J Neurochem, 1999 May, 72(5), 1991 - 8 Clostridium neurotoxins influence serotonin uptake and release differently in rat brain synaptosomes; Najib A et al.; Clostridium neurotoxins produce inhibition of both basal and K(+)-evoked serotonin release in rat brain synaptosomes . To produce these effects, tetanus toxin (TeTx), as well as botulinum neurotoxin type A (BoNT/A), added to brain synaptosomes, must be incubated at 37 degrees C over a long interval (hours) . This serotonin exocytosis inhibition was abolished with previous treatment with specific Zn2(+)-metalloprotease inhibitors . Nevertheless, a short incubation time produces different behavior of the indicated neurotoxins: TeTx significantly blocks the sodium-dependent, high-affinity serotonin uptake, whereas a small increase of this uptake was found with BoNT/A . Both Zn2(+)-metalloprotease active fragments, light chains of TeTx and BoNT/A, are unable to reproduce the block of the serotonin uptake, whereas the C-terminal portion of the TeTx heavy chain (Hc-TeTx), which binds specifically to the target tissue, inhibited the serotonin uptake in a dose-dependent manner . The IC50 of Hc-TeTx ranges from 0.62 to 2.08 nM . Binding of {3H}imipramine and {3H}serotonin did not change after toxin treatments, which indicates that these clostridium neurotoxins do not act on the serotonin high-affinity site at the serotonin transporter or at other serotonin high-affinity sites . These results could indicate that TeTx and Hc-TeTx bind to different targets than BoNT/A in the plasma membrane. Cancer Res, 1999 Apr 15, 59(8), 2004 - 10 Overexpression of small GTP-binding protein RhoA promotes invasion of tumor cells; Yoshioka K et al.; Adhesion of tumor cells to host cell layers and subsequent migration are pivotal steps in cancer invasion and metastasis . The small GTP-binding protein RhoA controls cell adhesion and motility through organization of the actin cytoskeleton and regulation of actomyosin contractility . Cultured rat MM1 hepatoma cells migrate through a mesothelial cell monolayer in vitro in a serum-dependent, RhoA-mediated manner (K . Yoshioka et al., J . Biol . Chem., 273: 5146-5154, 1998) . Furthermore, the ROCK family of RhoA-associated serine-threonine protein kinases is involved in this migration, and an inhibitor for these kinases effectively inhibits the invasion of MM1 cells in vitro and in vivo (K . Itoh et al., Nat . Med., 5: 221-225, 1999) . Although there have been no reports of genetic alterations directly affecting RhoA in human cancer, the expression level of RhoA in tumors has been several times higher than that of surrounding normal tissue; RhoA was especially highly expressed in the metastatic region . To determine whether RhoA is activated by its overexpression, we made stable transfectants of MM1 cells expressing various levels of wild-type human RhoA . These transfectants showed promoted invasive ability in vitro in the absence and presence of 1-oleoyl-lysophosphatidic acid, marked adherence to the plastic culture dish with scattered shape, elevated phosphorylation of Mr 20,000 myosin light chain, and translocation of RhoA protein from the cytosol to the membrane . All of these phenotypes were similar to those of active RhoA transfectants, correlated with the expression level of RhoA and reversed by the treatment of the cells with Clostridium botulinum exoenzyme C3 ADP-ribosyltransferase . In addition, overexpression of wild-type RhoA in MM1 cells also conferred invasive ability in vivo after the cells were transplanted into the syngeneic rats . Thus, high expression of RhoA in the cell facilitates the translocation of this protein to the membrane, where it is activated, resulting in the stimulation of the RhoA-ROCK-actomyosin system, leading to invasion. Trends Microbiol, 1999 Mar, 7(3), 104 - 10 Clostridium perfringens: toxinotype and genotype; Petit L et al.; Clostridium perfringens is a ubiquitous pathogen that produces many toxins and hydrolytic enzymes . Because the toxin-encoding genes can be located on extrachromosomal elements or in variable regions of the chromosome, several pathovars have arisen, each of which is involved in a specific disease . Pathovar identification is required for a precise diagnosis of associated pathologies and to define vaccine requirements . For these purposes, toxin genotyping is more reliable than the classical toxinotyping. Oncogene, 1999 Mar 18, 18(11), 1975 - 82 Rho-dependent and -independent tyrosine phosphorylation of focal adhesion kinase, paxillin and p130Cas mediated by Ret kinase; Murakami H et al.; Glial cell line-derived neurotrophic factor (GDNF) signals through a unique receptor system that includes Ret receptor tyrosine kinase and a glycosyl-phosphatidylinositol-linked cell surface protein . In the present study, we have identified several proteins in neuroblastoma cells that are phosphorylated on tyrosine in response to GDNF . The phosphorylated proteins include focal adhesion kinase (FAK), paxillin and Crk-associated substrate, p130Cas, all of which are known to be associated with focal adhesions . Of these, paxillin and p130Cas interacted with Crk proteins in GDNF-treated neuroblastoma cells . GDNF also induced reorganization of the actin cytoskelton . Tyrosine phosphorylation of FAK, paxillin and p130Cas was inhibited by cytochalasin D or two specific inhibitors of phosphatidylinositol-3' kinase (PI-3' kinase), wortmannin and LY294002, indicating that their tyrosine phosphorylation depends on the formation of actin stress fiber and activation of PI-3' kinase . In addition, phosphorylation of FAK but not of paxillin and p130Cas was markedly impaired by the Clostridium botulinum C3 exoenzyme that specifically ADP-ribosylates and inactivates Rho . These results suggested the presence of Rho-dependent and -independent signaling pathways downstream of PI-3' kinase that mediate tyrosine phosphorylation of FAK, paxillin and p130Cas through Ret kinase. JAMA, 1999 Apr 14, 281(14), 1334 - 8, 1340 Outbreak of type A botulism and development of a botulism surveillance and antitoxin release system in Argentina; Villar RG et al.; CONTEXT: Botulism is an important public health problem in Argentina, but obtaining antitoxin rapidly has been difficult because global supplies are limited . In January 1998, a botulism outbreak occurred in Buenos Aires . OBJECTIVES: To determine the source of the outbreak, improve botulism surveillance, and establish an antitoxin supply and release system in Argentina . DESIGN, SETTING, AND PARTICIPANTS: Cohort study in January 1998 of 21 drivers of a specific bus route in urban Buenos Aires . MAIN OUTCOME MEASURE: Occurrence of botulism and implication of a particular food as the vehicle causing this outbreak . RESULTS: Nine (43%) of 21 bus drivers developed botulism, presenting with gastroenteritis, symptoms of acute cranial nerve dysfunction including ptosis, dysphagia, blurred vision, and motor weakness . One driver experienced respiratory failure . Type A toxin was detected from 3 of 9 patients' serum samples . All drivers received botulism antitoxin; there were no fatalities . Consumption of matambre (Argentine meat roll) was significantly associated with illness . Among 11 persons who ate matambre, 9 developed illness, compared with none of those who did not eat it (P<.001) . The matambre had been cooked in water at 78 degrees C to 80 degrees C for 4 hours, sealed in heat-shrinked plastic wrap, and stored in refrigerators that did not cool adequately . Subsequently, a botulism surveillance and antitoxin release system was established . CONCLUSIONS: Insufficient cooking time and temperatures, storage in heat-shrinked plastic wrap, and inadequate refrigeration likely contributed to Clostridium botulinum spore survival, germination, and toxin production . A rapid-response botulism surveillance and antitoxin release system in Argentina should provide more timely distribution of antitoxin to patients and may serve as a model for other nations. J Comp Pathol, 1999 May, 120(4), 415 - 20 Neuronal damage produced in rat brains by Clostridium perfringens type D epsilon toxin; Finnie JW et al.; This paper reports neuronal changes in rat brains subacutely intoxicated with Copyright Clostridium perfringens type D epsilon toxin . Neuronal damage was characterized by either (1) progressive cytoplasmic vacuolation leading to necrosis, or (2) shrunken hyperchromatic cells with nuclear pyknosis . The neuronal injury was also often bilaterally symmetrical, particularly in the brainstem . These findings suggest that, after gaining access to brain tissue by producing an increase in vascular permeability, epsilon toxin later exerts a directly cytotoxic effect on neurons . 1999 W.B . Saunders and Company Ltd. J Parasitol, 1999 Feb, 85(1), 33 - 40 Sialic acid-specific lectin-mediated adhesion of Tritrichomonas foetus and Tritrichomonas mobilensis; Babal P et al.; Tritrichomonas foetus is an obligate parasite of the bovine urogenital tract producing infection associated with inflammatory changes, abortion, and infertility, Tritrichomonas mobilensis was isolated from squirrel monkey colon, and symptoms involve diarrheal complications . Both tritrichomonads produced hemagglutinins with the properties of sialic acid-specific lectins . Assays on the adherence of these protozoans to Chinese hamster ovary (CHO) cells and to bovine cervical and monkey colon mucus were performed to assess the function of the lectins in adhesion . Sialic acid at concentration as low as 2 mM inhibited the adhesion to CHO cells, less effectively to the mucus . Predigestion with Clostridium perfringens sialidase prevented the adhesion to both epithelial cells and the mucus . Inhibition of endogenous sialidases with 2,3-dehydro-2-deoxy-NeuAc increased the adhesion of T . mobilensis to CHO cells . Specific anti-T . foetus lectin (TFL) and anti-T . mobilensis lectin (TML) antibodies inhibited adhesion of the trichomonads to the epithelial cells and to the mucus . TFL histochemistry disclosed the presence of lectin ligands on keratinized vaginal epithelia, cervical mucosa, and mucin and on endometrial glands and their secretions . TML histochemistry showed reactivity with the luminal membranes of colonic glandular epithelium and less with the colonic mucin . Both lectins bound to the surface membrane of CHO cells . Anti-lectin antibodies showed granular cytoplasmic and strong membrane localization of the lectins in both tritrichomonads . Although the 2 tritrichomonads have different habitats, the results indicate that both these protozoa use lectins with sialic acid specificity for adhesion to mucosal surfaces. J Gastroenterol, 1999 Feb, 34(1), 41 - 5 Laboratory diagnosis of toxigenic Clostridium difficile by polymerase chain reaction: presence of toxin genes and their stable expression in toxigenic isolates from Japanese individuals; Karasawa T et al.; Clostridium difficile causes pseudomembranous colitis and antibiotic-associated diarrhea . The definitive diagnosis of C . difficile infection is finally accomplished by the isolation of toxigenic C . difficile . However, only a small number of Japanese clinical laboratories are able to reach a definitive diagnosis of C . difficile infection, probably because simple reliable assays for toxins in the isolates are not available . In this study, we examined the compatibility of a polymerase chain reaction (PCR) assay and tissue culture assay to identify toxigenic C . difficile, in toxigenic and nontoxigenic C . difficile isolates from Japanese patients and healthy carriers . The specificity of PCR primers was demonstrated by restriction endonuclease digestion and seminested PCR in C . difficile VPI 10463 strain . No PCR product was amplified in the eight other clostridial species used to check the specificity of the PCR assay . The detection limit was 10(3) cells . Both toxin A and toxin B genes (the genes encoding the major virulence factors of C . difficile) were detected in 58 toxigenic C . difficile isolates, which showed a wide range of cytotoxic activity in tissue culture assays . Neither of the toxin genes was carried by 40 nontoxigenic strains of C . difficile . The results of this study strongly suggest that a definitive diagnosis of C . difficile infection can be accomplished by PCR detection of the toxin genes rather than by tissue culture assay of isolates. J Hosp Infect, 1999 Mar, 41(3), 213 - 8 Clostridium difficile: an update on its epidemiology and role in hospital outbreaks in England and Wales; Djuretic T et al.; Data from the surveillance system of general outbreaks of infectious intestinal disease and from laboratory reports collated by the Communicable Disease Surveillance Centre (CDSC) and requests for outbreak investigation by the PHLS Anaerobe Reference Unit were used to evaluate the current epidemiology of Clostridium difficile infection in England and Wales . Between January 1992 and December 1996, CDSC received 10,220 laboratory reports of C difficile isolation from patient's faeces and 26,873 of toxin in faeces . Over 75% of all reports were of people aged 64 years and over . The surveillance system captured a minimum data set on 694 hospital outbreaks of infectious intestinal disease . C . difficile was responsible for 109 (15%) outbreaks affecting 1625 people, of whom 1152 were found to have a C . difficile toxin producing strain . The median duration of outbreaks was 11 days . Fingerprinting by Pyrolysis Mass Spectrometry (PMS) was performed by the PHLS Anaerobe Reference Unit in 60 outbreaks, and typing by Polymerase Chain Reaction ribotyping (PCR) in 14. Cell Death Differ, 1998 Sep, 5(9), 720 - 8 Bacterial toxins and the Rho GTP-binding protein: what microbes teach us about cell regulation; Fiorentini C et al.; In the present review activities of two bacterial toxins, Clostridium botulinum exoenzyme C3 and Escherichia coli CNF1, both acting on the GTP-binding protein Rho are analyzed . Proteins belonging to the Rho family regulate the actin cytoskeleton and act as molecular switches in a number of signal transduction pathways . C3 and CNF1 have opposite effects on Rho thus representing useful tools for studies on cell division, cell differentiation and apoptosis. J Cell Physiol, 1999 May, 179(2), 179 - 92 Cytoplasmic elongation and rupture in megakaryoblastic leukemia cells via activation of adhesion and motility by staurosporine on fibronectin-bound substratum; Yamazaki Y et al.; Human megakaryoblastic leukemia Meg-01 cells were attached to fibronectin (FN)-coated substratum, on which remarkable spreading and cytoplasmic elongation was induced by treatment with a protein kinase inhibitor, staurosporine (stp) . This effect was inhibited by RGDS and was also not seen on FN-lacking substratum . The extended cytoplasm had swollen terminals and nodes, which contained GpIIb and beta-thromboglobulin, occasionally included alpha granules, and tended to form particles (2-5 microm) after rupture of the narrowed cytoplasm . Among other protein kinase modulators tested, only K252a promoted the elongation, while calphostin, herbimycin, TPA, and calyculin suppressed it . The cells began to migrate soon after addition of stp, with attachment to the substratum held at some sites during the migration . This tethered movement seemed to cause the cytoplasmic elongation and the rupture into particles . The elongation was retarded by pretreating the cells with cytochalasin A and Clostridium C3 toxin but not with demecolcine . Actin microfilaments in the stp-treated Meg-01 cells accumulated in the filopodia and periphery of the extended cytoplasm, in which vinculin was colocalized as adhesion plaques . The microtubules were longitudinally oriented through the cytoplasmic extension and showed no ring profile in the nodes and particles . Thus, stp in the presence of FN appears to stimulate reorganization of actin-based cytoskeleton and formation of focal contacts in Meg-01 cells . This leads to the activation of cell adhesion and motility, and then cytoplasmic elongation and rupture into particles. Surg Neurol, 1999 Apr, 51(4), 448 - 50; discussion 450-1 Clostridium perfringens: a rare cause of postoperative spinal surgery meningitis; Kristopaitis T et al.; BACKGROUND: Clostridium perfringens is a rare cause of central nervous system infections, particularly meningitis . The case of a 76-year-old man who developed fatal C . perfringens meningitis after routine decompressive laminectomy for spinal stenosis is described . CASE REPORT: Twelve days after surgery the patient presented with pain and serosangiunous drainage from the surgical incision site . A swab of the drainage revealed Gram-positive bacilli; MRI of the lumbosacral spine showed the appearance of air around the laminectomy site . The patient died within 6 hours of presentation . Autopsy revealed acute cranial and spinal meningitis and choroid plexitis with organisms consistent with C . perfringens . CONCLUSION: No significant enteral pathology or source of endogenous infection was determined, suggesting postoperative wound contamination and meningeal seeding with this ubiquitous organism . Clostridial infection, although rare, should be considered in any patient with meningitis with a history of surgical intervention . Survival with minimal neurological deficits was achieved in half of the previously reported cases. Am J Physiol, 1999 Apr, 276(4 Pt 1), G915 - 23 Involvement of RhoA and its interaction with protein kinase C and Src in CCK-stimulated pancreatic acini; Nozu F et al.; We evaluated intracellular pathways responsible for the activation of the small GTP-binding protein Rho p21 in rat pancreatic acini . Intact acini were incubated with or without CCK and carbachol, and Triton X-100-soluble and crude microsomes were used for Western immunoblotting . When a RhoA-specific antibody was used, a single band at the location of 21 kDa was detected . CCK (10 pM-10 nM) and carbachol (0.1-100 microM) dose dependently increased the amount of immunodetectable RhoA with a peak increase occurring at 3 min . High-affinity CCK-A-receptor agonists JMV-180 and CCK-OPE (1-1,000 nM) did not increase the intensities of the RhoA band, suggesting that stimulation of RhoA is mediated by the low-affinity CCK-A receptor . Although an increase in RhoA did not require the presence of extracellular Ca2+, the intracellular Ca2+ chelator 1, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-AM abolished the appearance of the RhoA band in response to CCK and carbachol . The Gq protein inhibitor G protein antagonist-2A (10 microM) and the phospholipase C (PLC) inhibitor U-73122 (10 microM) markedly reduced RhoA bands in response to CCK . The protein kinase C (PKC) activator phorbol ester (10-1,000 nM) dose dependently increased the intensities of the RhoA band, which were inhibited by the PKC inhibitor K-252a (1 microM) . The pp60(c-src) inhibitor herbimycin A (6 microM) inhibited the RhoA band in response to CCK, whereas the calmodulin inhibitor W-7 (100 microM) and the phosphoinositide 3-kinase inhibitor wortmannin (6 microM) had no effect . RhoA was immunoprecipitated with Src, suggesting association of RhoA with Src . Increases in mass of this complex were observed with CCK stimulation . In permeabilized acini, the Rho inhibitor Clostridium botulinum C3 exoenzyme dose dependently inhibited amylase secretion evoked by a Ca2+ concentration with an IC50 of C3 exoenzyme at 1 ng/ml . We concluded that the small GTP-binding protein RhoA p21 exists in pancreatic acini and appears to be involved in the mediation of pancreatic enzyme secretion evoked by CCK and carbachol . RhoA pathways are involved in the activation of PKC and Src cascades via Gq protein and PLC. J Pharm Pharmacol, 1999 Jan, 51(1), 79 - 84 Reductive debromination of (alpha-bromoiso-valeryl)urea by intestinal bacteria; Kitamura S et al.; The reductive debromination of the hypnotic (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea by intestinal bacteria has been studied . The caecal contents of rats, mice, hamsters, guinea-pigs and rabbits had significant debrominating activity toward (alpha-bromoiso-valeryl)urea . The cell-free extract of intestinal bacteria from the caecal contents of rats had debrominating activity in the presence of both flavin mononucleotide (FMN) and NADH (or NADPH) under anaerobic conditions . Seven pure strains of intestinal bacteria were also tested and the highest activity was observed with Clostridium sporogenes . The cell-free extract of Clostridium sporogenes had debrominating activity in the presence of both FMN and NADH (or NADPH), and this activity was inhibited by sodium arsenite and potassium cyanide . The activity of the cell-free extract was also supported by the photochemically reduced form of FMN . The debromination in intestinal bacteria seems to proceed in two steps--reduction of flavins by bacterial flavin reductase(s) in the presence of NADPH or NADH, and then the reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea by bacterial dehalogenase(s) using the reduced flavins as an electron donor . These results indicate that intestinal bacteria play a role in the reductive debromination of (alpha-bromoiso-valeryl)urea to (3-methylbutyryl)urea in animals . The debromination is inhibited by oxygen and dependent on flavins. Postgrad Med J, 1998 Nov, 74(877), 677 - 8 Clostridium difficile-associated diarrhoea; Wight N et al.; At our hospital, the number of cases of Clostridium difficile-associated diarrhoea increased from 29 in 1993 to 210 in 1995 . The case notes of 110 patients with C difficile-associated diarrhoea during the first 6 months of 1995 were analysed retrospectively . The majority of the patients (106) had received antibiotics before the onset of diarrhoea; 46 had received three or more different antibiotics and 28 had received metronidazole . In 19 patients, the first stool sample after the onset of diarrhoea was negative for C difficile cytotoxin, with a mean delay of 8.2 days before a positive stool sample . We conclude that C difficile-associated diarrhoea was associated with the usage of multiple antibiotics, and that metronidazole did not protect against colonisation by C difficile . We also recommend that more than one stool sample should be tested for the C difficile cytotoxin. J Appl Microbiol, 1999 Mar, 86(3), 412 - 20 Isolation and characterization of two glycerol-fermenting clostridial strains from a pilot scale anaerobic digester treating high lipid-content slaughterhouse waste; Jarvis GN et al.; Two obligately anaerobic bacterial strains were isolated from the contents of a pilot scale, anaerobic digester treating slaughterhouse waste with a high protein and lipid content . The isolates, LIP1 and MW8, were characterized as spore-forming, Gram-positive rods, capable of fermenting glycerol . Isolate LIP1 was also observed to be lipolytic and was able to hydrolyse tallow and olive oil . Both isolates grew optimally at 37 degrees C and formed either acetate and formate (LIP1), or acetate and butyrate (MW8), as major glycerol fermentation products . Both isolates produced ethanol as the major reduced fermentation end-product . Neither MW8 nor LIP1 had growth and metabolism inhibited by the addition of stearic acid at concentrations normally considered bactericidal . Analysis of the 16S rRNA gene sequences, in conjunction with the phenotypic data, confirmed that the isolates are members of the genus Clostridium (sensu lato), clustering with species of clostridial clusters I (MW8) and XIVa (LIP1). J Biol Chem, 1999 Apr 16, 274(16), 11046 - 52 A novel cytotoxin from Clostridium difficile serogroup F is a functional hybrid between two other large clostridial cytotoxins; Chaves-Olarte E et al.; The large clostridial cytotoxins (LCTs) constitute a group of high molecular weight clostridial cytotoxins that inactivate cellular small GTP-binding proteins . We demonstrate that a novel LCT (TcdB-1470) from Clostridium difficile strain 1470 is a functional hybrid between "reference" TcdB-10463 and Clostridium sordellii TcsL-1522 . It bound to the same specific receptor as TcdB-10463 but glucosylated the same GTP-binding proteins as TcsL-1522 . All three toxins had equal enzymatic potencies but were equally cytotoxic only when microinjected . When applied extracellularly TcdB-1470 and TcdB-10463 were considerably more potent cytotoxins than TcsL-1522 . The small GTP-binding protein R-Ras was identified as a target for TcdB-1470 and also for TcsL-1522 but not for TcdB-10463 . R-Ras is known to control integrin-extracellular matrix interactions from inside the cell . Its glucosylation may be a major determinant for the cell rounding and detachment induced by the two R-Ras-attacking toxins . In contrast, fibroblasts treated with TcdB-10463 were arborized and remained attached, with phosphotyrosine containing structures located at the cell-to-cell contacts and beta3-integrin remaining at the tips of cellular protrusions . These components were absent from cells treated with the R-Ras-inactivating toxins . The novel hybrid toxin will broaden the utility of the LCTs for clarifying the functions of several small GTPases, now including also R-Ras. Biotechnol Bioeng, 1998 Apr 5, 58(2-3), 215 - 21 Genetic manipulation of acid and solvent formation in clostridium acetobutylicum ATCC 824 Green EM, Bennett GN. The genes coding for enzymes involved in butanol or butyrate formation were subcloned into a novel Escherichia coli-Clostridium acetobutylicum shuttle vector constructed from pIMP1 and a chloramphenicol acetyl transferase gene . The resulting replicative plasmids, referred to as pTHAAD (aldehyde/alcohol dehydrogenase) and pTHBUT (butyrate operon), were used to complement C . acetobutylicum mutant strains, in which genes encoding aldehyde/alcohol dehydrogenase (aad) or butyrate kinase (buk) had been inactivated by recombination with Emr constructs . Complementation of strain PJC4BK (buk mutant) with pTHBUT restored butyrate kinase activity and butyrate production during exponential growth . Complementation of strain PJC4AAD (aad mutant) with pTHAAD restored NAD(H)-dependent butanol dehydrogenase activity, NAD(H)-dependent butyraldehyde dehydrogenase activity and butanol production during solventogenic growth . The development of an alternative selectable marker makes it is possible to overexpress genes, via replicative plasmids, in mutant strains that lack specific enzyme activities, thereby expanding the number of possible genetic manipulations that can be performed in C . acetobutylicum . Eur J Clin Microbiol Infect Dis, 1999 Jan, 18(1), 46 - 54 Comparative activity of eighteen antimicrobial agents against anaerobic bacteria isolated in South Africa; Lubbe MM et al.; The in vitro activity of 18 antimicrobial agents was determined against 378 anaerobic bacteria isolated in Bloemfontein, South Africa, during 1996/97 . Against the gram-positive isolates, MICs of penicillin and cefoxitin were >0.5 microg/ml and >16 microg/ml, respectively, for five and three strains of non-perfringens Clostridium spp . Seventeen Peptostreptococcus anaerobius strains were resistant to penicillin (MIC > or = 2 microg/ml) . All gram-positive anaerobes tested except one Peptostreptococcus sp . and one Clostridium sp . were susceptible to dalfopristin-quinupristin (MICs < or = 1 microg/ml) . The carbapenems exhibited excellent activity against the gram-positive isolates and were effective against most gram-negative anaerobes, with the exception of the fusobacteria . Only seven strains exhibited decreased susceptibility to trovafloxacin (MICs > 2 microg/ml) . In mixed anaerobic/aerobic infections, carbapenems and the fourth-generation quinolone trovafloxacin were the agents most suitable for us as broad-spectrum monotherapy. Hosp Formul, 1990 Jul, 25(7), 746 - 8, 750-1 In-hospital anaerobic susceptibility testing: an aid to formulary decision making; Lebar WD et al.; In this study, the susceptibility of 116 recent anaerobic isolates, including the Bacteroides fragilis group (n = 22), Bacteroides spp (n = 65), gram-positive cocci (n = 13), Clostridium spp (n = 10), and Fusobacterium spp (n = 6) were tested by agar dilution against a variety of agents suggested for prophylaxis or therapy . These agents included ampicillin sodium and sulbactam sodium, cefotaxime, cefoxitin, ceftizoxime, clindamycin, imipenem-cilastatin, metronidazole, and ticarcillin and clavulanate potassium . All anaerobes demonstrated 100% susceptibility to imipenem-cilastatin, metronidazole, ampicillin sodium and sulbactam sodium, and ticarcillin and clavulate potassium . Varying degrees of susceptibility (ranging from 60% to 100%) of Bacteroides spp to the selected panel of antibiotics were seen . Fusobacterium spp and the gram-positive cocci were inhibited by all agents tested . Clostridium spp was 90% susceptible to cefoxitin, 80% susceptible to clindamycin, and 100% susceptible to the other six agents . Due to the varying activity of these agents, local susceptibility patterns, antimicrobic spectrum, and cost effectiveness must be considered in the choice of agents used for empiric therapy. Biotechnol Bioeng, 1998 Nov 20, 60(4), 498 - 507 Acetate production from whey lactose using co-immobilized cells of homolactic and homoacetic bacteria in a fibrous-bed bioreactor; Huang Y et al.; Acetate was produced from whey lactose in batch and fed-batch fermentations using co-immobilized cells of Clostridium formicoaceticum and Lactococcus lactis . The cells were immobilized in a spirally wound fibrous sheet packed in a 0.45-L column reactor, with liquid circulated through a 5-L stirred-tank fermentor . Industrial-grade nitrogen sources, including corn steep liquor, casein hydrolysate, and yeast hydrolysate, were studied as inexpensive nutrient supplements to whey permeate and acid whey . Supplementation with either 2.5% (v/v) corn steep liquor or 1.5 g/L casein hydrolysate was adequate for the cocultured fermentation . The overall acetic acid yield from lactose was 0.9 g/g, and the productivity was 0.25 g/(L h) . Both lactate and acetate at high concentrations inhibited the homoacetic fermentation . To overcome these inhibitions, fed-batch fermentations were used to keep lactate concentration low and to adapt cells to high-concentration acetate . The final acetate concentration obtained in the fed-batch fermentation was 75 g/L, which was the highest acetate concentration ever produced by C . formicoaceticum . Even at this high acetate concentration, the overall productivity was 0.18 g/(L h) based on the total medium volume and 1.23 g/(L h) based on the fibrous-bed reactor volume . The cells isolated from the fibrous-bed bioreactor at the end of this study were more tolerant to acetic acid than the original culture used to seed the bioreactor, indicating that adaptation and natural selection of acetate-tolerant strains occurred . This cocultured fermentation process could be used to produce a low-cost acetate deicer from whey permeate and acid whey . Biotechnol Bioeng, 1998 Jun 20, 58(6), 561 - 71 Metabolism analysis and on-line physiological state diagnosis of acetone-butanol fermentation; Chauvatcharin S et al.; Fermentation equations for acetone-butanol (AB) were applied in a metabolic analysis of the reaction network under various conditions; that is, at different pHs and a high NADH2 turnover rate using methyl viologen, in a Clostridium acetobutylicum culture . The results disclosed variations in the pattern of rate changes that reflected changes in the physiological state . A linear relationship was found to exist between NADH2 generation and butanol production rate . By coupling an automated measurement system with the fermentation model, on-line estimation of the culture state was accomplished . Based on the AB fermentation model, new parameters were defined for on-line diagnosis of the physiological state and determination of the best timing for amplifying NADH2 generation by the addition of methyl viologen to obtain a high level of butanol productivity . A potential means of achieving optimal control for a high level of solvent production, involving the correlation of certain rates, is proposed . Int J Food Microbiol, 1999 Feb 18, 46(3), 179 - 85 Feasibility of using food-grade additives to control the growth of Clostridium perfringens; Sikes A et al.; Previously, it was demonstrated that the combination of sucrose laurate (SL) ethylenediaminetetraacetate (E) and butylated hydroxyl anisole (B) (SLEB) was an effective antimicrobial agent against both gram-negative (aerobes) and gram-positive (facultative anaerobes) foodborne bacteria . This investigation examines the sensitivity of Clostridium perfringens to SLEB relative to: (1) the minimum inhibitory concentration (MIC) of SLEB required to inhibit the growth of C . perfringens and (2) the antibacterial effectiveness of different combination ratios of SLEB in fluid thioglycollate medium (FTM) . Results indicated that the MIC of SLEB (1:1:1, v/v/v) against C . perfringens on tryptose sulfite cycloserine (TSC) agar was > 150 ppm at 37 degrees C . However, in FTM, a SLEB (1:1:1, v/v/v) concentration of > 100 ppm inhibited C . perfringens during an incubation (anaerobic) period of 196 h at 37 degrees C . The sensitivity of C . perfringens to different combination ratios was also investigated in FTM . The results showed that, when the concentrations of SL and E were held at 75 ppm in the SLEB combination, and the concentration of B increased from 0 to 75 ppm, C . perfringens growth increased initially during the first 24 h of incubation (37 degrees C) but remained constant during the next 48 h . Similarly, when concentrations of SL and E were held constant at 150 ppm in the SLEB combination and the B ratio increased from 50 to 150 ppm in FTM, C . perfringens viability decreased in all of the treated samples during 72-h incubation at 37 degrees C . The results indicated that SLEB was an effective inhibitor of C . perfringens growth activities, and the ratios of the components of SLEB can be adjusted to meet specific preservation needs. Microbiol Immunol, 1999, 43(1), 1 - 8 Cloning and sequencing of the central region of the flagellin gene from the Gram-positive bacterium Clostridium tyrobutyricum ATCC 25755; Arnold F et al.; The purpose of this study was to sequence the central part of the coding region of the Clostridium tyrobutyricum fiagellin gene to improve the immunoenzymatic counting of cells after milk filtration . The coding region was amplified by PCR, and the amplified products were cloned . A DNA sequence analysis of positive clones gave us 1,131 nucleotides with a partial calculated flagellin molecular mass of 40,143 Da . The flagellar filament protein sequence exhibited high levels of homology to sequences of flagellin protein from other bacteria in both N- and C-terminal parts, but little homology in the central domain . A PCR-restriction fragment length polymorphism analysis of amplified C . tyrobutyricum flagellin gene products confirmed the variability of the central domain . The flagellin mRNA was determined to be 1.1 kb in size, which suggests a monocistronic mRNA . Furthermore, the deduced protein flagellin contains eleven potential N-glycosylation sites and one sequence rich in serine, which could be modified by O-glycosylation. Am J Ther, 1998 Sep, 5(5), 287 - 94 Clostridium difficile-associated diarrhea in acute and long-term care facilities; Khayr W et al.; This report presents an overview of the epidemiology, diagnosis, complications, and treatment of Clostridium difficile-associated diarrhea in acute and long-term care facilities . More studies are needed to understand the epidemiology of this disease in long-term care facilities, to identify the risk factors for its recurrence, and to evaluate new treatment modalities. Exp Cell Res, 1999 Apr 10, 248(1), 260 - 71 Activation of protein kinase C by phorbol esters modulates alpha2beta1 integrin on MCF-7 breast cancer cells; Rosfjord EC et al.; Cellular adhesions to other cells and to the extracellular matrix play crucial roles in the malignant progression of cancer . In this study, we investigated the role of protein kinase C (PKC) in the regulation of cell-substratum adhesion by the breast adenocarcinoma cell line MCF-7 . A PKC activator, 12-O-tetradecanoylphorbol-l, 3-acetate (TPA), stimulated cell adhesion to laminin and collagen I in a dose-dependent manner over a 1- to 4-h interval . This enhanced adhesion was mediated by alpha2beta1 integrin, since both anti-alpha2 and anti-beta1 blocking antibodies each completely abrogated the TPA-induced adhesion . FACS analysis determined that TPA treatment does not change the cell surface expression of alpha2beta1 integrin over a 4-h time interval . However, alpha2beta1 levels were increased after 24 h of TPA treatment . Thus, the enhanced avidity of alpha2beta1-dependent cellular adhesion preceded the induction of alpha2beta1 cell surface expression . Northern blot analysis revealed that mRNA levels of both alpha2 and beta1 subunits were increased after exposure to TPA for 4 h, indicating that the induction of alpha2beta1 mRNA preceded that of its cell surface expression . This further suggested that the TPA-induced avidity of alpha2beta1 was independent of increased expression of alpha2beta1 . Pretreatment of cells with the PKC inhibitor calphostin C partially antagonized the TPA-induced increase in expression of alpha2beta1 integrin expression and of alpha2beta1-mediated cellular adhesion . To identify a possible mechanism by which TPA could be acting to promote the rapid induction of alpha2beta1 adhesion, we treated the cells with the Rho-GTPase inhibitor Clostridium botulinumexotoxin C3 . C3 inhibited TPA-induced adhesion to laminin and collagen I in a dose-dependant manner, suggesting a likely role for Rho in TPA-induced adhesion . Together, these results suggest that PKC can modulate the alpha2beta1-dependent adhesion of MCF-7 cells by two distinct mechanisms: altering the gene expression of integrins alpha2 and beta1 and altering the avidity of the alpha2beta1 integrin by a Rho-dependant mechanism . Mol Microbiol, 1998 Nov, 30(4), 737 - 49 The S box regulon: a new global transcription termination control system for methionine and cysteine biosynthesis genes in gram-positive bacteria; Grundy FJ et al.; The molecular mechanisms for regulation of the genes involved in the biosynthesis of methionine and cysteine are poorly characterized in Bacillus subtilis . Analyses of the recently completed B . subtilis genome revealed 11 copies of a highly conserved motif . In all cases, this motif was located in the leader region of putative transcriptional units, upstream of coding sequences that included genes involved in methionine or cysteine biosynthesis . Additional copies were identified in Clostridium acetobutylicum and Staphylococcus aureus, indicating conservation in other Gram-positive genera . The motif includes an element resembling an intrinsic transcriptional terminator, suggesting that regulation might be controlled at the level of premature termination of transcription . The 5' portion of all of the leaders could fold into a conserved complex structure . Analysis of the yitJ gene, which is homologous to Escherichia coli metH and metF, revealed that expression was induced by starvation for methionine and that induction was independent of the promoter and dependent on the leader region terminator . Mutation of conserved primary sequence and structural elements supported a model in which the 5' portion of the leader forms an anti-antiterminator structure, which sequesters sequences required for the formation of an antiterminator, which, in turn, sequesters sequences required for the formation of the terminator; the anti-antiterminator is postulated to be stabilized by the binding of some unknown factor when methionine is available . This set of genes is proposed to form a new regulon controlled by a global termination control system, which we designate the S box system, as most of the genes are involved in sulphur metabolism and biosynthesis of methionine and cysteine. J Food Prot, 1999 Mar, 62(3), 262 - 7 Efficacy of oxonia active against selected spore formers; Blakistone B et al.; Alternatives to hydrogen peroxide are being sought for use in aseptic packaging systems because this sterilant is efficacious at temperatures higher than some of the newer packaging materials can tolerate . Earlier in this century, peracetic acid was known to be bactericidal, sporicidal, and virucidal but was not widely used because of handling, toxicity, and stability problems . Sanitizer suppliers have capitalized on the efficacy of hydrogen peroxide, acetic acid, and peracetic acid stabilized with a sequestering agent . Formulations have been improved and marketed as Oxonia Active, and its use as an alternative sterilant to hydrogen peroxide merits evaluation . Oxonia was assessed at a concentration of 2% and a temperature of 40 degrees C against a number of spore-forming organisms, including foodborne pathogens . Spores tested in aqueous suspension showed an order of sensitivity (least to greatest) to Oxonia as follows: Bacillus cereus > B . subtilis A > B . stearothermophilus > B . subtilis var . globigii > B . coagulans > Clostridium sporogenes (PA3679) > C . butyricum > C . botulinum type B (nonproteolytic) > C . botulinum type B (proteolytic) = C . botulinum type A = C . botulinum type E . B . subtilis A and B . stearothermophilus spores tested in the dry state were less sensitive to Oxonia than when tested in aqueous suspension . B . cereus, a foodborne pathogen, proved to be markedly less sensitive to Oxonia under the described test conditions . The decreased sensitivity to Oxonia by the foodborne pathogen B . cereus raises concern about the efficacy of the sterilant for aseptic packaging of low-acid foods . Further work will be needed to determine if this decreased sensitivity is an inherent property of the organism that affords unusual protection against Oxonia or if the challenge parameters selected were at the minimum conditions for efficacy. Acta Crystallogr D Biol Crystallogr, 1998 Nov 1, 54(Pt 6 Pt 2), 1425 - 8 Crystallization and preliminary X-ray diffraction studies of alpha-toxin from two different strains (NCTC8237 and CER89L43) of Clostridium perfringens; Basak AK et al.; The alpha-toxin of Clostridium perfringens is the major virulence determinant for gas gangrene in man . The gene encoding the alpha-toxin has been cloned into E . coli from two strains of the bacterium (NCTC8237 and CER89L43) and subsequently purified to homogeneity . The two strains of alpha-toxin differ by five amino acids, resulting in the toxin from NCTC8237 being sensitive to chymotrypsin digestion while that from CER89L43 is resistant . The alpha-toxin from each of these strains has been crystallized in two different forms by the hanging-drop vapour-diffusion method at 293 K . CER89L43 form I crystals belong to space group R32 and have two molecules in the crystallographic asymmetric unit and a unit cell with a = b = 151.4, c = 195.5 A, alpha = beta = 90, gamma = 120 degrees . The crystals diffracted to dmin = 1.90 A . The characteristics of the NCTC8237 form I crystals have already been reported . The form II crystals from both strains belong to space group C2221 with one molecule in the crystallographic asymmetric unit and, for strain CER89L43, have cell dimensions a = 61.05, b = 177.50, c = 79.05 A, alpha = beta = gamma = 90 degrees, while for strain NCTC8237 the cell dimensions are a = 60.50, b = 175.70, c = 80.20 A, alpha = beta = gamma = 90 degrees . The crystals diffracted to maximum resolutions of 1.85 and 2.1 A for the CER89L43 and the NCTC8237 strains, respectively. World J Surg, 1999 May, 23(5), 429 - 32; discussion 433 Antibiotic administration in patients undergoing common surgical procedures in a community teaching hospital: the chaos continues; Gorecki P et al.; The influence of recently published guidelines by the Surgical Infection Society (SIS) on current surgical practice are not well documented . The appropriateness of antibiotic administration in a cohort of surgical patients undergoing elective and emergency surgery in a department of surgery in an urban, community-based, private, 560-bed teaching hospital was retrospectively reviewed . The following were the criteria defining administration as appropriate as modified from SIS guidelines: Prophylactic use: (1) started prior to operation; (2) spectrum appropriate to the specific operation; (3) duration </= 24 hours . Therapeutic use: (1) started prior to operation; (2) spectrum appropriate to pathology; (3) Duration </= 24 hours for contamination or "resectable" infection and </= 5 days for established infection in the absence of clinical evidence of persisting infection . Any switchover from an appropriate agent to another appropriate or inappropriate agent in the same patient in the absence of microbiologic or clinical indication was considered inappropriate administration . We reviewed the charts of 211 randomly selected patients who underwent elective (n = 132) or emergency (n = 79) procedures during 1996 . The operations included gastrectomy (n = 22), appendectomy (n = 27), open (n = 5) or laparoscopic (n = 27) cholecystectomy, colectomy (n = 28), hysterectomy (n = 8), laparotomy for intestinal obstruction (n = 11), mastectomy (n = 26), and ventral hernia repair (n = 37) . A total of 17 antibiotics were used for prophylaxis and 21 for therapy . In 156 patients (74%) the administration was considered inappropriate . Eight patients in the inappropriate group developed diarrhea (two cases of Clostridium difficile-induced colitis) compared to two cases of diarrhea in the appropriate group (nonsignificant) . The average duration of administration after elective and emergency operations was 3.3 and 5 . 7 days, respectively . The total expense for excessive duration of administration was $18,533 . Many surgeons are not familiar with the spectrum of antimicrobials and often do not distinguish between prophylactic and therapeutic administration . Antibiotic usage in current surgical practice is often inappropriate, excessive, and chaotic. J Biol Chem, 1999 Mar 26, 274(13), 8445 - 54 Identification of D-proline reductase from Clostridium sticklandii as a selenoenzyme and indications for a catalytically active pyruvoyl group derived from a cysteine residue by cleavage of a proprotein; Kabisch UC et al.; Highly active D-proline reductase was obtained from Clostridium sticklandii by a modified purification scheme . The cytoplasmic enzyme had a molecular mass of about 870 kDa and was composed of three subunits with molecular masses of 23, 26, and 45 kDa . The 23-kDa subunit contained a carbonyl group at its N terminus, which could either be labeled with fluorescein thiosemicarbazide or removed by o-phenylenediamine; thus, N-terminal sequencing became feasible for this subunit . L-{14C}proline was covalently bound to the 23-kDa subunit if proline racemase and NaBH4 were added . Selenocysteine was detected in the 26-kDa subunit, which correlated with an observed selenium content of 10.6 g-atoms in D-proline reductase . No other non-proteinaceous cofactor was identified in the enzyme . A 4.8-kilobase pair (kb) EcoRI fragment was isolated and sequenced containing the two genes prdA and prdB . prdA coding for a 68-kDa protein was most likely translated as a proprotein that was posttranslationally cleaved at a threonine-cysteine site to give the 45-kDa subunit and most probably a pyruvoyl-containing 23-kDa subunit . The gene prdB encoded the 26-kDa subunit and contained an in frame UGA codon for selenocysteine insertion . prdA and prdB were transcribed together on a transcript of 4.5 kb; prdB was additionally transcribed as indicated by a 0.8-kb mRNA species. Immunobiology, 1999 Feb, 200(1), 106 - 19 Immunostimulatory properties of genomic DNA from different bacterial species; Neujahr DC et al.; Bacterial DNA has potent immunological properties because of its content of immunostimulatory sequences centering on CpG motifs . To investigate whether DNA from various bacterial species differ in these properties, the activity of a panel of DNA was assessed in in vitro cultures of murine spleen cells . This panel varied in base composition and included DNA from Clostridium perfringens (CP), Escherichia coli (EC), Micrococcus lysodeikticus (MC), Staphylococcus aureus (SA), and, as a mammalian DNA control, calf thymus (CT) DNA . In assays of IL-12 and IFN-gamma production as well as B cell mitogenesis, these DNA showed marked differences in their immunostimulatory activity . For both cytokine and B cell responses, EC DNA demonstrated the highest activity while CP DNA had the lowest activity among the bacterial DNA . To determine whether differences in stimulatory capacity resulted from differences in cell uptake, the activity of DNA complexed with lipofectin was tested . While the addition of lipofectin to DNA increased stimulation by all DNA, it did not change the relative potency of the DNA tested . These results indicate that bacterial DNA differ in their immunostimulatory capacity, most likely reflecting their content of CpG motifs . These differences could affect the induction of innate immunity as well as the consequences of infection. J Histochem Cytochem, 1999 Apr, 47(4), 481 - 8 Keratan sulfate glycosaminoglycans in murine eosinophil-specific granules; Ohmori J et al.; We examined the presence of sialyl glycoconjugates in specific granules from murine bone marrow eosinophils . Lectin cytochemistry using Maackia amurensis lectin II (MAL II) specific for sialyl alpha-2,3 galactose residues demonstrated positive labeling in both immature and mature specific granules . Pretreatment with Clostridium neuraminidase or keratanase II eliminated the positive labeling of MAL II in the specific granules . High iron diamine-thiocarbohydrazide-silver proteinate physical development (HID-TCH-SP-PD) staining, which is specific for sulfated glycoconjugates, also positively labeled immature specific granules lacking crystalloids but not mature granules with crystalloids . Pretreatment with a combination of chondroitinase ABC and keratanase, or a combination of chondroitinase ABC and keratanase II, eliminated the positive labeling obtained with HID-TCH-SP-PD . These results indicate that the sialyl residues detected by MAL II are expressed as terminal sugar residues of keratan sulfate proteoglycan, which appears to be of the corneal type in view of its sensitivity to keratanase and keratanase II . (J Histochem Cytochem 47:481-488, 1999) J Vet Med Sci, 1999 Feb, 61(2), 175 - 7 Hemorrhagic enteritis associated with Clostridium perfringens type A in a dog; Sasaki J et al.; A female Shetland sheep dog died suddenly with hemorrhagic diarrhea and vomitting, and was examined pathologically and microbiologically . Gross pathological change was restricted to the intestinal tract . The intestine contained watery, blood-stained fluid . Histopathologically, the principal intestinal lesion was superficial mucosal hemorrhagic necrosis at the jejunoileum . Many Gram-positive bacilli were found adhering to the necrotic mucosal surface in parts of the intestinal tract . Clostridium perfringens in pure culture were isolated from jejunal contents by anaerobic culture . These results suggested that the typical lesion of this case coincided with canine hemorrhagic enteritis and enterotoxemia due to C . perfringens infection could be the cause of sudden death. Toxicon, 1999 Mar, 37(3), 471 - 84 Detection of Clostridium perfringens alpha toxin using a capture antibody ELISA; Hale ML et al.; Clostridium perfringens phospholipase C (PLC), commonly known as alpha toxin, is the lethal, dermonecrotic toxin produced by all strains and is considered a major virulence factor in clostridial myonecrosis . We developed a capture antibody ELISA that accurately and specifically quantitates alpha toxin produced by C . perfringens . Another PLC, derived from Bacillus cereus, and culture filtrates from various bacterial species including Clostridium bifermentans and Clostridium novyi were not cross-reactive in this ELISA . Standard curves generated with homogenous C . perfringens alpha toxin revealed detection limits of 19 ng/ml . The ELISA was more sensitive in detecting alpha toxin than techniques such as PLC enzymatic activity and mouse lethality assays. Antibiot Khimioter, 1998, 43(12), 20 - 4 {Laboratory and clinical study of nitazole}; Blatun LA et al.; Nitazole, a drug from the nitrotiazole group, was shown to be active in vitro against bacteroides, peptococci, peptostreptococci, clostridia, staphylococci, colibacilli and streptococci . By its activity and antibacterial spectrum nitazole had some advantages over metronidazole, a drug from the nitroimidazole group . Experimental study of nitazole aerosole formulation in 4 models of purulent wounds of rabbits infected by Bacteroides fragilis, B . melaninogenicus, Clostridium perfringens 27 and Staphylococcus aureus 209P revealed its high therapeutic efficacy . In the treatment of 37 patients with purulent wounds of the soft tissues including 12 cases isolating anaerobic microbes, the clinical process of the acute suppuration in all the patients at the average reduced to the 5th-7th day . By the data of the bacteriological and cytological examinations the wound surface was ready for putting in stitches or free perforated cutaneous graft by the 10th-12th day . The drug tolerance was good . No adverse reaction were observed under the nitazole dressing in any case during the treatment of the wounds. FEMS Microbiol Lett, 1999 Mar 1, 172(1), 79 - 83 Prevalence of nim genes in anaerobic/facultative anaerobic bacteria isolated in South Africa; Lubbe MM et al.; This study investigated the prevalence of nim genes (proposed to encode a 5-nitroimidazole resistance product) in 64 anaerobic/facultative anaerobic bacteria . Employing universal nim gene primers, 458-bp amplified fragments were recorded as presumptive positives in 22/64 strains at an annealing temperature of 52 degrees C and 15/64 strains at 62 degrees C, of which seven were propionibacteria . DNA sequencing confirmed the presence of nimA genes in Propionibacterium spp . (five strains), Actinomyces odontolyticus (one strain), Prevotella bivia (one strain) and Clostridium bifermentans (one strain) and nimB genes from five strains of Bacteroides fragilis . nimA genes were predominant in propionibacteria indicating a potential nimA gene source in anaerobic environments. J Clin Invest, 1999 Mar, 103(6), 843 - 9 Neurotensin is a proinflammatory neuropeptide in colonic inflammation; Castagliuolo I et al.; The neuropeptide neurotensin mediates several intestinal functions, including chloride secretion, motility, and cellular growth . However, whether this peptide participates in intestinal inflammation is not known . Toxin A, an enterotoxin from Clostridium difficile, mediates pseudomembranous colitis in humans . In animal models, toxin A causes an acute inflammatory response characterized by activation of sensory neurons and intestinal nerves and immune cells of the lamina propria . Here we show that neurotensin and its receptor are elevated in the rat colonic mucosa following toxin A administration . Pretreatment of rats with the neurotensin receptor antagonist SR-48, 692 inhibits toxin A-induced changes in colonic secretion, mucosal permeability, and histologic damage . Exposure of colonic explants to toxin A or neurotensin causes mast cell degranulation, which is inhibited by SR-48,692 . Because substance P was previously shown to mediate mast cell activation, we examined whether substance P is involved in neurotensin-induced mast cell degranulation . Our results show that neurotensin-induced mast cell degranulation in colonic explants is inhibited by the substance P (neurokinin-1) receptor antagonist CP-96,345, indicating that colonic mast activation in response to neurotensin involves release of substance P . We conclude that neurotensin plays a key role in the pathogenesis of C . difficile-induced colonic inflammation and mast cell activation. J Bacteriol, 1999 Mar, 181(6), 1801 - 10 Sequence analysis of scaffolding protein CipC and ORFXp, a new cohesin-containing protein in Clostridium cellulolyticum: comparison of various cohesin domains and subcellular localization of ORFXp; Pages S et al.; The gene encoding the scaffolding protein of the cellulosome from Clostridium cellulolyticum, whose partial sequence was published earlier (S . Pages, A . Belaich, C . Tardif, C . Reverbel-Leroy, C . Gaudin, and J.-P . Belaich, J . Bacteriol . 178:2279-2286, 1996; C . Reverbel-Leroy, A . Belaich, A . Bernadac, C . Gaudin, J . P . Belaich, and C . Tardif, Microbiology 142:1013-1023, 1996), was completely sequenced . The corresponding protein, CipC, is composed of a cellulose binding domain at the N terminus followed by one hydrophilic domain (HD1), seven highly homologous cohesin domains (cohesin domains 1 to 7), a second hydrophilic domain, and a final cohesin domain (cohesin domain 8) which is only 57 to 60% identical to the seven other cohesin domains . In addition, a second gene located 8.89 kb downstream of cipC was found to encode a three-domain protein, called ORFXp, which includes a cohesin domain . By using antiserum raised against the latter, it was observed that ORFXp is associated with the membrane of C . cellulolyticum and is not detected in the cellulosome fraction . Western blot and BIAcore experiments indicate that cohesin domains 1 and 8 from CipC recognize the same dockerins and have similar affinity for CelA (Ka = 4.8 x 10(9) M-1) whereas the cohesin from ORFXp, although it is also able to bind all cellulosome components containing a dockerin, has a 19-fold lower Ka for CelA (2.6 x 10(8) M-1) . Taken together, these data suggest that ORFXp may play a role in cellulosome assembly. J Protein Chem, 1999 Jan, 18(1), 89 - 95 In vitro translation of type A Clostridium botulinum neurotoxin heavy chain and analysis of its binding to rat synaptosomes; Li L et al.; Botulinum neurotoxins (BoNTs) are highly potent toxins that inhibit neurotransmitter release from peripheral cholinergic synapses . BoNTs consist of a toxifying light chain (LC; 50 kDa) and a binding/translocating heavy chain (HC; 100 kDa) linked through a disulfide bond . A DNA fragment encoding type A Clostridium botulinum heavy chain (BoNT/A HC) was amplified by polymerase chain reaction and cloned into an E . coli PET-15b vector . In vitro translated {35S}BoNT/A HC was identified by anti-BoNT/A polyclonal antibodies, and was used to investigate the binding of the toxin to rat synaptosomes . The binding of {35S}BoNT/A HC to synaptosomes was abolished by 500-fold excess of cold BoNT/A, and by incubation with trypsin . Treatment of BoNT/A HC with anti-BoNT/A or G(T1b) blocked its binding to synaptosomes . The radioactive BoNT/A HC recognized three proteins corresponding to a molecular mass of 150 (P150), 120 (P120), and 75 (P75) kDa in rat and bovine synaptosomal preparations . These results represent the first successful expression of functional full-length BoNT heavy chain. J Protein Chem, 1999 Jan, 18(1), 29 - 38 Molecular properties of a hemagglutinin purified from type A Clostridium botulinum; Sharma SK et al.; Clostridium botulinum causes the food poisoning disease botulism by producing botulinum neurotoxin, the most potent toxin known . The neurotoxin is produced along with a group of neurotoxin-associated proteins, or NAPs, which protect it from the low pH and proteases of the gastrointestinal tract . Recently, we isolated one of the major components of NAPs, a 33-kDa hemagglutinin (Hn-33) {Fu et al . (1998), J . Protein Chem . 17, 53-60} . In this study, we present molecular properties of Hn-33 derived from several biochemical and biophysical techniques . Hn-33 in pure form requires a 66-fold lower concentration of sugar inhibition of its hemagglutination activity than in its complexed form with the neurotoxin and other NAPs . However, its protease resistance is not affected by sugar binding . Based on FT-IR and circular dichroism (CD) analysis, Hn-33 is a predominantly beta-sheet protein (74-77%) . Hn-33 analysis by laser desorption mass spectrometry and size exclusion column chromatography reveals that it exists predominantly in a dimeric form in the aqueous solution . Even a very low concentration of SDS (0.05%) irreversibly destroyed the biological activity of Hn-33 by changing its secondary structure as revealed by far-UV CD analysis. Am J Physiol, 1999 Mar, 276(3 Pt 1), C717 - 24 Monocytic cell necrosis is mediated by potassium depletion and caspase-like proteases; Warny M et al.; Apoptosis is a physiological cell death that culminates in mitochondrial permeability transition and the activation of caspases, a family of cysteine proteases . Necrosis, in contrast, is a pathological cell death characterized by swelling of the cytoplasm and mitochondria and rapid plasma membrane disruption . Necrotic cell death has long been opposed to apoptosis, but it now appears that both pathways involve mitochondrial permeability transition, raising the question of what mediates necrotic cell death . In this study, we investigated mechanisms that promote necrosis induced by various stimuli (Clostridium difficile toxins, Staphylococcus aureus alpha toxin, ouabain, nigericin) in THP-1 cells, a human monocytic cell line, and in monocytes . All stimuli induced typical features of necrosis and triggered protease-mediated release of interleukin-1beta (IL-1beta) and CD14 in both cell types . K+ depletion was actively implicated in necrosis because substituting K+ for Na+ in the extracellular medium prevented morphological features of necrosis and IL-1beta release . N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone, a broad-spectrum caspase inhibitor, prevented morphological features of necrosis, plasma membrane destruction, loss of mitochondrial membrane potential, IL-1beta release, and CD14 shedding induced by all stimuli . Thus, in monocytic cells, necrosis is a cell death pathway mediated by passive K+ efflux and activation of caspase-like proteases. Biol Cell, 1998 Nov, 90(8), 573 - 83 Ras family proteins: new players involved in the diplotene arrest of Xenopus oocytes; Jessus C et al.; Oogonia undergo numerous mitotic cell cycles before completing the last DNA replication and entering the meiotic prophase I . After chromosome pairing and chromatid exchanges between paired chromosomes, the oocyte I remains arrested at the diplotene stage of the first meiotic prophase . Oocyte growth then occurs independently of cell division; indeed, during this growth period, oocytes (4n DNA) are prevented from completing the meiotic divisions . How is the prophase arrest regulated? One of the players of the prophase block is the high level of intracellular cAMP, maintained by an active adenylate cyclase . By using lethal toxin from Clostridium sordellii (LT), a glucosyltransferase that glucosylates and inactivates small G proteins of the Ras subfamily, we have shown that inhibition of either Ras or Rap or both proteins is sufficient to release the prophase block of Xenopus oocytes in a cAMP-dependent manner . The implications of Ras family proteins as new players involved in the prophase arrest of Xenopus oocytes will be discussed here. J Physiol, 1999 Apr 1, 516 ( Pt 1), 67 - 74 Role of Rho and Rho kinase in the activation of volume-regulated anion channels in bovine endothelial cells; Nilius B et al.; 1 . We have studied the modulation of volume-regulated anion channels (VRACs) by the small GTPase Rho and by one of its targets, Rho kinase, in calf pulmonary artery endothelial (CPAE) cells . 2 . RT-PCR and immunoblot analysis showed that both RhoA and Rho kinase are expressed in CPAE cells . 3 . ICl,swell, the chloride current through VRACs, was activated by challenging CPAE cells with a 25 % hypotonic extracellular solution (HTS) or by intracellular perfusion with a pipette solution containing 100 microM GTPgammaS . 4 . Pretreatment of CPAE cells with the Clostridium C2IN-C3 fusion toxin, which inactivates Rho by ADP ribosylation, significantly impaired the activation of ICl,swell in response to the HTS . The current density at +100 mV was 49 +/- 13 pA pF-1 (n = 17) in pretreated cells compared with 172 +/- 17 pA pF-1 (n = 21) in control cells . 5 . The volume-independent activation of ICl,swell by intracellular perfusion with GTPgammaS was also impaired in C2IN-C3-pretreated cells (31 +/- 7 pA pF-1, n = 11) compared with non-treated cells (132 +/- 21 pA pF-1, n = 15) . 6 . Activation of ICl,swell was pertussis toxin (PTX) insensitive . 7 . Y-27632, a blocker of Rho kinase, inhibited ICl,swell and delayed its activation . 8 . Inhibition of Rho and of Rho kinase by the above-described treatments did not affect the extent of cell swelling in response to HTS . 9 . These experiments provide strong evidence that the Rho-Rho kinase pathway is involved in the VRAC activation cascade. Curr Opin Microbiol, 1998 Feb, 1(1), 66 - 74 Toxins from anaerobic bacteria: specificity and molecular mechanisms of action; Boquet P et al.; Major advances have been made in the past five years in the identification of cellular targets of toxins produced by anaerobic bacteria . These targets include the vesicular membrane docking and fusion apparatus, the actin cytoskeleton, the signal transduction machinery and the cell membrane . The recent discovery that large clostridial toxins (Clostridium difficile A and B toxins, C . sordellii lethal and hemorrhagic toxins, and alpha C . novyi toxin) are monoglucosyltransferases, together with the establishment of the perfringolysin crystal structure, has led to new insights in the field of toxins from anaerobic bacteria. J Cardiovasc Surg (Torino), 1996 Dec, 37(6 Suppl 1), 183 - 7 Wound infection after median sternotomy during the war in Croatia; Jelic I et al.; From 1990 to 1994 at Clinical Hospital Center, Zagreb, 1904 median sternotomies were performed for cardiac operations . Patients shared the same intensive care unit (ICU) with the wounded persons, admitted to the hospital from battlefield . Infection developed in 124 patients, an incidence of 6.51% . Methicillin resistant Staphylococcus aureus (MRSA) was isolated from 90, methicillin resistant Staphylococcus epidermidis (MRSE) from 19, and gram negative bacilli (GNB) from 56 patients, Pseudomonas aeruginosa in 2, and Clostridium pneumoniae in 1 case . Ninety-six patients (5.04%) developed superficial localized infection of subcutaneous tissues and they were treated with frequent dressing changes with antibiotic-soaked gauze in combination with systemic antibiotics . Twenty-eight patients (1.47%) developed mediastinitis and sternal dehiscence . They were treated by operative debridement followed by reclosure of the sternum with continuous antibiotic irrigation . We obtained satisfactory results with our method of closure of sternum which is a modification of Robicsek's technique . Nine of them required further operation . In seven cases we performed muscle flaps and in two omentoplasty . One hundred and twenty patients were discharged in satisfactory condition . The uncontrolled mediastinal sepsis caused death in 4 patients . Higher infection rate after median sternotomy during 1991 and 1992 could be possibly explained with the war circumstances in Croatia, and especially with MRSA strain becoming endemic in surgical ICU. Lett Appl Microbiol, 1999 Feb, 28(2), 108 - 12 Psychrotrophic clostridia mediated gas and botulinal toxin production in vacuum-packed chilled meat; Moorhead SM et al.; A cocktail of washed spores from six psychrotrophic Clostridium strains isolated from blown vacuum-packed meats was inoculated onto lamb chumps . A second washed spore cocktail of four toxigenic reference Cl . botulinum strains, types A, B (two strains) and E, and a Cl . butyricum type E strain, was similarly inoculated onto lamb chumps . All inoculated lamb chumps were individually vacuum-packed and placed into storage at various temperatures typical of good to grossly abusive chilled storage (-1 degree C to 15 degrees C) . All packs were observed for gas production (pack-'blowing') over a 12 week storage period . On gas production, or after 12 weeks of storage, packs were examined by mouse bioassay for botulinum toxin production . The packs inoculated with the meat isolate cocktail showed evidence of gas production earlier than packs inoculated with reference strains . No botulinum toxin was recovered from the meat isolate inoculated packs, while botulinal toxin was detected in reference strain inoculated packs down to a nominal storage temperature of 2 degrees C. Lett Appl Microbiol, 1999 Feb, 28(2), 98 - 102 A new growth and in vitro sporulation medium for Clostridium perfringens; Meyer M et al.; Growth and in vitro sporulation capabilities of three related Clostridium perfringens strains (NCTC 8798, 8-6 and R3) were followed in a new sporulation medium (NSM), with notable changes from a maintenance medium originally designed for strictly anaerobic bacteria . Compared with thioglycollate (FTG) medium, the new sporulation medium promoted growth of Cl . perfringens with a shorter lag phase and a 20% higher biomass production . The age of inoculum did not change Cl . perfringens growth kinetics . When compared with reference conditions, in vitro spore production kinetics were different in the new sporulation medium, but both conditions led routinely to 100% sporulation and spore counts of approximately 10(8) ml-1 . The ease of preparation of the NSM, and the use of the same culture medium for good growth, high sporulation yields and spore production, represent an attractive alternative to the complex media routinely used for in vitro studies of Cl . perfringens physiology. J Hosp Infect, 1999 Feb, 41(2), 145 - 9 Rapid detection of toxigenic Clostridium difficile from stool samples by a nested PCR of toxin B gene; Alonso R et al.; Toxigenic Clostridium difficile is the aetiologic agent of most cases of antibiotic-associated diarrhoea and pseudomembranous colitis . The present standard method for C . difficile diagnosis is a cytotoxicity assay, performed on human fibroblast cultures . It is time consuming and requires special facilities . A nested-PCR assay detecting toxin B gene within a few hours was designed . One hundred and two stool samples were collected during four months . All samples were processed for toxin B-PCR, cultured for C . difficile and tested for cytotoxicity . This approach achieved 99% concordance with the cytotoxic assay . The sensitivity and specificity for the new PCR assay were 96.3% and 100% respectively . The procedure described is easy to perform, does not require special equipment and has produced excellent results . It deserves serious consideration for routine clinical microbiology laboratory use. J Hosp Infect, 1999 Feb, 41(2), 101 - 5 Evaluation of microbicidal activity of a new disinfectant: Sterilox 2500 against Clostridium difficile spores, Helicobacter pylori, vancomycin resistant Enterococcus species, Candida albicans and several Mycobacterium species; Shetty N et al.; The microbicidal activity of a new disinfectant Sterilox, a super-oxidized water, containing a mixture of oxidizing substances, was tested against Clostridium difficile spores, Helicobacter pylori, vancomycin resistant Enterococcus species, Candida albicans and several Mycobacterium species using membrane filters . All tests were performed in duplicate with and without added horse serum at 1% and 5% v/v . Distilled water, 0.35% peracetic acid (Nu-Cidex) and 2% glutaraldehyde were included as controls . Sterilox: spore suspension (9:1 v/v) achieved log10 kill of > 5 with 5% horse serum in 2 min against H . pylori, vancomycin resistant Enterococcus species, C . albicans and four atypical Mycobacterium species: M . avium, M . chelonei, M . xenopi and M . smegmatis . Sporicidal activity of Sterilox against Clostridium difficile was markedly diminished in the presence of 5% horse serum . Sterilox may be an effective alternative in endoscopy units, as it is a potent microbicidal agent and the manufacturer claims it is not corrosive to metal and is nontoxic to biological tissues. J Nurse Midwifery, 1999 Jan-Feb, 44(1), 19 - 29 Clostridium difficile-associated disease . Implications for midwifery practice; Alef K; Clostridium difficile-associated disease (CDAD), a gastrointestinal infection with a wide range of manifestations whose primary symptom is diarrhea, occurs when antibiotic medications, or rarely other drugs or conditions, disrupt the normal colonic microflora, making it susceptible to the growth of toxigenic C difficile . It is a significant nosocomial infection and an increased incidence has been noted in recent years . Although infrequently seen in midwifery practices, it does occur and may increase with the growing usage of intrapartal antibiotics . Midwives may evaluate and treat a client with an initial episode of mild to moderate CDAD; they also may manage collaboratively or refer for medical management those clients with recurrent or severe disease . This article reviews the epidemiology, pathogenesis, clinical presentation, prevention, and midwifery management of initial and recurrent CDAD . The limitation in the use of oral vancomycin due to the emergence of vancomycin-resistant enterococcus, resulting in metronidazole becoming the primary agent for treatment of CDAD, and the implications of this in the treatment of CDAD during pregnancy and lactation are addressed. Biochem Biophys Res Commun, 1999 Feb 16, 255(2), 317 - 23 NADH peroxidase activity of rubrerythrin; Coulter ED et al.; P . S . Alban et al . (J . Appl . Microbiol . (1998) 85, 875-882) reported that a mutant H2O2-resistant strain of Spirullum (S.) volutans showed constitutive overexpression of a protein whose amino acid sequence and molecular weight closely resembled that of a subunit of rubrerythrin, a non-heme iron protein with no known function . They also reported that the mutant strain, but not the wild-type, showed NADH peroxidase activity . Here we demonstrate that rubrerythrin and nigerythrin from Desulfovibrio vulgaris and rubrerythrin from Clostridium perfringens show NADH peroxidase activities in an in vitro system containing NADH, hydrogen peroxide, and a bacterial NADH oxidoreductase . The peroxidase specific activities of the rubrerythrins with the "classical" heme peroxidase substrate, o-dianisidine, are many orders of magnitude lower than that of horseradish peroxidase . These results are consistent with the phenotype of the H2O2-resistant strain of S . volutans . The reaction of reduced (i.e., all-ferrous) rubrerythrin with excess O2 takes several minutes, whereas the anaerobic reaction of reduced rubrerythrin with hydrogen peroxide is on the millisecond time scale and results in full oxidation of all iron centers to their ferric states . Rubrerythrins could, thus, function as the terminal components of NADH peroxidases in air-sensitive bacteria and archaea . Poult Sci, 1999 Feb, 78(2), 182 - 9 Effect of betaine supplement on broiler performance during an experimental coccidial infection; Waldenstedt L et al.; A trial was conducted to investigate the effects of betaine as a feed supplement, given singly and in combination with the ionophore coccidiostat narasin, on broiler performance during an experimental coccidial infection . Five hundred and sixty female Ross broiler chickens were kept in floor pens and given a wheat-based diet . At 10 d of age, 420 chickens were individually inoculated with a mixture of Swedish chicken Eimeria isolates containing E . acervtulina, E . praecox, E . maxima, and E . tenella . Remaining birds were kept as uninoculated controls . The effects of betaine (0 or 1.0 g/kg) and narasin (0 or 70 ppm) added to the basal diet were evaluated . Overall, betaine as a single feed supplement improved live weight by 5.7, 5.4, and 5.6% at 22, 29, and 36 d, respectively, but had no positive effect in combination with narasin . A longer withdrawal period of the coccidiostat (10 vs 5 d) did not affect live weight, but significantly increased feed intake by 9.6% and feed conversion ratio by 12.6%, irrespective of betaine supplement . Inoculated birds had a 10% lower live weight than uninoculated chickens . Performance of uninoculated birds was similar to that of inoculated birds treated with narasin, except at 7 d after inoculation, when live weights of uninoculated birds were significantly higher . Chickens given coccidiostat had less Clostridium perfringens in their ceca, but the prevalence was not altered by betaine supplement . There was no difference in intestinal lesion scores between inoculated chickens given coccidiostat or not, despite the better performance of chickens given coccidiostat . Betaine did not affect Eimeria oocyst output or intestinal lesion scores. Appl Environ Microbiol, 1999 Mar, 65(3), 936 - 45 Antisense RNA strategies for metabolic engineering of Clostridium acetobutylicum; Desai RP et al.; We examined the effectiveness of antisense RNA (as RNA) strategies for metabolic engineering of Clostridium acetobutylicum . Strain ATCC 824(pRD4) was developed to produce a 102-nucleotide asRNA with 87% complementarity to the butyrate kinase (BK) gene . Strain ATCC 824(pRD4) exhibited 85 to 90% lower BK and acetate kinase specific activities than the control strain . Strain ATCC 824(pRD4) also exhibited 45 to 50% lower phosphotransbutyrylase (PTB) and phosphotransacetylase specific activities than the control strain . This strain exhibited earlier induction of solventogenesis, which resulted in 50 and 35% higher final concentrations of acetone and butanol, respectively, than the concentrations in the control . Strain ATCC 824(pRD1) was developed to putatively produce a 698-nucleotide asRNA with 96% complementarity to the PTB gene . Strain ATCC 824(pRD1) exhibited 70 and 80% lower PTB and BK activities, respectively, than the control exhibited . It also exhibited 300% higher levels of a lactate dehydrogenase activity than the control exhibited . The growth yields of ATCC 824(pRD1) were 28% less than the growth yields of the control . While the levels of acids were not affected in ATCC 824(pRD1) fermentations, the acetone and butanol concentrations were 96 and 75% lower, respectively, than the concentrations in the control fermentations . The lower level of solvent production by ATCC 824(pRD1) was compensated for by approximately 100-fold higher levels of lactate production . The lack of any significant impact on butyrate formation fluxes by the lower PTB and BK levels suggests that butyrate formation fluxes are not controlled by the levels of the butyrate formation enzymes. Protein Expr Purif, 1999 Mar, 15(2), 221 - 7 Recombinant and truncated tetanus neurotoxin light chain: cloning, expression, purification, and proteolytic activity; Tonello F et al.; Tetanus neurotoxin (TeNT) consists of two disulfide-linked polypeptide chains, heavy (H) and light (L) . The L chain is a zinc endopeptidase protein highly specific for vesicle-associated membrane protein (VAMP), which is an essential component of the exocytosis apparatus . Here we describe the cloning of the L chain of TeNT from Clostridium tetani strain Y-IV-3 (WS 15) and its expression in Escherichia coli as a glutathione S-transferase fusion protein . The full-length recombinant L chain, corresponding to residues 1-457, was obtained as a mixture of proteins of slightly different mass with identical N-terminal ends . To obtain a product useful for structural analysis and crystallization, a COOH-terminally truncated L chain (residues 1-427) was cloned, expressed, and purified with high yield . This truncated L chain is more active than the full-length and wild-type proteins in the hydrolysis of VAMP . Preliminary experiments of crystallization of the truncated recombinant L chain gave encouraging results . J Bacteriol, 1999 Mar, 181(5), 1489 - 95 Nitrate-dependent regulation of acetate biosynthesis and nitrate respiration by Clostridium thermoaceticum; Arendsen AF et al.; Nitrate has been shown to shunt the electron flow in Clostridium thermoaceticum from CO2 to nitrate, but it did not influence the levels of enzymes involved in the Wood-Ljungdahl pathway (J . M . Frostl, C . Seifritz, and H . L . Drake, J . Bacteriol . 178:4597-4603, 1996) . Here we show that under some growth conditions, nitrate does in fact repress proteins involved in the Wood-Ljungdahl pathway . The CO oxidation activity in crude extracts of nitrate (30 mM)-supplemented cultures was fivefold less than that of nitrate-free cultures, while the H2 oxidation activity was six- to sevenfold lower . The decrease in CO oxidation activity paralleled a decrease in CO dehydrogenase (CODH) protein level, as confirmed by Western blot analysis . Protein levels of CODH in nitrate-supplemented cultures were 50% lower than those in nitrate-free cultures . Western blots analyses showed that nitrate also decreased the levels of the corrinoid iron-sulfur protein (60%) and methyltransferase (70%) . Surprisingly, the decrease in activity and protein levels upon nitrate supplementation was observed only when cultures were continuously sparged . Northern blot analysis indicates that the regulation of the proteins involved in the Wood-Ljungdahl pathway by nitrate is at the transcriptional level . At least a 10-fold decrease in levels of cytochrome b was observed with nitrate supplementation whether the cultures were sparged or stoppered . We also detected nitrate-inducible nitrate reductase activity (2 to 39 nmol min-1 mg-1) in crude extracts of C . thermoaceticum . Our results indicate that nitrate coordinately represses genes encoding enzymes and electron transport proteins in the Wood-Ljungdahl pathway and activates transcription of nitrate respiratory proteins . CO2 also appears to induce expression of the Wood-Ljungdahl pathway genes and repress nitrate reductase activity. Antimicrob Agents Chemother, 1999 Mar, 43(3), 582 - 8 Antimicrobial activities of synthetic bismuth compounds against Clostridium difficile; Mahony DE et al.; Clostridium difficile is a major nosocomial pathogen responsible for pseudomembranous colitis and many cases of antibiotic-associated diarrhea . Because of potential relapse of disease with current antimicrobial therapy protocols, there is a need for additional and/or alternative antimicrobial agents for the treatment of disease caused by C . difficile . We have synthesized a systematic series of 14 structurally simple bismuth compounds and assessed their biological activities against C . difficile and four other gastrointestinal species, including Helicobacter pylori . Here, we report on the activities of six compounds that exhibit antibacterial activities against C . difficile, and some of the compounds have MICs of less than 1 microgram/ml . Also tested, for comparison, were the activities of bismuth subcitrate and ranitidine bismuth citrate obtained from commercial sources . C . difficile and H . pylori were more sensitive both to the synthetic bismuth compounds and to the commercial products than were Escherichia coli, Pseudomonas aeruginosa, and Proteus mirabilis, and the last three species were markedly resistant to the commercial bismuth salts . Testing with human foreskin fibroblast cells revealed that some of the synthetic compounds were more cytotoxic than others . Killing curves for C . difficile treated with the more active compounds revealed rapid death, and electron microscopy showed that the bismuth of these compounds was rapidly incorporated by C . difficile . Energy dispersive spectroscopy X-ray microanalysis of C . difficile cells containing electron-dense material confirmed the presence of internalized bismuth . Internalized bismuth was not observed in C . difficile treated with synthetic bismuth compounds that lacked antimicrobial activity, which suggests that the uptake of the metal is required for killing activity . The nature of the carrier would seem to determine whether bismuth is transported into susceptible bacteria like C . difficile. Mol Microbiol, 1999 Feb, 31(3), 785 - 94 Expression and properties of an aerolysin--Clostridium septicum alpha toxin hybrid protein; Diep DB et al.; Aerolysin is a bilobal channel-forming toxin secreted by Aeromonas hydrophila . The alpha toxin produced by Clostridium septicum is homologous to the large lobe of aerolysin . However, it does not contain a region corresponding to the small lobe of the Aeromonas toxin, leading us to ask what the function of the small lobe is . We fused the small lobe of aerolysin to alpha toxin, producing a hybrid protein that should structurally resemble aerolysin . Unlike aerolysin, the hybrid was not secreted when expressed in Aeromonas salmonicida . The purified hybrid was activated by proteolytic processing in the same way as both parent proteins and, after activation, it formed oligomers that corresponded to the aerolysin heptamer . Like aerolysin, the hybrid was far more active than alpha toxin against human erythrocytes and mouse T lymphocytes . Both aerolysin and the hybrid bound to human glycophorin, and both were inhibited by preincubation with this erythrocyte glycoprotein, whereas alpha toxin was unaffected . We conclude that aerolysin contains two receptor binding sites, one for glycosyl-phosphatidylinositol-anchored proteins that is located in the large lobe and is also found in alpha toxin, and a second site, located in the small lobe, that binds a surface carbohydrate determinant. J Cell Biol, 1999 Feb 22, 144(4), 745 - 54 Activation of G12/G13 results in shape change and Rho/Rho-kinase-mediated myosin light chain phosphorylation in mouse platelets; Klages B et al.; Platelets respond to various stimuli with rapid changes in shape followed by aggregation and secretion of their granule contents . Platelets lacking the alpha-subunit of the heterotrimeric G protein Gq do not aggregate and degranulate but still undergo shape change after activation through thromboxane-A2 (TXA2) or thrombin receptors . In contrast to thrombin, the TXA2 mimetic U46619 led to the selective activation of G12 and G13 in Galphaq-deficient platelets indicating that these G proteins mediate TXA2 receptor-induced shape change . TXA2 receptor-mediated activation of G12/G13 resulted in tyrosine phosphorylation of pp72(syk) and stimulation of pp60(c-src) as well as in phosphorylation of myosin light chain (MLC) in Galphaq-deficient platelets . Both MLC phosphorylation and shape change induced through G12/G13 in the absence of Galphaq were inhibited by the C3 exoenzyme from Clostridium botulinum, by the Rho-kinase inhibitor Y-27632 and by cAMP-analogue Sp-5,6-DCl-cBIMPS . These data indicate that G12/G13 couple receptors to tyrosine kinases as well as to the Rho/Rho-kinase-mediated regulation of MLC phosphorylation . We provide evidence that G12/G13-mediated Rho/Rho-kinase-dependent regulation of MLC phosphorylation participates in receptor-induced platelet shape change. Medsurg Nurs, 1998 Dec, 7(6), 348 - 9, 352-6 Recognizing and managing Clostridium difficile-associated diarrhea; Miller JM et al.; Clostridium difficile-associated diarrhea poses a significant physical risk and cost to the recovery of hospitalized older adults . C . difficile is responsible for 75% or more of the diarrhea-associated enteric infections acquired during a hospital stay (Gerding, Johnson, Peterson, Mulligan, & Silva, 1995) . C . difficile is easily spread by direct or indirect contact, therefore placing other patients at great risk for contamination by this organism . Nursing plays a significant role in early identification, management, and control of the spread of this potentially lethal infection. Rinsho Biseibutshu Jinsoku Shindan Kenkyukai Shi, 1998, 9(2), 97 - 104 Human diseases caused by exotoxins produced by anaerobes and their rapid detection; Kato N et al.; Major human diseases caused by exotoxins produced by anaerobes include botulisms, tetanus, foodborne illness caused by enterotoxin-producing Clostridium perfringens, and diarrhea/colitis caused by toxigenic Clostridium difficile . Recently, enterotoxigenic Bacteroides fragilis (ETBF) has been recognized, that may be related to childhood diarrheal disease . Detection test of botulinal neurotoxin is hardly performed at clinical laboratories since the most reliable means of detection and identification of botulinal toxin is by using mouse toxicity and neutralization tests . Clinical laboratories should request the tests to a reference laboratory . Since tetanus is easily diagnosed clinically on the basis of its unique, recognizable sings, the bacteriological tests is not usually requested . C . perfringens foodborne illness can be confirmed by testing stool specimens or the suspect food(s) for enterotoxin by the reversed passive latex agglutination test or counting > 10 5 C . perfringens per g of suspected food or > 10 6 C . perfringens spores per g of stool . Diagnosis of C . difficile-associated diarrhea/colitis is confirmed by detection of toxins A or B of C . difficile and/or recovery of toxigenic C . difficile . Isolation of C . difficile strains or detection C . difficile-speciffic antigen from stool specimens is less diagnostic since nontoxic or toxin A positive-toxin B negative strains are prevalent in Japan . Reliable laboratory tests for ETBF-associated childhood diarrhea are not established yet although ETBF can be proved by polymerase chain reaction for detection of the enterotoxin gene. FEMS Immunol Med Microbiol, 1999 Jan, 23(1), 45 - 8 Direct detection of Clostridium perfringens enterotoxin in patients' stools during an outbreak of food poisoning; Arcieri R et al.; An outbreak of diarrhoea in a hotel affected 25 time keepers attending the 1997 Mediterranean Games . Epidemiological investigation implicated a 'pasta al ragu' consumed at the hotel's restaurant and Clostridium perfringens food poisoning was identified by direct detection of C . perfringens enterotoxin in patients' stools . This report confirms that a careful evaluation of epidemiological features, together with the availability of direct and rapid laboratory methods, may lead to a prompt identification of C . perfringens food poisoning. Int J Syst Bacteriol . 1999 Jan;49 Pt 1:339. Rejection of Clostridium putrificum and conservation of Clostridium botulinum and Clostridium sporogenes-Opinion 69 . Judicial Commission of the International Committee on Systematic Bacteriology; Structural similarities between the N-terminal domain of Clostridium pasteurianum hydrogenase and plant-type ferredoxins; Departement de Biologie Moleculaire et Structurale, CEA-Grenoble, FranceAn N-terminal domain of Clostridium pasteurianum hydrogenase I, encompassing 76 residues out of the 574 composing the full-size enzyme, had previously been overproduced in Escherichia coli and shown to form a stable fold around a {2Fe-2S} cluster . This domain displays only marginal sequence similarity with {2Fe-2S} proteins of known structure, and therefore, two-dimensional 1H NMR has been implemented to elucidate features of the polypeptide fold . Despite the perturbing presence of the paramagnetic {2Fe-2S} cluster, 57 spin systems were detected in the TOCSY spectra, 52 of which were sequentially assigned through NOE connectivities . Several secondary structure elements were identified . The N terminus of the protein consists of two antiparallel beta strands followed by an alpha helix contacting both strands . Two additional antiparallel beta strands, one of them at the C terminus of the sequence, form a four-stranded beta sheet together with the two N-terminal strands . The proton resonances that can be attributed to this beta2alphabeta2 structural motif undergo no paramagnetic perturbations, suggesting that it is distant from the {2Fe-2S} cluster . In plant- and mammalian-type ferredoxins, a very similar structural pattern is found in the part of the protein farthest from the {2Fe-2S} cluster . This indicates that the N-terminal domain of C . pasteurianum hydrogenase folds in a manner very similar to those of plant- and mammalian-type ferredoxins over a significant part (ca . 50%) of its structure . Even in the vicinity of the metal site, where 1H NMR data are blurred by paramagnetic interactions, the N-terminal domains of hydrogenase and mammalian- and plant-type ferredoxins most likely display significant structural similarity, as inferred from local sequence alignments and from previously reported circular dichroism and resonance Raman spectra . These data afford structural information on a kind of {2Fe-2S} cluster-containing domain that occurs in a number of redox enzymes and complexes . In addition, together with previously published sequence alignments, they highlight the widespread distribution of the plant-type ferredoxin fold in bioenergetic systems encompassing anaerobic metabolism, photosynthesis, and aerobic respiratory chains. Bioorg Med Chem Lett, 1999 Jan 4, 9(1), 109 - 12 Cleavage of L-leucine-containing dipeptides by Clostridium butyricum; Khelifa N et al.; The ability of Clostridium butyricum cultures to hydrolyze three L-leucine-containing dipeptides (Leu-Leu, Leu-Gly and Gly-Leu) in a synthetic minimal medium is demonstrated by using gas chromatography coupled with mass spectrometry . The 13C nuclear magnetic resonance and a labeled dipeptide L-{1-13C}Leu-Gly were used to confirm this activity. J Med Microbiol, 1999 Feb, 48(2), 133 - 7 Isolation and characterisation of neurotoxigenic Clostridium butyricum from soil in China; Meng X et al.; Soil specimens collected from a site around the home of patients with food-borne type E . botulism probably caused by neurotoxigenic Clostridium butyricum in Guanyun, Jiangsu province, China, were examined for the presence of neurotoxigenic C . butyricum . Five lakeside sites of Weishan lake, in an area near to the sites where the type E . botulism outbreaks caused by neurotoxigenic C . butyricum occurred were also surveyed . Type E toxin-producing C . butyricum was isolated from soil from four sites including the site in Guanyun . Polymerase chain reaction assay demonstrated the presence of the type E toxin gene in all the toxigenic isolates . The biochemical properties of the isolates from the Guanyun soil and the lakeside soil were identical except for inulin fermentation and starch hydrolysis properties . These results indicate that neurotoxigenic C . butyricum has its principal habitat in soil. Biochim Biophys Acta, 1999 Jan 11, 1429(2), 307 - 16 Investigation of the role of surface residues in the ferredoxin from Clostridium pasteurianum; Brereton PS et al.; Eleven mutant forms of the ferredoxin from Clostridium pasteurianum (CpFd; 2 Fe4S4; 6200 Da) have been isolated in which six surface carboxylates are changed systematically to their uncharged but stereochemically equivalent carboxamide analogues . Such changes provide molecules which vary in overall charge and its surface distribution but vary minimally in structure and reduction potential . Glu-17 and Asp-6, -27, -33, -35, and -39 were converted providing six single mutants, four double mutants and one triple mutant . The proteins were characterised by UV-visible spectroscopy, square-wave voltammetry and 1H NMR . Their ability to mediate electron transfer between spinach NADH:ferredoxin oxidoreductase and horse heart cytochrome c was assessed . Each mutant is 30-100% as active as the recombinant protein with the triple mutant D33,35,39N being least active . Second-order rate constants k2 for the oxidation of reduced mutant ferredoxins by {Co(NH3)6}3+ were measured at 25 degrees C and I = 0.1 M by stopped-flow techniques . Each mutant displayed saturation kinetics with k2 being 30-100% of that for the recombinant protein . The rates were moderately sensitive to ionic strength . Variation in association constant K could not be detected within the confidence limits of the data . Overall the effects of the mutations were minor . In contrast to human and Anabaena 7120 {Fe2S2}-ferredoxins, electron transfer does not appear to rely on the presence of one or two specific surface carboxylate residues . It may occur from multiple sites on the surface of CpFd with recognition processes for its many physiological redox partners being controlled by relative reduction potentials, in addition to unidentified criteria . The conclusions are consistent with previous results for another series of mutant CpFd proteins interacting with physiological redox partners pyruvate: Fd oxidoreductase and hydrogenase (J.M . Moulis, V . Davasse (1995) Biochemistry 34, 16781-16788). Mol Gen Mikrobiol Virusol, 1998, (4), 22 - 8 {Extrachromosomal genetic elements of Clostridium botulinum . II . Isolation and analysis of DNA from bacteriophages of Clostridium botulinum types C and A}; Rudoi BA et al.; The DNAs of bacteriophage c-st, known to realize the lysogenic conversion of toxinogenicity among C . botulinum types C and D strains, and the nucleic acid of a virulent mutant of bacteriophage CB propagated in type A C . botulinum cells were purified and examined . Heterogeneity of phage c-st preparations was observed during purification, manifesting by formation of several bands in isopiknic CsCl gradient during centrifuging . An extra nucleic acid fraction was detected in some DNA preparations of phage c-st; the origin of this fraction is discussed . Plasmid extrachromosomal elements were for the first time found in the cells of nontoxigenic type C C . botulinum A02 strain, known as the indicator for c-st phage . The sensitivity of phage c-st DNA to 25 restriction endonucleases was examined . Analysis of the results of restriction analysis of c-st and CB phage DNAs and plasmid nucleic acids, revealed earlier in type A C . botulinum strains, disclosed several DNA modification enzymes with different recognition sites in type C C . botulinum . At least two of these activities are not found in type A strains . According to restriction analysis, total size of phage c-st DNA is about 160 kbp and of phage CB DNA 35 kbp . Individual EcoRI and HindIII restricts of phage c-st DNA, containing the initial site of botulinum toxin CI gene, were recognized by radioisotope labeled oligonucleotide probe Enzyme immunoassay revealed slight expression of the N-terminal region of bntc I gene in E . coli recombinant variants . These data can be used in further investigation of C . botulinum genetics. Am J Physiol, 1999 Feb, 276(2 Pt 1), G485 - 90 Participation of reactive oxygen metabolites in Clostridium difficile toxin A-induced enteritis in rats; Qiu B et al.; Reactive oxygen metabolites (ROMs) contribute to the pathophysiology of intestinal inflammation . Our aim was to ascertain the involvement of ROMs in experimental ileitis in rats produced by toxin A of Clostridium difficile . Intraluminal toxin A caused a significant increase in hydroxyl radical and hydrogen peroxide production by ileal microsomes starting 1 h following toxin exposure and peaking at 2-3 h, and this was inhibited by pretreatment with DMSO, a ROM scavenger, or superoxide dismutase (SOD), which inactivates ROMs . In contrast, mucosal xanthine oxidase increased only slightly after toxin A exposure, and allopurinol, an inhibitor of xanthine oxidase, had no effect on toxin A-associated intestinal responses . Induction of neutropenia resulted in reduction of toxin-mediated free radical formation, fluid secretion, and permeability . The enterotoxic effects of C . difficile toxin A were associated with increased ROM release in ileal tissues, and the ROM inhibitors DMSO and SOD inhibited these effects . This suggests that ROMs released during toxin A enteritis are released primarily from neutrophils invading the inflamed bowel segment. AJR Am J Roentgenol, 1999 Feb, 172(2), 517 - 21 Atypical presentation of Clostridium difficile colitis in patients with cystic fibrosis; Binkovitz LA et al.; OBJECTIVE: This report describes the unusual presentation of Clostridium difficile colitis in five patients with cystic fibrosis and the role of CT in first suggesting the correct diagnosis in this group of patients . Because of the absence of watery diarrhea and the presence of abdominal bloating and decreased stooling, cystic fibrosis patients with C . difficile colitis will be treated for stool impaction, meconium ileus equivalent, or distal intestinal obstruction syndrome . CT of the abdomen, performed in these five patients because of their lack of improvement after standard therapy for stool impaction, showed an extensive pancolitis later confirmed to be caused by C . difficile infection . CONCLUSION: In patients with cystic fibrosis, imaging findings of a pancolitis should raise the possibility of C . difficile colitis despite the lack of watery diarrhea . Anticlostridial treatment can be initiated before bacteriologic confirmation is obtained. Rev Soc Bras Med Trop, 1999 Jan-Feb, 32(1), 47 - 52 {Clostridium difficile as an inducer of inflammatory diarrhea}; Rocha MF et al.; Clostridium difficile has been pointed out as an important agent of diarrheal diseases associated with antibiotic use . However, due to its complexity, the physiopathology of these diseases is only partially elucidated, although a series of scientific works has demonstrated the importance of toxins A and B in the pathogenesis of the inflammatory diarrhea induced by this microorganism . The inflammatory mechanisms involved in the biological activities of these toxins are complex . There are some studies demonstrating that toxin B has no enterotoxic activity in vivo . However, this toxin causes dose-dependent eletrophysiologic and morphologic modifications of human colonic mucosa in vitro . In addition, toxin B stimulates the synthesis of potent inflammatory mediators by monocytes and macrophages . The effects provoked by toxin A on the intestinal mucosa are quite evident and are characterized by intense fluid secretion and by inflammatory cell accumulation, such as macrophages, mast cells, lymphocytes and neutrophils, with the consequent release of mediators such as prostaglandins, leukotrienes, platelet activating factor, nitric oxide and cytokines. Infect Control Hosp Epidemiol, 1999 Jan, 20(1), 43 - 50 Recurrent Clostridium difficile disease: epidemiology and clinical characteristics; McFarland LV et al.; OBJECTIVE: To describe the epidemiology, diagnosis, risk factors, patient impact, and treatment strategies for recurrent Clostridium difficile-associated disease (CDAD) . DESIGN: Data were collected as part of a blinded, placebo-controlled clinical trial testing a new combination treatment for recurrent CDAD . Retrospective data regarding prior CDAD episodes were collected from interviews and medical-chart review . Prospective data on the current CDAD episode, risk factors, and recurrence rates were collected during a 2-month follow-up . SETTINGS: National referral study . PARTICIPANTS: Patients with recurrent CDAD . INTERVENTIONS: Treatment with a 10-day course of low-dose (500 mg/d) or high-dose (2 g/d) vancomycin or metronidazole (1 g/d) . RESULTS: Recurrent CDAD was found to have a lengthy course involving multiple episodes of diarrhea, abdominal cramping, nausea, and fever . CDAD may recur over several years despite frequent treatment with antibiotics . Recurrence rates were similar regardless of the choice or dose of antibiotic . Recurrent CDAD is not a trivial disease: patients may have multiple episodes (as many as 14), may require hospitalization, and the mean lifetime cost of direct medical care was $10,970 per patient . Fortunately, the disease does not become progressively more severe as the number of episodes increase . Two risk factors predictive for recurrent CDAD were found: increasing age and a decreased quality-of-life score at enrollment . CONCLUSIONS: Recurrent CDAD is a persistent disease that may result in prolonged hospital stays, additional medical costs, and rare serious complications. Acta Vet Scand, 1998, 39(4), 461 - 71 Comparison between effects of standard feed and whole wheat supplemented diet on experimental Eimeria tenella and Eimeria maxima infections in broiler chickens; Waldenstedt L et al.; The effects of experimental infections with Eimeria tenella (Experiment 1, n = 144) or E . maxima (Experiment 2, n = 216) in broiler chickens fed whole wheat, with or without access to grit, as compared to a standard pelleted feed were studied . Inclusion of whole wheat was gradually increased up to 30% at 3 weeks of age . Grit was given separately . The chickens were kept on litter in a parasite-free environment with free access to water and feed . At 3 weeks of age half the number of chickens were individually inoculated with 500 sporulated oocysts of E . tenella (Experiment 1) or 3,000 sporulated oocysts of Eimeria maxima (Experiment 2), and the remaining birds were kept separate as uninfected controls . Neither coccidiostats nor growth enhancers were used . Oocyst concentration was determined from each group separately . Intestinal lesions were scored on 6 birds per feed regime 7 d postinoculation, and on the remaining birds at slaughter . Diet had no significant effect or bird performance during infection . However, there was an indication that the E . maxima infection had more negative effect on weight gain in birds given standard feed than in those given whole wheat supplement, but the difference was not significant (p < 0.09) . The number of oocysts shed or mean intestinal lesion scores did not differ between diets in either experiment . In both experiments, the number of Clostridium perfringens was higher in the caeca of inoculated birds, but there were no differences between diets. Acta Vet Scand, 1998, 39(4), 433 - 41 Effect of antibiotic growth promoters and anticoccidials on growth of Clostridium perfringens in the caeca and on performance of broiler chickens; Elwinger K et al.; The effects of the growth promoters avoparcin and avilamycin and the ionophore anticoccidials maduramicin, narasin and monensin on the growth of Clostridium perfringens (Cp) in the caeca and on performance of broiler chickens were tested in 2 experiments . The supplements were fed as single feed additives or in some combinations . No clinical signs or lesions caused by coccidia were observed in any of the studies . All supplements had an antibacterial effect on Cp and improved growth rate significantly . Carcass yield of birds fed growth promoters avilamycin or avoparcin was significantly higher compared with birds fed anticoccidials . These data indicate that, what concerns bird performance, during good hygienic conditions supplementation with antibiotic growth promoters may not be necessary when the diet is supplemented with an anticoccidial with antibacterial effects. J Am Vet Med Assoc, 1999 Jan 15, 214(2), 229 - 32, 205 Association of Clostridium difficile with enterocolitis and lactose intolerance in a foal; Weese JS et al.; Diagnoses of Clostridium difficile enterocolitis and lactose intolerance were made in a neonatal foal with persistent diarrhea . It was determined that the foal had lactose intolerance on the basis of the results of a lactose tolerance test, and a diagnosis of C difficile enterocolitis was subsequently made . The foal responded to oral administration of metronidazole and lactase . Lactose intolerance is a secondary problem most commonly associated with rotavirus infection, but it can be caused by any condition affecting the small intestine . Because C difficile can affect the small intestine in foals, it was presumably the cause of the lactose intolerance in this foal with persistent diarrhea . Oral administration of lactase was not initially successful in this foal, most likely because of ongoing C difficile enterocolitis . Presumably, metronidazole was an effective treatment for C difficile enterocolitis and administration of lactase allowed for normal digestion of milk until endogenous lactose production returned . Clostridium difficile enterocolitis and lactose intolerance should be considered as differential diagnoses in neonatal foals with diarrhea, especially when the foal is bright and alert. Appl Environ Microbiol, 1999 Feb, 65(2), 499 - 505 Effect of acetate on molecular and physiological aspects of clostridium beijerinckii NCIMB 8052 solvent production and strain degeneration; Chen CK et al.; The addition of sodium acetate to chemically defined MP2 medium was found to increase and stabilize solvent production and also increase glucose utilization by Clostridium beijerinckii NCIMB 8052 . RNA and enzyme analyses indicated that coenzyme A (CoA) transferase was highly expressed and has higher activity in C . beijerinckii NCIMB 8052 grown in MP2 medium containing added sodium acetate than in the microorganism grown without sodium acetate . RNA analysis suggested the existence of a sol operon and confirmed the presence of a ptb-buk operon in C . beijerinckii NCIMB 8052 . In addition to CoA transferase, C . beijerinckii NCIMB 8052 grown in MP2 medium containing added acetate demonstrated higher acetate kinase- and butyrate kinase-specific activity than when the culture was grown in MP2 medium containing no added acetate . Southern blot analysis with chromosomal DNA isolated from solventogenic and degenerated C . beijerinckii NCIMB 8052 indicated that C . beijerinckii NCIMB 8052 strain degeneration does not involve loss of the CoA transferase genes . The addition of acetate to MP2 medium may induce the expression of the sol operon, which ensures solvent production and prevents strain degeneration in C . beijerinckii NCIMB 8052. Appl Environ Microbiol, 1999 Feb, 65(2), 659 - 64 Characterization of fatty acid composition, spore germination, and thermal resistance in a nisin-resistant mutant of Clostridium botulinum 169B and in the wild-type strain; Mazzotta AS et al.; The membrane fatty acids, thermal resistance, and germination of a nisin-resistant (Nisr) mutant of Clostridium botulinum 169B were compared with those of the wild-type (WT) strain . In the membranes of WT cells, almost 50% of the total fatty acids were unsaturated, but in those of Nisr cells, only 23% of the fatty acids were unsaturated . WT and Nisr spores contained similar amounts (approximately 23%) of unsaturated fatty acids, but the saturated straight-chain/branched-chain ratio was significantly higher in Nisr spores than in WT spores . These fatty acid differences suggest that Nisr cell and spore membranes may be more rigid, a characteristic which would interfere with the pore-forming ability of nisin . Nisr C . botulinum did not produce an extracellular nisin-degrading enzyme, nor were there any differences in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis patterns of coat proteins extracted from WT and Nisr spores, eliminating these as possible reasons for nisin resistance . Nisr spores had thermal resistance parameters similar to those of WT spores . In WT spores, but not in Nisr spores, nisin caused a 40% reduction in thermal resistance and a twofold increase in the germination rate . Because the nisin-induced increase in the germination rate of WT spores occurred only in the presence of a germinant (a molecule that triggers germination), nisin can be classified as a progerminant (a molecule that stimulates germination only in the presence of a germinant). Eur J Clin Microbiol Infect Dis, 1998 Nov, 17(11), 788 - 90 Multicentre evaluation of a commercial test for the rapid diagnosis of Clostridium difficile-mediated antibiotic-associated diarrhoea; Bentley AH et al.; An immunoassay for t