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Sample records for clostridium perfringens strain

  1. Genetic characterization of type A enterotoxigenic Clostridium perfringens strains.

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    Agi Deguchi

    Full Text Available Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE. The gene (cpe encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i assemble into one definitive cluster ii lack pfoA and iii lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s and/or the conjugative transfer of cpe-plasmid(s into unrelated C. perfringens strains.

  2. In-vitro growth characteristics of commercial probiotic strains and their potential for inhibition of Clostridium difficile and Clostridium perfringens

    DEFF Research Database (Denmark)

    Schoster, A.; Kokotovic, Branko; Permin, A.

    2012-01-01

    Probiotics have gained importance in human and veterinary medicine to prevent enteric disease. Little information is available on commercial probiotic strains regarding their growth characteristics and inhibition of equine enteric pathogens such as Clostridium difficile and Clostridium perfringens...

  3. In vitro inhibition of Clostridium difficile and Clostridium perfringens by commercial probiotic strains

    DEFF Research Database (Denmark)

    Schoster, A.; Kokotovic, Branko; Permin, Anders

    2013-01-01

    Probiotics have gained importance in human and veterinary medicine to prevent and control clostridial enteric disease. Limited information is available on the ability of different probiotic bacteria used in food products to inhibit Clostridium difficile and Clostridium perfringens. The objective......) on the reference strains of C. difficile and C. perfringens were assessed by an agar well diffusion assay and by a broth culture inhibition assay using cell-free supernatant harvested at different growth phases, with and without pH neutralization. To study growth characteristics, probiotic strains were cultivated...... in different acid and bile environments, and growth in the modified media was compared to growth in standard medium.In the agar well diffusion assay, supernatant obtained from two probiotic strains inhibited the growth of both reference and clinical strains of C. perfringens. This effect as seen when...

  4. NetB, a pore-forming toxin from necrotic enteritis strains of Clostridium perfringens.

    Science.gov (United States)

    Keyburn, Anthony L; Bannam, Trudi L; Moore, Robert J; Rood, Julian I

    2010-07-01

    The Clostridium perfringens necrotic enteritis B-like toxin (NetB) is a recently discovered member of the β-barrel pore-forming toxin family and is produced by a subset of avian C. perfringens type A strains. NetB is cytotoxic for avian cells and is associated with avian necrotic enteritis. This review examines the current state of knowledge of NetB: its role in pathogenesis, its distribution and expression in C. perfringens and its vaccine potential.

  5. CLOSTRIDIUM PERFRINGENS

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    The incidence of C. perfringens food-poisoning is quite common and costly. Although somewhat fastidious in growth characteristics using synthetic laboratory media, the microorganism is very prolific when found in food products. Despite the pathogen’s ubiquity in the natural environment, foodborne i...

  6. NetB, a Pore-Forming Toxin from Necrotic Enteritis Strains of Clostridium perfringens

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    Anthony L. Keyburn

    2010-07-01

    Full Text Available The Clostridium perfringens necrotic enteritis B-like toxin (NetB is a recently discovered member of the β-barrel pore-forming toxin family and is produced by a subset of avian C. perfringens type A strains. NetB is cytotoxic for avian cells and is associated with avian necrotic enteritis. This review examines the current state of knowledge of NetB: its role in pathogenesis, its distribution and expression in C. perfringens and its vaccine potential.

  7. NetB, a Pore-Forming Toxin from Necrotic Enteritis Strains of Clostridium perfringens

    OpenAIRE

    Keyburn, Anthony L.; Bannam, Trudi L.; Robert J Moore; Rood, Julian I.

    2010-01-01

    The Clostridium perfringens necrotic enteritis B-like toxin (NetB) is a recently discovered member of the β-barrel pore-forming toxin family and is produced by a subset of avian C. perfringens type A strains. NetB is cytotoxic for avian cells and is associated with avian necrotic enteritis. This review examines the current state of knowledge of NetB: its role in pathogenesis, its distribution and expression in C. perfringens and its vaccine potential.

  8. Characterization of Clostridium perfringens strains isolated from clinically healthy and necrotic enteritis-afflicted broiler chickens

    Science.gov (United States)

    Clostridium perfringens (CP) Type A strains are the main etiological factor for necrotic enteritis (NE), one of the important enteric diseases in poultry, which has gained worldwide attention during the last decade and is responsible for the annual loss of 6 billion dollars in US poultry industry. ...

  9. Toxin genotyping of Clostridium perfringens strains using a polymerase chain reaction protocol

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    Elisabetta Di Giannatale

    2010-03-01

    Full Text Available A polymerase chain reaction protocol consisting of a multiplex to identify the cpa, cpb1, cpetx, cpi genes and a duplex to identify the cpe and cpb2 genes encoding for a, b1, e, i, enterotoxin and b2 toxins, respectively, was applied to DNA extracted from two collections of Clostridium perfringens strains. The first collection involved 19 isolates from rabbits. The second collection of 41 isolates came from routine necropsies. The cpa gene alone, or in association with the cpb2 gene, was detected in all DNA samples examined. The cpa gene, together with cpb2 gene, were detected in seven of the rabbit C. perfringens strains (36.8% and in nine isolates from necropsies (21.9%. The cpa gene was found in 63.2% of rabbit strains and 76.9% of strains from other animal species. In rabbits, the pathological lesions associated with C. perfringens detection were predominantly forms of non-inflammatory enteropathies. In other species, C. perfringens was mainly associated with congestive-haemorrhagic enteropathy, but also with fatal traumatic lesions, degenerative diseases and organs with post-mortem autolysis. No clear correlation was observed between detection of b2 toxin gene and species-specific pathological features.

  10. Tips to Prevent Illness from Clostridium Perfringens

    Science.gov (United States)

    ... C. perfringens Recommend on Facebook Tweet Share Compartir Clostridium perfringens ( C. perfringens ) is one of the most ... gov More Information More Information Learn more about Clostridium perfringens Find out safe minimum cooking temperatures for ...

  11. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens Sensibilidade antimicrobiana de estirpes de Clostridium perfringens isoladas de aves de corte

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    R. O. S. Silva

    2009-06-01

    Full Text Available 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.Clostridium perfringens é um habitante normal da microbiota intestinal de frangos, sendo um agente potencialmente patogênico, causador de enterite necrótica e colangio-hepatite. A concentração inibitória mínima (CIM de sete drogas utilizadas na terapêutica, como agentes promotores de crescimento ou na prevenção de coccidiose foi determinada pelo método de diluição em ágar para 55 estirpes de C. perfringens isoladas do intestino de frangos de corte. Todas as estirpes revelaram alta sensibilidade à penicilina, avilamicina, narasin e monensina, apenas 7,3% demonstraram CIM intermediário para lincomicina e 89.1% foram consideradas sensíveis. Para tetraciclina e bacitracina, 41,8% e 47.3% das amostras, respectivamente, foram consideradas resistentes.

  12. Bacteriophages of Clostridium perfringens

    Science.gov (United States)

    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...

  13. Functional analysis of an feoB mutant in Clostridium perfringens strain 13.

    Science.gov (United States)

    Awad, Milena M; Cheung, Jackie K; Tan, Joanne E; McEwan, Alastair G; Lyras, Dena; Rood, Julian I

    2016-10-01

    Bacterial pathogens have adopted numerous mechanisms for acquiring iron from host proteins during an infection, including the direct acquisition of ferric iron from heme-associated proteins or from iron-scavenging siderophores. Ferric iron then is transported into the cytosol, where it can be utilized by the bacterial pathogen. Under anaerobic conditions bacteria can also transport ferrous iron using the transmembrane complex FeoAB, but little is known about iron transport systems in anaerobic bacteria such as the pathogenic clostridia. In this study we sought to characterize the iron acquisition process in Clostridium perfringens. Bioinformatic analysis of the Clostridium perfringens strain 13 genome sequence revealed that it has seven potential iron acquisition systems: three siderophore-mediated systems, one ferric citrate uptake system, two heme-associated acquisition systems and one ferrous iron uptake system (FeoAB). The relative level of expression of these systems was determined using quantitative real-time RT-PCR assays that were specific for one gene from each system. Each of these genes was expressed, with the feoAB genes generating the most abundant iron-uptake related transcripts. To further examine the role of this system in the growth of C. perfringens, insertional inactivation was used to isolate a chromosomal feoB mutant. Growth of this mutant in the presence and absence of iron revealed that it had altered growth properties and a markedly reduced total iron and manganese content compared to the wild type; effects that were reversed upon complementation with the wild-type feoB gene. These studies suggest that under anaerobic conditions FeoB is the major protein required for the uptake of iron into the cell and that it may play an important role in the pathogenesis of C. perfringens infections.

  14. Collaborative study with reference materials containing Clostridium perfringens (strain D10) for water microbiology

    NARCIS (Netherlands)

    Manavakis M; van Dommelen JA; Mooijman KA; Havelaar AH; LWL

    1995-01-01

    In december 1993 werd een internationaal ringonderzoek voor watermicrobiologische laboratoria georganiseerd om een nieuw ontwikkeld referentiemateriaal met Clostridium perfringens (stam D10) uit te testen. Aan dit ringonderzoek namen 24 Europese laboratoria deel. Elk laboratorium testte 8 capsules

  15. Toxin Plasmids of Clostridium perfringens

    Science.gov (United States)

    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

  16. Sequence variation in the alpha-toxin encoding plc gene of Clostridium perfringens strains isolated from diseased and healthy chickens

    DEFF Research Database (Denmark)

    Abildgaard, L; Engberg, RM; Pedersen, Karl

    2009-01-01

    The aim of the present study was to analyse the genetic diversity of the alpha-toxin encoding plc gene and the variation in a-toxin production of Clostridium perfringens type A strains isolated from presumably healthy chickens and chickens suffering from either necrotic enteritis (NE) or cholangio......-hepatitis. The a-toxin encoding plc genes from 60 different pulsed-field gel electrophoresis (PFGE) types (strains) of C perfringens were sequenced and translated in silico to amino acid sequences and the a-toxin production was investigated in batch cultures of 45 of the strains using an enzyme...

  17. Biochemistry and physiology of the β class carbonic anhydrase (Cpb) from Clostridium perfringens strain 13.

    Science.gov (United States)

    Kumar, R Siva Sai; Hendrick, William; Correll, Jared B; Patterson, Andrew D; Melville, Stephen B; Ferry, James G

    2013-05-01

    The carbonic anhydrase (Cpb) from Clostridium perfringens strain 13, the only carbonic anhydrase encoded in the genome, was characterized both biochemically and physiologically. Heterologously produced and purified Cpb was shown to belong to the type I subclass of the β class, the first β class enzyme investigated from a strictly anaerobic species of the domain Bacteria. Kinetic analyses revealed a two-step, ping-pong, zinc-hydroxide mechanism of catalysis with Km and kcat/Km values of 3.1 mM CO₂ and 4.8 × 10⁶ s⁻¹ M⁻¹, respectively. Analyses of a cpb deletion mutant of C. perfringens strain HN13 showed that Cpb is strictly required for growth when cultured in semidefined medium and an atmosphere without CO₂. The growth of the mutant was the same as that of the parent wild-type strain when cultured in nutrient-rich media with or without CO₂ in the atmosphere, although elimination of glucose resulted in decreased production of acetate, propionate, and butyrate. The results suggest a role for Cpb in anaplerotic CO₂ fixation reactions by supplying bicarbonate to carboxylases. Potential roles in competitive fitness are discussed.

  18. Identification of novel pathogenicity loci in Clostridium perfringens strains that cause avian necrotic enteritis.

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    Dion Lepp

    Full Text Available Type A Clostridium perfringens causes poultry necrotic enteritis (NE, an enteric disease of considerable economic importance, yet can also exist as a member of the normal intestinal microbiota. A recently discovered pore-forming toxin, NetB, is associated with pathogenesis in most, but not all, NE isolates. This finding suggested that NE-causing strains may possess other virulence gene(s not present in commensal type A isolates. We used high-throughput sequencing (HTS technologies to generate draft genome sequences of seven unrelated C. perfringens poultry NE isolates and one isolate from a healthy bird, and identified additional novel NE-associated genes by comparison with nine publicly available reference genomes. Thirty-one open reading frames (ORFs were unique to all NE strains and formed the basis for three highly conserved NE-associated loci that we designated NELoc-1 (42 kb, NELoc-2 (11.2 kb and NELoc-3 (5.6 kb. The largest locus, NELoc-1, consisted of netB and 36 additional genes, including those predicted to encode two leukocidins, an internalin-like protein and a ricin-domain protein. Pulsed-field gel electrophoresis (PFGE and Southern blotting revealed that the NE strains each carried 2 to 5 large plasmids, and that NELoc-1 and -3 were localized on distinct plasmids of sizes approximately 85 and approximately 70 kb, respectively. Sequencing of the regions flanking these loci revealed similarity to previously characterized conjugative plasmids of C. perfringens. These results provide significant insight into the pathogenetic basis of poultry NE and are the first to demonstrate that netB resides in a large, plasmid-encoded locus. Our findings strongly suggest that poultry NE is caused by several novel virulence factors, whose genes are clustered on discrete pathogenicity loci, some of which are plasmid-borne.

  19. Characterization of Clostridium perfringens Strains Isolated from Healthy and Necrotic Enteritis-Afflicted Broiler Chickens.

    Science.gov (United States)

    Li, Charles; Lillehoj, Hyun S; Gadde, Ujvala Deepthi; Ritter, Don; Oh, SungTaek

    2017-06-01

    Necrotic enteritis (NE) is an important enteric disease in poultry, and Clostridium perfringens (CP) type A strains are the primary etiology. NE is responsible for annual losses of US $6 billion to the poultry industry in the United States. An increase in the incidence of NE has been also associated with withdrawal of antibiotic growth promoters from poultry feed. In this study, CP strains isolated from healthy and NE-afflicted birds were characterized microbiologically and molecularly, and their virulence was experimentally tested in chickens. All strains were hemolytic, lecithinase positive, and identified as CP by biochemical tests. Three distinct colony morphologies were seen in brain-heart infusion media with 0.3% agarose, FeSO4, and ZnCl2. The CP strains responded differently to iron chelation with 2,2'-bidypinol. PCR toxinotyping showed that all tested strains were alpha toxin-positive, seven (N11, N10, CP1, CP5, CP13, JGS, and Del1) were beta2-toxin-positive, and only one (Del1) was necrotic enteritis toxin B-like-positive. In vivo studies indicated that most isolates, including strain N11 isolated from the normal chicken gut, were sufficiently virulent to produce NE disease in the Eimeria/CP dual infection model. The Del1 and N11 strains merit further investigation to identify their virulence factors and immune-protective antigens.

  20. Experimental infections with rifampicin-resistant Clostridium perfringens strains in broiler chickens using isolator facilities

    DEFF Research Database (Denmark)

    Pedersen, Karl; Bjerrum, Lotte; Nauerby, Birgitte

    2003-01-01

    old. At 22 days none of the strains, given in doses of approximately 10(10) colony-forming units, caused mortality or clinical necrotic enteritis. None was able to colonize the intestine permanently and all were eliminated within 9 days. One strain given to groups of 9-day-old birds was recovered only...... of establishing a reproducible model for infection with C. perfringens in broiler chickens....

  1. Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates.

    Science.gov (United States)

    Baums, Christoph G; Schotte, Ulrich; Amtsberg, Gunter; Goethe, Ralph

    2004-05-20

    In this study we provide a protocol for genotyping Clostridium perfringens with a new multiplex PCR. This PCR enables reliable and specific detection of the toxin genes cpa, cpb, etx, iap, cpe and cpb2 from heat lysed bacterial suspensions. The efficiency of the protocol was demonstrated by typing C. perfringens reference strains and isolates from veterinary bacteriological routine diagnostic specimens.

  2. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Perfringens Type C... REQUIREMENTS Antibody Products § 113.454 Clostridium Perfringens Type C Antitoxin. Clostridium Perfringens Type... Clostridium perfringens Type C. Each serial shall be tested as provided in this section. Any serial found...

  3. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Science.gov (United States)

    2010-01-01

    ... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Clostridium Perfringens Type D... REQUIREMENTS Antibody Products § 113.455 Clostridium Perfringens Type D Antitoxin. Clostridium Perfringens Type... Clostridium perfringens Type D. Each serial shall be tested as provided in this section. Any serial found...

  4. Toxinas de Clostridium perfringens Toxins of Clostridium perfringens

    Directory of Open Access Journals (Sweden)

    W. E. Morris

    2009-12-01

    Full Text Available Clostridium perfringens es un bacilo grampositivo anaerobio con capacidad de formar esporas. Es uno de los patógenos bacterianos con mayor distribución en el medio ambiente, ya que puede ser aislado de muestras de suelo y de agua y además forma parte de la microbiota intestinal de animales y humanos. Sin embargo, en ciertas ocasiones puede actuar como patógeno oportunista y causar enfermedades como la gangrena gaseosa, la enterotoxemia del ovino y del caprino y la disentería del cordero, entre otras. En humanos, está asociado a enfermedades como la intoxicación por alimentos, la enterocolitis necrotizante en niños y la enteritis necrótica o pigbel de las tribus de Papúa-Nueva Guinea. El renovado interés que existe actualmente en el estudio de C. perfringens como patógeno veterinario y humano, junto con el avance de la biología molecular, han hecho posible que la ciencia tenga hoy un conocimiento más profundo sobre la biología y la patogenia de esta bacteria. En esta revisión bibliográfica se discuten y actualizan los principales aspectos de la patogenia intestinal de C. perfringens teniendo en cuenta las toxinas con mayor importancia médica descritas hasta el presente.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

  5. Sialidases affect the host cell adherence and epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718.

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    Jihong Li

    2011-12-01

    Full Text Available Clostridium perfringens type B or D isolates, which cause enterotoxemias or enteritis in livestock, produce epsilon toxin (ETX. ETX is exceptionally potent, earning it a listing as a CDC class B select toxin. Most C. perfringens strains also express up to three different sialidases, although the possible contributions of those enzymes to type B or D pathogenesis remain unclear. Type D isolate CN3718 was found to carry two genes (nanI and nanJ encoding secreted sialidases and one gene (nanH encoding a cytoplasmic sialidase. Construction in CN3718 of single nanI, nanJ and nanH null mutants, as well as a nanI/nanJ double null mutant and a triple sialidase null mutant, identified NanI as the major secreted sialidase of this strain. Pretreating MDCK cells with NanI sialidase, or with culture supernatants of BMC206 (an isogenic CN3718 etx null mutant that still produces sialidases enhanced the subsequent binding and cytotoxic effects of purified ETX. Complementation of BMC207 (an etx/nanH/nanI/nanJ null mutant showed this effect is mainly attributable to NanI production. Contact between BMC206 and certain mammalian cells (e.g., enterocyte-like Caco-2 cells resulted in more rapid sialidase production and this effect involved increased transcription of BMC206 nanI gene. BMC206 was shown to adhere to some (e.g. Caco-2 cells, but not all mammalian cells, and this effect was dependent upon sialidase, particularly NanI, expression. Finally, the sialidase activity of NanI (but not NanJ or NanH could be enhanced by trypsin. Collectively these in vitro findings suggest that, during type D disease originating in the intestines, trypsin may activate NanI, which (in turn could contribute to intestinal colonization by C. perfringens type D isolates and also increase ETX action.

  6. Genotyping of Clostridium perfringens associated with sudden death in cattle

    OpenAIRE

    2009-01-01

    Toxigenic types of Clostridium perfringens are significant causative agents of enteric disease in domestic animals, although type E is presumably rare, appearing as an uncommon cause of enterotoxemia of lambs, calves and rabbits. We report herein the typing of 23 C. perfringens strains, by the polymerase chain reaction (PCR) technique, isolated from small intestine samples of bovines that have died suddenly, after manifesting or not enteric or neurological disorders. Two strains (8.7%) were i...

  7. Clostridium perfringens isolate typing by multiplex PCR

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    MR Ahsani

    2010-01-01

    Full Text Available Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

  8. Alternative medium for Clostridium perfringens sporulation.

    OpenAIRE

    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.

  9. Dynamics of plc gene transcription and α-toxin production during growth of Clostridium perfringens strains with contrasting α-toxin production

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Schramm, Andreas; Rudi, Knut

    2009-01-01

    The aim of the present study was to investigate transcription dynamics of the α-toxin-encoding plc gene relative to two housekeeping genes (gyrA and rplL) in batch cultures of three Clostridium perfringens strains with low, intermediate, and high levels of α-toxin production, respectively. The plc...... transcript level was always low in the low α-toxin producing strain. For the two other strains, plc transcription showed an inducible pattern and reached a maximum level in the late exponential growth phase. The transcription levels were however inversely correlated to α-toxin production for the two strains....... We propose that this discrepancy is due to differences in plc translation rates between the strains and that strain-specific translational rates therefore must be determined before α-toxin production can be extrapolated from transcript levels in C. perfringens....

  10. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    Science.gov (United States)

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

    2015-05-14

    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.

  11. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    Directory of Open Access Journals (Sweden)

    Stefanie Verherstraeten

    2015-05-01

    Full Text Available 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.

  12. Presence and molecular characterization of Clostridium difficile and Clostridium perfringens in intestinal compartments of healthy horses

    DEFF Research Database (Denmark)

    Schoster, Angelika; Arroyo, Luis Guillermo; Staempfli, Henry Rolf

    2012-01-01

    BACKGROUND: Clostridium difficile and Clostridium perfringens are commonly associated with colitis in equids, but healthy carriers exist. Scarce information is available on the prevalence of Clostridium spp. in gastrointestinal compartments other than faeces in healthy horses, and it is unknown...... colon and rectum. When multiple compartments were positive in a single horse, two different C. difficile ribotypes were always present. Clostridium perfringens Type A (CPE, beta2 toxin gene negative) was recovered from the left ventral colon of one horse (0.74%, 1/135 samples). Agreement between faeces...... and overall C. difficile carrier status was good. CONCLUSIONS: Clostridium difficile can be found in different compartments of the gastrointestinal tract of healthy horses, and multiple strains can be present in an individual horse. The prevalence of C. perfringens in healthy adult hoses was low, consistent...

  13. Reproducible Infection Model for Clostridium perfringens in Broiler Chickens

    DEFF Research Database (Denmark)

    Pedersen, Karl; Friis-Holm, Lotte Bjerrum; Heuer, Ole Eske

    2008-01-01

    Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments w...

  14. Genotyping of Clostridium perfringens associated with sudden death in cattle

    Directory of Open Access Journals (Sweden)

    S Miyashiro

    2009-01-01

    Full Text Available Toxigenic types of Clostridium perfringens are significant causative agents of enteric disease in domestic animals, although type E is presumably rare, appearing as an uncommon cause of enterotoxemia of lambs, calves and rabbits. We report herein the typing of 23 C. perfringens strains, by the polymerase chain reaction (PCR technique, isolated from small intestine samples of bovines that have died suddenly, after manifesting or not enteric or neurological disorders. Two strains (8.7% were identified as type E, two (8.7% as type D and the remainder as type A (82.6%. Commercial toxoids available in Brazil have no label claims for efficacy against type E-associated enteritis; however, the present study shows the occurrence of this infection. Furthermore, there are no recent reports on Clostridium perfringens typing in the country.

  15. Heterologous production and secretion of Clostridium perfringens beta-toxoid in closely related Gram-positive hosts

    NARCIS (Netherlands)

    Nijland, Reindert; Lindner, Cordula; van Hartskamp, Mariska; Hamoen, Leendert W.; Kuipers, Oscar P.

    2007-01-01

    The spore forming bacterium Clostridium perfringens is a widely occurring pathogen. Vaccines against C perfringens type B and C are currently manufactured using beta-toxin secreted by virulent C perfringens strains. Large-scale production of vaccines from virulent strains requires stringent safety

  16. Comparative Analysis of Clostridium perfringens Bacteriophage

    Science.gov (United States)

    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 perfringens type A–E toxin plasmids

    Science.gov (United States)

    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

  18. Diffusion of Clostridium perfringens NetB positive strains in healthy and diseased chickens

    Directory of Open Access Journals (Sweden)

    Luca Bano

    2010-01-01

    Full Text Available For over 30 years α toxin was considered the key virulence factor responsible for the appearance of necrotic enteritis (NE in chickens but, recently, a new toxin related to the occurrence of NE, called NetB, has been described. The aim of this work was to evaluate the CP toxin-type and the NetB gene presence in strains collected from chickens affected or not by enteric diseases. 107 strains were tested: 30 isolated from chickens affected by NE, 54 from subjects affected by other enteric pathologies and 22 from healthy animals. All strains resulted toxin-type A and 26.17% of these was positive also for β2 toxin gene. No strains were positive for cpe gene. 27% (29/107 of CP was NetB positive and 93% (27/29 of these was isolated from birds affected by intestinal disorders. 16 NetB positive strains were obtained from chickens affected by NE (16/30, 9 from animals affected by other intestinal disorders (9/54 and 4 from healthy animals (4/22. A significant difference between the number of NetB positive strains isolated from animals affected by NE and healthy chickens has been observed (P=0.014. However, the finding that the 17.4% of strains isolated from healthy chickens was also positive for NetB, confirm that other virulence factors could play an important role on NE appearance.

  19. Incidence and tracking of Clostridium perfringens through an integrated broiler chicken operation

    Science.gov (United States)

    Clostridium perfringens has been shown to be widespread in the broiler chicken hatchery, grow-out, and processing operations. In a previous study, ribotypes of certain strains of C. perfringens isolated from processed chicken carcasses were shown to match ribotypes isolated from paper pad lining tra...

  20. Perfringolysin O: the underrated Clostridium perfringens toxin?

    OpenAIRE

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

    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 theta 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 membr...

  1. Four phage endolysins that are lytic for clostridium perfringens

    Science.gov (United States)

    Clostridium perfringens is a bacterial pathogen and the cause of necrotic enteritis in poultry, and a source of food poisoning and gas gangrene in people. C. perfringens can also cause mild to severe enteritis in pigs. In the EU, the occurrence of C. perfringens-associated necrotic enteritis in pou...

  2. Effect of cooling on Clostridium perfringens in pea soup

    NARCIS (Netherlands)

    Jong, de A.E.I.; Rombouts, F.M.; Beumer, R.R.

    2004-01-01

    Foods associated with Clostridium perfringens outbreaks are usually abused after cooking. Because of their short generation times, C. perfringens spores and cells can grow out to high levels during improper cooling. Therefore, the potential of C. perfringens to multiply in Dutch pea soup during diff

  3. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications.

    Science.gov (United States)

    Freedman, John C; Shrestha, Archana; McClane, Bruce A

    2016-03-16

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe) is located on either the chromosome (for most C. perfringens type A food poisoning strains) or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains). In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination.

  4. Clostridium perfringens Enterotoxin: Action, Genetics, and Translational Applications

    Directory of Open Access Journals (Sweden)

    John C. Freedman

    2016-03-01

    Full Text Available Clostridium perfringens enterotoxin (CPE is responsible for causing the gastrointestinal symptoms of several C. perfringens food- and nonfood-borne human gastrointestinal diseases. The enterotoxin gene (cpe is located on either the chromosome (for most C. perfringens type A food poisoning strains or large conjugative plasmids (for the remaining type A food poisoning and most, if not all, other CPE-producing strains. In all CPE-positive strains, the cpe gene is strongly associated with insertion sequences that may help to assist its mobilization and spread. During disease, CPE is produced when C. perfringens sporulates in the intestines, a process involving several sporulation-specific alternative sigma factors. The action of CPE starts with its binding to claudin receptors to form a small complex; those small complexes then oligomerize to create a hexameric prepore on the membrane surface. Beta hairpin loops from the CPE molecules in the prepore assemble into a beta barrel that inserts into the membrane to form an active pore that enhances calcium influx, causing cell death. This cell death results in intestinal damage that causes fluid and electrolyte loss. CPE is now being explored for translational applications including cancer therapy/diagnosis, drug delivery, and vaccination.

  5. Influence of elevated temperature on starch hydrolysis by enterotoxin-positive and enterotoxin-negative strains of Clostridium perfringens type A.

    Science.gov (United States)

    García-Alvarado, J S; Rodriguez, M A; Labbé, R G

    1992-01-01

    Enterotoxin-positive (Ent+) and enterotoxin-negative (Ent-) strains of Clostridium perfringens were cultured in Duncan-Strong sporulation medium containing starch at 37 and 46 degrees C. At 37 degrees C, all strains degraded starch and sporulated well. However, only Ent- strains could hydrolyze starch, grow extensively, and sporulate at 46 degrees C. Growth, sporulation, and starch hydrolysis by Ent+ strains at 46 degrees C were equivalent to those obtained at 37 degrees C when alpha-amylase was added to the cultures during growth. The total amount of extracellular plus intracellular amylase in cultures of Ent+ strains was significantly less in cells incubated at 46 degrees C than in cells incubated at 37 degrees C. These results contradict an earlier report that Ent+ strains cannot sporulate well near their optimal growth temperature (R. G. Labbe and C. L. Duncan, Can. J. Microbiol. 20:1493-1501, 1974) and suggest that synthesis of alpha-amylase in Ent+ strains is regulated by temperature.

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

    Science.gov (United States)

    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.

  7. Enumeration and isolation of cpe-positive Clostridium perfringens spores from feces.

    Science.gov (United States)

    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.

  8. IN-VITRO GROWTH CHARACTERISTICS OF COMMERCIAL PROBIOTIC STRAINS AND THEIR POTENTIAL FOR INHIBITION OF CLOSTRIDIUM DIFFICILE AND CLOSTRDIDUM PERFRINGENS

    DEFF Research Database (Denmark)

    Schoster, Angelika; Kokotovic, Branko; Permin, Anders

    aerobic conditions was assessed. To evaluate inhibition of C. difficile and C. perfringens sterile supernatant of the probiotic culture was added to BHI inoculated with a standard C. difficile or C. perfringens suspension. Growth was measured spectrophotometrically at 0 and 24h and compared to the control...

  9. Comparison of media for enumeration of Clostridium perfringens from foods

    NARCIS (Netherlands)

    Jong, A.E.I. de; Eijhusen, G.P.; Brouwer-Post, E.J.F.; Grand, M.; Johansson, T.; Kärkkäinen, T.; Marugg, J.; Veld, P.H. in 't; Warmerdam, F.H.M.; Wörner, G.; Zicavo, A.; Rombouts, F.M.; Beumer, R.R.

    2003-01-01

    Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin p

  10. Comparison of media for enumeration of Clostridium perfringens from foods

    NARCIS (Netherlands)

    Jong, A.E.I. de; Eijhusen, G.P.; Brouwer-Post, E.J.F.; Grand, M.; Johansson, T.; Kärkkäinen, T.; Marugg, J.; Veld, P.H. in 't; Warmerdam, F.H.M.; Wörner, G.; Zicavo, A.; Rombouts, F.M.; Beumer, R.R.

    2003-01-01

    Many media have been developed to enumerate Clostridium perfringens from foods. In this study, six media [iron sulfite (IS) agar, tryptose sulfite cycloserine (TSC) agar, Shahidi Ferguson perfringens (SFP) agar, sulfite cycloserine azide (SCA), differential clostridial agar (DCA), and oleandomycin p

  11. [Clostridium perfringens septicemia associated with foodborne toxic infection and abortion].

    Science.gov (United States)

    Lantelme, P; Mohammedi, I; Duperret, S; Vedrinne, J M; Allaouchiche, B; Motin, J

    1995-01-01

    A 32-year-old pregnant woman with poor life and hygiene conditions presented with premature labour, fever and diarrhoea. After admission she gave birth to a stillborn child. The examination revealed a septicaemia with massive haemolysis and renal failure. Six blood cultures obtained on admission yielded Clostridium perfringens. The outcome was favourable after an adapted antibiomicrobial therapy. This case illustrates the potential severity of Clostridium perfringens foodborne toxi-infection which can lead to abortion and septicaemia with massive haemolysis.

  12. The Tcp conjugation system of Clostridium perfringens.

    Science.gov (United States)

    Wisniewski, Jessica A; Rood, Julian I

    2017-03-07

    The Gram-positive pathogen Clostridium perfringens possesses a family of large conjugative plasmids that is typified by the tetracycline resistance plasmid pCW3. Since these plasmids may carry antibiotic resistance genes or genes encoding extracellular or sporulation-associated toxins, the conjugative transfer of these plasmids appears to be important for the epidemiology of C. perfringens-mediated diseases. Sequence analysis of members of this plasmid family identified a highly conserved 35kb region that encodes proteins with various functions, including plasmid replication and partitioning. The tcp conjugation locus also was identified in this region, initially based on low-level amino acid sequence identity to conjugation proteins from the integrative conjugative element Tn916. Genetic studies confirmed that the tcp locus is required for conjugative transfer and combined with biochemical and structural analyses have led to the development of a functional model of the Tcp conjugation apparatus. This review summarises our current understanding of the Tcp conjugation system, which is now one of the best-characterized conjugation systems in Gram-positive bacteria.

  13. Clostridium perfringens strains from bovine enterotoxemia cases are not superior in in vitro production of alpha toxin, perfringolysin O and proteolytic enzymes

    OpenAIRE

    2014-01-01

    Background: Bovine enterotoxemia is a major cause of mortality in veal calves. Predominantly veal calves of beef cattle breeds are affected and losses due to enterotoxemia may account for up to 20% of total mortality. Clostridium perfringens type A is considered to be the causative agent. Recently, alpha toxin and perfringolysin O have been proposed to play an essential role in the development of disease. However, other potential virulence factors also may play a role in the pathogenesis of b...

  14. Distribution of Clostridium perfringens Isolates from Piglets in South Korea

    OpenAIRE

    LEE, Ki-Eun; Lim, Seong-In; SHIN, Seong-Ho; Kwon, Yong-Kuk; Kim, Ha-Young; Song, Jae-Young; An, Dong-Jun

    2014-01-01

    ABSTRACT Clostridium perfringens causes various digestive system disease symptoms in pigs. In the present study, 11 C. perfringens isolates were obtained from diarrheic piglets and 18 from healthy piglets. All of the C. perfringens isolates were shown to be type A using a multiplex PCR assay. The β2 toxin gene was detected in 27/29 C. perfringens isolates, i.e., 81% (9/11) of diarrheic piglets and 100% (18/18) of healthy piglets, and all of the genes had the same sequence. In conclusion, the ...

  15. Clostridium perfringens type A enteritis in blue and yellow macaw (Ara ararauna).

    Science.gov (United States)

    Guimarães, Marta Brito; Torres, Luciana Neves; Mesquita, Ramon Gomes; Ampuero, Fernanda; Cunha, Marcos Paulo Vieira; Ferreira, Thais Sebastiana Porfida; Ferreira, Antonio José Piantino; Catão-Dias, José Luiz; Moreno, Andrea Micke; Knöbl, Terezinha

    2014-12-01

    This study describes an outbreak of necrotic enteritis caused by Clostridium perfringens type A in captive macaws (Ara ararauna). Two psittacine birds presented a history of prostration and died 18 hr after manifestation of clinical signs. The necropsy findings and histopathologic lesions were indicative of necrotic enteritis. Microbiologic assays resulted in the growth of large gram-positive bacilli that were identified as C. perfringens. PCR was used to identify clostridium toxinotypes and confirmed the identification of isolated strains as C pefringens type A, positive to gene codifying beta 2 toxin. The infection source and predisposing factors could not be ascertained.

  16. The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603.

    Science.gov (United States)

    Li, Jihong; Chen, Jianming; Vidal, Jorge E; McClane, Bruce A

    2011-06-01

    Clostridium perfringens type A strains producing enterotoxin (CPE) cause one of the most common bacterial food-borne illnesses, as well as many cases of non-food-borne human gastrointestinal disease. Recent studies have shown that an Agr-like quorum-sensing system controls production of chromosomally encoded alpha-toxin and perfringolysin O by C. perfringens, as well as sporulation by Clostridium botulinum and Clostridium sporogenes. The current study explored whether the Agr-like quorum-sensing system also regulates sporulation and production of two plasmid-encoded toxins (CPE and beta2 toxin) that may contribute to the pathogenesis of non-food-borne human gastrointestinal disease strain F5603. An isogenic agrB null mutant was inhibited for production of beta2 toxin during vegetative growth and in sporulating culture, providing the first evidence that, in C. perfringens, this system can control production of plasmid-encoded toxins as well as chromosomally encoded toxins. This mutant also showed reduced production of alpha-toxin and perfringolysin O during vegetative growth. Importantly, when cultured in sporulation medium, the mutant failed to efficiently form spores and was blocked for CPE production. Complementation partially or fully reversed all phenotypic changes in the mutant, confirming that they were specifically due to inactivation of the agr locus. Western blots suggest that this loss of sporulation and sporulation-specific CPE production for the agrB null mutant involves, at least in part, Agr-mediated regulation of production of Spo0A and alternative sigma factors, which are essential for C. perfringens sporulation.

  17. Genome analysis of Clostridium perfringens isolates from healthy and necrotic enteritis infected chickens and turkeys

    DEFF Research Database (Denmark)

    Ronco, Troels; Stegger, Marc; Ng, Kim Lee

    2017-01-01

    Objective: Clostridium perfringens causes gastrointestinal diseases in both humans and domestic animals. Type A strains expressing the NetB toxin are the main cause of necrotic enteritis (NE) in chickens, which has remarkable impact on animal welfare and production economy in the international...

  18. Reproducible infection model for Clostridium perfringens in broiler chickens

    DEFF Research Database (Denmark)

    Pedersen, Karl; Friis-Holm, Lotte Bjerrum; Heuer, Ole Eske

    2008-01-01

    Experiments were carried out to establish an infection and disease model for Clostridium perfringens in broiler chickens. Previous experiments had failed to induce disease and only a transient colonization with challenge strains had been obtained. In the present study, two series of experiments w...

  19. BEC, a novel enterotoxin of Clostridium perfringens found in human clinical isolates from acute gastroenteritis outbreaks.

    Science.gov (United States)

    Yonogi, Shinya; Matsuda, Shigeaki; Kawai, Takao; Yoda, Tomoko; Harada, Tetsuya; Kumeda, Yuko; Gotoh, Kazuyoshi; Hiyoshi, Hirotaka; Nakamura, Shota; Kodama, Toshio; Iida, Tetsuya

    2014-06-01

    Clostridium perfringens is a causative agent of food-borne gastroenteritis for which C. perfringens enterotoxin (CPE) has been considered an essential factor. Recently, we experienced two outbreaks of food-borne gastroenteritis in which non-CPE producers of C. perfringens were strongly suspected to be the cause. Here, we report a novel enterotoxin produced by C. perfringens isolates, BEC (binary enterotoxin of C. perfringens). Culture supernatants of the C. perfringens strains showed fluid-accumulating activity in rabbit ileal loop and suckling mouse assays. Purification of the enterotoxic substance in the supernatants and high-throughput sequencing of genomic DNA of the strains revealed BEC, composed of BECa and BECb. BECa and BECb displayed limited amino acid sequence similarity to other binary toxin family members, such as the C. perfringens iota toxin. The becAB genes were located on 54.5-kb pCP13-like plasmids. Recombinant BECb (rBECb) alone had fluid-accumulating activity in the suckling mouse assay. Although rBECa alone did not show enterotoxic activity, rBECa enhanced the enterotoxicity of rBECb when simultaneously administered in suckling mice. The entertoxicity of the mutant in which the becB gene was disrupted was dramatically decreased compared to that of the parental strain. rBECa showed an ADP-ribosylating activity on purified actin. Although we have not directly evaluated whether BECb delivers BECa into cells, rounding of Vero cells occurred only when cells were treated with both rBECa and rBECb. These results suggest that BEC is a novel enterotoxin of C. perfringens distinct from CPE, and that BEC-producing C. perfringens strains can be causative agents of acute gastroenteritis in humans. Additionally, the presence of becAB on nearly identical plasmids in distinct lineages of C. perfringens isolates suggests the involvement of horizontal gene transfer in the acquisition of the toxin genes.

  20. Detection of toxigenic Clostridium perfringens and Clostridium botulinum from food sold in Lagos, Nigeria.

    Science.gov (United States)

    Chukwu, Emelda E; Nwaokorie, Francisca O; Coker, Akitoye O; Avila-Campos, Mario J; Solis, Rosa L; Llanco, Luis A; Ogunsola, Folasade T

    2016-12-01

    Food-borne diseases contribute to the huge burden of sickness and death globally and in the last decade, have become more frequently reported in Africa. In line with this, food safety is becoming a significant and growing public health problem in Nigeria. Diarrhoea is a common problem in Nigeria and has been reported but there has been little data on the possibility of clostridia as aetiological agents. Clostridium species are ubiquitous in the environment and in the gastrointestinal tract of man and animals and can serve as a marker for faecal contamination. We set out to determine the potential of these foods to transmit Clostridium species. A total of 220 food commodities from six local governments in Lagos State were sampled. Isolates obtained were identified based on cultural, morphological and biochemical characteristics. Toxinotyping was done using multiplex-PCR with primers specific for alpha, beta, epsilon and iota-toxin genes, enterotoxigenic cpe gene and neurotoxigenic BoNt gene. Fifty (22.7%) clostridial species were isolated of which 29 (58%) were identified as C. perfringens. Toxinotyping of the 29 strains showed that 28 (96.6%) were toxin producing C. perfringens type A while one (3.4%) was C. perfringens type D. Two (4%) C. botulinum species were isolated and identified by 16S rRNA sequencing, both harbouring BoNt/A gene. The contamination rates of food with Clostridium species show that food hygiene is a problem and Clostridium species may be a source of food borne disease in Lagos State, Nigeria.

  1. Pancreatitis caused by Clostridium perfringens infection with extensive retropneumoperitoneum; Pancreatitis por Clostridium perfringens con retroneumoperitoneo extenso

    Energy Technology Data Exchange (ETDEWEB)

    Merchante, E.; Garcia, F. J.; Perez, H.; Marquez, J. L. [Hospital Universitario Virgen del Rocio. Sevilla (Spain)

    2001-07-01

    We present a case of primary emphysematous pancreatitis caused by Clostridium perfringens infection (also Known as spontaneous pancreatic gas gangrene) in a 66-year-old man with diabetes and a history of recurrent pancreatitis. One notable feature is the absence of a focal distribution, which is seen on radiological studies to be accompanied by extensive retropneumoperitoneum, with dissemination of the gas toward the mesenteric root and pelvic extra peritoneal spaces. This wide diffusion is aided by the C. perfringens toxins and the pancreatic enzymes released, leading to a fulminate course, an elevated rate of early mortality among the cases reviewed. The early diagnosis of this disease is fundamental, enabling aggressive medical treatment and emergency surgery. Diabetes is a known risk factor for anaerobic infection, including C. perfringens, as in the case of emphysematous cholecystitis. A diseased pancreas or pancreatic duct facilitates the development of infections since it eliminates poorly the microorganisms that reach it from the duodenum. Gas gangrene secondary to necrosis-related super infection or pancreatic collections is uncommon, and spontaneous or primary cases are exceptionally are. (Author) 13 refs.

  2. Predisposing factors and prevention of Clostridium perfringens-associated enteritis.

    Science.gov (United States)

    Allaart, Janneke G; van Asten, Alphons J A M; Gröne, Andrea

    2013-09-01

    Clostridium perfringens is one of the major causes of intestinal disease in humans and animals. Its pathogenicity is contributed to by the production of a variety of toxins. In addition, predisposing environmental factors are important for the induction of C. perfringens-associated enteritis as shown by infection models. Environmental contamination, gastric and intestinal pH, intestinal microflora, nutrition, concurrent infections, and medical interventions may influence the intestinal colonization, growth, and toxin production by C. perfringens. Prevention of C. perfringens-associated enteritis may be mediated by the use of feed additives like probiotics, prebiotics, organic acids, essential oils, bacteriophages, lysozymes, bacteriocins, and antimicrobial peptides. Here we summarize and discuss published data on the influence of different environmental predisposing factors and preventive measures. Further research should focus on feed composition and feed additives in order to prevent C. perfringens-associated enteritis.

  3. EtfA catalyses the formation of dipicolinic acid in Clostridium perfringens.

    Science.gov (United States)

    Orsburn, Benjamin C; Melville, Stephen B; Popham, David L

    2010-01-01

    Dipicolinic acid (DPA) is a major component of bacterial endospores, comprising 5-15% of the spore dry weight, and is important for spore stability and resistance properties. The biosynthetic precursor to DPA, dihydro-dipicolinic acid (DHDPA), is produced by DHDPA synthase within the lysine biosynthesis pathway. In Bacillus subtilis, and most other bacilli and clostridia, DHDPA is oxidized to DPA by the products of the spoVF operon. Analysis of the genomes of the clostridia in Cluster I, including the pathogens Clostridium perfringens, Clostridium botulinum and Clostridium tetani, has shown that no spoVF orthologues exist in these organisms. DPA synthase was purified from extracts of sporulating C. perfringens cells. Peptide sequencing identified an electron transfer flavoprotein, EtfA, in this purified protein fraction. A C. perfringens strain with etfA inactivated is blocked in late stage sporulation and produces < or = 11% of wild-type DPA levels. C. perfringens EtfA was expressed in and purified from Escherichia coli, and this protein catalysed DPA formation in vitro. The sequential production of DHDPA and DPA in C. perfringens appears to be catalysed by DHDPA synthase followed by EtfA. Genome sequence data and the taxonomy of spore-forming species suggest that this may be the ancestral mechanism for DPA synthesis.

  4. ISOLATION AND GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FREE-LIVING SOUTH AMERICAN COATI (NASUA NASUA).

    Science.gov (United States)

    Silva, Rodrigo O S; Almeida, Lara R; Oliveira Junior, Carlos A; Lima, Paula C S; Soares, Danielle F M; Pereira, Pedro L L; Silva, Israel J; Lobato, Francisco C F

    2016-03-01

    The importance of Clostridium perfringens for most wild animal species remains unclear. This study aimed to isolate and genotype C. perfringens in stool samples from free-living South American coati (Nasua nasua) in Brazil. Forty-six free-living N. nasua were trapped and stool samples were collected. Two different protocols for C. perfringens isolation were tested: direct plating onto selective agar and pre-enrichment in broth followed by plating in selective agar. Clostridium perfringens type A was isolated from 15 (32.6%) animals by direct plating and 36 (78.3%) animals by broth PE, and the rate of isolation was significantly different between these two methods (P < 0.01). Twelve of the 36 (33.3%) isolated strains by the PE protocol were positive for the β-2 toxin-encoding gene (cpb2) whereas the enterotoxin-encoding gene (cpe) and necrotic enteritis like-B toxin gene (netb) were not found. These results suggest that C. perfringens is commonly part of the microbiota of free-living coatis. Additionally, the use of a PE protocol appears to be essential for studies on C. perfringens in this species.

  5. Metataxonomics reveal vultures as a reservoir for Clostridium perfringens

    Science.gov (United States)

    Meng, Xiangli; Lu, Shan; Yang, Jing; Jin, Dong; Wang, Xiaohong; Bai, Xiangning; Wen, Yumeng; Wang, Yiting; Niu, Lina; Ye, Changyun; Rosselló-Móra, Ramon; Xu, Jianguo

    2017-01-01

    The Old World vulture may carry and spread pathogens for emerging infections since they feed on the carcasses of dead animals and participate in the sky burials of humans, some of whom have died from communicable diseases. Therefore, we studied the precise fecal microbiome of the Old World vulture with metataxonomics, integrating the high-throughput sequencing of almost full-length small subunit ribosomal RNA (16S rRNA) gene amplicons in tandem with the operational phylogenetic unit (OPU) analysis strategy. Nine vultures of three species were sampled using rectal swabs on the Qinghai-Tibet Plateau, China. Using the Pacific Biosciences sequencing platform, we obtained 54 135 high-quality reads of 16S rRNA amplicons with an average of 1442±6.9 bp in length and 6015±1058 reads per vulture. Those sequences were classified into 314 OPUs, including 102 known species, 50 yet to be described species and 161 unknown new lineages of uncultured representatives. Forty-five species have been reported to be responsible for human outbreaks or infections, and 23 yet to be described species belong to genera that include pathogenic species. Only six species were common to all vultures. Clostridium perfringens was the most abundant and present in all vultures, accounting for 30.8% of the total reads. Therefore, using the new technology, we found that vultures are an important reservoir for C. perfringens as evidenced by the isolation of 107 strains encoding for virulence genes, representing 45 sequence types. Our study suggests that the soil-related C. perfringens and other pathogens could have a reservoir in vultures and other animals. PMID:28223683

  6. Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals.

    OpenAIRE

    1987-01-01

    Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animal...

  7. Genotyping and antimicrobial susceptibility of Clostridium perfringens isolated from Tinamidae, Cracidae and Ramphastidae species in Brazil

    Directory of Open Access Journals (Sweden)

    Rodrigo Otávio Silveira Silva

    2014-03-01

    Full Text Available The aim of this study was to isolate, genotype and evaluate the antimicrobial susceptibility of Clostridium perfringens found in species Tinamidae, Cracidae and Ramphastidae in Brazil. C. perfringens was isolated in 13 (5% out of 260 swab samples and five (8.3% out of 60 stool samples. All strains were classified as C. perfringens type A, and nine (50% were positive for the beta-2 toxin-encoding gene. No strains were positive for the necrotic enteritis toxin B-like (NetB-encoding gene. All isolates were susceptible to penicillin, metronidazole and vancomycin, whereas four (22.2%, five (27.8% and 13 (72.2% strains were considered resistant to erythromycin, oxytetracycline and lincomycin, respectively.

  8. Toxigenic characteristics of Clostridium perfringens type C in enterotoxemia of domestic animals.

    Science.gov (United States)

    Niilo, L

    1987-04-01

    Eleven Clostridium perfringens type C strains isolated from fatal cases of hemorrhagic enterotoxemia of Canadian calves, a piglet, and a foal were studied for the production of soluble antigens. All the isolates from calves and a foal failed to produce delta toxin, but were capable of producing large amounts of lethal beta toxin. A strain isolated from a piglet produced delta, but very little beta toxin. Other differences were relatively minor. The results indicated that young domestic animals may be susceptible to all subtypes of C. perfringens type C. A simple method of using blood agar plates coated with type A antiserum for demonstration of hemolytic patterns was found advantageous in differentiation of C. perfringens strains.

  9. Clostridium perfringens associated with food borne disease : final report

    NARCIS (Netherlands)

    Wijnands LM; van der Mey-Florijn A; Delfgou-van Asch EHM; LZO

    2011-01-01

    Mensen die voedsel eten dat de bacterie Clostridium perfringens bevat, kunnen daar diarree van krijgen. Deze bacterie komt vooral voor in producten die vlees bevatten, zoals soepen en stoofschotels, maar ook in kruiden en specerijen. Mensen worden voornamelijk ziek na het eten van vleesbevattende pr

  10. Binding of Clostridium perfringens to collagen correlates with the ability to cause necrotic enteritis in chickens.

    Science.gov (United States)

    Wade, B; Keyburn, A L; Seemann, T; Rood, J I; Moore, R J

    2015-11-18

    This study investigated the ability of Clostridium perfringens isolates derived from chickens to bind to collagen types I-V and gelatin. In total 21 strains from three distinct backgrounds were studied: (i) virulent strains isolated from birds suffering from necrotic enteritis, (ii) avirulent strains isolated from birds suffering from necrotic enteritis and (iii) strains isolated from healthy birds. All strains isolated from diseased birds had been assessed for virulence in a disease induction model. The virulent isolates all displayed collagen binding ability. However, most strains in the other two classes showed negligible binding to collagen. The prevalence of a previously described C. perfringens putative collagen adhesin-encoding gene was investigated by PCR screening. It was found that five of the strains carried the putative collagen adhesin-encoding gene and that all of these strains were virulent isolates. Based on these studies it is postulated that collagen adhesion may play a role in the pathogenesis of necrotic enteritis.

  11. Clostridium perfringens Sialidases: Potential Contributors to Intestinal Pathogenesis and Therapeutic Targets

    Directory of Open Access Journals (Sweden)

    Jihong Li

    2016-11-01

    Full Text Available Clostridium perfringens is a major cause of histotoxic and intestinal infections of humans and other animals. This Gram-positive anaerobic bacterium can produce up to three sialidases named NanH, NanI, and NanJ. The role of sialidases in histotoxic infections, such as gas gangrene (clostridial myonecrosis, remains equivocal. However, recent in vitro studies suggest that NanI may contribute to intestinal virulence by upregulating production of some toxins associated with intestinal infection, increasing the binding and activity of some of those toxins, and enhancing adherence of C. perfringens to intestinal cells. Possible contributions of NanI to intestinal colonization are further supported by observations that the C. perfringens strains causing acute food poisoning in humans often lack the nanI gene, while other C. perfringens strains causing chronic intestinal infections in humans usually carry a nanI gene. Certain sialidase inhibitors have been shown to block NanI activity and reduce C. perfringens adherence to cultured enterocyte-like cells, opening the possibility that sialidase inhibitors could be useful therapeutics against C. perfringens intestinal infections. These initial in vitro observations should be tested for their in vivo significance using animal models of intestinal infections.

  12. Growth of Clostridium perfringens in food proteins previously exposed to proteolytic bacilli.

    Science.gov (United States)

    Schroder, D J; Busta, F F

    1973-11-01

    Proteolytic sporeforming bacteria capable of surviving processing heat treatments in synthetic or fabricated protein foods exhibited no antagonistic effects on growth of Clostridium perfringens, but instead shortened the lag of subsequent growth of C. perfringens in sodium caseinate and isolated soy protein. Bacillus subtilis A cells were cultured in 3% sodium caseinate or isolated soy protein solutions. The subsequent effect on the lag time and growth of C. perfringens type A (strain S40) at 45 C was measured by colony count or absorbance at 650 nm, or both. B. subtilis incubation for 12 h or more in sodium caseinate reduced the C. perfringens lag by 3 h. Incubation of 8 h or more in isolated soy protein reduced the lag time by 1.5 h. Molecular sieving of the B. subtilis-treated sodium caseinate revealed that all molecular sizes yielded a similar reduced lag time. Diethylaminoethyl-Sephadex ion exchange fractionation and subsequent amino acid analysis indicated that the lag time reduction caused by B. subtilis incubation was not related to charge of the peptides nor to their amino acid composition. Apparently the shortened C. perfringens lag in these B. subtilis-hydrolyzed food proteins was a result of the protein being more readily available for utilization by C. perfringens.

  13. Structural Insights into Clostridium perfringens Delta Toxin Pore Formation.

    Science.gov (United States)

    Huyet, Jessica; Naylor, Claire E; Savva, Christos G; Gibert, Maryse; Popoff, Michel R; Basak, Ajit K

    2013-01-01

    Clostridium perfringens Delta toxin is one of the three hemolysin-like proteins produced by C. perfringens type C and possibly type B strains. One of the others, NetB, has been shown to be the major cause of Avian Nectrotic Enteritis, which following the reduction in use of antibiotics as growth promoters, has become an emerging disease of industrial poultry. Delta toxin itself is cytotoxic to the wide range of human and animal macrophages and platelets that present GM2 ganglioside on their membranes. It has sequence similarity with Staphylococcus aureus β-pore forming toxins and is expected to heptamerize and form pores in the lipid bilayer of host cell membranes. Nevertheless, its exact mode of action remains undetermined. Here we report the 2.4 Å crystal structure of monomeric Delta toxin. The superposition of this structure with the structure of the phospholipid-bound F component of S. aureus leucocidin (LukF) revealed that the glycerol molecules bound to Delta toxin and the phospholipids in LukF are accommodated in the same hydrophobic clefts, corresponding to where the toxin is expected to latch onto the membrane, though the binding sites show significant differences. From structure-based sequence alignment with the known structure of staphylococcal α-hemolysin, a model of the Delta toxin pore form has been built. Using electron microscopy, we have validated our model and characterized the Delta toxin pore on liposomes. These results highlight both similarities and differences in the mechanism of Delta toxin (and by extension NetB) cytotoxicity from that of the staphylococcal pore-forming toxins.

  14. Clostridium perfringens enterotoxicosis in two Amur leopards (Panthera pardus orientalis).

    Science.gov (United States)

    Neiffer, D L

    2001-03-01

    Two 6-yr-old male sibling Amur leopards (Panthera pardus orientalis) housed together at the Pittsburgh Zoo presented for acute onset of diarrhea with no changes in appetite or behavior. Heat-fixed modified Wright-stained and Gram-stained fecal smears revealed a mixed bacterial population with a large number of gram-positive Clostridium perfringens-like spores (>20 per high-power oil immersion field). In addition, C. perfringens enterotoxin was isolated from one leopard at 1:256, confirming the presence of C. perfringens enterotoxicosis. Treatment with oral metronidazole, tylosin tartrate, and psyllium fiber was prescribed, with return of more normal stool by the third day of treatment. Fecal consistency steadily improved and was considered normal by the time all prescribed treatments were complete. Diarrhea has not recurred. Partially thawed meat in the leopards' diet may have precipitated the production of an endogenous clostridial enterotoxicosis by disrupting digestive tract flora with resultant clostridial overgrowth and sporulation.

  15. Hazard analysis of Clostridium perfringens in the Skylab Food System

    Science.gov (United States)

    Bourland, C. T.; Huber, C. S.; Kiser, P. R.; Heidelbaugh, N. D.; Rowley, D. B.

    1974-01-01

    The Skylab Food System presented unique microbiological problems because food was warmed in null-gravity and because the heat source was limited to 69.4 C (to prevent boiling in null-gravity). For these reasons, the foods were manufactured using critical control point techniques of quality control coupled with appropriate hazard analyses. One of these hazard analyses evaluated the threat from Clostridium perfringens. Samples of food were inoculated with C. perfringens and incubated for 2 h at temperatures ranging from 25 to 55 C. Generation times were determined for the foods at various temperatures. Results of these tests were evaluated taking into consideration: food-borne disease epidemiology, the Skylab food manufacturing procedures, and the performance requirements of the Skylab Food System. Based on this hazard analysis, a limit for C. perfringens of 100/g was established for Skylab foods.

  16. [Study of the role of Clostridium perfringens in bovine enterotoxemia].

    Science.gov (United States)

    Manteca, C; Daube, G; Mainil, J

    1999-01-01

    Bovine enterotoxaemia is an acute to peracute syndrome occurring mainly in calves and characterized by the sudden or very rapid death of the calf, with colics, convulsions and nervous disorders as clinical signs, if any. The most pronounced lesion is a necrohaemorrhagic enteritis of the jejunum, the ileum, and sometimes the colon. Suckling beef calves are the most frequently affected ones. In 67% of the 78 field cases investigated, some kind of stress was observed 24 to 36 hours prior to the death: change in diet or pasture, vaccination... The most frequently isolated bacteria, and the one isolated in highest numbers, was non-sporulated non-enterotoxigenic toxinotype A Clostridium perfringens. Reproduction of the lesions was successful in a ligated intestinal loop assay in one calf with a few of these strains, more especially with one of them, which was shown later to produce another recently described toxin, the beta 2 toxin. A role for this beta 2 toxin in bovine enterotoxaemia is thus speculated for future research.

  17. Comparative genome analysis of clostridium perfringens isolates from healthy and necrotic enteritis infected poultry and diseased pigs

    DEFF Research Database (Denmark)

    Ronco, Troels; Lyhs, Ulrike; Stegger, Marc;

    2015-01-01

    Introduction: Clostridium perfringens causes gastrointestinal diseases in both humans and domestic animals. Type A strains are the main cause of necrotic enteritis (NE) in chickens, which is a significant economic issue in the international poultry industry. The NetB and Cpb2 toxins seem to be im...

  18. A rapid qualitative assay for detection of Clostridium perfringens in canned food products.

    Science.gov (United States)

    Dave, Gayatri Ashwinkumar

    2017-01-01

    Clostridium perfringens (MTCC 1349) is a Gram-positive, anaerobic, endospore forming, and rod-shaped bacterium. This bacterium produces a variety of toxins under strict anaerobic environment. C. perfringens can grow at temperatures ranging between 20°C and 50°C. It is the major causetive agent for gas gangrene, cellulitis, septicemia, necrotic enteritis and food poisoning, which are common toxin induced conditions noted in human and animals. C. perfringens can produce produce four major types of toxins that are used for the classification of strains, classified under type A-E. Across the globe many countries, including the United States, are affected by C. perfringens food poisonings where it is ranked as one of the most common causes of food borne infections. To date, no direct one step assay for the detection of C. perfringens has been developed and only few methods are known for accurate detection of C. perfringens. Long detection and incubation time is the major consideration of these reporter assays. The prensent study proposes a rapid and reliable colorimetric assay for the detection of C. perfringens. In principale, this assay detects the para nitrophenyl (yellow colour end product) liberated due to the hydrolysis of paranitrophenyl phosphetidyl choline (PNPC) through phospholipase C (lecithinase). Constitutive secretion of phospholipase C is a charactristic feature of C. perfringens. This assay detects the presence of the extracellular lecithinse through the PNPC impragnated impregnated probe. The probe is impregnated with peranitrophenyl phosphotidyl choline ester, which is colourless substrate used by lecithinase. The designed assay is specific towards PNPC and detectes very small quantites of lecithinase under conditions used. The reaction is substrate specific, no cross reaction was observed upon incubation with other substrates. In addition, this assay gave negative results with other clostridium strains, no cross reactions were observed with other

  19. Clostridium Perfringens Toxins Involved in Mammalian Veterinary Diseases

    Science.gov (United States)

    Uzal, F. A.; Vidal, J. E.; McClane, B. A.; Gurjar, A. A.

    2013-01-01

    Clostridium perfringens is a gram-positive anaerobic rod that is classified into 5 toxinotypes (A, B, C, D, and E) according to the production of 4 major toxins, namely alpha (CPA), beta (CPB), epsilon (ETX) and iota (ITX). However, this microorganism can produce up to 16 toxins in various combinations, including lethal toxins such as perfringolysin O (PFO), enterotoxin (CPE), and beta2 toxin (CPB2). Most diseases caused by this microorganism are mediated by one or more of these toxins. The role of CPA in intestinal disease of mammals is controversial and poorly documented, but there is no doubt that this toxin is essential in the production of gas gangrene of humans and several animal species. CPB produced by C. perfringens types B and C is responsible for necrotizing enteritis and enterotoxemia mainly in neonatal individuals of several animal species. ETX produced by C. perfringens type D is responsible for clinical signs and lesions of enterotoxemia, a predominantly neurological disease of sheep and goats. The role of ITX in disease of animals is poorly understood, although it is usually assumed that the pathogenesis of intestinal diseases produced by C. perfringens type E is mediated by this toxin. CPB2, a necrotizing and lethal toxin that can be produced by all types of C. perfringens, has been blamed for disease in many animal species, but little information is currently available to sustain or rule out this claim. CPE is an important virulence factor for C. perfringens type A gastrointestinal disease in humans and dogs; however, the data implicating CPE in other animal diseases remains ambiguous. PFO does not seem to play a direct role as the main virulence factor for animal diseases, but it may have a synergistic role with CPA-mediated gangrene and ETX-mediated enterotoxemia. The recent improvement of animal models for C. perfringens infection and the use of toxin gene knock-out mutants have demonstrated the specific pathogenic role of several toxins of C

  20. Blocking the butyrate-formation pathway impairs hydrogen production in Clostridium perfringens

    Institute of Scientific and Technical Information of China (English)

    Ruisong Yu; Ruofan Wang; Ting Bi; Weining Sun; Zhihua Zhou

    2013-01-01

    Inactivating competitive pathways will improve fermentative hydrogen production by obligate anaerobes,such as those of genus Clostridium.In our previous study,the hydrogen yield of Clostridium perfringens W13 in which L-lactate dehydrogenase was inactivated increased by 44% when compared with its original strain W12.In this study,we explored whether blocking butyrate formation pathway would increase hydrogen yield.The acetyl-CoA acetyltransferase gene (atoB) encodes the first enzyme in this pathway,which ultimately forms butyrate.Clostridium perfringens W14 and W15 were constructed by inactivating atoB in W13 and W12,respectively.The hydrogen yield of W14 and W15 was 44% and 33% of those of W13 and W12,respectively.Inactivation of atoB decreased the pyruvate synthesis and its conversion to acetyl-CoA in both mutants,and increased ethanol formation in W14 and W15.Proteomic analysis revealed that the expressions of five proteins involved in butyrate formation pathway were up-regulated in W14.Our results suggest that butyrate formation deficiency improved ethanol production but not hydrogen production,indicating the importance of butyrate formation pathway for hydrogen production in C.perfringens.

  1. Experimental Clostridium perfringens type D enterotoxemia in goats.

    Science.gov (United States)

    Uzal, F A; Kelly, W R

    1998-03-01

    The effects of intraduodenal administration of Clostridium perfringens cultures and culture products in goats were evaluated to develop a reliable experimental model of enterotoxemia in this species. Five conventionally reared, 11-16-week-old Angora goat kids were dosed intraduodenally with whole cultures of C. perfringens type D; five similar animals were dosed with C. perfringens type D filtered culture supernatant; and a third group of five kids was dosed with C. perfringens type D washed cells. Two kids were used as controls and received sterile, nontoxic culture medium intraduodenally. All animals received starch solution into the abomasum. All five kids inoculated with whole culture and three of five dosed with culture supernatant and with washed cells developed central nervous system signs. Diarrhea was observed in two of five kids inoculated with whole culture, in all five of those dosed with culture supernatant, and in three of five of those that received washed cells. The most striking postmortem findings consisted of lung edema, necrotizing pseudomembranous colitis, and cerebral vasogenic edema. The protocol thus provided a reasonable model of naturally occurring enterotoxemia in goats, producing a range of clinical signs and postmortem changes similar to those observed in the natural disease.

  2. Recent Insights into Clostridium perfringens Beta-Toxin

    Directory of Open Access Journals (Sweden)

    Masahiro Nagahama

    2015-02-01

    Full Text Available Clostridium perfringens beta-toxin is a key mediator of necrotizing enterocolitis and enterotoxemia. It is a pore-forming toxin (PFT that exerts cytotoxic effect. Experimental investigation using piglet and rabbit intestinal loop models and a mouse infection model apparently showed that beta-toxin is the important pathogenic factor of the organisms. The toxin caused the swelling and disruption of HL-60 cells and formed a functional pore in the lipid raft microdomains of sensitive cells. These findings represent significant progress in the characterization of the toxin with knowledge on its biological features, mechanism of action and structure-function having been accumulated. Our aims here are to review the current progresses in our comprehension of the virulence of C. perfringens type C and the character, biological feature and structure-function of beta-toxin.

  3. Comparative genomic hybridization analysis shows different epidemiology of chromosomal and plasmid-borne cpe-carrying Clostridium perfringens type A.

    Directory of Open Access Journals (Sweden)

    Päivi Lahti

    Full Text Available Clostridium perfringens, one of the most common causes of food poisonings, can carry the enterotoxin gene, cpe, in its chromosome or on a plasmid. C. perfringens food poisonings are more frequently caused by the chromosomal cpe-carrying strains, while the plasmid-borne cpe-positive genotypes are more commonly found in the human feces and environmental samples. Different tolerance to food processing conditions by the plasmid-borne and chromosomal cpe-carrying strains has been reported, but the reservoirs and contamination routes of enterotoxin-producing C. perfringens remain unknown. A comparative genomic hybridization (CGH analysis with a DNA microarray based on three C. perfringens type A genomes was conducted to shed light on the epidemiology of C. perfringens food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains by comparing chromosomal and plasmid-borne cpe-positive and cpe-negative C. perfringens isolates from human, animal, environmental, and food samples. The chromosomal and plasmid-borne cpe-positive C. perfringens genotypes formed two distinct clusters. Variable genes were involved with myo-inositol, ethanolamine and cellobiose metabolism, suggesting a new epidemiological model for C. perfringens food poisonings. The CGH results were complemented with growth studies, which demonstrated different myo-inositol, ethanolamine, and cellobiose metabolism between the chromosomal and plasmid-borne cpe-carrying strains. These findings support a ubiquitous occurrence of the plasmid-borne cpe-positive strains and their adaptation to the mammalian intestine, whereas the chromosomal cpe-positive strains appear to have a narrow niche in environments containing degrading plant material. Thus the epidemiology of the food poisonings caused by two populations appears different, the plasmid-borne cpe-positive strains probably contaminating foods via humans and the chromosomal strains being connected to plant material.

  4. Characterization of genes encoding for acquired bacitracin resistance in Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Audrey Charlebois

    Full Text Available Phenotypic bacitracin resistance has been reported in Clostridium perfringens. However, the genes responsible for the resistance have not yet been characterized. Ninety-nine C. perfringens isolates recovered from broilers and turkeys were tested for phenotypic bacitracin resistance. Bacitracin MIC(90 (>256 µg/ml was identical for both turkey and chicken isolates; whereas MIC(50 was higher in turkey isolates (6 µg/ml than in chicken isolates (3 µg/ml. Twenty-four of the 99 isolates showed high-level bacitracin resistance (MIC breakpoint >256 µg/ml and the genes encoding for this resistance were characterized in C. perfringens c1261_A strain using primer walking. Sequence analysis and percentages of amino acid identity revealed putative genes encoding for both an ABC transporter and an overproduced undecaprenol kinase in C. perfringens c1261_A strain. These two mechanisms were shown to be both encoded by the putative bcrABD operon under the control of a regulatory gene, bcrR. Efflux pump inhibitor thioridazine was shown to increase significantly the susceptibility of strain c1261_A to bacitracin. Upstream and downstream from the bcr cluster was an IS1216-like element, which may play a role in the dissemination of this resistance determinant. Pulsed-field gel electrophoresis with prior double digestion with I-CeuI/MluI enzymes followed by hybridization analyses revealed that the bacitracin resistance genes bcrABDR were located on the chromosome. Semi-quantitative RT-PCR demonstrated that this gene cluster is expressed under bacitracin stress. Microarray analysis revealed the presence of these genes in all bacitracin resistant strains. This study reports the discovery of genes encoding for a putative ABC transporter and an overproduced undecaprenol kinase associated with high-level bacitracin resistance in C. perfringens isolates from turkeys and broiler chickens.

  5. Use of an indirect haemagglutination test, for the detection of Clostridium perfringens type A enterotoxin

    Directory of Open Access Journals (Sweden)

    Marlene B. Serafim

    1991-03-01

    Full Text Available An indirect haemagglutination (IH test is described for the detection of Clostridium perfringens type A enterotoxin, produced by strains isolated from human cases of food poisoning and from contaminated food. Though no strict relationship could be observed between titers in the IH test and the time it took mice to die from the intravenous inoculation of mice (IIM, results of the supernatants examined by both methods demonstrated that the IH test was more sensitive than the ILM one. No unspecific reaction was obtanined int he IH wirh a negative control and the inhibitions of the IH and IIM tests by specific antiserum against C. perfringens enterotoxin showed that the IH test is very spcific. The IH assay is recommended for its sensitivity and easy performance by less-equipped laboratories, by these and other data.

  6. Antimicrobial susceptibility of Clostridium perfringens isolated from domestic and wild animal species in Brazil

    Directory of Open Access Journals (Sweden)

    Carlos Augusto de Oliveira Júnior

    2016-02-01

    Full Text Available Clostridium perfringens is a microorganism commonly found in the microbiota of humans and animals and a potential cause of enteric, muscle or nervous diseases. The treatment of these diseases is based on antimicrobial therapy and it is extremely important to know the antimicrobial susceptibility profile of the strains present in the region. The aim of this study was to evaluate the antimicrobial susceptibility of C. perfringens isolated from domestic and wild animals in Brazil against seven different antimicrobials. Forty-one strains from the stool samples of cattle (n = 12, buffalo (n = 2, goat (n = 3, dogs (n = 12 and wild carnivores (n = 12 were examined. The minimum inhibitory concentration was determined by the agar dilution method using Brucella agar supplemented with 5% of sheep blood, 0.1% of vitamin K, 0.1% of hemin and concentrations ranging from 0,25 to 256,0 mg L-1 of the following antibiotics: erythromycin, florfenicol, metronidazole, oxytetracycline, penicillin, tylosin, and vancomycin. All C. perfringens strains were susceptible to florfenicol, metronidazole, penicillin and vancomycin. Two strains (4.9% were resistant to erythromycin and tylosin, while five (12.2% were resistant to oxytetracycline, one of which (2.4% from an ocelot.

  7. NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Anthony L Keyburn

    2008-02-01

    Full Text Available For over 30 years a phospholipase C enzyme called alpha-toxin was thought to be the key virulence factor in necrotic enteritis caused by Clostridium perfringens. However, using a gene knockout mutant we have recently shown that alpha-toxin is not essential for pathogenesis. We have now discovered a key virulence determinant. A novel toxin (NetB was identified in a C. perfringens strain isolated from a chicken suffering from necrotic enteritis (NE. The toxin displayed limited amino acid sequence similarity to several pore forming toxins including beta-toxin from C. perfringens (38% identity and alpha-toxin from Staphylococcus aureus (31% identity. NetB was only identified in C. perfringens type A strains isolated from chickens suffering NE. Both purified native NetB and recombinant NetB displayed cytotoxic activity against the chicken leghorn male hepatoma cell line LMH; inducing cell rounding and lysis. To determine the role of NetB in NE a netB mutant of a virulent C. perfringens chicken isolate was constructed by homologous recombination, and its virulence assessed in a chicken disease model. The netB mutant was unable to cause disease whereas the wild-type parent strain and the netB mutant complemented with a wild-type netB gene caused significant levels of NE. These data show unequivocally that in this isolate a functional NetB toxin is critical for the ability of C. perfringens to cause NE in chickens. This novel toxin is the first definitive virulence factor to be identified in avian C. perfringens strains capable of causing NE. Furthermore, the netB mutant is the first rationally attenuated strain obtained in an NE-causing isolate of C. perfringens; as such it has considerable vaccine potential.

  8. On the Interaction of Clostridium perfringens Enterotoxin with Claudins

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    Anna Veshnyakova

    2010-06-01

    Full Text Available Clostridium perfringens causes one of the most common foodborne illnesses, which is largely mediated by the Clostridium perfringens enterotoxin (CPE. The toxin consists of two functional domains. The N-terminal region mediates the cytotoxic effect through pore formation in the plasma membrane of the mammalian host cell. The C-terminal region (cCPE binds to the second extracellular loop of a subset of claudins. Claudin-3 and claudin-4 have been shown to be receptors for CPE with very high affinity. The toxin binds with weak affinity to claudin-1 and -2 but contribution of these weak binding claudins to CPE-mediated disease is questionable. cCPE is not cytotoxic, however, it is a potent modulator of tight junctions. This review describes recent progress in the molecular characterization of the cCPE-claudin interaction using mutagenesis, in vitro binding assays and permeation studies. The results promote the development of recombinant cCPE-proteins and CPE-based peptidomimetics to modulate tight junctions for improved drug delivery or to treat tumors overexpressing claudins.

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

    Science.gov (United States)

    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.

  10. Epsilon-toxin plasmids of Clostridium perfringens type D are conjugative.

    Science.gov (United States)

    Hughes, Meredith L; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A; McClane, Bruce A; Rood, Julian I

    2007-11-01

    Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of "unmarked" native epsilon-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional epsilon-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative.

  11. Epsilon-Toxin Plasmids of Clostridium perfringens Type D Are Conjugative▿ †

    Science.gov (United States)

    Hughes, Meredith L.; Poon, Rachael; Adams, Vicki; Sayeed, Sameera; Saputo, Juliann; Uzal, Francisco A.; McClane, Bruce A.; Rood, Julian I.

    2007-01-01

    Isolates of Clostridium perfringens type D produce the potent epsilon-toxin (a CDC/U.S. Department of Agriculture overlap class B select agent) and are responsible for several economically significant enterotoxemias of domestic livestock. It is well established that the epsilon-toxin structural gene, etx, occurs on large plasmids. We show here that at least two of these plasmids are conjugative. The etx gene on these plasmids was insertionally inactivated using a chloramphenicol resistance cassette to phenotypically tag the plasmid. High-frequency conjugative transfer of the tagged plasmids into the C. perfringens type A strain JIR325 was demonstrated, and the resultant transconjugants were shown to act as donors in subsequent mating experiments. We also demonstrated the transfer of “unmarked” native ɛ-toxin plasmids into strain JIR325 by exploiting the high transfer frequency. The transconjugants isolated in these experiments expressed functional ɛ-toxin since their supernatants had cytopathic effects on MDCK cells and were toxic in mice. Using the widely accepted multiplex PCR approach for toxin genotyping, these type A-derived transconjugants were genotypically type D. These findings have significant implications for the C. perfringens typing system since it is based on the toxin profile of each strain. Our study demonstrated the fluid nature of the toxinotypes and their dependence upon the presence or absence of toxin plasmids, some of which have for the first time been shown to be conjugative. PMID:17720791

  12. Identification of a two-component signal transduction system that regulates maltose genes in Clostridium perfringens.

    Science.gov (United States)

    Hiscox, Thomas J; Ohtani, Kaori; Shimizu, Tohru; Cheung, Jackie K; Rood, Julian I

    2014-12-01

    Clostridium perfringens is a Gram-positive rod that is widely distributed in nature and is the etiological agent of several human and animal diseases. The complete genome sequence of C. perfringens strain 13 has been determined and multiple two-component signal transduction systems identified. One of these systems, designated here as the MalNO system, was analyzed in this study. Microarray analysis was used to carry out functional analysis of a malO mutant. The results, which were confirmed by quantitative reverse-transcriptase PCR, indicated that genes putatively involved in the uptake and metabolism of maltose were up-regulated in the malO mutant. These effects were reversed by complementation with the wild-type malO gene. Growth of these isogenic strains in medium with and without maltose showed that the malO mutant recovered more quickly from maltose deprivation when compared to the wild-type and complemented strains, leading to the conclusion that the MalNO system regulates maltose utilization in C. perfringens. It is postulated that this regulatory network may allow this soil bacterium and opportunistic pathogen to respond to environmental conditions where there are higher concentrations of maltose or maltodextrins, such as in the presence of decaying plant material in rich soil. Copyright © 2014 Elsevier Ltd. All rights reserved.

  13. Cellular Entry of Clostridium perfringens Iota-Toxin and Clostridium botulinum C2 Toxin

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    Masaya Takehara

    2017-08-01

    Full Text Available Clostridium perfringens iota-toxin and Clostridium botulinum C2 toxin are composed of two non-linked proteins, one being the enzymatic component and the other being the binding/translocation component. These latter components recognize specific receptors and oligomerize in plasma membrane lipid-rafts, mediating the uptake of the enzymatic component into the cytosol. Enzymatic components induce actin cytoskeleton disorganization through the ADP-ribosylation of actin and are responsible for cell rounding and death. This review focuses upon the recent advances in cellular internalization of clostridial binary toxins.

  14. Four Foodborne Disease Outbreaks Caused by a New Type of Enterotoxin-Producing Clostridium perfringens

    OpenAIRE

    Monma, Chie; Hatakeyama, Kaoru; Obata, Hiromi; Yokoyama, Keiko; Konishi, Noriko; Itoh, Takeshi; Kai, Akemi

    2015-01-01

    The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 an...

  15. Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract

    DEFF Research Database (Denmark)

    Jozefiak, D; Kieronczyk, B; Rawski, M

    2014-01-01

    of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v......The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments...... fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C...

  16. Lyophilized Carnobacterium divergens AS7 bacteriocin preparation improves performance of broiler chickens challenged with Clostridium perfringens

    DEFF Research Database (Denmark)

    Jozefiak, D; Sip, A; Rutkowski, A;

    2012-01-01

    The present study aimed to investigate the effects of Carnobacterium divergens AS7 bacteriocin (divercin AS7) on growth performance, digestibility, fermentation processes, selected microbial populations, and histomorphology in broiler chickens challenged with a mixture of 3 Clostridium perfringens...

  17. Molecular and cellular basis of microvascular perfusion deficits induced by Clostridium perfringens and Clostridium septicum.

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    Michael J Hickey

    2008-04-01

    Full Text Available Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens alpha-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (theta-toxin is less well understood. Similarly, Clostridium septicum is a highly virulent causative agent of spontaneous gas gangrene, but its effect on the microcirculation has not been examined. Therefore, the aim of this study was to use intravital microscopy to examine the effects of C. perfringens and C. septicum on the functional microcirculation, coupled with the use of isogenic toxin mutants to elucidate the role of particular toxins in the resultant microvascular perfusion deficits. This study represents the first time this integrated approach has been used in the analysis of the pathological response to clostridial toxins. Culture supernatants from wild-type C. perfringens induced extensive cell death within 30 min, as assessed by in vivo uptake of propidium iodide. Furthermore, significant reductions in capillary perfusion were observed within 60 min. Depletion of either platelets or neutrophils reduced the alteration in perfusion, consistent with a role for these blood-borne cells in obstructing perfusion. In addition, mutation of either the alpha-toxin or perfringolysin O structural genes attenuated the reduction in perfusion, a process that was reversed by genetic complementation. C. septicum also induced a marked reduction in perfusion, with the degree of microvascular compromise correlating with the level of the C. septicum alpha-toxin. Together, these data indicate that as a result of its ability to produce alpha-toxin and perfringolysin O, C. perfringens rapidly induces irreversible cellular injury and a marked reduction in microvascular perfusion. Since C. septicum induces a similar reduction in microvascular perfusion, it is postulated that this function

  18. Purification and biochemical properties of Clostridium perfringens type A enterotoxin.

    Science.gov (United States)

    Stark, R L; Duncan, C L

    1972-11-01

    The sporulation-specific enterotoxin of Clostridium perfringens type A, which is the toxin active in human food poisoning, has been purified from extracts of sporulating cells. Highly purified enterotoxin was obtained by treatment of crude cell extract with ribonuclease for 30 min, followed by sequential chromatography on Sephadex G-100, Cellex T cellulose, and hydroxylapatite. Recovery was 65 to 75% of the initial activity. Enterotoxin purity was > 99% as indicated by sedimentation velocity, sedimentation equilibrium, disc electrophoresis, and serological methods. Purified enterotoxin focused at pH 4.3 during isoelectric focusing. Molecular weights of 34,000 and 35,000 were obtained by Sephadex G-100 chromatography and sedimentation equilibrium, respectively. An S(20,w) of 3.08 was obtained for the purified enterotoxin. The enterotoxin precipitated heavily at its isoelectric point and at concentrations greater than 4 mg/ml.

  19. Acute Hemolysis in the Emergency Department: Think about Clostridium perfringens!

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    Roustit Cécilia

    2013-01-01

    Full Text Available Clostridium perfringens (CP gives several clinical settings, from an asymptomatic to a massive intravascular hemolysis. We report a case of fatal intravascular hemolysis due to CP septicemia having a hepatic supposed starting point in the emergency department. Like in many cases, the diagnosis was made when patient had already gone into shock and died. The CP septicemia often complicated the course of the digestive or genital pathologies. The alpha toxin can damage the structural integrity of the red cell membrane by means of a phospholipase activity. Nevertheless, a massive intravascular hemolysis arises only rarely in this septicemia, only from 7 to 15% of the cases. The emergency physician has to think about this complication in case of hemoglobinuria and/or signs of hemolysis associated with a septic syndrome. An immediate antibiotic treatment adapted as well as the symptomatic treatment of the spread intravascular coagulation could improve the survival of these patients.

  20. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis.

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    Soshi Seike

    Full Text Available Clostridium perfringens delta-toxin is a β-pore-forming toxin and a putative pathogenic agent of C. perfringens types B and C. However, the mechanism of cytotoxicity of delta-toxin remains unclear. Here, we investigated the mechanisms of cell death induced by delta-toxin in five cell lines (A549, A431, MDCK, Vero, and Caco-2. All cell lines were susceptible to delta-toxin. The toxin caused rapid ATP depletion and swelling of the cells. Delta-toxin bound and formed oligomers predominantly in plasma membrane lipid rafts. Destruction of the lipid rafts with methyl β-cyclodextrin inhibited delta-toxin-induced cytotoxicity and ATP depletion. Delta-toxin caused the release of carboxyfluorescein from sphingomyelin-cholesterol liposomes and formed oligomers; toxin binding to the liposomes declined with decreasing cholesterol content in the liposomes. Flow cytometric assays with annexin V and propidium iodide revealed that delta-toxin treatment induced an elevation in the population of annexin V-negative and propidium iodide-positive cells. Delta-toxin did not cause the fragmentation of DNA or caspase-3 activation. Furthermore, delta-toxin caused damage to mitochondrial membrane permeability and cytochrome c release. In the present study, we demonstrate that delta-toxin produces cytotoxic activity through necrosis.

  1. A novel toxin homologous to large clostridial cytotoxins found in culture supernatant of Clostridium perfringens type C.

    Science.gov (United States)

    Amimoto, Katsuhiko; Noro, Taichi; Oishi, Eiji; Shimizu, Mitsugu

    2007-04-01

    An unknown cytotoxin was identified in the culture supernatant of Clostridium perfringens type C. The cytotoxin, named TpeL, which was purified using mAb-based affinity chromatography, had a lethal activity of 62 minimum lethal dose (MLD) mg(-1) in mice and a cytotoxic activity of 6.2x10(5) cytotoxic units (CU) mg(-1) in Vero cells. The nucleotide sequence of TpeL was determined. The entire ORF had a length of 4953 bases, and the same nucleotide sequence was not recorded in the GenBank/EMBL/DDBJ databases. The molecular mass calculated from the deduced amino acid sequence was 191 kDa, and a signal peptide region was not found within the ORF. The deduced amino acid sequence exhibited 30-39 % homology to Clostridium difficile toxins A (TcdA) and B (TcdB), Clostridium sordellii lethal toxin (TcsL) and Clostridium novyi alpha-toxin (TcnA). The amino acid sequence of TpeL is shorter than these toxins, and the homologous region was located at the N-terminal site. Eighteen strains of C. perfringens types A, B and C were surveyed for the presence of the tpeL gene by PCR. The tpeL gene was detected in all type B (one strain) and C strains (five strains), but not in any type A strains (12 strains). TpeL was detected in culture filtrates of the five type C strains by dot-blot analysis, but not in the type B strain. It was concluded that TpeL is a novel toxin similar to the known large clostridial cytotoxins. Furthermore, the data indicated that TpeL is produced by many C. perfringens type C strains.

  2. Antibiotic resistance of Clostridium perfringens isolates from broiler chickens in Egypt.

    Science.gov (United States)

    Osman, K M; Elhariri, M

    2013-12-01

    The use of antibiotic feed additives in broiler chickens results in a high prevalence of resistance among their enteric bacteria, with a consequent emergence of antibiotic resistance in zoonotic enteropathogens. Despite growing concerns about the emergence of antibiotic-resistant strains, which show varying prevalences in different geographic regions, little work has been done to investigate this issue in the Middle East. This study provides insight into one of the world's most common and financially crippling poultry diseases, necrotic enteritis caused by Clostridium perfringens. The study was designed to determine the prevalence of antibiotic resistance in C. perfringens isolates from clinical cases of necrotic enteritis in broiler chickens in Egypt. A total of 125 isolates were obtained from broiler flocks in 35 chicken coops on 17 farms and were tested using the disc diffusion method. All 125 isolates were resistant to gentamicin, streptomycin, oxolinic acid, lincomycin, erythromycin and spiramycin. The prevalence of resistance to other antibiotics was also high: rifampicin (34%), chloramphenicol (46%), spectinomycin (50%), tylosin-fosfomycin (52%), ciprofloxacin (58%), norfloxacin (67%), oxytetracycline (71%), flumequine (78%), enrofloxacin (82%), neomycin (93%), colistin (94%), pefloxacin (94%), doxycycline (98%) and trimethoprim-sulfamethoxazole (98%). It is recommended that C. perfringens infections in Egypt should be treated with antibiotics for which resistant isolates are rare at present; namely, amoxicillin, ampicillin, cephradine, fosfomycin and florfenicol.

  3. Clostridium perfringens type E enteritis in calves: two cases and a brief review of the literature.

    Science.gov (United States)

    Songer, J Glenn; Miskimmins, Dale W

    2004-08-01

    Toxigenic types of Clostridium perfringens are important causes of enteric disease in domestic animals, although type E is putatively rare, appearing as an uncommon cause of enterotoxemia of lambs, calves, and rabbits. We report here two geographically distinct cases of type E enterotoxemia in calves, and diagnostic findings which suggest that type E may play a significant role in enteritis of neonatal calves. The cases had many similarities, including a history of diarrhea and sudden death, abomasitis, and hemorrhagic enteritis. In both cases, anaerobic cultures of abomasum yielded heavy growth of C. perfringens genotype E. Four percent of > 1000 strains of C. perfringens from cases of enteritis in domestic animals were type E, and all (n=45) were from neonatal calves with hemorrhagic enteritis. Furthermore, type E isolates represented nearly 50% of all isolates submitted from similar clinical cases in calves. Commercial toxoids available in North America have no label claims for efficacy against type E infections. Consideration should be given to type E-associated enteritis when planning for the health care of calves.

  4. Regulation of sialidase production in Clostridium perfringens by the orphan sensor histidine kinase ReeS.

    Directory of Open Access Journals (Sweden)

    Thomas J Hiscox

    Full Text Available Clostridium perfringens is ubiquitous in nature and is often found as a commensal of the human and animal gastrointestinal tract. It is the primary etiological agent of clostridial myonecrosis, or gas gangrene, a serious infection that results in extensive tissue necrosis due to the action of one or more potent extracellular toxins. α-toxin and perfringolysin O are the major extracellular toxins involved in the pathogenesis of gas gangrene, but histotoxic strains of C. perfringens, such as strain 13, also produce many degradative enzymes such as collagenases, hyaluronidases, sialidases and the cysteine protease, α-clostripain. The production of many of these toxins is regulated either directly or indirectly by the global VirSR two-component signal transduction system. By isolating a chromosomal mutant and carrying out microarray analysis we have identified an orphan sensor histidine kinase, which we have named ReeS (regulator of extracellular enzymes sensor. Expression of the sialidase genes nanI and nanJ was down-regulated in a reeS mutant. Since complementation with the wild-type reeS gene restored nanI and nanJ expression to wild-type levels, as shown by quantitative reverse transcription-PCR and sialidase assays we concluded that ReeS positively regulates the expression of these sialidase genes. However, mutation of the reeS gene had no significant effect on virulence in the mouse myonecrosis model. Sialidase production in C. perfringens has been previously shown to be regulated by both the VirSR system and RevR. In this report, we have analyzed a previously unknown sensor histidine kinase, ReeS, and have shown that it also is involved in controlling the expression of sialidase genes, adding further complexity to the regulatory network that controls sialidase production in C. perfringens.

  5. Comparative transcriptome analysis by RNAseq of necrotic enteritis Clostridium perfringens during in vivo colonization and in vitro conditions.

    Science.gov (United States)

    Parreira, Valeria R; Russell, Kay; Athanasiadou, Spiridoula; Prescott, John F

    2016-08-12

    Necrotic enteritis (NE) caused by netB-positive type A Clostridium perfringens is an important bacterial disease of poultry. Through its complex regulatory system, C. perfringens orchestrates the expression of a collection of toxins and extracellular enzymes that are crucial for the development of the disease; environmental conditions play an important role in their regulation. In this study, and for the first time, global transcriptomic analysis was performed on ligated intestinal loops in chickens colonized with a netB-positive C. perfringens strain, as well as the same strain propagated in vitro under various nutritional and environmental conditions. Analysis of the respective pathogen transcriptomes revealed up to 673 genes that were significantly expressed in vivo. Gene expression profiles in vivo were most similar to those of C. perfringens grown in nutritionally-deprived conditions. Taken together, our results suggest a bacterial transcriptome responses to the early stages of adaptation, and colonization of, the chicken intestine. Our work also reveals how netB-positive C. perfringens reacts to different environmental conditions including those in the chicken intestine.

  6. Four foodborne disease outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens.

    Science.gov (United States)

    Monma, Chie; Hatakeyama, Kaoru; Obata, Hiromi; Yokoyama, Keiko; Konishi, Noriko; Itoh, Takeshi; Kai, Akemi

    2015-03-01

    The epidemiological and bacteriological investigations on four foodborne outbreaks caused by a new type of enterotoxin-producing Clostridium perfringens are described. C. perfringens isolated from patients of these outbreaks did not produce any known enterotoxin and did not carry the C. perfringens enterotoxin gene. However, the culture filtrates of these isolates induced the accumulation of fluid in rabbit ileal loop tests. The molecular weight of the new enterotoxin may be between 50,000 and 100,000, although the known C. perfringens enterotoxin is ca. 35,000. This new enterotoxin was heat labile, and its biological activities were inactivated by heating for 5 min at 60°C. The new enterotoxin was sensitive to pH values higher than 11.0 and protease treatment but was resistant to trypsin treatment. These results suggest that the new enterotoxin may be a protein. Although C. perfringens enterotoxin induced morphological changes in Vero cells, the changes induced by the new enterotoxin differed from those by the known C. perfringens enterotoxin. The new enterotoxin also induced morphological changes in L929 cells, whereas the known C. perfringens enterotoxin did not, because L929 cells lacked an appropriate enterotoxin receptor. Although C. perfringens enterotoxin is recognized as the only diarrheagenic toxin responsible for C. perfringens foodborne outbreaks, the results of the present study indicate that C. perfringens isolated from these four outbreaks produced a new type of enterotoxin.

  7. The NEAT Domain-Containing Proteins of Clostridium perfringens Bind Heme.

    Science.gov (United States)

    Choo, Jocelyn M; Cheung, Jackie K; Wisniewski, Jessica A; Steer, David L; Bulach, Dieter M; Hiscox, Thomas J; Chakravorty, Anjana; Smith, A Ian; Gell, David A; Rood, Julian I; Awad, Milena M

    2016-01-01

    The ability of a pathogenic bacterium to scavenge iron from its host is important for its growth and survival during an infection. Our studies on C. perfringens gas gangrene strain JIR325, a derivative of strain 13, showed that it is capable of utilizing both human hemoglobin and ferric chloride, but not human holo-transferrin, as an iron source for in vitro growth. Analysis of the C. perfringens strain 13 genome sequence identified a putative heme acquisition system encoded by an iron-regulated surface gene region that we have named the Cht (Clostridium perfringens heme transport) locus. This locus comprises eight genes that are co-transcribed and includes genes that encode NEAT domain-containing proteins (ChtD and ChtE) and a putative sortase (Srt). The ChtD, ChtE and Srt proteins were shown to be expressed in JIR325 cells grown under iron-limited conditions and were localized to the cell envelope. Moreover, the NEAT proteins, ChtD and ChtE, were found to bind heme. Both chtDE and srt mutants were constructed, but these mutants were not defective in hemoglobin or ferric chloride utilization. They were, however, attenuated for virulence when tested in a mouse myonecrosis model, although the virulence phenotype could not be restored via complementation and, as is common with such systems, secondary mutations were identified in these strains. In summary, this study provides evidence for the functional redundancies that occur in the heme transport pathways of this life threatening pathogen.

  8. Characterization of Clostridium perfringens isolates obtained from 2010 to 2012 from chickens with necrotic enteritis in Korea.

    Science.gov (United States)

    Park, Ji Young; Kim, Sara; Oh, Jae Young; Kim, Hye Ryoung; Jang, Il; Lee, Hee Soo; Kwon, Yong Kuk

    2015-06-01

    Clostridium perfringens produces diverse virulent toxins that cause necrotic enteritis in poultry, resulting in a great negative impact on the poultry industry. To study the characteristics of C. perfringens in chickens, we isolated 88 strains from chickens (1 strain per flock) with necrotic enteritis. The isolated bacterial strains were screened for toxin type and antimicrobial susceptibility. Necropsy of 17 chickens that died from necrotic enteritis revealed that their intestines were dilated with inflammatory exudates and characterized by mucosal necrosis. All the isolated strains were identified as toxin type A using multiplex PCR for toxin typing. We found that the rate of netB-positive strains isolated from dead chickens was significantly higher (8 of 17) than the rate among healthy chickens (2 of 50). We performed antimicrobial susceptibility test with 20 selected antimicrobial agents using the disk diffusion test and found that 30 tested strains were completely resistant to 5 antibiotics and partially resistant to 6 antibiotics whereas all the strains were susceptible to 9 antimicrobial agents. Using pulsed-field gel electrophoresis analysis, the 17 strains were divided into 13 genetic clusters showing high genetic diversity. In conclusion, C. perfringens strains isolated from Korean poultry showed a high resistance to antimicrobial drugs and high genetic diversity, suggesting that continuous monitoring is essential to prevent outbreaks of necrotic enteritis in chickens.

  9. Effect of a probiotic on prevention of diarrhea and Clostridium difficile and Clostridium perfringens shedding in foals

    DEFF Research Database (Denmark)

    Schoster, Angelika; Staempfli, H R; Abrahams, M;

    2015-01-01

    of incidence and duration of diarrhea and fecal shedding of Clostridium perfringens and Clostridium difficile between treatment and age groups. RESULTS: The overall incidence of diarrhea was 41 of 72 (59%) and did not differ (P = 0.37) between treatment groups. Foals treated with probiotics were more likely...... of C. perfringens shedding was 55% with no difference between treatment groups (P = 0.23). The prevalence of C. difficile shedding was 11%. CONCLUSION AND CLINICAL IMPORTANCE: There was no benefit of administering a 3-week course of probiotics, but potential adverse effects were noted. Whether...

  10. Clostridium perfringens type A netF and netE positive and Clostridium difficile co-infection in two adult dogs.

    Science.gov (United States)

    Diniz, Amanda Nádia; Silva, Rodrigo Otávio Silveira; Oliveira Junior, Carlos Augusto; Pierezan, Felipe; Lobato, Francisco Carlos Faria

    2016-04-01

    The aim of this study was to report two cases of Clostridium perfringens type A and Clostridium difficile co-infection in adult dogs. Both animals were positive for A/B toxin. Toxigenic C. difficile and C. perfringens type A positive for NetE and NetF-encoding genes were isolated. This report reinforces the necessity of studying a possible synergism of C. difficile and C. perfringens in enteric disorders.

  11. Differential responses of cecal microbiota to fishmeal, Eimeria and Clostridium perfringens in a necrotic enteritis challenge model in chickens.

    Science.gov (United States)

    Stanley, Dragana; Wu, Shu-Biao; Rodgers, Nicholas; Swick, Robert A; Moore, Robert J

    2014-01-01

    Clostridium perfringens causes enteric diseases in animals and humans. In poultry, avian-specific C. perfringens strains cause necrotic enteritis, an economically significant poultry disease that costs the global industry over $2 billion annually in losses and control measures. With removal of antibiotic growth promoters in some countries this disease appears to be on the rise. In experimental conditions used to study disease pathogenesis and potential control measures, reproduction of the disease relies on the use of predisposing factors such as Eimeria infection and the use of high protein diets, indicating complex mechanisms involved in the onset of necrotic enteritis. The mechanisms by which the predisposing factors contribute to disease progression are not well understood but it has been suggested that they may cause perturbations in the microbiota within the gastrointestinal tract. We inspected changes in cecal microbiota and short chain fatty acids (SCFA) induced by Eimeria and fishmeal, in birds challenged or not challenged with C. perfringens. C. perfringens challenge in the absence of predisposing factors did not cause significant changes in either the alpha or beta diversity of the microbiota nor in concentrations of SCFA. Moreover, there was no C. perfringens detected in the cecal microbiota 2 days post-challenge without the presence of predisposing factors. In contrast, both fishmeal and Eimeria caused significant changes in microbiota, seen in both alpha and beta diversity and also enabled C. perfringens to establish itself post challenge. Eimeria had its strongest influence on intestinal microbiota and SCFA when combined with fishmeal. Out of 6 SCFAs measured, including butyric acid, none were significantly influenced by C. perfringens, but their levels were strongly modified following the use of both predisposing factors. There was little overlap in the changes caused following Eimeria and fishmeal treatments, possibly indicating multiple routes for

  12. Prevalence of Clostridium perfringens type A isolates in different tissues of broiler chickens

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    A. Doosti

    2017-03-01

    Full Text Available Clostridium perfringens (C. perfringens is an important pathogen in both human and veterinary medicine. Necrotic enteritis is the most severe clinical bacterial enteric disease of poultry induced by C. perfringens.Study was done on 100 broiler chickens (400 different tissues of chickens in Southwest Iran.C. perfringens was isolated from different tissues of chickens (meats, liver, gizzard and intestine using bacterial culture methods. DNA extraction from grown colonies was carried out by boiling method and finally, PCR assay was used for definitive diagnosis of type AC. perfringens. The results of present study showed that from 400 different tissues of chickens 169(42.25%samples were positive for C. perfringens infection that is a comparatively high prevalence of C. perfringens. The highest rate of C. perfringens infection in tissues was in intestine (55% and meat 42%. The high prevalence of type A C. perfringens in different tissues observed in the present study is very disturbing, as it can cause irreparable damage to the poultry industry and human health.

  13. The interaction of Clostridium perfringens enterotoxin with receptor claudins.

    Science.gov (United States)

    Shrestha, Archana; Uzal, Francisco A; McClane, Bruce A

    2016-10-01

    Clostridium perfringens enterotoxin (CPE) has significant medical importance due to its involvement in several common human gastrointestinal diseases. This 35 kDa single polypeptide toxin consists of two domains: a C-terminal domain involved in receptor binding and an N-terminal domain involved in oligomerization, membrane insertion and pore formation. The action of CPE starts with its binding to receptors, which include certain members of the claudin tight junction protein family; bound CPE then forms a series of complexes, one of which is a pore that causes the calcium influx responsible for host cell death. Recent studies have revealed that CPE binding to claudin receptors involves interactions between the C-terminal CPE domain and both the 1st and 2nd extracellular loops (ECL-1 and ECL-2) of claudin receptors. Of particular importance for this binding is the docking of ECL-2 into a pocket present in the C-terminal domain of the toxin. This increased understanding of CPE interactions with claudin receptors is now fostering the development of receptor decoy therapeutics for CPE-mediated gastrointestinal disease, reagents for cancer therapy/diagnoses and enhancers of drug delivery.

  14. Comparison of methods for the enumeration of Clostridium perfringens spores in water.

    Science.gov (United States)

    Junqueira, Valéria Christina Amstalden; Neto, Romeu Cantúsio; da Silva, Neusely; Terra, Juliana Hirata

    2012-01-01

    Four methods for enumerating Clostridium perfringens spores in water were evaluated: (1) the IMM (Iron Milk Medium) method (MPN); (2) the LS (Lactose Sulfite Broth) method (MPN); (3) the m-CP (membrane filtration Clostridium perfringens Agar) method (membrane filtration); and (4) the TSC (Tryptose Sulfite Cycloserine Agar) method (membrane filtration). The performance of these methods was compared with that of the DRCM (Differential Reinforced Clostridium Medium) method (MPN) as adopted by CETESB (Brazil's Environmental Sanitation Technology Company) for the analysis of C. perfringens spores in water. Statistical analysis was performed according to ISO 17994:2004 (Water Quality - Criteria for Establishing Equivalence between Microbiological Methods). The LS, m-CP, and TSC methods were considered not equivalent to the DRCM method, as they gave significantly lower results. The IMM showed inconclusive results and, according to ISO 17994:2004, analysis of a greater number of samples is needed to draw definitive conclusions comparing IMM and DRCM.

  15. Sudden death syndrome in adult cows associated with Clostridium perfringens type E.

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    Redondo, L M; Farber, M; Venzano, A; Jost, B H; Parma, Y R; Fernandez-Miyakawa, M E

    2013-04-01

    Clostridium perfringens type E is considered a rare toxinotype and an infrequent cause of enterotoxemia of lambs, calves, and rabbits. Until now, only cases of young animal of C. perfringens type E bovine enterotoxemia, characterized by hemorrhagic enteritis and sudden death, have been reported. The present report details the genotypic characterization of C. perfringens type E isolates obtained from intestinal samples of adult cattle during an outbreak of enterotoxemia in Argentina. The sequences of several housekeeping genes of these isolates were analyzed and compared with those obtained from calves in North America showing a clonal unique lineage.

  16. Oral immunization of mice against Clostridium perfringens epsilon toxin with a Lactobacillus casei vector vaccine expressing epsilon toxoid.

    Science.gov (United States)

    Alimolaei, Mojtaba; Golchin, Mehdi; Daneshvar, Hamid

    2016-06-01

    Clostridium perfringens type D infects ruminants and causes the enterotoxemia disease by ε-toxin. A mutated ε-toxin gene lacking toxicity was designed, synthesized, and cloned into the pT1NX vector and electroporated into Lactobacillus casei competent cells to yield LC-pT1NX-ε recombinant strain. BALB/c mice, immunized orally with this strain, highly induced mucosal, humoral, and cell-mediated immune responses and developed a protection against 200 MLD/ml of the activated ε-toxin. This study showed that the LC-pT1NX-ε could be a promising vaccine candidate against the enterotoxemia disease.

  17. Growth potential of Clostridium perfringens from spores in acidified beef, pork, and poultry products during chilling.

    Science.gov (United States)

    Juneja, Vijay K; Baker, David A; Thippareddi, H; Snyder, O Peter; Mohr, Tim B

    2013-01-01

    The ability of Clostridium perfringens to germinate and grow in acidified ground beef as well as in 10 commercially prepared acidified beef, pork, and poultry products was assessed. The pH of ground beef was adjusted with organic vinegar to achieve various pH values between 5.0 and 5.6; the pH of the commercial products ranged from 4.74 to 6.35. Products were inoculated with a three-strain cocktail of C. perfringens spores to achieve ca. 2-log (low) or 4-log (high) inoculum levels, vacuum packaged, and cooled exponentially from 54.4 to 7.2°C for 6, 9, 12, 15, 18, or 21 h to simulate abusive cooling; the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS) recommends a cooling time of 6.5 h. Total germinated C. perfringens populations were determined after plating on tryptose-sulfite-cycloserine agar and incubating the plates anaerobically at 37°C for 48 h. In addition, C. perfringens growth from spores was assessed at an isothermal temperature of 44°C. Growth from spores was inhibited in ground beef with a pH of 5.5 or below, even during extended cooling from 54.4 to 7.2°C in 21 h. In ground beef with a pH of 5.6, the growth was >1 log after 18 h of cooling from 54.4 to 7.2°C. However, 15 h of cooling controlled the growth to product with a pH ranging from 4.74 to 5.17, both during exponential abusive cooling periods of up to 21 h and during storage for 21 h at 44°C. While product cooled exponentially from 54.4 to 7.2°C in 15 h or less, the pH 6.35 product supported growth, even after 6 h of cooling from 54.4 to 7.2°C. These challenge tests demonstrate that adjustment of ground beef to pH of 5.5 or less and of barbeque products to pH of 5.63 or less inhibits C. perfringens spore germination and outgrowth during extended cooling periods from 54.4 to 7.2°C up to 15 h. Therefore, safe cooling periods for products with homogeneous, lower pHs can be substantially longer.

  18. Clostridium perfringens bacteremia caused by choledocholithiasis in the absence of gallbladder stones

    Institute of Scientific and Technical Information of China (English)

    Antwan Atia; Tejas Raiyani; Pranav Patel; Robert Patton; Mark Young

    2012-01-01

    A 67-years-old male presented with periumbilical abdominal pain,fever and jaundice.His anaerobic blood culture was positive for clostridium perfringens.Computed tomogram scan of the abdomen and abdominal ultrasound showed normal gallbladder and common bile duct (CBD).Subsequently magnetic resonance cholangiopancreaticogram showed choledocholithiasis.Endoscopic retrograde cholangiopancreaticogramwith sphincterotomy and CBD stone extraction was performed.The patient progressively improved with antibiotic therapy Choledocholithiasis should be considered as a source of clostridium perfringens bacteremia especially in the setting of elevated liver enzymes with cholestatic pattern.

  19. Antimicrobial activities of six essential oils commonly used as condiments in Brazil against Clostridium perfringens

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    Marcela Radaelli

    2016-06-01

    Full Text Available Abstract Despite recent advances in food production technology, food-borne diseases (FBD remain a challenging public health concern. In several countries, including Brazil, Clostridium perfringens is among the five main causative agents of food-borne diseases. The present study determines antimicrobial activities of essential oils of six condiments commonly used in Brazil, viz., Ocimum basilicum L. (basil, Rosmarinus officinalis L. (rosemary, Origanum majorana L. (marjoram, Mentha × piperita L. var. Piperita (peppermint, Thymus vulgaris L. (thyme and Pimpinella anisum L. (anise against C. perfringens strain A. Chemical compositions of the oils were determined by GC–MS (gas chromatography–mass spectrometry. The identities of the isolated compounds were established from the respective Kováts indices, and a comparison of mass spectral data was made with those reported earlier. The antibacterial activity was assessed from minimum inhibitory concentration (MIC and minimum bactericidal concentration (MBC using the microdilution method. Minimum inhibitory concentration values were 1.25 mg mL-1 for thyme, 5.0 mg mL-1 for basil and marjoram, and 10 mg mL-1 for rosemary, peppermint and anise. All oils showed bactericidal activity at their minimum inhibitory concentration, except anise oil, which was only bacteriostatic. The use of essential oils from these common spices might serve as an alternative to the use of chemical preservatives in the control and inactivation of pathogens in commercially produced food systems.

  20. Antimicrobial activities of six essential oils commonly used as condiments in Brazil against Clostridium perfringens.

    Science.gov (United States)

    Radaelli, Marcela; da Silva, Bárbara Parraga; Weidlich, Luciana; Hoehne, Lucélia; Flach, Adriana; da Costa, Luiz Antonio Mendonça Alves; Ethur, Eduardo Miranda

    2016-01-01

    Despite recent advances in food production technology, food-borne diseases (FBD) remain a challenging public health concern. In several countries, including Brazil, Clostridium perfringens is among the five main causative agents of food-borne diseases. The present study determines antimicrobial activities of essential oils of six condiments commonly used in Brazil, viz., Ocimum basilicum L. (basil), Rosmarinus officinalis L. (rosemary), Origanum majorana L. (marjoram), Mentha × piperita L. var. Piperita (peppermint), Thymus vulgaris L. (thyme) and Pimpinella anisum L. (anise) against C. perfringens strain A. Chemical compositions of the oils were determined by GC-MS (gas chromatography-mass spectrometry). The identities of the isolated compounds were established from the respective Kováts indices, and a comparison of mass spectral data was made with those reported earlier. The antibacterial activity was assessed from minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) using the microdilution method. Minimum inhibitory concentration values were 1.25mgmL(-1) for thyme, 5.0mgmL(-1) for basil and marjoram, and 10mgmL(-1) for rosemary, peppermint and anise. All oils showed bactericidal activity at their minimum inhibitory concentration, except anise oil, which was only bacteriostatic. The use of essential oils from these common spices might serve as an alternative to the use of chemical preservatives in the control and inactivation of pathogens in commercially produced food systems. Copyright © 2016 Sociedade Brasileira de Microbiologia. Published by Elsevier Editora Ltda. All rights reserved.

  1. Adhesive properties of Clostridium perfringens to extracellular matrix proteins collagens and fibronectin.

    Science.gov (United States)

    Hitsumoto, Yasuo; Morita, Naomi; Yamazoe, Ryosuke; Tagomori, Mika; Yamasaki, Tsutomu; Katayama, Seiichi

    2014-02-01

    The adhesive properties of Clostridium perfringens to collagens, gelatin, fibronectin (Fn), Fn-prebound collagens, and Fn-prebound gelatin were investigated. C. perfringens could bind to Fn-prebound collagen type II, type III, and gelatin, but not to gelatin or collagens except for collagen type I directly. Recombinant Fn-binding proteins of C. perfringens, rFbpA and rFbpB, were used to examine Fn-mediated bacterial adherence to collagen type I. In the presence of rFbps, C. perfringens adherence to Fn-prebound collagen type I was inhibited in a dose-dependent manner. Fn was not released from the coated collagen type I by the presence of rFbps, and rFbps did not bind to collagen type I. Thus, the inhibition of C. perfringens binding to Fn-prebound collagen type I by rFbps could not be explained by the removal of Fn from collagen or by the competitive binding of rFbps to collagen. Instead, both rFbps were found to bind to C. perfringens. These results suggest the possibility that rFbps may bind to the putative Fn receptor expressed on C. perfringens and competitively inhibit Fn binding to C. perfringens.

  2. Occurrence of Clostridium perfringens Types A, E, and C in Fresh Fish and Its Public Health Significance.

    Science.gov (United States)

    Sabry, Maha; Abd El-Moein, Khaled; Hamza, Eman; Abdel Kader, Fatma

    2016-06-01

    Fish remains among the most traded of food commodities, and Egypt is one of the emerging countries being recognized as an important world fish exporter. Clostridium perfringens is an important foodborne pathogen to consider in fish trade, as it has been implicated as the causative organism of two fish outbreaks. The aim of the present study was to investigate the occurrence and toxin diversity of C. perfringens associated with fresh and canned fish and to examine the public health significance of C. perfringens infection in fish. Isolation and identification of C. perfringens showed a significantly higher prevalence of the bacterium in fresh fish collected from aquaculture (54.5%) and from markets (71%) as well as in humans in contact with fish (63%) compared with water used for keeping fresh fish (27.3%) and water used in canned fish (17.8%). The isolation level was significantly higher in samples from the external surface of fresh fish (31.8% in aquaculture, 45.6% in markets) than from the intestinal contents of the same fish (9.1% in aquaculture, 6.7% in markets). Thus, markets represent a risk factor for contamination of the external surface of fish from the surrounding environment. Genotyping of the C. perfringens-positive isolates by using multiplex PCR revealed that type A enterotoxin-negative (CPE(-)) is the predominant strain among fish (fresh and canned), humans, and water in contact with fresh fish. Interestingly, C. perfringens types A enterotoxin-positive (CPE(+)) and C were found only in fresh fish, and these two strains have great health importance in humans. Strikingly, C. perfringens type E strain was detected for the first time in fish, humans, and water in contact with fresh fish. Our results demonstrate for the first time that fish act as a reservoir for C. perfringens, particularly for types A CPE(+), C, and E. The external surface of fish represents a vehicle for contamination of fish from the surrounding environment as well as a source of

  3. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

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    Archana Shrestha

    2016-12-01

    Full Text Available Clostridium perfringens enterotoxin (CPE binds to claudin receptors, e.g., claudin-4, and then forms a pore that triggers cell death. Pure cultures of host cells that do not express claudin receptors, e.g., fibroblasts, are unaffected by pathophysiologically relevant CPE concentrations in vitro. However, both CPE-insensitive and CPE-sensitive host cells are present in vivo. Therefore, this study tested whether CPE treatment might affect fibroblasts when cocultured with CPE-sensitive claudin-4 fibroblast transfectants or Caco-2 cells. Under these conditions, immunofluorescence microscopy detected increased death of fibroblasts. This cytotoxic effect involved release of a toxic factor from the dying CPE-sensitive cells, since it could be reproduced using culture supernatants from CPE-treated sensitive cells. Supernatants from CPE-treated sensitive cells, particularly Caco-2 cells, were found to contain high levels of membrane vesicles, often containing a CPE species. However, most cytotoxic activity remained in those supernatants even after membrane vesicle depletion, and CPE was not detected in fibroblasts treated with supernatants from CPE-treated sensitive cells. Instead, characterization studies suggest that a major cytotoxic factor present in supernatants from CPE-treated sensitive cells may be a 10- to 30-kDa host serine protease or require the action of that host serine protease. Induction of caspase-3-mediated apoptosis was found to be important for triggering release of the cytotoxic factor(s from CPE-treated sensitive host cells. Furthermore, the cytotoxic factor(s in these supernatants was shown to induce a caspase-3-mediated killing of fibroblasts. This bystander killing effect due to release of cytotoxic factors from CPE-treated sensitive cells could contribute to CPE-mediated disease.

  4. Enteric Diseases of Poultry with Special Attention to Clostridium perfringens

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    Hafez Mohamed Hafez

    2011-06-01

    Full Text Available The enteric heath of growing poultry is imperative to success of the production. The basic role of poultry production is turning feed stuffs into meat. Any changes in this turning process, due to mechanical, chemical or biological disturbance of digestive system (enteric disorders is mostly accompanied with high economic losses due to poor performance, increased mortality rates and increased medication costs. The severity of clinical signs and course of the disorders are influenced several factors such as management, nutrition and the involved agent(s. Several pathogens (viruses, bacteria and parasites are incriminated as possible cause of enteric disorders either alone (mono-causal, in synergy with other micro-organisms (multi-causal, or with non-infectious causes such as feed and /or management related factors. In addition, excessive levels of mycotoxins and biogenic amines in feed lead to enteric disorders. Also factors such as high stocking density, poor litter conditions, poor hygiene and high ammonia level and other stressful situation may reduce the resistance of the birds and increases their susceptibility to infections. Under field conditions, however, under filed conditions it is difficult to determine whether the true cause of enteric disorders, is of infectious or non-infectious origin. In recent years and since the ban of use of antimicrobial growth promoters in several countries the incidence of intestinal disorders especially those caused by clostridial infection was drastically increased. The present review described in general the several factors involved in enteric disorders and summarized the available literatures about Clostridium perfringens infection in poultry.

  5. Global regulation of gene expression in response to cysteine availability in Clostridium perfringens

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    André Gaelle

    2010-09-01

    Full Text Available Abstract Background Cysteine has a crucial role in cellular physiology and its synthesis is tightly controlled due to its reactivity. However, little is known about the sulfur metabolism and its regulation in clostridia compared with other firmicutes. In Clostridium perfringens, the two-component system, VirR/VirS, controls the expression of the ubiG operon involved in methionine to cysteine conversion in addition to the expression of several toxin genes. The existence of links between the C. perfringens virulence regulon and sulfur metabolism prompted us to analyze this metabolism in more detail. Results We first performed a tentative reconstruction of sulfur metabolism in C. perfringens and correlated these data with the growth of strain 13 in the presence of various sulfur sources. Surprisingly, C. perfringens can convert cysteine to methionine by an atypical still uncharacterized pathway. We further compared the expression profiles of strain 13 after growth in the presence of cystine or homocysteine that corresponds to conditions of cysteine depletion. Among the 177 genes differentially expressed, we found genes involved in sulfur metabolism and controlled by premature termination of transcription via a cysteine specific T-box system (cysK-cysE, cysP1 and cysP2 or an S-box riboswitch (metK and metT. We also showed that the ubiG operon was submitted to a triple regulation by cysteine availability via a T-box system, by the VirR/VirS system via the VR-RNA and by the VirX regulatory RNA. In addition, we found that expression of pfoA (theta-toxin, nagL (one of the five genes encoding hyaluronidases and genes involved in the maintenance of cell redox status was differentially expressed in response to cysteine availability. Finally, we showed that the expression of genes involved in [Fe-S] clusters biogenesis and of the ldh gene encoding the lactate dehydrogenase was induced during cysteine limitation. Conclusion Several key functions for the

  6. Bacillus subtilis and yeast cell wall improve the intestinal health of broilers challenged by Clostridium perfringens.

    Science.gov (United States)

    Li, Z; Wang, W; Lv, Z; Liu, D; Guo, Y

    2017-08-25

    1. The objective was to investigate the effects of Bacillus subtilis, yeast cell wall (YCW) and their combination on intestinal health of broilers challenged by Clostridium perfringens over a 21-d period. 2. Using a 5 × 2 factorial arrangement of treatments, 800 1-d-old male Cobb 500 broilers were used to study the effects of feed additives (without additive or with zinc bacitracin, B. subtilis, YCW, and the combination of B. subtilis and YCW), pathogen challenge (without or with Clostridium perfringens challenge), and their interactive effects. 3. C. perfringens infection increased intestinal lesions scores, damaged intestinal histomorphology, increased serum endotoxin concentration, cytokine mRNA expression and intestinal population of C. perfringens and Escherichia coli, and decreased ileal Bifidobacteria numbers. The 4 additives decreased serum endotoxin. Zinc bacitracin tended to decrease cytokine mRNA expression and the intestinal number of C. perfringens and E. coli. B. subtilis, YCW and their combination increased cytokine mRNA expression. B. subtilis and YCW decreased the number of C. perfringens and E. coli in the ileum, and their combination decreased pathogens numbers in the ileum and caecum. 4. In conclusion, B. subtilis, YCW and their combination improved the intestinal health of NE-infected broilers, and could be potential alternatives to antibiotics.

  7. Characterization of Clostridium perfringens isolated from mammals and birds from Guwahati city, India

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    Mafruza S Rahman

    2012-01-01

    Full Text Available Of the 102 samples collected from mammals and birds, both domestic and captive wild, 48 were found to be positive for Clostridium perfringens. Most of the mammal isolates (84.38% appeared to have been collected from clinically affected animals, while 33.33% of the bird samples were from clinically affected and 21.43% from apparently healthy birds infected with C. perfringens. Isolates revealed high sensitivity to ciprofloxacin, enrofloxacin and norfloxacin. Among the isolated C. perfringens, 30 (62.50% showed DNase production. Hemolytic activity was recorded in 14 (24.16% of the isolates and 28 (58.33% showed phospholipase C production. All the phospholipase C positive isolates revealed the presence of cpa gene encoding alpha (α toxin. Of the 102 samples collected from mammals and birds, both domestic and captive wild, 48 were found to be positive for Clostridium perfringens. Most of the mammal isolates (84.38% appeared to have been collected from clinically affected animals, while 33.33% of the bird samples were from clinically affected and 21.43% from apparently healthy birds infected with C. perfringens. Isolates revealed high sensitivity to ciprofloxacin, enrofloxacin and norfloxacin. Among the isolated C. perfringens, 30 (62.50% showed DNase production. Hemolytic activity was recorded in 14 (24.16% of the isolates and 28 (58.33% showed phospholipase C production. All the phospholipase C positive isolates revealed the presence of cpa gene encoding α toxin.

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

    Science.gov (United States)

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

    2016-04-01

    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.

  9. Evaluation of the microbial quality of Tajik sambusa and control of Clostridium perfringens germination and outgrowth by buffered sodium citrate and potassium lactate.

    Science.gov (United States)

    Yarbaeva, Shakhlo N; Velugoti, Padmanabha R; Thippareddi, Harshavardhan; Albrecht, Julie A

    2008-01-01

    Clostridium perfringens spore destruction, aerobic plate counts (APCs), and counts of Enterobacteriaceae, coliforms, and Escherichia coli during baking of sambusa (a traditional Tajik food) were evaluated. Control of germination and outgrowth of C. perfringens spores in sambusa during cooling at room or refrigerated temperatures was evaluated using organic acid salts (buffered sodium citrate [Ional] and 1 and 2% potassium lactate, wt/wt). Sambusa were prepared with 40 g of either inoculated or noninoculated meat and baked for 45 min at 180 degrees C. For evaluation of destruction of C. perfringens spores during heating and germination and outgrowth of spores during cooling, ground beef was inoculated and mixed with a three-strain cocktail of C. perfringens spores. Aerobic bacteria, Enterobacteriaceae, coliforms, and E. coli were enumerated in noninoculated sambusa before and after baking and after cooling at room or refrigeration temperatures. After baking, APCs and Enterobacteriaceae and coliform counts were reduced by 4.32, 2.55, and 1.96 log CFU/g, respectively. E. coli counts were below detectable levels in ground beef and sambusa samples. Enterobacteriaceae, coliform, and E. coli counts were below detectable levels (sodium citrate controlled C. perfringens spore germination and outgrowth (0.25 log CFU/g), whereas incorporation of up to 2% (wt/wt) potassium lactate did not prevent C. perfringens spore germination and outgrowth. Incorporation of organic acid salts at appropriate concentrations can prevent germination and outgrowth of C. perfringens in improperly cooled sambusa.

  10. NetB-producing and beta2-producing Clostridium perfringens associated with subclinical necrotic enteritis in laying hens in the Netherlands.

    Science.gov (United States)

    Allaart, Janneke G; de Bruijn, Naomi D; van Asten, Alphons J A M; Fabri, Teun H F; Gröne, Andrea

    2012-12-01

    Since 2006 increasing numbers of laying hen flocks with decreased production have been reported in the Netherlands. At necropsy, birds from affected flocks showed multifocal areas of necrosis in the duodenum. Histologically the duodenum had moderate to marked villus atrophy and fusion with crypt hyperplasia and a mixed inflammatory infiltrate within the lamina propria underlying focal areas of degenerative epithelium. Multifocally, free within the intestinal lumen and associated with epithelial necrosis, were marked numbers of large rod-shaped bacteria. Anaerobic culturing and subsequent toxin typing revealed, in 19 out of 73 affected birds, the presence of Clostridium perfringens strains, either type A or type C harbouring the atypical allele of cpb2 and netB. Eighteen out of these 19 birds carried C. perfringens strains capable of producing beta2 toxin in vitro and all of these birds harboured C. perfringens strains capable of producing NetB toxin in vitro. In contrast, specific pathogen free (SPF) birds lacked gross or histological lesions in their duodenum, and C. perfringens type C was isolated from four out of 15 SPF birds tested. One of these isolates harboured the consensus three allele of cpb2 that produced beta2 toxin in vitro. None of the C. perfringens isolates originating from SPF birds harboured netB. These findings might indicate that the NetB toxin produced by C. perfringens is associated with subclinical necrotic enteritis in layers, whereas the involvement of beta2 toxin in subclinical necrotic enteritis, if any, might be variant dependent.

  11. Tolerance of Clostridium perfringens biofilms to disinfectants commonly used in the food industry.

    Science.gov (United States)

    Charlebois, Audrey; Jacques, Mario; Boulianne, Martine; Archambault, Marie

    2017-04-01

    Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Recently, it was shown to form mono-species biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. Biofilms have been associated with tolerance to antibiotics, disinfectants, and physical and environmental stresses. Very little is known about the tolerance of C. perfringens biofilm toward disinfectants. In the present study, susceptibilities of C. perfringens biofilms to five types of commonly used disinfectants on farms and in food processing environments were analysed. In this paper, we show that C. perfringens mono-species biofilms can protect the bacterial cells from the action of potassium monopersulfate, quaternary ammonium chloride, hydrogen peroxide and glutaraldehyde solutions. However, sodium hypochlorite solution was shown to be effective on C. perfringens biofilms. Our investigation of dual-species biofilms of C. perfringens with the addition of Staphylococcus aureus or Escherichia coli demonstrated that overall, the mono-species biofilm of C. perfringens was more tolerant to all disinfectants than the dual-species biofilms. For the anaerobic grown biofilms, the mono-species biofilm of C. perfringens was more tolerant to sodium hypochlorite and quaternary ammonium chloride than the dual-species biofilms of C. perfringens with S. aureus or E. coli. This study demonstrates that C. perfringens biofilm is an effective protection mechanism to disinfectants commonly used on farms and in food processing environments.

  12. Control of Clostridium perfringens spores by plant-derived antimicrobials during cooling of cooked ground beef

    Science.gov (United States)

    Inhibition of Clostridium perfringens spore germination and outgrowth by carvacrol, cinnamaldehyde, thymol, oregano oil and two green tea extracts with low (green tea leaf powder (GTL); 141 mg of total catechins/g of green tea extract) and high (green tea leaf extract (GTE); 697 mg of total catechin...

  13. Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin.

    Science.gov (United States)

    Alves, Guilherme Guerra; Machado-de-Ávila, Ricardo Andrez; Chávez-Olórtegui, Carlos Delfin; Silva, Rodrigo Otávio Silveira; Lobato, Francisco Carlos Faria

    2017-02-03

    The epsilon toxin, produced by Clostridium perfringens, is responsible for enterotoxemia in ruminants and is a potential bioterrorism agent. In the present study, 15 regions of the toxin were recognized by antibodies present in the serum, with different immunodominance scales, and may be antigen determinants that can be used to formulate subunit vaccines.

  14. Clostridium perfringens infection complicating periprosthetic fracture fixation about the hip: successful treatment with early aggressive debridement.

    LENUS (Irish Health Repository)

    Baker, Joseph F

    2012-07-13

    Periprosthetic fracture and infection are both challenges following hip arthroplasty. We report the case of an 87 year old female who underwent open reduction and internal fixation of a periprosthetic femoral fracture. Her post-operative course was complicated by infection with Clostridium perfringens. Early aggressive antibiotic treatment and surgical debridement were successful, and allowed retention of the original components.

  15. Molecular Characterization of Podoviridae Bacteriophages Virulent for Clostridium perfringens and Comparison of Their Predicted Lytic Proteins

    Science.gov (United States)

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control ba...

  16. 9 CFR 113.112 - Clostridium Perfringens Type D Toxoid and Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Inactivated Bacterial Products § 113.112 Clostridium Perfringens Type D... completed product from each serial shall be tested for potency using the Epsilon toxin-neutralization test...

  17. 9 CFR 113.111 - Clostridium Perfringens Type C Toxoid and Bacterin-Toxoid.

    Science.gov (United States)

    2010-01-01

    ... INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE VIRUSES, SERUMS, TOXINS, AND ANALOGOUS PRODUCTS; ORGANISMS AND VECTORS STANDARD REQUIREMENTS Inactivated Bacterial Products § 113.111 Clostridium Perfringens Type C... completed product from each serial shall be tested for potency using the Beta toxin-neutralization test...

  18. Behaviour of Clostridium perfringens in simulated gastrointestinal conditions. An interim report

    NARCIS (Netherlands)

    Wijnands LM; van der Meij-Florijn A; Mensink ME; de Jonge R; van Leusden FM; LZO

    2009-01-01

    Het RIVM onderzoekt hoe mensen ziek worden als zij voedsel eten dat besmet is met de bacterie Clostridium perfringens. Dit wordt gedaan met experimenten die de omstandigheden in maag en darmen nabootsen.
    Doel van het onderzoek is om een schatting te maken van het aantal ziektegevallen in N

  19. Complete genome sequence of the podoviral bacteriophage CP24R virulent for Clostridium perfringens

    Science.gov (United States)

    Bacteriophage 'CP24R was isolated from raw sewage of a waste treatment plant and lytic activity was observed against a type C Clostridium perfringens isolate. Electron microscopy revealed a small virion (44nm diameter icosahedral capsid) with a short, non-contractile tail, indicative of the family ...

  20. Evidence that Clostridium perfringens theta-toxin induces colloid-osmotic lysis of erythrocytes.

    OpenAIRE

    Harris, R W; Sims, P J; Tweten, R K

    1991-01-01

    Clostridium perfringens theta-toxin was shown to lyse target erythrocytes by a colloid-osmotic mechanism. Analysis showed the onset of lysis of erythrocytes by theta-toxin could be temporarily stabilized with 0.3 M sucrose. Flow cytometry analysis of the size distribution of theta-toxin-treated erythrocytes showed swelling of the erythrocytes prior to lysis.

  1. Toxin types of Clostridium perfringens isolated from free-ranging, semi-domesticated reindeer in Norway.

    Science.gov (United States)

    Aschfalk, A; Valentin-Weigand, P; Müller, W; Goethe, R

    2002-08-17

    Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence of Clostridium perfringens. The organism was isolated from 98 (59 per cent) of the reindeer. The isolates were classified into C perfringens toxin types by PCR analysis specific for the genes encoding the four major toxins (alpha, beta, epsilon and tau) and were subclassified by the detection of the genes encoding C perfringens beta2-toxin and enterotoxin. All the isolates belonged to C perfringens toxin type A. In addition, 15 of the 98 isolates were PCR-positive for the beta2-toxin gene, and two of the isolates had the the gene encoding for enterotoxin.

  2. Bacillus subtilis PB6 improves intestinal health of broiler chickens challenged with Clostridium perfringens-induced necrotic enteritis.

    Science.gov (United States)

    Jayaraman, Sathishkumar; Thangavel, Gokila; Kurian, Hannah; Mani, Ravichandran; Mukkalil, Rajalekshmi; Chirakkal, Haridasan

    2013-02-01

    Necrotic enteritis (NE) is an enterotoxemic disease caused by Clostridium perfringens that results in significant economic losses, averaging damage of $0.05 per bird. The present study investigated the influence of a dietary supplement, Bacillus subtilis PB6, on performance, intestinal health, and gut integrity against C. perfringens-induced NE in broiler birds. Bacillus subtilis PB6 (ATCC-PTA 6737) is a natural strain isolated from healthy chicken gut that has been shown in in vitro to produce antimicrobial substances with broad activity against various strains of Campylobacter and Clostridium species. The animal study was conducted on broiler chickens (Cobb 400) for the period of 35 d using a completely randomized design. The experimental design included 3 treatments groups. Each treatment group contained 6 replicates, 3 male and 3 female, with 12 birds in each replicate. The 3 treatment groups were an uninfected control, an infected control, and an infected group supplemented with B. subtilis PB6 at 500 g/t of feed, containing 5 × 10(11) cfu/kg. Necrotic enteritis was induced in the broiler birds via oral inoculation of 30,000 oocysts of mixed strains of Eimeria species on d 14 followed by C. perfringens (10(8) cfu/mL) on d 19 through 21 of trial. The birds were analyzed for BW gain, mortality, feed conversion ratio (FCR), intestinal lesion score, intestinal C. perfringens counts, and villus histomorphometry. The infected control group showed markedly thickened mucosa, hemorrhages, intestinal lesions, and ballooning of intestine. The supplementation of B. subtilis PB6 reduced the FCR (P < 0.05) and intestinal C. perfringens counts significantly (P < 0.05) compared with the infected control group. It was also observed that B. subtilis PB6 improved villi length by 10.88 and 30.46% (P < 0.05) compared with uninfected and infected control groups, respectively. The group supplemented with B. subtilis PB6 significantly (P < 0.05) increased the villi length to crypt

  3. Chitosan inhibits enterotoxigenic Clostridium perfringens type A in growth medium and chicken meat.

    Science.gov (United States)

    Alnoman, Maryam; Udompijitkul, Pathima; Sarker, Mahfuzur R

    2017-06-01

    Clostridium perfringens is a spore-forming bacterium and a major cause of bacterial food-borne illness. In this study, we evaluated the inhibitory effects of chitosan against spore germination, spore outgrowth and vegetative growth of C. perfringens food poisoning (FP) isolates. Chitosan of differing molecular weights inhibited germination of spores of all tested FP isolates in a KCl germinant solution containing 0.1 mg/ml chitosan at pH 4.5. However, higher level (0.25 mg/ml) of chitosan was required to effectively arrest outgrowth of the germinated C. perfringens spores in Tripticase-yeast extract-glucose (TGY) medium. Furthermore, chitosan (1.0 mg/ml) was bacteriostatic against vegetative cells of C. perfringens in TGY medium. Although chitosan showed strong inhibitory activities against C. perfringens in laboratory medium, higher levels (2.0 mg/g) were required to achieve similar inhibition of spores inoculated into chicken meat. In summary, the inhibitory effects of chitosan against C. perfringens FP isolates was concentration dependent, and no major difference was observed when using different molecule weight chitosan as an inhibitor. Our results contribute to a better understanding on the potential application of chitosan in cooked meat products to control C. perfringens-associated disease. Copyright © 2016 Elsevier Ltd. All rights reserved.

  4. In vitro cytotoxicity induced by Clostridium perfringens isolate carrying a chromosomal cpe gene is exclusively dependent on sporulation and enterotoxin production.

    Science.gov (United States)

    Yasugi, Mayo; Sugahara, Yuki; Hoshi, Hidenobu; Kondo, Kaori; Talukdar, Prabhat K; Sarker, Mahfuzur R; Yamamoto, Shigeki; Kamata, Yoichi; Miyake, Masami

    2015-08-01

    Clostridium perfringens type A is a common source of food poisoning (FP) and non-food-borne (NFB) gastrointestinal diseases in humans. In the intestinal tract, the vegetative cells sporulate and produce a major pathogenic factor, C. perfringens enterotoxin (CPE). Most type A FP isolates carry a chromosomal cpe gene, whereas NFB type A isolates typically carry a plasmid-encoded cpe. In vitro, the purified CPE protein binds to a receptor and forms pores, exerting a cytotoxic activity in epithelial cells. However, it remains unclear if CPE is indispensable for C. perfringens cytotoxicity. In this study, we examined the cytotoxicity of cpe-harboring C. perfringens isolates co-cultured with human intestinal epithelial Caco-2 cells. The FP strains showed severe cytotoxicity during sporulation and CPE production, but not during vegetative cell growth. While Caco-2 cells were intact during co-culturing with cpe-null mutant derivative of strain SM101 (a FP strain carrying a chromosomal cpe gene), the wild-type level cytotoxicity was observed with cpe-complemented strain. In contrast, both wild-type and cpe-null mutant derivative of the NFB strain F4969 induced Caco-2 cell death during both vegetative and sporulation growth. Collectively, the Caco-2 cell cytotoxicity caused by C. perfringens strain SM101 is considered to be exclusively dependent on CPE production, whereas some additional toxins should be involved in F4969-mediated in vitro cytotoxicity.

  5. Clostridium perfringens and Clostridium difficile in cooked beef sold in Côte d'Ivoire and their antimicrobial susceptibility.

    Science.gov (United States)

    Kouassi, Kra Athanase; Dadie, Adjéhi Thomas; N'Guessan, Kouadio Florent; Dje, Koffi Marcellin; Loukou, Yao Guillaume

    2014-08-01

    The aim of this study was to evaluate the prevalence of Clostridium difficile and Clostridium perfringens in cooked beef sold in the streets in Côte d'Ivoire and their antimicrobial susceptibility. A total of 395 kidney and flesh samples of cooked beef were collected from vendors at Abidjan and subjected to C. difficile and C. perfringens isolation and identification by using biochemical tests, API 20A system and PCR detection. Subsequently, the antimicrobial susceptibility test was performed for confirmed isolates. Our results showed the prevalence of 12.4% for C. difficile (11.04% in kidney and 13.45% in flesh) and 5.06% for C. perfringens (2.32% in kidney and 7.17% in flesh). Metronidazole and vancomycin remained the most potent antimicrobial agents against C. difficile while metronidazole and penicillin G were the most potent agents against C. perfringens. The resistance rates to tetracycline, doxycycline, chloramphenicol and erythromycin against C. difficile and C. perfringens isolates ranged from 2.05% to 8.16% and from 20% to 50%, respectively. Among all antimicrobial agents tested against C. difficile, percentages of resistance to quinolones ciprofloxacin, norfloxacin and nalidixic acid as well as to gentamicin and cefotaxime were the highest. Eight resistant phenotypes were defined for C. difficile isolates and eleven resistant phenotypes for C. perfringens isolates. Clindamycin/gentamicin/cefotaxime/ciprofloxacin/norfloxacin/nalidixic acid resistance was the most common phenotype for C. difficile (55.10% of isolates) while norfloxacin/nalidixic acid resistance was the most common phenotype for C. perfringens (20% of isolates).

  6. Structural and functional analysis of the pore-forming toxin NetB from Clostridium perfringens.

    Science.gov (United States)

    Yan, Xu-Xia; Porter, Corrine J; Hardy, Simon P; Steer, David; Smith, A Ian; Quinsey, Noelene S; Hughes, Victoria; Cheung, Jackie K; Keyburn, Anthony L; Kaldhusdal, Magne; Moore, Robert J; Bannam, Trudi L; Whisstock, James C; Rood, Julian I

    2013-02-05

    Clostridium perfringens is an anaerobic bacterium that causes numerous important human and animal diseases, primarily as a result of its ability to produce many different protein toxins. In chickens, C. perfringens causes necrotic enteritis, a disease of economic importance to the worldwide poultry industry. The secreted pore-forming toxin NetB is a key virulence factor in the pathogenesis of avian necrotic enteritis and is similar to alpha-hemolysin, a β-barrel pore-forming toxin from Staphylococcus aureus. To address the molecular mechanisms underlying NetB-mediated tissue damage, we determined the crystal structure of the monomeric form of NetB to 1.8 Å. Structural comparisons with other members of the alpha-hemolysin family revealed significant differences in the conformation of the membrane binding domain. These data suggested that NetB may recognize different membrane receptors or use a different mechanism for membrane-protein interactions. Consistent with this idea, electrophysiological experiments with planar lipid bilayers revealed that NetB formed pores with much larger single-channel conductance than alpha-hemolysin. Channel conductance varied with phospholipid net charge. Furthermore, NetB differed in its ion selectivity, preferring cations over anions. Using hemolysis as a screen, we carried out a random-mutagenesis study that identified several residues that are critical for NetB-induced cell lysis. Mapping of these residues onto the crystal structure revealed that they were clustered in regions predicted to be required for oligomerization or membrane binding. Together these data provide an insight into the mechanism of NetB-mediated pore formation and will contribute to our understanding of the mode of action of this important toxin. IMPORTANCE Necrotic enteritis is an economically important disease of the worldwide poultry industry and is mediated by Clostridium perfringens strains that produce NetB, a β-pore-forming toxin. We carried out

  7. Complementation of a Clostridium perfringens spo0A mutant with wild-type spo0A from other Clostridium species.

    Science.gov (United States)

    Huang, I-Hsiu; Sarker, Mahfuzur R

    2006-09-01

    To evaluate whether C. perfringens can be used as a model organism for studying the sporulation process in other clostridia, C. perfringens spo0A mutant IH101 was complemented with wild-type spo0A from four different Clostridium species. Wild-type spo0A from C. acetobutylicum or C. tetani, but not from C. botulinum or C. difficile, restored sporulation and enterotoxin production in IH101. The ability of spo0A from C. botulinum or C. difficile to complement the lack of spore formation in IH101 might be due, at least in part, to the low levels of spo0A transcription and Spo0A production.

  8. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    Directory of Open Access Journals (Sweden)

    Jihong Li

    2008-05-01

    Full Text Available Clostridium perfringens is a major cause of food poisoning (FP in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  9. A novel small acid soluble protein variant is important for spore resistance of most Clostridium perfringens food poisoning isolates.

    Science.gov (United States)

    Li, Jihong; McClane, Bruce A

    2008-05-02

    Clostridium perfringens is a major cause of food poisoning (FP) in developed countries. C. perfringens isolates usually induce the gastrointestinal symptoms of this FP by producing an enterotoxin that is encoded by a chromosomal (cpe) gene. Those typical FP strains also produce spores that are extremely resistant to food preservation approaches such as heating and chemical preservatives. This resistance favors their survival and subsequent germination in improperly cooked, prepared, or stored foods. The current study identified a novel alpha/beta-type small acid soluble protein, now named Ssp4, and showed that sporulating cultures of FP isolates producing resistant spores consistently express a variant Ssp4 with an Asp substitution at residue 36. In contrast, Gly was detected at Ssp4 residue 36 in C. perfringens strains producing sensitive spores. Studies with isogenic mutants and complementing strains demonstrated the importance of the Asp 36 Ssp4 variant for the exceptional heat and sodium nitrite resistance of spores made by most FP strains carrying a chromosomal cpe gene. Electrophoretic mobility shift assays and DNA binding studies showed that Ssp4 variants with an Asp at residue 36 bind more efficiently and tightly to DNA than do Ssp4 variants with Gly at residue 36. Besides suggesting one possible mechanistic explanation for the highly resistant spore phenotype of most FP strains carrying a chromosomal cpe gene, these findings may facilitate eventual development of targeted strategies to increase killing of the resistant spores in foods. They also provide the first indication that SASP variants can be important contributors to intra-species (and perhaps inter-species) variations in bacterial spore resistance phenotypes. Finally, Ssp4 may contribute to spore resistance properties throughout the genus Clostridium since ssp4 genes also exist in the genomes of other clostridial species.

  10. The cysteine protease α-clostripain is not essential for the pathogenesis of Clostridium perfringens-mediated myonecrosis.

    Directory of Open Access Journals (Sweden)

    Anjana Chakravorty

    Full Text Available Clostridium perfringens is the causative agent of clostridial myonecrosis or gas gangrene and produces many different extracellular toxins and enzymes, including the cysteine protease α-clostripain. Mutation of the α-clostripain structural gene, ccp, alters the turnover of secreted extracellular proteins in C. perfringens, but the role of α-clostripain in disease pathogenesis is not known. We insertionally inactivated the ccp gene C. perfringens strain 13 using TargeTron technology, constructing a strain that was no longer proteolytic on skim milk agar. Quantitative protease assays confirmed the absence of extracellular protease activity, which was restored by complementation with the wild-type ccp gene. The role of α-clostripain in virulence was assessed by analysing the isogenic wild-type, mutant and complemented strains in a mouse myonecrosis model. The results showed that although α-clostripain was the major extracellular protease, mutation of the ccp gene did not alter either the progression or the development of disease. These results do not rule out the possibility that this extracellular enzyme may still have a role in the early stages of the disease process.

  11. A Five Site Clostridium Perfringens Food-Borne Outbreak: A Retrospective Cohort Study / Izbruh Okužb S Clostridium Perfringens, Prenesenih S Hrano, Na Petih Lokacijah: Retrospektivna Kohortna Raziskava

    Directory of Open Access Journals (Sweden)

    Fafangel Mario

    2015-03-01

    Full Text Available Uvod. V maju 2012 smo preiskovali izbruh okužb s Clostridium perfringens, prenesenih s hrano, v katerega so bile vpletene ena kuhinja in pet lokaciji s 477 izpostavljenimi osebami.

  12. Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable rates of evolution within a core genome

    Science.gov (United States)

    Background: Biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context. We sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricu...

  13. Growth promoting effects of prebiotic yeast cell wall products in starter broilers under an immune stress and Clostridium perfringens challenge

    Science.gov (United States)

    This study was designed to investigate the growth promoting effects of supplementing different sources and concentrations of prebiotic yeast cell wall (YCW) products containing mannanoligosaccharides in starter broilers under an immune stress and Clostridium perfringens challenge. Through a series ...

  14. Fusion of a thermophilic phage endolysin to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Science.gov (United States)

    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...

  15. A thermophilic phage endolysin fusion to a Clostridium perfringens-specific cell wall binding domain creates an anti-clostridium antimicrobial with improved thermostability

    Science.gov (United States)

    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...

  16. Implications of Decreased Nitrite Concentrations on Clostridium perfringens Outgrowth during Cooling of Ready-to-Eat Meats.

    Science.gov (United States)

    Myers, Megan I; Sebranek, Joseph G; Dickson, James S; Shaw, Angela M; Tarté, Rodrigo; Adams, Kristin R; Neibuhr, Steve

    2016-01-01

    Increased popularity of natural and organic processed meats can be attributed to the growing consumer demand for preservative-free foods, including processed meats. To meet this consumer demand, meat processors have begun using celery juice concentrate in place of sodium nitrite to create products labeled as no-nitrate or no-nitrite-added meat products while maintaining the characteristics unique to conventionally cured processed meats. Because of flavor limitations, natural cures with celery concentrate typically provide lower ingoing nitrite concentrations for ready-to-eat processed meats than do conventional cures, which could allow for increased growth of pathogens, such as Clostridium perfringens, during cooked product cooling such as that required by the U.S. Department of Agriculture. The objective of this study was to investigate the implications associated with reduced nitrite concentrations for preventing C. perfringens outgrowth during a typical cooling cycle used for cooked products. Nitrite treatments of 0, 50, and 100 ppm were tested in a broth system inoculated with a three-strain C. perfringens cocktail and heated with a simulated product thermal process followed by a typical cooling-stabilization process. The nitrite concentration of 50 ppm was more effective for preventing C. perfringens outgrowth than was 0 ppm but was not as effective as 100 ppm. The interaction between nitrite and temperature significantly affected (P 0.05) on spore outgrowth. Results indicate that decreased nitrite concentrations (50 ppm) have increased potential for total C. perfringens population outgrowth during cooling and may require additional protective measures, such as faster chilling rates.

  17. Mechanisms of Antibacterial Action of Quinoxaline 1,4-di-N-oxides against Clostridium perfringens and Brachyspira hyodysenteriae.

    Science.gov (United States)

    Xu, Fanfan; Cheng, Guyue; Hao, Haihong; Wang, Yulian; Wang, Xu; Chen, Dongmei; Peng, Dapeng; Liu, Zhenli; Yuan, Zonghui; Dai, Menghong

    2016-01-01

    Quinoxaline 1,4-di-N-oxides (QdNOs) are a class of bioreductive compounds, however, their antibacterial mechanisms are still unclarified. The aim of this study was to assess the ability of two representative QdNO drugs, cyadox (CYA) and olaquindox (OLA), to produce reactive oxide species (ROS) in Gram-positive anaerobe Clostridium perfringens CVCC1125 and Gram-negative anaerobe Brachyspira hyodysenteriae B204. In addition, the effects of QdNOs on the integrity of bacterial cell walls and membranes as well as the morphological alterations and DNA oxidative damage in C. perfringens and B. hyodysenteriae were analyzed. It was demonstrated that under anaerobic conditions, QdNOs were metabolized into the reduced products which did not show any antibacterial activity. A significant dose-related increase of intracellular ROS level and intracellular hydroxyl radicals were evident in bacteria exposed to QdNOs. The result of biochemical assay showed that the cell walls and membranes of the bacteria treated with QdNOs were damaged. After exposure to 1/2MIC to 4MIC of CYA and OLA, C. perfringens and B. hyodysenteriae became elongated and filamentous. Morphological observation with scanning and transmission electron microscopes revealed rupture, loss of cytoplasmic material and cell lysis in QdNO-treated bacteria, indicating serious damage of cells. There was an increase of 8-OHdG in the two strains treated by QdNOs, but it was lower in C. perfringens CVCC1125 than in B. hyodysenteriae B204. Agarose gel electrophoresis showed the degradation of chromosomal DNA in both of the two anaerobes treated by QdNOs. The results suggest that QdNOs may kill C. perfringens and B. hyodysenteriae via the generation of ROS and hydroxyl radicals from the bacterial metabolism of QdNOs, which cause oxidative damage in bacteria under anaerobic conditions.

  18. Transcription activation of a UV-inducible Clostridium perfringens bacteriocin gene by a novel sigma factor.

    Science.gov (United States)

    Dupuy, Bruno; Mani, Nagraj; Katayama, Seiichi; Sonenshein, Abraham L

    2005-02-01

    Expression of the plasmid-encoded Clostridium perfringens gene for bacteriocin BCN5 was shown to depend in vivo and in vitro on the activity of UviA protein. UviA, also plasmid-encoded, proved to be an RNA polymerase sigma factor and was also partly autoregulatory. The uviA gene has two promoters; one provided a UviA-independent, basal level of gene expression while the stronger, UviA-dependent promoter was only utilized after the cell experienced DNA damage. As a result, BCN5 synthesis is induced by treatment with UV light or mitomycin C. UviA is related to a special class of sigma factors found to date only in Clostridium species and responsible for activating transcription of toxin genes in Clostridium difficile, Clostridium tetani, and Clostridium botulinum.

  19. Clostridium perfringens: A review of enteric diseases in dogs, cats and wild animals.

    Science.gov (United States)

    Silva, Rodrigo Otávio Silveira; Lobato, Francisco Carlos Faria

    2015-06-01

    Clostridium perfringens is a gram-positive anaerobic bacillus that is commonly part of the microbiota of humans and animals. It is considered a common enteric pathogen, but the pathogenesis and the predisposing factors of the disease commonly differ between host species. Thus, specific research is necessary to understand the role of this pathogen, how to diagnose it, and which control measures are applicable. The aim of this paper is to review the current knowledge of C. perfringens infections in dogs, cats and wild animals.

  20. Fatal Plasmodium falciparum, Clostridium perfringens, and Candida spp. Coinfections in a Traveler to Haiti

    Science.gov (United States)

    Genrich, Gillian L.; Bhatnagar, Julu; Paddock, Christopher D.; Zaki, Sherif R.

    2009-01-01

    Malaria is one of the most common causes of febrile illness in travelers. Coinfections with bacterial, viral, and fungal pathogens may not be suspected unless a patient fails to respond to malaria treatment. Using novel immunohistochemical and molecular techniques, Plasmodium falciparum, Clostridium perfringens, and Candida spp. coinfections were confirmed in a German traveler to Haiti. Plasmodium falciparum-induced ischemia may have increased this patient's susceptibility to C. perfringens and disseminated candidiasis leading to his death. When a patient presents with P. falciparum and shock and is unresponsive to malaria treatment, secondary infections should be suspected to initiate appropriate treatment. PMID:20339463

  1. Enumeration and Isolation of cpe-Positive Clostridium perfringens Spores from Feces

    OpenAIRE

    Heikinheimo, A.; Lindström, M.; Korkeala, H

    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 ...

  2. GENOTYPING OF CLOSTRIDIUM PERFRINGENS FROM FRESH WATER FISH AND FISH PICKLES

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    Adarsh Jain

    2012-08-01

    Full Text Available This study aims to evaluate the genotypes of Clostridium perfringens in fish and fish based products from Tamil Nadu and Kerala states of India. A total of 301 samples consisting intestinal contents of freshwater fish (234 from various dams, freshwater lakes, ponds, retail shops and markets and fish pickles (67 obtained from randomly selected retail shops and supermarkets were investigated. Bacterial isolations, identifications and phenotypic characterization of virulence factors were carried out as per standard microbiological procedures. Genotyping of the C. perfringens isolates were done by amplifying four major lethal toxin genes namely- alpha toxin gene (cpa, beta toxin gene (cpb, epsilon toxin gene (etx, iota toxin gene (iA in a Thermal Cycler. Isolates were also screened for the presence of enterotoxin gene (cpe and beta2 toxin gene (cpb2 by single step PCR. Biochemical tests and phenotypic determination of virulence factors tentatively identified 82 (27.24% isolates of C. perfringens. In PCR assay, all 82 (100% isolates harbored cpa toxin genes of C. perfringens, however, 65 (79.26% isolates also carried additional cpb2 toxin genes. None of the isolates were found positive for beta, epsilon, iota and enterotoxin genes. Genotyping of the 82 isolates by PCR revealed that all the isolated bacteria were belonged to C. perfringens type A and both cpa and cpb2 toxin genes were prevalent among the isolates of C. perfringens type A, impending the risk of pathogenicity to human via freshwater fish and fish pickles.

  3. Aislamiento de Clostridium perfringens tipo D en un ternero lactante afectado con abomasitis ulcerativa Isolation of Clostridium perfringens type D from a suckling calve with ulcerative abomasitis

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    R. A ASSIS

    2002-01-01

    Full Text Available Se describe un brote de abomasitis ulcerativa asociada con infección por Clostridium perfringens tipo D en terneros lactantes. Seis terneros Holstein, de 2 semanas de edad, murieron después de presentar anorexia y apatía. Otros animales del mismo establecimiento habían muerto de la misma forma seis meses antes. A la necropsia el abomaso estaba muy distendido con contenido fluido y oscuro, la mucosa estaba edematosa y presentaba gran cantidad de úlceras milimétricas y habían depósitos de fibrina en la serosa. En el ciego de un animal se observaron extensas areas de infarto. En frotis de la mucosa abomasal se observaron bacilos cortos Gram positivos, no esporulados, aislándose de ella un cultivo rico de C. perfringens tipo D. Es probable que la baja inmunidad de los terneros debido a falta de calostro y estrés alimenticio, haya sido el predisponente para la infección por C. perfringens tipo DAn outbreak of ulcerative abomasitis in suckling calves associated with Clostridium perfrigens type D infection is described. Six twoweek old Holstein calves died after showing loss of appetite and lethargy. Other animals had died in similar circumstances during the previous six months. At necropsy, the abomasum was severely distended with dark fluid and the mucosa was oedematous and covered with many millimetric ulcers, while the serous surface of this organ was covered with fibrin. Several irregular black areas of infarcts were observed in the cecum of one animal. Histologically, the abomasal mucosa showed ulcers and haemorrhage, while the submucosa showed severe oedema and infiltration of neutrophils, lymphocytes and plasma cells. Short, thick, nonsporulated Gram positive rods were observed on smears of abomasal mucosa. C. perfringens type D was isolated from the abomasal mucosa. Low immunity and stress could have contributed to the pathogenesis of the lesions described

  4. Gut microbiome analysis in neuromyelitis optica reveals overabundance of Clostridium perfringens

    Science.gov (United States)

    Cree, Bruce A. C.; Spencer, Collin M.; Varrin‐Doyer, Michel; Baranzini, Sergio E.

    2016-01-01

    T cells from neuromyelitis optica (NMO) patients, which recognize the immunodominant epitope of aquaporin‐4, exhibit Th17 polarization and cross‐react with a homologous sequence of a Clostridium perfringens adenosine triphosphate‐binding cassette transporter. Therefore, this commensal microbe might participate in NMO pathogenesis. We examined the gut microbiome by PhyloChip G3 from 16 NMO patients, 16 healthy controls (HC), and 16 multiple sclerosis patients. A significant difference in the abundance of several microbial communities was observed between NMO and HC (Adonis test, p = 0.001). Strikingly, C. perfringens was overrepresented in NMO (p = 5.24 × 10−8). These observations support a potential role for C. perfringens in NMO pathogenesis. Ann Neurol 2016;80:443–447 PMID:27398819

  5. Quantitative Microbial Risk Assessment for Clostridium perfringens in Natural and Processed Cheeses.

    Science.gov (United States)

    Lee, Heeyoung; Lee, Soomin; Kim, Sejeong; Lee, Jeeyeon; Ha, Jimyeong; Yoon, Yohan

    2016-08-01

    This study evaluated the risk of Clostridium perfringens (C. perfringens) foodborne illness from natural and processed cheeses. Microbial risk assessment in this study was conducted according to four steps: hazard identification, hazard characterization, exposure assessment, and risk characterization. The hazard identification of C. perfringens on cheese was identified through literature, and dose response models were utilized for hazard characterization of the pathogen. For exposure assessment, the prevalence of C. perfringens, storage temperatures, storage time, and annual amounts of cheese consumption were surveyed. Eventually, a simulation model was developed using the collected data and the simulation result was used to estimate the probability of C. perfringens foodborne illness by cheese consumption with @RISK. C. perfringens was determined to be low risk on cheese based on hazard identification, and the exponential model (r = 1.82×10(-11)) was deemed appropriate for hazard characterization. Annual amounts of natural and processed cheese consumption were 12.40±19.43 g and 19.46±14.39 g, respectively. Since the contamination levels of C. perfringens on natural (0.30 Log CFU/g) and processed cheeses (0.45 Log CFU/g) were below the detection limit, the initial contamination levels of natural and processed cheeses were estimated by beta distribution (α 1 = 1, α 2 = 91; α 1 = 1, α 2 = 309)×uniform distribution (a = 0, b = 2; a = 0, b = 2.8) to be -2.35 and -2.73 Log CFU/g, respectively. Moreover, no growth of C. perfringens was observed for exposure assessment to simulated conditions of distribution and storage. These data were used for risk characterization by a simulation model, and the mean values of the probability of C. perfringens foodborne illness by cheese consumption per person per day for natural and processed cheeses were 9.57×10(-14) and 3.58×10(-14), respectively. These results indicate that probability of C. perfringens foodborne illness

  6. The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens.

    Science.gov (United States)

    Cheung, Jackie K; Keyburn, Anthony L; Carter, Glen P; Lanckriet, Anouk L; Van Immerseel, Filip; Moore, Robert J; Rood, Julian I

    2010-07-01

    Clostridium perfringens causes several diseases in domestic livestock, including necrotic enteritis in chickens, which is of concern to the poultry industry due to its health implications and associated economic cost. The novel pore-forming toxin NetB is a critical virulence factor in the pathogenesis of this disease. In this study, we have examined the regulation of NetB toxin production. In C. perfringens, the quorum sensing-dependent VirSR two-component signal transduction system regulates genes encoding several toxins and extracellular enzymes. Analysis of the sequence upstream of the netB gene revealed the presence of potential DNA binding sites, or VirR boxes, that are recognized by the VirR response regulator. In vitro binding experiments showed that purified VirR was able to recognize and bind to these netB-associated VirR boxes. Furthermore, using a reporter gene assay, the netB VirR boxes were shown to be functional. Mutation of the virR gene in two avian C. perfringens strains was shown to significantly reduce the production of the NetB toxin; culture supernatants derived from these strains were no longer cytotoxic to Leghorn male hepatoma cells. Complementation with the virRS operon restored the toxin phenotypes to wild type. The results also showed that the VirSR two-component system regulates the expression of netB at the level of transcription. We postulate that in the gastrointestinal tract of infected birds, NetB production is upregulated when the population of C. perfringens cells reaches a threshold level that leads to activation of the VirSR system.

  7. The pathology of enterotoxemia by Clostridium perfringens type C in calves.

    Science.gov (United States)

    Garcia, Jorge P; Anderson, Mark; Blanchard, Patricia; Mete, Asli; Uzal, Francisco A

    2013-05-01

    The pathology of Clostridium perfringens type C infection has been described with detail only in foals and piglets. The current report describes the diagnostic workup and detailed pathology of 3 cases of C. perfringens type C infection in calves. A 2-day-old Jersey calf and fresh and fixed tissues from a 4-week-old Angus calf and from a 1-week-old Jersey calf were received at the California Animal Health and Food Safety Laboratory System with a history of digestive disease and death. The gross changes in the gastrointestinal tract of 1 calf consisted of multifocal subserosal hemorrhages of the rumen, diffuse congestion and multifocal hemorrhages of the small intestinal mucosa, and dilated cecum with bloody liquid contents. In a second calf, a large segment of small intestine was hemorrhagic. The small intestine of the third calf was dilated and filled with abundant yellow fluid content. Microscopically, all 3 calves had diffuse coagulation necrosis of the intestinal mucosa. Clostridium perfringens type A was isolated from the intestinal content of 2 calves. In addition, enzyme-linked immunosorbent assay for Bovine rotavirus was positive on colonic content of 1 calf. Small numbers of cryptosporidia were seen in smears of colonic content of 2 calves, and Salmonella sp. group E was detected in the small intestinal content of another calf. Clostridium perfringens beta toxin was detected in the intestinal content of the 3 animals. A diagnosis of C. perfringens type C infection was confirmed based on pathological findings and detection of beta toxin in the intestinal content of the 3 animals.

  8. Expression, crystallization and preliminary X-ray diffraction studies of recombinant Clostridium perfringens β2-toxin

    Energy Technology Data Exchange (ETDEWEB)

    Gurjar, Abhijit A. [Department of Veterinary and Biomedical Science, The Pennsylvania State University (United States); Yennawar, Neela H.; Yennawar, Hemant P. [Macromolecular X-ray Crystallography Facility, The Pennsylvania State University (United States); Rajashankar, Kanagalaghatta R. [Argonne National Laboratory (United States); Hegde, Narasimha V.; Jayarao, Bhushan M., E-mail: bmj3@psu.edu [Department of Veterinary and Biomedical Science, The Pennsylvania State University (United States)

    2007-06-01

    The cloning, expression, purification and crystallization of recombinant Clostridium perfringens β2-toxin is described. The crystals diffracted to 2.9 Å resolution. Clostridium perfringens is a Gram-positive sporulating anaerobic bacterium that is responsible for a wide spectrum of diseases in animals, birds and humans. The virulence of C. perfringens is associated with the production of several enterotoxins and exotoxins. β2-toxin is a 28 kDa exotoxin produced by C. perfringens. It is implicated in necrotic enteritis in animals and humans, a disease characterized by a sudden acute onset with lethal hemorrhagic mucosal ulceration. The recombinant expression, purification and crystallization of β2-toxin using the batch-under-oil technique are reported here. Native X-ray diffraction data were obtained to 2.9 Å resolution on a synchrotron beamline at the F2 station at Cornell High Energy Synchrotron Source (CHESS) using an ADSC Quantum-210 CCD detector. The crystals belong to space group R3, with a dimer in the asymmetric unit; the unit-cell parameters are a = b = 103.71, c = 193.48 Å, α = β = 90, γ = 120° using the hexagonal axis setting. A self-rotation function shows that the two molecules are related by a noncrystallographic twofold axis with polar angles ω = 90.0, ϕ = 210.3°.

  9. Clostridium perfringens types A and D associated with enterotoxemia in an 18-month-old goat

    Directory of Open Access Journals (Sweden)

    S. Miyashiro

    2007-01-01

    Full Text Available Postmortem examination of a Boer buck that died peracutely revealed bowel and liver diffusely congested and edematous. Kidney was apparently edematous. Clostridium perfringens type A was isolated from bowel and type D from kidney. Microscopic examination revealed large areas of necrosis in the renal cortex and medulla (pulpy kidney disease, hyperemia and centrilobular necrosis of the liver, necrosis of the small-intestine wall, pulmonary edema and congestion, intense hyperemia of the cerebellum, hyperemia and edema of the brain.

  10. Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR.

    OpenAIRE

    1997-01-01

    Clostridium perfringens has been classified into five toxigenic types (A through E) on the basis of its capability to produce major lethal toxins (alpha, beta, epsilon, and iota toxins). Seroneutralization with mice or guinea pigs has been used to type each toxin, but this conventional method has some disadvantages. Therefore, we used a molecular biological technique to type the bacterium in the present study. A multiplex PCR was developed for this purpose. This method has several advantages ...

  11. Clostridium perfringens challenge and dietary fat type affect broiler chicken performance and fermentation in the gastrointestinal tract.

    Science.gov (United States)

    Józefiak, D; Kierończyk, B; Rawski, M; Hejdysz, M; Rutkowski, A; Engberg, R M; Højberg, O

    2014-06-01

    The aim of the present work was to examine how different fats commonly used in the feed industry affect broiler performance, nutrient digestibility and microbial fermentation in the gastrointestinal tract of broiler chickens challenged with virulent Clostridium perfringens strains. Two experiments were carried out, each including 480-day-old male broilers (Ross 308), which were randomly distributed to eight experimental groups using six replicate pens per treatment and 10 birds per pen. In Experiment 1, birds were fed diets containing soybean oil, palm kernel fatty acid distillers, rendered pork fat and lard. In Experiment 2, birds were fed diets containing rapeseed oil, coconut oil, beef tallow and palm oil. In both experiments, the birds were either not challenged or challenged with a mixture of three C. perfringens type A strains. Irrespective of the fat type present in the diet, C. perfringens did not affect broiler chicken body weight gain (BWG) and mortality in either of the two experiments. The BWG was affected by dietary fat type in both experiments, indicating that the fatty acid composition of the fat source affects broiler growth performance. In particular, the inclusion of animal fats tended to improve final BW to a greater extent compared with the inclusion of unsaturated vegetable oils. In Experiment 2, irrespective of the dietary fat type present in the diet, C. perfringens challenge significantly impaired feed conversion ratio in the period from 14 to 28 days (1.63 v. 1.69) and at 42 days (1.65 v. 1.68). In both experiments apparent metabolizable energy values were affected by dietary fat type. Irrespective of the fat type present in the diet, C. perfringens challenge decreased the digesta pH in the crop and ileum, but had no effect in cecal contents. Moreover, in Experiment 1, total organic acid concentration in the ileum was two to three times lower on soybean oil diets as compared with other treatments, indicating that C. perfringens as well as

  12. Clinicopathologic features of experimental Clostridium perfringens type D enterotoxemia in cattle.

    Science.gov (United States)

    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.

  13. Clostridium perfringens type-D enterotoxaemia in cattle: the diagnostic significance of intestinal epsilon toxin.

    Science.gov (United States)

    Jones, A L; Dagleish, M P; Caldow, G L

    2015-10-17

    The aims of this study were to describe 42 cases of Clostridium perfringens type-D enterotoxaemia in cattle seen between 2003 and 2014 and to determine the diagnostic value of detecting epsilon toxin in bovine intestinal content. All cases in the series had histological brain changes considered pathognomonic for C. perfringens type-D enterotoxaemia in sheep and goats and the epsilon toxin of C. perfringens was concurrently detected in the intestinal contents of 15 (36 per cent) cases. The data from the case series indicate that intestinal epsilon toxin has a sensitivity of 56 per cent compared with histology of the brain for diagnosis of bovine C. perfringens type-D enterotoxaemia. The diagnostic specificity of detecting epsilon toxin in bovine intestinal content was investigated by screening intestinal contents of 60 bovine carcases submitted for postmortem examination. Epsilon toxin was detected in 11 (18 per cent) carcases but no pathognomonic histological brain change was found in any. The specificity of intestinal epsilon toxin was estimated to be 80.4 per cent. These studies demonstrate that for a definitive diagnosis of C. perfringens type-D enterotoxaemia in cattle histological examination of the brain is essential as the presence of epsilon toxin in the intestinal contents alone is neither sensitive nor specific enough.

  14. ASSESSMENT OF ANTIMICROBIAL ACTIVITY OF PUNICA GRANATUM AGAINST ANTIBIOTIC-RESISTANT CLOSTRIDIUM PERFRINGENS TYPE (D

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    FRDOOS AL FADEL , SHAZA AL LAHAM, HASSANA CHOUR

    2015-06-01

    Full Text Available The search for new antibiotics and alternative products to solve the increasing number of bacterial resistance to customary antibiotics has become an urgent need. To investigate the effectiveness of the extracts prepared from different parts of Syrian Punica granatum Linn (family Punicaceae, against Clostridium perfringens type (D, which is resistant against many antibiotics, 684 samples were isolated from intestines and livers of death goats by using blood agar, and a selective agar for growing of Clostridium perfringens(SPS agar. The isolated bacteria were typed by using ELISA apparatus. Many parts of Punica granatum was extracted with water, absolute alcohol, then ether by using soxhlet apparatus and rotary evaporator. The Antibiotic susceptibility testing of many antibiotics was conducted by using disc diffusion method in anaerobic atmosphere and break points method. The alcoholic extracts prepared from many parts of punica granatum (pericarp, leaves, flowers, seeds showed different antibacterial effect against Clostridium perfringens type(D,whereas the studied antibiotics had not shown any antibacterial effect, except Clindamycin which showed partial effectiveness. 

  15. Mechanisms of antibacterial action of Quinoxaline 1,4-di-N-oxides against Clostridium perfringens and Brachyspira hyodysenteriae

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    Fanfan Xu

    2016-12-01

    Full Text Available Quinoxaline 1,4-di-N-oxides (QdNOs are a class of bioreductive compounds, however their antibacterial mechanisms are still unclarified. The aim of this study was to assess the ability of two representative QdNO drugs, cyadox (CYA and olaquindox (OLA, to produce reactive oxide species (ROS in Gram-positive anaerobe Clostridium perfringens CVCC1125 and Gram-negative anaerobe Brachyspira hyodysenteriae B204. In addition, the effects of QdNOs on the integrity of bacterial cell walls and membranes as well as the morphological alterations and DNA oxidative damage in C. perfringens and B. hyodysenteriae were analyzed. It was demonstrated that under anaerobic conditions, QdNOs were metabolized into the reduced products which did not show any antibacterial activity. A significant dose-related increase of intracellular ROS level and intracellular hydroxyl radicals were evident in bacteria exposed to QdNOs. The result of biochemical assay showed that the cell walls and membranes of the bacteria treated with QdNOs were damaged. After exposure to 1/2MIC to 4MIC of CYA and OLA, C. perfringens and B. hyodysenteriae became elongated and filamentous. Morphological observation with scanning and transmission electron microscopes revealed rupture, loss of cytoplasmic material and cell lysis in QdNO-treated bacteria, indicating serious damage of cells. There was an increase of 8-OHdG in the two strains treated by QdNOs, but it was lower in Gram-positive than in Gram-negative bacteria. Agarose gel electrophoresis showed the degradation of chromosomal DNA in both of the two anaerobes treated by QdNOs. The results suggest that QdNOs may kill C. perfringens and B. hyodysenteriae via the generation of ROS and hydroxyl radicals from the bacterial metabolism of QdNOs, which cause oxidative damage in bacteria under anaerobic conditions.

  16. Biofilm formation of Clostridium perfringens and its exposure to low-dose antimicrobials

    Directory of Open Access Journals (Sweden)

    Audrey eCharlebois

    2014-04-01

    Full Text Available Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxemia in animal species. Very little is known on the biofilm of C. perfringens and its exposure to subminimal inhibitory concentrations of antimicrobials. This study was undertaken to address these issues. Most of the C. perfringens human and animal isolates tested in this study were able to form biofilm (230/277. Porcine clinical isolates formed significantly more biofilm than the porcine commensal isolates. A subgroup of clinical and commensal C. perfringens isolates was randomly selected for further characterization. Biofilm was found to protect C. perfringens bacterial cells from exposure to high concentrations of tested antimicrobials. Exposure to low doses of some of these antimicrobials tended to lead to a diminution of the biofilm formed. However, a few isolates showed an increase in biofilm formation when exposed to low doses of tylosin, bacitracin, virginiamycin and monensin. Six isolates were randomly selected for biofilm analysis using scanning laser confocal microscopy. Of those, four produced more biofilm in presence of low doses of bacitracin whereas biofilms formed without bacitracin were thinner and less elevated. An increase in the area occupied by bacteria in the biofilm following exposure to low doses of bacitracin was also observed in the majority of isolates. Morphology examination revealed flat biofilms with the exception of one isolate that demonstrated a mushroom-like biofilm. Matrix composition analysis showed the presence of proteins, beta 1-4 linked polysaccharides and extracellular DNA, but no poly-beta-1,6-N-acetyl-D-glucosamine (PNAG. This study brings new information on the biofilm produced by C. perfringens and its exposure to low doses of antimicrobials.

  17. Purification and Properties of Clostridium perfringens Spore Lytic Enzymes.

    Science.gov (United States)

    1983-01-01

    sacs was effective. Further purification was obtained using carboxymethylcellulose and Sephadex G-100 chromatography. At this point the purified produce...Concentrated culture supernatant fluid (CSF) containing the initiation protein (IP) was prepared from 7 h cultures of C perfringens NCTC 8798 grown in DS...four different methods (a) 0.05 M DTT, (b) 0.05 M DTT plus 0.5% (w/v) SDS, both prepared in 0.05 M glycine-NaOH buffer, with the pH adjusted to 10.0

  18. A recombinant carboxy-terminal domain of alpha-toxin protects mice against Clostridium perfringens.

    Science.gov (United States)

    Nagahama, Masahiro; Oda, Masataka; Kobayashi, Keiko; Ochi, Sadayuki; Takagishi, Teruhisa; Shibutani, Masahiro; Sakurai, Jun

    2013-05-01

    Clostridium perfringens alpha-toxin (CP, 370 residues) is one of the main agents involved in the development of gas gangrene. In this study, the immunogenicity and protective efficacy of the C-terminal domain (CP251-370) of the toxin and phospholipase C (PLC; CB, 372 residues) of Clostridum bifermentans isolated from cases of clostridium necrosis were examined. The recombinant proteins were expressed as glutathione S-transferase (GST) fusion proteins. Antibodies that cross-reacted with alpha-toxin were produced after immunization with recombinant proteins including GST-CP251-370, GST-CP281-370, GST-CP311-370, CB1-372 and GST-CB251-372. Anti-GST-CP251-370, anti-GST-CP281-370 and anti-GST-CP311-370 sera neutralized both the PLC and hemolytic activities of alpha-toxin, whereas anti-CB1-372 and anti-GST-CB251-372 weakly neutralized these activities. Immunization with GST-CP251-370 and GST-CP281-370 provided protection against the lethal effects of the toxin and C. perfringens type A NCTC8237. Partial protection from the toxin and C. perfringens was elicited by immunization with GST-CP311-370 and CB1-372. GST-CP251-370 and GST-CP281-370 are promising candidates for vaccines for clostridial-induced gas gangrene.

  19. Antibody response to the epsilon toxin of Clostridium perfringens following vaccination of Lama glama crias.

    Science.gov (United States)

    Bentancor, Adriana B; Halperin, Pablo; Flores, Myriam; Iribarren, Fabián

    2009-09-15

    Enterotoxaemia produced by Clostridium perfringens A, C and D is an important cause of mortality in young llamas. There is no data on antibody responses following vaccination with epsilon toxin. Twenty-six L. glama crias were divided into four groups which were vaccinated with a commercial vaccine (Mancha Gangrena Enterotoxemia, Instituto Rosembusch Sociedad Anónima, Argentina) on days 0, 21 and 42 or left as unvaccinated controls. An indirect ELISA was compared with the mouse neutralization test (MNT) for measuring titers to C. perfringens type D epsilon toxin and used to determine titers in sera taken before vaccination and 16, 28, 49, 59, and 93 days later. The ELISA gave comparable results to the MNT and showed animals vaccinated once failed to develop raised titers. A week following a second vaccination, mean antibody titers rose significantly (P < 0.05) and 7/12 animals developed high titers which were present in only one animal at the end of the study (day 93). A third vaccination resulted in a decrease in mean antibody titers a week later. Llamas develop antibodies to Clostridium perfringens type D epsilon toxin after two vaccinations at a 21-day interval. Further studies are indicated to determine if these inoculations protect against enterotoxemia and the most appropriate vaccination schedule.

  20. Growth of Clostridium perfringens from spore inocula in sous-vide turkey products.

    Science.gov (United States)

    Juneja, V K; Marmer, B S

    1996-09-01

    Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag ground turkey (pH 6) that included 0.3% (w/w) sodium pyrophosphate, and sodium chloride at 0, 1, 2, or 3% (w/w). The packages were processed to an internal temperature of 71.1 degrees C, ice chilled and stored at various temperatures. The total C. perfringens population was determined by plating diluted samples on tryptose-sulfite-cycloserine agar followed by anaerobic incubation at 37 degrees C for 48 h. At 28 degrees C, the addition of 3% salt in turkey was effective in delaying growth for 12 h. At 15 degrees C, growth occurred at a relatively slow rate in the presence of 1-2% salt. Vegetative cells were not observed even after 28 days of storage in the presence of 3% salt. C. perfringens growth was not observed at 4 degrees C regardless of salt levels. The D-values ranged from 23.2 min (no salt) to 17.7 min (3% salt). Cyclic and static temperature abuse of refrigerated products for 8 h did not lead to growth by C. perfringens from a spore inoculum.

  1. Effects of thymol and carvacrol supplementation on intestinal integrity and immune responses of broiler chickens challenged with Clostridium perfringens

    OpenAIRE

    Du, Encun; Wang, Weiwei; Gan, Liping; Li, Zhui; Guo, Shuangshuang; Guo, Yuming

    2016-01-01

    Background Necrotic enteritis caused by Clostridium perfringens infection leads to serious economic losses in the global poultry production. In the present study, we investigated the protective effects of essential oils (EO, which contained 25 % thymol and 25 % carvacrol as active components) supplementation on growth performance, gut lesions, intestinal morphology, and immune responses of the broiler chickens infected with C. perfringens. A total of 448 1-day-old male broiler chicks were all...

  2. How do swine practitioners and veterinary pathologists arrive at a diagnosis of Clostridium perfringens type A enteritis in neonatal piglets?

    Science.gov (United States)

    Chan, Gloria; Farzan, Abdolvahab; Prescott, John F; Friendship, Robert

    2013-05-01

    A questionnaire was administered to 22 veterinary practitioners and 17 veterinary pathologists to investigate the methods used for diagnosis of Clostridium perfringens type A enteritis in neonatal pigs. Practitioners generally diagnosed C. perfringens type A associated enteritis by age of onset of diarrhea (between 1 to 7 days of age). Most practitioners (95%) were moderately to very confident in their diagnosis. Pathologists generally diagnosed C. perfringens type A associated enteritis by combinations of isolation of the organism, genotyping or detecting the toxins of the organism, and ruling out other pathogens through histopathology. Almost half (41%) of the pathologists were not confident of their diagnosis. This study reports that the current diagnostic method for C. perfringens type A enteritis is not specific, and although many pathologists expressed reservations about making a diagnosis of C. perfringens type A enteritis, most practitioners were confident in their diagnosis, even though reported clinical signs of clostridial diarrhea are similar to those of a number of other enteric diseases.

  3. The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens.

    Science.gov (United States)

    Antonissen, Gunther; Van Immerseel, Filip; Pasmans, Frank; Ducatelle, Richard; Haesebrouck, Freddy; Timbermont, Leen; Verlinden, Marc; Janssens, Geert Paul Jules; Eeckhaut, Venessa; Eeckhout, Mia; De Saeger, Sarah; Hessenberger, Sabine; Martel, An; Croubels, Siska

    2014-01-01

    Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON) is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (Penteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens.

  4. Effect of a probiotic on prevention of diarrhea and Clostridium difficile and Clostridium perfringens shedding in foals

    DEFF Research Database (Denmark)

    Schoster, Angelika; Staempfli, H R; Abrahams, M

    2015-01-01

    BACKGROUND: Up to 60% of foals develop diarrhea within 6 months after birth. Preventive measures are limited but potentially probiotics could be used. OBJECTIVE: To evaluate the effect of a newly designed probiotic on the incidence of foal diarrhea in a randomized field trial. ANIMALS: Seventy......-two healthy neonatal foals. METHODS: Randomized, placebo-controlled field trial. Foals were administered a placebo or probiotic for 3 weeks and monitored for an additional week. A total of 3 fecal samples were taken from each foal at biweekly intervals. Statistical modeling was applied for comparison...... of incidence and duration of diarrhea and fecal shedding of Clostridium perfringens and Clostridium difficile between treatment and age groups. RESULTS: The overall incidence of diarrhea was 41 of 72 (59%) and did not differ (P = 0.37) between treatment groups. Foals treated with probiotics were more likely...

  5. Prevalence of Clostridium perfringens toxin genotypes in enterotoxemia suspected sheep flocks of Andhra Pradesh

    Directory of Open Access Journals (Sweden)

    N. Vinod Kumar

    2014-12-01

    Full Text Available Aim: To identify the Clostridium perfringens toxin genotypes prevailing in enterotoxemia suspected sheep flocks in Andhra Pradesh by using multiplex PCR. Materials and Methods: Intestinal scrapings were collected from lambs below three months of age from flocks with similar management from different Districts of Andhra Pradesh, in India. A total of 208 samples were collected with 140 from enterotoxemia suspected flocks and 68 from healthy flocks. Following processing and culture of the samples, colonies were identified by morphological and biochemical tests. All the clostridial isolates were analyzed by multiplex PCR. Results: C. pefringens were isolated from 97 out of 140 enterotoxemia suspected flocks (69.29% and 27 out of 68 healthy flocks (39.71 %. Genotyping of the 97 iolates by multiplex PCR from enterotoxemia suspected flocks indicated C. perfringens type A, C and D 67.01% (65 out of 97; 11.34% (11 out of 97 and 21.65% (21 out of 97 respectively. Isolates from healthy flocks indicated the presence of type A and D 92.59% (25 out of 27 and 7.40% (2 out of 27 respectively. Number of isolates from enterotoxaemia suspected flocks were significantly high (P<0.001 than healthy flocks. Type A is found to be predominant in both enterotoxemia suspected and healthy flocks (67.01% and 92.59%. Conclusions: Prevalence of C. perfringens type C was reported for the first time in India. Clostridium perfringens type D and type C were found to be the major causative types for enterotoxemia.

  6. Associations between intestinal lesions and detection of Clostridium perfringens type A or beta-2 toxin in neonatal piglets with diarrhoea

    DEFF Research Database (Denmark)

    Kongsted, Hanne; Jonach, Beata Renata; Kokotovic, Branko

    Objective: To evaluate associations between gross and histopathological lesions and the presence of Clostridium perfringens type A (CpA) and beta-2 toxin in piglets from 4 herds with outbreaks of diarrhoea. Methods: Pathological examinations on 51 diarrhoeic piglets aged 3-7 days were carried out....... CpA was cultured and typed by PCR. A Clostridium perfringens spp targeted probe was used for flourescent in situ hybridization (FISH) and detection of beta-2 toxin in intestinal contents was performed by enzyme-linked immunosorbent assay (ELISA). Results: Piglets without intestinal lesions had...

  7. Phospholipase C Produced by Clostridium botulinum Types C and D:Comparison of Gene, Enzymatic, and Biological Activities with Those of Clostridium perfringens Alpha-toxin

    Directory of Open Access Journals (Sweden)

    Sakurai,Jun

    2013-02-01

    Full Text Available Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs, the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  8. Phospholipase C produced by Clostridium botulinum types C and D: comparison of gene, enzymatic, and biological activities with those of Clostridium perfringens alpha-toxin.

    Science.gov (United States)

    Fatmawati, Ni Nengah Dwi; Sakaguchi, Yoshihiko; Suzuki, Tomonori; Oda, Masataka; Shimizu, Kenta; Yamamoto, Yumiko; Sakurai, Jun; Matsushita, Osamu; Oguma, Keiji

    2013-01-01

    Clostridium botulinum type C and D strains recently have been found to produce PLC on egg yolk agar plates. To characterize the gene, enzymatic and biological activities of C. botulinum PLCs (Cb-PLCs), the cb-plc genes from 8 strains were sequenced, and 1 representative gene was cloned and expressed as a recombinant protein. The enzymatic and hemolytic activities of the recombinant Cb-PLC were measured and compared with those of the Clostridium perfringens alpha-toxin. Each of the eight cb-plc genes encoded a 399 amino acid residue protein preceded by a 27 residue signal peptide. The protein consists of 2 domains, the N- and C-domains, and the overall amino acid sequence identity between Cb-PLC and alpha-toxin was greater than 50%, suggesting that Cb-PLC is homologous to the alpha-toxin. The key residues in the N-domain were conserved, whereas those in the C-domain which are important in membrane interaction were different than in the alpha-toxin. As expected, Cb-PLC could hydrolyze egg yolk phospholipid, p-nitrophenylphosphorylcholine, and sphingomyelin, and also exhibited hemolytic activity;however, its activities were about 4- to over 200-fold lower than those of alpha-toxin. Although Cb-PLC showed weak enzymatic and biological activities, it is speculated that Cb-PLC might play a role in the pathogenicity of botulism or for bacterial survival.

  9. Isolation and characterization of multiply antibiotic-resistant Clostridum perfringens strains from porcine feces.

    Science.gov (United States)

    Rood, J I; Maher, E A; Somers, E B; Campos, E; Duncan, C L

    1978-05-01

    Multiply antibiotic-resistant strains of Clostridium perfringens were isolated from porcine feces. Strains that were resistant to tetracycline, erythromycin, clindamycin, and lincomycin were isolated, but no penicillin- or chloramphenicol-resistant strains were obtained. Typical minimal inhibitory concentrations for resistant strains were 16 to 64 mug of tetracycline per ml, 64 to >128 mug of erythromycin per ml, >/=128 mug of lincomycin per ml, and 16 to 128 mug of clindamycin per ml. Resistance to erythromycin was always associated with resistance to lincomycin and clindamycin. Minimal inhibitory concentrations were determined for 258 strains from six farms that used antibiotics in their feeds and 240 strains from five farms that did not use antibiotics. The results show that 77.9 and 22.7% of the strains from the former farms were resistant to tetracycline and erythromycin-clindamycin-lincomycin, respectively. The comparable data from the latter farms were 25.0 and 0.8%, respectively. Agarose gel electrophoresis failed to reveal a plasmid band that was common to the resistant strains but absent in the susceptible strains. Attempts to transfer tetracycline, erythromycin, and clindamycin resistance from one strain, CW459, were not successful. Antibiotic-susceptible mutants were not isolated from this strain, despite the use of a variety of curing agents.

  10. The VirS/VirR two-component system regulates the anaerobic cytotoxicity, intestinal pathogenicity, and enterotoxemic lethality of Clostridium perfringens type C isolate CN3685.

    Science.gov (United States)

    Ma, Menglin; Vidal, Jorge; Saputo, Juliann; McClane, Bruce A; Uzal, Francisco

    2011-01-25

    Clostridium perfringens vegetative cells cause both histotoxic infections (e.g., gas gangrene) and diseases originating in the intestines (e.g., hemorrhagic necrotizing enteritis or lethal enterotoxemia). Despite their medical and veterinary importance, the molecular pathogenicity of C. perfringens vegetative cells causing diseases of intestinal origin remains poorly understood. However, C. perfringens beta toxin (CPB) was recently shown to be important when vegetative cells of C. perfringens type C strain CN3685 induce hemorrhagic necrotizing enteritis and lethal enterotoxemia. Additionally, the VirS/VirR two-component regulatory system was found to control CPB production by CN3685 vegetative cells during aerobic infection of cultured enterocyte-like Caco-2 cells. Using an isogenic virR null mutant, the current study now reports that the VirS/VirR system also regulates CN3685 cytotoxicity during infection of Caco-2 cells under anaerobic conditions, as found in the intestines. More importantly, the virR mutant lost the ability to cause hemorrhagic necrotic enteritis in rabbit small intestinal loops. Western blot analyses demonstrated that the VirS/VirR system mediates necrotizing enteritis, at least in part, by controlling in vivo CPB production. In addition, vegetative cells of the isogenic virR null mutant were, relative to wild-type vegetative cells, strongly attenuated in their lethality in a mouse enterotoxemia model. Collectively, these results identify the first regulator of in vivo pathogenicity for C. perfringens vegetative cells causing disease originating in the complex intestinal environment. Since VirS/VirR also mediates histotoxic infections, this two-component regulatory system now assumes a global role in regulating a spectrum of infections caused by C. perfringens vegetative cells.

  11. Caracterización molecular y resistencia antimicrobiana de aislamientos de Clostridium perfringens de diferentes orígenes en Costa Rica

    Directory of Open Access Journals (Sweden)

    María del Mar Gamboa-Coronado

    2011-12-01

    Full Text Available Clostridium perfringens es un bacilo Gram positivo, esporulado, anaerobio, ampliamente distribuido en la naturaleza, que produce cuatro toxinas principales α, β, ε y ι, las cuales permiten su clasificación en cinco toxinotipos (A-E. Algunas cepas producen una enterotoxina (CPE, codificada por el gen cpe, que causa diarrea en seres humanos y en algunos animales. La presencia de los genes de estas toxinas y la sensibilidad a los antibióticos se determinó en 81 cepas de C. perfringens previamente aisladas y que habían sido mantenidas a -80°C; 20 de suelos, 20 de origen animal, 20 de origen humano y 21 de alimentos cocidos no relacionados con brotes alimentarios. De acuerdo con los resultados de PCR, todas las cepas fueron clasificadas como C. perfringens tipo A, debido a que solo se les detectó el gen de la toxina α, mientras que el gen de la enterotoxina (cpe se detectó en dos cepas (2.5% aisladas de alimentos, tal como ha sido descrito en otras regiones del mundo. El 44% de las cepas fue resistente a algún antibiótico; clindamicina (41%, cloranfenicol (25%, penicilina (22% y metronidazol (20%. En general, las cepas provenientes de suelos presentaron los mayores porcentajes de resistencia a casi todos los antibióticos. El 40% de las cepas de suelo presentó multiresistencia (a tres o más grupos de antibióticos, el 30% de las de origen humano, el 14% de las de alimentos y el 5% de las de origen animal. Las altas tasas de resistencia encontradas podrían deberse al amplio uso de antibióticos como promotores de crecimiento de plantas y animales y esas cepas resistentes podrían actuar como reservorio de genes de resistencia que pueden transferirse entre bacterias de diversos ambientes.Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica. Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their

  12. Alphitobius diaperinus spp como veiculador de Clostridium perfringens em granjas avícolas do interior paulista - Brasil Alphitobius diaperinus spp as a vector of Clostridium perfringens in broiler houses in the state of São Paulo - Brazil

    Directory of Open Access Journals (Sweden)

    Juliano Vittori

    2007-06-01

    Full Text Available O besouro Alphitobius diaperinus spp (cascudinho é visto como uma importante praga da avicultura mundial. Por suas características comportamentais e hábitos biológicos que dificultam seu controle, é considerado um vetor de agentes patogênicos. O objetivo desta pesquisa foi investigar o cascudinho como possível vetor de Clostridium perfringens em granjas avícolas industriais, localizadas em diferentes regiões do interior Paulista. Através de métodos bacteriológicos convencionais, em 40 amostras analisadas, foram encontradas contagens significativas de Clostridium perfringens em todas elas. A partir dos resultados obtidos, pôde-se demonstrar o potencial deste inseto como vetor do agente responsável pela enterite necrótica.The Alphitobius diaperinus spp (lesser mealworm is considered an important world poultry plague. Due to its behavior characteristics and biological habits that make its control difficult it is considered a vector of pathogenic agents. The objective of this research was to investigate the little mealworm as possible vector of Clostridium perfringens in broiler houses, located in different parts of the state of São Paulo. Through conventional bacteriological methods, 40 samples of little mealworm collected were analyzed. Clostridium perfringens was found in all of the samples and the potential of this insect as vector of the necrotic enteritis was demonstrated.

  13. Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.

    Science.gov (United States)

    Mehdizadeh Gohari, Iman; Kropinski, Andrew M; Weese, Scott J; Parreira, Valeria R; Whitehead, Ashley E; Boerlin, Patrick; Prescott, John F

    2016-01-01

    The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT) technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb) and 81 (~430 kb) regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb) were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a large and

  14. Plasmid Characterization and Chromosome Analysis of Two netF+ Clostridium perfringens Isolates Associated with Foal and Canine Necrotizing Enteritis.

    Directory of Open Access Journals (Sweden)

    Iman Mehdizadeh Gohari

    Full Text Available The recent discovery of a novel beta-pore-forming toxin, NetF, which is strongly associated with canine and foal necrotizing enteritis should improve our understanding of the role of type A Clostridium perfringens associated disease in these animals. The current study presents the complete genome sequence of two netF-positive strains, JFP55 and JFP838, which were recovered from cases of foal necrotizing enteritis and canine hemorrhagic gastroenteritis, respectively. Genome sequencing was done using Single Molecule, Real-Time (SMRT technology-PacBio and Illumina Hiseq2000. The JFP55 and JFP838 genomes include a single 3.34 Mb and 3.53 Mb chromosome, respectively, and both genomes include five circular plasmids. Plasmid annotation revealed that three plasmids were shared by the two newly sequenced genomes, including a NetF/NetE toxins-encoding tcp-conjugative plasmid, a CPE/CPB2 toxins-encoding tcp-conjugative plasmid and a putative bacteriocin-encoding plasmid. The putative beta-pore-forming toxin genes, netF, netE and netG, were located in unique pathogenicity loci on tcp-conjugative plasmids. The C. perfringens JFP55 chromosome carries 2,825 protein-coding genes whereas the chromosome of JFP838 contains 3,014 protein-encoding genes. Comparison of these two chromosomes with three available reference C. perfringens chromosome sequences identified 48 (~247 kb and 81 (~430 kb regions unique to JFP55 and JFP838, respectively. Some of these divergent genomic regions in both chromosomes are phage- and plasmid-related segments. Sixteen of these unique chromosomal regions (~69 kb were shared between the two isolates. Five of these shared regions formed a mosaic of plasmid-integrated segments, suggesting that these elements were acquired early in a clonal lineage of netF-positive C. perfringens strains. These results provide significant insight into the basis of canine and foal necrotizing enteritis and are the first to demonstrate that netF resides on a

  15. Recombinant Alpha, Beta, and Epsilon Toxins of Clostridium perfringens: Production Strategies and Applications as Veterinary Vaccines

    Science.gov (United States)

    Ferreira, Marcos Roberto A.; Moreira, Gustavo Marçal S. G.; da Cunha, Carlos Eduardo P.; Mendonça, Marcelo; Salvarani, Felipe M.; Moreira, Ângela N.; Conceição, Fabricio R.

    2016-01-01

    Clostridium perfringens is a spore-forming, commensal, ubiquitous bacterium that is present in the gastrointestinal tract of healthy humans and animals. This bacterium produces up to 18 toxins. The species is classified into five toxinotypes (A–E) according to the toxins that the bacterium produces: alpha, beta, epsilon, or iota. Each of these toxinotypes is associated with myriad different, frequently fatal, illnesses that affect a range of farm animals and humans. Alpha, beta, and epsilon toxins are the main causes of disease. Vaccinations that generate neutralizing antibodies are the most common prophylactic measures that are currently in use. These vaccines consist of toxoids that are obtained from C. perfringens cultures. Recombinant vaccines offer several advantages over conventional toxoids, especially in terms of the production process. As such, they are steadily gaining ground as a promising vaccination solution. This review discusses the main strategies that are currently used to produce recombinant vaccines containing alpha, beta, and epsilon toxins of C. perfringens, as well as the potential application of these molecules as vaccines for mammalian livestock animals. PMID:27879630

  16. Benthic distribution of sewage sludge indicated by Clostridium perfringens at a deep-ocean dump site

    Energy Technology Data Exchange (ETDEWEB)

    Hill, R.T.; Anikis, M.S.; Colwell, R.R. (Univ. of Maryland, Baltimore (United States)); Knight, I.T. (James Madison Univ., Harrisonburg, VA (United States))

    1993-01-01

    Since 1986, sewage sludge from New York and northern New Jersey has been dumped 196 km off the coast of New Jersey at the Deep Water Municipal Sewage Sludge Disposal Site. This study determines the distribution of sludge contamination of the benthic environment in the area, by using Clostridium perfringens as an indicator. The counts of C. perfringens confirm a previous report that sewage sludge is reaching the ocean floor at the disposal site as a result of the sludge dumping. C. perfringes counts within the dump site and to the south and west of the dump site are considerably elevated compared to counts east of the site. The distribution pattern of C. perfringes is broadly consistent with the estimates of the sea floor area impacted in the most recent computer model. However, the area of maximum desposition of sludge may be slightly further north than predicted. Use of C. perfringens has proven to be an efficient and reliable method for tracing sewage contamination of deep ocean sediments. 18 refs., 4 figs., 3 tabs.

  17. Toxinotyping and antimicrobial susceptibility of enterotoxigenic Clostridium perfringens isolates from mutton, beef and chicken meat.

    Science.gov (United States)

    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.

  18. Development and application of an oral challenge mouse model for studying Clostridium perfringens type D infection.

    Science.gov (United States)

    Fernandez-Miyakawa, Mariano E; Sayeed, Sameera; Fisher, Derek J; Poon, Rachael; Adams, Vicki; Rood, Julian I; McClane, Bruce A; Saputo, Julian; Uzal, Francisco A

    2007-09-01

    Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections.

  19. Development and Application of an Oral Challenge Mouse Model for Studying Clostridium perfringens Type D Infection▿

    Science.gov (United States)

    Fernandez-Miyakawa, Mariano E.; Sayeed, Sameera; Fisher, Derek J.; Poon, Rachael; Adams, Vicki; Rood, Julian I.; McClane, Bruce A.; Saputo, Julian; Uzal, Francisco A.

    2007-01-01

    Clostridium perfringens type D isolates cause enterotoxemia in sheep, goats, and probably cattle. While the major disease signs and lesions of type D animal disease are usually attributed to epsilon toxin, a class B select agent, these bacteria typically produce several lethal toxins. Understanding of disease pathogenesis and development of improved vaccines are hindered by the lack of a small-animal model mimicking natural disease caused by type D isolates. Addressing this need, we developed an oral challenge mouse model of C. perfringens type D enterotoxemia. When BALB/c mice with a sealed anus were inoculated by intragastric gavage with type D isolates, 7 of 10 type D isolates were lethal, as defined by spontaneous death or severe clinical signs necessitating euthanasia. The lethalities of the seven type D isolates varied between 14 and 100%. Clinical signs in the lethally challenged mice included seizures, convulsions, hyperexcitability, and/or depression. Mild intestinal gas distention and brain edema were observed at necropsy in a few mice, while histology showed multifocal acute tubular necrosis of the kidney and edema in the lungs of most challenged mice that developed a clinical response. When the lethality of type D isolates in this model was compared with in vitro toxin production, only a limited correlation was observed. However, mice could be protected against lethality by intravenous passive immunization with an epsilon toxin antibody prior to oral challenge. This study provides an economical new model for studying the pathogenesis of C. perfringens type D infections. PMID:17562765

  20. Beta Lactamase Producing Clostridium perfringens Bacteremia in an Elderly Man with Acute Pancreatitis

    Directory of Open Access Journals (Sweden)

    Rashmi Mishra

    2016-01-01

    Full Text Available 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.

  1. Enforcement of science-using a Clostridium perfringens outbreak investigation to take legal action.

    Science.gov (United States)

    Acheson, Peter; Bell, Vikki; Gibson, Janet; Gorton, Russell; Inns, Thomas

    2016-09-01

    We report an outbreak of Clostridium perfringens in a care home in North East England. A retrospective cohort study was used to investigate this outbreak. Faecal samples were obtained from symptomatic residents. Environmental Health Officers carried out a food hygiene inspection and formal statements were taken. Fifteen residents reported illness and the epidemic curve was suggestive of a point source outbreak. Results suggest that illness was associated with consumption of mince & vegetable pie and/or gravy. There were a number of issues with food served, in particular the mince products had been cooked, cooled, reheated and served again over a period of several days. Faecal sampling revealed the presence of C.perfringens enterotoxin gene and four samples were indistinguishable by fluorescent amplified fragment length polymorphism, indicating a likely common source. The operator of the home was charged with three offences under the General Food Regulations 2004 and the Food Hygiene (England) Regulations 2006 and was convicted on all counts. An outbreak of C.perfringens occurred in a care home. The likely cause was consumption of mince & vegetable pie and/or gravy. Epidemiological evidence can be used to help prosecute businesses with food safety offences in such circumstances. © The Author 2015. Published by Oxford University Press on behalf of Faculty of Public Health. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  2. Identification, Isolation and characterization of a novel azoreductase from Clostridium perfringens.

    Science.gov (United States)

    Morrison, Jessica M; Wright, Cristee M; John, Gilbert H

    2012-04-01

    Azo dyes are used widely in the textile, pharmaceutical, cosmetic and food industries as colorants and are often sources of environmental pollution. There are many microorganisms that are able to reduce azo dyes by use of an azoreductase enzyme. It is through the reduction of the azo bonds of the dyes that carcinogenic metabolites are produced thereby a concern for human health. The field of research on azoreductases is growing, but there is very little information available on azoreductases from strict anaerobic bacteria. In this study, the azoreductase gene was identified in Clostridium perfringens, a pathogen that is commonly found in the human intestinal tract. C. perfringens shows high azoreductase activity, especially in the presence of the common dye Direct Blue 15. A gene that encodes for a flavoprotein was isolated and expressed in Escherichia coli, and further purified and tested for azoreductase activity. The azoreductase (known as AzoC) was characterized by enzymatic reaction assays using different dyes. AzoC activity was highest in the presence of two cofactors, NADH and FAD. A strong cofactor effect was shown with some dyes, as dye reduction occurred without the presence of the AzoC (cofactors alone). AzoC was shown to perform best at a pH of 9, at room temperature, and in an anaerobic environment. Enzyme kinetics studies suggested that the association between enzyme and substrate is strong. Our results show that AzoC from C. perfringens has azoreductase activity.

  3. Detection of Clostridium perfringens alpha toxin gene in lambs by loop mediated isothermal amplification

    Directory of Open Access Journals (Sweden)

    B. Radhika

    2016-01-01

    Full Text Available Aim: The loop mediated isothermal amplification (LAMP was standardized for rapid detection of Clostridium perfringens. Materials and Methods: A total of 120 fecal samples were collected from enterotoxemia suspected lambs were used for screening of C. perfringens cpa gene by LAMP. The specificity of the LAMP amplified products was tested by digesting with restriction enzyme XmnI for alpha toxin gene. Results: Out of 120 samples screened 112 (93.3% samples were positive by both LAMP and polymerase chain reaction (PCR for detection of cpa gene which indicated the equal sensitivity of both the tests. The enzyme produced single cut in 162 base pair amplified product of alpha toxin gene at 81 base pair resulting in a single band in gel electrophoresis. Conclusion: Both LAMP and PCR for detection of cpa gene indicated the equal sensitivity of both the tests. Standardization of LAMP reaction for amplification of epsilon and beta toxin genes will help to identify the C. perfringens toxin types from the clinical samples. The test could be a suitable alternative to the PCR in detection of toxin types without the help of sophisticated machinery like thermal cycler. Considering its simplicity in operation and high sensitivity, there is the potential use of this technique in clinical diagnosis and surveillance of infectious diseases.

  4. The Generation and Characterization of Recombinant Protein and Antibodies of Clostridium perfringens Beta2 Toxin

    Science.gov (United States)

    Zeng, Jin; Song, Fuyang; Yang, Yi; Ma, Chenjie; Deng, Guangcun; Li, Yong; Wang, Yujiong

    2016-01-01

    Introduction. Clostridium perfringens (C. perfringens) beta2 toxin (CPB2) is an important virulent factor of necrotic enteritis in both animals and humans. However, studies of its pathogenic roles and functional mechanisms have been hampered due to the difficulty of purification and lack of specific antibodies against this toxin. Methods. A recombinant His-tagged C. perfringens beta2 (rCPB2) toxin and monoclonal antibodies (McAbs) against CPB2 were generated and characterized by assays of cytotoxicity, immunoblotting, ELISA, neutralization, and immunofluorescence. Results. A His-tagged rCPB2 with integrity and cytotoxicity of native CPB2 was purified from E. coli expressing system, which exhibited a moderate cytotoxicity on NCM460 human intestinal epithelial cells. The rCPB2 could induce apoptotic cell death rather than necrotic death in part through a pathway involved in caspase-3 signaling. Mechanistically, rCPB2 was able to first bind to cell membrane and dynamically translocate into cytoplasm for its cytotoxic activity. Three McAbs 1E23, 2G7 and 2H7 were characterized to be able to immunologically react with CPB2 and neutralize rCPB2 cytotoxicity on NCM460 cells. Conclusion. These results indicated the rCPB2 and antibodies generated in this study are useful tools for studies of biological functions and pathogenic mechanisms of CPB2 in future, which warrants for further investigations. PMID:27672668

  5. Impedance Analysis of Ovarian Cancer Cells upon Challenge with C-terminal Clostridium Perfringens Enterotoxin

    Science.gov (United States)

    Gordon, Geoffrey; Lo, Chun-Min

    2007-03-01

    Both in vitro and animal studies in breast, prostate, and ovarian cancers have shown that clostridium perfringens enterotoxin (CPE), which binds to CLDN4, may have an important therapeutic benefit, as it is rapidly cytotoxic in tissues overexpressing CLDN4. This study sought to evaluate the ability of C-terminal clostridium perfringens enterotoxin (C-CPE), a CLDN4-targetting molecule, to disrupt tight junction barrier function. Electric cell-substrate impedance sensing (ECIS) was used to measure both junctional resistance and average cell-substrate separation of ovarian cancer cell lines after exposure to C-CPE. A total of 14 ovarian cancer cell lines were used, and included cell lines derived from serous, mucinous, and clear cells. Our results showed that junctional resistance increases as CLDN4 expression increases. In addition, C-CPE is non-cytotoxic in ovarian cancer cells expressing CLDN4. However, exposure to C-CPE results in a significant (p<0.05) dose- and CLDN4-dependent decrease in junctional resistance and an increase in cell-substrate separation. Treatment of ovarian cancer cell lines with C-CPE disrupts tight junction barrier function.

  6. Effects of Tylosin on Bacterial Mucolysis, Clostridium perfringens Colonization, and Intestinal Barrier Function in a Chick Model of necrotic Enteritis

    NARCIS (Netherlands)

    Collier, C.T.; Klis, van der J.D.; Deplancke, B.; Anderson, D.B.; Gaskins, G.R.

    2003-01-01

    Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and

  7. Necrotizing enterocolitis and death in a goat kid associated with enterotoxin (CPE)-producing Clostridium perfringens type A.

    Science.gov (United States)

    Miyakawa, Mariano E Fernandez; Saputo, Julian; Leger, Judy St; Puschner, Birgit; Fisher, Derek J; McClane, Bruce A; Uzal, Francisco A

    2007-12-01

    A goat kid died after being depressed for several days. No significant gross abnormalities were observed at postmortem examination, while histopathological analysis revealed diffuse necrotizing enterocolitis. Isolation of Clostridium perfringens type A secreting enterotoxin (CPE) and presence of CPE in the small intestine suggest that CPE contributed to the death of this kid.

  8. Necrotizing enterocolitis and death in a goat kid associated with enterotoxin (CPE)-producing Clostridium perfringens type A

    OpenAIRE

    2007-01-01

    A goat kid died after being depressed for several days. No significant gross abnormalities were observed at postmortem examination, while histopathological analysis revealed diffuse necrotizing enterocolitis. Isolation of Clostridium perfringens type A secreting enterotoxin (CPE) and presence of CPE in the small intestine suggest that CPE contributed to the death of this kid.

  9. Literatuuronderzoek naar gegevens betreffende de betekenis van een aantal verwekkers van zoonosen in verband met de vleesconsumptie. VII: Clostridium perfringens

    NARCIS (Netherlands)

    Bos; J.M.; Engel; H.W.B.; Groothuis; D.G.; Knapen; F.van; Weiss; J.W.

    1986-01-01

    Besmettingen met Clostridium perfringens kunnen bij de mens leiden tot uiteenlopende ziektebeelden. Deze kunnen varieren van necrotiserende wondinfecties en necrotiserende enteritis met toxinemie tot relatief milde zelflimiterende enteritis. E.e.a. hangt af van de plaats van infectie en de

  10. Molecular Characterization of Podoviral Bacteriophages Virulent for Clostridium perfringens and Their Comparison with Members of the Picovirinae

    Science.gov (United States)

    Clostridium perfringens is a Gram positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal, and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control b...

  11. Effects of Tylosin on Bacterial Mucolysis, Clostridium perfringens Colonization, and Intestinal Barrier Function in a Chick Model of necrotic Enteritis

    NARCIS (Netherlands)

    Collier, C.T.; Klis, van der J.D.; Deplancke, B.; Anderson, D.B.; Gaskins, G.R.

    2003-01-01

    Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats

  12. Bacteriological investigation of an outbreak of Clostridium perfringens food poisoning caused by Japanese food without animal protein.

    Science.gov (United States)

    Miwa, N; Masuda, T; Terai, K; Kawamura, A; Otani, K; Miyamoto, H

    1999-08-01

    An outbreak of Clostridium perfringens food poisoning occurred in a senior citizen's home in Japan. Japanese food, spinach boiled with fried bean curd, was considered to be the causative food as a result of the detection of the C. perfringens enterotoxin gene by nested PCR. The number of enterotoxin-positive C. perfringens was enumerated as 4.3 x 10(5)/g in the causative food by the MPN method combined with nested PCR. By cultivation, enterotoxin-positive C. perfringens was isolated from all the fecal specimens of patients tested and the causative food. The isolates from patients were serotypable, heat-resistant and the majority produced enterotoxin, however most isolates from the causative food were nonserotypable, enterotoxin-negative and heat-sensitive.

  13. Induction of potential protective immunity against enterotoxemia in calves by single or multiple recombinant Clostridium perfringens toxoids.

    Science.gov (United States)

    Jiang, Zhigang; De, Yanyan; Chang, Jitao; Wang, Fang; Yu, Li

    2014-11-01

    Cattle enterotoxemia caused by Clostridium perfringens toxins is a noncontagious, sporadic, and fatal disease characterized by sudden death. Strategies for controlling and preventing cattle enterotoxemia are based on systematic vaccination of herds with toxoids. Because the process of producing conventional clostridial vaccines is dangerous, expensive, and time-consuming, the prospect of recombinant toxoid vaccines against diseases caused by C. perfringens toxins is promising. In this study, nontoxic recombinant toxoids derived from α-, β- and ε-toxins of C. perfringens, namely, rCPA247-370 , rCPB and rEtxHP, respectively, were expressed in Escherichia coli. High levels of specific IgG antibodies and neutralizing antibodies against the toxins were detected in sera from calves vaccinated with either a single recombinant toxoid or a mixed cocktail of all three recombinant toxoids, indicating the potential of these recombinant toxoids to provide calves with protective immunity against enterotoxemia caused by C. perfringens.

  14. Structural and biochemical analyses of a Clostridium perfringens sortase D transpeptidase

    Energy Technology Data Exchange (ETDEWEB)

    Suryadinata, Randy, E-mail: randy.suryadinata@csiro.au; Seabrook, Shane A.; Adams, Timothy E.; Nuttall, Stewart D.; Peat, Thomas S., E-mail: randy.suryadinata@csiro.au [Commonwealth Scientific and Industrial Research Organisation, 343 Royal Parade, Parkville, Victoria 3052 (Australia)

    2015-06-30

    The structure of C. perfringens sortase D was determined at 1.99 Å resolution. Comparative biochemical and structural analyses revealed that this transpeptidase may represent a new subclass of the sortase D family. The assembly and anchorage of various pathogenic proteins on the surface of Gram-positive bacteria is mediated by the sortase family of enzymes. These cysteine transpeptidases catalyze a unique sorting signal motif located at the C-terminus of their target substrate and promote the covalent attachment of these proteins onto an amino nucleophile located on another protein or on the bacterial cell wall. Each of the six distinct classes of sortases displays a unique biological role, with sequential activation of multiple sortases often observed in many Gram-positive bacteria to decorate their peptidoglycans. Less is known about the members of the class D family of sortases (SrtD), but they have a suggested role in spore formation in an oxygen-limiting environment. Here, the crystal structure of the SrtD enzyme from Clostridium perfringens was determined at 1.99 Å resolution. Comparative analysis of the C. perfringens SrtD structure reveals the typical eight-stranded β-barrel fold observed in all other known sortases, along with the conserved catalytic triad consisting of cysteine, histidine and arginine residues. Biochemical approaches further reveal the specifics of the SrtD catalytic activity in vitro, with a significant preference for the LPQTGS sorting motif. Additionally, the catalytic activity of SrtD is most efficient at 316 K and can be further improved in the presence of magnesium cations. Since C. perfringens spores are heat-resistant and lead to foodborne illnesses, characterization of the spore-promoting sortase SrtD may lead to the development of new antimicrobial agents.

  15. Prevalence and characterization of enterotoxin gene-carrying Clostridium perfringens isolates from retail meat products in Japan.

    Science.gov (United States)

    Miki, Yasuhiro; Miyamoto, Kazuaki; Kaneko-Hirano, Ikuko; Fujiuchi, Kanako; Akimoto, Shigeru

    2008-09-01

    Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. It is thought that C. perfringens food poisoning isolates typically carry the enterotoxin gene (cpe) on their chromosome, while isolates from other GI diseases, such as antibiotic-associated diarrhea, carry cpe on a transferable plasmid. However, food-borne GI disease outbreaks associated with C. perfringens isolates carrying plasmid-borne cpe (plasmid cpe isolates) were recently reported in Japan and Europe. To investigate whether retail food can be a reservoir for food poisoning generally, we evaluated Japanese retail meat products for the presence of two genotypes of enterotoxigenic C. perfringens. Our results demonstrated that approximately 70% of the Japanese retail raw meat samples tested were contaminated with low numbers of C. perfringens bacteria and 4% were contaminated with cpe-positive C. perfringens. Most of the cpe-positive C. perfringens isolates obtained from Japanese retail meat carried cpe on a plasmid. The plasmid cpe isolates exhibited lower spore heat resistance than did chromosomal cpe isolates. Collectively, these plasmid cpe isolates might be causative agents of food poisoning when foods are contaminated with these isolates from equipment and/or the environment after cooking, or they may survive in food that has not been cooked at a high enough temperature.

  16. Prevalence and Characterization of Enterotoxin Gene-Carrying Clostridium perfringens Isolates from Retail Meat Products in Japan▿

    Science.gov (United States)

    Miki, Yasuhiro; Miyamoto, Kazuaki; Kaneko-Hirano, Ikuko; Fujiuchi, Kanako; Akimoto, Shigeru

    2008-01-01

    Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. It is thought that C. perfringens food poisoning isolates typically carry the enterotoxin gene (cpe) on their chromosome, while isolates from other GI diseases, such as antibiotic-associated diarrhea, carry cpe on a transferable plasmid. However, food-borne GI disease outbreaks associated with C. perfringens isolates carrying plasmid-borne cpe (plasmid cpe isolates) were recently reported in Japan and Europe. To investigate whether retail food can be a reservoir for food poisoning generally, we evaluated Japanese retail meat products for the presence of two genotypes of enterotoxigenic C. perfringens. Our results demonstrated that approximately 70% of the Japanese retail raw meat samples tested were contaminated with low numbers of C. perfringens bacteria and 4% were contaminated with cpe-positive C. perfringens. Most of the cpe-positive C. perfringens isolates obtained from Japanese retail meat carried cpe on a plasmid. The plasmid cpe isolates exhibited lower spore heat resistance than did chromosomal cpe isolates. Collectively, these plasmid cpe isolates might be causative agents of food poisoning when foods are contaminated with these isolates from equipment and/or the environment after cooking, or they may survive in food that has not been cooked at a high enough temperature. PMID:18606797

  17. The NanI and NanJ Sialidases of Clostridium perfringens Are Not Essential for Virulence▿

    Science.gov (United States)

    Chiarezza, Martina; Lyras, Dena; Pidot, Sacha J.; Flores-Díaz, Marietta; Awad, Milena M.; Kennedy, Catherine L.; Cordner, Leanne M.; Phumoonna, Tongted; Poon, Rachael; Hughes, Meredith L.; Emmins, John J.; Alape-Girón, Alberto; Rood, Julian I.

    2009-01-01

    The essential toxin in Clostridium perfringens-mediated gas gangrene or clostridial myonecrosis is alpha-toxin, although other toxins and extracellular enzymes may also be involved. In many bacterial pathogens extracellular sialidases are important virulence factors, and it has been suggested that sialidases may play a role in gas gangrene. C. perfringens strains have combinations of three different sialidase genes, two of which, nanI and nanJ, encode secreted sialidases. The nanI and nanJ genes were insertionally inactivated by homologous recombination in derivatives of sequenced strain 13 and were shown to encode two functional secreted sialidases, NanI and NanJ. Analysis of these derivatives showed that NanI was the major sialidase in this organism. Mutation of nanI resulted in loss of most of the secreted sialidase activity, and the residual activity was eliminated by subsequent mutation of the nanJ gene. Only a slight reduction in the total sialidase activity was observed in a nanJ mutant. Cytotoxicity assays using the B16 melanoma cell line showed that supernatants containing NanI or overexpressing NanJ enhanced alpha-toxin-mediated cytotoxicity. Finally, the ability of nanI, nanJ, and nanIJ mutants to cause disease was assessed in a mouse myonecrosis model. No attenuation of virulence was observed for any of these strains, providing evidence that neither the NanI sialidase nor the NanJ sialidase is essential for virulence. PMID:19651873

  18. Evidence That the Enterotoxin Gene Can Be Episomal in Clostridium perfringens Isolates Associated with Non-Food-Borne Human Gastrointestinal Diseases

    OpenAIRE

    1998-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for the diarrheal and cramping symptoms of human C. perfringens type A food poisoning. CPE-producing C. perfringens isolates have also recently been associated with several non-food-borne human gastrointestinal (GI) illnesses, including antibiotic-associated diarrhea and sporadic diarrhea. The current study has used restriction fragment length polymorphism (RFLP) and pulsed-field gel electrophoresis (PFGE) analyses to compare the genoty...

  19. Survival of Clostridium perfringens During Simulated Transport and Stability of Some Plasmid-borne Toxin Genes under Aerobic Conditions

    Directory of Open Access Journals (Sweden)

    Johansson K-E

    2005-12-01

    Full Text Available Clostridium perfringens is a pathogen of great concern in veterinary medicine, because it causes enteric diseases and different types of toxaemias in domesticated animals. It is important that bacteria in tissue samples, which have been collected in the field, survive and for the classification of C. perfringens into the correct toxin group, it is crucial that plasmid-borne genes are not lost during transportation or in the diagnostic laboratory. The objectives of this study were to investigate the survival of C. perfringens in a simulated transport of field samples and to determine the stability of the plasmid-borne toxin genes cpb1 and etx after storage at room temperature and at 4°C. Stability of the plasmid-borne genes cpb1 and etx of C. perfringens CCUG 2035, and cpb2 from C. perfringens CIP 106526, JF 2255 and 6 field isolates in aerobic atmosphere was also studied. Survival of C. perfringens was similar in all experiments. The cpb1 and etx genes were detected in all isolates from samples stored either at room temperature or at 4°C for 24–44 h. Repeated aerobic treatment of C. perfringens CCUG 2035 and CIP 106526 did not result in the loss of the plasmid-borne genes cpb1, cpb2 or etx. Plasmid-borne genes in C. perfringens were found to be more stable than generally reported. Therefore, C. perfringens toxinotyping by PCR can be performed reliably, as the risk of plasmid loss seems to be a minor problem.

  20. Development and Application of New Mouse Models To Study the Pathogenesis of Clostridium perfringens Type C Enterotoxemias▿

    Science.gov (United States)

    Uzal, Francisco A.; Saputo, Juliann; Sayeed, Sameera; Vidal, Jorge E.; Fisher, Derek J.; Poon, Rachael; Adams, Vicki; Fernandez-Miyakawa, Mariano E.; Rood, Julian I.; McClane, Bruce A.

    2009-01-01

    Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality. PMID:19805537

  1. Development and application of new mouse models to study the pathogenesis of Clostridium perfringens type C Enterotoxemias.

    Science.gov (United States)

    Uzal, Francisco A; Saputo, Juliann; Sayeed, Sameera; Vidal, Jorge E; Fisher, Derek J; Poon, Rachael; Adams, Vicki; Fernandez-Miyakawa, Mariano E; Rood, Julian I; McClane, Bruce A

    2009-12-01

    Clostridium perfringens type C isolates cause enterotoxemias and enteritis in humans and livestock. While the major disease signs and lesions of type C disease are usually attributed to beta toxin (CPB), these bacteria typically produce several different lethal toxins. Since understanding of disease pathogenesis and development of improved vaccines is hindered by the lack of small animal models mimicking the lethality caused by type C isolates, in this study we developed two mouse models of C. perfringens type C-induced lethality. When inoculated into BALB/c mice by intragastric gavage, 7 of 14 type C isolates were lethal, whereas when inoculated intraduodenally, these strains were all lethal in these mice. Clinical signs in intragastrically and intraduodenally challenged mice were similar and included respiratory distress, abdominal distension, and neurological alterations. At necropsy, the small, and occasionally the large, intestine was dilated and gas filled in most mice developing a clinical response. Histological changes in the gut were relatively mild, consisting of attenuation of the mucosa with villus blunting. Inactivation of the CPB-encoding gene rendered the highly virulent type C strain CN3685 avirulent in the intragastric model and nearly nonlethal in the intraduodenal model. In contrast, inactivation of the genes encoding alpha toxin and perfringolysin O only slightly decreased the lethality of CN3685. Mice could be protected against lethality by intravenous passive immunization with a CPB antibody prior to intragastric challenge. This study proves that CPB is a major contributor to the systemic effects of type C infections and provides new mouse models for investigating the pathogenesis of type C-induced lethality.

  2. Expression of a Clostridium perfringens genome-encoded putative N-acetylmuramoyl-L-alanine amidase as a potential antimicrobial to control the bacterium

    Science.gov (United States)

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a substantial role in non-foodborne human, animal and avian diseases as well as human foodborne disease. Previously discovered C. perfringens bacteriophage lytic enzyme amino acid sequences were utilized to iden...

  3. Evaluating the performance of a new model for predicting the growth of Clostridium perfringens in cooked, uncured meat and poultry products under isothermal, heating, and dynamically cooling conditions

    Science.gov (United States)

    Clostridium perfringens Type A is a significant public health threat and may germinate, outgrow, and multiply during cooling of cooked meats. This study evaluates a new C. perfringens growth model in IPMP Dynamic Prediction using the same criteria and cooling data in Mohr and others (2015), but inc...

  4. Mucin gene mRNA levels in broilers challenged with eimeria and/or Clostridium perfringens.

    Science.gov (United States)

    Kitessa, Soressa M; Nattrass, Gregory S; Forder, Rebecca E A; McGrice, Hayley A; Wu, Shu-Biao; Hughes, Robert J

    2014-09-01

    The effects of Eimeria (EM) and Clostridium perfringens (CP) challenges on the mRNA levels of genes involved in mucin (Muc) synthesis (Muc2, Muc5ac, Muc13, and trefoil family factor-2 [TFF2]), inflammation (tumor necrosis factor alpha [TNF-alpha] and interleukin-18 [IL-18]), and metabolic processes (cluster of differentiation [CD]36) in the jejunum of broilers were investigated. Two parallel experiments involving 1) EM challenge and 2) EM and CP challenges were conducted. The first experiment was a 2 X 2 study with 12 birds per treatment (N = 48) involving fishmeal substitution (25%) in the diet (FM) and EM challenge. The treatments were: Control (FM-, EM-), Fishmeal (FM+, EM-), EM challenge (FM-, EM+), and fishmeal substitution and EM challenge (FM+, EM+). The second experiment was a 2 X 2 X 2 experiment with six birds per treatment (N = 48) involving fishmeal (FM-, FM+), Eimeria (EM-, EM+), and C perfringens (CP-, CP+). In both arms of the study, male broilers were given a starter diet for the whole period of 16 days, except those assigned to FM+, where 25% of the starter ration was replaced with fishmeal from days 8 to 14. EM inoculation was performed on day 9 and CP inoculation on days 14 and 15. The EM challenge birds were euthanatized for sampling on day 13; postmortem examination and sampling for the Eimeria plus C perfringens challenge arm of the study were on day 16. In the Eimeria challenge arm of the study, fishmeal supplementation significantly suppressed the mRNA levels of TNF-alpha, TFF2, and IL-18 pre-CP inoculation but simultaneously increased the levels of Muc13 and CD36 mRNAs. Birds challenged with Eimeria exhibited increased mRNA levels of Muc13, Muc5ac, TNF-alpha, and IL-18. In the Eimeria and C. perfringens challenge arm, birds exposed to EM challenge exhibited significantly lower mRNA levels of Muc2 and CD36. The mRNA levels of CD36 were also significantly suppressed by CP challenge. Our results showed that the transcription of mucin synthesis

  5. Oral Multiple Sclerosis Drugs Inhibit the In vitro Growth of Epsilon Toxin Producing Gut Bacterium, Clostridium perfringens

    Science.gov (United States)

    Rumah, Kareem R.; Vartanian, Timothy K.; Fischetti, Vincent A.

    2017-01-01

    There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod, and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium, Clostridium perfringens, may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro. Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens. Sphingosine, a Fingolimod homolog with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs. PMID:28180112

  6. Oral Multiple Sclerosis Drugs Inhibit the In vitro Growth of Epsilon Toxin Producing Gut Bacterium, Clostridium perfringens.

    Science.gov (United States)

    Rumah, Kareem R; Vartanian, Timothy K; Fischetti, Vincent A

    2017-01-01

    There are currently three oral medications approved for the treatment of multiple sclerosis (MS). Two of these medications, Fingolimod, and Teriflunomide, are considered to be anti-inflammatory agents, while dimethyl fumarate (DMF) is thought to trigger a robust antioxidant response, protecting vulnerable cells during an MS attack. We previously proposed that epsilon toxin from the gut bacterium, Clostridium perfringens, may initiate newly forming MS lesions due to its tropism for blood-brain barrier (BBB) vasculature and central nervous system myelin. Because gut microbiota will be exposed to these oral therapies prior to systemic absorption, we sought to determine if these compounds affect C. perfringens growth in vitro. Here we show that Fingolimod, Teriflunomide, and DMF indeed inhibit C. perfringens growth. Furthermore, several compounds similar to DMF in chemical structure, namely α, β unsaturated carbonyls, also known as Michael acceptors, inhibit C. perfringens. Sphingosine, a Fingolimod homolog with known antibacterial properties, proved to be a potent C. perfringens inhibitor with a Minimal Inhibitory Concentration similar to that of Fingolimod. These findings suggest that currently approved oral MS therapies and structurally related compounds possess antibacterial properties that may alter the gut microbiota. Moreover, inhibition of C. perfringens growth and resulting blockade of epsilon toxin production may contribute to the clinical efficacy of these disease-modifying drugs.

  7. Effect of tannins on the in vitro growth of Clostridium perfringens.

    Science.gov (United States)

    Elizondo, Ana M; Mercado, Elsa C; Rabinovitz, Bettina C; Fernandez-Miyakawa, Mariano E

    2010-10-26

    Vegetable tannins are water-soluble polyphenolic compounds of varying molecular weights that occur abundantly in nature. The diet of many free-ranging wild animals contains significant amounts of tannins. Also, commercial tannins are used in animal industry as food additives to improve animal performance. In order to further determine the capacity of tannins to inhibit the development of intestinal diseases produced by Clostridium pefringens, we evaluated here the effect of tannins from quebracho, chestnut or combinations of both on C. perfringens and their toxins. The C. perfringens (types A, B, C, D and E) growth obtained from the intestine of healthy and diseased animals was reduced in a dose-dependent manner in the presence of quebracho tannins, chestnut tannins, combinations of both or a commercial formula based in these tannins. Although the minimal inhibitory concentration of both tannins varied between isolates, no statistically significant differences were observed between isolates from healthy or sick animals. Comparative analysis showed that the concentrations of quebracho tannin inhibiting the growth of C. perfringens were higher than chestnut tannin. In fact, antibacterial effect of quebracho tannin was increased up to 20 times with the addition of 25% of chestnut tannin and 85 times with 75% of chestnut tannin. Antibacterial activity of the commercial product was up to ~50 times higher than quebracho tannin alone. Quebracho tannin showed partial bactericidal activity, whereas chestnut tannin activity was stronger. Both tannins were able to reduce the alpha toxin lecithinase activity and epsilon toxin cytotoxicity in MDCK cells. These results suggest that tannin-supplemented diet could be useful to prevent some clostridial diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Claudins Overexpression in Ovarian Cancer: Potential Targets for Clostridium Perfringens Enterotoxin (CPE Based Diagnosis and Therapy

    Directory of Open Access Journals (Sweden)

    Diana P. English

    2013-05-01

    Full Text Available Claudins are a family of tight junction proteins regulating paracellular permeability and cell polarity with different patterns of expression in benign and malignant human tissues. There are approximately 27 members of the claudin family identified to date with varying cell and tissue-specific expression. Claudins-3, -4 and -7 represent the most highly differentially expressed claudins in ovarian cancer. While their exact role in ovarian tumors is still being elucidated, these proteins are thought to be critical for ovarian cancer cell invasion/dissemination and resistance to chemotherapy. Claudin-3 and claudin-4 are the natural receptors for the Clostridium perfringens enterotoxin (CPE, a potent cytolytic toxin. These surface proteins may therefore represent attractive targets for the detection and treatment of chemotherapy-resistant ovarian cancer and other aggressive solid tumors overexpressing claudin-3 and -4 using CPE-based theranostic agents.

  9. Carriage of Clostridium perfringens by benthic crabs in a sewage-polluted estuary.

    Science.gov (United States)

    La Sala, Luciano F; Redondo, Leandro M; Díaz Carrasco, Juan M; Pereyra, Ana María; Farber, Marisa; Jost, Helen; Fernández-Miyakawa, Mariano E

    2015-08-15

    The Estuary of Bahía Blanca (EBB), Argentina, is an important wetland under intense sewage pollution. We investigated the occurrence of Clostridium perfringens (CP) in populations of two benthic crabs (Neohelice granulata and Cyrtograpsus angulatus) and in sediment from the EBB. CP was found in 49.1% of the crabs and all of the isolates were identified as type A. The alpha (cpa) and enterotoxin (cpe) encoding genes were identified. Genetic analyses identified 13 novel sequence types, and found no clustering among isolates, suggesting that CP is not part of the crabs' commensal flora. CP carriage was 51 times more likely in crabs from the area nearest sewage outfalls compared with crabs from a reference site. Our in vitro experiments suggest that the carriage of CP in crabs is transient. The use of these benthic crabs as monitoring organisms of sewage pollution in coastal habitats is proposed.

  10. Molecular typing and epidemiological survey of prevalence of Clostridium perfringens types by multiplex PCR.

    Science.gov (United States)

    Yoo, H S; Lee, S U; Park, K Y; Park, Y H

    1997-01-01

    Clostridium perfringens has been classified into five toxigenic types (A through E) on the basis of its capability to produce major lethal toxins (alpha, beta, epsilon, and iota toxins). Seroneutralization with mice or guinea pigs has been used to type each toxin, but this conventional method has some disadvantages. Therefore, we used a molecular biological technique to type the bacterium in the present study. A multiplex PCR was developed for this purpose. This method has several advantages in comparison with seroneutralization with mice or guinea pigs. By this method, we also investigated the most prevalent type(s) of the organism in Korean calves, piglets, and chickens showing clinical symptoms such as diarrhea, enterotoxemia, and necrotic enteritis. Only type A was isolated from calves and chickens, while type C (2 of 14 isolates), in addition to type A, was isolated from piglets. These results suggested that seroneutralization could be replaced by our new method and that type A of C. perfringens is the most prevalent type in livestock in Korea.

  11. Analysis of the virulence-associated RevSR two-component signal transduction system of Clostridium perfringens.

    Science.gov (United States)

    Cheung, Jackie K; Wisniewski, Jessica A; Adams, Vicki M; Quinsey, Noelene S; Rood, Julian I

    2016-09-01

    Clostridium perfringens is a Gram-positive, anaerobic, spore-forming bacterium that causes human gas gangrene (clostridial myonecrosis) and food poisoning. Early studies showed that virulence was regulated by the VirSR two-component signal transduction system. However, our identification of the RevR orphan response regulator indicated that more than one system was involved in controlling virulence. To further characterize this virulence-associated regulator, gel mobility shift experiments, coupled with DNase I footprinting, were used to identify the RevR DNA binding sequence. Bioinformatics analysis suggested that an orphan sensor histidine kinase, CPE1757 (renamed RevS), was the cognate sensor of RevR. Interaction between RevS and RevR was demonstrated by use of a bacterial two-hybrid system and validated by protein-protein interaction studies using biolayer interferometry. To assess the involvement of RevS in virulence regulation, the revS gene was inactivated by Targetron insertion. When isogenic wild-type, revS and complemented revS strains were tested in a mouse myonecrosis model, the revS mutant was found to be attenuated in virulence, which was similar to the attenuation observed previously with the revR mutant. However, transcriptional analysis of selected RevR-regulated genes in the revS mutant revealed a different pattern of expression to a revR mutant, suggesting that the RevSR system is more complex than originally thought. Taken together, the results have led to the identification and characterization of the two essential parts of a new regulatory network that is involved in the regulation of virulence in C. perfringens.

  12. Comparison of the Effect of Curing Ingredients Derived from Purified and Natural Sources on Inhibition of Clostridium perfringens Outgrowth during Cooling of Deli-Style Turkey Breast.

    Science.gov (United States)

    King, Amanda M; Glass, Kathleen A; Milkowski, Andrew L; Sindelar, Jeffrey J

    2015-08-01

    The antimicrobial impact of purified and natural sources of both nitrite and ascorbate were evaluated against Clostridium perfringens during the postthermal processing cooling period of deli-style turkey breast. The objective of phase I was to assess comparable concentrations of nitrite (0 or 100 ppm) and ascorbate (0 or 547 ppm) from both purified and natural sources. Phase II was conducted to investigate concentrations of nitrite (50, 75, or 100 ppm) from cultured celery juice powder and ascorbate (0, 250, or 500 ppm) from cherry powder to simulate alternative curing formulations. Ground turkey breast (75% moisture, 1.2% salt, pH 6.2) treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in 10 additional hours. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar; experiments were replicated three times. In phase I, uncured, purified nitrite, and natural nitrite treatments without ascorbate had 5.3-, 4.2-, and 4.4-log increases in C. perfringens, respectively, at 15 h, but <1-log increase was observed at the end of chilling in treatments containing 100 ppm of nitrite and 547 ppm of ascorbate from either source. In phase II, 0, 50, 75, and 100 ppm of nitrite and 50 ppm of nitrite plus 250 ppm of ascorbate supported 4.5-, 3.9-, 3.5-, 2.2-, and 1.5-log increases in C. perfringens, respectively. In contrast, <1-log increase was observed after 15 h in the remaining phase II treatments supplemented with 50 ppm of nitrite and 500 ppm of ascorbate or ≥75 ppm of nitrite and ≥250 ppm of ascorbate. These results confirm that equivalent concentrations of nitrite, regardless of the source, provide similar inhibition of C. perfringens during chilling and that ascorbate enhances the antimicrobial effect of nitrite on C

  13. Primeiro relato no Brasil de mastite necrótica bovina por Clostridium perfringens tipo A First report in Brazil of bovine necrotic mastitis due to Clostridium perfringens type A

    Directory of Open Access Journals (Sweden)

    Luciana Aramuni Gonçalves

    2006-08-01

    Full Text Available Relata-se o primeiro caso no Brasil de mastite bovina por Clostridium perfringens tipo A. O quadro clínico caracterizou-se por necrose da papila mamária e porção ventral do quarto afetado. O agente foi isolado em cultura pura e identificado como tipo A por PCR a partir do leite do quarto mamário afetado.This report describes a case of bovine mastitis due to Clostridium perfringens type A for first time in Brazil. The unical case showed necrosis of papilla mammary and ventral portion of the affected quarter. The microorganism was isolated in pure culture and identified as type A by PCR from milk of the affected mammary quarter.

  14. Produção e caracterização de anticorpos monoclonais contra toxina épsilon de Clostridium perfringens Tipo D Production and characterization of monoclonal antibodies against Clostridium perfringens Type D epsilon toxin

    Directory of Open Access Journals (Sweden)

    Theonys Diógenes Freitas

    2009-02-01

    Full Text Available Clostridium perfringens tipo D é o agente etiológico da enterotoxemia em ruminantes, causada pela toxina épsilon e caracterizada por edema cardíaco, pulmonar, renal e cerebral. Anticorpos monoclonais contra toxina épsilon de C. perfringens tipo D foram produzidos a partir da fusão da linhagen de mieloma P3-X63-Ag8 653 com células do baço de camundongos Balb/c imunizados com o toxóide épsilon. Seis linhagens de híbridos secretores de anticorpos monoclonais das classes e IgM e IgG foram estabelecidas.Clostridium perfringens type D is the aetiological agent of enterotoxemia in ruminants. The disease is caused by epsilon toxin characterized by cardiac, pulmonary, kidney and brain edema. Monoclonal antibodies were produced by using myeloma cell line P3-X63-Ag8 653 fused with spleen cells from Balb/c mice, immunized with epsilon toxoid of C. perfringens type D. Six hybrids were established secreting monoclonal antibodies of the IgM class and IgG3 subclass.

  15. Necrotizing Enterocolitis in Preterm Pigs Is Associated with Increased Density of Intestinal Mucosa-Associated Bacteria Including Clostridium perfringens

    DEFF Research Database (Denmark)

    Støy, Ann Cathrine Findal; Mølbak, Lars; Delègue, Camilla Lindholm;

    2015-01-01

    Background: Necrotizing enterocolitis (NEC) is associated with changes in the luminal gut microbiota. It is not known whether the mucosa-associated microbiota is affected by NEC and stimulates inflammatory lesions. Objective: We hypothesized that the density of the mucosa-associated microbiota...... correlates with NEC severity in preterm pigs and that in vitro infection with increasing densities of Clostridium perfringens, which has been associated with NEC in preterm infants, would lead to a transcriptional response related to the inflammatory conditions of NEC. Methods: First, we determined...... the density of total bacteria and C. perfringens in the distal small intestinal mucosa of 58 NEC and healthy preterm pigs using quantitative PCR. Next, we analyzed in IPEC-J2 cells the effect of different infection densities of C. perfringens type A on the expression of genes related to intestinal function...

  16. An unusual gangrenous goat mastitis caused by Staphylococcus aureus, Clostridium perfringens and Escherichia coli co-infection Mastite gangrenosa caprina atípica causada por co-infecção por Staphylococcus aureus, Clostridium perfringens e Escherichia coli

    Directory of Open Access Journals (Sweden)

    M.G. Ribeiro

    2007-06-01

    Full Text Available Relata-se, pela primeira vez no Brasil, a ocorrência de mastite gangrenosa caprina atípica causada pela co-infecção por Staphylococcus aureus, Clostridium perfringens e Escherichia coli em uma cabra da raça Boer, na segunda semana de lactação. Descrevem-se os achados clínicos, os procedimentos de diagnóstico microbiológico e a conduta terapêutica.

  17. Evidence that the Agr-like quorum sensing system regulates the toxin production, cytotoxicity and pathogenicity of Clostridium perfringens type C isolate CN3685.

    Science.gov (United States)

    Vidal, Jorge E; Ma, Menglin; Saputo, Julian; Garcia, Jorge; Uzal, Francisco A; McClane, Bruce A

    2012-01-01

    Clostridium perfringens possesses at least two functional quorum sensing (QS) systems, i.e. an Agr-like system and a LuxS-dependent AI-2 system. Both of those QS systems can reportedly control in vitro toxin production by C. perfringens but their importance for virulence has not been evaluated. Therefore, the current study assessed whether these QS systems might regulate the pathogenicity of CN3685, a C. perfringens type C strain. Since type C isolates cause both haemorrhagic necrotic enteritis and fatal enterotoxemias (where toxins produced in the intestines are absorbed into the circulation to target other internal organs), the ability of isogenic agrB or luxS mutants to cause necrotizing enteritis in rabbit small intestinal loops or enterotoxemic lethality in mice was evaluated. Results obtained strongly suggest that the Agr-like QS system, but not the LuxS-dependent AI-2 QS system, is required for CN3685 to cause haemorrhagic necrotizing enteritis, apparently because the Agr-like system regulates the production of beta toxin, which is essential for causing this pathology. The Agr-like system, but not the LuxS-mediated AI-2 system, was also important for CN3685 to cause fatal enterotoxemia. These results provide the first direct evidence supporting a role for any QS system in clostridial infections.

  18. Clostridium perfringens removal in different stages in a Drinking Water Treatments plant; Eliminacion de Clostidium perfringens en diversas etapas de una estacion de tratamiento de aguas potables

    Energy Technology Data Exchange (ETDEWEB)

    Ormad, M. P.; Lanao, M.; Goni, P.; Ibarz, C.; Ovelleiro, J. L.

    2008-07-01

    The purpose of this research is to evaluate the effectiveness of different stages, which take part in the conventional treatments used in the drinking water treatment plants in Spain, in the removal of a microbiological indicator of faecal pollution, Clostridium perfringens. The stages studied are pre oxidation with chlorine and ozone, chemical precipitation, adsorption with activated coal and filtration sand. The pre oxidation, either with sodium hypochlorite or with ozone, gets final recounts below the detection limit with the conditions studied (> 8 log). In the rest of stages, the removal is minimal, achieving 1,32 logarithmic units at best case. (Author) 6 refs.

  19. Disruption in the cecal microbiota of chickens challenged with Clostridium perfringens and other factors was alleviated by Bacillus licheniformis supplementation

    Science.gov (United States)

    Ni, Xueqin; Zhou, Mengjia; Zeng, Yan; Wang, Hesong; Zhou, Yi; Zhu, Hui; Pan, Kangcheng; Li, Guangyao

    2017-01-01

    Clostridium perfringens can induce necrotic enteritis of chickens, which causes large economic losses every year. Bacillus licheniformis, a probiotic, can inhibit the growth of pathogenic bacteria such as Clostridium perfringens, thereby improving the health status of chickens. However, from a microbial ecology perspective, the mechanisms by which alterations to the gut microbiota improve health remain unknown. In this study, we used Illumina MiSeq sequencing to investigate the cecal microbiota of a negative control group (NC), a C. perfringens and Eimeria challenge group with fishmeal supplementation (PC), a group supplemented with fishmeal and infected with coccidia (FC), and group PC with B. licheniformis supplementation (BL). We found that the health status of C. perfringens-challenged chickens was compromised, and that B. licheniformis improved the growth of the chickens challenged with pathogens. Microbial diversity analysis and taxonomic profiling of groups NC, PC, and FC revealed a disturbed cecal microflora of the birds with C. perfringens. We also characterized the microbiota of the chickens in the BL group using several methods. Principal coordinate analysis demonstrated that, compared with group PC, the bacterial community structure of group BL was more similar to that of group NC. Linear discriminant analysis with effect size revealed less differentially represented bacterial taxa between groups BL and NC than between groups PC and NC. In addition, groups BL and NC appeared to have similar overrepresented microbial taxa (such as Bacteroides, Helicobacter, Megamonas, and Akkermansia) compared with group PC. Finally, a phylogenetic investigation of communities by reconstruction of unobserved states analysis indicated that large differences existed between group PC and groups NC and BL. In conclusion, pre-treatment with B. licheniformis reduced the disturbance of the cecal microbiome induced by challenge with C. perfringens and other factors in broiler

  20. Ulcerative enterocolitis in two goats associated with enterotoxin- and beta2 toxin-positive Clostridium perfringens type D.

    Science.gov (United States)

    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.

  1. B-cell epitope of beta toxin of Clostridium perfringens genetically conjugated to a carrier protein: expression, purification and characterization of the chimeric protein.

    Science.gov (United States)

    Bhatia, Bharti; Solanki, Amit Kumar; Kaushik, Himani; Dixit, Aparna; Garg, Lalit C

    2014-10-01

    Beta toxin (btx) is the prime virulence factor for the pathogenesis of Clostridium perfringens type C strain, known to cause necrotic enteritis and enterotoxaemia in mammalian species. The existing vaccines targeting btx are formaldehyde inactivated culture filtrates of Clostridium. These filtrates raise antigenic load in the host leading to nonspecific and poor responses. The present study aimed to overcome these drawbacks and generate a chimeric protein carrying in silico identified B-cell epitope of btx fused with a carrier protein as a vaccine candidate. Using bioinformatic tools, three stretches of amino acids were predicted as putative B-cell epitopes. One of the epitopes spanning 140-156 amino acid residues was genetically conjugated with B-subunit of heat labile enterotoxin (LTB) of Escherichia coli and expressed as a translational fusion in Vibrio cholerae secretory expression system. High level expression of the recombinant fusion protein rLTB-Btx140-156 was obtained and the protein was successfully purified. The recombinant protein retained the native LTB property to pentamerize and bind to GM1 ganglioside receptor of LTB. The antigenicity of both the epitope and the carrier protein was maintained in fusion protein as indicated by immunoblotting against anti-LTB and anti-btx antibody. The rLTB-Btx140-156 fusion protein therefore can be evaluated as a potential vaccine candidate against C. perfringens.

  2. The mycotoxin deoxynivalenol predisposes for the development of Clostridium perfringens-induced necrotic enteritis in broiler chickens.

    Directory of Open Access Journals (Sweden)

    Gunther Antonissen

    Full Text Available Both mycotoxin contamination of feed and Clostridium perfringens-induced necrotic enteritis have an increasing global economic impact on poultry production. Especially the Fusarium mycotoxin deoxynivalenol (DON is a common feed contaminant. This study aimed at examining the predisposing effect of DON on the development of necrotic enteritis in broiler chickens. An experimental Clostridium perfringens infection study revealed that DON, at a contamination level of 3,000 to 4,000 µg/kg feed, increased the percentage of birds with subclinical necrotic enteritis from 20±2.6% to 47±3.0% (P<0.001. DON significantly reduced the transepithelial electrical resistance in duodenal segments (P<0.001 and decreased duodenal villus height (P = 0.014 indicating intestinal barrier disruption and intestinal epithelial damage, respectively. This may lead to an increased permeability of the intestinal epithelium and decreased absorption of dietary proteins. Protein analysis of duodenal content indeed showed that DON contamination resulted in a significant increase in total protein concentration (P = 0.023. Furthermore, DON had no effect on in vitro growth, alpha toxin production and netB toxin transcription of Clostridium perfringens. In conclusion, feed contamination with DON at concentrations below the European maximum guidance level of 5,000 µg/kg feed, is a predisposing factor for the development of necrotic enteritis in broilers. These results are associated with a negative effect of DON on the intestinal barrier function and increased intestinal protein availability, which may stimulate growth and toxin production of Clostridium perfringens.

  3. Delayed Clostridium perfringens growth from a spore inocula by sodium lactate in sous-vide chicken products.

    Science.gov (United States)

    Juneja, Vijay K

    2006-04-01

    Clostridium perfringens growth from a spore inoculum was investigated in vacuum-packaged, cook-in-bag marinated chicken breast that included 0%, 1.5%, 3%, or 4.8% sodium lactate (NaL; w/w). The packages were processed to an internal temperature of 71.1 degrees C, ice chilled and stored at 4, 19, and 25 degrees C. The total C. perfringens population was determined by plating diluted samples on Tryptose-sulfite-cycloserine agar followed by anaerobic incubation for 48 h at 37 degrees C. At 25 degrees C, addition of 1.5% NaL was effective in delaying growth for 29 h. Increasing the NaL level to 4.8%, C. perfringens growth from a spore inoculum during storage at 25 degrees C for 480 h was not observed. At 19 degrees C, the growth was > 6 log 10 cfu/g by 288 h in control samples. In samples with 3.0% or 4.8% NaL, the growth of C. perfringens from spores was dramatically restricted with little or no growth in 648 h at 19 degrees C. C. perfringens growth was not observed at 4 degrees C regardless of NaL concentration. The D-values at 55 degrees C ranged from 47.40 (no NaL) to 57.58 min (1.5% NaL). Cyclic and static temperature abuse of refrigerated products for 20 h did not permit C. perfringens growth. However, temperature abuse of products for periods 24 h or longer in the absence of NaL led to growth of C. perfringens from a spore inoculum. An extra degree of safety may be assured in such products by supplementation with NaL at 1.5-4.8% NaL level.

  4. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin.

    Directory of Open Access Journals (Sweden)

    Alexander Belyy

    Full Text Available Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia.

  5. Alterations in DNA Gyrase and Topoisomerase IV in Resistant Mutants of Clostridium perfringens Found after In Vitro Treatment with Fluoroquinolones

    Science.gov (United States)

    Rafii, Fatemeh; Park, Miseon; Novak, John S.

    2005-01-01

    To compare mutations in the DNA gyrase (gyrA and gyrB) and topoisomerase IV (parC and parE) genes of Clostridium perfringens, which are associated with in vitro exposure to fluoroquinolones, resistant mutants were selected from eight strains by serial passage in the presence of increasing concentrations of norfloxacin, ciprofloxacin, gatifloxacin, or trovafloxacin. The nucleotide sequences of the entire gyrA, gyrB, parC, and parE genes of 42 mutants were determined. DNA gyrase was the primary target for each fluoroquinolone, and topoisomerase IV was the secondary target. Most mutations appeared in the quinolone resistance-determining regions of gyrA (resulting in changes of Asp-87 to Tyr or Gly-81 to Cys) and parC (resulting in changes of Asp-93 or Asp-88 to Tyr or Ser-89 to Ile); only two mutations were found in gyrB, and only two mutations were found in parE. More mutants with multiple gyrA and parC mutations were produced with gatifloxacin than with the other fluoroquinolones tested. Allelic diversity was observed among the resistant mutants, for which the drug MICs increased 2- to 256-fold. Both the structures of the drugs and their concentrations influenced the selection of mutants. PMID:15673722

  6. Stabilization of Clostridium perfringens collagenase mRNA by VR-RNA-dependent cleavage in 5' leader sequence.

    Science.gov (United States)

    Obana, Nozomu; Shirahama, Yu; Abe, Kimihiro; Nakamura, Kouji

    2010-09-01

    The small RNA (sRNA), VR-RNA that is directly regulated by the VirR/VirS two-component system, regulates many genes including toxin genes such as collagenase (colA) and phospholipase C (plc) in Clostridium perfringens. Although the VR-RNA 3' region is sufficient to regulate the colA and plc genes, the molecular mechanism of toxin gene regulation by VR-RNA remains unclear. Here, we found that colA mRNA is cleaved at position -79 and -78 from the A of the first codon (ATG) in the presence of VR-RNA. The processed transcripts were stable compared with longer intact transcripts. On the other hand, colA mRNA was labile in a VR-RNA-deficient strain, and processed transcripts were undetectable. The stability and processing of colA mRNA were restored by transformation of the 3' region of VR-RNA-expression vector. The 3' region of VR-RNA and colA mRNA had significant complementation and interacted in vitro. These results show that VR-RNA base pairs with colA mRNA and induces cleavage in the 5' untranslated region (UTR) of colA mRNA, which leads to the stabilization of colA mRNA and the activation of colA expression. © 2010 Blackwell Publishing Ltd.

  7. Interaction of Clostridium perfringens epsilon-toxin with biological and model membranes: A putative protein receptor in cells.

    Science.gov (United States)

    Manni, Marco M; Sot, Jesús; Goñi, Félix M

    2015-03-01

    Epsilon-toxin (ETX) is a powerful toxin produced by some strains of Clostridium perfringens (classified as types B and D) that is responsible for enterotoxemia in animals. ETX forms pores through the plasma membrane of eukaryotic cells, consisting of a β-barrel of 14 amphipathic β-strands. ETX shows a high specificity for certain cell lines, of which Madin-Darby canine kidney (MDCK) is the first sensitive cell line identified and the most studied one. The aim of this study was to establish the role of lipids in the toxicity caused by ETX and the correlation of its activity in model and biological membranes. In MDCK cells, using cell counting and confocal microscopy, we have observed that the toxin causes cell death mediated by toxin binding to plasma membrane. Moreover, ETX binds and permeabilizes the membranes of giant plasma membrane vesicles (GPMV). However, little effect is observed on protein-free vesicles. The data suggest the essential role of a protein receptor for the toxin in cell membranes.

  8. Clostridium Perfringens Epsilon Toxin Binds to Membrane Lipids and Its Cytotoxic Action Depends on Sulfatide.

    Directory of Open Access Journals (Sweden)

    Carles Gil

    Full Text Available Epsilon toxin (Etx is one of the major lethal toxins produced by Clostridium perfringens types B and D, being the causal agent of fatal enterotoxemia in animals, mainly sheep and goats. Etx is synthesized as a non-active prototoxin form (proEtx that becomes active upon proteolytic activation. Etx exhibits a cytotoxic effect through the formation of a pore in the plasma membrane of selected cell targets where Etx specifically binds due to the presence of specific receptors. However, the identity and nature of host receptors of Etx remain a matter of controversy. In the present study, the interactions between Etx and membrane lipids from the synaptosome-enriched fraction from rat brain (P2 fraction and MDCK cell plasma membrane preparations were analyzed. Our findings show that both Etx and proEtx bind to lipids extracted from lipid rafts from the two different models as assessed by protein-lipid overlay assay. Lipid rafts are membrane microdomains enriched in cholesterol and sphingolipids. Binding of proEtx to sulfatide, phosphatidylserine, phosphatidylinositol (3-phosphate and phosphatidylinositol (5-phosphate was detected. Removal of the sulphate groups via sulfatase treatment led to a dramatic decrease in Etx-induced cytotoxicity, but not in proEtx-GFP binding to MDCK cells or a significant shift in oligomer formation, pointing to a role of sulfatide in pore formation in rafts but not in toxin binding to the target cell membrane. These results show for the first time the interaction between Etx and membrane lipids from host tissue and point to a major role for sulfatides in C. perfringens epsilon toxin pathophysiology.

  9. Organization of the cpe locus in CPE-positive clostridium perfringens type C and D isolates.

    Directory of Open Access Journals (Sweden)

    Jihong Li

    Full Text Available Clostridium perfringens enterotoxin (encoded by the cpe gene contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644 found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity.

  10. Effects of Clostridium perfringens Beta-Toxin on the Rabbit Small Intestine and Colon▿

    Science.gov (United States)

    Vidal, Jorge E.; McClane, Bruce A.; Saputo, Juliann; Parker, Jaquelyn; Uzal, Francisco A.

    2008-01-01

    Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 μg of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates. PMID:18625730

  11. Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon.

    Science.gov (United States)

    Vidal, Jorge E; McClane, Bruce A; Saputo, Juliann; Parker, Jaquelyn; Uzal, Francisco A

    2008-10-01

    Clostridium perfringens type B and type C isolates, which produce beta-toxin (CPB), cause fatal diseases originating in the intestines of humans or livestock. Our previous studies demonstrated that CPB is necessary for type C isolate CN3685 to cause bloody necrotic enteritis in a rabbit ileal loop model and also showed that purified CPB, in the presence of trypsin inhibitor (TI), can reproduce type C pathology in rabbit ileal loops. We report here a more complete characterization of the effects of purified CPB in the rabbit small and large intestines. One microgram of purified CPB, in the presence of TI, was found to be sufficient to cause significant accumulation of hemorrhagic luminal fluid in duodenal, jejunal, or ileal loops treated for 6 h with purified CPB, while no damage was observed in corresponding loops receiving CPB (no TI) or TI alone. In contrast to the CPB sensitivity of the small intestine, the colon was not affected by 6 h of treatment with even 90 mug of purified CPB whether or not TI was present. Time course studies showed that purified CPB begins to induce small intestinal damage within 1 h, at which time the duodenum is less damaged than the jejunum or ileum. These observations help to explain why type B and C infections primarily involve the small intestine, establish CPB as a very potent and fast-acting toxin in the small intestines, and confirm a key role for intestinal trypsin as an innate intestinal defense mechanism against CPB-producing C. perfringens isolates.

  12. Functional analysis of the VirSR phosphorelay from Clostridium perfringens.

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    Jackie K Cheung

    Full Text Available Toxin production in Clostridium perfringens is controlled by the VirSR two-component signal transduction system, which comprises the VirS sensor histidine kinase and the VirR response regulator. Other studies have concentrated on the elucidation of the genes controlled by this network; there is little information regarding the phosphorelay cascade that is the hallmark of such regulatory systems. In this study, we have examined each step in this cascade, beginning with autophosphorylation of VirS, followed by phosphotransfer from VirS to VirR. We also have studied the effects of gene dosage and phosphorylation in vivo. We have used random and site-directed mutagenesis to identify residues in VirS that are important for its function and have identified a region in the putative sensory domain of VirS that appeared to be essential for function. In vitro phosphorylation studies showed that VirSc, a truncated VirS protein that lacked the N-terminal sensory domain, was capable of autophosphorylation and could subsequently act as a phosphodonor for its cognate response regulator, VirR. Conserved residues of both VirS and VirR, including the D57 residue of VirR, were shown to be essential for this process. By use of Targetron technology, we were able to introduce a single copy of virR or virR(D57N onto the chromosome of a virR mutant of C. perfringens. The results showed that in vivo, when virR was present in single copy, the production of wild-type levels of perfringolysin O was dependent on the presence of virS and an unaltered D57 residue in VirR. These results provide good evidence that phosphorylation is critical for VirR function.

  13. Organization of the cpe Locus in CPE-Positive Clostridium perfringens Type C and D Isolates

    Science.gov (United States)

    Li, Jihong; Miyamoto, Kazuaki; Sayeed, Sameera; McClane, Bruce A.

    2010-01-01

    Clostridium perfringens enterotoxin (encoded by the cpe gene) contributes to several important human, and possibly veterinary, enteric diseases. The current study investigated whether cpe locus organization in type C or D isolates resembles one of the three (one chromosomal and two plasmid-borne) cpe loci commonly found amongst type A isolates. Multiplex PCR assays capable of detecting sequences in those type A cpe loci failed to amplify products from cpe-positive type C and D isolates, indicating these isolates possess different cpe locus arrangements. Therefore, restriction fragments containing the cpe gene were cloned and sequenced from two type C isolates and one type D isolate. The obtained cpe locus sequences were then used to construct an overlapping PCR assay to assess cpe locus diversity amongst other cpe-positive type C and D isolates. All seven surveyed cpe-positive type C isolates had a plasmid-borne cpe locus partially resembling the cpe locus of type A isolates carrying a chromosomal cpe gene. In contrast, all eight type D isolates shared the same plasmid-borne cpe locus, which differed substantially from the cpe locus present in other C. perfringens by containing two copies of an ORF with 67% identity to a transposase gene (COG4644) found in Tn1546, but not previously associated with the cpe gene. These results identify greater diversity amongst cpe locus organization than previously appreciated, providing new insights into cpe locus evolution. Finally, evidence for cpe gene mobilization was found for both type C and D isolates, which could explain their cpe plasmid diversity. PMID:20532170

  14. Clostridium perfringens delta toxin is sequence related to beta toxin, NetB, and Staphylococcus pore-forming toxins, but shows functional differences.

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    Maria Manich

    Full Text Available Clostridium perfringens produces numerous toxins, which are responsible for severe diseases in man and animals. Delta toxin is one of the three hemolysins released by a number of C. perfringens type C and possibly type B strains. Delta toxin was characterized to be cytotoxic for cells expressing the ganglioside G(M2 in their membrane. Here we report the genetic characterization of Delta toxin and its pore forming activity in lipid bilayers. Delta toxin consists of 318 amino acids, its 28 N-terminal amino acids corresponding to a signal peptide. The secreted Delta toxin (290 amino acids; 32619 Da is a basic protein (pI 9.1 which shows a significant homology with C. perfringens Beta toxin (43% identity, with C. perfringens NetB (40% identity and, to a lesser extent, with Staphylococcus aureus alpha toxin and leukotoxins. Recombinant Delta toxin showed a preference for binding to G(M2, in contrast to Beta toxin, which did not bind to gangliosides. It is hemolytic for sheep red blood cells and cytotoxic for HeLa cells. In artificial diphytanoyl phosphatidylcholine membranes, Delta and Beta toxin formed channels. Conductance of the channels formed by Delta toxin, with a value of about 100 pS to more than 1 nS in 1 M KCl and a membrane potential of 20 mV, was higher than those formed by Beta toxin and their distribution was broader. The results of zero-current membrane potential measurements and single channel experiments suggest that Delta toxin forms slightly anion-selective channels, whereas the Beta toxin channels showed a preference for cations under the same conditions. C. perfringens Delta toxin shows a significant sequence homolgy with C. perfringens Beta and NetB toxins, as well as with S. aureus alpha hemolysin and leukotoxins, but exhibits different channel properties in lipid bilayers. In contrast to Beta toxin, Delta toxin recognizes G(M2 as receptor and forms anion-selective channels.

  15. Adhesive properties of an outer structure of Clostridium perfringens type A isolated from piglets with with catarrhal enteritis Propriedades adesivas de uma estrutura externa de Clostridium perfringens tipo A isolada de leitões com enterite catarral

    Directory of Open Access Journals (Sweden)

    Elizabeth Pelosi Teixeira

    1999-07-01

    Full Text Available One strain (S32 of Clostridium perfringens type A was isolated from a case of catarrhal enteritis of piglets. This strain was able to adhere to HeLa cells showing an adherence index (AI of 25.15 ± 1.26 (mean ± 1 standard error of the mean. Treatment of the bacterial cells with trypsin (0.25mg/ml decreased in 70%-80% the AI and metaperiodate (10mg/ml abolished completely the adherence, suggesting that the structure responsible for this phenomenon was probably a glycoprotein. Heating of bacterial suspensions (100ºC/5 min before carrying out the adhesion test decreased the AI rendering it equal to the negative controls. Rabbit homologous S32 antiserum inhibited the adherence up to dilutions of 1: 640, at least. The piglet ileal loop assay, carried out with strains S32 and Jab-1 (negative control demonstrated that the strain S32 was able to adhere to the intestinal epithelial cells when examined after Gram staining. Transmission electron microcopy (TEM demonstrated that S32 strain displayed a loose fibrillar material not seen with Jab-1. Stabilization of the bacterial cells with homologous antiserum of strain S32, followed by staining with rhuteniun red, revealed loose long fibrillar material on the outer surface of the cells, that sometimes could be seen spreading out from the cells and linking bacterial cells. The question whether this structure might be an adhesin for this strain of Cl. perfringes type A, perhaps playing a role in the pathogenesis of the catarrhal enteritis of piglets, is dependent on further studies.Uma amostra (S32 de Clostridium perfringens tipo A foi isolada de um caso de enterite catarral em leitões. Esta amostra foi capaz de aderir a células HeLa mostrando um índice de adesão (AI de 25,15 ± 1,26 (media ± 1 erro padrão da media. Tratamento das células bacterianas com tripsina (0,25mg/ml diminuiu 70%-80% e metaperiodato (10mg/ml aboliu significantemente a adesão, sugerindo que a estrutura responsável por esta

  16. Effects of Tylosin on Bacterial Mucolysis, Clostridium perfringens Colonization, and Intestinal Barrier Function in a Chick Model of Necrotic Enteritis

    Science.gov (United States)

    Collier, C. T.; van der Klis, J. D.; Deplancke, B.; Anderson, D. B.; Gaskins, H. R.

    2003-01-01

    Necrotic enteritis (NE) is a worldwide poultry disease caused by the alpha toxin-producing bacterium Clostridium perfringens. Disease risk factors include concurrent coccidial infection and the dietary use of cereal grains high in nonstarch polysaccharides (NSP), such as wheat, barley, rye, and oats. Outbreaks of NE can be prevented or treated by the use of in-feed antibiotics. However, the current debate regarding the prophylactic use of antibiotics in animal diets necessitates a better understanding of factors that influence intestinal colonization by C. perfringens as well as the pathophysiological consequences of its growth. We report a study with a chick model of NE, which used molecular (16S rRNA gene [16S rDNA]) and culture-based microbiological techniques to investigate the impact of the macrolide antibiotic tylosin phosphate (100 ppm) and a dietary NSP (pectin) on the community structure of the small intestinal microbiota relative to colonization by C. perfringens. The effects of tylosin and pectin on mucolytic activity of the microbiota and C. perfringens colonization and their relationship to pathological indices of NE were of particular interest. The data demonstrate that tylosin reduced the percentage of mucolytic bacteria in general and the concentration of C. perfringens in particular, and these responses correlated in a temporal fashion with a reduction in the occurrence of NE lesions and an improvement in barrier function. The presence of pectin did not significantly affect the variables measured. Thus, it appears that tylosin can control NE through its modulation of C. perfringens colonization and the mucolytic activity of the intestinal microbiota. PMID:14506046

  17. Clostridium perfringens growth from spore inocula in sous-vide processed pork-based Mexican entrée.

    Science.gov (United States)

    Miguel-Garcia, Denise Y; Juneja, Vijay K; Valenzuela-Melendrez, Martin; Díaz-Cinco, Martha E; Thippareddi, H; Aida Peña-Ramos, E

    2009-01-01

    The combined effect of Citricidal wih irradiation on Clostridium perfringens growth from spores in a sous-vide processed marinated pork meat Mexican entrée was investigated. Citricidal was added at 200 or 800 ppm after mixing pork meat with tomatillo sauce and inoculated with 3 log(10) CFU/g of C. perfringens spores. Samples were irradiated at either 0 or 2 kGy, heated to an internal temperature of 71 degrees C, and stored at 4 degrees C for 28 d, 15 degrees C for 45 d, and 25 degrees C for 26 h. To simulate the conditions that may occur during transportation, distribution, storage, or handling in supermarkets or by consumers, the effect of static temperature abuse on C. perfringens growth was assessed by transferring samples stored at 4 to 25 degrees C for 13 and 15 h. Total C. perfringens populations were determined by plating diluted samples on tryptose-sulfite-cycloserine agar. Growth was not observed up to 45 d of storage at 15 degrees C in samples supplemented with 800 ppm of Citricidal. At 25 degrees C, no significant differences (P > 0.05) on the lag phase duration due to antimicrobial treatments was observed. The temperature abuse of refrigerated products for up to 15 h did not lead to C. perfringens growth to high infective dose levels of 1 million cells required to cause food poisoning. The results suggest that 800 ppm Citricidal can have significant bacteriostatic activity against C. perfringens and may provide a degree of protection against this pathogen in sous-vide processed marinated pork meat Mexican entrée, under mild temperature abuse (

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

    Science.gov (United States)

    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

    2015-06-12

    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 ɸGVE2 to the cell-wall binding domain (CWB) from the endolysin of the C. perfringens-specific bacteriophage ɸCP26F. 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.

  19. TcpM: a novel relaxase that mediates transfer of large conjugative plasmids from Clostridium perfringens.

    Science.gov (United States)

    Wisniewski, Jessica A; Traore, Daouda A; Bannam, Trudi L; Lyras, Dena; Whisstock, James C; Rood, Julian I

    2016-03-01

    Conjugative transfer of toxin and antibiotic resistance plasmids in Clostridium perfringens is mediated by the tcp conjugation locus. Surprisingly, neither a relaxase gene nor an origin of transfer (oriT) has been identified on these plasmids, which are typified by the 47 kb tetracycline resistance plasmid pCW3. The tcpM gene (previously called intP) encodes a potential tyrosine recombinase that was postulated to be an atypical relaxase. Mutagenesis and complementation studies showed that TcpM was required for wild-type transfer of pCW3 and that a tyrosine residue, Y259, was essential for TcpM activity, which was consistent with the need for a relaxase-mediated hydrophilic attack at the oriT site. Other catalytic residues conserved in tyrosine recombinases were not required for TcpM activity, suggesting that TcpM was not a site-specific recombinase. Mobilization studies led to the identification of the oriT site, which was located in the 391 bp intergenic region upstream of tcpM. The oriT site was localized to a 150 bp region, and gel mobility shift studies showed that TcpM could bind to this region. Based on these studies we postulate that conjugative transfer of pCW3 involves the atypical relaxase TcpM binding to and processing the oriT site to initiate plasmid transfer.

  20. Investigating an outbreak of Clostridium perfringens gastroenteritis in a school using smartphone technology, London, March 2013.

    Science.gov (United States)

    Simone, B; Atchison, C; Ruiz, B; Greenop, P; Dave, J; Ready, D; Maguire, H; Walsh, B; Anderson, S

    2014-05-15

    On 22 March 2013, 150 of 1,255 students (13–17 years) and staff at a school in London reported gastrointestinal symptoms; onset peaked 8 to 12 hours after a lunch served in the school on 21 March. We performed a retrospective cohort study of all students and staff. We defined cases as school attenders on 20 and 21 March with onset of gastrointestinal symptoms between 20 and 23 March. We tested food, environmental and stool samples of cases for common pathogens and bacterial toxins. We administered an online questionnaire via email, encouraging the use of smartphones to respond, to measure risk of illness for food items eaten at school on 20 and 21 March. Survey response was 45%. Adjusted risk ratios were generated in a multivariable analysis. Those who ate chicken balti on 21 March were 19.3 times more likely to become ill (95% confidence interval: 7.3–50.9). Clostridium perfringens was detected in all 19 stool samples collected. Within eight school hours of its launch, 412 of 561 (73%) responders had completed the survey. Hygienic standards in the kitchen were satisfactory. The investigation was done rapidly due to smartphone technology and we recommend considering this technology in future outbreaks.

  1. Fast kinetics of nucleotide binding to Clostridium perfringens family II pyrophosphatase containing CBS and DRTGG domains.

    Science.gov (United States)

    Jämsen, J; Baykov, A A; Lahti, R

    2012-02-01

    We earlier described CBS-pyrophosphatase of Moorella thermoacetica (mtCBS-PPase) as a novel phosphohydrolase that acquired a pair of nucleotide-binding CBS domains during evolution, thus endowing the protein with the capacity to be allosterically regulated by adenine nucleotides (Jämsen, J., Tuominen, H., Salminen, A., Belogurov, G. A., Magretova, N. N., Baykov, A. A., and Lahti, R. (2007) Biochem. J., 408, 327-333). We herein describe a more evolved type of CBS-pyrophosphatase from Clostridium perfringens (cpCBS-PPase) that additionally contains a DRTGG domain between the two CBS domains in the regulatory part. cpCBS-PPase retained the ability of mtCBS-PPase to be inhibited by micromolar concentrations of AMP and ADP and activated by ATP and was additionally activated by diadenosine polyphosphates (AP(n)A) with n > 2. Stopped-flow measurements using a fluorescent nucleotide analog, 2'(3')-O-(N-methylanthranoyl)-AMP, revealed that cpCBS-PPase interconverts through two different conformations with transit times on the millisecond scale upon nucleotide binding. The results suggest that the presence of the DRTGG domain affords greater flexibility to the regulatory part, allowing it to more rapidly undergo conformational changes in response to binding.

  2. C-Terminal Clostridium perfringens Enterotoxin-Mediated Antigen Delivery for Nasal Pneumococcal Vaccine.

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    Hidehiko Suzuki

    Full Text Available Efficient vaccine delivery to mucosal tissues including mucosa-associated lymphoid tissues is essential for the development of mucosal vaccine. We previously reported that claudin-4 was highly expressed on the epithelium of nasopharynx-associated lymphoid tissue (NALT and thus claudin-4-targeting using C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE effectively delivered fused antigen to NALT and consequently induced antigen-specific immune responses. In this study, we applied the C-CPE-based vaccine delivery system to develop a nasal pneumococcal vaccine. We fused C-CPE with pneumococcal surface protein A (PspA, an important antigen for the induction of protective immunity against Streptococcus pneumoniae infection, (PspA-C-CPE. PspA-C-CPE binds to claudin-4 and thus efficiently attaches to NALT epithelium, including antigen-sampling M cells. Nasal immunization with PspA-C-CPE induced PspA-specific IgG in the serum and bronchoalveolar lavage fluid (BALF as well as IgA in the nasal wash and BALF. These immune responses were sufficient to protect against pneumococcal infection. These results suggest that C-CPE is an efficient vaccine delivery system for the development of nasal vaccines against pneumococcal infection.

  3. Inactivation and ultrastructure analysis of Bacillus spp. and Clostridium perfringens spores.

    Science.gov (United States)

    Brantner, Christine A; Hannah, Ryan M; Burans, James P; Pope, Robert K

    2014-02-01

    Bacterial endospores are resistant to many environmental factors from temperature extremes to ultraviolet irradiation and are generally more difficult to inactivate or kill than vegetative bacterial cells. It is often considered necessary to treat spores or samples containing spores with chemical fixative solutions for prolonged periods of time (e.g., 1-21 days) to achieve fixation/inactivation to enable electron microscopy (EM) examination outside of containment laboratories. Prolonged exposure to chemical fixatives, however, can alter the ultrastructure of spores for EM analyses. This study was undertaken to determine the minimum amount of time required to inactivate/sterilize and fix spore preparations from several bacterial species using a universal fixative solution for EM that maintains the ultrastructural integrity of the spores. We show that a solution of 4% paraformaldehyde with 1% glutaraldehyde inactivated spore preparations of Bacillus anthracis, Bacillus cereus, Bacillus megaterium, Bacillus thuringiensis, and Clostridium perfringens in 30 min, and Bacillus subtilis in 240 min. These results suggest that this fixative solution can be used to inactivate and fix spores from several major groups of bacterial spore formers after 240 min, enabling the fixed preparations to be removed from biocontainment and safely analyzed by EM outside of biocontainment.

  4. Oligomerization of Clostridium perfringens epsilon toxin is dependent upon caveolins 1 and 2.

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    Christine M Fennessey

    Full Text Available Evidence from multiple studies suggests that Clostridium perfringens ε-toxin is a pore-forming toxin, assembling into oligomeric complexes in the plasma membrane of sensitive cells. In a previous study, we used gene-trap mutagenesis to identify mammalian factors contributing to toxin activity, including caveolin-2 (CAV2. In this study, we demonstrate the importance of caveolin-2 and its interaction partner, caveolin-1 (CAV1, in ε-toxin-induced cytotoxicity. Using CAV2-specific shRNA in a toxin-sensitive human kidney cell line, ACHN, we confirmed that cells deficient in CAV2 exhibit increased resistance to ε-toxin. Similarly, using CAV1-specific shRNA, we demonstrate that cells deficient in CAV1 also exhibit increased resistance to the toxin. Immunoprecipitation of CAV1 and CAV2 from ε-toxin-treated ACHN cells demonstrated interaction of both CAV1 and -2 with the toxin. Furthermore, blue-native PAGE indicated that the toxin and caveolins were components of a 670 kDa protein complex. Although ε-toxin binding was only slightly perturbed in caveolin-deficient cells, oligomerization of the toxin was dramatically reduced in both CAV1- and CAV2-deficient cells. These results indicate that CAV1 and -2 potentiate ε-toxin induced cytotoxicity by promoting toxin oligomerization - an event which is requisite for pore formation and, by extension, cell death.

  5. Efecto Antagónico del Kefir sobre Endosporas y Células Vegetativas de Bacillus Cereus y Clostridium Perfringens Antagonistic Effect of the Kefir on Endospores and Vegetative Cells of Bacillus Cereus and Clostridium Perfringens

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    Ricardo J Anselmo

    2010-01-01

    Full Text Available Se estudió el efecto bactericida del kéfir sobre endosporas y células vegetativas de Bacillus cereus y Clostridium perfringens inoculados en kéfir durante su almacenamiento. Muestras de kéfir de dos procedencias, uno de origen italiano y otro peruano, fueron inoculadas con una población conocida de B. cereus y Cl. perfringens (10(6 UFC/mL y conservados a 4ºC durante 30 días. Cada dos días se realizó un recuento de bacterias lácticas, de levaduras, de los patógenos agregados y se determinó el pH. La población de células vegetativas de B. cereus se redujo a niveles no detectables en 14 días de almacenamiento y la de sus endosporas en 20 días de almacenamiento. Respecto a Cl. perfringens se redujo la población de células vegetativas y de endosporas a niveles no detectables en 14 y 18 días de almacenamiento, respectivamente. Los resultados confirman los efectos bactericidas de los cultivos iniciadores del kéfir.The bactericide effect of the kefir was studied on endospores and vegetative cells of Bacillus cereus and Clostridium perfringens inoculated in kefir during its storage. Samples of kefir of two different origins, one Italian and the other Peruvian were inoculated with a well-known population of B. cereus and Cl. perfringens (10(6 UFC/mL and conserved at 4ºC during 30 days. Every two days it was carried out a recount of lactic bacteria, of yeasts, of the pathogenic added and the pH was determined. The population of vegetative cells of B. cereus reduced to non detectable levels in 14 days of storage and of its endospores in 20 days of storage. Regarding Cl. perfringens the population of vegetative cells and its endospores reduced to non detectable levels in 14 and 18 days of storage, respectively. The results confirm the bactericide effects of the starter cultures of kefir.

  6. Estudo da aderencia "in vitro" e "in vivo" de amostras de clostridium perfringens tipos A e C isoladas de diferentes origens

    OpenAIRE

    1991-01-01

    Resumo: A intoxicação alimentar no homem, causada pelo Clostridium perfringens tipo A e a enterocolite necrotizante, doença letal para animais recém-nascidos, produzida provavelmente pelo C. perfringens tipo C são patologias atrbuídas a produção de toxinas. No entanto, nos estudos sobre estas doenças há uma lacuna no que fiz respeito a possíveis mecanismos de aderência de formas vegetativas do C. perfringens que lhe permitiriam colonizar a mucosa intestinal do homem e animais. Assim, o presen...

  7. Hemorrhagic enterocolitis and death in two felines (Panthera tigris altaica and Panthera leo) associated with Clostridium perfringens type A.

    Science.gov (United States)

    Zhang, Yanlong; Hou, Zhijun; Ma, Jianzhang

    2012-06-01

    Severe hemorrhagic enterocolitis was observed in a Siberian tiger (Panthera tigris altaica) and a lion (Panthera leo). Both animals developed acute depression, anorexia, and bloody diarrhea several days before death. Small and large intestines were diffusely congested, edematous, necrotic, and filled with hemorrhagic fluid, and mesenteric lymph nodes were enlarged and congested. Pure and abundant growth of gram-positive bacilli was obtained in culture under anaerobic conditions from the livers of both felines. Identification of highly virulent Clostridium perfringens Type A was based on pathologic lesions, hemolytic patterns, morphologic structure, and polymerase chain reaction. Animal inoculation assays indicated that C. perfringens Type A played an important role in the pathogenesis of both felines.

  8. Residues involved in the pore-forming activity of the Clostridium perfringens iota toxin.

    Science.gov (United States)

    Knapp, Oliver; Maier, Elke; Waltenberger, Eva; Mazuet, Christelle; Benz, Roland; Popoff, Michel R

    2015-02-01

    Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334-359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β-strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the β-pore-forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic β-strands affected pore formation, single-channel conductance and ion selectivity (S339E-S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ-clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single-channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ-clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single-channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E-S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.

  9. Embryonated chicken eggs as an alternative model for mixed Clostridium perfringens and Eimeria tenella infection in chickens.

    Science.gov (United States)

    Alnassan, Alaa Aldin; Shehata, Awad Ali; Kotsch, Marianne; Lendner, Matthias; Daugschies, Arwid; Bangoura, Berit

    2013-06-01

    The chorioallantoic membrane (CAM) of chicken embryo eggs is a suitable model for viral and bacterial infections. In the present study, a new approach for testing the pathogenesis and virulence of Clostridium perfringens and Eimeria tenella dual infections as a model using the CAM of embryonated chicken eggs was developed. For this purpose, 24 specific pathogen-free (SPF) embryonated chicken eggs were divided into four groups (n = 6) and designated group E, group CP, group CPE, and NC. Sporozoites of E. tenella (20,000 sporozoites) were inoculated into 10-day-old embryonated SPF chicken eggs (groups E and CPE) via allantoic sac route. At 15-day-old, eggs of groups CP and CPE were infected with 10 (4)  cfu C. perfringens via the same route. Assessment of pathogenicity was assessed using gross and histopathological lesions. Embryo mortality reached 17 % after mono-infection with C. perfringens and/or E. tenella and 50 % in the mixed-infected group. Lesions in the CAMs were most numerous and most severe in co-infected eggs (group CPE), reaching the maximum score of 3 in 50 % of the inoculated eggs (P < 0.01). In Eimeria spp.-infected eggs (group E), lesions of score were between 1 and 2. Mono-infection with C. perfringens did not lead to a significant occurrence of lesions. Histopathological investigations of the CAM revealed clusters of Gram-positive bacteria, infiltration with leukocytes, lymphocytes, and developmental stages of E. tenella in the co-infected group. These data suggest that embryonated eggs could be an in ovo model for studying the pathogenesis of mixed infection with Eimeria and C. perfringens.

  10. Characterization of polymorphisms and isoforms of the Clostridium perfringens phospholipase C gene (plc) reveals high genetic diversity.

    Science.gov (United States)

    Siqueira, Flávia F; Almeida, Marcelle O; Barroca, Tatiana M; Horta, Carolina C R; Carmo, Anderson O; Silva, Rodrigo O S; Pires, Prhiscylla S; Lobato, Francisco C F; Kalapothakis, Evanguedes

    2012-10-12

    Clostridium perfringens phospholipase C (Cp-PLC), also called alpha-toxin, is encoded by the plc gene and has been implicated in several diseases; however, only a few studies have described polymorphisms in this gene. The aim of this study was to analyze polymorphisms in the Cp-PLC nucleotide and amino acid sequences obtained from isolates from different regions and to compare them to Clostridium phospholipase C sequences deposited in the NCBI database. Environmental samples (sediment, poultry feed, sawdust) and stool samples (from poultry, bovine, swine, horse, caprine, bird, dog, rabbit, toucan) were collected from healthy and sick animals. A total of 73 isolates were analyzed with the majority of samples belonging to the toxin type A subtype and possessing the gene encoding for the beta-2 toxin. Comparison of plc gene sequences from respective isolates revealed a high genetic diversity in the nucleotide sequences of mature Cp-PLC. Sequence comparisons identified 30 amino acid substitutions and 34 isoforms including some isoforms with substitutions in amino acids critical to toxin function. Comparison of sequences obtained in this study to Cp-PLC sequences obtained from the NCBI database resulted in the identification of 11 common haplotypes and 22 new isoforms. Phylogenetic analysis of phospholipase C sequences obtained from other Clostridium species identified relationships previously described. This report describes a broad characterization of the genetic diversity in the C. perfringens plc gene resulting in the identification of various isoforms. A better understanding of sequences encoding phospholipase C isoforms may reveal changes associated with protein function and C. perfringens virulence.

  11. Evaluation of a new mathematical model to describe Clostridium perfringens growth during the cooling of cooked ground beef

    Directory of Open Access Journals (Sweden)

    Marcelo Decker

    2013-09-01

    Full Text Available A mathematical model previously developed to study microbial growth in food products under an isothermal environment was adapted to a time-varying temperature regime. The resulting model was applied to study the growth of Clostridium perfringens in meat products. This micro-organism is of particular relevance to public health and economy due to the loss of productivity caused by it. Results showed a similar performance of the model used compared to the Baranyi model under an isothermal situation and a slightly better performance under a non-isothermal temperature profile.

  12. Screening Escherichia coli, Enterococcus faecalis, and Clostridium perfringens as Indicator Organisms in Evaluating Pathogen-Reducing Capacity in Biogas Plants

    DEFF Research Database (Denmark)

    Watcharasukarn, Montira; Kaparaju, Prasad Laxmi-Narasimha; Steyer, Jean-Philippe

    2009-01-01

    perfringens strain were exposed to 37A degrees C for 15 days, 55A degrees C for 48 h, and 70A degrees C for 24 h. C. perfringens was the most heat-resistant organism followed by E. faecalis, while E. coli was the most heat-sensitive organism. E. coli was reduced below detection limit at all temperatures...... with log(10) reductions of 4.94 (10 s), 4.37 (40 min), and 2.6 (5 days) at 70A degrees C, 55A degrees C, and 37A degrees C, respectively. Maximum log(10) reductions for E. faecalis were 1.77 at 70A degrees C (1 day), 1.7 at 55A degrees C (2 days) and 3.13 at 37A degrees C (15 days). For C. perfringens......, maximum log(10) reduction at 37A degrees C was 1.35 log(10) units (15 days) compared to less than 1 unit at 55 and 70A degrees C. Modeling results showed that E. faecalis and C. perfringens had higher amount of heat-resistant fraction than E. coli. Thus, E. faecalis and C. perfringens can be used...

  13. Gene-trap mutagenesis identifies mammalian genes contributing to intoxication by Clostridium perfringens ε-toxin.

    Directory of Open Access Journals (Sweden)

    Susan E Ivie

    Full Text Available The Clostridium perfringens ε-toxin is an extremely potent toxin associated with lethal toxemias in domesticated ruminants and may be toxic to humans. Intoxication results in fluid accumulation in various tissues, most notably in the brain and kidneys. Previous studies suggest that the toxin is a pore-forming toxin, leading to dysregulated ion homeostasis and ultimately cell death. However, mammalian host factors that likely contribute to ε-toxin-induced cytotoxicity are poorly understood. A library of insertional mutant Madin Darby canine kidney (MDCK cells, which are highly susceptible to the lethal affects of ε-toxin, was used to select clones of cells resistant to ε-toxin-induced cytotoxicity. The genes mutated in 9 surviving resistant cell clones were identified. We focused additional experiments on one of the identified genes as a means of validating the experimental approach. Gene expression microarray analysis revealed that one of the identified genes, hepatitis A virus cellular receptor 1 (HAVCR1, KIM-1, TIM1, is more abundantly expressed in human kidney cell lines than it is expressed in human cells known to be resistant to ε-toxin. One human kidney cell line, ACHN, was found to be sensitive to the toxin and expresses a larger isoform of the HAVCR1 protein than the HAVCR1 protein expressed by other, toxin-resistant human kidney cell lines. RNA interference studies in MDCK and in ACHN cells confirmed that HAVCR1 contributes to ε-toxin-induced cytotoxicity. Additionally, ε-toxin was shown to bind to HAVCR1 in vitro. The results of this study indicate that HAVCR1 and the other genes identified through the use of gene-trap mutagenesis and RNA interference strategies represent important targets for investigation of the process by which ε-toxin induces cell death and new targets for potential therapeutic intervention.

  14. Predictive model for growth of Clostridium perfringens during cooling of cooked uncured beef.

    Science.gov (United States)

    Juneja, Vijay K; Marks, Harry; Thippareddi, Harshavardhan

    2008-02-01

    This paper considers growth models including one based on Baranyi's equations for growth and the other based on the logistic function. Using a common approach for constructing dynamic models for predicting Clostridium perfringens growth in ready-to-eat uncured beef during cooling, there was no appreciable difference between the models' predictions when the population of cells was within the lag or exponential phases of growth. The developed models can be used for designing safe cooling processes; however, the discrepancies between predicted and observed growths obtained in this study, together with discrepancies reported in other papers using the same, or similar methodology as used in this paper, point to a possible inadequacy of the derived models. In particular, the appropriateness of the methodology depends on the appropriateness of using estimated growth kinetics obtained from experiments conducted in isothermal environments for determining coefficients of differential equations that are used for predicting growth in constantly changing (dynamic) environments. The coefficients are interpreted as instantaneous specific rates of change that are independent of prior history. However, there is no known scientific reason that would imply the truth of this assumption. Incorporating a different, less restrictive assumption, allowing for a dependency on the prior history of cells for these kinetic parameters, might lead to models that provide more accurate estimates of growth. For example, a cooling scenario of 54.4-27 degrees C in 1.5h, the average predicted and observed log(10) relative growths were 1.1log(10) and 0.66log(10), respectively, a difference of 0.44log(10,) whereas, when assuming a particular dependency of history, the predicted value was 0.8log(10). More research is needed to characterize the behavior of growth kinetic parameters relative to prior history in dynamic environments.

  15. Expression of Clostridium perfringens enterotoxin receptors claudin-3 and claudin-4 in prostate cancer epithelium.

    Science.gov (United States)

    Long, H; Crean, C D; Lee, W H; Cummings, O W; Gabig, T G

    2001-11-01

    The mRNA for Rvp.1 (rat ventral prostate) increases in abundance before gland involution after androgen deprivation. Rvp.1 is homologous to CPE-R, the high-affinity intestinal epithelial receptor for Clostridium perfringens enterotoxin (CPE), and is sufficient to mediate CPE binding and trigger subsequent toxin-mediated cytolysis. Rvp.1 (claudin-3) and CPE-R (claudin-4) are members of a larger family of transmembrane tissue-specific claudin proteins that are essential components of intercellular tight junction structures regulating paracellular ion flux. However, claudin-3 and claudin-4 are the only family members capable of mediating CPE binding and cytolysis. The present study was designed to study the expression of claudin-3 and claudin-4 in human prostate tissue as potential targets for CPE toxin-mediated therapy for prostate cancer. On human multiple-tissue Northern blot analysis, mRNAs for both claudin-3 and claudin-4 were expressed at high levels in prostate tissue. In normal prostate tissue, expression of claudin-3 was localized exclusively within acinar epithelial cells by in situ mRNA hybridization. Compared with expression within prostate epithelial cells in surrounding normal glandular tissue, expression of claudin-3 mRNA remained high in the epithelium of prostate adenocarcinoma (10 of 10) and prostatic intraepithelial neoplasia (five of five). Prostate adenocarcinoma cells metastatic to bone were obtained from a patient with disease progression during antiandrogen therapy. These metastatic cells were prostate-specific antigen-positive by immunohistochemical staining and also expressed functional CPE receptors as measured by sensitivity to CPE-induced cell lysis. The persistent high level of claudin-3 expression in prostate adenocarcinoma and functional cytotoxicity of CPE in metastatic androgen-independent prostate adenocarcinoma suggests a new potential therapeutic strategy for prostate cancer.

  16. Occurrence of Clostridium perfringens in the broiler chicken processing plant as determined by recovery in iron milk medium.

    Science.gov (United States)

    Craven, S E

    2001-12-01

    Over 30 years ago, Clostridium perfringens was reported as a contaminant of the processing plant and processed carcasses of broiler chickens. Poultry processing procedures and methods for detecting C. perfringens have changed since that time. Therefore, a study was conducted to determine the incidence and numbers of C. perfringens in the water of the scald tank, the water of the chill tank, and the rinse water of the processed carcasses from modern broiler chicken processing plants. In trial 1, collected samples were inoculated into iron milk medium (IMM) and incubated at 46 degrees C for 18 h (the traditional method) or at 37 degrees C for 3 h followed by incubation at 46 degrees C for 15 h (an injury recovery method). Each of three preselected broiler chicken flocks from two integrators were the first processed for that processing shift. The overall incidence of confirmed C. perfringens in samples associated with the three flocks was 40% of postprocessing scald water samples, 13% of preprocessing chill water samples, 13% of postprocessing chill water samples, and 19% of carcass rinses. The incidence of C. perfringens in samples incubated in IMM using the injury recovery procedure was significantly higher than in samples incubated in IMM by the traditional method, but only when all samples associated with the three flocks were pooled. In trial 2, water samples from each tank of a three-tank counterflow scalder, water samples from the prechill and chill tank, and samples of carcass rinses were collected in the middle of a processing shift during multiple visits to a processing plant. Samples were inoculated into IMM with neomycin and polymyxin B sulfate (IMMA) and incubated using the traditional and injury recovery procedures. The incidence of C. perfringens in water samples was 100% from scald tank 1, 100% from scald tank 2, 100% from scald tank 3, 88% from the prechill tank, and 63% from the chill tank. The incidence in carcass rinse samples was 67%. The mean

  17. An Intelligent Procedure for the Detection and Classification of Chickens Infected by Clostridium Perfringens Based on their Vocalization

    Directory of Open Access Journals (Sweden)

    M Sadeghi

    2015-12-01

    Full Text Available ABSTRACT In this study, an intelligent method was implemented for the detection and classification of chickens by infected Clostridium perfringens type A based on their vocalization. To this aim, the birds were first divided into two groups that were placed in separate cages with 15 chickens each. Chickens were inoculated with Clostridium perfringens type A on day 14. In order to ensure the absence of secondary diseases and their probable effect on bird vocalization, vaccines for common diseases were administered. During 30 days of the experiment, chicken vocalization was recorded every morning at 8 a.m. using a microphone and a data collection card under equal and controlled conditions. Sound signals were investigated in time domains, and 23 features were selected. Using Fisher Discriminate Analysis (FDA, five of the most important and effective features were chosen. Neural Network Pattern Recognition (NNPR structure with one hidden layer was applied to detect signals and classifying healthy and unhealthy chickens. Firstly, this neural network was trained with 34 samples, after which eight samples were tested for accuracy. Classification accuracy was 66.6 and 100% for days 16 and 22; i.e., two and eight days after the disease, respectively. The results of this study demonstrated the usefulness and effectiveness of intelligent methods for diagnosing diseases in chickens.

  18. Characterization of Bacteriophages Virulent for Clostridium perfringens and Identification of Phage Lytic Enzymes as Alternatives to Antibiotics for Potential Control of the Bacterium

    Science.gov (United States)

    Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne disease as well as non-food-borne human, animal, and poultry diseases. There has been a resurgent interest in the use of bacteriophages or their gene products to control b...

  19. Riverbed sediments in the Apies River, South Africa: recommending the use of both Clostridium perfringens and Escherichia coli as indicators of faecal pollution

    CSIR Research Space (South Africa)

    Abia, ALK

    2015-12-01

    Full Text Available to be assessed in order to monitor faecal pollution in sediments do not exist. In this study, Escherichia coli (a well-established indicator) and Clostridium perfringens were monitored to determine their suitability as indicators for faecal pollution of sediments...

  20. Clostridium Perfringens a-Toxin and NetB Toxin Antibodies and their possible role in protection against Necrotic Enteritis and Gangrenous Dermatitis in broiler chickens

    Science.gov (United States)

    Necrotic enteritis (NE) and gangrenous dermatitis (GD) are important infectious diseases of poultry. Although NE and GD share a common pathogen, Clostridium perfringens, they differ in other important aspects, such as clinical signs, pathologic symptoms, and age of onset. The primary virulence facto...

  1. Inhibition of clostridium perfringens spore germination and outgrowth by buffered vinegar and lemon juice concentrate during chilling.....of ground turkey road containing minimal ingredients

    Science.gov (United States)

    Inhibition of Clostridium perfringens spore germination and outgrowth in ground turkey roast containing minimal ingredients (salt and sugar), by buffered vinegar (MoStatin V) and a blend (buffered) of lemon juice concentrate and vinegar (MoStatin LV) was evaluated. Ground turkey roast was formulat...

  2. Inhibition of Clostridium perfringens spore germination and outgrowth by lemon juice and vinegar product in reduced NaCl roast beef

    Science.gov (United States)

    Inhibition of Clostridium perfringens spore germination and outgrowth in reduced sodium roast beef by a blend of buffered lemon juice concentrate and vinegar (MoStatin LV) during abusive exponential cooling was evaluated. Roast beef containing salt (NaCl; 1, 1.5, or 2%, wt/wt), blend of sodium pyro-...

  3. Endothelial binding of beta toxin to small intestinal mucosal endothelial cells in early stages of experimentally induced Clostridium perfringens type C enteritis in pigs.

    Science.gov (United States)

    Schumacher, V L; Martel, A; Pasmans, F; Van Immerseel, F; Posthaus, H

    2013-07-01

    Beta toxin (CPB) is known to be an essential virulence factor in the development of lesions of Clostridium perfringens type C enteritis in different animal species. Its target cells and exact mechanism of toxicity have not yet been clearly defined. Here, we evaluate the suitability of a neonatal piglet jejunal loop model to investigate early lesions of C. perfringens type C enteritis. Immunohistochemically, CPB was detected at microvascular endothelial cells in intestinal villi during early and advanced stages of lesions induced by C. perfringens type C. This was first associated with capillary dilatation and subsequently with widespread hemorrhage in affected intestinal segments. CPB was, however, not demonstrated on intestinal epithelial cells. This indicates a tropism of CPB toward endothelial cells and suggests that CPB-induced endothelial damage plays an important role in the early stages of C. perfringens type C enteritis in pigs.

  4. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies.

    Science.gov (United States)

    Alam, Syed Imteyaz; Bansod, Sunita; Singh, Lokendra

    2008-11-11

    Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs) have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65-78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3) was the most abundant protein (43.3%), followed by methylaspartate ammonia-lyase (36.8%) and 2-phosphoglycerate dehydratase (35.6%). All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG) functional category, either of posttranslational modification, protein turnover, and chaperones (O) or energy production and conversion (C). The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

  5. Immunization against Clostridium perfringens cells elicits protection against Clostridium tetani in mouse model: identification of cross-reactive proteins using proteomic methodologies

    Directory of Open Access Journals (Sweden)

    Singh Lokendra

    2008-11-01

    Full Text Available Abstract Background Clostridium tetani and Clostridium perfringens are among the medically important clostridial pathogens causing diseases in man and animals. Several homologous open reading frames (ORFs have been identified in the genomes of the two pathogens by comparative genomic analysis. We tested a likelihood of extensive sharing of common epitopes between homologous proteins of these two medically important pathogens and the possibility of cross-protection using active immunization. Results Eight predominant cross-reactive spots were identified by mass spectrometry and had hits in the C. tetani E88 proteome with significant MOWSE scores. Most of the cross-reactive proteins of C. tetani shared 65–78% sequence similarity with their closest homologues in C. perfringens ATCC13124. Electron transfer flavoprotein beta-subunit (CT3 was the most abundant protein (43.3%, followed by methylaspartate ammonia-lyase (36.8% and 2-phosphoglycerate dehydratase (35.6%. All the proteins were predicted to be cytoplasmic by PSORT protein localization algorithm. Active immunization with C. perfringens whole cells elicited cross-protective immunity against C. tetani infection in a mouse model. Conclusion Most of the dominant cross-reactive proteins of C. tetani belonged to the cluster of orthologous group (COG functional category, either of posttranslational modification, protein turnover, and chaperones (O or energy production and conversion (C. The homologs of the identified proteins have been shown to play role in pathogenesis in other Gram-positive pathogenic bacteria. Our findings provide basis for the search of potential vaccine candidates with broader coverage, encompassing more than one pathogenic clostridial species.

  6. Roles of DacB and spm proteins in clostridium perfringens spore resistance to moist heat, chemicals, and UV radiation.

    Science.gov (United States)

    Paredes-Sabja, Daniel; Sarker, Nahid; Setlow, Barbara; Setlow, Peter; Sarker, Mahfuzur R

    2008-06-01

    Clostridium perfringens food poisoning is caused mainly by enterotoxigenic type A isolates that typically possess high spore heat resistance. Previous studies have shown that alpha/beta-type small, acid-soluble proteins (SASP) play a major role in the resistance of Bacillus subtilis and C. perfringens spores to moist heat, UV radiation, and some chemicals. Additional major factors in B. subtilis spore resistance are the spore's core water content and cortex peptidoglycan (PG) structure, with the latter properties modulated by the spm and dacB gene products and the sporulation temperature. In the current work, we have shown that the spm and dacB genes are expressed only during C. perfringens sporulation and have examined the effects of spm and dacB mutations and sporulation temperature on spore core water content and spore resistance to moist heat, UV radiation, and a number of chemicals. The results of these analyses indicate that for C. perfringens SM101 (i) core water content and, probably, cortex PG structure have little if any role in spore resistance to UV and formaldehyde, presumably because these spores' DNA is saturated with alpha/beta-type SASP; (ii) spore resistance to moist heat and nitrous acid is determined to a large extent by core water content and, probably, cortex structure; (iii) core water content and cortex PG cross-linking play little or no role in spore resistance to hydrogen peroxide; (iv) spore core water content decreases with higher sporulation temperatures, resulting in spores that are more resistant to moist heat; and (v) factors in addition to SpmAB, DacB, and sporulation temperature play roles in determining spore core water content and thus, spore resistance to moist heat.

  7. Use of single-enzyme amplified fragment length polymorphism for typing Clostridium perfringens isolated from diarrheic piglets Uso do polimorfismo do comprimento de fragmentos amplificados para tipagem de Clostridium perfringens isolados de suínos com diarréia

    Directory of Open Access Journals (Sweden)

    Luciane Tieko Shinya

    2006-09-01

    Full Text Available Clostridium perfringens is an important pathogen in human and veterinary medicine. In swine, the agent is responsible for necrotic enteritis and enterotoxemia characterized by diarrhea, weight loss, delayed development and, in some cases, death. In the present study amplified fragment length polymorphism analyses (AFLP was used to characterize 54 C. perfringens strains isolated from swine presenting diarrhea. Analysis of the results showed 29 distinct profiles with discriminatory index equal to 0.97. Partial correlation between the origin of the isolates and groups was drawn, and correlation was possible in only 18.5% of the samples. Characterization of the strains in biotypes (A, B, C, D and E, production of beta-2 toxin and enterotoxin were performed by means of the polymerase chain reaction (PCR. Biotypes A, C and D were observed among the strains analyzed. All samples were positive for presence of the gene encoding beta-2 toxin and negative for the gene encoding enterotoxin. AFLP have shown to be a simple, fast, low cost method with high discriminative power and good reproducibility, presenting a great potential in epidemiological studies involving C. perfringens strains of animal origin.Clostridium perfringens é um importante agente infeccioso em medicina veterinária e humana. Em suínos, o agente é responsável pela enterite necrótica e enterotoxemia, caracterizadas por diarréia, perda de peso, atraso no desenvolvimento e morte. No presente estudo foi utilizado o polimorfismo do comprimento de fragmentos amplificados (AFLP, para caracterizar 54 isolados de C. perfringens obtidos de suínos com diarréia. A análise dos resultados do AFLP demonstrou 29 perfis distintos com índice discriminatório igual a 0,97. A correlação entre a origem dos isolados e os agrupamentos obtidos foi parcial, sendo apenas possível a correlação total de 18,5% das amostras estudadas. A caracterização das cepas em biotipos (A, B, C, D e E, produ

  8. Diarréia em leitões lactentes por Clostridium perfringens tipo A em granjas tecnificadas nos estados de Minas Gerais e São Paulo Clostridium perfringens type A diarrhea in suckling piglets in industrial swine farms in the states of Minas Gerais and São Paulo

    Directory of Open Access Journals (Sweden)

    G.M. Costa

    2004-06-01

    Full Text Available Diarrhea in suckling piglets caused by Clostridium perfringens type A was diagnosed in industrial (technified swine farms of the states of Minas Gerais and São Paulo (Brazil, based on isolation and identification of bacterium by biochemical tests, detection of alpha toxin in animal bioassays, and PCR. This seems to be the first report of clostridial enterotoxaemia in piglets by C. perfringens type A in Brazil and allowed specific procedures to control the disease.

  9. Selection of Bacillus spp. for cellulase and xylanase production as direct-fed microbials to reduce digesta viscosity and Clostridium perfringens proliferation using an in vitro digestive model in different poultry diets

    Directory of Open Access Journals (Sweden)

    Juan D Latorre

    2015-08-01

    Full Text Available Previously, our laboratory has screened and identified Bacillus spp. isolates as direct-fed microbials (DFM. The purpose of the present study was to evaluate the cellulase and xylanase production of these isolates and select the most appropriate Bacillus spp. candidates for DFM. Furthermore, an in vitro digestive model, simulating different compartments of the gastrointestinal tract, was used to determine the effect of these selected candidates on digesta viscosity and Clostridium perfringens proliferation in different poultry diets. Production of cellulase and xylanase were based on their relative enzyme activity. Analysis of 16S rRNA sequence classified two strains as B. amyloliquefaciens and one of the strains as B. subtilis. The DFM was included at a concentration of 108 spores/g of feed in 5 different sterile soybean-based diets containing corn, wheat, rye, barley, or oat. After digestion time, supernatants from different diets were collected to measure viscosity, and C. perfringens proliferation. Additionally, from each in vitro simulated compartment, samples were taken to enumerate viable Bacillus-spores using a plate count method after heat-treatment. Significant (P<0.05 DFM-associated reductions in supernatant viscosity and C. perfringens proliferation were observed for all non-corn diets. These results suggest that antinutritional factors such as non-starch polysaccharides from different cereals can enhance viscosity and C. perfringens growth. Remarkably, dietary inclusion of the DFM that produce cellulase and xylanase reduced both viscosity and C. perfringens proliferation compared with control diets. Regardless of diet composition, 90% of the DFM spores germinated during the first 30 min in the crop compartment of the digestion model, followed by a noteworthy increased in the intestine compartment by ~2 log10, suggesting a full-life cycle development. Further studies to evaluate in vivo necrotic enteritis effects are in progress.

  10. Impact of Clean-Label Antimicrobials and Nitrite Derived from Natural Sources on the Outgrowth of Clostridium perfringens during Cooling of Deli-Style Turkey Breast.

    Science.gov (United States)

    King, Amanda M; Glass, Kathleen A; Milkowski, Andrew L; Sindelar, Jeffrey J

    2015-05-01

    Organic acids and sodium nitrite have long been shown to provide antimicrobial activity during chilling of cured meat products. However, neither purified organic acids nor NaNO2 is permitted in products labeled natural and both are generally avoided in clean-label formulations; efficacy of their replacement is not well understood. Natural and clean-label antimicrobial alternatives were evaluated in both uncured and in alternative cured (a process that uses natural sources of nitrite) deli-style turkey breast to determine inhibition of Clostridium perfringens outgrowth during 15 h of chilling. Ten treatments of ground turkey breast (76% moisture, 1.2% salt) included a control and four antimicrobials: 1.0% tropical fruit extract, 0.7% dried vinegar, 1.0% cultured sugar-vinegar blend, and 2.0% lemon-vinegar blend. Each treatment was formulated without (uncured) and with nitrite (PCN; 50 ppm of NaNO2 from cultured celery juice powder). Treatments were inoculated with C. perfringens spores (three-strain mixture) to yield 2.5 log CFU/g. Individual 50-g portions were vacuum packaged, cooked to 71.1°C, and chilled from 54.4 to 26.7°C in 5 h and from 26.7 to 7.2°C in an additional 10 h. Triplicate samples were assayed for growth of C. perfringens at predetermined intervals by plating on tryptose-sulfite-cycloserine agar. Uncured control and PCN-only treatments allowed for 4.6- and 4.2-log increases at 15 h, respectively, and although all antimicrobial treatments allowed less outgrowth than uncured and PCN, the degree of inhibition varied. The 1.0% fruit extract and 1.0% cultured sugar-vinegar blend were effective at controlling populations at or below initial levels, whether or not PCN was included. Without PCN, 0.7% dried vinegar and 2.0% lemon-vinegar blend allowed for 2.0- and 2.5-log increases, respectively, and ∼1.5-log increases with PCN. Results suggest using clean-label antimicrobials can provide for safe cooling following the study parameters, and greater

  11. Clostridium perfringens enterotoxin as a potential drug for intravesical treatment of bladder cancer.

    Science.gov (United States)

    Gabig, Theodore G; Waltzer, Wayne C; Whyard, Terry; Romanov, Victor

    2016-09-16

    The current intravesical treatment of bladder cancer (BC) is limited to a few chemotherapeutics that show imperfect effectiveness and are associated with some serious complications. Thus, there is an urgent need for alternative therapies, especially for patients with high-risk non-muscle invasive (NMIBC). Clostridium perfringens enterotoxin (CPE), cytolytic protein binds to its receptors: claudin 3 and 4 that are expressed in epithelial cells. This binding is followed by rapid cell death. Claudin 4 is present in several epithelial tissue including bladder urothelium and its expression is elevated in some forms of BC. In addition to directly targeting BC cells, binding of CPE to claudins increases urothelium permeability that creates conditions for better accession of the tumor. Therefore, we evaluated CPE as a candidate for intravesical treatment of BC using a cellular model. We examined cytotoxicity of CPE against BC cells lines and 3D cultures of cells derived from surgical samples. To better elucidate cellular mechanisms, activated by CPE and to consider the use of CPE non-toxic fragment (C-CPE) for combination treatment with other drugs we synthesized C-CPE, compared its cytotoxic activity with CPE and examined claudin 4 expression and intracellular localization after C-CPE treatment. CPE induced cell death after 1 h in low aggressive RT4 cells, in moderately aggressive 5637 cells and in the primary 3D cultures of BC cells derived from NMIBC. Conversely, non-transformed urothelial cells and cells derived from highly aggressive tumor (T24) survived this treatment. The reason for this resistance to CPE might be the lower expression of CLDNs or their inaccessibility for CPE in these cells. C-CPE treatment for 48 h did not affect cell viability in tested cells, but declined expression of CLDN4 in RT4 cells. C-CPE increased sensitivity of RT4 cells to Mitommycin C and Dasatinib. To better understand mechanisms of this effect we examined expression and

  12. EPIDEMIOLOGIC INVESTIGATION OF CLOSTRIDIUM DIFFICILE AND CLOSTRIDIUM PERFRINGENS IN HEALTHY HORSES

    DEFF Research Database (Denmark)

    Schoster, Angelika; Arroyo, Luis; Staempfli, Henry

    than feces and variability in shedding over time. The objectives were to investigate the presence of C. difficile and C. perfringens in healthy horses over time and assess prevalence in different gastrointestinal compartments. Feces were collected monthly from 25 horses for one year. Ingesta were...... collected from nine GI compartments of a separate group of 15 euthanized horses. Selective enrichment culture was performed, followed by toxin gene detection and ribotyping (C. difficile) and multiplex PCR (C. perfringens). Toxigenic C. difficile was isolated from 15/275 (5.5%) samples from 10/25 (40......%) horses over one year. Three horses were positive in consecutive months, but different ribotypes were found in 2/3. Ribotypes included 078 (n=6), 001 (n=5) and an A+B+CDT+ ribotype previously identified as toxinotype IX in this laboratory (n=4). C. perfringens was not recovered despite a detection...

  13. NanR, a Transcriptional Regulator That Binds to the Promoters of Genes Involved in Sialic Acid Metabolism in the Anaerobic Pathogen Clostridium perfringens.

    Directory of Open Access Journals (Sweden)

    Blair Therit

    Full Text Available Among many other virulence factors, Clostridium perfringens produces three sialidases NanH, NanI and NanJ. NanH lacks a secretion signal peptide and is predicted to be an intracellular enzyme, while NanI and NanJ are secreted. Previously, we had identified part of an operon encoding NanE (epimerase and NanA (sialic acid lyase enzymes. Further analysis of the entire operon suggests that it encodes a complete pathway for the transport and metabolism of sialic acid along with a putative transcriptional regulator, NanR. The addition of 30 mM N-acetyl neuraminic acid (Neu5Ac to a semi-defined medium significantly enhanced the growth yield of strain 13, suggesting that Neu5Ac can be used as a nutrient. C. perfringens strain 13 lacks a nanH gene, but has NanI- and NanJ-encoding genes. Analysis of nanI, nanJ, and nanInanJ mutants constructed by homologous recombination revealed that the expression of the major sialidase, NanI, was induced by the addition of Neu5Ac to the medium, and that in separate experiments, the same was true of a nanI-gusA transcriptional fusion. For the nanI and nanJ genes, primer extension identified three and two putative transcription start sites, respectively. Gel mobility shift assays using purified NanR and DNA from the promoter regions of the nanI and nanE genes showed high affinity, specific binding by NanR. We propose that NanR is a global regulator of sialic acid-associated genes and that it responds, in a positive feedback loop, to the concentration of sialic acid in the cell.

  14. Selection for pro-inflammatory mediators produces chickens more resistant to Clostridium perfringens-induced necrotic enteritis.

    Science.gov (United States)

    Swaggerty, C L; McReynolds, J L; Byrd, J A; Pevzner, I Y; Duke, S E; Genovese, K J; He, H; Kogut, M H

    2016-02-01

    We developed a novel selection method based on an inherently high and low phenotype of pro-inflammatory mediators and produced "high" and "low" line chickens. We have shown high line birds are more resistant to Salmonella enterica serovar Enteritidis and Eimeria tenella compared to the low line. Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illness, and is also an economically important poultry pathogen and known etiologic agent of necrotic enteritis (NE). The objective of this study was to determine if high line birds were also more resistant to NE than low line birds using an established model. Birds were reared in floor pens and challenges were conducted twice (high line = 25/trial, 50 birds total; low line = 26/trial, 52 birds total). Day-old chicks were provided a 55% wheat-corn-based un-medicated starter diet. A bursal disease vaccine was administered at 10× the recommended dose via the ocular route at 14-d-of-age. Birds were challenged daily for 3 d beginning at 16-d-of-age by oral gavage (3 mL) with 10(7) colony forming units (cfu) of C. perfringens/mL then necropsied at 21-d-of-age. All birds had sections of the intestine examined and scored for lesions while the first 10 necropsied also had gut content collected for C. perfringens enumeration. Chickens from the high line were more resistant to C. perfringens-induced NE pathology compared to the low line, as indicated by reduced lesion scores. Ninety percent of the high line birds had lesions of zero or one compared to 67% of the low line birds. Wilcoxon rank sum test showed significantly higher lesion scores in the low line birds compared to the high line (P < 0.0001). There were no differences in the C. perfringens recovered (P = 0.83). These data provide additional validation and support selection based on elevated levels of pro-inflammatory mediators produces chickens with increased resistance against foodborne and poultry pathogens.

  15. Incidência de escherichia coli enterotoxigênica (ETEC, rotavirus e Clostridium perfringens de casos de diarréia em crianças, na região de Campinas, SP, Brasil Incidence of enterotoxigenic Escherichia coli (ETEC, rotaviruses and Clostridium perfringens from cases of diarrhea among children, in the region of Campinas, SP, Brazil

    Directory of Open Access Journals (Sweden)

    Maria Silvia V. Gatti

    1989-12-01

    Full Text Available Foi realizada uma pesquisa na região de Campinas, SP, Brasil, sobre a presença de Escherichia coli enterotoxigênica (ETEC, rotavírus e Clostridium perfringens enterotoxigênico em fezes diarréicas de crianças com até 2 anos de idade. Dos 132 espécimens fecais examinados quanto à presença de ETEC 27 (20,45% foram positivos. Destes foram isoladas 41 amostras de ETEC, das quais 40 produziram apenas a enterotoxina termolábil (LT detectada pelo teste de imuno hemólise radial modifi cado. Entre as 183 amostras de fezes examinadas para rotavírus, 29 (15,84% foram positivas pelas técnicas de eletroforese em gel de poliacrilamida (PAGE e ensaio imunoenzimático (EIE, sendo que destas, 15 (51,7% foram provenientes de materiais coletados nos meses de inverno. Todas as amostras pertenciam ao grupo A e, através da técnica de PAGE, pode-se observar que o tipo eletroforético mais freqüente (9 amostras foi designado Ib, IIc, Illb, IVa, de acordo com a classificação por nós adotada. Apenas 113 amostras de fezes foram examinadas para a presença de C. perfringens enterotoxigênico. Para a detecção da enterotoxina nos sobrenadantes das culturas foram utilizadas as técnicas de hemaglutinação passiva reversa e inoculação intravenosa em camundongos, sendo encontradas 12 (10,61% amostras entero-toxigênicas. Diante destes resultados é chamada a atenção sobre o valor apenas relativo de uma coprocultura convencional para fins de diagnóstico, ressaltando-se a importância da criação de métodos simplificados que favoreçam a detecção e identificação dos grupos de agentes enteropatogênicos estudados na presente pesquisa.A survey for the detection of enterotoxigenic Escherichia coli (ETEC, rotavirus and enterotoxigenic Clostridium perfringens in diarrheic stools of children up to 2 years old was carried out in the region of Campinas, SP, Brazil. Twenty-seven (20.45% faecal specimens were positive for ETEC. From these samples 41

  16. An atypical Clostridium strain related to the Clostridium botulinum group III strain isolated from a human blood culture.

    Science.gov (United States)

    Bouvet, Philippe; Ruimy, Raymond; Bouchier, Christiane; Faucher, Nathalie; Mazuet, Christelle; Popoff, Michel R

    2014-01-01

    A nontoxigenic strain isolated from a fatal human case of bacterial sepsis was identified as a Clostridium strain from Clostridium botulinum group III, based on the phenotypic characters and 16S rRNA gene sequence, and was found to be related to the mosaic C. botulinum D/C strain according to a multilocus sequence analysis of 5 housekeeping genes.

  17. Genotipicação de Clostridium perfringens isolados de frangos de corte através da PCR múltipla Genotyping Clostridium perfringens broiler chickens isolates by multiplex PCR products analyses

    Directory of Open Access Journals (Sweden)

    Alexis de Matos Gomes

    2008-10-01

    Full Text Available Clostridium perfringens (Cp é uma bactéria aneróbica gram positiva que, além de provocar gangrena gasosa e enterotoxemia em humanos e animais, constitui-se na principal causa de enterite necrótica em aves de criações intensificadas. A identificação dos isolados foi realizada pela reação de lecitinase em ágar TSC-gema de ovo, colônias com dupla hemólise em ágar sangue desfibrinado de eqüino, coloração de Gram e provas bioquímicas. Das amostras analisadas, 171Cp foram isolados em jejuno e íleo de frangos de corte provenientes de um frigorífico da região de Pará de Minas-MG. Cp foi isolado em 62 (49,6% de 125 amostras de conteúdo lumenal de jejunos e em 109 (87,2% de igual número de íleos dos frangos de corte. Utilizando-se a técnica da PCR múltipla para genotipicacão das estirpes de Cp, de acordo com os genes para as toxinas principais e letais (cpa, cpb, etx e iA, da toxina cpb2 (cpb2 e enterotoxina (cpe, as estirpes de Cp isoladas foram classificadas em cinco tipos toxigênicos (A-E. Das 62 estirpes de Cp isoladas do jejuno, foram obtidos 42/62 (67,7% tipo A, 1/62 (1,6% tipo A com produto de amplificação para o gene da toxina beta2, 0/62 (0% tipo B, 17/62 (27,4% tipo C, 1/62 (1,6% tipo D. Das 109 amostras de Cp isolados do íleo das aves foram obtidos 62/109 (56,9% tipo A, 3/109 (2,7% tipo A com produto de amplificação para o gene da toxina beta2, 1/62 (0,9% tipo B, 38/109 (34,9% tipo C, 1/109 (0,9% tipo D. Cp A (60,8% e Cp C (32,2% foram os tipos toxigênicos predominantes em conteúdo intestinal de frango de corte. Cinco (2,9% das 171 amostras de Cp isolados não foram tipificadas. Não foram detectados os genes codificadores das toxinas iota (iA e enterotoxina (cpe em nenhuma das 171 estirpes de Cp caracterizados.Clostridium perfringens (Cp is an anaerobic gram-positive bacterium which causes gaseous gangrene and enterotoxaemias in humans and domestic animals, besides being the primary cause of necrotic

  18. Immunogenicity of a Trivalent Recombinant Vaccine Against Clostridium perfringens Alpha, Beta, and Epsilon Toxins in Farm Ruminants.

    Science.gov (United States)

    Moreira, Gustavo Marçal Schmidt Garcia; Salvarani, Felipe Masiero; da Cunha, Carlos Eduardo Pouey; Mendonça, Marcelo; Moreira, Ângela Nunes; Gonçalves, Luciana Aramuni; Pires, Prhiscylla Sadanã; Lobato, Francisco Carlos Faria; Conceição, Fabricio Rochedo

    2016-03-23

    Clostridium perfringens is an anaerobic bacterium that produces several toxins. Of these, the alpha, beta, and epsilon toxins are responsible for causing the most severe C. perfringens-related diseases in farm animals. The best way to control these diseases is through vaccination. However, commercially available vaccines are based on inactivated toxins and have many production drawbacks, which can be overcome through the use of recombinant antigens. In this study, we produced recombinant alpha, beta, and epsilon toxins in Escherichia coli to formulate a trivalent vaccine. Its effectiveness was evaluated through a potency test in rabbits, in which the vaccine generated 9.6, 24.4, and 25.0 IU/mL of neutralizing antibodies against the respective toxins. Following this, cattle, sheep, and goats received the same formulation, generating, respectively, 5.19 ± 0.48, 4.34 ± 0.43, and 4.70 ± 0.58 IU/mL against alpha toxin, 13.71 ± 1.17 IU/mL (for all three species) against beta toxin, and 12.74 ± 1.70, 7.66 ± 1.69, and 8.91 ± 2.14 IU/mL against epsilon toxin. These levels were above the minimum recommended by international protocols. As such, our vaccine was effective in generating protective antibodies and, thus, may represent an interesting alternative for the prevention of C. perfringens-related intoxications in farm animals.

  19. Development and application of a mouse intestinal loop model to study the in vivo action of Clostridium perfringens enterotoxin.

    Science.gov (United States)

    Caserta, Justin A; Robertson, Susan L; Saputo, Juliann; Shrestha, Archana; McClane, Bruce A; Uzal, Francisco A

    2011-08-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of C. perfringens type A food poisoning, the second most commonly identified bacterial food-borne illness in the United States. CPE is produced by sporulating C. perfringens cells in the small intestinal lumen, where it then causes epithelial cell damage and villous blunting that leads to diarrhea and cramping. Those effects are typically self-limiting; however, severe outbreaks of this food poisoning, particularly two occurring in psychiatric institutions, have involved deaths. Since animal models are currently limited for the study of the CPE action, a mouse ligated intestinal loop model was developed. With this model, significant lethality was observed after 2 h in loops receiving an inoculum of 100 or 200 μg of CPE but not using a 50-μg toxin inoculum. A correlation was noted between the overall intestinal histological damage and lethality in mice. Serum analysis revealed a dose-dependent increase in serum CPE and potassium levels. CPE binding to the liver and kidney was detected, along with elevated levels of potassium in the serum. These data suggest that CPE can be absorbed from the intestine into the circulation, followed by the binding of the toxin to internal organs to induce potassium leakage, which can cause death. Finally, CPE pore complexes similar to those formed in tissue culture cells were detected in the intestine and liver, suggesting that (i) CPE actions are similar in vivo and in vitro and (ii) CPE-induced potassium release into blood may result from CPE pore formation in internal organs such as the liver.

  20. Development and Application of a Mouse Intestinal Loop Model To Study the In Vivo Action of Clostridium perfringens Enterotoxin ▿

    Science.gov (United States)

    Caserta, Justin A.; Robertson, Susan L.; Saputo, Juliann; Shrestha, Archana; McClane, Bruce A.; Uzal, Francisco A.

    2011-01-01

    Clostridium perfringens enterotoxin (CPE) is responsible for causing the gastrointestinal symptoms of C. perfringens type A food poisoning, the second most commonly identified bacterial food-borne illness in the United States. CPE is produced by sporulating C. perfringens cells in the small intestinal lumen, where it then causes epithelial cell damage and villous blunting that leads to diarrhea and cramping. Those effects are typically self-limiting; however, severe outbreaks of this food poisoning, particularly two occurring in psychiatric institutions, have involved deaths. Since animal models are currently limited for the study of the CPE action, a mouse ligated intestinal loop model was developed. With this model, significant lethality was observed after 2 h in loops receiving an inoculum of 100 or 200 μg of CPE but not using a 50-μg toxin inoculum. A correlation was noted between the overall intestinal histological damage and lethality in mice. Serum analysis revealed a dose-dependent increase in serum CPE and potassium levels. CPE binding to the liver and kidney was detected, along with elevated levels of potassium in the serum. These data suggest that CPE can be absorbed from the intestine into the circulation, followed by the binding of the toxin to internal organs to induce potassium leakage, which can cause death. Finally, CPE pore complexes similar to those formed in tissue culture cells were detected in the intestine and liver, suggesting that (i) CPE actions are similar in vivo and in vitro and (ii) CPE-induced potassium release into blood may result from CPE pore formation in internal organs such as the liver. PMID:21628512

  1. Cost-effective screening of pooled faecal specimens from patients with nosocomial diarrhoea for Clostridium perfringens enterotoxin

    Directory of Open Access Journals (Sweden)

    C Vaishnavi

    2011-01-01

    Full Text Available Purpose: Clostridium perfringens is a significant cause of nosocomial AAD. The prevalence of C. perfringens enterotoxin (CPE-positive stool specimens in hospitalised patients is very low in the Indian setting making the diagnostics very expensive. Therefore, a cost-effective diagnostic approach to screen faecal specimens for CPE was devised. Materials and Methods: Faecal specimens from 540 hospitalised patients with various ailments and from 340 healthy subjects were investigated for CPE. An aliquot of pooled faecal supernatants was made by mixing 100 μl each of 10 specimens to be tested. Each aliquot was investigated for the presence of CPE by an enzyme immunoassay. A repetition of the assay was done with individual specimens of the pooled aliquots from each positive well as seen visually by colour development. Results: Of the 540 patient specimens tested, 405 (75% patients were on antibiotics, the predominant ones being cephalosporins, penicillin, quinolones, aminoglycosides, etc. During the time of sampling, diarrhoea was present in 481 (89%, abdomen pain in 203 (37.6% and fever in 242 (44.8% patients. C. perfringens enterotoxin was positive in nine wells of the 540 pooled test specimens whereas all of the pooled 340 control samples were negative. Repeat of individual specimens comprising the nine wells with positive samples helped to identify the individual patients positive for CPE. Conclusion: Only two CPE kits were needed for a total of 880 faecal specimens tested. The cost-effective diagnostic approach to screen faecal specimens for CPE, as described herein will help to save institutional resources.

  2. Identification of a key residue for oligomerisation and pore-formation of Clostridium perfringens NetB.

    Science.gov (United States)

    Fernandes da Costa, Sérgio P; Savva, Christos G; Bokori-Brown, Monika; Naylor, Claire E; Moss, David S; Basak, Ajit K; Titball, Richard W

    2014-03-12

    Necrotic enteritis toxin B (NetB) is a β-pore-forming toxin produced by Clostridium perfringens and has been identified as a key virulence factor in the pathogenesis of avian necrotic enteritis, a disease causing significant economic damage to the poultry industry worldwide. In this study, site-directed mutagenesis was used to identify amino acids that play a role in NetB oligomerisation and pore-formation. NetB K41H showed significantly reduced toxicity towards LMH cells and human red blood cells relative to wild type toxin. NetB K41H was unable to oligomerise and form pores in liposomes. These findings suggest that NetB K41H could be developed as a genetic toxoid vaccine to protect against necrotic enteritis.

  3. Predicting outgrowth and inactivation of Clostridium perfringens in meat products during low temperature long time heat treatment

    DEFF Research Database (Denmark)

    Duan, Zhi; Hansen, Terese Holst; Hansen, Tina Beck

    2016-01-01

    With low temperature long time (LTLT) cooking it can take hours for meat to reach a final core temperature above 53 °C and germination followed by growth of Clostridium perfringens is a concern. Available and new growth data in meats including 154 lag times (tlag), 224 maximum specific growth rates.......5 log CFU/g. If at least 85% of the observed log-counts were below the FSAZ, the model was considered fail-safe. The two models showed similar performance but none of them performed satisfactorily for all conditions. It is recommended to use the models without a lag phase until more precise lag time...... (μmax) and 25 maximum population densities (Nmax) were used to developed a model to predict growth of C. perfringens during the coming-up time of LTLT cooking. New data were generate in 26 challenge tests with chicken (pH 6.8) and pork (pH 5.6) at two different slowly increasing temperature (SIT...

  4. Structure of the food-poisoning Clostridium perfringens enterotoxin reveals similarity to the aerolysin-like pore-forming toxins.

    Science.gov (United States)

    Briggs, David C; Naylor, Claire E; Smedley, James G; Lukoyanova, Natalya; Robertson, Susan; Moss, David S; McClane, Bruce A; Basak, Ajit K

    2011-10-14

    Clostridium perfringens enterotoxin (CPE) is a major cause of food poisoning and antibiotic-associated diarrhea. Upon its release from C. perfringens spores, CPE binds to its receptor, claudin, at the tight junctions between the epithelial cells of the gut wall and subsequently forms pores in the cell membranes. A number of different complexes between CPE and claudin have been observed, and the process of pore formation has not been fully elucidated. We have determined the three-dimensional structure of the soluble form of CPE in two crystal forms by X-ray crystallography, to a resolution of 2.7 and 4.0 Å, respectively, and found that the N-terminal domain shows structural homology with the aerolysin-like β-pore-forming family of proteins. We show that CPE forms a trimer in both crystal forms and that this trimer is likely to be biologically relevant but is not the active pore form. We use these data to discuss models of pore formation.

  5. Molecular cloning and expression of epsilon toxin from Clostridium perfringens type D and tests of animal immunization.

    Science.gov (United States)

    Souza, A M; Reis, J K P; Assis, R A; Horta, C C; Siqueira, F F; Facchin, S; Alvarenga, E R; Castro, C S; Salvarani, F M; Silva, R O S; Pires, P S; Contigli, C; Lobato, F C F; Kalapothakis, E

    2010-02-18

    Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-epsilon) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the toxin (as an antigen), rabbits were immunized with 50, 100 or 200 microg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-epsilon is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.

  6. A novel pore-forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis.

    Science.gov (United States)

    Mehdizadeh Gohari, Iman; Parreira, Valeria R; Nowell, Victoria J; Nicholson, Vivian M; Oliphant, Kaitlyn; Prescott, John F

    2015-01-01

    A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute hemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.

  7. A novel pore-forming toxin in type A Clostridium perfringens is associated with both fatal canine hemorrhagic gastroenteritis and fatal foal necrotizing enterocolitis.

    Directory of Open Access Journals (Sweden)

    Iman Mehdizadeh Gohari

    Full Text Available A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related to the pore-forming Leukocidin/Hemolysin Superfamily; these were designated netE, netF, and netG. netE and netF were located on one large conjugative plasmid, and netG was located with a cpe enterotoxin gene on a second large conjugative plasmid. Mutation and complementation showed that only netF was associated with the cytotoxicity. Although netE and netG were not associated with cytotoxicity, immunoblotting with specific antisera showed these proteins to be expressed in vitro. There was a highly significant association between the presence of netF with type A strains isolated from cases of canine acute hemorrhagic gastroenteritis and foal necrotizing enterocolitis. netE and netF were found in all cytotoxic isolates, as was cpe, but netG was less consistently present. Pulsed-field gel electrophoresis showed that netF-positive isolates belonged to a clonal population; some canine and equine netF-positive isolates were genetically indistinguishable. Equine antisera to recombinant Net proteins showed that only antiserum to rNetF had high supernatant cytotoxin neutralizing activity. The identifica-tion of this novel necrotizing toxin is an important advance in understanding the virulence of type A C. perfringens in specific enteric disease of animals.

  8. Effect of rooibos (Aspalathus linearis) on growth control of Clostridium perfringens and lipid oxidation of ready-to-eat Jokbal (pig's trotters).

    Science.gov (United States)

    Park, Hee Jin; Park, Keun-Cheol; Yoon, Ki Sun

    2014-12-01

    This study investigated the antimicrobial effects of rooibos (tea extract), potassium lactate (PL) and sodium diacetate (SDA) mixture alone or in combinations on the growth of Clostridium perfringens vegetative cell and spore in ready-to-eat (RTE) Jokbal (pig's trotters). Addition of a combination of 10% rooibos and 4% PL + SDA inhibit growth of C. perfringens vegetative cell in Jokbal at 24 °C and 36 °C. The significant inhibition on germination and growth of C. perfringens spores was also observed in Jokbal with a combination of 10% rooibos and 4% PL + SDA (PL: 2.24%, SDA: 0.16%) at 24 °C. The Jokbal treated with 10% rooibos and 4% PL + SDA mixture had significantly (P food preservative in meat products.

  9. Assessing the performance of Clostridium perfringens cooling models for cooked, uncured meat and poultry products

    Science.gov (United States)

    Heat-resistant spores of C. perfringens may germinate and multiply in cooked meat and poultry products if the rate and extent of cooling does not occur in a timely manner. Therefore, six cooling models (PMP 7.0 broth model; PMIP Uncured Beef, Chicken, and Pork Models; Smith-Schaffner (version 3); a...

  10. A Novel Pore-Forming Toxin in Type A Clostridium perfringens Is Associated with Both Fatal Canine Hemorrhagic Gastroenteritis and Fatal Foal Necrotizing Enterocolitis

    OpenAIRE

    Iman Mehdizadeh Gohari; Valeria R Parreira; Victoria J Nowell; Nicholson, Vivian M.; Kaitlyn Oliphant; Prescott, John F.

    2015-01-01

    A role for type A Clostridium perfringens in acute hemorrhagic and necrotizing gastroenteritis in dogs and in necrotizing enterocolitis of neonatal foals has long been suspected but incompletely characterized. The supernatants of an isolate made from a dog and from a foal that died from these diseases were both found to be highly cytotoxic for an equine ovarian (EO) cell line. Partial genome sequencing of the canine isolate revealed three novel putative toxin genes encoding proteins related t...

  11. Genomics of Clostridium botulinum group III strains.

    Science.gov (United States)

    Sakaguchi, Yoshihiko; Suzuki, Tomonori; Yamamoto, Yumiko; Nishikawa, Atsushi; Oguma, Keiji

    2015-05-01

    In Clostridium botulinum, the characteristics of type C and D strains are quite different from other types, and they are classified as group III. They produce C2 binary toxin and C3 exoenzyme in addition to type C and D neurotoxins. Two different phages and many plasmids are identified in the organisms. The genes of neurotoxin and C3 exoenzyme are converted from toxigenic strains to non-toxigenic strains by the specific bacteriophages (phages), whereas, the C2 toxin gene is carried by large or small plasmids. Classification of type C and D strains has been in confusion because 1) antigenicity of type C and D neurotoxins is complex, 2) the cells produce two types of toxins, neurotoxin and C2 toxin, and 3) some non-toxigenic strains can be converted to produce C or D neurotoxin by the infection with phages. Until now, entire nucleotide sequences of cell chromosomes, phages, and plasmids have been determined. Since both genetic and protein-chemical analyses have been clarifying the above confusions, these data are reviewed historically.

  12. Gas gangrene caused by clostridium perfringens involving the liver, spleen, and heart in a man 20 years after an orthotopic liver transplant: a case report.

    Science.gov (United States)

    Kitterer, Daniel; Braun, Niko; Jehs, Margit C; Schulte, Bernhard; Alscher, M Dominik; Latus, Joerg

    2014-04-01

    Despite advances in immunosuppression and liver transplant in the past, mortality and morbidity caused by infections remain major problems. We present a 71-year-old man who was admitted to our internal intensive care unit with septicemia. Upon admission, he had poorly localized epigastric pain and fever of 2 days ' duration. Twenty years earlier, he had undergone an orthotopic liver transplant. Testing revealed a high C-reactive protein level, elevated liver enzymes, and an acute kidney injury. A computer tomography scan showed 2 circular, non--rim-enhancing, totally emphysematous intrahepatic lesions. Additionally, gas could be seen in the portal veins mainly, as well as in the biliary system, in the right auricle, and the splenic veins. To the best of our knowledge, he showed no malignant lesion or predisposing trauma. Empirically, treatment with broad-spectrum antibiotics was begun, and the patient was transferred to the operating suite. When surgery began, blood cultures revealed the presence of gram-positive bacilli, which were identified as Clostridium perfringens. Seven hours after the surgery, the patient developed asystole and died. In septic patients presenting with severe hemolysis, Clostridium perfringens infection must be considered in the absence of a malignant lesion or a predisposing trauma; a previous episode of gastroenteritis might be a predisposing trauma by impairing the barrier of the intestinal flora, leading to Clostridium perfringens infection.

  13. A novel watery diarrhoea caused by the co-infection of neonatal piglets with Clostridium perfringens type A and Escherichia coli (K88, 987P).

    Science.gov (United States)

    Wang, Xiaocen; Ren, Wenzhi; Nie, Ying; Cheng, Liqing; Tan, Wei; Wang, Chong; Wei, Libin; Zhang, Rui; Yan, Guangmou

    2013-09-01

    In 2011, a novel watery diarrhoea in 1-7 day-old piglets occurred in Changchun, China, characterized by high pathogenicity and mortality. Investigation of clinical signs, examination for viruses, and isolation and identification of bacteria showed that co-infection by Clostridium perfringens type A and Escherichia coli (K88, 987P) was the most likely cause of the disease. Newborn piglets challenged with a mixture of Clostridium perfringens type A and Escherichia coli (K88, 987P) died within 3 days with clinical signs and gross lesions similar to those in the piglets that died in the outbreak. A subsequent study showed that the use in sows of an inactivated vaccine against the two causal bacteria was effective at reducing the incidence of the watery diarrhoea in piglets. Piglets from sows given the inactivated vaccine had a incidence of watery diarrhoea of 8% (14/175), much lower than the 95% (192/201) seen in piglets from control sows. This is the first report of diarrhoea in piglets resulting from co-infection of Clostridium perfringens type A and Escherichia coli (K88, 987P). Further studies are required to better understand the pathogenesis of this disease. Copyright © 2013 Elsevier Ltd. All rights reserved.

  14. Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model.

    Science.gov (United States)

    Sayeed, Sameera; Uzal, Francisco A; Fisher, Derek J; Saputo, Juliann; Vidal, Jorge E; Chen, Yue; Gupta, Phalguni; Rood, Julian I; McClane, Bruce A

    2008-01-01

    Clostridium perfringens type C isolates, which cause enteritis necroticans in humans and enteritis and enterotoxaemias of domestic animals, typically produce (at minimum) beta toxin (CPB), alpha toxin (CPA) and perfringolysin O (PFO) during log-phase growth. To assist development of improved vaccines and therapeutics, we evaluated the contribution of these three toxins to the intestinal virulence of type C disease isolate CN3685. Similar to natural type C infection, log-phase vegetative cultures of wild-type CN3685 caused haemorrhagic necrotizing enteritis in rabbit ileal loops. When isogenic toxin null mutants were prepared using TargeTron technology, even a double cpa/pfoA null mutant of CN3685 remained virulent in ileal loops. However, two independent cpb null mutants were completely attenuated for virulence in this animal model. Complementation of a cpb mutant restored its CPB production and intestinal virulence. Additionally, pre-incubation of wild-type CN3685 with a CPB-neutralizing monoclonal antibody rendered the strain avirulent for causing intestinal pathology. Finally, highly purified CPB reproduced the intestinal damage of wild-type CN3685 and that damage was prevented by pre-incubating purified CPB with a CPB monoclonal antibody. These results indicate that CPB is both required and sufficient for CN3685-induced enteric pathology, supporting a key role for this toxin in type C intestinal pathogenesis.

  15. Characterization of Clostridium perfringens isolates from healthy turkeys and from turkeys with necrotic enteritis

    DEFF Research Database (Denmark)

    Lyhs, Ulrike; Perko-Mäkelä, P.; Kallio, H.;

    2013-01-01

    from 1998 to 2012. Furthermore, C. perfringens isolates from healthy and diseased turkeys were characterized and their genetic diversity was investigated using pulsed-field gel electrophoresis (PFGE). Isolates (n = 212) from birds with necrotic gut lesions and from healthy flocks of 30 commercial...... turkey farms were characterized for the presence of cpa, cpb, iA, etx, cpb2, and cpe and netB genes. A total of 93 C. perfringens isolates, including 55 from birds with necrotic gut lesions and 38 from healthy birds from 13 different farms, were analyzed with PFGE. All contract turkey farmers (n = 48......) of a turkey company that produces 99% of domestic turkey meat in Finland were interviewed about background information, management at the farm, and stress factors related to NE outbreaks. Pulsed-field gel electrophoresis analysis with SmaI restriction enzyme resulted in 30 PFGE patterns among the 92 C...

  16. Engineering clostridium strain to accept unmethylated DNA.

    Directory of Open Access Journals (Sweden)

    Hongjun Dong

    Full Text Available It is difficult to genetically manipulate the medically and biotechnologically important genus Clostridium due to the existence of the restriction and modification (RM systems. We identified and engineered the RM system of a model clostridial species, C. acetobutylicum, with the aim to allow the host to accept the unmethylated DNA efficiently. A gene CAC1502 putatively encoding the type II restriction endonuclease Cac824I was identified from the genome of C. acetobutylicum DSM1731, and disrupted using the ClosTron system based on group II intron insertion. The resulting strain SMB009 lost the type II restriction endonuclease activity, and can be transformed with unmethylated DNA as efficiently as with methylated DNA. The strategy reported here makes it easy to genetically modify the clostridial species using unmethylated DNA, which will help to advance the understanding of the clostridial physiology from the molecular level.

  17. Administration of non-pathogenic isolates of Escherichia coli and Clostridium perfringens type A to piglets in a herd affected with a high incidence of neonatal diarrhoea.

    Science.gov (United States)

    Unterweger, C; Kahler, A; Gerlach, G-F; Viehmann, M; von Altrock, A; Hennig-Pauka, I

    2017-04-01

    A bacterial cocktail of living strains of Clostridium perfringens type A (CPA) without β2-toxin gene and non-pathogenic Escherichia coli was administered orally to newborn piglets before first colostrum intake and on 2 consecutive days on a farm with a high incidence of diarrhoea and antibiotic treatment in suckling piglets associated with E. coli and CPA. This clinical field study was driven by the hypothetic principle of competitive exclusion of pathogenic bacteria due to prior colonization of the gut mucosal surface by non-pathogenic strains of the same bacterial species with the aim of preventing disease. Although CPA strains used in this study did not produce toxins in vitro, their lack of pathogenicity cannot be conclusively confirmed. The health status of the herd was impaired by a high incidence of postpartum dysgalactia syndrome in sows (70%) and a high incidence of neonatal diarrhoea caused by enterotoxigenic E. coli and CPA during the study. No obvious adverse effect of the bacterial treatment occurred. On average, more piglets were weaned in litters treated (P=0.009). Visual pathological alterations in the small intestinal wall were more frequent in dead piglets of the control group (P=0.004) and necrotizing enteritis was only found in that group. A higher average daily weight gain of piglets in the control group (P<0.001) may be due to an increased milk uptake due to less competition in the smaller litters. The bacterial cocktail was tested under field conditions for its potential to stabilize gut health status in suckling piglets before disease development due to colibacillosis and clostridial infections; however, the gut flora stabilizing effect of the bacterial cocktail was not clearly discernible in this study. Further basic research is needed to confirm the positive effects of the bacterial treatment used and to identify additional potential bacterial candidates for competitive exclusion.

  18. Effect of yeast-derived products and distillers dried grains with solubles (DDGS) on growth performance and local innate immune response of broiler chickens challenged with Clostridium perfringens.

    Science.gov (United States)

    Alizadeh, M; Rogiewicz, A; McMillan, E; Rodriguez-Lecompte, J C; Patterson, R; Slominski, B A

    2016-06-01

    This study evaluated the effect of yeast-derived products on growth performance, gut lesion score, intestinal population of Clostridium perfringens, and local innate immunity of broiler chickens challenged with C. perfringens. One-day-old broiler chickens were randomly assigned to eight dietary treatments providing six replicate pens of 55 birds each per treatment. Dietary treatments consisted of Control diets without and with C. perfringens challenge, and diets containing bacitracin methylene disalicylate (BMD, 55 g/tonne), nucleotides (150 g/tonne), yeast cell wall (YCW, 300 g/tonne), and a commercial product Maxi-Gen Plus (1 kg/tonne) fed to chickens challenged with C. perfringens. Diets containing 10% distillers dried grains with solubles without and with C. perfringens challenge were also used. Birds were orally challenged with C. perfringens (10(8) colony-forming units (cfu)/bird) on day 14. On day 21, intestinal samples were collected for gene expression analysis. Pathogen challenge significantly (P intestinal lesion scores were observed for challenged birds except the BMD-containing diet. Over the entire trial (1-35 days), no difference in growth performance was observed except the BMD diet which improved FCR over the Control, challenged group. Birds receiving nucleotides showed increased expression of toll-like receptors and cytokines interleukin (IL)-4 and IL-18 compared to the Control, challenged group. Expression of macrophage mannose receptor and IL-18 was upregulated in birds receiving YCW. Increased expression of cytokines and receptors involved in innate immunity in broilers receiving nucleotides and YCW suggests the immunomodulatory properties of these products under pathogen challenge conditions.

  19. Use of Plant Extracts as an Effective Manner to Control Clostridium perfringens Induced Necrotic Enteritis in Poultry

    Science.gov (United States)

    Dominguez, J. E.; Chacana, A. P.

    2016-01-01

    Necrotic enteritis (NE) is an important concern in poultry industry since it causes economic losses, increased mortality, reduction of bird welfare, and contamination of chicken products for human consumption. For decades, the use of in-feed antimicrobial growth promoters (AGPs) has been the main strategy to control intestinal pathogens including Clostridium perfringens (CP), the causative agent of NE. However, the use of AGPs in animal diet has been linked to the emergence and transmission of antimicrobial resistance through food-borne microorganisms, which has led to the ban of AGPs in many countries. This scenario has challenged the poultry industry to search for safer alternative products in order to prevent NE. In this context, the utilization of natural plant extracts with antimicrobial properties appears as a promising and feasible tool to control NE in chicken. In this paper, we review the scientific studies analyzing the potential of plant extracts as alternative feed additives to reduce NE in poultry, with focus on two types of plant products that arise as promising candidates: tannins and essential oils. Some of these products showed antimicrobial activity against CP and coccidia in vitro and in vivo and are able to increase productive performance, emulating the bioactive properties of AGPs. PMID:27747227

  20. Specific binding of Clostridium perfringens enterotoxin fragment to Claudin-b and modulation of zebrafish epidermal barrier.

    Science.gov (United States)

    Zhang, Jingjing; Ni, Chen; Yang, Zhenguo; Piontek, Anna; Chen, Huapu; Wang, Sijie; Fan, Yiming; Qin, Zhihai; Piontek, Joerg

    2015-08-01

    Claudins (Cldn) are the major components of tight junctions (TJs) sealing the paracellular cleft in tissue barriers of various organs. Zebrafish Cldnb, the homolog of mammalian Cldn4, is expressed at epithelial cell-cell contacts and is important for regulating epidermal permeability. The bacterial toxin Clostridium perfringens enterotoxin (CPE) has been shown to bind to a subset of mammalian Cldns. In this study, we used the Cldn-binding C-terminal domain of CPE (194-319 amino acids, cCPE 194-319 ) to investigate its functional role in modulating zebrafish larval epidermal barriers. In vitro analyses show that cCPE 194-319 removed Cldn4 from epithelial cells and disrupted the monolayer tightness, which could be rescued by the removal of cCPE 194-319. Incubation of zebrafish larvae with cCPE 194-319 removed Cldnb specifically from the epidermal cell membrane. Dye diffusion analysis with 4-kDa fluorescent dextran indicated that the permeability of the epidermal barrier increased due to cCPE 194-319 incubation. Electron microscopic investigation revealed reversible loss of TJ integrity by Cldnb removal. Collectively, these results suggest that cCPE 194-319 could be used as a Cldnb modulator to transiently open the epidermal barrier in zebrafish. In addition, zebrafish might be used as an in vivo system to investigate the capability of cCPE to enhance drug delivery across tissue barriers.

  1. Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia.

    Science.gov (United States)

    Bokori-Brown, Monika; Hall, Charlotte A; Vance, Charlotte; Fernandes da Costa, Sérgio P; Savva, Christos G; Naylor, Claire E; Cole, Ambrose R; Basak, Ajit K; Moss, David S; Titball, Richard W

    2014-05-13

    Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT50) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD50 dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.

  2. Use of Plant Extracts as an Effective Manner to Control Clostridium perfringens Induced Necrotic Enteritis in Poultry

    Directory of Open Access Journals (Sweden)

    J. M. Diaz Carrasco

    2016-01-01

    Full Text Available Necrotic enteritis (NE is an important concern in poultry industry since it causes economic losses, increased mortality, reduction of bird welfare, and contamination of chicken products for human consumption. For decades, the use of in-feed antimicrobial growth promoters (AGPs has been the main strategy to control intestinal pathogens including Clostridium perfringens (CP, the causative agent of NE. However, the use of AGPs in animal diet has been linked to the emergence and transmission of antimicrobial resistance through food-borne microorganisms, which has led to the ban of AGPs in many countries. This scenario has challenged the poultry industry to search for safer alternative products in order to prevent NE. In this context, the utilization of natural plant extracts with antimicrobial properties appears as a promising and feasible tool to control NE in chicken. In this paper, we review the scientific studies analyzing the potential of plant extracts as alternative feed additives to reduce NE in poultry, with focus on two types of plant products that arise as promising candidates: tannins and essential oils. Some of these products showed antimicrobial activity against CP and coccidia in vitro and in vivo and are able to increase productive performance, emulating the bioactive properties of AGPs.

  3. Clostridium perfringens phospholipase C induced ROS production and cytotoxicity require PKC, MEK1 and NFκB activation.

    Directory of Open Access Journals (Sweden)

    Laura Monturiol-Gross

    Full Text Available Clostridium perfringens phospholipase C (CpPLC, also called α-toxin, is the most toxic extracellular enzyme produced by this bacteria and is essential for virulence in gas gangrene. At lytic concentrations, CpPLC causes membrane disruption, whereas at sublytic concentrations this toxin causes oxidative stress and activates the MEK/ERK pathway, which contributes to its cytotoxic and myotoxic effects. In the present work, the role of PKC, ERK 1/2 and NFκB signalling pathways in ROS generation induced by CpPLC and their contribution to CpPLC-induced cytotoxicity was evaluated. The results demonstrate that CpPLC induces ROS production through PKC, MEK/ERK and NFκB pathways, the latter being activated by the MEK/ERK signalling cascade. Inhibition of either of these signalling pathways prevents CpPLC's cytotoxic effect. In addition, it was demonstrated that NFκB inhibition leads to a significant reduction in the myotoxicity induced by intramuscular injection of CpPLC in mice. Understanding the role of these signalling pathways could lead towards developing rational therapeutic strategies aimed to reduce cell death during a clostridialmyonecrosis.

  4. Molecular architecture and functional analysis of NetB, a pore-forming toxin from Clostridium perfringens.

    Science.gov (United States)

    Savva, Christos G; Fernandes da Costa, Sérgio P; Bokori-Brown, Monika; Naylor, Claire E; Cole, Ambrose R; Moss, David S; Titball, Richard W; Basak, Ajit K

    2013-02-01

    NetB is a pore-forming toxin produced by Clostridium perfringens and has been reported to play a major role in the pathogenesis of avian necrotic enteritis, a disease that has emerged due to the removal of antibiotics in animal feedstuffs. Here we present the crystal structure of the pore form of NetB solved to 3.9 Å. The heptameric assembly shares structural homology to the staphylococcal α-hemolysin. However, the rim domain, a region that is thought to interact with the target cell membrane, shows sequence and structural divergence leading to the alteration of a phosphocholine binding pocket found in the staphylococcal toxins. Consistent with the structure we show that NetB does not bind phosphocholine efficiently but instead interacts directly with cholesterol leading to enhanced oligomerization and pore formation. Finally we have identified conserved and non-conserved amino acid positions within the rim loops that significantly affect binding and toxicity of NetB. These findings present new insights into the mode of action of these pore-forming toxins, enabling the design of more effective control measures against necrotic enteritis and providing potential new tools to the field of bionanotechnology.

  5. A claudin 3 and claudin 4-targeted Clostridium perfringens protoxin is selectively cytotoxic to PSA-producing prostate cancer cells.

    Science.gov (United States)

    Romanov, Victor; Whyard, Terry C; Waltzer, Wayne C; Gabig, Theodore G

    2014-09-01

    Prostate cancer is the second leading cause of non-cutaneous cancer-related death in males, and effective strategies for treatment of metastatic disease are currently limited. The tight junction proteins, claudin 3 and claudin 4, serve as cell-surface receptors for the pore-forming Clostridium perfringens enterotoxin [CPE]. Most prostate cancer cells overexpress claudin 3 and claudin 4, and claudins are aberrantly distributed over the plasma membrane, making these cells particularly sensitive to cytolysis by CPE. Prostate cancer cells secrete PSA locally that is proteolytically active; however, circulating PSA is inactivated via binding to protease inhibitors. To overcome systemic toxicity of CPE, a modified protoxin was constructed with a tethered ligand attached to the C-terminus connected by a flexible linker containing a PSA-specific protease cleavage site. This engineered protoxin selectively and efficiently lyses PSA-producing prostate cancer cells whereas CLDN3 and CLDN4 positive cells that do not express PSA are resistant to cytolysis.

  6. Clostridium perfringens TpeL Induces Formation of Stress Fibers via Activation of RhoA-ROCK Signaling Pathway.

    Science.gov (United States)

    Nagahama, Masahiro; Ohkubo, Akiko; Kinouchi, Yoshihito; Kobayashi, Keiko; Miyamoto, Kazuaki; Takehara, Masaya; Sakurai, Jun

    2015-01-01

    Clostridium perfringens TpeL belongs to a family of large clostridial glucosylating cytotoxins. TpeL modifies Rac1 and Ras subfamily proteins. Herein we report TpeL-induced formation of stress fibers via RhoA-Rho kinase (ROCK) signaling. A recombinant protein (TpeL1-525) derived from the TpeL N-terminal catalytic domain in the presence of streptolysin O (SLO) induced the formation of actin stress fibers in Madin-Darby canine kidney (MDCK) cells in a dose-dependent manner. The RhoA/ROCK pathway is known to control the formation of stress fibers. We examined the role of the RhoA/ROCK pathway in TpeL-induced formation of stress fibers. TpeL1-525-induced formation of stress fibers was inhibited by the ROCK inhibitor, Y27632 and Rho protein inhibitor, C3 transferase. TpeL1-525 activated RhoA and ROCK in a dose-dependent manner. C3 transferase blocked TpeL1-525-induced activation of RhoA and ROCK whereas Y27632 inhibited TpeL-induced activation of ROCK. These results demonstrate for the first time that TpeL induces the formation of stress fibers by activating the RhoA/ROCK signaling pathway.

  7. Clostridium perfringens sporulation, germination and outgrowth in food: a functional genomics approach

    NARCIS (Netherlands)

    Xiao, Y.

    2014-01-01

    At the heart of foodborne disease caused by Clostridium perfringenslays its ability to form spores. The ubiquitous presence of C. perfringensis due to the fact that spores are resilient and are able to survive harsh environmental conditions. As a result, spores of C. perfringenscan be found in many

  8. Fatal Clostridium perfringens sepsis due to emphysematous gastritis and literature review.

    Science.gov (United States)

    Sarvari, Karoly Peter; Vasas, Bela; Kiss, Ildiko; Lazar, Andrea; Horvath, Istvan; Simon, Marianna; Peto, Zoltan; Urban, Edit

    2016-08-01

    A 76-year-old female patient was admitted to the Level I Emergency Department of University of Szeged with severe abdominal pain and vomiting. The clinical assessment with laboratory tests and radiological investigations confirmed severe sepsis associated with intravascular hemolysis and multiorgan failure and acute pancreatitis. On the abdominal CT, besides of other abnormalities, the presence of gas bubbles in the stomach, small intestines and liver were seen. The gastric alterations pointed to emphysematous gastritis. Despite of the medical treatment, the patient's condition quickly deteriorated and eight hours after admission the patient died. The autopsy evaluation revealed systemic infection of abdominal origin caused by gas-producing Gram-positive bacteria, and the post-mortem microbiological cultures confirmed the presence of Cloctridium perfringens in many abdominal organs. Emphysematous gastritis seemed to be the primary infectious focus. Copyright © 2016 Elsevier Ltd. All rights reserved.

  9. Clostridium perfringens em rações e águas fornecidos a frangos de corte em granjas avícolas do interior paulista: Brasil Clostridium perfringens search in water and ration used in the raising of broiler in sheds of São Paulo State: Brazil

    Directory of Open Access Journals (Sweden)

    Rubén Pablo Schocken-Iturrino

    2010-02-01

    Full Text Available Através de métodos bacteriológicos convencionais, avaliou-se a contaminação por Clostridium perfringens na ração e água utilizadas na alimentação e dessedentação de frangos de corte em diferentes regiões avícolas do interior paulista. C. perfringens esteve presente em 42 e 30% das amostras de ração e águas analisadas, respectivamente. As médias das contagens foram 6,7 x 10-2UFC mL para as amostras de água e 3,69 x 10-2UFC g para as de rações. As altas freqüências e contagens de C. perfringens verificadas nas rações e nas águas podem estar associadas à falta de higiene geral na manipulação e armazenamento dos mesmos. Sugere-se o monitoramento periódico da presença de C. perfringens nestas fontes, com a finalidade de evitar tal patógeno, em vista que o mesmo pode causar um surto de enterite necrótica levando, assim a grandes prejuízos na produção avícola.Through conventional bacteriological methods, the contamination by Clostridium perfringens was evaluated in the ration and water used in the feeding of poultry chickens from different region of the interior from São Paulo. C. perfringens was present in 42 and 30% of the ration samples and waters analyzed respectively. The averages of the countings were 6.7 x 10-2CFU mL for the samples of water and 3.69 x 10-2CFU g for rations. The high frequencies and countings of C. perfringens verified in the rations and in the waters may be associated to the lack of general hygiene in the manipulation and storage of the same ones. These suggests a periodic monitoration of the presence of C. perfringens in these sources, with the purpose of avoiding such pathogen, in view that this organism can provoke an outbreak of necrotic enteritis, and cause great damages in the poultry production.

  10. Clostridium perfringens bacteriophages ΦCP39O and ΦCP26F: genomic organization and proteomic analysis of the virions.

    Science.gov (United States)

    Seal, Bruce S; Fouts, Derrick E; Simmons, Mustafa; Garrish, Johnna K; Kuntz, Robin L; Woolsey, Rebekah; Schegg, Kathleen M; Kropinski, Andrew M; Ackermann, Hans-W; Siragusa, Gregory R

    2011-01-01

    Poultry intestinal material, sewage and poultry processing drainage water were screened for virulent Clostridium perfringens bacteriophages. Viruses isolated from broiler chicken offal washes (O) and poultry feces (F), designated ΦCP39O and ΦCP26F, respectively, produced clear plaques on host strains. Both bacteriophages had isometric heads of 57 nm in diameter with 100-nm non-contractile tails characteristic of members of the family Siphoviridae in the order Caudovirales. The double-strand DNA genome of bacteriophage ΦCP39O was 38,753 base pairs (bp), while the ΦCP26F genome was 39,188 bp, with an average GC content of 30.3%. Both viral genomes contained 62 potential open reading frames (ORFs) predicted to be encoded on one strand. Among the ORFs, 29 predicted proteins had no known similarity while others encoded putative bacteriophage capsid components such as a pre-neck/appendage, tail, tape measure and portal proteins. Other genes encoded a predicted DNA primase, single-strand DNA-binding protein, terminase, thymidylate synthase and a transcription factor. Potential lytic enzymes such as a fibronectin-binding autolysin, an amidase/hydrolase and a holin were encoded in the viral genomes. Several ORFs encoded proteins that gave BLASTP matches with proteins from Clostridium spp. and other Gram-positive bacterial and bacteriophage genomes as well as unknown putative Collinsella aerofaciens proteins. Proteomics analysis of the purified viruses resulted in the identification of the putative pre-neck/appendage protein and a minor structural protein encoded by large open reading frames. Variants of the portal protein were identified, and several mycobacteriophage gp6-like protein variants were detected in large amounts relative to other virion proteins. The predicted amino acid sequences of the pre-neck/appendage proteins had major differences in the central portion of the protein between the two phage gene products. Based on phylogenetic analysis of the large

  11. Quantitative Detection of Clostridium perfringens in Broiler Chickens by Real-Time PCR Targeting the Alpha-Toxin Gene

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Engberg, Ricarda M.; Schramm, Andreas

    2006-01-01

    was developed by sequencing the α-toxin gene from ~60 strains of C. perfringens, isolated from diseased as well as healthy broilers. For its application to the chicken gastrointestinal tract (i.e., ileum), DNA extraction efficiency and potential inhibition of the real-time PCR process by ileum content...... was evaluated by (i) analysing ileum samples spiked with C. perfringens cells, and (ii) comparing DNA extractions/real-time PCR of pure cultures with and without ileum content added. No inhibition was observed, and the quantitative real-time PCR approach correlated well with counts in pure cultures and spiked...... ileum content. The protocol had a detection limit of ~1000 cells spiked into 200 mg ileal material. This matched the sensitivity obtained using published primers targeting C. perfringens 16S rDNA. However, targeting the functional α-toxin gene offers the possibility to assess not only abundance of C...

  12. Tissue distribution and safety evaluation of a claudin-targeting molecule, the C-terminal fragment of Clostridium perfringens enterotoxin.

    Science.gov (United States)

    Li, Xiangru; Saeki, Rie; Watari, Akihiro; Yagi, Kiyohito; Kondoh, Masuo

    2014-02-14

    We previously found that claudin (CL) is a potent target for cancer therapy using a CL-3 and -4-targeting molecule, namely the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE). Although CL-3 and -4 are expressed in various normal tissues, the safety of this CL-targeting strategy has never been investigated. Here, we evaluated the tissue distribution of C-CPE in mice. Ten minutes after intravenous injection into mice, C-CPE was distributed to the liver and kidney (24.0% and 9.5% of the injected dose, respectively). The hepatic level gradually fell to 3.2% of the injected dose by 3 h post-injection, whereas the renal C-CPE level gradually rose to 46.5% of the injected dose by 6 h post-injection and then decreased. A C-CPE mutant protein lacking the ability to bind CL accumulated in the liver to a much lesser extent (2.0% of the dose at 10 min post-injection) than did C-CPE, but its renal profile was similar to that of C-CPE. To investigate the acute toxicity of CL-targeted toxin, we intravenously administered C-CPE-fused protein synthesis inhibitory factor to mice. The CL-targeted toxin dose-dependently increased the levels of serum biomarkers of liver injury, but not of kidney injury. Histological examination confirmed that injection of CL-targeted toxin injured the liver but not the kidney. These results indicate that potential adverse hepatic effects should be considered in C-CPE-based cancer therapy.

  13. Comparative genomics of four closely related Clostridium perfringens bacteriophages reveals variable evolution among core genes with therapeutic potential

    Directory of Open Access Journals (Sweden)

    Siragusa Gregory R

    2011-06-01

    Full Text Available Abstract Background Because biotechnological uses of bacteriophage gene products as alternatives to conventional antibiotics will require a thorough understanding of their genomic context, we sequenced and analyzed the genomes of four closely related phages isolated from Clostridium perfringens, an important agricultural and human pathogen. Results Phage whole-genome tetra-nucleotide signatures and proteomic tree topologies correlated closely with host phylogeny. Comparisons of our phage genomes to 26 others revealed three shared COGs; of particular interest within this core genome was an endolysin (PF01520, an N-acetylmuramoyl-L-alanine amidase and a holin (PF04531. Comparative analyses of the evolutionary history and genomic context of these common phage proteins revealed two important results: 1 strongly significant host-specific sequence variation within the endolysin, and 2 a protein domain architecture apparently unique to our phage genomes in which the endolysin is located upstream of its associated holin. Endolysin sequences from our phages were one of two very distinct genotypes distinguished by variability within the putative enzymatically-active domain. The shared or core genome was comprised of genes with multiple sequence types belonging to five pfam families, and genes belonging to 12 pfam families, including the holin genes, which were nearly identical. Conclusions Significant genomic diversity exists even among closely-related bacteriophages. Holins and endolysins represent conserved functions across divergent phage genomes and, as we demonstrate here, endolysins can have significant variability and host-specificity even among closely-related genomes. Endolysins in our phage genomes may be subject to different selective pressures than the rest of the genome. These findings may have important implications for potential biotechnological applications of phage gene products.

  14. Removal and fate of Cryptosporidium parvum, Clostridium perfringens and small-sized centric diatoms (Stephanodiscus hantzschii) in slow sand filters.

    Science.gov (United States)

    Hijnen, Wim A M; Dullemont, Yolanda J; Schijven, Jack F; Hanzens-Brouwer, Anke J; Rosielle, Martine; Medema, Gertjan

    2007-05-01

    The decimal elimination capacity (DEC) of slow sand filtration (SSF) for Cryptosporidium parvum was assessed to enable quantitative microbial risk analysis of a drinking water production plant. A mature pilot plant filter of 2.56m(2) was loaded with C. parvum oocysts and two other persistent organisms as potential surrogates; spores of Clostridium perfringens (SCP) and the small-sized (4-7microm) centric diatom (SSCD) Stephanodiscus hantzschii. Highly persistent micro-organisms that are retained in slow sand filters are expected to accumulate and eventually break through the filter bed. To investigate this phenomenon, a dosing period of 100 days was applied with an extended filtrate monitoring period of 150 days using large-volume sampling. Based on the breakthrough curves the DEC of the filter bed for oocysts was high and calculated to be 4.7log. During the extended filtrate monitoring period the spatial distribution of the retained organisms in the filter bed was determined. These data showed little risk of accumulation of oocysts in mature filters most likely due to predation by zooplankton. The DEC for the two surrogates, SCP and SSCD, was 3.6 and 1.8log, respectively. On basis of differences in transport behaviour, but mainly because of the high persistence compared to the persistence of oocysts, it was concluded that both spores of sulphite-reducing clostridia (incl. SCP) and SSCD are unsuited for use as surrogates for oocyst removal by slow sand filters. Further research is necessary to elucidate the role of predation in Cryptosporidium removal and the fate of consumed oocysts.

  15. The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

    Directory of Open Access Journals (Sweden)

    Kareem Rashid Rumah

    2015-05-01

    Full Text Available Clostridium perfringens ε-toxin (ETX is a potent pore-forming toxin responsible for a central nervous system (CNS disease in ruminant animals with characteristics of blood-brain barrier (BBB dysfunction and white matter injury. ETX has been proposed as a potential causative agent for Multiple Sclerosis (MS, a human disease that begins with BBB breakdown and injury to myelin forming cells of the CNS. The receptor for ETX is unknown. Here we show that both binding of ETX to mammalian cells and cytotoxicity requires the tetraspan proteolipid Myelin and Lymphocyte protein (MAL. While native Chinese Hamster Ovary (CHO cells are resistant to ETX, exogenous expression of MAL in CHO cells confers both ETX binding and susceptibility to ETX-mediated cell death. Cells expressing rat MAL are ~100 times more sensitive to ETX than cells expressing similar levels of human MAL. Insertion of the FLAG sequence into the second extracellular loop of MAL abolishes ETX binding and cytotoxicity. ETX is known to bind specifically and with high affinity to intestinal epithelium, renal tubules, brain endothelial cells and myelin. We identify specific binding of ETX to these structures and additionally show binding to retinal microvasculature and the squamous epithelial cells of the sclera in wild-type mice. In contrast, there is a complete absence of ETX binding to tissues from MAL knockout (MAL-/- mice. Furthermore, MAL-/- mice exhibit complete resistance to ETX at doses in excess of 1000 times the symptomatic dose for wild-type mice. We conclude that MAL is required for both ETX binding and cytotoxicity.

  16. In vivo antimicrobial potentials of garlic against Clostridium perfringens and its promotant effects on performance of broiler chickens.

    Science.gov (United States)

    Jimoh, A A; Ibitoye, E B; Dabai, Y U; Garba, S

    2013-12-15

    This study was conducted to investigate in vivo antimicrobial potential of garlic against Clostridium perferinges and resultant promotant effects on performance of the broiler chickens. Garlic powder was used as an alternative to GPAs (Growth Promotant Antibiotics) to prevent subclinical Necrotic Enteritis (NE) due to C. perferinges. 120 day-old broiler chicks were randomly distributed to six treatment groups of 20 chicks each (2 replicates(-10) chicks). Six isonutrient diets supplemented with garlic at graded levels of 0.0, 0.5, 1.0, 1.5, 2.0 and 2.5 g kg(-1) were fed to the birds for seven weeks. Data were collected weekly on performance parameters including feed intake, weight gain and feed conversion ratio (FCR). Also, on the 21 35 and 49th days of the study, two birds per group were randomly selected, slaughtered and dissected. 1 g of caecal contents per each bird were sampled into labelled sterile sample bottles. The samples were subjected to culturing, bacterial identification and colony counting. All data were subjected to analysis of variance. Results showed that garlic significantly (p > 0.05) depressed feed intake (3310 g feed/bird at 1.0 g kg(-1) supplementation) but improved FCR. The supplement has no significant effect on weight gain but C. perfringens colony counts in the treated groups, were numerically reduced (lowest count, 0.93 x 10(5) cfu g(-1) at 1.0 g kg(-1) supplementation), as compared to the control. It is therefore concluded that diets could be supplemented with garlic at dose range of 1.0 to 1.5 g kg(-1) to prevent subclinical NE and achieve improved performance in birds.

  17. Bacterial spores as possible contaminants of biomedical materials and devices. [Bacillus anthracis, clostridium botulinum, C. perfringens, C. tetani

    Energy Technology Data Exchange (ETDEWEB)

    Grecz, N.; Kang, T.

    1973-01-01

    Destruction of spores on biomedical devices in drugs, and biologicals is essential for prevention of infection of patients with pathogenic sporeformers. Of particular concern are Clostridium tetani, C. perfringens, C. botulinum, Bacillus anthracis and other sporeforming pathogens. Spores are ubiquitous in nature and contamination of biomedical devices varies depending on manufacturing process, handling, raw materials and other variables. In the last 20 years the number of cases per year of specific notifiable diseases in the United States was as follows: tetanus, 120 to 500 cases, botulism, 7 to 47 cases, and anthrax, 2 to 10 cases. Gas gangrene is caused by a mixed flora consisting predominantly of sporeformers. C botulinum, which usually acts as saprophytic agent of food poisoning, may also initiate pathogenic processes; there are nine cases on record in the United States of botulism wound infections almost half of which ended in death. The spores of these organisms are distinguished by high radiation resistance and their erradication often requires severe radiation treatments. Representative bacterial spores in various suspending media show D/sub 10/ values (dose necessary to destroy 90 percent of a given population) ranging from approximately 0.1 to 0.4 Mrad. Some viruses show D/sub 10/ values up to greater than 1 Mrad. The D/sub 10/-values of spores vary depending on physical, chemical and biological factors. This variability is important in evaluation and selection of biological indicator organisms. Radiation sterilization of biomedical devices and biomedical materials must provide safety from infectious microorganisms including radiation resistant spores and viruses.

  18. The effect of calcium and sodium lactates on growth from spores of Bacillus cereus and Clostridium perfringens in a 'sous-vide' beef goulash under temperature abuse.

    Science.gov (United States)

    Aran, N

    2001-01-22

    The effect of calcium and sodium lactates on growth from spores of Bacillus cereus and Clostridium perfringens at three different concentrations (0, 1.5 and 3% w/w) and at different temperatures (10, 15 and 20 degrees C for B. cereus and 15, 20 and 25 degrees C for C. perfringens) was investigated, using beef goulash as a model system for pasteurised vacuum-packaged convenience foods. Calcium lactate at a level of 3% reduced the pH values of the samples from 6.0 to 5.5. No B. cereus growth was observed at 10 degrees C, but after 7 days at an incubation temperature of 15 degrees C, cell number increased by 1 log cfu/g in the control samples. At this temperature, lactates were seen to be effective at inhibiting growth. Calcium lactate was more inhibitory than sodium lactate as the growth of B. cereus was inhibited at 1.5 and 3% concentrations at 20 degrees C, respectively. Growth of C. perfringens was arrested in the presence of 1.5% calcium lactate at all storage temperatures, whereas growth was inhibited by 3% sodium lactate only at 15 degrees C.

  19. Relative disease susceptibility and clostridial toxin antibody responses in three commercial broiler lines co-infected with Clostridium perfringens and Eimeria maxima using an experimental model of necrotic enteritis

    Science.gov (United States)

    Necrotic enteritis is an enteric disease of poultry resulting from infection by Clostridium perfringens with co-infection by Eimeria spp. constituting a major risk factor for disease pathogenesis. This study compared three commercial broiler chicken lines using an experimental model of necrotic ente...

  20. Development of an integrated model for heat transfer and dynamic growth of Clostridium perfringens during the cooling of cooked boneless ham.

    Science.gov (United States)

    Amézquita, A; Weller, C L; Wang, L; Thippareddi, H; Burson, D E

    2005-05-25

    Numerous small meat processors in the United States have difficulties complying with the stabilization performance standards for preventing growth of Clostridium perfringens by 1 log10 cycle during cooling of ready-to-eat (RTE) products. These standards were established by the Food Safety and Inspection Service (FSIS) of the US Department of Agriculture in 1999. In recent years, several attempts have been made to develop predictive models for growth of C. perfringens within the range of cooling temperatures included in the FSIS standards. Those studies mainly focused on microbiological aspects, using hypothesized cooling rates. Conversely, studies dealing with heat transfer models to predict cooling rates in meat products do not address microbial growth. Integration of heat transfer relationships with C. perfringens growth relationships during cooling of meat products has been very limited. Therefore, a computer simulation scheme was developed to analyze heat transfer phenomena and temperature-dependent C. perfringens growth during cooling of cooked boneless cured ham. The temperature history of ham was predicted using a finite element heat diffusion model. Validation of heat transfer predictions used experimental data collected in commercial meat-processing facilities. For C. perfringens growth, a dynamic model was developed using Baranyi's nonautonomous differential equation. The bacterium's growth model was integrated into the computer program using predicted temperature histories as input values. For cooling cooked hams from 66.6 degrees C to 4.4 degrees C using forced air, the maximum deviation between predicted and experimental core temperature data was 2.54 degrees C. Predicted C. perfringens growth curves obtained from dynamic modeling showed good agreement with validated results for three different cooling scenarios. Mean absolute values of relative errors were below 6%, and deviations between predicted and experimental cell counts were within 0.37 log10

  1. Effects of heat stress on the formation of splenic germinal centres and immunoglobulins in broilers infected by Clostridium perfringens type A.

    Science.gov (United States)

    Calefi, Atílio Sersun; de Siqueira, Adriana; Namazu, Lilian Bernadete; Costola-de-Souza, Carolina; Honda, Bruno Bueno Takashi; Ferreira, Antonio José Piantino; Quinteiro-Filho, Wanderley Moreno; da Silva Fonseca, Juliana Garcia; Palermo-Neto, João

    2016-03-01

    Avian necrotic enteritis (NE) induced by Clostridium perfringens is a disease that affects mainly the first weeks of poultry's life. The pathogenesis of NE is complex and involves the combination of several factors, such as co-infection with different species of coccidia, immunosuppression and stress. Stress is one of the main limiting factors in poultry production. Although several studies emphasized the effects of stress on immunity, few works analyzed these effects on immunoglobulins and on germinal centres (GCs), which are specialized microenvironments, responsible for generating immune cells with high affinity antibodies and memory B-lymphocytes. Thus, the effects of heat stress associated or not with thioglycolate broth culture medium intake and/or C. perfringens infection on corticosterone serum levels, spleen GCs development and immunoglobulin production in broilers were evaluated. Results showed that heat stress, thioglycolate and C. perfringens per se increased corticosterone serum levels, although this was not observed in heat stressed and thioglycolate and C. perfringens-treated chickens. The serum levels of IgA, IgM and IgY were differently affected by heat stress and/or infection/thioglycolate. Heat stress decreased the duodenal concentrations of sIgA, which was accompanied by a reduction in GCs number in the duodenal lamina propria; a trend to similar findings of sIgA concentrations was observed in the chickens' jejunum. Changes in spleen and Bursa of Fabricius relative weights as well as in spleen morphometry were also noted in heat stressed animals, infected or not. Together, these data suggest that heat stress change GCs formation in chickens infected or not, which that may lead to failures in vaccination protocols as well as in the poultries' host resistance to infectious diseases during periods of exposure to heat stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  2. Effects of Temperature and Packaging on the Growth Kinetics of Clostridium perfringens in Ready-to-eat Jokbal (Pig's Trotters).

    Science.gov (United States)

    Park, Hee-Jin; Na, Yu-Jin; Cho, Joon-Il; Lee, Soon-Ho; Yoon, Ki-Sun

    2014-01-01

    Ready-to-eat (RTE) Jokbal (Pig's trotter), which consists of pig's feet cooked in soy sauce and various spices, is a very popular and widely sold in Korean retail markets. Commercially, the anaerobically packed Jokbal have also become a popular RTE food in several convenience stores. This study evaluates the effects of storage temperature and packaging methods for the growth of C. perfringens in Jokbal. Growth kinetic parameters of C. perfringens in aerobically and anaerobically packed Jokbals are determined at each temperature by the modified Gompertz equation. The lag time, specific growth rate, and maximum population density of C. perfringens are being analyzed as a function of temperature and packaging method. The minimum growth temperature of C. perfringens in aerobically and anaerobically packed Jokbal is 24℃ and 18℃, respectively. The C. perfringens in Jokbal did not grow under conditions of over 50℃ regardless of the packaging methods, indicating that the holding temperature of Jokbal in markets must be maintained at above 50℃ or below 18℃. Growth of C. perfringens in anaerobically packed Jokbal is faster than in aerobically packed Jokbal when stored under the same conditions. This indicates that there are a higher risks associated with C. perfringens for anaerobically packed meat products.

  3. Isolation,Identification and Drug Sensitivity Test of Clostridium perfringens%鸡产气荚膜梭菌的分离鉴定及药敏试验

    Institute of Scientific and Technical Information of China (English)

    陈海宁; 廖申权; 戚南山; 李娟; 吕敏娜; 孙铭飞; 吴彩艳

    2013-01-01

    为确定广东韶关地区两个养殖密度较大鸡场病死鸡的患病病原,根据临床症状、剖检病变疑似坏死性肠炎,每场各分离到1株细菌,通过对细菌分离株进行形态培养、生化鉴定、牛乳汹涌发酵试验、PCR种特异性和毒素型鉴定等试验,鉴定2个分离株均为 A 型产气荚膜梭菌。对分离株进行药敏试验,测定了临床上常用的抗菌药物添加剂对两株产气荚膜梭菌的最小抑菌浓度(MIC),结果显示恩拉霉素和维吉尼亚霉素对两分离株具有良好的抑菌效果,黄霉素、吉他霉素、硫酸黏杆菌素对个别菌株具有抑菌作用,而那西肽则无明显抑菌作用。研究结果可为鸡坏死性肠炎的临床防控提供参考。%Two strains of bacteria were isolated from resembled necrotic enteritis infectious cases basing on clinical symptom and pathological lesions in two scaled chicken farms in Shaoguan area,Guang dong prov-ince.According to the bacterial morphology,biochemical properties,milk fermentation tests,species-spe-cific and toxin PCR tests,the pathogen was identified as A type Clostridium perfringens .We determined Minimum Inhibitory Concentration (MIC)of antibiotic additives used commonly for two strains.The re-sults showed that enramycin and virginiamycin had a better antibacterial effect than flavomycin,kitasamy-cin and colistin sulfate,and nosiheptide didn′t have an obvious inhibitory effect.This results may provide a guiding significance for the prevention and control of necrotic enteritis in chickens.

  4. Clostridium perfringens enterotoxin carboxy-terminal fragment is a novel tumor-homing peptide for human ovarian cancer

    Directory of Open Access Journals (Sweden)

    Azoudi Masoud

    2010-07-01

    Full Text Available Abstract Background Development of innovative, effective therapies against recurrent/chemotherapy-resistant ovarian cancer remains a high priority. Using high-throughput technologies to analyze genetic fingerprints of ovarian cancer, we have discovered extremely high expression of the genes encoding the proteins claudin-3 and claudin-4. Methods Because claudin-3 and -4 are the epithelial receptors for Clostridium perfringens enterotoxin (CPE, and are sufficient to mediate CPE binding, in this study we evaluated the in vitro and in vivo bioactivity of the carboxy-terminal fragment of CPE (i.e., CPE290-319 binding peptide as a carrier for tumor imaging agents and intracellular delivery of therapeutic drugs. Claudin-3 and -4 expression was examined with rt-PCR and flow cytometry in multiple primary ovarian carcinoma cell lines. Cell binding assays were used to assess the accuracy and specificity of the CPE peptide in vitro against primary chemotherapy-resistant ovarian carcinoma cell lines. Confocal microscopy and biodistribution assays were performed to evaluate the localization and uptake of the FITC-conjugated CPE peptide in established tumor tissue. Results Using a FITC-conjugated CPE peptide we show specific in vitro and in vivo binding to multiple primary chemotherapy resistant ovarian cancer cell lines. Bio-distribution studies in SCID mice harboring clinically relevant animal models of chemotherapy resistant ovarian carcinoma showed higher uptake of the peptide in tumor cells than in normal organs. Imunofluorescence was detectable within discrete accumulations (i.e., tumor spheroids or even single chemotherapy resistant ovarian cancer cells floating in the ascites of xenografted animals while a time-dependent internalization of the FITC-conjugated CPE peptide was consistently noted in chemotherapy-resistant ovarian tumor cells by confocal microscopy. Conclusions Based on the high levels of claudin-3 and -4 expression in chemotherapy

  5. Haemorrhagic bowel syndrome in dairy cattle: possible role of Clostridium perfringens type A in the disease complex.

    Science.gov (United States)

    Ceci, L; Paradies, P; Sasanelli, M; de Caprariis, D; Guarda, F; Capucchio, M T; Carelli, G

    2006-12-01

    A survey based on clinical, pathological and microbiological investigations was performed on 11 Brown Swiss cattle affected with depression, anorexia, agalaxia, ruminal hypomotility, abdominal pain and melaena. In eight animals, macroscopical lesions consisted in haemorrhagic enteritis in the small intestine. Seven of eight isolates from tissue samples were identified as Clostridum perfringens type A, and four were identified as C. perfringens type A with the beta2 toxin gene. Based on these observations, animals were considered affected with haemorrhagic bowel syndrome.

  6. Experimental induction of abdominal tympany, abomasitis, and abomasal ulceration by intraruminal inoculation of Clostridium perfringens type A in neonatal calves.

    Science.gov (United States)

    Roeder, B L; Chengappa, M M; Nagaraja, T G; Avery, T B; Kennedy, G A

    1988-02-01

    The etiologic role of Clostridum perfringens type A in the acute abdominal syndrome characterized by abomasal and rumen tympany, abomasitis, and abomasal ulceration was investigated in neonatal calves. Eight calves, 4 to 12 days old, were inoculated intraruminally with toxigenic C perfringens type A. Before and after C perfringens inoculation, blood samples were collected from all calves for blood gas and serum biochemical analysis and for determination of serum copper concentration; ruminal fluid was obtained for isolation of C perfringens. Calves were monitored daily for clinical signs of the syndrome and, depending on the severity of clinical signs, they were either euthanatized or redosed within 4 to 7 days. After necropsy, specimens obtained from the abomasum and rumen for macroscopic and microscopic examination and for anaerobic bacteriologic culture were processed in routine manner. Intraruminal inoculation of C perfringens type A into healthy calves induced anorexia, depression, bloat, diarrhea, and in some calves, death. Serum copper concentration was within normal range. Necropsy revealed variable degrees of abomasitis, petechial and ecchymotic hemorrhages, and ulcers (ranging from pinpoint to nearly perforate) in the abomasum. Seven of those calves also had multiple trichobezoars in the rumen. These necropsy findings were not seen in calves (controls) given distilled H2O only. In affected calves, acute abdominal syndrome was unrelated to copper deficiency, and C perfringens type A given intraruminally was able to induce clinical signs similar to those of the naturally acquired disease.

  7. Study of Real- time PCR Assay for Rapid Detection of Alpha Toxin Gene from Clostridium perfringens in Food%食品中产气荚膜梭菌α毒素基因快速检测方法的研究

    Institute of Scientific and Technical Information of China (English)

    贺连华; 扈庆华; 王冰; 吴平芳; 石晓路; 陈妙玲

    2011-01-01

    Objective To rapidly detect Clostridium perfringens by developing a molecular - beacon - based PCR assay, and the established method was applied to rapid diagnosis of food poisoning. Methods Based on the core sequence of gene published on GenBank, the primers and modified molecular beacons were designed to detect alpha toxin gene from Clostridium perfringens. The probe was labeled with FAM in 5' end. The molecular beacons and primer set were tested against numerous strains from 10 different bacterial species. Results With the PCR assay based on the molecular beacon, Clostridium perfringens and no false signals were observed. There was no cross - reaction with other bacteria as control. 40 food samples were tested, 3 samples were found Clostridium perfringens positive by the PCR assay. Among the tested positive samples, 2 samples were detected Clostridium perfringens positive by traditional culture method. The overall test could be finished within 2 hours to one day starting from sample preparation. Conclusions The molecular - beacon - based PCR assay is rapid, sensitive, and specific. It could be applied to the rapid diagnosis of Clostridium perfringens food poisoning.%目的 建立分子信标荧光PCR体系检测产气荚膜梭菌的快速检测方法,应用于食物中毒快速诊断和食品微生物检验.方法 根据GenBank公布的保守序列,针对α毒素基因设计一对引物和分子信标探针,用FAM荧光剂标记探针的5端,并进行特异性和灵敏度分析,同时以10种细菌作对照.结果 分子信标荧光PCR反应体系检测10种细菌,只有产气荚膜梭菌出现特异荧光信号,其他均无荧光信号,而且与其他细菌无交叉反应.对40份食品样品进行检测,3份产气荚膜梭菌PCR阳性,其余样品为阴性,检测仅需2 h.3份阳性样本经传统方法培养,2份有检出产气荚膜梭菌.结论 分子信标荧光PCR检测体系快速、灵敏度高,特异性强,可用于产气荚膜梭菌食物中毒的快

  8. Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica.

    Science.gov (United States)

    Lisle, John T; Smith, James J; Edwards, Diane D; McFeters, Gordon A

    2004-12-01

    McMurdo Station, Antarctica, has discharged untreated sewage into McMurdo Sound for decades. Previous studies delineated the impacted area, which included the drinking water intake, by using total coliform and Clostridium perfringens concentrations. The estimation of risk to humans in contact with the impacted and potable waters may be greater than presumed, as these microbial indicators may not be the most appropriate for this environment. To address these concerns, concentrations of these and additional indicators (fecal coliforms, Escherichia coli, enterococci, coliphage, and enteroviruses) in the untreated wastewater, water column, and sediments of the impacted area and drinking water treatment facility and distribution system at McMurdo Station were determined. Fecal samples from Weddell seals in this area were also collected and analyzed for indicators. All drinking water samples were negative for indicators except for a single total coliform-positive sample. Total coliforms were present in water column samples at higher concentrations than other indicators. Fecal coliform and enterococcus concentrations were similar to each other and greater than those of other indicators in sediment samples closer to the discharge site. C. perfringens concentrations were higher in sediments at greater distances from the discharge site. Seal fecal samples contained concentrations of fecal coliforms, E. coli, enterococci, and C. perfringens similar to those found in untreated sewage. All samples were negative for enteroviruses. A wastewater treatment facility at McMurdo Station has started operation, and these data provide a baseline data set for monitoring the recovery of the impacted area. The contribution of seal feces to indicator concentrations in this area should be considered.

  9. Caracterización molecular y resistencia antimicrobiana de aislamientos de Clostridium perfringens de diferentes orígenes en Costa Rica

    Directory of Open Access Journals (Sweden)

    María del Mar Gamboa-Coronado

    2011-12-01

    Full Text Available Clostridium perfringens es un bacilo Gram positivo, esporulado, anaerobio, ampliamente distribuido en la naturaleza, que produce cuatro toxinas principales α, β, ε y ι, las cuales permiten su clasificación en cinco toxinotipos (A-E. Algunas cepas producen una enterotoxina (CPE, codificada por el gen cpe, que causa diarrea en seres humanos y en algunos animales. La presencia de los genes de estas toxinas y la sensibilidad a los antibióticos se determinó en 81 cepas de C. perfringens previamente aisladas y que habían sido mantenidas a -80°C; 20 de suelos, 20 de origen animal, 20 de origen humano y 21 de alimentos cocidos no relacionados con brotes alimentarios. De acuerdo con los resultados de PCR, todas las cepas fueron clasificadas como C. perfringens tipo A, debido a que solo se les detectó el gen de la toxina α, mientras que el gen de la enterotoxina (cpe se detectó en dos cepas (2.5% aisladas de alimentos, tal como ha sido descrito en otras regiones del mundo. El 44% de las cepas fue resistente a algún antibiótico; clindamicina (41%, cloranfenicol (25%, penicilina (22% y metronidazol (20%. En general, las cepas provenientes de suelos presentaron los mayores porcentajes de resistencia a casi todos los antibióticos. El 40% de las cepas de suelo presentó multiresistencia (a tres o más grupos de antibióticos, el 30% de las de origen humano, el 14% de las de alimentos y el 5% de las de origen animal. Las altas tasas de resistencia encontradas podrían deberse al amplio uso de antibióticos como promotores de crecimiento de plantas y animales y esas cepas resistentes podrían actuar como reservorio de genes de resistencia que pueden transferirse entre bacterias de diversos ambientes.

  10. Immunoprophylactic strategies against enterotoxemia caused by Clostridium perfringens type D in goats Estratégias imunoprofiláticas contra enterotoxemia causada por Clostridium perfringens tipo D em caprinos

    Directory of Open Access Journals (Sweden)

    Josir Laine A. Veschi

    2006-03-01

    Full Text Available The serological response to an experimental vaccine against Clostridium perfringens type D enterotoxemia was evaluated in four groups of goats. Group 1 received colostrum from unvaccinated cows and no vaccine. Groups 2, 3 and 4 received colostrum from vaccinated cows. In addition, Groups 3 and 4 received a vaccine dose at 80 days of age, and Group 4 received a second vaccine dose at 120 days of age. Serum antibody levels were determined by ELISA in cows before and after calving, and in goats at 3, 80, 120 and 160 days of age. No significant difference in serum antibody levels was observed between vaccinated and unvaccinated cows, or between the four groups of goats evaluated at 3 days of life. Groups 3 and 4 presented mean antibody titers of 0.6 and 1.1 IU/ml, respectively, 40 days after first vaccination. The vaccine response of Group 4 was 1.8 IU/ml 40 days after the booster dose and was higher than that observed for Group 3 (0.2 IU/ml. Thus, in the proposed regimen the use of heterologous colostrum did not induce passive immunization in goat kids. However, first vaccination and a booster dose after 40 days triggered satisfactory antibody levels.Foi avaliada a resposta sorológica de vacina experimental contra a enterotoxemia em quatro grupos de caprinos. O Grupo 1 recebeu colostro de vacas não vacinadas e nenhuma dose de vacina. Os Grupos 2, 3 e 4 receberam colostro de vacas vacinadas, e uma dose de vacina aos 80 dias de idade nos Grupos 3 e 4. O Grupo 4 recebeu a segunda dose de vacina aos 120 dias de idade. Os níveis de anticorpos séricos foram avaliados pelo ELISA nas vacas antes e depois do parto e nos caprinos aos 3, 80, 120 e 160 dias de idade. Não houve diferença significativa nos níveis de anticorpos séricos das vacas vacinadas e não vacinadas, assim como entre os quatro grupos de caprinos avaliados aos três dias de vida. Os Grupos 3 e 4 apresentaram títulos médios de anticorpos de 0,6 UI/mL e 1,1 UI/mL, respectivamente

  11. Crystallization and preliminary X-ray diffraction studies of θ-toxin (perfringolysin O), a pore-forming cytolysin of Clostridium perfringens

    Science.gov (United States)

    Sugahara, Mitsuaki; Sekino-Suzuki, Naoko; Ohno-Iwashita, Yoshiko; Miki, Kunio

    1996-10-01

    θ-Toxin (perfringolysin O), a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens type A was crystallized by the vapor diffusion procedure using polyethyleneglycol 4000 and sodium chloride as precipitants in 2-(cyclohexylamino)ethanesulfonic acid (CHES) buffer at pH 9.5. The diffraction patterns of precession photographs indicated that the crystals belong to the orthorhombic system and the space group C222 1 with unit-cell dimensions of a = 47.7 Å, b = 182.0 Å and c = 175.8 Å. Assuming that the asymmetric unit contains one or two molecules (Mw 52 700), the Vm value is calculated as 3.6 or 1.8 Å 3/dalton, respectively. The crystals diffract X-rays to at least 3 Å resolution and are suitable for high resolution X-ray crystal structure determination.

  12. Comparison of toxicity neutralization-, ELISA- and PCR tests for typing of Clostridium perfringens and detection of the enterotoxin gene by PCR

    DEFF Research Database (Denmark)

    Møller, Kristian; Ahrens, Peter

    1996-01-01

    A polymerase chain reaction (PCR) was developed for the specific amplification of a part of each of the five Clostridium perfringens toxin genes: alpha (alpha), beta (beta), epsilon (epsilon), iota (iota), and enterotoxin (CPE). While the toxicity neutralization test (TNT) only showed limited...... ability to detect the or toxin, the lecithinase test and PCR test (PCR(alpha)) concordantly detected the ct toxin and the alpha toxin gene, respectively. A monoclonal enzyme linked immunosorbent assay (ELISA) and a PCR(beta) test were compared and were in accordance for the detection of the beta toxin...... (gene) from pure and mixed cultures from piglets suffering from necrotizing enteritis. However, the PCR(beta) test was superior to the ELISA for detection of the beta toxin (gene) in necrotic intestinal mucosa without culturing. An internal standard to be co-amplified with the beta toxin gene...

  13. Hydrogen production at high Faradaic efficiency by a bio-electrode based on TiO2 adsorption of a new [FeFe]-hydrogenase from Clostridium perfringens.

    Science.gov (United States)

    Morra, Simone; Valetti, Francesca; Sarasso, Veronica; Castrignanò, Silvia; Sadeghi, Sheila J; Gilardi, Gianfranco

    2015-12-01

    The [FeFe]-hydrogenase CpHydA from Clostridium perfringens was immobilized by adsorption on anatase TiO2 electrodes for clean hydrogen production. The immobilized enzyme proved to perform direct electron transfer to and from the electrode surface and catalyses both H2 oxidation (H2 uptake) and H2 production (H2 evolution) with a current density for H2 evolution of about 2 mA cm(-1). The TiO2/CpHydA bioelectrode remained active for several days upon storage and when a reducing potential was set, H2 evolution occurred with a mean Faradaic efficiency of 98%. The high turnover frequency of H2 production and the tight coupling of electron transfer, resulting in a Faradaic efficiency close to 100%, support the exploitation of the novel TiO2/CpHydA stationary electrode as a powerful device for H2 production.

  14. Relative disease susceptibility and clostridial toxin antibody responses in three commercial broiler lines coinfected with Clostridium perfringens and Eimeria maxima using an experimental model of necrotic enteritis.

    Science.gov (United States)

    Jang, Seung I; Lillehoj, Hyun S; Lee, Sung-Hyen; Lee, Kyung Woo; Lillehoj, Erik P; Hong, Yeong Ho; An, Dong-Jun; Jeoung, D Hye-Young; Chun, Ji-Eun

    2013-09-01

    Necrotic enteritis is an enteric disease of poultry resulting from infection by Clostridium perfringens with coinfection by Eimeria spp. constituting a major risk factor for disease pathogenesis. This study compared three commercial broiler chicken lines using an experimental model of necrotic enteritis. Day-old male Cobb, Ross, and Hubbard broilers were orally infected with viable C. perfringens and E. maxima and fed a high-protein diet to promote the development of experimental disease. Body weight loss, intestinal lesions, and serum antibody levels against alpha-toxin and necrotic enteritis B-like (NetB) toxin were measured as parameters of disease susceptibility and host immune response. Cobb chickens exhibited increased body weight loss compared with Ross and Hubbard breeds and greater gut lesion severity compared with Ross chickens. NetB antibody levels were greater in Cobb chickens compared with the Ross or Hubbard groups. These results suggest that Cobb chickens may be more susceptible to necrotic enteritis in the field compared with the Ross and Hubbard lines.

  15. 动物源产气荚膜梭菌耐药性的研究进展%Research progress on antimicrobial resistance in Clostridium perfringens of animal origin

    Institute of Scientific and Technical Information of China (English)

    董卫超; 刘凌; 杜向党

    2012-01-01

    Clostridium perfringens is an important zoonotic pathogen that can cause; various diseases in animals and hu -mans . The antimicrobial resistance in Clostridium perfringens is common in animal production . M ost of the resistant genes in Clostridium perfringens locate on the conjngative plasm ids or mobile genetic elements , which accelerate its dissemination . In this article , the resistance mechanism of Clostridium perfringens to the commonly used antimicrobials including tetracyclines , macrolides , lincosamides , streptogramins , cloramphenicol and bacitracin was summarized . In addition , the role the conjngative plasmids or the mobilized transposons played in the horizontal dissemination of the resistant genes in Clostridium perfringens was clarified . The deep understanding on the resistance mechanism of Clostridium perfringens of animal origin can aid the control of this pathogen , which provides the guarantee for the food safety and human health .%目的 产气荚膜梭菌是可引起多种动物和人类疾病的重要人兽共患病原菌.其抗生素耐药性在动物生产中较为普遍,多数耐药基因常位于接合型质粒或流动遗传因子上,加速了产气荚膜梭菌耐药性的扩散.本文概述了动物源产气荚膜梭菌对常用抗生素(四环素类、大环内酯类、林可胺类、链阳霉素类以及氯霉素和杆菌肽锌等)的耐药机制.在此基础上,阐述了接合型质粒和转座子等在产气荚膜梭菌耐药基因水平扩散中的作用.对动物源产气荚膜梭菌耐药机制及扩散机理的深入认识有助于该菌的控制,为食品安全和人类健康提供重要保障.

  16. Inhibition of C. difficile and C. perfringens by commercial and potential probiotic strains and their in-vitro growth characteristics

    DEFF Research Database (Denmark)

    Schoster, A.; Kokotovic, Branko; Permin, A.;

    2012-01-01

    and their growth characteristics. The objective of this study was to determine the inhibitory effect of commercial and potential probiotic on C. difficile and C. perfringens and assess their growth characteristics in-vitro. The inhibitory effect of a cell free probiotic supernatant of 17 commercial bacterial...... strains (Lactobacilli n=16, Bifidobacteria n=1) on growth of clostridia spp was assessed in an agar well diffusion assay and broth co-culture experiment, using supernatant harvested at different growth phases and with and without pH adjustment. To study growth characteristics MRS broth was adjusted to pH2...... it was harvested. 10/17 probiotic supernatants inhibited C. difficile in a pH dependant manner when harvested in the stationary growth phase. In the broth co-culture 5/17 probiotics inhibited C. perfringens and 10/17 inhibited C. difficile both in a pH dependant manner. All probiotic strains were able to grow at p...

  17. Finished Whole-Genome Sequences of Clostridium butyricum Toxin Subtype E4 and Clostridium baratii Toxin Subtype F7 Strains.

    Science.gov (United States)

    Halpin, Jessica L; Hill, Karen; Johnson, Shannon L; Bruce, David Carlton; Shirey, T Brian; Dykes, Janet K; Lúquez, Carolina

    2017-07-20

    Clostridium butyricum and Clostridium baratii species have been known to produce botulinum toxin types E and F, respectively, which can cause botulism, a rare but serious neuroparalytic disease. Here, we present finished genome sequences for two of these clinically relevant strains. Copyright © 2017 Halpin et al.

  18. Evaluating the Performance of a New Model for Predicting the Growth of Clostridium perfringens in Cooked, Uncured Meat and Poultry Products under Isothermal, Heating, and Dynamically Cooling Conditions.

    Science.gov (United States)

    Huang, Lihan

    2016-07-01

    Clostridium perfringens type A is a significant public health threat and its spores may germinate, outgrow, and multiply during cooling of cooked meats. This study applies a new C. perfringens growth model in the USDA Integrated Pathogen Modeling Program-Dynamic Prediction (IPMP Dynamic Prediction) Dynamic Prediction to predict the growth from spores of C. perfringens in cooked uncured meat and poultry products using isothermal, dynamic heating, and cooling data reported in the literature. The residual errors of predictions (observation-prediction) are analyzed, and the root-mean-square error (RMSE) calculated. For isothermal and heating profiles, each data point in growth curves is compared. The mean residual errors (MRE) of predictions range from -0.40 to 0.02 Log colony forming units (CFU)/g, with a RMSE of approximately 0.6 Log CFU/g. For cooling, the end point predictions are conservative in nature, with an MRE of -1.16 Log CFU/g for single-rate cooling and -0.66 Log CFU/g for dual-rate cooling. The RMSE is between 0.6 and 0.7 Log CFU/g. Compared with other models reported in the literature, this model makes more accurate and fail-safe predictions. For cooling, the percentage for accurate and fail-safe predictions is between 97.6% and 100%. Under criterion 1, the percentage of accurate predictions is 47.5% for single-rate cooling and 66.7% for dual-rate cooling, while the fail-dangerous predictions are between 0% and 2.4%. This study demonstrates that IPMP Dynamic Prediction can be used by food processors and regulatory agencies as a tool to predict the growth of C. perfringens in uncured cooked meats and evaluate the safety of cooked or heat-treated uncured meat and poultry products exposed to cooling deviations or to develop customized cooling schedules. This study also demonstrates the need for more accurate data collection during cooling. Published 2016. This article is a U.S. Government work and is in the public domain in the USA.

  19. Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens.

    Science.gov (United States)

    Skarin, Hanna; Segerman, Bo

    2014-01-01

    Clostridium botulinum (group III), Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular prophages. The plasmidome of C. novyi sensu lato has so far been poorly characterized. In this study we explored the genomic relationship of a wide range of strains of C. novyi sensu lato with a special focus on the dynamics of the plasmidome. Twenty-four genomes were sequenced from strains selected to represent as much as possible the genetic diversity in C. novyi sensu lato. Sixty-one plasmids were identified in these genomes and 28 of them were completed. The genomic comparisons revealed four separate lineages, which did not strictly correlate with the species designations. The plasmids were categorized into 13 different plasmid groups on the basis of their similarity and conservation of plasmid replication or partitioning genes. The plasmid groups, lineages and species were to a large extent entwined because plasmids and toxin genes had moved across the lineage boundaries. This dynamic process appears to be primarily driven by phages. We here present a comprehensive characterization of the complex species group C. novyi sensu lato, explaining the intermixed genetic properties. This study also provides examples how the reorganization of the botulinum toxin and the novyi alpha toxin genes within the plasmidome has affected the pathogenesis of the strains.

  20. Plasmidome interchange between Clostridium botulinum, Clostridium novyi and Clostridium haemolyticum converts strains of independent lineages into distinctly different pathogens.

    Directory of Open Access Journals (Sweden)

    Hanna Skarin

    Full Text Available Clostridium botulinum (group III, Clostridium novyi and Clostridium haemolyticum are well-known pathogens causing animal botulism, gas gangrene/black disease, and bacillary hemoglobinuria, respectively. A close genetic relationship exists between the species, which has resulted in the collective term C. novyi sensu lato. The pathogenic traits in these species, e.g., the botulinum neurotoxin and the novyi alpha toxin, are mainly linked to a large plasmidome consisting of plasmids and circular prophages. The plasmidome of C. novyi sensu lato has so far been poorly characterized. In this study we explored the genomic relationship of a wide range of strains of C. novyi sensu lato with a special focus on the dynamics of the plasmidome. Twenty-four genomes were sequenced from strains selected to represent as much as possible the genetic diversity in C. novyi sensu lato. Sixty-one plasmids were identified in these genomes and 28 of them were completed. The genomic comparisons revealed four separate lineages, which did not strictly correlate with the species designations. The plasmids were categorized into 13 different plasmid groups on the basis of their similarity and conservation of plasmid replication or partitioning genes. The plasmid groups, lineages and species were to a large extent entwined because plasmids and toxin genes had moved across the lineage boundaries. This dynamic process appears to be primarily driven by phages. We here present a comprehensive characterization of the complex species group C. novyi sensu lato, explaining the intermixed genetic properties. This study also provides examples how the reorganization of the botulinum toxin and the novyi alpha toxin genes within the plasmidome has affected the pathogenesis of the strains.

  1. Draft Genome Sequence of the Cellulolytic and Xylanolytic Thermophile Clostridium clariflavum Strain 4-2a.

    Science.gov (United States)

    Rooney, Elise A; Rowe, Kenneth T; Guseva, Anna; Huntemann, Marcel; Han, James K; Chen, Amy; Kyrpides, Nikos C; Mavromatis, Konstantinos; Markowitz, Victor M; Palaniappan, Krishna; Ivanova, Natalia; Pati, Amrita; Liolios, Konstantinos; Nordberg, Henrik P; Cantor, Michael N; Hua, Susan X; Shapiro, Nicole; Woyke, Tanja; Lynd, Lee R; Izquierdo, Javier A

    2015-07-23

    Clostridium clariflavum strain 4-2a, a novel strain isolated from a thermophilic biocompost pile, has demonstrated an extensive capability to utilize both cellulose and hemicellulose under thermophilic anaerobic conditions. Here, we report the draft genome of this strain.

  2. Molecular diversity of Clostridium botulinum and phenotypically similar strains.

    Science.gov (United States)

    Grenda, T; Kukier, E; Sieradzki, Z; Goldsztejn, M; Kwiatek, K

    2016-12-01

    This study was undertaken to examine phenotypic and genetic features of strains preliminary classified as Clostridium botulinum species. The phenotypic characteristics were assessed with different culture media and biochemical tests. The genetic characterization included detection of botulinum toxin genes by PCR and macrorestriction analysis with SmaI, XhoI and SacII by PFGE (Pulsed-field Gel Electrophoresis). Despite similar biochemical properties of all analysed strains, only 47% of them contained genes determining toxicity specific to C. botulinum species. The most valuable differentiation of C. botulinum and C. botulinum-like strains was obtained after SmaI digestion. The highest affinity was observed among C. botulinum type B profiles which was even up to 100%. It was found 100% of affinity between C. botulinum and C. botulinum-like strains, however, the similarity among C. botulinum and C. botulinum-like was generally lower than 80%.

  3. Predicting outgrowth and inactivation of Clostridium perfringens in meat products during low temperature long time heat treatment

    DEFF Research Database (Denmark)

    Duan, Zhi; Holst Hansen, Terese; Hansen, Tina Beck

    ), was developed from literature data (215 isothermal growth data), and was combined with a linear inactivation model developed from data at 53°C from this study for completely predicting fate of Cl. perfringens during LTLT treatment. RESULTS Very short lag times were observed in most of the challenge tests......, a new growth model and a new inactivation model were developed and combined to predict the overall fate of Cl. Perfringens during LTLT profiles at 53°C. However, predicted lag time was still much longer than the observed lag time in our challenge tests, which caused a general underestimation of growth...... of meats. Very short lag times were observed during LTLT treatments, which were not observed in isothermal or cooling conditions previously. The reason for short lag time during slowly increasing temperature conditions, and how to predict it, should be interesting for future studies....

  4. Traits of selected Clostridium strains for syngas fermentation to ethanol.

    Science.gov (United States)

    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.

  5. Growth of group II Clostridium botulinum strains at extreme temperatures.

    Science.gov (United States)

    Derman, Yağmur; Lindström, Miia; Selby, Katja; Korkeala, Hannu

    2011-11-01

    The minimum and maximum growth temperatures and the maximum growth rates at 10, 30, 37, and 40°C were determined for 24 group II Clostridium botulinum strains. Genetic diversity of the strains was revealed by amplified fragment length polymorphism (AFLP) analysis. The minimum growth temperatures ranged from 6.2 to 8.6°C, and the maximum growth temperatures ranged from 34.7 to 39.9°C. The mean maximum growth temperatures and mean maximum growth rates of type E strains at 37°C were significantly higher than those of type B and type F strains. A significant correlation between maximum growth rates at 37°C and maximum growth temperatures was found for all strains. Some type E strains with a high minimum growth temperature also had a higher maximum growth rate at 37°C than at 30°C, which suggests that some group II C. botulinum strains are more mesophilic in their growth properties than others. We found relatively small differences between AFLP clusters, indicating that diverse genetic background among the strains was not reflected in the growth properties. The growth characteristics of group II C. botulinum and some type E strains with mesophilic growth properties may have an impact on inoculation studies and predictive modeling for assessing the safety of foods.

  6. Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model.

    Science.gov (United States)

    Shreya, Das; Uppalapati, Siva R; Kingston, Joseph J; Sripathy, Murali H; Batra, Harsh V

    2015-05-01

    Clostridium perfringens type A, an anaerobic pathogen is the most potent cause of soft tissue infections like gas gangrene and enteric diseases like food poisoning and enteritis. The disease manifestations are mediated via two important exotoxins, viz. myonecrotic alpha toxin (αC) and enterotoxin (CPE). In the present study, we synthesized a bivalent chimeric protein r-Cpae comprising C-terminal binding regions of αC and CPE using structural vaccinology rationale and assessed its protective efficacy against both alpha toxin (αC) and enterotoxin (CPE) respectively, in murine model. Active immunization of mice with r-Cpae generated high circulating serum IgG (systemic), significantly increased intestinal mucosal s-IgA antibody titres and resulted in substantial protection to the immunized animals (100% and 75% survival) with reduced tissue morbidity when administered with 5×LD(100) doses of αC (intramuscular) and CPE (intra-gastric gavage) respectively. Mouse RBCs and Caco-2 cells incubated with a mixture of anti-r-Cpae antibodies and αC and CPE respectively, illustrated significantly higher protection against the respective toxins. Passive immunization of mice with a similar mixture resulted in 91-100% survival at the end of the 15 days observation period while mice immunized with a concoction of sham sera and respective toxins died within 2-3 days. This work demonstrates the efficacy of the rationally designed r-Cpae chimeric protein as a potential sub unit vaccine candidate against αC and CPE of C. perfringens type A toxemia.

  7. In ovo vaccination using Eimeria profilin and Clostridium perfringens NetB proteins in Montanide IMS adjuvant increases protective immunity against experimentally-induced necrotic enteritis.

    Science.gov (United States)

    Lillehoj, Hyun Soon; Jang, Seung Ik; Panebra, Alfredo; Lillehoj, Erik Peter; Dupuis, Laurent; Ben Arous, Juliette; Lee, Seung Kyoo; Oh, Sung Taek

    2017-10-01

    The effects of vaccinating 18-day-old chicken embryos with the combination of recombinant Eimeria profilin plus Clostridium perfringens (C. perfringens) NetB proteins mixed in the Montanide IMS adjuvant on the chicken immune response to necrotic enteritis (NE) were investigated using an Eimeria maxima (E. maxima)/C. perfringens co-infection NE disease model that we previously developed. Eighteen-day-old broiler embryos were injected with 100 μL of phosphate-buffered saline, profilin, profilin plus necrotic enteritis B-like (NetB), profilin plus NetB/Montanide adjuvant (IMS 106), and profilin plus Net-B/Montanide adjuvant (IMS 101). After post-hatch birds were challenged with our NE experimental disease model, body weights, intestinal lesions, serum antibody levels to NetB, and proinflammatory cytokine and chemokine mRNA levels in intestinal intraepithelial lymphocytes were measured. Chickens in ovo vaccinated with recombinant profilin plus NetB proteins/IMS106 and recombinant profilin plus NetB proteins/IMS101 showed significantly increased body weight gains and reduced gut damages compared with the profilin-only group, respectively. Greater antibody response to NetB toxin were observed in the profilin plus NetB/IMS 106, and profilin plus NetB/IMS 101 groups compared with the other three vaccine/adjuvant groups. Finally, diminished levels of transcripts encoding for proinflammatory cytokines such as lipopolysaccharide-induced tumor necrosis factor-α factor, tumor necrosis factor superfamily 15, and interleukin-8 were observed in the intestinal lymphocytes of chickens in ovo injected with profilin plus NetB toxin in combination with IMS 106, and profilin plus NetB toxin in combination with IMS 101 compared with profilin protein alone bird. These results suggest that the Montanide IMS adjuvants potentiate host immunity to experimentally-induced avian NE when administered in ovo in conjunction with the profilin and NetB proteins, and may reduce disease pathology by

  8. Efectividad de diferentes combinaciones de tratamientos con antibióticos en laa mortalidad por Clostridium Perfringens en una empresa avícola

    Directory of Open Access Journals (Sweden)

    Ervelio Olazábal, Roberto González, Sailí Flores, Lizzett Alcina

    2005-02-01

    Full Text Available Esta investigación se desarrolló en una empresa de reproductores ligeros Leghorns en el municipio de Santa Clara, provincia Villa Clara con 20 000 aves y una proporción de hembras y machos de 6:1. Para declarar la presencia de la enfermedad, enteritis necrótica hemorrágica, producida por Clostridium perfringens en las aves muertas, se siguió un esquema que contempló el seguimiento de la mortalidad, los síntomas clínicos, la necropsia, evaluación anatomopatológica, emisión de un diagnóstco presuntivo, corroboración del diagnóstico por el laboratorio y conclusiones de las muertes ocurridas. Se realizaron un grupo de actividades de habilitación sanitaria, las que se evaluaron atendiendo a la calidad de las mismas. Se evaluó la eficacia de 4 esquemas de tratamiento con antibióticos aplicados a las aves enfermas separadas en cuatro naves, evaluando la influencia de cada uno sobre la mortalidad, durante los primeros 30 días de la aparición de las muertes. Se obtuvo que no existieron diferencias significativas entre los resultados de las aplicaciones de los diferentes esquemas, todos los tratamientos fueron efectivos para controlar el brote y que la aplicación de Amprolium no evitó la aparición de las muertes por la enfermedad en las aves.A research on hemorrhagic necrotic enteritis produced by Clostridium perfringens in 20 000 Leghorn laying hens with a 6:1 female:male proportion was performed in the municipality of Santa Clara, province of Villa Clara. A scheme, which includes mortality, clinical symptoms, necropsy, anatomopathological evaluation, emission of a presumptive diagnosis, corroboration of the laboratory diagnosis and conclusions about death, was used to diagnose the disease. A series of sanitary activities, which were evaluated according to their quality, were also carried out. The efficacy that four antibiotic treatment schemes had on mortality was evaluated during a 30 days period in sick hens separated in four

  9. Effect of the cortex-lytic enzyme SleC from non-food-borne Clostridium perfringens on the germination properties of SleC-lacking spores of a food poisoning isolate.

    Science.gov (United States)

    Paredes-Sabja, Daniel; Sarker, Mahfuzur R

    2010-11-01

    The hallmark of bacterial spore germination is peptidoglycan cortex hydrolysis by cortex-lytic enzymes. In spores of Clostridium perfringens wild-type strain SM101, which causes food poisoning, the sole essential cortex-lytic enzyme SleC is activated by a unique serine protease CspB. Interestingly, the non-food-borne wild-type strain F4969 encodes a significantly divergent SleC variant (SleCF4969) and 3 serine proteases (CspA, CspB, and CspC). Consequently, in this study we evaluated the functional compatibility of SleCF4969 and SleCSM101 by complementing the germination phenotypes of SM101ΔsleC spores with sleCF4969. Our results show that although pro-SleCF4969 was processed into mature SleCF4969 in the SM101ΔsleC spores, it partially restored spore germination with nutrient medium, with a mixture of ʟ-asparagine and KCl, or with a 1:1 chelate of Ca2+ and dipicolinic acid. While the amount of dipicolinic acid released was lower, the amount of hexosamine-containing material released during germination of SM101ΔsleC(sleCF4969) spores was similar to the amount released during germination of SM101 wild-type spores. The viability of SM101ΔsleC(sleCF4969) spores was 8- and 3-fold lower than that of SM101 and F4969 spores, respectively. Together, these data indicate that the peptidoglycan cortex hydrolysis machinery in the food poisoning isolate SM101 is functionally divergent than that in the non-food-borne isolate F4969.

  10. Noncytotoxic Clostridium perfringens enterotoxin (CPE) variants localize CPE intestinal binding and demonstrate a relationship between CPE-induced cytotoxicity and enterotoxicity.

    Science.gov (United States)

    Smedley, James G; Saputo, Juliann; Parker, Jacquelyn C; Fernandez-Miyakawa, Mariano E; Robertson, Susan L; McClane, Bruce A; Uzal, Francisco A

    2008-08-01

    Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE(168-319). Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE(168-319) binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE(168-319) was able to partially inhibit CPE-induced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE.

  11. Comparison of two methods of bacterial DNA extraction from human fecal samples contaminated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni.

    Science.gov (United States)

    Kawase, Jun; Kurosaki, Morito; Kawakami, Yuta; Kashimoto, Takashi; Tsunomori, Yoshie; Sato, Koji; Ikeda, Tetsuya; Yamaguchi, Keiji; Watahiki, Masanori; Shima, Tomoko; Kameyama, Mitsuhiro; Etoh, Yoshiki; Horikawa, Kazumi; Fukushima, Hiroshi; Goto, Ryoichi; Shirabe, Komei

    2014-01-01

    In this study, 2 methods of DNA extraction were evaluated for use in conjunction with the screening system Rapid Foodborne Bacterial Screening 24 (RFBS24), which employs multiplex real-time SYBR Green polymerase chain reaction (SG-PCR) and can simultaneously detect 24 target genes of foodborne pathogens in fecal DNA samples. The QIAamp DNA Stool mini kit (Qkit) and Ultra Clean Fecal DNA Isolation Kit (Ukit) were used for bacterial DNA extraction from fecal samples artificially inoculated with Clostridium perfringens, Staphylococcus aureus, Salmonella Typhimurium, and Campylobacter jejuni. SG-PCR and simplex real-time quantitative PCR (S-qPCR) analyses revealed higher copy numbers (8-234 times) of DNA in samples obtained using Ukit compared with those obtained using Qkit, resulting in lower cycle threshold values for the Ukit samples of the 4 bacteria on SG-PCR analysis. Fecal DNA samples from patients infected during foodborne outbreaks of Salmonella and Campylobacter were also prepared by Qkit and Ukit methods and subjected to RFBS24 analyses. Higher numbers of RFBS24 bacterial target genes were detected in DNA samples obtained using Ukit compared with those obtained using Qkit. Thus, the higher DNA extraction efficiency of the Ukit method compared with Qkit renders the former more useful in achieving improved detection rates of these 4 bacteria in fecal samples using SG-PCR.

  12. Release of Glycoprotein (GP1 from the Tegumental Surface of Taenia solium by Phospholipase C from Clostridium perfringens Suggests a Novel Protein-Anchor to Membranes

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    Abraham Landa

    2010-01-01

    Full Text Available In order to explore how molecules are linked to the membrane surface in larval Taenia solium, whole cysticerci were incubated in the presence of phospholipase C from Clostridium perfringens (PLC. Released material was collected and analyzed in polyacrylamide gels with sodium dodecyl sulfate. Two major bands with apparent molecular weights of 180 and 43 kDa were observed. Western blot of released material and localization assays in cysticerci tissue sections using antibodies against five known surface glycoproteins of T. solium cysticerci indicated that only one, previously called GP1, was released. Similar localization studies using the lectins wheat-germ-agglutinin and Concanavalin A showed that N-acetyl-D-glucosamine, N-acetylneuraminic, sialic acid, αmethyl-D-mannoside, D-manose/glucose, and N-acetyl-D-glucosamine residues are abundantly present on the surface. On the other hand, we find that treatment with PLC releases molecules from the surface; they do not reveal Cross Reacting Determinant (CRD, suggesting a novel anchor to the membrane for the glycoprotein GP1.

  13. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

    Science.gov (United States)

    Sonoda, N; Furuse, M; Sasaki, H; Yonemura, S; Katahira, J; Horiguchi, Y; Tsukita, S

    1999-10-04

    Claudins, comprising a multigene family, constitute tight junction (TJ) strands. Clostridium perfringens enterotoxin (CPE), a single approximately 35-kD polypeptide, was reported to specifically bind to claudin-3/RVP1 and claudin-4/CPE-R at its COOH-terminal half. We examined the effects of the COOH-terminal half fragment of CPE (C-CPE) on TJs in L transfectants expressing claudin-1 to -4 (C1L to C4L, respectively), and in MDCK I cells expressing claudin-1 and -4. C-CPE bound to claudin-3 and -4 with high affinity, but not to claudin-1 or -2. In the presence of C-CPE, reconstituted TJ strands in C3L cells gradually disintegrated and disappeared from their cell surface. In MDCK I cells incubated with C-CPE, claudin-4 was selectively removed from TJs with its concomitant degradation. At 4 h after incubation with C-CPE, TJ strands were disintegrated, and the number of TJ strands and the complexity of their network were markedly decreased. In good agreement with the time course of these morphological changes, the TJ barrier (TER and paracellular flux) of MDCK I cells was downregulated by C-CPE in a dose-dependent manner. These findings provided evidence for the direct involvement of claudins in the barrier functions of TJs.

  14. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

    Science.gov (United States)

    Kominsky, Scott L; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G; Weitzman, Sigmund A; Argani, Pedram; Sukumar, Saraswati

    2004-05-01

    Clostridium perfringens enterotoxin (CPE) induces cytolysis very rapidly through binding to its receptors, the tight junction proteins CLDN 3 and 4. In this study, we investigated CLDN 3 and 4 expression in breast cancer and tested the potential of CPE-mediated therapy. CLDN 3 and 4 proteins were detected in all primary breast carcinomas tested (n = 21) and, compared to normal mammary epithelium, were overexpressed in approximately 62% and 26%, respectively. Treatment of breast cancer cell lines in culture with CPE resulted in rapid and dose-dependent cytolysis exclusively in cells that expressed CLDN 3 and 4. Intratumoral CPE treatment of xenografts of T47D breast cancer cells in immunodeficient mice resulted in a significant reduction in tumor volume (P = 0.007), with accompanying necrosis. Necrotic reactions were also seen in three freshly resected primary breast carcinoma samples treated with CPE for 12 hours, while isolated primary breast carcinoma cells underwent rapid and complete cytolysis within 1 hour. Thus, expression of CLDN 3 and 4 sensitizes primary breast carcinomas to CPE-mediated cytolysis and emphasizes the potential of CPE in breast cancer therapy.

  15. Recombination and Insertion Events Involving the Botulinum Neurotoxin Complex Genes in Clostridium botulinum Types A, B, E and F and Clostridium butyricum Type E Strains

    Science.gov (United States)

    2009-10-05

    Bruggemann H, Baumer S, Fricke WF, Wiezer A, Liesegang H, Decker I, et al.: The genome sequence of Clostridium tetani , the caus- ative agent of... Clostridium botulinum types A, B, E and F and Clostridium butyricum type E strains Karen K Hill*1, Gary Xie2, Brian T Foley3, Theresa J Smith4, Amy C Munk2...ornl.gov; John C Detter - cdetter@lanl.gov * Corresponding author Abstract Background: Clostridium botulinum is a taxonomic designation for at least

  16. DOMESTIC BUTANOL-PRODUCING STRAINS OF THE Clostridium GENUS

    Directory of Open Access Journals (Sweden)

    O. O. Tigunova

    2017-02-01

    Full Text Available The aim of the work was to summarize the results of own research concerning obtaining butanol producing strains of Clostridium genus, to identify them by physiological, morphological and genetic methods. Further study of characteristics and biological features of the strains, and various approaches in biotechnological process of butanol production are discussed. The work includes methods to increase butanol accumulation by producer strains. Perspectives of using chemical mutagenesis in Clostridia as a method of increasing butanol production are considered. The feasibility of using non-food raw material as a substrate for fermentation is discussed. Different methods of pretreatment and their impact on the accumulation of butanol in the liquid medium are compared. Butanol accumulation is shown to increase significantly if the synthesis precursors are added as components of enzymatic medium, and the “reverse bard” is used to reduce waste production without affecting the level of butanol synthesis. The problem of conservation of producing strains is given, and protective medium for microorganisms during the freeze-drying is defined.

  17. Hydrogen production from wastewater sludge using a Clostridium strain.

    Science.gov (United States)

    Wang, C C; Chang, C W; Chu, C P; Lee, D J; Chang, B V; Liao, C S

    2003-09-01

    Limited data in literature revealed a relatively low hydrogen yield from wastewater sludge, ca. 0.16 mg/g-dried solids, using anaerobic fermentation. We demonstrated in this work a much higher hydrogen yield, around 1.1 mg-H2/g-dried solids using a clostridium strain isolated from the sludge sample. The formed hydrogen would be consumed after passing the peak value at around 30-36 h of fermentation. We examined the effects of employing five different pre-treatments on substrate sludge, but noted no appreciable enhancement in hydrogen yield as commonly expected for methane production. Since a vast amount of organic matters had been released to water after hydrogen fermentation, we externally dosed methanogenic bacteria to the fermented liquor to produce methane. The fermented liquor could produce more methane than the non-fermented sample, indicating that the dosed methanogenic bacteria readily utilized the organic matters derived from the fermentation test.

  18. Avaliação da resposta de antitoxinas beta e épsilon de Clostridium perfringens induzidas em bovinos e coelhos por seis vacinas comerciais no Brasil

    Directory of Open Access Journals (Sweden)

    Lobato F.C.F.

    2000-01-01

    Full Text Available Avaliou-se a resposta de antitoxinas beta e épsilon de Clostridium perfringens em bovinos vacinados contra clostridioses com seis vacinas disponíveis no mercado. Quarenta e oito bezerros de seis a sete meses de idade foram divididos em oito grupos (T1 a T8 de seis animais cada. Os grupos de número 2 a 7 receberam as vacinas T2 a T7 nos dias 0 e 42 com a dose e via recomendadas pelos fabricantes. Solução salina e toxóide padrão foram usados nos mesmos dias nos grupos 1 e 8 (T1 e T8, respectivamente, como controles negativo e positivo. Amostras de sangue foram coletadas nos dias 0, 42 e 56 pós-vacinação (PV, para titulação de anticorpos no soro. As vacinas e os controles foram também testados em oito coelhos cada, inoculados nos dias 0 e 21 com metade da dose indicada para bovinos. Os coelhos foram sangrados no dia 35 e os soros foram misturados em partes iguais para cada vacina para a titulação de anticorpos. Os soros dos bovinos foram titulados individualmente contra as toxinas beta e épsilon de C. perfringens pelo método de soroneutralização em camundongos. A vacina T2 apresentou títulos de anticorpos de 22,6 e 5,6 UI/ml e a vacina T4 11,2 e 7,0 UI/ml, respectivamente, contra toxinas beta e épsilon em coelhos. Os títulos do toxóide padrão (T8 foram 45,2 UI/ml contra ambas as toxinas. Em bovinos, as médias dos títulos de anticorpos contra a toxina beta nos dias 42 e 56 PV com a vacina T2 (1,15 UI/ml e 8,0 UI/ml foram similares ao toxóide padrão (2,02 e 10,03 UI/ml. A vacina T4 (0,73 e 4,54 UI/ml teve títulos menores (P<0,05 que o toxóide padrão e similares a T2. Contra a toxina épsilon, o toxóide padrão teve média de título (0,97 UI/ml no dia 42 que foi significativamente maior (P<0,05 do que T4 (0,15 UI/ml e similar a T2 (0,42 UI/ml. No dia 56, T2 (4,27 UI/ml teve títulos significativamente maiores (P<0,05 do que T4 (0,68 UI/ml e similares ao toxóide padrão (4,98 UI/ml. Em cada tratamento, a resposta aos

  19. Clostridium perfringens promotes the growth and development of Drosophila melanogaster%产气荚膜梭菌促进黑腹果蝇的生长和发育

    Institute of Scientific and Technical Information of China (English)

    刘威; 李玉娟; 刘晓梁; 卓萍; 姚红

    2016-01-01

    [目的]黑腹果蝇Drosophila melanogaster肠道中栖生着众多微生物,通过分离和研究其内共生菌,以研究肠道菌群的多态性和作用.[方法]利用Hungate滚管技术从黑腹果蝇成虫肠道分离厌氧细菌;通过记录果蝇的发育历期和生长速率,检测该细菌对果蝇发育和生长的影响.[结果]首次从黑腹果蝇肠道内分离到一株产气荚膜梭茵Clostridium perfringens.该茵能够有效地定植到果蝇肠道内,是果蝇肠道共生菌.产气荚膜梭菌显著地缩短无菌果蝇的发育历期,将无菌果蝇成蛹天数由20 d缩短到8.1d,羽化天数由30 d缩短到12.7 d.该菌还可以提高果蝇生长速率.[结论]本研究揭示了产气荚膜梭菌是果蝇的内共生菌,可以通过提高生长速率而有效地促进果蝇的生长和发育.%[Aim] To isolate and investigate the microbiota from Drosophila melanogaster gut harboring substantial numbers of commensal microorganisms,and to further reveal the diversity and functions of microbial communities.[Methods] Anaerobic bacteria from the gut of D.melanogaster adults were isolated with Hungate roll tube technique.The symbiotic bacteria were assayed with colonization testing.The effects of the bacteria on developmental time and growth rate of D.melanogaster were detected.[Results] We first isolated one strain of the anaerobic bacteria (Clostridium pefringens) from D.melanogaster gut.C.perfingens is able to colonize the fly gut and could be maintained in fly breeding medium,suggesting that it is a commensal bacterium of Drosophila.Furthermore,C.perfringensstimulated the development of germ-free D.melanogaster by shortening its developmental time:shortening pupal formation from 20 d to 8.1 d and adult eclosion from 30 d to 12.7 d,respectively.C.peqfringens also accelerated the growth rate.[Conclusion] The study revealed that C.perfringens is the commensal bacteria of D.melanogaster,and has the promoting effect on the growth and development of D

  20. Noncytotoxic Clostridium perfringens Enterotoxin (CPE) Variants Localize CPE Intestinal Binding and Demonstrate a Relationship between CPE-Induced Cytotoxicity and Enterotoxicity▿

    Science.gov (United States)

    Smedley, James G.; Saputo, Juliann; Parker, Jacquelyn C.; Fernandez-Miyakawa, Mariano E.; Robertson, Susan L.; McClane, Bruce A.; Uzal, Francisco A.

    2008-01-01

    Clostridium perfringens enterotoxin (CPE) causes the symptoms of a very common food poisoning. To assess whether CPE-induced cytotoxicity is necessary for enterotoxicity, a rabbit ileal loop model was used to compare the in vivo effects of native CPE or recombinant CPE (rCPE), both of which are cytotoxic, with those of the noncytotoxic rCPE variants rCPE D48A and rCPE168-319. Both CPE and rCPE elicited significant fluid accumulation in rabbit ileal loops, along with severe mucosal damage that starts at villus tips and then progressively affects the entire villus, including necrosis of epithelium and lamina propria, villus blunting and fusion, and transmural edema and hemorrhage. Similar treatment of ileal loops with either of the noncytotoxic rCPE variants produced no visible histologic damage or fluid transport changes. Immunohistochemistry revealed strong CPE or rCPE168-319 binding to villus tips, which correlated with the abundant presence of claudin-4, a known CPE receptor, in this villus region. These results support (i) cytotoxicity being necessary for CPE-induced enterotoxicity, (ii) the CPE sensitivity of villus tips being at least partially attributable to the abundant presence of receptors in this villus region, and (iii) claudin-4 being an important intestinal receptor for CPE. Finally, rCPE168-319 was able to partially inhibit CPE-induced histologic damage, suggesting that noncytotoxic rCPE variants might be useful for protecting against some intestinal effects of CPE. PMID:18505809

  1. Clostridium perfringens enterotoxin C-terminal domain labeled to fluorescent dyes for in vivo visualization of micrometastatic chemotherapy-resistant ovarian cancer.

    Science.gov (United States)

    Cocco, Emiliano; Shapiro, Erik M; Gasparrini, Sara; Lopez, Salvatore; Schwab, Carlton L; Bellone, Stefania; Bortolomai, Ileana; Sumi, Natalia J; Bonazzoli, Elena; Nicoletti, Roberta; Deng, Yang; Saltzman, W Mark; Zeiss, Caroline J; Centritto, Floriana; Black, Jonathan D; Silasi, Dan-Arin; Ratner, Elena; Azodi, Masoud; Rutherford, Thomas J; Schwartz, Peter E; Pecorelli, Sergio; Santin, Alessandro D

    2015-12-01

    Identification of micrometastatic disease at the time of surgery remains extremely challenging in ovarian cancer patients. We used fluorescence microscopy, an in vivo imaging system and a fluorescence stereo microscope to evaluate fluorescence distribution in Claudin-3- and -4-overexpressing ovarian tumors, floating tumor clumps isolated from ascites and healthy organs. To do so, mice harboring chemotherapy-naïve and chemotherapy-resistant human ovarian cancer xenografts or patient-derived xenografts (PDXs) were treated with the carboxyl-terminal binding domain of the Clostridium perfringens enterotoxin (c-CPE) conjugated to FITC (FITC-c-CPE) or the near-infrared (NIR) fluorescent tag IRDye CW800 (CW800-c-CPE) either intraperitoneally (IP) or intravenously (IV). We found tumor fluorescence to plateau at 30 min after IP injection of both the FITC-c-CPE and the CW800-c-CPE peptides and to be significantly higher than in healthy organs (p < 0.01). After IV injection of CW800-c-CPE, tumor fluorescence plateaued at 6 hr while the most favorable tumor-to-background fluorescence ratio (TBR) was found at 48 hr in both mouse models. Importantly, fluorescent c-CPE was highly sensitive for the in vivo visualization of peritoneal micrometastatic tumor implants and the identification of ovarian tumor spheroids floating in malignant ascites that were otherwise not detectable by conventional visual observation. The use of the fluorescent c-CPE peptide may represent a novel and effective optical approach at the time of primary debulking surgery for the real-time detection of micrometastatic ovarian disease overexpressing the Claudin-3 and -4 receptors or the identification of residual disease at the time of interval debulking surgery after neoadjuvant chemotherapy treatment.

  2. Identification of Small Molecule Inhibitors of Clostridium perfringens ε-Toxin Cytotoxicity Using a Cell-Based High-Throughput Screen

    Directory of Open Access Journals (Sweden)

    Mark S. McClain

    2010-07-01

    Full Text Available The Clostridium perfringens epsilon toxin, a select agent, is responsible for a severe, often fatal enterotoxemia characterized by edema in the heart, lungs, kidney, and brain. The toxin is believed to be an oligomeric pore-forming toxin. Currently, there is no effective therapy for countering the cytotoxic activity of the toxin in exposed individuals. Using a robust cell-based high-throughput screening (HTS assay, we screened a 151,616-compound library for the ability to inhibit e-toxin-induced cytotoxicity. Survival of MDCK cells exposed to the toxin was assessed by addition of resazurin to detect metabolic activity in surviving cells. The hit rate for this screen was 0.6%. Following a secondary screen of each hit in triplicate and assays to eliminate false positives, we focused on three structurally-distinct compounds: an N-cycloalkylbenzamide, a furo[2,3-b]quinoline, and a 6H-anthra[1,9-cd]isoxazol. None of the three compounds appeared to inhibit toxin binding to cells or the ability of the toxin to form oligomeric complexes. Additional assays demonstrated that two of the inhibitory compounds inhibited ε-toxin-induced permeabilization of MDCK cells to propidium iodide. Furthermore, the two compounds exhibited inhibitory effects on cells pre-treated with toxin. Structural analogs of one of the inhibitors identified through the high-throughput screen were analyzed and provided initial structure-activity data. These compounds should serve as the basis for further structure-activity refinement that may lead to the development of effective anti-ε-toxin therapeutics.

  3. Gene expression profiling within the spleen of Clostridium perfringens-challenged Broilers fed antibiotic-medicated and non-medicated diets

    Directory of Open Access Journals (Sweden)

    Yu Hai

    2009-06-01

    Full Text Available Abstract Background Clostridium perfringens (Cp is a Gram-positive anaerobic bacterium that causes necrotic enteritis (NE in poultry when it overgrows in the small intestine. NE disease has previously been controlled through the use of growth-promoting antibiotics. This practice was recently banned in European countries, leading to significantly increased incidence of NE threatening the poultry industry. Control strategies and technology as substitutes to dietary antibiotics are therefore urgently required. To develop the substitutes, it is important to understand host immune responses to Cp infection. However, the knowledge is still lacking. We therefore investigated gene expression profiles within immunologically-relevant tissue, the spleen, in order to identify factors that are involved in immunity to NE and have potential as therapeutic targets. Results Use of a 44 K Agilent chicken genome microarray revealed significant up-regulation of many immune-associated genes in Cp-challenged chickens, including galectin 3, IFNAR1, IgY-receptor, TCRγ, granzyme A, and mannose-6-P-R, which were subsequently validated by quantitative PCR assays. Functional annotation of differentially expressed genes was conducted using the High Throughput Gene Ontology Functional Annotation database. Medicated and Non-medicated chickens had similar annotation profiles with cell activities and regulation being the most dominant biological processes following Cp infection. Conclusion Broiler chickens demonstrated an intricate and holistic magnitude of host response to Cp challenge and the development of NE. Although the influence of dietary antibiotics appeared to be less significant than the disease process, both had a considerable impact on the host response. Markers previously identified in intestinal inflammatory diseases of other species, including humans, and indicators of enhanced antibody responses, appeared to be involved in the chicken response to Cp challenge

  4. Reclassification of non-type strain Clostridium pasteurianum NRRL B-598 as Clostridium beijerinckii NRRL B-598.

    Science.gov (United States)

    Sedlar, Karel; Kolek, Jan; Provaznik, Ivo; Patakova, Petra

    2017-02-20

    The complete genome sequence of non-type strain Clostridium pasteurianum NRRL B-598 was introduced last year; it is an oxygen tolerant, spore-forming, mesophilic heterofermentative bacterium with high hydrogen production and acetone-butanol fermentation ability. The basic genome statistics have shown its similarity to C. beijerinckii rather than the C. pasteurianum species. Here, we present a comparative analysis of the strain with several other complete clostridial genome sequences. Besides a 16S rRNA gene sequence comparison, digital DNA-DNA hybridization (dDDH) and phylogenomic analysis confirmed an inaccuracy of the taxonomic status of strain Clostridium pasteurianum NRRL B-598. Therefore, we suggest its reclassification to be Clostridium beijerinckii NRRL B-598. This is a specific strain and is not identical to other C. beijerinckii strains. This misclassification explains its unexpected behavior, different from other C. pasteurianum strains; it also permits better understanding of the bacterium for a future genetic manipulation that might increase its biofuel production potential. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Genomic characterization of Italian Clostridium botulinum group I strains.

    Science.gov (United States)

    Giordani, Francesco; Fillo, Silvia; Anselmo, Anna; Palozzi, Anna Maria; Fortunato, Antonella; Gentile, Bernardina; Azarnia Tehran, Domenico; Ciammaruconi, Andrea; Spagnolo, Ferdinando; Pittiglio, Valentina; Anniballi, Fabrizio; Auricchio, Bruna; De Medici, Dario; Lista, Florigio

    2015-12-01

    Clostridium botulinum is a gram-positive bacterium capable of producing the botulinum neurotoxin, a powerful poison that causes botulism, a severe neuroparalytic disease. Its genome has been sequenced entirely and its gene content has been analyzed. To date, 19 full genomes and 64 draft genomes are available. The geographical origin of these genomes is predominantly from the US. In the present study, 10 Italian genomes of C. botulinum group I were analyzed and compared with previously sequenced group I genomes, in order to genetically characterize the Italian population of C. botulinum group I and to investigate the phylogenetic relationships among different lineages. Using the suites of software ClonalFrame and ClonalOrigin to perform genomic analysis, we demonstrated that Italian C. botulinum group I population is phylogenetically heterogeneous encompassing different and distant lineages including overseas strains, too. Moreover, a high recombination rate was demonstrated in the evolution of C. botulinum group I species. Finally, genome sequencing of the strain 357 led us to identify a novel botulinum neurotoxin subtype, F8.

  6. Influence of high gas production during thermophilic anaerobic digestion in pilot-scale and lab-scale reactors on survival of the thermotolerant pathogens Clostridium perfringens and Campylobacter jejuni in piggery wastewater.

    Science.gov (United States)

    Skillman, L C; Bajsa, O; Ho, L; Santhanam, B; Kumar, M; Ho, G

    2009-07-01

    Safe reuse of animal wastes to capture energy and nutrients, through anaerobic digestion processes, is becoming an increasingly desirable solution to environmental pollution. Pathogen decay is the most important safety consideration and is in general, improved at elevated temperatures and longer hydraulic residence times. During routine sampling to assess pathogen decay in thermophilic digestion, an inversely proportional relationship between levels of Clostridium perfringens and gas production was observed. Further samples were collected from pilot-scale, bench-scale thermophilic reactors and batch scale vials to assess whether gas production (predominantly methane) could be a useful indicator of decay of the thermotolerant pathogens C. perfringens and Campylobacter jejuni. Pathogen levels did appear to be lower where gas production and levels of methanogens were higher. This was evident at each operating temperature (50, 57, 65 degrees C) in the pilot-scale thermophilic digesters, although higher temperatures also reduced the numbers of pathogens detected. When methane production was higher, either when feed rate was increased, or pH was lowered from 8.2 (piggery wastewater) to 6.5, lower numbers of pathogens were detected. Although a number of related factors are known to influence the amount and rate of methane production, it may be a useful indicator of the removal of the pathogens C. perfringens and C. jejuni.

  7. International Clostridium difficile animal strain collection and large diversity of animal associated strains

    DEFF Research Database (Denmark)

    Janezic, Sandra; Zidaric, Valerija; Pardon, Bart

    2014-01-01

    Background: Clostridium difficile is an important cause of intestinal infections in some animal species and animals might be a reservoir for community associated human infections. Here we describe a collection of animal associated C. difficile strains from 12 countries based on inclusion criteria...... of one strain (PCR ribotype) per animal species per laboratory. Results: Altogether 112 isolates were collected and distributed into 38 PCR ribotypes with agarose based approach and 50 PCR ribotypes with sequencer based approach. Four PCR ribotypes were most prevalent in terms of number of isolates...... animal associated C. difficile strains worldwide. The widespread dissemination of toxigenic C. difficile and the considerable overlap in strain distribution between species furthers concerns about interspecies, including zoonotic, transmission of this critically important pathogen....

  8. Comparative in vitro activities of SMT19969, a new antimicrobial agent, against 162 strains from 35 less frequently recovered intestinal Clostridium species: implications for Clostridium difficile recurrence.

    Science.gov (United States)

    Goldstein, Ellie J C; Citron, Diane M; Tyrrell, Kerin L

    2014-01-01

    We determined the comparative activity of SMT19969 (SMT) against 162 strains representing 35 well-characterized Clostridium species in clusters I to XIX and 13 Clostridium species that had no 16S rRNA match. SMT MICs ranged from 0.06 to >512 μg/ml and were not species related. SMT might have less impact on normal gut microbiota than other Clostridium difficile infection (CDI) antimicrobials.

  9. Intoxicación alimentaria por Clostridium perfringens en el Centro Penitenciario de Atención Institucional de San José: estudio de cohorte retrospectivo

    Directory of Open Access Journals (Sweden)

    Roy Wong-Mc Clure

    2004-06-01

    Full Text Available Las enfermedades transmitidas por los alimentos se presentan anualmente en Costa Rica y por lo general se estudian hasta identificar el agente etiológico, pero no hasta determinar su fuente de contaminación. No se ha documentado la importancia de fortalecer la inocuidad de alimentos, lo cual produce eventos que comprometen la salud pública cada año, por lo que fue importante estudiar el presente brote. En este estudio se analiza el brote diarreico de un centro penitenciario de San José, en 2003, procurando determinar el probable alimento contaminado. Materiales y métodos: El presente estudio corresponde a una cohorte retrospectiva que identifica exposición a los alimentos preparados dentro del Centro, en las 72 horas previas al pico epidémico. La descripción de la cohorte y el cálculo del (RR se realizó posteriormente para determinar la posible\tasociación causal. Las muestras de heces fueron procesadas en el laboratorio de referencia del INCIENSA. Resultados: La totalidad de los individuos pertenecían al sexo masculino, todos privados de libertad, con un promedio de edad de 31.1años (IC95%=28.9-33.4. La sintomatología más frecuente fue la diarrea (92.3% Y dolor abdominal tipo cólico (84.6%. El resultado de laboratorio aisló como agente etiológico el Clostridium perfringens enterotoxigénico (CPE. Al analizar los alimentos suministrados, pico epidémico y período de incubación, se tiene que el salchichón (RR=1.1;IC95%0.5-2.7 y la preparación con carne molida (RR=2.1,IC95%:0.6-7.4 mostraron ser los únicos alimentos capaces de transmitir el CPE. Discusión: El brote del Centro evidencia que la sintomatología descrita corresponde con lo documentado para la intoxicación por CPE y que, tomando en cuenta las condiciones necesarias para la transmisión, solo el salchichón y la preparación con carne molida cuentan con esta plausibilidad biológica. Al considerar el periodo de incubación, los valores de riesgo relativo

  10. Controlling of growth performance, lipid deposits and fatty acid composition of chicken meat through a probiotic, Lactobacillus johnsonii during subclinical Clostridium perfringens infection.

    Science.gov (United States)

    Wang, Hesong; Ni, Xueqin; Liu, Lei; Zeng, Dong; Lai, Jing; Qing, Xiaodan; Li, Guangyao; Pan, Kangcheng; Jing, Bo

    2017-02-10

    Meat is considered as a major source of polyunsaturated fatty acid (PUFA) which is essential for humans, therefore its lipid level and fatty acid composition have drawn great attention. As no clinical sign can be found in chicks subclinically infected by Clostridium perfringens (CP), the meat may be purchased and eaten. The objective of the present study was to determine whether Lactobacillus johnsonii (LJ) can control the CP-caused impact on growth, lipid levels, fatty acid composition and other flavor or nutritional quality in the meat. 480 one-day-old chicks were divided into four groups and fed with basal diet (control and CP group). Supplemented with 1 × 10(5) (L-LJ) and 1 × 10(6) (H-LJ) colony-forming unit (cfu), CP diet was fed for 42 days. From day 19 to 22, birds of CP and LJ groups were administered with CP twice per day and the control was administered with liver broth. LJ-treated chickens were free from negative influences on growth performance and significant decrease of abdominal fat deposit., LJ inhibited CP-caused shearing force and drip loss increase and pH 40 min and 24 h decrease after sacrifice. In addition, LJ exhibited a positive effect on muscle lipid peroxidation by significantly increasing SOD, CAT and GSH-Px activity and decreasing MDA level. Besides, LJ attenuated the decrease of intramuscular fat, total cholesterol and triglyceride contents caused by CP infection. However, levels of total protein and most of amino acids were not changed. CP infection decreased C18:3n-3 (α-LA), C20:4n-6, C20:5n-3(EPA), C22:4n-6, C22:5n-3, C22:6n-3(DHA), total PUFA, n-3 PUFA and PUFA:SFA ratio and increased C14:0, total SFA and n-6:n-3 ratio. LJ was found to protect the muscle from these changes. Meanwhile, the 28-day gut permeability level was higher in CP group. These findings suggest that CP may affect the growth performance of chicks and negatively influence lipid content and fatty acid composition in chicken meat. Meanwhile, LJ

  11. Isolation of Clostridium absonum and its cultural and biochemical properties.

    Science.gov (United States)

    Hayase, M; Mitsui, N; Tamai, K; Nakamura, S; Nishida, S

    1974-01-01

    A new procedure for isolation of Clostridium absonum was devised. Sixtyseven strains of C. absonum were isolated from 135 soil samples, but no strain of C. absonum could be found from human fecal samples. The lecithinase, hemolysin, and lethal toxin in the culture filtrates of this species exhibited low avidity for C. perfringens type A antitoxin. The three activities were inseparable by the present method of purification. A reinvestigation of biochemical properties revealed that incomplete suppression of lecithinase reaction by C. perfringens type A antitoxin and no fermentation of raffinose, melibiose, and starch are useful criteria to differentiate C. absonum from C. perfringens, and that positive, although weak, gelatin liquefaction and fermentation of trehalose are useful to differentiate it from C. paraperfringens.

  12. Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives.

    OpenAIRE

    OGUMA, K.; Yamaguchi, T.; Sudou, K; Yokosawa, N; Fujikawa, Y.

    1986-01-01

    The biochemical properties of 11 toxigenic and 10 nontoxigenic type C and D strains of Clostridium botulinum were studied. All of the strains examined were motile and hemolytic and produced lipase and liquid gelatin. Fermentation of several sugars and the production of lecithinase, indole, and hydrogen sulfide varied with the strain. The strains were classified into four groups based on their sugar fermentation profiles. The resulting classification was identical to the classification which h...

  13. Draft Genome Sequence of Clostridium tyrobutyricum Strain DIVETGP, Isolated from Cow's Milk for Grana Padano Production

    DEFF Research Database (Denmark)

    Soggiu, Alessio; Piras, Cristian; Gaiarsa, Stefano;

    2015-01-01

    We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%....

  14. Draft Genome Sequence of Clostridium tyrobutyricum Strain DIVETGP, Isolated from Cow's Milk for Grana Padano Production

    OpenAIRE

    2015-01-01

    We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow’s milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%.

  15. Draft Genome Sequence of Clostridium tyrobutyricum Strain DIVETGP, Isolated from Cow's Milk for Grana Padano Production.

    Science.gov (United States)

    Soggiu, Alessio; Piras, Cristian; Gaiarsa, Stefano; Bendixen, Emøke; Panitz, Frank; Bendixen, Christian; Sassera, Davide; Brasca, Milena; Bonizzi, Luigi; Roncada, Paola

    2015-03-26

    We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow's milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%.

  16. FERMENTATION OF INULIN BY A NEW STRAIN OF CLOSTRIDIUM-THERMOAUTOTROPHICUM ISOLATED FROM DAHLIA TUBERS

    NARCIS (Netherlands)

    DRENT, WJ; GOTTSCHAL, JC

    1991-01-01

    A new inulin-fermenting strain of Clostridium thermoautotrophicum was isolated through enrichment on dahlia tubers, and subsequent plating on agar media with undissolved inulin. Both the cell-bound and cell-free inulinase(s) functioned optimally at 60-degrees-C and at neutral pH. This new strain I1

  17. FERMENTATION OF INULIN BY A NEW STRAIN OF CLOSTRIDIUM-THERMOAUTOTROPHICUM ISOLATED FROM DAHLIA TUBERS

    NARCIS (Netherlands)

    DRENT, WJ; GOTTSCHAL, JC

    1991-01-01

    A new inulin-fermenting strain of Clostridium thermoautotrophicum was isolated through enrichment on dahlia tubers, and subsequent plating on agar media with undissolved inulin. Both the cell-bound and cell-free inulinase(s) functioned optimally at 60-degrees-C and at neutral pH. This new strain I1

  18. Study on Culture Technology of Escherichia coli and Clostridium Perfringens Type C of Piglets%仔猪大肠杆菌·C型产气荚膜梭菌菌液的培养工艺

    Institute of Scientific and Technical Information of China (English)

    刘泽文; 袁芳艳; 周丹娜; 梁望旺; 段正赢; 杨克礼; 徐涤平; 田永祥

    2012-01-01

    [目的]研究仔猪大肠杆菌、C型产气荚膜梭菌菌液的培养工艺.[方法]分别试验不同的培养时间、培养温度和接种剂量对大肠杆菌纤毛表达量和C型产气荚膜梭菌产生毒素的影响.[结果]大肠杆菌在接种剂量为3%~5%,在36~37℃条件下培养7h,可产生较高含量的纤毛抗原;当接种剂量为1%~2%,C型产气荚膜梭菌C59-2菌株在35℃条件下培养16~20 h,可产生大量致死性毒素.[结论]该研究为研制具有较好效果的仔猪红痢、黄痢预防疫苗提供了依据.%[Objective] The aim was to study the culture technology of Escherichia coli and clostridium perfringens type C of piglets. [ Method ]Effects of different culture time, culture temperature and inoculation dosage on the cilia expression of E. coli and toxin production of clostridium perfringens type C were studied respectively, [Result] The results showed that the content of fimbriae antigen was higher when inoculation dosage of E. coli was i%-5% ,and cultured at 36 -37 ℃ for 7 h. There would produce huge amounts lethal toxin when inoculation dosage of clostridium perfringens type C was 1% -2% ,and cultured at 35 ℃, for 16 -20 h. [Conclusion] The study provides a basis for development with good effect prevention vaccine of red scour of piglets and white scour of piglets.

  19. Intoxicación alimentaria por Clostridium perfringens en el Centro Penitenciario de Atención Institucional de San José: estudio de cohorte retrospectivo

    Directory of Open Access Journals (Sweden)

    Roy Wong-Mc Clure

    2004-06-01

    Full Text Available Las enfermedades transmitidas por los alimentos se presentan anualmente en Costa Rica y por lo general se estudian hasta identificar el agente etiológico, pero no hasta determinar su fuente de contaminación. No se ha documentado la importancia de fortalecer la inocuidad de alimentos, lo cual produce eventos que comprometen la salud pública cada año, por lo que fue importante estudiar el presente brote. En este estudio se analiza el brote diarreico de un centro penitenciario de San José, en 2003, procurando determinar el probable alimento contaminado. Materiales y métodos: El presente estudio corresponde a una cohorte retrospectiva que identifica exposición a los alimentos preparados dentro del Centro, en las 72 horas previas al pico epidémico. La descripción de la cohorte y el cálculo del (RR se realizó posteriormente para determinar la posible\tasociación causal. Las muestras de heces fueron procesadas en el laboratorio de referencia del INCIENSA. Resultados: La totalidad de los individuos pertenecían al sexo masculino, todos privados de libertad, con un promedio de edad de 31.1años (IC95%=28.9-33.4. La sintomatología más frecuente fue la diarrea (92.3% Y dolor abdominal tipo cólico (84.6%. El resultado de laboratorio aisló como agente etiológico el Clostridium perfringens enterotoxigénico (CPE. Al analizar los alimentos suministrados, pico epidémico y período de incubación, se tiene que el salchichón (RR=1.1;IC95%0.5-2.7 y la preparación con carne molida (RR=2.1,IC95%:0.6-7.4 mostraron ser los únicos alimentos capaces de transmitir el CPE. Discusión: El brote del Centro evidencia que la sintomatología descrita corresponde con lo documentado para la intoxicación por CPE y que, tomando en cuenta las condiciones necesarias para la transmisión, solo el salchichón y la preparación con carne molida cuentan con esta plausibilidad biológica. Al considerar el periodo de incubación, los valores de riesgo relativo

  20. The effect of Artemisia annua on broiler performance, on intestinal microbiota and on the course of a Clostridium perfringens infection applying a necrotic enteritis disease model

    DEFF Research Database (Denmark)

    Engberg, Ricarda M; Grevsen, Kai; Ivarsen, Elise

    2012-01-01

    The aerial parts of the plant Artemisia annua contain essential oils having antimicrobial properties against Clostridium perfringens Type A, the causal agent for necrotic enteritis in broilers. In two experiments, the influence of increasing dietary concentrations of dried A. annua leaves (0, 5, 10...... and 20 g/kg) and n-hexane extract from fresh A. annua leaves (0, 125, 250 and 500 mg/kg) on broiler performance was investigated. Dried plant material decreased feed intake and body weight in a dose-dependent manner, and 10 and 20 g/kg diet tended to improve the feed conversion ratio. The n......-hexane extract also reduced feed intake, but broiler weight tended to decrease only at the highest dietary concentration. The feed conversion ratio tended to improve when birds received 250 and 500 mg/kg n-hexane extract. In a third experiment, a necrotic enteritis disease model was applied to investigate...

  1. Clonagem e expressão do gene da toxina épsilon de clostridium perfringens tipo D e sua aplicação na imunização de animais

    OpenAIRE

    2008-01-01

    A toxina épsilon é produzida pelo Clostridium perfringens tipos B ou D, sendo responsável pela enterotoxemia em ovinos, caprinos e bovinos. A doença é de caráter agudo ou super agudo, causando morte súbita e grandes perdas econômicas. A evolução da doença dificulta qualquer medida terapêutica, e o processo pode ser prevenido através de imunizações com vacinas comprovadamente eficientes. O gene etx, que codifica a toxina épsilon, foi clonado no plasmídeo TOPO TA e subclonado em pET-11a, utiliz...

  2. 抗脆弱类杆菌和产气荚膜杆菌McAb池在外科感染快速诊断中的应用%Application of monoclonal antibody pool against Bacteroides fragilis and Clostridium perfringens in rapid diagnosis in surgical infection

    Institute of Scientific and Technical Information of China (English)

    张雅萍; 甘露; 常山

    2001-01-01

    目的 制备抗脆弱类杆菌和抗产气荚膜杆菌单克隆抗体池,并用于快速诊断及时指导临床治疗。方法 采用间接免疫荧光抗体染色法(IFA)和免疫酶标抗体染色法(ELA)对我院1998~1999年191例外科感染患者的标本进行细菌学检测,并与常规培养法进行比较。结果 3种方法从191份标本中分别检出脆弱类杆菌53株(27.7%)和55株(28.8%)以及25株(13.1%);检出产气荚膜杆菌12株(6.3%)和11株(5.8%)以及6株(3.1%)。IFA和ELA法2种厌氧菌检出率明显高于CM法。但IFA和ELA法之间检出率差异无显著性。结论 自制抗脆弱类杆菌和抗产气荚膜杆菌的McAb池,检测平时常见的脆弱类杆菌和战时常见的产气荚膜杆菌,敏感性高,特异性强,简便,快速,便于推广。%Objective To prepare monoclonal antibody (McAb) pool against Bacteroides fragilis and Clostridium perfringens and evaluated its effect in rapid diagnosis in surgical infection. Methods After the preparation of monoclonal antibodies against B. fragilis and C. perfringens, 191 specimens collected from infectious patients was detected with indirect immunofluorescent antibody (IFA) and enzyme labeled antibody (ELA). The results were compared with routine cultural method (CM). Results Among the 191 specimens, 53 (27.7%), 55 (28.8%) and 25 (13.1%)strains of B. fragilis were detected with IFA, ELA and CM respectively, and for C.perfringens, 12(6.3%), 11(5.8%), and 6(3.1%) strains were found. The detectable rate of anaerobic bacteria by IFA and ELA was higher than by CM, but no difference was found between these 2. Conclusion The self-made McAb pool against B.fagilis and C.perfringens might be a rapid, specific, sensitive and simple McAb pool for detecting B.fragilis in surgical infection and C.perfringens in wartime.

  3. The ability of Clostridium bifermentans strains to lactic acid biosynthesis in various environmental conditions

    OpenAIRE

    Leja, Katarzyna; Myszka, Kamila; Czaczyk, Katarzyna

    2013-01-01

    Clostridium bifermentans strains, isolated from a manure, were examinated for their ability to produce lactic acid from PY medium with glycerol under different pH conditions and when PY medium was supplemented with saccharides such as fructose, sorbitol, glucose, mannose, mannitol, maltose, xylose, raffinose, and arabinose. In the last test performed, the ability of investigated strains to produce lactic acid from mixed carbon source (glycerol plus saccharide) was checked. The strains of Cl. ...

  4. Isolation and characterization of a hydrogen- and ethanol-producing Clostridium sp. strain URNW.

    Science.gov (United States)

    Ramachandran, Umesh; Wrana, Nathan; Cicek, Nazim; Sparling, Richard; Levin, David B

    2011-03-01

    Identification, characterization, and end-product synthesis patterns were analyzed in a newly identified mesophilic, anaerobic Clostridium sp. strain URNW, capable of producing hydrogen (H₂) and ethanol. Metabolic profiling was used to characterize putative end-product synthesis pathways of the Clostridium sp. strain URNW, which was found to grow on cellobiose; on hexose sugars, such as glucose, sucrose, and mannose; and on sugar alcohols, like mannitol and sorbitol. When grown in batch cultures on 2 g cellobiose·L⁻¹, Clostridium sp. strain URNW showed a cell generation time of 1.5 h, and the major end-products were H2, formate, carbon dioxide (CO₂), lactate, butyrate, acetate, pyruvate, and ethanol. The total volumetric H₂ production was 14.2 mmol·(L culture)⁻¹ and the total production of ethanol was 0.4 mmol·(L culture)⁻¹. The maximum yield of H₂ was 1.3 mol·(mol glucose equivalent)⁻¹ at a carbon recovery of 94%. The specific production rates of H₂, CO₂, and ethanol were 0.45, 0.13, and 0.003 mol·h⁻¹·(g dry cell mass)-1, respectively. BLAST analyses of 16S rDNA and chaperonin 60 (cpn60) sequences from Clostridium sp. strain URNW revealed a 98% nucleotide sequence identity with the 16S rDNA and cpn60 sequences from Clostridium intestinale ATCC 49213. Phylogenetic analyses placed Clostridium sp. strain URNW within the butyrate-synthesizing clostridia.

  5. Characterization of the spore surface and exosporium proteins of Clostridium sporogenes; implications for Clostridium botulinum group I strains.

    Science.gov (United States)

    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. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

  6. Genome Resequencing of the Virulent and Multidrug-Resistant Reference Strain Clostridium difficile 630

    OpenAIRE

    Riedel, Thomas; Bunk, Boyke; Thürmer, Andrea; Spröer, Cathrin; Brzuszkiewicz, Elzbieta; Abt, Birte; Gronow, Sabine; Liesegang, Heiko; Daniel, Rolf; Overmann, Jörg

    2015-01-01

    We resequenced the complete genome of the virulent and multidrug-resistant pathogen Clostridium difficile strain 630. A combination of single-molecule real-time and Illumina sequencing technology revealed the presence of an additional rRNA gene cluster, additional tRNAs, and the absence of a transposon in comparison to the published and reannotated genome sequence.

  7. Complete Genome Sequence of the Clostridium difficile Type Strain DSM 1296T

    Science.gov (United States)

    Bunk, Boyke; Wittmann, Johannes; Thürmer, Andrea; Spröer, Cathrin; Gronow, Sabine; Liesegang, Heiko; Daniel, Rolf; Overmann, Jörg

    2015-01-01

    In this study, we sequenced the complete genome of the Clostridium difficile type strain DSM 1296T. A combination of single-molecule real-time (SMRT) and Illumina sequencing technology revealed the presence of one chromosome and two extrachromosomal elements, the bacteriophage phiCDIF1296T and a putative plasmid-like structure harboring genes of another bacteriophage. PMID:26450746

  8. Genome Resequencing of the Virulent and Multidrug-Resistant Reference Strain Clostridium difficile 630

    Science.gov (United States)

    Bunk, Boyke; Thürmer, Andrea; Spröer, Cathrin; Brzuszkiewicz, Elzbieta; Abt, Birte; Gronow, Sabine; Liesegang, Heiko; Daniel, Rolf; Overmann, Jörg

    2015-01-01

    We resequenced the complete genome of the virulent and multidrug-resistant pathogen Clostridium difficile strain 630. A combination of single-molecule real-time and Illumina sequencing technology revealed the presence of an additional rRNA gene cluster, additional tRNAs, and the absence of a transposon in comparison to the published and reannotated genome sequence. PMID:25858846

  9. Complete Genome Sequence of the Clostridium difficile Type Strain DSM 1296T

    OpenAIRE

    Riedel, Thomas; Bunk, Boyke; Wittmann, Johannes; Thürmer, Andrea; Spröer, Cathrin; Gronow, Sabine; Liesegang, Heiko; Daniel, Rolf; Overmann, Jörg

    2015-01-01

    In this study, we sequenced the complete genome of the Clostridium difficile type strain DSM 1296T. A combination of single-molecule real-time (SMRT) and Illumina sequencing technology revealed the presence of one chromosome and two extrachromosomal elements, the bacteriophage phiCDIF1296T and a putative plasmid-like structure harboring genes of another bacteriophage.

  10. Selection of an Asporogenous Strain of Clostridium acetobutylicum in Continuous Culture Under Phosphate Limitation

    OpenAIRE

    Meinecke, Beatrix; Bahl, Hubert; Gottschalk, Gerhard

    1984-01-01

    Based on the observation that cells of Clostridium acetobutylicum unable to store granulose do not initiate sporulation, a staining procedure was developed for the detection of asporogenous mutants. By application of this procedure it was shown that an asporogenous strain of C. acetobutylicum was selected in continuous culture under phosphate limitation.

  11. Draft Genome Sequence of the Butyric Acid Producer Clostridium tyrobutyricum Strain CIP I-776 (IFP923).

    Science.gov (United States)

    Wasels, François; Clément, Benjamin; Lopes Ferreira, Nicolas

    2016-03-03

    Here, we report the draft genome sequence of Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain.

  12. Draft Genome Sequence of the Butyric Acid Producer Clostridium tyrobutyricum Strain CIP I-776 (IFP923)

    OpenAIRE

    2016-01-01

    Here, we report the draft genome sequence of Clostridium tyrobutyricum CIP I-776 (IFP923), an efficient producer of butyric acid. The genome consists of a single chromosome of 3.19 Mb and provides useful data concerning the metabolic capacities of the strain.

  13. Complete Genome Sequence of the Hypervirulent Bacterium Clostridium difficile Strain G46, Ribotype 027

    Science.gov (United States)

    Gaulton, Tom; Rose, Graham; Baybayan, Primo; Hall, Richard; Freeman, Jane; Turton, Jane; Picton, Steve; Korlach, Jonas; Gharbia, Saheer; Shah, Haroun

    2015-01-01

    Clostridium difficile is one of the leading causes of antibiotic-associated diarrhea in health care facilities worldwide. Here, we report the genome sequence of C. difficile strain G46, ribotype 027, isolated from an outbreak in Glamorgan, Wales, in 2006. PMID:25814591

  14. Genome Sequence of Clostridium acetobutylicum GXAS18-1, a Novel Biobutanol Production Strain

    OpenAIRE

    Mo, Xinchun; Pei, Jianxin; Guo, Yuan; Lin, Lihua; Peng, Lixin; Kou, Chan; Fan, Danmin; Pang, Hao

    2015-01-01

    Clostridium acetobutylicum is an organism involved in the production of acetone and butanol by traditional acetone-butanol-ethanol fermentation (ABE). We report the draft genome sequence of C.?acetobutylicum strain GXAS18-1, which can produce ABE directly from cassava flour.

  15. Genome Sequence of Clostridium acetobutylicum GXAS18-1, a Novel Biobutanol Production Strain

    OpenAIRE

    Mo, Xinchun; Pei, Jianxin; Guo, Yuan; Lin, Lihua; Peng, Lixin; Kou, Chan; Fan, Danmin; Pang, Hao

    2015-01-01

    Clostridium acetobutylicum is an organism involved in the production of acetone and butanol by traditional acetone-butanol-ethanol fermentation (ABE). We report the draft genome sequence of C. acetobutylicum strain GXAS18-1, which can produce ABE directly from cassava flour.

  16. Draft Genome Sequence of Clostridium ultunense Strain Esp, a Syntrophic Acetate-Oxidizing Bacterium.

    Science.gov (United States)

    Manzoor, Shahid; Müller, Bettina; Niazi, Adnan; Bongcam-Rudloff, Erik; Schnürer, Anna

    2013-03-28

    Clostridium ultunense strain Esp belongs to the functional group of syntrophic acetate-oxidizing bacteria (SAOB), which have been identified as key organisms for efficient biogas production from protein-rich materials. Genome analysis and comparative genomics might aid us to define physiological features that are essential for maintaining this particular syntrophic lifestyle.

  17. Characterisation of non-toxigenic Clostridium spp. strains, to use as surrogates for non-proteolytic Clostridium botulinum in chilled food challenge testing.

    Science.gov (United States)

    Parker, M D; Barrett, P I; Shepherd, J; Price, L J; Bull, S D

    2015-01-01

    Under many of the conditions studied, a two-strain cocktail of non-toxigenic Clostridium spp. was found to be suitable as a surrogate for non-proteolytic Clostridium botulinum, and has the potential for use in chilled food challenge tests measuring growth. Non-toxigenic surrogates could also be used in thermal process screening studies. Copyright © 2014 Elsevier B.V. All rights reserved.

  18. [Engineering and metabolic characteristics of a Clostridium tyrobutyricum strain].

    Science.gov (United States)

    Yang, Guiqing; Liu, Gang; Yang, Changde

    2010-02-01

    Clostridium tyrobutyricum is suitable for simultaneous saccharification and fermentation of lignocellulosic. It can produce butyric acid, acetic acid as its main fermentation products from a wide variety of carbohydrates such as glucose, xylose, cellobiose and arabinose. In order to decrease acetic acid content and increase butyric acid content in C. tyrobutyricum, we replaced genes on the acetic acid fermentation pathway with genes on the butyric acid fermentation pathway. Three genes were selected. They were acetyl-CoA acetylrtansfers gene (thl) which is the key enzyme gene associated with acetic acid fermentation pathway from Clostridium acetobutylicum, erythromycin gene (em) from plasmid pIMP1 and phosphotransacetylase gene (pta) which is the key enzyme gene associated with butyric acid fermentation pathway from C. tyrobutyricum. We fused these genes with pUC19 to construct nonreplicative integrated plasmids pUC19-EPT. Then we transformed pUC19-EPT into C. tyrobutyricum through electroporation. The recombinant transformants grown on plates containing erythromycin were validated by PCR. A mutant whose pta gene was displaced by thl gene on the chromosome was selected. In the fermentation from glucose, the mutant's yield of butyric acid is 0.47, increased by 34% compared with wild type; and the yield of acetic acid is 0.05, decreased by 29% compared with wild type.

  19. Clostridium strain co-cultures for biohydrogen production enhancement from condensed molasses fermentation solubles

    Energy Technology Data Exchange (ETDEWEB)

    Hsiao, Chin-Lang; Wu, Jou-Hsien; Lin, Chiu-Yue [BioHydrogen Lab, Graduate Institute of Civil and Hydraulic Engineering, Feng Chia University, Taichung (China); Chang, Jui-Jen [Genomics Research Center, Academia Sinica, Taipei (China); Department of Life Sciences, National Chung Hsing University, Taichung (China); Chin, Wei-Chih; Wen, Fu-Shyan; Huang, Chieh-Chen [Department of Life Sciences, National Chung Hsing University, Taichung (China); Chen, Chin-Chao [Environmental Resources Laboratory, Department of Landscape Architecture, Chungchou Institute of Technology, Changhwa (China)

    2009-09-15

    An anaerobic continuous-flow hydrogen fermentor was operated at a hydraulic retention time of 8 h using condensed molasses fermentation solubles (CMS) substrate of 40 g-COD/L. Serum bottles were used for seed micro-flora cultivation and batch hydrogen fermentation tests (CMS substrate concentrations of 10-160 g-COD/L). Three hydrogen-producing bacterial strains Clostridium sporosphaeroides F52, Clostridium tyrobutyricum F4 and Clostridium pasteurianum F40 were isolated from the seed fermentor and used as the seeding microbes in single and mixed-culture cultivations for determining their hydrogen productivity. These strains possessed specific hydrogenase genes that could be detected from CMS-fed hydrogen fermentors and were major hydrogen producers. C. pasteurianum F40 was the dominant strain with a high hydrogen production rate while C. sporosphaeroides F52 may play a main role in degrading carbohydrate and glutamate. These strains could be co-cultivated as a symbiotic mixed-culture process to enhance hydrogen productivity. C. pasteurianum F40 or C. tyrobutyricum F4 co-culture with the glutamate-utilizing strain C. sporosphaeroides F52 efficiently enhanced hydrogen production by 12-220% depending on the substrate CMS concentrations. (author)

  20. [Enterotoxemia in newborn calves due to Cl. perfringens types A, C and D].

    Science.gov (United States)

    Lulov, R; Angelov, A K

    1986-01-01

    The clinical symptoms and the morphologic picture of calf enterotoxemia are described. Studied were a total of thirty-two dead and slaughtered animals. Bacteriologically, the disease was shown to be caused by types A, D, and C of Clostridium perfringens. Types C and D proved pathogenic for guinea pigs, while type A did not. Isolated was a strain of Clostridium perfringens, which had high toxigenicity. It was found that calves were fairly often affected with the disease. Most severe were the infections caused by type C of Cl. perfringens, with most pronounced morphologic lesions. There were differences in the changes caused by all three types of the organism in calves, which made it possible to distinguish them as causative agents. Type A induced slight icterus and slightly manifested hemosiderosis of the liver, kidneys, and spleen; type D was responsible for severe injury and hyalin dystrophy of the kidneys; and type C caused necrotic enteritis, pronounced hemorrhagic diathesis, degenerative changes in the ganglial cells, and demyelinization of the brain.

  1. Genomic analysis of carbon monoxide utilization and butanol production by Clostridium carboxidivorans strain P7.

    Directory of Open Access Journals (Sweden)

    Guillaume Bruant

    Full Text Available Increasing demand for the production of renewable fuels has recently generated a particular interest in microbial production of butanol. Anaerobic bacteria, such as Clostridium spp., can naturally convert carbohydrates into a variety of primary products, including alcohols like butanol. The genetics of microorganisms like Clostridium acetobutylicum have been well studied and their solvent-producing metabolic pathways characterized. In contrast, less is known about the genetics of Clostridium spp. capable of converting syngas or its individual components into solvents. In this study, the type of strain of a new solventogenic Clostridium species, C. carboxidivorans, was genetically characterized by genome sequencing. C. carboxidivorans strain P7(T possessed a complete Wood-Ljungdahl pathway gene cluster, involving CO and CO(2 fixation and conversion to acetyl-CoA. Moreover, with the exception of an acetone production pathway, all the genetic determinants of canonical ABE metabolic pathways for acetate, butyrate, ethanol and butanol production were present in the P7(T chromosome. The functionality of these pathways was also confirmed by growth of P7(T on CO and production of CO(2 as well as volatile fatty acids (acetate and butyrate and solvents (ethanol and butanol. P7(T was also found to harbour a 19 Kbp plasmid, which did not include essential or butanol production related genes. This study has generated in depth knowledge of the P7(T genome, which will be helpful in developing metabolic engineering strategies to improve C. carboxidivorans's natural capacity to produce potential biofuels from syngas.

  2. A型产气荚膜梭菌经小鼠传代的毒力变化%Investigation on Virulence of Clostridium perfringens Type A by Transfer of Culture in Mice

    Institute of Scientific and Technical Information of China (English)

    孙朝晖; 徐少珊; 石玉玲; 唐玲玲

    2011-01-01

    anarobic condiction.The colony is proved to be C.perfringens by APl 20A test.The survival time had increased by transfer culture in abdominal while first generation's is the short and the sequence generations' is longer with no statistics difference.Conclusion: The virulence of Clostridium perfringens type A can not increased according transfer of culture in mice.

  3. Draft Genome Sequence of Clostridium bifermentans Strain WYM, a Promising Biohydrogen Producer Isolated from Landfill Leachate Sludge.

    Science.gov (United States)

    Wong, Y M; Juan, J C; Gan, H M; Austin, C M

    2014-03-06

    Clostridium bifermentans strain WYM is an effective biohydrogen producer isolated from landfill leachate sludge. Here, we present the assembly and annotation of its genome, which may provide further insights into the metabolic pathways involved in efficient biohydrogen production.

  4. Diversity of Group I and II Clostridium botulinum Strains from France Including Recently Identified Subtypes.

    Science.gov (United States)

    Mazuet, Christelle; Legeay, Christine; Sautereau, Jean; Ma, Laurence; Bouchier, Christiane; Bouvet, Philippe; Popoff, Michel R

    2016-06-13

    In France, human botulism is mainly food-borne intoxication, whereas infant botulism is rare. A total of 99 group I and II Clostridium botulinum strains including 59 type A (12 historical isolates [1947-1961], 43 from France [1986-2013], 3 from other countries, and 1 collection strain), 31 type B (3 historical, 23 recent isolates, 4 from other countries, and 1 collection strain), and 9 type E (5 historical, 3 isolates, and 1 collection strain) were investigated by botulinum locus gene sequencing and multilocus sequence typing analysis. Historical C. botulinum A strains mainly belonged to subtype A1 and sequence type (ST) 1, whereas recent strains exhibited a wide genetic diversity: subtype A1 in orfX or ha locus, A1(B), A1(F), A2, A2b2, A5(B2') A5(B3'), as well as the recently identified A7 and A8 subtypes, and were distributed into 25 STs. Clostridium botulinum A1(B) was the most frequent subtype from food-borne botulism and food. Group I C. botulinum type B in France were mainly subtype B2 (14 out of 20 historical and recent strains) and were divided into 19 STs. Food-borne botulism resulting from ham consumption during the recent period was due to group II C. botulinum B4. Type E botulism is rare in France, 5 historical and 1 recent strains were subtype E3. A subtype E12 was recently identified from an unusual ham contamination. Clostridium botulinum strains from human botulism in France showed a wide genetic diversity and seems to result not from a single evolutionary lineage but from multiple and independent genetic rearrangements.

  5. Molecular characterization of Clostridium tetani strains by pulsed-field gel electrophoresis and colony PCR.

    Science.gov (United States)

    Plourde-Owobi, Lucile; Seguin, Delphine; Baudin, Marie-Anne; Moste, Catherine; Rokbi, Bachra

    2005-09-01

    Pulsed-field gel electrophoresis and PCR were applied for the first time to the molecular characterization of Clostridium tetani. Among five strains tested, one (CN1339) turned out to contain a mixture of two genetically different clones and two (D11 and G761) to contain bacteria differing by the presence or absence of the 74-kb plasmid harboring the tetX gene.

  6. Whole-genome single-nucleotide-polymorphism analysis for discrimination of Clostridium botulinum group I strains.

    Science.gov (United States)

    Gonzalez-Escalona, Narjol; Timme, Ruth; Raphael, Brian H; Zink, Donald; Sharma, Shashi K

    2014-04-01

    Clostridium botulinum is a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA(+) OrfX(-)) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA(-) OrfX(+)) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producing C. botulinum strains: two strains with the HA(+) OrfX(-) cluster (69A and 32A) and one strain with the HA(-) OrfX(+) cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly available C. botulinum group I strains revealed five distinct lineages. Strains 69A and 32A clustered with the C. botulinum type A1 Hall group, and strain CDC297 clustered with the C. botulinum type Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination of C. botulinum group I strains and demonstrates the utility of this analysis in quickly differentiating C. botulinum strains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

  7. Prevention of Clostridium difficile infection in hamsters using a non-toxigenic strain

    Directory of Open Access Journals (Sweden)

    Carlos Augusto de Oliveira Júnior

    2016-05-01

    Full Text Available ABSTRACT: The present study aimed to evaluate five non-toxigenic strains of Clostridium difficile (NTCD in vitro and to select one strain to prevent C. difficile (CDI infection in hamsters ( Mesocricetus auratus . The NTCD strains were evaluated for spore production in vitro, antimicrobial susceptibility and presence of antimicrobial resistance genes. Approximately 107 spores of the selected strain (Z31 were administered by esophageal gavage in hamsters pretreated with 30mg kg-1 of clindamycin. The challenge with a toxigenic strain of C. difficile was conducted at 36 and 72h, and the animals were observed for 28 days. The NTCD strain of C. difficile (Z31 was able to prevent CDI in all animals that received it.

  8. The same clade of Clostridium botulinum strains is causing avian botulism in southern and northern Europe.

    Science.gov (United States)

    Anza, Ibone; Skarin, Hanna; Vidal, Dolors; Lindberg, Anna; Båverud, Viveca; Mateo, Rafael

    2014-04-01

    Avian botulism is a paralytic disease caused by Clostridium botulinum-produced botulinum neurotoxins (BoNTs), most commonly of type C/D. It is a serious disease of waterbirds and poultry flocks in many countries in Europe. The objective of this study was to compare the genetic relatedness of avian C. botulinum strains isolated in Spain with strains isolated in Sweden using pulsed-field gel electrophoresis (PFGE). Fifteen strains were isolated from Spanish waterbirds using an immunomagnetic separation technique. Isolates were characterized by PCR, and all were identified as the genospecies Clostridium novyi sensu lato and eight harboured the gene coding for the BoNT type C/D. PFGE analysis of the strains revealed four highly similar pulsotypes, out of which two contained strains from both countries. It also showed that outbreaks in wild and domestic birds can be caused by the same strains. These results support a clonal spreading of the mosaic C. botulinum type C/D through Europe and give relevant information for future epidemiological studies.

  9. Claudin-4 Overexpression in Epithelial Ovarian Cancer Is Associated with Hypomethylation and Is a Potential Target for Modulation of Tight Junction Barrier Function Using a C-Terminal Fragment of Clostridium perfringens Enterotoxin

    Directory of Open Access Journals (Sweden)

    Babak Litkouhi

    2007-04-01

    Full Text Available BACKGROUND: Claudin-4, a tight junction (TJ protein and receptor for the C-terminal fragment of Clostridium perfringens enterotoxin (C-CPE, is overexpressed in epithelial ovarian cancer (EOC. Previous research suggests DNA methylation is a mechanism for claudin-4 overexpression in cancer and that C-CPE acts as an absorption-enhancing agent in claudin-4expressing cells. We sought to correlate claudin-4 overexpression in EOC with clinical outcomes and TJ barrier function, investigate DNA methylation as a mechanism for overexpression, and evaluate the effect of C-CPE on the TJ. METHODS: Claudin-4 expression in EOC was quantified and correlated with clinical outcomes. Claudin-4 methylation status was determined, and claudin-4-negative cell lines were treated with a demethylating agent. Electric cell-substrate impedance sensing was used to calculate junctional (paracellular resistance (Rb in EOC cells after claudin-4 silencing and after C-CPE treatment. RESULTS: Claudin4 overexpression in EOC does not correlate with survival or other clinical endpoints and is associated with hypomethylation. Claudin-4 overexpression correlates with Rb and C-CPE treatment of EOC cells significantly decreased Rb in a dose- and claudin-4-dependent noncytotoxic manner. CONCLUSIONS: C-CPE treatment of EOC cells leads to altered TJ function. Further research is needed to determine the potential clinical applications of C-CPE in EOC drug delivery strategies.

  10. Butyric acid fermentation from pre-treated wheat straw by a mutant clostridium tyrobutyricum strain

    DEFF Research Database (Denmark)

    Baroi, George Nabin; Baumann, Ivan; Westermann, Peter;

    ’s platform for a variety of products for industrial use. Butyric acid is considered as a potential chemical building-block for the production of chemicals for e.g. polymeric compounds and the aim of this work was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces less acetic...... acid (higher selectivity), has a higher yield and a higher productivity of butyric acid from pre-treated lignocellulosic biomass. Pre-treated wheat straw was used as the main carbon source. After one year of serial adaptation and selection a mutant strain of C. tyrobutyricum was developed. This new...

  11. Acetone-butanol-ethanol production from substandard and surplus dates by Egyptian native Clostridium strains.

    Science.gov (United States)

    Abd-Alla, Mohamed Hemida; Zohri, Abdel-Naser Ahmed; El-Enany, Abdel-Wahab Elsadek; Ali, Shimaa Mohamed

    2015-04-01

    One hundred and seven mesophilic isolates of Clostridium were isolated from agricultural soils cultivated with different plants in Assuit Governorate, Egypt. Eighty isolates (out of 107) showed the ability to produce ABE (Acetone, butanol and ethanol) on T6 medium ranging from 0.036 to 31.89 g/L. The highest numbers of ABE producing isolates were obtained from soil samples of potato contributing 27 isolates, followed by 18 isolates from wheat and 10 isolates from onion. On the other hand, there were three native isolates that produced ABE more than those produced by the reference isolate Clostridium acetobutylicum ATCC 824 (11.543 g/L). The three isolates were identified based on phenotypic and gene encoding 16S rRNA as Clostridium beijerinckii ASU10 (KF372577), Clostridium chauvoei ASU55 (KF372580) and Clostridium roseum ASU58 (KF372581). The highest ABE level from substandard and surplus dates was produced by C. beijerinckii ASU10 (24.07 g/L) comprising butanol 67.15% (16.16 g/L), acetone 30.73% (7.4 g/L) and ethanol 2.12% (0.51 g/L), while C. roseum ASU58 and C. chauvoei ASU55 produced ABE contributing 20.20 and 13.79 g/L, respectively. ABE production by C. acetobutylicum ATCC 824 was 15.01 g/L. This study proved that the native strains C. beijerinckii ASU10 and C. roseum ASU58 have high competitive efficacy on ABE production from economical substrate as substandard and surplus date fruits. Additionally, using this substrate without any nutritional components is considered to be a commercial substrate for desired ABE production. Copyright © 2014 Elsevier Ltd. All rights reserved.

  12. 魏氏梭菌α毒素快速诊断金标试纸条的研制%Development of colloidal gold immunochromatographic test strip for detection of Clostridium perfringensα toxin

    Institute of Scientific and Technical Information of China (English)

    刘晓敏; 柴同杰; 邹明强; 黄蓉; 张明亮

    2012-01-01

    【Objective】 The study developed a colloidal gold immunochromatographic test strip for detection of Clostridium perfringens α toxin and provided a new method for detection of C.perfringens.【Method】 C.perfringens α toxin was developed by the method of molecular biology,and injected into rabbits to prepare anti-α polyclonal antibody.According to the immune colloidal gold technique principle,we prepared detection dipsticks using anti-α polyclonal antibody as gold labeled antibody and capture antibody of the detection line to detect the C.perfringens α toxin.【Result】 The anti-α polyclonal antibody and colloidal gold immunochromatographic test strip for detection CPT α toxin was developed successfully and the suitable concentration of antibody on test line was 1 mg/mL;Tests on serum samples showed that only C.perfringens α toxin positive serum produced a positive signal on the IC strip;The results revealed that the test strip had good specificity and reproducibility for the same result of different batches of test strips;The detection limit of α toxin was 1.40 mg/mL and the correspondence rate between the colloidal gold strip and PCR was 80.77%,meeting the requirement of field application basically.【Conclusion】 The colloidal gold immunochromatographic test strip for detection of C.perfringens α toxin was developed successfully.All these results showed that the strip was sensitive,specific,and rapid for the detection of C.perfringens,and had good application prospect.%【目的】研制魏氏梭菌α毒素快速诊断金标试纸条,为魏氏梭菌病的快速诊断提供新方法。【方法】采用分子生物学方法制备魏氏梭菌α毒素,将其免疫家兔制备多克隆抗体。用制备的抗魏氏梭菌α毒素多克隆抗体作为胶体金标记抗体和检测线上的捕获抗体,制备魏氏梭菌α毒素快速诊断金标试纸条,对其特异性、敏感性、稳定性和重复性进行检测。用制备的魏氏

  13. Effects of Megaplasmid Loss on Growth of Neurotoxigenic Clostridium butyricum Strains and Botulinum Neurotoxin Type E Expression

    OpenAIRE

    Concetta eScalfaro; Angelo eIacobino; Laura eGrande; Stefano eMorabito; Giovanna eFranciosa

    2016-01-01

    Clostridium butyricum strains that atypically produce the botulinum neurotoxin type E (BoNT/E) possess a megaplasmid of unknown functions in their genome. In this study, we cured two botulinum neurotoxigenic C. butyricum type E strains of their megaplasmids, and compared the obtained megaplasmid-cured strains to their respective wild-type parental strains. Our results showed that the megaplasmids do not confer beta-lactam resistance on the neurotoxigenic C. butyricum type E strains, although ...

  14. Process engineering and scale-up of autotrophic Clostridium strain P11 syngas fermentation

    Science.gov (United States)

    Kundiyana, Dimple Kumar Aiyanna

    Scope and Method of Study. Biomass gasification followed by fermentation of syngas to ethanol is a potential process to produce bioenergy. The process is currently being researched under laboratory- and pilot-scale in an effort to optimize the process conditions and make the process feasible for commercial production of ethanol and other biofuels such as butanol and propanol. The broad research objectives for the research were to improve ethanol yields during syngas fermentation and to design a economical fermentation process. The research included four statistically designed experimental studies in serum bottles, bench-scale and pilot-scale fermentors to screen alternate fermentation media components, to determine the effect of process parameters such as pH, temperature and buffer on syngas fermentation, to determine the effect of key limiting nutrients of the acetyl-CoA pathway in a continuous series reactor design, and to scale-up the syngas fermentation in a 100-L pilot scale fermentor. Findings and Conclusions. The first experimental study identified cotton seed extract (CSE) as a feasible medium for Clostridium strain P11 fermentation. The study showed that CSE at 0.5 g L-1 can potentially replace all the standard Clostridium strain P11 fermentation media components while using a media buffer did not significantly improve the ethanol production when used in fermentation with CSE. Scale-up of the CSE fermentation in 2-L and 5-L stirred tank fermentors showed 25% increase in ethanol yield. The second experimental study showed that syngas fermentation at 32°C without buffer was associated with higher ethanol concentration and reduced lag time in switching to solventogenesis. Conducting fermentation at 40°C or by lowering incubation pH to 5.0 resulted in reduced cell growth and no production of ethanol or acetic acid. The third experiment studied the effect of three limiting nutrients, calcium pantothenate, vitamin B12 and CoCl2 on syngas fermentation. Results

  15. Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Veen, Douwe [ORNL; Lo, Jonathan [Dartmouth College; Brown, Steven D [ORNL; Johnson, Courtney M [ORNL; Tschaplinski, Timothy J [ORNL; Martin, Madhavi Z [ORNL; Engle, Nancy L [ORNL; Van den Berg, Robert A [Katholieke University Leuven, Belgium; Argyros, Aaron [Mascoma Corporation; Caiazza, Nicky [Mascoma Corporation; Guss, Adam M [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    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.

  16. Characterization of Clostridium thermocellum strains with disrupted fermentation end product pathways

    Energy Technology Data Exchange (ETDEWEB)

    Van Der Veen, Douwe [ORNL; Lo, Jonathan [Dartmouth College; Brown, Steven D [ORNL; Johnson, Courtney M [ORNL; Tschaplinski, Timothy J [ORNL; Martin, Madhavi Z [ORNL; Engle, Nancy L [ORNL; Argyros, Aaron [Mascoma Corporation; Van den Berg, Robert A [Katholieke University Leuven, Belgium; Caiazza, Nicky [Mascoma Corporation; Guss, Adam M [ORNL; Lynd, Lee R [Thayer School of Engineering at Dartmouth

    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.

  17. Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test.

    Science.gov (United States)

    Rosskopf-Streicher, Ute; Volkers, Peter; Noeske, Kerstin; Werner, Esther

    2004-01-01

    Clostridium (C.) perfringens is a Gram-positive anaerobic spore-forming bacterium. Disease caused by C. perfringens infection is called enterotoxaemia. C. perfringens strains are classified on the basis of the lethal exotoxins formed by the bacteria. Epsilon toxin is one of the major lethal toxins and is formed by C. perfringens types B and D. C. perfringens is an ubiquitous bacterium. Infection occurs via food, water, animal litter or soil. Affected animals include mainly sheep, pigs and cattle. C. perfringens infection manifests as pulpy kidney disease and diarrhoea in suckling lambs. Enterotoxaemia development is peracute in most cases. Animals die suddenly while grazing on the pasture, without any prior signs of disease. Therefore, treatment is possible only in very rare cases. Suitable immunoprophylactic measures are the treatment of choice to combat the disease: Vaccines and immunosera have therefore been used extensively for a long time. The requirements for quality, efficacy and safety testing of the inactivated vaccines are laid down in the Ph. Eur. in the monograph: Clostridium perfringens vaccines for veterinary use. After a marketing authorisation is attained, the product batches must be tested in laboratory animal models for their potency against all vaccine components (Pharmeuropa, 1997). For potency testing (batch control) of C. perfringens types B and D, the induction of specific antibodies against epsilon toxin in rabbits must be verified. For this purpose, 10 rabbits are immunised twice with the product to be tested. Their blood is taken 14 days after the last immunisation and the serum is pooled. The pooled serum is then tested for its protective effect. This is done by means of the toxin neutralisation test in mice (optionally also in guinea pigs) in comparison with an international reference serum. The evaluation criterion is the death rate of the mice in the test and reference groups after administration of lethal doses of epsilon toxin. The

  18. Biofilms of Clostridium species.

    Science.gov (United States)

    Pantaléon, Véronique; Bouttier, Sylvie; Soavelomandroso, Anna Philibertine; Janoir, Claire; Candela, Thomas

    2014-12-01

    The biofilm is a microbial community embedded in a synthesized matrix and is the main bacterial way of life. A biofilm adheres on surfaces or is found on interfaces. It protects bacteria from the environment, toxic molecules and may have a role in virulence. Clostridium species are spread throughout both environments and hosts, but their biofilms have not been extensively described in comparison with other bacterial species. In this review we describe all biofilms formed by Clostridium species during both industrial processes and in mammals where biofilms may be formed either during infections or associated to microbiota in the gut. We have specifically focussed on Clostridium difficile and Clostridium perfringens biofilms, which have been studied in vitro. Regulatory processes including sporulation and germination highlight how these Clostridium species live in biofilms. Furthermore, biofilms may have a role in the survival and spreading of Clostridium species. Copyright © 2014 Elsevier Ltd. All rights reserved.

  19. Draft Genome Sequence of Clostridium tyrobutyricum Strain UC7086, Isolated from Grana Padano Cheese with Late-Blowing Defect.

    Science.gov (United States)

    Bassi, Daniela; Fontana, Cecilia; Gazzola, Simona; Pietta, Ester; Puglisi, Edoardo; Cappa, Fabrizio; Cocconcelli, Pier Sandro

    2013-08-15

    Clostridium tyrobutyricum is considered the main agent of late-blowing defect in the production of hard cheese. Here, we described the draft genome sequences and annotation of C. tyrobutyricum strain UC7086, which was isolated from Grana Padano cheese with blowing defect, and C. tyrobutyricum DSM 2637 type strain in a comparative study.

  20. Draft Genome Sequence of Clostridium tyrobutyricum Strain DIVETGP, Isolated from Cow’s Milk for Grana Padano Production

    Science.gov (United States)

    Piras, Cristian; Gaiarsa, Stefano; Bendixen, Emøke; Panitz, Frank; Bendixen, Christian; Sassera, Davide; Brasca, Milena; Bonizzi, Luigi; Roncada, Paola

    2015-01-01

    We announce the draft genome sequence of Clostridium tyrobutyricum strain DIVETGP. This strain was isolated from cow’s milk used for Grana Padano cheese production. The genome was obtained using Illumina HiSeq technology and comprises 45 contigs for 3,018,999 bp, with a G+C content of 30.8%. PMID:25814611

  1. Draft Genome Sequence of Clostridium tyrobutyricum Strain UC7086, Isolated from Grana Padano Cheese with Late-Blowing Defect

    OpenAIRE

    Bassi, Daniela; Fontana, Cecilia; Gazzola, Simona; Pietta, Ester; Puglisi, Edoardo; Cappa, Fabrizio; Cocconcelli, Pier Sandro

    2013-01-01

    Clostridium tyrobutyricum is considered the main agent of late-blowing defect in the production of hard cheese. Here, we described the draft genome sequences and annotation of C. tyrobutyricum strain UC7086, which was isolated from Grana Padano cheese with blowing defect, and C. tyrobutyricum DSM 2637 type strain in a comparative study.

  2. Genome Sequence of the Solvent-Producing Clostridium beijerinckii Strain 59B, Isolated from Staffordshire Garden Soil

    OpenAIRE

    Little, Gareth T.; Winzer, Klaus; Minton, Nigel P.

    2015-01-01

    The genome sequence of the solvent-producing, spore-forming, saccharolytic, mesophilic bacterium Clostridium beijerinckii strain 59B, isolated from Staffordshire garden soil, was obtained via a combination of sequencing with the 454 and Illumina platforms. This information will allow for metabolic engineering of a potentially industrially useful strain.

  3. Variations in Virulence and Molecular Biology among Emerging Strains of Clostridium difficile

    Science.gov (United States)

    Hunt, Jonathan J.

    2013-01-01

    SUMMARY Clostridium difficile is a Gram-positive, spore-forming organism which infects and colonizes the large intestine, produces potent toxins, triggers inflammation, and causes significant systemic complications. Treating C. difficile infection (CDI) has always been difficult, because the disease is both caused and resolved by antibiotic treatment. For three and a half decades, C. difficile has presented a treatment challenge to clinicians, and the situation took a turn for the worse about 10 years ago. An increase in epidemic outbreaks related to CDI was first noticed around 2003, and these outbreaks correlated with a sudden increase in the mortality rate of this illness. Further studies discovered that these changes in CDI epidemiology were associated with the rapid emergence of hypervirulent strains of C. difficile, now collectively referred to as NAP1/BI/027 strains. The discovery of new epidemic strains of C. difficile has provided a unique opportunity for retrospective and prospective studies that have sought to understand how these strains have essentially replaced more historical strains as a major cause of CDI. Moreover, detailed studies on the pathogenesis of NAP1/BI/027 strains are leading to new hypotheses on how this emerging strain causes severe disease and is more commonly associated with epidemics. In this review, we provide an overview of CDI, discuss critical mechanisms of C. difficile virulence, and explain how differences in virulence-associated factors between historical and newly emerging strains might explain the hypervirulence exhibited by this pathogen during the past decade. PMID:24296572

  4. Subinhibitory concentrations of metronidazole increase biofilm formation in Clostridium difficile strains.

    Science.gov (United States)

    Vuotto, Claudia; Moura, Ines; Barbanti, Fabrizio; Donelli, Gianfranco; Spigaglia, Patrizia

    2016-03-01

    Resistance mechanism to metronidazole is still poorly understood, even if the number of reports on Clostridium difficile strains with reduced susceptibility to this antibiotic is increasing. In this study, we investigated the ability of the C. difficile strains 7032994, 7032985 and 7032989, showing different susceptibility profiles to metronidazole but all belonging to the PCR ribotype 010, to form biofilm in vitro in presence and absence of subinhibitory concentrations of metronidazole. The quantitative biofilm production assay performed in presence of metronidazole revealed a significant increase in biofilm formation in both the susceptible strain 7032994 and the strain 7032985 exhibiting a reduced susceptibility to this antibiotic, while antibiotic pressure did not affect the biofilm-forming ability of the stable-resistant strain 7032989. Moreover, confocal microscopy analysis showed an abundant biofilm matrix production by the strains 7032994 and 7032885, when grown in presence of metronidazole, but not in the stable-resistant one. These results seem to demonstrate that subinhibitory concentrations of metronidazole are able to enhance the in vitro biofilm production of the above-mentioned PCR ribotype 010 C. difficile strains, susceptible or with reduced susceptibility to this antibiotic, suggesting a possible role of biofilm formation in the multifactorial mechanism of metronidazole resistance developed by C. difficile.

  5. Systemic colonization of clover (Trifolium repens by Clostridium botulinum strain 2301

    Directory of Open Access Journals (Sweden)

    Matthias eZeiller

    2015-10-01

    Full Text Available In recent years, cases of botulism in cattle and other farm animals and also in farmers increased dramatically. It was proposed, that these cases could be affiliated with the spreading of compost or other organic manures contaminated with Clostridium botulinum spores on farm land. Thus, soils and fodder plants and finally farm animals could be contaminated. Therefore, the colonization behavior and interaction of the botulinum neurotoxin (BoNT D producing C. botulinum strain 2301 and the non-toxin producing Clostridium sporogenes strain 1739 were investigated on clover (Trifolium repens in a field experiment as well as in phytochamber experiments applying axenic and additionally soil based systems under controlled conditions. Plants were harvested and divided into root and shoot parts for further DNA isolation and PCR assays; subsamples were fixed for fluorescence in situ hybridization (FISH analysis in combination with confocal laser scanning microscopy (CLSM. To target C. botulinum and C. sporogenes, 16S rDNA directed primers were used and to specifically detect C. botulinum, BoNT D toxin genes targeted primers, using a multiplex PCR approach, were applied. Our results demonstrate an effective colonization of roots and shoots of clover by C. botulinum strain 2301 and C. sporogenes strain 1739. Detailed analysis of colonization behavior showed that C. botulinum can occur as individual cells, in cell clusters and in microcolonies within the rhizosphere, lateral roots and within the roots tissue of clover. In addition, we observed significant differences in the growth behavior of clover plants when inoculated with Clostridia spores, indicating a plant growth promoting effect. Inoculated plants showed an increased growth index (shoot size, wet and dry weight and an enlarged root system, which suggests the involvement of phytohormonal effects induced by the systemic colonization of clover by C. botulinum strain 2301.

  6. Clostridium botulinum strains producing BoNT/F4 or BoNT/F5.

    Science.gov (United States)

    Raphael, Brian H; Bradshaw, Marite; Kalb, Suzanne R; Joseph, Lavin A; Lúquez, Carolina; Barr, John R; Johnson, Eric A; Maslanka, Susan E

    2014-05-01

    Botulinum neurotoxin type F (BoNT/F) may be produced by Clostridium botulinum alone or in combination with another toxin type such as BoNT/A or BoNT/B. Type F neurotoxin gene sequences have been further classified into seven toxin subtypes. Recently, the genome sequence of one strain of C. botulinum (Af84) was shown to contain three neurotoxin genes (bont/F4, bont/F5, and bont/A2). In this study, eight strains containing bont/F4 and seven strains containing bont/F5 were examined. Culture supernatants produced by these strains were incubated with BoNT/F-specific peptide substrates. Cleavage products of these peptides were subjected to mass spectral analysis, allowing detection of the BoNT/F subtypes present in the culture supernatants. PCR analysis demonstrated that a plasmid-specific marker (PL-6) was observed only among strains containing bont/F5. Among these strains, Southern hybridization revealed the presence of an approximately 242-kb plasmid harboring bont/F5. Genome sequencing of four of these strains revealed that the genomic backgrounds of strains harboring either bont/F4 or bont/F5 are diverse. None of the strains analyzed in this study were shown to produce BoNT/F4 and BoNT/F5 simultaneously, suggesting that strain Af84 is unusual. Finally, these data support a role for the mobility of a bont/F5-carrying plasmid among strains of diverse genomic backgrounds.

  7. Antibacterial activity against Clostridium genus and antiradical activity of the essential oils from different origin.

    Science.gov (United States)

    Kačániová, Miroslava; Vukovič, Nenad; Horská, Elena; Salamon, Ivan; Bobková, Alica; Hleba, Lukáš; Fiskelová, Martina; Vatľák, Alexander; Petrová, Jana; Bobko, Marek

    2014-01-01

    In the present study, the antimicrobial and antiradical activities of 15 essential oils were investigated. The antimicrobial activities were determined by using agar disc diffusion and broth microdilution methods against Clostridium genus and antioxidant properties of essential oils by testing their scavenging effect on DPPH radicals activities. We determined the antibacterial activity of Clostridium butyricum, Clostridium hystoliticum, Clostridium intestinale, Clostridium perfringens and Clostridium ramosum. We obtained the original commercial essential oils samples of Lavandula angustifolia, Carum carvi, Pinus montana, Mentha piperita, Foeniculum vulgare Mill., Pinus sylvestris, Satureia montana, Origanum vulgare L. (2 samples), Pimpinella anisum, Rosmarinus officinalis L., Salvia officinalis L., Abies alba Mill., Chamomilla recutita L. Rausch and Thymus vulgaris L. produced in Slovakia (Calendula a.s., Nova Lubovna, Slovakia). The results of the disk diffusion method showed very high essential oils activity against all tested strains of microorganisms. The best antimicrobial activity against C. butyricum was found at Pimpinella anisum, against C. hystoliticum was found at Pinus sylvestris, against C. intestinale was found at Satureia hortensis L., against C. perfringens was found at Origanum vulgare L. and against C. ramosum was found at Pinus sylvestris. The results of broth microdilution assay showed that none of the essential oils was active against C. hystoliticum. The best antimicrobial activity against C. butyricum was found at Abies alba Mill., against C. intestinale was found at Abies alba Mill., against C. perfringens was found at Satureia montana and against C. ramosum was found at Abius alba and Carum carvi. Antioxidant DPPH radical scavenging activity was determined at several solutions of oil samples (50 μL.mL(-1)-0.39 μL.mL(-1)) and the best scavenging effect for the highest concentration (50 μL.mL(-1)) was observed. The antioxidant properties

  8. Meta-analysis of D-values of proteolytic Clostridium botulinum and its surrogate strain Clostridium sporogenes PA 3679.

    Science.gov (United States)

    Diao, Mamadou Moctar; André, Stéphane; Membré, Jeanne-Marie

    2014-03-17

    Foodborne botulism is a serious disease resulting from ingestion of preformed Clostridium botulinum neurotoxin in foodstuff. Since the 19th century, the heat resistance of this spore forming bacteria has been extensively studied in order to guarantee the public health associated with low acidic, ambient stable products. The most largely used heat resistance parameters in thermal settings of such products are the D121.1°C values (time required to have a 10-fold decrease of the spore count, at 121.1°C) and the z-values (temperature increase to have a 10-fold decrease of D-values). To determine D121.1°C and z-values of proteolytic C. botulinum and its nontoxigenic surrogate strain C. sporogenes PA3679, a dataset of 911 D-values was collected from 38 scientific studies. Within a meta-analysis framework, a mixed-effect linear model was developed with the log D-value (min) as response and the heat treatment temperature as explicative variable. The studies (38), the C. botulinum strains (11), and the heat treatment media (liquid media and various food matrices, split into nine categories in total) were considered as co-variables having a random effect. The species (C. botulinum and C. sporogenes) and the pH (five categories) were considered as co-variables having a fixed effect. Overall, the model gave satisfactory results with a residual standard deviation of 0.22. The heat resistance of proteolytic C. botulinum was found significantly lower than the C. sporogenes PA 3679 one: the mean D-values at the reference temperature of 121.1°C, in liquid media and pH neutral, were estimated to 0.19 and 1.28min for C. botulinum and C. sporogenes, respectively. On the other hand, the mean z-values of the two species were similar: 11.3 and 11.1°C for C. botulinum and C. sporogenes, respectively. These results will be applied to thermal settings of low-acid ambient stable products. Copyright © 2014. Published by Elsevier B.V.

  9. Microbial Enhanced Oil Recovery-Laboratory Experiments with a Strain of Clostridium tyrobutyricum

    DEFF Research Database (Denmark)

    Jimoh, Ismaila Adetunji

    It is well known that each microbial enhanced oil recovery (MEOR) method has its own selection criteria in order to match different reservoir conditions. Therefore successful application of MEOR method depends on the ability of microbes to adapt to extreme reservoir conditions and to produce...... that the strain of Clostridium tyrobutyricum adapted to 10, 30, 50, and 90 g/l before the start of the experiments produce more gas with an increase factor of between 0.39-6.9 for the same salinity condition than the pure culture. The adaptation process also led to the production of a strain 90F which can grow...... of the tubes. The mean porosity of carbonate rock samples increase from 42% to 48% when treated in microbial media leading to increase void ratio and the release of calcium ions; conditions that are favorable for biofilms formation in porous rock. These experimental results suggested that adaptation...

  10. Direct bioconversion of raw corn stalk to hydrogen by a new strain Clostridium sp. FS3.

    Science.gov (United States)

    Song, Zhao-Xia; Li, Xiao-Hu; Li, Wei-Wei; Bai, Yan-Xia; Fan, Yao-Ting; Hou, Hong-Wei

    2014-04-01

    A new strain FS3 which could achieve an efficient bioconversion of raw corn stalk to hydrogen had been isolated from anaerobic acclimated sludge, and identified as Clostridium butyricum on the basis of a series of physiological and biochemical experiments and 16S rDNA gene sequence. The strain could utilize various carbon sources to produce hydrogen. On the basis of single-factor experiments, the response surface methodology (RSM) was performed to optimize the media for hydrogen production. The maximum hydrogen yield of 92.9ml/g was observed under the optimal conditions: 20g/l raw corn stalk, 1.76g/l NH4HCO3, 0.91g/l KH2PO4 and 10.4ml/l nutrient solution. This finding opens a new avenue for direct conversion of raw cellulosic biomass to bio-hydrogen.

  11. Spread and Pathogenic Mechanism of Clostridium perfringens%产气荚膜梭状芽孢杆菌病的流行与致病机制

    Institute of Scientific and Technical Information of China (English)

    柴同杰; 马瑞华; 常维山; 张绍学

    2001-01-01

    产气荚膜梭状芽孢杆菌是人和动物肠道的正常菌群,亦是条件性致病菌。该菌感染主要由毒素导致的毒血症致病。因此,有针对地选用类毒素预防接种,才能防止本病流行。%Clostridium perffringens belong to normal bacteria in intestine in both man and animal.They are also conditional pathogenic. bacteria and can cause disease through their toxin,e.g. toxaemia.Therefore,using toxoid to make preventive inoculation can avoid spread of this infectious disease.

  12. A LITHOTROPHIC CLOSTRIDIUM STRAIN WITH EXTREMELY THERMORESISTANT SPORES ISOLATED FROM A PECTIN-LIMITED CONTINUOUS CULTURE OF CLOSTRIDIUM-THERMOSACCHAROLYTICUM STRAIN HAREN

    NARCIS (Netherlands)

    VANRIJSSEL, M; VANDERVEEN, [No Value; HANSEN, TA

    1992-01-01

    A thermophilic Clostridium sp. with extremely thermoresistant spores was isolated from a pectin-limited continuous culture of Clostridium thermosaccharolyticum. The decimal reduction time of the spores was 70 min at 121-degrees-C. Because of the ability of the bacterium to grow both heterotrophicall

  13. Intoxicación alimentaria por clostridium perfringens en el centro penitenciario cocori, cartago, costa rica, del 4 al 5 de septiembre del 2002

    Directory of Open Access Journals (Sweden)

    A Espinoza

    2004-07-01

    Full Text Available Introducción El 5 de setiembre del 2002 el Centro Penitenciario Cocorí en Cartago, notificó al Sistema de Vigilancia del Ministerio de Salud, la presencia de 133 casos de diarrea. Ante esta situación, las autoridades de salud plantearon la investigación, para identificar los factores asociados a la presencia del brote. Material y métodos Mediante la revisión de los expedientes de los casos atendidos por diarrea en el Centro Penitenciario, se identificaron los que cumplieran con la siguiente definición: persona con deposiciones líquidas que podían acompañarse de náuseas, vómitos, dolor de cabeza, dolor abdominal, tenesmo y meteorismo, y que consumieron alimentos en el Centro Penitenciario el 4 de setiembre del 2002. Para determinar el mecanismo de transmisión, comparamos la exposición de los alimentos servidos durante el desayuno, almuerzo y cena del 4 de setiembre, de los casos con 41 prisioneros no enfermos. Se aislaron enteropatógenos, de muestras del pollo servidos y de heces de los casos seleccionados al azar. Resultados Se identificaron 133 casos ocurridos en el centro penitenciario. El primer caso ocurrió 4:30 horas después de servido el almuerzo. El 100% presentó diarrea, ninguno tuvo fiebre, ni recibió antibióticos. Las personas que consumieron pollo en salsa presentaron un RR = 2,92 (95% CI: 1,217,04. El laboratorio aisló Cl. perfringens en una concentración de 10 5 u.f.c. por gramo de heces y 10 4 u.f.c. por gramo de carne de pollo. Conclusiones La presencia de CI. perfringes en el pollo y heces de los casos demuestran la transmisión del agente en la población del Centro Penitenciario (fuente común.

  14. Identification and genetic characterization of Clostridium botulinum serotype A strains from commercially pasteurized carrot juice.

    Science.gov (United States)

    Marshall, Kristin M; Nowaczyk, Louis; Raphael, Brian H; Skinner, Guy E; Rukma Reddy, N

    2014-12-01

    Clostridium botulinum is an important foodborne pathogen capable of forming heat resistant endospores and producing deadly botulinum neurotoxins (BoNTs). In 2006, C. botulinum was responsible for an international outbreak of botulism attributed to the consumption of commercially pasteurized carrot juice. The purpose of this study was to isolate and characterize strains of C. botulinum from the adulterated product. Carrot juice bottles retrieved from the manufacturing facility were analyzed for the presence of BoNT and BoNT-producing isolates using DIG-ELISA. Toxigenic isolates from the carrot juice were analyzed using pulsed-field gel electrophoresis (PFGE) and DNA microarray analysis to determine their genetic relatedness to the original outbreak strains CDC51348 and CDC51303. PFGE revealed that isolates CJ4-1 and CJ10-1 shared an identical pulsotype with strain CDC51303, whereas isolate CJ5-1 displayed a unique restriction banding pattern. DNA microarray analysis identified several phage related genes unique to strain CJ5-1, and Southern hybridization analysis of XhoI digested and nondigested DNA showed their chromosomal location, while a homolog to pCLI_A009 of plasmid pCLI of C. botulinum serotype Langeland F, was located on a small plasmid. The acquisition or loss of bacteriophages and other mobile genetic elements among C. botulinum strains has epidemiological and evolutionary implications.

  15. Butyric acid fermentation from pretreated and hydrolysed wheat straw by an adapted Clostridium tyrobutyricum strain.

    Science.gov (United States)

    Baroi, G N; Baumann, I; Westermann, P; Gavala, H N

    2015-09-01

    Butyric acid is a valuable building-block for the production of chemicals and materials and nowadays it is produced exclusively from petroleum. The aim of this study was to develop a suitable and robust strain of Clostridium tyrobutyricum that produces butyric acid at a high yield and selectivity from lignocellulosic biomasses. Pretreated (by wet explosion) and enzymatically hydrolysed wheat straw (PHWS), rich in C6 and C5 sugars (71.6 and 55.4 g l(-1) of glucose and xylose respectively), was used as substrate. After one year of serial selections, an adapted strain of C. tyrobutyricum was developed. The adapted strain was able to grow in 80% (v v(-1) ) PHWS without addition of yeast extract compared with an initial tolerance to less than 10% PHWS and was able to ferment both glucose and xylose. It is noticeable that the adapted C. tyrobutyricum strain was characterized by a high yield and selectivity to butyric acid. Specifically, the butyric acid yield at 60-80% PHWS lie between 0.37 and 0.46 g g(-1) of sugar, while the selectivity for butyric acid was as high as 0.9-1.0 g g(-1) of acid. Moreover, the strain exhibited a robust response in regards to growth and product profile at pH 6 and 7.

  16. Comparison of mutant and parent strains of Clostridium acetobutylicum: butyrate uptake at different temperatures

    Energy Technology Data Exchange (ETDEWEB)

    Soni, B.K. [Institute of Gas Technology, 1700 S. Mount Prospect Road, Des Plaines, IL 60018 (United States); Jain, M.K. [MBI International, P.O. Box 27609, 3900 Collins Road, Lansing, MI 48909 (United States)

    1997-10-01

    A high butanol producing mutant strain of Clostridium acetobutylicum ATCC 4259 was obtained by chemical mutagenesis. Both mutant and parent strains were evaluated for butyrate uptake using the culture effluents of solventogenic fermentor-2 of the two-fermentor continuous system. Batch incubation of fermentor-2 culture effluents at 37 C indicated lower butyrate uptake rates for mutant and parent strain, at 0.05 and 0.03 g l{sup -1} h{sup -1}, respectively. Increased butyrate uptake rates of 0.33 and 0.26 g l{sup -1} h{sup -1} for mutant and parent strain, respectively, were observed when effluents were batch incubated at lower temperature of 30 C. Butyrate conversion efficiency, at 5 {+-} 0.1 g l{sup -1} of externally added butyrate, were 98.8% and 96.9% for mutant and parent strain, respectively. Butyrate up to the externally added concentration of 11.4 g l{sup -1} did not inhibit butyrate uptake. The maximum butyrate consumption at a slightly reduced uptake rate was seen at 10.2 g l{sup -1} butyrate concentration at 27 C. Based on the results under different temperatures, the electron flow pattern has been computed and the mechanism for butyrate uptake has been hypothesized. (orig.) With 6 figs., 4 tabs., 26 refs.

  17. Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese

    OpenAIRE

    2015-01-01

    Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively.

  18. Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

    Science.gov (United States)

    Storari, Michelangelo; Wüthrich, Daniel; Bruggmann, Rémy; Berthoud, Hélène; Arias-Roth, Emmanuelle

    2015-03-12

    Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively.

  19. Draft Genome Sequences of Clostridium tyrobutyricum Strains FAM22552 and FAM22553, Isolated from Swiss Semihard Red-Smear Cheese.

    OpenAIRE

    2015-01-01

    Clostridium tyrobutyricum is the main microorganism responsible for late blowing defect in cheeses. Here, we present the draft genome sequences of two C. tyrobutyricum strains isolated from a Swiss semihard red-smear cheese. The two draft genomes comprise 3.05 and 3.08 Mbp and contain 3,030 and 3,089 putative coding sequences, respectively.

  20. Analysing the dhaT gene in Colombian Clostridium sp. (Clostridia 1,3-propanediol-producing strains

    Directory of Open Access Journals (Sweden)

    Diana Milena Quilaguy-Ayure

    2010-04-01

    Full Text Available To analyze the dhaT gene, one of the genes responsible for the 1,3-propanediol (1,3-PD production, in two native Clostridiumstrains. Materials and methods: The dhaT gene was amplified by Polimerase Chain Reaction with specific primers designed fromClostridium butyricum VPI1718 operon. Bioinformatics tools like BLASTN, ORF finder, BLASTP and ClustalW were used to determinethe identity of the sequence and to assign a function. Results: DNA amplification products were obtained from Colombian Clostridium sp.native strains (IBUN 13A and IBUN 158B and the Clostridium butyricum DSM 2478 strain, which were sequenced. According to thebioinformatics analysis of the above sequences, a high degree of similarity was found with the dhaT gene of different bacterial species. Thehighest percentage of identity was obtained with the Clostridium butyricum VPI 1718 strain. Conclusion: knowledge of the physicalstructure of the 1,3-PD operon in native strains opens the way for developing genetic and metabolic engineering strategies for improvingprocesses productivity.

  1. Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains

    NARCIS (Netherlands)

    Collas, Florent; Kuit, Wouter; Clément, Benjamin; Marchal, Rémy; López-contreras, Ana M.; Monot, Frederic

    2012-01-01

    Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase

  2. Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains

    NARCIS (Netherlands)

    Collas, Florent; Kuit, Wouter; Clément, Benjamin; Marchal, Rémy; López-contreras, Ana M.; Monot, Frederic

    2012-01-01

    Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase

  3. Genomic Fingerprinting of the Vaccine Strain of Clostridium Tetani by Restriction Fragment Length Polymorphism Technique

    Directory of Open Access Journals (Sweden)

    Naser Harzandi

    2013-05-01

    Full Text Available Background: Clostridium tetani or Nicolaier’s bacillus is an obligatory anaerobic, Gram-positive, movable with terminal or sub terminal spore. The chromosome of C. tetani contains 2,799,250 bp with a G+C content of 28.6%. The aim of this study was identification and genomic fingerprinting of the vaccine strain of C. tetani.Materials and Methods: The vaccine strain of C. tetani was provided by Razi Vaccine and Serum Research Institute. The seeds were inoculated into Columbia blood agar and grown for 72 h and transferred to the thioglycolate broth medium for further 36 h culturing. The cultures were incubated at 35ºC in anaerobic conditions. DNA extraction with phenol/ chloroform method was performed. After extraction, the consistency of DNA was assayed. Next, the vaccine strain was digested using pvuII enzyme and incubated at 37ºC for overnight. The digested DNA was gel-electrophoresed by 1% agarose for a short time. Then, the gel was studied with Gel Doc system and transferred to Hybond N+membrane using standard DNA blotting techniques.Results: The vaccine strain of C. tetani genome was fingerprinted by RFLP technique. Our preliminary results showed no divergence exists in the vaccine strain used for the production tetanus toxoid during the periods of 1990-2011.Conclusion: Observation suggests that there is lack of significant changes in RFLP genomic fingerprinting profile of the vaccine strain. Therefore, this strain did not lose its efficiency in tetanus vaccine production. RFLP analysis is worthwhile in investigating the nature of the vaccine strain C. tetani.

  4. Isolation,Identification and Drug Sensitivity Test of Clostridium perfringens from Broiler Chickens%鸡产气荚膜梭菌的分离鉴定及药敏试验

    Institute of Scientific and Technical Information of China (English)

    艾地云; 温国元; 邵华斌; 张腾飞; 詹丽超; 罗玲; 张蓉蓉; 汪宏才; 王红琳; 罗青平

    2014-01-01

    For investigating the prevalence and genetypes of C.perfringens in healthy chickens in Hubei province,120 anus cotton swab samples were collected from large-scale broiler chicken farms in Hubei. After culturing in anaerobic meat liver soup and CW agar culture-medium,9 suspected C.perfringens were isolated and then identified with purple milk test,Gram stain test and biochemical test.The isolation rate was 7.5 %.9 strains were genotyped as type A by PCR.These strains were tested for susceptibility to common antimicrobials.All of the isolates were resistant to streptomycin,neomycin,ciprofloracin,cip-rofloxacin,and were sensitive to ampicillin,amoxicillin,cefotaxime,tetracycline,amikacin and clindamy-cin.%为调查湖北地区健康鸡群中产气荚膜梭菌的分布状况,从规模化养鸡场采集鸡肛门棉拭子样品,应用厌气肉肝汤和 CW 琼脂培养基培养、紫牛乳反应、革兰染色及生化试验进行细菌的分离鉴定,应用多重 PCR 方法对分离菌株进行基因分型,并对分离菌株进行体外药敏试验。结果从120份样品中分离到9株(7.5%)产气荚膜梭菌,多重 PCR 都只扩增出α条带,毒素基因分型结果均为 A 型。药敏试验结果显示,分离菌株对链霉素、新霉素、丙氟哌酸、环丙沙星耐药,对丁胺卡那霉素,氯洁霉素中度敏感,对氨苄西林、阿莫西林、四环素、头孢噻肟高度敏感。

  5. Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion.

    Science.gov (United States)

    Fraga, Edmir Geraldo; Nicodemo, Antonio Carlos; Sampaio, Jorge Luiz Mello

    2016-01-01

    Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125μg/mL for both antimicrobials. The MIC90 were 4μg/mL and 2μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

  6. Isolation and characterization of a new cellulosome-producing Clostridium thermocellum strain.

    Science.gov (United States)

    Tachaapaikoon, Chakrit; Kosugi, Akihiko; Pason, Patthra; Waeonukul, Rattiya; Ratanakhanokchai, Khanok; Kyu, Khin Lay; Arai, Takamitsu; Murata, Yoshinori; Mori, Yutaka

    2012-02-01

    The anaerobic thermophilic bacterium, Clostridium thermocellum, is a potent cellulolytic microorganism that produces large extracellular multienzyme complexes called cellulosomes. To isolate C. thermocellum organisms that possess effective cellulose-degrading ability, new thermophilic cellulolytic strains were screened from more than 800 samples obtained mainly from agriculture residues in Thailand using microcrystalline cellulose as a carbon source. A new strain, C. thermocellum S14, having high cellulose-degrading ability was isolated from bagasse paper sludge. Cellulosomes prepared from S14 demonstrated faster degradation of microcrystalline cellulose, and 3.4- and 5.6-fold greater Avicelase activity than those from C. thermocellum ATCC27405 and JW20 (ATCC31449), respectively. Scanning electron microscopic analysis showed that S14 had unique cell surface features with few protuberances in contrast to the type strains. In addition, the cellulosome of S14 was resistant to inhibition by cellobiose that is a major end product of cellulose hydrolysis. Saccharification tests conducted using rice straw soaked with sodium hydroxide indicated the cellulosome of S14 released approximately 1.5-fold more total sugars compared to that of ATCC27405. This newly isolated S14 strain has the potential as an enzyme resource for effective lignocellulose degradation.

  7. Physiology, Genomics, and Pathway Engineering of an Ethanol-Tolerant Strain of Clostridium phytofermentans.

    Science.gov (United States)

    Tolonen, Andrew C; Zuroff, Trevor R; Ramya, Mohandass; Boutard, Magali; Cerisy, Tristan; Curtis, Wayne R

    2015-08-15

    Novel processing strategies for hydrolysis and fermentation of lignocellulosic biomass in a single reactor offer large potential cost savings for production of biocommodities and biofuels. One critical challenge is retaining high enzyme production in the presence of elevated product titers. Toward this goal, the cellulolytic, ethanol-producing bacterium Clostridium phytofermentans was adapted to increased ethanol concentrations. The resulting ethanol-tolerant (ET) strain has nearly doubled ethanol tolerance relative to the wild-type level but also reduced ethanol yield and growth at low ethanol concentrations. The genome of the ET strain has coding changes in proteins involved in membrane biosynthesis, the Rnf complex, cation homeostasis, gene regulation, and ethanol production. In particular, purification of the mutant bifunctional acetaldehyde coenzyme A (CoA)/alcohol dehydrogenase showed that a G609D variant abolished its activities, including ethanol formation. Heterologous expression of Zymomonas mobilis pyruvate decarboxylase and alcohol dehydrogenase in the ET strain increased cellulose consumption and restored ethanol production, demonstrating how metabolic engineering can be used to overcome disadvantageous mutations incurred during adaptation to ethanol. We discuss how genetic changes in the ET strain reveal novel potential strategies for improving microbial solvent tolerance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Mechanisms of microbial oil recovery by Clostridium acetobutylicum and Bacillus strain JF-2

    Energy Technology Data Exchange (ETDEWEB)

    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.

  9. Antimicrobial susceptibility of Brazilian Clostridium difficile strains determined by agar dilution and disk diffusion

    Directory of Open Access Journals (Sweden)

    Edmir Geraldo Fraga

    Full Text Available Abstract Clostridium difficile is a leading cause of diarrhea in hospitalized patients worldwide. While metronidazole and vancomycin are the most prescribed antibiotics for the treatment of this infection, teicoplanin, tigecycline and nitazoxanide are alternatives drugs. Knowledge on the antibiotic susceptibility profiles is a basic step to differentiate recurrence from treatment failure due to antimicrobial resistance. Because C. difficile antimicrobial susceptibility is largely unknown in Brazil, we aimed to determine the profile of C. difficile strains cultivated from stool samples of inpatients with diarrhea and a positive toxin A/B test using both agar dilution and disk diffusion methods. All 50 strains tested were sensitive to metronidazole according to CLSI and EUCAST breakpoints with an MIC90 value of 2 μg/mL. Nitazoxanide and tigecycline were highly active in vitro against these strains with an MIC90 value of 0.125 μg/mL for both antimicrobials. The MIC90 were 4 μg/mL and 2 μg/mL for vancomycin and teicoplanin, respectively. A resistance rate of 8% was observed for moxifloxacin. Disk diffusion can be used as an alternative to screen for moxifloxacin resistance, nitazoxanide, tigecycline and metronidazole susceptibility, but it cannot be used for testing glycopeptides. Our results suggest that C. difficile strains from São Paulo city, Brazil, are susceptible to metronidazole and have low MIC90 values for most of the current therapeutic options available in Brazil.

  10. DETECTION, RIBOTYPING AND ANTIMICROBIAL RESISTANCE PROPERTIES OF CLOSTRIDIUM DIFFICILE STRAINS ISOLATED FROM THE CASES OF DIARRHEA

    Science.gov (United States)

    Kouzegaran, Samaneh; Ganjifard, Mahmood; Tanha, Amir Saber

    2016-01-01

    Background: Clostridium difficile is the most prevalent cause of antibiotic-associated infectious diarrhea al-around the world. Prevalence of virulent and resistant strains of Clostridium difficile is increasing now a day. The present investigation was carried out to study the prevalence, ribotyping and antibiotic resistance pattern of C. difficile isolated from diarrheic and non-diarrheic pediatrics. Materials and methods: Four-hundred stool specimens were collected from the diarrheic and non-diarrheic pediatrics hospitalized due to the diseases other than diarrhea. Samples were cultured and their positive results were subjected to disk diffusion and PCR-based ribotyping. Results: Thirty-five out of 400 (8.75%) samples were positive for C. difficile. Prevalence of C. difficile in diarrheic and non-diarrheic pediatrics were 11.25% and 4.16%, respectively. Male had the higher prevalence of bacteria than female (P < 0.05). eight to twelve months old pediatrics were the most commonly infected group. R27 (14.28%), R1 (10.71%), R12 (7.14%), R13 (7.14%) and R18 (7.14%) were most commonly detected ribotypes. There were no positive results for studied ribotypes in non-diarrheic pediatrics. C. difficile strains had the highest levels of resistance against tetracycline (71.42%), erythromycin (57.14%), moxifloxacin (48.57%), metronidazole (28.57%) and clindamycin (22.85%) antibiotics. Conclusion: Prescription of antibiotics in diarrheic pediatrics, males and also 8-12 months old pediatrics should be done in a regular and cautious manner. PMID:27999477

  11. 产气荚膜梭菌α毒素突变体构建及其卵黄抗体制备%Construction of Clostridium perfringens alpha toxin mutant and preparation of egg yolk IgY antibody

    Institute of Scientific and Technical Information of China (English)

    李箐; 辛文文; 康琳; 高姗; 王景林

    2013-01-01

    目的 制备特异性的产气荚膜梭菌α毒素(Clostridium perfringens alpha-toxin,CPA)卵黄抗体(yolk immunoglobulin,IgY).方法 利用定点突变技术,将CPA第56位天冬氨酸和第68位组氨酸分别突变为丝氨酸,构建了重组表达载体pTIG-mCPAD56S和pTIG-mCPAH68S,将其转化入E.coli Origami中进行诱导表达.用亲和层析的方法对突变体蛋白进行纯化并对其活性及抗原性进行检测,将获得的突变体蛋白分别免疫健康母鸡,收集鸡蛋;经水稀释法纯化卵黄中IgY;用酶联免疫吸附试验检测IgY效价;通过筛选保护剂而选择最佳的IgY冻干条件.结果 与结论 pTIG-mCPA在Origami表达菌株中得到高效表达,经验证,mCPAD56S和mCPAH68S均完全失去生物学活性同时保留抗原性;纯化卵黄后得到较高纯度的特异性IgY,其效价可达到1∶200 000;经过IgY冻干条件的筛选,最终选择不添加保护剂大量冻干IgY作为抗体的储备.该研究为研制基于IgY抗体的检测方法奠定了基础.

  12. Impacts of infection with different toxigenic Clostridium difficile strains on faecal microbiota in children

    Science.gov (United States)

    Ling, Zongxin; Liu, Xia; Jia, Xiaoyun; Cheng, Yiwen; Luo, Yueqiu; Yuan, Li; Wang, Yuezhu; Zhao, Chunna; Guo, Shu; Li, Lanjuan; Xu, Xiwei; Xiang, Charlie

    2014-12-01

    Increasing evidence suggests that altered intestinal microbial composition and function result in an increased risk of Clostridium difficile-associated diarrhoea (CDAD); however, the specific changes of intestinal microbiota in children suffering from CDAD and their associations with C. difficile strain toxigenicity are poorly understood. High-throughput pyrosequencing showed that reduced faecal bacterial diversity and dramatic shifts of microbial composition were found in children with CDAD. The Firmicutes/Bacteroidetes ratio was increased significantly in patients with CDAD, which indicated that dysbiosis of faecal microbiota was closely associated with CDAD. C. difficile infection resulted in an increase in lactate-producing phylotypes, with a corresponding decrease in butyrate-producing bacteria. The decrease in butyrate and lactate buildup impaired intestinal colonisation resistance, which increased the susceptibility to C. difficile colonisation. Strains of C. difficile which were positive for both toxin A and toxin B reduced faecal bacterial diversity to a greater degree than strains that were only toxin B-positive, and were associated with unusually abundant Enterococcus, which implies that the C. difficile toxins have different impacts on the faecal microbiota of children. Greater understanding of the relationships between disruption of the normal faecal microbiota and colonisation with C. difficile that produces different toxins might lead to improved treatment.

  13. Converting Carbon Dioxide to Butyrate with an Engineered Strain of Clostridium ljungdahlii

    Energy Technology Data Exchange (ETDEWEB)

    Ueki, T; Nevin, KP; Woodard, TL; Lovley, DR

    2014-08-26

    Microbial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogen Clostridium ljungdahlii has suggested that C. ljungdahlii may be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product of C. ljungdahlii metabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into the C. ljungdahlii chromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70% of the carbon and electron flow was diverted to the production of butyrate with H-2 or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide with C. ljungdahlii as the catalyst. IMPORTANCE The development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities. Clostridium ljungdahlii is an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors

  14. Converting carbon dioxide to butyrate with an engineered strain of Clostridium ljungdahlii.

    Science.gov (United States)

    Ueki, Toshiyuki; Nevin, Kelly P; Woodard, Trevor L; Lovley, Derek R

    2014-10-21

    Microbial conversion of carbon dioxide to organic commodities via syngas metabolism or microbial electrosynthesis is an attractive option for production of renewable biocommodities. The recent development of an initial genetic toolbox for the acetogen Clostridium ljungdahlii has suggested that C. ljungdahlii may be an effective chassis for such conversions. This possibility was evaluated by engineering a strain to produce butyrate, a valuable commodity that is not a natural product of C. ljungdahlii metabolism. Heterologous genes required for butyrate production from acetyl-coenzyme A (CoA) were identified and introduced initially on plasmids and in subsequent strain designs integrated into the C. ljungdahlii chromosome. Iterative strain designs involved increasing translation of a key enzyme by modifying a ribosome binding site, inactivating the gene encoding the first step in the conversion of acetyl-CoA to acetate, disrupting the gene which encodes the primary bifunctional aldehyde/alcohol dehydrogenase for ethanol production, and interrupting the gene for a CoA transferase that potentially represented an alternative route for the production of acetate. These modifications yielded a strain in which ca. 50 or 70% of the carbon and electron flow was diverted to the production of butyrate with H2 or CO as the electron donor, respectively. These results demonstrate the possibility of producing high-value commodities from carbon dioxide with C. ljungdahlii as the catalyst. Importance: The development of a microbial chassis for efficient conversion of carbon dioxide directly to desired organic products would greatly advance the environmentally sustainable production of biofuels and other commodities. Clostridium ljungdahlii is an effective catalyst for microbial electrosynthesis, a technology in which electricity generated with renewable technologies, such as solar or wind, powers the conversion of carbon dioxide and water to organic products. Other electron donors

  15. Flagellin Diversity in Clostridium botulinum Groups I and II: a New Strategy for Strain Identification▿

    Science.gov (United States)

    Paul, Catherine J.; Twine, Susan M.; Tam, Kevin J.; Mullen, James A.; Kelly, John F.; Austin, John W.; Logan, Susan M.

    2007-01-01

    Strains of Clostridium botulinum are traditionally identified by botulinum neurotoxin type; however, identification of an additional target for typing would improve differentiation. Isolation of flagellar filaments and analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) showed that C. botulinum produced multiple flagellin proteins. Nano-liquid chromatography-tandem mass spectrometry (nLC-MS/MS) analysis of in-gel tryptic digests identified peptides in all flagellin bands that matched two homologous tandem flagellin genes identified in the C. botulinum Hall A genome. Designated flaA1 and flaA2, these open reading frames encode the major structural flagellins of C. botulinum. Colony PCR and sequencing of flaA1/A2 variable regions classified 80 environmental and clinical strains into group I or group II and clustered isolates into 12 flagellar types. Flagellar type was distinct from neurotoxin type, and epidemiologically related isolates clustered together. Sequencing a larger PCR product, obtained during amplification of flaA1/A2 from type E strain Bennett identified a second flagellin gene, flaB. LC-MS analysis confirmed that flaB encoded a large type E-specific flagellin protein, and the predicted molecular mass for FlaB matched that observed by SDS-PAGE. In contrast, the molecular mass of FlaA was 2 to 12 kDa larger than the mass predicted by the flaA1/A2 sequence of a given strain, suggesting that FlaA is posttranslationally modified. While identification of FlaB, and the observation by SDS-PAGE of different masses of the FlaA proteins, showed the flagellin proteins of C. botulinum to be diverse, the presence of the flaA1/A2 gene in all strains examined facilitates single locus sequence typing of C. botulinum using the flagellin variable region. PMID:17351097

  16. Structure and genetic content of the megaplasmids of neurotoxigenic clostridium butyricum type E strains from Italy.

    Science.gov (United States)

    Iacobino, Angelo; Scalfaro, Concetta; Franciosa, Giovanna

    2013-01-01

    We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated (cas) genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain or to lose the

  17. Structure and genetic content of the megaplasmids of neurotoxigenic clostridium butyricum type E strains from Italy.

    Directory of Open Access Journals (Sweden)

    Angelo Iacobino

    Full Text Available We determined the genetic maps of the megaplasmids of six neutoroxigenic Clostridium butyricum type E strains from Italy using molecular and bioinformatics techniques. The megaplasmids are circular, not linear as we had previously proposed. The differently-sized megaplasmids share a genetic region that includes structural, metabolic and regulatory genes. In addition, we found that a 168 kb genetic region is present only in the larger megaplasmids of two tested strains, whereas it is absent from the smaller megaplasmids of the four remaining strains. The genetic region unique to the larger megaplasmids contains, among other features, a locus for clustered regularly interspaced short palindromic repeats (CRISPR and CRISPR associated (cas genes, i.e. a bacterial adaptive immune system providing sequence-specific protection from invading genetic elements. Some CRISPR spacer sequences of the neurotoxigenic C. butyricum type E strains showed homology to prophage, phage and plasmid sequences from closely related clostridia species or from distant species, all sharing the intestinal habitat, suggesting that the CRISPR locus might be involved in the microorganism adaptation to the human or animal intestinal environment. Besides, we report here that each of four distinct CRISPR spacers partially matched DNA sequences of different prophages and phages, at identical nucleotide locations. This suggests that, at least in neurotoxigenic C. butyricum type E, the CRISPR locus is potentially able to recognize the same conserved DNA sequence of different invading genetic elements, besides targeting sequences unique to previously encountered invading DNA, as currently predicted for a CRISPR locus. Thus, the results of this study introduce the possibility that CRISPR loci can provide resistance to a wider range of invading DNA elements than previously appreciated. Whether it is more advantageous for the peculiar neurotoxigenic C. butyricum type E strains to maintain

  18. Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile.

    Directory of Open Access Journals (Sweden)

    Kate E Mackin

    Full Text Available Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.

  19. Spo0A differentially regulates toxin production in evolutionarily diverse strains of Clostridium difficile.

    Science.gov (United States)

    Mackin, Kate E; Carter, Glen P; Howarth, Pauline; Rood, Julian I; Lyras, Dena

    2013-01-01

    Clostridium difficile is an important pathogen of humans and animals, representing a significant global healthcare problem. The last decade has seen the emergence of epidemic BI/NAP1/027 and ribotype 078 isolates, associated with the onset of more severe disease and higher rates of morbidity and mortality. However, little is known about these isolates at the molecular level, partly due to difficulties in the genetic manipulation of these strains. Here we report the development of an optimised Tn916-mediated plasmid transfer system, and the use of this system to construct and complement spo0A mutants in a number of different C. difficile strain backgrounds. Spo0A is a global regulator known to control sporulation, but may also be involved in the regulation of potential virulence factors and other phenotypes. Recent studies have failed to elucidate the role of Spo0A in toxin A and toxin B production by C. difficile, with conflicting data published to date. In this study, we aimed to clarify the role of Spo0A in production of the major toxins by C. difficile. Through the construction and complementation of spo0A mutants in two ribotype 027 isolates, we demonstrate that Spo0A acts as a negative regulator of toxin A and toxin B production in this strain background. In addition, spo0A was disrupted and subsequently complemented in strain 630Δerm and, for the first time, in a ribotype 078 isolate, JGS6133. In contrast to the ribotype 027 strains, Spo0A does not appear to regulate toxin production in strain 630Δerm. In strain JGS6133, Spo0A appears to negatively regulate toxin production during early stationary phase, but has little effect on toxin expression during late stationary phase. These data suggest that Spo0A may differentially regulate toxin production in phylogenetically distinct C. difficile strain types. In addition, Spo0A may be involved in regulating some aspects of C. difficile motility.

  20. Butyric acid from anaerobic fermentation of lignocellulosic biomass hydrolysates by Clostridium tyrobutyricum strain RPT-4213.

    Science.gov (United States)

    Liu, Siqing; Bischoff, Kenneth M; Leathers, Timothy D; Qureshi, Nasib; Rich, Joseph O; Hughes, Stephen R

    2013-09-01

    A novel Clostridium tyrobutyricum strain RPT-4213 was found producing butyrate under strict anaerobic conditions. This strain produced 9.47 g L(-1) butyric acid from MRS media (0.48 g/g glucose). RPT-4213 was also used to ferment dilute acid pretreated hydrolysates including wheat straw (WSH), corn fiber (CFH), corn stover (CSH), rice hull (RHH), and switchgrass (SGH). Results indicated that 50% WSH with a Clostridia medium (Ct) produced the most butyric acid (8.06 g L(-1), 0.46 g/g glucose), followed by 50% SGH with Ct (6.01 g L(-1), 0.44 g/g glucose), however, 50% CSH Ct showed growth inhibition. RPT-4213 was then used in pH-controlled bioreactor fermentations using 60% WSH and SGH, with a dilute (0.5×) Ct medium, resulting 9.87 g L(-1) butyric acid in WSH (yield 0.44 g/g) and 7.05 g L(-1) butyric acid in SGH (yield 0.42 g/g). The titer and productivity could be improved through process engineering.

  1. Consolidated bioprocessing of transgenic switchgrass by an engineered and evolved Clostridium thermocellum strain

    Energy Technology Data Exchange (ETDEWEB)

    Yee, Kelsey L [ORNL; Rodriguez Jr, Miguel [ORNL; Thompson, Olivia A [ORNL; Fu, Chunxiang [Noble Foundation; Wang, Zeng-Yu [Noble Foundation; Davison, Brian H [ORNL; Mielenz, Jonathan R [ORNL

    2014-01-01

    Background: Switchgrass is an abundant and dedicated bioenergy feedstock however its inherent recalcitrance is one of the economic hurdles for producing biofuels. The down-regulation of the caffeic acid O-methyl transferase (COMT) gene in the lignin pathway of switchgrass reduced lignin content and S/G ratio, and the transgenic lines showed improved fermentation yield with S. cerevisiae and C. thermocellum (ATCC 27405) in comparison to the wild-type switchgrass. Results: Here we examine the fermentation potential of the COMT transgenic switchgrass and its wild-type line, with an engineered and evolved Clostridium thermocellum (M1570) strain. The fermentation of the transgenic switchgrass had superior conversion relative to the control line with an increase of 20% and ethanol was the primary metabolite accounting for 90% of the total metabolites measured by HPLC. Conclusions: The down-regulation of the COMT gene in switchgrass reduced recalcitrance and improved microbial bioconversion yield. Moreover, these results showed ethanol as the main fermentation metabolite produced by an engineered and evolved C. thermocellum strain grown on a transgenic switchgrass.

  2. Simultaneous production of isopropanol, butanol, ethanol and 2,3-butanediol by Clostridium acetobutylicum ATCC 824 engineered strains.

    OpenAIRE

    Collas, Florent; Kuit, Wouter; Clément, Benjamin; Marchal, Rémy; López-Contreras, Ana M.; Monot, Frederic

    2012-01-01

    International audience; Isopropanol represents a widely-used commercial alcohol which is currently produced from petroleum. In nature, isopropanol is excreted by some strains of Clostridium beijerinckii, simultaneously with butanol and ethanol during the isopropanol butanol ethanol (IBE) fermentation. In order to increase isopropanol production, the gene encoding the secondary-alcohol dehydrogenase enzyme from C. beijerinckii NRRL B593 (adh) which catalyzes the reduction of acetone to isoprop...

  3. Distinctive Profiles of Infection and Pathology in Hamsters Infected with Clostridium difficile Strains 630 and B1 ▿

    OpenAIRE

    Goulding, David; Thompson, Harold; Emerson, Jenny; Fairweather, Neil F.; Dougan, Gordon; Douce, Gill R.

    2009-01-01

    Currently, the Golden Syrian hamster is widely considered an important model of Clostridium difficile disease, as oral infection of this animal pretreated with antibiotics reproduces many of the symptoms observed in humans. Two C. difficile strains, B1 and 630, showed significant differences in the progression and severity of disease in this model. B1-infected hamsters exhibited more severe pathology and a shorter time to death than hamsters infected with 630. Histological changes in the gut ...

  4. Direct fermentation of xylan by Clostridium strain BOH3 for the production of butanol and hydrogen using optimized culture medium.

    Science.gov (United States)

    Rajagopalan, Gobinath; He, Jianzhong; Yang, Kun Lin

    2014-02-01

    Clostridium strain BOH3 is able to utilize 10 g/l of xylan in reinforced clostridial medium (RCM) and produce butanol and hydrogen. However, increasing xylan concentration to 30 g/l does not enhance the production of butanol and hydrogen due to insufficient expression of xylanase enzyme (27.5 U/mg). To enhance the xylanase activity, an optimized culture medium (OCM), which consists of sugarcane bagasse hydrolysate (11.75 g/l), ammonium sulfate (8.92 g/l) and iron (III) chloride (1.45 mM) is designed. In the optimized OCM, Clostridium strain BOH3 expresses more xylanase and shows higher xylanase activity (44.05 ± 0.25 U/mg) in the OCM. This activity is about 1.6-fold higher than that in the original RCM. Employing OCM as a medium, Clostridium strain BOH3 effectively ferments high concentration (30 and 50 g/l) of xylan and produces 12.05 ± 0.15 and 14.80 ± 0.15 g/l of butanol and 1.78 ± 0.08 and 2.65 ± 0.15 l/l of hydrogen, respectively. Copyright © 2013 Elsevier Ltd. All rights reserved.

  5. Purification and characterization of a primary-secondary alcohol dehydrogenase from two strains of Clostridium beijerinckii.

    Science.gov (United States)

    Ismaiel, A A; Zhu, C X; Colby, G D; Chen, J S

    1993-01-01

    Two primary alcohols (1-butanol and ethanol) are major fermentation products of several clostridial species. In addition to these two alcohols, the secondary alcohol 2-propanol is produced to a concentration of about 100 mM by some strains of Clostridium beijerinckii. An alcohol dehydrogenase (ADH) has been purified to homogeneity from two strains (NRRL B593 and NESTE 255) of 2-propanol-producing C. beijerinckii. When exposed to air, the purified ADH was stable, whereas the partially purified ADH was inactivated. The ADHs from the two strains had similar structural and kinetic properties. Each had a native M(r) of between 90,000 and 100,000 and a subunit M(r) of between 38,000 and 40,000. The ADHs were NADP(H) dependent, but a low level of NAD(+)-linked activity was detected. They were equally active in reducing aldehydes and 2-ketones, but a much lower oxidizing activity was obtained with primary alcohols than with secondary alcohols. The kcat/Km value for the alcohol-forming reaction appears to be a function of the size of the larger alkyl substituent on the carbonyl group. ADH activities measured in the presence of both acetone and butyraldehyde did not exceed activities measured with either substrate present alone, indicating a common active site for both substrates. There was no similarity in the N-terminal amino acid sequence between that of the ADH and those of fungi and several other bacteria. However, the N-terminal sequence had 67% identity with those of two other anaerobes, Thermoanaerobium brockii and Methanobacterium palustre. Furthermore, conserved glycine and tryptophan residues are present in ADHs of these three anaerobic bacteria and ADHs of mammals and green plants. Images PMID:8349550

  6. In vitro activity of MCB3681 against Clostridium difficile strains.

    Science.gov (United States)

    Rashid, Mamun-Ur; Dalhoff, Axel; Weintraub, Andrej; Nord, Carl Erik

    2014-08-01

    One hundred fourteen Clostridium difficile strains were collected from 67 patients and analyzed for the presence of C. difficile toxin B by the cell cytotoxoicity neutralization assay, genes for toxin A, toxin B, binary toxin and TcdC deletion by PCR. All strains were also PCR-ribotyped. The MICs of the isolates were determined against MCB3681 and nine other antimicrobial agents by the agar dilution method. All isolates were positive for toxin B as well as for toxin A and B genes. In addition, 13 isolates were positive for the binary toxin genes. Thirty-two different ribotypes were identified. No strain of ribotype 027 was found. All 114 isolates were sensitive to MCB3681 (0.008-0.5 mg/l), cadazolid (0.064-0.5 mg/l), fidaxomicin (0.008-0.125 mg/l), metronidazole (0.125-2 mg/l), vancomycin (0.125-1 mg/l) and tigecycline (0.032-0.25 mg/l). Three isolates were resistant to linezolid (8 mg/l), 12 isolates were resistant to moxifloxacin (8-32 mg/l), 87 isolates were resistant to clindamycin (8-256 mg/l) and 107 isolates were resistant to ciprofloxacin (8-256 mg/l). No association between toxins A, B and binary toxin, ribotypes and the sensitivity to MCB3681 could be found. MCB3681 has a potent in vitro activity against C. difficile.

  7. The effect of feeding a commercial essential oil product on Clostridium perfringens numbers in the intestine of broiler chickens measured by real-time PCR targeting the α-toxin-encoding gene (plc)

    DEFF Research Database (Denmark)

    Abildgaard, Lone; Højberg, Ole; Schramm, Andreas

    2010-01-01

    concentrations of 100 and 200 mg/kg feed of the essential oil blend did not reduce the intestinal numbers of C. perfringens compared to a non-supplemented control group (P>0.05). Further, the essential oil blend failed to improve (P>0.05) both the growth and feed conversion ratio of the broilers. For rapid...... quantification of C. perfringens type A in broilers, a real-time PCR assay, targeting the α-toxin-encoding plc gene, was developed for use in ileal and caecal samples and was shown to be a fast and reliable alternative to conventional plate counting....

  8. Effect of tcdR Mutation on Sporulation in the Epidemic Clostridium difficile Strain R20291.

    Science.gov (United States)

    Girinathan, Brintha P; Monot, Marc; Boyle, Daniel; McAllister, Kathleen N; Sorg, Joseph A; Dupuy, Bruno; Govind, Revathi

    2017-01-01

    Clostridium difficile is an important nosocomial pathogen and the leading cause of hospital-acquired diarrhea. Antibiotic use is the primary risk factor for the development of C. difficile-associated disease because it disrupts normally protective gut flora and enables C. difficile to colonize the colon. C. difficile damages host tissue by secreting toxins and disseminates by forming spores. The toxin-encoding genes, tcdA and tcdB, are part of a pathogenicity locus, which also includes the tcdR gene that codes for TcdR, an alternate sigma factor that initiates transcription of tcdA and tcdB genes. We created a tcdR mutant in epidemic-type C. difficile strain R20291 in an attempt to identify the global role of tcdR. A site-directed mutation in tcdR affected both toxin production and sporulation in C. difficile R20291. Spores of the tcdR mutant were more heat sensitive than the wild type (WT). Nearly 3-fold more taurocholate was needed to germinate spores from the tcdR mutant than t