Clostridia

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 LD50/mg nitrogen . The molecular weight of the toxin was about 500,000, corresponding to that of L toxin of the other types . No M nor LL toxin was detected.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Apr, 268(2), 180 - 92
Purification and partial characterization of two proteinases from Clostridium butyricum M 55; Ciborowski P et al.; Clostridium butyricum M55 proteinases were purified by application of a multistep procedure involving ethanol precipitation, DEAE cellulose chromatography and molecular sieving . The purified enzymes obtained were called proteinase I and proteinase II . They appeared to be homogeneous when examined by molecular sieving and polyacrylamide gel electrophoresis . The highly purified proteinases were studied for their physico-chemical properties . The influences of pH, temperature, ionic strength and amino acids composition were investigated . The effects of metal ions and of protein-structure-modifying agents support views suggesting the character of these enzymes.

Dis Colon Rectum, 1988 Apr, 31(4), 306 - 10
Occult gastrointestinal carcinoma causing metastatic clostridial soft-tissue infection . Report of two cases; Buckley D et al.; The association between nontraumatic, metastatic, clostridial soft-tissue infection and malignancy is well recognized, particularly when Clostridium septicum is involved . This report presents two patients with nontraumatic, metastatic, soft-tissue infection due to C . septicum and reviews the English medical literature . Eighty-four percent of the reported patients were found to have colon carcinoma, which was postulated to be the portal of entry . Prognosis is poor, but early recognition and antimicrobial therapy with prompt and aggressive surgical intervention, both for the soft-tissue infection and the abdominal process, may lower the high mortality.

Pathology, 1988 Apr, 20(2), 194 - 7
Clostridium septicum infection in neutropenic enterocolitis; Yeong ML et al.; A 36-year-old woman developed neutropenia following chemotherapy for inoperable carcinoma of the cervix . She suffered acute abdominal pain, nausea, vomiting, and peritonitis of rapid onset . The right hemicolon and 15 cm of terminal ileum were resected at laparotomy and this showed marked edema of the cecum and ileo-cecal valve associated with superficial ulceration of the valve . There was necrosis of submucosal tissues and the muscle wall which contained a large number of Gram-positive bacilli . These showed positive membrane immunofluorescence with specific anti-Clostridium septicum antisera . We identify a case of enterocolitis due to Clostridium septicum infection . This is associated with neutropenia and is often fatal due to the rapid course of and failure to recognize the infection.

Pathology, 1988 Apr, 20(2), 188 - 90
Acute hepatic failure precipitated in a patient with subclinical liver disease by vibrionic and clostridial septicemia; Simon TP et al.; A case of fatal septicemia caused by Vibrio vulnificus and Clostridium bifermentans in a patient with subclinical liver disease is described . The patient appeared to recover from the infection initially after antibiotic therapy but finally succumbed to rapidly developing hepatic failure . Postmortem examination revealed hemochromatosis . The pathogenesis of the polymicrobial septicemia and hepatic failure is discussed in the light of the published literature.

Comput Appl Biosci, 1988 Apr, 4(2), 275 - 9
A general purpose computer analysis system for chromatographic data; Santangelo JD; A manual integration system for the analysis of chromatographic data is described . The analog output produced by an HPLC absorbance monitor is passed to a non-inverting signal amplifier . This amplified signal is sent to an IBM PC where an analog to digital converter is used to digitize the data . A set of six computer programs which collect, store and analyze these data are presented . This system was used to analyze the nucleotide content of the anaerobic organism Clostridium acetobutylicum by strong anion-exchange HPLC.

Antimicrob Agents Chemother, 1988 Apr, 32(4), 601 - 4
In vitro activity of cefotetan compared with that of other antimicrobial agents against anaerobic bacteria; Wexler HM et al.; The activity of cefotetan against 430 strains of anaerobic bacteria was compared with that of cefoxitin, ceftizoxime, clindamycin, metronidazole, and chloramphenicol . Percent susceptible values for the Bacteroides fragilis group were 60, 80, 29, 86, 100, and 100%, respectively . Percent susceptible values for the B . fragilis species were 91, 92, 46, 98, 100, and 100%, respectively . Non-B . fragilis-group Bacteroides species were inhibited very well (90 to 100%) by all drugs except ceftizoxime (80%) . Cefotetan and metronidazole were the most active agents against Clostridium difficile . Percent susceptible values for all strains were 72, 79, 44, 82, 93, and 98%, respectively.

Arch Biochem Biophys, 1988 Apr, 262(1), 59 - 66
Damaging effects of Clostridium perfringens delta toxin on blood platelets and their relevance to ganglioside GM2; Jolivet-Reynaud C et al.; The lytic effect of Clostridium perfringens delta toxin was investigated on goat, human, rabbit, and guinea pig platelets . In contrast to erythrocytes from the latter three species, which are insensitive to the toxin, the platelets were equally lysed by the same amount of toxin . These results suggest the presence of GM2 or GM2-like ganglioside(s) as a specific recognition site of the toxin on platelet plasmic membrane as previously established for sensitive erythrocytes . Plasmic membrane damage of human platelets was evidenced by the release of entrapped alpha-{14C}aminoisobutyric acid used as a cytoplasmic marker . The specific binding of hemolytically active 125I-delta toxin by human and rabbit platelets was practically identical, dose dependent, and inhibitable by GM2 . Labeled toxin was also bound by various subcellular organelles separated from rabbit platelets except the 5-hydroxytryptamine (5-HT)-containing dense bodies, suggesting the absence or inaccessibility of GM2 on the surface of the latter organelles . This result correlates with the low amounts of 5-{3H}HT liberated after platelet challenge with delta toxin whereas this mediator was massively liberated upon lysis by the sulfhydryl-activated toxin alveolysin . The levels of M and P forms of phenol sulfotransferase (PST), involved in 5-HT catabolism, were determined in human platelet lysates after challenge with delta toxin, alveolysin, and other disruptive treatments . The low PST-M activities detected after lysis by delta toxin suggest that this isoenzyme is very likely associated to dense bodies in contrast to PST-P which is cytoplasmic . Platelet lysis by the toxin allows easy separation of these organelles.

Eur J Clin Microbiol Infect Dis, 1988 Apr, 7(2), 312 - 5
Clostridium difficile plasmid isolation as an epidemiologic tool; Clabots C et al.; A large hospital outbreak of Clostridium difficile diarrhea at the Minneapolis Veterans Administration Medical Center (MVAMC) was studied by plasmid profile typing . Plasmids were obtained from 30 (37%) of 82 clinical isolates from MVAMC patients and 10 (67%) of 15 non-MVAMC isolates . While bacteriophage plus bacteriocin typing and polyacrylamide gel electrophoresis (PAGE) plus bacterial agglutination typing proved more universally applicable, plasmid profiles may be useful for tracing isolated epidemic outbreaks, reinfections and relapses caused by plasmid-bearing strains.

Eur J Clin Microbiol Infect Dis, 1988 Apr, 7(2), 285 - 90
Computer-aided densitometric analysis of protein patterns of Clostridium difficile; Ehret W et al.; The applicability of whole-cell protein patterns obtained by sodium dodecylsulfate-polyacrylamide gel electrophoresis as a typing method for Clostridium difficile was examined using a total of 227 strains isolated from 191 patients and their surroundings . Computer-aided densitometric analysis was used to establish a reliable standardization technique with which a large number of protein patterns could be efficiently classified . The normalized tracks could be electronically superimposed and compared to give reproducible results . The influence of sample preparation for electrophoresis was found to be negligible . By this technique 35 subgroups could be differentiated, and further discrimination was possible within each subgroup using minor, even very weak protein bands . Epidemiologically related strains of Clostridium difficile yielded absolutely identical patterns, allowing unequivocal identification . Thus protein electrophoresis combined with densitometric analysis was shown to be useful for typing Clostridium difficile isolates.

Eur J Clin Microbiol Infect Dis, 1988 Apr, 7(2), 274 - 8
Incidence and significance of Clostridium difficile in hospitalized cancer patients; Gerard M et al.; The aim of the study was to assess the incidence and clinical significance of Clostridium difficile in patients in our cancer center . Over a period of seven consecutive months, 557 stools samples obtained from 156 hospitalized cancer patients (37 leukemic patients receiving oral antimicrobial prophylaxis and 119 patients from whom a stool sample was sent to the laboratory) were analyzed for the presence of Clostridium difficile . Clostridium difficile and/or its toxin was recovered from 13 (35%) of the 37 patients receiving oral antimicrobial prophylaxis, and from 15 (12%) of the other 119 patients (p less than 0.05) . Isolation of Clostridium difficile was associated with diarrhoea in 13 (46%) of 28 patients but specific treatment was initiated only in 7 (25%) of the 28 patients in whom Clostridium difficile was isolated . The wide distribution of the serotypes identified in our patients does not suggest an epidemic situation in our hospital.

Infect Immun, 1988 Apr, 56(4), 926 - 9
Comparison of toxins of Clostridium butyricum and Clostridium botulinum type E; Gimenez JA et al.; The toxin of Clostridium butyricum strains isolated from two infants with botulism is neutralized by antitoxin for type E botulinum toxin . This toxin and that of a C . botulinum type E strain were purified by the same protocol . Both toxins were Mr 145,000 proteins which, when activated with trypsin, were composed of an H subunit of Mr 105,000 and an L subunit of Mr 50,000 . The activated specific toxicity of purified butyricum toxin based on an intravenous assay was 2 X 10(8) mouse 50% lethal doses (LD50s)/mg of protein, but that based on an intraperitoneal assay was 7 X 10(7) LD50s/mg, compared with 6 X 10(7) LD50s/mg for type E toxin as determined by both methods . Immunodiffusion tests with antitoxin raised with type E toxin indicated that the two toxins were serologically very similar except for a spur formed by type E toxin . The close similarities of the two toxins suggest that toxigenic C . butyricum could arise when a wild-type strain, which is normally nontoxigenic, acquires the toxin gene of a C . botulinum type E strain.

J Appl Bacteriol, 1988 Apr, 64(4), 285 - 91
Evaluation of a monoclonal antibody-based immunoassay for detecting type B Clostridium botulinum toxin produced in pure culture and an inoculated model cured meat system; Gibson AM et al.; A monoclonal antibody-based amplified ELISA method for detecting Clostridium botulinum type B toxin was evaluated for its ability to detect the toxin in the supernatant fluid of pure cultures and after growth from Cl . botulinum spores inoculated into pork slurries . Slurries containing NaCl (1.5-4.5% w/v) and polyphosphate (0.3% w/v) were either unheated or heated 80 degrees C/5 min followed by 70 degrees C/2 h before incubation at 15 degrees, 20 degrees or 27 degrees C . Presence of specific toxin was confirmed by mouse bioassay and results were compared with those of the amplified ELISA method . A total of 48 strains, consisting of 38 Cl . botulinum and 10 Cl . sporogenes (putrefactive anaerobes), and 140 slurry samples were tested . Cultures of eight out of nine strains of type B Cl botulinum and 73 of 101 slurry samples containing type B toxin were positive by ELISA; the remaining 28 slurry samples contained type B toxin at levels below or close to the detection limit (20 LD50/ml) of the type B ELISA . No false-positive reactions occurred with Cl . botulinum types A, C, D, E or F, or with the 10 strains of Cl . sporogenes . Toxin produced by one strain of Cl . botulinum type B (NCTC 3807) was not detected by this single monoclonal antibody-based amplified ELISA . With a mixture of two monoclonal antibodies, however, the toxin from NCTC 3807 could be detected without reducing the sensitivity of the ELISA.

J Biochem (Tokyo), 1988 Apr, 103(4), 583 - 4
Rubredoxin as an intermediary electron carrier for nitrate reduction by NAD(P)H in Clostridium perfringens; Seki S et al.; The NAD(P)H-dependent nitrate reductase system in Clostridium perfringens was reconstituted with rubredoxin (Rd), nitrate reductase (NaR), and an unadsorbed fraction, on a DEAE-cellulose column, of the extract (designated as fraction A), under nitrogen gas . Ferredoxin in place of Rd was not effective as an electron carrier in this reconstituted system . NAD(P)H-dependent nitrate reducing activity was also obtained by replacing fraction A with ferredoxin-NADP+ reductase from spinach . We propose the following scheme for the electron transfer in this NAD(P)H dependent nitrate reduction system . NAD(P)H----NAD(P)H-Rd reductase----Rd----NaR----NO3-.

J Hosp Infect, 1988 Apr, 11(3), 209 - 19
Microbiological evaluation of a hospital delivered meals service using precooked chilled foods; Sandys GH et al.; A delivered meals service supplying centrally produced, precooked, chilled foods to 24 hospitals was introduced in Plymouth Health District between August 1985 and July 1986 . Over 18 months, 3393 food items were examined microbiologically, using the criteria recommended by the Department of Health and Social Security (DHSS) (1980) . No Salmonella spp., Staphylococcus aureus or Clostridium perfringens were detected . Seventy-five (8.6%) of 876 cooked vegetable items had total viable counts (TVC) greater than 1 x 10(5) cfu g-1 (the recommended limit) after aerobic incubation at 37 degrees C for 48 h, whereas only 2.6% (66) of 2517 foods other than vegetables had TVC above this limit . Reasons for high counts were investigated and mostly corrected so that, whereas 8.4% of samples had TVC above recommended limits in the first 4 months of the study, only 1.6% exceeded this limit in the last 3 months . The value of microbiological standards for cook-chill food and their use in hygiene control are assessed.

Clin Orthop, 1988 Apr, (229), 228 - 31
Hematogenously acquired infection of a total knee arthroplasty by Clostridium perfringens; Wilde AH et al.; A 64-year-old man with total knee arthroplasty for tricompartmental osteoarthritis had a postoperative course complicated by an attack of acute cholecystitis and was treated with cholecystectomy . The TKA was hematogenously seeded with Clostridium perfringens, which necessitated emergency removal of the implants, debridement, and ultimately arthrodesis.

J Bacteriol, 1988 Apr, 170(4), 1820 - 4
Light-driven amino acid uptake in Streptococcus cremoris or Clostridium acetobutylicum membrane vesicles fused with liposomes containing bacterial reaction centers; Crielaard W et al.; Reaction centers of the phototrophic bacterium Rhodopseudomonas palustris were introduced as proton motive force-generating systems in membrane vesicles of two anaerobic bacteria . Liposomes containing reaction center-light-harvesting complex I pigment protein complexes were fused with membrane vesicles of Streptococcus cremoris or Clostridium acetobutylicum by freeze-thawing and sonication . Illumination of these fused membranes resulted in the generation of a proton motive force of approximately -110 mV . The magnitude of the proton motive force in these membranes could be varied by changing the light intensity . As a result of this proton motive force, amino acid transport into the fused membranes could be observed . The initial rate of leucine transport by membrane vesicles of S . cremoris increased exponentially with the proton motive force . An H+/leucine stoichiometry of 0.8 was determined from the steady-state level of leucine accumulation and the proton motive force, and this stoichiometry was found to be independent of the magnitude of the proton motive force . These results indicate that the introduction of bacterial reaction centers in membrane vesicles by the fusion procedure yields very attractive model systems for the study of proton motive force-consuming processes in membrane vesicles of (strict) anaerobic bacteria.

Infect Immun, 1988 Apr, 56(4), 898 - 902
Establishment of a monoclonal antibody recognizing an antigenic site common to Clostridium botulinum type B, C1, D, and E toxins and tetanus toxin; Tsuzuki K et al.; The partial amino acid sequence of the light-chain (Lc) component of Clostridium botulinum type C1 toxin was determined . The sequence was quite similar to those of the other types of botulinum and tetanus toxins . Nine monoclonal antibodies against botulinum type E toxin were established by immunizing BALB/c mice with type E toxoid or its Lc component . Six antibodies reacted with the heavy-chain component and three reacted with the Lc component of the toxin . One of the latter three antibodies reacted with botulinum type B, C1, and D toxins and tetanus toxin, as well as botulinum type E toxin . This antibody recognized the Lc components of these toxins, indicating that there exists one common antigenic determinant on the Lc regions of these toxins.

Biochem Biophys Res Commun, 1988 Mar 30, 151(3), 1371 - 7
The effect of a Clostridium perfringens 8-6 enterotoxin on viability and macromolecular synthesis in Vero cells; Lindsay JA; Clostridium perfringens type A 8-6 enterotoxin causes gross morphological damage to Vero cells grown in tissue culture . Damage was observed to occur after only 30 minutes exposure at concentrations as low as 20 ng/ml, and within 60 minutes 95% of the cells had detached . Concentrations as low as 0.01 ng were able to cause detectable inhibition of plating efficiency . The enterotoxin inhibited DNA, RNA and protein synthesis and caused the reversal of glucose transport . Heat inactivated enterotoxin had no effect on cell function or morphology.

Biochem J, 1988 Mar 15, 250(3), 813 - 8
Purification and some properties of the extracellular alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum; Melasniemi H; The novel alpha-amylase-pullulanase produced by Clostridium thermohydrosulfuricum E 101-69 was purified as two forms (I and II) from culture medium, by using gel filtration in 6 M-guanidine hydrochloride as the final step . Renatured alpha-amylase-pullulanase I and II had apparent Mr values of 370,000 +/- 85,000 and 330,000 +/- 85,000 respectively, as determined by native polyacrylamide-gradient-gel electrophoresis . Both forms appear to be dimers of two similar subunits, with Mr values of 190,000 +/- 30,000 for enzyme I and 180,000 +/- 30,000 for enzyme II according to SDS/polyacrylamide-gradient-gel electrophoresis . The two forms had similar amino acid compositions, the same N-terminal sequence (Glu-Ile-Asp-Thr-Ala-Pro-Ala-Ile) and the same pI of 4.25 . Both forms contained sugars having mobilities identical with those of rhamnose, glucose, galactose and mannose . The amount of neutral hexoses relative to protein was 11-12% (w/w) for both forms.

Eur J Biochem, 1988 Mar 15, 172(3), 647 - 52
Attachment of human placental-type alkaline phosphatase via phosphatidylinositol to syncytiotrophoblast and tumour cell plasma membranes; Webb PD et al.; Phosphatidylinositol anchors human placental-type alkaline phosphatase (PLAP) to both syncytiotrophoblast and tumour cell plasma membranes . PLAP activity was released from isolated human placental syncytiotrophoblast plasma membranes and the surface of tumour cells with a phospholipase C from Bacillus cereus . This was a specific event, not the result of proteolysis or membrane perturbation, but the action of a phosphatidylinositol-specific phospholipase C in the preparation . Soluble PLAP, released with B . cereus phospholipase C and purified by immunoaffinity chromatography, ran on SDS-PAGE as a 66-kDa band . This corresponded to intact PLAP molecules . The protease bromelain cleaved lower-molecular-mass PLAP (64 kDa) from the membranes . Flow cytometry demonstrated that B . cereus phospholipase C released human tumour cell membrane PLAP in preference to other cell-surface molecules . This was in contrast to the non-specific proteolytic action of bromelain or Clostridium perfringens phospholipase C, which had no effect on membrane PLAP expression . Radiolabelling of tumour cells with fatty acids indicated PLAP to be labelled with both {3H}myristic and {3H}palmitic acid . This fatty-acid--PLAP bond was sensitive to pH 10 hydroxylamine treatment indicating an O-ester linkage.

Eur J Biochem, 1988 Mar 15, 172(3), 761 - 6
New thiol inhibitors of Clostridium histolyticum collagenase . Importance of the P3' position; Yiotakis A et al.; An extensive series of synthetic mercaptotripeptides (HS-CH2-CH2-CO-Pro-Yaa) was prepared, and the inhibitory constants were determined on the Clostridium histolyticum collagenase . Among the factors which control the optimal binding of these inhibitors, we found that the presence of a free C-terminal carboxylate group in the position P3' of the compounds is of primary importance . In general, the esterification of this carboxylate group decreased the potency of the inhibitors by two orders of magnitude . We observed also that the enzyme favored the inhibitors having a long linear apolar or basic side-chain at the position P3' . The present data suggest a large S3' subsite of the C . histolyticum collagenase . The compound which contains a homoarginine residue at the P3' position with a Ki of 0.2 microM proved to be the most potent synthetic inhibitor known to date for the C . histolyticum collagenase.

Biochemistry, 1988 Mar 8, 27(5), 1636 - 42
Isolation and characterization of rubrerythrin, a non-heme iron protein from Desulfovibrio vulgaris that contains rubredoxin centers and a hemerythrin-like binuclear iron cluster; LeGall J et al.; A new non-heme iron protein from the periplasmic fraction of Desulfovibrio vulgaris (Hildenbourough NCIB 8303) has been purified to homogeneity, and its amino acid composition, molecular weight, redox potential, iron content, and optical, EPR, and Mossbauer spectroscopic properties have been determined . This new protein is composed of two identical subunits with subunit molecular weight of 21,900 and contains four iron atoms per molecule . The as-purified oxidized protein exhibits an optical spectrum with absorption maxima at 492, 365, and 280 nm, and its EPR spectrum shows resonances at g = 4.3 and 9.4, characteristic of oxidized rubredoxin . The Mossbauer data indicate the presence of approximately equal amounts of two types of iron; we named them the Rd-like and the Hr-like iron due to their similarity to the iron centers of rubredoxins (Rds) and hemerythrins (Hrs), respectively . For the Rd-like iron, the measured fine and hyperfine parameters (D = 1.5 cm-1, E/D = 0.26, delta EQ = -0.55 mm/s, delta = 0.27 mm/s, Axx/gn beta n = -16.5 T, Ayy/gn beta n = -15.6 T, and Azz/gn beta n = -17.0 T) are almost identical with those obtained for the rubredoxin from Clostridium pasteurianum . Redox-titration studies monitored by EPR, however, showed that these Rd-like centers have a midpoint redox potential of +230 +/- 10 mV, approximately 250 mV more positive than those reported for rubredoxins . Another unusual feature of this protein is the presence of the Hr-like iron atoms.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1988 Mar 5, 263(7), 3142 - 9
Intracellular processing of cytidylyltransferase in Krebs II cells during stimulation of phosphatidylcholine synthesis . Evidence that a plasma membrane modification promotes enzyme translocation specifically to the endoplasmic reticulum; Terce F et al.; After a 3-h incubation of Krebs II ascitic cells in the presence of phospholipase C from Clostridium welchii under nonlytic conditions, the incorporation of {3H} choline into phosphatidylcholine was increased 1.7-fold as compared to untreated cells . The total amounts of phosphatidylcholine, phosphatidylethanolamine, and sphingomyelin were unchanged up to 3 h of incubation . The limiting step in phosphatidylcholine biosynthesis was the formation of CDP-choline catalyzed by CTP:choline-phosphate cytidylyltransferase (EC 2.7.7.15) as monitored by the decrease in phosphocholine labeling following phospholipase C treatment of cells prelabeled with {3H}choline . The specific activity of homogenate cytidylyltransferase was increased about 1.6-fold in phospholipase C-treated cells . Specific activity of the membrane fraction was increased 2-fold, whereas cytosolic specific activity decreased in phospholipase C-treated cells . The activation of cytidylyltransferase was concomitant with translocation of the enzyme from the cytosol to the membrane fraction . The latter was further fractionated using a Percoll gradient that allowed an efficient separation between endoplasmic reticulum and other subcellular membranes . In control cells, particulate cytidylyltransferase activity co-migrated with the endoplasmic reticulum and ribosome markers and not with the plasma membrane . Also, in treated cells, the stimulation of cytidylyltransferase activity occurred at the endoplasmic reticulum level and did not involve either the external cell membrane or other cellular organelles including the Golgi apparatus, lysosomes, or mitochondria . Thus, our results demonstrate that a stimulus acting on the plasma membrane promotes the translocation of the soluble form of cytidylyltransferase specifically to the endoplasmic reticulum.

Immunobiology, 1988 Mar, 176(4-5), 394 - 409
The second component of human complement: use of glycosidases and glucosylation to distinguish the two forms; Schultz DR et al.; The two forms of human plasma C2 that were described in the preceding report (1) were investigated for their functional and biochemical differences . Incubation with the neuraminidase (NAN'dase) of Clostridium perfringens at 37 degrees C resulted in a four- to fivefold increase in the hemolytic activity of both forms . The increase in activity was different than the increase caused by treatment with iodine . The mechanism of increased activity of NAN'dase-treated C2 was the generation of increased molecules of activated C3 (C3b), resulting in more molecules of C5 binding to (C4b, 2a, 3b)n . Removal of N-acetyl-neuraminate from C2 did not alter its binding to a cationic exchanger . Nonenzymatic glucosylation was used to distinguish the two forms of C2 . Incubation of highly pure C2 with 14C-D-glucose resulted in the gradual accumulation of radioactivity in acid-precipitable material . The two forms of C2 were glucosylated in vitro for seven days with 14C-D-glucose in phosphate-buffered saline at 25 degrees C . Form 2 bound twice as much 14C-D-glucose as form 1 . Glucosylated form 2, but not form 1, lost some of its affinity to bind to a cationic exchanger . Since the interaction between glucose and protein occurs at free amino groups, we conclude that form 2 of C2 has approximately twice as many free amino groups as form 1 . This explains the reason for the existence of two forms of C2 in plasma independent of the allelic variant.

Res Vet Sci, 1988 Mar, 44(2), 270 - 1
Detection of Clostridium perfringens beta toxin by enzyme-linked immunosorbent assay; Martin PK et al.; An enzyme-linked immunosorbent assay (ELISA) for the detection of Clostridium perfringens beta toxin in intestinal contents has been developed by a modification of the method reported for epsilon toxin . Although the test results for beta and epsilon toxins cannot be directly compared, lower levels of beta toxin were generally demonstrated in the samples examined . The use of the ELISA for beta toxin in conjunction with that for epsilon toxin allows the differential diagnosis of C perfringens type B, C and D enterotoxaemias in the laboratory.

Infection, 1988 Mar-Apr, 16(2), 131 - 4
In vitro activity of flomoxef in comparison to other cephalosporins; Simon C et al.; Flomoxef and cefazolin had nearly the same activity against staphylococci, which was stronger than that of other cephalosporins . Against Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus pneumoniae, cefotaxime and cefazolin were more active than flomoxef, but the other cephamycins were less active than flomoxef . In comparison to the other cephalosporins, latamoxef and flomoxef had higher activity against Branhamella catarrhalis, whereas cefotaxime, latamoxef and cefotetan were more active against Haemophilus influenzae . Flomoxef was the only drug exhibiting activity against Clostridium difficile . The activity of flomoxef and latamoxef against Bacteroides fragilis was stronger than that of the other cephalosporins, but Bacteroides bivius was resistant to each of these antibiotics.

J Clin Microbiol, 1988 Mar, 26(3), 540 - 3
Electrophoretic characterization of Clostridium difficile strains isolated from antibiotic-associated colitis and other conditions; Pantosti A et al.; Clostridium difficile has been recognized as the cause of antibiotic-associated pseudomembranous colitis and of less severe diarrheal diseases associated with the use of antimicrobial agents . However, healthy carriers of this microorganism have been found, particularly healthy neonates and small children . Various typing systems have been used to clarify the epidemiology of C . difficile . We used the electrophoretic patterns of EDTA-extracted proteins to characterize C . difficile strains from various sources . Altogether, 110 strains were studied, including 2 reference strains, and 21 different protein profiles were obtained . However, two patterns were the most common: the group 2 pattern, characterized by a major 35-kilodalton polypeptide band, and the group 5 pattern, identified by principal bands of 37 and 56 kilodaltons . The group 2 pattern was characteristic of strains isolated during hospital outbreaks and from sporadic cases of pseudomembranous colitis and antibiotic-associated diarrhea . The group 5 pattern was obtained only from isolates from healthy neonates and children . A correlation between electrophoretic characteristics and virulence can be hypothesized, namely that group 2 strains are more prone to induce diseases and cause outbreaks . It is noteworthy that strains isolated from children with diarrhea of unknown etiology, not related to antibiotic use, belong to the "virulent" group 2; strains from leukemic patients showed a variety of different patterns, and only two belong to group 2 . This characterization can be used to aid studies on the virulence and clinical significance of C . difficile.

Eur J Biochem, 1988 Mar 1, 172(2), 459 - 64
Diversity of corrinoids in acetogenic bacteria . P-cresolylcobamide from Sporomusa ovata, 5-methoxy-6-methylbenzimidazolylcobamide from Clostridium formicoaceticum and vitamin B12 from Acetobacterium woodii; Stupperich E et al.; The Co beta-cyanocobamides obtained by cyanide extractions from several acetogenic bacteria were structurally characterized by ultraviolet/visible spectra, proton-nuclear-magnetic-resonance spectra and fast-atom-bombardment mass spectra . p-Cresolycobamide was detected as a major corrinoid from Sporomusa ovata . This 'complete' corrinoid was isolated from an organism for the first time . Instead of the common Co alpha bases of the known and biologically active cobamides, p-cresolylcobamide contained a glycosidically bound cresolyl function that was unable to coordinate to the cobalt of the corrin ring . An additional, previously unknown corrinoid from natural sources, Co alpha-{alpha-(5-methoxy-6-methylbenzimidazolyl)}-Co beta-cyanocobamide, was isolated along with vitamin B12 from Clostridium formicoaceticum . Both homoacetogenic eubacteria were grown on methanol and contained high amounts of corrinoids (greater than 950 nmol/g cell dry mass) . Less corrinoid was isolated from Acetobacterium woodii and characterized as vitamin B12.

J Med Microbiol, 1988 Mar, 25(3), 191 - 6
Mucosal association by Clostridium difficile in the hamster gastrointestinal tract; Borriello SP et al.; For many organisms, mucosal association is an important virulence determinant . Although studied in detail for other intestinal pathogens, this aspect of pathogenicity has not been studied for Clostridium difficile . We compared the ability of an avirulent non-toxigenic strain (M-1), a highly virulent toxigenic strain (B-1), and a poorly virulent toxigenic strain (BAT) of C . difficile to adhere to different regions of the gastrointestinal tract of hamsters pre-treated with clindamycin . Strain B-1 associated with the gut mucosa significantly better than strain M-1 (p less than 0.001) for all sites other than the caecum, and achieved significantly higher levels in the caecal contents (p less than 0.001) . The same was true when strain B-1 was compared with strain BAT except that there was no significant difference for the large bowel mucosa . To assess the possible role of toxin in promoting mucosal association, e.g., by compromising host defences or exposing masked adherence sites, strain M-1 was given to animals after intra-caecal administration of crude toxin preparations from strain-B1, which were heat-inactivated in control experiments . The addition of this toxin increased significantly the mucosal association of M-1 for the small bowel only, whereas the inactivated toxin had no significant effect . These results imply that there may be intrinsic differences between strains in their ability to colonise and associate with the gut mucosa, which may partly depend on their ability to produce toxin . These differences do not correlate with cell-surface hydrophobicity or the presence of plasmids, flagella or fimbriae.

Am J Gastroenterol, 1988 Mar, 83(3), 304 - 7
Absence of diarrhea in toxic megacolon complicating Clostridium difficile pseudomembranous colitis; Burke GW et al.; We describe a patient with Clostridium difficile-associated pseudomembranous colitis who presented with toxic megacolon without diarrhea . The discussion includes a brief review of the literature, and suggests an important role for endoscopy in the diagnosis of pseudomembranous colitis and, possibly, as part of the therapy for toxic megacolon associated with Clostridium difficile colitis . The unusual combination of toxic megacolon without antecedent diarrhea should be recognized as a possible manifestation of antibiotic-associated pseudomembranous colitis, especially in the setting of simultaneous antimicrobial and opiate administration . Early diagnosis and disease-specific intervention can be lifesaving.

Surg Gynecol Obstet, 1988 Mar, 166(3), 197 - 9
Diffuse spreading Clostridium septicum infection, malignant disease and immune suppression; Bretzke ML et al.; Nineteen patients have been treated with gas gangrene infection proved on cultures to be caused by Clostridium septicum . Infection occurred after trauma in three patients and occurred spontaneously in 16, including six patients with peritonitis . None of the instances occurred as a postoperative infection . Ten of the patients had an associated malignant disease including carcinoma of the colon and rectum in six, hematologic malignant conditions in three and carcinoma of the gallbladder in one . Of the remaining six patients, four had evidence of immune suppression (two had leukopenia, one was preleukemic and one had undergone a postcadaver renal transplant), one had diabetes and one had overwhelming sepsis due to a perforated gallbladder with peritonitis . Only three of the 19 patients were otherwise healthy, and infection occurred after trauma in all of these patients . Four patients were moribund upon admission and died before initial therapy was completed . In the other 15 patients, treatment consisted of administration of penicillins and broad spectrum antibiotics and debridement, as well as hyperbaric oxygen in 13 patients . Thirteen of 19 patients died and eight of ten with malignant disease died . The data show a striking relationship between Clostridium septicum and both malignant disease and immune suppression.

Infect Immun, 1988 Mar, 56(3), 582 - 8
Effects of Clostridium difficile toxins A and B in rabbit small and large intestine in vivo and on cultured cells in vitro; Lima AA et al.; Clostridium difficile is recognized as the major cause of antibiotic-associated colitis . C . difficile produces two toxins, A (enterotoxin) and B (cytotoxin), that are implicated in the pathogenesis of the colitis . We examined the dose responses, time course, and synergism of these two toxins in ligated rabbit intestinal loops and in tissue culture . In rabbit small intestinal loops, toxin A caused histologically demonstrable intestinal tissue damage as early as 2 h . The secretory response greater than or equal to 8 h was similar to that of a cholera toxin control . The effect of toxin A on tissue damage or secretion was seen even if toxin was removed after 5 min . Purified toxin A caused significant net accumulation of sodium, chloride, potassium, and total protein and slightly increased osmolality of the fluid content at 6 h; these effects were similar to those caused by crude C . difficile culture filtrates containing toxins A and B . Crude C . difficile toxin caused fluid accumulation with a delayed time course in the rabbit large intestine, and in contrast to its effect in small intestine, crude toxin caused net accumulation of bicarbonate and increased pH . In tissue culture, toxin A caused a rounding up of CHO and T-84 colonic carcinoma cells . A monoclonal antibody (PCG-4) that has no effect on tissue culture cytotoxicity with toxins A and B completely inhibited the secretory and tissue-damaging effects in the intestine . Toxins A and B were synergistic in the gut only at high doses of toxin B (greater than or equal to 10 micrograms/ml), and they were additive in tissue culture . The cytopathic effect in tissue culture was not consistently associated with trypan blue uptake . The cytopathic effect of toxin A in tissue culture did not appear to involve inhibitable Ca2+-dependent or prostaglandin synthesis pathways or intact microfilament or microtubule function for its activity and was not inhibited by reducing or lysosomotropic agents . Our results suggest that toxins A and B have independent and distinct effects in vivo and in vitro.

Arch Surg, 1988 Mar, 123(3), 377 - 9
Emphysematous pyelonephritis in a xanthogranulomatous kidney . An unusual cause of pneumoperitoneum; Langdale LA et al.; Emphysematous pyelonephritis is a rare, life-threatening suppurative infection of the renal parenchyma and perinephric tissues . The disease is encountered primarily in patients with diabetes mellitus or ureteral obstruction associated with perinephric and intrarenal gas . Causative organisms are those normally found in the urinary and gastrointestinal tracts; however, anaerobic bacteria have been demonstrated in only 1% of cases . We describe a case of emphysematous pyelonephritis, which presented as an acute abdomen with pneumoperitoneum in a nondiabetic patient . No visceral injury was found at laparotomy . Multiple gas-producing organisms, including Clostridium ramosum (not previously reported, to our knowledge), were the cause of the free intraperitoneal and perinephric air . Subsequent radical nephrectomy revealed a xanthogranulomatous kidney . An aggressive surgical approach combined with intensive antibiotic therapy, after aerobic and anaerobic culture of excised tissue, is lifesaving.

J Appl Bacteriol, 1988 Mar, 64(3), 241 - 6
Growth and formation of toxin by Clostridium botulinum in peeled, inoculated, vacuum-packed potatoes after a double pasteurization and storage at 25 degrees C; Lund BM et al.; A process that claims to use a double pasteurization to produce vacuum-packed potatoes for storage at ambient temperature has been evaluated . After the first pasteurization, potatoes are vacuum-packed and stored at 25 degrees-35 degrees C for up to 24 h, which is intended to allow germination of bacterial spores, and are then pasteurized again . When potatoes were inoculated with spores of Clostridium botulinum and subjected to this double-pasteurization process a high proportion of spores remained viable and resulted in growth and formation of toxin within 5-9 d at 25 degrees C . To provide an appropriate reduction in the risk o survival and growth of Cl . botulinum, peeled, vacuum-packed potatoes for storage at ambient temperature should be given a heat treatment equivalent to an F(0)3 process . If they are not given such a heat treatment they should be stored at a temperature below 4 degrees C.

Appl Environ Microbiol, 1988 Mar, 54(3), 753 - 9
Development of improved defined media for Clostridium botulinum serotypes A, B, and E; Whitmer ME et al.; The minimal nutritional growth requirements were determined for strains Okra B and Iwanai E, which are representatives of groups I and II, respectively, of Clostridium botulinum . These type B and E strains differed considerably in their nutrient requirements . The organic growth factors required in high concentrations by the Okra B strain (group I) were arginine and phenylalanine . Low concentrations (less than or equal to 0.1 g/liter) of eight amino acids (methionine, leucine, valine, isoleucine, glycine, histidine, tryptophan, and tyrosine) and of five vitamins (pyridoxamine, p-aminobenzoic acid, biotin, nicotinic acid, and thiamine) were also essential for biosynthesis . The 10 required amino acids could be replaced by intact protein of known composition by virtue of the bacterium's ability to synthesize proteases . Glucose or other carbohydrates were not essential for Okra B, although they did stimulate growth . Quantitatively, the most essential nutrients for Okra B were arginine and phenylalanine . In contrast, the nonproteolytic strain, Iwanai E (group II), did not require either arginine or phenylalanine . It required glucose or another carbohydrate energy source for growth and did not utilize arginine or intact protein as a substitute source of energy . Iwanai E utilized ammonia as a nitrogen source, although growth was stimulated significantly by organic nitrogenous nutrients, especially glutamate and asparagine . Iwanai E also required biosynthesis levels of seven amino acids (histidine, isoleucine, leucine, tryptophan, tyrosine, valine, and serine), adenine, and six vitamins (biotin, thiamine, pyridoxamine, folic acid, choline, and nicotinamide) . Calcium pantothenate also stimulated growth . On the basis of the nutritional requirements, chemically defined minimal media have been constructed for C . botulinum serotypes A, B, E, and F (proteolytic).(ABSTRACT TRUNCATED AT 250 WORDS)

Am J Obstet Gynecol, 1988 Mar, 158(3 Pt 2), 728 - 35
Comparative study of cefotetan and cefoxitin in the treatment of intra-abdominal infections; Lewis RT et al.; One hundred eighty-eight patients were enrolled in a multicenter, randomized clinical trial to compare the safety and effectiveness of 1 to 2 gm cefotetan every 12 hours with those of 1 to 2 gm cefoxitin every 6 hours in patients with intra-abdominal infections . Most of the infections were community acquired, were associated with gastrointestinal tract perforation, and were caused by both anaerobic and aerobic bacteria . The median duration of therapy was 6 days for each group . The clinical response rate for the 95 evaluable patients in the cefotetan group was 98%, and that for the 43 evaluable patients in the cefoxitin group was 95% . Bacteriologically, 97% of the 58 evaluable patients in the cefotetan group and 89% of the 27 evaluable patients in the cefoxitin group had a satisfactory or presumed satisfactory response; two patients in the cefotetan group and three in the cefoxitin group were considered bacteriologic failures . Cefotetan was as effective as cefoxitin in eradicating Bacteroides fragilis and other species of Bacteroides, Clostridium sp., and gram-negative bacilli . The incidence of treatment-related adverse reactions for cefotetan (27%) was not statistically different from that for cefoxitin (17%) . No clinically significant differences were detected between the treatment groups in changes in the results of clinical laboratory tests performed before and after treatment; a decrease in hematocrit among the cefotetan group was statistically greater (p = 0.04) than that for the cefoxitin group, and a decrease in serum creatinine level for the cefoxitin group was greater than that for the cefotetan group (p = 0.02) . Cefotetan may represent an effective, safe, and cost-saving alternative to cefoxitin for the prompt treatment of community-acquired intra-abdominal infections.

Arch Surg, 1988 Mar, 123(3), 322 - 6
Prospective study comparing imipenem-cilastatin with clindamycin and gentamicin for the treatment of serious surgical infections; Hackford AW et al.; Surgical infection remains a leading cause of hospital morbidity and mortality . We compared the efficacy and toxicity of imipenem-cilastatin sodium in 32 patients with that of clindamycin phosphate and gentamicin sulfate in 25 patients . In the imipenem-cilastatin group, 87.5% had a favorable outcome, with a 12.5% failure rate and 13 adverse reactions . In the clindamycin-gentamicin group, 80% had a favorable outcome, with a 20% failure rate and ten adverse reactions . Two significant superinfections with Pseudomonas and Candida were noted in patients treated with impenem-cilastatin . Each group had one case of Clostridium difficile-associated colitis . Cost analysis showed no differences between treatment arms, except in the appendicitis subgroup . For serious surgical infections, single-agent therapy with imipenem-cilastatin appears to be as efficacious as combination therapy with clindamycin and gentamicin.

J Rheumatol, 1988 Mar, 15(3), 520 - 2
Reactive arthritis associated with Clostridium difficile enteritis; Atkinson MH et al.; Reactive arthritis of one or more peripheral joints developed after an enteric infection with Clostridium difficile in 2 adult patients . Other reactive signs such as conjunctivitis, mucous membrane lesions and urethritis were absent . One patient had the HLA-B27 antigen . Short term followup showed a benign course of the arthritis . Three other cases of arthritis associated with C . difficile were reviewed.

J Biochem (Tokyo), 1988 Mar, 103(3), 504 - 7
Purification and properties of 3 alpha-hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum from human intestine; Akao T et al.; 3 alpha-Hydroxyglycyrrhetinate dehydrogenase of Clostridium innocuum, isolated from human intestinal bacteria, was capable of converting 3-ketoglycyrrhetic acid to 3 alpha-hydroxyglycyrrhetic acid . The enzyme was purified to homogeneity by means of butyl-Toyopearl 650M, Sephadex G-150, Matrex Red A, Toyopearl HW-55S, and isoelectric focusing column chromatographies . The purified enzyme showed a specific activity of 156 mumol/min.mg toward 3 alpha-hydroxyglycyrrhetic acid, and showed a single band on SDS-polyacrylamide gel electrophoresis . The apparent molecular weight was 53,000, as estimated by gel filtration, and 30,000, as judged by SDS-polyacrylamide gel electrophoresis . Its isoelectric point was 5.2 . The enzyme showed absolute specificity for the 3 alpha-hydroxyl and 3-ketonic groups of 18 alpha- or 18 beta-glycyrrhetic acid and required NADP+ and NADPH as cosubstrates . The enzyme did not act on any 3 alpha-hydroxyl or 3-ketonic group of steroids or bile acids . The enzyme is a novel type of enzyme, defined as 3 alpha-hydroxy-glycyrrhetinate dehydrogenase, being quite different from 3 alpha-hydroxysteroid dehydrogenase {EC 1.1.1.50}.

Am J Clin Pathol, 1988 Mar, 89(3), 407 - 9
Acute ileotyphlitis as presenting manifestation of acute myelogenous leukemia; Ahsan N et al.; A case of acute myelogenous leukemia is reported in a child who presented with acute ileotyphlitis and died of an over-whelming Clostridium septicum sepsis before the chemotherapy was administered . The pathogenesis of acute ileotyphlitis, especially the role of granulocytopenia is discussed.

J Clin Microbiol, 1988 Mar, 26(3), 397 - 400
Characterization of cross-reactive proteins detected by Culturette Brand Rapid Latex Test for Clostridium difficile; Lyerly DM et al.; Clostridium sporogenes, Peptostreptococcus anaerobius, and Bacteroides asaccharolyticus have been reported to react in the Culturette Brand Rapid Latex Test (Marion Scientific, Div . Marion Laboratories, Inc., Kansas City, Mo.) for Clostridium difficile . From the results of this study we showed that C . sporogenes and P . anaerobius produce a protein which is very similar biochemically and immunologically to the protein of C . difficile that is detected by the test . Thus, the positive latex reactions observed with C . sporogenes and P . anaerobius are due to a cross-reactive protein . We did not detect this cross-reactive protein in filtrates from B . asaccharolyticus, indicating that this bacterium reacts with the latex reagent by some other mechanism . We cloned the C . difficile gene that codes for the cross-reactive protein and showed that the protein produced by the recombinant organism is nontoxic and distinct from toxin A, thus confirming our earlier findings.

Eur J Biochem, 1988 Mar 1, 172(2), 445 - 50
Botulinum ADP-ribosyltransferase C3 . Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes; Aktories K et al.; A novel ADP-ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion-exchange chromatography and heat treatment . The molecular mass of botulinum ADP-ribosyltransferase C3 was found to be 25 kDa . In the presence of {32P}NAD but not with {carbonyl-14C}NAD, C3 labelled 21-24-kDa protein(s) in membranes of human platelets and other tissues . The Km value of the ADP-ribosylation reaction for NAD was about 2 microM . Labelling of the 21-24-kDa protein(s) by C3 was largely reduced by addition of nicotinamide . Snake venom phosphodiesterase cleaved the ADP-ribose attached to the 21-24-kDa protein(s) by C3 and released 5'AMP . C3 catalyzed hydrolysis of {carbonyl-14C}NAD and released {carbonyl-14C}nicotinamide . ADP-ribosylation of 21-24-kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+ . In the absence of free divalent cations GTP, GTP{gamma S} and GDP but not GDP{beta S}, GMP, ATP or ATP{gamma S} increased labelling by C3 . In the presence of Mg2+, GTP{gamma S} was inhibitory . Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP{gamma S} = GTP = GDP greater than GDP{beta S} greater than GMP much greater than ATP = GMP = ATP{gamma S} . The data support the view that the novel ADP-ribosyltransferase C3 modifies eukaryotic 21-24-kDa GTP-binding protein(s).

Aust Vet J, 1988 Mar, 65(3), 78 - 80
A Clostridium botulinum type B vaccine for prevention of shaker foal syndrome; Thomas RJ et al.; A toxoid was prepared from type B toxin of Clostridium botulinum by treatment with 0.6% formalin for 6 weeks . The toxoid was adsorbed to aluminium hydroxide and this vaccine was evaluated for safety in guinea pigs, mice and horses, and for immunogenicity in guinea pigs and horses . Neutralising antitoxin was demonstrated in adult horses receiving two 2 ml subcutaneous doses 6 weeks apart, and in a foal which suckled its vaccinated dam . Another vaccinated mare and the passively immunised foal were protected against subcutaneous injection of 1600 and 2000 mouse lethal doses of toxin per kg respectively.

Plasmid, 1988 Mar, 19(2), 164 - 8
The Clostridium perfringens chloramphenicol resistance transposon Tn4451 excises precisely in Escherichia coli; Abraham LJ et al.; Nucleotide sequence analysis of the Tn4451-deletion derivatives, pJIR47 and pJIR86, which were derived from Escherichia coli and Clostridium perfringens, respectively, showed that the deletion events that led to the formation of these plasmids were identical and precise . The results also showed that the termini of this C . perfringens-derived transposon contained imperfect 12-bp inverted repeat sequences which had some sequence similarity with the termini of Tn3-like transposons.

Plasmid, 1988 Mar, 19(2), 151 - 60
Identification and molecular genetic analysis of replication functions of the bacteriocinogenic plasmid pIP404 from Clostridium perfringens; Garnier T et al.; The replication functions of the bacteriocinogenic plasmid pIP404, from Clostridium perfringens, were localized to a 2.8-kb EcoRI-EcoRV fragment by cloning into a vector deficient for replication in Bacillus subtilis . This fragment contains two genes, cop and rep, which encode proteins and an 800-bp noncoding segment of complex structure consisting of multiple tandemly repeated sequences . The Cop protein is involved in copy number control, whereas the rep gene product is essential for plasmid replication . By deletion analysis the minimal origin of replication was defined as the rep gene plus most of the repeated sequences . A powerful promoter producing a 150-nucleotide RNA molecule, RNA1, that could act as an anti-sense RNA to the rep gene was detected in the "origin-like" region . In contrast to most other small plasmids of gram-positive bacteria, pIP404, and its derivatives, does not appear to replicate via a single stranded intermediate in either C . perfringens or B . subtilis.

Plasmid, 1988 Mar, 19(2), 134 - 50
Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens; Garnier T et al.; The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined . The plasmid genome comprises 10,207 bp and has a dA + dT content of 75% . Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified . The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content . Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.

Plasmid, 1988 Mar, 19(2), 113 - 20
Hybridization analysis of the class P tetracycline resistance determinant from the Clostridium perfringens R-plasmid, pCW3; Abraham LJ et al.; The tetracycline resistance determinant from pCW3, a conjugative plasmid from Clostridium perfringens, has been identified and the structural gene localized to within a 1.4-kb region . Hybridization analysis, which utilized an internal 0.8-kb specific gene probe, showed that eight nonconjugative tetracycline resistant C . perfringens strains all carried homologous resistance determinants . No homology was detected in DNA prepared from tetracycline resistant isolates of Clostridium difficile or Clostridium sporogenes . However, the one strain of Clostridium paraputrificum that was tested did contain an homologous determinant . No homology was found to any of the recognized classes of tetracycline resistance determinants . The C . perfringens tetracycline resistance determinant represents a new hybridization group, Class P.

Onderstepoort J Vet Res, 1988 Mar, 55(1), 47 - 50
The passive protection of lambs against Clostridium perfringens type D with semi-purified hyperimmune serum; Odendaal MW et al.; Weaned lambs, having a detectable level of maternal antibodies (1-2 units/ml) against C . perfringens type D, showed protective antitoxin levels lasting for 29 days after receiving a single parenteral dose of 200 units/kg hyperimmune serum . Lambs, having no maternal antibodies (less than 0,07 units/ml) to C . perfringens type D but receiving the same dose of hyperimmune serum, maintained protective antibody levels for only 21 days . Three weeks after the titres fell below the minimum protective level of 0,15 units/ml, both these groups were treated again in the same manner . The passive immunity conferred in both groups now lasted for 42 days . When the hyperimmune serum was administered to lambs already immunized by vaccination, a slight increase was noted in the antibody titre.

Am J Epidemiol, 1988 Mar, 127(3), 605 - 11
A large Clostridium perfringens foodborne outbreak with an unusual attack rate pattern; Petersen LR et al.; On November 7, 1985, a Clostridium perfringens gastroenteritis outbreak occurred in approximately 44% of the 1,362 employees at a Connecticut factory . Although the same foods were served to all three shifts at an employee banquet on November 6, the attack rate was almost twice as high for those who ate on the day shift (attack rate = 50%) than for those on the evening shift (attack rate = 20%) or night shift (attack rate = 29%) . Among employees of the day shift, attack rates were highest for those who ate during the first 30 minutes of the 2.5-hour day shift serving period and decreased throughout the serving period . The one-hour evening shift serving period had a similar trend toward higher attack rates earlier in the serving period . Four main-course foods were significantly associated with illness, and over 95% of the employees had eaten each of them . Stratified analysis indicated that gravy was the responsible food and, furthermore, that the decreasing attack rate pattern within serving periods occurred only for those who ate gravy . The gravy had been prepared 12-24 hours in advance of banquet service . After it was prepared, the gravy was improperly cooled and was reheated shortly before and throughout the serving periods . Persons who ate gravy that had been reheated for the longest period of time had the lowest attack rate, probably because they were exposed to a lower concentration of organisms . This outbreak underscores the need for properly reheating food to prevent C . perfringens gastroenteritis and suggests that analysis of attack rate trends may provide important epidemiologic clues to understanding the causes of foodborne disease outbreaks.

J Clin Microbiol, 1988 Mar, 26(3), 426 - 8
Serotyping of Clostridium difficile; Toma S et al.; A total of 246 live Clostridium difficile cultures were serotyped by a slide agglutination technique . Fifteen grouping antisera were produced which serotyped 98% of the cultures (241 of 246) . Our results indicated that certain serogroups may have specific pathogenicity . Strains of serogroups A, G, H, K, S1, and S4 were cytotoxigenic and were isolated mainly from adult patients with pseudomembranous colitis or antibiotic-associated diarrhea . Nontoxigenic strains of serogroups D and Cd-5 were isolated mainly from asymptomatic neonates and small children . Some cross-reactions occurred among some strains of serogroups A, Cd-5, G, and K . These strains were further examined by analysis of protein profiles and restriction endonuclease patterns to elucidate their serology . Typing of C . difficile by using slide agglutination is a simple technique suitable for routine examination . Serogrouping may be a useful epidemiological marker and could help in elucidating the medical relevance of some C . difficile isolates.

J Neurochem, 1988 Mar, 50(3), 722 - 9
Release of proteins from the surface of bovine central nervous system myelin by salts and phospholipases; Smith R et al.; Incubation of bovine CNS myelin with phospholipase C from Bacillus cereus under conditions that lead to extensive phospholipid degradation caused 10% of the myelin protein to be released from the membrane . The myelin basic protein (MBP) was a major component of the dissolved protein . Comparable incubations with phospholipase C from Clostridium perfringens, phosphatidylinositol-specific phospholipase C from Staphylococcus aureus, or cabbage phospholipase D removed little MBP . However, concentrations of sodium chloride near 1 M and concentrations of divalent metal ions between 50 and 600 mM released typically 9-12% of the total myelin protein, with MBP again as the predominant component . Repetitive washing with calcium chloride solutions resulted in dissolution of over 90% of the MBP . When myelin was incubated in 1.0 M sodium chloride or 25 mM calcium chloride, the MBP was cleaved largely into two major peptides with apparent molecular weights near 14,000 and 12,000, but with 200 mM or higher concentrations of calcium chloride most of this protein remained intact . With appropriate manipulation of the ionic milieu, it is thus possible to remove most of this extrinsic protein from the myelin surface under relatively mild conditions . The conditions that release the protein suggest that it is held at the membrane surface by ionic interactions.

Biochem Biophys Res Commun, 1988 Feb 15, 150(3), 1122 - 30
Subcellular distribution and isoelectric heterogeneity of the substrate for ADP-ribosyl transferase from Clostridium botulinum; Narumiya S et al.; When the homogenate of bovine adrenal gland was subjected to subcellular fractionation, an Mr 21,000 substrate for botulinum ADP-ribosyl transferase was found not only in the membrane fractions but also in the cytosol; the amounts in the 10,000 x g precipitates and the 100,000 x g supernatant were about 21 and 56% of the total amount, respectively . Each fraction gave a single ADP-ribosylated protein band on SDS-polyacrylamide gel electrophoresis, but yielded on isoelectric focusing at least three bands between pH 5.5 and 6.0, suggesting the presence of multiple forms of the substrate of a similar molecular weight but different isoelectric points . ADP-ribosylated protein bands from the membrane and cytosol overlapped each other on both electrophoreses . After ammonium sulfate fractionation, the substrate from the cytosol showed requirement of divalent cations or guanine nucleotides for the reaction . Among cations tested, calcium, magnesium and manganese stimulated, whereas cadmium and lanthanum inhibited the reaction . Guanine nucleotides such as GTP, GDP and GTP-gamma-S also stimulated the substrate activity in the cytosol as that in the membrane fraction . However, no additive effects were observed when the nucleotides and cations were added together.

J Biol Chem, 1988 Feb 15, 263(5), 2423 - 35
Divalent cation involvement in the action of Clostridium perfringens type A enterotoxin . Early events in enterotoxin action are divalent cation-independent; McClane BA et al.; Clostridium perfringens type A enterotoxin (CPE) is a membrane-active cytotoxin . There are a number of recognized early steps in CPE cytotoxicity including binding of CPE to a protein receptor, insertion of CPE into membranes, and CPE-mediated induction of changes in membrane permeability for small molecules such as ions and amino acids . Further support for the existence of these early steps and further characterization of these events are presented in this report . We now report that these early steps in CPE action are largely independent of extracellular divalent cations . It is also shown that 3H-nucleotide release, known to be a later CPE effect, is Ca2+-dependent . A model for CPE cytotoxicity is suggested involving CPE action as a two-step process with Ca2+-independent early steps and Ca2+-dependent late steps.

J Immunol, 1988 Feb 15, 140(4), 1223 - 7
Nonimmune binding of Ig to Clostridium perfringens . Preferential binding of IgM and aggregated IgG; Lindahl G et al.; In an attempt to find a bacterial IgM receptor, a large number of bacterial strains of different species were screened for the ability to bind human IgM . Certain strains of the anaerobic bacterium Clostridium perfringens were found to bind a major fraction of polyclonal IgM . One bacterial strain showed a particularly high binding capacity and was studied in more detail . This strain is also able to bind a minor fraction of polyclonal IgA and IgG . Inhibition experiments indicate that the different Ig classes bind to one and the same R structure . The ability of the strain to bind polyclonal Ig is correlated to the number of subunits in the Ig . This correlation can most simply be explained by increasing avidity with increasing number of subunits . In agreement with this hypotheses, experiments with aggregated IgG show that binding ability increases with aggregate size . Experiments with Ig fragments indicate that the binding structure in Ig is located in the F(ab')2 region . The ability of this bacterial strain to bind a majority of IgM molecules as well as aggregated IgG is potentially useful in immunologic work and represents a new type of Ig binding to bacteria.

JAMA, 1988 Feb 12, 259(6), 860 - 4
Infectious complications in four long-term recipients of the Jarvik-7 artificial heart; Kunin CM et al.; This article describes the infectious complications that occurred among four of the longest-term recipients of the Jarvik-7 artificial heart . Infection arising from the drive lines, with spread to the mediastinal periprosthetic space, was the major limiting factor in long-term use of the device in these patients . Periprosthetic infections were due to coagulase-negative staphylococci, Staphylococcus aureus, Pseudomonas aeruginosa, and other Pseudomonas species . Other infectious complications incurred by some of the patients included pneumonia, empyema, urinary tract infection, and intravascular line sepsis with Candida . Intensive antimicrobial therapy for prolonged periods seemed to suppress but not to eradicate infection and was accompanied by the appearance of multiresistant bacterial strains . Complications of antimicrobial therapy included diarrhea secondary to overgrowth with Clostridium difficile in two patients . Use of the current device for more than 30 days should be considered extraordinary and should be reserved for patients for whom no other form of life support is available.

FEBS Lett, 1988 Feb 8, 228(1), 89 - 93
5-Aminolevulinic acid formation from glutamate via the C5 pathway in Clostridium thermoaceticum; Oh-hama T et al.; A cell-free extract of the anaerobic eubacterium, Clostridium thermoaceticum, catalyzes the synthesis of 5-aminolevulinic acid (ALA) from glutamate via the C5 pathway . The enzyme reaction resembles that of higher plants and algae in cofactor requirements and sensitivity to ribonuclease . From the phylogenetic distribution it is proposed that the C5 pathway evolved earlier than the ALA synthase pathway.

Mol Cell Biochem, 1988 Feb, 79(2), 153 - 9
Circular dichroic and fluorescence spectroscopic study of the conformation of botulinum neurotoxin types A and E; Datta A et al.; Botulinum neurotoxin (NT) is synthesized by Clostridium botulinum in any of seven antigenically distinct forms, called types A through G . Protease(s) endogenous to the bacteria, or trypsin, nicks the single chain protein to a dichain molecule which generally is more toxic . The conformation of dichain type A (nicked by endogenous protease), single chain type E, and dichain type E NT (nicked by trypsin) have been determined using circular dichroism (CD) and fluorescence spectroscopy . The high degree of ordered secondary structure (alpha helix 28%, beta sheet 42%, total 70%) found in type A NT at pH 6.0 was similar to that found at pH 9.0 (alpha 22%, beta 47%, total 69%) . The secondary structure of the single chain type E NT at pH 6.0 (alpha 18%, beta 37%, total 55%) differed somewhat from these values at pH 9.0 (alpha 22%, beta 43%, total 65%) . The dichain type E NT at pH 6.0 assumed a secondary structure (alpha 20%, beta 47%, total 67%) more similar to that of dichain type A than the single chain type E NT . Examination with the fluorogenic probe toluidine napthalene sulfonate revealed that the hydrophobicity of the type A and E NTs were higher at pH 9.0 than at pH 6.0 . Also, the hydrophobicity of the dichain type E NT was higher than its precursor the single chain protein and appeared similar to that of the dichain type A NT . The CD and fluorescence studies indicate that conversion of the single chain type E NT to the dichain form (i.e . nicking by trypsin) induced changes in conformation.(ABSTRACT TRUNCATED AT 250 WORDS)

Klin Monatsbl Augenheilkd, 1988 Feb, 192(2), 141 - 2
{Gas gangrene of the orbit}; Speiser P et al.; This case report describes a penetrating injury of the orbit damaging the inferior division of the oculomotor nerve and causing a Clostridium perfringens inflammation of the orbit within 24 hours.

J Clin Microbiol, 1988 Feb, 26(2), 225 - 30
Clinical evaluation of the Vitek ANI card for identification of anaerobic bacteria; Schreckenberger PC et al.; An evaluation of the Vitek Anaerobe Identification (ANI) card was performed with 341 bacterial isolates, including 313 clinical isolates and 28 stock strains of anaerobic microorganisms . Identifications obtained with the ANI card were compared with those determined by conventional methods . The card identified 73.2% of 149 anaerobic gram-negative bacilli, 63.6% of 44 Clostridium spp., 65.8% of 38 anaerobic nonsporeforming gram-positive bacilli, and 69.1% of 110 anaerobic cocci, with no further testing required . When genus-level identifications were included, 83.9% of the anaerobic gram-negative bacilli, 70.5% of Clostridium spp., 73.7% of the anaerobic nonsporeforming gram-positive bacilli, and 73.6% of the anaerobic cocci were identified . Nineteen isolates (5.6%) produced identifications of good confidence but marginal separation or questionable biotype, in which the correct identification was listed with one or two other possible choices and extra tests were required and suggested . A total of 28 (8.2%) were not identified and 29 isolates (8.5%) were misidentified by the ANI card . Among the commonly isolated clinically significant anaerobes, the ANI card identified 100% of 55 Bacteroides fragilis and 100% of 8 Clostridium perfringens . Use of supplemental tests and expansion of the data base to include additional strains of organisms that are difficult to separate even with conventional methods may improve the accuracy of the ANI card as a method for identification of anaerobic bacteria in the clinical laboratory.

Am J Clin Pathol, 1988 Feb, 89(2), 228 - 33
Evaluation of a latex agglutination test for diagnosis of Clostridium difficile-associated colitis; Sherman ME et al.; Current methods for diagnosis of Clostridium difficile-associated colitis (CAC) based on detection of cytotoxin B by a tissue culture assay (TCA) require technical expertise and up to 48 hours incubation . Recently, a latex agglutination (LA) test (Marion Laboratories) for rapid diagnosis of CAC has become available . Although early evaluations have been favorable, new evidence suggests that the LA reagent binds a soluble bacterial antigen that is not unique to toxigenic strains of C . difficile . The authors examined 201 stools received for CAC testing by LA and a reference TCA and investigated discrepant results . They obtained 29 LA(+)/TCA(+) and 155 LA(-)/TCA(-) results . Eleven patients had LA(+)/TCA(+) and 155 LA(-)/TCA(-) results . Eleven patients had LA(+)/TCA(-) results and 6 had LA(-)/TCA(+) results . The sensitivity and specificity of the LA were 83% and 93%, respectively, compared with TCA . The predictive values of positive and negative results obtained with the LA were 72% and 96%, respectively . Concentrated broth supernatants and live suspensions of three C . difficile isolates with LA(+)/TCA(-) results were tested in a rabbit ileal loop assay . All failed to demonstrate ability to produce an enterotoxin . The authors conclude that the LA method is suitable for rapid screening, but LA(+) results require confirmation by testing with other methods.

South Med J, 1988 Feb, 81(2), 283 - 4
Necrotizing enterocolitis associated with Clostridium paraputrificum septicemia; Shandera WX et al.; A 38-year-old woman with chronic, noncyclic neutropenia had an episode of acute abdominal pain associated with clostridial septicemia . Clostridium paraputrificum was isolated from blood and peritoneal cultures . The pathogenic potential of C paraputrificum was established by surgical biopsy specimens which demonstrated necrotizing enterocolitis with the typical gram-positive rods . This report strengthens a recognized, established association between neutropenia and clostridial infection.

J Bacteriol, 1988 Feb, 170(2), 751 - 6
Purification and characterization of Clostridium sticklandii D-selenocystine alpha, beta-lyase; Esaki N et al.; We have found a novel enzyme that decomposes D-selenocystine into pyruvate, ammonia, and elemental selenium in extracts of Clostridium sticklandii and C . sporogenes . The enzyme of C . sticklandii has been purified to homogeneity . It has a molecular weight of 74,000 and consists of two subunits identical in molecular weight (35,000) . Pyridoxal 5'-phosphate is required as a cofactor . In addition to D-selenocystine, D-cystine, D-lanthionine, meso-lanthionine, and D-cysteine serve as substrates . However, D-selenocysteine, D-serine, DL-selenohomocystine, and L-amino acids are inert . The enzyme also catalyzes the beta-replacement reaction between D-selenocystine and a thiol to produce S-substituted D-cysteine . L-Selenohomocysteine also can serve as a substituent donor in the beta-replacement reaction to yield selenocystathionine.

Scand J Dent Res, 1988 Feb, 96(1), 50 - 5
Ultrastructure of a novel anaerobic gram-positive nonsporing rod from dental root canal; Kerosuo E et al.; A novel anaerobic Gram-positive rod, strain ES4C, was isolated from a dental root canal infection . The isolate did not produce acids from carbohydrates and showed no glycosidase activity . Most biochemical reactions were identical to Clostridium malenominatum with the exception of the production of three aminopeptidases . In addition, no spores were detected . A tetragonally arranged surface layer was consistently found by electron microscopy . The ultrastructure of closely related Eubacterium spp . was also studied, but no crystalline surface structures were found . The physiologic and ultrastructural characteristics of ES4C did not allow identification as any known species . The periapical lesion responded to routine root canal therapy, but after 18 months observation the radiologic signs indicated partial healing only.

Int J Food Microbiol, 1988 Feb, 6(1), 19 - 24
Effect of reduced water activity on vegetative growth of cells and on germination of endospores of Sporolactobacillus; Botha SJ et al.; Growth of vegetative cells and germination of endospores of Sporolactobacillus at reduced water activity (aw) values were studied with NaCl and glycerol as humectants, and compared with Bacillus cereus and Clostridium sporogenes . With NaCl the highest aw for complete inhibition of both growth of vegetative cells and germinated spores of Sporolactobacillus was 0.955 . With glycerol, growth of vegetative cells was completely inhibited at 0.905 as compared to 0.880 for vegetative growth resulting from germinated endospores . The mechanism of this phenomenon cannot be explained yet . NaCl appeared to have a toxic effect, in addition to inhibition by reduced aw per se.

Antimicrob Agents Chemother, 1988 Feb, 32(2), 216 - 23
In vitro antibacterial activity of trospectomycin (U-63366F), a novel spectinomycin analog; Zurenko GE et al.; Trospectomycin (U-63366F) is a novel spectinomycin analog with broad-spectrum antibacterial activity . The in vitro activity of this analog was compared with that of spectinomycin and other reference antibiotics against 411 clinical isolates of aerobic and anaerobic bacteria . MICs were determined by agar or broth dilution methods . The stability of trospectomycin in the presence of an enzyme extract derived from spectinomycin-resistant Escherichia coli was determined . Trospectomycin was more active than spectinomycin (4- to 32-fold) against strains of numerous bacterial species, including staphylococci, streptococci, Haemophilus influenzae, Branhamella catarrhalis, Neisseria gonorrhoeae, Proteus species, Bacteroides species, Clostridium difficile, Clostridium species, and Chlamydia trachomatis . Trospectomycin demonstrated a moderate level of activity (comparable to that of spectinomycin) for most species of the family Enterobacteriaceae tested and was generally cross resistant with spectinomycin . Trospectomycin was susceptible to inactivation by crude enzyme preparations from spectinomycin-inactivating strains of E . coli . Trospectomycin inhibited a variety of clinically important organisms, including agents of sexually transmitted diseases and pelvic inflammatory disease . Clinical studies with this novel aminocyclitol antibiotic are in progress.

Jpn J Med Sci Biol, 1988 Feb, 41(1), 27 - 30
Clostridium absonum from gas gangrene; Masaki T et al.; A Clostridium perfringens-like strain was isolated from a case of gas gangrene . The morphological properties and the lecithinase reaction of the isolate were very similar to those of C . perfringens; however, the lecithinase reaction was only slightly suppressed by C . perfringens alpha-antitoxin serum and the organism was identified as Clostridium absonum from its biochemical properties.

Poult Sci, 1988 Feb, 67(2), 261 - 9
Egg yolk paste for determining some food poisoning bacteria; Imai C et al.; Egg yolk, aseptically prepared from fresh eggs, was partially dehydrated with a 40% high fructose corn syrup solution, and 10% salt was added . This salted yolk paste was added to mannitol salt agar for the detection of Staphylococcus aureus, to NaCl-glycine Kim and Goepfert medium for detection of Bacillus cereus, to Clostridium welchii agar for detection of C . perfringens, and to Gifu anaerobic medium for detection of C . botulinum . These food poisoning bacteria showed the same lecithovitellin (LV) reaction on these media as on the same media prepared with fresh egg yolk . The yolk paste could be stored at -20 C without freezing and did not show any bacterial growth after holding at 25 C for 30 days . The increased salt content resulted from the addition of salted yolk paste to the media did not inhibit the growth of the food poisoning bacteria used in these experiments . For the identification of the food poisoning bacteria used in this work, and which give a LV reaction, salted yolk paste is more convenient to use than yolk separated from fresh shell eggs.

J Hosp Infect, 1988 Feb, 11 Suppl A, 378 - 85
Clostridium difficile as a nosocomial pathogen; Silva J Jr et al.; Patients admitted to a 19-bed floor with intermediate nursing care were studied for the onset of Clostridium difficile-associated diarrhoea during a six-month period (181 calendar days) in 1986-87 . All admitted patients were reviewed weekly and followed after discharge from the study unit to other inpatient services . Multiple items in the environment of five patients' rooms were sampled bacteriologically for the presence of C . difficile weekly during the study period . Three of the rooms were selected for study because of a higher prevalence of C . difficile associated diarrhoea in the prior three years and two were selected because no cases had been discovered previously in these rooms ('control rooms') . Nine of 521 patients admitted to this unit developed C . difficile diarrhoea (1.73 cases/100 patients admitted) versus 0.30/100 patients admitted to all other sites in our hospital (24 of 7970 other patients) . This represented respectively 3.91 cases per 1000 patient days on this floor versus 0.37 patients/1000 patient days throughout the hospital . Seven of the C . difficile diarrhoea cases were associated with stay in the C . difficile associated rooms, versus two cases in the two 'control rooms' . C . difficile was isolated from the toilet seats, bedpan hopper, night stands or food trays . Of some 1955 cultures taken, only 1.9% overall were positive for C . difficile.

Arthritis Rheum, 1988 Feb, 31(2), 295 - 8
Clostridial septic arthritis: case report and review of the literature; Fauser DJ et al.; We describe a patient who had septic arthritis caused by Clostridium perfringens . Clostridial organisms are very uncommon causes of septic arthritis . Only 13 cases have been reported previously . The diagnosis should be suspected in patients with a history of penetrating joint trauma and in immunocompromised patients . Successful treatment has usually consisted of surgical synovectomy in combination with high-dose intravenous penicillin therapy . Multiple aspirations of affected joints as a definitive treatment should be used with caution and only in patients who are not candidates for surgery.

Am J Vet Res, 1988 Feb, 49(2), 201 - 7
Experimental induction of abdominal tympany, abomasitis, and abomasal ulceration by intraruminal inoculation of Clostridium perfringens type A in neonatal calves; Roeder BL et al.; The etiologic role of Clostridum perfringens type A in the acute abdominal syndrome characterized by abomasal and rumen tympany, abomasitis, and abomasal ulceration was investigated in neonatal calves . Eight calves, 4 to 12 days old, were inoculated intraruminally with toxigenic C perfringens type A . Before and after C perfringens inoculation, blood samples were collected from all calves for blood gas and serum biochemical analysis and for determination of serum copper concentration; ruminal fluid was obtained for isolation of C perfringens . Calves were monitored daily for clinical signs of the syndrome and, depending on the severity of clinical signs, they were either euthanatized or redosed within 4 to 7 days . After necropsy, specimens obtained from the abomasum and rumen for macroscopic and microscopic examination and for anaerobic bacteriologic culture were processed in routine manner . Intraruminal inoculation of C perfringens type A into healthy calves induced anorexia, depression, bloat, diarrhea, and in some calves, death . Serum copper concentration was within normal range . Necropsy revealed variable degrees of abomasitis, petechial and ecchymotic hemorrhages, and ulcers (ranging from pinpoint to nearly perforate) in the abomasum . Seven of those calves also had multiple trichobezoars in the rumen . These necropsy findings were not seen in calves (controls) given distilled H2O only . In affected calves, acute abdominal syndrome was unrelated to copper deficiency, and C perfringens type A given intraruminally was able to induce clinical signs similar to those of the naturally acquired disease.

J Infect Dis, 1988 Feb, 157(2), 272 - 9
Lethal effects and cardiovascular effects of purified alpha- and theta-toxins from Clostridium perfringens; Stevens DL et al.; Shock, a common and frequently fatal manifestation of gas gangrene caused by Clostridium perfringens, is probably mediated by extracellular toxins . Previous studies implicating alpha-toxin as the major lethal factor were frequently done with preparations contaminated with a second lethal factor, theta-toxin . We purified alpha- and theta-toxins from C . perfringens and demonstrated that both were lethal to mice . We investigated the effects of these purified toxins on cardiovascular function in intact rabbits; both toxins caused profound hypotension and bradycardia within 40 min . Reduced cardiac output preceded the development of hypotension and bradycardia . Purified alpha-toxin produced a dose-dependent reduction in myocardial function in isolated rabbit atrial preparations . Purified theta-toxin did not directly inhibit myocardial function . Shock induced by alpha-toxin may be partly mediated by direct depression of myocardial function . theta-Toxin reduced cardiac output in intact animals but had no direct effects on isolated heart preparations at concentrations that induced shock in intact animals . These data suggest that theta-toxin-induced shock could be mediated by an endogenous myocardial depressant factor.

Diagn Microbiol Infect Dis, 1988 Feb, 9(2), 79 - 85
In vitro activity of LY146032 against gram-positive bacteria; Silva M et al.; The activity of LY146032 (LY) was evaluated against 269 clinical isolates: 150 Staphylococcus spp . (Staph), 45 enterococci, 51 Clostridium spp., and 23 peptostreptococci . LY was compared to penicillin, metronidazole, imipenem, clindamycin, oxacillin, ciprofloxacin, vancomycin, and ampicillin . LY and oxacillin were tested against Staph by microdilution in cation-supplemented Mueller-Hinton broth (CSMHB), and in unsupplemented Mueller-Hinton broth (MHB) . For LY, the MIC 90s in CSMHB were 16-32 dilutions lower . Among the Staph, the MIC 90s for LY, vancomycin, and ciprofloxacin were 4 micrograms/ml, 4 micrograms/ml, and 2 micrograms/ml respectively . The MIC 90s for enterococci by agar dilution were as follows: LY 8 micrograms/ml; ampicillin 4 micrograms/ml; imipenem 4 micrograms/ml; vancomycin 4 micrograms/ml; and ciprofloxacin 2 micrograms/ml . Clindamycin and penicillin were the most effective drugs against peptostreptococci and Clostridia spp., but LY was the most active drug against Clostridium difficile . The bactericidal activity of LY was determined by 24-hr time-kill curves in MHB . These showed a bactericidal effect against enterococci, and a bacteriostatic effect against three of four strains of Staph . Synergy was demonstrated against enterococci and Staph when LY was tested with aztreonam, ceftriaxone, or tobramycin . LY is a promising new agent against gram-positive bacteria, including methicillin resistant strains of staphylococci and enterococci.

J Bacteriol, 1988 Feb, 170(2), 817 - 20
Amino acid transport by membrane vesicles of an obligate anaerobic bacterium, Clostridium acetobutylicum; Driessen AJ et al.; Membrane vesicles were isolated from the obligate anaerobic bacterium Clostridium acetobutylicum . Beef heart mitochondrial cytochrome c oxidase was inserted in these membrane vesicles by membrane fusion by using the freeze-thaw sonication technique (A . J . M . Driessen, W . de Vrij, and W . N . Konings, Proc . Natl . Acad . Sci . USA 82:7555-7559, 1985) to accommodate them with a functional proton motive force-generating system . With ascorbate-N,N,N',N'-tetramethyl-p-phenylenediamine-cytochrome c as the electron donor, a proton motive force (delta p) of -80 to -120 mV was generated in these fused membranes . This delta p drove the accumulation of leucine and lysine up to 40- and 100-fold, respectively . High transport activities were observed in fused membranes containing Escherichia coli lipids, whereas the transport activities in fused membranes containing mainly soybean lipids or phosphatidylcholine were low . It is suggested that branched-chain amino acids and lysine were taken up by separate systems . The effects of the ionophores nigericin and valinomycin indicated that lysine and leucine were translocated in symport with a proton.

Nucleic Acids Res, 1988 Jan 25, 16(2), 439 - 54
The presence of five nifH-like sequences in Clostridium pasteurianum: sequence divergence and transcription properties; Wang SZ et al.; The nifH gene encodes the iron protein (component II) of the nitrogenase complex . We have previously shown the presence in Clostridium pasteurianum of two nifH-like sequences in addition to the nifH1 gene which codes for a protein identical to the isolated iron protein . In the present study, we report that there are at least five nifH-like sequences in C . pasteurianum . DNA sequencing data indicate that the six nifH (nifH1) and nifH-like (nifH2, nifH3, nifH4, nifH5 and nifH6) sequences are not identical and vary from each other to different extents with sequence identity ranging between 68 to 99.9% within the nifH coding regions . Under normal N2-fixing growth conditions (molybdenum-containing medium), transcripts of nifH1 and most of the nifH-like sequences accumulate . The above results suggest the functioning of more than one "nifH" gene under N2-fixing growth conditions for C . pasteurianum . A common sequence was found around the -100 regions of all nif or nif-like transcription units . Sequences identical to or very similar to the consensus Escherichia coli promoter were found in the -35 and -10 regions.

Thromb Res, 1988 Jan 15, 49(2), 225 - 39
Human platelet activation by bacterial phospholipase C: mechanism of inhibition by flurazepam; Anwer K et al.; We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes platelet activation possibly by inducing turnover of phosphoinositides and phosphorylation of a 47,000 Dalton protein (P47) . Moreover, only 15 microM and 11 microM flurazepam inhibits PLC-induced platelet aggregation and serotonin secretion by 50% respectively . This study was conducted to better understand the mechanism of platelet activation by PLC and its inhibition by flurazepam . Incubation of (14C)-arachidonic acid labelled platelets with PLC produced diacylglycerol in a time- and concentration-dependent manner . Flurazepam did not inhibit diacylglycerol production by PLC . Paranitrophenolphosphorylcholine and prostaglandin E1 inhibited diacylglycerol production by 75% and 20% respectively . In a platelet-free system PLC hydrolyzed 14C-choline-phosphatidylcholine (14C-PC) in a time- and calcium ions-dependent manner . Flurazepam had no effect on PLC-induced hydrolysis of 14C-PC . Platelet cytosolic fraction (PCF), containing phosphatidylinositol-specific PLC (PI-PLC), hydrolyzed (3H-inositol)-phosphatidylinositol (3H-PI) in a platelet-free system . Flurazepam did not inhibit hydrolysis of 3H-PI by PCF . Phospholipase C caused phosphorylation of P47 in 32P-labelled platelets . Flurazepam did not block phosphorylation of P47 in the first three minutes and had very little inhibitory effect by five minutes . However, flurazepam completely blocked phosphorylation of P47 by seven minutes . Platelet aggregation induced by ionomycin, a calcium ionophore, was completely inhibited by 100 microM flurazepam whereas platelet aggregation induced by 12-O-Tetradecanoylphorbol-13-acetate (TPA), which mimics the action of diacylglycerol, was partially inhibited by 300 microM flurazepam . These findings suggest that PLC induced platelet activation depends, at least in part, on diacylglycerol production and phosphorylation of P47 . These data also suggest that flurazepam does not inhibit PLC-induced platelet activation by inhibiting: (a) the production of diacylglycerol from phosphatidylcholine; and (b) the action of PI-PLC on phosphatidylinositol . The ability of flurazepam to inhibit ionomycin-induced platelet aggregation indicates that flurazepam is able to block platelet activation by inhibiting the increase in free cytosolic calcium ions in platelets or by inhibiting a step subsequent to the rise in intraplatelet calcium ions.

Eur J Biochem, 1988 Jan 15, 171(1-2), 225 - 9
ADP-ribosylation of skeletal muscle and non-muscle actin by Clostridium perfringens iota toxin; Schering B et al.; The enzymatically active component ia of Clostridium perfringens iota toxin ADP-ribosylated actin in human platelet cytosol and purified platelet beta/gamma-actin, in a similar way to that been reported for component I of botulinum C2 toxin . ADP-ribosylation of cytosolic and purified actin by either toxin was inhibited by 0.1 mM phalloidin indicating that monomeric G-actin but not polymerized F-actin was the toxin substrate . Perfringens iota toxin and botulinum C2 toxin were not additive in ADP-ribosylation of platelet actin . Treatment of intact chicken embryo cells with botulinum C2 toxin decreased subsequent ADP-ribosylation of actin in cell lysates by perfringens iota or botulinum C2 toxin . In contrast to botulinum C2 toxin, perfringens iota toxin ADP-ribosylated skeletal muscle alpha-actin with a potency and efficiency similar to non-muscle actin . ADP-ribosylation of purified skeletal muscle and non-muscle actin by perfringens iota toxin led to a dose-dependent impairment of the ability of actin to polymerize.

Eye, 1988, 2 ( Pt 1), 16 - 23
Clostridium botulinum toxins: nature and preparation for clinical use; Melling J et al.; C . botulinum neurotoxins are acutely toxic materials and act by inhibiting release of the neurotransmitter acetylcholine . The specific nature of this inhibition is discussed and the preparation and purification of Type A toxin specifically for clinical use is described.

Arch Microbiol, 1988, 149(6), 527 - 33
Surface properties from the S-layer of Clostridium thermosaccharolyticum D120-70 and Clostridium thermohydrosulfuricum L111-69; Sara M et al.; Labelling experiments using a positively charged topographical marker for electron microscopy, polycationized ferritin, showed that the S-layers of two closely related clostridia Clostridium thermohydrosulfuricum L111-69 and C . thermosaccharolyticum D120-70 do not exhibit a net negative charge, as usually observed for bacterial cell surfaces . Chemical modification of reactive sites confirmed that amino and carboxyl groups are exposed on the S-layer surface of both strains . Amino-specific, bifunctional agents crosslinked both S-layer lattices . Studies with carbodiimides revealed that only the S-layer surface of C . thermohydrosulfuricum L111-69 had amino and carboxyl groups closely enough aligned to permit electrostatic interactions between the constituent protomers . The regular structure of this S-layer lattice was lost upon converting the carboxyl groups into neutral groups by amidation . Disintegration of both S-layer lattices occurred upon N-acetylation or N-succinylation of the free amino groups . Adhesion experiments showed that in neutral and weakly alkaline environment whole cells of C . thermosaccharolyticum D120-70 exhibited a stronger tendency to bind to charged surfaces than whole cells of C . thermohydrosulfuricum L111-69, but showed a lower tendency to bind to hydrophobic materials.

Gene, 1988, 63(1), 23 - 30
Nucleotide sequence of the celC gene encoding endoglucanase C of Clostridium thermocellum; Schwarz WH et al.; The nucleotide sequence of the cellulase gene celC, encoding endoglucanase C of Clostridium thermocellum, has been determined . The coding region of 1032 bp was identified by comparison with the N-terminal amino acid (aa) sequence of endoglucanase C purified from Escherichia coli . The ATG start codon is preceded by an AGGAGG sequence typical of ribosome-binding sites in Gram-positive bacteria . The derived amino acid sequence corresponds to a protein of Mr 40,439 . Amino acid analysis and apparent Mr of endoglucanase C are consistent with the amino acid sequence as derived from the DNA sequencing data . A proposed N-terminal 21-aa residue leader (signal) sequence differs from other prokaryotic signal peptides and is non-functional in E . coli . Most of the protein bears no resemblance to the endoglucanases A, B, and D of the same organism . However, a short region of homology between endoglucanases A and C was identified, which is similar to the established active sites of lysozymes and to related sequences of fungal cellulases.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Jan, 267(3), 383 - 94
Studies on the resistance of Clostridium difficile to antimicrobial agents; Wust J et al.; The susceptibility of C . difficile isolated at the Department of Medical Microbiology of the University of Zurich to a wide selection of antibacterial, antimycobacterial and antifungal agents was tested in vitro . Great differences in susceptibility were found against chloramphenicol, clindamycin, erythromycin, rifamycin, and tetracycline . Resistance to clindamycin and erythromycin could always be transferred jointly to a susceptible C . difficile strain by mixed culture on filters at low frequencies (1 X 10(-8) to 4 X 10(-8) per donor cell) . Transfer of tetracycline resistance occurred at frequencies of 3 X 10(-7) to 5 X 10(-7) . Chloramphenicol and rifamycin resistance could not be transferred in the system used (frequencies less than 10(-8)) . Although a total of 38,000 colonies was screened by various methods known to affect plasmid replication, resistance to chloramphenicol, clindamycin, erythromycin, rifamycin, and tetracycline could not be eliminated . No connection between plasmid DNA and antimicrobial resistance could be established . Especially, no plasmid DNA was involved in the transfer of resistance determinants from resistant to susceptible strains.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Jan, 267(3), 367 - 78
{Morphological changes in human embryonic lung fibroblasts caused by cytotoxins of various Clostridium species}; Schallehn G et al.; A total of 243 strains of 35 Clostridium species were tested for cytotoxin production in cooked meat medium or liver broth within 48-72 h at 37 degrees C, using human embryonal lung fibroblasts in tissue-culture as indicator cells . Cytotoxin could be detected in the culture-filtrates of all toxigenic strains of C . chauvoei, C . difficile, C . histolyticum, C . novyi types A and B, C . septicum and C . tetani, but not in the atoxigenic ones . The cytotoxin of C . novyi correlated with alpha-toxin in the culture filtrate . All strains of C . perfringens and C . novyi D tested were not cytotoxic for lung fibroblasts despite their pathogenicity for guinea-pigs . Further cytotoxigenic strains were found among C . hastiforme, C . limosum, C . oceanicum, C . putrificum, C . ramosum, C . sordellii, C . sporogenes, and C . subterminale . The morphological changes in lung fibroblasts caused by the culture filtrates were characteristic and species-specific and corresponded with pathogenicity for guinea-pigs and/or mice . No cytotoxin was produced by C . absonum, C . barati, C . bifermentans, C . botulinum (atoxic), C . butyricum, C . cadaveris, C . carnis, C . clostridioforme, C . cochlearium, C . glycolicum, C . innocuum, C . malenominatum, C . mangenotii, C . paraputrificum, C . putrefaciens, C . rectum, C . tertium, and C . tyrobutyricum.

APMIS, 1988 Jan, 96(1), 50 - 5
Characterization of the dermal lesions induced by a purified protein from toxigenic Pasteurella multocida; Elling F et al.; The dermonecrotic effect of purified Pasteurella multocida toxin (PMT) was studied sequentially in guinea pigs and rats . The skin reaction was initially an acute inflammatory reaction, with edema and emigration of neutrophils and a few eosinophils and diapedesis of some erythrocytes . Four hours after intracutaneous injection the vessels were congested and thrombocytes were focally attached to the endothelial wall . Twenty-four h after the injection the inflammatory reaction appeared more severe and venules and arterioles were thrombosed . Necrotic changes were seen in hair follicles and in striated muscle fibers . Crude extracts from P . multocida and Clostridium perfringens injected intracutaneously into guinea pigs induced skin lesions qualitatively similar to the lesions induced by the purified PMT, indicating that dermonecrotic bacterial toxins may share similar biochemical properties.

Scand J Gastroenterol, 1988 Jan, 23(1), 83 - 90
Beta-glucuronidase activity related to bacterial growth in common bile duct bile in gallstone patients; Skar V et al.; Beta-glucuronidase activity in the bile may be of importance in the etiology of pigment gallstones . This enzyme is of hepatic or bacterial origin . We have described a method to measure the activity of bacterial beta-glucuronidase in human bile, using 4-nitrophenyl-beta-D-glucopyranosiduronic acid as substrate . The method was used to measure the beta-glucuronidase activity in the bile from 51 patients with gallstone disease . This activity was related to the presence of beta-glucuronidase-producing bacteria in the bile . Escherichia coli, Bacteroides species, and Clostridium perfringens were the only species found to produce beta-glucuronidase . Patients with beta-glucuronidase-producing bacteria had on an average significantly higher enzyme activity in the bile than patients without such bacteria (p less than 0.01) . The limitations of using artificial substrates in this type of studies are discussed.

J Clin Microbiol, 1988 Jan, 26(1), 41 - 6
Immunoblots and plasmid fingerprints compared with serotyping and polyacrylamide gel electrophoresis for typing Clostridium difficile; Mulligan ME et al.; Two new methods for typing Clostridium difficile, immunoblotting and plasmid fingerprinting, were compared with serotyping and polyacrylamide gel electrophoresis (PAGE) . Of these methods, immunoblotting was found to be the most valuable for use in a comprehensive typing system . More groups could be distinguished by immunoblotting than by serotyping or PAGE . Immunoblotting results were also more reproducible and distinctive than results by PAGE . Plasmid fingerprinting was an excellent marker for plasmid-bearing strains, but it had limited use because many isolates lacked plasmids . A unique plasmid profile observed for one group of isolates correlated with differences in phenotypic characteristics resolved by immunoblot analysis but not by serotyping or PAGE . Preliminary attempts to correlate typing results with pathogenicity of isolates were not successful but underscored the need for future studies to include careful assessment of the clinical significance of isolates.

Ann Plast Surg, 1988 Jan, 20(1), 39 - 42
Studies on the endogenous flora of the human breast; Thornton JW et al.; Identification of the endogenous microbiological flora of the human breast and its role in breast infections following subglandular augmentation or reduction mammaplasty was undertaken . A total of 231 cultures were performed on 59 breasts in 30 patients . Patients were followed for 12 months . No fungus was cultured from any specimen . Of the breasts cultured 53% were positive for coagulase-negative staphylococcus . Other aerobes found included diphtheroids, lactobacillus, D-enterococcus, micrococcus, and alpha-hemolytic streptococcus . Propionibacterium acne was the most frequent anaerobic bacteria cultured . Other anaerobes included peptococcus and clostridium sporogenes . There was no correlation with respect to the type of bacterium and the depth within the breast where the culture specimens were taken . Postoperative wound infections developed in 2 of 19 patients undergoing reduction mammaplasty . Bacteria identical to those cultured at the time of surgery were again cultured from the wound . Twenty subglandular augmentation mammaplasties were performed with a 25% capsular rate at one year . Two capsules were associated with no bacterial growth at the time of mammaplasty surgery, whereas three were associated with coagulase-negative staphylococcus, Propionibacterium acne, and diphtheroids, respectively . Of the 15 breasts with no capsular contracture after one year, operative culture revealed coagulase-negative staphylococcus in 8 and no bacterial growth in 7 . Even breast tissue located deep within the gland away from the nipple contains a flora that is similar to that of normal skin . Cases of infection in which the endogenous bacteria were correlated with later infection was documented.

J Bacteriol, 1988 Jan, 170(1), 234 - 8
Energy-dependent transport of nickel by Clostridium pasteurianum; Bryson MF et al.; The mechanism of nickel transport by Clostridium pasteurianum was investigated by using 63NiCl2 and a microfiltration transport assay . Nickel transport was energy dependent, requiring either glucose or sucrose; xylose and o-methyl glucose did not support growth, butyrogenesis, or transport . Transport was optimum at pH 7 and 37 degrees C, and early-stationary-phase cells had the highest propensity for nickel transport . The apparent Km and Vmax for nickel transport approximated 85 microM Ni and 1,400 pmol of Ni transported per min per mg (dry weight) of cells, respectively . On the basis of metal specificity, nickel appears to be transported primarily by a magnesium transporter, although an alternative nickel transporter may also be involved . ATPase inhibitors (N,N'-dicyclohexylcarbodiimide, tributyltin chloride, 7-chloro-4-nitrobenz-2-oxa-1,3-diazole, and quercetin), protonophores (carbonyl cyanide m-chlorophenylhydrazone, 2,4-dinitrophenol, and gramicidin D), metal ionophores (valinomycin, monensin, and nigericin), benzyl viologen, carbon monoxide, and oxygen inhibited nickel transport . Nickel transport was coupled indirectly to butyrogenesis and was dependent on the energy state of the cell.

Folia Microbiol (Praha), 1988, 33(2), 148 - 54
Production of toxic antigens in dialyzed cultures of microorganisms; Vrany B et al.; Data on the production of clostridial toxins in dialyzed cultures are summarized . Principal modifications of this cultivation technique suitable for both research and production are shown . If toxins are released from the cells by autolysis (neurotoxins of Clostridium tetani, C . botulinum; lethal factors of C . novyi and C . sordellii), a 10-fold increase of the antigen concentration in filtrates of dialyzed cultures is found in comparison with normal cultures . If toxins are excreted already during growth (lethal factors of C . perfringens type A, C . septicum), the positive effect of the technique is less significant . A dialyzed culture ensures a well-balanced production of toxic filtrates that contain highly concentrated, relatively pure and strongly immunogenic antigens.

Infection, 1988, 16(1), 36 - 41
Infections after experimental cadaver bone marrow transplantation in beagle dogs . Transplantations with and without selective gastrointestinal decontamination; Haralambie E et al.; Experimental transplantations of cadaver bone marrow (BMT) in beagle dogs were performed to evaluate the problems and potentials in a preclinical setting . The effectiveness of selective decontamination of the gut (SD) and gnotobiotic surveillance in preventing infections during longer aplastic periods was investigated . Three groups of dogs were compared . Group A: controls . Group B: dogs with BMT, without SD and irregular gnotobiotic surveillance . Group C: dogs with BMT, with SD and regular gnotobiotic surveillance . The intestinal colonization of normal healthy beagles shows similarities as well as dissimilarities to the human intestinal microflora . Aerobic potentially pathogenic organisms do not colonize the gut of healthy beagles under our keeping conditions . SD resulted in a significant decrease in infections with Escherichia coli and Plesiomonas . Infections with anaerobes, as well as bacterial infections of the respiratory tract were, however, not prevented . The intestinal colonization in dogs of group C with Clostridium difficile is another obvious effect of SD . The infections encountered during the study indicate the importance of the "take" for the clinical significance and outcome of intestinal colonization with potentially pathogenic organisms . In order to reduce the drug burden of BMT patients, we consider the elimination of routine SD after BMT not to be superior to gnotobiotic surveillance and germ-specific short term decontamination.

Appl Environ Microbiol, 1988 Jan, 54(1), 69 - 73
Characterization of bacteriophage nucleic acids obtained from Clostridium botulinum types C and D; Fujii N et al.; Nontoxigenic strains of Clostridium botulinum types C and D are converted to toxigenic strains by infection with specific Tox+ bacteriophages . The nucleic acids were extracted from five converting phages, c-st, c-468, c-203, c-d6f, and d-1873, and one nonconverting phage, c-n71, and treated with nucleases . The nucleic acids isolated were not digested by RNase A, but were digested by DNase I and exonuclease III, indicating that they were double-stranded DNA . On the basis of the restriction endonuclease digestion patterns on 0.8% agarose gel electrophoresis, the length of c-st, c-n71, c-468, and c-d6f phage DNAs was estimated to be about 110 kilobase pairs and that of c-203 and d-1873 was about 150 kilobase pairs . The digestion patterns of c-st, c-468, and c-n71 phage DNAs by PstI and HindIII were very similar . High homology was observed in the dot hybridization test . For other phages and nucleases, a good similarity was not observed . Only a little similarity was observed between c-203 and c-d6f phages . The existence of the structural genes for the toxin in both c-st and c-n71 phages was confirmed by the hybridization test with these phage DNAs and the oligonucleotide probe which represented the DNA sequence predicted for the N-terminal amino acids (2 to 17) of C . botulinum type C toxin . The loss of the converting ability of c-n71 phage may be caused not by the deletion of the tox+ gene but rather by the base mutation in c-st phage DNA.

J Clin Microbiol, 1988 Jan, 26(1), 144 - 6
Comparison of API ZYM system with API AN-Ident, API 20A, Minitek Anaerobe II, and RapID-ANA systems for identification of Clostridium difficile; Head CB et al.; The API ZYM system was compared with four anaerobe identification systems for the definitive identification of Clostridium difficile by using 88 cultures of C . difficile grown on Mueller-Hinton blood agar medium . The API ZYM system yielded a distinct and consistent enzyme profile for all test strains, whereas the sensitivities of the other systems in identifying C . difficile ranged from 78 to 96% (AN-Ident, 77.9%; RapID-ANA, 88.6%; Minitek Anaerobe II, 90.9%; and API 20A, 95.5%) . The API ZYM system is highly reliable in identifying C . difficile accurately, is rapid, and is relatively simple to use.

Drugs, 1988, 35 Suppl 2, 14 - 21
In vitro activity of cefotaxime against clinically significant pathogens; Nakashio S et al.; The present in vitro antibacterial activities of cefotaxime and 8 other cephalosporins (cefoperazone, cefmenoxime, cefpiramide, latamoxef, cefamandole, cefmetazole, cefotiam and cephazolin) were evaluated simultaneously in 384 strains of Gram-positive cocci, 595 strains of Enterobacteriaceae, 240 strains of non-fermenters and 143 strains of anaerobes and miscellaneous organisms . The results were expressed as minimum inhibitory concentration (MIC) range, MIC50 and MIC90 . Of the beta-lactams, cefotaxime and latamoxef exhibited the highest activity against a wide variety of Gram-positive and Gram-negative bacteria . MIC90 of cefotaxime, however, for species of Pseudomonas aeruginosa, Xanthomonas maltophilia, enterococci, Bacteroides sp . and Clostridium difficile were more than 100 mg/L . Cefpiramide and cefoperazone were generally less active than these 2 agents . All strains were tested for beta-lactamase production by the cefinase disc method and the relationship of susceptibility to beta-lactams was evaluated in each species . The need was demonstrated for periodic susceptibility testing to be performed to better guide empirical antimicrobial therapy.

Acta Neurochir (Wien), 1988, 95(1-2), 53 - 6
Gas gangrene occurring soon after compound depressed skull fracture; Sutcliffe JC et al.; Two cases of Clostridium perfringens infection occurring less than 24 hours after compound depressed skull fracture are reported . The infection was principally intracranial in the first and extracranial in the second; both required surgical debridement and antibiotic treatment . Attention is drawn to the rapidity with which a potentially life-threatening infection can develop in civilian head injury and to the implications for acute management of patients with compound depressed fractures.

Toxicon, 1988, 26(9), 817 - 25
Evidence that subunits of type A botulinum toxin need not be linked by disulfide; Bhattacharyya SD et al.; Type A neurotoxin of Clostridium botulinum strain 62A was purified by a modification of the procedure of TSE et al . (1982) . Electrophoresis in sodium dodecyl sulfate - polyacrylamide gels (SDS - PAGE) indicated the mol . wt of the intact dichain molecule is 140,000 and that of its L subunit is 52,000, both expected from published values . However the mol . wt of 83,000 for the H subunit was lower than the mol . wt of 97,000 in the literature . The purified toxin separated in SDS-PAGE into H and L subunits when pretreated with 2-mercaptoethanol but it unexpectedly behaved similarly without the pretreatment . Specific toxicity (approximately 3 x 10(8) mouse LD50/mg protein) was not affected by the spontaneous molecular change that made dissociation into subunits possible . The subunits of dichain botulinum toxins are believed to be covalently joined by intersubunit disulfide(s) since they have been demonstrated only when samples are treated with 2-mercaptoethanol or dithiothreitol . Since it is not always needed, the pretreatment is apparently not reducing a disulfide that connects the subunits . The strong chelating activity also possessed by the pretreating agents suggest that the subunits may be joined by a metallic divalent cation.

Microbiol Immunol, 1988, 32(6), 551 - 64
Purification and some properties of cytotoxin produced by Clostridium difficile; Katoh T et al.; The cytotoxin produced by Clostridium difficile was highly purified by using ammonium sulfate fractionation and successive column chromatographies of DEAE-Sephadex A-25, hydroxyapatite, Bio-Gel A-0.5m, Phenyl-Sepharose CL-4B, and Mono Q . The purified cytotoxin gave a single band on conventional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence of dithiothreitol . Its molecular weight was estimated to be 260,000 and 50,000 by gel filtration and sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis in the presence of dithiothreitol, respectively . Thus it was supposed that the toxin consists of 5 subunits having molecular weight of approximately 50,000 . It had an isoelectric point of 6.6 . The toxin was heat-labile (60 C for 10 min) and inactivated by treatment with trypsin and pronase, or at pH below 4 or over 10 . The minimum cytotoxic dose of the cytotoxin against Chinese hamster ovary cells was 3 ng . It was also demonstrated that the toxin is antigenically different from enterotoxin of C . difficile.

Reprod Nutr Dev, 1988, 28(6A), 1455 - 64
Production of volatile fatty acids as a result of bacterial interactions in the cecum of gnotobiotic rats and chickens fed a lactose-containing diet; Szylit O et al.; Volatile fatty acid (VFA) productions from lactose and lactate by a Clostridium butyricum and a Veillonella alcalescens strain, alone or in combination with a Lactobacillus acidophilus strain, were determined both in vitro in culture media and in vivo in the ceca of gnotobiotic animals . Gnotobiotic rats, which possess intestinal lactase, and chickens, which are devoid of it, were used . Both animal species were fed a diet containing 4% lactose . The in vitro results showed that the C . butyricum strain fermented lactose and D-lactic acid to butyric and acetic acids, whereas L-lactic acid was not fermented . The V . alcalescens strain did not ferment lactose and fermented L better than D-lactic acid . The in vivo results showed that high VFA concentrations were obtained in the ceca of chickens either disassociated with V . alcalescens or C . butyricum and Lactobacillus strains or monoassociated with C . butyricum . VFA concentrations in the ceca of rats were low, whatever strain the rats harbored . In addition, an antagonistic effect of the C . butyricum strain against the Lactobacillus strain was evidenced both in rats and chickens . It is suggested that the absence of a host lactase makes the chick a good model for lactose intolerance studies in human newborns.

Clin Microbiol Rev, 1988 Jan, 1(1), 1 - 18
Clostridium difficile: its disease and toxins; Lyerly DM et al.; Clostridium difficile is the etiologic agent of pseudomembranous colitis, a severe, sometimes fatal disease that occurs in adults undergoing antimicrobial therapy . The disease, ironically, has been most effectively treated with antibiotics, although some of the newer methods of treatment such as the replacement of the bowel flora may prove more beneficial for patients who continue to relapse with pseudomembranous colitis . The organism produces two potent exotoxins designated toxin A and toxin B . Toxin A is an enterotoxin believed to be responsible for the diarrhea and mucosal tissue damage which occur during the disease . Toxin B is an extremely potent cytotoxin, but its role in the disease has not been as well studied . There appears to be a cascade of events which result in the expression of the activity of these toxins, and these events, ranging from the recognition of a trisaccharide receptor by toxin A to the synergistic action of the toxins and their possible dissemination in the body, are discussed in this review . The advantages and disadvantages of the various assays, including tissue culture assay, enzyme immunoassay, and latex agglutination, currently used in the clinical diagnosis of the disease also are discussed.

Mol Cell Biol, 1988 Jan, 8(1), 418 - 26
Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum; Rubin EJ et al.; Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot . Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein . When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function . NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins . When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins . Nevertheless, p21.bot was immunologically distinct from p21ras.

Infect Immun, 1988 Jan, 56(1), 24 - 7
Partial characterization of the enzymatic activity associated with the binary toxin (type C2) produced by Clostridium botulinum; Simpson LL et al.; Clostridium botulinum produces a binary toxin that possesses a heavy chain (approximately 100,000 daltons) and a light chain (approximately 50,000 daltons) . The heavy chain is a binding component that directs the toxin to vulnerable cells, and the light chain is an enzyme that has mono(ADP-ribosyl)ating activity . A number of experiments have been done to help characterize the enzymatic activity of the toxin . The data reveal that the enzyme has a pH optimum within the range of 7.0 to 8.0 . It is not inhibited or stimulated by physiological concentrations of sodium, potassium, calcium, or magnesium . The enzyme is inhibited by high concentrations of salt, however, as well as high concentrations of nicotinamide, thymidine, theophylline, and histamine; and it is stimulated by histone and lysolecithin . Boiling irreversibly denatures the light chain of the toxin, but denaturation caused by guanidine and urea is substantially reversible . Enzymatic activity is not altered by short exposure to lysosomal proteases, including cathepsin B, cathepsin H, dipeptidyl aminopeptidase, and catheptic carboxypeptidase B.

Exp Cell Res, 1988 Jan, 174(1), 215 - 29
Clostridium difficile toxin B induces reorganization of actin, vinculin, and talin in cultured cells; Ottlinger ME et al.; Clostridium difficile toxin B is a powerful cytopathic agent which causes animal cells in culture to become rounded and arborized, an effect similar to that induced by the cytochalasins . In this study, we demonstrated that the morphological effects of the toxin are directed specifically against the actin and related components of the cytoskeleton . Dramatic disruption and reorganization of the actin stress fibers were detectable prior to significant changes in cell shape and alterations in the microtubular and intermediate filament networks . Along with F-actin, the adhesion plaque proteins, vinculin and talin were localized in intoxicated cells in a patchy pattern reminiscent of that seen in cells treated with phorbol esters or transformed by oncogenic viruses . A quantitative fluorescence assay for cellular F-actin showed that these morphological changes were accompanied by a modest net depolymerization of only 15 to 20% of the actin filaments in the cell, and that depolymerization was closely correlated with changes in cell shape . In complementary studies on cells spreading on a substrate, we found that the toxin affected the actin content and the shape of the processes extended from the cell body . As in cells treated with cytochalasin, there was a differential response between normal and virally transformed cells spreading in the presence of the toxin . The results of this study support the view that C . difficile toxin B affects one or more cellular components that regulate the structure and function of the actin cytoskeleton, and that its predominant effect is to cause a dramatic disruption of stress fibers and relocalization of the F-actin.+

Arch Microbiol, 1988, 150(5), 460 - 4
Purification, properties, and metabolic roles of NAD+-glutamate dehydrogenase in Clostridium botulinum 113B; Hammer BA et al.; Cell-free extracts of proteolytic strains of Clostridium botulinum types A, B and F (group I) were found to have unusually high specific activities of NAD+-dependent L-glutamate dehydrogenase (NAD-GDH) . In comparison, nonproteolytic strains of types B, E and F (group II) had low specific activities . The enzyme was purified 131-fold from C . botulinum 113B to a final specific activity of greater than 1,092 mumol x min-1 x mg protein-1 . The enzyme is a hexamer of a polypeptide of Mr = 42,500, and the native molecular weight is 250,800 . The apparent Km values for substrates were 5.3 mM for glutamate and 0.028 mM for NAD+ in the deamination reaction, and 7.2 mM for alpha-ketoglutarate, 243 mM for NH4+ and 0.028 mM for NADH in the reverse reaction . NADP+ did not serve as a hydrogen acceptor for the enzyme . Activity in the animation direction was inhibited by fumarate, oxalacetate, aspartate, glutamate and glutamine . The results suggest that GDH is important in group I (proteolytic) C . botulinum to generate alpha-ketoglutarate as a substrate for transamination reactions . We have also found that the high activity decreases significantly when cells are exposed to sodium chloride . Therefore GDH probably has several important physiological roles in group I proteolytic C . botulinum.

Crit Rev Microbiol, 1988, 15(4), 297 - 338
The phosphoenolpyruvate:sugar phosphotransferase system in gram-positive bacteria: properties, mechanism, and regulation; Reizer J et al.; This review consists of three major sections . The first and largest section reviews the protein constituents and known properties of the phosphotransferase systems present in well-studied Gram-positive bacteria . These bacteria include species of the following genera: (1) Staphylococcus, (2) Streptococcus, (3) Bacillus, (4) Lactobacillus, (5) Clostridium, (6) Arthrobacter, and (7) Brochothrix . The properties of the different systems are compared . The second major section deals with the regulation of carbohydrate uptake . There are four parts: (1) inhibition by intracellular sugar phosphates in Staphylococcus aureus, (2) PTS-mediated regulation of glycerol uptake in Bacillus subtilis, (3) competition for phospho-HPr in Streptococcus mutans, and (4) the possible involvement of protein kinases in the regulation of sugar uptake via the phosphotransferase system . The third section deals with the phenomenon of inducer expulsion . The first part is concerned with the physiological characterization of the phenomenon; then the consequences of unregulated uptake and expulsion, a futile cycle of energy expenditure, are considered . Finally, the biochemistry of the protein kinase and the protein phosphate phosphatase system, which appears to regulate sugar transport via the phosphotransferase system, is defined . The review, therefore, concentrates on the phosphotransferase system, its functions in carbohydrate transport and phosphorylation, the mechanisms of its regulation, and the mechanism by which it participates in the regulation of other physiological processes in the bacterial cell.

Rev Argent Microbiol, 1988 Jan-Mar, 20(1), 17 - 24
{Growth of Clostridium botulinum in media with garlic (Allium sativum)}; Gimenez MA et al.; The effect of garlic on the growth and toxin formation of Clostridium botulinum (GT) was studied in A) crude juice obtained from a pool of cloves by i) crushing, ii) pressing out and iii) filtration, and B) minced garlic (6 to 8 pieces per clove) . For both, "white" and "red" garlic varieties were used . The juice (pH 5.7 to 6.0, for different batches) was activated 30 min at 37 degrees C and diluted (log 2) in PGY broth (g%: peptone (Difco) 1.0; glucose 0.5; yeast extract (Difco) 0.5; pH 7.3) . A small drop from a 18 h at 37 degrees C chopped meat medium culture of a highly toxigenic autochthonous strain (110) of C . botulinum type A, was transferred to the juice dilutions, incubating anaerobically 15d at 37 degrees C . As a control of the inhibitory effect of the juice, four microorganisms were cultured 48 h at 37 degrees C in the juice dilutions (Table 1) . Clove pieces were suspended to 50% (w/v) either in PGY broth or distilled water without pH adjustment . Aliquots were heated in water bath 15 min at 100 degrees C . After seeded with the A 110 strain, duplicate tubes and their controls were incubated 15 d at 37 degrees C in aerated and anaerobic conditions (Table 2) . Titers of botulinum toxin were empirically estimated by the time to death of a pair of mice injected with 0.5 ml each, via IP, observed 72 h . Results are shown in tables 1 and 2 . Garlic reduces (in undiluted juice, traces or 3 to 5 DL50/ml were recorded in separate experiments) but not inhibit GT.(ABSTRACT TRUNCATED AT 250 WORDS)

Pediatrician, 1988, 15(1-2), 45 - 57
Advances in ulcerative colitis; Ament ME et al.; Ulcerative colitis is one of the two common chronic inflammatory bowel diseases which affect the colon of children . The disease can occur at any time during infancy and childhood and is far commoner than Crohn's disease of the colon in children less than 6 years old . The Jewish population outside of Israel is at far greater risk of developing the condition than any other ethnic group . The reason for this is unknown . The chances of a family member developing the condition is 2-3 times as great as in the general population . The etiology of the condition remains unknown; however, recent advances in the understanding of the immune mechanisms in the bowel and circulation indicate there are major immunological differences between ulcerative colitis and Crohn's disease . Intestinal B cells secrete enormously increased amounts of IgG1 and a lesser increase in IgG3 in ulcerative colitis whereas in Crohn's disease, all IgG subclasses are increased, but especially IgG2 . Failure of the gut immune system to control antigen crossing the colonic mucosa may be the basis for the condition . The disease is classified as moderate to severe two thirds of children as opposed to less than one third of adults . Diagnostic testing must include 3 stool cultures negative for bacterial and viral pathogens, 3 stools negative for amebiasis, trichuriasis and other intestinal parasites and absence of clostridium difficile and its toxin in the stool . Flexible proctosigmoidoscopy and/or colonoscopy should be done in every case with biopsies . Barium enema is contraindicated in the severely ill patient . Major improvements in medical treatment being tested involve the development of nonabsorbable corticosteroid enemas and sulfapyridene-free forms of salicylazosulfapyridene for use in enema and oral form . Surgery for ulcerative colitis has made major advances with the development of the Koch pouch (internal ileostomy) and ileoproctostomy . Both procedures although associated with relatively high complication rates, are esthetically and psychologically better than standard ileostomy because in neither procedure must the patient wear an ileostomy appliance . However these advanced surgical procedures are typically not done until adolescence is reached.

Microbiol Immunol, 1988, 32(6), 579 - 87
Distribution of Clostridium botulinum in Japan and in Shinkiang district of China; Yamakawa K et al.; Soil specimens obtained from several areas of Japan, which are closely located to or facing the Continental land of China, were examined for the distribution of Clostridium botulinum, especially pertaining to types A and B . A total of 266 specimens of Japan, when cultured, showed no type A or B toxicity, although 30 (11.3%), 4 (1.5%), and 10 (3.8%) of the specimens showed C1, C2, and type E toxicities, respectively . On the contrary, types A and/or B toxicities were shown, by the same method, in 14 of 20 specimens of Shinkiang district, China . The highest number of C . botulinum cells found in one gram of soil specimen was 25 for type A and 10 for type B.

Chemotherapy, 1988, 34(6), 472 - 7
Chemotherapy of experimental (murine) Clostridium perfringens type A gas gangrene; Traub WH; Five antimicrobial drugs (ciprofloxacin, clindamycin, imipenem, penicillin G, and rifampin) were examined for therapeutic efficacy in a murine model gas gangrene due to Clostridium perfringens type A, following infection with large bacterial inocula . Imipenem was effective against all 6 strains of C . perfringens; conversely, penicillin G failed against these 6 strains . Ciprofloxacin, clindamycin, and rifampin occupied intermediate positions.

Antonie Van Leeuwenhoek, 1988, 54(6), 497 - 507
Purification and characterisation of glutamine synthetase from Nocardia corallina; Illing N et al.; Glutamine synthetase (GS) (EC 6.3.1.2) has been purified 67-fold from Nocardia corallina . The apparent Mr of the GS subunit was approximately 56,000 . Assuming the enzyme is a typical dodecamer this indicates a particle mass for the undissociated enzyme of 672,000 . The GS is regulated by adenylylation and deadenylylation, and subject to feedback inhibition by alanine and glycine . The pH profiles assayed by the gamma-glutamyl transferase method were similar for NH+4-treated and untreated cell extracts and an isoactivity point was not obtained from these curves . GS activity was repressed by (NH4)2SO4 and glutamate . Cells grown in the presence of glutamine, alanine, proline and histidine had enhanced levels of GS activity . The GS of N . corallina cross-reacted with antisera prepared against GS from a Gram-negative Thiobacillus ferrooxidans strain but not with antisera raised against GS from a Gram-positive Clostridium acetobutylicum strain.

J Arthroplasty, 1988, 3 Suppl, S37 - 40
Clostridium perfringens infection in a total knee arthroplasty . A case report; Stern SH et al.; A 66-year-old woman with a painful total knee arthroplasty and turbid fluid aspirates had her prosthesis removed for a presumed diagnosis of infection . The intraoperative cultures were positive for Clostridium perfringens, and the patient did well after a course of intravenous antibiotics prior to reimplantation . Clostridium perfringens is a rare cause of pyarthrosis in both nonoperative and prosthetic joints . This report details the first case of clostridial infection involving a patient with a total knee arthroplasty.

J Hyg Epidemiol Microbiol Immunol, 1988, 32(2), 219 - 26
On the in vitro neutralization test of Clostridium perfringens type A toxin; Shemanova GF et al.; Data are presented on the detection in crude animal and human sera of Cl . perfringens phospholipase C (PLC) inhibitor . When the level of Cl . perfringens type A antitoxin is determined in the in vitro toxin neutralization test the inhibitor is found to decrease PLC activity in the test dose of experimental homologous toxin . The extent of decrease accounts for the variation of results obtained in the in vitro and in vivo toxin neutralization tests . The variation may be cancelled out by introducing a corresponding coefficient to calculate the level of alpha-antitoxin . It is suggested that the isolation and investigation of the PLC inhibitor will contribute to the development of preparations for treatment of gas gangrene due to Cl . perfringens type A.

Can J Microbiol, 1988 Jan, 34(1), 78 - 9
A modified m-CP medium for enumerating Clostridium perfringens from water samples; Armon R et al.; Medium m-CP, designed for the isolation of Clostridium perfringens from water samples, contains indoxyl beta-D-glucoside, an expensive chemical that is present at a high concentration in this medium . The use of m-CP with three concentrations of indoxyl beta-D-glucoside was tested at 0, 60, and 600 mg/L . Lowering the amount of indoxyl beta-D-glucoside to 60 mg/L (1/10 the recommended concentration) reduced the cost of this medium without affecting its sensitivity.

Res Vet Sci, 1988 Jan, 44(1), 68 - 70
Influence of Clostridium perfringens and its toxin in germ-free chickens; Fukata T et al.; Twenty-one of 56 germ-free chickens died after receiving an oral inoculation of a broth culture of Clostridium perfringens . At necropsy there was detachment and disruption of the epithelial layer at the tips of villi and sloughed epithelial cells in the duodenum . These are characteristic lesions of necrotic enteritis . Germ-free chickens receiving either purified alpha-toxin or the supernatant of broth cultures of C perfringens died, but no bird died after receiving supernatant of broth cultures neutralised by anti-alpha-toxin serum . It was concluded that alpha-toxin of C perfringens was the cause of death in young germ-free chickens.

Appl Environ Microbiol, 1988 Jan, 54(1), 268 - 70
Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens; Roberts I et al.; We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E . coli plasmid (pJIR62) which provides an E . coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin . The shuttle plasmid transformed E . coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C . perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells . Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C . perfringens.

Appl Environ Microbiol, 1988 Jan, 54(1), 264 - 7
Transformation of Clostridium perfringens L forms with shuttle plasmid DNA; Mahony DE et al.; L-form (L-phase) cultures of Clostridium perfringens were tested for their transformability with plasmid DNA . Three L-form strains were transformable, but one, strain L-13, was superior to the others . This strain was easily and reproducibly transformed with previously described shuttle vectors which were derived from either C . perfringens or Escherichia coli . Strain L-13 was transformable by a variety of methods, and a new micromethod worked well under both aerobic and anaerobic conditions . The maximal number of transformants was attained after strain L-13 was exposed for 4 h to the transforming DNA and polyethylene glycol . Viable counts determined in tubes of semisolid brain heart infusion medium containing 10% sucrose, with or without 2 micrograms of tetracycline per ml, showed a transformation rate of 3.9 X 10(-5) (transformants per viable cells).

J Bacteriol, 1988 Jan, 170(1), 400 - 8
Molecular analysis and regulation of the glnA gene of the gram-positive anaerobe Clostridium acetobutylicum; Janssen PJ et al.; The nucleotide sequence of a 2.0-kilobase DNA segment containing the Clostridium acetobutylicum glnA gene was determined . The upstream region of the glnA gene contained two putative extended promoter consensus sequences (p1 and p2), characteristic of gram-positive bacteria . A third putative extended gram-positive promoter consensus sequence (p3), oriented towards the glnA gene, was detected downstream of the structural gene . The sequences containing the proposed promoter regions p1 and p2 or p3 were shown to have promoter activity by subcloning into promoter probe vectors . The complete amino acid sequence (444 residues) of the C . acetobutylicum glutamine synthetase (GS) was deduced, and comparisons were made with the reported amino acid sequences of GS from other organisms . To determine whether the putative promoter p3 and a downstream region with an extensive stretch of inverted repeat sequences were involved in regulation of C . acetobutylicum glnA gene expression by nitrogen in Escherichia coli, deletion plasmids were constructed lacking p3 and various downstream sequences . Deletion of the putative promoter p3 and downstream inverted repeat sequences affected the regulation of GS and reduced the levels of GS approximately fivefold under nitrogen-limiting conditions but did not affect the repression of GS levels in cells grown under nitrogen-excess conditions.

Microbiol Immunol, 1988, 32(12), 1201 - 10
Adsorption of LLCMK2 cell-grown Sendai virus onto human red blood cells and its release from the virus adsorbed cells; Komatsu H et al.; An early stage of virus adsorption was studied in a system of Sendai virus metabolically labeled with {3H}leucine in LLCMK2 cells and of human red blood cells (RBCs) . The efficiency of viral release from the virus-bound RBCs by incubation at 37 C depended on the number of virus particles which had been used for adsorption onto the RBC at 4 C . When 7.8 x 10(2) virus particles were previously adsorbed onto the RBC at 4 C, most of the viruses were dissociated from the RBC at 37 C . In the case of adsorption of 3 to 12 virus particles per RBC, however, most of the viruses were not dissociated from the RBC by incubation at 37 C . Such RBC-bound viruses were released by incubation with various bacterial neuraminidases (Clostridium perfringens, etc.) or with a large number of LLCMK2 cell-grown Sendai virus (LLCMK2-Sendai) particles, but not released by treatment with hemagglutinin-neuraminidase protein (Sendai-gp) isolated from egg-grown Sendai virus.

Arch Microbiol, 1988, 149(4), 280 - 5
Cloning and expression in Escherichia coli of the Clostridium thermoaceticum gene encoding thermostable formyltetrahydrofolate synthetase; Lovell CR et al.; Formyltetrahydrofolate synthetase (FTHFS) (EC 6.3.4.3), a thermostable protein of four identical subunits from Clostridium thermoaceticum was cloned into Escherichia coli SK1592 . The clone (CRL47) contained a 9.5 kb EcoRI fragment of C . thermoaceticum DNA ligated into pBR322 . It produced catalytically active, thermostable FTHFS, that was not found in E . coli SK1592 containing native pBR322 . The identity of the expressed enzyme was confirmed by specific binding of rabbit polyclonal anti-FTHFS serum produced against C . thermoaceticum FTHFS . The specific activities (mumol.min-1.mg-1) of FTHFS in cell free extracts of CRL47 were 28-89 when assayed at 50 degrees C and pH 8 . This was from 3-10-fold higher than in C . thermoaceticum extracts . FTHFS was purified to homogeneity from CRL47 . The purified enzyme behaved during electrophoresis and gel chromatography and it had similar specific activity and thermostability as the enzyme purified from C . thermoaceticum.

Toxicon, 1988, 26(6), 583 - 97
Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies; Rothman SW et al.; Clostridium difficile toxins A and B were shown to share immunochemical and structural features, including shared sequential epitopes . Nineteen hybridomas generated after immunization of mice with a mixture of toxoids produced monoclonal antibodies, all IgM(x), which bound to toxin A and toxin B in a solid-phase radioimmunoassay (RIA) . None of the antibodies neutralized the cytotoxicity of either toxin, alone or in pairs, nor did they neutralize mouse lethality . The antibodies did not inhibit hemagglutination by toxin A, and none of those tested neutralized the toxin's enterotoxic activity . Studies of binding of antibodies to native toxins in the RIA showed that the antibodies differed in their recognition of the toxins . Many of the antibodies bound with higher avidity to toxin A than to toxin B . In Western blots, all the antibodies recognized both toxins in the native state; in addition, some antibodies recognized the minor cytotoxic species of toxin B . When the toxins were denatured and reduced, five antibodies bound to both toxins, five to A only, and nine to neither, demonstrating that the antibodies had different epitope specificities . Further structural comparisons were made by investigation of mol . wts, subunit structures and amino acid compositions . The native mol . wts of toxin A and toxin B, as determined by electrophoresis to equilibrium in 4-30% polyacrylamide gel electrophoresis (PAGE), were 430,000 and 368,000, respectively . Denatured and reduced toxins each had a single subunit of 315,000 . Both toxins had about 50% hydrophobic amino acids.

Microbios, 1988, 54(219), 87 - 99
Isolation of promoters from two anaerobic bacteria; Roberts I et al.; Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli . An E . coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron . Random restriction fragments of chromosomal DNA from these organisms were fused to the galactokinase (galK) gene of E . coli within a plasmid vector . Approximately 10% of these fragments functioned as promoters in E . coli, and a broad range of activities was evident . A single 88 base pair (bp) C . absonum DNA fragment yielded, in the E . coli plasmid vector, approximately the same high activity as that provided by the E . coli galK promoter . Sequence analysis of this fragment showed typical -35 and -10 sequences, with about five -10-like sequences closely flanking each other, some overlapping, and this appears to result in multiple start sites for transcription . The transcriptions of E . coli plasmid fragments in vitro with both E . coli RNA polymerase and C . absonum RNA polymerase showed pairs of transcripts corresponding to two start sites . By colony hybridization with the 88 bp fragment, radioactively labelled, as a probe, a 4.2 kilobase segment of C . absonum chromosomal DNA containing the 88 bp fragment was isolated . About 375 bp of this fragment was sequenced . A putative Shine-Dalgarno sequence and ATG start site were detected, followed by an opening reading frame . Using a sequence about 100 bp downstream from the 88 bp sequence, a 17-base oligonucleotide was synthesized to serve as a primer . With C . absonum RNA as a template, a reverse transcriptase primer extension assay located a pair of transcription start sites just downstream from the 88 bp sequence, proving that the 88 bp sequence functions as a promoter in C . absonum.

Biochem Biophys Res Commun, 1987 Dec 16, 149(2), 424 - 30
Electron paramagnetic resonance studies of the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum; Deaton JC et al.; The redox centers in the tungsten-containing formate dehydrogenase from Clostridium thermoaceticum were examined by potentiometric titration and electron paramagnetic resonance spectroscopy . At low temperature two overlapping iron-sulfur signals which correlated with enzymatic activity were observed with formal potentials near -400 mV vs . SHE . Based on their temperature dependences, one signal is assigned to a reduced Fe2S2 cluster and one to a reduced Fe4S4 cluster . Quantitation of signal intensity suggests two Fe2S2 and two Fe4S4 clusters per formate dehydrogenase molecule . Another signal (g = 2.101, 1.980, 1.950) present in low concentrations at more negative potentials was observable up to 200 degrees K and is not attributed to any iron-sulfur cluster . The possible origin of this signal is analyzed using ligand field theory, and the redox behavior is considered with respect to possible ligation at the active site.

Pharm Weekbl Sci, 1987 Dec 11, 9 Suppl, S45 - 7
Ciprofloxacin for infection prevention in patients with acute leukemia; Rozenberg-Arska M et al.; Ciprofloxacin, a new quinolone derivative, was given prophylactically (500 mg twice daily) to 15 patients with acute leukemia during remission induction treatment . The effect on the microbial flora of the alimentary tract was evaluated . A rapid elimination of Enterobacteriaceae was observed . Bacteriodes and Clostridium species were not affected . Few ciprofloxacin resistant strains were isolated but did not lead to colonization . In a randomized study 56 patients with acute leukemia received either ciprofloxacin or trimethoprim-sulfamethoxazole plus colistin for prevention of infections . Six major infections occurred in 28 patients receiving ciprofloxacin, and 11 major infections in 28 patients receiving trimethoprim-sulfamethoxazole plus colistin . No infections caused by Gram-negative bacilli were seen in the ciprofloxacin group compared to 17 in the other group (p less than 0.02) . Ciprofloxacin prevented colonization with resistant Gram-negative bacilli while 12 resistant colonizing strains were isolated from 10 patients receiving trimethoprim-sulfamethoxazole (p less than 0.01) . Ciprofloxacin was better tolerated than trimethoprim-sulfamethoxazole + colistin; fewer side effects occurred.

FEBS Lett, 1987 Dec 10, 225(1-2), 82 - 6
Molecular cloning and expression of Clostridium difficile toxin A in Escherichia coli K12; Wren BW et al.; Clostridium difficile toxin A was purified to homogeneity and was used to raise monospecific antiserum in rabbits . A gene bank of C . difficile DNA in Escherichia coli was constructed by cloning Sau3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3 . Out of 4500 plaques screened with antitoxin A, 9 clones were positively identified . One of these clones lambda tA5 expressed a 235 kDa protein which exhibited a cytotonic effect on Chinese hamster ovary cells, and had the ability to haemagglutinate rabbit erythrocytes, both properties characteristic of toxin A . The size of the lambda tA5 insert DNA was 14.3 kb.

FEBS Lett, 1987 Dec 10, 225(1-2), 48 - 52
Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177; Vandekerckhove J et al.; Clostridium perfringens iota toxin ADP-ribosylates actin . Substrates of C . perfringens toxin are both non-muscle beta/gamma-actin and skeletal muscle actin . This finding suggests that C . perfringens iota ADP-ribosylates the same amino acid in skeletal muscle and non-muscle actin as does C . botulinum C2 toxin in non-muscle actin . Protein chemical analysis involving thermolysin cleavage on {32P}ADP-ribosylated actin or tryptic digestion followed by a secondary thermolysin cleavage of the radiolabelled fragments showed one major site of ADP-ribosylation . From its amino acid composition and sequence, the radiolabelled peptide was identified as peptide 175-177, locating the acceptor ADP-ribosyl amino acid as Arg-177.

Eur J Biochem, 1987 Dec 1, 169(2), 441 - 8
Purification of 2-hydroxyglutaryl-CoA dehydratase from Acidaminococcus fermentans . An iron-sulfur protein; Schweiger G et al.; 1 . The (R)-2-hydroxyglutaryl-CoA dehydratase system from Acidaminococcus fermentans was separated by chromatography of cell-free extracts on Q-Sepharose into two components, an activator and the actual dehydratase . The latter enzyme was further purified to homogeneity by chromatography on blue-Sepharose . It is an iron-sulfur protein (Mr 210,000) consisting of two different polypeptides (alpha, Mr 55,000, and beta, Mr 42,000) in an alpha 2 beta 2 structure with probably two {4Fe-4S} centers . After activation this purified enzyme catalysed the dehydration of (R)-2-hydroxyglutarate only in the presence of acetyl-CoA and glutaconate CoA-transferase, demonstrating that the thiol ester and not the free acid is the substrate of the dehydration . The result led to a modification of the hydroxyglutarate pathway of glutamate fermentation . 2 . The activation of the dehydratase by the flow-through from Q-Sepharose concentrated by ultrafiltration required NADH, MgCl2, ATP and strict anaerobic conditions . This fraction was designated as Ao . Later when the concentration was performed by chromatography on phenyl-Sepharose, an NADH-independent form of the activator, designated as A*, was obtained . This enzyme, which required only ATP for activation of the dehydratase, was purified further by affinity chromatography on ATP-agarose . It contains neither iron nor inorganic sulfur . A*, as well as the activated dehydratase, were irreversibly inactivated by exposure to air within less than 15 min . The activated dehydratase but not A* was also inactivated by 1 mM hydroxylamine or by 0.1 mM 2,4-dinitrophenol . 3 . The (R)-2-hydroxyglutaryl-CoA dehydratase system is closely related the that of (R)-lactoyl-CoA dehydratase from Clostridium propionicum as described by R . D . Kuchta and R . H . Abeles {(1985) J . Biol . Chem . 260, 13,181-13,189}.

J Bacteriol, 1987 Dec, 169(12), 5845 - 7
Carbon monoxide-driven electron transport in Clostridium thermoautotrophicum membranes; Hugenholtz J et al.; Membrane vesicles of Clostridium thermoautotrophicum prepared by osmotic lysis after lysozyme treatment contained carbon monoxide dehydrogenase and methylenetetrahydrofolate dehydrogenase with specific activities three- to fourfold higher than the specific activity of the cytoplasm . The membrane-associated carbon monoxide dehydrogenase mediated the reduction with CO or the oxidation with CO2 of b-type cytochromes and other electron carriers in the membrane.

Infect Immun, 1987 Dec, 55(12), 2984 - 92
Immunization of adult hamsters against Clostridium difficile-associated ileocecitis and transfer of protection to infant hamsters; Kim PH et al.; In this investigation, the role of antibodies against Clostridium difficile toxins A and B in protecting hamsters against C . difficile-associated ileocecitis was examined . We also studied the transfer of protection against C . difficile-associated intestinal disease from immunized female hamsters to their infants . Adult female hamsters immunized parenterally with toxoid A or a mixture containing both toxoids A and B were protected against clindamycin-induced C . difficile-associated fatal ileocecitis . On the other hand, hamsters immunized with toxoid B or a broth filtrate from a nontoxigenic strain of C . difficile were not protected against C . difficile-induced ileocecitis . Antibody against the immunizing toxoid could be demonstrated in both the serum and the cecal contents of hamsters . Some infant hamsters from mothers immunized with toxoid A or AB were protected against C . difficile-associated ileocecitis, while infant hamsters from mothers immunized with toxoid B or a nontoxigenic broth filtrate were not protected against disease . Neutralizing antibodies to toxins A and B could be demonstrated in both maternal milk and serum, as well as in infant serum and intestinal contents . Foster-mothering experiments demonstrated that maternal protection of infants against C . difficile-associated ileocecitis was transferred to infant hamsters through breast milk . These results suggest that toxin A may play a more important role in the pathogenesis of C . difficile-associated ileocecitis in hamsters than toxin B . Furthermore, variations in the severity of C . difficile-associated illness in infants and adults may reflect the lack or presence of passively or actively acquired immunity against C . difficile toxins.

Antimicrob Agents Chemother, 1987 Dec, 31(12), 2010 - 2
Activity of cefmetazole against anaerobic bacteria; Cornick NA et al.; The in vitro activity of cefmetazole versus that of other antimicrobial drugs was assessed against 374 clinical isolates of Bacteroides spp., Clostridium spp., and anaerobic gram-positive cocci . Compared with cefoxitin, cefmetazole showed good activity against Bacteroides fragilis, other Bacteroides species, and anaerobic cocci . It was somewhat less active than cefoxitin against Bacteroides thetaiotaomicron, B . ovatus, B . distasonis, and B . vulgatus and somewhat more active against Clostridium spp.

Mol Cell Probes, 1987 Dec, 1(4), 337 - 45
Monoclonal antibody for the detection of Clostridium botulinum type A toxin; Ferreira JL et al.; Hybridomas producing monoclonal antibodies against type A Hall strain Clostridium botulinum toxin were generated by fusing mouse myeloma cell line P3-X63-Ag8.653 with spleen cells of Balb/c mice immunized with C . botulinum type A toxoid . The monoclonal antibody from one hybridoma, identified as No . 424, was selected from 61 others for its high antibody titre . This monoclonal antibody was used in a double-sandwich enzyme-linked immunosorbent assay (ELISA) system to detect type A toxin in culture fluids and in foods . The monoclonal antibody did not react with either C . botulinum toxin types B, C, D, E and F or with other clostridial species tested . This particular monoclonal antibody (No . 424) did not neutralize type A toxin in the mouse bioassay procedure but detected approximately 10 mouse lethal doses of type A toxin/ml culture fluid . Monoclonal antibody and rabbit antitoxin to type A C . botulinum toxin were useful in a double-sandwich ELISA for the rapid and specific detection of type A toxic fluids in culture and in food samples.

Diagn Microbiol Infect Dis, 1987 Dec, 8(4), 203 - 14
Effect of age on the sensitivity of cell cultures to Clostridium difficile toxin; Tichota-Lee J et al.; The effect of age on the sensitivity of four cell lines, human foreskin fibroblasts (HFS), CHO-K1, HEp-2, and WI-38 to detect Clostridium difficile toxin was tested . This study also addressed the sensitivity of these cell lines as expressed by early toxin detection . Twenty-eight positive and 13 negative patient specimens were tested . Cell cultures were inoculated at ages 3, 4, 5, 6, 7, 9 and 14 days and examined for cytopathic effects at 4, 24, and 48 hours post-inoculation . The sensitivity of three of the four cell cultures to C . difficile toxin decreased as the age of the cell cultures increased . However, the sensitivity of the HFS cell line was not influenced by the age of the culture or the time the assay was read in comparison to the other three cell lines . Five- to six-day-old HFS cell cultures detected 22/28 positive samples within 4 hr after inoculation and 28/28 positive samples by 24 hr post-inoculation.

Eur J Clin Microbiol, 1987 Dec, 6(6), 623 - 7
Epidemiology and prevention of Clostridium difficile infections in a leukemia unit; Delmee M et al.; A 29-month prospective study was carried out in a leukemia unit with the aim of investigating the epidemiology of Clostridium difficile infections and limiting their spread . Systematic cultures of stools and assays for cytotoxin were performed on patient admission and at weekly intervals, yielding 1,355 cultures and assays . The study period was divided in period A, before total unit renovation, and period B, afterwards . During period B all patient carriers of Clostridium difficile received vancomycin . A comparison of the two periods showed that the percentage of positive cultures fell from 16.6% to 3.6% and the positive toxin assays from 9.9% to 1.2% . It was concluded that colonization by Clostridium difficile can be prevented in hospital wards with generally high rates of infection by a combination of decontamination of the environment, introduction of preventive measures and treatment of Clostridium difficile carriage with vancomycin.

Appl Environ Microbiol, 1987 Dec, 53(12), 2827 - 30
Simplified purification method for Clostridium botulinum type E toxin; Gimenez JA et al.; Clostridium botulinum type E toxin was purified in three chromatography steps . Toxin extracted from cells was concentrated by precipitation and dissolving in a small volume of citrate buffer . When the extract was chromatographed on DEAE-Sephadex without RNase or protamine treatment, the first protein peak had most of the toxin but little nucleic acid . When the toxic pool was applied to a carboxymethyl Sepharose column, toxin was recovered in the first protein peak in its bimolecular complex form . The final chromatography step at 4 degrees C on a DEAE-Sephacel column at a slightly alkaline pH purified the toxin (Mr, 145,000) by separating the nontoxic protein from the complex . At least 1.5 mg of pure toxin was obtained from each liter of culture, and the toxicity was 6 X 10(7) 50% lethal doses per mg of protein . These values are significantly higher than those previously reported.

Jpn J Exp Med, 1987 Dec, 57(6), 315 - 20
Colonization of the intestinal tract of mice with Clostridium tetani; Ebisawa I et al.; Colonization of intragastrically inoculated Clostridium tetani in intestinal tract of mice was investigated by cultivation of daily stool samples and quantification of the bacilli over a period of 3 weeks . We found that most of the inoculum was excreted in the stool during the first 24 hr . The amount of C . tetani recovered on the second day after inoculation was very small . C . tetani apparently thrived in the intestinal tract when more than 6 log colony forming units (CFU) of tetanus spores had been inoculated . Otherwise, C . tetani colonized poorly in intestinal tract . Tetanus toxin was not detected in the stool nor in the contents of the small and large intestines 24 hr after 6.23 log CFU of tetanus spores had been inoculated . We concluded that the intestinal cavity of mouse is not a favorable environment for colonization of C . tetani under natural conditions.

J Clin Microbiol, 1987 Dec, 25(12), 2334 - 8
Examination of feces and serum for diagnosis of infant botulism in 336 patients; Hatheway CL et al.; In the 12-year period 1975 to 1987, feces from 336 infants were examined for botulinal neurotoxin and Clostridium botulinum . All the infants had illnesses which prompted their physicians to consider infant botulism in the diagnosis . Stool specimens from 113 of the infants yielded organisms that produced botulinal neurotoxins assumed to be responsible for the illness . The types of botulinal toxin in the confirmed cases were distributed as follows: 38 A, 69 B, 2 atypical B, 1 E, 1 F, 1 A + B, and 1 B + F . The type A and B toxins in a single infant were produced by two different strains of organism, and the type B and F toxins in another infant were produced by a single strain . The physiological characteristics of all the isolated toxigenic organisms except two were consistent with those of group I (proteolytic) C . botulinum . The toxigenic isolate from the infant with type E botulism was identified as C . butyricum, and that from the infant with type F botulism was identified as C . barati . Toxin of the same type as produced by the isolated organisms was identified in feces of 98 of 111 culture-positive infants . Botulinal toxin was identified in the serum of 9 of 67 culture-positive infants (8 of 22 infants with type A organisms; 1 of 43 infants with type B organisms; neither of 2 infants with A + B or atypical type B organisms) . Botulinal toxin was not detected in feces (206 infants) or in serum (114 infants) of the culture-negative infants . The culture-positive infants had clinical features and a course of illness consistent with those of infant botulism . Most of the culture-negative infants probably had illnesses other than botulism, but specimens might have been obtained late in some infants' illnesses, when the organism had disappeared.

J Clin Pathol, 1987 Dec, 40(12), 1397 - 401
Association between production of toxins A and B and types of Clostridium difficile; Wren B et al.; One hundred and seventy two strains of Clostridium difficile isolated from 62 patients with antibiotic associated diarrhoea or pseudomembranous colitis were analysed for the production of toxins A and B and typed using 35S-methionine labelling followed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) . There was a correlation between production of toxins A and B and the type of C difficile . One hundred and forty four of 172 strains were either high or low producers of both toxins . Toxins were not detected in 28 of 172 strains . Types A and Y were consistently non-toxin producers; types B and E were high toxin producers . Type X, the epidemic strain, showed variable toxin production . Symptoms of 49 patients with haematological malignant pathology who were part of a documented outbreak of antibiotic associated diarrhoea, were analysed in relation to toxin production and type of the clinical strains isolated . In general, there was a correlation between symptoms of antibiotic associated diarrhoea, the type of C difficile, and its potential for producing toxins.

J Am Coll Nutr, 1987 Dec, 6(6), 517 - 23
Diarrhea in the intensive care unit: the role of hypoalbuminemia and the response to a chemically defined diet (case reports and review of the literature); Brinson RR et al.; We describe five patients who developed acute kwashiorkor-like hypoalbuminemia during their hospitalization in the intensive care unit . With the initiation of enteral alimentation, diarrhea ensued and continued for at least 48 hours . Routine evaluation for the cause of diarrhea including stool culture for enteric pathogens, white blood cells, ova and parasites, Clostridium difficile cytotoxin, and flexible sigmoidoscopy was negative . When a peptide based formula (Vital HN, Ross Laboratories, Columbus, OH) was initiated, there was a significant reduction in stool output within 24 hours . In three of the five cases, hypoalbuminemia corrected within 2 weeks . Four of the five patients were discharged from the hospital . Although several reports have acknowledged the association between hypoalbuminemia and impaired gastrointestinal absorption, no previous enteral formula has been tolerated in these acutely ill patients until serum albumin levels had improved . Further studies will be required to confirm the better gastrointestinal tolerance of this peptide based formula in patients with severe hypoalbuminemia.

Microb Pathog, 1987 Dec, 3(6), 469 - 74
Carboxyl groups in Clostridium perfringens epsilon toxin; Sakurai J et al.; The maximal number of norleucine methyl ester (NME) incorporated into carboxyl groups in epsilon toxin of Clostridium perfringens by the carbodiimide-nucleophile procedure was 7 and 17 in the absence and presence of 8 M urea, respectively . The introduction of 3-4 nucleophilic modifying agents such as NME, glycine methyl ester or taurine into carboxyl groups of the toxin reduced the lethality to approximately 10% of the original activity . The incorporation of 6-7 of these agents resulted in complete loss of the activity . On the other hand, circular dichroism spectra and the reaction between the toxin or the NME-incorporated toxin and antiepsilon toxin reaction showed no difference between the intact toxin and the NME-incorporated toxin . The data suggested that at least 4 out of 7 carboxyl groups on the surface of the toxin are important in maintaining the lethal activity of toxin.

Infect Immun, 1987 Dec, 55(12), 2912 - 5
Isolation and function of a Clostridium perfringens enterotoxin fragment; Horiguchi Y et al.; A fragment was obtained by treating Clostridium perfringens enterotoxin with 2-nitro-5-thiocyanobenzoic acid, a reagent which specifically cleaves the amino-terminal peptide bond of cysteine residues . The fragment (molecular weight, 15,000) was purified by high-performance liquid chromatography . The fragment had no cytotoxic effect on Vero cells but competitively inhibited enterotoxin-induced 51Cr release . Binding of 125I-labeled fragment to Vero cells was comparable to that of enterotoxin . Moreover, 125I-labeled fragment did not bind to FL cells, which lack receptor for enterotoxin . We conclude that the fragment contains the binding domain of enterotoxin . The amino acid composition of the fragment suggests that it is located on the carboxyl-terminal part of enterotoxin.

J Clin Microbiol, 1987 Dec, 25(12), 2402 - 4
Restriction endonuclease DNA analysis of Clostridium difficile; Wren BW et al.; HindIII restriction enzyme digests of genomic DNA from nine distinct strains of Clostridium difficile were undertaken, and the results were related to those of a previously established typing method based on {35S}methionine-labeled protein profiles . Each of the typed strains identified by its protein profile could also be distinguished by its unique DNA digestion pattern . Analysis of strains isolated from 10 patients during a hospital outbreak of antibiotic-associated colitis revealed identical DNA profiles, confirming a single strain as the source of cross-infection . Characterization of isolates from worldwide sources revealed similar digestion patterns within the same strain type . Restriction endonuclease DNA analysis provides a sensitive and useful technique for studying the epidemiology of C . difficile.

Infect Immun, 1987 Dec, 55(12), 3051 - 6
Antigenic structure of Clostridium botulinum type B neurotoxin and its interaction with gangliosides, cerebroside, and free fatty acids; Kozaki S et al.; A fragment distinct from the heavy and light chains was obtained by treatment of Clostridium botulinum type B neurotoxin with chymotrypsin . Enzyme-linked immunosorbent assay and immunoblotting analysis with monoclonal antibodies showed that the fragment consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond . Monoclonal antibodies reacting to the heavy chain but not to the fragment were thought to recognize the epitopes on the remaining portion (H-2 fragment) of the heavy chain, being easily digested by chymotrypsin . Thus, the antigenic structure of type B neurotoxin resembles those of type A and E neurotoxins . The chymotrypsin-induced fragment bound to cerebroside and free fatty acids but not to gangliosides . The manner of binding of type B neurotoxin to gangliosides and free fatty acids was different from those of type A and E neurotoxins . Such differences in the reactivities to lipids may be related to the finding that each neurotoxin binds to a type-specific site on the neural membrane.

Biochim Biophys Acta, 1987 Nov 13, 904(2), 283 - 9
Lipid shape as a determinant of lipid composition in Clostridium butyricum . The effects of incorporation of various fatty acids on the ratios of the major ether lipids; Goldfine H et al.; The lipid composition of Clostridium butyricum is strongly influenced by the aliphatic chain compositions of the membrane lipids . Growth on cis-monounsaturated fatty acids in the absence of biotin was shown to affect the relative proportions of phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine most strongly, with smaller effects on the acidic lipids, phosphatidylglycerol and cardiolipin . The ratio of the glycerol acetal of plasmenylethanolamine to total phosphatidylethanolamine in cells grown on a series of fatty acids is shown to decrease in the following order; cis-vaccenic acid greater than or equal to oleic acid = C19-cyclopropane fatty acid greater than linoleic acid greater than petroselinic acid greater than elaidic acid greater than 14-methylhexadecanoic acid (anteiso-C17) greater than 12-methyltridecanoic acid (iso-C14) . All fatty acids were extensively incorporated into the lipid acyl, alkenyl, and alkyl chains . There was considerable chain-elongation of the iso-C14 to iso-C16 . The results are consistent with the hypothesis that the membrane lipid composition is strongly influenced by lipid shape and that the observed changes in lipid composition serve to stabilize the bilayer arrangement of the cell membrane.

N Z Med J, 1987 Nov 11, 100(835), 685 - 6
Ileal infarction: a rare cause of tetanus in the elderly; Holdaway CM et al.; We report the case of a 70 year-old woman who presented with clinical tetanus following a four day history of abdominal pain and vomiting . The source of Clostridium tetani was an infarcted loop of ileum resulting from pelvic adhesions . The treatment of tetanus and the incidence of endogenous sources of Clostridium tetani are reviewed . Health Department figures for the decade 1973-1982 show that 70% of reported cases of clinical tetanus occur in those aged 45 years or older.

J Biol Chem, 1987 Nov 5, 262(31), 15054 - 61
The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum; Adams MW; Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO . These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type {4Fe-4S} clusters (F) . The results show that the H clusters of these two hydrogenases have very different properties . The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent . Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum . The H cluster of reduced hydrogenase II (Hred-II) does not react with CO . In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH . Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal . Hred-I also binds CO irreversibly . The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment . Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis . Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO) . These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation . The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.

J Clin Microbiol, 1987 Nov, 25(11), 2241 - 2
Rectal swab cultures for Clostridium difficile surveillance studies; McFarland LV et al.; We compared the recovery of Clostridium difficile from hospitalized patients by two collection methods: rectal swabs and stool cultures . Rectal swab cultures were as sensitive as stool cultures and were more easily obtained . Transport of swabs in an anaerobic VACUTAINER system resulted in longer survival times compared with transport in Amies medium.

J Med Microbiol, 1987 Nov, 24(3), 197 - 203
Quantitative cell-adhesion assay for Clostridium difficile cytotoxin; Mayall BC et al.; A quantitative assay for Clostridium difficile cytotoxin has been developed, based on the observation that suspended fibroblasts exposed to cytotoxin fail to adhere to plastic . A dye-binding technique was used to quantitate adherent cells, in order to obviate microscopy . Adherent BHK cells were fixed with glutaraldehyde and cell protein was stained with Coomassie blue R-250 . Cell-bound dye was eluted and estimated spectrophotometrically . The amount of eluted dye was proportional to the number of adherent cells and cell staining was time dependent . Cytotoxin was purified by gel-permeation and ion-exchange chromatography and migrated as a single band on SDS-PAGE . After exposure of suspended BHK cells to purified cytotoxin, their adhesion to plastic was inhibited in a manner which depended on concentration of cytotoxin and on time and temperature of exposure . This study provides the basis for a C . difficile cytotoxin assay that is quantitative, rapid and reproducible and may have wider applicability in the study of other toxins or agents that inhibit cell adhesion.

Postgrad Med J, 1987 Nov, 63(745), 993 - 4
Clostridium difficile colitis following treatment with metronidazole and vancomycin; Bingley PJ et al.; A 25 year old woman developed Clostridium difficile colitis following a course of vancomycin and metronidazole prescribed for pelvic inflammatory disease . The condition resolved after treatment with vancomycin given alone . Colitis following this combination of antibiotics has not been described previously.

J Antimicrob Chemother, 1987 Nov, 20 Suppl B, 13 - 9
In-vitro comparison of roxithromycin and erythromycin against 900 anaerobic bacterial strains; Dubreuil L; The in-vitro antibacterial activity of roxithromycin was assessed by an agar dilution method against 900 recent clinical anaerobic isolates by five laboratories in England, France, Germany and Japan . Roxithromycin had similar activity to erythromycin against most anaerobic bacteria, the latter being slightly more active against Gram-negative bacilli . Roxithromycin inhibited 53% of the Bacteroides fragilis group strains; the noticeable exception was Bact . thetaiotaomicron, only 17% of strains of which were inhibited by 4 mg/l roxithromycin . In contrast, all isolates of Bacteroides, other than the Bact . fragilis group, and all Mobiluncus isolates were inhibited by 2 mg/l of roxithromycin or less . This compound was inactive against half the Fusobacterium strains . Roxithromycin and erythromycin inhibited 65 and 72%, respectively, of 436 strains of Gram-negative anaerobes (MIC less than or equal to 4 mg/l) . Roxithromycin suppressed 97% of non-sporulating Gram-positive to bacilli, 86% of Peptococcoaceae and 95% of clostridia . Two-thirds of Clostridium difficile strains were susceptible to roxithromycin (MIC less than or equal to 1 mg/l) . The activity of roxithromycin against Gram-positive bacilli was identical to that of erythromycin . Overall a concentration of 4 mg/l roxithromycin and erythromycin inhibited 79 and 83%, respectively, of the strains investigated.

Allergy, 1987 Nov, 42(8), 591 - 6
Cell-mediated immunity assessed by skin testing (Multitest) . I . Normal values in healthy Danish adults; Moesgaard F et al.; The Multitest CMI system consists of a disposable multiple puncture device that simultaneously applies seven standardized recall antigens for assessment of cell-mediated immunity (CMI) . The seven antigens included were toxoid from Clostridium tetani and Corynebacterium diphtheriae, tuberculin, plus antigens from streptococcus (group C), Candida albicans, Trichophyton mentagrophytes, and Proteus mirabilis . A population of 352 healthy Danish adults, aged between 17 and 90 years, was tested to determine the incidence and size of delayed-type hypersensitivity (DTH) responses . All but six healthy adults (98%) responded to one or more antigens, the median number of positive responses being four in males and three in females . The incidence of positive responses ranged from 91% for tuberculin to 11% for trichophyton . The number of positive responses declined with age, being somewhat faster in females than males . Six of the seven antigen response rates were significantly lower in the over 65-years-olds, the only exception being trichophyton, and for four of the seven antigens significantly lower in females compared to males . When correlated to age and sex no major differences were found with regard to the size of response to the seven antigens except that the tuberculin response was larger in males . A scoring system based on both number and size of positive responses revealed significant age and sex related differences . The median "score" in 17-65-year-old males and females were, respectively, 17 mm and 14 mm compared to 13 mm and 8 mm in those over 65 years old (P less than 0.001 for both comparisons).

J Appl Bacteriol, 1987 Nov, 63(5), 443 - 8
Antibody response to bacterial antigens covalently bound to biodegradable polymerized serum albumin beads; Langhein C et al.; Model vaccines have been made by covalently linking Clostridium botulinum type D toxin and Klebsiella pneumoniae capsular polysaccharide antigen to polymerized rabbit serum albumin beads . When injected into rabbits these bead vaccines induced an enhanced production of specific humoral antibody without causing adverse reactions . The adjuvant effect is due to a slow release from the bead structure and offers an alternative to oil emulsions and mineral salt adsorbents.

J Appl Bacteriol, 1987 Nov, 63(5), 387 - 93
The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum; Graham AF et al.; Low-acid foods (pH greater than or equal to 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions . The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions . The combined effect of incubation temperatures of 12 degrees and 16 degrees C and pH values between 5.2 and 5.5 on growth and toxin production from spores of Cl . botulinum during incubation for 28 d has been investigated . Growth and formation of toxin (type B) were detected only in medium at pH 5.5 and incubated at 16 degrees C, corresponding to a probability of growth from a single spore within 14 d of 1.6 x 10(-5) . The probability of growth in 28 d in the remaining conditions was less than 9 x 10(-6) . After transfer of inoculated media from 12 degrees to 30 degrees C growth occurred at pH 5.2-5.5 within 19 d . After transfer of inoculated media from 12 degrees to 20 degrees C growth occurred at pH 5.5 and 5.4 but not at pH 5.3 or 5.2 in 40 d . Growth at pH 5.2-5.5 was accompanied by formation of toxin, in most cases of types A or B . In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.

Mayo Clin Proc, 1987 Nov, 62(11), 1013 - 7
Symposium on antimicrobial agents . Metronidazole; Rosenblatt JE et al.; Metronidazole, a nitroimidazole derivative, is a unique antimicrobial agent that is active against both bacterial and parasitic organisms, although only the anaerobic members of these groups are susceptible . It has been used for the treatment of trichomoniasis for almost 30 years and is also effective in amebiasis and giardiasis . More recently, metronidazole has emerged as a principal agent for the treatment of anaerobic infections . It is highly effective against all species of anaerobes except certain non-spore-forming gram-positive bacilli and cocci and is the only agent rapidly bactericidal against the Bacteroides fragilis group . The hydroxy metabolite is 65% as effective as metronidazole and may play a major therapeutic role . Clinical studies have substantiated its efficacy for prophylaxis during elective colorectal surgical procedures and the treatment of deep abdominal sepsis (usually in combination with another agent such as an aminoglycoside) . Metronidazole is the treatment of choice for bacterial vaginosis and seems to be as effective as vancomycin for treatment of Clostridium difficile-related diarrhea and colitis . Good blood levels are produced after both oral and intravenous administration, and side effects are infrequent and minimal . Metronidazole should not be taken during the first trimester of pregnancy because of concerns about mutagenicity . Tinidazole and ornidazole are recently developed nitroimidazole derivatives that have even greater antimicrobial activity than metronidazole.

Biochimie, 1987 Nov-Dec, 69(11-12), 1183 - 90
Effect of pyruvate on glucose metabolism in Clostridium acetobutylicum; Junelles AM et al.; Pyruvate effects on the metabolism of Clostridium acetobutylicum during glucose fermentation were studied . After addition to the culture medium, the pyruvate was rapidly used, provoking several changes in the metabolic pattern of the bacteria . When pyruvate addition occurred early in the fermentation, the glucose utilization decreased and the solventogenic phase was not induced . When pyruvate was added during solventogenesis, glucose consumption was slightly affected and the cells fermented both substrates simultaneously: however, the acidogenic phase started again to the detriment of solvent formation . Usually, during the solvent phase, the cells remetabolized acetic and butyric acids into solvents, but when pyruvate was added, the utilization of acids was stopped and the specific rates of acetate and butyrate formation increased immediately . The acidogenic growth phase was characterized by high levels of acetate and butyrate kinase which dropped during the solvent phase . Addition of pyruvate limited the down shift of these two enzymes and the levels of the activities remained constant during the course of the fermentation . Conversely, the acetoacetate decarboxylase, which is characteristic of the solvent phase, decreased sharply in the presence of pyruvate . The fact that the specific rate of glucose consumption was not decreased by the pyruvate metabolism, a cosubstrate, proves that the phosphoroclastic reaction is not a limiting step . Furthermore, the pyruvate utilization represented a promising approach to obtain useful data on the intracellular compounds implicated in the mechanism for switching from the acidogenic to the solventogenic phase.

Ann Inst Pasteur Microbiol, 1987 Nov-Dec, 138(6), 597 - 608
Mode of action of Clostridium perfringens initiation protein (spore-lytic enzyme); Tang SS et al.; The extracellular initiation protein (IP; spore germination enzyme) produced by Clostridium perfringens was further purified and characterized . IP hydrolysed spore cortical fragments with the release of free amino groups . End group analysis of hydrolysed fragments indicated the presence of N-terminal alanine but no reducing sugars . Molecular weight analysis of IP- and lysozyme-treated fluorescamine-labelled cortical fragments indicated that IP acts only on peptidoglycan chains containing cross-linked peptide subunits . IP failed to hydrolyse a number of nitrophenyl-conjugated glucopyranosides and galactopyranosides . The results indicate that IP is an N-acetylmuramyl-L-alanine amidase.

Postgrad Med J, 1987 Nov, 63(745), 955 - 7
Clostridium difficile, sulphasalazine, and ulcerative colitis; Burke DA et al.; Clostridium difficile has been implicated in the relapse of ulcerative colitis . Controversy exists over this role and its relationship to sulphasalazine exposure . Sixty two of 77 patients with a documented relapse of ulcerative colitis were investigated for the presence of Clostridium difficile, or its toxin, prior to hospitalization . There was a low incidence of detection which was related to antibiotic exposure (2/62) . Sampling during the treatment period showed that the occurrence of Clostridium difficile in the stool was related to antibiotic treatment (2/66) . Fifty six percent of patients were taking sulphasalazine, none of whom became culture or toxin positive . This study demonstrates that Clostridium difficile is not related to relapse of ulcerative colitis and is not secondarily acquired during relapse unless the patient is exposed to antibiotics . Sulphasalazine does not predispose to acquisition of Clostridium difficile . There is no role for routine screening or treatment of Clostridium difficile in ulcerative colitis.

J Assoc Off Anal Chem, 1987 Nov-Dec, 70(6), 994 - 6
Enumeration of Clostridium perfringens spores in human feces: comparison of four culture media; Harmon SM et al.; Enumeration of Clostridium perfringens spores was compared using 4 culture media . Duplicate 1 g portions of 35 stools (25 from C . perfringens food poisoning outbreaks and 10 from normal stools) were heat treated 20 min at 75 degrees C and tested on tryptose-sulfite-cycloserine (TSC) agar, trypticase-soy-blood (TSB) agar, lactose-sulfite (LS) medium, and iron milk (IM) medium . Dilutions were plated directly onto TSB and TSC, and a 3-tube most probable number determination was made with each specimen in LS and IM incubated at 45 degrees C . TSB was easiest to use and nonhemolytic food poisoning strains were readily differentiated from the normal hemolytic biotype on this medium . Confirmed counts on TSC and TSB were similar for all specimens, but counts of 8 of 25 outbreak specimens were 2-4 log units lower in LS and IM than on plating media; spores in specimens associated with 2 of 5 outbreaks were intolerant of the elevated temperatures . Results showed that elevated temperature MPN methods in LS and IM are inappropriate for the examination of outbreak stools.

J Hosp Infect, 1987 Nov, 10(3), 243 - 7
Investigation of Clostridium difficile diarrhoea in a district general hospital: room for improvement?
Lewis R.
A retrospective survey of case-notes was carried out on all patients investigated for possible Clostridium difficile diarrhoea during a 1-month period at a district general hospital . Seven of 29 patients probably had the disease, but no case of pseudomembranous colitis was documented . Delay in reporting and discrepancies between culture and cytotoxin results contributed in several cases to mismanagement . In 14 of 16 cases negative by culture and cytotoxin, a plausible non-microbiological case for diarrhoea was found, including aperients in six.

Arch Biochem Biophys, 1987 Nov 1, 258(2), 504 - 9
The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine; Riedy GP et al.; Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity . These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme . The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed . Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom . These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate . The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period . The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.

J Clin Microbiol, 1987 Nov, 25(11), 2168 - 72
Restriction endonuclease analysis of nosocomial isolates of Clostridium difficile; Devlin HR et al.; A total of 110 clinical isolates of Clostridium difficile were analyzed by agarose gel electrophoresis by using both bacterial restriction endonuclease analysis (REA) and plasmid profiles . A total of 72 isolates were divided into 12 groups according to their REA patterns . Some 38 isolates exhibited unique patterns . Pattern A occurred in 20% of isolates . Isolates with patterns B, E, and G were cytotoxin negative . The remaining groups were cytotoxin positive . Multiple isolates obtained from two stool specimens were studied to examine the variation in REA profiles found in single specimens . In these specimens no variation in REA profiles was found . The stability of C . difficile was studied by examining sequential in vitro subcultures of a single isolate and strains isolated over a 4-month period from two long-term carriers . REA patterns were stable over time, both in vitro and in vivo . Because plasmid DNA was observed in 53% of isolates, plasmid profiles alone could not be used to study the spread of C . difficile; however, they were necessary for the interpretation of REA patterns in some instances.

Biochem J, 1987 Oct 15, 247(2), 363 - 8
ADP-ribosylation of a 21-24 kDa eukaryotic protein(s) by C3, a novel botulinum ADP-ribosyltransferase, is regulated by guanine nucleotide; Aktories K et al.; Besides botulinum C2 toxin, Clostridium botulinum type C produces another ADP-ribosyltransferase, which we termed 'C3' . ADP-ribosyltransferase C3 has a molecular mass of 25 kDa and modifies 21-24 kDa protein(s) in platelet and brain membranes . C3 was about 1000 times more potent than botulinum C1 toxin in ADP-ribosylation of membrane proteins . C3-catalysed ADP-ribosylation of the 21-24 kDa protein(s) was decreased by stable guanosine triphosphates, with the potency order GTP{S} much greater than p{NH}ppG greater than p{CH2}ppG . GTP{S} inhibited the ADP-ribosylation caused by C3 by maximally 70-80%, with half-maximal and maximal effects occurring at 0.3 and 10 microM-GTP{S} respectively . The concomitant addition of GTP decreased the inhibitory effect of GTP{S} . GTP{S}-induced inhibition of ADP-ribosylation was resistant to washing of pretreated platelet membranes . The data suggest that the novel botulinum ADP-ribosyltransferase C3 modifies eukaryotic 21-24 kDa guanine nucleotide-binding protein(s).

J Biol Chem, 1987 Oct 15, 262(29), 14289 - 97
Mössbauer, EPR, and optical studies of the corrinoid/iron-sulfur protein involved in the synthesis of acetyl coenzyme A by Clostridium thermoaceticum; Ragsdale SW et al.; We have purified to homogeneity the 88-kDa corrinoid protein from Clostridium thermoaceticum which acts as a methyl carrier in the synthesis of acetyl-CoA . As shown here, this protein contains a {4Fe-4S}1+/2+ cluster in addition to a corrinoid . The corrinoid is 5-methoxybenzimidazolylcobamide, with an OH- group probably present as the upper axial ligand . Co+ is present in the reduced form, Co2+ in the as-isolated form, and Co3+ in the methylated form of the protein . The as-isolated corrinoid/Fe-S protein exhibits a Co2+ EPR signal lacking nitrogen superhyperfine splittings, indicating that the benzimidazole base is uncoordinated ("base-off") in the Co2+ state . Optical studies suggest that the Co3+-CH3 corrinoid is also base-off . In the as-isolated and methylated forms, the iron-sulfur cluster is diamagnetic, with quadrupole splittings and isomer shifts characteristic of {4Fe-4S}2+ clusters . The protein can be reduced by CO and CO dehydrogenase in the absence of ferredoxin . The EPR spectra of the reduced cluster exhibit two components: one with principal g-values at 2.07, 1.93, and 1.82 and the other at 2.02, 1.94, and 1.86 . The Mossbauer data show that these signals result from {4Fe-4S}1+ clusters . Chemical analysis shows that the iron:cobalt atomic ratio is close to 4:1, suggesting that a single {4Fe-4S}1+ cluster occurs in two distinct S = 1/2 spin states in the reduced state . Treatment with 1-2.5 M urea converts the two cluster forms into a single one, with EPR and Mossbauer spectra of typical {4Fe-4S}1+ clusters . A 27-kDa corrinoid protein (Ljungdahl, L.G., LeGall, J., and Lee, J.P . (1973) Biochemistry 12, 1802-1808) also was purified and found to be inactive in the synthesis of acetyl-CoA, contrary to the suggestion of Ljungdahl et al . (1973).

J Mol Biol, 1987 Oct 5, 197(3), 525 - 41
Rubredoxin from Desulfovibrio gigas . A molecular model of the oxidized form at 1.4 A resolution; Frey M et al.; The crystal structure of rubredoxin from the sulfate-reducing bacterium Desulfovibrio gigas has been determined at 1.4 A resolution (1 A = 0.1 nm) by X-ray diffraction methods; starting with a model of the isostructural rubredoxin from Desulfovibrio vulgaris . Refinement of the molecular model has been carried out by restrained least-squares techniques and Fourier series calculations . The present model includes a formyl at the N-terminal end and 121 possible sites for solvent molecules with full or partial occupancy, which corresponds to the modeling of nearly all the solvent medium . The crystallographic R factor against the data with 10 A greater than d greater than 1.4 A with F greater than 2 sig(F), is 0.136; and R = 0.140 when all the data are considered . The estimated average root-mean-square (r.m.s.) error on the positional parameters is about 0.12 A . The overall structural features of this molecule are close to those of the two highly refined rubredoxins from Clostridium pasteurianum and D . vulgaris . Superposition of these two molecules on the rubredoxin from D . gigas shows in both cases an overall r.m.s . deviation of 0.5 A for the atoms in the main-chain and of 0.4 A for the atoms in the side-chains that make up the hydrophobic core . The iron atom is co-ordinated to four cysteine sulfur atoms forming an almost regular tetrahedron, with Fe-SG distances ranging from 2.27 A to 2.31 A and angles varying from 103 degrees to 115 degrees . The intramolecular hydrogen-bonding pattern is quite comparable to those found in other proteins refined at high resolution . All the polar groups are involved in hydrogen bonds: intramolecular, intermolecular or with solvent molecules . The main structural differences from the other rubredoxins are in the nature and the distribution of some of the charged residues over the molecular surface . The possible influence of several structural factors on the intramolecular and intermolecular electron transfer properties such as the NH...SG bonds, the solvent exposure of the redox center, and the aromatic core is discussed . The conservation, during evolution, of a ring of acidic residues in the proximity of the FeSG4 center suggests that this ring may be implicated in the recognition processes between rubredoxins and their functional partners.

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