ribosome-binding site, 5'-AGGAGG . The cloned beta Gal was found to be indistinguishable from the native enzyme . The Mr of the active beta Gal was 170,000, as determined by Superose 12HR gel filtration and gradient gel electrophoresis . This indicated that this enzyme is composed of two identical subunits . Comparison of the aa sequences of different beta Gal revealed that five large regions of similarity with the enzymes from E . coli (lacZ, ebgA), Klebsiella pneumoniae (lacZ), and Lactobacillus bulgaricus are present in the beta Gal of C . thermosulfurogenes EM1 and that the putative active site residues (Glu461 and Tyr503 in the E . coli lacZ-encoded beta Gal) are conserved (Glu389 and Tyr429) . Therefore, the thermostable beta Gal of C . thermosulfurogenes EM1 is more closely related to the enzyme of E . coli than to the likewise thermostable one of Bacillus stearothermophilus.(ABSTRACT TRUNCATED AT 250 WORDS) Am J Med, 1991 Sep 16, 91(3B), 138S - 144S Control of nosocomial transmission of Clostridium difficile based on sporadic case surveillance; Struelens MJ et al.; The recognition of a cluster of antibiotic-associated nosocomial Clostridium difficile disease (NCDD) caused by serotype C in a surgical ward led to a hospital-wide NCDD surveillance and control program . The initial step included: (a) gas-liquid chromatography screening of inpatients' diarrheal stools; (b) enteric isolation precautions, cohorting and terminal room disinfection in wards with a cluster of two or more NCDD cases per month . During a 12-month period, the quarterly incidence of NCDD remained unchanged and six new clusters of serotype C, K, and H infections occurred, giving a global incidence of 1.5/1,000 admissions . C . difficile spores were recovered from 36.7% surfaces of case patient rooms versus 6.7% in control rooms . More intensive control measures were evaluated: (a) culture screening of inpatients' diarrheal stools; (b) early therapy, enteric isolation precautions, and daily meticulous room disinfection for each sporadic NCDD case . Surface disinfection reduced the contamination level four-fold (p = 0.04) . In the following 12 months, no cluster occurred and the incidence of NCDD fell to 0.3/1,000 admission (protective efficacy 73%, 95% confidence interval: 46-87%) . These observations suggest that early therapy, isolation precautions, and surface disinfection, focused on patients with sporadic NCDD detected by active surveillance, can prevent nosocomial transmission of C . difficile. FEBS Lett, 1991 Sep 9, 289(2), 253 - 6 2D 1H NMR studies of oxidized 2(Fe4S4) ferredoxin from Clostridium pasteurianum; Bertini I et al.; Oxidized ferredoxin from Clostridium pasteurianum, containing two Fe4S4 clusters, has been investigated using 2D 1H NMR spectroscopy at 600 MHz . 2D NMR experiments allowed complete assignment of the sixteen isotropically shifted signals corresponding to the beta-CH2 protons of the eight metal coordinated cysteines . Geminal connectivities of Cys beta-CH2 protons were identified through magnitude COSY experiments and confirmed through 2D NOESY experiments . A few additional signals could be assigned to the corresponding alpha-CH protons . The importance of 2D experiments to achieve firm assignments of isotropically shifted signals in paramagnetic metalloproteins is stressed. Anal Biochem, 1991 Sep 2, 197(2), 290 - 5 Endoglucanase A gene fusion vectors for monitoring protein secretion and glycosylation in yeast; Silva A et al.; We have constructed a set of replicating and integrating vectors that allow expression and secretion of Clostridium thermocellum endoglucanase A in Saccharomyces cerevisiae using the alpha-factor or the invertase promoters and secretion signals . The enzyme expressed in yeast was enzymatically active regardless of its degree of glycosylation and was released into the culture medium . One advantage of using this experimental system is that secretion of the reporter enzyme can be detected in individual colonies facilitating the isolation of mutants . A second advantage is that transit through the secretory pathway, as judged from the extent of glycosylation, can be easily monitored by staining for enzyme activity in agar replicas of polyacrylamide gels. Neurosci Lett, 1991 Sep 2, 130(1), 5 - 8 Ultrastructure of botulinum type-A poisoned frog motor nerve terminals after enhanced quantal transmitter release caused by carbonyl cyanide m-chlorophenylhydrazone; Pecot-Dechavassine M et al.; The effects of carbonyl cyanide m-chlorophenylhydrazone (CCCP) on spontaneous quantal transmitter release and nerve terminal ultrastructure were studied on isolated cutaneous pectoris nerve-muscle preparations from frogs that were completely paralysed by a single sublethal dose of Clostridium botulinum type A toxin (BoTx) . CCCP enhanced miniature endplate potential frequency at poisoned junctions and caused a reduction in the density of clear synaptic vesicles and of large dense core vesicles in motor nerve terminals . However, the intensity of these effects was much less important than that previously reported at unpoisoned junctions . The moderate depletion of synaptic vesicles can be related to the low levels of transmitter release detected with CCCP at BoTx-poisoned terminals. Br Vet J, 1991 Sep-Oct, 147(5), 484 - 5 Clostridium perfringens type A enterotoxaemia in pigs: a report of five cases; Okewole PA et al.; Enterotoxaemia caused by Clostridium perfringens type A in five intensively managed pigs is reported . The condition was precipitated by constipative digestive disturbance . Diarrhoea was not observed in the five animals before death. Drug Saf, 1991 Sep-Oct, 6(5), 339 - 49 The clinical significance of antibiotic-associated pseudomembranous colitis in the 1990s; Andrejak M et al.; Antibiotic-associated pseudomembranous colitis is an uncommon but potentially serious adverse reaction, resulting in acute diarrhoea and characterised by colonic pseudomembranes . A direct relationship between the disease, recent antibiotic therapy and proliferation of Clostridium difficile in the colonic lumen was established in the late 1970s . It is thought that antibiotic therapy may alter the enteric flora, enabling C . difficile to proliferate and produce toxins with cytopathic (toxin B or cytotoxin) and hypersecretory (toxin A or enterotoxin) effects on the mucosa . Apart from clindamycin, the first antibiotic recognised to be clearly associated with pseudomembranous colitis, the antimicrobial agents most commonly responsible are cephalosporins and ampicillin (or amoxicillin) . However, virtually all antibiotics except parenterally administered aminoglycosides can cause the disease . Vancomycin and metronidazole, 2 drugs used to treat antibiotic-associated pseudomembranous colitis, have also been reported to be responsible for the complication when used parenterally . Pseudomembranous colitis may develop after perioperative prophylactic antibiotic therapy with cephalosporins . Antibiotic-associated pseudomembranous colitis is most frequent in elderly and debilitated patients and in intensive care units . Nosocomial acquisition of C . difficile has been documented . Therefore it has been recommended that enteric isolation precautions should be taken with patients with this disease . The clinical symptoms include watery diarrhoea, abdominal cramping, and frequently fever, leucocytosis and hypoalbuminaemia . Toxic megacolon and acute peritonitis secondary to perforation of the colon are the most serious complications . The pseudomembranes are usually seen during endoscopic procedures, sigmoidoscopy or, if possible, colonoscopy; the most useful microbiological tests for confirmation of the diagnosis include cycloserine cefoxitin fructose agar (CCFA) stool cultures and stool toxin assays on tissues or by immunological techniques . However, cultures and toxin tests may be positive in patients without pseudomembranous colitis or C . difficile-associated diarrhoea . Mild cases may respond to discontinuation of the drug responsible, but therapy with an anticlostridial antibiotic is often necessary: a 10-day course of oral vancomycin, metronidazole or bacitracin should be given . Relapses are seen in 5 to 50% of patients treated . Antibiotic treatment should avoid sporulation leading to other relapses . 'Biotherapy' (lactobacilli, Saccharomyces) has also been proposed. Eur J Biochem, 1991 Sep 1, 200(2), 301 - 9 Structure of the beta-glucosidase gene bglA of Clostridium thermocellum . Sequence analysis reveals a superfamily of cellulases and beta-glycosidases including human lactase/phlorizin hydrolase; Grabnitz F et al.; The nucleotide sequence of the Clostridium thermocellum gene bglA, coding for the thermostable beta-glucosidase A, has been determined . The coding region of 1344 bp was identified by comparison with the N-terminal amino acid squence of recombinant beta-glucosidase A purified from Escherichia coli . The deduced amino acid sequence corresponds to a protein of 51,482 Da . The coding region is flanked by putative promoter and transcription terminator sequences . The protein is unrelated to beta-glucosidase B of C . thermocellum, but has a high level of similarity with other bacterial beta-glucosidases and phospho-beta-glucosidases . Similarity is also observed with the beta-galactosidase of the archaebacterium Sulfolobus solfataricus . Unexpectedly, it was found that human lactase-phlorizin hydrolase contains three copies of a sequence closely related to C . thermocellum beta-glucosidase A (up to 40% sequence identity) . These diverse beta-glucosidases can therefore be grouped into an enzyme family (BGA) of common structural design . Sequence comparison by hydrophobic cluster analysis revealed that all BGA enzymes share a well conserved region which is homologous to the catalytic domain of the widely distributed cellulase family A . A distinctive feature of this domain is the sequence motif His-Asn-Glu-Pro in which the catalytic residues His and Glu are separated by 35-55 amino acid residues . The cellulase family A and the beta-glucosidase family BGA might thus be considered as members of a protein super-family comprising beta-glucanases and beta-glycosidases from all three primary kingdoms of living organisms. J Bacteriol, 1991 Sep, 173(17), 5431 - 8 Cloning, mapping, and molecular characterization of the rRNA operons of Clostridium perfringens; Garnier T et al.; All 10 rRNA operons have been situated on the genome map of the anaerobic pathogen Clostridium perfringens . Four of these have been cloned and partially sequenced, and their transcriptional patterns in vivo and in vitro have been examined . Expression of rrnA, rrnB, and rrnE is directed by tandem promoters, P1 and P2, whereas rrnH is the only one to be expressed from a single promoter, which resembles P1 . On inspection of the nucleotide sequences of the control regions, several sites which might be involved in the regulation of rrn expression were identified . These include a possible upstream activating region which could be recognized by the C . perfringens equivalent of the Escherichia coli Fis protein and a stringent response target site . Studies of maturation of 16S RNA identified two 5' cleavage sites and sequence analysis showed the dG+dC content of its gene, rrs, to be 52%, which is twice that of the genome. Dis Mon, 1991 Sep, 37(9), 545 - 603 Tetanus: pathophysiology, management, and prophylaxis; Bleck TP; As tetanus has become a rare disease in the developed world, physicians have become less comfortable with its diagnosis and management . The extent of adequate antitetanus immunity in the adult population, especially the elderly, is waning, in great measure because primary care physicians have not made prophylaxis a priority in their routine encounters with patients . Furthermore, as the population of immunocompromised hosts grows, an increasing percentage of our patients may not respond to standard active immunization routines . Unless these trends are reversed, we face a substantial increase in the incidence of this dread disorder . Tetanus is also of interest as a relatively simple model of disordered motor control that can instruct us in the management of the many more common causes of neurogenic muscular rigidity . The toxin produced by Clostridium tetani finds increasing use in laboratories investigating brain function as well . Clinical tetanus is divided into four symptomatic types: generalized tetanus, local tetanus, cephalic tetanus, and neonatal tetanus . This monograph discusses the diagnostic aspects of each type of tetanus, its pathophysiology, diagnosis, differential diagnosis, and treatment . Preventing tetanus should be a high priority for all primary care physicians . Active immunization with tetanus toxoid is remarkably effective and safe . Passive immunization with human tetanus immune globulin is indicated in certain circumstances, which are discussed below. Gastroenterology, 1991 Sep, 101(3), 685 - 91 Acute abdomen as the first presentation of pseudomembranous colitis; Triadafilopoulos G et al.; Acute abdomen was the presenting manifestation of pseudomembranous colitis in six men who had previously been treated with antibiotics and presented with abdominal distention, pain, fever, and leukocytosis with absent or mild diarrhea . Plain abdominal radiographs revealed megacolon in two, combined small and large bowel dilation in three, with one of them showing volvuluslike pattern, and isolated small bowel ileus in one . Emergency colonoscopy was performed successfully in all patients and revealed pseudomembranes in five and nonspecific colitis in one . All patients had positive latex test results for Clostridium difficile, and two tested positive for cytotoxicity . All patients were treated with IV metronidazole, resulting in resolution of symptoms and abdominal findings . In addition, two patients underwent colonoscopic decompression with improvement . Endoscopically, complete resolution of the pseudomembranes occurred at 4 weeks in all cases . No patient had a recurrence . It is concluded that (a) pseudomembranous colitis may present as abdominal distention mimicking small bowel ileus . Ogilvie's syndrome, volvulus, or ischemia; (b) in such cases, emergency colonoscopy is safe and useful for diagnosis and therapeutic decompression and may obviate the need for surgery; and (c) treatment with IV metronidazole is effective . Colitis due to C . difficile should be considered in the differential diagnosis of acute abdomen in patients previously treated with antibiotics. J Assoc Physicians India, 1991 Sep, 39(9), 683 - 4 Clinical manifestation of Clostridium difficile enteritis in Calcutta; Bhattacharya MK et al.; 233 cases with acute diarrhoea investigated, Clostridium difficile was isolated as a sole pathogen from 17 (7.3%) cases . The Major clinical features of these cases were watery diarrhoea (82.4%), bloody stool (17.6%), vomiting (64.8%), fever (17.6%) and abdominal pain (2.5%) . Fourteen (82.4%) of 17 C difficile isolates were found to produce cytotoxin as detected by Verocell assay. Eur J Clin Microbiol Infect Dis, 1991 Sep, 10(9), 770 - 2 Usefulness of semi-quantitative cultures in the diagnosis of Clostridium difficile associated disease; Poirier L et al.; Semi-quantitative stool cultures on CCFA were compared to cytotoxic assays for the diagnosis of Clostridium difficile associated disease (CAD) . There was a significant correlation between the amount of Clostridium difficile growth on CCFA, the presence of cytotoxin and a clinical diagnosis of CAD in the 541 initial stool specimens tested. Clin Ther, 1991 Sep-Oct, 13(5), 579 - 88 Comparison of oral cefpodoxime proxetil and penicillin V potassium in the treatment of group A streptococcal pharyngitis/tonsillitis . The Cefpodoxime Pharyngitis Study Group; Brown RJ et al.; Ninety-three patients with a diagnosis of acute pharyngitis/tonsillitis due to Streptococcus pyogenes were randomly assigned to receive 100 mg of cefpodoxime proxetil orally with food every 12 hours or 250 mg of penicillin V potassium orally on an empty stomach every six hours . Treatment efficacy was evaluated in 30 cefpodoxime-treated and 33 penicillin-treated patients . After 10 days of treatment, S pyogenes was eradicated from the throat culture in 29 of the 30 cefpodoxime-treated patients and in 30 of the 33 penicillin-treated patients . Twenty days after treatment termination, infection recurred in one patient of each treatment group . Clinical cure or improvement was found in 97% of the patients in each group . Adverse medical events occurred in nine of the 48 cefpodoxime-treated patients and in four of the 45 penicillin-treated patients; these were probably related to the study drug in seven and two patients, respectively . The most common adverse events were nausea (in three cefpodoxime and one penicillin patient) and diarrhea (in three and two) . No patient showed colitis related to Clostridium difficile . No clinically significant abnormal laboratory test results were found in either treatment group . It is concluded that cefpodoxime proxetil is as effective and safe as penicillin V potassium in the treatment of pharyngitis due to S pyogenes. Stomatologiia (Mosk), 1991 Sep-Oct, (5), 20 - 1 {Clostridium perfringens in the oral saliva in galvanism}; Freidin LI et al.; Clostridia perfringens are much more frequently isolated from galvanism patients than other microorganisms . This fact should be not omitted by physicians, for these bacteria may induce a specific infectious process. J Biochem (Tokyo), 1991 Sep, 110(3), 369 - 75 A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region; Ohno-Iwashita Y et al.; We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens {Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y . (1988) Eur . J . Biochem . 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S . (1990) Biochim . Biophys . Acta 1023, 441-448} . To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol) . Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites . The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin . Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes. Appl Environ Microbiol, 1991 Sep, 57(9), 2699 - 702 Purification and properties of 4-hydroxybutyrate coenzyme A transferase from Clostridium aminobutyricum; Scherf U et al.; A new coenzyme A (CoA)-transferase from the anaerobe Clostridium aminobutyricum catalyzing the formation of 4-hydroxybutyryl-CoA from 4-hydroxybutyrate and acetyl-CoA is described . The enzyme was purified to homogeneity by standard techniques, including fast protein liquid chromatography under aerobic conditions . Its molecular mass was determined to be 110 kDa, and that of the only subunit was determined to be 54 kDa, indicating a homodimeric structure . Besides acetate and acetyl-CoA, the following substrates were detected (in order of decreasing kcat/Km): 4-hydroxybutyryl-CoA, butyryl-CoA and propionyl-CoA, vinyl-acetyl-CoA (3-butenoyl-CoA), and 5-hydroxyvaleryl-CoA . In an indirect assay the corresponding acids were also found to be substrates; however, DL-lactate, DL-2-hydroxybutyrate, DL-3-hydroxybutyrate, crotonate, and various dicarboxylates were not. Appl Environ Microbiol, 1991 Sep, 57(9), 2534 - 9 Properties of the glucose phosphotransferase system of Clostridium acetobutylicum NCIB 8052; Mitchell WJ et al.; The glucose phosphotransferase system (PTS) of Clostridium acetobutylicum was studied by using cell extracts . The system exhibited a Km for glucose of 34 microM, and glucose phosphorylation was inhibited competitively by mannose and 2-deoxyglucose . The analogs 3-O-methylglucoside and methyl alpha-glucoside did not inhibit glucose phosphorylation significantly . Activity showed no dependence on Mg2+ ions or on pH in the range 6.0 to 8.0 . The PTS comprised both soluble and membrane-bound proteins, which interacted functionally with the PTSs of Clostridium pasteurianum, Bacillus subtilis, and Escherichia coli . In addition to a membrane-bound enzyme IIGlc, sugar phosphorylation assays in heterologous systems incorporating extracts of pts mutants of other organisms provided evidence for enzyme I, HPr, and IIIGlc components . The HPr was found in the soluble fraction of C . acetobutylicum extracts, whereas enzyme I, and probably also IIIGlc, was present in both the soluble and membrane fractions, suggesting a membrane location in the intact cell. Appl Environ Microbiol, 1991 Sep, 57(9), 2544 - 8 Isolation and characterization of Clostridium acetobutylicum mutants with enhanced amylolytic activity; Annous BA et al.; Clostridium acetobutylicum mutants BA 101 (hyperamylolytic) and BA 105 (catabolite depressed) were isolated by using N-methyl-N'-nitro-N-nitrosoguanidine together with selective enrichment on the glucose analog 2-deoxyglucose . Amylolytic enzyme production by C . acetobutylicum BA 101 was 1.8- and 2.5-fold higher than that of the ATCC 824 strain grown in starch and glucose, respectively . C . acetobutylicum BA 105 produced 6.5-fold more amylolytic activity on glucose relative to that of the wild-type strain . The addition of glucose at time zero to starch-based P2 medium reduced the total amylolytic activities of C . acetobutylicum BA 101 and BA 105 by 82 and 25%, respectively, as compared with the activities of the same strains grown on starch alone . Localization studies demonstrated that the amylolytic activities of C . acetobutylicum BA 101 and BA 105 were primarily extracellular on all carbohydrates tested. Infect Immun, 1991 Sep, 59(9), 3151 - 5 Antigenic cross-reactivity and functional inhibition by antibodies to Clostridium difficile toxin A, Streptococcus mutans glucan-binding protein, and a synthetic peptide; Wren BW et al.; A 10-amino-acid repeating sequence of the hemagglutinating portion of Clostridium difficile toxin A has been synthesized and used to produce antisera in rabbits . Antipeptide antibody inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity . Immunoblot analysis with the antipeptide antibody revealed cross-reactivity with native toxin, a recombinant protein containing the toxin A repeats, and a glucan-binding protein from Streptococcus mutans whose primary structure has repeating amino acid motifs similar to those of the synthetic peptide . A polyclonal antibody against the glucan-binding protein, which cross-reacted with purified toxin A, also inhibited toxin A-mediated hemagglutination and neutralized cytotoxic activity . We recently identified toxin A and the glucan-binding protein as members of a novel family of clostridial and streptococcal binding proteins based on conserved repeating amino acid motifs at the C-terminal region of the molecules . This study provides immunological and functional evidence of the predicted relationship between toxin A and the glucan-binding protein and further implicates the repeating subunits as ligand-binding domains in this family of proteins. Appl Environ Microbiol, 1991 Sep, 57(9), 2735 - 41 Cloning of the Clostridium acetobutylicum ATCC 824 acetyl coenzyme A acetyltransferase (thiolase; EC 2.3.1.9) gene; Petersen DJ et al.; Thiolase (acetyl coenzyme A acetyltransferase; EC 2.3.1.9) from Clostridium acetobutylicum is a key enzyme in the production of acids and solvents in this organism . The purification and properties of the enzyme have already been described (D . P . Wiesenborn, F . B . Rudolph, and E.T . Papoutsakis, Appl . Environ . Microbiol . 54:2717-2722, 1988) . The thl gene encoding the thiolase has been cloned by using primary antibodies raised to the purified enzyme . A bacteriophage lambda EMBL3 library of C . acetobutylicum DNA was prepared and screened by immunoblots with the antithiolase antibodies . Phage DNA was purified from positive plaques, and restriction enzyme digests identified an approximately 4.8-kb AccI fragment common to all positive plaques . A corresponding fragment was also found in AccI digests of C . acetobutylicum chromosomal DNA . The fragment was purified and EcoRI linkers were attached before being subcloned into pUC19 . Maxicell analysis showed the production of an approximately 42-kDa protein, whose size corresponded to the molecular size of the purified thiolase, from the clostridial insert . Enzyme activity assays and Western blot (immunoblot) analysis of sodium dodecyl sulfate-polyacrylamide gel electrophoresis-separated whole-cell extracts of Escherichia coli harboring the cloned thl confirmed the presence of the thiolase encoded within the cloned DNA. Yakugaku Zasshi, 1991 Sep, 111(9), 538 - 41 {Studies on collagenase inhibitors . IV . Inhibitors of bacterial collagenase in Coptidis rhizoma}; Tanaka T et al.; A hot aqueous extract of Coptidis Rhizoma had an inhibitory effect on the bacterial collagenase from Clostridium histolyticum . Active principles were isolated by silica gel column chromatography from the CHCl3 extract . Consequently, two inhibitors obtained were identified with the chloride of berberine and coptisine . The concentrations of the berberine and coptisine in the assay mixture to give 50% inhibition (IC50) were 0.73 mM and 0.16 mM, respectively . The type of inhibition by coptisine chloride was shown to be a mixture type from Lineweaver-Burk plots . Tetrahydroberberine, a reduction product of berberine chloride, had no inhibitory effect . This result suggests that the quaternary nitrogen of the alkaloids plays an important role in inhibitory activity. Vet Microbiol, 1991 Aug 30, 28(4), 391 - 7 In vitro susceptibility of rabbit strains of Clostridium spiroforme to antimicrobial agents; Carman RJ et al.; Using an agar dilution method we measured the minimum inhibitory concentration (MIC) of 12 antimicrobial agents against 11 strains of iota-toxigenic strains of Clostridium spiroforme . Each strain was isolated from a separate outbreak of toxic diarrhoea of rabbits . Vancomycin and bacitracin, both agents used to treat intestinal clostridioses of humans and other animals, had a relatively high MIC (8 micrograms/ml or more) . Metronidazole was uniformly active against C . spiroforme . With MIC of 8 micrograms/ml or more, both lincomycin (11 strains) and erythromycin (9 strains) were relatively inactive against C . spiroforme, conversely, penicillin G was active (MIC for 8 strains was 0.5 micrograms/ml or less) . Exposure to any one of these drugs has been implicated as a predisposing factor for C . spiroforme mediated diarrhoea of rabbits . The greatest variation in MIC was seen for erythromycin (8-fold), penicillin G (8-fold) and tetracycline (16-fold). J Am Vet Med Assoc, 1991 Aug 15, 199(4), 471 - 2 Botulism associated with feeding alfalfa hay to horses; Wichtel JJ et al.; Botulism was believed to be the cause of progressive symmetric myasthenia in 8 horses on a farm in North Carolina . One horse was found dead, 6 were euthanatized after becoming recumbent, and 1 affected horse recovered . Cecal and colonic contents of 2 horses were determined to contain Clostridium botulinum spores . Alfalfa hay that was fed to the horses contained spores and toxin. Eur J Pharmacol, 1991 Aug 6, 200(2-3), 267 - 76 Phospholipase C-induced anion secretion and its interaction with carbachol in the rat colonic mucosa; Diener M et al.; Phospholipase C (PLC) from clostridium perfringens induced a biphasic increase in short-circuit current (Isc) in the rat colon . The Isc rose rapidly to a transient peak, before it increased again to a plateau lasting for several hours . Ion replacement experiments and sensitivity to furosemide or a Cl- channel blocker indicated that PLC induced Cl- secretion . The first peak was suppressed by indomethacin, indicating mediation by prostaglandins . In contrast, the second phase was only partially sensitive to the cyclooxygenase blocker . The long-time action of PLC was dependent on intra- and extracellular Ca2+, although PLC did not induce an increase in the intracellular Ca2+ concentration of the enterocytes . The effect of PLC was blocked by the protein kinase inhibitor, staurosporine . Carbachol, when added during the second phase of the PLC response, induced a 'paradox' change in Isc: a rapid, transient increase in Isc was followed by a long-lasting decrease . This inhibition of the PLC response was more pronounced after elevation of the external Ca2+ concentration . A Ca2+ ionophore, ionomycin, and a Ca2+ channel activator, BAY K 8644, also inhibited the PLC response . The results suggest dual dependence of the action of PLC on the intracellular Ca2+ concentration. Appl Biochem Biotechnol, 1991 Aug, 30(2), 129 - 36 Anomalous dissociative behavior of the major glycosylated component of the cellulosome of Clostridium thermocellum; Morag E et al.; The cellulosome of Clostridium thermocellum is a highly cohesive multienzyme complex that is capable of completely solubilizing insoluble cellulose . One of the major cellulosomal components, the glycosylated S1 subunit, is believed to play an important structural role and normally migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 210,000 . It is shown here that by simply altering the conditions (pH or ionic strength) of the environment prior to electrophoresis, a different migratory profile for S1 emerges, yielding a collection of bands, all of which migrate faster than the parent band . The original electrophoretic behavior of S1 can be reproduced on restoration of the original pH and ionic strength . These results may bear important significance for the physiological role of the S1 subunit in facilitating the observed synergistic action of the other (cellulolytic) components of the cellulosome. Antimicrob Agents Chemother, 1991 Aug, 35(8), 1551 - 6 Nucleotide sequence and phylogeny of a chloramphenicol acetyltransferase encoded by the plasmid pSCS7 from Staphylococcus aureus; Schwarz S et al.; The nucleotide sequence of the chloramphenicol acetyltransferase gene (cat) and its regulatory region, encoded by the plasmid pSCS7 from Staphylococcus aureus, was determined . The structural cat gene encoded a protein of 209 amino acids, which represented one monomer of the enzyme chloramphenicol acetyltransferase (CAT) . Comparisons between the amino acid sequences of the pSCS7-encoded CAT from S . aureus and the previously sequenced CAT variants from S . aureus, Staphylococcus intermedius, Staphylococcus haemolyticus, Bacillus pumilis, Clostridium difficile, Clostridium perfringens, Escherichia coli, Shigella flexneri, and Proteus mirabilis were performed . An alignment of CAT amino acid sequences demonstrated the presence of 34 conserved amino acids among all CAT variants . These conserved residues were considered for their possible roles in the structure and function of CAT . On the basis of the alignment, a phylogenetic tree was constructed . It demonstrated relatively large evolutionary distances between the CAT variants of enteric bacteria, Clostridium, Bacillus, and Staphylococcus species. Mol Gen Genet, 1991 Aug, 228(1-2), 320 - 3 Sequence of a cellulase gene from the rumen anaerobe Ruminococcus flavefaciens 17; Cunningham C et al.; A cellulase gene (endA) was isolated from a library of Ruminococcus flavefaciens strain 17 DNA fragments inserted in pUC13 . The endA product showed activity against acid-swollen cellulose, carboxymethyl-cellulose, lichenan, cellopentaose and cellotetraose, but showed no activity against cellotriose or binding to avicel . Nucleotide sequencing indicated an encoded product of 455 amino acids which showed significant sequence similarity (ranging from 56% to 61%) with three endoglucanases from Ruminococcus albus, and with Clostridium thermocellum endoglucanase E . Little relatedness was found with a cellodextrinase previously isolated from R . flavefaciens FD1. APMIS, 1991 Aug, 99(8), 711 - 20 Susceptibility testing of anaerobic bacteria . Regression lines for six antibiotics determined with and without prediffusion; Jansen JE et al.; The relationship between susceptibility testing by an agar dilution test and a tablet diffusion test was studied for 60 anaerobic bacteria (20 B . fragilis, 20 anaerobic cocci, 20 Clostridium species) . For cefoxitin, no prediffusion and prediffusion times of one h, three h, 12 h, 24 h and 48 h were examined . For metronidazole, erythromycin, clindamycin, penicillin and imipenem, only 24 h prediffusion and no prediffusion were studied . Measurements were made after incubation for 24 h and 48 h . Prediffusion improved the correlation for all antibiotics tested, and 24 h prediffusion gave the best results . The slope of the regression line increased and the influence of the individual growth parameters on zone size was reduced . Prediction of susceptibility based on three zone breakpoints to estimate MIC was also better with 24 h prediffusion . However, the variation about the regression line for many of the antibiotics was still extremely high . Measurements after 24 h and 48 h incubation times showed almost identical regression equations, except for erythromycin, where the regression lines differed. Chem Phys Lipids, 1991 Aug, 59(1), 69 - 74 A continuous fluorometric assay for phospholipase C from Clostridium perfringens; Thuren T et al.; A fluorescent assay for Clostridium perfringens phospholipase C is described using 1-palmitoyl-2-{6(pyren-1-yl)hexanoyl}-sn-glycero-3- phospho-N-(trinitrophenyl)aminoethanol (PPHTE) as the substrate . This method is based on the decrease of the quenching of pyrene monomer fluorescence when phospholipase C hydrolyzes PPHTE into pyrenediglyceride and phospho(trinitrophenyl)-aminoethanol . The hydrolysis of egg lecithin/PPHTE (25:1 molar ratio) substrate by C . perfringens phospholipase C was linear with time for at least 2 min . Optimal conditions for the hydrolysis by phospholipase C were 50 mM Tris-HCl pH 7.0-30 mM CaCl2/63 microM egg lecithin and 2.5 microM PPHTE . The Km and Vmax values for the hydrolysis of egg lecithin/PPHTE vesicles were 28 microM and 280 pmol min-1, respectively . The detection limit of the assay was 40 microU of C . perfringens phospholipase C . When diglyceride was included into egg lecithin/PPHTE vesicles up to 30 mol% the reaction velocity increased 13-fold . Higher molar proportions of diglyceride were inhibitory . When the hydrolysis of mixtures of different naturally occurring phospholipids and PPHTE was studied egg lecithin was found to be the best substrate . When dipalmitoylphospholipids with different polar head groups were used the reaction velocity decreased in the order egg lecithin greater than or equal to dipalmitoylphosphatidylserine greater than dipalmitoylphosphatidic acid greater than dipalmitoylphosphatidylcholine greater than dipalmitoylphosphatidylglycerol. J Protein Chem, 1991 Aug, 10(4), 415 - 25 Clostridium botulinum types A, B, C1, and E produce proteins with or without hemagglutinating activity: do they share common amino acid sequences and genes? Somers E, DasGupta BR. Clostridium botulinum produce the antigenically distinct 150 kD neurotoxin serotypes (e.g., A, B, C1, and E) and simultaneously proteins, A Hn+, B Hn+, C Hn+, and E Hn-, that have high, low, and no hemagglutinating activity . A Hn+ and B Hn+ are serologically cross-reactive . A Hn+, B Hn+, and C Hn+ found as large aggregates (900-220 kD) can be dissociated on SDS-PAGE into multiple subunits, the smallest for A Hn+, B Hn+ is 17 kD and 27 kD for C Hn+ . The 116 kD E Hn- does not aggregate . We determined the sequences of 10-33 amino terminal residues of the 17, 21.5, 35, and 57 kD subunits of A Hn+ and B Hn+ . Each of these subunits have unique sequences, indicating that the larger units studies are not homomers or heteromers of smaller units . The subunits of A Hn+ and B Hn+ of comparable size have striking sequence identity (e.g., 21.5 kD subunits from the two are identical and 57 kD subunits have 80% identity) . In vitro proteolysis of 116 kD E Hn- with different proteases did not impart hemagglutinating activity to the fragments . The 116 kD E Hn- and one of its proteolytic fragments (87 kD) were partially sequenced . Sixty-two base pairs downstream from the termination codon of the cloned 33 kD subunit of C Hn+, there is an initiation codon followed by an open reading frame for at least 34 amino acid residues (Tsuzuki et al., 1990) . The derived amino acid sequence of this open reading frame, we found, has 73-84% sequence identity with those of the 17 kD subunits of A Hn+ and B Hn+ and significant identity with the N-terminal of E Hn- . These highly conserved sequences show existence of genetic linkage among the Hn+ and Hn- proteins. J Antimicrob Chemother, 1991 Aug, 28(2), 221 - 8 Physiological effects of metronidazole on Clostridium pasteurianum; Church DL et al.; The physiological effects of metronidazole on the growth, viability, fermentation end-product production and cellular morphology of Clostridium pasteurianum cells growing logarithmically were studied . Metronidazole (a 5-nitroimidazole) was found to be the most potent of the nitroimidazole compounds tested against C . pasteurianum . It inhibited the growth rate of C . pasteurianum cultures by varying degrees over a range of drug concentrations (2.5-10 mg/L) . Metronidazole had an immediate bactericidal effect at a concentration of 10 mg/L, killing 99.9% of cells within 5 min of drug addition . The same concentration caused an immediate cessation of fermentation end-product (acetate and butyrate) production in these cultures . These observations may be relevant to a proposed cell lysing mechanism which may form an additional mode of action of this important antibiotic. Sangre (Barc), 1991 Aug, 36(4), 315 - 7 {Massive intravascular hemolysis in septicemia caused by Clostridium perfringens}; Borrego D et al.; A case of massive haemolytic anaemia in the course of a C . perfringens sepsis of hepatic origin is presented . The diagnosis was strongly suggested by the presence of intragranulocytic capsulated bacilli in a Giemsa stained peripheral blood smear . The patient developed disseminated intravascular coagulation . The outcome was fatal and the patient died eight hours after admission . We review the aetiopathogenesis, diagnosis and therapy of haemolysis in Clostridium perfringens infections. PCR Methods Appl, 1991 Aug, 1(1), 51 - 6 The 16s/23s ribosomal spacer region as a target for DNA probes to identify eubacteria; Barry T et al.; Variable regions of the 16s ribosomal RNA have been frequently used as the target for DNA probes to identify microorganisms . In some situations, however, there is very little sequence variation observed between the 16s rRNA genes of closely related microorganisms . This study presents a general method to obtain species-specific probes using the spacer (intergenic) region between the 16s and 23s rRNA genes . The overall strategy is analogous to that which has previously been developed for the variable regions of the 16s rRNA genes . Sequence analysis of the 16s rRNA and 23s rRNA coding sequences flanking the spacer regions resulted in the design of PCR primers that can be used to amplify the spacer regions of a wide range of eubacteria . Sequencing the amplified spacer region then gives rise to the information that can be used to select specific DNA sequences for use as a DNA probe or for the generation of specific PCR primers to a microorganism of interest . In this study the approach to develop specific DNA markers for members of the genus Clostridium is described in detail . A specific DNA oligonucleotide probe and PCR primers have been designed for Clostridium perfringens that distinguish it from other organisms in the genus. Antonie Van Leeuwenhoek, 1991 Aug, 60(2), 109 - 14 Degradation of galactomannan by a Clostridium butyricum strain; Dong XZ et al.; An anaerobic, sporeforming, galactomannan-degrading bacterium was isolated from methanogenic granular sludge of an UASB reactor used for treatment of wastewater from a sugar refinery . The isolate degraded the polymer rapidly (doubling time was 55 min) and completely in mineral media . The bacterium was classified as Clostridium butyricum; the main products were acetate, butyrate, hydrogen, formate, and presumably CO2 . The strain produced extracellular endo-mannanase, cell-associated exo-mannanase and intracellular alpha-galactosidase activity . The mannanases were present when grown on galactomannan, but not after growth on glucose, galactose, or mannose. Gene, 1991 Jul 31, 104(1), 33 - 8 Nucleotide sequence analysis of the endoglucanase-encoding gene, celCCD, of Clostridium cellulolyticum; Shima S et al.; The nucleotide sequence of the Clostridium cellulolyticum endo-beta-1,4- glucanase (EGCCD)-encoding gene, celCCD, and its flanking regions, was determined . The open reading frame encodes a protein (Mr 66,061) which consists of 584 amino acids (aa) . The N terminus shows the features of the typical signal peptide, with a cleavage site after Gly24 . The protein could be divided into N-terminal and C-terminal regions by an intermediate Pro + Thr-rich sequence . Deletion analysis suggests the C-terminal region is not necessary for EG activity . The predicted aa sequence of the mature protein was similar to those of the central catalytic and the following C-terminal regions of the C . thermocellum endoglucanase H (EGH; identity, 58.8%) . The N-terminal region resembled that of the endoglucanase, EGCCA, from C . cellulolyticum (identity, 24.7%; 336 aa) and the endoglucanase, EGE, from C . thermocellum (identity, 31.4%; 373 aa) . The C-terminal regions ended with two conserved 21-aa stretches which had close similarity to each other . The C-terminal sequence was also highly similar to the reiterated domain of several EG and a xylanase from C . thermocellum, and of an EG from C . cellulolyticum. Gene, 1991 Jul 31, 104(1), 25 - 31 Sequence of the lyc gene encoding the autolytic lysozyme of Clostridium acetobutylicum ATCC824: comparison with other lytic enzymes; Croux C et al.; The lyc gene, encoding an autolytic lysozyme from Clostridium acetobutylicum ATCC824, has been cloned . The nucleotide sequence of the lyc gene has been determined and found to encode a protein of 324 amino acids (aa) with a deduced Mr of 34,939 . The lyc gene is preceded by two open reading frames with unknown functions, suggesting that this gene is part of an operon . Comparison between the deduced aa sequence of the lyc gene and the directly determined N-terminal sequence of the extracellular clostridial lysozyme suggests that the enzyme is synthesized without a cleavable signal peptide . Moreover, the comparative analyses between the clostridial lysozyme and other known cell-wall lytic enzymes revealed a significant similarity with the N-terminal portion of the lysozymes of Streptomyces globisporus, the fungus Chalaropsis, the Lactobacillus bulgaricus bacteriophage mv1, and the Streptococcus pneumoniae bacteriophages of the Cp family (CPL lysozymes) . In addition, the analyses showed that the C-terminal half of the clostridial lysozyme was homologous to the N-terminal domain of the muramoyl-pentapeptide-carboxypeptidase of Streptomyces albus, suggesting a role in substrate binding . The existence of five putative repeated motifs in the C-terminal region of the autolytic lysozyme suggests that this region could play a role in the recognition of the polymeric substrate. FEBS Lett, 1991 Jul 29, 286(1-2), 100 - 4 Autoregulatory control of actin synthesis in cultured rat hepatocytes; Reuner KH et al.; ADP-ribosylation of actin by Clostridium botulinum C2 toxin resulted in a depolymerization of filamentous F-actin and an increase of monomeric G-actin in cultured hepatocytes . Simultaneously the de novo synthesis of actin was largely reduced, while the synthesis of albumin and of other proteins was not significantly impaired . The specific decrease of actin mRNA to 30% of the control indicates a down-regulation of actin synthesis at a pretranslational level . On the other hand, treatment with the mycotoxin phalloidin resulted in an increase of F-actin and a decrease of monomeric G-actin . Under this condition the de novo synthesis of actin was specifically enhanced and the level of actin mRNA was increased to 600% of the control . The data suggest an autoregulatory control of the actin synthesis. J Biol Chem, 1991 Jul 5, 266(19), 12449 - 54 Isolation of a tryptic fragment from Clostridium perfringens theta-toxin that contains sites for membrane binding and self-aggregation; Tweten RK et al.; Trypsin cleaves Clostridium perfringens theta-toxin (perfringolysin O or PFO) at a single site between residues 303 and 304 (Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Kawasaki, H., and Ando, S . (1986) Biochemistry 25, 6048-6053; Tweten, R . K . (1988b) Infect . Immun . 56, 3228-3234) and yields an amino-terminal fragment of 30,208 Da (T1) and a carboxyl-terminal fragment of 22,268 Da (T2) . Both peptides were purified by reverse phase chromatography of trypsin-nicked PFO . Neither peptide retained hemolytic activity . Peptide T1 had no apparent effect on the hemolytic activity of PFO, whereas T2 was found to inhibit the hemolytic activity of PFO and was analyzed further . The order of binding of T2 and PFO to membranes did not alter the inhibitory effect of T2 on PFO-induced hemolysis, indicating that competitive binding by T2 for PFO membrane binding sites was not the basis for the observed inhibition . Further analysis showed that T2 could inhibit membrane-dependent fluorescence energy transfer (FET) between PFO molecules labeled with fluorescein (fluorescent donor) or tetramethylrhodamine (fluorescent acceptor) . This provided evidence that T2 could complex with PFO . T2 was also found to be incapable of self-aggregation (as opposed to PFO), since preincubation of T2 with either erythrocytes or erythrocyte ghost membranes did not affect the T2-dependent inhibition of hemolysis or FET . These data indicate that T2 inhibits PFO-dependent hemolysis by forming a complex with PFO, which inhibits aggregation and that the membrane binding site and a single aggregation site remain intact on T2. Med J Aust, 1991 Jul 1, 155(1), 47 - 50 Pig-bel but no pig: enteritis necroticans acquired in Australia; Watson DA et al.; OBJECTIVE: To report a case of enteritis necroticans acquired in Australia, and to review the history, epidemiology, pathogenesis, clinical features, management and prevention of this disease . CLINICAL FEATURES: A 44-year-old diabetic and alcoholic restaurateur of Chinese-Malay origin, who had been living in Australia for over 20 years, was admitted to hospital with bloody diarrhoea which progressed to fulminant toxaemia and circulatory collapse, and ultimately required laparotomy . Typical pathological features and the isolation of Clostridium perfringens type C from faeces confirmed the diagnosis of enteritis necroticans . INTERVENTION AND OUTCOME: He was treated initially with ampicillin, gentamicin, metronidazole and chloramphenicol, and later with penicillin and metronidazole, and he required large volumes of intravenously administered fluid and blood for his toxaemic, hypotensive state . Laparotomy was performed as a life-saving procedure . Despite a lengthy convalescence, the patient recovered . CONCLUSIONS: Enteritis necroticans is a rare disease in developed countries, however it is likely to be underdiagnosed . Clinicians are encouraged to be on the alert for signs of severity that may indicate the need for laparotomy in a predisposed individual with features of this condition. Infect Immun, 1991 Jul, 59(7), 2499 - 501 Evidence that Clostridium perfringens theta-toxin induces colloid-osmotic lysis of erythrocytes; Harris RW et al.; Clostridium perfringens theta-toxin was shown to lyse target erythrocytes by a colloid-osmotic mechanism . Analysis showed the onset of lysis of erythrocytes by theta-toxin could be temporarily stabilized with 0.3 M sucrose . Flow cytometry analysis of the size distribution of theta-toxin-treated erythrocytes showed swelling of the erythrocytes prior to lysis. Infect Immun, 1991 Jul, 59(7), 2456 - 62 Correlation of immunoblot type, enterotoxin production, and cytotoxin production with clinical manifestations of Clostridium difficile infection in a cohort of hospitalized patients; McFarland LV et al.; To determine whether strain-specific differences in immunoblot type, enterotoxin production, or cytotoxin production correlated with clinical presentation of Clostridium difficile infection, we evaluated isolates obtained from 428 prospectively studied hospitalized patients . Of 99 isolates available for immunoblot typing, 61 were recovered from asymptomatic carriers and 38 were from patients with C . difficile-associated diarrhea . Of 17 immunoblot types, the seven types comprising the majority of isolates (82 of 99; 83%) were variably associated with disease . Neither the presence of cytotoxin in the stool nor the production of cytotoxin or enterotoxin by isolates in vitro was significantly different for symptomatic versus asymptomatic patients . Selected host factors were more predictive of symptomatic disease than was the specific infecting C . difficile strain . These results suggest that variations in the clinical severity of C . difficile infection in different patients are not solely strain-specific phenomena related to immunoblot type or to the production of cytotoxin or enterotoxin. J Gen Microbiol, 1991 Jul, 137 ( Pt 7), 1729 - 36 Nucleotide sequence and characteristics of endoglucanase gene engB from Clostridium cellulovorans; Foong F et al.; An endoglucanase gene, engB, from Clostridium cellulovorans, previously cloned into pUC19, has been further characterized and its product investigated . The enzyme, EngB, encoded by the gene was secreted into the periplasmic space of Escherichia coli . The enzyme was active against carboxymethylcellulose, xylan and lichenan but not Avicel (crystalline cellulose) . The sequenced gene showed an open reading frame of 1323 base pairs and coded for a protein with a molecular mass of 48.6 kDa . The mRNA contained a typical Gram-positive ribosome-binding site sequence GGAGG and a sequence coding for a putative signal peptide . There is high amino acid and base sequence homology between the N-terminal regions of EngB and another C . cellulovorans endoglucanase, EngD, but they differ significantly in their C-termini . Deletion analyses revealed that up to 32 amino acids of the N-terminus and 52 amino acids of the C-terminus were not required for catalytic activity . The conserved reiterated domains at the C-terminus of EngB were similar to those from endoglucanases from other cellulytic bacteria . According to our deletion analyses, this region is not needed for catalytic activity. Antimicrob Agents Chemother, 1991 Jul, 35(7), 1370 - 5 In vitro activity of YM133, a new semisynthesized macrolide; Terasawa T et al.; YM133, the 4"-O-(4-methoxyphenyl)acetyltylosin, is a new macrolide . The in vitro activity of YM133 was compared with those of erythromycin, josamycin, and rokitamycin by an agar dilution method . YM133 inhibited 90% of the tested isolates of Streptococcus pneumoniae, Legionella spp., and anaerobic bacteria at less than or equal to 1.56 micrograms/ml . The drug inhibited 90% of erythromycin-resistant staphylococci and Streptococcus pyogenes at less than or equal to 50 micrograms/ml . YM133 showed activity against erythromycin-, josamycin-, and rokitamycin-resistant (MIC greater than or equal to 100 micrograms/ml) strains of staphylococci, streptococci, Bacteroides spp., and Clostridium spp . Enterococci were less susceptible to other YM133-like macrolides . Unlike other macrolides, YM133 showed killing activity, and the MBC/MIC ratios of YM133 for several strains were 1:32, whereas those of erythromycin were 4:1,024 . In a time-kill curve study, the reduction of viable cells started within 2 h after the addition of YM133. Ther Umsch, 1991 Jul, 48(7), 508 - 14 {Antibiotic-associated colitis--the dark side of antibiotic therapy}; Marbet UA; Intestinal side effects after antibiotic therapy are frequent . Mostly, harmless diarrhea disappears after cessation of therapy without inducing colitis; however, changing of the intestinal flora sometimes leads to colonization of the colon by toxin-producing strains of Clostridium difficile, inducing sometimes severe pseudomembranous colitis . A rapid correct diagnosis by anamnesis, clinical signs, endoscopical aspect and demonstration of toxin in the stool allows an efficient treatment . The therapeutic modalities, especially in recurrency, are delineated . In addition, the unusual and etiologically still unknown illness of penicillin-induced segmental hemorrhagic colitis will be discussed as well. Rev Infect Dis, 1991 Jul-Aug, 13 Suppl 8, S657 - 64 Adherence of Helicobacter pylori to gastric carcinoma cells: analysis by flow cytometry; Dunn BE et al.; An in vitro assay using immunofluorescence and flow cytometry for quantitative assessment of the adherence of Helicobacter pylori to cultured human gastric carcinoma (KATO III) cells was developed . Adherence was rapid, saturable, energy dependent, mannose resistant, and significantly inhibited by fetuin, a glycoprotein containing N-acetylneuraminyllactose . Pretreatment of KATO cells with neuraminidase from Clostridium perfringens, however, did not reduce adherence of H . pylori . Ultrastructurally, adherent cells of H . pylori were associated with indentations of KATO cell membranes . KATO cells should prove useful in the investigation of mechanisms of adherence of H . pylori to mammalian cells . Ultimately, this flow cytometric assay may be helpful in assessment of the adherence of laboratory strains of H . pylori directly to surface mucous cells dissociated from biopsied human gastric tissue. J Pediatr Gastroenterol Nutr, 1991 Jul, 13(1), 39 - 41 Clostridium difficile in inflammatory bowel disease relapse; Gryboski JD; Stools of 65 patients with exacerbation of symptoms of inflammatory bowel disease were examined for the presence of enteric pathogens and Clostridium difficile . Ten (16%) had C . difficile toxin . Symptoms in all patients cleared after therapy, with improvement correlating with elimination of toxin from the stool. FEMS Microbiol Lett, 1991 Jul 1, 65(3), 311 - 5 Production of monoclonal antibody to Clostridium difficile toxin A which neutralizes enterotoxicity but not haemagglutination activity; Kamiya S et al.; Nine monoclonal antibodies (mAb) to Clostridium difficile toxin A were produced . The isotype of one mAb (37B5) was IgG2b, kappa, and that of the other eight mAbs was IgM, kappa . Immunoblot analysis after non-denatured PAGE showed that with the exception of one mAb (112G6) all mAbs gave a positive reaction with the 540 kDa band of toxin A . Immunoblot analysis showed that four mAbs (2E15, 3B4, 37B5 and 49C4) gave a positive reaction with the 240 kDa major band of toxin A . In neutralisation tests with these mAbs for enterotoxicity, mouse lethality, haemagglutination activity and cytotoxicity, 37B5 neutralised enterotoxicity in a rabbit ileal loop response test but did not neutralise any other biological activities . None of the other eight mAbs showed any neutralising activities at all. Eur J Epidemiol, 1991 Jul, 7(4), 434 - 6 New Clostridium difficile serotypes in Poland; Meisel-Mikolajczyk F et al.; Polish isolates of Clostridium difficile strains of different origin were studied . All strains were serotyped according to the Delmee scheme . Twenty-one strains remained untypeable . By typing them with antisera against 5 Polish strains we observed 5 new serotypes. J Vet Diagn Invest, 1991 Jul, 3(3), 215 - 7 Suitability of the broth-disk elution test for evaluating susceptibility of obligate anaerobes to trimethoprim-sulfonamides; Indiveri MC et al.; Species of anaerobic bacteria isolated from clinical veterinary specimens were tested for susceptibility to trimethoprim-sulfonamides by the broth-disk elution technique . Three different media were used for each organism: prereduced anaerobically sterilized (PRAS) brain-heart infusion broth (BHI), thioglycollate broth, and a semidefined PRAS medium . Susceptibility results from these media were compared with those determined by interpreting the minimal inhibitory concentration obtained using an agar dilution technique . Results from broth-disk testing in semidefined medium agreed in 68.7% of the cases, in 53.7% for thioglycollate broth, and in 36.9% for BHI . The greatest deviation between techniques occurred with isolates belonging to the genus Bacteroides, followed by those of the genus Clostridium and those of the genus Fusobacterium . This deviation was directly proportional to increasing concentrations of thymidine in the BHI and thioglycollate broths but not with the semidefined medium . We conclude that the broth-disk elution method for measuring susceptibility of obligate anaerobes to trimethoprim-sulfonamides is unsuitable. J Appl Bacteriol, 1991 Jul, 71(1), 78 - 85 Expression of a beta-galactosidase gene from Clostridium acetobutylicum in Lactococcus lactis subsp . lactis; Pillidge CJ et al.; A beta-galactosidase gene from Clostridium acetobutylicum NCIB 2951 was expressed after cloning into pSA3 and electroporation into derivatives of Lactococcus lactis subsp . lactis strains H1 and 7962 . When the clostridial gene was introduced into a plasmid-free derivative of the starter-type Lact . lactis subsp . lactis strain H1, the resulting construct had high beta-galactosidase activity but utilized lactose only slightly faster than the recipient . beta-galactosidase activity in the construct decreased by over 50% if the 63 kb Lac plasmid pDI21 was also present with the beta-galactosidase gene . Growth rates of Lac+ H1 and 7962 derivatives were not affected after introduction of the clostridial beta-galactosidase, even though beta-galactosidase activity in a 7962 construct was more than double that of the wild-type strain . When pDI21 was electroporated into a plasmid-free variant of strain 7962, the recombinant had high phospho-beta-galactosidase activity and a growth rate equal to that of the H1 wild-type strain . The H1 plasmid-free strain grew slowly in T5 complex medium, utilized lactose and contained low phospho-beta-galactosidase activity . We suggest that beta-galactosidase expression can be regulated by the lactose phosphotransferase system-tagatose pathway and that Lact . lactis subsp . lactis strain H1 has an inefficient permease for lactose and contains chromosomally-encoded phospho-beta-galactosidase genes. J Med Microbiol, 1991 Jul, 35(1), 40 - 4 Purification and characterisation of toxin B from a strain of Clostridium difficile that does not produce toxin A; Torres JF; Most toxigenic strains of Clostridium difficile produce both toxin A and toxin B . The toxin produced by C . difficile strain 8864 was characterised and compared with those produced by C . difficile strain 10463 . Toxin A was not detected by immunoassay in cultures from strain 8864 and all the cytotoxic activity produced by this strain was neutralised by antiserum to toxin B . Toxin B from strain 8864 was purified and compared with toxin B from strain 10463 . The size of the purified subunits of toxin B from strain 8864 differed slightly from those of strain 10463 and there were small immunological differences . The effect on fibroblast cells was more like that of C . sordellii cytotoxin than of toxin B from strain 10463 . These results suggest that C . difficile strain 8864 produces a modified toxin B and does not produce toxin A. J Clin Invest, 1991 Jul, 88(1), 119 - 25 Characterization of rabbit ileal receptors for Clostridium difficile toxin A . Evidence for a receptor-coupled G protein; Pothoulakis C et al.; The purpose of this study was to characterize the surface receptor for toxin A, the enterotoxin from Clostridium difficile, on rabbit intestinal brush borders (BB) and on rat basophilic leukemia (RBL) cells . Purified toxin A was radiolabeled using a modified Bolton-Hunter method to sp act 2 microCi/micrograms, with retention of full biologic activity . 3H-Toxin A bound specifically to a single class of receptors on rabbit BB and on RBL cells with dissociation constants of 5.4 x 10(-8) and 3.5 x 10(-8) M, respectively . RBL cells were highly sensitive to toxin A (cell rounding) and had 180,000 specific binding sites per cell, whereas IMR-90 fibroblasts were far less sensitive to toxin A and lacked detectable specific binding sites . Exposure of BB to trypsin or chymotrypsin significantly reduced 3H-toxin A specific binding . Preincubation of BB with Bandeirea simplicifolia (BS-1) lectin also reduced specific binding, and CHAPS-solubilized receptors could be immobilized with WGA-agarose . The addition of 100 nM toxin A accelerated the association of 35S-GTP gamma S with rabbit ileal BB, and preincubation of BB with the GTP analogues GTP gamma S or Gpp(NH)p, significantly reduced 3H-toxin A specific binding . Our data indicate that the membrane receptor for toxin A is a galactose and N-acetyl-glucosamine-containing glycoprotein which appears to be coupled to a G protein. Appl Environ Microbiol, 1991 Jul, 57(7), 1873 - 9 Development and application of a multiple typing system for Clostridium difficile; Mahony DE et al.; A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile . A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C . difficile were screened for the ability to produce bacteriocin . These strains were used to type a collection of 114 isolates of C . difficile . Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable . Plasmid typing of the same 114 isolates of C . difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids . Twenty different plasmid typing patterns were observed among the isolates . A combination of bacteriocin and plasmid typing provided 77% typeability . Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8% . Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production . This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included . We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species. Am J Vet Res, 1991 Jul, 52(7), 1147 - 52 Differences in signs and lesions in sheep and goats with enterotoxemia induced by intraduodenal infusion of Clostridium perfringens type D; Blackwell TE et al.; Enterotoxemia was induced in 4 lambs and 4 goat kids by continuous intraduodenal infusion of a whole culture of Clostridium perfringens type D . Clinical signs, hematologic values, biochemical alterations, and postmortem lesions in the lambs and goat kids were compared . The 4 lambs and 4 goat kids died within 25 hours of beginning the infusions . Lesions were not observed in the gastrointestinal tract of the 4 lambs; however, severe hemorrhagic enterocolitis was found in the 4 goat kids . This difference between the lambs and goat kids in the lesions caused by experimentally induced enterotoxemia may explain the discrepancies reported between sheep and goats in clinical signs, response to treatment, and efficacy of vaccination observed in naturally induced enterotoxemia in the 2 species. Diagn Microbiol Infect Dis, 1991 Jul-Aug, 14(4), 331 - 5 Antibacterial activity of rokitamycin compared with that of other macrolides; Yu KW et al.; The activity of rokitamycin, a 16-membered macrolide, was compared with other macrolides and agents used to treat respiratory infections . Rokitamycin had in vitro activity against streptococci and Streptococcus pneumoniae comparable to the other macrolides, inhibiting most organisms at less than 0.03 to 0.5 microgram/ml . It was the most active macrolide agent against Staphylococcus aureus, inhibiting 90% at 1 microgram/ml . Macrolide-resistant streptococci and staphylococci in which resistance was inducible were inhibited, but constitutively resistant Gram-positive bacteria were resistant . Rokitamycin was less active than erythromycin against Haemophilus influenzae, but had activity comparable to erythromycin against Moraxella catarrhalis and Neisseria gonorrhoeae . It inhibited Clostridium spp . and peptostreptococci, but had poor activity against Bacteroides species . Rokitamycin was bactericidal for streptococci and staphylococci, but not for enterococci . Overall rokitamycin has in vitro activity comparable to currently available 14-membered macrolides . Its clinical utility will be influenced by the degree of metabolism to less active metabolites since 70% is rapidly metabolized. Nippon Geka Gakkai Zasshi, 1991 Jul, 92(7), 889 - 92 {A case of gas gangrene following operation of cervical esophageal carcinoma}; Mori T et al.; A 74-year-old woman underwent surgery for cervical esophageal carcinoma . After operation, she had abdominal distension, high fever, and suddenly fell into shock and died . Autopsy revealed necrosis and multiple gas blebs in the entire digestive tract and liver . Direct smear specimens Gram-stained disclosed clostridium septicum and the cause of death was found to be gas gangrene . This is the first of clostridium infection localized in the digestive tract and liver after operation . Abdominal radiogram showed massive gas in the intestine, intramural bowel gas and gas in the portal vein, and these are very suggestive of gas gangrene of the intestinal tract. Int J Syst Bacteriol, 1991 Jul, 41(3), 355 - 7 Clostridium orbiscindens sp . nov., a human intestinal bacterium capable of cleaving the flavonoid C-ring; Winter J et al.; Clostridium orbiscindens sp . nov . is an obligate anaerobe that is capable of cleaving the C-3-C-4 bond of the natural anticarcinogen quercetin . The metabolic products, 3,4-dihydroxyphenylacetic acid and presumably phlorglucinol, are not known to possess anticarcinogen properties . This organism was isolated from human feces . On sheep blood agar plates C . orbiscindens forms minute, irregular, convex, gray or white, shiny, smooth, nonhemolytic colonies . It is beta-hemolytic on rabbit blood agar . The motile peritrichous rods are gram variable . Subpolar spores are common . Cultures are resistant to 80 degrees C for 10 min . Capsules are absent . This asaccharolytic organism does not metabolize esculin, urea, meat, gelatin, casein, or nitrate . The G + C content is 56 to 57 mol% . DNA hybridization experiments did not reveal relatedness to phenotypically similar Clostridium strains . Strain 265 (= ATCC 49531) is the type strain. Angiology, 1991 Jul, 42(7), 585 - 9 Mycotic aortic aneurysm infected by Clostridium septicum--a case history; Hurley L et al.; The authors describe a sixty-seven-year-old hypertensive, diabetic man with a mycotic abdominal aortic aneurysm infected with Clostridium septicum . The patient had colonic polyps but no malignant disease . They could find only one other report of a mycotic aneurysm infected with C . septicum . In that case, as in most other cases of C . septicum bacteremia, the patient had gastrointestinal cancer . Their case suggests that treatment for a clostridial infection should be considered in patients with known gastrointestinal disease, signs and symptoms of sepsis, and abdominal pain . Conversely, patients known to have a C . septicum infection should be evaluated for gastrointestinal lesions. Gut, 1991 Jul, 32(7), 791 - 5 Clostridium difficile toxin A induces a specific antisecretory factor which protects against intestinal mucosal damage; Torres J et al.; Peroral challenge with toxin A from Clostridium difficile induced the formation of antisecretory factor in rats . The animals were given 100 micrograms of the toxin, which was followed by a pronounced diarrhoea and by the appearance of antisecretory factor in the pituitary gland . In electrofocusing, the induced antisecretory factor separated in two peaks (pI 5.4 and 5.0); both fractions showed a lectin-like binding to agarose . The pI 5.4 fraction inhibited cholera toxin as well as toxin A induced fluid secretion, while pI 5.0 inhibited toxin A induced secretion only . Immunohistochemistry showed that an antisecretory factor of pI 5.0 protected the mucosa from the cytotoxic effect of toxin A, but did not affect the binding of toxin A to the intestinal epithelium . Sodium dodecyl-sulphate-polyacrylamide gel electrophoresis of the pI 5.0 protein showed two major fractions to be present, one of molecular weight 60 kDa, the other of 30 kDa, the latter probably being a degradation product of the former. J Exp Med, 1991 Jul 1, 174(1), 179 - 91 The Trypanosoma cruzi neuraminidase contains sequences similar to bacterial neuraminidases, YWTD repeats of the low density lipoprotein receptor, and type III modules of fibronectin; Pereira ME et al.; Trypanosoma cruzi expresses a developmentally regulated neuraminidase (TCNA) implicated in parasite invasion of cells . We isolated full-length DNA clones encoding TCNA . Sequence analysis demonstrated an open reading frame coding for a polypeptide of 1,162 amino acids . In the N-terminus there is a cysteine-rich domain containing a stretch of 332 amino acids nearly 30% identical to the Clostridium perfringens neuraminidase, three repeat motifs highly conserved in bacterial and viral neuraminidases, and two segments with similarity to the YWTD repeats found in the low density lipoprotein (LDL) receptor and in other vertebrate and invertebrate proteins . This domain is connected by a structure characteristic of type III modules of fibronectin to a long terminal repeat (LTR) consisting of 44 full length copies of twelve amino acids rich (75%) in serine, threonine, and proline . LTR is unusual in that it contains at least 117 potential phosphorylation sites . At the extreme C-terminus is a hydrophobic segment of 35 amino acids, which could mediate anchorage of TCNA to membranes via a glycosylphosphatidylinositol linkage . This is the first time a protozoan protein has been found to contain a YWTD repeat and a fibronectin type III module . The domain structure of TCNA suggests that the enzyme may have functions additional to its catalytic activity such as in protein-protein interaction, which could play a role in T . cruzi binding to host cells. FEBS Lett, 1991 Jun 24, 284(2), 165 - 8 Resonance Raman studies of the {4Fe-4S} to {2Fe-2S} cluster conversion in the iron protein of nitrogenase; Fu WG et al.; Resonance Raman spectroscopy has been used to investigate the Fe-S stretching modes of the {4Fe-4S}2+ cluster in the oxidized iron protein of Clostridium pasteurianum nitrogenase . The results are consistent with a cubane {4Fe-4S} cluster having effective Td symmetry with cysteinyl coordination for each iron . In accord with previous optical and EPR studies {(1984) Biochemistry 23, 2118-2122}, treatment with the iron chelator alpha, alpha'-dipyridyl in the presence of MgATP is shown to effect cluster conversion to a {2Fe-2S}2+ cluster . Resonance Raman data also indicate that partial conversion to a {2Fe-2S}2+ cluster is induced by thionine-oxidation in the presence of MgATP in the absence of an iron chelator . This result suggests new explanations for the dramatic change in the CD spectrum that accompanies MgATP-binding to the oxidized Fe protein and the anomalous resonance Raman spectra of thionine-oxidized Clostridium pasteurianum bidirectional hydrogenase. FEMS Microbiol Lett, 1991 Jun 15, 65(2), 221 - 6 Overproduction of a Clostridium cellulolyticum endoglucanase by mutant strains of Escherichia coli; Blanco A et al.; We have studied the expression of an endoglucanase from Clostridium cellulolyticum in mutant strains of Escherichia coli that overproduce haemolysin . When these mutants were transformed with plasmids encoding the endoglucanase, they showed a significantly enhanced endoglucanase activity, compared to transformed parental strains . Among the mutants, strain Hha-2 showed the highest production . We have identified the endoglucanase gene product synthesized in E . coli Hha-2/pBP8 and detected an increased amount of the enzyme parallel to the increase of endoglucanase activity . This was mainly localized in the periplasm and only a small percentage of it was found in the culture fluid. J Biol Chem, 1991 Jun 15, 266(17), 11037 - 43 Localization of the receptor-binding region of Clostridium perfringens enterotoxin utilizing cloned toxin fragments and synthetic peptides . The 30 C-terminal amino acids define a functional binding region; Hanna PC et al.; In this study a short sequence encoding the receptor-binding activity of the much larger 35-kDa enterotoxin elaborated by Clostridium perfringens was localized by recombinant DNA techniques . Defined fragments corresponding to portions of the enterotoxin gene were cloned into an Escherichia coli expression vector system, and these lysates were analyzed for their ability to compete for binding with native C . perfringens enterotoxin (CPE) . The lysate containing CPE290-319 (CPE sequence encompassing residues 290-319) was shown to compete with 125I-CPE for specific binding sites on rabbit intestinal brush border membranes . To confirm this finding, a peptide corresponding to the CPE amino acid sequence 290-319 was synthesized and found to completely block CPE specific binding . To demonstrate directly that CPE290-319 can act as a competitive antagonist of CPE cytotoxicity for physiologic receptors, Vero cells were preincubated with either E . coli lysates containing CPE290-319 or the synthetic peptide corresponding to this sequence . Preincubation of Vero cells with either the lysate or the peptide completely protected these cells from CPE challenge . This information localizes the C-terminal 30 residues of CPE (CPE290-319) as a linear sequence sufficient for recognition and binding to the eukaryotic CPE receptor. J Biol Chem, 1991 Jun 5, 266(16), 10313 - 8 Identification of a histidyl residue in the active center of endoglucanase D from Clostridium thermocellum; Tomme P et al.; Diethylpyrocarbonate modification of endoglucanase D from Clostridium thermocellum, cloned in Escherichia coli, resulted in a rapid but partial (maximally 70-80%) loss of activity . The second-order rate constant of inactivation proved to be exceptionally high (3210 M-1.min-1) . A 3-fold reduction of the kcat and a 2-fold increase of the Km for 2'-chloro-4'-nitrophenyl beta-cellobioside were observed . Spectrophotometric analysis indicate the presence of one rapidly (k = 0.45 min-1) and two slower (k = 0.23 min-1) reacting histidyl residues . In the presence of 50 mM methyl beta-cellotrioside, the rate of inactivation was reduced 16-fold, and the kinetics of modification were compatible with the protection of 1 histidyl residue . Since peptide analysis was inconclusive, identification of the critical residue was attempted by site-directed mutagenesis . Each of the 12 histidyl residues present in the endoglucanase D sequence was mutated into either Ala or Ser . Seven of the mutant enzymes had specific activities lower than 50% of the wild-type . Only in the case of the Ser-516 mutant, however, was the residual activity not affected by diethyl pyrocarbonate . These findings suggest an important functional or structural role for His-516 in the wild-type enzyme. J Biol Chem, 1991 Jun 5, 266(16), 10062 - 5 Identification of a botulinum C3-like enzyme in bovine brain that catalyzes ADP-ribosylation of GTP-binding proteins; Maehama T et al.; A novel enzyme activity was found in bovine brain cytosol that transfers the ADP-ribosyl moiety of NAD to proteins with Mr values of 22,000 and 25,000 . The substrates were the same GTP-binding proteins serving as the substrate of an ADP-ribosyltransferase C3 which was produced by a type C strain of Clostridium botulinum . The brain enzyme was partially purified from the cytosol and had a molecular mass of approximately 20,000 on a gel filtration column . The brain endogenous enzyme displayed unique properties similar to those observed with botulinum C3 enzyme . The enzyme activity was markedly stimulated by a protein factor that had been initially found in the cytosol as an activator for botulinum C3-catalyzed ADP-ribosylation (Ohtsuka, T., Nagata, K., Iiri, T., Nozawa, Y., Ueno, K., Ui, M., and Katada, T . (1989) J . Biol . Chem . 264, 15000-15005) . The activity of the brain enzyme was also affected by certain types of detergents or phospholipids . The substrate of the brain enzyme was specific for GTP-binding proteins serving as the substrate of botulinum C3 enzyme; the alpha-subunits of trimeric GTP-binding proteins which served as the substrate of cholera or pertussis toxin were not ADP-ribosylated by the endogenous enzyme . Thus, this is the first report showing an endogenous enzyme in mammalian cells that catalyzes ADP-ribosylation of small molecular weight GTP-binding proteins. Infect Control Hosp Epidemiol, 1991 Jun, 12(6), 345 - 8 Risk factors for the development of Clostridium difficile-associated diarrhea during a hospital outbreak; Thibault A et al.; OBJECTIVE: To evaluate the risk factors associated with a nosocomial outbreak of Clostridium difficile-associated diarrhea . DESIGN: Case-control study with two control groups . SETTING: University-affiliated urban hospital . PATIENTS: A convenience sample of 26 patients was chosen out of a total of 78 hospitalized patients with C difficile-associated diarrhea, defined as the presence of diarrhea and a positive C difficile cytotoxin assay or stool culture . Twenty-six controls were matched for age, gender, ward, and date of admission; 18 additional controls were matched to surgical patients for date and ward of admission, as well as for the type of surgical procedure performed . RESULTS: Significant risk factors for the development of C difficile-associated diarrhea were gastrointestinal surgery (exposure odds ratio {EOR} = 7.9, p = .004, 95% confidence interval {CI} = 1.9, 35), use of neomycin (EOR = 15.6, p = .012, 95% CI = 1.7, 92), clindamycin (EOR = 15.6, p = .005, 95% CI = 1.7, 92), metronidazole (EOR = 5.7, p = .02, 95% CI = 1.4, 25), and excess antibiotic use (mean number of antibiotics = 4.2 versus 1.4, p less than .00005) . The presence of systemic disease and the use of antacids or immunosuppressive drugs were similar in cases and controls . In surgical patients, no specific antibiotic could be linked to C difficile-associated diarrhea because of uniform perioperative antibiotic use . There was a significant difference in the number of antibiotics administered to cases and controls (mean = 3.1 versus 1.9, p less than .005) . CONCLUSIONS: The results suggest that control of nosocomial C difficile-associated diarrhea may be attained by minimizing the administration of antibiotics, avoidance of high-risk antibiotics, and having a high index of suspicion of C difficile-associated diarrhea in patients who develop diarrhea after gastrointestinal surgery . Perioperative administration of metronidazole, when given with other antibiotics, failed to protect against the development of C difficile-associated diarrhea. Immunology, 1991 Jun, 73(2), 235 - 8 Actions of three clostridial IgA proteases on distinct forms of immunoglobulin A molecules; Hashim OH et al.; Three bacterial species of Clostridium (septicum, tertium and sporogenes) were identified to produce extracellular proteases cleaving IgA to Fab and Fc fragments, as demonstrated by SDS-PAGE and immunoelectrophoretic procedures . These enzymes acted on monometric IgA1 paraproteins and normal serum IgA1 but had no activity on IgA2 paraproteins and intact secretory IgA1 from human colostrum . Their action on polyclonal serum IgA1 suggested the absence of neutralizing anti-clostridial IgA protease activity . Although the enzymes were shown not to act on secretory IgA1, they were, however, able to digest free alpha-heavy chains of the dimeric IgA molecules . Susceptibility of the alpha-heavy chain to the proteases was more likely due to the change to a more accessible conformation than because of the absence of neutralizing anti-enzymic activity. Clin Radiol, 1991 Jun, 43(6), 397 - 9 Sonographic demonstration of septicaemia with gas-forming organisms after liver transplantation; Jantsch H et al.; Sepsis with gas-forming organisms, e.g . Clostridium perfringens and anaerobic streptococci occurred in three of 120 liver-transplant recipients (2.5%) . The diagnosis was made in all three patients by bedside ultrasound, before blood cultures revealed bacterial growth . Floating high amplitude echoes within the extra- and intrahepatic portal veins and persistent small high amplitude echoes in the non-dependent portion of the liver are indicative of portal venous gas and should prompt further laboratory investigations. Am J Gastroenterol, 1991 Jun, 86(6), 685 - 9 Positive role of Clostridium difficile infection in diarrhea in infants and children; Gryboski JD et al.; A retrospective review of children with Clostridium difficile infection and diarrhea identified 43 patients . Fifteen (35%) had immunoglobulins below the normal range for age, and typified those with transient hypogammaglobulinemia of infancy, because all increased their levels over the following 12 months . The age of those with hypogammaglobulinemia was significantly younger (p less than 0.01), averaging 18.8 months, than those with normal immunoglobulins who had a mean age of 4.6 yr . The number with recurrent C . difficile infection was significantly greater (p less than 0.001) in the hypogammaglobulinemic group than in those with normal immunoglobulins (46% vs 18%) . Eleven children were identified who had C . difficile toxin in the stool after antibiotic therapy, but who had no diarrhea at the time of study . All had normal immunoglobulins . Control patients (20 without and 40 with diarrhea) had no evidence of C . difficile infection, and all but three had normal immunoglobulins . Three (7%) of those with diarrhea had IgA deficiency, and all had a diagnosis of milk protein allergy . These data suggest that immunoglobulin values be obtained in infants and children who have problematic C . difficile diarrhea in order to identify disorders of immunoglobulin synthesis which may be the underlying cause of repeated upper respiratory disease requiring antibiotic usage. Infect Immun, 1991 Jun, 59(6), 2215 - 8 Passive immunization of hamsters against disease caused by Clostridium difficile by use of bovine immunoglobulin G concentrate; Lyerly DM et al.; Gestating Holstein cows were vaccinated with Clostridium difficile toxoid prepared from the culture filtrate of a strain that produces high levels of toxins A and B and other antigens . A bovine immunoglobulin G (IgG) concentrate was prepared from colostrum collected at parturition . The results of our studies showed that hamsters treated prophylactically with the hyperimmune bovine IgG concentrate were protected against C . difficile disease . These results suggest that orally administered hyperimmune bovine IgG specific for C . difficile culture filtrate may be useful in prophylaxis against C . difficile disease. Xenobiotica, 1991 Jun, 21(6), 737 - 50 Metabolism of levamisole, an anti-colon cancer drug, by human intestinal bacteria; Shu YZ et al.; 1 . Anaerobic incubation of levamisole with human intestinal flora resulted in the formation of three thiazole ring-opened metabolites, namely, levametabol-I, II and III . These new hydroxamic lactam-type metabolites were isolated and characterized by various spectroscopic methods . 2 . Various pure cultures of human intestinal bacterial strains were shown, by quantitative h.p.l.c . analysis, to have ring-opening ability . Strong metabolizers include Bacteroides and Clostridium spp . Bacterial mixtures prepared from human faeces showed much greater ability to transform levamisole (74% in 48 h) than any pure strain culture . 3 . Greatly decreased levamisole-transforming activity was observed with autoclaved bacterial cultures, and no activity was found with broth medium alone . This indicates that metabolism requires the presence of anaerobic bacteria and involves, at least in part, a non-enzymic process. Antimicrob Agents Chemother, 1991 Jun, 35(6), 1108 - 11 In vitro and in vivo evaluation of tiacumicins B and C against Clostridium difficile; Swanson RN et al.; Tiacumicins B and C are members of a novel group of 18-membered macrolide antibiotics with in vitro activity against Clostridium difficile . The MICs against 15 strains of C . difficile were 0.12 to 0.25 microgram/ml for tiacumicin B, 0.25 to 1 microgram/ml for tiacumicin C, and 0.5 to 1 microgram/ml for vancomycin . The resistance frequency for both compounds against C . difficile was less than 2.8 x 10(-8) at four and eight times the MIC . The in vivo activities of the tiacumicins against two strains of C . difficile were compared with that of vancomycin in a hamster model of antibiotic-associated colitis . Oral therapy with 0.2, 1, or 5 mg of tiacumicin B or C per kg of body weight protected 100% of clindamycin-treated hamsters exposed to C . difficile ATCC 9689 . Oral treatment with identical doses of vancomycin produced a prolonged, dose-dependent survival of hamsters, but it did not prevent the development of fatal colitis at doses of up to 5 mg/kg . When clindamycin-treated animals were exposed to another strain of C . difficile, both tiacumicin B and vancomycin were protective at 5 mg/kg, but not at lower doses . Tiacumicin C was not tested in vivo against the second strain of C . difficile . No tiacumicin B or C was detected in the sera of hamsters treated with single oral doses of 25 mg/kg, while antibiotic levels in the ceca of these hamsters reached 248 micrograms/ml and 285 mg/ml for tiacumicins B and C, respectively . The tiacumicins demonstrated in vitro and in vivo potencies against C . difficile and achieved high concentrations in the cecum, but not the serum, of hamsters after oral administration. Int J Epidemiol, 1991 Jun, 20(2), 521 - 6 Risk factors of neonatal tetanus in Senegal; Leroy O et al.; A case-control study for evaluating the risk factors of neonatal tetanus was conducted in a rural area of Senegal under demographic surveillance (Niakhar) . Some 45 neonatal tetanus deaths that occurred in the study area between March 1983 and March 1986 were investigated . They were matched with 187 controls . Neonatal tetanus accounted for one-third of all neonatal deaths; mortality from neonatal tetanus was 16/1000 livebirths . The effect of various demographic, socioeconomic, epidemiological and behavioural factors was investigated in a multivariate analysis using linear logistic regression . There was no difference associated with socioeconomic factors between cases and controls . Factors associated with the skill and behaviour of birth attendant and mother were highly significant and were associated with high odds ratio (OR) and included whether the hands of the person cutting the cord were washed with soap (OR = 5.22; p = 0.001); whether the person who dressed the cord was skilled (OR = 4.71; p = 0.012); whether the age of the mother was less than 18 years (OR = 7.03; p = 0.027) and whether the birth attendant arrived before delivery (OR = 4.15, p = 0.023) . Conversely, the type of tool used to cut the cord did not have a significant effect (p = 0.239) . Data analysis suggests that a main source of Clostridium tetani may be the hands of the birth attendant and that the main mode of contamination may be the dressing of the wound stump . Results suggest that teaching mothers and birth attendants simple hygienic principles and basic techniques may have a significant impact on neonatal tetanus mortality. Int J Food Microbiol, 1991 Jun, 13(2), 143 - 55 The occurrence and growth of microorganisms during the fermentation of fish sausage; Aryanta RW et al.; Minced fish (mullet) sausage mixes containing added sugar, salt, nitrate, nitrite and spices were fermented (48 h, 30 degrees C) by indigenous flora or by a starter culture (Pediococcus acidilactici) and the microbial ecology and behaviour of various bacteria was monitored . Pediococcus pentosaceus and Lactobacillus plantarum dominated the indigenous fermentation, achieving populations of 10(7)-10(8) cfu/g by 48 h, and decreasing the pH of the mix to 4.5-4.7 . Significant growth (10(5)-10(7) cfu/g) of Staphylococcus warneri, Staphylococcus aureus, Staphylococcus epidermidis . Micrococcus varians and Micrococcus luteus also occurred during this fermentation . Less growth was exhibited by Bacillus megaterium and yeasts . Pediococcus acidilactici dominated the fermentation when it was inoculated as a starter culture, but indigenous lactic acid bacteria (P . pentosaceus and L . plantarum) also grew to 10(7)-10(8) cfu/g . The growth of other bacteria and yeasts was restricted during fermentation with starter culture . Inoculated Escherichia coli, Salmonella typhimurium, Salmonella sofia, and Staphylococcus aureus grew to 10(6)-10(7