Clostridia

Biochem Biophys Res Commun, 1986 Nov 14, 140(3), 1015 - 9
Evidence for direct binding of Clostridium botulinum type E derivative toxin and its fragments to gangliosides and free fatty acids; Kamata Y et al.; Clostridium botulinum type E derivative toxin and its heavy chain bound to gangliosides GT1b, GD1a and GQ1b and saturated and unsaturated free fatty acids with chain lengths of 14-20 carbons . The L-H-1 fragment lacking the carboxyl-terminal portion of the heavy chain bound to free fatty acids but not to gangliosides . These observations led us to a new hypothesis on the mechanism of binding between botulinum toxin and gangliosides; the carboxyl-terminal portion (H-2 fragment) of the heavy chain binds to an oligosaccharide residue of gangliosides and then the amino-terminal portion (H-1 fragment) interacts with the hydrophobic portion of gangliosides consisting of fatty acids.

Nucleic Acids Res, 1986 Nov 11, 14(21), 8605 - 13
Nucleotide sequence of the cellulase gene celD encoding endoglucanase D of Clostridium thermocellum; Joliff G et al.; The nucleotide sequence of the celD gene, encoding the previously crystallized endoglucanase D of Clostridium thermocellum, is reported . The enzyme shares a conserved, reiterated domain with the COOH-terminal end of endoglucanases A and B from the same organism . The overexpression in Escherichia coli of celD subcloned in pUC8 appears to result from a translational fusion of the NH2-terminal end of the endoglucanase with the NH2-terminal end of beta-galactosidase.

J Immunol Methods, 1986 Nov 6, 93(2), 225 - 30
Enzyme-linked immunosorbent assay (ELISA) for the detection and differentiation of Clostridium botulinum toxins type A and B; Michalik M et al.; Affinity chromatography has been used for a two-step purification of commercial horse botulinum antitoxic globulins type A and B . The first step performed using CH-Sepharose 4B conjugated to toxin type A (or B), permitted the removal of non-botulinal antibodies from antitoxic globulin type A (or B) . The anti-botulinal antibodies obtained from the first step were cross-absorbed in the second affinity chromatography using CH-Sepharose 4B conjugated to toxin B (for the purification of antibodies to type A) or to toxin A (for antibodies to type B) . The antibodies obtained were used to coat polystyrene wells in an ELISA for the detection of botulinum toxin type A and type B . The first purification step increases the sensitivity of such an ELISA whereas the second step improved the specificity of the test . Only slight cross-reactions were observed between the type A and type B detection systems . The sensitivity achieved with ELISA was 100 and 300 DLM (dosis lethalis minima) for type A and B respectively.

J Biol Chem, 1986 Nov 5, 261(31), 14551 - 6
The hormone-sensitive hepatic Na+-pump . Evidence for regulation by diacylglycerol and tumor promoters; Lynch CJ et al.; Ouabain-sensitive 86Rb+ uptake by isolated rat hepatocytes was studied to elucidate how Ca2+-mobilizing hormones stimulate the Na+-pump . Stimulation of this uptake was observed with concentrations of vasopressin ({8-arginine}vasopressin, AVP), angiotensin II, and norepinephrine which elicited Ca2+ mobilization and phosphorylase activation . These results suggested that changes in cytosolic Ca2+, mediated by inositol trisphosphate, might trigger sodium pump stimulation by AVP . However, in hepatocytes incubated in Ca2+-free Krebs-Henseleit buffer, Na+-pump activity was not altered over 15 min by either 1.5 mM EGTA or 1.5 mM Ca2+ . Furthermore, incubation of cells in 5 mM EGTA for 15-30 min drastically impaired the ability of AVP to increase cytosolic Ca2+, but only modestly attenuated AVP-stimulated Na+-pump activity . Two tumor promoters, phorbol myristate acetate (PMA) and mezerein, stimulated Na+/K+-ATPase-mediated transport activity . Similarly, addition of synthetic diacylglycerols or of exogenous phospholipase C from Clostridium perfringens to increase endogenous diacylglycerol levels also resulted in a stimulation of the Na+-pump in the absence of changes in cytosolic or total cellular Ca2+ levels . Stimulation of the Na+-pump by the combination of maximal concentrations of PMA and AVP did not produce an additive response, and both agents displayed a transient time course, suggesting that the two agents share a common mechanism . Stimulation of the Na+-pump by AVP and PMA was not blocked by amiloride analogs which inhibit Na+/H+ exchange, but these compounds blocked the action of insulin . These data suggest that the elevated Na+/K+-ATPase-mediated transport activity observed in hepatocytes following exposure to Ca2+-mobilizing hormones is a consequence of stimulated diacylglycerol formation and may involve protein kinase C.

Biochemistry, 1986 Nov 4, 25(22), 6789 - 99
A comparative carbon-13, nitrogen-15, and phosphorus-31 nuclear magnetic resonance study on the flavodoxins from Clostridium MP, Megasphaera elsdenii, and Azotobacter vinelandii; Vervoort J et al.; The flavodoxins from Megasphaera elsdenii, Clostridium MP, and Azotobacter vinelandii were studied by 13C, 15N, and 31P NMR techniques by using various selectivity enriched oxidized riboflavin 5'-phosphate (FMN) derivatives . It is shown that the pi electron distribution in protein-bound flavin differs from that of free flavin and depends also on the apoflavoprotein used . In the oxidized state Clostridium MP and M . elsdenii flavodoxins are very similar with respect to specific hydrogen bond interaction between FMN and the apoprotein and the electronic structure of flavin . A . vinelandii flavodoxin differs from these flavodoxins in both respects, but it also differs from Desulfovibrio vulgaris flavodoxin . The similarities between A . vinelandii and D . vulgaris flavodoxins are greater than the similarities with the other two flavodoxins . The differences in the pi electron distribution in the FMN of reduced flavodoxins from A . vinelandii and D . vulgaris are even greater, but the hydrogen bond patterns between the reduced flavins and the apoflavodoxins are very similar . In the reduced state all flavodoxins studied contain an ionized prosthetic group and the isoalloxazine ring is in a planar conformation . The results are compared with existing three-dimensional data and discussed with respect to the various possible mesomeric structures in protein-bound FMN . The results are also discussed in light of the proposed hypothesis that specific hydrogen bonding to the protein-bound flavin determines the specific biological activity of a particular flavoprotein.

Ann Neurol, 1986 Nov, 20(5), 641 - 3
Botulism in a patient with jejunoileal bypass; Freedman M et al.; A 45-year-old woman was diagnosed as having the unclassified form of botulism . Her intestines may have been predisposed to colonization with Clostridium botulinum because of a jejunoileal bypass procedure that had been done several years earlier . One other similar case has been reported.

J Bacteriol, 1986 Nov, 168(2), 688 - 93
Purification and characterization of a molybdenum-pterin-binding protein (Mop) in Clostridium pasteurianum W5; Hinton SM et al.; A large-scale fractionation scheme purified the major molybdenum(Mo)-binding protein (Mop) from crude extracts of Clostridium pasteurianum, with a 10 and 0.2% yield of Mo and protein, respectively . The apparent molecular weight of the purified molybdoprotein is 5,700, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The protein contains 0.7 mol of Mo per mol of protein with a molecular weight of 5,700 . Mop, as isolated, has a peak absorbency at 293 nm . Denaturation and oxidation of the molybdoprotein released multiple pterin like fluorescent compounds . Mop appears to contain a pterin derivative and Mo, but phosphate analysis indicated that the pterin at the very least is not phosphorylated; phosphorylation is required for functional molybdenum cofactor . All treatments used to release the putative Mo-pterin species from Mop failed to yield a molybdopterin that had detectable molybdenum cofactor activity.

Arch Biochem Biophys, 1986 Nov 1, 250(2), 440 - 5
Conversion of purines to xanthine by Methanococcus vannielii; DeMoll E et al.; Based on the finding that Methanococcus vannielii can employ any of several purines as the sole nitrogen source, an investigation was undertaken to elucidate the pathways of purine metabolism in this organism . Cell-free extracts of M . vannielii converted guanine, uric acid, and hypoxanthine to xanthine and also formed guanine from guanine nucleotides or guanosine . The conversions of guanine and uric acid to xanthine appear to occur by pathways similar to those described in clostridia . The conversion of hypoxanthine to xanthine, however, is different than that described for Clostridium cylindrosporum and C . acidiurici, but is similar to that of C . purinolyticum, and apparently involves the direct oxidation of hypoxanthine to xanthine.

Gastroenterology, 1986 Nov, 91(5), 1147 - 53
Clostridium difficile cytotoxin inhibits protein synthesis in fibroblasts and intestinal mucosa; Pothoulakis C et al.; The pathophysiology of Clostridium difficile colitis is thought to be mediated by release of toxin A, an enterotoxin, and toxin B, a cytotoxin . We compared the differential effects of toxin B on protein synthesis in IMR-90 fibroblasts and in hamster esophagus, stomach, gallbladder, small intestine, and cecum in organ culture . Toxin B in low concentrations stimulated (p less than 0.001) incorporation of {3H}leucine into fibroblast proteins, whereas at higher dosages it inhibited incorporation (p less than 0.001) . This biphasic effect was independent of cell rounding and was not caused by a change in uptake of precursor . Purified toxin B had no effect on protein synthesis in a cell-free rabbit reticulocyte translation system, indicating that inhibition of protein synthesis in intact fibroblast monolayers and intestinal explants is a consequence of toxin B effect on some other cellular target . Toxin B significantly inhibited protein synthesis in hamster cecal explants in a dose-dependent fashion . Again, this inhibition was not mediated by altered precursor uptake . Toxin B significantly inhibited in vitro protein synthesis in hamster terminal ileum, cecum, and sigmoid colon, but not in esophagus, gallbladder, stomach, or duodenum . These results suggest that toxin B-mediated inhibition of protein synthesis may be a generalized toxic effect in tissue culture cells and intestinal epithelium . Inhibition of protein synthesis in the distal intestinal epithelium may contribute to the pathophysiology of colitis caused by this organism.

Pathol Biol (Paris), 1986 Nov, 34(9), 977 - 82
{Clostridium difficile and its cytotoxin in the stools of young hospitalized children . Influence of antibiotic treatment}; Collignon A et al.; Clostridium difficile has been searched in 153 stool samples from 138 children aged 0 to 12 months . We divided the population in two groups depending on the antibiotic treatment . We have found C difficile in 39 samples (25%) . The colonization rate increases with age ranging from 5% before 1 month, to 36% between 1 and 6 months and 54% between 6 and 13 months . An environmental sampling yielded once C difficile . Contamination may be related to the environment . 29% of the isolates produced a cytopathic toxin . Toxin titers in infants' stools range from 1/160 to 1/10240 . One only of these children had diarrhea . C difficile and its toxin does not seem to infer any signs of enteric illness with infants . The results obtained with the group of non treated infants are not significantly different from the ones of the other group: the colonization rates are 21% in the non treated group and 29% in the other group . The rate of strains yielding a cytophatic toxin is similar in the 2 groups . It seems reasonable to agree that antibiotics do not influence the settlement of C difficile in infants' intestine.

Rev Infect Dis, 1986 Nov-Dec, 8 Suppl 5, S543 - 8
Mechanisms of beta-lactam resistance in anaerobic bacteria; Nord CE; The known mechanisms of beta-lactam resistance in anaerobic bacteria involve production of beta-lactamases, alteration of penicillin-binding proteins, and blocking of the penetration of beta-lactams through the outer membranes . The most important factor in beta-lactam resistance is the production of beta-lactamase . beta-Lactamases in various Bacteroides, Fusobacterium, and Clostridium species have been described . beta-Lactam resistance in Bacteroides fragilis is most commonly mediated by the production of beta-lactamase, primarily of the cephalosporinase type . Studies have also shown that B . fragilis can produce a penicillinase that inactivates piperacillin and carbenicillin . Enzymes that inactivate cefoxitin and imipenem have also been found in B . fragilis . The nonfragilis Bacteroides species produce beta-lactamases mainly of the penicillinase type . Recently a penicillinase from Fusobacterium nucleatum has been characterized . Among the clostridia, Clostridium butyricum, clostridium clostridiiforme and Clostridium ramosum have been shown to produce penicillinases.

Antimicrob Agents Chemother, 1986 Nov, 30(5), 749 - 55
Inactivation of cefoxitin and moxalactam by Bacteroides bivius beta-lactamase; Malouin F et al.; Moxalactam and cefoxitin are known for their high stability against Bacteroides beta-lactamases . We investigated the beta-lactamase activity of crude extracts obtained from three strains of Bacteroides bivius and two strains of Bacteroides fragilis against cefoxitin and moxalactam . In a spectrophotometric antibiotic assay with a 24-h incubation period, B . bivius extracts decreased the initial concentration (10 micrograms/ml) of moxalactam and cefoxitin by 60%, whereas B . fragilis extracts had no effect . In a microbiological assay, when B . bivius or B . fragilis extracts were added to cephalothin (10 micrograms/ml) or cefamandole (4 micrograms/ml), we observed complete disappearance of the inhibitory zones against the indicator strain (Clostridium perfringens ATCC 13124) . Only the B . bivius extracts were able to decrease the inhibitory activity (from 10 to 100%) of cefoxitin and moxalactam (each at 10 micrograms/ml) . Prior addition of clavulanic acid to crude extracts prevented the losses of antibacterial activity . Furthermore, the inhibition of the beta-lactamase hydrolysis of nitrocefin by cefoxitin or moxalactam was prevented by a 12-h preincubation of the beta-lactam with the B . bivius extracts but not with the B . fragilis extracts . Finally, with the B . bivius strain producing the most beta-lactamase, we showed an effect of inoculum size on the MICs of cefoperazone, cefoxitin, and moxalactam with a broth dilution technique . Increasing the inoculum size with the B . fragilis strains had no effect on the MISs of cefoxitin and moxalactam . These results indicate a slow and clavulanate-sensitive beta-lactamase activity of B . bivius extracts against cefoxitin and moxalactam.

Steroids, 1986 Nov-Dec, 48(5-6), 381 - 94
The 21-acetylation of corticosteroids by Clostridium sporogenes; Winter J et al.; A strain of Clostridium sporogenes, an anaerobic bacterium, isolated from sewage in New York City synthesizes two constitutive enzymes with action on steroid molecules: (i) an enzyme capable of selectively acetylating the 21-hydroxyl function of certain steroids and (ii) the corresponding esterase . Under our experimental conditions the enzymes have a strict structural requirement for 3-keto-4-ene and C-20-keto or 20 alpha-hydroxyl group and convert their respective substrates to a mixture of free and acetylated products.

Ann Inst Pasteur Microbiol, 1986 Nov-Dec, 137B(3), 271 - 82
Major protein components in the cell envelope of Clostridium tyrobutyricum; Bergere JL et al.; The overall composition of the Clostridium tyrobutyricum cell envelope did not vary significantly during cell growth and was characterized by a high protein content (about 40% dry weight) . Teichoic and teichuronic acids were absent and the neutral sugar content low . Insoluble peptidoglycan represented only 10-12% of the cell envelope (dry weight basis); it contained glucosamine, muramic acid, alanine, diaminopimelic acid and glutamic acid (molecular ratio 1/1/2/1/1) . SDS-PAGE revealed the presence of about 50 proteins in this cell envelope; however, one high molecular weight protein was largely predominant . They were not covalently bound to the peptidoglycan and their relative amounts were practically constant through cell growth and with various extraction treatments . A brief heat treatment of whole cells in PBS caused selective release of the major cell envelope proteins together with flagellin; this method was used to characterize these proteins in 37 strains of C . tyrobutyricum and some other clostridia . The major envelope proteins had molecular weights ranging from 96 to 145 Kd and the flagellins from 32 to 72 Kd.

J Infect, 1986 Nov, 13(3), 245 - 53
Pathogenicity of Clostridium species with other bacteria in mixed infections; Brook I et al.; The relationship of clostridial isolates with other bacteria in mixed infections was studied by means of a subcutaneous abscess model in mice . We used 26 isolates of seven clostridial species, two Bacteroides spp., eight Gram-positive facultative or anaerobic cocci and three enteric Gram-negative aerobic rods . Abscesses were induced by all seven Clostridium perfringens and three C . butyricum isolates and by some of the others . Selective antimicrobial therapy experiments showed that enteric Gram-negative rods were of equal or greater significance in the formation of abscesses than were clostridial strains in mixed infections . Enhancement or suppression of each component of the mixed infection was studied by comparing the number of each bacterium to its number when injected alone . Enhancement was observed mainly with C . perfringens in mixed infections . By contrast, other Clostridium spp . were less able to induce enhancement . Clostridium difficile and C . sporogenes often inhibited other bacterial species . This study demonstrated the synergistic and antagonistic relationship between clostridial species and other bacteria.

Biochim Biophys Acta, 1986 Oct 31, 889(1), 65 - 71
Effects of Ca2+ and other cations on the action of Clostridium perfringens enterotoxin; Horiguchi Y et al.; We investigated the role of extracellular Ca2+ in the Clostridium perfringens enterotoxin-induced alteration of the permeability of the plasma membrane . Enterotoxin released 86Rb and 51Cr from the Vero cells preloaded with the isotope . In the presence of EGTA, however, it released 86Rb but not 51Cr . The binding of enterotoxin to the cells was not influenced by Ca2+ or Mg2+ . The effects of various cations on the enterotoxin-induced 51Cr release was also studied . The release depended on extracellular Ca2+ but not on Mg2+; it was inhibited by each of Zn2+, La3+ and Co2+ . Zn2+ and Co2+ also inhibited 51Cr release caused by the enterotoxin previously bound to the cell membrane . In contrast, antibody against enterotoxin did not neutralize the toxin once it was bound to the Vero cells . When the cells were treated with enterotoxin, 45Ca influx occurred and reached the plateau in a few minutes, as did 86Rb release.

J Biol Chem, 1986 Oct 15, 261(29), 13536 - 41
An EPR and electron nuclear double resonance investigation of carbon monoxide binding to hydrogenase I (bidirectional) from Clostridium pasteurianum W5; Telser J et al.; Previous Mossbauer and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I (bidirectional) from Clostridium pasteurianum W5 demonstrated that this enzyme contains two diamagnetic {4Fe-4S}2+ clusters and an iron-sulfur center of unknown structure and composition that is characterized by its novel Mossbauer and ENDOR properties . In the present study we combine ENDOR and EPR measurements to show that the novel cluster contains 3-4 iron atoms . In addition, we have used EPR and ENDOR spectroscopies to investigate the effect of binding the competitive inhibitor carbon monoxide to oxidized hydrogenase I, using 13C-labeled CO and enzyme isotopically enriched in 57Fe . Treatment of oxidized enzyme with CO causes the g-tensor of the paramagnetic center to change from rhombic to axial symmetry . The observation of a 13C signal by ENDOR spectroscopy and analysis of the EPR broadening show that a single CO covalently binds to the paramagnetic center . The 13C hyperfine coupling constant (Ac approximately equal to 21 MHz) is within the range observed for inorganic iron-carbonyl clusters . The observation of 57Fe ENDOR signals from two types of iron site ({A1c} approximately 30-34 MHz; {A2c} approximately 6 MHz) and resolved 57Fe hyperfine interactions in the EPR spectrum from two nuclei characterized by {A1c} confirm that the iron-sulfur cluster remains intact upon CO coordination, but show that CO binding greatly changes the 57Fe hyperfine coupling constants.

Biochemistry, 1986 Oct 7, 25(20), 6054 - 61
Amino acid sequence of {2Fe-2S} ferredoxin from Clostridium pasteurianum; Meyer J et al.; The complete amino acid sequence of the {2Fe-2S} ferredoxin from the saccharolytic anaerobe Clostridium pasteurianum has been determined by automated Edman degradation of the whole protein and of peptides obtained by tryptic and by staphylococcal protease digestion . The polypeptide chain consists of 102 amino acids, including 5 cysteine residues in positions 11, 14, 24, 56, and 60 . The sequence has been analyzed for hydrophilicity and for secondary structure predictions . In its native state the protein is a dimer, each subunit containing one {2Fe-2S} cluster, and it has a molecular weight of 23,174, including the four iron and inorganic sulfur atoms . The extinction coefficient of the native protein is 19,400 M-1 cm-1 at 463 nm . The positions of the cysteine residues, four of which are most probably the ligands of the {2Fe-2S} cluster, on the polypeptide chain of this protein are very different from those found in other {2Fe-2S} proteins, and in other ferredoxins in general . In addition, whole sequence comparisons of the {2Fe-2S} ferredoxin from C . pasteurianum with a number of other ferredoxins did not reveal any significant homologies . The likely occurrence of several phylogenetically unrelated ferredoxin families is discussed in the light of these observations.

Biochemistry, 1986 Oct 7, 25(20), 6048 - 53
Cold-labile hemolysin produced by limited proteolysis of theta-toxin from Clostridium perfringens; Ohno-Iwashita Y et al.; A nicked toxin whose hemolytic activity is temperature dependent was obtained by limited proteolysis of theta-toxin (Mr 54,000) with subtilisin . The nicked toxin (C theta) is a complex of two fragments: the N-terminal fragment (Mr 15,000) with basic isoelectric point and the C-terminal fragment (Mr 39,000) with the single cysteinyl residue of the toxin whose reduced form is essential for the hemolytic activity . C theta hemolyzes erythrocytes only at temperatures above 25 degrees C, whereas the native toxin hemolyzes them even at 10 degrees C . At temperatures below 25 degrees C, C theta does not hemolyze them although it does bind to membrane cholesterol and although no distinct difference was observed between the secondary structure of C theta and that of native toxin . It was found that C theta binds to the cells only in a reversible manner at low temperature, while the native one binds irreversibly to the cells within 10 min, which explains the cold lability of C theta on hemolysis . The structural basis of the cold lability was discussed through comparison of C theta with another nicked derivative of theta-toxin that was also obtained.

J Steroid Biochem, 1986 Oct, 25(4), 561 - 6
Influence of 1-double bond and 11 beta-hydroxy group on stereospecific microbial reductions of 4-en-3-oxo-steroids; Kaufmann G et al.; The yeast Rhodotorula glutinis and the anaerobic bacterium Clostridium paraputrificum were used for stereospecific reductions of 4-chloro-11 beta-hydroxy-17 alpha-methyl-testosterone and the corresponding 1-dehydro compound to prepare 5 alpha- and 5 beta-H derivatives, respectively . C . paraputrificum was able to 5 beta-reduce both substances, whereas the 5 alpha-reduction by R . glutinis was inhibited by the structure elements 1-en and 11 beta-OH so that the substrate with both structure elements was not 5 alpha-reduced . The microbial conversion of the four steroids with and without 1-en and 11 beta-OH was compared in semiquantitative experiments . A number of new substances are described, 11 beta-hydroxy and 11-oxo derivatives of 5 alpha- and 5 beta-dihydro-4-chloro-17 alpha-methyltestosterone including some 3-OH compounds and characterized by NMR, mass spectrometric and further data.

Am J Med, 1986 Oct, 81(4), 596 - 600
Emergence of Clostridium tertium as a pathogen in neutropenic patients; Thaler M et al.; Although usually considered a non-pathogen, Clostridium tertium was isolated from 10 immunosuppressed patients including seven patients with bacteremia . This organism can grow aerobically and can be easily disregarded as a contaminant . It also has a somewhat unusual susceptibility pattern, with significant resistance to the penicillins, cephalosporins, and clindamycin, possibly explaining its emergence in immunocompromised patients already receiving multiple antibiotics.

Am J Gastroenterol, 1986 Oct, 81(10), 940 - 3
Clostridium difficile culture-positive toxin-negative diarrhea; Lashner BA et al.; Antibiotic-associated colitis (AAC) is confirmed by the isolation of Clostridium difficile cytotoxin from stool in patients with diarrhea . Culture of the organism has not been required to confirm the diagnosis . A review of cases of C . difficile culture-positive patients was performed in an attempt to clarify the significance of culture-positive toxin-negative (CPTN) compared to culture-positive toxin-positive (CPTP) disease . During an 11-month period, 45 patients were identified who had stool cultures positive for C . difficile . Sixteen of the patients studied were CPTP and 29 were CPTN . There were no major differences between the two groups for underlying diseases, antibiotic exposure, or diagnostic testing . Of the CPTP patients, 10 were treated for AAC and all responded . Two untreated patients resolved spontaneously . Of the CPTN patients, none was given specific antibiotic therapy, symptoms spontaneously resolved in 17, and symptoms were unresolved in five (colectomy or expired before resolution) . A prospective analysis was performed of all C . difficile isolated from stool samples by the microbiology laboratory . Isolates were incubated in vitro and cytotoxin production was measured . Of isolates from CPTP patients 97% produced cytotoxin compared to 67% of isolates from CPTN patients (p less than 0.005) . The results suggest that C . difficile, despite the absence of cytotoxin, may be an etiological factor in certain diarrheal syndromes . Until a randomized therapeutic trial for CPTN patients is conclusive, a positive culture should be considered evidence for treatment of patients with persistent diarrhea.

J Antimicrob Chemother, 1986 Oct, 18 Suppl C, 141 - 7
Some aspects of the occurrence of resistant bacteria in the normal animal flora; Wray C; The paper outlines some of the problems encountered in assessing the occurrence of antibiotic resistance in the normal flora of animals, using Escherichia coli as an example . Additional information is provided on the occurrence and mechanisms of resistance in Clostridium perfringens, Pasteurella haemolytica and Staphylococcus aureus, and some of the factors which may affect this resistance . The paper concludes with general considerations about the choice of organism, the sample size and the desirability of designing standard protocols.

EMBO J, 1986 Oct, 5(10), 2495 - 502
Tetanus toxin: primary structure, expression in E . coli, and homology with botulinum toxins; Eisel U et al.; A pool of synthetic oligonucleotides was used to identify the gene encoding tetanus toxin on a 75-kbp plasmid from a toxigenic non-sporulating strain of Clostridium tetani . The nucleotide sequence contained a single open reading frame coding for 1315 amino acids corresponding to a polypeptide with a mol . wt of 150,700 . In the mature toxin molecule, proline (2) and serine (458) formed the N termini of the 52,288 mol . wt light chain and the 98,300 mol . wt heavy chain, respectively . Cysteine (467) was involved in the disulfide linkage between the two subchains . The amino acid sequences of the tetanus toxin revealed striking homologies with the partial amino acid sequences of botulinum toxins A, B, and E, indicating that the neurotoxins from C . tetani and C . botulinum are derived from a common ancestral gene . Overlapping peptides together covering the entire tetanus toxin molecule were synthesized in Escherichia coli and identified by monoclonal antibodies . The promoter of the toxin gene was localized in a region extending 322 bp upstream from the ATG codon and was shown to be functional in E . coli.

Infect Immun, 1986 Oct, 54(1), 260 - 1
Lyophilized airborne Clostridium botulinum spores as inocula that intestinally colonize antimicrobially pretreated adult mice; Sugiyama H et al.; Adult mice, made susceptible to Clostridium botulinum by feedings of metronidazole, were immobilized with an anesthetic and held for 30 min in isolators in which a fine powder of lyophilized pathogen spores was made airborne . Exposed mice were surface decontaminated before being kept for 2 days in holding isolators . Mice were intestinally colonized by the pathogen . Colonization rates were related to spore numbers (10(4) to 10(7) type A or B) seeded into isolators.

Am J Dis Child, 1986 Oct, 140(10), 1013 - 4
Infant botulism . Three cases in a small town; Istre GR et al.; Through Dec 31, 1985, there have been six cases of infant botulism reported in Colorado . Three of these infants have lived in the same town of 800 people in western Colorado . Two of these three infants developed infant botulism within a six-month period in late 1981 . The infants lived approximately 400 m apart; they had used the same crib at the time each developed botulism . A specimen from the crib yielded Clostridium botulinum, as did four soil samples from the town and house-dust samples from the home of a relative of the second infant . The third infant developed infant botulism in September 1984 . This infant had not shared the crib . In this case, all seven samples of soil from various locations in the town yielded C botulinum, as did a sample of house dust from the home of this infant . The occurrence of these three cases in such a small town seems unlikely to be only coincidental . Investigations and reports of other such clusters may provide insight into modes of transmission of infant botulism.

Orthop Rev, 1986 Oct, 15(10), 658 - 63
Nontraumatic clostridial myonecrosis, a case report; Jamison JP et al.; Physicians are aware of the association between massively contaminated wounds and clostridial myonecrosis, or gas gangrene . A far less common but equally devastating presentation is that of nontraumatic or spontaneous gas gangrene . The most frequently encountered organism in this rare form of gangrene is Clostridium septicum, and there is a high correlation with hematologic or gastrointestinal malignancy . The mainstays of treatment are intravenous antibiotics, surgical debridement, and hyperbaric oxygen therapy . The prognosis is dependent upon early recognition and institution of treatment.

J Appl Bacteriol, 1986 Oct, 61(4), 331 - 7
Purification and characterization of a streptomycete collagenase; Chakraborty R et al.; A soil streptomycete designated as Streptomyces sp . A8 produced an extracellular collagen hydrolysing enzyme that appeared to be 'true collagenase' as it degraded native collagen under physiological conditions and cleaved the synthetic hexapeptide 4-phenylazobenzyloxycarbonyl-L-prolyl-L-leucyl-glycyl-L-prolyl-D-a rginine into two tripeptides . The enzyme was purified by diethyl aminoethyl cellulose chromatography and Sephadex G-150 gel filtration . The purified enzyme had an apparent molecular weight of about 75,000 by SDS-polyacrylamide gel electrophoresis . Treatment with lithium chloride did not dissociate it into subunits . A strong inhibition was observed with chelating agents such as alpha-alpha-dipyridyl and 8-hydroxyquinoline . Ethylene diamine tetraacetate completely inhibited the enzyme activity . Among the cations tested only Ca2+ and Mg2+ enhanced the collagenase activity . Heavy metal ions like Pb2+, Ag+, Cu2+ and Zn2+ strongly inhibited the enzyme . The EDTA inhibition could be reversed with Ca2+ . Cysteine and reduced glutathione caused significant reduction in enzyme activity . Parachloromercuribenzoate and iodoacetamide had no effect on the collagenase . Amino acid analysis revealed the absence of cysteine and tyrosine . Many of the properties were the same as collagenases of Clostridium histolyticum and Vibrio alginolyticus.

J Neurochem, 1986 Oct, 47(4), 1098 - 105
Effect of phospholipases on chronic opiate action in neuroblastoma X glioma NG108-15 hybrid cells; Griffin MT et al.; Chronic treatment of neuroblastoma X glioma NG108-15 hybrid cells with opiate agonist resulted in loss of the acute opiate inhibition of adenylate cyclase activity with a concomitant increase in the enzymatic activity observable on addition of the antagonist naloxone . The role of membrane lipids in the cellular expression of these chronic opiate effects was investigated by the hydrolysis of phospholipids with various lipases . Treatment with phospholipase C from Clostridium welchii produced an enzyme concentration-dependent decrease of prostaglandin E1-stimulated adenylate cyclase activity in control or etorphine-treated (1 microM for 4 h) hybrid cells . In addition, incubation of hybrid cells with phospholipase C concentrations of greater than or equal to 0.5 U/ml completely abolished the compensatory increase in adenylate cyclase activity after chronic opiate treatment . This attenuation of the increase in adenylate cyclase activity by phospholipase C could be prevented by inclusion of phosphatidylcholine but not of phosphatidic acid during the enzymatic incubations . The specificity of the phospholipids involved in expression of the chronic opiate effect could be demonstrated further by the absence of effect exhibited by phospholipase C from Bacillus cereus and phospholipase D . Hydrolysis of the acyl side chains of phospholipids with phospholipase A2 did not alter the chronic opiate effect after removal of lysophosphatides with bovine serum albumin . Because the guanylylimidodiphosphate- and NaF-sensitive adenylate cyclase activities were not affected by these phospholipase treatments, the expression of the compensatory increase in adenylate cyclase activity is mediated via an increase in the coupling between hormonal receptor and adenylate cyclase with the participation of the polar head groups of the phospholipids and not the hydrophobic side chains.

Microb Pathog, 1986 Oct, 1(5), 417 - 23
Amino groups in Clostridium perfringens epsilon prototoxin and epsilon toxin; Sakurai J et al.; Modification with succinic anhydride (SA) of Clostridium perfringens epsilon prototoxin or toxin resulted in a loss of activation by trypsin or lethal activity, respectively . The prototoxin was more sensitive to succinylation than the toxin . On the other hand, the succinylated prototoxin was activated and cleaved by chymotrypsin, but not by trypsin . The lethal activity of the toxin was also lost after treatment with 2,3-dimethylmaleic anhydride (DMA) or 2,4,6-trinitrobenzenesulfonic acid (TNBS) . When the DMA-treated toxin treated with SA or TNBS was incubated under acidic condition, it regained lethal activity . Thus modification of amino groups (lysine residues) prevented activation of the prototoxin by trypsin, and abolished lethal activity of the toxin.

J Gen Microbiol, 1986 Oct, 132 ( Pt 10), 2893 - 8
A scanning electron microscope study of the effect of an enterotoxin from Clostridium perfringens 8-6 on mice of different ages; Lindsay JA et al.; Intestinal damage to mice caused by an enterotoxin from a coatless spore mutant of Clostridium perfringens type A (8-6) was examined by scanning electron microscopy . Two distinct types of damage were observed, both of which could be correlated with animal age . Damage appeared to occur in a specific sequence similar to that found in previous studies in rabbits . We conclude that the type of ileal tissue damage reflects the mode of toxin incorporation from the gut, which is a function of animal age.

Anal Biochem, 1986 Oct, 158(1), 158 - 64
Analysis of sialidase and N-acetylneuraminate pyruvate-lyase substrate specificity by high-performance liquid chromatography; Shukla AK et al.; A rapid and sensitive assay by high-performance liquid chromatography for determination of the activity and substrate specificity of sialidase (EC 3.2.1.18) and N-acetylneuraminate lyase (EC 4.1.3.3) is described . Sialic acids were separated on a strong anion-exchange resin using 0.75 mM sodium sulfate as elution medium . This method allows the determination of a minimum amount of 200 pg (0.6 pmol) of sialic acid . Usually the enzyme mixtures were directly applied to the column without prior purification of substrates and products . The action of sialidase was studied either by the decrease of sialyllactose concentration or by the amount of sialic acid liberated . The relative hydrolysis rates of N-acetylneuraminyl-alpha(2-3)-lactose, N-glycolylneuraminyl-alpha(2-3)-lactose, N-acetylneuraminyl-alpha(2-6)-lactose, N-acetyl-9-O-acetylneuraminyl-alpha(2-3)-lactose, and N-acetyl-4-O-acetylneuraminyl-alpha(2-3)-lactose by Vibrio cholerae sialidase were 100, 88, 25, 12, and 0, respectively . The activity of N-acetylneuraminate lyase from Clostridium perfringens was determined by measuring the rate of disappearance of sialic acids or the formation of acylmannosamines, which is possible in the same chromatogram . Relative cleavage rates of N-acetylneuraminic acid, N-glycolylneuraminic acid, N-acetyl-9-O-acetylneuraminic acid, N-acetyl-7-O-acetylneuraminic acid, and N-acetyl-4-O-acetylneuraminic acid were found to be 100, 67, 24, 3, and 0, respectively . Comparison of the substrate specificities shows that substituents on the neuraminic acid molecule influence the reactions of both enzymes in a similar way.

Appl Environ Microbiol, 1986 Oct, 52(4), 969 - 70
Evaluation of the diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin; Jackson SG et al.; The diagnostic application of an enzyme immunoassay for Clostridium perfringens type A enterotoxin was evaluated . Test results from 100 individuals associated with C . perfringens gastroenteritis outbreaks and 111 control individuals were included . The assay sensitivity was 93.7%, and the assay specificity was 98.7%.

Am Fam Physician, 1986 Oct, 34(4), 130 - 6
Anaerobic infections in childhood; Brook I; Bacteroides melaninogenicus and Bacteroides oralis are predominant anaerobes in orofacial infections and aspiration pneumonia . Fusobacterium species are common pathogens in aspiration pneumonia, brain abscesses and orofacial infections . Clostridium perfringens can cause bacteremia and wound infections . Clostridium botulinum can produce a paralytic toxin that causes a paralytic syndrome in infants . Clostridium difficile can cause diarrhea or antibiotic-associated colitis.

Surg Gynecol Obstet, 1986 Oct, 163(4), 310 - 4
The significance of clostridial isolates in intra-abdominal sepsis; Jaggers RL et al.; In order to evaluate the significance of clostridial species in intra-abdominal infections, the bacteriology records of three hospitals were reviewed during a period of five years . Included in this report were 41 patients from whom clostridial species were recovered from specimens of free peritoneal fluid, abscess cavities or bile . Seven patients died for a mortality rate of 17.1 per cent . Most patients had polymicrobial infections of which clostridial organisms were one of the several anaerobes isolated . Clostridium perfringens was the single most frequently noted species, identified in 23 of the patients, but it was not associated with a different mortality rate than was observed for the other clostridial species . Clostridial bacteremia was uncommon and demonstrated in only one patient . The mean age of the patients was 56.4 years; 56.2 for males and 56.8 for females . Neither age nor sex of the patient influenced the likelihood of survival . The source of the clostridial isolates--bile, abscess cavity or free peritoneal fluid--had no effect upon the outcome . Several underlying conditions were responsible for the intraperitoneal clostridial organisms identified in this series . Only mesenteric infarction proved significantly predictive of a fatal result . Antibiotic coverage specifically directed against clostridia did not influence survival.

J Submicrosc Cytol, 1986 Oct, 18(4), 773 - 81
The surface charge of Toxoplasma gondii: a cytochemical and electrophoretic study; Cintra WM et al.; The surface charge of Toxoplasma gondii tachyzoites was evaluated by means of binding of colloidal iron hydroxide particles at pH 1.8, cationized ferritin particles at pH 7.2 and ruthenium red to the cell surface, as visualized by electron microscopy and by direct measurements of the electrophoretic mobility (EPM) of the cells suspended in solutions of different ionic strength and pH . At pH 7.2, T . gondii has a negative surface charge with a mean EPM of--1.1272 +/- 0.0917 micron.s-1 X V-1 X cm . No significant difference was observed between the EPM of living cells at 25 degrees C and that of glutaraldehyde-fixed cells . At lower pH, there is a decrease in the negative surface charge, with an isoelectric point at pH 3.5 . At higher pH (greater than 10), there is an increase in the surface charge reaching an EPM of--1.5675 +/- 0.0848 micron.s-1 X V-1 X cm at pH 7.2 . These results indicate that the surface of T . gondii contains both negatively and positively charged dissociating groups . Binding of cationized ferritin particles and ruthenium red throughout the cell surface of glutaraldehyde-fixed cells was observed . However, when living parasites were incubated at 4 degrees C in the presence of cationized ferritin some cells showed a uniform distribution of the label, others showed a patch-distribution and still in others no label was seen, indicating a process of mobility and shedding of surface anionic sites . Colloidal iron hydroxyde particles did not bind to the surface of T . gondii . Incubation of the parasites in the presence of neuraminidase from Clostridium perfringens or Vibrio cholerae or in the presence of proteolytic enzymes (trypsin or protease) did not interfere with the surface charge.(ABSTRACT TRUNCATED AT 250 WORDS)

J Appl Physiol, 1986 Oct, 61(4), 1418 - 30
Effect of proteolytic enzymes on transepithelial solute transport; Niewoehner DE et al.; The effects of proteases on air-space clearance (AC) of small ({14C}sucrose, 342 daltons) and large (125I-neutral dextran, 70,000 daltons) solutes were studied in isolated, fluid-filled hamster lungs that were perfused in a nonrecirculating system . When instilled into the air spaces, porcine pancreatic elastase (0.1-0.4 mg/ml) and bovine pancreatic trypsin (BPT) (0.5-2.0 mg/ml), but neither Clostridium histolyticum collagenase (5.0 mg/ml) nor phenylmethylsulfonyl fluoride-inactivated BPT caused large increases in the AC of both tracer molecules . BPT-induced solute clearance was further characterized functionally and morphologically . The functional characteristics of solute AC under steady-state conditions did not indicate that transepithelial transport was diffusion-limited . Inhibition by millimolar concentrations of Zn2+ and by lung cooling, along with electron microscopic studies employing horseradish peroxidase as a macromolecule tracer, were consistent with epithelial solute transport by a vesicular mechanism (transcytosis) . Solute transport from the interstitial compartment to the lung exterior was shown to occur via two pathways . By unknown mechanisms BPT caused small amounts of water to flow through an incompletely identified, extravascular pathway . In BPT-exposed lungs efflux of 125I-dextran 70 occurred almost exclusively through this pathway, whereas {14C}sucrose was transported to the lung exterior partly through this same pathway and partly through the vasculature . The large differences in the diffusion coefficients of the two tracers may have accounted for these observed patterns of solute efflux from the lung . The possible significance of our findings to the pathogenesis of experimental emphysema are discussed.

Infect Immun, 1986 Oct, 54(1), 70 - 6
Characterization of toxins A and B of Clostridium difficile with monoclonal antibodies; Lyerly DM et al.; Two monoclonal antibodies (MAbs) were used to learn more about the structures of Clostridium difficile toxins A and B . One of the antibodies, the PCG-4 MAb, reacted specifically with toxin A . This MAb precipitated toxin A and neutralized the enterotoxic but not the cytotoxic activity of the toxin . The site to which the antibody bound was resistant to denaturation with sodium dodecyl sulfate; however, it was destroyed by N-bromosuccinimide . Immunoblot analysis with the PCG-4 MAb revealed the presence of a large number of bands in preparations of denatured toxin A, suggesting that toxin A exists as an aggregate of smaller components . The antibody was covalently coupled to Affi-Gel 10, and the gel was used to purify toxin A from the culture filtrate of a highly toxigenic strain of C . difficile by immunoaffinity chromatography . The second antibody, the G-2 MAb, cross-reacted with toxins A and B . The cross-reaction was confirmed by immunoblot analysis . These results show that toxins A and B share an epitope and suggest that they have a common subunit . The G-2 MAb did not neutralize or precipitate either toxin . The site to which the G-2 MAb bound was partially destroyed by sodium dodecyl sulfate and was resistant to oxidation with N-bromosuccinimide.

Biochem Pharmacol, 1986 Sep 15, 35(18), 3031 - 8
Effects of phospholipases C on the beta-receptor-adenylate cyclase system of chick erythrocyte membranes; Nakajima M et al.; The beta-adrenergic receptor located in chick erythrocyte membranes was characterized using (-)-{3H}-dihydroalprenolol ({3H}-DHA) with rapid filtration techniques . The affinity of beta-adrenergic antagonist, (-)-propranolol, was approximately 100-fold higher than that of (+)-propranolol . Catecholamines were bound with the receptor in the following order, (-)-isoproterenol greater than (-)-norepinephrine greater than (-)-epinephrine, suggested the binding site to be beta 1-classification . When the membrane preparation was treated with phosphatidylcholine-hydrolyzing phospholipase C (PCase) of Clostridium perfringens or phosphatidylinositol-specific phospholipase C (PIase) of Bacillus thuringiensis, {3H}-DHA binding was decreased to the level of 66 or 86% of the control, respectively . The treatment with sphingomyelinase C (SMase) of Bacillus cereus, however, did not cause any appreciable reduction of {3H}-DHA binding . Throughout these experiments, the equilibrium dissociation constant (KD) of {3H}-DHA was not influenced by phospholipases C . The affinity of isoproterenol for beta-receptor was decreased in the absence of GTP, but not altered in the presence of GTP by PIase action . Treatment with PCase or SMase, however, did not affect the affinity of isoproterenol for beta-receptor . Treatment with PIase inhibited basal, isoproterenol-stimulated and forskolin-stimulated adenylate cyclase activities . On the other hand, PCase treatment inhibited only isoproterenol-stimulated adenylate cyclase activity, but not basal and forskolin-stimulated activities . These results suggest that membrane phospholipids, especially phosphatidylcholine (PC) and phosphatidylinositol (PI), are directly related to the receptor binding and that PI interacts with adenylate cyclase activity.

Biochim Biophys Acta, 1986 Sep 11, 860(3), 620 - 5
Difference in phospholipid dependence between two isozymes of brain (Na+ + K+)-ATPase; Matsuda T et al.; The effect of phospholipase C on two isozymes (alpha (+) and alpha forms) of rat brain (Na+ + K+)-ATPase and the temperature-dependence of their activities were investigated . Phospholipase C from Clostridium welchii inhibited the activities of the enzymes treated with and without pyrithiamin or N-ethylmaleimide, a preferential inhibitor of the alpha (+) form, but the extent of the inhibition was higher in the control enzyme than in the treated enzymes . The treatment of the (Na+ + K+)-ATPase with phospholipase C altered a ratio between high- and low-affinity components for ouabain inhibition . It also caused the similar change in a ratio between the alpha (+) and alpha forms of Na+-stimulated phosphorylation from {gamma-32P}ATP . These findings indicate that the alpha (+) form of rat brain (Na+ + K+)-ATPase is more sensitive to phospholipase C than the alpha form . Analysis of Arrhenius plots of the activities of the control and pyrithiamin-treated enzymes showed that there was a difference between the two enzymes in a break point . We suggest that two isozymes of rat brain (Na+ + K+)-ATPase differ in the interaction with phospholipids or in the lipid-environment.

Biochemistry, 1986 Sep 9, 25(18), 5189 - 95
Purification of human collagenases with a hydroxamic acid affinity column; Moore WM et al.; Human collagenase has been isolated from skin fibroblasts and rheumatoid synovium by using an affinity matrix, prepared by coupling Pro-Leu-Gly-NHOH to agarose . Following the methodology described herein, the skin enzyme was isolated in two steps in 76% yield and the synovial enzyme was purified in three steps in 71% yield . Importantly, each enzyme hydrolyzed collagen into 3/4-1/4 cleavage fragments, indicating that a true collagenase had been isolated . The column was specific for the human enzyme since the collagenase from Clostridium histolyticum did not bind . The affinity ligand was designed according to the formalism proposed by Holmquist and Vallee {Holmquist, B., & Vallee, B . L . (1979) Proc . Natl . Acad . Sci . U.S.A . 76, 6216} that effective metalloenzyme inhibitors can be synthesized by coupling a suitable metal-coordinating group to a substrate analogue . In this case, the hydroxamic acid probably coordinates to the active-site metal and the Pro-Leu-Gly moiety is similar to the carboxyl side of the cleavage site of collagen, the enzyme's substrate . The IC50 for N-(benzyloxycarbonyl)-Pro-Leu-Gly-NHOH is 4 X 10(-5) M for both enzymes . The affinity chromatographic procedures described here should aid in future studies on vertebrate collagenases.

Mikrobiologiia, 1986 Sep-Oct, 55(5), 804 - 7
{Biological activity of a polar lipid of Clostridium butyricum spores}; Gaenko GP et al.; A fraction of polar lipids was isolated from spores of the anaerobic bacterium Clostridium butyricum 35/11 exerting a noticeable radioprotective effect . The main biological activity of spore extracts was associated with this fraction . The fraction of polar lipids inhibited autolysis of the bacterial cell walls . The fraction was found to contain a phenolic glycolipid and a peptide component . The bacteriostatic and radiotherapeutic properties of the fraction are presumed to be due to its membranotropic activity.

J Antibiot (Tokyo), 1986 Sep, 39(9), 1205 - 10
Lustromycin, a new antibiotic produced by Streptomyces sp; Tomoda H et al.; A new antibiotic, lustromycin, was isolated from the cultured broth of Streptomyces sp . SK-1071 . It exhibits selective antibacterial activity against anaerobic bacteria including Clostridium sp . The molecular formula C32H38O13 as determined by high resolution mass spectrometry, and elemental analysis and the NMR spectrum suggest structural resemblance of this antibiotic to luminamicin, an anti-anaerobic antibiotic reported previously.

Pediatr Infect Dis, 1986 Sep-Oct, 5(5), 550 - 6
Anaerobic osteomyelitis in children; Brook I; Twenty-six pediatric patients with osteomyelitis caused by anaerobic bacteria are presented . The etiologic factors were chronic mastoiditis (7 patients), decubitus ulcers (5), chronic sinusitis (4), periodontal abscesses (3), bites (3), paronychia (2), trauma (1) and scalp infection after fetal monitoring (1) . Seventy-four organisms (2.8 isolates/specimen), including 63 anaerobes (2.4/specimen), and 11 facultative and aerobic bacteria (0.4/specimen) were recovered . The predominant organisms were anaerobic cocci (29 isolates), Bacteroides sp . (21), Fusobacterium sp . (8), Streptococcus sp . (5) and Clostridium sp . (4) . The organisms generally reflected the microbial flora of the mucous surface adjacent to the infected site . Ten beta-lactamase-producing organisms were recovered from 7 (27%) patients . These included all isolates of the Bacteroides fragilis (4) and of Staphylococcus aureus (3), 2 of the 12 Bacteroides melaninogenicus group and 1 of 3 Bacteroides oralis . The clinical, diagnostic and therapeutic aspects of anaerobic osteomyelitis in children are discussed.

J Bacteriol, 1986 Sep, 167(3), 828 - 36
Ultrastructure of the cell surface cellulosome of Clostridium thermocellum and its interaction with cellulose; Bayer EA et al.; The ultrastructural distribution of the cellulosome (a cellulose-binding, multicellulase-containing protein complex) on the cell surface of Clostridium thermocellum YS was examined by cytochemical techniques and immunoelectron microscopy . When cells of the bacterium were grown on cellobiose, cellulosome complexes were compacted into quiescent exocellular protuberant structures . However, when the same cells were grown on cellulose, these polycellulosomal organelles underwent extensive structural transformation; after attachment to the insoluble substrate, the protuberances protracted rapidly to form fibrous "contact corridors." The contact zones mediated physically between the cellulosome (which was intimately attached to the cellulose matrix) and the bacterial cell surface (which was otherwise detached from its substrate) . In addition, cell-free cellulosome clusters coated the surface of the cellulose substrate . The cellulose-bound cellulosome clusters appear to be the site of active cellulolysis, the products of which are conveyed subsequently to the cell surface via the exocellular contact zones.

Infect Immun, 1986 Sep, 53(3), 573 - 81
Cell surface binding site for Clostridium difficile enterotoxin: evidence for a glycoconjugate containing the sequence Gal alpha 1-3Gal beta 1-4GlcNAc; Krivan HC et al.; This study was undertaken to determine whether a binding site for Clostridium difficile enterotoxin (toxin A) exists in the brush border membranes (BBMs) of the hamster, an animal known to be extremely sensitive to the action of the toxin . Toxin A was the only antigen adsorbed by the BBMs from the culture filtrate of C . difficile . The finding that binding activity could not be destroyed by heat indicated that a carbohydrate moiety might be involved . We therefore examined erythrocytes from various animal species for binding activity since erythrocytes provide a variety of carbohydrate sequences on their cell surfaces . Only rabbit erythrocytes bound the toxin, and the cells agglutinated . A binding assay based on an enzyme-linked immunosorbent assay method for quantifying C . difficile toxin A was used to compare binding of the toxin to hamster BBMs, rabbit erythrocytes, and BBMs from rats, which are less susceptible to the action of C . difficile toxin A than hamsters . Results of this comparison indicated the following order of toxin-binding frequency: rabbit erythrocytes greater than hamster BBMs greater than rat BBMs . Binding of toxin A to hamster BBMs at 37 degrees C was comparable to what has been observed with cholera toxin, but binding was enhanced at 4 degrees C . A similar binding phenomenon was observed with rabbit erythrocytes . Examination of the cell surfaces of hamster BBMs and rabbit erythrocytes with lectins and specific glycosidases revealed a high concentration of terminal alpha-linked galactose . Treatment of both membrane types with alpha-galactosidase destroyed the binding activity . The glycoprotein, calf thyroglobulin, also bound the toxin and inhibited toxin binding to cells . Toxin A did not bind to human erythrocytes from blood group A, B, or O donors . However, after fucosidase treatment of human erythrocytes, only blood group B erythrocytes, which possess the blood group B structure Gal alpha 1-3{Fuc alpha 1-2}Gal beta 1-4GlcNAc-R, bound the toxin . This indicated that toxin A was likely binding to Gal alpha 1-3Gal beta 1-4GlcNAc, a carbohydrate sequence also found on calf thyroglobulin and rabbit erythrocytes . All of the results indicate that hamster BBMs contain a carbohydrate-binding site for toxin A that has at least a Gal alpha 1-3Gal beta 1-4GlcNAc nonreducing terminal sequence.

Obstet Gynecol, 1986 Sep, 68(3 Suppl), 26S - 28S
Clostridial myonecrosis arising from an episiotomy; Soper DE; A case of clostridial episiotomy infection is reported and the literature reviewed . Early recognition of the severity of this disease along with aggressive operative therapy is necessary to improve outcome . Clostridium sordellii is now the most common Clostridia species to be isolated from patients with these serious infections.

Can J Microbiol, 1986 Sep, 32(9), 743 - 50
Aerobic degradation of choline by Proteus mirabilis: enzymatic requirements and pathway; Sandhu SS et al.; Cleavage of choline to trimethylamine and acetaldehyde by extracts of Proteus mirabilis requires both particulate and soluble protein fractions, K+, and a bound divalent metal cation . The reaction shows a long lag period, abolished only by preincubation of the particulate fraction in the complete reaction system . The two-carbon fragment produced is acetaldehyde; choline cleavage appears to be tightly coupled to dismutation of the acetaldehyde to ethanol and acetate, as indicated by stimulation by NAD+, ADP, and Fe2+ and inhibition by reagents reacting with acetaldehyde . The system is thus similar to that previously described in anaerobes (Desulfovibrio, Clostridium) . Attempts to demonstrate a cobamide coenzyme requirement (as in the similar ethanolamine ammonia-lyase reaction) were unsuccessful; the reaction was carried out by fractions devoid of vitamin B12 activity (not supporting growth of Lactobacillus leichmannii) and insensitive to light.

J Am Vet Med Assoc, 1986 Sep 1, 189(5), 557 - 9
Splenic hematoma and abscess as a cause of chronic weight loss in a horse; Spier S et al.; An 8-year-old gelding with a 3-month history of anorexia and weight loss was found to have a massive subcapsular splenic hematoma . At flank laparotomy, 36 L of fluid was removed from the hematoma . The horse's condition improved after drainage . Fifteen months later, the horse became depressed and febrile . A splenic abscess containing Bacteroides ruminicola and Clostridium sporogenes was found at necropsy.

Appl Environ Microbiol, 1986 Sep, 52(3), 407 - 12
Enterotoxin synthesis by nonsporulating cultures of Clostridium perfringens; Goldner SB et al.; Chemostat-cultured Clostridium perfringens ATCC 3624 and NCTC 10240, and a nonsporulating mutant strain, 8-5, produced enterotoxin in the absence of sporulation when cultured in a chemically defined medium at a 0.084-h-1 dilution rate at 37 degrees C . The enterotoxin was detected by serological and biological assays . Examination of the chemostat cultures by electron microscopy did not reveal sporulation at any stage . The culture maintained enterotoxigenicity throughout cultivation in a continuous system . The enterotoxin was detected in batch cultures of each strain cultivated in fluid thioglycolate medium and a chemically defined medium . No heat-resistant or light-refractile spores were detected in batch cultures during the exponential growth.

J Am Vet Med Assoc, 1986 Sep 1, 189(5), 564 - 5
Clostridium perfringens type C enterotoxemia in a newborn foal; Howard-Martin M et al.; A 1-day old, full-term foal with a history of colic died 2 hours after admission . Necropsy revealed an extremely flaccid, fluid-filled intestinal tract . Histopathologically, the superficial intestinal mucosa was completely necrotic, with minimal inflammatory response . Numerous large, gram-positive rods covered the villi . Clostridium perfringens was isolated on bacteriologic culturing of the intestinal tract contents and was identified as type C by toxin neutralization tests.

J Clin Microbiol, 1986 Sep, 24(3), 384 - 7
Immunoblotting to demonstrate antigenic and immunogenic differences among nine standard strains of Clostridium difficile; Heard SR et al.; The epidemiology of Clostridium difficile-associated disease is being elucidated with the development of typing schemes for the organism . We recently described a new typing scheme based on the incorporation of {35S}methionine into bacterial proteins followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography . Nine standard strains were identified . We report here some observations on the antigenic differences among these nine strains when studied by immunoblotting . Type-specific rabbit antiserum was raised against each of the nine standard strains . Immunoblotting of the strains with these antisera demonstrated, in addition to the presence of shared, common proteins, a type-specific response with homologous antisera . When {35S}methionine-labeled C . difficile proteins were immunoblotted with homologous and heterologous antisera, both the immunoblots and the autoradiographs demonstrated the same strain-specific response . These strain-specific proteins, which have been so useful for epidemiological and typing purposes, were also immunogenic.

Biochem Biophys Res Commun, 1986 Aug 29, 139(1), 64 - 70
ADP-ribosylation in cultured cells treated with Clostridium difficile toxin B; Florin I et al.; In cultured fibroblasts intoxicated with Clostridium difficile toxin B, a radioactive moiety was transferred from {14C-adenosine}NAD, but not from {14C-nicotinamide} NAD, into a cellular protein (MW 90,000) . No labeling was detected in toxin-treated cultures not yet showing any toxin-induced cytopathogenic effect, whereas maximal labeling was obtained in cultures with about half of the cells showing a cytopathogenic effect . The radioactivity was removed from the substrate by treatment with snake venom phosphodiesterase . The results suggest that ADP-ribosylation of a cellular protein occurs in toxin B-treated cells and that this reaction may be responsible for development of the cytopathogenic effect.

N Z Med J, 1986 Aug 27, 99(808), 617 - 20
Hyperbaric oxygen therapy in the management of Clostridium perfringens infections; Gibson A et al.; Patients with Clostridium perfringens infections treated in Christchurch over a 14 year period to 1984 were reviewed retrospectively . Of the 46 documented cases, 21 died . Twenty-nine patients were treated with hyperbaric oxygen (HBO) therapy . Of these, nine died, whilst 12 of 17 other patients who did not receive HBO died, including five in whom the diagnosis was only made at postmortem . The clinical features and predisposing factors for clostridial infections are discussed . The importance of early vigorous treatment with a combination of surgical debridement, antibiotics and HBO to reduce the morbidity and mortality of this lethal infection is emphasised.

N Z Med J, 1986 Aug 27, 99(808), 620 - 2
Clostridium difficile toxin in chronic idiopathic colitis; Lee DK et al.; Clostridium difficile toxin was isolated from the stools of three patients with chronic idiopathic colitis . Two patients were known to have chronic idiopathic colitis before Cl difficile toxin was isolated . The third patient was subsequently found to have ulcerative colitis after presentation with Cl difficile toxin in the stool . Two patients were on sulphasalazine at the time of diagnosis of Cl difficile infection and one had taken sulphasalazine two months previously . Only one patients had antibiotic exposure and that was at least three months before presentation . In each patient, treatment with vancomycin was accompanied by symptomatic improvement and disappearance of the toxin . The underlying colitis remained unaffected . In patients with inflammatory bowel disease in relapse, the presence of Cl difficile toxin should be sought as this may be a factor in the relapse . In any patient presenting with diarrhoea, the presence of Cl difficile toxin may obscure the presence of underlying inflammatory bowel disease.

J Biol Chem, 1986 Aug 25, 261(24), 11409 - 15
In vivo and in vitro transcription of the Clostridium pasteurianum ferredoxin gene . Evidence for "extended" promoter elements in gram-positive organisms; Graves MC et al.; Analysis of Clostridium pasteurianum genomic DNA indicates that the ferredoxin (Fd) gene is present in a single copy . The cloned Fd gene previously described (Graves, M.C., Mullenbach, G . T., and Rabinowitz, J . C . (1985) Proc . Natl . Acad . Sci . U . S . A . 82, 1653-1657) was used to map in vivo and in vitro synthesized Fd transcripts . The in vivo mRNA was sized in two ways: by Northern hybridization analysis, and more directly from the known DNA sequence after the 5'- and 3'-termini were identified . The 5'-end was determined by primer extension-dideoxy sequencing and the 3'-end by S1 nuclease mapping . The monocistronic Fd mRNA contains about 255 nucleotides and, thus, is one of the shortest bacterial mRNAs yet described . We also examined the Fd transcripts produced by Escherichia coli transformed with the plasmid containing the Fd gene . E . coli RNA polymerase most likely recognizes the same promoter (P1) as the clostridial polymerase, and furthermore, efficiently uses an additional promoter (P2) that is poorly recognized by the normal host enzyme . For comparison, in vitro transcripts were generated by E . coli and Bacillus subtilis RNA polymerases . In vitro, only promoter P1 is used by either E . coli or B . subtilis RNA polymerase . The 3'-end of each of the four types of transcripts occurs essentially at the same location and maps to within a large dyad symmetry element . Comparison of the Fd promoter with other Gram-positive promoters reveals that some sequences outside of the traditional Pribnow and -35 regions are conserved . This analysis indicates that an "extended" promoter recognition site may be required in these organisms.

J Biol Chem, 1986 Aug 25, 261(24), 11049 - 55
Amino acid sequence of the pyruvate and the glyoxylate active-site lysine peptide of Escherichia coli 2-keto-4-hydroxyglutarate aldolase; Vlahos CJ et al.; Pure 2-keto-4-hydroxyglutarate aldolase of Escherichia coli, a "lysine-type" trimeric enzyme which has the unique properties of forming an "abortive" Schiff-base intermediate with glyoxylate (the aldehydic product/substrate) and of showing strong beta-decarboxylase activity toward oxalacetate, binds any one of its substrates (2-keto-4-hydroxyglutarate, pyruvate, or glyoxylate) in a competitive manner . To determine whether the substrates bind at the same or different (juxta-positioned) sites and what degree of homology might exist between the active-site lysine peptide of this enzyme and that of other lysine-type (Class I) aldolases or beta-decarboxylases, the azomethine formed separately by this aldolase with either {14C}pyruvate or {14C}glyoxylate was reduced with CNBH3- . After each enzyme adduct was digested with trypsin, the 14C-labeled peptide was isolated, purified, and subjected to amino acid analysis and sequence determination . In each case, the same 14-amino acid lysine-peptide was isolated and found to have the following primary sequence: Glu-Phe-*Lys-Phe-Phe-Pro-Ala-Glu-Ala-Asn-Gly-Gly-Val-Lys (where * = the active-site lysine) . Hence, glyoxylate competes for, and inhibits aldolase activity by reacting with, the one active-site lysine residue/subunit . This active-site lysine peptide has a high degree (65%) of homology with that of 2-keto-3-deoxy-6-phosphogluconate aldolase of Pseudomonas putida but is not similar to that of any Class I fructose-1,6-bisphosphate aldolase or of acetoacetate beta-decarboxylase of Clostridium acetobutylicum . Furthermore, it was found that extensive reaction of glyoxylate with the N-terminal amino group of this enzyme may well be general complicating factor in sequence studies with proteins plus glyoxylate.

Orv Hetil, 1986 Aug 10, 127(32), 1923 - 8
{The role of intrauterine contraceptive devices in the development of inflammatory processes in the small pelvis}; Batar I; PIP: The incidence of pelvic inflammatory disease (PID) attributable to IUD use has been increasing, especially after the removal of the Dalkon shield from the market, but this relationship has not been settled conclusively . In recent decades PID included a variety of infections, but lately the definition of PID has meant acute ascending infections of the female genital tract . Its most common risk factors include promiscuity of IUD use, although this can be reduced to one fourth by regular checkups and proper hygiene . The frequency of PID is estimated at 2-5% of IUD users . Microorganisms contributing to PID include Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Escherichia coli, Proteus, Staphylococcus epidermis, Haemophilus influenzae, Bacteroides, Peptococcus, Peptostreptococcus, Clostridium, and Actinomyces israelii, The differentiation of actinomycosis (AC) and pseudoactinomycosis (PAC) is well advised . The potential of IUD use in increasing the risk of AIDS should not be discounted . The clinical picture of PID is varied, it can be mild requiring conservative drug therapy; with medium severity requiring removal of the IUD and drug therapy; severe necessitating removal, antibiotics and sulfonamide treatment and laparotomy; and very severe with potentially fatal generalized sepsis . In addition to antibiotics, e.g., penicillin, treatment can include the so called catastrophy combination of Mandokef- Metronidazol-Gentamycin . An analysis of the data of 8536 IUD fittings in Debrecen, Hungary showed 1.4% removals due to PID after 4 years, 694 patients (8.1%) had lower abdominal pain 73 of which (0.9%) had palpable resistance, and suppuration occurred in only 30 cases (0.4%) . Treatment included Semicillin or Tetran, or removal of the IUD, and even surgery if no improvement resulted . Prevention of PID include elimination of risk factors, the careful selection of IUD users, regular checkups, the use of copper (Cu) T device, and strict adherence to professional standards .

S Afr Med J, 1986 Aug 2, 70(3), 137 - 42
Surgery in patients with heart transplants . Anaesthetic and operative considerations; Cooper DK et al.; As cardiac transplantation becomes more common, so an increasing number of patients with functioning heart transplants may require surgery for related or unrelated non-cardiac conditions . Fifteen patients who have undergone a total of 39 operations (excluding retransplantation) since heart transplantation were reviewed; 36% were for infective conditions and 23% each for gastro-intestinal and vascular lesions . There was one postoperative death in a patient undergoing leg amputation for overwhelming Clostridium welchii infection . There were no major non-fatal complications . The conditions for which operation may be necessary, the specific problems of anaesthesia and surgery in such patients, and the prophylactic measures which may be undertaken to ensure an uncomplicated clinical course are discussed . A clear understanding of the physiology and pharmacology of the denervated heart is essential if these patients are successfully to undergo major operations requiring general anaesthesia.

J Antibiot (Tokyo), 1986 Aug, 39(8), 1054 - 8
SQ 30,957, a new antibiotic produced by Penicillium funiculosum . Taxonomy, fermentation, isolation, structure determination, synthesis and antibacterial activity; Singh PD et al.; A new antibiotic, SQ 30,957, 4-diazo-3-methoxy-2,5-cyclohexadien-1-one, has been isolated from fermentation broths of Penicillium funiculosum . The structure (1) was deduced from its spectroscopic properties and its degradation reaction . SQ 30,957 has excellent activity against anaerobic bacteria such as Clostridium and Bacteroides and has moderate activity against aerobic bacteria . The compound has an LD50 of less than 17 mg/kg in mice by intraperitoneal administration.

Eur J Clin Microbiol, 1986 Aug, 5(4), 441 - 5
Hydrophobic and adherence properties of Clostridium difficile; Wood-Helie SJ et al.; Nine strains of Clostridium difficile isolated from symptomatic and asymptomatic patients and four other species of clostridia were tested for relative hydrophobicity by determining the degree of adherence to polystyrene . Under three different conditions of growth all strains of Clostridium difficile had high rates of adherence, whereas the other clostridial species showed no pronounced adherence . Isolates of Clostridium difficile were also tested for their ability to adhere to human embryonic intestinal cells and adult colon cells . All strains adhered to both cell lines, although the percentages of organisms adhering varied . Adherence was greatest at pH 5.5-6.0 but was not significantly altered at a pH of 7.0-7.8 (p = 0.15, p = 0.20); it decreased significantly upon washing with 1% Tween 80 but not with 0.1% Tween 80 . This capacity for adherence may play a part in the organism's colonization of the human intestinal tract.

J Clin Pathol, 1986 Aug, 39(8), 861 - 2
Isolation of Clostridium difficile from human jejunum: identification of a reservoir for disease?
Testore GP, Nardi F, Babudieri S, Giuliano M, Di Rosa R, Panichi G.
The possibility that the small intestine may represent a reservoir for Clostridium difficile was studied, using segments of human jejunum collected at necropsy . Our results (three of 100 specimens positive for C difficile culture) support the hypothesis that C difficile can be found in human jejunum and that it adheres to the normal mucosa as a resident bacterium . These findings suggest that gastrointestinal disease caused by C difficile has an endogenous origin.

J Clin Microbiol, 1986 Aug, 24(2), 181 - 5
Predicting the susceptibility of anaerobes to cefoperazone, cefotaxime, and cefoxitin with the thioglycolate broth disk procedure; Zabransky RJ et al.; A variety of clinical anaerobic isolates were tested against cefoperazone (216 strains), cefoxitin (120 strains), and cefotaxime (120 strains) by the thioglycolate anaerobic broth disk method, and the results were compared with the National Committee for Clinical Laboratory Standards reference agar dilution method . The broth disk and reference breakpoint concentrations were as follows: cefoperazone, 60 and 64 or 30 and 32 micrograms/ml; cefotaxime, 30 and 32 micrograms/ml; cefoxitin, 18 and 16 micrograms/ml, respectively . Discrepant results were retested to obtain a mode . There was 99% agreement between the broth disk and reference methods for cefotaxime, 98% for cefoperazone with 60- and 64-micrograms/ml breakpoints and 91% with 30- and 32-micrograms/ml breakpoints, and 75% for cefoxitin . All but one of the strains that produced false susceptibility results by broth disk were members of the Bacteroides fragilis group, 1 with cefoperazone using the 60-micrograms/ml concentration, 14 with cefoperazone at the 30-micrograms/ml concentration, and 27 with cefoxitin . One strain of Clostridium difficile produced false susceptibility results to cefoperazone at the 30-micrograms/ml concentration . The lack of agreement between the broth disk and reference methods with cefoxitin may be a reflection of the number of isolates at the 16-micrograms/ml level and that the broth disk breakpoint was slightly higher than this concentration . Increased incubation time did not improve the results significantly.

Br J Exp Pathol, 1986 Aug, 67(4), 617 - 21
Individual variation in botulism; Smith GR; Mice were treated per os with one oral LD100 of toxic filtrate from a culture of Clostridium botulinum type C . The period between dosing and the first appearance of clinical signs varied greatly (2-31 h) from one animal to another . The duration of the pre-clinical and clinical phases together ranged from 5.5 to greater than 55 h . The duration of the clinical phase alone ranged from 1.25 to greater than 24 h, except for a minority of mice in which death occurred suddenly from apparent heart failure with no premonitory signs 4.75-31 h after dosing . Toxaemia was demonstrable in all mice that had just begun to show a clinical response 3.75-6.5 h after dosing, and in some that had not . Outside these time limits toxaemia was demonstrable only rarely, and beyond 12 h after dosing never . Therefore the many (approximately 50%) mice that began to show clinical signs more than 12 h after dosing had no demonstrable toxaemia throughout the entire clinical phase of the disease . The concentrations of toxin demonstrated in the blood ranged from less than 5 to greater than or equal to 20 (but less than 40) intravenous mouse-lethal doses/ml.

Am J Clin Pathol, 1986 Aug, 86(2), 208 - 11
Detection of Clostridium difficile toxins A (enterotoxin) and B (cytotoxin) in clinical specimens . Evaluation of a latex agglutination test; Peterson LR et al.; A new latex test, Culturette Brand Rapid Latex Test for detection of Clostridium difficile toxin A, was tested on 408 stool samples . In 247 frozen tissue culture supernate specimens previously obtained from patients with C . difficile-associated diarrhea (CAD), the latex test (enterotoxin) was positive in 182 (74%) as compared with 194 (79%) for the repeat tissue culture (P greater than 0.1) cytotoxin (toxin B) test . Testing of 161 fresh stool samples found the latex test superior to tissue culture (P less than 0.05) in cases of CAD (90% positivity vs . 70%), with the two tests being equal in both non-CAD diarrheal and non-diarrheal control groups . In vitro evaluation of 61 C . difficile isolates found all (100%) to be producers of enterotoxin A, while only 53 (87%) produced toxin B . The latex test for C . difficile toxin detection is a rapid, simple test for use in the diagnosis in CAD.

J Med Microbiol, 1986 Aug, 22(1), 33 - 8
Sporogenesis and toxin A production by Clostridium difficile; Ketley JM et al.; The kinetics of spore production by Clostridium difficile were not paralleled by release of C . difficile toxin A in vitro . Toxin A was not found to be associated with either purified whole spores or spore coats . Residual traces of toxin A detected in spore contents were almost certainly derived from contaminating vegetative cell debris . Thus, toxin A is unlikely to be a spore constituent or associated with sporogenesis.

Clin Chem, 1986 Aug, 32(8), 1503 - 5
Effect of phospholipase C on high-molecular-mass alkaline phosphatase in serum; Sykes E et al.; Electrophoresis of some serum samples on polyacrylamide gel, followed by staining for alkaline phosphatase (EC 3.1.3.1), produces a band of activity at the gel origin . This high-Mr band consists of liver membrane fragments containing alkaline phosphatase and other enzymes . Alkaline phosphatase is closely associated with phosphatidylinositol in liver plasma membranes, and we have found that phospholipase C (EC 3.1.4.3) from Bacillus cereus, known to possess some phosphatidylinositol specificity, was able to release liver alkaline phosphatase from the high-Mr band . Two preparations of phospholipase C from Clostridium perfringens, however, which has no phosphatidylinositol specificity, had no effect on the alkaline phosphatase activity in the high-Mr band.

J Infect Dis, 1986 Aug, 154(2), 207 - 11
Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy; Aureli P et al.; The first two confirmed cases of type E infant botulism occurred in two 16-week-old girls in Rome, Italy . The original diagnosis for the first patient was intestinal blockage due to an ileocecal invagination, which was treated surgically . Postoperatively, the patient became unresponsive and required ventilatory assistance . A diagnosis of infant botulism was then made . The second infant presented to the same hospital 7 1/2 months later with profound weakness, hypotonicity, mydriasis, and areflexia . This case was recognized as possible botulism at admission . Both cases were confirmed by detection and identification of type E botulinal toxin in stool specimens and in enrichment cultures of those specimens . The toxigenic organisms isolated were quite different from Clostridium botulinum type E . The apparent causative organism in each case resembles Clostridium butyricum but produces a neurotoxin that is indistinguishable from type E botulinal toxin by its effects on mice and by its neutralization with type E botulinal antitoxin.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 Aug, 262(2), 179 - 88
Molecular characterization of a protein, insoluble at low temperature, produced by Clostridium botulinum type G; Gimenez JA et al.; A preliminary study of a low-toxicity protein, called cryoprotein, produced by Clostridium botulinum type G, led to a better characterization of this substance and to discriminate its relationship with type G botulinum toxin . This sparingly soluble protein has been characterized as an aggregated form of a soluble precursor with an Mr of 170,000 . This phenomenon is temperature-dependent . The monomeric protein is usually contaminated with a lower Mr form (150,000) quite probably originated by a limited proteolytic process . The amino acid composition of this protein is relatively analogous to that of the botulinum toxins A and B, the only notable exception being the absence of cysteine . The N-terminal amino acid is alanine and the C-terminal sequence is Val-Ala-Leu-OH . The low toxicity which is usually demonstrable in samples of this protein disappears after a reductive treatment, strongly suggesting that it is not an intrinsic property . Taking into account that some of its physiochemical properties are similar to those of the known botulinal toxins, it is quite probable that this substance accompanies G toxin preparations currently obtained by routine methods, increasing its non-toxic antigenic mass . This fact could be critical to the sensitivity and specificity of G toxin immunological detection methods.

Microb Pathog, 1986 Aug, 1(4), 373 - 85
Lysosomal involvement in cellular intoxication with Clostridium difficile toxin B; Florin I et al.; The process of internalisation of Clostridium difficile toxin B into human lung fibroblasts was further studied, with the aim of elucidating the fate of endocytosed toxin . Development of the toxin-induced cytopathogenic effect was reversibly inhibited at 18 degrees C and in the presence of 200 mM KCl or 1-20 mM benzyl alcohol, i.e . at conditions when the fusion between endosomes and lysosomes is prevented . Fibroblasts treated with toxin at 37 degrees C but transferred to 18 degrees C within 10 min were also completely protected, whereas transfer to 18 degrees C later during the latency resulted in only partial protection . KCl was also protective upon addition after the toxin binding step . Inhibitors of lysosomal proteases, such as chymostatin, leupeptin and antipain, prevented the appearance of the cytopathogenic effect, when present during toxin exposure or added after the toxin binding step . Chinese hamster ovary cell mutants, defective in acidification of their endosomes, were resistant to toxin B, whereas wildtype cells were sensitive . The resistance was not overcome by applying a low extracellular pH . The results suggest that exposure to a low pH compartment is necessary but not sufficient for entry of active toxin B to the cytosol . In addition to a low pH, a fusion of toxin-containing endosomes with lysosomes and a further processing of the toxin by lysosomal proteases is required for cellular intoxication.

Mol Gen Genet, 1986 Aug, 204(2), 317 - 21
Cloning of Clostridium acetobutylicum genes and their expression in Escherichia coli and Bacillus subtilis; Efstathiou I et al.; DNA from Clostridium acetobutylicum ABKn8 was partially digested with Sau3A and the fragments obtained were inserted into the unique BamHI site of the cloning vector pHV33 . The recombinant plasmids were used to transform Escherichia coli HB101 with selection for ampicillin resistance . A collection of ampicillin-resistant, tetracycline-sensitive clones representative of the Clostridium acetobutylicum genome was made . The clones were shown to carry recombinant plasmids each containing an insert of 2 to 16 kb in size . Several of them complemented the HB101 proA2 or leuB6 auxotrophic mutations . The cloned sequences were shown by Southern blot hybridization to be homologous to the corresponding ABKn8 DNA fragments.

J Biochem (Tokyo), 1986 Aug, 100(2), 407 - 13
NAD-glycohydrolase activity of botulinum C2 toxin: a possible role of component I in the mode of action of the toxin; Ohishi I; C2 toxin (C2T) elaborated by Clostridium botulinum types C and D is composed of two separate protein components, designated components I and II, which individually have little activity, but, when mixed and treated with trypsin, exert the potent activity . The present study provides the evidence that component I of the toxin catalyzes the hydrolysis of NAD into nicotinamide and ADP-ribose, whereas component II does not, indicating that component I of C2T has NAD-glycohydrolase activity, which ability is shared with cholera and diphtheria toxins . However, C2T affected neither glycerol production of fat cells nor protein synthesis in cell-free system . Component I of C2T in the presence of {alpha-32P}NAD radiolabeled a protein of Mr 46,000 in the supernatant fractions of mouse tissue homogenates; the protein was abundant in brain, lung and intestine, whereas there was little or none of the protein in muscle . These results indicate that component I can catalyze the covalent attachment of the ADP-ribose moiety of NAD to intracellular protein, which differs from those modified with cholera and diphtheria toxins . The present data, together with previous findings, suggest that the biological activity of C2T is elicited by ADP-ribosylation activity of component I, which is internalized into the cells after binding to the receptor site introduced with the binding of component II to the cell surface membrane.

J Am Acad Dermatol, 1986 Aug, 15(2 Pt 1), 180 - 5
Effects of topical clindamycin on intestinal microflora in patients with acne; Siegle RJ et al.; Thirty-two patients with acne completed a randomized, double-blind study using topical 1% clindamycin phosphate or its vehicle applied twice daily for 8 weeks for a study of its effects on the intestinal microflora . Two clindamycin patients and one vehicle patient had Clostridium difficile in stools prior to therapy . Of the remaining twenty-nine patients, four of nineteen patients who used clindamycin and none of ten patients who used vehicle had C . difficile detected during treatment; the difference was not statistically significant . There was no diarrhea in the clindamycin group, even though clostridial cytotoxin was found transiently in two patients . Self-limited diarrhea occurred in one vehicle-treated patient, whose stool culture was negative but whose stool specimen showed a positive reaction for C . difficile cytotoxin . With the use of a bioassay, clindamycin was not detected in urine or stool of any patient . No significant changes in Bacteroides fragilis counts in stools were observed in either group.

J Lipid Res, 1986 Aug, 27(8), 905 - 9
Reaffirmation of the validity of enzymatic cleavage of lithocholic acid from N-epsilon-lithocholyl-L-lysine and N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine; Nair PP et al.; N-epsilon-lithocholyl-L-lysine or N-alpha-CBZ-N-epsilon-lithocholyl-L-lysine when incubated overnight at 37 degrees C with 3 K units of clostridial cholanoylaminoacid hydrolase (from Clostridium perfringens ATCC 19574) in the presence of disodium EDTA (0.1 M), beta-mercaptoethanol (0.1 M), and sodium acetate buffer, pH 5.6, released free lithocholic acid . The latter material was isolated by thin-layer chromatography and identified by combined gas-liquid chromatography-mass spectrometry in the full scan and selected-ion mode . In order to maintain its activity, the enzyme was always stored in 1.0-ml aliquots at temperatures below -20 degrees C and each aliquot when thawed was used immediately; any left over enzyme was never reused . Contrary to the observations of Yanagisawa et al . (J . Lipid Res . 1984 . 25: 1263-1271) the results of this study reaffirm the validity of the original observations on the enzymatic cleavage of lithocholic acid from tissue-bound form.

J Hyg (Lond), 1986 Aug, 97(1), 71 - 80
Large outbreaks of Clostridium perfringens food poisoning associated with the consumption of boiled salmon; Hewitt JH et al.; Five large outbreaks of food poisoning are described in which clinical, epidemiological or laboratory data indicated Clostridium perfringens as the causative organism . The foodstuff common to all incidents was boiled salmon served cold as an hors d 'oeuvre . In all cases the fish had been subject to a long period of cooling or storage between boiling and consumption . It is thought that multiplication of the organism occurred during this time . Recommendations are made for the avoidance of further similar incidents.

Nature, 1986 Jul 24-30, 322(6077), 390 - 2
Botulinum C2 toxin ADP-ribosylates actin; Aktories K et al.; ADP-ribosylation of regulatory proteins is an important pathological mechanism by which various bacterial toxins affect eukaryotic cell functions . While diphtheria toxin catalyses the ADP-ribosylation of elongation factor 2, which results in inhibition of protein synthesis, cholera toxin and pertussis toxin ADP-ribosylate Ns and Ni, respectively, the GTP-binding regulatory components of the adenylate cyclase system, thereby modulating the bidirectional hormonal regulation of the adenylate cyclase . Botulinum C2 toxin is another toxin which has been reported to possess ADP-ribosyltransferase activity . This extremely toxic agent is produced by certain strains of Clostridium botulinum and induces hypotension, an increase in intestinal secretion, vascular permeability and haemorrhaging in the lungs . In contrast to botulinum neurotoxins, the botulinum C2 toxin apparently lacks any neurotoxic effects . Here we report that botulinum C2 toxin ADP-ribosylates a protein of relative molecular mass 43,000 (43K) in intact cells and in cell-free preparations . We present evidence that the 43K protein substrate is actin, which is apparently mono-ADP-ribosylated by the toxin . Botulinum C2 toxin also ADP-ribosylated purified liver G-actin, whereas liver F-actin was only poorly ADP-ribosylated and skeletal muscle actin was not ADP-ribosylated in either its G form or its F form . ADP-ribosylation of liver G-actin by botulinum C2 toxin resulted in a drastic reduction in viscosity of actin polymerized in vitro.

Lancet, 1986 Jul 5, 2(8497), 11 - 3
Is Clostridium difficile endemic in chronic-care facilities?
Bender BS, Bennett R, Laughon BE, Greenough WB 3rd, Gaydos C, Sears SD, Forman MS, Bartlett JG.
An apparent outbreak of Clostridium difficile diarrhoea on the chronic hospital ward of a long-term care facility prompted an investigation lasting seven months . Approximately a third of patients had stools that were positive for C difficile by either toxin or culture . Attempts to eradicate the infection by simultaneously treating all toxin-positive patients with metronidazole, limiting antibiotic use, and implementing enteric isolation were unsuccessful . New cases were both nosocomially acquired and imported into the facility . Of the C difficile toxin-positive patients, 34% had diarrhoea and 19/49 (38%) died during the study period . C difficile is not routinely sought by most clinical microbiology laboratories and may therefore be endemic in many long-term care facilities for the elderly.

Int J Pediatr Nephrol, 1986 Jul-Sep, 7(3), 173 - 6
Fever due to Clostridium difficile during hemodialytic treatment; Agrawal R et al.; A ten-year-old on hemodialysis had a prolonged unexplained fever secondary to Clostridium difficile antibiotic-associated colitis and posed a great diagnostic challenge.

Lab Anim, 1986 Jul, 20(3), 266 - 70
Isolation of Clostridium difficile from various colonies of laboratory mice; Itoh K et al.; An attempt was made to isolate Clostridium difficile from a total of 565 mice from nine different conventional mouse colonies and six different specified-pathogen-free mouse colonies . C . difficile was isolated from all the conventional colonies but from none of the specified-pathogen-free colonies . Ampicillin injected intraperitoneally increased the isolation rate of C . difficile from mouse faeces to 63.6% compared with 19.4% from untreated mice.

J Biochem (Tokyo), 1986 Jul, 100(1), 27 - 33
Binding of Clostridium botulinum neurotoxin to gangliosides; Ochanda JO et al.; The binding characteristics of Clostridium botulinum neurotoxins of types B, C1, and F to gangliosides was studied by thin layer chromatography plate and microtiter plate methods at low (10 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) or high (150 mM NaCl in 10 mM Tris-HCl buffer, pH 7.2) ionic strengths and at 0 or 37 degrees C . The three types of toxins bound exclusively to three kinds of gangliosides, GD1a, GD1b, and GT1b, in both the thin layer chromatography plate and the microtiter plate methods . Type C1 toxin bound to the three gangliosides under all the conditions, while type B and F toxins bound only at low ionic strength and 37 degrees C . At low ionic strength, the binding kinetics for the three toxins was monophasic in Scatchard plots, and the association constants obtained in the microtiter plate system were 2-4 X 10(8) M-1 . In contrast, the binding kinetics of type C1 toxin in high ionic strength was biphasic in the Scatchard plot, and two association constants were obtained in the microtiter plate system . The heavy chain facilitated the binding of the toxin to the gangliosides . These results indicate that different types of botulinum toxins bind to the gangliosides under different optimal conditions and that gangliosides may not be the common receptor for all types of botulinum toxins . The gangliosides may bind to type C1 toxin together with other potential receptor(s) on synaptosomal membranes.

J Appl Bacteriol, 1986 Jul, 61(1), 81 - 6
Stoichiometry of glucose and starch splitting by strains of amylolytic bacteria from the rumen and anaerobic digester; Marounek M et al.; The stoichiometry of glucose and starch splitting by the amylolytic bacteria Streptococcus bovis, Selenomonas ruminantium, Butyrivibrio fibrisolvens, Eubacterium ruminantium and Clostridium sp . was followed . There were many differences in the ratios of metabolites and in growth yields, as well as in the cell composition, between the growth on glucose and starch . The bacteria employ different nutritional strategies with respect to both energy sources.

Ann Gastroenterol Hepatol (Paris), 1986 Jul-Sep, 22(4), 235 - 40
{Pseudomembranous rectocolitis}; Debat J et al.; The following conclusions were drawn from a study of 15 cases of pseudo-membranous coloproctitis (PMCP): PMCP was seen in subjects of both sexes and all ages . The causative agent was found in all antibiotic classes . Clinical signs comprised constant diarrhea, fever, abdominal pain, toxic shock and, more rarely, pseudo-occlusive, pseudo-perforative surgical evidence . Diagnosis involved visualization of pseudo-membranes by endoscopy . Lesions were most frequent in the left colon and increased in severity towards the distal end . Three stages were distinguished by histological examination: superficial necrosis of the mucous membranes, interruption of glands, complete necrosis of the mucous membrane . Without preparation the abdomen did not provide specific information; nor did barium enema which revealed lesions that were frequently diffuse but more marked in the left colon . Conventional coprocultures did not provide diagnostic information . Only a more sophisticated technique will be capable of detecting the pathogen currently considered to be the cause of PMCP: Clostridium difficile . The course of the disorder is generally satisfactory under medical treatment (parenteral feeding, vancomycin) but may sometimes call for surgery.

Ann Gastroenterol Hepatol (Paris), 1986 Jul-Sep, 22(4), 217 - 22
{Undesirable colorectal effects of drugs}; Cheymol G et al.; The most severe adverse reactions associated with medicinal treatment of the colon involve pseudomembranous colitis following antibiotic treatment, notably with clindamycin, lincomycin and betalactamine . The frequency of this adverse reaction is poorly defined: 1 per 100 to 1 per 5 000 treatments depending on the study . Lesions are explained by the cytotoxic effect of Clostridium difficile toxin . Necrotizing anorectitis has been seen in cases of abuse of suppositories containing propoxyphene . The mechanism involved is still unknown.

Br J Pharmacol, 1986 Jul, 88(3), 531 - 9
Excitatory effect of Clostridium perfringens alpha toxin on the rat isolated aorta; Fujii Y et al.; Clostridium perfringens alpha toxin caused contraction of the isolated aorta of the rat in a dose-dependent manner . The contractile action caused by the toxin was inhibited or abolished by calcium antagonists such as nifedipine, verapamil and cinnarizine, or a Ca-free medium, but was not affected by phentolamine, chlorpheniramine, atropine, tetrodotoxin or a low Na medium . The toxin stimulated Ca uptake into the aorta in a dose-dependent manner . 8-N,N'-diethylaminooctyl-3,4,5-trimethoxybenzoate (TMB-8) blocked significantly both the toxin- and noradrenaline (NA)-induced contractions . Trifluoperazine (TFP) and N-(6-aminohexyl)-5-chloro-1-naphtharene sulphonamide (W-7) did not affect the contractile activity of the toxin but blocked the NA-induced contraction . The toxin also stimulated the 32P phosphate labelling of phosphatidylinositol (PI) and phosphatidic acid (PA) in the preparation . These results indicate that the toxin-induced contraction, which is different from that induced by NA, is the result of a direct action of the toxin on the aorta and is due to an increased Ca2+ permeability across the smooth muscle membrane . It is suggested that the contractile response to the toxin is associated with activation of phospholipid metabolism and enhanced entry of Ca into the aorta.

J Infect, 1986 Jul, 13(1), 5 - 9
Application of a technique for serogrouping Clostridium difficile in an outbreak of antibiotic-associated diarrhoea; Delmee M et al.; A severe outbreak of Clostridium difficile antibiotic-associated diarrhoea (AAD) in an orthopaedic surgical unit is reported . Thirty-seven cases and eight relapses were observed . The 45 related strains together with another 13 strains of C . difficile isolated during the same period in other wards of the same hospital were typed by detection of cytotoxin production, determination of sorbitol fermentation and serogrouping by agglutination with six rabbit antisera defining the serogroups A, B, C, D, F and G . All the strains from the outbreak belonged to serogroup C, were toxigenic and fermented sorbitol . In contrast, four different patterns were observed in seven isolates from cases of AAD in other wards . Finally, six strains isolated from four asymptomatic adults, one adult suffering from shigellosis and one child with salmonellosis demonstrated two patterns different from that of the epidemic isolates . The data strongly suggest nosocomial spread of this micro-organism.

J Appl Bacteriol, 1986 Jul, 61(1), 39 - 49
The effect of citric acid on growth of proteolytic strains of Clostridium botulinum; Graham AF et al.; In strictly anaerobic conditions in a culture medium adjusted to pH 5.2 with HCl and incubated at 30 degrees C, inocula containing less than 10 vegetative bacteria of Clostridium botulinum ZK3 (type A) multiplied to give greater than 10(8) bacteria per ml in 3 d . Growth from an inoculum of between 10 and 100 spores occurred after a delay of 10-20 weeks . Citric acid concentrations of 10-50 mmol/l at pH 5.2 inhibited growth from both vegetative bacteria and spore inocula, a concentration of 50 mmol/l increasing the number of vegetative bacteria or of spores required to produce growth by a factor of approximately 10(6) . The citric acid also reduced the concentration of free Ca2+ in the medium . The inhibitory effect of citric acid on vegetative bacteria at pH 5.2 could be prevented by the addition of Ca2+ or Mg2+ and greatly reduced by Fe2+ and Mn2+ . The addition of Ca2+, but not of the remaining divalent metal ions, restored the concentration of free Ca2+ in the medium to that in the citrate-free medium . The inhibitory effect of citric acid on growth from a spore inoculum was only partially prevented by Ca2+ . Citric acid (50 mmol/l) did not inhibit growth of strain ZK3 at pH 6 despite the greater chelating activity of citrate at pH 6 than at pH 5.2 . The effect of citric acid and Ca2+ at pH 5.2 on vegetative bacteria of strains VL1 (type A) and 2346 and B6 (proteolytic type B) was similar to that on strain ZK3.

Ann Inst Pasteur Microbiol, 1986 Jul-Aug, 137B(1), 113 - 21
Ability of two Clostridium difficile strains from man and hare to produce cytotoxin in vitro and in gnotobiotic rodent intestines; Corthier G et al.; Cytotoxin production by human (VP1) and hare (FD) strains of Clostridium difficile were compared both in vitro in a broth culture and in vivo in intestinal contents of gnotobiotic rodents . Strain VP1 produced about 1,000 times more cytotoxin than the FD strain, both in vitro and in vivo, although the population levels of the two strains were not significantly different either in vitro or in vivo . Ninety percent of gnotobiotic rats and 100% of gnotobiotic mice established with the VP1 strain died within 3 days, whereas no mortality in rats or mice was observed with the FD strain . The cytotoxin titre increased during the 3 weeks following establishment of the FD strain in mice, decreasing thereafter . Mice previously established with the FD strain were protected from VP1 strain challenge . Cytotoxin production was greatly decreased in diassociated mice, although the population levels of the two strains did not differ to a great extent.

Biochem J, 1986 Jul 1, 237(1), 235 - 42
Mechanism of hepatic glycogen synthase inactivation induced by Ca2+-mobilizing hormones . Studies using phospholipase C and phorbol myristate acetate; Blackmore PF et al.; Incubation of hepatocytes with the protein kinase C activator and tumour promoter 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) produced a time- and concentration-dependent inactivation of glycogen synthase, but no change in phosphorylase . The same rate and extent of inactivation occurred in hepatocytes depleted of Ca2+ by treatment with the Ca2+ chelator EGTA . When hepatocytes were treated with the Ca2+-mobilizing hormone vasopressin (10 nM), the rate of glycogen synthase inactivation was similar to that observed with PMA (1 microM) . Depletion of intracellular Ca2+ stores with EGTA abolished the ability of vasopressin to mobilize Ca2+ and activate phosphorylase without abolishing its ability to inactivate glycogen synthase and increase 1,2-diacylglycerol (DAG), the endogenous activator of protein kinase C . Protein kinase C, either in membranes or after partial purification, was shown to be activated in vitro by PMA in the presence of very low concentrations of Ca2+ . Exogenous phospholipase C from Clostridium perfringens, at low concentrations, inactivated glycogen synthase and increased DAG without affecting cell Ca2+ or phosphorylase . It is proposed that the inactivation of glycogen synthase elicited by the Ca2+-mobilizing hormones is due, at least in part, to generation of DAG and activation of protein kinase C.

Avian Dis, 1986 Jul-Sep, 30(3), 620 - 2
Subcutaneous clostridial infection in broilers; Hofacre CL et al.; A flock of 12,500 broilers 36 days of age experienced a sudden increase in mortality . Post-mortem lesions were emphysema, severe enteritis, and a serosanguineous fluid in the subcutaneous tissue of the breast and thighs; there was no evidence of a loss in the integrity of the skin . Clostridium perfringens and C . septicum were isolated from the affected subcutaneous tissue . Histopathological and serological examination indicated previous infection with infectious bursal disease virus . The subsequent immunosuppression and severe enteritis may have permitted the clostridia access to the circulatory system, with localization in the subcutaneous areas of the breast and thighs . Mortality returned to normal 48 hours after potassium penicillin G was administered via the drinking water.

J Bacteriol, 1986 Jul, 167(1), 205 - 9
Cloning and expression in Escherichia coli of the gene for 10-formyltetrahydrofolate synthetase from Clostridium acidiurici ("Clostridium acidi-urici"); Whitehead TR et al.; The gene for 10-formyltetrahydrofolate synthetase (EC 6.3.4.3) from the purinolytic anaerobic bacterium Clostridium acidiurici ("Clostridium acidi-urici") was cloned into Escherichia coli JM83 with plasmid pUC8 . A C . acidiurici genomic library was prepared in E . coli from a partial Sau3A digest and screened with antibody against the synthetase . Of 10 antibody-positive clones, 1 expressed a high level of synthetase activity . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblot analysis demonstrated that the protein synthesized in E . coli had the same subunit molecular weight as the C . acidiurici enzyme . The gene was located on an 8.3-kilobase genomic insert and appeared to be transcribed from its own promoter . Analysis of genomic digests with a fragment of the synthetase gene indicated that one copy of the gene was present in the C . acidiurici chromosome.

Avian Dis, 1986 Jul-Sep, 30(3), 617 - 9
Necrotic enteritis in cage-reared commercial layer pullets; Broussard CT et al.; Necropsy of five 12-week-old pullets from a flock of 99,300 suffering from an increased mortality rate revealed enlarged, gas-filled intestines, the mucosal surfaces of which had the "dirty turkish towel" appearance typical of necrotic enteritis . Although the pullets had been raised entirely in cages, intestinal scrapings revealed the presence of Eimeria maxima . Histopathological findings were compatible with necrotic enteritis . Clostridium perfringens was isolated by anaerobic culture from the intestines . Mortality returned to normal after bacitracin and amprolium were added to the feed.

Appl Environ Microbiol, 1986 Jul, 52(1), 214 - 6
Lethal effect of butylated hydroxyanisole as related to bacterial fatty acid composition; Post LS et al.; Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Pseudomonas fluorescens, Pseudomonas fragi, Escherichia coli, and Salmonella "anatum" were challenged with butylated hydroxyanisole (BHA) . Susceptibility was measured as the concentration of BHA required to cause a 90% reduction in bacterial survivors . Staphylococcus aureus LP and P . fragi were two of the most resistant species examined; C . perfringens and P . fluorescens were the most susceptible . Gram stain reaction was found not to be a strict indicator of bacterial susceptibility to BHA . There was no obvious relationship between individual fatty acids and susceptibility . The ratio of saturated to unsaturated fatty acids in the total lipid fraction of only the gram-positive species was related to susceptibility . The ratios of saturated to unsaturated fatty acids of other fractions were not related to susceptibility.

Appl Environ Microbiol, 1986 Jul, 52(1), 197 - 9
Modified plasmid isolation method for Clostridium perfringens and Clostridium absonum; Roberts I et al.; A rapid plasmid isolation procedure for Clostridium perfringens and C . absonum is described . The ratio of culture volume to lysis buffer volume was found to be crucial for efficient plasmid isolation . The method can be scaled up, without difficulty, for large-scale plasmid preparation.

J Leukoc Biol, 1986 Jul, 40(1), 65 - 72
Ganglioside identification on human monocyte membrane with Clostridium perfringens delta-toxin; Cavaillon JM et al.; Clostridium perfringens delta-toxin was first described as a hemolysin with a restricted lytic spectrum . A selective cytotoxicity of the delta-toxin was then found on rabbit leukocytes: peritoneal and alveolar macrophages were uniformly killed, whereas thymocytes were essentially resistant . The toxin was shown to be specific for GM2 ganglioside or a GM2-like structure . In the present study we report the interaction of delta-toxin with human monocytes . A specific, saturable, and irreversible binding of 125I-delta-toxin was demonstrated . Binding was inhibited by preincubation of the radiolabeled toxin with GM2 and with high amount of GM1 ganglioside . As judged by dye exclusion, no cytotoxicity was observed on freshly isolated monocytes, but when added at the beginning of a culture of human adherent cells, the cytotoxic effect was detected after 48 hours of culture . Taken together, these data indicate the presence of monosialoganglioside(s) at the surface of human monocytes, and suggest a possible reorganisation of such structure into the cell membrane when monocytes mature in vitro toward macrophage-like cells.

J Gen Microbiol, 1986 Jul, 132 ( Pt 7), 1981 - 8
Activation of Clostridium botulinum type E toxin purified by two different procedures; Yokosawa N et al.; Clostridium botulinum type E toxin was purified from culture supernates and from cell extracts by two methods . The specific activity {2 X 10(4) mouse LD50 (mg protein)-1} of the toxin purified from cell extract under slightly acidic conditions was lower than that {3 X 10(5) LD50 (mg protein)-1} of the toxin purified from culture supernate under slightly alkaline conditions . Both toxin preparations were activated by trypsin treatment, but to different extents, the degree of activation of the toxin from cell extract being about 30-fold higher than that of the toxin from culture supernate . The two toxin preparations had the same electrophoretic mobility on SDS-polyacrylamide gels and antigenic specificity as revealed by agar gel double-immunodiffusion tests . The antigenic specificity of the two toxin preparations was unaltered by trypsin treatment . In SDS-polyacrylamide gel electrophoresis, a single band of Mr 144,000 was demonstrated before trypsin treatment and two bands of Mr 100,000 and 55,000 appeared after trypsin treatment . The two toxin preparations were labelled with 125I and chymotryptic peptide maps were obtained before and after trypsin treatment . The two toxin preparations without trypsin treatment demonstrated many differences in their peptide maps, but the preparations after trypsin activation had similar peptide maps . These results indicate that the toxin obtained from culture fluid was a partially activated form, and that its molecular conformation was different from that of the toxin from cell extract . Differences in specific activity and activation ratio by trypsin treatment may be due to differences in the conformation of the toxin molecules.

FEBS Lett, 1986 Jun 9, 201(2), 229 - 32
TLC immunostaining characterization of Clostridium botulinum type A neurotoxin binding to gangliosides and free fatty acids; Takamizawa K et al.; The receptor structure of Clostridium botulinum neurotoxin type A was analysed by TLC immunostaining . GQ1b was found to be the most potent receptor, and the neurotoxin also bound to GT1b and GD1a, but not to GM3, GM2, GM1, GD3, GD1b and GT1a . Optimum binding of neurotoxin to the ganglioside appeared in 0.01 M phosphate buffer (pH 7.2) containing 0.2% NaCl . Higher and lower NaCl concentrations diminished neurotoxin binding to the ganglioside . In addition, the neurotoxin was able to bind to free fatty acids . Maximum binding was observed on stearic acid and neurotoxin binding to free fatty acids was not affected by NaCl concentration.

Biochemistry, 1986 Jun 3, 25(11), 3318 - 28
Kinetic studies of reduction of a 1:1 cytochrome c-flavodoxin complex by free flavin semiquinones and rubredoxin; Hazzard JT et al.; The kinetics of reduction by free flavin semiquinones and reduced rubredoxin of the individual components of the 1:1 complex formed between horse heart cytochrome c and Clostridium pasteurianum flavodoxin have been studied . Complex formation did not affect the rate constant for reduction of flavodoxin by 5-deazariboflavin semiquinone, indicating that the accessibility of the flavin mononucleotide (FMN) of complexed flavodoxin is the same as in the free protein . Reduction of the complexed cytochrome c by the neutral flavin semiquinones of lumiflavin and riboflavin was significantly affected by complex formation (2-3-fold rate constant decrease), indicating that there are steric constraints on the accessibility of the cytochrome heme to small exogenous reductants . Reduction of complexed cytochrome c by the negatively charged semiquinones of FMN and Cl2FMN was also characterized . A repulsive electrostatic interaction between the reductants and complexed cytochrome was observed, whereas with free cytochrome an attractive interaction had previously been found . This is consistent with the presence of negative electrostatic potential at the protein interface due to uncompensated flavodoxin carboxylates, as predicted by Matthew et al . {Matthew, J . B., Weber, P . C., Salemme, F . R., & Richards, F . M . (1983) Nature (London) 301, 169-171} . Further, pseudo-first-order rate constants for the reduction of complexed cytochrome by these flavins had a nonlinear concentration dependence, rather than obeying simple second-order kinetics . This is interpreted by using a mechanism involving a rate-determining structural isomerization of the protein complex prior to the second-order electron-transfer step . The magnitude of the decrease in the rate constant for reduction of complexed cytochrome c by the negatively charged reduced rubredoxin was approximately the same as observed for free flavins . Furthermore, simple second-order kinetics were obtained, and the apparent electrostatic interaction between rubredoxin and the complex was attractive . These results suggest that flavodoxin was partially displaced from its complex with cytochrome c by a collisional interaction with rubredoxin . The effects of complexation on the kinetics have been correlated with a solvent-accessible surface representation of the computer-generated model of the flavodoxin-cytochrome c complex {Simondsen, R . P., Weber, P . C., Salemme, F . R., & Tollin, G . (1982) Biochemistry 21, 6366-6375} . The experimental observations are generally consistent with the structural model but clearly require the invocation of dynamic motions at the protein-protein interface.

Arch Microbiol, 1986 Jun, 145(1), 85 - 90
Degradation of various amine compounds by mesophilic clostridia; Moller B et al.; From 60 species of the genus Clostridium tested 26 species were able to degrade one to three of the following compounds: betaine, choline, creatine, and ethanolamine . Degradation of betaine and choline was always associated with the formation of trimethylamine as one of the products . Creatine was converted to N-methylhydantoin and with one species (Clostridium sordellii) to sarcosine in addition . The diagnostic value of the ability of clostridial species to degrade the compounds mentioned is discussed . N,N-dimethylglycine, N,N-dimethylethanolamine or sarcosine were not metabolized by the strains tested.

Vet Microbiol, 1986 Jun, 12(1), 25 - 31
Significance of Clostridium spiroforme in the enteritis-complex of commercial rabbits; Peeters JE et al.; Commercial rabbits showing clinical signs of enteritis-complex were examined for the presence of Clostridium spiroforme and its iota-like toxin . The bacterium was detected by Gram stain in 52.4% of 149 cecal samples and iota-like toxin in 7.4% . From 29 strains of C . spiroforme tested, 26 were toxigenic, originating from 24 of 29 rabbitries . In 13.4% of the samples, C . spiroforme was present as the only known disease agent . Gross and microscopic lesions were similar to those described in the literature . In the other samples, C . spiroforme was associated with attaching effacing Escherichia coli (29.5%), Bacillus piliformis (10.3%), rotaviruses (25.6%), coronavirus (2.6%), Eimeria spp . (44.9%) and cryptosporidia (6.4%) . In 33.3% of C . spiroforme-containing samples, more than one of these agents was present . There was no significant difference between the presence of these organisms in C . spiroforme-positive and negative samples . On the basis of these results as well as that of previous data, we suggest that C . spiroforme-mediated diarrhea is favoured by maldigestion, initiated by infectious agents and/or nutritional factors.

Br J Surg, 1986 Jun, 73(6), 457 - 60
Changing epidemiology, diagnosis, and treatment of Clostridium difficile toxin-associated colitis; Talbot RW et al.; One hundred and ninety patients with Clostridium difficile toxin-associated colitis (CTAC) or pseudomembranous colitis (PMC) were identified, from microbiology records, disease index and proctoscopy service records, and studied retrospectively . CTAC was associated with cephalosporin antibiotic administration in 70 per cent of the patients . CTAC developed postoperatively in 108 patients after all types of surgery with no preponderance for abdominal surgery . Identification of cytotoxin in stool samples was the primary diagnostic test in 81 per cent of patients but cytotoxin was isolated in 98 per cent of all patients . Pseudomembranes visible on proctoscopy established the diagnosis in 19 per cent of patients and were more commonly seen in severe colitis (71 per cent) than in mild colitis (23 per cent) . CTAC responded similarly to oral vancomycin and metronidazole with a relapse rate of 20-23 per cent, respectively . With its association with cephalosporin administration, CTAC is likely to occur with increasing frequency in surgical practice . Oral metronidazole is an effective, cheap, alternative to vancomycin therapy.

Food Chem Toxicol, 1986 Jun-Jul, 24(6-7), 481 - 94
Evaluation of in vitro predictive tests for irritation and allergic sensitization; Parish WE; Investigations of in vitro procedures to predict the potential of substances as skin irritants and as allergens inducing delayed hypersensitivity (contact dermatitis) are described, with indications of possible advances and known limitations . The examination of keratome slices of skin for release of enzymes, for changed histochemistry and for utilization of radioisotope-labelled amino acids will detect weak irritants but is of doubtful value for moderate irritants and will detect corrosive substances only through their inhibition of all cell activities . Fibroblast cultures, tested with Clostridium perfringens toxin and chemicals, show similar limitations in detecting moderate or severe irritants . Fibroblast cultures can be made more relevant to epidermal exposure by an overlying layer of agar containing keratin . In vitro tests to detect induction of sensitizing potential for delayed hypersensitivity have made little progress . The most promising approach is to treat antigen-presenting Langerhans cells with antigen and co-culture with lymphocytes . The lymphocytes may be examined for changes in receptor expression, for synthesis of interleukin 2, and possibly for responses to allergen if sufficient cells become specifically sensitized . There are several in vitro techniques to detect responses of in vivo- or in vitro-sensitized lymphocytes treated with antigen.

Pathol Biol (Paris), 1986 Jun, 34(5 Pt 2), 672 - 5
{Isolation of Clostridium difficile from the stools of hospitalized patients with diarrhea}; de Barbeyrac B et al.; 408 stool samples from 354 hospitalized patients with diarrhea were evaluated for the presence of Clostridium difficile . C . difficile was detected in stools of 42 patients (12%), 19 of them being hospitalized in neurosurgery units . The strains were cytotoxigenic in 31 cases and non cototoxigenic in 11 cases . The diagnosis of C . difficile induced diarrhea was based on the clinical setting {presence of diarrhea that could be attributed to antimicrobial therapy and endoscopy for detection of pseudomembranous colitis (PMC)}, laboratory studies (cytotoxigenic strains of C . difficile) and therapeutic response (oral vancomycin or metronidazole) . C . difficile was implicated as a cause of PMC (2 cases) and antimicrobial associated diarrhea (8 cases) (PMC not documented) . The pathogenic role of C . difficile was questionable in 24 cases (no specific therapy) and unlikely in 8 cases . In fact C . difficile could be implicated as a cause of diarrhea in 24% of the cases.

Pathol Biol (Paris), 1986 Jun, 34(5 Pt 2), 645 - 7
{Sensitivity of anaerobes to 8 antibiotics}; Sedallian A; We studied the susceptibility of 282 obligate anaerobes to 8 antibiotics . The strains were isolated from clinical specimens taken during 1983 and 1984 . Minimal inhibitory concentrations (MICs) were determined using a microplate method for 245 strains and agar dilution for 33 Bacteroides asaccharolyticus strains . Tested strains were as follows: 85 Bacteroides fragilis, 71 Clostridium, 19 Fusobacterium nucleatum, 35 Peptostreptococcus, 39 Bacteroides oralis and Bacteroides bivius and 33 Bacteroides asaccharolyticus . Tested antibiotics were: metronidazole, clindamycin, minocycline, cefoxitin, cefotaxime, moxalactam, keflin and carbenicillin . For Bacteroides fragilis, the three most active antibiotics were metronidazole (inhibition of all strains with 8 micrograms/ml), minocycline and clindamycin (inhibition of 95% of strains by MICs less than or equal to 0.5 microgram/ml) . However, four strains were resistant to clindamycin (MICs greater than 8 micrograms/ml) . The other anaerobic strains tested were susceptible . However, a few Bacteroides bivius strains were resistant to keflin (MIC greater than or equal to 64 micrograms/ml for 5% of strains).

J Appl Bacteriol, 1986 Jun, 60(6), 483 - 90
Identification and grouping of Clostridium botulinum strains by numerical analysis of their electrophoretic protein patterns; Bom IJ et al.; Strains of Clostridium botulinum type A, type E and both non-proteolytic and proteolytic types B and F were characterized by their electrophoretic protein patterns . As the protein pattern changes during sporulation, special attention was paid to the prevention of sporulation by selecting an appropriate medium (Strasdine's medium plus 1% w/v glucose) and a scheme of repeated subculturing . Ribosomal proteins, evolutionarily conservative and hence relatively similar in all types of bacteria, were removed to optimize the resolving power of the electrophoretic technique . Protein patterns were compared by computing correlation coefficients of normalized densitometric tracings . The method is highly reproducible and its resolving power is high: all protein patterns found were specific . The strains tested fall into two main groups: the proteolytic and the non-proteolytic cluster . Type A strains form a separate subgroup within the proteolytic cluster, the same applies to type E strains within the non-proteolytic group . Although time-consuming for spore-forming bacteria, this method is, to our knowledge, the only technique that recognizes individual strains of Cl . botulinum . For non-spore-forming micro-organisms the method is certainly much simpler and hence even more valuable.

Am J Infect Control, 1986 Jun, 14(3), 99 - 109
Review of Clostridium difficile-associated diseases; McFarland LV et al.; Clostridium difficile has recently become recognized as an important nosocomial pathogen . This review summarizes what is known about the isolation of the organism, the spectrum of clinical disease, virulence factors, treatments, and methods of prevention . Risk factors for C . difficile disease are also discussed . The most important risk factor is the use of certain antibiotics (ampicillin, cephalosporins, and clindamycin) . C . difficile is associated with 96% to 100% of cases of pseudomembraneous colitis, 60% to 75% of antibiotic-associated cases of colitis, and 11% to 33% of antibiotic-associated cases of diarrhea . Other risk factors include gastrointestinal manipulations, advanced age, female sex, inflammatory bowel disease, cancer chemotherapy, and renal disorders . Hospital outbreaks of C . difficile disease are examined . Data from nosocomial outbreaks support transmission of C . difficile by contaminated fomites and hand carriage by hospital personnel.

Arch Intern Med, 1986 Jun, 146(6), 1101 - 4
Oral bacitracin vs vancomycin therapy for Clostridium difficile-induced diarrhea . A randomized double-blind trial; Dudley MN et al.; The effectiveness of a ten-day course of either oral bacitracin or oral vancomycin hydrochloride for treatment of Clostridium difficile-induced antibiotic-associated diarrhea was compared in a randomized double-blind study . Bacitracin was as effective as vancomycin in resolving diarrhea; most patients responded within five days of therapy with either drug . Three patients receiving bacitracin worsened during therapy; two of these were considered treatment failures . Neither C difficile nor its toxin was detected in stool samples collected on the final day of therapy in 71% of patients (10/14) receiving vancomycin and in 30% (3/10) receiving bacitracin . Five patients receiving bacitracin and three receiving vancomycin had at least one recurrence . Low but nontoxic concentrations of bacitracin were detected in serum samples collected from 11 patients . Oral bacitracin at this dosage level was as effective as vancomycin in resolving the symptoms of C difficile-induced antibiotic-associated diarrhea in most patients but was less effective in eradicating C difficile and its toxin from patients' stools.

Am J Clin Pathol, 1986 Jun, 85(6), 716 - 8
Comparison of Minitek Anaerobe II, API An-Ident, and RapID ANA systems for identification of Clostridium difficile; Bate G; Three commercial anaerobic systems, Minitek (BBL Microbiology Systems, Cockeysville, MD), API An-Ident (Analytab Products, Plainview, NY), and RapID ANA (Innovative Diagnostic Systems, Decatur, GA) were evaluated for ability to identify 45 Clostridium difficile isolates accurately . Minitek correctly identified 66% of C . difficile isolates to species, 27% were incompletely identified, and 7% were misidentified . Most of the discrepancies with Minitek were due to false negative biochemical results . API An-Ident correctly identified 9% C . difficile isolates to species, 89% were incompletely identified, and 2% were misidentified . Most of the API An-Ident discrepancies were due to the data base, which distinguished poorly between C . difficile, Clostridium hastiforme, and Clostridium sporogenes . RapID ANA correctly identified 100% of C . difficile isolates.

Chemioterapia, 1986 Jun, 5(3), 177 - 84
In vitro activity of flurithromycin, a novel macrolide antibiotic; Gialdroni Grassi G et al.; Flurithromycin is an (8,S)-8-fluoroerythromycin isolated from the fermentation broth of Streptomyces erythraeus ATCC 31772, a blocked mutant of a strain producer of erythromycin . Its in vitro antibacterial activity has been determined on recent clinical isolates of respiratory pathogens . The range of MIC for Streptococcus pneumoniae and Streptococcus beta-haemolyticus group A is from 0.0015 to 0.006 microgram/ml, for Haemophilus influenzae from 0.012 to 0.4 microgram/ml, for Staphylococcus aureus from 0.1 to 3.1 micrograms/ml . Its action is bacteriostatic and increases at alkaline pH . Among anaerobes Clostridium perfringens, Bacteroides fragilis, other species of Bacteroides and Peptostreptococcus are particularly susceptible . Flurithromycin also showed some activity on Mycobacterium bovis, M . scrofulaceum and M . phley . The determination of killing curves indicated that in most cases a killing effect was obtained at 4 X MIC . A combination of flurithromycin with ampicillin or doxycycline sometimes was synergic, but more often additive and never antagonistic . The possible interference of flurithromycin on some parameters of the natural system of defense was determined . At concentrations equal to therapeutic levels in blood and tissues, flurithromycin did not influence chemotaxis, phagocytosis, metabolic activation and the killing activity of neutrophils.

J Med Microbiol, 1986 Jun, 21(4), 293 - 7
Growth of Clostridium difficile and production of toxins A and B in complex and defined media; Haslam SC et al.; The ability of several strains of Clostridium difficile to grow and to produce toxins A and B in complex and defined culture media has been studied with special reference to the amino-acid composition of the medium . The production of these toxins varied with the strain used and with the composition of the growth medium . Toxin A production was not inextricably linked to production of toxin B since conditions were found in which only one or other toxin was produced.

Pathol Biol (Paris), 1986 Jun, 34(5 Pt 2), 643 - 4
{Activity of cefotetan against anaerobic bacteria}; Sedallian A; We determined the susceptibility to antibiotics of 64 strains of obligate anaerobes . Tested bacterial strains were as follows: 28 Bacteroides fragilis, 22 Fusobacterium nucleatum, 10 Clostridium perfringens, and 4 Bacteroides bivius . Tested antibiotics were cefotetan, cefoxitin and piperacillin . Cefotaxime was tested against 20 Bacteroides fragilis strains . Bacteroides fragilis, which is the most resistant species, was susceptible to cefotetan, cefoxitin and piperacillin, with comparable MICs for these three drugs (0.5 to 16 micrograms/ml); cefotaxime was less effective (MICs = 0.5 to 128 micrograms/ml) . Fusobacterium nucleatum and Clostridium perfringens were highly susceptible to cefotetan, cefoxitin and piperacillin.

J Appl Bacteriol, 1986 Jun, 60(6), 517 - 25
Scaled-up production and purification of Clostridium perfringens type A enterotoxin; Reynolds D et al.; Methods for small-scale production of Clostridium perfringens type A enterotoxin were unsuitable for large-scale culture of this organism . Rapid, efficient harvesting of 40 1 batch culture of Cl . perfringens was achieved by tangential flow micro-filtration with the Millipore Pellicon cassette system . Enterotoxin-containing extracts were prepared by passing concentrated suspensions of the harvested cells through a French pressure cell . The overall yield of purified enterotoxin was 38.8% . The toxin gave a single band on native polyacrylamide gels but formed high molecular weight aggregates in the presence of sodium dodecyl sulphate . These aggregates frequently occurred during storage of non-sterile enterotoxin preparations but could be separated from the monomer toxin by gel filtration on Sephadex G-100 . Purified monomer enterotoxin had biological activities of 119.3 micrograms/kg mouse lethal dose when injected intraperitoneally and 3333 capillary permeability increasing units/mg protein in guinea pig skin . Thirty micrograms of the enterotoxin caused fluid accumulation in ligated rabbit ileal loops . Aggregated enterotoxin had no demonstrable biological or immunological activity.

Vet Microbiol, 1986 Jun, 12(1), 93 - 6
Toxin-types of Clostridium perfringens strains isolated from sheep, cattle and paddock soils in Nigeria; Itodo AE et al.; Two-hundred and forty-five strains of Clostridium perfringens isolated from the faeces of apparently healthy sheep and cattle and from their environments (paddock soils) in Kano and Kaduna States of Nigeria were studied . The isolates were examined by the toxin-antitoxin neutralisation tests performed intradermally in depilated albino guinea-pigs . One-hundred and twenty-seven (53.1%) of the isolates were type A, 17 (7.1%) were type B, 14 (4.9%) were type C, 44 (18.4%) were type D and 19 (7.9%) were type E . Eighteen (7.5%) were not typable while six were lost during storage . The significance of this distribution and the potential danger to animal health, especially as regards enterotoxaemias, cannot be over-emphasized.

J Am Vet Med Assoc, 1986 Jun 1, 188(11), 1309 - 10
Hemorrhagic enteritis caused by Clostridium perfringens type C in a foal; Pearson EG et al.; A 4-day-old foal died with bloody diarrhea . Using a mouse neutralization test, Clostridium perfringens type C was isolated from intestinal contents, and alpha and beta toxins were identified . About 4 m of the jejunum had severe necrohemorrhagic enteritis . Microscopically, large, rod-shaped, gram-positive bacteria were seen on necrotic intestinal villi by use of Brown and Hopp's stains.

J Bacteriol, 1986 Jun, 166(3), 1128 - 30
Macromolecular organization of F1-ATPase isolated from Clostridium thermoaceticum as revealed by electron microscopy; Mayer F et al.; Membrane vesicles and the F1-ATPase from Clostridium thermoaceticum were examined by electron microscopy . F1-ATPase particles projecting from the vesicles have a diameter of 10 to 12 nm . The F1-ATPase has an alpha 3 beta 3 gamma delta structure . The alpha and beta subunits are most likely arranged in an alternating sequence around a central protein mass consisting of the gamma and delta subunits.

Infect Immun, 1986 Jun, 52(3), 786 - 91
The use of monoclonal antibodies to analyze the structure of Clostridium botulinum type E derivative toxin; Kozaki S et al.; Six monoclonal antibodies against Clostridium botulinum type E derivative toxin were prepared . Three of the five binding to the heavy chain neutralized the derivative toxin; the other one binding to the light chain did not . Immunoblotting analysis with the monoclonal antibodies showed that the fragment obtained by tryptic digestion consisted of the light chain and part of the heavy chain (H-1 fragment) linked together by a disulfide bond(s) and that the antigenic determinants common between type E and F derivative toxins were located on both the heavy and light chains . The fragment induced by chymotrypsin treatment, like the tryptic fragment, bound to four monoclonal antibodies . The mild tryptic treatment and reduction resulted in separation of the chymotryptic fragment into two smaller fragments corresponding to the light chain and H-1 fragment . These results indicate that H-1 fragment contains the amino-terminal portion of the heavy chain . The monoclonal antibody neutralizing the toxin and probably recognizing the epitope on the carboxyl-terminal portion (H-2 fragment) of the heavy chain effectively competed for binding of 125I-labeled derivative toxin to synaptosomes . Of the two monoclonal antibodies neutralizing the toxin and recognizing the epitopes on H-1 fragment, one partially inhibited binding, but the other did not . This suggests that the binding of 125I-labeled derivative toxin depends mainly on the carboxyl-terminal region of the heavy chain and that interference with binding is not the only means of toxin neutralization.

FEBS Lett, 1986 May 26, 201(1), 5 - 8
Solubilization of trehalase from rabbit renal and intestinal brush-border membranes by a phosphatidylinositol-specific phospholipase C; Takesue Y et al.; Trehalase (EC 3.2.1.28) associated with renal and intestinal brush-border membranes was solubilized by highly purified phosphatidylinositol-specific phospholipase C (EC 3.1.4.10) from Bacillus thuringiensis, but not by phosphatidylcholine-hydrolyzing phospholipase C (EC 3.1.4.3) from Clostridium welchii or phospholipase D (EC 3.1.4.4) from cabbage . The solubilized trehalase was not adsorbed on phenyl-Sepharose, indicating that it was hydrophilic . Phosphatidylinositol-specific phospholipase C also converted Triton X-100-solubilized amphipathic trehalase into a hydrophilic form . These results suggest that trehalase is bound to the membrane through a direct and specific interaction with phosphatidylinositol.

J Mol Biol, 1986 May 5, 189(1), 249 - 50
Crystallization and preliminary X-ray diffraction study of an endoglucanase from Clostridium thermocellum; Joliff G et al.; Endoglucanase D, a cellulose degradation enzyme from Clostridium thermocellum has been cloned in Escherichia coli, purified and crystallized . The crystals are trigonal, space group P3(1)12 (or P3(2)12) with a = 57.7 (+/- 0.1) A, c = 192.1 (+/- 0.2) A, and diffract X-rays to a resolution of 2.8 A . They are suitable for a high-resolution X-ray diffraction analysis.

Acta Pathol Jpn, 1986 May, 36(5), 757 - 64
A case of nontraumatic clostridial gas gangrene occurring in a patient with colon adenocarcinoma, liver cirrhosis, and diabetes mellitus; Takeyama M et al.; An autopsy case of clostridial gas gangrene occurring in a 54-year-old man with colon adenocarcinoma, liver cirrhosis, and diabetes mellitus is reported . The patient died 4 days after the onset of symptoms with episodes of vomiting and abdominal pain . Gangrene of both hips and perineum, hemolysis, renal failure, and disseminated intravascular coagulation were the dominant clinical features . Clostridium septicum was isolated from the subcutaneous tissue fluid . Adenocarcinoma of the ascending colon with ulceration found at autopsy was supposed to be an entry of the organism . Histologically, lesions of subcutaneous tissue and muscles were characterized by the absence of inflammatory infiltrates in spite of extensive necrosis . A summary of 35 cases of gas gangrene hospitalized to the Osaka University Hospital for the past 16 years indicates that clostridial gas gangrene patients with underlying diseases such as malignant neoplasm, diabetes, liver cirrhosis or immunodeficiency have a relatively poor prognosis.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 May, 261(3), 345 - 9
Antibacterial susceptibility of Clostridium sordellii strains; Nakamura S et al.; A total of 24 Clostridium sordellii strains was tested for susceptibility to 24 antibacterial agents . All strains exhibited a high degree of resistance to streptomycin, kanamycin, sulfamethoxazole, and trimethoprim.

J Clin Microbiol, 1986 May, 23(5), 954 - 5
Evaluation of a commercial cytotoxicity assay for detection of Clostridium difficile toxin; Nachamkin I et al.; A comparative study was performed to determine the accuracy of Clostridium difficile toxin detection . A commercial cytotoxicity assay (Bartels Immunodiagnostic Supply, Bellevue, Wash.) was compared with conventional microcytotoxicity assays, using Vero and MRC-5 cells . The Bartels system was found to be essentially equivalent to conventional cytotoxicity assays currently being performed for routine C . difficile toxin detection.

J Neurosurg, 1986 May, 64(5), 813 - 5
Delayed brain abscess related to a retained foreign body with culture of Clostridium bifermentans . Case report; Pencek TL et al.; Although it is well documented that retained foreign bodies are associated with delayed intracranial abscess, there are few reports of anaerobic organism growth . A case is presented in which a left parieto-occipital abscess surrounded a metallic fragment implanted when a mortar shell exploded in Vietnam 15 years before . The diagnostic evaluation and surgical management of this case are presented.

Dig Dis Sci, 1986 May, 31(5), 548 - 51
Yersinia colitis masquerading as pseudomembranous colitis; Brown R et al.; We describe a 15-month-old male who presented with fever and diarrhea 24 hr after receiving antibiotics for otitis media . A flexible sigmoidoscopy was initially interpreted endoscopically as antibiotic-associated pseudomembranous colitis, and the patient was treated with vancomycin . The diagnosis of antibiotic-associated colitis was excluded in our patient by the negative stool examination for Clostridium difficile toxin, the failure to obtain supportive features on rectal biopsy, and the failure to demonstrate sigmoidoscopic improvement with vancomycin therapy . Thirteen days later, Y . enterocolitica was cultured from the initial stool specimens . In this case, the raised central whitish area on an erythematous base was misinterpreted as pseudomembranous colitis.

Zh Mikrobiol Epidemiol Immunobiol, 1986 May, (5), 49 - 52
{Immunocytochemistry of Clostridium novyi antigens . Electron microscopic research}; Vaisman ISh et al.; The dynamics of the synthesis, transfer and excretion of toxin in C . novyi, growing in a liquid culture medium, have been studied on the level of bacterial ultrastructure by means of immunoferritin techniques modified by the authors . As revealed in this study, the basic mechanism of toxin excretion is realized by the active transfer of toxin through the enveloping structures after its accumulation in the periplasmatic space . In ageing cultures toxin may also be released in the process of bacteriolysis with the degradation of bacterial structures.

J Clin Microbiol, 1986 May, 23(5), 863 - 8
Relationships between rotavirus diarrhea and intestinal microflora establishment in conventional and gnotobiotic mice; Moreau MC et al.; Intestinal microflora did not play a role in the intensity or course of EDIM rotavirus-induced diarrhea, since similar results were observed in axenic and conventional mice . In conventional mice, rotavirus-induced diarrhea did not modify the establishment of Lactobacillus spp . and Escherichia coli before weaning . The consequences of diarrhea on the establishment of strictly anaerobic bacteria after weaning were studied through the measurement of two bacterial functions, the microbial barrier effect against E . coli and the development of the immunoglobulin A intestinal immune system . These two bacterial functions were expressed in a similar way in diarrheic and control mice . In young gnotobiotic mice inoculated with Clostridium perfringens or C . difficile, rotavirus infection led to an earlier development of both strains, as compared with controls . This effect was more pronounced with C . difficile . These results suggest that rotavirus infections might enhance opportunistic bacterial infections.

Ann Inst Pasteur Microbiol, 1986 May-Jun, 137A(3), 287 - 94
Urease activity in gastrointestinal tract of rabbit and electrophoretic behaviour of urease; Crociani F et al.; Urease activity in the stomach (fundus and antrum), caecal content and soft faeces of rabbit was studied . Significant differences between fundus and antral content (P less than 0.01) and between caecum and soft faeces (P less than 0.05) were observed . The urease zymograms from caecal content and soft faeces of rabbit presented two different bands . The fundus content and the caecal ureolytic Clostridium innocuum bacterium exhibited only one band . Band B of caecal content was not evident at pH 4, whereas band A, also present in the stomach, was observed both at pH 4 and at pH 6 . The optimal pH of urease activity of stomach and caecal content was in the range of 4-5 and 5-6, respectively . A comparison of intestinal urease zymograms with those of the single ureolytic bacterial species was suggested in order to clarify their role in urea metabolism.

Diagn Microbiol Infect Dis, 1986 May, 5(1), 61 - 9
Comparison of culture, cytotoxicity assays, and enzyme-linked immunosorbent assay for toxin A and toxin B in the diagnosis of Clostridium difficile-related enteric disease; Walker RC et al.; Clostridium difficile culture, test tube, and microtiter cytotoxicity assays, and enzyme-linked immunosorbent assays (ELISAs) for toxin A and toxin B, were simultaneously performed on 113 fresh diarrheal stool specimens randomly selected from those submitted to our clinical laboratory for routine C . difficile testing . The performance of these tests in diagnosing C . difficile-related enteric disease (CDRED) was based on a clinical assessment of the likelihood of CDRED as determined by a systematic review of case histories blinded from the test results . Among 61 antibiotic recipients, both the microtiter cytotoxicity assay and the toxin A ELISA were highly specific for CDRED (95% and 100%, respectively) . Specificities for the other procedures were much lower (tube cytotoxicity assay, 79%; culture, 74%; and toxin B ELISA, 56%) . The high sensitivities of the culture (89%) and toxin B ELISA (83%) were somewhat negated by their low specificities . The only test that was both specific and had acceptable sensitivity (78%) was the microtiter cytotoxicity assay . This study indicates that ELISAs for detection of C . difficile toxins are not as reliable as the cytotoxicity assay in the laboratory diagnosis of CDRED, and that clinical correlation is essential in the evaluation of any new test for CDRED.

J Gen Microbiol, 1986 May, 132 ( Pt 5), 1367 - 72
Cloning and expression of Clostridium acetobutylicum endoglucanase, cellobiase and amino acid biosynthesis genes in Escherichia coli; Zappe H et al.; Clostridium acetobutylicum P262 endoglucanase and cellobiase genes, cloned on a 4.9 kb DNA fragment in the recombinant plasmid pHZ100, were expressed from their own promoter in Escherichia coli . Active carboxymethylcellulase and cellobiase enzymes were produced, but there was no degradation of Avicel . The endoglucanase activities observed in cell extracts of E . coli HB101(pHZ100) differed in their pH and temperature optima from those previously reported for C . acetobutylicum P270 . Complementation of E . coli arg and his mutations by cloned C . acetobutylicum DNA was also observed.

Biochimie, 1986 May, 68(5), 687 - 95
Purification and properties of the endoglucanase C of Clostridium thermocellum produced in Escherichia coli; Petre D et al.; The celC gene, which codes for a new endoglucanase of Clostridium thermocellum, termed endoglucanase C, was found to be expressed when cloned in Escherichia coli . The enzyme was purified to electrophoretic homogeneneity from E . coli and its biochemical properties were studied . It differs from the previously studied endoglucanases A and B . In particular, endoglucanase C displays features common to endo- and exoglucanases, since it had a high activity on carboxymethylcellulose and on p-nitrophenyl-beta-D-cellobioside where only the agluconic bond was split . In addition, the enzyme was able to release cellobiose units from G3, G4 and G5 cellodextrins . Endoglucanase C was characterized by Western blot in a culture supernatant from C . thermocellum grown on cellulose, using an antiserum raised against the enzyme produced by E . coli.

Arch Dermatol, 1986 May, 122(5), 583 - 4
Pseudomembranous colitis caused by topical clindamycin phosphate; Parry MF et al.; Pseudomembranous colitis was observed on two occasions in the same patient and was associated with the topical administration of clindamycin phosphate . Assay for Clostridium difficile toxin was positive, and the patient was ultimately cured by oral vancomycin hydrochloride and the withdrawal of clindamycin therapy.

Am J Vet Res, 1986 May, 47(5), 1132 - 3
Colostral transfer of Clostridium perfringens type C beta antitoxin in swine; Matisheck PH et al.; Nine bred gilts were vaccinated with 2 doses of a Clostridium perfringens type C toxoid at a 5-week interval . Time of vaccination during gestation differed among the gilts . Clostridium perfringens beta antitoxin in colostral samples and in serum samples was titrated in mice . Blood was collected from 2 to 5 neonatal pigs from each dam (total = 32 pigs) when the pigs were 36 to 48 hours old . Antitoxin titers in colostrum were 123 to 4.5 IU/ml, indicating considerable variation in individual responses of the gilts to toxoid . Serum titers of neonatal pigs reflected colostrum titers of their dams . This colostrum-to-serum titer correlation was essentially a straight-line fit by least-squares linear regression analysis, establishing a direct proportional relationship between colostrum titers and serum titers of neonatal pigs . In the dams, a correlation was not found between colostral titers and serum titers of blood samples collected 2 weeks after collection of colostrum.

Zh Mikrobiol Epidemiol Immunobiol, 1986 May, (5), 41 - 5
{Antigenic interrelations within the species Clostridium novyi, C . histolyticum and C . septicum as a basis for developing an immunofluorescence method}; Baraitseva VA et al.; The cellular and antigenic interrelations within each Clostridium species have been studied with a view to the selection of strains for the preparation of specific rabbit antisera possessing a wide serological spectrum of action within the species . The indirect immunofluorescence test making it possible to determine the species of clostridia in native pathological material from wounds or in cultures, subjected to short-term incubation, within 1-1.5 hours has been developed . This test can be used in laboratory practice for the diagnosis of gas gangrene in humans, as well as for the identification of pathogenic clostridia.

Biochem Biophys Res Commun, 1986 Apr 29, 136(2), 802 - 6
ADP-ribosylation of nonmuscle actin with component I of C2 toxin; Ohishi I et al.; C2 toxin elaborated by Clostridium botulinum type C is composed of two dissimilar protein components, designated components I and II . Component I of the toxin caused ADP-ribosylation of a protein of Mr 45,000 in chicken tissue homogenates and also purified nonmuscle but not muscle actin . The endogenous ADP-ribosylation of intracellular actin with C2 toxin was correlated with the morphological change in intact culture cells caused by the toxin . These results indicate that the biological activity of the toxin involves a novel enzymatic activity of component I, which catalyzes the preferential ADP-ribosylation of nonmuscle actin of the target cells.

J Biol Chem, 1986 Apr 25, 261(12), 5487 - 95
Novel fucolipids of human adenocarcinoma: disialosyl Lea antigen (III4FucIII6NeuAcIV3NeuAcLc4) of human colonic adenocarcinoma and the monoclonal antibody (FH7) defining this structure; Nudelman E et al.; A new fucoganglioside, disialosyl Lea, has been found in the disialoganglioside fraction of human colonic adenocarcinoma . The ganglioside has been isolated from four other disialogangliosides by high-performance liquid chromatography followed by preparative high-performance thin-layer chromatography . The structure of the antigen was characterized by its conversion to lactofucopentaosyl(II)ceramide (Lea-active ceramide pentasaccharide), methylation analysis, and high-mass range electron-impact as well as field-desorption mass spectrometry of the permethylated derivative, as shown below: (Formula; see text) Specific removal of alpha 2----3-linked sialosyl residue by influenza virus A2 sialidase, or preferential hydrolysis of the same residue by Clostridium perfringens sialidase in the absence of detergent, resulted in the formation of an intermediate product, monosialosyl LeaII (III4FucIII6NeuAcLc4), which reacted with anti-Lea antibody and with monoclonal antibody FH7 and may have a sialic acid linked at the 6 position of GlcNAc . The IgG3 monoclonal antibody FH7 was established, which reacts specifically with disialosyl Lea and monosialosyl LeaII as above, but does not react with disialosyllactotetraosylceramide (IV3NeuAcIII6Neu-AcLc4), monosialosyl LeaI (IV3NeuAcIII4FucLc4), and other mono- and disialogangliosides isolated from the same cancer tissue . The antibody FH7 may be useful in the detection of human cancer.

Eur J Biochem, 1986 Apr 15, 156(2), 301 - 4
On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri; Hartrampf G et al.; The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration . This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer . In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face . 2-Methylene{2-2H1}glutarate was synthesized via (R)-3-methyl{3-2H1}itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O . The predominantly monodeuterated compound was oxidized to (S)-{2-2H1}succinate as analysed by circular dichroism . The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon.

Biochemistry, 1986 Apr 8, 25(7), 1589 - 99
Purification and characterization of glutamine synthetase from Clostridium pasteurianum; Krishnan IS et al.; Glutamine synthetase from Clostridium pasteurianum grown on molasses as the sole carbon source and ammonium chloride as the nitrogen source has been purified to homogeneity (45-fold) with 32% recovery . The procedure involves ammonium sulfate precipitation and chromatography on a combined Sepharose 4B/DEAE-Sephadex A-50 column . The purified enzyme being very unstable was stabilized by the addition of 25% (v/v) glycerol . The enzyme has an unusually high molecular weight of 1 X 10(6) and 20 subunits of Mr 50 000 each, as determined by gel filtration and sodium dodecyl sulfate gel electrophoresis, respectively . It has an absorption maximum at 280 nm and a fluorescence emission maximum at 380 nm when excited at 280 nm . Its substrate binding pattern as studied by fluorescence quenching studies is different from that of the Escherichia coli enzyme . Both the gamma-glutamyltransferase and synthetase activities reside in the same protein as the ratio of the two activities at each step of purification remains constant and the enzyme exhibits optimal transferase and synthetase activities at the same pH (7.2) and temperature (50 degrees C) . The thermal stabilities of both activities were also similar, and decay of both the activities at 50 degrees C ran parallel . The enzyme shows stabilization by substrates, as L-glutamate, Mg2+, and ATP + Mg2+ protected both the synthetase and gamma-glutamyltransferase activities against thermal inactivation . Storage in 25% (v/v) glycerol enhanced the thermal stability of glutamine synthetase . Metal ion requirement and substrate specificity of the enzyme have been examined . Maximum synthetase activity occurs when {Mg2+}: {ATP} = 2 . The Km app values are as follows (in parentheses): ATP (0.34 mM), NH2OH (0.4 mM in the synthetase reaction and 4.1 mM in the transferase reaction), glutamine (14.7 mM), ADP (3.8 X 10(-4) mM), arsenate (2.5 mM), and L-glutamate (3.4 mM, 22.2 mM) . The enzyme exhibits negative cooperativity in the binding of glutamate . Amino acids such as L-serine, glycine, L-alanine, and L-aspartic acid inhibit the enzyme.

J Clin Microbiol, 1986 Apr, 23(4), 792 - 3
Evaluation of a commercial kit for the routine detection of Clostridium difficile cytotoxin by tissue culture; Wu TC et al.; The Toxi-titer microtiter plate system (Bartels Immunodiagnostic Supplies, Inc., Bellevue, Wash.) is a simplified procedure for detecting the cytotoxin produced by Clostridium difficile in stool filtrates . In a parallel study of 74 stool specimens, results from the Toxin-titer system compared favorably with those from the conventional system . Our experience with the Toxin-titer system in testing 540 stool specimens was, in general, satisfactory, although a few problems with toxin control did occur . The method requires no specialized experience in tissue culture techniques and can therefore be adopted for routine use in clinical laboratories.

Jpn J Exp Med, 1986 Apr, 56(2), 69 - 74
Density and distribution of Clostridium tetani in the soil; Ebisawa I et al.; Clostridium tetani or its toxin was recovered from a minute amount of soil samples collected on the surface of the ground of various places . C . tetani was recovered from 10% and tetanus toxin alone from another 1% of soil samples employing 1 mg of the soil . C . tetani or its toxin alone was recovered, employing 1 mg or more of soil, from the wet shores of ponds and rivers, fields and rice fields (85%), the yards of farmers and non-farmers (53%), school and hospital grounds (30%) and on the roadside (20%) . Two tetanus patients were injured in their yards where 1 mg soil samples yielded C . tetani . 12 of 13 soil samples collected at the yard of one of these patients at 3 different times yielded C . tetani . C . tetani did not remain at the same place in the same quantity . It was isolated more readily from the soil samples collected on the surface of than deep in the ground.

Immun Infekt, 1986 Apr, 14(2), 63 - 7
{Clostridium difficile-induced enterocolitis: pathogenesis, clinical course, epidemiology and laboratory diagnosis}; Meyer-Kawohl R et al.; Clostridium difficile-induced enterocolitis almost exclusively occurs associated with antibiotic exposure . The organisms produce several exotoxins of which toxins A (enterotoxin) and B (cytotoxin) are of primary importance . It is assumed that preceding antibiotic therapy creates an ecological niche allowing massive proliferation of the organisms and production of their toxins . The clinical course varies from mild diarrhoea to severe pseudomembranous colitis . Patients over 40 years of age are primarily at risk, especially when suffering from severe underlying disease or abdominal surgery . The therapy includes discontinuation of the actual antibiotic treatment, substitution of fluid and electrolytes, and causal therapy with vancomycin . In contrast to adult persons 25-60% of healthy infants excrete C . difficile and/or its toxins with their faeces . Exogenous infection by direct contact or contaminated tools and equipment is common in the hospital . The diagnosis includes: antibiotic history of the patient, clinical diagnosis (endoscopy), isolation of the organisms and demonstration of toxin production, detection of C . difficile toxins in the patient's stool . For isolation and identification appropriate methods are available . Cell culture tests for toxin detection are reliable but require experienced personnel . Immunological methods (ELISA, latex agglutination test) have been described but are not yet commercially available.

Appl Environ Microbiol, 1986 Apr, 51(4), 844 - 8
Effect of water activity and pH on growth and toxin production by Clostridium botulinum type G; Briozzo J et al.; The combined effect of water activity (aw) and pH on growth and toxin production by Clostridium botulinum type G strain 89 was investigated . The minimum aw at which growth and toxin formation occurred was 0.965, for media in which the pH was adjusted with either sodium chloride or sucrose . The minimum pH (at the optimum aw) for growth and toxin production of C . botulinum type G was found to be 5.6 . Optimum conditions for toxin activation were a trypsin concentration of 0.1%, a pH of the medium of 6.5, and an incubation for 45 min at 37 degrees C . These data did not show evidence of heat-labile spores, since a heat shock of 75 degrees C for 10 min did not significantly decrease the spore count of strain 89G in media at pH 7.0 or 5.6 . It was frequently observed that cells grown at reduced aw or pH experienced severe morphological changes.

Compr Ther, 1986 Apr, 12(4), 12 - 21
Tetanus; Edlich RF et al.; Tetanus is produced by the action of the potent neurotoxin, tetanospasmin, which is elaborated during the growth of Clostridium tetani . The objectives of management of tetanus are to provide supportive care until the tetanospasmin that is fixed in tissue has been metabolized, to neutralize circulating toxin, and to remove the source of tetanospasmin . This disease, which is frequently fatal, is prevented by immunization.

Gut, 1986 Apr, 27(4), 411 - 7
Effect of clindamycin on the ability of a continuous culture of colonic bacteria to ferment carbohydrate; Edwards CA et al.; A continuous culture model of the proximal colon was used to study the effect of clindamycin on the ability of colonic bacteria to ferment carbohydrate . Six steady state anaerobic cultures of human faeces, in a medium simulating ileostomy effluent, were treated with 26 micrograms/ml clindamycin . They were paired with six untreated cultures, run under identical conditions . Clindamycin treatment eliminated the anaerobic bacteria, significantly decreased osmolality and the output of volatile fatty acids, particularly propionic acid and increased the residual carbohydrate concentration . Doubling the amount of carbohydrate in the medium increased osmolality and the production of volatile fatty acid, though the response of clindamycin treated cultures was less than that of untreated cultures . Attempts to introduce Clostridium difficile into three pairs of cultures were successful in only two cultures after administration with clindamycin and when a heavy inoculum (10(6)-10(9) organisms) had been used.

Diagn Microbiol Infect Dis, 1986 Apr, 4(4), 359 - 63
Activity of cefotetan against anaerobic bacteria; Dias MB et al.; The in vitro activity of cefotetan, a new cephamycin, was compared with that of cefoxitin, cefotaxime, moxalactam, and piperacillin against 368 strains of anaerobic bacteria . Cefotetan was similar to cefoxitin, moxalactam, and piperacillin in its activity against B . fragilis, B . vulgatus, Bacteroides species, anaerobic gram-positive cocci, Fusobacterium and Clostridium species . High resistance rates for cefotetan, however, were demonstrated among B . thetaiotaomicron-ovatus group and B . distasonis . Increasing inoculum concentrations had little effect on the activity of cefotetan.

J Bacteriol, 1986 Apr, 166(1), 162 - 72
Structural features of multiple nifH-like sequences and very biased codon usage in nitrogenase genes of Clostridium pasteurianum; Chen KC et al.; The structural gene (nifH1) encoding the nitrogenase iron protein of Clostridium pasteurianum has been cloned and sequenced . It is located on a 4-kilobase EcoRI fragment (cloned into pBR325) that also contains a portion of nifD and another nifH-like sequence (nifH2) . C . pasteurianum nifH1 encodes a polypeptide (273 amino acids) identical to that of the isolated iron protein, indicating that the smaller size of the C . pasteurianum iron protein does not result from posttranslational processing . The 5' flanking region of nifH1 or nifH2 does not contain the nif promoter sequences found in several gram-negative bacteria . Instead, a sequence resembling the Escherichia coli consensus promoter (TTGACA-N17-TATAAT) is present before C . pasteurianum nifH2, and a TATAAT sequence is present before C pasteurianum nifH1 . Codon usage in nifH1, nifH2, and nifD (partial) is very biased . A preference for A or U in the third position of the codons is seen . nifH2 could encode a protein of 272 amino acid residues, which differs from the iron protein (nifH1 product) in 23 amino acid residues (8%) . Another nifH-like sequence (nifH3) is located on a nonadjacent EcoRI fragment and has been partially sequenced . C . pasteurianum nifH2 and nifH3 may encode proteins having several amino acids that are conserved in other proteins but not in C . pasteurianum iron protein, suggesting a possible role for the multiple nifH-like sequences of C . pasteurianum in the evolution of nifH . Among the nine sequenced iron proteins, only the C . pasteurianum protein lacks a conserved lysine residue which is near the extended C terminus of the other iron proteins . The absence of this positive charge in the C . pasteurianum iron protein might affect the cross-reactivity of the protein in heterologous systems.

Biochimie, 1986 Apr, 68(4), 575 - 80
{Role of acetate and butyrate in the induction of NADH: rubredoxin oxidoreductase in Clostridium acetobutylicum}; Ballongue J et al.; Study of the biosynthesis of NADH: rubredoxin oxidoreductase in resting cells of Clostridium acetobutylicum shows that this enzyme is synthesized at a maximal rate in the presence of acetic acid at a concentration of 3 g . l-1 and at pH 4.8 . Protons do not play any role in this biosynthesis since no induction is observed in a medium without acetate for the same values of pH . Butyric acid at a concentration of 0.5 g . l-1 gives 50% induction and formic acid, isobutyric acid and propionic acid have no inductive action on NADH: rubredoxin oxidoreductase . These results are confirmed by studies using a dialysis bag . Only a culture against acetic acid at an initial concentration of 2 g . l-1 gives maximal biosynthesis of the enzyme, whereas a culture in which all products of metabolism are eliminated gives an activity which is 80% lower.

Poult Sci, 1986 Apr, 65(4), 729 - 37
Effect of an elevated level of carbon dioxide containing atmosphere on the growth of spoilage and pathogenic bacteria at 2, 7, and 13 C; Baker RC et al.; The effect of 80% CO2 (balance air) on the survival and growth of microorganisms most often associated with spoilage and foodborne disease in poultry carcasses was compared to air at 2, 7, and 13 C . The CO2 atmosphere substantially retarded the growth of the total bacterial load in uninoculated ground chicken meat and parts at all temperatures when compared to air; however, temperature had a larger overall effect than atmosphere . Ground chicken meat and synthetic broth were inoculated (greater than 10(4) cells/ml or g) with Pseudomonas fragi, Salmonella typhimurium, Staphylococcus aureus, or Clostridium sporogenes and the influence of 80% CO2 and incubation temperature studied . With the exception of Cl . sporogenes, 80% CO2 was inhibitory when compared to air at any given temperature . In most cases, CO2 was more inhibitory at 2 C than at 7 or 13 C . The Cl . sporogenes inoculum failed to grow above initial inoculum levels in any combination of temperature and atmosphere, but samples packed in 80% CO2 had higher numbers of colony forming units than air-packaged samples . This study does not indicate that modified atmosphere packaging of refrigerated poultry in elevated CO2 atmospheres increases the microbial hazards when compared to air at the same temperature.

Anal Biochem, 1986 Apr, 154(1), 26 - 8
Use of specific collagenases for the isolation of rat liver cells with preserved lipase activities; Quibel JR et al.; The heparin-releasable neutral lipase (EC 3.1.1.3) from rat liver is inactivated by the common preparations of collagenases (EC 3.4.24.3) used for the isolation of liver cells . We show that two collagenases purified from Clostridium histolyticum allow both the complete preservation of this lipolytic activity and a good viability of liver cells isolated by the usual perfusion protocol.

J Antibiot (Tokyo), 1986 Apr, 39(4), 550 - 6
Effects of streptonigrin derivatives and sakyomicin A on the respiration of isolated rat liver mitochondria; Inouye Y et al.; Though all the streptonigrin derivatives with modifications in the carboxyl group on C2' were active as electron acceptors in the oxidation of NADH by Clostridium kluyveri diaphorase as well as streptonigrin, the glycine derivative did not show any marked effect on the KCN-insensitive oxidation of glutamate by rat liver mitochondria, suggesting a poor membrane transport of the glycine derivative . Sakyomicin A also induced the KCN-insensitive oxidation of glutamate by mitochondria, while a trace activity was observed by mitomycin C and the effect of doxorubicin was negligible . Like streptonigrin, the autoxidation of a reduced form (hydroquinone) of sakyomicin A to a quinone accompanied the generation of H2O2 . However, exogenous NADH was oxidized by mitochondria in the presence of sakyomicin A but not streptonigrin.

Lab Anim, 1986 Apr, 20(2), 114 - 7
Spontaneous necrotic enteritis in young RFM/Ms mice; Matsushita S et al.; Fatal necrotic enteritis was observed in mice 24-52 days old in the RFM/Ms breeding colony maintained in a clean conventional condition in the National Institute of Radiological Sciences . Gross lesions included hyperaemia, petechiae, erosion and the occasional formation of pseudomembranes in the mucosa of the ileum and caecum . Histologically, there was necrotic enteritis with numerous Gram-positive bacilli-forming spores but no inflammatory cell reaction . Non-type-A Clostridium perfringens was isolated from the intestinal contents . This disease cleared after the addition of chlortetracycline hydrochloride (11 mg/l) to the drinking water.

Diagn Microbiol Infect Dis, 1986 Apr, 4(4), 307 - 13
Detection of Clostridium perfringens enterotoxin in stool specimens and culture supernatants by enzyme-linked immunosorbent assay; Wimsatt JC et al.; An enzyme-linked immunosorbent assay was developed to detect and quantitate Clostridium perfringens enterotoxin A in culture supernatants and in stool specimens from cases of diarrhea in which high numbers of enterotoxin-producing Clostridium perfringens were isolated . To analyze for enterotoxin A, polyvinyl chloride microtiter plates were coated with dilute immune whole rabbit serum . Enterotoxin A standards and samples were allowed to react with sensitized wells . The presence of the immobilized antigen in the wells was detected by the binding of immune rabbit immunoglobulin conjugated with peroxidase . Nanograms of enterotoxin were detectable . Four enterotoxin-positive and seven enterotoxin-negative cultures grown in Duncan-Strong medium gave expected results . Eighteen of 23 diarrheal stool specimens obtained after a food-poisoning outbreak at a state hospital were found to contain microgram quantities of enterotoxin per gram of stool, whereas five control diarrheal specimens contained less than 0.6 ng enterotoxin per gram of stool . These results indicate that the enzyme-linked immunosorbent assay technique is useful for differentiating enterotoxigenic strains and for diagnosing diarrhea caused by enterotoxigenic Clostridium perfringens.

Infect Immun, 1986 Apr, 52(1), 31 - 5
Production and characterization of monoclonal antibodies to Clostridium perfringens enterotoxin; Horiguchi Y et al.; Four hybridoma cell lines producing monoclonal antibodies to Clostridium perfringens enterotoxin were established by fusion of mouse myeloma and spleen cells obtained from mice immunized with the enterotoxin and its toxoid . An enzyme-linked immunosorbent assay indicated that the two antibodies, 2-B-4 and 3-G-10, bound to those regions that were located close each other; the others, 3-B-2 and 2-H-2, bound to other independent regions on the enterotoxin . Release of 51Cr from Vero cells with the enterotoxin was inhibited by either 2-B-4 or 3-G-10, both of which inhibited the binding of 125I-labeled enterotoxin to the cells . Neither binding nor cytotoxicity of the enterotoxin was affected by 2-H-2; 3-B-2 only barely inhibited the binding but neutralized the enterotoxin shown by 51Cr release . It seems justified to conclude that 3-B-2 blocks the toxic action after the enterotoxin has bound to Vero cells.

J Antibiot (Tokyo), 1986 Apr, 39(4), 557 - 63
Role of the naphthoquinone moiety in the biological activities of sakyomicin A; Take Y et al.; The naphthoquinone moiety was proven to be essential to the biological activities of sakyomicin A using various naphthoquinone derivatives . Among the naphthoquinones tested, juglone (5-hydroxy-1,4-naphthoquinone) which resembles the partial structure of sakyomicin A was the most active in cytotoxicity against murine lymphosarcoma L5178Y cells, electron acceptor function in the oxidation of NADH by Clostridium kluyveri diaphorase or rat liver mitochondria and inhibition against avian myeloblastosis virus reverse transcriptase . The significantly lower cytotoxicity of sakyomicin A as compared with juglone was attributable to its poor membrane transport . The inhibition of reverse transcriptase activity may result from the interaction between a sulfhydryl group in the active center of the enzyme and quinone groups of the naphthoquinones and sakyomicin A.

Med J Aust, 1986 Mar 17, 144(6), 303 - 6
Antibiotic-associated colitis caused by Clostridium difficile: relapse and risk factors; Young GP et al.; Relapse is a common sequel of antibiotic-associated colitis due to Clostridium difficile . It has been suggested that Cl . difficile may persist in the stools in spite of the resolution of symptoms after treatment and this may cause the relapse . Our study was designed to define the factors that predispose to relapse and to determine if prolonging treatment to clear Cl . difficile from the stools might prevent relapse . Of 60 consecutive patients, 36 with more severe disease required treatment . Treatment with either vancomycin or bacitracin was continued until the results of the examination of stools for cytotoxin became negative and Cl . difficile could no longer be cultured (sensitivity of culture was 10-100 organisms/mL) . This was achieved in 35 patients who were then followed for one month . Symptoms reappeared in 10 (28.6%) of the treated patients while Cl . difficile reappeared in the stools of an additional seven patients (20%) without the recurrence of diarrhoea . On comparing those who relapsed with those who did not, the age (67.3 +/- 5.5 years in those who relapsed compared with 51.6 +/- 4.4 years; P less than 0.025, means +/- SE) and a history of recent abdominal surgery (59% of those who relapsed compared with 17%; P less than 0.05) were significantly different . Although those who relapsed had received therapy with multiple antibiotic agents more often, this was not statistically significant . Disease was not more severe in patients who relapsed, nor was it more difficult to clear the pathogen from these patients . The 24 untreated patients did not suffer symptomatic relapse . Continuation of treatment until Cl . difficile apparently is absent from the stools is expensive and does not prevent relapse . Elderly patients and those who have recently undergone abdominal surgery are more likely to suffer a relapse.

Biochemistry, 1986 Mar 11, 25(5), 1002 - 8
The antitumor agent mitoxantrone binds cooperatively to DNA: evidence for heterogeneity in DNA conformation; Rosenberg LS et al.; The equilibrium binding of the antitumor compound DHAQ, or mitoxantrone {1,4-dihydroxy-5,8-bis{{2-{(2-hydroxyethyl)amino}ethyl}amino}-9,10- anthracenedione}, to various DNAs has been examined by optical titration and equilibrium dialysis methods . At low r (bound drug/DNA base pair) values, r less than 0.03, DHAQ binds, in a highly cooperative manner, to calf thymus and Micrococcus lysodeikticus DNAs . The binding isotherms for the interaction of DHAQ with Clostridium perfringens DNA and poly(dA-dT).poly(dA-dT) exhibit a small positive slope at low r values, suggestive of cooperative binding . In contrast, the binding of DHAQ to poly(dG-dC).poly(dG-dC) shows no evidence of cooperative binding even at very low r values . At higher r values (r greater than 0.05), the binding of DHAQ to all the DNAs studied is characterized by a neighbor-exclusion process . A model is proposed to account for the two modes of binding exhibited in the cooperative binding isotherms . The main feature of the proposed model is that local sequence and structural heterogeneity of the DNA give rise to sets of binding sites to which DHAQ binds in a highly cooperative manner, while the majority of the DNA sites bind DHAQ via a neighbor-exclusion process . This two-site model reproduces the observed binding isotherms and leads to the conclusion that DHAQ binds in clusters to selected regions of DNA . It is suggested that clustering may play a role in the physiological activity of drugs.

Onderstepoort J Vet Res, 1986 Mar, 53(1), 51 - 3
Immunization of guinea-pigs and cattle with a reduced dose Clostridium chauvoei vaccine produced in a semi-synthetic medium; Cameron CM et al.; A semi-synthetic culture medium and method are described for the production of a reduced dose Clostridium chauvoei vaccine . The vaccine gave excellent results in guinea-pigs, and 2 injections of 2.0 ml protected cattle against challenge with 2 M.L.D . of a virulent culture for at least 12 months . The suitability of C . chauvoei Strain OP64 as a vaccine strain was confirmed.

Aust Vet J, 1986 Mar, 63(3), 68 - 70
Standards for Clostridium chauvoei vaccine--the relationship between the response of guinea pigs and sheep following vaccination and challenge with virulent C . chauvoei; Crichton R et al.; Twelve commercial 5-component clostridial vaccines with known variations in potency of the blackleg (Clostridium chauvoei) component, were simultaneously tested in sheep and guinea pigs . Controlled challenge experiments provided evidence of a highly significant correlation in the response of the 2 species . The guinea pig laboratory model is considered to be a valid indicator of field performance for vaccines containing blackleg antigen.

Acta Chir Belg, 1986 Mar-Apr, 86(2), 63 - 71
{Gas gangrene: a military disease?}; Pailler JL et al.; Gas gangrene is a general toxi-infection, which is mostly the result of a contamination of the muscles from traumatic or post-operative origin . Muscular necrosis expands very quickly, causes mutilations, hits several organs and leads to shock . The spontaneous evolution is mostly mortal within a couple of hours . For a long time it was considered to be a typical military disease: during World War I some 100,000 German soldiers lost their lives as a direct result . It represented then 10 to 12% of all deads of wounds . This mortality rate decreased between 0.3 and 1.5% during World War II and to 0.016% during the Vietnam WAr . Although gas gangrene is no longer a typical military disease, the holy principles of war surgery must be respected . All war wounds, and in particular those caused by high velocity missiles are contaminated, a.o . by clostridium spores . An immediate suture of those wounds, through which necrotized tissue is covered (an ideal anaerobic environment), furthers the development of gas gangrene . The only prevention consists of debriding those wounds, leaving them opened, followed by a delayed primary suture between the 4th and 7th day after the debridement . Not properly treated will gas gangrene always be mortal . In some instances, an emergency amputation will be the only way to save the patient's life . Antibiotherapy, hyperbaric oxygenotherapy and most of all surgery from the medical triade which is imperative to treat this disease . Thanks to these measures, the mortality rate decreased from 70% before 1960 to 41% . Nonetheless the most important act remains the early, complete, rational and rigorous debridement of the wounds . Far from being an obsolete attitude, this is one of humility, wisdom .. . and with future prospects.

Scand J Gastroenterol, 1986 Mar, 21(2), 253 - 6
Beta-glucuronidase-producing bacteria in bile from the common bile duct in patients treated with endoscopic papillotomy for gallstone disease; Skar V et al.; This paper reports the occurrence of beta-glucuronidase-producing bacteria in the bile in gallstone patients treated with endoscopic papillotomy (EPT) . The study included 36 patients--18 women and 18 men, aged 43-87 years, with a median of 72.5 years . Bile sampling was done with an endoscopic technique . All bacterial strains were tested for beta-glucuronidase activity with a rapid chromogenic tablet test, using 4-nitrophenyl-beta-D-glucuronic acid as substrate . Bacterial growth was found in the bile in 35 patients . Of 103 strains isolated, 30 produced beta-glucuronidase . Twenty-five of the patients had at least one beta-glucuronidase-producing strain in the bile . All 26 strains of Escherichia coli were producing the enzyme . Both strains in the Bacteroides fragilis group and one out of two strains of Clostridium perfringens were producing beta-glucuronidase . The activity of the bacterial beta-glucuronidase was found within the pH range of the bile in these patients . A relationship between the presence of beta-glucuronidase-producing bacteria in the bile and pigment gallstone is suggested.

J Biochem (Tokyo), 1986 Mar, 99(3), 961 - 9
Purification and properties of nitrate reductase from Mitsuokella multiacidus; Yamamoto I et al.; Nitrate reductase of Mitsuokella multiacidus (formerly Bacteroides multiacidus) was solublized from the membrane fraction with 1% sodium deoxycholate and purified 40-fold by immunoaffinity chromatography on the antibody-Affi-Gel 10 column . The preparation showed a major band (86% of total protein) with enzyme activity and a minor band on polyacrylamide gel after disc electrophoresis in the presence of 0.1% Triton X-100 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a major band, the relative mobility of which corresponded to a molecular weight of 160,000, and two minor bands . The molecular weight of the enzyme was determined to be 160,000 by gel filtration on Bio-Gel A-1.5 m in the presence of 0.1% deoxycholate . Molybdenum cofactor was detected in the enzyme by fluorescence spectroscopy and by complementation of nitrate reductase from the nit-1 mutant of Neurospora crassa . The M . multiacidus enzyme catalyzed reduction of nitrate, chlorate, and bromate using methyl viologen as an electron donor . The maximal activity was found at pH 6.2-7.5 for nitrate reduction . Either methyl or benzyl viologen served well as the electron donor, but FAD, FMN, and horse heart cytochrome c were not effective . Ferredoxin from Clostridium pasteurianum supplied electron to the nitrate reductase . The purified enzyme had Km values of 0.13 mM, 0.12 mM, and 0.22 mM for nitrate, methyl viologen, and ferredoxin, respectively . The enzyme activity was inhibited by cyanide (85% at 1 mM), azide (88% at 0.1 mM), and thiocyanate (75% at 10 mM).

Diagn Microbiol Infect Dis, 1986 Mar, 4(3 Suppl), 87S - 91S
Clinical considerations in the diagnosis of antimicrobial agent-associated gastroenteritis; Finegold SM; Most gastrointestinal infections secondary to the use of antimicrobial agents that have been documented are related to overgrowth of Clostridium difficile which produces a spectrum from severe pseudomembranous colitis to mild diarrhea or asymptomatic carriage . The most common inducers of pseudomembranous colitis or antimicrobial agent-associated diarrhea are ampicillin, clindamycin, and various cephalosporins, but almost all antimicrobials may cause this problem . Symptoms vary from watery to bloody diarrhea; the extent and severity of the diarrhea, fever, and abdominal cramps and the incidence of complications (such as toxic megacolon and perforation of the bowel) and of fatality are variable . Normal carriage of C . difficile in infants and asymptomatic carriage in adults who have received antimicrobial therapy make it impossible to rely on culture for diagnosis . The presence of cytotoxin or enterotoxin produced by C . difficile is much more reliable diagnostically, but there may be false-positives with this as well, particularly in infants . However, the combination of the appropriate clinical picture and background and presence of toxin usually permit accurate diagnosis . The definitive method of diagnosis, often not feasible to employ, is demonstration by colonoscopy or sigmoidoscopy of the pathognomonic yellow, elevated plaques on the colonic mucosa . Colonoscopy is preferred since the plaques may be restricted to the right colon, particularly in early cases . From the practical standpoint, the best diagnostic test is demonstration of C . difficile toxin.

Zh Mikrobiol Epidemiol Immunobiol, 1986 Mar, (3), 82 - 6
{Characteristics of the immune response of inoculated and intact animals to the administration of toxigenic and nontoxigenic strains of Clostridium tetani}; Shvarts SA et al.; In experiments on guinea pigs the immune reactions of the animals immunized and not immunized against tetanus in response to the injection of C . tetani spores were studied . In the immunized animals an increase in the production of tetanus antitoxin, the development of delayed hypersensitivity and the activation of the mechanisms of cell-mediated immunity were observed . The nonimmunized animals showed specific changes in the T-system of immunity without the appearance of the clinical symptoms of tetanus, which is, probably, one of the mechanisms of natural immunity.

J Infect Dis, 1986 Mar, 153(3), 547 - 51
Gnotobiotic models for study of the microbial ecology of Clostridium difficile and Escherichia coli; Wilson KH et al.; Hamster flora introduced into germfree mice reduced the cecum to conventional size, suppressed populations of Escherichia coli and Clostridium difficile to the same degree that mouse flora did, and corrected the hypocellularity that is characteristic of the small bowel of germfree mice . A highly toxigenic strain of C . difficile readily induced cecitis in germfree and antibiotic-treated conventional mice, and histological examination frequently revealed pseudomembranes . Toxins A and B were both detected in ceca of animals with colitis . Gnotobiotic mice provide a model in which to study the role of the indigenous microflora in protecting against antibiotic-associated colitis.

J Clin Microbiol, 1986 Mar, 23(3), 622 - 3
Commercial latex test for Clostridium difficile toxin A does not detect toxin A; Lyerly DM et al.; The rapid latex test recently marketed by Marion Scientific (Div . Marion Laboratories, Inc., Kansas City, Mo.) for the detection of Clostridium difficile toxin A does not react with the toxin, based on the following findings: culture filtrates from nontoxigenic strains of C . difficile gave positive reactions in the test, culture filtrate in which toxin A had been removed gave positive reactions, purified toxin A did not react in the test, and the latex reagent bound an antigen which is distinct from toxin A and which is produced in various amounts by both toxigenic and nontoxigenic strains of C . difficile.

Spine, 1986 Mar, 11(2), 170 - 2
Discitis due to Clostridium perfringens; Beguiristain JL et al.; Diagnosis of the infectious process of the intervertebral disc is often delayed due to the lack of specifity of the clinical picture . This delay in diagnosis and consequently in treatment can lead to severe neurologic sequela . In these cases, punction biopsy of the intervertebral disc is of special value . The authors found no mention in the literature concerning Chlostridium perfringens discitis verified by aspiration and fluid culture extracted by intervertebral disc puncture.

Appl Environ Microbiol, 1986 Mar, 51(3), 521 - 3
Rapid extraction of plasmids from Clostridium perfringens; Mahony DE et al.; Two rapid methods were evaluated for their extraction of plasmids from Clostridium perfringens . The first method involved lysis of 1 to 2 ml of C . perfringens culture by treatment with hyaluronidase, lysozyme, and sarcosyl . DNA, extracted with phenol-chloroform, was treated with RNase, boiled, and electrophoresed in a 1.2% agarose gel . The second method involved lysis of 2 ml of culture by lysozyme treatment and extraction with alkaline sodium dodecyl sulfate (SDS) . Extracted DNA was treated with RNase, boiled, and electrophoresed in a 0.7% agarose gel . Of 57 strains of C . perfringens analyzed by both extraction procedures, 11 were shown to have plasmids by the alkaline SDS method which were missed by the phenol-chloroform extraction method . These new plasmids were of higher molecular mass and ranged up to 68 megadaltons . Use of the DNase inhibitor diethyl pyrocarbonate did not further improve the yield of plasmid DNA . An additional 159 isolates of C . perfringens screened by the alkaline SDS method revealed plasmids up to 80 megadaltons in mass and an overall plasmid carriage rate of 69%.

Transfusion, 1986 Mar-Apr, 26(2), 182 - 5
Uneventful administration of plasma products in a recipient with T-activated red cells; Eversole M et al.; A patient with T-polyagglutinable red cells and a severe coagulopathy provided an opportunity to observe the results of plasma transfusion in the face of T-activation . The patient was a 52-year-old Navajo Indian with a perforated gall bladder and related sepsis due to Clostridium perfringens . The gall bladder was removed surgically . Postoperatively, he had severe thrombocytopenia, and prolonged partial thromboplastin and prothrombin times . The patient's red cells were agglutinated by Arachis hypogaea and Glycine soja lectins but were unagglutinated by extracts of Salvia horminum, Salvia sclarea, and Bandeiraea simplicifolia . No untoward reactions or any evidence of hemolysis were observed when the patient was given platelet concentrates and 4 units of single-donor plasma . Serial plasma hemoglobin and haptoglobin levels documented that there was no hemolysis . His coagulopathy responded, and he had a successful surgical re-exploration and recovery . This case documents that serious adverse consequences do not necessarily follow transfusion of plasma in a recipient with T-activated red cells . T-activation is a relative but not absolute contraindication to plasma transfusion.

Nucleic Acids Res, 1986 Feb 25, 14(4), 1791 - 9
Sequence of the cellulase gene of Clostridium thermocellum coding for endoglucanase B; Grepinet O et al.; The nucleotide sequence of the CelB gene, encoding the extracellular endoglucanase B of Clostridium thermocellum, is reported . The putative start of the 1689 bp coding sequence was assigned to an ATG codon which is preceded by an AGGAGG sequence typical of ribosomal binding sites in Gram-positive bacteria . The amino-terminal end of the deduced protein sequence is similar to signal peptides described for other bacterial secretory proteins . The carboxy-terminal ends of endoglucanases A and B appear to be remarkably homologous . A striking feature of the conserved region is that both proteins contain two reiterated stretches of 23 aminoacids each, separated by 9 residues.

Cancer, 1986 Feb 15, 57(4), 885 - 9
Distant nontraumatic clostridial myonecrosis and malignancy; Kaiser CW et al.; Distant nontraumatic clostridial myonecrosis in association with malignancy is an uncommon disorder, with only 14 well-documented cases previously reported in the English literature . Clostridium perfringens and C . septicum are the most common organisms, usually gaining access to the circulation through an ulcerated lesion of the small bowel or colon . A case report of this syndrome caused by C . histolyticum is presented with a review of the literature.

J Am Vet Med Assoc, 1986 Feb 15, 188(4), 382 - 6
Clostridium botulinum type B toxicosis in a herd of cattle and a group of mules; Divers TJ et al.; Clostridium botulinum type B toxicosis was diagnosed as the cause of generalized weakness and death in a group of cows and mules fed from the same batch of rye silage . One severely affected cow was treated and recovered, as did other less severely affected cows . All affected mules died . The remaining cattle in the herd were then vaccinated before continued feeding of the silage.

Arch Biochem Biophys, 1986 Feb 15, 245(1), 144 - 52
Diol metabolism and diol dehydratase in Clostridium glycolicum; Hartmanis MG et al.; Levels of the five enzymes involved in the fermentation of 1,2-ethanediol and 1,2-propanediol in the strictly anaerobic bacterium, Clostridium glycolicum, were investigated . All enzymes with the exception of the first enzyme in the pathway, diol dehydratase, were found to be constitutive, stable to exposure to oxygen, and present in the cytosol . Diol dehydratase was found to be extremely oxygen sensitive and strongly associated with the cell membrane . Treatment with ionic and nonionic detergents, butanol, phospholipase A2, or osmotic shock procedures failed to solubilize any diol dehydratase activity . Limited proteolysis using subtilisin released small amounts of activity . Diol dehydratase was found to be specific for 1,2-ethanediol and 1,2-propanediol and required the addition of a reducing agent for maximal activity . The enzyme was strongly inhibited by low concentrations of EDTA, ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, o-phenanthroline, hydroxylamine, hydroxyurea, and sulfhydryl reagents . Addition of adenosylcobalamin or high levels of intrinsic factor did not affect the reaction rate . Irradiation with light also did not inhibit the enzyme activity . These results suggest that the catalytic mechanism of diol dehydratase from C . glycolicum does not involve a cobamide coenzyme.

J Chromatogr, 1986 Feb 14, 375(1), 11 - 25
Headspace gas chromatographic-mass spectrometric analysis of light hydrocarbons and volatile organosulphur compounds in reduced-pressure cultures of Clostridium; Rimbault A et al.; A static headspace gas chromatographic method for the simultaneous separation of trace light hydrocarbons and volatile organosulphur compounds in gases of nineteen Clostridium cultures at reduced pressure is described . The separation was achieved on n-octane-Porasil C after sampling of the gaseous compounds in a PTFE loop without any pretreatment . Most peaks were identified by gas chromatography-mass spectrometry . The presence of methane and ethylene sulphide among Clostridium volatiles is confirmed and 3-methyl-1-butene, 2-methyl-2-butene, dimethyl trisulphide and S-methyl thioacetate are reported for the first time in the Clostridium group.

Vet Rec, 1986 Feb 8, 119(6), 151 - 2
Pathological changes in the pericardium and meninges of cattle associated with Clostridium chauvoei; Malone FE et al.; Twenty-nine cases of Clostridium chauvoei infection in cattle were investigated over a two-year period . Fourteen had lesions of myositis only, eight had lesions of both myositis and fibrinous pericarditis, six had lesions of fibrinous pericarditis only and one had lesions of purulent meningitis only . Cl chauvoei was identified in all the lesions using the fluorescent antibody technique.

J Biol Chem, 1986 Feb 5, 261(4), 1609 - 15
The autotrophic pathway of acetogenic bacteria . Role of CO dehydrogenase disulfide reductase; Pezacka E et al.; An enzyme from Clostridium thermoaceticum has been isolated which reduces disulfides of carbon monoxide dehydrogenase and it has been named CO dehydrogenase disulfide reductase . The enzyme is a tetramer of molecular weight 225,000 made up of four apparently identical monomers . It does not contain methionine or tryptophan and contains 2 calcium and 1 zinc/monomer . NADP or ferredoxin serves as an electron carrier . This enzyme is part of the system that permits certain bacteria to grow with CO or CO2 and H2 as the source of carbon and energy . The portion of the pathway which is being investigated is the conversion of methyltetrahydrofolate, CO, and CoASH to acetyl-CoA . All the enzymes required for this synthesis have now been purified . In combination with CO dehydrogenase, CO dehydrogenase disulfide reductase with NADP or ferredoxin catalyzes a reversible exchange of {3H}CoASH with acetyl-CoA . The disulfide reductase apparently is involved in the portion of the pathway in which CoASH is introduced into the acetyl-CoA . In addition, the reductase activates CO dehydrogenase in the overall synthesis of acetyl-CoA from methyltetrahydrofolate, CO, and CoASH by reducing about one disulfide group/monomer of the alpha 3 beta 3 CO dehydrogenase . The above exchange reaction in combination with the observation that {14C}acetate is formed from CO and the 14CH3-{Co}corrinoid enzyme in the absence of CoASH have permitted ordering of the sequence of reactions by which CO dehydrogenase plays a central role in the autotrophic synthesis of acetyl-CoA.

J Hyg (Lond), 1986 Feb, 96(1), 13 - 7
Clostridium difficile in general practice and community health; Riley TV et al.; The isolation rate for Clostridium difficile in diarrhoeal stools was investigated in patients from general practice and community health centres over a 14-month period . C . difficile or its cytotoxin was detected in specimens from 89 (4.7%) of 1882 patients studied and accounted for 30.3% of all enteropathogenic micro-organisms isolated . Overall C . difficile was second only to Giardia lamblia in frequency . Recovery rates in the different groups of patients surveyed varied from 3.6 to 27.5% . The relationship between stool culture results and stool cytotoxin assay also varied considerably between groups of patients studied . Coincident infections with a variety of enteropathogenic bacteria and intestinal parasites were diagnosed in 14 of the 89 patients . It was concluded that laboratories servicing this type of practice should be aware that C . difficile may be a cause of diarrhoea . An adequate clinical history should facilitate proper processing of the specimen.

J Clin Pathol, 1986 Feb, 39(2), 212 - 4
Latex particle agglutination for detecting and identifying Clostridium difficile; Bowman RA et al.; A total of 329 selective enrichment broth cultures were tested for detection of Clostridium difficile by latex particle agglutination (LPA), gas-liquid chromatography, and bacterial culture . Of 53 broths positive by LPA, 36 were positive by gas-liquid chromatography, and 42 were positive by bacterial culture . The sensitivity and specificity of LPA relative to bacterial culture was 95.6% and 96.3%, respectively, while the sensitivity and specificity of gas-liquid chromatography relative to bacterial culture was 84.6% and 100%, respectively . The high predictive value of a negative test (99%) should make LPA on broth cultures a good screening test for detecting C difficile . Of several other Clostridium spp tested in pure culture, strains of C sordellii and C bifermentans also gave a positive result by LPA . These results, together with the low cost and simple facilities required, suggest that the LPA test will be a useful procedure for the presumptive identification of C difficile in selective enrichment broths and for the identification of pure cultures.

Infect Immun, 1986 Feb, 51(2), 707 - 11
Ability of oral bacteria to degrade fibronectin; Wikstrom M et al.; The fibronectin-degrading ability of 116, mainly oral, strains was assayed by using plasma-derived fibronectin adsorbed to a polystyrene surface . Ability to degrade fibronectin was revealed in strains of Bacteroides gingivalis, Bacteroides intermedius, Bacteroides loeschii, Staphylococcus aureus, Staphylococcus epidermidis, Peptococcus prevotii, Clostridium sporogenes, and Propionibacterium acnes . The fibronectinolytic activity of subgingival bacteriological samples was found to be related to the presence of B . gingivalis and B . intermedius . In addition, strains of the nonoral Bacteroides species B . asaccharolyticus and B . fragilis showed fibronectin-degrading ability . No such ability was detected in the oral strains tested of Streptococcus, Veillonella, Actinomyces, Lactobacillus, Actinobacillus, Capnocytophaga, Fusobacterium, or Haemophilus species.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1986 Feb, 261(1), 29 - 42
Detection of Clostridium difficile Toxin A by immunoblotting; Rautenberg P et al.; A clinical isolate of Clostridium difficile has been tested for its toxin production . Both toxins, toxin A and toxin B, could be detected by tissue culture and in animal models as well . Antibodies against a crude toxin A preparation have been prepared . These antibodies are able to neutralize the toxin both in the mouse lethality test and tissue culture test systems . The specificity of this antiserum has been analysed by electroimmunoprecipitation methods . Using immunoblotting, it could be demonstrated that the antigenicity of toxin A after SDS polyacrylamide gel electrophoresis under denaturing and reducing conditions was still preserved . The molecular weight of toxin A has been estimated to be 250000 . Immunoblotting offers a simple and reliable procedure for toxin A detection from culture supernatants of C . difficile.

Can J Microbiol, 1986 Feb, 32(2), 132 - 6
{Transfer of the cecal flora of the hamster to the germfree C3H mouse: use of this model to study the flora of the anti-Clostridium difficile barrier}; Su WJ et al.; The purpose of this work was the research and development of an experimental model to study anti-Clostridium difficile caecal microflora in the hamster . First the existence of this "barrier" was verified in conventional hamsters . Then, the caecal flora from these animals was orally transferred to C3H germfree mice . The barrier effect was maintained in the axenic mice . The comparative bacteriological analysis of hamster and mouse feces did not reveal important variations in the dominant anaerobic flora (P less than 0.01) . After treatment with erythromycin, the barrier effect was maintained and while the disappearance of Escherichia coli was observed, the dominant anaerobic flora persisted . After dilution (10(-2} and subsequent heating (70 degrees C, 10 min) of caecal contents, the inhibitory activity against C . difficile was maintained although the number of aerobic and aerotolerant bacteria was reduced . The isolation from caecal microflora of anaerobic strains implicated in the resistance to colonization is presently underway in Freter anaerobic chambers.

Appl Environ Microbiol, 1986 Feb, 51(2), 256 - 60
Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives; Oguma K et al.; The biochemical properties of 11 toxigenic and 10 nontoxigenic type C and D strains of Clostridium botulinum were studied . All of the strains examined were motile and hemolytic and produced lipase and liquid gelatin . Fermentation of several sugars and the production of lecithinase, indole, and hydrogen sulfide varied with the strain . The strains were classified into four groups based on their sugar fermentation profiles . The resulting classification was identical to the classification which had been proposed from the relationship between toxin production and phages and similar to the grouping based on the nature of toxin antigenic structures . Lecithinase production was negative in the cells belonging to group III, and indole and hydrogen sulfide production was negative in the cells of groups III and IV . Strains belonging to groups III and IV have many characteristics different from those of groups I and II.

Mol Immunol, 1986 Feb, 23(2), 147 - 50
The site of cleavage in human alpha chains of IgA1 and IgA2:A2m(1) allotype paraproteins by the clostridial IGA protease; Fujiyama Y et al.; Fc fragments of human immunoglobulin A(IgA) of IgA1 subclass and IgA2 subclass of A2m(1) allotype were prepared from IgA paraproteins by digestion with a protease from Clostridium sp . (M.O.-6) . The N-terminal tetrapeptide of Val-Pro-Ser-Thr- for the Fc of IgA1 subclass, and that of Val-Pro-Pro-Pro- for the Fc of IgA2:A2m(1) allotype, were identified by sequence analysis . The site of cleavage by the protease was defined to be at the Pro-Val peptide bond, which is a common peptide bond present at 221-222 in both alpha chains . IgA of IgA2 subclass of A2m(2) allotype is resistant to the protease due to the different, Arg-Val, peptide bond at the same position.

Gastrointest Endosc, 1986 Feb, 32(1), 7 - 9
Efficacy of routine fiberoptic endoscope cleaning and disinfection for killing Clostridium difficile; Hughes CE et al.; We have evaluated a standard procedure for cleaning and disinfection of endoscopes for efficacy in eradicating a spore-forming bacterial organism, Clostridium difficile . Initially, 23 endoscopes were cultured for the presence of C . difficile after hanging in storage for at least 24 hours after cleaning and disinfection . All cultures were negative . Subsequently, endoscopes used in 15 patients who had stool cultures positive for C . difficile were cultured immediately after use and again after cleaning and disinfection with 2% alkaline glutaraldehyde for 5 min . Ten of 15 (67%) endoscopes were culture positive for C . difficile immediately after use . After cleaning and disinfection, all of the endoscopes were culture negative except one, which yielded two negative cultures and two cultures showing late growth of rare C . difficile colonies, but contamination could not be ruled out . In vitro exposure to 2% alkaline glutaraldehyde for 5 min resulted in 99% or greater killing of C . difficile spores . We conclude that cleaning and a minimum of 5 min of disinfection with 2% alkaline glutaraldehyde are likely to be effective in killing C . difficile vegetative organisms and spores on endoscopes.

J Infect Dis, 1986 Feb, 153(2), 267 - 71
Bacterial agglutination and polyacrylamide gel electrophoresis for typing Clostridium difficile; Mulligan ME et al.; Bacterial agglutination and polyacrylamide gel electrophoresis (PAGE) were methods evaluated for typing strains of Clostridium difficile . A panel of four antisera, obtained by immunizing rabbits with washed whole cells of different strains of C . difficile, produced distinctive patterns of agglutination . Ethylenediaminetetraacetate (EDTA) extracts subjected to PAGE also produced distinctive protein profiles . Excellent correlation between the two methods was observed when geographically distant isolates were typed without knowledge of their clinical origin . Both typing methods should receive further evaluation for their value as tools for epidemiological studies.

Mol Gen Genet, 1986 Feb, 202(2), 251 - 4
Mapping of mRNA encoding endoglucanase A from Clostridium thermocellum; Beguin P et al.; The size and location of the 5' end of celA mRNA encoding endoglucanase A of Clostridium thermocellum were investigated in C . thermocellum and in an Escherichia coli clone that carries and expresses the celA gene . In E . coli, the 5' end of celA mRNA was located 134 bp upstream from the initiation codon and 10 bp downstream from a sequence homologous to the consensus sequence of E . coli sigma 70 and Bacillus subtilis sigma 43 (formerly sigma 55) vegetative promoters . In C . thermocellum, a minor transcript appeared to start from the same site, but a major species started 57 bp upstream from the coding sequence . The 5' end of this mRNA was preceded by a sequence reminiscent of B . subtilis sigma 28 vegetative promoters . In both organisms, the size of the transcript suggested that celA belongs to a monocistronic unit of transcription.

Antimicrob Agents Chemother, 1986 Feb, 29(2), 374 - 5
Comparative in vitro activity of seven quinolones against 100 clinical isolates of Clostridium difficile; Delmee M et al.; The in vitro activity of seven quinolone derivatives against 100 clinical isolates of Clostridium difficile was determined . CI934 was the most active, inhibiting 90% of the strains at 4 micrograms/ml and 100% at 8 micrograms/ml . Ofloxacin and ciprofloxacin had moderate activity (16 and 32 micrograms/ml) whereas enoxacin, pefloxacin, norfloxacin, and nalidixic acid had poor activity (128 micrograms/ml).

J Med Chem, 1986 Feb, 29(2), 181 - 5
Synthesis and investigation of the beta-adrenoceptor agonist and platelet antiaggregatory properties of 1,7,8-trisubstituted 2,3,4,5-tetrahydro-1H-2-benzazepine analogues of trimetoquinol; Clark MT et al.; The synthesis and biological evaluation of 7,8-dihydroxy (2) and 7,8-methylenedioxy (3) analogues of 1-{(3,4,5-trimethyoxyphenyl)methyl}-2,3,4,5-tetradhyo-1H-2-b enzazepine on beta-adrenoceptor systems and human platelets were undertaken and compared with trimetoquinol (TMQ, 1) . Whereas 1 is a potent beta-adrenoceptor agonist in guinea pig atria and trachea (pD2 = 8.2), analogue 2 was marginally effective at relaxing guinea pig tracheal smooth muscle (pD2 = 4.4) and inactive as an agonist on guinea pig atria . Analogues 2 and 3 were inhibitors of phospholipase C (PLC; from Clostridium perfringens) induced and secondary wave of ADP-induced aggregation responses and inactive against low-dose thrombin-induced or stable endoperoxide (U46619) induced human platelet aggregation . Against ADP-induced serotonin secretion, 3 was 9-fold more active than analogue 2 . Further, the rank order of TMQ isomers and 3 as inhibitors of PLC-induced platelet aggregation, serotonin secretion, and phosphatidylinositol degradation was identical (3 greater than (S)-(-)-1 greater than (R)-(+)-1) . The results suggest that these compounds are blocking the action of PLC by interfering with phosphatidylinositol turnover in platelet membranes . The inhibition of ADP-induced responses in human platelets by analogues 2 and 3 also suggests a site of inhibition at a level of arachidonic acid release . Thus, ring expansion of 1 as in the benzazepine analogues 2 and 3 has allowed us to develop selective inhibitors of platelet function that lack significant beta-adrenoceptor activity.

J Bacteriol, 1986 Feb, 165(2), 542 - 8
Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A; Loffler A et al.; Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized . IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride . Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium . Denaturing and reducing agents were necessary to solubilize the IB . An alkylating agent was required to prevent reaggregation of the subunits . Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C . perfringens . IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly . Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C . perfringens, trypsin, or trypsin plus bile salts.

Microb Pathog, 1986 Feb, 1(1), 89 - 100
Comparison of receptors for Clostridium perfringens type A and cholera enterotoxins in isolated rabbit intestinal brush border membranes; Wnek AP et al.; The rabbit intestinal brush border membrane (BBM) receptors for Clostridium perfringens type A (CPE) and cholera (CT) enterotoxins were compared . Initial studies characterized binding of 125I-CPE to isolated BBMs as specific, saturable, and irreversible . BBMs appear to contain a single type of CPE binding site . Protease pretreatment of BBMs strongly reduced subsequent specific binding of 125I-CPE but not 125I-CT, while neuraminidase pretreatment had little effect on binding of either enterotoxin . Proteases did not significantly release pre-bound 125I-CPE . Preincubation of CPE with an affinity-purified preparation containing a previously identified (Biochem . Biophys . Res . Commun . 112, 1099-105) CPE-binding protein resulted in reduced specific binding of 125I-CPE and an inhibition of CPE biologic activity . Similar experiments showed that CPE-binding protein had no effect on CT binding or biologic activity . Gangliosides had no significant effect on specific binding or biologic activity of CPE but reduced CT binding and biologic activity . Lipids, including gangliosides, separated by thin layer chromatography specifically bound CT but not CPE . Preincubation of BBMs with CT did not reduce subsequent binding of 125I-CPE; conversely, prebound CPE did not affect subsequent 125I-CT binding . These results strongly suggest that CPE does not share the CT BBM receptor ganglioside GM1, and support a role for the CPE-binding protein in CPE binding.

Vet Microbiol, 1986 Feb, 11(1-2), 191 - 5
Cellular morphology of Clostridium spiroforme; Borriello SP et al.; The helically-coiled bacterium, Clostridium spiroforme, has been shown to consist of an ordered aggregation of numerous individual semi-circular cells joined end to end.

Biochemistry, 1986 Jan 28, 25(2), 464 - 8
High-multiplicity spin states of 2{4Fe-4Se}+ clostridial ferredoxins; Gaillard J et al.; The electron paramagnetic resonance (EPR) spectra of the reduced selenium-substituted 2-{4Fe-4Se}+ ferredoxins from three bacteria of the Clostridium genus display low-field signals at g = 5.17, g = 10.11, and g = 12.76 . The positions, shapes, and temperature dependencies of these signals have allowed their assignments to the three excited states of an S = 7/2 spin multiplet, the fundamental state of which is observed as unusual features in low-temperature (T less than or equal to 20 K) Mossbauer spectra . The S = 7/2 spin state is present in 2{4Fe-4Se}+ clostridial ferredoxins together with the classical S = 1/2 state and with a S = 3/2 state, the fundamental doublet of which is observed as a broad signal in the g = 3-4 region . The relative intensities of the EPR signals corresponding to these spin states depend on the species of Clostridium that the ferredoxin is extracted from . In contrast with clostridial ferredoxins, the reduced selenium-substituted ferredoxin from Bacillus stearothermophilus, which differs significantly from the clostridial proteins by its primary structure and by its containing only one tetranuclear cluster, displays only the S = 1/2 state . Thus, the high-multiplicity spin states arise from a specific interaction between the clostridial ferredoxin polypeptide chain and the reduced {4Fe-4Se}+ clusters.

J Biol Chem, 1986 Jan 25, 261(3), 1316 - 21
Purification and properties of Clostridium difficile cytotoxin B; Pothoulakis C et al.; Toxin B, a potent cytotoxin produced by Clostridium difficile, was purified to homogeneity from 6-day broth cultures of a toxigenic isolate . Cytotoxin was purified approximately 4000-fold by sequential ammonium sulfate precipitation, DEAE-Sepharose chromatography, and high performance liquid chromatography on a Mono Q anion-exchange column . The molecular weight of reduced purified toxin was 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, compared to 150,000 for unreduced toxin . Dose-response studies indicated that subpicogram concentrations of purified toxin caused rounding of approximately 20,000 IMR-90 fibroblasts . The phenomenon of cell rounding caused by toxin B was correlated with the ratio of globular to filamentous actin in fibroblasts as measured by two techniques . The toxin caused a significant increase in the ratio of globular to filamentous actin which was nearly completed prior to the onset of rounding . We conclude that cell rounding of fibroblasts exposed to toxin B is related to an increase in the ratio of globular to filamentous actin which is produced by small numbers of toxin molecules/cell.

Eur J Biochem, 1986 Jan 15, 154(2), 409 - 16
Botulinum neurotoxin type B . Its purification, radioiodination and interaction with rat-brain synaptosomal membranes; Evans DM et al.; Neurotoxin from Clostridium botulinum type B was purified to homogeneity by by affinity and ion-exchange chromatography; specific neurotoxicity of this protein (Mr of approximately equal to 155 000) following trypsinisation attained a level of 2 X 10(8) mouse LD50 units/mg protein . 125I-iodination of the toxin to high specific radioactivities (19-63 TBq/mmol) yielded typically greater than 65% of its original toxicity; dodecyl sulphate gel electrophoresis under reducing conditions, after trypsinisation, showed that the larger polypeptide (Mr of approximately equal to 101 000) was labelled preferentially . Saturable binding of the 125I-labelled neurotoxin to rat cerebrocortical synaptosomes was observed and Scatchard analysis showed a low content of acceptors with high affinity (Kd = 0.3-0.5 nM;Bmax approximately equal to 30-60 fmol/mg protein, together with a much larger population of weak-affinity sites . No significant differences in binding affinity were seen in competition experiments using native or fully activated (trypsinized) neurotoxin, indicating that chain cleavage is not essential for acceptor-toxin interaction . Type A botulinum neurotoxin showed a limited capacity to inhibit the synaptosomal binding of labelled type B toxin, even at high concentrations (1 muM), and other neurotoxins were without effect, emphasising the acceptor selectivity . Near-complete loss of specific toxin binding was produced by preincubation of synaptosomes with neuraminidase whereas inhibition of the low-affinity sites with wheat-germ agglutinin was less pronounced; such inactivation was prevented by inclusion of selective inhibitors (2,3-dehydro-2-deoxy-N-acetylneuraminic acid and N-acetylglucosamine, respectively) . These observations implicate N-acetylneuraminic acid and, possibly, other sugar moieties as constituents of the toxin acceptors . Trypsinisation of synaptosomes gave incomplete inhibition of binding when assayed with 1 nM or 10 nM 125I-iodinated toxin . Detailed analysis of the actions of neuraminidase, trypsin and heat treatment on the concentration dependence of toxin binding suggest the existence of at least two distinguishable populations of sites that contain N-acetylneuraminic acid, with a protein component being associated with the acceptors of lower affinity . These findings are discussed in relation to those previously reported for type A neurotoxin and to the possible physiological significance of such membrane acceptors.

J Am Vet Med Assoc, 1986 Jan 15, 188(2), 188 - 90
Clostridial peritonitis associated with a mast cell tumor in a dog; Clark DM et al.; Recurrent peritonitis caused by Clostridium limosum was associated with a mast cell tumor of a cranial mesenteric lymph node in a dog . The diagnosis of mast cell tumor was obscured because of the peritonitis and the appearance and location of the mass, which resembled an abscess . Since clostridial infections frequently are associated with neoplasia in man, veterinarians should be aware of the possibility of a similar relationship in animals.

Biochemistry, 1986 Jan 14, 25(1), 50 - 4
Hydrogen-1 nuclear magnetic resonance investigation of Clostridium pasteurianum rubredoxin: previously unobserved signals; Krishnamoorthi R et al.; Previously unobserved signals were located in the 470-MHz 1H NMR spectra of oxidized and reduced rubredoxin (Rd) from Clostridium pasteurianum . When the protein was oxidized, some of the resonances broadened beyond detection . Longitudinal relaxation (T1) measurements identified a number of these peaks as arising from residues close to the paramagnetic iron; these resonances exhibited short T1 values attributable to the dominant electron-nuclear dipolar relaxation mechanism . The chemical shifts of these peaks were not strongly dependent on the oxidation state of the protein, although relative ratios of line widths of several peaks in the spectra of oxidized and reduced Rd suggested localized conformational changes of the protein as a result of oxidation . Furthermore, spectra of the oxidized protein collected in the range 8-60 degrees C revealed no appreciable changes in the chemical shifts of these peaks with temperature . These results seem to point out a negligible dipolar contribution, due to either magnetic anisotropy or zero field splitting, to the observed shifts in the spectrum of oxidized Rd . Resonances were assigned to tyrosine-11 or phenylalanine-49 (but not to either specifically) on the basis of their T1 values and the X-ray diffraction data of the protein molecule {Watenpaugh, K . D., Sieker, L . C., Herriott, J . R., & Jensen, L . H . (1973) Acta Crystallogr., Sect . B: Struct . Crystallogr . Cryst . Chem . B29, 943-956; and a further refinement deposited with the Protein Data Bank} . An upfield-shifted peak at about -1.1 ppm in the spectra of both oxidized and reduced Rd was assigned to a methyl group.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Orthop Trauma Surg, 1986, 104(6), 374 - 6
Gas-producing infections after lower-limb amputation because of ischemia; Krebs B et al.; During the period 1972-1982, 22 patients with gas-producing infections after lower-limb amputation because of ischemia were reviewed . In 16 cases bacteria of the genus Clostridium could be cultured . Treatment consisted of hyperbaric oxygen, surgical debridement, and antibiotics . The incubation period was 1-11 days after the primary amputation, and the duration of the infection 2-31 days, significantly lower among 14 patients with diabetes mellitus . There was no difference in the course of clostridial and nonclostridial infections . No deaths were attributable to gas-forming infection.

Digestion, 1986, 33(4), 225 - 8
Recurrent Clostridium difficile-associated colitis responding to cholestyramine; Kunimoto D et al.; We describe a patient with relapses of Clostridium difficile cytotoxin-positive pseudomembranous colitis (PMC) after treatment with vancomycin, a course of metronidazole and a trial of bacitracin . She remains free of disease after a prolonged course of cholestyramine . We suggest there may be a role for anion-exchange resins in patients with PMC relapsing after vancomycin therapy.

Br J Hosp Med, 1986 Jan, 35(1), 37 - 42
Pseudomembranous colitis in children and adults; Bolton RP et al.; Pseudomembranous colitis remains a potentially lethal complication of antibiotic usage . Identification of Clostridium difficile as the major pathogen has led to a rational successful approach to therapy and has widened the spectrum of associated disease . The frequent asymptomatic colonization of healthy neonates by C . difficile remains an enigma.

Gut, 1986 Jan, 27(1), 78 - 85
Effect of toxin A and B of Clostridium difficile on rabbit ileum and colon; Mitchell TJ et al.; The effect of purified toxin A and partially purified toxin B on rabbit ileum and colon was investigated . Toxin A caused tissue damage which was followed by permeability changes and fluid accumulation in both tissues . Toxin A did not increase the permeability of the colon to the extent observed for ileum; secreted fluid contained less protein of plasma origin . Toxin B had no effect on either tissue . Secretory and tissue damaging properties of crude C difficile toxins were found to be due to toxin A.

Arch Intern Med, 1986 Jan, 146(1), 95 - 100
Clostridium difficile-associated diarrhea and colitis in adults . A prospective case-controlled epidemiologic study; Gerding DN et al.; In a one-year period, 149 adult cases of Clostridium difficile-associated diarrhea and colitis were compared with 148 diarrhea-free controls . Eighty-seven percent were nosocomial and 75% were on surgical services . Endoscopy revealed pseudomembranes in 51% of the 109 cases in which stool cytotoxin was present, compared with 11% of the 40 cases that were culture-positive but cytotoxin-negative . Cases diagnosed only by stool culture showed essentially no differences from controls, 21% of whom had asymptomatic stool colonization . We estimate that only 20% of these cases had diarrhea due to C difficile . Compared with controls, cases diagnosed by the presence of cytotoxin or pseudomembranes were found to have been hospitalized longer at diarrhea onset, to have had more antecedent infections, and to have received clindamycin, multiple antimicrobials, and therapeutic antimicrobials more often than controls, but controls received prophylactic antimicrobials more frequently than cases . Cultures of the environment, patients, and personnel failed to detect a mechanism of acquisition.

J Bacteriol, 1986 Jan, 165(1), 319 - 20
Clostridium thermosaccharolyticum strain deficient in acetate production; Rothstein DM; A mutant of Clostridium thermosaccharolyticum that is blocked in acetate production was isolated after treatment with nitrosoguanidine and selection for fluoroacetate resistance . The mutant produced more ethanol than the parent strain did.

J Bacteriol, 1986 Jan, 165(1), 315 - 8
Adaptation of the acetogen Clostridium thermoautotrophicum to minimal medium; Savage MD et al.; Clostridium thermoautotrophicum was adapted to minimal medium and cultivated at the expense of glucose, methanol, or H2-CO2 . No supplemental amino acids were required for growth of the adapted strain, and nicotinic acid was the sole essential vitamin . Neither N2 nor nitrate could replace ammonium as the nitrogen source, and biotin was preferentially stimulatory for glucose cell lines . Growth in minimal medium yielded substantially higher acetate concentrations per unit of biomass formed than did growth in undefined medium.

Am J Clin Pathol, 1986 Jan, 85(1), 82 - 6
Agar medium for gas-liquid chromatography of anaerobes; Pankuch GA et al.; This study evaluates a method of performing gas-liquid chromatography (GLC) by direct extraction of fatty acids from agar for identification of clinically significant anaerobic bacteria . The potential use of agar cultures for GLC was studied by comparing chromatograms of 117 clinically isolated anaerobes grown in peptone yeast glucose broth and chopped meat carbohydrate broth, and on enriched brucella blood agar . For 98 of 117 anaerobes, fatty acid patterns from agar cultures were similar to those in broth . Significant differences were only found with Streptococcus intermedius, Clostridium perfringens, Clostridium tertium, and Actinomyces species, which produced less of certain fatty acids on agar than in broth . Results of this study indicate that GLC of short chain fatty acids produced on agar medium by anaerobes, combined with simple tests such as Gram's stain and colonial morphology, may allow fir direct presumptive genus identification from an initial pure agar culture.

Cancer Res, 1986 Jan, 46(1), 375 - 80
Effects of 12-O-tetradecanoylphorbol-13-acetate on adhesiveness and lung-colonizing ability of Lewis lung carcinoma cells; Takenaga K et al.; The potent tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) enhanced the adherence of low-metastatic Lewis lung carcinoma cells (P-29) to the surface of plastic culture dishes and to monolayers of endothelial cells . This effect was transient, being apparent within 15 min and maximal within 1 h after treatment with TPA . Biologically active analogues of TPA and mezerein also enhanced attachment of P-29 cells, whereas inactive analogues of TPA did not . TPA-treated P-29 cells formed many more pulmonary nodules than did untreated P-29 cells when injected i.v . into C57BL/6 mice . The kinetics of enhancement of attachment of P-29 cells after TPA treatment coincided well with that of enhancement of their lung-colonizing ability . Addition of TPA to P-29 cells stimulated phosphorylation of a cellular protein with a molecular weight of 54,000 . The possibility that this phosphorylation was related to activation of Ca2+ phospholipid-dependent protein kinase was suggested by the fact that phospholipid breakdown induced by exogenous treatment of the cells with Clostridium perfringens phospholipase C and 1-oleoyl-2-acetylglycerol also enhanced Mr 54,000 cellular protein phosphorylation . However, neither phospholipase C nor 1-oleoyl-2-acetylglycerol enhanced attachment of P-29 cells or their lung-colonizing ability.

Arch Geschwulstforsch, 1986, 56(6), 401 - 6
Conception and stages of development of the microbiological cancer test; Schneeweiss U et al.; Pathology and experimental bacteriology have, in scientific medicine, created fundamental methodical presuppositions for the description and recognition of etiologic entities . With the tumour-tetanus phenomen, two pathogenic principles--cancer growth and tetanus infection--coincide in a so far unknown manner . Analytical experiments have led to the novel idea of the mitosis-stimulated anaerobic tetanus infection . This specific pathomechanism and its mathematical model formed the basis for the development of the serological Clostridium-tumour test with nontoxic, apathogenic Butyricus Clostridia as test microbes . As viable diagnostic agents the test spores are subject to the 12th Regulation of the Drug Act of the GDR . Only the histopathological judgement allows for the detection of micro-tissue alterations following i.v . overdoses of spores . The test spores were confirmed as safe and effective, and the microbiological cancer test approved to be appropriate for a clinical trial.

Toxicon, 1986, 24(10), 967 - 73
Scombroid poisoning: mini-review with case histories; Russell FE et al.; Scombroid poisoning has become an almost world-wide medical problem . It is probably the most common cause of fish poisoning, although frequently misdiagnosed as "Salmonella infection' . While there remains some question as to the definitive etiology, there is little doubt that the poisoning is caused by the ingestion of certain mackerel-like fishes whose tissues have undergone a number of changes provoked by bacteria, and involving the conversion of histidine to histamine, potentiated by diamines . Improper storage of the fishes, usually at temperatures above 20 degrees C, appears to be the most important predisposing factor . The organisms most commonly involved are Proteus sp., Clostridium sp., Escherichia sp., Salmonella sp . and Shigella sp . Twenty-five cases of scombroid poisoning are presented . The clinical manifestations were very similar in most cases, consisting of: alterations in taste; anxiety; hyperemia, particularly of the face and neck; nausea; pruritis; headache; certain other symptoms and signs . Most patients responded to antihistamitics, and all cases were self-limiting.

Dev Biol Stand, 1986, 64, 141 - 5
In vitro assays for botulinum toxin and antitoxins; Shone C et al.; Clostridium botulinum produces several powerful neuroparalytic toxins which, although rare in food-poisoning instances, are generally fatal . A considerable amount of effort has therefore been made by the food industry to ensure that food treatment processes adequate to prevent growth and toxin production by Cl . botulinum . Laboratory mice and guinea-pigs are presently used extensively both for the assay of botulinum toxins and for the development and assessment of vaccines used to protect laboratory workers . An amplified ELISA, using a monoclonal antibody, has been developed for botulinum type A toxin with a sensitivity similar to that of the mouse acute toxicity test . The immunoassay has been found to be applicable to the detection of toxin in foodstuffs and could replace the currently used mouse bioassay in many laboratories . Immunoassays have also been developed for the detection of antibodies to botulinum toxins . A preliminary study has shown that antibody titres to botulinum types A and B toxins in sera taken from immunised personnel, as measured by ELISA, showed limited correlation with those measured by the toxin neutralisation test in mice . A more extensive study should determine whether the latter test can be replaced by the ELISA.

Dev Biol Stand, 1986, 64, 137 - 40
Mass production and standardization of Clostridium oedematiens vaccine against black disease (infectious necrotic hepatitis) of sheep; Ardehali M et al.; The object of this study was to prepare a potent vaccine against black disease of sheep . Attempts were made to prepare and formulate the ingredients in order to obtain high yield of toxin . A 600 litre batch of mass production of Clostridium oedematiens (Cl . novyi) vaccine was prepared with ingredients consisting of 4% peptone, 0.25% NaCl, 0.5% liver extract, 0.1% L-cysteine, 1% maltose, 0.02% sodium dithionite and 0.25% ferrous sulphate solution . The prepared vaccine was diluted to concentrations of 20, 40, 60 and 80 per cent of antigens . Potassium alum was added as an adjuvant . The potency test of the prepared vaccine was determined in a group of forty rabbits according to the British Pharmacopoeia (Veterinary) . Maximum titre was obtained at 80 per cent with the level of 33 units per ml of alpha antitoxin in rabbits pooled serum . 20, 16 and 8 units per ml of alpha antitoxin were obtained respectively for 60, 40 and 20 per cent of diluted antigen in rabbits pooled serum . Sheep have been vaccinated in black disease areas with this vaccine . Reports obtained from the field indicate that black disease in sheep could be effectively controlled by this vaccine in Iran.

Pathology, 1986 Jan, 18(1), 141 - 4
A comparison of selective media for the isolation of anaerobic bacteria from clinical material; Downes J et al.; Clinical specimens submitted for anaerobic culture to a Melbourne teaching hospital microbiology laboratory were plated onto 3 types of selective media, to determine which would allow the optimal recovery of anaerobic organisms . The 3 media employed were kanamycin agar (KA), neomycin agar (NA) and nalidixic acid-Tween 80 agar (NAT) . The highest isolation rate was achieved on NAT, 89% of the total of all anaerobes isolated being recovered on this medium . A recovery rate of 69% was achieved using NA, while use of KA allowed the isolation of only 56% of all strains . The major difference between 3 media was in the recovery of anaerobic Gram-positive cocci, which accounted for 40% of the total isolates on NAT, 25% on NA, and only 11% on KA . The NAT was also more successful in the isolation of Fusobacterium and Veillonella species . The NAT medium failed, however, to recover Clostridium spp . that were isolated on both NA and KA . There was no significant difference between the 3 media in regard to the recovery of Bacteroides spp.

J Clin Microbiol, 1986 Jan, 23(1), 197 - 8
Method for the typing of Clostridium difficile based on polyacrylamide gel electrophoresis of {35S}methionine-labeled proteins; Tabaqchali S et al.; A typing method for Clostridium difficile based on the incorporation of {35S}methionine into cellular proteins, their separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and their visualization by autoradiography is described . On analysis of the radiolabeled-protein profiles, nine distinct groups were observed (A to E and W to Z) . The method, which is simple, reproducible, and readily expandable, has been applied in epidemiological studies to demonstrate cross-infection and hospital acquisition of C . difficile.

Rev Argent Microbiol, 1986, 18(1), 49 - 51
{Formation and regeneration of protoplasts of a native strain of Clostridium acetobutylicum}; Floccari ME et al.; A procedure was optimized for the formation and regeneration of protoplasts in a Clostridium acetobutylicum strain isolated from cassava roots in Misiones, Argentina . The regeneration frequency was calculated as the ratio of regenerants (colony forming units on regeneration plates minus colony forming units from osmotically resistant cells) per initial cell number (colony forming units before protoplast formation) . The percentage regeneration varied from 10 to 15%.

Ann Inst Pasteur Microbiol, 1986 Jan-Feb, 137A(1), 89 - 96
{Evaluation of an experimental animal model allowing the study of the cecal microflora in the hamster, antagonistic to clostridium difficile}; Su WJ et al.; The purpose of this study was the development and evaluation of an experimental model allowing the investigation of hamster anti-Clostridium difficile coecal microflora . The existence of this "barrier" was verified in conventional hamsters . Such hamster coecal flora was then orally transferred to C3H germ-free mice . In such animals, the "barrier effect" was maintained . After treatment with erythromycin, the colonization resistance was always maintained; despite two subsequent processes, dilutions of coecal contents (10(-2} and subsequent heating of this fluid (70 degrees C, 10 min), the inhibitory activity against C . difficile was partially maintained (10(4) UFC/g faeces) . The isolation of anaerobic strains implicated in colonization resistance will next be carried out in an anaerobic chamber using this microflora.

Ann Inst Pasteur Microbiol, 1986 Jan-Feb, 137A(1), 33 - 43
{Description of 23 cellulolytic or non-cellulolytic clostridia isolated from a marine milieu}; Marty DG; Twenty-three obligately anaerobic mesophilic bacteria were isolated from marine environments . The isolates were rod-shaped, spore-forming bacteria and were placed in the genus Clostridium . They could be divided into three groups: 9 non-cellulolytic strains which used cellobiose as sole energy and carbon source; 6 pseudo-cellulolytic strains which fermented carboxymethyl-cellulose but degraded cellulose very slowly, and 8 cellulolytic bacteria . The morphological, physiological and biochemical characteristics indicated that, except for one strain which could be identified with C . aminovalerium and three strains which resembled C . sphenoides, these marine clostridia did not correspond to any previously described species.

Scand J Infect Dis Suppl, 1986, 49, 160 - 4
Anaerobic infections and Clostridium difficile colitis emerging during antibacterial therapy; Finegold SM; Almost all cases of Clostridium difficile-related pseudomembranous colitis are related to antimicrobial therapy . Virtually all antibacterial agents have been implicated, notable exceptions being vancomycin and parenterally administered aminoglycosides . The most prominent causes of colitis are ampicillin, clindamycin and various cephalosporins . In general, this complication is related to suppression of indigenous flora and overgrowth of C . difficile . In the case of ampicillin, however, C . difficile is always susceptible . Beta-lactamase production by elements of the bowel flora leads to destruction of ampicillin and subsequently to increased counts of C . difficile and colitis . Much less well-appreciated, and much less studied, is overgrowth and subsequent infection by other types of anaerobic bacteria . Superinfection by anaerobes may follow the use of "intestinal antiseptics" such as oral neomycin; indeed, that is the rationale for the current practice in the U.S . of combining erythromycin or tetracycline with the oral aminoglycoside . Superinfection with anaerobes may also follow systemic administration of various antimicrobial compounds . Such superinfections may involve any site in the body, although sepsis and intraabdominal infection have been noted most commonly; all major types of anaerobes have been involved . A wide variety of antimicrobial compounds has been implicated in predisposing to anaerobic infection.

Folia Microbiol (Praha), 1986, 31(5), 382 - 6
Preparation of Clostridium septicum antigen for hyperimmunization of horses using a dialyzed culture; Hnatkova Z et al.; The preparation of toxic cultures of Clostridium septicum is described, using an apparatus with a straight dialysis tubing, where the medium is filled both into the nutrition and cultivation space of the apparatus . Using the cultivation to nutrition volume rate 1:2, mean titre of lethal antigen in filtrates 3.86 limes mortis per mL and 300 dosis lethalis minima per Lm was obtained in comparison with the values of 2.22 and 150 respectively in flask filtrates . Native filtrates of dialyzed cultures were better antigens for hyperimmunization of horses than the culture filtrates from flasks.

Crit Rev Clin Lab Sci, 1986, 24(3), 235 - 61
Diagnosis of Clostridium difficile-associated intestinal disease; Rolfe RD; Toxigenic Clostridium difficile is the major cause of antimicrobial agent-associated pseudomembranous colitis and is the etiological agent of approximately 30% of cases of nonspecific colitis and diarrhea (without colitis) induced by antimicrobial agents . In addition, C . difficile has been implicated in certain intestinal diseases not related to prior antimicrobial administration . C . difficile has been reported to be one of the most common enteropathogens isolated from stool specimens submitted to hospital laboratories . Thus, diagnosis of C . difficile-associated intestinal disease should now be routinely performed in diagnostic clinical laboratories . The diagnosis of C . difficile-associated intestinal disease relies on the demonstration of either the organism or the toxin(s) in stool specimens or antibody response in serum to the toxin(s) . Several selective medium are available for the recovery of C . difficile from stool specimens . The toxin(s) of C . difficile can be demonstrated using a variety of techniques, including biological assays as well as immunological assays . This article will review the techniques currently available to aid in the diagnosis of C . difficile-associated intestinal disease.

Arch Immunol Ther Exp (Warsz), 1986, 34(2), 183 - 7
Blocking of antibody passive hemagglutination reaction (Bl-AbHAp) as a test for in vitro recognition of type-specific soluble antigens of Clostridium botulinum; Ligieza J et al.; The blocking of hemagglutination reaction (Bl-AbHAp) was used as a new indicating version of the HAp-test applied for rapid, type-specific in vitro identification of the Cl . botulinum A, B, E and F toxic culture supernatants . The laboratory diagnostic system based on the described method and a special computation table is proposed for simple indicating set with 30 minutes results reading of the test.

J Clin Microbiol, 1986 Jan, 23(1), 201 - 2
Characterization of an organism that produces type E botulinal toxin but which resembles Clostridium butyricum from the feces of an infant with type E botulism; McCroskey LM et al.; The apparent causative organism from the only reported case of type E infant botulism was isolated and characterized . Except for its ability to produce type E botulinal toxin, this organism (strain 5262) would be unquestionably identified as Clostridium butyricum . This is the second time an organism resembling a defined Clostridium species other than a member of the C . botulinum group has been implicated in infant botulism.

J Bacteriol, 1986 Jan, 165(1), 21 - 7
Cloning, nucleotide sequencing, and expression of tetanus toxin fragment C in Escherichia coli; Fairweather NF et al.; The amino acid sequence of the first 30 residues of fragment C of tetanus toxin was determined, and a mixture of 32 complementary oligonucleotides, each 17 bases long, was synthesized . A 2-kilobase (kb) EcoI fragment of Clostridium tetani DNA was identified by Southern blotting and was cloned into the Escherichia coli plasmid vector pAT153 with the 32P-labeled oligonucleotide mixture as a probe . A second 3.2-kb Bg/II fragment was identified and cloned with the 2-kb EcoRI fragment as a probe . The nucleotide sequence of 1.8 kb of this DNA was determined and was shown to encode the entire fragment C and a portion of fragment B of tetanus toxin . The tetanus DNA was expressed in E . coli with pWRL507, a plasmid vector containing the trp promoter and a portion of the trpE gene . The trpE-tetanus fusion proteins were visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were shown to react with anti-fragment C antibody.

Antimicrob Agents Chemother, 1986 Jan, 29(1), 158 - 60
Comparative in vitro activities of amoxicillin-clavulanic acid and imipenem against anaerobic bacteria isolated from community hospitals; Goldstein EJ et al.; The susceptibilities of recent clinical isolates of anaerobic bacteria from two community hospitals were determined . Thirty percent of pigmented Bacteroides species and bile-sensitive, nonpigmented Bacteroides species produced penicillinase and were resistant to amoxicillin . Cefoxitin and clindamycin showed good activity against most strains, with the exception of rare isolates of the Bacteroides fragilis group and some strains of Clostridium species . While amoxicillin was not active against B . fragilis and other members of the B . fragilis group, amoxicillin-clavulanic acid was very active against almost all of these organisms . Imipenem was the most potent agent against all strains tested.

Rev Argent Microbiol, 1986, 18(1), 29 - 31
{First outbreak of botulism caused by Clostridium botulinum subtype Af}; Fernandez RA et al.; In December 1982 an outbreak of foodborne botulism presumably produced by the ingestion of home-made pickled trout occurred in San Rafael, province of Mendoza, Argentina . The toxin detected in blood serum and feces samples of the sole affected patient was preliminarily typed as a plain type A . A strain of Clostridium botulinum was isolated from feces which, after culture by the dialysis method, produced 1 x 10(7) LD50/mouse per ml . Quantitative neutralization tests carried out at different levels of toxin concentration, showed that this toxin consists of a major type A antigenic component (about 99% of the complex) and a minor type F component, defining its identity as a subtype Af . A rabbit antiserum obtained from this toxin neutralizes A and F reference toxins . Despite the antitoxic and supportive treatment, the patient died as a consequence of the poisoning . The polyvalent antiserum administered contained A, B and E antitoxins . Death could be due to the lethal effect of the A fraction, the F fraction or to a combined effect of both toxic components of the toxin . This is the first detection of an outbreak produced by C . botulinum subtype Af, serotype described in Argentina fifteen years ago, and not detected until now in other parts of the world.(ABSTRACT TRUNCATED AT 250 WORDS)

Ann Inst Pasteur Microbiol, 1986 Jan-Feb, 137A(1), 79 - 87
{Implantation of a mutant of Escherichia coli requiring diaminopimelic acid in the digestive tract of gnotobiotic mice}; Ducluzeau R et al.; A DAP- auxotroph mutant of Escherichia coli DP50 requiring DAP and thymidine for growth was used as the receptor strain in genetic engineering . It failed to be implanted in axenic mice . However, when an inoculum containing more than 10(7) bacteria/ml was used, the DAP+ reverse mutant devoid of requirement for DAP became implanted . When axenic mice were previously associated with Clostridium difficile containing DAP in the cell wall, the strain DAP- became implanted even when the inoculum was too small to permit implantation in axenic mice . Conversely, C . butyricum and C . perenne, whose cell walls also contain DAP, did not allow the establishment of a DAP- mutant . In animals associated with complex human flora without enterobacteria, neither of the 2 DAP- and DAP+ mutants became implanted.

Microbiol Sci, 1986 Jan, 3(1), 19 - 22
Gene transfer, recombination and gene cloning in Clostridium acetobutylicum; Jones DT et al.; Although Clostridium acetobutylicum has been used for over 70 years for the industrial production of solvents, it is only recently that studies on the genetics of this organism have been initiated . Recent advances in the development of genetic transfer systems as well as the cloning and expression of genes from this organism in Escherichia coli are reviewed.

Biochimie, 1986 Jan, 68(1), 217 - 21
Light induced H2 evolution in a hydrogenase-TiO2 particle system by direct electron transfer or via rhodium complexes; Cuendet P et al.; Three different hydrogenases (isolated from Clostridium pasteurianum, Desulfovibrio desulfuricans strain Norway 4 and D . baculatus 9974) added to a suspension of TiO2 (anatase) powder are able to catalyze H2 evolution under band gap illumination of the semiconducting particles, and in the presence of EDTA or methanol as electron donor . This H2 production can be obtained by the direct electron transfer from the conduction band of the TiO2 particles to the active site of the enzyme at pHs higher than 7 . This mediator-independent charge transfer is more efficient with C . pasteurianum and D . baculatus 9974 hydrogenases, and in the presence of methanol . Rhodium tris- and bis-bipyridyl complexes can act efficiently as electron carriers from the supporting particles to the adsorbed enzyme molecules in cases where the direct transfer is inefficient.

J Gen Microbiol, 1986 Jan, 132 ( Pt 1), 125 - 31
Heterogeneities of two components of C2 toxin produced by Clostridium botulinum types C and D; Ohishi I et al.; Botulinum C2 toxin (C2T) is composed of two dissimilar protein components, designated components I and II, which are linked with neither covalent nor noncovalent bonds . The heterogeneity of these two components of C2T produced by Clostridium botulinum type C and D strains was examined . Of 21 strains examined, 19 strains produced the two components, while the others produced neither component I nor component II . The 19 producers of C2T could be divided into three groups based on the differences in antigenicity, molecular weight and biological activity of components I and II . The results provide evidence of heterogeneity in the molecular structure of the two components of C2T, which is possibly a cause of the differences in the biological activity of the toxin observed in different strains.

Appl Environ Microbiol, 1986 Jan, 51(1), 52 - 6
Production of toxin by Clostridium botulinum type A strains cured by plasmids; Weickert MJ et al.; Twelve strains of Clostridium botulinum type A and seven strains of Clostridium sporogenes were screened for plasmids by agarose gel electrophoresis of cleared lysates of cells from 5 ml of mid-log-phase culture . Nine type A strains had one or more plasmids of 4.3, 6.8, or 36 megadaltons (MDa); several strains showed a large plasmid of 61 MDa, but it was not consistently recovered . Four C . sporogenes strains had one or more plasmids of 4.3, 5.6 or 36 MDa . Isolates obtained from cultures of plasmid-containing C . botulinum type A strains grown in ionic detergent broth and from spontaneously arising variants were screened both for toxin production and for plasmid content . Toxigenicity of C . botulinum could not be correlated with the presence of any one plasmid.

Biochimie, 1986 Jan, 68(1), 35 - 42
On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases; Adams MW et al.; The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel . They are the most active hydrogenases known . All four enzymes in their reduced states give rise to EPR signals typical of {4Fe-4S}1+ clusters but exhibit novel EPR signals in their oxidized states . For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal . A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO . EPR, Mossbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center . Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I . From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species . The data are most consistent with the S = 1/2 species being a low spin Fe(III) center . The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme . We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.

Nauchnye Doki Vyss Shkoly Biol Nauki, 1986, (4), 6 - 19
{Role of temperate phage in bacterial dissociation}; Mil'ko ES et al.; The analysis of literary and own data testifies that the dissociants may appear in bacteria population from spontaneous mutations and transfer of genetic material (conjugation, transformation, transduction) . The phage conversion and different DNA reorganizations within a cell where prophage plays an active role, probably introduce the largest contribution into the dissociative transitions of variants which occur with high frequency (about 10(-2)-10(-4) . The dissociation of various bacteria has been studied with different degree . The role of temperate phage has been shown in splitting of bacteria into variants in the genera Mycobacterium, Corynebacterium, some Bacillus, Clostridium, Staphylococcus, some enterobacteria, Yersinia, Vibrio Pseudomonas, Rhizobium, Nostoc; the participation of prophage in dissociation of bacteria of the genera Xanthomonas, Erwinia, Bacteroides is proposed . A method for obtaining the nondissociating S-variants for stability of biologically active substances synthesized by cells has been suggested.

Neurochirurgia (Stuttg), 1986 Jan, 29(1), 9 - 14
The glioblastoma multiforme: a lifelong challenge to the neurosurgeon; Heppner F; During a period of 34 years 1316 gliomas of the brain have been treated, among them 508 glioblastomas (= 39%) . In addition to the operative removal and postoperative X-ray therapy of glioblastomas various attempts have been undertaken to prevent recurrences: Intracerebral application of Cobalt60 (6 patients); locally applied antimitotic agents (76 patients); bacterial liquefaction induced by intracarotid administration of Clostridium butyricum M 55 spores ("Oncolysis", 67 patients); circumscribed heating of the extirpation cavity with metal and high-frequency electromagnetic field (85 patients); vaporization of the tumour bed with the defocussed CO2- or Neodymium-YAG-Laser beam (177 patients) . Permanent cure has been attained only in single cases so that the problem does not yet seem to be definitely solved by any of these methods . However, bacterial oncolysis in combination with the periodic postoperative generation of heat locally in the excision cavity of the tumour, might justify cautious optimism about future developments.

Z Naturforsch {C}, 1986 Jan-Feb, 41(1-2), 172 - 8
Degradation of NAD(H) by endogenous enzymes of yeasts and Clostridia; Schuetz HJ et al.; The time courses of degradation of exogenous NAD and NADH (2.5 mM) catalyzed by endogenous enzymes present in Saccharomyces cerevisiae, Candida utilis, Clostridium spec . La 1, Clostridium kluyveri, and Clostridium sporogenes have been determined . The half lives of the pyridine nucleotides depend extremely on the organism and, for the same organism, on the growth conditions . C . spec . La 1 as well as C . kluyveri possess only negligible enzyme activities for NAD degradation . However, C . sporogenes shows activities leading to half lives of less than 2 h for NAD and 5 h for NADH . At 25 degrees C half lives in the order of 5-17 h have been observed for Candida utilis under different conditions . The half lives of NAD are roughly 5 times higher in the presence of Saccharomyces cerevisiae.

Scand J Infect Dis, 1986, 18(2), 149 - 51
Comparative in vitro activities of ciprofloxacin, enoxacin, norfloxacin, ofloxacin and pefloxacin against Bacteroides fragilis and Clostridium difficile; Edlund C et al.; The in vitro activity of ciprofloxacin, enoxacin, norfloxacin, ofloxacin and pefloxacin against 99 Bacteroides fragilis strains and 105 Clostridium difficile strains were determined by the agar dilution method . Ofloxacin was the most potent agent . The MIC for 90% of the B . fragilis and C . difficile strains was 8 mg/l . Ciprofloxacin had MIC90's of 16 mg/l for B . fragilis and C . difficile . The MIC90's of pefloxacin against B . fragilis and C . difficile were 32 mg/l . 90% of the B . fragilis strains were inhibited by 32 mg/l enoxacin, while the MIC90's of enoxacin as well as of norfloxacin against C . difficile were 64 mg/l . The lowest activity against B . fragilis was obtained with norfloxacin (MIC90's of 128 mg/l) . Since serum levels of these agents are reported to be between 1 and 3 mg/l after orally administered doses, B . fragilis and C . difficile should be regarded as insusceptible to these quinolone compounds.

Dev Biol Stand, 1986, 64, 129 - 36
The titration of clostridial toxoids and antisera in cell culture; Knight PA et al.; The practicability of cell culture titration methods for antigens and antitoxins derived from Clostridium perfringens, Cl . septicum and Cl . novyi was investigated . Toxin neutralization could not be demonstrated with Cl . perfringens filtrates but assays for antitoxin based on the use of cultures of Vero cells were practicable for Cl . novyi and Cl . septicum . In the case of the latter, the precision and reproducibility of the test was sufficient for quality control purposes.

Toxicon, 1986, 24(8), 767 - 73
Clostridium perfringens iota toxin: synergism between two proteins; Stiles BG et al.; The iota toxin of Clostridium perfringens type E is a guinea pig dermonecrotic, mouse lethal toxin which cross-reacts with the iota-like toxin of Clostridium spiroforme . Antiserum raised against C . spiroforme or C . perfringens type E neutralizes the toxin from both species . By using C . spiroforme antiserum and crossed immunoelectrophoresis, we have found that there are two cross-reacting proteins, designated iota a (ia) and iota b (ib) in the culture filtrate of C . perfringens type E . Both proteins of C . perfringens were separated by preparative isoelectric focusing and had very little toxic activity when tested alone . However, when they were recombined there were 8- and 25-fold increases in bioactivity as determined by mouse lethal and guinea pig dermonecrotic assays, respectively . These results demonstrate that the iota toxin of C . perfringens requires two immunologically and biochemically different proteins for maximum activity.

J Hyg Epidemiol Microbiol Immunol, 1986, 30(3), 323 - 9
Comparative study on the immunogenic properties of Clostridium perfringens type A toxoid; Shemanova GF et al.; The paper presents the results of a study on the immunogenic properties of toxoid preparations from Cl . perfringens type A obtained using the routine method of detoxifying alpha = toxin in the culture medium (commercial preparations) and by means of detoxifying a previously purified alpha = toxin (experimental preparations) . When tested in immunized guinea pigs, the immunogenicity of experimental preparations was found to be 4.5 to 6 times that of commercial preparations . In mice, there was no difference in the immunogenic properties of the two types of preparations as determined by the ED30 of the antigen and the serum levels of Cl . perfringens antitoxin . The possibility is discussed of using the guinea pig as a laboratory animal model due to its ability to reflect most clearly the differences in the immunogenicity of Cl . perfringens type A toxoid preparations.

Antonie Van Leeuwenhoek, 1986, 52(4), 273 - 80
Changes in the cell wall of Clostridium species following passage in animals; Brook I et al.; Morphological changes in clostridial isolates after animal passage with other flora in mixed infections were studied by utilizing a subcutaneous abscess model in mice . We used 26 isolates of 7 clostridial species, and one isolate each of Bacteroides fragilis and Klebsiella pneumoniae . Abscesses were induced by all 7 Clostridium perfringens and 3 Clostridium butyricum isolates and by some of the other isolates . A thick granular wall prior to animal inoculation was shown only in C . perfringens, C . butyricum, and C . difficile . This structure was observed in other clostridia only following their animal passage alone or when co-inoculated with K . pneumoniae or B . fragilis.

Vet Med Nauki, 1986, 23(5), 20 - 8
{Enterotoxemia in newborn calves due to Cl . perfringens types A, C and D}; Lulov R et al.; The clinical symptoms and the morphologic picture of calf enterotoxemia are described . Studied were a total of thirty-two dead and slaughtered animals . Bacteriologically, the disease was shown to be caused by types A, D, and C of Clostridium perfringens . Types C and D proved pathogenic for guinea pigs, while type A did not . Isolated was a strain of Clostridium perfringens, which had high toxigenicity . It was found that calves were fairly often affected with the disease . Most severe were the infections caused by type C of Cl . perfringens, with most pronounced morphologic lesions . There were differences in the changes caused by all three types of the organism in calves, which made it possible to distinguish them as causative agents . Type A induced slight icterus and slightly manifested hemosiderosis of the liver, kidneys, and spleen; type D was responsible for severe injury and hyalin dystrophy of the kidneys; and type C caused necrotic enteritis, pronounced hemorrhagic diathesis, degenerative changes in the ganglial cells, and demyelinization of the brain.

Can J Vet Res, 1986 Jan, 50(1), 32 - 5
Experimental production of hemorrhagic enterotoxemia by Clostridium perfringens type C in maturing lambs; Niilo L; Maturing lambs, eight to nine months old, were dosed by the intraduodenal route with various preparations of Clostridium perfringens type C . Whole cultures of this organism or cells suspended in fresh medium, both supplemented with soybean flour as a protease inhibitor, produced acute fatal hemorrhagic enterotoxemia in these animals . The latter preparation was more effective than the former in causing disease . Without the soybean supplement the inocula did not produce fatal disease . Dosing with toxic cell-free culture supernatant fluid, with or without soybean supplement, had no lethal effect . Animals that died showed severe hemorrhagic enteritis with necrosis and sloughing of the mucosal epithelium, involving jejunum, ileum and part of duodenum . These lesions were similar to those seen in natural cases of hemorrhagic enterotoxemia in neonatal animals . This experiment demonstrated that nonimmune animals are normally protected against C . perfringens type C enterotoxemia by adequate levels of pancreatic proteases in the intestine, and that factors which inhibit or reduce these enzymes predispose animals for the development of this disease.

Microbiol Immunol, 1986, 30(2), 89 - 95
Biochemical differentiation between enterotoxigenic heat-sensitive and heat-resistant Clostridium perfringens strains; Tortora JC et al.; Some biochemical characteristics of 37 enterotoxigenic Clostridium perfringens strains isolated from human feces, ground beef, and soil samples by heat-selection methods and of two NCTC strains were studied . Two different biochemical patterns closely related to the heat resistance of the strains were found . The strains placed into group 1 were trehalose, inositol, and sorbitol negative and synthesized heat-resistant spores, while those placed into group 2 were trehalose and inositol positive and synthesized heat-sensitive spores . Sorbitol fermentation was variable among the strains of this last group . The strains of group 1 were more cellobiose, melibiose, and salicin fermentative than those of group 2 . Only the strains placed into group 2 synthesized toxins of sufficient levels for typing . In spite of having been isolated by mild heat treatment of the specimens, two strains showed the same biochemical and toxigenic characteristics of the strains of group 1 . The heating of these two strains did not modify their characteristics . We conclude that enterotoxigenic C . perfringens strains showing the two different toxigenic and biochemical patterns are present in the human gut, ground beef, and, probably, in soil . These strains may be differentiated on the basis of their capacity to produce acid from trehalose, inositol, and sorbitol, heat resistance of the spores and grade of toxigenicity . The heat-selection methods used for isolation of C . perfringens strains from different sources exerted a selection of strains from one or another group, but had no influence on their toxigenic and biochemical properties.

Med Hypotheses, 1986 Jan, 19(1), 27 - 39
A review of the sheep-multiple sclerosis connection; Murrell TG et al.; This paper reviews a notion that the prevalence of multiple sclerosis is high in global areas where sheep populations are concentrated . Pilot studies are reported to serum antibodies in humans to three sheep diseases; focal symmetrical encephalomalacia (FSE), maedi visna and sarcocystis . In MS patients and controls antibodies were not found to the epsilon neurotoxin of the FSE organism, Clostridium welchii type D and to a caprine retrovirus that is closely related to maedi-visna virus . However, 34% of MS and control patients had antibodies to the protozoan parasite Sarcocystis spp., tissue cysts of which contain a powerful neurotoxin, sarcocystin . It is suggested that epidemiological MS prevalence rates for country areas of southern Australia require further study along with an examination for the prevalence of MS in vegetarians.

J Bacteriol, 1986 Jan, 165(1), 252 - 7
Purification and characterization of the F1-ATPase from Clostridium thermoaceticum; Ivey DM et al.; The F1 portion of the H+-ATPase from Clostridium thermoaceticum was purified to homogeneity by solubilization at low ionic strength, ion-exchange chromatography, and gel filtration . The last indicated the Mr to be 370,000 . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the pure enzyme revealed four bands with Mr corresponding to 60,000, 55,000, 37,000, and 17,000 in an apparent molar ratio of 3:3:1:1 . The purified enzyme would bind to stripped membranes to reconstitute dicyclohexylcarbodiimide-sensitive ATPase activity . Phosphohydrolase activity, measured at 58 degrees C, was optimal at pH 8.5 . In the presence of a 1 mM excess of Mg2+ over the concentration of ATP, the Km for ATP was 0.4 mM, and the Vmax was 6.7 mumol min-1 mg-1 . Unlike the membrane-bound F1F0 complex, the F1-ATPase was relatively insensitive to the inhibitors dicyclohexylcarbodiimide and tributyltin chloride . Both the complex and the F1-ATPase were inhibited by quercetin, azide, 7-chloro-4-nitro-benz-2-oxa-1,3-diazole, and free magnesium, and both were stimulated by primary alcohols and sulfite . In whole cells, the F1F0-ATPase catalyzed the synthesis of ATP in response to a pH gradient.

Arch Immunol Ther Exp (Warsz), 1986, 34(2), 189 - 95
Determination of soluble antigens of Clostridium botulinum A by chemiluminescent--immunosorbent assay (CLISA); Ligieza J et al.; The chemiluminescent--immunosorbent assay (CLISA) was adopted for Cl . botulinum A soluble toxic antigens determination . Luminol (ABEI) labelled botulinum antitoxin globulins showed a strongly positive specific immunochemiluminescent reactions with the native botulinum toxin preparations coupled (adsorbed) on polystyrene balls . The sensitivity of the reaction reached 20 DLM/ml (5,000 light impulses per 40 sec) in comparison with 2 X 10(6) DLM (57,000 impulses), control preparations (1,500 impulses) and the background (150-300 impulses) . The results give a perspective further investigations with the CLISA for rapid indication of Cl . botulinum toxic antigens.

J Biochem (Tokyo), 1986 Jan, 99(1), 219 - 26
Biochemical properties of N-methylamides of sialic acids in gangliosides; Nakamura K et al.; A simple and rapid method for the preparation of N-methylamides ( - CONHCH3) of sialic acids in gangliosides and biochemical properties of the modified gangliosides are described . The sialic acid carboxyl groups of gangliosides were esterified with CH3I-dimethylsulfoxide, followed by heating with monomethylamine . The modified gangliosides were chemically identified by TLC, IR spectroscopy, GLC-mass spectrometry and NMR spectroscopy . The N-methylamide derivative of GM1 produced a high titer IgG antibody . The antibody weakly cross-reacted with the methylester of GM1 and its reductive derivative but did not react with the intact GM1 . A monoclonal antibody (M2590) specific for GM3 did not react with carboxyl-modified GM3 (methylester, N-methylamide, and reduced GM3), but it reacted with modified GM3 which contains the C7-analog of the sialic acid . Clostridium perfringens and Arthrobacter ureafaciens sialidases did not hydrolyze the N-methylamide derivatives, methylesters or reductive derivatives of the gangliosides and, furthermore, these derivatives did not inhibit the actions of these sialidases.

Arch Biochem Biophys, 1986 Jan, 244(1), 137 - 46
Characterization of selenium-containing tRNAGlu from Clostridium sticklandii; Ching WM; A selenium-containing tRNA from Clostridium sticklandii has been shown to be an isoaccepting tRNAGlu (W.-M . Ching and T . C . Stadtman (1982) Proc . Natl . Acad . Sci . USA 79, 374-377) . Not only is this tRNAGlu one of the most abundant selenium-containing tRNA species but it is also the major glutamate isoacceptor in this organism . The selenonucleoside, which is located at the first position of the anticodon, was identified as 5-methylaminomethyl-2-selenouridine (A . J . Wittwer, L . Tsai, W.-M . Ching, and T . C . Stadt (1984) Biochemistry 23, 4650-4655) . Other modified nucleosides present in this tRNA include 4-thiouridine, pseudouridine, ribothymidine, modified guanosine, and two different modified adenosines . When this seleno-tRNAGlu is incubated in 1.0 M Tris X HCl, pH 8.5, partial deselenization occurs . Moreover, treatment with cyanogen bromide almost completely removes the selenium . The presence of selenium in this tRNAGlu is essential for its enzymatic acylation with glutamate . This seleno-tRNAGlu recognizes both GAA and GAG codons . However, at 10 mM magnesium, which is near the physiological range, the GAA codon is slightly favored . In a cell free translation system, the acylated seleno-tRNAGlu is a very active glutamate donor.

Am J Obstet Gynecol, 1985 Dec 15, 153(8), 835 - 8
Clostridium difficile-associated diarrhea follows perioperative prophylaxis with cefoxitin; Block BS et al.; Clostridium difficile-associated diarrhea during prolonged therapy of obstetric and gynecologic infections is known to occur with use of all classes of antibiotics except vancomycin and the aminoglycosides . We present 11 cases of C . difficile-associated diarrhea which followed a short course of perioperative prophylaxis with cefoxitin during a 1-year period . Nine of the cases of C . difficile-associated diarrhea were among 162 women who received cefoxitin perioperative prophylaxis for cesarean section or hysterectomy, but none occurred in 85 women who received one of four other antibiotics for perioperative prophylaxis (p = 0.024, Fisher's exact test) . The two other occurrences of C . difficile-associated diarrhea following perioperative prophylaxis with cefoxitin were in women who underwent exploratory laparotomy . We conclude that C . difficile-associated diarrhea is related to perioperative prophylaxis with cefoxitin.

Biochem J, 1985 Dec 15, 232(3), 891 - 6
The base sequence of the nifF gene of Klebsiella pneumoniae and homology of the predicted amino acid sequence of its protein product to other flavodoxins; Drummond MH; The nucleotide sequence of a 629 base-pair segment of DNA spanning the nifF gene of Klebsiella pneumoniae is presented . The structural gene comprises 531 base-pairs (175 codons, excluding the translational initiator and terminator) encoding an acidic polypeptide of 18950 Da . The nifF product thus belongs to the long-chain class of flavodoxins . It shows some sequence homology to the short-chain flavodoxins from Desulfovibrio vulgaris, Clostridium MP and Megasphaera elsdenii, and much stronger homology to long-chain flavodoxins from Azotobacter vinelandii and Anacystis nidulans . The long chain flavodoxins thus seem to constitute a well-conserved sub-group . The homology with the A . vinelandii flavodoxin is particularly strong, which may reflect their common function in nitrogen fixation.

J Mol Biol, 1985 Dec 5, 186(3), 533 - 45
Cloning and expression of Clostridium pasteurianum galactokinase gene in Escherichia coli K-12 and nucleotide sequence analysis of a region affecting the amount of the enzyme; Daldal F et al.; The Clostridium pasteurianum galactokinase gene was cloned by complementation, of the galK locus, into Escherichia coli . Restriction enzyme analysis subcloning and Tn5 mutagenesis indicated that the gene was located on a 1.8 X 10(3) base-pair ClaI-Sau3A fragment that encoded a polypeptide of approximately 40 Mr . Although the C . pasteurianum and the E . coli galactokinases have similar subunit molecular weights, Southern hybridization analysis indicated no strong homology between their genes . Even though this clone showed a low level of galactokinase expression, the Gal+ phenotype, provided by the clostridial galactokinase, was unstable in E . coli, and the gene was frequently inactivated by the spontaneous acquisition of insertion sequences . A second clone containing this gene on a large restriction fragment was isolated by hybridization . This clone was unable to grow on galactose-containing media due to the overproduction of galactokinase . Comparison of the plasmids from these two clones revealed that the second contained an additional 300 base-pairs located at one end of the galactokinase gene . Appropriate operon fusions with a promoter-less E . coli galactokinase gene indicated that these additional 300 base-pairs had promoter activity in E . coli . The DNA sequence of this region which lies upstream of the C . pasteurianum galactokinase gene was determined and compared with that from several clones producing high, low or undetectable amounts of galactokinase . The reasons for the high and low level expression and for the instability of the C . pasteurianum galactokinase in E . coli are discussed . The presence of the galactokinase suggests that galactose is used in C . pasteurianum through the Leloir pathway via galactose 1-phosphate.

Eur J Biochem, 1985 Dec 2, 153(2), 413 - 20
Covalent modification of citrate lyase ligase from Clostridium sphenoides by phosphorylation/dephosphorylation; Antranikian G et al.; Citrate lyase ligase was shown to be present in Clostridium sphenoides actively degrading citrate . In contrast to citrate lyase ligase from C . sporosphaeroides and Streptococcus lactis, the enzyme from C . sphenoides was under stringent regulatory control . The alteration of the kinetic properties of the enzyme after depletion of citrate suggested the presence of two different enzyme species in different phases of growth: active and partially active citrate lyase ligase . These enzymes were purified from in vivo 32P-labeled C . sphenoides cells, which were grown on low-phosphate medium containing 40 mM citrate and 1 mCi {32}orthophosphate . During enzyme purification only the active form of citrate lyase ligase was shown to be radioactively labeled . Growth experiments with 14C-labeled precursors of purines and pyrimidines and subsequent purification of active citrate lyase ligase indicated that the 32P labeling of the enzyme was not due to the incorporation of a nucleotide . Inactivation of the ligase after its treatment with acid phosphatase also suggested that the active form of the enzyme is phosphorylated . Citrate lyase ligase, therefore, is the first known enzyme in an anaerobic bacterium whose activity is modulated by phosphorylation/dephosphorylation.

Appl Environ Microbiol, 1985 Dec, 50(6), 1349 - 56
Nine-year microflora study of an isolator-maintained immunodeficient child; Taylor GR et al.; A male child, maintained in a controlled environment, was monitored each month for bacteria, yeasts, and filamentous fungi recovered from the mouth, nasal passages, feces, and nine body surface sites . Three natural microbial categories became apparent . Incident microorganisms were recovered from within the isolator but did not establish permanent residence . Of the 53 incident types isolated, 20 were filamentous fungi and 4 were yeasts . Some genera, such as Fusobacterium, Lactobacillus, Neisseria, and Rothia, which were commonly found in the reference group, did not become permanent inhabitants . Transient microorganisms were repeatedly recovered but could not be demonstrated within the isolated environment at the end of the study . The loss of only a few of the 19 transient species could be associated with antimicrobial therapy . Permanent microorganisms consisted of Pencillium citrinum and 17 bacterial types, of which alpha-hemolytic streptococci, Staphylococcus edpidermidis subgroups II and V, Micrococcus groups 1 and 2, Clostridium bifermentans, and Propionibacterium acnes were recovered throughout the entire 9 years of the study . The number of CFUs recovered from each sample type was generally not unlike that from the reference group of healthy male adults . Also, the number of different aerobic species recovered from the feces was within the normal range of that of the reference group . In contrast, the number of different species recovered from all other samples was less than that commonly found in the reference group.

Dig Dis Sci, 1985 Dec, 30(12), 1149 - 55
Chronic diarrhea associated with hypogammaglobulinemia and enteropathy in infants and children; Perlmutter DH et al.; In order to define the gastrointestinal manifestations and small intestinal structure and function in a group of infants with chronic nonspecific diarrhea and hypogammaglobulinemia, we retrospectively identified 55 such patients from a population of 518 children evaluated for chronic diarrhea over a 6-year span (10.6%) . All patients had IgG levels 2.0 SD or more below the mean values for age . Patients with biochemical evidence of protein loss (enteropathy or nephropathy) were excluded . There was a 50% incidence of small intestinal mucosal injury among these patients, with a spectrum of morphological findings ranging from healing enteritis to severe active enteritis . Carbohydrate malasorption, and infection with Giardia lamblia or Clostridium difficile occurred in 34% and 24% of patients tested, respectively . These structural, functional, and infectious complications were all statistically more common in patients than in a control group of children with chronic diarrhea, normal growth, and normal immunoglobulin levels . This study suggests that immunoglobulin determination, in children who would otherwise carry a diagnosis of chronic nonspecific diarrhea, identifies a group with hypogammaglobulinemia, having an increased incidence of treatable intestinal dysfunction or infection, and a spectrum of small intestinal histologic abnormalities.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1985 Dec, 182(1), 25 - 32
{Demonstration of Clostridium difficile and toxin B in patients at a university clinic}; Heizmann W et al.; 107 stool specimens from not hospitalized individuals and 69 stool specimens from 61 hospitalized patients (Internal Medicine) were examined for C . difficile and toxin B . Included were 42 specimens of 33 female patients of the department of maxillo-facial surgery, receiving prophylactic clindamycin and 7 samples from the ward staff . From the samples of the first group, C . difficile was isolated in one case (0.93%), whereas 8 out of 61 internal patients (13%) were positive for C . difficile by culture and/or toxin test . In 12 out of 33 surgical patients (36%), C . difficile and/or toxin could be detected in the stool . Examination of the ward staff was negative . Environmental samplings on the ward, comprehending 246 contact cultures, resulted in the isolation of C . difficile from 25 plates (10.1%) . The majority of the patients showed no symptoms or slight diarrhea . Only 5 patients required vancomycin therapy . Frequency and clinical significance of C . difficile isolation is discussed under these aspects.

J Appl Bacteriol, 1985 Dec, 59(6), 549 - 53
The influence of the host on expression of intestinal microbial enzyme activities involved in metabolism of foreign compounds; Cole CB et al.; The activities of four enzymes (beta-glucuronidase, nitrate reductase and nitroreductase) in selected intestinal bacteria (Escherichia coli, Clostridium sp., Streptococcus sp., Bacteroides sp . and Lactobacillus salivarius) were measured after growth in vitro and in vivo . The five strains differed in their activities with Clostridium sp . being the most active for beta-glucosidase, beta-glucuronidase and nitroreductase, and E . coli the most active producer of nitrate reductase . Enzyme activity in vivo tended to be higher than in vitro but there were instances where the comparative activities were reversed.

Arch Microbiol, 1985 Dec, 143(3), 311 - 2
p-Cresol formation by cell-free extracts of Clostridium difficile; D'Ari L et al.; Cell-free extracts of Clostridium difficile were shown to form p-cresol by decarboxylation of p-hydroxyphenylacetic acid . This activity required both high and low molecular weight fractions . The active component of the low molecular weight fraction had properties of an amino acid and could be replaced by serine, threonine or the corresponding alpha keto acids . Pyruvate was shown to function catalytically . Since the high molecular weight fraction was O2-sensitive and since dithionite was as effective as pyruvate with some high molecular weight fractions, the alpha keto acids probably serve as low potential reducing agents in this system . Because of instability, the p-cresol-forming enzyme could not be purified.

J Bacteriol, 1985 Dec, 164(3), 1153 - 61
Differential amylosaccharide metabolism of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum; Hyun HH et al.; Clostridium thermosulfurogenes displayed faster growth on either glucose, maltose, or starch than Clostridium thermohydrosulfuricum . Both species grew faster on glucose than on starch or maltose . The fermentation end product ratios were altered based on higher ethanol and lactate yields on starch than on glucose . In C . thermohydrosulfuricum, glucoamylase, pullulanase, and maltase were mainly responsible for conversion of starch and maltose into glucose, which was accumulated by a putative glucose permease . In C . thermosulfurogenes, beta-amylase was primarily responsible for degradation of starch to maltose, which was accumulated by a putative maltose permease and then hydrolyzed by glucoamylase . Regardless of the growth substrate, the rates of glucose, maltose, and starch transformation were higher in C . thermosulfurogenes than in C . thermohydrosulfuricum . Both species had a functional Embden-Meyerhof glycolytic pathway and displayed the following catabolic activities: ferredoxin-linked pyruvate dehydrogenase, acetate kinase, NAD(P)-ethanol dehydrogenase, NAD(P)-ferredoxin oxidoreductase, hydrogenase, and fructose-1,6-diphosphate-activated lactate dehydrogenase . Ferredoxin-NAD reductase activity was higher in C . thermohydrosulfuricum than NADH-ferredoxin oxidase activity, but the former activity was not detectable in C . thermosulfurogenes . Both NAD- and NADP-linked ethanol dehydrogenases were unidirectional in C . thermosulfurogenes but reversible in C . thermohydrosulfuricum . The ratio of hydrogen-producing hydrogenase to hydrogen-consuming hydrogenase was higher in C . thermosulfurogenes . Two biochemical models are proposed to explain the differential saccharide metabolism on the basis of species enzyme differences in relation to specific growth substrates.

J Bacteriol, 1985 Dec, 164(3), 1146 - 52
Regulation and genetic enhancement of glucoamylase and pullulanase production in Clostridium thermohydrosulfuricum; Hyun HH et al.; We studied the general mechanism for regulation of glucoamylase and pullulanase synthesis in Clostridium thermohydrosulfuricum . These amylases were expressed only when the organism was grown on maltose or other carbohydrates containing maltose units . Amylase synthesis was more severely repressed by glucose than by xylose . Catabolite repression-resistant mutants were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained glucose-starch agar plates . Amylases were produced in both wild-type and mutant strains when starch was added to cells growing on xylose but not when starch was added to cells growing on glucose . In both wild-type and mutant strains, glucoamylase and pullulanase were produced at high levels in starch-limited chemostats but not in glucose- or xylose-limited chemostats . Therefore, we concluded that amylase synthesis in C . thermohydrosulfuricum was inducible and subject to catabolite repression . The mutants produced about twofold more glucoamylase and pullulanase, and they were catabolite repression resistant for production of glucose isomerase, lactase, and isomaltase . The mutants displayed improved starch metabolism features in terms of enhanced rates of growth, ethanol production, and starch consumption.

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