Neurochem Res, 1988 Aug, 13(8), 737 - 42 Membrane phospholipid polar heads influence the coupling of M2 muscarinic receptors to G proteins; Gies JP et al.; Treating membranes from rat heart with phospholipase C (phosphatidylcholine choline phosphohydrolase) from Clostridium perfringens increased the affinity of muscarinic acetylcholine receptors (M2) for the agonists carbachol and oxotremorine . The affinity for antagonists was not affected . Phospholipase C activity, i.e., the cleavage of polar heads of membrane phospholipids, led to the disappearance of the guanine nucleotide-dependent rightward shift of the isotherm for agonist binding . The treatment of tracheal smooth muscle with phospholipase C led to a decrease in the maximum contractile effect of muscarinic (M2) stimulation with no modification of the agonist EC50, i.e., to the uncoupling of the stimulation-contraction process . These results demonstrate that when phospholipid polar heads are hydrolysed by phospholipase C, M2 receptors are uncoupled from G proteins, which enhances their affinity for agonists but prevents information transfer. J Biochem (Tokyo), 1988 Aug, 104(2), 242 - 6 Pseudomonas stutzeri ferredoxin: close similarity to Azotobacter vinelandii and Pseudomonas ovalis ferredoxins; Saeki K et al.; The complete primary structure of Pseudomonas stutzeri strain ZoBell ferredoxin was determined by a combination of protease digestion, Edman degradation, and carboxypeptidase digestion and was: TFVVTDNCIKCKYTDCVEVCPVDCFYEGPNFLVIH PDECIDCALCEPECPAQAIFSEDEVPEDQQEFIELNADLAEVWPNITE KKDALADAEEWDGVKDKLQYLER . The calculated molecular weight was 12,110 excluding iron and sulfur atoms . The amino acid sequence was highly homologous to those of Azotobacter vinelandii and Pseudomonas ovalis ferredoxins . It showed, like the other two, a Tyr-Thr insertion between the second and third Cys, and extra Cys at position 24 and, compared to Clostridium- and Bacillus-type ferredoxins, an extended C-terminal sequence. J Hosp Infect, 1988 Aug, 12(2), 125 - 9 A microbiological study of absorbent pads; Sprott MS et al.; A study was carried out of the microbial content of three types of incontinence underpads and a clinical absorbent protection pad . Coagulase-negative staphylococci and Bacillus spp . were isolated from unused samples of all makes of pad examined . Clostridium spp., including C . tetani and C . perfringens, were isolated from a proportion of pads containing re-cycled waste material . We recommend that incontinence underpads are used solely for the purpose for which they were marketed, namely, the containment of excreta. J Lipid Res, 1988 Aug, 29(8), 1079 - 85 Purification and characterization of bile salt hydrolase from Clostridium perfringens; Gopal-Srivastava R et al.; Bile salt hydrolase (cholylglycine hydrolase, EC 3.5.1.24) has been purified to homogeneity (792-fold) from Clostridium perfringens using high performance DEAE-chromatography . The purified enzyme showed a single detectable protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with a relative molecular weight ca . 56,000 . The intact enzyme had a relative molecular weight (Mr) of ca . 250,000 as determined by nondenaturing PAGE . The NH2-terminal sequence of bile salt hydrolase was determined to be Met-(Ser/Cys)-Arg-Thr-Lys-Leu-Val-Ileu-Thr-Ileu-Gly-Ala-Ser . The purified enzyme was active towards both glycine and taurine conjugates of cholate . The apparent Km and Vmax of the enzyme for glycocholate was estimated to be 0.5 mM and 107 nmol/min.mg protein, respectively . The pH optimum was in the range of 5.8 to 6.4 . The enzyme was inhibited 85%, 81%, and 83% by 2 mM iodoacetate, p-chloromercuribenzoate, and phenylmethanesulfonylfluoride, respectively . Rabbit polyclonal antibody was prepared and used to demonstrate a single form of the enzyme in crude cell extracts. Chemioterapia, 1988 Aug, 7(4), 264 - 6 The in-vitro activity of three long-acting cephalosporins against Bacteroides fragilis, Peptostreptococcus species and Clostridium perfringens; Watt B et al.; The in-vitro activity of three long-acting cephalosporins (cefotetan, cefonicid and ceftriaxone) was compared against 206 clinical isolates of Bacteroides fragilis, Peptostreptococcus species and Clostridium perfringens, using an agar dilution procedure to determine minimum inhibitory concentrations (MICs) . Clindamycin was included as a comparator . Cefotetan was much more active than the two other cephalosporins against strains of Bacteroides fragilis (MIC90 16 mg/l compared with greater than 128 mg/l for the other two agents) . Cefotetan also demonstrated superior activity against anaerobic cocci . All three compounds showed good activity against strains of Clostridium perfringens . Clindamycin was active against all of the test strains. Appl Environ Microbiol, 1988 Aug, 54(8), 2042 - 8 Activation of Clostridium perfringens spores under conditions that disrupt hydrophobic interactions of biological macromolecules; Craven SE; The effect of hydrophobic interactions on the activation of C . perfringens NCTC 8679 spores was examined by heating spores under conditions that modify the hydrophobic properties of biological macromolecules . After the activation treatment and a washing procedure, germination was determined by measuring the decrease in optical density of spores suspended in an enriched germination medium . Activation was inhibited for spores that were treated under conditions that strengthen hydrophobic interactions, i.e., a decrease in pH or the presence of structure-stabilizing neutral salts . Activation was enhanced by treatment under conditions that disrupt hydrophobic interactions, i.e., an increase in pH or the presence of urea, dibucaine, or denaturing neutral salts . A deactivation treatment with the antichaotropic salt (NH4)2SO4 reversed activation by the chaotropic salt CaCl2 and to a lesser extent reversed activation by sublethal heat (75 degrees C) or urea . Most treatments that enhanced activation increased spore injury at higher temperatures, which resulted in decreased germination . However, (NH4)2SO4 and a decrease in pH from 5.6 to 3.8, which inhibited activation, also favored injury . The results suggest that activation involves a conformational change of a spore protein(s) through weakening of hydrophobic molecular forces and that activation and injury occur at different spore sites. Antimicrob Agents Chemother, 1988 Aug, 32(8), 1213 - 7 Molecular cloning and genetic analysis of a chloramphenicol acetyltransferase determinant from Clostridium difficile; Wren BW et al.; A gene bank from a clinical isolate of Clostridium difficile expressing high chloramphenicol acetyltransferase activity was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the plasmid vector pUC13 . Among 1,020 clones tested, 11 were resistant to chloramphenicol; 1 of these, with an insert size of 1.9 kilobases (pPPM9), was studied further . The clone pPPM9 was mapped using a variety of restriction enzymes, and a 0.27-kilobase EcoRV-TaqI restriction fragment was shown to be within the chloramphenicol resistance (Cmr) gene by using transposon (Tn1000) mutagenesis . The 0.27-kilobase fragment and the 1.9-kilobase insert were radiolabeled and used as DNA probes in hybridization studies . Southern blot analysis with the gene probes against chromosomal DNA from Cmr strains of C . difficile obtained from five distinct geographical locations revealed that at least two copies of the same chloramphenicol acetyltransferase gene were present for each strain . Hybridization of the gene probes against Cmr strains of Staphylococcus epidermidis, Staphylococcus aureus, Klebsiella edwardsii, Escherichia coli, and to four other clostridial species revealed no homology even under conditions of low stringency. Biochim Biophys Acta, 1988 Jul 7, 942(1), 125 - 30 Topological distribution of choline phospholipid fatty acids in trout intestinal brush-border membrane; Pelletier X et al.; The transbilayer distribution of choline phospholipids in trout intestinal brush-border membrane has been investigated using phospholipase C (from Clostridium welchii) . In the middle intestine, 84% of phosphatidylcholine (PC) and 60% of sphingomyelin (SP) are located in the outer membrane leaflet . In the posterior intestine, 89% of PC and 52% of SP are located in the outer membrane leaflet . The externally located PC molecular species are (n - 3) fatty acid-rich in both parts of the intestine . While the sphingomyelin molecular species containing 24:1(n - 9) are exclusively located in the outer leaflet in the middle intestine, those containing 14:0 are more abundant in the same leaflet but in the posterior intestine . This strongly asymmetric distribution of both choline phospholipids may have numerous consequences on the brush-border membrane characteristics. Aust Vet J, 1988 Jul, 65(7), 208 - 9 Clostridial myocarditis in lambs; Glastonbury JR et al.; Five outbreaks of myocarditis were investigated in young sheep . They occurred during late winter and spring when there was lush growth of pasture following a prolonged period of drought . Clinically the disease was characterised by sudden death and pathological findings were dominated by acute multifocal locally extensive necrotising and haemorrhagic myocarditis . A fluorescent antibody technique was used to demonstrate the presence of Clostridium chauvoei in paraffin embedded sections of myocardium from 4 of the outbreaks. J Wildl Dis, 1988 Jul, 24(3), 471 - 6 An outbreak of type E botulism among common loons (Gavia immer) in Michigan's upper peninsula; Brand CJ et al.; An epizootic of type E botulism (Clostridium botulinum) occurred among common loons (Gavia immer) along the Lake Michigan shore of Michigan's Upper Peninsula (USA) during October and November 1983 . An estimated 592 dead loons washed ashore along the Garden Peninsula . Type E botulinal toxin was demonstrated in blood samples and stomach contents of dead loons, and in samples of three species of dead fish found on the Lake Michigan shore . We suspect that loons acquired botulism by ingesting sick or dead fish containing type E toxin. J Steroid Biochem, 1988 Jul, 31(1), 97 - 105 Preparation of 3 beta, 5 alpha-, 3 alpha, 5 alpha- and 3 alpha, 5 beta-tetrahydro derivatives of 19-noraldosterone by chemical synthesis and microbial bioconversion; Harnik M et al.; The 3 beta, 5 alpha-, 3 alpha, 5 alpha- and 3 alpha, 5 beta-tetrahydro derivatives 19, 20 and 27 of 19-noraldosterone (1) were prepared to facilitate the search for these compounds in urine . The diketal 4, consisting of a 2:1 mixture of the 5,6- and 5(10)-ene isomers, was hydrogenated with Pd-C and partially hydrolyzed to 5 alpha, 10 alpha- and 5 alpha, 10 beta-dihydroketals 8 and 10 in a 1:2.5 ratio . Assignment of protons was done with aid of COSY 45 experiments . Compound 10 was reduced with diisobutylaluminum hydride (DIBAH) to 4 products: the 3 alpha- and 3 beta-ol hemiacetals 16 and 15, and the corresponding tetraols 14 and 13 . Alternatively, hydrogenation of the 4-en-3-one 2 gave 10, its 5 beta, 10 beta-isomer 21 and the tetrahydro compound 22, in a 4:2:1 ratio . A better way to prepare the 5 beta, 10 beta-series involved microbial conversion of 2 with Clostridium paraputrificum, and the resulting tetrahydrolactone 23 was reduced with DIBAH to the hemiacetal 24 . Acid hydrolysis of 16, 15 and 24 afforded 20, 19 and 27, respectively . According to {1H}-NMR, in solution 20 and 24 exist as mixtures of isomers, while 19 appears in one form only . Periodate oxidation converted 19 and 27 into their gamma-etiolactones 18 and 28 . EI MS base peaks are different and characteristic for 19, 20 and 27. Arch Biochem Biophys, 1988 Jul, 264(1), 281 - 7 pKa values of the 8 alpha-imidazole substituents in selected flavoenzymes containing 8 alpha-histidylflavins; De Francesco R et al.; Difference absorption spectroscopy as a function of pH is described as a probe to determine the pKa values of the 8 alpha-imidazole substituent in flavoenzymes containing 8 alpha-histidylflavin coenzymes . Reversible absorption difference spectra are observed in the pH range 5.5 to 8.5 when synthetic 8 alpha-imidazolyl-FMN is bound to the apoflavodoxins from Azotobacter vinelandii and from Clostridium pasterianum . The observed spectral perturbations of these two flavodoxin complexes follow a single proton ionization dependence with respective pKa values of 6.7 and 6.8 . No pH-induced spectral perturbations were observed when 8 alpha-(N-CH3)-imidazolium FMN was bound to either flavodoxin . Similar approaches are described to determine the 8 alpha-imidazolyl pKa values of the 8 alpha-histidyl-FAD coenzyme of the cholesterol oxidases from Schizophyllum commune and from Gleocystidium chrysocreas . Previous work has shown the former enzyme contains an 8 alpha-N1-histidyl-FAD (W . C . Kenney et al . (1979) J . Biol . Chem . 254, 4689-4690) while experiments reported here show the latter enzyme also contains one 8 alpha-N1-histidyl-FAD per mole of enzyme . The pKa value for the 8 alpha-imidazole substituent on the flavin of S . commune cholesterol oxidase is 5.4 while that determined for the G . chrysocreas enzyme is 6.2 . These results demonstrate that the pKa of the 8 alpha-imidazole substituent can be determined in enzymes containing an 8 alpha-histidylflavin, provided that the enzyme is stable in the pH range required to observe ionization . Furthermore it is shown this the pKa value can differ even on comparison of enzymes from different sources that catalyze the same reaction. J Bacteriol, 1988 Jul, 170(7), 2971 - 6 Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum; Palosaari NR et al.; The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity . The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth . The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity . The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production . Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose . A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer . Kinetic constants were determined in both the forward and reverse directions . In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA . These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases. Infect Immun, 1988 Jul, 56(7), 1708 - 14 Purification and characterization of toxin B from Clostridium difficile; Meador J 3rd et al.; Toxin B from Clostridium difficile was purified to homogeneity and characterized . Purification of toxin B was achieved by gel filtration, chromatography on two consecutive anion-exchange columns, and chromatography on a high-resolution anion-exchange column in the presence of 50 mM CaCl2 . The molecular weight of toxin B was estimated to be 250,000 by denaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 500,000 by gel filtration . No subunits were apparent when the toxin was reduced and analyzed by SDS-PAGE . The estimated molecular weight of native toxin B indicated that dimers may form in solution . Toxin B was homogeneous by SDS-PAGE, native PAGE, and high-resolution anion-exchange chromatography . No secondary sequences were detected when the amino terminus of the toxin was sequenced, which also indicated that contaminating peptides were absent from the preparation . The amino terminus of toxin B was determined to be NH3-Trp-Leu-Val-Asn-Arg-Lys-Gln-Leu-Glu-Lys-Met-Ala-Asn-Val-ARg-Phe-Arg . One cytotoxic unit of toxin B was estimated to be 0.2 to 0.8 pg. Biochim Biophys Acta, 1988 Jul 1, 961(1), 1 - 12 Isolation and characterization of a novel four-chain ether lipid from Clostridium butyricum: the phosphatidylglycerol acetal of plasmenylethanolamine; Johnston NC et al.; We describe the isolation and characterization of a novel four-chain ether phospholipid in Clostridium butyricum grown on petroselinic acid in the absence of biotin . The results of quantitative analyses of hydrolytic products, selective hydrolysis by mild acid or phospholipase C, 1H-, 13C-, and 31P-NMR, and fast atom bombardment-mass spectroscopy (FABMS) have resulted in the determination of the structure of this lipid as a phosphatidylglycerol acetal of plasmenylethanolamine . Smaller amounts of this lipid have been found in cells grown under similar conditions in the presence of oleic, cis-vaccenic, elaidic or dihydrosterculic acids . It also appears to be present in small amounts in cells grown with biotin . This lipid is structurally related to the more plentiful glycerol acetal of plasmenylethanolamine found in these cells. J Am Vet Med Assoc, 1988 Jul 1, 193(1), 76 - 9 Hemorrhagic necrotizing enterocolitis associated with Clostridium difficile infection in four foals; Jones RL et al.; Severe hemorrhagic necrotizing enterocolitis was determined to be the cause of death for 4 foals . Toxigenic Clostridium difficile was isolated form the intestine of each foal, and large, gram-positive, rod-shaped bacteria lined the surface of necrotic villi . This finding of toxigenic C difficile associated with enteritis in foals adds another possible cause to the list of infectious agents that should be considered when evaluating foals with enteritis . Definitive diagnosis requires a thorough diagnostic evaluation, including procedures that will identify the organism and demonstrate its toxigenicity. Bioorg Khim, 1988 Jul, 14(7), 944 - 51 {Identification of 5-aminovaleric acid as a characteristic product of metabolism of various Clostridium species}; Rodionov AV et al.; An unusual ninhydrin-positive compound has been isolated from feces of accidentally contaminated Sprague-Dawley autbred rats and identified by mass spectrometry and 1H-NMR spectroscopy techniques as 5-aminovaleric acid . The described procedure of isolation, purification and structure determination can be recommended as a general method of identification of unusual ninhydrin-positive compounds in complex mixtures of biological origin . The 5-aminovaleric acid was found to be produced by an anaerobic bacterium Clostridium bifermentans . It is shown that not all Clostridium spp . excrete his amino acid and that the majority of microorganisms, normally inhabiting intestine of rats, simians and man, do not possess this ability . On the basis of data obtained, the test for 5-aminovaleric acid is proposed to be included into the taxonomy of bacteria. Zh Mikrobiol Epidemiol Immunobiol, 1988 Jul, (7), 3 - 6 {Comparative characterization of the morphologic changes induced by the cytotoxic action of a filtrate of Clostridium difficile strain B in cell cultures}; Chervonskaia GP et al.; Investigations carried out by the authors have demonstrated the possibility of the simultaneous evaluation of the results of the quantitative and qualitative characterization of the cytotoxic action of the filtrate of C . difficile strain B in the cultures of diploid human cells and cells Fl . The action of the filtrate used in the same dilution (1:1,000) over equal incubation periods (15 minutes) has resulted in the appearance of different morphological changes in each of these cultures . The degree of the manifestation of the cytotoxic action of the filtrate and the consequences of this action depend not only on the dose of the filtrate and the duration of the contact, but also on the kind of cell cultures used in the experiment . The 15-minute contact of human diploid cell culture with the filtrate leads to irreversible lesions of the cells . The rounding of cells Fl, observed during the first 15 minutes of the action of the filtrate, is not fatal for them; in the overwhelming majority of these cells the capacity for proliferation restores at the expiration of a certain period (about 3 weeks), and their adhesive properties increase. Microbiologica, 1988 Jul, 11(3), 259 - 61 Clostridium difficile in preterm neonates; Cardines R et al.; Stool specimens from premature neonates over the first month of life were examined for the presence of toxigenic Clostridium difficile and to evaluate a possible correlation between colonization and bowel disorders or prior antibiotic administration . Results showed a high isolation rate (63%) of Clostridium difficile with similar incidence in infants treated or not with antibiotics and with or without bowel disorders . Differentiation among strains according to SDS-PAGE, antibiotic susceptibility patterns and toxin production were useful to reveal cross-contamination . Both toxin-producing and non toxigenic strains were found in the infants' intestines . However, toxigenic strains were only present in infants suffering from bowel disorders and thus treated with oral antibiotics, suggesting that these factors may favour colonization by toxigenic strains. Microbiologica, 1988 Jul, 11(3), 179 - 99 Characterization of some Clostridium species by gas-liquid chromatography using numerical analysis; Cresci A et al.; We studied 42 strains of Clostridia belonging to 20 different species . All the strains were examined for morphological characters, biochemical reactions, and analyzed by means of gas-liquid chromatography (GLC) to determine metabolic patterns of short chain fatty acids and alcohols . To increase the number of criteria for the differentiation, specimens were grown on Peptone Yeast Extract medium (PY) with the addition of 13 different carbohydrates . The strains were compared using numerical taxonomic techniques based upon 20 unit qualitative and 224 quantitative characters . Data were examined using the simple matching coefficient (SSM) for qualitative characters, and degree of overlap between superimposed trace (So) for qualitative characters, and clustering was achieved using the unweighted pair group method with arithmetic averages (UPGMA) technique . DNA base composition was also determined . Numerical analysis showed remarkable difference between phenograms derived from SSM and So coefficients . The phenogram (SSM) is formed by 11 clusters and eight of these include strains from only one species . Only three clusters contained strains from different species . The cluster variability range of G + C base composition was never higher than 4 mol% except for one cluster where it reached 7 mol% G + C . In the phenogram (So) instead, there are 8 clusters and in only one case are strains from one species aggregated . In the remaining 7 clusters strains belonging to two or more species aggregated . The range values of base composition are over 4 mol% in three of the eight clusters. Can J Microbiol, 1988 Jul, 34(7), 916 - 8 Effect of toxins produced by various Clostridium difficile strains on cecum size reduction in gnotobiotic mice; Mahe S et al.; Inoculation of axenic mice with Clostridium difficile strains induced a significant reduction in ceca weight (dry or wet), whereas a nontoxinogenic strain led to a partial reduction . A strain, which produces cytotoxin and no enterotoxin in vivo, caused a reduction similar to that observed with a nontoxinogenic strain . Simultaneous cytotoxin and enterotoxin production by various C . difficile strains caused the cecum size to diminish to that observed for conventional control mice. Plasmid, 1988 Jul, 20(1), 17 - 22 Construction of shuttle cloning vectors for Bacteroides fragilis and use in assaying foreign tetracycline resistance gene expression; Guiney DG et al.; Shuttle vectors capable of replication in both Escherichia coli and Bacteroides fragilis have been developed . Conjugal transfer of these plasmids from E . coli to B . fragilis is facilitated by inclusion of the origin of transfer of the IncP plasmid RK2 . The vectors pDK1 and pDK2 provide unique sites for cloning selectable markers in Bacteroides . pOA10 is a cosmid vector containing the replication region of pCP1 necessary for maintenance in Bacteroides . pDK3, pDK4.1, and pDK4.2 contain the Bacteroides clindamycin resistance gene allowing selection and maintenance in B . fragilis of plasmids containing inserted DNA fragments . pDK3 was used to test the expression in B . fragilis of five foreign tetracycline resistance (TcR) genes . The tetA, -B, and -C markers from facultative gram-negative bacteria, as well as a TcR determinant from Clostridium perfringens, did not express TcR in B . fragilis . The tetM gene, originally described in streptococci, encoded a small but reproducible increase of TcR in Bacteroides . These studies demonstrate the utility of shuttle vectors for introducing cloned genes into Bacteroides and underscore the differences in gene expression in these anaerobes. Pathology, 1988 Jul, 20(3), 256 - 9 Evaluation of the RapID ANA system as a four-hour method for anaerobe identification; Downes J et al.; The RapID ANA system (Innovative Diagnostic Systems Inc., Atlanta, Ga, USA), a 4-hour micromethod for identifying clinically important anaerobic bacteria, was evaluated using 196 anaerobic clinical isolates and the results were compared with those obtained by applying conventional Virginia Polytechnic Institute (VPI) methodology . The identifications achieved by the RapID ANA system for 141 (72%) of these isolates agreed with the species identifications obtained using VPI methodology, without the need for additional tests . A further 40 (20%) were in agreement after performance of additional suggested tests, 11 (6%) were misidentified and 4 (2%) could not be identified using the RapID ANA system . Excellent agreement between the two methods was demonstrated for the Gram-positive cocci and good agreement for the Gram-negative bacilli . The RapID ANA system was suboptimal in the identification of Clostridium species other than C . perfringens. J Infect, 1988 Jul, 17(1), 35 - 42 Evaluation of a computer-assisted method of analysing SDS-PAGE protein profiles in tracing a hospital outbreak of Serratia marcescens; Arzese A et al.; Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) profiles of bacterial proteins have been successfully used for taxonomical purposes . More recently this technique has been applied to epidemiological investigations in respect of various micro-organisms including Neisseria meningitidis, Staphylococcus aureus and Clostridium difficile . The main limitations of the methods so far described are lack of standardisation in extraction and separation as well as in the analysis of results . Although reproducibility in the same laboratory has been shown to be satisfactory, comparison of results among laboratories is still difficult . Moreover, assessment of differences and/or similarities among chromatograms or autoradiographs showing many bands depends upon qualitative descriptions . Interpretation of densitometric scannings is laborious and time-consuming . In this paper we present our experience of a completely standardised, fully computer-controlled procedure for SDS-PAGE (AMBIS System) in analysing 35S-methionine-labelled total proteins . The methodology proved very useful in monitoring a hospital outbreak of Serratia marcescens . It allowed us to make quantitative comparison in a shorter time as well as to handle easily a great amount of data and usefully integrate it with those obtained with other systems such as serotyping . Furthermore, when the two systems are used together, more precise information can be gained . In this epidemic, serotyping indicated the presence of two groups which would have been missed by PAGE analysis alone . Electrophoretotyping, however, focused on similarities of cellular proteins among the epidemic strains . This allowed us to distinguish them from epidemiologically unrelated strains of the same serogroup.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Stand, 1988 Jul, 16(3), 207 - 18 The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin; Boarer CD et al.; The production of five monoclonal antibodies to the epsilon prototoxin of Clostridium perfringens is described . All five monoclonal antibodies located three proteins in a trypsinized preparation of epsilon prototoxin . These proteins were located at 37.6 kDal, 35.6 kDal and 33.7 kDal by Western blots . Two of the monoclonal antibodies, M26/2 and M27/12, neutralized epsilon toxin in the mouse lethality assay . Four of the five monoclonal antibodies are considered suitable as reagents to detect epsilon toxin in assay procedures. Biofactors, 1988 Jul, 1(2), 147 - 52 Role of carbon monoxide dehydrogenase in acetate synthesis by the acetogenic bacterium, Acetobacterium woodii; Shanmugasundaram T et al.; Carbon monoxide dehydrogenase (CODH) plays a key role in acetate synthesis by the acetogenic bacterium, Clostridium thermoaceticum . Acetobacterium woodii, like C . thermoaceticum contains high levels of CODH . In this work we show that crude extracts of A . woodii synthesize acetate from methyl tetrahydrofolate or methyl iodide, carbon monoxide and coenzyme A (CoA) . The purified CODH from A . woodii catalyzes an exchange reaction between CO and the carbonyl group of acetyl-CoA even faster than the C . thermoaceticum enzyme, indicating the CODH of A . woodii, like that of C . thermoaceticum is an acetyl-CoA synthetase . Fluorescence and EPR studies further support this postulate by demonstrating that CODH binds CoA near the CO binding site involving a tryptophan residue . The UV absorption spectra and the amino acid compositions of A . woodii and C . thermoaceticum CODHs are very similar . Evidence is presented using purified enzymes from A . woodii that the synthesis of acetyl-CoA occurs by a pathway similar to that utilized by C . thermoaceticum. J Bacteriol, 1988 Jul, 170(7), 3255 - 61 Nucleotide sequence of the Clostridium acidiurici ("Clostridium acidi-urici") gene for 10-formyltetrahydrofolate synthetase shows extensive amino acid homology with the trifunctional enzyme C1-tetrahydrofolate synthase from Saccharomyces cerevisiae; Whitehead TR et al.; The nucleotide sequence of the gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from Clostridium acidiurici ("Clostridium acidi-urici") was determined . The synthetase mRNA initiation and termination regions were determined by primer extension and S1 nuclease mapping . Two potential -10 and -35 promoter regions were identified upstream of mRNA initiation . The terminator region was found to be in a large region of dyad symmetry . A comparison of the amino acid sequences of the monofunctional synthetase and the eucaryotic trifunctional enzyme, C1-tetrahydrofolate synthase, from Saccharomyces cerevisiae demonstrated a region of strong homology. Gastroenterology, 1988 Jul, 95(1), 11 - 7 In vivo profiles of eicosanoids in ulcerative colitis, Crohn's colitis, and Clostridium difficile colitis; Lauritsen K et al.; To compare the local release of arachidonic acid metabolites in inflammatory diarrheal disease, in vivo equilibrium dialysis of the rectum was done in consecutive untreated patients with ulcerative colitis (n = 20), Crohn's colitis (n = 10), and Clostridium difficile colitis (n = 7) . All patients had endoscopically proven rectal inflammation . Eicosanoid profiles were determined in rectal dialysates by radioimmunoassay after preliminary purification . Concentrations of prostaglandin E2, prostaglandin F2 alpha, and thromboxane B2, but not 6-keto-prostaglandin F1 alpha, were raised in all groups and compared with healthy controls . The highest levels within each group were obtained in patients with widespread epithelial damage, as judged by endoscopy . In patients with ulcerative colitis, an extreme rise in prostaglandin E2 and thromboxane B2 were observed . Similarly, concentrations of leukotriene B4 were substantially increased in ulcerative colitis, but in Crohn's colitis and Clostridium difficile colitis only those patients with rectal ulcerations showed elevations . These findings probably reflect more severe tissue damages in ulcerative colitis, but differences between disease groups in cell-to-cell interaction may also contribute . The data suggest, therefore, that therapeutic inhibition of lipoxygenase pathways may prove more effective in ulcerative colitis than in Crohn's disease. FEBS Lett, 1988 Jun 20, 233(2), 417 - 20 Purification and characterisation of two forms of toxin B produced by Clostridium difficile; Torres JF et al.; Toxin B from Clostridium difficile was purified to homogeneity by gel filtration and high resolution ion exchange chromatography . Two forms of toxin B were found . Form 1 which seemed to consist of two identical subunits of 220-300 kDa; femtogram amounts of this toxin induced rounding of fibroblast cells . Form 2 contained subunits of 43 kDa and 105 kDa; the stoichiometric ratio probably being 4:1; picogram amounts were needed to induce rounding of fibroblast cells . Immunological studies suggested that both subunit types were antigenic and had epitopes which were identical with those of form 1. S Afr Med J, 1988 Jun 18, 73(12), 718 - 20 Occurrence of Clostridium tetani in soil and horses; Wilkins CA et al.; The annual incidence of tetanus in the RSA is up to 300 cases with more than 50% of these coming from Natal/KwaZulu . The condition of playing fields and the excretion of Clostridium tetani by horses was therefore investigated . The overall contamination rate of soils in the Durban area is lower than that of published data from other parts of the world, for instance 28% for Durban in comparison with 31-42% for Japan and Quebec . A rugby field in the Transvaal showed 40% contamination and a pasture used for horses for more than 20 years 65% . No case of human or equine tetanus has ever been reported from either the playing field or the pasture . A permanent carrier state in horses could not be established; the organisms were only excreted for 3-4 days . At any one time only 2 out of 27 horses in a stable were excreting C . tetani . Only 7 of 118 faeces samples were positive over a period of 4 months (5-9%). Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 699 - 707 The effects of Clostridium perfringens enterotoxin on intracellular levels or transport of uridine, thymidine and leucine do not fully explain enterotoxin-induced inhibition of macromolecular synthesis in Vero cells; Hulkower KI et al.; Clostridium perfringens type A enterotoxin (CPE) has been shown previously to inhibit the incorporation of radiolabeled precursors into acid-insoluble material but the mechanism of inhibition is unknown . It has also been shown that extracellular calcium is required for some CPE effects . In this report, it is shown that CPE completely and virtually simultaneously inhibits incorporation of precursors into RNA, DNA and protein in either the presence or absence of extracellular divalent cations and that changes in intracellular precursor levels did not consistently correlate with this CPE-induced inhibition of incorporation . These results strongly suggest that CPE can inhibit macromolecular synthesis, not just inhibit precursor transport . It is inferred from this that CPE can affect DNA and RNA synthesis, and possibly protein synthesis, by altering other cellular processes besides, or in addition to, precursor transport and these effects then lead to a shutdown of macromolecular synthesis. Rev Fr Gynecol Obstet, 1988 Jun 15, 83(6), 407 - 9 {Clostridium perfringens septicemia}; Jemni L et al.; We report 3 cases of Clostridium perfringens bacteremia with uterine gas gangrene . Clinical presentation included severe infectious syndrome, hemoglobinemia and hemoglobinuria, jaundice, uterine tenderness and hypertension . All 3 cases were first seen with installed renal failure . Diagnosis and modalities of therapy were reviewed . Clostridium perfringens bacteremia with uterine gas gangrene still occur in developing countries. Biochemistry, 1988 Jun 14, 27(12), 4279 - 83 Interaction of N-acetyl-4-epi-D-neuraminic acid with key enzymes of sialic acid metabolism; Gross HJ et al.; In spite of the axially orientated hydroxy group at C-4, the benzyl alpha-glycoside of N-acetyl-4-epi-D-neuraminic acid (4-epi-NeuAc) is a substrate for sialidases from Vibrio cholerae, Clostridium perfringens, and Arthrobacter ureafaciens, although to an extent which differs depending on the enzyme . Surprisingly, V . cholerae sialidase is by far the slowest acting enzyme; this is in contrast to its usual behavior . Fowl plague virus sialidase and bovine testis sialidase also cleave this glycoside slowly . 4-Epi-NeuAc is not a substrate for N-acetylneuraminic acid aldolase from C . perfringens but reversibly inhibits the enzyme with a Ki = 2.3 mM . The N-acetylneuraminic acid analogue is not converted to the corresponding CMP-glycoside by CMP-sialic acid synthase from bovine brain; however, it is an effective reversible inhibitor of the enzyme . The kinetic properties were analyzed with an assay system at pH 9 as well as an assay system at pH 7.5 . The results from Dixon and Hanes plots did not agree . Therefore, no conclusions about the mechanism of the inhibition could be reached . This is the first reported sialic acid analogue which can act as an inhibitor of CMP-sialic acid synthase. Biochemistry, 1988 Jun 14, 27(12), 4299 - 304 Ketone-substrate analogues of Clostridium histolyticum collagenases: tight-binding transition-state analogue inhibitors; Mookhtiar KA et al.; A series of ketone-substrate analogues has been synthesized for the two classes of collagenases from Clostridium histolyticum and shown to be competitive inhibitors . These compounds have sequences that match those of specific peptide substrates for these enzymes . The best inhibitor is the ketone analogue of cinnamoyl-Leu-Gly-Pro-Pro, which has a KI value of 18 nM for epsilon-collagenase, a class II enzyme . This is the tightest binding inhibitor reported for any collagenase to date . Plots of log KI for the inhibitors vs log KM/kcat for the matched substrates for both collagenases are linear with slopes near unity, indicating that the ketones are transition-state analogues . This strongly implies that the ketone carbon atoms of these inhibitors are tetrahedral when bound to the enzymes. Vet Rec, 1988 Jun 11, 122(24), 579 - 81 A major outbreak of botulism in cattle being fed ensiled poultry litter; McLoughlin MF et al.; Eighty of a group of 150 housed beef cattle showed classical signs of botulism after eating a batch of ensiled poultry litter . Sixty-eight of the animals died and Clostridium botulinum type C toxin was detected in 18 of 22 sera examined . C botulinum organisms were isolated from the ensiled litter and type C toxin was demonstrated in samples of decomposed poultry carcases present in the litter . This outbreak of bovine botulism was the most serious to have been recorded in Europe and was the first associated with feeding ensiled poultry litter. Biochem J, 1988 Jun 1, 252(2), 343 - 8 Purification and characterization of a highly thermostable novel pullulanase from Clostridium thermohydrosulfuricum; Saha BC et al.; Clostridium thermohydrosulfuricum mutant Z 21-109 produced intracellular thermostable pullulanase and glucoamylase activities . The glucoamylase activity was inactivated by treating C . thermohydrosulfuricum cells with 10% (v/v) propan-1-ol at 85 degrees C in the presence of 5 mM-CaCl2 . Pullulanase activity was selectively solubilized from cells by treatment with detergent and lipase . The solubilized pullulanase was purified by treatment with streptomycin sulphate and (NH4)2SO4 and by DEAE-Sephacel, octyl-Sepharose and pullulan-Sepharose chromatography . Pullulanase was purified 3511-fold and displayed homogeneity on SDS/polyacrylamide-gel electrophoresis . The pullulanase was a monomeric glycoprotein with an apparent Mr of about 136,500, and it displayed a pI of 5.9 . The enzyme was enriched in both acidic and hydrophobic amino acids . The purified pullulanase was stable and optimally active at 90 degrees C . The optimum pH for activity and pH-stability ranges were 5.0-5.5 and 3.0-5.0 respectively . The enzyme was inhibited by cyclodextrins, EDTA and N-bromosuccinimide, but not by p-chloromercuribenzoate and acarbose . The pullulanase displayed a relative substrate specificity for hydrolysis of pullulan (100%) versus 75% for glycogen and 50% for soluble starch . The apparent Km, Vmax . and Kcat . values for enzyme activity on pullulan at 60 degrees C were 0.675 mg/ml, 122.5 mumol of reducing sugar formed/min per mg of protein and 16,240 min-1 respectively . The novel properties of this extremely thermostable pullulanase are discussed in relation to other purified starch-debranching enzymes. J Med Microbiol, 1988 Jun, 26(2), 125 - 8 Evidence for cross-infection in an outbreak of Clostridium difficile-associated diarrhoea in a surgical unit; Testore GP et al.; Environmental studies were performed in a hospital outbreak of Clostridium difficile-associated diarrhoea . Transmission was associated with the sluice room and the storage room where medical equipment was found to be contaminated with C.difficile . Typing of isolates by antibiotic-susceptibility patterns and profiles of EDTA-extracted proteins showed the presence of an "epidemic" strain common to the majority of patients and environmental sites . Control of the outbreak was achieved by improvement of environmental hygiene and use of disposable equipment. J Clin Microbiol, 1988 Jun, 26(6), 1181 - 8 Recovery of anaerobic bacteria from clinical specimens in 12 years at two military hospitals; Brook I; Examination of 15,844 clinical specimens submitted over 12 years (1973 to 1985) to the anaerobic microbiology laboratories in two military hospitals demonstrated the recovery of anaerobic bacteria in 4,458 (28.1%) specimens . The specimens yielded 6,557 anaerobic isolates (1.47 isolates per specimen) . Bacteroides spp . accounted for 43% of all isolates; anaerobic gram-positive cocci, 26%; Clostridium spp., 7%; and Fusobacterium spp., 4% . Bacteroides spp . predominated in abscesses, obstetrical and gynecological (OBG) infections, abdominal infections, cysts, wounds, and tumors . Members of the Bacteroides fragilis group accounted for 44% of all Bacteroides spp., and of them, B . fragilis was mostly isolated in abscesses, wounds, abdomen, and blood . Pigmented Bacteroides spp . accounted for 21% of all Bacteroides sp . isolates and were mostly isolated in sinus, eye, chest, bone, and ear infections . Bacteroides melaninogenicus accounted for 42% of this group's isolates . Bacteroides bivius accounted for 9% of Bacteroides spp., and most isolates were found in OBG infections . Anaerobic gram-positive cocci were mostly isolated in OBG infections, abscesses, and wounds . The predominant anaerobic gram-positive cocci were Peptostreptococcus magnus (18%), Peptostreptococcus asaccharolyticus (17%), Peptostreptococcus anaerobius (16%), and Peptostreptococcus prevotii (13%) . Clostridium spp . were mostly isolated from wounds, abscesses, abdominal infections, and blood . The predominant strain was Clostridium perfringens (48%) . Fusobacterium spp . were recovered in abscesses and abdominal and OBG infections . The predominant isolate was Fusobacterium nucleatum (47%) . These data illustrate the relative frequency of the different anaerobic bacteria in a variety of infections and demonstrate the predominance of certain isolates at different sites. Proc Natl Acad Sci U S A, 1988 Jun, 85(12), 4526 - 9 Human and bovine coronaviruses recognize sialic acid-containing receptors similar to those of influenza C viruses; Vlasak R et al.; Human coronavirus OC43 and bovine coronavirus elute from agglutinated chicken erythrocytes when incubated at 37 degrees C, suggesting the presence of a receptor-destroying enzyme . Moreover, bovine coronavirus exhibits an acetylesterase activity in vitro using bovine submaxillary mucin as substrate similar to the enzymatic activity found in influenza C viruses . Furthermore, pretreatment of erythrocytes with either influenza C virus or bovine coronavirus eliminates subsequent binding and agglutination by either coronaviruses or influenza C virus, whereas binding of influenza A virus remains intact . In addition, hemagglutination by coronaviruses can be inhibited by pretreatment of erythrocytes with Arthrobacter ureafaciens or Clostridium perfringens neuraminidase or by addition of sialic acid-containing gangliosides . These results suggest that, like influenza C viruses, human coronavirus OC43 and bovine coronavirus recognize O-acetylated sialic acid or a similar derivative as cell receptor. Infect Immun, 1988 Jun, 56(6), 1500 - 4 Emergence in gnotobiotic mice of nontoxinogenic clones of Clostridium difficile from a toxinogenic one; Corthier G et al.; In previous studies, we showed that diet composition or Saccharomyces boulardii ingestion could protect gnotobiotic mice against lethal Clostridium difficile infection . Using an original method, we detected nontoxinogenic clones from feces of protected mice challenged with a toxinogenic clone of C . difficile . These clones became established at the same level as the toxinogenic one after about 30 days . In these protected mice bearing nontoxinogenic clones, no enterotoxin production could be detected and cytotoxin titers were highly reduced . These nontoxinogenic clones were genetically stable because nontoxinogenic clones and clones that produce intermediate levels of toxins in vivo did not revert to toxin production, even after repeated culture in vitro . Furthermore, the nontoxinogenic clones were shown to arise from a single toxinogenic clone and were identical to that clone in metabolic patterns and antibiotic sensitivity tests . When mice fed a nonprotective diet were challenged with a nontoxinogenic or intermediate clone, they remained healthy and no toxin production could be detected in their feces . Moreover, these mice were protected against further infections with toxinogenic strains of C . difficile, and a strong antagonism between nontoxinogenic and toxinogenic clones was observed. Epidemiol Infect, 1988 Jun, 100(3), 399 - 405 Factors affecting the toxicity of rotting carcasses containing Clostridium botulinum type E; Smith GR et al.; Mice killed shortly after receiving c . 2000 spores of a type E strain of Clostridium botulinum per os were incubated at one of five chosen temperatures together with bottles of cooked meat medium seeded with a similar inoculum . After incubation the rotting carcasses were homogenized . Sterile membrane filtrates of the homogenates (10%, w/v) and pure cultures were then titrated for toxicity . Some of the main findings were confirmed with two further type E strains . Toxicity produced at 37 degrees C was poor in both carcasses and cultures (200-20,000 mouse intraperitoneal LD/g or ml) . It was good in both systems at 30 and 23 degrees C, usually reaching 20,000-200,000 LD/g or ml, and in carcasses occasionally more; at 30 degrees C maximal toxicity was reached more quickly in carcasses than in cultures . Prolonged incubation (36-118 days) at 30 or 23 degrees C resulted in complete loss of toxicity in virtually all carcasses but not in cultures . At 16 degrees C the development of toxicity in carcasses was strikingly greater than in cultures . At 9 degrees C neither system produced more than slight toxicity after prolonged incubation . Trypsinization increased the toxicity of cultures but not usually of carcasses . Unfiltered carcass homogenate (10%, w/v) with maximal intraperitoneal toxicity was harmless for mice by mouth in doses of 0.25 ml . These findings differed in important respects from those made earlier with a type C strain. Infect Immun, 1988 Jun, 56(6), 1655 - 7 Cloning and expression of secreted antigens of Clostridium difficile in Escherichia coli; Dailey DC et al.; The feasibility of the cloning and expression of Clostridium difficile antigens in Escherichia coli was investigated . The expression of a limited number of cloned clostridial antigens under the control of clostridial promoter elements in E . coli was observed. Biol Chem Hoppe Seyler, 1988 Jun, 369(6), 451 - 60 Reductions of 2-enals, dehydrogenation of saturated aldehydes and their racemisation; Thanos I et al.; Enoate reductase or clostridia containing this enzyme (Clostridium tyrobutyricum or C . kluyveri) catalyse the reduction of alpha,beta-unsaturated aldehydes (enals) . The enantiomeric purity of the saturated aldehydes obtained from alpha-substituted enals is usually rather low and depends heavily on the reaction conditions . The reduction of the corresponding allyl alcohols to the saturated alcohols leads to much higher enantiomeric purities, though the reduction of the enal corresponding to the allyl alcohol to the saturated aldehyde is an intermediary step in the reaction sequence allyl alcohol----saturated alcohol . The explanation seems to be the racemisation of saturated aldehydes caused by enoate reductase . This is illustrated by the reduction of (E)-2-methylcinnamyl aldehyde to (R)-2-methyl-3-phenylpropanal or (R)-2-methyl-3-phenylpropanol under different conditions and measuring the racemisation of the aldehyde as well as the hydrogen-deuterium exchange of 3-phenylpropanal . In contrast to saturated carboxylates saturated aldehydes can be dehydrogenated to alpha,beta-unsaturated aldehydes (enals) by enoate reductase in the presence of electron acceptors such as oxygen or dichlorophenol indophenol . Under these conditions enoate reductase shows in the presence of oxygen a surprisingly high half life (greater than 20 h) as compared to that which is observed when the enzyme was used as a reductase with NADH in the presence of oxygen . In this case the enzyme is inactivated within a few minutes. Biochem Int, 1988 Jun, 16(6), 1145 - 51 Phosphatidylcholine synthesis in platelets is stimulated by diacylglycerol but not by phorbol ester; Muir JG et al.; The biosynthesis of phosphatidylcholine (PC) in platelets was followed by measuring the incorporation of 32Pi . Incorporation into PC was stimulated by treatment with Clostridium perfringens phospholipase C or with the synthetic diacylglycerol sn-1,2-dioctanoylglycerol . However, neither the phorbol ester tumour promoter 12-O-tetradecanoylphorbol-13-acetate or thrombin stimulated 32Pi incorporation into PC . We conclude that phorbol ester does not stimulate the hydrolysis of PC to diacylglycerol in platelets. Biochimie, 1988 Jun, 70(6), 811 - 7 Botulinum neurotoxin type B (strain 657): partial sequence and similarity with tetanus toxin; Dasgupta BR et al.; The type B neurotoxin (NT) isolated from Clostridium botulinum (strain 657) behaved as a mixture of single (unnicked) and dichain (nicked) proteins, both of Mr approximately 150 kDa . When the dichain NT was reduced by mercaptoethanol, the two chains migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as separate polypeptides of Mr approximately 100 and 50 kDa that appeared similar to the heavy and light chains of other serotypes of botulinum NT . The N-terminal amino acid sequences of the two chains were determined . They were as follows: light chain: Pro-Val-Thr-Ile-Asn-Asn-Phe-Asn-Tyr-Asn-Asp-Pro-Ile-Asp-Asn-Asn-Asn-Ile- Ile-Met - Met-Glu-Pro-Pro-Phe-Ala-Arg-Gly-Met-Gly-Arg-Tyr-Tyr-Lys-Ala-Phe-Lys-Ile- Thr-Asp - Arg-Ile-Trp-Ile-; and heavy chain: Ala-Pro-Gly-Ile-X-Ile-Asp-Val-Asp-Asn-Glu-Asp-Leu-Phe-Phe-Ile-Ala-Asp-Ly s-Asn- Ser-Phe-Arg-Asp-Asp-Leu- . These two sequences matched exactly with those of the light and heavy chains of type B NT (strain Okra) of which only 16 and 18 residues were known (J . Biol . Chem . (1985) 260, 10461) . The above sequences were different from those of type A NT . Immunoprecipitation reactions of type B NT isolated from strains 657 and Okra were indistinguishable against polyclonal anti-type B NT serum . These two preparations did not produce precipitin reactions with polyclonal anti-type A NT serum.(ABSTRACT TRUNCATED AT 250 WORDS) Diagn Microbiol Infect Dis, 1988 Jun, 10(2), 85 - 91 Results of a prospective, 18-month clinical evaluation of culture, cytotoxin testing, and culturette brand (CDT) latex testing in the diagnosis of Clostridium difficile-associated diarrhea; Peterson LR et al.; An 18-mo evaluation of culture, cytotoxin, and latex testing for Clostridium difficile was performed between July 1, 1985, and December 31, 1986, on 1,536 specimens from 1,406 patients during evaluation of diarrhea . All cases with at least one test positive were investigated for clinical status . There were 144 Clostridium difficile-associated diarrhea (CAD) patients; 139 (97%) were positive by culture, 96 (67%) by cytotoxin, and 98 (68%) by latex testing . In the 1,262 non-CAD patients with diarrheal stool, 89 (7.1%) were positive by culture, 18 (1.4%) by cytotoxin, and 68 (5.4%) by the latex test . No CAD patient was positive by cytotoxin testing only, and two were positive by latex testing only . The culture and cytotoxin positivity were similar to our previous reports of 90-97% and 70-73%, respectively . Latex sensitivity (68%) was comparable to that of cytotoxin testing in this large group of patients (p greater than 0.5) . Overall, in the 1,262 patients without clinical evidence of Clostridium difficile disease, positive tests by latex testing (5.4%) were intermediate between those of culture (7.1%, p less than 0.1) and cytotoxin (1.4%, p less than 0.001). Pathol Biol (Paris), 1988 Jun, 36(5 Pt 2), 754 - 8 {Clostridium difficile in children and adolescents undergoing anticancer and antimicrobial chemotherapy . The possibility of nosocomial acquisition}; Collignon A et al.; We looked for C . difficile and its cytotoxin among children and adolescents treated with diverse kinds of cancer chemotherapy in an oncologic ward of a pediatric hospital . Most of them were also given multiple antibiotic treatments, susceptible of making C . difficile colonization easier due to the modification of the intestinal ecosystem . As the colonization may have an external origin, we also looked for C . difficile in the environment . 14 patients have been studied and we found cytotoxic strains in four of them and non cytotoxic ones in two . This paper discusses the influence of cancer chemotherapy and antibiotherapy on C . difficile colonization among those patients, and the role played by this bacteria and its toxin on the diarrhea cycle . The facts that several patients have been colonized together and that environmental samples showed up positive, lead us to suspect a nosocomial spread . To confirm this thesis, strains originating from patients and environments were compared. Pathol Biol (Paris), 1988 Jun, 36(5 Pt 2), 682 - 4 {In vitro activity of beta-lactams, clindamycin and metronidazole on Clostridium difficile}; Sedallian A et al.; In one year and a half period 47 Clostridium difficile were isolated from stool samples . Isolates with questionable morphologic features were identified using sugars fermentation, gaz liquid chromatography (GLC) . Cytotoxin assay was performed on Mac Coy cells . The comparative susceptibilities to amoxicillin-clavulanic acid (AMC), cefotetan (CTT), cefoxitin (FOX), cefotaxime (CTX), latamoxef (MOX), mezlocillin (MEZ), piperacillin (PIP), clindamycin (CLIN) and metronidazole was tested using minimal inhibition concentration (MIC) determination on Wilkins-Chalgren blood agar . CLIN, FOX, CTX, MOX were inactive, CTT is moderately inactive . All strains were susceptible to AMX, MEZ, PIP and metronidazole. Pathol Biol (Paris), 1988 Jun, 36(5 Pt 2), 678 - 81 {In vitro activity of a combination of amoxicillin-clavulanic acid on anaerobic bacteria}; Sedallian A et al.; One hundred and one anaerobes isolated from clinical specimen were examined for their susceptibility to amoxicillin, alone or in combination with clavulanic acid, cefoxitin, cefotetan, cefotaxime, piperacillin and metronidazole . Tested strains were as follow: 90 Bacteroides fragilis group, 16 Bacteroides bivius and 5 Clostridium difficile . For 54 strains combination amoxicillin-sulbactam were also studied . Minimal inhibitory concentrations (MICs) were determined by agar dilution method on Wilkins-Chalgren agar . All the strains were susceptible to the combination amoxicillin-clavulanic acid . The mode MIC was 0.5 microgram/ml and 16 micrograms/ml for amoxicillin alone. Mol Cell Biochem, 1988 Jun, 81(2), 187 - 94 Botulinum neurotoxin types A, B & E: pH induced difference spectra; Datta A et al.; The alkaline pH induced difference spectra (270-310 nm) of three antigenically distinct forms of the botulinum neurotoxin (NT) types A, B and E were examined . When isolated from the cultures of Clostridium botulinum, type A NT is a fully toxic dichain (nicked) protein, type E is a mildly toxic single chain (unnicked) protein, and type B NT is a mixture of single and dichain proteins and near fully toxic . Trypsin nicks the single chain protein to the dichain and increases its toxicity (up to about 100 fold in type E) . A strong difference spectrum peak at approximately 296 nm was found when types A, B or E NT were in the alkaline pH region . This peak was not observed at pH 4.0 . For types A and B NT plots of difference absorptivity vs . pH were simple sigmoidal curves . The pK of phenolic moieties of tyrosine residues in both proteins were 10.9 . Nearly all tyrosine residues in both proteins were ionized . The single chain type E, unlike type A and B NT, yielded a two step titration curve and pK values 11.3 and less than 7.5; about 60% of the total tyrosine residues present were ionized . The two step titration curve was not observed when the single chain protein was nicked with trypsin to the dichain type E NT . The titration curve of dichain type E NT, although complex, was more like those of type A and B NT. Appl Environ Microbiol, 1988 Jun, 54(6), 1446 - 50 Factors influencing Clostridium botulinum spore germination, outgrowth, and toxin formation in acidified media; Wong DM et al.; Clostridium botulinum type A spores were inoculated at a level of 10(7) spores per ml into sterile beef media with protein concentrations of 1, 2, 3, 4, or 6% and acidified to pH values of 2.01 to 4.75 with hydrochloric acid or 4.19 to 4.60 with citric acid . All experimental manipulations, including blending, acidification, inoculation, incubation (30 degrees C), and analyses, were conducted in an anaerobic chamber-incubator in which atmospheric oxygen levels were maintained below 2 ppm (2 microliters/liter) . Under these strict anaerobic conditions (oxidation-reduction values in media ranging from -370 to -391 mV), C . botulinum spores were consistently found to germinate, grow, and produce toxin below pH 4.6 . The boundary between toxic and atoxic samples in HC1-acidified beef media was mediated by titratable acidity, pH, and protein concentration . A limiting acidity was not established for the citrate-acidified samples; all blends tested (1, 2, 3, and 4% protein and titratable acidities of 0.091 to 0.453%) became toxic within 5 weeks . At the same pH and protein concentration, citric acid was less effective than HC1 in preventing the germination of C . botulinum spores . Higher levels of cell proliferation in the beef protein, as well as enhanced gas production and putrefactive degradation, indicated that beef was a better substrate than soy for C . botulinum spores under these conditions . Reducing the inoculum to 10(4) delayed but did not prevent spore outgrowth and toxin release at pH levels below 4.6. Appl Environ Microbiol, 1988 Jun, 54(6), 1405 - 8 Evidence for plasmid-mediated toxin and bacteriocin production in Clostridium botulinum type G; Eklund MW et al.; A single 81-megadalton plasmid was previously isolated from each of six toxigenic strains of Clostridium botulinum type G (M . S . Strom, M . W . Eklund, and F . T . Poysky, Appl . Environ . Microbiol . 48:956-963, 1984) . In this study, nontoxigenic derivatives isolated from each of the toxigenic strains following consecutive daily transfers in Trypticase (BBL Microbiology Systems, Cockeysville, Md.)-yeast extract-glucose broth at 44 degrees C simultaneously ceased to produce type G neurotoxin and to harbor the resident 81-megadalton plasmid . The nontoxigenic derivatives also ceased to produce bacteriocin and lost their immunity to the bacteriocin produced by the toxigenic strains . In contrast, all of the toxigenic isolates continued to carry the resident plasmid and to produce both bacteriocin and type G neurotoxin . This is the first evidence suggesting that the production of neurotoxin and bacteriocin by C . botulinum is mediated by a plasmid. J Inorg Biochem, 1988 Jun, 33(2), 111 - 20 Comparison of redox and EPR properties of the molybdenum iron proteins of Clostridium pasteurianum and Azotobacter vinelandii nitrogenases; Morgan TV et al.; Both heterologous crosses of the Clostridium pasteurianum and Azotobacter vinelandii nitrogenase components are completely inactive, although the reasons for this incompatibility are not known . We have compared a number of properties of the MoFe proteins from these organisms (Cp1 and Av1, respectively) in an attempt to find differences that could explain this lack of functional activity . Optical and CD spectroscopic titrations are similar for both Av1 and Cp1, but EPR titrations are significantly different, suggesting different chemical reactivity patterns and/or magnetic interaction behavior . Similarly, reduction measurements on the six-electron-oxidized state of Cp1 and Av1 at controlled potentials indicate a difference in both the relative reduction sequence of the redox centers and the numerical values for their measured midpoint potentials . EPR measurements as a function of temperature also demonstrate that the relaxation behavior of the S = 3/2 MoFe centers associated with the proteins differ markedly . The Cp1 EPR signal only begins to undergo broadening above 65 K, whereas the Av1 signal is severely broadened above 25 K . These variations in the EPR properties for the two proteins are not likely to be due to differences in the stoichiometry and/or geometry of the MoFe cluster units themselves since similar EPR studies of the isolated cofactors showed them to be essentially identical . Thus, the different EPR behavior of the two proteins seems to arise either from protein constraints imposed on identical cofactors, and/or from magnetic interactions due to neighboring metal clusters. J Clin Invest, 1988 Jun, 81(6), 1741 - 5 Clostridium difficile toxin A stimulates intracellular calcium release and chemotactic response in human granulocytes; Pothoulakis C et al.; Clostridium difficile, a common enteric pathogen, mediates tissue damage and intestinal fluid secretion by release of two protein exotoxins: toxin A, an enterotoxin, and toxin B, a cytotoxin . Because toxin A elicits an intense inflammatory reaction in vivo, we studied the effects of highly purified C . difficile toxins on activation of human granulocytes . Toxin A at concentrations of 10(-7) to 10(-6) M, but not toxin B, elicited a significant chemotactic and chemokinetic response by granulocytes that was comparable with that induced by the chemotactic factor N-FMLP (10(-7) M) . Neither toxin stimulated release of superoxide anion from granulocytes . Toxin A produced a rapid, transient rise in cytosolic {Ca2+}i, as measured by quin 2 fluorescence . Pertussis toxin and depletion of intra- and extracellular calcium blocked the toxin A effect on cytosolic {Ca2+}i . These findings suggest that the inflammatory effects of C . difficile toxin A in the intestine may be related to its ability to mobilize intracellular Ca2+ and elicit a chemotactic response by granulocytes. Am J Epidemiol, 1988 Jun, 127(6), 1289 - 94 Acquisition of Clostridium difficile from the hospital environment; Kaatz GW et al.; An outbreak of antibiotic-associated colitis that occurred on a ward of a Michigan hospital during February-April, 1984, was studied by bacteriophage-bacteriocin typing . Stools from the seven involved patients yielded Clostridium difficile isolates of types B1537 or Cld7;B1537 . C . difficile was recovered from 31.4% of environmental cultures obtained on the ward, and the majority of isolates were types B1537 or Cld7;B1537 . When the ward was disinfected with unbuffered hypochlorite (500 parts per million (ppm) available chlorine), surface contamination decreased to 21% of initial levels and the outbreak subsequently ended . Phosphate buffered hypochlorite (1,600 ppm available chlorine, pH 7.6) was even more effective; its use resulted in a 98% reduction in surface contamination . These findings suggest that environmental contamination with C . difficile is important in the epidemiology of antibiotic-associated colitis, and that hypochlorite is effective in eliminating C . difficile from the hospital environment. Am J Surg, 1988 May 31, 155(5A), 47 - 51 A multicenter study of the in vitro antianaerobic activity of cefotetan compared with other antimicrobial agents; Zabransky RJ et al.; The in vitro antianaerobic activity of cefotetan was compared with that of chloramphenicol, clindamycin, cefoxitin, and penicillin in a multicenter study . Both agar dilution and broth microdilution testing procedures, as described by the National Committee for Clinical Laboratory Standards (NCCLS), were employed; a total of 1,377 strains were examined . Results were interpreted using the U.S . Food and Drug Administration- and NCCLS-recommended criteria . This study indicates that Bacteroides fragilis, Clostridium difficile, and most other clinically significant anaerobic bacteria are susceptible to cefotetan. Biochem Biophys Res Commun, 1988 May 16, 152(3), 1361 - 8 Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin; Popoff MR et al.; We have purified from Clostridium spiroforme strain 246 an heterogeneous population of proteins (Sa) ranging from 43 to 47 kilodaltons exhibiting ADP-ribosyl transferase activity as do C . botulinum C2 toxin component I or the ia chain of C . perfringens E iota toxin . C . spiriforme Sa had alone no activity upon injection in mice or inoculated to Vero cells . When spiroforme ADP ribosyl transferase were mixed with a trypsin activated protein (Sb) separated from C . spiroforme bacterial supernatant, a lethal effect in mice and cytotoxicity on Vero cells were recorded . The Sa cross-reacted immunologically with either the light chain of C . perfringens E iota toxin or the ADP-ribosyl transferase from C . difficile 196 strain . No immunological relatedness was observed between Sa and C2 toxin component I . C . spiroforme toxin is thus another binary toxin close to iota. Gene, 1988 May 15, 65(1), 51 - 8 Cloning and expression of two cellulase genes of Clostridium cellulolyticum in Escherichia coli; Faure E et al.; Two cellulase genes isolated from Clostridium cellulolyticum strain ATCC3519 were cloned in Escherichia coli using plasmid pACYC184 . Plasmids pB52 and pB43 were isolated from the transformants producing carboxymethylcellulase (CMCase) and the two cloned CMCase-coding genes were found to be included in two EcoRI fragments of 5.7 kb and 2.6 kb, respectively . These two genes showed no homology . The CMCase-coding genes were found to be contained in a 1.8-kb KpnI-HindIII fragment and a 2.05-kb HindIII-PvuII fragment of the DNA donor strain . Expression of these genes in E . coli was found not to depend on their orientation in the cloning vector . Hybridization experiments between these two fragments and Clostridium thermocellum NCIB10682 DNA fragments carrying genes celA, celB, celC and celD were carried out and some homologies were detected. Gut, 1988 May, 29(5), 598 - 602 Pathogenesis of postantibiotic diarrhoea caused by Clostridium difficile: an in vitro study in the rabbit intestine; Guandalini S et al.; To elucidate the pathophysiological changes leading to postantibiotic diarrhoea caused by Clostridium difficile and its cytotoxin, oral ampicillin was given to rabbits, and jejunal, ileal, and caecal segments of those that developed diarrhoea were investigated in vitro . The rabbits that, in response to treatment, harboured Clostridium difficile in their colonic lumen were studied, and the results expressed according to the presence or absence of Clostridium difficile and/or its cytotoxin . Thus, we refer to either CD+ or CD- segments . The influx of glucose, phenylalanine, glycylphenylalanine, and lysine across the brush border of jejunum and ileum of CD+ segments was severely impaired, while only slightly blunted in CD- . No significant change was detected in the influx of glutamic acid in the jejunum of all treated animals and in the CD- ilea . Morphologic damage in ileum and caecum of CD+ was also more evident than in CD- . Transepithelial ion transport across short circuited ileal mucosa (CD+ and CD-) revealed secretory changes in Cl net transport that were more marked in CD- . We conclude that: (1) Clostridium difficile may also colonise the upper intestinal tract, where it induces morphological and functional damage, severely impairing nutrient absorption; and (2) the ileum contributes to the diarrhoea caused by CD even when the micro-organism is confined to the more distal gut by showing moderate impairment of nutrient absorption and marked electrolyte secretion. Br J Ophthalmol, 1988 May, 72(5), 380 - 5 Sudden visual loss associated with clostridial bacteraemia; Cannistra AJ et al.; A patient with endogenously acquired Clostridium septicum panophthalmitis is presented . The patient exhibited a striking sequence of signs and symptoms associated with this devastating ocular infection . Intensive antibiotic therapy was ineffective and enucleation of the globe was required . The microscopic pathology of the enucleated globe showed extensive infarction and necrosis of ocular structures in association with the panophthalmitis . In addition thrombosis of the central retinal artery and of choroidal vessels was observed. Arch Dis Child, 1988 May, 63(5), 543 - 5 Clostridium difficile and acute enterocolitis; Price EH et al.; Clostridium difficile belonging to groups not normally detected in infancy was the only potential pathogen detected in the stools of two infants with severe enterocolitis . Further information regarding the virulence of this organism was obtained by use of a recently introduced typing scheme. Appl Environ Microbiol, 1988 May, 54(5), 1289 - 92 Structure of an endo-beta-1,4-glucanase gene from Clostridium acetobutylicum P262 showing homology with endoglucanase genes from Bacillus spp; Zappe H et al.; The nucleotide sequence of an endo-beta-1,4-glucanase gene of Clostridium acetobutylicum contained two putative extended promoter consensus sequences, a Shine-Dalgarno sequence and a TTG initiation codon . The nucleotide sequence of the gene coding for the C-terminal region of this enzyme was not required for activity . Extensive homology in the nucleotide and amino acid sequences of the endoglucanase genes from C . acetobutylicum and Bacillus spp . was demonstrated. Appl Environ Microbiol, 1988 May, 54(5), 1254 - 7 Isolation and characterization of an anaerobic dehydrodivanillin-degrading bacterium; Chen W et al.; A novel, strictly anaerobic, gram-negative, non-spore-forming, fusiform, rod-shaped bacterium having high dehydrodivanillin (DDV)-degrading activity was isolated from cow ruminal fluid . This strain degraded a range of six main lignin-related compounds such as DDV, ferulic acid, dehydrodiisoeugenol, guaiacoxyacetic acid, vanillin, and veratrylglycerol-beta-guaiacyl ether to the extent of 14 to 83% within 2 days under strictly anaerobic conditions . As DDV degradation intermediates, three aromatic compounds (dehydrodivanillic acid, vanillic acid, and 5-carboxyvanillic acid) and two alicyclic compounds (cyclohexanecarboxylic acid and cyclohexanol) were detected by thin-layer, high-performance liquid, and gas chromatography and mass spectrometry . The addition of 1% glucose and peptone in a synthetic medium stimulated growth of the strain but slowed down DDV degradation . The presence of 0.1% yeast extract increased both cell growth and DDV degradation . The growth yield in defined medium was 151.5 g (dry weight) of cells per mol of DDV utilized . Characterization of the strain indicated that it was distinct from known Fusobacterium and Clostridium species . The bacterium was easily induced to form protoplasts after treatment with either penicillin or lysozyme . The frequencies of protoplast formation and regeneration in the strain were 94 and 18%, respectively. Appl Environ Microbiol, 1988 May, 54(5), 1237 - 42 Metabolism of the 18O-methoxy substituent of 3-methoxybenzoic acid and other unlabeled methoxybenzoic acids by anaerobic bacteria; DeWeerd KA et al.; O-methyl substituents of aromatic compounds can provide C1 growth substrates for facultative and strict anaerobic bacteria isolated from diverse environments . The mechanism of the bioconversion of methoxylated benzoic acids to the hydroxylated derivatives was investigated with a model substrate and cultures of one anaerobic consortium, eight strict anaerobic bacteria, and one facultative anaerobic microorganism . Using high-pressure liquid chromatography and gas chromatography-mass spectral analysis, we found that a haloaromatic dehalogenating consortium, a dehalogenating isolate from that consortium, Eubacterium limosum, and a strain of Acetobacterium woodii metabolized 3-{methoxy-18O}methoxybenzoic acid (3-anisic acid) to 3-{hydroxy-18O}hydroxybenzoic acid stoichiometrically at rates of 1.5, 3.2, 52.4, and 36.7 nmol/min per mg of protein, respectively . A different strain of Acetobacterium and strains of Syntrophococcus, Clostridium, Desulfotomaculum, Enterobacter, and an anaerobic bacterium, strain TH-001, were unable to transform this compound . The O-demethylating ability of E . limosum was induced only with appropriate methoxylated benzoates but not with D-glucose, lactate, isoleucine, or methanol . Cross-acclimation and growth experiments with E . limosum showed a rate of metabolism that was an order of magnitude slower and showed no growth with either 4-methoxysalicylic acid (2-hydroxy-4-methoxybenzoic acid) or 4-anisic acid (4-methoxybenzoic acid) when adapted to 3-anisic acid . However, A . woodii NZva-16 showed slower rates and no growth with 3- or 4-methoxysalicylic acid when adapted to 3-anisic acid in similar experiments . The results clearly indicate a methyl rather than methoxy group removal mechanism for such reactions. J Bacteriol, 1988 May, 170(5), 2301 - 5 Metabolism of adenylylated nucleotides in Clostridium acetobutylicum; Balodimos IA et al.; In response to the stresses imposed by temperature upshift or addition of butanol, Clostridium acetobutylicum cultures accumulated diadenosine-5',5'''-P1,P4-tetraphosphate (Ap4A) and adenosine 5'-P1,P4-tetraphospho-5'-guanosine (Ap4G) to high levels . The two adenylylated nucleotides were also accumulated in batch culture in the absence of imposed stresses when the clostridia switched from the acidogenic phase of growth to the solventogenic phase . Most of the adenylylated nucleotides were extracellular . The intracellular concentrations of these compounds were low throughout batch growth and in cells stressed by added butanol . In contrast to other procaryotes, these clostridia did not possess enzymes to degrade the dinucleotides, as shown with both intact cells and cell-free preparations . Our findings are consistent with the hypothesis that endogenously produced solvents are stressful to the cells, stimulating the synthesis of adenylylated nucleotides . The nucleotides accumulate extracellularly because they cannot be degraded and because the cell membranes are permeabilized by the solvents produced. J Clin Microbiol, 1988 May, 26(5), 1052 - 4 Laboratory findings in four cases of adult botulism suggest colonization of the intestinal tract; McCroskey LM et al.; There was laboratory evidence of intestinal colonization in four cases of adult botulism confirmed by the Centers for Disease Control . No performed toxin was detected in available foods, but Clostridium botulinum was isolated from foods in two instances . Botulinal toxin was detected in the sera of all four patients, in one case at 47 days after ingestion of suspected food . C . botulinum was demonstrated in the stool of all four patients and persisted for 119 days after the onset of illness in one patient . Two patients had surgical alterations of the gastrointestinal tract, which may have promoted the colonization . The apparent lack of ingestion of performed toxin in these cases and the persistence of botulinal toxin or C . botulinum, or both, for long periods in three of the patients suggest that colonization of the intestinal tract occurred. J Appl Bacteriol, 1988 May, 64(5), 389 - 94 Post-processing microflora and the shelf stability of gari marketed in Port Harcourt; Ofuya CO et al.; Gari was examined for its post-processing microbial content . Aerobic mesophilic bacteria and fungi were isolated from all samples . The total viable bacterial counts ranged from 2.0 X 10(2) to 8.0 X 10(4) cfu/g . Fungal counts ranged from 1.0 X 10(2) to 1.5 X 10(4) cfu/g . The total viable counts of fresh samples were much lower than those of market and packaged samples . Bacillus, Micrococcus and Proteus spp . were the bacteria isolated, Aspergillus niger, Aspergillus flavus and Penicillium spp . the fungi . Food borne parasites and pathogens such as Staph . aureus and Clostridium perfringens were not found . The gari samples were quite stable, having a shelf life of 3-6 months . The water activities of the samples ranged from 0.52 to 0.68 . Based on the microbial counts of the samples, the critical upper limit for the safety of gari was set at 10(4) cfu/g dry sample. Scand J Gastroenterol, 1988 May, 23(4), 413 - 21 Phospholipase activation and arachidonic acid release in isolated intestinal epithelial cells; Gustafson C et al.; A novel method for studying the mobilization of free arachidonic acid (AA) in isolated intestinal epithelial cells is described . The method is based on labeling the cellular phospholipids with 14C-AA and studying the release of this 14C-AA on subsequent phospholipase activation . Cells of high viability were isolated from the small intestine of guinea pigs and incubated with 14C-AA for 2 h; most of the incorporated 14C-AA was then esterified into phosphatidylethanolamine and phosphatidylcholine . When the labeled cells were stimulated with the calcium ionophore A23187 in the presence of external calcium, they released significant amounts of AA . In contrast, the cells released no AA when stimulated with A23187 in the absence of external calcium or in the presence of chlorpromazine or 4-bromophenacyl bromide, both of which are known to inhibit phospholipase A2 activity . On the other hand, the cells released significant AA in response to exogenous phospholipase C from Clostridium perfringens . These findings indicate that AA release in intestinal cells may be caused by calcium-mediated phospholipase A2 activation or by products of microbial phospholipase C activity . They also suggest the further use of 14C-AA-labeled cells for studying agents and mechanisms that may influence the release of AA in the gastrointestinal tract. Infect Immun, 1988 May, 56(5), 1215 - 21 Purification and characterization of Clostridium sordellii hemorrhagic toxin and cross-reactivity with Clostridium difficile toxin A (enterotoxin); Martinez RD et al.; Hemorrhagic toxin (toxin HT) was purified from Clostridium sordellii culture filtrate . The purification steps included ultrafiltration through an XM-100 membrane filter and immunoaffinity chromatography, using a monoclonal antibody to toxin A of Clostridium difficile as the ligand . Toxin HT migrated as a major band with a molecular weight of 525,000 and a minor band at 450,000 on nondenaturing gradient polyacrylamide gel electrophoresis . The molecular weight was estimated at 300,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Isoelectric focusing indicated an apparent pI of 6.1 . Toxin HT was cytotoxic for cultured cells and lethal for mice by intraperitoneal injection, and it elicited an accumulation of hemorrhagic fluid in rabbit ileal loops . Immunodiffusion analysis revealed a reaction of partial identity between toxins A and HT . Immunological cross-reactivity between these toxins was further demonstrated by immunoblotting and by neutralization of toxin HT biological activity with antibodies to toxin A . A sensitive indirect enzyme-linked immunosorbent assay was used to examine the affinity involved in homologous and heterologous antigen-antibody interactions . Our findings show that toxin HT has biological activities and immunological properties similar to those of toxin A; however, the toxins are not identical. Infect Immun, 1988 May, 56(5), 1107 - 12 Clostridium difficile toxins A and B inhibit human immune response in vitro; Daubener W et al.; Two Clostridium difficile toxins isolated from strain VPI 10463 were tested for their effect on different human T-cell proliferation systems . In mitogen- and antigen-driven T-cell proliferation systems, toxins inhibited the proliferative response in a dose-dependent fashion . In interleukin-2-driven culture systems, no effect of toxins could be found on preactivated T cells . We suspected that monocytes were the influenced cells, since in antigen- and mitogen-driven systems monocytes were necessary for the proliferative response, whereas the interleukin-2-driven system was independent of monocytes . To prove this concept, purified monocytes were treated with toxins . The treatment was found to markedly reduce the capacity of monocytes to stimulate T-cell proliferation . No inhibition of the proliferative response was measured when, instead of monocytes, resting or preactivated T cells were treated with toxins . These experiments clearly show that C . difficile toxins interact with monocytes and not with T cells . The effect of toxins on cells of the immune system might be one factor in the development of pseudomembranous colitis. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 May, 268(3), 347 - 56 Recombinant plasmid DNA variation of Clostridium oncolyticum--model experiments of cancerostatic gene transfer; Schlechte H et al.; The specific germinating capacity of spores of Clostridium oncolyticum in tumours in vivo, and various reports on concomitant partial oncolysis (tumour lysis) have prompted us to propose a concept of equipping C . oncolyticum with genes of other organisms producing cancerostatics . As an example we used Colicin E3, whose structural genes lies in the E . coli plasmid pCo1E3-CA38 . After in vitro recombination of restrictase EcoRI-fragmented pCo1E3-CA38 DNA with an uncharacterized plasmid fraction from C . concolyticum a plasmid-free strain of C . oncolyticum was infected with recombinant DNA . A special microbiological selection system allows the identification of C . oncolyticum clones with Colicin E3-similar formation of cleared haloes. Microb Pathog, 1988 May, 4(5), 317 - 23 Clostridium perfringens enterotoxin; McClane BA et al.; Current knowledge of CPE action is briefly summarized in Figure 1 . After specific binding to a protein receptor(s), the entire CPE molecule rapidly inserts into membranes forming a complex of 150,000 Mr . Almost simultaneously with insertion, there is a sudden change in ion fluxes . The molecular events behind the induction of ion flux changes remain undefined, but might involve either direct formation of membrane pores by CPE or activation of pre-existing membrane pores . As intracellular ion levels change, cellular metabolism is affected and processes such as macromolecular syntheses are inhibited . One of the ion flux effects resulting from CPE treatment involves increased Ca2+ influx; as more Ca2+ enters the cell, morphologic damage and permeability alterations for larger molecules occur . It remains to be determined if both morphologic damage and larger permeability alterations are necessarily linked but, for example, it could be envisioned that CPE-induced Ca2+ influx causes a cytoskeletal collapse leading to altered membrane permeability . The cytoskeleton has been shown to be sensitive to intracellular Ca2+ levels and is important in normal membrane structure/function relationships . As the cumulative effects of CPE inhibit cellular metabolism, cell death occurs . The precise irreversible CPE lethal action still must be identified . As CPE-treated intestinal epithelial cells die in vivo, histopathologic damage appears . This damage results in loss of normal intestinal function causing secretion of fluids and electrolytes . This effect is clinically manifested as diarrhea . The strongly cytotoxic action of CPE clearly distinguished the action enterotoxin from STa or CT.(ABSTRACT TRUNCATED AT 250 WORDS) J Biochem (Tokyo), 1988 May, 103(5), 840 - 2 Inhibitory effect of taurolipids on Clostridium perfringens sialidase; Nohara-Uchida K et al.; Taurolipids A and B, which are detergent-type compounds isolated from protozoan Tetrahymena cells, were demonstrated to inhibit strongly the activity of Clostridium perfringens sialidase . On addition of 280 pmol of taurolipid B to 20 mU of the enzyme, the sialidase activity was decreased to 7% of the original activity at pH 5.1 as the optimum pH . The inhibition was non-competitive . Effective inhibition was observed at the acidic region from the isoelectric point of the sialidase, and at a low ionic strength . Both the long chain acyl and sulfonic acid groups of taurolipids were required for the inhibition of the sialidase activity . A mechanism is postulated for the inhibition. Prikl Biokhim Mikrobiol, 1988 May-Jun, 24(3), 305 - 9 {Isolation and purification of phospholipase C from Clostridium perfringens and antibodies against it using polymeric biospecific adsorbents}; Shemanova GF et al.; A new polymeric biospecific adsorbent intended for isolation of Clostridium perfringens phospholipase C (PLC) and PLC-specific antibodies is discussed . It was obtained by radical copolymerization of acrylamide, methylenbisacrylamide, and the acryl acid chloranhydride-acylated substrate of PLC (chicken yolk lecithovitellin) . Maximal adsorption of PLC was observed in the presence of the enzyme activator, and the highest amount of PLC was eluted in case of its minimal adsorption . The "residual" activity of PLC on the adsorbent was used to isolate homologous antibodies from the anti-perfringens serum . PLC and PLC-specific antibodies were serologically pure in the agar precipitation test with the anti-perfringens serum and the primary PLC concentrate, respectively; the antibodies gave only one zone in PAAG-electrophoresis. Pathol Biol (Paris), 1988 May, 36(5), 420 - 4 {In vitro activity of roxithromycin against hospital bacteria and the concordance curve}; Soussy CJ et al.; This study was set up to establish the regression curve for roxithromycin inhibition zone diameters (disks 15 micrograms) and MIC to create a strain distribution plot, in order to allow accurate interpretation of the disk diffusion method for testing susceptibility to roxithromycin . 373 bacterial strains were studied in three university hospital . Roxithromycin was active against erythromycin sensitive Staphylococcus aureus and coagulase negative Staphylococci at concentrations of 0.06 to 4 micrograms/ml (mode 0.5) . Erythromycin resistant strains were also resistant to roxithromycin . Enterococci could be divided into two populations, one resistant (MIC greater than 128 micrograms/ml) and the other with MIC of 0.5 to 32 (mode 1-2) . This was also the case for Streptococci and Pneumococci with MIC lower for susceptible strains (mode 0.06-0.12) . Roxithromycin was active on Haemophilus at concentrations of 0.12 to 32 micrograms/ml; MIC for beta-lactamase producing strains were comparable to those of strains not producing . MIC for Gonococci were low (less than 0.008 to 0.12), except for three strains . They were higher for Meningococci (0.03 to 32) with a majority of strains inhibited by 0.5 to 4 micrograms/ml . MIC were 4 for Clostridium perfringens; Bacteroides fragilis strains were inhibited by 0.5 to 2 micrograms/ml . The correlation coefficient for regression curve was 0.79; for critical concentrations less than or equal to 1 and greater than 4 micrograms/ml, critical diameters are greater than or equal to 22 and less than 17 mm. J Hosp Infect, 1988 May, 11(4), 335 - 9 Gastrointestinal carriage rate of Clostridium difficile in elderly, chronic care hospital patients; Cefai C et al.; The carriage rate of Clostridium difficile in patients at a chronic care hospital was determined by two point prevalence surveys at 6-monthly intervals . In the first survey C . difficile or its toxin was present in stool samples from five symptomless patients on three of the four wards studied . All of these colonized patients had been in hospital for at least 2 months, but there was no relationship between carriage of the organism and antibiotic use . When the survey was repeated 6 months later, no symptomless carriers were found but one symptomatic patient had C . difficile and its toxin present in the stool . The results suggest that C . difficile should always be considered as a possible cause of diarrhoea in long-stay hospitalized patients. Appl Environ Microbiol, 1988 May, 54(5), 1104 - 8 Growth of Clostridium perfringens in cooked chili during cooling; Blankenship LC et al.; U.S . Department of Agriculture regulations require that brick chili be cooled from 48.9 degrees C to 4.4 degrees C within 2 h of cooking, but processors may not always be able to comply . Studies were conducted to evaluate the extent of bacterial multiplication resulting from outgrowth of germinated Clostridium perfringens spores experimentally inoculated into chili and incubated at various temperatures . Inoculated samples were heated (75 degrees C for 20 min) to activate spores, quickly equilibrated, and held at one of five desired temperatures for 6 h . No growth was observed for C . perfringens in samples held at 26.7 degrees C and below for 6 h, but growth was observed by 6 h in samples held at 32.2 degrees C and after 2 h in samples held at temperatures between 37.8 degrees C and 48.9 degrees C . Using isothermal growth data, we developed a simple model for predicting the growth of bacteria with time under exponential cooling conditions . The model predicts both the lag phase and the numbers of bacteria at specific times during the growth phase . It was developed by using isothermal growth data and tested by using temperature-varying growth data from experiments with spores of C . perfringens in chili . Actual data agreed closely with predicted results . The results should be useful for evaluating the hazard potential for growth of C . perfringens in chili. Clin Chim Acta, 1988 Apr 29, 173(3), 251 - 62 Early diagnosis of clostridial gas gangrene using sialidase antibodies; Roggentin P et al.; In order to improve the diagnosis of gas gangrene, especially at an early stage of infection, new ways for the detection of the responsible Clostridia were investigated . Sialidase, known to be excreted in large amounts by the most frequently occurring myonecrotizing clostridial species, Clostridium perfringens, Clostridium septicum, and Clostridium sordellii, was isolated . With polyclonal antibodies raised against these enzymes, two immunological assays were established, which are directed against the sialidase activity (sialidase inhibition test) and the enzyme protein ('sandwich'-ELISA), respectively . Using these assays, species-specific information about the presence of clostridial sialidase was obtained within 50 min or 6 h . Animal tests revealed that both assays are applicable 8-12 h after clostridial infection, using resected tissues or wound fluids for estimations . The assays allow specific, sensitive, and quantitative measurement of clostridial sialidases, and no significant interference by sialidases from other microbes or from host tissues occurred . The applicability of the new assays for an early diagnosis of gas gangrene in human patients is discussed. Biochemistry, 1988 Apr 19, 27(8), 2800 - 10 Distinct structural features of the alpha and beta subunits of nitrogenase molybdenum-iron protein of Clostridium pasteurianum: an analysis of amino acid sequences; Wang SZ et al.; Nitrogenase is composed of two separately purified proteins, a molybdenum-iron (MoFe) protein and an iron (Fe) protein . Structural genes (nifD and nifK) encoding alpha and beta subunits of the MoFe protein of Clostridium pasteurianum (Cp) have been cloned and sequenced . The deduced amino acid sequences were analyzed for structures that could be related to the unique properties of the Cp protein, particularly its low capacity to form an active enzyme with a heterologous Fe protein . Cp nifK is located immediately downstream from Cp nifD, with the start codon of nifK overlapping by one base with the stop codon of nifD . An open reading frame following nifK was identified as nifE . The amino acid sequence deduced from nifK encompasses the partial amino acid sequences previously reported from the isolated beta subunit . Cp nifK encodes a polypeptide of 458 amino acid residues (Mr 50 115) whose amino-terminal region is about 50 residues shorter than the otherwise conserved corresponding polypeptides from four other organisms . In contrast, Cp alpha subunit (nifD product) contains an additional stretch of 50 amino acid residues in the 380-430 region, which is unique to the Cp protein . It therefore appears that the combined size of the alpha and beta subunits could be important to nitrogenase function . An analysis of the predicted secondary structure from the amino acid sequence of each subunit from three species (C . pasteurianum, Azotobacter vinelandii, and Rhizobium japonicum) further revealed structural features, including regions adjacent to some of the conserved cysteine residues, differentiating the Cp MoFe protein from others . These different regions may be further tested for correlation with distinct properties of Cp nitrogenase. Biochem J, 1988 Apr 15, 251(2), 379 - 83 Interaction of botulinum and tetanus toxins with the lipid bilayer surface; Montecucco C et al.; The interaction of botulinum neurotoxins serotypes A, B and E (from Clostridium botulinum) and of tetanus neurotoxin (from Clostridium tetani) with the surface of liposomes made of different lipid compositions was studied by photolabelling with a radioiodinated photoactive phosphatidylethanolamine analogue {125I-dipalmitoyl (3,4-azidosalicylamido)phosphatidylethanolamine} . When the vesicles were made of negatively charged lipids (asolectin), each of these neurotoxic proteins was radioiodinated, thus providing evidence for their attachment to the membrane surface . The presence of gangliosides on liposome membranes enhanced fixation of the neurotoxic proteins to the lipid vesicle surface . Both the heavy and light chains of the clostridial neurotoxins were involved in the attachment to the lipid bilayer surface . Each of the toxins tested here attached poorly to liposomes made of zwitterionic lipids (egg phosphatidylcholine), even when polysialogangliosides were present . The data suggest that the binding of botulinum and tetanus neurotoxins to their target neuronal cells involves negatively charged lipids and polysialogangliosides on the cell membrane. Biochem Pharmacol, 1988 Apr 15, 37(8), 1525 - 34 Role of hydrogenase 1 of Clostridium pasteurianum in the reduction of metronidazole; Church DL et al.; Competition studies between the phosphoroclastic reaction and the metronidazole reduction reaction using dialyzed crude cell-free extracts of Clostridium pasteurianum which were essentially devoid of Hydrogenase 1 activity demonstrated that this enzyme plays an important role in the reduction of metronidazole . To determine further the exact function for Hydrogenase 1 in the reduction of the drug, this enzyme was highly purified from C . pasteurianum . Metronidazole reduction activity copurified with Hydrogenase 1 specific activity throughout the purification procedure . Drug reduction required the presence of an electron carrier and could not be accomplished by the enzyme alone . Ferredoxin, and also the low potential electron carrier dyes, methyl and benzyl viologen, and the flavin coenzymes, FAD and flavin mononucleotide (FMN), could couple the reduction of metronidazole . Hydrogenase 1 activity and its metronidazole reduction activity were inactivated irreversibly in the presence of oxygen . Metronidazole could be reduced only by an electron carrier-Hydrogenase 1 mechanism or directly by sodium dithionite. Biochem J, 1988 Apr 15, 251(2), 609 - 12 ADP-ribosylation of dinitrogenase reductase from Clostridium pasteurianum prevents its inhibition of nitrogenase from Azotobacter vinelandii; Murrell SA et al.; The effect of ADP-ribosylation of dinitrogenase reductase on its binding to dinitrogenase was investigated . Dinitrogenase reductase from Clostridium pasteurianum (Cp2) was a substrate for the ADP-ribosyltransferase and the dinitrogenase-reductase-activating glycohydrolase from Rhodospirillum rubrum . ADP-ribosylation inactivated Cp2 and prevented its formation of a tight complex with dinitrogenase from Azotobacter vinelandii (Av1) . The complex between Cp2 and Av1 could not be ADP-ribosylated once it formed. Thromb Haemost, 1988 Apr 8, 59(2), 236 - 9 Phospholipase C from clostridium perfringens induces human platelet aggregation in plasma; Barzaghi G et al.; We studied the aggregating effect of different concentrations of phospholipase C (PLC) (extracted from Clostridium perfringens) on human platelet-rich plasma (PRP) . PRP was preincubated with PLC for 3 min at 37 degrees C and the platelet aggregation was followed for 10 min . The threshold aggregating concentration (TAC) of PLC was 3-4 U/ml . We also studied the potentiation of PLC with other stimuli on platelet aggregation . Potentiating stimuli, such as arachidonic acid (AA), ADP . Platelet Activating Factor (PAF) and U-46619 (a stable analogue of cyclic endoperoxides) were all used at subthreshold concentrations . We also studied the possible inhibitory effect of aspirin, apyrase, TMQ, a prostaglandin endoperoxide/thromboxane receptor antagonist and BN-52021, a PAF receptor antagonist . Only aspirin and apyrase were able to reduce aggregation induced by PLC alone and PLC + AA and PLC + ADP respectively . TMQ and BN-52021 were inactive . In ex vivo experiments oral aspirin (500 mg) partially inhibited platelet aggregation induced by PLC alone, PLC + AA and PLC + ADP 2 and 24 h after administration . Aspirin 20 mg for 7 days also reduced aggregation induced by PLC + AA. Biochemistry, 1988 Apr 5, 27(7), 2319 - 23 A new kinetic approach for studying phospholipase C (Clostridium perfringens alpha toxin) activity on phospholipid monolayers; Moreau H et al.; The enzymatic activity of purified phospholipase C (alpha toxin) from Clostridium perfringens was investigated with various phospholipid monolayers . A two-step reaction was used . Enzymatic hydrolysis of insoluble lecithin films by phospholipase C, generating 1,2-diacylglycerol and water-soluble phosphocholine, was coupled with the action of pancreatic lipase in order to give rise to fatty acid and 2-monoacylglycerol, which are rapidly desorbed from the interface . With this new procedure, it is possible to obtain continuous and accurate kinetic measurements of the phospholipase C catalyzed reaction with phospholipid monolayers as the substrate . It is thus possible to avoid the use of radiolabeled substrates as necessary in previous studies, and the difficulties caused by diacylglycerol accumulation in the lipid film are minimized . No hydrolysis was detected when either phosphatidylethanolamine, phosphatidylserine, or phosphatidylglycerol films were used as substrates . By means of a film transfer technique, Ca2+ and Zn2+ ions were found to play a specific and critical role . The present study demonstrates clearly for the first time that Ca2+ is essential for enzyme binding to lipid films, whereas Zn2+ is specifically involved in the catalytic hydrolysis of the substrate. Presse Med, 1988 Apr 2, 17(12), 564 - 7 {Purulent pleurisy caused by anaerobic bacteria . A retrospective study of 19 cases}; Dalphin JC et al.; A retrospective clinical, biological, radiological and evolutive study of 19 cases of pleural empyema caused by anaerobic organisms diagnosed between 1980 and 1986 was carried out . These 19 cases accounted for 30.6 p . 100 of all cases of pleural empyema diagnosed during the same period . A local or general contributory factor was found in all patients; false passage, gastrointestinal pathology and buccal or dental diseases were the most frequent aetiological circumstances . The clinical picture was rather torpid, with a body temperature below 38 degrees C in 42 p . 100 of the cases, which delayed the diagnosis: the mean time interval between onset and diagnosis was 20 days . In nearly one half of the cases, blood stained expectoration was present and air-fluid levels were visualized at standard radiography and computerized tomography, which is the best exploratory method to evaluate the size and appearance of the pleural lesion, to guide percutaneous drainage and later to assess possible sequelae . The predominant anaerobic flora consisted of Gram-positive cocci and Bacteroides spp; in 70 p . 100 of the cases the anaerobic organism(s) was (were) sensitive to penicillin G . The course of the disease was favourable in all patients . Surgery was performed in 3 of the 19 patients on account of chest wall gangrene due to Clostridium perfringens in 1 case and of the presence of multiple fluid pockets making drainage ineffective in 2 cases. J Antimicrob Chemother, 1988 Apr, 21(4), 425 - 38 European collaborative study of reproducibility of quantitative sensitivity testing of anaerobes; King A et al.; Minimum inhibitory concentrations (MICs) of ampicillin, cefoxitin, cefbuperazone, latamoxef, metronidazole, clindamycin and chloramphenicol were determined for 15 different anaerobic bacteria including Bacteroides spp., anaerobic cocci and Clostridium spp., in 18 European laboratories, who used their own methodology . The degree of intra- and inter-laboratory reproducibility was surprisingly good--87% of results fell on the modal MIC or were within one dilution of it and only 4.4% of the results differed by three or more dilutions . Results for clindamycin were the least reproducible, as were those for clostridia . Of the organisms that we tested Bacteroides fragilis ATCC 25285, NCTC 9343 emerged as the most suitable for use in quality control, and Peptococcus variabilis ATCC 14956 the most appropriate if a control for more slowly-growing species is required. Appl Environ Microbiol, 1988 Apr, 54(4), 872 - 7 Enrichment and isolation of a ruminal bacterium with a very high specific activity of ammonia production; Russell JB et al.; When mixed ruminal bacteria were inoculated into semicontinuous cultures (25% transfer every other day) containing lactate, dulcitol, pectin, or xylose and Trypticase (1 g/liter) as the sole nitrogen source, the specific activity of ammonia production increased . The greatest enrichment was observed with lactate and xylose, and in these cases the specific rate of ammonia production was eightfold higher than that of the ruminal fluid control (approximately 35 nmol of ammonia per mg of protein per min) . Isolates with different morphologies were obtained from each of the enrichments, but in no case did the specific activity of any isolate exceed that of the mixed ruminal bacteria . If Trypticase (15 g/liter) was used as the only energy and nitrogen source, there was an even greater increase in ammonia production, and two monensin-sensitive bacteria, a Peptostreptococcus species and a Clostridium species, were obtained . The Peptostreptococcus species was unable to grow on any of 25 carbohydrate or carbohydrate derivatives tested; but the Clostridium species was able to use glucose, maltose, fructose, cellobiose, trehalose, sorbitol, and salicin as energy sources . Neither organism was able to grow in the absence of an amino acid source, but growth rates on Trypticase were greater than 0.35/h . The specific activities of ammonia production were 346 and 427 nmol/mg of protein per min for strains of Peptostreptococcus and Clostridium, respectively . Megasphaera elsdenii and Bacteroides ruminicola, previously isolated ruminal ammonia producers, had specific activities of only 11 and 19 nmol of ammonia per mg of protein per min, respectively . The most probable number of Clostridium species in ruminal fluid was less than 10(3)/ml, but the Peptostreptococcus species was present at 10(8)/ml.(ABSTRACT TRUNCATED AT 250 WORDS) Antimicrob Agents Chemother, 1988 Apr, 32(4), 580 - 3 In vitro activities of two oxazolidinone antimicrobial agents, DuP 721 and DuP 105; Neu HC et al.; The antibacterial activities of DuP 105 and DuP 721, new oxazolidinone antimicrobial agents, were compared with those of beta-lactams and glycopeptides . Ninety percent of Staphylococcus aureus and Staphylococcus epidermidis isolates, including methicillin-resistant isolates, were inhibited by 4 micrograms of DuP 105 and 1 microgram of DuP 721 per ml . DuP 721 inhibited hemolytic streptococcus groups A, B, C, F, and G at a concentration of less than or equal to 1 microgram/ml, and it inhibited viridans group streptococci at a concentration of 2 micrograms/ml . Both agents inhibited Listeria monocytogenes, Corynebacterium group JK species, anaerobic cocci, and Clostridium spp . including Clostridium difficile . They did not inhibit members of the family Enterobacteriaceae or Pseudomonas aeruginosa, but the MIC for 90% of Bacteroides fragilis isolates was 8 micrograms of DuP 721 per ml. Lab Anim Sci, 1988 Apr, 38(2), 167 - 8 Isolation of Clostridium spiroforme from rabbits; Holmes HT et al.; The isolation of Clostridium spiroforme from intestinal contents of rabbits was achieved by sampling the supernatant-pellet interphase of centrifuged specimens processed for routine toxin analysis . High-speed centrifugation at 20,000x for 15 minutes provided a rapid and effective means of separating this anaerobic pathogen from the majority of both indigenous and non-indigenous intestinal microbial flora . The unusual helically-coiled, semicircular shape of the microorganism is considered, at least in part, responsible for this phenomenon. J Wildl Dis, 1988 Apr, 24(2), 240 - 5 Effects of botulism on ducks drinking saline water; Wobeser G; Mallard (Anas platyrhynchos) ducklings (2 wk old) were given water from natural saline wetlands or fresh water as drinking water for 1 or 2 wk prior to, and after, receiving material containing Clostridium botulinum type C toxin . Water with conductivity ranging from 3,460 to 6,690 mu mhos/cm had no detectable effect on the occurrence or severity of clinical signs of botulism . Ducks drinking water with conductivity of 7,130 mu mhos/cm for 1 wk prior to receiving toxin had more severe clinical signs and greater mortality than did birds drinking fresh water . Ducks given the same water for 2 wk prior to receiving toxin did not differ from the controls in response to toxin . Fewer ducks in groups drinking the most saline water tested (conductivity = 13,500 mu mhos/cm) had clinical signs of botulism than in groups drinking fresh water. Am J Clin Pathol, 1988 Apr, 89(4), 525 - 7 Stool caproic acid for screening of Clostridium difficile; Madan E et al.; Clostridium difficile is the prime etiologic agent in the production of pseudomembranous colitis by its powerful cytotoxin . The most common test for the toxin is a tissue culture method with neutralization of cytopathic effect by a C . difficile antiserum . This method is expensive and requires a minimum of 72 hours before results can be obtained . Attempts to create a rapid method, counterimmunoelectrophoresis, enzyme-linked immunosorbent, latex agglutination, and fluorescent antibody test are fraught with many problems . This report describes a rapid method for the identification of C . difficile, using gas-liquid chromatography (GLC) for the demonstration of caproic acid, a product of the organisms fatty acid metabolism. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Apr, 268(2), 220 - 7 Purification of Clostridium botulinum type G progenitor toxin; Nukina M et al.; Clostridium botulinum type G cultured for 6 days at 30 degrees C in proteose peptone-yeast extract-glucose medium produced toxin of 1.3 x 10(4) LD50/ml . The toxin was precipitated at pH 4.0, extracted with 0.2 M phosphate buffer, pH 6.0, and activated with trypsin . Sonic treatment and trypsinization of the residual precipitate released additional toxin, the toxicity of which corresponded to that detected in whole culture . Activated toxin obtained from the first extract and that from the residual precipitate were combined and purified by salting out, acid precipitation, gel filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second gel filtration on Sephadex G-200 . The yield of purified toxin from 10 liters of culture was 22.9 mg an 1.1 X 10(8) mouse ip LD50 with a specific toxicity of 3.0 X 10(7) mouse ip LD