Clostridia

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 the detection of Clostridium difficile toxin A in stool samples (Clearview C . DIFF A; Unipath, UK) was evaluated against the cell cytotoxicity assay using 407 stool samples from patients suspected to have, or considered at risk of, antibiotic-associated diarrhoea . Of the samples tested, 98 were positive and 280 were negative by both tests (sensitivity 83.1%, specificity 96.9%) . Following resolution of the 29 discrepant results, the sensitivity and specificity of the immunoassay were 91% and 98%, respectively, and the sensitivity for the cell cytotoxicity assay was calculated as 91.5%, with a specificity of 99% . The Clearview C . DIFF A test proved to be a rapid simple assay for the detection of Clostridium difficile toxin A in stool samples . The test was equally suited to single or batch testing, required minimal sample handling, and provided results within 30 min of applying the sample to the test unit.

Rev Rhum Engl Ed, 1998 Dec, 65(12), 795 - 8
Reactive oligoarthritis in a patient with Clostridium difficile pseudomembranous colitis . Review of the literature; Veillard E et al.; A 57-year-old man developed oligoarthritis of the right sacroiliac joint, knee and elbow in the wake of Clostridium difficile pseudomembranous colitis . He was HLA B27-positive and had a history of Reiter's syndrome . His joint manifestations resolved after a course of nonsteroidal antiinflammatory drug therapy and injection of the right knee with triamcinolone acetonide . Clostridium difficile should be recognized as a rare cause of reactive arthritis.

J Bacteriol, 1999 Feb, 181(3), 923 - 33
Gene duplication and multiplicity of collagenases in Clostridium histolyticum; Matsushita O et al.; Clostridium histolyticum collagenase contains a number of different active components . Previously we have shown that colH encodes a 116-kDa collagenase (ColH) and a 98-kDa gelatinase . We purified a different 116-kDa collagenase (ColG) from the culture supernatant and sequenced its gene (colG) . We also identified four other gelatinases (105, 82, 78, and 67 kDa) and determined their N-terminal amino acid sequences, all of which coincided with that of either ColG or ColH . Hybridization experiments showed that each gene is present in a single copy and each gene is transcribed into a single mRNA . These results suggest that all the gelatinases are produced from the respective full-length collagenase by the proteolytic removal of C-terminal fragments . The substrate specificities of the enzymes suggest that colG and colH encode class I and class II enzymes, respectively . Analysis of their DNA locations by pulsed-field gel electrophoresis and nucleotide sequencing of their surrounding regions revealed that the two genes are located in different sites on the chromosome . C . histolyticum colG is more similar to C . perfringens colA than to colH in terms of domain structure . Both colG and colA have a homologous gene, mscL, at their 3' ends . These results suggest that gene duplication and segment duplication have occurred in an ancestor cell common to C . histolyticum and C . perfringens and that further divergence of the parent gene produced colG and colA.

J Bacteriol, 1999 Feb, 181(3), 781 - 90
A four-dimensional view of assembly of a morphogenetic protein during sporulation in Bacillus subtilis; Price KD et al.; We report the use of a fusion to the green fluorescent protein to visualize the assembly of the morphogenetic protein SpoIVA around the developing forespore during the process of sporulation in the bacterium Bacillus subtilis . Using a deconvolution algorithm to process digitally-collected optical sections, we show that SpoIVA, which is synthesized in the mother cell chamber of the sporangium, assembled into a spherical shell around the outer surface of the forespore . Time-lapse fluorescence microscopy showed that this assembly process commenced at the time of polar division and seemed to continue after engulfment of the forespore was complete . SpoIVA remained present throughout the late stages of morphogenesis and was present as a component of the fully mature spore . Evidence indicates that assembly of SpoIVA depended on the extreme C-terminal region of the protein and an additional region that directly or indirectly facilitated interaction among SpoIVA molecules . The N- and C-terminal regions of SpoIVA, including the extreme C terminus, are highly similar to the corresponding regions of the homologous protein from the distantly related endospore-forming bacterium Clostridium acetobutylicum, attesting to their importance in the function of the protein . Finally, we show that proper localization of SpoIVA required the expression of one or more genes which, like spoIVA, are under the control of the mother cell transcription factor sigmaE . One such gene was spoVM, whose product was required for efficient targeting of SpoIVA to the outer surface of the forespore.

J Bacteriol, 1999 Feb, 181(3), 718 - 25
Purification and properties of NADH-dependent 5, 10-methylenetetrahydrofolate reductase (MetF) from Escherichia coli; Sheppard CA et al.; A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity . A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme . The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD) . No additional cofactors or metals have been detected . The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant . Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1 . NADPH also serves as a reductant, but exhibits a much higher Km . MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor . The properties of MetF from E . coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 281 - 6
Evidence that Tn5565, which includes the enterotoxin gene in Clostridium perfringens, can have a circular form which may be a transposition intermediate; Brynestad S et al.; The Clostridium perfringens enterotoxin gene is on a transposon-like element, Tn5565, integrated in the chromosome in human food poisoning strains . The flanking IS elements, IS1470 A and B, are related to IS30 . The IS element found in the transposon, IS1469, is related to IS200 and has been found upstream of cpe in all Type A strains . PCR and sequencing studies from cell extracts and plasmid isolations of C . perfringens indicate that Tn5565 can form a circular form with the tandem repeat (IS1470)2, similar to the transposition intermediates described for a number of IS elements.

FEMS Microbiol Lett, 1999 Jan 1, 170(1), 119 - 23
Functional characterisation of the chaperones DnaK, DnaJ, and GrpE from Clostridium acetobutylicum; Rungeling E et al.; The DnaK chaperone system is involved in various cellular processes such as the control of the folded and oligomeric state of proteins under stress and non-stress conditions . In this study we functionally characterised the homologues of the DnaK system from Clostridium acetobutylicum DnaK, DnaJ, GrpE and OrfA were heterologously synthesised in Escherichia coli and affinity purified via a His-tag . By optimising the stoichiometry, we were able to refold guanidinium hydrochloride-denatured firefly luciferase in vitro with 22% of the yield obtained with the E . coli DnaK system . In addition, C . acetobutylicum DnaJ could stimulate the E . coli DnaK ATPase by a factor of 55 . Furthermore, the DnaK system from C . acetobutylicum was able to prevent the aggregation of OrfA from C . acetobutylicum, which is similar to the repressor HrcA of CIRCE-regulated heat shock genes in Bacillus subtilis.

Dev Biol, 1999 Jan 15, 205(2), 322 - 31
A role for Rho-like GTPases in the polarisation of mouse eight-cell blastomeres; Clayton L et al.; Polarisation of cells during mouse preimplantation development first occurs within blastomeres at the eight-cell stage, as part of a process called compaction . Cell-cell contact mediated by the cell adhesion molecule uvomorulin (E-cadherin) and the activity of the microfilament cytoskeleton are important in the development of compaction, which is crucial for establishment of trophoblast and pluriblast (inner cell mass) lineages and for subsequent development . Members of the Rho family of p21 GTPases have been shown to regulate the organisation of the actin cytoskeleton and adhesion in other cell types . The potential role of these proteins in compaction was investigated . Inhibition of Rho with Clostridium botulinum C3-transferase disturbed intercellular flattening at compaction and prevented cytocortical microfilament polarisation of eight-cell blastomeres, in contrast to cytochalasin D which inhibited only adhesion . Microinjection of a constitutively activated recombinant Rho protein into four-cell blastomeres induced cortical microfilament disruption and apical displacement of nuclei associated with polarised clustering of microtubules . Interblastomere adhesion was reduced and E-cadherin was aberrently clustered at remaining cell-cell contacts . Similarly, activated Cdc42 protein induced nuclear displacement with additional cytoplasmic actin bundle formation between nucleus and cell-cell contacts . The effects produced by both of the activated GTPase proteins are indicative of prematurely induced but aberrently organised polarity . These results suggest that Rho family GTPases are involved in the polarisation of early mouse blastomeres .

Infect Immun, 1999 Feb, 67(2), 527 - 38
Serum antitoxin antibodies mediate systemic and mucosal protection from Clostridium difficile disease in hamsters; Giannasca PJ et al.; Clostridium difficile is the bacterial pathogen identified as the cause of pseudomembranous colitis and is principally responsible for nosocomial antibiotic-associated diarrhea and colitis . The pathologic findings associated with this infection are believed to be caused by two large (approximately 300-kDa) exotoxins, toxins A and B . Because of the mucosal nature of this infection, vaccination strategies aimed at providing prophylactic or therapeutic immune protection have included immunization by mucosal routes . Using the hamster model of C . difficile infection, we examined the protective efficacy of inactivated toxin (toxoid) vaccine formulations prepared as either culture filtrate or partially purified toxoid . We compared combination parenteral and mucosal vaccination regimens involving intranasal, intragastric, or rectal routes of immunization and found that rectal immunization in conjunction with intramuscular (i.m.) vaccination provided full protection of hamsters from death and diarrhea while the other mucosal routes did not . Protection was associated with high levels of toxin-neutralizing antibodies in serum . The requirement for adjuvants for protection was assessed by using sequential i.m . and rectal or i.m . vaccination regimens . Unexpectedly, i.m . immunization without adjuvant conferred the highest protection from death and diarrhea; this regimen elicited the highest serum anti-toxin B titers as well as toxin B neutralizing titers . Passive transfer of mouse antitoxin antibodies protected hamsters in a dose-dependent manner, demonstrating the principal role of circulating antitoxin antibodies in immunity from this toxin-mediated mucosal disease . These results suggest that prophylactic parenteral vaccination or intravenous immunotherapy could provide protection from C . difficile disease in humans.

Int J Antimicrob Agents, 1998 Nov, 10(4), 321 - 4
Comparative anti-anaerobic activity of Men 10700, a penem antibiotic; Hamilton-Miller JM et al.; The in vitro activity of Men 10700, a new penem, has been compared with that of metronidazole, clindamycin, ciprofloxacin, co-amoxiclav, imipenem and three third generation cephalosporins against 120 strains of anaerobes . The organisms tested comprised Clostridium perfringens, Clostridium difficile, Bacteroides fragilis and speciated members of the genera Fusobacterium, Veillonella and Peptostreptococcus . Men 10700 showed activity similar to that of imipenem, and was more potent than metronidazole against all species except C . difficile and P . anaerobius . The spectrum of activity of Men 10700 suggests this agent may be useful for treating infections caused by anaerobes.

Zentralbl Veterinarmed B, 1998 Dec, 45(10), 595 - 602
Strain differentiation of Clostridium perfringens by bacteriocin typing, plasmid profiling and ribotyping; Schalch B et al.; Bacteriocin typing, plasmid profiling and ribotyping were used to type 34 food and patient Clostridium perfringens isolates from 10 food poisoning cases, respectively, outbreaks . In nine cases/outbreaks bacteriocin patterns showed identical main groups . Subgroups differed within all cases/outbreaks . Plasmid profiles were identical for all isolates within each of three outbreaks . In eight food poisoning cases and outbreaks, all the ribotypes of each food and stool isolate were found to be identical . All three typing methods give valuable results for the characterization of C . perfringens beyond the species level . Bacteriocin typing represents a suitable addition to plasmid typing, particularly since the results do not show any correlation between losses of plasmids and changes in bacteriocin sensitivity patterns . Ribotyping was found to be a suitable tool to determine the genetic relationship of C . perfringens isolates in the context of food-borne poisoning.

Science, 1999 Jan 22, 283(5401), 537 - 40
A tobacco syntaxin with a role in hormonal control of guard cell ion channels; Leyman B et al.; The plant hormone abscisic acid (ABA) regulates potassium and chloride ion channels at the plasma membrane of guard cells, leading to stomatal closure that reduces transpirational water loss from the leaf . The tobacco Nt-SYR1 gene encodes a syntaxin that is associated with the plasma membrane . Syntaxins and related SNARE proteins aid intracellular vesicle trafficking, fusion, and secretion . Disrupting Nt-Syr1 function by cleavage with Clostridium botulinum type C toxin or competition with a soluble fragment of Nt-Syr1 prevents potassium and chloride ion channel response to ABA in guard cells and implicates Nt-Syr1 in an ABA-signaling cascade.

Pediatr Surg Int, 1999, 15(1), 75 - 6
Thermography of Clostridium perfringens infection in childhood; Saxena AK et al.; Gas gangrene is not a frequently encountered toxic wound infection in childhood . We present a case of postoperative Clostridium perfringens infection with proximal forearm myonecrosis . In order to reveal the full extent of tissue viability in the right upper extremity, infrared thermography was performed . Although dyschromia was evident in the proximal forearm, thermographs revealed viable tissue only up to the supracondylar region . Angiography, which provided valuable clues to the patency of the vascular supply, and subsequent intraoperative findings confirmed the extent of tissue perfusion as revealed by infrared thermography.

Gut, 1999 Feb, 44(2), 212 - 7
Bovine immunoglobulin concentrate-clostridium difficile retains C difficile toxin neutralising activity after passage through the human stomach and small intestine; Warny M et al.; BACKGROUND: Bovine immunoglobulin concentrate (BIC)-Clostridium difficile is prepared from the colostrum of cows immunised against C difficile toxins and contains high concentrations of neutralising IgG antitoxin . AIMS: To determine the proportion of BIC-C difficile which survives passage through the human stomach and small intestine . METHODS: Six volunteers with an end ileostomy took 5 g of BIC-C difficile containing 2.1 g of bovine IgG on four occasions: alone, with an antacid, during treatment with omeprazole, and within enteric coated capsules . RESULTS: When BIC-C difficile was taken alone, a mean (SEM) of 1033 (232) mg of bovine IgG was recovered in the ileal fluid representing 49% of the total ingested dose . Bovine IgG recovery was not significantly increased by antacid (636 (129) mg) or omeprazole (1052 (268) mg) . The enteric capsules frequently remained intact or only partially opened in the ileal effluent and free bovine IgG levels were low in this treatment group (89 (101) mg) . Bovine IgG recovery was higher in volunteers with shorter (less than two hours) mouth to ileum transit times (68% versus 36%, p<0 . 05) . Specific bovine IgG against C difficile toxin A was detected in ileal fluid following oral BIC . Toxin neutralising activity was also present and correlated closely with bovine IgG levels (r=0.95, p<0 . 001) . Conclusion: BIC-C difficile resists digestion in the human upper gastrointestinal tract and specific anti-C difficile toxin A binding and neutralising activity was retained . Passive oral immunotherapy with anti-C difficile BIC may be a useful non-antibiotic approach to the prevention and treatment of C difficile antibiotic associated diarrhoea and colitis.

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 511 - 6
Claudin multigene family encoding four-transmembrane domain protein components of tight junction strands; Morita K et al.; Two related integral membrane proteins, claudin-1 and -2, recently were identified as novel components of tight junction (TJ) strands . Here, we report six more claudin-like proteins, indicating the existence of a claudin gene family . Three of these were reported previously as RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF, but their physiological functions were not determined . Through similarity searches followed by PCR, we isolated full length cDNAs of mouse RVP1, Clostridium perfringens enterotoxin receptor, and TMVCF as well as three mouse claudin-like proteins and designated them as claudin-3 to -8, respectively . All of these claudin family members showed similar patterns on hydrophilicity plots, which predicted four transmembrane domains in each molecule . Northern blotting showed that the tissue distribution pattern varied significantly, depending on claudin species . Similarly to claudin-1 and -2, when these claudins were HA-tagged and introduced into cultured Madin-Darby canine kidney cells, all showed a tendency to concentrate at TJs . Immunofluorescence and immunoelectron microscopy with polyclonal antibodies specific for claudin-3, -4, or -8 revealed that these molecules were exclusively concentrated at TJs in the liver and/or kidney . These findings indicated that multiple claudin family members are involved in the formation of TJ strands in various tissues.

Annu Rev Microbiol, 1998, 52, 333 - 60
Virulence genes of Clostridium perfringens; Rood JI; Clostridium perfringens causes human gas gangrene and food poisoning as well as several enterotoxemic diseases of animals . The organism is characterized by its ability to produce numerous extracellular toxins including alpha-toxin or phospholipase C, theta-toxin or perfringolysin O, kappa-toxin or collagenase, as well as a sporulation-associated enterotoxin . Although the genes encoding the alpha-toxin and theta-toxin are located on the chromosome, the genes encoding many of the other extracellular toxins are located on large plasmids . The enterotoxin gene can be either chromosomal or plasmid determined . Several of these toxin genes are associated with insertion sequences . The production of many of the extracellular toxins is regulated at the transcriptional level by the products of the virR and virS genes, which together comprise a two-component signal transduction system.

Tidsskr Nor Laegeforen, 1998 Nov 20, 118(28), 4357 - 9
{Wound botulism in heroin addiction}; Holmaas G et al.; Botulism is a rare disease which usually is caused by preformed botulinum toxin in food . However, this article describes a case of wound botulism in a 29-year-old male heroin addict who developed progressive diplopia, dysphagia and proximal weakness of skeletal limb muscles . He needed mechanical ventilation for two weeks . The clinical diagnosis of botulism was supported by neurophysiological tests . Assays for detection of botulinum toxin and Clostridium botulinum were negative . The patient had not eaten any contaminated food the last two weeks before symptoms appeared, but he had multiple contaminated skin wounds . After treatment with botulinum antitoxins and antibiotics he gradually recovered, and six weeks later he was discharged from hospital in good condition . To the best of our knowledge this is the first case of wound botulism reported in Norway.

Tidsskr Nor Laegeforen, 1998 Nov 20, 118(28), 4355 - 6
{Botulism in newborn infants}; Tollofsrud PA et al.; Infant botulism, first described in 1976, is the most common form of botulism . The majority of cases are reported from the USA . The disease is rare in Europe, and this article describes the first patient reported in Norway . A three-month-old boy of Norwegian origin who had been fed Argentinian honey developed symptoms of botulism . Electromyography showed presynaptic neuromuscular dysfunction . The diagnosis was confirmed by the demonstration of Clostridium botulinum type A neurotoxin in the faeces . After supportive treatment, breast-milk feeding and lactobacillus supplementation he made a complete recovery . If spores of C . botulinum are ingested, they can bind to the epithelium, germinate and produce toxin which causes botulism . Because of the composition of their intestinal flora, children below one year of age are at risk . Ingestion of honey is a well known risk factor . Contamination of Norwegian honey has never been reported but we recommend that honey should not be given to children during their first year of life.

J Clin Microbiol, 1999 Feb, 37(2), 461 - 3
PCR targeted to the 16S-23S rRNA gene intergenic spacer region of Clostridium difficile and construction of a library consisting of 116 different PCR ribotypes; Stubbs SL et al.; A reference library of types of Clostridium difficile has been constructed by PCR ribotyping isolates (n = 2,030) from environmental (n = 89), hospital (n = 1,386), community practitioner (n = 395), veterinary (n = 27), and reference (n = 133) sources . The library consists of 116 distinct types identified on the basis of differences in profiles generated with PCR primers designed to amplify the 16S-23S rRNA gene intergenic spacer region . Isolates from 55% of infections in hospitals in the United Kingdom belonged to one ribotype (type 1), but this type was responsible for only 7 . 5% of community infections.

Gynecol Oncol, 1999 Jan, 72(1), 116 - 9
Clostridium septicum myonecrosis and ovarian cancer: a case report and review of literature; Prinssen HM et al.; A case report of lethal distant myonecrosis with gas gangrene is presented . Cultures from blood and suppuration of our patient revealed Clostridium septicum . At obduction the patient appeared to have a necrotic metastasis of a known ovarian carcinoma in the cecum wall . Predisposing conditions for this type of infection are hematologic malignancies, colon carcinoma, neutropenia, diabetes mellitus, and disruption of the bowel mucosa . Clostridium septicum is highly associated with the presence of a malignancy, either known or occult at the time infection occurs . Occult tumors are mostly situated in the cecal area of the bowel . Clinical symptoms of the syndrome and therapeutic options are discussed .

Gynecol Oncol . 1998 Dec;71(3):481.
Papers to appear in forthcoming issues
Exogenously acquired Clostridium septicum gas gangrene--a case report.
Berufsgenossenschaftliche Unfallklinik Tuebingen, Department of Trauma Surgery, University of TubingenWe report on a 72-year-old woman suffering from gas gangrene of the right leg, which developed 3 days after getting injured while gardening . Due to rapid microbiological diagnosis-direct microscopical examination of muscle biopsies revealed numerous grampositive rods-, immediately performed amputation of the leg and antibiotic treatment with penicillin and metronidazole the patient survived . From the muscle biopsies Clostridium septicum, which is found ubiquitously, was isolated in pure culture.

Br J Pharmacol, 1998 Dec, 125(8), 1651 - 60
Tyrosine phosphorylation as a convergent pathway of heterotrimeric G protein- and rho protein-mediated Ca2+ sensitization of smooth muscle of rabbit mesenteric artery; Sasaki M et al.; 1 . The aim of this study was to determine whether different signal transduction mechanisms underlie the Ca2+ sensitizing effects of guanosine 5'-O-(3-thiotriphosphate) (GTP(gamma)S) and receptor agonists on beta-escin-skinned smooth muscle of rabbit mesenteric artery . 2 . In the homogenate of the beta-escin-skinned arterial strip, C3 exoenzyme of Clostridium botulinum catalyzed the {32P}-ADP-ribosylation of only one protein that had the same molecular mass as the protein detected in Western blots with anti-rho p21 antibody . Pretreatment of preparations with C3 resulted in great inhibition of GTP(gamma)S-induced Ca2+ sensitization, although the effect of GTP(gamma)S at higher concentrations (> or = 30 microM) was not completely blocked by this treatment . In contrast, the enhancement by phenylephrine and histamine, in the presence of guanosine 5'-triphosphate, of the Ca2+-induced contraction was not affected by C3 pretreatment . 3 . The protein kinase C (PKC) inhibitors calphostin C and staurosporine completely eliminated the enhancement by phorbol ester 12,13-dibutyrate of the Ca2+-induced contraction . However, these PKC inhibitors had no effect on GTP(gamma)S- and receptor agonist-induced Ca2+ sensitization . 4 . The tyrosine kinase inhibitors genistein and tyrphostin 25 caused an irreversible and complete block of the enhancement by GTP(gamma)S of the Ca2+-induced contraction without affecting this Ca2+ contraction . The inactive genistein analogue daidzein did not modify the effect of GTP(gamma)S . The Ca2+ sensitizing effects of phenylephrine and histamine were also blocked by these tyrosine kinase inhibitors . 5 . These results suggest that rho p21 predominantly mediates GTP(gamma)S-induced Ca2+ sensitization of beta-escin-skinned smooth muscle of rabbit mesenteric artery, while the Ca2+ sensitizing actions of heterotrimeric G protein-coupled receptor agonists do not involve this small G protein . However, it seems that tyrosine phosphorylation, but not PKC activation, plays an important role in both of the rho p21 protein- and heterotrimeric G protein-mediated Ca2+ sensitization mechanisms.

Dev Biol, 1998 Dec 15, 204(2), 592 - 602
Inhibition of small G proteins by clostridium sordellii lethal toxin activates cdc2 and MAP kinase in Xenopus oocytes; Rime H et al.; The lethal toxin (LT) from Clostridium sordellii is a glucosyltransferase that modifies and inhibits small G proteins of the Ras family, Ras and Rap, as well as Rac proteins . LT induces cdc2 kinase activation and germinal vesicle breakdown (GVBD) when microinjected into full-grown Xenopus oocytes . Toxin B from Clostridium difficile, that glucosylates and inactivates Rac proteins, does not induce cdc2 activation, indicating that proteins of the Ras family, Ras and/or Rap, negatively regulate cdc2 kinase activation in Xenopus oocyte . In oocyte extracts, LT catalyzes the incorporation of {14C}glucose into a group of proteins of 23 kDa and into one protein of 27 kDa . The 23-kDa proteins are recognized by anti-Rap1 and anti-Rap2 antibodies, whereas the 27-kDa protein is recognized by several anti-Ras antibodies and probably corresponds to K-Ras . Microinjection of LT into oocytes together with UDP-{14C}glucose results in a glucosylation pattern similar to the in vitro glucosylation, indicating that the 23- and 27-kDa proteins are in vivo substrates of LT . In vivo time-course analysis reveals that the 27-kDa protein glucosylation is completed within 2 h, well before cdc2 kinase activation, whereas the 23-kDa proteins are partially glucosylated at GVBD . This observation suggests that the 27-kDa Ras protein could be the in vivo target of LT allowing cdc2 kinase activation . Interestingly, inactivation of Ras proteins does not prevent the phosphorylation of c-Raf1 and the activation of MAP kinase that occurs normally around GVBD .

Burns, 1998 Nov, 24(7), 676 - 9
Silver sulfadiazine induced Clostridium difficile toxic megacolon in a burn patient: case report; Jennings LJ et al.; A 53 yr old diabetic male presented with a 34% total body surface area (TBSA) deep partial- and full-thickness burns . On post burn days 4 and 9, all of his burns were excised and grafted . Although he had only been treated with topical antibiotics, he developed Clostridium difficile colitis after his second surgery that progressed to Toxic Megacolon and perforation . The incidence and treatment of Toxic Megacolon secondary to C . difficile is reviewed . Early diagnosis and treatment with colonoscopic decompression may obviate the need for surgery.

Aliment Pharmacol Ther, 1998 Dec, 12(12), 1217 - 23
Prospective study of the risk of Clostridium difficile diarrhoea in elderly patients following treatment with cefotaxime or piperacillin-tazobactam; Settle CD et al.; BACKGROUND: Rates of Clostridium difficile diarrhoea have recently been rising, with the elderly being at highest risk . AIM: To compare the incidence of C . difficile colonization and diarrhoea in elderly patients treated for presumed infection with either empirical cefotaxime (CTX) or piperacillin-tazobactam (PT) . METHODS: A prospective, ward-based, crossover study was carried out on two well-matched care of the elderly wards at a UK tertiary care hospital, in patients requiring empirical broad-spectrum antibiotic treatment . RESULTS: There was a highly significant increased incidence of C . difficile colonization (26/34 vs . 3/14, P=0.001) and diarrhoea (18/34 vs . 1/14, P=0.006) in patients who received CTX as opposed to PT . DNA fingerprinting suggested that most infections arose from strains acquired from the hospital environment . CONCLUSIONS: Elderly patients are significantly less likely to develop C . difficile diarrhoea after treatment with PT than after CTX . The source of C . difficile appears to be predominantly from the ward environment.

Med Hypotheses, 1998 Aug, 51(2), 133 - 44
Autism and Clostridium tetani; Bolte ER; Autism is a severe developmental disability believed to have multiple etiologies . This paper outlines the possibility of a subacute, chronic tetanus infection of the intestinal tract as the underlying cause for symptoms of autism observed in some individuals . A significant percentage of individuals with autism have a history of extensive antibiotic use . Oral antibiotics significantly disrupt protective intestinal microbiota, creating a favorable environment for colonization by opportunistic pathogens . Clostridium tetani is an ubiquitous anaerobic bacillus that produces a potent neurotoxin . Intestinal colonization by C . tetani, and subsequent neurotoxin release, have been demonstrated in laboratory animals which were fed vegetative cells . The vagus nerve is capable of transporting tetanus neurotoxin (TeNT) and provides a route of ascent from the intestinal tract to the CNS . This route bypasses TeNT's normal preferential binding sites in the spinal cord, and therefore the symptoms of a typical tetanus infection are not evident . Once in the brain, TeNT disrupts the release of neurotransmitters by the proteolytic cleavage of synaptobrevin, a synaptic vesicle membrane protein . This inhibition of neurotransmitter release would explain a wide variety of behavioral deficits apparent in autism . Lab animals injected in the brain with TeNT have exhibited many of these behaviors . Some children with autism have also shown a significant reduction in stereotyped behaviors when treated with antimicrobials effective against intestinal clostridia . When viewed as sequelae to a subacute, chronic tetanus infection, many of the puzzling abnormalities of autism have a logical basis . A review of atypical tetanus cases, and strategies to test the validity of this paper's hypothesis, are included.

Microbios, 1998, 95(380), 7 - 13
Detection of cytotoxic effects of Clostridium novyi type A on bovine kidney cells; Kanoe M et al.; The cytotoxic effects of a toxic preparation from Clostridium novyi type A were demonstrated on tissue-cultured bovine kidney cells . The cytotoxic response was dose-dependent and could be neutralized by homologous antiserum . Scanning electron microscopy revealed damaged kidney cell surfaces . These findings indicated that the cytotoxicity may contribute to the formation of the foci in bovine tissue during an infection with C . novyi.

Berl Munch Tierarztl Wochenschr, 1998 Nov-Dec, 111(11-12), 418 - 21
{Experimental investigations on the effect of Escherichia coli and Clostridium perfringens on the steroid 4-pregnen-20beta-ol-3-one}; Schlenker G et al.; In the context of investigations about the stability of sexual steroids, which are excreted from animals to the environment, it was checked whether microorganisms of the intestinal microflora influence the degradation of these compounds . The germs used were Escherichia coli or Clostridium perfringens . The hormone investigated was the 4-pregnene-20 beta-ol-3-one (20 beta P4) . 20 beta P4 was incubated together with the microbes (experimental groups) and without these (control groups) in nutrient medium for the bacteria over 48 hours at 37 degrees C or 42 degrees C . As a result no differences in concentration were found between the experimental and control groups after incubation . Neither an effect of Escherichia coli nor one of Clostridium perfringens was detectable on the concentration of 20 beta P4 . However there was a significant decrease of the 20 beta P4-concentration after the incubation over 48 hours at 37 degrees C or 42 degrees C in both the experimental and control groups . That could indicate a thermal degradation of the compound tested.

J Med Microbiol, 1998 Feb, 47(2), 117 - 21
PCR-ribotyping and pyrolysis mass spectrometry fingerprinting of environmental and hospital isolates of Clostridium difficile; Al-Saif NM et al.; The relationships between environmental isolates of Clostridium difficile were examined by two typing methods, PCR ribotyping and pyrolysis mass spectrometry (PyMS) . The 184 isolates were divided into 23 different PCR ribotypes, 13 of which were producers of toxins A and B; the remaining 10 types did not produce either toxin A or B . PyMS analysis resolved 31 groups with 60 (32.5%) isolates in one group (group 9) . In both methods most of the isolates showed similar clustering . PCR ribotypes of the environmental isolates were compared with those of clinical isolates that had been typed previously . Seventeen PCR types (13 toxigenic PCR types and four non-toxigenic types) were found in both sets of isolates.

Prev Vet Med, 1998 Dec 1, 37(1-4), 173 - 83
Incidence risk and aetiology of mammary abnormalities in dry ewes in 10 flocks in southern Greece; Saratsis P et al.; In a field investigation of 10 flocks in Southern Greece, 3367 dairy ewes were examined twice, in order to estimate the incidence risk and the aetiology of mammary abnormalities during the dry-period . Abnormal secretion, lumps, nodules, diffuse hardness, abscesses and cysts were the abnormalities detected . The cumulative incidence of mammary abnormalities during the dry-period was 5.1% (95% confidence interval: 4.4-5.8%); 47% of the cases detected developed during the first three weeks after cessation of lactation . Despite variation in the flock size, there was no between-flock variation in the risk of a ewe developing mammary abnormalities . Staphylococci (Staphylococcus aureus and coagulase-negative isolates) were the most frequently isolated bacteria from mammary samples; Actinomyces pyogenes, Clostridium perfringens, streptococci and Escherichia coli were also isolated . Resistance was encountered among the staphylococcal isolates.

J Vet Med Sci, 1998 Dec, 60(12), 1357 - 9
Flow cytometric analysis for enterotoxin exposed on Clostridium perfringens spores; Kusunoki H et al.; Flow cytometric method (FCM) with fluorescent-labeled anti-CPE antibody was applied to develop a rapid, specific, and convenient method to detect enterotoxin (CPE) exposed on the surface of spores of Clostridium perfringens . The results obtained indicate that FCM can specifically detect CPE exposed on C . perfringens spores for a short time . Thus, FCM is found to be a rapid, specific, and convenient assay method for detection of CPE exposed on C . perfringens spores, suggesting that it will be hopefully useful to diagnose food poisoning caused by C . perfringens.

J Mol Biol, 1999 Jan 15, 285(2), 875 - 85
Insights into the mechanism of domain closure and substrate specificity of glutamate dehydrogenase from Clostridium symbiosum; Stillman TJ et al.; Comparisons of the structures of glutamate dehydrogenase (GluDH) and leucine dehydrogenase (LeuDH) have suggested that two substitutions, deep within the amino acid binding pockets of these homologous enzymes, from hydrophilic residues to hydrophobic ones are critical components of their differential substrate specificity . When one of these residues, K89, which hydrogen-bonds to the gamma-carboxyl group of the substrate l-glutamate in GluDH, was altered by site-directed mutagenesis to a leucine residue, the mutant enzyme showed increased substrate activity for methionine and norleucine but negligible activity with either glutamate or leucine . In order to understand the molecular basis of this shift in specificity we have determined the crystal structure of the K89L mutant of GluDH from Clostridium symbiosum . Analysis of the structure suggests that further subtle differences in the binding pocket prevent the mutant from using a branched hydrophobic substrate but permit the straight-chain amino acids to be used as substrates.The three-dimensional crystal structure of the GluDH from C . symbiosum has been previously determined in two distinct forms in the presence and absence of its substrate glutamate . A comparison of these two structures has revealed that the enzyme can adopt different conformations by flexing about the cleft between its two domains, providing a motion which is critical for orienting the partners involved in the hydride transfer reaction . It has previously been proposed that this conformational change is triggered by substrate binding . However, analysis of the K89L mutant shows that it adopts an almost identical conformation with that of the wild-type enzyme in the presence of substrate . Comparison of the mutant structure with both the wild-type open and closed forms has enabled us to separate conformational changes associated with substrate binding and domain motion and suggests that the domain closure may well be a property of the wild-type enzyme even in the absence of substrate .

Genomics, 1998 Dec 15, 54(3), 453 - 9
Genes for the CPE receptor (CPETR1) and the human homolog of RVP1 (CPETR2) are localized within the Williams-Beuren syndrome deletion; Paperna T et al.; Williams-Beuren syndrome (WBS) is a neurodevelopmental disorder affecting multiple systems . Haploinsufficiency of genes deleted in chromosomal region 7q11.23 is the likely cause for this syndrome . We now report the localization of the genes for the CPE-R (Clostridium perfringens enterotoxin receptor, CPETR1) and the human homolog of RVP1 (rat ventral prostate 1 protein, CPETR2), both previously mapped to 7q11, to the WBS critical region . A single nucleotide polymorphism (SNP) present in CPETR1 has been identified and was used to determine parental origin of the deleted allele in five informative families . The mouse homologs Cpetr1 and Cpetr2 were identified and mapped to the conserved syntenic region on mouse chromosome 5 . Northern blot analysis of CPETR1 demonstrates tissue specificity, with expression in kidney, lung, thyroid, and gastrointestinal tissues . In mouse, Cpetr1 is expressed in the early embryo, appears to be developmentally upregulated during gestation, and is present in adult tissues . Our results suggest a role for CPE-R in internal organ development and function during pre- and postnatal life .

Bioorg Med Chem Lett, 1998 Dec 1, 8(23), 3429 - 34
Synthesis of 2-hydroxy acid from 2-amino acid by Clostridium butyricum; Khelifa N et al.; Cultures of Clostridium butyricum type strain in synthetic medium supplemented with various L-2-amino acids revealed the presence of the corresponding 2-hydroxy acid . This metabolite is able to produce the polyester poly(2-hydroxyalkanoic acid) . The bioconversion is not stereoselective since D-2-amino acids were also converted . Chiral GC analysis demonstrated that only D-enantiomer is formed from L-leucine.

J Biol Chem, 1999 Jan 8, 274(2), 1050 - 7
Monocyte adherence induced by lipopolysaccharide involves CD14, LFA-1, and cytohesin-1 . Regulation by Rho and phosphatidylinositol 3-kinase; Hmama Z et al.; Mechanisms regulating lipopolysaccharide (LPS)-induced adherence to intercellular adhesion molecule (ICAM)-1 were examined using THP-1 cells transfected with CD14-cDNA (THP-1wt) . THP-1wt adherence to ICAM-1 was LPS dose-related, time-dependent, and inhibited by antibodies to either CD14 or leukocyte function associated antigen (LFA)-1, but was independent of any change in the number of surface expressed LFA-1 molecules . A potential role for phosphatidylinositol (PI) 3-kinase (PI 3-kinase) in LPS-induced adherence was examined using the PI 3-kinase inhibitors LY294002 and Wortmannin . Both inhibitors selectively attenuated LPS-induced, but not phorbol 12-myristate 13-acetate-induced adherence . Inhibition by these agents was unrelated to any changes in either LPS binding to or LFA-1 expression by THP-1wt cells . LPS-induced adherence was also abrogated in U937 cells transfected with a dominant negative mutant of of PI 3-kinase . Toxin B from Clostridium difficile, an inhibitor of the Rho family of GTP-binding proteins, abrogated both PI-3 kinase activation and adherence induced by LPS . Cytohesin-1, a phosphatidylinositol 3,4,5-triphosphate-regulated adaptor molecule for LFA-1 activation, was found to be expressed in THP-1wt cells . In addition, treatment of THP-1wt with cytohesin-1 antisense attenuated LPS-induced adherence . These findings suggest a model in which LPS induces adherence through a process of "inside-out" signaling involving CD14, Rho, and PI 3-kinase . This converts low avidity LFA-1 into an active form capable of increased binding to ICAM-1 . This change in LFA-1 appears to be cytohesin-1-dependent.

Appl Environ Microbiol, 1999 Jan, 65(1), 189 - 97
Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains; Ohura T et al.; The extracellular polyhydroxybutyrate (PHB) depolymerase gene (phaZPst) of Pseudomonas stutzeri was cloned and sequenced . phaZPst was composed of 1,728 bp encoding a protein of 576 amino acids . Analyses of the N-terminal amino acid sequence and the matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrum of the purified enzyme showed that the mature enzyme consisted of 538 amino acids with a deduced molecular mass of 57,506 Da . Analysis of the deduced amino acid sequence of the protein revealed a domain structure containing a catalytic domain, putative linker region, and two putative substrate-binding domains (SBDI and SBDII) . The putative linker region was similar to the repeating units of the cadherin-like domain of chitinase A from Vibrio harveyi and chitinase B from Clostridium paraputrificum . The binding characteristics of SBDs to poly({R}-3-hydroxybutyrate) {P(3HB)} and chitin granules were characterized by using fusion proteins of SBDs with glutathione S-transferase (GST) . These GST fusion proteins with SBDII and SBDI showed binding activity toward P(3HB) granules but did not bind on chitin granules . It has been suggested that the SBDs of the depolymerase interact specifically with the surface of P(3HB) . In addition, a kinetic analysis for the enzymatic hydrolysis of 3-hydroxybutyrate oligomers of various sizes has suggested that the catalytic domain of the enzyme recognizes at least two monomeric units as substrates.

FEBS Lett, 1998 Dec 4, 440(3), 287 - 90
Regulation of phospholipase D activity in synaptosomes permeabilized with Staphylococcus aureus alpha-toxin; Sarri E et al.; In order to investigate the regulation of presynaptic phospholipase D (PLD) activity by calcium and G proteins, we established a permeabilization procedure for rat cortical synaptosomes using Staphylococcus aureus alpha-toxin (30-100 microg/ml) . In permeabilized synaptosomes, PLD activity was significantly stimulated when the concentration of free calcium was increased from 0.1 microM to 1 microM . This activation was inhibited in the presence of KN-62 (1 microM), an inhibitor of calcium/calmodulin-dependent kinase II (CaMKII), but not by the protein kinase C inhibitor, Ro 31-8220 (1-10 microM) . Synaptosomal PLD activity was also stimulated in the presence of 1 microM GTPgammaS . When Rho proteins were inhibited by pretreatment of the synaptosomes with Clostridium difficile toxin B (TcdB; 1-10 ng/ml), the effect of GTPgammaS was significantly reduced; in contrast, brefeldin A (10-100 microM), an inhibitor of ARF activation, was ineffective . Calcium stimulation of PLD was inhibited by TcdB, but GTPgammaS-dependent activation was insensitive to KN-62 . We conclude that synaptosomal PLD is activated in a pathway which sequentially involves CaMKII and Rho proteins.

Lett Appl Microbiol, 1998 Dec, 27(6), 323 - 7
Evaluation of two media for the membrane filtration enumeration of Clostridium perfringens from water; Sartory DP et al.; Two media (mCP medium and Tryptose Sulphite Cycloserine (TSC) agar) were evaluated for recovery of Clostridium perfringens in environmental and part-treated drinking water . For laboratory strains of Clostridium, mCP was more selective and specific for Cl . perfringens than TSC, but was markedly less efficient for the enumeration of both vegetative cells and spores . For samples of river water and part-treated drinking water, TSC recovered significantly greater numbers of Cl . perfringens than mCP . In contrast to previous reports, there was a significant number of false presumptive positive and negative isolates on mCP . TSC is a more suitable medium for the routine monitoring of water supplies for the presence of Cl . perfringens.

Curr Microbiol, 1999 Feb, 38(2), 96 - 100
Evidence for Clostridium perfringens enterotoxin (CPE) inducing a mitogenic and cytokine response in vitro and a cytokine response in vivo; Wallace FM et al.; We investigated some immunogenic properties of Clostridium perfringens enterotoxin (CPE) in vitro using murine J774A macrophages (MPhi) and in vivo using Swiss Webster (SW) mice . CPE was a potent mitogen in vitro, where cell proliferation increased with CPE concentration . CPE was nonmitogenic when MPhi were concurrently incubated with CPE and interferon gamma (IFN-gamma) . MPhi incubated in the presence of CPE induced the synthesis of interleukin-1 (IL-1), interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), but not interleukin-2 (IL-2) . In vivo, CPE induced a pro-inflammatory cytokine response with striking production of IFN-gamma, IL-1, and IL-6 . Regardless of route of CPE entry, serum cytokine levels generally peaked within 1 h of administration and were maintained for 4-8 h . Although CPE engenders an intense immune response during toxicosis, the toxin does not appear to be a superantigen . Death from CPE-induced shock appears to result from various interrelating immunological mechanisms.

Glycoconj J, 1998 Aug, 15(8), 769 - 75
Effect of cysteine modifications on the activity of the 'small' Clostridium perfringens sialidase; Kruse S et al.; The 'small' (43 kDa) sialidase of Clostridium perfringens is inhibited by low concentrations of mercury ions . For the investigation of possible functional roles of the enzyme's four cysteine residues at the amino acid positions 2, 282, 333 and 349, they were separately altered to serine by site-directed mutagenesis . The four mutant sialidases expressed in E . coli and purified by metal chelate chromatography were markedly reduced in specific activity when compared to the wild-type enzyme but with the exception of C282S exhibited similar K(M)-values indicating an unchanged mode of substrate binding . The substrate specificity was also conserved for C2S, C282S, and C333S . Only the C349S sialidase exhibited a higher relative activity with colominic acid and the alpha2,6-linked sialic acid of sialyllactose compared to the alpha2,3-linked isomer than the other mutants . Chemical modifications with the thiol-blocking reagents N-ethylmaleimide (NEM), p-chloromercuribenzoate (pCMB) and HgCl2 had little effect on the C282S sialidase, e.g., 6% inhibition by 5 mM NEM compared to reductions in activity between 65 and 90% for the wild-type and other mutant enzymes, supporting the idea that among the enzyme's cysteines, Cys-282 has the highest structural or functional significance . The results also explain the higher mercury tolerance of Salmonella typhimurium and Clostridium tertium sialidases, which have the positions equivalent to Cys-282 altered to Val and Thr, respectively, indicating that the thiol group of Cys-282, despite being situated near the active site, is not involved in catalysis.

Ann Intern Med, 1998 Dec 15, 129(12), 1012 - 9
Acquisition of Clostridium difficile and Clostridium difficile-associated diarrhea in hospitalized patients receiving tube feeding; Bliss DZ et al.; BACKGROUND: Clostridium difficile is the most common infectious cause of nosocomial diarrhea, but its role in diarrhea associated with tube feeding has not been rigorously investigated . OBJECTIVE: To determine the incidence of C . difficile acquisition and C . difficile-associated diarrhea in tube-fed and non-tube-fed patients . DESIGN: Prospective cohort study . SETTING: A university-affiliated Veterans Affairs Medical Center . PATIENTS: 76 consecutive hospitalized, tube-fed patients and 76 hospitalized, non-tube-fed patients . The two cohorts were matched for age, unit location, duration of hospitalization before surveillance, and severity of illness . MEASUREMENTS: Incidence of C . difficile acquisition, incidence of C . difficile-associated diarrhea, and C . difficile restriction endonuclease analysis typing results . RESULTS: More tube-fed patients than non-tube-fed patients acquired C . difficile (15 of 76 patients {20%} compared with 6 of 76 patients {8%}; P=0.03) and developed C . difficile-associated diarrhea (7 of 76 patients {9%} compared with 1 of 76 patients {1%}; P=0.03) . The mean proportion (+/-SD) of surveillance days with diarrhea was greater for tube-fed patients after the development of C . difficile-associated diarrhea than for tube-fed patients without this diarrhea (0.68+/-0.4 compared with 0.22+/-0.2 {95% CI for the mean difference, 0.08 to 0.84}) . Postpyloric tube feeding (odds ratio, 3.14 {CI, 1.008 to 9.77}) and duration of surveillance (odds ratio, 1.08 {CI, 1.0009 to 1.16}) were risk factors for the acquisition of C . difficile . Nineteen restriction endonuclease analysis types of C . difficile were identified from 20 patients . CONCLUSIONS: Hospitalized, tube-fed patients, especially those receiving postpyloric tube feeding, are at greater risk for the acquisition of C . difficile and the development of C . difficile-associated diarrhea than are hospitalized, non-tube-fed patients . Clinicians should test for C . difficile in tube-fed patients with diarrhea.

Emerg Infect Dis, 1998 Oct-Dec, 4(4), 619 - 25
Increasing hospitalization and death possibly due to Clostridium difficile diarrheal disease; Frost F et al.; This study calculated yearly estimated national hospital discharge (1985 to 1994) and age-adjusted death rates (1980 to 1992) due to bacterial, viral, protozoal, and ill-defined enteric pathogens . Infant and young child hospitalization (but not death) rates in each category increased more than 50% during 1990 to 1994 . Age-adjusted death and hospitalization rates due to enteric bacterial infections and hospitalizations due to enteric viral infections have increased since 1988 . The increases in hospitalization and death rates from enteric bacterial infections were due to a more than eightfold increase in rates for specified enteric bacterial infections that were uncoded during this period (ICD9 00849) . To identify bacterial agents responsible for most of these infections, hospital discharges and outpatient claims (coded with more detail after 1992) were examined for New Mexico's Lovelace Health Systems for 1993 to 1996 . Of diseases due to uncoded enteric pathogens, 73% were due to Clostridium difficile infection . Also, 88% of Washington State death certificates (1985 to 1996) coded to unspecified enteric pathogen infections (ICD0084) listed C . difficile infection.

Appl Microbiol Biotechnol, 1998 Nov, 50(5), 564 - 7
Electroporation of, plasmid isolation from and plasmid conservation in Clostridium acetobutylicum DSM 792; Nakotte S et al.; Procedures have been developed allowing recombinant DNA work with Clostridium acetobutylicum DSM 792 . Electroporation was used to introduce plasmid DNA into exponentially growing clostridial cells and 6 x 10(2) transformants/microgram DNA could be obtained at a time constant of 5.5 ms, 1.8 kV, 50 microF, and 600 omega . The method also allowed the taxonomic group IV strain NI-4082 to be transformed (10(1) transformants/microgram DNA) . Plasmid preparation from recombinant clostridia was optimal when a modification of the alkaline lysis method was employed . It was also important to use cells from the mid-logarithmic growth phase . Recombinant strains could be easily preserved as spore suspensions; under all conditions tested plasmids were maintained.

Ned Tijdschr Geneeskd, 1998 Oct 24, 142(43), 2361 - 3
{A patient with tetanus without an obvious point of entry}; Stassen PM et al.; A 59-years-old man with oesophageal cancer (T3NXMo) presented with trismus, dysarthria and diaphoresis . Later, he developed opisthotonus and generalized spasms . Despite negative blood cultures and sufficiently high anti-tetanus-titres, tetanus was suspected, on clinical grounds . He was intubated and treated with tetanus toxoid, human antitetanus immunoglobulin, benzylpenicillin, propofol, benzodiazepines, vecuronium, and sufentanil, and recovered gradually . Tetanus is caused by Clostridium tetani, a Gram-positive rod capable of remaining present latently in the body for years . Absence of a visible external wound suggests that the oesophageal mucosal cancer lesion could have served as portal of entry or that endogenous reactivation of latent tetanus bacteria had taken place.

J Bacteriol, 1999 Jan, 181(1), 319 - 30
Regulation of the sol locus genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 by a putative transcriptional repressor; Nair RV et al.; A gene (orf1, now designated solR) previously identified upstream of the aldehyde/alcohol dehydrogenase gene aad (R . V . Nair, G . N . Bennett, and E . T . Papoutsakis, J . Bacteriol . 176:871-885, 1994) was found to encode a repressor of the sol locus (aad, ctfA, ctfB and adc) genes for butanol and acetone formation in Clostridium acetobutylicum ATCC 824 . Primer extension analysis identified a transcriptional start site 35 bp upstream of the solR start codon . Amino acid comparisons of SolR identified a potential helix-turn-helix DNA-binding motif in the C-terminal half towards the center of the protein, suggesting a regulatory role . Overexpression of SolR in strain ATCC 824(pCO1) resulted in a solvent-negative phenotype owing to its deleterious effect on the transcription of the sol locus genes . Inactivation of solR in C . acetobutylicum via homologous recombination yielded mutants B and H (ATCC 824 solR::pO1X) which exhibited deregulated solvent production characterized by increased flux towards butanol and acetone formation, earlier induction of aad, lower overall acid production, markedly improved yields of solvents on glucose, a prolonged solvent production phase, and increased biomass accumulation compared to those of the wild-type strain.

Infect Immun, 1999 Jan, 67(1), 302 - 7
Saccharomyces boulardii protease inhibits the effects of Clostridium difficile toxins A and B in human colonic mucosa; Castagliuolo I et al.; Saccharomyces boulardii is a nonpathogenic yeast used in the treatment of Clostridium difficile diarrhea and colitis . We have reported that S . boulardii inhibits C . difficile toxin A enteritis in rats by releasing a 54-kDa protease which digests the toxin A molecule and its brush border membrane (BBM) receptor (I . Castagliuolo, J . T . LaMont, S . T . Nikulasson, and C . Pothoulakis, Infect . Immun . 64:5225-5232, 1996) . The aim of this study was to further evaluate the role of S . boulardii protease in preventing C . difficile toxin A enteritis in rat ileum and determine whether it protects human colonic mucosa from C . difficile toxins . A polyclonal rabbit antiserum raised against purified S . boulardii serine protease inhibited by 73% the proteolytic activity present in S . boulardii conditioned medium in vitro . The anti-protease immunoglobulin G (IgG) prevented the action of S . boulardii on toxin A-induced intestinal secretion and mucosal permeability to {3H}mannitol in rat ileal loops, while control rabbit IgG had no effect . The anti-protease IgG also prevented the effects of S . boulardii protease on digestion of toxins A and B and on binding of {3H}toxin A and {3H}toxin B to purified human colonic BBM . Purified S . boulardii protease reversed toxin A- and toxin B-induced inhibition of protein synthesis in human colonic (HT-29) cells . Furthermore, toxin A- and B-induced drops in transepithelial resistance in human colonic mucosa mounted in Ussing chambers were reversed by 60 and 68%, respectively, by preexposing the toxins to S . boulardii protease . We conclude that the protective effects of S . boulardii on C . difficile-induced inflammatory diarrhea in humans are due, at least in part, to proteolytic digestion of toxin A and B molecules by a secreted protease.

J Pharmacol Exp Ther, 1999 Jan, 288(1), 36 - 42
RhoA-sensitive trafficking of muscarinic acetylcholine receptors; Vogler O et al.; The clathrin-mediated sequestration pathway is used by non-G protein-coupled receptors (e.g., transferrin receptors) and a large number of G protein-coupled receptors, including beta-2 adrenoceptors and various muscarinic acetylcholine receptor (mAChR) subtypes . Recently, the ubiquitously expressed small GTPase RhoA has been implicated as a negative regulator of transferrin receptor internalization . Because mAChRs and other G protein-coupled receptors are able to activate RhoA, we investigated in HEK-293 cells whether RhoA regulates the sequestration of m1 and m2 mAChRs, which internalize via clathrin-coated and nonclathrin-coated vesicles in HEK-293 cells, respectively . Overexpression of wild-type RhoA inhibited agonist-induced sequestration of both m1 and m2 mAChRs by as much as 70% . Inhibition could be reversed by coexpression of Clostridium botulinum C3 transferase, which inactivates RhoA by ADP-ribosylation . Overexpression of C3 transferase alone had no effect on m1 and m2 mAChR sequestration . In addition, overexpression of RhoA inhibited m1 and m2 mAChR transport to the plasma membrane by 60 and 31%, respectively, which was blocked by coexpression of C3 transferase . We conclude that RhoA is not an endogenous regulator of mAChR sequestration, but when overexpressed, strongly inhibits mAChR trafficking (i.e., sequestration and transport to the plasma membrane) in HEK-293 cells.

Anesthesiology, 1998 Dec, 89(6), 1543 - 52
Halothane attenuates calcium sensitization in airway smooth muscle by inhibiting G-proteins; Kai T et al.; BACKGROUND: Halothane directly relaxes airway smooth muscle partly by decreasing the Ca2+ sensitivity . In smooth muscle, receptor stimulation is thought to increase Ca2+ sensitivity via a cascade of heterotrimeric and small monomeric guanine nucleotide-binding proteins (G-proteins) . Whether this model is applicable in the airway and where halothane acts in this pathway were investigated . METHODS: A beta-escin-permeabilized canine tracheal smooth muscle preparation was used . Exoenzyme C3 of Clostridium botulinum, which inactivates Rho monomeric G-proteins, was used to evaluate the involvement of this protein in the Ca2+ sensitization pathway . The effects of halothane on different stimulants acting at different levels of signal transduction were compared: acetylcholine on the muscarinic receptor, aluminum fluoride (AIF4-) on heterotrimeric G-proteins, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) on all G-proteins . RESULTS: Exoenzyme C3 equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization, suggesting that these pathways are both mediated by Rho . Halothane applied before stimulation equally attenuated acetylcholine- and AIF4--induced Ca2+ sensitization . However, when added after Ca2+ sensitization was established, the effect of halothane was greater during Ca2+ sensitization induced by acetylcholine compared with AIF4-, which, along with the previous result, suggests that halothane may interfere with dissociation of heterotrimeric G-proteins . Halothane applied during GTPgammaS-induced Ca2+ sensitization had no significant effect on force, suggesting that halothane has no effect downstream from monomeric G-proteins . CONCLUSION: Halothane inhibits increases in Ca2+ sensitivity of canine tracheal smooth muscle primarily by interfering with the activation of heterotrimeric G-proteins, probably by inhibiting their dissociation.

J Med Microbiol, 1998 Dec, 47(12), 1075 - 80
Microbiology of liver and spleen abscesses; Brook I et al.; To study the aerobic and anaerobic microbiology of liver and spleen abscesses and correlate the results with predisposing factors, potential causes and routes of infection, clinical and laboratory data of 48 patients with liver abscesses and 29 with spleen abscesses treated between 1970 and 1990 were reviewed retrospectively . In liver abscesses, a total of 116 isolates (2.4 isolates/specimen) was obtained; 43 were aerobic and facultative species (0.9 isolates/specimen) and 73 were anaerobic species or microaerophilic streptococci (1.5 isolates/specimen) . Aerobic bacteria only were isolated from 12 (25%) abscesses, anaerobic bacteria only from eight (17%), and mixed aerobic and anaerobic bacteria from 28 (58%); polymicrobial infection was present in 38 (79%) . The predominant aerobic and facultative isolates were Escherichia coli (11 isolates), Streptococcus group D (8), Klebsiella pneumoniae (5) and Staphylococcus aureus (4) . The predominant anaerobes were Peptostreptococcus spp . (18 isolates), Bacteroides spp . (13), Fusobacterium spp . (10), Clostridium spp . (10) and Prevotella spp . (4) . There were 12 isolates of micro-aerophilic streptococci . S . aureus and beta-haemolytic streptococci were associated with trauma; Streptococcus group D, K . pneumoniae and Clostridium spp . with biliary disease; and Bacteroides spp . and Clostridium spp . with colonic disease . In splenic abscesses, a total of 56 isolates (1.9 isolates/specimen) was obtained; 23 were aerobic and facultative species (0.8 isolates/specimen), 31 were anaerobic species or micro-aerophilic streptococci (1.1 isolates/specimen) and two were Candida albicans . Aerobic bacteria only were isolated from nine (31%) abscesses, anaerobic bacteria from eight (28%), mixed aerobic and anaerobic bacteria from 10 (34%) and C . albicans in two (7%); polymicrobial infection was present in 16 (55%) . The predominant aerobic and facultative isolates were E . coli (5 isolates), Proteus mirabilis (3), Streptococcus group D (3), K . pneumoniae (3) and S . aureus (4) . The predominant anaerobes were Peptostreptococcus spp . (11 isolates), Bacteroides spp . (5), Fusobacterium spp . (3) and Clostridium spp . (3) . S . aureus, K . pneumoniae and Streptococcus group D were associated with endocarditis, E . coli with urinary tract and abdominal infection, Bacteroides spp . and Clostridium spp . with abdominal infection and Fusobacterium spp . with respiratory infection.

Commun Dis Public Health, 1998 Dec, 1(4), 229 - 30
Guidelines for optimal surveillance of Clostridium difficile infection in hospitals; Brazier JS et al.; The availability of surveillance data on C . difficile infection in hospitals in England and Wales is being jeopardised by the trend not to culture the organism for diagnostic purposes . NHS trust laboratories that no longer have the ability to isolate C . difficile cannot investigate putative outbreaks or monitor antimicrobial susceptibilities . These laboratories may now need to rely on their local public health laboratory for such investigations . Recent recommendations from the Department of Health(DH)/PHLS have highlighted the need for culture in outbreak investigations, for surveillance purposes, and for monitoring antimicrobial susceptibilities . It is important, therefore, that NHS diagnostic laboratories and public health laboratories, in particular, retain the ability to isolate C . difficile . A cost-effective approach is described that will facilitate surveillance by typing of strains and also enable their antimicrobial susceptibilities to be monitored.

Indian J Exp Biol, 1998 Sep, 36(9), 911 - 5
Effect of bacterial association on virulence of Entamoeba histolytica to baby hamster kidney cell monolayers; Ghosh PK et al.; Axenic E . histolytica trophozoite strain NIH:200 and HMI:IMSS when co-associated with aerobic bacteria Escherichia coli strain K12 and serotype 056 showed marked increase in virulence as observed by destruction of baby hamster kidney (BHK) monolayers . However, when incubated with anaerobic bacterial strains Clostridium perfringens and Bacteroides fragilis virulence remained unaltered . Further, adherence of E . histolytica to BHK monolayer was found to be mediated by N-acetyl-D-galactosamine.

Microbios, 1998, 94(379), 183 - 92
Evidence for an heterogeneous glycosylation of the Clostridium tyrobutyricum ATCC 25755 flagellin; Bedouet L et al.; Glycosylation analysis of the flagellin from the Gram-positive species Clostridium tyrobutyricum has been supplemented . Amino acid analysis of the glycopeptides obtained after pronase digestion of flagellin indicated that O-glycosylation which was previously demonstrated after nonreductive beta-elimination, probably occurred via the hydroxyl group of serine . Otherwise, beta-elimination partly deglycosylated flagellin . After this treatment carbohydrates were still linked to protein as shown by a digoxigenin-hydrazide labelling . Therefore, in addition to linkages via serine, alkaline resistant linkages exist on the flagellin and some glycans may be linked to the protein core via the amide nitrogen of asparagine or via the hydroxyl group of tyrosine . Furthermore, according to an immunological analysis, glycans attached to flagellin via alkaline sensitive linkages may be different from those attached via alkaline resistant linkages.

J Bacteriol, 1998 Dec, 180(24), 6581 - 5
Involvement of both dockerin subdomains in assembly of the Clostridium thermocellum cellulosome; Lytle B et al.; Clostridium thermocellum produces an extracellular cellulase complex termed the cellulosome . It consists of a scaffolding protein, CipA, containing nine cohesin domains and a cellulose-binding domain, and at least 14 different enzymatic subunits, each containing a conserved duplicated sequence, or dockerin domain . The cohesin-dockerin interaction is responsible for the assembly of the catalytic subunits into the cellulosome structure . Each duplicated sequence of the dockerin domain contains a region bearing homology to the EF-hand calcium-binding motif . Two subdomains, each containing a putative calcium-binding motif, were constructed from the dockerin domain of CelS, a major cellulosomal catalytic subunit . These subdomains, called DS1 and DS2, were cloned by PCR and expressed in Escherichia coli . The binding of DS1 and DS2 to R3, the third cohesin domain of CipA, was analyzed by nondenaturing gel electrophoresis . A stable complex was formed only when R3 was combined with both DS1 and DS2, indicating that the two halves of the dockerin domain interact with each other and such interaction is required for effective binding of the dockerin domain to the cohesin domain.

Dev Biol, 1998 Dec 1, 204(1), 151 - 64
Cellularization in Drosophila melanogaster is disrupted by the inhibition of rho activity and the activation of Cdc42 function; Crawford JM et al.; Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis . Drosophila melanogaster embryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellularization, a specialized form of cytokinesis . During cellularization, the actomyosin cytoskeleton forms a hexagonal array and drives invagination of the plasma membrane between the nuclei located at the cortex of the syncytial blastoderm . Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenance of the actin cytoskeleton throughout phylogeny and in D . melanogaster . To investigate how Rho subfamily activity affects the cytoskeleton during cellularization stages, embryos were microinjected with C3 exoenzyme from Clostridium botulinum or with wild-type, constitutively active, or dominant negative versions of Rho, Rac, and Cdc42 proteins . C3 exoenzyme ADP-ribosylates and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate downstream effectors . Dominant negative mutations likely inhibit endogenous small G protein activity by sequestering exchange factors . Of the 10 agents microinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negative Rho have a specific and indistinguishable effect: the actomyosin cytoskeleton is disrupted, cellularization halts, and embryogenesis arrests . Time-lapse video records of DIC imaged embryos show that nuclei in injected regions move away from the cortex of the embryo, thereby phenocopying injections of cytochalasin or antimyosin . Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is disrupted in a dose-dependent fashion . Additionally, DNA stain reveals that nuclei in the microinjected embryos aggregate in regions that correspond to actin disruption . These embryos halt in cellularization and do not proceed to gastrulation . We conclude that Rho activity and Cdc42 regulation are required for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning .

J Mol Biol, 1998 May 22, 278(5), 967 - 81
NADP-dependent bacterial alcohol dehydrogenases: crystal structure, cofactor-binding and cofactor specificity of the ADHs of Clostridium beijerinckii and Thermoanaerobacter brockii; Korkhin Y et al.; We have determined the X-ray structures of the NADP(H)-dependent alcohol dehydrogenase of Clostridiim beijerinckii (CBADH) in the apo and holo-enzyme forms at 2.15 A and 2.05 A resolution, respectively, and of the holo-alcohol dehydrogenase of Thermoanaerobacter brockii (TBADH) at 2.5 A . These are the first structures of prokaryotic alcohol dehydrogenase to be determined as well as that of the first NADP(H)-dependent alcohol dehydrogenase . CBADH and TBADH 75% have sequence identity and very similar three-dimensional structures . Both are tetramers of 222 symmetry . The monomers are composed of two domains: a cofactor-binding domain and a catalytic domain . These are separated by a deep cleft at the bottom of which a single zinc atom is bound in the catalytic site . The tetramers are composed of two dimers, each structurally homologous to the dimer of alcohol dehydrogenases of vertebrates . The dimers form tetramers by means of contacts between surfaces opposite the interdomain cleft thus leaving it accessible from the surface of the tetramer . The tetramer encloses a large internal cavity with a positive surface potential . A molecule of NADP(H) binds in the interdomain cleft to the cofactor-binding domain of each monomer . The specificity of the two bacterial alcohol dehydrogenases toward NADP(H) is determined by residues Gly198, Ser199, Arg200 and Tyr218, with the latter three making hydrogen bonds with the 2'-phosphate oxygen atoms of the cofactor . Upon NADP(H) binding to CBADH, Tyr218 undergoes a rotation of approximately 120 degrees about chi1 which facilitates stacking interactions with the adenine moiety and hydrogen bonding with one of the phosphate oxygen atoms . In apo-CBADH the catalytic zinc is tetracoordinated by side-chains of residues Cys37, His59, Asp150 and Glu60; in holo-CBADH, Glu60 is retracted from zinc in three of the four monomers whereas in holo-TBADH, Glu60 does not participate in Zn coordination . In both holo-enzymes, but not in the apo-enzyme, residues Ser39 and Ser113 are in the second coordination sphere of the catalytic zinc . The carboxyl group of Asp150 is oriented with respect to the active carbon of NADP(H) so as to form hydrogen bonds with both pro-S and pro-R hydrogen atoms.

Am J Physiol, 1998 Dec, 275(6 Pt 1), G1454 - 62
Rho A regulates sustained smooth muscle contraction through cytoskeletal reorganization of HSP27; Wang P et al.; The ras-related protein Rho p21 regulates various actin-dependent functions, including smooth muscle contraction . However, the precise mechanism of action of Rho p21 is still not clear . We report here that Rho A is a key regulator of agonist-induced contractile effects in rabbit colonic smooth muscle . Endothelin-1 and C2 ceramide were used . Both seem to activate phosphoinositide 3-kinase (PI 3-kinase) through G protein and pp60(src), respectively . Immunoprecipitation and immunoblotting revealed one form of 21-kDa Rho A that translocated from the cytosol to the membrane in response to stimulation by either endothelin (10(-7) M) or ceramide (10(-7) M) ( approximately 30% increase at 30 s that was sustained at 4 min) . The translocation of Rho A to the membrane was confirmed by immunostaining . The translocation of Rho A was inhibited by Clostridium botulinum C3 exoenzyme, which ADP ribosylated Rho A, but was not inhibited by the pp60(src) inhibitor herbimycin A or by the protein kinase C (PKC) inhibitor calphostin C, suggesting that Rho A may be upstream of pp60(src) and PKC or may belong to a different pathway than these proteins . Both ceramide- and endothelin-induced PI 3-kinase activation was inhibited by C3 exoenzyme pretreatment . However, the C3 exoenzyme inhibited endothelin- but not ceramide-induced mitogen-activated protein kinase phosphorylation, indicating that Rho regulates ceramide- and endothelin-induced contraction through different pathways . Furthermore, the dominant negative form of Rho (N19Rho) inhibited the actin binding protein, 27-kDa heat shock protein (HSP27), reorganization in response to ceramide and endothelin observed under confocal microscopy.

Mol Cell Probes, 1998 Dec, 12(6), 359 - 65
Controlled multiplex PCR of enterotoxigenic Clostridium perfringens strains in food samples; Schoepe H et al.; A controlled multiplex polymerase chain reaction (PCR) for the detection of Clostridium(C.) perfringens enterotoxin gene (cpe) was established and compared with an in vitro antigenic detection method . Thecpe PCR and the classical method of electric immunodiffusion gave identical results . The predicted specific amplicon of the cpe gene was generated from all of the tested enterotoxigenic C . perfringens strains . In contrast, cultures of any other Clostridium species tested by PCR were negative (100% sensitivity, 100% specificity) . Addition of an alphatoxin (plc) gene specific PCR as an in process control reaction was performed in order to prevent false negative PCR results . The PCR detection limit was 0.5 ng of genomic C . perfringens DNA per ml of bouillon culture . By contaminating minced meat with C . perfringens reference strains, the multiplex PCR was established as a tool for routine diagnostic laboratories . The detection limit was approximately 3.0x10(5)C . perfringens cells per gram meat . The results demonstrate the multiplex PCR as an easy, specific, sensitive and time saving diagnostic procedure . Application of this improved method should enhance the knowledge concerning epidemiological aspects of food borne diseases caused by enterotoxigenic C . perfringens .

Biochemistry, 1998 Nov 10, 37(45), 15974 - 80
Heterologous biosynthesis and characterization of the {2Fe-2S}-containing N-terminal domain of Clostridium pasteurianum hydrogenase; Atta M et al.; The primary structure of Clostridium pasteurianum hydrogenase I appears to be composed of modules suggesting that the various iron-sulfur clusters present in this enzyme might be segregated in structurally distinct domains . On the basis of this observation, a gene fragment encoding the 76 N-terminal residues of this enzyme has been expressed in Escherichia coli . The polypeptide thus produced contains a {2Fe-2S}n+ cluster of which the oxidized level (n = 2) has been monitored by UV-visible absorption, circular dichroism, and resonance Raman spectroscopy . This cluster can be reduced by dithionite or electrochemically to the n = 1 level which has been investigated by EPR and by low-temperature magnetic circular dichroism . The redox potential of the +2 to +1 transition is -400 mV (vs the normal hydrogen electrode) . The spectroscopic and redox results indicate a {2Fe-2S}2+/+ chromophore coordinated by four cysteine ligands in a protein fold similar to that found in plant- and mammalian-type ferredoxins . Among the five cysteines present in the N-terminal hydrogenase fragment, four (in positions 34, 46, 49, and 62) are conserved in other sequences and are therefore the most likely ligands of the {2Fe-2S} site . The fifth cysteine, in position 39, can be dismissed on the grounds that the Cys39Ala mutation does not alter any of the properties of the iron-sulfur cluster . The spectroscopic signatures of this chromophore are practically identical with some of those reported for full-size hydrogenase . This confirms that C . pasteurianum hydrogenase I contains a {2Fe-2S} cluster and indicates that the polypeptide fold around the metal site of the N-terminal fragment is very similar, if not identical, to that occurring in the full-size protein . The N-terminal sequence of this hydrogenase is homologous to sequences of a number of proteins or protein domains, including a subunit of NADH-ubiquinone oxidoreductase of respiratory chains . From that, it can be anticipated that the structural domain isolated and described here is a building block of electron transfer complexes involved in various bioenergetic processes.

Curr Microbiol, 1999 Jan, 38(1), 37 - 42
Cell surface hydrophobicity of sucrose fermenting and nonfermenting Corynebacterium diphtheriae strains evaluated by different methods; Mattos-Guaraldi AL et al.; Corynebacterium diphtheriae strains expressed variation in hydrophobic characteristics dependent on the method used . Results of single assays are not a reliable representation of C . diphtheriae hydrophobicity . All 12 strains adhered to polystyrene surfaces; three showed spontaneous aggregation (SA) in Trypticase Soy Broth (TSB) medium, and eight exhibited autoagglutination in phosphate-buffered saline (PBS; AA-positive) . The salt aggregation test (SAT) values </=0.002 or >/=1.6 represented breakpoints for groups of strains with differing hydrophobicity . C . diphtheriae strains showed affinity towards n-hexadecane . Percentages of adhesion varied from 31% to 63% and were not directly related to morphological n-hexadecane adhesion patterns . Diffuse and localized adhesion patterns were noted predominantly among sucrose-positive and sucrose-negative strains, respectively . Strains of the sucrose-negative biotype expressed a higher degree of hydrophobicity . The choice of the growth medium influenced the hydrophobicity, not the hemagglutinating activity (HA) of C . diphtheriae . Heating bacterial suspensions at 121 degrees C decreased both HA and hydrophobicity of three strains . However, hydrophobins and hemagglutinins were trypsin and detergent resistant . The treatment of microorganisms with Clostridium perfringens neuraminidase increased the hydrophobicity but not the HA titers of strains tested . Hemagglutinins were partially responsible for hydrophobicity . Hydrophilic AA-negative strains adhered strongly to glass but expressed weak HA . Sialylglycoconjugates functioned as hydrophilins on C . diphtheriae surfaces.

Naunyn Schmiedebergs Arch Pharmacol, 1998 Nov, 358(5), 509 - 17
Activation of a Ca2+-dependent K+ current in mouse fibroblasts by lysophosphatidic acid requires a pertussis toxin-sensitive G protein and Ras; Repp H et al.; Lysophosphatidic acid (LPA) is a bioactive lipid that acts through G protein-coupled plasma membrane receptors and mediates a wide range of cellular responses . Here we report that LPA activates a K+ current in NIH3T3 mouse fibroblasts that leads to membrane hyperpolarization . The activation occurs with an EC50 value of 1.7 nM LPA . The K+ current is Ca2+-dependent, voltage-independent, and completely blocked by the K+ channel blockers charybdotoxin, margatoxin, and iberiotoxin with IC50 values of 1.7, 16, and 62 nM, respectively . The underlying K+ channels possess a single channel conductance of 33 pS in symmetrical K+ solution . Pretreatment of cells with pertussis toxin (PTX), Clostridium sordellii lethal toxin, or a farnesyl protein transferase inhibitor reduced the K+ current amplitude in response to LPA to about 25% of the control value . Incubation of cells with the protein tyrosine kinase inhibitor genistein or microinjection of the neutralizing anti-Ras monoclonal antibody Y13-259 reduced it by more than 50% . In contrast, the phospholipase C inhibitor U-73122 and the protein kinase A activator 8-bromo-cAMP had no effect . These results indicate that the K+ channel activation by LPA is mediated by a signal transduction pathway involving a PTX-sensitive G protein, a protein tyrosine kinase, and Ras . LPA is already known to activate Cl- channels in various cell types, thereby leading to membrane depolarization . In conjunction with our results that demonstrate LPA-induced membrane hyperpolarization by activation of K+ channels, LPA appears to be significantly involved in the regulation of the cellular membrane potential.

J Med Microbiol, 1998 Jul, 47(7), 591 - 8
Bacterial translocation, intestinal microflora and morphological changes of intestinal mucosa in experimental models of Clostridium difficile infection; Naaber P et al.; Bacteraemia and subsequent sepsis is one possible complication of Clostridium difficile infection . The aim of this study was to examine a correlation between bacterial translocation with morphological changes of intestinal mucosa and shifts of intestinal microflora in experimental models of C . difficile infection . A mouse model was used to study post-antibiotic shifts and mild C . difficile infection, and hamsters were used to study fatal enterocolitis . The influence of pro- and pre-biotics (lactobacilli and xylitol) were also studied in the hamster model . The quantitative composition of luminal and mucosal microflora was evaluated in different intestinal loci, inflammatory changes of mucosa were estimated in histological sections and bacterial translocation was detected in samples from blood, liver, spleen and mesenteric lymph nodes . In cases of mild C . difficile infection, the extent of disturbance of intestinal microflora appeared to be a more important promoting factor in translocation than inflammatory activity in the mucosa . Translocation was frequent in fatal enterocolitis, with facultative species predominating in the intestinal mucosa and also C . difficile in some cases . The combination of lactobacilli and xylitol had some protective effect against C . difficile infection in these models.

Acta Microbiol Pol, 1998, 47(2), 177 - 83
Dioctahedral smectite neutralization activity of Clostridium difficile and Bacteroides fragilis toxins in vitro; Martirosian G et al.; The neutralization activity of dioctahedral smectite for ten toxigenic Clostridium difficile and eight enterotoxigenic Bacteroides fragilis strains was studied using McCoy and HT 29/C1 cell lines, respectively . Minimalization of the cytopathic effect of C . difficile toxin B on McCoy cell lines by dioctahedral smectite dissolved in PBS was observed . After incubation with dioctahedral smectite the toxic effects of B . fragilis enterotoxins on HT/29C1 (human colon adenocarcinoma cell line) were eliminated . Best neutralization of B . fragilis toxin was achieved using dioctahedral smectite dissolved in BHI.

Pathology, 1998 Nov, 30(4), 402 - 4
Isolation of Clostridium tetani from anaerobic empyema; Mayall BC et al.; We report the isolation of Clostridium tetani (along with Fusobacterium mortiferum) from empyema pus . The patient, a 68 year old retired farmer from rural NSW, had recently undergone cholecystectomy, had heart failure and developed an empyema . He improved after drainage of the empyema and penicillin therapy, but died suddenly during convalescence.

Am J Infect Control, 1998 Dec, 26(6), 588 - 93
Effectiveness of infection control program in controlling nosocomial Clostridium difficile; Zafar AB et al.; OBJECTIVE: To report the effectiveness of use of comprehensive infection control measures to reduce the incidence of Clostridium difficile (CD) in an acute-care teaching hospital . METHODS: All CD infections were reviewed by the infection control coordinator from 1987 to 1996 . The Centers for Disease Control and Prevention's nosocomial infection definition was used . CD-inclusion criteria remained unchanged during the study period . Interventions were started in 1990 . INTERVENTIONS: The interventions used were: (1) Isolation policy-revision and enforcement, which included universal precautions policy, (2) educational program-monthly to all health care workers, (3) phenolic disinfectant for environmental cleaning, (4) triclosan (0.03%) soap for handwashing, (5) centralization of sterilization department, (6) cart-washer installation, and (7) aggressive surveillance activity . RESULTS: From 1987 to 1989, before the interventions, a total of 466 CD infections (mean 155 per year) occurred . From 1990 to 1996, after the interventions, 475 infections (mean 67 per year) occurred . Incidence of CD decreased by 60% from 1990 to 1996 . CONCLUSION: The sustained decrease of nosocomial CD during the 7-year period demonstrated the effectiveness of aggressive infection control measures that involve multiple disciplines.

Am J Infect Control, 1998 Dec, 26(6), 584 - 7
Evaluation of an interdisciplinary re-isolation policy for patients with previous Clostridium difficile diarrhea; Boone N et al.; BACKGROUND: Diarrhea caused by Clostridium difficile is increasingly recognized as a nosocomial problem . The effectiveness and cost of a new program to decrease nosocomial spread by identifying patients scheduled for readmission who were previously positive for toxin was evaluated . METHODS: The Memorial Sloan-Kettering Cancer Center is a 410-bed comprehensive cancer center in New York City . Many patients are readmitted during their course of cancer therapy . In 1995 as a result of concern about the nosocomial spread of C difficile, we implemented a policy that all patients who were positive for C difficile toxin in the previous 6 months with no subsequent toxin-negative stool as an outpatient would be placed into contact isolation on readmission pending evaluation of stool specimens . Patients who were previously positive for C difficile toxin were identified to infection control and admitting office databases via computer . Admitting personnel contacted infection control with all readmissions to determine whether a private room was required . RESULTS: Between July 1, 1995, and June 30, 1996, 47 patients who were previously positive for C difficile toxin were readmitted . Before their first scheduled readmission, the specimens for 15 (32%) of these patients were negative for C difficile toxin . They were subsequently cleared as outpatients and were readmitted without isolation . Workup of the remaining 32 patients revealed that the specimens for 7 patients were positive for C difficile toxin and 86 isolation days were used . An additional 25 patients used 107 isolation days and were either cleared after a negative specimen was obtained in-house or discharged without having an appropriate specimen sent . Four patients (9%) had reoccurring C difficile after having toxin-negative stools . We estimate (because outpatient specimens were not collected) the cost incurred at $48,500 annually, including the incremental cost of hospital isolation and equipment . CONCLUSION: Our policy to control the spread of nosocomial C difficile required interdisciplinary cooperation between infection control and the admitting department . By identifying patients who were positive for toxin through admitting, we were able to place all potentially infected patients into isolation . Our positivity rate of 15% on readmission demonstrates the importance of this policy . The cost of controlling C difficile can be significantly lowered by clearing patients who were previously positive for toxin before hospital readmission.

Science, 1998 Dec 4, 282(5395), 1853 - 8
X-ray crystal structure of the Fe-only hydrogenase (CpI) from Clostridium pasteurianum to 1.8 angstrom resolution; Peters JW et al.; A three-dimensional structure for the monomeric iron-containing hydrogenase (CpI) from Clostridium pasteurianum was determined to 1.8 angstrom resolution by x-ray crystallography using multiwavelength anomalous dispersion (MAD) phasing . CpI, an enzyme that catalyzes the two-electron reduction of two protons to yield dihydrogen, was found to contain 20 gram atoms of iron per mole of protein, arranged into five distinct {Fe-S} clusters . The probable active-site cluster, previously termed the H-cluster, was found to be an unexpected arrangement of six iron atoms existing as a {4Fe-4S} cubane subcluster covalently bridged by a cysteinate thiol to a {2Fe} subcluster . The iron atoms of the {2Fe} subcluster both exist with an octahedral coordination geometry and are bridged to each other by three non-protein atoms, assigned as two sulfide atoms and one carbonyl or cyanide molecule . This structure provides insights into the mechanism of biological hydrogen activation and has broader implications for {Fe-S} cluster structure and function in biological systems.

Appl Environ Microbiol, 1998 Dec, 64(12), 4757 - 66
Genetic characterization and heterologous expression of brochocin-C, an antibotulinal, two-peptide bacteriocin produced by Brochothrix campestris ATCC 43754; McCormick JK et al.; Brochocin-C, produced by Brochothrix campestris ATCC 43754, is active against many strains of the closely related meat spoilage organism Brochothrix thermosphacta and a wide range of other gram-positive bacteria, including spores of Clostridium botulinum . Purification of the active compound and genetic characterization of brochocin-C revealed that it is a chromosomally encoded, two-peptide nonlantibiotic bacteriocin . Both peptides of brochocin-C are ribosomally synthesized as prepeptides that are typical of class II bacteriocins . They are cleaved following Gly-Gly cleavage sites to yield the mature peptides, BrcA and BrcB, containing 59 and 43 amino acids, respectively . Fusion of the nucleotides encoding the signal peptide of the bacteriocin divergicin A in front of the structural genes for either BrcA or BrcB allowed independent expression of each component by the general protein secretion pathway . This revealed the two-component nature of brochocin-C and the necessity for both peptides for activity . A 53-amino-acid peptide encoded downstream of brcB functions as the immunity protein (BrcI) for brochocin-C . In addition, the cloned chromosomal fragment revealed open reading frames downstream of brcI, designated brcT and brcD, that encode proteins with homology to ATP-binding cassette translocator and accessory proteins, respectively, involved in the secretion of Gly-Gly-type bacteriocins.

Enferm Infecc Microbiol Clin, 1998 Oct, 16(8), 359 - 63
{Diarrhea associated with Clostridium difficile . One-year retrospective study at a tertiary hospital}; Barreiro PM et al.; BACKGROUND: Diarrhea associated with Clostridium difficile is a health care problem of growing importance in the last few years specially in the hospital environment . The epidemiologic data and factors associated with this disease have not, to date, been sufficiently studied in Spain . METHODS: The cases of diarrhea associated with C . difficile reported in 1996 in a tertiary hospital of 1,500 beds were retrospectively reviewed, collecting clinical and epidemiologic data . The technique used for the detection of the C . difficile toxin was EIA Premier . RESULTS: One hundred thirty-two patients were included in the study, 83.3% of whom were over the age of 65 years, who had had 148 episodes of diarrhea associated with C . difficile . Most had been admitted into internal medicine (36%) or in the geriatric department (25%) and the remaining in the surgical departments (16.4%) or others (22.6%) . The most frequently prescribed antibiotics were third generation cephalosporins (28.6%), clindamycin (17%), quinolones (11.1%) and macrolides (9.1%) . Most of the patients received from 2 to 4 antibiotics prior to presenting diarrhea . Thirteen percent of the episodes of diarrhea associated with C . difficile were exclusively treated with withdrawal of the prescribed antibiotic, while the remaining cases were also given specific treatment which in 68.6% of the cases was with metronidazole and 31.4% with vancomycin . No significant difference was observed in the evolution of the patients according to the antibiotic prescribed . CONCLUSIONS: Diarrhea associated with C . difficile should be taken into account as a frequent complication of wide spectrum antibiotic treatment, specially in the elderly, immunosuppressed or in patients with pluripathology . With this study the authors wish to underline the need for the judicious use of antibiotics in the hospital environment and aid in the rapid diagnosis of this entity.

FEMS Microbiol Lett, 1998 Nov 15, 168(2), 319 - 24
In vitro synthesis of poly(3-hydroxybutyric acid) by using an enzymatic coenzyme A recycling system; Jossek R et al.; Purified recombinant poly(hydroxyalkanoic acid), PHA, synthase from Chromatium vinosum was used to examine in vitro poly(3-hydroxybutyric acid) (P(3HB)) formation . In combination with purified propionyl-coenzyme A transferase of Clostridium propionicum a two-enzyme in vitro P(3HB) biosynthesis system was established which allowed the synthesis of P(3HB) from free D-(-)-3-hydroxybutyric acid as substrate . The coenzyme A residue for the activation of this hydroxyacid was provided by acetyl-coenzyme A . By adding acetyl-coenzyme A synthetase to this system, a three-enzyme in vitro P(3HB) biosynthesis system was established . Coenzyme A that was released during the polymerization reaction was coupled to acetate which again served as the coenzyme A donor for the activation of 3-hydroxybutyric acid . The energy for the in vitro P(3HB) synthesis was provided by ATP hydrolyses resulting in acetyl-coenzyme A synthesis catalyzed by the acetyl coenzyme A synthetase . In this way the in vitro synthesis of P(3HB) became independent of the consumption of the expensive coenzyme A . By this procedure a handy system is available to produce in vitro PHA on a semipreparative scale.

Dermatol Surg, 1998 Nov, 24(11), 1249 - 54
Complications of botulinum A exotoxin for hyperfunctional lines; Matarasso SL; Clostridium botulinum type A exotoxin is one of the recent advances for treatment of the aging face . Due to the sudden and exponential surge in popularity, there is little precise consensus regarding its safety and efficacy . Many of the reported complications associated with its aesthetic use are few and anecdotal . As we gain more experience and long-term follow-up with this procedure, complications and their treatment can be better documented . As most of the salutary effects of Botulinum toxin are temporary, fortunately, so too are the complications associated with this form of therapy.

Dermatol Surg, 1998 Nov, 24(11), 1208 - 12
Oculoplastic experience with the cosmetic use of botulinum A exotoxin; Edelstein C et al.; BACKGROUND: Botulinum toxin is a neuromuscular blocking agent produced by the bacterium Clostridium botulinum . It has been used as a therapeutic agent in ophthalmology for over 18 years . OBJECTIVE: To present the cosmetic use of botulinum toxin in oculoplastic surgery . METHODS: We present our experience with botulinum toxin as a cosmetic agent . RESULTS: Botulinum toxin was successful in treating hyperfunctional lines of the face and neck . CONCLUSIONS: Botulinum toxin is a safe and effective method of treating dynamic lines of the face and neck.

Gastroenterology, 1998 Dec, 115(6), 1329 - 34
Single toxin detection is inadequate to diagnose Clostridium difficile diarrhea in pediatric patients; Kader HA et al.; BACKGROUND & AIMS: Clostridium difficile is an important cause of symptomatic diarrhea in pediatric patients . The bacterium produces two toxins, although many laboratories assay for only one . We questioned this diagnostic approach when patients had positive results for C . difficile at our institution, but initially had tested negative at outside laboratories . METHODS: We retrospectively analyzed relative frequencies of C . difficile toxin A alone, toxin B alone, and toxins A and B from pediatric patients with diarrhea . Results were stratified according to toxin detection and patient age . RESULTS: Of 1061 specimens, 276 (26.8%) were positive for C . difficile toxin(s) . Fifty-one (18.5%) were positive for toxin A alone, 133 (48.2%) for toxin B alone, and 92 (33.3%) for both toxins . Assaying for toxin B identified C . difficile infection more frequently than did assaying for toxin A (P < 0.0001) . The frequency of toxin B detection was significantly higher for older children but not for infants . CONCLUSIONS: Testing for C . difficile toxin A or toxin B alone will result in more frequent misdiagnosis than testing for both toxins . This practice may lead to inappropriate further invasive investigations in children, although this finding may not be applicable to adults.

J Biomol Struct Dyn, 1998 Oct, 16(2), 223 - 35
Molecular dynamics simulation of interaction of histone-like protein of mycobacterium tuberculosis (Hlpmt) and histone of clostridium pasteurianum (DBHclopa) with 35 based paired GC rich U-bend DNA; Kothekar V et al.; Three dimensional structure of the first ninety two residues of histone like protein from Mycobacterium tuberculosis (Hlpmt) and histone of Clostridium pasteurianum (DBHclopa) are obtained here, on the basis of amino acid sequences of the two proteins, making use of secondary structure prediction programs, sequence search and HOMOLOGY based modeling tools available on Internet . The proteins were docked to a 35 base paired GC rich U bend DNA (U35DNA) . Structures of proteins Hlpmt and DBHclopa; U35DNA; and complexes: Hlpmt-U35DNA and DBHclopa-U35DNA were optimized by molecular mechanics (MM) and simulated for 260 pico seconds (ps) in vacuum by molecular dynamics (MD) technique using AMBER 4.0 package with Cornell et al force field . The proteins, when simulated alone, showed compaction . DBHclopa showed larger compaction compared with Hlpmt . U35DNA when simulated alone straightened out and assumed a B-form . In the complexes, Hlpmt showed same order of compaction as in absence of DNA, while DBHclopa showed reduced compaction . In the presence of Hlpmt two ends of helicoidal axis of U35DNA came closer, but slightly out of plane, indicative of its role in overwinding and packaging double stranded DNA . DBHclopa did not give rise to DNA overwinding . The results show architectural role of Hlpmt and DBHclopa in DNA packaging and its sequence dependence.

J Biochem (Tokyo), 1998 Dec 1, 124(6), 1101 - 10
Enzymatic and molecular properties of the Clostridium tertium sialidase; Grobe K et al.; Clostridium tertium metabolizes sialoglycoconjugates via a secreted sialidase {EC 3.2.1.18} and an intracellular acylneuraminate pyruvate lyase {EC 4.1.3.3} . The sialidase was enriched 1,900-fold from the culture medium with a specific activity of 0.7 U per mg protein . It exhibits a temperature optimum of 50 degreesC and tolerates mercury ions at relatively high concentrations (50% inhibition at 5.2 mM Hg2+) . The sialidase gene was detected on two restriction fragments (HincII, HindIII) of chromosomal DNA and their correct recombination resulted in an active enzyme expressed by Escherichia coli . The structural gene is represented by 2,319 bp encoding a protein of 773 amino acids with a molecular mass of 85.4 kDa . The first 28 amino acids possess the character of a signal peptide . The protein reveals a FRIP-region and four Asp-boxes common in all bacterial sialidases . It has 42.6% identical amino acids when compared with the sialidase of Clostridium septicum and 64.8% with a sialidase gene amplified from Macrobdella decora . A further open reading frame was detected 30 bp downstream from the sialidase gene, which exhibits significant homology with acylneuraminate pyruvate lyases . For the first time, both genes were found in close proximity on a bacterial chromosome, probably being part of one operon.

Arch Latinoam Nutr, 1998 Jun, 48(2), 152 - 5
{Microwave effect on survival of sporulated bacteria inoculated in minced meat}; Arias ML et al.; Due to the current tendency of cooking and heating meat prepared foods in microwave ovens and the possibility that they transmit bacterial diseases, the survival rate of spore-forming bacteria was evaluated in minced meat samples . Meat was innoculated with a known number of Bacillus cereus and Clostridium perfringens spores, and laterly thawed and cooked in an Amana microwave oven (2450 Hz) . Survival rate was determined according to the methodology described by Vanderzant & Splittstoesser, and the activity of the enzyme acid phosphatase was determined as cooking parameter . B . cereus spore showed a decrease in its number as the time of exposition increased, but without fully disappearing . C . perfringens spores also decreased in number, but showed a later increase, associated with the germination of survival spores.

J Appl Microbiol, 1998 Nov, 85(5), 875 - 82
Identification of a gene for a rubrerythrin/nigerythrin-like protein in Spirillum volutans by using amino acid sequence data from mass spectrometry and NH2-terminal sequencing; Alban PS et al.; A hydrogen peroxide-resistant mutant of the catalase-negative microaerophile, Spirillum volutans, constitutively expresses a 21.5 kDa protein that is undetectable and non-inducible in the wild-type cells . Part of the gene that encodes the protein was cloned using amino acid sequence data obtained by both mass spectrometry and NH2-terminal sequencing . The deduced 158 amino acid polypeptide shows high relatedness to rubrerythrin and nigerythrin previously described in the anaerobes Clostridium perfringens and Desulfovibrio vulgaris . The protein also shows high similarity to putative rubrerythrin proteins found in the anaerobic archeons Archaeoglobus fulgidus, Methanococcus jannaschii and Methanobacterium thermoautotrophicum . This is the first report of this type of protein in an organism that must respire with oxygen . It seems likely that the novel combination of methodologies used in this study could be applied to the rapid cloning of other genes in bacteria for which no genomic library yet exists.

Can J Microbiol, 1998 Aug, 44(8), 759 - 67
The enzymatic activity of phosphoglycerate mutase from gram-positive endospore-forming bacteria requires Mn2+ and is pH sensitive; Chander M et al.; The enzymatic activity of phosphoglycerate mutase (Pgm) from three gram-positive endospore-forming bacteria (Bacillus subtilis, Clostridium perfringens, and Sporosarcina ureae) requires Mn2+ and is very sensitive to pH; at low concentrations of Mn2+, a pH change from 8 to 6 resulted in greater than 30- to 200-fold decreases in the activity of these Pgms . However, Pgm deactivation at pH 6 was reversed by shifting the enzyme to pH 7 or 8 . Free Mn2+ was not directly involved in Pgm catalysis, although enzyme-bound Mn2+ may be involved . The rate of catalysis by Mn(2+)-containing Pgm was also slightly pH dependent, although the Km for 3-phosphoglyceric acid appeared to be the same at pH 6, 7, and 8 . These findings suggest that Mn2+ binds to catalytically inactive Pgm and converts it to a catalytically competent form, and further, that pH influences the efficiency with which the enzyme binds Mn2+ . The extreme pH sensitivity of the Mn(2+)-dependent Pgms supports a model in which this enzyme is inhibited during sporulation by acidification of the forespore, thus allowing accumulation of the spore's large depot of 3-phosphoglyceric acid . The activity of Pgm from two closely related gram-positive bacteria that do not form spores (Planococcus citreus and Staphylococcus saprophyticus) also requires Mn2+ and is pH sensitive . In contrast, the Pgm activities from two more distantly related non-endospore-forming gram-positive bacteria (Micrococcus luteus and Streptomyces coelicolor) are neither dependent on metal ions nor particularly sensitive to pH.

Appl Microbiol Biotechnol, 1998 Oct, 50(4), 426 - 8
Solvent production by Clostridium beijerinckii NRRL B592 growing on different potato media; Nimcevic D et al.; Very good solvent formation rates were observed when Clostridium beijerinckii NRRL B592 was cultivated on different whole potato media . The increase in whole potato concentration contributed to the increased final solvent concentrations, while the addition of yeast extract or mineral salts gave negative effects . To obtain good solvent productivities and high final solvent concentrations during batch fermentation, no enzymatic hydrolysis of the potato starch was necessary, indicating high activity of the clostridial amylases produced by the strain applied.

Nephrol Dial Transplant, 1998 Nov, 13(11), 2842 - 6
Clostridium difficile colitis associated with chronic renal failure; Cunney RJ et al.; BACKGROUND: Clostridium difficile-associated diarrhoea (CDAD) is a potentially life-threatening illness which has been shown to be more common and more severe in patients with chronic renal failure (CRF) than in other groups . A review of CDAD in our nephrology unit was carried out . METHODS: A review of microbiology and histology records identified 32 cases of CDAD in the nephrology unit over a 24-month period . Patient notes were reviewed to identify risk factors, clinical features and outcome . Available isolates of C . difficile underwent 16S ribosomal RNA typing . RESULTS: The incidence of CDAD in the nephrology unit was 10.7 per 1000 admissions, compared to 2.7 per 1000 in other areas of the hospital (P<0.0001) . CDAD was considered the sole or principal cause of death in six (19%) and was considered a contributing factor in a further seven (22%) . Mortality was significantly higher among patients with established CRF (P=0.04) . Seven cases occurred as a cluster, over a 1-month period . Isolates from this cluster, along with comparative strains from other areas of the hospital, were found to be PCR type 1 . Diarrhoea occurred in 28 (89%) of cases, pyrexia in 17 (53%) and ileus or abdominal pain in 14 (44%) . Six patients responded to discontinuation of antibiotics alone and 22 required metronidazole and/or vancomycin . Three patients had colectomy and one caecostomy because of toxic megacolon . Four patients died before specific therapy could be given and in two of these cases the diagnosis was made at autopsy . Twenty-six patients had a record of recent antibiotic therapy . Of these, 15 had at least one agent considered to be inappropriate (excessively broad spectrum agent in 11, excessive duration of therapy in four) . Nine patients had only received antibiotics prior to admission . CONCLUSIONS: CDAD carries a high mortality in nephrology patients, especially those with established CRF . The diagnosis may be missed if a careful antibiotic history is not taken, including agents received prior to admission . Rational antibiotic prescribing and adherence to infection control measures are vital to reduce the incidence of this serious condition.

Vet Rec, 1998 Oct 24, 143(17), 472 - 4
Variability of serum antibody responses of goat kids to a commercial Clostridium perfringens epsilon toxoid vaccine; Uzal FA et al.; Twenty-nine Angora goats were used in a trial of a commercial enterotoxaemia (pulpy kidney disease) vaccine . The animals were allocated to four groups, of which three received an initial dose of vaccine, two also received a booster of the same vaccine either 28 or 42 days after the first vaccination, and the fourth remained as an unvaccinated control group . An indirect ELISA technique was used to measure the titres of Clostridium perfringens type D epsilon antitoxin in serum samples taken before vaccination and 17, 28, 42, 59, 70, 86, 98 and 128 days after vaccination . There was a wide range of antibody titres after vaccination, and the great majority of the vaccinated animals had titres below the protective level, arbitrarily set at 0.25 iu/ml, by day 98.

Muscle Nerve Suppl, 1997, 6, S146 - 68
Botulinum toxin: chemistry, pharmacology, toxicity, and immunology; Brin MF; The seven serotypes of botulinum toxin (BTX) produced by Clostridium botulinum exert their paralytic effect by inhibiting acetylcholine release at the neuromuscular junction . Each of these zinc endopeptidases cleaves one or more proteins involved in vesicle transport and membrane fusion . The extent of paralysis depends on both doses and volume; the duration of paralysis is further dependent on the serotype employed . Restoration of neuromuscular function follows axon terminal sprouting . The two major commercial preparations of BTX-A appear to differ in their relative potencies, despite a common unit labeling system . Adverse effects are a consequence of the drug's mechanism of action, and can usually be tolerated or mitigated through dosing changes . Patients who are pregnant or lactating, or who have a neuromuscular disease, may not be appropriate candidates for BTX therapy . Development of resistance to BTX-A therapy, characterized by absence of any beneficial effect and by lack of muscle atrophy following the injection, is an important clinical issue . The incidence of antibody-mediated resistance, as determined by the mouse lethality assay, is reported between 3% and 10% . Use of the smallest possible effective dose and longer treatment intervals may reduce the likelihood of antibody development . Other serotypes may benefit those who have developed antibody resistance.

Nucleic Acids Res, 1998 Dec 1, 26(23), 5300 - 9
The hyperthermophilic bacterium Thermotoga maritima has two different classes of family C DNA polymerases: evolutionary implications; Huang YP et al.; Bacterial DNA polymerase III (family C DNA polymerase), the principal chromosomal replicative enzyme, is known to occur in at least three distinct forms which have provisionally been classified as class I ( Escherichia coli DNA pol C-type), class II ( Bacillus subtilis DNA pol C-type) and class III (cyanobacteria DNA pol C-type) . We have identified two family C DNA polymerase sequences in the hyperthermophilic bacterium Thermotoga maritima . One DNA polymerase consisting of 842 amino acid residues and having a molecular weight of 97 213 belongs to class I . The other one, consisting of 1367 amino acid residues and having a molecular weight of 155 361, is a member of class II . Comparative sequence analyses suggest that the class II DNA polymerase is the principal DNA replicative enzyme of the microbe and that the class I DNA polymerase may be functionally inactive . A phylogenetic analysis using the class II enzyme indicates that T.maritima is closely related to the low G+C Gram-positive bacteria, in particular to Clostridium acetobutylicum, and mycoplasmas . These results are in conflict with 16S rRNA-based phylogenies, which placed T.maritima as one of the deepest branches of the bacterial tree.

Infect Immun, 1998 Dec, 66(12), 5897 - 905
Characterization of membrane-associated Clostridium perfringens enterotoxin following pronase treatment; Wieckowski EU et al.; After binding, Clostridium perfringens enterotoxin (CPE) initially localizes in a small (approximately 90-kDa) complex in plasma membranes . This event is followed by formation of a second membrane complex, referred to as large (160-kDa) complex . Contrary to a previous hypothesis proposing that CPE inserts into intestinal brush border membranes (BBMs) when this toxin is localized in the small complex, this study shows that BBMs do not offer CPE localized in the small complex protection from pronase . However, our experiments indicate that BBMs do substantially protect CPE from pronase when this toxin is localized in large complex . Since the onset of CPE-induced permeability alterations closely coincides with large-complex formation, these new results suggest that CPE-induced alterations in permeability may result from pore formation due to the partial membrane insertion of CPE when this toxin is present in large complex.

Infect Immun, 1998 Dec, 66(12), 5698 - 702
TetR is a positive regulator of the tetanus toxin gene in Clostridium tetani and is homologous to botR; Marvaud JC et al.; The TetR gene immediately upstream from the tetanus toxin (TeTx) gene was characterized . It encodes a 21,562-Da protein which is related (50 to 65% identity) to the equivalent genes (botR) in Clostridium botulinum . TetR has the feature of a DNA binding protein with a basic pI (9.53) . It contains a helix-turn-helix motif and shows 29% identity with other putative regulatory genes in Clostridium, i.e., uviA from C . perfringens and txeR from C . difficile . We report for the first time the transformation of C . tetani by electroporation, which permitted us to investigate the function of tetR . Overexpression of tetR in C . tetani induced an increase in TeTx production and in the level of the corresponding mRNA . This indicates that TetR is a transcriptional activator of the TeTx gene . Overexpression of botR/A (60% identity with TetR at the amino acid level) in C . tetani induced an increase in TeTx production comparable to that for overexpression of tetR . However, botR/C (50% identity with TetR at the amino acid level) was less efficient . This supports that TetR positively regulates the TeTx gene in C . tetani and that a conserved mechanism of regulation of the neurotoxin genes is involved in C . tetani and C . botulinum.

Dis Colon Rectum, 1998 Nov, 41(11), 1435 - 49
Clostridium difficile-associated diarrhea and colitis: clinical manifestations, diagnosis, and treatment; Cleary RK; PURPOSE: This review examines the pathogenesis, clinical manifestations, diagnosis, and current medical and operative strategies in the treatment of Clostridium difficile diarrhea and colitis . Prevention and future avenues of research are also investigated . METHODS: A review of the literature was conducted with the use of MEDLINE . RESULTS: C . difficile is a gram-positive, spore-forming bacterium capable of causing toxigenic colitis in susceptible patients, usually those receiving antibiotics . Overgrowth of toxigenic strains may result in a spectrum of disease, including becoming an asymptomatic carrier, diarrhea, self-limited colitis, fulminant colitis, and toxic megacolon . Diagnosis requires a high index of suspicion and depends on clinical data, laboratory stool studies (enzyme-linked immunoabsorbent assay and cytotoxin test), and endoscopy in selected cases . Protocols for treatment of primary and relapsing infections are provided in algorithm format . Discontinuation of antibiotics may be enough to resolve symptoms . Medical management with oral metronidazole or vancomycin is the first-line therapy for those with symptomatic colitis . Teicoplanin, Saccharomyces spp . and Lactobacillus spp., and intravenous IgG antitoxin are reserved for more recalcitrant cases . Refractory or relapsing infections may require vancomycin given orally or other newer modalities . Fulminant colitis and toxic megacolon warrant subtotal colectomy . Cost, in terms of extended hospital stay, medical and surgical management, and, in some cases, ward closure, is thought to be formidable . Review of perioperative antibiotic policies and analysis of hospital formularies may contribute to prevention and decreased costs . CONCLUSION: C . difficile diarrhea and colitis is a nosocomial infection that may result in significant morbidity, mortality, and medical costs . Standard laboratory studies and endoscopic evaluation assist in the diagnosis of clinically suspicious cases . Appropriate perioperative antibiotic dosing, narrowing the antibiotic spectrum when treating infections, and discontinuing antibiotics at appropriate intervals prevent toxic sequelae.

Mol Cell Biochem, 1998 Nov, 188(1-2), 217 - 23
On the mechanism of the phospholipase C-mediated attenuation of cardiolipin biosynthesis in H9c2 cardiac myoblast cells; Xu FY et al.; The effect of phospholipase C treatment on cardiolipin biosynthesis was investigated in intact H9c2 cardiac myoblasts . Treatment of cells with phosphatidylcholine-specific Clostridium welchii phospholipase C reduced the pool size of phosphatidylcholine compared with controls whereas the pool size of cardiolipin and phosphatidylglycerol were unaffected . Pulse labeling experiments with {1,3-3H}glycerol and pulse-chase labeling experiments with {1,3-3H}glycerol were performed in cells incubated or pre-incubated in the absence or presence of phospholipase C . In all experiments, radioactivity incorporated into cardiolipin and phosphatidylglycerol were reduced in phospholipase C-treated cells with time compared with controls indicating attenuated de novo biosynthesis of these phospholipids . Addition of 1,2-dioctanoyl-sn-glycerol, a cell permeable 1,2-diacyl-sn-glycerol analog, to cells mimicked the inhibitory effect of phospholipase C on cardiolipin and phosphatidylglycerol biosynthesis from {1,3-3H}glycerol indicating the involvement of 1,2-diacyl-sn glycerol . The mechanism for the reduction in cardiolipin and phosphatidylglycerol biosynthesis in phospholipase C-treated cells appeared to be a decrease in the activities of phosphatidic acid:cytidine-5'triphosphate cytidylyltransferase and phosphatidylglycerolphosphate synthase, mediated by elevated 1,2-diacylsn-glycerol levels . Upon removal of phospholipase C from the incubation medium, phosphatidylcholine biosynthesis from {methyl-3H}choline was markedly stimulated . These data suggest that de novo phosphatidylglycerol and cardiolipin biosynthesis may be regulated by 1,2-diacyl-sn-glycerol and support the notion that phosphatidylglycerol and cardiolipin biosynthesis may be coordinated with phosphatidylcholine biosynthesis in H9c2 cardiac myoblast cells.

Am J Phys Anthropol, 1998 Nov, 107(3), 285 - 95
Sequence analysis of bacterial DNA in the colon of an Andean mummy; Ubaldi M et al.; We have isolated DNA from 14 tissue samples from the internal organs of an Andean human mummy (10th-11th century A.D.) and have checked the persistence of the original human and bacterial templates using the following main approaches: 1) amino acid racemization test; 2) quantification of mitochondrial DNA copy number; 3) survey of bacterial DNA in the different organs; 4) sequence analysis of bacterial amplicons of different lengths . The results demonstrate that both the original human DNA and the DNA of the bacteria of the mummy gut are preserved . In particular, sequence analysis of two (respectively 100 and 196 bp in length) libraries of bacterial 16s ribosomal RNA gene amplicons from the mummy colon shows that while the shortest amplicons give only modest and biased indications about the bacterial taxa, the longer amplicons allow the identification several species of the genus Clostridium which are typical of the human colon . This work represents a first example of a methodological approach which is applicable, in principle, to many other natural and artificial mummies and might open the way to the study of the structure of the human microbial ecosystem from prehistory to present.

MMWR Morb Mortal Wkly Rep, 1998 Nov 6, 47(43), 928 - 30
Neonatal tetanus--Montana, 1998; Pasteurella multocida toxin increases endothelial permeability via Rho kinase and myosin light chain phosphatase; Institut fur Prophylaxe und Epidemiologie der Kreislaufkrankheiten, Universitat Munchen, Germany . messler@klp.med.uni-muenchen.de

Pasteurella multocida toxin (PMT) has been shown to induce actin reorganization through activation of the GTPase Rho . Here we investigated the involvement of the Rho target proteins Rho kinase and myosin light chain (MLC) phosphatase in the PMT-induced increase in endothelial permeability and the underlying actin reorganization of endothelial cells . Stimulation of endothelial layers with PMT enhanced transendothelial permeability > 10-fold, and this was abolished by pretreatment with the specific Rho inactivator C3 transferase from Clostridium botulinum . The PMT-induced increase in endothelial permeability was associated with 1) inactivation of MLC phosphatase, 2) an increase in MLC phosphorylation, and 3) endothelial cell retraction and actin stress fiber formation . PMT-stimulated actin reorganization could be prevented by 1) pretreatment of cells with C3 transferase, 2) microinjection of the Rho binding domain and the pleckstrin homology domain of Rho kinase, and 3) microinjection of constitutively active MLC phosphatase . Together, these results suggest that PMT activates Rho/Rho kinase, which inactivates MLC phosphatase . The resulting increase in MLC phosphorylation causes endothelial cell retraction and a rise in endothelial permeability.

Rofo, 1998 Oct, 169(4), 402 - 7
{Bacteremia in intra-arterial angiography, percutaneous transluminal angioplasty and percutaneous transhepatic cholangio-drainage}; Wagner HJ et al.; PURPOSE: Prospective evaluation of the rate of bacteremia attributed to invasive radiological techniques . METHODS: Aerobic and anerobic blood cultures were obtained in 100 patients (62 men, 38 women; mean age 65 +/- 14 years) undergoing intra-arterial angiography (N = 50), PTA (N = 30) or percutaneous transhepatic biliary drainage (PTCD; N = 20) . Samples were taken before the treatment (T0), immediately after puncture of the vessel or bile duct (T2), and 30 min after the termination of the procedure (T3) . RESULTS: The overall rate of bacteremia was 18% . During diagnostic angiography a 16% rate of temporary bacteremia (no positive T3 samples) was observed . During PTA the rate was 27% (no clinically significant infectious disease) and during PTCD the rate was 10% (5% cholangitis with septicemia) . We isolated staphylococci (S . epidermidis: N = 7, S . species: N = 3, S . aureus: N = 1), streptococci (N = 2), Propionibacterium acnes (N = 5), E . coli (N = 1), Enterococcus faecium (N = 1), Enterobacter species (N = 1), and Clostridium perfringens (N = 1) . Apart from the one patient with cholangitis no clinical infectious complication occurred . CONCLUSION: Temporary bacteremia is rather frequent during invasive radiological procedures . Strictly aseptic conditions and antibiotic prophylaxis, specially in case of implantation of a permanent foreign body, is warranted.

J Infect, 1998 Jul, 37 Suppl 1, 51 - 4
Implementation of sequential therapy programs--a microbiologist's view; Wilcox MH; Sequential antimicrobial therapy is not new, but confusion about the timing and nature of the switch often negates perceived advantages . A common problem is the choice of oral antibiotic to follow empirical administration of an intravenous second or third generation cephalosporin . Where guidelines do not exist, particularly when data are lacking as the the best option, the Delphi technique of obtaining a consensus agreement by review of a series of case histories is recommended . Majority verdicts are used to determine what is acceptable practice, and as such the approach is also suitable for audit . Savings through reduced drug acquisition costs and shorter lengths of stay have been highlighted . However, other less obvious potential benefits of sequential antimicrobial therapy include reduced incidence of intravascular catheter infection because of shorter line dwell times and less endoluminal contamination . Sequential antimicrobial therapy may also be used as part of a policy to reduce the selective pressure, particularly due to cephalosporin use, for endemic hospital pathogens such as Clostridium difficile and extended spectrum producing gram-negative baccilli.

J Infect, 1998 Jul, 37(1), 48 - 53
Chronic diarrhoea among HIV-infected adult patients in Nairobi, Kenya; Mwachari C et al.; OBJECTIVES: Chronic diarrhoea and wasting are well recognized features of AIDS in Africa . However, because of resource constraints few comprehensive aetiological studies have been conducted in sub-Saharan Africa which have included a broad range of microbiological investigations . We undertook a prospective cross-sectional study of adult patients admitted to a government hospital in Nairobi, Kenya, to determine possible bacterial, mycobacterial, parasitic and viral causes of diarrhoea; to consider which may be treatable; and to relate microbiological findings to clinical outcome . METHODS: Stool specimens from 75 consecutive HIV-seropositive patients with chronic diarrhoea admitted to a Nairobi hospital were subjected to microbiological investigation and results were compared with clinical findings and outcome . Stool samples were cultured for bacteria and mycobacteria and underwent light and electron microscopy; lawns of Escherichia coli were probed for pathogenic types and aliquots were tested for the presence of Clostridium difficile cytotoxin . Blood cultures for mycobacteria and other bacterial pathogens were performed as clinically indicated . RESULTS: Thirty-nine (52%) patients yielded putative pathogens, the most common being Cryptosporidium sp . (17%), Salmonella typhimurium (13%), and Mycobacterium tuberculosis (13%) . Of 41 patients investigated for pathogenic Escherichia coli, enteroaggregative E . coli and diffusely adherent E . coli were each found in four patients . Thirty-one (41%) patients died . Detection of cryptosporidium cysts was the single most significant predictor of death (X2 = 5.2, P<0.05) . Many patients did not improve (21; 28%) or self-discharged whilst still sick (5; 7%) but five (7%) were diagnosed ante mortem with tuberculosis and treated and a further 13 (17%) showed improvement by time of discharge . CONCLUSIONS: HIV-infected patients with chronic diarrhoea in Nairobi have a poor outcome overall, and even with extensive investigation a putative pathogen was identified in only just over half the patients . The most important step is to exclude tuberculosis; and the most useful investigation appears to be Ziehl-Neelsen staining . Other potentially treatable gram-negative bacterial pathogens, S . typhimurium, Shigella sp . and adherent E . coli were, however, common but require culture facilities which are not widely accessible for definitive identification . Further studies focussing on simple ways to identify sub-groups of patients with treatable infections are warranted.

J Infect, 1998 May, 36(3), 287 - 8
Community-acquired Clostridium difficile infection; Kyne L et al.; Clostridium difficile-associated disease (CDAD) is primarily a nosocomial condition . Community-acquired disease has been reported but the incidence is felt to be low and the rate of disease resulting in hospitalization is reported as negligible . We recently experienced a 6-month outbreak of CDAD (January to June 1995): 139 patients were involved and four deaths were attributable to pseudomembranous colitis . Early in the outbreak period we were aware that many new admissions presented with C . difficile cytotoxin B positive diarrhoea: in some cases this was the sole reason for hospitalization . This observation forms the basis of this report.

J Infect, 1998 Mar, 36(2), 171 - 4
The lack of therapeutic effect of Saccharomyces boulardii in the prevention of antibiotic-related diarrhoea in elderly patients; Lewis SJ et al.; Diarrhoea is a common side effect of antibiotic therapy, especially in the elderly . Saccharomyces boulardii is a non-pathogenic yeast which has been demonstrated to reduce the frequency of diarrhoea in patients due to a variety of causes . We set out to assess its role in preventing antibiotic-related diarrhoea . Consecutive patients over the age of 65 admitted to medical wards, and who were being prescribed antibiotics, were randomized to receive either S . boulardii 113 g twice daily or placebo for as long as they received antibiotics . Bowel habit was monitored using a record of interdefaecatory intervals (IDI) and stool form graded 1-4 (hard to liquid) . Stool samples were tested every fourth day for Clostridium difficile toxin . Of the 72 patients randomized, 69 completed the study . There was no difference in sex, age, duration of antibiotic use, length of hospital stay, IDI, stool form, the proportion of patients receiving laxatives, the number of patients experiencing watery stools (seven vs . five), or the presence of C . difficile toxin (five vs . three) . No side effects were attributable to S . boulardii . There was no evidence that the concomitant use of S . boulardii with antibiotics alters patients' bowel habits or prevents the appearance of C . difficile toxin in the stool . Thus, S . boulardii cannot be recommended as a 'natural' way to prevent antibiotic-related diarrhoea . This highlights the need for proper evaluation of probiotics before their unrestricted use in medical practice.

Dtsch Med Wochenschr, 1998 Oct 23, 123(43), 1274 - 8
{Probiotic therapy of pseudomembranous colitis . Combination of intestinal lavage and oral administration of Escherichia coli}; Goerg KJ et al.; HISTORY AND FINDINGS: An 82-year-old woman was admitted because of acute left heart failure with pulmonary oedema . There was a right basal pneumonia with congestion which was treated with amoxycillin and clavulanic acid . Severe watery diarrhoea with more than 10 stools daily occurred . Clostridium difficile was not isolated . INVESTIGATIONS: Coloscopy as far as the right flexure revealed proximally increasing whitish coatings, like those in severe pseudomembranous colitis (PMC), revealed as acute erosive colitis histologically . The plate-like incomplete erosions contained fibrin and granulocyte deposits so typical of PMC . Clostridium difficile could again not be demonstrated . TREATMENT AND COURSE: As intestinal lavage, already undertaken in preparation of the coloscopy, markedly improved the symptoms it was repeated for therapeutic purposes . This brought about further improvement and a fall in granulocyte count from 29,500 G/l to 10,700 G/l . Subsequently live E . coli bacteria (nissle 1917 strain) were administered . After 5 days stool frequency had fallen to one or two daily, each soft or partly formed . Stools were normal after a further week . Coloscopy 3 weeks after onset of treatment showed almost complete healing of the PMC . CONCLUSION: The successful treatment of PMC with intestinal lavage and physiological E . coli administration agrees with the results of animal experiments and clinical experience . Whether it is an effective alternative to primary standard treatment with vancomycin or metronidazole remains to be tested by further experience, preferably properly controlled therapeutic trials.

Clin Cancer Res, 1997 Dec, 3(12 Pt 1), 2337 - 45
High-dose infusional doxorubicin and cyclophosphamide: a feasibility study of tandem high-dose chemotherapy cycles without stem cell support; Morgan RJ Jr et al.; The purpose of this study was to determine the maximally tolerated dose of doxorubicin administered during two cycles of intensive chemotherapy with cyclophosphamide and doxorubicin without stem cell support in patients with advanced cancer and to assess the cumulative cardiac toxicity of the regimen by noninvasive radionuclide imaging and by pre-and postchemotherapy endomyocardial biopsies . Thirty-eight patients (thirty-six with high risk or metastatic breast cancer) were treated in a dose-escalation trial using a fixed dose of i.v . cyclophosphamide (4.2 g/m2) administered over 2 h on day 5 and escalating doses of doxorubicin (50-175 mg/m2) given as a 96-h continuous i.v . infusion on days 1-4, using Filgrastim (granulocyte colony-stimulating factor) for hematological support beginning on day 6 . All patients underwent pretreatment, and 28 patients underwent postchemotherapy endomyocardial biopsies . Twenty-nine of 38 patients received two cycles of treatment (median number of days between cycles, 44; range, 34-62) . Twenty-one patients had received doxorubicin previously at cumulative dose levels </=150 mg/m2; all patients had pretreatment endomyocardial biopsy scores less than 1 . One patient treated at the highest dose level of doxorubicin (175 mg/m2) developed symptoms of mild congestive heart failure following two cycles of chemotherapy . Pre- and posttreatment radionuclide ejection fractions were 65 and 45%, respectively; this patient had a posttreatment endomyocardial biopsy score of 1 (damage to <5% of myocytes) . One additional patient at this dose level had an asymptomatic biopsy score of 1, with a decrease in ejection fraction from 62 to 43%; this recovered to 58% 5 months after completion of chemotherapy . Six additional patients treated at lower dose levels had abnormal posttreatment endomyocardial biopsies without abnormal posttreatment ejection fractions . Nine patients received only one cycle of chemotherapy: five patients due to decreased cardiac ejection fraction following cycle 1 (two of these patients had normal endomyocardial biopsies, and two patients had biopsy scores of 1); one patient secondary to tumor progression following cycle one; one patient due to persistently detectable Clostridium difficile toxin in the stool; one patient refused cycle two; and one patient died following cycle one of complications related to sepsis . A single patient experienced a grand mal seizure associated with orthostatic hypotension, which was considered the dose-limiting toxicity . The median duration (over two cycles) of granulocytopenia (absolute granulocyte count <500/microliter) at the maximally tolerated dose level of 150 mg/m2 was 8.5 days (range, 5-13 days), and the median duration of thrombocytopenia (platelets <20,000/microliter) was 2.5 days (range, 0-9 days) . The median duration of hospitalization including chemotherapy administration was 23 days (range, 19-36 days) . Other toxicities included stomatitis, fever, diarrhea, and emesis . One patient developed acute leukemia 54 months posttreatment . We conclude that two courses of high-dose cyclophosphamide and doxorubicin using granulocyte colony-stimulating factor are feasible and safe with tolerable myocardial toxicity as evidenced by serial endomyocardial biopsies . The dose-limiting toxicity encountered was a grand mal seizure . The recommended Phase II dose is doxorubicin 150 mg/m2 administered as a 96-h infusion on days 1-4, with cyclophosphamide 4 . 2 g/m2 on day 5 and G-CSF 5 microgram/kg/day started on day 6 and administered until the total WBC is above 10,000/microliter for three consecutive days.

Chem Res Toxicol, 1998 Nov, 11(11), 1361 - 7
Nitroreduction of nitrated and C-9 oxidized fluorenes in vitro; Ritter CL et al.; Widespread environmental pollution with mutagenic and carcinogenic nitrofluorenes contributes to human health risks . Since nitroreduction leads to activation of many nitro compounds, nitroreduction of the nitrofluorene (NF) derivatives by one- and two-electron reductants was examined . Rates of nitroreduction catalyzed by xanthine oxidase (XO)/hypoxanthine and measured via stimulation of acetylated cytochrome c reduction increased with the number of nitro groups and oxidation at C-9: 9-oxo-2,4,7-triNF > 9-oxo-2,7-diNF > 2,7-diNF > 9-oxo-2-NF = 2,5-diNF > 9-hydroxy-2-NF > 2-NF . Ascorbate catalyzed one-electron reduction to nitro anion radicals which reacted with molecular O2 to yield superoxide . Rates of O2 uptake with 9-oxo-2,4,7-triNF and 9-oxo-2,7-diNF were 63 and 0.17 times those, respectively, with equivalent concentrations of nitrofurazone, a classical substrate . Superoxide formation was indicated by the approximately 75% regeneration of O2 upon addition of superoxide dismutase and catalase . 9-Oxo-2,4,7-triNF stimulated O2 uptake in the presence of XO/NADH with typical Michaelis-Menten kinetics with an apparent Km of 0.476 +/- 0.054 microM versus a Km of 6.18 +/- 0.719 microM for nitrofurazone . HPLC analyses of products from reduction catalyzed by XO or diaphorase of Clostridium with NADH showed the following trends for the rates of amine formation from 9-oxo-2,7-diNF > 2,7-diNF; 9-oxo-2-NF > 9-hydroxy-2-NF > 2-NF; 2,7-diNF > 2-NF; and 9-oxo-2,7-diNF > 9-oxo-2-NF . Little or no amine was formed in 95% O2, suggesting O2-labile intermediates . The data herein suggest that oxidation at C-9 and multiple nitro groups increase the potential for nitroreduction of the nitrofluorenes in vivo which may lead to genotoxic effects.

J Wildl Dis, 1998 Oct, 34(4), 830 - 3
The inhibition of Clostridium botulinum type C by other bacteria in wetland sediments; Sandler RJ et al.; Bacteria with inhibitory activity against Clostridium botulinum type C were isolated from 32% of sediment samples (n = 1600) collected from 10 marshes in a northern California wetland over a 12 mo period . Aerobic and anaerobic bacteria with inhibitory activity were isolated from 12% and 23% of the samples, respectively . Bacteria with inhibitory activity were isolated from all 10 study sites and throughout the year . This study demonstrates that bacteria with inhibitory activity against C . botulinum type C occur naturally in wetland sediments.

J Wildl Dis, 1998 Oct, 34(4), 744 - 51
Preliminary evaluation of a simple in vitro test for the diagnosis of type C botulism in wild birds; Rocke TE et al.; An enzyme-linked immunosorbent assay (ELISA) was developed for the detection of type C botulinum toxin (Clostridium botulinum) in wild birds . This simple, antigen-capture ELISA utilizes polystyrene immunosticks as the solid substrate, chicken antitoxin (IgY) as the coating antibody, rabbit antitoxin as the primary antibody, and peroxidase-labeled goat-anti-rabbit as the secondary antibody . To evaluate the immunostick ELISA as a diagnostic test for avian botulism, known concentrations of toxin were added to heparinized blood collected from healthy birds and tested by both the ELISA and mouse bioassay . Also, blood samples from 236 bird carcasses submitted to the National Wildlife Health Center (NWHC) for cause of death determinations were tested by both procedures . Using < or = 0.5 ml as the test volume for both procedures, the ELISA was less sensitive, detecting 0.25 ng/ml of toxin compared to 0.12 ng/ml for the mouse bioassay . Using the same volume of test sample for diagnostic submissions (< or = 0.5 ml), the ELISA was positive for 60% of the 149 clinically-diagnosed cases of botulism, whereas the mouse bioassay was positive for 79% . However, we demonstrated that with larger sample volumes (> or = 1.0 ml), the sensitivity of the ELISA may be equivalent or better than the mouse test due to the concentrating effect of the ELISA procedure . These preliminary results suggest that when adequate sample volumes are available, the immunostick ELISA can replace the mouse test for the diagnosis of botulism in wild birds.

Lett Appl Microbiol, 1998 Oct, 27(4), 219 - 23
A PCR survey of psychrotrophic Clostridium botulinum-like isolates for the presence of BoNT genes; Broda DM et al.; Isolates (259) of psychrotrophic Clostridium spp . associated with either blown pack spoilage (five isolates) or slaughter stock (254 isolates) were screened for the presence of botulinum neurotoxin (BoNT) genes using degenerate PCR primers capable of amplifying A, B, E, F and G BoNT genes . No BoNT gene amplification products were detected using DNA templates from the 259 psychrotrophic isolates, including 249 isolates that showed the same 16S rRNA gene Restriction Fragment Length Polymorphism (RFLP) patterns as authentic Cl . botulinum type B . It is concluded that although the growth of such micro-organisms in vacuum-packed chilled meat leads to product spoilage, it does not prejudice product safety.

Zentralbl Bakteriol, 1995 Oct, 282(4), 442 - 8
Fibronectin and laminin binding of eighteen Clostridium species; Kreutz C et al.; The ability of Clostridium difficile, Clostridium perfringens, Clostridium sporogenes and fifteen other Clostridium species to bind to human serum fibronectin or laminin was tested by using protein-coated latex particles . Three groups of Clostridium species were formed, namely the pseudomembranous colitis-causing species Clostridium difficile, the gas gangrene-causing Clostridium species and other Clostridium species, which are infrequently found in human infections . Significantly more strains of gas gangrene-causing Clostridium species, and strains of Clostridium species other than Clostridium difficile recognized fibronectin or laminin than did Clostridium difficile . Experiments with monoclonal antibodies revealed the specificity of the bacterial binding to fibronectin or laminin.

J Am Vet Med Assoc, 1998 Nov 1, 213(9), 1305 - 7, 1280
Enterotoxigenic Clostridium perfringens type A necrotic enteritis in a foal; Bueschel D et al.; A Thoroughbred-Quarter Horse crossbred foal developed hemorrhagic enteritis and died < 48 hours after birth . Gross and histologic findings were suggestive of Clostridium perfringens type C infection, and large numbers of C perfringens were isolated from intestinal contents . However, genotyping of isolates indicated that they were enterotoxigenic C perfringens type A, and isolates were found to produce C perfringens enterotoxin in vitro . This case suggests that enterotoxigenic C perfringens type A may cause enteric disease in horses.

Orv Hetil, 1998 Oct 18, 139(42), 2495 - 500
{Botulism . Summary based on six cases}; Adorjan T et al.; Botulism is a rare neuroparalytic disease caused by neurotoxins of Clostridium species . In Hungary it most commonly occurs as a foodborne illness with ocular and bulbar paralysis, muscle weakness and gastrointestinal symptoms . Six cases of botulism were observed by the authors, first in 1993 five members of a family, then in 1997 a patient with sporadic illness . The diagnosis was confirmed by toxin tests in addition to the symptoms and food history . Recognition of the epidemiologic associations proved very useful in the confirmation of outbreak-related cases . The illness was moderately serious at three patients and mild at two patients . One of the patients had a cirrhosis of the liver, and her status became critical because of the repeated bleeding from oesophagus varicose vein . The patient with sporadic illness had a serious gastric dilatation and palsy of bowels causing paralytic ileus at the start of the illness . The symptoms regressed slowly, roughly in three weeks, at all patients . Death did not happened . After the case reports the authors review the disease-microorganism, toxin, clinical entities, incidence, symptoms, diagnosis, differential-diagnosis, and finally the treatment.

Scand J Rheumatol, 1998, 27(5), 357 - 62
Clostridium difficile infection in patients with reactive arthritis of undetermined etiology; Kocar IH et al.; In this study Clostridium difficile infection, which has been reported to induce reactive arthritis (ReA), was investigated in patients with ReA of undetermined etiology . One hundred patients with acute arthritis were included to in the study . The diagnosis of arthritis and/or infectious agents that are capable of causing ReA were determined in 69 of them . The remaining 31 patients (study group) with ReA of undetermined etiology were further investigated for C . difficile Toxin A (CDTA) . The control groups were consisted of hospitalized patients and outpatients who had no history of diarrhea, arthritis, and antibiotic use . CDTA positive patients (19.4% of the study group) were treated only with oral vancomycin and evaluated for the prognosis of diarrhea and/or arthritis . The results strongly suggested C . difficile infection can induce ReA, especially in patients with antibiotic-associated colitis.

Plasmid, 1998 Nov, 40(3), 233 - 7
Conjugative transfer of the Escherichia coli-Clostridium perfringens shuttle vector pJIR1457 to Clostridium botulinum type A strains; Bradshaw M et al.; An RP4-oriT shuttle vector pJIR1457 originally developed for Clostridium perfringens was successfully transferred by conjugation from Escherichia coli to Clostridium botulinum type A strains and to a nontoxigenic C . botulinum type A-transposon Tn916 mutant strain lacking the entire toxin gene cluster . The light chain (LC) of botulinum toxin was highly expressed in the toxin deletion mutant strain from a pJIR1457 construct containing the recombinant botulinal gene for LC . This shuttle vector system will be valuable for genetic analysis of C . botulinum and will enable genetic manipulation and recombinant expression studies of botulinum neurotoxins as pharmaceutical agents .

J Biol Chem, 1998 Nov 13, 273(46), 30836 - 41
ADP-ribosylation factor proteins mediate agonist-induced activation of phospholipase D; Shome K et al.; The role of small G proteins of the ADP-ribosylation factor (ARF) and Rho families on the activation of phospholipase D (PLD) by platelet-derived growth factor (PDGF) and phorbol esters (PMA) has been investigated . The activation of PLD by PDGF and PMA was blocked by brefeldin A (BFA), an inhibitor of ARF activation, but not by Clostridium botulinum C3 exotoxin, an inhibitor of the activity of Rho . PDGF and PMA, in the presence of GTPgammaS, promoted the association of ARF and RhoA with cell membranes . Cells depleted of ARF and Rho by digitonin permeabilization showed a significant reduction of the activity of phospholipase D . Recombinant ARF was sufficient to restore agonist-induced PLD activity to digitonin-permeabilized, cytoplasm-depleted cells . In contrast, isoprenylated recombinant RhoA had no effects in this reconstitution assay . HIRcB cells were transiently transfected with wild-type and dominant-negative mutants of ARF1 and ARF6 . Neither wt-ARF1 nor wt-ARF6 had any effects on agonist-dependent PLD activity . However, dominant-negative ARF1 and ARF6 mutants blocked the stimulation of PLD by PDGF but only partially inhibited the effects of PMA . These results demonstrate that ARF rather than Rho proteins mediate the activation of PLD by PDGF and phorbol esters in HIRcB fibroblasts.

Chirality, 1998, 10(8), 727 - 33
Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor; Bear MM et al.; beta-methylaspartate ammonia-lyase, EC 4.3.1.2, (beta-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b, respectively, from mesaconic acid 1 as substrate, on a large scale . To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated . After three further steps, (2S,3S)-3a yielded the alpha, beta-substituted beta-lactone (3R,4R) 6 with a very high diastereoisomeric excess (> 95% by chiral gas chromatography) . The corresponding crystalline polymer, poly{benzyl beta-(2R,3S)-3-methylmalate} 8, prepared by an anionic ring opening polymerization, was highly isotactic as determined by 13C NMR . Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7, to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications . Because of the ability of beta-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions . These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions.

Microbiol Immunol, 1998, 42(9), 599 - 605
Molecular composition of the 16S toxin produced by a Clostridium botulinum type D strain, 1873; Nakajima H et al.; The 16S toxin was purified from a Clostridium botulinum type D strain 1873 (D-1873) . Furthermore, the entire nucleotide sequences of the genes coding for the 16S toxin were determined . It became clear that the purified D-1873 16S toxin consists of neurotoxin, nontoxic nonhemagglutinin (NTNH), and hemagglutinin (HA), and that HA consists of four subcomponents, HA1, HA2, HA3a, and HA3b, the same as type D strain CB16 (D-CB16) 16S toxin . The nucleotide sequences of the nontoxic components of these two strains were also found to be identical except for several bases . However, the culture supernatant and the purified 16S toxin of D-1873 showed little HA activity, unlike D-CB16, though the fractions successively eluted after the D-1873 16S toxin peak from an SP-Toyopearl 650S column showed a low level of HA activity . The main difference between D-1873 and D-CB16 HA molecules was the mobility of the HA1 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . Therefore it was presumed that the loss of HA activity of D-1873 16S toxin might be caused by the differences of processing HA after the translation.

Appl Microbiol Biotechnol, 1998 Sep, 50(3), 331 - 8
Two novel gene expression systems based on the yeasts Schwanniomyces occidentalis and Pichia stipitis; Piontek M et al.; Two non-Saccharomyces yeasts have been developed as hosts for heterologous gene expression . The celD gene from Clostridium thermocellum, encoding a heat-stable cellulase, served as the test sequence . The first system is based on the amylolytic species Schwanniomyces occidentalis, the second on the xylolytic species Pichia stipitis . The systems comprise auxotrophic host strains (trp5 in the case of S . occidentalis; trp5-10, his3 in the case of P . stipitis) and suitable transformation vectors . Vector components consist of an S . occidentalis-derived autonomously replicating sequence (SwARS) and the Saccharomyces cerevisiae-derived TRP5 sequence for plasmid propagation and selection in the yeast hosts, an ori and an ampicillin-resistance sequence for propagation and selection in a bacterial host . A range of vectors has been engineered employing different promoter elements for heterologous gene expression control in both species . Homologous elements derived from highly expressed genes of the respective hosts appeared to be of superior quality: in the case of S . occidentalis that of the GAM1 gene, in the case of P . stipitis that of the XYL1 gene . Further elements tested are the S . cerevisiae-derived ADH1 and PDC1 promoter sequences.

Int J Food Microbiol, 1998 Sep 8, 43(3), 231 - 5
Use of a broad-host-range bacteriocin-producing Lactococcus lactis transconjugant as an alternative starter for salami manufacture; Coffey A et al.; Lactococcus lactis DPC4268 is widely used for Cheddar cheese manufacture in Ireland where it is recognised for its reliable fast acid producing ability in dairy environments . A transconjugant of this strain, L . lactis DPC4275, was generated which produces the broad spectrum, two-component bacteriocin lacticin 3147, which is inhibitory to a variety of undesirable gram-positive bacteria including Clostridium, Bacillus, Enterococcus, Listeria, Staphylococcus and Streptococcus . Both DPC4268 and DPC4275 were used as single strain starters for manufacture of salamis . These products were compared with a salami manufactured with a conventional starter (Lactobacillus sake and Staphylococcus carnosus) in terms of pH development, a(w)-value, weight loss, colour development and sensory characteristics . Salamis produced with either lactococcal culture exhibited pH values below 5.1 and a(w)-values below 0.90 which is favourable for preservation and hygienic stability . In addition, these salamis had good sensory and colorimetric qualities . A minimum lacticin 3147 concentration of 640 AU/g was detected in the salamis which were produced with L . lactis DPC4275.

Int J Food Microbiol, 1998 Sep 8, 43(3), 173 - 83
Differentiation of lactate-fermenting, gas-producing Clostridium spp . isolated from milk; Ingham SC et al.; Endospores of Clostridium spp . capable of producing gas in a lactate-containing medium were enumerated from 14 pasteurized milk samples from Wisconsin cheese plants . Concentrations of endospores of lactate-fermenting, gas-producing Clostridium spp . were between 5.0 x 10(-2) and 1.7 x 10(0) MPN ml(-1) . Concentrations of presumptive C . tyrobutyricum endospores (defined by subterminal endospore position and lactate dehydrogenase activity) were lower, not exceeding 2.0 x 10(-2) MPN ml(-1) . Based on subterminal endospore position, lactate dehydrogenase activity, and a carbohydrate fermentation profile identical to C . tyrobutyricum strain ATCC 25755, five isolates (Ct) were initially characterized as C . tyrobutyricum, a known cause of late-blowing in high-pH cheeses . Twenty-eight other isolates (Cx) produced gas from lactate, but differed from ATCC 25755 in either endospore position, lactate dehydrogenase activity or carbohydrate fermentation profile . When inoculated at high concentrations in Gouda cheese, strain ATCC 25755, two Ct isolates and 18 Cx isolates tested produced gas during ripening . Among the five Ct isolates obtained and two reference strains confirmed as C . tyrobutyricum, there were four qualitatively different volatile organic acid byproduct profiles . Each of the two confirmed C . tyrobutyricum reference strains and five Ct isolates had distinct quantitative cell membrane fatty acid (CMFA) profiles . The Cx isolates represented 14 different volatile organic acid byproduct profiles and each isolate had a unique CMFA profile . Pulsed field gel electrophoresis (PFGE) of DNA from the two confirmed reference C . tyrobutyricum strains, four Ct and three Cx isolates, showed a low degree of relatedness . The results of this study suggest that a heterogeneous group of lactate-fermenting, gas-producing Clostridium spp . may be found in milk . Gas chromatographic analysis of volatile organic acid byproducts or CMFA, and PFGE of DNA are highly discriminating methods for differentiating Clostridium spp . that may cause late blowing in high-pH cheeses.

Clin Geriatr Med, 1998 Nov, 14(4), 681 - 98
Prevention of iatrogenic illness: adverse drug reactions and nosocomial infections in hospitalized older adults; Riedinger JL et al.; Adverse drug reactions and nosocomial infections are two important aspects of iatrogenesis in hospitalized older adults . Adverse drug reactions are related to changes in pharmacodynamics and pharmacokinetics that occur with aging as well as polypharmacy . Strategies for limiting iatrogenesis due to medications are discussed . Nosocomial infections continue to complicate older inpatients' care despite advances in understanding and treating institutional infections . In particular, urinary tract infections, nosocomial pneumonias, and Clostridium difficile-associated diarrhea are potentially preventable.

Arch Microbiol, 1998 Nov, 170(6), 442 - 50
Sarcosine reductase of Tissierella creatinophila: purification and characterization of its components; Harms C et al.; Sarcosine reductase is the only reductase system present in Tissierella creatinophila when grown on creatinine plus formate . The acetyl-phosphate-forming component protein C was purified to homogeneity . SDS-PAGE of the purified protein revealed two protein bands with apparent mol . masses of 62 and 50 kDa . The N-terminal amino acid sequence of the two subunits was determined . Antibodies raised against each of the subunits of protein C from Eubacterium acidaminophilum cross-reacted with the corresponding protein present in T . creatinophila, Clostridium litorale and Clostridium sporogenes . The arsenate-dependent hydrolysis of acetyl phosphate catalyzed by protein C was partly inhibited by antibodies directed against the large subunit . Antibodies raised against the small subunit were twice as effective, which indicates that this subunit is the primary site of acetyl transfer from acetyl phosphate . The protein A component of the sarcosine reductase of T . creatinophila was purified to homogeneity by cochromatography with thioredoxin reductase on DEAE-Sephacel, hydroxylapatite, Q-Sepharose, and Sephacryl 100-HR . Protein A had an apparent mol . mass of 21 kDa . Its N-terminal amino acid sequence showed high similarities to that of other proteins A . Initial steps for the purification and preliminary characterization of the sarcosine-specific, substrate-binding protein Bsarcosine component of T . creatinophila indicated the involvement of a 50-kDa protein.

Arch Microbiol, 1998 Nov, 170(6), 427 - 34
Metabolism of aromatic aldehydes as cosubstrates by the acetogen Clostridium formicoaceticum; Frank C et al.; When the acetogen Clostridium formicoaceticum was cultivated on mixtures of aromatic compounds (e.g., 4-hydroxybenzaldehyde plus vanillate), the oxidation of aromatic aldehyde groups occurred more rapidly than did O-demethylation . Likewise, when fructose and 4-hydroxybenzaldehyde were simultaneously provided as growth substrates, fructose was utilized only after the aromatic aldehyde group was oxidized to the carboxyl level . Aromatic aldehyde oxidoreductase activity was constitutive (activities approximated 0 . 8 U mg-1), and when pulses of 4-hydroxybenzaldehyde were added during fructose-dependent growth, the rate at which fructose was utilized decreased until 4-hydroxybenzaldehyde was consumed . Although 4-hydroxybenzaldehyde inhibited the capacity of cells to metabolize fructose, lactate or gluconate were consumed simultaneously with 4-hydroxybenzaldehyde, and lactate or aromatic compounds lacking an aldehyde group were utilized concomitantly with fructose . These results demonstrate that (1) aromatic aldehydes can be utilized as cosubstrates and have negative effects on the homoacetogenic utilization of fructose by C . formicoaceticum, and (2) the consumption of certain substrates by this acetogen is not subject to catabolite repression by fructose.

EMBO J, 1998 Nov 2, 17(21), 6219 - 29
Fc receptor-mediated phagocytosis requires CDC42 and Rac1; Massol P et al.; At the surface of phagocytes, antibody-opsonized particles are recognized by surface receptors for the Fc portion of immunoglobulins (FcRs) that mediate their capture by an actin-driven process called phagocytosis which is poorly defined . We have analyzed the function of the Rho proteins Rac1 and CDC42 in the high affinity receptor for IgE (FcepsilonRI)-mediated phagocytosis using transfected rat basophil leukemia (RBL-2H3) mast cells expressing dominant inhibitory forms of CDC42 and Rac1 . Binding of opsonized particles to untransfected RBL-2H3 cells led to the accumulation of F-actin at the site of contact with the particles and further, to particle internalization . This process was inhibited by Clostridium difficile toxin B, a general inhibitor of Rho GTP-binding proteins . Dominant inhibition of Rac1 or CDC42 function severely inhibited particle internalization but not F-actin accumulation . Inhibition of CDC42 function resulted in the appearance of pedestal-like structures with particles at their tips, while particles bound at the surface of the Rac1 mutant cell line were enclosed within thin membrane protrusions that did not fuse . These phenotypic differences indicate that Rac1 and CDC42 have distinct functions and may act cooperatively in the assembly of the phagocytic cup . Inhibition of phagocytosis in the mutant cell lines was accompanied by the persistence of tyrosine-phosphorylated proteins around bound particles . Phagocytic cup closure and particle internalization were also blocked when phosphotyrosine dephosphorylation was inhibited by treatment of RBL-2H3 cells with phenylarsine oxide, an inhibitor of protein phosphotyrosine phosphatases . Altogether, our data show that Rac1 and CDC42 are required to coordinate actin filament organization and membrane extension to form phagocytic cups and to allow particle internalization during FcR-mediated phagocytosis . Our data also suggest that Rac1 and CDC42 are involved in phosphotyrosine dephosphorylation required for particle internalization.

Clin Infect Dis, 1998 Oct, 27(4), 702 - 10
Mechanisms and management of antibiotic-associated diarrhea; Hogenauer C et al.; Only 10%-20% of all cases of antibiotic-associated diarrhea (AAD) are caused by infection with Clostridium difficile . Other infectious organisms causing AAD include Clostridium perfringens, Staphylococcus aureus, Klebsiella oxytoca, Candida species, and Salmonella species . Most of the clinically mild AAD cases are due to functional disturbances of intestinal carbohydrate or bile acid metabolism, to allergic and toxic effects of antibiotics on intestinal mucosa, or to pharmacological effects on motility . Saccharomyces boulardii and Enterococcus SF68 can reduce the risk of developing AAD . Patients receiving antibiotic treatment should avoid food containing high amounts of poorly absorbable carbohydrates . Mild cases of AAD that may or may not be caused by C . difficile can be resolved by discontinuation of antibiotic therapy and by dietary carbohydrate reduction . Only severe AAD caused by C . difficile requires specific antibiotic treatment.

Infection, 1998 Sep-Oct, 26(5), 309 - 10
Clostridium septicum myonecrosis presenting as a parapharyngeal abscess in a patient with aplastic anemia; Fowler VG Jr et al.; The first reported case of Clostridium septicum myonecrosis in an adult with aplastic anemia is described . The patient presented with sepsis, a parapharyngeal abscess that necessitated emergent intubation, and severe intravascular hemolysis attributed to clostridial alpha-toxin production . Despite prompt recognition and treatment, the patient died of his infection . C . septicum myonecrosis should be considered in any immunocompromised patient with sepsis, especially when accompanied by evidence of multiple sites of tissue infection.

Appl Environ Microbiol, 1998 Nov, 64(11), 4416 - 22
Analysis of the influence of environmental parameters on Clostridium botulinum time-to-toxicity by using three modeling approaches; Schaffner DW et al.; This study used the technique of waiting time modeling to analyze the combined effects of temperature, pH, carbohydrate, protein, and lipid on the time-to-toxicity of Clostridium botulinum 56A . Waiting time models can be used whenever the time to the occurrence of some event is the variable of interest . In the case of the time-to-toxicity data, the variable is the time from the beginning of an experiment until a tube is identified as positive . The statistical analysis used the SAS procedure LIFEREG and included determination of the form of the response surface, identification of the error distribution, and simplification of the response surface . We found that increasing the macromolecule concentration decreased the probability of toxin formation . The probability of toxin formation also decreased at lower temperatures and at pHs further from the optimum . The waiting time modeling approach to developing models for botulinal toxin formation compared favorably with other approaches but had one specific advantage . Waiting time models have the inherent advantage that safety concerns regarding predictions are automatically quantified in the analysis by formally identifying a distribution of times-to-toxicity . The use of this time-to-toxicity distribution permits a customizable margin of safety (e.g., one in a million) not possible with other approaches.

Appl Environ Microbiol, 1998 Nov, 64(11), 4161 - 7
Prevalence of Clostridium botulinum in Finnish trout farms: pulsed-field gel electrophoresis typing reveals extensive genetic diversity among type E isolates; Hielm S et al.; The distribution of Clostridium botulinum serotypes A, B, E, and F in Finnish trout farms was examined . A total of 333 samples were tested with a neurotoxin-specific PCR assay . C . botulinum type E was found in 68% of the farm sediment samples, in 15% of the fish intestinal samples, and in 5% of the fish skin samples . No other serotypes were found . The spore counts determined by the most-probable-number method were considerably higher for the sediments than for the fish intestines and skin; the average values were 2,020, 166, and 310 C . botulinum type E spores kg-1, respectively . The contamination rates in traditional freshwater ponds and marine net cages were high, but in concrete ponds equipped with sediment suction devices the contamination rates were significantly lower . Pulsed-field gel electrophoresis (PFGE) typing of 42 isolates obtained in this survey and 12 North American reference strains generated 28 pulsotypes upon visual inspection, suggesting that there was extensive genetic diversity and that the discriminatory power of PFGE typing in C . botulinum type E was high . A numerical analysis of SmaI-XmaI macrorestriction profiles confirmed these findings, as it divided the 54 isolates into 15 clusters at a similarity level of 76% . For this material, this level of similarity corresponded to a three-band difference in the macrorestriction profiles, which indicated that there is no genotypic proof of a close epidemiological relationship among the clusters.

Vaccine, 1998 Nov, 16(19), 1850 - 6
Identifying the principal protective antigenic determinants of type A botulinum neurotoxin; Bavari S et al.; The neurotoxins from Clostridium botulinum (BoNT serotypes A-G) exert their lethal effect by preventing the release of acetylcholine at the neuromuscular junction . As with tetanus toxin, immunization with a non-toxic fragment, the 50 kDa C-terminal portion of BoNT/A (Hc; residues 861-1296), protects mice against lethal challenges with the intact toxin . To locate the neutralizing epitopes, several protective monoclonal antibodies (mAbs) against BoNT/A-Hc were isolated and cloned . Specific binding of the mAbs to BoNT/A-Hc was demonstrated by surface plasmon resonance, with Kas in the range of 10(-10) to 10(-11) M . These antibodies recognized a genetically engineered polypeptide (1150-1289) that was previously shown to induce protective immunity . Prior to the determination of the X-ray crystal structure of the tetanus neurotoxin Hc fragment, molecular modelling studies indicated that it contained two highly solvent-exposed loops . Based on these predictions, two 25-mer Hc-peptides corresponding to these two regions were synthesized and were demonstrated to bind the neutralizing mAbs . Mice immunized with the Hc-peptides had high levels of antibodies that recognized BoNT/A-Hc . However, immunizations with only one of the Hc peptides protected when mice were challenged with BoNT/A . On the basis of these analyses, it should be possible to develop small peptides that could be useful in the design of future vaccines against these neurotoxins.

Rev Esp Quimioter, 1998 Sep, 11(3), 221 - 8
Antimicrobial susceptibility of anaerobic bacteria from the intestinal microflora of healthy children and antimicrobial-treated children in Nicaragua; Caceres M et al.; The objective of this study was to determine the antimicrobial susceptibility of anaerobic bacteria from the intestinal microflora of healthy children who had not been treated with antimicrobial agents since birth or at 1, 3, 6, 12, 18 and 24 months of age, as well as from children of the same ages treated with the most commonly used antimicrobial agents in Nicaragua . A total of 947 Bacteroides and 745 Clostridium strains were isolated from 67 healthy and 94 antimicrobial-treated children . The minimal inhibitory concentrations of ampicillin, cefoxitin, imipenem, clindamycin, metronidazole and chloramphenicol were determined by the agar dilution method . Detection of ss-lactamase was made by the nitrocefin assay . No bacterial strains resistant to imipenem, clindamycin, metronidazole or chloramphenicol were found . The susceptibility of Bacteroides species to ampicillin and cefoxitin isolated from antimicrobial-treated children decreased progressively as the children reached 24 months of age, from 88% to 78% and from 94% to 81%, respectively . All the Bacteroides strains isolated from the healthy children were 100% susceptible to cefoxitin when they were <=12 months and 92% susceptible after this age; the susceptibility of Bacteroides strains to ampicillin in these children was from 91% at 1 month to 86% at 24 months of age . All Clostridium strains were susceptible to ampicillin and cefoxitin . The ss-lactamase production was seen only in Bacteroides species . These data indicate that a rational use of antimicrobial agents is needed to avoid the development of resistance in anaerobic bacteria.

Vestn Otorinolaringol, 1998, (5), 43 - 5
{Clinical aspects and treatment of purulent anaerobic non-Clostridium ethmoid sinusitis}; Mirazizov KD et al.; The ethmoidal sinus was punctured for pus samples in 120 patients with purulent ethmoiditis . The sinus of 47.5% of the patients contained non-clostridial anaerobic infection . The species of the microflora and its sensitivity to antibiotics were defined . An effective system of examination and treatment of purulent ethmoiditis caused by anaerobic infection is proposed . The essential elements of this system are: aeration of the paranasal sinuses, immunostimulation and immunomodulation . For non-responders and subjects with frequent recurrences surgical intervention is indicated.

J Biol Chem, 1998 Nov 6, 273(45), 29506 - 11
Characterization of the catalytic site of the ADP-ribosyltransferase Clostridium botulinum C2 toxin by site-directed mutagenesis; Barth H et al.; The actin ADP-ribosylating Clostridium botulinum C2 toxin is a binary toxin composed of the binding component C2II and the enzyme component C2I . C2I ADP-ribosylates G-actin at arginine 177, resulting in the depolymerization of the actin cytoskeleton . Here, we studied the structure-function relationship of C2I by site-directed mutagenesis . Exchange of Glu389 to glutamine caused the complete loss of ADP-ribosyltransferase and NAD-glycohydrolase activities of C2I . In contrast, exchange of Glu387 to glutamine blocked ADP-ribosyltransferase but not NAD-glycohydrolase activity . Whereas photoaffinity labeling of the double mutant E387Q/E389Q C2I with {carbonyl-14C}NAD was blocked, labeling of the single C2I mutants was reduced (E389Q) or not changed (E387Q) . Exchange of the STS motif (amino acid residues 348-350) of C2I caused a decrease in transferase activity by more than 99 (S348A) and 90% (T349V), or did not affect activity (S350A) . Exchange of Arg299 and Arg300 to lysine reduced transferase activity to <0.1 and approximately 35% of wild-type activity . The data indicate that the amino acid residues Glu389, Glu387, Ser348, and Arg299, which are conserved in various prokaryotic and eukaryotic arginine-modifying ADP-ribosyltransferases, are essential for ADP-ribosyltransferase activity of the enzyme component of C . botulinum C2 toxin.

Protein Sci, 1998 Oct, 7(10), 2081 - 8
Structure of the xylanase from Penicillium simplicissimum; Schmidt A et al.; Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching . We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases . Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence . The xylanase was crystallized from ammonium sulfate at pH 8.4, and X-ray data were collected at cryo-temperature to a crystallographic resolution of 1.75 A . The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (R(free) = 23%) for data between 10 and 1.75 A . The xylanase folds in an (alpha/beta)8 barrel (TIM-barrel), with additional helices and loops arranged at the "top" forming the active site cleft . In its overall shape, the P . simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider . This probably accounts for the differences in catalysis and in the mode of action of this enzyme . Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues . It appears that they occupy putative xylose binding subsites.

Biochem Biophys Res Commun, 1998 Oct 9, 251(1), 100 - 5
The enzymatic domain of Clostridium difficile toxin A is located within its N-terminal region; Faust C et al.; Clostridium difficile, an anaerobic pathogen encountered in human enteric disease, produces two major virulence factors, toxins A and B, which are members of a clostridial family of large cytotoxins . These are glucosyltransferases, which use a UDP-sugar as co-substrate to glucosylate and inactivate small GTPases of the Rho or Ras families, culminating in cytotoxicity . Clinically, toxin A is perhaps the most important family member, because it causes major tissue damage in the course of disease, leading to a potentially lethal, pseudomembranous colitis . The location of the enzymatic domain of toxin A and mechanistic details of its action are not yet known, so we wished to localize this domain using gene deletion constructions from the full-length gene and by monitoring glucosylation activity of encoded protein products . Toxin A deletions were obtained by successively truncating the C-terminal coding region . These were transformed into E . coli, cell lysates were prepared and they were assayed for their ability to glucosylate Rho A protein, using an in vitro enzymatic assay . We report that the UDP-glucose binding site, the catalytic site for glucose transfer and the Rho A interaction site occur within the first 659 N-terminal amino acids of toxin A, i.e., less than 25% of the length of holotoxin A . Localization of the enzymatic domain of toxin A to these 659 N-terminal amino acids should greatly simplify studies on mechanistic details of this clinically important toxin .

Drugs Aging, 1998 Sep, 13(3), 245 - 53
Mechanisms of drug-induced diarrhoea in the elderly; Ratnaike RN et al.; In the rapidly increasing elderly population, diarrhoea as a result of drug therapy is an important consideration . The elderly consume a disproportionately large number of drugs for multiple acute and chronic diseases . Drugs can compromise both immune and nonimmune responses . Aging decreases the quality and proportion of T cells which in turn reduces the production of secretory IgA, the primary immune response of the gut . Acid production in the stomach decreases with increasing age and this compromise its vital 'self-sterilising' function, thus increasing the risk of diarrhoea due to viral, bacterial and protozoal pathogens . Other nonimmune defence mechanisms include the motility of the small intestine and the host-protective commensal bacteria of the colon . Drug induced hypomotility may result in bacterial overgrowth, deconjugation of bile salts and diarrhoea . Less commonly, diarrhoea may occur due to hypermotility because of a cholinergic-like syndrome . In the colon the host-protective commensal bacteria provide a powerful defence against pathogens . Disruption of this commensal population by antibiotic therapy may result in Clostridium difficile supra-infection which causes diarrhoea through toxin production . This is especially important in the elderly patient on chemotherapy for malignancy and those with multiple diseases . The organism responds to vancomycin, metronidazole and bacitracin . Metronidazole is the suggested drug of choice, with vancomycin reserved for relapses . Drugs also cause diarrhoea by interfering with normal physiological processes . Drugs impair fluid absorption by activating adenylate cyclase within the small intestinal enterocyte which increases the level of cyclic AMP . This causes active secretion of Cl- and HCO3-, passive efflux of Na+, K+ and water and inhibition of Na+ and Cl- into the enterocyte . Examples of these drugs (secretagogues) are bisacodyl, misoprostol and chenodeoxycholic acid (used to dissolve cholesterol gallstones) . Drugs may also affect a second mechanism that regulates water and electrolyte transport, the Na+, K+ exchange pump . The energy for this pump is provided by the ATPase mediated breakdown of ATP . ATPase may be inhibited by digoxin, auranofin, colchicine and olsalazine . A number of drugs cause osmotic diarrhoea including antacids containing magnesium trisilicate or hydroxide . Lactulose is being used increasingly in compensated liver disease to increase protein tolerance and prevent hepatic encephalopathy . Sorbitol, an osmotic laxative agent also used in some liquid pharmaceutical preparations, induces diarrhoea by virtue of its osmotic potential . Another mechanism by which drugs cause diarrhoea is by mucosal damage of the small and large bowel . In the small intestine mucosal damage causes diarrhoea and fat malabsorption, as may occur with neomycin and colchicine . In the colon, for example, gold salts and penicillamine cause colitis of varying severity . Though the causes of diarrhoea are diverse, a drug-associated aetiology should always be considered and actively sought and addressed to prevent the complications of dehydration, electrolyte imbalance and undernutrition.

J Med Microbiol, 1998 Oct, 47(10), 879 - 88
Binding of Clostridium difficile toxin A to human milk secretory component; Dallas SD et al.; Toxigenic Clostridium difficile is isolated from a majority of healthy human infants . The exact mechanism of asymptomatic colonisation is unclear; however, previous studies in this laboratory have shown that components of both the immunoglobulin and non-immunoglobulin fractions of human milk bind to toxin A and prevent its interaction with hamster intestinal brush border membranes (BBMs) . Secretory IgA (sIgA) is the primary immunoglobulin found in human milk . As sIgA resists digestion in the infant stomach and passes at high levels into the colon, its ability to bind toxin A was the subject of this investigation . Purified sIgA in concentrations at and below those found in human milk inhibited the binding of toxin A to purified BBM receptors . Heating sIgA to 100 degrees C for 5 min did not affect its inhibitory activity . IgM, IgG and serum IgA did not appreciably inhibit the binding of toxin A to BBM receptors . SDS-PAGE separated sIgA into three major bands: secretory component, heavy chains and light chains . Autoradiography with radiolabelled toxin A revealed that toxin A bound to the secretory component (SC) of sIgA . When the three purified subunits of sIgA were coated on to microtitration wells, SC bound significantly more toxin A than the heavy or light chains of sIgA . Purified SC also inhibited toxin binding to receptors in a dose-dependent fashion similar to sIgA . The heavy and light chains of sIgA did not inhibit toxin A receptor binding . Removing carbohydrates from sIgA and SC by enzymic digestion showed that toxin A binds much less to deglycosylated SC than to glycosylated SC . These data suggest that SC in human milk binds to toxin A and may function as a receptor analogue, protecting human infants against C . difficile-associated disease.

Mayo Clin Proc, 1998 Oct, 73(10), 943 - 7
Extraintestinal Clostridium difficile: 10 years' experience at a tertiary-care hospital; Wolf LE et al.; OBJECTIVE: To determine the clinical characteristics of patients with extraintestinal Clostridium difficile (ECD) . MATERIAL AND METHODS: All cultures obtained during a 10.5-year period (from Jan . 1, 1985, to Jun . 30, 1995) at a tertiary-care hospital were retrospectively examined . The medical records of patients from whom ECD was isolated were then reviewed . RESULTS: Fourteen patients from whom ECD was cultured were identified . Thirteen of these patients (93%) had underlying systemic disease . All but one patient had recent exposure to antibiotics, and all had major bowel pathologic conditions . Nine patients had colon perforation . Of the eight patients in whom the colonic mucosa was directly inspected at operation or endoscopy, only two had evidence of pseudomembranous colitis . Five patients (36%) had documentation of recent diarrhea . ECD was isolated from intraperitoneal sites (in nine patients), blood cultures (in three), a perianal abscess, and a prosthetic hip joint . In 13 patients (93%), the infection was polymicrobial . Seven of the 13 inpatients (54%) survived to dismissal . CONCLUSION: C . difficile is a rare isolate outside of the gastrointestinal tract . ECD is found in patients with systemic illness who have been hospitalized (usually for an extended period), have intestinal pathologic conditions, and have received antibiotics . The isolation of ECD portends a poor prognosis.

Am J Emerg Med, 1998 Oct, 16(6), 585 - 91
Aerobic and anaerobic microbiology of infection after trauma; Brook I et al.; Clinical and laboratory data from 1973 to 1988 were retrospectively reviewed to study the microbiology of infection following trauma . A total of 368 specimens obtained from 340 trauma patients showed bacterial growth . The traumas included lacerations (163), blunt trauma (76), penetrating trauma (65), bites (20), and open fractures (10) . Anaerobic bacteria only were isolated in 119 (32%) specimens, aerobic bacteria only in 58 (16%), and mixed aerobic-anaerobic flora in 191 (52%) . A total of 444 anaerobic (1.2 isolates per specimen) and 267 aerobic or facultative (0.7 per specimen) were recovered . The predominant anaerobic bacteria included Bacteroides fragilis group (119 isolates), Peptostreptococcus spp (113), Clostridium spp (78), Prevotella spp (58), and Fusobacterium spp (23) . The predominant aerobic bacteria included Escherichia coli (83), Staphylococcus aureus (61), Streptococcus pyogenes (27), Streptococcus group D (16), and Klebsiella pneumoniae (16) . The types of infections included abscesses (109), bacteremia (32), bites (13), empyema (10), osteomyelitis (21), peritonitis (52), thrombophlebitis (12), and wounds (116, including posttraumatic wounds, cellulitis, stump wound, decubitus ulcers, myositis, and fasciitis) . S . aureus was isolated at all sites . However, organisms of the oropharyngeal flora predominated in infections that originated from that location (ie, head and neck wounds, and abscesses or bites), and those of the gastrointestinal flora predominated in infections that originated from that site (ie, peritonitis, abdominal abscesses, decubitus ulcers) . This study showed the polymicrobial nature of many infections that follow trauma.

Biochim Biophys Acta, 1998 Oct 21, 1405(1-3), 161 - 70
Role of rho proteins in agonist regulation of phospholipase D in HL-60 cells; Guillemain I et al.; Rho family GTP-binding proteins have been demonstrated to play a role in the regulation of phospholipase D (PLD) activity . In the present study, we examined the role of Rho proteins in PLD activation in differentiated HL-60 cells using C3 exoenzyme from Clostridium botulinum, which ADP-ribosylates and inactivates Rho proteins . Introduction of C3 exoenzyme into differentiated HL-60 cells by electroporation resulted in complete inhibition of PLD activity stimulated by formyl methionine-leucine-phenylalanine (fMLP) and ATP, two receptor agonists . Phorbol myristate acetate-induced PLD activation was also inhibited in C3 exoenzyme-treated cells, but the inhibition was only partial . GTPgammaS-dependent activation of PLD, measured in the absence or presence of ATP in permeabilized cells, was also partially affected by C3 exoenzyme treatment . Thus, these results indicate that Rho proteins play a key role in receptor-mediated PLD regulation in differentiated HL-60 cells, but play a partial role in the in vivo action of PMA and in vitro action of GTPgammaS on PLD . ATP produced a significant enhancement of the in vitro effect of GTPgammaS on PLD activity, but the effect of ATP was not altered by inhibitors of serine/threonine and tyrosine kinases . However, it was markedly reduced by neomycin and accompanied by an increase in phosphatidylinositol 4,5-bisphosphate (PtdInsP2) synthesis . These data indicate that in permeabilized HL-60 cells, the stimulatory effect of ATP on PLD does not involve protein phosphorylation but is due to an increase in PtdInsP2.

Infect Immun, 1998 Nov, 66(11), 5462 - 9
Effect of Clostridium difficile toxin A on human colonic lamina propria cells: early loss of macrophages followed by T-cell apoptosis; Mahida YR et al.; We have previously shown that Clostridium difficile toxin A induces detachment of human colonic epithelial cells from the basement membrane and subsequent cell death by apoptosis . Because these cells require adhesion-dependent signalling from the extracellular matrix for survival, their detachment from the basement membrane by other means also induces apoptosis . The role of toxin A in the induction of apoptosis therefore remains to be determined . In addition, sensitivities to C . difficile toxin A of lamina propria lymphocytes, macrophages, and eosinophils, which lie below the surface epithelium, are not known . In contrast to epithelial cells, these lamina propria cells do not require adhesion-dependent signalling from the extracellular matrix for survival, and this may allow the mechanisms of toxin A-induced cell death to be further investigated . The aim of this study was to investigate the effect of purified C . difficile toxin A on human colonic lamina propria T cells, macrophages, and eosinophils . We show that C . difficile toxin A induces loss of viability in isolated colonic lamina propria cell preparations containing the three different cell types in a dose- and time-dependent fashion . Exposure to high concentrations of the toxin led to loss of macrophages within 72 h . T-lymphocyte and eosinophil cell death was prominent at later time points and occurred by apoptosis . Exposure to toxin A also induced the production of tumor necrosis factor alpha by the isolated colonic lamina propria cells . However, the presence of neutralizing antibodies to this cytokine did not influence C . difficile toxin A-induced T-cell apoptosis . Moreover, purified T cells also underwent apoptosis following exposure to toxin A, implying that apoptosis occurred as a consequence of a direct interaction between T cells and the toxin . Our studies suggest that C . difficile toxin A is capable of suppressing human colonic mucosal immune responses by inducing early loss of macrophages followed by T-cell apoptosis.

Infect Immun, 1998 Nov, 66(11), 5125 - 31
Activation of Rho GTPases by Escherichia coli cytotoxic necrotizing factor 1 increases intestinal permeability in Caco-2 cells; Gerhard R et al.; The cytotoxic necrotizing factor 1 (CNF1) activates Rho GTPases by deamidation of glutamine-63 and thereby induces redistribution of the actin cytoskeleton and formation of stress fibers . Here, we have studied the effects of CNF1 on the transepithelial resistance of Caco-2 cells, a human intestinal epithelial cell line, in comparison with the Rho-inactivating toxin B of Clostridium difficile . Whereas toxin B decreased the transepithelial resistance of Caco-2 cells by about 80% after 4 h, CNF1 reduced it by about 40% . Significant changes of the transepithelial resistance induced by CNF1 were detected after 3 h of incubation . Half-maximal effects were observed with 10 and 41 ng of CNF1 and toxin B per ml, respectively . Flux measurement revealed no CNF1-induced increase of fluorescein isothiocyanate-dextran permeation within the first 4 h of incubation and a 2.9-fold increase after 24 h of incubation . In contrast, toxin B induced a 28-fold increase of permeation after 24 h . As detected by rhodamine-phalloidin staining, CNF1 increased polymerization of F actin at focal contacts of adjacent cells and induced formation of stress fibers . The data indicate that not only depolymerization but also polymerization of actin and subsequent reorganization of the actin cytoskeleton alter the barrier function of intestinal tight junctions.

Gynecol Oncol, 1998 Oct, 71(1), 104 - 7
Gastrointestinal toxicity and Clostridium difficile diarrhea in patients treated with paclitaxel-containing chemotherapy regimens; Husain A et al.; OBJECTIVE: The objective of this study was to determine the incidence of grade 3 and 4 gastrointestinal toxicity and the prevalence of Clostridium difficile-associated diarrhea (CDAD) in patients with gynecologic malignancies treated with paclitaxel-based chemotherapy regimens . METHODS: We retrospectively reviewed the medical records of all patients treated on the Gynecology Service at Memorial Sloan-Kettering Cancer Center from January 1, 1993 to July 1, 1996 . We identified all patients treated with paclitaxel during this period and determined the total number of patients hospitalized for symptoms of gastrointestinal toxicity, including nausea, vomiting, diarrhea, and dehydration, within 4 weeks of chemotherapy, as well as the incidence of CDAD among these patients . RESULTS: Six hundred and twenty-four patients were treated with paclitaxel-containing chemotherapy regimens during the study period, including 55 patients who were treated on a "dose-dense" high-dose protocol for advanced ovarian cancer . Among these, 149 patients (24%) were hospitalized for symptoms of gastrointestinal toxicity . During the study period, a total of 40 cases of CDAD were reported among hospitalized patients on the Gynecology Service and 24 (60%) of these cases occurred in patients who had received paclitaxel within the prior 4 weeks . CONCLUSIONS: The occurrence of CDAD in patients receiving paclitaxel-containing chemotherapy is not rare and can result in severe dehydration requiring hospitalization . The risk of C . difficile colitis appears to be 2.2% in patients receiving standard-dose regimens and as high as 20% in patients receiving high-dose regimens . This etiology should be considered and treated early in patients presenting with symptoms of gastrointestinal toxicity subsequent to chemotherapy treatments .

J Nat Toxins, 1998 Oct, 7(3), 291 - 302
Assembly of Clostridium perfringens epsilon-toxin on MDCK cell membrane; Nagahama M et al.; Clostridium perfringens epsilon-toxin bound to the Madin Darby canine kidney (MDCK) cells and aggregated . The complex of the toxin was formed in a dose- and a time-dependent manner . The formation of the complex increased with a decrease in viable counts of MDCK cells and with increasing K+ release from the cells . The inactivated toxin heated at 100 degrees C did not aggregate under the condition . In addition, the prototoxin dose-dependently bound to the cells, but did not form the complex . Incubation of the toxin with MDCK cell membranes also showed the formation of the complex, but that with membrane preparations prepared from Vero cells or sheep erythrocytes, which are insensitive for the toxin, showed no formation of the complex . Incubation of the toxin with mouse brain homogenates resulted in formation of the complex, but that with brain homogenates heated at 80 degrees C or mouse liver homogenates showed no formation of the complex . These observations show that the complex formation of epsilon-toxin is essential for toxicity of the toxin.

J Nat Toxins, 1998 Oct, 7(3), 239 - 53
Hemagglutinin binding mediated protection of botulinum neurotoxin from proteolysis; Sharma SK et al.; Type A Clostridium botulinum, the causative agent of the food poisoning botulism disease, secretes botulinum neurotoxins along with seven neurotoxin associated proteins (NAPs) . The function of NAPs has been shown to protect the neurotoxin from acidity, heat, and proteolytic attack in the environmental and gastrointestinal tract during the toxicogenesis of the botulism disease . One of the NAPs, purified from type A botulinum neurotoxin complex, showed hemagglutination activity . A direct interaction has been demonstrated between purified NAP, a 33-kDa hemagglutinin or Hn-33, and the neurotoxin by using Sephadex G-200 column chromatography . Furthermore, Hn-33 has complete resistance against proteolytic attack at pH 2.0 as well as at normal physiological pH . We have investigated digestion of the neurotoxin in the presence and absence of Hn-33 . The neurotoxin alone has been found to be more susceptible to the enzymatic digestion than neurotoxin with Hn-33 . The presence of Hn-33 changes the proteolytic fragmentation pattern of the neurotoxin . It seems that Hn-33 protects the neurotoxin from proteolysis either by structural modification of the neurotoxin or by blocking the protease accessible sites of the neurotoxin.

J Nat Toxins, 1998 Oct, 7(3), 215 - 26
Isolation of synaptotagmin as a receptor for types A and E botulinum neurotoxin and analysis of their comparative binding using a new microtiter plate assay; Li L et al.; Clostridium botulinum neurotoxin acts on nerve endings to block acetylcholine release . Binding of the neurotoxin to a membrane receptor through its heavy chain is the first essential step in its mode of toxin action . Type E botulinum neurotoxin (BoNT/E) or type A botulinum neurotoxin (BoNT/A) receptor was purified from rat brain synaptosomes employing a neurotoxin affinity column chromatography . The protein fraction eluted from the affinity column with 0.5 M NaCl contained a 57 kDa protein as a major eluant . Immunoblotting the eluant with anti-synaptotagmin antibodies revealed that the 57 kDa protein was synaptotagmin I . Rat synaptotagmin I has been suggested as the receptor for BoNT/B (Nishiki et al., J . Biol . Chem . 269, 10498-10503, 1994) in rat brain . In this study, binding of BoNT/A and BoNT/E to synaptotagmin I was studied by a microtiter plate-based method . Binding of synaptotagmin I to BoNT/A coated on the plate was competitively reduced upon preincubation of the proteins with BoNT/E, suggesting a competitive binding of BoNT/A and BoNT/E to the receptor . Taken together, these results suggest that the same receptor protein binds to all three BoNT serotypes tested.

Nippon Rinsho, 1998 Sep, 56(9), 2382 - 6
{Drug-associated hemorrhagic enteritis}; Sakurai Y; Drug-associated hemorrhagic colitis are divided into antibiotic associated hemorrhagic colitis (AAHC) and other drug associated hemorrhagic colitis . AAHC are mainly caused by oral usage of Ampicillin and its derivatives (85%) . Initially AAHC are believed to be caused by Klebsiella oxytoca overgrowth . However, these organisum has no exotoxin like Clostridium difficile and pathogenesis of AAHC are still unresolved . Typical AAHC are diagnosed by colonoscopy with diffuse hemorrhage and edema mainly found in descending colon and transverse colon . NSAIDs are also the cause of hemorrhagic colitis like AAHC . Mephenamic acid are famous for this complication . Diarrhea is one of the main complication of oral 5-fluorouracil administration and even causes hemorrhagic colitis . Its histology are characteristic in gland atrophy . Gold colitis are reported 36 cases in rheumatoid arthritis patients . Exact mechanism of bleeding are not understood . NSAIDs may cause collagenous colitis and or lymphocytic colitis in RA patients . Other rare hemorrhagic colitis are associated with azathioprine, methyl dopa, interferon alfa etc . NSAIDs and anticoagulants are well known drugs for complication of GI bleeding making hemorrhagic enteritis.

J Hosp Infect, 1998 Sep, 40(1), 1 - 15
Risk factors for Clostridium difficile infection; Bignardi GE; A systematic review of the literature to identify risk factors associated with Clostridium difficile infection was conducted . Two main outcomes were considered: C . difficile diarrhoea and C . difficile carriage . A qualitative assessment, based on a set of defined and consistently applied criteria, appeared to be the best approach for risk factors other than antibiotic use, as an approach based on meta-analysis would have utilized only the information provided by a minority of the studies . Risk factors for which there was evidence suggestive or consistent with an association with C . difficile diarrhoea were: increasing age (excluding infancy), severity of underlying diseases, non-surgical gastrointestinal procedures, presence of a nasogastric tube, anti-ulcer medications, stay on ITU, duration of hospital stay, duration of antibiotic course, administration of multiple antibiotics . For malignant haematological disorders there was evidence of an association only with C . difficile carriage, but there were no suitable studies to explore a possible association of this risk factor with symptomatic infection . Antibiotic use lent itself to quantitative assessment with meta-analysis using logistic regression . Exposure to an antibiotic was shown to be statistically significantly associated with both C . difficile diarrhoea and C . difficile carriage . The meta-analysis approach enabled the ranking of individual antibiotics in relation to the risk of C . difficile infection, though the 95% confidence intervals were often wide and overlapping . Antibiotics associated with a lower risk of C . difficile diarrhoea should be considered, especially when attempting to control a C . difficile outbreak or when prescribing for a patient with other C . difficile risk factors . This systematic review of the literature enabled the identification of features it would be desirable to consider in future epidemiological studies.

Avian Dis, 1998 Jul-Sep, 42(3), 579 - 84
Use of Aviguard and other intestinal bioproducts in experimental Clostridium perfringens-associated necrotizing enteritis in broiler chickens; Hofacre CL et al.; Clostridium perfringens-associated necrotic enteritis (CPANE) is a common problem among rapidly growing broiler strains of chickens that are raised intensively in modern microenvironments . The purpose of this study was to compare the use of Aviguard and three other intestinal bioproducts (two normal gut flora {NGF} products and one probiotic product) in experimental CPANE in broiler chickens . Male broiler chicks were housed in the same environmentally controlled facility and given one of six treatments . The necrotic enteritis infection model (NEIM) used in the present study was effective in inducing CPANE intestinal gross lesions in broiler chickens . Equally important, Aviguard was found to be significantly more effective than either the other two NGF products or the probiotic for reducing gross lesions induced by the NEIM . In addition, Aviguard/NEIM-treated chicks ate more feed and had better feed efficiency than their NGF- or probiotic/NEIM-treated counterparts . Other significant differences among these four reconstituted microbial preparations were not found . Results from this study have additional importance because they further support the use of reconstituted microbial preparations as novel and effective alternatives to antibiotics that can reduce the severity of C . perfringens-associated necrotic enteritis challenge in broilers.

Microbiol Immunol, 1998, 42(8), 533 - 8
Mechanism of membrane damage by Clostridium perfringens alpha-toxin; Nagahama M et al.; The effect of Clostridium perfringens alpha-toxin on liposomes prepared from phosphatidylcholine (PC) containing the fatty acyl residues of 18 carbon atoms was investigated . The toxin-induced carboxyfluorescein (CF) leakage and phosphorylcholine release from multilamellar liposomes increased as the phase transition temperature of the phosphatidylcholines containing unsaturated fatty acyl residues decreased . However, there was no difference between the sensitivity of the different phosphatidylcholines solubilized by deoxycholate to the phospholipase C (PLC) activity of the toxin . However, the toxin did not hydrolyze solubilized distearoyl-L-alpha-phosphatidylcholine (DSPC) or phosphatidylcholine containing saturated fatty acyl residue, and caused no effect on liposomes composed of DSPC . These results suggest that the activity of the toxin is closely related to the membrane fluidity and double bond in PC . The N-terminal domain of alpha-toxin (AT1-246) and variant H148G did not induce CF leakage from liposomes composed of dioleoyl-L-alpha-phosphatidylcholine (DOPC) . H148G bound to the liposomes, but AT1-246 did not . However, the C-terminal domain (AT251-370) conferred binding to liposomes and the membrane-damaging activity on AT1-246 . These observations suggest that the membrane-damaging action of alpha-toxin is due to the binding of the C-terminal domain of the toxin to the double bond in the PC in the bilayer and hydrolysis of the PC by the N-terminal domain.

Lakartidningen, 1998 Sep 9, 95(37), 3932 - 4, 3937-9
{Choose preparations that affect the intestinal ecosystem as little as possible!}; Edlund C et al.; The administration of antimicrobials often causes ecological disturbances in the normal microflora, such as decreased colonisation resistance which may result in overgrowth of potentially pathogenic micro-organisms such as yeasts and Clostridium difficile . Another possible consequence is the establishment of resistant strains which may spread within the host, or from person to person, causing infection . Resistant bacteria can also transfer resistance genes to their own or other species . The article consists in a review of studies performed by various investigators during the past 20 years, and illustrating the impact of different antimicrobial agents on the human gastrointestinal microflora.

Biochemistry, 1998 Oct 13, 37(41), 14563 - 74
Identification of a membrane-spanning domain of the thiol-activated pore-forming toxin Clostridium perfringens perfringolysin O: an alpha-helical to beta-sheet transition identified by fluorescence spectroscopy; Shepard LA et al.; Clostridium perfringens perfringolysin O (PFO or theta-toxin) is a cytolytic toxin that binds to cholesterol-containing membranes and then self-associates to spontaneously form aqueous pores of varying size in the bilayer . In this study, a membrane-spanning domain has been identified in PFO by a combination of fluorescence spectroscopic methods using the fluorescent dye N, N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa-1, 3-diazolyl)ethylenediamine (NBD) whose emission properties are sensitive to water . PFO was substituted with a single cysteine at most of the residues between amino acids K189 and N218, and then each cysteine was modified with NBD . Each purified NBD-labeled PFO was then bound to membranes, and the probe's environment was ascertained by measuring its fluorescence lifetime, emission intensity, and collisional quenching with either aqueous (iodide ions) or nonaqueous (nitroxide-labeled phospholipids) quenchers . Lifetime and intensity measurements revealed that the amino acid side chains in this region of the membrane-bound PFO polypeptide alternated between being in an aqueous or a nonaqueous environment . This pattern indicates that this portion of the membrane-bound PFO spans the membrane in an antiparallel beta-sheet conformation . The alternating exposure of these residues to the hydrophobic interior of the bilayer was demonstrated by their susceptibility to quenching by nitroxide moieties attached to phospholipid acyl chains . Residues K189-N218 therefore form a two-stranded, amphipathic beta-sheet in the membrane-bound PFO that creates a stable interface between the pore and the membrane . This same region packs as three short alpha-helices in the soluble, monomeric form of PFO, and therefore, the cholesterol-dependent conversion of PFO to a membrane-bound oligomer involves a major structural transition in which three alpha-helices unfold to form a membrane-spanning amphipathic beta-sheet.

Am J Gastroenterol, 1998 Oct, 93(10), 1873 - 6
Clostridium difficile colitis: factors influencing treatment failure and relapse--a prospective evaluation; Nair S et al.; OBJECTIVE: The aim of this study was to identify patient related factors that may influence the treatment response and relapse following Clostridium difficile (C . difficile) colitis . METHODS: A total of 36 patients with C . difficile colitis were followed for 3 months . Age, sex, place of residence, severity of infection, treatment, underlying medical condition, and treatment were compared in patients who failed to respond to treatment in 14 days and in patients who relapsed after a successful treatment . Student's t test and Fisher's exact test were used to compare the groups . A p value of <0.05 was considered significant . RESULTS: A low serum albumin (p=0.016) and continuation of systemic antibiotic treatment were found to be associated with refractoriness to treatment . Continuation or restarting of antibiotics after successful treatment increases the risk of relapse (p=0.003) . The age, sex place of residence, underlying medical condition, and type of precipitating antibiotics had no effect on the treatment response and relapse . The severity of colitis and the type of therapy (metronidazole vs vancomycin) did not influence the treatment response or relapse . CONCLUSION: Low serum albumin serves as a useful marker for patients who require prolonged treatment for C . difficile colitis . It is prudent to review the need for the continuation of systemic antibiotic treatment, as it adversely affects the treatment response and increases the risk of relapse.

Oncogene, 1998 Sep 10, 17(10), 1235 - 43
The Bcl-2 gene is differentially regulated by IL-2 and IL-4: role of the transcription factor NF-AT; Gomez J et al.; The murine TS1alphabeta T cell line expresses the anti-apoptotic protein Bcl-2 upon IL-2 stimulation, whereas IL-4-mediated growth of this cell line proceeds in the absence of Bcl-2 expression . In addition, IL-4 stimulation inhibits Bcl-2 expression and modulates its mRNA level . IL-2-induced DNA binding activity for these transcription factors is sensitive to phosphatidylinositol 3 kinase inhibitor wortmannin and to Rho inhibitor Clostridium difficile toxin B, which inhibit IL-2-induced Bcl-2 expression . NF-AT transcription factor appears to be the most important in the control Bcl-2 expression, since inhibition of the calcium-calmodulin-dependent phosphatase calcineurin, which regulates NF-AT activity, downregulates Bcl-2 expression in IL-2-stimulated cells . Constitutive expression of this phosphatase also upregulates Bcl-2 expression in IL-4-stimulated cells . In addition, a dominant negative NF-AT expression vector downregulates Bcl-2 expression in IL-2-stimulated cells . These results suggest that IL-2 induction of Bcl-2 expression may be directly or indirectly mediated by NF-AT.

Clin Infect Dis, 1998 Sep, 27(3), 487 - 93
Diarrhea and Clostridium difficile infection in Latin American patients with AIDS . Working Group on AIDS in Peru; Willingham FF et al.; Diarrhea and wasting are among the most debilitating and deadly manifestations of AIDS, yet only limited information is available regarding the etiology, clinical consequences, and immunologic effects of infection with diarrheal agents . Peruvian AIDS patients presenting with and without diarrhea were followed prospectively to examine the relations among diarrheal pathogens, clinical presentations, CD4 lymphocyte count, weight loss, and survival . Patients with chronic diarrhea had lower CD4 lymphocyte counts (P = .001) and lost more weight (P < .001) . Weight loss and a decreased CD4 lymphocyte count were associated with increased mortality (P = .011 and P = .003, respectively) . Mean CD4 lymphocyte count varied significantly by diarrheal agent . Clostridium difficile was the most prevalent pathogen and was associated with significantly increased mortality before and after adjustment for coinfection, length of follow-up, CD4 lymphocyte count, and weight loss (P = .006) . C . difficile may be a more important and more prevalent etiologic agent in AIDS than previously recognized and may represent a preventable cause of death in patients with immunosuppression.

Pathol Biol (Paris), 1998 Jun, 46(6), 395 - 7
{Systematic detection of toxigenic strains of Clostridium difficile--is it useful?}; Honderlick P et al.; The incidence of Clostridium difficile (Cd) infection is rising and Cd in fact is now endemic in many hospitals . During the past 4 years we analyzed our data concerning diarrhea caused by Cd in our 700 beds hospital . A positive case was defined as a Cd cytotoxine positive with or without positive culture for Cd . In the present study 120 episodes of Cd associated diarrhea occurred in 102 patients . 1101 stools were cultured from 921 patients . Since 1995 we choose to systematically evaluate Cd in diarrheal stools from hospitalized patients . 120 stool were positives (102 patients), we observed a significant difference between the 2 study periods: Cd was recovered from 16.9% of stool specimen during 1993-1994 and from 9.6% since 1995 . This study clearly confirm the common role of Cd in our hospitalized patients as in all positive case, Cd was the only enteropathogen isolated . We suggest the systematic investigation of Cd in hospitalized patients.

Aliment Pharmacol Ther, 1998 Sep, 12(9), 807 - 22
Review article: the use of biotherapeutic agents in the prevention and treatment of gastrointestinal disease; Lewis SJ et al.; There is presently a lack of well conducted clinical trials demonstrating any significant benefits of probiotics in humans . With the exception of diarrhoea due to rotavirus infection in children there is little evidence from randomized, double-blind, placebo-controlled studies that bacterial probiotics have a significant beneficial action in preventing diarrhoea of any cause . The yeast Saccharomyces boulardii has been shown to be of benefit in the prevention of antibiotic-associated diarrhoea but not in preventing infection with Clostridium difficile . S . boulardii may also be of benefit in preventing relapse of C . difficile infection . Because of the simplicity of in vitro systems and some animal models, beneficial characteristics of probiotics such as the ability of bacteria to bind to epithelial surfaces are not always transferable to humans . Thus any postulated benefit from consumption of probiotic bacteria should only be accepted as fact after testing in clinical studies . This review outlines our present knowledge of the mode of action of probiotics and presents the data from clinical trials on their use.

Curr Microbiol, 1998 Nov, 37(5), 312 - 8
Characterization of the genes encoding the botulinum neurotoxin complex in a strain of Clostridium botulinum producing type B and F neurotoxins; Santos-Buelga JA et al.; The organization of the clusters of genes encoding proteins of the botulinum neurotoxin (BoNT) progenitor complex was elucidated in a strain of Clostridium botulinum producing type B and F neurotoxins . With PCR and sequencing strategies, the type B BoNT-gene cluster was found to be composed of genes encoding BoNT/B, nontoxic nonhemagglutinin component (NTNH), P-21, and the hemagglutinins HA-33, HA-17, and HA-70, whereas the type F BoNT-gene cluster has genes encoding BoNT/F, NTNH, P-47, and P-21 . Comparative sequence analysis showed that BoNT/F in type BF strain 3281 shares highest homology with BoNT/F of non-proteolytic (group II) C . botulinum whereas NTNH and P-21 in the type F cluster of strain 3281 are more similar to the corresponding proteins in proteolytic (group I) type F C . botulinum . These findings indicate diverse evolutionary origins for genes encoding BoNT/F and its associated non-toxic proteins, although the genes are contiguous . By contrast, sequence comparisons indicate that genes encoding BoNT/B and associated non-toxic proteins in strain 3281 possess a similar evolutionary origin . It was demonstrated that the genes present in the BoNT/B gene cluster of this type BF strain show exceptionally high homology with the equivalent genes in the silent BoNT/B gene cluster of C . botulinum type A(B), possibly indicating their common ancestry.

Mol Microbiol, 1998 Aug, 29(4), 1009 - 18
botR/A is a positive regulator of botulinum neurotoxin and associated non-toxin protein genes in Clostridium botulinum A; Marvaud JC et al.; The genes of the botulinum neurotoxin A (BoNT) complex are clustered in a locus consisting of two divergent polycistronic operons, one containing the non-toxic, non-haemagglutinin (NTNH) component and bontA genes, the other containing the haemagglutinin (HA) component genes . The two operons are separated by a gene (botR/A, previously called orf21) encoding a 21 kDa protein . A recombinant Clostridium botulinum A strain that overexpresses botR/A was constructed by electroporating strain 62 with the vector pAT19 containing botR/A under the control of its own promoter . The transformed strain produced more BoNT/A and associated non-toxic proteins (ANTPs) and the corresponding mRNAs than the non-transformed strain . Partial inhibition of botR/A by antisense mRNA resulted in lower levels of BoNT/A, NTNH and HA70 and the levels of the corresponding mRNAs . Gel mobility shift assays and immunoprecipitations showed that BotR/A bound to the DNA promoter region upstream from the two BoNT/A complex operons . These results show that botR/A activated transcription of the genes encoding BoNT/A and ANTPs in C . botulinum A by interacting directly with the region promoter, and that the homologous genes in C . botulinum B, C and D presumably have the same function.

Res Microbiol, 1998 Jun, 149(6), 417 - 27
Cellulolytic enzymes of rumen anaerobic fungi Orpinomyces joyonii and Caecomyces communis; Hodrova B et al.; The rumen anaerobic fungi Orpinomyces joyonii A4 and Caecomyces communis JB1 were grown on microcrystalline cellulose (MC) and alfalfa hay . The cellular distribution of cellulases produced by these organisms was monitored . Fungal cultures were separated into extracellular, intracellular and cell wall fractions and assayed for endoglucanase (EG) and beta-glucosidase activity . In both fungal isolates, EG activity was the highest in the extracellular fraction regardless of the substrate used . The beta-glucosidase activity produced by O . joyonii was mainly found in the cell wall fraction . On the contrary, the same enzyme activity in C . communis predominated in the extracellular fraction . The polycentric isolate A4 more efficiently utilized both substrates, produced more short chain fatty acids (up to 31 mmol/l) and showed higher total levels of EG (2744 nmol glucose/h/ml) than the monocentric strain JB1 . On the other hand, beta-glucosidase (9033 nmol glucose/h/ml) activity was the highest in cultures of C . communis grown on cellulose . In cultures of O . joyonii grown on MC, the production of yellow affinity substance (YAS) with similar properties compared with yellow substance from Clostridium thermocellum was observed . This compound increased the adsorption of fungal cellulases to MC the temperature and pH range tested.

J Food Prot, 1998 Sep, 61(9), 1154 - 60
Conservative prediction of time to Clostridium botulinum toxin formation for use with time-temperature indicators to ensure the safety of foods; Skinner GE et al.; Integrating-type time-temperature indicators (TTIs) may be utilized to warn food processors and consumers about storage conditions that may have rendered a food potentially hazardous . As an example of how integrated TTIs could be manufactured to emulate an infinite set of time-temperature situations, a set of conditions which have supported C . botulinum growth and toxin production was compiled . The time-temperature curve representing conservative times required for toxin formation was constructed with data from literature relating to toxin formation as a function of temperature in any media or food product . This set of critical time-temperature data is fit by a conservative empirical relationship that can be used to predict combinations of incubation times and storage temperatures that represent a potential health risk from C . botulinum in foods . A TTI could be constructed to indicate deviation from such a given set of conditions to bring attention to foods that may have been exposed to potentially hazardous temperatures with respect to C . botulinum toxin formation.

J Food Prot, 1998 Sep, 61(9), 1148 - 53
Microbiological quality and the inability of proteolytic Clostridium botulinum to produce toxin in film-packaged fresh-cut cabbage and lettuce; Hao YY et al.; The production of toxin by a 10-strain mixture of proteolytic Clostridium botulinum in fresh produce packaged in polyethylene films having high (7,000 cc/m2/24 h; HOTR) and low (3,000 cc/m2/24 h; LOTR) relative oxygen permeability was determined . Shredded cabbage and lettuce inoculated with approximately 10(2) spores/g were placed in bags composed of the two films (1.4 kg/bag), and the bags were then vacuum sealed . Produce was stored at 4, 13, and 21 degrees C for up to 21 (cabbage) or 28 (lettuce) days and analyzed periodically . At each sampling time, the gas composition within the bags, pH of the produce, and microbial populations (total aerobic and anaerobic microorganisms, lactic acid bacteria, psychrotrophic bacteria, and yeasts and molds) were determined . In addition, the presence of botulinal toxin was determined using the standard U.S . Food and Drug Administration mouse bioassay protocol . Bags made of HOTR film prolonged sensory quality of cabbage and lettuce, especially at 13 and 4 degrees C . Packaging material had an effect on the growth of various groups of microorganisms; however, there was not a general trend . For example, lettuce packaged in HOTR bags had higher aerobic microbial populations than that packed in LOTR, but no significant difference (P < or = 0.05) was observed with cabbage . Growth of psychrotrophic bacteria was greater in vegetables packaged in HOTR film while growth of yeasts and molds was not affected by either packaging film . Most differences in microbial populations in produce packaged in LOTR and HOTR films were less than 1 log10 CFU/g . Botulinal toxin was not detected in cabbage or lettuce packaged in either film or stored under any test condition.

J Food Prot, 1998 Sep, 61(9), 1143 - 7
Alteration in sporulation, enterotoxin production, and protein synthesis by Clostridium perfringens type A following heat shock; Heredia NL et al.; Application of a heat shock (43 to 50 degrees C) applied early during the sporulation process of Clostridium perfringens delayed spore and enterotoxin production . Final levels of heat-resistant spores were similar to the control, but enterotoxin levels were reduced when the heat shock was applied at the third hour of incubation . The response of the microorganism to the heat shock was also examined by analysis of pulse-labeled proteins . Seven heat shock proteins (HSPs) associated with vegetative cells were identified by polyacrylamide gel electrophoresis . Most were localized mainly in the membrane, although one small protein was mostly present in the cytoplasm . Fewer HSPs were detected during sporulation . Two HSPs were immunologically related to the GroEL and DnaK HSPs from Lactobacillus lactis and Escherichia coli, respectively.

J Food Prot, 1998 Sep, 61(9), 1109 - 18
Bacterial profile of ground beef made from carcass tissue experimentally contaminated with pathogenic and spoilage bacteria before being washed with hot water, alkaline solution, or organic acid and then stored at 4 or 12 degrees C; Dorsa WJ et al.; The long-term effectiveness of several beef-carcass surface-tissue (BCT) wash interventions on the microbiology of ground beef produced from this tissue was determined . BCT was inoculated with bovine feces containing one of two different levels (ca . 4 or 6 log CFU/ml) of Escherichia coli O157:H7, Listeria innocua, Salmonella typhimurium, and Clostridium sporogenes . The BCT was then subjected to one of several treatment washes: 2% (vol/vol) DL-lactic acid (LA), 2% (vol/vol) acetic acid (AA), 12% (wt/vol) trisodium phosphate (TSP), hot water (HW; 74 +/- 2 degrees C at the tissue surface), or water (WW; 32 +/- 2 degrees C at the tissue surface) . A control group was left untreated . After treatments, BCT was held at 4 degrees C for 24 h and then ground . The ground beef was packaged and incubated at 4 degrees C for 21 days or 12 degrees C for 3 days . AA-treated samples held at 12 degrees C for 3 days yielded significantly lower aerobic plate counts than the control and also yielded the lowest levels of pseudomonads when compared to other sample groups . After being held at 4 degrees C for 21 days or 12 degrees C for 3 days, samples treated with antimicrobial compounds had lower or no detectable (< 1 CFU/g) levels of E . coli O157:H7, L . innocua, S . typhimurium, and C . sporogenes than beef treated with a WW or the control . Ground beef produced from tissue treated with HW yielded lower populations of these bacteria when compared to WW or untreated control beef, but the populations were generally higher than those observed in any of the antimicrobial chemical-treated samples . These trends continued throughout all storage conditions over time . Results from this study indicate that the use of carcass interventions, especially antimicrobial compounds, presently available to the slaughter industry will lower bacterial counts in ground beef.

J Virol, 1998 Nov, 72(11), 8806 - 12
Adenovirus endocytosis requires actin cytoskeleton reorganization mediated by Rho family GTPases; Li E et al.; Adenovirus (Ad) endocytosis via alphav integrins requires activation of the lipid kinase phosphatidylinositol-3-OH kinase (PI3K) . Previous studies have linked PI3K activity to both the Ras and Rho signaling cascades, each of which has the capacity to alter the host cell actin cytoskeleton . Ad interaction with cells also stimulates reorganization of cortical actin filaments and the formation of membrane ruffles (lamellipodia) . We demonstrate here that members of the Rho family of small GTP binding proteins, Rac and CDC42, act downstream of PI3K to promote Ad endocytosis . Ad internalization was significantly reduced in cells treated with Clostridium difficile toxin B and in cells expressing a dominant-negative Rac or CDC42 but not a H-Ras protein . Viral endocytosis was also inhibited by cytochalasin D as well as by expression of effector domain mutants of Rac or CDC42 that impair cytoskeletal function but not JNK/MAP kinase pathway activation . Thus, Ad endocytosis requires assembly of the actin cytoskeleton, an event initiated by activation of PI3K and, subsequently, Rac and CDC42.

J Vet Med Sci, 1998 Aug, 60(8), 981 - 3
Gastric mucormycosis in a sika deer (Cervus nippon) associated with proliferation of Clostridium perfringens; Sato Y et al.; Seven sika deer (Cervus nippon) died in a park where 30 deer were kept . One adult female deer died suddenly was necropsied . Severe hemorrhages were noted beneath the serous membranes of the forestomach and abomasum . Hyphal proliferation with neutrophil infiltration was observed in the mucous membranes of the stomaches, and the hyphae showed characteristics of order Mucorales . Catarrhal enteritis with hemorrhages was also observed . A large number of Clostridium perfringens was isolated from the contents of the abomasum and small intestine . The case was diagnosed as gastric mucormycosis associated with proliferation of Clostridium perfringens . The incidence occurred during breeding season and incorrect management was considered to be a predisposing factor for the infection.

J Exp Med, 1998 Oct 5, 188(7), 1211 - 21
Adenosine diphosphate (ADP)-ribosylation of the guanosine triphosphatase (GTPase) rho in resting peripheral blood human T lymphocytes results in pseudopodial extension and the inhibition of T cell activation; Woodside DG et al.; Scrape loading Clostridium botulinum C3 exoenzyme into primary peripheral blood human T lymphocytes (PB T cells) efficiently adenosine diphosphate (ADP)-ribosylates and thus inactivates the guanosine triphosphatase (GTPase) Rho . Basal adhesion of PB T cells to the beta1 integrin substrate fibronectin (Fn) was not inhibited by inactivation of Rho, nor was upregulation of adhesion using phorbol myristate acetate (PMA; 10 ng/ml) or Mn++ (1 mM) affected . Whereas untreated PB T cells adherent to Fn remain spherical, C3-treated PB T cells extend F-actin-containing pseudopodia . Inactivation of Rho delayed the kinetics of PMA-dependent PB T cell homotypic aggregation, a process involving integrin alphaLbeta2 . Although C3 treatment of PB T cells did not prevent adhesion to the beta1 integrin substrate Fn, it did inhibit beta1 integrin/CD3-mediated costimulation of proliferation . Analysis of intracellular cytokine production at the single cell level demonstrated that ADP-ribosylation of Rho inhibited beta1 integrin/ CD3 and CD28/CD3 costimulation of IL-2 production within 6 h of activation . Strikingly, IL-2 production induced by PMA and ionomycin was unaffected by C3 treatment . Thus, the GTPase Rho is a novel regulator of T lymphocyte cytoarchitecture, and functional Rho is required for very early events regulating costimulation of IL-2 production in PB T cells.

Acta Crystallogr D Biol Crystallogr, 1998 Jan 1, 54 ( Pt 1), 114 - 8
Crystallization of the catalytic domain of Clostridium cellulolyticum CeLF cellulase in the presence of a newly synthesized cellulase inhibitor; Reverbel-Leroy C et al.; The catalytic domain of the CeIF processive endocellulase, a family 48 glycosyl hydrolase from Clostridium cellulolyticum has been crystallized in the presence of a newly synthesized inhibitor (methyl 4-S-beta-cellobiosyl-4-thio-beta-cellobioside), by vapour diffusion, using PEG as a precipitant . The protein crystallizes in the orthorhombic P212121 space group and diffracts to a resolution of 2.0 A . The unit-cell parameters are a = 61.4, b = 84.5, c = 121.9 A.

Biotechnol Prog, 1998 Sep, 14(5), 800 - 6
Acetic acid production from fructose by clostridium formicoaceticum immobilized in a fibrous-Bed bioreactor
Huang YL, Mann K, Novak JM, Yang ST.
The fermentation kinetics of acetic acid production from fructose by Clostridium formicoaceticum was studied at pH 7.6 and 37 degreesC . Recycle batch, fed-batch, and continuous fermentations using immobilized cells in a fibrous-bed bioreactor were studied for their potential application in producing acetic acid from fructose, a fermentable sugar commonly found in corn steep liquor and many other food processing wastes . For the immobilized cell fermentation, acetic acid yield from fructose was approximately 1.0 g/g, with a final acetate concentration of approximately 78 g/L and the overall reactor productivity (based on the fibrous bed bioreactor volume) of approximately 0.95 g/(L.h) in the fed-batch fermentation . For a similar fed-batch fermentation with free cells, acetic acid yield was approximately 0.9 g/g, the highest final acetate concentration was approximately 46 g/L, and the overall productivity was approximately 0.12 g/(L.h) . In the continuous fermentation with immobilized cells, the reactor productivity decreased from 3.2 to 1 . 3 g/(L.h) as retention time increased from 16 to 72 h to reach 100% conversion . Compared to free-cell fermentations, the superior performance of the fibrous-bed bioreactor can be attributed to the high density (>30 g/L) of viable cells immobilized in the fibrous bed . The fermentation product, acetic acid, was found to be a noncompetitive inhibitor to the cells . However, the immobilized cells had a higher maximum production rate (pmax) and a higher value for the inhibition rate constant (Kp) than those for the free cells, suggesting that the immobilized cells in the fibrous-bed bioreactor were less sensitive to acetic acid inhibition than the free cells . This improvement in kinetic behaviors for immobilized cells confirms that the fibrous-bed bioreactor can be used as an effective tool for adapting and screening for acetate-tolerant strains.

J Surg Res, 1998 Oct, 79(2), 170 - 8
In vitro effects of Clostridium difficile toxins on hepatocytes; Mazuski JE et al.; BACKGROUND: Clostridium difficile infections are associated with development of the systemic inflammatory response, including the production of hepatic acute phase proteins . Lipopolysaccharide (LPS) directly stimulates the production of at least one of these proteins, a 23-kDa acute phase protein (the LPS-induced protein, or LIP) by murine hepatocytes in vitro . The aim of the present study was to determine if C . difficile toxins also stimulated the synthesis of this protein in vitro . METHODS: Cultured murine hepatocytes were treated for 24 h with various concentrations of C . difficile culture extract or purified toxins A and B in the presence or absence of dexamethasone or interleukin-1 (IL-1) receptor antagonist (IL-1 RA) . The cells were then metabolically radiolabeled with {35S}methionine . Secretory proteins were identified using electrophoresis and autoradiography, and their synthesis was quantitated by image analysis of the autoradiograms . RESULTS: The C . difficile culture extract, at dilutions as low as 1:200,000, significantly stimulated LIP synthesis in vitro . Toxins A and B, at concentrations as low as 1.6 and 0.02 pg/ml, respectively, also induced production of this protein . Dexamethasone further augmented C . difficile toxin-stimulated synthesis of LIP, but IL-1 RA inhibited the effects of these toxins on the synthesis of this protein . Only minimal quantities of IL-1 were found in culture supernatants following treatment with the toxins . CONCLUSIONS: C . difficile toxins A and B, at very low concentrations, stimulate hepatocyte acute phase protein synthesis . Even though IL-1 RA inhibits this process, it does not appear that local production of IL-1 mediates the action of these toxins .

Acta Crystallogr D Biol Crystallogr, 1998 Sep 1, 54 ( Pt 5), 1039 - 42
Crystallization and preliminary X-ray analysis of recombinant glutamate mutase and of the isolated component S from Clostridium cochlearium; Reitzer R et al.; Glutamate mutase {varepsilon2sigma2(B12)1} was reconstituted by incubating purified components E (varepsilon2) and S (sigma2) from Clostridium cochlearium, both produced in Escherichia coli, with either aquo- or cyanocobalamin . The inactive glutamate mutase obtained was crystallized with polyethyleneglycol 4000 as precipitant . Crystals are monoclinic with space group P21 and have cell dimensions a = 64.6, b = 113.2, c = 108.4 A and beta = 96.0 degrees for the glutamate mutase reconstituted with aquocobalamin . They diffract to a resolution of at least 2.7 A . Isolated component S was crystallized in the presence of an excess of cyanocobalamin, yielding red crystals of space group I422 with unit-cell dimensions of a = b = 69.9 and c = 107.1 A . The crystals diffract to about 3.2 A resolution . Native data sets were collected for both crystal forms.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1488 - 91
Novel histamine measurement by HPLC analysis used to assay histidine decarboxylase inhibitory activity of shoyuflavones from soy sauce; Kinoshita E et al.; An easy and highly sensitive method for measuring histamine by HPLC analysis coupled with precolumn derivatization was established . The amino group of histamine was completely colorimetrically labelled with 4-N,N-dimethylamino-azobenzene-4'-isothiocyanate (DABITC) in the presence of sodium bicarbonate at 90 degrees C for 5 min . The derivative was sensitively and easily analyzed by HPLC on a Cosmosil 5SL column using CHCl3/N,N-dimethylformamide/H2O (210:90:4) containing 0.4% acetic acid . Using the established method, histidine decarboxylase (HDC) inhibitory activities of three tartaric acid isoflavone derivatives, named shoyuflavones, isolated from soy sauce were examined in vitro by measuring the histamine produced by HDC . They showed intense inhibition of the activities of HDC from both mouse mastocytoma P-815 cells and Clostridium perfringens.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1476 - 82
Binding characteristics of bovine lactoferrin to the cell surface of Clostridium species and identification of the lactoferrin-binding protein; Tomita S et al.; The binding characteristics of bovine lactoferrin (bLf) to cells of the Clostridium species were observed by using a horseradish peroxidase-bLf conjugate . A bLf-binding protein (BP) having a relative molecular mass of about 33 kDa was confirmed in the surface layer components from 7 strains of the Clostridium species . The binding of the conjugate to bLf-BP or C . perfringens was strongly blocked by intact Lfs, lysine or arginine residues modified bLf, and deglycosylated bLf, but was not by other milk proteins or by the constituent sugars of glycan . Bacterial growth was inhibited by bLf, but was slightly inhibited by lysine residues modified bLf or deglycosylated bLf . Lactoferricin B did not block the binding of the conjugate, but strongly inhibited the bacterial growth . This suggests that the lysine or arginine residues and glycan of bLf hardly participated in binding bLf to the bacterial cells, but that the amino acid residues and glycan played an important role in inhibiting the growth of bacteria.

Biosci Biotechnol Biochem, 1998 Aug, 62(8), 1471 - 5
Binding of lactoferrin to bacterial cells of the Clostridium species and their agglutination; Tomita S et al.; Cell agglutination in cell suspensions of 10 strains of Clostridium by lactoferrin (Lf) was observed by obtaining the ratio of increased absorbance (RIA) at 450 nm . The RIA values were very different among the species, being higher in the cell suspensions with bovine Lf (bLf) than in those with human Lf . The binding ability of bLf to the bacterial cells was also observed by an enzyme-linked ligand-binding assay, using the conjugate of iron-free or iron-saturated bLf with horseradish peroxidase (HRPO) . The binding ability of bLf was very different among the 10 species, and showed a significant correlation with the cell agglutination of each strain . bLf formed a complex with the cells of C . perfringens, did not dissociate in 2 M NaCl or 4 M urea, but did dissociate in 1 M KSCN . These results suggest that the agglutination of cells of the Clostridium species by bLf is probably caused by the cooperative action of at least electrostatic and hydrophobic interactions between bLf and certain components of the cell surface.

EMBO J, 1998 Oct 1, 17(19), 5551 - 62
The crystal structure of the processive endocellulase CelF of Clostridium cellulolyticum in complex with a thiooligosaccharide inhibitor at 2.0 A resolution; Parsiegla G et al.; The mesophilic bacterium Clostridium cellulolyticum exports multienzyme complexes called cellulosomes to digest cellulose . One of the three major components of the cellulosome is the processive endocellulase CelF . The crystal structure of the catalytic domain of CelF in complex with two molecules of a thiooligosaccharide inhibitor was determined at 2.0 A resolution . This is the first three-dimensional structure to be solved of a member of the family 48 glycosyl hydrolases . The structure consists of an (alpha alpha)6-helix barrel with long loops on the N-terminal side of the inner helices, which form a tunnel, and an open cleft region covering one side of the barrel . One inhibitor molecule is enclosed in the tunnel, the other exposed in the open cleft . The active centre is located in a depression at the junction of the cleft and tunnel regions . Glu55 is the proposed proton donor in the cleavage reaction, while the corresponding base is proposed to be either Glu44 or Asp230 . The orientation of the reducing ends of the inhibitor molecules together with the chain translation through the tunnel in the direction of the active centre indicates that CelF cleaves processively cellobiose from the reducing to the non-reducing end of the cellulose chain.

Am J Physiol, 1998 Oct, 275(4 Pt 1), L748 - 55
Role of G proteins in agonist-induced Ca2+ sensitization of tracheal smooth muscle; Croxton TL et al.; Increased sensitivity to intracellular Ca2+ concentration ({Ca2+}) is an important mechanism for agonist-induced contraction of airway smooth muscle, but the signal transduction pathways involved are uncertain . We studied Ca2+ sensitization with acetylcholine (ACh) and endothelin (ET)-1 in porcine tracheal smooth muscle by measuring contractions at a constant {Ca2+} in strips permeabilized with alpha-toxin or beta-escin . The peptide inhibitor G protein antagonist 2A (GP Ant-2A), which has selectivity for Gq over Gi, inhibited contractile responses to ET-1, ACh, and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS), but the proportional inhibition of ACh responses was less than that of ET-1 . Pretreatment with pertussis toxin reduced ACh contractions but had no effect on those of ET-1 or GTPgammaS . Clostridium botulinum C3 exoenzyme, which inactivates Rho family monomeric G proteins, caused similar reductions in contractile responses to ACh, ET-1, and GTPgammaS . Farnesyltransferase inhibition, which inhibits Ras G proteins, reduced responses to ET-1 . We conclude that the heterotrimeric G proteins Gq and Gi both contribute to Ca2+ sensitization by ACh, whereas ET-1 responses involve Gq but not Gi . Both Gq and Gi pathways likely involve Rho family small G proteins . A Ras-mediated pathway also contributes to Ca2+ sensitization by ET-1 in airway smooth muscle.

Biochemistry, 1998 Sep 29, 37(39), 13463 - 74
Location of the phosphate binding site within Clostridium symbiosum pyruvate phosphate dikinase; McGuire M et al.; Pyruvate phosphate dikinase (PPDK) catalyzes the interconversion of ATP, Pi, and pyruvate with AMP, PPi, and PEP in three partial reactions: (1) E + ATP --> E.ATP --> E-PP.AMP, (2) E-PP.AMP + Pi --> E-PP.AMP.Pi --> E-P.AMP.PPi, and (3) E-P + pyruvate --> E-P.pyruvate --> E.PEP . The Clostridium symbiosum PPDK structure consists of N-terminal, central, and C-terminal domains . The N-terminal and central domains catalyze partial reactions 1 and 2 whereas the C-terminal and central domains catalyze partial reaction 3 . The goal of the present work is to determine where on the N-terminal domain catalysis of partial reactions 1 and 2 occurs and, in particular, where the Pi binding site is located . Computer modeling studies implicated Arg337 as a key residue for Pi binding . This role was tested by site-directed mutagenesis . The R337A PPDK was shown to be impaired in catalysis of the forward (kcat 300-fold lower) and reverse (kcat 30-fold lower) full reactions . Time courses for the single turnover reactions were measured to show that catalysis of partial reaction 1 is 5-fold slower in the mutant, catalysis of the second partial reaction is 140-fold slower in the mutant, and catalysis of the third partial reaction is unaffected . With the exception of the mutation site, the crystal structure of the R337A PPDK closely resembles the structure of the wild-type protein . Thus, the altered kinetic properties observed for this mutant are attributed solely to the elimination of the interaction between substrate and the guanidinium group of the Arg337 side chain . On the basis of these findings we propose that the Pi binding site is located within the crevice of the PPDK N-terminal domain, at a site that is flanked by the ATP beta-P and the Mg2+ cofactor.

Dig Dis Sci, 1998 Sep, 43(9), 2055 - 60
Brewer's yeast and Saccharomyces boulardii both attenuate Clostridium difficile-induced colonic secretion in the rat; Izadnia F et al.; Saccharomyces boulardii (Sb), a nonpathogenic yeast, has been used to prevent recurrences of Clostridium difficile (C.diff) -associated diarrhea . A single report suggested that treatment with Saccharomyces cerevisiae (Sc), commonly called brewer's yeast (BY), facilitates treatment of persistent C.diff infection . We conducted this experiment to determine whether C.diff toxin A-induced colonic secretion in the rat is blunted by pretreatment with either Sb or BY . We employed closed cecal pouches in two groups of five adult male Sprague-Dawley rats fed with standard chow for five days prior to the experiment, another group whose water was supplemented with 20 x 10(9) colony-forming units (CFU) of Sb per day for five days, and another group whose water was supplemented with 20 x 10(9) CFU of Sc per day for five days . Cecal pouches were infused for 3 hr with one of the following: (1) normal saline alone for a control group, or (2) normal saline plus 5 microg of C.diff toxin A (for the other control group and for the two experimental groups) . Water movement was measured by a nonabsorbable marker technique . Sodium movement and permeability to mannitol were also measured . Prior to the infusion, cecal contents were quantitatively cultured . In the three animals whose ceca were colonized with less than 10(6) CFU of either yeast per gram wet cecal content, toxin A-induced secretion could not be attenuated . In contrast, animals whose ceca were colonized with more than 10(6) CFU of either yeast per gram of wet cecal content showed significantly less secretion after toxin A application than those which were not fed yeast . S . cerevisiae reduced secretion by half (N = 5, P = 0.039 for water, 0.044 for sodium) and Sb by 75% (N = 4, P = 0.015 for water, 0.034 for sodium) . Toxin-induced increases in permeability to {3H}mannitol from systemic circulation to cecum could not be blunted by either yeast . We conclude that rat ceca can be colonized by either organism and that both organisms reduce C.diff toxin A-mediated secretion . We speculate that both organisms might have benefit in human C.diff-associated enterocolitis . Further studies of their mechanisms of action as well as clinical trials for the prevention and treatment of human C.diff infections should be pursued.

Wiad Lek, 1998, 51(7-8), 360 - 7
{Some aspects of the Clostridium difficile infection}; Wojtowicz A; Clostridium difficile is now regarded as the most common nosocomial enteric pathogen . C . difficile infection has a wide spectrum of a clinical presentation ranging from asymptomatic carriage to the fulminant colitis . Antibiotic therapy is the most important risk factor in pathogen contagion, however other factors are also involved . Typical pathophysiology: 1 . alteration of the indigenous colonic flora by antibodies, 2 . ingestion of spores, 3 . colonization by Clostridium difficile, 4 . production of its toxins . Both entherotoxin A and cytotoxin B are active in human colon . The mode of action of these toxins is already quite well known . The main treatment includes withdrawal of the inducing agents, supported occasionally by oral Vancomycin and Metronidazole . Relapse is a major complication.

Infect Immun, 1998 Oct, 66(10), 4942 - 6
Interleukin-12 has a role in mediating resistance of murine strains to Tyzzer's disease; Van Andel RA et al.; Clostridium piliforme induces enterohepatic disease in many domestic and laboratory animal species . Susceptibility to infection is known to vary with the immune status and strain of the host, but little is known about specific immune mechanisms that regulate this disease . To evaluate host control of C . piliforme infection, we examined the role of interleukin-12 (IL-12) both in the control of and in the response to murine C . piliforme infection . For this study, 3-week-old C . piliforme-resistant C57BL/6 or -susceptible DBA/2 mice were infected intravenously with either the toxic H1 or the nontoxic M1 C . piliforme isolate . Serum and liver samples were collected prior to C . piliforme inoculation (day 0) and at days 1, 3, 7, 14, and 28 postinoculation . Evaluation of hepatic IL-12 p40 mRNA expression by reverse transcription-PCR and of total-IL-12 protein levels in serum by enzyme-linked immunosorbent assay revealed that C . piliforme induced elevations in both hepatic p40 mRNA and serum total-IL-12 levels at all times postinoculation . Elevations were similar with both toxic and nontoxic C . piliforme isolates . Levels of total IL-12 in serum were significantly (P < 0.05) higher in C57BL/6 mice than in DBA/2 mice . Additional experiments were performed in which polyclonal antibody treatment was used to neutralize IL-12 in mice of both strains prior to intravenous inoculation with toxic C . piliforme H1 . IL-12 neutralization increased the severity of Tyzzer's disease at day 3 postinoculation in both mouse strains, but the degree of increase was greater in C57BL/6 mice than in DBA/2 mice.

Infect Immun, 1998 Oct, 66(10), 4910 - 6
Intestinal secretory factor released by macrophages stimulated with Clostridium difficile toxin A: role of interleukin 1beta; Rocha MF et al.; Clostridium difficile toxin A is associated with enterocolitis in animals and humans . However, the mechanisms of its secretory and damaging effects are not totally understood . In this work, we examined the intestinal secretion of electrolytes and water caused by supernatants from macrophages stimulated with toxin A in rabbit ileal mucosa mounted in Ussing chambers . We also investigated the mechanism by which the intestinal secretory factor (ISF) is released from stimulated macrophages . Supernatants from macrophages stimulated with toxin A caused potent intestinal secretion (change in short-circuit current {DeltaIsc}, 76 microA x cm-2; P < 0.01) . The release of the ISF was pertussis toxin sensitive (reduction, 61%; P < 0.01) and was also reduced (P < 0.05) by a protein synthesis inhibitor (67%), protease inhibitors (57%), a phospholipase A2 inhibitor (54%), a cyclo-oxygenase inhibitor (62%), a dual cyclo- and lipoxygenase inhibitor (48%), a platelet-activating factor (PAF) receptor antagonist (55%), and tumor necrosis factor alpha (TNF-alpha) synthesis inhibitors (48%) . However, this release was not inhibited by a lipo-oxygenase inhibitor . Monoclonal anti-interleukin 1beta (IL-1beta) but not anti-IL-1alpha antibody blocked (72%; P < 0.01) the secretory action of the ISF, as did recombinant human IL-1 receptor antagonist (80%; P < 0.01) . High levels of IL-1beta (3,476 pg/ml) were detected by an enzyme-linked immunosorbent assay in the above supernatants . Furthermore, the addition of IL-1beta to the serosal side caused a potent secretory effect (DeltaIsc, 80 microA x cm-2; P < 0.01) . These results show that macrophages stimulated with toxin A release an ISF capable of provoking intestinal secretion . The regulation of this factor is dependent upon the activation of the G protein . In addition, prostaglandins, PAF, and TNF-alpha are involved in the release of the ISF . We conclude that IL-1beta is probably the ISF released by macrophages in response to toxin A.

Infect Immun, 1998 Oct, 66(10), 4823 - 31
Modulation of enzymatic activity and biological function of Listeria monocytogenes broad-range phospholipase C by amino acid substitutions and by replacement with the Bacillus cereus ortholog; Zuckert WR et al.; The secreted broad-range phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC) of Listeria monocytogenes plays a role in the bacterium's ability to escape from phagosomes and spread from cell to cell . Based on comparisons with two orthologs, Clostridium perfringens alpha-toxin and Bacillus cereus PLC (PLCBc), we generated PC-PLC mutants with altered enzymatic activities and substrate specificities and analyzed them for biological function in tissue culture and mouse models of infection . Two of the conserved active-site zinc-coordinating histidines were confirmed by single amino acid substitutions H69G and H118G, which resulted in proteins inactive in broth culture and unstable intracellularly . Substitutions D4E and H56Y remodeled the PC-PLC active site to more closely resemble the PLCBc active site, while a gene replacement resulted in L . monocytogenes secreting PLCBc . All of these mutants yielded similar amounts of active enzyme as wild-type PC-PLC both in broth culture and intracellularly . D4E increased activity on and specificity for PC, while H56Y and D4E H56Y showed higher activity on both PC and sphingomyelin, with reduced specificity for PC . As expected, PLCBc expressed by L . monocytogenes was highly specific for PC . During early intracellular growth in human epithelial cells, the D4E mutant and the PLCBc-expressing strain performed significantly better than the wild type, while the H56Y and D4E H56Y mutants showed a significant defect . In assays for cell-to-cell spread, the H56Y and D4E mutants had close to wild-type characteristics, while the spreading efficiency of PLCBc was significantly lower . These studies emphasize the species-specific features of PC-PLC important for growth in mammalian cells.

Infect Immun, 1998 Oct, 66(10), 4811 - 6
Characterization of Clostridium botulinum type B neurotoxin associated with infant botulism in japan; Kozaki S et al.; The neurotoxin of strain 111 (111/NT) associated with type B infant botulism showed antigenic and biological properties different from that (Okra/NT) produced by a food-borne botulism-related strain, Okra . The specific toxicity of 111/NT was found to be about 10 times lower than that of Okra/NT . The monoclonal antibodies recognizing the light chain cross-reacted with both neurotoxins, whereas most of the antibodies recognizing the carboxyl-terminal half of the heavy chain of Okra/NT did not react to 111/NT . Binding experiments with rat brain synaptosomes revealed that 125I-labeled 111/NT bound to a single binding site with a dissociation constant (Kd) of 2.5 nM; the value was rather lower than that (0.42 nM) of 125I-Okra/NT for the high-affinity binding site . In the lipid vesicles reconstituted with ganglioside GT1b, 125I-Okra/NT interacted with the amino-terminal domain of synaptotagmin 1 (Stg1N) or synaptotagmin 2 (Stg2N), fused with the maltose-binding protein, in the same manner as the respective full-length synaptotagmins, and the Kd values accorded with those of the low- and high-affinity binding sites in synaptosomes . However, 125I-111/NT only exhibited a low capacity for binding to the lipid vesicles containing Stg2N, but not Stg1N, in the presence of ganglioside GT1b . Moreover, synaptobrevin-2, an intracellular target protein, was digested to the same extent by the light chains of both neurotoxins in a concentration-dependent manner . These findings indicate that the 111/NT molecule possesses the receptor-recognition site structurally different from Okra/NT, probably causing a decreased specific toxicity.

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