Clostridia

Nippon Juigaku Zasshi, 1989 Jun, 51(3), 582 - 6
Implication of coprophagy in pathogenesis of chicken botulism; Hyun SH et al.; Oral administration of 1 x 10(7) viable spores of Clostridium botulinum type C killed the chickens kept on a board floor to allow them coprophagy, whereas the same dose of the spores failed to develop symptoms in those kept on a wire-net floor not to allow them coprophagy . Type C toxin was detected in the cecal droppings of the chickens of both the groups after feeding the spores and also in serum of symptomatic as well as asymptomatic chickens kept on a board floor . Thus, coprophagy, by which chickens ingest type C toxin (C1 L toxin) and the bacterial cells, seems to be a prerequisite for development of chicken botulism.

J Antimicrob Chemother, 1989 Jun, 23(6), 929 - 31
Extended spectrum cephalosporins and Clostridium difficile; Golledge CL et al.; There is little information about how commonly the newer cephalosporins cause diarrhoea due to Clostridium difficile . In this study of 111 patients with C . difficile-associated diarrhoea, 106 had received antimicrobial agents in the four weeks before detection of C . difficile . The relative risk for each antimicrobial agent was greatest with clindamycin, followed by cefotaxime, cephamandole and ceftriaxone . There was no statistically significant difference in risk between the cephalosporins evaluated . Narrower spectrum penicillins, anti-pseudomonal penicillins and aminoglycosides were not potent inciting agents.

J Bacteriol, 1989 Jun, 171(6), 2925 - 32
Purification and characterization of a novel form of 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens; Krafft AE et al.; We have purified a steroid-inducible 20 alpha-hydroxysteroid dehydrogenase from Clostridium scindens to apparent homogeneity . The final enzyme preparation was purified 252-fold, with a recovery of 14% . Denaturing and nondenaturing polyacrylamide gradient gel electrophoresis showed that the native enzyme (Mr, 162,000) was a tetramer composed of subunits with a molecular weight of 40,000 . The isoelectric point was approximately pH 6.1 . The purified enzyme was highly specific for adrenocorticosteroid substrates possessing 17 alpha, 21-dihydroxy groups . The purified enzyme had high specific activity for the reduction of cortisone (Vmax, 280 nmol/min per mg of protein; Km, 22 microM) but was less reactive with cortisol (Vmax, 120 nmol/min per mg of protein; Km, 32 microM) at pH 6.3 . The apparent Km for NADH was 8.1 microM with cortisone (50 microM) as the cosubstrate . Substrate inhibition was observed with concentrations of NADH greater than 0.1 mM . The purified enzyme also catalyzed the oxidation of 20 alpha-dihydrocortisol (Vmax, 200 nmol/min per mg of protein; Km, 41 microM) at pH 7.9 . The apparent Km for NAD+ was 526 microM . The initial reaction velocities with NADPH were less than 50% of those with NADH . The amino-terminal sequence was determined to be Ala-Val-Lys-Val-Ala-Ile-Asn-Gly-Phe-Gly-Arg . These results indicate that this enzyme is a novel form of 20 alpha-hydroxysteroid dehydrogenase.

J Gen Microbiol, 1989 Jun, 135 ( Pt 6), 1755 - 62
Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli; Melasniemi H et al.; An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector . The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E . coli . Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities . Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000 . Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host . Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.

Arch Dis Child, 1989 Jun, 64(6), 871 - 2
Infantile botulism; Smith GE et al.; A 4 month old boy presented with respiratory difficulty and hypotonia . Clostridium botulinum and its toxin were isolated from his faeces and he had electromyographic changes typical of infantile botulism . This is only the second case in the United Kingdom: unfamiliarity with the presentation could result in misdiagnosis.

Anal Biochem, 1989 Jun, 179(2), 341 - 6
Determination of ursodeoxycholic acid in serum by a new fluorometric enzymatic method using 7 beta-hydroxysteroid dehydrogenase from Clostridium absonum; Lianidou ES et al.; A fluorometric enzymatic method for the determination of ursodeoxycholic acid (UDCA) and its glycine and taurine conjugates in human serum has been developed . A simple and fast purification and preconcentration procedure using Sep Pak C18 cartridges was employed for the UDCA extraction from human serum . UDCA and its conjugates were determined in the extracted sample by an equilibrium method based on the enzymatic conversion of the 7 alpha-hydroxy group into 7-oxo group by beta-nicotinamide adenine dinucleotide phosphate in the presence of 7 beta-hydroxysteroid dehydrogenase (7 beta-HSD) and the produced NADPH was monitored fluorometrically . The 7 beta-HSD, which is not yet commercially available, was isolated from Clostridium absonum cultures (ATCC No . 27555) and purified by affinity chromatography . The method has a limit of detection of 0.8 microM in serum and the precision varied from 6.1 to 2.0% for low and high concentrations, respectively . The recovery of UDCA from serum samples was about 99% (range 85-105%) . The method was successfully applied to UDCA determination in serum samples from patients treated with UDCA for primary biliary cirrhosis.

Appl Environ Microbiol, 1989 Jun, 55(6), 1544 - 8
Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine; Patterson-Curtis SI et al.; Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A . Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine . Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine . Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation . In contrast, certain other nutritional conditions stimulated protease and toxin formation in C . botulinum and counteracted the repression by arginine . Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers . Casein supplementation of a medium containing high levels of arginine prevented protease inactivation . High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine . These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C . botulinum.

Helv Paediatr Acta, 1989 Jun, 43(5-6), 521 - 30
{Botulism in infancy}; Gautier E et al.; The authors describe a case of botulism in a 3-month-old infant infected with Clostridium botulinum type A . Symptomatology developed within four days, persisted for two weeks, then regressed . Symptoms were paresis of face muscles, hyporeactive pupils, loss of succion and deglutition, axial hypotonia, weakness of peripheral muscles, lability of the autonomic nervous system with acute episodes of bradycardia and constipation . Anomalies of the electroen-cephalogram and of the auditory evoked responses suggest that the toxin penetrated the central nervous system . Treatment was symptomatic, without need for assisted ventilation . It was not possible to detect the source of infection.

Epidemiol Infect, 1989 Jun, 102(3), 467 - 71
The production of Clostridium botulinum toxin in mammalian, avian and piscine carrion; Smith GR et al.; Mice, birds (chicks, quail) and fish (rudd, goldfish) killed shortly after receiving 1300-2000 spores of Clostridium botulinum per os were incubated, usually at 23 degrees C for 7 days . A 10% (w/v) homogenate of each rotting carcass was then prepared, sterilized by membrane filtration, and assayed for toxin . In mouse carcasses a type C strain of C . botulinum usually produced greater than 2 X 10(5) mouse intraperitoneal LD/g; in fish carcasses it usually produced less--often much less--than 2 X 10(4) LD/g . Avian carcasses appeared to be intermediate between those of mice and fish in their ability to support toxigenesis . A type E strain of C . botulinum, unlike type C, produced toxin equally well in fish and mouse carrion, usually at a concentration of between 2 X 10(4) and 2 X 10(5) LD/g.

Showa Shigakkai Zasshi, 1989 Jun, 9(2), 97 - 102
Novel method for the preparation of sterile collagen-agarose-plates for isolating collagenolytic bacteria; Takahashi M et al.; An assay method for bacterial collagenase using sterile collagen-agarose plates was developed to isolate collagenolytic bacteria . In order to avoid heat-denaturation of collagen, the plates were made at 37 degrees C mixing microwave-sterilized collagen with low melting point-agarose . Sensitivities to various proteases were tested on the plates; it was shown that whereas the collagen in these plates was digested only by the collagenases, the collagen could also be hydrolyzed by other proteolytic enzymes if it had been either sterilized by ethylene oxide or solidified with conventional agar at a higher temperature . Colonies of Clostridium histolyticum cells grown on the plates had clear zones around them and their collagenase activities could be readily detected.

Food Chem Toxicol, 1989 Jun, 27(6), 399 - 402
Phospholipase C-mediated intestinal mucosal damage is ameliorated by quinacrine; Otamiri T; Phospholipase C from Clostridium perfringens, when injected into a closed loop of the rat small intestine in vivo, caused an increase in the activity of intraluminal N-acetyl-beta-glucosaminidase and mucosal permeability to sodium fluorescein, indicating damage to the mucosa . Phospholipase C also caused an influx of granulocytes (neutrophils) into the mucosa, as shown by the myeloperoxidase activity--a granulocyte neutrophil marker, and increased localized lipid peroxidation . Pretreatment of animals with quinacrine, a known inhibitor of phospholipase A2, prevented the increases in the luminal N-acetyl-beta-glucosaminidase activity, mucosal permeability, malondialdehyde and myeloperoxidase activity after deposition of phospholipase C in the gut lumen . It is concluded that phospholipase C might impair the function of the mucosal barrier and increase the permeability of the gut to undesirable molecules and pathogens . Part of its action may be mediated via phospholipase A2 activation since pretreatment with quinacrine afforded protection.

Eur J Clin Microbiol Infect Dis, 1989 Jun, 8(6), 550 - 1
Comparative antibacterial activity of the new cephalosporin cefcanel against anaerobic bacteria; Nord CE et al.; The activity of cefcanel against anaerobic cocci, Clostridium perfringens, Bacteroides fragilis, Bacteroides spp . and fusobacteria was determined by the agar dilution method and compared with the activity of cefaclor, cephalexin, cefadroxil, phenoxymethylpenicillin and ampicillin . Cefcanel showed good activity against Clostridium perfringens, Bacteroides spp . and fusobacteria (MIC90 = 1-4 mg/l) . Against anaerobic cocci its MIC90 value was 16 mg/l, and against Bacteroides fragilis, 32 mg/l . Cefcanel has an antibacterial activity that warrants investigation in clinical trials.

J Bacteriol, 1989 Jun, 171(6), 3433 - 9
A novel and remarkably thermostable ferredoxin from the hyperthermophilic archaebacterium Pyrococcus furiosus; Aono S et al.; The archaebacterium Pyrococcus furiosus is a strict anaerobe that grows optimally at 100 degrees C by a fermentative-type metabolism in which H2 and CO2 are the only detectable products . A ferredoxin, which functions as the electron donor to the hydrogenase of this organism was purified under anaerobic reducing conditions . It had a molecular weight of approximately 12,000 and contained 8 iron atoms and 8 cysteine residues/mol but lacked histidine or arginine residues . Reduction and oxidation of the ferredoxin each required 2 electrons/mol, which is consistent with the presence of two {4Fe-4S} clusters . The reduced protein gave rise to a broad rhombic electronic paramagnetic resonance spectrum, with gz = 2.10, gy = 1.86, gx = 1.80, and a midpoint potential of -345 mV (at pH 8) . However, this spectrum represented a minor species, since it quantitated to only approximately 0.3 spins/mol . P . furiosus ferredoxin is therefore distinct from other ferredoxins in that the bulk of its iron is not present as iron-sulfur clusters with an S = 1/2 ground state . The apoferredoxin was reconstituted with iron and sulfide to give a protein that was indistinguishable from the native ferredoxin by its iron content and electron paramagnetic resonance properties, which showed that the novel iron-sulfur clusters were not artifacts of purification . The reduced ferredoxin also functioned as an electron donor for H2 evolution catalyzed by the hydrogenase of the mesophilic eubacterium Clostridium pasteurianum . P . furiosus ferredoxin was resistant to denaturation by sodium dodecyl sulfate (20%, wt/vol) and was remarkably thermostable . Its UV-visible absorption spectrum and electron carrier activity to P . furiosus hydrogenase were unaffected by a 12-h incubation of 95 degrees C.

Gene, 1989 May 30, 78(2), 355 - 64
Molecular analysis and nucleotide sequence of the adh1 gene encoding an NADPH-dependent butanol dehydrogenase in the Gram-positive anaerobe Clostridium acetobutylicum; Youngleson JS et al.; The nucleotide sequence of a 2081-bp fragment of Clostridium acetobutylicum DNA containing the adh1 gene was determined . The butanol dehydrogenase gene is referred to as the adh1 gene since it was shown to have activity using butanol and ethanol as substrates . The adh1 gene consisted of 1164 bp and encoded an alcohol dehydrogenase (ADH) enzyme of 388 aa residues with an Mr of 43,274 . The adh1 gene was separated from an upstream open reading frame by an intergenic region of 354 bp . No promoter consensus sequences were identified in the intergenic upstream region and the adh1 gene did not appear to be expressed off its own promoter in Escherichia coli . Three separate types of ADH have been recognized . The ADH1 from C . acetobutylicum exhibited 39% homology with the Fe-containing ADH2 from Zymomonas mobilis and 37% homology with the ADH4 from Saccharomyces cerevisiae, but showed little or no homology with the other characterised types of ADH.

Biochemistry, 1989 May 30, 28(11), 4675 - 80
Kinetic characterization of the {3'-32P}coenzyme A/acetyl coenzyme A exchange catalyzed by a three-subunit form of the carbon monoxide dehydrogenase/acetyl-CoA synthase from Clostridium thermoaceticum; Ramer SE et al.; The ability of acetyl coenzyme A synthesizing carbon monoxide dehydrogenase isolated from Clostridium thermoaceticum to catalyze the exchange of {3'-32P}coenzyme A with acetyl coenzyme A is studied . This exchange is found to have a rate exceeding that of the acetyl coenzyme A carbonyl exchange also catalyzed by CO dehydrogenase ({1-14C}acetyl coenzyme A + CO in equilibrium acetyl coenzyme A + 14CO) . These two exchanges are diagnostic of the ability of CO dehydrogenase to synthesize acetyl coenzyme A from a methyl group, coenzyme A, and carbon monoxide . The kinetic parameters for the coenzyme A exchange have been determined: Km(acetyl coenzyme A) = 1500 microM, Km(coenzyme A) = 50 microM, and Vmax = 2.5 mumol min-1 mg-1 . Propionyl coenzyme A is shown to be a substrate (Km approximately 5 mM) for the coenzyme A exchange, with a rate 1/15 that of acetyl coenzyme A, but is not a substrate for the carbonyl exchange . CO dehydrogenase capable of catalyzing both these two exchanges, and the oxidation of CO to CO2, is isolated as a complex of molecular weight 410,000 consisting of three proteins in an alpha 2 beta 2 gamma 2 stoichiometry . The proposed gamma subunit, not previously reported as part of CO dehydrogenase, copurifies with the enzyme and has the same molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as the disulfide reductase previously separated from CO dehydrogenase in a final chromatographic step.

Vet Rec, 1989 May 27, 124(21), 558 - 60
Type C botulism in cattle being fed ensiled poultry litter; Neill SD et al.; A botulinum toxin from ensiled poultry litter which caused a major outbreak of bovine botulism was characterised as type C1 . The litter produced transient ataxia when fed to two experimental calves and the clinical signs were accompanied by a transient appearance of serum toxin . Type C1 toxin was demonstrated in muscle tissues which had been taken during the outbreak from an affected animal with high circulating serum toxin, and held frozen for seven months . Clostridium botulinum type C organisms were demonstrated in faeces from another affected animal and also in kidney tissue from a third animal . These observations have implications for the diagnosis and management of future outbreaks of botulism and for the potential health risk from the meat of affected animals.

Lancet, 1989 May 27, 1(8648), 1156 - 60
Bacteriotherapy for chronic relapsing Clostridium difficile diarrhoea in six patients; Tvede M et al.; Six patients with chronic relapsing diarrhoea caused by Clostridium difficile were treated with rectal instillation of homologous faeces (one patient) or a mixture of ten different facultatively aerobic and anaerobic bacteria diluted in sterile saline (five patients) . The mixture led to a prompt loss of Cl difficile and its toxin from the stools and to bowel colonisation by Bacteroides sp, which had not been present in pre-treatment stool samples . Strains of Escherichia coli, Cl bifermentans, and Peptostreptococcus productus in the mixture inhibited the in-vitro growth of Cl difficile, which in turn inhibited the growth of Bacteroides ovatus, Bacteroides vulgatus, and Bacteroides thetaiotaomicron . The finding that Bacteroides sp had been absent during the patients' illness but was present after recovery suggests that the absence of Bacteroides sp may result in chronic relapsing Cl difficile diarrhoea, and that its presence may prevent colonisation by Cl difficile.

Wien Med Wochenschr, 1989 May 15, 139(9), 202 - 6
{Diarrhea induced by antibiotics}; Mittermayer H; The most frequent cause of antibiotic-associated colitis is Clostridium difficile . This gram-positive, spore-forming anaerobic bacillus releases toxins, which produce diarrhea and damage the colonic mucosa . Endoscopy shows a wide range of alterations, "unspecific colitis" with reddening or edema, ulcerations or at the worst pseudomembranous colitis . Nearly all antibiotics are able to trigger Clostridium difficile colitis . An enhanced risk is exerted by broad spectrum substances, which act also on the anaerobic flora protecting the gastrointestinal tract from unphysiological colonization . Clusters of cases were observed in hospitalized patients . The patients risk factors coincide with the administration of antibiotics . Furthermore Clostridium difficile is likely to be spread as a nosocomial infection in many instances . Less often colitis is observed in connection with oral antibiotics outside the hospital . However, substantial underreporting of cases has to be considered . Clinical symptoms usually start 4 to 10 days after first administration of the antibiotic . Leading symptoms are frequent profuse watery stools . Abdominal cramps and tenderness as well as fever and leukocytosis are common . Intense symptoms can simulate serious conditions like perforation . Upon clinical suspicion the diagnosis is made by endoscopy, stool culture and possibly demonstration of toxin . The predictive value of the stool culture equals that of toxin detection . In adult patients there is a good correlation between positive stool culture and clinical presentation . Infants can carry Clostridium difficile as part of their normal flora, therefore positive stool culture or toxin detection in an infant cannot necessarily be linked to clinical symptoms . In some cases Clostridium difficile has to be regarded as etiologic organism also in infants.(ABSTRACT TRUNCATED AT 250 WORDS)

J Chromatogr, 1989 May 5, 490(1), 91 - 100
Clostridium difficile toxin B: characterization and sequence of three peptides; Bisseret F et al.; The cytotoxin, also named toxin B, was isolated from a toxigenic strain of Clostridium difficile, purified to homogeneity and partially characterized . The purification procedure included ultrafiltration followed by anion-exchange chromatography . We noticed that a non-specific nucleic material eluted with the protein during the purification . The presence of these nucleic acids appeared to be important for the toxic activity of the protein . Some characteristics of the cytotoxin were examined, especially the amino acid composition and the sequence of three tryptic fragments.

East Afr Med J, 1989 May, 66(5), 319 - 23
Feacal carriage of cytotoxigenic strains of Clostridium difficile by adult Nigerians; Rotimi VO et al.; The isolation rate of Clostridium difficile and the presence of its cytotoxin in stool were studied in adults with diarrhoea and healthy normal individuals over a period of one year . C . difficile was isolated from 23(56%) out of 41 patients with diarrhoea who gave history of antibiotic exposure prior to the development of the disease from 12(31.6%) of the 38 healthy controls with no history of antibiotic exposure . Of the 23(31.6%) C . difficile isolated from the patients, 16(69.6%), and only 8(66.6%) of the 12 isolates from the healthy adults, were cytotoxigenic . Only one stool specimen from a diarrhoeic patient, was positive for cytotoxin assay . The clinical significance of this finding is discussed.

J Clin Microbiol, 1989 May, 27(5), 889 - 93
Evaluation of a latex agglutination test for Clostridium difficile in two nursing home outbreaks; Bennett RG et al.; The Culturette Brand Clostridium difficile test (CDT; Marion Laboratories, Inc., Kansas City, Mo.) is a latex agglutination test for C . difficile . The recent controversy involving the identity of antigens detected by CDT has made decisions on its use difficult . We compared the test results with those of selective culture and stool cytotoxin assays in investigations of two nursing home outbreaks of C . difficile-associated disease in order to formulate usage recommendations . Selective culture for C . difficile identified 27 (19%) of 142 subjects as carriers . CDT and the stool cytotoxin assay identified only 52 and 48% of these carriers, respectively . Compared with the stool cytotoxin assay, CDT had a high sensitivity (92%) and specificity (89%) for the detection of C . difficile disease, but the positive predictive value of the test was only 17% when the prevalence of disease was 2% . We conclude that the CDT should not be used to identify carriers but that it is a sufficiently sensitive and specific screening test for diagnosing C . difficile disease . However, since the positive predictive value of the CDT is low when the prevalence of disease is low, positive test results should be confirmed by the stool cytotoxin assay.

J Clin Microbiol, 1989 May, 27(5), 806 - 7
Microbiology of postthoractomy sternal wound infection; Brook I; Specimens from 74 patients with postthoractomy sternal wound infection were studied for aerobic and anaerobic bacteria . Bacterial growth was obtained in specimens from 65 patients (88%) . Aerobic or facultative bacteria only were recovered in 50 specimens (77%), anaerobic bacteria only in 6 (9%), and mixed aerobic, facultative, and anaerobic bacteria in 9 (14%) . Eighty-seven isolates were recovered (1.3 per specimen): 68 aerobic or facultative (1.0 per specimen) and 19 anaerobic (0.3 per specimen) . The predominant aerobes were Staphylococcus epidermidis (28 isolates), Staphylococcus aureus (21 isolates), and members of the family Enterobacteriaceae (14 isolates) . The predominant anaerobes were Peptostreptococcus spp . (10 isolates), Bacteroides spp . (4 isolates), and Clostridium spp . (3 isolates) . Polymicrobial infection occurred in 18 instances (28%) . A single organisms was recovered in 47 instances (72%); these included 20 isolates of S . epidermidis, 15 of S . aureus, 5 of Enterobacteriaceae, and 4 of anaerobes . These data highlight the previously unrecognized polymicrobial aerobic and anaerobic bacteriology in a percentage of patients with postthoractomy sternal wound infection.

J Clin Microbiol, 1989 May, 27(5), 1137 - 8
Liver abscess caused by Clostridium bifermentans following blunt abdominal trauma; Nachman S et al.; A case of hepatic abscess and subsequent septicemia caused by Clostridium bifermentans is described . The abscess manifested itself on the third day after blunt trauma to the torso . The patient had nausea, vomiting, fever, evidence of hepatic dysfunction, and subphrenic gas . This case illustrates the association of hepatic abscess and blunt trauma to the torso.

Br Vet J, 1989 May-Jun, 145(3), 291 - 2
Double intussusception fatally complicated by clostridial infection in a dog (a case report); Okewole PA et al.; A 10-month-old Alsation dog with history of anorexia, diarrhoea, dehydration and vomition developed a double intussusception which affected the distal jejunum and proximal ileum . Necropsy revealed the intussusception to be swollen and congested with fibrinous adhesions between the intussusceptum and intussuscepiens . Two pieces of bone believed to be the inciting cause were found within the intussusceptum . Clostridium welchii and Clostridium bifermentans were isolated from the lesions.

J Clin Pathol, 1989 May, 42(5), 511 - 5
Typing of Clostridium difficile causing diarrhoea in an orthopaedic ward; McKay I et al.; In an outbreak of diarrhoeal disease in an orthopaedic ward Clostridium difficile was isolated from all six patients with diarrhoea . Attempts were made to type these isolates by means of antibiogram, detection of pre-formed enzymes, analysis of surface proteins by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting, and plasmid profile analysis . This showed that a single strain (type E) indistinguishable by the four distinct methods of typing, was isolated from all six patients at some time during their episodes of diarrhoea . Relapse was caused by the acquisition of a new strain in two patients, and by re-emergence or reacquisition of the original strain in two patients . The immunochemical method was the most sensitive and discriminatory of the typing strategies adopted.

Medicine (Baltimore), 1989 May, 68(3), 151 - 62
Bacteremia caused by non-sporulating anaerobes in cancer patients . A 12-year experience; Fainstein V et al.; The clinical, epidemiological and laboratory characteristics of bacteremia caused by anaerobic organisms other than Clostridium spp . in cancer patients are described and compared to other previously reported series . Of the 315 episodes, 246 (78%) were caused by a single organism and 69 (22%) were polymicrobial . The most common underlying malignancies were genitourinary and gynecological tumors, acute leukemia, and gastrointestinal malignancies . Most patients (94%) were febrile, and septic shock was documented in 24% of monomicrobial episodes and in 58% of those with polymicrobial infection . Soft-tissue infection was present in 44% of the cases, and it presented as tissue necrosis in 11% . The most common sites identified as the portal of entry were intra-abdominal abscesses, soft tissue, and the oropharynx . The most common organisms were Bacteroides fragilis (57%) and other Bacteroides spp . (22%) . Most polymicrobial infections were caused by 2 organisms, the second most commonly another anaerobe or an aerobic gram-negative bacillus . The most active antibiotic in vitro was chloramphenicol . High rates of resistance to penicillin were observed not only among B . fragilis, but also among Bacteroides spp . The frequency of penicillin resistance increased throughout the study years . The overall survival was 70% . The cure rate for monomicrobial bacteremias was 76% vs . 51% for polymicrobial episodes . Infection was the cause of death in 20 and 16 episodes, respectively . The response rate for patients in septic shock was 47% in contrast to an 85% recovery rate for those without it . Ninety-five patients had documented abscesses accompanying the bacteremic episode . The most effective antibiotics were clindamycin and chloramphenicol . Overall response to penicillin was only 13% . Suboptimal responses were also observed for the antipseudomonal penicillins . High response rates (82%) were also obtained with cefoxitin, metronidazole, and moxalactam.

J Bacteriol, 1989 May, 171(5), 2873 - 5
Electron transport and electrochemical proton gradient in membrane vesicles of Clostridium thermoautotrophicum; Hugenholtz J et al.; Membrane vesicles of Clostridium thermoautotrophicum containing carbon monoxide dehydrogenase generated a proton motive force when exposed to CO . This proton motive force, with a value of -140 mV, consisted of only an electrical potential at pH 7.5 and above and of an electrical potential and pH gradient at a lower pH . The proton motive force drove the uptake of L-alanine by the vesicles to a concentration of 300 times that of the medium.

FEMS Microbiol Lett, 1989 May, 50(1-2), 135 - 40
Cloning and expression of a Clostridium acetobutylicum alpha-amylase gene in Escherichia coli; Verhasselt P et al.; A gene library of Clostridium acetobutylicum ATCC824 was constructed in the plasmid vector pEcoR251 . The library was tested for the presence of starch hydrolyzing clones . One clone in which the recombinant plasmid, pVP101, conferred alpha-amylase activity to the Escherichia coli host cell, was detected . The gene is carried on a 3.45-kbp BglII restriction fragment . A detailed physical map of pVP101 is presented.

Int J Food Microbiol, 1989 May, 8(2), 121 - 32
Spoilage of an acid food product by Clostridium perfringens, C . barati and C . butyricum; de Jong J; Spoilage of canned pasteurized brined mung bean sprouts, acidified with citric acid to pH 4.0-4.5, was found to be caused by acid tolerant Clostridium spp . including the species barati, perfringens and butyricum . The pH limit for growth in the brine used were estimated 3.7, 3.7 and 4.0 respectively . Some of the isolated C . perfringens strains produced enterotoxins in sporulation media . The spores of the isolated anaerobes appeared to originate from mung beans, but C . barati and C . perfringens strains freshly isolated from dry beans, were unable to grow in acidified brine . During germination and sprouting of mung beans, the oxygen concentration decreased, while carbon dioxide concentration increased considerably, due to respiration of the sprouts and actively growing Enterobacteriaceae and lactobacilli . It was assumed that this allowed C . barati and C . perfringens strains to grow and acquire the observed unusual acid tolerance . After increasing aerobicity during sprouting, no growth of Clostridium spp . was observed, substantiating the assumption.

Trans R Soc Trop Med Hyg, 1989 May-Jun, 83(3), 344 - 5
In vitro activity of nalidixic acid and its iron (III) complex on Entamoeba histolytica; Anaya-Velazquez F et al.; The in vitro activity of the antibacterial agent nalidixic acid (HNal) and its iron (III) complex (FeNal) against Entamoeba histolytica HM1 strain trophozoites in axenic or monoxenic (associated with Clostridium symbiosum) cultures was investigated . Using a dilution test with TYI-S-33 medium, this protozoan was found to be susceptible to both drugs, but FeNal showed amoebicidal activity only at concentrations higher than those used with HNal.

Biokhimiia, 1989 May, 54(5), 804 - 10
{Study on the effect of some group-specific agents on clostridiopeptidase}; Balaevskaia TO et al.; The interaction of clostridiopeptidase of Clostridium histolyticum with EDC, TNM and MA, the specific reagents for COOH-groups, tyrosine and lysine residues was studied . It was shown that at pH 6.0 EDC inactivates the enzyme . The inactivation process follows the pseudo-first order kinetics and is described by a second order rate constant equal to 1 M-1 min-1 . The synthetic substrate does not prevent, in practical terms, the enzyme inactivation by EDC . At pH 8.0 TNM modifies about 19 tyrosine residues in the clostridiopeptidase molecule which is accompanied by marked inhibition of the enzyme activity (down to 70-90%) . In this case, the inactivation process is not described by simple pseudo-first order kinetics but is characterized by two steps (fast and slow) with second order rate constants of approximately 14 and 3.5 M-1 min-1, respectively . The synthetic substrate partly prevents the inactivation of the enzyme by TNM and protects 11 tyrosine residues . The MA-induced incorporation of 13 +/- 3 maleyl groups into the clostridiopeptidase molecule in partially prevented by the synthetic substrate with protects the enzyme against inactivation . The data obtained suggest that lysine residues are seemingly included into the active center of clostridiopeptidase, whereas tyrosine residues provide for the maintenance of active conformation of the enzyme.

Rev Infect Dis, 1989 May-Jun, 11(3), 470 - 3
Postpartum uterine infection with Clostridium perfringens; Dylewski J et al.; Clostridium perfringens is commonly present in the female genital tract . Uterine infection with this organism is a potentially fatal disease infrequently seen in obstetric practice . The manifestations of C . perfringens uterine infection are variable, ranging from endometritis to gas gangrene with fulminant septicemia . The usual precipitating event has been septic abortion, but such infections can also occur spontaneously in uterine tumors and after complicated deliveries requiring mechanical intervention . Diagnosis may be aided by radiologic techniques, and treatment involves high-dose penicillin and possibly surgery . We report two cases and review the clinical presentation and the diagnostic and therapeutic aspects of this disease.

FEMS Microbiol Lett, 1989 May, 50(1-2), 221 - 4
Stimulation of growth and sporulation of Clostridium perfringens by its homologous enterotoxin; Dillon ME et al.; C . perfringens enterotoxin shortened the lag phase and time of onset of sporulation of the same organism in a dose-dependent manner . The toxin stimulated macromolecular synthesis of pre-exponential phase cells.

FEMS Microbiol Lett, 1989 May, 50(1-2), 173 - 6
Monoclonal antibodies against alpha toxin of Clostridium perfringens; Sato H et al.; Ten distinct monoclonal antibodies (MAbs) against alpha toxin of Clostridium perfringens were produced by the fusion of SP2/O with spleen cells of mice immunized with alpha toxoid, and alpha toxin mixed with or without ethylenediamine-tetraacetate (EDTA) . The antibody activity was evaluated by antigen-binding activity in an enzyme linked immunosorbent assay (ELISA), by phospholipase C (PLC)-neutralizing activity using both egg yolk lecithin and p-nitrophenylphosphoryl-choline (PNPPC) hydrolysis reactions and by anti-lethal activity in mice . Since the toxin-neutralizing activities of each MAb were not parallel, it has been suggested that the three biological activities may not be located in the same site in the toxin molecule . This report also describes the development of a simple purification of the toxin by affinity chromatography and a sensitive immunoassay for quantitation of the toxin using the monoclonal antibody.

Arch Pathol Lab Med, 1989 May, 113(5), 534 - 5
Fatal Clostridium perfringens septicemia associated with gastrointestinal arteriovenous malformations (vascular ectasias); Craven CM; Clostridial infections usually occur in association with trauma, malignancy, or intra-abdominal disease . A 72-year-old previously healthy man presented with abdominal distress and fever . He developed a hemolytic anemia, coagulopathy, and fulminant clostridial septicemia . The patient died less than 24 hours after presentation . At autopsy, no malignancy was detected . The patient had an acute clostridial hepatic abscess and multiple arteriovenous malformations (vascular ectasias) of the large and small bowel . The case suggests that these mucosal and submucosal vascular lesions, which usually cause hemorrhage, may also predispose to infection.

Scand J Gastroenterol, 1989 May, 24(4), 475 - 84
Phospholipase activation and arachidonic acid release in cultured intestinal epithelial cells (INT 407); Gustafson C et al.; The release of free arachidonic acid (AA) in cultured intestinal epithelial cells (INT 407) was investigated . INT-407 cells were first incubated overnight with radiolabeled 14C-AA, and most of the incorporated 14C-AA esterified into phosphatidylethanolamine, phosphatidylcholine, and phosphatidylinositol . Labeled cells were then exposed to different stimulating agents and the release of free 14C-AA determined . The calcium ionophore A23187 caused a dose-dependent AA release that was preceded by a rapid uptake and a subsequent efflux of 45Ca2+ . By contrast, phospholipase C from Clostridium perfringens caused a great AA release that was accompanied by an apparent uptake and a sustained intracellular accumulation of 45Ca2+ . The cells alos released AA when exposed to the protein kinase C activator, 4 beta-phorbol-12-myristate-13-acetate (PMA), and this agent, like the diacylglycerol 1-oleoyl-2-acetyl-rac-glycerol, significantly potentiated the AA release caused by A23187 . Not only A23187-mediated but also phospholipase C- and PMA-mediated AA release was inhibited by 4-bromophenacyl bromide, a known phospholipase A2 inhibitor . These findings, taken together, indicate that AA release in intestinal epithelial cells can be caused by (i) Ca2+-mediated phospholipase activation, (ii) products of phospholipase C activity, and (iii) stimulation of protein kinase C . It is suggested, therefore, that AA release in intestinal epithelial cells is governed by intracellular Ca2+, protein kinase C-mediated protein phosphorylation, and activation of phospholipase A2.

Br J Pharmacol, 1989 May, 97(1), 119 - 24
Contraction of the rat isolated aorta caused by Clostridium perfringens alpha toxin (phospholipase C): evidence for the involvement of arachidonic acid metabolism; Fujii Y et al.; 1 . Alpha toxin produced by Clostridium perfringens contracted the rat isolated aorta and stimulated release of arachidonic acid in the tissue . 2 . Quinacrine did not inhibit contraction caused by the toxin . 3 . Indomethacin blocked contraction caused by the toxin in a dose-dependent manner and markedly increased levels of arachidonic acid released by the toxin . 4 . The toxin-induced contraction was blocked by the thromboxane synthetase inhibitor OKY-046 and the thromboxane A2 (TXA2) antagonist ONO-3708 . 5 . The toxin stimulated production of TXB2 and this was blocked by pretreatment with either indomethacin or OKY-046 . 6 . Toxin-induced contraction was diminished by pretreating aorta with collagenase or by rubbing the intimal surface to remove the endothelium . 7 . These data suggest that the contractile response to the toxin is associated with stimulation of TXA2 production from arachidonic acid released by the toxin in the endothelial cells of the aorta.

J Biol Chem, 1989 Apr 15, 264(11), 6325 - 33
The regulation of platelet-activating factor production in endothelial cells . The role of calcium and protein kinase C; Whatley RE et al.; Endothelial cells (EC) synthesize platelet-activating factor (PAF) when stimulated with agonists that bind to cell-surface receptors . We examined events that link receptor binding to synthesis of PAF by EC . Bovine EC stimulated with agonists that interact with specific cell-surface receptors accumulated PAF only in the presence of extracellular calcium . Hormonal stimulation of EC resulted in Ca2+ entry characteristic of that seen with receptor-operated calcium channels; Indo-1 measurements demonstrated that this inward flux of Ca2+ caused prolonged elevated levels of intracellular Ca2+ . EC were exposed to melittin or theta toxin from Clostridium perfringens (pore-forming peptides that increase the permeability of the plasma membrane for small molecules) resulting in an inward flux of Ca2+ and accumulation of PAF . Ca2+ appears to be regulatory for PAF production at the level of phospholipase A2-mediated production of the PAF precursor 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine, as Ca2+ was required for the stimulated hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine . PAF accumulation in EC is also regulated by protein kinase C . Pretreatment of EC with phorbol esters that activate protein kinase C or with dioctanoylglycerol, followed by stimulation, resulted in a 2-fold increase in stimulated PAF production . The regulatory effect of protein kinase C also appears to be at a phospholipase A2-mediated hydrolysis of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.

Biochem J, 1989 Apr 15, 259(2), 597 - 600
X-ray-absorption-spectroscopic evidence for a novel iron cluster in hydrogenase II from Clostridium pasteurianum; George GN et al.; Hydrogenase II from Clostridium pasteurianum contains three different iron-sulphur clusters . Two are {4Fe-4S}(2+.1+) clusters, whereas the other, which is thought to be the site of interaction with H2 and is known as the 'H cluster', is of unknown structure and possesses unusual spectroscopic properties . Analysis of the iron e.x.a.f.s . spectra shows that the H cluster contains iron co-ordinated mostly to sulphur and possesses 2.8 A (1 A = 0.1 nm) Fe--Fe separations when oxidized and 3.3 A Fe--Fe separations when reduced with H2 . The data suggest that the reduced H cluster represents a new structural type of iron-sulphur cluster.

Biochem Biophys Res Commun, 1989 Apr 14, 160(1), 33 - 9
Cloning and sequencing of a phospholipase C gene of Clostridium perfringens; Okabe A et al.; The gene encoding phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into lambda gt10 . The maximal size of the coding region was 1.4 kb and the minimum was 1.1 kb as determined by subcloning into the vector pBR322 and testing for activity . The nucleotide sequence of this region contained a single open reading frame of 1194 bp corresponding to a protein of Mr 45473 with a possible N-terminal signal sequence of 28 amino acids which when removed, would give a mature protein of Mr 42521 . This is in good agreement with the reported size of 43 kDa . The coding region has a dG + dC content of 33.7%, and the codon usage displays a pronounced preference for codons with the lowest dG + dC content.

Am J Gastroenterol, 1989 Apr, 84(4), 379 - 82
Evaluation of antibiotic-associated diarrhea with a latex agglutination test and cell culture cytotoxicity assay for Clostridium difficile; Biddle WL et al.; Diagnosis of Clostridium difficile (C . difficile) by its antigen or toxin has improved treatment for patients who have antibiotic-associated diarrhea and opportunistic colonization of the colon with C . difficile . Unfortunately, results from the tissue culture cytotoxicity assay are not available for 48 h . We prospectively compared a latex agglutination test with the tissue culture cytotoxicity assay in 83 patients (15 with antibiotic-associated diarrhea, 23 with non-antibiotic-associated diarrhea, and 45 without diarrhea) . In the antibiotic-associated diarrhea group, 43% of patients with pseudomembranes and 25% without pseudomembranes tested positive with both tests . In the non-antibiotic groups 92% with diarrhea and 90% without diarrhea were negative with both tests . The number of discordant results illustrates the value of using two tests to identify C . difficile, since additional patients can be identified by using two tests . We found false positives were rare, and C . difficile toxin or antigen could not be detected in more than half the patients with pseudomembranes . The latex agglutination test for C . difficile is reliable, specific, and faster than the tissue culture cytotoxicity assay . The data suggest that other organisms may also be responsible for pseudomembranous colitis, and that negative tests do not obviate the need for visual evaluation of colonic mucosa in suspected cases of antibiotic-associated diarrhea.

Can J Microbiol, 1989 Apr, 35(4), 481 - 6
Evidence for proton motive force dependent transport of selenite by Clostridium pasteurianum; Bryant RD et al.; The proton motive force mediated the transport of selenite (SeO3(2-)) in Clostridium pasteurianum cells by proton symport . The proton conductor, carbonyl cyanide m-chlorophenylhydrazone, inhibited SeO3(2-) uptake while N,N'-dicyclohexylcarbodiimide prevented SeO3(2-) uptake by presumably inhibiting the unidirectional ATPase . Acid pulse studies and antibiotic experiments with valinomycin suggest that the transmembrane delta pH component of the proton motive force mediated the transport of SeO3(2-) into the cells . The SeO3(2-) porter system in C . pasteurianum was found to be dependent upon energy source, temperature, and medium pH.

Zh Mikrobiol Epidemiol Immunobiol, 1989 Apr, (4), 22 - 4
{Preparation of diagnostic antitoxic serum to Clostridium difficile}; Alchinbaeva VM et al.; The results of the studies on the preparation of diagnostic antitoxic sera to C . difficile, intended for use in biological assays with the aim of the laboratory diagnosis of clostridial enteric infections, are presented . The conditions for the detoxification of C . difficile native toxin have been established, the optimum schedules for the immunization of rabbits have been selected and specific antitoxic sera to C . difficile have been obtained . The neutralizing activity of these sera has been evaluated in the lethality test and in the cytotoxic test on human embryo dermo-muscular fibroblast cells M-19.

Appl Environ Microbiol, 1989 Apr, 55(4), 845 - 51
Kinetics of biotransformation of 1,1,1-trichloroethane by Clostridium sp . strain TCAIIB; Galli R et al.; Batch experiments were conducted to examine the effects of high concentrations of 1,1,1-trichloroethane (TCA) on the biotransformation of TCA by Clostridium sp . strain TCAIIB . The biotic dehalogenation of TCA to 1,1-dichloroethane by nongrowing cells was measured at 35 degrees C, and the data were used to obtain the kinetic parameters of the Monod relationship half-velocity coefficient Ks (31 microM) and the coefficient of maximum rate of TCA biotransformation (kTCA; 0.28 mumol per mg per day) . The yield of biomass decreased with an increase in the TCA concentration, although TCA concentrations up to 750 microM did not completely inhibit bacterial growth . Also, kTCA was higher in the presence of high concentrations of TCA . A mathematical model based on a modified Monod equation was used to describe the biotransformation of TCA . The abiotic transformation of TCA to 1,1-dichloroethene was measured at 35 degrees C, and the first-order formation rate coefficient for 1,1-dichloroethene (ke) was determined to be 0.86 per year.

Appl Environ Microbiol, 1989 Apr, 55(4), 837 - 44
Biotransformation of 1,1,1-trichloroethane, trichloromethane, and tetrachloromethane by a Clostridium sp; Galli R et al.; A gram-positive, strictly anaerobic, motile, endospore-forming rod, tentatively identified as a proteolytic Clostridium sp., was isolated from the effluent of an anaerobic suspended-growth bioreactor . The organism was able to biotransform 1,1,1-trichloroethane, trichloromethane, and tetrachloromethane . 1,1,1-Trichloroethane was completely transformed (greater than or equal to 99.5%) by reductive dehalogenation to 1,1-dichloroethane (30 to 40%) and, presumably by other mechanisms, to acetic acid (7%) and unidentified products . The reductive dehalogenation of tetrachloromethane led to the intermediate trichloromethane, which was further transformed to dichloromethane (8%) and unidentified products . The biotransformation occurred during the exponential growth phase, as well as during the stationary phase . Tetrachlorethene, trichloroethene, 1,1-dichloroethene, chloroethane, 1,1-dichloroethane, and dichloromethane were not biotransformed significantly by the organism.

Antimicrob Agents Chemother, 1989 Apr, 33(4), 560 - 1
Antibacterial activity of the new glycopeptide antibiotic SKF104662; Jorgensen JH et al.; The inhibitory activity of the new glycopeptide antibiotic SKF104662 was generally equivalent (+/- 1 concentration increment) to the activities of vancomycin, teicoplanin, and daptomycin against selected gram-positive bacteria . However, SKF104662 demonstrated greater activity against Staphylococcus epidermidis and S . haemolyticus than did teicoplanin and was more active than the other drugs against Clostridium difficile isolates . SKF104662 possessed bactericidal activity quite similar to that of vancomycin against selected isolates of Staphylococcus, Streptococcus, and Enterococcus species.

Am J Infect Control, 1989 Apr, 17(2), 77 - 82
Diagnostic studies of nosocomial diarrhea in children: assessing their use and value; Brady MT et al.; During a 17-month period (01/11/85-05/31/86) 225 cases of nosocomial diarrhea were identified in a children's hospital . Diarrhea was considered to be nosocomial if it began at least 72 hours after the patient's hospital admission or within 3 days after discharge . One or more routine diagnostic studies for identification of a pathogen were performed in 195 (87%) cases . The most commonly performed test was the bacterial stool culture . None of these samples yielded a bacterial pathogen . The only pathogens detected by routine laboratory studies were rotavirus (61/137 {45%} samples were positive for rotavirus by ELISA) and Clostridium difficile (9/54 {17%} positive for toxin) . Of the patients whose tests were positive for rotavirus 56 were younger than 2 years of age, and all were identified in the winter and spring . When multiple stool samples were tested by the diagnostic laboratory, rotavirus was identified in an additional 14 patients whose initial stool samples were negative for rotavirus . All patients whose tests were positive for C . difficile toxin had received antibiotics within the previous 3 months . Ova/parasites were not detected in 53 of the tested stools . We also identified enteric adenovirus in six patients . Viruses were identified in 95 (42%) of the 225 cases of nosocomial gastroenteritis . Nosocomial diarrhea is common in a children's hospital . Rotavirus is the most commonly identified pathogen . Rotavirus testing is valuable in children with nosocomial diarrhea who are younger than 2 years of age, especially in the winter and spring . Multiple samples may be necessary to identify rotavirus . C . difficile toxin assay should be considered for patients who are receiving or who have received antibiotics.(ABSTRACT TRUNCATED AT 250 WORDS)

Mayo Clin Proc, 1989 Apr, 64(4), 392 - 9
Antimicrobial susceptibilities of anaerobic bacteria isolated at the Mayo Clinic during 1982 through 1987: comparison with results from 1977 through 1981; Musial CE et al.; Results of antimicrobial susceptibility testing of anaerobic bacteria isolated at the Mayo Clinic were reviewed for 1982 through 1987 and compared with a previous survey during 1977 through 1981 at this institution . Between the earlier and the later period, clindamycin resistance increased in the Bacteroides fragilis group (from 4% of isolates to 8%) . We noted continuing penicillin resistance among Bacteroides species other than B . fragilis and rare penicillin resistance among Fusobacterium organisms, with four isolates during the 1982 through 1987 period being beta-lactamase producers . The high levels of resistance to some agents seen in certain Clostridium species in 1977 through 1981 were not as great in the current survey . No major changes were noted in the susceptibilities of C . perfringens, anaerobic non-spore-forming gram-positive bacilli, and anaerobic gram-positive cocci.

J Biol Stand, 1989 Apr, 17(2), 117 - 24
The detection of Clostridium perfringens epsilon antitoxin in rabbit serum by monoclonal antibody based competition ELISA; Sojka MG et al.; A competitive enzyme-linked immunosorbent assay (CELISA) has been developed, standardized and compared with the toxin neutralization (TN) test performed in mice for the measurement of antibody responses in rabbits vaccinated with clostridial vaccines . In CELISA, sera were tested at a single dilution for their ability to compete with the reaction between a monoclonal antibody, which neutralizes epsilon toxin, and epsilon toxoid coated on to a solid phase . The results of the two tests correlated well . CELISA was specific, rapid, reproducible and simple to perform and offered an alternative to the TN test that reduced the requirement for experimental animals in the potency testing of clostridial vaccines.

Rev Argent Microbiol, 1989 Apr-Jun, 21(2), 47 - 53
{Type D and A Clostridium botulinum in necropsy samples from bovines with "mal de Aguapey"}; Fernandez RA et al.; Bacteriological studies were carried out on several necropsy samples from five animals whose deaths had been attributed to bovine botulism . This disease, regionally called Mal de Aguapey, enzootically affects animals from a wide area of the north-east of Argentina (Province of Corrientes) with a bovine population estimated at near to 2,500,000 . Either C . botulinum type D, its toxin or both were identified in all animal samples, alternatively in contents of rumen, jejunum, ileum, caecum and in samples of spleen, liver and kidney (Table 1) . C . botulinum type A was isolated respectively from the liver and the kidney of two animals . Cultures of 100 soil samples taken in the enzootic area were positive only for C . botulinum type A (3%) . These results enlarge and confirm previous findings and lend support to the botulinic etiology of the Mal de Aguapey.

J Antimicrob Chemother, 1989 Apr, 23(4), 623 - 31
Third generation cephalosporins as a risk factor for Clostridium difficile-associated disease: a four-year survey in a general hospital; de Lalla F et al.; The main clinical features of patients who developed pseudomembranous colitis (PMC) or Clostridium difficile-associated diarrhoea (CDAD) during their stay at the S . Anna General Hospital, Como, over the period February 1984 to May 1988, are reported . Forty patients developed either CDAD (ten cases) or PMC (30 cases) . Twenty-seven (65.7%) had undergone surgery and 32 (80.0%) had received prolonged antibiotic treatment . Three patients (7.5%) were given three doses only of ceftriaxone . Five patients (12.5%) had not received any antibiotic treatment; but three were nursed in a bed next to a patient with PMC-CDAD . The number of cases diagnosed were correlated retrospectively with the cumulative consumption of different groups of antibiotics on wards in which PMC or CDAD occurred . A significant difference (P less than 0.01) between third generation cephalosporins (16 cases) and ureidopenicillins (one case), was found . Twenty-five patients were treated with oral vancomycin . Two died of the underlying disease and 23 were cured . The disease recurred clinically in three, and follow-up cultures were positive in another asymptomatic case . Fifteen patients (all PMC cases) were treated with oral teicoplanin . All were clinically cured and remained asymptomatic and all but one were also cleared of C . difficile . No adverse reactions were observed in patients given either drug . Third generation cephalosporins, even when administered as short-term perioperative prophylaxis, but not ureidopenicillins, are significantly associated with C . difficile-related diseases . Teicoplanin proved to be very effective and safe in the treatment of PMC, and should be further evaluated there.

Postgrad Med J, 1989 Apr, 65(762), 208 - 10
Effect of treatment with botulinum toxin on spasticity; Das TK et al.; Botulinum toxin, a product of Clostridium botulinum, produces presynaptic neuromuscular block by preventing release of acetylcholine from nerve endings . The toxin was injected directly into the skeletal muscles of six patients with severe spasticity due to stroke-related hemiplegia . It produced both subjective and objective improvement . The toxin injections were well tolerated and no significant side effect was reported.

J Hosp Infect, 1989 Apr, 13(3), 309 - 14
Outbreak of Clostridium difficile diarrhoea in an orthopaedic unit: evidence by phage-typing for cross-infection; Degl'Innocenti R et al.; In a three-week period five patients had diarrhoea in an orthopaedic unit . The first case was clinically diagnosed as pseudomembranous colitis but the causative agent was not sought . Of the remaining cases, two were Clostridium difficile positive . The outbreak then apparently ceased, but during the following several days two of seven stool samples taken at random from asymptomatic patients yielded C . difficile . Phage-typing of the isolates showed that all apparently belonged to the same strain.

J Bacteriol, 1989 Apr, 171(4), 2148 - 54
Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum; Kanamori K et al.; Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase . C . kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+ . The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account . In C . butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation . Under these growth conditions, C . butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+ . However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C . butyricum after growth on complex nitrogen and carbon sources . The ammonia-assimilating pathway of N2-fixing C . butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways.

J Vet Diagn Invest, 1989 Apr, 1(2), 170 - 3
A bacteriologic study of scabby-hip lesions from broiler chickens in Texas; Scanlan CM et al.; Broilers from commercial flocks experiencing a 10-60% incidence of scabby-hip lesions at processing were examined, and selected skin lesions were cultured . Over 70% of the lesions were associated with traumatic excoriations, particularly on the caudal dorsal convexity of the birds . Most lesions were observed on birds that were 5 weeks of age or older . From the 27 specimens cultured, Clostridium perfringens was isolated in pure culture from 4 lesions and Staphylococcus species from 10 lesions . Pure cultures of staphylococci were recovered from 4 lesions, and 2-5 different staphylococci were isolated from 6 lesions . Eight staphylococci were identified as S . sciuri, 8 as S . simulans, 2 as S . epidermidis, 2 as S . lentus, 2 as S . warneri, 1 as S . cohnii, and 1 as S . intermedius . Fifty cutaneous specimens from 10 5-week-old normal broilers were cultured . A total of 197 isolates were identified including 65 S . sciuri, 52 S . lentus, 24 S . simulans, 12 S . hyicus, 11 S . warneri, 9 S . cohnii, 9 S . gallinarium, 8 S . xylosus, and 7 S . epidermidis.

J Gen Microbiol, 1989 Apr, 135 ( Pt 4), 903 - 9
Cloning in Escherichia coli of the enterotoxin gene from Clostridium perfringens type A; Iwanejko LA et al.; A 26 bp DNA probe has been constructed with minimal degeneracy to the protein sequence for Clostridium perfringens enterotoxin . The probe has been hybridized against a 6-10 kb chromosomal bank from C . perfringens 8239, prepared as a HindIII partial digest in pHG165 . From this survey a clone has been identified containing a 6.8 kb DNA insert with strong hybridization to the probe . Direct plasmid sequencing has identified a translational reading frame within this clone which correlates with the known protein sequence for the type A enterotoxin . DNA sequences 5' to this open reading frame and containing the putative transcriptional control regions show areas of significant homology with regions upstream from the ATG codon of the tetanus toxin gene.

Kansenshogaku Zasshi, 1989 Apr, 63(4), 410 - 6
{An outbreak of enterocolitis due to Clostridium perfringens in a hospital for the severely disabled}; Machida Y et al.; We had an outbreak of 14 cases of enterocolitis due to Clostridium perfringens (Cl . perfringens) in a hospital for the severe multiply-disabled, where the 100 disabled were admitted, in summer in 1985 . The signs and symptoms shown by this enterocolitis were primarily diarrhea without fever and loss of appetite . The feces of 10 cases were examined bacteriologically . The test showed 10(3) to 10(6) cells of Cl . perfringens per one gram of their feces and all the strains isolated were untypable by the classification of Hobbs . Nine out of 10 cases were randomly selected and all of the 9 cases were proved to have enterotoxin producing strains . All the strains were highly sensitive to many kinds of antibiotics except kanamycin and gentamicin . Eleven out of the 14 cases were admitted in the same ward and the 7 out of the 11 cases were in the same room of this ward . Considering the spreading route of this infection, it is unlikely that this outbreak occurred due to food supplied from kitchen in this hospital, because all of the disabled, admitted in this hospital, had little chance by which some of the disabled only in a specific ward or room were supplied with bacteriologically contaminated meals from the point of view of cooking and supplying system of this hospital . Adding to this fact, if this outbreak was due to food-born infection, the symptoms of most patients should occur within 1-2 days, because the incubation period of this disease is within a day, however, the patients increased day by day for more than a week.(ABSTRACT TRUNCATED AT 250 WORDS)

J Appl Bacteriol, 1989 Apr, 66(4), 263 - 70
Bacterial overgrowth in the jejunum of ICR mice and Wistar rats orally administered with a single lethal dose of fusarenon-X, a trichothecene mycotoxin; Morishita Y et al.; A single oral dose of fusarenon-X (F-X), a trichothecene mycotoxin, resulted in abnormal microflora in the jejunum in ICR mice and Wistar rats with some differences in dose response between the species . In the acute phase, enterobacteria, streptococci, Clostridium perfringens and bacteroides showed remarkably increased counts in the jejunum of mice and rats dosed with F-X while lactobacilli showed a decrease in count . F-X brought an invasion of Pseudomonas aeruginosa into the livers, lungs, kidneys and spleens of ICR mice . Changes in the jejunal microflora appeared after 7 h in ICR mice and after 24 h in Wistar rats after a single oral dose of F-X of 7.5 and 4.0 mg/kg b.w., respectively, and the microflora returned to its normal state at 72 h in mice and 96 h in rats . The changes of intestinal microflora were followed by alterations in the growth curves of both animal species . The pH in the glandular stomach was also greatly enhanced before changes in the jejunal microflora . Acute F-X intoxication may be an involved manifestation of essential cytotoxicity of F-X mycotoxin alone and secondary bacterial overgrowth in the bowel.

FEMS Microbiol Lett, 1989 Apr, 49(2-3), 269 - 72
Contraction induced by Clostridium perfringens epsilon toxin in the isolated rat ileum; Sakurai J et al.; Clostridium perfringens epsilon toxin caused contraction of the isolated ileum of the rat in a dose-dependent manner . The contraction caused by the toxin was inhibited by a low Na medium, tetrodotoxin (TTX), atropine, mecamylamine or tetraethylammonium (TEA) . Furthermore, the contractile response induced by the toxin was abolished by incubation in Ca-free medium, and completely restored by and addition of Ca2+ . In addition, verapamil inhibited contraction induced by the toxin in a dose-dependent manner . These data suggest that epsilon toxin induces contraction of the isolated ileum and that the toxin-elicited contraction is the result of an indirect action mediated through the nervous systems.

Eur J Cell Biol, 1989 Apr, 48(2), 191 - 202
Mechanism of action of Clostridium difficile toxin B: role of external medium and cytoskeletal organization in intoxicated cells; Ciesielski-Treska J et al.; Toxin B, an exotoxin produced by Clostridium difficile, induces the rounding-up and arborization of cultured mammalian cells, a typical effect which resembles that provoked by cytochalasins . In this study, the effect of toxin B was examined on astroglial cells grown in primary culture . A specific antiserum to toxin B was used to investigate its mechanisms of action . We found that the toxin exerts its effects on cell morphology after its incorporation into cells . The internalization of toxin B requires the presence of calcium ions in the extracellular medium . Replacement of NaCl with sucrose or with potassium glutamate prevents the internalization of the toxin . The direct introduction of calcium ions into cells by the calcium ionophore A23187 stimulates toxin-induced morphological changes . In contrast, toxin-induced morphological transformations were prevented in cells treated with tumor-promoting phorbol . esters or with dibutyryl-cAMP, although such treatment did not abolish the internalization of the toxin . As in the other cell types, the earliest effect of toxin B on astrocyte cytoskeleton is the disruption of actin filaments, without no visible alteration of intermediate filament nor microtubule networks . As astrocytes with toxin-induced stellate morphology survive toxin treatment, the progression of cell morphology and cytoskeleton organization were followed for several weeks . Twenty-six days after exposure to toxin B, stellate astrocytes have processes which were markedly longer and much more branched than those of cells freshly exposed to toxin . At that time, cells are still devoid of F-actin as assessed with rhodamine-conjugated phalloidin and only 70% contain vimentin while all astrocytes present in control cultures express vimentin . Some flat epithelioid astrocytes with prominent bundles of microfilaments reappear during the second week after toxin treatment . Our results show that Clostridium difficile toxin B is internalized into brain astrocytes in culture where it acts by modifying cytoskeletal elements . Its cytopathic effects are reversible . Although actin-related components of the cytoskeleton are the major target of toxin B, other cytoskeletal elements also seem to be affected.

Clin Orthop, 1989 Apr, (241), 245 - 7
Clostridium perfringens and Staphylococcus epidermidis polymicrobial septic arthritis . A case report; McAllister CM et al.; Septic arthritis caused by Clostridium perfringens is extremely rare . Previously there has been only one report of Clostridium perfringens in combination with an aerobe causing septic arthritis . This report presents a 23-year-old man with a mixed aerobic/anaerobic septic arthritis treated with intravenous antibiotics and repeated surgical drainage . It is the only report to date of a polymicrobial septic arthritis involving both Staphylococcus epidermidis and Clostridium perfringens . This report and a review of the literature demonstrate the existence of polymicrobial septic arthritis and illustrate its fulminant nature.

EMBO J, 1989 Apr, 8(4), 1087 - 92
The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells; Chardin P et al.; Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily . We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells . C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin . Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

Am J Physiol, 1989 Apr, 256(4 Pt 1), G767 - 72
Effect of purified Clostridium difficile toxins on intestinal smooth muscle . II . Toxin B; Gilbert RJ et al.; In the companion paper {Am . J . Physiol . 256 (Gastrointest . Liver Physiol . 19): G759-G766, 1989} we showed that highly purified Clostridium difficile toxin A had a profound effect on intestinal smooth muscle after in vivo but not in vitro exposure . In this study we assessed the effects of in vivo and in vitro exposure to C . difficile toxin B on simultaneous measurements of intracellular membrane potential and contractility in rabbit ileal smooth muscle . Direct exposure of ileal smooth muscle to toxin B (0.1-60 micrograms/ml) in vitro caused membrane depolarization and inhibition of action potential frequency, amplitude, and peak voltage, but no effect on slow wave frequency or amplitude was seen . Toxin exposure also resulted in inhibition of the amplitude of carbachol-induced contractions, with phasic contractions being significantly more sensitive to the effect of toxin B than tonic contractions over the complete dose range . The electromechanical effects of toxin B were not affected by prior administration of tetrodotoxin, atropine, hexamethonium, or phentolamine . In contrast, toxin B administered in vivo into an isolated ileal loop had no effect on spontaneous electromechanical properties of excised smooth muscle strips . Our results indicate that direct exposure in vitro of ileal smooth muscle to C . difficile toxin B causes membrane depolarization in association with inhibition of electromechanical activity . This effect, in combination with the indirect effects of toxin A, may contribute to altered intestinal motility during diarrhea caused by C . difficile.

Am J Physiol, 1989 Apr, 256(4 Pt 1), G759 - 66
Effect of purified Clostridium difficile toxins on intestinal smooth muscle . I . Toxin A; Gilbert RJ et al.; In these studies we determined the effects of purified Clostridium difficile toxin A, an enterotoxin, on the electrophysiological and contractile properties of rabbit intestinal circular smooth muscle and correlated these effects with changes of smooth muscle morphology . Simultaneous measurements of intracellular membrane potential and contractility were determined in excised ileal muscle strips after administration of toxin A in vivo (60 micrograms/ml) into an isolated rabbit ileal loop or directly in vitro (0.1-60 micrograms/ml) to a normal muscle strip . Toxin A injection in vivo resulted in membrane depolarization and increased slow wave and action potential frequency . Toxin A injection in vivo also caused increased amplitude of spontaneous and carbachol-induced phasic contractions . The electrophysiological effects of in vivo administration of toxin A were correlated with an inflammatory infiltrate of the lamina propria, but no light or electron microscopic evidence of injury to smooth muscle cells was seen . In contrast to the in vivo studies, direct in vitro exposure of normal ileal muscle strips to toxin A had no effect on either spontaneous or carbachol-induced electromechanical activity . Our results indicate that in vivo administration of C . difficile toxin A into a rabbit ileal loop, but not direct in vitro exposure, causes significant alterations of smooth muscle excitation-contraction coupling that may be mediated by products of local inflammatory cells.

J Bacteriol, 1989 Apr, 171(4), 2209 - 15
Purification and partial characterization of the glycine decarboxylase multienzyme complex from Eubacterium acidaminophilum; Freudenberg W et al.; The proteins P1, P2, and P4 of the glycine cleavage system have been purified from the anaerobic, glycine-utilizing bacterium Eubacterium acidaminophilum . By gel filtration, these proteins were determined to have Mrs of 225,000, 15,500, and 49,000, respectively . By sodium dodecyl sulfate-polyacrylamide gel electrophoresis, protein P1 was determined to have two subunits with Mrs of 59,500 and 54,100, indicating an alpha 2 beta 2 tetramer, whereas the proteins P2 and P4 showed only single bands with estimated Mrs of 15,500 and 42,000, respectively . In reconstitution assays, proteins P1, P2, P4 and the previously reported lipoamide dehydrogenase (P3) had to be present to achieve glycine decarboxylase or synthase activity . All four glycine decarboxylase proteins exhibited highest activities when NADP+ was used as the electron acceptor or when NADPH was used as the electron donor in the glycine synthase reaction . The oxidation of glycine depended on the presence of tetrahydrofolate, dithioerythreitol, NAD(P)+, and pyridoxal phosphate . The latter was loosely bound to the purified protein P1, which was able to catalyze the glycine-bicarbonate exchange reaction only in combination with protein P2 . Protein P2 could not be replaced by lipoic acid or lipoamide, although lipoic acid was determined to be a constituent (0.66 mol/mol of protein) of protein P2 . Glycine synthase activity of the four isolated proteins and in crude extracts was low and reached only 12% of glycine decarboxylase activity . Antibodies raised against P1 and P2 showed cross-reactivity with crude extracts of Clostridium cylindrosporum.

Gastroenterology, 1989 Apr, 96(4), 981 - 8
Prevention of antibiotic-associated diarrhea by Saccharomyces boulardii: a prospective study; Surawicz CM et al.; Saccharomyces boulardii, a nonpathogenic yeast, has been widely used in Europe to prevent antibiotic-associated diarrhea (AAD) . We performed a prospective double-blind controlled study to investigate AAD in hospitalized patients and to evaluate the effect of S . boulardii, a living yeast, given in capsule form concurrently with antibiotics . Over 23 mo, 180 patients completed the study . Of the patients receiving placebo, 22% experienced diarrhea compared with 9.5% of patients receiving S . boulardii (p = 0.038) . Risk factors found to be associated with AAD were multiple antibiotic combinations (containing clindamycin, cephalosporins, or trimethoprim-sulfamethoxazole) and tube feeding . Clostridium difficile, an anaerobe found in the stools of most patients with pseudomembranous colitis, was variably associated with AAD . We evaluated the role of C . difficile in AAD in the study population and found no significant association between the presence of C . difficile or cytotoxin with AAD . Approximately 33% of the patients without diarrhea harbored at least one C . difficile-positive stool and nearly 50% of these patients had detectable cytotoxin . Similar values were obtained in patients with diarrhea . Of C . difficile-positive patients, 31% (5/16) on placebo developed diarrhea compared with 9.4% (3/32) on S . boulardii; this difference was not statistically significant (p = 0.07) . There were no discernable adverse effects of yeast administration . We conclude that S . boulardii reduces the incidence of antibiotic-associated diarrhea in hospitalized patients.

Biochim Biophys Acta, 1989 Mar 16, 995(1), 17 - 20
Coenzyme non-specific glutamate dehydrogenase from Chlorella pyrenoidosa 82T: electron microscopic studies; Shatilov VR et al.; The constitutive coenzyme non-specific glutamate dehydrogenase (GDH) from Chlorella pyrenoidosa 82T was purified to homogeneity by column immunoaffinity chromatography and examined by an electron microscope . The enzyme molecule was found to be a hexameric oligomer composed of monomers arranged in three 2-point group symmetry in two layers slightly twisted round the 3-fold axis . The molecule is 8 +/- 1 nm in diameter and 10 +/- 1 nm in height . The enzyme molecules appear both to dissociate into trimers and to associate along the 3-fold axis forming linear aggregates under certain conditions . A tentative model of the Chlorella GDH molecule is proposed, which is very similar to those described for bovine liver GDH and GDH from Clostridium symbiosum.

Biochim Biophys Acta, 1989 Mar 16, 995(1), 59 - 63
Enzymatic cleavage of Clostridium pasteurianum apoferredoxin and reconstitution of the cleaved products; Skjeldal L et al.; Native ferredoxin from Clostridium pasteurianum proved to be resistant to proteolytic cleavage under anaerobic conditions, but was digested in the presence of air . Apoferredoxin was hydrolyzed by the proteinases used, while cobalt-substituted ferredoxin was resistant both under anaerobic and aerobic conditions . These studies indicate that metal binding of the protein stabilizes the folded state, which is extremely resistant to proteolytic attack . Sulfitolyzed apoferredoxin was subjected to specific cleavage by pepsin at pH 3.2, yielding two fragments . The fragments could be reconstituted to an unstable holoprotein with UV-visible absorption features like that of the native form.

Biochem Biophys Res Commun, 1989 Mar 15, 159(2), 426 - 31
The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro is a tetrahedral transition state analog inhibitor of C . histolyticum collagenase; Grobelny D et al.; The ketone cinnamoyl-(1-13C-Phe)-CGly-Pro-Pro {(4-13C-5-cinnamido-4-oxo-6-phenylhexanoyl)-Pro-Pro 2} competitively inhibits a mixture of collagenases from Clostridium histolyticum with a Ki of 40 +/- 6 nM . 13C-nmr spectroscopy of the ketone in the presence of this collagenase shows a bound 13C resonance at 102.6 ppm and the resonance of the free ketone at 212 ppm . Ketone alone shows no trace (less than 0.5%) of a resonance in the region around 100 ppm . The bound resonance is displaceable by another competitive inhibitor . This ketone is thus a transition state analog which is rehybridized from trigonal planar to tetrahedral upon binding to collagenase.

J Biol Chem, 1989 Mar 15, 264(8), 4342 - 8
Electron paramagnetic resonance studies of the low temperature photolytic behavior of oxidized hydrogenase I from Clostridium pasteurianum; Kowal AT et al.; The effects of CO and O2 on the EPR spectrum of oxidized Clostridium pasteurianum hydrogenase I have been investigated both before and after prolonged exposure to white light at 8 K and 30 K . Low concentrations of O2 were found to induce analogous changes in the EPR spectrum as CO, i.e . conversion of the rhombic signal with g approximately 2.10, 2.04, 2.00, a characteristic of the novel H2-activating center in oxidized Fe-hydrogenases, to an axial signal with g approximately 2.07, 2.01, 2.01 . The results suggest a common binding site and mode of coordination for CO and O2 and permit rationalization of conflicting reports from different laboratories concerning the EPR properties of oxidized Fe-hydrogenases . The CO- and O2-induced axial EPR signals were found to be light-sensitive at low temperatures . Moreover, they exhibited indistinguishable and unusual photolysis behavior with the dominant photo-product being dependent on the temperature at which illumination was performed . At 8 K, photodissociation of CO or O2 occurs, resulting in an EPR signal identical with that of the oxidized enzyme in the absence of CO or O2 . However, at 30 K, the dominant photoproduct is a rhombic EPR signal with g approximately 2.26, 2.12, 1.89 . While the origin of this new EPR signal is uncertain, the g-value anisotropy and relaxation characteristics resemble those of a low spin Fe(III) center . These two photoproducts cannot be thermally or photolytically interconverted, but both are quantitatively reconverted to the original axial EPR signal on warming in the dark to 200 K . A tentative working hypothesis for the nature of the H2-activating center of Fe-hydrogenases is presented that is consistent with the available physiochemical data and permits rationalization of the novel photolysis behavior.

Biochim Biophys Acta, 1989 Mar 14, 1002(1), 37 - 44
NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities from a lecithinase-lipase-negative Clostridium species 25.11.c; Edenharder R et al.; A lecithinase-lipase-negative Clostridium sp . 25.11.c., not fitting in any of the species of Clostridia described so far as judged by morphological, physiological, and biochemical data, was shown to contain NADP-dependent 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenases . The three hydroxysteroid dehydrogenases could be demonstrated in the supernatant and in the membrane fraction after solubilization with Triton X-100, suggesting enzymes which were originally membrane bound . The 3 beta-hydroxysteroid dehydrogenase was synthesized constitutively, and the specific enzyme activity was significantly reduced by growth medium supplementation with 3-keto bile acids and trisubstituted bile acids . A pH optimum of 7.5 and a molecular weight of approx . 104,000 were estimated by molecular sieve chromatography . The enzyme reduced the 3-keto group of bile acids; an oxidation of a 3 beta-hydroxyl function could not be demonstrated . The lowest Km values were found for disubstituted bile acids, trisubstituted and conjugated bile acids having higher Km values . 7 alpha-Hydroxysteroid dehydrogenase, but not 7 beta-hydroxysteroid dehydrogenase, was already present in uninduced cells . The specific activities, however, were greatly enhanced when cells were grown in the presence of chenodeoxycholic acid or 3 alpha-hydroxy-7-keto-5 beta-cholanoic acid . Ursodeoxycholic acid with its 7 beta-hydroxyl group was ineffective as an inducer . Molecular weights of approx . 82,000 and 115,000 were found for the 7 alpha-hydroxysteroid dehydrogenase and the 7 beta-hydroxysteroid dehydrogenase, respectively . In contrast to the in vivo situation, the reaction could only be demonstrated in the reductive direction in vitro . Here, the pH optimum for the overall reaction was 8.5-8.7 . 3 beta-, 7 alpha- and 7 beta-hydroxysteroid dehydrogenase activities were readily demonstrated for at least 48 h when preparations were stored at 4 degrees C, but were found to be heat-sensitive.

Orv Hetil, 1989 Mar 12, 130(11), 551 - 6
{Gas gangrene cases in Hungary 1979-1986}; Sere G et al.; The authors evaluate epidemiologic data of 182 patients suffering from gas gangrene during an eight year period from 1979 to 1986 . Of the patients surveyed 127 died; thus lethality reached 69.2% . The average age of the survivors was 49.9 year as opposed to the 66.1 year of the fatal cases . More than half of the illnesses followed amputation of extremities, and a quarter of them was a consequence of an accident . Samples of the bacteriologically examined wound discharges yielded in 93.1% bacteria from the Clostridium genus . Hygiene was poor in operation theatres and in the hospital environment in 1/4-th of the cases . Sixty six patients died within 24 hours after diagnosis . The presented data suggest that in Hungary the number of gas gangrene cases and deaths surpass those of tetanus.

J Biol Chem, 1989 Mar 5, 264(7), 4082 - 7
Purification, microheterogeneity, and stability of human lipid transfer protein; Kato H et al.; A method for the purification of lipid transfer protein (LTP) from human plasma was developed with the aid of succinylated low density lipoprotein-Sepharose affinity column chromatography . The purified LTP exhibited a single main band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis . However, upon isoelectric focusing on polyacrylamide gel, the preparations consistently showed nine bands with isoelectric points ranging from 4.6 to 5.4 . The treatment of LTP with Clostridium perfringens neuraminidase shifted these multiple bands toward higher pH regions due to the release of sialic acid . Extensive treatment with neuraminidase resulted in the appearance of a major band with the isoelectric point of 5.6 . The purified LTP was rapidly inactivated upon incubation at 37 degrees C due to the denaturation at the "air"-water interface . Various factors promoting or preventing this interfacial denaturation were elucidated . When purified LTP was stored at 4 degrees C, plasma neuraminidase co-purified with LTP became activated, resulting in the gradual desialylation of LTP . It seemed that the LTP preparations of apparent homogeneity are associated with a trace amount of an inactive form of plasma neuraminidase . The inclusion of 4 mM 2-mercaptoethanol or 0.2% EDTA in the storage media completely prevented the activation of plasma neuraminidase . These agents, however, did not significantly inhibit the already activated neuraminidase . When LTP was stored at -20 degrees C in very low ionic strength media, such as 0.001% EDTA (pH 7.4) and at high protein concentrations, the loss of the activity was minimal even after prolonged storage.

J Med Microbiol, 1989 Mar, 28(3), 217 - 21
Recovery of spores of Clostridium difficile altered by heat or alkali; Kamiya S et al.; The effect of heating or alkali-treatment on spore recovery in ordinary growth medium was examined for four strains of Clostridium difficile . Heating spores at 80 degrees C for 10 min produced 95.50-99.95% decreases in the recovery rates . Treatment with 0.1 N NaOH for 15 min produced 99.47 and 99.83% decreases in spore recovery rates for two of the four strains . The influence of either addition of lysozyme after treatment with sodium thioglycollate (thioglycollate-lysozyme method) or addition of sodium taurocholate (taurocholate method) on recovery of heat- or alkali-treated C . difficile spores was also examined . Viable spores of all strains altered by heating at 90 degrees C or 100 degrees C for 10 min could not be recovered at all by the taurocholate method . Nor did this method allow recovery of alkali-altered spores treated with greater than 0.2 N NaOH for 15 min . On the other hand, 10-47% of altered spores heated at 90 degrees C for 10 min were recovered by the thioglycollate-lysozyme method, and alkali-altered spores treated with 0.1-0.3 N NaOH for 15 min were as completely recovered by this method as untreated spores . These results indicate that the thioglycollate-lysozyme method is more effective than the taurocholate method for recovery of the heat- or alkali-altered C . difficile spores.

Infect Immun, 1989 Mar, 57(3), 932 - 6
Experimental cecitis in gnotobiotic quails monoassociated with Clostridium butyricum strains isolated from patients with neonatal necrotizing enterocolitis and from healthy newborns; Bousseboua H et al.; Using axenic quails fed a diet containing lactose, we have investigated the potentially pathogenic roles of six Clostridium butyricum strains of human origin . Three strains (CB155-3, CB1002, and CB203-1) isolated from neonatal necrotizing enterocolitis patients and two of three strains (CB19-1 and CB25-2) isolated from healthy newborns led to cecal or crop lesions or both similar to those observed in human neonatal necrotizing enterocolitis: thickening of the cecal wall with gas cysts, hemorrhagic ulcerations, and necrotic areas . The lactose-negative strain (CB46-1) did not develop any lesions . The neuraminidase-producing strain (CB155-3) caused lesions in all monoassociated quails, whereas the other strains caused lesions in 28 to 85% of animals . Removal of dietary lactose suppressed all pathological incidence . These results show that lactose fermentation is a prerequisite in these pathological changes and stress the roles played by both the strain and the host in the expression of C . butyricum enteropathogenicity.

Br Poult Sci, 1989 Mar, 30(1), 81 - 9
Influence of peas (Pisum sativum) as a dietary ingredient and flavomycin supplementation on the performance and intestinal microflora of broiler chicks; Brenes A et al.; 1 . Two experiments were carried out to study the effects of diets containing various concentrations of pea meal (Pisum sativum L.), with or without flavomycin supplementation, on the performance and intestinal microflora of broiler chicks . 2 . During the 7 to 28-d period, chicks fed on diets containing 300, 600 and 800 g pea meal/kg consumed more food and gained more weight than chicks receiving a maize-isolated soyabean protein control diet . The addition of flavomycin to the diets had similar effects on the performance of both the control and the pea groups . 3 . Pea diets, with and without supplemental flavomycin, had little influence on the composition of intestinal microflora . The counts of enterococci in the small intestine and Clostridium perfringens and coliforms in the caeca of pea-fed chicks exceeded those of control chicks.

Can J Microbiol, 1989 Mar, 35(3), 388 - 98
Morphological changes in putrefactive anaerobe 3679 (Clostridium sporogenes) induced by sorbate, hydrochloric acid, and nitrite; Ronning IE et al.; Putrefactive anaerobe 3679 (Clostridium sporogenes), a gram-positive bacterium, was examined by light and electron microscopy during normal growth and in a medium containing sorbate (50 mM, pH 6.5), hydrochloric acid (pH of medium adjusted from 7 to 5 with HCl), or nitrite (1 mM, pH 7) . During the early exponential growth phase, untreated cells were filamentous and nonseptate, but became septate later and divided when the culture entered the stationary phase . Untreated short and filamentous cells had a double-layered cell wall . Sorbate-treated cells were usually filamentous and nonseptate, but with distorted shapes characterized by numerous bends and bulges . Septation, when present, resulted in minicells . The inner cell wall appeared to be thickened and the outer wall was absent in many areas . Acid-treated cells were similar to sorbate-treated cells but contained septa . Considerable cellular debris was present in the suspension . Nitrite-treated cells were also filamentous, bent, and bulged but the cell wall appeared normal . Considerable cellular debris was also present in suspensions of nitrite-treated cells . Changes in morphology are discussed in relation to possible mechanisms of cell growth regulation and the inhibitory action of sorbate, acid, and nitrite.

FEMS Microbiol Lett, 1989 Mar, 49(1), 15 - 20
Construction of shuttle vectors useful for transforming Clostridium acetobutylicum; Truffaut N et al.; Plasmids pIM13, pT127 and pBC16 delta 1, introduced by transformation into Clostridium acetobutylicum N1-4081, were shown to replicate in, and to confer antibiotic resistance upon this new host . Recombinant plasmids were constructed by inserting erythromycin-resistant plasmid pIM13 into the unique ClaI site of pBR322 or by ligating a tetracycline-resistant determinant of plasmid pT127 to HindIII-linearized pIM13 . The hybrid plasmids replicated and expressed erythromycin resistance in C . acetobutylicum strain N1-4081 and in Escherichia coli or Bacillus subtilis, indicating that they might be useful as shuttle vectors for transferring genes between these strains . The efficiency and stability of different replicons in C . acetobutylicum were compared.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1989 Mar, 270(4), 456 - 61
Demonstration of capsules in Clostridium difficile; Strelau E et al.; In four strains of Clostridium difficile the formation of capsules was demonstrated by light and electron microscopy.

Biofactors, 1989 Mar, 2(1), 57 - 63
A general method for generation and analysis of defined mutations in enzymes involved in a tetrahydrofolate-interconversion pathway; Barlowe CK et al.; In eukaryotes, 10-formyltetrahydrofolate (THF) synthetase, 5,10-methenyl-THF cyclohydrolase and 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase . These reactions are generally catalyzed by three separate monofunctional enzymes in prokaryotic cells . In this report a general method for the generation, detection and analysis of specific mutations affecting the catalytic activity of any of the reactions catalyzed by C1-THF synthase or its monofunctional counterparts is described . The method relies on plasmid-borne expression of genes in strains of the yeast Saccharomyces cerevisiae that are missing one or more of the activities of C1-THF synthase . Specific segments of the gene are subjected in vitro to random mutagenesis, the mutant genes expressed in yeast and screened by phenotype for inactivating mutations . Plasmids encoding mutant enzymes are recovered for sequence analysis . One-step purification of C1-THF synthase from the yeast expression system is demonstrated . The feasibility and versatility of the method is shown with the yeast ADE3 gene encoding the cytoplasmic C1-THF synthase and the gene encoding the monofunctional 10-formyl-THF synthetase from Clostridium acidiurici.

Biokhimiia, 1989 Mar, 54(3), 387 - 95
{A new type of Clostridium thermocellum endoglucanase produced by the recombinant strain of E . coli . Some properties and identification in donor cells}; Mel'nik MS et al.; The properties of endoglucanase produced by the recombinant strain of E . coli carrying plasmid pCU 104 with a 2.9 kb insert of chromosomal DNA of C . thermocellum encoding the multiple forms of the 35.5 kD polypeptide (pI 4.3-4.7) were studied . The enzyme has a broad pH optimum of activity (6.0-7.5) . The half-inactivation time for different forms of the enzyme at 65 degrees C is similar and is equal to 25-30 minutes . The enzyme is related to endoglucanases weakly adsorbed on cellulose (Kp = 0.065 1/g) . Hydrolysis of microcrystalline cellulose is completed within 7 days (7-9%) and is accompanied by the formation of cellobiose and cellotriose . The enzyme splits dyed lichenan (mixed 1,3-1,4-beta-glucane) at a higher rate than the dyed CM-cellulose . A guinea pig antiserum to enzyme isoforms with a pI of 4.46-4.54 was obtained . Using direct solid phase immunoenzymatic analysis, it was demonstrated that all the enzyme isoforms under study (pI 4.3-4.7) are immunologically related (serum titers for different enzyme isoforms vary from 1:20,000 to 1:50,000) . In the original culture fluid of C . thermocellum, the antigen related to the enzyme isolated from the recombinant strain was unobserved . However, SDS-PAAG electrophoresis of SDS- and mercaptoethanol-treated culture fluids revealed among 11 protein bands at least 4 antigens interacting with antibodies (Mr = 107, 76, 67 and 37 kD), although their antibody titers were far lower and did not exceed 1:300-1:500 . The cumulative data suggest that the endoglucanase under study is not identical to the earlier described enzymes encoded by the cel A- and ceI B-genes of C . thermocellum.

Dtsch Tierarztl Wochenschr, 1989 Mar, 96(3), 140 - 3
{Penile inflammation in ganders}; Behr KP et al.; Since 1987 penis-inflammation and -prolapse were observed in north-german breeder-geese . Up to 28% of the ganders showed local symptoms . In the females no clinical signs of cloaca-inflammation were seen . The general condition of the birds was good and there was no increased mortality . Egg production and fertility were not influenced . Bacteriological examinations of the altered penis-tissues revealed in different frequency microorganisms belonging to the genera Mycoplasma, Candida, Bacteroides, Clostridium, Streptococcus, Micrococcus, Staphylococcus, Corynebacterium, Escherichia, Proteus and Pseudomonas . Within 70 days only one third of the diseased ganders recovered completely, the others still showed penis-necrosis or -deformation . It is recommended to examine all ganders prior to each sexual season and to eliminate affected birds.

Appl Environ Microbiol, 1989 Mar, 55(3), 656 - 60
Combined effect of water activity and pH on inhibition of toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes; Dodds KL; The effects of water activity (aw, 0.955 to 0.970), pH (4.75 to 5.75), and storage time (up to 60 days) on toxin production by Clostridium botulinum in cooked, vacuum-packed potatoes were studied by using factorial design experiments and most-probable-number methodology . Samples were inoculated with 10(3), 10(4), or 10(5) spores of a mixture of five type A and five proteolytic type B strains, incubated at 25 degrees C, and analyzed for toxin production . Toxin was produced at pH levels of greater than or equal to 4.75 when the aw was greater than or equal to 0.970, pH greater than 5.25 when the aw was 0.965, and pH greater than or equal to 5.75 at an aw of 0.960 . No toxin was detected when the aw was 0.955 . The probability of toxigenesis was significantly affected (P less than 0.0001) by storage time, aw, pH, and the interactions aw.pH and aw.storage time . The response to a decrease in pH was linear, while the response to a decrease in aw was curvilinear . Using multiple linear regression, equations were derived which could predict the length of time until toxin production and the probability of toxigenesis by a single spore under defined conditions.

Appl Environ Microbiol, 1989 Mar, 55(3), 559 - 67
Occurrence and distribution of Vibrio spp., Listonella spp., and Clostridium botulinum in the Seto Inland Sea of Japan; Venkateswaran K et al.; The distribution of Vibrio species in samples of surface water, bottom water (water 2 m above the sediment), and sediment from the Seto Inland Sea was studied . A simple technique using a membrane filter and short preenrichment in alkaline peptone water was developed to resuscitate the injured cells, followed by plating them onto TCBS agar . In addition, a survey was conducted to determine the incidence of Clostridium botulinum in sediment samples . Large populations of heterotrophs were found in surface water, whereas large numbers of total vibrios were found in bottom water . In samples from various water sampling regions, high counts of all bacterial populations were found in the inner regions having little exchange of seawater when compared with those of the open region of the inland sea . In the identification of 463 isolates, 23 Vibrio spp . and 2 Listonella spp . were observed . V . harveyi was prevalent among the members of the Vibrio genus . Vibrio species were categorized into six groups; an estimated 20% of these species were in the so-called "pathogenic to humans" group . In addition, a significant proportion of this group was hemolytic and found in the Bisan Seto region . V . vulnificus, V . fluvialis, and V . cholerae non-O1 predominated in the constricted area of the inland sea, which is eutrophic as a result of riverine influence . It was concluded that salinity indirectly governs the distribution of total vibrios and analysis of variance revealed that all bacterial populations were distributed homogeneously and the variance values were found to be significant in some water sampling regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Vet Clin North Am Food Anim Pract, 1989 Mar, 5(1), 145 - 57
Infectious diseases of New-World camelids (NWC); Thedford TR et al.; Although there are notable infectious conditions that are capable of producing clinical disease in the NWC, overall, these species are quite healthy . Of the bacterial diseases, enterotoxemia caused by Clostridium perfringens types C and D would be deemed the most significant in North America, while type A also would be regarded as important in South America . Other important bacterial infections of potential concern are tuberculosis, Johne's disease, anthrax, malignant edema, actinomycosis, tetanus, and the South American condition referred to as alpaca fever, which, to date, has not been observed in North America . Fungal infections include classical ringworm, principally caused by Trichophyton spp., and the cases of coccidioidomycosis that are associated with the arid desert lands of the southwestern United States . Most notable of naturally occurring viral infections in the NWC would be rabies, ecthyma, and a recently described blindness neuropathy that has been associated with the equine herpesvirus I . NWC can be infected experimentally with agents causing hoof-and-mouth disease and vesicular stomatitis, but naturally occurring cases do not seem to occur . Serological evidence of exposure to many viral agents, including blue tongue, parainfluenza 3, bovine respiratory syncytial virus, bovine herpesvirus I, bovine viral diarrhea, influenza A, and rotavirus, has been demonstrated; however, no clinical disease associated with these agents, as yet, is apparent.

Biol Reprod, 1989 Mar, 40(3), 615 - 21
Alteration of the spermatozoal glycocalyx and its effect on duration of fertility in the fowl (Gallus domesticus); Froman DP et al.; The hypothesis that sialic acid has a role in spermatozoal sequestration within the hen's oviduct was tested by treating spermatozoa with Clostridium perfringens neuraminidase . Spermatozoal content of sialic acid ranged from 94 to 135 micrograms per 10(9) spermatozoa (n = 12 roosters) . Spermatozoa contained 80% of total seminal sialic acid (coefficient of variation = 4.6%) . Spermatozoal sialic acid content was reduced by 18% when 10(9) spermatozoa were incubated at pH 6.5 with 10 IU neuraminidase activity (Type V, Sigma Chemical Co.) . Such treatment had no effect on spermatozoal viability as evidenced by ethidium bromide uptake . However, treatment of spermatozoa with neuraminidase prior to intravaginal insemination reduced fertility by 24 percentage units (p less than 0.001) . In contrast, when similarly treated spermatozoa were deposited in the magnum via laparatomy, fertility was not affected (p greater than 0.05) . The preceding work was done with neuraminidase prepared by salt fractionation (Type V, Sigma Chemical Co.) . Type V neuraminidase was absorbed to diethylaminoethyl-Sephacel and then eluted with a stepwise KCl gradient . Treatment of spermatozoa with this preparation of neuraminidase (10 IU/10(9) spermatozoa) prior to intravaginal insemination reduced fertility by 19 percentage units (p less than 0.001) . Decreased fertility could not be attributed to contamination of neuraminidase preparation with proteolytic activity . We conclude that spermatozoal sialic acid has a role in spermatozoal sequestration within the hen's utero-vaginal glands.

Mol Microbiol, 1989 Mar, 3(3), 383 - 92
Phospholipase C and haemolytic activities of Clostridium perfringens alpha-toxin cloned in Escherichia coli: sequence and homology with a Bacillus cereus phospholipase C; Leslie D et al.; The Clostridium perfringens alpha-toxin (phospholipase C) gene (cpa) has been cloned and expressed in Escherichia coli . The biological activities of the cloned gene product have been analysed and the complete nucleotide sequence of the cpa gene has been determined . The cloned cpa gene product, which is exported to the periplasm in E . coli, possesses both phospholipase C and haemolytic activities . Haemolysis is not apparent when cell extracts are incubated with isotonic suspensions of sheep erythrocytes, but can be detected and quantified readily when dilutions of the same extracts are placed in wells in sheep-blood agar plates . Like other sequenced clostridial genes, the cpa gene has a high AT content (66.4%), exhibits a strong bias for using codons with A or T in the wobble position, and the 350 base pairs upstream from the gene have a significantly higher AT content (79.5%) than the coding region . The cpa gene encodes a 398 amino acid polypeptide with a deduced molecular weight of 45,481 D . This is very similar to the estimated molecular weight (Mr) of the cpa primary gene product expressed in an in vitro transcription-translation system (Mr 46,000), but larger than the cpa gene product detected in E . coli minicells, E . coli whole cells or in C . perfringens cells (Mr 43,000), suggesting post-translational processing . The 28 N-terminal residues of the deduced alpha-toxin sequence possess the consensus features of a signal peptide and may be removed during secretion . The deduced alpha-toxin sequence shares significant structural homology with the phosphatidylcholine-preferring phospholipase C of Bacillus cereus.

J Bacteriol, 1989 Mar, 171(3), 1346 - 54
Isolation of an atypically small lipoamide dehydrogenase involved in the glycine decarboxylase complex from Eubacterium acidaminophilum; Freudenberg W et al.; The lipoamide dehydrogenase of the glycine decarboxylase complex was purified to homogeneity (8 U/mg) from cells of the anaerobe Eubacterium acidaminophilum that were grown on glycine . In cell extracts four radioactive protein fractions labeled with D-{2-14C}riboflavin could be detected after gel filtration, one of which coeluted with lipoamide dehydrogenase activity . The molecular mass of the native enzyme could be determined by several methods to be 68 kilodaltons, and an enzyme with a molecular mass of 34.5 kilodaltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . Immunoblot analysis of cell extracts separated by sodium dodecyl sulfate-polyacrylamide or linear polyacrylamide gel electrophoresis resulted in a single fluorescent band . NADPH instead of NADH was the preferred electron donor of this lipoamide dehydrogenase . This was also indicated by Michaelis constants of 0.085 mM for NADPH and 1.1 mM for NADH at constant lipoamide and enzyme concentrations . The enzyme exhibited no thioredoxin reductase, glutathione reductase, or mercuric reductase activity . Immunological cross-reactions were obtained with cell extracts of Clostridium cylindrosporum, Clostridium sporogenes, Clostridium sticklandii, and bacterium W6, but not with extracts of other glycine- or purine-utilizing anaerobic or aerobic bacteria, for which the lipoamide dehydrogenase has already been characterized.

Kansenshogaku Zasshi, 1989 Mar, 63(3), 268 - 72
{Infant botulism was confirmed in Ehime Prefecture}; Nabeya T et al.; The third case of infant botulism in Japan is reported . A four-month-old baby boy suddenly had weakness of suckling force, constipation and generalized hypotonicity . He was the product of normal gestation, labor and delivery . Growth and development were normal until he was nineteen weeks old . He received fruit-juice and honey daily . By bacteriological examination, Clostridium botulism type A was isolated from his feces and the honey which he had received . Type A toxin was detected from his feces but not from his serum.

Gene, 1989 Feb 20, 75(2), 349 - 54
Nucleotide sequence analysis and expression studies of a chloramphenicol-acetyltransferase-coding gene from Clostridium perfringens; Steffen C et al.; The nucleotide sequence of a CmR determinant, located on the Clostridium perfringens plasmid pIP401, was determined and its gene product was identified as chloramphenicol acetyltransferase (CAT) . The cat structural gene is preceded by transcription-initiation signals characteristic for Escherichia coli sigma 70 or Bacillus subtilis sigma 43 promoters . By promoter probing in the heterologous hosts the direction of transcription of the clostridial cat gene was analysed and the cat mRNA start point was determined in vitro using the RNA polymerases of E . coli and B . subtilis . Comparison of the amino acid sequences of C . perfringens CAT and other CAT proteins of Gram-positive and Gram-negative origin shows a remarkable degree of homology between the various enzymes.

Anaesthesist, 1989 Feb, 38(2), 89 - 94
{Monitoring critically ill patients during transport by helicopter using a patient with abdomen apertum as an example}; Mertzlufft FO et al.; Noninvasive continuous monitoring systems are newly emerging as an important means of monitoring during transports in emergency care, e.g . transportation by helicopter . While automatic oscillometric blood pressure monitors have been used in the perioperative area for some time, a similar development can be observed in the field of emergency care and transportation with the availability of light, portable and battery operated systems . For monitoring adequate oxygenation, pulse oximeters have recently been brought into discussion for both the perioperative period and the transport of critically ill patients . In contrast to well-established monitoring techniques during helicopter transports (ECG, inspection, manually measured blood pressure (BP), pulse oximetry reveals an oxygen deficiency due to respiratory and cardiocirculatory problems, enabling precious time to be saved . This concept is illustrated during the helicopter transport of a critically ill patient with abdomen apertum caused by Clostridium perfringens infection . Even with a critical look at the already described mishaps of this method--e.g . overestimation of true O2 saturation (sO2) and additional overestimation caused by Hb-derivatives--pulse oximetry was found to be superior to the established monitoring techniques . Furthermore, oscillometric blood pressure detection was very satisfactory during the 30-min helicopter transport . Based on our results, we believe pulse oximetry and automatic oscillometric BP-measurement to be useful for monitoring during transports in helicopters, thus improving patient safety.

Appl Environ Microbiol, 1989 Feb, 55(2), 323 - 9
Coenzyme A transferase from Clostridium acetobutylicum ATCC 824 and its role in the uptake of acids; Wiesenborn DP et al.; Coenzyme A (CoA) transferase from Clostridium acetobutylicum ATCC 824 was purified 81-fold to homogeneity . This enzyme was stable in the presence of 0.5 M ammonium sulfate and 20% (vol/vol) glycerol, whereas activity was rapidly lost in the absence of these stabilizers . The kinetic binding mechanism was Ping Pong Bi Bi, and the Km values at pH 7.5 and 30 degrees C for acetate, propionate, and butyrate were, respectively, 1,200, 1,000, and 660 mM, while the Km value for acetoacetyl-CoA ranged from about 7 to 56 microM, depending on the acid substrate . The Km values for butyrate and acetate were high relative to the intracellular concentrations of these species; consequently, in vivo enzyme activity is expected to be sensitive to changes in those concentrations . In addition to the carboxylic acids listed above, this CoA transferase was able to convert valerate, isobutyrate, and crotonate; however, the conversion of formate, n-caproate, and isovalerate was not detected . The acetate and butyrate conversion reactions in vitro were inhibited by physiological levels of acetone and butanol, and this may be another factor in the in vivo regulation of enzyme activity . The optimum pH of acetate conversion was broad, with at least 80% of maximal activity from pH 5.9 to greater than 7.8 . The purified enzyme was a heterotetramer with subunit molecular weights of about 23,000 and 25,000.

Appl Environ Microbiol, 1989 Feb, 55(2), 317 - 22
Phosphotransbutyrylase from Clostridium acetobutylicum ATCC 824 and its role in acidogenesis; Wiesenborn DP et al.; Phosphotransbutyrylase (phosphate butyryltransferase {EC 2.3.1.19}) from Clostridium acetobutylicum ATCC 824 was purified approximately 200-fold to homogeneity with a yield of 13% . Steps used in the purification procedure were fractional precipitation with (NH4)2SO4, Phenyl Sepharose CL-4B chromatography, DEAE-Sephacel chromatography, high-pressure liquid chromatography with an anion-exchange column, and high-pressure liquid chromatography with a hydrophobic-interaction column . Gel filtration and denaturing gel electrophoresis data were consistent with a native enzyme having eight 31,000-molecular-weight subunits . Within the physiological range of pH 5.5 to 7, the enzyme was very sensitive to pH change in the butyryl phosphate-forming direction and showed virtually no activity below pH 6 . This finding indicates that a change in internal pH may be one important factor in the regulation of the enzyme . The enzyme was less sensitive to pH change in the reverse direction . The enzyme could use a number of substrates in addition to butyryl coenzyme A (butyryl-CoA) but had the highest relative activity with butyryl-CoA, isovaleryl-CoA, and valeryl-CoA . The Km values at 30 degrees C and pH 8.0 for butyryl-CoA, phosphate, butyryl phosphate, and CoASH (reduced form of CoA) were 0.11, 14, 0.26, and 0.077 mM, respectively . Results of product inhibition studies were consistent with a random Bi Bi binding mechanism in which phosphate binds at more than one site.

Antimicrob Agents Chemother, 1989 Feb, 33(2), 215 - 22
In vitro antibacterial activity of SM-7338, a carbapenem antibiotic with stability to dehydropeptidase I; Edwards JR et al.; SM-7338, a new carbapenem antibiotic, was demonstrated to have potent antibacterial activity against a broad spectrum of aerobes, including Staphylococcus aureus, beta-hemolytic streptococci, Streptococcus pneumoniae, Haemophilus influenzae, Neisseria spp., members of the family Enterobacteriaceae, Pseudomonas spp., and gram-positive and gram-negative anaerobes in a collection of 1,102 unselected clinical isolates . At a concentration of 0.5 micrograms/ml, SM-7338 inhibited 90% of these strains . The spectrum of activity of ceftazidime and cefotaxime was more limited, and many of the Enterobacteriaceae and Pseudomonas spp . were resistant to these agents, piperacillin, or gentamicin . A collection of ofloxacin-resistant strains was inhibited by SM-7338 or imipenem at 4 micrograms/ml . SM-7338 was more active than metronidazole and clindamycin against anaerobes . Of the carbapenems, imipenem had greater activity against staphylococci but SM-7338 was much more active against Haemophilus, Branhamella, and Neisseria spp . and all genera of Enterobacteriaceae tested . The MIC of SM-7338 for 90% of these strains ranged from less than or equal to 0.008 to 0.13 micrograms/ml . When tested against 124 strains of Pseudomonas aeruginosa, SM-7338 inhibited 76% at 0.5 microgram/ml but imipenem inhibited only 15% at this concentration . Both carbapenems exhibited similar activities against Bacteroides spp., but SM-7338 was more active than imipenem against Clostridium spp . The MBC of SM-7338 was most commonly the same as or twice the MIC . SM-7338 and imipenem showed excellent activities against bacteria elaborating chromosome- or plasmid-mediated beta-lactamases, including those conferring resistance to broad-spectrum cephalosporins . The activity of SM-7338 was generally unaffected by the culture medium used, pH, 25% human serum, and inoculum size, but the susceptibility of Xanthomonas maltophilia was medium dependent.

Nippon Juigaku Zasshi, 1989 Feb, 51(1), 169 - 76
Association between the cell-wall peptidoglycan and the progenitor toxin of Clostridium botulinum type C; Hyun SH et al.; Clostridium botulinum type C progenitor toxin with a molecular weight of 500 k daltons (C1 L toxin), purified from the bacterial cells, bound at pH 2 to the cell-wall peptidoglycan derived from certain strains . The carbohydrate moiety of the peptidoglycan contained arabinose and galactose at a certain ratio, both of which may directly be associated with the binding . The binding, being dependent on the quality and quantity of the sugars, enhanced the oral toxicity of the toxin to the chicken as well as the mouse.

Infect Control Hosp Epidemiol, 1989 Feb, 10(2), 65 - 9
Anaerobic bacteremia in patients with acute leukemia; Brown EA et al.; We reviewed 402 hospital admissions of patients with acute leukemia to define the frequency and characteristics of anaerobic bacteremia in this patient population . Six (5.2%) of the 116 septicemia episodes documented in these patients were caused by anaerobes (Bacteroides species, 3; Fusobacterium species, 2; and Clostridium tertium, 1); two of these episodes were polymicrobial . Five patients had had prior bacteremia . All six patients were receiving broad-spectrum antibiotics, including an anti-pseudomonal penicillin, at the time of the episode . In each instance, the absolute granulocyte count was 0/mm3 . Five patients had clinically apparent sources of infection, including perirectal abscess, gastrointestinal bleeding, or Clostridium difficile-associated diarrhea . Anaerobic bacteremia is an infrequent occurrence in granulocytopenic patients with acute leukemia, but may occur when there is obvious disruption of normal gastrointestinal anatomic barriers.

Arch Intern Med, 1989 Feb, 149(2), 455 - 6
Clostridial endocarditis . Report of a case caused by Clostridium bifermentans and review of the literature; Kolander SA et al.; A case of Clostridium bifermentans endocarditis occurred in a 23-year-old man who was an intravenous drug user . There was no history of preexisting valvular heart disease . He was initially treated with high-dose penicillin G potassium but remained bacteremic for a ten-day period . The bacteremia resolved when the therapy was changed to metronidazole hydrochloride . A review of the 16 reported cases of clostridial endocarditis showed no predisposing host factor to the development of the disease . Penicillin is the treatment of choice for clostridial endocarditis, but metronidazole should be considered as an alternate therapy for treatment that fails.

J Clin Microbiol, 1989 Feb, 27(2), 361 - 3
Characterization of anaerobic bacteria by using a commercially available rapid tube test for glutamic acid decarboxylase; Banks ER et al.; A rapid glutamic acid decarboxylase microdilution test for presumptive identification of certain anaerobic bacteria was marketed recently by Carr-Scarborough Microbiologicals, Inc., Stone Mountain, Ga . The test was evaluated with 474 clinical isolates, representing 11 genera and 54 species, and was found to be a useful aid in the presumptive identification of Bacteroides fragilis, B . distasonis, B . vulgatus, B . thetaiotaomicron, B . ovatus, B . uniformis, B . eggerthii, Clostridium perfringens, C . barati, C . sordellii, and Eubacterium limosum.

Appl Environ Microbiol, 1989 Feb, 55(2), 360 - 5
Construction of an Escherichia coli-Clostridium perfringens shuttle vector and plasmid transformation of Clostridium perfringens; Kim AY et al.; A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C . perfringens plasmid pHB101 (3.1 kb) . The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106 . The resulting shuttle vector, designated pAK201, is 8 kb in size and codes for resistance to 20 micrograms of chloramphenicol per ml in both E . coli and C . perfringens . Following shuttle vector construction in E . coli, plasmid pAK201 was transformed into E . coli HB101 and C . perfringens ATCC 3624A, using intact cell electroporation . The transformation frequencies were 10(6) and 10(4) transformants per microgram of DNA in E . coli and C . perfringens, respectively . Restriction enzyme analysis of the chimera isolated from transformants of both microorganisms suggested that the plasmids were identical . Reciprocal transformation experiments in E . coli and C . perfringens indicated no difference in transformation frequency . Plasmid pAK201 was stable in C . perfringens following repeated transfer in the absence of chloramphenicol pressure . The restriction map of plasmid pAK201 shows six unique cut sites which should be useful for future genetic analysis and C . perfringens gene library construction.

Biol Chem Hoppe Seyler, 1989 Feb, 370(2), 141 - 9
{Cleavage and synthesis of sialic acids with aldolase . NMR studies of stereochemistry, kinetics, and mechanisms}; Baumann W et al.; 1H-NMR spectroscopy was used to study cleavage and synthesis of N-acetyl- and N-glycoloyl-D-neuraminic acid by Clostridium perfringens aldolase . Whereas the alpha-anomers of Neu5Ac and Neu5Gc serve as substrate in the cleavage reaction, alpha-ManNAc and alpha-ManNGc are its primary products . The same alpha-anomers are needed by the aldolase for the synthesis of Neu5Ac and Neu5Gc . During the enzyme reaction in D2O both H-atoms at C-3 of Neu5Ac are exchanged by deuterium, H-3e reacting faster than H-3a . Rate constants and concentrations at equilibrium of reactants are temperature- and pH-dependent: The amount of Neu5Ac in equilibrium increases with decreasing temperature and increasing pH-value . Based on these results a mechanism of aldolase action is discussed.

Infect Immun, 1989 Feb, 57(2), 574 - 81
Preliminary evidence that Clostridium perfringens type A enterotoxin is present in a 160,000-Mr complex in mammalian membranes; Wnek AP et al.; Clostridium perfringens type A 125I-enterotoxin (125I-CPE) was bound to rabbit intestinal brush border membranes (BBMs) or Vero cells and then solubilized with 3-{(3-cholamidopropyl)dimethyl-ammonio}-1-propanesulfonate (CHAPS) . Solubilized radioactivity was analyzed by gel filtration chromatography on a Sepharose 4B column or by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) without sample boiling and autoradiography . Specifically bound 125I-CPE extracted from either BBMs or Vero cells was primarily associated with a complex of approximately 160,000 Mr . The CPE complex was partially purified by gel filtration or SDS-PAGE without sample boiling . SDS-PAGE analysis with sample boiling of the partially purified 125I-CPE complex from Vero cells or BBMs suggested that CPE complex contains both a 50,000-Mr protein and a 70,000-Mr protein in approximately equimolar amounts . This result is supported by affinity chromatography with CPE immobilized on Sepharose 4B, which showed the specific interaction of similar size proteins with CPE . The simplest explanation for these results is that CPE (Mr 35,000) interacts with 50,000-Mr and 70,000-Mr eucaryotic proteins to form a membrane-dependent complex of approximately 160,000 Mr . These results suggest that the receptor or target site(s) or both for CPE are similar in both BBMs and Vero cells . The significance of these findings in terms of CPE binding, insertion, and biologic action is discussed.

Infect Immun, 1989 Feb, 57(2), 468 - 76
Cloning and expression of the phospholipase C gene from Clostridium perfringens and Clostridium bifermentans; Tso JY et al.; The phospholipase C gene from Clostridium perfringens was isolated, and its sequence was determined . It was found that the structural gene codes for a protein of 399 amino acid residues . The NH2-terminal residues have the typical features of a signal peptide and are probably cleaved after secretion . Escherichia coli cells harboring the phospholipase C gene-containing plasmid expressed high levels of this protein in the periplasmic space . Phospholipase C purified from E . coli transformants was enzymatically active, hemolytic to erythrocytes, and toxic to animals when injected intravenously . The phospholipase C gene from a related organism, Clostridium bifermentans, was also isolated . The two phospholipase C genes were found to be 64% homologous in coding sequence . The C . bifermentans protein, however, was 50-fold less active enzymatically than the C . perfringens enzyme.

Infect Immun, 1989 Feb, 57(2), 367 - 76
Molecular cloning and nucleotide sequence of the alpha-toxin (phospholipase C) of Clostridium perfringens; Titball RW et al.; A fragment of DNA containing the gene coding for the phospholipase C (alpha-toxin) of Clostridium perfringens was cloned into Escherichia coli . The cloned DNA appeared to code only for the alpha-toxin and contained both the coding region and its associated gene promoter . The nucleotide sequence of the cloned DNA was determined, and an open reading frame was identified which encoded a protein with a molecular weight of 42,528 . By comparison of the gene sequence with the N-terminal amino acid sequence of the protein, a 28-amino-acid signal sequence was identified . The gene promoter showed considerable homology with the E . coli sigma 55 consensus promoter sequences, and this may explain why the gene was expressed by E . coli . The cloned gene product appeared to be virtually identical to the native protein . A 77-amino-acid stretch that was close to the N terminus of the alpha-toxin showed considerable homology with similarly located regions of the Bacillus cereus phosphatidylcholine, preferring phospholipase C and weaker homology with the phospholipase C from Pseudomonas aeruginosa.

N Engl J Med, 1989 Jan 26, 320(4), 204 - 10
Nosocomial acquisition of Clostridium difficile infection; McFarland LV et al.; We studied the acquisition and transmission of Clostridium difficile infection prospectively on a general medical ward by serially culturing rectal-swab specimens from 428 patients admitted over an 11-month period . Immunoblot typing was used to differentiate individual strains of C . difficile . Seven percent of the patients (29) had positive cultures at admission . Eighty-three (21 percent) of the 399 patients with negative cultures acquired C . difficile during their hospitalizations . Of these patients, 52 (63 percent) remained asymptomatic and 31 (37 percent) had diarrhea; none had colitis . Patient-to-patient transmission of C . difficile was evidenced by time-space clustering of incident cases with identical immunoblot types and by significantly more frequent and earlier acquisition of C . difficile among patients exposed to roommates with positive cultures . Of the hospital personnel caring for patients with positive cultures, 59 percent (20) had positive cultures for C . difficile from their hands . The hospital rooms occupied by symptomatic patients (49 percent) as well as those occupied by asymptomatic patients (29 percent) were frequently contaminated . Eighty-two percent of the infected cohort still had positive cultures at hospital discharge, and such patients were significantly more likely to be discharged to a long-term care facility . We conclude that nosocomial C . difficile infection, which was associated with diarrhea in about one third of cases, is frequently transmitted among hospitalized patients and that the organism is often present on the hands of hospital personnel caring for such patients . Effective preventive measures are needed to reduce nosocomial acquisition of C . difficile.

Biochim Biophys Acta, 1989 Jan 16, 978(1), 85 - 90
Curvature and composition-dependent lipid asymmetry in phosphatidylcholine vesicles containing phosphatidylethanolamine and gangliosides; Thomas PD et al.; The effect of curvature on transbilayer lipid asymmetry in vesicles is investigated using vesicles of different sizes (30-140 nm) prepared by sonication and polycarbonate filter extrusion techniques . The transbilayer distributions of phosphatidylethanolamine and gangliosides are measured using 2,4,6-trinitrobenzenesulphonic acid and Clostridium perfringens neuraminidase as non-penetrating probes, respectively . The distribution of phosphatidylethanolamine in a phosphatidylcholine/phosphatidylethanolamine (4:1, molar ratio) system is more or less symmetric and curvature seems to have little effect . However, the distribution of gangliosides in a phosphatidylcholine/ganglioside (10:1, molar ratio) system is asymmetric in favour of the outer layer in smaller vesicles, the asymmetry disappearing as the degree of curvature decreases . In a phosphatidylcholine/phosphatidylethanolamine/ganglioside (8:2:1, molar ratio) system, both phosphatidylethanolamine and gangliosides distribute asymmetrically, indicating a composition-dependent asymmetric distribution of phosphatidylethanolamine . In this system asymmetry also increases with increasing curvature . The asymmetric distribution of gangliosides in vesicles of low curvature may be due to their long headgroup and larger headgroup surface area in accordance with the theoretical predictions of Israelachvili et al . (Biochim . Biophys . Acta 470 (1977) 185-201).

J Am Vet Med Assoc . 1989 Jan 15;194(2):272.
Treatment of a calf with Clostridium chauvoei infection; Hall KE; In a small beef cattle herd, two 2- to 3-month-old calves had died suddenly . A 2.5-month-old calf, with Clostridium chauvoei infection of the right hip and stifle region, was treated successfully with procaine penicillin G . The herd had not been vaccinated against any disease.

J Biol Chem, 1989 Jan 15, 264(2), 1027 - 35
Novel O-linked carbohydrate chains in the cellulase complex (cellulosome) of Clostridium thermocellum . 3-O-Methyl-N-acetylglucosamine as a constituent of a glycoprotein; Gerwig GJ et al.; Alkaline borohydride treatment of the cellulosome of Clostridium thermocellum yielded two major oligosaccharide-alditols, namely D-Galp-beta(1----4)-D-GalOH and (formula; see text) The compounds, isolated via gel permeation chromatography and high performance liquid chromatography, were analyzed by monosaccharide analysis, methylation analysis, gas-liquid chromatography/mass spectrometry, fast atom bombardment/mass spectrometry, and one- and two-dimensional 500-MHz (COSY, HOHAHA, ROESY) 1H NMR spectroscopy . Sodium dodecyl sulfate-polyacrylamide gel electrophoresis combined with blotting technology indicated that the tetrasaccharide is mainly associated with one of the cellulosome subunits.

FEMS Microbiol Lett, 1989 Jan 15, 48(2), 213 - 7
Influence of substrate carbon on the metabolism of Clostridium thermohydrosulfuricum; Donaduzzi L et al.; The concentration of carbon sources has a significant influence on the growth, carbohydrate uptake and metabolite distribution in Clostridium thermohydrosulfuricum . The growing concentrations of glucose or starch increase the production of ethanol and lactate, the intracellular fructose-1,6-diphosphate (FDP) and the specific activity of lactate dehydrogenase (LDH), but decrease the ethanol/lactate ratio.

Eur J Biochem, 1989 Jan 15, 179(1), 229 - 32
ADP-ribosylation of actin causes increase in the rate of ATP exchange and inhibition of ATP hydrolysis; Geipel U et al.; ADP-ribosylation of skeletal muscle actin by Clostridium perfringens iota toxin increased the rate of exchange of actin-bound {gamma-32P}ATP by unlabelled ATP about twofold . Increased exchange rates were observed with ATP and ATP{gamma S}, much less with ADP but not with AMP or NAD . ADP-ribosylation of skeletal muscle actin reduced "basal" and Mg2+ (1 mM)-induced ATP hydrolysis by about 80% . Similar inhibition of ATP hydrolysis was observed with liver actin ADP-ribosylated by Clostridium botulinum C2 toxin . The data indicate that ADP-ribosylation of actin at Arg-177 largely affects the ATP-binding and ATPase activity.

J Biol Chem, 1989 Jan 15, 264(2), 706 - 12
ADP-ribosylation of 24-26-kDa GTP-binding proteins localized in neuronal and non-neuronal cells by botulinum neurotoxin D; Matsuoka I et al.; Clostridium botulinum D (strain South Africa) produces ADP-ribosyltransferase which modifies eukaryotic 24-26-kDa proteins . ADP-ribosyltransferase activity was associated with a neurotoxin of 150 kDa (Dsa toxin) as confirmed by the elution profile of Dsa toxin from high performance anion-exchange column . The 24-kDa substrate of Dsa toxin-catalyzed ADP-ribosylation was detected in several tissues examined including rat brain, heart, and liver; bovine adrenal medulla; sea urchin eggs; electric organs of electric fish; and cell lines of neural (N18, N1E115, NS20Y, NG108, PC12, and C6) and non-neural (3T3) origins, suggesting its ubiquitous localization in eukaryotic cells . On the other hand, the 26-kDa substrate was detected only in membrane fractions of neural tissues and neuronal cells, suggesting its specific localization in membrane of nerve terminals . ADP-ribosylation of both the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane was potentiated by either divalent cations or guanine nucleotides, whereas adenine nucleotides did not affect the ADP-ribosylation reaction . Trypsin digestion of the 24-kDa substrate in PC12 membrane and the 24-26-kDa substrates in rat brain membrane extract produced different tryptic fragments indicative of the structural difference between the 24- and 26-kDa substrates . Both the 24- and 26-kDa substrates were less sensitive to trypsin digestion before being ADP-ribosylated by Dsa toxin than after, suggesting the conformational alterations of the 24-26-kDa proteins induced by ADP-ribosylation . These results suggest that Dsa toxin modifies two distinct low molecular mass GTP-binding proteins by ADP-ribosylation to alter their putative function(s).

J Biol Chem, 1989 Jan 5, 264(1), 393 - 401
Human fibroblast collagenase-alpha-macroglobulin interactions . Localization of cleavage sites in the bait regions of five mammalian alpha-macroglobulins; Sottrup-Jensen L et al.; The interaction between human fibroblast collagenase and five mammalian alpha-macroglobulins (human alpha 2-macroglobulin and pregnancy zone protein, rat alpha 1- and alpha 2-macroglobulin, and rat alpha 1-inhibitor 3) differing in primary and quaternary structure has been investigated . Complex formation with each of these alpha-macroglobulins follows the course identified for many other proteinases, i.e . specific limited proteolysis in their bait regions inducing a set of conformational changes resulting in activation of the internal beta-cysteinyl-gamma-glutamyl thiol esters and covalent complex formation . At collagenase: alpha-macroglobulin molar ratios of less than 1:1 3.2-3.6 mol of SH groups appear for 1 mol of collagenase bound to human and rat alpha 2-macroglobulin and to rat alpha 1-macroglobulin . For these alpha-macroglobulins it can be estimated that the overall rate constant of complex formation is greater than 1.10(6) M-1 s-1 while it is much lower for human pregnancy zone protein and rat alpha 1-inhibitor 3 . More than 95% of the complexed collagenase is covalently bound, and sodium dodecyl sulfate gel electrophoresis shows the typical pattern of bands corresponding to reaction products of very high apparent molecular weight . The same pattern is also seen in the covalent (greater than 98%) complex very slowly formed from Clostridium histolyticum collagenase and human alpha 2-macroglobulin . The identification of the sites of specific limited proteolysis in the bait regions of the five alpha-macroglobulins shows that cleavage may take place in sequences that are not related to those identified earlier in the collagens . These results greatly expand the repertoire of sequences known to be cleaved by fibroblast collagenase and suggest that this proteinase has a primary substrate specificity resembling that of the microbial proteinase thermolysin, as it preferentially cleaves at the NH2-terminal side of large hydrophobic residues . In addition, the results highlight the unique structure of the flexible alpha-macroglobulin bait region in that it can accommodate a conformation required by the highly restrictive fibroblasts collagenase . It is suggested that alpha-macroglobulins may play an important role in locally controlling the activity of collagenases and perhaps other proteinases of the extracellular matrix.

Eur J Biochem, 1989 Jan 2, 178(3), 763 - 70
Characterization and application of a thermostable primary transport system: cytochrome-C oxidase from Bacillus stearothermophilus; De Vrij W et al.; Cytochrome-c oxidase from Bacillus stearothermophilus has been purified to homogeneity by detergent extraction followed by DEAE-cellulose, hydroxyapatite- and gel-filtration chromatography . The enzyme is a typical cytochrome-aa3-type oxidase which binds carbon monoxide and is sensitive to classical oxidase inhibitors like cyanide and azide . The purified enzyme is composed of three different subunits (57, 37 and 22 kDa) . The subunit with intermediate molecular mass contains a covalently attached heme-c moiety . The enzyme appeared to be extremely thermostable (inactivation temperature = 81 degrees C) . Highest turnover rates of the reconstituted enzyme were obtained with Saccharomyces cerevisiae cytochrome c or reduced forms of non-physiological electron donors like N,N,N',N'-tetramethyl-p-phenylenediamine and phenazine methosulphate . The reconstituted enzyme can generate a proton-motive force consisting of a high membrane potential and trans-membrane pH gradient . The high electro-motive force of the enzyme (delta p = -180 to -200 mV) indicates that this enzyme functions as a high-capacity electrogenic proton pump . Liposomes containing the purified thermostable and thermoactive cytochrome-c oxidase were fused with membranes from the fermentative bacterium Clostridium acetobutylicum . In the hybrid system a high proton-motive force can be generated upon oxidation of reduced N,N,N',N'-tetramethyl-p-phenylenediamine by the incorporated oxidase which subsequently can be used to drive secondary transport of amino acids . This demonstrates the applicability of the cytochrome-c oxidase to study solute transport in membranes of fermentative bacteria.

J Antibiot (Tokyo), 1989 Jan, 42(1), 94 - 101
Phenelfamycins, a novel complex of elfamycin-type antibiotics . III . Activity in vitro and in a hamster colitis model; Swanson RN et al.; Phenelfamycins A, B, C, E, F and unphenelfamycin make up a recently isolated group of elfamycin-type antibiotics . All of the phenelfamycins were active against Gram-positive anaerobes, including Clostridium difficile . Phenelfamycin A was also active in vitro against Neisseria gonorrhoeae and Streptococci . Phenelfamycin A was found to be effective in prolonging the survival of hamsters in an animal model of C . difficile enterocolitis . After oral administration of phenelfamycin A to hamsters, antibiotic was detected in the caecal contents but not in the blood.

J Am Vet Med Assoc, 1989 Jan 1, 194(1), 69 - 70
Clostridial myositis in a dog; Poonacha KB et al.; Myositis caused by Clostridium septicum was diagnosed in a 3-year-old Doberman Pinscher . The illness was characterized by signs of pain, swelling, and lameness of left forelimb . Despite treatment, the dog died . Necropsy revealed crepitant swelling over the entire left forelimb, thoracic and abdominal wall, and lumbosacral area . Subcutaneous edema and black, emphysematous muscles also were found . Histologically, hemorrhages, congested vessels, and degeneration and necrosis of myofibers with scattered infiltration of neutrophils were seen in the affected muscles.

Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 32 - 6
Cloning and expression of the gene cluster encoding key proteins involved in acetyl-CoA synthesis in Clostridium thermoaceticum: CO dehydrogenase, the corrinoid/Fe-S protein, and methyltransferase; Roberts DL et al.; Acetogenic bacteria fix CO or CO2 by a pathway of autotrophic growth called the acetyl-CoA (or Wood) pathway . Key enzymes in the pathway are a methyltransferase, a corrinoid/Fe-S protein, a disulfide reductase, and a carbon monoxide dehydrogenase . This manuscript describes the isolation of the genes that code for the methyltransferase, the two subunits of the corrinoid/Fe-S protein, and the two subunits of carbon monoxide dehydrogenase . These five genes were found to be clustered within an approximately 10-kilobase segment on the Clostridium thermoaceticum genome . The proteins were expressed at up to 5-10% of Escherichia coli cell protein, and isopropyl beta-D-thiogalactopyranoside had no effect on the levels of expression, implying that the C . thermoaceticum inserts contained transcriptional and translational signals that were recognized by E . coli . The methyltransferase is expressed in E . coli in a fully active dimeric form with a specific activity and heat stability similar to the enzyme expressed in C . thermoaceticum . However, both the corrinoid/Fe-S protein and carbon dioxide dehydrogenase, although expressed in high amounts and with identical subunit molecular weights in E . coli, are inactive and less heat stable than are the native enzymes from C . thermoaceticum.

Dig Dis Sci, 1989 Jan, 34(1), 148 - 9
Clostridium difficile colitis secondary to intravenous vancomycin; Hecht JR et al.; Nearly every known antibiotic has been implicated as a cause of Clostridium difficile colitis . We report the first case resulting from monotherapy with intravenous vancomycin . The patient was on chronic hemodialysis and was treated with intravenous vancomycin for presumed cervical osteomyelitis . After 29 days of therapy he developed abdominal pain and diarrhea and his stool was found to contain both C . difficile and cytotoxin . The patient responded with symptomatic and microbiological recovery to withdrawal of the drug and treatment with oral metronidazole . The prolonged elevation of serum vancomycin levels in patients with renal failure may predispose them to the development of C . difficile colitis.

Am J Med, 1989 Jan, 86(1), 15 - 9
Treatment of antibiotic-associated Clostridium difficile colitis with oral vancomycin: comparison of two dosage regimens; Fekety R et al.; PURPOSE: High-dose (500 mg orally four times daily) vancomycin is considered by many investigators to be the most effective treatment for antibiotic-associated Clostridium difficile colitis . However, a lower dosage of 125 or 150 mg given three or four times a day has become popular, has been shown to be effective, and is less expensive than the high-dose regimen . We therefore decided to compare two vancomycin dosage regimens in a randomized trial . PATIENTS AND METHODS: The study involved 46 hospitalized patients with serious underlying diseases complicated by C . difficile diarrhea or colitis . Patients were assigned (according to a table of random numbers) to treatment with either 125 or 500 mg of vancomycin orally four times daily for an average of 10 days . RESULTS: No significant differences in measurable responses to the two regimens were noted . There were no treatment failures . The mean duration of diarrhea after initiation of therapy was about four days, and almost all patients had no diarrhea after one week . The organism continued to be demonstrated in the stools of about 50 percent of patients for the first few weeks after completion of therapy, and nine (20 percent) patients developed a recurrence of their diarrheal illness . Vancomycin was well tolerated by all patients . CONCLUSION: Since the dose of 125 mg appeared to be as effective as the 500-mg dose, which is more expensive, the 125-mg dose is preferred when vancomycin is used in treatment of this disease, unless the patient is critically ill.

Am J Clin Pathol, 1989 Jan, 91(1), 104 - 6
Fatal acute spontaneous endometritis resulting from Clostridium sordelli; Hogan SF et al.; Intrauterine clostridial infections have historically been associated with puerperal sepsis, often subsequent to instrumented abortions . Isolated reports have been associated with a malignant neoplasm or degenerating leiomyoma, most often after instrumentation, radiation therapy, or chemotherapy . In these cases, well-known risk factors or obvious niduses for growth and causation of disease were identified . The authors report a case of fatal spontaneous endometritis resulting from Clostridum sordellii, which, to their knowledge, has not been previously reported in the literature . Characteristics of this rare virulent human pathogen are discussed.

Gerontology, 1989, 35(2-3), 130 - 6
Anaerobic bacteremia in the elderly; Terpenning MS; Anaerobic bacteremia occurred in 68 patients over the age of 60 in a university hospital . These elderly patients were more likely than younger patients to have an underlying malignancy . Anaerobes involved included Bacteroides fragilis group (64 isolates), Bacteroides melaninogenicus group (11) and Clostridium species (11) . Polymicrobial bacteremia was common, occurring in 32.3% of patients . Mortality in patients who had surgery to remove the source of anaerobes was 43.3%, compared to 81.7% in patients with no surgical treatment . Overall mortality was 66.1%, much higher than noted in younger populations.

Arch Microbiol, 1989, 152(3), 244 - 50
Purification and characterization of the pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum; Meinecke B et al.; The pyruvate-ferredoxin oxidoreductase from Clostridium acetobutylicum was purified to homogeneity and partially characterized . A 9.2-fold purification was achieved in a three step purification procedure: ammonium sulfate fractionation, chromatography on Phenyl Sepharose and on Procion Blue H-EGN12 . The pure enzyme exhibited a specific activity of 25 U/mg of protein . Homogeneity of the pyruvate-ferredoxin oxidoreductase was confirmed by native polyacrylamide gel electrophoresis and sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis . The molecular weight was determined to be 123,000/monomer . The subunit composition of the native enzyme could not be determined because of the instability of the pure enzyme . The pyruvate-ferredoxin oxidoreductase is sensitive to oxygen and dilution during purification . The dilution inactivation could be partially overcome by the addition of 300 microM coenzyme A or 50% ethyleneglycol . A thiamine pyrophosphate content of 0.39 mol per mol of enzyme monomer was found, the iron and sulfur content was 4.23 and 0.91, respectively . The pH-optimum was at pH 7.5 and the temperature optimum was at 60 degrees C . Kinetic constants were measured in the forward reaction . The apparent Km for pyruvate and coenzyme A were 322 microM and 3.7 microM, respectively . With 2-ketobutyrate the pyruvate-ferredoxin oxidoreductase showed 12.5% of the activity compared to pyruvate . No activity was found with 2-ketoglutarate . Ferredoxin from Clostridium pasteurianum could be used as physiological electron acceptor.

Arch Microbiol, 1989, 152(3), 209 - 14
Reisolation and characterization of Clostridium longisporum, a ruminal sporeforming cellulolytic anaerobe; Varel VH; Two strictly anaerobic strains of ruminal cellulolytic bacteria were isolated which are very similar to the original description given for Clostridium longisporum . Vegetative cells were 1 micron wide by 5 to 15 microns long . Subterminal spores were observed only when an insoluble carbon source was provided for growth . Besides cellulose, the organisms fermented cellobiose, glucose, galactose, fructose, mannose, pectin, salicin and sucrose . Xylan and xylose were not fermented . Fermentation products from glucose or alfalfa cell walls included formate, acetate, butyrate, ethanol, H2 and CO2 . The GC content was 23% for one strain and 33% for the other . These isolates hydrolyzed cell wall fractions of alfalfa, in particular, hemicellulose, more rapidly and extensively than other ruminal cellulolytic species examined.

Microbiol Immunol, 1989, 33(4), 287 - 98
Characterization and reassembly of a regular array in the cell wall of Clostridium difficile GAI 4131; Masuda K et al.; The cell wall of Clostridium difficile GAI 4131 was revealed by electron microscopy to have an outer layer composed of a nearly square array and contained the two major proteins with molecular weights of 38 kDa and 42 kDa . The properties and reassembly of the two major proteins into the regular array were investigated . When the isolated cell walls were treated with hydrophobic bond-disrupting agents or a chelating agent specific for Ca2+, the two major proteins were effectively removed and the regularly arranged outer layer disappeared . The amino acid composition of the two major proteins differed from each other . The two major proteins also gave different peptide maps from each other upon proteolysis with Staphylococcus aureus V8 protease . The major proteins solubilized from the isolated cell walls with 8 M urea or 4 M guanidine hydrochloride could be reassembled into open-ended cylinders possessing the native regular pattern by dialysis against neutral buffer containing 5 mM CaCl2 . The reassembled cylinders purified by centrifugation on a Percoll density gradient were composed of almost equal amounts of the 38 kDa and 42 kDa proteins and freed from the other proteins . These results suggest that the regular array in the outer cell wall layer is constructed from the two major cell wall proteins and requires Ca2+ for its assembly.

Arch Microbiol, 1989, 151(4), 287 - 93
Clostridium methylpentosum sp . nov.: a ring-shaped intestinal bacterium that ferments only methylpentoses and pentoses; Himelbloom BH et al.; An intestinal bacterium isolated from a human subject utilized only two methylpentoses (L-rhamnose and L-fucose) and two pentoses (L-lyxose and D-arabinose) as fermentable substrates, among many compounds tested . The isolate was obligately anaerobic and had a distinctive morphology, its cells being rods bent in the shape of rings with the ends slightly overlapping . Single ring-shaped cells and left-handed helical chains of cells were present in cultures . The cells were surrounded by large capsules which appeared as thick, fibrous masses when examined by electron microscopy . Capsules were formed by cells growing in media containing any one of the four fermentable substrates . Terminally located, heat-resistant endospores were formed on plates of an enriched agar medium supplemented with L-rhamnose . End products of L-rhamnose or L-fucose fermentation included acetate, propionate, n-propanol, CO2, and H2 . The isolate represented a new species of Clostridium for which the name Clostridium methylpentosum (type strain R2, ATCC 43829) is proposed . This organism may participate in intestinal digestive processes by metabolizing rhamnose released via the enzymatic depolymerization of dietary pectin.

Toxicon, 1989, 27(4), 403 - 10
Molecular differences between type A botulinum neurotoxin and its toxoid; Singh BR et al.; The neurotoxins (seven serotypes, Mr approximately 150,000) produced by Clostridium botulinum cause the neuroparalytic disease botulism . Prophylaxis, definitive diagnosis and the only effective therapy for botulism depend, at present, on chemically detoxified form(s) of the neurotoxins, i.e . toxoids (immunogens), and the antisera raised with the immunogens . And yet, the toxoids currently used for immunization of humans and animals and for raising antibody are very crude preparations (approximately 90% impure) of the neurotoxins . Hence, the highly heterogenous toxoids were not suitable for physicochemical studies . We have detoxified a pure (greater than 99%) neurotoxin (serotype A) with formaldehyde . The native neurotoxin is composed of two subunit chains (Mr 53,000 and 97,000) . The physicochemical properties of the toxoid (immunogenic in rabbits) were analyzed . The chemical modification produced inter- and intrasubunit covalent links at multiple sites and thus extensive aggregation of the neurotoxin . The secondary structure parameters (alpha-helix, beta-sheet, beta-turn and random coil) of the native protein were not significantly altered . Tertiary structure, as measured by exposure of tyrosine residues and fluorescence quantum yield of tryptophan residues, was considerably altered . The data imply that conformational (topographical) antigenic determinants may not contribute significantly to the serological property of the neurotoxin.

Toxicon, 1989, 27(3), 317 - 23
Dissociation of various biological activities of Clostridium perfringens alpha toxin by chemical modification; Sakurai J et al.; The effect of N-acetylimidazole, tetranitromethane, maleic anhydride and N-ethylmaleimide on various biological activities of Clostridium perfringens alpha (alpha)-toxin was investigated . Treatment of the toxin with N-acetylimidazole, tetranitromethane or maleic anhydride resulted in significant reduction of lethal, hemolytic and platelet-aggregating activities and phospholipase C activity (EY activity), as measured by increased turbidity in egg yolk emulsions . However, EY activity was more resistant to these reagents than lethal, hemolytic or aggregating activities . Phospholipase C activity (PN activity) as measured by hydrolysis of p-nitrophenylphosphorylcholine was retained after treatment with N-acetylimidazole, tetranitromethane or maleic anhydride . The activities of the toxin were not inactivated by treatment with N-ethylmaleimide . These data suggest that alpha-toxin contains multiple sites for biological activities of the toxin.

Mikrobiyol Bul, 1989 Jan, 23(1), 51 - 7
{The distribution of clostridia in soil samples from Bursa}; Senyuz O et al.; In the soil samples taken from different places of Bursa, distribution of the Clostridia were searched by anaerobic jar and immunofluorescence reaction (FAT) . In our study, 122 bacteria belonging to 11 Clostridium species were isolated in 35 soil samples . These strains were identified by studying morphological and biochemical properties, lecithinase C and lipase activities, toxin neutralization characteristics . In addition, FAT were used for 4 Clostridium species.

Arch Microbiol, 1989, 152(4), 377 - 81
Identification of three distinct Clostridium thermocellum xylanase genes by molecular cloning; MacKenzie CR et al.; Three genes coding for xylanase synthesis in Clostridium thermocellum were cloned and expressed in Escherichia coli . Genomic DNA from Clostridium thermocellum was digested to completion with HindIII, BamHI, and SalI . The fragments were ligated into the corresponding sites of pUC19 and transformed into Escherichia coli . Two of the genes encoded for xylanases which depolymerized xylans but were unable to extensively convert these substrates to reducing sugar . The third gene encoded for an enzyme that extensively hydrolyzed xylan . The insert containing the latter gene was subjected to extensive mapping and was found to encode for a xylanase with a molecular weight of approximately 25,000 . The protein product of the cloned gene was obtained in a relatively pure form by heat treatment, ion exchange and gel permeation steps . The enzyme was quite stable to high temperatures with a half-life of 24 h at 70 degrees C.

Langenbecks Arch Chir, 1989, 374(5), 272 - 9
{Duration of the preventive use of antibiotics in colorectal surgery--single administration versus short-term prevention}; Bittner R et al.; The effect of a combination of 4 g mezlocillin and 0.5 g metronidazole for the prophylaxis against infections in a one-shot dose immediately preoperatively compared to a short-time dose of 2 days given to 90 patients with resection of colorectal carcinoma was investigated in a prospective and randomized study . 6 patients developed a wound infection in the early postoperative phase; 4 of these infections (3 were severe, 1 was mild) occurred in the one-shot group and 2 in the short-time prophylaxis group . After more than 20 days postoperatively 3 late infections were observed which had a mild course (2 cases in the one-shot group, 1 case in the short-time prophylaxis group) . All infections were localized in the sacral wound region in patients with abdominoperineal resection . The abdominal wounds healed per primam in each case . Besides those, 26 infections of the urinary tract were observed, which occurred significantly more often after the one-shot dose (40.9%) than with the short-time prophylaxis (18.6%) . Intraoperative smears of the lumen of the bowels showed a remaining bacterial settlement . Besides Bacteroides species, especially Escherichia coli were found among the isolates . Moreover in some cases Clostridium, Klebsiella, Proteus and Pseudomonas could be identified . Smears of the site of operation (sacral/peritoneal cavity) were contaminated in over 50%, above all by Bacteroides species; besides those, E . coli were found most often . The subcutaneous smears showed a growth of the germs only in a few cases . Aerobic bacteria in 93.8%, anaerobic bacteria except for thetaiotaomicron and B . asaccharolyticus in 85.1%.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppl Int J Gynecol Obstet, 1989, 2, 7 - 12; discussion 47-8
A multicenter international study on the activity of sulbactam/ampicillin, ampicillin, and cefoxitin against anaerobic bacteria and introduction of a new model of susceptibility testing in mixed infections; Heizmann WR et al.; Susceptibility of anaerobic clinical isolates from the United States, Canada, and Germany to sulbactam/ampicillin (1 + 2) (SBT/AMP), ampicillin alone (AMP), and cefoxitin (CFX) was determined with a standard agar dilution test . The isolates included 192 strains of Bacteroides fragilis, 132 strains of other Bacteroides spp., and 19 strains of Clostridium spp . Against all species tested, SBT/AMP was more active than AMP or CFX . Results obtained by a new model of associative susceptibility testing indicated that susceptibility testing of single pathogens from polymicrobial infections is not necessarily reflective of the susceptibility of the pathogens at the site of infection . The results suggest that sulbactam is effective in associations of pathogens producing beta-lactamases of Richmond-Sykes types II-V and beta-lactamases of anaerobic bacteria.

Ann Biol Clin (Paris), 1989, 47(2), 67 - 70
Clostridium difficile and its cytotoxin in diarrhoeic stools of hospitalized patients . Toxigenic potential of the isolates; De Barbeyrac B et al.; Cytotoxin assay and culture for Clostridium difficile were performed on 303 diarrhoeic stools from 261 hospitalized patients . Specimens from 42 patients were positive by at least one of the methods, and 40 of them had an antibiotic-associated diarrhoea . The cytotoxin assay was positive in 5 of 7 patients with pseudomembranous colitis . Thirteen had an appropriate response to specific therapy and the remainder have resolved of diarrhoea without C . difficile directed chemotherapy . These findings show the lack of reliability of the cytotoxin assay for the diagnosis of C . difficile antibiotic-associated diarrhoea . The 6 strains isolated from patients with pseudomembranous colitis were examined for enterotoxin by the rabbit ileal loop test: 4 produced both toxins, 2 only enterotoxin . Both toxins could therefore not be essential for the clinical expression of the disease.

Toxicon, 1989, 27(2), 221 - 8
Clostridium botulinum type D neurotoxin: purification and detection; De Jongh KS et al.; A method is reported for the purification of type D botulinum toxin using a combination of low and high pressure ion exchange chromatography . The procedure produced homogeneous toxin in its free form in 3 days, with a specific toxicity in mice of 5.4 x 10(7) LD50/mg protein . Polyclonal antibodies against the pure toxin were raised in rabbits and detected the toxin in both ELISA and western blotting . The antibodies also detected type C1 botulinum toxin using these techniques, confirming the presence of cross-reacting antigenic determinants in these two proteins.

J Rheumatol, 1989 Jan, 16(1), 133 - 5
Clostridium difficile associated reactive arthritis in an HLA-B27 positive female: report and literature review; Mermel LA et al.; A case of Clostridium difficile associated reactive arthritis in an HLA-B27 positive female is reported and compared to 9 other cases . The clinical course of C . difficile associated reactive arthritis is similar to that caused by other enteric pathogens . Therefore, C . difficile should be considered in the differential diagnosis of the reactive arthritides.

J Clin Microbiol, 1989 Jan, 27(1), 190 - 1
Quality control guidelines for testing cefotetan in the reference agar dilution procedure for susceptibility testing of anaerobic bacteria; Zabransky RJ et al.; Reference values for quality control of in vitro susceptibility tests with cefotetan against anaerobic bacteria were determined in two independent multilaboratory studies with the approved National Committee for Clinical Laboratory Standards agar dilution method and three control strains (Bacteroides fragilis ATCC 25285, Bacteroides thetaiotaomicron ATCC 29741, and Clostridium perfringens ATCC 13124) . The results of the two studies were in agreement . The recommended MIC control limits for B . fragilis ATCC 25285 and B . thetaiotaomicron ATCC 29741 are 4.0 to 16 micrograms/ml and 32 to 128 micrograms/ml, respectively . MICs for C . perfringens ATCC 13124 were too variable to be useful for controlling tests with cefotetan.

Diabetes, 1989 Jan, 38 Suppl 1, 126 - 8
Protease activity in pancreatic islet isolation by enzymatic digestion; McShane P et al.; Commercial Collagenase* prepared from Clostridium histolyticum is widely used in isolation of pancreatic islets . It is known that the enzyme is very impure and that there are substantial variations in effectiveness between batches . Our studies suggest that one of the impurities of importance in islet isolation is a protease that has not been very well characterized . Comparison of two batches of enzyme, one of which was known to give good yields of islets and the other poor yields, showed that they had very similar activity against collagen (measured by digestion of insoluble collagen followed by assay of soluble products with ninhydrin) but substantially different activities against azocasein as measured by optical density increase (measured by release of dye) . Eighteen batches of Collagenase were examined for efficiency in islet isolation, and the yields obtained correlated with manufacturer's data of activity against casein . The data show that low caseinase activity is associated with performance in islet isolation (r = .5 after adjusting for collagenase activity) . The effect of supplementing a batch of collagenase, known to be poor in isolating islets, with proteolytic enzymes was investigated . Trypsin and papain had apparently no effect, but dispase significantly increased yield . Dispase alone failed to digest pancreas . Size-exclusion high-performance liquid chromatography identified a peak associated with high protease activity and efficiency in islet isolation, having an Mr of approximately 30,000, compared to 78,000 for collagenase . The protease, like collagenase, is inhibited by EDTA . Increased Ca2+ and Mg2+ (up to 10 mM) did not affect activity . Both the protease and collagenase are stable under normal use but are inactivated by heating at 56 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Medicine (Baltimore), 1989 Jan, 68(1), 30 - 7
Clostridium septicum infection and associated malignancy . Report of 2 cases and review of the literature; Kornbluth AA et al.; We report 2 patients with myonecrosis due to Clostridium septicum and associated colon carcinoma and have reviewed the English language literature for all reported cases of atraumatic C . septicum infection . A total of 162 cases of C . septicum infection have been reported . Eighty-one percent of these patients had an associated malignancy . Thirty-four percent of all patients had associated colon carcinoma, while 40% had a hematologic malignancy . Thirty-seven percent of reported patients had an occult malignancy at the time of their infection with C . septicum . In many patients, the portal of entry was found in the large intestine . In a particularly lethal form (79% mortality) of C . septicum infection, known as "distant myonecrosis," infection metastatic from the initial site of infection causes severe myonecrosis, gangrene, and often death within hours of clinical detection . Overall, survival of patients with C . septicum infection is only 35% . Review of all cases of C . septicum infection suggests several conclusions . 1) Patients with malignancy, particularly colonic or hematologic, and patients with cyclic neutropenia who develop signs and symptoms of sepsis, especially with associated findings of abdominal pain or pain in an extremity, should be treated for possible clostridial infection . 2) C . septicum infection does not appear to be a result of a single specific defect in either humoral or cell-mediated immunity . Rather, it may occur in patients who are granulocytopenic and therefore prone to an enterocolitis . 3) Patients in whom an infection with C . septicum is found must undergo a vigorous search for malignancy following acute therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

Rev Elev Med Vet Pays Trop, 1989, 42(2), 169 - 72
Changes in clinical values of cattle infected with Clostridium chauvoei CH3 strain and a local Kad1 strain . Haematological values; el Sawi Mohamed O et al.; Clostridium chauvoei CH3 and Kad1 strains were found to cause marked changes in the blood parameters during the course of blackleg disease . These changes displayed by CH3 were found to be more marked than the local Kad1 strain . Results of changes in the haematological values in calves infected with blackleg organisms, showed an increase in RBC, PCV, Hb and the total leukocyte count . MCHC and MCH remained within normal range values, however, a terminal significant increase of MCV was obtained . Thrombocytes showed a steady drop after infection to the time of death of the animals.

Acta Clin Belg, 1989, 44(4), 228 - 36
In vitro activity of amoxycillin plus clavulanic acid and ticarcillin plus clavulanic acid compared with that of other antibiotics against anaerobic bacteria; Pierard D et al.; The activity of amoxycillin/clavulanic acid (Augmentin) and ticarcillin/clavulanic acid (Timentin) was tested against 303 unselected clinical anaerobic isolates recently collected in seven Belgian university hospitals and compared with that of 11 other antimicrobial agents . Bacteroides spp . accounted for 52.1% of the isolates, Clostridium spp . for 23.4%, anaerobic cocci for 15.5%, nonsporeforming gram-positive bacilli for 4.6% and Fusobacterium spp . for 3.3% . Ticarcillin/clavulanic acid (fixed clavulanic acid concentration of 2 mg/l) was the most active drug with an overall susceptibility rate of 99.7% . Amoxycillin/clavulanic acid (fixed ratio of 2:1) and chloramphenicol inhibited 97.4% of the isolates, metronidazole 95.4%, piperacillin 92.4%, ticarcillin 91.4%, clindamycin 87.8%, cefotetan 81.2%, cefazolin 63.0%, cefuroxime 60.4%, erythromycin 57.8%, penicillin 57.1% and doxycycline 52.1% . beta-lactamases were detected exclusively in Bacteroides spp . isolates (79.1% positive).

Scand J Infect Dis, 1989, 21(6), 733 - 4
Medical implications of nosocomial infection with Clostridium difficile; Eriksson S et al.; 88 patients above the age of 60 years who contracted Clostridium difficile associated diarrhoea (CDAD) between 1979 and 1986, mostly during their stay in hospital were studied retrospectively as regards nursing time and clinical outcome . These patients were compared with 176 control patients who were matched according to diagnosis on admission to hospital, sex, age and underlying disease . Thromboembolic complications occurred in 14% in CDAD patients (controls 4%) (p = 0.0042) . The mortality rate was 21% in CDAD and 7% in controls (p = 0.0009) . The median time in hospital for CDAD patients was 50 days (14 days for controls).

Ann Ist Super Sanita, 1989, 25(2), 327 - 32
{Hospital epidemic of Clostridium difficile diarrhea: demonstration of cross-infection using a typing technic}; Cerquetti M et al.; Two hospital outbreaks of Clostridium difficile diarrhoea in two general surgery units of different hospitals are described . Moreover, the results of a study on the circulation of C . difficile in a neurosurgery unit following two cases of colitis are reported . C . difficile strains isolated from patients and environment have been typed using antimicrobial susceptibility testing and electrophoretic profiles of EDTA-extracted proteins . The majority of strains isolated in each hospital shared the same protein profile and the same pattern of antibiotic susceptibility . This provides strong evidence of cross-infection and hospital acquisition of C . difficile.

FEMS Microbiol Lett, 1989 Jan 1, 48(1), 81 - 6
The complete nucleotide sequence of the glutamine synthetase gene (glnA) of Bacillus subtilis; Nakano Y et al.; The glutamine synthetase (GS) gene from Bacillus subtilis PCI 219 was cloned in Escherichia coli using the vector pBR329 . A plasmid, pSGS2, was isolated from a glnA+ transformant and the cloned GS gene was found to be located in a 3.6 kb DNA fragment . The nucleotide sequence of a 1.8 kb segment encoding the GS was determined . This segment showed an open reading frame which would encode a polypeptide of 444 amino acids . The amino acid sequence of this GS gene product has higher homology with that of the Clostridium acetobutylicum GS than that of the E . coli GS.

Glycoconj J, 1989, 6(3), 349 - 53
Conserved sequences in bacterial and viral sialidases; Roggentin P et al.; The genes of the bacterial sialidases from Clostridium sordellii G12, C . perfringens A99, Salmonella typhimurium LT-2 and Vibrio cholerae 395 sequenced so far were examined for homologies and were compared with sequences of viral sialidases . Each of the bacterial sialidases contains a short sequence of twelve amino-acids, which is repeated at four positions in the protein . All these sequences exhibit significant similarities . Comparing the repeated sequences of the four sialidases, five amino-acids were found to be highly conserved at defined positions: Ser-X-Asp-X-Gly-X-Thr-Trp . Additionally, most of the distances between the four repeated regions are also conserved among the different sialidases . The conserved bacterial sequences show similarity with sialidases of influenza A H7N1 and H13N9.

Biol Met, 1989, 2(3), 135 - 41
Spectroscopic studies of cobalt-substituted ferredoxin from Clostridium pasteurianum; Skjeldal L et al.; Ferredoxin from Clostridium pasteurianum substituted with two Co atoms did not give any cobalt EPR signal at 8 K as isolated, but upon reduction with sodium dithionite, a broad signal appeared with g values that indicate highspin (S = 3/2) Co(II) . These signals were distinct from Co(II)-dithiothreitol signals, and disappeared upon reoxidation with air . Under anaerobic incubation of apoferredoxin with Co(II), a green derivative showed a visible spectrum typical of tetrahedral Co(II)-thiolate coordination, which shifted dramatically upon exposure to air . The 1H-NMR spectrum of the aerobically isolated protein is reported at 300 MHz; magnetic susceptibility measurements were indicative of a diamagnetic species . These spectroscopic studies indicate that Co(II)-substituted ferredoxin is oxidized to low-spin Co(III)-ferredoxin in the presence of sulfide and oxygen . The diamagnetic Co(III) state could reversibly be reduced to highspin Co(II) by sodium dithionite.

Med Dosw Mikrobiol, 1989, 41(3-4), 166 - 9
{Detection of toxins of Clostridium perfringens type A in infected animals by using the ELISA test}; Nowakowska M et al.; The aim of this study was to evaluate the usefulness of ELISA in toxin detection in guinea pigs experimentally infected with toxinogenic strain of Clostridium perfringens type A . The toxin was detected in blood serum and muscles from 12 hours after infection . The results obtained indicate the advantage of ELISA over to date methods used as immunofluorescence or microscopic examination of muscle exudate or sections . ELISA due to its high sensitivity rapidity and specificity allows to detect toxin in guinea pigs before clinical symptoms of gas gangrene are developed.

Nahrung, 1989, 33(9), 895 - 900
The microbiological quality of Erzincan (Savak) Tulum cheese from Turkish retail markets; Kivanc M; Twenty samples of Erzincan (Savak) Tulum cheese were investigated for the microbiological quality and some chemical analyses . Cheese was characterized by moisture content means = 45.0%; 3.27% sodium chloride and 2.14% acidity . Significant variation was found in the major compositional factors indicating a general lack of quality and/or extreme diversity of the manufacturing conditions . Microbiological test revealed the presence of very high count of total coliforms, psychrotrophic bacteria, yeasts and moulds, high numbers of Staphylococcus aureus and Clostridium perfringens but no salmonellas . Statistical relationship between growth and counts, and the presence of other indicators, pathogens, and compositional factors was investigated.

Scand J Infect Dis, 1989, 21(4), 467 - 71
Clostridium perfringens septicemia with massive hemolysis; Tsai IK et al.; Massive hemolysis with acute renal failure occurred in a previously healthy 69-year-old patient as a complication of Clostridium perfringens septicemia secondary to gall bladder empyema . To our knowledge, this is one of the few patients with C . perfringens septicemia and massive intravascular hemolysis who survived the episode and regained a normal renal function.

C R Seances Soc Biol Fil, 1989, 183(2), 153 - 6
{Activation in vivo of the collagenase of Clostridium histolyticum by erythromycin lactobionate}; Warter A et al.; The authors demonstrate a previously not described increase, in vivo, in a mean proportion of 70%, of the enzymatic activity of the clostridium collagenase in rats by erythromycin lactobionate . The pathogenesis of this phenomenon cannot yet be explained.

Toxicon, 1989, 27(10), 1113 - 26
Interaction of Clostridium perfringens delta toxin with erythrocyte and liposome membranes and relation with the specific binding to the ganglioside GM2; Jolivet-Reynaud C et al.; The specific interaction of the cytolytic Clostridium perfringens delta toxin with membrane GM2 was indicated by: (i) characterization of this glycolipid in the membrane of sheep and goat erythrocytes, which are lysed by the toxin, whereas GM2 was undetectable in insensitive rabbit erythrocytes, (ii) demonstration of 125I-toxin binding to GM2, by autoradiography, following incubation with thin-layer chromatograms containing separated neuroblastoma gangliosides, and (iii) toxin fixation by phospholipid-cholesterol unilamellar vesicles containing either sheep gangliosides or GM2 . In order to investigate the intramembrane events leading to membrane disruption following toxin binding, the photoreactive probe 12(4-azido-2-nitrophenoxy)stearoyl 1-14C glucosamine, which inserts into the outer layer and labels integral membrane proteins, was used to establish whether delta toxin penetrates into target cell membrane . No toxin labeling was found, suggesting that toxin action takes place at the membrane surface . This contention is supported by the observation that despite toxin binding, GM2 liposomes did not release entrapped 14C-glucose . Treatment of toxin with carboxypeptidases, but not aminopeptidases, abolished both toxin binding capacity onto erythrocytes and its combination with antitoxin neutralizing antibodies, suggesting that the carboxy terminal end of the toxin is critical for binding to cell membrane.

Folia Microbiol (Praha), 1989, 34(3), 202 - 8
Effect of N,N'-bis(alkyldimethyl)-alpha, omega-alkanediammonium dibromides on bacteria of the genus Clostridium; Ciganekova V et al.; Antibacterial effect of 17 ammonium compounds of the type of N,N'-bis(alkyldimethyl)-alpha, omega-alkanediammonium dibromides was tested on anaerobically sporulating bacteria of the genus Clostridium . A sizable antibacterial activity was displayed by five N,N'-bis(alkyldimethyl)-1,6-hexanediammonium dibromides and by four N,N'-bis(decyldimethyl)-alpha, omega-alkanediammonium dibromides . These compounds exhibited activity higher than, or comparable with, that of the reference standards Ajatin and Septonex . The maximum antibacterial activity was found in compounds whose alkyl chain contained 9-12 carbon atoms . Compounds with a lower number of carbon atoms in the chain (less than 8) exhibited a low activity.

Gastroenterol J, 1989, 49(2), 59 - 62
{Therapeutic study of the effect of selective decontamination on microbial overgrowth syndrome of the small intestine}; Knoke M et al.; The microbial overgrowth syndrome of the small bowel (MOS) is characterized by clinically found symptoms of increased metabolic activities of microorganisms existing in a great number in the intestinal juice of these patients . We treated 15 patients who suffered from MOS following a modified scheme of selective intestinal decontamination (SID) with oral doses of BerlocombinR (trimethoprim/sulfamerazin) and polymyxin for three weeks . Comparative investigations of duodenal juices before and after treatment showed unchanged high germ-counts (total aerobes and total anaerobes up to 10(7) germs/ml juice) . Only the increase of Clostridium (up to 10(6)/ml) was statistically significant which has to be disapproved . In most of the patients there was no subjective improvement . SID in the examined manner is not able to cure a microbial overgrowth in the upper gastrointestinal tract or to reduce facultatively pathogenic microorganisms.

Microb Pathog, 1989 Jan, 6(1), 17 - 28
Characterization of calcium involvement in the Clostridium perfringens type A enterotoxin-induced release of 3H-nucleotides from Vero cells; McClane BA; This report characterizes the involvement of Ca2+ in the release of nucleotides from Vero cells caused by Clostridium perfringens enterotoxin (CPE) . A positive linear correlation was observed between increased CPE-induced nucleotide-release and increased extracellular calcium over the range 0.01 to 10 mM calcium . Above 5 mM Ca2+, CPE-specific lysis (i.e . disintegration of cells as monitored by light microscopy) was observed . Addition of 1.7 mM Ca2+ to Vero cells previously CPE-treated in Ca2+-free buffer rapidly increased nucleotide-release, even when cells had been previously incubated for 1 h at 37 degrees C in Ca2+-free buffer . Withdrawal of Ca2+, even after the onset of nucleotide-release, halted further CPE-induced nucleotide-release . These results indicate that Ca2+ must be continuously present for significant CPE-induced nucleotide-release . However, withdrawal of Ca2+ did not reverse membrane bleb formation by CPE . This differentiates bleb formation and nucleotide-release (both Ca2+-dependent CPE effects) and suggests that nucleotide-release does not result simply from bleb formation . Lastly, it was shown that other ions besides physiologic Ca2+ (1.7 mM) are required for CPE-induced nucleotide-release . Interestingly, a role for other ions (but not physiologic Ca2+) is also shown for 86Rb-release by CPE (an early Ca2+-independent CPE effect) . This indicates that extracellular ions other than physiologic Ca2+ can be required for both Ca2+-independent and Ca2+-dependent CPE effects.

J Appl Bacteriol, 1989 Jan, 66(1), 69 - 75
Antimicrobial activity of clove oil dispersed in a concentrated sugar solution; Briozzo J et al.; Essential oil of clove, dispersed (0.4% v/v) in a concentrated sugar solution, had a marked germicidal effect against various bacteria and Candida albicans . Staphylococcus aureus (five strains), Klebsiella pneumoniae, Pseudomonas aeruginosa, Clostridium perfringens, and Escherichia coli inoculated at a level of 10(7) cfu/ml, and C . albicans (inoculum 4.0 x 10(5) cfu/ml) were killed (greater than 99.999%) after 2-7 min in a laboratory broth supplemented with 63% (v/w) of sugar, and containing 0.4% (v/w) of essential oil of clove . Added organic matter (i.e . human or bovine serum) did not impair its antimicrobial activity . Sugar was not necessary for the antimicrobial activity of clove oil, but the concentrated sugar solution provided a good vehicle for obtaining an oil dispersion that is relatively stable for certain practical applications.

Infect Immun, 1989 Jan, 57(1), 272 - 7
Binding of Clostridium botulinum type C neurotoxin to different neuroblastoma cell lines; Yokosawa N et al.; Binding of type C neurotoxin (C1 toxin) from Clostridium botulinum (strain Stockholm) to neuroblastoma cell lines was studied by using biotinylated anti-toxin antibody and avidin-biotinylated peroxidase complex . The neurotoxin bound with high efficiency to mouse neuroblastoma (NS-20Y and NIE-115) cells and to hybridomas of rat glioblastoma and mouse neuroblastoma (NG108-C15) cells . The toxin bound little to human neuroblastoma, rat astrocytoma, and nonneural cell lines . Binding of the neurotoxin to NG108-C15 cells was inhibited by gangliosides (GT1b and GM1) and by monoclonal antibodies (CA-12 and C-9), although inhibition was not complete . Sequential preincubation of C1 toxin with GT1b and CA-12 caused complete inhibition . A Scatchard plot of binding of 125I-labeled C1 toxin to NG108-C15 cells showed a hyperbolic curve . Monoclonal antibody CA-12 but not C-9 neutralized the lethal activity of the toxin toward mice . Only C-9 clearly inhibited toxin binding to GT1b . These results suggest that NG108-C15 cells have at least two kinds of receptors for C1 toxin . From the results of binding tests with neuraminidase-, pronase-, and trypsin-treated NG108-C15 cells, the chemical nature of the high-affinity site was presumed to be a glycoprotein containing sialic acid . GT1b may have an important role in low-affinity sites.

J Antimicrob Chemother, 1989 Jan, 23(1), 131 - 42
Teicoplanin in the treatment of infections by staphylococci, Clostridium difficile and other gram-positive bacteria; de Lalla F et al.; Eighty-three episodes of Gram-positive infection in 82 patients were treated with teicoplanin in an open study . Infectious episodes included endocarditis (6 cases), bacteraemia (7), osteomyelitis (8), pseudomembranous colitis (13), cellulitis (11), urinary tract infection (5), pneumonia (1), wound and post-surgical infections (9) and erysipelas (23) . Four patients affected by an overwhelming Gram-positive infection as well as eight cases of Gram-positive-Gram-negative mixed infections received teicoplanin in combination with other antibiotics . The average duration of treatment was 16 days (range 5-70) . In pseudomembranous colitis teicoplanin was given by mouth for ten days . Staphylococcus aureus (11 methicillin-sensitive and 13 methicillin-resistant strains) and Clostridium difficile (13 isolates) were the most frequent pathogens . Overall 89% (74/83) of the infections were cured, 3.6% (3/83) improved and 3.6% (3/83) failed . Relapse and superinfection were observed in 2.4% (2/83) and 1.2% (1/83) episodes respectively . All pseudomembranous colitis cases were clinically cured and C . difficile was eradicated in all but one patient . The MIC range, MIC50 and MIC90 (mg/l) of teicoplanin for C . difficile were less than 0.125-0.250, less than 0.125 and 0.250 respectively . Pharmacokinetic studies in patients given a single iv daily maintenance dose of 400 mg showed that the steady-state trough teicoplanin concentrations in serum were reached on day 8 . Assays of skin-subcutaneous tissue biopsies showed that teicoplanin penetrated well into these structures . Side effects were observed in six of the 82 treated patients (7.3%) and teicoplanin had to be discontinued in four cases . The results of the study show that teicoplanin is a safe and useful new agent for the treatment of infections caused by Gram-positive organisms, including methicillin-resistant staphylococci and C . difficile.

Infect Immun, 1989 Jan, 57(1), 255 - 61
Production by Clostridium spiroforme of an iotalike toxin that possesses mono(ADP-ribosyl)transferase activity: identification of a novel class of ADP-ribosyltransferases; Simpson LL et al.; Clostridium spiroforme iotalike toxin produced time- and concentration-dependent incorporation of ADP-ribose into homo-poly-L-arginine . Polyasparagine, polyglutamic acid, polylysine, and agmatine were poor substrates . Enzyme activity was associated with the light-chain polypeptide of the toxin . The heavy chain did not possess ADP-ribosyltransferase activity, nor did it enhance or inhibit activity of the light chain . In broken-cell assays, the toxin acted mainly on G-actin, rather than F-actin . A single ADP-ribose group was transferred to each substrate molecule (G-actin) . The enzyme was heat sensitive, had a pH optimum in the range of 7 to 8, was inhibited by high concentrations of nicotinamide, and was reversibly denatured by urea and guanidine . Physiological levels of nucleotides (AMP, ADP, ATP, and ADP-ribose) and cations (Na+, K+, Ca2+, and Mg2+) were not very active as enzyme inhibitors . The toxin was structurally and functionally similar to Clostridium botulinum type C2 toxin and Clostridium perfringens iota toxin . When combined with previous findings, the data suggest that a new class of mono(ADP-ribosyl)ating toxins has been found and that these agents belong to a related and possibly homologous series of binary toxins.

Toxicon, 1989, 27(11), 1209 - 18
Effects of Clostridium difficile toxins A and B on cytoskeleton organization in HEp-2 cells: a comparative morphological study; Fiorentini C et al.; A comparative study on the effects of toxin A and toxin B from Clostridium difficile on HEp-2 cells was carried out . Both toxins caused cell retraction and rounding and seemed to exert their effect on cell morphology via a rearrangement of actin and alpha-actinin microfilaments . Such a rearrangement occurred at an early stage, when no change in microtubular and cytokeratin systems was detectable . Nevertheless, several structural modifications accompanying the cytopathological process induced by toxins A and B appeared to be quite different . In particular, toxin B-treated cells showed an arborized phenotype as a result of cell retraction and rounding, whereas toxin A caused cell rounding without arborization . Moreover, nuclear polarization following disorganization of the microfilament system was only observed in toxin A-treated cells . The structural features distinguishing intoxication processes induced by the two toxins probably reflect a different mechanism of action and suggest the presence of a distinct subcellular component as a primary target for each toxin.

J Gen Microbiol, 1989 Jan, 135 ( Pt 1), 55 - 64
Cloning and characterization of overlapping DNA fragments of the toxin A gene of clostridium difficile; von Eichel-Streiber C et al.; Clostridium difficile, a human pathogen, produces two very large protein toxins, A and B (250-600 kDa), which resist dissociation into subunits . To clone the toxin A gene, a genomic library of 3-8 kb chromosomal DNA fragments of C . difficile strain VPI 10463 established in pUC12 was screened with a rabbit polyclonal toxin A antiserum . Thirty-five clones were isolated which carried 2.5-7.0 kb inserts representing a 10 kb region of the C . difficile genome . All the inserts were oriented in the same direction, suggesting that toxin A gene expression was under control of the lac promoter of the pUC12 vector . Western blot experiments revealed the presence of low amounts of fusion proteins of variable size (30-170 kDa) in Escherichia coli strains harbouring recombinant plasmids . As deduced from subcloning experiments, the DNA sequences encoding toxin A comprised about 4 kb, corresponding to about 140 kDa of the 300-600 kDa protein . This was either due to incomplete cloning of the gene or it might indicate a subunit composition of toxin A . No additional gene(s) with homology to the cloned toxin A gene was detected.

Boll Ist Sieroter Milan, 1989, 68(2), 142 - 4
{2 cases of arthralgia associated with infection by Clostridium difficile}; Limonta M et al.; The Clostridium difficile is the etiologic agent most often isolated in patients with antibiotic-associated colitis . Rarely this symptomatology is complicated by postinfection arthritis . The following describes 2 cases of acute colitis by clostridium difficile associated with acute polyarthritis.

G Batteriol Virol Immunol, 1989 Jan-Dec, 82(1-12), 152 - 64
{Isolation of anaerobes during a 30-month observation at a hospital microbiology laboratory}; Pistono PG et al.; The authors evaluate retrospectively the results obtained from the research of anaerobial bacteria on 1313 samples received at the Microbiology Laboratory of the "Ospedale Civile di Ivrea" over a period of 31 months (6/1/86-12/31/88) . From this evaluation, high percentages of detection of anaerobic bacteria are emerging in the following infections: appendiculare abscesses (60%), intestinal operations (71%), wounds (57%), tubovarian abscesses (100%), as well as thoracic empyema (50%) . Also relevant are the isolations from skin and subcutaneous tissues: breast infections (50%) preputial infections (60%), perineal and perirectal abscesses (60%) . The incident of anaerobic bacteria in bacteriemia is 17% . The most representative anaerobic bacteria group are: Bacteroides spp . (56%), Peptostreptococcus spp . (12%), Propionibacterium spp . (9%), Fusobacterium spp . (7%) Clostridium spp . (6%), Veillonella spp . and Eubacterium spp . (3%) . In the intraabdominal infections prevails the Bacteroides group, particularly fragilis species, while in the skin and subcutaneous infections prevails the Peptostreptococcus group.

Arch Inst Pasteur Alger, 1989, 57, 277 - 84
{Enumeration and identification of Clostridium from stools treated with the thioglycollate-lysozyme method}; Merad S et al.; We have used spore isolation by the sodium thioglycolate-lysozyme technique on collected stools . Of the 51 stools studied, we found 41% of Clostridium perfringens with an average ratio of 10(4) germs/gr . 15 strains were typical double hemolysis and trehalose positive-5 presented only one hemolysis and were trehalose negative . We only found a single strain of Clostridium difficile with a rate of 10(4) germs/gr in the stools of a 10 months infant.

Ophthal Plast Reconstr Surg, 1989, 5(3), 212 - 5
Eyelid gas gangrene; Lyon DB et al.; A case of eyelid gas gangrene caused by Clostridium hastiforme is reported . The infection followed an alkali injury that probably aided proliferation of the organism by inducing local tissue ischemia . Debridement combined with antibiotic therapy controlled the infection, and the large lid defect healed satisfactorily by secondary intention.

Rev Elev Med Vet Pays Trop, 1989, 42(3), 391 - 2
Note on an association of Clostridium novyi type A and Clostridium sordellii with a case of gas-gangrene in a Zebu cow; el Sanousi SM et al.; A case of gas-gangrene myositis on a Zebu cow with association of Clostridium novyi type A and Clostridium sordellii is described . The occurrence of both organisms in the same lesion has been rarely reported and differential diagnosis with blackleg is difficult in the absence of bacteriological tests . An iatrogenic cause cannot be overruled as routine mass vaccination against blackleg are practiced in the area with possible introduction of spores through infection material . Full recovery occurred after four days intramuscular Terramycin shot.

Folia Haematol Int Mag Klin Morphol Blutforsch, 1989, 116(3-4), 569 - 76
Bacteriological findings in patients with bone marrow transplantation (Karl Marx University Leipzig, 1985-1987); Wonitzki C et al.; The results of the bacteriological surveillance cultures for 26 patients with bone marrow transplantation (Karl Marx University Leipzig, G.D.R., 1985-1987) are presented . 5.9% of all surveillance cultures contained facultatively pathogenic germs (with Pseudomonas aeruginosa as the most frequent representative, which was the reason of a sepsis in two patients) . Coagulasenegative Staphylococci and other germs with an obscure pathogenicity were isolated upon a large scale, especially from the mucous membrane regions . There are hints, that above all special strains of coagulasenegative Staphylococci "colonize" the patient's body (also for longer periods) and turn into the blood too . During the total decontamination intestinal anaerobic flora is absent . After closing of total decontamination Clostridium perfringens is the first detectable anaerobic species . During the selective decontamination systemic applications of antibiotics are able to obliterate anaerobic findings for certain periods . Recommendations for an effective arrangement of the surveillance cultures of bone marrow transplantation patients are given.

Scand J Infect Dis Suppl, 1989, 62, 7 - 14
Application of chemotaxonomic techniques to the taxonomy of anaerobic bacteria; Hardie JM; The use of chemical characters in bacterial classification and identification has proved to be an essential component of modern systematics . Several clinically important anaerobic genera, such as Bacteroides, Clostridium, Eubacterium, Fusobacterium and Peptostreptococcus, are known to be heterogeneous of the basis of chemotaxonomic and genetic data and are in need of further examination . Recent work on bacteriodes has led to the genus being redefined and restricted to species within the former Bacteroides fragilis group, and a number of new genera have been proposed . It is important that suitable phenotypic characters are identified so that newly-defined genospecies can be differentiated in diagnostic laboratories.

J Bacteriol, 1989 Jan, 171(1), 430 - 5
Immunological relationship among hydrogenases; Kovacs KL et al.; We examined the immunological cross-reactions of 11 different hydrogenase antigens with 9 different hydrogenase antibodies . Included were antibodies and antigens of both subunits of the hydrogenases of Bradyrhizobium japonicum and Thiocapsa roseopersicina . The results showed a strong relationship among the Ni-Fe dimeric hydrogenases . The two subunits of Ni-Fe dimeric hydrogenases appeared immunologically distinct: specific interactions occurred only when antibodies to the 60- and 30-kilodalton subunits reacted with the 60- and 30-kilodalton-subunit antigens . The interspecies cross-reactions suggested that at least one conserved protein region exists among the large subunits of these enzymes, whereas the small subunits are less conserved . Antibodies to the Fe-only bidirectional hydrogenase of Clostridium pasteurianum reacted with the Desulfovibrio vulgaris bidirectional hydrogenase . Surprisingly, antibodies to the clostridial uptake hydrogenase did not react with any of the Fe-only bidirectional hydrogenases but did react with several of the Ni-Fe dimeric hydrogenases . The two hydrogenases from C . pasteurianum were found to be quite different immunologically . The possible relationship of these findings to the structure and catalytic functions of hydrogenase are discussed.

J Biol Chem, 1988 Dec 25, 263(36), 19552 - 7
High resolution deuterium NMR studies of bacterial metabolism; Aguayo JB et al.; High resolution deuterium NMR spectra were obtained from suspensions of five bacterial strains: Escherichia coli, Clostridium perfringens, Klebsiella pneumoniae, Proteus mirabilis, and Staphylococcus aureus . Deuterium-labeled D-glucose at C-1, C-2, and C-6 was used to monitor dynamically anaerobic metabolism . The flux of glucose through the various bacterial metabolic pathways could be determined by following the disappearance of glucose and the appearance of the major end products in the 2H NMR spectrum . The presence of both labeled and unlabeled metabolites could be detected using 1H NMR spectroscopy since the proton resonances in the labeled species are shifted upfield due to an isotopic chemical shift effect . The 1H-1H scalar coupling observed in both the 2H and 1H NMR spectra was used to assign definitively the resonances of labeled species . An increase in the intensity of natural abundance deuterium signal of water can be used to monitor pathways in which a deuteron is lost from the labeled metabolite . The steps in which label loss can occur are outlined, and the influence these processes have on the ability of 2H NMR spectroscopy to monitor metabolism are assessed.

J Biol Chem, 1988 Dec 5, 263(34), 18430 - 6
Crystal structure of Clostridium acidi-urici ferredoxin at 5-A resolution based on measurements of anomalous X-ray scattering at multiple wavelengths; Murthy HM et al.; The crystal structure of Clostridium acidi-urici ferredoxin has been determined using multiple wavelength anomalous diffraction (MAD) techniques at 5.0-A resolution . The electron density map shows striking similarity to a map of Peptococcus aerogenes ferredoxin computed at the same resolution from the atomic coordinates reported by Adman et al . (Adman, E . T., Sieker, L . C., and Jensen, L . H . (1973) J . Biol . Chem . 248, 3987-3996) . Such similarity is expected from the high degree of identity between amino acid sequences of the two proteins . The use of MAD methods has in the relatively recent past become a practical possibility due to instrumental advances enabling the collection of accurate data at several wavelengths at synchrotrons and due to theoretical and computational advances that facilitate the analysis of these data for the determination of phases . These methods hold great promise as an alternative to the multiple isomorphous replacement method in macromolecular structure determination . The present report represents one of the first applications of the MAD techniques to the determination of the structure of a protein which was previously unknown in detail.

FEMS Microbiol Immunol, 1988 Dec, 1(3), 163 - 8
Identification and molecular cloning of a 70 kDa species-specific antigen common to Clostridium difficile; Wren BW et al.; Three common antigens (CB 1, 2 and 3), characteristic of Clostridium difficile species were identified by immunoblot analysis using homologous and heterologous rabbit antisera, raised against whole cells from 9 distinct strains of C . difficile . A gene library of C . difficile genomic DNA was constructed in Escherichia coli by cloning in Sau 3A-cleaved clostridial DNA fragments into the bacteriophage vector lambda EMBL3 . OUt of 3000 plaques screened using the whole cell antisera, 27 clones were positively identified . One of these clones, designated gamma Cd21, expressed high levels of an antigen which could be immunologically identified using whole cell antisera against the 9 C . difficile strains . Antiserum raised against the clone gamma Cd21 identified a 70 kDa antigen (previously named CB1) as demonstrated by immunoblot analysis . Monospecific antiserum against gamma Cd21 recognises the 70 kDa antigen in all 97 strains of C . difficile derived from worldwide sources and does not cross-react with 17 strains from 13 other clostridial species.

J Biomol Struct Dyn, 1988 Dec, 6(3), 443 - 58
Dynamics of drug-DNA interactions: a comparative temperature jump study of ellipticinium and 9-hydroxy ellipticinium; Schwaller MA et al.; The temperature-jump method has been used to compare the binding of 2-N methyl ellipticinium (NME) and 2-N methyl 9 hydroxy ellipticinium (NMHE) to three natural DNA's of different AT/GC composition . The relaxation signals, analyzed by the Pade-Laplace method, are characterized by two distinct relaxation times, tau 1 and tau 2, respectively in the 1-4 ms and 20-80 ms range . In the case of the NMHE/DNA interaction, the slower relaxation time tau 2 depends on the DNA composition, as follows: tau 2 (Micrococcus lysodeikticus) greater than tau 2 (Calf thymus) greater than tau 2 (Clostridium perfringens) . Contrary to NMHE, NME which does not possess an OH group at the C-9 position, shows no relaxation time dependence upon DNA base composition . The observation of two relaxation times indicates that the binding equilibria are associated with at least two distinct drug/DNA complexes (probably arising from two distinct DNA binding sites) . Three kinetic models, involving the formation of a weak intermediate ionic complex, are given to explain the binding reaction between these cationic drugs and the DNA . They allow the determination of the four rate constants associated with the two binding steps and lead to equilibrium association constants in agreement with those obtained from spectroscopic studies . The validity of the models is discussed and it is shown that the best kinetic scheme, for either NMHE or NME, could be that in which the ionic step is not a prerequiste to intercalation . The kinetic results show that the residence time of 9 hydroxy ellipticinium is markedly increased in GC rich DNA's and this could be related to the higher in vitro and in vivo cytotoxic properties of 9 hydroxy substituted ellipticines.

J Gen Microbiol, 1988 Dec, 134 ( Pt 12), 3151 - 7
Restriction endonucleases in Clostridium pasteurianum ATCC 6013 and C . thermohydrosulfuricum DSM 568; Richards DF et al.; A small collection of clostridia was surveyed for type II restriction endonucleases . Enzymes were detected in two organisms . Clostridium pasteurianum ATCC 6013 contains an isoschizomer of ThaI (FnuDII) {5'-CGCG-3'} and preliminary evidence suggests that cleavage generates blunt-ended fragments . Clostridium thermohydrosulfuricum DSM 568 contains an isoschizomer of MboI (Sau3A) {5'-GATC-3'} that is inactive on dam methylated substrates . The DNA of this latter organism shows dam methylation.

Biofactors, 1988 Dec, 1(4), 293 - 6
Selenium-dependent glycine reductase: differences in physicochemical properties and biological activities of selenoprotein A components isolated from Clostridium sticklandii and Clostridium purinolyticum; Sliwkowski MX et al.; The selenoprotein A component of the glycine reductase complex of Clostridium sticklandii was shown to differ in certain properties from the selenoprotein A produced by a purine-fermenting organism, Clostridium purinolyticum . Both proteins contain one selenocysteine and two cysteine residues.

J Clin Microbiol, 1988 Dec, 26(12), 2484 - 8
Species-specific oligonucleotide probes for rRNA of Clostridium difficile and related species; Wilson KH et al.; The large copy number of rRNA makes it an appealing target for oligonucleotide probes designed to identify microorganisms . Given that nucleotide sequences in rRNA are known to reflect phylogeny, species-specific rRNA probes should be feasible if the sequences found in closely related species are different . We sequenced portions of the 16S rRNA of three closely related clostridia found in the human colonic microflora: Clostridium bifermentans, C . sordellii, and C . difficile . The rRNAs of these three species showed 97 to 98% sequence similarity . Five oligonucleotide probes complementary to unique segments of the sequences were end labeled with 32P and hybridized on a nylon filter to the immobilized rRNA of each clostridium . Each probe efficiently hybridized only to the rRNA of the species to which it was directed . Complementary probes emitted a signal that exceeded by a factor of 100 to 1,000 the signal of probes that mismatched the target rRNA by 2 to 5 bases . Even a 1-base difference in rRNA sequence allowed a clear distinction between species . A systematic approach can efficiently yield taxon-specific oligonucleotide probes directed at rRNA.

J Infect Dis, 1988 Dec, 158(6), 1336 - 40
Clostridium tertium septicemia in patients with neutropenia; Speirs G et al.; Eighteen adult patients with hematologic malignancy developed bacteremia due to Clostridium tertium while neutropenic . Fifteen had accompanying abdominal pain, colonic bleeding, or diarrhea, and three had perianal cellulitis . Fourteen recovered with antibiotic therapy alone; no patient was treated by surgery . C . tertium is an unusual Clostridium because it is resistant to many beta-lactam antibiotics and to metronidazole but is susceptible to vancomycin, trimethoprim-sulfamethoxazole, and ciprofloxacin . It is possible that use of third-generation cephalosporins (cefotaxime, ceftizoxime, ceftazidime) for treating febrile episodes in the absence of any selective intestinal decontamination with trimethoprim-sulfamethoxazole or ciprofloxacin may have resulted in selection for C . tertium in our patients.

J Bacteriol, 1988 Dec, 170(12), 5747 - 50
Characterization of a CO-dependent O-demethylating enzyme system from the acetogen Clostridium thermoaceticum; Wu ZR et al.; An inducible O-demethylating enzyme system was characterized from Clostridium thermoaceticum cultivated at the expense of syringate . Glucose and methanol, but not CO, partially repressed its expression . Induced whole cells catalyzed the carbon monoxide (CO)-dependent O demethylation of methoxylated aromatic compounds with the concomitant formation of acetate . Pyruvate and, to a lesser extent, H2-CO2 could replace CO in these reactions . KCN inhibited pyruvate-dependent activity but not the CO-dependent activity . The ATPase inhibitor N,N'-dicyclohexylcarbodiimide, the protonophore carbonyl cyanide m-chlorophenylhydrazone, and methyl viologen did not appreciably inhibit O demethylation by induced cells, whereas Triton X-100 was inhibitory . The enzyme system appeared to convert syringate sequentially to 5-hydroxyvanillate and gallate . The proposed overall reaction stoichiometry was as follows: syringate + 2CO + 2H2O----gallate + 2 acetates . Growth-supportive methoxylated aromatic compounds were O demethylated by syringate-cultivated cells and inhibitory to syringate O demethylation.

J Bacteriol, 1988 Dec, 170(12), 5705 - 8
Energy-dependent, high-affinity transport of nickel by the acetogen Clostridium thermoaceticum; Lundie LL Jr et al.; The nickel transport system of Clostridium thermoaceticum was investigated with 63NiCl2 and an anaerobic microfiltration transport assay . Transport was optimal at pH 7 to pH 7.5 and 65 degrees C and decreased in the presence of metabolic uncouplers and inhibitors . Exogenous nickel was concentrated 3,000-fold over the apparent nickel concentration gradient during typical transport assays . Stored cellular energy appeared to provide a short-term energy source to power nickel transport, and starvation experiments demonstrated external energy source stimulation of nickel translocation . The apparent Km and Vmax for nickel transport by carbon monoxide-dependent chemolithotrophic cells approximated 3.2 microM Ni and 400 pmol of Ni transported per min per mg of cells (dry weight), respectively . Magnesium, calcium, cobalt, iron, manganese, and zinc did not inhibit the transport of nickel.

J Bacteriol, 1988 Dec, 170(12), 5895 - 900
Bacteria of the genus Bacillus have a hydrolase stereospecific to the D isomer of benzoyl-arginine-p-nitroanilide; Gofshtein-Gandman LV et al.; A stereospecific enzyme activity capable of cleaving the amide bond of the synthetic substrate N-benzoyl-D-arginine-p-nitroanilide (D-BAPA) has been found in all aerobic and anaerobic members of the family Bacillaceae tested by us . Cells of nonsporeforming gram-positive or gram-negative bacteria contain a hydrolase activity stereospecific to N-benzoyl-L-arginine-p-nitroanilide . The D-BAPA-hydrolyzing enzymes (D-BAPAases) of mid-logarithmic-phase cells of Bacillus subtilis 168 and B . cereus T were compared . These enzymes had the same molecular weight of approximately 66,000 in gel filtration and the same electrophoretic mobility after electrophoresis on polyacrylamide gels . The D-BAPAases of B . subtilis 168 and B . cereus T differed in the effect of inhibitors on enzymatic activity . While both hydrolases were inhibited by tosyl-L-lysine chloromethyl ketone and tosyl-L-arginine-methyl ester as well as leupeptin, only the D-BAPAase of B . cereus T was inhibited by p-chloromercuribenzene sulfonic acid . The D-BAPAases of B . subtilis and B . cereus T had a Michaelis constant for D-BAPA of 2.9 x 10(-5) M and 1.4 x 10(-4) M, respectively . D-BAPAase is an intracellular enzyme localized in the protoplast (80 to 90% in soluble form in the cytoplasm) . The ability to cleave D-BAPA is suggested as an additional chemotaxonomic characteristic of sporeforming bacteria of the genera Bacillus and Clostridium.

Arch Otolaryngol Head Neck Surg, 1988 Dec, 114(12), 1407 - 12
Management of facial spasm with Clostridium botulinum toxin, type A (Oculinum)
Biglan AW, May M, Bowers RA.
One hundred five patients received 391 graded injections of Clostridium botulinum type A toxin (Oculinum) to treat uncontrollable facial muscle spasm . Patients had essential blepharospasm (n = 61), hemifacial spasm (n = 24), or aberrant regeneration of the seventh cranial nerve (n = 20) . Muscle spasms were reduced within two days of the first injection of toxin and, in most cases, the drug effect lasted three to four months . Control of facial muscle spasm was achieved in all patients . Complications related to treatment included transient blepharoptosis (n = 7), diplopia (n = 2), and altered facial expression (n = 11) . Systemic side effects were not observed . Select chemodenervation of facial muscles with graded injections of botulinum toxin is a useful adjunct to control blepharospasm, hemifacial spasm, and facial spasm due to aberrant regeneration of the facial nerve.

Eur J Clin Microbiol Infect Dis, 1988 Dec, 7(6), 798 - 802
Comparative in vitro activity of A-56268; Sefton AM et al.; The comparative in vitro activity of A-56268 was studied using 1,006 clinical isolates including streptococci, enterococci, staphylococci, Neisseria gonorrhoeae, Haemophilus influenzae and anaerobes . A-56268 showed activity comparable to that of erythromycin and was more active than josamycin and roxithromycin against erythromycin-sensitive aerobic and facultatively anaerobic gram-positive cocci . A-56268 was the most active macrolide against Clostridium spp . and Neisseria gonorrhoeae . Josamycin was more active than either A-56268 or erythromycin against the anaerobic gram-positive cocci and the Bacteroides fragilis group . Staphylococci moderately resistant or resistant to erythromycin (MIC 3.12-50 mg/l) remained susceptible to josamycin but not the other macrolides.

Jpn J Exp Med, 1988 Dec, 58(6), 233 - 41
Some factors affecting isolation of Clostridium tetani from human and animal stools; Ebisawa I et al.; Clostridium tetani was isolated from human and animal stools at the following rates {95% confidence interval (CI)}: Human, 0% (1.5-0); horse, 1% (5-0); cow in cowshed, 4% (10-1); cow in pasture, 8.3% (17-1), calf in pasture, 0% (7-0); dog, 2% (11-0) and sheep in pasture, 25% (44-14) . Quantification of C . tetani in 16 animal stools positive for the bacillus was impossible in most cases, as the number of tetanus bacilli present was not large enough for this purpose . Contaminating anaerobic and facultative anaerobic bacteria in human and animal stools, i.e., C . perfringens and Streptococcus sp., Group G, inhibited isolation of C . tetani from these materials, particularly at the step of isolation employing its swarming character.

Epidemiol Infect, 1988 Dec, 101(3), 661 - 7
Food poisoning in hospitals in Scotland, 1978-87; Collier PW et al.; During the 10-year period 1978-87 there were 48 outbreaks of food poisoning in Scottish hospitals affecting a total of 2287 persons of whom 12 died . This compared with 50 outbreaks during the previous 5 years (1973-77) when over 1500 persons and 7 deaths were recorded . Although the incidence of outbreaks has decreased the average number of persons affected in outbreaks has increased . A marked reduction was seen in the incidence of outbreaks due to Clostridium perfringens, in contrast to foodborne salmonellosis which remains a problem . Thirty-four hospitals, of which 10 reported two or more outbreaks, were involved . The type of hospitals most frequently affected were general (14), psychiatric (13), geriatric (9) and hospitals for the mentally subnormal (7) . Meat, including poultry meat, was incriminated in over 90% of outbreaks where a food vehicle was identified . In modern or re-equipped kitchens cooking in advance with subsequent reheating is being progressively discontinued as more food is being cooked on the day of consumption, a practice which may readily explain the decreasing incidence of outbreaks due to Cl . perfringens . Bacterial cross-contamination from poultry-meat and other raw foods, compounded by inadequate temperature control, however, continues to be a problem in some hospitals . It is too early as yet to determine whether the removal of Crown immunity will have any effect on the future incidence of hospital 'food poisoning'.

Infect Immun, 1988 Dec, 56(12), 3235 - 40
Nucleotide sequence of the gene for perfringolysin O (theta-toxin) from Clostridium perfringens: significant homology with the genes for streptolysin O and pneumolysin; Tweten RK; The nucleotide sequence was determined for the gene encoding the thiol-activated cytolysin, perfringolysin O (theta-toxin), from Clostridium perfringens . The nucleotide-sequence-derived primary structure of perfringolysin O is 499 residues long and exhibits a 27-amino-acid signal peptide . The calculated molecular weight of the secreted (mature) form of perfringolysin O is 52,469 . The deduced amino-terminal sequence of perfringolysin O is identical to that determined for purified perfringolysin O . Hydropathy analysis indicated that, except for the signal peptide, no major stretches of hydrophobic residues are present . Extensive amino acid sequence homology (65%) was detected with the low-molecular-weight form of streptolysin O, and a lesser amount (42%) was detected with pneumolysin . The nucleotide sequence of the perfringolysin O gene (pfo) exhibits approximately 60% homology with the streptolysin O gene (slo) and 48% homology with the pneumolysin gene (ply) . All three toxins contain an identical region of 12 amino acids, which includes the essential cysteine of all three toxins . The location of these 12 residues was conserved in all three toxins when the primary sequences were aligned for maximum homology.

Infect Immun, 1988 Dec, 56(12), 3228 - 34
Cloning and expression in Escherichia coli of the perfringolysin O (theta-toxin) gene from Clostridium perfringens and characterization of the gene product; Tweten RK; The gene encoding perfringolysin O, the thiol-activated hemolysin from Clostridium perfringens (ATCC 13124), was cloned and expressed in Escherichia coli . A gene library of C . perfringens chromosomal DNA was constructed in bacteriophage lambda EMBL3 . A recombinant was identified that produced a hemolysin that was inhibited by cholesterol and was tentatively identified as perfringolysin O . Subcloning experiments localized the perfringolysin O gene (pfo) to a 1.8-kilobase region on the cloned chromosomal fragment . E . coli which carried a plasmid subclone of pfo (pRT1B) expressed perfringolysin O and secreted it into the periplasm . The amino-terminal sequence of the pfo gene product was identical with that determined for perfringolysin O purified from C . perfringens, indicating that E . coli correctly removed the signal peptide during secretion . Purification of the pfo product was accomplished by high-resolution gel filtration and anion-exchange chromatography . Analysis of the pfo product by sodium dodecyl sulfate gel electrophoresis showed that it comigrated with authentic perfringolysin O; both had an estimated molecular weight of 54,000 . Two-dimensional tryptic peptide maps of the pfo product and of authentic perfringolysin O purified from C . perfringens were identical . The hemolytic activity of the pfo product was similar to that of authentic perfringolysin O; one hemolytic unit (HU) of the cloned gene product or authentic perfringolysin O corresponded to approximately 1 ng or a hemolytic activity of 10(6) HU per mg.

Anal Biochem, 1988 Dec, 175(2), 569 - 72
Raw starch adsorption-desorption purification of a thermostable beta-amylase from Clostridium thermosulfurogenes; Saha BC et al.; The beta-amylase from Clostridium thermosulfurogenes was readily adsorbed onto raw starch . The adsorbed beta-amylase was eluted from raw starch by using boiled soluble starch solution as an elutant . The soluble starch treated beta-amylase could not adsorb onto raw starch which indicates that the soluble and insoluble substrate binding sites of the beta-amylase may be the same . The beta-amylase was purified to homogeneity by raw starch adsorption-desorption techniques and octyl-Sepharose chromatography . It had a specific activity of 4188 units/mg protein . The insoluble substrate adsorption-desorption technique may be used for the purification of other enzymes.

Gene, 1988 Nov 30, 71(2), 257 - 65
Sequence of the Bacillus subtilis glutamine synthetase gene region; Strauch MA et al.; The nucleotide sequence of the glutamine synthetase (GS) region of Bacillus subtilis has been determined and found to contain several unique features . An open reading frame (ORF) upstream of the GS structural gene is part of the same operon as GS and is involved in regulation . Two downstream ORFs are separated from glnA by an apparent Rho-independent termination site . One of the downstream ORFs encodes a very hydrophobic polypeptide and contains its own potential RNA polymerase and ribosome-binding sites . The derived amino acid (aa) sequence of B . subtilis GS is similar to that of several other prokaryotes, especially to the GS of Clostridium acetobutylicum . The B . subtilis and C . acetobutylicum enzymes differ from the others in the lack of a stretch of about 25 aa as well as the presence of extra cysteine residues in a region known to contain regulatory as well as catalytic mutations . The region around the tyrosine residue that is adenylylated in GS from many species is fairly similar in the B . subtilis GS despite its lack of adenylylation.

Gene, 1988 Nov 30, 71(2), 247 - 56
Cloning and nucleotide sequence of the Streptomyces coelicolor gene encoding glutamine synthetase; Wray LV Jr et al.; The Streptomyces coelicolor glutamine synthetase (GS) structural gene (glnA) was cloned by complementing the glutamine growth requirement of an Escherichia coli strain containing a deletion of its glnALG operon . Expression of the cloned S . coelicolor glnA gene in E . coli cells was found to require an E . coli plasmid promoter . The nucleotide sequence of an S . coelicolor 2280-bp DNA segment containing the glnA gene was determined and the complete glnA amino acid sequence deduced . Comparison of the derived S . coelicolor GS protein sequence with the amino acid sequences of GS from other bacteria suggests that the S . coelicolor GS protein is more similar to the GS proteins from Gram-negative bacteria than it is with the GS proteins from two Gram-positive bacteria, Bacillus subtilis and Clostridium acetobutylicum.

J Biol Chem, 1988 Nov 25, 263(33), 17255 - 7
Substrate for botulinum ADP-ribosyltransferase, Gb, has an amino acid sequence homologous to a putative rho gene product; Narumiya S et al.; Gb (b for botulinum) is an Mr 21,000-22,000 guanine nucleotide binding protein which is specifically ADP-ribosylated by an ADP-ribosyltransferase produced by types C1 and D Clostridium botulinum (Morii, N., Sekine, A., Ohashi, Y., Nakao, K., Imura, H., Fujiwara, M., and Narumiya, S . (1988) J . Biol . Chem . 263, 12420-12426) . In this study, we have identified the partial amino acid sequence of this protein . Purified Gb was digested with various proteases, and the proteolytic fragments were separated by high performance liquid chromatography . Isolated peptides were subjected to protein sequencing by an automated pulse-liquid phase protein sequenator . Eight partial amino acid sequences consisting of a total of 105 amino acid residues were obtained from 16 peptides . Computerized homology analysis revealed that they are homologous to the sequence deduced from cDNA of the rho genes . About 88% of the amino acid residues was identical between the sequenced Gb fragments and the corresponding regions of the Aplysia rho gene product . The result strongly suggests that Gb is a putative rho gene product.

J Biol Chem, 1988 Nov 15, 263(32), 16714 - 9
Purification and properties of dinitrogenase reductase ADP-ribosyltransferase from the photosynthetic bacterium Rhodospirillum rubrum; Lowery RG et al.; The enzyme that catalyzes the ADP-ribosylation and concomitant inactivation of dinitrogenase reductase in Rhodospirillum rubrum has been purified greater than 19,000-fold to near homogeneity . We propose dinitrogenase reductase ADP-ribosyltransferase (DRAT) as the working name for the enzyme . DRAT activity is stabilized by NaCl and ADP . The enzyme is a monomer with a molecular mass of 30 kDa and is a different polypeptide than dinitrogenase reductase activating glycohydrolase . NAD (Km = 2 mM), etheno-NAD, nicotinamide hypoxanthine dinucleotide, and nicotinamide guanine dinucleotide will serve as donor molecules in DRAT-catalyzed ADP-ribosylation reaction, and dinitrogenase reductases from R . rubrum, Azotobacter vinelandii, Klebsiella pneumoniae, and Clostridium pasteurianium will serve as acceptors . No other proteins or small molecules, including water, have been found to be effective as acceptors . Nicotinamide is released stoichiometrically with formation of the ADP-ribosylated product . DRAT is inhibited by NaCl and has maximal activity at a pH of 7.0.

Przegl Dermatol, 1988 Nov-Dec, 75(6), 439 - 43
{Aerobic and anaerobic bacterial flora of crural ulcers}; Lawrynowicz R; In a group of 600 patients treated in the Metropolitan Dermatological Hospital in Warsaw bacteriological examination were carried out of swabs from the untreated crural ulcers . In 95% of these cultures growth of pathological aerobic organisms was obtained . Coagulase-positive staphylococci (St . aureus) and Gram-negative bacteria (Pseudomonas aeruginosa, Proteus vulgaris, Enterobacter sp and E . coli) prevailed . In 27% of cases the cultured strains were resistant to the generally available antibiotics . In the second group in 70 patients no growth of anaerobes exclusively was noted . Pathological aerobes and anaerobes in the same case were found in 45% of cultures . In the remaining ones pathogenic aerobes were present with a similar frequency as in the preceding group . Of the anaerobes the most frequently cultured species were Gram-negative bacteria such as Bacteroides melaninogenicus, Bacteroides sp, and Bacteroides fragilis . Among pathogenic anerobic cocci Peptostreptococcus and Peptococcus were most frequent . In 2 cases spore-forming anaerobic bacteria (Clostridium perfringens) were obtained . Forty-eight percent of anaerobes were resistant to the commonly used antibiotics.

Ann Inst Pasteur Microbiol, 1988 Nov-Dec, 139(6), 683 - 8
Regulation of coenzyme A transferase and acetoacetate decarboxylase activities in Clostridium acetobutylicum; Janati-Idrissi R et al.; The activity of two enzymes involved in acetone production in Clostridium acetobutylicum, acetoacetate decarboxylase and coenzyme A transferase, was studied under acidogenic or solventogenic conditions . Acetoacetate decarboxylase activity was low under acidogenic conditions and after pyruvate addition . Under the same conditions, coenzyme A transferase activity was high . A mutant which lacked acetoacetate decarboxylase activity but was positive for coenzyme A transferase activity was isolated.

J Clin Microbiol, 1988 Nov, 26(11), 2452 - 5
Evaluation of a commercial latex test for Clostridium difficile for reactivity with C . difficile and cross-reactions with other bacteria; Miles BL et al.; Seventy-eight species of bacteria (739 isolates) were tested for reactivity with a commercial latex test for Clostridium difficile . All noncytotoxic as well as cytotoxic strains of C . difficile reacted positively . Immuno-specific cross-reactions were found only with C . sporogenes, proteolytic C . botulinum, and Peptostreptococcus anaerobius.

Arzneimittelforschung, 1988 Nov, 38(11), 1553 - 6
In vitro activity of flomoxef compared to moxalactam, cefoxitin, cefotaxime, and clindamycin against anaerobes; Werner H et al.; To assess the in vitro activity of flomoxef (6315-S), moxalactam, cefoxitin, cefotaxime, and clindamycin against anaerobes 197 clinical isolates (27 Bacteroides fragilis, 42 B . thetaiotaomicron, 10 B . vulgatus, 7 B . ovatus, 6 B . uniformis, 6 B . distasonis, 7 Bacteroides melaninogenicus group, 11 Bacteroides oralis group, 21 Clostridium difficile, 7 C . perfringens, 3 C . sporogenes, 3 Clostridium spp., 33 Propionibacterium acnes, 14 Peptococcaceae) were studied by means of agar dilution tests . The MIC90 of B . fragilis was less than 2 micrograms/ml for flomoxef, less than 4 micrograms/ml for moxalactam, less than 16 micrograms/ml for cefoxitin, less than 128 micrograms/ml for cefotaxime and less than 2 micrograms/ml for clindamycin . The respective MIC90's of B . thetaiotaomicron were less than 64, less than 128, less than 32, less than 256 and 8 micrograms/ml . Strains of the other Bacteroides species and groups were more susceptible to flomoxef and the other antibiotics than B . thetaiotaomicron . Against Clostridium difficile flomoxef (MIC90 less than 4 micrograms/ml) proved to be superior to the other agents tested . Most of the Clostridium strains other than C . difficile were also susceptible to flomoxef; anaerobic grampositive cocci and Propionibacterium acnes were very sensitive (MIC90's less than 1 and less than or equal to 0.125 micrograms/ml, respectively) . Its anti-anaerobic activity, together with its efficacy against aerobes, should make flomoxef a useful adjunct to the arsenal of modern antibiotic therapy.

Clin Orthop, 1988 Nov, (236), 23 - 35
Two-stage reimplantation in infected total knee arthroplasty; Wilde AH et al.; Twenty-one infected total knee arthroplasties (TKA) in 21 patients were treated from September 1980 through October 1987 . Of these, 15 were followed for more than one year . Treatment of these patients consisted of thorough debridement of all infected tissue and components; a cement spacer was used in ten patients . The cement was impregnated with antibiotics . This procedure was followed for an average of 4.2 weeks with intravenous antibiotics and TKA utilizing antibiotic-impregnated cement . Five patients had rheumatoid arthritis and ten had osteoarthritis . The organisms included Staphylococcus coagulase negative (seven patients), Streptococcus group B (two patients), Streptococcus bovis (one patient), Enterococcus (one patient), Staphylococcus coagulase positive and Bacillus circulans (one patient), Staphylococcus coagulase negative and Enterococcus (one patient), Staphylococcus coagulase negative and Pseudomonas aeuriginosa (one patient), and Clostridium perfringens (one patient) . Of the 15 patients, 12 appeared to be free of infection, two were obvious failures and required knee fusion, and one was suspected of having continued infection at five years and was treated elsewhere . Eleven patients with revision TKA were available for follow-up examinations at an average of 2.9 years (range, one to six years) . One patient died five years after reimplantation but had been functioning well . One patient functioning at three years postreimplantation did not return for a later follow-up examination . The average knee score (modification of the Hospital for Special Surgery Knee Score) was 75.5 points (range, 48-94); average flexion was 81 degrees (range, 52 degrees-120 degrees), and average extension was +6 degrees (range, 0 degrees-30 degrees).(ABSTRACT TRUNCATED AT 250 WORDS)

Gastroenterology, 1988 Nov, 95(5), 1403 - 8
Chemical colitis due to endoscope cleaning solutions: a mimic of pseudomembranous colitis; Jonas G et al.; A unique form of colitis was observed during endoscopy of the lower gastrointestinal tract in 21 patients . The patients were prepared using either tap-water enemas or standard lavage solutions . Patients were found to have discrete or confluent white plaques adherent to the colonic mucosa, mild to severe erythema of the surrounding mucosa, and variable amounts of foamy liquid upon withdrawal of the endoscope . Stool assays for Clostridium difficile toxin and bacterial cultures were negative . Mucosal biopsies revealed vacuolar changes in the lamina propria, with slight to moderate vascular congestion and foci of intramucosal hemorrhage . Five patients developed rectal bleeding, tenesmus, and increased frequency of stools, lasting up to 12 days . We believe these cases were due to contamination of the endoscope's air-water channel with solutions used during endoscope cleaning . Recognition of this entity is important, as it is preventable and may mimic pseudomembranous colitis.

Appl Environ Microbiol, 1988 Nov, 54(11), 2819 - 24
Transformation of tetrachloromethane to dichloromethane and carbon dioxide by Acetobacterium woodii; Egli C et al.; Five anaerobic bacteria were tested for their abilities to transform tetrachloromethane so that information about enzymes involved in reductive dehalogenations of polychloromethanes could be obtained . Cultures of the sulfate reducer Desulfobacterium autotrophicum transformed some 80 microM tetrachloromethane to trichloromethane and a small amount of dichloromethane in 18 days under conditions of heterotrophic growth . The acetogens Acetobacterium woodii and Clostridium thermoaceticum in fructose-salts and glucose-salts media, respectively, degraded some 80 microM tetrachloromethane completely within 3 days . Trichloromethane accumulated as a transient intermediate, but the only chlorinated methanes recovered at the end of the incubation were 8 microM dichloromethane and traces of chloromethane . Desulfobacter hydrogenophilus and an autotrophic, nitrate-reducing bacterium were unable to transform tetrachloromethane . Reduction of chlorinated methanes was thus observed only in the organisms with the acetyl-coenzyme A pathway . Experiments with {14C}tetrachloromethane were done to determine the fate of this compound in the acetogen A . woodii . Radioactivity in an 11-day heterotrophic culture was largely (67%) recovered in CO2, acetate, pyruvate, and cell material . In experiments with cell suspensions to which {14C}tetrachloromethane was added, 14CO2 appeared within 20 s as the major transformation product . A . woodii thus catalyzes reductive dechlorinations and transforms tetrachloromethane to CO2 by a series of unknown reactions.

J Clin Invest, 1988 Nov, 82(5), 1516 - 24
Clostridium difficile toxin A perturbs cytoskeletal structure and tight junction permeability of cultured human intestinal epithelial monolayers; Hecht G et al.; Toxin A of Clostridium difficile causes severe inflammatory enterocolitis in man and animals that appears to be mediated in part by acute inflammatory cells that migrate into the toxin A-exposed mucosa . To determine the direct effects of toxin A on intestinal epithelial permeability and structure in the absence of other modulating factors, we used cultured monolayers of a human intestinal epithelial cell line (T84) . A toxin A concentration of 7 x 10(-1) micrograms/ml (3 x 10(-9) M) nearly abolished monolayer transepithelial resistance within 6-8 h . This marked permeability defect occurred while the monolayers were still confluent . Dual sodium-mannitol flux studies localized the permeability defect to the intercellular tight junction . Cytotoxicity assays and morphological evaluation using Nomarski optics and electron microscopy failed to demonstrate any evidence of cell damage at the time the maximum resistance response was observed . Fluorescent staining for F actin, however, revealed a marked decrease in fluorescent intensity in toxin-treated monolayers versus controls . These data show that toxin A can directly affect the barrier function of this model intestinal epithelium and initially does so by selectively enhancing tight junction permeability . Furthermore, cytoskeletal structure is markedly altered over the same time course, although the integrity of individual cells is maintained . Because the cytoskeleton of intestinal epithelial cells is known to be capable of regulating tight junction permeability, we speculate that the above effects of toxin A on epithelial barrier function result from alterations of the cytoskeleton.

J Clin Microbiol, 1988 Nov, 26(11), 2447 - 9
Multilocus enzyme electrophoresis of Clostridium argentinense (Clostridium botulinum toxin type G) and phenotypically similar asaccharolytic clostridia; Altwegg M et al.; Twenty-three strains of Clostridium argentinense, C . subterminale, C . hastiforme, and other phenotypically similar asaccharolytic clostridia recently placed in seven DNA hybridization groups were compared by multilocus enzyme electrophoresis . The three nontoxigenic strains of C . argentinense were most closely related to the toxigenic strains of this species . All nine toxigenic strains of C . argentinense belonging to a single DNA hybridization group had identical enzyme types on the basis of nine enzymes . All other strains except for two derived from the type strain of C . subterminale were differentiable . Overall, there was excellent agreement between DNA relatedness and multilocus enzyme electrophoresis results.

Res Vet Sci, 1988 Nov, 45(3), 337 - 40
In vitro lecithinase activity and sensitivity to 22 antimicrobial agents of Clostridium perfringens isolated from necrotic enteritis of broiler chickens; Kondo F; Viable Clostridium perfringens ranging from 10(5) to 10(8) g-1 was detected in all of 88 intestinal content specimens of necrotic enteritis in broiler chickens . In vitro lecithinase activity and sensitivity to 22 antimicrobial agents were determined for the 88 isolates . The activities of lecithinase in the culture filtrate of isolates were estimated to be 0.5 to 4.0 AE ml-1 as alpha-antitoxin equivalent . With reference to antimicrobial activity penicillins and cephazolin showed excellent activity and no resistance; peptides, of the agents used as growth promoters, showed that all except bacitracin had low minimum inhibitory concentration levels (1.6 micrograms ml-1 or less) against this organism; polyethers of monensin, salinomycin and lasalocid were generally adequate in low concentrations while there was a high level of resistance to three tetracyclines in 90 per cent of the strains and all isolates were insusceptible to streptomycin of the aminoglycoside antibiotics.

Gene, 1988 Oct 30, 70(2), 343 - 50
Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli; Dailey DC et al.; The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized . A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46 . Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments . The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements . This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E . coli and C . difficile, can be used in the construction of a shuttle vector capable of gene expression in E . coli and C . difficile.

Tijdschr Diergeneeskd, 1988 Oct 15, 113(20), 1135 - 8
{A case of food poisoning caused by C . perfringens}; Mol H et al.; A case of food borne infection among a hundred inhabitants of a home for the old aged, caused by Clostridium perfringens (Clostridium welchii) following consumption of a filled veal roll is reported.

Biochim Biophys Acta, 1988 Oct 14, 962(3), 362 - 70
Characterization of NADP-dependent 12 beta-hydroxysteroid dehydrogenase from Clostridium paraputrificum; Edenharder R et al.; Clostridium paraputrificum D 762-06 was found to contain an NADP-dependent 12 beta-hydroxysteroid dehydrogenase, already present in uninduced cells . Its specific activity could, however, be enhanced up to about 3-fold by the inclusion of bile acids with a 12-keto group or a 12 beta-hydroxy group in the growth medium . 3 alpha-Hydroxy-12-keto-5 beta-cholanoic acid was the most effective inducer . A pH optimum of 10.0 and a molecular weight of 126,000 were estimated by molecular sieve chromatography . The enzyme preparation reduced 12-keto groups in conjugated and unconjugated bile acids and oxidized a 12 beta-hydroxy function, but oxidative activity was only about 25% of the reductive one . Disubstituted bile acids showed lower Km values than the corresponding trisubstituted ones, the lowest Km values being those observed for 3,12- and 7,12-5 beta-cholanoic acids . No measurable activity against 12 alpha-hydroxyl groups could be detected . The enzyme was found to be heat-labile (95% inactivation at 50 degrees C for 10 min), but the activity was maintained for about 4 weeks when lyophilized preparations were stored at -20 degrees C . 12 beta-Hydroxysteroid dehydrogenase activity was also demonstrated in the membrane fraction after solubilization with Triton X-100, suggesting that it was originally a membrane-bound enzyme.

Biochem Biophys Res Commun, 1988 Oct 14, 156(1), 551 - 6
Enterotoxin of Clostridium perfringens type A forms ion-permeable channels in a lipid bilayer membrane; Sugimoto N et al.; The enterotoxin of Clostridium perfringens type A was found to form ion-permeable channels in a lipid bilayer . A patch clamp technique was used to detect channel activities in an asolectin bilayer with incorporated enterotoxin . About 20% of the lipid bilayer patches examined showed rectangular or stepwise shift of membrane current . The shifts indicated the gating of ion-permeable channels in the patches . The channels showed high conductance (40-450 pS), no rectification in current-voltage curves and occasional long-lasting events . The significance of these findings is discussed in relation to the mechanism of action of the toxin.

Biochemistry, 1988 Oct 4, 27(20), 7698 - 702
Kinetic characterization of the carbon monoxide-acetyl-CoA (carbonyl group) exchange activity of the acetyl-CoA synthesizing CO dehydrogenase from Clostridium thermoaceticum; Raybuck SA et al.; CO dehydrogenase from Clostridium thermoaceticum is a nickel-containing enzyme that catalyzes both the reversible conversion of CO2 to CO (for incorporation into the carbonyl group of acetate) and the synthesis of acetyl-CoA from methyl corrinoid, CO, and CoASH . The latter activity is conveniently assayed by monitoring the exchange of {1-14C}acetyl-CoA (carbonyl group) with 12CO . Kinetic parameters for the highly oxygen sensitive exchange activity have been determined: Km (acetyl-CoA) = 600 microM; Vmax = 440 min-1 . In addition, coenzyme A analogues have been tested as inhibitors of the exchange to probe the active site of the enzyme; each has no effect on the CO2 in equilibrium CO activity of CO dehydrogenase . Coenzyme A, the substrate for acetate biosynthesis, is a potent competitive inhibitor, KI = 7 microM . Comparison of this value with that for desulfo-CoA (KI = 6000 microM) suggests that a key mode of binding is through the sulfur atom, possibly to a metal site on the enzyme . The relatively high affinity of the enzyme for CoASH relative to acetyl-CoA is consistent with its proposed operation in the acetogenic direction . The differential sensitivity to oxygen and storage of the two activities of CO dehydrogenase as well as the contrasting effect of coenzyme A inhibitors suggests that acetate assemblage occurs at a site distinct from that for CO dehydrogenation.

Int J Food Microbiol, 1988 Oct, 7(2), 169 - 72
Resistance of vegetative cells and endospores of Sporolactobacillus to gamma-irradiation; Botha SJ et al.; Vegetative cells of Sporolactobacillus showed average resistance to gamma-irradiation compared to other vegetative bacteria with D10 values ranging from 0.350 to 0.525 kGy . Endospores of Sporolactobacillus showed higher resistance to gamma-irradiation than most Bacillus species but were close to that of Clostridium species . The average D10 value for Sporolactobacillus endospores was 2.5 kGy.

Pathology, 1988 Oct, 20(4), 349 - 52
An evaluation of a rapid latex test for the diagnosis of Clostridium difficile-associated diarrhea; Munro R et al.; We present here the results of an evaluation of a rapid latex test for detection of Cl . stridium difficile-associated in comparison with our standard cytotoxin assay and culture for C . difficile . Some 515 diarrheal stools were examined . C . difficile was cultured from 70 specimens (13.5%); 53 specimens (10.2%) were positive with the latex test, and 50 (9.6%) by cytotoxin assay . The latex test did not differ significantly from the cytotoxin assay in sensitivity or specificity compared to culture results . There was also no significant difference in the specificity of the latex test compared to cytotoxin assay in patients in whom the diagnosis of C . difficile-associated diarrhea was negative . Positive and negative predictive values of the latex test for C . difficile-associated diarrhea were similar to those of cytotoxin assay . The latex test thus appears to be a rapid and practical test for the laboratory diagnosis of C . difficile-associated diarrhea . To optimize specificity and sensitivity its use should be restricted to patients where the diagnosis is strongly suspected and a rapid answer is required . As it does not distinguish between toxigenic virulent C . difficile strains and non-toxigenic avirulent strains, it would seem prudent to confirm positive results subsequently by demonstrating in-vivo or in-vitro cytotoxin production.

Poult Sci, 1988 Oct, 67(10), 1424 - 30
Ulcerative enteritis in broiler chickens caused by Clostridium colinum and in vitro activity of 19 antimicrobial agents in tests on isolates; Kondo F et al.; Ulcerative enteritis in broiler chickens occurred at five poultry farms in Kagoshima Prefecture, southern Japan, in February and March, 1987 . This is the first incidence of this disease reported for chickens in Japan . The mortality rate was estimated to be 1 to 5% . Ulcerative enteritis in the intestines, and necrosis in the liver and spleen, were observed mainly in autopsied broilers . Identification involving tests of biochemical properties and production of metabolic endproducts using gas-liquid chromatography were consistent with an identification of Clostridium colinum . Antimicrobial agent susceptibility tests on the isolates showed that all were highly sensitive to the agents, with the exception of aminoglycoside antibiotics . Minimum inhibitory concentrations of penicillin-G and ampicillin ranged from .025 microgram/mL to .05 microgram/mL . No resistant strains were isolated.

Arch Biochem Biophys, 1988 Oct, 266(1), 142 - 51
Botulinum neurotoxin type A: cleavage of the heavy chain into two halves and their partial sequences; Sathyamoorthy V et al.; The 145-kDa type A botulinum neurotoxin (NT) is produced by the bacteria Clostridium botulinum (strain, Hall) . The heavy (H) and light (L) chains (97- and 53-kDa, respectively) of this protein are linked by at least one disulfide bond . The N- and C-terminal halves of the H chain appear to have different functions in the mechanism of action of the NT {1987) FEBS Lett . 226, 115-120) . Well-characterized and highly purified preparations of the two halves of the H chain are needed for such studies . Two different approaches were taken to cut the H chain with trypsin and isolate the fragments . In one method the cleavage products were: (i) 94-kDa fragment made of the L chain linked to the N-terminal half of the H chain (49 kDa) by a disulfide bond(s), and (ii) the C-terminal 44-kDa fragment . The N-terminal half of H chain was separated from the L chain by reducing the disulfide bond(s) linking them and then purified by ion-exchange chromatography . The 1-27 residues of 49-kDa N-terminal half of the H chain were Ala-Leu-Asn-Asp-Leu-Cys-Ile-Lys-Val-Asn-Asn-Trp-Asp-Leu-Phe-Phe-Ser-Pro- Ser-Glu - Asp-Asn-Phe-Thr-Asn-Asp-Leu- . The sequence of the other half of the H chain (44 kDa) was X-Ile-Ile-Asn-Leu-X-Ile-Leu-Asn-Leu-Arg-Tyr-Glu-X-Asn-His-Leu-Ile-Asp-Le u-Lys- X-Tyr-Ala-Ser- . In the second method, the H chain was first separated from the L chain, purified, and then cleaved . One product of cleavage, the 44-kDa fragment, was partially sequenced; the first 25 residues were identical to the sequence of the 44-kDa fragment generated by the first method . The present work also demonstrated that (i) The cysteine residue(s) located on the N-terminal half of the H chain form the -S-S- link(s) with the L chain . (ii) The other half of the H chain (44-kDa fragment, apparently the C-terminal half) is not linked via -S-S- to the L-chain or to the N-terminal half (49-kDa fragment) of the H chain.(ABSTRACT TRUNCATED AT 400 WORDS)

Pediatr Nephrol, 1988 Oct, 2(4), 415 - 8
Haemolytic uraemic syndrome and pseudomembranous colitis; Rooney N et al.; Two cases of haemolytic uraemic syndrome (HUS) associated with pseudomembranous colitis (PMC) are described . The toxin of Clostridium difficile was detected post mortem in the stool of one patient and the other patient showed a good therapeutic response to oral vancomycin, an antibiotic with established efficacy in the management of PMC . When associated with HUS, PMC is probably an independent specific disease that, in common with many other infections, may activate HUS.

Mol Gen Genet, 1988 Oct, 214(2), 328 - 32
Cloning of two chloramphenicol acetyltransferase genes from Clostridium butyricum and their expression in Escherichia coli and Bacillus subtilis; Dubbert W et al.; Two non-homologous chloramphenicol (Cm) acetyltransferase (CAT) genes, designated catA and catB, were cloned from Clostridium butyricum type strains and characterized by restriction mapping . Both genes are efficiently expressed in Escherichia coli and Bacillus subtilis . In contrast to analogous genes from staphylococci and bacilli, gene expression is not dependent on induction by Cm . The genes are considered as chromosomal, since no association with endogenous plasmids was detectable . Southern hybridization revealed a homology between catA and the staphylococcal Cm resistance plasmid, pC194 . The subunit size of the clostridial CAT enzymes expressed in E . coli was determined as 22.5 kDa (catA) and 24 kDa (catB), respectively . The C . butyricum cat genes provide potentially useful selection markers for the construction of cloning vectors from cryptic clostridial plasmids.

J Antimicrob Chemother, 1988 Oct, 22 Suppl D, 115 - 8
The penetration of fleroxacin into intra-abdominal abscesses; Youngs DJ et al.; Using a recently developed, low mortality model of an intra-abdominal abscess in the Wistar rat, we have studied the penetration of fleroxacin into the abscess . Maximum serum concentration was 1.83 +/- 0.39 mg l and occurred 1 h after iv injection (20 mg/kg), but even at 4 h after administration the mean serum level was 1.21 +/- 0.27 mg/l . By contrast, levels in pus were 6.27 +/- 0.83 mg/l at 1 h rising steadily to a value of 12.7 +/- 3.69 mg/l at 4 h . The study has confirmed exceptional antibiotic penetration into the abscess, with levels at all time intervals between 0.5 and 8 h after administration in excess of the MIC50 for Escherichia coli, Proteus vulgaris and Clostridium perfringens.

J Bacteriol, 1988 Oct, 170(10), 4582 - 8
Nucleotide sequence and deletion analysis of the xylanase gene (xynZ) of Clostridium thermocellum; Grepinet O et al.; The nucleotide sequence of the xynZ gene, encoding the extracellular xylanase Z of Clostridium thermocellum, was determined . The putative xynZ gene was 2,511 base pairs long and encoded a polypeptide of 837 amino acids . A region of 60 amino acids containing a duplicated segment of 24 amino acids was found between residues 429 and 488 of xylanase Z . This region was strongly similar to the conserved domain found at the carboxy-terminal ends of C . thermocellum endoglucanases A, B, and D . Deletions removing up to 508 codons from the 5' end of the gene did not affect the activity of the encoded polypeptide, showing that the active site was located in the C-terminal half of the protein and that the conserved region was not involved in catalysis . Expression of xylanase activity in Escherichia coli was increased up to 220-fold by fusing fragments containing the 3' end of the gene with the start of lacZ present in pUC19 . An internal translational initiation site which was efficiently recognized in E . coli was tentatively identified 470 codons downstream from the actual start codon.

J Bacteriol, 1988 Oct, 170(10), 4576 - 81
Purification of Clostridium thermocellum xylanase Z expressed in Escherichia coli and identification of the corresponding product in the culture medium of C . thermocellum; Grepinet O et al.; An endoxylanase encoded by the xynZ gene of Clostridium thermocellum was purified from Escherichia coli harbouring a fragment of the gene cloned in pUC8 . The purified enzyme showed two active bands of Mr 41,000 and 39,000, the latter one presumably derived from the former through proteolysis . The enzyme was highly active on xylan and para-nitrophenyl-beta-D-xylobioside . The major end product of xylan hydrolysis was xylobiose . With an antiserum raised against the enzyme purified from E . coli, an immunoreactive polypeptide of Mr 90,000, corresponding to the entire xynZ gene product, was detected in a culture supernatant from C . thermocellum grown on cellulose . By immunological detection, xylanase Z was shown to be associated with a cellulose-binding, high-molecular-weight fraction whose properties coincided with those described previously for the cellulose-degrading complex of C . thermocellum known as the cellulosome.

Microbiol Sci, 1988 Oct, 5(10), 310 - 5
Host: vector systems for gene cloning in Clostridium; Minton NP et al.; The genus Clostridium contains some of the most toxic bacteria known to man (e.g., Clostridium tetani, Clostridium botulinum) as well as solvent-producing organisms of biotechnological interest (e.g., Clostridium acetobutylicum) . Developments within the areas of plasmid vector construction and plasmid delivery methodology now provide the opportunity for applying recombinant DNA technology to this important group of bacteria.

Hinyokika Kiyo, 1988 Oct, 34(10), 1833 - 6
{A case of Fournier's gangrene}; Yonezu M et al.; A 59-year-old man was admitted to our hospital complaining of perineal pain and scrotal redness . He was diagnosed with diabetes mellitus 12-years ago but had not received treatment . The scrotum soon showed necrosis and pus discharge . A culture study revealed Clostridium, Bacteroides and many other bacteria . Antibiotics chemotherapy (CMZ, AMK, MINO) was done but because necrotizing fasciitis progressed, we performed surgical treatment of necrosis . The skin defect was closed naturally with wound irrigation . The patient is doing well and his diabetes is under control . We found 13 other cases of Fournier's gangrene in the Japanese literature and their age, culture study and complications are discussed.

Epidemiol Infect, 1988 Oct, 101(2), 321 - 5
Thermal sensitivity of Clostridium botulinum type C toxin; Hubalek Z et al.; A sterile suspension containing 950 mouse LD50 per ml of type C botulinum toxin was exposed for various periods to different temperatures . The time required for the 99% (hundred-fold) reduction of toxicity was more than 5 years at -70 degrees C or -20 degrees C, 6 months at +5 degrees C, 3 weeks at +20 degrees C, 2 weeks at +28 degrees C, 2 days at +37 degrees C, 9 h at +42 degrees C, less than 30 min at +56 degrees C, less than 20 min at +60 degrees C, and below 5 min at +80 degrees C . The results suggest that Clostridium botulinum type C toxin, if produced in an ecosystem of the mild climatic zone, might persist there over the winter season and cause the intoxication of vertebrates next early spring in the absence of further microbial toxigenesis.

Postgrad Med J, 1988 Oct, 64(756), 812 - 3
Gas gangrene in a diabetic after intramuscular injection; Kershaw CJ et al.; A 47 year old male insulin-dependent diabetic developed two synchronous life-threatening Clostridium perfringens infections under unusual circumstances . Numerous sterile abscesses were also present, being the result of regular pentazocine intramuscular injections . Extensive surgical debridement and parenteral antibiotic therapy proved effective in treating the septicaemia and the large soft tissue abscesses in the buttock and thigh . The patient made an excellent functional recovery . The pathogenesis of gas gangrene after injection is discussed with particular reference to the diabetic patient.

J Appl Bacteriol, 1988 Oct, 65(4), 261 - 8
Purification, characterization and antimicrobial spectrum of a bacteriocin produced by Pediococcus acidilactici; Bhunia AK et al.; An antimicrobial peptide designated pediocin AcH was isolated from Pediococcus acidilactici strain H . The pediocin AcH was purified by ion exchange chromatography . The molecular weight of pediocin AcH was determined by SDS-PAGE to be about 2700 daltons . Pediocin AcH was sensitive to proteolytic enzymes resistant to heat and organic solvents, and active over a wide range of pH . Pediocin AcH exhibited inhibition against several food spoilage bacteria and foodborne pathogens including Staphylococcus aureus, Clostridium perfringens and Listeria monocytogenes . It was bactericidal to sensitive cells and acted very rapidly . The bactericidal effect was not produced by either cell lysis or apparent loss of membrane permeability.

J Infect Dis, 1988 Oct, 158(4), 731 - 6
Characterization of a nosocomial Clostridium difficile outbreak by using plasmid profile typing and clindamycin susceptibility testing; Clabots CR et al.; The mean number of cases of Clostridium difficile diarrhea at the Minneapolis Veterans Administration Medical Center increased to 17.3 per month in June-August 1985, compared with 7.1 per month in the previous 17 mo . Plasmid profiles and clindamycin susceptibility were used as markers to evaluate the increase in cases . Ninety clindamycin-resistant and 22 clindamycin-susceptible isolates of C . difficile from 1985 were examined for plasmids . A clindamycin-resistant organism contained a cryptic plasmid of 3.1 kilobases (kb) . None of the clindamycin-susceptible isolates contained the 3.1-kb plasmid, as compared with 40 of 90 clindamycin-resistant isolates (P less than .005) . Restriction endonuclease digestion and Southern blot hybridization were used to confirm the identity of the 3.1-kb plasmid between strains . Isolates retained clindamycin resistance after plasmid curing . It could not be determined if the organism responsible was an indigenous C . difficile strain that acquired a plasmid or was a new strain introduced from outside the hospital.

J Bacteriol, 1988 Oct, 170(10), 4613 - 8
Cloning and expression of Clostridium acetobutylicum phosphotransbutyrylase and butyrate kinase genes in Escherichia coli; Cary JW et al.; A 13.6-kilobase (kb) Sau3AI restriction endonuclease fragment of Clostridium acetobutylicum DNA cloned into pBR322 enabled Escherichia coli ato mutants to grow on butyrate as a sole carbon source (But+) . Complementation of the ato defect by the recombinant plasmid pJC6 was due to expression of the genes for phosphotransbutyrylase (PTB) and butyrate kinase (BK) . Both genes were efficiently expressed in E . coli, as their products were readily detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of whole-cell extracts . PTB was found to have a polypeptide subunit molecular weight of approximately 31,000, while that of BK was approximately 39,000 . Deletion analysis and Tn5 mutagenesis of plasmid pJC7 (a But+ subclone containing a 4.4-kb BamHI fragment from the insert of pJC6) localized the PTB and BK genes within a region spanning approximately 2.9 kb . Preliminary evidence suggests that the two genes may form an operon that is transcribed as a single unit from a promoter of clostridial origin within the 4.4-kb insert of pJC7.

J Antibiot (Tokyo), 1988 Oct, 41(10), 1471 - 8
Mechanism of inhibition of reverse transcriptase by quinone antibiotics . II . Dependence on putative quinone pocket on the enzyme molecule; Hafuri Y et al.; Inhibition of avian myeloblastosis virus (AMV) reverse transcriptase by natural and synthetic quinones including antibiotics could be accounted for by an oxidation-reduction reaction . The quinones were shown to function as electron acceptors as revealed by the catalytic oxidation of NADH by Clostridium kluyveri diaphorase which was in excellent agreement with enzyme inhibition activity . The kinetics of inhibition of AMV reverse transcriptase by three synthetic quinones with different core structures, i.e., 6-methoxy-5,8-dihydroquinoline-5,8- dione, 5,8-dihydroisoquinoline-5,8-dione and 1,4-naphthoquinone, were studied . These quinones inhibited reverse transcriptase in the same manner as streptonigrin (STN) and were shown to act at a single class of reaction site(s) on the enzyme molecule . In contrast, the quinones with bulky substituents, i.e., 7-(2-nitrophenethylamino)-5,8-dihydroisoquinoline-5,8-dione and 7-methoxy-6-methyl-3-piperidino-5,8-dihydroisoquinoline-5,8-dione, were inactive as inhibitors of reverse transcriptase, whereas they retained competent catalytic activities in the oxidation of NADH by C . kluyveri diaphorase . Based on these observations, the existence of a specific site of interaction on the enzyme molecule, referred to as a quinone pocket, was proposed . The quinone pocket might play a crucial role in the early sequence of events leading to the inhibition of reverse transcriptase by quinones including STN and sakyomicin A (SKM) . Access of SKM to a quinone pocket might be restricted due to its bulky structure in the vicinity of the quinone group . This is inferred from unsuccessful inhibition of reverse transcriptase by the quinones with bulky substituents, resulting in much poorer inhibition of reverse transcriptase in spite of more potent electron acceptor activity in the oxidation-reduction system as compared with those of STN.

FEBS Lett, 1988 Sep 26, 238(1), 31 - 4
Cloning and sequencing of a Clostridium perfringens sialidase gene; Roggentin P et al.; Escherichia coli was transformed with pUC vectors containing Sau3A restriction fragments (RF) of Clostridium perfringens DNA . Two clones expressed sialidase activity when assayed with the fluorogenic substrate 4-methylumbelliferyl-alpha-D-N-acetylneuraminic acid . A synthetic oligonucleotide representing the N-terminus of the expressed enzyme hybridized with the clostridial insert and with a corresponding 2.1 kb Sau3A RF of the C . perfringens genome . The insert reduced to 1.4 kb, which still encoded active sialidase, has been sequenced . The structural gene encodes 382 amino acids representing an Mr of 42770 . A hydrophobic leader sequence is absent . Upstream from the initiation codon ATG, a GA-rich region is found and considered as the Shine-Dalgarno sequence . Homology with the N-terminus of the Vibrio cholerae sialidase gene and with viral sialidase sequences was not found.

FEBS Lett, 1988 Sep 26, 238(1), 22 - 6
A 22 kDa ras-related G-protein is the substrate for an ADP-ribosyltransferase from Clostridium botulinum; Quilliam LA et al.; A ribosyltransferase from C . botulinum type D ADP-ribosylated a protein of 22 kDa (p22) in human astrocytoma (1321N1) cells . ADP-ribosylation of membrane-bound p22 was potentiated by 2 mM MgCl2 or guanine nucleotides but was much reduced in the presence of 10 mM Mg2+ plus GTP gamma S . p22 was immunoprecipitated by a monoclonal antibody (142-24E05) raised against a peptide sequence common to the ras gene family but not by other ras or G-protein antibodies . p22 was also ADP-ribosylated in Drosophila but was not detected in Dictyostelium . These data suggest that the 22 kDa botulinum toxin substrate is a GTP-binding protein and a member of the ras protein family.

Lancet, 1988 Sep 24, 2(8613), 714 - 7
Treatment of anismus in intractable constipation with botulinum A toxin; Hallan RI et al.; In seven patients with anismus the striated sphincter muscle complex was selectively weakened by local injection of Clostridium botulinum type A toxin . Symptom scores improved significantly and correlated with a significant reduction in the maximum voluntary and canal squeeze pressure and a significant increase in the anorectal angle on straining . Botulinum A toxin seems to be promising treatment for some patients with anismus.

Biochemistry, 1988 Sep 20, 27(19), 7413 - 8
Preparation by direct metal exchange and kinetic study of active site metal substituted class I and class II Clostridium histolyticum collagenases; Angleton EL et al.; Active site metal substitutions for both gamma- and zeta-collagenases from Clostridium histolyticum have been made by direct metal exchange . The incubation of Co(II), Cu(II), Ni(II), Cd(II), and Hg(II) with these native collagenases results in changes in activity that parallel those observed for the reconstitution of the respective apoenzymes with these metal ions . For both collagenases, the exchange reactions with Co(II) and Cu(II) are complete within 1 min . However, the changes in activity observed on addition of Ni(II), Cd(II), and Hg(II) to gamma-collagenase and Cd(II) and Hg(II) to zeta-collagenase are time dependent . The kinetic parameters Kcat and KM have been determined for each of the active metallospecies . The substitution of the active-site metal ion in gamma-collagenase results in changes in both kcat and KM, while the effect observed in zeta-collagenase is primarily on KM . This suggests that there are differences in the mechanisms of these two collagenases, at least with respect to the role of the zinc ion in catalysis.

Biochemistry, 1988 Sep 20, 27(19), 7406 - 12
Preparation and reconstitution with divalent metal ions of class I and class II Clostridium histolyticum apocollagenases; Angleton EL et al.; Both gamma- and zeta-collagenases from Clostridium histolyticum are fully and reversibly inhibited by 1,10-phenanthroline at pH 7.5 in the presence of 10 mM CaCl2 with KI values of 0.11 and 0.040 mM, respectively . The inhibition is caused by removal of the single, active-site Zn(II) present in each of these enzymes . The nonchelating analogue 1,5-phenanthroline has no effect on the activity of either enzyme . Dialysis of the enzymes in the presence of 1,10-phenanthroline, followed by back dialysis against buffer containing no chelating agent, gives the respective apocollagenases . Both apoenzymes can be instantaneously and fully reactivated by the addition of 1 equiv of Zn(II) . Variable amounts of activity are restored to both apocollagenases by Co(II) and Ni(II) and to gamma-apocollagenase by Cu(II) . The activity titration curve for gamma-apocollagenase with Co(II) and Scatchard plots for the reconstitution of gamma-apocollagenase with Cu(II) and Ni(II) and of zeta-apocollagenase with Ni(II) and Co(II) indicate that all activity changes are the result of binding of a single equivalent of these divalent metal ions at the active site of the collagenases . Cd(II) and Hg(II) do not restore measurable activity to either apoenzyme.

Gene, 1988 Sep 15, 69(1), 29 - 38
Conserved reiterated domains in Clostridium thermocellum endoglucanases are not essential for catalytic activity; Hall J et al.; The complete nucleotide sequence of the Clostridium thermocellum celE gene, coding for an endo-beta-1,4-glucanase (endoglucanase E; EGE) with xylan-hydrolysing activity has been determined . The structural gene consists of an open reading frame (ORF) of 2442 bp commencing with a GTG start codon and followed by a TAA stop codon . The nucleotide sequence obtained has been confirmed by comparing the predicted amino acid sequence with that derived by N-terminal amino acid sequencing of the purified protein . The EGE sequence contains a region homologous to the reiterated domain found at the C terminus of other endoglucanases from the same organism . BAL 31 deletions of the structural gene have revealed the extent to which this conserved sequence is necessary for endoglucanase and xylanase activity . A region of DNA, upstream from the structural gene has also been sequenced and a ribosome-binding site and putative promoter sequences have been identified . A second ORF which ends 349 bp 5' to the GTG start codon of the celE gene has also been identified . The encoded product contains a C terminus homologous to other C . thermocellum endoglucanases.

Biochem J, 1988 Sep 15, 254(3), 835 - 40
Purification and characterization of a novel thermostable beta-amylase from Clostridium thermosulphurogenes; Shen GJ et al.; An extracellular beta-amylase from Clostridium thermosulphurogenes was purified 811-fold to homogeneity, and its general molecular, physico-chemical and catalytic properties were determined . The native enzyme was a tetramer of 210 kDa composed of a single type subunit; its 20 amino acid N-terminus displayed 45% homology with Bacillus polymyxa beta-amylase . The beta-amylase was enriched in both acidic and hydrophobic amino acids . The pure enzyme displayed an isoelectric point of 5.1 and a pH activity optimum of 5.5 . The optimum temperature for beta-amylase activity was 75 degrees C, and enzyme thermostability at 80 degrees C was enhanced by substrate and Ca2+ addition . The beta-amylase hydrolysed amylose to maltose and amylopectin and glycogen to maltose and limit dextrins, and it was inhibited by alpha- and beta-cyclodextrins . The enzyme displayed kcat . and Km values for boiled soluble starch of 400,000 min-1 per mol and 1.68 mg/ml, respectively . The enzyme was antigenically distinct from plant beta-amylases.

J Biol Chem, 1988 Sep 5, 263(25), 12420 - 6
Purification and properties of the cytosolic substrate for botulinum ADP-ribosyltransferase . Identification as an Mr 22,000 guanine nucleotide-binding protein; Morii N et al.; The substrate for ADP-ribosyltransferase from Clostridium botulinum was purified from the cytosol of bovine adrenal gland . Purification procedures consisted of ammonium sulfate fractionation, chromatographies on columns of DEAE-Sepharose and phenyl-Sepharose, gel filtration on a TSK-gel G3000SW column, and Mono Q fast protein liquid chromatography . On DEAE-Sepharose chromatography, the substrate activity was eluted in two separate peaks, and electrophoretic analyses revealed that the substrates in the two peaks are of similar molecular weight but different isoelectric points . The major peak of the substrate was further purified . It was purified about 1,800-fold with a recovery of 2.2% by the above procedures . On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the final preparation showed a single protein band at Mr 22,000 . The purified protein served as a substrate for botulinum ADP-ribosyltransferase and was maximally ADP-ribosylated to the extent of about 0.7 mol of ADP-ribose/mol of protein . A guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) binding activity was co-purified with the ADP-ribosylation substrate, and the purified protein maximally bound about 0.5 mol of GTP gamma S/mol . GTP gamma S binding was effectively competed by GTP and GDP but not by GMP, ATP, and ADP . Thus, the ADP-ribosylation substrate is a GTP-binding protein . This protein, designated Gb (b for botulinum), is widely distributed in various tissues . It was rich in brain, pituitary, and adrenal glands, and poor in heart, smooth, and skeletal muscles.

Biochim Biophys Acta, 1988 Sep 2, 962(1), 116 - 21
Lyophilized Clostridium perfringens 3 alpha- and Clostridium bifermentans 7 alpha-hydroxysteroid dehydrogenases: two new stable enzyme preparations for routine bile acid analysis; Sutherland JD et al.; Preparations of 3 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.50) from Clostridium perfringens were successfully lyophilized into a stable powder form . Purification of the enzyme was achieved using triazine dye affinity chromatography . C . perfringens 3 alpha-hydroxysteroid dehydrogenase was purified 24-fold using Reactive Red 120 (Procion Red) -cross-linked agarose (70% yield) . Quantitative measurement of bile acids with the purified enzymes, 3 alpha-hydroxysteroid dehydrogenase and 7 alpha-hydroxysteroid dehydrogenase (EC 1.1.1.159) from Clostridium bifermentans (strain F-6), was achieved spectrophotometrically . Standard curves with chenodeoxycholic acid (CDC) and cholic acid were linear within a concentration range of 20-100 microM . Analysis of mixtures of ursodeoxycholic acid and CDC showed the additive nature of the 3 alpha-hydroxysteroid dehydrogenase and showed also that 7 alpha-hydroxyl groups were independently quantified by the 7 alpha-hydroxysteroid dehydrogenase . Bile acids in Folch extracts of human bile samples were measured using purified preparations of Pseudomonas testosteroni 3 alpha-hydroxysteroid dehydrogenase, C . perfringens 3 alpha-hydroxysteroid dehydrogenase, Escherichia coli 7 alpha-hydroxysteroid dehydrogenase and C . bifermentans (strain F-6) 7 alpha-hydroxysteroid dehydrogenase . Statistical comparison validated the use of C . perfringens 3 alpha- and C . bifermentans 7 alpha-hydroxysteroid dehydrogenases for the quantification of bile acids in bile.

Appl Environ Microbiol, 1988 Sep, 54(9), 2305 - 10
Classification and distribution of large intestinal bacteria in nonhibernating and hibernating leopard frogs (Rana pipiens); Banas JA et al.; The large intestinal flora of the leopard frog, Rana pipiens, was examined to determine whether differences existed between the nonhibernating and hibernating states of the animal and to determine the relative concentrations and proportions of potential frog pathogens . Hibernators had a logarithmic decrease of bacteria per milligram of intestine averaging one, and significantly greater proportions of facultative bacteria and psychrophiles relative to nonhibernators . The predominant anaerobic bacteria were gram-positive Clostridium species and gram-negative Bacteroides and Fusobacterium species . The predominant facultative bacteria were enterobacteria in nonhibernators but Pseudomonas species in hibernators . Many species of Pseudomonas are pathogenic for frogs, and thus the intestinal flora in hibernators may be a potential source of infectious disease.

J Appl Bacteriol, 1988 Sep, 65(3), 223 - 9
Studies on the irradiation of toxins of Clostridium botulinum and Staphylococcus aureus; Rose SA et al.; The effects of irradiation of Clostridium botulinum neurotoxin type A (BNTA) and staphylococcal enterotoxin A (SEA) in gelatin phosphate buffer and cooked mince beef slurries were investigated . Estimation of toxins by immunoassays showed that in buffer, toxins were destroyed by irradiation at 8.0 kGy; in mince slurries however, 45% of BTNA and 27-34% of SEA remained after this level of irradiation . At 23.7 kGy, over twice the dose of irradiation proposed for legal acceptance in the UK, 15% of BNTA and 16-26% of SEA still remained . Increasing concentrations of mince conferred increased protection against the effect of irradiation on both toxins . The biological activity of BNTA was more sensitive to irradiation than the immunological activity . Staphylococcal enterotoxin was more resistant to irradiation than BNTA . Irradiation should therefore only be used in conjunction with good manufacturing practices to prevent microbial proliferation and toxin production prior to irradiation.

Mol Gen Genet, 1988 Sep, 214(1), 177 - 9
Introduction of genes for leucine biosynthesis from Clostridium pasteurianum into C . acetobutylicum by cointegrate conjugal transfer; Oultram JD et al.; A Clostridium pasteurianum gene bank was constructed in Escherichia coli, using plasmid pAT153, and several chromosomal fragments found which complemented both leuB and leuC mutations in auxotrophic E . coli K12 strains . No fragments capable of complementing leuA or leuD mutations were identified . Conjugal transfer of the leuB/leuC genes from Bacillus subtilis into two different Leu- C . acetobutylicum auxotrophic strains was elicited by their incorporation into a large plasmid cointegrate composed of the conjugal plasmid pAM beta 1 and a specially constructed gram-positive, replication-deficient plasmid, pMTL21 EC . Inheritance of the cointegrate plasmid restored one of the auxotrophic C . acetobutylicum strains to prototrophy . The second strain remained Leu-.

Infect Immun, 1988 Sep, 56(9), 2299 - 306
Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain; Popoff MR et al.; By screening possible ADP-ribosyltransferase activities in culture supernatants from various Clostridium species, we have found one Clostridium difficile strain (CD196) (isolated in our laboratory) that is able to produce, in addition to toxins A and B, a new ADP-ribosyltransferase that was shown to covalently modify cell actin as Clostridium botulinum C2 or Clostridium perfringens E iota toxins do . The molecular weight of the CD196 ADP-ribosyltransferase (CDT) was determined to be 43 kilodaltons, and its isoelectric point was 7.8 . No cytotoxic activity on Vero cells or lethal activity upon injection in mice was associated with this enzyme . CDT was neither related to C . difficile A or B toxins nor to C . botulinum C2 toxin component I . However, Vero cells cultivated in the presence of C . difficile B toxin had a lower amount of actin able to be ADP-ribosylated by CDT or C2 toxin in vitro . Antibodies raised against CDT reacted by immunoblot analysis with a 43-kilodalton protein of C . perfringens type E culture supernatant producing the iota toxin.

J Antimicrob Chemother, 1988 Sep, 22(3), 325 - 31
Correlation between serogroup and susceptibility to chloramphenicol, clindamycin, erythromycin, rifampicin and tetracycline among 308 isolates of Clostridium difficile; Delmee M et al.; The susceptibility to chloramphenicol, clindamycin, erythromycin, rifampicin and tetracycline of 308 isolates of Clostridium difficile from various origins was determined by a disc diffusion susceptibility testing and the results were compared with the serogroup of the strains . For the five antimicrobials, there was a clear-cut separation between susceptible and resistant strains . Some correlation between resistance and serogroup was found . Almost all of the 161 isolates of serogroups A, F, G, H and X were susceptible to all antibiotics . The 32 toxigenic isolates of serogroup C were characterized by a typical resistance pattern which could be used for typing purposes . Other serogroups showed variable patterns . The review of 64 cases of antibiotic associated diarrhoea showed that these differences in susceptibility could have clinical implications: all seven cases due to clindamycin were caused by a clindamycin resistant strain of serogroup C, whereas cases associated with other antibiotics were distributed among various serogroups.

Hum Pathol, 1988 Sep, 19(9), 1102 - 8
The histopathology of the hemolytic uremic syndrome associated with verocytotoxin-producing Escherichia coli infections; Richardson SE et al.; Verocytotoxin-producing Escherichia coli (VTEC) infection was present in three cases of hemolytic uremic syndrome (HUS), two fatal and one non-fatal, in which detailed histopathologic investigations were conducted . Two patients had a prodrome of bloody diarrhea, one of whom required a hemicolectomy for severe bleeding . The renal histopathology was characterized primarily by glomerular thrombotic microangiopathy (TMA) with greater than 95% of glomeruli showing changes of capillary wall thickening, endothelial cell swelling, and narrowing or thrombosis of the capillary lumen . Preglomerular arterioles were frequently thrombosed, and abnormalities of the medium-sized vessels, including endothelial cell damage and thrombosis, were also commonly observed . Gastrointestinal involvement was prominent in all three cases . The colon was most severely involved, with marked mucosal and submucosal edema and hemorrhage, in the absence of significant inflammation or widespread ulceration . Microvascular angiopathy was present in all cases, with changes ranging from endothelial cell damage to overt thrombosis . Similar pathology was seen throughout the small bowel, including the presence of TMA . In one patient, typical morphologic changes of pseudomembranous enterocolitis were found in the absence of infection with Clostridium difficile . The nature of vascular involvement in the kidneys and intestinal tract supports the hypothesis that HUS is mediated by systemic toxemia, and that endothelial cells are the primary target cells for the action of verocytotoxin.

Infect Immun, 1988 Sep, 56(9), 2491 - 4
Isolation of Shiga toxin-resistant Vero cells and their use for easy identification of the toxin; Kongmuang U et al.; Shiga toxin-resistant Vero cells were isolated by treatment of the cells with nitrosoguanidine . These mutant cells were not affected by Shiga toxin at more than 1 microgram/ml, although the parent Vero cells were sensitive to 25 pg of the toxin per ml . Immunofluorescence studies showed that all the mutant cells had lost toxin-binding capacity . The cytotoxic activities of various bacterial cultures against the parent and mutant cells were compared . All samples from 10 strains of Shigella dysenteriae type 1 and all three strains of Escherichia coli O157:H7 tested showed cytotoxicity to the parent cells but not to the mutant cells . Samples from other organisms, such as Shigella flexneri, Shigella sonnei, Clostridium difficile, Aeromonas hydrophila, Aeromonas sobria, and other E . coli strains, either had no effect or were cytotoxic on both the parent and mutant cells . Thus, these mutant cells could be used to identify Shiga-like toxin and distinguish it from other cytotoxins . The results also suggest the presence of a receptor for Shiga-like toxin on Vero cells that is essential for expression of the cytotoxicity of Shiga toxin but is not essential for growth of Vero cells.

Res Vet Sci, 1988 Sep, 45(2), 219 - 21
Effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens introduced into chickens; Baba E et al.; The effect of Eimeria tenella infection on the caecal population of lincomycin-resistant Clostridium perfringens (KGW 851) newly introduced into chickens was studied . Four groups of chickens consisted of: (1) Uninoculated controls; (2) inoculated with C perfringens (KGW 851); (3) inoculated with E tenella; and (4) inoculated with C perfringens (KGW 851) followed by E tenella . Five chickens in each group were necropsied on each of days 0, 3, 5, 7, 10 and 14 after the E tenella inoculation . The mean total C perfringens counts in the caecal contents increased at five days after E tenella inoculation and reached maximum counts at seven days after the inoculation . Also lincomycin-resistant C perfringens readily established itself at approximately one 10th of the total C perfringens population in the presence or absence of E tenella infection.

Appl Environ Microbiol, 1988 Sep, 54(9), 2322 - 4
Electroporation-induced transformation of intact cells of Clostridium perfringens; Allen SP et al.; Electroporation-induced transformation of intact cells of Clostridium perfringens 3624A with plasmids pAMB1 and pHR106 resulted in 3.8 X 10(-5) and 4.2 X 10(-4) transformants per viable cell, respectively . With respect to shuttle plasmid pHR106, these values represent a greater than 100-fold increase in transformation frequency when compared with the values reported with polyethylene glycol-induced L-phase variants.

Poult Sci, 1988 Sep, 67(9), 1269 - 73
Effect of product form on the microbiological growth support characteristics of turkey meat products; Denton JH et al.; Effects of product form classifications, consisting of intact turkey drumsticks, dark meat trim tissue, and mechanically deboned meat, on microbiological concentrations were evaluated . Samples were inoculated with actively growing cultures of Salmonella and Clostridium perfringens to increase naturally low bacterial populations prior to being placed at 1 C and 10 C for storage evaluations . Growth support potential for all product forms stored at 10 C was extensive, making it difficult to characterize individual product form effects . Results from samples stored at 1 C indicated that emulsion and trim tissue afforded the greatest growth support potential, probably due to the increased nutrient availability and potential protective effects of the environment associated with the increased surface area of these tissues.

Eur J Biochem, 1988 Sep 1, 176(1), 95 - 101
Protease-nicked theta-toxin of Clostridium perfringens, a new membrane probe with no cytolytic effect, reveals two classes of cholesterol as toxin-binding sites on sheep erythrocytes; Ohno-Iwashita Y et al.; A nicked theta-toxin (C theta), obtained by limited proteolysis with subtilisin Carlsberg, causes almost no hemolysis while it retains a nearly intact cholesterol binding site below 20 degrees C . Neither electron microscopic evidence for the formation of arc- and ring-shaped structures on the membrane nor toxin-stimulated influx of extracellular Ca2+ are detected in C theta-treated cells below 20 degrees C . Thus, event(s) in the lytic process are responsible for the temperature dependency of hemolysis, which is also supported by the observation that C theta requires higher Arrhenius activation energy for hemolysis than the native toxin . Using C theta as a probe due to its high affinity for membrane cholesterol without causing any obvious membrane changes, we demonstrated the possible existence of high- and low-affinity sites for theta-toxin on sheep erythrocytes . Both binding sites disappear by simultaneous treatment of the cells with sublytic doses of digitonin . Furthermore, C theta binds only to cholesterol among the chloroform/methanol-extractable, lipid components of sheep and human erythrocytes but not to the protein components derived from them . These results strongly suggest that cholesterol is an essential component of the both high- and low-affinity sites, and also imply that the modes of existence of cholesterol in the red cell membrane are heterogeneous.

J Pharmacol Exp Ther, 1988 Sep, 246(3), 1183 - 9
Actions of the Clostridium botulinum binary toxin on the structure and function of Y-1 adrenal cells; Zepeda H et al.; The binary toxin produced by Clostridium botulinum, also known as type C2 toxin, was examined for its ability to alter the structure and function of Y-1 cells, a murine adrenocortical tumor line . The toxin produced time- and concentration-dependent changes in morphology, characterized by retraction of cell extensions and by rounding of the cell body . These changes were not accompanied by increases or decreases in tissue levels of c-AMP or c-GMP, although there was an increase in the release of total steroids . When cells were exposed to toxin for 24 hr there was no evidence of cell death or lysis . Total nucleic acid content and the rate of incorporation of 14C-leucine into protein were comparable in control cells and intoxicated cells . The toxin has been shown to possess mono(ADP-ribosyl)ating activity, and actin is the presumed substrate . When Y-1 cells were ruptured and then exposed to the toxin in the presence of 32P-NAD, actin was ADP-ribosylated . When cells were exposed to the toxin before being ruptured, there was a subsequent loss in the amount of actin that was available for ADP-ribosylation (32P-NAD) in the broken cell assay . The data suggest that Y-1 cells can survive challenge with the botulinum binary toxin, and thus they are a suitable tissue in which to use the toxin as a pharmacological tool.

Mol Microbiol, 1988 Sep, 2(5), 607 - 14
Studies of UV-inducible promoters from Clostridium perfringens in vivo and in vitro; Garnier T et al.; Expression of a 4 kb segment of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens is inducible by UV-irradiation . DNA sequence analysis revealed that this region contains three genes: uviA, uviB and bcn encoding the bacteriocin BCN5 . Biochemical studies with mRNAs showed that expression was controlled at the transcriptional level and that the genes were organized in two independent transcriptional units, uviAB and bcn, both directed by tandem promoters inducible by UV light . The bcn gene is transcribed from three promoters (P1, P2, P3) while transcription of uviAB is directed by two promoters (P4, P5) . With the exception of P4, which bears some resemblance to the consensus eubacterial promoter sequence, none of these promoters was recognized in vitro by the major forms of RNA polymerase from C . perfringens, Bacillus subtilis or Escherichia coli . Promoters P1, P3 and P5, which show striking homology with each other, contain unusual sequences in the '-35' and '-10' regions known to be recognized by RNA polymerase and this might indicate positive control.

Biochemistry, 1988 Aug 23, 27(17), 6499 - 503
Involvement of tryptophan residues at the coenzyme A binding site of carbon monoxide dehydrogenase from Clostridium thermoaceticum; Shanmugasundaram T et al.; Carbon monoxide dehydrogenase (CODH) from Clostridium thermoaceticum plays a central role in the newly discovered acetyl-CoA pathway {Wood, H.G., Ragsdale, S.W., & Pezacka, E . (1986) FEMS Microbiol . Rev . 39, 345-362} . The enzyme catalyzes the formation of acetyl-CoA from methyl, carbonyl, and CoA groups, and it has specific binding sites for these moieties . In this study, we have determined the role of tryptophans at these subsites . N-Bromosuccinimide (NBS) oxidation of the exposed and reactive tryptophans (5 out of a total of approximately 20) of CODH at pH 5.5 results in the partial inactivation of the exchange reaction (approximately 50%) involving carbon monoxide and the carbonyl group of the acetyl-CoA . Also, about 70% of the acetyl-CoA synthesis was abolished as a result of NBS modification . The presence of CoA (10 microM) produced complete protection against the partial inhibition of the exchange activity and the overall synthesis of acetyl-CoA caused by NBS . Additionally, none of the exposed tryptophans of CODH was modified in the presence of CoA . Ligands such as the methyl or the carbonyl groups did not afford protection against these inactivations or the modification of the exposed tryptophans . A significant fraction of the accessible fluorescence of CODH was shielded in the presence of CoA against acrylamide quenching . On the basis of these observations, it appears that certain tryptophans are involved at or near the CoA binding site of CODH.

Biochemistry, 1988 Aug 9, 27(16), 6150 - 6
Hydrogen-1 nuclear magnetic resonance of the nitrogenase iron protein (Cp2) from Clostridium pasteurianum; Meyer J et al.; Proton NMR spectra (250 MHz) of the nitrogenase iron protein from Clostridium pasteurianum (Cp2) were found to display 9 or 10 paramagnetically shifted resonances in the 15-50 ppm range . The most shifted resonances belonged to two approximately equal subsets having temperature dependences of opposite sign . The latter occurrence is consistent with the interaction of the corresponding protons with an antiferromagnetically coupled metal center . The number of proton resonances of Cp2, their positions, and their temperature dependences were similar to those observed in spectra of (4Fe-4S)+ ferredoxins, particularly those of the latter that contain a single tetranuclear cluster, such as the ferredoxin from Bacillus stearothermophilus . The effects of several adenine nucleotides on the paramagnetically shifted proton resonances of Cp2 have been investigated . Whereas MgAMP had no effect at all, MgADP and MgATP were found to induce different modifications, which in both cases involved approximately half only of the shifted proton resonances . These data suggest that nucleotide binding affects mainly one part of the iron-sulfur cluster . A remarkable feature of the spectra of Cp2 in the presence of MgATP is the grouping of the shifted proton resonances in sets of two or four having identical chemical shifts and temperature dependences . A nearly perfect 2-fold symmetry is thus suggested for the arrangement of the cysteine protons around the active site . These observations lend support to the proposal that the (4Fe-4S) cluster is held symmetrically between the two identical subunits and are consistent with the existence of two MgATP binding sites on nitrogenase iron proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Dis Child, 1988 Aug, 63(8), 979 - 81
Clostridium difficile in an oncology unit; Brunetto AL et al.; In one year 21 new cases of Clostridium difficile infection occurred on a paediatric oncology unit . Eleven cases were in a two month period . This infection should be regarded as a communicable disease . Investigations to detect C difficile should be carried out in children with malignant disease who have diarrhoea.

J Infect Dis, 1988 Aug, 158(2), 349 - 54
Epidemiology of Clostridium difficile colonization in newborns: results using a bacteriophage and bacteriocin typing system; Bacon AE et al.; We used a typing system based on bacteriophage and bacteriocin susceptibility to study the epidemiology of Clostridium difficile colonization of newborn infants . C . difficile was found in the stools of 30 (16.0%) of 187 infants who were screened . Increased length of stay in the nursery (P less than .001) and delivery by cesarian section (P less than .001) were associated with higher rates of colonization . The isolates initially detected from the environment and the infants were strain B1811-1700 . Strain B1537/Cld7 became the predominant isolate obtained from the infants; positive cultures were also obtained from the environment and the hands of personnel who worked in the nursery and had strain B1537/Cld7 . Our results suggest that the infants acquired C . difficile through transfer from the hands of hospital staff.

J Bone Joint Surg Br, 1988 Aug, 70(4), 600 - 2
Pseudomembranous colitis associated with antibiotic prophylaxis in orthopaedic surgery; Cannon SR et al.; We report 16 orthopaedic patients who had antibiotic-associated diarrhoea (pseudomembranous colitis) after operation . There was an association with the use of cephradine and with the prolongation of prophylaxis for more than three peri-operative doses . Five cases occurred as a cluster, suggesting that the causative agent, Clostridium difficile, may be infectious in some situations.

J Antimicrob Chemother, 1988 Aug, 22(2), 167 - 73
Antimicrobial susceptibility of Clostridium difficile determined by disc diffusion and breakpoint methods; Levett PN; The susceptibility of 160 isolates of Clostridium difficile to eight antimicrobial agents was studied by two methods . There was generally good agreement between the results obtained with the disc diffusion and breakpoint methods . More than 90% of isolates studied were considered sensitive by both methods . However there was a major difference between the results obtained with the two methods for penicillin G and clindamycin, resistance to both agents being overestimated by the disc diffusion method . Several isolates exhibited multiple resistance to penicillin G, tetracycline, erythromycin and clindamycin . All isolates were inhibited by both metronidazole and vancomycin . None of the isolates produced beta-lactamase.

Mol Gen Genet, 1988 Aug, 213(2-3), 238 - 46
Conservation of nif sequences in Frankia; Normand P et al.; Southern blots of Frankia total DNAs were hybridized with nifHDK probes from Rhizobium meliloti, Klebsiella pneumoniae and Frankia strain Arl3 . Differences between strains were noted in the size of the hybridizing restriction fragments . These differences were more pronounced among Elaeagnus-compatible strains than among Alnus- or Casuarina-compatible strains . Gene banks constructed for Frankia strains EUN1f, HRN18a, CeD and ACoN24d were used to isolate nif-hybridizing restriction fragments for subsequent mapping and comparisons . The nifH zone had the highest sequence conservation and the nifH and nifD genes were found to be contiguous . The complete nucleotide sequence of the nifH open reading frame (ORF) from Frankia strain Arl3 is 861 bp in length and encodes a polypeptide of 287 amino acids . Comparisons of these nucleic acid and amino acid sequences with other published nifH sequences suggest that Frankia is most similar to Anabaena and Azotobacter spp . and K . pneumoniae and least similar to the Gram-positive Clostridium pasteurianum and to the archaebacterium Methanococcus voltae.

J Antibiot (Tokyo), 1988 Aug, 41(8), 1057 - 65
Studies on the biosynthesis of bialaphos (SF-1293) . 8 . Purification and characterization of 2-phosphinomethylmalic acid synthase from Streptomyces hygroscopicus SF-1293; Shimotohno KW et al.; 2-Phosphinomethylmalic acid (PMM) synthase catalyzes the condensation of phosphinopyruvic acid (PPA), an analog of oxalacetic acid, and acetyl-CoA to form PMM . The enzyme was purified approximately 700-fold from a cell-free extract of Streptomyces hygroscopicus SF-1293, a bialaphos producing organism, to an electrophoretically homogeneous state . The purified PMM synthase has a subunit molecular weight of 48,000 by SDS-polyacrylamide gel electrophoresis and a native molecular weight of 90,000 approximately 98,000 by gel filtration . PMM synthase was relatively unstable, showed maximum activity at pH 8.0 and 30 degrees C, and was inhibited strongly by p-chloromercuribenzoate, iodoacetamide and EDTA . Enzyme activity suppressed by EDTA was completely restored by adding Co++ or Mn++ and partially restored by addition of Ca++, Fe++ or Mg++ . The specific substrates of this enzyme are PPA or oxalacetic acid in addition to acetyl-CoA . The enzyme does not catalyze the liberation of CoA from acetyl-CoA in the presence of alpha-keto acids, such as pyruvate, alpha-ketoglutarate, deamino-alpha-ketodemethylphosphinothricin or phosphonopyruvate . The condensation reaction did not take place when propionyl-CoA or butyryl-CoA was used as a substrate in place of acetyl-CoA . The Km values of the enzyme were 0.05 mM for acetyl-CoA, 0.39 mM for PPA and 0.13 mM for oxalacetate . PMM synthase is very similar to (R)-citrate synthase of Clostridium in the inhibition pattern by sulfhydryl compounds, its metal ion requirement and stereospecificity; unlike (R)-citrate synthase PMM synthase was not inhibited by oxygen.

Eur J Clin Microbiol Infect Dis, 1988 Aug, 7(4), 528 - 9
Pancreatic abscess caused by Clostridium difficile; Sofianou DC; The first known case of pancreatic abscess caused by Clostridium difficile in a patient with no history of diarrhea or previous antibiotic therapy is presented . After surgical intervention and antibiotic therapy with metronidazole (500 mg every 8 h) and cefotaxime (1 g every 8 h) the patient recovered completely.

Eur J Clin Microbiol Infect Dis, 1988 Aug, 7(4), 476 - 84
Laboratory diagnosis of Clostridium difficile-associated diarrhoea; Bowman RA et al.; This paper reviews the various laboratory procedures available for the isolation and identification of Clostridium difficile and the detection of toxins produced by this organism . Laboratories should be selective in determining which patients require investigation for Clostridium difficile-associated diarrhoea . Transport and storage of stool specimens at 4 degrees C is recommended when delays in processing may occur . Tissue culture techniques are still the best method for detection of cytotoxin and a variety of cell lines can be used . Other methods for detecting cytotoxin, and methods for detecting other toxins are not sufficiently developed yet to warrant introduction into diagnostic laboratories . Culture techniques remain the most sensitive for diagnosis, particularly since the development of a variety of enrichment techniques . Cycloserine cefoxitin fructose agar is still adequate, although reduced concentrations of antimicrobial agents are necessary, and improvements, such as the addition of sodium taurocholate, increase the recovery of spores . Enrichment cultures have markedly increased isolation rates for Clostridium difficile but the significance of these isolates needs to be carefully evaluated . Until simpler and more reliable tests are available in clinical laboratories for the detection of toxins, the isolation of Clostridium difficile from patients with diarrhoeal disease should be considered paramountPublication Types:

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