Microbiol Immunol, 1979, 23(6), 443 - 54 Isolation and partial characterization of exosporium from spores of a highly sporogenic mutant of Clostridium botulinum type A; Takumi K et al.; Homogeneous fragments of exosporium were isolated and purified from mature spores of a highly sporogenic mutant derived from Clostridium botulinum type A strain 190L . The exosporium was composed of three lamellae and showed a hexagonal array when negatively stained . The hexagonal array of isolated exosporium was resistant to sodium dodecyl sulfate, urea, dithiothreitol, and proteolytic enzymes such as trypsin, pronase, and nagarse, except for pepsin . The hexagonal array was partially disintegrated with 5 M guanidine-HCl and almost completely disrupted with 8 M urea in combination with 1% mercaptoethanol under alkaline conditions . The purified exosporium fraction was composed mainly of protein (69.1%) and lipids (13.8%) . A small amount of amino sugars (2.5%) was present, but neutral sugars could not be detected . The exosporium protein had a predominantly acidic amino acid composition accompanied by low levels of cystine, methionine, and histidine. Am J Clin Nutr, 1979 Jan, 32(1), 251 - 7 Diarrhea and colitis associated with antimicrobial therapy in man and animals; George WL et al.; Antimicrobial agent-induced ileocecitis of laboratory animals and colitis of man share common features . The significance of a newly described toxin in these two entities, the apparent source of the toxin (Clostridium difficile) and characteristics of the toxin are reviewed . Methods of toxin detection, isolation and rapid identification of C . difficile, and possible modes of therapy for antimicrobial agent-associated colitis of man are discussed. Am J Clin Nutr, 1979 Jan, 32(1), 113 - 8 Introduction to mechanisms of association of indigenous microbes; Savage DC; Indigenous microorganisms of numerous types associate with epithelial surfaces in the gastrointestinal tracts of mammals and birds . Some of the microbial types, e.g., Lactobacillus sp . in the stomachs of laboratory rodents, adhere to a surface without altering it ultrastructurely . In such cases, the adherence is mediated undoubtedly by macromolecules on the bacterial surfaces, possibly polysaccharides in most cases, interacting in specific ways with receptor macromolecules on the epithelial surface . Other microbial types that associate with epithelial surfaces without altering them ultrastructurally, e.g., Clostridium 109-2 in the mouse large bowel, may adhere to the surface only weakly or not at all, and maintain the association because they are motile and attracted to the epithelium by chemotactic substances . Microbial types that alter the ultrastructure of the epithelial cells to which they attach interact intimately with the membranes of the epithelial cells . In such cases, the microbes have specialized segments or ends for adhering to the membranes, and probably elaborate systems for stabilizing the membranes and cytoplasm at the site in the epithelial cell to which they attach . Some such organisms may have evolved unique reproductive mechanisms to maintain their populations on the epithelial surface. Rev Infect Dis, 1979 Jan-Feb, 1(1), 218 - 23 In vitro activity of cefoxitin and parenterally administered cephalosporins against anaerobic bacteria; Sutter VL et al.; The in vitro activity of cefoxitin against 221 recent isolates of anaerobic bacteria was compared with that of cefamandole and cefuroxime by the agar dilution technique . At achievable serum levels of 32 micrograms/ml, all three drugs were active against most groups of anaerobic bacteria tested . Cefoxitin was active against most strains of the Bacteroides fragilis group, whereas cefamandole and cefuroxime were relatively ineffective . Cefoxitin was least active against Clostridium species other than Clostridium perfringens, whereas cefamandole was very active and cefuroxime moderately active . A review of recent in vitro studies with cefoxitin and parenterally administered cephalosporins indicates that cefoxitin is the most active compound against the B . fragilis group and is least active against Clostridium species other than C . perfringens. Arch Exp Veterinarmed, 1979, 33(4), 621 - 37 {Studies of necrotizing enteritis of suckling piglets (Clostridium perfringens type C enterotoxemia) in industrialized sow breeding units . 5 . Control of the disease}; Kohler B et al.; Recent methods used and experience obtained in the control of necrotising enteritis are reported in this paper, with reference being made to both the pathogenesis and epizootiology of the disease . Two inoculations of the sows, using "Enterotoxamievakzine Dessau bivalent" five and three weeks before parturition, have worked well for prophylaxis . Oral treatment was applied to nursed piglets, using 40,000 I.U . of "Aviapen" and "V-Tablopen" penicillin per animal and day over periods between two and four days, helped to minimise piglet loss, particularly in the period between a fresh outbreak and full effectiveness of immunoprophylactic action . Such treatment was conducted metaphylactically and therapeutically . The first metaphylactic treatment was given within 24 hours from parturition . Combination of mother animal vaccination with the above therapeutic use of those two penicillin preparations worked extremely well in enzootically contaminated stocks and proved to be the most effective approach, for the time being, to controlling necrotising enteritis of nursed piglets . Yet, all those control measures failed to bring about full stock sanitation on industrialised units . Sow trading was not permitted until at least four weeks had elapsed from full effectiveness of mother animal vaccination, with the view to reducing the proliferation of Clostridium perfringens Type C via sales of breeding animals . All sows were given two "Enterotoxamievakzine Dessau bivalent" vaccinations, prior to sale . The animals were sold only to smaller farms (less than 500 sows for breeding) with concentional keeping patterns which were kept under constant diagnostic supervision . Neomycin, oxytetracycline, chloramphenicol, and other antibiotics against which Clostridium perfringens was resistant or in a position to assume resistance were used on endangered stocks only in conjunction with penicillin or not at all . This programme of control has proved to be efficient through a period of more than three years. Arch Exp Veterinarmed, 1979, 33(4), 595 - 619 {Studies of necrotizing enteritis of suckling piglets (Clostridium perfringens type C enterotoxemia) in industrialized sow breeding units . 4 . Epizootiology}; Kohler B et al.; Necrotising enteritis had been the cause of death of 4.9 per cent in 5,177 nursed piglets, which was established by pathological examination . The number of piglets, in that context, which had come from industrialised sow breeding units was equivalent to 92 per cent . The nursed piglet held the third position, next to smaller ruminants (19.4 per cent) and fowl (6.0 per cent), with regard to the occurrence of Clostridium perfringens enterotoxemia or necrotising enteritis in 112,218 animals which were pathologically examined after death . Necrotising enteritis so far has been rare in the GDR . No regional accumulation has been observed . Several outbreaks on industrialised sow breeding units actually remained stationary . The occurrence of the disease may be favoured by a number of factors which are conducive to accumulation of Clostridium perfringens Type C in a given stock . Group keeping of pregnant sows, simultaneous farrowing of larger groups of sows, group treatment of nursed piglets, using neomycin, chloramphenicol, oxytetracycline, and other antibiotics to which Clostridium perfringens is primarily resistant or has acquired resistance in the course of time are some of those contributive factors . Transmission of Clostridium perfringens Type C through feedstuff is possible, though it would lead to a real outbreak only by high intensity of the contamination, and it played a minor role in proliferation of the disease . 3479 Clostridium perfringens strains were isolated from 9,481 animals, both clinically intact and after death, with 30 species being included . Type classification revealed 2454 strains of Type A (70 per cent), 204 of Type D (5.88 per cent), 164 of Type C (four per cent), and 48 of Type B (1.34 per cent) . There were 688 atoxic strains (17 per cent) . Swine is the major carrier of Clostridium perfringens Type C, with 87 per cent of all Clostridium perfringens Type C strains having been isolated from swine . Swine was followed by fowl (four per cent), sheep (four per cent), cattle, rabbit, and dog (1.27 per cent each) . Clostridium perfringens Type C was obtained from the faeces of clinically intact sows in seven instances, including two cases with sows (0.46 per cent) from farms with no previous record of necrotising enteritis. Microbios, 1979, 25(100), 85 - 91 Sensitivity of chemically treated spores of Clostridium perfringens type A to an initiation protein; Franceschini TJ et al.; Extraction of Clostridium perfringens type A spores with dithiothreitol (DTT), DTT plus sodium dodecyl sulphate (DTT-SDS), urea-mercaptoethanol (UME), or alkali, solubilized from 18.6 to 46.5 of the total dry weight of spores . The initiation of germination and lysis of such treated spores with lysozyme and an initiation protein (IP) from the culture supernatant fluid of sporulating cells of C . perfringens was studied under various conditions . The ability of lysozyme and the crude IP to induce germination and lysis of extracted spores was concentration dependent up to 0.5 microgram/ml and 5.6 mg/ml respectively . IP showed an optimum of activity between pH 7 and 8 for DTT-SDS and DTT extracted spores, and between pH 6 and 9 for UME extracted spores . The optimum temperature of activity for IP was 55 degrees C . Dissimilarities in the extent to which lysozyme and the IP initiated germination and lysis of spores extracted by various methods may have been a reflection of the differences in amounts of protein solubilized by each treatment. J Biol Stand, 1979, 7(4), 373 - 81 Development, preparation and safety testing of a Clostridium welchii type C toxoid . II . Laboratory evaluation of potency; Knight PA et al.; The results obtained with four laboratory tests on four candidate formulations of Clostridium welchii type C vaccine for use in man have been compared with clinical responses to the same vaccines . Quantal response assays in mice appeared to reflect the ranking of the four vaccines in human subjects better than did the guinea pig tests . They also enabled the potency of the vaccine preparations to be related to an existing International Reference Preparation . Mouse assays in which the animals received two spaced doses of vaccine prior to challenge yielded marginally more satisfactory results in terms of precision and reflection of human responses than did assays involving a single dose of vaccine. J Biol Stand, 1979, 7(4), 315 - 23 Development, preparation and safety testing of a Clostridium welchii type C toxoid . I: preliminary observations in man in Papua New Guinea; Walker PD et al.; The reactogenicity and immunogenicity of various strengths of plain and adsorbed Cl . welchii type C toxoid have been evaluated in laboratory tests and in man in Papua New Guinea . The greater antigenicity and acceptable clinical reactivity of a vaccine containing 50 total combining power units per 0.5 mm of adsorbed toxoid resulted in its selection for further field studies. Arch Exp Veterinarmed, 1979, 33(2), 313 - 33 {Studies of necrotizing enteritis of suckling piglets (Cl . perfringens typc C enterotoxemia) in industrialized sow breeding units . 3 . Experimental reproduction of the disease}; Kohler B et al.; Experimental reproduction of necrotising enteritis of sucking pigs was successfully achieved by using both Clostridium perfringens Type C strains, which had been isolated from sucking pigs with necrotising enteritis, and Type C strain 3628 of A.T.C.C . (sub-type C1) . The lethal dose for sucking pigs was between 20 X 10(6) and 12 X 10(7) pathogens per animal . The disease could not even be induced by repeated application of no-bacterial toxin of Cl . perfringens Type C nor by administration of Cl . perfringens Type A strains which had been cultured from broilers with necrotising enteritis . Necrotising enteritis was found to develop in two phases in sucking pigs . First, the pathogen will deposit to the villous epithelium and then penetrate the superficial strata of the mocous membrane . In the second phase, the villous structure will be destroyed by the lethal, haemolysing, and necrotising toxins of Cl . perfringens . The role played by individual toxin fractions is discussed together with the importance of humoral and localised infection defence . Sucking pigs may be sufficiently protected against infection based on single or ten-fold lethal infectious dosage by two vaccinations of the mother animal, five and three weeks prior to parturition, using "Enterotoxaemia Vaccine Dessau bivalent" . Infection then would not occur unless a hundredfold lethal dose was applied . Characteristics include diarrhoea, apathy, exhaustion, and death. Microbiol Immunol, 1979, 23(4), 213 - 21 Pathogenesis of Hobbs' heat-sensitive spore forming Clostridium perfringens type A strain; Chakrabarty AK et al.; Food poisoning in man due to heat-sensitive strains of Cl . perfringens type A appeared to be mediated through enterotoxin synthesized in vivo during sporulation . A minimum of 2.0 X 10(5) vegetative cells suspended in sporulation medium was sufficient to elicit gut-loop response in rabbits . The functional disturbance in the gut as well as the structural changes were progressive and proportional to the size of the inoculum up to a dose limit of 2.0 X 10(7) vegetative cells and beyond this the changes remained steady. Can J Comp Med, 1979 Jan, 43(1), 98 - 101 Identification of enterotoxigenic Clostridium perfringens type A in mixed cultures; Niilo L; Three known enterotoxigenic Clostridium perfringens type A strains were mixed in various combinations with three nonenterotoxigenic strains and three lots of animal intestinal contents . They were grown as mixed cultures and tested for the presence of enterotoxin by the fluorescent antibody, reversed passive hemagglutination and immunodiffusion techniques . The fluorescent antibody and reversed passive hemagglutination tests detected enterotoxin in all 16 cultures prepared but the immunodiffusion test failed on two cultures . Attempts to reisolate the enterotoxigenic strains from the mixed cultures was successful in 12 cases when two of five isolated colonies were selected. Zh Mikrobiol Epidemiol Immunobiol, 1979 Jan, (1), 43 - 6 {Electron microscopic study of several features of spore formation in type B Clostridium perfringens}; Shakhbanov AA; The authors carried out electron microscopy of the thin sections of Cl . perfringens, type B (strain No . 89) . Material of middle electron density was revealed on the cell wall surface from the first hours of the culture growing; the cytoplasm displayed both rod-like incorporations with transverse striations, and phage particles . Different spore formation disturbances were revealed in the strain under study . In the majority of cells spore formation was blocked at the III--V stage . Besides, there were pseudospores, whereas mature spores were rarely encountered, and even those which did occur, were at the stage of growing. Appl Environ Microbiol, 1979 Jan, 37(1), 55 - 66 Membrane filter enumeration method for Clostridium perfringens; Bisson JW et al.; A membrane filter procedure has been developed for the rapid quantitation of C . perfringens in the aquatic environment . Background growth is inhibited by the use of D-cycloserine, polymyxin B sulfate, and incubation at 45 degrees C . Differential characteristics include the fermentation of sucrose, production of acid phosphatase, and the absence of beta-D-glucosidase activity . The medium is prepared as follows (in grams per 100 ml of distilled water): tryptose, 3.0; yeast extract, 2.0; sucrose, 0.5; L-cysteine, 0.1; MgSO4 . 7H2O, 0.01; bromocresol purple, 0.004; and agar, 1.5 . The ingredients are dissolved, and the pH is adjusted to 7.6 . After autoclaving at 121 degrees C for 15 min, the medium is allowed to cool at 50 degrees C, and the following are added per 100 ml: D-cycloserine, 40 mg; polymyxin B sulfate, 2.5 mg; indoxyl-beta-D-glucoside, 60 mg; 2.0 ml of a filter-sterilized 0.5% phenolpthalein diphosphate solution; and 0.2 ml of a filter-sterilized 4.5% FeCl3.6H2O solution . Enumeration of C . perfringens in a water sample is completed within 18 to 24 h . The verification of typical colonies was 93% . The average recovery from peptone-water spore suspensions of five strains was 79%, and that from filter-sterilized seawater suspensions was 90% . The precision of the method was approximately equal to that expected from random error alone . Confirmed recoveries of C . perfringens from water and sewage samples generally were greater than those by the Bonde pour tube method. Am J Clin Nutr, 1979 Jan, 32(1), 210 - 8 The molecular mode of action of Clostridium perfringens enterotoxin; McDonel JL; While certain strains of Clostridium perfringens have been associated with food poisoning outbreaks for the past 30 years, it has been only during the past 10 years that progress has been made in describing the disease process . And only within the past 5 years has meaningful progress been made in understanding the mechanism by which the disease is caused . Early observations, that the protein enterotoxin can cause erythema, increase capillary permeability, and exhibit parasympathomimetic properties, have been greatly added to in more recent studies . It is now believed tht the enterotoxin can alter intestinal transport of fluid, ions, and glucose, cause tissue damage in the gut and inhibit metabolic processes in intestinal tissue . Furthermore, the enterotoxin is thought to act very quickly (in a matter of minutes, compared to hours for other known enteropathogenic factors) and to affect basic function (macromolecular synthesis) and structure (membrane damage to microvillus brush borders) of individual cells . These findings have opened up many new questions that hopefully, when answered, will further the understanding of how this enterotoxin acts, as well as other enterotoxins being studied today. Microbios, 1979, 24(97-98), 151 - 7 The formation of serine from glycine and formaldehyde by cell free extracts of Clostridium acidi-urici; Hougland AE et al.; Cells of Clostridium acidi-urici which were grown in a medium containing uric acid were harvested, disrupted by sonication and centrifuged . After centrifugation the supernatant which served as the cell free extract was used to study the synthesis of serine from 2-14C glycine and formaldehyde . Serine was isolated from the reaction mixture by column chromatography . After identification by paper chromatography, serine was degraded carbon by carbon to locate the position of the labelled carbon . Radioactivity was confined almost exclusively to the alpha carbon of serine which was derived from the alpha carbon of glycine . Formaldehyde, therefore, binds at the alpha carbon of glycine to form serine . Conversion of serine to pyruvate was prevented by adding EDTA to the reaction mixture. Infect Immun, 1979 Jan, 23(1), 168 - 74 Microbial interference and colonization of the murine gastrointestinal tract by Listeria monocytogenes; Zachar Z et al.; Two strains of Listeria monocytogenes, one that formed smooth colonies on agar surfaces and a varient of it that formed rough colonies, colonized the gastrointestinal tracts of germfree mice . Within 24 h after mice were inoculated orally with about 100 bacteria, the population levels per gram (wet weight) of tissue of both strains were 10(5) to 10(7) in the stomach and ileum and 10(8) to 10(9) in the cecum and colon, respectively . As detected in Gram-stained histological sections, in such gnotobiotes, the bacteria colonized the lumen in all areas of the tract and much of the mucus layer on the epithelial surface in the proximal colon . The strain that formed smooth colonies did not colonize the tracts of specific-pathogen-free mice, but did colonize, to the same levels as in germfree mice, the stomachs and bowels of ex-germfree mice previously associated with two members of the indigenous flora (Bacteroides and Clostridium) . In the latter animals, however, the listeria did not form layers on the colonic epithelium as efficiently as they did in monoassociated gnotobiotes. J Wildl Dis, 1979 Jan, 15(1), 25 - 31 Techniques for evaluating humoral and cell-mediated immunity in mule deer fawns (Odocoileus hemionus); Trindle BD et al.; Twenty mule deer fawns (Odocoileus hemionus) were removed from their dams 48 h after birth, and hand-reared . Methods for monitoring their immune capability are described . Passive humoral immunity was determined by serum protein electrophoresis . Active humoral immunity following Clostridium toxoid vaccination was determined by immunodiffusion . Cell-mediated immunity was assayed using contact sensitization to 1-nitro, 2,4-dichlorobenzene (DNCB). Zentralbl Bakteriol {Orig A}, 1979, 245(3), 332 - 44 {Studies of the purification of the exotoxin of Bacillus cereus (author's transl)}; Katsaras K et al.; The exotoxins of Bacillus cereus show haemolytic, lethal, Phospholipase-C- and enterotoxigenic activities . The enterotoxigenic activity is regarded to be the factor which causes food poisoning in man . Efforts to purify the B . cereus exotoxins by precipitation with ammonium sulphate and subsequent chromatography on Sephadex-G-75 and Biogel-P-60 columns were partially successfull . Haemolysin and Phospholipase-C could be separated by gel-chromatography, they demonstrated partial identity on gel-diffusion agar . The lethal and enterotoxigenic activities could not be separated from the other toxins and remained heterogenous . No immunological relationship was found between Clostridium Type A alpha toxin and enterotoxin and the exotoxins of B . cereus. J Hyg Epidemiol Microbiol Immunol, 1979, 23(3), 266 - 72 Clostridium perfringens type A: certain characters of epidemiologic significance; Chakrabarty AN et al.; Survival of spores of Cl . perfringens type A was significantly greater than that of vegetative cells in acid pH (pH 1.2) . Survival of spores in soil varied from strain to strain . Time required for 5 million spores to be reduced to 500 per gram of soil varied from 2 to 8 months with an average of 4.5+/-2.3 months . Quantitative and qualitative heat resistance studies revealed that a majority of the Indian and all the American strains tested were heat-sensitive . These characters of Cl . perfringens have an important bearing on the epidemiology of food poisoning due to this agent. Microbiol Immunol, 1979, 23(5), 313 - 8 Effect of carbohydrates and control of culture pH on beta toxin production by Clostridium perfringens type C; Sakurai J et al.; Clostridium perfringens type C strain CN 5384 produced a higher level of beta toxin in a controlled pH medium containing 1% glucose, starch, or sucrose than in media with dextrin, fructose, or raffinose . Toxin synthesis was not related to the growth yield . The effect of glucose on beta toxin production by 11 strains was investigated with and without control of the culture pH at 7.5 . Strain CN 5386 produced distinctly higher toxin when the pH of the culture was maintained at 7.5, compared with uncontrolled pH. Biochim Biophys Acta, 1978 Dec 20, 537(2), 185 - 207 Nitrogenase X: Mössbauer and EPR studies on reversibly oxidized MoFe protein from Azotobacter vinelandii OP . Nature of the iron centers; Zimmermann R et al.; Under anaerobic conditions the molybdenum-iron protein (MoFe protein) from Azotobacter vinelandii can be reversibly oxidized with thionine . Electron paramagnetic resonance studies reveal that the oxidation proceeds in two distinct phases: the MoFe protein can be oxidized by four electrons without loss of the EPR signal from the S = 3/2 cofactor centers . A second oxidation step, involving two electrons, leads to the disappearance of the cofactor EPR signal . In order to correlate the events during the thionine titration with redox reactions involving individual iron centers we have studied the MoFe proteins from A vinelandii and Clostridium pasteurianum with Mossbauer spectroscopy . Spectra were taken in the temperature range from 1.5 K to 200 K in applied magnetic fields of up to 54 kG . Analysis of the Mossbauer data allows us to draw three major conclusions: (1) the holoprotein contains 30 +/- 2 iron atoms . (2) Most probably, 12 iron atoms belong to two, apparently identical, iron clusters (labeled M) which we have shown previously to be structural components of the iron and molybdenum containing cofactor of nitrogenase . The M-centers can be stabilized in three distinct oxidation states, MOXe- in equilibrium MNe- in equilibrium MR . The diamagnetic (S = 0) state MOX is attained by oxidation of the native state MN with either thionine or oxygen . MR is observed under nitrogen fixing conditions . (3) The data strongly suggest that 16 iron atoms are associated with four iron centers which we propose to call P-clusters . Each P-cluster contains four spin-coupled iron atoms . In the native protein the P-clusters are in the diamagnetic state PN, yielding the Mossbauer signature which we have labeled previously 'components D and Fe2+' . Three irons of the D-type and one iron of the Fe2+-type appear to comprise a P-cluster . A one-electron oxidation yields the paramagnetic state POX . Although the state POX is characterized by half-integral electronic spin a peculiar combination of zero-field splitting parameters and spin relaxation renders this state EPR-silent . Spectroscopically, the P-clusters are novel structures; there is, however, evidence that they are closely related to familiar 4Fe-4S centers. Biochim Biophys Acta, 1978 Dec 20, 537(2), 501 - 6 Isoelectric focusing of ferredoxins, flavordoxins and a rubredoxin; Dutton JE et al.; Isoelectric points of ferredoxins, flavodoxins and a rubredoxin from a range of sources were measured by electrofocusing over the pH range between 2.5 and 5.0 on thin layers of polyacrylamide gel . The pH gradient along the gel was measured directly by a surface electrode . The isoelectric points of the plant-type ferredoxins were between approx . 3.15 and 3.35, and those of the flavodoxins close to 3.5 . Ferredoxin, rubredoxin and flavodoxin from Clostridium pasteurianum had isolectric points of the of 2.75, 2.9, and 3.1, respectively . The values for the isoelectric points ferredoxins are significantly lower than previous results in the literature suggest. Tijdschr Diergeneeskd, 1978 Dec 15, 103(24), 1327 - 33 {Incidence of Clostridium botulinum in the rumen contents and faeces of cattle fed brewers' grains naturally contaminated with Clostridium botulinum (author's transl)}; Notermans S et al.; The number of Clostridium botulinum type B organisms excreted by cattle fed brewers' grains in which these organisms were found to be present and the period for which they were excreted, were studied . Large numbers (10(5) - 10(7) per gramme) of these organisms were detected in the rumen contents and faeces of the animals . When feeding brewers' grains was discontinued, Cl . botulinum type B was still detectable in the faeces for a considerable period (greater than eight weeks) . There was evidence to suggest that the number of Cl . botulinum organisms multiplies in the gastrointestinal tract of cattle. J Chromatogr Sci, 1978 Dec 10, 16(12), 623 - 9 Botulism: a pyrolysis-gas-liquid chromatographic study; Reiner E et al.; Forty samples of dried Clostridia bacteria were subjected to pyrolysis-gas-liquid chromatography (PGLC) . Examination of the key fingerprint peaks enabled the analyst to differentiate the samples into their respective antigenic groups . Peaks occurring at the high boiling end of profile could be used to distinguish proteolytic from non-proteolytic strains of C-botulinum . PGLC has proven to be a highly reproducible as well as a rapid specific method for differentiating and identifying samples of Clostridium botulinum. Biochim Biophys Acta, 1978 Dec 8, 527(2), 359 - 69 Nitrogenase: properties of the catalytically inactive complex between the Azotobacter vinelandii MoFe protein and the Clostridium pasteurianum Fe protein; Emerich DW et al.; The catalytically inactive complex generated by the combination of the Azotobacter vinelandii MoFe protein (Av1) and the Clostridium pasteurianum Fe protein (Cp2) inhibits N2 reduction, C2H3 reduction, H+ reduction and ATP hydrolysis catalyzed by the homologous nitrogenases . Kinetic data indicate that the inactive complex consists of two molecules of Cp2 to one molecule of Av1, with values for the inhibitor constant in the range of 1--10 nM . Inhibition of C . pasteurianum nitrogenase by Av1 produces a lag phase in acetylene reduction that increases with increasing Av1 . The lag phase is found only at levels of Av1 sufficient to keep the ratio of Cp2 : Cp1 lower than 2 . Gel filtration of a mixture of Av1 and Cp2 provides evidence for complex formation and indicates that each Av1 molecule binds more than one Cp2 molecule . The Av1-Cp2 complex binds two molecules of MgATP per molecule of Cp2 . MgATP is not required for complex formation, but complex formation lowers the MgATP-Cp2 dissociation constant approx . 3-fold . Av1 protects the iron-sulfur center in Cp2 completely against the MgATP-induced reaction with chelators . This provides additional evidence for formation of the Av1-Cp2 complex and together with the results of the MgATP-binding studies suggests that the two binding sites for MgATP are some distance away from the iron-sulfur site on Cp2. Eur J Biochem, 1978 Dec, 92(2), 449 - 54 FAD is covalently attached to peptidyl-tRNA during cell-free synthesis of 6-hydroxy-D-nicotine oxidase; Hamm HH et al.; The process, by which FAD is attached covalently to the 6-hydroxy-D-nicotine oxidase apoprotein in D-nicotine-induced cells of Arthrobacter oxidans was studied in vitro . {3H}Adenine-labelled FAD prepared biosynthetically in Clostridium kluyveri was incorporated into the 6-hydroxy-D-nicotine oxidase molecule during cell-free translation . FAD rather than FMN or riboflavin was thus shown to be the flavin derivative transferred to the polypeptide chain . After short-term protein synthesis on ribosomes from induced A . oxidans cells in the presence of an Escherichia coli MRE 600 supernatant fraction and {adenine-2-3H}FAD, THE PEPTIDYL-TRNA fraction was separated from completed polypeptides . Labelled FAD was found to be covalently attached to the tRNA-bound polypeptides . Cleavage of the tRNA-peptide bond released labelled polypeptides the largest of which migrated as authentic 6-hydroxy-D-nicotine oxidase during dodecylsulfate/polyacrylamide gel electrophoresis . These results strongly suggest that FAD is incorporated into the nascent polypeptide chains of 6-hydroxy-D-nicotine oxidase during ribosomal translation. J Med Chem, 1978 Dec, 21(12), 1301 - 7 Synthesis and quantitative structure--activity relationships of some antibacterial 3-formylrifamycin SV N-(4-substituted phenyl)piperazinoacethydrazones; Kiritsy JA et al.; A series of 14 3-formylrifamycin SV N-(4-substituted phenyl)piperazinoacethydrazones has been synthesized and evaluated for their antimicrobial activity . The compounds were found active against Bacillus subtilis, Staphylococcus aureus, Mycobacterium phlei, and Mycobacterium tuberculosis but not as active as rifampin . The compounds also exhibited significant activity against Clostridium perfringens and in this bacterial system some were more active than rifampin . The QSAR showed that the activity against B . subtilis depended only on lipophilicity, and the regression equation was linear . A parabolic relationship between the antibacterial activity and lipophilicity of the compounds was found in Staph . aureus . Additionally, the activity was dependent upon the electronic and steric effects of the phenyl substituents . The sensitivity of M . phlei to the compounds was found to correlate well with a linear combination of hydrophobic, electronic, and steric parameters . No statistically significant correlation was possible between the physicochemical parameters studied and the activity of the compounds against C . perfringens and M . tuberculosis. Can J Biochem, 1978 Dec, 56(12), 1141 - 8 Bile acids of a 3200-year-old Egyptian mummy; Kuksis A et al.; The bile acids of the gall bladder and hepatic tissue of a 3200-year-old Egyptian mummy were isolated by thin-layer chromatography and identified by combined gas-liquid chromatrography and mass spectrometry . Except for complete deconjugation and extensive dehydration, the bile acids were found to correspond in their qualitative and quantitative composition to the gall bladder bile acids of modern man . The secondary bile acids constituted about 50% of the total and were identified as the normal bacterial oxidoreduction products of the primary bile acids and their dehydration products . In addition a series of unsaturated bile acids were identified, which corresponded to the dehydration products of cholic and chenodeoxycholic acids . It is suggested that both bile acid deconjugation and the limited oxidoreduction were probably brought about by the Clostridium organisms identified in the tissue . On the basis of the bile acid composition it is concluded that the ancient man metabolized cholesterol along the same pathways as modern man. Can J Microbiol, 1978 Dec, 24(12), 1602 - 6 Endotoxin in germfree, gnotobiotic, or conventional-flora Sprague-Dawley rats; McLeod JC et al.; The Limulus assay for bacterial endotoxin was performed on serum and (or) plasma from animals monoassociated with Clostridium species, Staphylococcus aureus, Escherichia coli, Proteus mirabilis, Enterobacter agglomerans, Bacteroides fragilis, Klebsiella pneumoniae, or Candida albicans . Plasma from animals monoassociated with the gram-negative bacteria or C . albicans consistently showed a positive Limulus test while conventional-flora controls, germfree rats, and gnotobiotic animals monoassociated with gram-positive bacteria or E . agglomerans were negative . Germfree and conventional rats were injected (intraperitoneal (i.p.)) with Salmonella typhosa lipopolysaccharide (LPS) . Although no endotoxin was detectable in either group prior to the injection, by 1 h post injection endotoxin was in the plasma of all groups . The germfree rats appeared to clear the LPS quicker than their conventional-flora counterparts . Generally, LPS-injected rats (conventional and germfree) showed clumping and decreased number of platelets, a decrease in their lymphocyte counts, and increased polymorphonuclear leukocyte (PMN) counts. Eur J Biochem, 1978 Nov 15, 91(2), 345 - 50 Isolation of thiomolybdate compounds from the molybdenum-iron protein of clostridial nitrogenase; Zumft WG; Acid/base treatment of the molybdenum-iron protein of the nitrogenase from Clostridium pasteurianum 25 yields low-molecular-weight compounds of molybdenum, which can be separated from the protein by gel chromatography . Elementary analysis and spectral properties relate these compounds to thiomolybdate anions . It is proposed that in its native state nitrogenase contains a thio complex of molybdenum coupled to iron-sulfur clusters. Lancet, 1978 Nov 11, 2(8098), 1014 - 6 Fulminant necrotising enterocolitis associated with Clostridia; Kosloske AM et al.; 5 infants with no growth of bacteria on cultures of blood and peritoneal fluid recovered from necrotising enterocolitis after medical treatment alone . 12 infants with positive cultures required surgery . 5 of these 12, who did not harbour clostridia, had a mild clinical course and all 5 survived segmental bowel resection . The 7 infants who harboured clostridia had a more severe clinical course and 4 died . In 3 of 4 infants with Clostridium perfringens, the necrotising enterocolitis was fulminant, characterised by severe pneumatosis intestinalis, extensive gangrene, early intestinal perforation, and a fatal outcome. J Biol Chem, 1978 Nov 10, 253(21), 7722 - 30 Assignment of the cysteinyl 13C nuclear magnetic resonances and comparison of other aliphatic amino acid resonances of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins; Packer EL et al.; 13C NMR spectra of Clostridium acidi-urici, Clostridium pasteurianum, and Peptococcus aerogenes ferredoxins show that some 13C resonances of the aliphatic amino acid residues are shifted significantly from their corresponding resonance positions in the spectra of model polypeptides or apoferredoxin . Thirteen 13C resonances are shifted into the 80- to 120-ppm (from CS2) region, and have been assigned to the cysteinyl alpha and beta carbon atoms . The remaining shifted resonances in the 120- to 190-ppm region are tentatively assigned to amino acid residues that may be close to {4Fe-4S} clusters of the oxidized and reduced ferredoxins . The similarity in the shift pattern of the corresponding 13C resonances of the cysteinyl alpha and beta carbon atoms in the three ferredoxins studied suggests that the three-dimensional amino acid environments of the corresponding {4Fe-4S} clusters in each protein are similar. Can Med Assoc J, 1978 Nov 4, 119(9), 1058 - 60 Pseudomembranous colitis: isolation of two species of cytotoxic clostridia and successful treatment with vancomycin; Marrie TJ et al.; Lincomycin-resistant Clostridium sporogenes obtained from the stools of a patient with lincomycin-associated pseudomembranous colitis produced a heat-stable cytotoxin in low titre when grown in chopped meat medium . Vancomycin eradicated this strain and all other clostridia, and controlled the symptoms . When diarrhea recurred 7 days after treatment with vancomycin was stopped, clostridia including C . sporogenes and C . difficile were again isolated . The C . difficile produced a heat-labile cytotoxin in high titre that was unaffected by growth in various media and induced colitis in hamsters . Treatment with vancomycin, to which all the clostridia were sensitive, eradicated both toxic species and controlled the diarrhea . Antibiotic-induced pseudomembranous colitis may be associated with more than one species of toxin-producing clostridia . Vancomycin therapy should be continued for 10 days or more in patients with severe disease to eradicate the responsible organism. J Am Vet Med Assoc, 1978 Nov 1, 173(9), 1131 - 3 Hazards of disease transfer from marine mammals to land mammals: review and recent findings; Smith AW et al.; In a 5-year study (1972-1977) of microbial agents isolated from both clinically normal and diseased marine mammals, it was shown that certain disease agents are widespread in a diversity of ocean populations and that some are also transmissible to a number of terrestrial mammal species . Leptospira interrogans serovar pomona has been isolated repeatedly from 2 species of pinnipeds (Zalophus californianus califonianus and Callorhinus ursinus) . Some of the more important bacterial pathogens for land mammals that were isolated from wild marine mammals are Pseudomonas mallei, Clostridium chauvoei, C novyi, Neisseria mucosa var heidelbergensis, Klebsiella pneumoniae, Salmonella spp, and Pasteurella multocida . Numerous serotypes of viruses classified as caliciviruses were isolated from a variety of marine mammals . Some of these are known to infect several land mammal species including swine horses, and primates . For this reason., precautions should be taken to ensure that disease agents shed by captive marine mammals are not transmitted to susceptible terrestrial mammals, including animal handlers and other human beings. J Clin Microbiol, 1978 Nov, 8(5), 509 - 11 Clostridium pseudotetanicum bacteremia in a patient with Pasteurella multocida conjunctivitis; Eschete ML et al.; Clostridium pseudotetanicum only once previously has been identified as causing disease . Pasteurella multocida has been identified only three times as the cause of purulent conjunctivitis . A very debilitated patient had C . pseudotetanicum bacteremia and P . multocida conjunctivitis from which she recovered only to die of a nosocomial Staphylococcus epidermidis septicemia, originating in a site for the administration of intravenous fluids. Infect Immun, 1978 Nov, 22(2), 418 - 22 Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxins; Chang TW et al.; Neutralization of Clostridium difficile toxin by Clostridium sordellii antitoxin was studied by cytotoxicity assay in tissue culture . The sources of toxin were stools from two patients with pseudomembranous colitis and a culture filtrate of C . difficile isolated from one of the patients . C . sordellii antitoxin was available either in monovalent form or as gas gangrene polyvalent antitoxin . The potency of antitoxins against C . difficile determined by cytotoxicity assay did not correlate with the established values reported for mouse protection tests against C . sordellii toxin . An equivalent zone of optimal neutralization was demonstrated for stool toxin, and a slightly different one for culture toxin . The rate of neutralization appeared to be instantaneous, either at 24 or at 37 degrees C . The efficacy of antitoxin in preventing cytotoxicity in cultured cells preexposed to toxin decreased rapidly with preexposure time . The union between toxin and antitoxin could be readily dissociated by simple dilution or by ammonium sulfate precipitation followed by dissociated by simple dilution or by ammonium sulfate precipitation followed by dilution . Continued incubation of toxin-antitoxin mixture did not increase the firmness of the union; on the contrary, more dissociation occurred . The unusual looseness of the toxin-antitoxin union is probably relatd to lack of serological specificity or affinity . Based on these observations, a practical diagnostic method for antibiotic-induced colitis is outlined. Zh Mikrobiol Epidemiol Immunobiol, 1978 Nov, (11), 59 - 62 {Mechanism of segregation of bacterial chromosomes following division of anaerobic clostridia}; Vaisman ISh; Morphological events during division in Clostridium oedematiens, strains A-277 and A-79, were studied on ultrathin sections . The bacterial cells of the species under investigation containing numerous and various intracytoplasmic membranous structures were practically devoid of the "nuclear mesosomes" type structures . In these anaerobic Clostridia the segregation of bacterial chromosomes after the replication was effected by means of direct connections between the DNA filaments and the cytoplasmic membrane of the bacterial cell . These connections were multiple, up to 250 nm in length, possibly ensuring a proper mechanical strength needed for translocation of the chromosomes into the daughter cells . It is suggested that multiple contact zones between the cytoplasmic membrane and the DNA filaments of the bacterial chromosome arose after the triggering of their DNA replication. Arch Intern Med, 1978 Nov, 138(11), 1618 - 20 Ticarcillin disodium in anaerobic infections; Webb D et al.; Twenty-five patients were treated with ticarcillin disodium, 18 of whom had anaerobic infections that included pleuropulmonary infections (seven), mandibular osteomyelitis (four), perirectal abscess (two), sepsis, primary site unknown (one), liver abscess (one), pelvic abscess (one), decubitus ulcer (one), and synergistic gangrene (one) . Seven had no anaerobic infections . Three had anaerobic septicemia . Culture results included anaerobes: peptococci (ten), peptostreptococci (ten), Bacteroides fragilis (six), Bacteroides not fragilis (ten), eubacteria (three), fusobacteria (two), Clostridium (one), Veillonella (one), and acidaminococcus (one); aerobes: Proteus (three), Klebsiella (two), Escherichia coli (two), and streptococci (two) . Six patients with mixed aerobic infections initially received gentamicin sulfate in addition . The serum levels were 110 +/- 20 microgram/ml one hour after intravenous infusion of 5 g of ticarcillin disodium . All anaerobic isolates were susceptible at less than or equal to 100 microgram/ml and 85% by less than or equal to 25 microgram/ml of ticarcillin . Sixteen patients responded well to ticarcillin and two failed to respond . Our study suggests that ticarcillin is useful in the treatment of anaerobic infections. Gastroenterology, 1978 Nov, 75(5), 778 - 82 Role of Clostridium difficile in antibiotic-associated pseudomembranous colitis; Bartlett JG et al.; Tissue cultures were performed on stools from 189 patients to detect a cytopathic toxin which is neutralized by Clostridium sordellii antitoxin . Specimens satisfying these criteria were considered positive in the tissue culture assay . Stools from 26 of 27 patients with antibiotic-associated pseudomembranous colitis were positive and 16 of these specimens showed toxin titers of 10(-3) dilutions or greater . The tissue culture assay was positive with specimens from 9 of 63 patients with antibiotic-associated diarrhea without documented pseudomembrane formation . Stools from patients with neonatal necrotizing enterocolitis, ulcerative colitis, and healthy controls were uniformly negative in this assay . Cultures were performed on stools from 38 patients with antibiotic-associated diarrhea or colitis to detect clostridia which produce a cytopathic toxin in vitro . Clostridium difficile was recovered from 6 of 8 specimens which were positive in the tissue culture assay and 5 of 30 which were negative in this assay . C . sordellii was recovered in a single specimen . One hundred and nine clostridia strains were tested in the tissue culture assay and C . difficile was the only species which produced a cytopathic toxin . All strains of this organism were positive in the tissue culture assay and, in each instance, cytotoxicity was neutralized by C . sordellii antitoxin . These results indicate that C . difficile is the major cause of antibiotic-associated pseudomembranous colitis and offer an explanation for previous studies showing that the cytotoxin of stools from these patients is neutralized by C, sordellii antitoxin. Gastroenterology, 1978 Nov, 75(5), 791 - 5 Pathogenesis of mucosal injury in the blind loop syndrome; Jonas A et al.; Bacterial extracts were prepared from cultures originating in chronic self-filling intestinal blind loops in rats . Their ability to remove active maltase molecules from isolated brush border membranes was studied in vitro . Twelve strains in 51 tested, belonging to one of three species, Bacteroides fragilis, Clostridium perfringens, and Streptococcus fecalis, possessed maltase-releasing activity . The ability to remove maltase correlated well with the ability to hydrolyze p-nitrophenyl-tert-butyloxycarbonyl-l-alaninate (NBA), an ester substrate rapidly hydrolyzed by elastase, but not with substrated favored by tryhsin and chymotrypsin . Maltase-releasing activity from C . perfringens was strongly inhibited by soybean trypsin inhibitor and to a lesser extent by lima bean trypsin inhibitor . Of four chloromethylketone active-site directed inhibitors tested with specificities for elastase, trypsin, and chymotrypsin, inhibition was maximal with elastase-specific inhibitors . In two species, activity was shown to be heat sensitive, and to be inhibited by concentration of the extract . In one species maltase-releasing activity was shown to be due to an enzyme of molecular weight at least 66,000 with the capacity to remove lactase, sucrase, and alkaline phosphatase, as well as maltase . The results indicate that anaerobic or facultatively anaerobic species, previously identified with the pathology of of the blind loop syndrome, contain proteases which are capable of removing components of the intestinal surface membrane . These proteases appear to have elastase-like substrate specificity and may be involved in the etiology of disaccharidase deficiency in bacterial overgrowth syndromes. Proc Natl Acad Sci U S A, 1978 Nov, 75(11), 5273 - 5 Cluster characterization in iron-sulfur proteins by magnetic circular dichroism; Stephens PJ et al.; We report magnetic circular dichroism (MCD) spectra of 4-Fe iron-sulfur clusters in the iron-sulfur proteins Chromatium high-potential iron protein (HIPIP), Bacillus stearothermophilus ferredoxin and Clostridium pasteurianum ferredoxin . The MCD is found to vary significantly with cluster oxidation state but is relatively insensitive to the nature of the protein . The spectra obtained are compared with the corresponding spectra of iron-sulfur proteins containing 2-Fe clusters . It is concluded that MCD is useful for the characterization of iron-sulfur cluster type and oxidation state in iron-sulfur proteins and is superior for this purpose to absorption and natural circular dichroism spectroscopy. Aust Vet J, 1978 Nov, 54(11), 541 - 4 Reverse phase passive haemagglutination and single radial immunodiffusion to detect epsilon antigen of Clostridium perfringens type D; Beh KJ et al.; Two in vitro immunological assays were developed for detection of the epsilon (epsilon) antigen of Cl . perfringens type D . It was found that the reverse phase passive haemagglutination assay (RPHA) was able to detect concentrations of epsilon-antigen as low as 6 x 10-7 mg/ml whereas the single radial immunodiffusion techniques (SRID) was capable of detecting concentrations of epsilon-antigen above 0.01 mg/ml . When applied to gut contents from freshly dead infected sheep the RPHA test was found to be more sensitive than mouse toxicity assay in detecting the presence of epsilon-antigen . However, very low titres were detected in gut contents from normal sheep which meant that in a diagnostic situation interpretation of RPHA titres would be difficult . No epsilon-antigen was detected by SRID in gut contents from normal sheep or in gut contents from freshly dead infected sheep . The SRID assay could detect epsilon-antigen in gut contents from infected sheep allowed to decompose for 20 h post-mortem. Endokrinologie, 1978 Nov, 72(3), 363 - 4 Specificity of action of neuraminidase, according to its bacteriological origin; Finne E; Neuraminidase from Vibrio Cholerae selectively cleaves sialic acid from FSH, but leaves the LH sialic acid, not influencing the biological activity of the latter hormone . On the other hand, Neuraminidase from Clostridium perfringens does not possess this specific action and destroys the biological activity of LH as is suggested by Parlow's OAAD-test. J Clin Microbiol, 1978 Nov, 8(5), 480 - 8 Synergistic hemolysis phenomenon shown by an alpha-toxin-producing Clostridium perfingens and streptococcal CAMP factor in presumptive streptococcal grouping; Gubash SM; A new phenomenon of synergistic hemolysis by Clostridium perfringens alpha-toxin and the streptococcal CAMP factor on human and guinea pig erythrocytes is described . A possible mode of action of the CAMP factors is suggested . On human blood agar all of the tested isolates of group B streptococci gave an arrowhead-shaped zone of hemolysis; 74% of group A gave a crescent-shaped lytic zone, whereas all isolates of groups C and G and the remaining 26% of group A streptococci gave a bullet-shaped lytic zone . By comparison, in the CAMP test incubated aerobically and anaerobically, 70 and 91%, respectively, of streptococci other than group B gave positive, arrowhead-shaped lytic zones . If all intermediate positive reactions in the CAMP tests were read as negative after aerobic incubation, only 89% of group B streptococci would be properly identified . The synergistic hemolysis phenomenon, using an alpha-toxin-producing C . perfringens and human blood agar, provided a reliable test for presumptive identification of group B streptococci, with promising potential to differentiate in the same test group A streptococci from other groups. Mikrobiologiia, 1978 Nov-Dec, 47(6), 1124 - 6 {Number of butyric acid bacteria belonging to the genus Clostridium in the slimy sediments of Volga reservoirs}; Dziuban AN; The number of Clostridium pasteurianum, Cl . butyricum, and Cl . acetobutylicum was determined in ooze deposits of the Volga River reservoirs using enriched nutrient media . The bacterial number for the two former species was about 1 + 10(6) cells per 1 ml of ooze with a high content of easily assimilated organic substances, thus being by 1--3 orders of magnitude higher than for Cl . pasteurianum on media without nitrogen . The bacterial number for Cl . acetobutylicum was 0.1--1 + 10(3) cells per 1 ml. Biochemistry, 1978 Oct 31, 17(22), 4770 - 8 Circular dichroism and magnetic circular dichroism of iron-sulfur proteins; Stephens PJ et al.; Circular dichroism (CD) and magnetic circular dichroism (MCD) spectra are reported for the 2-Fe ferredoxins from Pseudomonas putida and Spirulina maxima, Chromatium HIPIP, the 4-Fe ferredoxin from Bacillus stearothermophilus, and the 8-Fe ferredoxin from Clostridium pasteurianum . The spectral range spans the near-infrared, visible, and near ultraviolet . In all cases except oxidized 2-Fe ferredoxins, electronic absorption is observed continuously from less than 5000 cm-1 to above 30,000 cm-1 . The CD spectra of the two 2-Fe ferredoxins are similar . In contrast, the CD of the 4-Fe and 8-Fe proteins, for a given 4-Fe cluster oxidation level, varies considerable with protein . MCD is less sensitive to protein environment than is CD . In the 2-Fe proteins, MCD at 5 T is appreciably smaller than the CD; in the 4-Fe and 8-Fe proteins, MCD and CD are comparable in magnitude . Both CD and MCD are more highly structured than the corresponding absorption spectra . The CD and MCD spectra reported provide a broader base than heretofore available for the characterization of iron-sulfur proteins containing 2-Fe and 4-Fe clusters and for the evaluation of electronic structural models for these clusters. Wien Klin Wochenschr, 1978 Oct 27, 90(20), 733 - 6 {Detection of collagenase in passive haemagglutination using collagen-coated erythrocytes (author's transl)}; Kojer M et al.; Human rheumatoid arthritis (RA) collagenase and bacterial collagenase were shown to agglutinate collagen-coated erythrocytes . Native collagens of type I and type II reacted equally well, while denatured collagens showed less distinct agglutination activity . The sensitivity of the method for the detection of purified bacterial collagenase from Clostridium histolyticum is very high (100 pg) . It is, however, low for human RA collagenase . The agglutination reaction is not inhibited by concentrations of native collagen causing distinct inhibition of anticollagen sera (2mg%) . EDTA inhibits the agglutination completely. Arch Microbiol, 1978 Oct 4, 119(1), 7 - 11 Fumarate reductase of Clostridium formicoaceticum . A peripheral membrane protein; Dorn M et al.; When Clostridium formicoaceticum was grown on fumarate or L-malate crude cell extracts contained a high fumarate reductase activity . Using reduced methyl viologen as electron donor the specific activity amounted to 2-3.5 U per mg of protein . Reduced benzyl viologen, FMNH2 and NADH could also serve as electron donors but the specific activities were much lower . The NADH-dependent activity was strictly membrane-bound and rather labile . Its specific activity did not exceed 0.08 U per mg of particle protein . Fumarate reductase activity was also found in cells of C . formicoaceticum grown on fructose, gluconate, glutamate and some other substrates . The methyl viologen-dependent fumarate reductase activity could almost completely be measured with intact cells whereas only about 25% of the cytoplasmic acetate kinase activity was detected with cell suspensions . The preparation of spheroplasts from cells of C . formicoaceticum in 20 mM HEPES-KOH buffer containing 0.6 M sucrose and 1 mM dithioerythritol resulted in the specific release of 88% of the fumarate reductase activity into the spheroplast medium . Only small amounts of the cytoplasmic proteins malic enzyme and acetate kinase were released during this procedure . There results indicate a peripheral location of the fumarate reductase of C . formicoaceticum on the membrane. Biochim Biophys Acta, 1978 Oct 3, 543(2), 226 - 34 Studies on the characterization of the Rho(D) antigen; Litten J et al.; The Rho(D) antigen of red cell membranes was solubilized using ethylene-diamine tetraacetic acid (EDTA) and 2-mercaptoethanol . The solubilized antigen was partially separated from other solubilized membrane components using molecular filtration . The antigen was treated with various enzymes to learn some of the chemical characteristics . It was found that the activity of the antigen, as measured by hemagglutination inhibition, was not affected by bee venom phospholipase A, Clostridium welchii phospholipase C, calf-intestinal alkaline phosphatase, Vibrio cholerae neuraminidase, pig kidney leucine aminopeptidase, bovine pancreatic carboxypeptidase A, and pig pancreatic carboxypeptidase B . However, the proteolytic enzymes, pronase, trypsin, chymotrypsin and papain, did destroy Rho(D) activity as measured by hemagglutination inhibition . These results indicate that protein is an important part of the active determinant of the Rho(D) antigen . The experiments by other investigators have shown that lipid is important to maintain the Rho(D) activity in the intact membrane; lipid probably helps to maintain the structural conformation of the Rho(D) molecule in its natural environment . The solubilized Rho(D) molecules are apparently not dependent on lipid for their Rho(D) activity. Am J Clin Nutr, 1978 Oct, 31(10 Suppl), S243 - S247 Characterization of 7-alpha-dehydroxylase in Clostridium leptum; Stellwag EJ et al.; 7-alpha-Dehydroxylation of primary bile acids was demonstrated radiochromatographically in whole cells of Clostridium leptum but was not observed in intestinal Bacteroides species . Activity of 7-alpha-Dehydroxylase was detected within a pH range of 5 to 9 and was 8-fold higher in specific activity in cell cultures in the presence of 0.1 mM sodium cholate than in its absence . 7-alpha-Dehydroxylase activity in whole cells was markedly inhibitied by 2,4-dinitrophenol, carbonyl-cyanide-m-chlorophenylhydrazine, and dicyclohexylcarbodiimide . A hypothesis concerning the dietary regulation of 7-alpha-dehydroxylating intestinal anaerobic bacteria is presented. Ann Microbiol (Paris), 1978 Oct, 129 B(3), 437 - 49 {Investigation of the immunity status towards tetanus of a population of mechanics at the car-factory "Renault" (author's transl)}; Bizzini B et al.; Tetanus immunity has been studied in a population of mechanics working at the car-factory "Renault" . For the study, 283 individuals were divided into 3 groups . The first group consisted of non-vaccined individuals, the second of vaccinated individuals and the third of individuals who had been boostered . The influence of different parameters on tetanus immunity status was considered, i . e . the age of the subjects, the time at which boostering was given, the serotherapy (when it was administered) and the contact with machine lubricating and cooling fluids . Clostridium tetani bacilli and spores were frequently found in aqueous machine fluids . Their presence is a potential hazard for non-vaccinated mechanics coming to contact with the fluids . Tetanus antibody levels in the sera of the tested population were determined in vivo by the toxin neutralization test . The influence on tetanus immunity of the different parameters considered in this paper was subjected to statistical analysis . From the whole population, 27% of the individuals were found to have no protection against tetanus . When age was taken into account, 53% of the individuals over 45 years old and 10% of those under 45 years old were shown to be devoid of tetanus immunity . It could be shown that younger individuals were better protected than older ones, because the formers had been immunized with adsorbed tetanus toxoid and most of the latters with fluid toxoid . Of the individuals in the third group who had received a booster injection within 15 years after primo-vaccination, 98% showed protective tetanus antibody levels in their sera in contrast to 25% when boostering had occurred more than 15 years after primo-vaccination . Contact with machine fluids was found to influence the degree of immunity of only those individuals whose boostering dated back to more than 25 years . Unexpectedly, 3 mechanics seemed to develop immunity after coming into contact with machine fluids . From the results reported here, it is concluded that tetanus immunity in vaccinated individuals should be renewed by a compulsory booster injection given every 5, 10 or, at the minimum, 15 years . Moreover, high-risk populations such as that represented by the mechanics should be immunized or boostered on commencing employment. Zentralbl Bakteriol {Orig A}, 1978 Oct, 241(4), 438 - 47 {Characterization of plasmid DNA in a lecithinase-positive and in a lecithinase-negative strain of Clostridium perfringens (author's transl)}; Kramer J et al.; A non pathogenic variant of Clostridium perfringens and the wild type strain were characterized . The strains agreed in most of the biochemical properties, in susceptibility against antibiotics and in bacteriocin production . Contrary to the wild type the variant did not produce lecithinase and gelatinase . In deoxyribonucleic acid (DNA) of both strains centrifuged in cesiumchlorid-ethidiumbromide equilibrium there was found a satellite peak containing three distinct, covalently closed circular (CCC) DNA elements . The sum of the average molecular weight or contour length of the two small circular molecules was equal to the average molecular weight or contour length of the third . The presence of the plasmids in the variant indicated that the synthesis of lecithinase might not be coded by a plasmid. Lab Anim Sci, 1978 Oct, 28(5), 536 - 40 Enterotoxemia in rabbits; Patton NM et al.; The presence of Clostridium perfringens Type E iota toxin was confirmed from the cecal contents of 23 of 46 rabbits which died of enteritis complex . The most consistent lesions observed were hemorrhage and edema in the cecum . Rabbit toxicity tests showed the toxic cecal contents were lethal for young rabbits unless incubated with Clostridium perfringens Type E antiserum. Nord Vet Med, 1978 Oct, 30(10), 430 - 3 {A simple device for cultivation of anaerobic bacteria (author's transl)}; Melinder PC; A new concept for isolation and enumeration of anaerobic bacteria in food is presented . The sample is collected and diluted under anaerobic conditions in a specially designed syringe (M.O.S.), in which cultivation also takes place . A comparison between this method and cultivation in anaerobic jars (GasPak) from a common inoculum revealed a higher number of Clostridium perfringens with the M.O.S.-technique. Appl Environ Microbiol, 1978 Oct, 36(4), 567 - 71 New medium for rapid screening and enumeration of Clostridium perfringens in foods; Erickson JE et al.; A rapid and sensitive procedure for estimating low numbers of Clostridium perfringens has been investigated and compared to methods used currently in the food industry . The new liquid medium, RPM (rapid perfringens medium), was compared with sulfite-polymyxin-sulfadiazine agar and tryptose-sulfite-cycloserine agar in recovery studies with naturally contaminated and with inoculated foods . The medium consists of a mixture of litmus milk and fluid thioglycolate medium fortified with glucose, peptone, gelatin, yeast extract, sodium chloride, and ferrous sulfate . Selectivity is based on an antibiotic system (polymyxin B sulfate and neomycin sulfate) incorporated into the medium, coupled with an incubation temprature of 46 to 48 degrees C for 24 h . Tubes were scored as positive if a stormy fermentation was observed . All tubes demonstrating stormy fermentation were confirmed as containing C . perfringens . Of a total of 774 naturally contaminated food samples, 546 samples (71%) were found to contain C . perfringens with RPM, whereas only 168 (22%) of the samples were positive using sulfite-polymyxin-sulfadiazine agar . C . perfringens was isolated from 71% of 85 other samples using RPM as compared to 14% with tryptose-sulfite-cycloserine agar . Enumeration studies on 14 individual samples using the most probable number technique also demonstrated greater sensitivity with RPM. Fed Proc, 1978 Oct, 37(12), 2577 - 81 Foodborne hazards of microbial origin; Foster EM; Foods can serve as vehicles of many pathogenic and toxigenic agents of disease . Bacterial agents comprise three groups: 1) those that grow in the food and produce an active toxin before consumption (e.g., clostridium botulinium); 2) those that merely exist as contaminants in the food but are able to initiate infection when swallowed (e.g., Salmonella spp.); and 3) those that multiply and produce large numbers of vegetative cells in the food, then release an active enterotoxin when they sporulate in the gut . A few parasitic (e.g., Trichinella spiralis) and viral agents (e.g., hepatitis A) also can be transmitted by food . Botulinum poisoning is the deadliest foodborne disease . The potential danger of botulism from cured meats is a major factor in the argument over use of nitrites as meat curing agents . A new disease called infant botulism has been recognized since 1976 . Apparently it is not foodborne but results from intraintestinal growth of C . botulinum in very young infants . Salmonellosis is the most important of the foodborne diseases from the standpoint of overall human health . The primary vehicles are contaminated raw meat, poultry, and eggs . Faulty food handling practices are responsible for most food poisoning in the United States. Biochimie, 1978 Sep 29, 60(6-7), 653 - 61 Phosphatidic and lysophosphatidic acid production in phospholipase C-and thrombin-treated platelets . Possible involvement of a platelet lipase; Mauco G et al.; Incubation of 32P-labelled platelets with Clostridium welchii phospholipase C greatly stimulates 32P-incorporation into phosphatidic and lysophosphatidic acids . A net synthesis is demonstrated for both phospholipids, which exhibit identical specific radioactivities . Phosphatidic acid production roughly parallels the phospholipase C-induced aggregation, whereas lysophosphatidic acid appears secondarily during cell lysis . The same qualitative variations are observed during thrombin-induced aggregation . At the physiological pH used throughout the incubations, platelets display no phospholipase A activity towards phosphatidic acid, whereas diglycerides are deacylated by platelet lysates . On the basis of these findings, a mechanism for phosphatidic and lysophosphatidic acid production is proposed, involving a phosphorylation of the di- and monoglycerides formed upon phospholipase C and lipase action . The possible role of such a pathway in regulating arachidonic acid release from phospholipids during platelet activation is discussed. C R Acad Sci Hebd Seances Acad Sci D, 1978 Sep 25, 287(6), 659 - 61 {Initiation of germination of Clostridium difficile spores by lysozyme}; Ionesco H; The germination rate of spores of C . difficile which is usually lower than 10(-5) is raised to about 5.10(-3) in presence of lysozyme . All spores are initiated by lysozyme when previously treated by sodium thioglycolate . These spores are indeed lysozyme-dependent for germination. Biochim Biophys Acta, 1978 Sep 11, 526(1), 34 - 41 Hydrogen bonding of flavoprotein . I . Effect of hydrogen bonding on electronic spectra of flavoprotein; Nishimoto K et al.; The effect of hydrogen bonding on the transition energy and the oscillator strength of the isoalloxazine nucleus of flavins was studied by the molecular orbital method . Among the possible hydrogen bondings examined, characteristic spectral shifts were found for the hydrogen bondings at N(1) and N(5) of the nucleus . The hydrogen bonding at N(1) resulted in the shift of the first absorption band towards blue and that of the second one towards red . On the other hand, the hydrogen bonding at N(5) resulted in the shifts of both the first and the second band towards red . The spectral characteristics reported on Clostridium MP and Desulfovibrio vulgaris flavodoxin coincided with the calculated results . The application of the calculated results to D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating), EC 1.4.3.3) led to the conclusion that hydrogen bonding occurs at O(12), N(3)H, O(14) and N(5) of the isoalloxazine nucleus . The occurrence of hydrogen bondings at O(12), N(3)H, and O(14) is favorable for N(5) of the isoalloxazine nucleus to accept electron from an electron donor. Infect Immun, 1978 Sep, 21(3), 989 - 93 Cytotoxic activity of Aeromonas hydrophila; Donta ST et al.; Most strains of Aeromonas hydrophila tested demonstrated cytotoxic activity on several tissue-cultured cell lines . The cytotoxin is heat-labile, non-dialyzable, and immunologically distinct from that of Shigella dysenteriae and Clostridium perfringens . None of the aeromonas isolates was found to be enterotoxigenic by either tissue culture or rabbit ileal loop assays. Am J Vet Res, 1978 Sep, 39(9), 1525 - 30 Antibiotic-induced lethal enterocolitis in hamsters: studies with eleven agents and evidence to support the pathogenic role of toxin-producing Clostridia; Bartlett JG et al.; Clindamycin-induced enterocolitis in hamsters was studied, using a tissue culture assay to detect clostridial toxin . It was found that animals with lethal enterocolitis had a cytopathogenic substance in cecal contents and blood that was neutralized by clostridial antitoxins . Cultures of the cecal flora yielded numerous species of clostridia, but only 1 organism was detected which produced a toxin which was cytopathic in tissue culture . This organism, Clostridium difficile, was consistently present in high concentrations, and the cell-free supernate of these strains caused enterocolitis if injected intracecally into hamsters . Ten additional antimicrobials were tested ih hamsters . Ampicillin, vancomycin, erythromycin, cephalosporins, and oral gentamicin caused lethal enterocolitis in most recipients, and all animals which died had evidence of clostridia toxin in cecal contents at necropsy . Tetracycline and metronidazole were well tolerated, and the animals given these antimicrobials had no evidence of the toxin . We conclude that toxin-producing clostridia are responsible for lethal enterocolitis due to a variety of antimicrobials in hamsters. Appl Environ Microbiol, 1978 Sep, 36(3), 403 - 7 Sensitization of Clostridium perfringens spores to heat by gamma radiation; Gombas DE et al.; Spores of Clostridium perfringens, type A, were given separate or sequential treatments of gamma radiation (0 to 0.7 Mrad) and/or high temperature (93 to 103 degrees C) . Prior heating, sufficient to inactivate 40 to 99% of the viable spores, had no effect on the subsequent radiation inactivation rate . Prior irradiation had a sensitizing effect on subsequently heated spores . The degree of sensitization to heat, as measured by thermal inactivation rate, increased with increased radiation pretreatment dose. Lab Invest, 1978 Sep, 39(3), 210 - 8 The effects of Clostridium perfringens enterotoxin on rat and rabbit ileum: an electron microscopic study; McDonel JL et al.; Intestinal epithelial damage caused by Clostridium perfringens enterotoxin in rats and rabbits was identified by light microscopy and compared at the surface (scanning electron microscopy), and the ultrastructural (transmission electron microscopy) levels . Under the light microscope damage to the epithelial layer of villus tips was clearly evident in cross-sections . Whole tissue viewed under the scanning electron microscope showed comparable tip localization of morpholigic damage in the form of collapsed tips and a dense covering of rounded blebs on the tips . Ulstructuctural observations included partial and sometimes complete disappearance of microvilli structures, budding of the terminal web region into the lumen, and even complete destruction of epithelial cells . These data suggest that C . perfringens enterotoxin attacks the epithelial cells with a preference for cells at the villus tips and causes damage at least in part by altering the cells' apical membranes . This then leads to cellular sloughing, death, and lysing. Biochem J, 1978 Sep 1, 173(3), 831 - 9 Electron-paramagnetic-resonance studies on the redox properties of the molybdenum-iron protein of nitrogenase between +50 and -450 mV; O'Donnell MJ et al.; The midpoint potentials, Em, for the oxidation of the characteristic e.p.r . signal with g values near 4.3, 3.7 and 2.01, of the nitrogenase Mo-Fe proteins from a number of bacteria were measured . They were 0mV for Clostridium pasteurianum, -42mV for Azotobacter chroococcum and Azotobacter vinelandii, -95mV for Bacillus polymyxa and -180mV for Klebsiella pneumoniae Mo-Fe proteins at pH 7.9 . The oxidations were thermodynamically reversible for the proteins from A . chroococcum, A . vinelandii and K . pneumoniae and the Em was independent of protein activity for this last protein . The protein from C . pasteurianum required a lower potential for reduction than for oxidation, and the oxidation of the protein from B . polymyxa was only 70% reversible . The apparent Em of the latter protein was decreased by 40mV in the presence of 60mM-MgCl2 . The pH-dependence of the Em of the protein from K . pneumoniae was interpreted in terms of a single ionization, not directly associated with the e.p.r.-active centre, with a pKa of 7.0 in the oxidized form of the protein and a pH-independent region at low pH (Em = 118 +/- 6.3 mV) . Approx . 20% increase in activity after oxidation was observed for the proteins from B . polymyxa, A . chroococcum and K . pneumoniae . The significance of the above results and their relationship to other published data are discussed. J Biol Chem, 1978 Aug 25, 253(16), 5832 - 8 Purification of the "corrinoid" enzyme involved in the synthesis of acetate by Clostridium thermoaceticum; Welty FK et al.; A corrinoid enzyme has been purified to approximately 80% homogeneity from Clostridium thermoaceticum . It catalyzes the formation of acetate from N5-methyltetrahydrofolate and pyruvate in combination with the required supplementary enzymes which are supplied by an extract that has been treated with propyl iodide . The enzyme was purified by chromatography on a folate affinity column and a DEAE-Bio-Gel column and by ultrafiltration . The molecular weight as determined by sedimentation equilibrium is 158,000 and the sedimentation coefficient is 10.5 S . By gel electrophoresis in sodium dodecyl sulfate, the subunit molecular weight was found to be 40,000, thus, the enzyme may be a tetramer of four similar subunits . The results of electron microscopy confirmed the tetrameric structure . In the absence of sodium dodecyl sulfate, two bands of similar intensity were observed by electrophoresis, but both yielded the 40,000 molecular weight subunit in the presence of sodium dodecyl sulfate . These results indicate the two bands represent either two different molecular weight forms of the enzyme or two differently charged isoenzymes . The enzyme is quite labile being sensitive to dilution, aerobic conditions, and light . Dithiothreitol and glycerol were found to stabilize the enzyme . The cofactor requirements for acetate synthesis have been determined . ATP, thiamin pyrophosphate, S-adenosylmethionine, and Fe2+ were found to be required for maximum activity and the Km values were determined . High concentrations of methyltetrahydrofolate, pyruvate, and S-adenosylmethionine were found to inhibit the synthesis of acetate. Fortschr Med, 1978 Aug 10, 96(30), 1510 - 7 {Microbiological evaluation of cefoxitin, a new beta-lactamase resistant cephamycin antibiotic, compared to cephalothin, cephaloridine and cefazolin}; Schassan HH et al.; Cefoxitin (Mefoxitin), Cephaloridin, Cephalothin and Cefazolin were tested in vitro against 380 clinical isolates of Staph . aureus and Enterobacteriaceae using the tube dilution procedure . Of the gramnegative microorganisms a selection of 150 strains was examined regarding susceptibility to the antibiotic combinations cefoxitin/gentamicin and cefoxitin/sisomicin in comparison to cephalothin/gentamicin and cephalothin/sisomicin . Cefoxitin has a broad antimicrobial spectrum and a high beta-lactamase-resistance . Cefoxitin was more active than the other antibiotics tested against E . coli, Klebsiella, Serratia, Enterobacter, indole-negative and -positive Proteus species . The high effectiveness of cefoxitin against anaerobic bacteria such as Bacteroides fragilis and Clostridium perfringens is discussed . Cefoxitin has an excellent bactericidal potency . Among the combinations tested cefoxitin/sisomicin was most active and obtained synergistic effects in most species . The resistance rate of cefoxitin of 8.7% was reduced to 0.7% with cefoxitin/sisomicin. Vet Rec, 1978 Aug 5, 103(6), 116 - 7 Haemorrhagic gastroenteritis in the dog associated with Clostridium welchii; Prescott JF et al.; Two cases of peracute haemorrhagic enteritis in the dog are reported . Gram-positive bacilli, which were shown in one case to be Clostridium welchii were found adhering to the necrotic epithelial surfaces in parts of the gastrointestinal tract in both cases . Large numbers of C welchii were recovered from the intestines of both dogs. J Mol Evol, 1978 Aug 2, 11(3), 233 - 43 The evolution of protein sequences by repetitious gene duplication: clostridial flavodoxin; Kobayashi K et al.; Internal regularities of amino acid sequences of flavodoxins, FMN-containing, low molecular weight flavoproteins, were statistically examined using the minimum mutation method . The sequence of Clostridium pasteurianum flavodoxin shows statistically significant evidence of repetitious internal gene duplications at different levels of structure . Peptide pairs with a low chance probabilitiy of occurrence were frequently observed at a shift of 5 residues . The pairs with the lowest chance probabilities are a pair of heptapeptides at positions39--45 vs . 44--50, a 5 residue shift (p = 9 x 10(-6)) . Most of the related pairs are consistent and could best be explained by the repeating pentapeptide sequence: (Lys-Gly-Ala-Asp-Val-)n and appropriate gaps . Internal repetitions with longer shifts were also suggested for other flavodoxins . Repetitious gene duplication is proposed for the early stages of flavodoxin evolution. Zentralbl Bakteriol {B}, 1978 Aug, 167(1-2), 22 - 8 {Investigation of the hygienic standard in two hospitals including the control of disinfection (author's transl)}; Pfanzelt R et al.; In two operative departments with different architectural presuppositions, the hygienic standard was checked up . Under favourable conditions in clinic B (Hosch-filter, sluice-systems) the relative frequency of demonstrable bacteria amounted to 55% . In clinic A, where these conditions failed, it amounted to 80% . Among the non pathogenic bacteria DNase-negative staphylococci were demonstrated more frequently than others . 13.4% and 18.9% resp . of the bacteria were DNase-positive staphylococci . We used Clostridium perfringens for detecting invasion-paths of germs . The most important ones are leaky windows, air conditioning and insufficient sluice-systems . The success of desinfection was examined . It fluctuates from 67% to 100% . One control amounted to 42% . The results show, that it is impossible to establish sterile rooms for common operative departments . But they show as well that a satisfying hygienic standard cannot be arrived without sluice-systems and appropriate air conditioning. J Lipid Res, 1978 Aug, 19(6), 757 - 62 Effects of small amounts of pentadecan-2-one on the growth of Clostridium butyricum; Gilbertson JR et al.; Primary alcohols occur as trace lipids and are the only long-chain alcohol species present in Clostridium butyricum . Secondary alcohols do not occur physiologically in this microorganism . Exposure of these cells to the methyl ketone, pentadecan-2-one, results in a marked decrease in the primary alcohol content with the secondary alcohol, pentadadecan-2-ol, becoming the major alcohol present . This change in lipid composition is associated with a significant decrease in growth rate that is proportional to the log of the pentadecan 2-one concentration of the incubation medium . When these cells are incubated with pentadecan-2-ol alone, growth is unaffected . Simultaneous exposure of the bacteria to pentadecan-2-one and a mixture of primary alcohols results in a partial relief of the growth inhibition observed with the ketone alone . These observations indicate that pentadecan-2-one inhibits the formation of primary alcohols that are important for normal growth of this bacterium. J Antibiot (Tokyo), 1978 Aug, 31(8), 756 - 60 In vitro activity of tiamulin (81.723 HFU), a new pleuromulin derivative, against clinically significant anaerobes; Werner H et al.; The susceptibility of more than 40 strains of Gram-negative and Gram-positive anaerobes to tiamulin (Sandoz 81.723 hfu), a new pleuromulin (pleuromutilin) derivative, was determined by broth dilution and agar dilution tests . The influences of density of the inoculum upon MICs was studied by a specially designed pour plate-technique . Bacteroides fragilis, B . vulgatus, B . splanchnicus, B . oralis, B . asaccharolyticus, B . melaninogenicus, Fusobacterium fusiforme (F . nucleatum), Sphaerophorus necrophorus, Clostridium perfringens, C . fallax, Propionibacterium acnes and several species of Peptococcaceae showed broth dilution MICs of 0.03 similar to 1 microgram/ml . Members of B . thetaiotaomicron, B . distasonis and S . freundii (F . mortiferum) were inhibited by 8 similar to 32 microgram/ml and 2 strains of S . varius had a broth dilution MIC of 256 microgram/ml . With most strains, the agar dilution MICs were 2 similar to 4 similar to 8 times the broth dilution MICs . In pour plate-tests, the MICs were not considerably influenced influenced by varying initial concentrations of viable cells . With most anaerobes, the MBCs of tiamulin were more than 100-fold higher than the MICs . The results obtained indicated that, apart from S . varius, B . thetaiotaomicron, B . distasonis and S . freundii (F . mortiferum), members of 16 other anaerobic species including B . fragilis were without exception sensitive to tiamulin. Acta Pathol Microbiol Scand {B}, 1978 Aug, 86(4), 207 - 13 Analysis of amines and other bacterial products by head-space gas chromatography; Larsson L et al.; A gas chromatographic (GC) head-space technique is presented, which is suitable for the analysis of volatile products in bacterial broth cultures . This is exemplified by studies on Clostridium septicum, Klebsiella pneumoniae and Proteus mirabilis . The media were acidified or made alkaline and after heating, samples of the gas phase above the media were directly injected into the gas chromatograph . A gas chromatograph equipped with dual columns and flame ionization detectors was used, employing Porapak Q and Chromosorb 103 as stationary phases . Analysis of acidified media, using Porapak Q, gave chromatograms representing acidic and neutral volatile products, while when analysing samples made alkaline, using Chromosorb 103, alkaline and neutral compounds could be detected . Interest was particularly concentrated on the analysis of bacterial amines . P . mirabilis was found to produce isobutylamine and isopentylamine, which were identified by mass spectrometry and GC retention times C . septicum produced ethylamine . The GC head-space technique described constitutes a means for rapid identification of microorganisms . It is adaptable for use on a routine basis in the clinical microbiological laboratory. Proc Natl Acad Sci U S A, 1978 Aug, 75(8), 3640 - 3 A rationale for stabilization of oxygen-labile enzymes: application to a clostridial hydrogenase; Klibanov AM et al.; A general procedure for stabilization of O2-labile enzymes exploiting "salting out" of oxygen from the microenvironment in the molecular layers immediately adjacent to charged surfaces of polyionic solid adsorbents has been developed . Empirical verification of this rationale is provided . The half-life of air inactivation of the O2-labile hydrogenase (EC 1.12.7.1) from Clostridium pasteurianum is increased 20- to 25-fold simply by adsorption (noncovalent binding) in dilute Tris.HCl buffer on common anion exchange supports such as DEAE-cellulose or Dowex 1-X2 . Predicted increases in degree of stabilization by using more densely charged adsorbents (such as polyethyleneimine-cellulose), as well as bulkier solvent counter-anions, are found; half-lives for air inactivation for the bound hydrogenase can be increased to 3000-fold longer than that of the free enzyme . Most of the total catalytic activity, assayed as H2 evolution from dithionite mediated by methyl viologen or ferredoxin, is retained, whereas the expected suppression of H2 uptake in the reverse reaction is observed. Age Ageing, 1978 Aug, 7(3), 161 - 4 Age changes in collagen characteristics of bone and skin of a short-lived species of reptile; Panigrahy GK et al.; Decalcified bone collagen of older male garden lizards, Calotes versicolor, was less susceptible to digestion by collagenase from Clostridium histolyticum than that from younger individuals . In aged skin the percentage solubility and the soluble/insoluble collagen ratio decreased, with a concomitant rise in insoluble and total collagen . Collagen/unit area increased with advancing age in both dorsal and ventral skin . These results from a non-mammalian poikilothermic vertebrate provide additional evidence in favour of the cross-linkage theory of ageing and suggest a common pattern of collagen ageing in vertebrates. J Clin Microbiol, 1978 Aug, 8(2), 238 - 41 Synergistic lysis of erythrocytes by Propionibacterium acnes; Choudhury TK; Sheep and human erythrocytes, partially processed by Staphylococcus aureus or Clostridium perfringens, were susceptible to lysis in the presence of Propionibacterium acnes . P . acnes liberated a lipase that was detected on Tween 80 agar and also on phospholipase C-precipitated egg yolk agar . Such a lipase might have contributed in the process of an intensified cellular lysis . Similar reactions were attempted with Lactobacillus acidophilus, known to possess a nondiffusible lipase, and failed to produce any such reactions . The synergistic reactions, between P . acnes and C . perfringens, were compared with The classical CAMP reaction in an attempt to find a correlation with the established membrane composition of the erythrocytes involved . Synergistic reactions observed do seem to reflect the membrane composition . Such findings, besides being contributory to an understanding of the role of these organisms in the process of pathogenesis, are of importance in the elucidation of molecular organization of biomembranes . Detailed studies, involving a large number of representative anaerobic bacteria, may also help provide an avenue in anaerobic species identification. J Am Vet Med Assoc, 1978 Aug 1, 173(3), 306 - 7 Enterotoxemia in two foals; Dickie CW et al.; Two Quarter Horse foals from different premises died from enterotoxemia . Clostridium perfringens toxins alpha and beta were demonstrated in the foal's intestines by mouse protection tests . Clostridium perfringens type C was isolated from the intestines of each foal . Histologic examination revealed hemorrhage, necrosis, and massive numbers of C perfringens. Infect Immun, 1978 Aug, 21(2), 678 - 80 Some properties of beta-toxin produced by Clostridium perfringens type C; Sakurai J et al.; Purified beta-toxin from Clostridium perfringens type C was found to be a single polypeptide chain protein with a molecular weight of approximately 30,000 . The toxin was heat labile, with 75% of its activity being inactivated by incubation at 50 degrees C for 5 min . Biological activity of the purified toxin was completely destroyed on exposure to trypsin for 30 min at 37 degrees C . The 50% lethal dose for mice was 1.87 microgram of purified toxin. J Med Microbiol, 1978 Aug, 11(3), 299 - 302 In-vitro effects of Clostridium welchii type-D epsilon toxin on guinea-pig, mouse, rabbit and sheep cells; Buxton D; Epsilon toxin, at relatively low concentrations, killed guinea-pig peritoneal macrophages in vitro . The cells became swollen, the nuclear and cytoplasmic membranes "blistered" and discontinuous, and the cytoplasm appeared structureless . Formalinised epsilon prototoxin was shown to bind closely to the outer surface of the cells and it is concluded that this site represents the location of the receptors for epsilon toxin . In addition the toxin at higher concentrations killed rabbit peritoneal macrophages after increased periods of incubation, but had no demonstrable effect on other cells from guinea-pigs, rabbits, mice and sheep. J Med Microbiol, 1978 Aug, 11(3), 289 - 92 The use of an immunoperoxidase technique to investigate by light and electron microscopy the sites of binding of Clostridium welchii type-D epsilon toxin in mice; Buxton D; Mice were given an intravenous dose of formalinised C . welchii type-D epsilon prototoxin and an immunoperoxidase technique was used to demonstrate this antigen in the tissues . The antigen was found to bind to the luminal surface of the endothelial lining of certain blood vessels, to the luminal surface of the cells lining the loops of Henle and distal convoluted tubules in the kidney, and to the hepatic sinusoids . As it has been shown previously that formalinised epsilon prototoxin and epsilon toxin can compete for the same receptor sites it is postulated that the binding sites demonstrated represent the location of the receptors for C . welchii type-D epsilon toxin. J Med Microbiol, 1978 Aug, 11(3), 269 - 80 Neuraminidase production by clostridia; Fraser; The production of neuraminidase (EC 3.2.1.18) by a range of clostridial species was investigated with techniques previously developed to distinguish neuraminidase-negative and neuraminidase-positive strains of Clostridium perfringens (welchii) . Large amounts of extracellular neuraminidase were produced by representative strains of C . perfringens and C . septicum in the test media . Under similar conditions, two strains each of C . chauvoei and C . tertium were found to produce small amounts of the enzyme . All of 12 strains of C . sordellii were clearly shown to produce neuraminidase, often in large amounts, but none of five strains of the closely related but non-pathogenic C . bifermentans had demonstrable neuraminidase activity . No neuraminidase was produced by C . novyi (oedematiens) types A-D (10 strains), C . tetani (6), C . botulinum types A, B, C or E (4), C . sporogenes (4), C . histolyticum (4) or by single strains of five other clostridial species . Clostridial neuraminidase was predominantly extracellular and was not calcium-dependent . The investigation took account of variations in growth and enzyme production in different media . It was necessary to prolong the neuraminidase-assay reaction time to 24 h and to monitor for the presence of NAN-aldolase (EC 4.1.3.3) to define true negatives . It is suggested that neuraminidase production may be of value in taxonomic studies and that its production by several pathogenic species of clostridia may be of interest in studies of pathogenicity and virulence. J Infect Dis, 1978 Aug, 138(2), 257 - 9 Effects of gentamicin on trypsin, chymotrypsin, and collagenase; Asch HL et al.; The effects of gentamicin on three proteolytic enzymes were studied . Gentamicin was tested at concentrations of 0.5-500 microgram/ml . Trypsin was tested at 0.5 microgram/ml using p-tosyl-L-arginine methyl ester and at 0.1 and 0.5 microgram/ml using azocoll as the substrate . Chymotrypsin was tested at 0.1 and 0.5 microgram/ml with azocoll . A soluble {14C}collagen assay was used to measure activity of collagenase derived from Clostridium histolyticum . The profiles of proteolytic activity vs . gentamicin concentration were similar for all three enzymes . At lower concentrations of gentamicin (less than 70 microgram/ml), there were two peaks of enhanced protease activity generally followed by inhibition . These unusual multiphasic effects of gentamicin on three different proteases are not presently understood, but they imply a previously unreported mode of action for this antibiotic. Cancer Res, 1978 Aug, 38(8), 2295 - 300 Growth retardation and prevention of Ehrlich solid tumor by Clostridium perfringens type A spores and culture supernatant; Lapointe JR et al.; When given by direct s.c . injection into the Ehrlich solid carcinoma 1 week after s.c . tumor transfer, viable crude spores of Clostridium perfringens type A (attenuated mutant strain LNG11 ATCC 29348) inhibited tumor growth and significantly prolonged the life span of male outbred Swiss mice . Under these conditions a concentrated sterile supernatant of a C . perfringens culture proved to be slightly more effective than were viable crude spores . In contrast viable crude spores were ineffective in the treatment of female Swiss mice, but the sterile supernatant retained significant activity . When given at the time and site of s.c . grafting of Ehrlich tumor cells, a concentrated sterile supernatant of a C . perfringens culture prevented tumor growth in 80% of male outbred Swiss mice . Under these conditions viable crude spores prevented tumor growth in 70% of mice and significantly prolonged the life span in the other 30% . When given by i.p . injection and before i.p . grafting of tumor cells, viable crude spores of C . perfringens prevented Ehrlich ascites tumor in 5 of 12 Swiss mice and prolonged life span in the other 7 . In contrast concentrated sterile supernatant and viable purified spores were ineffective in the prevention or delay of the growth of Ehrlich ascites tumor . These data indicate that C . perfringens can be a potent antitumor agent without producing the harmful anaerobic infection of solid tumors (clostridial oncolysis. J Med Microbiol, 1978 Aug, 11(3), 293 - 8 Further studies on the mode of action of Clostridium welchii type-D epsilon toxin; Buxton D; Intradermal injection of Clostridium welchii type-D epsilon toxin increased the permeability of blood vessels in guinea-pig skin to Evans blue dye by a mechanism not dependent on the release of histamine . The toxin was also found to raise the plasma concentration of cyclic adenosine 3', 5'-monophosphate in mice . It is concluded that epsilon toxin is an enterotoxin capable of causing widespread damage, after binding to receptor sites on the surface of certain cells, through a mechanism mediated by an adenyl cyclase-cAMP system. Appl Environ Microbiol, 1978 Aug, 36(2), 386 - 8 Establishment of a heat inactivation curve for Clostridium botulinum 62A toxin in beef broth; Losikoff ME; A procedure is described for establishing a heat inactivation curve for the toxin of Clostridium botulinum 62A in beef broth . The effect of toxin titer, pH, and the type of acid employed for pH adjustment on the heat stability of the toxin is described. Biochemistry, 1978 Jul 25, 17(15), 2943 - 8 Role of hydrophobicity in the binding of coenzymes . Appendix . Translational and rotational contribution to the free energy of dissociation; Janin J et al.; We calculate the loss of surface area accessible to solvent associated with coenzyme binding in Clostridium flavodoxin, in dogfish lactate dehydrogenase, and in lobster glyceraldehyde-3-phosphate dehydrogenase . The coenzymes are nearly buried in the complexes and lose on the order of 600 A2, while the proteins lose a similar amount of accessible surface area . Some of the loss can be attributed to conformation changes in the protein, at least in the case of lactate dehydrogenase, where we show that the apoenzyme has a larger accessible surface area than the holoenzyme . Using known correlations with the hydrophobic contribution to the free energy, we demonstrate that hydrophobicity is the major source of stabilization free energy in FMN binding to flavodoxin and in NAD binding to the two dehydrogenases: it contributes 25 to 30 kcal/mol to the free energy of dissociation, more than required in order to compensate for the loss of six degrees of translational/rotational freedom by the coenzyme. Biochemistry, 1978 Jul 11, 17(14), 2857 - 63 Purification and characterization of a marine bacterial collagenase; Merkel JR et al.; A true collagenase was isolated from the culture fluid of a marine bacterium which has been designated Vibrio B-30 (ATCC 21250) . Collagenase production was obtained only in media containing collagen or certain degradation products of collagen . Partial purification on DEAE-cellulose and Sephadex G-200 columns produced active enzyme which was free of nonspecific proteases but which contained two collagenases . The two collagenases have the same apparent molecular size, and evidence is presented to support the theory that one collagenase is derived from the other . Vibrio B-30 collagenase appears to be a tetramer with a molecular weight of about 105 000 composed of two different subunits (mol wt 24 000 and 28 000) . Some of the properties of the Vibrio collagenase are compared with those of Clostridium histolyticum collagenase . Molecular weights, subunit structures, specificity and mode of collagen hydrolysis, insensitivity to diisopropyl fluorophosphate and calf serum, and sensitivity to certain metal ion complexing agents and isopropyl alcohol are similar for the collagenases from both organisms . However, Vibrio B-30 collagenase and Clostridium collagenase differ immunologically and electrophoretically. J Biol Chem, 1978 Jul 10, 253(13), 4525 - 9 A pyruvate-containing peptide of proline reductase in Clostridium sticklandii; Seto B; Proline reductase in Clostridium sticklandii is composed of 10 apparently identical subunits . Each subunit contains a pyruvate residue that became labeled when the cell culture was supplemented with {14C}serine . No NH2-terminal amino acid was detected either by dansylation, by Edman degradation, or by aminopeptidase M digestion . The results suggest that the NH2 terminus may be blocked by pyruvate . A pyruvate-containing peptide, also blocked at the NH2 terminus, was isolated from the NH2-terminal portion of proline reductase . From amino acid analysis the peptide was found to be rich in basic amino acids and to have a molecular weight of 4621 . Its COOH-terminal amino acid was found to be serine and since the peptide was released from proline reductase by very mild alkali hydrolysis, it is suspected that an ester bond is involved. Biochim Biophys Acta, 1978 Jul 7, 525(1), 45 - 54 The kinetics of methyl viologen oxidation and reduction by the hydrogenase from Clostridium pasteurianum; Erbes DL et al.; A mechanism for the reduction and oxidation of methyl viologen by Clostridium pasteurianum hydrogenase (hydrogen:ferredoxin oxidoreductase, EC 1.12.7.1) is proposed . Double reciprocal plots for methyl viologen reduction and oxidation at pH values 7.0-9.85 are linear, and the plots for reduction and oxidation are intersecting . Such data are consistent with a mechanism in which the H2 and one methyl viologen bind (either in order or randomly) with subsequent reduction and release of the methyl viologen . A second methyl viologen then is bound, reduced and released . Comparison of the calculated Keq' with the Haldane expression in which both methyl viologens react at the same rate show a large difference . This difference indicates that the two methyl viologens react at different rates . Addition of oxidized electron carriers inhibits the hydrogen-deuterium exchange reaction (i.e., the exchange of protons between H2 and 2H2O) . CO reversibly inhibits methyl viologen reduction and is competitive vs . H2 . O2 acts as an irreversible inhibitor. Biochem J, 1978 Jul 1, 173(1), 129 - 44 Interaction between synthetic analogues of quinoxaline antibiotics and nucleic acids . Changes in mechanism and specificity related to structural alterations; Lee JS et al.; The interaction with DNA of six chemically synthesized derivatives of the quinoxaline antibiotics was investigated . Five of the compounds bound only weakly to DNA or not at all; for these substances spectrophotometric measurements, sedimentation studies with closed circular duplex bacteriophage-PM2 DNA and thermal-denaturation profiles were used to determine limits fot the binding constants . No interaction could be detected with two products of degradation of echinomycin (quinomycin A), one of which, echinomycinic acid dimethyl ester, had the lactone linkages opened, whereas the other retained an intact octapeptide ring but had a broken cross-bridge . The other compounds studied were des-N-tetramethyl-triostin A ('TANDEM') and its derivatives . A derivative of 'TANDEM' IN WHICH benzyloxycarbonyl moieties replace both quinoxaline chromophores had binding constants to nucelic acids in the range 10(2)--10(3)-1, whereas no interaction could be detected for a benzyloxycarbonyl derivative that, in addition, had the cross-bridge broken . The derivative of 'TANDEM' with L-serine in place of D-serine in both positions showed no detectable interaction with Clostridium perfringens DNA, whereas the binding constant to poly(dA-dT) was approx 2 X 10(3)M-1 . 'TANDEM' itself bound strongly to DNA, and the bathochromic and hypochromic shifts in its u.v.-absorption spectrum in the presence of DNA were similar to those seen with echinomycin . From the effect on the sedimentation coefficient of closed circular duplex bacteriophage-PM2 DNA the mechanism of binding was shown to involve bifunctional intercalation, typical of the naturally occurring quinoxaline antibiotics . Solvent-partition analysis was used to determine binding constants for the interaction between 'TANDEM' and a variety of natural and synthetic DNA species . The pattern of specificity thus revealed differed markedly from that previously found with the naturally occurring quinoxaline antibiotics . Most striking was the evident large preference for (A + T)-rich DNA species, in complete contrast with echinomycin and triostin A . The highest binding constant was found for poly(dA-dT), the interaction with which appeared highly co-operative in character . The conformations adopted by those quinoxaline compounds that bind strongly to DNA were examined withe aid of molecular models on the basis of results derived from n.m.r . and computer studies . It appears that the observed patterns of base-sequence specificity are determined, at least in part, by the structure and conformation of the sulphur-containing cross-bridge. J Gen Microbiol, 1978 Jul, 107(1), 85 - 91 Proteases produced by a proteolytic mutant of Clostridium botulinum type E; Nakane A; A proteolytic mutant from Clostridium botulinum type E produced extracellular proteases after the end of exponential growth coinciding with the period of sporulation . Proteases were separated into four fractions by chromatography on a DEAE-cellulose column . One was a sulphydryl-dependent protease that also apparently required a divalent cation for enzyme activity since it was inhibited by EDTA . This enzyme hydrolysed synthetic amide and ester compounds containing an arginine residue, and showed some activity towards L-lysine methyl ester . It appeared that two of the other proteases were serine proteases and the fourth was a metal protease . These last three proteases did not require a thiol agent and did not hydrolyse any of the synthetic amides or esters examined . Only the sulphydryl-dependent protease could activate C . botulinum type B, E and F toxins . The ability of this enzyme to activate type B and E toxins was markedly lower than that of trypsin . The susceptibility of type B toxin to this protease was lower than that of type E toxin . C2 toxin was not activated by this enzyme . It is suggested that the sulphydryl-dependent protease in this proteolytic mutant of C . botulinum type E has properties similar to those of proteases from C . botulinum types B and F. Infect Immun, 1978 Jul, 21(1), 59 - 63 Intraintestinal toxin in infant mice challenged intragastrically with Clostridium botulinum spores; Sugiyama H et al.; Conventionally raised suckling mice were injected intragastrically with 10(5) spores of a Clostridium botulinum type A culture . Botulism was not observed, but 80% or more of mice challenged when 8 to 11 days old had botulinum toxin in the large intestine 3 days later . Mice younger than 7 days or older than 15 days were resistant to the challenge . When in vivo toxin production was started by spores given to 9-day-old mice, toxin was present in the intestine at 1 through 7 days postchallenge but with greatest consistency between 1 and 4 days . Total toxin in an intestine ranged up to 1,920 50% lethal doses as titrated intraperitoneally in adult mice . The dose infecting 50% of a group of 9-day-old mice was 700 (95% confidence limits of 170 to 3,000) spores per animal . Toxin was formed in the lumen of the large intestine; it was not associated with the ileum . Injection of 10(5) spores intraperitoneally into 9-day-old mice resulted in toxin production in the large intestines of 30% of the test animals. Appl Environ Microbiol, 1978 Jul, 36(1), 210 - 1 System for evaluating clostridial inhibition in cured meat products; Robach MC et al.; A method for evaluating inhibition of Clostridium botulinum, C . sporogenes, and C . perfringens in cured meat products was developed . This system can easily be used in the microbiology laboratory using aluminum ointment tubes as the product container . Swells caused by gas production by the organism are easily observed by using the aluminum tubes . Results obtained confirmed earlier work on the inhibitory effect of sodium nitrite and sorbic acid against the clostridia in cured meat products. Can J Comp Med, 1978 Jul, 42(3), 357 - 63 Enterotoxigenic Clostridium perfringens type A isolated from intestinal contents of cattle, sheep and chickens; Niilo L; One hundred and fourteen strains of Clostridium perfringens, isolated from the intestinal contents of cattle, sheep, and chickens with enteritis or other disease conditions were studied for their ability to produce enterotoxin . Reversed passive hemagglutination, fluorescent antibody and immunodiffusion tests were used . On the basis of the reversed passive hemagglutination titres, supported by the other two tests, enterotoxigenicity of the strains was arbitrarily classified into two categories: highly enterotoxigenic and potentially enterotoxigenic, with 12% falling into each category . All the highly enterotoxigenic strains originated from cases of enteritis and included all three animal species . Apart from enterotoxigenicity, one C . perfringens strain produced beta toxin (type C) and 21 strains produced large amounts of alpha-toxin . The latter strains were predominantly associated with necrotic enteritis in chickens. J Assoc Off Anal Chem, 1978 Jul, 61(4), 785 - 8 Method for maintaining viability of Clostridium perfringens in foods during shipment and storage: collaborative study; Harmon SM et al.; A collaborative study was conducted in 12 laboratories to determine the effectiveness of a new method for maintaining vegetative cells of Clostridium perfringens in viable condition during storage and transport of food specimens to the laboratory . The collaborative results showed that treatment of brown gravy and roast beef samples with an equal amount by weight of sterile buffered glycerol-sodium chloride solution to give a final 10% glycerol concentration and storage with Dry Ice for 10 days at -56 degrees C resulted in plate counts of C . perfringens which were 2-4 log cycles higher with 2 different strains than counts with untreated specimens stored by the usual method at -20 degrees C . Plate counts obtained with the treated specimens stored with Dry Ice were less than 1 log cycle lower than counts made with identical specimens before freezing for storage and shipment to the collaborators . The results with treated specimens were also more uniform among the different laboratories . Because the new method for storage and shipment of food samples was so effective for maintaining viability of the organism, the official first action method for C . perfringens (46.B01) was changed to incorporate these procedures as part of the method. J Pharm Sci, 1978 Jul, 67(7), 900 - 5 Synthesis and quantitative structure-activity relationships of antibacterial 1-(substituted benzhydryl)-4-(5-nitro-2-furfurylideneamino) piperazines; Yung DK et al.; 1-Benzhydryl -4- (5-nitro-2-furfurylideneamino) piperazine and 11 substituted analogs were prepared and examined for in vitro antimicrobial activity . The compounds were active against Bacillus cereus 7, Bacillus megaterium 122, Bacillus subtilis 104, Clostridium perfringens 13, and the tetracycline-resistant Clostridium perfringens 37 . Regression analyses on the antibacterial activity data based on the Hansch approach, using pi, pi2, and sigma parameters, yielded several statistically significant correlation equations . 1-Benzhydryl-4-(5-nitro-2-furfurylideneamino) piperazine stopped the protein and DNA syntheses in C . perfringens 13, as indicated by precipitable radioactivity . The compound, however, showed no effect on the cell wall synthesis in the bacteria. Mol Cell Biochem, 1978 Jun 15, 20(1), 25 - 40 Binding of protein chemotactic factors to the surfaces