Clostridia

Microbiol Immunol, 1985, 29(12), 1185 - 95
Solubilization and partial properties of receptor substance for bacteriophage alpha 2 induced from Clostridium botulinum type A 190L; Takumi K et al.; Bacteriophage alpha 2, one of the two inducible phages from Clostridium botulinum type A 190L, had a latent period of 55 min and an average burst size of 75 in C . botulinum type A Hall used as the host bacterium . The phage particles were adsorbed on the cell walls extracted with hot trichloroacetic acid (TCA-walls) . The receptor substance for the phage was solubilized from the TCA-walls with Achromopeptidase and fractionated by gel filtration on Sephadex G-150 . The fraction having the highest level of receptor activity for the phage contained large amounts of muramic acid and glucosamine . Both authentic muramic acid and glucosamine significantly inactivated the phage, whereas glucose, galactose, L-and D-alanine, diaminopimeric acid, or D-glutamic acid did not exhibit similar activity . There results strongly suggest that the receptor site for phage alpha 2 is closely associated with glycan moieties of the cell wall peptidoglycan.

Scand J Infect Dis Suppl, 1985, 46, 47 - 56
Pathogenesis and diagnosis of clostridium difficile enterocolitis; Mollby R et al.; Antibiotic associated Clostridium difficile enterocolitis is an infectious disease with symptoms ranging from self-limiting diarrhoea to severe colitis with bloody stools and formation of pseudomembranes . The carrier rate of C . difficile in a general Swedish population was found to be low (2%; 11/594) . In patients with acute diarrhoea unrelated to antibiotics the bacterium or its toxin was found in 3% (12/398) . In patients with diarrhoea associated with antibiotics C . difficile or its toxin was demonstrated in 18% (873/4 793) during 1980-1982 . Local outbreaks reported recently from different hospital wards in Sweden suggest that nosocomial spread of C . difficile takes place among patients on antibiotic treatment . Immunochemical fingerprinting of the isolates from one outbreak showed that one specific strain of C . difficile had spread among the patients in one hospital ward . C . difficile produces at least 2 toxins: a cytotoxin currently used in the diagnosis of C . difficile enterocolitis, and an enterotoxin . A "sandwich" enzyme-linked immunosorbent assay (ELISA) was used for the detection of enterotoxin in stool specimens . The enterotoxin was demonstrated in 80% (57/71) of patients with cytotoxin in stools . In an additional 5 patients with colitis the immunoassay was positive while the cytotoxin assay remained negative . An immunoassay demonstrating circulating antibodies to C . difficile toxins seemed to be positive in about half of the patients with verified C . difficile infection . It was notable that most patients suffering from repeated episodes of colitis did not develop an antibody response until after final recovery.

Toxicon, 1985, 23(2), 235 - 46
A sensitive and useful radioimmunoassay for neurotoxin and its haemagglutinin complex from Clostridium botulinum; Ashton AC et al.; A sensitive radioimmunoassay for the detection of botulinum toxin, produced by Clostridium botulinum, was developed . This employs homogeneous botulinum neurotoxin type A and its 125I-labelled derivative of high specific radioactivity, rather than its complex with haemagglutinin as used hitherto . The sensitivity of the assay is 1 ng of neurotoxin per ml, which is equivalent to 80 LD50 units (half-lethal doses) in mice . Neurotoxin and its complex with haemagglutinin were measurable with equal sensitivity when using antibodies against botulinum neurotoxin type A . Specificity of the assay was demonstrated by the lack of response to type B and E botulinum toxins and to heat-inactivated botulinum toxin or extracts of Clostridium sporogenes strain BL46, which contains many surface antigenic determinants common to Clostridium botulinum . Using appropriate conditions, neurotoxin added to fish extract could be quantified accurately, proportionality being observed between the amounts of standard toxin added . In addition, the amounts of toxin species produced by culturing Clostridium botulinum in canned fish was measurable; the values obtained were comparable to those observed by the mouse bioassay . Moreover, the fish samples gave a dose-response curve in the competition radioimmunoassay which was paralleled by the response of botulinum neurotoxin standards . This assay offers the most sensitive, reliable immunological method available for the quantitation of molecular forms of botulinum toxin . As the technique can be used with unpurified fish extracts, it should be widely applicable to different types of samples contaminated with botulinum toxin; furthermore, the clinical diagnosis of human botulism could be substantiated with this method.

Ciba Found Symp, 1985, 111, 97 - 111
Chiral products from non-pyridine nucleotide-dependent reductases and methods for NAD(P)H regeneration; Simon H et al.; Enoate reductase (EC 1.3.1.31) from a Clostridium tyrobutyricum strain catalyses the stereospecific reduction of many different alpha, beta-unsaturated carboxylates, aldehydes and even some ketones . The enzyme accepts electrons from NADH and, 1.5 times faster, from reduced methyl viologen (1,1'-dimethyl-4,4'-bipyridinium) . Another new type of non-pyridine nucleotide-dependent reductase has an extremely broad substrate specificity for 2-oxo-carboxylates and 2-oxo-dicarboxylates . In crude extracts from Proteus mirabilis and Proteus vulgaris, specific activities of 2-12 mumol product formed per mg protein per min can be found when reduced methyl or benzyl viologen is used as electron donor . The products are (2R)-hydroxy acids . Enoate reductase and 2-oxo-carboxylate reductase are suitable for electro-enzymic reductions in which catalytic amounts of viologens are continuously reduced in an electrochemical cell . This procedure has three advantages: (1) regeneration of NAD(P)H by a second enzyme and substrate is not required, (2) the unstable pyridine nucleotides are not required in the reaction mixture, and (3) the rate of the reaction can be observed continuously by measuring an electric current . Several yeasts, as well as aerobic and anaerobic bacteria, catalyse the reduction of NAD(P)+ by reduced methyl viologen . Such cells can be used for electro-microbial reductions when only pyridine nucleotide-dependent reductases are present . Information about the enzymes which catalyse the reduction of NAD(P)+ at the expense of reduced methyl viologen is given.

Zentralbl Allg Pathol, 1985, 130(1), 45 - 50
Pathogenesis of pseudomembranous colitis; Pesce CM et al.; This work is concerned with new morphologic data pointing to an immune component in the pathogenesis of pseudomembranous colitis . The focal distribution of the pseudomembranes suggests selective damage induced by Clostridium difficile toxins . The sites of attachment to the mucosa correspond anatomically to the intestinal structures specialized for immune information and response . Furthermore, viable IgA production supports the view that toxins are carried to lymphoid aggregates where plasma cell proliferation takes place . A sharp increase in the mast cell population of the colon is also reported . Mast cells, whose role in the pathogenesis of intestinal diseases is still obscure, are diffusely distributed, irrespective of the focal lesions of pseudomembranous colitis.

J Clin Microbiol, 1985 Jan, 21(1), 122 - 6
Comparison of PRAS II, RapID ANA, and API 20A systems for identification of anaerobic bacteria; Karachewski NO et al.; This study evaluated the PRAS II, RapID ANA, and API 20A systems for the identification of anaerobic bacteria . A total of 80 isolates (68 fresh clinical isolates and 12 stock cultures) were examined and included 25 Bacteriodes spp., 7 Fusobacterium spp., 12 Clostridium spp., 2 Veillonella spp., 16 gram-positive cocci, and 18 gram-positive nonsporeforming bacilli . All isolates were initially identified by the procedures outlined in Holdeman et al . (ed.), Anaerobe Laboratory Manual, Virginia Polytechnic Institute and State University, Blacksburg, Va., 1977; identifications from the PRAS II, RapID ANA, and API 20A systems were compared with these initial identifications . If no supplemental tests were required, the RapID ANA and API 20A systems had incubation times of 4 and 24 h, respectively; the PRAS II system generally required 2 to 5 days of incubation, depending on the growth rate of the isolate . PRAS II identified 74% correct to species level, 14% correct to genus only, and 6% incorrect; 6% could not be identified . PRAS II data were reevaluated according to a revised data base that was provided after completion of the study; PRAS II (revised) identified 82% correct to species, 12% correct to genus only, and 6% incorrect . RapID ANA identified 62% correct to the species level, 28% correct to genus only, and 10% incorrect . API 20A identified 71% correct to the species level, 10% correct to genus only, and 3% incorrect; 16% could not identified . The API 20A is a more established system for identification of anaerobic bacteria; PRAS II and RapID ANA appear to be promising new methods for the identification of anaerobic bacteria.

Scand J Infect Dis Suppl, 1985, 46, 57 - 63
Beta-lactamases in anaerobic bacteria; Nord CE et al.; The known mechanisms of beta-lactam resistance in anaerobic bacteria involve production of beta-lactamases, alteration of penicillin-binding proteins and blocked penetration of beta-lactams through the outer membranes . The most important factor in beta-lactam resistance is production of beta-lactamase . Beta-lactamases in various Bacteroides, Fusobacterium and Clostridium species have been described . Beta-lactam resistance in Bacteroides fragilis is most commonly mediated by beta-lactamase production mainly of cephalosporinase character . Recent studies have also shown that B . fragilis can produce a penicillinase which inactivates piperacillin and carbenicillin . Enzymes inactivating cefoxitin and imipenem have also been isolated from B . fragilis . The Bacteroides non-fragilis species produce beta-lactamases of mainly penicillinase character . Recently a penicillinase from Fusobacterium nucleatum has been characterized . Among the clostridia, Clostridium butyricum, C . clostridiiformis and C . ramosum have been shown to produce penicillinases.

Drugs Exp Clin Res, 1985, 11(7), 431 - 4
The comparative activity of twelve 4-quinolone antimicrobials against gram-positive and gram-negative anaerobes; Robbins MJ et al.; The minimal inhibitory concentrations (MICs) of twelve 4-quinolone antimicrobials were determined for the Bacteroides fragilis group (50), Bacteroides melaninogenicus (20), Bacteroides bivius (10), Fusobacterium spp . (10), anaerobic Gram-positive cocci (50) and Clostridium spp . (20) . MICs were determined using an agar dilution technique in Mueller-Hinton agar supplemented with 10% lysed horse blood . The inoculum used was approximately 10(4) colony-forming units, contained in 10 microliter of Mueller-Hinton broth, which was applied to the agar plates using a multipoint inoculator . Following inoculation, plates were incubated at 37 degrees C for 48 h in an anaerobic atmosphere . The MIC of each antimicrobial for each isolate examined was determined as the lowest concentration of the antimicrobial which completely inhibited growth of the inoculum . The minimum concentrations required to inhibit the growth of 50% (MIC50) and 90% (MIC90) of the organism examined were also determined . All of the more recently synthesised 4-quinolones showed increased activity against the anaerobic bacteria used in this study . Ciprofloxacin and ofloxacin were the most active compounds examined (Bacteroides fragilis group MIC90 ciprofloxacin 4 micrograms/ml; ofloxacin 4 microgram/ml; Bacteroides melaninogenicus MIC90 ciprofloxacin 2 micrograms/ml, ofloxacin 2 micrograms/ml; Bacteroides bivius MIC90 ciprofloxacin 16 micrograms/ml, ofloxacin 32 micrograms/ml; Fusobacterium spp . MIC90 ciprofloxacin 2 micrograms/ml, ofloxacin 4 micrograms/ml; Clostridium spp . MIC90 ciprofloxacin 1 microgram/ml, ofloxacin 1 microgram/ml and anaerobic Gram-positive cocci MIC90 ciprofloxacin 4 micrograms/ml, ofloxacin 4 micrograms/ml).

Rev Argent Microbiol, 1985, 17(1), 55 - 8
{Specificity against group A, C, and G streptococci of the reverse CAMP test for identifying Clostridium perfringens}; Martini P et al.; We studied "reverse CAMP" test specificity for the identification of Clostridium perfringens, and the probable existence of cross-reactions with groups A, C and G streptococci . Ninety eight Clostridium strains were tested with ten S . agalactiae, ten S . pyogenes, two group C streptococci and one group G . All the strains of S . agalactiae yielded a positive "reverse CAMP" reaction when it was performed with C . perfringens strains . Cross-reactions were not detected when C . perfringens were tested with groups A, C or G streptococci . The "reverse CAMP" test proved to be specific for all the C . perfringens strains used.

Microbios, 1985, 44(181S), 271 - 9
Isolation of Clostridium species from herbage; Princewill TJ et al.; A total of fourteen herbage samples were collected from paddocks on six farms from two States of Nigeria . These samples yielded twenty two clostridial isolates out of which eleven were identified as Clostridium perfringens and five as Clostridium bifermentans . The other species identified were less frequently isolated . No farm yielded all the species isolated . The farm with the highest distribution of the species of Clostridium was Government Farm, Butura, Plateau State, with a frequency distribution of 50% of the species isolated . In each of two other farms the distribution was 37.5%; and in each of the remaining farms it was less than 30% in frequency . The effect of ubiquitous clostridial infections on ruminants is discussed.

Arch Orthop Trauma Surg, 1985, 104(4), 262 - 4
Wound infection after lower extremity amputation because of ischemia; Moller BN et al.; The importance of postoperative wound infection in major amputations was elucidated by recording the organisms isolated in preoperatively infected gangrene and in postoperatively infected wounds of patients undergoing lower-limb amputations for ischemia . Sixty-four amputations were performed on 61 patients . The frequency of coexisting diabetes mellitus was 34% . Postoperative infections occurred in nearly two-thirds of the 19 cases of infected gangrene, as compared with less than one-third of cases of noninfected gangrene . The presence of diabetes mellitus did not significantly influence the infection rate . Preoperatively as well as postoperatively, the most frequently isolated bacterium was Staphylococcus aureus . Clostridium perfringens was cultured in four cases . Postoperative wound infection following lower-limb amputation for ischemia is the main reason for reamputation, especially in patients with infected gangrene.

Acta Med Scand, 1985, 218(3), 341 - 3
Clostridium perfringens septicemia and acute leukemia; Grutzmeier S; Three cases of fatal clostridial septicemia in patients with acute leukemia are described . Predisposing factors and treatment are discussed . Clostridium septicemia should always be suspected when a patient with neutropenia suddenly develops diffuse abdominal pain, fever, and tachycardia over 120/min . The importance of early treatment with penicillin or another adequate antibiotic is discussed.

Microbiol Immunol, 1985, 29(6), 509 - 16
The suitability of Tórtora's medium for the production of enterotoxin in Clostridium perfringens strains; Tortora JC et al.; Examination of 200 samples from soil and the same number of samples from healthy human feces yielded 49 (24.5%) and 105 (52.5%) strains of heat-resistant Clostridium perfringens respectively . Fourteen (7.0%) strains isolated from soil and 37 (18.5%) from feces synthesized enterotoxin, as demonstrated by Tortora's method, at sufficient levels to permit its detection by mouse lethality, microslide double gel diffusion or counterimmunoelectrophoresis tests . By using the Duncan-Strong (DS) method, only four (2%) enterotoxigenic strains from soil and 14 (7.0%) from feces were obtained . The supernatant fluid from two enterotoxigenic-negative strains grown in DS medium gave a false-positive reaction when they were injected intravenously into mice . Tortora's medium was preferable because a larger number of isolated strains produced spores and enterotoxin to permit their recognition as enterotoxigenic strains.

Microbios, 1985, 42(169-170), 155 - 62
Animal feeds as likely vehicles of clostridial infections in livestock; Princewill TJ et al.; A total of sixteen samples were collected from various food items on seven farms in three States of Nigeria . These samples yielded thirty one clostridial isolates which were identified as eleven species . The species most frequently isolated was Clostridium perfringens representing 35.5% of the isolates . The next highest was C . bifermentans with a frequency of 22.6% . The other nine species identified were each isolated at frequencies less than 10% . No farm yielded all the species isolated . The farms with the highest distribution of the species of Clostridium were Government Farm, Buruku (Kaduna State), Kano Farm Centre and Government Farm, Butura (Plateau State) with frequency distributions of 36.4% each of the species isolated . Except at the Dairy Cattle Multiplication Centre, Shika (Kaduna State), where the frequency distribution was 27.3%, at none of the other three farms was the distribution more than 20%.

Comp Biochem Physiol A, 1985, 81(4), 713 - 39
Effectors of amino acid transport processes in animal cell membranes; Lerner J; Various effectors, which act upon ion gradients, protein synthesis, membrane components or cellular functional groups, have been employed to provide insights into the nature of amino acid-membrane transport processes in animal cells . Such effectors, for example, include ions, hormones, metabolites and various organic reagents and their judicious use has allowed the following list of conclusions . Sodium ion has been found to stimulate amino acid transport in a wide variety of cell systems, although depending on the tissue and/or substrate, this ion may have no effect on such transport, or even inhibit it . Amino acid transport can be stimulated in some cell systems by other ions such as K+, Li+, H+ or Cl- . Both H+ and K+ have been found to be inhibitory in other systems . Amino acid transport is dependent in many cell systems upon an inwardly directed Na+ gradient and is stimulated by a membrane potential (negative cell interior) . In some cell systems an inwardly directed Cl- and H+ gradient or an outwardly directed K+ gradient can energize transport . Structurally dissimilar effectors such as ouabain, Clostridium enterotoxin, aspirin and amiloride inhibit amino acid transport presumably through dissipation of the Na+ gradient . Inhibition by certain sugars or metabolic intermediates of the tricarboxylic acid cycle may compete with the substrate for the energy of the Na+ gradient or interact with the substrate at the carrier level either allosterically or at a common site . Stimulation of transport by other sugars or intermediates may result from their catabolism to furnish energy for transport . Insulin and glucagon stimulate transport of amino acids in a variety of cell systems by a mechanism which involves protein synthesis . Microtubules may be involved in the regulation of transport by insulin or glucagon . Some reports also suggest that insulin has a direct effect on membranes . In addition, a number of growth hormones and factors have stimulatory effects on amino acid transport which are also mediated by protein synthesis . Steroid hormones have been noted to enhance or diminish transport of amino acids depending on the nature of the hormone . These agents appear to function at the level of protein synthesis . While stimulation may involve increased carrier synthesis, inhibition probably involves synthesis of a labile protein which either decreases the rate of synthesis or increases the rate of degradation of a component of the transport system.(ABSTRACT TRUNCATED AT 400 WORDS)

Toxicon, 1985, 23(3), 449 - 55
Effect of Clostridium perfringens alpha toxin on the isolated rat vas deferens; Sakurai J et al.; Alpha toxin produced by Clostridium perfringens potentiated norepinephrine-evoked contraction in the isolated rat vas deferens, but itself caused no contraction within 60 min . The potentiating activity was dependent on the dose of the toxin and was quantitatively related to the phospholipase C activity of the toxin preparation.

Prog Clin Biol Res, 1985, 181, 65 - 8
Steam quality and effective sterilization; Sedlacek RS et al.; Faced with using steam from a commercial utility having boilers greater than 5 miles distant and being the last user on the system resulted in ineffective sterilization . A three phase testing program was established utilizing: Direct physical measurements - an Ellison model 915A portable steam calorimeter . Direct microbiology - Autoclaved feed pellets were aseptically placed in fluid thioglycolate medium and incubated at 37 degrees C . Indirect microbiology - Feces from "defined flora" mice fed the autoclaved pelleted feed were tested . Colorimetric measurements verified that the steam sometimes contained greater than 5% entrained water . During periods of wet steam it was impossible to maintain consistent sterility of the mouse pellets even using a cycle of 126 degrees C for 60 minutes . One spore-forming Gram positive rod, Clostridium perfringens type D was the predominant bacterium isolated . Lactating mice, or mice stressed experimentally came down with diarrhea within days of eating pellets treated with wet steam (calorimetric measurements) and a subsequent positive culture . These mice voided stools predominantly showing Clostridium perfringens type D.

Microbiol Immunol, 1985, 29(4), 317 - 26
Heat resistance, spore germination, and enterotoxigenicity of Clostridium perfringens; Ando Y et al.; Heat resistance at 95 C, heat activation at 75 C, and germination response were determined for spores of 10 serotype strains of Clostridium perfringens type A, including five heat-resistant and five heat-sensitive strains . The D95-values ranged from 17.6 to 63.0 and from 1.3 to 2.8 for the heat-resistant and the heat-sensitive strains, respectively . The heat-activation values, the ratios between the heated and unheated viable counts of spore suspensions, ranged from 0.0035 to 0.65 and from 6.5 to 60.0 for the heat-sensitive and the heat-resistant strains, respectively . Spores of these strains were divided into two distinct germination types on the basis of their germination response; spores of the heat-resistant strains germinated in KC1 medium after heat activation (K-type), and spores of the heat-sensitive strains germinated in a mixture of L-alanine, inosine, and CaCl2 in the presence of CO2 without heat activation (A-type) . The strains were tested for enterotoxigenicity by a reversed passive latex-agglutination (RPLA) test . All the heat-resistant strains were RPLA-positive, whereas the heat-sensitive strains were all RPLA-negative . A total of 37 strains of the organism isolated from food-poisoning outbreaks were tested for spore germination and enterotoxin formation . All of the 20 heat-resistant strains showed K-type spore germination and, except for three strains, were RPLA-positive, whereas all of the 17 heat-sensitive strains showed A-type spore germination and, except for only one strain, were RPLA-negative.

Ciba Found Symp, 1985, 112, 230 - 41
Clostridial toxins active locally in the gastrointestinal tract; Wilkins T et al.; Clostridium difficile and Clostridium spiroforme have only in recent years been recognized as intestinal pathogens . They both produce toxins that are also produced by other clostridia . C . difficile toxins A and B are produced by C . sordellii and a few strains of C . perfringens whereas C . spiroforme produces the same toxins as C . perfringens Type E (iota toxin) . Iota toxin activity may be the product of two proteins . Toxigenic strains of C . spiroforme and Type E produce two antigens which possess much more biological activity when administered together than when given alone . C . difficile was thought for some time to produce only a single toxin, but then the enterotoxic activity was shown to be due to a separate toxin (toxin A) . This toxin increases the oral toxicity of toxin B (the main cytotoxin) and may increase the permeability of the colon . Toxin A binds to a specific receptor in hamster brush border membranes and in the membranes of rabbit erythrocytes . This receptor appears to be a glycoprotein . The receptor can be extracted from the membrane with Triton and binds to immobilized toxin A . The receptor can be extracted and used to coat plastic plates as a first phase in an ELISA assay . Another assay has been developed in which the toxin A binds to the red cells and then the erythrocytes are agglutinated with antitoxin . An even more sensitive assay consists of using rabbit erythrocyte ghosts to bind the toxin and then precipitating the ghosts with antibody to toxin A attached to latex beads . Monoclonal antibodies to toxin A also have been developed and are used in these and other assays.

Infect Immun, 1985 Jan, 47(1), 260 - 3
Tryptophan content of Clostridium perfringens epsilon toxin; Sakurai J et al.; The tryptophan content of Clostridium perfringens epsilon toxin was investigated . When the tryptophan content was determined by amino acid analysis after the hydrolysis of epsilon prototoxin with methanesulfonic acid containing 3-(2-aminoethyl)indole and by the spectrophotometric method with N-bromosuccinimide, the number of tryptophan residues was calculated at 1/mol of the protein . Cleavage of the prototoxin or the toxin with N-bromosuccinimide in the presence of urea gave two new fragments on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . There was only a tyrosine residue as the new N-terminal amino acid after the cleavage of the prototoxin or the toxin with N-bromosuccinimide . The data showed that epsilon prototoxin or epsilon toxin contained only one tryptophan residue.

J Biochem (Tokyo), 1985 Jan, 97(1), 213 - 8
The structural relation between the antigenic determinants to monoclonal antibodies and binding sites to rat brain synaptosomes and GT1b ganglioside in Clostridium botulinum type C neurotoxin; Agui T et al.; The inhibition of the binding of 125I-labeled Clostridium botulinum type C neurotoxin to synaptosomes by unlabeled toxin indicated that there were two kinds of receptors on the synaptosomal membrane . The dissociation constants (Kd) were calculated as 79 pM and 35 nM from the concentration of unlabeled toxin that induced half-displacement of bound 125I-toxin . These values agree satisfactorily with the values obtained from direct binding experiments (Agui, T, Syuto, B., Oguma, K., Iida, H., & Kubo, S . (1983) J . Biochem . 94, 521-527) . The inhibition of the binding of 125I-toxin to synaptosomes and N-acetylneuraminyl(alpha 2-3)galactosyl(beta 1-3)N-acetylgalactosaminyl(beta 1-4) {N-acetylneuraminyl(alpha 2-8) N-acetylneuraminyl(alpha 2-3)}galactosyl(beta 1-4)glucosyl(beta 1-1)ceramide (GT1b) by unlabeled heavy chain indicated that heavy chain facilitates the binding of toxin to synaptosomes and GT1b . The synaptosomal and heavy chain complex Kd values were estimated as 12 nM and 24 microM . Monoclonal antibodies C-9 and CA-12 recognized the binding sites to GT1b and synaptosomes, respectively . Antigenic determinants against the two antibodies are presumably partially overlapping, and the overlapping area seems to be essential to the reaction between toxin and C-9 antibody.

Biol Cell, 1985, 53(1), 23 - 32
Effect of the cytotoxin of Clostridium difficile on cultured hepatoma cells; Rihn B et al.; Clostridium difficile is the major etiologic agent of human pseudomembranous colitis . It produces two toxins: an enterotoxin and a cytotoxin . In cultured hepatoma cells, at very low doses, the cytotoxin inhibits the incorporation of precursors into biological macromolecules . Protein synthesis is more affected than RNA and DNA synthesis . The toxin also induces severe alterations of the cell morphology consisting in damages to the cytoskeleton and to the cell shape.

Biochemistry, 1984 Dec 18, 23(26), 6345 - 9
Electron transfer between flavodoxin semiquinone and c-type cytochromes: correlations between electrostatically corrected rate constants, redox potentials, and surface topologies; Tollin G et al.; We have measured the ionic strength dependence of the rate constants for electron transfer from the semiquinone of Clostridium pasteurianum flavodoxin to 12 c-type cytochromes and several inorganic oxidants using stopped-flow methodology . The experimental data were fit quite well by an electrostatic model that represents the interaction domains as parallel disks with a point charge equal to the charge within this region of the protein . The analysis provides an evaluation of the electrostatic interaction energy and the rate constant at infinite ionic strength (k affinity) . The electrostatic charge on the oxidant within the interaction site can be obtained from the electrostatic energy, and for most of those reactants for which structures are available, the results are in good agreement with expectation . The k affinity values were found to correlate with redox potential differences, as expected from the theory of adiabatic (or nonadiabatic) outer-sphere electron-transfer reactions . Deviations from the theoretical curves are interpreted in terms of the influence of surface topology on reaction rate constants . In general, we find that electrostatic effects, steric influences, and redox potential all exert a much larger effect on reaction rate constants for the flavodoxin-cytochrome system than has been previously observed for free flavin-cytochrome interactions . The implications of this for determining biological specificity are discussed.

J Biol Chem, 1984 Dec 10, 259(23), 14328 - 31
Mössbauer and electron nuclear double resonance study of oxidized bidirectional hydrogenase from Clostridium pasteurianum W5; Wang G et al.; The bidirectional hydrogenase from Clostridium pasteurianum W5 is an iron-sulfur protein containing approximately 12 Fe atoms and 12 labile sulfides . We have studied oxidized samples of the enzyme with Mossbauer and electron nuclear double resonance (ENDOR) spectroscopy to elucidate the nature of the center that gives rise to the EPR signal with principal g-values at 2.10, 2.04, and 2.01 . The g = 2.10 center exhibits two well-resolved 57Fe ENDOR resonances . One is isotropic with A1 = 9.5 MHz; the other is nearly isotropic with A2 = 17 MHz . These magnetic hyperfine coupling constants are substantially (approximately 50%) smaller than those observed for {2Fe-2S}, {3Fe-4S}, and {4Fe-4S} clusters . The Mossbauer and ENDOR data, taken together, suggest that the g = 2.10 center contains at least two but not more than four iron atoms . Comparison of our data with recent results reported for Escherichia coli sulfite reductase and the ferricyanide-treated {4Fe-4S} cluster from Azotobacter vinelandii ferredoxin I suggests that the g = 2.10 center may possibly be formed, by oxidation, from a structure with a {4Fe-4S} core . The Mossbauer spectra give evidence that at least 8 of the 12 Fe atoms of oxidized hydrogenase are organized in two ferredoxin-type {4Fe-4S} clusters, supporting conclusions derived previously from EPR studies of the reduced enzyme.

Chemioterapia, 1984 Dec, 3(6), 365 - 7
In vitro activity of rifaximin and rifampicin against some anaerobic bacteria; Lamanna A et al.; Activity of rifampicin and of a new rifamycin, rifaximin, was tested in strains of anaerobic bacteria belonging to the Bacteroides genus (75 B . fragilis group and 17 Bacteroides non-fragilis group) and in Clostridium perfringens (15 strains) . It turned out that the bactericidal activity of both rifamycins could be overlapped and that it equalled 100% in the case of the non-fragilis Bacteroides and Clostridium species.

J Antimicrob Chemother, 1984 Dec, 14 Suppl D, 85 - 95
Diagnosis and epidemiology of Clostridium difficile enterocolitis in Sweden; Aronsson B et al.; Experience of the diagnosis and epidemiology of Clostridium difficile in Sweden is reviewed . Samples from 5885 patients have been investigated at the National Bacteriological Laboratory in Stockholm from 1978-1983 . Patients originate from all parts of the country and their number continues to increase . Cl . difficile seem to be of growing importance, especially in nosocomial infections . Most patients with antibiotic-associated diarrhoea and colitis (AAD/AAC) and Cl . difficile in their stools were above 60 years of age (63%) and there was a significant preponderance of females over males in the age groups 21-50 and above 60 years of age . The antibiotics most commonly associated with Cl . difficile enterocolitis (CDE) were penicillins, cephalosporins and clindamycin/lincomycin . On the basis of consumption clindamycin/lincomycin and cephalosporins are associated 70 and 40 times, respectively, more often than penicillins in CDE . Diagnosis of CDE relies mainly on detection of the cytotoxin (toxin B) in stool specimens . It was present in 873/4793 (18.2%) patients whereas the bacterium was found in only 12% . An immunoassay for detection of the enterotoxin (toxin A) of Cl . difficile seems to be a useful alternative to the cytotoxin assay, but some stool specimens with a low toxin B titre were negative . Five specimens negative for toxin B were positive for toxin A and came from patients where additional information suggested CDE . A serological assay for demonstration of circulating antibodies to Cl . difficile toxins has also been evaluated and is positive in about half of the cases of CDE . Antibody response seems to be absent or delayed in patients with relapse of colitis after antibiotic treatment.

Vet Med (Praha), 1984 Dec, 29(12), 747 - 52
{Botulism of aquatic birds at the Starý u Pohorelic pond (Breclav District)}; Hubalek Z et al.; In the years 1981 and 1982 when a mass mortality of wild and domestic water birds was observed on the Stary pond, eleven diseases or just died birds were examined by the neutralization test for botulotoxin and four sludge samples for the presence of Clostridium botulinum . C . Botulinum toxin of C type was detected in four wild ducks (Anas platyrhynchos) from September 1981 and July 1982 and in one gull (Larus ridibundus) and July 1982 . The highest titres (of mice i . p . LD50/g) of botulotoxin in ducks were 10(6.3) in the intestinal contents, 10(6.1) in the liver tissue and 10(5.1) in the stomach contents . In one duck we detected, besides the C type, a smaller botulotoxin amount of A type . Therefore we can recommend a complete serotypifying for the cases of botulism in water birds . Out of four sludge samples collected in September 1982 one sample was negative, whereas in three samples the C botulotoxin was demonstrated after propagation and one C . botulinum strain of C type was isolated.

J Antimicrob Chemother, 1984 Dec, 14 Suppl D, 7 - 18
Mode of action and in-vitro activity of vancomycin; Watanakunakorn C; Vancomycin is a unique glycopeptide structurally unrelated to any currently available antibiotic . It also has a unique mode of action inhibiting the second stage of cell wall synthesis of susceptible bacteria . There is also evidence that vancomycin alters the permeability of the cell membrane and selectively inhibits ribonucleic acid synthesis . Induction of bacterial L-phase variants from susceptible organisms with vancomycin is extremely difficult, and such variants are unstable . Stable L-phase variants induced by other agents are susceptible to vancomycin . Vancomycin is active against a large number of species of Gram-positive bacteria, such as Staphylococcus aureus (including methicillin-resistant strains), Staph . epidermidis (including multiple-resistant strains), Streptococcus pneumoniae (including multiple-resistant strains), Str . pyogenes, Str . agalactiae, Str . bovis, Str . mutans, viridans streptococci, enterococci, Clostridium species, diphtheroids, Listeria monocytogenes, Actinomyces species and Lactobacillus species . There has been no increase in resistance to vancomycin during the past three decades . Enhancement of antimicrobial activity has been demonstrated with the combination of vancomycin and an aminoglycoside against Staph . aureus, Str . bovis, enterococci and viridans streptococci . The combination of vancomycin and rifampicin are antagonistic to most strains of Staph . aureus, though indifference and occasionally synergism have been shown, but is synergistic against strains of Staph . epidermidis . It shows indifference against enterococci . Vancomycin and fusidic acid are indifferent against Staph . aureus.

Onderstepoort J Vet Res, 1984 Dec, 51(4), 249 - 52
Bacteriological findings regarding the hygienic safety of poultry litter intended as an ingredient of feeds for ruminants; Ogonowski K et al.; An investigation of poultry litter intended for use in farm feeds showed that 0,37%, 0,49%, 0,25% and 12,3% of the 813 samples tested were contaminated with Clostridium spp., haemolytic Escherichia coli, Staphylococcus aureus and 21 different species of Salmonella . The findings clearly underline the hygienically dangerous nature of crude poultry litter . The practical implications of the results are briefly discussed, particularly in view of current regulations.

J Clin Microbiol, 1984 Dec, 20(6), 1209 - 12
Evaluation of fluorescent-antibody tests as a means of confirming infant botulism; Glasby C et al.; Fluorescent-antibody techniques were evaluated for confirming infant botulism . Seventy-seven stool specimens from suspected cases were examined . All 34 specimens containing viable Clostridium botulinum at time of study gave positive results (29 on direct smears and 34 on enrichments) . Two false-positive reactions were observed.

J Clin Microbiol, 1984 Dec, 20(6), 1145 - 53
Rapid differentiation of enterotoxigenic Escherichia coli that produce heat-stable and heat-labile toxins by frequency-pulsed electron capture gas-liquid chromatography analysis of diarrheal stool specimens; Brooks JB et al.; Thirty-three stool specimens from infants in the village of Tamooh near Cairo, Egypt, were studied by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC) . In 13 of the diarrheal cases, the suspected causative agent isolated was Escherichia coli which produced heat-stable toxin (ST), and in 10 other cases E . coli that produced heat-labile toxin (LT) were isolated . Ten control stool samples, collected from infants from whom no pathogenic organisms were isolated, were analyzed at the same time . Comparisons also were made against healthy control stools from individuals in the United States who had been previously analyzed by FPEC-GLC (Brooks et al., J . Clin . Microbiol . 20:549-560, 1984) . The stools were suspended in water and centrifuged, and the supernatant was extracted with organic solvents and derivatized to form electron-capturing derivatives of carboxylic acids, hydroxy acids, alcohols, and amines . Results from the study showed distinct differences among the FPEC-GLC profiles of E . coli ST-positive stools, of E . coli LT-positive stools, and of the control stool samples . An unidentified compound appearing in the ether-soluble hydroxy acid fraction from E . coli ST-positive stools was tentatively identified by mass spectrometry as 6-methoxy-2-hydroxyhexanoic acid . 6-Methoxy-2-hydroxyhexanoic acid was found in all stools that contained E . coli ST but was not present either in stools from which E . coli LT was isolated or in control samples . 6-Methoxy-2-hydroxyhexanoic acid may prove to be an important marker for use in the identification of E . coli ST . In addition to 6-methoxy-2-hydroxyhexanoic acid, the carboxylic acid, alcohol, and amine FPEC-GLC profiles obtained from stools were very different between these two organisms . The data indicate that FPEC-GLC analysis of diarrheal stool specimens might be a rapid way to distinguish diarrhea caused by E . coli ST, E . coli LT, Clostridium difficile, and rotavirus.

J Antimicrob Chemother, 1984 Dec, 14 Suppl D, 103 - 9
Treatment of pseudomembranous colitis and antibiotic-associated diarrhoea; Burdon DW; Pseudomembranous colitis is caused by release of toxins from Clostridium difficile when it colonizes the large intestine . This clostridium is susceptible to concentrations of vancomycin which are readily attained in the colon after oral administration . When vancomycin is given orally to infected patients in a dose of 125 mg every 6 h, a rapid clinical cure can be expected . Some patients may relapse after the vancomycin is stopped, but a further course of treatment will control symptoms.

J Med Microbiol, 1984 Dec, 18(3), 385 - 91
Production and release of toxins A and B by Clostridium difficile; Ketley JM et al.; The production and release of toxins A and B by Clostridium difficile during in-vitro culture was investigated . Cell-associated toxin A was detected by immunoelectrophoresis of bacterial extracts released by ultrasonication and by fluorescent antibody labelling of whole cells . Extracellular toxin A was detected by immunoelectrophoresis and by enzyme-linked immunosorbent assay; extracellular toxin B was detected by cytotoxin assay . Both toxins A and B were produced and released during the decline phase of the bacterial growth cycle . The possible significance of these results in relation to the pathogenesis of pseudomembranous colitis is discussed.

Infect Immun, 1984 Dec, 46(3), 715 - 9
Botulism in metronidazole- treated conventional adult mice challenged orogastrically with spores of Clostridium botulinum type A or B; Wang Y et al.; Conventional adult mice were pretreated with metronidazole to make their intestinal tracts receptive to colonization by Clostridium botulinum . These mice, in groups of 10, were fed 0 (controls), 10(2), 10(3), 10(4), or 10(5) C . botulinum type B spores and were placed for observation in filter-lid cages whose screen floors minimized the amounts of feces available for coprophagy . The opportunity to eat feces was made equal for all mouse groups by placing one mouse of every group in each of 10 cages . Mice given a spore inoculum began to develop botulism after incubation periods of slightly less than 2.75 days . Morbidity rates, which reached maxima within 5 days of challenge, were related to inocula levels . Mortality rates were also dose related . Mice given 10(5) spores and then type B antitoxin intraperitoneally, a treatment not affecting intraintestinal toxin production, remained healthy . Morbidity among control mice was seldom more than 10% and could be ascribed to toxin ingested with feces . A C . botulinum type A spore suspension gave similar results, although morbidity and mortality rates were generally lower than after challenge with a comparable number of type B spores . Mice challenged with 10(2) or 10(5) spores had similar toxin levels in their large intestines 48 h later . Morbidity rates correlated better with toxin levels in the small intestines.

J Immunol, 1984 Dec, 133(6), 3250 - 4
Lysine residues, but not carbohydrates, are required for the regulatory function of H on the amplification C3 convertase of complement; Jouvin MH et al.; Lysine epsilon-amino groups of human factor H were selectively converted to guanidino groups by treatment with 0.1 M O-methylisourea at pH 10.4 . Guanidination resulted in a dose-dependent decrease in the capacity of the regulatory protein to accelerate decay dissociation of P-stabilized amplification C3 convertase sites, to serve as a co-factor for cleavage of cell-bound C3b by I, and to compete for binding of 125I-untreated H to C3b . Modification of approximately 75% lysine epsilon-amino groups suppressed 97% of H functional activity . Biochemical analysis of native H demonstrated a total carbohydrate content of 18.5% (w/w) and the presence in the molecule of 11 biantennary oligosaccharidic chains of the N-acetyl-lactosaminic type . Total desialation of H by using Clostridium perfringens neuraminidase, and total deglycosylation of desialated H by using beta-endo-N-acetylglucosaminidase resulted in a 1.5- to 2-fold increase in H activity on a weight basis . Deglycosylation did not alter the capacity of H to discriminate between activating and nonactivating surfaces of the alternative pathway . Thus, lysine residues are important determinants of the binding capacity of H for cell-bound C3b, whereas the carbohydrate portion of the molecule is not required for the regulatory function of the protein on the amplification C3 convertase.

J Immunol, 1984 Dec, 133(6), 2898 - 903
Immune response to ferredoxin; nonresponder status is not due to suppression; Sikora LK et al.; Ferredoxin (Fd), a small protein from Clostridium pasteurianum, has been selected for immunologic studies because of its limited number (two) of antigenic determinants . Functionally (as determined by antibody binding), monodeterminant fragments of Fd can be generated enzymatically, leaving molecules only a few amino acids smaller than the native protein, with unaltered solid phase binding properties . These fragments were used to assess the immune response to each of the two determinants . Clear differences in immunologic properties can be assigned to sequences within Fd: the amino terminal tripeptide is responsible for inducing a proliferative response and limited antibody production, whereas the carboxy terminal dipeptide accounts for most of the antibody activity, yet little, if any, T-proliferative activity . Studies with the enzyme-generated fragments of Fd have unmasked a sequence proximal to the amino terminal that represents a second determinant for T cell proliferation but does not have any demonstrable antibody-inducing activity . This third determinant is shown to induce responsiveness to Fd in nonresponder animals after the removal of the amino terminal tripeptide . The results indicate that nonresponsiveness to this molecule in H-2d mice is not a direct effect of suppression.

Gastroenterology, 1984 Dec, 87(6), 1344 - 50
Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity . Assessment of inflammation in rat and hamster models; Krawisz JE et al.; An assay was devised to quantitate acute intestinal inflammation based on the assessment of myeloperoxidase activity . Myeloperoxidase is an enzyme found in neutrophils and, in much smaller quantities, in monocytes and macrophages . Myeloperoxidase was solubilized with hexadecyltrimethylammonium bromide and myeloperoxidase activity was measured with a dianisidine-H2O2 assay . In neutrophil suspensions, myeloperoxidase activity was directly related to cell number down to as few as 500 cells . Myeloperoxidase activity was assayed in two animal models of inflammation: acetic acid-induced colitis in rats and Clostridium difficile enterotoxin-induced enteritis in hamsters . In both models, the activity of myeloperoxidase solubilized from the inflamed tissue was directly proportional to the number of neutrophils seen in histologic sections . Histologic evaluation of neutrophil accumulation was performed by counting the number of neutrophils in a histologic section 0.18 mm long and 5 micron thick . In both animal models, myeloperoxidase activity was linearly related to neutrophil number from 400 and 4000 cells/mm . Myeloperoxidase activity from chronically inflamed colon, in which both neutrophils and histiocytes were present, was directly related to neutrophil content . Histiocytes did not contribute significantly to myeloperoxidase activity . The determination of myeloperoxidase activity in the intestine is a simple biochemical assay that can be used to quantitate inflammation.

Wien Med Wochenschr, 1984 Nov 30, 134(22), 509 - 13
{Tinidazole in the therapy of anaerobic pulmonary infections}; Vetter N et al.; The purpose of the study in 10 patients with anaerobic lung infections was to demonstrate the efficacy of therapy with Tinidazole . In 9 patients, the clinical evaluation as well as X-ray and bacteriological evidence showed positive results of therapy, while in one patient a mixed anaerobic infection with Streptococcus an., Bacteroides frag., and Clostridium sp . was still present after the end of therapy . Side effects were not observed with Tinidazole therapy.

Biochem Biophys Res Commun, 1984 Nov 30, 125(1), 413 - 9
Different susceptibility of alkylacyl--versus diacyl--and alkenylacyl--phosphatidylcholine subclasses to stimulation of biosynthesis by phospholipase C; Terce F et al.; Krebs II ascites cells were incubated with {3H} or {14C} choline in the presence or in the absence of Clostridium welchii phospholipase C (PLC) . At enzyme concentrations where cell lysis remained limited, PLC specifically enhanced phosphatidylcholine (PC) biosynthesis, as shown by comparison with {14C} ethanolamine . Further analysis revealed that the stimulating effect of PLC remained limited to 1,2-diacyl-sn-glycero-3-phosphocholine (diacyl-GPC) and 1-alkenyl-2-acyl-GPC, whereas the biosynthesis of 1-alkyl-2-acyl-GPC, the putative precursor of platelet activating factor (PAF-acether) remained unchanged . These differences reflect different localizations of the three PC subclasses in the plasma membrane and are discussed in relation to the regulation mechanism of PC biosynthesis.

Biochem Biophys Res Commun, 1984 Nov 14, 124(3), 690 - 5
A new purification procedure for Clostridium difficile enterotoxin; Rihn B et al.; Clostridium difficile produces two toxins, an enterotoxin and a cytotoxin . The enterotoxin was purified using fast methods (tangential flow filtration, fast protein liquid chromatography) . The purified enterotoxin is composed of two subunits (A1 = 41,500, A2 = 16,000) and its pI is 3.5.

Antimicrob Agents Chemother, 1984 Nov, 26(5), 785 - 6
In vitro susceptibility of anaerobic bacteria to ciprofloxacin (Bay o 9867); Prabhala RH et al.; About 80% of 70 clinical isolates of Bacteroides fragilis were inhibited by 4 micrograms of ciprofloxacin (Bay o 9867) per ml . The 90% MIC of ciprofloxacin was 8 micrograms/ml for other Bacteroides species, 2 micrograms/ml for Peptococcus species, 8 micrograms/ml for Peptostreptococcus species, and 16 micrograms/ml for Clostridium and Eubacterium species.

Antimicrob Agents Chemother, 1984 Nov, 26(5), 665 - 9
Ferredoxin-linked reduction of metronidazole in Clostridium pasteurianum; Lockerby DL et al.; Clostridium pasteurianum cell-free extracts enzymatically reduced metronidazole when coupled by hydrogenase via reduced ferredoxin . A 5 mM concentration of methyl viologen, flavin adenine dinucleotide, or flavin mononucleotide could completely replace ferredoxin (0.05 mM) in the in vitro reduction assay system, whereas 5 mM benzyl viologen was less effective . However, when these electron carriers were used at a concentration of 0.05 mM, there was a drastic loss in their abilities to couple the metronidazole reduction system compared with the comparable concentration of ferredoxin . It is not understood why these flavin coenzymes participate in this enzymatic reaction . NAD and NADP had no activity when substituted for ferredoxin in the enzyme system . Two reduced ferredoxin-linked pathways, "metronidazole reductase" and the inducible dissimilatory sulfite reductase system, when combined in a single in vitro competition experiment demonstrated a preferential flow of electrons to metronidazole away from sulfite . A proposed bactericidal mechanism for metronidazole against C . pasteurianum incorporating the above findings is discussed.

Age Ageing, 1984 Nov, 13(6), 363 - 6
Clostridium difficile diarrhoea: a highly infectious organism; Bennett GC et al.; This paper describes an outbreak of Clostridium difficile diarrhoea in a ward for the elderly in a 550-bedded District General Hospital . The measures taken to contain it and clinical features, previously undescribed, are highlighted.

Appl Environ Microbiol, 1984 Nov, 48(5), 951 - 5
Acute toxicity of aminoglycoside antibiotics as an aid in detecting botulism; Wang YC et al.; Gentamicin sulfate or neomycin sulfate injected intraperitoneally into 24- to 27-g mice at a dose of 6.2 mg per mouse elicited botulism-like responses in less than 30 min, but a dose of 3.1 mg per mouse had no observable effect . The normally nontoxic 3.1-mg aminoglycoside dose aggravated the illness induced by an earlier injection of Clostridium botulinum type A or B toxin; it was usually lethal in 2 to 20 min if the preexisting illness was moderate to severe and worsened the condition of mice for about 30 min if the preexisting botulism was mild . The aminoglycoside had no effect when given shortly after the botulinum toxin was injected intraperitoneally; the sensitized state followed a latent period . It rapidly produced botulism-like effects when given to mice which had responded to a mixture of botulinum toxin and another mouse toxic agent with an illness that did not include signs of botulism . An unexpected illness devoid of botulism-like effects was encountered during intestinal colonization of mice by C . botulinum . The appearance of botulism-like signs soon after 3.1 mg of gentamicin sulfate was injected supported other suggestions that this illness included botulism that was masked by the effects of a second cause.

Infect Immun, 1984 Nov, 46(2), 324 - 31
Differential cytotoxic effects of toxins A and B isolated from Clostridium difficile; Rothman SW et al.; Toxin A and toxin B preparations of Clostridium difficile have been shown to affect metabolic functions of intact HeLa cells with different kinetics . The cytotoxins were purified from dialyzed filtrates of C . difficile strain VPI 10463 by hydrophobic interaction chromatography and ion-exchange chromatography and were concentrated by dialysis or by ultrafiltration . The toxins, which are immunologically unrelated, were analyzed by polyacrylamide gel electrophoresis and by immunochemistry with the Western blot technique . Toxin A was resolved into one major cytotoxic protein and a minor, rapidly migrating species that did not comigrate with toxin B . Toxin B was resolved into one major and three minor cytotoxic proteins . One protein comigrating with toxin A had no cytotoxic activity . The highly purified toxin A at 1.0 mg/ml caused loss of intracellular K+ and inhibition of protein synthesis in HeLa cells within 1 h . These effects correlated with morphological changes indicating cytotoxicity . At lower protein concentrations of toxin A (10- to 100-fold less), however, cytotoxic effects were seen at 120 min, whereas no changes in K+ levels or protein synthesis were yet evident . The toxin B preparation, 1,000-fold more toxic than toxin A, was diluted to equivalent cytotoxicity as measured in the overnight assay . Toxin B caused loss of K+ and inhibition of protein synthesis well after cytotoxic morphological changes were complete . In contrast, at higher protein concentrations (2- to 2,000-fold more), intracellular K+ was lost completely by 120 min . The effects on cell rounding and protein synthesis were incomplete at 120 min, but increased with the toxin B concentration.

Equine Vet J, 1984 Nov, 16(6), 519 - 21
Outbreak of botulism in horses; Kelly AP et al.; An outbreak of nervous disease in Standardbred horses occurred near Bendigo, in south-eastern Australia, in October 1980 . Over a two week period 11 horses in four training stables were affected with gait abnormalities, depression and recumbency . Eight of the 11 died . The results of an investigation implicated Clostridium botulinum toxin as the cause . The toxin was food-borne as a contaminant of oaten chaff.

Equine Vet J, 1984 Nov, 16(6), 515 - 8
Thirteen cases of botulism in horses fed big bale silage; Ricketts SW et al.; An outbreak of pharyngeal and limb paresis involving four horses and nine ponies in the south east of England is described . Nine of the animals died or were destroyed on humane grounds . The clinical features suggested a diagnosis of botulism and mouse innoculation tests confirmed the presence of type B toxin in the serum of one case . All animals were fed big bale silage . It is describe how, in plastic wrapped silage manufacture, conditions of fermentation may be inadequate to prevent the growth of Clostridium botulism . Examination of a sample of silage fed to the affected horses suggested that this was probably the source of the toxin.

Arch Microbiol, 1984 Nov, 139(4), 361 - 5
Hydrogenase from Acetobacterium woodii; Ragsdale SW et al.; Hydrogenase from fructose-grown cells of Acetobacterium woodii has been purified 70-fold to a specific activity of 3,500 mumol hydrogen oxidized per min per mg of protein measured at 35 degrees C and pH 7.6 with methyl viologen as electron acceptor . At the same conditions with reduced methyl viologen as electron donor the enzyme catalyzes the evolvement of 440 mumol of H2 per min per mg of protein . The enzyme was found in the soluble portion of the cell, indicating that it is either not membrane-bound or is loosely associated with the membrane . The purified enzyme, which does not contain nickel, exhibits spectroscopic properties similar to the iron-sulfur hydrogenase of Clostridium pasteurianum . The enzyme is strongly inhibited by carbon monoxide, with 50% inhibition occurring at approximately 7 nM CO . Ferredoxin, flavodoxin, and carbon monoxide dehydrogenase are reduced in hydrogen-dependent reaction by the A . woodi hydrogenase.

Appl Environ Microbiol, 1984 Nov, 48(5), 956 - 63
Plasmids in Clostridium botulinum and related Clostridium species; Strom MS et al.; Toxigenic Clostridium botulinum and nontoxigenic C . sporogenes, C . subterminale, and C . botulinum-like organisms from a variety of sources were screened for plasmids . Of the 68 toxigenic C . botulinum isolates, 56% carried one or more plasmids, ranging in mass from 2.1 to 81 megadaltons . Within individual groups (based on the type of neurotoxin produced), many strains showed identical plasmid banding patterns on agarose gels . Of the 15 nontoxigenic strains tested, 40% also carried one or more plasmids ranging from 1.7 to 25.0 megadaltons, with both unique and common banding patterns represented . A total of 67 plasmids from both toxigenic and nontoxigenic strains were detected . At this time, no phenotypic functions have been assessed for these plasmids, and they must therefore be considered cryptic . A variety of lysing and extraction techniques were necessary to detect plasmids in the different C . botulinum groups.

Ann Microbiol (Paris), 1984 Nov-Dec, 135B(3), 269 - 82
Physical characterization of the Clostridium perfringens tetracycline-chloramphenicol resistance plasmid pIP401; Magot M; Four restriction endonucleases were used to construct a physical map of the tetracycline-chloramphenicol resistance plasmid pIP401, which had been isolated from Clostridium perfringens . Twenty-seven restriction sites were placed within the 52-kb map, including 1 site for AvaI, 2 sites for KpnI, 10 sites for EcoRI and 14 sites for PstI . The loss of chloramphenicol resistance in the derived pIP406 plasmid was associated with a deletion of a 6.2-kb DNA segment located in a 10.55 EcoRI-PstI fragment . Two 1.4-kb inverted repeats were also characterized in both pIP401 and pIP406.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Nov, (11), 65 - 8
{Economic coefficients during batch and multicyclic cultivation of Clostridium perfringens type A}; Mel'nikova VA et al.; The analysis of the accomplished processes of cultivation of C . perfringens, type A, has shown that at all cycles of the multicyclic process and during large-scale submerged cultivation in reactors the culture of this micro-organism remains in a physiologically active state . This is confirmed by high values of economic coefficients and by the consumption of keto acids.

Poult Sci, 1984 Nov, 63(11), 2241 - 6
The iron milk most probable number method for enumeration of Clostridium perfringens in the diet and the intestine of the chick; Stutz MW et al.; Seven experiments were conducted to evaluate the iron milk most probable number method for enumeration of Clostridium perfringens in the diet and the intestine of broiler chicks . Levels of 50 ppm of neomycin and 20 ppm of polymixin improved the iron milk tube method when compared to the TSN (tryptone-sulfite-neomycin) agar plate method . Low numbers of the organism, approximately 5 per gram, were detected in the practical diet fed to the chicks . Vegetative cell numbers of C . perfringens increased from 1.7 log10 in the duodenum of chicks to greater than 9.2 log10 in the ceca . Spores of the organism were detected in the ileum and ceca . Results of two experiments demonstrated that C . perfringens became established in the ileum of chicks early in life, before initiation of feeding at 2 days of age.

Biochemistry, 1984 Oct 23, 23(22), 5175 - 81
Purification and characterization of three forms of collagenase from Clostridium histolyticum; Sugasawara R et al.; Three collagenases from Clostridium histolyticum, designated C1, C2, and C3, with apparent molecular weights of 96 000, 92 000, and 76 000 were purified . Peptide maps of the enzymes prepared by digestion with Staphylococcus aureus V-8 protease were found to be similar . Cleavage of native C1 with alpha-chymotrypsin or V-8 protease yielded C2 and C3 . This suggested that proteolysis of the Mr 96 000 collagenase may have occurred in vivo, producing the other two lower molecular weight enzymes . Previously prepared antiserum directed against a form of the bacterial enzyme similar by molecular weight and charge to collagenase C3 and Fab' fragments generated from this antiserum inhibited the collagenolytic activity . C1, C2, and C3 were immunologically identical by Ouchterlony double diffusion, and C3 was able to compete with C1 for the antiserum binding site . The ability of each enzyme to bind to antiserum raised against the bacterial collagenase supported the hypothesis that these three proteins were closely related . Zinc analyses of C1 and C3 resulted in a value of 1.14 mol of zinc/mol of C1 and 0.82 mol of zinc/mol of C3 . C1 did not contain carbohydrate as measured by gas-liquid chromatography or periodic acid-Schiff staining.

Biochim Biophys Acta, 1984 Oct 17, 777(1), 99 - 106
Osmotic stabilizers differentially inhibit permeability alterations induced in Vero cells by Clostridium perfringens enterotoxin; McClane BA; Using a sensitive Vero (African green monkey kidney) cell model system, studies were performed to further investigate whether Clostridium perfringens enterotoxin acts via disruption of the colloid-osmotic equilibrium of sensitive cells . Enterotoxin was shown to cause a rapid loss of intracellular 86Rb+ (Mr approx . 100) with time- and dose-dependent kinetics . The enterotoxin-induced release of intracellular 86Rb+ preceded the loss of two larger labels, 51Cr label (Mr approx . 3500) and 3H-labeled nucleotides (Mr less than 1000) . The osmotic stabilizers, sucrose and poly(ethylene glycol), differentially inhibited enterotoxin-induced larger label loss versus 86Rb+ loss . Further, enterotoxin was shown to cause a rapid influx of 24Na+ that was not significantly inhibited by osmotic stabilizers . Additional studies demonstrated that lysosomotropic agents were not protective against characteristic enterotoxin-induced membrane permeability alterations or morphological damage . Taken collectively, these results are consistent with an action for enterotoxin which involves a disruption of the osmotic equilibrium.

Biochim Biophys Acta, 1984 Oct 12, 805(2), 131 - 6
Polyphosphate-mediated protection from cellular intoxication with Clostridium difficile toxin B; Florin I et al.; The influence of polyphosphorylated compounds on intoxication of human lung fibroblasts with Clostridium difficile toxin B was studied . ATP, as well as other nucleoside di-, tri-, and tetraphosphates, inorganic polyphosphates and polyphosphorylated sugars, caused a dose-dependent (1-5 mM range) delay in the appearance of the cytopathogenic effect . With a longer phosphate chain, the delay was more pronounced, although the cytopathogenic effect always developed finally, reaching the level of the control within 20 h . Toxin preparations contained one fraction of molecules able to bind ATP, besides one non-binding fraction . The protective effect of ATP did not depend on its energy producing ability . Neither was the protective effect due to an inactivation of the toxin per se, or to an interference with binding of the toxin to the cells . ATP was protective even upon addition 10 min after the toxin binding step . In the presence of ATP, the toxin remained accessible to neutralization with antitoxin . In analogy with the P-site on diphtheria toxin, we postulate that C . difficile toxin B contains a polyphosphate-binding site . This site is separate from the receptor-binding site, but involved in the interaction of toxin B with the cell surface shortly after the binding step.

Aust N Z J Med, 1984 Oct, 14(5), 606 - 10
Clostridium difficile colitis; Rocca JM et al.; We reviewed all rectal biopsies performed on patients with proven C . difficile infection between 1977 and 1982 (36 patients) . All patients were symptomatic and all had received antibiotic treatment recently, the commonest antibiotic treatment being ampicillin or amoxycillin . There was poor correlation between the histological appearances and the severity of symptoms . A range of histological appearances was observed: normal (8%), congestion and edema (8%), nonspecific colitis (3%), infective colitis (28%) and pseudomembranous colitis (53%) (PMC) . Most cases of PMC showed 'early' features, involving predominantly the surface epithelium, where attenuation and inflammation, intraepithelial microabscesses, and small eruptive lesions were seen . Recognition of these features, in the context of an acute infective-type colitis, may lead to early diagnosis of C . difficile colitis.

FEBS Lett, 1984 Oct 1, 175(2), 208 - 12
The primary structure of the DNA-binding protein II from Clostridium pasteurianum; Kimura M et al.; The complete amino acid sequence of the Clostridium pasteurianum DNA-binding protein II (DNAb-II) has been determined . The molecule contains 91 amino acid residues and has an Mr of 10 133 . Sequence data were obtained from manual Edman degradation, using the DABITC/PITC double-coupling of the tryptic, peptic, chymotryptic and Staphylococcus protease peptides . A comparison of the amino acid sequence of the C . pasteurianum DNAb-II with those of the DNAb-II from Escherichia coli, Bacillus stearothermophilus, Thermoplasma acidophilum and Pseudomonas aeruginosa shows that the C . pasteurianum protein is more homologous to that of B . stearothermophilus (60%) than to that of E . coli (45%) . All DNAb-II proteins have identical sequences Gly-Phe-Gly-X-Phe at positions 46-50 and Arg-Asn-Pro-X-Thr at positions 61-65.

J Wildl Dis, 1984 Oct, 20(4), 267 - 71
Internal temperature of decomposing duck carcasses in relation to botulism; Wobeser G et al.; Under spring conditions (mean daily maximum 22 C, mean daily minimum 9 C), the temperature within duck carcasses paralleled air temperature for 3 days; on days 4 and 5 the internal temperature rose above 30 C for approximately 30 hr and maximum temperatures of 40-47 C occurred . This coincided with the period of maximum blowfly maggot activity in the carcasses . Carcasses screened from blowflies did not experience this period of high internal temperature . Under autumn conditions (mean daily maximum 13 C, mean daily minimum 1 C), the internal temperature of carcasses paralleled air temperature for approximately 2 wk . Following a warm day (23.5 C), maggots appeared in the carcasses and the internal temperature rose markedly higher than air temperature . Maggots moved into the soil on cold nights and reinhabited the carcasses during the day . The microclimate within maggot-infested carcasses appeared very suitable for growth and toxin production by Clostridium botulinum and this phenomenon may help explain the occurrence of botulism outbreaks during cool weather.

Antimicrob Agents Chemother, 1984 Oct, 26(4), 552 - 6
Prevention of clindamycin-induced mortality in hamsters by Saccharomyces boulardii; Toothaker RD et al.; Saccharomyces boulardii, a yeast used in a number of countries for general and antibiotic-associated gastrointestinal illnesses, was examined for possible application in the prevention of clindamycin-induced mortality in the hamster colitis model . Hamsters were given free access to an aqueous 5% suspension of lyophilized yeast for 3 days before and 10 days after administration of a single oral clindamycin dose of from 0.2 to 0.8 mg/kg . Mortality was recorded in groups of 7 to 20 animals every 24 h for 10 to 30 days . Mean cecal concentrations of S . boulardii were greater than 10(6) CFU/ml throughout the yeast administration period . Yeast treatment significantly decreased cumulative percent mortality by an average of 29% . Death onset was not affected by yeast treatment . Cecitis was present in 86% of moribund animals (N = 95) and was absent in all surviving animals examined (N = 27) . Toxigenic Clostridium difficile was isolated from 13 of 14 moribund hamsters examined . No adverse effects of the yeast treatment were observed in animals receiving S . boulardii without clindamycin . The results suggest that S . boulardii warrants further evaluation for the prevention of antibiotic-associated colitis.

Anal Biochem, 1984 Oct, 142(1), 226 - 31
Chromatographic separation of extruded iron-sulfur cores from the apoproteins of Clostridium pasteurianum and spinach ferredoxins in aqueous Triton X-100/urea; Bonomi F et al.; Iron-sulfur core extrusions from spinach {( 2Fe-2S}) and Clostridium pasteurianum (2{4Fe-4S}) ferredoxins in aqueous Triton X-100/urea containing excess benzenethiol yield quantitatively {FenSn(SPh)4}2- with n = 2 and n = 4, respectively . The iron-sulfur cluster can be separated from the corresponding apoprotein by rapid passage of the extrusion mixture over a small anaerobic column of Whatman DE-52 anion-exchange cellulose . Essentially quantitative recovery of {FenSn (SPh)4}2- is achieved in the eluate . The apoprotein remaining on the column can be eluted with 0.5 M NaCl . Most of the residual Triton X-100 and benzenethiol can be removed by passage of the apoprotein eluate over a small column of Bio-Beads SM-2, a hydrophobic polystyrene adsorbent . Apoprotein recovery is comparable to that obtained by other chromatographic methods . At least with spinach ferredoxin, the apoprotein prepared in this fashion can be reconstituted . The procedures developed in this work are potentially most applicable to selective removal of {2Fe-2S} and {4Fe-4S} centers from a multicenter enzyme without irreversible denaturation.

Microbiologica, 1984 Oct, 7(4), 375 - 9
Cytotoxin and enterotoxin production by Clostridium difficile; Gianfrilli P et al.; 30 strains of Cl . difficile isolated from faeces of patients with pseudomembranous colitis (PMC), antibiotic associated diarrhoea (AAC) and other intestinal disorders and from faeces of asymptomatic carriers were studied for production of toxins . Tissue culture assay was used for the detection of cytotoxin (toxin B) and ileal loop test for enterotoxin (toxin A) . All Cl . difficile isolates from patients with PMC and AAC were found to produce cytotoxin, whereas enterotoxin was demonstrated only in approximately 70% of strains.

J Lipid Res, 1984 Oct, 25(10), 1124 - 31
Mode of action of steroid desmolase and reductases synthesized by Clostridium "scindens" (formerly Clostridium strain 19); Winter J et al.; A recently isolated hitherto unknown Clostridium from human feces, designated Clostridium "scindens" (formerly strain 19), synthesizes at least two enzymes active on the side-chain of the steroid molecule and two enzymes active on the hydroxyl groups of the 7-position of bile acids . Steroid desmolase, responsible for side-chain cleavage of corticoids, and 20 alpha-hydroxysteroid dehydrogenase have not been detected in any other bacterial species of the resident colonic flora . Steroid desmolase is Eh-dependent (optimum ca . -130 mV), requires a hydroxy group at C-17, and preferably an alpha-ketol group in the side-chain; an alpha-hydroxy group at C-20 reduces and a beta-hydroxy group at C-20 prevents side-chain cleavage . With suitable substrates, the yield of C-19 steroids is proportional to the bacterial multiplication rate . 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSDH) is also Eh-dependent (optimum ca . -300 mV) and reduces the C-20 keto function to an alpha-hydroxy group, regardless of the presence or absence of a hydroxy group at C-17 . 7 alpha-Dehydroxylase metabolizes cholic and chenodeoxycholic acid, while 7 beta-hydroxysteroid dehydrogenase acts upon ursodeoxycholic acid . The latter two enzymes are not specific for C . scindens.

J Lipid Res, 1984 Oct, 25(10), 1084 - 9
Formation of urso- and ursodeoxy-cholic acids from primary bile acids by a Clostridium limosum soil isolate; Sutherland JD et al.; A gram-positive, rod-shaped anaerobe (isolate F-14) was isolated from soil . This organism was identified by cellular morphology as well as by fermentative and biochemical data as Clostridium limosum . Isolate F-14 formed ursocholic acid (UC) and 7-ketodeoxycholic acid (7-KDC) from cholic acid (CA), and ursodeoxycholic acid (UDC) and 7-ketolithocholic acid (7-KLC) from chenodeoxycholic acid (CDC) in whole cell cultures, but did not transform deoxycholic acid (DC) . No hydrolysis or transformation occurred when either taurine- or glycine-conjugated bile acids were incubated with F-14 . The type stain of Clostridium limosum (American Type Culture Collection 25620) did not transform bile acids . The structures of ursocholic, ursodeoxycholic, 7-ketodeoxycholic, and 7-ketolithocholic acids were verified by mass spectroscopy and by thin-layer chromatography using Komarowsky's spray reagent . The organism transformed cholic and chenodeoxycholic acids at concentrations of 20 mM and 1 mM, respectively; higher concentrations of bile acids inhibited growth . Optimal yields of ursocholic and ursodeoxycholic acids were obtained at 9-24 hr of incubation and depended upon the substrate used . Increasing yields of 7-ketodeoxycholic and 7-ketolithocholic acids, and decreasing yields of ursocholic and ursodeoxycholic acids were observed with longer periods of incubation . Culture pH changed with time and was characterized by a small initial drop (0.2-0.4 pH units) and a subsequent increase to a pH (8.1-8.2) that was above the starting pH (7.4).(ABSTRACT TRUNCATED AT 250 WORDS)

J Am Vet Med Assoc, 1984 Oct 1, 185(7), 798 - 801
Catastrophic death losses in a dairy herd attributed to type D botulism; Abbitt B et al.; Clostridium botulinum type D intoxication was diagnosed as the cause of death of 42 of 67 lactating cows in a southeast Texas dairy herd over an 11-day period . By necessity, the diagnosis was based on clinicopathologic findings, as the toxin could not, by standard laboratory tests, be demonstrated in affected cattle . The predominant clinical findings were hindlimb weakness/ataxia rapidly progressing to persistent recumbency . Affected cattle were alert until just before death, which occurred without notable agonal movements or respirations after 6 to 72 hours' recumbency . Abnormal laboratory findings included neutrophilic leukocytosis (all affected cattle), proteinuria (most affected cattle), slight elevations of serum aspartate transaminase and low serum inorganic phosphorus (some affected cattle), and patchy areas of hyperemia/congestion of the mucosa in the small intestine (postmortem examination of 3 affected cattle) . This report confirms the findings of others with regard to the difficulty of demonstrating the causative toxin in C botulinum type D-intoxicated cattle and presents available information on the clinicopathologic features of this intoxication that may aid in the differentiation of this condition from other causes of down cows.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Oct, 258(1), 62 - 8
{Formation of alpha-aminobutyric acid in Clostridium sordellii}; Huckenbeck W et al.; After a modified growth C . sordellii is able to ferment threonine as mono-substrate . It could be demonstrated for the first time that alpha-aminobutyric acid is definitely formed from threonine by clostridia . The pH-optimum of this reaction is greater than 8, the temperature optimum is greater than 28 degrees C . Further fermenting products are: glycine and presumably acetaldehyde.

J Bacteriol, 1984 Oct, 160(1), 466 - 9
Effects of cultivation gas phase on hydrogenase of the acetogen Clostridium thermoaceticum; Kellum R et al.; The effect of cultivation gas phase on the expression and activity of hydrogenase in heterotrophic cultures of Clostridium thermoaceticum was examined . Of the five gas phases tested, hydrogenase was maximal from cells cultivated under CO . Correlations were observed between the level of hydrogenase and the evolution of H2 by growing cultures . Activity stains of polyacrylamide gels revealed a single hydrogenase band in CO2 cells and multiple hydrogenase bands in CO cells.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Oct, 258(1), 51 - 61
{Degradation of glutamic acid and proline in Clostridium sordellii under cadaveric bacteriological inspection}; Huckenbeck W et al.; Glutamic acid (GLU) is decarboxylated to gamma-aminobutyric acid (GABA) by Clostridium sordellii isolated from a decaying human brain . The dependence of this reaction on temperature, pH and substrate concentration has been established . The pH-optimum is in the range of 5.0 to 5.2 . Near optimal pH the temperature optimum is greater than 28 degrees C . At higher pH-values (6 to 7) activity is relatively independent of temperature . The similarity to results in decaying human brains (10, 11, 12) shows a good correlation between postmortem bacterial flora and GLU-degradation . Furthermore it is shown that C . sordellii produces delta-aminovaleric acid (AVA) from proline (PRO).

Avian Dis, 1984 Oct-Dec, 28(4), 1120 - 4
The occurrence of Clostridium perfringens in the intestine of chicks; Shane SM et al.; Commercial broiler chicks brooded either on wire or on used or new litter demonstrated a 75% (62/75) incidence of recovery of "perfringens-like" colonies from the intestine during a 5-week period . Eleven Clostridium spp . were identified from among these "perfringens-like" organisms, which were cultured on SPS selective agar medium . Clostridium perfringens was positively identified only infrequently (five isolates) from among the "perfringens-like" colonies . In contrast, "perfringens-like" colonies were not recovered from the intestinal contents of specific-pathogen-free chicks reared in an isolation unit . However, C . perfringens was isolated from the yolk sac of one embryonated egg and from the intestine of a single 7-day-old chick, indicating the possibility of vertical transmission of this potential pathogen.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Oct, (10), 20 - 4
{Effect of iron on the growth and toxin formation of Clostridium perfringens type A cultures}; Artemenko VD et al.; The influence of iron at different concentrations on the growth and toxin formation of the cultures of C . perfringens strain 28 BP6K, type A, in media having different composition and protein base has been studied . As revealed by the results of these studies, bivalent iron at concentrations of 1-30 mg%, while having no essential influence on the growth and development of the culture, not only produces no stimulating effect on toxin formation, but even inhibits it . The concentrations of iron, both producing the optimum effect on toxin formation and inhibiting it, are not identical for different kinds of culture media and depend on their nature and composition . In culture media with fodder yeast extract added, and especially in media based on fermentative fodder yeast hydrolysate, the cultures have shown the increased stability of toxin formation in the presence of higher concentrations of iron.

Lab Anim Sci, 1984 Oct, 34(5), 443 - 52
Experimental and spontaneous clostridial enteropathies of laboratory and free living lagomorphs; Carman RJ et al.; The enteric diseases of hares, European and cottontail rabbits, which are caused by members of the genus Clostridium are reviewed . Disease caused by C . perfringens Types A and E, C . spiroforme, C . difficile, C . sordellii, C . tympany cuniculi and clostridial enterotoxins are included . Tyzzer's disease also is discussed.

Poult Sci, 1984 Oct, 63(10), 2036 - 42
Effects of diet and antimicrobials on growth, feed efficiency, intestinal Clostridium perfringens, and ileal weight of broiler chicks; Stutz MW et al.; Five experiments were conducted to evaluate the effects of diet and antimicrobials on weight gain, feed efficiency, ileal weight, and Clostridium perfringens in the ileum of broiler chicks . In the first experiment, glucose, sucrose, and fructose were added to a semipurified diet and the results were compared with those from a practical corn and soybean meal diet . All of the diets were fed with and without bacitracin at a level of 55 ppm . Fructose resulted in the greatest depression in weight gain, followed by sucrose . Bacitracin significantly improved weight gain and feed efficiency of chicks fed the fructose, sucrose, and practical diets . Highly significant inverse correlations were obtained between ileal weight and weight gain and the number of C . perfringens in the ileum and weight gain . In other experiments bacitracin, penicillin, chlortetracycline, oxytetracycline, erythromycin, tylosin, virginiamycin, lincomycin, bambermycins, and carbadox, all at a level of 55 ppm, improved weight gain and feed efficiency and significantly reduced the weight of the ileum and the number of C . perfringens in the ileum of chicks fed the practical diet . The antibacterial agents 3-nitro-4-hydroxy-phenylarsonic acid, arsanilic acid, furazolidone, and sulfathiazole had little to no effect on the 4 parameters evaluated . Virginiamycin and lincomycin at 16.5 and 4.4 ppm, respectively, were shown to be effective . In vitro activities of the antimicrobials against C . perfringens did not directly relate to in vivo activities and the effects on growth and feed efficiency . The results of these experiments support the concept of antimicrobials as growth permittants and provide further evidence for C . perfringens as a causative bacteria for growth depression.

Pflugers Arch, 1984 Oct, 402(2), 171 - 5
Ca2+- and H+-dependent effects of crude bacterial phospholipase C on the hydroosmotic response of toad urinary bladder to serosal hypertonicity; Hardy MA; Phospholipase C (EC 3.1.4.3.) from Clostridium perfringens (crude extracts) was used to study the role of phospholipids in the osmotic permeability of the urinary bladder of the toad . When added to the serosal bath (430 mU/ml) it inhibited the effects of antidiurectic hormone (ADH) and exogenous cyclic AMP . Under the same conditions the increase in osmotic flow produced by serosal hypertonicity (SH) was slightly enhanced by the lipase . The hydroosmotic effect of SH was greatly potentiated by the lipase by decreasing 10-fold the Ca2+ concentration . The SH-induced flow was inhibited by the lipase if the Ca2+ or the H+ concentration was increased 10-fold, but not if the increase in positive charges was produced by a concentration of Mg2+ . Phospholipase C had no effect on the action of either ADH or SH if added to the mucosal bath . Serosal neuraminidase or phospholipase A2 could not mimic the effect of phospholipase C on SH . The effect of phospholipase C on the response to SH was not modified if fatty acid-free bovine serum albumin was added to the bath . Therefore, the release of products of lipolysis into the bath do not seem to be responsible for the effects of phospholipase C on SH-induced water flow . The results suggest that the effects of the enzyme on the composition and rearrangement of lipids at the basolateral membrane produce modifications of the water flow . Ca2+ and H+ may modify the enzyme-substrate interaction, suggesting that different phospholipids may be differentially involved in the control of water permeability of the basolateral membrane.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1984 Sep 25, 23(20), 4650 - 5
Identification and synthesis of a naturally occurring selenonucleoside in bacterial tRNAs: 5-{(methylamino)methyl}-2-selenouridine; Wittwer AJ et al.; Escherichia coli, Clostridium sticklandii, and Methanococcus vannielii synthesize 75Se-labeled amino acid transfer ribonucleic acids {( 75Se}tRNAs) when grown with low levels (approximately equal to 1 microM) of 75SeO32- . When E . coli {75Se}tRNA was digested to nucleosides and analyzed by reversed-phase high-performance liquid chromatography, a single selenonucleoside accounted for 70-90% of the 75Se label in the bulk tRNA . This nucleoside was shown to be indistinguishable in a number of its properties from authentic 5-{(methylamino)methyl}-2-selenouridine . Preparation of the authentic selenonucleoside was accomplished and the synthetic compound characterized by its UV and 1H NMR spectral properties . The new selenonucleoside also accounted for 40-60% of the 75Se found in {75Se}tRNA from C . sticklandii or M . vannielii . Each of these anaerobic bacteria contains one additional selenonucleoside in their tRNA populations distinct from 5-{(methylamino)methyl}-2-selenouridine . Pure seleno-tRNAGlu isolated from C . sticklandii contains one 5-{(methylamino)methyl}-2-selenouridine and one 4-thiouridine per tRNA molecule.

J Biol Chem, 1984 Sep 25, 259(18), 11396 - 402
Unusual features in EPR and Mössbauer spectra of the 2{4Fe-4Se}+ ferredoxin from Clostridium pasteurianum; Moulis JM et al.; The electronic and magnetic properties of the selenium-substituted 2{4Fe-4Se}2+/+ ferredoxin (Fd) from Clostridium pasteurianum have been investigated by EPR and Mossbauer spectroscopy . The {4Fe-4Se}2+ clusters of oxidized Fd are diamagnetic and the Mossbauer spectra are nearly identical to those of oxidized 2{4Fe-4S}2+ Fd . The addition of 2e- per molecule of Se-substituted Fd causes the simultaneous appearance of three EPR signals: one (g1,2,3 = 2.103, 1.940, 1.888) is reminiscent of {4Fe-4S}+ EPR spectra and accounts for 0.7 to 0.8 spin/molecule . The two others consist of a broad signal with g = 4.5, 3.5, and approximately 2 (0.7 to 0.8 spin/molecule) and of a narrow peak at g = 5.172 which is observed up to 60 K . Peculiar features are also present in the Mossbauer spectra of 2{4Fe-4Se}+ Fd below 20 K: a subcomponent with lines near to +/- 4 mm/s and accounting for 20% of the total iron corresponds to two antiferromagnetically coupled sites in approximately a 3:1 ratio and displays fully developed paramagnetic hyperfine interactions at 4.2 K without any applied field . At 77 K, however, the reduced Se-substituted Fd yields a Mossbauer spectrum similar to that of 2{4Fe-4S}+ Fd . The new EPR and Mossbauer spectroscopic features of the 2{4Fe-4Se}+ Fd are attributed to S = 3/2 and S = 7/2 spin states which accompany the classical S = 1/2 state of {4Fe-4X}+ (X = S, Se) structures.

Biochem Biophys Res Commun, 1984 Sep 17, 123(2), 463 - 7
Energy-dependent activation of spore-lytic enzyme precursor by germinated spores of Clostridium perfringens; Ando Y et al.; A precursor of the spore-lytic enzyme of Clostridium perfringens was extracted with alkali from dormant spores of the organism . The enzyme precursor was activated by incubating it with germinated spores which had been treated with alkali . The activation was greatly enhanced by the addition of 3-phosphoglycerate, suggesting that the conversion of precursor to active enzyme depends on endogenous energy-producing metabolism during germination.

Biochemistry, 1984 Sep 11, 23(19), 4309 - 17
23Na NMR relaxation study of the effects of conformation and base composition on the interactions of counterions with double-helical DNA; Nordenskiold L et al.; NMR relaxation rates (T1(-1) and T2(-1)) have been determined for 23Na in aqueous salt solutions containing various types of helical double-stranded deoxyribonucleic acids . These measurements were performed on three synthetic polynucleotides having different overall conformations, poly-(dA-dT).poly(dA-dT) (alternating B-DNA), poly(dG-dC).poly(dG-dC) at low salt (B-DNA), and Br-poly(dG-dC).Br-poly(dG-dC) (left-handed Z-DNA), and on four types of natural DNA differing in base composition, Clostridium perfringens (26% GC), calf thymus (40% GC), Escherichia coli (50% GC), and Micrococcus lysodeikticus (72% GC) . For all types of DNA investigated, except poly(dA-dT).poly(dA-dT), the 23Na NMR spectra measured at 21 degrees C and an applied field of 4.7 T are non-Lorentzian . These non-Lorentzian spectra were analyzed on the basis of the two-state model and the standard theory of nonexponential quadrupolar relaxation processes in order to obtain estimates of the correlation times (tau c) characteristic of the sodium nuclei associated with the various nucleic acids . All of the correlation times estimated in this way are in the range of nanoseconds . The magnitudes of these correlation times show a significant dependence on the overall conformation of the nucleic acid (B vs . Z) but not on its base composition . To investigate the concentration dependence of tau c, sodium or magnesium salts were added to solutions of Br-poly(dG-dC).Br-poly(dG-dC) (Z-DNA).(ABSTRACT TRUNCATED AT 250 WORDS)

J Biol Chem, 1984 Sep 10, 259(17), 10845 - 9
Purification and properties of 5,10-methylenetetrahydrofolate reductase, an iron-sulfur flavoprotein from Clostridium formicoaceticum; Clark JE et al.; Methylenetetrahydrofolate reductase in Clostridium formicoaceticum has been purified to a specific activity of 140 mumol min-1 mg-1 when assayed at 37 degrees C, pH 7.2, in the direction of oxidation of 5-methyltetrahydrofolate with benzyl viologen as electron acceptor . The purified enzyme is judged to be homogeneous by polyacrylamide disc-gel electrophoresis and gel filtration . The enzyme which is an octamer has a molecular weight of about 237,000 and consists of four each of two different subunits having the molecular weights 26,000 and 35,000 . The octameric enzyme contains per mol 15.2 +/- 0.3 iron, 2.3 +/- 0.2 zinc, 19.5 +/- 1.3 acid-labile sulfur, and 1.7 FAD . The UV-visible absorbance spectrum has a peak at 385 nm and a shoulder at 430 nm and is that of a flavoprotein containing iron-sulfur centers . The reductase, which is sensitive to oxygen, must be handled anaerobically and is stabilized by 2 mM dithionite . It catalyzes the reduction of methylene blue, menadione, benzyl viologen, rubredoxin, and FAD with 5-methyltetrahydrofolate and the oxidation of reduced ferredoxin and FADH2 with 5,10-methylenetetrahydrofolate . No activity was observed with pyridine nucleotides . It is suggested that the physiologically important reaction catalyzed by the enzyme is the reduced ferredoxin-dependent reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate.

FEBS Lett, 1984 Sep 3, 174(2), 284 - 8
A high-molecular-mass cell wall protein released from Clostridium tyrobutyricum by heat treatment; Hayes H et al.; Analysis of the cell wall of 4 strains of Clostridium tyrobutyricum reveals an unusually high protein content (35-40% dry weight) . Brief heat treatment of whole cells of these stains causes release of two proteins, flagellin and a cell wall component of high molecular mass (110-125 kDa in the different strains) . This component represents approx . 5% of the dry cell weight.

J Antimicrob Chemother, 1984 Sep, 14 Suppl C, 7 - 17
The activity of enoxacin against clinical bacterial isolates in comparison with that of five other agents, and factors affecting that activity; Reeves DS et al.; The activity of enoxacin against 362 clinical bacterial isolates in comparison with norfloxacin, nalidixic acid, ampicillin, latamoxef (moxalactam) and gentamicin was tested by an agar dilution method . Typical MICs for enterobacteria lay between 0.12 and 1.0 mg/l . Enterobacter spp . and Serratia spp . tended to be more resistant . Enoxacin was also active against Pseudomonas aeruginosa (mean MIC 0.5 mg/l) and highly active against fastidious Gram-negative aerobes . Typical MICs for Staphylococcus aureus were 1-2 mg/l while streptococci were more resistant (16-32 mg/l) . Enoxacin had no useful activity against Bacteroides fragilis and Clostridium perfringens . Enoxacin was generally more active at pH 8 than pH 6, and in broth than in urine . It was bactericidal in its action . Daily serial passage from growth in broth containing enoxacin caused decreased sensitivity which was limited to four- to 16-fold greater than the original MIC . Enoxacin was about half as active as norfloxacin against enterobacteria, equally active against staphylococci, and some two to four times less active against streptococci.

Ann Microbiol (Paris), 1984 Sep-Oct, 135B(2), 219 - 22
{Biosynthesis of toluene in Clostridium aerofoetidum strain WS}; Pons JL et al.; Formation of toluene in growing cultures of Clostridium aerofoetidum strain WS was enhanced when the medium was supplemented with phenylacetic acid or with L-phenylalanine and L-methionine together . Evidence for the role of L-phenylalanine was shown by the detection of {2H2}-methyl{2,3,4,5,6-2H5}benzene ("heptadeuterotoluene") in growing cultures with L-{2',3',4',5',6'-2H5}phenyl{2,3-2H3}alanine and L-methionine.

J Antibiot (Tokyo), 1984 Sep, 37(9), 949 - 57
Empedopeptin (BMY-28117), a new depsipeptide antibiotic . I . Production, isolation and properties; Konishi M et al.; Empedopeptin is a new antibiotic produced by empedobacter haloabium nov . sp . (ATCC 31962) . It is a water-soluble depsipeptide antibiotic containing eight amino acid residues and a C14-fatty acid moiety in the molecule . Although structurally unrelated, empedopeptin and vancomycin have similar antimicrobial spectra against aerobic and anaerobic Gram-positive bacteria including antibiotic-resistant strains . Empedopeptin is highly active in vivo in mice against systemic infections of Staphylococcus aureus, Streptococcus pyogenes and Clostridium perfringens . Empedopeptin is not absorbed orally.

Vet Microbiol, 1984 Sep, 9(5), 497 - 502
Infectious nature of Clostridium spiroforme-mediated rabbit enterotoxaemia; Carman RJ et al.; Newly weaned rabbits had diarrhoea only if they were infected with Clostridium spiroforme . In adult rabbits exposure to both clindamycin and C . spiroforme was necessary to induce disease . All diseased animals harboured C . spiroforme and its toxin . Adult rabbits given a course of clindamycin survived when held in a protected environment as did those challenged with C . spiroforme alone . At necropsy none of these apparently healthy animals showed signs of diarrhoea or caecitis . These findings suggest that, in the development of enterotoxaemia, weaning or clindamycin treatment and infection with C . spiroforme are separate events and that disease follows infection with this organism from the environment, as opposed to overgrowth by undetectable levels of C . spiroforme resident in the gut . Our data indicate that C . spiroforme is not a normal component of the rabbit gut flora and that the normal bowel ecology of the adult must be disrupted before C . spiroforme will colonize.

Pediatr Infect Dis, 1984 Sep-Oct, 3(5), 429 - 32
Nosocomial Clostridium difficile reservoir in a neonatal intensive care unit; Zedd AJ et al.; A new bacteriophage/bacteriocin typing system was used to study Clostridium difficile colonization in a neonatal intensive care unit . C . difficile was isolated from 21 of 62 (34%) stools from 15 of 37 (41%) infants . Colonization was reduced during antimicrobial therapy and for about 1 week thereafter . One of five nurses and one of two parents studied were carriers . Eight isolates were cultured from environmental surfaces . Thirty of 31 C . difficile isolates were found to be a single type, Cld 6,9,10,13; bacteriocin 1320,1537,2304 . No C . difficile was found in 29 specimens obtained in the delivery room from mothers and infants, and there was no association of early colonization with vaginal delivery . The data provide strong evidence for nosocomial acquisition of C . difficile by infants in the neonatal intensive care unit . No obvious pathologic role for C . difficile could be identified among colonized infants . Among 22 C . difficile isolates from 7 adult inpatients with diarrhea and 13 healthy infants attending the center's well baby clinic, 4 were the same type as the strain found in the intensive care nursery . Only one of these patients had had direct contact with the neonatal intensive care unit, indicating that the nursery strain may also be found elsewhere in the community.

J Clin Microbiol, 1984 Sep, 20(3), 549 - 60
Studies of stools from pseudomembranous colitis, rotaviral, and other diarrheal syndromes by frequency-pulsed electron capture gas-liquid chromatography; Brooks JB et al.; Thirty-five patients with various diarrheal syndromes and 22 controls were studied . All stool samples were carefully cultured for Clostridium difficile, using selective isolation media . Cytotoxin assays with proper antitoxin neutralization were done in MRC-5 cells . The stool samples were extracted four times, three times at pH 2 and once at pH 10, using CHCl3 or ether . Derivatizations of extracts were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol, and all derivatives were analyzed by frequency-pulsed electron capture gas-liquid chromatography (FPEC-GLC) . A dedicated computer was used to assist in both qualitative and quantitative data analysis . Isocaproic acid (iC6) was always found in stool from which C . difficile was isolated and was absent in C . difficile-negative specimens . p-Cresol was found frequently in both persons with pseudomembranous colitis and controls . Tryptamine was found in stool containing C . bifermentans . The FPEC-GLC profiles of persons with acute diarrhea were very different from those of normal persons . Diarrhea associated with adenovirus and rotavirus, Klebsiella spp., and Escherichia spp . showed different FPEC-GLC patterns . Stools from well persons consistently contained full-scale peaks of pyruvic, acetic, propionic, isobutyric, butyric, isovaleric, and valeric acids . In rotavirus stools isobutyric, isovaleric, and valeric acids were reduced in quantity from those found in control stools, whereas propionic and butyric acids were increased.

J Clin Microbiol, 1984 Sep, 20(3), 539 - 48
Frequency-pulsed electron capture gas-liquid chromatographic analysis of metabolites produced by Clostridium difficile in broth enriched with amino acids; Brooks JB et al.; Clostridium difficile strain CDC A-567 was cultured in Trypticase (BBL Microbiology Systems)-yeast-salt broth supplemented with 0.2% L-leucine, L-norleucine, L-isoleucine, L-tyrosine, or L-tryptophan . Four extractions were done on the spent medium, three at pH 2 and one at pH 10, using CHCL3 or ether . Derivatizations were done with trichloroethanol, heptafluorobutyric anhydride, and heptafluorobutyric anhydride-ethanol . All samples were analyzed with frequency-pulsed electron capture gas-liquid chromatography . A dedicated computer was used to assist in data analysis . C . difficile produced both short-chain and aromatic acids in Trypticase-yeast-salt broth; hydroxy acids were also detected . p-Cresol, indoleacetic acid, 4-methylthio-2-hydroxybutyric acid, and some unidentified alcohols were observed . The basic chloroform extraction contained cadaverine and putrescine . Leucine, norleucine, and isoleucine influenced the production of C5 and C6 acids and alcohols . L-Tyrosine underwent successive degradation to produce p-cresol and aromatic acids as final products . Tryptophan increased the production of indoleacetic, indolepropionic, and indolebutyric acids . Isocaproic acid was produced in relatively high concentrations regardless of medium substitution . The consistent production of iC6 under various substrate conditions indicates that the production of this compound might be consistent enough in vitro to form the basis of a rapid test for detection of C . difficile in stool specimens by frequency-pulsed electron capture gas-liquid chromatography.

J Clin Microbiol, 1984 Sep, 20(3), 339 - 41
Latex agglutination test for detection of Clostridium difficile toxin in stool samples; Shahrabadi MS et al.; A total of 163 stool specimens were tested for detection of Clostridium difficile and its toxin by cytotoxicity assay with tissue culture, latex agglutination test, and isolation of the organism . From 33 specimens which were positive for toxin by cytotoxicity, 30 were positive by the latex agglutination test; the organism was isolated from 21 . The total number of samples which were positive with the latex agglutination test was 44 . The predictive value of a positive latex agglutination result relative to the cytotoxicity test was 68%, and the predictive value of a negative result was 97.5% . The specificity and sensitivity of the latex agglutination test relative to the cytotoxicity assay and the low cost and simple facilities required indicate that the latex agglutination test is a useful procedure for screening for C . difficile toxins, provided that positive latex results are confirmed by cytotoxicity assay.

Tijdschr Diergeneeskd, 1984 Sep 1, 109(17), 669 - 71
{Liability, prostaglandins and Clostridium}; van Nie GJ; A case of malignant oedema following injection of fenprostalene in cattle is reported . The question is asked whether the vasoconstrictive action of prostaglandins may predispose to the establishment of anaerobes.

Can J Surg, 1984 Sep, 27(5), 435 - 7
Clostridium difficile in Crohn's disease; Wright JM et al.; Clostridium difficile has been detected in the stools of some patients with relapse of Crohn's disease . The authors looked prospectively for present or previous exposure to C . difficile cytotoxin in 10 patients with mild to severe Crohn's disease . None of 25 stool samples from these 10 patients was positive for C . difficile cytotoxin . These negative stool ultrafiltrates had mild cytotoxin neutralizing activity, but this finding did not differ from that in 30 cytotoxin-negative stools from patients with other diarrheal diseases . Serum from these patients also showed no cytotoxin neutralizing activity . Review of the literature reveals that C . difficile can cause complications ranging from diarrhea to toxic megacolon in a small but variable proportion of patients with Crohn's disease . There is no evidence that C . difficile plays a part in the pathogenesis of the disease.

Biochem J, 1984 Sep 1, 222(2), 535 - 40
A transient increase in diacylglycerols is associated with the action of vasopressin on hepatocytes; Hughes BP et al.; Vasopressin induced a transient increase of 50% in the total concentration of diacylglycerols (determined by g.l.c.) in isolated hepatocytes . The increase was maximal at 0.25 min, and the concentration of diacylglycerols in cells treated with vasopressin had returned to the basal value by 4 min . No change in the concentration of diacylglycerols was observed after the treatment of cells with glucagon . The dependency of this effect on the concentration of vasopressin was similar to that of the effect of the hormone on 45Ca2+ efflux measured at 0.1 mM extracellular Ca2+ . Vasopressin increased the proportion of arachidonic acid and stearic acid and decreased the proportion of oleic acid present in the diacylglycerols . In hepatocytes prelabelled with {14C}arachidonic acid, vasopressin increased the amount of {14C}diacylglycerol . The effects of vasopressin on the total concentration of diacylglycerols and {14C}diacylglycerol were mimicked by an exogenous phospholipid phosphodiesterase (phospholipase C) from Clostridium perfringens . The results are consistent with the conclusion that the transient increase in diacylglycerols induced by vasopressin is caused by the rapid hydrolysis of both the phosphoinositides and one or more other phospholipids.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Sep, (9), 97 - 9
{Comparative study of resistance to tetanus and indices of the passive hemagglutination reaction in experimental animals}; Stovbun SF et al.; The article presents the results of the study of resistance to tetanus in 450 guinea pigs immunized against tetanus in a single injection and having antitoxin titers in their blood, as determined in the passive hemagglutination test, from less than or equal to 0.01 IU/ml to 1.6 IU/ml and more . The degree of protection in the immunized animals was determined by their challenge with Clostridium tetani spores in DCL and LD50.

J Anim Sci, 1984 Sep, 59(3), 813 - 22
Thiamin and niacin in the rumen; Brent BE et al.; Thiamin analogs, produced in the rumen by thiaminase I, in the presence of a cosubstrate appear to be responsible for the central nervous system disorder, polioencephalomalacia (PEM) . For PEM to occur, an analog must be produced that inhibits an essential thiamin-requiring reaction, and results from a cosubstrate present in the rumen . In high concentrate diets, thiaminase I is produced by rumen microbes . However, PEM can also be caused by thiaminase I of plant origin . Based on physical characteristics and cosubstrate specificity, the thiaminase I enzymes produced by Bacillus thiaminolyticus and Clostridium sporogenes appear to be different from the enzyme produced by the rumen . Because niacin and certain antihelmentics are thiaminase I cosubstrates, they should be used cautiously . Supplementary niacin increased microbial protein synthesis in vitro and in vivo, and was more effective with urea than soybean meal . Supplementary niacin (5 to 6 g X cow-1 X d-1) increased milk production in postpartum cows but not in those in mid-lactation, and in cows fed soybean meal but not in those fed urea . We believe the heating of soybean meal during commercial processing decreased the availability of niacin for rumen protozoa . Supplementary niacin for postpartum cows increased blood glucose, decreased blood ketones and reduced the incidence of ketosis . Niacin flow to the small intestine and its absorption from the small intestine increased with niacin supplementation . Supplemental niacin prevented the postpartum decrease in red blood cell niacin observed in control cows.

Pediatr Infect Dis, 1984 Sep-Oct, 3(5), 433 - 6
Occurrence of Clostridium difficile toxin-associated gastroenteritis following antibiotic therapy for otitis media in young children; Hyams JS et al.; The pathogenesis of diarrhea following antibiotic therapy for otitis media in young children remains unknown . We performed a prospective study evaluating the incidence of diarrhea and Clostridium difficile toxin in 115 outpatients (ages 6 months to 6 years) with acute otitis media treated with ampicillin, amoxicillin or trimethoprim-sulfamethoxazole . In 21 patients younger than one year of age six of 11 developing diarrhea had toxin-positive stools compared with three of 10 without diarrhea (P = 0.39) . In 94 patients between 13 months and 6 years of age three of 12 with diarrhea had toxin-positive stools compared with five of 82 without diarrhea (P = 0.06) . Diarrhea was self-limited in all cases . Although the data suggest that C . difficile might have been associated with diarrhea in the older children, further studies will be required to confirm this finding.

J Pharmacol Exp Ther, 1984 Sep, 230(3), 665 - 9
Molecular basis for the pharmacological actions of Clostridium botulinum type C2 toxin; Simpson LL; The light chain of type C2 toxin produced by Clostridium botulinum was isolated by high-performance liquid chromatography . The protein eluted as a single peak; as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, it had an apparent molecular weight of 51,000 daltons . The light chain was an enzyme that possessed ADP-ribosylating activity . In experiments with synthetic substrates (homo-poly-L-amino acids; alanine, arginine, asparagine, aspartic acid, histidine, leucine, lysine, methionine, phenylalanine, proline, serine and tryptophan), only poly-L-arginine was ADP-ribosylated by the enzyme . In experiments with endogenous substrates (50,000 X g pellet and 50,000 X g supernatant from homogenates of mouse brain, liver and lung), the enzyme ADP-ribosylated proteins or polypeptides in both the particulate and soluble fractions . ADP-ribosylation of the soluble substrate was antagonized by adenine (K1 approximately 2.1 X 10(-5) M) and by adenosine (K1 approximately 2.7 X 10(-4) M); the reaction was reversed by a large molar excess of nicotinamide (0.1 M) . ADP-ribosylation of soluble substrate was diminished when the substrate had been pretreated with 1,2-cyclohexane-dione (0.1 M), a site reactive reagent that modified selectively arginine residues . Neither the light chain nor the heavy chain of the binary toxin possessed adenylate cyclase activity . Tissue fractions did possess endogenous adenylate cyclase activity, but the toxin did not stimulate this activity . The data indicate that the binary toxin produced by Clostridium botulinum resembles other protein toxins.

Anal Biochem, 1984 Sep, 141(2), 344 - 7
Simultaneous single-step purification of thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri; Sliwkowski MX et al.; Thiolase and NADP-dependent 3-hydroxybutyryl-CoA dehydrogenase from Clostridium kluyveri were purified by ion-exchange chromatography to near homogeneity in a simultaneous, single-step procedure . The yield of both enzymes was greater than 80% . Thiolase was purified approximately 8-fold with sp act 115 units/mg, whereas 3-hydroxybutyryl-CoA dehydrogenase was purified 14-fold with sp act 292 units/mg . Isoelectric points of the enzymes are 7.7 for thiolase and 7.8 for 3-hydroxybutyryl-CoA dehydrogenase . Milligram quantities of each of these enzymes are readily obtained from this fatty acid-producing organism.

Rev Infect Dis, 1984 Sep-Oct, 6(5), 715 - 9
Spontaneous clostridial empyema and pyopneumothorax; Raff MJ et al.; Five patients developed pleural empyema due to Clostridium perfringens in the absence of penetration of the thorax; two of the patients presented with pyopneumothorax . Thirteen additional cases from the literature are reviewed . Predisposing factors to the development of pleural empyema appear to include aspiration pneumonia, pulmonary embolization and infarction, and bacteremia from other foci . Pleural disease and pulmonary tuberculosis may also predispose patients to pleural empyema . Treatment consists of drainage and antimicrobial chemotherapy.

Biochem Biophys Res Commun, 1984 Aug 30, 123(1), 33 - 40
Steroidogenic effect of exogenous phospholipase C on bovine adrenal fasciculata cells; Hadjian AJ et al.; Phospholipase C (Bacillus cereus) added to the incubation medium stimulated the steroidogenic activity of bovine adrenal zona fasciculata cell suspensions to a level similar to that induced by optimal concentration of ACTH . This effect was not related to an increase of cyclic AMP; it was calcium-dependent and was also induced by an other bacterial phospholipase C (from Clostridium perfringens) whereas phospholipases A2 and D were ineffective . Phospholipid metabolism was examined in these cells after radiolabeling with {14C}-glycerol or {32P}orthophosphate . Phospholipase C induced a very fast (5 seconds) increase in cellular {14C}-1,2-diacylglycerol followed by {32P} labeling of phosphatidic acid and phosphatidylinositol . These events preceded the stimulation of steroidogenesis which was detectable after 2 minutes of incubation . These observations suggest that activation of an endogenous phospholipase C activity may be considered as an early event in the response of bovine adrenocortical cells to steroidogenic effectors such as angiotensin II and acetylcholine.

Life Sci, 1984 Aug 20, 35(8), 849 - 54
Inhibition of the autoxidation of ascorbate and norepinephrine by extracts of Clostridium butyricum, Megasphaera elsdenii and Escherichia coli; Mishra OP et al.; The autoxidation of ascorbate and of norepinephrine in Krebs Ringer phosphate medium, pH 7.4, was studied . The autoxidation of the two substances was determined spectrophotometrically at 265 and 480 nm respectively . The effect of dialyzed extracts (m.w . greater than 12,000) from Escherichia coli (aerobe), Megasphaera elsdenii, and Clostridium butyricum (obligate anaerobes) was examined and compared to similarly prepared extracts from rat serum and cerebral cortex . The assay medium contained cellular components diluted 10(3)-10(6)-fold . Up to 10(4)-fold dilution there was a substantial reduction in the rate of both autoxidation reactions, but the preparations from M . elsdenii and C . butyricum were conspicuously less effective . After 5 min heat treatment at 100 degrees C the anaerobic preparations produced less than 20% inhibition, while the activity of the other preparations remained unchanged at 75-95% inhibition . These and earlier experiments involving additional mammalian species (Mishra and Kovachich, Neurosci . Lett., 43: 103-108, 1983) and plants (Mishra and Kovachich, Life Sci., 34: 2207-2212, 1984) suggest that a high level of heat-stable antioxidant activity in one or both of these autoxidation tests (denatured plant extracts only inhibit ascorbate autoxidation) is a general characteristic of organisms that thrive in oxygen-rich atmosphere.

J Hyg (Lond), 1984 Aug, 93(1), 17 - 25
Prospective study of Clostridium difficile colonization and paracresol detection in the stools of babies on a special care unit; Phua TJ et al.; Infants' stools were examined for the presence of Clostridium difficile and its cytotoxin in a study performed over a one-year period on a special care baby unit . Overall, 21% of infants were colonized, but the organism was only recovered in a seven-month period during which its weekly prevalence in the group varied from zero to 44%, with a distinct clustering of colonized infants being observed . Tests for the presence of cytotoxin in the stools and in supernatants of broth that had been inoculated with each isolate were negative . The factors predisposing to colonization were a prolonged stay in the unit, low birth weight, younger gestational age and being nursed in an incubator . The organism was recovered only once from an environmental screen . An antibiogram, used in conjunction with toxin production, was helpful in distinguishing these isolates from a collection obtained from other units in the hospital . We conclude that Cl . difficile was acquired by nosocomial spread although we did not establish the precise mechanism involved . The detection of para-cresol by gas-liquid chromatography was found to be specific but insufficiently sensitive as a screening test for the organism's presence in the stools . It could only be demonstrated in infants whose birth-weights were less than 2500 g, and no association was observed between the type of feed and para-cresol presence in stools.

J Bacteriol, 1984 Aug, 159(2), 700 - 3
Development of a minimally defined medium for the acetogen Clostridium thermoaceticum; Lundie LL Jr et al.; A minimally defined medium was developed for the cultivation of the acetogen Clostridium thermoaceticum . The medium contained glucose as the carbon and energy source, ammonium sulfate as the nitrogen source, nicotinic acid as the sole essential vitamin, reductant, a phosphate-bicarbonate buffer, mineral salts and chelator, and a CO2 gas phase . Adaptation of C . thermoaceticum from undefined medium containing yeast extract and tryptone to the minimally defined medium required sequential passage on defined medium supplemented with amino acids and vitamins . Growth and cell yields were reduced on the minimal medium, but the activities of carbon monoxide dehydrogenase, hydrogenase, and formate dehydrogenase were comparable between undefined and minimal media.

J Bacteriol, 1984 Aug, 159(2), 597 - 604
Biosynthesis of phospholipids in Clostridium butyricum: kinetics of synthesis of plasmalogens and the glycerol acetal of ethanolamine plasmalogen; Koga Y et al.; The biosynthesis of the plasmalogen forms of phosphatidylethanolamine (plasmenylethanolamine) and phosphatidylglycerol (plasmenylglycerol) and of the glycerol acetal of plasmenylethanolamine has been studied in cultures of Clostridium butyricum IFO 3852 . When growing cells were pulsed with {32P}orthophosphate, there was a lag of 5 to 7 min between the rapid incorporation of label into the acylphosphatides and the rapid incorporation of label into the corresponding plasmalogens . The labeling of the glycerol acetal of plasmenylethanolamine was even slower . In pulse-chase experiments with 32Pi, the kinetics of labeling indicated precursor-product relationships between phosphatidylethanolamine and plasmenylethanolamine and between the latter and its glycerol acetal . A precursor-product relationship was also seen between phosphatidylglycerol and cardiolipin, but the kinetics of labeling of the alkenyl-containing forms of these lipids were not consistent with direct precursor-product relationships with the acyl lipids . In the presence of hydroxylamine and 32Pi, both phosphatidylserine and plasmenylserine accumulated 32P in a ratio of ca . 15:1 . Upon release of the inhibition of phosphatidylserine decarboxylase, label appeared in the following sequence: phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine . Acyl phosphatidylglycerol was identified as a major phospholipid (17% of lipid phosphorus) in C . butyricum grown in low-phosphate (1.13 mM) medium with 50 mM Tris buffer . Of the acyl phosphatidylglycerol, 13% was acid labile . There appear to be two plasmalogen forms of acyl phosphatidylglycerol . One of these has a single alkenyl ether group, and the other has alkenyl ether groups on both glycerols.

J Clin Microbiol, 1984 Aug, 20(2), 274 - 5
Prevalence of Clostridium difficile and its cytotoxin in infants in Mexico; Torres JF et al.; The incidence of Clostridium difficile and its cytotoxic activity were determined in the feces of 122 children under 1 year of age . Samples were obtained from children receiving antibiotics and with (52 cases) or without (26 cases) diarrhea, from children with diarrhea who did not receive antibiotics (22 cases), and from healthy children (22 cases) . Isolation of C . difficile in feces from children in all groups was similar (mean 23.4%) except for the group with non-antibiotic-associated diarrhea (4.5%) . In both groups of children receiving antibiotics, with or without diarrhea, the cytotoxin was detected in 7.6% of the cases . In the group with non-antibiotic-associated diarrhea, none of the samples was positive for cytotoxicity . In healthy children, cytotoxin was positive in 4.5% of the cases.

J Appl Bacteriol, 1984 Aug, 57(1), 83 - 8
Anaerobic ureolytic bacteria from caecal content and soft faeces of rabbit; Crociani F et al.; Forty strains of ureolytic bacteria were isolated from the caecal content and soft faeces of seven rabbits by the anaerobic roll tube method and were characterized . The isolates were identified with Clostridium coccoides, Cl . innocuum, Peptostreptococcus productus, P . micros, Peptococcus magnus, Fusobacterium russii and Fusobacterium sp . Urease activity of representative strains of the various species was also determined . The study indicated that strongly-ureolytic anaerobic bacteria are present in the caecum of the rabbit.

Hoppe Seylers Z Physiol Chem, 1984 Aug, 365(8), 847 - 57
Nicotinic acid metabolism . Dimethylmaleate hydratase; Kollmann-Koch A et al.; The partial enrichment of a new enzyme, dimethylmaleate hydratase from Clostridium barkeri and some of its characteristics are described . The unstable and oxygen-sensitive hydratase depends on ferrous ions and is induced during growth of C . barkeri on nicotinic acid . The enzyme uses both dimethylmaleate and the hydration product, 2,3-dimethylmalate, as substrates to establish an equilibrium that is 70% in favour of the latter acid; dimethylfumarate is not attacked . A 2,3-dimethyl{3-3H}malate specimen was prepared from dimethylmaleate with the hydratase in tritiated water . Based on proton attack at the re-face of the double bond, experimental results indicate the (2R,3S)-configuration for this malate . The hydration reaction takes an anti-course . The tritium label was lost in the sequence (2R,3S)-2,3-dimethyl{3-3H}malate----(R)-{2-3H1}-propionate----(2R) - {2-3H1}propionyl-CoA----(2S)-methylmalonyl-CoA . This result confirms the stereochemical course of the 2,3-dimethylmalate lyase reaction, inversion of configuration, by an independent approach . The hydratase reaction completes the degradation scheme of nicotinic acid by C . barkeri . The pathway is briefly reviewed.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Aug, 257(3), 317 - 22
{A simple method for the rapid detection of bacterial hyaluronidase in K hyaluronate-containing gel}; Balke E et al.; For detection of hyaluronidase activities we investigated several groups of bacteria . The bacteria were inoculated on a 1,5% agarose gel in Petri plates of 4 cm diameter or gel discs of 7 mm diameter, containing 0,1% of K-hyaluronate as well as nutritient medium, and were incubated for 2-20 h at 37 degrees C in a moist chamber . Subsequently some ml of a 10% solution of cetylpyridiniumchloride were poured on the gel to precipitate the polymere hyaluronate . If the hyaluronate was depolymerized by hyaluronidase, a translucent area was visible around the colonies . We found out, that a gel layer of 1 mm was sufficient to detect the small amounts of hyaluronidase, which were produced by bacteria within an incubation time of 2 h . These results were confirmed by incubation for 20 h and in some cases 36 h . The hyaluronidase production by different anaerobic Clostridium strains was always proved after a 20 h growth period . The bacteria were inoculated with the whole loop of a self made platin sowing wire loop . By this method quantitative differences of hyaluronidase activities between different strains of bacteria could be detected.

Arch Microbiol, 1984 Aug, 138(4), 345 - 53
Comparative studies on physiology and taxonomy of obligately purinolytic clostridia; Schiefer-Ullrich H et al.; Eleven strains of obligately purinolytic clostridia have been studied with respect to their assignment to the three type strains of Clostridium acidiurici, C . cylindrosporum, and C . purinolyticum . DNA/DNA-hybridization proved to be the method of choice for differentiation whereas phenotypic characteristics such as spore morphology, substrate spectra, nutritional requirements, product formation, and sensitivity against various antibiotics did not allow unequivocal identification . All strains depended on selenite for growth.

Eur J Biochem, 1984 Aug 1, 142(3), 487 - 92
Comparison of Clostridium botulinum toxins type D and C1 in molecular property, antigenicity and binding ability to rat-brain synaptosomes; Murayama S et al.; Botulinum type D neurotoxin was purified 950-fold from the culture supernatant with an overall yield of 32% . The purified toxin had a specific toxicity of 5.8 X 10(7) mouse minimal lethal dose per mg of protein and a relative molecular mass of 140000 . The purified toxin had a di-chain structure consisting of heavy and light chains with relative molecular masses of 85000 and 55000, respectively, linked by one disulfide bond . These subunits had different amino acid compositions and antigenicities . A similarity in molecular constructions and amino acid compositions was observed between type D and type C1 toxins as well as between their subunits . Among the seven kinds of monoclonal antibodies against type D toxin, six reacted with the heavy chain of type D toxin, while one of the six also reacted with the heavy chain of type C1 toxin and neutralized the toxicities of the two toxins . The other one of monoclonal antibodies reacted with the light chains of both toxins . This evidence indicates that both toxins have common antigenic sites on their heavy and light chains and that the antigenic site on the heavy chain may contribute to the neutralization of both toxins by antibody . The binding of type D toxin to rat brain synaptosomes was examined by use of 125I-labelled type D toxin . The binding was competitively inhibited not only by unlabelled type D and C1 toxins, but also by the heavy chains of both toxins, however, it was not inhibited by the light chain of type D toxin . These results suggest that the toxin receptors on synaptosomal membrane are common for type D and C1 toxins, and that the heavy chain contributes to the binding of toxin to synaptosomes and the structure of the binding sites on the heavy chains of both toxins is quite similar.

Arch Neurol, 1984 Aug, 41(8), 882 - 4
CNS infection and bacteremia due to clostridium septicum; Gorse GJ et al.; Central nervous system infection with Clostridium septicum is rare . We report two fulminant cases of such infection with accompanying bacteremia . The presence of extensive brain necrosis was striking in our two cases . The association of C septicum bacteremia with hematologic disease, and with solid tumors, was present in our cases . We conclude that C septicum should be considered as a potential cause of life-threatening bacteremia and meningitis in the compromised host.

Eur J Clin Microbiol, 1984 Aug, 3(4), 294 - 300
Rapid enzymatic characterization of clinically encountered anaerobic bacteria with the API ZYM system; Marler L et al.; The purpose of this study was to determine if enzyme profiles of anaerobic bacteria, obtained with the API ZYM system, are sufficiently distinctive and reproducible to merit future development of the system and an expanded data base for the four major groups of clinically encountered anaerobes . Of the total 155 clinical isolates and reference strains that were tested, 88% had distinctive patterns . It was possible to differentiate between 89% of the anaerobic gram-negative bacilli, 64% of anaerobic cocci, 100% of anaerobic gram-positive non-sporeforming bacilli and 100% of the Clostridium spp . using only the API ZYM plus Gram reaction, morphology and relation to oxygen . Overall reproducibility for 1,330 enzyme-substrate reactions was 97% . It was concluded that the API ZYM could be a practical system for rapid identification of clinical anaerobe isolates provided that an expanded data base is developed and that certain additional recommended tests are used.

Appl Environ Microbiol, 1984 Aug, 48(2), 311 - 6
Characterization of a halo-acid-tolerant variant of Clostridium botulinum B-aphis; Montville TJ; Clostridium botulinum B-aphis spores plated on medium containing 4% salt at pH 6.0 yielded colonies at a frequency of ca . 1 in 10(6) . A subculture of one of these colonies, designated strain Ba410, was compared with the parent strain, B-aphis, for a variety of traits . After 7 days of incubation at 37 degrees C, strain Ba410 grew in medium containing 7% NaCl, whereas strain B-aphis could not grow in salt concentrations greater than 5% . The strains also differed in cellular and colonial morphology . After exponential growth in the basal medium was completed, lysis of both strains was pH dependent; in media containing salt, lysis of Ba410 cells was pH independent . Strain Ba410 was more proteolytic than strain B-aphis in conditions of low pH and high salt, so that its toxin could be detected by the mouse assay . In a medium containing alanine and cysteine, the germination rate of B-aphis was 0.77% min-1, whereas that of Ba410 was 0.14% min-1; 2% salt inhibited the germination of Ba410 but not B-aphis.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 Aug, 257(3), 308 - 16
Beta-glucuronidases of clostridium perfringens; Sakaguchi Y et al.; GAM broth was cultured for 5 days at 37 degrees C to obtain maximum yields of extracellular beta-glucuronidase from smooth colonies of Clostridium perfringens (Hobbs' type 4) isolated from the feces of a patient . A crude enzyme preparation was obtained by 20-80% ammonium sulfate precipitation of the broth . The beta-glucuronidase was purified using DEAE cellulose column chromatography, gel filtration on Sephadex G-200, and affinity chromatography on Sepharose 4B-bound glucuronolactone . We obtained two kinds of beta-glucuronidase . Properties of the purified beta-glucuronidase I were an optimum pH of 7.2, a pH stability range below 7.0, and a molecular weight of 115,000 . The purified beta-glucuronidase II had an optimum pH of 6.0, pH stability at around 6.0, and a molecular weight of 195,000 . Cu++ and Hg++ were strong inhibitors, which inhibition was restored by cysteine . EDTA did not influence enzyme activity . The Michaelis constants of beta-glucuronidase I and beta-glucuronidase II for p-nitrophenyl glucuronide were 1.25 X 10(-3) M and 4.17 X 10(-4) M, for naphthol AS-BI glucuronide 1.35 X 10(-4) M and 1.14 X 10(-4) M, and for phenolphthalein glucuronide 7.46 X 10(-5) M and 2.50 X 10(-4) M.

J Clin Pathol, 1984 Aug, 37(8), 942 - 4
Clostridium perfringens type C causing necrotising enteritis; Severin WP et al.; A rapidly fatal case of enteritis necroticans in a 24 year old man with diabetes was caused by Clostridium perfringens type C . The role of beta toxin in the disease is discussed . This type has not been previously described as a causative agent in necrotising bowel disease of man outside endemic areas.

J Bacteriol, 1984 Aug, 159(2), 465 - 71
Shuttle plasmids for Escherichia coli and Clostridium perfringens; Squires CH et al.; Small plasmids which replicate in both Escherichia coli and Clostridium perfringens were made by recombining E . coli plasmid pBR322 with three different small (less than 4 kilobases) plasmids native to C . perfringens . Subsequently, two homologous, though distinct, tetracycline resistance determinants (tet) from other C . perfringens plasmids were cloned into them . Both tet systems made E . coli resistant to at least 5 micrograms of tetracycline per ml when resident on the shuttle plasmids . The shuttle vectors have been used to transform L-phase variants and autoplasts of C . perfringens . In the latter case, the intact transforming plasmid could be isolated from walled cells after cell wall regeneration . Reciprocal transformation experiments in which plasmid DNAs derived from E . coli or C . perfringens were used suggest that restriction barriers exist between these two organisms . The plasmids contain restriction enzyme recognition sites in locations which are useful for cloning experiments.

J Bacteriol, 1984 Aug, 159(2), 460 - 4
Transformation of Clostridium perfringens; Heefner DL et al.; Clostridium perfringens 11268 CDR (Rifr Tcs), the strain transformed in our experiments, was generated by curing a spontaneous, rifampicin-resistant mutant of C . perfringens 11268 (Rifr Tcr) . High-temperature growth yielded tetracycline-sensitive, rifampicin-resistant cells which no longer contained pCW3, a 42.8-kilobase plasmid . The tetracycline-sensitive, rod-shaped cell was then converted to an L-phase variant by growth in the presence of penicillin G (10 micrograms/ml) and 0.4 M sucrose . After several passages, the antibiotic was removed from the medium, and cells continued to grow as L-phase variants . Another large plasmid, pJU124 (38.8 kilobases), which confers tetracycline resistance, was used for transformation . Transformation of L-phase variants of C . perfringens 11268 CDR (Rifr Tcs) was mediated by polyethylene glycol . Transformation frequency is a nonlinear function of DNA concentration . Restriction analysis showed that the plasmid isolated from the transformants was identical to that supplied . Stable L-phase variants do not revert to rod-shaped cells, but autoplasts can be both transformed and reverted.

Am J Med, 1984 Jul 31, 77(1B), 3 - 10
Selected aspects of nosocomial infections in the 1980s; Harris AA et al.; Unusual or rare pathogens and syndromes may become significant problems in nosocomial infection . Pathogens that usually produce community-onset disease, particularly respiratory viruses, Legionella, and atypical mycobacteria, also cause nosocomial infection . Conversely, nosocomial pathogens may also produce disease in the community, as has been seen with Clostridium difficile . Contamination of parenteral and antiseptic solutions continues to be a problem in hospitals . Hospital-acquired viral infections are receiving increasing recognition . Nosocomial gastrointestinal infections, although of low frequency, are of major import because of their epidemic potential . Airborne transmission of pathogens is becoming more apparent at the same time that recognition of the importance of hand transmission creates hope for infection control . Antibiotics influence the type of microorganisms that colonize patients, but the host determines superinfection.

J Biol Chem, 1984 Jul 25, 259(14), 8892 - 7
Acetate synthesis from carbon monoxide by Clostridium thermoaceticum . Purification of the corrinoid protein; Hu SI et al.; A corrinoid protein has been purified from Clostridium thermoaceticum which is required for the synthesis of acetyl-CoA from carbon monoxide and methyltetrahydrofolate . The purified protein is an alpha beta dimer with subunit molecular weights of 34,000 and 55,000, respectively, and contains 0.69 mol of corrinoid/mol of dimer . The corrinoid protein is methylated in the presence of methyltransferase and methyltetrahydrofolate; methylation is on the cobalt of the corrinoid moiety of the protein . When 14C-methylated protein is incubated with Fraction F3, ATP, CoASH, and CO, {14C}acetyl-CoA is formed . Methylation of cobalamin (B12) is catalyzed by the methyltransferase but methylcobalamin does not substitute for the methylated corrinoid protein as the source of methyl in the formation of acetyl-CoA.

Biochem Biophys Res Commun, 1984 Jul 18, 122(1), 9 - 16
Purification and properties of the H2-oxidizing (uptake) hydrogenase of the N2-fixing anaerobe Clostridium pasteurianum W5; Chen JS et al.; Clostridium pasteurianum has two distinct hydrogenases, the bidirectional hydrogenase and the H2-oxidizing (uptake) hydrogenase . The H2-oxidizing hydrogenase has been purified (up to 970-fold) to a specific activity of 17,600 mumol H2 oxidized/min X mg protein (5 mM methylene blue) or 3.5 mumol H2 produced/min X mg protein (1 mM methyl viologen) . The uptake hydrogenase has a Mr of 53,000 (one polypeptide chain) . Depending upon how protein was measured, the Fe and S = contents (gatom/mol) were 4.7 and 5.2 (by the dye-binding assay) or 7.2 and 8.0 (by the Lowry method) . Both reduced and oxidized forms of the enzyme gave electron paramagnetic resonance signals . The activation energy for H2-production and H2-oxidation by the uptake hydrogenase was 59.1 and 31.2 kJ/mol, respectively . In the exponential phase of growth, the ratio of uptake hydrogenase/bidirectional hydrogenase in NH3-grown cells was much lower than that in N2-fixing cells.

Biochim Biophys Acta, 1984 Jul 16, 800(1), 96 - 101
Induction of ornithine decarboxylase in guinea-pig lymphocytes and its relation to phospholipid metabolism; Otani S et al.; Treatment of lymphocytes with exogenous phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3.) derived from Clostridium perfringens at concentrations similar to those which induced ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activity produced diacylglycerol and phosphatidate . A divalent cation ionophore, A23187, and phytohemagglutinin induced not only diacylglycerol formation, but also ornithine decarboxylase activity . Dibutyryl cAMP inhibited both diacylglycerol formation and ornithine decarboxylase induction to a similar extent in phytohemagglutinin-stimulated lymphocytes, but stimulated them somewhat in ionophore A23187-activated lymphocytes . This suggests that the activation of intracellular phospholipase C and the formation of diacylglycerol is involved in ornithine decarboxylase induction in lymphocytes.

J Biol Chem, 1984 Jul 10, 259(13), 8252 - 9
Distinct reduction of nitrofurans and metronidazole to free radical metabolites by Tritrichomonas foetus hydrogenosomal and cytosolic enzymes; Moreno SN et al.; Anaerobic Tritrichomonas foetus hydrogenosomes supplemented with pyruvate and CoA effectively reduce nitrofurans and metronidazole to their respective anion free radicals . Addition of purified ferredoxins from Clostridium pasteurianum or Spinacia oleracea to these preparations causes a great stimulation of metronidazole reduction, but does not affect nitrofuran reduction . A similar stimulatory effect of ferredoxin on metronidazole reduction, but not on nitrofuran reduction, is observed in incubations containing purified NADPH:ferredoxin oxidoreductase from S . oleracea . NADH is less effective than pyruvate as a reducing cofactor for metronidazole and nitrofuran reduction by the hydrogenosomes, and these activities are not modified by the addition of ferredoxins . In contrast to the results observed with hydrogenosomes, the T . foetus soluble fraction supplemented with NADH or NADPH is able to reduce nitrofurans, but not metronidazole . Under aerobic conditions, the anion free radical metabolites generated from metronidazole and nitrofurans are oxidized, resulting in catalytic superoxide anion formation as detected by spin-trapping experiments . Oxygen consumption and H2O2 formation by T . foetus hydrogenosomes and NADPH:ferredoxin oxidoreductase are also stimulated by nitrofurans and high concentrations of metronidazole . Addition of ferredoxin enhances metronidazole-stimulated, but not nitrofuran-stimulated, oxygen consumption and H2O2 formation in both systems . These results support the role of air oxidation as a detoxification reaction of the metronidazole anion radical and the involvement of ferredoxin in its formation . On the other hand, redox cycling of nitrofurans with formation of high steady state concentrations of oxygen-derived radicals might be of toxicological significance.

Mol Pharmacol, 1984 Jul, 26(1), 105 - 11
DT-diaphorase and peroxidase influence the covalent binding of the metabolites of phenol, the major metabolite of benzene; Smart RC et al.; The role of various enzymes and biological molecules on the activation and deactivation of the metabolites of phenol was investigated in vitro . Phenol, the major metabolite of benzene, is metabolized to hydroquinone and catechol . Activation of these metabolites and deactivation of their oxidized forms was assessed by the amount of covalent binding to microsomal protein . {14C}Phenol and NADPH were incubated with hepatic microsomes isolated from phenobarbital-pretreated guinea pigs, and 2.33 nmoles of hydroquinone and 0.12 nmole of catechol were formed per minute per milligram of microsomal protein . Covalent binding of the metabolites to microsomal protein incubated with microsomes isolated from guinea pigs pretreated with phenobarbital was 252 pmoles bound/min/mg; with microsomes from untreated guinea pigs, covalent binding was 146 pmoles bound/min/mg . Covalent binding was inhibited greater than 90% with the addition of N-octylamine, ascorbate, or GSH . The addition of superoxide dismutase inhibited covalent binding with microsomes isolated from phenobarbital-pretreated guinea pigs 35% but did not inhibit it with microsomes isolated from untreated animals . Partially purified guinea pig hepatic DT-diaphorase {NAD(P)H (quinone acceptor) oxidoreductase, EC 1.6.99.2} inhibited covalent binding 70% . This effect was reversed in the presence of dicumarol, a specific inhibitor of DT-diaphorase . DT-diaphorase present in the 10(5) X g supernatant fraction was also active in inhibiting covalent binding but only after the removal of endogenous reduced glutathione . This effect could also be reversed by dicumarol . The addition of diaphorase (NADH:lipoamide oxidoreductase, EC 1.6.4.3) partially purified from Clostridium kluyveri inhibited covalent binding 86% . The addition of hydrogen peroxide and horseradish peroxidase (peroxidase, EC 1.11.17) or myeloperoxidase(s) increased covalent binding 30-fold and 6-fold, respectively . Ascorbate decreased this binding greater than 95% . These results indicate that hydroquinone, catechol, and phenol as well as their oxidized forms can be activated or deactivated by several of the above model systems . These systems may play a role in the myelotoxicity of benzene by modulating covalent binding.

J Infect Dis, 1984 Jul, 150(1), 57 - 62
In vitro and in vivo neutralizing activity of human colostrum and milk against purified toxins A and B of Clostridium difficile; Kim K et al.; The neutralizing activity (NA) of supernates of colostral samples collected postpartum from 55 women and tested against a 50% cytopathic dose of purified toxins A and B of Clostridium difficile was evaluated in Y1 adrenal cells . Thirty-one (56%) of the samples had NA against one or both toxins . Samples of breast milk were collected postpartum from five women-three had colostral NA and two did not . All milk specimens from the three women with colostral NA had NA titers of 1:1-1:4 throughout the study (609 days in one case) . Samples from the two women without colostral NA did not exhibit NA during a 60-day follow-up period . In suckling mice either toxin plus human milk with in vitro NA elicited significantly less fluid accumulation than did toxin plus diluent or toxin plus milk without in vitro NA (P less than .025 to P less than .05) . Twelve (63%) of 19 milk samples with in vitro NA against toxin A and 15 (65%) of 23 with in vitro NA against toxin B inhibited fluid accumulation caused by the corresponding toxin . In vitro NA against toxin A appeared to reside in the secretory IgA fraction of one milk sample assessed by immune precipitation . The results suggest that human milk may protect newborn infants against toxins A and B of C . difficile.

Infect Immun, 1984 Jul, 45(1), 185 - 91
Role of volatile fatty acids in colonization resistance to Clostridium difficile; Rolfe RD; The in vitro inhibition of Clostridium difficile by volatile fatty acids was correlated with the pH and concentrations of volatile fatty acids in the ceca of hamsters of different ages . The concentrations of cecal volatile fatty acids increased with the age of the animals . Maximum concentrations of individual volatile fatty acids were attained when the animals were ca . 19 days old, with acetic, propionic, and butyric acids occurring in the highest concentrations (72, 16, and 32 microequivalents/g of cecum, respectively) . The cecal pH was approximately the same in hamsters of all ages (pH 6.6 to 7.0) . Only butyric acid reached a concentration in the ceca of hamsters which was inhibitory to the in vitro multiplication of C . difficile . This inhibitory concentration was attained when the animals were ca . 19 days of age . When mixtures of volatile fatty acids were prepared at concentrations equal to those present in the ceca of hamsters, there was a direct correlation between the in vitro inhibitory activity of the volatile fatty acids and the susceptibility of hamsters 4 days of age or older to C . difficile intestinal colonization . The resistance of hamsters less than 4 days of age to C . difficile intestinal colonization appears to be due to factors other than volatile fatty acids.

Gastroenterology, 1984 Jul, 87(1), 213 - 5
Systemic absorption of oral cholestyramine; McDonald GB et al.; A patient with Clostridium difficile -toxin colitis was treated with oral cholestyramine, but died of other causes 15 days later . At autopsy, the colitis had resolved, but cholestyramine particles were found within the vessels of most body tissues, most prominently in his ulcerated distal esophagus . Clusters of bacteria were found adjacent to some of the cholestyramine particles, suggesting a common portal of entry.

J Bacteriol, 1984 Jul, 159(1), 375 - 80
Formate dehydrogenase of Clostridium pasteurianum; Liu CL et al.; Formate dehydrogenase was purified to electrophoretic homogeneity from N2-fixing cells of Clostridium pasteurianum W5 . The purified enzyme has a minimal Mr of 117,000 with two nonidentical subunits with molecular weights of 76,000 and 34,000, respectively . It contains 2 mol of molybdenum, 24 mol of nonheme iron, and 28 mol of acid-labile sulfide per mol of enzyme; no other metal ions were detected . Analysis of its iron-sulfur centers by ligand exchange techniques showed that 20 iron atoms of formate dehydrogenase can be extruded as Fe4S4 centers . Fluorescence analysis of its isolated molybdenum centers suggests it is a molybdopterin . The clostridial formate dehydrogenase has a pH optimum between 8.3 and 8.5 and a temperature optimum of 52 degrees C . The Km for formate is 1.72 mM with a Vmax of 551 mumol of methyl viologen reduced per min per mg of protein . Sodium azide competes competitively with formate (K1 = 3.57 microM), whereas the inactivation by cyanide follows pseudo-first-order kinetics with K = 5 X 10(2) M-1 s-1.

Acta Gastroenterol Belg, 1984 Jul-Aug, 47(4), 396 - 402
{Severe digestive complications of AIDS in a group of patients from Zaire}; Jonas C et al.; PIP: Severe digestive complications of acquired immune deficiency syndrome (AIDS) were observed in 9 patients among a group of 17 patients from Zaire treated for AIDS in Belgium between May 1979-April 1983 . Among the 9 cases, there were 10 ailments of the upper digestive tract, 7 of intestinal disorders, 3 of hepatic disorders, and 2 of pancreatic disorders . The average age of affected patients was 35 years . 4 men averaged 32 years and 5 women averaged 39 years . Their average stay in Belgium was 8 months . All 9 were anorexic and had lost at least 10 kg over the past year . 6 were pyretic and developed palpable adenopathies . 7 patients had episodic or continuous diarrhea in the early stages of illness and 8 had diarrhea in the later phase . 1 patient had bloody diarrhea . None were homosexual or drug addicted or had histories of transfusions . None was dysphagic . The patients exhibited lymphopenia affecting primarily the helper T lymphocytes . 7 patients had Candida albicans infections of upper digestive tract . 1 patient had an esophageal herpes infection . 4 patients had enterocolitis caused by opportunistic organisms: Cryptosporidium, Isospora Belli, cytomegalovirus, Clostridium Difficile, or Salmonella . 2 patients had septicemia caused by Salmonella and 1 had septicemia caused by Shigella . All 9 patients had at least 1 of the markers of hepatitis B . By April 1984, 8 patients had died and 1 who returned to Zaire had been lost to follow-up . The cause of death of the 3 patients for whom it was known was generally a nondigestive complication . Analysis of stool samples was found to be most useful means of diagnosing digestive complications of AIDS . Systemic infection with cytomegalovirus is very frequent in AIDS . The case in this series was diagnosed after discovery of inclusions in the intestinal mucus after repeated noncontributory analyses of the stools . In cases of enterocolitis, the endoscopic appearance of the mucus is not very specific and colposcopy is less useful than of stool samples . Upper endoscopy is very useful in diagnosis of Candida, which responds well to treatment . Hepatic biopsy and laparoscopy appear to be of limited usefulness, since liver and pancreatic involvement are usually self-limited with slight clinical manifestations . Endoscopic examinations pose the problem of possible contaminatin . The endoscope and all accessories should be systematically disinfected before and after use .

Infection, 1984 Jul-Aug, 12(4), 276 - 9
Fusidic acid for the treatment of antibiotic-associated colitis induced by Clostridium difficile; Cronberg S et al.; Twenty courses of fusidic acid were given to 16 patients with antibiotic-associated colitis caused by Clostridium difficile . Fusidic acid was given in a dose of 0.5-1.5 g daily for seven to 21 days . Diarrhoea disappeared rapidly . Clinical relapse occurred after five courses and once when the patient was still on treatment . Clinical cure with persistence or reappearance of toxin occurred in four further patients . Nineteen courses of metronidazole were given to 19 patients who experienced six failures or relapses . Seven courses of vancomycin were given to five patients, three of whom had had relapse . Five patients healed without treatment . The relapses occurred only in old and prostrated patients . They often recurred several times in the same patient . 0.5 g of fusidic acid daily appears to be as effective as vancomycin and metronidazole for the treatment of C . difficile-induced colitis.

Can J Microbiol, 1984 Jul, 30(7), 874 - 83
Interaction of Clostridium difficile toxin A with L cells in culture; Shahrabadi MS et al.; Toxin A of Clostridium difficile was purified by column chromatography and acetic acid precipitation . Cells exposed to toxin A showed polarization of nuclei towards one pole of the cells . Toxin A was conjugated to ferritin and applied to L cells to localize binding sites of this toxin to the cell surface . It was found that toxin A conjugate attached to the cell membrane in aggregated form . Antibody specific to toxin A was prepared and used for localization of intracellular toxins in intoxicated cells . Toxin A was found inside the cytoplasm 6 h after cell treatment, mainly in the form of aggregates inside the cytoplasmic vacuoles . At 24 h after exposure, toxin A could be detected within the cytoplasm . Tunicamycin treatment of cells reduced the cell-binding efficiency of toxin A to 50%, but neuraminidase did not effect toxin binding significantly.

Prikl Biokhim Mikrobiol, 1984 Jul-Aug, 20(4), 522 - 7
{Minimal synthetic nutrient medium for Clostridium stricklandii bacteria}; Golovchenko NP et al.; A composition of minimal culture medium for the anaerobic bacterium Clostridium sticklandii strain CSG was determined . A fully synthetic culture medium promoting the cell yield up to 1 g dry biomass per 1 1 was found . It is composed of 13 amino acids, sodium formiate, four vitamins, microelements and salts . The strain under study does not utilize glucose as a carbon and energy source.

Arch Biochem Biophys, 1984 Jul, 232(1), 414 - 21
Presence of a nonlysosomal endo-beta-N-acetylglucosaminidase in the cellular slime mold Dictyostelium discoideum; Freeze HH et al.; Vegetative cells of the cellular slime mold Dictyostelium discoideum have been found to contain an endo-beta-N-acetylglucosaminidase (EC 3.2.1.96) activity which hydrolyzes the di-N-acetylchitobiosyl linkage found in asparagine-linked oligosaccharides . In contrast to other previously characterized glycosyl hydrolases of Dictyostelium, this endoglycosidase is not secreted during vegetative growth or development nor is it developmentally regulated . Cellular fractionation studies showed that the endoglycosidase activity is not associated with lysosomes and remains soluble after centrifugation at 180,000g for 1 h . The enzyme has been partially purified (350-fold) from cell lysates, and its substrate specificity has been examined by its ability to hydrolyze several glycopeptides prepared from ovalbumin and from slime mold lysosomal enzymes . These preliminary studies revealed that the enzyme, called endoglycosidase S, has a substrate specificity similar to that of endo-beta-N-acetylglucosaminidase CII secreted by Clostridium perfringens.

J Environ Pathol Toxicol Oncol, 1984 Jul, 5(4-5), 79 - 87
Survival of pathogenic bacteria in crude oil; Myers GE; The survival of three strains of Staphylococcus aureus and one strain each of Pseudomonas aeruginosa, Bacillus subtilis and Clostridium sporogenes has been investigated in samples of seven different crude oils obtained from Alberta oil fields . One strain each of Staphylococcus aureus, Pseudomonas aeruginosa, Clostridium sporogenes and Bacillus subtilis survived for longer than three years in three of the seven crude oils tested . The majority (four out of six) of the species of test micro-organisms remained viable for over two years in six out of the seven crude oils tested . Paraffinic or asphaltic-naphthenic base crude oils were better suited for the survival of the bacteria employed in these tests than were naphthenic-paraffinic or aromatic-paraffinic base oils . Only Clostridium sporogenes survived longer than three days in one of the crude oils (No . 7) which contained considerably more aromatic material than any of the other oils tested . The public health implications of the experimental results are discussed.

Am J Dis Child, 1984 Jul, 138(7), 686 - 8
Necrotizing enterocolitis and hemolysis associated with Clostridium perfringens; Warren S et al.; Two newborns had necrotizing enterocolitis (NEC) and severe hemolytic anemia . Clostridium perfringens was identified in the peritoneal fluid of both infants, supporting the previous association of C perfringens with hemolysis reported in adult patients . Infants with NEC and hemolytic anemia should be aggressively treated with surgical debridement and high-dose parenteral penicillin G potassium . Similarly, Gram's stain of peritoneal fluid and resected bowel at laparotomy for NEC may be useful for early identification of the primary organism.

Zh Mikrobiol Epidemiol Immunobiol, 1984 Jul, (7), 106 - 10
{Morphological and histochemical study of the action of a native Clostridium perfringens type A enterotoxin on a model of the ligated ileal loop in the rabbit}; Ermakova MP et al.; Morphological studies carried out in the igated loop of the rabbit small intestine, used as a model, have revealed that the action of the enterotoxins of C . perfringens type A reference strain and C . perfringens type A strains isolated in the USSR on the intestinal mucosa is absolutely identical . This action is manifested by the destruction of epithelial cells on the tops of the villi, inflammatory infiltration and considerable exudation of fluid into the lumen of the intestinal loop . Early lesions of the brush border of enterocytes, accompanied by the loss of alkaline phosphatase activity and the disappearance of the glycocalix, have-been histochemically established . C . perfrinegens antienterotoxic serum has proved to be capable of completely neutralizing the histopathological activity of C . perfringens enterotoxin.

Appl Environ Microbiol, 1984 Jul, 48(1), 178 - 81
Role of DNase in recovery of plasmid DNA from Clostridium perfringens; Blaschek HP et al.; Recovery of plasmid DNA from Clostridium perfringens 10543A and 3626B cleared lysates was significantly improved by the addition of 0.2% (vol/vol) diethylpyrocarbonate (DEP) before protoplast disruption in the cleared lysate protocol . Three previously undetected, large-molecular-mass plasmids (45.2, 51.9, and 68.2 megadaltons) were isolated from modified DEP-treated cleared lysates of C . perfringens 3626B . Two plasmids (9.4 and 30 megadaltons) were recovered from C . perfringens 10543A modified DEP-treated cleared lysates which previously required dye-buoyant density gradient centrifugation for visualization on agarose gels . Unsuccessful attempts to isolate plasmid DNA from Brij 58 cleared lysates of extracellular DNase-negative mutants of C . perfringens suggested the deleterious DNase activity was not extracellular . Cellular localization studies indicated that the cell wall-compartmentalized cell fraction contained 72.2% of the total DNase activity, whereas the extracellular and intracellular fractions demonstrated much less (26.8 and 1.0%, respectively) . Cleared lysates prepared with DEP demonstrated much less DNase activity than cleared lysates prepared without DEP . The variable and irreproducible recovery of plasmid DNA from C . perfringens cleared lysates was attributed to cell wall-compartmentalized DNase.

J Comp Pathol, 1984 Jul, 94(3), 445 - 52
Ultrastructural changes in the brain of mice given Clostridium perfringens type D epsilon toxin; Finnie JW; Mice were given lethal and sublethal doses of Clostridium perfringens Type D epsilon toxin and the early morphological changes in perfusion-fixed intoxicated brains were examined from 30 min to 6 h post-inoculation . The initial ultrastructural finding was swelling of astrocytes, especially the perivascular extensions of these cells . Astrocytes in the cerebellum appeared to be particularly sensitive to this toxin . These changes were quickly followed by evidence of severe endothelial damage, with the endothelial cytoplasm becoming attenuated, vacuolated and very electron-dense . A pathogenetic sequence of events leading to malacia, derived from ultrastructural observations, is proposed.

J Comp Pathol, 1984 Jul, 94(3), 363 - 70
Histopathological changes in the brain of mice given Clostridium perfringens type D epsilon toxin; Finnie JW; The distribution, severity and frequency of brain lesions produced in mice by the administration of Clostridium perfringens Type D epsilon toxin were examined by light microscopy . The granular layer of the cerebellum was the area most frequently affected in mice given single doses of toxin . Sequential changes in brain morphology were examined from 1 h to 7 days after injection of toxin . Lesions progressed from an initial vasogenic oedema to malacic foci which commonly were focal and bilaterally symmetrical, with a predilection for white matter . The topographical distribution of these malacic areas is discussed.

Biochem Biophys Res Commun, 1984 Jun 29, 121(3), 1042 - 7
Activation of nit-1 nitrate reductase by W-formate dehydrogenase; Deaton JC et al.; Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme . FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively . Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added . These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase . The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.

Biochemistry, 1984 Jun 19, 23(13), 3092 - 9
Relationship between the individual collagenases of Clostridium histolyticum: evidence for evolution by gene duplication; Bond MD et al.; The relationship between the six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) isolated and characterized in the preceding papers {Bond, M.D., & Van Wart, H.E . (1984) Biochemistry (preceding two papers in this issue)} has been investigated . Chemical modification reactions establish that all six enzymes contain essential carboxyl, tyrosine, and lysine residues . Circular dichroism spectra of the peptide bond region show that the secondary structures of the collagenases are very similar . Ouchterlony double-immunodiffusion experiments carried out with antiserum prepared against beta-collagenase indicate that all six collagenases are cross-reactive . Reverse-phase high-pressure liquid chromatography elution profiles of tryptic digests of these collagenases and sodium dodecyl sulfate electrophoresis gels of the peptides formed on reaction with cyanogen bromide have been obtained . The results indicate that the class I collagenases have extensive sequence homology with each other and that the class II collagenases have extensive sequence homology with each other but that the enzymes in the two classes have substantially different sequences . In addition, the data show that beta-collagenase probably consists of domains that have homologous amino acid sequences, which may have arisen by full or partial intragenic gene duplication . This may account for the unusually high molecular weight of this and the other collagenases . Finally, on the basis of the similarities between the collagenases in the two classes, it is suggested that one class evolved from the other by gene duplication followed by independent evolution by point mutations to yield enzymes with different substrate specificities.

Biochemistry, 1984 Jun 19, 23(13), 3085 - 91
Characterization of the individual collagenases from Clostridium histolyticum; Bond MD et al.; The six collagenases (alpha, beta, gamma, delta, epsilon, and zeta) from Clostridium histolyticum isolated in the preceding paper {Bond, M . D., & Van Wart, H . E . (1984) Biochemistry (first paper of three in this issue)} have been characterized in detail . The molecular weights determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis range from 68 000 to 125 000 . Isoelectric focusing experiments demonstrate that the isoelectric points of the collagenases are in the 5.35-6.20 range . These experiments also reveal that the subspecies of alpha- and gamma-collagenases (alpha1 vs . alpha 2 and gamma 1 vs . gamma 2) have different isoelectric points but the same molecular weights . Microheterogeneity is also observed for the beta- and epsilon-collagenases . The amino acid compositions of all six collagenases have been determined, and analysis for neutral sugars and hexosamines shows that none of the enzymes have a significant carbohydrate content . Zinc and calcium are the only metals that copurify with the collagenases . The purified enzymes contain approximately 1 mol of zinc/mol of protein and a calcium content that varies from about 2 mol/mol for alpha-collagenase to about 7 mol/mol for beta-collagenase . All of the collagenases are 5-10 times more active against gelatin than collagen . The alpha-, beta-, and gamma-collagenases are significantly less active toward the synthetic peptide substrates examined than the delta-, epsilon, and zeta-collagenases . This property, taken together with data on the stabilities and amino acid compositions of these enzymes, strongly supports their assignment to two distinct classes . This establishes clearly that C . histolyticum does, indeed, produce more than one different type of collagenase.

Biochemistry, 1984 Jun 19, 23(13), 3077 - 85
Purification and separation of individual collagenases of Clostridium histolyticum using red dye ligand chromatography; Bond MD et al.; Six collagenases present in the culture filtrate of Clostridium histolyticum have been purified to homogeneity . Chromatography over hydroxylapatite, Sephacryl S-200, and L-arginine-Affi-Gel 202 removes the brown pigment and the great majority of the contaminating proteinases active against casein, benzoyl-L-arginine ethyl ester, and elastin . Reactive Red 120 dye ligand chromatography subdivides the collagenases, which have very similar physicochemical properties, among four fractions . The final purification is achieved by chromatography over DEAE-cellulose and SP-Sephadex . All six collagenases, designated alpha, beta, gamma, delta, epsilon and zeta by the order of their purification, are highly active against collagen and devoid of other proteolytic activities . Each exhibits a single band on sodium dodecyl sulfate-polyacrylamide gels . Two distinct subspecies of the alpha and gamma enzymes have been isolated, which have the same molecular weight and activity but different isoelectric points . There is some less pronounced microheterogeneity for the other collagenases . On the basis of their activities toward native collagen and the synthetic peptide 2-furanacryloyl-L-leucylglycyl-L-prolyl-L-alanine (FALGPA), the six collagenases are divided into two classes . Class I collagenases (alpha, beta, and gamma) have high collagenase activity and moderate FALGPA activity while the class II collagenases (sigma, epsilon, and sigma) have moderate collagenase and high FALGPA activities . The relationship between these six collagenases and other reported to have been isolated in the literature has also been examined.

J Biol Chem, 1984 Jun 10, 259(11), 7045 - 55
The physical and catalytic properties of hydrogenase II of Clostridium pasteurianum . A comparison with hydrogenase I; Adams MW et al.; Hydrogenase II of Clostridium pasteurianum is a monomeric protein of Mr = 53,000 containing 8 iron and 8 acid-labile sulfide atoms/mol . It is distinct from hydrogenase I from the same organism (Mr = 60,000 12 Fe and 12 S2-/mol) . Metal analyses showed that neither hydrogenase contains nickel or any other metals in significant amounts . The iron atoms of hydrogenase II resisted chelation by 2,2'-bipyridyl but all were susceptible when the enzyme was treated with ferricyanide . Core extrusion indicated the presence of two {4Fe-4S} clusters in hydrogenase II and EPR spectroscopy showed two distinct paramagnetic species which could be interpreted as one {4Fe-4S}2+(2+,1+) and one {4Fe-4S}2+(2+,3+) per molecule . The absorption coefficient of H2-reduced hydrogenase II at 420 nm was 23,000 M-1 cm-1 with a A420 / A275 ratio of 0.27 . There were large differences between hydrogenase I and hydrogenase II in the absorption spectra of the air-oxidized, H2-reduced, and dithionite-reduced forms of the enzymes . Hydrogenase II catalyzed H2 evolution with methyl viologen or ferredoxin as the electron carrier, and H2 oxidation with methylene blue or methyl viologen as the electron acceptor . Apparent Km values were determined for all these reactions with both hydrogenases . Hydrogenase II is a relatively inactive enzyme, except in the reduction of methylene blue by H2 . The pH dependencies of H2 oxidation were similar for both hydrogenases but were very different in H2 evolution . The activation energy values were much higher for H2 catalysis by hydrogenase II than for hydrogenase I . The two hydrogenases have the same sensitivity to inactivation by O2 but differ in their sensitivity to metal-chelating reagents and to CO . Hydrogenase I is more readily inhibited by CO but hydrogenase II binds CO irreversibly . From the above data, a mechanism is proposed to account for the observed differences in the catalytic activities of hydrogenase I and hydrogenase II.

Biochemistry, 1984 Jun 5, 23(12), 2745 - 52
Mechanistic studies with deuterated dihydroorotates on the dihydroorotate oxidase from Crithidia fasciculata; Pascal RA Jr et al.; Deuterium-labeled dihydroorotates bearing one, two, or three deuteriums at the pair of C4 and C5 positions have been synthesized in high isotopic and chiral purity and characterized by NMR and mass spectroscopy . These substrates have been used with the FMN-containing biosynthetic dihydroorotate oxidase from Crithidia fasciculata {Pascal, R., Trang, N., Cerami, A., & Walsh, C . (1983) Biochemistry 22, 171} to probe stereochemistry and mechanism . At pH 6.0 the (4RS)-{5,5-2H2}dihydroorotate shows a Vmax isotope effect (DV) of 2.83; since the (4S,5R)-{5-2H}dihydroorotate shows a DV of no more than 1.1, a secondary effect, the overall stereochemistry of desaturation is anti as previously reported for the degradative orotate reductase from Clostridium oroticum . The (4RS)-{4-2H}dihydroorotate shows a DV of 2.97, indicating removal of the C4-H is also partially rate limiting at pH 6.0 . When trideuterio (4RS)-{4,5,5-2H3}dihydroorotate was tested, a DV of 8.0, a value close to the product of the separate isotope effects at the 4- and 5S-positions, was observed . At this pH then, both C-H cleavage steps are partly rate limiting in catalysis . Under anaerobic conditions without an electron acceptor the enzyme catalyzes the preferential exchange of the 5S hydrogen with solvent protons . The aggregate isotope effects on Vmax (DV) and on Vmax/Km {D(V/K)} are analyzed and suggest a stepwise rather than a concerted mechanism for this biosynthetic desaturation in pyrimidine biosynthesis.

FEBS Lett, 1984 Jun 4, 171(1), 73 - 8
The stereochemistry of the formation of the methyl group in the glutamate mutase-catalysed reaction in Clostridium tetanomorphum; Hartrampf G et al.; The adenosylcobalamin-dependent enzyme glutamate mutase from Clostridium tetanomorphum catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3- methylaspartate . In this conversion 6 carbon centers are involved . The stereochemistry of 4 has been elucidated whereas the formation of the methyl group from the methylene group remains to be established . To solve this problem, (2S,3R)- and (2S,3S)-{3,3-2H1,3H}glutamates were prepared via the 2-oxo{3,3-2H2 or 3H} glutarates by incubation with isocitrate dehydrogenase in deuterium oxide or tritiated water . The labelled glutamates were fermented with growing cells of C . tetanomorphum to butyrate and acetate . Butyrate was further degraded to acetate in which methyl group over 90% of the tritium of the starting glutamate was retained . The chirality of the acetates was determined with malate synthase and fumarase . In both samples complete racemisation was found . This result confirms the rule that racemisation occurs in all adenosylcobalamin-dependent rearrangements in which methyl groups are formed . A methylene radical as intermediate could explain these observations . In a control experiment inversion of configuration in the formation of the methine group of (2S,3S)-3-methylaspartate from the methylene group of (2S)-glutamate was confirmed . Glutamates stereospecifically labelled at C-4 were synthesized from chiral acetates via citrate.

FEBS Lett, 1984 Jun 4, 171(1), 79 - 84
On the dehydration of (R)-lactate in the fermentation of alanine to propionate by Clostridium propionicum; Schweiger G et al.; All the enzymes of the pathway of (S)-alanine fermentation to acetate and propionate were detected in cell-free extracts of Clostridium propionicum . Among these (S)-glutamate dehydrogenase (NAD), (R)-lactate dehydrogenase (NAD) and propionate CoA-transferase were purified to apparent homogeneity . Their structures were presumably alpha 6, alpha 2 and alpha 4, respectively . The latter enzyme was specific for short-chain monocarboxylic acids with a pronounced preference for (R)-lactate over the (S)-enantiomer . The key step of the pathway, the dehydration of (R)-lactate required acetyl phosphate and CoASH under anaerobic conditions . It was inhibited by hydroxylamine, arsenate, azide (1 mM each) or by 0.1 mM 2,4-dinitrophenol . Thus it closely resembled the dehydration of (R)-2-hydroxyglutarate in Acidaminococcus fermentans , although an activation was not necessary.

Eur J Biochem, 1984 Jun 1, 141(2), 323 - 30
The use of two-dimensional nuclear-magnetic-resonance spectroscopy and two-dimensional difference spectra in the elucidation of the active center of Megasphaera elsdenii flavodoxin; Moonen CT et al.; 1H-1H 'through bond' correlated (COSY) and 1H-1H 'through space' (NOESY) two-dimensional NMR techniques were applied to study the structure of Megasphaera elsdenii flavodoxin in the oxidized and reduced state . It is shown that two-dimensional NOESY difference spectra between spectra of flavodoxin in the reduced and semiquinone state are sensitive to the active center of the fully reduced state . The sphere of the active center observed in the difference spectra can be varied easily by changing the relative amount of flavodoxin semiquinone in the second sample . The difference NOESY spectra simplified the analysis of the complex spectra . Resonances could be assigned to Ala-56, Tyr-89 and Trp-91, which are located in the direct vicinity of the protein-bound flavin . The relative positions and side-chain dihedral angles of these residues are compared for the two redox states . Ala-56 and Tyr-89 show identical relative positions and dihedral angles in the two redox states, although the rotational motion of Tyr-89 is enhanced in the oxidized state . In both redox states Trp-91 is immobilized and extremely close to the prosthetic group . However, a small displacement of Trp-91 towards the (N(5) atom of the flavin occurs upon reduction . The results obtained for Trp-91 are in excellent agreement with crystallographic results of the related flavodoxin from Clostridium MP . However, the latter studies showed a somewhat different position of the tyrosine residue compared with our results.

J Med Microbiol, 1984 Jun, 17(3), 317 - 24
Immunochemical fingerprinting of Clostridium difficile strains isolated from an outbreak of antibiotic-associated colitis and diarrhoea; Poxton IR et al.; Twenty eight strains of Clostridium difficile , isolated from an outbreak of antibiotic-associated colitis and diarrhoea in an orthopaedic ward and from sporadic cases throughout Sweden, were sent to Edinburgh for immunochemical fingerprinting without information about their origin . EDTA extracts of the organisms were examined by crossed immunoelectrophoresis (CIE), polyacrylamide gel electrophoresis (PAGE) and electroblot transfer . Two patterns were revealed by CIE: group A (18 strains) and group B (10 strains) . PAGE and electroblot transfer revealed one major group of 10 strains (group 1), six small groups of two or three strains and six strains which were unlike any other strain . The CIE group B and PAGE- electroblot group 1 were identical . Nine of the 10 strains in this group were from patients in the outbreak . These findings indicate that a single strain spread in the orthopaedic ward as a nosocomial infection and that this strain differed from most other strains investigated . The PAGE- electroblot technique should, therefore, greatly aid investigations into the epidemiology of C . difficile infections.

Jpn J Med Sci Biol, 1984 Jun, 37(3), 131 - 5
Titration of botulinum toxins for lethal toxicity by intravenous injection into mice; Kondo H et al.; Clostridium botulinum type A - F toxins can be titrated by the time-to-death method by iv injection into mice . The time to death is not dependent upon the molecular size, but upon the immunological type of the toxin . It is necessary to assure complete activation of the activable toxin produced by nonproteolytic as well as a certain proteolytic strains before subjecting to titration by the iv injection method.

Can J Biochem Cell Biol, 1984 Jun, 62(6), 398 - 408
The determination of calcium-binding sites of human erythrocyte membranes; Moore RB et al.; Calcium binding to leaky erythrocyte plasma membranes was measured by three different procedures: Millipore filtration, equilibrium dialysis, and partition centrifugation . The curve derived from the binding equation, which best fit the means of the raw data, was used to estimate the association constants and capacities of the binding sites . A computer program (Gaushaus) which uses a nonlinear, least-squares regression protocol was also used to confirm these estimates . On the basis of these analyses we propose the presence of three classes of calcium-binding sites with the following apparent association constants and capacities: site 1, Ka = 3 X 10(4) M-1 and n = 30 nmol/mg protein; site 2, Ka = 3 X 10(3) M-1 and n = 200 nmol/mg protein; site 3, Ka = approximately 10(2) M-1 and n = approximately 200 nmol/mg protein . Calcium binding to erythrocyte membranes sealed in a high-salt solution showed the presence of site 3, but not site 2 . The influence of phospholipids on the binding of calcium was evaluated by pretreating ghosts with phospholipase C (Clostridium welchii, EC 3.1.4.3) . Treatment with this enzyme removed 80% of the total membrane phosphorus, predominantly from sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine . By the method of partition centrifugation two classes of binding sites were identified by computer analysis . Their association constants and capacities are, respectively, 1.1 X 10(5) M-1 and 20 nmol/mg protein for site 1 and 4.4 X 10(3) M-1 and 200 nmol/mg protein for site 2 . We speculate that calcium-binding site 1 is composed of acidic phospholipids, calcium-binding site 2 is composed of spectrin and actin, and calcium-binding site 3 is composed of sialic acid.

Pathol Biol (Paris), 1984 Jun, 32(5 Pt 2), 540 - 3
{In vitro activity of the amoxicillin-clavulanic acid combination on anaerobic bacteria . Comparison with amoxicillin, lamoxactam and metronidazole}; Deforges L et al.; Minimal inhibitory concentrations (MIC) of the combination amoxicillin + clavulanic acid (AAC) (2/1 w/w) on 84 strains of obligate anaerobes were measured by the agar diffusion method and compared to those of amoxicillin (AMO), lamoxactam (LAM) and metronidazole (MET) . For the 47 Bacteroides fragilis strains tested, the mode MIC was 0.5 microgram/ml with AAC, 1-2 with MET, 0.5-1 with LAM and 8-16 with AMO . A similar range of activity was found on the 16 other strains of Bacteroides sp. . AAC and AMO have a comparable activity on Clostridium perfringens (mode MIC 0.016-0.032 microgram/ml), superior to that of LAM (mode MIC 0.125) and MET (mode MIC 0.5) . AAC and AMO have an almost identical activity on Peptococci and Peptostreptococci, the MIC for both drugs being less than 0.125 microgram/ml . A similar activity was found for LAM and MET.

J Infect Dis, 1984 Jun, 149(6), 924 - 8
Synergistic effect of bacteroides, Clostridium, Fusobacterium, anaerobic cocci, and aerobic bacteria on mortality and induction of subcutaneous abscesses in mice; Brook I et al.; The potential for synergy between aerobic, facultative, and anaerobic bacteria was studied by subcutaneous inoculation of mixtures of these organisms into mice and observation of subsequent mortality and abscess formation . The anaerobic bacteria tested included 12 strains of gram-positive cocci and two strains each of Bacteroides species, Clostridium species, and Fusobacterium species . The facultative and aerobic bacteria included one strain each of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Proteus mirabilis . Mortality increased significantly when each aerobic organism was inoculated along with either of the Bacteroides species . A similar increase occurred when the anaerobic gram-positive cocci were inoculated along with P . aeruginosa (four of six combinations) or S . aureus (four of six) . The rate of abscess induction increased significantly when 10 of the 12 strains of anaerobic gram-positive cocci were injected along with B . fragilis and when nine of these strains were inoculated along with Bacteroides asaccharolyticus . The results demonstrate synergistic potential between Bacteroides species and all aerobic bacteria tested, between Bacteroides species and most anaerobic gram-positive cocci, and between most anaerobic gram-positive cocci and P . aeruginosa or S . aureus.

Jpn J Med Sci Biol, 1984 Jun, 37(3), 137 - 40
Use of monoclonal antibodies in enzyme linked immunosorbent assay (ELISA) for detection of botulinum type B toxins; Notermans S et al.; Use of polyclonal antibodies failed to correlate mouse assay with enzyme linked immunosorbent assay (ELISA) in titration of culture fluid of different strains of Clostridium botulinum type B . If ELISA is performed with such a monoclonal antibody that is capable of neutralizing the toxin, however, the lethal toxicity can be determined quantitatively.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1984 Jun, 179(3), 225 - 34
Evaluation of the ELISA as tool in diagnosing Clostridium perfringens enterotoxins; Notermans S et al.; Detecting Clostridium perfringens enterotoxin (CPE) using the enzyme linked linked immunosorbent assay (ELISA) was evaluated as a tool for diagnosing enterotoxicosis caused by C . perfringens . This method was assessed using a number of different food poisoning outbreaks with possible C . perfringens associations . CPE can easily be detected in faeces of patients involved in food-borne disease caused by C . perfringens . In stools of patients with diarrhoea 0.01-10 micrograms/g of CPE is detectable, however not all samples examined are found to contain CPE . CPE in faeces maintains its immunological stability over a long period (greater than 20 days at room temperature) enabling samples to be stored for some time before assay . The ELISA technique is also useful for the detection of CPE in culture fluids of C . perfringens strains isolated from faeces and from any remaining food considered to have caused the food poisoning outbreak . Detection of CPE in stools combined with testing for CPE production in C . perfringens strains isolated both from faeces and from the suspect food seems to give good evidence linking a food-borne disease outbreak with C . perfringens.

J Clin Microbiol, 1984 Jun, 19(6), 915 - 6
Evaluation of the 24-h API 20A anaerobe system for identification of Clostridium difficile; Gresser ME et al.; Accurate identification of Clostridium difficile is important when antibiotic-associated diarrhea or pseudomembranous colitis is suspected . Presumptive identification of C . difficile was made on the basis of microscopic features and colony characteristics on cycloserine, cefoxitin, fructose, and egg yolk agar medium . We studied the reliability of the 24-h API 20A anaerobe system for definitive identification of C . difficile . This system showed low dependability after the recommended 24 h of incubation by confirming the identity of only 54% of the isolates presumptively identified as C . difficile . There was a marked improvement in the system's capability after 48 h of incubation, when the identity of 95% of the isolates was confirmed.

J Bacteriol, 1984 Jun, 158(3), 1061 - 9
Thioredoxin system of the photosynthetic anaerobe Chromatium vinosum; Johnson TC et al.; Chromatium vinosum, an anaerobic photosynthetic purple sulfur bacterium, resembles aerobic bacterial cells in that it has an NADP-thioredoxin system composed of a single thioredoxin which is reduced by NADPH via NADP-thioredoxin reductase . Both protein components were purified to homogeneity, and some of their properties were determined . Chromatium vinosum thioredoxin was slightly larger than other bacterial thioredoxins (13 versus 12 kilodaltons) but was similar in its specificity (ability to activate chloroplast NADP-malate dehydrogenase more effectively than chloroplast fructose-1,6-bisphosphatase) and immunological properties . As in other bacteria, Chromatium vinosum NADP-thioredoxin reductase was an arsenite-sensitive flavoprotein composed of two 33.5-kilodalton subunits, that required thioredoxin for the NADPH-linked reduction of 5,5'-dithiobis(2-nitrobenzoic acid) . Chromatium vinosum NADP-thioredoxin reductase very effectively reduced several different bacterial-type thioredoxins (Escherichia coli, Chlorobium thiosulfatophilum (this name has not been approved by the International Committee of Systematic Bacteriology), Rhizobium meliloti) but not others (Clostridium pasteurianum, spinach chloroplast thioredoxin m) . The results show that Chromatium vinosum contains an NADP-thioredoxin system typical of evolutionarily more advanced microorganisms.

Arch Biochem Biophys, 1984 Jun, 231(2), 400 - 10
Phospholipase C from Clostridium perfringens: preparation and characterization of homogeneous enzyme; Krug EL et al.; A new procedure for the purification of phospholipase C from Clostridium perfringens has been devised that results in essentially pure enzyme . The procedure consists of ammonium sulfate fractionation, ion-exchange chromatography on QAE-Sephadex, and affinity chromatography on phosphatidylcholine linked to Sepharose . The molecular weight of the enzyme, determined by sodium dodecyl sulfate-gel electrophoresis, amino acid analysis, and gel filtration, is 43,000; and the isoelectric point is pH 5.4 . The enzyme was optimally active with phosphatidylcholine dispersed in sodium deoxycholate, although appreciable activity was observed with either phosphatidylcholine or sphingomyelin dispersed with ethanol . The requirement for metal ions in the assay could be met by a number of different ions . The pure enzyme was found to contain 2 mol zinc per mol enzyme, thus implicating it as a zinc metalloenzyme.

Appl Environ Microbiol, 1984 Jun, 47(6), 1319 - 22
Comparison of antigenicity of toxins produced by Clostridium botulinum type C and D strains; Ochanda JO et al.; C1 neurotoxin of Clostridium botulinum strains C-Stockholm (C-ST), C beta-Yoichi, C-468, CD6F, and C-CB19 and type D toxin of strains D-1873 and D-CB16 were purified by gel filtration, ion exchange, and affinity chromatographies . The purified toxins had di-chain structure made of heavy and light chains . The toxins of C beta-Yoichi, C-468, CD6F, and C-CB19 reacted with anti-C-ST heavy chain and anti-C-ST light chain in immunodiffusion tests and enzyme-linked immunosorbent assay, whereas D-CB16 toxin reacted with anti-D-1873 heavy chain and anti-D-1873 light chain . However, C-6813 toxin reacted with anti-D-1873 heavy chain and anti-C-ST light chain but not with anti-C-ST heavy chain or anti-D-1873 light chain immunoglobulin G . These results indicate common antigens in the heavy chains of C-6813 and D-1873 toxins and in the light chains of C-6813 and C-ST toxins . Further, they provide evidence for heterogeneity within type C1 toxin subunits.

J Neurol Sci, 1984 Jun, 64(3), 259 - 64
A functional membrane repair system in Duchenne muscular dystrophy fibroblasts; Wright PS et al.; Experiments have been performed to determine if fibroblasts from patients with Duchenne muscular dystrophy (DMD) are defective in a process of membrane repair . Normal and DMD fibroblasts were treated with phospholipase C from Clostridium perfringens to degrade plasma membrane phosphatidylcholine, and then phosphatidylcholine synthesis was measured as the incorporation of {3H} choline into lipid . Phosphatidylcholine synthesis was stimulated by phospholipase C treatment to a similar extent in normal and DMD fibroblasts . The activity of CTP: phosphocholine cytidylyltransferase, the enzyme regulating phosphatidylcholine synthesis in phospholipase C-treated mammalian cells, was also stimulated to the same extent in both cell types . The subcellular location of the cytidylyltransferase was changed by phospholipase C treatment from mostly cytosolic to mostly particulate in both normal and DMD fibroblasts . It appears, therefore, that at least one type of membrane repair system functions normally in DMD fibroblasts.

J Biochem (Tokyo), 1984 Jun, 95(6), 1593 - 602
Structural studies of Fc receptors . V . Effect of phospholipase C treatment on the binding activities of the Fc receptor of macrophage or its isolated plasma membrane; Aida Y et al.; The effect of phospholipase C treatment on the binding activity of the Fc receptor of guinea pig macrophage was studied to analyze the interaction of the Fc receptor with membrane phospholipids necessary for the activity . It was confirmed by subcellular fractionation that the receptor is localized on the plasma membrane . Treatment of the whole cell or isolated plasma membrane with phospholipase C of Clostridium perfringens diminished the binding of soluble IgG2-immune complex to Fc receptors on the cell or membrane . On the other hand, phospholipase C of Bacillus cereus did not affect the activity when it acted on the whole cell but it did diminish the activity when it acted on the isolated plasma membrane . Analysis of the phospholipids of untreated and treated macrophages or plasma membrane showed that phosphatidylcholine molecules, particularly those located in the membrane (not accessible to attack from the cell surface by phospholipase C of B . cereus), appear to be crucial for efficient interaction of macrophage Fc receptors with immune complex . Ligand-binding experiments with macrophages showed that the diminished binding activity was due to a decrease of the avidity for immune complex, but did not seem to be due to a decrease in the number or affinity of Fc receptors for monomeric IgG2 . Taken together with the previous results which demonstrated that Fc receptors which had apparently lost the activity due to delipidation could be reconstituted with phosphatidylcholine but not with most other phospholipids, the results seem to indicate that the diminution of the binding activity to the immune complex of macrophage or its plasma membrane caused by phospholipase C treatment is due to the impairment of multivalent interaction between Fc receptor molecules on the membrane and IgG2 molecules in the immune complex, probably as a result of the loss of interaction of the head groups of phospholipids with Fc receptor molecules and the change in membrane properties resulting from the increase of diglycerides.

J Biol Chem, 1984 May 25, 259(10), 6085 - 9
Glycine synthase of the purinolytic bacterium, Clostridium acidiurici . Purification of the glycine-CO2 exchange system; Gariboldi RT et al.; When the growth medium of Clostridium acidiurici was supplemented with trace metals, glycine synthase and glycine-CO2 exchange activities in cell-free extracts were found to increase significantly . The glycine-CO2 exchange system was purified and shown to consist of a heat-labile component and a heat-stable component . By gel filtration, heat-labile component had an estimated native Mr = 230,000 and contained two subunits of Mr = 65,000 and 58,000 on sodium dodecyl sulfate-polyacrylamide gels, indicating an alpha 2 beta 2 tetramer . Heat-stable component had an estimated Mr = 20,000 and could not be replaced by lipoic acid in reaction mixtures . Pyridoxal phosphate was not bound to either of the purified components but was essential for glycine-CO2 exchange . By spectral analysis, heat-labile component was shown to interact with pyridoxal phosphate and that reductant influenced this interaction.

Science, 1984 May 25, 224(4651), 881 - 4
The structural gene for tetanus neurotoxin is on a plasmid; Finn CW Jr et al.; A pool of synthetic oligonucleotides was prepared based on the amino terminal amino acid sequence of tetanus toxin . This probe hybridized to plasmid DNA isolated from three toxigenic strains of Clostridium tetani but not to plasmid DNA from a nontoxigenic strain . These results show that the structural gene for the toxin is on the plasmid . The pCL1 plasmid from one of the toxigenic strains spontaneously deleted 22 kilobase pairs of DNA to form pCL2 . Strains harboring this deleted plasmid are nontoxigenic . However, the probe mixture hybridized to pCL2, indicating that the DNA encoding the amino terminus of the toxin had not been deleted . Restriction endonuclease cleavage maps of pCL1 and pCL2 were constructed and indicate the approximate location and orientation of the structural gene for tetanus toxin.

Tijdschr Diergeneeskd, 1984 May 15, 109(10), 394 - 401
{Coccidiosis during the rearing of broiler breeding stock in Limburg}; Braunius WW et al.; Increasing complaints concerning the rearing of broiler breeding stock in the Province of Limburg were the reason for collecting data and examining the possibilities of prevention more closely . The first batches of broiler breeders submitted to the Animal Health Service in Heythuysen were examined for the presence of coccidiosis throughout the year 1982 . Of 363 groups submitted, 1,484 birds about which there were complaints, were examined . Of these, 153 flocks were found to be positive for coccidiosis, that is 42 per cent . The proportion of birds found to be positive was highest during the third quarter of 1982, being 50.52 per cent; two-thirds of these were birds under 3 months . Eimeria necatrix was the most common, being mainly present in the younger birds during the third quarter of the year, and the highest score was also recorded in this group . Coccidiosis may be associated with necrotic enteritis due to Clostridium perfringens . When these two are combined, this may cause very severe inflammatory lesions which may complicate treatment . Cl . perfringens was also found to be present in 11.6 per cent of the birds submitted, being particularly associated with E . necatrix and E . maxima . Amprol plus was found to be the anti-coccidial agent giving rise to the majority of problems, particularly in chickens under three months . Pancoxin plus and the shuttle monensin- Pancoxin plus were found to prevent more cases of coccidiosis, though they did so to a lesser degree in the older birds . Attention is drawn to the possible effect of feed-restriction on the prevention of coccidiosis in broiler breeding stock.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochem J, 1984 May 1, 219(3), 829 - 32
Characterization of {4Fe-4S}2+, {4Fe-4Se}2+ and hybrid (S, Se) clusters in Clostridium pasteurianum ferredoxin . A resonance Raman study; Moulis JM et al.; Resonance Raman spectra of native 2{4Fe-4S} ferredoxin from Clostridium pasteurianum and of its selenium-substituted analogue 2{4Fe-4Se} are compared . The experimental conditions used in this study included low temperature (approx . 25K) and the absence of a glass sample cell, thus ensuring high signal-to-noise ratios . The spectra of the 2{4Fe-4S} and 2{4Fe-4Se} ferredoxins display similar numbers of bands, but the resonance Raman patterns differ largely, except for two bands observed at 353 cm-1 and 365 cm-1 in spectra of the native ferredoxin, which are only moderately shifted upon S----Se substitution and are attributed to Fe-S(cysteine) stretching modes . The activities of the latter modes, enhanced by 457.9 nm excitation, are nearly equal in both ferredoxins . These data were used to demonstrate the presence of hybrid clusters {4Fe-(4-n)S-nSe} (n = 1, 2, 3) in a ferredoxin the active sites of which had been reconstituted in the presence of both S and Se.

Arch Microbiol, 1984 May, 138(1), 72 - 8
Purification and characterization of an inducible dissimilatory type sulfite reductase from Clostridium pasteurianum; Harrison G et al.; An inducible sulfite reductase was purified from Clostridium pasteurianum . The pH optimum of the enzyme is 7.5 in phosphate buffer . The molecular weight of the reductase was determined to be 83,600 from sodium dodecyl sulfate gel electrophoresis with a proposed molecular structure: alpha 2 beta 2 . Its absorption spectrum showed a maximum at 275 nm, a broad shoulder at 370 nm and a very small absorption maximum at 585 nm . No siroheme chromophore was isolated from this reductase . The enzyme could reduce the following substrates in preferential order: NH2OH greater than SeO3 (2-) greater than NO(2-) 2 at rates 50% or less of its preferred substrate SO3(2-) . The proposed dissimilatory intermediates, S3O6 (2-) or S2O3(2-), were not utilized by this reductase while KCN inhibited its activity . Varying the substrate concentration {SO3(2-)} from 1 to 2.5 mumol affected the stoichiometry of the enzyme reaction by alteration of the ratio of H2 uptake to S2- formed from 2.5:1 to 3.1:1 . The inducible sulfite reductase was found to be linked to ferredoxin which could be completely replaced by methyl viologen or partially by benzyl viologen . Some of the above-mentioned enzyme properties and physiological considerations indicated that it was a dissimilatory type sulfite reductase.

Scand J Gastroenterol, 1984 May, 19(3), 441 - 4
Clostridium difficile isolation in neonates in a special care unit . Lack of correlation with necrotizing enterocolitis; Lishman AH et al.; The stools of 78% of 45 infants in a Special Care Baby Unit yielded Clostridium difficile on culture, and in 67% of these it was possible to detect C . difficile toxin by means of a tissue culture technique . The stools of six of the seven infants with necrotizing enterocolitis were positive for C . difficile, but neither of the two most severely affected contained C . difficile toxin . The incidence of C . difficile isolation was similar in infants treated by exchange transfusion, those treated with antibiotics, those of low birth weight, and those with respiratory distress . The serum of only 2 of 28 infants and 1 of 20 mothers contained a neutralizing factor to C . difficile toxin . The present study does not support a role for C . difficile in neonatal disorders and in particular necrotizing enterocolitis . The reason for the apparent tolerance of the neonatal bowel to C . difficile toxin remains to be explained.

Mod Vet Pract, 1984 May, 65(5), A9 - 12
Some nutritional aspects of colic in horses; Hintz HF; Consistency of exercise and diet are important in colic prevention . Water should be offered before and after feeding . Fast-growing foals suckling heavily lactating mares may overeat grain at weaning . Creep feeding to accustom the foal to eating grain and gradually increasing the grain intake after weaning are helpful in preventing colic in foals . Stallions may overeat grain when taken off pasture in hot weather . Feeding hay initially and grain later helps avoid colic in these stallions . Type-D Clostridium perfringens may cause enterotoxemia in foals . Corn should be fed in moderation . High-Mg diets, ingestion of sand, and pelleted feed have been associated with colic . Endoparasitism is the most important cause of colic in horses.

J Antimicrob Chemother, 1984 May, 13(5), 521 - 4
Effect of therapy with latamoxef (moxalactam) on carriage of Clostridium difficile; Deery HG et al.; Twenty-seven patients receiving latamoxef (moxalactam) as a single antimicrobial agent were studied prospectively for Clostridium difficile carriage and development of diarrhoea or colitis . Stools were available prior to therapy from only seven patients, one of whom (14.3%) was an asymptomatic carrier . None of twelve patients studied during therapy were carriers . Seven of 27 patients (25.9%) were colonized with Cl . difficile after completion of latamoxef therapy, and three patients had cytotoxin positive stools . Two patients with cytotoxin grew Cl difficile from stools and one patient was culture negative . Only one patient, who had both culture and cytotoxin positive stools, had profuse diarrhoea . Cl . difficile clinical isolates were only moderately susceptible to latamoxef in vitro . Hamsters given moxalactam developed caecitis . Patients receiving latamoxef, or third generation cephalosporins, may be at increased risk of development of Cl . difficile associated diarrhoea and should be followed closely for this complication, especially after therapy has been discontinued.

Arch Surg, 1984 May, 119(5), 546 - 50
Surgical aspects of Clostridium septicum septicemia; Pelfrey TM et al.; Clostridium septicum is a virulent cause of gas gangrene and sepsis . Although thought to be rare, a survey of our affiliated hospitals for a recent five-year period disclosed eight cases . Seven of the eight had an occult malignant neoplasm . The eighth patient was thought to be preleukemic . All seven malignant neoplasms involved the gastrointestinal tract . Four patients were admitted with gangrene of an extremity, three with abdominal pain, and one with both . In four patients, C septicum septicemia appeared in an extremity before the underlying gastrointestinal malignant neoplasm was recognized . Four patients had surgical therapy and two survived; four received medical therapy and one survived . Patients who have C septicum septicemia should be assumed to harbor an underlying malignant neoplasm until proved otherwise.

J Infect Dis, 1984 May, 149(5), 775 - 80
Epidemiology of colitis induced by Clostridium difficile in hamsters: application of a bacteriophage and bacteriocin typing system; Hawkins CC et al.; The epidemiology of colitis induced by Clostridium difficile in hamsters was studied with a new bacteriophage and bacteriocin typing system . Fatal enterocolitis was induced by administration of N-formimidoyl thienamycin . Environmental cultures were obtained repeatedly throughout the experiments . Thirteen percent of 90 healthy hamsters were already colonized with C difficile on arrival from the supplier . Mortality from enterocolitis after antibiotic administration was 75% and was not diminished by use of a laminar-flow facility . The same uncommon bacteriocin type (83/1309/2329) of toxigenic C difficile that colonized hamsters on arrival was recovered from the cecal contents of all hamsters dying with enterocolitis and from most environmental isolates . Previously uncolonized , antibiotic-treated hamsters placed into cages where animals had died from enterocolitis also developed enterocolitis with the same bacteriocin type (83/1309/2329), an outcome suggesting acquisition of C difficile from the environment.

Ann Microbiol (Paris), 1984 May-Jun, 135A(3), 443 - 56
{Antibiogram of bacteria of the Clostridium genus}; Magot M; The disc-agar diffusion method for antibiotic sensitivity testing was adapted to Clostridium . The main feature of the method described here resided in the use of an inoculum which varied according to the physiological properties of the strain tested, so that the same critical time was obtained in all cases . This technique employs commercially available media and reduced equipment . Statistical analysis of the results indicated a strong correlation between disc-agar sensitivity testing and MIC determined by the agar dilution method.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1984 May, 257(1), 68 - 72
Clostridium botulinum subtype Ba; Gimenez DF; Strain 657 has been described as a toxin variant of Clostridium botulinum type B . Neutralization tests performed with types A and B botulinal antitoxins of known potency and avidity at 20, 25, 50, 100, 200, 2,000 and 20,000 mouse LD50 levels of testing, have shown that 657 toxin is a mixture of B (approximately 95% of the complex) and A antigenic fractions . The possibility of a cross-contamination between A and B serotypes has been practically ruled out through the serologic screening of the toxins from 100 well isolated colonies taken from two colony variants of this strain . Strain 657 rabbit antitoxin possesses A and B neutralizing activities . Strain B 657 produces a hitherto undescribed complex toxin and it represents the prototype of a new C . botulinum serotype, subtype Ba.

J Clin Microbiol, 1984 May, 19(5), 645 - 8
Detection of Clostridium botulinum type A toxin by enzyme-linked immunosorbent assay with antibodies produced in immunologically tolerant animals; Dezfulian M et al.; Immunological tolerance is a state of unresponsiveness to foreign substances (antigens) which can develop in human and animal species as the result of continued exposure to antigens early in life . We utilized this principle for the preparation of antibodies against Clostridium botulinum type A toxin . By selective suppression of the immunological response of rabbits to unwanted antigens and subsequent immunization with a toxoid, we were able to produce a specific type A antitoxin without the need to purify the toxin . Despite cross-reactivity with C . botulinum type B, our type A antitoxin was otherwise specific since it did not react with culture filtrates of nontoxigenic variants of type B, any other C . botulinum type (C, D, E, F, and G), nor with 18 other Clostridium species, including Clostridium sporogenes . Using this antitoxin, we developed a sensitive enzyme-linked immunosorbent assay for detection of C . botulinum type A toxin.

J Clin Microbiol, 1984 May, 19(5), 592 - 3
Rapid glutamic acid decarboxylase test for identification of Bacteroides and Clostridium spp; Jilly BJ et al.; A rapid 4-h test for glutamic acid decarboxylase is described for the identification of certain anaerobic bacteria . The test substrate consisted of 1.0 g of L-glutamic acid, 0.3 ml of Triton X-155, and 0.05 g of bromcresol green sodium salt in 1 liter of water . The substrate was dispensed in 0.5-ml amounts into test tubes, and a turbid suspension was made with the test organism . The test was then incubated aerobically at 35 degrees C for 4 h . The development of a blue color was considered positive . A total of 345 strains of clinically isolated anaerobic bacteria were tested . All isolates of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis . Clostridium perfringens, and Clostridium sordellii gave a positive reaction . Some isolates of Bacteroides distasonis and Bacteroides vulgatus were also positive . The use of this rapid test in conjunction with other rapid methods, such as the spot indol test, will enable laboratory workers to report these pathogens on the same day on which an inoculum of pure culture growth on agar is available.

Arch Intern Med, 1984 May, 144(5), 967 - 9
Clostridium difficile . Colonization and toxin production in a cohort of patients with malignant hematologic disorders; Morris JG Jr et al.; We examined 45 (80%) of 56 consecutive adult patients with malignant hematologic disorders who were hospitalized during a 15-week period at Emory University Hospital, Atlanta . Stool samples for Clostridium difficile culture and cytotoxin assay were obtained on admission and then weekly during each patient's hospitalization . On admission, four patients had detectable C difficile in their stool samples, which was associated with prior antimicrobial use but not with prior cancer chemotherapy . One of the four patients with positive stool samples also had toxin present in the stool sample and was the only one with diarrhea . Eight (36%) of 22 patients hospitalized for one or more weeks had C difficile isolated from at least one stool specimen . The positive cultures showed no clustering in time, and no risk factors were identified for colonization . Only seven of 15 culture-positive stool samples and three of seven toxin-positive samples were associated with diarrhea.

Appl Environ Microbiol, 1984 May, 47(5), 1172 - 4
Alternative medium for Clostridium perfringens sporulation; Tortora JC; A medium containing 0.50 g of thiotone peptone, 0.30 g of soluble starch, 0.02 g of MgSO4 X 7H2O, 0.90 g of Na2HPO4 X 2H2O, 100.00 ml of distilled water, and optionally , 166 micrograms of dichloridric thiamine supported sporulation of 138 out of 141 Clostridium perfringens strains . Comparatively this medium gave a greater percentage of sporulation than five other media described previously.

Prikl Biokhim Mikrobiol, 1984 May-Jun, 20(3), 349 - 54
{Phospholipase C inhibitor from Streptoverticillium mycoheptinicum}; Iakovleva EP et al.; Streptoverticillium mycoheptinicum, a producer of the antifungal antibiotic mycoheptin, was found to produce an inhibitor of Clostridium perfringens phospholipase C . Dynamics of the enzyme accumulation in the culture liquid filtrate was studied . A technique was developed for purification of the inhibitor, which includes adsorption, selective precipitation, ultrafiltration, gel chromatography and isoelectric focusing . The inhibitor was found to be of the peptide nature with the molecular weight of 3500-4000 and to exist in two isoforms with the isoelectric points of 8.15 and 8.50.

Lancet, 1984 Apr 28, 1(8383), 935 - 8
Typing scheme for Clostridium difficile: its application in clinical and epidemiological studies; Tabaqchali S et al.; Epidemiological studies of Clostridium difficile diarrhoeal disease have been hindered by the lack of a typing scheme for this organism . A typing method based on the incorporation of sulphur-35-labelled methionine into cellular proteins and their separation by sodium dodecylsulphate/polyacrylamide gel electrophoresis showed clear pattern differences between strains, and nine distinct groups within the C difficile species were established . 98% of 250 clinical strains derived from four hospitals were typable . Group X was the commonest group and was associated with outbreaks of pseudomembranous colitis and antibiotic-associated colitis in two hospitals . Groups A-D were isolated predominantly from mothers and newborn infants . In outbreaks of antibiotic-associated colitis in oncology and orthopaedic wards the same strains, group X and group E, respectively, were isolated from patients and their environment, providing strong evidence of cross-infection between patients and of hospital acquisition of C difficile.

J Wildl Dis, 1984 Apr, 20(2), 86 - 9
An outbreak of botulism in waterfowl and fly larvae in New York State; Shayegani M et al.; In October 1982 the death of approximately 1,500 wild ducks, mostly mallards (Anas platyrhynchos), and about 100 shore birds including greater yellowlegs (Tringa melanoleuca) was observed in the New York State Oak Orchard Wildlife Management Area . The lack of gross pathology, the signs exhibited by the moribund ducks, and the ecologic conditions indicated possible botulinal intoxication . Clostridium botulinum toxin type C was demonstrated in duck serum (approximately 5 X 10(4) mouse intraperitoneal LD50 of toxin per ml of serum) and in an extract from fly larvae (Lucilia spp.) taken from the same area (approximately 1 X 10(6) mouse intraperitoneal LD50 of toxin per gram of larvae).

Am J Surg, 1984 Apr, 147(4), 486 - 91
Clostridium difficile colitis in surgical patients; Rosenberg JM et al.; The clinical course of 75 patients with diarrhea and positive C . difficile toxin stool assays has been examined . The mean age of the patients was 68 years . Five of 25 surgical nursing units accounted for two thirds of the cases . Many patients were immuno-suppressed with cancer, sepsis, or diabetes mellitus . The median onset of diarrhea was 2.7 days after initial administration of antibiotics . Fever and leukocytosis were frequently seen . Diarrhea ceased in 30 percent of the patients after withdrawal of the offending antibiotics . The remainder required specific therapy with vancomycin, bacitracin, or metronidazole . Two deaths were directly attributable to C . difficile colitis . The hospital stay was prolonged in many patients . C . difficile colitis should be suspected in any patient in whom diarrhea develops during or after a course of antibiotics . Enteric precautions may prevent clustering in these cases and colonization in other susceptible patients.

Radiology, 1984 Apr, 151(1), 53 - 4
Secondary infection of an endometrioma following fine-needle aspiration; Martino CR et al.; A Clostridium infection occurred in an endometrioma following fine-needle aspiration . Fine-needle aspirations are clinically useful and safe . The authors conclude, however, that this procedure may rarely result in a secondary infection . Specific recommendations to avoid such complications are stated.

J Gen Microbiol, 1984 Apr, 130 ( Pt 4), 975 - 81
Demonstration of the common antigens of Clostridium botulinum, C . sporogenes and C . novyi by an enzyme-linked immunosorbent assay and electroblot transfer; Poxton IR; EDTA extracts were prepared from whole cells of 16 strains of Clostridium botulinum (types A-E), 6 strains of C . novyi (types A-D) and 3 strains of C . sporogenes . They were reacted in an enzyme-linked immunosorbent assay (ELISA) with antisera raised against whole, UV-killed cells of C . sporogenes and C . novyi type A . Results showed significant cross-reactions between C . sporogenes antiserum and the C . botulinum type A (three out of four strains), proteolytic type B (all strains) and one type E strain, and between C . novyi type A antiserum and C . botulinum types C and D . All the C . sporogenes and C . novyi strains reacted with their homologous antiserum; these two species showed no cross-reactions . All the reactions were investigated further by running the EDTA extracts on SDS-polyacrylamide gels . The separated molecules were electrophoretically transferred to nitrocellulose membranes, reacted with antiserum and complexes visualized with horseradish peroxidase conjugate reagents . Only those extracts that reacted significantly in the ELISA gave a pattern of cross-reactive antigen bands and the number of bands and intensity of stain closely paralleled the strength of the ELISA reaction.

Arch Intern Med, 1984 Apr, 144(4), 849 - 50
Clostridium perfringens bacteremia in prosthetic valve endocarditis . Diagnosis by peripheral blood smear; Alvarez-Elcoro S et al.; A patient with two prosthetic valves had clinical evidence of infectious endocarditis caused by Clostridium perfringens . The diagnosis was made by routine examination of the peripheral blood smear . To our knowledge, no previous reports have been made of clostridial endocarditis in prosthetic valves with the presence of clostridia in the peripheral blood smear.

J Med Microbiol, 1984 Apr, 17(2), 171 - 6
Demonstration of shared antigens in the genus Clostridium by an enzyme-linked immunosorbent assay; Poxton IR et al.; Antigens prepared from several strains of each of 10 Clostridium species were used in an indirect enzyme-linked immunosorbent assay with antisera raised against whole cells of a representative strain from each of the 10 species killed by ultra-violet irradiation . With the exception of C . cadaveris, the antisera gave similar results with antigens prepared from all strains of the homologous species . Antigens prepared from 13 other clostridial species were then investigated in an ELISA system with the 10 representative antisera . The results showed many cross-reactions, particularly in the C . perfringens group, the C . difficile/sordelli group and the C . botulinum/novyi/sporogenes group.

Infect Immun, 1984 Apr, 44(1), 188 - 93
Generation of leukotrienes from human granulocytes by alveolysin from Bacillus alvei; Bremm KD et al.; We investigated the effect of alveolysin on human granulocytes . Alveolysin is an exoprotein produced by Bacillus alvei and belongs to the group of sulfhydryl-activated cytolysins . Other members of this group are streptolysin O and theta-toxin from Clostridium perfringens . It is demonstrated that alveolysin leads to leukotriene generation from human granulocytes, which exert chemotactic (leukotriene B4) and slow-reacting substance (leukotriene C4, D4, and E4) activity under sublytic concentrations.

J Med Microbiol, 1984 Apr, 17(2), 151 - 8
The influence of drugs on the response of a cell culture preparation to bacterial toxins; Giugliano LG et al.; The influence was studied of lanthanum chloride, chlorpromazine hydrochloride, indomethacin and sodium cromoglycate on the morphological changes induced in Vero cells by the action of the cholera toxin, the thermolabile enterotoxin (LT) and the Vero cell cytotoxin (VT) of Escherichia coli, the enterotoxin of Clostridium perfringens, and the cytotoxin of Clostridium difficile . These drugs were able to inhibit the effects produced by C . difficile cytotoxin but not by the other toxins examined.

Jpn J Antibiot, 1984 Apr, 37(4), 555 - 7
Cefotaxime-associated diarrhea and Clostridium difficile; Funada H et al.; A patient with rheumatoid arthritis-associated interstitial pneumonitis, treated with prednisolone, developed mild colitis due to Clostridium difficile in association with the use of cefotaxime (CTX) . Diarrhea was successfully treated with the discontinuation of CTX and initiation of oral vancomycin.

Biochem Biophys Res Commun, 1984 Mar 30, 119(3), 828 - 35
Structural differences between {2Fe-2S} clusters in spinach ferredoxin and in the "red paramagnetic protein" from Clostridium pasteurianum . A resonance Raman study; Meyer J et al.; The {2Fe-2S} ferredoxin ("Red paramagnetic protein", RPP) from C . pasteurianum has been found to be composed of two identical subunits of 10,000 +/- 2 000 daltons, each containing a {2Fe-2S} cluster . Resonance Raman (RR) spectra of RPP have been obtained at 23 degrees K, and compared to those of spinach ferredoxin (Sp Fd) . Ten modes of the {2Fe-2S} chromophore were observed in the 100-450 cm-1 range . Assignments of non fundamental modes in the 500-900 cm-1 range allowed correlations between fundamental stretching modes of RPP and Sp Fd . Although assuming a {2Fe-2S} structure, the chromophore of RPP differs from that of Sp Fd by its conformation and by a slight weakening of Fe-S bonds, involving both the inorganic core and the cysteine ligands.

Biochemistry, 1984 Mar 27, 23(7), 1442 - 8
Modulation of neuraminidase activity by the physical state of phospholipid bilayers containing gangliosides Gd1a and Gt1b; Myers M et al.; The thermotropic behavior of large unilamellar dipalmitoylphosphatidylcholine vesicles containing the disialoganglioside Gd1a and the trisialoganglioside Gt1b on their outer surface has been studied as a function of the ganglioside molar fraction and Ca2+ concentration by using high-sensitivity differential scanning calorimetry and steady-state fluorescence spectroscopy . These studies indicate that both gangliosides have an ordering effect on the hydrocarbon region of the bilayer and that this effect is enhanced by the presence of Ca2+ ions . The calorimetric experiments also indicate that ganglioside Gt1b has an intrinsic tendency to phase separate into compositional-rich ganglioside domains even in the absence of Ca2+ . Ganglioside Gd1a, on the other hand, only phase separates at Ca2+ concentrations equal to or higher than 10 mM . These studies have allowed us to identify and evaluate the factors affecting the rates of hydrolysis of gangliosides by the soluble neuraminidase from Clostridium perfringens . The data presented in this paper indicate that the rates of hydrolysis of membrane-bound gangliosides are correlated to the physical state of the membrane and the state of aggregation of the ganglioside molecules within the lipid bilayer . For membrane-bound gangliosides, maximal activation energies were found at temperatures slightly below the lipid phase transition temperature . The rates of hydrolysis of the soluble substrate sialyllactose or that of the micellar ganglioside is independent of Ca2+ concentration, whereas the rates of hydrolysis of membrane-bound ganglioside are inhibited by Ca2+ especially under conditions in which the clustering effect of Ca2+ is maximal . These studies suggest that the soluble neuraminidases from Clostridium perfringens prefer ganglioside substrates that are dispersed within the membrane and not forming part of largely aggregated clusters.

Z Gesamte Inn Med, 1984 Mar 15, 39(6), 85 - 92
{Recent advances in the molecular mechanism of action of bacterial toxins, in particular of diphtheria, cholera, coli, botulinum and shigella toxins as well as tetanospasmin and the toxins of staphylococcus aureus}; Kolb E; Great progress was achieved in the clarification of the molecular structure and the mechanism of action of the toxins of pathogenic forms of bacteria . Proportions of toxins of Corynebacterium diphtheriae and of Pseudomonas aeruginosa transfer from the NAD and ADP-ribose protein to an amino acid of the elongation factor 2 . Thus the protein synthesis is much inhibited . The cholera toxin and the L-toxin from Escherichia coli have a similar structure . They transfer an ADP-ribose portion from NAD to the GTP-protein of the adenylate cyclase complex, by which means the GTPase activity is reduced . The increase of the cAMP content leads to an increase of the permeability of the cells of the intestinal epithelium . The tetanospasmin decreases the production of the inhibitingly acting neurotransmitters ( glycin ) from intermediate neurons and thus evokes spasms . The botulinum toxin inhibits the release of acetylcholine from the motor end-plates and leads to paralyses . Staphylococcus aureus and Clostridium perfringens form among others cytolysins which are injurious to membranes.

J Biol Chem, 1984 Mar 10, 259(5), 3167 - 72
Effects of deglycosylation on the architecture of ovine submaxillary mucin glycoprotein; Rose MC et al.; The structural features of native and deglycosylated ovine submaxillary mucin (OSM) were determined by electron microscopy of platinum unidirectionally shadowed preparations and by ultracentrifugation . Thin filamentous molecules, of which 90% were 100-230 nm in length with estimated diameters of 1.0-1.4 nm, were observed with dilute samples of OSM in high ionic strength solvents (5-30 micrograms/ml in 0.8 M NaCl or NH4Ac) . Ultracentrifugation studies indicated that these filamentous structures were monomers and/or dimers . At higher mucin concentrations or in lower ionic strength solvents, OSM molecules were oligomers that appeared as long rope-like strands . Removal of sialic acid residues by incubation with Clostridium perfringens neuraminidase yielded filamentous structures similar to those observed with OSM and some smaller less extended structures . Subsequent removal of the GalNAc residues of asialo-OSM with C . perfringens alpha-N-acetylgalactosaminidase resulted in a dramatic change in appearance, from an extended filament to a globular form . The frictional ratios of OSM and deglycosylated OSM were consistent with the marked structural differences of these molecules . Native OSM had a frictional ratio of 3.09, comparable to that of highly asymmetric tropomyosin (3.22); deglycosylated OSM had a frictional ratio of 1.11, comparable to that of globular ovalbumin (1.08).

Antimicrob Agents Chemother, 1984 Mar, 25(3), 342 - 3
Effect of inoculum, pH, and medium on the activity of ciprofloxacin against anaerobic bacteria; Borobio MV et al.; The in vitro activity of ciprofloxacin against 30 Bacteroides fragilis group, 30 Clostridium, and 30 Peptococcaceae strains was determined by the agar dilution method in two different culture media at three pH and in three inoculum densities . In Wilkins-Chalgren agar the MICs for 90% of the strains were 0.12 micrograms/ml for B . fragilis, 0.5 micrograms/ml for Clostridium spp., and 2 micrograms/ml for Peptococcaceae . The pH and medium composition affected the MICs of ciprofloxacin.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S235 - 41
Treatment of antibiotic-associated pseudomembranous colitis; Bartlett JG; The experience of this laboratory with the treatment of Clostridium-induced colitis in experimental animals and in patients was reviewed . Optimal results in hamsters were achieved with the antibiotics vancomycin, metronidazole, and tetracycline . Cholestyramine was less effective . The outcome for animals given corticosteroids and Clostridium sordellii antitoxin systemically was not different from that for untreated control animals . The second facet of the study was a retrospective review of therapy in 272 patients with C . difficile-induced diarrhea or colitis . No specific therapy was given to 56 patients who had mild symptoms or were improving at the time the toxin was detected . The therapy most frequently used was oral vancomycin, which was given to 189 patients, including 100 with confirmed pseudomembranous colitis . The response rate was 97%, but 46 patients (24%) relapsed when treatment was discontinued . Response to cholestyramine was favorable in 12 of 19 patients . The results with metronidazole and bacitracin were uniformly good, although the experience was limited.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S229 - 34
Polyacrylamide gel electrophoresis patterns produced by Clostridium difficile; Wexler H et al.; Clostridium difficile is a major cause of pseudomembranous colitis following antimicrobial therapy . There is evidence to suggest that this organism may be hospital acquired . Polyacrylamide gel electrophoretic (PAGE) analysis of protein profiles of C . difficile cell extracts was examined for possible usefulness in epidemiologic studies . At least 50 bands could be distinguished in soluble cell extracts of C . difficile . Freeze-thawing of extracts and/or length of storage time did not affect the protein profiles . While all strains tested were nearly identical, several strains were unique in their lack of a 34,000-dalton polypeptide . Protein patterns of C . difficile could easily be distinguished from those of Clostridium sordellii . Additionally, surface antigens extracted from several strains of C . difficile and from a few strains of C . sordellii revealed marked differences . Ethylenediaminetetraacetate (EDTA) extracts from C . difficile showed two or three major bands in PAGE analysis; strains could be divided into two major subgroups on the basis of band distribution . EDTA extracts from C . sordellii bore no similarity to those of C . difficile.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S214 - 21
Inciting and etiologic agents of colitis; Silva J et al.; Since 1979, 3,115 stool samples were tested for detection of Clostridium difficile and its cytotoxin; these were obtained from patients who had drug-related diarrhea . Presumed or proven colitis due to C . difficile was diagnosed in 130 patients . Drugs implicated most commonly as causing or associated with the onset of enterocolitis due to C . difficile were ampicillin (38 episodes), cephalosporins (71), clindamycin (36), and the aminoglycosides (45) . The hamster model of colitis was employed to explore the role of other inducing agents . Altering the usual diet of hamsters to one with a higher protein content decreased the time to death due to C . difficile cecitis following the administration of cefazolin (10 mg) . Several cathartics also were studied for their effect on the lethality of antibiotic-induced cecitis . Daily administrations of castor oil (0.5 ml per day) and vegetable oil (1.0 ml per day) improved survival against lethal doses of clindamycin . Milk of magnesia or mineral oil provided no protection . Four patients with C . difficile colitis induced by therapy with cytotoxic drugs also were identified . Methotrexate induced cecitis when administered orally and daily to hamsters, and C . difficile and its cytotoxin were identified in the hamsters' stools . Death due to methotrexate-induced cecitis was prevented by daily administration of folinic acid or vancomycin . These data demonstrate that a variety of antibiotics, antineoplastic agents, cathartics, and diet changes can induce C . difficile colitis in humans and hamsters.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S171 - 6
The infected foot of the diabetic patient: quantitative microbiology and analysis of clinical features; Sapico FL et al.; The quantitative deep-tissue microbiology of the infected feet of 32 patients with diabetes mellitus was studied, and the clinical features of the patients were analyzed . Techniques of specimen collection designed to avoid contamination from surface flora were used to study amputated lower limbs . Cultures of deep tissue from six patients yielded only aerobes, and for one patient, only anaerobes . Cultures for 25 patients yielded a mixture of aerobes and anaerobes . A mean of 4.81 species (2.84 aerobes and 1.97 anaerobes) were isolated from each patient . The density of growth of anaerobes, however, was significantly higher than that of aerobes . Culture specimens obtained by curettage of the base of the ulcer correlated better with results of deep-tissue culture than did those obtained by needle aspiration or swab of the ulcers . The most frequently isolated organisms were Bacteroides species, anaerobic streptococci, group D streptococci, Clostridium species, and Proteus species . The presence of anaerobes was associated with a higher frequency of fever and foul-smelling lesions and with the presence of a foot ulcer . Prior antibiotic therapy did not appear to influence the nature of the microorganisms isolated . The polymicrobial nature of this disease should be considered when antimicrobial therapy is indicated.

Surgery, 1984 Mar, 95(3), 281 - 3
The significance of anaerobic bacteria in biliary tract infection after hepatic portoenterostomy for biliary atresia; Brook I et al.; Aspiration of the biliary atrium was performed in six children who developed cholangitis after hepatic portoenterostomy (Kasai procedure) . All aspirates were cultured for aerobic and anaerobic bacteria . Aerobic bacteria were recovered in all six specimens and anaerobic organisms were recovered in three . The predominant aerobic organisms were Klebsiella pneumoniae (four isolates), enterococcus (three isolates, and Escherichia coli (two isolates) . The anaerobes recovered were Bacteroides fragilis (two) and Clostridium perfringens (one) . These findings demonstrate the recovery of anaerobic as well as aerobic organisms in cholangitis after hepatic portoenterostomy.

J Surg Oncol, 1984 Mar, 25(3), 188 - 91
Non-spore-forming clostridial bacteremia; Grasberger RC et al.; The significance of the non-spore-forming clostridium as a blood-borne pathogen has been underemphasized . Over a 7-year period, these bacteria were cultured 40 times in 30 patients . Overall mortality rate for non-spore-forming clostridial bacteremia was 39% . Patients with polymicrobial infections had a 58% mortality rate, while those with only clostridium in their blood had a mortality of 23% . Mortality increased as the number of positive cultures . Patients who were clinically septic at the time of their cultures fared poorly . Advanced age and underlying malignant disease were associated with increased mortality . Patients who have positive blood cultures for non-spore-forming clostridium are at a significant risk and should be treated aggressively.

J Clin Pathol, 1984 Mar, 37(3), 335 - 43
Neutropenic enterocolitis due to Clostridium septicum infection; King A et al.; Three cases of neutropenic enterocolitis are described . This unusual condition occurs almost exclusively in neutropenic patients and has a fulminating course which is almost invariably fatal without surgical intervention . The lesion is centered on the caecum and hitherto its pathogenesis has been unclear, partly because most studies have been performed on material obtained at necropsy . In these three patients, all of whom were treated surgically, Clostridium septicum was identified by specific immunofluorescence in the bowel wall of the resected specimens . Two patients also had C septicum septicaemia . Various forms of mucosal damage can be identified which predispose towards invasion of the bowel wall by this organism . These cases provide further confirmation of a primary role for C septicum in the pathogenesis of neutropenic enterocolitis.

J Pediatr, 1984 Mar, 104(3), 454 - 9
Moxalactam therapy of Haemophilus influenzae type b meningitis in children; Chartrand SA et al.; Thirty-four children with Haemophilus influenzae type b meningitis were given prospectively either moxalactam (200 mg/kg/day) or ampicillin (400 mg/kg/day) plus chloramphenicol (75 mg/kg/day) . One patient in each group died . The mean duration of fever, clinical response, sequential cerebrospinal fluid findings, and incidence of neurologic sequelae were similar between groups . Moxalactam cerebrospinal fluid bioactivity was significantly greater than that of ampicillin or chloramphenicol throughout therapy . Neutropenia, liver enzyme abnormalities, and diarrhea were not significantly different . In eight of 11 patients given moxalactam (versus one of 14 controls) there was complete elimination of gram-negative aerobic flora in the stools by day 10 (P = 0.002); however, none acquired Clostridium difficile . Moxalactam in effective therapy for H . influenzae type b meningitis.

Arch Intern Med, 1984 Mar, 144(3), 617 - 9
Acute oligoarthritis associated with Clostridium difficile pseudomembranous colitis; Lofgren RP et al.; The abrupt onset of a sterile inflammatory oligoarthritis developed in a patient with active Clostridium difficile pseudomembranous colitis . The arthritis affected a hip and a knee . Leukocyte counts of synovial fluid obtained from the patient's left hip and knee were elevated . He was haplotyped as HLA-B27 antigen-positive . The colitis and arthritis promptly abated after treatment with oral vancomycin hydrochloride . Three other cases of arthritis associated with antibiotic-induced colitis were reviewed . It seems as if treatment of the colitis leads to resolution of the arthritis.

J Diarrhoeal Dis Res, 1984 Mar, 2(1), 41 - 2
Significance of stool toxin determination to Clostridium difficile diarrhoea; Chang TW; PIP: Clostridium difficile toxin in stool from patients with antibiotic-associated diarrhea was titered and correlated with stool consistency, presence of white cells, epithelial cells and occult blood . It was found that watery stools contained more toxin, with a higher % of positive occult blood and leukocytes than did soft or formed stools, suggesting a correlation between toxic titer and the severity of the pathologic processes of the disease . Of 71 toxin-positive stools from patients with antimicrobial-associated diarrhea, more than 1/2 were watery in consistency . About 1/4 were yellow and the remaining were dark brown . C . difficle toxin titer in watery stools was approximately 5 times higher than that in soft or formed stools . Toxin titration is useful for following the clinical response to vancomycin therapy . The persistence of toxin after vancomycin therapy is an indication of bacterial persistence in the colon and carries a 50% chance of relapse within the next 2-4 weeks .

Aust Paediatr J, 1984 Mar, 20(1), 29 - 33
Necrotizing enterocolitis in very low birthweight infants: a four-year experience; Yu VY et al.; Fifty (13%) of 375 infants who weighed 1500 g or less at birth had necrotizing enterocolitis (NEC) . Haematological changes suggestive of sepsis occurred in 83% and positive bacteriological cultures were found in 38%, the most common organism isolated being Clostridium perfringens . Complications included intestinal perforation in six patients and recurrence of NEC in five, of whom one subsequently developed an intestinal stricture . Five of the eight nursery deaths were secondary to peritonitis and overwhelming sepsis from NEC . In spite of the discontinuation of milk feeds for prolonged periods, satisfactory caloric intake and weight gain were achieved with parenteral nutrition in the survivors . Of the 41 long-term survivors, six (15%) were found to have a disability at 2 years of age, corrected for prematurity, compared with 48 (20%) of 241 very low birthweight survivors from the same study period who did not have NEC . None had evidence of gastrointestinal dysfunction . Six (15%) children remained below the 10th percentile for both weight and height . This study showed that early diagnosis and therapy for NEC in very low birthweight infants were associated with a favourable short- and long-term outcome.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S11 - 20
Biochemical characterization and biologic actions of two toxins (D-1 and D-2) from Clostridium difficile; Banno Y et al.; Two toxins were isolated from a toxigenic strain of Clostridium difficile . The toxins were purified by gel filtration and ion-exchange column chromatography to homogeneity as judged from polyacrylamide gel electrophoresis and were designated D-1 and D-2 . Toxin D-1 was lethal for mice, increased vascular permeability, and induced fluid accumulation in ligated rabbit ileal loops, and toxin D-2 displayed cytotoxicity in HeLa cells with a minimum of 1 pg of toxin . The molecular weights of toxins D-1 and D-2, as estimated by gel filtration, ranged from 550,000 to 600,000 and from 450,000 to 500,000, respectively . These toxins were heat labile and inactivated by pronase and trypsin . Amino acid analyses of both toxins showed them to be of relatively similar composition . Antisera prepared against purified toxin D-1 neutralized all of the biologic activities of toxin D-1, whereas it did not affect any of the biologic activities of toxin D-2 . A sensitive latex-agglutination immunoassay was developed for screening for C . difficile toxin D-1 in patients with pseudomembranous colitis.

J Trauma, 1984 Mar, 24(3), 267 - 70
Bacillus cereus-induced myonecrosis; Johnson DA et al.; A patient with an incomplete amputation due to a crush injury to his arm developed a myonecrosis with Gram-positive rods noted on muscle and wound aspirates . The patient was treated for a probable Clostridium perfringens infection but culture results proved the organism to be Bacillus cereus . In light of the reported resistance of Bacillus cereus to penicillin, this case serves to emphasize the importance of expanded empiric coverage with high-dose penicillin and an aminoglycoside pending the return of culture and sensitivity results.

Thromb Res, 1984 Mar 1, 33(5), 499 - 510
Human platelet activation by bacterial phospholipase C is mediated by phosphatidylinositol hydrolysis but not generation of phosphatidic acid: inhibition by a selective inhibitor of phospholipase C; Navran SS et al.; We have shown earlier that phospholipase C (PLC) from Clostridium perfringens causes human platelet aggregation and secretion in a concentration dependent manner . The present study was undertaken to further characterize the specificity of the effects of PLC and to better understand the mechanism of the action of this inducer . A methylene-dioxybenzazepine (MDBA) analog of trimetoquinol was synthesized and tested for antiplatelet activity . MDBA (3-30 microM) inhibited PLC-induced aggregation in a concentration dependent manner . Whereas up to 200 microM MDBA did not inhibit aggregation induced by either thrombin, arachidonic acid, or U46619 . Effects of PLC (0.05 U/ml) on hydrolysis of phosphatidylinositol, production of phosphatidic acid and thromboxane B2 (TXB2) synthesis were investigated using {32P}-phosphate and {14C}-arachidonic acid labeled platelets . PLC (0.05 U/ml) caused a time dependent decrease in platelet phosphatidylinositol . Up to 50% of labeled phosphatidylinositol was lost from platelets in five minutes . MDBA (3-30 microM) inhibited PLC-induced loss of phosphatidylinositol in a concentration dependent manner . An increase in phosphatidic acid was also observed in PLC-stimulated platelets . Up to 100 microM MDBA did not inhibit production of phosphatidic acid . PLC-treated platelets did not produce any TXB2 . In other experiments possible protease contamination of PLC preparations was tested by incubating PLC (0.03-0.5 U/ml) with {14C}-casein . PLC in concentrations up to ten times higher than the concentrations used in aggregation studies did not cause hydrolysis of {14C}-casein, whereas more than 30% of {14C}-casein was hydrolyzed by trypsin . PLC-induced aggregation was not inhibited by up to 300 microM adenosine or ATP . In other experiments, platelet aggregation by ADP was inhibited by adenosine and ATP in a concentration dependent manner . The addition of calcium (0.5- 2.0 mM) increased aggregation by PLC in a concentration dependent manner . These findings suggest that PLC-induced activation of platelets is: (a) dependent on phosphatidylinositol hydrolysis but not on the production of phosphatidic acid, TXB2 or secretion of ADP; (b) not caused by protease contaminants; (c) calcium dependent; and (d) MDBA inhibits PLC-induced aggregation by blocking phosphatidylinositol hydrolysis.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S59 - 61
Recovery of anaerobic bacteria from vented blood-culture bottles; Martin WJ et al.; From January 1979, to July 1981, blood from each patient suspected of having bacteremia was collected in two bottles of commercially prepared tryptic soy broth; one bottle was vented for 24 hr to the atmosphere . Bottles were incubated at 37 degrees C for a maximum of seven days and were examined daily for signs of growth . Blind subcultures onto chocolate-agar plates were performed 18-24 hr after collection and again at 48 hr . For 63,106 bottles inoculated, a total of 4,788 strains were isolated, for a rate of 6.4% (excluding contaminants) . Two hundred and thirty (4.8%) isolates were anaerobes, of which 75 (33%) were identified as Bacteroides fragilis . It is significant that 20 of these anaerobic isolates (approximately 9%) were recovered only from the vented bottles . These included eight strains of B . fragilis; three strains of Clostridium perfringens; and one strain each of Bacteroides distasonis, Fusobacterium nucleatum, Peptococcus saccharolyticus, and Eubacterium species . These results emphasize the need to check vented bottles for anaerobes, since significant numbers of these bacteria could be missed by failing to do so . These data further show the desirability of holding anaerobic blood cultures for a minimum of one week.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S222 - 8
Epidemiology of Clostridium difficile-induced intestinal disease; Mulligan ME; The epidemiology of Clostridium difficile-induced intestinal disease is an intriguing subject about which there are few answers but many remaining questions . Although it is accepted that altered intestinal microecology (usually the result of antimicrobial therapy) is a major predisposition to disease, the details of microbial interactions are not yet known and clearly involve more than simple overgrowth of a resistant member of the resident flora . A variety of reservoirs of C . difficile are recognized . These include endogenous carriage, environmental contamination, and zoonoses, but the relative epidemiologic importance of these varied sources is yet to be determined . Because minor variations in methods for cultivation of C . difficile can markedly affect the ability to detect the organism, even the prevalence of endogenous carriage by various populations is not fully defined . There is good evidence for nosocomial acquisition of disease, but the frequency of this event and the usefulness of preventive measures need to be determined . The development of a typing system would provide a valuable tool for investigating many of the remaining questions . Finally, in addition to the recognized risk factors, which include the apparently predisposing alteration in intestinal microecology and exposure to C . difficile, there appear to be other, as yet undefined, variables that help to determine whether disease will occur . Perhaps the elucidation of the details of the pertinent microbial interactions as well as an understanding of the relevant host-pathogen relationships will provide important insights into the epidemiology of C . difficile-induced disease.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S208 - 13
Antimicrobial agent-associated colitis and diarrhea: historical background and clinical aspects; George WL; In the late 1970s it was found that Clostridium difficile causes a lethal, clindamycin-induced ileocecitis in the Syrian hamster; this animal model has been an invaluable aid to our understanding of antimicrobial agent-induced diarrhea in humans . C . difficile is involved in almost all cases of pseudomembranous colitis and in approximately one-fourth of cases of antimicrobial agent-associated diarrhea in humans in which a pseudomembrane is not detected . The presenting signs and symptoms of C . difficile-induced diarrhea are quite variable . Mild diarrhea may be the only finding in the least severe form of disease, whereas patients with severe disease may have high fever, leukocytosis, severe abdominal cramping, marked abdominal tenderness, and profuse diarrhea . Occasionally, symptoms may be so marked as to simulate an acute intraabdominal catastrophe . Diagnosis of C . difficile-induced disease usually is made by detecting C . difficile cytotoxin in the feces of a patient . Assay for cytotoxin in feces of infants is not reliable for diagnosis, however, because of the high incidence of an asymptomatic carrier state in this group . Appropriate therapy includes discontinuation of the offending antimicrobial agent and administration of oral vancomycin when specific antibacterial treatment is indicated.

Rev Infect Dis, 1984 Mar-Apr, 6 Suppl 1, S193 - 201
Breast feeding and toxigenic intestinal infections: missing links in crib death?
Arnon SS.
Infant botulism results when Clostridium botulinum spores germinate, colonize the gut, and there produce botulinal toxin, which after absorption causes flaccid muscle paralysis . The observed variation in the severity of the disease was linked to the infant's milk source, in that all sudden death cases indistinguishable from typical crib death occurred in infants who had been formula-fed, whereas the more gradual onset, hospitalized cases occurred in infants who were predominantly breast-fed . Secretory IgA antibody against C . botulinum vegetative cell antigens was found in human milk from mothers of both healthy infants and patients, a finding that further confirms the functioning of the "mucosal immune system" in humans . The hypothesis that some crib deaths might also result from other intestinally produced bacterial toxins was investigated by injecting infant rhesus monkeys with microgram amounts of purified Clostridium difficile toxins A and B; quiet death pathologically consistent with human crib death occurred within 4 hr to 10 hr . This and other evidence suggest that infant botulism may be the prototype of a putative class of heretofore unrecognized diseases, the "toxigenic intestinal infections of infancy." Collectively, these illnesses may account for a modest proportion of crib death, against which human milk may provide relative protection.

Arch Dis Child, 1984 Mar, 59(3), 270 - 2
Clostridial toxins in neonatal necrotising enterocolitis; Thomas DF et al.; Clostridium difficile cytopathic toxin was found in the faeces or gut content of five of 39 neonates with necrotising enterocolitis (NEC) . Toxin concentrations were uniformly low and did not differ from those found in healthy neonates . C difficile is unlikely to be involved in the pathogenesis of NEC . Stools from 33 babies with NEC were also tested for C perfringens alpha toxin, with negative results.

Biochem Biophys Res Commun, 1984 Feb 29, 119(1), 236 - 44
Activation of the cyclic nucleotide phosphodiesterase from rat heart cytosol by phospholipase C; Prigent AF et al.; Phospholipase C (clostridium perfringens) significantly increased the cyclic nucleotide phosphodiesterase activity of a crude 105 000 g supernatant from rat heart . This activation only concerned the basal activity of cyclic GMP phosphodiesterase determined with 0.25 microM cyclic GMP as substrate, in the presence of EGTA, whereas stimulation was found to be independent of EGTA when phosphodiesterase activity was measured with 0.25 microM cyclic AMP . Similar qualitative results were found for the three cytosolic forms of phosphodiesterase separated from rat heart supernatant by isoelectric focusing . Supplementary experiments provided evidence that the activation of the cyclic AMP-specific phosphodiesterase was attributable to Phospholipase C activity and not to contaminating protease(s) . In contrast, the stimulation of the cyclic GMP phosphodiesterase activity appeared to be largely dependent on the proteolytic activity of commercial Phospholipase C . Phosphatidic acid also significantly increased the cyclic AMP phosphodiesterase activity of the rat heart cytosol . These results suggest that the activation of cardiac cyclic AMP phosphodiesterase may be related to changes in phospholipid metabolism, notably the accumulation of phosphatidate, and relevant to physiological regulatory processes.

Biochem J, 1984 Feb 15, 218(1), 69 - 74
Stimulation of prostaglandin E2 synthesis by exogenous phospholipase A2 and C in rabbit kidney medulla slices; Fujimoto Y et al.; We have investigated the effects of phospholipase A2 and C on the synthesis of prostaglandin E2 in rabbit kidney medulla and the release of fatty acids from the medulla slices . Exogenous phospholipase A2 {from Naja naja (Indian cobra) venom} and phospholipase C (from Clostridium welchii) stimulated prostaglandin E2 production in a dose-dependent manner . At the maximal effective concentrations (0.5 unit of phospholipase A2/ml, 2 units of phospholipase C/ml), phospholipase C increased prostaglandin E2 formation to the level observed with phospholipase A2 . Phospholipase A2 enhanced the release only of unsaturated fatty acids, whereas phospholipase C stimulated the release of individual free fatty acids (C 16:0, C 18:0, C 18:1, C 18:2 and C 20:4) . Moreover, p-bromophenacyl bromide inhibited phospholipase A2-stimulated prostaglandin E2 production and the release of fatty acids, but it had no influence on prostaglandin E2 formation and the release of fatty acids increased by phospholipase C, indicating that the stimulatory effect of phospholipase C is not mediated through the activation of endogenous phospholipase A2 . These results suggest the presence of diacylglycerol lipase and monoacylglycerol lipase in the kidney and the importance of this pathway in prostaglandin synthesis by the kidney.

Nature, 1984 Feb 2-8, 307(5950), 457 - 60
Acceptors for botulinum neurotoxin reside on motor nerve terminals and mediate its internalization; Dolly JO et al.; Botulinum neurotoxin (BoNY) type A, a causative agent of botulism, is a di-chain protein (molecular weight 140,000) from Clostridium botulinum, and the most neurotoxic substance known . Some cases of sudden infant cot deaths have been attributed to such a neuroparalytic condition . BoNT inhibits irreversibly the release of acetylcholine from peripheral nerves in a highly selective manner . Hence, it is potentially an invaluable probe for studying the mechanism of transmitter release . Here we demonstrate specific labelling of murine motor nerve terminals with neurotoxic, 125I-labelled BoNT (type A) by autoradiography . We observed saturable, temperature-sensitive binding of BoNT to sites which reside solely on the nerve terminal membrane; these were distributed on all unmyelinated areas, at an average density of 150-500 per micron2 of membrane . The binding was mediated by the larger subunit of the toxin and was inhibited partially by tetanus toxin, another microbial protein . No specific binding was detectable on any other cell types examined, including noradrenergic terminals . Following binding, internalization of radioactivity was observed; this process was energy-dependent as it could be prevented totally by azide or dinitrophenol (DNP) . This direct demonstration of separable steps, including highly selective binding and acceptor-mediated internalization, is reconcilable with the unique potency and the multiphasic inhibitory action of BoNT on transmitter release, as shown electrophysiologically.

Aust Vet J, 1984 Feb, 61(2), 57 - 61
Osteomyelitis in dogs and cats caused by anaerobic bacteria; Johnson KA et al.; Localised osteomyelitis was diagnosed in 2 dogs and 2 cats . The disease was caused by fight wounds in 3 cases . Radiographic examination demonstrated a circumscribed zone of cortical bone lysis, sequestra and periosteal new bone . Each case was treated surgically by sequestrectomy and debridement . Infection was due mainly to anaerobic bacteria . The pathogenic bacteria isolated from the lesions of dogs were Actinomyces viscosus, Fusobacterium nucleatum and Bacteroides spp, and from the lesions in cats were Clostridium villosum , Peptostreptococcus anaerobius, Wolinella recta and Bacteroides gingivalis . As all the bacteria were sensitive to penicillin, each case was treated with penicillin and by irrigation of the wound . This resulted in resolution of the disease, within 4 weeks, in all cases.

J Gen Microbiol, 1984 Feb, 130 ( Pt 2), 309 - 18
Isoleucine synthesis by Clostridium sporogenes from propionate or alpha-methylbutyrate; Monticello DJ et al.; Preliminary studies demonstrated that Clostridium sporogenes synthesized isoleucine by a pathway not involving threonine or threonine dehydratase . Radiotracer experiments with cells grown in a defined carbohydrate-free medium showed that radioactivity from {U-14C}serine, {3-14C}pyruvate, {14C}NaHCO3 and {1-}, {2-} and {3-14C}propionate was incorporated into isoleucine . Conversely, there was no detectable incorporation of 14C into isoleucine during growth with {U-14C}glutamate, {U-14C}threonine, {U-14C}valine, {U-14C}leucine or {U-14C}methionine . Crude extracts of the bacteria grown in a minimal medium contained levels of alpha-acetohydroxyacid synthase activities comparable to those in Escherichia coli K12 grown in minimal medium . Stepwise degradation of isoleucine obtained from C . sporogenes grown in the presence of specifically-labelled precursors indicated that C . sporogenes can make isoleucine via the reductive carboxylation of propionate to yield alpha-oxobutyrate, which is metabolized to isoleucine in the classical fashion . Isoleucine was also formed by C . sporogenes via the reductive carboxylation of alpha-methylbutyrate to alpha-oxo-beta-methylvalerate.

Antimicrob Agents Chemother, 1984 Feb, 25(2), 162 - 4
Comparative in vitro activities of cefpiramide and apalcillin against anaerobic bacteria; Wexler H et al.; The in vitro activities of two new antimicrobial agents, apalcillin and cefpiramide (SM-1652), were evaluated against 324 strains of anaerobic bacteria . Apalcillin (a penicillin derivative) and cefpiramide (a semisynthetic cephalosporin) were compared with piperacillin, moxalactam, and cefoxitin . Organisms studied included the Bacteroides fragilis group, other Bacteroides species, fusobacteria, clostridia, nonsporeforming gram-positive rods, and anaerobic cocci . Piperacillin was found to be the most active overall, inhibiting 96% of the strains tested at its achievable level in serum (128 micrograms/ml) . Apalcillin was comparable in activity to piperacillin, inhibiting 93% of anaerobes tested at this concentration . The other antibiotics inhibited ca . 80% of the strains at 32 micrograms/ml . In terms of activities against particular species, apalcillin was active against 75% of B . fragilis group strains and 97 to 100% of all other anaerobes . Cefpiramide inhibited 37% of B . fragilis group strains at 32 micrograms/ml and 68% at 64 micrograms/ml (a level that may be achievable with this drug) . Cefpiramide inhibited 92% of all other anaerobes at 32 micrograms/ml and 95% at 64 micrograms/ml . The clostridia other than Clostridium perfringens were the most resistant (84% inhibited at 32 micrograms/ml and 95% inhibited at 64 micrograms/ml).

Eur J Clin Microbiol, 1984 Feb, 3(1), 10 - 3
Clostridium difficile colitis associated with the use of antineoplastic agents; Miller SD et al.; Eight patients are presented in whom treatment with antineoplastic agents, in particular the folic acid antagonist methotrexate, precipitated Clostridium difficile-related diarrhoea and pseudomembranous colitis . The clinical presentation of these patients was identical to that encountered in patients developing antibiotic associated diarrhoea and colitis . Clostridium difficile-related diarrhoea and colitis should be suspected in any patient developing diarrhoea during the course of anti-neoplastic chemotherapy or within three weeks of its cessation . This complication is effectively treated with oral vancomycin.

J Clin Microbiol, 1984 Feb, 19(2), 294 - 5
Detection of Clostridium difficile toxin with McCoy cell monolayers and cell suspensions and comparison with HeLa cell assay; Maniar AC et al.; McCoy cell monolayers were compared with HeLa cell monolayers for the detection of Clostridium difficile toxin in 301 stool samples . Tests were positive (greater than or equal to 1/100 dilution) in 83 and 81 specimens tested with McCoy and HeLa cell monolayers, respectively . McCoy cell suspensions were compared with HeLa cell monolayers in 532 stool filtrates . Overall, 90 positive specimens were within one dilution and 432 filtrates were negative with either test, giving a correlation coefficient of r = 0.98 . McCoy cell monolayers or suspensions may be a satisfactory substitute for the detection of C . difficile toxin in clinical specimens.

Am J Dis Child, 1984 Feb, 138(2), 183 - 5
Bacterial-induced RBC alterations complicating necrotizing enterocolitis; Novak RW; Enzymes released from bacteria can alter the surfaces of RBCs rendering them susceptible to destruction by antibodies present in a high percentage of adult plasmas . Such RBC alterations were observed in four of 20 consecutive cases of radiologically proved necrotizing enterocolitis . Involved infants were seriously ill, three of four demonstrating bowel perforation . Bacteria of the genus Clostridium were isolated from blood or peritoneal fluid in three of four affected patients and elaborated appropriate RBC-altering enzymes in vitro . Two patients who had received plasma-containing products experienced notable hemolysis . Patients treated with washed products and plasma protein fractions lacking immunoglobulins had no hemolytic problems . The alteration of the RBC membrane is easily detected by a rapid, simple lectin agglutination test.

J Clin Pathol, 1984 Feb, 37(2), 117 - 9
Detection of Clostridium difficile in faeces by direct gas liquid chromatography; Levett PN; Stool specimens examined for the presence of Clostridium difficile and its cytotoxin were screened by gas liquid chromatography for the presence of volatile fatty acids and p-cresol . Twenty seven of 110 (25%) stools yielded C difficile or cytotoxin; iso-valeric acid was detected in 63/110 (57%) and iso-caproic acid in 18/110 (16%) stools . Para-cresol was found in 24/71 (34%) stools examined . Iso-valeric acid was detected in 85% of stools positive for C difficile, whereas iso-caproic acid (41%) and p-cresol (52%) were found in much lower numbers of C difficile-positive stools . It is concluded that gas chromatographic detection of volatile fatty acids or p-cresol in faeces are not satisfactory screening tests for the presence of C difficile.

Infect Immun, 1984 Feb, 43(2), 487 - 90
Oral toxicities of Clostridium botulinum type A and B toxins from different strains; Ohishi I; The production and the oral toxicity for mice of Clostridium botulinum type A and B toxins of different strains were studied . All five type B strains produced both 16S (large or L) and 12S (medium or M) toxins, although the relative amounts varied with the strains . The culture supernatant of type B Okra strain was the most potent in oral toxicity . The L toxin of this culture was about 700 times more toxic in feeding tests with mice than the L toxin from type B strain NH-2, whereas the M toxins of the two strains had the same oral toxicity . These results indicate that the oral toxicity of type B toxin varies with the culture strain . Oral toxicities of L toxin produced by type A strains 62A and 97 were comparable but were 10 times higher than those of their M toxins . Hybrids of toxic and nontoxic components separated from L toxins of type B strains Okra and NH-2 revealed that the high oral toxicity of the B-L toxin of strain Okra is attributable not to the toxic but to the nontoxic component of the toxin . The present study suggests that the 16S molecular-sized toxin elaborated by a certain strain of C . botulinum type B is implicated in the high fatality rate in type B human botulism.

Eur J Biochem, 1984 Feb 1, 138(3), 481 - 9
Nuclear-magnetic-resonance investigation of 15N-labeled flavins, free and bound to Megasphaera elsdenii apoflavodoxin; Franken HD et al.; Flavin derivatives, enriched with 15N (approximately equal to 95%) at the four nitrogen atoms of the isoalloxazine ring, have been investigated in the oxidized and the two-electron reduced state by the 15N nuclear magnetic resonance technique . The measurements were conducted with aqueous and chloroform solutions of flavin . A comparison of the chemical shifts of the N(1) and N(5) atoms of oxidized flavin in the two solvents revealed that these atoms are sensitive indicators for possible hydrogen-bridge formation to these atoms . The N(5) atom of oxidized flavin resonates at low field and shifts about 300 ppm upfield upon reduction . A pKa of 6.8 was determined from pH-dependent 15N NMR measurements of the two-electron reduced flavin molecule . In addition it is also shown that reduced flavin in aqueous solution possesses a more coplanar structure than in chloroform solution . The 15N chemical shifts of flavin bound to Megasphaera elsdenii apoflavodoxin indicate that various hydrogen bridges are formed between the prosthetic group and the apoprotein . Especially the N(1) atom of the prosthetic group in the oxidized state seems to form a strong hydrogen bond with the apoprotein . In the reduced state the prosthetic group is bound in the anionic form and possesses an almost coplanar structure . These results are in agreement with published crystallographic data on the related flavodoxin from Clostridium MP . Where possible 15N-1H, 15N-15N and 13C-15N coupling constants were determined . Some of the coupling constants are useful parameters for the elucidation of the planarity of free and protein-bound flavin and for the evaluation of the interaction between flavin and apoprotein . Spin-lattice relaxation measurements show that the relaxation of the 15N(3)H group of flavin is predominantly determined by dipole-dipole interaction . The calculated rotational correlation times of flavin in two different solvents were determined and are in good agreement with published results.

Chemioterapia, 1984 Feb, 3(1), 41 - 4
In vitro susceptibility of Clostridium difficile isolates to 12 antimicrobial agents; Gianfrilli P et al.; The "in vitro" susceptibility of 48 strains of "Clostridium difficile" to 12 antimicrobial agents was determined by agar dilution method . All isolates were susceptible to ampicillin, metronidazole, piperacillin, vancomycin and N-formimidoylthienamycin, resistant or intermediate to the new cephalosporins: cefoxitin, cefotaxime and moxalactam . For clindamycin, the MIC distribution appeared to be bimodal with 41.7% of the strains susceptible to 8 micrograms/ml and 58.3% resistant to 128 micrograms/ml; 90% of these strains were also resistant to erythromycin, tetracycline and chloramphenicol . The percentage of resistant strains in the isolates from patients with pseudomembranous colitis is significantly higher than in the isolates from patients with antibiotic-associated colitis.

Infect Immun, 1984 Feb, 43(2), 612 - 6
Effect of antiflagellar serum in the protection of mice against Clostridium chauvoei; Tamura Y et al.; Specific antiflagellar serum of Clostridium chauvoei showed a powerful protective effect which prevented bacterial growth in the liver, but not in infected muscle, against intramuscular challenge with calcium chloride-activated spores in normal mice . No protective effect was observed in mice with polymorphonuclear leucocytes depleted by cyclophosphamide treatment . The antiflagellar serum had approximately the same protective effect in mice with macrophages blocked selectively by carrageenan as it did in normal mice . We suggest that the antiflagellar serum exerted its effect by opsonic function and that opsonized C . chauvoei was eliminated mainly by polymorphonuclear leucocytes rather than by macrophages.

Infect Immun, 1984 Feb, 43(2), 482 - 6
Sealed adult mice: new model for enterotoxin evaluation; Richardson SH et al.; Outbred, inbred, and congenic strains of conventional mice which were ano-rectally occluded with cyanoacrylate ester glue and converted to sealed adult mice (SAM) were given, per os, crude cholera enterotoxin (CT) in 10% NaHCO3 . At 6 h when the response was maximal, mice were killed, the small intestines were removed, and gut weight/body weight ratios were calculated . Experimental mice gave a linear response after receiving 1.5 to 60 micrograms of CT . Purified heat-stable enterotoxin from Escherichia coli and purified heat-labile enterotoxins from E . coli, Vibrio cholerae, and Clostridium difficile all elicited vigorous fluid outpouring as did culture filtrates from Vibrio fluvialis with cytotoxic activity . Active and passive immunization with crude CT completely or partially neutralized fluid secretion due to CT . Monospecific anti-CT incubated with CT before feeding also eliminated the response . Mice pretreated with penicillin, held in barrier cages, converted to SAM, and fed live vibrios, showed fluid responses similar to those seen with low doses of CT . Each of six different strains of inbred mice fed a half-maximal fluid accumulation response dose of CT gave fluid accumulation ratios which varied fourfold . There was no correlation of fluid accumulation with body weight, gut length, age, or sex . All poor responders were of H-2k haplotype and all good responders were H-2b . BALB congenic mice which differed only in H-2 haplotypes showed the same correlations, and body weights and gut lengths of all haplotypes were not significantly different.

J Gen Microbiol, 1984 Feb, 130 ( Pt 2), 267 - 73
The role of surface charge in ionic germination of Clostridium perfringens spores; Ando Y et al.; The surface charge against pH behaviour of spores of Clostridium perfringens type A was determined using a colloid titration method in the pH range 4-9 . The native spores as well as various ionic forms of the spores loaded with divalent cations were found to be negatively charged . A positive colloid, MGCh, inhibited the ionic germination of the spores and this inhibition was highly dependent on pH . However, another positive colloid, GCh, did not inhibit the germination . Chemical modification of carboxyl groups on the spore surface by use of a water-soluble carbodiimide with two nucleophiles, glycine ethyl ester and taurine, caused spores not only to have little or no colloidal charge but also to lose the ability to germinate . The modification of carboxyl groups in the spore coat protein also resulted in elimination of, or marked reduction in its negative charge . These results suggest that carboxyl groups in the spore coat protein are the major negatively charged species on the spore surface . The role of surface charge in the ionic germination of the spores is discussed.

J Infect Dis, 1984 Feb, 149(2), 215 - 9
Inactivation of Clostridium difficile cytotoxin by the neutrophil myeloperoxidase system; Ooi W et al.; The cytotoxin of Clostridium difficile was examined for sensitivity to oxidant secretory products of neutrophils . Exposure to myeloperoxidase, H2O2, and a halide resulted in loss of toxin activity measured by tissue-culture cytotoxicity . The peroxide requirement was provided by reagent H2O2, a peroxide-generating enzyme (glucose oxidase), or a peroxide-producing intestinal microorganism, Lactobacillus acidophilus . Human neutrophils stimulated by phorbol myristate acetate caused similar toxin inactivation . In both the cell-free and the neutrophil systems, inactivation of toxin required halides and was abrogated by azide, cyanide, or catalase . Neutrophils from patients with lack of myeloperoxidase or failure to produce H2O2 were impaired in toxin inactivation unless myeloperoxidase or H2O2, respectively, was added . The reducing agent 2-mercaptoethanol enhanced toxin activity . These data suggest a similarity between C difficile cytotoxin and the classic thiol-activated cytolysins . Moreover, they raise the possibility that neutrophils are involved in oxidative detoxification of microbial products.

Infect Immun, 1984 Feb, 43(2), 584 - 8
Analysis of antigenicity of Clostridium botulinum type C1 and D toxins by polyclonal and monoclonal antibodies; Oguma K et al.; Clostridium botulinum type C1 toxin was purified from C-Stockholm (C-ST), and D toxin was purified from D-1873 and D-South African . Polyclonal antibodies against these toxins were prepared in rabbits . Twenty-eight monoclonal antibodies to these toxins were also prepared with BALB/c myeloma cells . The antibodies were analyzed by both enzyme-linked immunosorbent assay (ELISA) and a toxin neutralization test . ELISA was performed with the three purified toxins and heavy-chain (Hc) and light-chain (Lc) components derived from C-ST and D-1873 toxins . A neutralization test was carried out with 11 toxin preparations (7 from type C and 4 from type D cultures) . ELISA results indicated that there exists at least one common antigenic determinant on each of the Hc and Lc components of the three purified toxins . The results of the neutralization test also indicated that type C1 and D toxin preparations contain several common antigenic sites in their molecules . Some are common to toxins from several specific cultures, whereas others are common to toxins from a large number of cultures . It was speculated that toxins from two type C strains are composed of Hc and Lc components which are somewhat similar to those of D-1873 and C-ST toxins, respectively.

FEBS Lett, 1984 Jan 23, 166(1), 39 - 43
Structural homologies between the amino acid sequence of Clostridium pasteurianum MoFe protein and the DNA sequences of nifD and K genes of phylogenetically diverse bacteria; Hase T et al.; The complete amino acid sequence of the larger (alpha-) subunit and about 70% of the total sequence of the smaller (beta-) subunit of the MoFe protein from Clostridium pasteurianum was determined by analyses of peptides derived from BrCN cleavage and by digestions with trypsin, staphylococcal protease and lysylendo-peptidase of the separated subunits . The alpha-subunit has 529 amino acid residues, giving an Mr value of 58 774 . This is the first complete sequence for the alpha-subunit of an isolated MoFe protein . In comparing the sequences of both subunits to those from other sources, 5 out of 9 cysteines in the alpha-subunit and 3 out of 6 in the beta-subunit are invariant, thus suggesting a function as ligands to FeS and MoFeS clusters in the MoFe protein . All of these cysteines are located in the amino terminal halves of both subunits.

Scand J Infect Dis, 1984, 16(2), 211 - 5
Relapsing Clostridium difficile enterocolitis cured by rectal infusion of normal faeces; Schwan A et al.; Repeated recurrence of Clostridium difficile-associated enterocolitis is uncommon but troublesome for the afflicted patient . The patient described here received vancomycin treatment several times but always had a relapse of C . difficile enterocolitis 2-3 weeks after discontinuation of treatment . She did not form serum antibodies to C . difficile cytotoxin (toxin B) . Rectal infusion of enemas prepared from fresh faeces resulted in final cure.

Scand J Infect Dis, 1984, 16(2), 207 - 9
Gas gangrene of the omental bursa following acute pancreatitis; Raahave D et al.; Acute pancreatitis with necrosis was followed by infection with Clostridium perfringens and formation of gas in the omental bursa . This could be seen preoperatively on an abdominal roentgenogram and was verified at operation . The patient recovered after debridement, drainage and irrigation and antimicrobial therapy.

Toxicon, 1984, 22(2), 312 - 5
Amino acid composition of Clostridium botulinum type B neurotoxin; DasGupta BR et al.; To characterize type B botulinum neurotoxin based on reliable data on the amino acid composition, three batches of the neurotoxin were analyzed . Each batch was isolated from a separate neurotoxin producing bacterial culture (strain Okra) . Two batches were purified by the same method and one was purified by a different method . The toxin preparations were comparable in purity (judged by polyacrylamide gel--sodium dodecyl sulfate electrophoresis) and similar in amino acid composition . The best estimate of the number of amino acid residues per toxin molecule (mol . wt 152,000) was: Asp212,Thr54,Ser83,Glu130,Pro46,Gly61++ +,Ala44,Val54,CyS11,Met23,Ile144,Leu107 , Tyr81,Phe77,Lys118,His7,Arg39,Trp18.

Zentralbl Chir, 1984, 109(6), 402 - 17
{Gas gangrene--still a diagnostic and therapeutic problem}; Nier H et al.; From 1970 to 1980 132 patients were admitted to our Dept . under the diagnosis of gas gangrene . In 54 cases there was no bacteriological evidence of clostridium perfringens . In all patients surgery was performed immediately, followed by hyperbaric oxygen therapy . The overall mortality rate among 78 patients with proven gas gangrene came up to 38%, the amputation after limb injuries to 55% . In our experience we can not state a clear cut advantage of hyperbaric oxygenation as far as the death rate is concerned.

J Gen Microbiol, 1984 Jan, 130 ( Pt 1), 173 - 83
Motility as a factor in bowel colonization by Roseburia cecicola, an obligately anaerobic bacterium from the mouse caecum; Stanton TB et al.; Roseburia cecicola strain GM is a motile obligate anaerobe that was isolated from mouse caecal mucosa . Twenty-five strains of motility mutants were obtained from populations of strain GM (wild-type) that had been exposed to UV light . Unlike GM cells, mutant bacteria were either non-motile and non-flagellated (Fla-) or migrated slowly or atypically in semi-solid medium . Strain GM and two mutant strains, SLS (Fla-) and WES (atypically motile), were used in mouse colonization experiments . In separate experiments, each strain colonized (4.8 X 10(9) to 1.5 X 10(10) c.f.u . per g caecum) the caecum of germfree mice inoculated intragastrically with pure cultures of the bacteria . In mice mono-associated with either mutant strain, bacteria which were non-motile or atypically motile predominated in their caeca (greater than 99% of total bacteria recovered) . In mice mono-associated with motile cells of strain GM, mutant strains which had lost wild-type motility became predominant in the caecal populations (97% of total bacteria recovered at 48 to 70 days after inoculation) . Mice mono-associated with either strain SLS or strain GM were colonized by one strain each of Escherichia coli, Candida pintolopesii, a Bacteroides sp., and a Clostridium sp . Most (99%) of the R . cecicola cells recovered from the caeca of these animals had typical wild-type motility . Motility, although not essential for R . cecicola to colonize germfree mice, is apparently advantageous to this bacterium when other micro-organisms are present with it in the mouse caecum . Motility may thus be essential for R . cecicola to colonize conventional laboratory mice.

Arch Geschwulstforsch, 1984, 54(1), 25 - 32
{The macrophage adherence inhibition test in Wistar rats bearing Jensen tumors . II . Macrophage adherence inhibition test after intravenous administration of spores of the CNRZ 528 strain of Clostridium butyricum and incubation with clostridial antigens}; Neumeister B et al.; The tumor-specificity of tumor-clostridia-phenomenon in the model of Jensen-sarcoma of the white rat was detected with the help of humoral and cell-mediated immune reactions of the host towards intravenous application of 5 X 10(8) spores of clostridia, which do not dissolve the tumor . Contrary to weak responses in Widal-agglutination the MAI showed characteristic values of inhibition of peritoneal-cell-adherence in tumor-bearing animals after incubation with soluble clostridia antigens.

Acta Paediatr Scand, 1984 Jan, 73(1), 86 - 91
Clostridium difficile in young children . Association with antibiotic usage; Vesikari T et al.; Clostridium difficile was isolated from the stools of 11/52 (21%) of children aged 0 to 2 years hospitalized with diarrhoea, and from 17/52 (33%) of a control group of hospitalized children with no diarrhoea; this difference was not significant . Direct demonstration of C . difficile toxin from the stools was positive in 1 case with diarrhoea and in 5 control cases . The children with positive stool culture for C . difficile had had significantly more treatments with antibiotics or chemotherapeutics than those with negative C . difficile culture (3.3 +/- 2.7 vs . 1.6 +/- 1.8, p less than 0.001), but there was no significant difference in the incidence of diarrhoea in the past . During a 4-6-month follow-up, C . difficile disappeared from the stools of 24 out of 28 initially culture-positive children; 3 of the 4 children with persistent C . difficile had received antibiotics during the follow-up period . We conclude that the presence of C . difficile is common in the stools of young children up to the age of 2 years, and that C . difficile is more frequently found in children who have received antimicrobial therapy . Most cases of C . difficile carriage state are symptomless at this age.

Acta Paediatr Scand, 1984 Jan, 73(1), 135 - 7
Pseudomembranous colitis with recurring diarrhoea and prolonged persistence of Clostridium difficile in a 10-year-old girl; Vesikari T et al.; A 10-year-old girl developed, after treatment with amyxocillin, a clinically and sigmoidoscopically apparent pseudomembranous colitis with positive Clostridium difficile stool culture . Treatment with vancomycin resulted in rapid clinical cure, but there was a relapse of diarrhoea and reappearance of C . difficile, with no pseudomembranous colitis, within one month . Clinical symptoms subsided spontaneously but C . difficile persisted for 2 more months in the stools . This case reflects three different aspects of C . difficile infection in one child: pseudomembranous colitis, ordinary diarrhoea, and symptomless carrier state.

Prikl Biokhim Mikrobiol, 1984 Jan-Feb, 20(1), 3 - 8
{Biosynthesis of corrinoids and other tetrapyrrole compounds by an acetogenic Clostridium}; Bykhovskii VIa et al.; Biosynthesis of corrinoids and other tetrapyrrole pigments by the pure culture of the acetogenic Clostridium 99 was studied . When growing on media containing glucose or methanol, the physiological and biochemical characteristics of Clostridium 99 are very close to those of C . thermoautotrophicum . Methanol was shown to stimulate the corrinoid accumulation with the yield increasing from 154 micrograms/g dry biomass (glucose medium) up to 2250 micrograms/g dry biomass (methanol medium) . According to the paper chromatography the corrinoid accumulated in Clostridium 99 cells differed both from vitamin B12 and Factor III . A study on the composition of extracellular tetrapyrroles, accumulated when the culture grows on the medium containing glucose and delta-aminolevulinic acid, revealed that they are represented both by uroporphyrin III and sirohydrochlorine-like pigments . The latters differ by a number of properties from sirohydrochlorine (corrifirine-2) of propione acidic bacteria . These pigments appear to be involved as intermediants in biosynthesis of corrinoids and other tetrapyrroles.

Appl Environ Microbiol, 1984 Jan, 47(1), 193 - 4
Effect of butanol on lipid composition and fluidity of Clostridium acetobutylicum ATCC 824; Vollherbst-Schneck K et al.; Butanol, at sub-growth-inhibitory levels, caused a ca . 20 to 30% increase in fluidity of lipid dispersions from Clostridium acetobutylicum . When grown in the presence of butanol or into stationary phase, C . acetobutylicum synthesized increased levels of saturated acyl chains at the expense of unsaturated chains.

J Pediatr, 1984 Jan, 104(1), 34 - 40
Rapid death of infant rhesus monkeys injected with Clostridium difficile toxins A and B: physiologic and pathologic basis; Arnon SS et al.; Clostridium botulinum can colonize and produce botulinal toxin in the human infant intestine, which the toxin then permeates to cause generalized flaccid paralysis, and occasionally, sudden death . This study was undertaken to test the hypothesis that toxins produced by other intestinal clostridia, e.g., C . difficile, might also cause systemic illness and sometimes death in infants (J Pediatr 100:568, 1982) . Because this hypothesis could not be evaluated clinically until the systemic manifestations of C . difficile toxins in primates were known, infant rhesus monkeys were given 6 to 11 micrograms/kg of the recently purified C . difficile toxins A or B, either intravenously or intraperitoneally . The animals showed no abnormalities for several hours, but then developed lethargy, hypotonia, hypothermia, and, shortly before death, sudden elevation of serum concentrations of potassium, magnesium, and phosphorus and of enzymes that derived mainly from skeletal muscle, heart and brain . Five of six animals died quietly 3.5 to 8.0 hours after onset of symptoms . Death appeared to result from cessation of breathing, after which the sinus tachycardia then deteriorated to a flat ECG . Necropsy findings were insufficient to explain the cause of death . It appears that in infant monkeys microgram amounts of C . difficile toxins A and B can produce a rapid quiet death, the cause of which is undetectable at necropsy, a situation pathologically reminiscent of crib death in human infants, although the possible clinical identity of these two conditions has yet to be established.

J Clin Microbiol, 1984 Jan, 19(1), 77 - 8
Incidence and origin of Clostridium difficile in neonates; Al-Jumaili IJ et al.; The stools of 65 of 92 (71%) infants in a special care nursery yielded Clostridium difficile on culture . Ninety percent of stools collected after 6 to 35 days in the unit were positive, and 36% of these also contained toxin . When tested in vitro, 94% of the isolates produced toxin . Of 110 swabs collected from the environment of the unit, 9% were positive for C . difficile, but the stools of 12 nurses working on the unit were negative . Thirty-five vaginal swabs collected from mothers just before delivery were negative for C . difficile on culture, but 16 of their infants had C . difficile in their stools . It was concluded that there is a high carriage rate in the stools of neonates of C . difficile acquired progressively during the course of their stay in the special care unit . Infection is mainly from environmental sources rather than maternal transmission.

J Bacteriol, 1984 Jan, 157(1), 1 - 6
Characterization of ferredoxin, flavodoxin, and rubredoxin from Clostridium formicoaceticum grown in media with high and low iron contents; Ragsdale SW et al.; Ferredoxin, flavodoxin, and rubredoxin were purified to homogeneity from Clostridium formicoaceticum and characterized . Variation of the iron concentration of the growth medium caused substantial changes in the concentrations of ferredoxin and flavodoxin but not of rubredoxin . The ferredoxin has a molecular weight of 6,000 and is a four iron-four sulfur protein with eight cysteine residues . The spectrum is similar to that of other ferredoxins . The molar extinction coefficients are 22.6 X 10(3) and 17.6 X 10(3) at 280 and 390 nm, respectively . From 100 g wet weight of cells grown with 3.6 microM iron and with 40 microM iron, 5 and 20 mg offerredoxin were isolated, respectively . The molecular weight of rubredoxin is 5,800 and it contains one iron and four cysteines . The UV-visible absorption spectrum is dissimilar to those of other rubredoxins in that the 373 nm absorption peak is quite symmetric, lacking the characteristic 350-nm shoulder found in other rubredoxins . The flavodoxin is a 14,500-molecular-weight protein which contains 1 mol of flavin mononucleotide per mol of protein . It forms a stable, blue semiquinone upon light irradiation in the presence of EDTA or during enzymatic reduction . When cells were grown in low-iron medium, flavodoxin constituted at least 2% of the soluble cell protein; however, it was not detected in extracts of cells grown in high-iron medium . The rubredoxin and ferredoxin expressed during growth in low-iron and high-iron media are identical as judged by iron, inorganic sulfide, and amino acid analysis, as well as light absorption spectroscopy.

Scand J Infect Dis, 1984, 16(2), 157 - 9
Fulminating clostridial septicemia in children treated for lymphoproliferative disorders; Bekassy AN et al.; Overwhelming Clostridium septicum infection in 2 children, 1 and 4 yr old, with acute lymphoblastic leukemia and B-cell non-Hodgkin malignant lymphoma, respectively, as well as fatal C . perfringens infection in a 3-yr-old child with histiocytosis-X are reported . A neutropenic patient with fever, abdominal symptoms and hypotension--but otherwise being well--must be suspected of having clostridial disease . The most alarming feature is shock and rapid course.

Scand J Infect Dis Suppl, 1984, 43, 62 - 6
Clindamycin as an anti-staphylococcal agent--indications and limitations; Hedstrom SA; In a general survey it is stated that for single Staphylococcus aureus infections, clindamycin is not considered to be a first-line drug . Its chief indication is penicillin allergy . Penetration and accumulation of clindamycin within leukocytes demonstrated in vitro may be of value in the treatment of S . aureus diseases resulting in large abscesses . An insidious risk of the development of Clostridium difficile diarrhoea limits the use of clindamycin in ambulatory long-term treatment of diabetic osteitis and chronic osteomyelitis . Such patients must therefore be carefully checked during clindamycin therapy . In staphylococcal endocarditis treated with clindamycin, relapses and development of resistance have been reported . Mixed staphylococcal and anaerobic infections in skin, subcutaneous tissue, the diabetic foot, bone and joints are primary indications for clindamycin . S . epidermidis infections, especially septicemia and endocarditis, are not suitable for clindamycin therapy due to a high rate of resistance.

Can J Biochem Cell Biol, 1984 Jan, 62(1), 60 - 71
Relationship between cell surface asparagine-linked glycoproteins and myoblast differentiation . Analysis of wheat germ agglutinin-resistant mutants; Gilfix BM et al.; Alterations in the oligosaccharides of complex surface glycoproteins have been studied in wild-type rat skeletal myoblasts (L6-9/1) and two wheat germ agglutinin resistant mutants, selected for either high level of resistance (WGARI) or low level of resistance (WGARII) . All three lines possessed high mannose oligosaccharides of the structure Man(n)GlcNAc, where n = 6, 7, or 8 . L6-9/1 had complex oligosaccharides of the type (+/- NeuNAc alpha 2-3Gal beta GlcNAc beta)n-Man3GlcNAc(+/- Fuc)GlcNAc where n = 3 or 4, in addition to small amounts of biantennary and higher order structures . WGARII, the mutant with low resistance to wheat germ agglutinin, possessed similar complex oligosaccharides except that they had undergone some base-sensitive modification which rendered them resistant to Clostridium perfringens sialidase . WGARI, mutant of high resistance to wheat germ agglutinin, possessed complex oligosaccharides of the type (GlcNAc beta)nMan3GlcNAc(+/- Fuc)GlcNAc, with n = 2, 3, or 4 . Since the WGARI mutants were unaltered in differentiation, it is concluded that the terminal sialic acid and galactose residues on the protein-bound oligosaccharides are not required for differentiation.

Arch Pathol Lab Med, 1984 Jan, 108(1), 82 - 3
Clostridium difficile peritonitis in a neonate . A case report; Genta VM et al.; We describe a case of fatal peritonitis due to Clostridium difficile in a neonate . Although the patient had several clinical features that were compatible with the diagnosis of neonatal necrotizing enterocolitis, examination of the bowel at laparotomy disclosed that a mesenteric band caused the patient's underlying disease . Postmortem histopathologic tests revealed gram-positive rods in the wall of the small intestine . Clostridium difficile was the only organism recovered from an antemortem culture of peritoneal fluid and was also recovered from a postmortem blood culture.

G Batteriol Virol Immunol, 1984 Jan-Jun, 77(1-6), 19 - 32
{Characterization of species of the genus Clostridium by gas chromatographic analysis of the fatty acids produced during metabolism}; Cresci A et al.; Volatile and non volatile fatty acids deriving from the bacterial metabolism of different sugars were determined by gas-chromatography to better characterize Clostridium tertium and Clostridium ramosum . The data obtained were also used for numerical taxonomic analysis and dendrograms were elaborated to study the taxonomic relationships between the two species.

Boll Ist Sieroter Milan, 1984, 63(6), 505 - 9
{In vitro activity of several cytostatic drugs against aerobic and anaerobic intestinal bacteria}; Vetere A et al.; The human normal intestinal flora prevents the colonization of exogenous bacteria, maintaining a constant microecology: this property is called "colonization resistance" . In leukemia patients antibiotics used for prevention and/or therapy of infectious episodes can alter the intestinal microecology, so that the gut can represent the trigger zone for generalized septicemia . Moreover cytotoxic drugs used in these patients can favour intestinal disturbances . In our study we evaluated the in vitro activity of three commonly used antineoplastic drugs (Daunorubicin, Cytosine arabinoside, Methotrexate) against aerobic and anaerobic intestinal bacteria and Clostridium difficile that is the aetiological agent of pseudomembranous colitis . Daunorubicin proved to be the most active inhibiting, in concentration ranging from 16 to 128 micrograms/ml, 50% of Bacteroides strains and 90% of Clostridium difficile and Enterococci strains tested . Methotrexate showed activity only against some Bacteroides strains, while Cytosine arabinoside had no activity at all . We conclude that in these patients the use of these drugs may represent another factor of risk altering the intestinal flora and so lowering the colonization resistance.

Microbiol Immunol, 1984, 28(12), 1325 - 32
Demonstration of protective antigen carried by flagella of Clostridium chauvoei; Tamura Y et al.; The protective antigen present on the flagella of Clostridium chauvoei was studied by the mouse protection test . A partially purified flagella preparation (PPF) showed protective antigenicity after two intraperitoneal injections of 2 micrograms as protein, while the protective antigenicity of nonflagellated mutants (NFM) was 100-fold less than that of the flagellated parent strain . Although the protective effect of antisera against the whole cells and PPF, in terms of ED50 values, was mostly lost after absorption with the parent strain, that of antisera after absorption with NFMs showed no appreciable loss . These results suggest that the flagella of Cl . chauvoei play some role in inducing protective immunity in mice.

Antonie Van Leeuwenhoek, 1984, 50(4), 355 - 60
A screen for Clostridium difficile in the vagina: an out-patient study using and comparing selective media; Thirkell D et al.; Cycloserine-Cefoxitin-Fructose Agar (CCFA) gives good presumptive identification of Clostridium difficile after 1- or 2-day incubation whereas Reinforced Clostridial Medium (RCM)/p-cresol is not very selective for the organism from the vagina . The identification of 91.5% of the isolates from an initial screen subjected to biochemically based tests was achieved . Conventional screening of vaginal swabs failed to confirm any significant occurrence of Cl . difficile in the vagina of pregnant or non-pregnant women . The incorporation of an enrichment stage in the isolation procedure, however, did reveal a significant presence of the organism in the vagina of both pregnant and non-pregnant women.

Chemotherapy, 1984, 30(6), 392 - 7
Antibiotic sensitivity testing of anaerobic bacteria by the breakpoint method; Hoffler U et al.; A shortened form of the agar dilution procedure (breakpoint method) was studied for susceptibility testing of 363 strains of anaerobic bacteria under routine conditions . Mezlocillin inhibited 99%, piperacillin 96%, cefoxitin 99%, latamoxef 90%, clindamycin 96% and metronidazole 100% of Bacteriodaceae strains tested . Peptococcaceae were susceptible to penicillins, cephalosporins and metronidazole . We did not find noticeable resistance of Clostridium perfringens strains to beta-lactam antibiotics or metronidazole, but to tetracycline and clindamycin . Propionibacteria were fully susceptible to beta-lactam antibiotics, tetracycline, erythromycin and clindamycin.

Arch Geschwulstforsch, 1984, 54(5), 369 - 76
{Modification of selected leukocyte functions in albino rats caused by the transplantable Jensen sarcoma and by therapeutic trials of cyclophosphamide and vinblastine, Clostridium butyricum M55 and short-term hyperglycemia}; Hambsch K et al.; A significant increase in leukocyte adhesion and agglomeration was observed in white rats with Jensen sarcoma . Glucose induced short-term hyperglycemia temporarily stimulated these leukocyte functions in healthy rats . This effect was not notably influenced by administration of tumorlyzing spores . In contrast administration of the cytostatic agents vinblastine and cyclophosphamide demonstrated dose-related inhibition of leukocyte agglomeration and adhesion.

Chemotherapy, 1984, 30(5), 331 - 6
Comparative efficacy of four antibiotics in anaerobic pulmonary infection . An experimental model in rabbits; Chandrasekar PH et al.; The efficacy of cefoxitin, mezlocillin, latamoxef and metronidazole in anaerobic lung infection was studied using a rabbit model . A mixture of Bacteroides fragilis, Peptococcus morbillorum, Eubacterium lentum and Fusobacterium nucleatum was inoculated transtracheally to produce infection within the lung . Mezlocillin was most effective, achieving bacteriologic cure in 5 out of 8 animals . With cefoxitin therapy, 4 out of 8 became bacteriologically sterile . Severe diarrhea with elevated titers of Clostridium difficile toxin was noted in most cefoxitin-treated animals . Latamoxef- and metronidazole-treated animals had apparently healed lesions, but cultures were positive in 6 and 7 out of 8 in each group, respectively . The commonest pathogen isolated in the last two groups was P . morbillorum . The therapeutic superiority of mezlocillin over metronidazole and latamoxef was statistically significant (p less than or equal to 0.05).

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