J Clin Microbiol, 1985 Dec, 22(6), 980 - 3 Evaluation of the RapID-ANA system for identification of anaerobic bacteria of veterinary origin; Adney WS et al.; This study evaluated the ability of the RapID-ANA system (Innovative Diagnostic Systems, Inc., Atlanta, Ga.) to accurately identify a spectrum of freshly isolated veterinary anaerobes . A total of 183 isolates were tested and included 7 Actinomyces spp., 53 Bacteroides spp., 32 Clostridium spp., 2 Eubacterium spp., 65 Fusobacterium spp., 1 Peptococcus spp., 22 Peptostreptococcus spp., and 1 Propionibacterium spp . All isolates were initially identified by conventional biochemical testing and gas-liquid chromatography of short-chain fatty acid metabolites . Additional tests were performed as required by the RapID-ANA system . Of these isolates, 81.4% were correctly identified to the genus level, including 59.6% to the species level, 14.2% were incorrectly identified at the genus level, and 4.4% were not identified . Initially, 20.2% of the strains were not identified because the microcodes were not in the code book . The majority of the incorrect identifications were caused by the misidentification of Fusobacterium spp . as Bacteroides spp . Errors also occurred when veterinary anaerobes not included in the data base were assigned an identification from the existing data base . The RapID-ANA system appears to be a promising new method for rapid identification of veterinary anaerobes; however, further evaluation with an extended data base is needed before the system can accurately identify all clinically significant anaerobes. J Clin Microbiol, 1985 Dec, 22(6), 962 - 7 Rapid identification of Clostridium species by high-pressure liquid chromatography; Harpold DJ et al.; High-pressure liquid chromatography was evaluated as a rapid means of identifying various species of clostridia . Isolates were inoculated into a defined medium and incubated aerobically for 1 h at 35 degrees C . The organisms were removed, and the supernatants were derivatized for 1 min at room temperature by the addition of o-phthalaldehyde . The total time required to run each chromatogram was approximately 50 min . Standardized peak heights for each medium component and any new peaks formed were calculated for each isolate and compared with those for uninoculated control medium . Multiple isolates of various Clostridium species gave consistent patterns of medium utilization that could be used for identification . This rapid method can easily be adapted for laboratory use and has the potential for automation. Antimicrob Agents Chemother, 1985 Dec, 28(6), 832 - 3 Pharmacokinetic and therapeutic trial of sultamicillin in acute sinusitis; Jones S et al.; Sultamicillin, an antibiotic combining ampicillin and the beta-lactamase inhibitor sulbactam, was administered to 13 patients diagnosed as having acute sinusitis . Specimens from sinus were obtained for all 13 patients by transantral puncture . Pharmacokinetics, bacteriology, and therapeutic efficacy were assessed . Eighty-five percent (11 of 13) were cured; two treatment failures were subsequently shown to have chronic (rather than acute) sinusitis during surgical exploration . Diarrhea was frequently encountered, and Clostridium difficile-associated enteritis was documented for one patient . Beta-lactamase-producing organisms were not encountered in this study; however, this study provides impetus for further controlled clinical trials. Arch Biochem Biophys, 1985 Dec, 243(2), 447 - 53 Enzyme elements involved in the interconversion of L-carbamylaspartate and L-dihydroorotate by dihydroorotase from Clostridium oroticum; Pettigrew DW et al.; Enzyme elements that are involved in the reversible cyclization of L-carbamylaspartate to L-dihdroorotate catalyzed by dihydroorotase (EC 3.5.2.3) from Clostridium oroticum (ATCC 25750) have been studied . Removal of Zn(II) from the enzyme by chelators followed by incubation of apoenzyme with Co(II) results in replacement of two to three of the four Zn(II) ions per molecule by Co(II) . The catalytic properties of the Zn(II)Co(II) dihydroorotase are different from those of native enzyme . The Vmax is increased for both the synthesis and hydrolysis of L-dihydroorotate . The Km for L-dihydroorotate is unchanged, while the Km for L-carbamylaspartate is increased more than twofold . On the other hand, the kinetic properties of Zn(II)-reconstituted dihydroorotase are indistinguishable from those of native enzyme . The pH dependence of Vmax is also altered by the Co(II) substitution . For both Zn(II)- and Zn(II)Co(II)-dihydroorotase, this pH dependence is well described by a single ionization and the pK's for L-dihydroorotate synthesis and hydrolysis are different . Substitution with Co(II) increases the pK for both reaction directions to different extents . These results strongly support a role for the tightly bound metals in the catalytic mechanism . In addition, diethylpyrocarbonate rapidly inactivates the enzyme . The inactivation is prevented by L-dihydroorotate . This result is consistent with a role for at least one histidine in catalysis . The possibility that C . oroticum dihydroorotase may be useful model for the more complex mammalian enzyme is considered. Infect Immun, 1985 Dec, 50(3), 844 - 51 Effect of thiol-activated toxins (streptolysin O, alveolysin, and theta toxin) on the generation of leukotrienes and leukotriene-inducing and -metabolizing enzymes from human polymorphonuclear granulocytes; Bremm KD et al.; The generation of leukotrienes (LTC4, LTD4, LTE4, and LTB4; 12-epi-LTB4 isomer) from human granulocytes by thiol-activated toxins (streptolysin O, alveolysin from Bacillus alvei, and theta toxin from Clostridium perfringens) is described . The release occurs under noncytolytic conditions . Although LTB4 is the major component after calcium ionophore stimulation, more LTC4 as compared with LTB4 is released with the toxins . The 5-lipoxygenase pathway of toxin-mediated activation can effectively be inhibited by caffeic acid, a lipoxygenase inhibitor . The toxins also induce the release of leukotriene-metabolizing enzymes such as gamma-glutamyltranspeptidase, which transfers LTC4 into LTD4, and dipeptidase, which metabolizes LTD4, into LTE4 . Dipeptidase activity is more pronounced than the gamma-glutamyltranspeptidase activity but still does not reach the levels obtained when cells were triggered with opsonized zymosan. CMAJ, 1985 Dec 1, 133(11), 1141 - 6 Food-borne botulism in Canada, 1971-84; Hauschild AH et al.; Sixty-one outbreaks of food-borne botulism involving a total of 122 cases, of which 21 were fatal, were recorded from 1971 to 1984 in Canada . Most occurred in northern Quebec, the Northwest Territories or British Columbia . Of the 122 victims 113 were native people, mostly Inuit . Most of the outbreaks (59%) were caused by raw, parboiled or "fermented" meats from marine mammals; fermented salmon eggs or fish accounted for 23% of the outbreaks . Three outbreaks were attributed to home-preserved foods, and one outbreak was attributed to a commercial product . The causative Clostridium botulinum type was determined in 58 of the outbreaks: the predominant type was E (in 52 outbreaks), followed by B (in 4) and A (in 2) . Renewed educational efforts combined with a comprehensive immunization program would significantly improve the control of botulism in high-risk populations. Eur J Epidemiol, 1985 Dec, 1(4), 264 - 73 Morphological alterations and changes in cellular cations induced by Clostridium perfringens type A enterotoxin in tissue culture cells; Sugimoto N et al.; The morphological alterations (bleb-balloon formation) induced by Clostridium perfringens type A enterotoxin in HeLa and Vero cells were studied under defined extracellular conditions . The action of enterotoxin was found to depend on the temperature but not on energy metabolism . The morphological alterations by the enterotoxin occurred in phosphate buffered saline containing Ca2+ and Mg2+ . Of the constituents of the buffered saline, Ca2+ was essential for the morphological alterations and other ions were interchangeable . The morphological alterations by the enterotoxin occurred also in 10 mM Hepes-Na buffer, pH 7.2 containing NaCl, KCl or choline chloride at a concentration of over ca . 50 mM and in 10 mM Hepes-Ca buffer, pH 7.2 containing CaCl2 at a concentration of over ca . 50 mM . Addition of sucrose to the medium prevented induction of the morphological alterations . The amount of sucrose necessary to protect the cells increased with increase in NaCl, KCl or CaCl2 concentration in the medium . A calcium ionophore A23187 mimicked the action of enterotoxin . Examination of the cation contents of the cells by atomic absorption spectrophotometry showed early and rapid increase of Ca2+ during intoxication with concomitant changes in Na+, K+ and Mg2+ that reduced the ion concentration gradients between inside and outside of the cell present before toxin treatment . The mechanism of action of C . perfringens type A enterotoxin is discussed on the basis of these findings. J Bacteriol, 1985 Dec, 164(3), 1162 - 70 Regulation and genetic enhancement of beta-amylase production in Clostridium thermosulfurogenes; Hyun HH et al.; We studied the general mechanism for regulation of beta-amylase synthesis in Clostridium thermosulfurogenes . beta-Amylase was expressed at high levels only when the organism was grown on maltose or other carbohydrates containing maltose units . Three kinds of mutants altered in beta-amylase production were isolated by using nitrosoguanidine treatment, enrichment on 2-deoxyglucose, and selection of colonies with large clear zones on iodine-stained starch-glucose agar plates . beta-Amylase was produced only when maltose was added to cells growing on sucrose in wild-type and catabolite repression-resistant mutant strains, but the differential rate of enzyme synthesis in constitutive mutants was constant regardless of the presence of maltose . In carbon-limited chemostats of wild-type and catabolite repression-resistant mutant stains, beta-amylase was expressed on maltose but not on glucose or sucrose . beta-Amylase synthesis was immediately repressed by the addition of glucose . Therefore, we concluded that beta-amylase synthesis in C . thermosulfurogenes was inducible and subject to catabolite repression . The addition of cAMP did not eliminate the repressive effect of glucose . The mutants were generally characterized in terms of beta-amylase production, growth properties, fermentation product formation, and alterations in glucose isomerase and glucoamylase activities . A hyperproductive mutant produced eightfold more beta-amylase on starch medium than the wild type and more rapidly fermented starch to ethanol. Experientia, 1985 Nov 15, 41(11), 1435 - 7 Increased formation of arginine deiminase by Clostridium perfringens FD-1 growing in the presence of caffeine; Sacks LE; Caffeine slowed growth and markedly increased the formation of arginine deiminase in growing C . perfringens FD-1 when dextrin, but not maltose or maltotriose, served as the energy source . It is postulated that the ability of caffeine to induce arginine deiminase is related to an inhibition of polysaccharide utilization, resulting in a shift-down condition known to induce arginine deiminase and other enzymes in bacteria. S Afr Med J, 1985 Nov 9, 68(10), 760 - 2 {Gas gangrene: A discussion of 3 cases and review of the literature}; du Toit DF et al.; Three patients with gas gangrene of the lower limbs are presented . In 2 of the 3 patients gas gangrene developed after lower-limb amputation, indications for amputation being atherosclerotic and diabetic gangrene . In the third patient associated leukaemia was diagnosed . All 3 patients presented with the typical clinical manifestations of gas gangrene . Clostridium perfringens was isolated from the affected leg in each patient . The current application of surgery and hyperbaric oxygen therapy in the treatment of gas gangrene is discussed. J Immunol Methods, 1985 Nov 7, 83(2), 241 - 8 An immunochemical method for fingerprinting Clostridium difficile; Sharp J et al.; The use of sodium dodecyl sulphate-polyacrylamide gel electrophoresis in association with electrophoretic transfer of proteins to nitrocellulose and subsequent probing with antisera appears useful as a method for fingerprinting Clostridium difficile . Thorough testing of the stability of the antigenic nature of isolates of the organism during subculture and antigen preparation has shown it to be remarkably stable both in vitro and in vivo . Minor differences in the method of antigen extraction do not markedly alter the immunoblot patterns produced . It has also been demonstrated that an individual may harbour more than one strain of the organism at any one time . Results show the possible usefulness of this technique in studying the epidemiology of diarrhoeal disease known to be associated with C . difficile . It is suggested that for any serious study several colonies should be subcultured from the primary isolation plate. J Biol Chem, 1985 Nov 5, 260(25), 13509 - 12 13C NMR studies of butyric fermentation in Clostridium kluyveri; Smith GM et al.; The fermentation of 13C-labeled ethanol and acetate into butyrate and caproate by Clostridium kluyveri has been studied by using 13C NMR . The pathway involves the conversion of both ethanol and acetate into acetyl coenzymes A, two of which condense to form CoA-linked precursors of butyrate . If butyryl-CoA is involved in the condensation, caproate is the ultimate product . ATP is produced from acetyl-CoA via the reactions catalyzed by phosphotransacetylase and acetate kinase with acetate, a required carbon source, as a co-product . In spectra of whole cells incubated with the labeled carbon sources, label from ethanol appears rapidly in acetate, which then reaches a lower, steady-state concentration due to its re-entry into the pathway . The rapid initial production of acetate indicates equally rapid production of ATP . Label from acetate appears in ethanol only if ethanol is already present, indicating that this process is one of isotopic equilibration rather than net synthesis of ethanol from acetate . The ratio of butyrate to caproate produced depends strongly on the initial ratio of ethanol to acetate in the medium . The relative rates of utilization of ethanol and acetate vary as the fermentation proceeds . 13C-13C coupling in the butyrate and caproate produced from {1-13C}ethanol and {2-13C}acetate can be used to determine if the acetyl-CoA molecules arising from ethanol and acetate enter the same pool or if they remain separated . The data are consistent with random mixing of the acetyl-CoA produced from the two carbon sources. Biochemistry, 1985 Nov 5, 24(23), 6527 - 33 Mode of hydrolysis of collagen-like peptides by class I and class II Clostridium histolyticum collagenases: evidence for both endopeptidase and tripeptidylcarboxypeptidase activities; Mookhtiar KA et al.; The action of three class I (beta, gamma, and eta) and three class II (delta, epsilon, and zeta) collagenases from Clostridium histolyticum on two series of peptides with collagen-like sequences has been examined . The peptides in the first series all contain 4-nitrophenylalanyl-Gly-Pro-Ala in subsites P1 through P3', but each is successively lengthened in the N-terminal direction by addition of an appropriate residue until subsite P5 is occupied . The second group of peptides all have cinnamoyl-Leu in subsites P2 and P1, respectively, but each is successively lengthened in the C-terminal direction by partial additions of the Gly-Pro-Leu triplet until subsite P6' is occupied . N-Terminal elongation causes the kcat/KM values to rise markedly and to level off after occupancy of subsite P6 for the class I enzymes and subsite P3 for the class II enzymes . C-Terminal elongation produces the best substrates for both classes of enzymes when subsites P3' or P4' are occupied by amino acids with free carboxyl groups . The kcat/KM values for the hydrolysis of both Leu-Gly bonds of cinnamoyl-Leu-Gly-Pro-Leu-Gly-Pro-Leu have been measured for both classes of enzymes . Both rates are large, but both classes preferentially hydrolyze the Leu-Gly bond of the C-terminal triplet . Thus, both classes of enzymes exhibit both endopeptidase and tripeptidylcarboxypeptidase activities. Biochemistry, 1985 Nov 5, 24(23), 6520 - 6 Complementary substrate specificities of class I and class II collagenases from Clostridium histolyticum; Van Wart HE et al.; The substrate specificities of three class I (beta, gamma, and eta) and three class II (sigma, epsilon, and zeta) collagenases from Clostridium histolyticum have been investigated by quantitating the kcat/KM values for the hydrolysis of 53 synthetic peptides with collagen-like sequences covering the P3 through P3 subsites of the substrate . For both classes of collagenases, there is a strong preference for Gly in subsites P1' and P3 . All six enzymes also prefer substrates that contain Pro and Ala in subsites P2 and P2' and Hyp, Ala, or Arg in subsite P3' . This agrees well with the occupancies of these sites by these residues in type I collagen . However, peptides with Glu in subsites P2 or P2' are not good substrates, even though Glu occurs frequently in these positions in collagen . Conversely, all six enzymes prefer aromatic amino acids in subsite P1, even though such residues do not occur in this position in type I collagen . In general, the class II enzymes have a broader specificity than the class I enzymes . However, they are much less active toward sequences containing Hyp in subsites P1 and P3' . Thus, the two classes of collagenases have similar but complementary sequence specificities . This accounts for the ability of the two classes of enzymes to synergistically digest collagen. Appl Environ Microbiol, 1985 Nov, 50(5), 1258 - 61 Inhibitory effect of a copper-dipeptide complex on the establishment of a Clostridium perenne strain in the intestinal tract of gnotobiotic mice; Dubos F et al.; A semisynthetic diet fed to axenic mice was found to prevent the establishment of a Clostridium perenne strain in their intestinal tract . This inhibitory effect did not occur when axenic mice were preinoculated with a strain of Clostridium difficile . The inhibitory effect was related to the presence in the intestinal contents of axenic mice of both dietary copper and a dipeptide, aspartic-epsilon-lysine . When C . difficile was inoculated into axenic mice, the dipeptide disappeared from the digesta, and C . perenne became established even in the presence of high concentrations of copper. Appl Environ Microbiol, 1985 Nov, 50(5), 1238 - 43 Isolation and characterization of butanol-resistant mutants of Clostridium acetobutylicum; Hermann M et al.; In a wild-type strain of Clostridium acetobutylicum isolated from soil, solvent production appeared limited by butanol toxicity . Butanol-resistant mutants have been obtained which produced significantly higher solvent concentrations (about 30%) than the wild-type strain . Some other physiological differences were observed between a selected resistant mutant and the wild-type strain at the level of solvent resistance and sporulation. Clin Nephrol, 1985 Nov, 24(5), 242 - 8 Clostridium difficile-associated colitis in uremic patients; Leung AC et al.; Five uremic patients managed in a renal unit developed Clostridium difficile-associated colitis . Four cases occurred in a cluster at about the same time . All patients had previously received or were on antibiotic therapy at the onset of diarrhea and one patient was also on oral steroid therapy . Cefotaxime, a third generation cephalosporin was involved in all five cases . All patients had severe diseases with explosive diarrhea and systemic toxicity . The diagnosis was confirmed in all cases by culture of C . difficile and demonstration of high titers of C . difficile cytotoxin in the stool . Histology from rectal biopsy in one patient showed classical pseudomembranous colitis . Response to treatment with vancomycin was generally good though one patient had two relapses . Uremic patients have impaired immune response and intestinal motility and are predisposed to C . difficile infection . Cross-infection can occur and the isolation of affected patients seems prudent. Biol Chem Hoppe Seyler, 1985 Nov, 366(11), 1057 - 62 Observations on the elimination of water from 2-hydroxy acids in the metabolism of amino acids by Clostridium sporogenes; Machacek-Pitsch C et al.; Cell-free extracts of Clostridium sporogenes catalyse the water elimination from (2R)-phenyllactate in the presence of one of the energy-rich compounds acetyl-CoA, acetylphosphate or ATP and coenzyme A . Water is eliminated from (2R)-phenyllactoyl-CoA without any of the aforementioned additions . Cinnamoyl-CoA also acts catalytically . One molecule of cinnamoyl-CoA causes the elimination of water from more than 8 molecules phenyllactate . This is important from an energetic point of view since less than 2 mol ATP are formed per 2-3 mol metabolized amino acids . An activation of the hydroxy group of the alpha-hydroxy acid in form of a phosphate ester can also be excluded for energetic reasons. Obstet Gynecol, 1985 Nov, 66(5), 737 - 8 Clostridium difficile colitis associated with single-dose cefazolin prophylaxis; McNeeley SG Jr et al.; Diarrhea and pseudomembranous colitis are associated with antimicrobial therapy and prophylaxis . Clostridium difficile colitis occurred in a patient who received a single dose of cefazolin for prophylaxis at cesarean section . Prompt remission occurred after treatment with oral vancomycin. J Clin Microbiol, 1985 Nov, 22(5), 873 - 6 Survey of neuraminidase production by Clostridium butyricum, Clostridium beijerinckii, and Clostridium difficile strains from clinical and nonclinical sources; Popoff MR et al.; Neuraminidase production was investigated in 57 Clostridium butyricum strains, 16 Clostridium beijerinckii strains, and 25 Clostridium difficile strains . Neuraminidase activity was found only in C . butyricum strains originating from one human newborn with neonatal necrotizing enterocolitis, two newborns with hemorrhagic colitis, one infected placenta, and one adult with peritonitis, It was concluded that neuraminidase was not a major virulence factor in C . butyricum strains. J Neurochem, 1985 Nov, 45(5), 1487 - 94 Solubilization of membrane-bound acetylcholinesterase by a phosphatidylinositol-specific phospholipase C; Futerman AH et al.; Phosphatidylinositol-specific phospholipase C (PIPLC) quantitatively solubilizes acetylcholinesterase (AChE) from purified synaptic plasma membranes and intact synaptosomes of Torpedo ocellata electric organ . The solubilized AChE migrates as a single peak of sedimentation coefficient 7.0S upon sucrose gradient centrifugation, corresponding to a subunit dimer . The catalytic subunit polypeptide of AChE is the only polypeptide detectably solubilized by PIPLC . This selective removal of AChE does not affect the amount of acetylcholine released from intact synaptosomes upon K+ depolarization . PIPLC also quantitatively solubilizes AChE from the surface of intact bovine and rat erythrocytes, but only partially solubilizes AChE from human and mouse erythrocytes . The AChE released from rat and human erythrocytes by PIPLC migrates as a approximately 7S species on sucrose gradients, corresponding to a catalytic subunit dimer . PIPLC does not solubilize particulate AChE from any of the brain regions examined of four mammalian species . Several other phospholipases tested, including a nonspecific phospholipase C from Clostridium welchii, fail to solubilize AChE from Torpedo synaptic plasma membranes, rat erythrocytes, or rat striatum. Gastroenterology, 1985 Nov, 89(5), 1038 - 45 Antibiotic-associated colitis due to Clostridium difficile: double-blind comparison of vancomycin with bacitracin; Young GP et al.; A randomized double-blind study was carried out in patients with unresolving antibiotic-associated colitis due to Clostridium difficile, to compare the effect of bacitracin (80,000 U/day) with vancomycin (500 mg/day) on the resolution of symptoms, clearance of organism, and prevention of relapse . Forty-two patients with colitis, 9 of whom had a pseudomembrane, were randomized, 21 patients to each treatment group . The two groups were comparable in age, disease severity, and antibiotic exposure . For a 50% reduction in stool frequency the mean times (+/- SE) were 4.1 +/- 0.4 days for bacitracin and 4.2 +/- 0.4 days for vancomycin . Sixteen patients (76%) had symptom resolution after 7 days of treatment with bacitracin, compared with 18 patients (86%) given vancomycin . Patients who failed to respond were crossed over (blind) to the alternative antibiotic, but tended to be refractory to the alternative medication as well . Vancomycin-treated patients had negative toxin (83% vs . 53%, p = 0.04) and negative stool cultures (81% vs . 52%, p = 0.02) more frequently than did those patients given bacitracin . Similar numbers of patients in each group had symptomatic relapse during 1 mo of follow-up, but most of them relapsed yet again after blinded crossover therapy . Although bacitracin was significantly less effective than vancomycin in clearing C . difficile from the stools, both were of similar value in the control of symptoms in a group of patients with predominantly nonpseudomembranous colitis . In view of its low cost, bacitracin is a reasonable first-line alternative to vancomycin in the treatment of antibiotic-associated colitis. J Appl Bacteriol, 1985 Nov, 59(5), 469 - 78 Multiple modes of inhibition of spore germination and outgrowth by reduced pH and sorbate; Blocher JC et al.; Germination and outgrowth of three strains of Clostridium botulinum in PYEG medium were measured by phase contrast microscopy . Reduction in pH from 7 to 5.5 completely inhibited germination of strain 12885A, reduced the extent of germination of strain 62A and had no effect on the extent of germination of strain 53B . At pH 5.5, 225 mg/l of undissociated sorbic acid had no effect on the germination of strain 53B, while at pH 6.5, 225 mg/l of undissociated sorbic acid completely inhibited germination of strains 62A and 12885A . Outgrowth of germinated spores of strains 62A and 53B was not inhibited at pH 5.5, but the addition of sorbate (225 mg/l undissociated sorbic acid) completely inhibited outgrowth . Sorbate inhibited germination of Cl . botulinum and Bacillus cereus spores triggered to germinate by amino acids . Inhibition occurred after germinant binding, as measured by commitment to germinate. Cancer Res, 1985 Nov, 45(11 Pt 2), 5714 - 21 Similar effects of phospholipase C and phorbol ester tumor promoters on primary mouse epidermal cells; Jeng AY et al.; Interaction of tumor promoting phorbol esters with specific high affinity receptors is probably essential for many of the biological responses elicited by these agents . Since diacylglycerols which can be produced enzymatically from phospholipids by phospholipase C are postulated to be the physiological ligands for the phorbol ester receptor, we have examined primary cultures of mouse epidermal basal cells exposed to phospholipase C (Clostridium perfringens) for several biological and biochemical responses characteristic of treatment with 12-O-tetradecanoyl-phorbol-13-acetate, the most potent phorbol ester tumor promoter . Formation of diacylglycerols by treatment with phospholipase C was demonstrated by the dose-dependent release of radioactive diacylglycerols in cells prelabeled with {3H}arachidonic acid . Treatment with phospholipase C at 0.05 units/ml for 30 min led to the morphological changes and to the reduction in epidermal growth factor binding (90%) associated with 12-O-tetradecanoylphorbol-13-acetate treatment . Continuous treatment at the same dose led to the induction of the enzymes ornithine decarboxylase and transglutaminase with a time course and extent similar to the inductions by 12-O-tetradecanoylphorbol-13-acetate . Treatment with phospholipase C at 0.1 enzyme unit/ml yielded substantial suppression of the binding affinity of phorbol-12,13-dibutyrate for its receptors without reduction in total number of binding sites, consistent with the production by phospholipase C of a competitive inhibitor of phorbol ester binding . Several diacylglycerols at concentrations of 250 microM and above effectively competed for phorbol-12,13-dibutyrate binding, reduced epidermal growth factor binding, and to a lesser extent induced ornithine decarboxylase and transglutaminase . These results support the hypothesis that diacylglycerols can act through the phorbol ester receptors and thus produce biological and biochemical responses similar to those of the phorbol esters. Arch Surg, 1985 Nov, 120(11), 1321 - 2 Clostridium difficile colitis mimicking acute peritonitis; Drapkin MS et al.; Five patients receiving penicillin V potassium or a cephalosporin antibiotic for 18 hours to 22 days developed fever, marked leukocytosis, and signs and symptoms that suggested right-lower-quadrant peritoneal irritation . All underwent emergency laparotomy, at which dilatation and inflammation of the ascending colon were found . Only one of the patients had profuse diarrhea, and two patients had no diarrhea prior to laparotomy . Postoperatively, Clostridium difficile colitis was diagnosed by stool toxin assay and was confirmed in one case by proctosigmoidoscopic biopsy results . Antibiotic-associated colitis must be considered in any patient who develops peritoneal signs while or after receiving antibiotics . Over a two-year period, the "acute abdomen" presentation accounted for 5.2% of all patients with C difficile colitis at our institutions . Early proctosigmoidoscopy or stool examination for C difficile or its toxin may avoid unnecessary laparotomy in such patients. Zentralbl Bakteriol Mikrobiol Hyg {A}, 1985 Nov, 260(3), 319 - 28 Correlative properties for a differentiation of two Clostridium sordellii phenotypes and their distinction from Clostridium bifermentans; Roggentin P et al.; As the present classification (19) of Clostridium sordellii and C . bifermentans is based on properties which are not conclusive for most of our strains, we investigated 80 strains from various origin of this group regarding 30 selected properties . Four of these properties were correlative and therefore particularly important for a distinct differentiation of the strains investigated: urease activity (U), growth inhibition by 1% mannose (M), arginine deaminase activity (A), and sialidase (EC 3.2.1.18) activity (S) . Concerning these four characters three clusters were formed: cluster I was positive for U, M, A, and S and comprised 36 strains including C . sordellii type strain (ATCC 9714T); cluster II was positive for M and S and negative for U and A and comprised twelve strains including strain ATCC 35392; and cluster III was positive for A and negative for U, M, and S and comprised 32 strains including C . bifermentans type strain (ATCC 638T) . Only two of the correlative properties (U and S, U and A, A and M, or A and S) needed to be tested to determine the affiliation of any strain of the C . sordellii/bifermentans group to one of the three clusters . Clusters I and II, representing two phenotypes of C . sordellii, can now clearly be distinguished from C . bifermentans . Sialidase formed by cluster I and II strains was inhibited by antibodies produced against cluster I strain sialidase . No cross reaction was found with other clostridial sialidases . Pathogenicity, hitherto considered as one of the distinctive properties of C . sordellii and C . bifermentans, was found with various strains of all the three clusters . Therefore, in the case of an infection caused by these two species, care should be taken as to the pathogenicity especially of C . bifermentans and treatment should be accordingly. Rev Infect Dis, 1985 Nov-Dec, 7 Suppl 4, S789 - 93 Clinical experience with aztreonam in the treatment of gram-negative bacteremia; Scully BE et al.; Aztreonam was used for the treatment of gram-negative bacteremia in 101 patients . In 34 instances a second antibiotic was prescribed for the treatment of suspected or documented gram-positive or anaerobic infection . The sources of bacteremia were the urinary tract (50 patients), an intraabdominal site (17), the respiratory tract (8), an intravascular site (9), and an unknown site (17) . The clinical response rate was 92% (91 of 99 patients) . The bacteriologic response rate was 97% (98 of 101 patients) . In six of seven patients, Pseudomonas aeruginosa bacteremia was cured . Twelve patients developed superinfection with gram-positive cocci or Candida, and one patient developed diarrhea associated with Clostridium difficile . No other serious toxic effects were noted. Rev Infect Dis, 1985 Nov-Dec, 7 Suppl 4, S579 - 93 Discovery and development of the monobactams; Sykes RB et al.; A novel procedure designed to detect naturally occurring beta-lactam-containing molecules led to isolation of the monobactams - structurally unique, bacterially produced, monocyclic beta-lactam antibiotics . Although none of these monobactams exhibited impressive antimicrobial activity, side-chain variation - as with the penicillins and cephalosporins - resulted in potently active compounds . Aztreonam was chosen from hundreds of compounds for extended laboratory studies . In addition to a unique chemical structure, aztreonam has biologic properties that are unique in comparison with those of the classical penicillins and cephalosporins . Aztreonam is relatively inactive against gram-positive bacteria and anaerobes but is extremely effective against aerobic gram-negative bacteria, including Pseudomonas aeruginosa . The drug is highly resistant to enzymatic hydrolysis by beta-lactamases, particularly those known to be mediated by R plasmids, and is a poor inducer of chromosomal beta-lactamases . In the majority of drug combinations tested, aztreonam exhibits additive or synergistic activity . In a series of animal-model infections, the drug showed a high degree of efficacy that was consistent with findings in studies in vitro . In a hamster model for Clostridium difficile-induced pseudomembranous colitis, aztreonam did not induce any significant changes. Pathol Biol (Paris), 1985 Nov, 33(9), 899 - 903 {Sensitivity of strict anaerobic bacteria to metronidazole, cefoxitin, and clindamycin}; Dubreuil L et al.; The minimal inhibitory concentrations of three antimicrobial agents were determined by two fold dilution in Wilkins Chalgren agar for over 569 anaerobes isolated and collected from 20 French hospitals . Metronidazole, cefoxitin and clindamycin inhibited respectively 95, 97 and 91% of tested strains . No metronidazole resistant strains could be found among Bacteroides fragilis group, Fusobacterium and Clostridium other than C . perfringens . Cefoxitin was the most active against C . perfringens, Gram positive cocci and non sporulated bacilli . Clindamycin resistance was observed mainly with B . fragilis and Clostridium other than C perfringens . This phenomenon occurred in most hospitals where strains were isolated. Pathol Biol (Paris), 1985 Nov, 33(9), 891 - 5 {Comparative sensitivity of 207 strains of strict anaerobic bacteria to piperacillin and 4 other antibiotics}; Reynaud A et al.; The MIC of two hundred and seven anaerobic bacterial strains was determined by an agar dilution method for five antibiotics (piperacillin, ampicillin, carbenicillin, cefalotin, metronidazole) . 100% of the strains were susceptible to carbenicillin (MIC less than or equal to 128 mg/l), 97% to piperacillin (MIC less than or equal to 16), 86% to metronidazole (MIC less than or equal to 4), 70% to ampicillin (MIC less than or equal to 4) and 68% to cefalotin (MIC less than or equal to 8) . Amongst beta-lactam compounds, piperacillin and ampicillin determined the lowest MIC for Bacteroides fragilis, Fusobacterium, Clostridium and Gram-positive cocci . Amongst Bacteroides fragilis strains, the lowest MIC were obtained with metronidazole. Vet Clin North Am Food Anim Pract, 1985 Nov, 1(3), 509 - 14 Enterotoxemia in neonatal calves; Fleming S; The incidence, bacterial characteristics, disease syndromes, diagnosis, treatment, and prevention of enterotoxemia of neonatal calves caused by Clostridium perfringens (Types A, B, C, D, and E) are reviewed. Dis Colon Rectum, 1985 Nov, 28(11), 765 - 9 The significance of quantitative results of C . difficile cultures and toxin assays in patients with diarrhea; Church JM et al.; The clinical courses of 114 patients with positive Clostridium difficile cultures or toxin assays performed between 1981 and 1984 were reviewed to determine the relationship between outcome of treatment and quantitative bacteriologic test results . C . difficile culture was positive in 60 of 91 patients while toxin assay was positive in 99 of 114 . One third of the patients received supportive therapy only, and 30 percent of these failed to resolve their symptoms . Ninety-one percent of the patients treated with vancomycin resolved, although 11 percent of these suffered relapse . Patients with high toxin titers receiving supportive treatment alone showed a lower response rate than patients with lower toxin titers . This effect was not seen in patients treated with specific therapy nor with different culture quantities . C . difficile colitis has a range of clinical and microbiologic manifestations . Endoscopy is not always diagnostic, both culture and toxin assays are needed for diagnosis, and toxin titer may help in planning treatment . Patients with low toxin titers may be treated supportively, but high toxin titers are an indication for specific therapy . Quantitative culture results have little diagnostic or therapeutic value. Appl Environ Microbiol, 1985 Nov, 50(5), 1165 - 70 Effects of butanol on Clostridium acetobutylicum; Bowles LK et al.; The internal pH of Clostridium acetobutylicum was determined at various stages during the growth of the organism . Even in the presence of significant quantities of acetic, butyric, and lactic acids, an internal pH of 6.2 was maintained . Experiments using N,N'-dicyclohexylcarbodiimide indicated that a functioning H+-ATPase is necessary for internal pH control . Butanol, one of the end products of the fermentation, had numerous harmful effects on C . acetobutylicum . At a concentration high enough to inhibit growth, butanol destroyed the ability of the cell to maintain internal pH, lowered the intracellular level of ATP, and inhibited glucose uptake . Experiments done at two different external pH values suggested that the butanol-mediated decrease in ATP concentration was independent of the drop in internal pH . Glucose uptake was not affected by arsenate, suggesting that uptake was not ATP dependent . The effects of butanol on C . acetobutylicum are complex, inhibiting several interrelated membrane processes. Zh Mikrobiol Epidemiol Immunobiol, 1985 Nov, (11), 37 - 41 {Use of an experimental analytical method for equilibrating nutrient broths for Clostridium perfringens type A growth and toxin formation}; Artemenko VD et al.; A successful attempt to use analytico-experimental approach to the evaluation of experimental data for the scientifically based calculation of the composition of complex culture media, intended for growing pathogenic microorganisms, has been made . The method is based on the evaluation of the specific growth-stimulating and toxin-forming activity of the components of a given culture medium, which are determined by the number of cells grown in the variants of the medium with the limited amount of one of its components . The use of the analytico-experimental balancing method makes it possible to develop culture media with the optimal composition ensuring the definite yield of the target product rather quickly and economically by experimenting on the minimal number of variants equal to the number of the components of the medium . The investigation carried out by means of the analytico-experimental method has revealed that on the basis of peptic serum albumin hydrolysate, pancreatic casein hydrolysate and fodder yeast extract, alongside the culture medium described in an earlier work and containing these components in the proportion 4:2:1, two other media, containing the above components in the proportion 2:4:1 and 3:4:2, can be obtained, these media providing the optimal conditions for, respectively, the toxin formation and growth of C . perfringens, type A. Hepatology, 1985 Nov-Dec, 5(6), 1126 - 31 Transformation of bile acids into iso-bile acids by Clostridium perfringens: possible transport of 3 beta-hydrogen via the coenzyme; Batta AK et al.; We have examined the mechanism for the bacterial transformation of chenodeoxycholic acid and lithocholic acid into the corresponding 3 beta-hydroxy epimers with the use of 3 alpha- and 3 beta-tritiated bile acids . The 3-oxo bile acids were transformed into the 3 alpha- (85%) and 3 beta- (15%) hydroxy bile acids after 20-hr incubation with Clostridium perfringens . Approximately 75% radioactivity was recovered in the aqueous medium when {3 beta-3H}chenodeoxycholic acid or {3 beta-3H}lithocholic acid was incubated with the bacteria, and approximately 15% of radioactivity in the bile acid fraction was associated with the 3 alpha-position of the iso-bile acids . When {3 beta-3H}chenodeoxycholic acid was incubated with unlabeled 3-oxo-5 beta-cholanoic acid, tritiated litho- and iso-lithocholic acids were recovered . These results can be explained only when a 3-oxo intermediate is postulated, and the 3 beta-hydrogen in the bile acids is transferred by the bacterial coenzyme (NAD+ or NADP+) to the 3 alpha-position in the iso-bile acids during the reduction of the 3-oxo compounds. Infect Immun, 1985 Nov, 50(2), 442 - 8 Production and characterization of monoclonal antibodies against Clostridium perfringens type A enterotoxin; Wnek AP et al.; Hybridomas secreting monoclonal antibodies (MABs) specific for Clostridium perfringens type A enterotoxin were produced by fusion of P3X63Ag8.653 myeloma cells with spleen cells from BALB/c mice immunized with purified enterotoxin . Wells containing hybridomas secreting immunoglobulin G (IgG) antibodies against enterotoxin were specifically identified by an indirect enzyme-linked immunosorbent assay (ELISA), and 10 ELISA-positive hybridomas were selected and cloned twice by limiting dilution . All 10 hybridomas produced MABs containing immunoglobulin G1 heavy chains and kappa (kappa) light chains . These hybridomas were then grown as ascitic tumors in mice, and MABs were purified from the ascites fluids with DEAE Affi-gel blue . The specificity of the MABs for enterotoxin was demonstrated by immunoblotting and ELISA . Competitive radioimmunoassay with 125I-MABs suggests that these MABs recognized at least four epitopes on the enterotoxin molecule . The enterotoxin-neutralizing ability of MABs from both hybridoma culture supernatants and ascites fluids was assessed by using a 3H-nucleotide-release Vero (African green monkey kidney) cell assay . Only 2 of the 10 hybridomas produced MABs which completely (greater than 90%) neutralized the biologic activity of enterotoxin . Preincubation of 125I-enterotoxin with MABs demonstrated that MAB neutralizing ability correlated with MAB-specific inhibition of specific binding of enterotoxin to intestinal brush border membranes. J Biol Chem, 1985 Oct 25, 260(24), 13181 - 9 Lactate reduction in Clostridium propionicum . Purification and properties of lactyl-CoA dehydratase; Kuchta RD et al.; Clostridium propionicum converts lactate to propionate (Cardon, B.P., and Barker, H.A . (1947) Arch . Biochem . Biophys . 12, 165-171) . We have obtained a soluble system that carries out this conversion as well as the hydration of acrylate to lactate and the reduction of acrylate to propionate . 3-Pentynyl-CoA inhibits reduction of acrylate and lactate to propionate, but not hydration of acrylate to lactate by cell extracts . The conversion probably involves CoA esters . When {beta-2H3} lactate is used as a substrate, the rate of propionate formation is reduced 1.8-fold, and the methyl group of the resulting propionate has lost 1.4 deuterium atoms . These results are consistent with the intermediate formation of acrylate (acrylyl-CoA) in the conversion of D-lactate to propionate . Two proteins, which we designate E I and E II, were purified to greater than 90% homogeneity . Together, they catalyze the hydration of acrylyl-CoA to lactyl-CoA . E I has an apparent molecular mass of 27,000 daltons and is rapidly and irreversibly inactivated by O2 . E II consists of two subunits of molecular mass 41,000 and 48,000 daltons and contains equal amounts of riboflavin and flavin mononucleotide . Hydration of acrylyl-CoA to lactyl-CoA requires Mg2+ and catalytic quantities of ATP . GTP can replace ATP, but ADP and adenylyl imidodiphosphate cannot . We were unable to detect any stable intermediate during acrylyl-CoA hydration . Finally, we proposed a mechanism for this reaction. J Immunol Methods, 1985 Oct 24, 83(1), 141 - 50 A double antibody sandwich enzyme-immunoassay for Clostridium perfringens type A enterotoxin detection in stool specimens; Jackson SG et al.; A double antibody sandwich enzyme-immunoassay has been developed for detection of Clostridium perfringens enterotoxin . Anti-enterotoxin immunoglobulin G-alkaline phosphatase conjugates were prepared using a rapid minicolumn procedure . The assay can achieve a sensitivity of greater than or equal to 1 ng/ml with purified enterotoxin . Sensitivity for detection of cases of C . perfringens enteritis in a C . perfringens outbreak (86 individuals tested) was between 85.7 and 98.0 per cent depending upon stringency of criteria for defining positive cases . Specificity of the assay was demonstrated by the lack of positive results in 53 individuals involved in a gastroenteritis outbreak of unknown etiology. Biochemistry, 1985 Oct 22, 24(22), 6145 - 52 Aldehyde and ketone substrate analogues inhibit the collagenase of Clostridium histolyticum; Grobelny D et al.; The collagenase from Clostridium histolyticum is a mixture of several collagenases, all of which are zinc metalloproteases . This enzyme catalyzes the cleavage of the X-Gly peptide bond in the repeating sequence of collagen: -Gly-Pro-X-Gly-Pro-X- . Thus the S3, S2, and S1 subsites on the enzyme appear to be occupied by the sequence -Gly-Pro-X- and the S1', S2', and S3' subsites also by -Gly-Pro-X- . Short peptides up to and including N alpha-acyltetrapeptides containing the repeat sequence do not detectably inhibit the enzyme (IC50 greater than 10 mM) . However, peptide aldehydes of the form aminoacyl-X-glycinal, presumably occupying the S1, S2, ..., Sn subsites, are inhibitors . The most potent of these was Pro6-Gly-Pro-glycinal, with an IC50 of 340 +/- 70 microM . The single peptide aldehyde investigated, which could occupy the S1' and S2' subsites, 4-oxobutanoyl-L-proline, did not inhibit collagenase (IC50 greater than 20 mM) . The peptide ketone 5-benzamido-4-oxo-6-phenylhexanoyl-Pro-Ala (XXV), which could occupy the S1-S3' subsites, inhibits collagenase with an IC50 of 120 +/- 50 microM, over 80-fold more potently than its parent peptide analogue benzoyl-Phe-Gly-Pro-Ala (XXIII) . The alcohol analogue of XXV, 5-benzamido-4-hydroxy-6-phenylhexanoyl-Pro-Ala (XXVI), is over 60-fold less potent with an IC50 of 8 +/- 2mM . Extending the peptide ketone XXV to occupy the S2-S3' subsites gave 5-(N alpha-carbobenzoxy-L-prolinamido)-4-oxo-6-phenylhexanoyl-Pro -Ala (XXVII) . Surprisingly, XXVII had an IC50 of only 5.2 +/- 2 mM.(ABSTRACT TRUNCATED AT 250 WORDS) Vet Rec, 1985 Oct 19, 117(16), 408 - 13 Protection of the nursing pig against experimentally induced enteric colibacillosis by vaccination of dam with fimbrial antigens of E coli (K88, K99 and 987P); Nagy LK et al.; Pregnant gilts were vaccinated with two doses of alhydrogel adsorbed fimbrial antigens of Escherichia coli (K88ab, K88ac, K99 and 987P) supplemented with beta toxoid of Clostridium perfringens type C . Their piglets, and piglets of nonvaccinated gilts, were subsequently orogastrically challenged with one or other of the four fimbrial types of enteropathogenic E coli . Some of the vaccinated animals were reinjected with a single dose of the vaccine during second gestation and their piglets, and piglets of non-vaccinated sows, were challenged the same way as were litters of gilts . Blood serum and colostra were examined for antibodies to the four fimbrial antigens of E coli and for antitoxin to beta toxin of C perfringens type C . It was found that: (1) a highly significant reduction in mortality and morbidity was achieved in vaccinated litters against all four challenge strains of E coli; (2) excretion of K88ab and K88ac but not of K99 and 987P challenge strains was significantly reduced; (3) revaccination of sows by a single dose of the vaccine during second gestation conferred complete protection against mortality and highly significant protection against morbidity; (4) no correlation was noted between colostral or seroagglutinins to fimbrial antigens of E coli and mortality rates in litters challenged with homologous fimbrial types of E coli, but good correlation was found between colostral precipitins to K88 antigens and mortality rates in litters; (5) antitoxin value in 97 per cent of colostrum of vaccinated sows was 10 iu equivalent of C perfringens type C toxin or more per ml of colostrum. Appl Environ Microbiol, 1985 Oct, 50(4), 1043 - 7 Transmembrane pH gradient and membrane potential in Clostridium acetobutylicum during growth under acetogenic and solventogenic conditions; Huang L et al.; The proton motive force and its electrical and chemical components were determined in Clostridium acetobutylicum, grown in a phosphate-limited chemostat, using {14C}dimethyloxazolidinedione and {14C}benzoic acid as transmembrane pH gradient (delta pH) probes and {14C}triphenylmethylphosphonium as a membrane potential (delta psi) indicator . The cells maintained an internal-alkaline pH gradient of approximately 0.2 at pH 6.5 and 1.5 at pH 4.5 . The delta pH was essentially constant between pH 6.5 and 5.5 but increased considerably at lower extracellular pH values down to 4.5 . Hence, the intracellular pH fell from 6.7 to 6.0 as the external pH was lowered from 6.5 to 5.5 but did not decrease further when the external pH was decreased to 4.5 . The transmembrane electrical potential decreased as the external pH decreased . At pH 6.5, delta psi was approximately -90 mV, whereas no negative delta psi was detectable at pH 4.5 . The proton motive force was calculated to be -106 mV at pH 6.5 and -102 mV at pH 4.5 . The ability to maintain a high internal pH at a low extracellular pH suggests that C . acetobutylicum has an efficient deacidification mechanism which expresses itself through the production of neutral solvents. Eur J Cancer Clin Oncol, 1985 Oct, 21(10), 1159 - 63 Clostridium difficile colitis in leukemia patients; Panichi G et al.; Leukemia patients with diarrhea or other abdominal symptoms have been investigated for the presence of Clostridium difficile and its cytotoxin in stools . Of the patients studied 19% had C . difficile, in most cases together with cytotoxin . All patients but one had received antibiotics, while one had been treated with cytotoxic agents only . Symptoms of colitis were most often abdominal pain and distension rather than diarrhea . Owing to the not infrequent fatal evolution, it is recommended that routine search for C . difficile in leukemia patients with abdominal symptoms be performed and appropriate therapy started immediately. J Clin Gastroenterol, 1985 Oct, 7(5), 387 - 90 Lack of relationship between Clostridium difficile toxin and inflammatory bowel disease in children; Hyams JS et al.; Conflicting reports have appeared concerning the role of Clostridium difficile toxin in chronic inflammatory bowel disease . Therefore, we prospectively evaluated the incidence of C . difficile toxin in 44 children with inflammatory bowel disease of variable clinical severity over a 1-year period . Only 3/128 stool specimens provided by these patients were found to be toxin-positive . These three stool specimens were from three different patients with Crohn's disease of moderate severity who had no recent hospitalization or antibiotic exposure . None received vancomycin therapy and their stools became toxin-negative over 3 weeks with no apparent change in the patients' clinical condition . No patient with severe disease or recent exposure to antibiotics or sulfasalazine was found to have toxin-positive stools . Routine screening for C . difficile toxin in children with inflammatory bowel disease appears unwarranted. Eur J Clin Microbiol, 1985 Oct, 4(5), 505 - 7 Identification of Clostridium difficile using the API ZYM system; Levett PN; The use of the API ZYM system for the identification of Clostridium difficile was investigated . The enzyme profiles generated by this system readily distinguished strains of Clostridium difficile from other clostridia commonly isolated from faeces . Enzyme activity of Clostridium difficile was influenced by the composition of the culture medium but appeared to be independent of the age of the culture . Given careful standardisation of techniques the API ZYM system is a suitable alternative to conventional techniques for identification of Clostridium difficile. Biol Chem Hoppe Seyler, 1985 Oct, 366(10), 953 - 61 Purification and some properties of an acryloyl-CoA reductase of Clostridium kluyveri; Sedlmeier H et al.; Acryloyl-CoA reductase, a presumably previously unknown soluble enzyme, is present in Clostridium kluyveri . It catalyses the reduction of the carbon-carbon double bond of acryloyl-CoA or ethyl vinyl ketone and other alpha, beta-unsaturated carbonyl compounds at the expense of reduced methylviologen . On the basis of a Vmax/Km ratio, which is at least 18 times higher than that for the next best substrate (E)-2-butenoyl-CoA, the enzyme is called acryloyl-CoA reductase . A purity of over 90% was achieved . The apparent molecular mass, as determined by gel chromatography, is 28.4 kDa . Dodecyl sulfate gel electrophoresis shows subunits with a molecular mass of 14.2 kDa . Based on a molecular mass of 28.4 kDa about 1.5 mol FMN have been observed . Less than 0.2 g-atom iron per mol protein were determined . Ferredoxin or flavodoxin seem to be able to carry electrons from hydrogenase to the acryloyl-CoA reductase . The addition of hydrogen to the alpha-carbon of ethyl vinyl ketone occurs from the re-side. Br J Ophthalmol, 1985 Oct, 69(10), 774 - 7 Clostridium septicum panophthalmitis with systemic complications; Insler MS et al.; A fulminant case of endophthalmitis due to Clostridium septicum is described . The patient presented with spontaneous gas gangrene panophthalmitis, with early visual loss and an air bubble in the anterior chamber . Death ensued, and necropsy revealed changes consistent with severe arterosclerotic cardiovascular disease, a relationship not uncommon in patients with clostridium sepsis . This association as well as the histopathology of the globe are discussed. Cancer Res, 1985 Oct, 45(10), 4986 - 9 Effects of the antitumor drug Adriamycin on human red blood cell discocyte-echinocyte transitions; Chahwala SB et al.; The antitumor drug Adriamycin, when preincubated with human red blood cells (discocytes) for 10 min, prevented the formation of echinocytes induced by the calcium ionophore A23187 in the presence of 0.2 mM calcium . The degree of protection was concentration dependent and was greater than 90% at 10 microM Adriamycin . Adriamycin did not interfere with the accumulation of calcium induced by a 5 microM concentration of the ionophore . Adriamycin reversed echinocyte morphology to the discocyte form in echinocytes which had been formed by adenosine triphosphate depletion but not those formed after treatment with A23187 and Ca2+ . Its ability to protect against Ca2+-induced echinocyte formation contrasts with the failure of the local anesthetic procaine to exert such an effect, even at 45 mM (J . Palek et al., Blood, 50: 155-164, 1977), and this difference suggests that Adriamycin may not be acting simply as a chaotropic agent . This hypothesis was supported by the observation that Adriamycin alone did not induce a cup-form morphology in discocytes (stomatocytosis) . Wheat germ agglutinin protection of echinocyte formation induced by calcium loading was reversed by 30 mM N-acetylglucosamine, which partially reversed the Adriamycin protection of echinocyte formation . However, desialylation of human red blood cells with Clostridium perfringens type V neuraminidase, while preventing the protection of echinocyte formation by wheat germ agglutinin, had no effect on the protection afforded by Adriamycin . This suggests that Adriamycin does not prevent echinocyte formation via binding to the sialic acid residues of the transmembrane protein glycophorin and that another mechanism or mechanisms are involved in its action to modulate morphological transitions of the red blood cell membrane. Appl Environ Microbiol, 1985 Oct, 50(4), 795 - 800 Germination of spores from Clostridium botulinum B-aphis and Ba410; Montville TJ et al.; The germination of spores from Clostridium botulinum B-aphis and Ba410 was examined . In a complex medium, heat activation of spores from both strains doubled the germination rates and was required for germination in the presence of 2% NaCl . In a defined medium (CTB {D . B . Rowley and F . Feeherry, J . Bacteriol . 104:1151-1157, 1970}), the parent strain B-aphis germinated at a rate of 0.77% min-1 in the absence of NaCl and was not affected by 2% NaCl . A salt-tolerant derivative, strain Ba410, germinated at rates of 0.16% min-1 in CTB and 0.04% min-1 in CTB containing 2% NaCl . L-Alanine-triggered spores germinated faster than did L-cysteine-triggered spores from both strains . When both amino acids were present, B-aphis germinated rapidly in the absence of NaCl and had biphasic kinetics in the presence of NaCl . Strain Ba410 had biphasic kinetics in the absence of NaCl and germinated slowly with single-phase kinetics in the presence of NaCl . L-Alanine- and L-cysteine-triggered germinations were each inhibited by both D-alanine and D-cysteine, indicating a common germinant-binding site for both alanine and cysteine . Attempts to select for variants with amino acid-specific germinant-binding sites were unsuccessful . Differences in the germination kinetics of both strains could not be explained by ultrastructural differences . Transmission electron micrographs revealed striking similarities between the strains. J Antibiot (Tokyo), 1985 Oct, 38(10), 1322 - 6 Luminamicin, a new antibiotic . Production, isolation and physico-chemical and biological properties; Omura S et al.; A new antibiotic, luminamicin, was isolated from the culture broth of an actinomycete strain OMR-59 . It exhibits antibacterial activity against anaerobic bacteria, especially against Clostridium sp . The molecular formula of the antibiotic was determined as C32H38O12 on the basis of high resolution mass spectrum, elemental analysis and NMR spectrum. Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6811 - 4 Evidence that an iron-nickel-carbon complex is formed by reaction of CO with the CO dehydrogenase from Clostridium thermoaceticum; Ragsdale SW et al.; The interaction between carbon monoxide and the CO dehydrogenase from Clostridium thermoaceticum was studied by electron spin resonance (ESR) techniques . When the enzyme reacts with CO, a paramagnetic complex is formed which previously was shown, by isotope substitution, to be due to a nickel-carbon species . In this paper, we demonstrate that iron is also a component of this ESR-detectable complex . When the iron in the enzyme is replaced with 57Fe, a broadening of 18 G in the g parallel and 7 G in the g perpendicular region is seen . This hyperfine interaction is probably due to more than one iron atom in the complex . Coenzyme A influences this ESR spectrum . In the absence of CoA, the ESR spectrum consists of two superimposed signals, which were simulated using the following ESR parameters: signal 1, with g = 2.074 and g = 2.028, and signal 2 with gx = 2.062, gy = 2.047, and gz = 2.028 . CoA converts signal 2 into signal 1 . Since iron, nickel, and carbon all are part of this ESR-detectable complex, we propose that these atoms exist in a spin-coupled complex with net spin = 1/2, analogous to other iron-sulfur centers in which the metals are bridged by acid-labile sulfide. Zh Mikrobiol Epidemiol Immunobiol, 1985 Oct, (10), 22 - 5 {Differentiation of the main species of Clostridium by gas chromatography}; Bychenko BD et al.; For the first time in the USSR the properties of microorganisms of the genus Clostridium have been studied with the use of the gas-chromatographic techniques . The analysis of the quantitative and qualitative composition of extracellular alcohols and carboxylic acids in 99 museum and newly isolated strains of 18 Clostridium species has made it possible to classify these microorganisms with 7 sharply differing groups . The above techniques permit the classification of clostridia with one of the groups within 2 hours if the microbial cultures have been grown in glucose-containing peptone yeast medium. Appl Environ Microbiol, 1985 Oct, 50(4), 1110 - 1 Selective and differential medium for detecting Clostridium botulinum; Silas JC et al.; A selective and differential growth medium was developed for detection of Clostridium botulinum types A, B, and F . The medium consisted of peptone-glucose-yeast extract agar supplemented with cycloserine, 250 micrograms/ml; sulfamethoxazole, 76 micrograms/ml; and trimethoprim, 4 micrograms/ml as selective inhibitors and various types and levels of botulinal antibodies for type differentiation in the immunodiffusion reaction . Growth of proteolytic types of C . botulinum were not affected by the incorporation of the selective agents, but some nonproteolytic types were suppressed . Cross-reactions between types A and B were visually distinguishable, whereas cross-reactions between type F and Clostridium sporogenes did not occur at the optimum antibody titer . Optimum antibody titer varied with toxin type . The proposed selective differential medium should be valuable in isolating and typing of proteolytic C . botulinum types A, B, and F from samples containing mixed microbial populations. Appl Environ Microbiol, 1985 Oct, 50(4), 1097 - 9 Protoplast formation and cell wall regeneration in Clostridium perfringens; Stal MH et al.; A protocol was developed for the efficient production and regeneration of Clostridium perfringens protoplasts . Cell wall regeneration frequencies of up to 5% were obtained. Am J Vet Res, 1985 Oct, 46(10), 2147 - 8 Enteropathogenicity of purified Clostridium perfringens enterotoxin in the pig; Popoff MR et al.; In an assay procedure, purified Clostridium perfringens enterotoxin induced the accumulation of fluid in the ileal loop of the axenic pig . The smallest amount of enterotoxin causing a positive response was 25 micrograms (1:32 titer by counterimmunoelectrophoresis {CIEP} ), and 100 micrograms (1:128 CIEP titer) caused a marked response . Possibly, diarrhea in pigs with 1:32 CIEP titer or more of enterotoxin in the feces may be associated with enterotoxigenic C perfringens. Biochemistry, 1985 Sep 24, 24(20), 5647 - 52 Kinetics of reduction of high redox potential ferredoxins by the semiquinones of Clostridium pasteurianum flavodoxin and exogenous flavin mononucleotide . Electrostatic and redox potential effects; Przysiecki CT et al.; We have measured the ionic strength dependence of the rate constants for the electron-transfer reactions of flavin mononucleotide (FMN) and flavodoxin semiquinones with 10 high redox potential ferredoxins (HiPIP's) . The rate constants were extrapolated to infinite ionic strength by using a theoretical model of electrostatic interactions developed in our laboratory . In all cases, the sign of the electrostatic interaction was the same as the protein net charge, but the magnitudes were much smaller . The results are consistent with a model in which the electrical charges are approximately uniformly distributed over the HiPIP surface and in which there are both short- and long-range electrostatic interactions . An electrostatic field calculation for Chromatium vinosum HiPIP is consistent with this . The presumed site of electron transfer includes that region of the protein surface to which the iron-sulfur cluster is nearest and appears to be relatively hydrophobic . The principal short-range electrostatic interaction would involve the negative charge on the iron-sulfur cluster . For some net negatively charged proteins, this effect is magnified, and for net positively charged HiPIP's, it is counterbalanced . The rate constants extrapolated to infinite ionic strength can be correlated with redox potential differences between the reactants, as has previously been shown for cytochrome-flavin semiquinone reactions . Both electrostatic and redox potential effects are magnified for the flavodoxin semiquinone as compared to the FMN semiquinone-HiPIP reactions . This was also observed previously for the flavin semiquinone-cytochrome reactions.(ABSTRACT TRUNCATED AT 250 WORDS) Biochim Biophys Acta, 1985 Sep 20, 831(1), 159 - 60 Evidence for a pH-dependent isomerization of Clostridium oroticum dihydroorotase; Bidigare RR et al.; Analytical gel permeation chromatography on both Sephadex and polyacrylamide columns shows that Clostridium oroticum dihydroorotase (L-5,6-dihydroorotate amidohydrolase, EC 3.5.2.3) undergoes a large decrease in molecular size when the pH is decreased from 8 to 6 . The Stokes radius decreases from about 40 A to 36 A . Neither the molecular size nor kinetic properties are dependent on protein concentration . Thus, the decreased molecular size reflects a pH dependent isomerization of the enzyme. Biochim Biophys Acta, 1985 Sep 11, 836(2), 255 - 61 Characterization of delta 4-3-ketosteroid-5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase in cell extracts of Clostridium innocuum; Stokes NA et al.; Cell extracts prepared anaerobically from Clostridium innocuum and Clostridium paraputrificum reduced delta 4-3-ketosteroids to 3 beta 5 beta and 3 alpha 5 beta derivatives, respectively . delta 4-3-Ketosteroid-5 beta-reductase (5 beta-reductase) from both organisms required NADH for activity . 5 beta-Reductase from C . innocuum had a pH optimum of 5.0 . The substrate concentration at half-maximal reaction velocity was 4.2 microM, and a specific activity of 17 nmol product formed/h per mg protein was determined using 4-pregnen-3,20-dione (progesterone) as a substrate . delta 4-3-Ketosteroid-5 beta-reductase from C . innocuum reduced progesterone and testosterone, but not 4-cholesten-3-one, to corresponding 3-keto-5 beta derivatives . A relative molecular (Mr) weight of 80 000 was estimated for 5 beta-reductase using HPLC-gel filtration chromatography . 3 beta-Hydroxysteroid dehydrogenase in cell extracts of C . innocuum was oxygen sensitive and required NADH for activity . An Mr of 80 000 was estimated for 3 beta-hydroxysteroid dehydrogenase . However, 5 beta-reductase and 3 beta-hydroxysteroid dehydrogenase activities were separated using an HPLC-DEAE chromatography technique. Biochim Biophys Acta, 1985 Sep 6, 841(3), 306 - 17 Amidation of, and (R)-1-amino-2-propanol attachment to, the corrin ring during vitamin B-12 biosynthesis by Clostridium tetanomorphum extracts; Ford SH; Two intermediate stages in cobalamin biosynthesis, amidation of carboxylic acid groups in the corrin ring and (R)-1-amino-2-propanol attachment at propionic acid position f, have been studied using cell-free extracts from the obligate anaerobe Clostridium tetanomorphum . The preparation of an incomplete corrinoid, probably cobinic acid-a,c,d,e,g-pentaamide, as an in vitro amidation substrate was accomplished via mild acid hydrolysis of cobinamide . Weak, but reproducible activities for both amidation and (R)-1-amino-2-propanol attachment were found in crude, nucleic acid-free and DE-52 column-purified protein fractions . The amidation reaction was glutamine-dependent in crude fractions, but became ammonium ion-dependent in more purified fractions . Significant problems encountered were (a) the weak and unstable character of both enzyme activities, and (b) the irreversible changes in the visible spectra of the incomplete corrinoids employed as substrates caused by use of thiol-reducing agents in the buffers and assays. J Biol Chem, 1985 Sep 5, 260(19), 10461 - 6 Separation, purification, partial characterization and comparison of the heavy and light chains of botulinum neurotoxin types A, B, and E; Sathyamoorthy V et al.; Clostridium botulinum produces botulinum neurotoxin (NT) in antigenically distinct forms . When isolated from bacterial cultures type E is a single chain, type B is a mixture of single and two-chain molecules, and type A is essentially a two-chain molecule (Mr approximately 150,000) . Protease(s) in the cultures or trypsin nick single-chain NT to the two-chain form . The heavy (Mr approximately 100,000) and light (Mr approximately 50,000) chains of the two-chain molecule remain held together by -S-S-bond(s) . The two chains are presumed to have different functions . NT binds to nerve cells via the heavy chain and then light chain enters the cell and blocks release of acetylcholine (Simpson, L . L . (1981) Pharmacol . Rev . 33, 155-188) . We nicked single-chain NT to form the two-chain form with trypsin, minimizing secondary cleavages, then separated and purified the heavy and light chains using ion-exchange chromatography . The technique, with minor modifications, is a generalized method for types A, B, and E . These subunit chains (each a single band in sodium dodecyl sulfatepolyacrylamide gel electrophoresis) were analyzed for their complete amino acid compositions . The amino acid contents of the heavy and light chains agreed well with the parent two-chain molecule . This affirms that NT is composed of two chains . The two subunit chains are now usable for amino acid sequence and other studies . Comparison of the amino acid contents indicates more similarity among the light chains than the heavy chains of the three NT types, a similarity that agrees with our published partial amino acid sequences (first 13-18 residues) of these chains . Several (up to 9) different amino acid residues of the heavy chain (which is twice the size of the light chain) are present in double the number of corresponding residues in the light chain. Arch Microbiol, 1985 Sep, 142(4), 375 - 82 Metabolic and energetic aspects of the growth of Clostridium butyricum on glucose in chemostat culture; Crabbendam PM et al.; The influence of a number of environmental parameters on the fermentation of glucose, and on the energetics of growth of Clostridium butyricum in chemostat culture, have been studied . With cultures that were continuously sparged with nitrogen gas, glucose was fermented primarily to acetate and butyrate with a fixed stoichiometry . Thus, irrespective of the growth rate, input glucose concentration, specific nutrient limitation and, within limits, the culture pH value, the acetate/butyrate molar ratio in the culture extracellular fluids was uniformly 0.74 +/- 0.07 . Thus, the efficiency with which ATP was generated from glucose catabolism also was constant at 3.27 +/- 0.02 mol ATP/mol glucose fermented . However, the rate of glucose fermentation at a fixed growth rate, and hence the rate of ATP generation, varied markedly under some conditions, leading to changes in the Y glucose and YATP values . In general, glucose-sufficient cultures expressed lower yield values than a corresponding glucose-limited culture, and this was particularly marked with a potassium-limited culture . However, with a glucose-limited culture increasing the input glucose concentration above 40 g glucose X 1(-1) also led to a significant decrease in the yield values that could be partially reversed by increasing the sparging rate of the nitrogen gas . Finally glucose-limited cultures immediately expressed an increased rate of glucose fermentation when relieved of their growth limitation . Since the rate of cell synthesis did not increase instantaneously, again the yield values with respect to glucose consumed and ATP generated transiently decreased . Two conditions were found to effect a change in the fermentation pattern with a lowering of the acetate/butyrate molar ratio . First, a significant decrease in this ratio was observed when a glucose-limited culture was not sparged with nitrogen gas; and second, a substantial (and progressive) decrease was observed to follow addition of increasing amounts of mannitol to a glucose-limited culture . In both cases, however, there was no apparent change in the YATP value.(ABSTRACT TRUNCATED AT 250 WORDS) Leber Magen Darm, 1985 Sep, 15(5), 192 - 7 {Disorders of intestinal flora in intensive care patients}; Graninger W; The intestinal flora under normal conditions prevents colonisation of the intestinal mucosa with pathogenic bacteria . Various diseases as well as antibiotics may disturb the host/bacteria balance . If patients are in addition immunocompromised, otherwise commensal bacteria may cause life threatening infections . Treatment of intensive care patients with antibiotics thus should account for preservation of resistance against colonization . Antibiotics active against anaerobes or poorly absorbed from the gastrointestinal tract, or excreted in the bile should be avoided . In patients with colitis induced by antibiotics the number of clostridium difficile with subsequent toxin production are greatly increased as a consequence of the killing of the normal anaerobic colon bacterial flora; in these patients vancomycin has to be applied . Mostly "dysbiosis" caused by antibiotics does not need any treatment . Therapeutic adjuncts like the administration of bacterial preparations e.g . lactobacilli are of no value. Can J Surg, 1985 Sep, 28(5), 432 - 3 Pseudomembranous colitis and wound infection following perioperative use of multiple antibiotics; Bohnen JM et al.; The prophylactic use of antibiotics in elective surgery of the colon is accepted practice, but it has inherent risks . The authors report the case of a 70-year-old woman who had wound infection and severe, relapsing pseudomembranous colitis due to Clostridium difficile after a short course of antibiotics given orally and parenterally at the time of elective resection of the colon . Perioperatively, she received erythromycin base and neomycin orally, plus netilmicin and metronidazole intravenously . Although the concomitant administration of parenteral antibiotics may enhance the benefit of antibiotics given orally before operation, this does not entirely prevent wound infection . Until the relation between the number of drugs and risk of antibiotic-associated colitis is more clearly defined, caution should be exercised in the use of multiple antibiotics in elective colonic surgery. J Antimicrob Chemother, 1985 Sep, 16(3), 305 - 13 The in-vitro activity of a novel penem FCE 22101 compared to other beta-lactam antibiotics; Neu HC et al.; FCE 22101 is a penem antibiotic which inhibits the majority of Enterobacteriaceae, Haemophilus influenzae, and Neisseria gonorrhoeae at concentrations of 0.5-4 mg/l . It inhibits staphylococci, haemolytic streptococci and Streptococcus pneumoniae at less than or equal to 0.25 mg/l . Pseudomonas aeruginosa and other Pseudomonas species are resistant . Bacteroides fragilis and Clostridium species are inhibited by less than or equal to 1 mg/l . FCE 22101 is not hydrolyzed by the common plasmid and chrosmosomal beta-lactamases . It shows minimal discrepancy between MIC and MBC values and there is minimal effect of inoculum size . Although FCE 22101 is generally less active against Enterobacteriaceae than are cefotaxime and ceftazidime, it does inhibit some Enterobacter spp . resistant to these agents . FCE 22101 and imipenem are similar in activity against Gram-positive and anaerobic species. Vet Res Commun, 1985 Sep, 9(4), 269 - 87 Etiology and pathogenesis of necrotic enteritis; Shane SM et al.; Sporulated oocysts of Eimeria acervulina were administered orally to cage-housed broilers at a dose of 3.5 X 10(5) resulted in mild subclinical coccidiosis . Clostridium perfringens incorporated in feed at a level of 2.5 X 10(8) organisms/g . produced lesions characteristic of necrotic enteritis . Mortality of 8% (7/80) occurred in birds fed a ration inoculated with Cl . perfringens alone . Mortality of 35% (28/80) was observed in birds which received an oral dose of E . acervulina and which were fed simultaneously with a ration containing Cl . perfringens . Birds which were fed an inoculated ration two days after an oral dose of E . acervulina showed 41% (33/80) mortality . Birds which received an inoculated ration for two days before administration of an oral dose of E . acervulina demonstrated 18% mortality (15/80) . Birds which were fed an inoculated ration four days after an oral dose of E . acervulina showed 10% mortality . Infection with E . acervulina reduced the pH of intestinal contents with a simultaneous depression in serum protein . A 39% increase in intestinal passage time from 178 to 248 minutes occurred on the fifth day after infection with E . acervulina . These experiments suggest that necrotic enteritis, attributed to proliferation of a toxigenic strain of Cl . perfringens, followed intestinal stasis and minimal lesions induced by mild intestinal coccidiosis. J Hosp Infect, 1985 Sep, 6(3), 312 - 22 A hospital outbreak of Clostridium difficile? Hall SM, Calver GP, Williams M. An increase in numbers of patients with Clostridium difficile and its toxin in their stools at a hospital in South-west London led to closure of a ward to admissions and to an investigation of a possible nosocomial outbreak . The findings suggested that the increase was not due to an outbreak of related cases but to increased investigation . The cost of the episode both in financial terms and in the effect on patient care, was considerable . This study highlights the need for caution in interpreting the significance of Cl . difficile in stool specimens . Laboratory data can only alert clinicians to the possibility of colitis; decisions about treatment and control of spread of infection should also be based on clinical criteria. J Assoc Off Anal Chem, 1985 Sep-Oct, 68(5), 881 - 3 Rapid detection of Clostridium perfringens: comparison of lactose sulfite broth with tryptose-sulfite-cycloserine agar; Neut C et al.; The lactose sulfite (LS) medium recommended for the detection and identification of Clostridium perfringens in foods was compared with a reference method using tryptose-sulfite-cycloserine (TSC) agar for the enumeration of this organism in a variety of foods and food ingredients . C . perfringens was detected and enumerated in 17 of the 54 samples examined with LS broth, but its presence could be confirmed in only 9 of the samples with TSC agar . In only 2 instances, C . perfringens was detected on TSC agar but not in LS broth . A positive response (FeS + and gas +) in LS broth incubated at 46 degrees C always corresponded to the presence of C . perfringens; whereas the black colonies formed on TSC agar incubated at 37 degrees C were frequently found to be Clostridium species other than C . perfringens . Thus, because of its highly selective nature, LS broth was superior to TSC agar for enumerating and confirming the small numbers of C . perfringens that were present in a majority of the samples . This was especially true when other clostridia were also present . Besides its greater selectivity and sensitivity, LS broth had the additional advantages of requiring less work and giving confirmed results within 24-48 h compared with 3 days for the TSC agar method. Biochem J, 1985 Sep 1, 230(2), 451 - 5 A study on the mechanism of the epimerization at C-3 of chenodeoxycholic acid by Clostridium perfringens; Aragozzini F et al.; The mechanism of 3-hydroxy epimerization of chenodeoxycholic acid by Clostridium perfringens was investigated in 3 alpha, 7 alpha-dihydroxy-{2,2,4,4-2H4}-, 3 alpha, 7 alpha-dihydroxy-{3 beta-2H}- and 3 beta, 7 alpha-dihydroxy-{3 alpha-2H}-5 beta-cholanoic acid transformations . Our findings rule out a dehydration-rehydration pathway and agree with a redox mechanism involving 3-oxochenodeoxycholic acid as intermediate. Age Ageing, 1985 Sep, 14(5), 296 - 302 Diarrhoea due to enterotoxigenic Clostridium perfringens: clinical features and management of a cluster of ten cases; Williams R et al.; Clostridium perfringens has recently been shown to be associated with antibiotic-associated diarrhoea . We describe here the clinical features and management of an outbreak of diarrhoea in a Geriatric Unit . Ten cases were due to enterotoxigenic C . perfringens and in these cases there was a highly significant correlation with recent antibiotic administration (P = 0.0001) . The importance of early recognition of C . perfringens as a cause of infective diarrhoea in the elderly is stressed. Eur J Biochem, 1985 Aug 15, 151(1), 75 - 82 Inactivation of Clostridium botulinum type A neurotoxin by trypsin and purification of two tryptic fragments . Proteolytic action near the COOH-terminus of the heavy subunit destroys toxin-binding activity; Shone CC et al.; Limited treatment of Clostridium botulinum type A neurotoxin with trypsin resulted in the cleavage of the heavy (95000 Da) subunit at approximately the mid-position and a loss of toxic activity . The rate of toxicity loss was considerably faster than that of mid-chain cleavage; thus a loss of toxicity in excess of 90% was accompanied by only 30-35% mid-chain cleavage of the heavy subunit . A study of the binding of 125I-labelled neurotoxin to rat brain synaptosomes showed the loss of toxicity on trypsin treatment to be paralleled by a loss of toxin binding to rat brain synaptosomes suggesting the presence of at least two sites of tryptic action on the 95000-Da binding subunit . Prolonged treatment of the neurotoxin with trypsin resulted in the complete digestion of a 46000-Da fragment of the heavy subunit, leaving intact a soluble fragment of approximately 105000 Da containing the light subunit linked to the remaining (49000-Da) portion of the heavy subunit . This fragment exhibited less than 0.01% of the original toxicity and gave immunoprecipitation reactions indistinguishable from the native toxin . The 49000-Da portion of the heavy chain was purified from the 105000-Da fragment of the toxin and the sequence of the first 35 amino acids determined . The sequence of the first 10 residues was found to be identical to that previously reported for the heavy subunit showing that the 49000-Da fragment represents the NH2-terminal portion of the heavy chain and that this region is resistant to tryptic action . It is suggested that the primary site(s) of tryptic action on the heavy subunit of botulinum type A neurotoxin is close to the COOH terminus and that cleavage of the polypeptide chain in this region results in a loss of toxic activity mediated by the destruction of the neurotoxin-binding site. Biochem J, 1985 Aug 15, 230(1), 101 - 8 Dihydro-orotase from Clostridium oroticum . Purification and reversible removal of essential zinc; Pettigrew DW et al.; A new purification procedure involving five column-chromatography steps is described for dihydro-orotase (L-5,6-dihydro-orotate amidohydrolase, EC 3.5.2.3) from Clostridium oroticum (A.T.C.C . 25750) . The native purified enzyme is a dimer of Mr 102 000 and contains 4.0 +/- 0.3 g-atoms of zinc/mol of dimer . These observations agree with those reported previously {Taylor, Taylor, Balch & Gilchrist (1976) J . Bacteriol . 127, 863-873} . It is conclusively demonstrated that dihydro-orotase is a zinc metalloenzyme . Zinc is reversibly removed by treatment with chelators in phosphate buffer at pH 6.5, as demonstrated by atomic absorption spectrophotometry and decrease of enzyme activity . The specific activity is linearly dependent on zinc content . Addition of ZnSO4 to the chelator-treated enzyme results in regain of the normal complement of zinc and enzyme activity . Kinetic properties of the reconstituted enzyme are indistinguishable from those of the native enzyme . The amino acid composition of the homogeneous enzyme suggests that the zinc atoms occupy different environments. Angew Parasitol, 1985 Aug, 26(3), 151 - 5 Connections between Ascaridia galli and the bacterial flora in the intestine of hens; Okulewicz A et al.; Parasitological dissections of 502 intestinal tracts of hens deriving from big private chicken-farms have been done . In the jejunum of 146 hosts (ext . 29.1%) from 1 to 21 individuals of A . galli were detected . Using bacterial selective media and biochemical tests, the microorganisms from the hen's intestinal tracts as well as from the cuticle surface of the nematodes were identified . Among them were: grampositive (+) Lactobacillus, Bacillus, Staphylococcus, Streptococcus, Micrococcus, Sarcina, Clostridium, Corynebacterium; gramnegative (-) Enterobacteriaceae, Pseudomonas, Pasteurella, and fungi Candida and others . The lower frequency of microorganisms and the smaller amount of bacteria in the intestinal content in infected hens than in uninfected show that A . galli has antibacterial properties. J Pediatr Gastroenterol Nutr, 1985 Aug, 4(4), 563 - 7 Differential effect of botulinal toxin on esophageal motor function in infants; Cannon RA; The effect of Clostridium botulinum toxin on esophageal motor function was studied in four infants (ages 5-9 months) with confirmed infant botulism . Esophageal motility studies using a perfused catheter assembly were performed during the acute phase in all patients, and during the recovery phase in one patient . Motor function of the proximal esophagus and upper esophageal sphincter was abnormal in each, while motor function of the distal esophagus and lower esophageal sphincter was normal . Mean lower esophageal sphincter pressure was 24 mm Hg for the group (normal: 15-30 mm Hg) . Sequential studies of proximal esophageal motility in one infant revealed a return of normal motor function which correlated with recovery of peripheral muscle strength and gag reflexes . C . botulinum toxin impairs proximal, but not distal, esophageal motor function in the infant botulism model . This effect appears to be consistent with the known action of the toxin on synaptic acetylcholine release and current concepts regarding distribution of cholinergic and noncholinergic neurotransmitter receptors in the esophagus. Arch Otolaryngol, 1985 Aug, 111(8), 550 - 3 Clostridium difficile colitis following head and neck surgery . Report of cases; Griebie M et al.; Clostridium difficile, a toxin-producing, gram-positive anaerobe, has been implicated as the causative agent of pseudomembranous colitis, an acute inflammatory bowel disease that generally occurs in association with antimicrobial therapy . This subject has received extensive review in the general surgical, medical, and pediatric literature but has not been specifically addressed in the literature of our specialty . We present the report of four recent cases, including one that progressed to the clinical picture of peritonitis and toxin megacolon . The literature is reviewed regarding presentation, diagnosis, and treatment of C difficile colitis . Familiarity with this disease process may minimize morbidity and prevent disastrous complications following major head and neck surgery. Am J Med, 1985 Aug, 79(2), 256 - 8 Clostridium septicum septicemia with identical metastatic myonecroses in a granulocytopenic patient . Infectious disease emergency; Tikko SK et al.; Clostridium septicum is a gram-positive, sporulating spindle-shaped rod . Gas gangrene secondary to trauma is not uncommon . However, nontraumatic clostridial infection causing myonecrosis is quite unusual . This is a unique case report of Clostridium septicum bacteremia with two simultaneously evolving metastatic foci of myonecrosis of the left arm and right thigh that developed in a patient with lymphoma when he became granulocytopenic during his hospital course. J Med Microbiol, 1985 Aug, 20(1), 17 - 26 The antagonism of tetracycline and ferric iron in vivo; Miles AA et al.; To test the hypothesis that the in-vivo antibiotic action of tetracycline might be affected by ferric iron and the enhancement of infection by ferric iron by tetracycline, the actions of intraperitoneal antibiotic and local ferric ammonium citrate, given separately and together, were measured in the dorsal skin of guinea-pigs bearing lesions due to staphylococci, streptococci, a Proteus sp., an Erysipelothrix sp., Clostridium perfringens, Pseudomonas aeruginosa, Aeromonas hydrophila and Klebsiella pneumoniae . Tetracycline, given in two intraperitoneal doses of 25 mg/kg at 0 and 2 h after intracutaneous challenge, maintained plasma concentrations of 4-6 micrograms/ml for more than the first 4 h of infection, after which the local lesions had become largely insusceptible to the antibiotic . The intracutaneous injection of Fe 10 micrograms in a volume of 0.1 ml containing the bacteria was sufficient to enhance infection by those strains susceptible to this effect . The in-vivo efficacy of tetracycline was not always related to low MIC; a low MIC was sometimes associated with little action and a high MIC with moderate action . Sixteen organisms were tested . The iron diminished the tetracycline effect only feebly with one staphylococcal strain and the strain of E . rhusiopathiae . In only one case, with a strain of Proteus sp., was the tetracycline action grossly diminished . On the other hand, tetracycline diminished the enhancement effect of iron moderately with three strains of staphylococci and one strain each of K . pneumoniae, P . aeruginosa and C . perfringens, and strongly with two strains of staphylococci, a group-C streptococcus and one strain each of K . pneumoniae, E . rhusiopathiae and A . hydrophila . It is evident that the diminution of tetracycline action by moderate excess of readily available Fe , whether endogenous or administered, is an unlikely event (three instances among the 16 tested) whereas the diminution of the infection-enhancing effect of iron by tetracycline is much more likely (12 instances among the 16) . Insofar as a decrease in iron available for enhancement of infection is valid evidence of a diminution of the iron available for necessary physiological processes of the subject treated, our results suggest that these processes might be affected by tetracycline. J Bacteriol, 1985 Aug, 163(2), 552 - 9 Organization and distribution of the cellulosome in Clostridium thermocellum; Bayer EA et al.; The properties of the cellulosome (the cellulose-binding, multicellulase-containing protein complex) in Clostridium thermocellum were examined by comparing the cellulase systems derived from the wild type and an adherence-defective mutant . The growth conditions--specifically, growth either on cellulose (Avicel) or on cellobiose as insoluble or soluble carbon sources, respectively--were found to be critical to the distribution of the cellulosome in the mutant system: the cellobiose-grown mutant (in contrast to the wild type) lacked the cellulosome on its surface and produced only minor quantities of the extracellular cellulosome accompanied by other relatively low-molecular-weight cellulases . The polypeptide composition of the respective purified cellulosome was dependent on the nature of the carbon source and was similar for both wild-type and mutant cells . Ultrastructural analysis revealed the presence of novel polycellulosomal protuberances on the cell surface of the cellobiose-grown wild type which were absent in the mutant. Appl Environ Microbiol, 1985 Aug, 50(2), 274 - 9 Inhibition of germinant binding by bacterial spores in acidic environments; Blocher JC et al.; Commitment to germinate occurred in both Clostridium botulinum and Bacillus cereus spores during 0.5 min of exposure to 100 mM L-alanine or L-cysteine, measured by the inability of germination inhibitors (D form of amino acid) to inhibit germination . Spore germination at pH 4.5 was inhibited because the germinant did not bind to the trigger sites . C . botulinum spores exposed to 100 mM L-alanine or L-cysteine at pH 4.5 remained sensitive to D-amino acid inhibition at pH 7, indicating that no germinants had bound to the trigger site at pH 4.5 . Inhibition of germinant binding at pH 4.5 was reversible but lagged in commitment to germinate upon transfer to pH 7 . Spores sequentially exposed to pH 4.5 buffer and pH 7 buffer with the germinant also demonstrated a lag in commitment to germinate . The pH at which binding was inhibited was not significantly affected by composition of the buffer or by reduced germinant concentrations (10 mM) . Nonspecific uptake of L-{3H}alanine by C . botulinum spores was not inhibited at pH 4.5 . Inhibition of germinant binding in acidic environments appeared to be due to protonation of a functional group in or near the trigger site . This may represent a general mechanism for inhibition of spore germination in acidic environments. Infect Immun, 1985 Aug, 49(2), 452 - 4 Kinetics of growth and toxigenicity of Clostridium botulinum in experimental wound botulism; Dezfulian M et al.; An animal model of wound botulism was developed in mice using an inoculum of Clostridium botulinum type A spores . The number of C . botulinum in infected wounds was quantitated by culturing on egg yolk agar, and the level of C . botulinum toxin in infected wound tissue was measured by a bioassay in mice and by an enzyme-linked immunosorbent assay . All infected mice receiving no further treatment developed neuroparalytic symptoms consistent with botulism after an incubation period of ca . 48 h, and all of these animals died . Serotherapy with C . botulinum type A antitoxin initiated 24 h postchallenge reduced the mortality rate to 5% . Treatment with metronidazole 2 to 24 h postchallenge resulted in recovery rates of 40 to 91%. J Gen Microbiol, 1985 Aug, 131 ( Pt 8), 2097 - 105 Identification of restriction fragments from two cryptic Clostridium butyricum plasmids that promote the establishment of a replication-defective plasmid in Bacillus subtilis; Collins ME et al.; Clostridium butyricum NCIB 7423 carries two cryptic plasmids, pCB101 (6.05 kbp) and pCB102 (7.8 kbp) . Sites for the restriction enzymes EcoRI, EcoRV, HindIII, ClaI and PstI have been found in one or both of these plasmids and their relative positions determined . Restriction fragments from both plasmids have been inserted into a vector plasmid (pJAB1) that is able to replicate in Escherichia coli but not in Bacillus subtilis and the recombinant plasmids have been established in E . coli . A 3.3 kbp Sau3A fragment of pCB101 conferred upon the vector the ability to transform both Rec+ and Rec- strains of B . subtilis . Plasmid pRB1, a representative chimaera carrying only the 3.3 kbp Sau3A fragment of pCB101, was successfully transferred from B . subtilis back to E . coli . Plasmid pRB1 was readily lost from B . subtilis in the absence of selection . This evidence, together with the results of hybridization experiments, suggests that pRB1 is present as a weakly replicating autonomous element in B . subtilis . A recombinant plasmid carrying a 2.0 kbp Sau3A fragment of pCB102 underwent integration into the B . subtilis chromosome. Biomed Mass Spectrom, 1985 Aug, 12(8), 359 - 63 Gas chromatography/mass spectrometry of bacterial amines; Tavakkol A et al.; Bacterial amines were examined by gas chromatography/mass spectrometry . Under electron impact all trifluoroacetamides exhibited peaks at m/z 69 due to {CF3}+ . Many trifluoroacetamides also showed peaks at m/z 97 corresponding to the {COCF3}+ ion fragment . The spectra of n-alkyl and aralkyl trifluoroacetamides were consistent with the spectra and their interpretations in the earlier literature . Molecular ions were of low abundance for all alkyl trifluoroacetamides having alkyl chains longer than two carbon atoms . Chemical ionization gave molecular weight information in all cases . Most peaks observed were molecular addition products, e.g . {M + H}+ and {M + NH4}+ . Application of chemical ionization mass spectrometry to analysis of bacterial amines revealed the production of beta-phenylethylamine, n-decylamine, 1,4-diaminobutane and 1,5-diaminopentane by Clostridium histolyticum; whereas both Clostridium bifermentans and Clostridium oedematiens produced beta-phenylethylamine . The latter organism also produced a peak with a retention time similar to that of an authentic amylamine derivative. Appl Environ Microbiol, 1985 Aug, 50(2), 249 - 56 Activation and injury of Clostridium perfringens spores by alcohols; Craven SE et al.; The activation properties of Clostridium perfringens NCTC 8679 spores were demonstrated by increases in CFU after heating in water or aqueous alcohols . The temperature range for maximum activation, which was 70 to 80 degrees C in water, was lowered by the addition of alcohols . The response at a given temperature was dependent on the time of exposure and the alcohol concentration . The monohydric alcohols and some, but not all, of the polyhydric alcohols could activate spores at 37 degrees C . The concentration of a monohydric alcohol that produced optimal spore activation was inversely related to its lipophilic character . Spore injury, which was manifested as a dependence on lysozyme for germination and colony formation, occurred under some conditions of alcohol treatment that exceeded those for optimal spore activation . Treatment with aqueous solutions of monohydric alcohols effectively activated C . perfringens spores and suggests a hydrophobic site for spore activation. Appl Environ Microbiol, 1985 Aug, 50(2), 202 - 6 Development of a minimal medium for Clostridium perfringens by using an anaerobic chemostat; Goldner SB et al.; A minimal medium was developed for the cultivation of Clostridium perfringens in an anaerobic chemostat . Cultures of C . perfringens ATCC 3624 and NCTC 10240 were grown at 46 and 43 degrees C, respectively, in a glucose-limited, chemically defined medium at pH 7.2 . The concentrations of amino acids, minerals, nucleotides, and vitamins, initially present in excess, were varied independently . The minimum concentration of each nutrient which would support 3 X 10(8) CFU/ml with a generation time of less than 40 min was determined and used to develop a reformulated defined medium . Atomic absorption spectroscopy and amino acid analyses of the reformulated medium indicated additional adjustments in nutrient content which led to the development of a minimal medium for each strain . The nutritional profile for each strain was similar . A decrease in the concentration of arginine, histidine, and tyrosine for strain 3624 and of arginine, histidine, and isoleucine for strain 10240 resulted in an increase in the optical density of each culture. Biochem Biophys Res Commun, 1985 Jul 31, 130(2), 904 - 9 The cellulolytic enzyme complex of Clostridium thermocellum is very large; Coughlan MP et al.; The cellulolytic enzyme system bound to cellulose during the early stages of growth of C . thermocellum on this substrate was resolved into two major complexes . These complexes, as viewed by electron microscopy, are spherical particles with diameters of 210 A and 610 A and calculated molecular weights of 4.2 million and 102 million daltons, respectively. Vet Rec, 1985 Jul 20, 117(3), 58 - 60 Diagnosis and treatment of botulism in lions; Greenwood AG; Six circus lions (Panthera leo) showed neurological and gastrointestinal signs after consuming casualty broiler chickens . Signs included ataxia, hindlimb paralysis and recumbency . Neurological examination of two affected males showed paralysis of extraocular muscles, fixed dilated pupils and inability to swallow . Replacement fluids and antibiotics were given and Clostridium botulinum type C antitoxin was found in serum samples . Type C antitoxin was not then available and therapy was started in one lioness with guanidine hydrochloride . Convulsions were controlled by diazepam but this animal died . One of the two males was given type C antitoxin; both were given anabolic steroids . All the remaining animals made slow recoveries over varying periods; one lion was recumbent for 41 days . No lion developed respiratory paralysis; other animals which had consumed the chickens remained healthy . Aspects of the treatment of botulism in animals are discussed. Biochim Biophys Acta, 1985 Jul 9, 835(2), 304 - 14 Loss of stereospecificity of phospholipases C and D upon introduction of a 2-alkyl group into rac-1,2-diacylglycero-3-phosphocholine; Bugaut M et al.; rac-1-{1-14C}Lauroyl-2-oleylglycero-3-phospho{methyl-3H}choline and rac-1-lauroyl-2-{1-14C}oleoylglycero-3-phospho{methyl-3H}choline along with rac-1-palmitoyl-2-oleylglycero-3-phosphocholine and sn-1-palmitoyl-2-oleylglycero-3-phosphocholine were synthesized and subjected to hydrolysis with phospholipase C (EC 3.1.4.3) from Clostridium perfringens and phospholipase D (EC 3.1.4.4) from cabbage . Kinetics of hydrolysis of the radioactive substrates were determined by measuring the 3H radioactivity retained in the aqueous phase due to free choline and phosphocholine and the 3H and 14C radioactivity recovered in the organic phase due to the released diacylglycerols and phosphatidic acids and the residual phosphatidylcholines . The rate of hydrolysis of the unlabelled substrates by phospholipase C was determined by thin-layer chromatography and gas-liquid chromatography of the methanolysis products . The relative initial rates of hydrolysis of sn-1,2,- and sn-2,3-enantiomers were 100-200:1 for phospholipase C and 40-50:1 for phospholipase D using rac-1-lauroyl-2-oleoylglycero-3-phosphocholine as the substrate . The substitution of the 2-acyl group by an alkyl group resulted in a loss of stereospecificity, which was partial for phospholipase C (relative rates equal to 8-13:1) and total for phospholipase D . There was a parallel dramatic decrease (500-1000-fold) in the initial rate of hydrolysis with phospholipase C but the activity of phospholipase D was only moderately reduced (18-fold) . These findings are consistent with the earlier observed loss of the stereospecificity of lipoprotein lipase following introduction of a 2-alkyl group into triacylycerols, and point to a general unsuitability of 2-alkyl-linked acylglycerols as substrates for the assay of the stereospecificity of lipases, as well as for the isolation of enantiomeric 2-alkylacylglycerols by means of stereospecific lipases. J Gen Microbiol, 1985 Jul, 131 ( Pt 7), 1697 - 703 Taxonomic position of lecithinase-negative strains of Clostridium sordellii; Popoff MR et al.; Eleven out of 43 strains of Clostridium sordellii from clinical sources did not produce lecithinase activity and were not toxic to mice . However, these strains did belong to the C . sordellii group and could readily be differentiated from C . bifermentans and C . difficile on the basis of DNA-DNA homologies, carbohydrate fermentation patterns, enzyme activities, GLC analysis of fatty acid fermentation products and the electrophoretic analysis of whole cell protein extracts. Arch Microbiol, 1985 Jul, 142(2), 128 - 35 A sodium ion gradient as energy source for Peptostreptococcus asaccharolyticus; Wohlfarth G et al.; The determination of enzymatic activities in cell-free extracts of Acidaminococcus fermentans and Peptostreptococcus asaccharolyticus led to a refined scheme for the pathway of glutamate fermentation via (R)-2-hydroxyglutarate to acetate and butyrate . From the ratio of these products the amount of ATP generated by substrate level phosphorylation was calculated . Growth experiments with the organisms including Clostridium symbiosum and Clostridium tetanomorphum indicated that a sodium gradient contributed additional energy for growth . The high growth yields found in organisms containing the biotin dependent sodium pump glutaconyl-CoA decarboxylase could be reduced by the sodium ionophor monensin . In P . asaccharolyticus energy equivalent up to 0.6 mol ATP per mol of glutaconyl-CoA decarboxylated was conserved via the Na+ gradient . The data may explain the growth promoting effects of monensin in cattle. J Assoc Off Anal Chem, 1985 Jul-Aug, 68(4), 626 - 31 Gas chromatographic detection of D-(-)-2,3-butanediol and butyric acid produced by sporeformers in cream-style corn and canned beef noodle soup: collaborative study; Schafer ML et al.; A gas chromatographic method that identifies sporeformers as the cause of spoilage in swollen cans of low-acid foods was collaboratively studied in 2 stages . Two organic compounds produced by sporeformers, D-(-)-2,3-butanediol and butyric acid, are measured in the upper phase after centrifugation of the liquid portion of the can contents . Each sample is assayed on 2 packed columns designed for the assay of aqueous solutions of volatile fatty acids, using flame ionization detectors . For study 1, 16 duplicate inoculated cans of cream-style corn and beef noodle soup were sent to 9 collaborators . For study 2, 7 collaborators received 11 duplicate inoculated cans of the 2 foods . Duplicate uninoculated cans of each food served as negative controls . The inocula were 6 sporeforming organisms (4 Clostridium and 2 gas-forming Bacillus species) and 2 nonsporeformers . After the deletion of marginal samples, the percentages of correctly identified sporeformers and nonsporeformers in beef noodle soup were 83 (110/132) and 90 (54/60), respectively; corresponding percentages for cream-style corn were 80 (98/123) and 100 (35/35) . The method has been adopted official first action. J Antimicrob Chemother, 1985 Jul, 16 Suppl A, 137 - 49 Evolution and epidemiology of MLS resistance; Duval J; Within the framework of this symposium, it is not feasible to present an exhaustive description of the present state of knowledge regarding the sensitivity and resistance of bacterial species to macrolides, lincosamides and streptogramins (MLS) . This paper is limited to a description of the evolution of different types of resistance in the light of decisive factors described in previous papers, in order to deduce, if at all possible, trends in future strategy in therapeutics . Only acquired resistance lends itself to epidemiological study, in contrast to natural resistance which is, by definition, characteristic of a species or a genus, and not liable to change . Three groups will therefore be studied in turn: Staphylococcus aureus, streptococci and Bacteroides fragilis . There is as yet insufficient accumulated data to draw conclusions regarding the epidemiology and evolution of MLSB resistance observed in Clostridium perfringens and Corynebacterium diphtheriae, or regarding the high-level resistance to erythromycin due to enzymatic inactivation recently described in Escherichia coli. Appl Environ Microbiol, 1985 Jul, 50(1), 63 - 7 Monoclonal antibody-based immunoassay for type A Clostridium botulinum toxin is comparable to the mouse bioassay; Shone C et al.; A monoclonal antibody (BA11) has been produced against Clostridium botulinum type A neurotoxin by the fusion of myeloma cells (P3 NS1/1-Ag4-1) with spleen cells from BALB/c mice immunized with botulinum type A neurotoxoid . The antibody bound specifically to botulinum type A neurotoxin, showing no cross-reactivity with types B and E botulinum toxins or with any of several other bacterial toxins tested . The monoclonal antibody did not bind to botulinum type A neurotoxin which had been denatured with sodium dodecyl sulfate and bound only weakly to each of the separated heavy and light subunits of the neurotoxin, suggesting a conformational requirement for the antigenic determinant of the antibody . A sensitive immunoassay for C . botulinum type A toxin with monoclonal antibody BA11 in conjunction with an enzyme amplication system has been developed which allows detection of 5 to 10 mouse 50% lethal doses ml-1 of purified neurotoxin . The assay was equally sensitive when applied to the detection of crude toxin in food stuffs; the average value for the minimum level of detectable toxin in extracts of tinned salmon or corned beef was 9 +/- 3.1 mouse 50% lethal doses ml-1. Rev Infect Dis, 1985 Jul-Aug, 7 Suppl 3, S417 - 25 In vitro activity of imipenem against anaerobic bacteria; Wexler HM et al.; The in vitro activity of imipenem, metronidazole, clindamycin, moxalactam, and cefoxitin against 203 strains of anaerobic bacteria isolated from patients at the Veterans Administration Wadsworth Medical Center in Los Angeles was studied . Imipenem and metronidazole were the most active agents overall, inhibiting 98% and 99%, respectively, of all anaerobes tested . At breakpoint levels all of the agents tested were very active against anaerobic cocci . Clostridium perfringens, and Bacteroides species other than those of the Bacteroides fragilis group . Imipenem, metronidazole, and clindamycin were the most active agents against the B . fragilis group in this study, although more recent experience with clindamycin indicates less potency . Marked variation among the susceptibility results obtained at various centers may be due to differences in technique, including inoculum size, media, and incubation time . In all instances, however, imipenem has clearly been the most active of the beta-lactam agents. Curr Eye Res, 1985 Jul, 4(7), 803 - 6 Inhibition of collagenase activity by extracts of bovine ocular tissues; Harper J et al.; Bovine eyes were dissected and separate pools of lens, lens capsule, cornea and vitreous were extracted in guanidine, subjected to ultrafiltration, and examined for their effects on collagenolytic activity . Although lens extract was not inhibitory, the cornea and vitreous both