Science.gov

Sample records for culture clostridium bifermentans

  1. A novel insecticidal serotype of Clostridium bifermentans.

    PubMed

    Seleena, P; Lee, H L; Lecadet, M M

    1997-12-01

    A novel Clostridium bifermentans strain toxic to mosquito larvae on ingestion was isolated from a soil sample collected from secondary forest floor. This strain was designated as serovar paraiba (C. b. paraiba) according to its specific H antigen. Clostridium bifermentans paraiba is most toxic to Anopheles maculatus Theobald larvae (LC50 = 0.038 mg/liter), whereas toxicity to Aedes aegypti (Linn.) (LC50 = 0.74 mg/liter) and Culex quinquefasciatus Say (LC50 = 0.11 mg/liter) larvae was 20 and 3 times lower, respectively. The toxicity to An. maculatus larvae is as high as that of Bacillus thuringiensis serovar israelensis. C. b. paraiba was also found to exhibit significant per os insecticidal activity toward adult Musca domestica (Linn.). PMID:9474569

  2. Biodegradation of trinitrotoluene (TNT) by a strain of Clostridium bifermentans

    SciTech Connect

    Shin, C.Y.; Crawford, D.L.

    1995-12-31

    A Clostridium capable of degrading 2,4,6-trinitrotoluene (TNT) cometabolically was isolated from a mixed culture obtained from a bioreactor fed TNT. This bacterium, identified as a strain of Clostridium bifermentans, and designated strain CYS-1, was able to degrade TNT via 4-amino-2,6-dinitrotoluene (4-ADNT) and 2,4-diamino-6-nitrotoluene (2,4-DANT) to aliphatic polar products which are now being identified and are assumed to be organic acids. CYS 1 cells are tolerant of TNT and capable of degrading it at starting concentrations of up to {ge}100 mg/L TNT. The number of cells inoculated and the availability of cosubstrate nutrients are significant factors influencing TNT degradation, as are TNT tolerance and survival of the cells at high TNT concentrations. In liquid media, at high TNT concentrations, TNT toxicity could be overcome by increasing the amount of inoculum and supplementing the culture with appropriate rich organic cosubstrates. Under these conditions, the reduction of 4-ADNT to 2,4-DANT occurred very fast, whereas the further degradation of 2,4-DANT proceeded more slowly.

  3. Fatal Spontaneous Clostridium bifermentans Necrotizing Endometritis: A Case Report and Literature Review of the Pathogen

    PubMed Central

    Hale, Andrew; Kirby, James E.; Albrecht, Mary

    2016-01-01

    Clostridium bifermentans is a rare pathogen in humans. A fatal case of fulminant endometritis with toxic shock and capillary leak secondary to C bifermentans infection in a young woman is described, and this is compared to all 13 previously described cases of C bifermentans infection. PMID:27419167

  4. Production of Clostridium bifermentans Spores as Inoculum for Bioremediation of Nitroaromatic Contaminants

    PubMed Central

    Sembries, S.; Crawford, R. L.

    1997-01-01

    Spores of Clostridium bifermentans KMR-1 were produced for use as a microbial inoculum for bioremediation and were preserved in both liquid and dry forms. All spore formulations showed good viability and ability to biodegrade the target compound, 2,4,6-trinitrotoluene (TNT), after 4 months of storage. For low-cost bulk spore production, several medium compositions, based on soy peptone, corn steep liquor, and meat peptone, were tested and yielded 10(sup7) spores per ml. A medium pH above 7.0, a low glucose concentration, and a sufficient concentration of protein favored the sporulation of C. bifermentans KMR-1. PMID:16535620

  5. Degradation of 2-sec-butyl-4,6-dinitrophenol (dinoseb) by Clostridium bifermentans KMR-1.

    PubMed Central

    Hammill, T B; Crawford, R L

    1996-01-01

    A strain of Clostridium bifermentans, KMR-1, degraded 2-sec-butyl-4,6-dinitrophenol (dinoseb) to a level below the limit of detection by high-performance liquid chromatography (0.5 mg/liter) within 96 h, with no accumulation of aromatic intermediates. KMR-1 could not utilize dinoseb as a sole carbon or energy source, and degradation occurred via cometabolism in the presence of a fermentable carbon source. KMR-1 mineralized some dinoseb in anaerobic cultures, evolving 7.2% of the radioactive label in U-ring 14C-labeled dinoseb as 14CO2. The remaining anaerobic degradation products were incubated with aerobic soil bacteria, and 35.4% of this residual radioactive label was evolved as 14CO2. During this mineralization experiment, 38.9% of the initial label was evolved as 14CO2 after both anaerobic and aerobic phases. This is the first demonstration of dinoseb degradation by a pure microbial culture. PMID:8633886

  6. First Report of Clostridium lavalense Isolated in Human Blood Cultures

    PubMed Central

    Bourque, Christine; Thibault, Louise; Côté, Jean-Charles; Domingo, Marc-Christian

    2016-01-01

    An 88-year-old man was admitted to the hospital with worsening malaise, fever, and weakness. Anaerobic blood culture bottles revealed the presence of an anaerobic, Gram-positive sporulated bacillus. Empirical antibiotherapy with intravenous piperacillin-tazobactam was initiated. The patient defervesced after four days and was switched to oral amoxicillin on his 6th day of antibiotic therapy and later discharged from the hospital. Four months later, he had recovered. The bacterium was initially identified as Clostridium butyricum using anaerobic manual identification panel. 16S rRNA gene sequence and phylogenetic analysis showed the bacterium to be Clostridium lavalense, a recently described species with no previously published case of isolation in human diagnostic samples so far. This is the first report of Clostridium lavalense isolation from human blood cultures. Further studies are needed in order to elucidate the role of Clostridium lavalense in human disease and its virulence factors. PMID:27478446

  7. Physiology, biochemistry, and genetics of a pure culture of an obligatory anaerobic bacterium that utilizes 2,4,-6-trinitrotoluene (TNT) and biodegradation of RDX by pure cultures of obligatory anaerobic bacteria of the genus clostridium. Final report, 1 September 1993-31 August 1996

    SciTech Connect

    Crawford, R.L.; Crawford, D.L.

    1996-09-01

    In work supported by the US AFOSR (grant F49620-94-1-0306) we are conducting detailed biochemical and genetic studies of three strains of Clostridium bifernientans, obligatory anaerobic bacteria that appear to completely degrade a variety of nitroaromatic compounds, including 2,4,6-trinitrotoluene (TNT). We are determining the optimal physiological conditions for the degradative activities of C. bifermentans strains; and identifying and characterizing enzymes and genes involved in the biotransformation of nitroaromatic compounds by C. bifermentans. In our AASERT supplemental grant(AFOSR-93-1-O464) we expanded these goals to the explosive RDX (1,3,5-triaza-1, 3,5-trinitrocyclohexane). The AASERT grant funded two graduate students, who characterized the ability of C. bifermentans to degrade RDX (Regan, K. N., and R.L. Crawford, 1994. Biotechnol. Kett. 16: 1081- 1086), and prepared both genomic and plasmid DNA libraries from C. bifermentans. This genetic work will accelerate our progress toward our goal of characterizing the genetics of TNT/RDx degradation by our clostridia (K. Diedrich, M.S. thesis, University of Idaho; in preparation).

  8. Culturing and Maintaining Clostridium difficile in an Anaerobic Environment

    PubMed Central

    Edwards, Adrianne N.; Suárez, Jose M.; McBride, Shonna M.

    2013-01-01

    Clostridium difficile is a Gram-positive, anaerobic, sporogenic bacterium that is primarily responsible for antibiotic associated diarrhea (AAD) and is a significant nosocomial pathogen. C. difficile is notoriously difficult to isolate and cultivate and is extremely sensitive to even low levels of oxygen in the environment. Here, methods for isolating C. difficile from fecal samples and subsequently culturing C. difficile for preparation of glycerol stocks for long-term storage are presented. Techniques for preparing and enumerating spore stocks in the laboratory for a variety of downstream applications including microscopy and animal studies are also described. These techniques necessitate an anaerobic chamber, which maintains a consistent anaerobic environment to ensure proper conditions for optimal C. difficile growth. We provide protocols for transferring materials in and out of the chamber without causing significant oxygen contamination along with suggestions for regular maintenance required to sustain the appropriate anaerobic environment for efficient and consistent C. difficile cultivation. PMID:24084491

  9. Cultures of "Clostridium acetobutylicum" from various collections comprise Clostridium acetobutylicum, Clostridium beijerinckii, and two other distinct types based on DNA-DNA reassociation.

    PubMed

    Johnson, J L; Toth, J; Santiwatanakul, S; Chen, J S

    1997-04-01

    The best-known acetone-butanol (solvent)-producing bacterium is the Weizmann organism, Clostridium acetobutylicum, which was used for starch-based industrial fermentation. In the past two decades, cultures of "C. acetobutylicum" from various culture collections have included organisms that were isolated for sugar (molasses)-based industrial solvent production. Recent biochemical and genetic studies have revealed significant differences among some of these "C. acetobutylicum" strains. We used DNA-DNA reassociation to analyze 39 cultures of "C. acetobutylicum" and phenotypically similar organisms from major collections. The results of this study clearly identified four groups intergroup reassociation values of less than 30%. All of the intragroup values except the value for one strain were 68% or more, which supported species status for each group. The C. acetobutylicum group (with ATCC 824 as the type strain) consisted of 17 cultures and had average reassociation values of 10% with the other three groups. All strains of C. acetobutylicum produced riboflavin in milk, and the cultures were bright yellow, which is useful for differentiating this species from the other three groups. The Clostridium beijerinckii group (with VPI 5481 [= ATCC 25752] as the type strain) consisted of 16 cultures and included strains NCIMB 8052 and NCP 270. Strains NCP 262 and NRRL B643 constituted the third group, whereas strain N1-4 ("Clostridium saccharoperbutylacetonicum") and its derivative, strain N1-4081, formed the fourth group. At present, the last two groups are each represented by only one independent strain; definitive descriptions of these two groups as two new or revived species will require further phenotypic characterization, as well as identification of additional strains. C. beijerinckii NCP 270, Clostridium sp. strain NRRL B643, and "C. saccharoperbutylacetonicum" were used in industrial solvent production from molasses, which confirms that the new organisms used for the

  10. Evaluation of CP Chromo Select Agar for the enumeration of Clostridium perfringens from water.

    PubMed

    Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

    2013-10-01

    The European Directive on drinking water quality has included mCP agar as the reference method for recovering Clostridium perfringens from drinking waters. In the present study, three media (mCP, TSCF and CP Chromo Select Agar) were evaluated for recovery of C. perfringens in different surface water samples. Out of 139 water samples, using a membrane filtration technique, 131 samples (94.2%) were found to be presumptively positive for C. perfringens in at least one of the culture media. Green colored colonies on CP Chromo Select Agar (CCP agar) were counted as presumptive C. perfringens isolates. Out of 483 green colonies on CCP agar, 96.3% (465 strains, indole negative) were identified as C. perfringens, and 15 strains (3.1%) were indole positive and were identified as Clostridium sordellii, Clostridium bifermentans or Clostridium tetani. Only 3 strains (0.6%) gave false positive results and were identified as Clostridium fallax, Clostridium botulinum, and Clostridium tertium. Variance analysis of the data obtained shows statistically no significant differences in the counts obtained between media employed in this work. The mCP method is very onerous for routine screening and bacterial colonies could not be used for further biochemical testing. The colonies on CCP and TSCF were easy to count and subculture for confirmation tests. TSCF detects sulfite-reducing clostridia, including species other than C. perfringens, and in some cases excessive blackening of the agar frustrated counting of the colonies. If the contamination was too high, TSCF did not consistently produce black colonies and as a consequence, the colonies were white and gave false negative results. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that CCP agar was the most useful medium for C. perfringens recovery in water samples. PMID:23816139

  11. Switchgrass (Panicum virgatum) fermentation by sequential culture of Clostridium thermocellum and Clostridium beijerinckii: effect of particle size on gas production

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fuel alcohols can be produced by fermenting cellulosic biomass. Clostridium beijerinckii produces both ethanol and butanol, but it is non-cellulolytic. Cellulose requires saccharification prior to fermentation by C. beijerinckii. In contrast, the thermophile, Clostridium thermocellum, is highly ce...

  12. Decreased competiveness of the foodborne pathogen, Campylobacter jejuni, co-culture with the hyper-ammonia anaerobe, Clostridium aminophilum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Campylobacter spp. are a leading bacterial cause of human foodborne illness. When co-cultured in anaerobic Bolton broth with the hyper-ammonia-producing bacterium, Clostridium aminophilum, ammonia accumulation was greater (P 1...

  13. Use of gas-liquid chromatography as a screening test for toxigenic Clostridium difficile in diarrhoeal stools.

    PubMed Central

    Pepersack, F; Labbe, M; Nonhoff, C; Schoutens, E

    1983-01-01

    In order to determine if gas-liquid chromatography (GLC) on concentrated stool extracts could be substituted to cell culture assay for cytotoxicity, we prospectively studied 154 diarrhoeal stools submitted for detection of Clostridium difficile toxin. Isocaproic-positive samples were cultured on egg yolk agar supplemented with cycloserine, cefoxitin and fructose for isolation of C difficile, and on egg yolk agar plus kanamycin for isolation of other clostridium species. Of the 154 samples, 129 were GLC-negative (height of the isocaproic peak less than 1.2 cm) and were toxin-negative. Twenty-five stools yielded isocaproic acid; C difficile isolated from 13 of them, six of which were also toxin-positive. Four other isocaproic-positive samples yielded C bifermentans and C sordellii; all were toxin-negative. These results indicate that a negative GLC is an excellent screening test for excluding C difficile infection; positive results must be checked by toxin testing and culture since they are not necessarily associated with the presence of C difficile or its toxin. PMID:6630574

  14. Specific detection of toxigenic strains of Clostridium difficile in stool specimens.

    PubMed Central

    Gumerlock, P H; Tang, Y J; Weiss, J B; Silva, J

    1993-01-01

    Clostridium difficile is the infectious agent responsible for antibiotic-associated colitis. We report the use of the polymerase chain reaction technique to identify toxigenic strains of C. difficile in human stool specimens. A set of primers based on the nucleotide sequence of the toxin B gene, which amplified a 399-bp fragment from isolates producing toxin B, was designed. We examined 28 known toxigenic strains, which were all positive by this assay. DNAs from the nontoxigenic strains examined and from strains of Clostridium sordellii and C. bifermentans were not amplified with these primers. The sensitivity of this assay allowed us to identify as little as 10% toxigenic C. difficile cells in the presence of 90% nontoxigenic cells and to detect the toxin B gene in 1 pg of DNA from a toxigenic strain. DNAs extracted from 18 clinical stool specimens that were positive for toxin B by the tissue culture cytotoxicity assay were also positive by this assay. In addition, we detected toxin B sequences in DNA from 2 of 18 stool specimens that were negative for toxin B by the cytotoxicity assay. These two stool specimens were from patients who had a clinical pattern of colitis that was compatible with C. difficile causation. This rapid, sensitive assay will be useful for specific identification of toxigenic C. difficile and for revealing cases that are undetected by analysis of fecal samples for toxin B alone. Images PMID:8458943

  15. Clostridium perfringens type A toxin production in 3 commonly used culture media.

    PubMed

    Fernandez-Miyakawa, Mariano E; Marcellino, Romanella; Uzal, Francisco A

    2007-03-01

    In vitro toxin production is an important tool not only for diagnostic purposes but also for the study of pathogenesis of Clostridium perfringens infections. The present study was carried out to compare the level of toxin production by several strains of C. perfringens type A, isolated from the intestine of animals, when cultured in 3 different conventional culture media. Six strains of C. perfringens type A isolated from the small intestine of healthy sheep were cultured in commercial cooked meat medium (CMM), brain heart infusion (BHI), and tryptone glucose yeast (TGY). Intravenous lethality in mice and phospholipase C (PLC) activity were measured in filtered culture supernatants. Lethality of culture supernatants was highest for all isolates when grown in BHI, followed by CMM. No supernatants from any isolates grown in TGY produced lethality in mice. Phospholipase C activity was highest when the isolates were grown in BHI and CMM and significantly lower when grown in TGY. PMID:17402614

  16. Regulation of acetone butanol production in batch and continuous cultures of Clostridium acetobutylicum

    SciTech Connect

    Monot, F.; Engasser, J.M.; Petitdemange, H.

    1983-01-01

    The influence of pH and glucose concentration in batch and continuous cultures of Clostridium acetobutylicum is examined. At high pH and low glucose concentration only acids are produced. At low pH and high initial or feed glucose concentration, butanol and acetone are the main metabolites produced. According to a detailed kinetic analysis of the different fermentations, solvents are only produced if the concentration of undissociated butyric acid in the medium reaches a critical level. 10 references, 9 figures, 1 table.

  17. Defined bacterial culture development for methane generation from lactose. [Streptococcus lactis; Clostridium formicoaceticum; Methanococcus mazei

    SciTech Connect

    Yang, S.T.; Tang, I.C.; Okos, M.R.

    1988-06-20

    The defined microbial cultures for methane generation from lactose were investigated. A mixed culture consisting of homolactic (Streptococcus lactis), homoacetic (Clostridium formicoaceticum), and acetate-utilizing methanogenic (Methanococcus mazei) bacteria was used to convert lactose and whey permeate to methane at mesophilic temperatures (35-37/sup 0/C) and a pH around 7.0. Lactose was first converted to lactic acid by S. lactis, then to acetic acid by C. formicoaceticum, and finally to methane and CO/sub 2/ by M. mazei. About 5.3 mol methane were obtained from each mole of lactose consumed, and the conversion of acetate to methane was the rate-limiting step for this mixed-culture fermentation.

  18. Switchgrass (Panicum virgatum) fermentation by Clostridium thermocellum and Clostridium saccharoperbutylacetonicum sequential culture in a continuous flow reactor

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The study was conducted to evaluate fermentation by Clostridium thermocellum and C. saccharoperbutylacetonicum in a continuous-flow, high-solids reactor. Liquid medium was continuously flowed through switchgrass (2 mm particle size) at one of three flow rates: 83.33 mL h-1 (2 L d-1), 41.66 mL h-1(1 ...

  19. Evaluation of a Chromogenic Culture Medium for the Detection of Clostridium difficile

    PubMed Central

    Yang, John Jeongseok; Nam, You Sun; Kim, Min Jin; Cho, Sun Young; You, Eunkyung; Soh, Yun Soo

    2014-01-01

    Purpose Clostridium difficile (C. difficile) is an important cause of nosocomial diarrhea. Diagnostic methods for detection of C. difficile infection (CDI) are shifting to molecular techniques, which are faster and more sensitive than conventional methods. Although recent advances in these methods have been made in terms of their cost-benefit, ease of use, and turnaround time, anaerobic culture remains an important method for detection of CDI. Materials and Methods In efforts to evaluate a novel chromogenic medium for the detection of C. difficile (chromID CD agar), 289 fecal specimens were analyzed using two other culture media of blood agar and cycloserine-cefoxitin-fructose-egg yolk agar while enzyme immunosorbent assay and polymerase chain reaction-based assay were used for toxin detection. Results ChromID showed the highest detection rate among the three culture media. Both positive rate and sensitivity were higher from chromID than other culture media. ChromID was better at detecting toxin producing C. difficile at 24 h and showed the highest detection rate at both 24 h and 48 h. Conclusion Simultaneous use of toxin assay and anaerobic culture has been considered as the most accurate and sensitive diagnostic approach of CDI. Utilization of a more rapid and sensitive chromogenic medium will aid in the dianogsis of CDI. PMID:24954329

  20. Dissimilation of Carbon Monoxide to Acetic Acid by Glucose-Limited Cultures of Clostridium thermoaceticum

    PubMed Central

    Martin, Douglas R.; Misra, Arun; Drake, Harold L.

    1985-01-01

    Clostridium thermoaceticum was cultivated in glucose-limited media, and the dissimilation of CO to acetic acid was evaluated. We found that cultures catalyzed the rapid dissimilation of CO to acetic acid and CO2, with the stoichiometry obtained for conversion approximating that predicted from the following reaction: 4CO + 2H2O → CH3CO2H + 2CO2. Growing cultures formed approximately 50 mmol (3 g) of CO-derived acetic acid per liter of culture, with the rate of maximal consumption approximating 9.1 mmol of CO consumed/h per liter of culture. In contrast, resting cells were found not to dissimilate CO to acetic acid. 14CO was incorporated, with equal distribution between the carboxyl and methyl carbons of acetic acid when the initial cultivation gas phase was 100% CO, whereas 14CO2 preferentially entered the carboxyl carbon when the initial gas phase was 100% CO2. Significantly, in the presence of saturating levels of CO, 14CO2 preferentially entered the methyl carbon, whereas saturating levels of CO2 yielded 14CO-derived labeling predominantly in the carboxyl carbon. These findings are discussed in relation to the path of carbon flow to acetic acid. PMID:16346807

  1. Sensitive and selective culture medium for detection of environmental Clostridium difficile isolates without requirement for anaerobic culture conditions.

    PubMed

    Cadnum, Jennifer L; Hurless, Kelly N; Deshpande, Abhishek; Nerandzic, Michelle M; Kundrapu, Sirisha; Donskey, Curtis J

    2014-09-01

    Effective and easy-to-use methods for detecting Clostridium difficile spore contamination would be useful for identifying environmental reservoirs and monitoring the effectiveness of room disinfection. Culture-based detection methods are sensitive for detecting C. difficile, but their utility is limited due to the requirement of anaerobic culture conditions and microbiological expertise. We developed a low-cost selective broth medium containing thioglycolic acid and l-cystine, termed C. difficile brucella broth with thioglycolic acid and l-cystine (CDBB-TC), for the detection of C. difficile from environmental specimens under aerobic culture conditions. The sensitivity and specificity of CDBB-TC (under aerobic culture conditions) were compared to those of CDBB (under anaerobic culture conditions) for the recovery of C. difficile from swabs collected from hospital room surfaces. CDBB-TC was significantly more sensitive than CDBB for recovering environmental C. difficile (36/41 [88%] versus 21/41 [51%], respectively; P = 0.006). C. difficile latex agglutination, an enzyme immunoassay for toxins A and B or glutamate dehydrogenase, and a PCR for toxin B genes were all effective as confirmatory tests. For 477 total environmental cultures, the specificity of CDBB-TC versus that of CDBB based upon false-positive yellow-color development of the medium without recovery of C. difficile was 100% (0 false-positive results) versus 96% (18 false-positive results), respectively. False-positive cultures for CDBB were attributable to the growth of anaerobic non-C. difficile organisms that did not grow in CDBB-TC. Our results suggest that CDBB-TC provides a sensitive and selective medium for the recovery of C. difficile organisms from environmental samples, without the need for anaerobic culture conditions. PMID:24958803

  2. Fermentative hydrogen production from glucose and starch using pure strains and artificial co-cultures of Clostridium spp.

    PubMed Central

    2012-01-01

    Background Pure bacterial strains give better yields when producing H2 than mixed, natural communities. However the main drawback with the pure cultures is the need to perform the fermentations under sterile conditions. Therefore, H2 production using artificial co-cultures, composed of well characterized strains, is one of the directions currently undertaken in the field of biohydrogen research. Results Four pure Clostridium cultures, including C. butyricum CWBI1009, C. pasteurianum DSM525, C. beijerinckii DSM1820 and C. felsineum DSM749, and three different co-cultures composed of (1) C. pasteurianum and C. felsineum, (2) C. butyricum and C. felsineum, (3) C. butyricum and C. pasteurianum, were grown in 20 L batch bioreactors. In the first part of the study a strategy composed of three-culture sequences was developed to determine the optimal pH for H2 production (sequence 1); and the H2-producing potential of each pure strain and co-culture, during glucose (sequence 2) and starch (sequence 3) fermentations at the optimal pH. The best H2 yields were obtained for starch fermentations, and the highest yield of 2.91 mol H2/ mol hexose was reported for C. butyricum. By contrast, the biogas production rates were higher for glucose fermentations and the highest value of 1.5 L biogas/ h was observed for the co-culture (1). In general co-cultures produced H2 at higher rates than the pure Clostridium cultures, without negatively affecting the H2 yields. Interestingly, all the Clostridium strains and co-cultures were shown to utilize lactate (present in a starch-containing medium), and C. beijerinckii was able to re-consume formate producing additional H2. In the second part of the study the co-culture (3) was used to produce H2 during 13 days of glucose fermentation in a sequencing batch reactor (SBR). In addition, the species dynamics, as monitored by qPCR (quantitative real-time PCR), showed a stable coexistence of C. pasteurianum and C. butyricum during this

  3. Fermentation of Cellulosic Substrates in Batch and Continuous Culture by Clostridium thermocellum

    PubMed Central

    Lynd, Lee Rybeck; Grethlein, Hans E.; Wolkin, Richard H.

    1989-01-01

    Fermentation of dilute-acid-pretreated mixed hardwood and Avicel by Clostridium thermocellum was compared in batch and continuous cultures. Maximum specific growth rates per hour obtained on cellulosic substrates were 0.1 in batch culture and >0.13 in continuous culture. Cell yields (grams of cells per gram of substrate) in batch culture were 0.17 for pretreated wood and 0.15 for Avicel. Ethanol and acetate were the main products observed under all conditions. Ethanol:acetate ratios (in grams) were approximately 1.8:1 in batch culture and generally slightly less than 1:1 in continuous culture. Utilization of cellulosic substrates was essentially complete in batch culture. A prolonged lag phase was initially observed in batch culture on pretreated wood; the length of the lag phase could be shortened by addition of cell-free spent medium. In continuous culture with ∼5 g of glucose equivalent per liter in the feed, substrate conversion relative to theoretical ranged from 0.86 at a dilution rate (D) of 0.05/h to 0.48 at a D of 0.167/h for Avicel and from 0.75 at a D of 0.05/h to 0.43 at a D of 0.11/h for pretreated wood. At feed concentrations of <4.5 g of glucose equivalent per liter, conversion of pretreated wood was 80 to 90% at D = 0.083/h. Lower conversion was obtained at higher feed substrate concentrations, consistent with a limiting factor other than cellulose. Free Avicelase activities of 12 to 84 mU/ml were observed, with activity increasing in this order: batch cellobiose, batch pretreated wood < batch Avicel, continuous pretreated wood < continuous Avicel. Free cellulase activity was higher at increasing extents of substrate utilization for both pretreated wood and Avicel under all conditions tested. The results indicate that fermentation parameters, with the exception of free cellulase activity, are essentially the same for pretreated mixed hardwood and Avicel under a variety of conditions. Hydrolysis yields obtained with C. thermocellum cellulase acting

  4. Comparison of real-time PCR techniques to cytotoxigenic culture methods for diagnosing Clostridium difficile infection.

    PubMed

    Knetsch, C W; Bakker, D; de Boer, R F; Sanders, I; Hofs, S; Kooistra-Smid, A M D; Corver, J; Eastwood, K; Wilcox, M H; Kuijper, E J

    2011-01-01

    In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027. PMID:20980562

  5. Direct glucose production from lignocellulose using Clostridium thermocellum cultures supplemented with a thermostable β-glucosidase

    PubMed Central

    2013-01-01

    Background Cellulases continue to be one of the major costs associated with the lignocellulose hydrolysis process. Clostridium thermocellum is an anaerobic, thermophilic, cellulolytic bacterium that produces cellulosomes capable of efficiently degrading plant cell walls. The end-product cellobiose, however, inhibits degradation. To maximize the cellulolytic ability of C. thermocellum, it is important to eliminate this end-product inhibition. Results This work describes a system for biological saccharification that leads to glucose production following hydrolysis of lignocellulosic biomass. C. thermocellum cultures supplemented with thermostable beta-glucosidases make up this system. This approach does not require any supplementation with cellulases and hemicellulases. When C. thermocellum strain S14 was cultured with a Thermoanaerobacter brockii beta-glucosidase (CglT with activity 30 U/g cellulose) in medium containing 100 g/L cellulose (617 mM initial glucose equivalents), we observed not only high degradation of cellulose, but also accumulation of 426 mM glucose in the culture broth. In contrast, cultures without CglT, or with less thermostable beta-glucosidases, did not efficiently hydrolyze cellulose and accumulated high levels of glucose. Glucose production required a cellulose load of over 10 g/L. When alkali-pretreated rice straw containing 100 g/L glucan was used as the lignocellulosic biomass, approximately 72% of the glucan was saccharified, and glucose accumulated to 446 mM in the culture broth. The hydrolysate slurry containing glucose was directly fermented to 694 mM ethanol by addition of Saccharomyces cerevisiae, giving an 85% theoretical yield without any inhibition. Conclusions Our process is the first instance of biological saccharification with exclusive production and accumulation of glucose from lignocellulosic biomass. The key to its success was the use of C. thermocellum supplemented with a thermostable beta-glucosidase and cultured

  6. Enumeration of Clostridium perfringens spores in human feces: comparison of four culture media.

    PubMed

    Harmon, S M; Kautter, D A

    1987-01-01

    Enumeration of Clostridium perfringens spores was compared using 4 culture media. Duplicate 1 g portions of 35 stools (25 from C. perfringens food poisoning outbreaks and 10 from normal stools) were heat treated 20 min at 75 degrees C and tested on tryptose-sulfite-cycloserine (TSC) agar, trypticase-soy-blood (TSB) agar, lactose-sulfite (LS) medium, and iron milk (IM) medium. Dilutions were plated directly onto TSB and TSC, and a 3-tube most probable number determination was made with each specimen in LS and IM incubated at 45 degrees C. TSB was easiest to use and nonhemolytic food poisoning strains were readily differentiated from the normal hemolytic biotype on this medium. Confirmed counts on TSC and TSB were similar for all specimens, but counts of 8 of 25 outbreak specimens were 2-4 log units lower in LS and IM than on plating media; spores in specimens associated with 2 of 5 outbreaks were intolerant of the elevated temperatures. Results showed that elevated temperature MPN methods in LS and IM are inappropriate for the examination of outbreak stools. PMID:2893783

  7. Artificial symbiosis for acetone-butanol-ethanol (ABE) fermentation from alkali extracted deshelled corn cobs by co-culture of Clostridium beijerinckii and Clostridium cellulovorans

    PubMed Central

    2014-01-01

    Background Butanol is an industrial commodity and also considered to be a more promising gasoline substitute compared to ethanol. Renewed attention has been paid to solvents (acetone, butanol and ethanol) production from the renewable and inexpensive substrates, for example, lignocellulose, on account of the depletion of oil resources, increasing gasoline prices and deteriorating environment. Limited to current tools for genetic manipulation, it is difficult to develop a genetically engineered microorganism with combined ability of lignocellulose utilization and solvents production. Mixed culture of cellulolytic microorganisms and solventogenic bacteria provides a more convenient and feasible approach for ABE fermentation due to the potential for synergistic utilization of the metabolic pathways of two organisms. But few bacteria pairs succeeded in producing biobutanol of high titer or high productivity without adding butyrate. The aim of this work was to use Clostridium cellulovorans 743B to saccharify lignocellulose and produce butyric acid, instead of adding cellulase and butyric acid to the medium, so that the soluble sugars and butyric acid generated can be subsequently utilized by Clostridium beijerinckii NCIMB 8052 to produce butanol in one pot reaction. Results A stable artificial symbiotic system was constructed by co-culturing a celluloytic, anaerobic, butyrate-producing mesophile (C. cellulovorans 743B) and a non-celluloytic, solventogenic bacterium (C. beijerinckii NCIMB 8052) to produce solvents by consolidated bioprocessing (CBP) with alkali extracted deshelled corn cobs (AECC), a low-cost renewable feedstock, as the sole carbon source. Under optimized conditions, the co-culture degraded 68.6 g/L AECC and produced 11.8 g/L solvents (2.64 g/L acetone, 8.30 g/L butanol and 0.87 g/L ethanol) in less than 80 h. Besides, a real-time PCR assay based on the 16S rRNA gene sequence was performed to study the dynamics of the abundance of each strain

  8. A Cost-Effective Approach for Detection of Toxigenic Clostridium difficile: Toxigenic Culture Using ChromID Clostridium difficile Agar

    PubMed Central

    To, Wing Kin; Ng, Tak Keung; Hui, Wai Ting; Lee, Wing Keung; Lau, Florence; Ching, Almond Man Wai

    2014-01-01

    We evaluated the performance and the cost of toxigenic culture using a commercial chromogenic medium (CDIF) for 538 stool specimens. Compared with real-time PCR, this method was found to detect an additional 9% of positive specimens and result in 61% reduction in material costs, with a trade-off increase in turnaround time of 1 day. PMID:24478510

  9. Clostridium perfringens

    PubMed Central

    Clifford, Walter J.; Anellis, Abe

    1971-01-01

    A biphasic culture medium suitable for cultivation and sporulation of Clostridium perfringens, C. botulinum, and C. sporogenes was devised. The medium designed for use in a disposable, compartmented, plastic film container contained peptones, yeast extract, minerals, an anion exchange resin, and glucose in 4% agar as the solid phase and (NH4)2SO4 and 0.1% agar as the liquid phase. With the biphasic system, it was not necessary to use active cultures as inocula. Growth was at least equal to that obtained in conventional media, and spore production of 9 out of 12 strains of C. perfringens equalled or usually exceeded that of conventional media. Images PMID:4332043

  10. Draft Genome Sequence of Clostridium ultunense Strain BS (DSMZ 10521), Recovered from a Mixed Culture.

    PubMed

    Wei, Yongjun; Zhou, Haokui; Zhang, Lei; Zhang, Jun; Wang, Yuezhu; Wang, Shengyue; Zhou, Zhihua; Yan, Xing

    2014-01-01

    Clostridium ultunense BS is the first isolated strain (type strain) of C. ultunense that was identified as a mesophilic syntrophic acetate-oxidizing bacterium (SAOB). Here, we report the draft genome sequence of this strain, which will help us to elucidate the mechanism of syntrophic acetate oxidization. PMID:24504003

  11. Draft Genome Sequence of Clostridium ultunense Strain BS (DSMZ 10521), Recovered from a Mixed Culture

    PubMed Central

    Wei, Yongjun; Zhou, Haokui; Zhang, Lei; Zhang, Jun; Wang, Yuezhu; Wang, Shengyue

    2014-01-01

    Clostridium ultunense BS is the first isolated strain (type strain) of C. ultunense that was identified as a mesophilic syntrophic acetate-oxidizing bacterium (SAOB). Here, we report the draft genome sequence of this strain, which will help us to elucidate the mechanism of syntrophic acetate oxidization. PMID:24504003

  12. Contributions to the taxonomy and biology of Clostridium difficile.

    PubMed

    Bittner, J; Macovei, A; Lemeni, D; Arboreanu, D; Potorac, E

    1992-01-01

    Clostridium difficile was incriminated by Hughes and Jarvis (1987) as a cause of intestinal infections in USA in the 1980-1984 period in 45 p. 100 of cases, whereas Salmonellae only in 12 p. 100. Four strains of this organism are studied in this paper in comparison with ten strains of C. bifermentans and six of C. sordellii, because the three species share a common antigen and have other common characteristics, as well. However, spores of C. difficile swell the bacteria and those of other bacteria (C. bifermentans and C. sordellii) do not; C. difficile does not produce indole, whereas the other species produce it. We confirmed the selective capacity of the medium of George et al. (1979) using the "alcohol shock" and as selective agents cycloserine D and cefoxitin. C. difficile proved to be most susceptible to metronidazole and rifampin. Whereas the former antibiotic was considered as a cause of post-antibiotic intestinal infections by different authors, the second was not, to our knowledge. The strain 10463 has a considerable toxicity (1000 DLM/ml for the white mouse, and pathogenicity--2000--5000 DCL for the white mouse, as compared to 25 DCL of the other three strains). Using this toxin an antitoxic serum was obtained in horse, with a capacity of neutralizing the action of the toxin up to a dilution of 1 p. 1000. PMID:1304829

  13. Rapid and Sensitive Loop-Mediated Isothermal Amplification Test for Clostridium difficile Detection Challenges Cytotoxin B Cell Test and Culture as Gold Standard▿

    PubMed Central

    Norén, Torbjörn; Alriksson, Ingegärd; Andersson, Josefin; Åkerlund, Thomas; Unemo, Magnus

    2011-01-01

    Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings. PMID:21106782

  14. Rapid and sensitive loop-mediated isothermal amplification test for Clostridium difficile detection challenges cytotoxin B cell test and culture as gold standard.

    PubMed

    Norén, Torbjörn; Alriksson, Ingegärd; Andersson, Josefin; Akerlund, Thomas; Unemo, Magnus

    2011-02-01

    Compared to the composite gold standard cytotoxin B assay and toxigenic culture, the loop-mediated isothermal amplification (LAMP) test for Clostridium difficile had a sensitivity and specificity of 98%, positive predictive value of 92%, and negative predictive value of >99%. A one-hour turnaround time for the LAMP test provides rapid diagnosis and cost savings. PMID:21106782

  15. Predictive modeling in Clostridium acetobutylicum fermentations employing Raman spectroscopy and multivariate data analysis for real-time culture monitoring

    NASA Astrophysics Data System (ADS)

    Zu, Theresah N. K.; Liu, Sanchao; Germane, Katherine L.; Servinsky, Matthew D.; Gerlach, Elliot S.; Mackie, David M.; Sund, Christian J.

    2016-05-01

    The coupling of optical fibers with Raman instrumentation has proven to be effective for real-time monitoring of chemical reactions and fermentations when combined with multivariate statistical data analysis. Raman spectroscopy is relatively fast, with little interference from the water peak present in fermentation media. Medical research has explored this technique for analysis of mammalian cultures for potential diagnosis of some cancers. Other organisms studied via this route include Escherichia coli, Saccharomyces cerevisiae, and some Bacillus sp., though very little work has been performed on Clostridium acetobutylicum cultures. C. acetobutylicum is a gram-positive anaerobic bacterium, which is highly sought after due to its ability to use a broad spectrum of substrates and produce useful byproducts through the well-known Acetone-Butanol-Ethanol (ABE) fermentation. In this work, real-time Raman data was acquired from C. acetobutylicum cultures grown on glucose. Samples were collected concurrently for comparative off-line product analysis. Partial-least squares (PLS) models were built both for agitated cultures and for static cultures from both datasets. Media components and metabolites monitored include glucose, butyric acid, acetic acid, and butanol. Models were cross-validated with independent datasets. Experiments with agitation were more favorable for modeling with goodness of fit (QY) values of 0.99 and goodness of prediction (Q2Y) values of 0.98. Static experiments did not model as well as agitated experiments. Raman results showed the static experiments were chaotic, especially during and shortly after manual sampling.

  16. Contributing factors in the improvement of cellulosic H2 production in Clostridium thermocellum/Thermoanaerobacterium co-cultures.

    PubMed

    Wang, Mingyu; Zhao, Qi; Li, Ling; Niu, Kangle; Li, Yi; Wang, Fangzhong; Jiang, Baojie; Liu, Kuimei; Jiang, Yi; Fang, Xu

    2016-10-01

    Lignocellulosic biohydrogen is a promising renewable energy source that could be a potential alternative to the unsustainable fossil fuel-based energy. Biohydrogen production could be performed by Clostridium thermocellum that is the fastest known cellulose-degrading bacterium. Previous investigations have shown that the co-culture of C. thermocellum JN4 and a non-cellulolytic bacterium Thermoanaerobacterium thermosaccharolyticum GD17 produces more hydrogen than the C. thermocellum JN4 mono-culture, but the mechanism of this improvement is unknown. In this work, we carried out genomic and evolutionary analysis of hydrogenase-coding genes in C. thermocellum and T. thermosaccharolyticum, identifying one Ech-type [NiFe] hydrogenase complex in each species, and, respectively, five and four monomeric or multimeric [FeFe] hydrogenases in the two species. Further transcriptional analysis showed hydrogenase-coding genes in C. thermocellum are regulated by carbon sources, while hydrogenase-coding genes in T. thermosaccharolyticum are not. However, comparison between transcriptional abundance of hydrogenase-coding genes in mono- and co-cultures showed the co-culturing condition leads to transcriptional changes of hydrogenase-coding genes in T. thermosaccharolyticum but not C. thermocellum. Further metabolic analysis showed T. thermosaccharolyticum produces H2 at a rate 4-12-fold higher than C. thermocellum. These findings lead to the suggestion that the improvement of H2 production in the co-culture over mono-culture should be attributed to changes in T. thermosaccharolyticum but not C. thermocellum. Further suggestions can be made that C. thermocellum and T. thermosaccharolyticum perform highly specialized tasks in the co-culture, and optimization of the co-culture for more lignocellulosic biohydrogen production should be focused on the improvement of the non-cellulolytic bacterium. PMID:27538932

  17. Simultaneous determination of amino acids and carbohydrates in culture media of Clostridium thermocellum by valve-switching ion chromatography.

    PubMed

    Fa, Yun; Yang, Haiyan; Ji, Chengshuai; Cui, He; Zhu, Xinshu; Du, Juan; Gao, Jun

    2013-10-10

    An improved method for the simultaneous determination of 20 amino acids and 7 carbohydrates using one-valve switching after injection, ion chromatography, and integrated pulsed amperometric detection is proposed. The resolution of the amino acids and carbohydrates in the cation trap column was investigated. In addition, parameters including flow liquid type, flow rate, concentration, and valve-switch timing were optimized. The method is time-saving, effective, and accurate for the simultaneous separation of amino acids and carbohydrates, with a mean correlation coefficient of >0.99 and repeatability of 0.5-4.6% for eight replicates. The method was successfully applied in the analysis of amino acids and carbohydrates in aseptic media and in extracellular culture media of three phenotypes of Clostridium thermocellum. PMID:24070489

  18. Co-culturing a novel Bacillus strain with Clostridium tyrobutyricum ATCC 25755 to produce butyric acid from sucrose

    PubMed Central

    2013-01-01

    Background Currently, the most promising microorganism used for the bio-production of butyric acid is Clostridium tyrobutyricum ATCC 25755T; however, it is unable to use sucrose as a sole carbon source. Consequently, a newly isolated strain, Bacillus sp. SGP1, that was found to produce a levansucrase enzyme, which hydrolyzes sucrose into fructose and glucose, was used in a co-culture with this strain, permitting C. tyrobutyricum ATCC 25755T to ferment sucrose to butyric acid. Results B. sp. SGP1 alone did not show any butyric acid production and the main metabolite produced was lactic acid. This allowed C. tyrobutyricum ATCC 25755T to utilize the monosaccharides resulting from the activity of levansucrase together with the lactic acid produced by B. sp. SGP1 to generate butyric acid, which was the main fermentative product within the co-culture. Furthermore, the final acetic acid concentration in the co-culture was significantly lower when compared with pure C. tyrobutyricum ATCC 25755T cultures grown on glucose. In fed-batch fermentations, the optimum conditions for the production of butyric acid were around pH 5.50 and a temperature of 37°C. Under these conditions, the final butyrate concentration was 34.2±1.8 g/L with yields of 0.35±0.03 g butyrate/g sucrose and maximum productivity of 0.3±0.04 g/L/h. Conclusions Using this co-culture, sucrose can be utilized as a carbon source for butyric acid production at a relatively high yield. In addition, this co-culture offers also the benefit of a greater selectivity, with butyric acid constituting 92.8% of the acids when the fermentation was terminated. PMID:23452443

  19. Effect of Bifidobacterium upon Clostridium difficile Growth and Toxicity When Co-cultured in Different Prebiotic Substrates.

    PubMed

    Valdés-Varela, L; Hernández-Barranco, Ana M; Ruas-Madiedo, Patricia; Gueimonde, Miguel

    2016-01-01

    The intestinal overgrowth of Clostridium difficile, often after disturbance of the gut microbiota by antibiotic treatment, leads to C. difficile infection (CDI) which manifestation ranges from mild diarrhea to life-threatening conditions. The increasing CDI incidence, not only in compromised subjects but also in traditionally considered low-risk populations, together with the frequent relapses of the disease, has attracted the interest for prevention/therapeutic options. Among these, probiotics, prebiotics, or synbiotics constitute a promising approach. In this study we determined the potential of selected Bifidobacterium strains for the inhibition of C. difficile growth and toxicity in different carbon sources. We conducted co-cultures of the toxigenic strain C. difficile LMG21717 with four Bifidobacterium strains (Bifidobacterium longum IPLA20022, Bifidobacterium breve IPLA20006, Bifidobacterium bifidum IPLA20015, and Bifidobacterium animalis subsp. lactis Bb12) in the presence of various prebiotic substrates (Inulin, Synergy, and Actilight) or glucose, and compared the results with those obtained for the corresponding mono-cultures. C. difficile and bifidobacteria levels were quantified by qPCR; the pH and the production of short chain fatty acids was also determined. Moreover, supernatants of the cultures were collected to evaluate their toxicity using a recently developed model. Results showed that co-culture with B. longum IPLA20022 and B. breve IPLA20006 in the presence of short-chain fructooligosaccharides, but not of Inulin, as carbon source significantly reduced the growth of the pathogen. With the sole exception of B. animalis Bb12, whose growth was enhanced, the presence of C. difficile did not show major effects upon the growth of the bifidobacteria. In accordance with the growth data, B. longum and B. breve were the strains showing higher reduction in the toxicity of the co-culture supernatants. PMID:27242753

  20. Effect of Bifidobacterium upon Clostridium difficile Growth and Toxicity When Co-cultured in Different Prebiotic Substrates

    PubMed Central

    Valdés-Varela, L.; Hernández-Barranco, Ana M.; Ruas-Madiedo, Patricia; Gueimonde, Miguel

    2016-01-01

    The intestinal overgrowth of Clostridium difficile, often after disturbance of the gut microbiota by antibiotic treatment, leads to C. difficile infection (CDI) which manifestation ranges from mild diarrhea to life-threatening conditions. The increasing CDI incidence, not only in compromised subjects but also in traditionally considered low-risk populations, together with the frequent relapses of the disease, has attracted the interest for prevention/therapeutic options. Among these, probiotics, prebiotics, or synbiotics constitute a promising approach. In this study we determined the potential of selected Bifidobacterium strains for the inhibition of C. difficile growth and toxicity in different carbon sources. We conducted co-cultures of the toxigenic strain C. difficile LMG21717 with four Bifidobacterium strains (Bifidobacterium longum IPLA20022, Bifidobacterium breve IPLA20006, Bifidobacterium bifidum IPLA20015, and Bifidobacterium animalis subsp. lactis Bb12) in the presence of various prebiotic substrates (Inulin, Synergy, and Actilight) or glucose, and compared the results with those obtained for the corresponding mono-cultures. C. difficile and bifidobacteria levels were quantified by qPCR; the pH and the production of short chain fatty acids was also determined. Moreover, supernatants of the cultures were collected to evaluate their toxicity using a recently developed model. Results showed that co-culture with B. longum IPLA20022 and B. breve IPLA20006 in the presence of short-chain fructooligosaccharides, but not of Inulin, as carbon source significantly reduced the growth of the pathogen. With the sole exception of B. animalis Bb12, whose growth was enhanced, the presence of C. difficile did not show major effects upon the growth of the bifidobacteria. In accordance with the growth data, B. longum and B. breve were the strains showing higher reduction in the toxicity of the co-culture supernatants. PMID:27242753

  1. Inhibitory activity of Lactobacillus plantarum TF711 against Clostridium sporogenes when used as adjunct culture in cheese manufacture.

    PubMed

    González, Lorena; Zárate, Victoria

    2015-05-01

    Bacteriocins produced by lactic acid bacteria are of great interest to the food-processing industry as natural preservatives. This work aimed to investigate the efficacy of bacteriocin-producing Lactobacillus plantarum TF711, isolated from artisanal Tenerife cheese, in controlling Clostridium sporogenes during cheese ripening. Cheeses were made from pasteurised milk artificially contaminated with 10(4) spores m/l C. sporogenes. Experimental cheeses were manufactured with Lb. plantarum TF711 added at 1% as adjunct to commercial starter culture. Cheeses made under the same conditions but without Lb. plantarum TF711 served as controls. Evolution of microbiological parameters, pH and NaCl content, as well as bacteriocin production was studied throughout 45 d of ripening. Addition of Lb. plantarum TF711 did not bring about any significant change in starter culture counts, NaCl content and pH, compared with control cheese. In contrast, clostridial spore count in experimental cheeses were significantly lower than in control cheeses from 7 d onwards, reaching a maximum reduction of 2·2 log units on day 21. Inhibition of clostridia found in experimental cheeses was mainly attributed to plantaricin activity, which in fact was recovered from these cheeses. PMID:25702615

  2. Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus.

    PubMed

    Wang, Han; Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J

    2015-01-01

    The draft genome sequence of Clostridium sp. Ne2 was reconstructed from a metagenome of a hydrogenogenic microbial consortium. The organism is most closely related to Clostridium magnum and is a strict anaerobe that is predicted to ferment a range of simple sugars. PMID:25908129

  3. Draft Genome Sequence of Clostridium sp. Ne2, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus

    PubMed Central

    Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J.

    2015-01-01

    The draft genome sequence of Clostridium sp. Ne2 was reconstructed from a metagenome of a hydrogenogenic microbial consortium. The organism is most closely related to Clostridium magnum and is a strict anaerobe that is predicted to ferment a range of simple sugars. PMID:25908129

  4. Enumeration of Clostridium perfringens spores in groundwater samples: comparison of six culture media.

    PubMed

    Araujo, M; Sueiro, R A; Gómez, M J; Garrido, M J

    2004-05-01

    In order to investigate the ability of Fluorocult-supplemented TSC agar (TSCF (Fluorocult supplemented TSC-agar): prepared from Tryptose Sulfite Cycloserine Agar Base (Merck), D-cycloserine (Fluka Chemika, USA), and fluorocult TSC-Agar supplement (Merck)) for detecting spores of Clostridium perfringens in water, we analyzed groundwater samples, pretreated by heating to 80 degrees C/5 min, using this fluorogenic medium together with five other media: mCP agar (Panreac; Cultimed), TSC agar (Merck, Germany), TSN agar (Merck), and SPS agar (BBL, USA) by the membrane filtration technique, and Wilson-Blair agar (WB) following the still-in-force Spanish official method. Variance analysis of the data obtained shows statistically significant differences in the counts obtained between media employed in this work. The C. perfringens spore counts on mCP agar were significantly lower (P<0.05) than the corresponding values of TSC, TSCF, SPS, and WB media. No statistically significant differences were found between C. perfringens spore counts on TSCF compared with those of other methods used. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for C. perfringens spore recovery in groundwater samples. Additionally, the results obtained indicate that mCP agar, which is the reference method in the European Union, is not suitable medium for recovering C. perfringens spores from groundwater samples. PMID:15063057

  5. Comparison of Real-Time PCR Techniques to Cytotoxigenic Culture Methods for Diagnosing Clostridium difficile Infection ▿

    PubMed Central

    Knetsch, C. W.; Bakker, D.; de Boer, R. F.; Sanders, I.; Hofs, S.; Kooistra-Smid, A. M. D.; Corver, J.; Eastwood, K.; Wilcox, M. H.; Kuijper, E. J.

    2011-01-01

    In the past decade, the incidence of Clostridium difficile infections (CDI) with a more severe course has increased in Europe and North America. Assays that are capable of rapidly diagnosing CDI are essential. Two real-time PCRs (LUMC and LvI) targeting C. difficile toxin genes (tcdB, and tcdA and tcdB, respectively) were compared with the BD GeneOhm PCR (targeting the tcdB gene), using cytotoxigenic culture as a gold standard. In addition, a real-time PCR targeting the tcdC frameshift mutation at position 117 (Δ117 PCR) was evaluated for detecting toxigenic C. difficile and the presence of PCR ribotype 027 in stool samples. In total, 526 diarrheal samples were prospectively collected and included in the study. Compared with those for cytotoxigenic culture, sensitivity, specificity, positive predicted value (PPV), and negative predicted value (NPV) were for PCR LUMC 96.0%, 88.0%, 66.0%, and 98.9%, for PCR LvI 100.0%, 89.4%, 69.7%, and 100.0%, for PCR Δ117 98.0%, 90.7%, 71.9%, and 99.5%, and for PCR BD GeneOhm 88.3%, 96.9%, 86.5%, and 97.4%. Compared to those with feces samples cultured positive for C. difficile type 027, the sensitivity, specificity, PPV, and NPV of the Δ117 PCR were 95.2%, 96.2%, 87.0%, and 98.7%. We conclude that all real-time PCRs can be applied as a first screening test in an algorithm for diagnosing CDI. However, the low PPVs hinder the use of the assays as stand-alone tests. Furthermore, the Δ117 PCR may provide valuable information for minimizing the spread of the epidemic C. difficile PCR ribotype 027. PMID:20980562

  6. Cloning of a genetic determinant from Clostridium difficile involved in adherence to tissue culture cells and mucus.

    PubMed Central

    Karjalainen, T; Barc, M C; Collignon, A; Trollé, S; Boureau, H; Cotte-Laffitte, J; Bourlioux, P

    1994-01-01

    Our laboratory has previously shown that Clostridium difficile adherence to Caco-2 cells is greatly enhanced after heat shock at 60 degrees C and that it is mediated by a proteinaceous surface component. The experiments described here show that C. difficile could adhere to several types of tissue culture cells (Vero, HeLa, and KB) after heat shock. The type of culture medium (liquid or solid, with or without blood) had little effect on adhesion. To clone the adhesin gene, polyclonal antibodies against C. difficile heated at 60 degrees C were used to screen a genomic library of C. difficile constructed in lambda ZapII. Ten positive clones were identified in the library, one of which (pCL6) agglutinated several types of erythrocytes in the presence of mannose. In Western blots (immunoblots), this clone expressed in Escherichia coli a 40- and a 27-kDa protein; a 27-kDa protein has been previously identified in the surface extracts of heat-shocked C. difficile as a possible adhesin. The clone adhered to Vero, Caco-2, KB, and HeLa cells; the adherence was blocked by anti-C. difficile antibodies, by a surface extract of C. difficile, and by mucus isolated from axenic mice. Furthermore, the clone could attach ex vivo to intestinal mucus isolated from axenic mice. Preliminary studies on the receptor moieties implicated in C. difficile adhesion revealed that glucose and galactose could partially block adhesion to tissue culture cells, as did di- or trisaccharides containing these sugars, suggesting that the adhesin is a lectin. In addition, N-acetylgalactosamine, a component of mucus, and gelatin partially impeded cell attachment. Images PMID:7927694

  7. Modelling the role of CtfA/B in reverse shift continuous culture experiments of Clostridium acetobutylicum.

    PubMed

    Thorn, Graeme J; King, John R

    2016-06-01

    In continuous phosphate-limited conditions, under pH control from high pH (pH ≳ 5.2) to low pH (pH ≲ 5.2), the metabolism of the Gram-positive bacterium Clostridium acetobutylicum,switches from acid to solvent production. Three main enzymes are responsible for the shift, acetoacetate decarboxylase (Adc), alcohol dehydrogenase (AdhE1/2) and a CoA-transferase (CtfA/B), which are produced in increased quantities during solventogenesis. A two-population model, Millat et al. (2013) and fitted to such 'forward'-shift data, can explain this, as well as observed changes in optical density immediately following the shift: an acidogenic subpopulation is washed out and a solventogenic subpopulation grows in its place, each with distinct physiologies and proteomes. We fit this model to a 'reverse'-shift experiment, where the pH is increased from solventogenic to acidogenic conditions. We find corresponding changes in reaction rates, with AdhE1 and Adc production falling, as in the 'forward' experiments; however, for CtfA/B, the best fit surprisingly arises from the same level of production in both conditions. We propose experiments that would test whether this is a model artefact or accurately reflects cultures shifted in this reverse direction, and, if true, may suggest that over-expressing CtfA/B in both solventogenic and acidogenic conditions could improve the efficiency of fermentation. PMID:26997560

  8. Combination of culture, antigen and toxin detection, and cytotoxin neutralization assay for optimal Clostridium difficile diagnostic testing

    PubMed Central

    Alfa, Michelle J; Sepehri, Shadi

    2013-01-01

    BACKGROUND: There has been a growing interest in developing an appropriate laboratory diagnostic algorithm for Clostridium difficile, mainly as a result of increases in both the number and severity of cases of C difficile infection in the past decade. A C difficile diagnostic algorithm is necessary because diagnostic kits, mostly for the detection of toxins A and B or glutamate dehydrogenase (GDH) antigen, are not sufficient as stand-alone assays for optimal diagnosis of C difficile infection. In addition, conventional reference methods for C difficile detection (eg, toxigenic culture and cytotoxin neutralization [CTN] assays) are not routinely practiced in diagnostic laboratory settings. OBJECTIVE: To review the four-step algorithm used at Diagnostic Services of Manitoba sites for the laboratory diagnosis of toxigenic C difficile. RESULT: One year of retrospective C difficile data using the proposed algorithm was reported. Of 5695 stool samples tested, 9.1% (n=517) had toxigenic C difficile. Sixty per cent (310 of 517) of toxigenic C difficile stools were detected following the first two steps of the algorithm. CTN confirmation of GDH-positive, toxin A- and B-negative assays resulted in detection of an additional 37.7% (198 of 517) of toxigenic C difficile. Culture of the third specimen, from patients who had two previous negative specimens, detected an additional 2.32% (12 of 517) of toxigenic C difficile samples. DISCUSSION: Using GDH antigen as the screening and toxin A and B as confirmatory test for C difficile, 85% of specimens were reported negative or positive within 4 h. Without CTN confirmation for GDH antigen and toxin A and B discordant results, 37% (195 of 517) of toxigenic C difficile stools would have been missed. Following the algorithm, culture was needed for only 2.72% of all specimens submitted for C difficile testing. CONCLUSION: The overview of the data illustrated the significance of each stage of this four-step C difficile algorithm and

  9. Enhanced isopropanol and n-butanol production by supplying exogenous acetic acid via co-culturing two clostridium strains from cassava bagasse hydrolysate.

    PubMed

    Zhang, Shaozhi; Qu, Chunyun; Huang, Xiaoyan; Suo, Yukai; Liao, Zhengping; Wang, Jufang

    2016-07-01

    The focus of this study was to produce isopropanol and butanol (IB) from dilute sulfuric acid treated cassava bagasse hydrolysate (SACBH), and improve IB production by co-culturing Clostridium beijerinckii (C. beijerinckii) with Clostridium tyrobutyricum (C. tyrobutyricum) in an immobilized-cell fermentation system. Concentrated SACBH could be converted to solvents efficiently by immobilized pure culture of C. beijerinckii. Considerable solvent concentrations of 6.19 g/L isopropanol and 12.32 g/L butanol were obtained from batch fermentation, and the total solvent yield and volumetric productivity were 0.42 g/g and 0.30 g/L/h, respectively. Furthermore, the concentrations of isopropanol and butanol increased to 7.63 and 13.26 g/L, respectively, under the immobilized co-culture conditions when concentrated SACBH was used as the carbon source. The concentrations of isopropanol and butanol from the immobilized co-culture fermentation were, respectively, 42.62 and 25.45 % higher than the production resulting from pure culture fermentation. The total solvent yield and volumetric productivity increased to 0.51 g/g and 0.44 g/L/h when co-culture conditions were utilized. Our results indicated that SACBH could be used as an economically favorable carbon source or substrate for IB production using immobilized fermentation. Additionally, IB production could be significantly improved by co-culture immobilization, which provides extracellular acetic acid to C. beijerinckii from C. tyrobutyricum. This study provided a technically feasible and cost-efficient way for IB production using cassava bagasse, which may be suitable for industrial solvent production. PMID:27116556

  10. Clostridium difficile and C. difficile Toxin Testing

    MedlinePlus

    ... C diff antigen; GDH Formal name: Clostridium difficile Culture; C. difficile Toxin, A and B; C. difficile Cytotoxin Assay; Glutamate Dehydrogenase Test Related tests: Stool Culture ; O&P At a Glance Test Sample The ...

  11. Draft Genome Sequence of Clostridium beijerinckii Ne1, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus.

    PubMed

    Wang, Han; Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J

    2015-01-01

    The draft genome of Clostridium beijerinckii strain Ne1 was reconstructed from the metagenomic sequence of a mixed-microbial consortium that produced commercially significant quantities of hydrogen from xylan as a sole feedstock. The organism possesses relatively limited hemicellulolytic capacity and likely requires the action of other organisms to completely degrade xylan. PMID:25908128

  12. Draft Genome Sequence of Clostridium beijerinckii Ne1, Clostridia from an Enrichment Culture Obtained from Australian Subterranean Termite, Nasutitermes exitiosus

    PubMed Central

    Lin, Hai; Tran-Dinh, Nai; Li, Dongmei; Greenfield, Paul; Midgley, David J.

    2015-01-01

    The draft genome of Clostridium beijerinckii strain Ne1 was reconstructed from the metagenomic sequence of a mixed-microbial consortium that produced commercially significant quantities of hydrogen from xylan as a sole feedstock. The organism possesses relatively limited hemicellulolytic capacity and likely requires the action of other organisms to completely degrade xylan. PMID:25908128

  13. Multiplex PCR Targeting tpi (Triose Phosphate Isomerase), tcdA (Toxin A), and tcdB (Toxin B) Genes for Toxigenic Culture of Clostridium difficile

    PubMed Central

    Lemee, Ludovic; Dhalluin, Anne; Testelin, Sabrina; Mattrat, Marie-Andre; Maillard, Karine; Lemeland, Jean-François; Pons, Jean-Louis

    2004-01-01

    A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections. PMID:15583303

  14. Regulation of Clostridium acetobutylicum metabolism as revealed by mixed-substrate steady-state continuous cultures: role of NADH/NAD ratio and ATP pool.

    PubMed Central

    Girbal, L; Soucaille, P

    1994-01-01

    Glycerol-glucose-fed (molar ratio of 2) chemostat cultures of Clostridium acetobutylicum were glucose limited but glycerol sufficient and had a high intracellular NADH/NAD ratio (I. Vasconcelos, L. Girbal, and P. Soucaille, J. Bacteriol. 176:1443-1450, 1994). We report here that the glyceraldehyde-3-phosphate dehydrogenase, one of the key enzymes of the glycolytic pathway, is inhibited by high NADH/NAD ratios. Partial substitution of glucose by pyruvate while maintaining glycerol concentration at a constant level allowed a higher consumption of glycerol in steady-state continuous cultures. However, glycerol-sufficient cultures had a constant flux through the glyceraldehyde-3-phosphate dehydrogenase and a constant NADH/NAD ratio. A high substitution of glucose by pyruvate [P/(G+P) value of 0.67 g/g] provided a carbon-limited culture with butanol and butyrate as the major end products. In this alcohologenic culture, the induction of the NADH-dependent butyraldehyde and the ferredoxin-NAD(P) reductases and the higher expression of alcohol dehydrogenases were related to a high NADH/NAD ratio and a low intracellular ATP concentration. In three different steady-state cultures, the in vitro phosphotransbutyrylase and butyrate-kinase activities decreased with the intracellular ATP concentration, suggesting a transcriptional regulation of these two genes, which are arranged in an operon (K. A. Walter, R. V. Nair, R. V. Carry, G. N. Bennett, and E. T. Papoutsakis, Gene 134:107-111, 1993). PMID:7961393

  15. Diagnosis of Clostridium difficile: real-time PCR detection of toxin genes in faecal samples is more sensitive compared to toxigenic culture.

    PubMed

    Jensen, M B F; Olsen, K E P; Nielsen, X C; Hoegh, A M; Dessau, R B; Atlung, T; Engberg, J

    2015-04-01

    The diagnosis of Clostridium difficile infection (CDI) requires the detection of toxigenic C. difficile or its toxins and a clinical assessment. We evaluated the performance of four nucleic acid amplification tests (NAATs) detecting toxigenic C. difficile directly from faeces compared to routine toxigenic culture. In total, 300 faecal samples from Danish hospitalised patients with diarrhoea were included consecutively. Culture was performed in duplicate (routine and 'expanded toxigenic culture': prolonged and/or re-culture) and genotypic toxin profiling by polymerase chain reaction (PCR), PCR ribotyping and toxinotyping (TT) were performed on culture-positive samples. In parallel, the samples were analysed by four NAATs; two targeting tcdA or tcdB (illumigene C. difficile and PCRFast C. difficile A/B) and two multi-target real-time (RT) PCR assays also targeting cdt and tcdC alleles characteristic of epidemic and potentially more virulent PCR ribotypes 027, 066 and 078 (GeneXpert C. difficile/Epi and an 'in-house RT PCR' two-step algorithm). The multi-target assays were significantly more sensitive compared to routine toxigenic culture (p < 0.05) and significantly more robust to inhibition compared to PCRFast (p < 0.001). Duplicate 'expanded toxigenic culture' increased the culture-positive rate by 29% compared to routine culture. The ability of the GeneXpert and in-house assays to correctly classify PCR ribotype 027 was high (>95%), and in-house PCR displayed 100% correct identification of PCR ribotypes 066 and 078. Furthermore, the presence of the PCR enhancer bovine serum albumin (BSA) was found to be related to high sensitivity and low inhibition rate. Rapid laboratory diagnosis of toxigenic C. difficile by RT PCR was accurate. PMID:25421216

  16. Yield of stool culture with isolate toxin testing versus a two-step algorithm including stool toxin testing for detection of toxigenic Clostridium difficile.

    PubMed

    Reller, Megan E; Lema, Clara A; Perl, Trish M; Cai, Mian; Ross, Tracy L; Speck, Kathleen A; Carroll, Karen C

    2007-11-01

    We examined the incremental yield of stool culture (with toxin testing on isolates) versus our two-step algorithm for optimal detection of toxigenic Clostridium difficile. Per the two-step algorithm, stools were screened for C. difficile-associated glutamate dehydrogenase (GDH) antigen and, if positive, tested for toxin by a direct (stool) cell culture cytotoxicity neutralization assay (CCNA). In parallel, stools were cultured for C. difficile and tested for toxin by both indirect (isolate) CCNA and conventional PCR if the direct CCNA was negative. The "gold standard" for toxigenic C. difficile was detection of C. difficile by the GDH screen or by culture and toxin production by direct or indirect CCNA. We tested 439 specimens from 439 patients. GDH screening detected all culture-positive specimens. The sensitivity of the two-step algorithm was 77% (95% confidence interval [CI], 70 to 84%), and that of culture was 87% (95% CI, 80 to 92%). PCR results correlated completely with those of CCNA testing on isolates (29/29 positive and 32/32 negative, respectively). We conclude that GDH is an excellent screening test and that culture with isolate CCNA testing detects an additional 23% of toxigenic C. difficile missed by direct CCNA. Since culture is tedious and also detects nontoxigenic C. difficile, we conclude that culture is most useful (i) when the direct CCNA is negative but a high clinical suspicion of toxigenic C. difficile remains, (ii) in the evaluation of new diagnostic tests for toxigenic C. difficile (where the best reference standard is essential), and (iii) in epidemiologic studies (where the availability of an isolate allows for strain typing and antimicrobial susceptibility testing). PMID:17804652

  17. Comparison of two rapid assays for Clostridium difficile Common antigen and a C difficile toxin A/B assay with the cell culture neutralization assay.

    PubMed

    Reller, Megan E; Alcabasa, Romina C; Lema, Clara A; Carroll, Karen C

    2010-01-01

    We compared 3 rapid assays for Clostridium difficile with a cell culture cytotoxicity neutralization assay (CCNA). Of 600 stool samples, 46 were positive for toxigenic C difficile. Both rapid common antigen assays were highly sensitive (91.3%-100%) and, therefore, were appropriate screening tests. The rapid toxin assay had poor sensitivity (61%) but excellent specificity (99.3%). Testing stools for glutamate dehydrogenase (step 1) and those positive with a rapid toxin assay (step 2) would correctly classify 81% of submitted specimens within 2 hours, including during periods of limited staffing (evenings, nights, and weekends). CCNA could then be used as a third step to test rapid toxin-negative samples, thereby providing a final result for the remaining 19% of samples by 48 to 72 hours. The use of rapid assays as outlined could enhance timely diagnosis of C difficile. PMID:20023265

  18. Comparison of a frozen human foreskin fibroblast cell assay to an enzyme immunoassay and toxigenic culture for the detection of toxigenic Clostridium difficile.

    PubMed

    Strachan, Alastair J; Evans, Natalie E; Williams, O Martin; Spencer, Robert C; Greenwood, Rosemary; Probert, Chris J

    2013-01-01

    This study set out to validate the Hs27 ReadyCell assay (RCCNA) as an alternative CCNA method compared against a commonly used commercial enzyme immunoassay (EIA) method and toxigenic culture (TC) reference standard. A total of 860 samples were identified from those submitted to the Health Protection Agency microbiology laboratories over a 30-week period. RCCNA performed much better than EIA when using TC as a gold standard, with sensitivities of 90.8% versus 78.6% and positive predictive value of 87.3% to 81.9%, respectively. The Hs27 Human Foreskin Fibroblast ReadyCells are an easy-to-use and a sensitive CCNA method for the detection of toxigenic Clostridium difficile directly from stool. A turnaround time of up to 48 h for a negative result and possible need for repeat testing make it an unsuitable method to be used in most clinical laboratory setting. PMID:23107315

  19. Production of medium-chain volatile fatty acids by mixed ruminal microorganisms is enhanced by ethanol in co-culture with Clostridium kluyveri.

    PubMed

    Weimer, Paul J; Nerdahl, Michael; Brandl, Dane J

    2015-01-01

    Mixed bacterial communities from the rumen ferment cellulosic biomass primarily to C2-C4 volatile fatty acids, and perform only limited chain extension to produce C5 (valeric) and C6 (caproic) acids. The aim of this study was to increase production of caproate and valerate in short-term in vitro incubations. Co-culture of mixed ruminal microbes with a rumen-derived strain of the bacterium Clostridium kluyveri converted cellulosic biomass (alfalfa stems or switchgrass herbage) plus ethanol to VFA mixtures that include valeric and caproic acids as the major fermentation products over a 48-72h run time. Concentrations of caproate reached 6.1gL(-1), similar to or greater than those reported in most conventional carboxylate fermentations that employ substantially longer run times. PMID:25459809

  20. Lignocellulosic hydrolysates and extracellular electron shuttles for H2 production using co-culture fermentation with Clostridium beijerinckii and Geobacter metallireducens.

    PubMed

    Zhang, Xinyu; Ye, Xiaofeng; Guo, Bin; Finneran, Kevin T; Zilles, Julie L; Morgenroth, Eberhard

    2013-11-01

    A co-culture of Clostridium beijerinckii and Geobacter metallireducens with AH2QDS produced hydrogen from lignocellulosic hydrolysates (biomass of Miscanthus prepared by hydrothermal treatment with dilute acids). This co-culture system enhanced hydrogen production from lignocellulosic hydrolysates by improving substrate utilization and diminishing acetate accumulation, despite the presence of fermentation inhibitors in the hydrolysates. The improvements were greater for xylose-rich hydrolysates. The increase in maximum cumulative hydrogen production for hydrolysates with glucose:xylose mass ratios of 1:0.2, 1:1 and 1:10 g/g was 0%, 22% and 11%, respectively. Alternative extracellular electron shuttles (EES), including indigo dye, juglone, lawsone, fulvic acids and humic acids, were able to substitute for AH2QDS, improving hydrogen production in the co-culture system using xylose as model substrate. Increased utilization of xylose-rich hydrolysates and substitution of alternative EES make the co-culture with EES system a more attractive strategy for industrial biohydrogen production. PMID:23994308

  1. Using Phenotype MicroArrays to Determine Culture Conditions That Induce or Repress Toxin Production by Clostridium difficile and Other Microorganisms

    PubMed Central

    Lei, Xiang-He; Bochner, Barry R.

    2013-01-01

    Toxin production is a central issue in the pathogenesis of Clostridium difficile and many other pathogenic microorganisms. Toxin synthesis is influenced by a variety of known and unknown factors of genetics, physiology, and environment. To facilitate the study of toxin production by C. difficile, we have developed a new, reliable, quantitative, and robust cell-based cytotoxicity assay. Then we combined this new assay with Phenotype MicroArrays (PM) technology which provides high throughput testing of culture conditions. This allowed us to quantitatively measure toxin production by C. difficile type strain ATCC 9689 under 768 culture conditions. The culture conditions include different carbon, nitrogen, phosphorus, and sulfur sources. Among these, 89 conditions produced strong toxin induction and 31 produced strong toxin repression. Strong toxin inducers included adenine, guanosine, arginine dipeptides, γ-D-Glu-Gly, methylamine, and others. Some leucine dipeptides and the triple-leucine tripeptide were among the strongest toxin repressors. While some results are consistent with previous observations, others are new observations that provide insights into toxin regulation and pathogenesis of C. difficile. Additionally, we have demonstrated that this combined assay technology can be applied broadly to a wide range of toxin producing microorganisms. This study is the first demonstration of simultaneous assessment of a large number of culture conditions influencing bacterial toxin production. The new functional cytotoxin quantitation method developed provides a valuable tool for studying toxigenic microorganisms and may also find applications in clinical and epidemiological research. PMID:23437164

  2. Comparison of real-time PCR for detection of the tcdC gene with four toxin immunoassays and culture in diagnosis of Clostridium difficile infection.

    PubMed

    Sloan, Lynne M; Duresko, Brian J; Gustafson, Daniel R; Rosenblatt, Jon E

    2008-06-01

    We have developed a rapid real-time PCR method using fluorescence resonance energy transfer probes and the LightCycler (Roche Diagnostics), which will detect the presence of the tcdC gene of Clostridium difficile in stool samples. Our PCR method also will identify the presence of base pair deletions, one of which (18 bp) has been associated with the "epidemic" toxin-hyperproducing strains. We compared the results of this PCR with those of three C. difficile toxin-detecting enzyme immunoassays (EIAs), an EIA for the detection of glutamate dehydrogenase (GDH), and culture of C. difficile. A total of 200 stool specimens were studied by the methods under comparison. C. difficile was isolated from 49 specimens by culture, and 44 of these were confirmed as containing one of the genes associated with toxin production ("toxigenic culture"). Using toxigenic culture as the "gold standard", the sensitivities, specificities, and positive and negative predictive values, respectively, of the assays were 48%, 98%, 88%, and 87% for the Premier toxin A and B test; 48%, 99%, 91%, and 87% for the ImmunoCard toxin A & B test; 48%, 84%, 46%, and 85% for the Xpect C. difficile toxin A/B test; 32%, 100%, 100%, and 84% for the Triage C. difficile panel (for toxin A); and 86%, 97%, 90%, and 96% for the LightCycler PCR. Thus, in comparison to the sensitivity of toxigenic culture, the sensitivities of the toxin immunoassays were unacceptably low, while the LightCycler real-time PCR assay for the detection of the tcdC gene of C. difficile is sensitive and specific. PMID:18434563

  3. Enhancing Butanol Production under the Stress Environments of Co-Culturing Clostridium acetobutylicum/Saccharomyces cerevisiae Integrated with Exogenous Butyrate Addition

    PubMed Central

    Luo, Hongzhen; Ge, Laibing; Zhang, Jingshu; Zhao, Yanli; Ding, Jian; Li, Zhigang; He, Zhenni; Chen, Rui; Shi, Zhongping

    2015-01-01

    In this study, an efficient acetone-butanol-ethanol (ABE) fermentation strategy integrating Clostridium acetobutylicum/Saccharomyces cerevisiae co-culturing system with exogenous butyrate addition, was proposed and experimentally conducted. In solventogenic phase, by adding 0.2 g-DCW/L-broth viable S. cerevisiae cells and 4.0 g/L-broth concentrated butyrate solution into C. acetobutylicum culture broth, final butanol concentration and butanol/acetone ratio in a 7 L anaerobic fermentor reached the highest levels of 15.74 g/L and 2.83 respectively, with the increments of 35% and 43% as compared with those of control. Theoretical and experimental analysis revealed that, the proposed strategy could, 1) extensively induce secretion of amino acids particularly lysine, which are favorable for both C. acetobutylicum survival and butanol synthesis under high butanol concentration environment; 2) enhance the utilization ability of C. acetobutylicum on glucose and over-produce intracellular NADH for butanol synthesis in C. acetobutylicum metabolism simultaneously; 3) direct most of extra consumed glucose into butanol synthesis route. The synergetic actions of effective amino acids assimilation, high rates of substrate consumption and NADH regeneration yielded highest butanol concentration and butanol ratio in C. acetobutylicum under this stress environment. The proposed method supplies an alternative way to improve ABE fermentation performance by traditional fermentation technology. PMID:26489085

  4. A systems biology approach to investigate the effect of pH-induced gene regulation on solvent production by Clostridium acetobutylicum in continuous culture

    PubMed Central

    2011-01-01

    Background Clostridium acetobutylicum is an anaerobic bacterium which is known for its solvent-producing capabilities, namely regarding the bulk chemicals acetone and butanol, the latter being a highly efficient biofuel. For butanol production by C. acetobutylicum to be optimized and exploited on an industrial scale, the effect of pH-induced gene regulation on solvent production by C. acetobutylicum in continuous culture must be understood as fully as possible. Results We present an ordinary differential equation model combining the metabolic network governing solvent production with regulation at the genetic level of the enzymes required for this process. Parameterizing the model with experimental data from continuous culture, we demonstrate the influence of pH upon fermentation products: at high pH (pH 5.7) acids are the dominant product while at low pH (pH 4.5) this switches to solvents. Through steady-state analyses of the model we focus our investigations on how alteration in gene expression of C. acetobutylicum could be exploited to increase butanol yield in a continuous culture fermentation. Conclusions Incorporating gene regulation into the model of solvent production by C. acetobutylicum enables an accurate representation of the pH-induced switch to solvent production to be obtained and theoretical investigations of possible synthetic-biology approaches to be pursued. Steady-state analyses suggest that, to increase butanol yield, alterations in the expression of single solvent-associated genes are insufficient; a more complex approach targeting two or more genes is required. PMID:21247470

  5. Integrative modelling of pH-dependent enzyme activity and transcriptomic regulation of the acetone–butanol–ethanol fermentation of Clostridium acetobutylicum in continuous culture

    PubMed Central

    Millat, Thomas; Janssen, Holger; Bahl, Hubert; Fischer, Ralf-Jörg; Wolkenhauer, Olaf

    2013-01-01

    Summary In a continuous culture under phosphate limitation the metabolism of Clostridium acetobutylicum depends on the external pH level. By comparing seven steady-state conditions between pH 5.7 and pH 4.5 we show that the switch from acidogenesis to solventogenesis occurs between pH 5.3 and pH 5.0 with an intermediate state at pH 5.1. Here, an integrative study is presented investigating how a changing external pH level affects the clostridial acetone–butanol–ethanol (ABE) fermentation pathway. This is of particular interest as the biotechnological production of n-butanol as biofuel has recently returned into the focus of industrial applications. One prerequisite is the furthering of the knowledge of the factors determining the solvent production and their integrative regulations. We have mathematically analysed the influence of pH-dependent specific enzyme activities of branch points of the metabolism on the product formation. This kinetic regulation was compared with transcriptomic regulation regarding gene transcription and the proteomic profile. Furthermore, both regulatory mechanisms were combined yielding a detailed projection of their individual and joint effects on the product formation. The resulting model represents an important platform for future developments of industrial butanol production based on C. acetobutylicum. PMID:23332010

  6. Acetate production from lactose by Clostridium thermolacticum and hydrogen-scavenging microorganisms in continuous culture--effect of hydrogen partial pressure.

    PubMed

    Collet, Christophe; Gaudard, Olivier; Péringer, Paul; Schwitzguébel, Jean-Paul

    2005-08-22

    The effect of the addition of hydrogen-consuming microorganisms on the metabolism of Clostridium thermolacticum was studied. By growing this bacterium in continuous culture at 58 degrees C, on 29 mmol lactose l(-1) (10 gl(-1)) in the feed, with the H2-consuming microorganisms Methanothermobacter thermoautotrophicus and Moorella thermoautotrophica, the volumetric productivity of acetate was increased up to 3.9 mmol l(-1)h(-1) at a dilution rate of 0.058 h(-1). This was about three times higher than the maximal acetate volumetric productivity quantified when C. thermolacticum was cultivated alone. In the consortium, C. thermolacticum was the only species able to metabolize lactose; it produced not only acetate, but also hydrogen, carbon dioxide and lactate. The other species of the consortium were growing on these by-products. Meth. thermoautotrophicus played an important role as a very efficient hydrogen scavenger and decreased the hydrogen partial pressure drastically: hydrogen was converted to methane. Moor. thermoautotrophica converted lactate as well as hydrogen and carbon dioxide into acetate. As a consequence, lactose was efficiently consumed and the only organic product in the liquid phase was acetate. PMID:15992956

  7. Enhancing acetone biosynthesis and acetone-butanol-ethanol fermentation performance by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae integrated with exogenous acetate addition.

    PubMed

    Luo, Hongzhen; Ge, Laibing; Zhang, Jingshu; Ding, Jian; Chen, Rui; Shi, Zhongping

    2016-01-01

    Acetone is the major by-product in ABE fermentations, most researches focused on increasing butanol/acetone ratio by decreasing acetone biosynthesis. However, economics of ABE fermentation industry strongly relies on evaluating acetone as a valuable platform chemical. Therefore, a novel ABE fermentation strategy focusing on bio-acetone production by co-culturing Clostridium acetobutylicum/Saccharomyces cerevisiae with exogenous acetate addition was proposed. Experimental and theoretical analysis revealed the strategy could, enhance C. acetobutylicum survival oriented amino acids assimilation in the cells; control NADH regeneration rate at moderately lower level to enhance acetone synthesis but without sacrificing butanol production; enhance the utilization ability of C. acetobutylicum on glucose and direct most of extra consumed glucose into acetone/butanol synthesis routes. By implementing the strategy using synthetic or acetate fermentative supernatant, acetone concentrations increased to 8.27-8.55g/L from 5.86g/L of the control, while butanol concentrations also elevated to the higher levels of 13.91-14.23g/L from 11.63g/L simultaneously. PMID:26476171

  8. Evaluation of 3 automated real-time PCR (Xpert C. difficile assay, BD MAX Cdiff, and IMDx C. difficile for Abbott m2000 assay) for detecting Clostridium difficile toxin gene compared to toxigenic culture in stool specimens.

    PubMed

    Yoo, Jaeeun; Lee, Hyeyoung; Park, Kang Gyun; Lee, Gun Dong; Park, Yong Gyu; Park, Yeon-Joon

    2015-09-01

    We evaluated the performance of the 3 automated systems (Cepheid Xpert, BD MAX, and IMDx C. difficile for Abbott m2000) detecting Clostridium difficile toxin gene compared to toxigenic culture. Of the 254 stool specimens tested, 87 (48 slight, 35 moderate, and 4 heavy growth) were toxigenic culture positive. The overall sensitivities and specificities were 82.8% and 98.8% for Xpert, 81.6% and 95.8% for BD MAX, and 62.1% and 99.4% for IMDx, respectively. The specificity was significantly higher in IMDx than BD MAX (P= 0.03). All stool samples underwent toxin A/B enzyme immunoassay testing, and of the 254 samples, only 29 samples were positive and 2 of them were toxigenic culture negative. Considering the rapidity and high specificity of the real-time PCR assays compared to the toxigenic culture, they can be used as the first test method for C. difficile infection/colonization. PMID:26081240

  9. Detection of toxigenic Clostridium difficile: comparison of the cell culture neutralization, Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays.

    PubMed

    Pancholi, P; Kelly, C; Raczkowski, M; Balada-Llasat, J M

    2012-04-01

    Clostridium difficile is the most important cause of nosocomial diarrhea. Several laboratory techniques are available to detect C. difficile toxins or the genes that encode them in fecal samples. We evaluated the Xpert C. difficile and Xpert C. difficile/Epi (Cepheid, CA) that detect the toxin B gene (tcdB) and tcdB, cdt, and a deletion in tcdC associated with the 027/NAP1/BI strain, respectively, by real-time PCR, and the Illumigene C. difficile (Meridian Bioscience, Inc.) that detects the toxin A gene (tcdA) by loop-mediated isothermal amplification in stool specimens. Toxigenic culture was used as the reference method for discrepant stool specimens. Two hundred prospective and fifty retrospective diarrheal stool specimens were tested simultaneously by the cell cytotoxin neutralization assay (CCNA) and the Xpert C. difficile, Xpert C. difficile/Epi, and Illumigene C. difficile assays. Of the 200 prospective stools tested, 10.5% (n = 23) were determined to be positive by CCNA, 17.5% (n = 35) were determined to be positive by Illumigene C. difficile, and 21.5% (n = 43) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 50 retrospective stools, previously determined to be positive by CCNA, 94% (n = 47) were determined to be positive by Illumigene C. difficile and 100% (n = 50) were determined to be positive by Xpert C. difficile and Xpert C. difficile/Epi. Of the 11 discrepant results (i.e., negative by Illumigene C. difficile but positive by Xpert C. difficile and Xpert C. difficile/Epi), all were determined to be positive by the toxigenic culture. A total of 21% of the isolates were presumptively identified by the Xpert C. difficile/Epi as the 027/NAP1/BI strain. The Xpert C. difficile and Xpert C. difficile/Epi assays were the most sensitive, rapid, and easy-to use assays for the detection of toxigenic C. difficile in stool specimens. PMID:22278839

  10. Detection of pathogenic clostridia in biogas plant wastes.

    PubMed

    Neuhaus, Jürgen; Shehata, Awad A; Krüger, Monika

    2015-01-01

    As the number of biogas plants has grown rapidly in the last decade, the amount of potentially contaminated wastes with pathogenic Clostridium spp. has increased as well. This study reports the results from examining 203 biogas plant wastes (BGWs). The following Clostridium spp. with different frequencies could be isolated via a new enrichment medium (Krüne medium) and detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS): Clostridium perfringens (58 %) then Clostridium bifermentans (27 %), Clostridium tertium (23 %) and Clostridium butyricum (19 %), Clostridium cadaveris (15 %), Clostridium parapurificum (6 %), Clostridium glycolicum (5 %), Clostridium baratii (4 %), Clostridium sporogenes (2 %), Clostridium sordellii (1 %) and Clostridium subterminale (0.5 %). The mean most probable number (MPN) count of sulfite reducing bacteria was between 10(3) and 10(4)/mL, and the higher the MPN, the more pathogenic Clostridium spp. were present. Also, real-time PCR was used to be compared with culture method for C. perfringens, C. bifermentans, C. butyricum, C. sporogenes/Clostridium botulinum and C. sordellii. Although real-time PCR was more sensitive than the culture method, both systems improve the recovery rate but in different ways and are useful to determine pathogenic clostridia in biogas plants. In conclusion, BGWs could present a biohazard risk of clostridia for humans and animals. PMID:24984829

  11. Prolonged conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum in a two-stage continuous culture with in-situ product removal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A 2-stage process was described for continuous bioconversion of n-butyrate into n-butanol with planktonic cells of Clostridium saccharoperbutylacetonicum N1-4. Online product removal via gas stripping was integrated within the system. Our work focused on a continuous fermentation system specifically...

  12. Clostridium perfringens infection after transarterial chemoembolization for large hepatocellular carcinoma.

    PubMed

    Li, Jing-Huan; Yao, Rong-Rong; Shen, Hu-Jia; Zhang, Lan; Xie, Xiao-Ying; Chen, Rong-Xin; Wang, Yan-Hong; Ren, Zheng-Gang

    2015-04-14

    We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2(nd) day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2(nd) TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found. PMID:25892893

  13. Clostridium perfringens infection after transarterial chemoembolization for large hepatocellular carcinoma

    PubMed Central

    Li, Jing-Huan; Yao, Rong-Rong; Shen, Hu-Jia; Zhang, Lan; Xie, Xiao-Ying; Chen, Rong-Xin; Wang, Yan-Hong; Ren, Zheng-Gang

    2015-01-01

    We report an unusual case of Clostridium perfringens liver abscess formation after transcatheter arterial chemoembolization (TACE) for large hepatocellular carcinoma. Severe deterioration in liver and renal function accompanied with hemocytolysis was found on the 2nd day after TACE. Blood culture found Clostridium perfringens and abdominal computed tomography revealed a gas-containing abscess in the liver. Following antibiotics administration and support care, the infection was controlled and the liver and renal function turned normal. The 2nd TACE procedure was performed 1.5 mo later and no recurrent Clostridium perfringens infection was found. PMID:25892893

  14. Clostridium Difficile Infections

    MedlinePlus

    Clostridium difficile (C. difficile) is a bacterium that causes diarrhea and more serious intestinal conditions such as colitis. Symptoms include Watery ... Nausea Abdominal pain or tenderness You might get C. difficile disease if you have an illness that ...

  15. Collagenase Clostridium Histolyticum Injection

    MedlinePlus

    ... disease (a thickening of tissue [plaque] inside the penis that causes the penis to curve). Collagenase Clostridium histolyticum injection is in ... the plaque of thickened tissue and allows the penis to be straightened.

  16. 3-Methylindole production is regulated in Clostridium scatologenes ATCC 25775

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Aims: 3-Methylindole (3-MI) is a degradation product of L-tryptophan and is both an animal waste malodorant and threat to ruminant health. Culture conditions which influence 3-MI production in Clostridium scatologenes ATCC 25775 were investigated. Methods and Results: Cells cultured in anaerobic ...

  17. Bacteriophages of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The specific aims of the book chapter are to: (1) Briefly review the nomenclature of bacteriophages and how these agents are classified. (2) Discuss the problems associated with addition/removal of antibiotics in commercial animal feeds. (3) Provide a brief overview of Clostridium perfringens biolog...

  18. Clostridium tetani bacteraemia.

    PubMed

    Hallit, Rabih Riad; Afridi, Muhammad; Sison, Raymund; Salem, Elie; Boghossian, Jack; Slim, Jihad

    2013-01-01

    Tetanus is a neuromuscular disease in which Clostridium tetani exotoxin (tetanospasmin) produces muscle spasms, incapacitating its host. To our knowledge, C. tetani bacteraemia has never been reported in the literature. The ideal management of this entity remains unresolved given that there is no literature to guide the therapy. PMID:22977074

  19. Clostridium glycolicum Isolated from a Patient with Otogenic Brain Abscesses▿

    PubMed Central

    Van Leer, C.; Wensing, A. M. J.; van Leeuwen, J. P.; Zandbergen, E. G. J.; Swanink, C. M. A.

    2009-01-01

    We describe a case of brain abscesses with gas formation following otitis media, for which the patient treated himself by placing clay in his ear. Several microorganisms, including Clostridium glycolicum, were cultured from material obtained from the patient. This is the first report of an infection in an immunocompetent patient associated with this microorganism. PMID:19109475

  20. BUTANOL PRODUCTION FROM WHEAT STRAW HYDROLYSATE USING CLOSTRIDIUM BEIJERINCKII

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In these studies, butanol (acetone butanol ethanol or ABE) was produced from wheat straw hydrolysate (WSH) in batch cultures using Clostridium beijerinckii P260. In control fermentation, 48.9 gL**-1 glucose was used to produce 20.1 gL**-1 ABE with a productivity and yield of 0.28 gL**-1h**-1 and 0....

  1. Clostridium difficile infection

    PubMed Central

    Viswanathan, VK; Mallozzi, MJ

    2010-01-01

    Clostridium difficile infection (CDI) is the primary cause of antibiotic-associated diarrhea and is a significant nosocomial disease. In the past ten years, variant toxin-producing strains of C. difficile have emerged, that have been associated with severe disease as well as outbreaks worldwide. This review summarizes current information on C. difficile pathogenesis and disease, and highlights interventions used to combat single and recurrent episodes of CDI. PMID:21327030

  2. Electrochemical detoxification of phenolic compounds in lignocellulosic hydrolysate for Clostridium fermentation.

    PubMed

    Lee, Kyung Min; Min, Kyoungseon; Choi, Okkyoung; Kim, Ki-Yeon; Woo, Han Min; Kim, Yunje; Han, Sung Ok; Um, Youngsoon

    2015-01-01

    Lignocellulosic biomass is being preferred as a feedstock in the biorefinery, but lignocellulosic hydrolysate usually contains inhibitors against microbial fermentation. Among these inhibitors, phenolics are highly toxic to butyric acid-producing and butanol-producing Clostridium even at a low concentration. Herein, we developed an electrochemical polymerization method to detoxify phenolic compounds in lignocellulosic hydrolysate for efficient Clostridium fermentation. After the electrochemical detoxification for 10h, 78%, 77%, 82%, and 94% of p-coumaric acid, ferulic acid, vanillin, and syringaldehyde were removed, respectively. Furthermore, 71% of total phenolics in rice straw hydrolysate were removed without any sugar-loss. Whereas the cell growth and metabolite production of Clostridium tyrobutyricum and Clostridium beijerinckii were completely inhibited in un-detoxified hydrolysate, those in detoxifying rice straw hydrolysate were recovered to 70-100% of the control cultures. The electrochemical detoxification method described herein provides an efficient strategy for producing butanol and butyric acid through Clostridium fermentation with lignocellulosic hydrolysate. PMID:25863199

  3. Clostridium difficile Enterocolitis and Reactive Arthritis: A Case Report and Review of the Literature

    PubMed Central

    Cappella, Michela; Pugliese, Fabrizio; Zucchini, Andrea; Marchetti, Federico

    2016-01-01

    Reactive arthritis is a rare complication of Clostridium difficile enterocolitis, especially in children. We review the 6 pediatric cases published in the English and non-English literature and discuss their clinical presentation, outcome, treatment, and pathophysiology. We also report the seventh case of Clostridium difficile reactive arthritis in a 6-year-old boy who was treated with amoxicillin-clavulanate for 10 days because of an upper respiratory infection. After the antibiotic course, the child developed at the same time diarrhea with positive stool culture for Clostridium difficile and an asymmetric polyarthritis. Nonsteroidal anti-inflammatory drugs and metronidazole completely resolved the pain, joint swelling, and diarrhea. After twelve months of follow-up there has been no recurrence. This report confirms the self-limiting course of Clostridium difficile reactive arthritis. Clostridium difficile testing in children with gastrointestinal symptoms and acute onset of joint pain should be always considered. PMID:27190666

  4. Clostridium difficile Enterocolitis and Reactive Arthritis: A Case Report and Review of the Literature.

    PubMed

    Cappella, Michela; Pugliese, Fabrizio; Zucchini, Andrea; Marchetti, Federico

    2016-01-01

    Reactive arthritis is a rare complication of Clostridium difficile enterocolitis, especially in children. We review the 6 pediatric cases published in the English and non-English literature and discuss their clinical presentation, outcome, treatment, and pathophysiology. We also report the seventh case of Clostridium difficile reactive arthritis in a 6-year-old boy who was treated with amoxicillin-clavulanate for 10 days because of an upper respiratory infection. After the antibiotic course, the child developed at the same time diarrhea with positive stool culture for Clostridium difficile and an asymmetric polyarthritis. Nonsteroidal anti-inflammatory drugs and metronidazole completely resolved the pain, joint swelling, and diarrhea. After twelve months of follow-up there has been no recurrence. This report confirms the self-limiting course of Clostridium difficile reactive arthritis. Clostridium difficile testing in children with gastrointestinal symptoms and acute onset of joint pain should be always considered. PMID:27190666

  5. [Oncologic aspects of Clostridium difficile].

    PubMed

    Telekes, András

    2016-07-01

    Clostridium difficile infection is one of the most frequent among cancer patients. Its diagnosis is complicated by the fact that the symptoms of the infection and the side effects of the anticancer treatments could be similar. Chemotherapy itself might facilitate Clostridium difficile infection. Several risk factors are known but Clostridium difficile infection can develop in the absence of these. Neutreopenia is a risk factor for fatal Clostridium difficile infection and also the side effect of chemotherapy. Therefore, if symptoms of the potential infection develop (eg. diarrhoea more than three times a day, fever above 38.5 °C, colitis, rapid increase of serum creatinin) Clostridium difficile infection should be excluded. If the infection is confirmed it should be managed in the most efficient way. Orv. Hetil., 2016, 157(28), 1110-1116. PMID:27397423

  6. Long-term conversion of n-butyrate to n-butanol with Clostridium saccharoperbutylacetonicum using a two-stage continuous culture and in-situ product removal

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We are operating anaerobic bioreactors with undefined mixed cultures to convert waste lignocellulosic biomass into useful products. A traditional product of anaerobic bioreactors (i.e., anaerobic digesters) is methane. Recently, we modified bioreactor conditions to more efficiently produce a range o...

  7. Blastocystis sp. Infection Mimicking Clostridium Difficile Colitis

    PubMed Central

    Gil, Gaby S.; Chaudhari, Shobhana; Shady, Ahmed; Caballes, Ana; Hong, Joe

    2016-01-01

    We report an unusual case of severe diarrhea related to Blastocystis sp. infection in a patient with end stage renal disease on hemodialysis. The patient was admitted due to profuse diarrhea associated with fever and leukocytosis. Pertinent stool work-up such as leukocytes in stool, stool culture, clostridium difficile toxin B PCR, and serology for hepatitis A, hepatitis B, and hepatitis C and cytomegalovirus screening were all negative. Ova and parasite stool examination revealed Blastocystis sp. The patient was given intravenous metronidazole with clinical improvement by day three and total resolution of symptoms by day ten. PMID:27247810

  8. Cellulolytic Activity of Clostridium acetobutylicum

    PubMed Central

    Lee, Song F.; Forsberg, Cecil W.; Gibbins, L. N.

    1985-01-01

    Clostridium acetobutylicum NRRL B527 and ATCC 824 exhibited extracellular and cell-bound endoglucanase and cellobiase activities during growth in a chemically defined medium with cellobiose as the sole source of carbohydrate. For both strains, the endoglucanase was found to be mainly extracellular (70 to 90%) during growth in continuous or batch cultures with the pH maintained at 5.2, whereas the cellobiase was mainly cell associated (60 to 90%). During continuous cultivation of strain B527 with cellobiose as the limiting nutrient, maximum production of the endoglucanase and cellobiase occurred at pH values of 5.2 and 4.8, respectively. In the carbon-limited continuous cultures, strain 824 produced similar levels of endoglucanase, cellobiosidase, and cellobiase activities regardless of the carbon source used. However, in ammonium- or phosphate-limited cultures, with an excess of glucose, only 1/10 of the endoglucanase was produced, and neither cellobiosidase nor cellobiase activities were detectable. A crude extracellular enzyme preparation from strain B527 hydrolyzed carboxymethylcellulose and phosphoric acid-swollen cellulose readily and microcrystalline cellulose (A vicel) to a lesser extent. Glucose accounted for more than 90% of the reducing sugar produced by the hydrolysis of acid-swollen cellulose and Avicel. Strain B527 did not grow in medium with acid-swollen cellulose as the sole source of carbohydrate, although it grew readily on the products obtained by hydrolyzing the cellulose in vitro with a preparation of extracellular cellulase derived from the same organism. PMID:16346847

  9. Emended descriptions of Clostridium acetobutylicum and Clostridium beijerinckii, and descriptions of Clostridium saccharoperbutylacetonicum sp. nov. and Clostridium saccharobutylicum sp. nov.

    PubMed

    Keis, S; Shaheen, R; Jones, D T

    2001-11-01

    On the basis of 16S rRNA gene sequencing and DNA-DNA reassociation, industrial solvent-producing clostridia have been assigned to four species. In this study, the phenotypic characteristics of Clostridium acetobutylicum, Clostridium beijerinckii, 'Clostridium saccharoperbutylacetonicum', and an unnamed Clostridium sp. represented by the strains NCP 262T and NRRL B643 are compared. In addition, a further 40 strains of solvent-producing clostridia have been classified by biotyping, DNA fingerprinting and 16S rRNA gene sequencing. These included 14 C. beijerinckii strains, two strains currently designated as 'Clostridium kaneboi' and 'Clostridium butanologenum', and 24 production strains used in the commercial acetone-butanol fermentation. All of the C. beijerinckii strains were confirmed to have been classified correctly. The 'C. kaneboi' and 'C. butanologenum' strains require reclassification as C. acetobutylicum and C. beijerinckii, respectively. The commercial production strains were found to belong either to C. beijerinckii or to the unnamed Clostridium sp. For the comparative phenotypic studies of the four species, representative strains were selected from each of the DNA-fingerprint subgroups within each species. These strains were analysed for their ability to utilize different carbohydrates, hydrolyse gelatin or aesculin, and produce indole, and were tested for the presence of catalase and urease. On the basis of these results, several phenotypic traits were found to be useful for differentiating between the four species. The descriptions of C. acetobutylicum and C. beijerinckii have been emended. The names Clostridium saccharoperbutylacetonicum sp. nov. [type strain = N1-4 (HMT) = ATCC 27021T] and Clostridium saccharobutylicum sp. nov. (type strain = DSM 13864T = ATCC BAA-117T) are proposed for the two new species. PMID:11760952

  10. Carbon Monoxide Oxidation by Clostridium thermoaceticum and Clostridium formicoaceticum

    PubMed Central

    Diekert, Gabriele B.; Thauer, Rudolf K.

    1978-01-01

    Cultures of Clostridium formicoaceticum and C. thermoaceticum growing on fructose and glucose, respectively, were shown to rapidly oxidize CO to CO2. Rates up to 0.4 μmol min−1 mg of wet cells−1 were observed. Carbon monoxide oxidation by cell suspensions was found (i) to be dependent on pyruvate, (ii) to be inhibited by alkyl halides and arsenate, and (iii) to stimulate CO2 reduction to acetate. Cell extracts catalyzed the oxidation of carbon monoxide with methyl viologen at specific rates up to 10 μmol min−1 mg of protein−1 (35°C, pH 7.2). Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate and ferredoxin from C. pasteurianum were ineffective as electron acceptors. The catalytic mechanism of carbon monoxide oxidation was “ping-pong,” indicating that the enzyme catalyzing carbon monoxide oxidation can be present in an oxidized and a reduced form. The oxidized form was shown to react reversibly with cyanide, and the reduced form was shown to react reversibly with alkyl halides: cyanide inactivated the enzyme only in the absence of carbon monoxide, and alkyl halides inactivated it only in the presence of carbon monoxide. Extracts inactivated by alkyl halides were reactivated by photolysis. The findings are interpreted to indicate that carbon monoxide oxidation in the two bacteria is catalyzed by a corrinoid enzyme and that in vivo the reaction is coupled with the reduction of CO2 to acetate. Cultures of C. acidi-urici and C. cylindrosporum growing on hypoxanthine were found not to oxidize CO, indicating that clostridia mediating a corrinoid-independent total synthesis of acetate from CO2 do not possess a CO-oxidizing system. PMID:711675

  11. Effects of culture conditions on the kinetic behavior of 1,3-propanediol fermentation by Clostridium butyricum with a kinetic model.

    PubMed

    Zhu, Chunjie; Fang, Baishan; Wang, Shizhen

    2016-07-01

    The effects of culture conditions on the kinetic behavior of 1,3-propanediol (PD) fermentation were investigated with a kinetic model. First, with initial glycerol concentration (S0) increasing, μmax and PD inhibition increased. Glycerol assimilation was harder and a little glycerol was consumed on cell maintenance at high S0. Second, with yeast extract concentration increasing, PD inhibition decreased. However, μmax decreased and glycerol assimilation became harder. It seems that the stimulus effect of yeast extract resulted from decreased PD inhibition. Glycerol amount consumed on cell maintenance also decreased. Third, with temperature decreasing, μmax and PD inhibition decreased. Glycerol assimilation was harder and a little more glycerol was consumed on cell maintenance at low temperature. Fourth, with pH increasing, μmax and PD inhibition decreased. Glycerol assimilation was harder and much more glycerol was consumed on cell maintenance at pH 6.5 and 7.5 than 7.0. This work facilitates further fermentation process optimization. PMID:27089428

  12. Vaccines against Clostridium difficile

    PubMed Central

    Leuzzi, Rosanna; Adamo, Roberto; Scarselli, Maria

    2014-01-01

    Clostridium difficile infection (CDI) is recognized as a major cause of nosocomial diseases ranging from antibiotic related diarrhea to fulminant colitis. Emergence during the last 2 decades of C. difficile strains associated with high incidence, severity and lethal outcomes has increased the challenges for CDI treatment. A limited number of drugs have proven to be effective against CDI and concerns about antibiotic resistance as well as recurring disease solicited the search for novel therapeutic strategies. Active vaccination provides the attractive opportunity to prevent CDI, and intense research in recent years led to development of experimental vaccines, 3 of which are currently under clinical evaluation. This review summarizes recent achievements and remaining challenges in the field of C. difficile vaccines, and discusses future perspectives in view of newly-identified candidate antigens. PMID:24637887

  13. Clostridium difficile recurrences in Stockholm.

    PubMed

    Sandell, Staffan; Rashid, Mamun-Ur; Jorup-Rönström, Christina; Ellström, Kristina; Nord, Carl Erik; Weintraub, Andrej

    2016-04-01

    Sixty-eight hospital-admitted patients with a first episode of Clostridium difficile infection (CDI) were included and followed up during 1 year. Faeces samples were collected at 1, 2, 6 and 12 months after inclusion and analyzed for the presence of C. difficile toxin B, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped and the MICs of the isolates were determined against eight antimicrobial agents. In 68 patients initially included, antibiotics, clinical signs and co-morbidities were analyzed and 56 were evaluable for recurrences. The mean number of different antibiotics given during 3 months prior to inclusion was 2.6 (range 0-6). Six patients had not received any antibiotics and three of them had diagnosed inflammatory bowel disease. Thirty-two patients (57%) had either a microbiological or clinical recurrence, 16 of whom had clinical recurrences that were confirmed microbiologically (13, 23%) or unconfirmed by culture (3, 5%). Twenty-nine patients were positive in at least one of the follow-up tests, 16 had the same ribotype in follow-up tests, i.e. relapse, and 13 a different ribotype, i.e., reinfection. Most common ribotypes were 078/126, 020, 023, 026, 014/077, 001 and 005. No strain of ribotype 027 was found. Strains ribotype 078/126 and 023 were positive for binary toxin and were the strains most prone to cause recurrence. All strains were sensitive to vancomycin and metronidazole. Patients with recurrences were significantly older (p = 0.02) and all patients had a high burden of comorbidities, which could explain the high fatality rate, 26 (38%) patients died during the 1-year follow-up. PMID:26802875

  14. Non-Clostridium perfringens infectious agents producing necrotic enteritis-like lesions in poultry.

    PubMed

    Uzal, F A; Sentíes-Cué, C G; Rimoldi, G; Shivaprasad, H L

    2016-06-01

    Necrotic enteritis (NE) produced by Clostridium perfringens is amongst the most prevalent enteric diseases of chickens and turkeys. However, several other bacterial, parasitic and viral agents can cause clinical signs, gross and microscopic lesions in poultry very similar to those of NE and the diseases produced by those agents need to be differentiated from NE. The main differential diagnoses for C. perfringens NE include bacterial (Clostridium colinum, Clostridium sordellii, Clostridium difficile, Pasteurella multocida, Brachyspira spp.), parasitic (Eimeria spp., Histomonas meleagridis) and viral (Duck Herpesvirus type 1, Avian Paramyxovirus type 1) diseases. Confirmation of the diagnosis of these diseases requires identification of the aetiological agents by morphological, cultural and/or molecular methods. PMID:27009483

  15. Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

    PubMed

    el Idrissi, A H; Ward, G E

    1992-06-15

    Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias. PMID:1496812

  16. Clostridium difficile Cell Attachment Is Modified by Environmental Factors

    PubMed Central

    Waligora, Anne-Judith; Barc, Marie-Claude; Bourlioux, Pierre; Collignon, Anne; Karjalainen, Tuomo

    1999-01-01

    Adherence of Clostridium difficile to Vero cells under anaerobic conditions was increased by a high sodium concentration, calcium-rich medium, an acidic pH, and iron starvation. The level of adhesion of nontoxigenic strains was comparable to that of toxigenic strains. Depending on the bacterial culture conditions, Vero cells could bind to one, two, or three bacterial surface proteins with molecular masses of 70, 50, and 40 kDa. PMID:10473442

  17. Clostridium difficile infection.

    PubMed

    Smits, Wiep Klaas; Lyras, Dena; Lacy, D Borden; Wilcox, Mark H; Kuijper, Ed J

    2016-01-01

    Infection of the colon with the Gram-positive bacterium Clostridium difficile is potentially life threatening, especially in elderly people and in patients who have dysbiosis of the gut microbiota following antimicrobial drug exposure. C. difficile is the leading cause of health-care-associated infective diarrhoea. The life cycle of C. difficile is influenced by antimicrobial agents, the host immune system, and the host microbiota and its associated metabolites. The primary mediators of inflammation in C. difficile infection (CDI) are large clostridial toxins, toxin A (TcdA) and toxin B (TcdB), and, in some bacterial strains, the binary toxin CDT. The toxins trigger a complex cascade of host cellular responses to cause diarrhoea, inflammation and tissue necrosis - the major symptoms of CDI. The factors responsible for the epidemic of some C. difficile strains are poorly understood. Recurrent infections are common and can be debilitating. Toxin detection for diagnosis is important for accurate epidemiological study, and for optimal management and prevention strategies. Infections are commonly treated with specific antimicrobial agents, but faecal microbiota transplants have shown promise for recurrent infections. Future biotherapies for C. difficile infections are likely to involve defined combinations of key gut microbiota. PMID:27158839

  18. Genomics of Clostridium tetani.

    PubMed

    Brüggemann, Holger; Brzuszkiewicz, Elzbieta; Chapeton-Montes, Diana; Plourde, Lucile; Speck, Denis; Popoff, Michel R

    2015-05-01

    Genomic information about Clostridium tetani, the causative agent of the tetanus disease, is scarce. The genome of strain E88, a strain used in vaccine production, was sequenced about 10 years ago. One additional genome (strain 12124569) has recently been released. Here we report three new genomes of C. tetani and describe major differences among all five C. tetani genomes. They all harbor tetanus-toxin-encoding plasmids that contain highly conserved genes for TeNT (tetanus toxin), TetR (transcriptional regulator of TeNT) and ColT (collagenase), but substantially differ in other plasmid regions. The chromosomes share a large core genome that contains about 85% of all genes of a given chromosome. The non-core chromosome comprises mainly prophage-like genomic regions and genes encoding environmental interaction and defense functions (e.g. surface proteins, restriction-modification systems, toxin-antitoxin systems, CRISPR/Cas systems) and other fitness functions (e.g. transport systems, metabolic activities). This new genome information will help to assess the level of genome plasticity of the species C. tetani and provide the basis for detailed comparative studies. PMID:25638019

  19. [Toxins of Clostridium perfringens].

    PubMed

    Morris, W E; Fernández-Miyakawa, M E

    2009-01-01

    Clostridium perfringens is an anaerobic gram-positive spore-forming bacillus. It is one of the pathogens with larger distribution in the environment; it can be isolated from soil and water samples, which also belongs to the intestinal flora of animals and humans. However, on some occasions it can act as an opportunistic pathogen, causing diseases such as gas gangrene, enterotoxemia in sheep and goats and lamb dysentery, among others. In human beings, it is associated to diseases such as food poisoning, necrotic enterocolitis of the infant and necrotic enteritis or pigbel in Papua-New Guinea tribes. The renewed interest existing nowadays in the study of C. perfringens as a veterinarian and human pathogen, together with the advance of molecular biology, had enabled science to have deeper knowledge of the biology and pathology of these bacteria. In this review, we discuss and update the principal aspects of C. perfringens intestinal pathology, in terms of the toxins with major medical relevance at present. PMID:20085190

  20. Isolation of Clostridium thermocellum auxotrophs

    SciTech Connect

    Mendez, B.S.; Gomez, R.F.

    1982-02-01

    The conversion of biomass of fuels and chemical feedstocks by microbial fermentation offers the potential of solving two of today's important problems: waste accumulation and exhaustion of fossil fuels. Microorganisms with the capabilities of converting biomass components such as cellulos and hemicellulose to chemicals and fuels in a single step are of particular interest. One such microorganism is Clostridium thermocellum, a thermophilic anaerobe which degrades cellulose to ethanol and organic acids. For efficient industrial use, the cellulolytic capacity of this strain must be improved by genetic means. Spontaneous and UV irradiation-induced auxotrophic mutants of Clostridium thermocellum, an anaerobic cellulolytic thermophile, were isolated after penicillin enrichment in a chemically defined medium.

  1. Clostridium difficile: clinical disease and diagnosis.

    PubMed Central

    Knoop, F C; Owens, M; Crocker, I C

    1993-01-01

    Clostridium difficile is an opportunistic pathogen that causes a spectrum of disease ranging from antibiotic-associated diarrhea to pseudomembranous colitis. Although the disease was first described in 1893, the etiologic agent was not isolated and identified until 1978. Since clinical and pathological features of C. difficile-associated disease are not easily distinguished from those of other gastrointestinal diseases, including ulcerative colitis, chronic inflammatory bowel disease, and Crohn's disease, diagnostic methods have relied on either isolation and identification of the microorganism or direct detection of bacterial antigens or toxins in stool specimens. The current review focuses on the sensitivity, specificity, and practical use of several diagnostic tests, including methods for culture of the etiologic agent, cellular cytotoxicity assays, latex agglutination tests, enzyme immunoassay systems, counterimmunoelectrophoresis, fluorescent-antibody assays, and polymerase chain reactions. PMID:8358706

  2. Anaerobic thermophilic culture system

    DOEpatents

    Ljungdahl, Lars G.; Wiegel, Jurgen K. W.

    1981-01-01

    A mixed culture system of the newly discovered microorganism Thermoanaerobacter ethanolicus ATCC31550 and the microorganism Clostridium thermocellum ATCC31549 is described. In a mixed nutrient culture medium that contains cellulose, these microorganisms have been coupled and cultivated to efficiently ferment cellulose to produce recoverable quantities of ethanol under anaerobic, thermophilic conditions.

  3. Autism and Clostridium tetani.

    PubMed

    Bolte, E R

    1998-08-01

    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. PMID:9881820

  4. Selective medium for isolation of Clostridium butyricum from human feces.

    PubMed Central

    Popoff, M R

    1984-01-01

    A selective medium, Clostridium butyricum isolation medium (BIM), is described for the isolation of C. butyricum from human feces. The BIM is a synthetic minimal medium and contains trimethoprim (16 micrograms/ml), D-cycloserine (10 micrograms/ml), and polymyxin B sulfate (20 micrograms/ml) as selective inhibitory agents. Qualitative tests indicated that C. butyricum and other butyric acid-producing clostridia grew on BIM, Clostridium sphenoides and Bacillus cereus produced small colonies, and other clostridia and other obligate anaerobic or facultatively anerobic bacteria were inhibited. Quantitative recovery of C. butyricum from cultures or seeded fecal samples was comparable with BIM and with complex medium, but the quantitative recovery of the other butyric acid-producing clostridia tested (C. beijerinckii, C. acetobutylicum) was lower with BIM than with complex medium. The BIM should aid the rapid isolation of C. butyricum from fecal samples and should be useful for bacteriological investigation of neonatal necrotizing enterocolitis. PMID:6490827

  5. Purification and characterization of konjac glucomannan degrading enzyme from anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group.

    PubMed

    Nakajima, N; Matsuura, Y

    1997-10-01

    Konjac glucomannan degrading enzyme was purified to homogeneity from the culture broth of an anaerobic human intestinal bacterium, Clostridium butyricum-Clostridium beijerinckii group. The enzyme was composed of a single polypeptide chain with a molecular weight of 50,000-53,000. The enzyme was an endo-beta-mannanase that acted specifically on the polysaccharides such as konjac glucomannan and coffee mannan, producing exclusively their smaller oligosaccharides and the monosaccharides. The optimal pH of the enzyme for the hydrolysis of konjac glucomannan was around 7-8 and the enzyme was stable in rather alkaline pH range of 8-10. The enzyme reaction was activated by the addition of CaCl2 and dithiothreitol. It was suggested that the enzyme might contribute to the decomposition of konjac glucomannan in human digestive tract. PMID:9362121

  6. Electrotransformation of Clostridium thermocellum.

    PubMed

    Tyurin, Michael V; Desai, Sunil G; Lynd, Lee R

    2004-02-01

    Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presumptive transformants of C. thermocellum with subsequent PCR specific to the mls gene on the plasmid, as well as by retransformation of Escherichia coli. Optimization carried out with strain DSM 1313 increased transformation efficiencies from <1 to (2.2 +/- 0.5) x 10(5) transformants per micro g of plasmid DNA. Factors conducive to achieving high transformation efficiencies included optimized periods of incubation both before and after electric pulse application, chilling during cell collection and washing, subculture in the presence of isoniacin prior to electric pulse application, a custom-built cuvette embedded in an ice block during pulse application, use of a high (25-kV/cm) field strength, and induction of the mls gene before plating the cells on selective medium. The protocol and preferred conditions developed for strain DSM 1313 resulted in transformation efficiencies of (5.0 +/- 1.8) x 10(4) transformants per micro g of plasmid DNA for strain ATCC 27405 and approximately 1 x 10(3) transformants per micro g of plasmid DNA for strains DSM 4150 and 7072. Cell viability under optimal conditions was approximately 50% of that of controls not exposed to an electrical pulse. Dam methylation had a beneficial but modest (7-fold for strain ATCC 27405; 40-fold for strain DSM 1313) effect on transformation efficiency. The effect of isoniacin was also strain specific. The results reported here provide for the first time a gene transfer method functional in C. thermocellum that is suitable for molecular manipulations involving either the introduction of genes associated with foreign

  7. Clostridium tertium Bacteremia in a Patient with Glyphosate Ingestion

    PubMed Central

    You, Myung-Jo; Shin, Gee-Wook; Lee, Chang-Seop

    2015-01-01

    Patient: Female, 44 Final Diagnosis: Clostridium tertium bacteremia Symptoms: Fever Medication: Ertapenem • Metronidazole Clinical Procedure: — Specialty: Infectious Disease Objective: Unknown etiology Background: Clostridium tertium is distributed in the soil and in animal and human gastrointestinal tracts. C. tertium has been isolated from patients with blood diseases, immune disorders, and abdominal surgeries. Glyphosate is toxic, causing cause eye and skin irritation, gastrointestinal pain, and vomiting. Ingestion of herbicides modifies the gastrointestinal environment, which stresses the living organisms. However, there has been little attention to cases of bacteremia in patients recovering from suicide attempt by ingesting herbicide. Case Report: Clostridium tertium was identified in a 44-year-old female who attempted suicide by glyphosate (a herbicide) ingestion. The 16S rRNA sequences from all colonies were 99% identical with that of C. tertium (AB618789) found on a BLAST search of the NCBI database. The bacterium was cultured on TSA under aerobic and anaerobic conditions. Antimicrobial susceptibility tests performed under both aerobic and anaerobic conditions showed that the bacterium was susceptible to penicillin, a combination of β-lactamase inhibitor and piperacillin or amoxicillin, and first- and second- generation cephalosporins. However, it was resistant to third- and fourth-generation cephalosporins. Conclusions: Glyphosate herbicide might be a predisposing factor responsible for the pathogenesis of C. tertium. The results highlight the need for careful diagnosis and selection of antibiotics in the treatment of this organism. PMID:25577783

  8. Fatal clostridial enterotoxemia (Clostridium glycolicum) in an ornate Nile monitor (Varanus ornatus).

    PubMed

    Bertelsen, Mads F; Weese, J Scott

    2006-03-01

    Enterotoxemia caused by Clostridium glycolicum was identified as the cause of circulatory collapse and death in a female, 3-yr-old, captive-bred ornate Nile monitor (Varanus ornatus). Anaerobic culture of fecal samples from 12 other monitor lizards from the collection failed to demonstrate the presence of this pathogen. PMID:17312813

  9. Clostridium perfringens in retail chicken.

    PubMed

    Nowell, Victoria J; Poppe, Cornelis; Parreira, Valeria R; Jiang, Yan-Fen; Reid-Smith, Richard; Prescott, John F

    2010-06-01

    Clostridium perfringens isolates were recovered by enrichment from retail grocery chicken samples (n = 88) in Ontario, Canada, with one sample per site. The gene associated with necrotic enteritis in chickens, netB, was found in 21% of the isolates. The tpeL gene was found in 2% and the cpb2 gene in 68% (95% "atypical" genes) of isolates. This study suggests that netB-positive C. perfringens can reach people through retail chicken. PMID:19961943

  10. Laboratory Diagnosis of Clostridium difficile Infection

    PubMed Central

    Tenover, Fred C.; Baron, Ellen Jo; Peterson, Lance R.; Persing, David H.

    2011-01-01

    The laboratory diagnosis of Clostridium difficile infection (CDI) continues to be challenging. Recent guidelines from professional societies in the United States note that enzyme immunoassays for toxins A and B do not have adequate sensitivity to be used alone for detecting CDI, yet the optimal method for diagnosing this infection remains unclear. Nucleic acid amplification tests (NAATs) that target chromosomal toxin genes (usually the toxin B gene, tcdB) show high sensitivity and specificity, provide rapid results, and are amenable to both batch and on-demand testing, but these tests were not universally recommended for routine use in the recent guidelines. Rather, two-step algorithms that use glutamate dehydrogenase (GDH) assays to screen for C. difficile in stool specimens, followed by either direct cytotoxin testing or culture to identify toxin-producing C. difficile isolates, were recommended in one guideline and either GDH algorithms or NAATs were recommended in another guideline. Unfortunately, neither culture nor direct cytotoxin testing is widely available. In addition, this two-step approach requires 48 to 92 hours to complete, which may delay the initiation of therapy and critical infection control measures. Recent studies also show the sensitivity of several GDH assays to be <90%. This review considers the role of NAATs for diagnosing CDI and explores their potential advantages over two-step algorithms, including shorter time to results, while providing comparable, if not superior, accuracy. PMID:21854871

  11. Regulation of protease production in Clostridium sporogenes.

    PubMed Central

    Allison, C; Macfarlane, G T

    1990-01-01

    The physiological and nutritional factors that regulate protease synthesis in Clostridium sporogenes C25 were studied in batch and continuous cultures. Formation of extracellular proteases occurred at the end of active growth and during the stationary phase in batch cultures. Protease production was inversely related to growth rate in glucose-excess and glucose-limited chemostats over the range D = 0.05 to 0.70 h-1. In pulse experiments, glucose, ammonia, phosphate, and some amino acids (tryptophan, proline, tyrosine, and isoleucine) strongly repressed protease synthesis. This repression was not relieved by addition of 4 mM cyclic AMP, cyclic GMP, or dibutyryl cyclic AMP. Protease formation was markedly inhibited by 4 mM ATP and ADP, but GTP and GDP had little effect on the process. It is concluded that protease production by C. sporogenes is strongly influenced by the amount of energy available to the cells, with the highest levels of protease synthesis occurring under energy-limiting conditions. PMID:2268158

  12. Parameters Affecting Solvent Production by Clostridium pasteurianum

    PubMed Central

    Dabrock, Birgit; Bahl, Hubert; Gottschalk, Gerhard

    1992-01-01

    The effect of pH, growth rate, phosphate and iron limitation, carbon monoxide, and carbon source on product formation by Clostridium pasteurianum was determined. Under phosphate limitation, glucose was fermented almost exclusively to acetate and butyrate independently of the pH and growth rate. Iron limitation caused lactate production (38 mol/100 mol) from glucose in batch and continuous culture. At 15% (vol/vol) carbon monoxide in the atmosphere, glucose was fermented to ethanol (24 mol/100 mol), lactate (32 mol/100 mol), and butanol (36 mol/100 mol) in addition to the usual products, acetate (38 mol/100 mol) and butyrate (17 mol/100 mol). During glycerol fermentation, a completely different product pattern was found. In continuous culture under phosphate limitation, acetate and butyrate were produced only in trace amounts, whereas ethanol (30 mol/100 mol), butanol (18 mol/100 mol), and 1,3-propanediol (18 mol/100 mol) were the major products. Under iron limitation, the ratio of these products could be changed in favor of 1,3-propanediol (34 mol/100 mol). In addition, lactate was produced in significant amounts (25 mol/100 mol). The tolerance of C. pasteurianum to glycerol was remarkably high; growth was not inhibited by glycerol concentrations up to 17% (wt/vol). Increasing glycerol concentrations favored the production of 1,3-propanediol. PMID:16348691

  13. Phylogenetic analysis and PCR detection of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum based on the flagellin gene.

    PubMed

    Sasaki, Yoshimasa; Kojima, Akemi; Aoki, Hiroshi; Ogikubo, Yasuaki; Takikawa, Noriyasu; Tamura, Yutaka

    2002-05-01

    The flagellin genes (fliC) of Clostridium chauvoei, Clostridium haemolyticum, Clostridium novyi types A and B, and Clostridium septicum were analysed by PCR amplification and DNA sequencing. The five Clostridium species have at least two copies of the flagellin gene (fliC) arranged in tandem on the chromosome. The deduced N- and C-terminal aminoacid sequences of the flagellin proteins (FliCs) of these clostridia are well conserved but their central region aminoacid sequences are not. Phylogenic analysis based on the N-terminal aminoacid sequence of the FliC protein revealed that these clostridia, which belong to Clostridium 16S rDNA phylogenic cluster I (), are more closely related to Bacillus subtilis than to Clostridium difficile, which belongs to the cluster XI. Moreover, a multiplex polymerase reaction (PCR) system based on the fliC sequence was developed to rapidly identify C. chauvoei, C. haemolyticum, C. novyi types A and B, and C. septicum. PCR of each Clostridium amplified a species-specific band. The multiplex PCR system may be useful for rapid identification of pathogenic clostridia. PMID:11900959

  14. Culture.

    ERIC Educational Resources Information Center

    1997

    Twelve conference papers on cultural aspects of second language instruction include: "Towards True Multiculturalism: Ideas for Teachers" (Brian McVeigh); Comparing Cultures Through Critical Thinking: Development and Interpretations of Meaningful Observations" (Laurel D. Kamada); "Authority and Individualism in Japan and the USA" (Alisa Woodring);…

  15. Citrate, a specific substrate for the isolation of Clostridium sphenoides.

    PubMed Central

    Walther, R; Hippe, H; Gottschalk, G

    1977-01-01

    With a medium containing citrate as the carbon and energy source, 10 clostridial strains were isolated from various mud samples. Characterization of these strains revealed that they all belonged to the same species, Clostridium sphenoides. Strains of this organism obtained from culture collections were also able to grow citrate, whereas 15 other clostridial species tested were not. Citrate was fermented by C. sphenoides to acetate, ethanol, carbon dioxide, and hydrogen. Experiments with stereospecifically 14C-labeled citrate indicated that citrate lyase was involved in citrate degradation. Images PMID:869540

  16. Rabbit Ileal Loop Response to Strains of Clostridium perfringens1

    PubMed Central

    Duncan, Charles L.; Sugiyama, H.; Strong, Dorothy H.

    1968-01-01

    The ligated loop of the rabbit intestine was investigated as a possible experimental model for the study of Clostridium perfringens food poisoning. The method of preparation of the challenge inoculum was important in determining whether a given strain would provoke a response. When cultures were grown for 4 hr at 37 C in Skim Milk (Difco), 14 of 29 type A strains isolated from food-poisoning outbreaks consistently produced exudation of fluid and consequent dilation of the ileal segments. In contrast, 15 of the 18 strains derived from other sources failed to elicit a response. By use of different inoculum preparations, nearly all strains could be made to give at least an occasional positive loop reaction. Diarrhea was not obtained in rabbits by intraluminal injection into the normal ileum or by per os administration of the cultures. Lecithinase, purified and in concentrated culture supernatant fractions, failed to produce a response in the isolated ileal loops. Images PMID:4297020

  17. Clostridium difficile and the microbiota

    PubMed Central

    Seekatz, Anna M.; Young, Vincent B.

    2014-01-01

    Clostridium difficile infection (CDI) is the leading health care–associated illness. Both human and animal models have demonstrated the importance of the gut microbiota’s capability of providing colonization resistance against C. difficile. Risk factors for disease development include antibiotic use, which disrupts the gut microbiota, leading to the loss of colonization resistance and subsequent CDI. Identification of the specific microbes capable of restoring this function remains elusive. Future studies directed at how microbial communities influence the metabolic environment may help elucidate the role of the microbiota in disease development. These findings will improve current biotherapeutics for patients with CDI, particularly those with recurrent disease. PMID:25036699

  18. Regulation of toxin synthesis in Clostridium botulinum and Clostridium tetani.

    PubMed

    Connan, Chloé; Denève, Cécile; Mazuet, Christelle; Popoff, Michel R

    2013-12-01

    Botulinum and tetanus neurotoxins are structurally and functionally related proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin (BoNT) associates with non-toxic proteins (ANTPs) to form complexes of various sizes, whereas tetanus toxin (TeNT) does not form any complex. The BoNT and ANTP genes are clustered in a DNA segment called the botulinum locus, which has different genomic localization (chromosome, plasmid, phage) in the various Clostridium botulinum types and subtypes. The botulinum locus genes are organized in two polycistronic operons (ntnh-bont and ha/orfX operons) transcribed in opposite orientations. A gene called botR lying between the two operons in C. botulinum type A encodes an alternative sigma factor which regulates positively the synthesis of BoNT and ANTPs at the late exponential growth phase and beginning of the stationary phase. In Clostridium tetani, the gene located immediately upstream of tent encodes a positive regulatory protein, TetR, which is related to BotR. C. botulinum and C. tetani genomes contain several two-component systems and predicted regulatory orphan genes. In C. botulinum type A, four two-component systems have been found that positively or negatively regulate the synthesis of BoNT and ANTPs independently of BotR/A. The synthesis of neurotoxin in Clostridia seems to be under the control of complex network of regulation. PMID:23769754

  19. Clostridium botulinum in cattle and dairy products.

    PubMed

    Lindström, Miia; Myllykoski, Jan; Sivelä, Seppo; Korkeala, Hannu

    2010-04-01

    The use of plastic-wrapped and nonacidified silage as cattle feed has led to an increasing number of botulism outbreaks due to Clostridium botulinum Groups I-III in dairy cattle. The involvement of Groups I and II organisms in cattle botulism has raised concern of human botulism risk associated with the consumption of dairy products. Multiplication of C. botulinum in silage and in the gastrointestinal tract of cattle with botulism has been reported, thus contamination of the farm environment and raw milk, and further transmission through the dairy chain, are possible. The standard milk pasteurization treatment does not eliminate spores, and the intrinsic factors of many dairy products allow botulinal growth and toxin production. Although rare, several large botulism outbreaks due to both commercial and home-prepared dairy products have been reported. Factors explaining these outbreaks include most importantly temperature abuse, but also unsafe formulation, inadequate fermentation, insufficient thermal processing, post-process contamination, and lack of adequate quality control for adjunct ingredients were involved. The small number of outbreaks is probably explained by a low incidence of spores in milk, the presence of competitive bacteria in pasteurized milk and other dairy products, and growth-inhibitory combinations of intrinsic and extrinsic factors in cultured and processed dairy products. PMID:20301016

  20. Clostridium difficile PCR Ribotypes in Calves, Canada

    PubMed Central

    Stämpfli, Henry R.; Duffield, Todd; Peregrine, Andrew S.; Trotz-Williams, Lise A.; Arroyo, Luis G.; Brazier, Jon S.; Weese, J. Scott

    2006-01-01

    We investigated Clostridium difficile in calves and the similarity between bovine and human C. difficile PCR ribotypes by conducting a case-control study of calves from 102 dairy farms in Canada. Fecal samples from 144 calves with diarrhea and 134 control calves were cultured for C. difficile and tested with an ELISA for C. difficile toxins A and B. C. difficile was isolated from 31 of 278 calves: 11 (7.6%) of 144 with diarrhea and 20 (14.9%) of 134 controls (p = 0.009). Toxins were detected in calf feces from 58 (56.8%) of 102 farms, 57 (39.6%) of 144 calves with diarrhea, and 28 (20.9%) of 134 controls (p = 0.0002). PCR ribotyping of 31 isolates showed 8 distinct patterns; 7 have been identified in humans, 2 of which have been associated with outbreaks of severe disease (PCR types 017 and 027). C. difficile may be associated with calf diarrhea, and cattle may be reservoirs of C. difficile for humans. PMID:17283624

  1. Clostridium difficile Is an Autotrophic Bacterial Pathogen

    PubMed Central

    Köpke, Michael; Straub, Melanie; Dürre, Peter

    2013-01-01

    During the last decade, Clostridium difficile infection showed a dramatic increase in incidence and virulence in the Northern hemisphere. This incessantly challenging disease is the leading cause of antibiotic-associated and nosocomial infectious diarrhea and became life-threatening especially among elderly people. It is generally assumed that all human bacterial pathogens are heterotrophic organisms, being either saccharolytic or proteolytic. So far, this has not been questioned as colonization of the human gut gives access to an environment, rich in organic nutrients. Here, we present data that C. difficile (both clinical and rumen isolates) is also able to grow on CO2+H2 as sole carbon and energy source, thus representing the first identified autotrophic bacterial pathogen. Comparison of several different strains revealed high conservation of genes for autotrophic growth and showed that the ability to use gas mixtures for growth decreases or is lost upon prolonged culturing under heterotrophic conditions. The metabolic flexibility of C. difficile (heterotrophic growth on various substrates as well as autotrophy) could allow the organism in the gut to avoid competition by niche differentiation and contribute to its survival when stressed or in unfavorable conditions that cause death to other bacteria. This may be an important trait for the pathogenicity of C. difficile. PMID:23626782

  2. Clostridium sordellii as a Cause of Fatal Septic Shock in a Child with Hemolytic Uremic Syndrome.

    PubMed

    Beyers, Rebekah; Baldwin, Michael; Dalabih, Sevilay; Dalabih, Abdallah

    2014-01-01

    Clostridium sordellii is a toxin producing ubiquitous gram-positive anaerobe, mainly associated with trauma, soft tissue skin infections, and gynecologic infection. We report a unique case of a new strain of Clostridium sordellii (not present in the Center for Disease Control (CDC) database) infection induced toxic shock syndrome in a previously healthy two-year-old male with colitis-related hemolytic uremic syndrome (HUS). The patient presented with dehydration, vomiting, and bloody diarrhea. He was transferred to the pediatric critical care unit (PICU) for initiation of peritoneal dialysis (PD). Due to increased edema and intolerance of PD, he was transitioned to hemodialysis through a femoral vascular catheter. He subsequently developed severe septic shock with persistent leukocytosis and hypotension, resulting in subsequent death. Stool culture confirmed Shiga toxin producing Escherichia coli 0157:H7. A blood culture was positively identified for Clostridium sordellii. Clostridium sordelli is rarely reported in children; to our knowledge this is the first case described in a pediatric patient with HUS. PMID:24891968

  3. Clostridium thermosaccharolyticum strain deficient in acetate production

    SciTech Connect

    Rothstein, D.M.

    1986-01-01

    A mutant of Clostridium thermosaccharolyticum that is blocked in acetate production was isolated after treatment with nitrosoguanidine and selection for fluoroacetate resistance. The mutant produced more ethanol than the parent strain did.

  4. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Screening for bacteriophages lytic for Clostridium perfringens was completed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Following limit dilution cloning and three rounds of plaque purification lytic phage preparations ...

  5. Impact of formate on the growth and productivity of Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 grown on syngas.

    PubMed

    Ramió-Pujol, Sara; Ganigué, Ramon; Bañeras, Lluís; Colprim, Jesús

    2014-12-01

    The current energy model based on fossil fuels is coming to an end due to the increase in global energy demand. Biofuels such as ethanol and butanol can be produced through the syngas fermentation by acetogenic bacteria. The present work hypothesizes that formate addition would positively impact kinetic parameters for growth and alcohol production in Clostridium ljungdahlii PETC and Clostridium carboxidivorans P7 by diminishing the need for reducing equivalents. Fermentation experiments were conducted using completely anaerobic batch cultures at different pH values and formate concentrations. PETC cultures were more tolerant to formate concentrations than P7, specially at pH 5.0 and 6.0. Complete growth inhibition of PETC occurred at sodium formate concentrations of 30.0 mM; however, no differences in growth rates were observed at pH 7.0 for the two strains. Incubation at formate concentrations lower than 2.0 mM resulted in increased growth rates for both strains. The most recognizable effects of formate addition on the fermentation products were the increase in the total carbon fixed into acids and alcohols at pH 5.0 and pH 6.0, as well as, a higher ethanol to total products ratio at pH 7.0. Taken all together, these results show the ability of acetogens to use formate diminishing the energy demand for growth, and enhancing strain productivity. PMID:26421736

  6. Clostridium difficile infections among Jordanian adult hospitalized patients.

    PubMed

    Nasereddin, Lina M; Bakri, Fares G; Shehabi, Asem A

    2009-12-01

    This prospective study investigated the important epidemiologic aspects of Clostridium difficile infections (CDIs) among Jordanian adult hospitalized patients. A total of 300 stool specimens were investigated using culture and polymerase chain reaction methods for detection of C difficile, its toxins, and fluoroquinolone resistance. C difficile-positive cultures were found in 13.7% of the patients, and 73% of the isolates carried tcdA and/or tcdB toxin genes, and all C difficile isolates were negative for binary toxin. The isolates showed moderate level of resistance to both ciprofloxacin and levofloxacin, whereas metronidazole and vancomycin were highly susceptible. This study indicates the need for early detection of CDIs and prevention of its severe disease in hospitalized patients. PMID:19712999

  7. Clostridium difficile in paediatric populations

    PubMed Central

    Allen, Upton D

    2014-01-01

    An increase in Clostridium difficile infection incidence has been observed among hospitalized children in the United States. The present statement, targeted at clinicians caring for infants and children in community and institutional settings, summarizes the relevant information relating to the role of C difficile in childhood diarrhea and provides recommendations for diagnosis, prevention and treatment. Significant differences between adult and paediatric risk factors and disease are discussed, along with emerging therapies. The relationship between age and disease severity in children with a newly emergent and more fluoroqinolone-resistant strain of C difficile (North American Pulse-field type-1 [NAP1]) remains unknown. The importance of antimicrobial stewardship as a preventive strategy is highlighted. This statement replaces a previous Canadian Paediatric Society position statement on C difficile published in 2000. PMID:24627655

  8. Clostridium perfringens Type C Enterotoxemia.

    PubMed

    Niilo, L

    1988-08-01

    Forms of enteric disease caused by Clostridium perfringens type C are critically reviewed with emphasis on practical aspects and recent research findings. Available data indicate that more animal species may be fatally infected by type C of this organism than by any other type of C. perfringens. Fatal cases have been recorded in pigs, cattle, sheep, horses and humans. Newborn animals are typically the most susceptible, possibly related to aspects of bacterial colonization, intestinal digestive functions, and to some other, unexplained, factors. Both beta toxin and the bacterial cells are required to initiate pathogenesis at the tips of jejunal villi, and subsequent massive adherence of these cells to necrotic mucosa is a characteristic feature. Although major lesions occur in the intestine, death is due to toxemia. The disease can be effectively controlled by vaccination of the dam. Epizootiology of this disease is a possible area for further studies. PMID:17423103

  9. Phosphotransacetylase from Clostridium acidiurici1

    PubMed Central

    Robinson, James R.; Sagers, Richard D.

    1972-01-01

    The phosphotransacetylase from Clostridium acidiurici has two properties not observed for this enzyme in other bacteria: (i) it requires a divalent metal for activity, and (ii) it is not subject to uncoupling by arsenate. The enzyme has been obtained in highly purified form, with a specific activity 500-fold higher than crude extracts. Ferrous or manganous ions are required for maximal activity, with Mn2+ being 50 to 75% as effective as Fe2+. The acetyl group can be transferred from acetyl phosphate to coenzyme A in 20 mm arsenate without a net decrease in high-energy acyl linkages. Likewise, H32PO42− will exchange with acetyl-PO42− in the presence of arsenate without loss of acetyl phosphate. This suggests that the active site on the enzyme is capable of discriminating between phosphate and arsenate while permitting the reversible transfer of acyl groups between CoA and phosphate. Images PMID:16559158

  10. Clostridium difficile infection in patients with inflammatory bowel disease

    PubMed Central

    Biesiada, Grażyna; Perucki, William; Mach, Tomasz

    2014-01-01

    Clostridium difficile is a bacterium widely distributed in the human environment. In the last decade the incidence and severity of Clostridium difficile infection has grown, particularly in Europe and North America, making it one of the more common nosocomial infections. A group particularly susceptible to Clostridium difficile infection are patients with inflammatory bowel disease, especially those with involvement of the colon. This paper presents relevant data on Clostridium difficile infections in inflammatory bowel disease patients, including epidemiology, pathogenesis, diagnosis and treatment. PMID:25097707

  11. Prospects for a vaccine for Clostridium difficile.

    PubMed

    Kyne, L; Kelly, C P

    1998-09-01

    Clostridium difficile diarrhoea and colitis is a new disease that is attributable to broad spectrum antibiotic therapy. During the past 2 decades C. difficile has become one of the most common nosocomial pathogens in the developed world. As changing demographics create an increasingly elderly population and the use of broad spectrum antimicrobials continues to expand, C. difficile is likely to become increasingly problematic. Disease caused by this organism is caused by the inflammatory actions of its 2 toxins, A and B, on the intestinal mucosa. Human antibody responses to these toxins are common in the general population and in patients with C. difficile-associated disease. There is substantial, albeit inconclusive, evidence to indicate that antitoxin antibodies provide protection against severe, prolonged or recurrent C. difficile diarrhoea. Immunity induced by oral or parenteral passive administration of antibody is protective in animal models of C. difficile infection. In humans, intravenous passive immunisation with pooled human immunoglobulin has been successful in the treatment of recurrent and severe C. difficile colitis. Human trials of oral passive immunotherapy with bovine immunoglobulin therapy are in progress. Formalin-inactivated culture filtrate from toxigenic C. difficile, as well as purified and inactivated toxins, have been used to successfully immunise animals. Similar preparations are under investigation as possible human vaccines. Antibiotic therapy is effective in treating most individual patients with C. difficile diarrhoea, but has proven ineffective in reducing the overall incidence of nosocomial infection. Active immunisation is probably the most promising approach to long term control of this difficult iatrogenic disease. PMID:18020593

  12. Characterization of a Symbiotic Coculture of Clostridium thermohydrosulfuricum YM3 and Clostridium thermocellum YM4.

    PubMed

    Mori, Y

    1990-01-01

    Clostridium thermohydrosulfuricum YM3 and C. thermocellum YM4 were isolated from a coculture which was obtained from an enrichment culture inoculated with volcanic soil in Izu Peninsula, Japan. Strain YM3 had advantages over reported C. thermohydrosulfuricum strains in that it fermented inulin and could accumulate ethanol up to 1.3% (wt/vol). The highest ethanol yield obtained was 1.96 mol/mol of anhydroglucose unit in cellobiose. Strain YM4 had features different from those reported in C. thermocellum strains: it formed spores rarely (at a frequency of <10), it required CO(2) and Na(2)CO(3) for growth, and it fermented sucrose. Strain YM4 completely decomposed 1% Avicel within 25 h when the inoculum constituted 2% of the culture medium volume, and it produced 0.22 U of Avicelase and 2.21 U of carboxymethylcellulase per ml of the medium. The doubling times on Avicel, cellobiose, and glucose were 2.7, 1.1, and 1.6 h, respectively. Reconstructed cocultures of strains YM3 and YM4 were very stable and degraded Avicel more rapidly than did strain YM4 monoculture. Without yeast extract, neither microorganism was able to grow. However, the coculture grew on cellulose without yeast extract and produced ethanol in high yield. Moreover, cell-free spent culture broth of strain YM3 could replace yeast extract in supporting the growth of strain YM4. The symbiotic relationship of the two bacteria in cellulose fermentation is probably a case of mutualism. PMID:16348106

  13. Evaluation of gas-liquid chromatography for the rapid diagnosis of Clostridium difficile associated disease.

    PubMed Central

    Gianfrilli, P; Pantosti, A; Luzzi, I

    1985-01-01

    Direct gas-liquid chromatography of faecal specimens with isocaproic acid as a marker was used for the rapid diagnosis of Clostridium difficile associated diarrhoeal diseases. Ninety stools were examined and results were compared with conventional culture on selective medium and cytotoxin assay in tissue culture. Using a combined analysis of isocaproic acid and butyric acid peak heights we defined three categories: positive, negative, and indeterminate. When the indeterminate group was excluded, the positive and negative predictive values of gas-liquid chromatography analysis were 86.9% and 85% respectively compared with culture and 71.4% and 95% respectively compared with cytotoxin assay. PMID:4008667

  14. A case of reactive arthritis due to Clostridium difficile colitis

    PubMed Central

    Essenmacher, Alex C.; Khurram, Nazish; Bismack, Gregory T.

    2016-01-01

    Reactive arthritis is an acute, aseptic, inflammatory arthropathy following an infectious process but removed from the site of primary infection. It is often attributed to genitourinary and enteric pathogens, such as Chlamydia, Salmonella, Shigella, Campylobacter, and Yersinia, in susceptible individuals. An uncommon and less recognized cause of this disease is preceding colonic infection with Clostridium difficile, an organism associated with pseudomembranous colitis and diarrhea in hospitalized patients and those recently exposed to antibiotics. Recognition of this association may be complicated by non-specific presentation of diarrhea, the interval between gastrointestinal and arthritic symptoms, and the wide differential in mono- and oligoarthritis. We present the case of a 61-year-old, hospitalized patient recently treated for C. difficile colitis who developed sudden, non-traumatic, right knee pain and swelling. Physical examination and radiographs disclosed joint effusion, and sterile aspiration produced cloudy fluid with predominant neutrophils and no growth on cultures. Diagnostic accuracy is enhanced by contemporaneous laboratory investigations excluding other entities such as gout and rheumatoid arthritis and other infections that typically precede reactive arthritis. Contribution of Clostridium infection to reactive arthritis is an obscure association frequently difficult to prove, but this organism is warranted inclusion in the differential of reactive arthritis. PMID:26908381

  15. A case of reactive arthritis due to Clostridium difficile colitis.

    PubMed

    Essenmacher, Alex C; Khurram, Nazish; Bismack, Gregory T

    2016-01-01

    Reactive arthritis is an acute, aseptic, inflammatory arthropathy following an infectious process but removed from the site of primary infection. It is often attributed to genitourinary and enteric pathogens, such as Chlamydia, Salmonella, Shigella, Campylobacter, and Yersinia, in susceptible individuals. An uncommon and less recognized cause of this disease is preceding colonic infection with Clostridium difficile, an organism associated with pseudomembranous colitis and diarrhea in hospitalized patients and those recently exposed to antibiotics. Recognition of this association may be complicated by non-specific presentation of diarrhea, the interval between gastrointestinal and arthritic symptoms, and the wide differential in mono- and oligoarthritis. We present the case of a 61-year-old, hospitalized patient recently treated for C. difficile colitis who developed sudden, non-traumatic, right knee pain and swelling. Physical examination and radiographs disclosed joint effusion, and sterile aspiration produced cloudy fluid with predominant neutrophils and no growth on cultures. Diagnostic accuracy is enhanced by contemporaneous laboratory investigations excluding other entities such as gout and rheumatoid arthritis and other infections that typically precede reactive arthritis. Contribution of Clostridium infection to reactive arthritis is an obscure association frequently difficult to prove, but this organism is warranted inclusion in the differential of reactive arthritis. PMID:26908381

  16. Evaluation of a Loop-Mediated Isothermal Amplification Assay for Diagnosis of Clostridium difficile Infections▿

    PubMed Central

    Lalande, Valérie; Barrault, Laurence; Wadel, Sophie; Eckert, Catherine; Petit, Jean-Claude; Barbut, Frédéric

    2011-01-01

    A new assay (illumigene C. difficile; Meridian Bioscience), based on the original loop-mediated isothermal amplification (LAMP) assay, was evaluated with 472 unformed stools from patients suspected of Clostridium difficile infection. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 91.8, 99.1, 91.8, and 99.1% for the illumigene C. difficile assay and 69.4, 100, 100, and 96.6% for the cytotoxicity assay, respectively. PMID:21525213

  17. Evaluation of a loop-mediated isothermal amplification assay for diagnosis of Clostridium difficile infections.

    PubMed

    Lalande, Valérie; Barrault, Laurence; Wadel, Sophie; Eckert, Catherine; Petit, Jean-Claude; Barbut, Frédéric

    2011-07-01

    A new assay (illumigene C. difficile; Meridian Bioscience), based on the original loop-mediated isothermal amplification (LAMP) assay, was evaluated with 472 unformed stools from patients suspected of Clostridium difficile infection. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 91.8, 99.1, 91.8, and 99.1% for the illumigene C. difficile assay and 69.4, 100, 100, and 96.6% for the cytotoxicity assay, respectively. PMID:21525213

  18. Production of butanol by fermentation in the presence of cocultures of clostridium

    NASA Technical Reports Server (NTRS)

    Bergstrom, S. L.; Foutch, G. L. (Inventor)

    1985-01-01

    Sugars are converted to a mixture of solvents including butanol by a fermentation process employing a coculture of microorganisms of the Clostridium genus, one of said microorganisms favoring the production of butyric acid and the other of which converts the butyric acid so produced to butanol. The use of a coculture substantially increases the yield of butanol over that obtained using a culture employing only one microorganism.

  19. Faecal carriage of Clostridium perfringens.

    PubMed Central

    Stringer, M. F.; Watson, G. N.; Gilbert, R. J.; Wallace, J. G.; Hassall, J. E.; Tanner, E. I.; Webber, P. P.

    1985-01-01

    The numbers and serotypes of Clostridium perfringens present in the faeces of three groups of hospital patients and young healthy laboratory workers were examined in studies lasting between 10 and 13 weeks. In one hospital some long-stay geriatric patients carried relatively high numbers of C. perfringens (greater than 10(7)/g) most of the time and it was not unusual in any one week for the majority of these patients to carry the same serotype(s). However, the numbers of C. perfringens in the faeces of young long-stay patients in the same hospital were in the range of 10(3)-10(4)/g and carriage of common serotypes was not observed. These results were similar to the findings with the young laboratory workers. This investigation indicates that two of the laboratory criteria often used in the investigation of C. perfringens food poisoning, i.e. faecal counts of greater than or equal to 10(5) C. perfringens/g and patients carrying the same serological type need to be interpreted with caution with suspected outbreaks involving some groups of geriatric long-stay hospital patients. PMID:2866214

  20. Clostridium difficile infections in China.

    PubMed

    Jin, Ke; Wang, Shixia; Huang, Zuhu; Lu, Shan

    2010-11-01

    Clostridium difficile (C. difficile) infection has become one of the major hospital-associated infections in Western countries in the last two decades. However, there is limited information on the status of C. difficile infection in Chinese healthcare settings. Given the large and increasing elderly population and the well-recognized problem of over-prescribing of broad spectrum antibiotics in China, it is critical to understand the epidemiology and potential risk factors that may contribute to C. difficile infection in China. A literature review of available published studies, including those in Chinese language-based journals, was conducted. A review of the currently available literature suggested the presence of C. difficile infections in China, but also suggested that these infections were not particularly endemic. This finding should lead to better designed and greatly expanded studies to provide a more reliable epidemiologically-based conclusion on the actual status of C. difficile infection in China, including the identification of any associated risk factors. Such information is ultimately valuable to develop appropriate strategies to prevent C. difficile infection and the vast negative impact of such infections in China and other developing countries. PMID:23554657

  1. Toxin plasmids of Clostridium perfringens.

    PubMed

    Li, Jihong; Adams, Vicki; Bannam, Trudi L; Miyamoto, Kazuaki; Garcia, Jorge P; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2013-06-01

    In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  2. Clostridium difficile infection in Thailand.

    PubMed

    Putsathit, Papanin; Kiratisin, Pattarachai; Ngamwongsatit, Puriya; Riley, Thomas V

    2015-01-01

    Clostridium difficile is the aetiological agent in ca. 20% of cases of antimicrobial-associated diarrhoea in hospitalised adults. Diseases caused by this organism range from mild diarrhoea to occasional fatal pseudomembranous colitis. The epidemiology of C. difficile infection (CDI) has changed notably in the past decade, following epidemics in the early 2000s of PCR ribotype (RT) 027 infection in North America and Europe, where there was an increase in disease severity and mortality. Another major event has been the emergence of RT 078, initially as the predominant ribotype in production animals in the USA and Europe, and then in humans in Europe. Although there have been numerous investigations of the epidemiology of CDI in North America and Europe, limited studies have been undertaken elsewhere, particularly in Asia. Antimicrobial exposure remains the major risk factor for CDI. Given the high prevalence of indiscriminate and inappropriate use of antimicrobials in Asia, it is conceivable that CDI is relatively common among humans and animals. This review describes the level of knowledge in Thailand regarding C. difficile detection methods, prevalence and antimicrobial susceptibility profile, as well as the clinical features of, treatment options for and outcomes of the disease. In addition, antimicrobial usage in livestock in Thailand will be reviewed. A literature search yielded 18 studies mentioning C. difficile in Thailand, a greater number than from any other Asian country. It is possible that the situation in Thailand in relation to CDI may mirror the situation in other developing Asians countries. PMID:25537687

  3. Production of hexanoic acid from D-galactitol by a newly isolated Clostridium sp. BS-1.

    PubMed

    Jeon, Byoung Seung; Kim, Byung-Chun; Um, Youngsoon; Sang, Byoung-In

    2010-11-01

    In a study screening anaerobic microbes utilizing D: -galactitol as a fermentable carbon source, four bacterial strains were isolated from an enrichment culture producing H₂, ethanol, butanol, acetic acid, butyric acid, and hexanoic acid. Among these isolates, strain BS-1 produced hexanoic acid as a major metabolic product of anaerobic fermentation with D: -galactitol. Strain BS-1 belonged to the genus Clostridium based on phylogenetic analysis using 16S rRNA gene sequences, and the most closely related strain was Clostridium sporosphaeroides DSM 1294(T), with 94.4% 16S rRNA gene similarity. In batch cultures, Clostridium sp. BS-1 produced 550 ± 31 mL L⁻¹ of H₂, 0.36 ± 0.01 g L⁻¹ of acetic acid, 0.44 ± 0.01 g L⁻¹ of butyric acid, and 0.98 ± 0.03 g L⁻¹ of hexanoic acid in a 4-day cultivation. The production of hexanoic acid increased to 1.22 and 1.73 g L⁻¹ with the addition of 1.5 g L⁻¹ of sodium acetate and 100 mM 2-(N-morpholino)ethanesulfonic acid (MES), respectively. Especially when 1.5 g L⁻¹ of sodium acetate and 100 mM MES were added simultaneously, the production of hexanoic acid increased up to 2.99 g L⁻¹. Without adding sodium acetate, 2.75 g L⁻¹ of hexanoic acid production from D-galactitol was achieved using a coculture of Clostridium sp. BS-1 and one of the isolates, Clostridium sp. BS-7, in the presence of 100 mM MES. In addition, volatile fatty acid (VFA) production by Clostridium sp. BS-1 from D-galactitol and D: -glucose was enhanced when a more reduced culture redox potential (CRP) was applied via addition of Na₂S·9H₂O. PMID:20721546

  4. The complete genome sequence of Clostridium indolis DSM 755T

    PubMed Central

    Leschine, Susan; Huntemann, Marcel; Han, James; Chen, Amy; Kyrpides, Nikos; Markowitz, Victor; Palaniappan, Krishna; Ivanova, Natalia; Mikhailova, Natalia; Ovchinnikova, Galina; Schaumberg, Andrew; Pati, Amrita; Stamatis, Dimitrios; Reddy, Tatiparthi; Lobos, Elizabeth; Goodwin, Lynne; Nordberg, Henrik P.; Cantor, Michael N.; Hua, Susan X.; Woyke, Tanja; Blanchard, Jeffrey L.

    2014-01-01

    Clostridium indolis DSM 755T is a bacterium commonly found in soils and the feces of birds and mammals. Despite its prevalence, little is known about the ecology or physiology of this species. However, close relatives, C. saccharolyticum and C. hathewayi, have demonstrated interesting metabolic potentials related to plant degradation and human health. The genome of C. indolis DSM 755T reveals an abundance of genes in functional groups associated with the transport and utilization of carbohydrates, as well as citrate, lactate, and aromatics. Ecologically relevant gene clusters related to nitrogen fixation and a unique type of bacterial microcompartment, the CoAT BMC, are also detected. Our genome analysis suggests hypotheses to be tested in future culture based work to better understand the physiology of this poorly described species. PMID:25197485

  5. Protein phosphorylation in response to stress in Clostridium acetobutylicum

    SciTech Connect

    Balodimos, I.A.; Rapaport, E.; Kashket, E.R. )

    1990-07-01

    The possible involvement of protein phosphorylation in the clostridial stress response was investigated by radioactively labeling growing cells of Clostridium acetobutylicum with {sup 32}P{sub i} or cell extracts with ({gamma}-{sup 32}P)ATP. Several phosphoproteins were identified; these were not affected by the growth stage of the culture. Although the extent of protein phosphorylation was increased by heat stress, the phosphoproteins did not correspond to known stress proteins seen in one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Purified clostridial DnaK, a stress protein, acted as a kinase catalyzing the phosphorylation of a 50-kilodalton protein. The phosphorylation of this protein was enhanced in extracts prepared from heat-stressed cells. Diadenosine-5{prime},5{double prime}{prime}-P{sup 1},P{sup 4}-tetraphosphate had no influence on protein phosphorylation.

  6. Clostridium difficile-associated reactive arthritis in two children.

    PubMed

    Löffler, Helga A; Pron, Benedicte; Mouy, Richard; Wulffraat, Nico M; Prieur, Anne-Marie

    2004-01-01

    In adults, reactive arthritis (ReA) following Clostridium difficile-enterocolitis has been documented. In children, only one case of C. difficile-associated ReA has been reported. We now describe two other cases of ReA associated with C. difficile in children. The characteristics of ReA due to C. difficile appear to be similar in adults and children. Both children show polyarthritis after an episode of diarrhoea with positive stool cultures for C. difficile. Arthritis is asymmetrical with a self-limiting course. Nonsteroidal antiinflammatory drug (NSAID) therapy is sufficient. One case is remarkable because of its prolonged course of ReA despite NSAID therapy, and its association with the presence of HLA-B27 antigen. PMID:14769523

  7. Clostridium perfringens Sepsis and Fetal Demise after Genetic Amniocentesis

    PubMed Central

    Hendrix, Nancy W.; Mackeen, A. Dhanya; Weiner, Stuart

    2011-01-01

    Clostridium perfringens is a rare cause of intrauterine infection. There have been five case reports concerning infection associated with invasive procedures. We report a woman who underwent a genetic amniocentesis due to her history of chronic granulomatous disease. She presented to the hospital ∼38 hours after the amniocentesis complaining of fever and chills. Due to acute decompensation, she underwent an emergent dilatation and evacuation. During her stay, blood cultures came back positive for C. perfringens. Gradual improvement with intensive monitoring led to hospital discharge 4 days after the procedure. Uterine infection due to C. perfringens leading to maternal sepsis is associated with a high morbidity and mortality rate. Our patient was able to survive without a hysterectomy due to the rapid administration of antibiotics and surgical intervention while being evaluated. PMID:23705080

  8. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin

    PubMed Central

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R.; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  9. EGA Protects Mammalian Cells from Clostridium difficile CDT, Clostridium perfringens Iota Toxin and Clostridium botulinum C2 Toxin.

    PubMed

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; Tehran, Domenico Azarnia; Montecucco, Cesare; Barth, Holger

    2016-01-01

    The pathogenic bacteria Clostridium difficile, Clostridium perfringens and Clostridium botulinum produce the binary actin ADP-ribosylating toxins CDT, iota and C2, respectively. These toxins are composed of a transport component (B) and a separate enzyme component (A). When both components assemble on the surface of mammalian target cells, the B components mediate the entry of the A components via endosomes into the cytosol. Here, the A components ADP-ribosylate G-actin, resulting in depolymerization of F-actin, cell-rounding and eventually death. In the present study, we demonstrate that 4-bromobenzaldehyde N-(2,6-dimethylphenyl)semicarbazone (EGA), a compound that protects cells from multiple toxins and viruses, also protects different mammalian epithelial cells from all three binary actin ADP-ribosylating toxins. In contrast, EGA did not inhibit the intoxication of cells with Clostridium difficile toxins A and B, indicating a possible different entry route for this toxin. EGA does not affect either the binding of the C2 toxin to the cells surface or the enzyme activity of the A components of CDT, iota and C2, suggesting that this compound interferes with cellular uptake of the toxins. Moreover, for C2 toxin, we demonstrated that EGA inhibits the pH-dependent transport of the A component across cell membranes. EGA is not cytotoxic, and therefore, we propose it as a lead compound for the development of novel pharmacological inhibitors against clostridial binary actin ADP-ribosylating toxins. PMID:27043629

  10. Toxin Plasmids of Clostridium perfringens

    PubMed Central

    Li, Jihong; Adams, Vicki; Bannam, Trudi L.; Miyamoto, Kazuaki; Garcia, Jorge P.; Uzal, Francisco A.; Rood, Julian I.

    2013-01-01

    SUMMARY In both humans and animals, Clostridium perfringens is an important cause of histotoxic infections and diseases originating in the intestines, such as enteritis and enterotoxemia. The virulence of this Gram-positive, anaerobic bacterium is heavily dependent upon its prolific toxin-producing ability. Many of the ∼16 toxins produced by C. perfringens are encoded by large plasmids that range in size from ∼45 kb to ∼140 kb. These plasmid-encoded toxins are often closely associated with mobile elements. A C. perfringens strain can carry up to three different toxin plasmids, with a single plasmid carrying up to three distinct toxin genes. Molecular Koch's postulate analyses have established the importance of several plasmid-encoded toxins when C. perfringens disease strains cause enteritis or enterotoxemias. Many toxin plasmids are closely related, suggesting a common evolutionary origin. In particular, most toxin plasmids and some antibiotic resistance plasmids of C. perfringens share an ∼35-kb region containing a Tn916-related conjugation locus named tcp (transfer of clostridial plasmids). This tcp locus can mediate highly efficient conjugative transfer of these toxin or resistance plasmids. For example, conjugative transfer of a toxin plasmid from an infecting strain to C. perfringens normal intestinal flora strains may help to amplify and prolong an infection. Therefore, the presence of toxin genes on conjugative plasmids, particularly in association with insertion sequences that may mobilize these toxin genes, likely provides C. perfringens with considerable virulence plasticity and adaptability when it causes diseases originating in the gastrointestinal tract. PMID:23699255

  11. Clostridium difficile phages: still difficult?

    PubMed Central

    Hargreaves, Katherine R.; Clokie, Martha R. J.

    2014-01-01

    Phages that infect Clostridium difficile were first isolated for typing purposes in the 1980s, but their use was short lived. However, the rise of C. difficile epidemics over the last decade has triggered a resurgence of interest in using phages to combat this pathogen. Phage therapy is an attractive treatment option for C. difficile infection, however, developing suitable phages is challenging. In this review we summarize the difficulties faced by researchers in this field, and we discuss the solutions and strategies used for the development of C. difficile phages for use as novel therapeutics. Epidemiological data has highlighted the diversity and distribution of C. difficile, and shown that novel strains continue to emerge in clinical settings. In parallel with epidemiological studies, advances in molecular biology have bolstered our understanding of C. difficile biology, and our knowledge of phage–host interactions in other bacterial species. These three fields of biology have therefore paved the way for future work on C. difficile phages to progress and develop. Benefits of using C. difficile phages as therapeutic agents include the fact that they have highly specific interactions with their bacterial hosts. Studies also show that they can reduce bacterial numbers in both in vitro and in vivo systems. Genetic analysis has revealed the genomic diversity among these phages and provided an insight into their taxonomy and evolution. No strictly virulent C. difficile phages have been reported and this contributes to the difficulties with their therapeutic exploitation. Although treatment approaches using the phage-encoded endolysin protein have been explored, the benefits of using “whole-phages” are such that they remain a major research focus. Whilst we don’t envisage working with C. difficile phages will be problem-free, sufficient study should inform future strategies to facilitate their development to combat this problematic pathogen. PMID:24808893

  12. Clostridium septicum Empyema in an Immunocompetent Woman

    PubMed Central

    Granok, Alexander B.; Mahon, Patrick A.; Biesek, Genesio W.

    2010-01-01

    We report a case of a Clostridium septicum empyema in an immunocompetent woman following operation for an incarcerated internal hernia. The patient was successfully treated with pleural decortication and an extended course of postoperative antibiotics. This is the first report of such an infection in the medical literature. PMID:20490275

  13. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enteritis infection among chickens ...

  14. Coculture Production of Butanol by Clostridium Bacteria

    NASA Technical Reports Server (NTRS)

    Bergstrom, S. L.; Foutch, G. L.

    1985-01-01

    Production of butanol by anaerobic fermentation of sugars enhanced by use of two Clostridium species, one of which feeds on metabolic product of other. Renewed interest in fermentation process for making butanol stimulated by potential use of butanol as surfactant in enhanced oil recovery. Butanol also used as fuel or as chemical feedstock and currently produced synthetically from petroleum.

  15. Clostridium difficile in poultry and poultry meat

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence and severity of disease associated with toxigenic Clostridium difficile have increased in hospitals in North America from the emergence of newer, more virulent strains. Toxigenic C. difficile has been isolated from food animals and retail meat with potential implications of transfer t...

  16. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Clostridium perfringens are Gram-positive bacteria that are a major bacterial cause of food-borne disease and gas gangrene among humans. These anaerobic bacteria are also the presumptive etiologic agent of necrotic enteritis among chickens. Pathogenesis and symptoms of a necrotic enterit...

  17. Clostridium acidurici Electron-Bifurcating Formate Dehydrogenase

    PubMed Central

    Wang, Shuning; Huang, Haiyan; Kahnt, Jörg

    2013-01-01

    Cell extracts of uric acid-grown Clostridium acidurici catalyzed the coupled reduction of NAD+ and ferredoxin with formate at a specific activity of 1.3 U/mg. The enzyme complex catalyzing the electron-bifurcating reaction was purified 130-fold and found to be composed of four subunits encoded by the gene cluster hylCBA-fdhF2. PMID:23872566

  18. Hemorrhagic enteritis associated with Clostridium perfringens type A in a dog.

    PubMed

    Sasaki, J; Goryo, M; Asahina, M; Makara, M; Shishido, S; Okada, K

    1999-02-01

    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. PMID:10081759

  19. Two cases of type E infant botulism caused by neurotoxigenic Clostridium butyricum in Italy.

    PubMed

    Aureli, P; Fenicia, L; Pasolini, B; Gianfranceschi, M; McCroskey, L M; Hatheway, C L

    1986-08-01

    The first two confirmed cases of type E infant botulism occurred in two 16-week-old girls in Rome, Italy. The original diagnosis for the first patient was intestinal blockage due to an ileocecal invagination, which was treated surgically. Postoperatively, the patient became unresponsive and required ventilatory assistance. A diagnosis of infant botulism was then made. The second infant presented to the same hospital 7 1/2 months later with profound weakness, hypotonicity, mydriasis, and areflexia. This case was recognized as possible botulism at admission. Both cases were confirmed by detection and identification of type E botulinal toxin in stool specimens and in enrichment cultures of those specimens. The toxigenic organisms isolated were quite different from Clostridium botulinum type E. The apparent causative organism in each case resembles Clostridium butyricum but produces a neurotoxin that is indistinguishable from type E botulinal toxin by its effects on mice and by its neutralization with type E botulinal antitoxin. PMID:3722863

  20. An unexpected negative influence of light intensity on hydrogen production by dark fermentative bacteria Clostridium beijerinckii.

    PubMed

    Zagrodnik, R; Laniecki, M

    2016-01-01

    The role of light intensity on biohydrogen production from glucose by Clostridium beijerinckii, Clostridium acetobutylicum, and Rhodobacter sphaeroides was studied to evaluate the performance and possible application in co-culture fermentation system. The applied source of light had spectrum similar to the solar radiation. The influence of light intensity on hydrogen production in dark process by C. acetobutylicum was negligible. In contrast, dark fermentation by C. beijerinckii bacteria showed a significant decrease (83%) in produced hydrogen at light intensity of 540W/m(2). Here, the redirection of metabolism from acetic and butyric acid formation towards lactic acid was observed. This not yet reported effect was probably caused by irradiation of these bacteria by light within UVA range, which is an important component of the solar radiation. The excessive illumination with light of intensity higher than 200W/m(2) resulted in decrease in hydrogen production with photofermentative bacteria as well. PMID:26602144

  1. Microbiology of wetwood: importance of pectin degradation and Clostridium species in living trees. [Eastern Cottonwood; Block Poplar; American Elm

    SciTech Connect

    Schink, B.; Ward, J.C.; Zeikus, J.G.

    1981-09-01

    Wetwood samples from standing trees of eastern cottonwood (Populus deltoides), black poplar (Populus nigra), and American elm (Ulmus americana) contained high numbers of aerobic and anaerobic pectin-degrading bacteria (10 to the power of 4 to 10 to the power of 6 cells per g of wood). High activity of polygalacturonate lyase (is less than or equal to 0.5 U/ml) was also detected in the fetid liquid that spurted from wetwood zones in the lower trunk when the trees were bored. A prevalent pectin-degrading obligately anaerobic bacterium isolated from these wetwoods was identified as Clostridium butyricum. Pectin decomposition by Clostridium butyricum strain 4P1 was associated with an inducible polygalacturonate lyase and pectin methylesterase, the same types of pectinolytic activity expressed in the wetwood of these trees. The pH optimum of the extracellular polygalacturonate lyase was alkaline (near pH 8.5). In vitro tests with sapwood samples from a conifer (Douglas fir, Pseudotsuga menziesii) showed that tori in membranes of bordered pits are degraded by pure cultures of strain 4P1, polygalacturonate lyase enzyme preparations of strain 4P1, and mixed methanogenic cultures from the tree samples of wetwood. These results provide evidence that pectin in xylem tissue is actively degraded by Clostridium butyricum strain 4P1 via polygalacturonate lyase activity. The importance of pectin degradation by bacteria, including Clostridium species, appears paramount in the formation and maintenance of the wetwood syndrome in certain living trees. (Refs. 38).

  2. Purification of Clostridium botulinum type G progenitor toxin.

    PubMed

    Nukina, M; Mochida, Y; Sakaguchi, S; Sakaguchi, G

    1988-04-01

    Clostridium botulinum type G cultured for 6 days at 30 degrees C in proteose peptone-yeast extract-glucose medium produced toxin of 1.3 x 10(4) LD50/ml. The toxin was precipitated at pH 4.0, extracted with 0.2 M phosphate buffer, pH 6.0, and activated with trypsin. Sonic treatment and trypsinization of the residual precipitate released additional toxin, the toxicity of which corresponded to that detected in whole culture. Activated toxin obtained from the first extract and that from the residual precipitate were combined and purified by salting out, acid precipitation, gel filtration on Sephadex G-200, chromatography on SP-Sephadex, and a second gel filtration on Sephadex G-200. The yield of purified toxin from 10 liters of culture was 22.9 mg an 1.1 X 10(8) mouse ip LD50 with a specific toxicity of 3.0 X 10(7) mouse ip LD50/mg nitrogen. The molecular weight of the toxin was about 500,000, corresponding to that of L toxin of the other types. No M nor LL toxin was detected. PMID:3293334

  3. Metabolic response of Clostridium ljungdahlii to oxygen exposure.

    PubMed

    Whitham, Jason M; Tirado-Acevedo, Oscar; Chinn, Mari S; Pawlak, Joel J; Grunden, Amy M

    2015-12-01

    Clostridium ljungdahlii is an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H2, CO, and fructose after 8% O2 exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and that C. ljungdahlii's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism of C. ljungdahlii and provides new genetic targets to generate C. ljungdahlii strains that produce more ethanol and are more tolerant to syngas contaminants. PMID:26431975

  4. Metabolic Response of Clostridium ljungdahlii to Oxygen Exposure

    PubMed Central

    Whitham, Jason M.; Tirado-Acevedo, Oscar; Chinn, Mari S.; Pawlak, Joel J.

    2015-01-01

    Clostridium ljungdahlii is an important synthesis gas-fermenting bacterium used in the biofuels industry, and a preliminary investigation showed that it has some tolerance to oxygen when cultured in rich mixotrophic medium. Batch cultures not only continue to grow and consume H2, CO, and fructose after 8% O2 exposure, but fermentation product analysis revealed an increase in ethanol concentration and decreased acetate concentration compared to non-oxygen-exposed cultures. In this study, the mechanisms for higher ethanol production and oxygen/reactive oxygen species (ROS) detoxification were identified using a combination of fermentation, transcriptome sequencing (RNA-seq) differential expression, and enzyme activity analyses. The results indicate that the higher ethanol and lower acetate concentrations were due to the carboxylic acid reductase activity of a more highly expressed predicted aldehyde oxidoreductase (CLJU_c24130) and that C. ljungdahlii's primary defense upon oxygen exposure is a predicted rubrerythrin (CLJU_c39340). The metabolic responses of higher ethanol production and oxygen/ROS detoxification were found to be linked by cofactor management and substrate and energy metabolism. This study contributes new insights into the physiology and metabolism of C. ljungdahlii and provides new genetic targets to generate C. ljungdahlii strains that produce more ethanol and are more tolerant to syngas contaminants. PMID:26431975

  5. Complete Genome Sequence of Clostridium clariflavum DSM 19732

    SciTech Connect

    Goodwin, Lynne A.; Davenport, Karen W.; Teshima, Hazuki; Bruce, David; Detter, J. Chris; Tapia, Roxanne; Han, Cliff; Land, Miriam L; Hauser, Loren John; Jeffries, Cynthia; Han, James; Pitluck, Sam; Nolan, Matt; Chen, Amy; Huntemann, Marcel; Mavromatis, K; Mikhailova, Natalia; Liolios, Konstantinos; Woyke, Tanja; Lynd, Lee R

    2012-01-01

    Clostridium clariflavum is a Cluster III Clostridium within the family Clostridiaceae isolated from thermophilic anaerobic sludge (Shiratori et al, 2009). This species is of interest because of its similarity to the model cellulolytic organism Clostridium thermocellum and for the ability of environmental isolates to break down cellulose and hemicellulose. Here we describe features of the 4,897,678 bp long genome and its annotation, consisting of 4,131 proteincoding and 98 RNA genes, for the type strain DSM 19732.

  6. Prevalence and Risk Factors for Asymptomatic Clostridium difficile Carriage

    PubMed Central

    Alasmari, Faisal; Seiler, Sondra M.; Hink, Tiffany; Burnham, Carey-Ann D.; Dubberke, Erik R.

    2014-01-01

    Background. Clostridium difficile infection (CDI) incidence has increased dramatically over the last decade. Recent studies suggest that asymptomatic carriers may be an important reservoir of C. difficile in healthcare settings. We sought to identify the prevalence and risk factors for asymptomatic C. difficile carriage on admission to the hospital. Methods. Patients admitted to Barnes-Jewish Hospital without diarrhea were enrolled from June 2010 through October 2011. Demographic information and healthcare and medication exposures 90 days prior to admission were collected. Stool specimens or rectal swabs were collected within 48 hours of admission and stored at −30°C until cultured. Clostridium difficile isolates were typed and compared with isolates from patients with CDI. Results. A stool/swab specimen was obtained for 259 enrolled subjects on admission. Two hundred four (79%) were not colonized, 40 (15%) had toxigenic C. difficile (TCD), and 15 (6%) had nontoxigenic C. difficile. There were no differences between TCD-colonized and -uncolonized subjects for age (mean, 56 vs 58 years; P = .46), comorbidities, admission from another healthcare facility (33% vs 24%; P = .23), or recent hospitalization (50% vs 50%; P = .43). There were no differences in antimicrobial exposures in the 90 days prior to admission (55% vs 56%; P = .91). Asymptomatic carriers were colonized with strains similar to strains from patients with CDI, but the relative proportions were different. Conclusions. There was a high prevalence of TCD colonization on admission. In contrast to past studies, TCD colonization was not associated with recent antimicrobial or healthcare exposures. Additional investigation is needed to determine the role of asymptomatic TCD carriers on hospital-onset CDI incidence. PMID:24755858

  7. A case if infant botulism due to neurotoxigenic Clostridium butyricum type E associated with Clostridium difficile colitis.

    PubMed

    Fenicia, L; Da Dalt, L; Anniballi, F; Franciosa, G; Zanconato, S; Aureli, P

    2002-10-01

    Reported here is the sixth case of intestinal toxemia botulism caused by Clostridium butyricum type E in Italy since 1984. In this case, the patient was concomitantly affected with colitis due to Clostridium difficile toxin. A review of previously reported cases revealed that some of these patients may also have had intestinal toxemia botulism associated with Clostridium difficile colitis, based on the reported symptoms. Given that this association has been shown to exist not only in Italy but also in the USA, it is recommended that individuals with intestinal botulism and symptoms of colitis undergo testing for Clostridium difficile and its toxins in fecal samples. PMID:12479171

  8. Removal of Fermentation Inhibitors from Alkaline Peroxide Pretreated and Enzymatically Hydrolyzed Wheat Straw: Production of Butanol from Hydrolysate Using Clostridium beijerinckii in Batch Reactors

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In these studies, alkaline peroxide pretreatment of wheat straw was investigated. Pretreated wheat straw was hydrolyzed using celluloytic and xylanolytic enzymes, and the hydrolysate was used to produce butanol using Clostridium beijerinckii P260. The culture produced less than 2.59 gL**-1 acetone...

  9. [Genetic modification systems for Clostridium acetobutylicum].

    PubMed

    Dong, Hongjun; Zhang, Yanping; Li, Yin

    2010-10-01

    Clostridium acetobutylicum, a biofuel-butanol producer, has attracted worldwide interests. Strain improvement is important for the process of biobutanol industrialization where efficient genetic modification systems are essential. In this review, the history of genetic modification systems of C. acetobutylicum was introduced, and the types and principles of these systems and their disadvantages are summarized and analysed. The development of updated genetic modification systems for C. acetobutylicum is also proposed. PMID:21218624

  10. Clostridium difficile Infection: A Worldwide Disease

    PubMed Central

    Burke, Kristin E.

    2014-01-01

    Clostridium difficile, an anaerobic toxigenic bacterium, causes a severe infectious colitis that leads to significant morbidity and mortality worldwide. Both enhanced bacterial toxins and diminished host immune response contribute to symptomatic disease. C. difficile has been a well-established pathogen in North America and Europe for decades, but is just emerging in Asia. This article reviews the epidemiology, microbiology, pathophysiology, and clinical management of C. difficile. Prompt recognition of C. difficile is necessary to implement appropriate infection control practices. PMID:24516694

  11. Persistent and Recurrent Clostridium difficile Colitis

    PubMed Central

    Cole, Shola A.; Stahl, Thomas J.

    2015-01-01

    Clostridium difficile infection (CDI) is the most frequent cause of nosocomial diarrhea. It has become a significant dilemma in the treatment of patients, and causes increasing morbidity that, in extreme cases, may result in death. Persistent and recurrent disease hamper attempts at eradication of this infection. Escalating levels of treatment and novel therapeutics are being utilized and developed to treat CDI. Further trials are warranted to definitively determine what protocols can be used to treat persistent and recurrent disease. PMID:26034401

  12. Multicenter evaluation of the Verigene Clostridium difficile nucleic acid assay.

    PubMed

    Carroll, Karen C; Buchan, Blake W; Tan, Sokha; Stamper, Paul D; Riebe, Katherine M; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V; Trevino, Ernest A; Weissfeld, Alice S; Ledeboer, Nathan A

    2013-12-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as "hypervirulent"; 53 were confirmed as ribotype

  13. Multicenter Evaluation of the Verigene Clostridium difficile Nucleic Acid Assay

    PubMed Central

    Buchan, Blake W.; Tan, Sokha; Stamper, Paul D.; Riebe, Katherine M.; Pancholi, Preeti; Kelly, Cheryl; Rao, Arundhati; Fader, Robert; Cavagnolo, Robert; Watson, Wendy; Goering, Richard V.; Trevino, Ernest A.; Weissfeld, Alice S.; Ledeboer, Nathan A.

    2013-01-01

    The Verigene Clostridium difficile Nucleic Acid test (Verigene CDF test) (Nanosphere, Northbrook, IL) is a multiplex qualitative PCR assay that utilizes a nanoparticle-based array hybridization method to detect C. difficile tcdA and tcdB in fecal specimens. In addition, the assay detects binary toxin gene sequences and the single base pair deletion at nucleotide 117 (Δ 117) in tcdC to provide a presumptive identification of the epidemic strain 027/NAP1/BI (referred to here as ribotype 027). This study compared the Verigene CDF test with anaerobic direct and enriched toxigenic culture on stool specimens from symptomatic patients among five geographically diverse laboratories within the United States. The Verigene CDF test was performed according to the manufacturer's instructions, and the reference methods performed by a central laboratory included direct culture onto cycloserine cefoxitin fructose agar (CCFA) and enriched culture using cycloserine cefoxitin mannitol broth with taurocholate and lysozyme. Recovered isolates were identified as C. difficile using gas liquid chromatography and were tested for toxin using a cell culture cytotoxicity neutralization assay. Strains belonging to ribotype 027 were determined by PCR ribotyping and bidirectional sequencing for Δ 117 in tcdC. A total of 1,875 specimens were evaluable. Of these, 275 specimens (14.7%) were culture positive by either direct or enriched culture methods. Compared to direct culture alone, the overall sensitivity, specificity, positive predictive value, and negative predictive value for the Verigene CDF test were 98.7%, 87.5%, 42%, and 99.9%, respectively. Compared to combined direct and enriched culture results, the sensitivity, specificity, positive predictive value, and negative predictive values of the Verigene CDF test were 90.9%, 92.5%, 67.6%, and 98.3%, respectively. Of the 250 concordantly culture-positive specimens, 59 (23.6%) were flagged as “hypervirulent”; 53 were confirmed as

  14. Growth from Spores of Nonproteolytic Clostridium botulinum in Heat-Treated Vegetable Juice

    PubMed Central

    Stringer, Sandra C.; Haque, Nuzrul; Peck, Michael W.

    1999-01-01

    Unheated spores of nonproteolytic Clostridium botulinum were able to lead to growth in sterile deoxygenated turnip, spring green, helda bean, broccoli, or potato juice, although the probability of growth was low and the time to growth was longer than the time to growth in culture media. With all five vegetable juices tested, the probability of growth increased when spores were inoculated into the juice and then heated for 2 min in a water bath at 80°C. The probability of growth was greater in bean or broccoli juice than in culture media following 10 min of heat treatment in these media. Growth was prevented by heat treatment of spores in vegetable juices or culture media at 80°C for 100 min. We show for the first time that adding heat-treated vegetable juice to culture media can increase the number of heat-damaged spores of C. botulinum that can lead to colony formation. PMID:10224012

  15. Physical map of the Clostridium beijerinckii (formerly Clostridium acetobutylicum) NCIMB 8052 chromosome.

    PubMed Central

    Wilkinson, S R; Young, M

    1995-01-01

    A combined physical and genetic map of the single, circular, 6.7-Mbp chromosome of the NCIMB 8052 strain of Clostridium beijerinckii (formerly Clostridium acetobutylicum) has been constructed by using a combination of cloned DNA fragments as hybridization probes and a bank of strains harboring insertions of the conjugative transposon Tn1545. The positions of 81 restriction endonuclease cleavage sites and 32 genes have been determined. Eight genes concerned with solventogenic fermentation are found at three different locations. The chromosome contains at least 13 rrn operons, 11 of which have been located on the map. Their transcriptional orientation diverges from the presumed location of the replication origin. PMID:7814334

  16. Genetic and biochemical analysis of solvent formation in Clostridium acetobutylicum

    SciTech Connect

    Bennett, G.N.; Rudolph, F.B.

    1998-05-01

    The anaerobic organism Clostridium acetobutylicum has been used for commercial production of important organic solvents due to its ability to convert a wide variety of crude substrates to acids and alcohols. Current knowledge concerning the molecular genetics, cell regulation and metabolic engineering of this organism is still rather limited. The objectives are to improve the knowledge of the molecular genetics and enzymology of Clostridia in order to make genetic alterations which will more effectively channel cell metabolism toward production of desired products. Two factors that limit butanol production in continuous cultures are: (1) The degeneration of the culture, with an increase in the proportion of cells which are incapable of solvent production. Currently isolated degenerate strains are being evaluated to analyze the molecular mechanism of degeneration to determine if it is due to a genetic loss of solvent related genes, loss of a regulatory element, or an increase in general mutagenesis. Recent studies show two general types of degenerates, one which seems to have lost essential solvent pathway genes and another which has not completely lost all solvent production capability and retains the DNA bearing solvent pathway genes. (2) The production of hydrogen which uses up reducing equivalents in the cell. If the reducing power were more fully directed to the reduction reactions involved in butanol production, the process would be more efficient. The authors have studied oxidation reduction systems related to this process. These studies focus on ferredoxin and rubredoxin and their oxidoreductases.

  17. Characteristics of patients with Clostridium difficile infection in Taiwan.

    PubMed

    Lin, Y-C; Huang, Y-T; Lee, T-F; Lee, N-Y; Liao, C-H; Lin, S-Y; Ko, W-C; Hsueh, P-R

    2013-10-01

    The medical records of 84 patients with stool cultures positive for Clostridium difficile during the period August 2007 to June 2009 were retrospectively reviewed. A case of confirmed (toxigenic)C. difficile infection (CDI) was defined by the presence of symptoms (fever, diarrhoea, abdominal discomfort or distension, ileus) and the presence of toxigenic C. difficile. Patients with compatible clinical symptoms and stool cultures positive for non-toxigenic C. difficile isolates were defined as probable (non-toxigenic) CDI cases. Of these 84 patients, 50 (59.5%) were diagnosed as confirmed CDI and 34 (40.5%) as probable CDI. Thirteen (15.5%) of the 84 patients died during their hospital stay. Usage of proton pump inhibitors was a significant independent risk factor for CDI (OR 3.21, P=0.014). Of the 50 isolates associated with confirmed CDI, seven (8.3%) carried binary toxin genes (cdtAB), and six (7.1%) had a deletion in the tcdC gene. The mortality rate in confirmed CDI patients with isolates exhibiting deletion in the tcdC gene (2/6, 33.3%), those with isolates harbouring binary toxin genes (2/7, 28.6%), and those with isolates containing mutations in gyrA (2/7, 28.6%) and gyrB (1/2, 50%) was higher than the overall mortality rate (10/50, 20%) in patients with confirmed CDI. PMID:23218131

  18. Clostridium difficile infection: a review of current and emerging therapies

    PubMed Central

    Ofosu, Andrew

    2016-01-01

    Clostridium difficile (C. difficile) infection (CDI) is the most common cause of ­healthcare-associated infections in US hospitals. The epidemic strain NAP1/BI/ribotype 027 accounts for outbreaks worldwide, with increasing mortality and severity. CDI is acquired from an endogenous source or from spores in the environment, most easily acquired during the hospital stay. The use of antimicrobials disrupts the intestinal microflora enabling C. difficile to proliferate in the colon and produce toxins. Clinical diagnosis in symptomatic patients requires toxin detection from stool specimens and rarely in combination with stool culture to increase sensitivity. However, stool culture is essential for epidemiological studies. Oral metronidazole is the recommended therapy for milder cases of CDI and oral vancomycin or fidaxomicin for more severe cases. Treatment of first recurrence involves the use of the same therapy used in the initial CDI. In the event of a second recurrence oral vancomycin often given in a tapered dose or intermittently, or fidaxomicin may be used. Fecal transplantation is playing an immense role in therapy of recurrent CDI with remarkable results. Fulminant colitis and toxic megacolon warrant surgical intervention. Novel approaches including new antibiotics and immunotherapy against CDI or its toxins appear to be of potential value. PMID:27065726

  19. Action of egg white lysozyme on Clostridium tyrobutyricum.

    PubMed Central

    Wasserfall, F; Teuber, M

    1979-01-01

    A 500-U ml-1 portion of egg white lysozyme was able to kill 99% of 5 X 10(5) resting vegetative cells of Clostridium tyrobutyricum within 24 h of incubation at 25 degrees C. Spores were completely resistant to lysozyme. Proliferating vegetative cells were severely inhibited, although lysozyme-resistant cells developed in growing cultures in the presence of lysozyme. Whereas early stages of spore germination (loss of optical refractility and heat resistance) were not inhibited by lysozyme, the overall outgrowth of spore cells into vegetative cells was delayed by 1 day in the presence of 500 U of lysosyme ml-1. This delay was independent of the lysozyme sensitivity or resistance of the mother culture of the used spores. It is suggested that this inhibition by lysozyme of the outgrowth of spore cells into vegetative cells of the lactate-fermenting C. tyrobutyricum is the basis for the observation that lysozyme can substitute for nitrate in preventing the "late gas" defect of Edam- and Gouda-type cheeses. PMID:518083

  20. Prevalence of Clostridium difficile colonization among healthcare workers

    PubMed Central

    2013-01-01

    Background Clostridium difficile infection (CDI) has increased to epidemic proportions in recent years. The carriage of C. difficile among healthy adults and hospital inpatients has been established. We sought to determine whether C. difficile colonization exists among healthcare workers (HCWs) in our setting. Methods A point prevalence study of stool colonization with C. difficile among doctors, nurses and allied health staff at a large regional teaching hospital in Geelong, Victoria. All participants completed a short questionnaire and all stool specimens were tested by Techlab® C.diff Quik Check enzyme immunoassay followed by enrichment culture. Results Among 128 healthcare workers, 77% were female, of mean age 43 years, and the majority were nursing staff (73%). Nineteen HCWs (15%) reported diarrhoea, and 12 (9%) had taken antibiotics in the previous six weeks. Over 40% of participants reported having contact with a patient with known or suspected CDI in the 6 weeks before the stool was collected. C. difficile was not isolated from the stool of any participants. Conclusion Although HCWs are at risk of asymptomatic carriage and could act as a reservoir for transmission in the hospital environment, with the use of a screening test and culture we were unable to identify C. difficile in the stool of our participants in a non-outbreak setting. This may reflect potential colonization resistance of the gut microbiota, or the success of infection prevention strategies at our institution. PMID:24090343

  1. Real-Time PCR Assay for Clostridium perfringens in Broiler Chickens in a Challenge Model of Necrotic Enteritis▿

    PubMed Central

    Wu, Shu-Biao; Rodgers, Nicholas; Choct, Mingan

    2011-01-01

    We compared ileal Clostridium perfringens quantification results produced by real-time PCR and culture-based methods in broiler chickens in a challenge model of necrotic enteritis. Assessment of the relative standard deviations (RSDs) revealed that the real-time PCR assay generated a smaller standard deviation and thus was more precise than the culture-based method. Linear regression analysis indicated that the bacterial counts of these two methods were highly correlated (R2 = 0.845). We suggest that real-time PCR could be a replacement of the culture method for quantifying C. perfringens in the intestinal tracts of broiler chickens. PMID:21148703

  2. Use of the cobas 4800 system for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus.

    PubMed

    Moure, Raquel; Cañizares, Ángeles; Muíño, María; Lobato, Margarita; Fernández, Ana; Rodríguez, María; Gude, Maria José; Tomás, Maria; Bou, Germán

    2016-01-01

    The new cobas® Cdiff and cobas® MRSA/SA tests were compared with conventional methods for the rapid detection of toxigenic Clostridium difficile and methicillin-resistant Staphylococcus aureus. The final concordance between cobas Cdiff Test and GDH/toxin gene screening was 97.62% and between cobas MRSA/SA Test and chromogenic culture, 91.30%, respectively. PMID:26611812

  3. Draft Genome Sequence of Clostridium butyricum Strain NOR 33234, Isolated from an Elderly Patient with Diarrhea

    PubMed Central

    Kwok, Jamie S. L.; Ip, Margaret; Chan, Ting-Fung; Lam, Wai-Yip

    2014-01-01

    Clostridium butyricum is one of the species frequently present in patients’ stool samples. However, the identification of this species is sometimes difficult. Here, we present the draft genome of Clostridium butyricum NOR 33234, which was isolated from a patient with suspected Clostridium difficile infection-associated diarrhea and resembles Clostridium clostridioforme in biochemical tests. PMID:25540356

  4. Clostridium difficile in the Long-Term Care Facility: Prevention and Management

    PubMed Central

    Jump, Robin L. P.; Donskey, Curtis J.

    2014-01-01

    Residents of long-term care facilities are at high risk for Clostridium difficile infection due to frequent antibiotic exposure in a population already rendered vulnerable to infection due to advanced age, multiple comorbid conditions and communal living conditions. Moreover, asymptomatic carriage of toxigenic C. difficile and recurrent infections are prevalent in this population. Here, we discuss epidemiology and management of C. difficile infection among residents of long-term care facilities. Also, recognizing that both the population and culture differs significantly from that of hospitals, we also address prevention strategies specific to LTCFs. PMID:25685657

  5. Diarrhea associated with enterotoxigenic Clostridium perfringens in a red-footed tortoise (Geochelone carbonaria).

    PubMed

    Weese, J S; Staempfli, H R

    2000-06-01

    Enterotoxigenic Clostridium perfringens was associated with diarrhea in a 4-yr-old female captive-bred red-footed tortoise (Geochelone carbonaria). Diagnosis was based on bacterial culture, detection of C. perfringens enterotoxin in feces, and exclusion of commonly recognized pathogens. After treatment with metronidazole, normal feces were passed and C. perfringens enterotoxin was no longer detected in the feces. Although the role of C. perfringens cannot be determined definitively from this case, this pathogen should be considered in cases of diarrhea in tortoises and, perhaps, other reptiles. PMID:10982148

  6. Application of Lactobacillus johnsonii expressing phage endolysin for control of Clostridium perfringens.

    PubMed

    Gervasi, T; Lo Curto, R; Minniti, E; Narbad, A; Mayer, M J

    2014-10-01

    Clostridium perfringens is frequently found in food and the environment and produces potent toxins that have a negative impact on both human and animal health and particularly on the poultry industry. Lactobacillus johnsonii FI9785, isolated from the chicken gastrointestinal tract, has been demonstrated to exclude Cl. perfringens in poultry. We have investigated the interaction of wild-type Lact. johnsonii FI9785 or an engineered strain expressing a cell wall-hydrolysing endolysin with Cl. perfringens in vitro, using a batch culture designed to simulate human gastrointestinal tract conditions. Co-culture experiments indicated that acid production by Lact. johnsonii is important in pathogen control. The co-culture of the endolysin-secreting Lact. johnsonii with Cl. perfringens showed that the engineered strain had the potential to control the pathogen, but the ability to reduce Cl. perfringens numbers was not consistent. Results obtained indicate that survival of high numbers of Lact. johnsonii will be essential for effective pathogen control. Significance and impact of the study: The bacterium Lactobacillus johnsonii FI9785 reduces numbers of the pathogen Clostridium perfringens in vitro. Biocontrol was improved by engineering the strain to produce and export a cell wall-hydrolysing endolysin, but good survival of the producer strain is essential. The production of bacteriophage endolysins by commensal bacteria has the potential to improve competitive exclusion of pathogens in the gastrointestinal tract. PMID:24961379

  7. Physical and genetic map of the Clostridium saccharobutylicum (formerly Clostridium acetobutylicum) NCP 262 chromosome.

    PubMed

    Keis, S; Sullivan, J T; Jones, D T

    2001-07-01

    A physical and genetic map of the Clostridium saccharobutylicum NCP 262 chromosome was constructed. The order of macrorestriction fragments was determined by analysing fragments generated after single and double digestion with the restriction enzymes BssHII, I-CeuI, Sse8387I, RsrII and SfiI and separation by PFGE. The I-CeuI backbone of C. saccharobutylicum was constructed by indirect end-labelling with rrs- and 3' rrl-specific probes located on either side of the I-CeuI site in the rrn operon, and reciprocal separation of BssHII and I-CeuI digestion products by two-dimensional PFGE. The positions of BssHII fragments on the physical map were determined using a library of linking clones containing BssHII cleavage sites. The size of the circular genome was estimated to be 5.3 Mb with a mean resolution of approximately 140 kb. The chromosome of C. saccharobutylicum contains 12 rrn operons, located on 46% of the chromosome, which are transcribed divergently from the deduced origin of replication. The genetic map was constructed by determining the location of 28 genes involved in house-keeping, heat-shock response, sporulation, electron transfer and acid- and solvent-formation. Comparison of the C. saccharobutylicum genetic map with those of the spore-forming bacteria Bacillus subtilis, Clostridium acetobutylicum, Clostridium perfringens and Clostridium beijerinckii indicated C. saccharobutylicum to be most similar to the latter two Clostridium species, with the order of the genes within the gyrAB and recA loci being conserved. PMID:11429467

  8. An Update on Clostridium difficile Toxinotyping

    PubMed Central

    Janezic, Sandra

    2015-01-01

    Toxinotyping is a PCR-restriction fragment length polymorphism (RFLP)-based method for differentiation of Clostridium difficile strains according to the changes in the pathogenicity locus (PaLoc), a region coding for toxins A and B. Toxinotypes are a heterogenous group of strains that are important in the development of molecular diagnostic tests and vaccines and are a good basis for C. difficile phylogenetic studies. Here we describe an overview of the 34 currently known toxinotypes (I to XXXIV) and some changes in nomenclature. PMID:26511734

  9. Alternative medium for Clostridium perfringens sporulation.

    PubMed Central

    Tórtora, J C

    1984-01-01

    A medium containing 0.50 g of thiotone peptone, 0.30 g of soluble starch, 0.02 g of MgSO4 X 7H2O, 0.90 g of Na2HPO4 X 2H2O, 100.00 ml of distilled water, and optionally , 166 micrograms of dichloridric thiamine supported sporulation of 138 out of 141 Clostridium perfringens strains. Comparatively this medium gave a greater percentage of sporulation than five other media described previously. PMID:6331307

  10. Clostridium difficile colitis: pathogenesis and host defence.

    PubMed

    Abt, Michael C; McKenney, Peter T; Pamer, Eric G

    2016-10-01

    Clostridium difficile is a major cause of intestinal infection and diarrhoea in individuals following antibiotic treatment. Recent studies have begun to elucidate the mechanisms that induce spore formation and germination and have determined the roles of C. difficile toxins in disease pathogenesis. Exciting progress has also been made in defining the role of the microbiome, specific commensal bacterial species and host immunity in defence against infection with C. difficile. This Review will summarize the recent discoveries and developments in our understanding of C. difficile infection and pathogenesis. PMID:27573580

  11. Inducing and Quantifying Clostridium difficile Spore Formation.

    PubMed

    Shen, Aimee; Fimlaid, Kelly A; Pishdadian, Keyan

    2016-01-01

    The Gram-positive nosocomial pathogen Clostridium difficile induces sporulation during growth in the gastrointestinal tract. Sporulation is necessary for this obligate anaerobe to form metabolically dormant spores that can resist antibiotic treatment, survive exit from the mammalian host, and transmit C. difficile infections. In this chapter, we describe a method for inducing C. difficile sporulation in vitro. This method can be used to study sporulation and maximize spore purification yields for a number of C. difficile strain backgrounds. We also describe procedures for visualizing spore formation using phase-contrast microscopy and for quantifying the efficiency of sporulation using heat resistance as a measure of functional spore formation. PMID:27507338

  12. Clostridium difficile: from obscurity to superbug.

    PubMed

    Brazier, J S

    2008-01-01

    According to the UK media and popular press, Clostridium difficile is now a fully fledged member of that notorious but ill-defined group of microorganisms portrayed to the general public as superbugs. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous superbug responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This review tracks the rise in scientific knowledge and public awareness of this organism. PMID:18476496

  13. Regulation of Toxin Production in Clostridium perfringens

    PubMed Central

    Ohtani, Kaori; Shimizu, Tohru

    2016-01-01

    The Gram-positive anaerobic bacterium Clostridium perfringens is widely distributed in nature, especially in soil and the gastrointestinal tracts of humans and animals. C. perfringens causes gas gangrene and food poisoning, and it produces extracellular enzymes and toxins that are thought to act synergistically and contribute to its pathogenesis. A complicated regulatory network of toxin genes has been reported that includes a two-component system for regulatory RNA and cell-cell communication. It is necessary to clarify the global regulatory system of these genes in order to understand and treat the virulence of C. perfringens. We summarize the existing knowledge about the regulatory mechanisms here. PMID:27399773

  14. Annotation of the Clostridium Acetobutylicum Genome

    SciTech Connect

    Daly, M. J.

    2004-06-09

    The genome sequence of the solvent producing bacterium Clostridium acetobutylicum ATCC824, has been determined by the shotgun approach. The genome consists of a 3.94 Mb chromosome and a 192 kb megaplasmid that contains the majority of genes responsible for solvent production. Comparison of C. acetobutylicum to Bacillus subtilis reveals significant local conservation of gene order, which has not been seen in comparisons of other genomes with similar, or, in some cases, closer, phylogenetic proximity. This conservation allows the prediction of many previously undetected operons in both bacteria.

  15. Novel Risk Factors for Recurrent Clostridium difficile Infection in Children

    PubMed Central

    Nicholson, Maribeth R.; Thomsen, Isaac P.; Slaughter, James C.; Creech, C. Buddy; Edwards, Kathryn M.

    2014-01-01

    Objectives Clostridium difficile, a common cause of antibiotic-associated diarrhea, has been reported to recur in high rates in adults. The rates and risk factors for recurrent Clostridium difficile infection (rCDI) in children have not been well established. Methods We conducted a retrospective cohort study of 186 pediatric patients seen at a tertiary care referral center over a 5-year period diagnosed with a primary infection with Clostridium difficile. Children with recurrent disease, defined as return of symptoms of Clostridium difficile infection and positive testing ≤60 days after the completion of therapy, were compared to children who did not experience an episode of recurrence. Results Of the 186 pediatric patients included in this study, 41 (22%) experienced recurrent Clostridium difficile infection. On univariable analysis, factors significantly associated with recurrent Clostridium difficile infection included malignancy, recent hospitalization, recent surgery, antibiotic use, number of antibiotic exposures by class, acid blocker use, immunosuppressant use, and hospital acquired disease. On multivariable analysis, malignancy (OR=3.39, 95% CI=1.52–7.85), recent surgery (OR=2.40, 95% CI=1.05–5.52), and the number of antibiotic exposures by class (OR=1.33, 95% CI=1.01–1.75) were significantly associated with recurrent disease in children. Conclusions The rate of recurrent Clostridium difficile infection in children was 22%. Recurrence was significantly associated with the risk factors of malignancy, recent surgery, and the number of antibiotic exposures by class. PMID:25199038

  16. Tests for the diagnosis of Clostridium difficile infection: the next generation.

    PubMed

    Carroll, Karen C

    2011-08-01

    Clostridium difficile (C. difficile) causes 25-30% of cases of antibiotic associated diarrhea and most cases of pseudomembranous colitis. Patients presenting with diarrhea after hospitalization for 3 or more days should be tested for C. difficile. There are many options available for testing, each of which has inherent advantages and disadvantages. Most laboratories perform toxin testing using an enzyme immunoassay method. In general these tests have sensitivities ranging from 60 to 70% and specificities of 98%. When using these methods, symptomatic patients with negative tests should be tested by another more sensitive method. Until recently, cell culture cytotoxicity neutralization assays (CCNAs) were considered the gold standard in the U.S. A two-step algorithm using an EIA for glutamate dehydrogenase detection followed by testing positives using CCNA, offered an improved alternative until the availability of molecular assays. Although early studies that compared the GDH assay to CCNA demonstrated high sensitivity and negative predictive values, more recent comparisons to toxigenic culture and PCR have shown the sensitivity to be in the mid to high 80's. When testing using a sensitive assay, repeat testing is not cost-effective. Outbreaks caused by a toxin variant epidemic strain have renewed interest in bacterial culture. Toxigenic culture has emerged as the new gold standard against which newer assays should be compared. However, there is no agreed upon standard method for culture performance. At least 4 FDA cleared nucleic acid amplification assays are available to clinical laboratories and several of these have been well evaluated in the literature. Because these assays detect a gene that encodes toxin and not the toxin itself it is important that laboratories test only patients with diarrhea. These molecular assays have been shown to be superior to toxin EIAs, CCNA and 2-step algorithms, but not to toxigenic culture. More studies are needed to assess the

  17. Comparison of Media for the Enumeration of Clostridium perfringens

    PubMed Central

    Harmon, Stanley M.; Kautter, Donald A.; Peeler, James T.

    1971-01-01

    For the enumeration of viable vegetative cells and spores of Clostridium perfringens, noncommercial (laboratory prepared) sulfite-polymyxin-sulfadiazine (SPS) agar, tryptone-sulfite-neomycin (TSN) agar, and Shahidi-Ferguson-perfringens (SFP) agar were statistically compared to SPS agar without antibiotics. The selectivities of these four media were also evaluated on the basis of their ability to inhibit the growth of pure cultures of a variety of other organisms. The average recovery of vegetative cells of 10 strains of C. perfringens with SFP agar was not significantly higher than with SPS agar with 104 organisms per g, but with 106 organisms per g it yielded significantly higher recoveries than SPS agar. TSN agar yielded significantly lower recoveries at both inoculum levels. SFP agar gave significantly higher recoveries of spores than SPS and TSN agars. Average plate counts of spores in SFP agar were 75% as high as in SPS agar without antibiotics, but only 45% of the spores grew in SPS agar and 25% in TSN agar. TSN agar was the most selective of the three media, but the selectivity of SPS agar approached that of TSN agar under the test conditions. SFP agar, which was the least selective of the media, allowed growth to some extent of nearly all of the facultative anaerobes tested. PMID:4324885

  18. Sporulation, Heat Resistance, and Biological Properties of Clostridium perfringens

    PubMed Central

    Nishida, S.; Seo, N.; Nakagawa, M.

    1969-01-01

    A sporulation medium for 134 Clostridium perfringens strains, including types A, B, C, D, E, and F, was devised according to Grelet's observation that sporulation occurred when cultural environment became limited in any nutritional requirement indispensable for the growth of the organism. Sporulation took place most prominently when 10% cooked-meat broth (pH 7.2) containing 3% Proteose Peptone and 1% glucose was used for the preculture and 2% Poli Peptone medium (pH 7.8) was used for the subculture medium. Sometimes, terminal spores could be observed. A correlation between sporulation and heat resistance was examined by use of C. perfringens strains isolated from samples heated at different temperatures. Almost all strains isolated from unheated samples and from those heated at lower temperatures gave rise to spores in our sporulation medium, but the spores were weakly heat-resistant, whereas strains isolated from samples heated at 100 C for 60 min were highly heat-resistant but sporulated poorly. A majority of these heat-resistant strains were non-gelatinolytic and definitely salicin-fermenting. Images PMID:4304763

  19. Case of Clostridium perfringens bacteremia after routine colonoscopy and polypectomy.

    PubMed

    Kunz, Anjali N; Riera, Diana; Hickey, Patrick

    2009-10-01

    Bacteremia is an uncommon complication after polypectomy and colonoscopy. We report one of the first cases of Clostridium perfringens bacteremia after polypectomy. Our patient was a four years old boy with congenital polyposis, who underwent colonoscopy and polypectomy without complication. Approximately 12h later he developed a fever and tachycardia with no other clinical symptoms. His blood cultures grew out penicillin susceptible C. perfringens and Enterococcus faecalis. He responded to antibiotic therapy and remained clinically asymptomatic for the duration of his course. There are a few reports of bacteremia after routine polypectomy, but no reported cases of C. perfringens bacteremia in the pediatric population. Clostridial sp. bacteremia can be fatal with devastating consequences if appropriate antibiotics and/or surgical debridement are delayed. Polymicrobial infection, as illustrated in our patient, is also common and can be a poor prognostic risk factor. Therefore, for patients with a history of polypectomy and new onset fever, anaerobic infections should be considered and empiric antibiotic therapy should include coverage for these organisms. PMID:19324098

  20. Genotypic investigation of Clostridium difficile in Prince Edward Island.

    PubMed

    Martin, H; Abbott, L P; Low, D E; Willey, B; Mulvey, M; Weese, J Scott

    2008-11-01

    Clostridium difficile is an important cause of disease in Canada; however, little information is available about the disease in the Maritime provinces. The objective of the present study was to characterize C difficile isolates obtained from people hospitalized with C difficile infection in Prince Edward Island. One hundred twenty-six C difficile ELISA toxin-positive stool samples were obtained and cultured using an enrichment protocol. C difficile was isolated from 105 of 126 (83%) samples. Twenty-two different ribotypes were identified. The most common ribotype, ribotype W, was a North American pulsotype 2 (NAP2), toxinotype 0 strain, which represented 18% of isolates. The next most common ribotype was a NAP1, toxinotype III strain, which accounted for 11% of isolates. Ribotype 027/NAP1 only accounted for five (4.7%) isolates. Forty-five per cent of isolates possessed genes encoding production of binary toxin. Three different ribotypes, all NAP1, toxinotype III strains, had a frameshift mutation in the tcdC gene (Delta117), while one isolate (ribotype 078, NAP4, toxinotype V) had a truncating mutation (C184T) in the tcdC gene. PMID:19436570

  1. Clostridium butyricum: from beneficial to a new emerging pathogen.

    PubMed

    Cassir, N; Benamar, S; La Scola, B

    2016-01-01

    Clostridium butyricum, a strictly anaerobic spore-forming bacillus, is a common human and animal gut commensal bacterium, and is also frequently found in the environment. Whereas non-toxigenic strains are currently used as probiotics in Asia, other strains have been implicated in pathological conditions, such as botulism in infants or necrotizing enterocolitis in preterm neonates. In terms of the latter, within the same species, different strains have antagonist effects on the intestinal mucosa. In particular, short-chain fatty acids, which are products of carbohydrate fermentation, have a dose-dependent paradoxical effect. Moreover, toxin genes have been identified by genome sequencing in pathological strains. Asymptomatic carriage of these strains has also been reported. Herein, we provide an overview of the implications of C. butyricum for human health, from the beneficial to the pathogenic. We focus on pathogenic strains associated with the occurrence of necrotizing enterocolitis. We also discuss the need to use complementary microbiological methods, including culture, in order to better assess gut bacterial diversity and identify new emergent enteropathogens at the strain level. PMID:26493849

  2. Heat treatment adaptations in Clostridium perfringens vegetative cells.

    PubMed

    Novak, J S; Tunick, M H; Juneja, V K

    2001-10-01

    Vegetative cells of Clostridium perfringens enterotoxigenic strains NCTC 8679, NCTC 8238. and H6 were grown at 37 degrees C followed by a 60-min exposure to 28 degrees C or 46 degrees C. D10-values, as a measure of thermal resistance at 60 degrees C, were significantly lower for 28 degrees C exposures as compared with cultures given 37 and 46 degrees C exposures. Following refrigeration at 4 degrees C for 24 h, D10-values for the 37 and 46 degrees C samples could not be differentiated from 28 degrees C samples. Western immunoblot analyses of lysates from heat-adapted cells also detected the increased expression of proteins reacting with antiserum directed against the molecular chaperonins from Escherichia coli; GroEL, DnaJ, and the small acid soluble protein from Bacillus subtilis, SspC. Differential scanning calorimetry (DSC) identified thermal transitions corresponding to ribosomal protein denaturations at 72.1 +/- 0.5 degrees C. Any cellular heat adaptations in the DSC profiles were lost following refrigeration for several days to simulate minimally processed food storage conditions. Further analyses of high-speed pellets from crude cell extract fractions using two-dimensional gel electrophoresis detected the differential gene expression of at least four major proteins in heat-adapted vegetative cells of C. perfringens. N-terminal amino acid analyses identified two of the proteins as glyceraldehyde 3-phosphate dehydrogenase and rubrerythrin. Both appear to have roles in this anaerobe under stressful conditions. PMID:11601701

  3. Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials

    PubMed Central

    Barker, Gary C.; Malakar, Pradeep K.; Plowman, June

    2016-01-01

    We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg−1. Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments. PMID:26729721

  4. Clostridium sordellii in a brown bear (Ursus arctos) from Spain.

    PubMed

    Balseiro, Ana; Oleaga, Álvaro; Polledo, Laura; Aduriz, Gorka; Atxaerandio, Raquel; Kortabarria, Nekane; Marín, Juan F García

    2013-10-01

    Clostridium sordellii is found in the environment and occasionally in animal (including human) intestines and may cause myonecrosis and large outbreaks of enterotoxemia. A few cases of fatal clostridial infection in bears (Ursus spp.) have been described worldwide but none attributed to C. sordellii. We describe a fatal case of septicemia caused by C. sordellii in an illegally trapped brown bear (Ursus arctos). At necropsy, acute gangrenous myositis was the primary lesion. Serohemorrhagic edema was observed in the abdominal cavity, thorax, pericardium, and skeletal muscle, mostly affecting femoral, humeral, and scapular muscles. Hemorrhage was observed in the heart, skeletal muscles, stomach, and intestine. Liver, spleen, and kidney appeared with loss of consistency, hemorrhages, and edema. Microscopically, primary lesions were in skeletal muscle, stomach, and small intestine, with gram-positive, clostridial-like bacilli. Biochemical and molecular tests identified C. sordellii in cultures from liver, muscle, and intestine. Sequences showed a homology of >99% with the 16S rRNA gene sequence of C. sordellii. The severity of effects of the C. sordellii infection reveal the importance of this pathogen as a wildlife health risk with conservation concerns, as well as the need to consider possible infection with this pathogen in management actions involving immobilization, stress, or severe muscular activity of wild brown bears. PMID:24502739

  5. Thermostable amylolytic enzymes from a new Clostridium isolate

    SciTech Connect

    Madi, E.; Antranikian, G.; Ohmiya, K.; Gottschalk, G.

    1987-07-01

    A new Clostridium strain was isolated on starch at 60 degrees C. Starch, pullulan, maltotriose, and maltose induced the synthesis of alpha-amylase and pullulanase, while glucose, ribose, fructose, and lactose did not. The formation of the amylolytic enzymes was dependent on growth and occurred predominantly in the exponential phase. The enzymes were largely cell bound during growth of the organism with 0.5% starch, but an increase of the starch concentration in the growth medium was accompanied by the excretion of alpha-amylase and pullulanase into the culture broth; but also by a decrease of total activity. Alpha-amylase, pullulanase, and alpha-glucosidase were active in a broad temperature range (40 to 85 degrees C) and displayed temperature optima for activity at 60 to 70 degrees C. During incubation with starch under aerobic conditions at 75 degrees C for 2 hours, the activity of both enzymes decreased to only 90 or 80%. The apparent Km values of alpha-amylase, pullulanase, and alpha-glucosidase for their corresponding substrates, starch, pullulan, and maltose were 0.35 mg/ml, 0.63 mg/ml, and 25 mM, respectively. (Refs. 31).

  6. Prospective assessment of two-stage testing for Clostridium difficile.

    PubMed

    Arnold, A; Pope, C; Bray, S; Riley, P; Breathnach, A; Krishna, S; Planche, T

    2010-09-01

    Commonly used immunoassays have limitations as stand-alone tests for the diagnosis of Clostridium difficile infection (CDI). In particular, the specificity of these assays means that these tests generate a relatively large number of false-positive results. We introduced a two-stage regimen for CDI as routine. Unformed stool samples received in our laboratory were initially tested with a Meridian Premier enzyme immunoassay (EIA) and positive samples were retested with reference testing methods (toxigenic culture and cell cytotoxicity assay). Clinicians received diagnostically useful information on the day that the sample arrived in the laboratory, with definitive negative and provisional positive results made available. We reviewed the first 3643 unformed stool specimens of which 158/3643 (4.3%) were provisionally positive by EIA. Of the 158 samples that were EIA positive, 119 were confirmed as being positive by at least one of the reference methods, giving a positive predictive value in this population of 75% (95% confidence interval: 67.6-81.7%). Comparison of the optical density values of the EIA lying between true and false-positive results suggests that the introduction of a second cut-off value would improve diagnostics. A test with two cut-offs would give the following results: 'positive', 'negative' and 'indeterminate result, please perform confirmatory test'. This algorithm was a simple and cost-effective method to immediately improve diagnostics, but there is an urgent need for further research in laboratory diagnosis for CDI. PMID:20638749

  7. Quantification of Nonproteolytic Clostridium botulinum Spore Loads in Food Materials.

    PubMed

    Barker, Gary C; Malakar, Pradeep K; Plowman, June; Peck, Michael W

    2016-01-01

    We have produced data and developed analysis to build representations for the concentration of spores of nonproteolytic Clostridium botulinum in materials that are used during the manufacture of minimally processed chilled foods in the United Kingdom. Food materials are categorized into homogenous groups which include meat, fish, shellfish, cereals, fresh plant material, dairy liquid, dairy nonliquid, mushroom and fungi, and dried herbs and spices. Models are constructed in a Bayesian framework and represent a combination of information from a literature survey of spore loads from positive-control experiments that establish a detection limit and from dedicated microbiological tests for real food materials. The detection of nonproteolytic C. botulinum employed an optimized protocol that combines selective enrichment culture with multiplex PCR, and the majority of tests on food materials were negative. Posterior beliefs about spore loads center on a concentration range of 1 to 10 spores kg(-1). Posterior beliefs for larger spore loads were most significant for dried herbs and spices and were most sensitive to the detailed results from control experiments. Probability distributions for spore loads are represented in a convenient form that can be used for numerical analysis and risk assessments. PMID:26729721

  8. Clostridium novyi, sordellii, and tetani: mechanisms of disease.

    PubMed

    Aronoff, David M

    2013-12-01

    Clostridia represent a diverse group of spore-forming gram positive anaerobes that include several pathogenic species. In general, diseases caused by clostridia are a result of intoxication of the infected host. Thus, clostridial toxins have been targeted for diagnostic, therapeutic, and preventive strategies against infection. Studying the mechanisms of action of clostridial toxins has not only shed light on the pathogenesis of infection but has provided important new insights into cell biology and immunology. A primary purpose of this manuscript is to provide a succinct review on the mechanisms of disease caused by intoxication by the pathogens Clostridium tetani, Clostridium novyi, and Clostridium sordellii. PMID:24036420

  9. Clostridium to treat cancer: dream or reality?

    PubMed Central

    Lambin, Philippe

    2015-01-01

    In their paper “Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses”, Roberts et al. describe the induction of antitumor responses following local spore administration of an attenuated C. novyi strain (C. novyi-NT). Stereotactic intratumoral spore injection led to significant survival advantages in a murine orthotopic brain model and local bacterial treatment produced robust responses in a set of spontaneous canine soft tissue carcinomas. Their preclinical findings in both models, provided the basis for a phase 1 investigational clinical study in patients with solid tumors that were either refractory to standard treatment or without an available standard treatment available (NCT01924689). The results of the first patient enrolled in this trial, a 53-year-old female with a retroperitoneal leiomyosarcoma, are described. Next to the non-armed C. novyi-NT described in this paper, very potent genetically modified Clostridium expressing anti-cancer therapeutic genes are also being developed. Are treatments with these non-pathogenic clostridia a viable alternative cancer treatment? PMID:26046067

  10. Secretion of clostridium cellulase by E. coli

    DOEpatents

    Yu, Ida Kuo

    1998-01-01

    A gene, encoding an endocellulase from a newly isolated mesophilic Clostridium strain IY-2 which can digest bamboo fibers, cellulose, rice straw, and sawdust, was isolated by shotgun cloning in an E. coli expression plasmid pLC2833. E. coli positive clones were selected based on their ability to hydrolyze milled bamboo fibers and cellulose present in agar plates. One clone contained a 2.8 kb DNA fragment that was responsible for cellulase activity. Western blot analyses indicated that the positive clone produced a secreted cellulase with a mass of about 58,000 daltons that was identical in size to the subunit of one of the three major Clostridium cellulases. The products of cellulose digestion by this cloned cellulase were cellotetraose and soluble higher polymers. The cloned DNA contained signal sequences capable of directing the secretion of heterologous proteins from an E. coli host. The invention describes a bioprocess for the treatment of cellulosic plant materials to produce cellular growth substrates and fermentation end products suitable for production of liquid fuels, solvents, and acids.

  11. Effective detection of toxigenic Clostridium difficile by a two-step algorithm including tests for antigen and cytotoxin.

    PubMed

    Ticehurst, John R; Aird, Deborah Z; Dam, Lisa M; Borek, Anita P; Hargrove, John T; Carroll, Karen C

    2006-03-01

    We evaluated a two-step algorithm for detecting toxigenic Clostridium difficile: an enzyme immunoassay for glutamate dehydrogenase antigen (Ag-EIA) and then, for antigen-positive specimens, a concurrent cell culture cytotoxicity neutralization assay (CCNA). Antigen-negative results were > or = 99% predictive of CCNA negativity. Because the Ag-EIA reduced cell culture workload by approximately 75 to 80% and two-step testing was complete in < or = 3 days, we decided that this algorithm would be effective. Over 6 months, our laboratories' expenses were US dollar 143,000 less than if CCNA alone had been performed on all 5,887 specimens. PMID:16517916

  12. Fermentation of cellulose and cellobiose by Clostridium thermocellum in the absence of Methanobacterium thermoautotrophicum.

    PubMed Central

    Weimer, P J; Zeikus, J G

    1977-01-01

    The fermentation of cellulose and cellobiose by Clostridium thermocellum monocultures and C. thermocellum/Methanobacterium thermoautotrophicum cocultures was studied. All cultures were grown under anaerobic conditions in batch culture at 60 degrees C. When grown on cellulose, the coculture exhibited a shorter lag before initiation and growth and celluloysis than did the monoculture. Cellulase activity appeared earlier in the coculture than in the monoculture; however, after growth had ceased, cellulase activity was greater in the monoculture. Monocultures produced primarily ethanol, acetic acid, H2 and CO2. Cocultures produced more H2 and acetic acid and less ethanol than did the monoculture. In the coculture, conversion of H2 to methane was usually complete, and most of the methane produced was derived from CO2 reduction rather than from acetate conversion. Agents of fermentation stoppage were found to be low pH and high concentrations of ethanol in the monoculture and low pH in the coculture. Fermentation of cellobiose was more rapid than that of cellulose. In cellobiose medium, the methanogen caused only slight changes in the fermentation balance of the Clostridium, and free H2 was produced. PMID:848953

  13. Microbiology of Wetwood: Importance of Pectin Degradation and Clostridium Species in Living Trees

    PubMed Central

    Schink, Bernhard; Ward, James C.; Zeikus, J. Gregory

    1981-01-01

    Wetwood samples from standing trees of eastern cottonwood (Populus deltoides), black poplar (Populus nigra), and American elm (Ulmus americana) contained high numbers of aerobic and anaerobic pectin-degrading bacteria (104 to 106 cells per g of wood). High activity of polygalacturonate lyase (≤0.5 U/ml) was also detected in the fetid liquid that spurted from wetwood zones in the lower trunk when the trees were bored. A prevalent pectin-degrading obligately anaerobic bacterium isolated from these wetwoods was identified as Clostridium butyricum. Pectin decomposition by C. butyricum strain 4P1 was associated with an inducible polygalacturonate lyase and pectin methylesterase, the same types of pectinolytic activity expressed in the wetwood of these trees. The pH optimum of the extracellular polygalacturonate lyase was alkaline (near pH 8.5). In vitro tests with sapwood samples from a conifer (Douglas fir, Pseudotsuga menziesii) showed that tori in membranes of bordered pits are degraded by pure cultures of strain 4P1, polygalacturonate lyase enzyme preparations of strain 4P1, and mixed methanogenic cultures from the tree samples of wetwood. These results provide evidence that pectin in xylem tissue is actively degraded by C. butyricum strain 4P1 via polygalacturonate lyase activity. The importance of pectin degradation by bacteria, including Clostridium species, appears paramount in the formation and maintenance of the wetwood syndrome in certain living trees. Images PMID:16345848

  14. Conditions associated with Clostridium sporogenes growth as a surrogate for Clostridium botulinum in nonthermally processed canned butter.

    PubMed

    Taylor, R H; Dunn, M L; Ogden, L V; Jefferies, L K; Eggett, D L; Steele, F M

    2013-05-01

    The objective of this study was to better understand the effect of butter composition and emulsion structure on growth and survival of Clostridium sporogenes, used as a surrogate for C. botulinum in canned butter. The lack of a thermal process step in commercially available canned butter raises questions of potential safety, because it is hermetically sealed and generally exhibits anaerobic growth conditions, which are optimal for Clostridium botulinum growth. Without thermal processing, low-acid canned foods must have inhibitory factors present to prevent C. botulinum growth. Some potential intrinsic inhibitory factors, or hurdles, within butter include: reduced water activity, acidity in cultured products, elevated salt content, and the micro-droplet nature of the aqueous phase in the butter emulsion. It was hypothesized that a normal, intact butter emulsion would have sufficient hurdles to prevent C. botulinum growth, whereas a broken butter emulsion would result in a coalesced aqueous phase that would allow for C. botulinum growth. Batch-churned butter was inoculated with C. sporogenes; butter samples with varying salt contents (0, 0.8, 1.6, and 2.4% wt/wt NaCl) were prepared and stored in coated steel cans for varying times (1 or 2 wk) and temperatures (22 or 41°C) to determine temperature and emulsion structure effects on C. sporogenes growth. Samples stored at 41°C showed a significant increase in C. sporogenes growth compared with those stored at 22°C. Furthermore, NaCl addition was found to have a significant effect on C. sporogenes growth, with 0.8% NaCl promoting more growth than 0%, but with decreases in growth observed at 1.6 and 2.4%. Uninoculated control plates were also found to have bacterial growth; this growth was attributed to other anaerobic bacteria present within the cream. It was concluded that removal of the hurdle created by the micro-droplet size of the emulsion aqueous phase could result in C. botulinum growth even at elevated salt

  15. Flooding and Clostridium difficile infection: a case-crossover analysis

    EPA Science Inventory

    Clostridium difficile is a bacterium that can spread by water. It often causes acute gastrointestinal illness in older adults who are hospttalized and/or receiving antibiotics; however, community­ associated infections affecting otherwise healthy individuals have become more ...

  16. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis

    PubMed Central

    Gohari, Iman Mehdizadeh; Arroyo, Luis; MacInnes, Janet I.; Timoney, John F.; Parreira, Valeria R.; Prescott, John F.

    2014-01-01

    Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in

  17. Characterization of Clostridium perfringens in the feces of adult horses and foals with acute enterocolitis.

    PubMed

    Gohari, Iman Mehdizadeh; Arroyo, Luis; Macinnes, Janet I; Timoney, John F; Parreira, Valeria R; Prescott, John F

    2014-01-01

    Up to 60% of cases of equine colitis have no known cause. To improve understanding of the causes of acute colitis in horses, we hypothesized that Clostridium perfringens producing enterotoxin (CPE) and/or beta2 toxin (CPB2) are common and important causes of severe colitis in horses and/or that C. perfringens producing an as-yet-undescribed cytotoxin may also cause colitis in horses. Fecal samples from 55 horses (43 adults, 12 foals) with clinical evidence of colitis were evaluated by culture for the presence of Clostridium difficile, C. perfringens, and Salmonella. Feces were also examined by enzyme-linked immunosorbent assay (ELISA) for C. difficile A/B toxins and C. perfringens alpha toxin (CPA), beta2 toxin (CPB2), and enterotoxin (CPE). Five C. perfringens isolates per sample were genotyped for the following genes: cpa, cpb, cpb2 consensus, cpb2 atypical, cpe (enterotoxin), etx (epsilon toxin), itx (iota toxin), netB (necrotic enteritis toxin B), and tpeL (large C. perfringens cytotoxin). The supernatants of these isolates were also evaluated for toxicity for an equine cell line. All fecal samples were negative for Salmonella. Clostridium perfringens and C. difficile were isolated from 40% and 5.4% of samples, respectively. All fecal samples were negative for CPE. Clostridium perfringens CPA and CPB2 toxins were detected in 14.5% and 7.2% of fecal samples, respectively, all of which were culture-positive for C. perfringens. No isolates were cpe, etx, netB, or tpeL gene-positive. Atypical cpb2 and consensus cpb2 genes were identified in 15 (13.6%) and 4 (3.6%) of 110 isolates, respectively. All equine C. perfringens isolates showed far milder cytotoxicity effects than a CPB-producing positive control, although cpb2-positive isolates were slightly but significantly more cytotoxic than negative isolates. Based on this studied population, we were unable to confirm our hypothesis that CPE and CPB2-producing C. perfringens are common in horses with colitis in

  18. Methylenetetrahydrofolate dehydrogenase from Clostridium formicoaceticum and methylenetetrahydrofolate dehydrogenase, methenyltetrahydrofolate cyclohydrolase (combined) from Clostridium thermoaceticum

    SciTech Connect

    Ljungdahl, L.G.; O'Brien, W.E.; Moore, M.R.; Liu, M.T.

    1980-01-01

    Methylenetetrahydrofolate dehydrogenase is widely distributed and has been found in every cell type investigated. The NAD-specific enzyme has been purified to homogeneity from Clostridium formicoaceticum and the NADP-specific enzyme has been obtained from Clostridium thermoaceticum. Other sources of the NADP-specific enzyme are Streptococcus species, Escherichia coli, Clostridium cylindrosporum, Salmonella typhimurium, yeast, liver from various animals, calf thymus, and plants. The NAD-specific enzyme has been demonstrated in Acetobacterium woodii, some methane bacteria, and in Ehrlich ascites tumor cells. Of considerable interest are the observations that in porcine and ovine livers, as well as in yeast, methylenetetrahydrofolate dehydrogenase purified to homogeneity also contains methylenetetrahydrofolate cyclohydrolase and formyltetrahydrofolate synthetase activities. Now it appears that the purified methylenetetrahydrofolate dehydrogenase from C. thermoaceticum also has cyclohydrolase but not synthetase activity. Methylenetetrahydrofolate dehydrogenase has been discussed previously in this series, as has methenyltetrahydrofolate cyclohydrolase. In C. formicoaceticum and C. thermoaceticum these tetrahydrofolate-dependent enzymes participate in a sequence of metabolic reactions by which carbon dioxide is reduced to the methyl group of 5-methyltetrahydrofolate which in turn is utilized for the synthesis of acetate. This pathway provides the mechanism for disposing of reducing equivalents generated in glycolysis.

  19. Small RNAs in the Genus Clostridium

    PubMed Central

    Chen, Yili; Indurthi, Dinesh C.; Jones, Shawn W.; Papoutsakis, Eleftherios T.

    2011-01-01

    The genus Clostridium includes major human pathogens and species important to cellulose degradation, the carbon cycle, and biotechnology. Small RNAs (sRNAs) are emerging as crucial regulatory molecules in all organisms, but they have not been investigated in clostridia. Research on sRNAs in clostridia is hindered by the absence of a systematic method to identify sRNA candidates, thus delegating clostridial sRNA research to a hit-and-miss process. Thus, we wanted to develop a method to identify potential sRNAs in the Clostridium genus to open up the field of sRNA research in clostridia. Using comparative genomics analyses combined with predictions of rho-independent terminators and promoters, we predicted sRNAs in 21 clostridial genomes: Clostridium acetobutylicum, C. beijerinckii, C. botulinum (eight strains), C. cellulolyticum, C. difficile, C. kluyveri (two strains), C. novyi, C. perfringens (three strains), C. phytofermentans, C. tetani, and C. thermocellum. Although more than one-third of predicted sRNAs have Shine-Dalgarno (SD) sequences, only one-sixth have a start codon downstream of SD sequences; thus, most of the predicted sRNAs are noncoding RNAs. Quantitative reverse transcription-PCR (Q-RT-PCR) and Northern analysis were employed to test the presence of a randomly chosen set of sRNAs in C. acetobutylicum and several C. botulinum strains, leading to the confirmation of a large fraction of the tested sRNAs. We identified a conserved, novel sRNA which, together with the downstream gene coding for an ATP-binding cassette (ABC) transporter gene, responds to the antibiotic clindamycin. The number of predicted sRNAs correlated with the physiological function of the species (high for pathogens, low for cellulolytic, and intermediate for solventogenic), but not with 16S rRNA-based phylogeny. PMID:21264064

  20. Butanol Production from Crystalline Cellulose by Cocultured Clostridium thermocellum and Clostridium saccharoperbutylacetonicum N1-4 ▿

    PubMed Central

    Nakayama, Shunichi; Kiyoshi, Keiji; Kadokura, Toshimori; Nakazato, Atsumi

    2011-01-01

    We investigated butanol production from crystalline cellulose by cocultured cellulolytic Clostridium thermocellum and the butanol-producing strain, Clostridium saccharoperbutylacetonicum (strain N1-4). Butanol was produced from Avicel cellulose after it was incubated with C. thermocellum for at least 24 h at 60°C before the addition of strain N1-4. Butanol produced by strain N1-4 on 4% Avicel cellulose peaked (7.9 g/liter) after 9 days of incubation at 30°C, and acetone was undetectable in this coculture system. Less butanol was produced by cocultured Clostridium acetobutylicum and Clostridium beijerinckii than by strain N1-4, indicating that strain N1-4 was the optimal strain for producing butanol from crystalline cellulose in this coculture system. PMID:21764954

  1. Improved Medium for Enumeration of Clostridium perfringens

    PubMed Central

    Harmon, Stanley M.; Kautter, Donald A.; Peeler, James T.

    1971-01-01

    An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 μg of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective. PMID:4331774

  2. Improved medium for enumeration of Clostridium perfringens.

    PubMed

    Harmon, S M; Kautter, D A; Peeler, J T

    1971-10-01

    An improved selective medium, Tryptose-sulfite-cycloserine (TSC) agar, for the enumeration of Clostridium perfringens is described. It consists of the same basal medium as Shahidi-Ferguson-perfringens (SFP) agar, but with 400 mug of D-cycloserine per ml substituted for the kanamycin and polymyxin. Tolerance of C. perfringens for D-cycloserine, its production of lecithinase, and its ability to reduce sulfite were used as the basis for development of this medium. Comparisons were made between TSC and SFP agars for the recovery of vegetative cells of C. perfringens by using statistical methods. The results showed that TSC allowed virtually complete recovery of most of the C. perfringens strains while inhibiting practically all facultative anaerobes tested. SFP agar allowed a slightly higher rate of recovery of C. perfringens but was found to be much less selective. PMID:4331774

  3. Isolating and Purifying Clostridium difficile Spores.

    PubMed

    Edwards, Adrianne N; McBride, Shonna M

    2016-01-01

    The ability for the obligate anaerobe, Clostridium difficile to form a metabolically dormant spore is critical for the survival of this organism outside of the host. This spore form is resistant to a myriad of environmental stresses, including heat, desiccation, and exposure to disinfectants and antimicrobials. These intrinsic properties of spores allow C. difficile to survive long-term in an oxygenated environment, to be easily transmitted from host-to-host, and to persist within the host following antibiotic treatment. Because of the importance of the spore form to the C. difficile life cycle and treatment and prevention of C. difficile infection (CDI), the isolation and purification of spores are necessary to study the mechanisms of sporulation and germination, investigate spore properties and resistances, and for use in animal models of CDI. Here we provide basic protocols, in vitro growth conditions, and additional considerations for purifying C. difficile spores for a variety of downstream applications. PMID:27507337

  4. Quantitation of Clostridium perfringens in foods.

    PubMed

    ANGELOTTI, R; HALL, H E; FOTER, M J; LEWIS, K H

    1962-05-01

    A procedure is described for identifying and enumerating Clostridium perfringens in foods by means of a simplified agar plating method, followed by confirmation of black colonies in tubes of motility-nitrate medium and sporulation broth. The test is routinely completed within 48 hr. Under experimental conditions, the procedure has been used to quantitatively recover various levels of C. perfringens contamination in a variety of foods and has recovered as few as ten C. perfringens per g without interference from food constituents and associated flora. Under practical conditions of field application, the method has been used to investigate five food-poisoning outbreaks, and C. perfringens was implicated as the etiological agent in two of these outbreaks. PMID:13861594

  5. Carbohydrate-based Clostridium difficile vaccines.

    PubMed

    Monteiro, Mario A; Ma, Zuchao; Bertolo, Lisa; Jiao, Yuening; Arroyo, Luis; Hodgins, Douglas; Mallozzi, Michael; Vedantam, Gayatri; Sagermann, Martin; Sundsmo, John; Chow, Herbert

    2013-04-01

    Clostridium difficile is responsible for thousands of deaths each year and a vaccine would be welcomed, especially one that would disrupt bacterial maintenance, colonization and persistence in carriers and convalescent patients. Structural explorations at the University of Guelph (ON, Canada) discovered that C. difficile may express three phosphorylated polysaccharides, named PSI, PSII and PSIII; this review captures our recent efforts to create vaccines based on these glycans, especially PSII, the common antigen that has precipitated immediate attention. The authors describe the design and immunogenicity of vaccines composed of raw polysaccharides and conjugates thereof. So far, it has been observed that anti-PSII antibodies can be raised in farm animals, mice and hamster models; humans and horses carry anti-PSII IgA and IgG antibodies from natural exposure to C. difficile, respectively; phosphate is an indispensable immunogenic epitope and vaccine-induced PSII antibodies recognize PSII on C. difficile outer surface. PMID:23560922

  6. [New aspects on Clostridium difficile infection].

    PubMed

    von Müller, Lutz

    2016-08-01

    Clostridium difficile infection (CDI) is a frequent and complex disease which is influenced by the repertoire of bacterial virulence factors, by host immunity and by the intestinal microbiome. These complex interaction opens a number of options which may be used for treatment in the future. One example for new treatment options is fecal microbiota transplantation (FMT). Driven by C. difficile related research activities the knowledge of protective microorganism is increasing and it may be assumed that bacteriotherapy by next-generation probiotics may be used very soon also for other diseases. Very often, CDI reflects to the clinician that antibiotic therapy is associated with side effects. Therefore, C. difficile is the guilty conscience which helps to implement targeted and restrictive antibiotic use in the daily practice. PMID:27509341

  7. Biotechnological potential of Clostridium butyricum bacteria

    PubMed Central

    Szymanowska-Powałowska, Daria; Orczyk, Dorota; Leja, Katarzyna

    2014-01-01

    In response to demand from industry for microorganisms with auspicious biotechnological potential, a worldwide interest has developed in bacteria and fungi isolation. Microorganisms of interesting metabolic properties include non-pathogenic bacteria of the genus Clostridium, particularly C. acetobutylicum, C. butyricum and C. pasteurianum. A well-known property of C. butyricum is their ability to produce butyric acid, as well as effectively convert glycerol to 1,3-propanediol (38.2 g/L). A conversion rate of 0.66 mol 1,3-propanediol/mol of glycerol has been obtained. Results of the studies described in the present paper broaden our knowledge of characteristic features of C. butyricum specific isolates in terms of their phylogenetic affiliation, fermentation capacity and antibacterial properties. PMID:25477923

  8. Current State of Clostridium difficile Treatment Options

    PubMed Central

    Venugopal, Anilrudh A.; Johnson, Stuart

    2012-01-01

    Recent reports of reduced response to standard therapies for Clostridium difficile infection (CDI) and the risk for recurrent CDI that is common with all currently available treatment agents have posed a significant challenge to clinicians. Current recommendations include metronidazole for treatment of mild to moderate CDI and vancomycin for severe CDI. Results from small clinical trials suggest that nitazoxanide and teicoplanin may be alternative options to standard therapies, whereas rifaximin has demonstrated success in uncontrolled trials for the management of multiple recurrences. Anecdotal reports have also suggested that tigecycline might be useful as an adjunctive agent for the treatment of severe complicated CDI. Reports of resistance will likely limit the clinical use of fusidic acid and bacitracin and, possibly, rifaximin if resistance to this agent becomes widespread. Treatment of patients with multiple CDI recurrences and those with severe complicated CDI is based on limited clinical evidence, and new treatments or strategies are needed. PMID:22752868

  9. SYBR Green Real-Time PCR Method To Detect Clostridium botulinum Type A▿

    PubMed Central

    Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

    2007-01-01

    Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 101 copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

  10. SYBR green real-time PCR method to detect Clostridium botulinum type A.

    PubMed

    Fenicia, Lucia; Anniballi, Fabrizio; De Medici, Dario; Delibato, Elisabetta; Aureli, Paolo

    2007-05-01

    Botulinum toxins (BoNTs) are classically produced by Clostridium botulinum but rarely also from neurotoxigenic strains of Clostridium baratii and Clostridium butyricum. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of C. botulinum. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting C. botulinum type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of C. botulinum type A, 4 strains of C. botulinum type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 x 10(1) copies of C. botulinum type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of C. botulinum types A and Ab (inclusivity and exclusivity, 100%). PMID:17369349

  11. Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks

    PubMed Central

    Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

    2015-01-01

    There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium. PMID:26584048

  12. Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

    PubMed

    Irikura, Daisuke; Monma, Chie; Suzuki, Yasunori; Nakama, Akiko; Kai, Akemi; Fukui-Miyazaki, Aya; Horiguchi, Yasuhiko; Yoshinari, Tomoya; Sugita-Konishi, Yoshiko; Kamata, Yoichi

    2015-01-01

    There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins' gene(s) among the Genus Clostridium. PMID:26584048

  13. Purification and characterization of Clostridium difficile toxin.

    PubMed Central

    Rolfe, R D; Finegold, S M

    1979-01-01

    Recent evidence indicates that toxigenic Clostridium difficile strains are a major cause of antimicrobial-associated ileocecitis in laboratory animals and pseudomembranous colitis in humans. C. difficile ATCC 9689 was cultivated in a synthetic medium to which 3% ultrafiltrated proteose peptone was added. Purification of the toxin from broth filtrate was accomplished through ultrafiltration (100,000 nominal-molecular-weight-limit membrane), precipitation with 75% (NH4)2SO4, and chromatographic separation using Bio-Gel A 5m followed by ion-exchange chromatography on a diethylaminoethyl-Sephadex A-25 column. The purified toxin displayed only one band on polyacrylamide gel electrophoresis, and approximately 170 pg was cytopathic for human amnion cells. The isolated toxin was neutralized by Clostridium sordelli antitoxin, heat labile (56 degrees C for 30 min), and inactivated at pH 4 and 9; it had an isoelectric point of 5.0, increased vascular permeability in rabbits, and caused ileocecitis in hamsters when injected intracecally. Treatment of the toxin with trypsin, chymotrypsin, pronase, amylase, or ethylmercurithiosalicylate caused inactivation, whereas lipase had no effect. By gel filtration, its molecular weight was estimated as 530,000. Upon reduction and denaturation, the toxin dissociated into 185,000- and 50,000-molecular-weight components, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Extensive dissociation yielded only the 50,000-molecular-weight component. The toxin appears to be protoplasmic and is released into the surrounding environment upon autolysis of the cells. Attempts to correlate specific enzymatic activity with the toxin have been unsuccessful. These studies will help delineate the role of C. difficile toxin in antimicrobial-associated colitis and diarrhea. Images PMID:478634

  14. Improved Medium for Sporulation of Clostridium perfringens1

    PubMed Central

    Duncan, Charles L.; Strong, Dorothy H.

    1968-01-01

    An improved sporulation medium has been developed in which all five strains of Clostridium perfringens tested exhibited a 100- to 10,000-fold increase in numbers of spores when compared with spore yields in SEC medium under comparable conditions. In addition, three of five strains produced a 100- to 1,000-fold increase, with the remaining two strains yielding approximately the same numbers of spores, when compared with strains cultured in Ellner medium. At the 40-hr sampling time, 18 of 27 strains produced a 10- to 100-fold increase in numbers of spores in our medium, when compared to spore production obtained in a medium recently reported by Kim et al. The new medium contained yeast extract, 0.4%; proteose peptone, 1.5%; soluble starch, 0.4%; sodium thioglycolate, 0.1%; and Na2HPO4. 7H2O, 1.0%. In some cases, the spore yield could be increased by the addition of activated carbon to the new medium. The inclusion of activated carbon in the medium resulted in spores with slightly greater heat resistance than spores produced in the new medium without added carbon or in SEC or in Ellner medium. The major differences in heat resistance of the various strains appeared to be genetically determined rather than reflections of a particular sporulation medium. A definite heat-shock requirement was shown for four of four strains, with the optimal temperature ranging from 60 C for a heat-sensitive strain to 80 C for a heat-resistant strain. Heating for 20 min at the optimal temperature resulted in a 100-fold increase over the viable count obtained after heating for 20 min at 50 C. PMID:4295179

  15. Ultrastructural Variability of the Exosporium Layer of Clostridium difficile Spores.

    PubMed

    Pizarro-Guajardo, Marjorie; Calderón-Romero, Paulina; Castro-Córdova, Pablo; Mora-Uribe, Paola; Paredes-Sabja, Daniel

    2016-01-01

    The anaerobic sporeformer Clostridium difficile is the leading cause of nosocomial antibiotic-associated diarrhea in developed and developing countries. The metabolically dormant spore form is considered the transmission, infectious, and persistent morphotype, and the outermost exosporium layer is likely to play a major role in spore-host interactions during the first contact of C. difficile spores with the host and for spore persistence during recurrent episodes of infection. Although some studies on the biology of the exosporium have been conducted (J. Barra-Carrasco et al., J Bacteriol 195:3863-3875, 2013, http://dx.doi.org/10.1128/JB.00369-13; J. Phetcharaburanin et al., Mol Microbiol 92:1025-1038, 2014, http://dx.doi.org/10.1111/mmi.12611), there is a lack of information on the ultrastructural variability and stability of this layer. In this work, using transmission electron micrographs, we analyzed the variability of the spore's outermost layers in various strains and found distinctive variability in the ultrastructural morphotype of the exosporium within and between strains. Through transmission electron micrographs, we observed that although this layer was stable during spore purification, it was partially lost after 6 months of storage at room temperature. These observations were confirmed by indirect immunofluorescence microscopy, where a significant decrease in the levels of two exosporium markers, the N-terminal domain of BclA1 and CdeC, was observed. It is also noteworthy that the presence of the exosporium marker CdeC on spores obtained from C. difficile biofilms depended on the biofilm culture conditions and the strain used. Collectively, these results provide information on the heterogeneity and stability of the exosporium surface of C. difficile spores. These findings have direct implications and should be considered in the development of novel methods to diagnose and/or remove C. difficile spores by using exosporium proteins as targets. PMID

  16. Clostridium clariflavum: Key Cellulosome Players Are Revealed by Proteomic Analysis

    PubMed Central

    Artzi, Lior; Morag, Ely; Barak, Yoav; Lamed, Raphael

    2015-01-01

    ABSTRACT Clostridium clariflavum is an anaerobic, cellulosome-forming thermophile, containing in its genome genes for a large number of cellulosomal enzyme and a complex scaffoldin system. Previously, we described the major cohesin-dockerin interactions of the cellulosome components, and on this basis a model of diverse cellulosome assemblies was derived. In this work, we cultivated C. clariflavum on cellobiose-, microcrystalline cellulose-, and switchgrass-containing media and isolated cell-free cellulosome complexes from each culture. Gel filtration separation of the cellulosome samples revealed two major fractions, which were analyzed by label-free liquid chromatography-tandem mass spectrometry (LC-MS/MS) in order to identify the key players of the cellulosome assemblies therein. From the 13 scaffoldins present in the C. clariflavum genome, 11 were identified, and a variety of enzymes from different glycoside hydrolase and carbohydrate esterase families were identified, including the glycoside hydrolase families GH48, GH9, GH5, GH30, GH11, and GH10. The expression level of the cellulosomal proteins varied as a function of the carbon source used for cultivation of the bacterium. In addition, the catalytic activity of each cellulosome was examined on different cellulosic substrates, xylan and switchgrass. The cellulosome isolated from the microcrystalline cellulose-containing medium was the most active of all the cellulosomes that were tested. The results suggest that the expression of the cellulosome proteins is regulated by the type of substrate in the growth medium. Moreover, both cell-free and cell-bound cellulosome complexes were produced which together may degrade the substrate in a synergistic manner. These observations are compatible with our previously published model of cellulosome assemblies in this bacterium. PMID:25991683

  17. Hydrolyzable and condensed tannins resistance in Clostridium perfringens.

    PubMed

    Redondo, L M; Dominguez, J E; Rabinovitz, B C; Redondo, E A; Fernández Miyakawa, M E

    2015-08-01

    Tannins added in the diet are being used to improve nutrition and health in farm animals as an alternative to antibiotic growth promoters and to control enteric clostridial diseases. However, the capacity of Clostridium perfringens to develop resistance under the selective pressure of tannins is unknown. The purpose of this study was to determine if C. perfringens possess the ability to develop resistance against tannins in comparison with antimicrobial agents. Susceptibility for 7 AGPs (antimicrobial growth promoters), 9 therapeutic antimicrobials and 2 tannin based extracts was determined for 30 C. perfringens strains isolated from poultry and cattle. Two susceptible strains were selected and cultured in presence of sub-inhibitory concentrations of tannins and AGPs for resistant sub-populations selection. Tannin resistance of C. perfringens isolates from both animal species revealed no statistically significant differences in MICs (minimum inhibitory concentration). Poultry isolates showed higher MICs to several AGPs compared with cattle isolates. All isolates were susceptible to the therapeutic antimicrobials tested, but avian isolates showed a significantly lower susceptibility to these antimicrobials which was highly correlated with an increased resistance to bacitracin and others AGPs. In-vitro selection of resistant clones suggests that C. perfringens was unable to develop resistance against tannins at least compared to AGPs like bacitracin and avilamycin. Avian origin strains, which were previously exposed to antibiotics showed higher resistance, compared to cattle origin strains. These results suggest that the evolution of resistance against tannins in C. perfringens would be more difficult and slower than to the determined AGPs. PMID:26037239

  18. Quantitative Detection of Clostridium perfringens in the Broiler Fowl Gastrointestinal Tract by Real-Time PCR

    PubMed Central

    Wise, Mark G.; Siragusa, Gregory R.

    2005-01-01

    Strains of Clostridium perfringens are a frequent cause of food-borne disease and gas gangrene and are also associated with necrotic enteritis in chickens. To detect and quantify the levels of C. perfringens in the chicken gastrointestinal tract, a quantitative real-time PCR assay utilizing a fluorogenic, hydrolysis-type probe was developed and utilized to assay material retrieved from the broiler chicken cecum and ileum. Primers and probe were selected following an alignment of 16S rDNA sequences from members of cluster I of the genus Clostridium, and proved to be specific for C. perfringens. The assay could detect approximately 50 fg of C. perfringens genomic DNA and approximately 20 cells in pure culture. Measurements of the analytical sensitivity determined with spiked intestinal contents indicated that the consistent limit of detection with ileal samples was approximately 102 CFU/g of ileal material, but only about 104 CFU/g of cecal samples. The decreased sensitivity with the cecal samples was due to the presence of an unidentified chemical PCR inhibitor(s) in the cecal DNA purifications. The assay was utilized to rapidly detect and quantify C. perfringens levels in the gut tract of broiler chickens reared without supplementary growth-promoting antibiotics that manifested symptoms of necrotic enteritis. The results illustrated that quantitative real-time PCR correlates well with quantification via standard plate counts in samples taken from the ileal region of the gastrointestinal tract. PMID:16000804

  19. Enhanced butanol production in a microbial electrolysis cell by Clostridium beijerinckii IB4.

    PubMed

    He, Ai-Yong; Yin, Chun-Yan; Xu, Hao; Kong, Xiang-Ping; Xue, Jia-Wei; Zhu, Jing; Jiang, Min; Wu, Hao

    2016-02-01

    Reducing power such as NADH is an essential factor for acetone/butanol/ethanol (ABE) fermentation using Clostridium spp. The objective of this study was to increase available NADH in Clostridium beijerinckii IB4 by a microbial electrolysis cell (MEC) with an electron carrier to enhance butanol production. First of all, a MEC was performed without electron carrier to study the function of cathodic potential applying. Then, various electron carriers were tested, and neutral red (NR)-amended cultures showed an increase of butanol concentration. Optimal NR concentration (0.1 mM) was used to add in a MEC. Electricity stimulated the cell growth obviously and dramatically diminished the fermentation time from 40 to 28 h. NR and electrically reduced NR improved the final butanol concentration and inhibited the acetone generation. In the MEC with NR, the butanol concentration, yield, proportion and productivity were increased by 12.2, 17.4, 7.2 and 60.3 %, respectively. To further understand the mechanisms of NR, cathodic potential applying and electrically reduced NR, NADH and NAD(+) levels, ATP levels and hydrogen production were determined. NR and electrically reduced NR also improved ATP levels and the ratio of NADH/NAD(+), whereas they decreased hydrogen production. Thus, the MEC is an efficient method for enhancing the butanol production. PMID:26615415

  20. Rapid glutamic acid decarboxylase test for identification of Bacteroides and Clostridium spp.

    PubMed Central

    Jilly, B J; Schreckenberger, P C; LeBeau, L J

    1984-01-01

    A rapid 4-h test for glutamic acid decarboxylase is described for the identification of certain anaerobic bacteria. The test substrate consisted of 1.0 g of L-glutamic acid, 0.3 ml of Triton X-155, and 0.05 g of bromcresol green sodium salt in 1 liter of water. The substrate was dispensed in 0.5-ml amounts into test tubes, and a turbid suspension was made with the test organism. The test was then incubated aerobically at 35 degrees C for 4 h. The development of a blue color was considered positive. A total of 345 strains of clinically isolated anaerobic bacteria were tested. All isolates of Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides uniformis. Clostridium perfringens, and Clostridium sordellii gave a positive reaction. Some isolates of Bacteroides distasonis and Bacteroides vulgatus were also positive. The use of this rapid test in conjunction with other rapid methods, such as the spot indol test, will enable laboratory workers to report these pathogens on the same day on which an inoculum of pure culture growth on agar is available. PMID:6376535

  1. Genotyping of Clostridium perfringens isolated from broiler meat in northeastern of Iran

    PubMed Central

    Afshari, Asma; Jamshidi, Abdollah; Razmyar, Jamshid; Rad, Mehrnaz

    2015-01-01

    Clostridium perfringens (C. perfringens) is an important cause of bacterial food poisoning worldwide. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe gene. The aim of this research was to identify the different types of C. perfringens and the presence of cpe gene in isolated bacteria from broilers’ meat marketed in retail meat shops of Mashhad city in Northeastern of Iran. After isolation of C. perfringens using conventional culture method and confirmation by specific 16S rDNA gene, a multiplex polymerase chain reaction assay with specific primers, were performed for toxin typing of isolates. Clostridium perfringens was isolated from 31 broilers’ meat samples (15.50%) out of 200 samples and for toxin typing the results showed 9 isolates as type A (29.03%) and 22 isolates as type C (70.96%). In this study, cpe-positive C. perfringens were detected in eight isolates of type C (25.00%). Our results indicated that C. perfringens type C is the most common type in broiler chicken carcasses. PMID:26973762

  2. Purification and characterization of a GH11 xylanase from biobutanol-producing Clostridium beijerinckii G117.

    PubMed

    Ng, Choong Hey; He, Jianzhong; Yang, Kun-Lin

    2015-03-01

    Most biobutanol-producing Clostridium strains are unable to ferment polysaccharides such as cellulose and xylan due to the lack of hydrolyzing enzymes. In this study, we show that Clostridium beijerinckii G117, a newly isolated biobutanol-producing strain, expresses xylanase enzyme in the presence of 1% beechwood xylan. The xylanase activity in the medium containing actively growing culture and 1% of beechwood xylan can reach up to 2.66 U/ml after 14 h of fermentation. Using salting-out and size-exclusion chromatography, we purify the crude xylanase by 8.7-fold from the supernatant with a yield of 32.2%. This purified xylanase has a molecular weight of 22.6 kDa, making it one of the smallest reported clostridial xylanases. Conserved domain analysis reveals that the xylanase belongs to glycoside hydrolase family 11 (GH11) but lacks a carbohydrate binding domain. When beechwood xylan is used as substrate for the xylanase, majority of the products are xylo-oligosaccharide (~98%), suggesting that this is an endo-1,4-β-xylanase. PMID:25564206

  3. Spontaneous Occurrence of Gangrene Due to Clostridium septicum in a Patient With Advanced Endometrial Carcinoma

    PubMed Central

    Ghosh, Kris; Sutton, Gregory P.

    1994-01-01

    Background: We report the first known case of spontaneous, atraumatic Clostridium septicum gangrene occurring in a patient with recurrent endometrial adenocarcinoma. Case: A 63-year-old white female undergoing chemotherapy for recurrent endometrial adenocarcinoma presented with right “arthritis-like” shoulder pain. She denied fever, chills, or shoulder trauma. The patient was afebrile and her blood pressure was 100/50. Her right shoulder and upper extremity were remarkable for an area of dark blue discoloration with crepitus. The white blood cell (WBC) count was 8,200/μl with left shift. Serum creatinine, platelet count, and coagulation studies were normal. Computed tomography revealed gas in the right shoulder tissues. A Gram stain of fluid aspirated from the shoulder demonstrated gram-positive spore-forming rods. She declined surgical intervention and expired within hours of admission. Cultures of the right shoulder eventually grew Clostridium septicum. Conclusion: It is imperative to consider clostridial gangrene in the differential diagnosis for any patient with cancer and a fever of unknown origin. PMID:18475364

  4. A recombinant Bacillus anthracis strain producing the Clostridium perfringens Ib component induces protection against iota toxins.

    PubMed Central

    Sirard, J C; Weber, M; Duflot, E; Popoff, M R; Mock, M

    1997-01-01

    The Bacillus anthracis toxinogenic Sterne strain is currently used as a live veterinary vaccine against anthrax. The capacity of a toxin-deficient derivative strain to produce a heterologous antigen by using the strong inducible promoter of the B. anthracis pag gene was investigated. The expression of the foreign gene ibp, encoding the Ib component of iota toxin from Clostridium perfringens, was analyzed. A pag-ibp fusion was introduced by allelic exchange into a toxin-deficient Sterne strain, thereby replacing the wild-type pag gene. This recombinant strain, called BAIB, was stable and secreted large quantities of Ib protein in induced culture conditions. Mice given injections of live BAIB spores developed an antibody response specific to the Ib protein. The pag-ibp fusion was therefore functional both in vitro and in vivo. Moreover, the immunized animals were protected against a challenge with C. perfringens iota toxin or with the homologous Clostridium spiroforme toxin. The protective immunity was mediated by neutralizing antibodies. In conclusion, B. anthracis is promising for the development of live veterinary vaccines. PMID:9169728

  5. Two simultaneous botulism outbreaks in Barcelona: Clostridium baratii and Clostridium botulinum.

    PubMed

    Lafuente, S; Nolla, J; Valdezate, S; Tortajada, C; Vargas-Leguas, H; Parron, I; Sáez-Nieto, J A; Portaña, S; Carrasco, G; Moguel, E; Sabate, S; Argelich, R; Caylà, J A

    2013-09-01

    Botulism is a severe neuroparalytic disorder that can be potentially life-threatening. In Barcelona, Spain, no outbreaks had been reported in the past 25 years. However, in September 2011, two outbreaks occurred involving two different families. A rare case of Clostridium baratii which produced a neurotoxin F outbreak was detected in five family members who had shared lunch, and several days before that another family was affected by C. botulinum toxin A which was probably present in homemade pâté. PMID:23158693

  6. Clostridium kogasensis sp. nov., a novel member of the genus Clostridium, isolated from soil under a corroded gas pipeline.

    PubMed

    Shin, Yeseul; Kang, Seok-Seong; Paek, Jayoung; Jin, Tae Eun; Song, Hong Seok; Kim, Hongik; Park, Hee-Moon; Chang, Young-Hyo

    2016-06-01

    Two bacterial strains, YHK0403(T) and YHK0508, isolated from soil under a corroded gas pipe line, were revealed as Gram-negative, obligately anaerobic, spore-forming and mesophilic bacteria. The cells were rod-shaped and motile by means of peritrichous flagella. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolates were members of the genus Clostridium and were the most closely related to Clostridium scatologenes KCTC 5588(T) (95.8% sequence similarity), followed by Clostridium magnum KCTC 15177(T) (95.8%), Clostridium drakei KCTC 5440(T) (95.7%) and Clostridium tyrobutyricum KCTC 5387(T) (94.9%). The G + C contents of the isolates were 29.6 mol%. Peptidoglycan in the cell wall was of the A1γ type with meso-diaminopimelic acid. The major polar lipid was diphosphatidylglycerol (DPG), and other minor lipids were revealed as phosphatidylglycerol (PG), phosphatidylethanolamine (PE), two unknown glycolipids (GL1 and GL2), an unknown aminoglycolipid (NGL), two unknown aminophospholipids (PN1 and PN2) and four unknown phospholipids (PL1 to PL4). Predominant fatty acids were C16:0 and C16:1cis9 DMA. The major end products from glucose fermentation were identified as butyrate (12.2 mmol) and acetate (9.8 mmol). Collectively, the results from a wide range of phenotypic tests, chemotaxonomic tests, and phylogenetic analysis indicated that the two isolates represent novel species of the genus Clostridium, for which the name Clostridium kogasensis sp. nov. (type strain, YHK0403(T) = KCTC 15258(T) = JCM 18719(T)) is proposed. PMID:26899448

  7. Phylogeny of the ammonia-producing ruminal bacteria Peptostreptococcus anaerobius, Clostridium sticklandii, and Clostridium aminophilum sp. nov

    NASA Technical Reports Server (NTRS)

    Paster, B. J.; Russell, J. B.; Yang, C. M.; Chow, J. M.; Woese, C. R.; Tanner, R.

    1993-01-01

    In previous studies, gram-positive bacteria which grew rapidly with peptides or an amino acid as the sole energy source were isolated from bovine rumina. Three isolates, strains C, FT (T = type strain), and SR, were considered to be ecologically important since they produced up to 20-fold more ammonia than other ammonia-producing ruminal bacteria. On the basis of phenotypic criteria, the taxonomic position of these new isolates was uncertain. In this study, the 16S rRNA sequences of these isolates and related bacteria were determined to establish the phylogenetic positions of the organisms. The sequences of strains C, FT, and SR and reference strains of Peptostreptococcus anaerobius, Clostridium sticklandii, Clostridium coccoides, Clostridium aminovalericum, Acetomaculum ruminis, Clostridium leptum, Clostridium lituseburense, Clostridium acidiurici, and Clostridium barkeri were determined by using a modified Sanger dideoxy chain termination method. Strain C, a large coccus purported to belong to the genus Peptostreptococcus, was closely related to P. anaerobius, with a level of sequence similarity of 99.6%. Strain SR, a heat-resistant, short, rod-shaped organism, was closely related to C. sticklandii, with a level of sequence similarity of 99.9%. However, strain FT, a heat-resistant, pleomorphic, rod-shaped organism, was only distantly related to some clostridial species and P. anaerobius. On the basis of the sequence data, it was clear that strain FT warranted designation as a separate species. The closest known relative of strain FT was C. coccoides (level of similarity, only 90.6%). Additional strains that are phenotypically similar to strain FT were isolated in this study.(ABSTRACT TRUNCATED AT 250 WORDS).

  8. Detection and characterization of Clostridium perfringens in the feces of healthy and diarrheic dogs

    PubMed Central

    Goldstein, Michael R.; Kruth, Stephen A.; Bersenas, Alexa M.E.; Holowaychuk, Marie K.; Weese, J. Scott

    2012-01-01

    Clostridium perfringens has been implicated as a cause of diarrhea in dogs. The objectives of this study were to compare 2 culture methods and to evaluate a multiplex polymerase chain reaction (PCR) assay to detect C. perfringens toxin genes alpha (α), beta (β ), beta 2 (β2), epsilon (ɛ), iota (ι), and C. perfringens enterotoxin (cpe) from canine isolates. Fecal samples were collected from clinically normal non-diarrheic (ND) dogs, (n = 105) and diarrheic dogs (DD, n = 54). Clostridium perfringens was isolated by directly inoculating stool onto 5% sheep blood agar (SBA) and enrichment in brain-heart infusion (BHI) broth, followed by inoculation onto SBA. Isolates were tested by multiplex PCR for the presence of α, β, β2, ɛ, ι, and cpe genes. C. perfringens was isolated from 84% of ND samples using direct culture and from 87.6% with enrichment (P = 0.79). In the DD group, corresponding isolation rates were 90.7% and 93.8% (P = 0.65). All isolates possessed the α toxin gene. Beta (β), β2, ɛ, ι, and cpe toxin genes were identified in 4.5%, 1.1%, 3.4%, 1.1%, and 14.8% of ND isolates, respectively. In the DD group, β and β2 were identified in 5%, ɛ and ι were not identified, and the cpe gene was identified in 16.9% of isolates. Enrichment with BHI broth did not significantly increase the yield of C. perfringens, but it did increase the time and cost of the procedure. C. perfringens toxin genes were present in equal proportions in both the ND and DD groups (P ≤ 0.15 to 0.6). Within the parameters of this study, culture of C. perfringens and PCR for toxin genes is of limited diagnostic usefulness due to its high prevalence in normal dogs and the lack of apparent difference in the distribution of toxin genes between normal and diarrheic dogs. PMID:23277693

  9. Carbon material distribution and flux analysis under varying glucose concentrations in hydrogen-producing Clostridium tyrobutyricum JM1.

    PubMed

    Jo, Ji Hye; Kim, Woong

    2016-06-20

    Anaerobic glucose metabolism in hydrogen-producing Clostridium tyrobutyricum was investigated in batch culture with varying initial glucose concentrations (27.8-333.6mM). To understand the regulation of metabolism, the carbon material and reduction balances were applied to estimate the carbon flux distribution for the first time, and metabolic flux analysis (MFA) was used to provide qualitative information and guidance for effective metabolic design. The overall flux distribution suggested that C. tyrobutyricum metabolism has a high capacity for the production of butyrate and hydrogen at an initial glucose concentration of 222.4mM, with balanced activities of NADH and ATP. PMID:27140868

  10. Detection of Clostridium tetani in human clinical samples using tetX specific primers targeting the neurotoxin.

    PubMed

    Ganesh, Madhu; Sheikh, Nasira K; Shah, Pooja; Mehetre, Gajanan; Dharne, Mahesh S; Nagoba, Basavraj S

    2016-01-01

    Tetanus resulting from ear injury remains an important health problem, particularly in the developing world. We report the successful detection of Clostridium tetani using tetX specific primers targeting the Cl. tetani neurotoxin. The sample was obtained from an ear discharge of a case of otogenic tetanus in a 2-year-old male child. Based on the culture results of the ear discharge, Gram staining and virulence testing by genotyping, a diagnosis of tetanus was confirmed. This is the first report from India on the successful detection of Cl. tetani in a human clinical sample using tetX specific primers targeting the Cl. tetani neurotoxin. PMID:26220795

  11. CRISPR-based genome editing and expression control systems in Clostridium acetobutylicum and Clostridium beijerinckii.

    PubMed

    Li, Qi; Chen, Jun; Minton, Nigel P; Zhang, Ying; Wen, Zhiqiang; Liu, Jinle; Yang, Haifeng; Zeng, Zhe; Ren, Xiaodan; Yang, Junjie; Gu, Yang; Jiang, Weihong; Jiang, Yu; Yang, Sheng

    2016-07-01

    Solventogenic clostridia are important industrial microorganisms that produce various chemicals and fuels. Effective genetic tools would facilitate physiological studies aimed both at improving our understanding of metabolism and optimizing solvent productivity through metabolic engineering. Here we have developed an all-in-one, CRISPR-based genome editing plasmid, pNICKclos, that can be used to achieve successive rounds of gene editing in Clostridium acetobutylicum ATCC 824 and Clostridium beijerinckii NCIMB 8052 with efficiencies varying from 6.7% to 100% and 18.8% to 100%, respectively. The plasmid specifies the requisite target-specific guide RNA, the gene encoding the Streptococcus pyogenes Cas9 nickase and the genome editing template encompassing the gene-specific homology arms. It can be used to create single target mutants within three days, with a further two days required for the curing of the pNICKclos plasmid ready for a second round of mutagenesis. A S. pyogenes dCas9-mediated gene regulation control system, pdCASclos, was also developed and used in a CRISPRi strategy to successfully repress the expression of spo0A in C. acetobutylicum and C. beijerinckii. The combined application of the established high efficiency CRISPR-Cas9 based genome editing and regulation control systems will greatly accelerate future progress in the understanding and manipulation of metabolism in solventogenic clostridia. PMID:27213844

  12. Group II Intron-Anchored Gene Deletion in Clostridium

    PubMed Central

    Jia, Kaizhi; Zhu, Yan; Zhang, Yanping; Li, Yin

    2011-01-01

    Clostridium plays an important role in commercial and medical use, for which targeted gene deletion is difficult. We proposed an intron-anchored gene deletion approach for Clostridium, which combines the advantage of the group II intron “ClosTron” system and homologous recombination. In this approach, an intron carrying a fragment homologous to upstream or downstream of the target site was first inserted into the genome by retrotransposition, followed by homologous recombination, resulting in gene deletion. A functional unknown operon CAC1493–1494 located in the chromosome, and an operon ctfAB located in the megaplasmid of C. acetobutylicum DSM1731 were successfully deleted by using this approach, without leaving antibiotic marker in the genome. We therefore propose this approach can be used for targeted gene deletion in Clostridium. This approach might also be applicable for gene deletion in other bacterial species if group II intron retrotransposition system is established. PMID:21304965

  13. Promoters and proteins from Clostridium thermocellum and uses thereof

    DOEpatents

    Wu, J. H. David; Newcomb, Michael

    2012-11-13

    The present invention relates to an inducible and a high expression nucleic acid promoter isolated from Clostridium thermocellum. These promoters are useful for directing expression of a protein or polypeptide encoded by a nucleic acid molecule operably associated with the nucleic acid promoters. The present invention also relates to nucleic acid constructs including the C. thermocellum promoters, and expression vectors and hosts containing such nucleic acid constructs. The present invention also relates to protein isolated from Clostridium thermocellum, including a repressor protein. The present invention also provides methods of using the isolated promoters and proteins from Clostridium thermocellum, including methods for directing inducible in vitro and in vivo expression of a protein or polypeptide in a host, and methods of producing ethanol from a cellulosic biomass.

  14. A prediction model for Clostridium difficile recurrence

    PubMed Central

    LaBarbera, Francis D.; Nikiforov, Ivan; Parvathenani, Arvin; Pramil, Varsha; Gorrepati, Subhash

    2015-01-01

    Background Clostridium difficile infection (CDI) is a growing problem in the community and hospital setting. Its incidence has been on the rise over the past two decades, and it is quickly becoming a major concern for the health care system. High rate of recurrence is one of the major hurdles in the successful treatment of C. difficile infection. There have been few studies that have looked at patterns of recurrence. The studies currently available have shown a number of risk factors associated with C. difficile recurrence (CDR); however, there is little consensus on the impact of most of the identified risk factors. Methods Our study was a retrospective chart review of 198 patients diagnosed with CDI via Polymerase Chain Reaction (PCR) from January 2009 to Jun 2013. In our study, we decided to use a machine learning algorithm called the Random Forest (RF) to analyze all of the factors proposed to be associated with CDR. This model is capable of making predictions based on a large number of variables, and has outperformed numerous other models and statistical methods. Results We came up with a model that was able to accurately predict the CDR with a sensitivity of 83.3%, specificity of 63.1%, and area under curve of 82.6%. Like other similar studies that have used the RF model, we also had very impressive results. Conclusions We hope that in the future, machine learning algorithms, such as the RF, will see a wider application. PMID:25656667

  15. The Changing Epidemiology of Clostridium difficile Infections

    PubMed Central

    Freeman, J.; Bauer, M. P.; Baines, S. D.; Corver, J.; Fawley, W. N.; Goorhuis, B.; Kuijper, E. J.; Wilcox, M. H.

    2010-01-01

    Summary: The epidemiology of Clostridium difficile infection (CDI) has changed dramatically during this millennium. Infection rates have increased markedly in most countries with detailed surveillance data. There have been clear changes in the clinical presentation, response to treatment, and outcome of CDI. These changes have been driven to a major degree by the emergence and epidemic spread of a novel strain, known as PCR ribotype 027 (sometimes referred to as BI/NAP1/027). We review the evidence for the changing epidemiology, clinical virulence and outcome of treatment of CDI, and the similarities and differences between data from various countries and continents. Community-acquired CDI has also emerged, although the evidence for this as a distinct new entity is less clear. There are new data on the etiology of and potential risk factors for CDI; controversial issues include specific antimicrobial agents, gastric acid suppressants, potential animal and food sources of C. difficile, and the effect of the use of alcohol-based hand hygiene agents. PMID:20610822

  16. Clostridium perfringens in Meat and Meat Products

    PubMed Central

    Hall, Herbert E.; Angelotti, Robert

    1965-01-01

    A total of 262 specimens of meat and meat dishes were examined for the presence of Clostridium perfringens. Of this total, 161 were raw, unprocessed beef, veal, lamb, pork, or chicken; 101 were processed meats and meat dishes. C. perfringens was isolated from 113 (43.1%) of these specimens. The highest percentage of contamination (82%) was found in veal cuts, and the lowest (4.7%) in sliced sandwich meats and spreads. Only 2 of the 113 isolates were shown to produce heat-resistant spores, which indicates a very low incidence (0.8%) of contamination. These findings indicate that outbreaks of C. perfringens food-borne disease in the Cincinnati area are caused principally by the contamination of the food with vegetative cells or spores of the organism after cooking. Studies of the effects of various holding temperatures on the growth of C. perfringens indicated that, in the range of 5 to 15 C, no multiplication would occur, but that viable cells would still be present at the end of a 5-day holding period. Extremely rapid growth occurred at temperatures around 45 C, and complete inhibition of growth was accomplished between 49 and 52 C. PMID:14325274

  17. Action of nitroheterocyclic drugs against Clostridium difficile

    PubMed Central

    Kumar, Manish; Adhikari, Sudip; Hurdle, Julian G.

    2014-01-01

    The nitroheterocyclic classes of drugs have a long history of use in treating anaerobic infections, as exemplified by metronidazole as a first-line treatment for mild-to-moderate Clostridium difficile infection (CDI). Since direct comparisons of the three major classes of nitroheterocyclic drugs (i.e. nitroimidazole, nitazoxanide and nitrofurans) and nitrosating agents against C. difficile are under-examined, in this study their actions against C. difficile were compared. Results show that whilst transient resistance occurs to metronidazole and nitazoxanide, stable resistance arises to nitrofurans upon serial passage. All compounds killed C. difficile at high concentrations in addition to the host defence nitrosating agent S-nitrosoglutathione (GSNO). This suggests that GSNO killing of C. difficile contributes to its efficacy in murine CDI. Although nitric oxide production could not be detected for the nitroheterocyclic drugs, the cellular response to metronidazole and nitrofurans has some overlap with the response to GSNO, causing significant upregulation of the hybrid-cluster protein Hcp that responds to nitrosative stress. These findings provide new insights into the action of nitroheterocyclic drugs against C. difficile. PMID:25129314

  18. Secretome analysis of Clostridium difficile strains.

    PubMed

    Boetzkes, Alexander; Felkel, Katharina Wiebke; Zeiser, Johannes; Jochim, Nelli; Just, Ingo; Pich, Andreas

    2012-08-01

    Clostridium difficile causes infections ranging from mild C. difficile-associated diarrhea to severe pseudomembranous colitis. Since 2003 new hypervirulent C. difficile strains (PCR ribotype 027) emerged characterized by a dramatically increased mortality. The secretomes of the three C. difficile strains CDR20291, CD196, and CD630 were analyzed and compared. Proteins were separated and analyzed by means of SDS--PAGE and LC-MS. MS data were analyzed using Mascot and proteins were checked for export signals with SecretomeP and SignalP. LC-MS analysis revealed 158 different proteins in the supernatant of C. difficile. Most of the identified proteins originate from the cytoplasm. Thirty-two proteins in CDR20291, 36 in CD196 and 26 in CD630 were identified to be secreted by C. difficile strains. Those were mainly S-layer proteins, substrate-binding proteins of ABC-transporters, cell wall hydrolases, pilin and unknown hypothetical proteins. Toxin A and toxin B were identified after growth in brain heart infusion medium using immunological techniques. The ADP-ribosyltransferase-binding component protein, which is a part of the binary toxin CDT, was only identified in the hypervirulent ribotype 027 strains. Further proteins that are secreted specifically by hypervirulent strains were identified. PMID:22398929

  19. Characterization of Functional Prophages in Clostridium difficile.

    PubMed

    Sekulović, Ognjen; Fortier, Louis-Charles

    2016-01-01

    Bacteriophages (phages) are present in almost, if not all ecosystems. Some of these bacterial viruses are present as latent "prophages," either integrated within the chromosome of their host, or as episomal DNAs. Since prophages are ubiquitous throughout the bacterial world, there has been a sustained interest in trying to understand their contribution to the biology of their host. Clostridium difficile is no exception to that rule and with the recent release of hundreds of bacterial genome sequences, there has been a growing interest in trying to identify and classify these prophages. Besides their identification in bacterial genomes, there is also growing interest in determining the functionality of C. difficile prophages, i.e., their capacity to escape their host and reinfect a different strain, thereby promoting genomic evolution and horizontal transfer of genes through transduction, for example of antibiotic resistance genes. There is also some interest in using therapeutic phages to fight C. difficile infections.The objective of this chapter is to share with the broader C. difficile research community the expertise we developed in the study of C. difficile temperate phages. In this chapter, we describe a general "pipeline" comprising a series of experiments that we use in our lab to identify, induce, isolate, propagate, and characterize prophages. Our aim is to provide readers with the necessary basic tools to start studying C. difficile phages. PMID:27507339

  20. Elimination of formate production in Clostridium thermocellum

    DOE PAGESBeta

    Rydzak, Thomas; Lynd, Lee R.; Guss, Adam M.

    2015-07-11

    We study the ability of Clostridium thermocellum to rapidly degrade cellulose and ferment resulting hydrolysis products into ethanol makes it a promising platform organism for cellulosic biofuel production via consolidated bioprocessing. Currently, however, ethanol yield are far below theoretical maximum due to branched product pathways that divert carbon and electrons towards formate, H2, lactate, acetate, and secreted amino acids. To redirect carbon and electron flux away from formate, pyruvate:formate lyase (pfl) and respective PFL-activating enzyme were deleted. Formate production in the resulting Δpfl strain was eliminated and acetate production decreased by 50% on both complex and defined medium. Growth ratemore » of Δpfl decreased by 2.9-fold on defined medium and diauxic growth was observed on complex medium. Supplementation of defined medium with 2 mM formate restored Δpfl growth rate to 80% of the parent strain. Finally, we discuss the role of pfl in metabolic engineering strategies and C1 metabolism.« less

  1. Biosynthesis of amino acids in Clostridium pasteurianum

    PubMed Central

    Dainty, R. H.; Peel, J. L.

    1970-01-01

    1. Clostridium pasteurianum was grown on a synthetic medium with the following carbon sources: (a) 14C-labelled glucose, alone or with unlabelled aspartate or glutamate, or (b) unlabelled glucose plus 14C-labelled aspartate, glutamate, threonine, serine or glycine. The incorporation of 14C into the amino acids of the cell protein was examined. 2. In both series of experiments carbon from exogenous glutamate was incorporated into proline and arginine; carbon from aspartate was incorporated into glutamate, proline, arginine, lysine, methionine, threonine, isoleucine, glycine and serine. Incorporations from the other exogenous amino acids indicated the metabolic sequence: aspartate → threonine → glycine ⇌ serine. 3. The following activities were demonstrated in cell-free extracts of the organism: (a) the formation of aspartate by carboxylation of phosphoenolpyruvate or pyruvate, followed by transamination; (b) the individual reactions of the tricarboxylic acid route to 2-oxoglutarate from oxaloacetate; glutamate dehydrogenase was not detected; (c) the conversion of aspartate into threonine via homoserine; (d) the conversion of threonine into glycine by a constitutive threonine aldolase; (e) serine transaminase, phosphoserine transaminase, glycerate dehydrogenase and phosphoglycerate dehydrogenase. This last activity was abnormally high. 4. The combined evidence indicates that in C. pasteurianum the biosynthetic role of aspartate and glutamate is generally similar to that in aerobic and facultatively aerobic organisms, but that glycine is synthesized from glucose via aspartate and threonine. PMID:5419750

  2. Crystal structure of Clostridium difficile toxin A.

    PubMed

    Chumbler, Nicole M; Rutherford, Stacey A; Zhang, Zhifen; Farrow, Melissa A; Lisher, John P; Farquhar, Erik; Giedroc, David P; Spiller, Benjamin W; Melnyk, Roman A; Lacy, D Borden

    2016-01-01

    Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon(1,2). The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host(3,4). The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics. PMID:27571750

  3. Crystal structure of Clostridium difficile toxin A

    PubMed Central

    Chumbler, Nicole M.; Rutherford, Stacey A.; Zhang, Zhifen; Farrow, Melissa A.; Lisher, John P.; Farquhar, Erik; Giedroc, David P.; Spiller, Benjamin W.; Melnyk, Roman A.; Lacy, D. Borden

    2016-01-01

    Clostridium difficile infection is the leading cause of hospital-acquired diarrhoea and pseudomembranous colitis. Disease is mediated by the actions of two toxins, TcdA and TcdB, which cause the diarrhoea, as well as inflammation and necrosis within the colon1,2. The toxins are large (308 and 270 kDa, respectively), homologous (47% amino acid identity) glucosyltransferases that target small GTPases within the host3,4. The multidomain toxins enter cells by receptor-mediated endocytosis and, upon exposure to the low pH of the endosome, insert into and deliver two enzymatic domains across the membrane. Eukaryotic inositol-hexakisphosphate (InsP6) binds an autoprocessing domain to activate a proteolysis event that releases the N-terminal glucosyltransferase domain into the cytosol. Here, we report the crystal structure of a 1,832-amino-acid fragment of TcdA (TcdA1832), which reveals a requirement for zinc in the mechanism of toxin autoprocessing and an extended delivery domain that serves as a scaffold for the hydrophobic α-helices involved in pH-dependent pore formation. A surface loop of the delivery domain whose sequence is strictly conserved among all large clostridial toxins is shown to be functionally important, and is highlighted for future efforts in the development of vaccines and novel therapeutics. PMID:27512603

  4. ClosTron-mediated engineering of Clostridium

    PubMed Central

    Kuehne, Sarah A.; Minton, Nigel P.

    2012-01-01

    Members of the genus Clostridium are of both medical and industrial importance. The molecular tools necessary to study and exploit their wide ranging physiological diversity through directed mutational analysis have until recently been lacking. The situation was transformed in the mid-2000s with the specific adaptation of intron re-targeting technology to the genus, through the development of the ClosTron. By making a handful of nucleotide changes to the group II intron encoding region, the intron can be directed to insert into almost any region within the genome. Through the use of a retrotransposition-activated marker (RAM), based on the ermB gene, successful insertion is selected on the basis of acquisition of resistance to erythromycin. The re-targeted region is designed using an online re-targeting algorithm (www.clostron.com), and then an order is placed with DNA2.0 for both the synthesis of the re-targeted region and its custom cloning into the ClosTron vector. Re-targeted ClosTrons are delivered ready for use in 10–14 days, allowing mutants to be isolated 5–7 days after receipt. Its availability has revolutionized clostridial molecular biology. PMID:22750794

  5. Cellulosomics of the cellulolytic thermophile Clostridium clariflavum

    PubMed Central

    2014-01-01

    Background Clostridium clariflavum is an anaerobic, thermophilic, Gram-positive bacterium, capable of growth on crystalline cellulose as a single carbon source. The genome of C. clariflavum has been sequenced to completion, and numerous cellulosomal genes were identified, including putative scaffoldin and enzyme subunits. Results Bioinformatic analysis of the C. clariflavum genome revealed 49 cohesin modules distributed on 13 different scaffoldins and 79 dockerin-containing proteins, suggesting an abundance of putative cellulosome assemblies. The 13-scaffoldin system of C. clariflavum is highly reminiscent of the proposed cellulosome system of Acetivibrio cellulolyticus. Analysis of the C. clariflavum type I dockerin sequences indicated a very high level of conservation, wherein the putative recognition residues are remarkably similar to those of A. cellulolyticus. The numerous interactions among the cellulosomal components were elucidated using a standardized affinity ELISA-based fusion-protein system. The results revealed a rather simplistic recognition pattern of cohesin-dockerin interaction, whereby the type I and type II cohesins generally recognized the dockerins of the same type. The anticipated exception to this rule was the type I dockerin of the ScaB adaptor scaffoldin which bound selectively to the type I cohesins of ScaC and ScaJ. Conclusions The findings reveal an intricate picture of predicted cellulosome assemblies in C. clariflavum. The network of cohesin-dockerin pairs provides a thermophilic alternative to those of C. thermocellum and a basis for subsequent utilization of the C. clariflavum cellulosomal system for biotechnological application. PMID:26413154

  6. Current status of Clostridium difficile infection epidemiology.

    PubMed

    Lessa, Fernanda C; Gould, Carolyn V; McDonald, L Clifford

    2012-08-01

    The dramatic changes in the epidemiology of Clostridium difficile infection (CDI) during recent years, with increases in incidence and severity of disease in several countries, have made CDI a global public health challenge. Increases in CDI incidence have been largely attributed to the emergence of a previously rare and more virulent strain, BI/NAP1/027. Increased toxin production and high-level resistance to fluoroquinolones have made this strain a very successful pathogen in healthcare settings. In addition, populations previously thought to be at low risk are now being identified as having severe CDI. Recent genetic analysis suggests that C. difficile has a highly fluid genome with multiple mechanisms to modify its content and functionality, which can make C. difficile adaptable to environmental changes and potentially lead to the emergence of more virulent strains. In the face of these changes in the epidemiology and microbiology of CDI, surveillance systems are necessary to monitor trends and inform public health actions. PMID:22752867

  7. Phosphorylation of proteins in Clostridium thermohydrosulfuricum

    SciTech Connect

    Londesborough, J.

    1986-02-01

    Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by (..gamma..-/sup 32/P)ATP of several endogenous proteins with M/sub r/s between 13,000 and 100,000. Serine and tyrosine were the main acceptors. Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of M/sub r/s 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity. The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor. Phosphorylation of p53 was promoted by 10..mu..M fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 ..mu..M brain (but not spinach) calmodulin. Polyamines, including the odd polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins. Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by /sub 32/P/sub i/. Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro.

  8. [Epidemiology of Clostridium difficile infection in Spain].

    PubMed

    Asensio, Angel; Monge, Diana

    2012-06-01

    There has been increasing interest in Clostridium difficile infection (CDI) due its association with healthcare and its impact on morbidity and mortality in the elderly. During the last few years there has been a growing increase in the number of published studies on the incidence, changes on the clinical presentation and on the epidemiology, with the description of new risk factors. The frequency of CDI in Spain is not sufficiently characterised. The available data indicates that incidence is within the range of that of surrounding countries but increasing. Furthermore, the high and growing use of broad spectrum antibiotics, both in our hospitals and in the community setting, are factors that favour the increase of the disease. The hyper-virulent ribotype 027 has not spread in our hospitals. We need to know with enhanced validity and accuracy the incidence of CDI, both community and healthcare-associated, the information on outbreaks, the incidence on certain population groups, the characterisation of circulating ribotypes and the impact of the disease in terms of mortality and health costs. We need to implement programs for the improvement of antibiotic therapy in the hospital, as well as in the community. Furthermore, the knowledge and the performance of standard precautions need to be improved, particularly hand hygiene, and the specific measures to limit the transmission of C. difficile among the healthcare institutions. PMID:22136747

  9. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    PubMed Central

    Verherstraeten, Stefanie; Goossens, Evy; Valgaeren, Bonnie; Pardon, Bart; Timbermont, Leen; Haesebrouck, Freddy; Ducatelle, Richard; Deprez, Piet; Wade, Kristin R.; Tweten, Rodney; Van Immerseel, Filip

    2015-01-01

    The anaerobic bacterium Clostridium perfringens expresses multiple toxins that promote disease development in both humans and animals. One such toxin is perfringolysin O (PFO, classically referred to as θ toxin), a pore-forming cholesterol-dependent cytolysin (CDC). PFO is secreted as a water-soluble monomer that recognizes and binds membranes via cholesterol. Membrane-bound monomers undergo structural changes that culminate in the formation of an oligomerized prepore complex on the membrane surface. The prepore then undergoes conversion into the bilayer-spanning pore measuring approximately 250–300 Å in diameter. PFO is expressed in nearly all identified C. perfringens strains and harbors interesting traits that suggest a potential undefined role for PFO in disease development. Research has demonstrated a role for PFO in gas gangrene progression and bovine necrohemorrhagic enteritis, but there is limited data available to determine if PFO also functions in additional disease presentations caused by C. perfringens. This review summarizes the known structural and functional characteristics of PFO, while highlighting recent insights into the potential contributions of PFO to disease pathogenesis. PMID:26008232

  10. Effects of butanol on Clostridium acetobutylicum.

    PubMed Central

    Bowles, L K; Ellefson, W L

    1985-01-01

    The internal pH of Clostridium acetobutylicum was determined at various stages during the growth of the organism. Even in the presence of significant quantities of acetic, butyric, and lactic acids, an internal pH of 6.2 was maintained. Experiments using N,N'-dicyclohexylcarbodiimide indicated that a functioning H+-ATPase is necessary for internal pH control. Butanol, one of the end products of the fermentation, had numerous harmful effects on C. acetobutylicum. At a concentration high enough to inhibit growth, butanol destroyed the ability of the cell to maintain internal pH, lowered the intracellular level of ATP, and inhibited glucose uptake. Experiments done at two different external pH values suggested that the butanol-mediated decrease in ATP concentration was independent of the drop in internal pH. Glucose uptake was not affected by arsenate, suggesting that uptake was not ATP dependent. The effects of butanol on C. acetobutylicum are complex, inhibiting several interrelated membrane processes. PMID:2868690

  11. Laboratory Detection of Clostridium difficile in Piglets in Australia

    PubMed Central

    Knight, Daniel R.; Squire, Michele M.

    2014-01-01

    Clostridium difficile is a well-known enteric pathogen of humans and the causative agent of high-morbidity enteritis in piglets aged 1 to 7 days. C. difficile prevalence in Australian piglets is as high as 70%. The current diagnostic assays have been validated only for human infections, and there are no published studies assessing their performance in Australian piglets. We evaluated the suitability of five assays for detecting C. difficile in 157 specimens of piglet feces. The assays included a loop-mediated isothermal amplification (LMIA)-PCR for tcdA (illumigene C. difficile; Meridian), a real-time PCR for tcdB (GeneOhm Cdiff; Becton Dickinson), two-component enzyme immunoassays (EIA) for C. difficile glutamate dehydrogenase (GDH) (EIA-GDH) and TcdA/TcdB (EIA-TcdA/TcdB) (C. diff Quik Chek; Alere), and direct culture (DC) (C. difficile chromID agar; bioMérieux). The assays for detection of the organism were compared against enrichment culture (EC), and assays for detection of toxins/toxin genes were compared against EC followed by PCR for toxin genes (toxigenic EC [TEC]). The recovery of C. difficile by EC was 39.5% (n = 62/157), and TEC revealed that 58.1% (n = 36/62) of isolates were positive for at least one toxin gene (tcdA/tcdB). Compared with those for EC/TEC, the sensitivities, specificities, positive predictive values, and negative predictive values were, respectively, as follows: DC, 91.9, 100.0, 100.0, and 95.0%; EIA-GDH, 41.9, 92.6, 78.8, and 71.0%; EIA-TcdA/TcdB, 5.6, 99.2, 66.7, and 77.9%; real-time PCR, 42.9, 96.7, 78.9, and 85.4% and LMIA-PCR, 25.0, 95.9, 64.3, and 81.1%. The performance of the molecular methods was poor, suggesting that the current commercially available assays for diagnosis of C. difficile in humans are not suitable for use in piglets. C. difficile recovery by the DC provides a cost-effective alternative. PMID:25122859

  12. Clostridium infection (jisizheng) in yaks in Qinghai, China.

    PubMed

    Changqing, Q; Xueli, Y

    2001-10-01

    Since the mid-1980s, outbreaks of a disease characterized by a sudden onset, acute deaths and extensive haemorrhages in the viscera and digestive tract of yaks have been prevalent in Qilian, Qinghai, China. The disease is known as jisiheng by local people. Virulent Clostridium perfringens type A and Clostridium haemolytica were isolated from yaks that had died of jisizheng. In 1996 and 1997, yaks were immunized with a polyvalent inactivated vaccine against C. perfringens and with an inactivated vaccine against C. haemolyticum, and this prevented the occurrence of jisizheng. PMID:11583378

  13. Clinical Utility of Laboratory Detection of Clostridium difficile Strain BI/NAP1/027

    PubMed Central

    Gerding, Dale N.

    2015-01-01

    Clostridium difficile strain BI/NAP1/027 is associated with increased C. difficile infection (CDI) rates and severity, and the efficacy of some CDI therapies may be strain dependent. Although cultured C. difficile isolates can be reliably subtyped by various methods, the long turnaround times, high cost, and limited availability of strain typing preclude their routine use. Nucleic acid amplification tests identify BI/NAP1/027 rapidly from stool, but the emergence of closely related strains compromises test specificity. Although detection of epidemiologically significant pathogens is generally useful for infection control programs, specific data supporting use of rapid detection of BI/NAP1/027 as an infection control tool are still awaited. PMID:26511742

  14. Oxidation-Reduction Potential and Growth of Clostridium perfringens and Pseudomonas fluorescens1

    PubMed Central

    Tabatabai, L. B.; Walker, H. W.

    1970-01-01

    A new apparatus was developed for measuring changes in Eh, pH, and cell numbers. With this apparatus, the relationships of these parameters were studied at initial Eh levels of 200 and 40 mv (pH 7.0), by using Clostridium perfringens and Pseudomonas fluorescens. One of the strains of C. perfringens grew more luxuriantly at the higher Eh, in the presence of small quantities of oxygen, than at the lower one in the absence of oxygen. P. fluorescens could grow at a relatively low Eh (40 mv, pH 7.0) in pure culture but not in the presence of C. perfringens under the same conditions. PMID:4320922

  15. Occurrence of human pathogenic Clostridium botulinum among healthy dairy animals: an emerging public health hazard.

    PubMed

    Abdel-Moein, Khaled A; Hamza, Dalia A

    2016-02-01

    The current study was conducted to investigate the occurrence of human pathogenic Clostridium botulinum in the feces of dairy animals. Fecal samples were collected from 203 apparently healthy dairy animals (50 cattle, 50 buffaloes, 52 sheep, 51 goats). Samples were cultured to recover C. botulinum while human pathogenic C. botulinum strains were identified after screening of all C. botulinum isolates for the presence of genes that encode toxins type A, B, E, F. The overall prevalence of C. botulinum was 18.7% whereas human pathogenic C. botulinum strains (only type A) were isolated from six animals at the rates of 2, 2, 5.8, and 2% for cattle, buffaloes, sheep, and goats, respectively. High fecal carriage rates of C. botulinum among apparently healthy dairy animals especially type A alarm both veterinary and public health communities for a potential role which may be played by dairy animals in the epidemiology of such pathogen. PMID:27077311

  16. Real-time cellular analysis for quantitative detection of functional Clostridium difficile toxin in stool.

    PubMed

    Huang, Bin; Li, Haijing; Jin, Dazhi; Stratton, Charles W; Tang, Yi-Wei

    2014-04-01

    Rapid and accurate diagnosis and monitoring of Clostridium difficile infection (CDI) is critical for patient care and infection control. We will briefly review current laboratory techniques for the diagnosis of CDI and identify aspects needing improvement. We will also introduce a real-time cellular analysis (RTCA) assay developed for the diagnosis and monitoring of CDI using electronic impedance to assess the cell status. The RTCA assay uses impedance measurement to detect minute physiological changes in cells cultured on gold microelectrodes embedded in glass substrates in the bottom of microtiter wells. This assay has been adapted for quantitative detection of C. difficile functional toxin directly from stool specimens. Compared to conventional techniques and molecular assays, the RTCA assay provides a valuable tool for the diagnosis of CDI as well as for the assessment of clinical severity and for monitoring therapeutic efficacies. PMID:24649817

  17. Fatal clostridium septicum myonecrosis in a captive canada lynx (Lynx canadensis).

    PubMed

    Izer, Jenelle M; Wilson, Ronald P; Cooper, Timothy K

    2014-09-01

    A 1-yr-old female Canada lynx (Lynx canadensis) presented for sudden onset of rapidly progressive bilateral pelvic limb paralysis. The lynx was chemically immobilized to perform a physical examination but expired shortly thereafter. On postmortem radiographs, there were myriad small irregular, round-to-spherical gas densities within the skeletal muscle of the right thigh and epaxial musculature. At gross necropsy, the muscles of the right thigh, right lateral abdominal wall, and epaxial region were emphysematous and necrohemorrhagic, with subcutaneous and muscular crepitant swelling. Multiple skin puncture wounds, consistent with bites, were present over the affected tissues. Clostridium septicum was isolated in pure anaerobic culture from the musculature of the right hind limb. Histopathologic examination confirmed the diagnosis of acute, severe necrohemorrhagic and gangrenous myositis and cellulitis. Gram stains demonstrated large gram-positive bacilli with subterminal spores. This is the first known documented case of C. septicum myonecrosis in a nondomestic felid. PMID:25314833

  18. Effect of exogenous electron shuttles on growth and fermentative metabolism in Clostridium sp. BC1

    SciTech Connect

    Yarlagadda V. N.; Francis A.; Gupta, A.; Dodge, C. J.

    2012-03-01

    In this study, the influence exogenous electron shuttles on the growth and glucose fermentative metabolism of Clostridium sp. BC1 was investigated. Bicarbonate addition to mineral salts (MS) medium accelerated growth and glucose fermentation which shifted acidogenesis (acetic- and butyric-acids) towards solventogenesis (ethanol and butanol). Addition of ferrihydrite, anthraquinone disulfonate, and nicotinamide adenine dinucleotide in bicarbonate to growing culture showed no significant influence on fermentative metabolism. In contrast, methyl viologen (MV) enhanced ethanol- and butanol-production by 28- and 12-fold, respectively with concomitant decrease in hydrogen, acetic- and butyric-acids compared to MS medium. The results show that MV addition affects hydrogenase activity with a significant reduction in hydrogen production and a shift in the direction of electron flow towards enhanced production of ethanol and butanol.

  19. Clinical Utility of Laboratory Detection of Clostridium difficile Strain BI/NAP1/027.

    PubMed

    Kociolek, Larry K; Gerding, Dale N

    2016-01-01

    Clostridium difficile strain BI/NAP1/027 is associated with increased C. difficile infection (CDI) rates and severity, and the efficacy of some CDI therapies may be strain dependent. Although cultured C. difficile isolates can be reliably subtyped by various methods, the long turnaround times, high cost, and limited availability of strain typing preclude their routine use. Nucleic acid amplification tests identify BI/NAP1/027 rapidly from stool, but the emergence of closely related strains compromises test specificity. Although detection of epidemiologically significant pathogens is generally useful for infection control programs, specific data supporting use of rapid detection of BI/NAP1/027 as an infection control tool are still awaited. PMID:26511742

  20. [Improvement of the method of isolation of hydrogen-forming bacteria of Clostridium genus].

    PubMed

    Pritula, I R; Tashirev, A B

    2012-01-01

    The method of isolation and quantitative account of pure cultures of obligate anaerobic hydrogen-forming clostridia is improved. A strain of hydrogen-forming bacteria Clostridium sp. BY-11 has been isolated from the association of sporulating bacteria. Quantitative indices of hydrogen synthesis and starch fermentation have been determined when growing the strain in the liquid medium. Concentration of H2 in the gas phase was 49%, microorganisms synthesized 128 1 of H2 from 1 kg of starch, the mass of starch decreased 7 times for 6 days. The mentioned indices for hydrogen synthesis and starch fermentation and for other organic model substrates in the future are the basis for creating the industrial biotechnology for production of hydrogen as the energy carrier under disposal of ecologically dangerous solid food waste. PMID:23293828

  1. Conversion of acids to alcohols by Clostridium ragsdalei strain P11: Process optimization and biochemistry

    NASA Astrophysics Data System (ADS)

    Isom, Catherine E.

    Research focus was directed toward the development of a biocatalyst that can be used to produce commodity chemicals and transportation fuels from volatile fatty acids ubiquitous in waste biomass. Clostridium ragsdalei was introduced to serve as an exemplar carboxidotrophic acetogen that reduces VFAs to alcohols of the same carbon structure with only acetate and ethanol as by-products of the fermentation. This dissertation developed a better understanding of this process in C. ragsdalei and, in turn, other similar bacteria and to supported previous discoveries as they relate to carboxylate reduction in acetogens. Additionally, pure culture studies allowed for a more detailed understanding of the biochemical behavior response to different compounds without skewing the results due to the influence of other species.

  2. Eradication of enteric helicobacters in Mongolian gerbils is complicated by the occurrence of Clostridium difficile enterotoxemia.

    PubMed

    Bergin, Ingrid L; Taylor, Nancy S; Nambiar, Prashant R; Fox, James G

    2005-06-01

    Outbred Mongolian gerbils from a United States commercial source were examined for colonization with naturally occurring enterohepatic Helicobacter spp. Helicobacter spp. were identified in the cecum and colon by culture and by using genus-specific primers in polymerase chain reaction (PCR) assays. Nutritionally balanced triple-antibiotic wafers (containing amoxicillin, metronidazole, and bismuth) used previously to eliminate helicobacter infections in mice were administered in an attempt to eradicate the naturally occurring novel helicobacters in the gerbils. After 7 days of antibiotic treatment, two of the experimental animals died due to Clostridium difficile-associated enterotoxemia. However, at 3 weeks after antibiotic cessation, the surviving three animals had no Helicobacter spp. in the cecum or colon according to PCR analysis. Eradication of Helicobacter spp. using dietary administration of antibiotics was complicated by the presence of toxin-producing C. difficile. An alternate method to develop helicobacter-free gerbils (such as Caesarian rederivation) may be necessary. PMID:16089175

  3. Symbiotic Relationship of Bacteroides cellulosolvens and Clostridium saccharolyticum in Cellulose Fermentation†

    PubMed Central

    Murray, William D.

    1986-01-01

    In coculture, Bacteroides cellulosolvens and Clostridium saccharolyticum fermented 33% more cellulose than did B. cellulosolvens alone. Also, cellulose digestion continued at a maximum rate 48 h longer in coculture. B. cellulosolvens hydrolyzes cellulose and supplies C. saccharolyticum with sugars and a growth factor replaceable by yeast extract. Alone, B. cellulosolvens exhibited an early cessation of growth which was not due to nutrient depletion, low pH, or toxic accumulation of acetic acid, ethanol, lactic acid, H2, CO2, cellobiose, glucose, or xylose. However, a 1-h incubation of B. cellulosolvens spent-culture medium with C. saacharolyticum cells starved for growth factor allowed a resumption of B. cellulosolvens growth. The symbiotic relationship of this naturally occurring coculture is one of mutualism, in which the cellulolytic microbe supplies the saccharolytic microbe with nutrients, and in turn the saccharolytic microbe removes a secondary metabolite toxic to the primary microbe. Images PMID:16347034

  4. Epidemiology of Clostridium difficile colonization in newborns: results using a bacteriophage and bacteriocin typing system.

    PubMed

    Bacon, A E; Fekety, R; Schaberg, D R; Faix, R G

    1988-08-01

    We used a typing system based on bacteriophage and bacteriocin susceptibility to study the epidemiology of Clostridium difficile colonization of newborn infants. C. difficile was found in the stools of 30 (16.0%) of 187 infants who were screened. Increased length of stay in the nursery (P less than .001) and delivery by cesarian section (P less than .001) were associated with higher rates of colonization. The isolates initially detected from the environment and the infants were strain B1811-1700. Strain B1537/Cld7 became the predominant isolate obtained from the infants; positive cultures were also obtained from the environment and the hands of personnel who worked in the nursery and had strain B1537/Cld7. Our results suggest that the infants acquired C. difficile through transfer from the hands of hospital staff. PMID:3403992

  5. Clostridium beijerinckii Cells Expressing Neocallimastix patriciarum Glycoside Hydrolases Show Enhanced Lichenan Utilization and Solvent Production

    PubMed Central

    López-Contreras, Ana M.; Smidt, Hauke; van der Oost, John; Claassen, Pieternel A. M.; Mooibroek, Hans; de Vos, Willem M.

    2001-01-01

    Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain. PMID:11679336

  6. Clostridium beijerinckii cells expressing Neocallimastix patriciarum glycoside hydrolases show enhanced lichenan utilization and solvent production.

    PubMed

    López-Contreras, A M; Smidt, H; van der Oost, J; Claassen, P A; Mooibroek, H; de Vos, W M

    2001-11-01

    Growth and the production of acetone, butanol, and ethanol by Clostridium beijerinckii NCIMB 8052 on several polysaccharides and sugars were analyzed. On crystalline cellulose, growth and solvent production were observed only when a mixture of fungal cellulases was added to the medium. On lichenan growth and solvent production occurred, but this polymer was only partially utilized. To increase utilization of these polymers and subsequent solvent production, the genes for two new glycoside hydrolases, celA and celD from the fungus Neocallimastix patriciarum, were cloned separately into C. beijerinckii. To do this, a secretion vector based on the pMTL500E shuttle vector and containing the promoter and signal sequence coding region of the Clostridium saccharobutylicum NCP262 eglA gene was constructed and fused either to the celA gene or the celD gene. Stable C. beijerinckii transformants were obtained with the resulting plasmids, pWUR3 (celA) and pWUR4 (celD). The recombinant strains showed clear halos on agar plates containing carboxymethyl cellulose upon staining with Congo red. In addition, their culture supernatants had significant endoglucanase activities (123 U/mg of protein for transformants harboring celA and 78 U/mg of protein for transformants harboring celD). Although C. beijerinckii harboring either celA or celD was not able to grow, separately or in mixed culture, on carboxymethyl cellulose or microcrystalline cellulose, both transformants showed a significant increase in solvent production during growth on lichenan and more extensive degradation of this polymer than that exhibited by the wild-type strain. PMID:11679336

  7. Rapid diagnosis of Clostridium difficile infection by multiplex real-time PCR.

    PubMed

    Barbut, F; Monot, M; Rousseau, A; Cavelot, S; Simon, T; Burghoffer, B; Lalande, V; Tankovic, J; Petit, J-C; Dupuy, B; Eckert, C

    2011-10-01

    The gold standards for the diagnosis of Clostridium difficile infections (CDIs) are the cytotoxicity assay and the toxigenic culture. However, both methods are time-consuming and the results are not available before 24-48 h. We developed and evaluated a multiplex in-house real-time polymerase chain reaction (PCR) assay for the simultaneous detection of toxigenic strains of C. difficile and the presumptive identification of the epidemic NAP1/027/BI strain from stools. Amplifications were performed using specific primers for tcdB and tcdC on an ABI Prism 7300 (Applied Biosystems). The detection of amplicons was done using TaqMan probes. The analytical sensitivity of the multiplex real-time PCR for detecting tcdB was estimated to 10 CFU/g of stools. This assay was assessed from 881 consecutive unformed stools from patients suspected of having CDI. The gold standard was the toxigenic culture for the diagnosis of CDI and PCR ribotyping for the identification of the NAP1/027/BI strain. The prevalence of positive toxigenic culture was 9.31%. Compared to the toxigenic culture, the sensitivity, specificity, and positive and negative predictive values were 86.59%, 97.43%, 78.02%, and 98.57%, respectively, for the real-time PCR and 70.73%, 100%, 100%, and 97.08%, respectively, for the cytotoxicity assay. PMID:21487764

  8. Comparison of ChromID Agar and Clostridium difficile Selective Agar for Effective Isolation of C. difficile from Stool Specimens

    PubMed Central

    Lee, Eun Joo

    2014-01-01

    Background ChromID Clostridium difficile agar (IDCd; bioMérieux SA, France) is a recently developed chromogenic medium for rapid and specific isolation of C. difficile. We compared the performance of IDCd with that of Clostridium difficile Selective Agar (CDSA). Methods A total of 530 fresh stool specimens were collected from patients with clinical signs compatible with C. difficile infection, and cultures for C. difficile were performed on IDCd and CDSA. C. difficile colonies were identified by spore staining, odor, use of an ANI identification test kit (bioMérieux SA), and multiplex PCR for tcdA, tcdB, and tpi. Results The concordance rate between IDCd and CDSA was 90.6% (480/530). The positivity rates on IDCd on days 1 and 2 (55.6% and 85.0%, respectively) were significantly higher than those on CDSA (19.4% and 75.6%, respectively) (P<0.001 for day 1 and P=0.02 for day 2), but the detection rates on IDCd and CDSA on day 3 were not different (89.4% vs. 82.8%, P=0.0914). On day 3, the recovery rates for non-C. difficile isolates on IDCd and CDSA were 30.2% (160/530) and 22.1% (117/530), respectively (P=0.0075). Clostridium spp. other than C. difficile were the most prevalent non-C. difficile isolates on both media. Conclusions The culture positivity rates on IDCd and CDSA were not different on day 3 but IDCd may allow for rapid and sensitive detection of C. difficile within 2 days of cultivation. PMID:24422190

  9. Adherence of Clostridium thermocellum to cellulose.

    PubMed Central

    Bayer, E A; Kenig, R; Lamed, R

    1983-01-01

    The adherence of Clostridium thermocellum, a cellulolytic, thermophilic anaerobe, to its insoluble substrate (cellulose) was studied. The adherence phenomenon was determined to be selective for cellulose. The observed adherence was not significantly affected by various parameters, including salts, pH, temperature, detergents, or soluble sugars. A spontaneous adherence-defective mutant strain (AD2) was isolated from the wild-type strain YS. Antibodies were prepared against the bacterial cell surface and rendered specific to the cellulose-binding factor (CBF) by adsorption to mutant AD2 cells. By using these CBF-specific antibodies, crossed immunoelectrophoresis of cell extracts revealed a single discrete precipitation peak in the parent strain which was absent in the mutant. This difference was accompanied by an alteration in the polypeptide profile whereby sonicates of strain YS contained a 210,000-molecular-weight band which was missing in strain AD2. The CBF antigen could be removed from cell extracts by adsorption to cellulose. A combined gel-overlay--immunoelectrophoretic technique demonstrated that the cellulose-binding properties of the CBF were accompanied by carboxymethylcellulase activity. During the exponential phase of growth, a large part of the CBF antigen and related carboxymethylcellulase activity was associated with the cells of wild-type strain YS. However, the amounts decreased in stationary-phase cells. Cellobiose-grown mutant AD2 cells lacked the cell-associated CBF, but the latter was detected in the extracellular fluid. Increased levels of CBF were observed when cells were grown on cellulose. In addition, mutant AD2 regained cell-associated CBF together with the property of cellulose adherence. The presence of the CBF antigen and related adherence characteristics appeared to be a phenomenon common to other naturally occurring strains of this species. Images PMID:6630152

  10. The emergence of 'hypervirulence' in Clostridium difficile.

    PubMed

    Cartman, Stephen T; Heap, John T; Kuehne, Sarah A; Cockayne, Alan; Minton, Nigel P

    2010-08-01

    The impact of Clostridium difficile-associated disease (CDAD) in healthcare settings throughout the developed world is considerable in terms of mortality, morbidity, and disease management. The incidence of CDAD has risen dramatically since the turn of this century, concomitant with the emergence of so-called hypervirulent strains which are thought to cause a more severe disease, higher relapse rates, and increased mortality. Pre-eminent amongst hypervirulent strains are those belonging to ribotype 027, which were first reported in Canada in 2003 and shortly thereafter in the UK. Since its arrival in Europe, it has spread rapidly and has now been reported in 16 member states and Switzerland. The physiological factors responsible for the rapid emergence of hypervirulent C. difficile strains remain unclear. It is known that they produce a binary toxin (CDT) in addition to toxins A and B, that they are resistant to fluoroquinolones due to mutations in gyrA, and that they are resistant to erythromycin. Representative strains have been suggested to produce more toxin A and B in the 'laboratory flask' (most likely due to a frameshift mutation in the repressor gene tcdC), to be more prolific in terms of spore formation, and also exhibit increased adherence to human intestinal epithelial cells due to altered surface proteins. However, the contribution of these and other as yet unidentified factors to the rapid spread of certain C. difficile variants (e.g., ribotypes 027 and 078) remains unclear at present. The advent of ClosTron technology means that it is now possible to construct genetically stable isogenic mutants of C. difficile and carry out reverse genetic studies to elucidate the role of specific gene loci in causing disease. The identification of virulence factors using this approach should help lead to the rational development of therapeutic countermeasures against CDAD. PMID:20547099

  11. Form and Function of Clostridium thermocellum Biofilms

    PubMed Central

    Dumitrache, Alexandru; Allen, Grant; Liss, Steven N.; Lynd, Lee R.

    2013-01-01

    The importance of bacterial adherence has been acknowledged in microbial lignocellulose conversion studies; however, few reports have described the function and structure of biofilms supported by cellulosic substrates. We investigated the organization, dynamic formation, and carbon flow associated with biofilms of the obligately anaerobic cellulolytic bacterium Clostridium thermocellum 27405. Using noninvasive, in situ fluorescence imaging, we showed biofilms capable of near complete substrate conversion with a characteristic monolayered cell structure without an extracellular polymeric matrix typically seen in biofilms. Cell division at the interface and terminal endospores appeared throughout all stages of biofilm growth. Using continuous-flow reactors with a rate of dilution (2 h−1) 12-fold higher than the bacterium's maximum growth rate, we compared biofilm activity under low (44 g/liter) and high (202 g/liter) initial cellulose loading. The average hydrolysis rate was over 3-fold higher in the latter case, while the proportions of oligomeric cellulose hydrolysis products lost from the biofilm were 13.7% and 29.1% of the total substrate carbon hydrolyzed, respectively. Fermentative catabolism was comparable between the two cellulose loadings, with ca. 4% of metabolized sugar carbon being utilized for cell production, while 75.4% and 66.7% of the two cellulose loadings, respectively, were converted to primary carbon metabolites (ethanol, acetic acid, lactic acid, carbon dioxide). However, there was a notable difference in the ethanol-to-acetic acid ratio (g/g), measured to be 0.91 for the low cellulose loading and 0.41 for the high cellulose loading. The results suggest that substrate availability for cell attachment rather than biofilm colonization rates govern the efficiency of cellulose conversion. PMID:23087042

  12. Colonic Immunopathogenesis of Clostridium difficile Infections

    PubMed Central

    Turnwald, Bradley P.; Koo, Hoonmo L.; Garey, Kevin W.; Jiang, Zhi-Dong; Aitken, Samuel L.; DuPont, Herbert L.

    2014-01-01

    There are major gaps in our understanding of the immunopathogenesis of Clostridium difficile infections (CDIs). In this study, 36 different biomarkers were examined in the stools of CDI and non-CDI patients using the Proteome Profiler human cytokine array assay and quantitative enzyme-linked immunosorbent assay. Diarrheal stools from patients with CDI (CDI-positive diarrheal stools) showed higher relative amounts of the following inflammatory markers than the diarrheal stools from CDI-negative patients (CDI-negative diarrheal stools): C5a, CD40L, granulocyte colony-stimulating factor, I-309, interleukin-13 (IL-13), IL-16, IL-27, monocyte chemoattractant protein 1, tumor necrosis factor alpha, and IL-8. IL-8 and IL-23 were present in a larger number of CDI-positive diarrheal stools than CDI-negative diarrheal stools. Th1 and Th2 cytokines were not significantly different between the CDI-positive and CDI-negative diarrheal stools. Lactoferrin and calprotectin concentrations were also higher in the CDI-positive diarrheal stools. Our results demonstrate that CDI elicits a proinflammatory host response, and we report for the first time that IL-23 is a major marker in CDI-positive diarrheal stools. IL-23 may explain the lack of a robust immunological response exhibited by a proportion of CDI patients and may relate to recurrence; the IL-23 levels induced during CDI in these patients may be inadequate to sustain the cellular immunity conferred by this cytokine in promoting the induction and proliferation of effector memory T cells. PMID:24477852

  13. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B

    PubMed Central

    Genth, Harald; Schelle, Ilona; Just, Ingo

    2016-01-01

    Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the “Large clostridial glycosylating toxins.” These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB—together with Toxin A (TcdA)—is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn2+ > Co2+ > Mg2+ >> Ca2+, Cu2+, Zn2+. TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn2+ and 180 µM for Mg2+. TcsL and TcdB further require co-stimulation by monovalent K+ (not by Na+). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K+ and Mg2+ (rather than Mn2+) in mammalian target cells. PMID:27089365

  14. Metal Ion Activation of Clostridium sordellii Lethal Toxin and Clostridium difficile Toxin B.

    PubMed

    Genth, Harald; Schelle, Ilona; Just, Ingo

    2016-01-01

    Lethal Toxin from Clostridium sordellii (TcsL) and Toxin B from Clostridium difficile (TcdB) belong to the family of the "Large clostridial glycosylating toxins." These toxins mono-O-glucosylate low molecular weight GTPases of the Rho and Ras families by exploiting UDP-glucose as a hexose donor. TcsL is casually involved in the toxic shock syndrome and the gas gangrene. TcdB-together with Toxin A (TcdA)-is causative for the pseudomembranous colitis (PMC). Here, we present evidence for the in vitro metal ion activation of the glucosyltransferase and the UDP-glucose hydrolysis activity of TcsL and TcdB. The following rating is found for activation by divalent metal ions: Mn(2+) > Co(2+) > Mg(2+) > Ca(2+), Cu(2+), Zn(2+). TcsL and TcdB thus require divalent metal ions providing an octahedral coordination sphere. The EC50 values for TcsL were estimated at about 28 µM for Mn(2+) and 180 µM for Mg(2+). TcsL and TcdB further require co-stimulation by monovalent K⁺ (not by Na⁺). Finally, prebound divalent metal ions were dispensible for the cytopathic effects of TcsL and TcdB, leading to the conclusion that TcsL and TcdB recruit intracellular metal ions for activation of the glucosyltransferase activity. With regard to the intracellular metal ion concentrations, TcsL and TcdB are most likely activated by K⁺ and Mg(2+) (rather than Mn(2+)) in mammalian target cells. PMID:27089365

  15. Antimicrobial susceptibilities of canine Clostridium difficile and Clostridium perfringens isolates to commonly utilized antimicrobial drugs.

    PubMed

    Marks, Stanley L; Kather, Elizabeth J

    2003-06-24

    Clostridium difficile and Clostridium perfringens are anaerobic, Gram-positive bacilli that are common causes of enteritis and enterotoxemias in both domestic animals and humans. Both organisms have been associated with acute and chronic large and small bowel diarrhea, and acute hemorrhagic diarrheal syndrome in the dog. The objective of this study was to determine the in vitro antimicrobial susceptibilities of canine C. difficile and C. perfringens isolates in an effort to optimize antimicrobial therapy for dogs with clostridial-associated diarrhea. The minimum inhibitory concentrations (MIC) of antibiotics recommended for treating C. difficile (metronidazole, vancomycin) and C. perfringens-associated diarrhea in the dog (ampicillin, erythromycin, metronidazole, tetracycline, tylosin) were determined for 70 canine fecal C. difficile isolates and 131 C. perfringens isolates. All C. difficile isolates tested had an MIC of or=256 microg/ml for both erythromycin and tylosin. A third C. perfringens isolate had an MIC of 32 microg/ml for metronidazole. Based on the results of this study, ampicillin, erythromycin, metronidazole, and tylosin appear to be effective antibiotics for the treatment of C. perfringens-associated diarrhea, although resistant strains do exist. However, because there is limited information regarding breakpoints for veterinary anaerobes, and because intestinal concentrations are not known, in vitro results should be interpreted with caution. PMID:12742714

  16. Ammonia assimilation pathways in nitrogen-fixing Clostridium kluyverii and Clostridium butyricum.

    PubMed Central

    Kanamori, K; Weiss, R L; Roberts, J D

    1989-01-01

    Pathways of ammonia assimilation into glutamic acid were investigated in ammonia-grown and N2-fixing Clostridium kluyverii and Clostridium butyricum by measuring the specific activities of glutamate dehydrogenase, glutamine synthetase, and glutamate synthase. C. kluyverii had NADPH-glutamate dehydrogenase with a Km of 12.0 mM for NH4+. The glutamate dehydrogenase pathway played an important role in ammonia assimilation in ammonia-grown cells but was found to play a minor role relative to that of the glutamine synthetase/NADPH-glutamate synthase pathway in nitrogen-fixing cells when the intracellular NH4+ concentration and the low affinity of the enzyme for NH4+ were taken into account. In C. butyricum grown on glucose-salt medium with ammonia or N2 as the nitrogen source, glutamate dehydrogenase activity was undetectable, and the glutamine synthetase/NADH-glutamate synthase pathway was the predominant pathway of ammonia assimilation. Under these growth conditions, C. butyricum also lacked the activity of glucose-6-phosphate dehydrogenase, which catalyzes the regeneration of NADPH from NADP+. However, high activities of glucose-6-phosphate dehydrogenase as well as of NADPH-glutamate dehydrogenase with a Km of 2.8 mM for NH4+ were present in C. butyricum after growth on complex nitrogen and carbon sources. The ammonia-assimilating pathway of N2-fixing C. butyricum, which differs from that of the previously studied Bacillus polymyxa and Bacillus macerans, is discussed in relation to possible effects of the availability of ATP and of NADPH on ammonia-assimilating pathways. PMID:2564848

  17. Characterization of microfouling and corrosive bacterial community of a firewater distribution system.

    PubMed

    Palaniappan, Balamurugan; Toleti, Subba Rao

    2016-04-01

    This investigation provides generic information on the culturable corrosive and the microfouling bacterial community in a firewater distribution system that uses freshwater. Conventional microbiological methods were used for the selective isolation of the major microfouling bacteria. The isolates were characterized by 16S rRNA gene sequencing and the biofilm as well as the corrosion characteristics of the isolates were evaluated. Pseudomonas aeruginosa and Bacillus cereus were predominantly observed in all the samples analysed. Denaturing gradient gel electrophoresis (DGGE) was carried out for the various samples of firewater system (FWS) and the high intensity bands were sequenced to identify the predominant bacteria. Bacterial groups such as Cyanobacteria, Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes were identified. Biofilm thickness was recorded using confocal scanning laser microscopy (CSLM). This was the first study to report Lysinibacillus fusiformis in a firewater system and its role in iron corrosion. Sulphidogenic bacteria Tissierella sp. and Clostridium bifermentans generated sulphides in the range of 400-900 ppm. Significant corrosion rates of carbon steel (CS) coupons were observed up to 4.3 mpy. C. bifermentans induced more localized corrosion in CS with a pit diameter of 50 μm. Overall, the data on the characterization of the fouling bacteria, their biofilm forming potential and subsequent metal deterioration studies supported in designing an effective water treatment program. PMID:26467696

  18. Clostridium difficile in mixed populations of animals and humans

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Objectives: Since 2003, there has been an emergence of BI/NAP1 strain of Clostridium difficile (Cd) in North American hospitals. The origins of this epidemic strain have yet to be determined. However, PFGE analysis has shown ~80% similarity between this strain and some swine isolates. The objecti...

  19. Clostridium difficile Ribotype 027, Toxinotype III, the Netherlands

    PubMed Central

    van den Berg, Renate J.; Debast, Sylvia; Visser, Caroline E.; Veenendaal, Dick; Troelstra, Annet; van der Kooi, Tjallie; van den Hof, Susan; Notermans, Daan W.

    2006-01-01

    Outbreaks due to Clostridium difficile polymerase chain reaction (PCR) ribotype 027, toxinotype III, were detected in 7 hospitals in the Netherlands from April 2005 to February 2006. One hospital experienced at the same time a second outbreak due to a toxin A–negative C. difficile PCR ribotype 017 toxinotype VIII strain. The outbreaks are difficult to control. PMID:16704846

  20. Clostridium novyi causing necrotising fasciitis in an injecting drug user

    PubMed Central

    Noone, M; Tabaqchali, M; Spillane, J B

    2002-01-01

    Necrotising fasciitis with pronounced local oedema is described in an injecting drug user. Clostridium novyi was an unexpected single pathogen isolated from infected tissue. The patient was among a cluster of cases, all injecting drug users, presenting with toxaemia and soft tissue infection. The causal role and pathogenicity of C novyi is discussed. PMID:11865011

  1. Clostridium difficile strains from community-associated infections.

    PubMed

    Limbago, Brandi M; Long, Cherie M; Thompson, Angela D; Killgore, George E; Hannett, George E; Havill, Nancy L; Mickelson, Stephanie; Lathrop, Sarah; Jones, Timothy F; Park, Mahin M; Harriman, Kathleen H; Gould, L Hannah; McDonald, L Clifford; Angulo, Frederick J

    2009-09-01

    Clostridium difficile isolates from presumed community-associated infections (n = 92) were characterized by toxinotyping, pulsed-field gel electrophoresis, tcdC and cdtB PCR, and antimicrobial susceptibility. Nine toxinotypes (TOX) and 31 PFGE patterns were identified. TOX 0 (48, 52%), TOX III (18, 20%), and TOX V (9, 10%) were the most common; three isolates were nontoxigenic. PMID:19571021

  2. Clostridium difficile in retail meat and processing plants in Texas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The incidence and severity of disease associated with toxigenic Clostridium difficile (Cd) have increased in hospitals in North America from the emergence of newer, more virulent strains of Cd. Toxigenic Cd has been isolated from food animals and retail meat with potential implications of transfer ...

  3. Genome of a chronic osteitis-causing Clostridium tetani.

    PubMed

    Fournier, P-E; Levy, P-Y; Million, M; Croce, O; Blanc-Tailleur, C; Brouqui, P; Raoult, D

    2014-01-01

    We sequenced the genome of a Clostridium tetani strain that caused chronic tibial osteitis without any clinical sign of tetanus in a 26-year-old man previously vaccinated against this disease. The genome contained a plasmid that harboured the tetX-tetR tetanospasmin operon, and was highly similar to that of a tetanus-causing strain. PMID:25356334

  4. Severe Clostridium difficile-associated disease in children.

    PubMed

    Pokorn, Marko; Radsel, Anja; Cizman, Milan; Jereb, Matjaz; Karner, Primoz; Kalan, Gorazd; Grosek, Stefan; Andlovic, Alenka; Rupnik, Maja

    2008-10-01

    Three cases of Clostridium difficile-associated disease in children were detected within a short time interval. Intensive therapy was required in 2 cases with colectomy in one of them. One of the severe cases was community-acquired. Two patients had underlying diseases (Hirschprung disease, Down syndrome) and also tested positive for enteric viruses (rotavirus, calicivirus). PMID:18756189

  5. Genome Sequence of the Autotrophic Acetogen Clostridium magnum DSM 2767

    PubMed Central

    Uhlig, Ronny; Fischer, Ralf-Jörg; Daniel, Rolf

    2016-01-01

    Here we report the draft genome sequence (6.6 Mbp) of the type strain Clostridium magnum, an acetogen with two operons coding for two separate Rnf complexes. C. magnum grows on a broad range of organic substrates and converts CO2 and H2 to acetate using the Wood-Ljungdahl pathway. PMID:27284147

  6. PREVALENCE OF CLOSTRIDIUM DIFFICILE IN AN INTEGRATED SWINE OPERATION

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the prevalence of Clostridium difficile among different age and production groups of swine in a vertically integrated swine operation in Texas in 2006 and to compare our isolates to other animal and human isolates. Isolation of C. difficile was performed u...

  7. Genome of a chronic osteitis-causing Clostridium tetani

    PubMed Central

    Fournier, P-E; Levy, P-Y; Million, M; Croce, O; Blanc-Tailleur, C; Brouqui, P; Raoult, D

    2014-01-01

    We sequenced the genome of a Clostridium tetani strain that caused chronic tibial osteitis without any clinical sign of tetanus in a 26-year-old man previously vaccinated against this disease. The genome contained a plasmid that harboured the tetX-tetR tetanospasmin operon, and was highly similar to that of a tetanus-causing strain. PMID:25356334

  8. Small Molecules Take A Big Step Against Clostridium difficile.

    PubMed

    Beilhartz, Greg L; Tam, John; Melnyk, Roman A

    2015-12-01

    Effective treatment of Clostridium difficile infections demands a shift away from antibiotics towards toxin-neutralizing agents. Work by Bender et al., using a drug that attenuates toxin action in vivo without affecting bacterial survival, demonstrates the exciting potential of small molecules as a new modality in the fight against C. difficile. PMID:26547239

  9. Clostridium difficile from healthy food animals: Optimized isolation and prevalence

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Two isolation methods were compared for isolation of Clostridium difficile from food animal feces. The single alcohol shock method (SS) used selective enrichment in cycloserine-cefoxitin fructose broth supplemented with 0.1% sodium taurocholate (TCCFB) followed by alcohol shock and isolation on tryp...

  10. Clostridium difficile prevalence in an integrated swine operation in Texas

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Recently there has been an epidemic of human disease in North America caused by the bacterium Clostridium difficile (Cd). It appears to be a new strain that is more virulent than previous strains, produces more toxins, and causes more severe disease (McDonald et al., 2005). The origin of the new s...

  11. Detection of Clostridium perfringens epsilon toxin by ELISA.

    PubMed

    Naylor, R D; Martin, P K; Sharpe, R T

    1987-03-01

    An enzyme-linked immunosorbent assay (ELISA) has been developed as an alternative to neutralisation tests in mice to detect Clostridium perfringens type D epsilon toxin in the intestinal contents of animals which have died from suspected enterotoxaemia. The test was sensitive and quantitative and gave excellent agreement with the mouse protection test. PMID:2884704

  12. Isolation of Clostridium difficile from healthy food animals

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: Clostridium difficile-associated disease is increasingly reported and studies indicate that food animals may be sources of human infections. Methods: The presence of C. difficile in 345 swine fecal, 1,325 dairy cattle fecal, and 371 dairy environmental samples were examined. Two isolati...

  13. ID Learning Unit: Understanding and Interpreting Testing for Clostridium difficile

    PubMed Central

    Solomon, Daniel A.; Milner, Danny A.

    2014-01-01

    Understanding and interpreting the molecular tests for Clostridium difficile is challenging because there are several different types of assays and most laboratories combine multiple tests in order to assess for presence of disease. This learning unit demonstrates the basic principles of each test along with its strengths and weaknesses, and illustrates how the tests are used in clinical practice. PMID:25734081

  14. Clostridium septicum brain abscesses in a premature neonate.

    PubMed

    Sadarangani, Sapna P; Batdorf, Rachel; Buchhalter, Lillian C; Mrelashvili, Anna; Banerjee, Ritu; Henry, Nancy K; Huskins, W Charles; Boyce, Thomas G

    2014-05-01

    Brain abscesses in neonates are typically caused by Gram-negative organisms. There are no previously described cases caused by Clostridium septicum. We present a case of a premature male infant who developed recurrent episodes of suspected necrotizing enterocolitis followed by brain abscesses, cerebritis and ventriculitis caused by C. septicum. PMID:24220230

  15. Varied prevalence of Clostridium difficile in an integrated swine operation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this study was to compare the prevalence of Clostridium difficile among different age and production groups of swine in a vertically integrated swine operation in Texas in 2006 and to compare our isolates to other animal and human isolates. Preliminary results are based on 131 C. d...

  16. Development of a triplex real-time PCR assay for the simultaneous detection of Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum in milk.

    PubMed

    Morandi, Stefano; Cremonesi, Paola; Silvetti, Tiziana; Castiglioni, Bianca; Brasca, Milena

    2015-08-01

    Clostridium beijerinckii, Clostridium sporogenes and Clostridium tyrobutyricum are considered the leading bacteria implicated in late blowing defects affecting semi-hard and hard cheese production. The aim of this study was to develop a multiplex Real-Time PCR (qPCR) analysis for a rapid and simultaneous detection of C. beijerinckii, C. sporogenes and C. tyrobutyricum, using specific primers respectively targeting the nifH, gerAA and enr genes. The limits of detection in raw milk were 300 CFU/50 mL in the case of C. beijerinckii, 2 CFU/50 mL for C. sporogenes and 5 CFU/50 mL for C. tyrobutyricum spores. The qPCR method was applied to artificially contaminated raw milk samples, and molecular quantification showed good correlation (R(2) = 0.978) with microbiological counting. Our results demonstrate that this method, combined with a DNA extraction protocol optimized for spore lysis, could be a useful tool for the direct quantification of the considered clostridia species. PMID:25870135

  17. The ClosTron: Mutagenesis in Clostridium refined and streamlined.

    PubMed

    Heap, John T; Kuehne, Sarah A; Ehsaan, Muhammad; Cartman, Stephen T; Cooksley, Clare M; Scott, Jamie C; Minton, Nigel P

    2010-01-01

    The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. PMID:19891996

  18. CLINICAL AND EPIDEMIOLOGIC CONSIDERATIONS OF CLOSTRIDIUM DIFFICILE IN HARBOR SEALS (PHOCA VITULINA) AT A MARINE MAMMAL REHABILITATION CENTER.

    PubMed

    Anderson, Chelsea E; Haulena, Martin; Zabek, Erin; Habing, Gregory; Raverty, Stephen

    2015-06-01

    Between 1998 and 2008, 15 cases of segmental to diffuse hemorrhagic to necrohemorrhagic enterocolitis were diagnosed in neonatal and weaned juvenile harbor seals (Phoca vitulina) presented from the Vancouver Aquarium Marine Mammal Rescue Centre for rehabilitation. Based on a combination of gross pathology, histopathology, bacterial isolation, and toxin testing, Clostridium difficile enterocolitis was diagnosed. Most pups were anorexic or inappetant and died acutely with few other premonitory signs. Due to ongoing clinical concerns and possible emergence of this pathogen at the facility, efforts to better characterize the disease and understand the epidemiology of C. difficile was initiated in 95 harbor seal pups presented for rehabilitation in a single stranding season. Fecal samples were collected on admission, following completion of antibiotic treatment, and also prerelease or postmortem. All samples were collected fresh and submitted either directly or stored frozen. Fecal samples were inoculated into selective media for culture and screened by enzyme-linked immunosorbant assay (ELISA) for C. difficile toxins A, B, or both. Results of the 95 seals in the study were as follows: on hospital admit 72 seals were sampled, 10 were culture positive, 12 were ELISA positive; following antibiotic therapy 46 seals were sampled noting three culture positive and nine ELISA positive; prior to release 58 seals were sampled noting zero culture positive and one ELISA positive; and on postmortem exam seven seals were sampled noting zero culture positive and two ELISA positive. Clostridium difficile was not deemed to be the cause of death in any of the animals. Although the exact mechanism of disease is unknown, this study suggests that C. difficile infection is not a significant cause of mortality and may be part of the normal flora in harbor seals undergoing rehabilitation. Morbidity and mortality from this bacterium can likely be minimized by judicious use of antibiotics

  19. Nitrate-Dependent Regulation of Acetate Biosynthesis and Nitrate Respiration by Clostridium thermoaceticum

    PubMed Central

    Arendsen, Alexander F.; Soliman, Mohsin Q.; Ragsdale, Stephen W.

    1999-01-01

    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. Fröstl, 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. PMID:10049380

  20. An environmental disinfection odyssey: evaluation of sequential interventions to improve disinfection of Clostridium difficile isolation rooms.

    PubMed

    Sitzlar, Brett; Deshpande, Abhishek; Fertelli, Dennis; Kundrapu, Sirisha; Sethi, Ajay K; Donskey, Curtis J

    2013-05-01

    OBJECTIVE. Effective disinfection of hospital rooms after discharge of patients with Clostridium difficile infection (CDI) is necessary to prevent transmission. We evaluated the impact of sequential cleaning and disinfection interventions by culturing high-touch surfaces in CDI rooms after cleaning. DESIGN. Prospective intervention. SETTING. A Veterans Affairs hospital. INTERVENTIONS. During a 21-month period, 3 sequential tiered interventions were implemented: (1) fluorescent markers to provide monitoring and feedback on thoroughness of cleaning facility-wide, (2) addition of an automated ultraviolet radiation device for adjunctive disinfection of CDI rooms, and (3) enhanced standard disinfection of CDI rooms, including a dedicated daily disinfection team and implementation of a process requiring supervisory assessment and clearance of terminally cleaned CDI rooms. To determine the impact of the interventions, cultures were obtained from CDI rooms after cleaning and disinfection. RESULTS. The fluorescent marker intervention improved the thoroughness of cleaning of high-touch surfaces (from 47% to 81% marker removal; P < .0001). Relative to the baseline period, the prevalence of positive cultures from CDI rooms was reduced by 14% (P=.024), 48% (P <.001), and 89% (P=.006) with interventions 1, 2, and 3, respectively. During the baseline period, 67% of CDI rooms had positive cultures after disinfection, whereas during interventions periods 1, 2, and 3 the percentages of CDI rooms with positive cultures after disinfection were reduced to 57%, 35%, and 7%, respectively. CONCLUSIONS. An intervention that included formation of a dedicated daily disinfection team and implementation of a standardized process for clearing CDI rooms achieved consistent CDI room disinfection. Culturing of CDI rooms provides a valuable tool to drive improvements in environmental disinfection. PMID:23571361

  1. Distribution of Neuraminidase among Food-poisoning Strains of Clostridium perfringens

    PubMed Central

    Moss, C. Wayne; Schekter, Marcia A.; Cherry, William B.

    1967-01-01

    A survey was made to determine the distribution of the enzyme neuraminidase among 76 strains of Clostridium perfringens. Representative strains from each toxigenic type (A to F) and atypical C. perfringens type A food-poisoning strains of both American and English (Hobbs types) origin were tested. Both the American food-poisoning and nonfood-poisoning associated cultures consisted of both neuraminidase-positive and -negative strains. Furthermore, American strains which could not be differentiated from the original Hobbs cultures consisted of both neuraminidase-positive and -negative representatives. In contrast, the English (Hobbs) strains uniformly failed to produce an active intracellular or extracellular neuraminidase. No enzyme activity was detected in these strains when cultures were grown in different growth media, when grown in the presence of substrate (neuraminlactose), or upon extended incubation of enzyme preparations with substrate. With the exception of a type F strain, representative strains of the other toxigenic types (A to F) produced neuraminidase; 85% of the typical type A strains contained the enzyme. PMID:4292823

  2. Mechanisms of enhanced cellulosic bioethanol fermentation by co-cultivation of Clostridium and Thermoanaerobacter spp.

    PubMed

    He, Qiang; Hemme, Christopher L; Jiang, Helong; He, Zhili; Zhou, Jizhong

    2011-10-01

    Engineering microbial consortia capable of efficient ethanolic fermentation of cellulose is a strategy for the development of consolidated bioprocessing for bioethanol production. Co-cultures of cellulolytic Clostridium thermocellum with non-cellulolytic Thermoanaerobacter strains (X514 and 39E) significantly improved ethanol production by 194-440%. Strain X514 enhanced ethanolic fermentation much more effectively than strain 39E in co-cultivation, with ethanol production in X514 co-cultures at least 62% higher than that of 39E co-cultures. Comparative genome sequence analysis revealed that the higher ethanolic fermentation efficiency in strain X514 was associated with the presence of a complete vitamin B(12) biosynthesis pathway, which is incomplete in strain 39E. The significance of the vitamin B(12)de novo biosynthesis capacity was further supported by the observation of improved ethanol production in strain 39E by 203% following the addition of exogenous vitamin B(12). The vitamin B(12) biosynthesis pathway provides a valuable biomarker for selecting metabolically robust strains for bioethanol production. PMID:21868218

  3. Isolation of Clostridium difficile from the environment and contacts of patients with antibiotic-associated colitis

    SciTech Connect

    Kim, K.H.; Fekety, R.; Batts, D.H.; Brown, D.; Cudmore, M.; Silva, J. Jr.; Waters, D.

    1981-01-01

    Clostridium difficile is the most important cause of antibiotic-associated colitis, but its epidemiology remains unknown. Using a selective medium for the isolation of C. difficile, cultures were obtained from the environment and contacts of hospitalized patients carrying C. difficile in their stools. In areas where carriers had diarrhea, 85 (9.3%) of 910 cultures of floors and other surfaces, especially those subject to fecal contamination, were positive. In areas where there were no known carriers, only 13 (2.6%) of 497 cultures of similar sites were positive (P less than 0.005). C difficile was isolated from hands and stools of asymptomatic hospital personnel, from sewage and soil, and from the home of a patient. Environmental isolates were toxigenic. C. difficile inoculated onto a floor persisted there for five months. Further studies are needed to document how often floor persisted there for five months. Further studies are needed to document how often C. difficile shed by patients with antibiotic-associated colitis is acquired by other persons and whether isolation precautions are capable of limiting the organism's spread.

  4. Characterization of Xylanolytic Enzymes in Clostridium cellulovorans: Expression of Xylanase Activity Dependent on Growth Substrates

    PubMed Central

    Kosugi, Akihiko; Murashima, Koichiro; Doi, Roy H.

    2001-01-01

    Xylanase activity of Clostridium cellulovorans, an anaerobic, mesophilic, cellulolytic bacterium, was characterized. Most of the activity was secreted into the growth medium when the bacterium was grown on xylan. Furthermore, when the extracellular material was separated into cellulosomal and noncellulosomal fractions, the activity was present in both fractions. Each of these fractions contained at least two major and three minor xylanase activities. In both fractions, the pattern of xylan hydrolysis products was almost identical based on thin-layer chromatography analysis. The major xylanase activities in both fractions were associated with proteins with molecular weights of about 57,000 and 47,000 according to zymogram analyses, and the minor xylanases had molecular weights ranging from 45,000 to 28,000. High α-arabinofuranosidase activity was detected exclusively in the noncellulosomal fraction. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that cellulosomes derived from xylan-, cellobiose-, and cellulose-grown cultures had different subunit compositions. Also, when xylanase activity in the cellulosomes from the xylan-grown cultures was compared with that of cellobiose- and cellulose-grown cultures, the two major xylanases were dramatically increased in the presence of xylan. These results strongly indicated that C. cellulovorans is able to regulate the expression of xylanase activity and to vary the cellulosome composition depending on the growth substrate. PMID:11717260

  5. Optimization of Influential Nutrients during Direct Cellulose Fermentation into Hydrogen by Clostridium thermocellum

    PubMed Central

    Islam, Rumana; Sparling, Richard; Cicek, Nazim; Levin, David B.

    2015-01-01

    Combinatorial effects of influential growth nutrients were investigated in order to enhance hydrogen (H2) production during direct conversion of cellulose by Clostridium thermocellum DSM 1237. A central composite face-centered design and response surface methodology (RSM) were applied to optimize concentrations of cellulose, yeast extract (YE), and magnesium chloride (Mg) in culture. The overall optimum composition generated by the desirability function resulted in 57.28 mmol H2/L-culture with 1.30 mol H2/mol glucose and 7.48 mmol/(g·cell·h) when cultures contained 25 g/L cellulose, 2 g/L YE, and 1.75 g/L Mg. Compared with the unaltered medium, the optimized medium produced approximately 3.2-fold more H2 within the same time-frame with 50% higher specific productivity, which are also better than previously reported values from similar studies. Nutrient composition that diverted carbon and electron flux away from H2 promoting ethanol production was also determined. This study represents the first investigation dealing with multifactor optimization with RSM for H2 production during direct cellulose fermentation. PMID:25647413

  6. Clostridium difficile associated infection, diarrhea and colitis

    PubMed Central

    Hookman, Perry; Barkin, Jamie S

    2009-01-01

    A new, hypervirulent strain of Clostridium difficile, called NAP1/BI/027, has been implicated in C. difficile outbreaks associated with increased morbidity and mortality since the early 2000s. The epidemic strain is resistant to fluoroquinolones in vitro, which was infrequent prior to 2001. The name of this strain reflects its characteristics, demonstrated by different typing methods: pulsed-field gel electrophoresis (NAP1), restriction endonuclease analysis (BI) and polymerase chain reaction (027). In 2004 and 2005, the US Centers for Disease Control and Prevention (CDC) emphasized that the risk of C. difficile-associated diarrhea (CDAD) is increased, not only by the usual factors, including antibiotic exposure, but also gastrointestinal surgery/manipulation, prolonged length of stay in a healthcare setting, serious underlying illness, immune-compromising conditions, and aging. Patients on proton pump inhibitors (PPIs) have an elevated risk, as do peripartum women and heart transplant recipients. Before 2002, toxic megacolon in C. difficile-associated colitis (CDAC), was rare, but its incidence has increased dramatically. Up to two-thirds of hospitalized patients may be infected with C. difficile. Asymptomatic carriers admitted to healthcare facilities can transmit the organism to other susceptible patients, thereby becoming vectors. Fulminant colitis is reported more frequently during outbreaks of C. difficile infection in patients with inflammatory bowel disease (IBD). C. difficile infection with IBD carries a higher mortality than without underlying IBD. This article reviews the latest information on C. difficile infection, including presentation, vulnerable hosts and choice of antibiotics, alternative therapies, and probiotics and immunotherapy. We review contact precautions for patients with known or suspected C. difficile-associated disease. Healthcare institutions require accurate and rapid diagnosis for early detection of possible outbreaks, to initiate

  7. Multicenter Evaluation of the Clostridium difficile TOX A/B TEST

    PubMed Central

    Lyerly, D. M.; Neville, L. M.; Evans, D. T.; Fill, J.; Allen, S.; Greene, W.; Sautter, R.; Hnatuck, P.; Torpey, D. J.; Schwalbe, R.

    1998-01-01

    Clostridium difficile, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the “gold standard” for C. difficile testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA−/toxB+) isolates were identified. One specimen was from a

  8. Unique regulatory mechanism of sporulation and enterotoxin production in Clostridium perfringens.

    PubMed

    Ohtani, Kaori; Hirakawa, Hideki; Paredes-Sabja, Daniel; Tashiro, Kosuke; Kuhara, Satoru; Sarker, Mahfuzur R; Shimizu, Tohru

    2013-06-01

    Clostridium perfringens causes gas gangrene and gastrointestinal (GI) diseases in humans. The most common cause of C. perfringens-associated food poisoning is the consumption of C. perfringens vegetative cells followed by sporulation and production of enterotoxin in the gut. Despite the importance of spore formation in C. perfringens pathogenesis, the details of the regulation of sporulation have not yet been defined fully. In this study, microarray and bioinformatic analyses identified a candidate gene (the RNA regulator virX) for the repression of genes encoding positive regulators (Spo0A and sigma factors) of C. perfringens sporulation. A virX mutant constructed in the food poisoning strain SM101 had a much higher sporulation efficiency than that of the wild type. The transcription of sigE, sigF, and sigK was strongly induced at 2.5 h of culture of the virX mutant. Moreover, the transcription of the enterotoxin gene was also strongly induced in the virX mutant. Western blotting confirmed that the levels of enterotoxin production were higher in the virX mutant than in the wild type. These observations indicated that the higher levels of sporulation and enterotoxin production in the virX mutant were specifically due to inactivation of the virX gene. Since virX homologues were not found in any Bacillus species but were present in other clostridial species, our findings identify further differences in the regulation of sporulation between Bacillus and certain Clostridium species. The virX RNA regulator plays a key role in the drastic shift in lifestyle of the anaerobic flesh eater C. perfringens between the vegetative state (for gas gangrene) and the sporulating state (for food poisoning). PMID:23585540

  9. Identification of risk factors influencing Clostridium difficile prevalence in middle-size dairy farms.

    PubMed

    Bandelj, Petra; Blagus, Rok; Briski, France; Frlic, Olga; Vergles Rataj, Aleksandra; Rupnik, Maja; Ocepek, Matjaz; Vengust, Modest

    2016-01-01

    Farm animals have been suggested to play an important role in the epidemiology of Clostridium difficile infection (CDI) in the community. The purpose of this study was to evaluate risk factors associated with C. difficile dissemination in family dairy farms, which are the most common farming model in the European Union. Environmental samples and fecal samples from cows and calves were collected repeatedly over a 1 year period on 20 mid-size family dairy farms. Clostridium difficile was detected in cattle feces on all farms using qPCR. The average prevalence between farms was 10% (0-44.4%) and 35.7% (3.7-66.7%) in cows and calves, respectively. Bacterial culture yielded 103 C. difficile isolates from cattle and 61 from the environment. Most C. difficile isolates were PCR-ribotype 033. A univariate mixed effect model analysis of risk factors associated dietary changes with increasing C. difficile prevalence in cows (P = 0.0004); and dietary changes (P = 0.004), breeding Simmental cattle (P = 0.001), mastitis (P = 0.003) and antibiotic treatment (P = 0.003) in calves. Multivariate analysis of risk factors found that dietary changes in cows (P = 0.0001) and calves (P = 0.002) increase C. difficile prevalence; mastitis was identified as a risk factor in calves (P = 0.001). This study shows that C. difficile is common on dairy farms and that shedding is more influenced by farm management than environmental factors. Based on molecular typing of C. difficile isolates, it could also be concluded that family dairy farms are currently not contributing to increased CDI incidence. PMID:26968527

  10. Evaluation of Clostridium novyi–NT spores in dogs with naturally occurring tumors

    PubMed Central

    Krick, Erika L.; Sorenmo, Karin U.; Rankin, Shelley C.; Cheong, Ian; Kobrin, Barry; Thornton, Katherine; Kinzler, Kenneth W.; Vogelstein, Bert; Zhou, Shibin; Diaz, Luis A.

    2015-01-01

    Objective To establish the maximum tolerated dose of Clostridium novyi–NT spores in tumor-bearing dogs and evaluate spore germination within tumors and tumor response. Animals 6 client-owned dogs. Procedures A standard dose-escalation study was planned, with maximum tolerated dose defined as the highest dose at which 0 or 1 of 6 dogs had dose-limiting toxicoses (DLT). Dogs received 1 dose of C novyi–NT spores IV. Toxicoses were graded and interventions performed according to specific guidelines. Grade 3 or higher toxicosis or any toxicosis combination that substantially affected patient status was considered DLT. Clinical response was measured by use of response evaluation criteria in solid tumors at 28 days. Results The first 2 dogs had DLT. The dose was decreased. Two of the next 4 dogs had DLT; therefore, dose administration was stopped because the study endpoint had been reached. The most common toxicosis was fever (n = 6 dogs). Two dogs developed abscesses (1 within a nasal carcinoma and 1 splenic abscess) attributable to C novyi–NT infection; both required surgical intervention. Clostridium novyi–NT was cultured from 1 of 6 tumors. Five dogs were available for response assessment (4 had stable disease; 1 had progressive disease). Conclusions and Clinical Relevance Results indicated that C novyi–NT can germinate within tumors of dogs. Toxicosis, although common and sometimes severe, was manageable with treatment. Further studies in dogs with superficial tumors may allow for continued dose escalation and provide information for use in clinical trials in veterinary and human oncology. PMID:22204296

  11. FT-IR spectroscopic analysis for studying Clostridium cell response to conversion of enzymatically hydrolyzed hay

    NASA Astrophysics Data System (ADS)

    Grube, Mara; Gavare, Marita; Nescerecka, Alina; Tihomirova, Kristina; Mezule, Linda; Juhna, Talis

    2013-07-01

    Grass hay is one of assailable cellulose containing non-food agricultural wastes that can be used as a carbohydrate source by microorganisms producing biofuels. In this study three Clostridium strains Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium tetanomorphum, capable of producing acetone, butanol and ethanol (ABE) were adapted to convert enzymatically hydrolyzed hay used as a growth media additive. The results of growth curves, substrate degradation kinetics and FT-IR analyses of bacterial biomass macromolecular composition showed diverse strain-specific cell response to the growth medium composition.

  12. Discrimination of clostridium species using a magnetic bead based hybridization assay

    NASA Astrophysics Data System (ADS)

    Pahlow, Susanne; Seise, Barbara; Pollok, Sibyll; Seyboldt, Christian; Weber, Karina; Popp, Jürgen

    2014-05-01

    Clostridium chauvoei is the causative agent of blackleg, which is an endogenous bacterial infection. Mainly cattle and other ruminants are affected. The symptoms of blackleg are very similar to those of malignant edema, an infection caused by Clostridium septicum. [1, 2] Therefore a reliable differentiation of Clostridium chauvoei from other Clostridium species is required. Traditional microbiological detection methods are time consuming and laborious. Additionally, the unique identification is hindered by the overgrowing tendency of swarming Clostridium septicum colonies when both species are present. [1, 3, 4] Thus, there is a crucial need to improve and simplify the specific detection of Clostridium chauvoei and Clostridium septicum. Here we present an easy and fast Clostridium species discrimination method combining magnetic beads and fluorescence spectroscopy. Functionalized magnetic particles exhibit plentiful advantages, like their simple manipulation in combination with a large binding capacity of biomolecules. A specific region of the pathogenic DNA is amplified and labelled with biotin by polymerase chain reaction (PCR). These PCR products were then immobilized on magnetic beads exploiting the strong biotin-streptavidin interaction. The specific detection of different Clostridium species is achieved by using fluorescence dye labeled probe DNA for the hybridization with the immobilized PCR products. Finally, the samples were investigated by fluorescence spectroscopy. [5

  13. Evaluation of the VIDAS glutamate dehydrogenase assay for the detection of Clostridium difficile.

    PubMed

    Shin, Bo-Moon; Lee, Eun Joo; Moon, Jung Wha; Lee, Seon Yeong

    2016-08-01

    We evaluated the performance of the VIDAS GDH assay for the detection of Clostridium difficile. In total, 350 fecal specimens collected from patients clinically suspected of having CDI were analyzed by C. difficile culture and enzyme-linked fluorescent immunoassay (VIDAS GDH); the results were compared with those of toxigenic C. difficile culture (TC), PCR (Xpert C. difficile assay), and toxin AB EIA (VIDAS CDAB). The numbers of culture-positive and culture-negative samples were 108 and 242, respectively. The concordance between the GDH assay and C. difficile culture was 90.3%. With PCR, 12 more samples were found to be positive in GDH-positive/C. difficile culture-negative specimens. Thus, the concordance between GDH assay and C. difficile culture/PCR was 93.7%. The sensitivity, specificity, positive predictive value, and negative predictive value of the VIDAS GDH assay were 97.2%, 87.2%, 77.2%, and 98.6%, respectively, based on the C. difficile culture, and 97.5%, 91.7%, 86.0%, and 98.6%, respectively, based on C. difficile culture/PCR. Positivity rates of the GDH assay were partially associated with those of semi-quantitative C. difficile cultures, which were maximized in grade 3 (>100 colony-forming unit [CFU]) compared with grade 1 (<10 CFU). We evaluated the two-step or three-step algorithm using GDH assay as a first step. No toxin EIA-positive case was found among GDH-negative samples, and 60.8% (48/79) were TC- and/or PCR-positive among the GDH-positive/toxin EIA-negative samples. Thus, approximately 25% of the 350 samples required a confirmatory test (TC or PCR) in the GDH-toxin EIA algorithm, whereas only 2.3% of the total samples in GDH-PCR algorithm was discrepant and required another confirmatory test like TC. PMID:27282799

  14. Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates

    PubMed Central

    Alvelo-Maurosa, Jesús G.; Lee, Scott J.; Hazen, Samuel P.

    2015-01-01

    A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass. PMID:26637592

  15. Direct Image-Based Enumeration of Clostridium phytofermentans Cells on Insoluble Plant Biomass Growth Substrates.

    PubMed

    Alvelo-Maurosa, Jesús G; Lee, Scott J; Hazen, Samuel P; Leschine, Susan B

    2016-02-01

    A dual-fluorescent-dye protocol to visualize and quantify Clostridium phytofermentans ISDg (ATCC 700394) cells growing on insoluble cellulosic substrates was developed by combining calcofluor white staining of the growth substrate with cell staining using the nucleic acid dye Syto 9. Cell growth, cell substrate attachment, and fermentation product formation were investigated in cultures containing either Whatman no. 1 filter paper, wild-type Sorghum bicolor, or a reduced-lignin S. bicolor double mutant (bmr-6 bmr-12 double mutant) as the growth substrate. After 3 days of growth, cell numbers in cultures grown on filter paper as the substrate were 6.0- and 2.2-fold higher than cell numbers in cultures with wild-type sorghum and double mutant sorghum, respectively. However, cells produced more ethanol per cell when grown with either sorghum substrate than with filter paper as the substrate. Ethanol yields of cultures were significantly higher with double mutant sorghum than with wild-type sorghum or filter paper as the substrate. Moreover, ethanol production correlated with cell attachment in sorghum cultures: 90% of cells were directly attached to the double mutant sorghum substrate, while only 76% of cells were attached to wild-type sorghum substrate. With filter paper as the growth substrate, ethanol production was correlated with cell number; however, with either wild-type or mutant sorghum, ethanol production did not correlate with cell number, suggesting that only a portion of the microbial cell population was active during growth on sorghum. The dual-staining procedure described here may be used to visualize and enumerate cells directly on insoluble cellulosic substrates, enabling in-depth studies of interactions of microbes with plant biomass. PMID:26637592

  16. Models for the study of Clostridium difficile infection

    PubMed Central

    Best, Emma L.; Freeman, Jane; Wilcox, Mark H.

    2012-01-01

    Models of Clostridium difficile infection (C. difficile) have been used extensively for Clostridium difficile (C. difficile) research. The hamster model of C. difficile infection has been most extensively employed for the study of C. difficile and this has been used in many different areas of research, including the induction of C. difficile, the testing of new treatments, population dynamics and characterization of virulence. Investigations using in vitro models for C. difficile introduced the concept of colonization resistance, evaluated the role of antibiotics in C. difficile development, explored population dynamics and have been useful in the evaluation of C. difficile treatments. Experiments using models have major advantages over clinical studies and have been indispensible in furthering C. difficile research. It is important for future study programs to carefully consider the approach to use and therefore be better placed to inform the design and interpretation of clinical studies. PMID:22555466

  17. Beneficial and harmful roles of bacteria from the Clostridium genus.

    PubMed

    Samul, Dorota; Worsztynowicz, Paulina; Leja, Katarzyna; Grajek, Włodzimierz

    2013-01-01

    Bacteria of the Clostridium genus are often described only as a biological threat and a foe of mankind. However, many of them have positive properties and thanks to them they may be used in many industry branches (e.g., in solvents and alcohol production, in medicine, and also in esthetic cosmetology). During the last 10 years interest in application of C. botulinum and C. tetani in medicine significantly increased. Currently, the structure and biochemical properties of neurotoxins produced by these bacterial species, as well as possibilities of application of such toxins as botulinum as a therapeutic factor in humans, are being intensely researched. The main aim of this article is to demonstrate that bacteria from Clostridium spp. are not only pathogens and the enemy of humanity but they also have many important beneficial properties which make them usable among many chemical, medical, and cosmetic applications. PMID:24432307

  18. Clostridium difficile spore biology: sporulation, germination, and spore structural proteins

    PubMed Central

    Paredes-Sabja, Daniel; Shen, Aimee; Sorg, Joseph A.

    2014-01-01

    Clostridium difficile is a Gram-positive, spore-forming obligate anaerobe and a major nosocomial pathogen of world-wide concern. Due to its strict anaerobic requirements, the infectious and transmissible morphotype is the dormant spore. In susceptible patients, C. difficile spores germinate in the colon to form the vegetative cells that initiate Clostridium difficile infections (CDI). During CDI, C. difficile induces a sporulation pathway that produces more spores; these spores are responsible for the persistence of C. difficile in patients and horizontal transmission between hospitalized patients. While important to the C. difficile lifecycle, the C. difficile spore proteome is poorly conserved when compared to members of the Bacillus genus. Further, recent studies have revealed significant differences between C. difficile and B. subtilis at the level of sporulation, germination and spore coat and exosporium morphogenesis. In this review, the regulation of the sporulation and germination pathways and the morphogenesis of the spore coat and exosporium will be discussed. PMID:24814671

  19. Clostridium perfringens in animal disease: a review of current knowledge.

    PubMed

    Niilo, L

    1980-05-01

    The diseases caused by various types of Clostridium perfringens are critically reviewed in the light of current knowledge. Particular emphasis is placed on information concerning these diseases in Canadian livestock. There are two etiologically clearly-defined acute C. perfringens diseases recognized in Canada: hemorrhagic enteritis of the new born calf, caused by C. perfringens type C, and enterotoxemia of sheep, caused by type D. Clostridium perfringens type A may play a role as a secondary pathological agent in various disease conditions, such as necrotic enteritis of chickens. It may also cause wound infections and may provide a source for human food poisoning outbreaks. There appears to be a considerable lack of knowledge regarding the distribution of C. perfringens types, their pathogenesis, diagnosis and the incidence of diseases caused by this organism. PMID:6253040

  20. Nosocomial outbreak of Clostridium difficile diarrhea in a pediatric service.

    PubMed

    Ferroni, A; Merckx, J; Ancelle, T; Pron, B; Abachin, E; Barbut, F; Larzul, J; Rigault, P; Berche, P; Gaillard, J L

    1997-12-01

    An outbreak of nosocomial diarrhea that occurred in a pediatric orthopedic service between 1 December 1993 and 15 April 1994 is reported. A total of 37 patients (mean age, 9.6 years; range, 2 months-19.3 years) were involved in the outbreak, including six patients with bacteriologically documented Clostridium difficile infection. A multivariate analysis identified lincomycin treatment for at least three days as the only significant risk factor. Stool samples from four asymptomatic patients were also positive for Clostridium difficile and its cytotoxins. Isolates from all patients belonged to serogroup C, were highly resistant to lincomycin, and exhibited the same restriction pattern by pulsed-field gel electrophoresis. The outbreak ended after treatment with lincomycin was discontinued and hygiene control measures were implemented. PMID:9495676

  1. The utilization of a commercial soil nucleic acid extraction kit and PCR for the detection of Clostridium tetanus and Clostridium chauvoei on farms after flooding in Taiwan.

    PubMed

    Huang, Shr-Wei; Chan, Jacky Peng-Wen; Shia, Wei-Yau; Shyu, Chin-Lin; Tung, Kwon-Chung; Wang, Chi-Young

    2013-05-01

    Clostridial diseases are zoonoses and are classified as soil-borne diseases. Clostridium chauvoei and Clostridium tetani cause blackleg disease and tetanus, respectively. Since bacteria and spores are re-distributed by floods and then, subsequently, contaminate soils, pastures and water; the case numbers associated with clostridial diseases usually increase after floods. Because Taiwan is often affected by flood damage during the typhoon season, possible threats from these diseases are present. Thus, this study's aim is to apply a combination of a commercial nucleic acid extraction kit and PCR to assess the prevalence of Clostridia spp. in soil and to compare the positivity rates for farms before and after floods. The minimum amounts of Clostridium tetanus and Clostridium chauvoei that could be extracted from soils and detected by PCR were 10 and 50 colony forming units (cfu), respectively. In total, 76 samples were collected from the central and southern regions of Taiwan, which are the areas that are most frequently damaged by typhoons. Noteworthy, the positive rates for Clostridium tetanus and Clostridium chauvoei in Pingtung county after the severe floods caused by a typhoon increased significantly from 13.73 and 7.84% to 53.85 and 50.00%, respectively. This study for the first time provides the evidence from surveillance data that there are changes in the environmental distribution of Clostridium spp. after floods. This study indicates that screening for soil-related zoonotic pathogens is a potential strategy that may help to control these diseases. PMID:23208321

  2. Closed Genome Sequence of Clostridium pasteurianum ATCC 6013

    PubMed Central

    Rotta, Carlo; Poehlein, Anja; Schwarz, Katrin; McClure, Peter; Daniel, Rolf

    2015-01-01

    We report here the closed genome of Clostridium pasteurianum ATCC 6013, a saccharolytic, nitrogen-fixing, and spore-forming Gram-positive obligate anaerobe. The organism is of biotechnological interest due to the production of solvents (butanol and 1,3-propanediol) but can be associated with food spoilage. The genome comprises a total of 4,351,223 bp. PMID:25700419

  3. Clostridium difficile in Children: To Treat or Not to Treat?

    PubMed Central

    2014-01-01

    Clostridium difficile infection has been increasing since 2000 in children and in adults. Frequent antibiotics use, comorbidity, and the development of hypervirulent strains have increased the risk of infection. Despite the high carriage rates of C. difficile, infants rarely develop clinical infection. Discontinuing antibiotics and supportive management usually leads to resolution of disease. Antibiotics use should be stratified depending on the patient's age and severity of the disease. PMID:25061582

  4. Clostridium chauvoei-associated meningoencephalitis in a calf.

    PubMed

    2016-01-16

    ·Meningoencephalitis in a calf associated with Clostridium chauvoei infection. ·Bovine papular stomatitis in calves. ·Otitis media due to Mycoplasma bovis in calves. ·Sporadic porcine abortion due to Nocardia species. ·Spotty liver disease in hens. These are among matters discussed in the disease surveillance report for September 2015 from SAC Consulting: Veterinary Services (SAC C VS). PMID:26769810

  5. Historical and current perspectives on Clostridium botulinum diversity.

    PubMed

    Smith, Theresa J; Hill, Karen K; Raphael, Brian H

    2015-05-01

    For nearly one hundred years, researchers have attempted to categorize botulinum neurotoxin-producing clostridia and the toxins that they produce according to biochemical characterizations, serological comparisons, and genetic analyses. Throughout this period the bacteria and their toxins have defied such attempts at categorization. Below is a description of both historic and current Clostridium botulinum strain and neurotoxin information that illustrates how each new finding has significantly added to the knowledge of the botulinum neurotoxin-containing clostridia and their diversity. PMID:25312020

  6. Clostridium perfringens Type A Food Poisoning II. Response of the Rabbit Ileum as an Indication of Enteropathogenicity of Strains of Clostridium perfringens in Human Beings

    PubMed Central

    Strong, Dorothy H.; Duncan, Charles L.; Perna, Giuseppe

    1971-01-01

    The effect of feeding human beings individual strains of Clostridium perfringens or culture filtrates thereof was examined. The strains selected for challenge included both those which had previously been shown to produce fluid accumulation in the ligated ileum or overt diarrhea when injected into the nonligated ileum of the rabbit, or had produced both, and those which did not regularly produce these responses. Challenge doses prepared by allowing each strain to grow in beef stew for 3 hr at 46 C resulted in a 61% incidence of diarrhea when rabbit-positive cells were used. No diarrhea occurred among the subjects fed rabbit-negative strains prepared in a similar manner. The procedures employed in preparing the challenge dose appeared to influence the results obtained. When cell-free filtrates were fed, 4 of 15 persons consuming filtrates from rabbit-positive strains developed diarrhea. All subjects fed filtrates from rabbit-negative strains remained free from diarrhea. Serological tests were carried out to compare the identity of the strains of C. perfringens consumed by the subjects and those excreted in the feces. Heat resistance measured as D100 values varied greatly among the rabbit-positive strains. PMID:16557937

  7. A Thermophilic Phage Endolysin Fusion to a Clostridium perfringens-Specific Cell Wall Binding Domain Creates an Anti-Clostridium Antimicrobial with Improved Thermostability

    PubMed Central

    Swift, Steven M.; Seal, Bruce S.; Garrish, Johnna K.; Oakley, Brian B.; Hiett, Kelli; Yeh, Hung-Yueh; Woolsey, Rebekah; Schegg, Kathleen M.; Line, John Eric; Donovan, David M.

    2015-01-01

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Many enzymes are added to animal feed during production and are subjected to high-heat stress during feed processing. To produce a thermostabile endolysin for treating poultry, an E. coli codon-optimized gene was synthesized that fused the N-acetylmuramoyl-l-alanine amidase domain from the endolysin of the thermophilic bacteriophage ΦGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ΦCP26F. The resulting protein, PlyGVE2CpCWB, lysed C. perfringens in liquid and solid cultures. PlyGVE2CpCWB was most active at pH 8, had peak activity at 10 mM NaCl, 40% activity at 150 mM NaCl and was still 16% active at 600 mM NaCl. The protein was able to withstand temperatures up to 50 °C and still lyse C. perfringens. Herein, we report the construction and characterization of a thermostable chimeric endolysin that could potentially be utilized as a feed additive to control the bacterium during poultry production. PMID:26075507

  8. [Massive intravascular hemolysis secondary to sepsis due to Clostridium perfringens].

    PubMed

    Pita Zapata, E; Sarmiento Penide, A; Bautista Guillén, A; González Cabano, M; Agulla Budiño, J A; Camba Rodríguez, M A

    2010-05-01

    Massive hemolysis secondary to sepsis caused by Clostridium perfringens is a rare entity but appears fairly often in the literature. In nearly all published reports, the clinical course is rapid and fatal. We describe the case of a 75-year-old woman with diabetes who was admitted with symptoms consistent with acute cholecystitis. Deteriorating hemodynamics and laboratory findings were consistent with intravascular hemolysis, coagulation disorder, and renal failure. Gram-positive bacilli of the Clostridium species were detected in blood along with worsening indicators of hemolysis. In spite of antibiotic and surgical treatment, hemodynamic support and infusion of blood products, the patient continued to decline and died in the postoperative recovery unit 14 hours after admission. Mortality ranges from 70% to 100% in sepsis due to Clostridium perfringens, and risk of death is greater if massive hemolysis is present, as in the case we report. Only a high degree of clinical suspicion leading to early diagnosis and treatment can improve the prognosis. This bacterium should therefore be considered whenever severe sepsis and hemolysis coincide. PMID:20527348

  9. [Toxins of Clostridium perfringens as a natural and bioterroristic threats].

    PubMed

    Omernik, Andrzej; Płusa, Tadeusz

    2015-09-01

    Clostridium perfringens is absolutely anaerobic rod-shaped, sporeforming bacterium. The morbidity is connected with producing toxins. Depending on the type of toxin produced Clostridium perfringens can be divided into five serotypes:A-E. Under natural conditions, this bacterium is responsible for local outbreaks of food poisoning associated with eating contaminated food which which was improperly heat treated. Some countries with lower economic level are endemic foci of necrotizing enteritis caused by Clostridium perfringens. The bacterium is also a major cause of gas gangrene. It is a disease, associated with wound infection, with potentially fatal prognosis in the case of treatment's delays. In the absence of early radical surgery, antibiotic therapy and (if available) hyperbaric treatment leads to the spread of toxins in the body causing shock, coma and death. Due to the force of produced toxins is a pathogen that poses a substrate for the production of biological weapons. It could potentially be used to induce outbreaks of food poisoning and by missiles contamination by spore lead to increased morbidity of gas gangrene in injured soldiers. C. perfringens types B and D produce epsilon toxin considered to be the third most powerful bacterial toxin. Because of the ability to disperse the toxin as an aerosol and a lack of methods of treatment and prevention of poisoning possible factors it is a potential tool for bioterrorism It is advisable to continue research into vaccines and treatments for poisoning toxins of C. perfringens. PMID:26449576

  10. Development of a minimal medium for Clostridium perfringens by using an anaerobic chemostat.

    PubMed Central

    Goldner, S B; Solberg, M; Post, L S

    1985-01-01

    A minimal medium was developed for the cultivation of Clostridium perfringens in an anaerobic chemostat. Cultures of C. perfringens ATCC 3624 and NCTC 10240 were grown at 46 and 43 degrees C, respectively, in a glucose-limited, chemically defined medium at pH 7.2. The concentrations of amino acids, minerals, nucleotides, and vitamins, initially present in excess, were varied independently. The minimum concentration of each nutrient which would support 3 X 10(8) CFU/ml with a generation time of less than 40 min was determined and used to develop a reformulated defined medium. Atomic absorption spectroscopy and amino acid analyses of the reformulated medium indicated additional adjustments in nutrient content which led to the development of a minimal medium for each strain. The nutritional profile for each strain was similar. A decrease in the concentration of arginine, histidine, and tyrosine for strain 3624 and of arginine, histidine, and isoleucine for strain 10240 resulted in an increase in the optical density of each culture. PMID:2864896

  11. Continuous lactose fermentation by Clostridium acetobutylicum--assessment of solventogenic kinetics.

    PubMed

    Procentese, Alessandra; Raganati, Francesca; Olivieri, Giuseppe; Russo, Maria Elena; Salatino, Piero; Marzocchella, Antonio

    2015-03-01

    This work reports the results of a series of tests on the specific butanol production rate by Clostridium acetobutylicum continuous cultures. The tests were carried out using lactose as carbon source to mimic cheese-whey. A continuous stirred tank reactor equipped with a microfiltration unit was used. The dilution rate (D) ranged between 0.02 and 0.15h(-1) and the ratio R of the permeate stream rate to the stream fed to the reactor ranged between 14% and 95%. For each set of D and R values, the continuous cultures were characterized in terms of concentration of cells, acids and solvents. Results were processed to assess the concentration of acidogenic cells, solventogenic cells, spores and the specific butanol production rate. The max butanol productivity was 0.5gL(-1)h(-1) at D=0.1h(-1) and R=95%. The butanol productivity referred to solventogenic cells was expressed as a function of concentration of lactose, acids and butanol. PMID:25621726

  12. Prevalence and Duration of Asymptomatic Clostridium difficile Carriage among Healthy Subjects in Pittsburgh, Pennsylvania

    PubMed Central

    Galdys, Alison L.; Nelson, Jemma S.; Shutt, Kathleen A.; Schlackman, Jessica L.; Pakstis, Diana L.; Pasculle, A. William; Marsh, Jane W.; Harrison, Lee H.

    2014-01-01

    Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens. PMID:24759727

  13. Prevalence and duration of asymptomatic Clostridium difficile carriage among healthy subjects in Pittsburgh, Pennsylvania.

    PubMed

    Galdys, Alison L; Nelson, Jemma S; Shutt, Kathleen A; Schlackman, Jessica L; Pakstis, Diana L; Pasculle, A William; Marsh, Jane W; Harrison, Lee H; Curry, Scott R

    2014-07-01

    Previous studies suggested that 7 to 15% of healthy adults are colonized with toxigenic Clostridium difficile. To investigate the epidemiology, genetic diversity, and duration of C. difficile colonization in asymptomatic persons, we recruited healthy adults from the general population in Allegheny County, Pennsylvania. Participants provided epidemiological and dietary intake data and submitted stool specimens. The presence of C. difficile in stool specimens was determined by anaerobic culture. Stool specimens yielding C. difficile underwent nucleic acid testing of the tcdA gene segment with a commercial assay; tcdC genotyping was performed on C. difficile isolates. Subjects positive for C. difficile by toxigenic anaerobic culture were asked to submit additional specimens. One hundred six (81%) of 130 subjects submitted specimens, and 7 (6.6%) of those subjects were colonized with C. difficile. Seven distinct tcdC genotypes were observed among the 7 C. difficile-colonized individuals, including tcdC genotype 20, which has been found in uncooked ground pork in this region. Two (33%) out of 6 C. difficile-colonized subjects who submitted additional specimens tested positive for identical C. difficile strains on successive occasions, 1 month apart. The prevalence of C. difficile carriage in this healthy cohort is concordant with prior estimates. C. difficile-colonized individuals may be important reservoirs for C. difficile and may falsely test positive for infections due to C. difficile when evaluated for community-acquired diarrhea caused by other enteric pathogens. PMID:24759727

  14. Confocal Laser Endomicroscopy for In Vivo Diagnosis of Clostridium difficile Associated Colitis — A Pilot Study

    PubMed Central

    Neumann, Helmut; Günther, Claudia; Vieth, Michael; Grauer, Martin; Wittkopf, Nadine; Mudter, Jonas; Becker, Christoph; Schoerner, Christoph; Atreya, Raja; Neurath, Markus F.

    2013-01-01

    Background Clostridium difficile infection (CDI) is one of the most dreaded causes of hospital-acquired diarrhea. Main objective was to investigate whether confocal laser endomicroscopy (CLE) has the capability for in vivo diagnosis of C. difficile associated histological changes. Second objective was to prove the presence of intramucosal bacteria using CLE. Methods 80 patients were prospectively included, 10 patients were diagnosed with CDI based on toxigenic culture. To validate the presence of intramucosal bacteria ex vivo, CLE was performed in pure C. difficile culture; additionally fluorescence in situ hybridization (FISH) was performed. Finally, CLE with fluorescence labelled oligonucleotide probe specific for C. difficile was performed ex vivo in order to prove the presence of bacteria. Results CLE identified CDI-associated histological changes in vivo (sensitivity and accuracy of 88.9% and 96.3%). In addition, intramucosal bacteria were visualized. The presence of these bacteria could be proven by CLE with labeled, specific molecular C. difficile probe and FISH-technique. Based on comparison between CLE and FISH analyses, sensitivity and specificity for the presence of intramucosal bacteria were 100%. Conclusion CLE has the potential for in vivo diagnosis of CDI associated colitis. In addition, CLE allowed the detection of intramucosal bacteria in vivo. PMID:23527018

  15. SpoIIE Regulates Sporulation but Does Not Directly Affect Solventogenesis in Clostridium acetobutylicum ATCC 824

    PubMed Central

    Scotcher, Miles C.; Bennett, George N.

    2005-01-01

    Using gene expression reporter vectors, we examined the activity of the spoIIE promoter in wild-type and spo0A-deleted strains of Clostridium acetobutylicum ATCC 824. In wild-type cells, the spoIIE promoter is active in a transient manner during late solventogenesis, but in strain SKO1, where the sporulation initiator spo0A is disrupted, no spoIIE promoter activity is detectable at any stage of growth. Strains 824(pMSpo) and 824(pASspo) were created to overexpress spoIIE and to decrease spoIIE expression via antisense RNA targeted against spoIIE, respectively. Some cultures of strains 824(pMSpo) degenerated during fermentations by losing the pSOL1 megaplasmid and hence did not produce the solvents ethanol, acetone, and butanol. The frequent degeneration event was shown to require an intact copy of spoIIE. Nondegenerate cultures of 824(pMSpo) exhibited normal growth and solvent production. Strain 824(pASspo) exhibited prolonged solventogenesis characterized by increased production of ethanol (225%), acetone (43%), and butanol (110%). Sporulation in strains harboring pASspo was significantly delayed, with sporulating cells exhibiting altered morphology. These results suggest that SpoIIE has no direct effect on the control of solventogenesis and that the changes in solvent production in spoIIE-downregulated cells are mediated by effects on the cell during sporulation. PMID:15743939

  16. Evaluation of an Automated Rapid Diagnostic Test for Detection of Clostridium difficile

    PubMed Central

    Tojo, Masayoshi; Nagamatsu, Maki; Hayakawa, Kayoko; Mezaki, Kazuhisa; Kirikae, Teruo; Ohmagari, Norio

    2014-01-01

    The Verigene Clostridium difficile Nucleic Acid Test (Verigene CDF Test) (Nanosphere, Northbrook, IL, USA) is a new multiplex qualitative polymerase chain reaction (PCR) test used to detect C. difficile toxin genes in fecal specimens. To evaluate the performance of the new method, we tested 69 fecal samples from patients with suspected C. difficile infection using the Verigene CDF test, an enzyme immunoassay (EIA) and PCR following anaerobic fecal culture. The sensitivity, specificity, and accuracy of the Verigene CDF test were 96.7% (29/30), 97.4% (38/39), and 97.1% (67/69) respectively, using PCR following fecal culture as a reference method. We also analyzed the potential clinical impact of the Verigene CDF test using chart reviews of the 69 patients with suspected C. difficile infection and found that 11 of the 69 patients were incorrectly diagnosed, and the Verigene CDF test would have led to them receiving more appropriate management including practice of treatment and contact precaution, although, of the 69 patients, there are two whose samples were incorrectly identified with the Verigene CDF test. The Verigene CDF test will have a positive impact on patient care. PMID:25170836

  17. Tracking Inhibitory Alterations during Interstrain Clostridium difficile Interactions by Monitoring Cell Envelope Capacitance

    PubMed Central

    2016-01-01

    Global threats arising from the increasing use of antibiotics coupled with the high recurrence rates of Clostridium difficile (C. difficile) infections (CDI) after standard antibiotic treatments highlight the role of commensal probiotic microorganisms, including nontoxigenic C. difficile (NTCD) strains in preventing CDI due to highly toxigenic C. difficile (HTCD) strains. However, optimization of the inhibitory permutations due to commensal interactions in the microbiota requires probes capable of monitoring phenotypic alterations to C. difficile cells. Herein, by monitoring the field screening behavior of the C. difficile cell envelope with respect to cytoplasmic polarization, we demonstrate that inhibition of the host-cell colonization ability of HTCD due to the S-layer alterations occurring after its co-culture with NTCD can be quantitatively tracked on the basis of the capacitance of the cell envelope of co-cultured HTCD. Furthermore, it is shown that effective inhibition requires the dynamic contact of HTCD cells with freshly secreted extracellular factors from NTCD because contact with the cell-free supernatant causes only mild inhibition. We envision a rapid method for screening the inhibitory permutations to arrest C. difficile colonization by routinely probing alterations in the HTCD dielectrophoretic frequency response due to variations in the capacitance of its cell envelope.

  18. Beta Lactamase Producing Clostridium perfringens Bacteremia in an Elderly Man with Acute Pancreatitis

    PubMed Central

    Mishra, Rashmi; Duncalf, Richard

    2016-01-01

    Clostridium perfringens bacteremia is associated with adverse outcomes. Known risk factors include chronic kidney disease, malignancy, diabetes mellitus, and gastrointestinal disease. We present a 74-year-old man admitted with confusion, vomiting, and abdominal pain. Exam revealed tachycardia, hypotension, lethargy, distended abdomen, and cold extremities. He required intubation and aggressive resuscitation for septic shock. Laboratory data showed leukocytosis, metabolic acidosis, acute kidney injury, and elevated lipase. CT scan of abdomen revealed acute pancreatitis and small bowel ileus. He was started on vancomycin and piperacillin-tazobactam. Initial blood cultures were positive for C. perfringens on day five. Metronidazole and clindamycin were added to the regimen. Repeat CT (day 7) revealed pancreatic necrosis. The patient developed profound circulatory shock requiring multiple vasopressors, renal failure requiring dialysis, and bacteremia with vancomycin-resistant enterococci. Hemodynamic instability precluded surgical intervention and he succumbed to multiorgan failure. Interestingly, our isolate was beta lactamase producing. We review the epidemiology, risk factors, presentation, and management of C. perfringens bacteremia. This case indicates a need for high clinical suspicion for clostridial sepsis and that extended spectrum beta lactam antibiotic coverage may be inadequate and should be supplemented with use of clindamycin or metronidazole if culture is positive, until sensitivities are known. PMID:26904307

  19. Clinicopathologic features of experimental Clostridium perfringens type D enterotoxemia in cattle.

    PubMed

    Filho, E J F; Carvalho, A U; Assis, R A; Lobato, F F; Rachid, M A; Carvalho, A A; Ferreira, P M; Nascimento, R A; Fernandes, A A; Vidal, J E; Uzal, F A

    2009-11-01

    This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep. PMID:19605912

  20. Experimental production of hemorrhagic enterotoxemia by Clostridium perfringens type C in maturing lambs.

    PubMed

    Niilo, L

    1986-01-01

    Maturing lambs, eight to nine months old, were dosed by the intraduodenal route with various preparations of Clostridium perfringens type C. Whole cultures of this organism or cells suspended in fresh medium, both supplemented with soybean flour as a protease inhibitor, produced acute fatal hemorrhagic enterotoxemia in these animals. The latter preparation was more effective than the former in causing disease. Without the soybean supplement the inocula did not produce fatal disease. Dosing with toxic cell-free culture supernatant fluid, with or without soybean supplement, had no lethal effect. Animals that died showed severe hemorrhagic enteritis with necrosis and sloughing of the mucosal epithelium, involving jejunum, ileum and part of duodenum. These lesions were similar to those seen in natural cases of hemorrhagic enterotoxemia in neonatal animals. This experiment demonstrated that nonimmune animals are normally protected against C. perfringens type C enterotoxemia by adequate levels of pancreatic proteases in the intestine, and that factors which inhibit or reduce these enzymes predispose animals for the development of this disease. PMID:2874878

  1. Effect of Potassium Sorbate on Salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum in Cooked, Uncured Sausage

    PubMed Central

    Tompkin, R. B.; Christiansen, L. N.; Shaparis, A. B.; Bolin, H.

    1974-01-01

    Skinless precooked, uncured sausage links with and without potassium sorbate (0.1% wt/wt) were inoculated with salmonellae, Staphylococcus aureus, Clostridium perfringens, and Clostridium botulinum and held at 27 C to represent temperature abuse of the product. Total counts of uninoculated product showed that the normal spoilage flora was delayed 1 day when sorbate was present. Growth of salmonellae was markedly retarded by sorbate. Growth of S. aureus was delayed 1 day in the presence of sorbate, after which growth occurred to the same level as in product without sorbate. C. perfringens declined to below detectable levels within the first day in product with and without sorbate. Sorbate retarded the growth of C. botulinum. Botulinal toxin was detected in 4 days in product without sorbate but not until after 10 days in product with sorbate. PMID:4368631

  2. Clostridium perfringens Iota Toxin: Binding Studies and Characterization of Cell Surface Receptor by Fluorescence-Activated Cytometry

    PubMed Central

    Stiles, Bradley G.; Hale, Martha L.; Marvaud, Jean-Christophe; Popoff, Michel R.

    2000-01-01

    The binding characteristics of iota toxin, a binary enterotoxin produced by Clostridium perfringens type E, were studied by fluorescence-activated cytometry. The proteolytically activated binding component of iota toxin, iota b (Ib), bound to various cell types when incubated at 4, 25, or 37°C for 10 min. The binding of Ib was inhibited by antisera against C. perfringens type E or Clostridium spiroforme culture supernatants, but not C. perfringens types C or D. Pretreatment of Vero cells with glycosidases or lectins did not affect Ib interactions, while pronase effectively prevented Ib binding to the cell surface. The Ib protomer (Ibp) bound to the cell surface, but trypsinization of Ibp was necessary for docking of the ADP-ribosylating component, iota a (Ia). Ia attached to cell-bound Ib within 10 min at 37°C, but surface levels of Ia decreased 90% after 30 min and were undetectable by 60 min. Detectable surface levels of Ib also diminished over time, and Western blot analysis suggested internalization or embedment of Ib into the membrane. PMID:10816501

  3. Detection of enterotoxigenic Clostridium perfringens in food and fecal samples with a duplex PCR and the slide latex agglutination test.

    PubMed Central

    Fach, P; Popoff, M R

    1997-01-01

    A duplex PCR procedure was evaluated for the detection of Clostridium perfringens in food and biological samples and for the identification of enterotoxigenic strains. This method uses two sets of primers which amplify in the same reaction two different DNA fragments simultaneously: the 283-bp C. perfringens phospholipase C gene fragment and the 426-bp enterotoxin gene fragment. Internal primers within the two primer sets confirmed the specificity of the method by DNA-DNA hybridization with the PCR products. No cross-reaction was observed with other Clostridium species or with other bacteria routinely found in food. The detection level was approximately 10(5) C. perfringens cells per g of stool or food sample. When overnight enrichment culture was used, 10 C. perfringens cells per g was detected in 57 artificially contaminated food samples. The duplex PCR is a rapid, sensitive, and reliable method for the detection and identification of enterotoxigenic C. perfringens strains in food samples. A slide latex agglutination test was also evaluated as a rapid, simple technique for the detection of C. perfringens enterotoxin in stool samples. PMID:9361409

  4. Expression of Clostridium difficile Toxins A and B and Their Sigma Factor TcdD Is Controlled by Temperature

    PubMed Central

    Karlsson, Sture; Dupuy, Bruno; Mukherjee, Kakoli; Norin, Elisabeth; Burman, Lars G.; Åkerlund, Thomas

    2003-01-01

    Growth temperature was found to control the expression of toxins A and B in Clostridium difficile VPI 10463, with a maximum at 37°C and low levels at 22 and 42°C in both peptone yeast (PY) and defined media. The up-regulation of toxin A and B mRNA and protein levels upon temperature upshift from 22 to 37°C followed the same kinetics, showing that temperature control occurred at the level of transcription. Experiments with Clostridium perfringens using gusA as a reporter gene demonstrated that both toxin gene promoters were temperature controlled and that their high activity at 37°C was dependent on the alternative sigma factor TcdD. Furthermore, tcdD was found to be autoinduced at 37°C. Glucose down-regulated all these responses in the C. perfringens constructs, similar to its impact on toxin production in C. difficile PY broth cultures. C. difficile proteins induced at 37°C and thus coregulated with the toxins by temperature were demonstrated by two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified as enzymes involved in butyric acid production and as electron carriers in oxidation-reduction reactions. The regulation of toxin production in C. difficile by temperature is a novel finding apparently reflecting an adaptation of the expression of its virulence to mammalian hosts. PMID:12654792

  5. Ulcerative enterocolitis in two goats associated with enterotoxin- and beta2 toxin-positive Clostridium perfringens type D.

    PubMed

    Uzal, Francisco A; Fisher, Derek J; Saputo, Juliann; Sayeed, Sameera; McClane, Bruce A; Songer, Glenn; Trinh, Hien T; Fernandez Miyakawa, Mariano E; Gard, Sharon

    2008-09-01

    Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa. PMID:18776108

  6. Evaluation of hydrogen peroxide vapour as a method for the decontamination of surfaces contaminated with Clostridium botulinum spores.

    PubMed

    Johnston, M D; Lawson, S; Otter, J A

    2005-03-01

    The aim of this study was to evaluate the efficacy of hydrogen peroxide vapour (HPV) against spores of Clostridium botulinum, for use as a method for decontaminating environments where this pathogen has been handled. Spores were dried onto stainless steel slides and exposed to HPV in a sealed glovebox enclosure, transferred to a quenching agent at timed intervals during the exposure period, before survivors were cultured and enumerated. D-values were calculated from graphs of log10 survivors plotted against time and were found to range from 1.41 to 4.38 min. HPV was found to be effective at deactivating spores of toxigenic Cl. botulinum, non-toxigenic Clostridium spp. and Geobacillus stearothermophilus dried onto stainless steel surfaces. HPV could be used to decontaminate cabinets and rooms where Cl. botulinum has been handled. The cycle parameters should be based on studies carried out with relevant spores of this organism, rather than based on inactivation data for G. stearothermophilus spores, which have been used in the past as a standard biological challenge for disinfection and sterilisation procedures. HPV could provide an attractive alternative to other decontamination methods, as it was rapid, residue-free and did not give rise to the health and safety concerns associated with other gaseous decontamination systems. PMID:15649542

  7. Complete Genome Sequence of Clostridium sp. Strain DL-VIII, a Novel Solventogenic Clostridium Species Isolated from Anaerobic Sludge

    PubMed Central

    Taghavi, Safiyh; Izquierdo, Javier A.

    2013-01-01

    We report the genome sequence of Clostridium sp. strain DL-VIII, a novel Gram-positive, endospore-forming, solventogenic bacterium isolated from activated anaerobic sludge of a wastewater treatment plant. Aside from a complete sol operon, the 6,477,357-bp genome of DL-VIII reveals genes for several unique enzymes with applications in lignocellulose degradation, including two phenolic acid decarboxylases. PMID:23929491

  8. Expression of Clostridium acetobutylicum ATCC 824 Genes in Escherichia coli for Acetone Production and Acetate Detoxification

    PubMed Central

    Bermejo, Lourdes L.; Welker, Neil E.; Papoutsakis, Eleftherios T.

    1998-01-01

    A synthetic acetone operon (ace4) composed of four Clostridium acetobutylicum ATCC 824 genes (adc, ctfAB, and thl, coding for the acetoacetate decarboxylase, coenzyme A transferase, and thiolase, respectively) under the control of the thl promoter was constructed and was introduced into Escherichia coli on vector pACT. Acetone production demonstrated that ace4 is expressed in E. coli and resulted in the reduction of acetic acid levels in the fermentation broth. Since different E. coli strains vary significantly in their growth characteristics and acetate metabolism, ace4 was expressed in three E. coli strains: ER2275, ATCC 11303, and MC1060. Shake flask cultures of MC1060(pACT) produced ca. 2 mM acetone, while both strains ER2275(pACT) and ATCC 11303(pACT) produced ca. 40 mM acetone. Glucose-fed cultures of strain ATCC 11303(pACT) resulted in a 150% increase in acetone titers compared to those of batch shake flask cultures. External addition of sodium acetate to glucose-fed cultures of ATCC 11303(pACT) resulted in further increased acetone titers. In bioreactor studies, acidic conditions (pH 5.5 versus 6.5) improved acetone production. Despite the substantial acetone evaporation due to aeration and agitation in the bioreactor, 125 to 154 mM acetone accumulated in ATCC 11303(pACT) fermentations. These acetone titers are equal to or higher than those produced by wild-type C. acetobutylicum. This is the first study to demonstrate the ability to use clostridial genes in nonclostridial hosts for solvent production. In addition, acetone-producing E. coli strains may be useful hosts for recombinant protein production in that detrimental acetate accumulation can be avoided. PMID:9501448

  9. Draft Genome Sequence of Clostridium sporogenes Strain UC9000 Isolated from Raw Milk

    PubMed Central

    La Torre, Angela; Zotta, Teresa; Orrù, Luigi; Lamontanara, Antonella; Cocconcelli, Pier Sandro

    2016-01-01

    Clostridium sporogenes is a causative agent of food spoilage and is often used as the nontoxigenic surrogate for Clostridium botulinum. Here, we described the draft genome sequence and annotation of C. sporogenes strain UC9000 isolated from raw milk. PMID:27081128

  10. Two Serious Cases of Infection with Clostridium celatum after 40 Years in Hiding?

    PubMed Central

    Hoegh, Silje Vermedal; Holt, Hanne Marie; Justesen, Ulrik Stenz

    2015-01-01

    Clostridium celatum [ce.la'tum. L. adj. celatum hidden] has been known since 1974, when it was isolated from human feces. In 40 years, no association with human infection has been reported. In this work, we present two serious cases of infection with the anaerobic Gram-positive rod Clostridium celatum. PMID:26560535

  11. Antimicrobial susceptibility of Clostridium difficile isolated from food animals on farms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium difficile is commonly associated with a spectrum of disease in humans referred to as C. difficile-associated disease (CDAD) and use of antimicrobials is considered a risk factor for development of disease in humans. Clostridium difficile can also inhabit healthy food animals and transmi...

  12. Mathematical modeling and growth kinetics of Clostridium sporogenes in cooked beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium sporogenes PA 3679 is a common surrogate for proteolytic Clostridium botulinum for thermal process development and validation. However, little information is available concerning the growth kinetics of C. sporogenes in food. Therefore, the objective of this study was to investigate the...

  13. Foot infection by Clostridium sordellii: case report and review of 15 cases in France.

    PubMed

    Bouvet, Philippe; Sautereau, Jean; Le Coustumier, Alain; Mory, Francine; Bouchier, Christiane; Popoff, Michel-R

    2015-04-01

    We report a case of foot infection by Clostridium sordellii and review 15 human infections registered at a Reference Center in France during the period 1998 to 2011. All strains were found nontoxigenic, lacking the lethal toxin gene coding for TcsL. Like Clostridium septicum, several C. sordellii infections were associated with intestinal neoplasms. PMID:25609723

  14. Foot Infection by Clostridium sordellii: Case Report and Review of 15 Cases in France

    PubMed Central

    Sautereau, Jean; Le Coustumier, Alain; Mory, Francine; Bouchier, Christiane; Popoff, Michel-R.

    2015-01-01

    We report a case of foot infection by Clostridium sordellii and review 15 human infections registered at a Reference Center in France during the period 1998 to 2011. All strains were found nontoxigenic, lacking the lethal toxin gene coding for TcsL. Like Clostridium septicum, several C. sordellii infections were associated with intestinal neoplasms. PMID:25609723

  15. Draft Genome Sequence of Clostridium sporogenes Strain UC9000 Isolated from Raw Milk.

    PubMed

    La Torre, Angela; Bassi, Daniela; Zotta, Teresa; Orrù, Luigi; Lamontanara, Antonella; Cocconcelli, Pier Sandro

    2016-01-01

    Clostridium sporogenesis a causative agent of food spoilage and is often used as the nontoxigenic surrogate forClostridium botulinum Here, we described the draft genome sequence and annotation ofC. sporogenesstrain UC9000 isolated from raw milk. PMID:27081128

  16. Genome sequence of Clostridium tunisiense TJ, isolated from drain sediment from a pesticide factory.

    PubMed

    Sun, Lili; Wang, Yu; Yu, Chunyan; Zhao, Yongqin; Gan, Yinbo

    2012-12-01

    Clostridium tunisiense is a Gram-positive, obligate anaerobe that was first isolated in an anaerobic environment under eutrophication. Here we report the first genome sequence of the Clostridium tunisiense TJ isolated from drain sediment of a pesticide factory in Tianjin, China. The genome is of great importance for both basic and application research. PMID:23209212

  17. Prevalence of Clostridium perfringens, Clostridium perfringens enterotoxin and dysbiosis in fecal samples of dogs with diarrhea.

    PubMed

    Minamoto, Yasushi; Dhanani, Naila; Markel, Melissa E; Steiner, Jörg M; Suchodolski, Jan S

    2014-12-01

    Clostridium perfringens has been suspected as an enteropathogen in dogs. However, its exact role in gastrointestinal (GI) disorders in dogs remains unknown. Recent studies suggest the importance of an altered intestinal microbiota in the activation of virulence factors of enteropathogens. The aim of this study was to evaluate the relationship between diarrhea, dysbiosis, and the presence of C. perfringens and its enterotoxin (CPE). Fecal samples were collected prospectively from 95 healthy control dogs and 104 dogs with GI disease and assessed for bacterial abundances and the presence of CPE using quantitative PCR and ELISA, respectively. C. perfringens was detected in all dogs. Potentially enterotoxigenic C. perfringens were detected in 33.7% (32/95) of healthy control dogs and 48.1% (50/104) diseased dogs, respectively. CPE was detected by ELISA in 1.0% (1/95) of control dogs and 16.3% (17/104) of diseased dogs. Abundances of Fusobacteria, Ruminococcaceae, Blautia, and Faecalibacterium were significantly decreased in diseased dogs, while abundances of Bifidobacterium, Lactobacillus, and Escherichia coli were significantly increased compared to control dogs. The microbial dysbiosis was independent of the presence of the enterotoxigenic C. perfringens or CPE. In conclusion, the presence of CPE as well as fecal dysbiosis was associated with GI disease. However, the presence of C. perfringens was not indicative of GI disease in all cases of diarrhea, and the observed increased abundance of enterotoxigenic C. perfringens may be part of intestinal dysbiosis occurring in GI disease. The significance of an intestinal dysbiosis in dogs with GI disease deserves further attention. PMID:25458422

  18. Intravascular Hemolysis and Septicemia due to Clostridium perfringens Emphysematous Cholecystitis and Hepatic Abscesses

    PubMed Central

    Cochrane, Justin; Bland, Lacie; Noble, Mary

    2015-01-01

    Context. Clostridium perfringens septicemia is often associated with translocation from the gastrointestinal or gastrourinary tract and occurs in patients who have malignancy or are immunocompromised. Clostridium perfringens septicemia is usually fatal without early identification, source control, and antibiotics. Case. We present a case of a 65-year-old female with Clostridium perfringens septicemia secondary to emphysematous cholecystitis, with progression to hepatic abscesses. Conclusion. Septicemia secondary to Clostridium perfringens is generally fatal if not detected early. Source control with surgery or percutaneous drainage and early antibiotic therapy is imperative. Hyperbaric oxygen therapy may reduce mortality. Clinicians caring for patients with sepsis and intravascular hemolysis must have Clostridium perfringens septicemia on their differential diagnosis with a low threshold for starting antibiotics and pursuing source of infection. PMID:26229537

  19. Fusion of a thermophilic phage endolysin to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  20. A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  1. First Complete Genome Sequence of Clostridium sporogenes DSM 795T, a Nontoxigenic Surrogate for Clostridium botulinum, Determined Using PacBio Single-Molecule Real-Time Technology

    PubMed Central

    Terabayashi, Yasunobu; Shiroma, Akino; Shimoji, Makiko; Tamotsu, Hinako; Ashimine, Noriko; Ohki, Shun; Shinzato, Misuzu; Teruya, Kuniko; Satou, Kazuhito; Hirano, Takashi

    2015-01-01

    The first complete genome sequence of Clostridium sporogenes DSM 795T, a nontoxigenic surrogate for Clostridium botulinum, was determined in a single contig using the PacBio single-molecule real-time technology. The genome (4,142,990 bp; G+C content, 27.98%) included 86 sets of >1,000-bp identical sequence pairs and 380 tandem repeats. PMID:26227598

  2. Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry for identification of Clostridium species isolated from Saudi Arabia.

    PubMed

    AlMogbel, Mohammed Suliman

    2016-01-01

    The aim of this study was to identify different Clostridium spp. isolated from currency notes from the Ha'il region of Saudi Arabia in September 2014 using MALDI-TOF-MS. Clostridium spp. were identified by Bruker MALDI-TOF-MS and compared with VITEK 2. The confirmation of the presence of different Clostridium spp. was performed by determining the sequence of the 16S ribosomal RNA gene. In this study, 144 Clostridium spp. were isolated. Among these specimens, MALDI-TOF-MS could identify 88.8% (128/144) of the isolates to the species level and 92.3% (133/144) to the genus level, whereas, VITEK 2 identified 77.7% of the (112/144) isolates. The correct identification of the 144 isolates was performed by sequence analysis of the 500bp 16S rRNA gene. The most common Clostridium spp. identified were Clostridium perfringens (67.36%), Clostridium subterminale (14.58%), Clostridium sordellii (9%) and Clostridium sporogenes (9%). The results of this study demonstrate that MALDI-TOF-MS is a rapid, accurate and user friendly technique for the identification of Clostridium spp. Additionally, MALDI-TOF-MS has advantages over VITEK 2 in the identification of fastidious micro-organisms, such as Clostridium spp. Incorporating this technique into routine microbiology would lead to more successful and rapid identification of pathogenic and difficult to identify micro-organisms. PMID:26991272

  3. Clostridium geopurificans strain MJ1 sp. nov., a strictly anaerobic bacterium that grows via fermentation and reduces the cyclic nitramine explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX).

    PubMed

    Kwon, Man Jae; Wei, Na; Millerick, Kayleigh; Popovic, Jovan; Finneran, Kevin

    2014-06-01

    A fermentative, non-spore forming, motile, rod-shaped bacterium, designated strain MJ1(T), was isolated from an RDX contaminated aquifer at a live-fire training site in Northwest NJ, United States. On the basis of 16S rRNA gene sequencing and DNA base composition, strain MJ1(T) was assigned to the Firmicutes. The DNA G+C content was 42.8 mol%. Fermentative growth was supported by glucose and citrate in a defined basal medium. The bacterium is a strict anaerobe that grows between at pH 6.0 and pH 8.0 and 18 and 37 °C. The culture did not grow with hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as the electron acceptor or mineralize RDX under these conditions. However, MJ1(T) transformed RDX into MNX, methylenedinitramine, formaldehyde, formate, ammonium, nitrous oxide, and nitrate. The nearest phylogenetic relative with a validly published name was Desulfotomaculum guttoideum (95 % similarity). However, MJ1(T) was also related to Clostridium celerecrescens DSM 5628 (95 %), Clostridium indolis DSM 755 (94 %), and Clostridium sphenoides DSM 632 (94 %). DNA:DNA hybridization with these strains was between 6.7 and 58.7 percent. The dominant cellular fatty acids (greater than 5 % of the total, which was 99.0 % recovery) were 16:0 fatty acid methyl ester (FAME) (32.12 %), 18:1cis 11 dimethyl acetal (DMA) (16.47 %), 16:1cis 9 DMA (10.28 %), 16:1cis 9 FAME (8.10 %), and 18:1cis 9 DMA (5.36 %). On the basis of morphological, physiological, and phylogenetic data, Clostridium geopurificans is proposed as a new species in genus Clostridium, with strain MJ1(T) as the type strain. PMID:24522483

  4. Cellulase production by a thermophilic clostridium species.

    PubMed

    Lee, B H; Blackburn, T H

    1975-09-01

    Strain M7, a thermophilic, anaerobic, terminally sporing bacterium (0.6 by 4.0 mum) was isolated from manure. It degraded filter paper in 1 to 2 days at 60 C in a minimal cellulose medium but was stimulated by yeast extract. It fermented a wide variety of sugars but produced cellulase only in cellulose or carboxymethyl-cellulose media. Cellulase synthesis not only was probably repressed by 0.4% glucose and 0.3% cellobiose, but also cellulase activity appeared to be inhibited by these sugars at these concentrations. Both C(1) cellulase (degrades native cellulose) and C(x) cellulase (beta-1,4-glucanase) activities in strain M7 cultures were assayed by measuring the liberation of reducing sugars with dinitrosalicylic acid. Both activities had optima at pH 6.5 and 67 C. One milliliter of a 48-h culture of strain M7 hydrolyzed 0.044-meq of glucose per min from cotton fibers. The cellulase(s) from strain M7 was extracellular, produced during exponential growth, but was not free in the growth medium until approximately 30% of the cellulose was hydrolyzed. Glucose and cellobiose were the major soluble products liberated from cellulose by the cellulase. ZnCl(2) precipitation appeared initially to be a good method for the concentration of cellulase activity, but subsequent purification was not successful. Isoelectric focusing indicated the presence of four C(x) cellulases (pI 4.5, 6.3, 6.8, and 8.7). The rapid production and high activity of cellulases from this organism strongly support the basic premise that increased hydrolysis of native cellulose is possible at elevated temperature. PMID:16350033

  5. [Tryptose sulphite cycloserine agar for the recovery of Clostridium perfringens in surface waters: a study of different modes of utilization].

    PubMed

    Nusca, A; Orefice, L; Paradiso, R

    2007-01-01

    In the recent European Drinking Water Directive, Clostridium perfringens has assumed increasing importance so as to be considered a primary contamination indicator. Therefore it emerged the necessity to make culture methods, aimed at its recovery, more specific and sensitive. In this study we have verified the ability of Tryptose Sulphite Cycloserine Agar plates (TSC Agar), prepared and stored before the use at refrigeration temperature (+4 degrees) for different times, to show typical colonies, using both, the single layer and double layer techniques. Results show that storage of the prepared medium, even for a few days, decrease the recovery of typical colonies although such negative effect is minimized by using the double layer technique. PMID:17405507

  6. Phosphoenolpyruvate-dependent phosphorylation of sucrose by Clostridium tyrobutyricum ZJU 8235: evidence for the phosphotransferase transport system.

    PubMed

    Jiang, Ling; Cai, Jin; Wang, Jufang; Liang, Shizhong; Xu, Zhinan; Yang, Shang-Tian

    2010-01-01

    The uptake and metabolism of sucrose, the major sugar in industrial cane molasses, by Clostridium tyrobutyricum ZJU 8235 was investigated and this study provided the first definitive evidence for phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) activity in butyric acid-producing bacteria. Glucose was utilized preferentially to sucrose when both substrates were present in the medium. The PEP-dependent sucrose: PTS was induced by growing C. tyrobutyricum on sucrose (but not glucose) as the sole carbon source. Extract fractionation and PTS reconstitution experiments revealed that both soluble and membrane components were required for bioactivity. Sucrose-6-phosphate hydrolase and fructokinase activities were also detected in sucrose-grown cultures. Based on these findings, a pathway of sucrose metabolism in this organism was proposed that includes the forming of sucrose-6-phosphate via the PTS and its further degradation into glucose-6-phosphate and fructose-6-phosphate. PMID:19726178

  7. Facultative Anaerobe Caldibacillus debilis GB1: Characterization and Use in a Designed Aerotolerant, Cellulose-Degrading Coculture with Clostridium thermocellum

    PubMed Central

    Wushke, Scott; Levin, David B.; Cicek, Nazim

    2015-01-01

    Development of a designed coculture that can achieve aerotolerant ethanogenic biofuel production from cellulose can reduce the costs of maintaining anaerobic conditions during industrial consolidated bioprocessing (CBP). To this end, a strain of Caldibacillus debilis isolated from an air-tolerant cellulolytic consortium which included a Clostridium thermocellum strain was characterized and compared with the C. debilis type strain. Characterization of isolate C. debilis GB1 and comparisons with the type strain of C. debilis revealed significant physiological differences, including (i) the absence of anaerobic metabolism in the type strain and (ii) different end product synthesis profiles under the experimental conditions used. The designed cocultures displayed unique responses to oxidative conditions, including an increase in lactate production. We show here that when the two species were cultured together, the noncellulolytic facultative anaerobe C. debilis GB1 provided respiratory protection for C. thermocellum, allowing the synergistic utilization of cellulose even under an aerobic atmosphere. PMID:26048931

  8. Comparison of Illumigene, Simplexa, and AmpliVue Clostridium difficile Molecular Assays for Diagnosis of C. difficile Infection

    PubMed Central

    Miller, S. A.; Humphries, R. M.

    2014-01-01

    We compared the performance of the Simplexa Universal Direct (Focus Diagnostics) and AmpliVue (Quidel Corporation) assays to that of the Illumigene assay (Meridian Bioscience, Inc.) for the diagnosis of Clostridium difficile infection. Two hundred deidentified remnant diarrheal stool specimens were tested by the Simplexa, AmpliVue, and Illumigene methods. Specimens with discrepant results among the three assays and a representative number of concordant specimens were further evaluated by toxigenic culture. The sensitivity and specificity were 98 and 100% and 96 and 100% for the Simplexa Universal Direct and AmpliVue assays, respectively. Both assays are easy to perform, with rapid turn-around-times, supporting their utility in the clinical laboratory as routine diagnostic platforms. PMID:24352999

  9. Evaluation of the rapid loop-mediated isothermal amplification assay Illumigene for diagnosis of Clostridium difficile in an outbreak situation.

    PubMed

    Norén, Torbjörn; Unemo, Magnus; Magnusson, Cecilia; Eiserman, Maud; Matussek, Andreas

    2014-02-01

    An outbreak of Clostridium difficile infection (CDI) at Höglandet Hospital Eksjö in southern Sweden in 2011 was mainly due to a multidrug-resistant PCR ribotype 046 (30% of all samples). Diagnostics used routinely was the Vidas CDAB assay, but to control the outbreak the rapid loop-mediated isothermal amplification (LAMP) assay Illumigene was introduced and both techniques were compared to Toxigenic culture (TC) prospectively. The LAMP assay had a superior sensitivity, that is, 98% compared to 79% for the Vidas CDAB assay. Most importantly, the mean turn-around-time from collecting sample to result was reduced from 59 h to 2 h enabling early isolation of patients and effective hygiene precautions. This may potentially decrease the morbidity and nosocomial transmissions of C. difficile. PMID:23758095

  10. Differences in signs and lesions in sheep and goats with enterotoxemia induced by intraduodenal infusion of Clostridium perfringens type D.

    PubMed

    Blackwell, T E; Butler, D G; Prescott, J F; Wilcock, B P

    1991-07-01

    Enterotoxemia was induced in 4 lambs and 4 goat kids by continuous intraduodenal infusion of a whole culture of Clostridium perfringens type D. Clinical signs, hematologic values, biochemical alterations, and postmortem lesions in the lambs and goat kids were compared. The 4 lambs and 4 goat kids died within 25 hours of beginning the infusions. Lesions were not observed in the gastrointestinal tract of the 4 lambs; however, severe hemorrhagic enterocolitis was found in the 4 goat kids. This difference between the lambs and goat kids in the lesions caused by experimentally induced enterotoxemia may explain the discrepancies reported between sheep and goats in clinical signs, response to treatment, and efficacy of vaccination observed in naturally induced enterotoxemia in the 2 species. PMID:1892271

  11. Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro.

    PubMed

    Uzal, F A; Rolfe, B E; Smith, N J; Thomas, A C; Kelly, W R

    1999-08-01

    Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro. PMID:10493114

  12. Nitrogen and Sulfur Requirements for Clostridium thermocellum and Caldicellulosiruptor bescii on Cellulosic Substrates in Minimal Nutrient Media

    SciTech Connect

    Kridelbaugh, Donna M; Nelson, Josh C; Engle, Nancy L; Tschaplinski, Timothy J; Graham, David E

    2013-01-01

    Growth media for cellulolytic Clostridium thermocellum and Caldicellulosiruptor bescii bacteria usually contain excess nutrients that would increase costs for consolidated bioprocessing for biofuel production and create a waste stream with nitrogen, sulfur and phosphate. C. thermocellum was grown on crystalline cellulose with varying concentrations of nitrogen and sulfur compounds, and growth rate and alcohol production response curves were determined. Both bacteria assimilated sulfate in the presence of ascorbate reductant, increasing the ratio of oxidized to reduced fermentation products. From these results, a low ionic strength, defined minimal nutrient medium with decreased nitrogen, sulfur, phosphate and vitamin supplements was developed for the fermentation of cellobiose, cellulose and acid-pretreated Populus. Carbon and electron balance calculations indicate the unidentified residual fermentation products must include highly reduced molecules. Both bacterial populations were maintained in co-cultures with substrates containing xylan or hemicellulose in defined medium with sulfate and basal vitamin supplements.

  13. Isolation of indigenous larvicidal microbial control agents of mosquitos: the Malaysian experience.

    PubMed

    Lee, H L; Seleena, P

    1990-06-01

    A nationwide screening program searching for microbial control agents of mosquitos was initiated in Malaysia in 1986. A total of 725 samples were collected and 2,394 bacterial colonies were isolated and screened for larvicidal activity. From such screening, 20 Bacillus thuringiensis, 6 B. sphaericus, 1 Clostridium bifermentans and 2 Pseudomonas pseudomallei larvicidal isolates were obtained. Of these, a new B. thuringiensis named as subspecies malaysianensis was found, while the C. bifermentans was also a new anaerobe individualized as serovar malaysia. It was concluded that this screening program was highly successful. PMID:2237596

  14. Reductive dissolution of Pu(IV) by Clostridium sp. under anaerobic conditions.

    PubMed

    Francis, Arokiasamy J; Dodge, Cleveland J; Gillow, Jeffrey B

    2008-04-01

    An anaerobic, gram positive, spore-forming bacterium Clostridium sp., common in soils and wastes, capable of reduction of Fe(III) to Fe(II), Mn(IV) to Mn(II), Tc(VII) to Tc(IV), and U(VI) to U(IV), reduced Pu(IV) to Pu(III). Addition of 242Pu (IV)-nitrate to the bacterial growth medium at pH 6.4 resulted in the precipitation of Pu as amorphous Pu(OH)4 due to hydrolysis and polymerization reactions. The Pu (1 x 10(-5) M) had no effect upon growth of the bacterium as evidenced by glucose consumption; carbon dioxide and hydrogen production; a decrease in pH of the medium from 6.4 to 3.0 due to production of acetic and butyric acids from glucose fermentation; and a change in the Eh of the culture medium from +50 to -180 mV. Commensurate with bacterial growth, Pu was rapidly solubilized as evidenced by an increase in Pu concentration in solution which passed through a 0.03 microm filtration. Selective solvent extraction of the culture by thenoyltrifluoroacetone (TTA) indicated the presence of a reduced Pu species in the soluble fraction. X-ray absorption near edge spectroscopic (XANES) analysis of Pu in the culture sample at the Pu LIII absorption edge (18.054 keV) showed a shift of -3 eV compared to a Pu(IV) standard indicating reduction of Pu(IV) to Pu(III). These results suggestthat, although Pu generally exists as insoluble Pu(IV) in the environment, under appropriate conditions, anaerobic microbial activity could affect the long-term stability and mobility of Pu by its reductive dissolution. PMID:18504965

  15. Clostridium difficile infection diagnosis in a paediatric population: comparison of methodologies.

    PubMed

    Hart, J; Putsathit, P; Knight, D R; Sammels, L; Riley, T V; Keil, A

    2014-09-01

    The increasing incidence of Clostridium difficile infection (CDI) in paediatric hospitalised populations, combined with the emergence of hypervirulent strains, community-acquired CDI and the need for prompt treatment and infection control, makes the rapid, accurate diagnosis of CDI crucial. We validated commonly used C. difficile diagnostic tests in a paediatric hospital population. From October 2011 to January 2012, 150 consecutive stools were collected from 75 patients at a tertiary paediatric hospital in Perth, Western Australia. Stools were tested using: C. Diff Quik Chek Complete, Illumigene C. difficile, GeneOhm Cdiff, cycloserine cefoxitin fructose agar (CCFA) culture, and cell culture cytotoxin neutralisation assay (CCNA). The reference standard was growth on CCFA or Cdiff Chromagar and PCR on isolates to detect tcdA, tcdB, cdtA, and cdtB. Isolates were PCR ribotyped. The prevalence of CDI was high (43 % of patients). Quik Chek Complete glutamate dehydrogenase (GDH) demonstrated a low negative predictive value (NPV) (93 %). Both CCNA and Quik Chek Complete toxin A/B had poor sensitivity (33 % and 29 % respectively). Molecular methods both had 89 % sensitivity. Algorithms using GDH + Illumigene or GeneOhm reduced the sensitivity to 85 % and 83 % respectively. Ribotype UK014/20 predominated. GDH NPV and GeneOhm and Illumigene sensitivities were reduced compared with adult studies. Quik Chek Complete and CCNA cannot reliably detect toxigenic CDI. A GDH first algorithm showed reduced sensitivity. In a high prevalence paediatric population, molecular methods alone are recommended over the use of GDH algorithm or culture and CCNA, as they demonstrate the best test performance characteristics. PMID:24781004

  16. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  17. Bile Salt Inhibition of Host Cell Damage by Clostridium Difficile Toxins

    PubMed Central

    Darkoh, Charles; Brown, Eric L.; Kaplan, Heidi B.; DuPont, Herbert L.

    2013-01-01

    Virulent Clostridium difficile strains produce toxin A and/or toxin B that are the etiological agents of diarrhea and pseudomembranous colitis. Treatment of C. difficile infections (CDI) has been hampered by resistance to multiple antibiotics, sporulation, emergence of strains with increased virulence, recurrence of the infection, and the lack of drugs that preserve or restore the colonic bacterial flora. As a result, there is new interest in non-antibiotic CDI treatments. The human conjugated bile salt taurocholate was previously shown in our laboratory to inhibit C. difficile toxin A and B activities in an in vitro assay. Here we demonstrate for the first time in an ex vivo assay that taurocholate can protect Caco-2 colonic epithelial cells from the damaging effects of the C. difficile toxins. Using caspase-3 and lactate dehydrogenase assays, we have demonstrated that taurocholate reduced the extent of toxin B-induced apoptosis and cell membrane damage. Confluent Caco-2 cells cultured with toxin B induced elevated caspase-3 activity. Remarkably, addition of 5 mM taurocholate reduced caspase-3 activity in cells treated with 2, 4, 6, and 12 µg/ml of toxin B by 99%, 78%, 64%, and 60%, respectively. Furthermore, spent culture medium from Caco-2 cells incubated with both toxin B and taurocholate exhibited significantly decreased lactate dehydrogenase activity compared to spent culture medium from cells incubated with toxin B only. Our results suggest that the mechanism of taurocholate-mediated inhibition functions at the level of toxin activity since taurocholate did not affect C. difficile growth and toxin production. These findings open up a new avenue for the development of non-antibiotic therapeutics for CDI treatment. PMID:24244530

  18. Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination

    PubMed Central

    Linden, Jennifer R.; Ma, Yinghua; Zhao, Baohua; Harris, Jason Michael; Rumah, Kareem Rashid; Schaeren-Wiemers, Nicole

    2015-01-01

    ABSTRACT Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. PMID:26081637

  19. Metabolic modelling of syntrophic-like growth of a 1,3-propanediol producer, Clostridium butyricum, and a methanogenic archeon, Methanosarcina mazei, under anaerobic conditions.

    PubMed

    Bizukojc, Marcin; Dietz, David; Sun, Jibin; Zeng, An-Ping

    2010-05-01

    Clostridium butyricum can convert glycerol into 1,3-propanediol, thereby generating unfortunately a high amount of acetate, formate and butyrate as inhibiting by-products. We have proposed a novel mixed culture comprising C. butyricum and a methane bacterium, Methanosarcina mazei, to relieve the inhibition and to utilise the by-products for energy production. In order to examine the efficiency of such a mixed culture, metabolic modelling of the culture system was performed in this work. The metabolic networks for the organisms were reconstructed from genomic and physiological data. Several scenarios were analysed to examine the preference of M. mazei in scavenging acetate and formate under conditions of different substrate availability, including methanol as a co-substrate, since it may exist in glycerol solution from biodiesel production. The calculations revealed that if methanol is present, the methane production can increase by 130%. M. mazei can scavenge over 70% of the acetate secreted by C. butyricum. PMID:19680695

  20. Traits of selected Clostridium strains for syngas fermentation to ethanol.

    PubMed

    Martin, Michael E; Richter, Hanno; Saha, Surya; Angenent, Largus T

    2016-03-01

    Syngas fermentation is an anaerobic bioprocess that could become industrially relevant as a biorefinery platform for sustainable production of fuels and chemicals. An important prerequisite for commercialization is adequate performance of the biocatalyst (i.e., sufficiently high production rate, titer, selectivity, yield, and stability of the fermentation). Here, we compared the performance of three potential candidate Clostridium strains in syngas-to-ethanol conversion: Clostridium ljungdahlii PETC, C. ljungdahlii ERI-2, and Clostridium autoethanogenum JA1-1. Experiments were conducted in a two-stage, continuously fed syngas-fermentation system that had been optimized for stable ethanol production. The two C. ljungdahlii strains performed similar to each other but different from C. autoethanogenum. When the pH value was lowered from 5.5 to 4.5 to induce solventogenesis, the cell-specific carbon monoxide and hydrogen consumption (similar rate for all strains at pH 5.5), severely decreased in JA1-1, but hardly in PETC and ERI-2. Ethanol production in strains PETC and ERI-2 remained relatively stable while the rate of acetate production decreased, resulting in a high ethanol/acetate ratio, but lower overall productivities. With JA1-1, lowering the pH severely lowered rates of both ethanol and acetate production; and as a consequence, no pronounced shift to solventogenesis was observed. The highest overall ethanol production rate of 0.301 g · L(-1)  · h(-1) was achieved with PETC at pH 4.5 with a corresponding 19 g/L (1.9% w/v) ethanol concentration and a 5.5:1 ethanol/acetate molar ratio. A comparison of the genes relevant for ethanol metabolism revealed differences between C. ljungdahlii and C. autoethanogenum that, however, did not conclusively explain the different phenotypes. PMID:26331212

  1. Isolation of Clostridium botulinum type G from Swiss soil specimens by using sequential steps in an identification scheme.

    PubMed Central

    Sonnabend, W F; Sonnabend, U P; Krech, T

    1987-01-01

    After Clostridium botulinum type G organisms and toxin were identified in necropsy specimens in cases of unexplained death in adults and infants (O. Sonnabend, W. Sonnabend, R. Heinzle, T. Sigrist, R Dirnhofer, and U. Krech, J. Infect. Dis. 143:22-27, 1981), extensive research to detect C. botulinum type G in soil samples from Switzerland was done. A total of 41 specimens from virgin soil and from cultivated land were examined for the presence of C. botulinum type G and other toxin types. Because of the lack of the lipase marker in type G, the detection of C. botulinum type G was based on the demonstration of type G organisms in enrichment cultures by a type G-specific enzyme-linked immunosorbent assay to detect both the type G toxin and antigen; enrichment cultures in which type G toxin or antigen was identified by enzyme-linked immunosorbent assay were then tested by a type G-specific gel immunodiffusion agar procedure. This method not only isolated strains of type G but also strains of Clostridium subterminale, a nontoxigenic variant of C. botulinum type G. As a consequence of the observed cross-reactions caused by strains of C. subterminale within this test system, all isolates of type G had to be definitively confirmed by mouse bioassay. The sequential steps of these methods seem to be very useful for detecting C. botulinum type G organisms. C. botulinum type G strains were isolated in five soil samples from different locations in close association with cultivated land.(ABSTRACT TRUNCATED AT 250 WORDS) Images PMID:3116935

  2. Impact of Pretreated Switchgrass and Biomass Carbohydrates on Clostridium thermocellum 27405 Cellulosome Composition- a Quantitative Proteomic Analysis

    SciTech Connect

    Raman, Babu; Pan, Chongle; Hurst, Gregory {Greg} B; Rodriguez, Jr., Miguel; McKeown, Catherine K; Lankford, Patricia K; Samatova, Nagiza F; Mielenz, Jonathan R

    2009-01-01

    The anaerobic thermophilic bacterium Clostridium thermocellum is a cellulolytic organism capable of hydrolyzing cellulose and fermenting the hydrolysis products to ethanol and other metabolic products. C. thermocellum achieves efficient cellulose hydrolysis using multiprotein extracellular enzymatic complexes, termed the cellulosomes. In this study, we used quantitative proteomics (multidimensional LC-MS/MS and 15N-metabolic labeling) to measure relative changes in levels of cellulosomal subunit proteins (per CipA scaffoldin basis) when C. thermocellum was grown on a variety of carbon sources [dilute-acid pretreated switchgrass, cellobiose, amorphous cellulose, crystalline cellulose (Avicel) and combinations of crystalline cellulose with pectin or xylan or both]. Cellulosome samples isolated from cultures grown on these carbon sources were compared to 15N labeled cellulosome samples isolated from crystalline cellulose grown cultures. In total from all samples, proteomic analysis identified 59 dockerin- and 8 cohesin-module containing components, including 15 previously undetected cellulosomal subunits. Many cellulosomal components showed differential protein abundance in the presence of non-cellulose substrates in the growth medium. Cellulosome samples from amorphous cellulose, cellobiose and pretreated switchgrass grown cultures displayed the most distinct differences in composition as compared to cellulosome samples from crystalline cellulose grown cultures. While Glycoside Hydrolase Family 9 enzymes showed increased levels in the presence of crystalline cellulose, and pretreated switchgrass in particular, GH5 enzymes showed increased levels in response to the presence of cellulose in general, amorphous or crystalline. Overall, the results suggest a coordinated substrate-specific regulation of cellulosomal composition in C. thermocellum.

  3. Controversies Surrounding Clostridium difficile Infection in Infants and Young Children

    PubMed Central

    Nicholson, Maribeth R.; Thomsen, Isaac P.; Edwards, Kathryn M.

    2014-01-01

    Clostridium difficile is a frequent cause of antibiotic-associated diarrhea in adults and older children. However, as many as 80% of infants can be asymptomatically colonized. The reasons for this have not been well established but are believed to be due to differences in toxin receptors or toxin internalization. Determining which children who test positive for C. difficile warrant treatment is exceedingly difficult, especially in the setting of increased rates of detection and the rising risk of disease in children lacking classic risk factors for C. difficile.

  4. On the difficulties of isolating Clostridium difficile from hospital environments.

    PubMed

    Malik, D J; Patel, K V; Clokie, M R J; Shama, G

    2013-06-01

    Spores of Clostridium difficile were deposited on to a stainless steel surface and subsequently exposed to a chlorine-releasing disinfectant (dichloroisocyanurate). Recovery of the spores was carried out using RODAC plates containing a variety of selective and non-selective agars. The non-selective agar media yielded higher recoveries of both control and chlorine-stressed spores. Our results show that the antibiotics used in selective media imposed an additional stress on both disinfectant-treated and untreated spores resulting in considerably reduced recoveries. This could lead to a serious underestimate of the extent of environmental contamination by this organism. PMID:23643391

  5. Infectious Diarrhea: Norovirus and Clostridium difficile in Older Adults.

    PubMed

    White, Mary B; Rajagopalan, Shobita; Yoshikawa, Thomas T

    2016-08-01

    Norovirus infection usually results in acute gastroenteritis, often with incapacitating nausea, vomiting, and diarrhea. It is highly contagious and resistant to eradication with alcohol-based hand sanitizer. Appropriate preventative and infection control measures can mitigate the morbidity and mortality associated with norovirus infection. Clostridium difficile infection is the leading cause of health care-associated diarrhea in the United States. Antibiotic use is by far the most common risk factor for C difficile colonization and infection. Appropriate preventive measures and judicious use of antibiotics can help mitigate the morbidity and mortality associated with C difficile infection. PMID:27394020

  6. Evaluation and Modifications of Media for Enumeration of Clostridium perfringens

    PubMed Central

    Hauschild, A. H. W.; Hilsheimer, R.

    1974-01-01

    The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. PMID:4358863

  7. Evaluation and modifications of media for enumeration of Clostridium perfringens.

    PubMed

    Hauschild, A H; Hilsheimer, R

    1974-01-01

    The suitability of the Shahidi-Ferguson perfringens, TSC (tryptose-sulfite-cycloserine), and oleandomycin-polymyxin-sulfadiazine perfringens agars for presumptive enumeration of Clostridium perfringens was tested. Of these, the TSC agar was the most satisfactory. The TSC agar method was improved by eliminating the egg yolk and using pour plates. The modified method allowed quantitative recoveries of each of 71 C. perfringens strains tested and is recommended. For confirmation of C. perfringens, the nitrite test in nitrate motility agar was unreliable, particularly after storage of the medium for a few days. In contrast, positive nitrite reactions were obtained consistently when nitrate motility agar was supplemented with glycerol and galactose. PMID:4358863

  8. Rapid Technique for the Enumeration of Clostridium perfringens

    PubMed Central

    Marshall, Robert S.; Steenbergen, J. Frank; McClung, L. S.

    1965-01-01

    A new medium, Tryptone-sulfite-neomycin (TSN) agar, and an incubation procedure for the enumeration of Clostridium perfringens are described. Tolerance to neomycin, optimal growth at 46 C, and sulfite-reducing properties of C. perfringens were used as a basis for development of the medium. Comparisons were made between sulfite-polymyxin-sulfadiazine (SPS) agar and TSN agar at 37 and 46 C with C. perfringens and other organisms. These studies indicate the quantitative and selective superiority of TSN agar, incubated at 46 C, over SPS agar. PMID:14339262

  9. Clostridium perfringens and other anaerobes isolated from bile.

    PubMed Central

    Sakaguchi, Y; Murata, K; Kimura, M

    1983-01-01

    Clostridium perfringens was isolated from bile in 13 cases of 150 patients examined. The serotypes of C perfringens strains isolated from bile and faeces were investigated using antisera to Hobbs' type 1-17. Two or more serological types were often found in a single specimen, but in the same patient the serotypes of C perfringens strains isolated from the bile were identical with those from the faeces. Beta-glucuronidase production in these C perfringens serotypes was tested with the API-Strep system. Strains agglutinated with Hobbs' antisera produced beta-glucuronidase, but non-agglutinated strains did not. PMID:6298284

  10. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  11. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens

    PubMed Central

    Silva, R. O. S.; Salvarani, F.M.; Assis, R.A.; Martins, N.R.S.; Pires, P.S.; Lobato, F.C.F.

    2009-01-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  12. [Characteristics of Clostridium tetani and laboratory diagnosis of tetanus].

    PubMed

    Smietańska, Karolina; Rokosz-Chudziak, Natalia; Rastawicki, Waldemar

    2013-01-01

    The causative agent of tetanus is the obligate anaerobic bacterium--Clostridium tetani. These bacteria form endospores that are able to survive long periods of exposure to air and other adverse environmental conditions. Infection generally occurs through wound contamination. We can distinguish several forms of tetanus: generalized, local and neonatal. Diagnosis of tetanus is based primarily on the patient's clinical symptoms (muscle cramps, painful back muscle spasms, generalized contractions of the arcuate curvature of the body) as well as on microbiological diagnosis. This article is a brief review of C. tetani and diagnosis of infections caused by these organisms in humans. PMID:24730217

  13. Pneumatosis intestinalis in a patient with recurrent Clostridium difficile infection

    PubMed Central

    Ha, Duc; Tsai, Chung-Jyi

    2012-01-01

    A 65-year-old man with long-standing diarrhoea, recurrent Clostridium difficile infection (CDI) in the previous 5 months presented to the gastroenterology clinic with recurrent diarrhoea and abdominal cramping. Physical examination was negative for signs of acute abdomen. Stool C difficile PCR was positive. Abdominal imaging demonstrated an extensive pneumatosis intestinalis involving the small bowel and a dilated small bowel loop. He was treated conservatively with oral vancomycin for recurrent CDI with resolution of diarrhoea and abdominal cramping on 1-month follow-up visit. PMID:23112256

  14. Clostridium thermocellum DSM 1313 transcriptional responses to redox perturbation

    DOE PAGESBeta

    Sander, Kyle B.; Wilson, Charlotte M.; M. Rodriquez, Jr.; Klingeman, Dawn Marie; Davison, Brian H.; Brown, Steven D.; Rydzak, T.

    2015-12-12

    Clostridium thermocellum is a promising consolidated bioprocessing candidate organism capable of directly converting lignocellulosic biomass to ethanol. Current ethanol yields, productivities, and growth inhibitions are industrial deployment impediments for commodity fuel production by this bacterium. Redox imbalance under certain conditions and in engineered strains may contribute to incomplete substrate utilization and may direct fermentation products to undesirable overflow metabolites. As a result, towards a better understanding of redox metabolism in C. thermocellum, we established continuous growth conditions and analyzed global gene expression during addition of two stress chemicals (methyl viologen and hydrogen peroxide) which changed the fermentation redox potential.

  15. Challenges for standardization of Clostridium difficile typing methods.

    PubMed

    Huber, Charlotte A; Foster, Niki F; Riley, Thomas V; Paterson, David L

    2013-09-01

    Typing of Clostridium difficile facilitates understanding of the epidemiology of the infection. Some evaluations have shown that certain strain types (for example, ribotype 027) are more virulent than others and are associated with worse clinical outcomes. Although restriction endonuclease analysis (REA) and pulsed-field gel electrophoresis have been widely used in the past, PCR ribotyping is the current method of choice for typing of C. difficile. However, global standardization of ribotyping results is urgently needed. Whole-genome sequencing of C. difficile has the potential to provide even greater epidemiologic information than ribotyping. PMID:23784128

  16. Community-Acquired Clostridium Difficile Infection: Awareness and Clinical Implications

    PubMed Central

    Juneau, Cheryl; Mendias, Elnora (Nonie) P.; Wagal, Nihas; Loeffelholz, Michael; Savidge, Tor; Croisant, Sharon; Dann, Sara

    2013-01-01

    The epidemiology of Clostridium difficile infection (CDI) is changing. CDI, usually depicted as a nosocomial infection in the elderly, is now occurring in community-dwelling persons who are younger and otherwise dissimilar. A more virulent isolate (North American Pulsed Field type 1 (NAP1) associated with increased morbidity and mortality, has been identified. In 2005, similar strains were associated with severe disease in community-dwelling patients at a rate of 7.6/100,000. Screening patients with potential CDI symptoms and implementing preventative measures, including judicious use of antibiotics, can reduce disease burden. PMID:23814528

  17. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens.

    PubMed

    Silva, R O S; Salvarani, F M; Assis, R A; Martins, N R S; Pires, P S; Lobato, F C F

    2009-04-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  18. New advances in the treatment of Clostridium difficile infection (CDI)

    PubMed Central

    Hedge, Dennis D; Strain, Joe D; Heins, Jodi R; Farver, Debra K

    2008-01-01

    Clostridium difficile infections (CDI) have increased in frequency throughout the world. In addition to an increase in frequency, recent CDI epidemics have been linked to a hypervirulent C. difficile strain resulting in greater severity of disease. Although most mild to moderate cases of CDI continue to respond to metronidazole or vancomycin, refractory and recurrent cases of CDI may require alternative therapies. This review provides a brief overview of CDI and summarizes studies involving alternative antibiotics, toxin binders, probiotics, and immunological therapies that can be considered for treatment of acute and recurrent CDI in severe and refractory situations. PMID:19209277

  19. Clostridium difficile Infection in Patients with Inflammatory Bowel Disease.

    PubMed

    Fu, Nancy; Wong, Titus

    2016-06-01

    Clostridium difficile infection (CDI) is now the leading cause of nosocomial infection. There has been an upsurge of CDI in patients with inflammatory bowel disease (IBD). IBD patients with CDI have increased morbidity and mortality. The establishment, proliferation, and recurrence of CDI in IBD patients form a complex interplay of microbial, environmental, and host-susceptibility factors. Different risk factors have been found predisposing IBD patients to CDI. Vancomycin performs better than metronidazole in treating IBD patients with CDI. Fecal microbiota transplantation continues to be a very effective therapy. New therapeutic modalities such as vaccinations and bile salts are currently being investigated. PMID:27137789

  20. Characteristic strategy of assimilation of various saccharides by Clostridium cellulovorans.

    PubMed

    Inamori, Takako; Aburaya, Shunsuke; Morisaka, Hironobu; Kuroda, Kouichi; Ueda, Mitsuyoshi

    2016-12-01

    Clostridium cellulovorans can effectively assimilate not only cellulose but also hemicellulose by producing cellulosomal and non-cellulosomal enzymes. However, little is known about how C. cellulovorans assimilates various saccharides in media containing polysaccharides and oligosaccharides. In this research, we investigated the property of saccharide incorporation and assimilation by C. cellulovorans. Faster growth in media containing xylan and cellulose was achieved by switching polysaccharides, in which xylan was first assimilated, followed by cellulose. Furthermore, the presence of polysaccharides that can be easily degraded might increase the assimilation rate of lignocellulose by promoting growth. These properties of C. cellulovorans could be suitable for the effective utilization of lignocellulosic biomass. PMID:27586595

  1. Clostridium difficile: the anaerobe that made the grade.

    PubMed

    Brazier, Jon S

    2012-04-01

    Unlike other anaerobic bacteria of clinical importance, Clostridium difficile has managed to enter into the realm of public awareness. Following the trail blazed by methicillin-resistant Staphylococcus aureus (MRSA), C. difficile has made the transition from being an obscure anaerobic bacterium, mainly of interest to specialist anaerobic microbiologists, to that of an infamous "superbug" responsible for outbreaks of hospital-acquired infection that commonly result in serious disease and death. This report picks out key moments, particularly in the UK, which tracked the rise in both the public and political awareness of this organism. PMID:22293217

  2. Acute aortic dissection caused by Clostridium septicum aortitis.

    PubMed

    Eplinius, Franziska; Hädrich, Carsten

    2014-11-01

    Clostridium septicum aortitis is a rare cause of aortic dissection. So far, only 28 cases have been described in literature before. Most of these cases occurred in elderly patients and an association to colonic neoplasms and/or atherosclerosis has been witnessed frequently. Here we report the case of a 32-year-old man with fatal aortic dissection due to aortic infection with C. septicum. Beside a case of a 22-year-old man who died of aortic dissection due to C. septicum aortitis this is the second case of C. septicum aortitis in a young individual with no signs of colonic neoplasms or atherosclerosis. PMID:25242573

  3. Structure, Function and Regulation of the Clostridium cellulovorans Cellulosome

    SciTech Connect

    Doi, Roy H

    2008-06-01

    Our major goal for this project (2004-2008) was to obtain an understanding ofthe structure, function, and regulation of the Clostridium cellulovorans cellulosomes. Our specific goals were to select genes for cellulosomal and non-cellulosomal enzymes and characterize their products, to study the synergistic action between cellulosomal and non-cellulosomal enzymes, to study the composition of cellulosomes when cells were grown with different carbon sources, continue our studies on the scaffolding protein and examine heterologous expression of cellulosomal genes in Bacillus subtilis. We fulfilled the specific goals of our proposal.

  4. Probiotics and Antibiotic-Associated Diarrhea and Clostridium difficile Infection

    NASA Astrophysics Data System (ADS)

    Surawicz, Christina M.

    Diarrhea is a common side effect of antibiotics. Antibiotics can cause diarrhea in 5-25% of individuals who take them but its occurrence is unpredictable. Diarrhea due to antibiotics is called antibiotic-associated diarrhea (AAD). Diarrhea may be mild and resolve when antibiotics are discontinued, or it may be more severe. The most severe form of AAD is caused by overgrowth of Clostridium difficile which can cause severe diarrhea, colitis, pseudomembranous colitis, or even fatal toxic megacolon. Rates of diarrhea vary with the specific antibiotic as well as with the individual susceptibility.

  5. Role of chemotaxis in solvent production by Clostridium acetobutylicum

    SciTech Connect

    Gutierrez, N.A.; Maddox, I.S.

    1987-08-01

    The motility of Clostridium acetobutylicum has been investigated during a typical batch fermentation process for solvent production. The motility is characterized by runs during the early phase of sugar utilization and acid production, but this changes to tumbles during the onset of solventogenesis. Sugars and undissociated acetic and butyric acids have been shown to be attractants for the bacterium, while acetone, butanol, ethanol, and dissociated acetate and butyrate are repellents. It is suggested that chemotactic responses explain why highly motile cells are strongly solventogenic.

  6. A fatal postpartum Clostridium sordellii associated toxic shock syndrome.

    PubMed Central

    Bitti, A; Mastrantonio, P; Spigaglia, P; Urru, G; Spano, A I; Moretti, G; Cherchi, G B

    1997-01-01

    Clostridium sordellii is an infrequent human pathogen. It has been demonstrated to be occasionally responsible for myonecrosis or gas gangrene. Interestingly, in the obstetric literature, some cases of postpartum maternal deaths have been associated with C sordellii infection causing a rapidly lethal toxin mediated syndrome. This is the first reported case of postpartum death in a 29 year old woman, in which a toxigenic C sordellii was isolated from the patient's blood antemortem during the fatal toxic shock, strongly indicating its role in this rare syndrome. PMID:9155682

  7. First Australian isolation of epidemic Clostridium difficile PCR ribotype 027.

    PubMed

    Riley, Thomas V; Thean, Sarah; Hool, Graham; Golledge, Clayton L

    2009-06-15

    We report the first isolation in Australia of a hypervirulent epidemic strain of Clostridium difficile, PCR ribotype 027. It was isolated from a 43-year-old woman with a permanent ileostomy, who appears to have been infected while travelling in the United States. The isolate was positive for toxin A, toxin B and binary toxin, and resistant to fluoroquinolone antimicrobials, and had characteristic deletions in the tcdC gene. All diagnostic laboratories and health care facilities in Australia should now be on high alert for this organism. PMID:19527210

  8. Characteristics of Clostridium botulinum Type F Isolated from the Pacific Coast of the United States

    PubMed Central

    Eklund, M. W.; Poysky, F. T.; Wieler, D. I.

    1967-01-01

    Some of the physiological and biochemical characteristics of a type F strain recently isolated from the United States were studied and compared with those of the prototype Langeland type F strain. The recent isolates were nonproteolytic, fermented sucrose and ribose, produced spores of low thermal resistance, produced a protoxin activated by trypsin, and grew and produced toxin at 38 F (3.3 C) from a spore inoculum. The prototype Langeland strain was proteolytic, did not ferment sucrose or ribose, and produced spores of relatively high thermal resistance, and the toxin of 3-day-old cultures was not activated by trypsin. Approximately two to three times the minimal lethal dose (MLD) of type F toxin from either Langeland or nonproteolytic strains was cross-neutralized by 1,000 anti-MLD of type E antitoxin. Antitoxin serums prepared by immunizing rabbits with the toxoid of the nonproteolytic type F isolate neutralized the toxin of the Langeland strain, but did not show cross-neutralization with the toxins of other types of Clostridium botulinum. Images Fig. 1 Fig. 2 PMID:4865980

  9. Identification of a glucose-mannose phosphotransferase system in Clostridium beijerinckii.

    PubMed

    Essalem, Mohemed E E; Mitchell, Wilfrid J

    2016-04-01

    Effective uptake of fermentable substrates is a fundamentally important aspect of any fermentation process. The solventogenic bacterium Clostridium beijerinckii is noted for its ability to ferment a wide range of carbohydrates, yet few of its sugar transport systems have been characterized. In common with other anaerobes, C. beijerinckii shows a marked dependence on the PEP-dependent phosphotransferase system (PTS) for sugar accumulation. In this study, the gene cbe0751 encoding the sugar-specific domains of a phosphotransferase belonging to the glucose family was cloned into an Escherichia coli strain lacking the ability to take up and phosphorylate glucose. Transformants gained ability to ferment glucose, and also mannose, and further analysis of a selected transformant demonstrated that it could take up and phosphorylate glucose, confirming that cbe0751 encodes a glucose PTS which also recognizes mannose as a substrate. RT-PCR analysis showed that cbe0751 was expressed in cultures grown on both substrates, but also to varying extents during growth on some other carbon sources. Although analogue inhibition studies suggested that Cbe0751 is not the only glucose PTS in C. beijerinckii, this system should nevertheless be regarded as a potential target for metabolic engineering to generate a strain showing improved sugar fermentation properties. PMID:26940293

  10. Enumeration and Isolation of cpe-Positive Clostridium perfringens Spores from Feces

    PubMed Central

    Heikinheimo, Annamari; Lindström, Miia; Korkeala, Hannu

    2004-01-01

    A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying (cpe-positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe-positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe-positive C. perfringens was successfully isolated from both of these samples. The detection of cpe-positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe-positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe-positive C. perfringens could be isolated if the ratio of cpe-positive C. perfringens spores to total C. perfringens spores was 6 × 10−5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe-positive C. perfringens. The method also offers a new approach in the investigation of the epidemiology of cpe-positive C. perfringens strains. PMID:15364981

  11. Enumeration and isolation of cpe-positive Clostridium perfringens spores from feces.

    PubMed

    Heikinheimo, Annamari; Lindström, Miia; Korkeala, Hannu

    2004-09-01

    A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying (cpe-positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe-positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe-positive C. perfringens was successfully isolated from both of these samples. The detection of cpe-positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe-positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe-positive C. perfringens could be isolated if the ratio of cpe-positive C. perfringens spores to total C. perfringens spores was 6 x 10(-5) or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe-positive C. perfringens. The method also offers a new approach in the investigation of the epidemiology of cpe-positive C. perfringens strains. PMID:15364981

  12. Cloning, characterization, and production of three α-l-fucosidases from Clostridium perfringens ATCC 13124.

    PubMed

    Fan, Shuquan; Zhang, Huaqin; Chen, Xiaodi; Lu, Lili; Xu, Li; Xiao, Min

    2016-04-01

    α-l-Fucosidases are key enzymes for the degradation of intestinal glycans by gut microbes. In this work, three putative α-l-fucosidases (Afc1, Afc2, and Afc3) genes from Clostridium perfringens ATCC 13124 were cloned and expressed in Escherichia coli. Afc1 had the α-l-fucosidase domain of glycoside hydrolase (GH) 29 family but showed no enzyme activity toward all the substrates examined. The putative acid/base residue of Afc1, Ser205, was replaced by a glutamic acid which is conserved in GH29-B α-l-fucosidases. However, the mutant Afc1-S205E still failed to show enzyme activity. Afc2 and Afc3 were determined to be 1,3-1,4-α-l-fucosidase of GH29-B subfamily and 1,2-α-l-fucosidase of GH95 family, respectively, and both of them could release fucose from porcine gastric mucin (PGM). When C. perfringens ATCC 13124 grew with the presence of PGM, the transcription of afc1 decreased slightly, while those of afc2 and afc3 increased to 2.2-fold and 1.4-fold, respectively, and the enzyme activities of Afc2 and Afc3 in the culture increased to 2.2-fold and 2.6-fold, respectively. These results suggest that Afc2 and Afc3 are involved in the degradation of intestinal fucosyl glycans by C. perfringens ATCC 13124. PMID:26663202

  13. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

    SciTech Connect

    Shao, Xiongjun; Raman, Babu; Zhu, Mingjun; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Lynd, Lee R

    2011-01-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

  14. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum

    SciTech Connect

    Lynd, Lee R; Shao, Xiongjun; Raman, Babu; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Zhu, Mingjun

    2011-01-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1 2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed.

  15. Mice and Monkeys as Assay Animals for Clostridium perfringens Food Poisoning1

    PubMed Central

    Weiss, K. F.; Strong, D. H.; Groom, R. A.

    1966-01-01

    Spores and vegetative cells of Clostridium perfringens, in combination with meat or starch paste, sterile culture filtrates, lecithinase, and phosphorylcholine, were administered to mice and rhesus monkeys in an attempt both to evaluate the animals as test agents and, if possible, to elucidate the active factors producing food-poisoning symptoms caused by this organsim. Some of the preparations were administered to the monkeys by stomach tube; others, in gelatin capsules which were treated with formaldehyde so that the release of their contents was delayed and presumably reached the intestines of the animals. Any changes in intestinal passage times and in consistency of stools of the animals were observed, and the counts of C. perfringens in the feces of the monkeys previous and subsequent to treatment were recorded. The results obtained were inconclusive. Diarrhea occurred only relatively infrequently in both species, regardless of the substance fed or the mode of administration. The changes in intestinal passage times were not great, although in the monkeys there appeared to be a slight trend toward reduction as the magnitude of the bacterial load increased. Phosphorylcholine appeared to have little, if any, effect in reducing intestinal passage time of mice or monkeys. No procedures explored in these experiments could be said to be satisfactory as a means of animal assay for food poisoning strains of C. perfringens since typical symptoms did not appear with regularity. PMID:4288826

  16. Induction of toxins in Clostridium difficile is associated with dramatic changes of its metabolism.

    PubMed

    Karlsson, Sture; Burman, Lars G; Akerlund, Thomas

    2008-11-01

    Certain amino acids, and cysteine in particular, promptly blocked toxin expression in Clostridium difficile strain VPI 10463 when added to late-exponential-phase peptone-yeast cultures, i.e. prior to normal induction of toxins A and B. Glucose reduced toxin yields by 80-fold, but only when supplemented at inoculation. Forty upregulated C. difficile proteins were identified during maximum toxin expression, and most of these were enzymes involved in energy exchange, e.g. succinate, CO/folate and butyrate metabolism. Transcription of tcdA (toxin operon) and folD (CO/folate operon) was induced by 20- and 10-fold, respectively, and with strikingly similar kinetics between OD 0.8 and 1.2. The sigma factors tcdR and sigH were upregulated simultaneously with tcdA and folD (3.5-fold increase of mRNA level), whereas transcription of tcdC, codY, sigB and sigL showed little or no correlation with that of tcdA and folD. The results suggest a connection between toxin expression, alternative energy metabolism and initial sporulation events in C. difficile. PMID:18957596

  17. Rapid molecular characterization of Clostridium difficile and assessment of populations of C. difficile in stool specimens.

    PubMed

    Wroblewski, Danielle; Hannett, George E; Bopp, Dianna J; Dumyati, Ghinwa K; Halse, Tanya A; Dumas, Nellie B; Musser, Kimberlee A

    2009-07-01

    Our laboratory has developed testing methods that use real-time PCR and pyrosequencing analysis to enable the rapid identification of potential hypervirulent Clostridium difficile strains. We describe a real-time PCR assay that detects four C. difficile genes encoding toxins A (tcdA) and B (tcdB) and the binary toxin genes (cdtA and cdtB), as well as a pyrosequencing assay that detects common deletions in the tcdC gene in less than 4 h. A subset of historical and recent C. difficile isolates (n = 31) was also analyzed by pulsed-field gel electrophoresis to determine the circulating North American pulsed-field (NAP) types that have been isolated in New York State. Thirteen different NAP types were found among the 31 isolates tested, 13 of which were NAP type 1 strains. To further assess the best approach to utilizing our conventional and molecular methods, we studied the populations of C. difficile in patient stool specimens (n = 23). Our results indicated that 13% of individual stool specimens had heterogeneous populations of C. difficile when we compared the molecular characterization results for multiple bacterial isolates (n = 10). Direct molecular analysis of stool specimens gave results that correlated well with the results obtained with cultured stool specimens; the direct molecular analysis was rapid, informative, and less costly than the testing of multiple patient stool isolates. PMID:19403775

  18. Regulation of neurotoxin and protease formation in Clostridium botulinum Okra B and Hall A by arginine.

    PubMed Central

    Patterson-Curtis, S I; Johnson, E A

    1989-01-01

    Supplementation of a minimal medium with high levels of arginine (20 g/liter) markedly decreased neurotoxin titers and protease activities in cultures of Clostridium botulinum Okra B and Hall A. Nitrogenous nutrients that are known to be derived from arginine, including proline, glutamate, and ammonia, also decreased protease and toxin but less so than did arginine. Proteases synthesized during growth were rapidly inactivated after growth stopped in media containing high levels of arginine. Separation of extracellular proteins by electrophoresis and immunoblots with antibodies to toxin showed that the decrease in toxin titers in media containing high levels of arginine was caused by both reduced synthesis of protoxin and impaired proteolytic activation. In contrast, certain other nutritional conditions stimulated protease and toxin formation in C. botulinum and counteracted the repression by arginine. Supplementation of the minimal medium with casein or casein hydrolysates increased protease activities and toxin titers. Casein supplementation of a medium containing high levels of arginine prevented protease inactivation. High levels of glucose (50 g/liter) also delayed the inactivation of proteases in both the minimal medium and a medium containing high levels of arginine. These observations suggest that the availability of nitrogen and energy sources, particularly arginine, affects the production and proteolytic processing of toxins and proteases in C. botulinum. Images PMID:2669631

  19. Proteome Alterations of Hippocampal Cells Caused by Clostridium botulinum C3 Exoenzyme.

    PubMed

    Schröder, Anke; Rohrbeck, Astrid; Just, Ingo; Pich, Andreas

    2015-11-01

    C3bot from Clostridium botulinum is a bacterial mono-ADP-ribosylating enzyme, which transfers an ADP-ribose moiety onto the small GTPases Rho A/B/C. C3bot and the catalytic inactive mutant (C3E174Q) cause axonal and dendritic growth as well as branching in primary hippocampal neurons. In cultured murine hippocampal HT22 cells, protein abundances were analyzed in response to C3bot or C3E174Q treatment using a shotgun proteomics approach. Proteome analyses were performed at four time points over 6 days. More than 4000 protein groups were identified at each time point and quantified in triplicate analyses. On day one, 46 proteins showed an altered abundance, and after 6 days, more than 700 proteins responded to C3bot with an up- or down-regulation. In contrast, C3E174Q had no provable impact on protein abundance. Protein quantification was verified for several proteins by multiple reaction monitoring. Data analysis of altered proteins revealed different cellular processes that were affected by C3bot. They are particularly involved in mitochondrial and lysosomal processes, adhesion, carbohydrate and glucose metabolism, signal transduction, and nuclear proteins of translation and ribosome biogenesis. The results of this study gain novel insights into the function of C3bot in hippocampal cells. PMID:26393427

  20. Diagnostic test accuracy of glutamate dehydrogenase for Clostridium difficile: Systematic review and meta-analysis.

    PubMed

    Arimoto, Jun; Horita, Nobuyuki; Kato, Shingo; Fuyuki, Akiko; Higurashi, Takuma; Ohkubo, Hidenori; Endo, Hiroki; Takashi, Nonaka; Kaneko, Takeshi; Nakajima, Atsushi

    2016-01-01

    We performed this systematic review and meta-analysis to assess the diagnostic accuracy of detecting glutamate dehydrogenase (GDH) for Clostridium difficile infection (CDI) based on the hierarchical model. Two investigators electrically searched four databases. Reference tests were stool cell cytotoxicity neutralization assay (CCNA) and stool toxigenic culture (TC). To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR) using a DerSimonian-Laird random-model and area the under hierarchical summary receiver operating characteristics (AUC) using Holling's proportional hazard models. The summary estimate of the sensitivity and the specificity were obtained using the bivariate model. According to 42 reports consisting of 3055 reference positive comparisons, and 26188 reference negative comparisons, the DOR was 115 (95%CI: 77-172, I(2) = 12.0%) and the AUC was 0.970 (95%CI: 0.958-0.982). The summary estimate of sensitivity and specificity were 0.911 (95%CI: 0.871-0.940) and 0.912 (95%CI: 0.892-0.928). The positive and negative likelihood ratios were 10.4 (95%CI 8.4-12.7) and 0.098 (95%CI 0.066-0.142), respectively. Detecting GDH for the diagnosis of CDI had both high sensitivity and specificity. Considering its low cost and prevalence, it is appropriate for a screening test for CDI. PMID:27418431

  1. Ethanol production by thermophilic bacteria: biochemical basis for ethanol and hydrogen tolerance in Clostridium thermohydrosulfuricum

    SciTech Connect

    Lovitt, R.W.; Shen, G.J.; Zeikus, J.G.

    1988-06-01

    The metabolic and enzymatic bases for growth tolerance to ethanol (4%) and H/sub 2/ (2 atm (1 atm = 101.29 kPa)) fermentation products in Clostridium thermohydrosulfuricum were compared in a sensitive wild-type strain and an insensitive alcohol-adapted strain. In the wild-type strain, ethanol (4%) and H/sub 2/ (2 atm) inhibited glucose but not pyruvate fermentation parameters (growth and end product formation). Inhibition of glucose fermentation by ethanol (4%) in the wild-type strain was reversed by addition of acetone (1%), which lowered H/sub 2/ and ethanol production while increasing isopropanol and acetate production. Pulsing cells grown in continuous culture on glucose with 5% ethanol or 1 atm of H/sub 2/ significantly raised the NADH/NAD ratio in the wild-type strain but not in the alcohol-adapted strain. Analysis of key oxidoreductases demonstrated that the alcohol-adapted strain lacked detectable levels of reduced ferredoxin-linked NAD reductase and NAD-linked alcohol dehydrogenase activities which are present in the wild-type strain. Differences in the glucose fermentation product ratios of the two strains were related to differences in lactate dehydrogenase and hydrogenase levels and sensitivity of glyceraldehyde 3-phosphate dehydrogenase activity to NADH inhibition. A biochemical model is proposed which describes a common enzymatic mechanism for growth tolerance of thermoanaerobes to moderate concentrations of both ethanol and hydrogen.

  2. Effect of Intramuscular or Intrahepatic Injections of Clostridium perfringens on Rabbit Serum Lactate Dehydrogenase 1

    PubMed Central

    Thomason, Dwayne

    1970-01-01

    Serum lactate dehydrogenase (LDH) activity, LDH isoenzyme pattern, phospholipase C activity, phosphorous level, hemoglobin, and erythrocyte osmotic fragility were followed in rabbits after intramuscular (IM) or intrahepatic (IH) injections of Clostridium perfringens. On the first day after IM injection, there was a drop in LDH activity; this was followed by an increase of LDH activity on the third and sixth day. On the seventh day, LDH activity began to decline, and by the ninth day it had almost returned to normal. On the sixth day after IM injection, there was an increase in serum LDH isoenzyme 5, hemoglobin, and erythrocyte osmotic fragility, but the increase of erythrocyte osmotic fragility and serum hemoglobin could not be attributed to phospholipase C activity since that enzyme was not detected nor was there an increase in serum phosphorus. C. perfringens was recovered by culturing the wound of IM-injected rabbits but not recovered from IH-injected rabbits. Rabbits injected IH showed no change from normal values in any of the tests performed. PMID:16557808

  3. Severe Sepsis due to Clostridium perfringens Bacteremia of Urinary Origin: A Case Report and Systematic Review

    PubMed Central

    Millard, Michael A.; McManus, Kathleen A.; Wispelwey, Brian

    2016-01-01

    Clostridium perfringens bacteremia is an uncommon yet serious clinical syndrome that typically arises from a gastrointestinal source. However, clinicians should consider nongastrointestinal sources as well. We present a rare case of C. perfringens bacteremia of urinary origin that required surgical intervention for definitive treatment. A 61-year-old male presented with acute nausea and vomiting, altered mental status, and chronic diarrhea. His physical exam revealed right costovertebral tenderness and his laboratory work-up revealed acute renal failure. Percutaneous blood cultures grew C. perfringens. Cross-sectional imaging revealed a right-sided ureteral stone with hydronephrosis, which required nephrostomy placement. On placement of the nephrostomy tube, purulent drainage was identified and Gram stain of the drainage revealed Gram-variable rods. A urinary source of C. perfringens was clinically supported. Although it is not a common presentation, nongastrointestinal sources such as a urinary source should be considered in C. perfringens bacteremia because failure to recognize a nongastrointestinal source can delay appropriate treatment, which may include surgical intervention. PMID:26998370

  4. Modulation of toxin production by the flagellar regulon in Clostridium difficile.

    PubMed

    Aubry, Annie; Hussack, Greg; Chen, Wangxue; KuoLee, Rhonda; Twine, Susan M; Fulton, Kelly M; Foote, Simon; Carrillo, Catherine D; Tanha, Jamshid; Logan, Susan M

    2012-10-01

    We show in this study that toxin production in Clostridium difficile is altered in cells which can no longer form flagellar filaments. The impact of inactivation of fliC, CD0240, fliF, fliG, fliM, and flhB-fliR flagellar genes upon toxin levels in culture supernatants was assessed using cell-based cytotoxicity assay, proteomics, immunoassay, and immunoblotting approaches. Each of these showed that toxin levels in supernatants were significantly increased in a fliC mutant compared to that in the C. difficile 630 parent strain. In contrast, the toxin levels in supernatants secreted from other flagellar mutants were significantly reduced compared with that in the parental C. difficile 630 strain. Transcriptional analysis of the pathogenicity locus genes (tcdR, tcdB, tcdE, and tcdA) revealed a significant increase of all four genes in the fliC mutant strain, while transcription of all four genes was significantly reduced in fliM, fliF, fliG, and flhB-fliR mutants. These results demonstrate that toxin transcription in C. difficile is modulated by the flagellar regulon. More significantly, mutant strains showed a corresponding change in virulence compared to the 630 parent strain when tested in a hamster model of C. difficile infection. This is the first demonstration of differential flagellum-related transcriptional regulation of toxin production in C. difficile and provides evidence for elaborate regulatory networks for virulence genes in C. difficile. PMID:22851750

  5. Mechanisms of microbial oil recovery by Clostridium acetobutylicum and Bacillus strain JF-2

    SciTech Connect

    Marsh, T.L.; Zhang, X.; Knapp, R.M.; McInerney, M.J.; Sharma, P.K.; Jackson, B.E.

    1995-12-31

    Core displacement experiments at elevated pressures were conducted to determine whether microbial processes are effective under conditions that simulate those found in an actual oil reservoir. The in-situ growth of Clostridium acetobutylicum and Bacillus strain JF-2 resulted in the recovery of residual oil. About 21 and 23% of the residual oil was recovered by C. acetobutylicum and Bacillus strain JF-2, respectively. Flooding cores with cell-free culture fluids of C. acetobutylicum with and without the addition of 50 mM acetone and 100 mM butanol did not result in the recovery of residual oil. Mathematical simulations showed that the amount of gas produced by the clostridial fermentation was not showed that the amount of gas produced by the clostridial fermentation was not sufficient to recover residual oil. Oil recovery by Bacillus strain JF-2 was highly correlated to surfactant production. A biosurfactant-deficient mutant of strain JF-2 was not capable of recovering residual oil. These data show that surfactant production is an important mechanism for microbially enhanced oil recovery. The mechanism for oil recovery by C. acetobutylicum is not understood at this time, but the production of acids, solvents, or gases alone cannot explain the observed increases in oil recovery by this organism.

  6. Isolation from soil and properties of the extreme thermophile Clostridium thermohydrosulfuricum.

    PubMed Central

    Wiegel, J; Ljungdahl, L G; Rawson, J R

    1979-01-01

    Thirteen strains of a strict anaerobic, extreme thermophilic bacterium were isolated from soil samples of moderate temperature, from a sewage plant in Georgia, and from hot springs in Utah and Wyoming. They were identified as strains of Clostridium thermohydrosulfuricum. The guanosine + cytosine content (moles percent) was 37.6 (determined by buoyant density) and 34.1 (determined by melting temperature). All strains required a factor present in yeast extract or tryptone growth. Growth characteristics were as follows: a pH range of 5 to 9, with the optimum between 6.9 to 7.5, in a temperature range of 40 to 78 degrees C, with the optimum at 68 degrees C. The doubling time, when grown on glucose at temperature and pH optima, was 1.2 h. The main products of glucose fermentation were ethanol, lactate, acetate, CO2, and H2. The fermentation was inhibited by H2. Formation of spores occurred easily on glucose-agar medium or when cultures growing at temperatures above 65 degrees C were allowed to cool to temperature below 55 degrees C. C. thermohydrosulfuricum occurs widely distributed in the natural environment. PMID:39062

  7. Cloning of thermostable cellulase genes of Clostridium thermocellum and their secretive expression in Bacillus subtilis.

    PubMed

    Liu, Jian-Ming; Xin, Xiu-Juan; Li, Chun-Xiu; Xu, Jian-He; Bao, Jie

    2012-02-01

    Screening for the powerful cellulase genes with improved activities remains a challenge for the biorefinery research. In this study, five cellobiohydrolase genes and one endoglucanase gene sourced from Clostridium thermocellum DSM 1237, cbhA, celK, celO, cel48Y, cel48S, and celA were cloned into a newly established tool vector pP43JM2 and expressed in two Bacillus subtilis strains, B. subtilis WB600 and B. subtilis WB800, respectively. Most of the cellulases produced in the B. subtilis recombinants were efficiently secreted into the culture medium. These secreted soluble proteins showed distinct cellulase activities using phosphoric acid swollen cellulose (PASC) as the substrate and they also demonstrated strong synergistic effects for PASC, Avicel cellulose, and the dilute acid pretreated corn stover. The current work provided a quick secretive cloning method for screening cellulase genes and may provide a host strain for constructing a consolidated bioprocessing platform with the capacity of secretive expression of multiple cellulases. PMID:22101447

  8. Clostridium difficile glutamate dehydrogenase is a secreted enzyme that confers resistance to H2O2

    PubMed Central

    Girinathan, Brintha Prasummanna; Braun, Sterling E.

    2014-01-01

    Clostridium difficile produces an NAD-specific glutamate dehydrogenase (GDH), which converts l-glutamate into α-ketoglutarate through an irreversible reaction. The enzyme GDH is detected in the stool samples of patients with C. difficile-associated disease and serves as one of the diagnostic tools to detect C. difficile infection (CDI). We demonstrate here that supernatant fluids of C. difficile cultures contain GDH. To understand the role of GDH in the physiology of C. difficile, an isogenic insertional mutant of gluD was created in strain JIR8094. The mutant failed to produce and secrete GDH as shown by Western blot analysis. Various phenotypic assays were performed to understand the importance of GDH in C. difficile physiology. In TY (tryptose yeast extract) medium, the gluD mutant grew slower than the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the growth defect in TY medium. The presence of extracellular GDH may have a functional role in the pathogenesis of CDI. In support of this assumption we found higher sensitivity to H2O2 in the gluD mutant as compared to the parent strain. Complementation of the gluD mutant with the functional gluD gene reversed the H2O2 sensitivity. PMID:24145018

  9. Mutant selection and phenotypic and genetic characterization of ethanol-tolerant strains of Clostridium thermocellum.

    PubMed

    Shao, Xiongjun; Raman, Babu; Zhu, Mingjun; Mielenz, Jonathan R; Brown, Steven D; Guss, Adam M; Lynd, Lee R

    2011-11-01

    Clostridium thermocellum is a model microorganism for converting cellulosic biomass into fuels and chemicals via consolidated bioprocessing. One of the challenges for industrial application of this organism is its low ethanol tolerance, typically 1-2% (w/v) in wild-type strains. In this study, we report the development and characterization of mutant C. thermocellum strains that can grow in the presence of high ethanol concentrations. Starting from a single colony, wild-type C. thermocellum ATCC 27405 was sub-cultured and adapted for growth in up to 50 g/L ethanol using either cellobiose or crystalline cellulose as the growth substrate. Both the adapted strains retained their ability to grow on either substrate and displayed a higher growth rate and biomass yield than the wild-type strain in the absence of ethanol. With added ethanol in the media, the mutant strains displayed an inverse correlation between ethanol concentration and growth rate or biomass yield. Genome sequencing revealed six common mutations in the two ethanol-tolerant strains including an alcohol dehydrogenase gene and genes involved in arginine/pyrimidine biosynthetic pathway. The potential role of these mutations in ethanol tolerance phenotype is discussed. PMID:21874277

  10. Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat.

    PubMed

    Khan, Madiha; Nazir, Jawad; Anjum, Aftab Ahmad; Ahmad, Mansur-Ud-Din; Nawaz, Muhammad; Shabbir, Muhammad Zubair

    2015-08-01

    A total of 300 meat samples comprising mutton, beef, and chicken meat (n = 100) collected from either local butcher shops or large meat outlets situated at various areas of Lahore City located in Punjab province of Pakistan were tested for the isolation of Clostridium perfringens. Prevalence of the organism was highest in the chicken (6 %) followed by mutton (5 %) and beef (1 %). Contamination level was high (10/150) in the samples collected from local butcher shops in comparison to the samples collected from large meat outlets (2/150). All of the raw meat samples were negative for the presence of alpha, beta and epsilon toxins of C. perfringens as detected through ELISA. Out of a total number of 12 isolates only half were capable of producing enterotoxins when cultured in trypticase glucose yeast (TGY) broth. Toxinotyping of the isolates showed that 3 were of type A while one each of the remaining three belonged to type B, C, and D. Antibiotic susceptibility testing of the toxin producing isolates revealed that C. perfringens were susceptible to chloramphenicol, ciprofloxacin, metronidazole, and ceftriaxone. All of the other drugs were relatively less effective with a least activity of amoxicillin against the isolates. PMID:26243960

  11. Diagnostic test accuracy of glutamate dehydrogenase for Clostridium difficile: Systematic review and meta-analysis

    PubMed Central

    Arimoto, Jun; Horita, Nobuyuki; Kato, Shingo; Fuyuki, Akiko; Higurashi, Takuma; Ohkubo, Hidenori; Endo, Hiroki; Takashi, Nonaka; Kaneko, Takeshi; Nakajima, Atsushi

    2016-01-01

    We performed this systematic review and meta-analysis to assess the diagnostic accuracy of detecting glutamate dehydrogenase (GDH) for Clostridium difficile infection (CDI) based on the hierarchical model. Two investigators electrically searched four databases. Reference tests were stool cell cytotoxicity neutralization assay (CCNA) and stool toxigenic culture (TC). To assess the overall accuracy, we calculated the diagnostic odds ratio (DOR) using a DerSimonian-Laird random-model and area the under hierarchical summary receiver operating characteristics (AUC) using Holling’s proportional hazard models. The summary estimate of the sensitivity and the specificity were obtained using the bivariate model. According to 42 reports consisting of 3055 reference positive comparisons, and 26188 reference negative comparisons, the DOR was 115 (95%CI: 77–172, I2 = 12.0%) and the AUC was 0.970 (95%CI: 0.958–0.982). The summary estimate of sensitivity and specificity were 0.911 (95%CI: 0.871–0.940) and 0.912 (95%CI: 0.892–0.928). The positive and negative likelihood ratios were 10.4 (95%CI 8.4–12.7) and 0.098 (95%CI 0.066–0.142), respectively. Detecting GDH for the diagnosis of CDI had both high sensitivity and specificity. Considering its low cost and prevalence, it is appropriate for a screening test for CDI. PMID:27418431

  12. In-vitro antimicrobial susceptibility of Clostridium perfringens from commercial turkey and broiler chicken origin.

    PubMed

    Watkins, K L; Shryock, T R; Dearth, R N; Saif, Y M

    1997-02-01

    The minimum inhibitory concentrations (MIC) of eight antibiotics and two anticoccidial agents were determined for Clostridium perfringens strains isolated from 26 commercial broiler farms and 22 commercial turkey farms. Isolates were obtained from the intestines of birds on the farm or as the processing plant using standard culture and identification techniques. The microbroth dilution test was used to determine the MIC for each compound. Most isolates from chickens had MICs in the range of 2-16 mg/L for tilmicosin, tylosin and virginiamycin, whereas the MICs for avilamycin, avoparcin, monensin, narasin and penicillin were < or = 1 mg/L. Most strains from chickens had high MICs (> or = 64 mg/L) and appeared to be resistant to bacitracin and lincomycin. Most turkey isolates had MICs in the range of 2-16 mg/L for bacitracin, tilmicosin, tylosin and virginiamycin, with strains exhibiting MICs < or = 1 mg/L for avilamycin, avoparcin, monensin, narasin and penicillin. Several turkey isolates had MICs > or = 64 mg/L to lincomycin. No attempt was made to associate farm usage of a particular antibiotic to the antibiograms. PMID:9057262

  13. Identification of an epithelial cell receptor responsible for Clostridium difficile TcdB-induced cytotoxicity

    PubMed Central

    LaFrance, Michelle E.; Farrow, Melissa A.; Chandrasekaran, Ramyavardhanee; Sheng, Jinsong; Rubin, Donald H.; Lacy, D. Borden

    2015-01-01

    Clostridium difficile is the leading cause of hospital-acquired diarrhea in the United States. The two main virulence factors of C. difficile are the large toxins, TcdA and TcdB, which enter colonic epithelial cells and cause fluid secretion, inflammation, and cell death. Using a gene-trap insertional mutagenesis screen, we identified poliovirus receptor-like 3 (PVRL3) as a cellular factor necessary for TcdB-mediated cytotoxicity. Disruption of PVRL3 expression by gene-trap mutagenesis, shRNA, or CRISPR/Cas9 mutagenesis resulted in resistance of cells to TcdB. Complementation of the gene-trap or CRISPR mutants with PVRL3 resulted in restoration of TcdB-mediated cell death. Purified PVRL3 ectodomain bound to TcdB by pull-down. Pretreatment of cells with a monoclonal antibody against PVRL3 or prebinding TcdB to PVRL3 ectodomain also inhibited cytotoxicity in cell culture. The receptor is highly expressed on the surface epithelium of the human colon and was observed to colocalize with TcdB in both an explant model and in tissue from a patient with pseudomembranous colitis. These data suggest PVRL3 is a physiologically relevant binding partner that can serve as a target for the prevention of TcdB-induced cytotoxicity in C. difficile infection. PMID:26038560

  14. DNA detection of Clostridium difficile infection based on real-time resistance measurement.

    PubMed

    Liu, C; Jiang, D N; Xiang, G M; Luo, F K; Liu, L L; Yu, J C; Pu, X Y

    2013-01-01

    We used a newly developed electrochemical method, real-time resistance measurement, based on loop-mediated isothermal amplification (LAMP), with real-time resistance monitoring and derivative analysis. DNA extracted from specimens was amplified through LAMP reaction. The 2 products of LAMP, DNA and pyrophosphate, both are negative ions; they combine with positive dye (crystal violet) and positive ions (Mg(2+)), which leads to an increase in the resistivity of the reaction liquid. The changes of resistivity were measured in real-time with a specially designed resistance electrode, to detect Clostridium difficile DNA. We found that electrochemical detection of C. difficile could be completed in 0.5-1 h, with a detection limit of 10(2) CFU/mL, with high accuracy (95.0%), sensitivity (91.1%), and specificity (97.3%) compared to PCR methods. C. difficile is commonly associated with antibiotic-induced diarrhea. Due to the difficulty in performing anaerobic culture and cytotoxicity neutralization assays, a simple, rapid, sensitive, and accurate method is preferred. We conclude that real-time resistance measurement is a rapid, sensitive, and stable method for the diagnosis of C. difficile infection that could be applied to gene chips and pocket instruments. PMID:24065671

  15. Optimization of the Production of Inactivated Clostridium novyi Type B Vaccine Using Computational Intelligence Techniques.

    PubMed

    Aquino, P L M; Fonseca, F S; Mozzer, O D; Giordano, R C; Sousa, R

    2016-07-01

    Clostridium novyi causes necrotic hepatitis in sheep and cattle, as well as gas gangrene. The microorganism is strictly anaerobic, fastidious, and difficult to cultivate in industrial scale. C. novyi type B produces alpha and beta toxins, with the alpha toxin being linked to the presence of specific bacteriophages. The main strategy to combat diseases caused by C. novyi is vaccination, employing vaccines produced with toxoids or with toxoids and bacterins. In order to identify culture medium components and concentrations that maximized cell density and alpha toxin production, a neuro-fuzzy algorithm was applied to predict the yields of the fermentation process for production of C. novyi type B, within a global search procedure using the simulated annealing technique. Maximizing cell density and toxin production is a multi-objective optimization problem and could be treated by a Pareto approach. Nevertheless, the approach chosen here was a step-by-step one. The optimum values obtained with this approach were validated in laboratory scale, and the results were used to reload the data matrix for re-parameterization of the neuro-fuzzy model, which was implemented for a final optimization step with regards to the alpha toxin productivity. With this methodology, a threefold increase of alpha toxin could be achieved. PMID:27003282

  16. Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

    SciTech Connect

    Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D; Johnson, Courtney M; Tschaplinski, Timothy J; Martin, Madhavi Z; Engle, Nancy L; Van den Berg, Robert A; Argyros, Aaron; Caiazza, Nicky; Guss, Adam M; Lynd, Lee R

    2013-01-01

    Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of L-lactate (Dldh) and/or acetate (Dpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Dldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Dhpt Dspo0A). The Dpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A Dldh Dpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Dpta. Free amino acids were secreted by all examined strains, with both Dpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Dldh Dpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Dpta and 16-fold in the Dldh Dpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP* through increased production of amino acids.

  17. Characterization of Clostridium thermocellum strains with disrupted fermentation end product pathways

    SciTech Connect

    Van Der Veen, Douwe; Lo, Jonathan; Brown, Steven D; Johnson, Courtney M; Tschaplinski, Timothy J; Martin, Madhavi Z; Engle, Nancy L; Argyros, Aaron; Van den Berg, Robert A; Caiazza, Nicky; Guss, Adam M; Lynd, Lee R

    2013-01-01

    Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of Llactate ( ldh) and/or acetate ( pta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end products. Cellobiose-grown cultures of the ldh strain had identical biomass accumulation, fermentation end products, transcription profile and intracellular metabolite concentrations compared to its parent strain (DSM1313 hpt spo0A). The pta-deficient strain grew slower and had 30% lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A ldh pta double mutant strain evolved for faster growth had growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to pta. Free amino acids were secreted by all examined strains, with both pta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for ldh pta reached 5 mM by the end of growth, or 2.7% of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to 6-fold in the pta and 16-fold in the ldh pta strain. We hypothesize that the deletions in fermentation end product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP+ through increased production of amino acids.

  18. Effects of end products on fermentation profiles in Clostridium carboxidivorans P7 for syngas fermentation.

    PubMed

    Zhang, Jie; Taylor, Steven; Wang, Yi

    2016-10-01

    Clostridium carboxidivorans P7 is a strict anaerobic bacterium capable of converting syngas to biofuels. However, its fermentation profiles is poorly understood. Here, various end-products, including acetic acid, butyric acid, hexanoic acid, ethanol and butanol were supplemented to evaluate their effects on fermentation profiles in C. carboxidivorans at two temperatures. At 37°C, fatty acids addition likely led to more corresponding alcohols production. At 25°C, C2 and C4 fatty acids supplementation resulted in more corresponding higher fatty acids, while supplemented hexanoic acid increased yields of C2 and C4 fatty acids and hexanol. Supplementation of ethanol or butanol caused increased production of C2 and C4 acids at both temperatures; however, long-chain alcohols were still more likely produced at lower temperature. In conclusion, fermentation profiles of C. carboxidivorans can be changed in respond to pre-added end-products and carbon flow may be redirected to desired products by controlling culture conditions. PMID:27459682

  19. Selection of a Clostridium perfringens type D epsilon toxin producer via dot-blot test.

    PubMed

    Gonçalves, Luciana A; Lobato, Zélia I P; Silva, Rodrigo O S; Salvarani, Felipe M; Pires, Prhiscylla S; Assis, Ronnie A; Lobato, Francisco C F

    2009-11-01

    Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin. PMID:19779698

  20. Functional analysis of neutralizing antibodies against Clostridium perfringens epsilon-toxin.

    PubMed

    McClain, Mark S; Cover, Timothy L

    2007-04-01

    The Clostridium perfringens epsilon-toxin causes a severe, often fatal illness (enterotoxemia) characterized by cardiac, pulmonary, kidney, and brain edema. In this study, we examined the activities of two neutralizing monoclonal antibodies against the C. perfringens epsilon-toxin. Both antibodies inhibited epsilon-toxin cytotoxicity towards cultured MDCK cells and inhibited the ability of the toxin to form pores in the plasma membranes of cells, as shown by staining cells with the membrane-impermeant dye 7-aminoactinomycin D. Using an antibody competition enzyme-linked immunosorbent assay (ELISA), a peptide array, and analysis of mutant toxins, we mapped the epitope recognized by one of the neutralizing monoclonal antibodies to amino acids 134 to 145. The antibody competition ELISA and analysis of mutant toxins suggest that the second neutralizing monoclonal antibody also recognizes an epitope in close proximity to this region. The region comprised of amino acids 134 to 145 overlaps an amphipathic loop corresponding to the putative membrane insertion domain of the toxin. Identifying the epitopes recognized by these neutralizing antibodies constitutes an important first step in the development of therapeutic agents that could be used to counter the effects of the epsilon-toxin. PMID:17261609

  1. Clostridium perfringens epsilon toxin is cytotoxic for human renal tubular epithelial cells.

    PubMed

    Fernandez Miyakawa, Mariano E; Zabal, Osvaldo; Silberstein, Claudia

    2011-04-01

    Clostridium perfringens epsilon toxin (ETX) is responsible for a fatal enterotoxemia in different animal species, producing extensive renal damage, neurological disturbance and edema of lungs, heart and kidneys. However, there is no information about the susceptibility of humans to ETX. Here, we report that primary cultures of human renal tubular epithelial cells (HRTEC) exposed to ETX showed a marked swelling with subsequent large blebs surrounding most cells. The incubation of HRTEC with ETX produced a reduction of cell viability in a dose- and time-dependent manner. The CD(50) after 1-hour and 24-hour incubation were 3 µg/mL and 0.5 µg/mL, respectively. The pulse with ETX for 3 min was enough to produce a significant cytotoxic effect on HRTEC after 1-hour incubation. ETX binds to HRTEC forming a large complex of about 160 kDa similar to what was found in the Madin-Darby canine kidney (MDCK) cell line. The HRTEC could be a useful cell model to improve the understanding of the mechanisms involved on the cell damage mediated by ETX. PMID:20488848

  2. Rapid Cytopathic Effects of Clostridium perfringens Beta-Toxin on Porcine Endothelial Cells▿ †

    PubMed Central

    Gurtner, Corinne; Popescu, Francesca; Wyder, Marianne; Sutter, Esther; Zeeh, Friederike; Frey, Joachim; von Schubert, Conrad; Posthaus, Horst

    2010-01-01

    Clostridium perfringens type C isolates cause fatal, segmental necro-hemorrhagic enteritis in animals and humans. Typically, acute intestinal lesions result from extensive mucosal necrosis and hemorrhage in the proximal jejunum. These lesions are frequently accompanied by microvascular thrombosis in affected intestinal segments. In previous studies we demonstrated that there is endothelial localization of C. perfringens type C β-toxin (CPB) in acute lesions of necrotizing enteritis. This led us to hypothesize that CPB contributes to vascular necrosis by directly damaging endothelial cells. By performing additional immunohistochemical studies using spontaneously diseased piglets, we confirmed that CPB binds to the endothelial lining of vessels showing early signs of thrombosis. To investigate whether CPB can disrupt the endothelium, we exposed primary porcine aortic endothelial cells to C. perfringens type C culture supernatants and recombinant CPB. Both treatments rapidly induced disruption of the actin cytoskeleton, cell border retraction, and cell shrinkage, leading to destruction of the endothelial monolayer in vitro. These effects were followed by cell death. Cytopathic and cytotoxic effects were inhibited by neutralization of CPB. Taken together, our results suggest that CPB-induced disruption of endothelial cells may contribute to the pathogenesis of C. perfringens type C enteritis. PMID:20404076

  3. The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep.

    PubMed

    Uzal, F A; Kelly, W R; Morris, W E; Bermudez, J; Baisón, M

    2004-09-01

    The pathological findings in sheep with peracute experimental Clostridium perfringens type D enterotoxemia are described. Of 16 animals inoculated intraduodenally with a whole culture of this microorganism and a starch solution in the abomasum, 12 developed clinical signs including increased respiratory efforts, recumbency, paddling, bleating, convulsions, blindness, and opisthotonus. Diarrhea was not observed in any of the animals. The time lapse between the beginning of intraduodenal infusion and onset of clinical signs varied between 30 minutes and 26 hours, and the clinical course varied between 1 and 9 hours. Gross postmortem changes were observed in these 12 animals and included pulmonary edema; excess pericardial, peritoneal, or pleural fluid with or without strands of fibrin; liquid small intestinal contents; leptomeningeal edema; cerebellar coning; and subcapsular petechiae on kidneys. Histological changes consisted of severe edema of pleura and interlobular septa and around blood vessels and airways and acidophilic, homogeneous, proteinaceous perivascular edema in the brain. Five of 12 animals (42%) with clinical signs consistent with enterotoxemia lacked specific histological lesions in the brain. None of the intoxicated or control animals developed nephrosis. Glucose was detected in the urine of 3 of 6 animals that were tested for this analyte. These results stress the importance of the use of histological examination of the brain, coupled with epsilon toxin detection, for a definitive diagnosis of C. perfringens type D enterotoxemia in sheep. PMID:15460322

  4. Purification and properties of the inducible coenzyme A-linked butyraldehyde dehydrogenase from Clostridium acetobutylicum.

    PubMed Central

    Palosaari, N R; Rogers, P

    1988-01-01

    The coenzyme A (CoA)-linked butyraldehyde dehydrogenase (BAD) from Clostridium acetobutylicum was characterized and purified to homogeneity. The enzyme was induced over 200-fold, coincident with a shift from an acidogenic to a solventogenic fermentation, during batch culture growth. The increase in enzyme activity was found to require new protein synthesis since induction was blocked by the addition of rifampin and antibody against the purified enzyme showed the appearance of enzyme antigen beginning at the shift of the fermentation and increasing coordinately with the increase in enzyme specific activity. The CoA-linked acetaldehyde dehydrogenase was copurified with BAD during an 89-fold purification, indicating that one enzyme accounts for the synthesis of the two aldehyde intermediates for both butanol and ethanol production. Butanol dehydrogenase activity was clearly separate from the BAD enzyme activity on TEAE cellulose. A molecular weight of 115,000 was determined for the native enzyme, and the enzyme subunit had a molecular weight of 56,000 indicating that the active form is a homodimer. Kinetic constants were determined in both the forward and reverse directions. In the reverse direction both the Vmax and the apparent affinity for butyraldehyde and caproaldehyde were significantly greater than they were for acetaldehyde, while in the forward direction, the Vmax for butyryl-CoA was fivefold that for acetyl-CoA. These and other properties of BAD indicate that this enzyme is distinctly different from other reported CoA-dependent aldehyde dehydrogenases. Images PMID:3384801

  5. Changes in Membrane Plasmalogens of Clostridium pasteurianum during Butanol Fermentation as Determined by Lipidomic Analysis

    PubMed Central

    Kolek, Jan; Patáková, Petra; Melzoch, Karel; Sigler, Karel; Řezanka, Tomáš

    2015-01-01

    Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content. PMID:25807381

  6. Cloning a neutral protease of Clostridium histolyticum, determining its substrate specificity, and designing a specific substrate.

    PubMed

    Maeda, Hiroshi; Nakagawa, Kanako; Murayama, Kazutaka; Goto, Masafumi; Watanabe, Kimiko; Takeuchi, Michio; Yamagata, Youhei

    2015-12-01

    Islet transplantation is a prospective treatment for restoring normoglycemia in patients with type 1 diabetes. Islet isolation from pancreases by decomposition with proteolytic enzymes is necessary for transplantation. Two collagenases, collagenase class I (ColG) and collagenase class II (ColH), from Clostridium histolyticum have been used for islet isolation. Neutral proteases have been added to the collagenases for human islet isolation. A neutral protease from C. histolyticum (NP) and thermolysin from Bacillus thermoproteolyicus has been used for the purpose. Thermolysin is an extensively studied enzyme, but NP is not well known. We therefore cloned the gene encoding NP and constructed a Bacillus subtilis overexpression strain. The expressed enzyme was purified, and its substrate specificity was examined. We observed that the substrate specificity of NP was higher than that of thermolysin, and that the protein digestion activities of NP, as determined by colorimetric methods, were lower than those of thermolysin. It seems that decomposition using NP does not negatively affect islets during islet preparation from pancreases. Furthermore, we designed a novel substrate that allows the measurement of NP activity specifically in the enzyme mixture for islet preparation and the culture broth of C. histolyticum. The activity of NP can also be monitored during islet isolation. We hope the purified enzyme and this specific substrate contribute to the optimization of islet isolation from pancreases and that it leads to the success of islet transplantation and the improvement of the quality of life (QOL) for diabetic patients. PMID:26307443

  7. Nisin is an effective inhibitor of Clostridium difficile vegetative cells and spore germination.

    PubMed

    Le Lay, Christophe; Dridi, Larbi; Bergeron, Michel G; Ouellette, Marc; Fliss, Ismaı L

    2016-02-01

    Clostridium difficile is the most frequently identified enteric pathogen in patients with nosocomial antibiotic-associated diarrhoea and pseudomembranous colitis. Several clinically isolated C. difficile strains are resistant to antibiotics other than metronidazole and vancomycin. Recently, bacteriocins of lactic acid bacteria have been proposed as an alternative or complementary treatment. The aim of this study was to investigate the inhibitory effect of nisin, a bacteriocin produced by several strains of Lactococcus lactis, against clinical isolates of C. difficile. Nisin Z obtained from culture of L. lactis subsp. lactis biovar. diacetylactis was tested along with commercial nisin A. The effect of nisin A on C. difficile spores was also examined. Nisin A and Z both inhibited the growth of all C. difficile isolates, and MICs were estimated at 6.2 μg ml(-1) for nisin Z and 0.8 μg ml(-1) for nisin A. In addition, C. difficile spores were also susceptible to nisin A (25.6 μg ml(-1)), which reduced spore viability by 40-50%. These results suggested that nisin and hence nisin-producing Lactococcus strains could be used to treat C. difficile-associated diarrhoea. PMID:26555543

  8. Prospects for flavonoid and related phytochemicals as nature-inspired treatments for Clostridium difficile infection

    PubMed Central

    Wu, Xiaoqian; Alam, Zahidul; Feng, Li; Tsutsumi, Lissa S.; Sun, Dianqing; Hurdle, Julian G.

    2013-01-01

    Aims There is a need for novel treatments for Clostridium difficile infection (CDI). Antibacterial flavonoids are part of a large family of polyphenol phytochemicals with a long history of use in ethnomedicine, but are unexamined against C. difficile. We explored their anti-difficile properties. Methods and Results Anti-difficile activities were determined for several naturally occurring flavonoids, olympicin A and synthetic 4-chromanone and chalcone analogs. With the exception of olympicin A, most naturally occurring phytochemicals tested were poorly active. Diversified synthetic flavonoids resembling olympicin A retained anti-difficile activity, suggesting olympicin A could act as a pharmacophore to obtain novel agents. They also demonstrated concentration dependent killing of logarithmic and stationary phase cultures and reduced sporulation and toxin production. Olympicin A and some synthetic flavonoids dissipated the bacterial transmembrane potential. Interestingly, mutants could only be selected with the analog 207 at a frequency of 10-9. Conclusions Based on the potent anti-difficile properties of olympicin A and modified flavonoids, further exploration of this class of phytochemicals is warranted. Significance and Impact of the Study CDI is a major problem in developed countries. These studies point to there being an avenue for optimizing plant-derived flavonoids, and related antibacterial phytochemicals, as nature-inspired approaches to treat CDI. PMID:24479135

  9. Contribution of Spores to the Ability of Clostridium difficile To Adhere to Surfaces

    PubMed Central

    Joshi, Lovleen Tina; Phillips, Daniel S.; Williams, Catrin F.; Alyousef, Abdullah

    2012-01-01

    Clostridium difficile is the commonest cause of hospital-acquired infection in the United Kingdom. We characterized the abilities of 21 clinical isolates to form spores; to adhere to inorganic and organic surfaces, including stainless steel and human adenocarcinoma cells; and to germinate. The composition of culture media had a significant effect on spore formation, as significantly more spores were produced in brain heart infusion broth (Student's t test; P = 0.018). The spore surface relative hydrophobicity (RH) varied markedly (14 to 77%) and was correlated with the ability to adhere to stainless steel. We observed no correlation between the ribotype and the ability to adhere to steel. When the binding of hydrophobic (DS1813; ribotype 027; RH, 77%) and hydrophilic (DS1748; ribotype 002; RH, 14%) spores to human gut epithelial cells at different stages of cell development was examined, DS1813 spores adhered more strongly, suggesting the presence of surface properties that aid attachment to human cells. Electron microscopy studies revealed the presence of an exosporium surrounding DS1813 spores that was absent from spores of DS1748. Finally, the ability of spores to germinate was found to be strain and medium dependent. While the significance of these findings to the disease process has yet to be determined, this study has highlighted the importance of analyzing multiple isolates when attempting to characterize the behavior of a bacterial species. PMID:22923404

  10. Severe Sepsis due to Clostridium perfringens Bacteremia of Urinary Origin: A Case Report and Systematic Review.

    PubMed

    Millard, Michael A; McManus, Kathleen A; Wispelwey, Brian

    2016-01-01

    Clostridium perfringens bacteremia is an uncommon yet serious clinical syndrome that typically arises from a gastrointestinal source. However, clinicians should consider nongastrointestinal sources as well. We present a rare case of C. perfringens bacteremia of urinary origin that required surgical intervention for definitive treatment. A 61-year-old male presented with acute nausea and vomiting, altered mental status, and chronic diarrhea. His physical exam revealed right costovertebral tenderness and his laboratory work-up revealed acute renal failure. Percutaneous blood cultures grew C. perfringens. Cross-sectional imaging revealed a right-sided ureteral stone with hydronephrosis, which required nephrostomy placement. On placement of the nephrostomy tube, purulent drainage was identified and Gram stain of the drainage revealed Gram-variable rods. A urinary source of C. perfringens was clinically supported. Although it is not a common presentation, nongastrointestinal sources such as a urinary source should be considered in C. perfringens bacteremia because failure to recognize a nongastrointestinal source can delay appropriate treatment, which may include surgical intervention. PMID:26998370

  11. Enhanced butanol production from cassava with Clostridium acetobutylicum by genome shuffling.

    PubMed

    Li, Shu-Bo; Qian, Yi; Liang, Zheng-Wu; Guo, Yuan; Zhao, Mou-Ming; Pang, Zong-Wen

    2016-04-01

    To obtain strains exhibiting high levels of solvent tolerance and butanol production, wild type strains of Clostridium acetobutylicum butanol-producing strain GX01 and Lactobacillus mucosae butanol-tolerant strain M26 were subjected to mutagenesis combining N-methyl-N-nitro-N-nitrosoguanidine induction with genome shuffling. After four successive rounds of genome shuffling, the C. acetobutylicum shuffled strain GS4-3 showing greater levels of fermentation performances (such as secreting a higher level of amylase, improving the thermal stability, and possessing greater environmental robustness) compared to the wild type strains was isolated. As a result, after optimization of culture conditions, mutant GS4-3 produced 32.6 g/L of total solvent, 20.1 g/L of butanol production, and 0.35 g/L/h of butanol productivity, which were, respectively, increased by 23.5, 23.3, and 40.0 % than the wild-type strain GX01, in a 10 L bioreactor. The enhanced production of butanol and tolerance of solvent of mutant associated with GS4-3 make it promising for acetone/butanol/ethanol fermentation from cassava (Manihot esculenta). PMID:26925615

  12. Specific immunotherapy in combination with Clostridium butyricum inhibits allergic inflammation in the mouse intestine

    PubMed Central

    Shi, Yanhong; Xu, Ling-Zhi; Peng, Kangsheng; Wu, Wei; Wu, Ruijin; Liu, Zhi-Qiang; Yang, Gui; Geng, Xiao-Rui; Liu, Jun; Liu, Zhi-Gang; Liu, Zhanju; Yang, Ping-Chang

    2015-01-01

    The current therapy on allergic inflammation is unsatisfactory. Probiotics improve the immunity in the body. This study aims to test a hypothesis that administration with Clostridium butyricum (C. butyricum) enforces the effect of specific immunotherapy (SIT) on intestinal allergic inflammation. In this study, an ovalbumin (OVA) specific allergic inflammation mouse model was created. The mice were treated with SIT or/and C. butyricum. The results showed that the intestinal allergic inflammation was only moderately alleviated by SIT, which was significantly enforced by a combination with C. butyricum; treating with C. butyricum alone did not show much inhibitory efficacy. The increase in the frequency of the interleukin (IL)-10-producing OVA-specific B cell (OVAsBC) was observed in mice in parallel to the inhibitory effect on the intestinal allergic inflammation. The in vitro treatment of the OVAsBCs with OVA increased the histone deacetylase-1 (HDAC1) phosphorylation, modulated the transcription of the Bcl6 gene, and triggered the OVAsBCs to differentiate to the IgE-producing plasma cells. Exposure to both OVA and butyrate sodium in the culture increased the expression of IL-10 in OVAsBCs. In conclusion, administration with C. butyricum enforces the inhibitory effect of SIT on allergic inflammation in the mouse intestine. PMID:26627845

  13. Clostridium Sacroiliitis (Gas Gangrene) Following Sacroiliac Joint Injection--Case Report and Review of the Literature.

    PubMed

    Kurnutala, Lakshmi N; Ghatol, Dipti; Upadhyay, Aman

    2015-01-01

    An 80-year-old woman presented with chronic lumbosacral pain since her laminectomy and instrumentation 10 years ago. Examination was consistent with left sacroiliitis, and the patient underwent an elective left sacroiliac joint injection. Two days following her procedure she fell and landed on her left hip and on the next day, she presented to the emergency room with acutely worsening left gluteal pain. On evaluation in the emergency department, she was found to be suffering from a fever, headache, nausea, vomiting, and dysuria. A computed tomography (CT) scan was performed and this showed extensive foci of gas throughout the posterior aspect of the left iliopsoas muscle, sacrum, and ileum surrounding the left sacroiliac joint. The patient underwent emergent surgical debridement. The microbiology report of blood culture revealed clostridium perfringens, while her pathology showed necrosis and acute inflammation of fibroadipose tissue, skeletal muscle, and fat. The patient's hospital course resulted in multi-organ failure and the family elected for comfort care measures only. Unfortunately, she passed away 36 hours later. Septic arthritis is a potential catastrophic complication following intra-articular steroid therapy. The cause of the septic joint can be multifactorial but is likely caused by one of the following processes: direct inoculation of bacteria by the injection, hematogenous seeding of the percutaneous injection tract, or due to activation of a quiescent infection by the injected steroid. Clostridial spores are very resistant to standard aseptic skin preparations, including chlorhexidine and betadine solutions. The only effective methods to eliminate the spores is to heat them at a temperature greater than 100 degrees Celsius for 10 minutes or with use of a 10% potassium hydroxide (KOH) solution. We hypothesize that clostridium spores were present on the patient's skin from previous stool soiling, and that these were introduced directly into the soft

  14. Reclassification of Clostridium difficile as Clostridioides difficile (Hall and O'Toole 1935) Prévot 1938.

    PubMed

    Lawson, Paul A; Citron, Diane M; Tyrrell, Kerin L; Finegold, Sydney M

    2016-08-01

    The recent proposal by Lawson and Rainey (2015) to restrict the genus Clostridium to Clostridium butyricum and related species has ramifications for the members of the genera that fall outside this clade that should not be considered as Clostridium sensu stricto. One such organism of profound medical importance is Clostridioides difficile that is a major cause of hospital-acquired diarrhea and mortality in individuals. Based on 16S rRNA gene sequence analysis, the closest relative of Clostridium difficile is Clostridium mangenotii with a 94.7% similarity value and both are located within the family Peptostreptococcaceae that is phylogenetically far removed from C. butyricum and other members of Clostridium sensu stricto. Clostridium difficile is Clostridium mangenotii each produce abundant H2 gas when grown in PYG broth and also produce a range of straight and branched chain saturated and unsaturated fatty acids with C16:0 as a major product. The cell wall peptidoglycan contains meso-DAP as the diagnostic diamino acid. Based on phenotypic, chemotaxonomic and phylogenetic analyses, novel genus Clostridioides gen. nov. is proposed for Clostridium difficile as Clostridioides difficile gen. nov. comb. nov. and that Clostridium mangenotii be transferred to this genus as Clostridioides mangenotii comb. nov. The type species of Clostridioides is Clostridioides difficile. PMID:27370902

  15. Type IV pili promote early biofilm formation by Clostridium difficile.

    PubMed

    Maldarelli, Grace A; Piepenbrink, Kurt H; Scott, Alison J; Freiberg, Jeffrey A; Song, Yang; Achermann, Yvonne; Ernst, Robert K; Shirtliff, Mark E; Sundberg, Eric J; Donnenberg, Michael S; von Rosenvinge, Erik C

    2016-08-01

    Increasing morbidity and mortality from Clostridium difficile infection (CDI) present an enormous challenge to healthcare systems. Clostridium difficile express type IV pili (T4P), but their function remains unclear. Many chronic and recurrent bacterial infections result from biofilms, surface-associated bacterial communities embedded in an extracellular matrix. CDI may be biofilm mediated; T4P are important for biofilm formation in a number of organisms. We evaluate the role of T4P in C. difficile biofilm formation using RNA sequencing, mutagenesis and complementation of the gene encoding the major pilin pilA1, and microscopy. RNA sequencing demonstrates that, in comparison to other growth phenotypes, C. difficile growing in a biofilm has a distinct RNA expression profile, with significant differences in T4P gene expression. Microscopy of T4P-expressing and T4P-deficient strains suggests that T4P play an important role in early biofilm formation. A non-piliated pilA1 mutant forms an initial biofilm of significantly reduced mass and thickness in comparison to the wild type. Complementation of the pilA1 mutant strain leads to formation of a biofilm which resembles the wild-type biofilm. These findings suggest that T4P play an important role in early biofilm formation. Novel strategies for confronting biofilm infections are emerging; our data suggest that similar strategies should be investigated in CDI. PMID:27369898

  16. Tequila vinasses acidogenesis in a UASB reactor with Clostridium predominance.

    PubMed

    Marino-Marmolejo, E N; Corbalá-Robles, L; Cortez-Aguilar, R C; Contreras-Ramos, S M; Bolaños-Rosales, R E; Davila-Vazquez, G

    2015-01-01

    Tequila vinasses represent an acidic, highly concentrated pollutant effluent generated during the distillation step of Tequila production. Although acidogenesis of Tequila vinasses has been reported for some reactor configurations, a characterization of the bacteria present during this metabolic process is lacking in the literature. Hydraulic retention times (HRT) between 36 and 6 h and organic loading rates (OLR) from 5 to 30 g COD L(-1) d(-1) were assessed in a UASB reactor fed with Tequila vinasses. Results showed that OLR excerted a stronger effect (p ≤ 0.0001) on parameters such as gas production rate, pH, and acidity than HRT. While it was clear that shorter HRT were related to higher volatile fatty acid production levels. Figures above 2 Lgas Lreactor (-1) d(-1) (where "gas" could be a mixture of methane and hydrogen) were attained only with an OLR as high as 30 g COD L(-1) d(-1). Bacterial identification of a sludge sample at the end of the experiment revealed that acid-tolerant microorganisms that remained in the reactor were exclusively affiliated to the Clostridium genera, being the first report of organisms identification for Tequila vinasses acidogenesis. These findings are relevant to the field of biotechnology since acidogenesis of Tequila vinasses using identified and studied microorganism abilities (i.e. Clostridium strains) presents the opportunity of optimizing processes intended for different metabolites production (butanol, volatile fatty acids, hydrogen, solvents). PMID:26301166

  17. Identification and Characterization of Clostridium sordellii Toxin Gene Regulator

    PubMed Central

    Sirigi Reddy, Apoorva Reddy; Girinathan, Brintha Parasumanna; Zapotocny, Ryan

    2013-01-01

    Toxigenic Clostridium sordellii causes uncommon but highly lethal infections in humans and animals. Recently, an increased incidence of C. sordellii infections has been reported in women undergoing obstetric interventions. Pathogenic strains of C. sordellii produce numerous virulence factors, including sordellilysin, phospholipase, neuraminidase, and two large clostridial glucosylating toxins, TcsL and TcsH. Recent studies have demonstrated that TcsL toxin is an essential virulence factor for the pathogenicity of C. sordellii. In this study, we identified and characterized TcsR as the toxin gene (tcsL) regulator in C. sordellii. High-throughput sequencing of two C. sordellii strains revealed that tcsR lies within a genomic region that encodes TcsL, TcsH, and TcsE, a putative holin. By using ClosTron technology, we inactivated the tcsR gene in strain ATCC 9714. Toxin production and tcsL transcription were decreased in the tcsR mutant strain. However, the complemented tcsR mutant produced large amounts of toxins, similar to the parental strain. Expression of the Clostridium difficile toxin gene regulator tcdR also restored toxin production to the C. sordellii tcsR mutant, showing that these sigma factors are functionally interchangeable. PMID:23873908

  18. Effective Sequestration of Clostridium difficile Protein Toxins by Calcium Aluminosilicate

    PubMed Central

    Pokusaeva, Karina; Carpenter, Robert

    2015-01-01

    Clostridium difficile is a leading cause of antibiotic-associated diarrhea and the etiologic agent responsible for C. difficile infection. Toxin A (TcdA) and toxin B (TcdB) are nearly indispensable virulence factors for Clostridium difficile pathogenesis. Given the toxin-centric mechanism by which C. difficile pathogenesis occurs, the selective sequestration with neutralization of TcdA and TcdB by nonantibiotic agents represents a novel mode of action to prevent or treat C. difficile-associated disease. In this preclinical study, we used quantitative enzyme immunoassays to determine the extent by which a novel drug, calcium aluminosilicate uniform particle size nonswelling M-1 (CAS UPSN M-1), is capable of sequestering TcdA and TcdB in vitro. The following major findings were derived from the present study. First, we show that CAS UPSN M-1 efficiently sequestered both TcdA and TcdB to undetectable levels. Second, we show that CAS UPSN M-1's affinity for TcdA is greater than its affinity for TcdB. Last, we show that CAS UPSN M-1 exhibited limited binding affinity for nontarget proteins. Taken together, these results suggest that ingestion of calcium aluminosilicate might protect gastrointestinal tissues from antibiotic- or chemotherapy-induced C. difficile infection by neutralizing the cytotoxic and proinflammatory effects of luminal TcdA and TcdB. PMID:26149988

  19. Role of collagenase clostridium histolyticum in Peyronie’s disease

    PubMed Central

    Peak, Taylor C; Mitchell, Gregory C; Yafi, Faysal A; Hellstrom, Wayne J

    2015-01-01

    Peyronie’s disease is a localized connective tissue disease characterized by an active, inflammatory phase and a stable, quiescent phase, with the eventual development of collagenous plaques within the tunica albuginea of the penis. Risk factors primarily associated with Peyronie’s disease include Dupuytren’s contracture, penile trauma, and family history. A variety of treatment strategies have been utilized, including oral and topical agents, electromotive drug administration, intralesional injections, extracorporeal shockwave therapy, penile traction, and surgery. However, most of these strategies are ineffective, with surgery being the only definitive treatment. Collagenase clostridium histolyticum is a newly US Food and Drug Administration-approved agent for intralesional injection. It is thought to downregulate many of the disease-related genes, cytokines, and growth factors and degrade collagen fibers. It also suppresses cell attachment, spreading, and proliferation. Collagenase clostridium histolyticum has been clinically proven to be a safe and effective therapeutic option, demonstrating decreases in penile curvature and plaque consistency, as well as increases in patient satisfaction. During clinical evaluation, the Peyronie’s Disease Questionnaire was validated as an effective tool for assessing treatment outcomes. PMID:26491251

  20. CRYSTAL STRUCTURE OF CLOSTRIDIUM BOTULINUM NEUROTOXIN SEROTYPE B.

    SciTech Connect

    SWAMINATHAN,S.; ESWARAMOORTHY,S.

    2001-11-19

    The toxigenic strains of Clostridium botulinum produce seven serologically distinct types of neurotoxins labeled A - G (EC 3.4.24.69), while Clostridium tetani produces tetanus neurotoxin (EC 3.4.24.68). Botulinum and tetanus neurotoxins (BoNTs and TeNT) are produced as single inactive chains of molecular mass of approximately 150 kDa. Most of these neurotoxins are released after being cleaved into two chains, a heavy chain (HI) of 100 kDa and a light chain (L) of 50 kDa held together by an interchain disulfide bond, by tissue proteinases. BoNT/E is released as a single chain but cleaved by host proteinases [1]. Clostvidium botulinum neurotoxins are extremely poisonous proteins with their LD{sub 50} for humans in the range of 0.1 - 1 ng kg{sup -1} [2]. Botulinum neurotoxins are responsible for neuroparalytic syndromes of botulism characterized by serious neurological disorders and flaccid paralysis. BoNTs block the release of acetylcholine at the neuromuscular junction causing flaccid paralysis while TeNT blocks the release of neurotransmitters like glycine and {gamma}-aminobutyric acid (GABA) in the inhibitory interneurons of the spinal cord resulting in spastic paralysis. In spite of different clinical symptoms, their aetiological agents intoxicate neuronal cells in the same way and these toxins have similar structural organization [3].

  1. Evaluation of the Cepheid Xpert Clostridium difficile Epi assay for diagnosis of Clostridium difficile infection and typing of the NAP1 strain at a cancer hospital.

    PubMed

    Babady, N Esther; Stiles, Jeffrey; Ruggiero, Phyllis; Khosa, Perminder; Huang, David; Shuptar, Susan; Kamboj, Mini; Kiehn, Timothy E

    2010-12-01

    Clostridium difficile is the most common cause of health care-associated diarrhea. Accurate and rapid diagnosis is essential to improve patient outcome and prevent disease spread. We compared our two-step diagnostic algorithm, an enzyme immunoassay for glutamate dehydrogenase (GDH) followed by the cytotoxin neutralization test (CYT) with a turnaround time of 24 to 48 h, versus the Cepheid Xpert C. difficile Epi assay, a PCR-based assay with a turnaround time of <1 h. In the first phase of the study, only GDH-positive stool samples were tested by both CYT and Xpert PCR. Discordant results were resolved by toxigenic culture. In the second phase, all stool samples were tested by GDH and Xpert PCR. Only GDH-positive stools were further tested by CYT. Genotypic characterization of 45 Xpert PCR-positive stools was performed by sequencing of the tcdC gene and PCR ribotyping. In phase 1, the agreement between the GDH-CYT and the GDH-Xpert PCR was 72%. The sensitivities and specificities of GDH-CYT and GDH-Xpert PCR were 57% and 97% and 100% and 97%, respectively. In phase 2, the agreement between GDH-CYT and Xpert PCR alone was 95%. As in phase 1, sensitivity of the Xpert PCR was higher than that of the GDH-CYT. The correlation between PCR-ribotyping, sequencing, and Xpert PCR for detection of NAP1 strains was excellent (>90%). The excellent sensitivity and specificity and the rapid turnaround time of the Xpert PCR assay as well as its strain-typing capability make it an attractive option for diagnosis of C. difficile infection. PMID:20943860

  2. Evaluation of the Cepheid Xpert Clostridium difficile Epi Assay for Diagnosis of Clostridium difficile Infection and Typing of the NAP1 Strain at a Cancer Hospital ▿

    PubMed Central

    Babady, N. Esther; Stiles, Jeffrey; Ruggiero, Phyllis; Khosa, Perminder; Huang, David; Shuptar, Susan; Kamboj, Mini; Kiehn, Timothy E.

    2010-01-01

    Clostridium difficile is the most common cause of health care-associated diarrhea. Accurate and rapid diagnosis is essential to improve patient outcome and prevent disease spread. We compared our two-step diagnostic algorithm, an enzyme immunoassay for glutamate dehydrogenase (GDH) followed by the cytotoxin neutralization test (CYT) with a turnaround time of 24 to 48 h, versus the Cepheid Xpert C. difficile Epi assay, a PCR-based assay with a turnaround time of <1 h. In the first phase of the study, only GDH-positive stool samples were tested by both CYT and Xpert PCR. Discordant results were resolved by toxigenic culture. In the second phase, all stool samples were tested by GDH and Xpert PCR. Only GDH-positive stools were further tested by CYT. Genotypic characterization of 45 Xpert PCR-positive stools was performed by sequencing of the tcdC gene and PCR ribotyping. In phase 1, the agreement between the GDH-CYT and the GDH-Xpert PCR was 72%. The sensitivities and specificities of GDH-CYT and GDH-Xpert PCR were 57% and 97% and 100% and 97%, respectively. In phase 2, the agreement between GDH-CYT and Xpert PCR alone was 95%. As in phase 1, sensitivity of the Xpert PCR was higher than that of the GDH-CYT. The correlation between PCR-ribotyping, sequencing, and Xpert PCR for detection of NAP1 strains was excellent (>90%). The excellent sensitivity and specificity and the rapid turnaround time of the Xpert PCR assay as well as its strain-typing capability make it an attractive option for diagnosis of C. difficile infection. PMID:20943860

  3. Identification and quantification of the caproic acid-producing bacterium Clostridium kluyveri in the fermentation of pit mud used for Chinese strong-aroma type liquor production.

    PubMed

    Hu, Xiao-long; Du, Hai; Xu, Yan

    2015-12-01

    Chinese strong-aroma type liquor (CSAL) is a popular distilled alcoholic beverage in China. It is produced by a complex fermentation process that is conducted in pits in the ground. Ethyl caproate is a key flavor compound in CSAL and is thought to originate from caproic acid produced by Clostridia inhabiting the fermentation pit mud. However, the particular species of Clostridium associated with this production are poorly understood and problematic to quantify by culturing. In this study, a total of 28 closest relatives including 15 Clostridia and 8 Bacilli species in pit muds from three CSAL distilleries, were detected by culture-dependent and -independent methods. Among them, Clostridium kluyveri was identified as the main producer of caproic acid. One representative strain C. kluyveri N6 could produce caproic, butyric and octanoic acids and their corresponding ethyl esters, contributing significantly to CSAL flavor. A real time quantitative PCR assay of C. kluyveri in pit muds developed showed that a concentration of 1.79×10(7) 16S rRNA gene copies/g pit mud in LZ-old pit was approximately six times higher than that in HLM and YH pits and sixty times higher than that in LZ-new pit respectively. This method can be used to improve the management of pit mud microbiology and its impact on CSAL quality. PMID:26267890

  4. Carbon isotope effects associated with mixed-acid fermentation of saccharides by Clostridium papyrosolvens

    NASA Astrophysics Data System (ADS)

    Penning, Holger; Conrad, Ralf

    2006-05-01

    In anoxic environments, microbial fermentation is the first metabolic process in the path of organic matter degradation. Since little is known about carbon isotope fractionation during microbial fermentation, we studied mixed-acid fermentation of different saccharides (glucose, cellobiose, and cellulose) in Clostridium papyrosolvens. The bacterium was grown anaerobically in batch under different growth conditions, both in pure culture and in co-culture with Methanobacterium bryantii utilizing H 2/CO 2 or Methanospirillum hungatei utilizing both H 2/CO 2 and formate. Fermentation products were acetate, lactate, ethanol, formate, H 2, and CO 2 (and CH 4 in methanogenic co-culture), with acetate becoming dominant at low H 2 partial pressures. After complete conversion of the saccharides, acetate was 13C-enriched ( αsacc/ac = 0.991-0.997), whereas lactate ( αsacc/lac = 1.001-1.006), ethanol ( αsacc/etoh = 1.007-1.013), and formate ( αsacc/form = 1.007-1.011) were 13C-depleted. The total inorganic carbon produced was only slightly enriched in 13C, but was more enriched, when formate was produced in large amounts, as 12CO 2 was preferentially converted with H 2 to formate. During biomass formation, 12C was slightly preferred ( αsacc/biom ≈ 1.002). The observations in batch culture were confirmed in glucose-limited chemostat culture at growth rates of 0.02-0.15 h -1 at both low and high hydrogen partial pressures. Our experiments showed that the carbon flow at metabolic branch points in the fermentation path governed carbon isotope fractionation to the accumulated products. During production of pyruvate, C isotopes were not fractionated when using cellulose, but were fractionated to different extents depending on growth conditions when using cellobiose or glucose. At the first catabolic branch point (pyruvate), the produced lactate was depleted in 13C, whereas the alternative product acetyl-CoA was 13C enriched. At the second branch point (acetyl-CoA), the ethanol

  5. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile.

    PubMed

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride ("DQAsomes") have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2'-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription-translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We describe

  6. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    PubMed Central

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  7. Calprotectin and lactoferrin faecal levels in patients with Clostridium difficile infection (CDI): a prospective cohort study.

    PubMed

    Swale, Andrew; Miyajima, Fabio; Roberts, Paul; Hall, Amanda; Little, Margaret; Beadsworth, Mike B J; Beeching, Nick J; Kolamunnage-Dona, Ruwanthi; Parry, Chris M; Pirmohamed, Munir

    2014-01-01

    Measurement of both calprotectin and lactoferrin in faeces has successfully been used to discriminate between functional and inflammatory bowel conditions, but evidence is limited for Clostridium difficile infection (CDI). We prospectively recruited a cohort of 164 CDI cases and 52 controls with antibiotic-associated diarrhoea (AAD). Information on disease severity, duration of symptoms, 30-day mortality and 90-day recurrence as markers of complicated CDI were recorded. Specimens were subject to microbiological culture and PCR-ribotyping. Levels of faecal calprotectin (FC) and lactoferrin (FL) were measured by ELISA. Statistical analysis was conducted using percentile categorisation. ROC curve analysis was employed to determine optimal cut-off values. Both markers were highly correlated with each other (r2 = 0.74) and elevated in cases compared to controls (p<0.0001; ROC>0.85), although we observed a large amount of variability across both groups. The optimal case-control cut-off point was 148 mg/kg for FC and 8.1 ng/µl for FL. Median values for FL in CDI cases were significantly greater in patients suffering from severe disease compared to non-severe disease (104.6 vs. 40.1 ng/µl, p = 0.02), but were not significant for FC (969.3 vs. 512.7 mg/kg, p = 0.09). Neither marker was associated with 90-day recurrence, prolonged CDI symptoms, positive culture results and colonisation by ribotype 027. Both FC and FL distinguished between CDI cases and AAD controls. Although FL was associated with disease severity in CDI patients, this showed high inter-individual variability and was an isolated finding. Thus, FC and FL are unlikely to be useful as biomarkers of complicated CDI disease. PMID:25170963

  8. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    DOE PAGESBeta

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel)more » initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.« less

  9. The exometabolome of Clostridium thermocellum reveals overflow metabolism at high cellulose loading

    SciTech Connect

    Holwerda, Evert K.; Thorne, Philip G.; Olson, Daniel G.; Amador-Noguez, Daniel; Engle, Nancy L.; Tschaplinski, Timothy J.; van Dijken, Johannes P.; Lynd, Lee R.

    2014-10-21

    Background: Clostridium thermocellum is a model thermophilic organism for the production of biofuels from lignocellulosic substrates. The majority of publications studying the physiology of this organism use substrate concentrations of ≤10 g/L. However, industrially relevant concentrations of substrate start at 100 g/L carbohydrate, which corresponds to approximately 150 g/L solids. To gain insight into the physiology of fermentation of high substrate concentrations, we studied the growth on, and utilization of high concentrations of crystalline cellulose varying from 50 to 100 g/L by C. thermocellum. Results: Using a defined medium, batch cultures of C. thermocellum achieved 93% conversion of cellulose (Avicel) initially present at 100 g/L. The maximum rate of substrate utilization increased with increasing substrate loading. During fermentation of 100 g/L cellulose, growth ceased when about half of the substrate had been solubilized. However, fermentation continued in an uncoupled mode until substrate utilization was almost complete. In addition to commonly reported fermentation products, amino acids - predominantly L-valine and L-alanine - were secreted at concentrations up to 7.5 g/L. Uncoupled metabolism was also accompanied by products not documented previously for C. thermocellum, including isobutanol, meso- and RR/SS-2,3-butanediol and trace amounts of 3-methyl-1-butanol, 2-methyl-1-butanol and 1-propanol. We hypothesize that C. thermocellum uses overflow metabolism to balance its metabolism around the pyruvate node in glycolysis. In conclusion: C. thermocellum is able to utilize industrially relevant concentrations of cellulose, up to 93 g/L. We report here one of the highest degrees of crystalline cellulose utilization observed thus far for a pure culture of C. thermocellum, the highest maximum substrate utilization rate and the highest amount of isobutanol produced by a wild-type organism.

  10. Characterization of the extracellular cellulase from a mesophilic clostridium (strain C7).

    PubMed Central

    Cavedon, K; Leschine, S B; Canale-Parola, E

    1990-01-01

    An extracellular, 700,000-Mr multiprotein complex that catalyzed the hydrolysis of crystalline cellulose (Avicel) was isolated from cultures of Clostridium sp. strain C7, a mesophile from freshwater sediment. In addition to cellulose (Avicel, ball-milled filter paper), the multiprotein complex hydrolyzed carboxymethylcellulose, cellodextrins, xylan, and xylooligosaccharides. Hydrolysis of cellulose or cellotetraose by the complex yielded cellobiose as the main product. Cellopentaose or cellohexaose was hydrolyzed by the complex to cellotriose or cellotetraose, respectively, in addition to cellobiose. Xylobiose was the main product of xylan hydrolysis, and xylobiose and xylotriose were the major products of xylooligosaccharide hydrolysis. Activity (Avicelase) resulting in hydrolysis of crystalline cellulose required Ca2+ and a reducing agent. The multiprotein complex had temperature optima for Avicelase, carboxymethylcellulase, and xylanase activities at 45, 55, and 55 degrees C, respectively, and pH optima at 5.6 to 5.8, 5.5, and 6.55, respectively. Electron microscopy of the 700,000-Mr enzyme complex revealed particles relatively uniform in size (12 to 15 nm wide) and apparently composed of subunit structures. Elution of strain C7 concentrated culture fluid from Sephacryl S-300 columns yielded an A280 peak in the 130,000-Mr region. Pooled fractions from the 130,000-Mr peak had carboxymethylcellulase activity but lacked Avicelase activity. Except for the inability to hydrolyze cellulose, the 130,000-Mr preparation had a substrate specificity identical to that of the 700,000-Mr protein complex. A comparison by immunoblotting techniques of proteins in the 130,000- and 700,000-Mr preparations, indicated that the two enzyme preparations had cross-reacting antigenic determinants. Images PMID:2376560

  11. Antibiotic Sensitivity of Clostridium perfringens Isolated From Faeces in Tabriz, Iran

    PubMed Central

    Akhi, Mohammad Taghi; Bidar Asl, Saeid; Pirzadeh, Tahereh; Naghili, Behruz; Yeganeh, Fatemeh; Memar, Yousef; Mohammadzadeh, Yalda

    2015-01-01

    Background: Clostridium perfringens, a Gram-positive, anaerobic bacterium that produces at least 16 virulence factors including 12 toxins (α-ν), enterotoxin, hemolysin and neuraminidase, can create variable pathogenic condition, ranging from a food poisoning to life-threatening myonecrosis. Among C. perfringens strains, resistance to the drug choices such as penicillin as well as to alternatives of penicillin like metronidazole and clindamycin has also been observed. Objectives: The aim of this study was to determine the resistance of isolated toxigenic and non-toxigenic C. perfringens strains against common antimicrobial agents. Materials and Methods: In this descriptive study, a total of 136 stool specimens were collected. At first, cooked meat medium enrichment method was performed on samples at 45°C. Thereafter, a loopful of the enriched culture was transferred to blood agar and incubated anaerobically at 37°C for 24-72 hours. Colonies with double zone of hemolysis were identified by different biochemical tests such as phospholipase C (lecithinase) test, indole and urease production. The Minimum Inhibitory Concentration (MIC) for common antibiotics was determined by Etests (Epsilometer) and duplex Polymerase Chain Reaction (PCR) reaction was performed with specific primers for amplification of cpe (426 bp) and plc (283 bp) Genes. Results: Of 136 stool samples including diarrhea [48] and non-diarrhea [88] ones, 83 (61.02%) C. perfringens were cultured. Of these 83, 79 C. perfringens isolates showed the alpha-toxin (phospholipase C) production gene by PCR. Respectively, 3 (9.09%) and 2 (4.34%) cpe genes were present in diarrhea and non-diarrhea samples. Of 79 isolates of C. perfringens, 34 (43.03%) cases showed no resistance, 18 (22.78%) had one resistance and 27 (34.17%) isolates had multiple resistance to imipenem, metronidazole, ceftriaxone, clindamycin, chloramphenicol, and penicillin. Conclusions: Periodic evaluation of antimicrobial susceptibility for C

  12. Characterization of a Cell Surface Protein of Clostridium difficile with Adhesive Properties

    PubMed Central

    Waligora, Anne-Judith; Hennequin, Claire; Mullany, Peter; Bourlioux, Pierre; Collignon, Anne; Karjalainen, Tuomo

    2001-01-01

    Our laboratory has previously shown that Clostridium difficile adherence to cultured cells is enhanced after heat shock at 60°C and that it is mediated by a proteinaceous surface component. The present study was undertaken to identify the surface molecules of this bacterium that could play a role in its adherence to the intestine. The cwp66 gene, encoding a cell surface-associated protein of C. difficile 79-685, was isolated by immunoscreening of a C. difficile gene library with polyclonal antibodies against C. difficile heated at 60°C. The Cwp66 protein (66 kDa) contains two domains, each carrying three imperfect repeats and one presenting homologies to the autolysin CwlB of Bacillus subtilis. A survey of 36 strains of C. difficile representing 11 serogroups showed that the 3′ portion of the cwp66 gene is variable; this was confirmed by sequencing of cwp66 from another strain, C-253. Two recombinant protein fragments corresponding to the two domains of Cwp66 were expressed in fusion with glutathione S-transferase in Escherichia coli and purified by affinity chromatography using gluthatione-Sepharose 4B. Antibodies raised against the two domains recognized Cwp66 in bacterial surface extracts. By immunoelectron microscopy, the C-terminal domain was found to be cell surface exposed. When used as inhibitors in cell binding studies, the antibodies and protein fragments partially inhibited adherence of C. difficile to cultured cells, confirming that Cwp66 is an adhesin, the first to be identified in clostridia. PMID:11254569

  13. Multiple product inhibition and growth modeling of Clostridium butyricum and Klebsiella pneumoniae in glycerol fermentation

    SciTech Connect

    Zeng, A.P.; Ross, A.; Biebl, H.; Tag, C.; Guenzel, B.; Deckwer, W.D. . Biochemical Engineering Division)

    1994-10-01

    The inhibition potentials of products and substrate on the growth of Clostridium butyricum and Klebsiella pneumoniae in the glycerol fermentation are examined from experimental data and with a mathematical model. Whereas the inhibition potential of externally added and self-produced 1,3-propanediol is essentially the same, butyric acid produced by the culture is more toxic than that externally added. The same seems to apply for acetic acid. The inhibitory effect of butyric acid is due to the total concentration instead of its undissociated form. For acetic acid, it cannot be distinguished between the total concentration and the undissociated form. The inhibition effects of products and substrate in the glycerol fermentation are irrespective of the strains, and, therefore, the same growth model can be used. The maximum product concentrations tolerated are 0.35 g/L for undissociated acetic acid, 10.1 g/L for total butyric acid, 16.6 g/L for ethanol, 71.4 g/L for 1,3-propanediol, and 187.6 g/L for glycerol, which are applicable to C. butyricum and K. pneumoniae growth under a variety of conditions. For 55 steady-states, which were obtained from different types of continuous cultures over a pH range of 5.3--8.5 and under both substrate limitation and substrate excess, the proposed growth model fits the experimental data with an average deviation of 17.0%. The deviation of model description from experimental values reduces of 11.4% if only the steady-states with excessive substrate are considered.

  14. Simultaneous and enhanced production of thermostable amylases and ethanol from starch by cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

    SciTech Connect

    Hyun, H.H.; Zeikus, J.G.

    1985-05-01

    Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of ..beta..-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60/sup 0/C and produced 9 U of ..beta..-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and > 120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed.

  15. Functional characterisation of germinant receptors in Clostridium botulinum and Clostridium sporogenes presents novel insights into spore germination systems.

    PubMed

    Brunt, Jason; Plowman, June; Gaskin, Duncan J H; Itchner, Manoa; Carter, Andrew T; Peck, Michael W

    2014-09-01

    Clostridium botulinum is a dangerous pathogen that forms the highly potent botulinum toxin, which when ingested causes a deadly neuroparalytic disease. The closely related Clostridium sporogenes is occasionally pathogenic, frequently associated with food spoilage and regarded as the non-toxigenic equivalent of Group I C. botulinum. Both species form highly resistant spores that are ubiquitous in the environment and which, under favourable growth conditions germinate to produce vegetative cells. To improve the control of botulinum neurotoxin-forming clostridia, it is imperative to comprehend the mechanisms by which spores germinate. Germination is initiated following the recognition of small molecules (germinants) by a specific germinant receptor (GR) located in the spore inner membrane. The present study precisely defines clostridial GRs, germinants and co-germinants. Group I C. botulinum ATCC3502 contains two tricistronic and one pentacistronic GR operons, while C. sporogenes ATCC15579 has three tricistronic and one tetracistronic GR operons. Insertional knockout mutants, allied with characterisation of recombinant GRs shows for the first time that amino acid stimulated germination in C. botulinum requires two tri-cistronic encoded GRs which act in synergy and cannot function individually. Spore germination in C. sporogenes requires one tri-cistronic GR. Two other GRs form part of a complex involved in controlling the rate of amino-acid stimulated germination. The suitability of using C. sporogenes as a substitute for C. botulinum in germination studies and food challenge tests is discussed. PMID:25210747

  16. Structure and function of a menaquinone involved in electron transport in membranes of Clostridium thermoautotrophicum and Clostridium thermoaceticum.

    PubMed Central

    Das, A; Hugenholtz, J; Van Halbeek, H; Ljungdahl, L G

    1989-01-01

    Clostridium thermoaceticum and Clostridium thermoautotrophicum contain the same menaquinone. Its structure, determined by thin-layer chromatography, UV absorption spectroscopy, mass spectrometry, and nuclear magnetic resonance spectroscopy, was found to be MK-7 (2-methyl-3-heptaprenyl-1,4-naphthoquinone). The menaquinone is located in the cytoplasmic membranes and is involved in redox reactions of two b-type cytochromes present in the clostridia. These reactions were studied with right-side-out membranes prepared from C. thermoautotrophicum by using CO as an electron donor. In intact membranes, both cytochromes were reduced, whereas after inactivation of the menaquinone by exposure of the membranes to UV irradiation, reduction of the low-potential cytochrome (Eo', -200 mV) but not of the high-potential cytochrome (Eo', -48 mV) occurred. The reduction of the high-potential cytochrome in UV-irradiated membranes was restored following the addition of oxidized menaquinone and with an excess of CO. The addition of oxidized menaquinone to reduced membranes resulted initially in a preferential oxidation of the low-potential cytochrome. The results obtained indicate that the menaquinone acts between the two b-type cytochromes in an electron transport chain. PMID:2808299

  17. Simultaneous and Enhanced Production of Thermostable Amylases and Ethanol from Starch by Cocultures of Clostridium thermosulfurogenes and Clostridium thermohydrosulfuricum

    PubMed Central

    Hyun, H. H.; Zeikus, J. G.

    1985-01-01

    Clostridium thermohydrosulfuricum and Clostridium thermosulfurogenes produced ethanol and amylases with different components as primary metabolites of starch fermentation. Starch fermentation parameters were compared in mono- and cocultures of these two thermoanaerobes to show that the fermentation was dramatically improved as a consequence of coordinate action of amylolytic enzymes and synergistic metabolic interactions between the two species. Under given monoculture fermentation conditions, neither species completely degraded starch during the time course of the study, whereas in coculture, starch was completely degraded. In monoculture starch fermentation, C. thermohydrosulfuricum produced lower levels of pullulanase and glucoamylase, whereas C. thermosulfurogenes produced lower levels of β-amylase and glucoamylase. In coculture fermentation, improvement of starch metabolism by each species was noted in terms of increased amounts and rates of increased starch consumption, amylase production, and ethanol formation. The single-step coculture fermentation completely degraded 2.5% starch in 30 h at 60°C and produced 9 U of β-amylase per ml, 1.3 U of pullulanase per ml, 0.3 U of glucoamylase per ml, and >120 mM ethanol with a yield of 1.7 mol/mol of glucose in starch. The potential industrial applications of the coculture fermentation and the physiological basis for the interspecies metabolic interactions are discussed. PMID:16346791

  18. Comparative analysis of the ability of Clostridium clariflavum strains and Clostridium thermocellumto utilize hemicellulose and unpretreated plant material

    SciTech Connect

    Izquierdo, Javier A.; Pattathil, Sivakumar; Guseva, Anna; Hahn, Michael G.; Lynd, Lee R.

    2014-11-18

    Among themophilic consolidated bioprocessing (CBP) candidate organisms, environmental isolates of Clostridium clariflavum have demonstrated the ability to grow on xylan, and the genome of C. clariflavum DSM 19732 has revealed a number of mechanisms that foster solubilization of hemicellulose that are distinctive relative to the model cellulolytic thermophile Clostridium thermocellum. Growth experiments on xylan, xylooligosaccharides, and xylose reveal that C. clariflavum strains are able to completely break down xylan to xylose and that the environmental strain C. clariflavum sp. 4-2a is able to grow on monomeric xylose. C. clariflavum strains were able to utilize a larger proportion of unpretreated switchgrass, and solubilize a higher proportion of glucan, xylan, and arabinan, with strain 4-2a reaching the highest extent of solubilization of these components (64.7 to 69.4%) compared to C. thermocellum (29.5 to 42.5%). In addition, glycome immunoanalyses of residual plant biomass reveal differences in the extent of degradation of easily accessible xylans, with C. clariflavum strains having increased solubilization of this fraction of xylans relative to C. thermocellum. In conclusion, C. clariflavum strains exhibit higher activity than C. thermocellum in the breakdown of hemicellulose and are capable of degrading xylan to xylooligomers and xylose. This capability seems to also play a role in the higher levels of utilization of unpretreated plant material.

  19. Comparative analysis of the ability of Clostridium clariflavum strains and Clostridium thermocellumto utilize hemicellulose and unpretreated plant material

    DOE PAGESBeta

    Izquierdo, Javier A.; Pattathil, Sivakumar; Guseva, Anna; Hahn, Michael G.; Lynd, Lee R.

    2014-11-18

    Among themophilic consolidated bioprocessing (CBP) candidate organisms, environmental isolates of Clostridium clariflavum have demonstrated the ability to grow on xylan, and the genome of C. clariflavum DSM 19732 has revealed a number of mechanisms that foster solubilization of hemicellulose that are distinctive relative to the model cellulolytic thermophile Clostridium thermocellum. Growth experiments on xylan, xylooligosaccharides, and xylose reveal that C. clariflavum strains are able to completely break down xylan to xylose and that the environmental strain C. clariflavum sp. 4-2a is able to grow on monomeric xylose. C. clariflavum strains were able to utilize a larger proportion of unpretreated switchgrass,more » and solubilize a higher proportion of glucan, xylan, and arabinan, with strain 4-2a reaching the highest extent of solubilization of these components (64.7 to 69.4%) compared to C. thermocellum (29.5 to 42.5%). In addition, glycome immunoanalyses of residual plant biomass reveal differences in the extent of degradation of easily accessible xylans, with C. clariflavum strains having increased solubilization of this fraction of xylans relative to C. thermocellum. In conclusion, C. clariflavum strains exhibit higher activity than C. thermocellum in the breakdown of hemicellulose and are capable of degrading xylan to xylooligomers and xylose. This capability seems to also play a role in the higher levels of utilization of unpretreated plant material.« less

  20. Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

    PubMed

    Janganan, Thamarai K; Mullin, Nic; Tzokov, Svetomir B; Stringer, Sandra; Fagan, Robert P; Hobbs, Jamie K; Moir, Anne; Bullough, Per A

    2016-10-01

    Clostridium sporogenes is a non-pathogenic close relative and surrogate for Group I (proteolytic) neurotoxin-producing Clostridium botulinum strains. The exosporium, the sac-like outermost layer of spores of these species, is likely to contribute to adhesion, dissemination, and virulence. A paracrystalline array, hairy nap, and several appendages were detected in the exosporium of C. sporogenes strain NCIMB 701792 by EM and AFM. The protein composition of purified exosporium was explored by LC-MS/MS of tryptic peptides from major individual SDS-PAGE-separated protein bands, and from bulk exosporium. Two high molecular weight protein bands both contained the same protein with a collagen-like repeat domain, the probable constituent of the hairy nap, as well as cysteine-rich proteins CsxA and CsxB. A third cysteine-rich protein (CsxC) was also identified. These three proteins are also encoded in C. botulinum Prevot 594, and homologues (75-100% amino acid identity) are encoded in many other Group I strains. This work provides the first insight into the likely composition and organization of the exosporium of Group I C. botulinum spores. PMID:27375261