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Sample records for netb-negative clostridium perfringens

  1. CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Bacteriophages of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  3. Toxin Plasmids of Clostridium perfringens

    PubMed Central

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

    2013-01-01

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

  4. Evidence for antibiotic induced Clostridium perfringens diarrhoea

    PubMed Central

    Modi, N; Wilcox, M

    2001-01-01

    Clostridium difficile is a well documented cause of antibiotic associated diarrhoea in hospitalised patients, but may account for only approximately 20% of all cases. This leader reviews the current knowledge and understanding of the pathogenesis, epidemiology, and diagnosis of non-food borne Clostridium perfringens diarrhoea. Although enterotoxigenic C perfringens has been implicated in some C difficile negative cases of antibiotic associated diarrhoea, C perfringens enterotoxin detection methods are not part of the routine laboratory investigation of such cases. Testing for C perfringens enterotoxin in faecal samples from patients with antibiotic associated diarrhoea and sporadic diarrhoea on a routine basis would have considerable resource implications. Therefore, criteria for initiating investigations and optimum laboratory tests need to be established. In addition, establishing the true burden of C perfringens antibiotic associated diarrhoea is important before optimum control and treatment measures can be defined. Key Words: Clostridium perfringensClostridium difficile • hospital acquired infective diarrhoea PMID:11577119

  5. Lytic Clostridium perfringens Bacteriophage 39-O Genomic

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  6. Regulation of Toxin Production in Clostridium perfringens

    PubMed Central

    Ohtani, Kaori; Shimizu, Tohru

    2016-01-01

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

  7. Comparative Analysis of Clostridium perfringens Bacteriophage

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

  8. Molecular genetics and pathogenesis of Clostridium perfringens.

    PubMed Central

    Rood, J I; Cole, S T

    1991-01-01

    Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals. Recently significant advances have been made in the development of C. perfringens genetics. Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C. perfringens genes and the construction of shuttle plasmids. The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated. A detailed physical map of the C. perfringens chromosome has been prepared, and numerous genes have been located on that map. Reproducible transformation methods for the introduction of plasmids into C. perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned. Now that it is possible to freely move genetic information back and forth between C. perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C. perfringens infections. PMID:1779929

  9. Perfringolysin O: The Underrated Clostridium perfringens Toxin?

    PubMed Central

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

    2015-01-01

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

  10. Prevalence of Clostridium perfringens, Clostridium perfringens enterotoxin and dysbiosis in fecal samples of dogs with diarrhea.

    PubMed

    Minamoto, Yasushi; Dhanani, Naila; Markel, Melissa E; Steiner, Jörg M; Suchodolski, Jan S

    2014-12-05

    Clostridium perfringens has been suspected as an enteropathogen in dogs. However, its exact role in gastrointestinal (GI) disorders in dogs remains unknown. Recent studies suggest the importance of an altered intestinal microbiota in the activation of virulence factors of enteropathogens. The aim of this study was to evaluate the relationship between diarrhea, dysbiosis, and the presence of C. perfringens and its enterotoxin (CPE). Fecal samples were collected prospectively from 95 healthy control dogs and 104 dogs with GI disease and assessed for bacterial abundances and the presence of CPE using quantitative PCR and ELISA, respectively. C. perfringens was detected in all dogs. Potentially enterotoxigenic C. perfringens were detected in 33.7% (32/95) of healthy control dogs and 48.1% (50/104) diseased dogs, respectively. CPE was detected by ELISA in 1.0% (1/95) of control dogs and 16.3% (17/104) of diseased dogs. Abundances of Fusobacteria, Ruminococcaceae, Blautia, and Faecalibacterium were significantly decreased in diseased dogs, while abundances of Bifidobacterium, Lactobacillus, and Escherichia coli were significantly increased compared to control dogs. The microbial dysbiosis was independent of the presence of the enterotoxigenic C. perfringens or CPE. In conclusion, the presence of CPE as well as fecal dysbiosis was associated with GI disease. However, the presence of C. perfringens was not indicative of GI disease in all cases of diarrhea, and the observed increased abundance of enterotoxigenic C. perfringens may be part of intestinal dysbiosis occurring in GI disease. The significance of an intestinal dysbiosis in dogs with GI disease deserves further attention.

  11. The Tcp conjugation system of Clostridium perfringens.

    PubMed

    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.

  12. Tips to Prevent Illness from Clostridium Perfringens

    MedlinePlus

    ... bacteria can be found on raw meat and poultry, in the intestines of animals and humans, and ... common food sources of C. perfringens ? Meat and poultry are common sources of C. perfringens infections. C. ...

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

    PubMed

    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.

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

    PubMed

    Omernik, Andrzej; Płusa, Tadeusz

    2015-09-01

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

  15. Clostridium perfringens type A-E toxin plasmids.

    PubMed

    Freedman, John C; Theoret, James R; Wisniewski, Jessica A; Uzal, Francisco A; Rood, Julian I; McClane, Bruce A

    2015-05-01

    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.

  16. Antimicrobial susceptibility of Clostridium perfringens strains isolated from broiler chickens

    PubMed Central

    Silva, R. O. S.; Salvarani, F.M.; Assis, R.A.; Martins, N.R.S.; Pires, P.S.; Lobato, F.C.F.

    2009-01-01

    Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant. PMID:24031355

  17. Clostridium perfringens type A–E toxin plasmids

    PubMed Central

    Freedman, John C.; Theoret, James R.; Wisniewski, Jessica A.; Uzal, Francisco A.; Rood, Julian I.; McClane, Bruce A.

    2014-01-01

    Clostridium perfringens relies upon plasmid-encoded toxin genes to cause intestinal infections. These toxin genes are associated with insertion sequences that may facilitate their mobilization and transfer, giving rise to new toxin plasmids with common backbones. Most toxin plasmids carry a transfer of clostridial plasmids locus mediating conjugation, which likely explains the presence of similar toxin plasmids in otherwise unrelated C. perfringens strains. The association of many toxin genes with insertion sequences and conjugative plasmids provides virulence flexibility when causing intestinal infections. However, incompatibility issues apparently limit the number of toxin plasmids maintained by a single cell. PMID:25283728

  18. Clostridium perfringens Sporulation and Sporulation-Associated Toxin Production

    PubMed Central

    Li, Jihong; Paredes-Sabja, Daniel; Sarker, Mahfuzur R.; McClane, Bruce A.

    2015-01-01

    The ability of Clostridium perfringens to form spores plays a key role during the transmission of this Gram-positive bacterium to cause disease. Of particular note, the spores produced by food poisoning strains are often exceptionally resistant to food environment stresses such as heat, cold and preservatives, which likely facilitates their survival in temperature-abused foods. The exceptional resistance properties of spores made by most type A food poisoning strains and some type C foodborne disease strains involves their production of a variant small acid soluble protein-4 that binds more tightly to spore DNA compared to the small acid soluble protein-4 made by most other C. perfringens strains. Sporulation and germination by C. perfringens and Bacillus spp. share both similarities and differences. Finally, sporulation is essential for production of C. perfringens enterotoxin, which is responsible for the symptoms of C. perfringens type A food poisoning, the second most common bacterial foodborne disease in the USA. During this foodborne disease, C. perfringens is ingested with food and then, using sporulation-specific alternate sigma factors, this bacterium sporulates and produces the enterotoxin in the intestines. PMID:27337447

  19. Identification of Clostridium Species and DNA Fingerprinting of Clostridium perfringens by Amplified Fragment Length Polymorphism Analysis▿

    PubMed Central

    Keto-Timonen, Riikka; Heikinheimo, Annamari; Eerola, Erkki; Korkeala, Hannu

    2006-01-01

    An amplified fragment length polymorphism (AFLP) method was applied to 129 strains representing 24 different Clostridium species, with special emphasis on pathogenic clostridia of medical or veterinary interest, to assess the potential of AFLP for identification of clostridia. In addition, the ability of the same AFLP protocol to type clostridia at the strain level was assessed by focusing on Clostridium perfringens strains. All strains were typeable by AFLP, so the method seemed to overcome the problem of extracellular DNase production. AFLP differentiated all Clostridium species tested, except for Clostridium ramosum and Clostridium limosum, which clustered together with a 45% similarity level. Other Clostridium species were divided into species-specific clusters or occupied separate positions. Wide genetic diversity was observed among Clostridium botulinum strains, which were divided into seven species-specific clusters. The same AFLP protocol was also suitable for typing C. perfringens at the strain level. A total of 29 different AFLP types were identified for 37 strains of C. perfringens; strains initially originating from the same isolate showed identical fingerprinting patterns and were distinguished from unrelated strains. AFLP proved to be a highly reproducible, easy-to-perform, and relatively fast method which enables high throughput of samples and can serve in the generation of identification libraries. These results indicate that the AFLP method provides a promising tool for the identification and characterization of Clostridium species. PMID:16971642

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

    NASA Technical Reports Server (NTRS)

    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.

  1. Comparative transcriptomic analysis of Clostridium perfringens biofilms and planktonic cells.

    PubMed

    Charlebois, Audrey; Jacques, Mario; Archambault, Marie

    2016-10-01

    Clostridium perfringens is an opportunistic pathogen that can cause food poisoning in humans and various enterotoxaemias in animal species. Recently, C. perfringens was shown to form biofilms, a structured community of bacterial cells enclosed in a self-produced extracellular matrix. However, very little is known on the subject and no information is available on gene expression in C. perfringens biofilms. To gain insights into the differences between free-living C. perfringens cells and those in biofilms, we used RNA sequencing. In total, 25.7% of genes showed differential expression in the two growth modes; about 12.8% of genes were up-regulated and about 12.9% were down-regulated in biofilms. We show that 772 genes were significantly differentially expressed between biofilms and planktonic cells from the supernatant of biofilms. Genes that were down-regulated in biofilm cells, relative to planktonic cells, included those involved in virulence, energy production, amino acid, nucleotide and carbohydrate metabolism, and in translation and ribosomal structure. Genes up-regulated in biofilm cells were mainly involved in amino acid and carbohydrate metabolism, transcription, inorganic ion metabolism and in defence mechanisms. This study provides new insights into the transcriptomic response of C. perfringens during biofilm formation.

  2. Growth of Clostridium perfringens during cooling of refried beans.

    PubMed

    Cevallos-Cevallos, Juan M; Akins, E Deann; Friedrich, Loretta M; Danyluk, Michelle D; Simonne, Amarat H

    2012-10-01

    Outbreaks of Clostridium perfringens have been associated with dishes containing refried beans from food service establishments. However, growth of C. perfringens in refried beans has not been investigated, and predictive models have not been validated in this food matrix. We investigated the growth of C. perfringens during the cooling of refried beans. Refried beans (pinto and black, with and without salt added) were inoculated with 3 log CFU/g C. perfringens spores and incubated isothermally at 12, 23, 30, 35, 40, 45, and 50°C. The levels of C. perfringens were monitored 3, 5, 8, and 10 h after inoculation, and then fitted to the Baranyi primary model and the Rosso secondary model prior to solving the Baranyi differential equation. The final model was validated by dynamic cooling experiments carried out in stockpots, thus mimicking the worst possible food service conditions. All refried beans samples supported the growth of C. perfringens, and all models fit the data with pseudo-R(2) values of 0.95 or greater and mean square errors of 0.3 or lower. The estimated maximum specific growth rates were generally higher in pinto beans, with or without salt added (2.64 and 1.95 h(-1), respectively), when compared with black beans, with or without salt added (1.78 and 1.61 h(-1), respectively). After 10 h of incubation, maximum populations of C. perfringens were significantly higher in samples with no salt added (7.9 log CFU/g for both pinto and black beans) than in samples with salt added (7.3 and 7.2 log CFU/g for pinto and black beans, respectively). The dynamic model predicted the growth of C. perfringens during cooling, with an average root mean squared error of 0.44. The use of large stockpots to cool refried beans led to an observed 1.2-log increase (1.5-log increase predicted by model) in levels of C. perfringens during cooling. The use of shallower pans for cooling is recommended, because they cool faster, therefore limiting the growth of C. perfringens.

  3. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats.

    PubMed

    Uzal, F A

    2004-04-01

    Clostridium perfringens produces disease in sheep, goats and other animal species, most of which are generically called enterotoxemias. This micro-organism can be a normal inhabitant of the intestine of most animal species including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates in large numbers and produces several potent toxins that are absorbed into the general circulation or act locally with usually devastating effects on the host. History, clinical signs and gross post-mortem findings are useful tools for establishing a presumptive diagnosis of enterotoxaemia by C. perfringens in sheep and goats, although no definitive diagnosis of these diseases can be made without laboratory confirmation. Because all types of C. perfringens can be normal inhabitants of the intestine of most animals, culture of this micro-organism from intestinal contents of animals has no diagnostic value unless a colony count is performed and large numbers (usually more than 10(4)-10(7)CFU/g) of C. perfringens are found. The most accepted criterion in establishing a definitive diagnosis of enterotoxaemia by C. perfringens is the detection of its toxins in intestinal contents. However, some of the major toxins of C. perfringens (i.e. epsilon toxin) can also be found, albeit in small amounts, in the small intestine of clinically normal sheep, and this poses a diagnostic challenge. In such cases the histopathology of the brain must be used as an alternative diagnostic tool, since the lesions produced by epsilon toxin in the brains of sheep and goats are unique and pathognomonic for C. perfringens type D enterotoxaemia. Ancillary tests, such as measurement of urine glucose or observation of Gram stained smears of intestinal mucosa can be used and, although they have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxaemia if they are negative. In conclusion, the

  4. Hydrolyzable and condensed tannins resistance in Clostridium perfringens.

    PubMed

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

    2015-08-01

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

  5. Naturally acquired antibodies against Clostridium perfringens epsilon toxin in goats.

    PubMed

    Veschi, Josir Laine A; Bruzzone, Octavio A; Losada-Eaton, Daniela M; Dutra, Iveraldo S; Fernandez-Miyakawa, Mariano E

    2008-09-15

    Clostridium perfringens type D-producing epsilon toxin is a common cause of death in sheep and goats worldwide. Although anti-epsilon toxin serum antibodies have been detected in healthy non-vaccinated sheep, the information regarding naturally acquired antibodies in ruminants is scanty. The objective of the present report was to characterize the development of naturally acquired antibodies against C. perfringens epsilon toxin in goats. The levels of anti-epsilon toxin antibodies in blood serum of goat kids from two different herds were examined continuously for 14 months. Goats were not vaccinated against any clostridial disease and received heterologous colostrums from cows that were not vaccinated against any clostridial disease. During the survey one of these flocks suffered an unexpectedly severe C. perfringens type D enterotoxemia outbreak. The results showed that natural acquired antibodies against C. perfringens epsilon toxin can appear as early as 6 weeks in young goats and increase with the age without evidence of clinical disease. The enterotoxemia outbreak was coincident with a significant increase in the level of anti-epsilon toxin antibodies.

  6. Diagnosis of Clostridium perfringens intestinal infections in sheep and goats.

    PubMed

    Uzal, Francisco A; Songer, J Glenn

    2008-05-01

    Clostridium perfringens produces enteric diseases, generically called enterotoxemias, in sheep, goats, and other animals. This microorganism can be a normal inhabitant of the intestine of most animal species, including humans, but when the intestinal environment is altered by sudden changes in diet or other factors, C. perfringens proliferates and produces potent toxins that act locally or are absorbed into the general circulation with usually devastating effects on the host. History, clinical signs, and gross postmortem findings are useful tools for establishing a presumptive diagnosis of clostridial enterotoxemia in sheep and goats. Definitive diagnosis requires laboratory confirmation. Isolation of some types of C. perfringens (e.g., B and C) can be of diagnostic value, but other types (e.g., A) are so commonly found in the intestine of normal animals that isolation is meaningless from a diagnostic point of view. The most accepted criterion in establishing a definitive diagnosis of enterotoxemia is detection of C. perfringens toxins in intestinal contents. Also, histopathological examination of brain is very useful for diagnosis of type D disease, as lesions produced by epsilon toxin in the brains of sheep and goats are pathognomonic for type D enterotoxemia. Ancillary tests, such as measuring urine glucose or observing Gram-stained smears of intestinal mucosa, can be used. However, although such tests have a presumptive diagnostic value when positive, they cannot be used to rule out a diagnosis of enterotoxemia when negative.

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

    PubMed

    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.

  8. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu and pyruvate:ferredoxin oxidoreductase of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by react...

  9. Prevalence and characterization of Clostridium perfringens from spices in Argentina.

    PubMed

    Aguilera, Milton Osmar; Stagnitta, Patricia Virginia; Micalizzi, Blas; de Guzmán, Ana María Stefanini

    2005-12-01

    Spices can present high microbial counts and Clostridium perfringens, Bacillus cereus, Salmonella and Shigella, among others have been isolated from spices. C. perfringens is an important pathogen agent causing, among other diseases, enteritis in humans caused by C. perfringens enterotoxin (CPE) which causes human food poisoning and enterotoxemia in domestic animals. The aims of the present work were (i) to establish the hygienic sanitary quality of some spices in San Luis, Argentina; (ii) to determine the presence of C. perfringens in these spices by means of the most probable number (MPN) and count on plate methods; (iii) to characterize the enterotoxigenic strains of C. perfringens by PCR and immunological methods such as reverse passive latex agglutination (RPLA) and (iv) to type by PCR C. perfringens strains isolated. A total of 115 samples of spices, 67 of which were purchased in local retail stores and 48 domestically collected were analysed. Total aerobe counts on tryptone glucose yeast extract agar medium of the 115 samples were between <10 and 10(6) CFU/g. The colifecal counts using Mac Conkey broth of the 115 samples were <4-10(3)CFU/g, with 28 samples (24.34%) exceeding the limit established by the Spanish Alimentary Code (10 CFU/g) while 2 samples (1.73%) had a sulfite reducing anaerobe load above standard limits. A total of 14 C. perfringens strains (12.17%) were isolated and characterized from 115 samples by the standard biochemical tests. Four of which (28.60%) turned out to be enterotoxigenic by PCR and RPLA. In order to type C. perfringens strains based on their main toxins, the 14 strains were analysed by PCR. All strains belonged to type A. All RPLA positive strains were cpe(+) by PCR. The percentage of enterotoxigenic strains was more elevated that those reported in other studies for this type of sample. These results indicate that sanitary conditions in different production stages of species must be improved to reduce health hazards. The high

  10. Recent Insights into Clostridium perfringens Beta-Toxin

    PubMed Central

    Nagahama, Masahiro; Ochi, Sadayuki; Oda, Masataka; Miyamoto, Kazuaki; Takehara, Masaya; Kobayashi, Keiko

    2015-01-01

    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. PMID:25654787

  11. Clostridium perfringens gas gangrene at a wrist intravenous line insertion.

    PubMed

    Determann, Catherine; Walker, Craig Andrew

    2013-10-09

    A patient admitted to the intensive care unit for management of hypotension following a multiple medications overdose subsequently deteriorated rapidly with sepsis. A cannula site was noted to be bruised, swollen and erythematous and the X-ray demonstrated gas sitting within the tissues surrounding the metacarpal bones. The patient was referred to the orthopaedic surgeons and quickly taken for debridement of the affected area and fasciotomies of the forearm. Microbiological investigation confirmed Clostridium perfringens to be present in multiple fluid samples taken from the affected site.

  12. Fulminant massive gas gangrene caused by Clostridium perfringens.

    PubMed

    Kuroda, Shoji; Okada, Yumi; Mita, Masaki; Okamoto, Yasuo; Kato, Hirotaka; Ueyama, Shigemitsu; Fujii, Ikuzo; Morita, Sumiharu; Yoshida, Yasuaki

    2005-05-01

    Clostridium perfringens (C.P) gas gangrene is one of the most fulminant infectious diseases. We encountered fulminant massive gas gangrene in a 56- year-old man with alcoholic liver cirrhosis. The patient died 14 hours after diagnosis of gas gangrene (54 hours after admission). Dramatic changes in abdominal CT imaging revealed development of a massive volume of gas in the intra-portal vein, retroperitoneum and abdominal subcutaneous tissue within 24 hours. We also proved C.P infection by immunohistological staining, leading to a diagnosis of C.P gas gangrene.

  13. Clostridium difficile and Clostridium perfringens from wild carnivore species in Brazil.

    PubMed

    Silva, Rodrigo Otávio Silveira; D'Elia, Mirella Lauria; Tostes Teixeira, Erika Procópio; Pereira, Pedro Lúcio Lithg; de Magalhães Soares, Danielle Ferreira; Cavalcanti, Álvaro Roberto; Kocuvan, Aleksander; Rupnik, Maja; Santos, André Luiz Quagliatto; Junior, Carlos Augusto Oliveira; Lobato, Francisco Carlos Faria

    2014-08-01

    Despite some case reports, the importance of Clostridium perfringens and Clostridium difficile for wild carnivores remains unclear. Thus, the objective of this study was to identify C. perfringens and C. difficile strains in stool samples from wild carnivore species in Brazil. A total of 34 stool samples were collected and subjected to C. perfringens and C. difficile isolation. Suggestive colonies of C. perfringens were then analyzed for genes encoding the major C. perfringens toxins (alpha, beta, epsilon and iota) and the beta-2 toxin (cpb2), enterotoxin (cpe) and NetB (netb) genes. C. difficile strains were analyzed by multiplex-PCR for toxins A (tcdA) and B (tcdB) and a binary toxin gene (cdtB) and also submitted to a PCR ribotyping. Unthawed aliquots of samples positive for C. difficile isolation were subjected to the detection of A/B toxins by a cytotoxicity assay (CTA). C. perfringens was isolated from 26 samples (76.5%), all of which were genotyped as type A. The netb gene was not detected, whereas the cpb2 and cpe genes were found in nine and three C. perfringens strains, respectively. C. difficile was isolated from two (5.9%) samples. A non-toxigenic strain was recovered from a non-diarrheic maned wolf (Chrysocyon brachyurus). Conversely, a toxigenic strain was found in the sample of a diarrheic ocelot (Leopardus pardallis); an unthawed stool sample was also positive for A/B toxins by CTA, indicating a diagnosis of C. difficile-associated diarrhea in this animal. The present work suggests that wild carnivore species could carry C. difficile strains and that they could be susceptible to C. difficile infection.

  14. Coat and enterotoxin-related proteins in Clostridium perfringens spores.

    PubMed

    Ryu, S; Labbe, R G

    1989-11-01

    Coat proteins from mature spores of two enterotoxin-positive (Ent+) and two enterotoxin-negative (Ent-) strains of Clostridium perfringens were solubilized using 50 mM-dithiothreitol and 1% sodium dodecyl sulphate at pH 9.7, and alkylated using 110 mM-iodoacetamide to prevent aggregation. The coat proteins and C. perfringens type A enterotoxin (CPE) were separated by SDS-PAGE and analysed by Western blotting using anti-CPE antibody. As previously reported, CPE aggregated in the presence of SDS, but no aggregation occurred at concentrations below 15 micrograms CPE ml-1. Two CPE-related proteins (34 and 48 kDa) were found in the solubilized spore coat protein of Ent+ strains while only the 48 kDa CPE-related protein was found in the spore coat fraction of Ent- strains. CPE-related proteins comprised 2.7% and 0.8% of the total solubilized coat protein of Ent+ and Ent- strains respectively. CPE-related proteins could be extracted from the spores with 1% SDS alone. They could also be released by disruption of whole spores, indicating that the CPE-related proteins may be in the spore core or trapped between the core and coat layers. The results suggest that CPE is not a major structural component of the coat fraction of C. perfringens spores.

  15. Plasmid partitioning systems of conjugative plasmids from Clostridium perfringens.

    PubMed

    Adams, Vicki; Watts, Thomas D; Bulach, Dieter M; Lyras, Dena; Rood, Julian I

    2015-07-01

    Many pathogenic strains of Clostridium perfringens carry several highly similar toxin or antibiotic resistance plasmids that have 35 to 40 kb of very closely related syntenous sequences, including regions that carry the genes encoding conjugative transfer, plasmid replication and plasmid maintenance functions. Key questions are how are these closely related plasmids stably maintained in the same cell and what is the basis for plasmid incompatibility in C. perfringens. Comparative analysis of the Rep proteins encoded by these plasmids suggested that this protein was not the basis for plasmid incompatibility since plasmids carried in a single strain often encoded an almost identical Rep protein. These plasmids all carried a similar, but not identical, parMRC plasmid partitioning locus. Phylogenetic analysis of the deduced ParM proteins revealed that these proteins could be divided into ten separate groups. Importantly, in every strain that carried more than one of these plasmids, the respective ParM proteins were from different phylogenetic groups. Similar observations were made from the analysis of phylogenetic trees of the ParR proteins and the parC loci. These findings provide evidence that the basis for plasmid incompatibility in the conjugative toxin and resistance plasmid family from C. perfringens resides in subtle differences in the parMRC plasmid partitioning loci carried by these plasmids.

  16. Effect of cooling on Clostridium perfringens in pea soup.

    PubMed

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

    2004-02-01

    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 different cooling times was investigated. Tubes of preheated pea soup (50 degrees C) were inoculated with cocktails of cells or heat-activated spores of this pathogen. The tubes were linearly cooled to 15 degrees C in time spans of 3, 5, 7.5, and 10 h and were subsequently stored in a refrigerator at 3 or 7 degrees C for up to 84 h. Cell numbers increased by 1-log cycle during the 3-h cooling period and reached their maximum after 10 h of cooling. Subsequent refrigeration hardly reduced cell numbers. Cooling of 3.75 liters of pea soup in an open pan showed that this amount of pea soup cooled from 50 to 15 degrees C in 5 h, which will allow a more than 10-fold increase in cell numbers. These findings emphasize the need of good hygienic practices and quick cooling of heated foods after preparation.

  17. Complex transcriptional regulation of citrate metabolism in Clostridium perfringens.

    PubMed

    Yuan, Yonghui; Ohtani, Kaori; Yoshizawa, Satoko; Shimizu, Tohru

    2012-02-01

    A Gram-positive, spore-forming bacterium, Clostridium perfringens, possesses genes for citrate metabolism, which might play an important role in the utilization of citrate as a sole carbon source. In this study, we identified a chromosomal citCDEFX-mae-citS operon in C. perfringens strain 13, which is transcribed on three mRNAs of different sizes. Expression of the cit operon was significantly induced when 5 mM extracellular citrate was added to the growth medium. Most interestingly, three regulatory systems were found to be involved in the regulation of the expression of cit genes: 1) the two upstream divergent genes citG and citI; 2) two different two-component regulatory systems, CitA/CitB (TCS6 consisted of CPE0531/CPE0532) and TCS5 (CPE0518/CPE0519); and 3) the global two-component VirR/VirS-VR-RNA regulatory system known to regulate various genes for toxins and degradative enzymes. Our results suggest that in C. perfringens the citrate metabolism might be strictly controlled by a complex regulatory system.

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

    PubMed

    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.

  19. The Genome Sequence of Bacteriophage CPV1 Virulent for Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Application of bacteriophages and their lytic enzymes to control Clostridium perfringens is one potential approach to reduce the pathogen on poultry farms and in poultry-processing facilities. Bacteriophages lytic for C. perfringens were isolated from sewage, feces and broiler intestinal contents. P...

  20. Clostridium perfringens in Long Island Sound sediments: An urban sedimentary record

    USGS Publications Warehouse

    Buchholtz ten Brink, M. R.; Mecray, E.L.; Galvin, E.L.

    2000-01-01

    Clostridium perfringens is a conservative tracer and an indicator of sewage-derived pollution in the marine environment. The distribution of Clostridium perfringens spores was measured in sediments from Long Island Sound, USA, as part of a regional study designed to: (1) map the distribution of contaminated sediments; (2) determine transport and dispersal paths; (3) identify the locations of sediment and contaminant focusing; and (4) constrain predictive models. In 1996, sediment cores were collected at 58 stations, and surface sediments were collected at 219 locations throughout the Sound. Elevated concentrations of Clostridium perfringens in the sediments indicate that sewage pollution is present throughout Long Island Sound and has persisted for more than a century. Concentrations range from undetectable amounts to 15,000 spores/g dry sediment and are above background levels in the upper 30 cm at nearly all core locations. Sediment focusing strongly impacts the accumulation of Clostridium perfringens spores. Inventories in the cores range from 28 to 70,000 spores/cm2, and elevated concentrations can extend to depths of 50 cm. The steep gradients in Clostridium perfringens profiles in muddier cores contrast with concentrations that are generally constant with depth in sandier cores. Clostridium perfringens concentrations rarely decrease in the uppermost sediment, unlike those reported for metal contaminants. Concentrations in surface sediments are highest in the western end of the Sound, very low in the eastern region, and intermediate in the central part. This pattern reflects winnowing and focusing of Clostridium perfringens spores and fine-grained sediment by the hydrodynamic regime; however, the proximity of sewage sources to the westernmost Sound locally enhances the Clostridium perfringens signals.

  1. Molecular and Cellular Basis of Microvascular Perfusion Deficits Induced by Clostridium perfringens and Clostridium septicum

    PubMed Central

    Hickey, Michael J.; Kwan, Rain Y. Q.; Awad, Milena M.; Kennedy, Catherine L.; Young, Lauren F.; Hall, Pam; Cordner, Leanne M.; Lyras, Dena; Emmins, John J.; Rood, Julian I.

    2008-01-01

    Reduced tissue perfusion leading to tissue ischemia is a central component of the pathogenesis of myonecrosis caused by Clostridium perfringens. The C. perfringens α-toxin has been shown capable of inducing these changes, but its potential synergy with perfringolysin O (θ-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 α-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 α-toxin. Together, these data indicate that as a result of its ability to produce α-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 is central to the pathogenesis of

  2. Metataxonomics reveal vultures as a reservoir for Clostridium perfringens.

    PubMed

    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

    2017-02-22

    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.

  3. Metataxonomics reveal vultures as a reservoir for Clostridium perfringens

    PubMed Central

    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

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

    PubMed

    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.

  5. Comparative Study of Ten Bacteriocins of Clostridium perfringens

    PubMed Central

    Mahony, D. E.; Li, A.

    1978-01-01

    Bacteriocins of Clostridium perfringens were prepared by ammonium sulfate precipitation of supernatant broth from 10 bacteriocinogenic strains. These bacteriocins were compared with respect to their ability to produce spheroplasts in a sensitive indicator strain; their inducibility; sensitivity to pH, proteolytic enzymes, and boiling; and their effect on macromolecular synthesis. Two bacteriocins were stable over a wide range of pH values and resisted boiling, and three bacteriocins were resistant to trypsin. Five bacteriocins shut down DNA, RNA, and protein synthesis; three bacteriocins had varying effects on DNA and RNA synthesis; and two bacteriocins had little effect on macromolecular synthesis. Antiserum prepared against one bacteriocin highly neutralized three bacteriocins with partial neutralization of five others; two bacteriocins were unaffected. Mutant strains selected for resistance to bacteriocin 28 also demonstrated coresistance to two other closely related bacteriocins and partial resistance to five others. PMID:217302

  6. Clostridium perfringens Delta-Toxin Induces Rapid Cell Necrosis

    PubMed Central

    Seike, Soshi; Miyamoto, Kazuaki; Kobayashi, Keiko; Takehara, Masaya; Nagahama, Masahiro

    2016-01-01

    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. PMID:26807591

  7. Clostridium perfringens epsilon toxin inhibits the gastrointestinal transit in mice.

    PubMed

    Losada-Eaton, D M; Fernandez-Miyakawa, M E

    2010-12-01

    Epsilon toxin produced by Clostridium perfringens type B and D is a potent toxin that is responsible for a highly fatal enterotoxemia in sheep and goats. In vitro, epsilon toxin produces contraction of the rat ileum as the result of an indirect action, presumably mediated through the autonomic nervous system. To examine the impact of epsilon toxin in the intestinal transit, gastric emptying (GE) and gastrointestinal transit (GIT) were evaluated after intravenous and oral administration of epsilon toxin in mice. Orally administered epsilon toxin produced a delay on the GIT. Inhibition of the small intestinal transit was observed as early as 1 h after the toxin was administered orally but the effects were not observed after 1 week. Epsilon toxin also produced an inhibition in GE and a delay on the GIT when relatively high toxin concentrations were given intravenously. These results indicate that epsilon toxin administered orally or intravenously to mice transitorily inhibits the GIT. The delay in the GIT induced by epsilon toxin could be relevant in the pathogenesis of C. perfringens type B and D enterotoxemia.

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

    PubMed

    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.

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

    PubMed Central

    Heikinheimo, Annamari; Lindström, Miia; Korkeala, Hannu

    2004-01-01

    A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying (cpe-positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe-positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe-positive C. perfringens was successfully isolated from both of these samples. The detection of cpe-positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe-positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe-positive C. perfringens could be isolated if the ratio of cpe-positive C. perfringens spores to total C. perfringens spores was 6 × 10−5 or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe-positive C. perfringens. The method also offers a new approach in the investigation of the epidemiology of cpe-positive C. perfringens strains. PMID:15364981

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

    PubMed

    Heikinheimo, Annamari; Lindström, Miia; Korkeala, Hannu

    2004-09-01

    A hydrophobic grid membrane filter-colony hybridization (HGMF-CH) method for the enumeration and isolation of cpe gene-carrying (cpe-positive) Clostridium perfringens spores from feces was developed. A 425-bp DNA probe specific for the cpe gene was sensitive and specific when tested with bacterial DNA and pure cultures. The enumeration of cpe-positive C. perfringens by the HGMF-CH method proved to be as sensitive as nested PCR combined with the most-probable number technique when tested with fecal samples from healthy individuals. With the aid of the HGMF-CH method, positive hybridization signals were detected from two out of seven fecal samples obtained from healthy individuals. Furthermore, cpe-positive C. perfringens was successfully isolated from both of these samples. The detection of cpe-positive C. perfringens by the HGMF-CH method is dependent on the ratio of cpe-positive C. perfringens colonies to total C. perfringens colonies growing on the HGMF-tryptose-sulfite-cycloserine plate. cpe-positive C. perfringens could be isolated if the ratio of cpe-positive C. perfringens spores to total C. perfringens spores was 6 x 10(-5) or higher. The HGMF-CH method provides an aid in the investigation of fecal samples of patients suffering from food poisoning or other diseases caused by cpe-positive C. perfringens. The method also offers a new approach in the investigation of the epidemiology of cpe-positive C. perfringens strains.

  11. Novel insights into the epidemiology of Clostridium perfringens type A food poisoning.

    PubMed

    Lindström, Miia; Heikinheimo, Annamari; Lahti, Päivi; Korkeala, Hannu

    2011-04-01

    Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. The disease is caused by C. perfringens enterotoxin (CPE) encoded by cpe and produced by less than 5% of C. perfringens type A strains. Molecular epidemiological research in the past 15 years has focused on the reservoirs and routes of cpe-positive C. perfringens aiming to clarify the role and epidemiology of chromosomal and plasmid-borne cpe-carrying strains. This literature review highlights novel aspects in the epidemiology of CPE-mediated diseases. We suggest that (1) chromosomal and plasmid-borne cpe-carrying C. perfringens strains are genetically and epidemiologically distinct and have adapted to different environments; (2) not only chromosomal but also plasmid-borne cpe-carrying C. perfringens strains cause food poisonings; (3) other CPE-mediated diseases, such as antibiotic-associated and sporadic diarrhea, associated with plasmid-borne cpe-positive strains, may be food-related; (4) the role of animals as the main reservoir of cpe-positive C. perfringens needs to be reconsidered; (5) humans serve as an important reservoir of cpe-positive C. perfringens, introducing a contamination risk into foods through handling; and (6) the current standard procedures to diagnose C. perfringens food poisoning fail to detect and isolate many C. perfringens strains, distorting the epidemiological understanding of C. perfringens food poisoning.

  12. Isolation and characterization of Clostridium perfringens from apparently healthy animals of the Shandong province of China.

    PubMed

    Chai, T; Wang, L; Wang, H; Duan, H; Müller, W; Zucker, B A

    2007-10-01

    In a pilot study the presence and frequency of Clostridium (C.) perfringens was investigated among apparently healthy farm animals in the Shandong province of China. 748 faecal samples were collected from 9 pig-, 4 sheep-, 7 cattle- and 5 rabbit farms. C. perfringens was isolated from 124 samples (16.6%). The isolates were classified into major toxin types by using PCR analysis detecting the genes encoding these toxins. All isolates were identified as C perfringens toxin type A. There are also some reports from different regions in China linking C. perfringens toxin type A strains to gastrointestinal diseases. Therefore further investigations about the epidemiologic role of C perfringens toxin type A strains in the Shandong region are necessary. Currently, cases of enterotoxemia from this region are investigated for the presence of C perfringens.

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

    PubMed

    Manafi, Mammad; Waldherr, Kerstin; Kundi, Michael

    2013-10-01

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

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    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.

  17. Occurrence and prevalence of Clostridium perfringens in polar bears from Svalbard, Norway.

    PubMed

    Jores, Joerg; Derocher, Andrew E; Staubach, Christoph; Aschfalk, Ansgar

    2008-01-01

    To obtain insight into the occurrence and prevalence of Clostridium perfringens and its major toxins in polar bears (Ursus maritimus), we took fecal samples for bacteriologic analysis from live-captured bears in the Svalbard Archipelago, Norway, in 2001. Clostridium perfringens was isolated from 40 of 92 samples (44%). Thirty strains were further characterized by determining toxin type and were classified to be type A, while one was also positive for the gene encoding beta2-toxin. Despite the fact that C. perfringens type A has been associated with fatal diseases in several animal species as well as in humans, our data indicate that C. perfringens type A is an normal inhabitant of the gastrointestinal tract of polar bears.

  18. Digestion of epithelial tight junction proteins by the commensal Clostridium perfringens.

    PubMed

    Pruteanu, Mihaela; Shanahan, Fergus

    2013-11-15

    The enteric microbiota contributes to the pathogenesis of inflammatory bowel disease, but the pathways involved and bacterial participants may vary in different hosts. We previously reported that some components of the human commensal microbiota, particularly Clostridium perfringens (C. perfringens), have the proteolytic capacity for host matrix degradation and reduce transepithelial resistance. Here, we examined the C. perfringens-derived proteolytic activity against epithelial tight junction proteins using human intestinal epithelial cell lines. We showed that the protein levels of E-cadherin, occludin, and junctional adhesion molecule 1 decrease in colonic cells treated with C. perfringens culture supernatant. E-cadherin ectodomain shedding in C. perfringens-stimulated intestinal epithelial cells was detected with antibodies against the extracellular domain of E-cadherin, and we demonstrate that this process occurs in a time- and dose-dependent manner. In addition, we showed that the filtered sterile culture supernatant of C. perfringens has no cytotoxic activity on the human intestinal cells at the concentrations used in this study. The direct cleavage of E-cadherin by the proteases from the C. perfringens culture supernatant was confirmed by C. perfringens supernatant-induced in vitro degradation of the human recombinant E-cadherin. We conclude that C. perfringens culture supernatant mediates digestion of epithelial cell junctional proteins, which is likely to enable access to the extracellular matrix components by the paracellular pathway.

  19. Rapid Confirmation of Clostridium perfringens by Using Chromogenic and Fluorogenic Substrates

    PubMed Central

    Adcock, Philip W.; Saint, Christopher P.

    2001-01-01

    The use of 4-methylumbelliferyl phosphate (MUP) and ortho-nitrophenyl-β-d-galactopyranoside (ONPG) for the identification of Clostridium perfringens was investigated. A liquid assay containing both MUP and ONPG was a highly specific alternative method for C. perfringens confirmation, reducing incubation time from 48 to only 4 h. The assay solution is easy to prepare, does not require anaerobic conditions for use, and has an extended shelf life. PMID:11526053

  20. Intracellular Trafficking of Clostridium perfringens Iota-Toxin b

    PubMed Central

    Umezaki, Mariko; Tashiro, Ryo; Oda, Masataka; Kobayashi, Keiko; Shibutani, Masahiro; Takagishi, Teruhisa; Ishidoh, Kazumi; Fukuda, Mitsunori; Sakurai, Jun

    2012-01-01

    Clostridium perfringens iota-toxin is composed of an enzymatic component (Ia) and a binding component (Ib). Ib binds to a cell surface receptor, undergoes oligomerization in lipid rafts, and binds Ia. The resulting complex is then endocytosed. Here, we show the intracellular trafficking of iota-toxin. After the binding of the Ib monomer with cells at 4°C, oligomers of Ib formed at 37°C and later disappeared. Immunofluorescence staining of Ib revealed that the internalized Ib was transported to early endosomes. Some Ib was returned to the plasma membrane through recycling endosomes, whereas the rest was transported to late endosomes and lysosomes for degradation. Degraded Ib was delivered to the plasma membrane by an increase in the intracellular Ca2+ concentration caused by Ib. Bafilomycin A1, an endosomal acidification inhibitor, caused the accumulation of Ib in endosomes, and both nocodazole and colchicine, microtubule-disrupting agents, restricted Ib's movement in the cytosol. These results indicated that an internalized Ia and Ib complex was delivered to early endosomes and that subsequent delivery of Ia to the cytoplasm occurs mainly in early endosomes. Ib was either sent back to the plasma membranes through recycling endosomes or transported to late endosomes and lysosomes for degradation. Degraded Ib was transported to plasma membranes. PMID:22825447

  1. Mechanistic Investigations of Unsaturated Glucuronyl Hydrolase from Clostridium perfringens*

    PubMed Central

    Jongkees, Seino A. K.; Yoo, Hayoung; Withers, Stephen G.

    2014-01-01

    Experiments were carried out to probe the details of the hydration-initiated hydrolysis catalyzed by the Clostridium perfringens unsaturated glucuronyl hydrolase of glycoside hydrolase family 88 in the CAZy classification system. Direct 1H NMR monitoring of the enzymatic reaction detected no accumulated reaction intermediates in solution, suggesting that rearrangement of the initial hydration product occurs on-enzyme. An attempt at mechanism-based trapping of on-enzyme intermediates using a 1,1-difluoro-substrate was unsuccessful because the probe was too deactivated to be turned over by the enzyme. Kinetic isotope effects arising from deuterium-for-hydrogen substitution at carbons 1 and 4 provide evidence for separate first-irreversible and overall rate-determining steps in the hydration reaction, with two potential mechanisms proposed to explain these results. Based on the positioning of catalytic residues in the enzyme active site, the lack of efficient turnover of a 2-deoxy-2-fluoro-substrate, and several unsuccessful attempts at confirmation of a simpler mechanism involving a covalent glycosyl-enzyme intermediate, the most plausible mechanism is one involving an intermediate bearing an epoxide on carbons 1 and 2. PMID:24573682

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

    PubMed

    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.

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

    PubMed

    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.

  4. Detection of Clostridium perfringens enterotoxin in human fecal samples and anti-enterotoxin in sera.

    PubMed Central

    Naik, H S; Duncan, C L

    1978-01-01

    By using counterimmunoelectrophoresis (CIEP), Clostridium perfringens enterotoxin was successfully demonstrated in fecal samples collected within 1 day of attack from sick individuals involved in a bacteriologically and epidemiologically proven outbreak of C. perfringens food poisoning. In contrast, enterotoxin was not demonstrable in fecal samples of apparently healthy individuals both at high- and low-risk exposure to the organism and enterotoxin or in fecal samples collected 4 to 5 days after a food poisoning outbreak. A 100% prevalence of C. perfringens anti-enterotoxin in sera of human volunteers at high- as well as low-risk exposure to the organism and enterotoxin was recorded with CIEP. PMID:211142

  5. Age related variations of serum concentrations of normally occurring IgG antibodies to Clostridium perfringens.

    PubMed Central

    Zarén, E; Schwan, A; Frenckner, B

    1987-01-01

    In studies using indirect immunofluorescence IgG antibodies to Clostridium perfringens were found in sera from healthy adults. Sera from 236 healthy children were examined. The normally occurring IgG antibodies to C perfringens were found to have an age related variation. Preliminary data suggest that they are not correlated to C perfringens alpha toxin. The antigen(s) against which the antibodies are directed is/are probably part of the cell wall, but its/their exact nature is not known. PMID:2881950

  6. Hemorrhagic Enterotoxemia Caused by Clostridium perfringens Type C in a Foal

    PubMed Central

    Niilo, L.; Chalmers, G. A.

    1982-01-01

    A four day old Appaloosa foal in Alberta died from hemorrhagic enterotoxemia. Beta-toxin of Clostridium perfringens was demonstrated in the intestinal contents of the foal and a pure culture of C. perfringens type C was grown from the small intestine. Histological examination showed hemorrhage and extensive necrosis of the small intestinal mucosa. Areas of necrotic tissue were surrounded by massive numbers of clostridia-like rods. There was also a moderate degree of thyroid hyperplasia. This is believed to be the first published report of hemorrhagic enterotoxemia associated with C. perfringens type C in the horse in Canada. ImagesFigure 1.Figure 2.Figure 3. PMID:17422190

  7. Antimicrobial susceptibility of Clostridium perfringens isolated from piglets with or without diarrhea in Brazil

    PubMed Central

    Salvarani, Felipe Masiero; Silveira Silva, Rodrigo Otávio; Pires, Prhiscylla Sadanã; da Costa Cruz Júnior, Eduardo Coulaud; Albefaro, Isabella Silva; de Carvalho Guedes, Roberto Maurício; Faria Lobato, Francisco Carlos

    2012-01-01

    The minimum inhibitory concentration (MIC) was determined for 13 antibiotics against Clostridium perfringens isolated from Brazilian piglets. The collection of isolates was performed in June to October 2010. All isolates were susceptible to amoxicillin and ceftiofur, whereas most were resistant to tetracycline and lincomycin. Avilamycin and narasin were more effective against isolates from non-diarrheic than from diarrheic piglets. The other antimicrobials were less active in need of high concentrations to inhibit the growth of the C. perfringens type A. These results suggest the need for further studies evaluating molecular factors related to the antimicrobial resistance of C. perfringens. PMID:24031924

  8. Clostridium perfringens toxin genotypes in the feces of healthy North Americans.

    PubMed

    Carman, Robert J; Sayeed, Sameera; Li, Jihong; Genheimer, Christopher W; Hiltonsmith, Megan F; Wilkins, Tracy D; McClane, Bruce A

    2008-04-01

    We investigated the frequency of Clostridium perfringens in the normal fecal flora of healthy North Americans. About half of 43 subjects were colonized with C. perfringens at levels of approximately 10(6)cfu/g feces. Only type A strains were recovered. Spores sometimes outnumbered vegetative cells. Several genotypes were found. Some donors carried two genotypes, some only one. We found no alpha, beta2 or enterotoxin in the stools of any donors. Though some isolates carried toxin genes (e.g. cpe and cpb2) on plasmids, we saw no indication that healthy humans are the reservoir for the chromosomally-borne cpe recovered from cases of C. perfringens food poisoning.

  9. A middle-aged lady with a pyogenic liver abscess caused by Clostridium perfringens

    PubMed Central

    Law, Siu-Tong; Lee, Ming Kai

    2012-01-01

    The pyogenic liver abscess caused by Clostridium perfringens (C. perfringens) is a rare, but rapidly fatal infection. It is usually associated with malignancy and immunosuppression. We report the case of 50-year-old lady with the secondary liver metastases from rectal cancer presented with fever and epigastric pain. The identification of Gram-positive bacilli septicaemia, the presence of gas-forming liver abscess and massive intravascular hemolysis should lead to the suspicion of C. perfringens infection. Here we review twenty cases published since 1990 and their clinical features are discussed. The importance of ”an aggressive treatment policy” with multidisciplinary team approach is emphasized. PMID:22993668

  10. Clostridium Perfringens Infection in a Febrile Patient with Severe Hemolytic Anemia

    PubMed Central

    Hashiba, Masamitsu; Tomino, Atsutoshi; Takenaka, Nobuyoshi; Hattori, Tomonori; Kano, Hideki; Tsuda, Masanobu; Takeyama, Naoshi

    2016-01-01

    Patient: Male, 82 Final Diagnosis: Clostridium perfringens infection Symptoms: Anemia • fever • shock Medication: — Clinical Procedure: Antimicrobial chemotherapy Specialty: Infectious Diseases Objective: Rare disease Background: Clostridium perfringens (C. perfringens) can cause various infections, including gas gangrene, crepitant cellulitis, and fasciitis. While C. perfringens sepsis is uncommon, it is often rapidly fatal because the alpha toxin of this bacterium induces massive intravascular hemolysis by disrupting red blood cell membranes. Case Report: We present the case of a male patient with diabetes who developed a fatal liver abscess with massive intravascular hemolysis and septic shock caused by toxigenic C. perfringens. The peripheral blood smear showed loss of central pallor, with numerous spherocytes. Multiplex PCR only detected expression of the cpa gene, indicating that the pathogen was C. perfringens type A. Conclusions: C. perfringens infection should be considered in a febrile patient who has severe hemolytic anemia with a very low MCV, hemolyzed blood sample, and negative Coombs test. The characteristic peripheral blood smear findings may facilitate rapid diagnosis. PMID:27049736

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

    PubMed

    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.

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

    PubMed Central

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

    2014-01-01

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

  13. Animal models to study the pathogenesis of human and animal Clostridium perfringens infections

    PubMed Central

    Uzal, Francisco A.; McClane, Bruce A.; Cheung, Jackie K.; Theoret, James; Garcia, Jorge P.; Moore, Robert J.; Rood, Julian I.

    2016-01-01

    The most common animal models used to study Clostridium perfringens infections in humans and animals are reviewed here. The classical C. perfringens-mediated histotoxic disease of humans is clostridial myonecrosis or gas gangrene and the use of a mouse myonecrosis model coupled with genetic studies has contributed greatly to our understanding of disease pathogenesis. Similarly, the use of a chicken model has enhanced our understanding of type A-mediated necrotic enteritis in poultry and has led to the identification of NetB as the primary toxin involved in disease. C. perfringens type A food poisoning is a highly prevalent bacterial illness in the USA and elsewhere. Rabbits and mice are the species most commonly used to study the action of enterotoxin, the causative toxin. Other animal models used to study the effect of this toxin are rats, non-human primates, sheep and cattle. In rabbits and mice, CPE produces severe necrosis of the small intestinal epithelium along with fluid accumulation. C. perfringens type D infection has been studied by inoculating epsilon toxin (ETX) intravenously into mice, rats, sheep, goats and cattle, and by intraduodenal inoculation of whole cultures of this microorganism in mice, sheep, goats and cattle. Molecular Koch's postulates have been fulfilled for enterotoxigenic C. perfringens type A in rabbits and mice, for C. perfringens type A necrotic enteritis and gas gangrene in chickens and mice, respectively, for C. perfringens type C in mice, rabbits and goats, and for C. perfringens type D in mice, sheep and goats. PMID:25770894

  14. Animal models to study the pathogenesis of human and animal Clostridium perfringens infections.

    PubMed

    Uzal, Francisco A; McClane, Bruce A; Cheung, Jackie K; Theoret, James; Garcia, Jorge P; Moore, Robert J; Rood, Julian I

    2015-08-31

    The most common animal models used to study Clostridium perfringens infections in humans and animals are reviewed here. The classical C. perfringens-mediated histotoxic disease of humans is clostridial myonecrosis or gas gangrene and the use of a mouse myonecrosis model coupled with genetic studies has contributed greatly to our understanding of disease pathogenesis. Similarly, the use of a chicken model has enhanced our understanding of type A-mediated necrotic enteritis in poultry and has led to the identification of NetB as the primary toxin involved in disease. C. perfringens type A food poisoning is a highly prevalent bacterial illness in the USA and elsewhere. Rabbits and mice are the species most commonly used to study the action of enterotoxin, the causative toxin. Other animal models used to study the effect of this toxin are rats, non-human primates, sheep and cattle. In rabbits and mice, CPE produces severe necrosis of the small intestinal epithelium along with fluid accumulation. C. perfringens type D infection has been studied by inoculating epsilon toxin (ETX) intravenously into mice, rats, sheep, goats and cattle, and by intraduodenal inoculation of whole cultures of this microorganism in mice, sheep, goats and cattle. Molecular Koch's postulates have been fulfilled for enterotoxigenic C. perfringens type A in rabbits and mice, for C. perfringens type A necrotic enteritis and gas gangrene in chickens and mice, respectively, for C. perfringens type C in mice, rabbits and goats, and for C. perfringens type D in mice, sheep and goats.

  15. Bystander Host Cell Killing Effects of Clostridium perfringens Enterotoxin

    PubMed Central

    Shrestha, Archana; Hendricks, Matthew R.; Bomberger, Jennifer M.

    2016-01-01

    ABSTRACT 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. PMID:27965452

  16. Influence of Water Activity on the Growth of Clostridium perfringens

    PubMed Central

    Strong, Dorothy H.; Foster, Edith F.; Duncan, Charles L.

    1970-01-01

    Each of four strains of Clostridium perfringens was grown in modified fluid thioglycolate medium which was adjusted to yield selected water activity (aw) levels. The adjustments to secure the desired aw levels were made with NaCl, KCl, or glucose. At each aw level, further modification was effected to produce four pH values. Cultures were incubated at either 37 or 46 C. The solute used to achieve the reduced aw levels appeared to have a definite effect on the magnitude of growth achieved, the rate of growth, and the limiting aw at which growth would occur. Use of glucose as the controlling solute permitted growth at the lowest aw level tested, 0.960, and yielded the greatest magnitude of growth as measured by turbidity values, at all of the aw levels investigated. Cultures grown in the medium with added KCl generally demonstrated the longest lag times and the least amount of growth. Regardless of specific solute used, as the aw level was lowered and the pH value decreased within each aw level, the rate and amount of growth were lessened. It appeared, however, that low pH values had less effect on inhibiting growth at low aw levels than at higher aw levels. Those cultures incubated at 46 C generally exhibited shorter lag periods than those at 37 C, although the maximal growth attained was somewhat less than that achieved at 37 C. The response to all of the investigated conditions was similar for each of the four strains tested. PMID:4318452

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

    PubMed

    Schnell, Leonie; Mittler, Ann-Katrin; Sadi, Mirko; Popoff, Michel R; Schwan, Carsten; Aktories, Klaus; Mattarei, Andrea; 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.

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

    PubMed

    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.

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

    PubMed

    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.

  20. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium-related diseases such as gangrenous dermatitis (GD) and necrotic enteritis (NE) are increasingly emerging as major diseases in recent years with high economic loss around the world. In this report, we characterized two immunogenic Clostridium perfringens (CP) proteins (e.g., elongation f...

  1. Clostridium perfringens Epsilon Toxin: A Malevolent Molecule for Animals and Man?

    PubMed Central

    Stiles, Bradley G.; Barth, Gillian; Barth, Holger; Popoff, Michel R.

    2013-01-01

    Clostridium perfringens is a prolific, toxin-producing anaerobe causing multiple diseases in humans and animals. One of these toxins is epsilon, a 33 kDa protein produced by Clostridium perfringens (types B and D) that induces fatal enteric disease of goats, sheep and cattle. Epsilon toxin (Etx) belongs to the aerolysin-like toxin family. It contains three distinct domains, is proteolytically-activated and forms oligomeric pores on cell surfaces via a lipid raft-associated protein(s). Vaccination controls Etx-induced disease in the field. However, therapeutic measures are currently lacking. This review initially introduces C. perfringens toxins, subsequently focusing upon the Etx and its biochemistry, disease characteristics in various animals that include laboratory models (in vitro and in vivo), and finally control mechanisms (vaccines and therapeutics). PMID:24284826

  2. Bacteriophages of the family siphoviridae contain amidase enzymes that lyse Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    *Agtech-Danisco, current address In chickens Clostridium perfringens (Cp) is the etiologic agent of necrotic enteritis and causes gas gangrene along with being the third leading cause of bacterial food-borne gastroenteritis in humans. While the disease in poultry can be controlled by antibiotics, th...

  3. BACTERIOPHAGES OF THE FAMILY SIPHOVIRIDAE CONTAIN AMIDASE ENZYMES THAT LYSE CLOSTRIDIUM PERFRINGENS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    In chickens Clostridium perfringens (Cp) is the etiologic agent of necrotic enteritis and causes gas gangrene along with being the third leading cause of bacterial food-borne gastroenteritis in humans. While the disease in poultry can be controlled by antibiotics, there is increasing pressure to ban...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed

    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.

  9. BEC, a Novel Enterotoxin of Clostridium perfringens Found in Human Clinical Isolates from Acute Gastroenteritis Outbreaks

    PubMed Central

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

    2014-01-01

    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. PMID:24664508

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

    PubMed

    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.

  11. Towards an understanding of the role of Clostridium perfringens toxins in human and animal disease

    PubMed Central

    Uzal, Francisco A; Freedman, John C; Shrestha, Archana; Theoret, James R; Garcia, Jorge; Awad, Milena M; Adams, Vicki; Moore, Robert J; Rood, Julian I; McClane, Bruce A

    2014-01-01

    Clostridium perfringens uses its arsenal of >16 toxins to cause histotoxic and intestinal infections in humans and animals. It has been unclear why this bacterium produces so many different toxins, especially since many target the plasma membrane of host cells. However, it is now established that C. perfringens uses chromosomally encoded alpha toxin (a phospholipase C) and perfringolysin O (a pore-forming toxin) during histotoxic infections. In contrast, this bacterium causes intestinal disease by employing toxins encoded by mobile genetic elements, including C. perfringens enterotoxin, necrotic enteritis toxin B-like, epsilon toxin and beta toxin. Like perfringolysin O, the toxins with established roles in intestinal disease form membrane pores. However, the intestinal disease-associated toxins vary in their target specificity, when they are produced (sporulation vs vegetative growth), and in their sensitivity to intestinal proteases. Producing many toxins with diverse characteristics likely imparts virulence flexibility to C. perfringens so it can cause an array of diseases. PMID:24762309

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

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

    2012-01-01

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

  15. Identification and cloning of two immunogenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of C. perfringens.

    PubMed

    Lee, Kyungwoo; Lillehoj, Hyun S; Li, Guangxing; Park, Myeong-Seon; Jang, Seung I; Jeong, Wooseog; Jeoung, Hye-Young; An, Dong-Jun; Lillehoj, Erik P

    2011-12-01

    Clostridium-related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic Clostridium perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with immune sera from commercial meat-type chickens with clinical outbreak of Clostridium infections. In addition to the genes encoding EF-Tu and PFO, C. perfringens alpha-toxin and necrotic enteritis B-like (NetB) toxin were also expressed in Escherichia coli and their corresponding recombinant proteins were purified. Using the four recombinant proteins as target antigens in ELISA immunoassays, high serum antibody titers were observed not only in chickens with clinical signs of Clostridium infections, but also in apparently healthy animals from the same disease-endemic farm. By contrast, no antibodies against any of the proteins were present in the serum of a specific pathogen-free bird. In ELISA using recombinant proteins of C. perfringens, the levels of anti-bacterial protein antibodies were also higher in chickens which were experimentally induced to show NE clinical signs after co-infection with C. perfringens and Eimeria maxima compared with uninfected controls. These results show that two antigenic C. perfringens proteins, EF-Tu and PFO can be useful detection antigens for C. perfringens-afflicted infections in commercial poultry.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the third leading cause of human foodborne bacterial disease and is the presumptive etiologic agent of Necrotic enteritis among chickens. Treatment of poultry with antibiotics is becoming less acceptable. Endolysin enzymes are potential replacements for antibiotics. Man...

  18. New Quantitative, Qualitative, and Confirmatory Media for Rapid Analysis of Food for Clostridium perfringens1

    PubMed Central

    Shahidi, Syed A.; Ferguson, Alphonza R.

    1971-01-01

    A selective and differential medium, Shahidi-Ferguson Perfringens agar (SFP agar), and a confirmatory medium, lactose-motility agar (LM agar), were developed for the enumeration and identification of Clostridium perfringens in foods. These media provide a rapid, specific, and direct diagnosis of C. perfringens. SFP agar contains sodium metabisulfite and ferric ammonium citrate to demonstrate H2S production and egg yolk to demonstrate lecithinase production by C. perfringens. On SFP agar, C. perfringens produces black colonies, 2 to 3 mm in diameter, surrounded by zones of opaque precipitate. The typical colonies are confirmed on LM agar. Enumeration and identification are completed within 48 hr. All of the ingredients of SFP agar are stable to heat and storage conditions. SFP agar also contains two antibiotics, kanamycin and polymyxin B, which are inhibitory to many bacteria commonly occurring in foods. A comparative study of SFP agar and noninhibitory media showed that SFP agar did not inhibit any of the 16 strains of C. perfringens tested. Recovery of C. perfringens added to foods averaged 90.6% for SFP agar as compared with 69.8% for sulfite polymyxin-sulfadiazine (SPS) agar (BBL) and 60.2% for SPS agar (Difco). The colonies on the SFP agar, were much larger and were consistently black. Of 464 food samples tested, C. perfringens was found in 27 samples with SFP agar and in 5 samples with SPS agar (Difco), with a recovery ratio considerably higher on SFP agar. SFP agar is a more specific presumptive medium for the enumeration of C. perfringens and in conjunction with LM agar should save considerable time, effort, and materials toward the final identification of the species. Images PMID:4324195

  19. Effect of direct-fed microbials on performance and Clostridium perfringens colonization of turkey poults.

    PubMed

    Rahimi, S; Kathariou, S; Grimes, J L; Siletzky, R M

    2011-11-01

    Clostridium perfringens is recognized as an enteric pathogen in humans, domestic animals, and livestock. This organism is associated with necrotic enteritis, gangrenous dermatitis, clostridial dermatitis (turkeys), and gizzard erosions in poultry. This study was conducted to evaluate the effectiveness of a direct-fed microbial (DFM), Primalac (Star Labs, Clarksdale, MO), in preventing intestinal colonization of turkey poults with C. perfringens. One-day-old turkey poults (n = 128) were randomly divided into 4 treatments with 4 replicates (8 birds/pen). Treatments were as follows: 1) basal diet without DFM (C); 2) basal diet supplemented with Primalac (1.5 kg/ton; PM); 3) basal diet with poults gavaged with C. perfringens (CCP); and 4) basal diet supplemented with Primalac and poults gavaged with C. perfringens (PMCP). Feed and water were provided ad libitum throughout the trials, and birds were inoculated with C. perfringens (10(8)cfu/mL) on d 3 and 7. On d 21, 2 birds/pen were killed, spleen and bursa of Fabricius were collected and weighed, and cecal contents were used for C. perfringens enumeration. Feed consumption, BW, and feed conversion were calculated throughout the trial (weekly and cumulatively). Data were analyzed using GLM of SAS (SAS Institute, Cary, NC; P < 0.05). Among the inoculated groups, birds fed the DFM-supplemented diet had significantly lower cecal C. perfringens counts than the birds fed the diet without the DFM. The C. perfringens (log(10) cfu/g) in ceca were as follows: C, 5.88; CCP, 7.26; PM, 5.35; PMCP, 6.19 ± 0.36. No differences were observed for BW (814 ± 11 g), feed conversion (1.33 ± 0.03), organ weights, or relative organ weights. Further studies are needed to fully ascertain the potential of using DFM to reduce the numbers of C. perfringens in the gastrointestinal tract of turkey poults.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  2. Clostridium perfringens bacteriophages FCP39O and FCP26F: genomic organization and proteomic analysis of the virions

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Initial screening for bacteriophages lytic for Clostridium perfringens was performed utilizing filtered samples obtained from poultry (intestinal material), soil, sewage and poultry processing drainage water. Lytic phage preparations were initially characterized by transmission electron microscopy ...

  3. BACTERIOCIN E1073 PRODUCED BY ENTEROCOCCUS FAECIUM LWP1073 IS EFFECTIVE FOR TREATING COMMENSAL CLOSTRIDIUM PERFRINGENS INFECTION IN BROILERS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Enterotoxin-producing Clostridium perfringens type A bacteria occupy a significant place in the etiological structure of food-borne infections in humans. One potential approach to minimize infections associated with food-borne pathogens is to control the carriage of C. perfringens in broilers. For ...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium perfringens is the fourth leading cause of bacterial-induced foodborne illnesses with an estimated economic burden of $342M USD per year. In addition to being a foodborne pathogen, C. perfringens is also an economically important poultry pathogen and is one of the known etiologic agents...

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    Li, Jihong; Uzal, Francisco A.; McClane, Bruce A.

    2016-01-01

    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. PMID:27869757

  8. Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in Thailand.

    PubMed

    Ngamwongsatit, Bhinyada; Tanomsridachchai, Wimonrat; Suthienkul, Orasa; Urairong, Supanee; Navasakuljinda, Wichian; Janvilisri, Tavan

    2016-04-01

    Clostridium perfringens causes diarrhea in neonatal piglets, thereby affecting commercial swine farming. The objective of this study was to determine the prevalence and characterize antimicrobial resistance in C. perfringens isolated from diarrheal neonatal piglets in Thailand. A total of 260 rectal swab samples were collected from 13 farms and were subjected to C. perfringens isolation. A total of 148 samples were PCR-positive for C. perfringens toxin genes, from which 122 were recovered. All isolates were cpb2-encoding C. perfringens type A and enterotoxin gene negative. Most of the isolates were susceptible to ampicillin, bacitracin, chlorotetracycline, doxycycline, and oxytetracycline with MIC50 values ranging from 0.32 to 8 μg/ml. The high resistance rates were observed for ceftiofur, enrofloxacin, erythromycin, lincomycin, and tylosin. Among resistant isolates, 82% were resistant to more than one type of antibiotics. The distinct pattern of multiple drug resistance in C. perfringens was observed in different regions, potentially reflecting the farm specific usage of these agents.

  9. Antimicrobial activity of butyrate glycerides toward Salmonella Typhimurium and Clostridium perfringens.

    PubMed

    Namkung, H; Yu, H; Gong, J; Leeson, S

    2011-10-01

    The antimicrobial activities of n-butyric acid and its derivatives against Salmonella Typhimurium and Clostridium perfringens were studied. n-Butyric acid and its derivatives (monobutyrin and a mixture of mono-, di-, and tri-glycerides of butyric acid) were added at different concentrations (ranging from 250 to 7,000 mg/kg to a media inoculated with either Salmonella Typhimurium or C. perfringens. The antimicrobial activity of butyric acid against C. perfringens was measured at 2 bacterium concentrations and 2 inoculations involving ambient aerobic or anaerobic conditions. The most effective antimicrobial activity for Salmonella Typhimurium was observed with n-butyric acid, with 90% inhibition rate at a concentration of 1,500 mg/kg. Although minimal inhibition for Salmonella Typhimurium was observed with butyric acid glycerides, lipase addition to a mixture of mono-, di-, and triglycerides of butyric acid increased (P < 0.01) antimicrobial activity of these derivatives. Antimicrobial activity of butyric acid and its derivative against C. perfringens was higher when using a moderate initial inoculation concentration (10(5)) compared with a higher initial concentration (10(7)) of this bacterium. At a lower inoculation of C. perfringens (10(5)), >90% inhibition rate by all butyric acid glycerides was observed with prior aerobic inoculation at 2,000 mg/kg, whereas using anaerobic inoculation, only 50% monobutyrin maintained >90% inhibitory effect at 3,000 mg/kg. The antimicrobial effect of monobutyrin against C. perfringens was generally higher (P < 0.01) for 50% monobutyrin than for 100% monobutyrin. Either a mixture of butyric acid derivatives or 50% monobutyrin decreased (P < 0.01) C. perfringens in a media containing intestinal contents whereas only 50% monobutyrin decreased (P < 0.01) Salmonella Typhimurium within a media containing cecal contents from mature Leghorns. These results show that n-butyric acid and 50% monobutyrin could be used to control Salmonella

  10. Survival and germination of Clostridium perfringens spores during heating and cooling of ground pork.

    PubMed

    Márquez-González, M; Cabrera-Díaz, E; Hardin, M D; Harris, K B; Lucia, L M; Castillo, A

    2012-04-01

    The effect of heating rate on the heat resistance, germination, and outgrowth of Clostridium perfringens spores during cooking of cured ground pork was investigated. Inoculated cured ground pork portions were heated from 20 to 75°C at a rate of 4, 8, or 12°C/h and then held at 75°C for 48 h. No significant differences (P > 0.05) in the heat resistance of C. perfringens spores were observed in cured ground pork heated at 4, 8, or 12°C/h. At heating rates of 8 and 12°C/h, no significant differences in the germination and outgrowth of spores were observed (P > 0.05). However, when pork was heated at 4°C/h, growth of C. perfringens occurred when the temperature of the product was between 44 and 56°C. In another set of experiments, the behavior of C. perfringens spores under temperature abuse conditions was studied in cured and noncured ground pork heated at 4°C/h and then cooled from 54.4 to 7.2°C within 20 h. Temperature abuse during cooling of noncured ground pork resulted in a 2.8-log CFU/g increase in C. perfringens. In cured ground pork, C. perfringens decreased by 1.1 log CFU/g during cooling from 54.4 to 36.3°C and then increased by 0.9 log CFU/g until the product reached 7.2°C. Even when the initial level of C. perfringens spores in cured ground pork was 5 log CFU/g, the final counts after abusive cooling did not exceed 3.4 log CFU/g. These results suggest that there is no risk associated with C. perfringens in cured pork products under the tested conditions.

  11. Effect of continuous sub-culturing on infectivity of Clostridium perfringens ATCC13124 in mouse gas gangrene model.

    PubMed

    Kumar, Ravi Bhushan; Alam, Syed Imteyaz

    2017-02-17

    Clostridium perfringens is a Validated Biological Agent and a pathogen of medical, veterinary, and military significance. Gas gangrene is the most destructive of all the clostridial diseases and is caused by C. perfringens type A strains wherein the infection spreads quickly (several inches per hour) with production of gas. Influence of repeated in vitro cultivation on the infectivity of C. perfringens was investigated by comparing the surface proteins of laboratory strain and repository strains of the bacterium using 2DE-MS approach. In order to optimize host-pathogen interaction during experimental gas gangrene infection, we also explored the role of particulate matrix on ability of C. perfringens to cause gas gangrene.

  12. Characterization of a parasporal inclusion body from sporulating, enterotoxin-positive Clostridium perfringens type A.

    PubMed Central

    Löffler, A; Labbé, R

    1986-01-01

    Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens NCTC 8239 and 8798 were isolated and characterized. IB were isolated by disruption of sporangia by sonication in the presence of tetrasodium EDTA and phenylmethylsulfonyl fluoride. Fractionation was carried out in a linear gradient of sodium bromide, sucrose, or diatrizoate sodium. Denaturing and reducing agents were necessary to solubilize the IB. An alkylating agent was required to prevent reaggregation of the subunits. Molecular weight, compositional, and serological analyses and peptide mapping revealed strong similarities between the IB subunits and the enterotoxin synthesized during sporulation by C. perfringens. IB appear to represent the structural component where overproduced enterotoxin accumulates intracellularly. Enterotoxin-like subunits in the IB appeared to be held together by noncovalent and disulfide bonds, which were generally resistant to the action of intracellular proteases of C. perfringens, trypsin, or trypsin plus bile salts. Images PMID:2867991

  13. Prevalence of β2-Toxigenic Clostridium perfringens in Horses with Intestinal Disorders

    PubMed Central

    Herholz, Cornelia; Miserez, Raymond; Nicolet, Jacques; Frey, Joachim; Popoff, Michel; Gibert, Maryse; Gerber, Heinz; Straub, Reto

    1999-01-01

    The incidence of a new, yet unassigned toxin type of Clostridium perfringens containing the genes for the α-toxin and the recently described β2-toxin in horses with intestinal disorders is reported. The study included 18 horses suffering from typical typhlocolitis, 7 horses with atypical typhlocolitis, 16 horses with other intestinal disorders, and 58 horses without intestinal disease. In total, 20 samples of ingesta of the small and large intestines, five biopsy specimens of the intestinal wall, and 74 fecal samples were analyzed bacteriologically. C. perfringens isolates were typed for the presence of the α-, β-, β2-, and ɛ-toxin and enterotoxin genes by PCR, including a newly developed PCR for the detection of the β2-toxin gene cpb2. β2-Toxigenic C. perfringens was detected in samples from 13 of 25 (52%) horses with typical or atypical typhlocolitis, with a particularly high incidence in specimens of ingesta and biopsy specimens (75%), whereas only 6 of 16 specimens from horses with other intestinal diseases yielded β2-toxigenic C. perfringens. No β2-toxigenic C. perfringens was found in the samples from the 58 control horses, of which only one fecal sample contained C. perfringens type A. Among the samples from the 15 horses with fatal cases of typical and atypical typhlocolitis 9 (60%) were positive for β2-toxigenic C. perfringens, whereas samples from only 4 of the 10 (40%) animals with nonfatal cases of infection were positive. We found an interesting correlation between the antibiotic-treated horses which were positive for β2-toxigenic C. perfringens and lethal progression of the disease. No C. perfringens strains isolated in this study contained genes for the β- and ɛ-toxins and enterotoxin. The high incidence of β2-toxigenic C. perfringens in samples of ingesta, biopsy specimens of the intestinal wall, and feces from horses suffering or dying from typhlocolitis together with the absence of this organism in healthy horses provides strong

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

    PubMed Central

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

    2016-01-01

    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 (α1 = 1, α2 = 91; α1 = 1, α2 = 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

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

    PubMed

    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

  16. Effect of 3',5'-cyclic diguanylic acid in a broiler Clostridium perfringens infection model.

    PubMed

    Fatima, Mussarat; Rempel, Heidi; Kuang, Xiaomei Tallie; Allen, Kevin J; Cheng, Kimberly M; Malouin, François; Diarra, Moussa S

    2013-10-01

    In an effort to explore strategies to control Clostridium perfringens, we investigated the synergistic effect of a ubiquitous bacterial second messenger 3',5'-cyclic diguanylic acid (c-di-GMP) with penicillin G in a broiler challenge model. All chicks were inoculated in the crop by gavage on d 14, 15, and 16 with a mixture of 4 C. perfringens strains. Birds were treated with saline (control group) or 20 nmol of c-di-GMP by gavage or intramuscularly (IM) on d 24, all in conjunction with penicillin G in water for 5 d. Weekly samplings of ceca and ileum were performed on d 21 to 35 for C. perfringens and Lactobacillus enumeration. On d 35 of age, the IM treatment significantly (P < 0.05) reduced C. perfringens in the ceca, suggesting possible synergistic activity between penicillin G and c-di-GMP against C. perfringens in broiler ceca. Moreover, analysis of ceca DNA for the presence of a series of C. perfringens virulence genes showed a prevalence of 30% for the Clostridium perfringens alpha-toxin gene (cpa) from d 21 to 35 in the IM-treated group, whereas the occurrence of the cpa gene increased from 10 to 60% in the other 2 groups (control and gavage) from d 21 to 35. Detection of β-lactamase genes (blaCMY-2, blaSHV, and blaTEM) indicative of gram-negative bacteria in the same samples from d 21 to 35 did not show significant treatment effects. Amplified fragment-length polymorphism showed a predominant 92% similarity between the ceca of 21-d-old control birds and the 35-d-old IM-treated c-di-GMP group. This suggests that c-di-GMP IM treatment might be effective at restoring the normal microflora of the host on d 35 after being challenged by C. perfringens. Our results suggest that c-di-GMP can reduce the colonization of C. perfringens in the gut without increasing the selection pressure for some β-lactamase genes or altering the commensal bacterial population.

  17. Spoilage of an acid food product by Clostridium perfringens, C. barati and C. butyricum.

    PubMed

    de Jong, J

    1989-05-01

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

  18. [A case of freeze-dried gas gangrene antitoxin for the treatment of Clostridium perfringens sepsis].

    PubMed

    Yoshida, Juichiro; Nakamura, Hideki; Yamada, Shinya; Sekoguchi, Satoru; Suzuki, Takahiro; Tomatsuri, Naoya; Sato, Hideki; Okuyama, Yusuke; Kimura, Hiroyuki; Yoshida, Norimasa

    2015-02-01

    A 66-year-old man was admitted to our hospital with high fever. We diagnosed a gas-containing liver abscess and performed percutaneous abscess drainage. However, 15 hours after admission, he developed massive intravascular hemolysis and acidosis. Sepsis due to Clostridium perfringens was suspected and we treated the patient intensively with multidisciplinary approaches, including antibiotics, mechanical ventilation, and renal replacement therapy. Furthermore, we administered freeze-dried gas gangrene antitoxin. Despite intensive care, the patient died 43 hours after admission.

  19. Evaluation of fluorogenic TSC agar for recovering Clostridium perfringens in groundwater samples.

    PubMed

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

    2001-01-01

    Clostridium perfringens is widely recognised as a reliable water pollution indicator. Since several media can be employed for the membrane filtration enumeration of this microorganism, the main aim of this work was to investigate the ability of fluorocult-supplemented TSC-agar (Merck) for recovering Cl. perfringens from public springs used for direct human consumption. Cl. perfringens recovery was also performed on mCP agar (Cultimed) according to Directive 98/83 as well as on TSC-Agar (Merck), TSN-Agar (Merck) and SPS-Agar (BBL) media. Variance analysis of data obtained showed no statistically significant differences in the counts obtained among all media employed in this work. However, the Cl. perfringens recovery efficiencies with TSC and fluorogenic TSC agars were significantly greater (P = < 0.05) than the corresponding values of mCP and TSN media. On the other hand, the identification of typical and atypical colonies isolated from all media demonstrated that fluorogenic TSC agar was the most specific medium for Cl. perfringens recovery in groundwater samples (85.3% of typical colonies and 82.8% of atypical colonies confirmed). In summary, the membrane filtration technique with fluorogenic TSC agar showed the best performance characteristics of all the media tested as judged by their recovery efficiency and specificity in these water samples.

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

    PubMed

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

    2004-05-01

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

  1. Severe Sepsis due to Clostridium perfringens Bacteremia of Urinary Origin: A Case Report and Systematic Review

    PubMed Central

    Millard, Michael A.; McManus, Kathleen A.; Wispelwey, Brian

    2016-01-01

    Clostridium perfringens bacteremia is an uncommon yet serious clinical syndrome that typically arises from a gastrointestinal source. However, clinicians should consider nongastrointestinal sources as well. We present a rare case of C. perfringens bacteremia of urinary origin that required surgical intervention for definitive treatment. A 61-year-old male presented with acute nausea and vomiting, altered mental status, and chronic diarrhea. His physical exam revealed right costovertebral tenderness and his laboratory work-up revealed acute renal failure. Percutaneous blood cultures grew C. perfringens. Cross-sectional imaging revealed a right-sided ureteral stone with hydronephrosis, which required nephrostomy placement. On placement of the nephrostomy tube, purulent drainage was identified and Gram stain of the drainage revealed Gram-variable rods. A urinary source of C. perfringens was clinically supported. Although it is not a common presentation, nongastrointestinal sources such as a urinary source should be considered in C. perfringens bacteremia because failure to recognize a nongastrointestinal source can delay appropriate treatment, which may include surgical intervention. PMID:26998370

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

    PubMed

    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.

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

    PubMed

    Filho, E J F; Carvalho, A U; Assis, R A; Lobato, F F; Rachid, M A; Carvalho, A A; Ferreira, P M; Nascimento, R A; Fernandes, A A; Vidal, J E; Uzal, F A

    2009-11-01

    This study was designed to experimentally reproduce enterotoxemia by Clostridium perfringens type D in cattle and to characterize the clinicopathologic findings of this disease. Fourteen 9-month-old calves were inoculated intraduodenally according to the following schedule: group 1 (n = 4), C. perfringens type D whole culture; group 2 (n = 3), C. perfringens type D washed cells; group 3 (n = 5), C. perfringens type D filtered and concentrated supernatant; group 4 (n = 2), sterile, nontoxic culture medium. In addition, all animals received a 20% starch solution in the abomasum. Ten animals from groups 1 (4/4), 2 (3/3), and 3 (3/5) showed severe respiratory and neurologic signs. Gross findings were observed in these 10 animals and consisted of acute pulmonary edema, excessive protein-rich pericardial fluid, watery contents in the small intestine, and multifocal petechial hemorrhages on the jejunal mucosa. The brain of one animal of group 2 that survived for 8 days showed multifocal, bilateral, and symmetric encephalomalacia in the corpus striatum. The most striking histologic changes consisted of perivascular high protein edema in the brain, and alveolar and interstitial proteinaceous pulmonary edema. The animal that survived for 8 days and that had gross lesions in the corpus striatum showed histologically severe, focal necrosis of this area, cerebellar peduncles, and thalamus. Koch's postulates have been met and these results show that experimental enterotoxemia by C. perfringens type D in cattle has similar clinical and pathologic characteristics to the natural and experimental disease in sheep.

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

    SciTech Connect

    Hill, R.T.; Knight, I.T.; Anikis, M.S.; Colwell, R.R.

    1993-01-01

    Clostridium perfringens in sediment samples collected at the Deep Water Municipal Sewage Sludge Disposal Site (also called the 106-Mile Site), off the coast of New Jersey, was enumerated. The counts of C. perfringens found in sediment samples collected within and to the southwest of the 106-Mile Site were significantly elevated (P. < 0.01) compared with counts of samples from reference stations of similar depth (2,400 to 2,700 m), topography, and distance from the continental shelf, indicating that the benthic environment was contaminated by sewage dumping at the site. Low counts of C. perfringens in sediment samples collected at stations between the base of the continental shelf and the 106-Mile Site indicated that coastal runoff was not a significant source of contamination. Elevated counts were observed for samples up to 92 km to the southwest, whereas low counts were obtained for samples from stations to the east of the 106-Mile Site. The distribution is consistent with previous model predictions of sludge deposition. In areas heavily impacted by sludge dumping, C. perfringens counts were generally highest in the top 1 cm of sediment and exceeded 9,000 CFU g (dry weight) of sediment. The patterns of C. perfringens dispersal observed in the study have proved useful for selection of heavily impacted areas and control stations for further ecological evaluation by a multidisciplinary research team.

  5. Non-classical azoreductase secretion in Clostridium perfringens in response to sulfonated azo dye exposure.

    PubMed

    Morrison, Jessica M; John, Gilbert H

    2015-08-01

    Clostridium perfringens, a strictly anaerobic microorganism and inhabitant of the human intestine, has been shown to produce an azoreductase enzyme (AzoC), an NADH-dependent flavin oxidoreductase. This enzyme reduces azo dyes into aromatic amines, which can be carcinogenic. A significant amount of work has been completed on the activity of AzoC. Despite this, much is still unknown, including whether azoreduction of these dyes occurs intracellularly or extracellulary. A physiological study of C. perfringens involving the effect of azo dye exposure was completed to answer this question. Through exposure studies, azo dyes were found to cause cytoplasmic protein release, including AzoC, from C. perfringens in dividing and non-dividing cells. Sulfonation (negative charge) of azo dyes proved to be the key to facilitating protein release of AzoC and was found to be azo-dye-concentration-dependent. Additionally, AzoC was found to localize to the Gram-positive periplasmic region. Using a ΔazoC knockout mutant, the presence of additional azoreductases in C. perfringens was suggested. These results support the notion that the azoreduction of these dyes may occur extracellularly for the commensal C. perfringens in the intestine.

  6. Humans as Reservoir for Enterotoxin Gene–carrying Clostridium perfringens Type A

    PubMed Central

    Lindström, Miia; Granum, Per Einar; Korkeala, Hannu

    2006-01-01

    We found a prevalence of 18% for enterotoxin gene–carrying (cpe+) Clostridium perfringens in the feces of healthy food handlers by PCR and isolated the organism from 11 of 23 PCR-positive persons by using hydrophobic grid membrane filter-colony hybridization. Several different cpe genotypes were recovered. The prevalence was 3.7% for plasmidial IS1151-cpe, 2.9% for plasmidial IS1470-like-cpe, 0.7% for chromosomal IS1470-cpe, and 1.5% for unknown cpe genotype. Lateral spread of cpe between C. perfringens strains was evident because strains from the same person carried IS1470-like cpe but shared no genetic relatedness according to pulsed-field gel electrophoresis analysis. Our findings suggest that healthy humans serve as a rich reservoir for cpe+ C. perfringens type A and may play a role in the etiology of gastrointestinal diseases caused by this organism. The results also indicate that humans should be considered a risk factor for spread of C. perfringens type A food poisoning and that they are a possible source of contamination for C. perfringens type A food poisoning. PMID:17283623

  7. [Cloning of Clostridium perfringens alpha-toxin gene and extracellular expression in Escherichia coli].

    PubMed

    Inoue, Masaharu; Kikuchi, Maho; Komoriya, Tomoe; Watanabe, Kunitomo; Kouno, Hideki

    2007-01-01

    Clostridium perfringens (C. perfringens) is a Gram-positive bacterial pathogen that widely propagets in the soil and the gastrointestinal tract of human and animals. This bacteria causes food poisoning, gas gangrene and other various range of infectious diseases. But there is no standard diagnosis method of C. perfringens. In order to develop a new type of immunoassay for clinical purpose, we studied expression and extracellular secretion of recombinant alpha-toxin having enzyme activity in E. coli expression system. Cloning was carried out after PCR amplification from C. perfringens GAI 94074 which was clinical isolate. Three kinds of fragment were cloned using pET100/D-TOPO vector. These fragments coded for ribosome binding site, signal peptide, and alpha-toxin gene respectively. Recombinant pET100 plasmid transformed into TOP 10 cells and the obtained plasmids were transformed into BL21 (DE3) cells. Then, the transformants were induced expression with IPTG. In conclusion, we successfully cloned, expressed and exteracellular secreted C. perfringens alpha-toxin containing signal peptide. Biologically, the obtained recombinant protein was positive for phospholipase C activity.

  8. Membrane vesicles of Clostridium perfringens Type A strains induce innate and adaptive immunity

    PubMed Central

    Jiang, Yanlong; Kong, Qingke; Roland, Kenneth L.; Curtiss, Roy

    2014-01-01

    Vesicle shedding from bacteria is a universal process in most Gram-negative bacteria and a few Gram-positive bacteria. In this report, we isolate extracellular membrane vesicles (MVs) from the supernatants of Gram-positive pathogen Clostridium perfringens (C. perfringens). We demonstrated vesicle production in a variety of virulent and nonvirulent type A strains. MVs did not contain alpha-toxin and NetB toxin demonstrated by negative reaction to specific antibody and absence of specific proteins identified by LC-MS/MS. C. perfringens MVs contained DNA components such as 16S ribosomal RNA gene (16S rRNA), alpha-toxin gene (plc) and the perfringolysin O gene (pfoA) demonstrated by PCR. We also identified a total of 431 proteins in vesicles by 1-D gel separation and LC-MS/MS analysis. In vitro studies demonstrated that vesicles could be internalized into murine macrophage RAW264.7 cells without direct cytotoxicity effects, causing release of inflammation cytokines including granulocyte colony stimulating factor (G-CSF), tumor necrosis factor-alpha (TNF-α) and interleukin-1 (IL-1), which could also be detected in mice injected with MVs through intraperitoneal (i.p.) route. Mice immunized with C. perfringens MVs produced high titer IgG, especially IgG1, antibodies against C. perfringens membrane proteins. However, this kind of antibody could not provide protection in mice following challenge, though it could slightly postpone the time of death. Our results indicate that release of MVs from C. perfringens could provide a previously unknown mechanism to induce release of inflammatory cytokines, especially TNF-α, these findings may contribute to a better understanding of the pathogenesis of C. perfringens infection. PMID:24631214

  9. Coccidia-induced mucogenesis promotes the onset of necrotic enteritis by supporting Clostridium perfringens growth.

    PubMed

    Collier, C T; Hofacre, C L; Payne, A M; Anderson, D B; Kaiser, P; Mackie, R I; Gaskins, H R

    2008-03-15

    This study tested the hypothesis that a host mucogenic response to an intestinal coccidial infection promotes the onset of necrotic enteritis (NE). A chick NE model was used in which birds were inoculated with Eimeria acervulina and E. maxima and subsequently with Clostridium perfringens (EAM/CP). A second group of EAM/CP-infected birds was treated with the ionophore narasin (NAR/EAM/CP). These groups were compared to birds that were either non-infected (NIF), or infected only with E. acervulina and E. maxima (EAM), or C. perfringens (CP). The impact of intestinal coccidial infection and anti-coccidial treatment on host immune responses and microbial community structure were evaluated with histochemical-, cultivation- and molecular-based techniques. Barrier function was compromised in EAM/CP-infected birds as indicated by elevated CFUs for anaerobic bacteria and C. perfringens in the spleen when compared to NIF controls at day 20, with a subsequent increase in intestinal NE lesions and mortality at day 22. These results correlate positively with a host inflammatory response as evidenced by increased ileal interleukin (IL)-4, IL-10 and IFN-gamma RNA expression. Concurrent increases in chicken intestinal mucin RNA expression, and goblet cell number and theca size indicate that EAM/CP induced an intestinal mucogenic response. Correspondingly, the growth of mucolytic bacteria and C. perfringens as well as alpha toxin production was greatest in EAM/CP-infected birds. The ionophore narasin, which directly eliminates coccidia, reduced goblet cell theca size, IL-10 and IFN-gamma expression, the growth of mucolytic bacteria including C. perfringens, coccidial and NE lesions and mortality in birds that were co-infected with coccidia and C. perfringens. Collectively the data support the hypothesis that coccidial infection induces a host mucogenic response providing a growth advantage to C. perfringens, the causative agent of NE.

  10. Identification and cloning of two immunogenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO) of Clostridium perfringens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Clostridium related poultry diseases such as necrotic enteritis (NE) and gangrenous dermatitis (GD) cause substantial economic losses on a global scale. Two antigenic C. perfringens proteins, elongation factor Tu (EF-Tu) and pyruvate:ferredoxin oxidoreductase (PFO), were identified by reaction with...

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

    PubMed Central

    Charlebois, Audrey; Jacques, Mario; Archambault, Marie

    2014-01-01

    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. This study brings new information on the biofilm produced by C. perfringens and its exposure to low doses of antimicrobials. PMID:24795711

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

    PubMed

    Charlebois, Audrey; Jacques, Mario; Archambault, Marie

    2014-01-01

    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. This study brings new information on the biofilm produced by C. perfringens and its exposure to low doses of antimicrobials.

  13. Transcriptional Profile during Deoxycholate-Induced Sporulation in a Clostridium perfringens Isolate Causing Foodborne Illness

    PubMed Central

    Okuzaki, Daisuke; Kuwana, Ritsuko; Takamatsu, Hiromu; Fujita, Masaya; Sarker, Mahfuzur R.; Miyake, Masami

    2016-01-01

    ABSTRACT Clostridium perfringens type A is a common source of foodborne illness (FBI) in humans. Vegetative cells sporulate in the small intestinal tract and produce the major pathogenic factor C. perfringens enterotoxin. Although sporulation plays a critical role in the pathogenesis of FBI, the mechanisms inducing sporulation remain unclear. Bile salts were shown previously to induce sporulation, and we confirmed deoxycholate (DCA)-induced sporulation in C. perfringens strain NCTC8239 cocultured with human intestinal epithelial Caco-2 cells. In the present study, we performed transcriptome analyses of strain NCTC8239 in order to elucidate the mechanism underlying DCA-induced sporulation. Of the 2,761 genes analyzed, 333 were up- or downregulated during DCA-induced sporulation and included genes for cell division, nutrient metabolism, signal transduction, and defense mechanisms. In contrast, the virulence-associated transcriptional regulators (the VirR/VirS system, the agr system, codY, and abrB) were not activated by DCA. DCA markedly increased the expression of signaling molecules controlled by Spo0A, the master regulator of the sporulation process, whereas the expression of spo0A itself was not altered in the presence or absence of DCA. The phosphorylation of Spo0A was enhanced in the presence of DCA. Collectively, these results demonstrated that DCA induced sporulation, at least partially, by facilitating the phosphorylation of Spo0A and activating Spo0A-regulated genes in strain NCTC8239 while altering the expression of various genes. IMPORTANCE Disease caused by Clostridium perfringens type A consistently ranks among the most common bacterial foodborne illnesses in humans in developed countries. The sporulation of C. perfringens in the small intestinal tract is a key event for its pathogenesis, but the factors and underlying mechanisms by which C. perfringens sporulates in vivo currently remain unclear. Bile salts, major components of bile, which is secreted

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

    PubMed

    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.

  15. [Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica].

    PubMed

    Gamboa-Coronado, María del Mar; Mau-Inchaustegui, Silvia; Rodríguez-Cavallini, Evelyn

    2011-12-01

    Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins alpha, beta, epsilon and iota, C. perfringens is classified into five toxinotypes (A-E). Some strains produce an enterotoxin (CPE), encoded by the cpe gene, which causes diarrhea in humans and some animals. C. perfringens strains that had been previously isolated and been kept at -80 degrees C were analyzed for the presence of toxin genes and for antimicrobial resistance: 20 from soils, 20 from animal, 20 from human origin and 21 from food non related to outbreaks. According to PCR results, all strains were classified as C. perfringens type A, since only alpha toxin gene was detected, while cpe was detected in two strains (2.5%) isolated from food, as it has been described in other world regions. Antibiotic resistance to at least one antibiotic was detected in 44% of the strains, 41% was resistant to clindamycin, 25% to chloramphenicol, 22% to penicillin and 20% to metronidazole. Soils strains showed the highest resistance percentages to almost all antibiotics. Multiresistance (to three or more antibiotic groups) was detected in the strains from soil (40%), human origin (30%), food (14%) and animal origin (5%). The high resistance rates found may be explained by the widespread use of antimicrobials as growth promoters in plants and animals; also these resistant strains may act as reservoir of resistance genes that may be transferred between bacteria in different environments.

  16. Development and application of an enzyme linked immunosorbent assay for Clostridium perfringens type A enterotoxin.

    PubMed Central

    Bartholomew, B A; Stringer, M F; Watson, G N; Gilbert, R J

    1985-01-01

    An enzyme linked immunosorbent assay (ELISA) has been developed to quantitate faecal Clostridium perfringens enterotoxin in the investigation of C perfringens food poisoning. The sandwich ELISA could be carried out in 24 h and was sensitive enough to detect as little as 5 ng/g of enterotoxin in faeces. Specificity of the assay was shown by comparing results with those obtained from other standard toxin assays, such as double gel diffusion and counterimmunoelectrophoresis, and by the assay of faecal material from control groups. By means of the ELISA method, 515 faecal samples from 50 separate outbreaks of C perfringens food poisoning were examined, together with 21 food samples from 12 of the outbreaks. A clear distinction was noted between faecal samples collected on the first two days of an outbreak, where 77% were enterotoxin positive, and those specimens collected later than the second day, when only 33% had detectable enterotoxin. The ELISA is recommended as a valuable tool in the investigation of C perfringens foodborne illness. PMID:2857184

  17. Gas gangrene due to Clostridium perfringens in two injecting drug users in Vienna, Austria.

    PubMed

    Assadian, Ojan; Assadian, Afshin; Senekowitsch, Christian; Makristathis, Athanasios; Hagmüller, George

    2004-04-30

    We describe two cases of severe myonecrotic infections caused by Clostridium perfringens in injecting drug users (IDUs) in Vienna, Austria. Clostridial myonecrosis, or gas gangrene, is a clostridial infection primarily of muscle tissue. C. perfringens is isolated in 90% of these infections. Other clostridial species isolated are C. novyi, C. septicum, C. histolyticum, C. fallax, and C. bifermentans. Classically, clostridial myonecrosis has an acute presentation and a fulminant clinical course. It is diagnosed mainly on a clinical basis. The infection may be so rapidly progressive that any delay in recognition or treatment may be fatal. The onset is sudden, often within 4 to 6 hours after an injury. An early clinical finding is sudden severe pain in the area of infection. Swelling and edema in the area of infection is pronounced. At surgery, the infected muscle is dark-red to black, is noncontractile, and does not bleed when cut. Crepitus, although not prominent, is sometimes detected. We were able to demonstrate spores that were morphologically indistinguishable from spores of C. perfringens in a drug sample obtained from case 2. General practitioners and accident and emergency staff should be aware of the possibility of C. perfringens infection in IDUs, especially if injection into soft tissue is suspected.

  18. Screening of Bacteriocin-producing Enterococcus faecalis Strains for Antagonistic Activities against Clostridium perfringens

    PubMed Central

    Kim, So-Young

    2014-01-01

    This study was conducted to isolate and characterize bacteriocin-producing bacteria against Clostridium perfringens (C. perfringens) from domestic animals to determine their usefulness as probiotics. Bacteriocin-producing bacteria were isolated from pig feces by the spot-on-lawn method. A total of 1,370 bacterial stains were isolated, and six were tentatively selected after identifying the inhibitory activity against the pathogenic indicator C. perfringens KCTC 3269 and KCTC 5100. The selected strains were identified as Enterococcus faecalis (E. faecalis) by 16s rRNA sequencing. Most of the isolated bacterial strains were resistant to 0.5% bile salts for 48 h and remained viable after 2 h at pH 3.0. Some E. faecalis also showed strong inhibitory activity against Listeria monocytogenes KCTC 3569, KCTC 3586 and KCTC 3710. In the present study, we finally selected E. faecalis AP 216 and AP 45 strain based on probiotic selection criteria such as antimicrobial activity against C. perfringens and tolerance to acid and bile salts. The bacteriocins of E. faecalis AP 216 and AP 45 strains were highly thermostable, showing anticlostridial activities even after incubation at 121℃ for 15 min. These bacteriocinproducing bacteria and/or bacteriocins could be used in feed manufacturing as probiotics as an alternative to antibiotics in the livestock industry. PMID:26761495

  19. Uterine Perforation with Intra-Abdominal Clostridium perfringens Gas Gangrene: A Rare and Fatal Infection.

    PubMed

    Kashan, David; Muthu, Nagarajan; Chaucer, Benjamin; Davalos, Fidencio; Bernstein, Michael; Chendrasekhar, Akella

    2016-06-01

    Background:Clostridium perfringens gas gangrene is an extremely rare and fatal infection. Necrosis of the myometrium is rarely seen and has only been recorded in 18 cases to date. Of these 18 reported cases, only 5 have occurred in nonpregnant women. This article presents the 6th case of myometrium necrosis from C. perfringens.Case: A 72-year-old woman, gravida 2, para 2, presented with abdominal pain and vaginal bleeding. After examinations, laboratory testing, and several surgical interventions, she was found to have C. perfringens infection and advanced high-grade serous adenocarcinoma of the endometrium with >50% invasion into the myometrium. Results: Despite the surgical interventions and use of several antibiotics, this patient did not improve. She was weaned from treatment per her advance directive and died after weaning. Conclusions: Awareness of the many etiologies for peritonitis is of great importance when a fatal infection may be the cause of the condition. Correct diagnosis and proper treatment is essential for the survival of patients infected with C. perfringens. (J GYNECOL SURG 32:182).

  20. Uterine Perforation with Intra-Abdominal Clostridium perfringens Gas Gangrene: A Rare and Fatal Infection

    PubMed Central

    Kashan, David; Muthu, Nagarajan; Davalos, Fidencio; Bernstein, Michael; Chendrasekhar, Akella

    2016-01-01

    Abstract Background: Clostridium perfringens gas gangrene is an extremely rare and fatal infection. Necrosis of the myometrium is rarely seen and has only been recorded in 18 cases to date. Of these 18 reported cases, only 5 have occurred in nonpregnant women. This article presents the 6th case of myometrium necrosis from C. perfringens. Case: A 72-year-old woman, gravida 2, para 2, presented with abdominal pain and vaginal bleeding. After examinations, laboratory testing, and several surgical interventions, she was found to have C. perfringens infection and advanced high-grade serous adenocarcinoma of the endometrium with >50% invasion into the myometrium. Results: Despite the surgical interventions and use of several antibiotics, this patient did not improve. She was weaned from treatment per her advance directive and died after weaning. Conclusions: Awareness of the many etiologies for peritonitis is of great importance when a fatal infection may be the cause of the condition. Correct diagnosis and proper treatment is essential for the survival of patients infected with C. perfringens. (J GYNECOL SURG 32:182) PMID:27274183

  1. Increase of Clostridium perfringens in association with Eimeria in haemorrhagic enteritis in Japanese beef cattle.

    PubMed

    Kirino, Y; Tanida, M; Hasunuma, H; Kato, T; Irie, T; Horii, Y; Nonaka, N

    2015-08-18

    A coprological survey with detailed clinical observation of naturally occurring haemorrhagic enteritis (HE) cases was conducted to understand the pathophysiology of HE by clarifying the infection status of Eimeria and enteropathogenic bacteria in cattle. Faecal samples from 55 cases of HE and 26 clinically normal animals were collected, and a quantitative examination of Eimeria and potential enteropathogenic bacteria was performed. The number of Eimeria species oocysts per gram of faeces (OPG) exceeded 10,000 in 69.1 per cent of HE cases with a maximum of 1,452,500 OPG and Eimeria zuernii was found to be overwhelmingly dominant. A significant increase in faecal coliform count was observed in HE cases compared with clinically normal animals. Among the animals shedding >10,000 OPG, 42.9 per cent showed a remarkable increase in Clostridium perfringens abundance (>10(4) CFU/g) in the faeces. In the cases with C. perfringens detected, its abundance was positively correlated with Eimeria OPG and high C. perfringens abundance was always accompanied by high Eimeria OPG. E. zuernii is likely to play a crucial role in massive multiplication of C. perfringens in HE in cattle.

  2. Use of power ultrasound to enhance the thermal inactivation of Clostridium perfringens spores in beef slurry.

    PubMed

    Evelyn; Silva, Filipa V M

    2015-08-03

    Clostridium perfringens is a pathogen of concern in pasteurised foods. The main objective of this study was to use power ultrasound to enhance the thermal inactivation of C. perfringens spores in beef slurry. The effect of simultaneous ultrasound and heat (TS, thermosonication) on the spore inactivation in beef slurry was first investigated. At 75 °C, a 60 min TS process (24 kHz, 0.33 W/g) resulted in a less than 1.5 log reduction for both C. perfringens NZRM 898 and NZRM 2621 spores. Then, the thermal inactivation first order kinetic parameters of C. perfringens spores in beef slurry were estimated for the two strains. The D105 °C- and z-values were 2.5 min and 10.6 °C for NZRM 898 and 1.8 min and 10.9 °C for NZRM 2621. After, the effect of a spore heat shock followed by ultrasound on its thermal inactivation in beef slurry was investigated. This heat shock+ultrasound pretreatment was able to double the spore thermal inactivation rate in beef slurry. For example at 95 °C D-value of 20.2 min decreased to 9.8 min, demonstrating that spore exposure to heat shock followed by ultrasonication enhanced its thermal inactivation.

  3. An investigation into the association between cpb2-encoding Clostridium perfringens type A and diarrhea in neonatal piglets

    PubMed Central

    Farzan, Abdolvahab; Kircanski, Jasmina; DeLay, Josepha; Soltes, Glenn; Songer, J. Glenn; Friendship, Robert; Prescott, John F.

    2013-01-01

    To investigate the possible role of cpb2-positive type A Clostridium perfringens in neonatal diarrheal illness in pigs, the jejunum and colon of matched normal and diarrheic piglets from 10 farms with a history of neonatal diarrhea were examined grossly and by histopathology, and tested for C. perfringens, for C. perfringens beta2 (CPB2) toxin, as well as for Clostridium difficile toxins, Salmonella, enterotoxigenic Escherichia coli, rotavirus, transmissible gastroenteritis (TGE) virus, and coccidia. Clostridium perfringens isolates were tested using a multiplex real-time polymerase chain reaction (PCR) to determine the presence of cpa, consensus and atypical cpb2, and other virulence-associated genes. The numbers of C. perfringens in the intestinal contents were lower in diarrheic piglets (log10 5.4 CFU/g) compared with normal piglets (log10 6.5 CFU/g) (P < 0.05). The consensus cpb2 was present in 93% of isolates in each group, but atypical cpb2 was less common (56% healthy, 32% diarrheic piglets isolates, respectively, P < 0.05). The presence of CPB2 toxin in the intestinal contents of normal and diarrheic piglets did not differ significantly. Clostridium difficile toxins and rotavirus were each detected in 7 of the 21 (33%) diarrheic piglets. Rotavirus, C. difficile toxins, Salmonella, or enterotoxigenic E. coli were concurrently recovered in different combinations in 4 diarrheic piglets. The cause of diarrhea in 8 of the 21 (38%) piglets on 6 farms remained unknown. The etiological diagnosis of diarrhea could not be determined in any of the piglets on 2 of the farms. This study demonstrated that the number of cpb2-positive type A C. perfringens in the intestinal contents was not a useful approach for making a diagnosis of type A C. perfringens enteritis in piglets. Further work is required to confirm whether cpb2-carrying type A C. perfringens have a pathogenic role in enteric infection in neonatal swine. PMID:23814355

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

    PubMed

    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.

  5. Comparison of different methods of cell lysis and protein measurements in Clostridium perfringens: application to the cell volume determination.

    PubMed

    Guerlava, P; Izac, V; Tholozan, J L

    1998-03-01

    Four cell lysis methods (NaOH-SDS solubilization, French press treatment, sonication, mutanolysin treatment) and three methods of protein assays (Lowry, Bradford, Pierce) were studied for their applicability to determination of cell volume in Clostridium perfringens NCTC 8798 cell suspensions. Protein contents were higher after a mechanical disruption of the cells than with the other techniques of lysis. The lowest concentrations of protein were obtained with the Bradford procedure. With each of the three protein assay methods, Clostridium perfringens NCTC 8798 protein cell contents were 45% to 58% of protein. Other factors possibly involved in variations of the intracellular volume measurements were examined. A control of the level of protein concentration in the test sample and the type of silicone oil used for the centrifugation were of prime importance during sample preparation. Under our conditions, an intracellular volume of 4 microl/(mg of protein) was routinely found for Clostridium perfringens NCTC 8798.

  6. Reversal of radiation-dependent heat sensitization of Clostridium perfringens spores.

    PubMed Central

    Gomez, R F; Gombas, D E; Herrero, A

    1980-01-01

    The effect of solute concentration on the sensitization of Clostridium perfringens spores to heat by ionizing radiation was investigated. As we have shown previously, spores of C. perfringens treated with gamma radiation are now sensitive to subsequent heat treatments than are spores that receive no radiation treatment. When gamma-irradiated spores were heated in the presence of increasing concentrations of glycerol or sucrose, the heat sensitivity induced by irradiation was progressively decreased. The magnitude of the increase in heat resistance induced by extracellular solutes was greater in gamma-irradiated spores than in nonirradiated spores. Based on these observations, it is proposed that the induction of heat sensitivity in spores by radiation is related to the loss of osmoregulatory or dehydrating mechanisms in irradiated spores. PMID:6247972

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

    PubMed

    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.

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

    PubMed

    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.

  9. [Studies of necrotizing enteritis of suckling piglets (Clostridium perfringens type C enterotoxemia) in industrialized sow breeding units. 4. Epizootiology].

    PubMed

    Köhler, B; Zabke, J; Sondermann, R; Pulst, H; Rummler, H J

    1979-01-01

    Necrotising enteritis had been the cause of death of 4.9 per cent in 5,177 nursed piglets, which was established by pathological examination. The number of piglets, in that context, which had come from industrialised sow breeding units was equivalent to 92 per cent. The nursed piglet held the third position, next to smaller ruminants (19.4 per cent) and fowl (6.0 per cent), with regard to the occurrence of Clostridium perfringens enterotoxemia or necrotising enteritis in 112,218 animals which were pathologically examined after death. Necrotising enteritis so far has been rare in the GDR. No regional accumulation has been observed. Several outbreaks on industrialised sow breeding units actually remained stationary. The occurrence of the disease may be favoured by a number of factors which are conducive to accumulation of Clostridium perfringens Type C in a given stock. Group keeping of pregnant sows, simultaneous farrowing of larger groups of sows, group treatment of nursed piglets, using neomycin, chloramphenicol, oxytetracycline, and other antibiotics to which Clostridium perfringens is primarily resistant or has acquired resistance in the course of time are some of those contributive factors. Transmission of Clostridium perfringens Type C through feedstuff is possible, though it would lead to a real outbreak only by high intensity of the contamination, and it played a minor role in proliferation of the disease. 3479 Clostridium perfringens strains were isolated from 9,481 animals, both clinically intact and after death, with 30 species being included. Type classification revealed 2454 strains of Type A (70 per cent), 204 of Type D (5.88 per cent), 164 of Type C (four per cent), and 48 of Type B (1.34 per cent). There were 688 atoxic strains (17 per cent). Swine is the major carrier of Clostridium perfringens Type C, with 87 per cent of all Clostridium perfringens Type C strains having been isolated from swine. Swine was followed by fowl (four per cent), sheep

  10. A Quantitative Electrochemiluminescence Assay for Clostridium perfringens alpha toxin

    DTIC Science & Technology

    2006-08-10

    is the predominant etiolog- ical agent responsible for gas gangrene [1], although other Clostridium species including C. novyi, C. bifermentans, and...great reduction of the inci- dence of gangrene [1]. Gas gangrene remains a potential problematic disease after traumatic injury such as burns and...is a bacterium associated with three disease syndromes: classic gas gangrene (a muscle tis- sue infection resulting in muscle necrosis), enteritis

  11. Genotyping of Clostridium perfringens isolated from domestic and exotic ruminants and swine.

    PubMed

    Sipos, W; Fischer, L; Schindler, M; Schmoll, F

    2003-09-01

    Clostridium perfringens types A, B, C, D and E are known to cause severe enteritis/enterotoxaemia and diseases (especially caused by type A) belonging to the gas oedema complex in many species. Samples from the small intestine as well as faeces of domestic and exotic animals suffering from enterotoxaemic signs or having died within days after first occurance of toxaemia were submitted for typing C. perfringens toxovars by multiplex PCR. The following species have been investigated: domestic sheep (Ovis ammon; n = 10), domestic goat (Capra aegagrus hircus; n = 26), Japanese serow (Capricornis sumatraensis; n = 4), lechwe waterbuck (Hydrotragus leche; n = 1), blackbuck (Antilope cervicapra; n = 1), European reindeer (Rangifer tarandus tarandus; n = 4), domestic swine (Sus scrofa; n = 52), and collared peccary (Tayassu albirostris; n = 1). Interestingly, the predominant C. perfringens toxovar in domestic sheep was type A. This toxovar could also be diagnosed in all reindeer, in three Japanese serows, one lechwe waterbuck and most pigs (n = 47), the majority of those being at suckling age. Type D was the most prevalent toxovar (n = 18) in domestic goats, but also types A and E could be identified as pathogens in this species. Type C could only be found in domestic swine (n = 5) and in one case of clostridiosis in a Japanese serow. Two cases of enterotoxaemia in goats, one case in reindeer, and a single case in blackbuck and collared peccary were caused by C. perfringens type E. Genotyping of C. perfringens is recommended before starting vaccination programmes as it could be shown, that the importance of specific toxovars has been underestimated in specific species and/or age groups.

  12. Epidemiology of foodborne disease outbreaks caused by Clostridium perfringens, United States, 1998-2010.

    PubMed

    Grass, Julian E; Gould, L Hannah; Mahon, Barbara E

    2013-02-01

    Clostridium perfringens is estimated to be the second most common bacterial cause of foodborne illness in the United States, causing one million illnesses each year. Local, state, and territorial health departments voluntarily report C. perfringens outbreaks to the U.S. Centers for Disease Control and Prevention through the Foodborne Disease Outbreak Surveillance System. Our analysis included outbreaks confirmed by laboratory evidence during 1998-2010. A food item was implicated if C. perfringens was isolated from food or based on epidemiologic evidence. Implicated foods were classified into one of 17 standard food commodities when possible. From 1998 to 2010, 289 confirmed outbreaks of C. perfringens illness were reported with 15,208 illnesses, 83 hospitalizations, and eight deaths. The number of outbreaks reported each year ranged from 16 to 31 with no apparent trend over time. The annual number of outbreak-associated illnesses ranged from 359 to 2,173, and the median outbreak size was 24 illnesses. Outbreaks occurred year round, with the largest number in November and December. Restaurants (43%) were the most common setting of food preparation. Other settings included catering facility (19%), private home (16%), prison or jail (11%), and other (10%). Among the 144 (50%) outbreaks attributed to a single food commodity, beef was the most common commodity (66 outbreaks, 46%), followed by poultry (43 outbreaks, 30%), and pork (23 outbreaks, 16%). Meat and poultry outbreaks accounted for 92% of outbreaks with an identified single food commodity. Outbreaks caused by C. perfringens occur regularly, are often large, and can cause substantial morbidity yet are preventable if contamination of raw meat and poultry products is prevented at the farm or slaughterhouse or, after contamination, if these products are properly handled and prepared, particularly in restaurants and catering facilities.

  13. Characterization of Genes Encoding for Acquired Bacitracin Resistance in Clostridium perfringens

    PubMed Central

    Charlebois, Audrey; Jalbert, Louis-Alexandre; Harel, Josée; Masson, Luke; Archambault, Marie

    2012-01-01

    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 MIC90 (>256 µg/ml) was identical for both turkey and chicken isolates; whereas MIC50 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. PMID:22970221

  14. Determination of the prevalence of antimicrobial resistance genes in canine Clostridium perfringens isolates.

    PubMed

    Kather, Elizabeth J; Marks, Stanley L; Foley, Janet E

    2006-03-10

    Clostridium perfringens is a well documented cause of a mild self-limiting diarrhea and a potentially fatal acute hemorrhagic diarrheal syndrome in the dog. A recent study documented that 21% of canine C. perfringens isolates had MIC's indicative of resistance to tetracycline, an antimicrobial commonly recommended for treatment of C. perfringens-associated diarrhea. The objective of the present study was to further evaluate the antimicrobial susceptibility profiles of these isolates by determining the prevalence of specific resistance genes, their expression, and ability for transference between bacteria. One hundred and twenty-four canine C. perfringens isolates from 124 dogs were evaluated. Minimum inhibitory concentrations of tetracycline, erythromycin, tylosin, and metronidazole were determined using the CLSI Reference Agar Dilution Method. All isolates were screened for three tetracycline resistance genes: tetA(P), tetB(P) and tetM, and two macrolide resistance genes: ermB and ermQ, via PCR using primer sequences previously described. Ninety-six percent (119/124) of the isolates were positive for the tetA(P) gene, and 41% (51/124) were positive for both the tetA(P) and tetB(P) genes. No isolates were positive for the tetB(P) gene alone. Highly susceptible isolates (MIC< or = 4 microg/ml) were significantly more likely to lack the tetB(P) gene. One isolate (0.8%) was positive for the ermB gene, and one isolate was positive for the ermQ gene. The tetM gene was not found in any of the isolates tested. Two out of 15 tested isolates (13%) demonstrated transfer of tetracycline resistance via bacterial conjugation. Tetracycline should be avoided for the treatment of C. perfringens-associated diarrhea in dogs because of the relatively high prevalence of in vitro resistance, and the potential for conjugative transfer of antimicrobial resistance.

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

    PubMed Central

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

    2015-01-01

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

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

    PubMed

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

    2015-01-01

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

  17. Role of Zinc in the Production of Clostridium perfringens Alpha Toxin

    PubMed Central

    Sato, Hiroko; Murata, Ryosuke

    1973-01-01

    Clostridium perfringens was found to produce alpha toxin in a synthetic medium containing zinc; in medium containing no zinc, a little toxin was detected in the early logarithmic phase of growth and it disappeared rapidly. No intracellular accumulation of alpha toxin protein occurred whether or not zinc was present in the medium. In zinc-deficient medium, the organisms produced and released into the surrounding medium the protein specifically precipitable with alpha antitoxin in an amount comparable to that of alpha toxin produced in the zinc-containing medium. The protein combined rapidly in some unknown way with zinc to form the active and stable alpha toxin. Images PMID:4354149

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

    PubMed Central

    Radhika, B.; Kumar, N. Vinod; Sreenivasulu, D.

    2016-01-01

    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. PMID:27051186

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

    PubMed

    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.

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

    PubMed Central

    Mishra, Rashmi; Duncalf, Richard

    2016-01-01

    Clostridium perfringens bacteremia is associated with adverse outcomes. Known risk factors include chronic kidney disease, malignancy, diabetes mellitus, and gastrointestinal disease. We present a 74-year-old man admitted with confusion, vomiting, and abdominal pain. Exam revealed tachycardia, hypotension, lethargy, distended abdomen, and cold extremities. He required intubation and aggressive resuscitation for septic shock. Laboratory data showed leukocytosis, metabolic acidosis, acute kidney injury, and elevated lipase. CT scan of abdomen revealed acute pancreatitis and small bowel ileus. He was started on vancomycin and piperacillin-tazobactam. Initial blood cultures were positive for C. perfringens on day five. Metronidazole and clindamycin were added to the regimen. Repeat CT (day 7) revealed pancreatic necrosis. The patient developed profound circulatory shock requiring multiple vasopressors, renal failure requiring dialysis, and bacteremia with vancomycin-resistant enterococci. Hemodynamic instability precluded surgical intervention and he succumbed to multiorgan failure. Interestingly, our isolate was beta lactamase producing. We review the epidemiology, risk factors, presentation, and management of C. perfringens bacteremia. This case indicates a need for high clinical suspicion for clostridial sepsis and that extended spectrum beta lactam antibiotic coverage may be inadequate and should be supplemented with use of clindamycin or metronidazole if culture is positive, until sensitivities are known. PMID:26904307

  1. The role of neutrophils and monocytic cells in controlling the initiation of Clostridium perfringens gas gangrene.

    PubMed

    O'Brien, David K; Therit, Blair H; Woodman, Michael E; Melville, Stephen B

    2007-06-01

    Clostridium perfringens is a common cause of the fatal disease gas gangrene (myonecrosis). Established gas gangrene is notable for a profound absence of neutrophils and monocytic cells (phagocytes), and it has been suggested that the bactericidal activities of these cells play an insignificant role in controlling the progression of the infection. However, large inocula of bacteria are needed to establish an infection in experimental animals, suggesting phagocytes may play a role in inhibiting the initiation of gangrene. Examination of tissue sections of mice infected with a lethal (1 x 10(9)) or sublethal (1 x 10(6)) inoculum of C. perfringens revealed that phagocyte infiltration in the first 3 h postinfection was inhibited with a lethal dose but not with a sublethal dose, indicating that exclusion of phagocytes begins very early in the infection cycle. Experiments in which mice were depleted of either circulating monocytes or neutrophils before infection with C. perfringens showed that monocytes play a role in inhibiting the onset of gas gangrene at intermediate inocula but, although neutrophils can slow the onset of the infection, they are not protective. These results suggest that treatments designed to increase monocyte infiltration and activate macrophages may lead to increased resistance to the initiation of gas gangrene.

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

    PubMed

    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. Development of a model to predict growth of Clostridium perfringens in cooked beef during cooling.

    PubMed

    Smith-Simpson, Sarah; Schaffner, Donald W

    2005-02-01

    The objective of this work was to develop a new model to predict the growth of Clostridium perfringens in cooked meat during cooling. All data were collected under changing temperature conditions. Individual growth curves were fit using DMFit. Germination outgrowth and lag (GOL) time was modeled versus temperature at the end of GOL using conservative assumptions. Each growth curve was used to estimate a series of exponential growth rates at a series of temperatures. The squareroot model was used to describe the relationship between the square root of the average exponential growth rate and effective temperature. Predictions from the new model were in close agreement with the data used to create the model. When predictions from the model were compared with new observations, fail-dangerous predictions were made a majority of the time. When GOL time was predicted exactly, many fail-dangerous predictions shifted toward the fail-safe direction. Two important facts regarding C. perfringens should impact future modeling research with this organism and may have broader food safety policy implications: (i) the normal variability in the response of the organism from replicate to replicate may be quite large (1 log CFU) and may exceed the current U.S. Food Safety Inspection Service performance standard, and (ii) the accuracy of the GOL time model has a profound influence upon the overall prediction, with small differences in GOL time prediction (approximately 1 h) having a very large effect on the predicted final concentration of C. perfringens.

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

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

    2016-11-21

    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.

  7. Growth conditions of clostridium perfringens type B for production of toxins used to obtain veterinary vaccines.

    PubMed

    Viana Brandi, Igor; Domenici Mozzer, Otto; Jorge, Edson Vander; Vieira Passos, Frederico Jose; Lopes Passos, Flavia Maria; Cangussu, Alex Sander Rodrigues; Macedo Sobrinho, Eliane

    2014-09-01

    The diseases caused for Clostridium perfringens are generically called enterotoxemias because toxins produced in the intestine may be absorbed into the general circulation. C. perfringens type B, grown in batch fermentation, produced toxins used to obtain veterinary vaccines. Glucose in concentrations of 1.4-111.1 mM was used to define the culture medium. The minimum concentration for a satisfactory production of vaccines against clostridial diseases was 55.6 mM. Best results were brought forth by meat and casein peptones, both in the concentration 5.0 g l(-1) in combination with glucose and a culture pH maintained at 6.5 throughout the fermentation process. The production of lactic, acetic and propionic organic acids was observed. Ethanol was the metabolite produced in the highest concentration when cultures maintained steady pH of 6.5 with exception of cultures with initial glucose concentration of 1.4 mM, where the highest production was of propionic acid. Maximal cell concentration and the highest toxin title concomitantly low yield coefficient to organic acids and ethanol were obtained using basal medium containing 111.1 mM glucose under a controlled pH culture (pH) 6.5 in batch fermentations of C. perfringens type B. These data contribute to improve process for industrial toxin production allowing better condition to produce a toxoid vaccine.

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

    PubMed

    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.

  9. Role of alpha-toxin in Clostridium perfringens infection determined by using recombinants of C. perfringens and Bacillus subtilis.

    PubMed Central

    Ninomiya, M; Matsushita, O; Minami, J; Sakamoto, H; Nakano, M; Okabe, A

    1994-01-01

    Clostridium perfringens type A strains which differed in alpha-toxin (phospholipase C [PLC]) productivity were inoculated intraperitoneally or intravenously into mice, and then their 50% mouse lethal doses (LD50) were determined. Strain NCTC 8237 produced ninefold higher PLC activity than strain 13. The mean LD50 for the former was 1 log unit lower than that for the latter. Two isogenic strains were constructed from strain 13: strain 13(pJIR418 alpha) (pJIR418 alpha contains the plc gene), which produced ninefold higher PLC activity than strain 13; and strain 13 PLC-, which showed no PLC productivity at all because of transformation-mediated gene disruption. The mean LD50 for strain 13(pJIR418 alpha) was 1 log unit lower than those for strain 13 PLC- and strain 13. These results indicate that PLC functions as a virulence-determining factor when it is produced in a sufficient amount. Such a difference in LD50 was also observed between Bacillus subtilis with and without the cloned plc gene. Inoculation of B. subtilis PLC+ intravenously into mice caused marked thrombocytopenia and leukocytosis. Mice inoculated with B. subtilis at 2 LD50 died because of circulatory collapse. Histological examination revealed that intravascular coagulation and vascular congestion occurred most prominently in the lungs. These results suggest that PLC plays a key role in the systemic intoxication of clostridial myonecrosis, probably by affecting the functions of platelets and phagocytes. Images PMID:7927785

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

    NASA Astrophysics Data System (ADS)

    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.

  11. Effect of phosphate and meat (pork) types on the germination and outgrowth of Clostridium perfringens spores during abusive chilling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of blends of phosphates and the pork meat type (pale, soft and exudative, PSE; normal; and dark, firm and dry, DFD) on the germination and outgrowth of Clostridium perfringens during abusive exponential chilling times was evaluated. Two different phosphates, tetrasodium pyrophosphate (TSP...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. Epidemiological and pathobiological profiles of Clostridium perfringens infections: review of consecutive series of 33 cases over a 13-year period

    PubMed Central

    Shindo, Yuji; Dobashi, Yoh; Sakai, Toshiyasu; Monma, Chie; Miyatani, Hiroyuki; Yoshida, Yukio

    2015-01-01

    Background: Although Clostridium perfringens (C. perfringens) is well known as the causative agent of several forms of enteric disease, precise epidemiological and pathobiological aspects are still unknown. Methods: We retrospectively reviewed the culture results of samples collected in our hospital from 2001 through 2013. In addition, for the detection and toxinogenic typing of C. perfringens, polymerase-chain-reaction amplification (PCR)-based rapid analysis was performed in 6 cases using DNA extracted from paraffin-embedded tissues. Results: A total of 35 samples from 33 cases were positive for C. perfringens, representing an incidence of 0.017% (35/205, 114). Among 33 patients, 21 patients manifested sepsis and 7 patients had bacteremia. One of the septic cases was complicated by fatal intravascular hemolysis and thus, the prevalence was estimated at 3.0% among C. perfringens infections (1/33). The direct causative disease or state for C. perfringens infection was identified in 18 patients: surgery or intervention for cancers, 8 patients; chemotherapy for cancer, 2 patients; surgery or intervention for non-neoplastic disease, 6 patients; liver cirrhosis, 3 patients, etc. PCR-based toxinogenic typing of C. perfringens detected the alpha-toxin gene only in tissue from a patient who died of massive hemolysis; none of the toxin genes could be amplified in the other 5 cases examined. Conclusions: The prevalence of overt C. perfringens infection is low, but upon detection, infected patients should be carefully monitored for fatal acute hemolysis caused by type A C. perfringens. Furthermore, PCR-based rapid detection of C. perfringens and toxinogenic typing by archival pathological material is applicable as a diagnostic tool. PMID:25755747

  14. Foodborne disease outbreaks caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus--United States, 1998-2008.

    PubMed

    Bennett, Sarah D; Walsh, Kelly A; Gould, L Hannah

    2013-08-01

    From 1998 to 2008, 1229 foodborne outbreaks caused by Bacillus cereus, Clostridium perfringens, and Staphylococcus aureus were reported in the United States; 39% were reported with a confirmed etiology. Vomiting was commonly reported in B. cereus (median, 75% of cases) and S. aureus outbreaks (median, 87%), but rarely in C. perfringens outbreaks (median, 9%). Meat or poultry dishes were commonly implicated in C. perfringens (63%) and S. aureus (55%) outbreaks, and rice dishes were commonly implicated in B. cereus outbreaks (50%). Errors in food processing and preparation were commonly reported (93%), regardless of etiology; contamination by a food worker was only common in S. aureus outbreaks (55%). Public health interventions should focus on these commonly reported errors to reduce the occurrence of outbreaks caused by B. cereus, C. perfringens, and S. aureus in the United States.

  15. Detection of Clostridium perfringens in yearling lamb meat (barbacoa), head, and gut tacos from public markets in Mexico City.

    PubMed

    Natividad-Bonifacio, Iván; Vázquez-Quiñones, Carlos R; Rodas-Suárez, Oscar R; Fernández, Francisco J; Rodríguez-Solis, Esteban; Quiñones-Ramírez, Elsa Irma; Vázquez-Salinas, Carlos

    2010-06-01

    No reports on the incidence of Clostridium perfringens in popularly-consumed food from Mexico City have been published; neither are there any reports that have analyzed food consumed in popular markets and less established restaurants. Therefore, this study is aimed at providing data to evaluate the relevance of C. perfringens as an etiologic agent of food-borne diseases. Of the 650 analyzed samples, 106 (16.3%) were positive for C. perfringens; 6.4% (16/250) isolates were from barbacoa, 19% (38/200) from head, and 13% (52/200) from gut tacos. The presence of C. perfringens in these popular-consumed foods demonstrates its relevance as an etiologic agent of food-borne diseases, and confirms the great sanitary risk involved in their consumption. These results may serve as a basis for the Mexican sanitary authorities to control the microbiological quality of street-made foods.

  16. Detection of enterotoxigenic Clostridium perfringens in spices used in Mexico by dot blotting using a DNA probe.

    PubMed

    Rodríguez-Romo, L A; Heredia, N L; Labbé, R G; García-Alvarado, J S

    1998-02-01

    Several reports on the microbiology of spices and herbs indicate the presence of Clostridium perfringens, a spore-forming foodborne pathogen responsible for gastrointestinal disease. In the present study, a total of 380 samples of spices and herbs (cumin seed, black pepper, oregano, garlic powder, and bay leaves) widely used in Mexico were analyzed for the presence of C. perfringens, and the enterotoxigenicity of the isolates was determined by a dot-blot technique using an enterotoxin degoxigenin-labeled DNA probe. C. perfringens counts varied from <100 to 433 CFU/g in garlic powder, from <100 to 200 CFU/g in black pepper, from <100 to 433 CFU/g in cumin seed, from <100 to 340 CFU/g in oregano, and from < 100 to 450 CFU/g in bay leaves. The dot-blot technique detected the enterotoxin gene in 8 (4.25%) of 188 confirmed isolates of C. perfringens. dot-blot.

  17. Comparative genomics of VirR regulons in Clostridium perfringens strains

    PubMed Central

    2010-01-01

    Background Clostridium perfringens is a Gram-positive anaerobic bacterium causing severe diseases such as gas gangrene and pseudomembranosus colitis, that are generally due to the secretion of powerful extracellular toxins. The expression of toxin genes is mainly regulated by VirR, the response regulator of a two-component system. Up to now few targets only are known for this regulator and mainly in one strain (Strain 13). Due to the high genomic and phenotypic variability in toxin production by different strains, the development of effective strategies to counteract C. perfringens infections requires methodologies to reconstruct the VirR regulon from genome sequences. Results We implemented a two step computational strategy allowing to consider available information concerning VirR binding sites in a few species to scan all genomes of the same species, assuming the VirR targets are at least partially conserved across these strains. Results obtained are in agreement with previous works where experimental validation of the promoters have been performed and showed the presence of a core and an accessory regulon of VirR in C. perfringens strains with three target genes also located on plasmids. Moreover, the type E strain JGS1987 has the largest predicted regulon with as many as 10 VirR targets not found in the other genomes. Conclusions In this work we exploited available experimental information concerning the targets of the VirR toxin regulator in one C. perfringens strain to obtain plausible predictions concerning target genes in genomes and plasmids of nearby strains. Our predictions are available for wet-lab researchers working on less characterized C. perfringens strains that can thus design focused experiments reducing the search space of their experiments and increasing the probability of characterizing positive targets with less efforts. Main result was that the VirR regulon is variable in different C. perfringens strains with 4 genes controlled in all but

  18. Synergistic Effects of Clostridium perfringens Enterotoxin and Beta Toxin in Rabbit Small Intestinal Loops

    PubMed Central

    Ma, Menglin; Gurjar, Abhijit; Theoret, James R.; Garcia, Jorge P.; Beingesser, Juliann; Freedman, John C.; Fisher, Derek J.; McClane, Bruce A.

    2014-01-01

    The ability of Clostridium perfringens type C to cause human enteritis necroticans (EN) is attributed to beta toxin (CPB). However, many EN strains also express C. perfringens enterotoxin (CPE), suggesting that CPE could be another contributor to EN. Supporting this possibility, lysate supernatants from modified Duncan-Strong sporulation (MDS) medium cultures of three CPE-positive type C EN strains caused enteropathogenic effects in rabbit small intestinal loops, which is significant since CPE is produced only during sporulation and since C. perfringens can sporulate in the intestines. Consequently, CPE and CPB contributions to the enteropathogenic effects of MDS lysate supernatants of CPE-positive type C EN strain CN3758 were evaluated using isogenic cpb and cpe null mutants. While supernatants of wild-type CN3758 MDS lysates induced significant hemorrhagic lesions and luminal fluid accumulation, MDS lysate supernatants of the cpb and cpe mutants caused neither significant damage nor fluid accumulation. This attenuation was attributable to inactivating these toxin genes since complementing the cpe mutant or reversing the cpb mutation restored the enteropathogenic effects of MDS lysate supernatants. Confirming that both CPB and CPE are needed for the enteropathogenic effects of CN3758 MDS lysate supernatants, purified CPB and CPE at the same concentrations found in CN3758 MDS lysates also acted together synergistically in rabbit small intestinal loops; however, only higher doses of either purified toxin independently caused enteropathogenic effects. These findings provide the first evidence for potential synergistic toxin interactions during C. perfringens intestinal infections and support a possible role for CPE, as well as CPB, in some EN cases. PMID:24778117

  19. Potential for growth of Clostridium perfringens from spores in pork scrapple during cooling.

    PubMed

    Juneja, Vijay K; Porto-Fett, Anna C S; Gartner, Kelly; Tufft, Linda; Luchansky, John B

    2010-02-01

    We conducted stabilization studies to determine the ability of Clostridium perfringens spores to germinate and grow during exponential cooling of a commercial formulation of pork scrapple. Scrapple was inoculated with a mixture of three strains of C. perfringens spores (NTCC 8238, NCTC 8239, and ATCC 10288), vacuum packaged, and reheated (20 min/93.3 degrees C) in a circulating water bath. The cooked samples were cooled (30 s) in an ice bath before being transferred to a programmable water bath to cool through the temperature range of 54.4 degrees C to 7.2 degrees C in 12, 14, or 21 h to simulate deviations from the required cooling time of 6.5 h. After cooling, the samples, in duplicate, were analyzed to determine if growth from spores had occurred. The samples were plated onto tryptose-sulfite-cycloserine agar and incubated anaerobically at 37 degrees C for 48 h before counting the colonies. Minimal growth (less than 1.0 log) was observed during a 12- or 14 h cooling period. However, when the time to achieve 7.2 degrees C was extended to 21 h, C. perfringens spores germinated and grew from an inoculum of approximately 3.0 log(10) to approximately 7.8 log(10) CFU/g. Thus, scrapple must be cooled after cooking to 7.2 degrees C within 6.5 h, but for no more than 14 h, to prevent a food safety hazard from outgrowth of C. perfringens spores during cooling.

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

    PubMed

    Hiscox, Thomas J; Harrison, Paul F; Chakravorty, Anjana; Choo, Jocelyn M; Ohtani, Kaori; Shimizu, Tohru; Cheung, Jackie K; Rood, Julian I

    2013-01-01

    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.

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

    SciTech Connect

    Suryadinata, Randy Seabrook, Shane A.; Adams, Timothy E.; Nuttall, Stewart D.; Peat, Thomas S.

    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.

  2. A Five Site Clostridium Perfringens Food-Borne Outbreak: A Retrospective Cohort Study

    PubMed Central

    FAFANGEL, Mario; UČAKAR, Veronika; VUDRAG, Marko; BERCE, Ingrid; KRAIGHER, Alenka

    2015-01-01

    Introduction In May of 2012, we investigated a food-borne Clostridium perfringens outbreak in Slovenia involving a single kitchen and five venues, with 477 exposed persons. Methods In order to identify the causative agent, vehicle of infection and source of contamination, we conducted microbiological and environmental investigations and an analytical cohort study (n = 138). Results The case definition in the outbreak was met by 104 persons. Predominant symptoms were diarrhoea, nausea and abdominal cramps. Median incubation time and duration of illness were 12 and 22.5 hours respectively. Stool samples were collected from 18 persons and in 13 C. perfringens spores were present; enterotoxin was detected in 9 persons. PCR and PFGE analysis of isolates from a cook with earlier onset time, who did not consume the implicated food, and cases from four venues showed the same strain of C. perfringens type A (with cpe-gene), indistinguishable by PFGE analysis. No food samples could be obtained. An analytical study showed that one food item (French salad) was the most likely vehicle of infection (RR: 6.35; 95% CI: 1.62–24.90). Conclusions This was the largest C. perfringens outbreak in Slovenia to date. Proper analytical study in combination with detailed laboratory investigation with genotypisation enabled us to identify a causative agent, vehicle of infection and possible source of contamination. Fast response and interdisciplinary collaboration led to timely implementation of control measures. These have led to the kitchen acquiring new equipment and improving staff knowledge of risks and processes, thus reducing the likelihood of future reoccurrences. PMID:27646622

  3. Regulation of Sialidase Production in Clostridium perfringens by the Orphan Sensor Histidine Kinase ReeS

    PubMed Central

    Hiscox, Thomas J.; Harrison, Paul F.; Chakravorty, Anjana; Choo, Jocelyn M.; Ohtani, Kaori; Shimizu, Tohru

    2013-01-01

    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. PMID:24023881

  4. Production of a bacteriocin by a poultry derived Campylobacter jejuni isolate with antimicrobial activity against Clostridium perfringens and other Gram positive bacteria.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have purified a bacteriocin peptide (termed CUV-3), produced by a poultry cecal isolate of Campylobacter jejuni (strain CUV-3) with inhibitory activity against Gram positive bacteria including Clostridium perfringens (38 strains), Staphylococcus aureus, Staphylococcus epidermidis and Listeria mon...

  5. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  7. Abilities of the mCP Agar method and CRENAME alpha toxin-specific real-time PCR assay to detect Clostridium perfringens spores in drinking water.

    PubMed

    Maheux, Andrée F; Bérubé, Eve; Boudreau, Dominique K; Villéger, Romain; Cantin, Philippe; Boissinot, Maurice; Bissonnette, Luc; Bergeron, Michel G

    2013-12-01

    We first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of a Clostridium perfringens-specific real-time PCR (rtPCR) assay based on the cpa gene (cpa rtPCR) by using a bacterial strain panel composed of C. perfringens and non-C. perfringens Clostridium strains. All non-C. perfringens Clostridium strains tested negative, whereas all C. perfringens strains tested positive with the cpa rtPCR, for an analytical specificity and ubiquity of 100%. The cpa rtPCR assay was then used to confirm the identity of 116 putative C. perfringens isolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar and cpa rtPCR were identified by sequencing the 16S rRNA and cpa genes. Four mCP(-)/rtPCR(+) colonies were identified as C. perfringens, whereas 3 mCP(+)/rtPCR(-) colonies were identified as non-C. perfringens. The cpa rtPCR was negative with all 51 non-C. perfringens strains and positive with 64 of 65 C. perfringens strains. Finally, we compared mCP agar and a CRENAME (concentration and recovery of microbial particles, extraction of nucleic acids, and molecular enrichment) procedure plus cpa rtPCR (CRENAME + cpa rtPCR) for their abilities to detect C. perfringens spores in drinking water. CRENAME + cpa rtPCR detected as few as one C. perfringens CFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME + cpa rtPCR also allows the simultaneous and sensitive detection of Escherichia coli and C. perfringens from the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

  8. The Clostridium perfringens enterotoxin from equine isolates; its characterization, sequence and role in foal diarrhoea.

    PubMed Central

    Netherwood, T.; Binns, M.; Townsend, H.; Wood, J. L.; Mumford, J. A.; Chanter, N.

    1998-01-01

    During a survey of foal diarrhoea between 1991 and 1994, Clostridium perfringens was significantly associated with disease with 56% of cases infected [1]. The contribution of enterotoxigenic C. perfringens to this association, was assessed by use of the reverse passive latex agglutination test for enterotoxin (RPLA; Oxoid Unipath) and vero cell toxicity neutralized by antitoxin on stored faecal samples and sporulated faecal isolates of C. perfringens. Polymerase chain reaction (PCR1) based on the DNA sequence for the whole enterotoxin gene [2] yielded a fragment from an equine isolate of the anticipated size which, cloned into plasmid M13 phage, had a sequence essentially identical to the published sequence. Consequently, all faecal isolates were also tested by PCR1 and for a part of the enterotoxin gene (PCR2). Significant association with diarrhoea (controls not in contact with cases) was found with positive RPLA tests on faeces (OR = 13, P = 0.002) and isolates (OR = 4.57, P = < 0.0001), vero cell toxicity of isolates (OR = 1.78, P = 0.026), and PCR1 (OR = nd, P = 0.029) but not PCR2 or vero cell toxicity of faeces. Significant association with diarrhoea was also found for isolates negative by RPLA (OR = 3.91; CI 2.05-7.57; P < 0.0001) or PCR1 (OR = 4.81; CI 2.84-8.20; P < 0.0001). Many of the isolates from RPLA positive faeces and verotoxic isolates were PCR negative and no evidence could be found for the presence of the enterotoxin gene in a random selection of RPLA positive/PCR negative isolates by gene probe on chromosomal DNA and PCR reaction product or vero cell toxicity neutralized by specific antiserum. Failure of the vero cell toxicity on faeces to be associated with diarrhoea or for cytotoxicity of cultures and RPLA on cultures to agree with the PCRs was believed to be related to the presence of other cytotoxins, the inherent cytotoxicity of equine faeces and to the poor specificity of the commercial antiserum used in the test. Enterotoxigenic C

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

    PubMed

    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

  10. [A patient with sepsis and a gas-forming liver abscess caused by Clostridium perfringens treated with continuous perfusion drainage].

    PubMed

    Kusumoto, Kiyonori; Hamada, Akihiko; Kusaka, Toshihiro; Yamaguchi, Daisuke; Yoshioka, Takuto; Nakai, Yoshitaka; Matsubara, Susumu; Azechi, Hidemasa; Fujii, Shigehiko; Kokuryu, Hiroyuki

    2014-07-01

    A 64-year-old man presented with diarrhea, fever, and disturbance of consciousness; he was subsequently diagnosed with acute renal and hepatic disorder. Abdominal computed tomography identified a gas-forming liver abscess, and the patient underwent emergency drainage. However, his condition did not improve, and Clostridium perfringens was observed in his blood culture. Continuous perfusion drainage was performed by placing an additional drainage tube, which resulted in abscess shrinkage and improved the patient's general condition. Despite the low survival rate in patients with gas-forming liver abscesses caused by C. perfringens, therapy was successful in this patient.

  11. Growth and physiology of Clostridium perfringens wild-type and ΔazoC knockout: an azo dye exposure study.

    PubMed

    Morrison, Jessica M; John, Gilbert H

    2016-02-01

    Clostridium perfringens, a strictly anaerobic micro-organism and inhabitant of the human intestine, has been shown to produce the azoreductase enzyme AzoC, an NAD(P)H-dependent flavin oxidoreductase. This enzyme reduces azo dyes to aromatic amines, which are carcinogenic in nature. A significant amount of work has been completed that focuses on the activity of this enzyme; however, few studies have been completed that focus on the physiology of azo dye reduction. Dye reduction studies coupled with C. perfringens growth studies in the presence of ten different azo dyes and in media of varying complexities were completed to compare the growth rates and dye-reducing activity of C. perfringens WT cells, a C. perfringens ΔazoC knockout, and Bifidobacterium infantis, a non-azoreductase-producing control bacterium. The presence of azo dyes significantly increased the generation time of C. perfringens in rich medium, an effect that was not seen in minimal medium. In addition, azo dye reduction studies with the ΔazoC knockout suggested the presence of additional functional azoreductases in this medically important bacterium. Overall, this study addresses a major gap in the literature by providing the first look, to our knowledge, at the complex physiology of C. perfringens upon azo dye exposure and the effect that both azo dyes and the azoreductase enzyme have on growth.

  12. Toxinotyping of Clostridium perfringens fecal isolates of reintroduced Père David's deer (Elaphurus davidianus) in China.

    PubMed

    Qiu, Huiling; Chen, Fu; Leng, Xinyan; Fei, Rongmei; Wang, Libo

    2014-10-01

    Clostridium perfringens is an important pathogen causing sudden death syndrome, necrotic enteritis, and gas gangrene in ruminants, especially some deer species. Père David's deer (Elaphurus davidianus) is one of the world's rare species and is an endangered and protected species in China. Some Père David's deer in the Chinese Shishou Père David's Deer Preserve died due to C. perfringens infection. We investigated the toxin types and C. perfringens enterotoxin-positive (cpe(+)) strains of isolated C. perfringens in Père David's deer in China. We collected 155 fecal samples from the Beijing Nanhaizi Père David's Deer Park and the Jiangsu Dafeng Père David's Deer National Nature Reserve between July 2010 and July 2011. Bacteria isolated using blood agar and mannitol agar plates were identified by Gram staining and nested PCR for 16S rRNA. We isolated C. perfringens from 41 fecal samples and used PCR amplification of five toxin genes to identify the toxinotypes and the cpe(+) strains of C. perfringens. Twenty-one isolates were type A, 15 were type E, and five were type D. Fifteen isolates were cpe(+) strains, including eight that were type A and seven that were type E.

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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.

  16. Characterization of Clostridium perfringens TpeL Toxin Gene Carriage, Production, Cytotoxic Contributions, and Trypsin Sensitivity

    PubMed Central

    Chen, Jianming

    2015-01-01

    Large clostridial toxins (LCTs) are produced by at least four pathogenic clostridial species, and several LCTs are proven pivotal virulence factors for both human and veterinary diseases. TpeL is a recently identified LCT produced by Clostridium perfringens that has received relatively limited study. In response, the current study surveyed carriage of the tpeL gene among different C. perfringens strains, detecting this toxin gene in some type A, B, and C strains but not in any type D or E strains. This study also determined that all tested strains maximally produce, and extracellularly release, TpeL at the late-log or early-stationary growth stage during in vitro culture, which is different from the maximal late-stationary-phase production reported previously for other LCTs and for TpeL production by C. perfringens strain JIR12688. In addition, the present study found that TpeL levels in culture supernatants can be repressed by either glucose or sucrose. It was also shown that, at natural production levels, TpeL is a significant contributor to the cytotoxic activity of supernatants from cultures of tpeL-positive strain CN3685. Lastly, this study identified TpeL, which presumably is produced in the intestines during diseases caused by TpeL-positive type B and C strains, as a toxin whose cytotoxicity decreases after treatment with trypsin; this finding may have pathophysiologic relevance by suggesting that, like beta toxin, TpeL contributes to type B and C infections in hosts with decreased trypsin levels due to disease, diet, or age. PMID:25824828

  17. Assessment of Clostridium perfringens spore response to high hydrostatic pressure and heat with nisin.

    PubMed

    Gao, Yulong; Qiu, Weifen; Wu, Ding; Fu, Qiang

    2011-08-01

    The elimination of spores from low-acid foods presents food-processing and food-safety challenges to high-pressure processing (HPP) developers as bacterial spores are extremely resistant to pressure. Therefore, the effects of pressure (400-800 MPa), temperature (35-95 °C), and nisin (0-496 IU/mL) on the inactivation of Clostridium perfringens AS 64701 spores at various pressure-holding times (7.5-17.5 min) were explored. A second-order polynomal equation for HPP- and nisin-induced inactivation of C. perfringens spores was constructed with response surface methodology. Experiment results showed that the experimental values were shown to be significantly in agreement with the predicted values because the adjusted determination coefficient (R (Adj)²) was 0.9708 and the level of significance was P < 0.0001. The optimum process parameters (obtained by solving the quadratic polynomal equation) for a six-log cycle reduction of C. perfringens AS 64701 spores were pressure of 654 Mpa, temperature of 74 °C, pressure-holding time of 13.6 min, and nisin concentration of 328 IU/mL. The validation of the model equation for predicting the optimum response values was verified effectively by ten test points that were not used in the establishment of the model. Compared with conventional HPP techniques, the main process advantages of HPP-nisin combination sterilization in the UHT milk are, lower pressure, temperature, natural preservative (nisin), and in a shorter treatment time. The synergistic inactivation of bacteria by HPP-nisin combination is a promising and natural method to increase the efficiency and safety of high-pressure pasteurization.

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

    PubMed Central

    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.

    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. PMID:27637108

  19. Necrotic enteritis potential in a model system using Clostridium perfringens isolated from field outbreaks.

    PubMed

    Chalmers, G; Bruce, H L; Toole, D L; Barnum, D A; Boerlin, P

    2007-12-01

    Necrotic enteritis is an enteric disease of avian species caused by the anaerobic bacterium Clostridium perfringens. The disease is regularly controlled in the broiler chicken industry with antimicrobials in feed but is reemerging in areas such as Europe where there is a ban on antimicrobials as growth promoters. To study prospective therapies, researchers must be able to reproduce this disease in a controlled environment, but this is not always possible because of differences in the pathogenicity of C. perfringens strains. Our objective was to test the potential of five isolates (SNECP43, 44, 47, 49, and 50), taken from field cases of necrotic enteritis, at recreating the disease in a controlled challenge experiment. SNECP43 and 50 were derived from a common clone, with SNECP50 passed in vivo and SNECP43 subcultured in vitro. Four hundred birds were divided into 16 pens, with three pens each receiving one of five treatments, with one control pen. Day-old birds were raised on a high wheat-based diet to promote necrotic enteritis development and were challenged with between 3.4 x 10(9) and 3.2 x 10(11) colony-forming units (cfu) of C. perfringens in feed for a period of 24 hr starting on day 13 of the challenge experiment. Lesion scores were assessed on two birds per pen sacrificed on day 17 and on any dead birds during the 25-day study. Growth performance was assessed up to 25 days, and mortality recorded throughout. Only SNECP50 produced necrotic enteritis mortalities significantly different (P < or = 0.05) from the control. The five isolates were also typed using pulsed-field gel electrophoresis to assess their genetic relatedness. All epidemiologically unrelated isolates were deemed genetically unrelated, whereas SNECP43 and 50 differed by only a single minor band. Toxin type was assessed using polymerase chain reaction (PCR), which was also used for the detection of the gene encoding the beta2-toxin.

  20. Structure-Function Analysis of Peptide Signaling in the Clostridium perfringens Agr-Like Quorum Sensing System

    PubMed Central

    Ma, Menglin; Li, Jihong

    2015-01-01

    ABSTRACT The accessory growth regulator (Agr)-like quorum sensing (QS) system of Clostridium perfringens controls the production of many toxins, including beta toxin (CPB). We previously showed (J. E. Vidal, M. Ma, J. Saputo, J. Garcia, F. A. Uzal, and B. A. McClane, Mol Microbiol 83:179–194, 2012, http://dx.doi.org/10.1111/j.1365-2958.2011.07925.x) that an 8-amino-acid, AgrD-derived peptide named 8-R upregulates CPB production by this QS system. The current study synthesized a series of small signaling peptides corresponding to sequences within the C. perfringens AgrD polypeptide to investigate the C. perfringens autoinducing peptide (AIP) structure-function relationship. When both linear and cyclic ring forms of these peptides were added to agrB null mutants of type B strain CN1795 or type C strain CN3685, the 5-amino-acid peptides, whether in a linear or ring (thiolactone or lactone) form, induced better signaling (more CPB production) than peptide 8-R for both C. perfringens strains. The 5-mer thiolactone ring peptide induced faster signaling than the 5-mer linear peptide. Strain-related variations in sensing these peptides were detected, with CN3685 sensing the synthetic peptides more strongly than CN1795. Consistent with those synthetic peptide results, Transwell coculture experiments showed that CN3685 exquisitely senses native AIP signals from other isolates (types A, B, C, and D), while CN1795 barely senses even its own AIP. Finally, a C. perfringens AgrD sequence-based peptide with a 6-amino-acid thiolactone ring interfered with CPB production by several C. perfringens strains, suggesting potential therapeutic applications. These results indicate that AIP signaling sensitivity and responsiveness vary among C. perfringens strains and suggest C. perfringens prefers a 5-mer AIP to initiate Agr signaling. IMPORTANCE Clostridium perfringens possesses an Agr-like quorum sensing (QS) system that regulates virulence, sporulation, and toxin production. The

  1. Macrolide-lincosamide-streptogramin resistance patterns in Clostridium perfringens from animals.

    PubMed Central

    Dutta, G N; Devriese, L A

    1981-01-01

    Different patterns of resistance against commonly used macrolide, lincosamide, and streptogramin antibiotics were found in Clostridium perfringens of animal origin. The patterns were designated as (i) macrolide-lincosamide-streptogramin group B generalized resistance, (ii) macrolide-lincosamide generalized resistance, (iii) macrolide-lincosamide inducible resistance, and (iv) macrolide-lincosamide-streptogramin low-level generalized resistance. The strains of the fourth pattern were able to inactivate pristinamycin and virginiamycin. The macrolide-susceptible strains showed a bimodal distribution of lincomycin and clindamycin susceptibility levels. The susceptible strains were inhibited by 0.25 micrograms of lincomycin per ml and 0.03 micrograms of clindamycin per ml. The low-level resistant strains were inhibited at concentrations of 2 to 4 micrograms of lincomycin per ml and 0.5 to 2 micrograms of clindamycin per ml. PMID:6289728

  2. Pathogenesis of brain damage produced in sheep by Clostridium perfringens type D epsilon toxin: a review.

    PubMed

    Finnie, J W

    2003-04-01

    Microvascular endothelial damage by the epsilon toxin of Clostridium perfringens type D appears to be the fundamental cause of cerebral parenchymal injury and lesions occur in a seemingly dose- and time-dependent manner. Large doses of circulating toxin produce a severe, generalised, vasogenic cerebral oedema and an acute or peracute clinical course to death. With lower doses of toxin, or in partially immune sheep, focal necrosis, often bilaterally symmetrical, occurs in certain selectively vulnerable brain regions, which appear to become fewer as the toxin dose is reduced. These cases follow a more protracted clinical course, but death is the usual outcome. The precise pathogenesis of the focal brain damage found in subacutely intoxicated sheep is unresolved, but several possible mechanisms are discussed.

  3. Specificity of Interaction between Clostridium perfringens Enterotoxin and Claudin-Family Tight Junction Proteins

    PubMed Central

    Mitchell, Leslie A.; Koval, Michael

    2010-01-01

    Clostridium perfringens enterotoxin (CPE), a major cause of food poisoning, forms physical pores in the plasma membrane of intestinal epithelial cells. The ability of CPE to recognize the epithelium is due to the C-terminal binding domain, which binds to a specific motif on the second extracellular loop of tight junction proteins known as claudins. The interaction between claudins and CPE plays a key role in mediating CPE toxicity by facilitating pore formation and by promoting tight junction disassembly. Recently, the ability of CPE to distinguish between specific claudins has been used to develop tools for studying roles for claudins in epithelial barrier function. Moreover, the high affinity of CPE to selected claudins makes CPE a useful platform for targeted drug delivery to tumors expressing these claudins. PMID:22069652

  4. Molecular typing and antimicrobial susceptibility of Clostridium perfringens from broiler chickens.

    PubMed

    Gharaibeh, Saad; Al Rifai, Rami; Al-Majali, Ahmad

    2010-12-01

    Clostridium perfringens (Cp) causes necrotic enteritis disease in commercial poultry. Antimicrobials are used to control and treat this disease and sometimes clinical outbreaks do not respond well to certain treatments. This study was designed to isolate Cp from clinical cases, type these isolates by multiplex PCR, and determine their antimicrobial susceptibility by micro-dilution method. A total of 67 Cp isolates were obtained from 155 broiler chicken flocks. All isolates were classified as type A and non-enterotoxin producers. Lincomycin, erythromycins, and tilmicosin showed very high minimal inhibitory concentration (MIC) 50 of ≥256 μg/ml. However, tylosin, amoxicillin, ampicillin, penicillin, florfenicol, danofloxacin, enrofloxacin, chlortetracycline, doxycycline, and oxytetracycline had variable MIC₅₀ of 64, 0.5, 1, 1, 8, 4, 8, 4, 8, 0.5 μg/ml, respectively. It is recommended that Cp infections in Jordan be treated with either penicillins or tetracyclines especially amoxicillin and oxytetracycline.

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

    PubMed Central

    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. PMID:8968913

  6. Potency against enterotoxemia of a recombinant Clostridium perfringens type D epsilon toxoid in ruminants.

    PubMed

    Lobato, Francisco C F; Lima, Catarina G R D; Assis, Ronnie A; Pires, Prhiscylla S; Silva, Rodrigo O S; Salvarani, Felipe M; Carmo, Anderson O; Contigli, Christiane; Kalapothakis, Evanguedes

    2010-08-31

    Enterotoxemia, a disease that affects domestic ruminants, is caused mainly by the epsilon toxin from Clostridium perfringens type D. Its eradication is virtually impossible, control and prophylaxis are based on systematic vaccination of herds with epsilon toxoids that are efficient in inducing protective antibody production. The use of recombinant toxins is one of the most promising of these strategies. This work evaluates the potency of a Cl. perfringens type D epsilon toxoid expressed by Escherichia coli administered to goats, sheep, and cattle. The etx gene was cloned into the pET-11a plasmid of E. coli strain BL21 to produce the recombinant toxin. Rabbits (n=8), goats, sheep, and cattle (n=5 for each species) were immunized with 0.2mg of the insoluble recombinant protein fraction to evaluate vaccine potency of the epsilon toxoid studied. Antibody titers were 40, 14.3, 26, and 13.1 IU/mL in the rabbit, goat, sheep, and cattle serum pools, respectively. The epsilon toxoid produced and tested in this work is adequate for immunization of ruminants against enterotoxemia.

  7. Clostridium perfringens epsilon toxin: the third most potent bacterial toxin known.

    PubMed

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

    2014-12-01

    Epsilon toxin (ETX) is produced by Clostridium perfringens type B and D strains and causes enterotoxemia, a highly lethal disease with major impacts on the farming of domestic ruminants, particularly sheep. ETX belongs to the aerolysin-like pore-forming toxin family. Although ETX has striking similarities to other toxins in this family, ETX is often more potent, with an LD50 of 100 ng/kg in mice. Due to this high potency, ETX is considered as a potential bioterrorism agent and has been classified as a category B biological agent by the Centers for Disease Control and Prevention (CDC) of the United States. The protoxin is converted to an active toxin through proteolytic cleavage performed by specific proteases. ETX is absorbed and acts locally in the intestines then subsequently binds to and causes lesions in other organs, including the kidneys, lungs and brain. The importance of this toxin for veterinary medicine and its possible use as a biological weapon have drawn the attention of researchers and have led to a large number of studies investigating ETX. The aim of the present work is to review the existing knowledge on ETX from C. perfringens type B and D.

  8. The pathology of peracute experimental Clostridium perfringens type D enterotoxemia in sheep.

    PubMed

    Uzal, F A; Kelly, W R; Morris, W E; Bermudez, J; Baisón, M

    2004-09-01

    The pathological findings in sheep with peracute experimental Clostridium perfringens type D enterotoxemia are described. Of 16 animals inoculated intraduodenally with a whole culture of this microorganism and a starch solution in the abomasum, 12 developed clinical signs including increased respiratory efforts, recumbency, paddling, bleating, convulsions, blindness, and opisthotonus. Diarrhea was not observed in any of the animals. The time lapse between the beginning of intraduodenal infusion and onset of clinical signs varied between 30 minutes and 26 hours, and the clinical course varied between 1 and 9 hours. Gross postmortem changes were observed in these 12 animals and included pulmonary edema; excess pericardial, peritoneal, or pleural fluid with or without strands of fibrin; liquid small intestinal contents; leptomeningeal edema; cerebellar coning; and subcapsular petechiae on kidneys. Histological changes consisted of severe edema of pleura and interlobular septa and around blood vessels and airways and acidophilic, homogeneous, proteinaceous perivascular edema in the brain. Five of 12 animals (42%) with clinical signs consistent with enterotoxemia lacked specific histological lesions in the brain. None of the intoxicated or control animals developed nephrosis. Glucose was detected in the urine of 3 of 6 animals that were tested for this analyte. These results stress the importance of the use of histological examination of the brain, coupled with epsilon toxin detection, for a definitive diagnosis of C. perfringens type D enterotoxemia in sheep.

  9. Experimental production of hemorrhagic enterotoxemia by Clostridium perfringens type C in maturing lambs.

    PubMed

    Niilo, L

    1986-01-01

    Maturing lambs, eight to nine months old, were dosed by the intraduodenal route with various preparations of Clostridium perfringens type C. Whole cultures of this organism or cells suspended in fresh medium, both supplemented with soybean flour as a protease inhibitor, produced acute fatal hemorrhagic enterotoxemia in these animals. The latter preparation was more effective than the former in causing disease. Without the soybean supplement the inocula did not produce fatal disease. Dosing with toxic cell-free culture supernatant fluid, with or without soybean supplement, had no lethal effect. Animals that died showed severe hemorrhagic enteritis with necrosis and sloughing of the mucosal epithelium, involving jejunum, ileum and part of duodenum. These lesions were similar to those seen in natural cases of hemorrhagic enterotoxemia in neonatal animals. This experiment demonstrated that nonimmune animals are normally protected against C. perfringens type C enterotoxemia by adequate levels of pancreatic proteases in the intestine, and that factors which inhibit or reduce these enzymes predispose animals for the development of this disease.

  10. In vivo studies of Clostridium perfringens in mouse gas gangrene model.

    PubMed

    Sengupta, Nabonita; Alam, Syed Imteyaz

    2011-03-01

    Understanding the pathogenesis of infectious diseases requires comprehensive knowledge of the proteins expressed by the pathogen during in vivo growth in the host. Proteomics provides the tools for such analyses but the protocols required to purify sufficient quantities of the pathogen from the host organism are currently lacking. In this study, we have separated Clostridium perfringens, a highly virulent bacterium and potential BTW agent, from the peritoneal fluid of infected mice using Percoll density gradient centrifugation. The bacterium could be isolated in quantities sufficient to carry out meaningful proteomic comparisons with in vitro grown bacteria. Furthermore, the isolates were found to be virtually free from contaminating host proteins. Microscopy revealed major morphological changes under host conditions at different stages of infection. Profile of immunogenic proteins from in vivo- and TPYG-grown whole cell lysate using mouse anti-gangrene serum indicated over-expression of several proteins especially in the low molecular weight region. Expression of two virulence determinants, ornithine carbamoyl transferase (cOTC), and cystathionine beta-lyase (CBL), under in vivo conditions has also been studied. Two-dimensional gel analysis revealed a host induced proteome which was apparently different in comparison to in vitro grown cells. Detailed proteomic elucidation of differentially expressed proteins shown here is likely to provide valuable insight towards understanding the complexity of the adaptive response of C. perfringens to the host environment.

  11. The control of necrotic enteritis in sucking piglets by means of a Clostridium perfringens toxoid vaccine.

    PubMed

    Springer, S; Selbitz, H J

    1999-07-01

    Necrotic enteritis in sucking piglets constitutes a serious problem in piglet rearing units because of the high morbidity and mortality associated with the disease. The primary causal agent is Clostridium perfringens type C. The beta-toxin plays a decisive role in the pathogenesis of this disease. A toxoid vaccine for use in sows has been developed and studied in field trials. The European Pharmacopoeia Monograph on vaccines for use in animals lays down a method of the efficacy testing based on the immunization of rabbits, the collection of pooled sera and the subsequent assay of anti-toxin antibodies in mice using an appropriate test toxin. The vaccine is regarded as effective if it induces a minimum of 10 IU of beta-anti-toxin per ml of rabbit serum. We have established a range of 17.14-98.23 IU beta-anti-toxin per ml rabbit serum induced by a sample of C. perfringens toxoid vaccine. The vaccine has been used under field conditions in different rearing units at the same time, mostly in the form of emergency vaccinations following the outbreak of disease. The outcome of vaccination was evaluated by recording the total numbers of piglets born alive and the piglet losses. Use of the vaccine, coupled with other measures, resulted in an approximately 30% reduction in the number of losses.

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

    PubMed

    Harmon, S M; Kautter, D A

    1987-01-01

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

  13. Influence of peptone source on sporulation of Clostridium perfringens type A.

    PubMed

    Hsieh, P Y; Labbe, R

    2007-07-01

    Clostridium perfringens is a foodborne disease agent that produces a sporulation-specific enterotoxin. To produce enterotoxin for experimental purposes or spores for challenge or physiological studies, the use of a convenient sporulation medium is required. The most commonly used is Duncan-Strong medium. Few isolates sporulate at high levels in this medium. We investigated the effectiveness of peptones from a variety of sources on the sporulation of this organism compared with the peptone in the original formulation, proteose peptone (control). Seven strains were used to screen 32 peptones, with starch or raffinose as the carbohydrate source. In most cases, raffinose was more effective than starch in stimulating sporulation, confirming our previous study. Two promising peptones, potato peptone, and Proteose Peptone no. 3, were selected and tested against 49 additional enterotoxin-positive and -negative strains, with raffinose as the carbohydrate. For 49 strains, 5 sporulated best (>10%) in the control peptone, 6 sporulated best in Peptone no. 3, and 23 sporulated best in the potato peptone. Of the 23 strains, 16 sporulated at levels 25% more than the control peptone. The increase in sporulation rates was reflected in the enterotoxin and heat-resistant spore levels. The methylxanthines caffeine and theobromine were effective in increasing the sporulation of less than half of 19 enterotoxin-positive strains. Our results suggest that the replacement of proteose peptone with potato peptone be considered if difficulty in obtaining spores of specific strains of C. perfringens is encountered.

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

    PubMed

    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.

  15. Lipidomic profile of GM95 cell death induced by Clostridium perfringens alpha-toxin.

    PubMed

    Manni, Marco M; Valero, Juan G; Pérez-Cormenzana, Miriam; Cano, Ainara; Alonso, Cristina; Goñi, Félix M

    2017-03-01

    Clostridium perfringens alpha-toxin (ATX) is considered as a prototype of cytotoxic bacterial phospholipases C, and is the major virulence factor in C. perfringens-induced gas gangrene. It is known that, depending on the dose, ATX causes membrane disruption and cytolysis or only limited hydrolysis of its substrates. In the latter case, toxin activity leads to the unregulated generation of bioactive lipids that can ultimately induce cell death. We have characterized apoptosis and necrosis in highly ATX-sensitive, ganglioside-deficient cells exposed to different concentrations of ATX and we have studied the lipidomic profile of cells treated with ATX as compared to native cells to detect the main changes in the lipidomic profile and the possible involvement of lipid signals in cell death. ATX causes both apoptosis and necrosis, depending on dose and time. ATX activates cell death, stimulating the release of cytochrome C from mitochondria and the consequent activation of caspases-3. Moreover GM95 cells treated with ATX showed important lipidomic alterations, among them we detected a general decrease in several phospholipid species and important changes in lipids involved in programmed cell death e.g. ceramide. The data suggest two different mechanisms of cell death caused by ATX, one leading to (mainly saturated) glycerophospholipid hydrolysis related to an increase in diacylglycerols and associated to membrane damage and necrosis, and a second mechanism involving chiefly sphingomyelin hydrolysis and generation of proapoptotic lipidic mediators such as ceramide, N-acylethanolamine and saturated non-esterified fatty acids.

  16. Genomic diversity of Clostridium perfringens strains isolated from food and human sources

    PubMed Central

    Afshari, A.; Jamshidi, A.; Razmyar, J.; Rad, M.

    2016-01-01

    Clostridium perfringens is a serious pathogen which causes enteric diseases in domestic animals and food poisoning in humans. Spores can survive cooking processes and play an important role in the possible onset of disease. In this study, RAPD-PCR and REP-PCR were used to examine the genetic diversity of 49 isolates of C. perfringens type A from three different sources. The results of RAPD-PCR revealed the most genetic diversity among poultry isolates, while human isolates showed the least genetic diversity. Cluster analysis obtained from RAPD-PCR and based on the genetic distances split the 49 strains into five distinct major clusters (A, B, C, D, and E). Cluster A and C were composed of isolates from poultry meat, cluster B was composed of isolates from human stool, cluster D was composed of isolates from minced meat, poultry meat and human stool and cluster E was composed of isolates from minced meat. Further characterization of these strains by using (GTG) 5 fingerprint repetitive sequence-based PCR analysis did not show further differentiation between various types of strains. In conclusion, RAPD-PCR method seems to be very promising for contamination source tracking in the field of food hygiene. PMID:27822244

  17. Role of RNase Y in Clostridium perfringens mRNA Decay and Processing.

    PubMed

    Obana, Nozomu; Nakamura, Kouji; Nomura, Nobuhiko

    2017-01-15

    RNase Y is a major endoribonuclease that plays a crucial role in mRNA degradation and processing. We study the role of RNase Y in the Gram-positive anaerobic pathogen Clostridium perfringens, which until now has not been well understood. Our study implies an important role for RNase Y-mediated RNA degradation and processing in virulence gene expression and the physiological development of the organism. We began by constructing an RNase Y conditional knockdown strain in order to observe the importance of RNase Y on growth and virulence. Our resulting transcriptome analysis shows that RNase Y affects the expression of many genes, including toxin-producing genes. We provide data to show that RNase Y depletion repressed several toxin genes in C. perfringens and involved the virR-virS two-component system. We also observe evidence that RNase Y is indispensable for processing and stabilizing the transcripts of colA (encoding a major toxin collagenase) and pilA2 (encoding a major pilin component of the type IV pili). Posttranscriptional regulation of colA is known to be mediated by cleavage in the 5' untranslated region (5'UTR), and we observe that RNase Y depletion diminishes colA 5'UTR processing. We show that RNase Y is also involved in the posttranscriptional stabilization of pilA2 mRNA, which is thought to be important for host cell adherence and biofilm formation.

  18. Crystal structure of the ADP-ribosylating component of BEC, the binary enterotoxin of Clostridium perfringens.

    PubMed

    Kawahara, Kazuki; Yonogi, Shinya; Munetomo, Ryota; Oki, Hiroya; Yoshida, Takuya; Kumeda, Yuko; Matsuda, Shigeaki; Kodama, Toshio; Ohkubo, Tadayasu; Iida, Tetsuya; Nakamura, Shota

    2016-11-11

    Binary enterotoxin of Clostridium perfringens (BEC), consisting of the components BECa and BECb, was recently identified as a novel enterotoxin produced by C. perfringens that causes acute gastroenteritis in humans. Although the detailed mechanism of cell intoxication by BEC remains to be defined, BECa shows both NAD(+)-glycohydrolase and actin ADP-ribosyltransferase activities in the presence of NAD(+). In this study, we determined the first crystal structure of BECa in its apo-state and in complex with NADH. The structure of BECa shows striking resemblance with other binary actin ADP-ribosylating toxins (ADPRTs), especially in terms of its overall protein fold and mechanisms of substrate recognition. We present a detailed picture of interactions between BECa and NADH, including bound water molecules located near the C1'-N glycosidic bond of NADH and the catalytically important ADP-ribosylating turn-turn (ARTT) loop. We observed that the conformational rearrangement of the ARTT loop, possibly triggered by a conformational change involving a conserved tyrosine residue coupled with substrate binding, plays a crucial role in catalysis by properly positioning a catalytic glutamate residue in the E-X-E motif of the ARTT loop in contact with the nucleophile. Our results for BECa provide insight into the common catalytic mechanism of the family of binary actin ADPRTs.

  19. Genome Sequencing and Analysis of a Type A Clostridium perfringens Isolate from a Case of Bovine Clostridial Abomasitis

    PubMed Central

    Nowell, Victoria J.; Kropinski, Andrew M.; Songer, J. Glenn; MacInnes, Janet I.; Parreira, Valeria R.; Prescott, John F.

    2012-01-01

    Clostridium perfringens is a common inhabitant of the avian and mammalian gastrointestinal tracts and can behave commensally or pathogenically. Some enteric diseases caused by type A C. perfringens, including bovine clostridial abomasitis, remain poorly understood. To investigate the potential basis of virulence in strains causing this disease, we sequenced the genome of a type A C. perfringens isolate (strain F262) from a case of bovine clostridial abomasitis. The ∼3.34 Mbp chromosome of C. perfringens F262 is predicted to contain 3163 protein-coding genes, 76 tRNA genes, and an integrated plasmid sequence, Cfrag (∼18 kb). In addition, sequences of two complete circular plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), and two incomplete plasmid fragments, pF262A (48.5 kb) and pF262B (50.0 kb), were identified. Comparison of the chromosome sequence of C. perfringens F262 to complete C. perfringens chromosomes, plasmids and phages revealed 261 unique genes. No novel toxin genes related to previously described clostridial toxins were identified: 60% of the 261 unique genes were hypothetical proteins. There was a two base pair deletion in virS, a gene reported to encode the main sensor kinase involved in virulence gene activation. Despite this frameshift mutation, C. perfringens F262 expressed perfringolysin O, alpha-toxin and the beta2-toxin, suggesting that another regulation system might contribute to the pathogenicity of this strain. Two complete plasmids, pF262C (4.8 kb) and pF262D (9.1 kb), unique to this strain of C. perfringens were identified. PMID:22412860

  20. Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron.

    PubMed

    Chen, Yue; McClane, Bruce A; Fisher, Derek J; Rood, Julian I; Gupta, Phalguni

    2005-11-01

    In developing Clostridium perfringens as a safe vaccine vector, the alpha toxin gene (plc) in the bacterial chromosome must be permanently inactivated. Disrupting genes in C. perfringens by traditional mutagenesis methods is very difficult. Therefore, we developed a new strategy using group II intron-based Target-Tron technology to inactivate the plc gene in C. perfringens ATCC 3624. Western blot analysis showed no production of alpha toxin protein in the culture supernatant of the plc mutant. Advantages of this technology, such as site specificity, relatively high frequency of insertion, and introduction of no antibiotic resistance genes into the chromosome, could facilitate construction of other C. perfringens mutants.

  1. Co-infection with Toxoplasma gondii and Clostridium perfringens in a postpartum woman with uterine gas gangrene: a case report.

    PubMed

    Alsammani, Mohamed Alkhatim; Ahmed, Salah Roshdy; Alsheeha, Muneera A; Saadia, Zaheera; Khairi, Somia A

    2012-07-01

    Toxoplasmosis is a protozoan infection caused by Toxoplasma gondii. We report a case of Toxoplasma gondii and Clostridium perfringens co-infection complicating uterine gas gangrene following a term pregnancy. The histological examination of the necrotic uterine tissues and uterine swab cultures obtained at laparotomy revealed T. gondii and C. perfringens, respectively. Treatment was administered with bactericidal activity against both pathogens and the patient had an uneventful post-operative recovery. Although there have been some cases that have documented an association between toxoplasmosis and non-uterine C. perfringens infection, such a relationship has not been established. It is of interest to determine if the presence of both organisms can explain the severe myonecrosis that occurs in some cases of uterine gas gangrene.

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

    PubMed

    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

  3. Assessing the Performance of Clostridium perfringens Cooling Models for Cooked, Uncured Meat and Poultry Products.

    PubMed

    Mohr, T B; Juneja, V K; Thippareddi, H H; Schaffner, D W; Bronstein, P A; Silverman, M; Cook, L V

    2015-08-01

    Heat-resistant spores of Clostridium perfringens may germinate and multiply in cooked meat and poultry products when 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; and UK IFR ComBase Perfringens Predictor) were evaluated for relative performance in predicting growth of C. perfringens under dynamic temperature conditions encountered during cooling of cooked, uncured meat and poultry products. The predicted growth responses from the models were extensively compared with those observed in food. Data from 188 time-temperature cooling profiles (176 for single-rate exponential cooling and 12 for dual-rate exponential cooling) were collected from 17 independent sources (16 peer-reviewed publications and one report) for model evaluation. Data were obtained for a variety of cooked products, including meat and poultry slurries, ground meat and poultry products with and without added ingredients (e.g., potato starch, sodium triphosphate, and potassium tetrapyrophosphate), and processed products such as ham and roast beef. Performance of the models was evaluated using three sets of criteria, and accuracy was defined within a 1- to 2-log range. The percentages of accurate, fail-safe, or fail-dangerous predictions for each cooling model differed depending on which criterion was used to evaluate the data set. Nevertheless, the combined percentages of accurate and fail-safe predictions based on the three performance criteria were 34.66 to 42.61% for the PMP 7.0 beef broth model, 100% for the PMIP cooling models for uncured beef, uncured pork and uncured chicken, 80.11 to 93.18% for the Smith-Schaffner cooling model, and 74.43 to 85.23% for the UK IFR ComBase Perfringens Predictor model during single-rate exponential chilling. Except for the PMP 7.0 broth model, the other five cooling models (PMIP, Smith-Schaffner, and UK IFR ComBase) are

  4. Clinico-pathological findings of Clostridium perfringens type D enterotoxaemia in goats and its hemolytic activity in different erythrocytes

    PubMed Central

    Ali Nasir, A.; Younus, M.; Rashid, A.; Abdul Khaliq, S.; Khan, E.; Shah, S. H.; Aslam, A.; Ghumman, M. A.; Joiya, M. H.

    2015-01-01

    The present investigation was conducted to study the effects of experimental Clostridium perfringens type D enterotoxaemia in teddy goats. Clinical signs started to appear after 30 min of experimental infection like anorexia, diarrhea, dehydration, frothing and dyspnea. Gross lesions consisted of severe congestion in tissues of varying intensity with enlarged mesenteric lymph nodes while histological examination revealed edema of lungs, kidney, and lymph nodes and to some extent in brain along with hemorrhages in lungs and intestines. Clostridium perfringens type D carrying alpha and epsilon toxin genes were amplified with amplicon size about 247 bp and 665 bp, respectively. Human erythrocytes showed the highest hemolysis, 68%, followed by mice, 57%, against culture supernatants. The percentage of hemolysis was significantly higher at 37°C as compared to 25°C except for rabbit and dog. PMID:27175159

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

    PubMed

    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 <1 log, regardless of the inoculum level. In addition, no growth was observed in any 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 <1-log growth of C. perfringens from spores was observed in the pH 5.63 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

  6. The Mycotoxin Deoxynivalenol Predisposes for the Development of Clostridium perfringens-Induced Necrotic Enteritis in Broiler Chickens

    PubMed Central

    Antonissen, Gunther; 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% (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. PMID:25268498

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

    PubMed

    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% (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.

  8. The impact of various browse feeds with different tannin content on the fecal shedding of Clostridium perfringens in West African dwarf sheep.

    PubMed

    Aschfalk, A; Müller, W; Drochner, W

    2000-01-01

    In 1994 and 1995 leaves from eight browse feeds, containing tannins in different amounts (BF), were fed to West African Dwarf Sheep in Benin to evaluate their impact on Clostridium perfringens in the intestinal tract. An inhibitory impact of various BF on the growth of C. perfringens was assessed in in-vitro assays before, and thus a potential use of these leaves as a preventive diet against C. perfringens enterotoxemia in small ruminants was assumed. Surprisingly, an inhibitory impact of the BF on the shedding of C. perfringens in the feces of West African Dwarf Sheep could not be shown in seven of the eight BF examined. However, the pattern of inhibition of unlike C. perfringens toxovars may differ and a selective inhibitory impact of the BF Dialium guineense on C. perfringens toxovar D may be assumed.

  9. Host cell-induced signaling causes Clostridium perfringens to upregulate production of toxins important for intestinal infections

    PubMed Central

    Chen, Jianming; Ma, Menglin; Uzal, Francisco A; McClane, Bruce A

    2014-01-01

    Clostridium perfringens causes enteritis and enterotoxemia in humans and livestock due to prolific toxin production. In broth culture, C. perfringens uses the Agr-like quorum sensing (QS) system to regulate production of toxins important for enteritis/enterotoxemia, including beta toxin (CPB), enterotoxin, and epsilon toxin (ETX). The VirS/VirR two-component regulatory system (TCRS) also controls CPB production in broth cultures. Both the Agr-like QS and VirS/VirR systems are important when C. perfringens senses enterocyte-like Caco-2 cells and responds by upregulating CPB production; however, only the Agr-like QS system is needed for host cell-induced ETX production. These in vitro observations have pathophysiologic relevance since both the VirS/VirR and Agr-like QS signaling systems are required for C. perfringens strain CN3685 to produce CPB in vivo and to cause enteritis or enterotoxemia. Thus, apparently upon sensing its presence in the intestines, C. perfringens utilizes QS and TCRS signaling to produce toxins necessary for intestinal virulence. PMID:24061146

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

    PubMed

    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) C. perfringens outgrowth in both total population and number of vegetative cells. Both temperature and nitrite concentration significantly affected (P < 0.05) C. perfringens spore survival, but the interaction between nitrite and temperature did not have a significant effect (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

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

    PubMed

    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. Differential Responses of Cecal Microbiota to Fishmeal, Eimeria and Clostridium perfringens in a Necrotic Enteritis Challenge Model in Chickens

    PubMed Central

    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

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

    PubMed Central

    Gil, Carles; Dorca-Arévalo, Jonatan; Blasi, Juan

    2015-01-01

    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. PMID:26452234

  14. Virulence for chickens of Clostridium perfringens isolated from poultry and other sources.

    PubMed

    Cooper, Kerry K; Theoret, James R; Stewart, Bernard A; Trinh, Hien T; Glock, Robert D; Songer, J Glenn

    2010-06-01

    Clostridium perfringens type A is the most common cause of poultry necrotic enteritis (NE). Of the four "major" toxins, type A strains produce only alpha toxin (CPA), which has long been considered a major factor in pathogenesis of NE. We investigated the virulence for poultry of type A strains from a variety of enteric sources. Newly-hatched CornishxRock chicks were fed a low protein diet for one week, a high protein diet for a second week, and then challenged with log-phase cultures of C. perfringens, mixed 3:4 (v/v) with high protein feed. Strain JGS4143 [genotype A, beta2 positive (cpb2(pos)), from a field case of NE] produced gross lesions compatible with NE in >85% of challenged birds. However, strains JGS1714 (enterotoxigenic genotype A, cpb2(pos), human food poisoning), JGS1936 (genotype A, cpb2(neg), bovine neonatal enteritis), JGS4142 (genotype A, cpb2(pos), bovine jejunal hemorrhage syndrome), JGS1473 (genotype A, cpb2(pos), chicken normal flora), JGS1070 (genotype C, cpb2(pos), porcine hemorrhagic enteritis), JGS1882 (genotype A, cpb2(pos), porcine neonatal enteritis), JGS1120 (ATCC 13124, genotype A, cpb2(neg), gas gangrene), JGS4151 (strain 13, genotype A, cpb2(pos), canine), and JGS4303 (SM101, enterotoxigenic genotype A, cpb2(neg), human food poisoning) failed to produce disease. In vivo passage failed to increase virulence of the non-NE strains. NE strains must have specific poultry-associated virulence attributes, such as the recently identified NetB and other factors, which allow for the development of disease.

  15. The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats

    PubMed Central

    Garcia, J. P.; Beingesser, J.; Fisher, D. J.; Sayeed, S.; McClane, B. A.; Posthaus, H.; Uzal, F. A.

    2012-01-01

    Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch’s postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24 h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24 h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis. PMID:22296994

  16. The effect of Clostridium perfringens type C strain CN3685 and its isogenic beta toxin null mutant in goats.

    PubMed

    Garcia, J P; Beingesser, J; Fisher, D J; Sayeed, S; McClane, B A; Posthaus, H; Uzal, F A

    2012-06-15

    Clostridium perfringens type C is an important cause of enteritis and/or enterocolitis in several animal species, including pigs, sheep, goats, horses and humans. The disease is a classic enterotoxemia and the enteric lesions and associated systemic effects are thought to be caused primarily by beta toxin (CPB), one of two typing toxins produced by C. perfringens type C. This has been demonstrated recently by fulfilling molecular Koch's postulates in rabbits and mice. We present here an experimental study to fulfill these postulates in goats, a natural host of C. perfringens type C disease. Nine healthy male or female Anglo Nubian goat kids were inoculated with the virulent C. perfringens type C wild-type strain CN3685, an isogenic CPB null mutant or a strain where the cpb null mutation had been reversed. Three goats inoculated with the wild-type strain presented abdominal pain, hemorrhagic diarrhea, necrotizing enterocolitis, pulmonary edema, hydropericardium and death within 24h of inoculation. Two goats inoculated with the CPB null mutant and two goats inoculated with sterile culture media (negative controls) remained clinically healthy during 24h after inoculation and no gross or histological abnormalities were observed in the tissues of any of them. Reversal of the null mutation to partially restore CPB production also increased virulence; 2 goats inoculated with this reversed mutant presented clinical and pathological changes similar to those observed in goats inoculated with the wild-type strain, except that spontaneous death was not observed. These results indicate that CPB is required for C. perfringens type C to induce disease in goats, supporting a key role for this toxin in natural C. perfringens type C disease pathogenesis.

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

    PubMed

    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 comparative study of the effects of Escherichia coli and Clostridium perfringens upon boar semen preserved in liquid storage.

    PubMed

    Pinart, Elisabeth; Domènech, Esther; Bussalleu, Eva; Yeste, Marc; Bonet, Sergi

    2017-02-01

    The present study compares the sperm quality of boar seminal doses artificially inoculated with Escherichia coli and Clostridium perfringens, and maintained in liquid storage at 15°C for a 9-day period. Seminal doses from 10 sexually mature Piétrain boars were diluted in a Beltsville Thawing Solution (BTS)-based extender and infected either with E. coli or C. perfringens, with bacterial loads ranging from 10(1) to 10(7)cfumL(-1). During storage, the changes in sperm quality were determined by assessing pH, sperm viability, sperm motiliy, sperm morphology, sperm agglutination degree, and sperm-bacteria interaction. The infection of seminal doses led to an alkalinization of the medium, which was of higher extend in doses infected with C. perfringens. The effect of contamination on sperm viability and motility relied on bacterial type and load. Therefore, while E. coli was more harmful than C. perfringens in bacterial loads ranging from 10(1) to 10(6)cfumL(-1), the detrimental impact of C. perfringens was more apparent than that of E. coli at a bacterial load of 10(7)cfumL(-1). Despite sperm morphology not being affected by either bacterial type or load, sperm agglutination and sperm-bacteria interaction were characteristic of doses infected with E. coli, and increased concomintantly with bacterial load and along storage period. In conclusion, the effects of infection by E. coli on sperm quality were dependent of both bacterial load and storage period, whereas the effects of C. perfringens were mainly dependent on the bacterial load, with a threshold at 10(7)cfumL(-1) from which the sperm quality of seminal doses was greatly impaired.

  19. Contact with enterocyte-like Caco-2 cells induces rapid upregulation of toxin production by Clostridium perfringens type C isolates

    PubMed Central

    Vidal, Jorge E.; Ohtani, Kaori; Shimizu, Tohru; McClane, Bruce A.

    2009-01-01

    Clostridium perfringens type C isolates cause necrotizing enteritis in humans and domestic animals. In vitro, type C isolates often produce beta toxin (CPB), beta2 toxin (CPB2), alpha toxin (CPA), perfringolysin O (PFO), and TpeL during (or after) late log-phase growth. In contrast, the current study found that many type C isolates respond to close contact with enterocyte-like Caco-2 cells by producing all toxins, except TpeL, much more rapidly than occurs during in vitro growth. This in vivo effect involves rapid transcriptional upregulation of the cpb, cpb2, pfoA and plc toxin genes. Rapid Caco-2 cell-induced upregulation of CPB and PFO production involves the VirS/VirR two-component system, since upregulated in vivo transcription of the pfoA and cpb genes was blocked by inactivating the virR gene and was reversible by complementation to restore VirR expression. However, the luxS quorum sensing system is not required for the rapid upregulation of type C toxin production induced by contact with Caco-2 cells. These results provide the first indication of host cell:pathogen cross-talk affecting toxin production kinetics by any pathogenic Clostridium spp., identify in vivo vs. in vitro differences in C. perfringens toxin expression, and implicate VirS/VirR as a possible contributor to some C. perfringens enteric diseases. PMID:19438515

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

    PubMed

    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.

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

    PubMed Central

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

    2015-01-01

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

  2. Development and application of a multiplex PCR assay for detection of the Clostridium perfringens enterotoxin-encoding genes cpe and becAB.

    PubMed

    Yonogi, Shinya; Kanki, Masashi; Ohnishi, Takahiro; Shiono, Masami; Iida, Tetsuya; Kumeda, Yuko

    2016-08-01

    Clostridium perfringens causes food-borne gastroenteritis following the consumption of contaminated food by producing C. perfringens enterotoxin (CPE) in the intestines. Recently, we reported a novel enterotoxin, binary enterotoxin of C. perfringens (BEC) in C. perfringens isolates, which caused two disease outbreaks in Japan. Consequently, in the event of food poisoning outbreaks caused by C. perfringens, it is now necessary to screen for both the cpe and becAB genes by diagnostic PCR. Here, we present a simple multiplex PCR method for simultaneous detection of cpe, becAB and a C. perfringens control locus, phospholipase C (plc). Applying this method, we investigated the prevalence of cpe- or becAB-carrying C. perfringens strains in human stool and bovine rectum swab samples. Using a total of 169 isolates, we found that the percentage of becAB-carrying strains was very small (0.59%), one-tenth that of cpe-carrying strains. The simple method presented in this study with high specificity and sensitivity to C. perfringens will be a useful tool to survey the global prevalence of becAB-carrying C. perfringens strains.

  3. Clostridium perfringens epsilon toxin induces permanent neuronal degeneration and behavioral changes.

    PubMed

    Morris, Winston E; Goldstein, Jorge; Redondo, Leandro M; Cangelosi, Adriana; Geoghegan, Patricia; Brocco, Marcela; Loidl, Fabián C; Fernandez-Miyakawa, Mariano E

    2017-05-01

    Clostridium perfringens epsilon toxin (ETX), the most potent toxin produced by this bacteria, plays a key role in the pathogenesis of enterotoxaemia in ruminants, causing brain edema and encephalomalacia. Studies of animals suffering from ETX intoxication describe severe neurological disorders that are thought to be the result of vasogenic brain edemas and indirect neuronal toxicity, killing oligodendrocytes but not astrocytes, microglia, or neurons in vitro. In this study, by means of intravenous and intracerebroventricular delivery of sub-lethal concentrations of ETX, the histological and ultrastructural changes of the brain were studied in rats and mice. Histological analysis showed degenerative changes in neurons from the cortex, hippocampus, striatum and hypothalamus. Ultrastructurally, necrotic neurons and apoptotic cells were observed in these same areas, among axons with accumulation of neurofilaments and demyelination as well as synaptic stripping. Lesions observed in the brain after sub-lethal exposure to ETX, result in permanent behavioral changes in animals surviving ETX exposure, as observed individually in several animals and assessed in the Inclined Plane Test and the Wire Hang Test. Pharmacological studies showed that dexamethasone and reserpine but not ketamine or riluzole were able to reduce the brain lesions and the lethality of ETX. Cytotoxicity was not observed upon neuronal primary cultures in vitro. Therefore, we hypothesize that ETX can affect the brain of animals independently of death, producing changes on neurons or glia as the result of complex interactions, independently of ETX-BBB interactions.

  4. The p38 MAPK and JNK Pathways Protect Host Cells against Clostridium perfringens Beta-Toxin

    PubMed Central

    Shibutani, Masahiro; Seike, Soshi; Yonezaki, Mami; Takagishi, Teruhisa; Oda, Masataka; Kobayashi, Keiko; Sakurai, Jun

    2013-01-01

    Clostridium perfringens beta-toxin is an important agent of necrotic enteritis and enterotoxemia. Beta-toxin is a pore-forming toxin (PFT) that causes cytotoxicity. Two mitogen-activated protein kinase (MAPK) pathways (p38 and c-Jun N-terminal kinase [JNK]-like) provide cellular defense against various stresses. To investigate the role of the MAPK pathways in the toxic effect of beta-toxin, we examined cytotoxicity in five cell lines. Beta-toxin induced cytotoxicity in cells in the following order: THP-1 = U937 > HL-60 > BALL-1 = MOLT-4. In THP-1 cells, beta-toxin formed oligomers on lipid rafts in membranes and induced the efflux of K+ from THP-1 cells in a dose- and time-dependent manner. The phosphorylation of p38 MAPK and JNK occurred in response to an attack by beta-toxin. p38 MAPK (SB203580) and JNK (SP600125) inhibitors enhanced toxin-induced cell death. Incubation in K+-free medium intensified p38 MAPK activation and cell death induced by the toxin, while incubation in K+-high medium prevented those effects. While streptolysin O (SLO) reportedly activates p38 MAPK via reactive oxygen species (ROS), we showed that this pathway did not play a major role in p38 phosphorylation in beta-toxin-treated cells. Therefore, we propose that beta-toxin induces activation of the MAPK pathway to promote host cell survival. PMID:23876806

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

    PubMed

    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.

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

    PubMed

    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.

  7. Clostridium perfringens infection among inmates at a county jail--Wisconsin, August 2008.

    PubMed

    2009-02-20

    On August 8, 2008, employees at a Wisconsin county jail noted nausea, vomiting, and diarrhea among more than 100 inmates during the early morning inspection. Seven inmates were seen by the jail nurse that morning. Following jail protocol, guards gave at least 60 inmates bismuth subsalicylate to relieve symptoms, and the jail nurse notified local health department staff members, who suspected a foodborne outbreak at the jail and initiated an investigation. This report summarizes the findings of an investigation by the Wisconsin Division of Public Health (WDPH) and the local health department, which determined the outbreak was caused by eating casserole containing ground turkey and beef (relative risk [RR] = 25.1) that was served during the evening meal on August 7. Clostridium perfringens enterotoxin was detected in stool samples collected from six ill inmates, and 43,000 CFU/g of the organism were isolated from a remaining sample of casserole. An environmental investigation determined the casserole was made with food items that were prepared and stored improperly. Proper food preparation and storage methods are especially important in large institutions such as jails and prisons, where large amounts of foods are prepared and served at one time.

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

    PubMed

    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

  9. Identification of a Lambda Toxin-Negative Clostridium perfringens Strain that Processes and Activates Epsilon Prototoxin Intracellularly

    PubMed Central

    Harkness, Justine M.; Li, Jihong; McClane, Bruce A.

    2012-01-01

    Clostridium perfringens type B and D strains produce epsilon toxin (ETX), which is one of the most potent clostridial toxins and is involved in enteritis and enterotoxemias of domestic animals. ETX is produced initially as an inactive prototoxin that is typically then secreted and processed by intestinal proteases or possibly, for some strains, lambda toxin. During the current work a unique C. perfringens strain was identified that intracellularly processes epsilon prototoxin to an active form capable of killing MDCK cells. This activated toxin is not secreted but instead is apparently released upon lysis of bacterial cells entering stationary phase. These findings broaden understanding of the pathogenesis of type B and D infections by identifying a new mechanism of ETX activation. PMID:22982043

  10. Detection of alpha- and epsilon-toxigenic Clostridium perfringens type D in sheep and goats using a DNA amplification technique (PCR).

    PubMed

    Miserez, R; Frey, J; Buogo, C; Capaul, S; Tontis, A; Burnens, A; Nicolet, J

    1998-05-01

    Clostridium perfringens isolated from sheep and goat with enterotoxaemia at necropsy and from healthy animals at slaughter were typed using specific PCR assays for the detection of the alpha-, beta- and epsilon-toxin genes. Clostridium perfringens isolated from all 52 animals with pathological signs of enterotoxaemia showed the presence of the alpha- and epsilon-toxin genes but were devoid of the beta-toxin gene. These strains could therefore be identified as type D, characteristic for clostridial enterotoxaemia of sheep, lambs and goats. In contrast, Cl. perfringens isolated from 11 of 13 healthy animals only contained the alpha-toxin gene which is typical for type A. Two of the healthy animals contained Cl. perfringens with the alpha- and epsilon-toxin genes. However, when several individual Cl. perfringens colonies were analysed from each of these two animals, only a small percentage was found to contain the epsilon-toxin gene, whereas the majority of the colonies were of type A with the alpha-toxin gene only. This is in contrast to the findings from the diseased animals which contained practically only type D Cl. perfringens. The beta-toxin gene was not found in any Cl. perfringens isolate from goat and sheep. Comparison of the PCR data with results obtained by the classical biological toxin assay using the mouse model showed a good correlation.

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

    PubMed

    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.

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

    PubMed

    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

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

    PubMed

    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.

  14. Exposure to β-lactams results in the alteration of penicillin-binding proteins in Clostridium perfringens.

    PubMed

    Park, Miseon; Rafii, Fatemeh

    2017-02-07

    Clostridium perfringens causes a variety of mild to severe infections in humans and other animals. A decrease in the affinity of penicillin-binding protein (PBP) transpeptidases for β-lactams is considered one of the mechanisms of β-lactam resistance in bacteria. Two strains of C. perfringens isolated from bovines and one isolated from a chicken, which had decreased susceptibility to β-lactams, had variations in the amino acid sequences of the central penicillin-binding regions of the PBPs. β-Lactam-resistant mutants of another C. perfringens strain, ATCC 13124, were selected in vitro to determine the effects of exposure to β-lactams on the PBP genes. Cultures of the wild type rapidly developed resistance to penicillin G, cephalothin and ceftriaxone. The susceptibilities of all of the selected mutants to some other β-lactams also decreased. The largest PBP found in C. perfringens, CPF_2395, appeared to be the primary target of all three drugs. Strain resistant to penicillin G had mutation resulting in the substitution of one amino acid within the central penicillin-binding/transpeptidase domain, but the ceftrioxane and cephalothin-resistant strains had mutations resulting in the substitution of two amino acids in this region. The cephalothin-resistant mutant also had additional mutations in the CPF_0340 and CPF_2218 genes in this critical region. No other mutations were observed in the three other PBPs of the in vitro resistant mutants. Resistance development also altered the growth rate and cell morphology of the mutants, so in addition to the PBPs, some other genes, including regulatory genes, may have been affected during the interaction with β-lactam antibiotics. This is the first study showing the effects of β-lactam drugs on the substitution of amino acids in PBPs of C. perfringens and points to the need for studies to detect other unknown alterations affecting the physiology of resistant strains.

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

    PubMed Central

    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. PMID:28018297

  16. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

    PubMed

    Payment, P; Franco, E

    1993-08-01

    To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

  17. Clostridium perfringens and somatic coliphages as indicators of the efficiency of drinking water treatment for viruses and protozoan cysts.

    PubMed Central

    Payment, P; Franco, E

    1993-01-01

    To find the most suitable indicator of viral and parasitic contamination of drinking water, large-volume samples were collected and analyzed for the presence of pathogens (cultivable human enteric viruses, Giardia lamblia cysts, and Cryptosporidium oocysts) and potential indicators (somatic and male-specific coliphages, Clostridium perfringens). The samples were obtained from three water treatment plants by using conventional or better treatments (ozonation, biological filtration). All samples of river water contained the microorganisms sought, and only C. perfringens counts were correlated with human enteric viruses, cysts, or oocysts. For settled and filtered water samples, all indicators were statistically correlated with human enteric viruses but not with cysts or oocysts. By using multiple regression, the somatic coliphage counts were the only explanatory variable for the human enteric virus counts in settled water, while in filtered water samples it was C. perfringens counts. Finished water samples of 1,000 liters each were free of all microorganisms, except for a single sample that contained low levels of cysts and oocysts of undetermined viability. Three of nine finished water samples of 20,000 liters each revealed residual levels of somatic coliphages at 0.03, 0.10, and 0.26 per 100 liters. Measured virus removal was more than 4 to 5 log10, and cyst removal was more than 4 log10. Coliphage and C. perfringens counts suggested that the total removal and inactivation was more than 7 log10 viable microorganisms. C. perfringens counts appear to be the most suitable indicator for the inactivation and removal of viruses in drinking water treatment.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:8368831

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

    PubMed

    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.

  19. Detergent-Resistant Membrane Microdomains Facilitate Ib Oligomer Formation and Biological Activity of Clostridium perfringens Iota-Toxin

    DTIC Science & Technology

    2004-04-01

    Activity of Clostridium perfringens Iota-Toxin Martha L. Hale,1* Jean -Christophe Marvaud,2 Michel R. Popoff,2 and Bradley G. Stiles1 Toxinology...are not necessarily endorsed by the U.S. Army. REFERENCES 1. Abrami, L., M. Fivaz , P. Glauser, N. Sugimoto, C. Zurzolo, and F. G. van der Goot. 2003...273:2355–2360. 10. Fivaz , M., L. Abrami, and F. G. van der Goot. 1999. Landing on lipid rafts. Trends Cell Biol. 9:212–213. 11. Gibert, M., L. Petit

  20. Binding of epsilon-toxin from Clostridium perfringens in the nervous system.

    PubMed

    Dorca-Arévalo, Jonatan; Soler-Jover, Alex; Gibert, Maryse; Popoff, Michel R; Martín-Satué, Mireia; Blasi, Juan

    2008-09-18

    Epsilon-toxin (epsilon-toxin), produced by Clostridium perfringens type D, is the main agent responsible for enterotoxaemia in livestock. Neurological disorders are a characteristic of the onset of toxin poisoning. Epsilon-Toxin accumulates specifically in the central nervous system, where it produces a glutamatergic-mediated excitotoxic effect. However, no detailed study of putative binding structures in the nervous tissue has been carried out to date. Here we attempt to identify specific acceptor moieties and cell targets for epsilon-toxin, not only in the mouse nervous system but also in the brains of sheep and cattle. An epsilon-toxin-GFP fusion protein was produced and used to incubate brain sections, which were then analyzed by confocal microscopy. The results clearly show specific binding of epsilon-toxin to myelin structures. epsilon-Prototoxin-GFP and epsilon-toxin-GFP, the inactive and active forms of the toxin, respectively, showed identical results. By means of pronase E treatment, we found that the binding was mainly associated to a protein component of the myelin. Myelinated peripheral nerve fibres were also stained by epsilon-toxin. Moreover, the binding to myelin was not only restricted to rodents, but was also found in humans, sheep and cattle. Curiously, in the brains of both sheep and cattle, the toxin strongly stained the vascular endothelium, a result that may explain the differences in potency and effect between species. Although the binding of epsilon-toxin to myelin does not directly explain its neurotoxic effect, this feature opens up a new line of enquiry into its mechanism of toxicity and establishes the usefulness of this toxin for the study of the mammalian nervous system.

  1. Clostridium perfringens Epsilon Toxin Causes Selective Death of Mature Oligodendrocytes and Central Nervous System Demyelination

    PubMed Central

    Linden, Jennifer R.; Ma, Yinghua; Zhao, Baohua; Harris, Jason Michael; Rumah, Kareem Rashid; Schaeren-Wiemers, Nicole

    2015-01-01

    ABSTRACT Clostridium perfringens epsilon toxin (ε-toxin) is responsible for a devastating multifocal central nervous system (CNS) white matter disease in ruminant animals. The mechanism by which ε-toxin causes white matter damage is poorly understood. In this study, we sought to determine the molecular and cellular mechanisms by which ε-toxin causes pathological changes to white matter. In primary CNS cultures, ε-toxin binds to and kills oligodendrocytes but not astrocytes, microglia, or neurons. In cerebellar organotypic culture, ε-toxin induces demyelination, which occurs in a time- and dose-dependent manner, while preserving neurons, astrocytes, and microglia. ε-Toxin specificity for oligodendrocytes was confirmed using enriched glial culture. Sensitivity to ε-toxin is developmentally regulated, as only mature oligodendrocytes are susceptible to ε-toxin; oligodendrocyte progenitor cells are not. ε-Toxin sensitivity is also dependent on oligodendrocyte expression of the proteolipid myelin and lymphocyte protein (MAL), as MAL-deficient oligodendrocytes are insensitive to ε-toxin. In addition, ε-toxin binding to white matter follows the spatial and temporal pattern of MAL expression. A neutralizing antibody against ε-toxin inhibits oligodendrocyte death and demyelination. This study provides several novel insights into the action of ε-toxin in the CNS. (i) ε-Toxin causes selective oligodendrocyte death while preserving all other neural elements. (ii) ε-Toxin-mediated oligodendrocyte death is a cell autonomous effect. (iii) The effects of ε-toxin on the oligodendrocyte lineage are restricted to mature oligodendrocytes. (iv) Expression of the developmentally regulated proteolipid MAL is required for the cytotoxic effects. (v) The cytotoxic effects of ε-toxin can be abrogated by an ε-toxin neutralizing antibody. PMID:26081637

  2. Proteolytic Processing and Activation of Clostridium perfringens Epsilon Toxin by Caprine Small Intestinal Contents

    PubMed Central

    Freedman, John C.; Li, Jihong; Uzal, Francisco A.

    2014-01-01

    ABSTRACT Epsilon toxin (ETX), a pore-forming toxin produced by type B and D strains of Clostridium perfringens, mediates severe enterotoxemia in livestock and possibly plays a role in human disease. During enterotoxemia, the nearly inactive ETX prototoxin is produced in the intestines but then must be activated by proteolytic processing. The current study sought to examine ETX prototoxin processing and activation ex vivo using the intestinal contents of a goat, a natural host species for ETX-mediated disease. First, this study showed that the prototoxin has a KEIS N-terminal sequence with a molecular mass of 33,054 Da. When the activation of ETX prototoxin ex vivo by goat small intestinal contents was assessed by SDS-PAGE, the prototoxin was processed in a stepwise fashion into an ~27-kDa band or higher-molecular-mass material that could be toxin oligomers. Purified ETX corresponding to the ~27-kDa band was cytotoxic. When it was biochemically characterized by mass spectrometry, the copresence of three ETX species, each with different C-terminal residues, was identified in the purified ~27-kDa ETX preparation. Cytotoxicity of each of the three ETX species was then demonstrated using recombinant DNA approaches. Serine protease inhibitors blocked the initial proteotoxin processing, while carboxypeptidase inhibitors blocked further processing events. Taken together, this study provides important new insights indicating that, in the intestinal lumen, serine protease (including trypsin and possibly chymotrypsin) initiates the processing of the prototoxin but other proteases, including carboxypeptidases, then process the prototoxin into multiple active and stable species. PMID:25336460

  3. New insights into Clostridium perfringens epsilon toxin activation and action on the brain during enterotoxemia.

    PubMed

    Freedman, John C; McClane, Bruce A; Uzal, Francisco A

    2016-10-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is responsible for diseases that occur mostly in ruminants. ETX is produced in the form of an inactive prototoxin that becomes proteolytically-activated by several proteases. A recent ex vivo study using caprine intestinal contents demonstrated that ETX prototoxin is processed in a step-wise fashion into a stable, active ∼27 kDa band on SDS-PAGE. When characterized further by mass spectrometry, the stable ∼27 kDa band was shown to contain three ETX species with varying C-terminal residues; each of these ETX species is cytotoxic. This study also demonstrated that, in addition to trypsin and chymotrypsin, proteases such as carboxypeptidases are involved in processing ETX prototoxin. Once absorbed, activated ETX species travel to several internal organs, including the brain, where this toxin acts on the vasculature to cross the blood-brain barrier, produces perivascular edema and affects several types of brain cells including neurons, astrocytes, and oligodendrocytes. In addition to perivascular edema, affected animals show edema within the vascular walls. This edema separates the astrocytic end-feet from affected blood vessels, causing hypoxia of nervous system tissue. Astrocytes of rats and sheep affected by ETX show overexpression of aquaporin-4, a membrane channel protein that is believed to help remove water from affected perivascular spaces in an attempt to resolve the perivascular edema. Amyloid precursor protein, an early astrocyte damage indicator, is also observed in the brains of affected sheep. These results show that ETX activation in vivo seems to be more complex than previously thought and this toxin acts on the brain, affecting vascular permeability, but also damaging neurons and other cells.

  4. Clostridium perfringens epsilon toxin H149A mutant as a platform for receptor binding studies

    PubMed Central

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

    2013-01-01

    Clostridium perfringens epsilon toxin (Etx) is a pore-forming toxin responsible for a severe and rapidly fatal enterotoxemia of ruminants. The toxin is classified as a category B bioterrorism agent by the U.S. Government Centres for Disease Control and Prevention (CDC), making work with recombinant toxin difficult. To reduce the hazard posed by work with recombinant Etx, we have used a variant of Etx that contains a H149A mutation (Etx-H149A), previously reported to have reduced, but not abolished, toxicity. The three-dimensional structure of H149A prototoxin shows that the H149A mutation in domain III does not affect organisation of the putative receptor binding loops in domain I of the toxin. Surface exposed tyrosine residues in domain I of Etx-H149A (Y16, Y20, Y29, Y30, Y36 and Y196) were mutated to alanine and mutants Y30A and Y196A showed significantly reduced binding to MDCK.2 cells relative to Etx-H149A that correlated with their reduced cytotoxic activity. Thus, our study confirms the role of surface exposed tyrosine residues in domain I of Etx in binding to MDCK cells and the suitability of Etx-H149A for further receptor binding studies. In contrast, binding of all of the tyrosine mutants to ACHN cells was similar to that of Etx-H149A, suggesting that Etx can recognise different cell surface receptors. In support of this, the crystal structure of Etx-H149A identified a glycan (β-octyl-glucoside) binding site in domain III of Etx-H149A, which may be a second receptor binding site. These findings have important implications for developing strategies designed to neutralise toxin activity. PMID:23504825

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

    PubMed

    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.

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

    PubMed

    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.

  7. Carbohydrate Recognition by an Architecturally Complex α-N-Acetylglucosaminidase from Clostridium perfringens

    PubMed Central

    Ficko-Blean, Elizabeth; Stuart, Christopher P.; Suits, Michael D.; Cid, Melissa; Tessier, Matthew; Woods, Robert J.; Boraston, Alisdair B.

    2012-01-01

    CpGH89 is a large multimodular enzyme produced by the human and animal pathogen Clostridium perfringens. The catalytic activity of this exo-α-d-N-acetylglucosaminidase is directed towards a rare carbohydrate motif, N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which is displayed on the class III mucins deep within the gastric mucosa. In addition to the family 89 glycoside hydrolase catalytic module this enzyme has six modules that share sequence similarity to the family 32 carbohydrate-binding modules (CBM32s), suggesting the enzyme has considerable capacity to adhere to carbohydrates. Here we suggest that two of the modules, CBM32-1 and CBM32-6, are not functional as carbohydrate-binding modules (CBMs) and demonstrate that three of the CBMs, CBM32-3, CBM32-4, and CBM32-5, are indeed capable of binding carbohydrates. CBM32-3 and CBM32-4 have a novel binding specificity for N-acetyl-β-d-glucosamine-α-1,4-d-galactose, which thus complements the specificity of the catalytic module. The X-ray crystal structure of CBM32-4 in complex with this disaccharide reveals a mode of recognition that is based primarily on accommodation of the unique bent shape of this sugar. In contrast, as revealed by a series of X-ray crystal structures and quantitative binding studies, CBM32-5 displays the structural and functional features of galactose binding that is commonly associated with CBM family 32. The functional CBM32s that CpGH89 contains suggest the possibility for multivalent binding events and the partitioning of this enzyme to highly specific regions within the gastrointestinal tract. PMID:22479408

  8. Epsilon Toxin Is Essential for the Virulence of Clostridium perfringens Type D Infection in Sheep, Goats, and Mice

    PubMed Central

    Garcia, J. P.; Adams, V.; Beingesser, J.; Hughes, M. L.; Poon, R.; Lyras, D.; Hill, A.; McClane, B. A.; Rood, J. I.

    2013-01-01

    Clostridium perfringens type D causes disease in sheep, goats, and other ruminants. Type D isolates produce, at minimum, alpha and epsilon (ETX) toxins, but some express up to five different toxins, raising questions about which toxins are necessary for the virulence of these bacteria. We evaluated the contribution of ETX to C. perfringens type D pathogenicity in an intraduodenal challenge model in sheep, goats, and mice using a virulent C. perfringens type D wild-type strain (WT), an isogenic ETX null mutant (etx mutant), and a strain where the etx mutation has been reversed (etx complemented). All sheep and goats, and most mice, challenged with the WT isolate developed acute clinical disease followed by death in most cases. Sheep developed various gross and/or histological changes that included edema of brain, lungs, and heart as well as hydropericardium. Goats developed various effects, including necrotizing colitis, pulmonary edema, and hydropericardium. No significant gross or histological abnormalities were observed in any mice infected with the WT strain. All sheep, goats, and mice challenged with the isogenic etx mutant remained clinically healthy for ≥24 h, and no gross or histological abnormalities were observed in those animals. Complementation of etx knockout restored virulence; most goats, sheep, and mice receiving this complemented mutant developed clinical and pathological changes similar to those observed in WT-infected animals. These results indicate that ETX is necessary for type D isolates to induce disease, supporting a key role for this toxin in type D disease pathogenesis. PMID:23630957

  9. The synergistic necrohemorrhagic action of Clostridium perfringens perfringolysin and alpha toxin in the bovine intestine and against bovine endothelial cells

    PubMed Central

    2013-01-01

    Bovine necrohemorrhagic enteritis is a major cause of mortality in veal calves. Clostridium perfringens is considered as the causative agent, but there has been controversy on the toxins responsible for the disease. Recently, it has been demonstrated that a variety of C. perfringens type A strains can induce necrohemorrhagic lesions in a calf intestinal loop assay. These results put forward alpha toxin and perfringolysin as potential causative toxins, since both are produced by all C. perfringens type A strains. The importance of perfringolysin in the pathogenesis of bovine necrohemorrhagic enteritis has not been studied before. Therefore, the objective of the current study was to evaluate the role of perfringolysin in the development of necrohemorrhagic enteritis lesions in calves and its synergism with alpha toxin. A perfringolysin-deficient mutant, an alpha toxin-deficient mutant and a perfringolysin alpha toxin double mutant were less able to induce necrosis in a calf intestinal loop assay as compared to the wild-type strain. Only complementation with both toxins could restore the activity to that of the wild-type. In addition, perfringolysin and alpha toxin had a synergistic cytotoxic effect on bovine endothelial cells. This endothelial cell damage potentially explains why capillary hemorrhages are an initial step in the development of bovine necrohemorrhagic enteritis. Taken together, our results show that perfringolysin acts synergistically with alpha toxin in the development of necrohemorrhagic enteritis in a calf intestinal loop model and we hypothesize that both toxins act by targeting the endothelial cells. PMID:23782465

  10. Evaluation of different fluids for detection of Clostridium perfringens type D epsilon toxin in sheep with experimental enterotoxemia.

    PubMed

    Layana, Jorge E; Fernandez Miyakawa, Mariano E; Uzal, Francisco A

    2006-08-01

    Enterotoxemia caused by Clostridium perfringens type D is a highly lethal disease of sheep, goats and other ruminants. The diagnosis of this condition is usually confirmed by detection of epsilon toxin, a major exotoxin produced by C. perfringens types B and D, in the intestinal content of affected animals. It has been suggested that other body fluids can also be used for detection of epsilon toxin. This study was performed to evaluate the usefulness of intestinal content versus other body fluids in detecting epsilon toxin in cases of sheep enterotoxemia. Samples of duodenal, ileal and colon contents, pericardial and abdominal fluids, aqueous humor and urine from 15 sheep with experimentally induced enterotoxemia, were analysed for epsilon toxin using a capture ELISA. Epsilon toxin was detected in 92% of the samples of ileal content, 64% of the samples of duodenal content, 57% of the samples of colon content and in 7% of the samples of pericardial fluid and aqueous humor. No epsilon toxin was found in samples of abdominal fluid or urine from the animals with enterotoxemia or in any samples from six clinically healthy sheep used as negative controls. The results of this study indicate that with the diagnostic capture ELISA used, intestinal content (preferably ileum) should be used for C. perfringens type D epsilon toxin detection in suspected cases of sheep enterotoxemia.

  11. The SpmA/B and DacF proteins of Clostridium perfringens play important roles in spore heat resistance.

    PubMed

    Orsburn, Benjamin; Sucre, Katie; Popham, David L; Melville, Stephen B

    2009-02-01

    Strains of Clostridium perfringens that cause acute food poisoning have been shown to produce spores that are significantly more heat resistant than those of other strains. Previous studies demonstrated that the spore core density and the ratio of spore cortex peptidoglycan relative to the germ cell wall were factors that correlated with the heat resistance of a C. perfringens spore. To further evaluate these relationships, mutant strains of C. perfringens SM101 were constructed with null mutations in dacF, encoding a D,D-carboxypeptidase, and in the spmA-spmB operon, which is involved in spore core dehydration. The dacF mutant was shown to produce less spore cortex peptidoglycan and had a corresponding decrease in spore heat resistance. The spmA-spmB strain produced highly unstable spores with significantly lower core densities and increased heat sensitivity, which were easily destroyed during treatments affecting the spore coat layers. These results support the previous assertion that a threshold core density as well as a high ratio of cortex peptidoglycan relative to the germ cell wall contribute to the formation of a more heat-resistant spore in this species.

  12. Cloning, recombinant production, crystallization and preliminary X-ray diffraction studies of a family 84 glycoside hydrolase from Clostridium perfringens

    SciTech Connect

    Ficko-Blean, Elizabeth; Boraston, Alisdair B.

    2005-09-01

    Crystallization of a family 84 glycoside hydrolase, a putative virulence factor, secreted by C. perfringens is reported. Clostridium perfringens is a ubiquitous environmental organism that is capable of causing a variety of diseases in mammals, including gas gangrene and necrotic enteritis in humans. The activity of a secreted hyaluronidase, attributed to the NagH protein, contributes to the pathogenicity of this organism. The family 84 catalytic module of one of the three homologues of NagH found in C. perfringens (ATCC 13124) has been cloned. The 69 kDa catalytic module of NagJ, here called GH84C, was overproduced in Escherichia coli and purified by immobilized metal-affinity chromatography (IMAC). Crystals belonging to space group I222 or I2{sub 1}2{sub 1}2{sub 1} with unit-cell parameters a = 130.39, b = 150.05, c = 155.43 Å were obtained that diffracted to 2.1 Å. Selenomethionyl crystals have also been produced, leading to the possibility of solving the phase problem by MAD using synchrotron radiation.

  13. Acid phosphatase test proves superior to standard phenotypic identification procedure for Clostridium perfringens strains isolated from water

    PubMed Central

    Ryzinska-Paier, G.; Sommer, R.; Haider, J.M.; Knetsch, S.; Frick, C.; Kirschner, A.K.T.; Farnleitner, A.H.

    2011-01-01

    Clostridium perfringens is used as an indicator for persistent faecal pollution as well as to monitor the efficacy of water treatment processes. For these purposes, differentiation between C. perfringens and other Clostridia is essential and is routinely carried out by phenotypic standard tests as proposed in the ISO/CD 6461-2:2002 (ISO_LGMN: lactose fermentation, gelatine liquidation, motility and nitrate reduction). Because the ISO_LGMN procedure is time consuming and labour intensive, the acid phosphatase test was investigated as a possible and much more rapid alternative method for confirmation. The aim of our study was to evaluate and compare confirmation results obtained by these two phenotypic methods using genotypically identified strains, what to our knowledge has not been accomplished before. For this purpose, a species specific PCR method was selected based on the results received for type strains and genotypically characterised environmental strains. For the comparative investigation type strains as well as presumptive C. perfringens isolates from water and faeces samples were used. The acid phosphatase test revealed higher percentage (92%) of correctly identified environmental strains (n = 127) than the ISO_LGMN procedure (83%) and proved to be a sensitive and reliable confirmation method. PMID:21872622

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

    PubMed

    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.

  15. Clostridium perfringens enterotoxin and Clostridium difficile toxin A/B do not play a role in acute haemorrhagic diarrhoea syndrome in dogs.

    PubMed

    Busch, K; Suchodolski, J S; Kühner, K A; Minamoto, Y; Steiner, J M; Mueller, R S; Hartmann, K; Unterer, S

    2015-03-07

    Although an association between clostridial pathogens and canine idiopathic acute haemorrhagic diarrhoea syndrome (AHDS) has been described, the relevance of those bacteria and their toxins remains unclear. The aim of this study was to evaluate the association between severity of clinical signs and presence of Clostridium perfringens enterotoxin (CPE) and Clostridium difficile toxin A/B (CDT A/B) in faeces of dogs with AHDS. Faecal samples of 54 dogs with idiopathic AHDS were tested by qualitative CPE and CDT A/B ELISA, and PCR was performed to detect enterotoxin genes of C. perfringens (cpe) and toxin B genes of C. difficile (cdt b). Prevalence of cdt b and CDT A/B in dogs with AHDS was 10/54 and 2/54 versus 3/23 and 0/23 in control dogs. Prevalence of cpe was 35/54 in affected versus 9/23 in control dogs. Prevalence of CPE in dogs with AHDS (13/54) was higher compared with control dogs (0/23). No significant difference was detected between CPE-positive and -negative and between cpe-positive and -negative dogs in severity of clinical signs, duration of hospitalisation, mortality rate and selected laboratory parameters. This study suggests that CPE and CDT A/B do not play a role in idiopathic AHDS, are not associated with clinical parameters in affected dogs and cannot be used to predict disease outcome.

  16. Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This study was performed to compare four Clostridium perfringens recombinant proteins as vaccine candidates using the Montanide™ ISA 71 VG adjuvant in an experimental model of necrotic enteritis. Broiler chickens were immunized with clostridial recombinant proteins with ISA 71 VG, and intestinal le...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  20. Effect of meat ingredients (sodium nitrite and erythorbate) and processing (vacuum storage and packaging atmosphere) on germination and outgrowth of Clostridium perfringens spores in ham during abusive cooling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effect of nitrite and erythorbate on Clostridium perfringens spore germination and outgrowth in ham during abusive cooling (15 h) was evaluated. Ham was formulated with ground pork, NaNO2 (0, 50, 100, 150 or 200 ppm) and sodium erythorbate (0 or 547 ppm). Ten grams of meat (stored at 5C for 3 or...

  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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne di...

  4. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently utilized antibiotics. Clostridium perfringens is a Gram-positive, spore-forming anaerobic bacterium that plays a significant role in human food-borne d...

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

    PubMed

    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.

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

    PubMed

    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.

  7. Necrotic enteritis-producing strains of Clostridium perfringens displace non-necrotic enteritis strains from the gut of chicks.

    PubMed

    Barbara, Angelique J; Trinh, Hien T; Glock, Robert D; Glenn Songer, J

    2008-01-25

    We inoculated broiler chicks with mixtures of Clostridium perfringens strains to investigate the single strain dominance observed in natural cases of necrotic enteritis (NE) [Nauerby, B., Pedersen, K., Madsen, M., 2003. Analysis by pulsed-field gel electrophoresis of the genetic diversity among Clostridium perfringens isolates from chickens. Vet. Microbiol. 94, 257-266]. Pre-inoculation bacteriologic culture of chick intestines yielded up to six pulsed-field gel electrophoresis (PFGE) types of C. perfringens. Birds developed typical NE lesions in response to administration (2x per day for 4 days) of a combined inoculum comprising one NE strain (JGS4143, PFGE pattern 8) and four non-NE strains (from piglet necrotizing enteritis, chicken normal flora, human gas gangrene, and bovine neonatal enteritis). After inoculation commenced, only the NE strain was recovered through the first post-inoculation day, in spite of intense efforts to recover pre-challenge flora strains and the other challenge strains. Thereafter, pre-inoculation and previously undetected PFGE types were found, and JGS4143 became undetectable. Birds inoculated simultaneously with five NE strains (from disease in chickens or turkeys, and including JGS4143) also developed lesions, but again only JGS4143 was recovered through the 1st day post-challenge. At that time, birds began to be repopulated with pre-challenge PFGE types. Two NE strains (JGS4143 and JGS4064) produced bacteriocins, which inhibited each other and normal flora strains (n=17), while normal flora strains inhibited neither NE strains nor each other. Thus, it appears that naturally occurring dominance of the gut by NE strains can be reproduced experimentally. Bacteriocins directed against normal flora could possibly provide the necessary advantage, although inhibition of one NE strain by another suggests that other factors may be partially or completely responsible for the dominance.

  8. Innate immune response to yeast-derived carbohydrates in broiler chickens fed organic diets and challenged with Clostridium perfringens.

    PubMed

    Yitbarek, A; Echeverry, H; Brady, J; Hernandez-Doria, J; Camelo-Jaimes, G; Sharif, S; Guenter, W; House, J D; Rodriguez-Lecompte, J C

    2012-05-01

    Necrotic enteritis (NE) caused by Clostridium perfringens is a reemerging disease of economic importance in areas of the world where antibiotic growth promoters have been banned. The effect of mannan-oligosaccharide (MOS) supplementation in organic diets of broilers challenged with C. perfringens on performance, gut morphology, and innate immunity was investigated. Three hundred Ross-308 broilers were fed antibiotic-free certified organic starter and grower diets. On d 14, birds were orally challenged with 1 mL of C. perfringens culture at 3 × 10(10) cfu/bird. Treatments consisted of a control no-challenge (CO; 0 g/kg of MOS in the basal diet), control challenge (COC, 0 g/kg of MOS in the basal diet), and MOS challenge (2 g/kg of MOS in the basal diet). Challenge of birds resulted in decreased feed intake and BW gain (P = 0.048 and P = 0.026, respectively). Even though supplementation of diet with MOS improved feed intake (P = 0.985), BW gain and G:F were not improved compared with those of the CO group (P = 0.026 and P = <0.001, respectively). There was no significant difference among treatments in jejunal and ileal villus height, crypt depth, and goblet cells/mm(2) (P > 0.05). Quantitative real-time PCR showed that, in the ileum, the MOS diet resulted in an upregulation of toll-like receptor (TLR)2b, TLR4, interleukin (IL)-12p35, and interferon (IFN)-γ compared with CO (P = 0.003, P = 0.018, and P = 0.024, respectively). In the cecal tonsil, challenging birds with C. perfringens resulted in an upregulation of TLR2b compared with CO (P = 0.036), and MOS resulted in an upregulation of TLR4 (P = 0.018). In conclusion, feeding a MOS-supplemented diet to C. perfringens-challenged broiler chickens did not improve performance and gut morphology-associated responses. However, MOS was capable of altering TLR and cytokine profiles, where dual TLR2 and TLR4 pathways were associated with MOS supplementation with subsequent upregulation of ileal IL-12p35 and IFN

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

    PubMed

    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

  10. Use of a mariner-based transposon mutagenesis system to isolate Clostridium perfringens mutants deficient in gliding motility.

    PubMed

    Liu, Hualan; Bouillaut, Laurent; Sonenshein, Abraham L; Melville, Stephen B

    2013-02-01

    Clostridium perfringens is an anaerobic Gram-positive pathogen that causes many human and animal diseases, including food poisoning and gas gangrene. C. perfringens lacks flagella but possesses type IV pili (TFP). We have previously shown that C. perfringens can glide across an agar surface in long filaments composed of individual bacteria attached end to end and that two TFP-associated proteins, PilT and PilC, are needed for this. To discover additional gene products that play a role in gliding, we developed a plasmid-based mariner transposon mutagenesis system that works effectively in C. perfringens. More than 10,000 clones were screened for mutants that lacked the ability to move away from the edge of a colony. Twenty-four mutants (0.24%) were identified that fit the criteria. The genes containing insertions that affected gliding motility fell into nine different categories. One gene, CPE0278, which encodes a homolog of the SagA cell wall-dependent endopeptidase, acquired distinct transposon insertions in two independent mutants. sagA mutants were unable to form filaments due to a complete lack of end-to-end connections essential for gliding motility. Complementation of the sagA mutants with a wild-type copy of the gene restored gliding motility. We constructed an in-frame deletion mutation in the sagA gene and found that this mutant had a phenotype similar to those of the transposon mutants. We hypothesize that the sagA mutant strains are unable to form the molecular complexes which are needed to keep the cells in an end-to-end orientation, leading to separation of daughter cells and the inability to carry out gliding motility.

  11. Experimental induction of necrotic enteritis in chickens by a netB-positive Japanese isolate of Clostridium perfringens

    PubMed Central

    TO, Ho; SUZUKI, Takayuki; KAWAHARA, Fumiya; UETSUKA, Koji; NAGAI, Shinya; NUNOYA, Tetsuo

    2016-01-01

    Necrotic enteritis (NE) is one of the most important bacterial diseases in terms of economic losses. Clostridium perfringens necrotic enteritis toxin B, NetB, was recently proposed as a new key virulent factor for the development of NE. The goal of this work was to develop a necrotic enteritis model in chickens by using a Japanese isolate of C. perfringens. The Japanese isolate has been found to contain netB gene, which had the same nucleotide and deduced amino acid sequences as those of prototype gene characterized in Australian strain EHE-NE18, and also expressed in vitro a 33-kDa protein identified as NetB toxin by nano-scale liquid chromatographic tandem mass spectrometry. In the challenge experiment, broiler chickens fed a commercial chicken starter diet for 14 days post-hatch were changed to a high protein feed mixed 50:50 with fishmeal for 6 days. At day 21 of age, feed was withheld for 24 hr, and each chicken was orally challenged twice daily with 2 ml each of C. perfringens culture (109 to 1010 CFU) on 5 consecutive days. The gross necrotic lesions were observed in 90 and 12.5% of challenged and control chickens, respectively. To our knowledge, this is the first study that demonstrated that a netB-positive Japanese isolate of C. perfringens is able to induce the clinical signs and lesions characteristic of NE in the experimental model, which may be useful for evaluating the pathogenicity of field isolates, the efficacy of a vaccine or a specific drug against NE. PMID:27980252

  12. Development of multiplex PCR for the detection of total coliform bacteria for Escherichia coli and Clostridium perfringens in drinking water.

    PubMed

    Tantawiwat, Suwalee; Tansuphasiri, Unchalee; Wongwit, Waranya; Wongchotigul, Varee; Kitayaporn, Dwip

    2005-01-01

    Multiplex PCR amplification of lacZ, uidA and plc genes was developed for the simultaneous detection of total coliform bacteria for Escherichia coli and Clostridium perfringens, in drinking water. Detection by agarose gel electrophoresis yielded a band of 876 bp for the lacZ gene of all coliform bacteria; a band of 147 bp for the uidA gene and a band of 876 bp for the lacZ gene of all strains of E. coli; a band of 280 bp for the p/c gene for all strains of C. perfringens; and a negative result for all three genes when tested with other bacteria. The detection limit was 100 pg for E. coli and C. perfringens, and 1 ng for coliform bacteria when measured with purified DNA. This assay was applied to the detection of these bacteria in spiked water samples. Spiked water samples with 0-1,000 CFU/ml of coliform bacteria and/or E. coli and/or C. perfringens were detected by this multiplex PCR after a pre-enrichment step to increase the sensitivity and to ensure that the detection was based on the presence of cultivable bacteria. The result of bacterial detection from the multiplex PCR was comparable with that of a standard plate count on selective medium (p=0.62). When using standard plate counts as a gold standard, the sensitivity for this test was 99.1% (95% CI 95.33, 99.98) and the specificity was 90.9 % (95% CI 75.67, 98.08). Multiplex PCR amplification with a pre-enrichment step was shown to be an effective, sensitive and rapid method for the simultaneous detection of these three microbiological parameters in drinking water.

  13. The CpAL quorum sensing system regulates production of hemolysins CPA and PFO to build Clostridium perfringens biofilms.

    PubMed

    Vidal, Jorge E; Shak, Joshua R; Canizalez-Roman, Adrian

    2015-06-01

    Clostridium perfringens strains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by the C. perfringens Agr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. Although C. perfringens strains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrB mutant strains were not able to produce biofilms, a ΔluxS mutant produced wild-type levels. The transcript levels of CpAL-regulated cpa and pfoA genes, but not cpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpa and ΔpfoA mutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpb made wild-type levels. Biofilm formation was restored in complemented Δcpa/cpa and ΔpfoA/pfoA strains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation in C. perfringens by increasing levels of certain toxins required to build biofilms.

  14. A sporulation factor is involved in the morphological change of Clostridium perfringens biofilms in response to temperature.

    PubMed

    Obana, Nozomu; Nakamura, Kouji; Nomura, Nobuhiko

    2014-04-01

    Biofilm formation has been associated with bacterial pathogenesis, such as nosocomial and chronic infections, as the resistance of biofilms to environmental stresses has increased. Clostridium perfringens is a Gram-positive spore-forming anaerobic pathogen. This organism survives antibiotic treatment through the formation of biofilms or spores, but the environmental and regulatory factors involved in the biofilm formation remain unclear. Here, we observed that temperature regulates C. perfringens biofilm morphology. At 37°C, C. perfringens adhered to the substrate surface and formed a flat, thin biofilm, herein referred to as adhered biofilm. However, at 25°C, this bacterium did not adhere and produced a threadlike extracellular matrix, forming a viscous, thick biofilm, herein referred to as pellicle biofilm. Pellicle biofilm formation requires the sporulation master regulator, Spo0A, and the toxin regulator, CtrAB, and is enhanced in the absence of the global repressor, AbrB. These transcriptional regulator genes are regulated by each other and temperature. Adhered-biofilm formation requires AbrB and pilA2, which encodes a component of type IV pili (TFP). TFP expression was activated at 37°C and regulated through Spo0A, AbrB, and CtrAB. These results indicate that the morphology of C. perfringens biofilm is dependent on temperature through the differential production of extracellular matrix and the activity of TFP. Moreover, pellicle biofilm formation is involved in sporulation and toxin production. Here, we demonstrated that clostridial biofilm formation is closely associated with sporulation and that the morphological change of the biofilms could play an important role in the pathogenesis of this organism.

  15. The CpAL Quorum Sensing System Regulates Production of Hemolysins CPA and PFO To Build Clostridium perfringens Biofilms

    PubMed Central

    Shak, Joshua R.; Canizalez-Roman, Adrian

    2015-01-01

    Clostridium perfringens strains produce severe diseases, including myonecrosis and enteritis necroticans, in humans and animals. Diseases are mediated by the production of potent toxins that often damage the site of infection, e.g., skin epithelium during myonecrosis. In planktonic cultures, the regulation of important toxins, such as CPA, CPB, and PFO, is controlled by the C. perfringens Agr-like (CpAL) quorum sensing (QS) system. Strains also encode a functional LuxS/AI-2 system. Although C. perfringens strains form biofilm-like structures, the regulation of biofilm formation is poorly understood. Therefore, our studies investigated the role of CpAL and LuxS/AI-2 QS systems and of QS-regulated factors in controlling the formation of biofilms. We first demonstrate that biofilm production by reference strains differs depending on the culture medium. Increased biomass correlated with the presence of extracellular DNA in the supernatant, which was released by lysis of a fraction of the biofilm population and planktonic cells. Whereas ΔagrB mutant strains were not able to produce biofilms, a ΔluxS mutant produced wild-type levels. The transcript levels of CpAL-regulated cpa and pfoA genes, but not cpb, were upregulated in biofilms compared to planktonic cultures. Accordingly, Δcpa and ΔpfoA mutants, in type A (S13) or type C (CN3685) backgrounds, were unable to produce biofilms, whereas CN3685Δcpb made wild-type levels. Biofilm formation was restored in complemented Δcpa/cpa and ΔpfoA/pfoA strains. Confocal microscopy studies further detected CPA partially colocalizing with eDNA on the biofilm structure. Thus, CpAL regulates biofilm formation in C. perfringens by increasing levels of certain toxins required to build biofilms. PMID:25824838

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

    PubMed

    Duan, Zhi; Hansen, Terese Holst; Hansen, Tina Beck; Dalgaard, Paw; Knøchel, Susanne

    2016-08-02

    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 (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 (pH6.8) and pork (pH5.6) at two different slowly increasing temperature (SIT) profiles (10°C to 53°C) followed by 53°C in up to 30h in total. Three inoculum types were studied including vegetative cells, non-heated spores and heat activated (75°C, 20min) spores of C. perfringens strain 790-94. Concentrations of vegetative cells in chicken increased 2 to 3logCFU/g during the SIT profiles. Similar results were found for non-heated and heated spores in chicken, whereas in pork C. perfringens 790-94 increased less than 1logCFU/g. At 53°C C. perfringens 790-94 was log-linearly inactivated. Observed and predicted concentrations of C. perfringens, at the time when 53°C (log(N53)) was reached, were used to evaluate the new growth model and three available predictive models previously published for C. perfringens growth during cooling rather than during SIT profiles. Model performance was evaluated by using mean deviation (MD), mean absolute deviation (MAD) and the acceptable simulation zone (ASZ) approach with a zone of ±0.5logCFU/g. The new model showed best performance with MD=0.27logCFU/g, MAD=0.66logCFU/g and ASZ=67%. The two growth models that performed best, were used together with a log-linear inactivation model and D53-values from the present study to simulate the behaviour of C. perfringens under the fast and slow SIT profiles investigated in the present study. Observed and predicted concentrations were compared using a new fail-safe acceptable

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

    PubMed

    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

  18. Clostridium perfringens α-Toxin Impairs Innate Immunity via Inhibition of Neutrophil Differentiation

    PubMed Central

    Takehara, Masaya; Takagishi, Teruhisa; Seike, Soshi; Ohtani, Kaori; Kobayashi, Keiko; Miyamoto, Kazuaki; Shimizu, Tohru; Nagahama, Masahiro

    2016-01-01

    Although granulopoiesis is accelerated to suppress bacteria during infection, some bacteria can still cause life-threatening infections, but the mechanism behind this remains unclear. In this study, we found that mature neutrophils in bone marrow cells (BMCs) were decreased in C. perfringens-infected mice and also after injection of virulence factor α-toxin. C. perfringens infection interfered with the replenishment of mature neutrophils in the peripheral circulation and the accumulation of neutrophils at C. perfringens-infected sites in an α-toxin-dependent manner. Measurements of bacterial colony-forming units in C. perfringens-infected muscle revealed that α-toxin inhibited a reduction in the load of C. perfringens. In vitro treatment of isolated BMCs with α-toxin (phospholipase C) revealed that α-toxin directly decreased mature neutrophils. α-Toxin did not influence the viability of isolated mature neutrophils, while simultaneous treatment of BMCs with granulocyte colony-stimulating factor attenuated the reduction of mature neutrophils by α-toxin. Together, our results illustrate that impairment of the innate immune system by the inhibition of neutrophil differentiation is crucial for the pathogenesis of C. perfringens to promote disease to a life-threatening infection, which provides new insight to understand how pathogenic bacteria evade the host immune system. PMID:27306065

  19. Isolation of Clostridium perfringens Type B in an Individual at First Clinical Presentation of Multiple Sclerosis Provides Clues for Environmental Triggers of the Disease

    PubMed Central

    Rumah, Kareem Rashid; Linden, Jennifer; Fischetti, Vincent A.; Vartanian, Timothy

    2013-01-01

    We have isolated Clostridium perfringens type B, an epsilon toxin-secreting bacillus, from a young woman at clinical presentation of Multiple Sclerosis (MS) with actively enhancing lesions on brain MRI. This finding represents the first time that C. perfringens type B has been detected in a human. Epsilon toxin’s tropism for the blood-brain barrier (BBB) and binding to oligodendrocytes/myelin makes it a provocative candidate for nascent lesion formation in MS. We examined a well-characterized population of MS patients and healthy controls for carriage of C. perfringens toxinotypes in the gastrointestinal tract. The human commensal Clostridium perfringens type A was present in approximately 50% of healthy human controls compared to only 23% in MS patients. We examined sera and CSF obtained from two tissue banks and found that immunoreactivity to ETX is 10 times more prevalent in people with MS than in healthy controls, indicating prior exposure to ETX in the MS population. C. perfringens epsilon toxin fits mechanistically with nascent MS lesion formation since these lesions are characterized by BBB permeability and oligodendrocyte cell death in the absence of an adaptive immune infiltrate. PMID:24146858

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

    PubMed

    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.

  1. Detection of toxins A/B and isolation of Clostridium difficile and Clostridium perfringens from dogs in Minas Gerais, Brazil

    PubMed Central

    Silva, Rodrigo Otávio Silveira; Santos, Renata Lara Resende; Pires, Prhiscylla Sadanã; Pereira, Luiz Carlos; Pereira, Silvia Trindade; Duarte, Marina Carvalho; de Assis, Ronnie Antunes; Lobato, Francisco Carlos Faria

    2013-01-01

    The objective of this study was to detect C. difficile A/B toxins and to isolate strains of C. perfringens and C. difficile from diarrheic and non-diarrheic dogs in Brazil. Stool samples were collected from 57 dogs, 35 of which were apparently healthy, and 22 of which were diarrheic. C. difficile A/B toxins were detected by ELISA, and C. perfringens and C. difficile were identified by multiplex PCR. C. difficile A/B toxins were detected in 21 samples (36.8%). Of these, 16 (76.2%) were from diarrheic dogs, and five (23.8%) were from non-diarrheic dogs. Twelve C. difficile strains (21.1%) were isolated, of which ten were A+B+ and two were A−B−. All non-toxigenic strains were isolated from non-diarrheic animals. The binary toxin gene cdtB was found in one strain, which was A+B+ and was derived from a non-diarrheic dog. C. perfringens strains were isolated from 40 samples (70.2%). Of these, 18 (45%) were from the diarrheic group, and 22 (55%) belonged to the non-diarrheic group. All isolates were classified as C. perfringens type A and there was an association between the detection of the cpe gene and the presence of diarrhea. Interestingly, ten strains (25%) were positive for the presence of the cpb2 gene. The high rate of detection of the A/B toxins in non-diarrheic dogs suggests the occurrence of subclinical disease in dogs or carriage of its toxins without disease. More studies are needed to elucidate the epidemiology of C. difficile and C. perfringens in dogs and to better our understanding of C. difficile as a zoonotic agent. This is the first study to report the binary toxin gene in C. difficile strains isolated from dogs in Brazil. PMID:24159295

  2. In Vitro Selective Growth-Inhibitory Effect of 8-Hydroxyquinoline on Clostridium perfringens versus Bifidobacteria in a Medium Containing Chicken Ileal Digesta

    PubMed Central

    Skrivanova, Eva; Van Immerseel, Filip; Hovorkova, Petra; Kokoska, Ladislav

    2016-01-01

    Clostridium perfringens-induced necrotic enteritis is generally controlled by antibiotics. However, because of increasing antibiotic resistance, other antibacterial agents are required, preferably ones that do not affect the beneficial intestinal microbiota of the host. This study evaluated the in vitro selective growth-inhibitory effect of 8-hydroxyquinoline (8HQ) on C. perfringens vs. bifidobacteria in a medium containing chicken ileal digesta. Prior to the experiments, the minimum inhibitory concentrations of 8HQ and penicillin G were determined by broth microdilution assay. The minimum inhibitory concentration values of 8HQ for C. perfringens were 16–32 times lower than the values for bifidobacteria. Treatment of autoclaved and non-autoclaved chicken ileal digesta with 8HQ showed a selective anticlostridial effect. After incubation of C. perfringens with autoclaved ileal digesta for 3 h, all 8HQ concentrations tested (32–2048 μg/mL) significantly reduced C. perfringens bacterial count. In contrast, the same treatment had no or only a slight effect on bifidobacteria counts. Unlike 8HQ, penicillin G did not exhibit any selectivity. Similar results were obtained after incubation for 24 h. In non-autoclaved ileal digesta, all 8HQ concentrations tested significantly reduced C. perfringens bacterial counts after incubation for 30 min and 3 h, while no effect was observed on bifidobacteria. These results suggest that 8HQ may serve as a prospective veterinary compound for use against necrotic enteritis in poultry. PMID:27936245

  3. CodY Is a Global Regulator of Virulence-Associated Properties for Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Ma, Menglin; Sarker, Mahfuzur R.; McClane, Bruce A.

    2013-01-01

    ABSTRACT CodY is known to regulate various virulence properties in several Gram-positive bacteria but has not yet been studied in the important histotoxic and intestinal pathogen Clostridium perfringens. The present study prepared an isogenic codY-null mutant in C. perfringens type D strain CN3718 by insertional mutagenesis using the Targetron system. Western blot analysis indicated that, relative to wild-type CN3718 or a complementing strain, this isogenic codY mutant produces reduced levels of epsilon toxin (ETX). Using supernatants from cultures of the wild-type, codY-null mutant, and complementing strains, CodY regulation of ETX production was shown to have cytotoxic consequences for MDCK cells. The CodY regulatory effect on ETX production was specific, since the codY-null mutant still made wild-type levels of alpha-toxin and perfringolysin O. Sialidase activity measurements and sialidase Western blot analysis of supernatants from CN3718 and its isogenic derivatives showed that CodY represses overall exosialidase activity due to a reduced presence of NanH in culture supernatants. Inactivation of the codY gene significantly decreased the adherence of CN3718 vegetative cells or spores to host Caco-2 cells. Finally, the codY mutant showed increased spore formation under vegetative growth conditions, although germination of these spores was impaired. Overall, these results identify CodY as a global regulator of many C. perfringens virulence-associated properties. Furthermore, they establish that, via CodY, CN3718 coordinately regulates many virulence-associated properties likely needed for intestinal infection. PMID:24105766

  4. Sialidases Affect the Host Cell Adherence and Epsilon Toxin-Induced Cytotoxicity of Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Sayeed, Sameera; Robertson, Susan; Chen, Jianming; McClane, Bruce A.

    2011-01-01

    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. PMID:22174687

  5. Direct Dynamic Kinetic Analysis and Computer Simulation of Growth of Clostridium perfringens in Cooked Turkey during Cooling.

    PubMed

    Huang, Lihan; Vinyard, Bryan T

    2016-03-01

    This research applied a new 1-step methodology to directly construct a tertiary model that describes the growth of Clostridium perfringens in cooked turkey meat under dynamically cooling conditions. The kinetic parameters of the growth models were determined by numerical analysis and optimization using multiple dynamic growth curves. The models and kinetic parameters were validated using independent growth curves obtained under various cooling conditions. The results showed that the residual errors (ε) of the predictions followed a Laplace distribution that is symmetric with respect to ε = 0. For residual errors, 90.6% are within ±0.5 Log CFU/g and 73.4% are ±0.25 Log CFU/g for all growth curves used for validation. For relative growth <1.0 Log CFU/g, 88.9% of the residual errors are within ±0.5 Log CFU/g, and 63.0% are within ±0.25 Log CFU/g. For relative growth of <2.0 Log CFU/g, 92.7% of the residual errors are within ±0.5 Log CFU/g, and 70.3% are within ±0.25 Log CFU/g. The scale and distribution of residual errors clearly suggests that the models and estimated kinetic parameters are reasonably accurate in predicting the growth of C. perfringens. Monte Carlo simulation was used to estimate the probabilities of >1.0 and 2.0 Log CFU/g relative growth of C. perfringens in the final products at the end of cooling. This probabilistic process analysis approach provides a new alternative for estimating and managing the risk of a product and can help the food industry and regulatory agencies assess the safety of cooked meat in the event of cooling deviation.

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

    PubMed Central

    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

    2016-01-01

    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. PMID:27004612

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

    PubMed

    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.

  8. Resistance of ovine, caprine and bovine endothelial cells to Clostridium perfringens type D epsilon toxin in vitro.

    PubMed

    Uzal, F A; Rolfe, B E; Smith, N J; Thomas, A C; Kelly, W R

    1999-08-01

    Ovine, caprine and bovine endothelial cells were grown in vitro and challenged with Clostridium perfringens type D epsilon toxin to compare their susceptibility to this toxin. Madin Darby canine kidney (MDCK) cells, which are known to be susceptible to epsilon toxin, were used as a positive control. No morphological alterations were observed in any of the endothelial cell cultures tested, even after challenging with doses as high as 1200 MLD50/ml of epsilon toxin. MDCK cells showed contour rounding and nuclear condensation as early as 30 min after exposure to 100 MLD50/ml of epsilon toxin and after 60 min of exposure to 12.5 MLD50/ml of the same toxin. All the MDCK cells were dead after 3 h of exposure to all concentrations of epsilon toxin. The results indicate that ovine, caprine and bovine endothelial cells are not morphologically responsive to the action of epsilon toxin in vitro.

  9. Differences in signs and lesions in sheep and goats with enterotoxemia induced by intraduodenal infusion of Clostridium perfringens type D.

    PubMed

    Blackwell, T E; Butler, D G; Prescott, J F; Wilcock, B P

    1991-07-01

    Enterotoxemia was induced in 4 lambs and 4 goat kids by continuous intraduodenal infusion of a whole culture of Clostridium perfringens type D. Clinical signs, hematologic values, biochemical alterations, and postmortem lesions in the lambs and goat kids were compared. The 4 lambs and 4 goat kids died within 25 hours of beginning the infusions. Lesions were not observed in the gastrointestinal tract of the 4 lambs; however, severe hemorrhagic enterocolitis was found in the 4 goat kids. This difference between the lambs and goat kids in the lesions caused by experimentally induced enterotoxemia may explain the discrepancies reported between sheep and goats in clinical signs, response to treatment, and efficacy of vaccination observed in naturally induced enterotoxemia in the 2 species.

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

    PubMed

    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.

  11. An unusual necrotic myositis by Clostridium perfringens in a German Shepherd dog: A clinical report, bacteriological and molecular identification

    PubMed Central

    Salari Sedigh, Hamideh; Rajabioun, Masoud; Razmyar, Jamshid; Kazemi Mehrjerdi, Hossein

    2015-01-01

    Clostridial myositis, considered to be rare in pet animals, is an acutely fatal toxaemic condition. Some species of clostridia are responsible for necrotic myositis. A 2-year-old male German shepherd dog was admitted with non-weight bearing lameness and massive swelling of the left hind limb. Clostridium perfringens type A with alpha toxin was diagnosed as a pathogenic agent. Based on the history, the bacteria were introduced inside the tissue via contaminated needle following intramuscular injection. Urgent medical therapy followed by surgical intervention was performed. The dog was discharged completely healthy after hospitalization for four weeks. The objective of this report was to describe necrotic myositis in a dog with an emphasis on clinical signs and treatment as well as bacteriological and molecular identification of the micro-organism. Because of the fatal entity of the disease, prompt diagnosis as well as proper and urgent treatment is very important for successful therapy. PMID:26973773

  12. Bilateral abscessed orchiepididymitis associated with sepsis caused by Veillonella parvula and Clostridium perfringens: case report and review of the literature.

    PubMed Central

    Arrosagaray, P M; Salas, C; Morales, M; Correas, M; Barros, J M; Cordon, M L

    1987-01-01

    Veillonella species is a gram-negative coccus which is part of the anaerobic normal flora in the oral cavity, small intestine, upper respiratory tract, vagina, and urinary tract. The role that this organism plays in infection is not well known, and it is generally associated with other bacteria. We present a case of bilateral abscessed orchiepididymitis associated with septicemia due to Veillonella parvula and, later, to Clostridium perfringens, with the development of severe renal insufficiency and septic shock, which resolved favorably with antibiotic therapy, treatment of shock, and hyperbaric oxygen therapy. In reviewing the literature, we have not found any other case of sepsis due to Veillonella sp. associated with urological disorders. PMID:2887584

  13. The early effects of Clostridium perfringens type D epsilon toxin in ligated intestinal loops of goats and sheep.

    PubMed

    Fernandez Miyakawa, M E; Uzal, F A

    2003-04-01

    Clostridium perfringens type D produces enterotoxaemia in goats, sheep and other animals. The disease is caused by C. perfringens epsilon toxin and, while enterotoxaemia in goats is usually characterized by enterocolitis, the disease in sheep is characterized by systemic lesions (such as lung and brain oedema) with minor and inconsistent changes observed in the intestine. A possible explanation for these differences is that epsilon toxin is more promptly absorbed by the ovine than by the caprine intestine. In an attempt to clarify this, we examined the early effects of epsilon toxin on caprine and ovine intestine. Intestinal loop assays were performed to analyse the physiological and morphological changes induced by epsilon toxin in the intestine of these species. Fluid accumulation was observed in caprine and ovine ileum and colon treated with epsilon toxin. Ileal loops from goats treated with epsilon toxin retained sodium and water earlier than ovine ileal loops treated with the same toxin. Histological analysis showed morphological alterations in the colon of both species as early as 2 h after the commencement of epsilon toxin treatment: these changes were more marked in goats than in sheep. No morphological changes were observed in the ileum of either species after 4 h incubation with epsilon toxin. These results suggest that epsilon toxin modifies ion and water transport in the small and the large intestine of goats and sheep through different mechanisms.

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

    PubMed

    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.

  15. Increased inactivation of ozone-treated Clostridium perfringens vegetative cells and spores on fabricated beef surfaces using mild heat.

    PubMed

    Novak, John S; Yuan, James T C

    2004-02-01

    Ozone treatment of beef surfaces enhanced the effectiveness of cooking temperatures ranging from 45 to 75 degrees C against enterotoxin-producing strains of Clostridium perfringens. Vegetative cells on beef surfaces at an initial concentration of 5.59 +/- 0.17 log CFU/g were reduced significantly (P < 0.05) to 4.09 +/- 0.72 log CFU/g and 3.50 +/- 0.90 log CFU/g after combined treatments with aqueous ozone (5 ppm) and subsequent heating at 45 and 55 degrees C, respectively. Spores on the beef surface were likewise significantly reduced from an initial concentration of 2.94 +/- 0.37 log spores per g to 2.07 +/- 0.38 log spores per g and 1.70 +/- 0.37 log spores per g after the combined treatment with aqueous ozone (5 ppm) and subsequent heating at 55 and 75 degrees C, respectively. Fluorescent nucleic acid stains were used with confocal fluorescence microscopy to show that spores remaining attached to the meat were protected from treatment-specific injury. This study provides evidence for the decreased resistance of both vegetative cells and spores of C. perfringens with ozone treatment that is followed by heat treatment at temperatures that would not otherwise be as effective, thus lowering the requirements for cooking beef while maintaining a margin of safety.

  16. Hypermotility in Clostridium perfringens strain SM101 is due to spontaneous mutations in genes linked to cell division.

    PubMed

    Liu, Hualan; McCord, Kristin D; Howarth, Jonathon; Popham, David L; Jensen, Roderick V; Melville, Stephen B

    2014-07-01

    Clostridium perfringens is a Gram-positive anaerobic pathogen of humans and animals. Although they lack flagella, C. perfringens bacteria can still migrate across surfaces using a type of gliding motility that involves the formation of filaments of bacteria lined up in an end-to-end conformation. In strain SM101, hypermotile variants are often found arising from the edges of colonies on agar plates. Hypermotile cells are longer than wild-type cells, and video microscopy of their gliding motility suggests that they form long, thin filaments that move rapidly away from a colony, analogously to swarmer cells in bacteria with flagella. To identify the cause(s) of the hypermotility phenotype, the genome sequences of normal strains and their direct hypermotile derivatives were determined and compared. Strains SM124 and SM127, hypermotile derivatives of strains SM101 and SM102, respectively, contained 10 and 6 single nucleotide polymorphisms (SNPs) relative to their parent strains. While SNPs were located in different genes in the two sets of strains, one feature in common was mutations in cell division genes, an ftsI homolog in strain SM124 (CPR_1831) and a minE homolog in strain SM127 (CPR_2104). Complementation of these mutations with wild-type copies of each gene restored the normal motility phenotype. A model explaining the principles underlying the hypermotility phenotype is presented.

  17. Effect of Trehalose and Trehalose Transport on the Tolerance of Clostridium perfringens to Environmental Stress in a Wild Type Strain and Its Fluoroquinolone-Resistant Mutant

    PubMed Central

    Park, Miseon; Mitchell, Wilfrid J.

    2016-01-01

    Trehalose has been shown to protect bacterial cells from environmental stress. Its uptake and osmoprotective effect in Clostridium perfringens were investigated by comparing wild type C. perfringens ATCC 13124 with a fluoroquinolone- (gatifloxacin-) resistant mutant. In a chemically defined medium, trehalose and sucrose supported the growth of the wild type but not that of the mutant. Microarray data and qRT-PCR showed that putative genes for the phosphorylation and transport of sucrose and trehalose (via phosphoenolpyruvate-dependent phosphotransferase systems, PTS) and some regulatory genes were downregulated in the mutant. The wild type had greater tolerance than the mutant to salts and low pH; trehalose and sucrose further enhanced the osmotolerance of the wild type to NaCl. Expression of the trehalose-specific PTS was lower in the fluoroquinolone-resistant mutant. Protection of C. perfringens from environmental stress could therefore be correlated with the ability to take up trehalose. PMID:28058047

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

    PubMed Central

    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

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

    PubMed

    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

  20. Identification and sequence determination of recombinant Clostridium perfringens alpha-toxin by use of electrospray ionization mass spectrometry.

    PubMed

    Saito, Hitoshi; Inoue, Masaharu; Tomiki, Masayoshi; Nemoto, Hiroshi; Komoriya, Tomoe; Kimata, Junko; Watanabe, Kunitomo; Kohno, Hideki

    2009-01-01

    Only a few methods exist for simple, sensitive and rapid detection of alpha-toxin in clinical and biological samples. The aim of our study was to establish a procedure for the production of an antibody against a recombinant antigen with confirmed sequence identity. We applied a noble approach based on proteomics using a mass spectrometer for the conclusive identification of the recombinant alpha-toxin that was subsequently used as an antigen. The recombinant alpha-toxin was produced in Escherichia coli. A clinical isolate of Clostridium perfringens GAI 94074 was amplified by polymerase chain reaction (PCR) and subsequently, cloning was performed. Three different fragments were cloned using a pET100/D-TOPO vector. These fragments coded for a ribosome binding site, a signal peptide and the alpha-toxin gene, respectively. Recombinant pET100 plasmids were cloned into TOP 10 cells and the isolated plasmids were transferred into BL21 Star (DE3) cells. Their expression was then induced with isopropyl-beta-D-thiogalactopyranoside (IPTG). Recombinant E. coli transformed with a plasmid encoding the alpha-toxin gene alone produced a biologically inactive protein. On the other hand, E. coli carrying the plasmid encoding the toxin sequence and its native signal peptide sequence, or the toxin sequence along with the ribosome binding sequence and the signal peptide sequence secreted an active alpha-toxin with phospholipase activity. Accordingly, the C. perfringens gene encoding the alpha-toxin protein along with its signal peptide was successfully cloned, expressed, and secreted by E. coli. Furthermore, without consideration of its activity, we used mass spectrometry to confirm that the expressed protein was indeed the alpha-toxin. Thus, the identification of alpha-toxin protein using both the biological activity testing and the mass spectrometry analysis is expected to verify the significant production of C. perfringens antibody. The study for the analysis of recombinant alpha

  1. Dynamic determination of kinetic parameters and computer simulation of growth of Clostridium perfringens in cooked beef

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The objective of this research was to develop a new one-step methodology that uses a dynamic approach to directly construct a tertiary model for prediction of the growth of C. perfringens in cooked beef. This methodology was based on numerical analysis and optimization of both primary and secondary...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The ability of C. perfringens to germinate and grow in acidified ground beef as well as in ten commercially prepared acidified beef, pork and poultry products was assessed. The pH of ground beef was adjusted using organic vinegar to achieve various pH values between 5.0 and 5.6; the pH of the commer...

  4. [The occurrence of Escherichia coli, Aeromonas hydrophila, Plesiomonas shigelloides and Clostridium perfringens in the intestinal flora of gray herons (Ardea cinerea)].

    PubMed

    Glünder, G

    1989-05-01

    The flora of the large intestine of 92 grey herons was examined for the frequency of aerobic and microaerobic growing bacteria. Clostridium perfringens, Aeromonas hydrophila, Plesiomonas shigelloides and E. coli were isolated from 55%, 48%, 14% and 35% of the birds, respectively. It could be demonstrated that the findings of these bacteria in the intestinal flora are depending on the age of the birds. The percentage of carriers of Clostridium perfringens, Aeromonas hydrophila and Plesiomonas shigelloides was highest in nestlings younger than 18 days, less high in older nestlings and lowest in adult grey herons. Contrary to those bacteria, E. coli was found more often in the intestinal flora at increasing age of the birds. Salmonella spp. were isolated from 6 birds. Two birds yielded positive for Yersinia enterocolitica and Campylobacter spp., respectively. Other aerobic and microaerobic bacteria play a less significant role as part of the intestinal flora.

  5. Entérite nécrotique chez le poulet de gril II. Caractères des souches de Clostridium perfringens isolées

    PubMed Central

    Bernier, G.; Filion, R.; Malo, R.; Phaneuf, J.-B.

    1974-01-01

    A Gram positive bacillus, strictly anaerobic, was isolated from the viscera of all diseased birds showing lesions of necrotic enteritis. Its morphology and biochemical reactions, the presence of alpha and thêta hemolysins and the production of a lecithinase-C in vitro, all these characteristics indicated a similarity to those belonging to the group of Clostridium perfringens. The two hemolysins were neutralized in vitro only by the antitoxin A. Broiler chickens injected I.V. with a Viande-Foie (VF) broth culture of Clostridium perfringens together with the antitoxin A survived, whereas those receiving antitoxin C died. These results seem to indicate that this organism belongs to the type A. This bacillus was sensitive to a great variety of antibiotics, except neomycin. PMID:4368193

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

    PubMed

    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.

  7. Impact of a drug-free program on broiler chicken growth performances, gut health, Clostridium perfringens and Campylobacter jejuni occurrences at the farm level.

    PubMed

    Gaucher, M-L; Quessy, S; Letellier, A; Arsenault, J; Boulianne, M

    2015-08-01

    The use of antimicrobial agents as feed additives in poultry production is a public health concern due to the overall increase in antimicrobial resistance. Although some alternative products are commercially available, little is known on their potential impact on flock health and productivity. A prospective study involving 1.55 million birds was conducted on eight commercial broiler farms in Québec, Canada, to evaluate the impact of replacing antibiotic growth promoters and anticoccidial drugs by a drug-free program including improved brooding conditions, anticoccidial vaccination, essential oil-based feed additives, and water acidification. Various productivity and health parameters were compared between barns allocated to the conventional and the drug-free program. Zootechnical performances were monitored as productivity criteria. Clinical necrotic enteritis and subclinical enteritis occurrences, litter and fecal moistures content were measured, and microscopic gut health was evaluated. Clostridium perfringens and Campylobacter spp. strains were recovered from fecal samples collected during farm visits. Clostridium perfringens counts were used as poultry health indicators and Campylobacter prevalence was noted as well. The drug-free program was associated with a significant increase in feed conversion ratio and a decrease in mean live weight at slaughter and in daily weight gain. An increased incidence of necrotic enteritis outbreaks and subclinical enteritis cases, as well as an increase in litter moisture content at the end of the rearing period were also observed for this program. Mean microscopic intestinal lesion scores and prevalence of Campylobacter colonization were not statistically different between the two groups but the drug-free program was associated with higher Clostridium perfringens isolation rates. According to the current study design, the results suggest that substitution of antibiotic growth promoters and anticoccidial drugs by a drug

  8. Cloning and expression of Clostridium perfringens type D vaccine strain epsilon toxin gene in E. coli as a recombinant vaccine candidate

    PubMed Central

    Aziminia, Parastoo; Pilehchian-Langroudi, Reza; Esmaeilnia, Kasra

    2016-01-01

    Background and Objectives: Clostridium perfringens, a Gram-positive obligate anaerobic bacterium, is able to form resistant spores which are widely distributed in the environment. C. perfringens is subdivided into five types A to E based on its four major alpha, beta, epsilon and iota toxins. The aim of the present study was cloning and expression of C. perfringens type D vaccine strain epsilon toxin gene. Materials and Methods: Genomic DNA was extracted and the epsilon toxin gene was amplified using Pfu DNA polymerase. The PCR product was cloned into pJET1.2/blunt cloning vector. The recombinant vector (pJETε) was sequenced using universal primers. At the next step epsilon toxin gene was subcloned into pET22b(+) expression vector and transformed into E. coli Rosetta (DE3) host strain. Results: The recombinant protein has been expressed in E. coli Rosetta (DE3) cells after subcloning of C. perfringens etx gene (1008 bp) into the expression vector. Conclusion: We concluded that E. coli Rosetta strain was suitable for the expression of recombinant C. perfringens epsilon toxin protein from pET22ε expression vector. This recombinant cell can be used for further research on recombinant vaccine development. PMID:28210460

  9. Most probable number method combined with nested polymerase chain reaction for detection and enumeration of enterotoxigenic Clostridium perfringens in intestinal contents of cattle, pig and chicken.

    PubMed

    Miwa, N; Nishina, T; Kubo, S; Atsumi, M

    1997-02-01

    The most probable number (MPN) method combined with a nested polymerase chain reaction (nested PCR) for the detection and enumeration of enterotoxigenic Clostridium perfringens in the intestinal contents of cattle, pig and chicken was examined. Ten-fold serial dilutions of samples were added to three tubes of enrichment medium, which were incubated at 37 degrees C for 20-24 hr, and the C. perfringens enterotoxin gene was detected by nested PCR from the enrichment culture without isolating the organism. The results obtained by this method with artificially contaminated intestinal contents were significantly correlated with those obtained by a plate count method. When the method was applied to the detection and enumeration of indigenous enterotoxigenic C. perfringens, the organism was found in two, two and three samples of 10 intestinal contents of cattle, pig and chicken, respectively. Most of the positive samples contained fewer than 10 MPN/g of enterotoxigenic C. perfringens, except one sample of chicken, which contained 1.5 x 10(2) MPN/g. The MPN method combined with nested PCR is easy to perform and may be a useful tool for the detection and enumeration of enterotoxigenic C. perfringens in intestinal contents.

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

    PubMed

    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 < 0.05) lower TBARS values than the control at 10 and 24 °C. The lipid oxidation inhibition effect was the highest (P < 0.05) in anaerobic packed Jokbal with 10% rooibos. The addition of a combination of 10% rooibos and 4% PL + SDA during the processing of Jokbal prevented the growth of C. perfringens and the germination and growth of C. perfringens spores at room temperature. This study shows rooibos tea as a valuable natural food preservative in meat products.

  11. Clostridium perfringens alpha-toxin and NetB toxin antibodies and their possible role in protection against necrotic enteritis and gangrenous dermatitis in broiler chickens.

    PubMed

    Lee, K W; Lillehoj, H S; Park, M S; Jang, S I; Ritter, G D; Hong, Y H; Jeong, W; Jeoung, H Y; An, D J; Lillehoj, E P

    2012-03-01

    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 factors of C perfringens are its four major toxins (alpha, beta, epsilon, iota) and the newly described NE B-like (NetB) toxin. While neutralizing antibodies against some C perfingens toxins are associated with protection against infection in mammals, the serologic responses of NE- and GD-afflicted birds to these toxins have not been evaluated. Therefore, we measured serum antibody levels to C perfringens alpha-toxin and NetB toxin in commercial birds from field outbreaks of NE and GD using recombinant toxin-based enzyme-linked immunosorbent assay (ELISA). Initially, we used this ELISA system to detect antibody titers against C perfringens alpha-toxin and NetB toxin that were increased in birds experimentally coinfected with Eimeria maxima and C perfringens compared with uninfected controls. Next, we applied this ELISA to field serum samples from flock-mated birds with or without clinical signs of NE or GD. The results showed that the levels of antibodies against both toxins were significantly higher in apparently healthy chickens compared to birds with clinical signs of NE or GD, suggesting that these antitoxin antibodies may play a role in protection against NE and GD.

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

    PubMed

    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.

  13. 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.

    PubMed

    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.

  14. Vaccination schedules to raise antibody concentrations against epsilon-toxin of Clostridium perfringens in ewes and their triplet lambs.

    PubMed

    de la Rosa, C; Hogue, D E; Thonney, M L

    1997-09-01

    The objective of this experiment was to compare vaccination schedules for ewes and their lambs to raise antibody concentrations to epsilon-toxin of Clostridium perfringens, the causative agent of enterotoxemia. Half of 200 Finnsheep x Dorset ewes were vaccinated with C. perfringens type D toxoid vaccine 3 wk before lambing. Serum samples were obtained from 20 ewes that were to be vaccinated and 20 ewes that would remain unvaccinated before treatment and at wk 2, 1, and 0 before the start of lambing. Antibody concentrations in sera of unvaccinated ewes remained at 2 IU/mL, but they peaked in vaccinated ewes at 15 IU/mL by wk 1 before lambing. Lambs from each of the first 13 and the first 14 sets of triplets from vaccinated and unvaccinated ewes, respectively, received one of three vaccination treatments: no vaccine (control), vaccination on d 1 and 21 of age, or vaccination on d 21 and 42 of age. Antibody concentrations declined in sera of vaccinated ewes from 8.5 IU/mL immediately after lambing to 3 IU/mL 12 wk later. Vaccination of lambs did not increase sera antibody concentration. However, prepartum vaccination of ewes significantly increased lamb antibody concentrations (19 IU/mL) compared with lambs reared by unvaccinated ewes (2 IU/mL). Vaccination of ewes resulted in lambs with higher antibody concentrations until wk 10 postpartum. Concentrations declined to .6 IU/mL in all lambs at 12 wk. Because concentrations of .2 IU/mL may be protective, these results indicate that vaccination of ewes before lambing imparts passive protection in lambs to 12 wk of age, whereas vaccination of young lambs provides no added protection.

  15. Genotypic and Phenotypic Characterization of Clostridium perfringens Isolates from Darmbrand Cases in Post-World War II Germany

    PubMed Central

    Ma, Menglin; Li, Jihong

    2012-01-01

    Clostridium perfringens type C strains are the only non-type-A isolates that cause human disease. They are responsible for enteritis necroticans, which was termed Darmbrand when occurring in post-World War II Germany. Darmbrand strains were initially classified as type F because of their exceptional heat resistance but later identified as type C strains. Since only limited information exists regarding Darmbrand strains, this study genetically and phenotypically characterized seven 1940s era Darmbrand-associated strains. Results obtained indicated the following. (i) Five of these Darmbrand isolates belong to type C, carry beta-toxin (cpb) and enterotoxin (cpe) genes on large plasmids, and express both beta-toxin and enterotoxin. The other two isolates are cpe-negative type A. (ii) All seven isolates produce highly heat-resistant spores with D100 values (the time that a culture must be kept at 100°C to reduce its viability by 90%) of 7 to 40 min. (iii) All of the isolates surveyed produce the same variant small acid-soluble protein 4 (Ssp4) made by type A food poisoning isolates with a chromosomal cpe gene that also produce extremely heat-resistant spores. (iv) The Darmbrand isolates share a genetic background with type A chromosomal-cpe-bearing isolates. Finally, it was shown that both the cpe and cpb genes can be mobilized in Darmbrand isolates. These results suggest that C. perfringens type A and C strains that cause human food-borne illness share a spore heat resistance mechanism that likely favors their survival in temperature-abused food. They also suggest possible evolutionary relationships between Darmbrand strains and type A strains carrying a chromosomal cpe gene. PMID:23027533

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

    PubMed

    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.

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

    PubMed Central

    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. Dynamic determination of kinetic parameters, computer simulation, and probabilistic analysis of growth of Clostridium perfringens in cooked beef during cooling.

    PubMed

    Huang, Lihan

    2015-02-16

    The objective of this research was to develop a new one-step methodology that uses a dynamic approach to directly construct a tertiary model for prediction of the growth of Clostridium perfringens in cooked beef. This methodology was based on simultaneous numerical analysis and optimization of both primary and secondary models using multiple dynamic growth curves obtained under different conditions. Once the models were constructed, the bootstrap method was used to calculate the 95% confidence intervals of kinetic parameters, and a Monte Carlo simulation method was developed to validate the models using the growth curves not previously used in model development. The results showed that the kinetic parameters obtained from this study accurately matched the common characteristics of C. perfringens, with the optimum temperature being 45.3°C. The results also showed that the predicted growth curves matched accurately with experimental observations used in validation. The mean of residuals of the predictions is -0.02logCFU/g, with a standard deviation of only 0.23logCFU/g. For relative growths <1logCFU/g, the residuals of predictions are <0.4logCFU/g. Overall, 74% of the residuals of predictions are <0.2logCFU/g, 7.7% are >0.4logCFU/g, while only 1.5% are >0.8logCFU/g. In addition, the dynamic model also accurately predicted four isothermal growth curves arbitrarily chosen from the literature. Finally, the Monte Carlo simulation was used to provide the probability of >1 and 2logCFU/g relative growths at the end of cooling. The results of this study will provide a new and accurate tool to the food industry and regulatory agencies to assess the safety of cooked beef in the event of cooling deviation.

  19. Detection of beta2 and major toxin genes by PCR in Clostridium perfringens field isolates of domestic animals suffering from enteritis or enterotoxaemia.

    PubMed

    Sting, Reinhard

    2009-01-01

    The production of Clostridium (C.) perfringens toxins in the intestine is an important cause of enteritis and enterotoxaemia in livestock. In the present study, the alpha toxin and the genes encoding beta2 and epsilon toxin could be frequently detected by means of phenotypical and PCR examinations in these bacteria. The C. perfringens isolates originated from 1213 field samples taken from diseased or perished livestock located in the north-eastern administrative districts of Baden-Württemberg (Germany) from 2005 to 2008. The beta2 toxin gene of C perfringens was detected in all animal species examined, comprising pigs, the small ruminants sheep and goats, cattle, horses, rabbits, alpacas and lamas, and fallow deer. Among all the animal species included in this study, pigs attracted attention by a high quota of 74.2% (610 of 822) cpb2-positive C. perfringens isolates in comparison to the other animal species tested, revealing a quota of 20.8% (72 of 346). Beta2 toxigenic isolates could be predominantly cultivated from the faeces of young piglets. The beta toxin gene was detected in isolates from piglets and small ruminants only, amounting to 82.5% (33 of 40) in piglets in combination with the cpb2 gene. In this context, cpb2/cpb-positive C. perfringens isolates of piglets could be clearly detected more often in the intestine of perished animals (18 of 158) than in faeces (15 of 629). Furthermore, cpb2-bearing C. perfringens isolates were detected in cattle, horses, rabbits, alpacas and lamas, and fallow deer to a notable degree. The detection of C. perfringens isolates carrying the epsilon toxin gene was restricted to sheep and goats. Of a total of 242 small ruminants that succumbed to sudden death, 71 (29.3%) harboured epsilon toxin-positive C. perfringens isolates in their intestines. These cases clustered seasonally in the second quarter (April, May, and June) of the year. Neither the isolates bearing the beta2 nor beta toxin gene nor those carrying the epsilon

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

    PubMed

    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.

  1. The CpAL system regulates changes of the trans-epithelial resistance of human enterocytes during Clostridium perfringens type C infection.

    PubMed

    Nava, Porfirio; Vidal, Jorge E

    2016-06-01

    Clostridium perfringens type C strains produce severe disease in humans and animals including enterotoxaemia and hemorrhagic diarrhea. Type C disease is mediated by production of toxins that damage the site of infection inducing loss of bloody fluids. Production of type C toxins, such as CPA, PFO, and, CPB is regulated by the C. perfringens Agr-like (CpAL) quorum sensing (QS) system. The CpAL system is also required to recapitulate, in vivo, intestinal signs of C. perfringens type C-induced disease, including hemorrhagic diarrhea and accumulation of fluids. The intestinal epithelium forms a physical barrier, made up of a series of intercellular junctions including tight junctions (TJs), adherens junctions (AJs) and desmosomes (DMs). This selective barrier regulates important physiological processes, including paracellular movement of ions and solutes, which, if altered, results in loss of fluids into the intestinal lumen. In this work, the effects of C. perfringens infection on the barrier function of intestinal epithelial cells was evaluated by measuring trans-epithelial resistance (TEER). Our studies demonstrate that infection of human enterocytes with C. perfringens type C strain CN3685 induced a significant drop on TEER. Changes in TEER were mediated by the CpAL system as a CN3685ΔagrB mutant did not induce such a drop. Physical contact between bacteria and enterocytes produced more pronounced changes in TEER and this phenomenon appeared also to be mediated by the CpAL system. Finally, immunofluorescence studies demonstrate that C. perfringens type C infection redistribute TJs protein occludin, and Claudin-3, and DMs protein desmoglein-2, but did not affect the AJs protein E-cadherin.

  2. Detection of a group II intron without an open reading frame in the alpha-toxin gene of Clostridium perfringens isolated from a broiler chicken.

    PubMed

    Ma, Menglin; Ohtani, Kaori; Shimizu, Tohru; Misawa, Naoaki

    2007-03-01

    A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.

  3. The VirS/VirR Two-Component System Regulates the Anaerobic Cytotoxicity, Intestinal Pathogenicity, and Enterotoxemic Lethality of Clostridium perfringens Type C Isolate CN3685

    PubMed Central

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

    2011-01-01

    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. PMID:21264065

  4. Two novel membrane proteins, TcpD and TcpE, are essential for conjugative transfer of pCW3 in Clostridium perfringens.

    PubMed

    Wisniewski, Jessica A; Teng, Wee L; Bannam, Trudi L; Rood, Julian I

    2015-02-15

    The anaerobic pathogen Clostridium perfringens encodes either toxin genes or antibiotic resistance determinants on a unique family of conjugative plasmids that have a novel conjugation region, the tcp locus. Studies of the paradigm conjugative plasmid from C. perfringens, the 47-kb tetracycline resistance plasmid pCW3, have identified several tcp-encoded proteins that are involved in conjugative transfer and form part of the transfer apparatus. In this study, the role of the conserved hypothetical proteins TcpD, TcpE, and TcpJ was examined. Mutation and complementation analyses showed that TcpD and TcpE were essential for the conjugative transfer of pCW3, whereas TcpJ was not required. To analyze the TcpD and TcpE proteins in C. perfringens, functional hemagglutinin (HA)-tagged derivatives were constructed. Western blots showed that TcpD and TcpE localized to the cell envelope fraction independently of the presence of other pCW3-encoded proteins. Finally, examination of the subcellular localization of TcpD and TcpE by immunofluorescence showed that these proteins were concentrated at both poles of C. perfringens donor cells, where they are postulated to form essential components of the multiprotein complex that comprises the transfer apparatus.

  5. Vegetative Bacillus amyloliquefaciens cells do not confer protection against necrotic enteritis in broilers despite high antibacterial activity of its supernatant against Clostridium perfringens in vitro.

    PubMed

    Geeraerts, S; Delezie, E; Ducatelle, R; Haesebrouck, F; Devreese, B; Van Immerseel, F

    2016-06-01

    In this study, the effect of Bacillus amyloliquefaciens on Clostridium perfringens was tested in vitro and in vivo. Using an agar well diffusion assay, the inhibitory activity of B. amyloliquefaciens supernatant was analysed against a large collection of netB-positive and netB-negative C. perfringens strains. Although strong growth inhibiting activity was detected against all C. perfringens isolates, it was significantly higher against virulent netB-positive C. perfringens strains compared with avirulent netB-negative isolates. Subsequently, the efficacy of in-feed administration of lyophilised vegetative cells of B. amyloliquefaciens to prevent necrotic enteritis was tested in vivo using an established experimental infection model in broilers. Ross 308 broilers received either B. amyloliquefaciens supplemented or unsupplemented feed throughout the experiment. No significant differences could be detected between the untreated positive control group and the B. amyloliquefaciens treated group in body weight, the number of chickens that developed necrotic lesions and in pathological lesion scores. These results demonstrate that despite its substantial inhibitory activity in vitro, lyophilised vegetative B. amyloliquefaciens cells had no beneficial effect against necrotic enteritis in the in vivo model used here.

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

    PubMed

    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. Effects of bilayer composition and physical properties on the phospholipase C and sphingomyelinase activities of Clostridium perfringens α-toxin.

    PubMed

    Urbina, Patricia; Flores-Díaz, Marietta; Alape-Girón, Alberto; Alonso, Alicia; Goñi, Félix M

    2011-01-01

    α-Toxin, a major determinant of Clostridium perfringens toxicity, exhibits both phospholipase C and sphingomyelinase activities. Our studies with large unilamellar vesicles containing a variety of lipid mixtures reveal that both lipase activities are enhanced by cholesterol and by lipids with an intrinsic negative curvature, e.g. phosphatidylethanolamine. Conversely lysophospholipids, that possess a positive intrinsic curvature, inhibit the α-toxin lipase activities. Phospholipids with a net negative charge do not exert any major effect on the lipase activities, and the same lack of effect is seen with the lysosomal lipid bis (monoacylglycero) phosphate. Ganglioside GT1b has a clear inhibitory effect, while the monosialic ganglioside GM3 is virtually ineffectual even when incorporated at 6mol % in the vesicles. The length of the lag periods appears to be inversely related to the maximum (post-lag) enzyme activities. Moreover, and particularly in the presence of cholesterol, lag times increase with pH. Both lipase activities are sensitive to vesicle size, but in opposite ways: while phospholipase C is higher with larger vesicles, sphingomyelinase activity is lower. The combination of our results with previous structural studies suggests that α-toxin lipase activities have distinct, but partially overlapping and interacting active sites.

  8. Correlation between In Vitro Cytotoxicity and In Vivo Lethal Activity in Mice of Epsilon Toxin Mutants from Clostridium perfringens

    PubMed Central

    Dorca-Arévalo, Jonatan; Pauillac, Serge; Díaz-Hidalgo, Laura; Martín-Satué, Mireia; Popoff, Michel R.; Blasi, Juan

    2014-01-01

    Epsilon toxin (Etx) from Clostridium perfringens is a pore-forming protein with a lethal effect on livestock, producing severe enterotoxemia characterized by general edema and neurological alterations. Site-specific mutations of the toxin are valuable tools to study the cellular and molecular mechanism of the toxin activity. In particular, mutants with paired cysteine substitutions that affect the membrane insertion domain behaved as dominant-negative inhibitors of toxin activity in MDCK cells. We produced similar mutants, together with a well-known non-toxic mutant (Etx-H106P), as green fluorescent protein (GFP) fusion proteins to perform in vivo studies in an acutely intoxicated mouse model. The mutant (GFP-Etx-I51C/A114C) had a lethal effect with generalized edema, and accumulated in the brain parenchyma due to its ability to cross the blood-brain barrier (BBB). In the renal system, this mutant had a cytotoxic effect on distal tubule epithelial cells. The other mutants studied (GFP-Etx-V56C/F118C and GFP-Etx-H106P) did not have a lethal effect or cross the BBB, and failed to induce a cytotoxic effect on renal epithelial cells. These data suggest a direct correlation between the lethal effect of the toxin, with its cytotoxic effect on the kidney distal tubule cells, and the ability to cross the BBB. PMID:25013927

  9. Identification of amino acids important for binding of Clostridium perfringens epsilon toxin to host cells and to HAVCR1

    PubMed Central

    Ivie, Susan E.; McClain, Mark S.

    2012-01-01

    Clostridium perfringens epsilon toxin belongs to the aerolysin-like family of pore-forming toxins and is one of the most potent bacterial toxins known. The epsilon toxin causes fatal enterotoxemia in sheep, goats, and possibly humans. Evidence indicates that the toxin binds to protein receptors including hepatitis A virus cellular receptor 1 (HAVCR1), but the region of the toxin responsible for cell binding has not been identified. In the present study, we identify amino acids within the epsilon toxin important for this cell interaction. Site-specific mutagenesis was used to investigate the role of a surface-accessible cluster of aromatic amino acids, and purified mutant proteins were tested in a series of cell-culture assays to assess cytotoxic activity and cell binding. When added to cells, four mutant proteins (Etx-Y29E, Etx-Y30E, Etx-Y36E and Etx-Y196E) were severely impaired in their ability to not only kill host cells, but also in their ability to permeabilize the plasma membrane. Circular dichroism spectroscopy and thermal stability studies revealed that the wild-type and mutant proteins were similarly folded. Additional experiments revealed that these mutant proteins were defective in binding to host cells and to HAVCR1. These data indicate that an amino acid motif including Y29, Y30, Y36, and Y196 is important for the ability of epsilon toxin to interact with cells and HAVCR1. PMID:22938730

  10. Attack of the nervous system by Clostridium perfringens Epsilon toxin: from disease to mode of action on neural cells.

    PubMed

    Wioland, Laetitia; Dupont, Jean-Luc; Bossu, Jean-Louis; Popoff, Michel R; Poulain, Bernard

    2013-12-01

    Epsilon toxin (ET), produced by Clostridium perfringens types B and D, ranks among the four most potent poisonous substances known so far. ET-intoxication is responsible for enterotoxaemia in animals, mainly sheep and goats. This disease comprises several manifestations indicating the attack of the nervous system. This review aims to summarize the effects of ET on central nervous system. ET binds to endothelial cells of brain capillary vessels before passing through the blood-brain barrier. Therefore, it induces perivascular oedema and accumulates into brain. ET binding to different brain structures and to different component in the brain indicates regional susceptibility to the toxin. Histological examination has revealed nerve tissue and cellular lesions, which may be directly or indirectly caused by ET. The naturally occurring disease caused by ET-intoxication can be reproduced experimentally in rodents. In mice and rats, ET recognizes receptor at the surface of different neural cell types, including certain neurons (e.g. the granule cells in cerebellum) as well as oligodendrocytes, which are the glial cells responsible for the axons myelination. Moreover, ET induces release of glutamate and other transmitters, leading to firing of neural network. The precise mode of action of ET on neural cells remains to be determined.

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

    PubMed Central

    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.

    2014-01-01

    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. PMID:24709588

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

    PubMed Central

    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

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    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.

  16. Clostridium perfringens Phospholipase C Induced ROS Production and Cytotoxicity Require PKC, MEK1 and NFκB Activation

    PubMed Central

    Monturiol-Gross, Laura; Flores-Díaz, Marietta; Pineda-Padilla, Maria Jose; Castro-Castro, Ana Cristina; Alape-Giron, Alberto

    2014-01-01

    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. PMID:24466113

  17. Crystal structure of the phosphate-binding protein (PBP-1) of an ABC-type phosphate transporter from Clostridium perfringens

    PubMed Central

    Gonzalez, Daniel; Richez, Magali; Bergonzi, Celine; Chabriere, Eric; Elias, Mikael

    2014-01-01

    Phosphate limitation is an important environmental stress that affects the metabolism of various organisms and, in particular, can trigger the virulence of numerous bacterial pathogens. Clostridium perfringens, a human pathogen, is one of the most common causes of enteritis necroticans, gas gangrene and food poisoning. Here, we focused on the high affinity phosphate-binding protein (PBP-1) of an ABC-type transporter, responsible for cellular phosphate uptake. We report the crystal structure (1.65 Å resolution) of the protein in complex with phosphate. Interestingly, PBP-1 does not form the short, low-barrier hydrogen bond with phosphate that is typical of previously characterized phosphate-binding proteins, but rather a canonical hydrogen bond. In its unique binding configuration, PBP-1 forms an unusually high number of hydrogen bonds (14) with the phosphate anion. Discrimination experiments reveal that PBP-1 is the least selective PBP characterised so far and is able to discriminate phosphate from its close competing anion, arsenate, by ~150-fold. PMID:25338617

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

    PubMed

    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.

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

    PubMed

    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.

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

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    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. PMID:26713879

  1. Molecular Architecture and Functional Analysis of NetB, a Pore-forming Toxin from Clostridium perfringens*

    PubMed Central

    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.

    2013-01-01

    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. PMID:23239883

  2. Influence of starch source on sporulation and enterotoxin production by Clostridium perfringens type A.

    PubMed

    Labbe, R; Somers, E; Duncan, C

    1976-03-01

    Of 16 different starch preparations tested, Clostridium perfringes NCTC 8798 yielded maximum sporulation and enterotoxin formation when ICN-soluble starch was included in Duncan and Strong sporulation medium. In general soluble starches were better than potato, corn, or arrowroot starch with regard to these two parameters.

  3. Application of a real-time PCR method for Salmonella spp., Escherichia coli, Staphylococcus aureus and Clostridium perfringens detection in water samples.

    PubMed

    Rozwadowska, Beata; Albertyńska, Marta; Hudzik, Grzegorz; Okła, Hubert; Jasik, Krzysztof P; Słodki, Jan

    2013-01-01

    The diagnostic assessment of water sanitary state is based mainly on the cultivation of bacteria retained on membrane filters. However classical microbiology methods have a lot of disadvantages. More and more frequently, rapid detection and identification of pathogens present in water is based on molecular biology techniques. The aim of this study was to determine the effectiveness and usefulness of a real-time PCR method, when compared to the recommended bacteria culture method, in diagnostics of pathogens in water samples. The research concerned the detection and identification of main sanitary indicators of water such as: Salmonella spp., Escherichia coli, Staphylococcus aureus and Clostridium perfringens. The analyses were conducted in water samples contaminated with the reference material (the aforementioned bacteria) and real environmental samples, which were examined for the presence of nucleic acid of: Salmonella spp., E. coli, S. aureus and C. perfringens using a real-time PCR method.

  4. Distribution of Clostridium perfringens and fecal sterols in a benthic coastal marine environment influenced by the sewage outfall from McMurdo Station, Antarctica.

    PubMed

    Edwards, D D; McFeters, G A; Venkatesan, M I

    1998-07-01

    The spatial distribution, movement, and impact of the untreated wastewater outfall from McMurdo Station, Antarctica, were investigated under early austral summer conditions. The benthic environment was examined to determine the distribution of Clostridium perfringens in sediment cores and the intestinal contents of native invertebrates and fish along a transect of stations. These stations extended ca. 411 m south of the outfall. The findings revealed that the concentration of C. perfringens decreased with depth in the sediment and distance from the outfall. High percentages of tunicates and sea urchins were colonized with this bacterium along the transect. Coprostanol concentrations were also measured in sediment samples taken from each of the transect stations, and a similar trend was observed. These results are in agreement with the findings of previous studies performed with the water column and collectively provide evidence that the disposal of domestic wastes deserves special consideration in polar marine environments.

  5. The Interaction of a Carbohydrate-Binding Module from a Clostridium perfringens N-Acetyl-beta-hexosaminidase with its Carbohydrate Receptor

    SciTech Connect

    Ficko-Blean,E.; Boraston, A.

    2006-01-01

    Clostridium perfringens is a notable colonizer of the human gastrointestinal tract. This bacterium is quite remarkable for a human pathogen by the number of glycoside hydrolases found in its genome. The modularity of these enzymes is striking as is the frequent occurrence of modules having amino acid sequence identity with family 32 carbohydrate-binding modules (CBMs), often referred to as F5/8 domains. Here we report the properties of family 32 CBMs from a C. perfringens N-acetyl-{beta}-hexosaminidase. Macroarray, UV difference, and isothermal titration calorimetry binding studies indicate a preference for the disaccharide LacNAc ({beta}-d-galactosyl-1,4-{beta}-d-N-acetylglucosamine). The molecular details of the interaction of this CBM with galactose, LacNAc, and the type II blood group H-trisaccharide are revealed by x-ray crystallographic studies at resolutions of 1.49, 2.4, and 2.3 Angstroms, respectively.

  6. [Production of a vaccine against enterotoxemia from Clostridium perfringens strains isolated in the field].

    PubMed

    Cherfaoui, S; Kadra, B

    1992-01-01

    We have isolated eight strains of C. perfringens from cases of enterotoxaemia. Five of these strains have revealed themselves toxic with respective types (type "A":2, type "C":2, type "D":1). In order to produce anti-enterotoxaemia vaccine, we have proceeded at the cultivation in fermenter of isolated strains and reference strains CWA 35, CWC and CWD AF. At the end of fermentation, we have evaluated the two following parameters: obtained biomass, and toxin titers. With the two classes of strains we reached an important biomass but toxins titers relatively weak comparatively to that which is usually required. It will be necessary then, to demonstrate the immunogen value of the produced vaccines by testing their efficacity.

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

    PubMed

    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.

  8. Characterization of bacteriophages virulent for Clostridium perfringens and identification of phage lytic enzymes as alternatives to antibiotics for potential control of the bacterium.

    PubMed

    Seal, Bruce S

    2013-02-01

    There has been a resurgent interest in the use of bacteriophages or their gene products to control bacterial pathogens as alternatives to currently used antibiotics. Clostridium perfringens is a gram-positive, spore-forming anaerobic bacterium that plays a significant role in human foodborne disease as well as non-foodborne human, animal, and avian diseases. Countries that have complied with the ban on antimicrobial growth promoters in feeds have reported increased incidences of C. perfringens-associated diseases in poultry. To address these issues, new antimicrobial agents, putative lysins encoded by the genomes of bacteriophages, are being identified in our laboratory. Poultry intestinal material, soil, sewage, and poultry processing drainage water were screened for virulent bacteriophages that could lyse C. perfringens and produce clear plaques in spot assays. Bacteriophages were isolated that had long noncontractile tails, members of the family Siphoviridae, and with short noncontractile tails, members of the family Podoviridae. Several bacteriophage genes were identified that encoded N-acetylmuramoyl-l-alanine amidases, lysozyme-endopeptidases, and a zinc carboxypeptidase domain that has not been previously reported in viral genomes. Putative phage lysin genes (ply) were cloned and expressed in Escherichia coli. The recombinant lysins were amidases capable of lysing both parental phage host strains of C. perfringens as well as other strains of the bacterium in spot and turbidity reduction assays, but did not lyse any clostridia beyond the species. Consequently, bacteriophage gene products could eventually be used to target bacterial pathogens, such as C. perfringens via a species-specific strategy, to control animal and human diseases without having deleterious effects on beneficial probiotic bacteria.

  9. Genome-Wide Transcriptional Profiling of Clostridium perfringens SM101 during Sporulation Extends the Core of Putative Sporulation Genes and Genes Determining Spore Properties and Germination Characteristics

    PubMed Central

    Xiao, Yinghua; van Hijum, Sacha A. F. T.; Abee, Tjakko; Wells-Bennik, Marjon H. J.

    2015-01-01

    The formation of bacterial spores is a highly regulated process and the ultimate properties of the spores are determined during sporulation and subsequent maturation. A wide variety of genes that are expressed during sporulation determine spore properties such as resistance to heat and other adverse environmental conditions, dormancy and germination responses. In this study we characterized the sporulation phases of C. perfringens enterotoxic strain SM101 based on morphological characteristics, biomass accumulation (OD600), the total viable counts of cells plus spores, the viable count of heat resistant spores alone, the pH of the supernatant, enterotoxin production and dipicolinic acid accumulation. Subsequently, whole-genome expression profiling during key phases of the sporulation process was performed using DNA microarrays, and genes were clustered based on their time-course expression profiles during sporulation. The majority of previously characterized C. perfringens germination genes showed upregulated expression profiles in time during sporulation and belonged to two main clusters of genes. These clusters with up-regulated genes contained a large number of C. perfringens genes which are homologs of Bacillus genes with roles in sporulation and germination; this study therefore suggests that those homologs are functional in C. perfringens. A comprehensive homology search revealed that approximately half of the upregulated genes in the two clusters are conserved within a broad range of sporeforming Firmicutes. Another 30% of upregulated genes in the two clusters were found only in Clostridium species, while the remaining 20% appeared to be specific for C. perfringens. These newly identified genes may add to the repertoire of genes with roles in sporulation and determining spore properties including germination behavior. Their exact roles remain to be elucidated in future studies. PMID:25978838

  10. The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin

    PubMed Central

    Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A.; Fischetti, Vincent A.; McClain, Mark S.; Vartanian, Timothy

    2015-01-01

    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. PMID:25993478

  11. ANTIBODY RESPONSE TO EPSILON TOXIN OF CLOSTRIDIUM PERFRINGENS IN CAPTIVE RED DEER (CERVUS ELAPHUS) OVER A 13-MONTH PERIOD.

    PubMed

    Scala, Christopher; Duffard, Nicolas; Beauchamp, Guy; Boullier, Séverine; Locatelli, Yann

    2016-03-01

    Deer are sensitive to clostridial diseases, and vaccination with clostridial toxoids is the method of choice to prevent these infections in ruminants. The purpose of this study was to evaluate the serologic responses in red deer (Cervus elaphus) over a 13-mo period after vaccination with a multivalent clostridial vaccine, containing an aluminium hydroxide adjuvant. Antibody production to the Clostridium perfringens type D epsilon toxin component of the vaccine was measured using an indirect enzyme-linked immunosorbent assay. Animals from group 1 (9 mo old; n = 6) were naïve and received an initial vaccination with a booster vaccine 4 wk apart and one annual booster. Animals from group 2 (21 mo old; n = 10) had been previously vaccinated 12 mo prior and received a first annual booster at the beginning of this study and a second annual booster 12 mo later. The multivalent clostridial vaccine induced a high antibody response that peaked after each injection and then slowly decreased with time. In group 1, a booster vaccine was required to obtain an initial high humoral response. The annual booster injection induced a strong, rapid, and consistent anamnestic response in both groups. The serologic responses persisted significantly over the baseline value for 9-12 mo in group 1, but more than 12 mo in group 2. It is unknown whether the measured humoral immune responses would have been protective as no challenge studies were performed. Further investigation is needed to determine the protective antibody titers to challenge and how long this immunity might persist after vaccination.

  12. The Myelin and Lymphocyte Protein MAL Is Required for Binding and Activity of Clostridium perfringens ε-Toxin.

    PubMed

    Rumah, Kareem Rashid; Ma, Yinghua; Linden, Jennifer R; Oo, Myat Lin; Anrather, Josef; Schaeren-Wiemers, Nicole; Alonso, Miguel A; Fischetti, Vincent A; McClain, Mark S; Vartanian, Timothy

    2015-05-01

    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.

  13. Evidence for coupling of Clostridium perfringens alpha-toxin-induced hemolysis to stimulated phosphatidic acid formation in rabbit erythrocytes.

    PubMed Central

    Sakurai, J; Ochi, S; Tanaka, H

    1993-01-01

    When rabbit erythrocytes were exposed to low concentrations of Clostridium perfringens alpha-toxin, hot-cold hemolysis was observed. The toxin induced production of phosphatidic acid (PA) in a dose-dependent manner when incubated with erythrocytes at 37 degrees C. When erythrocyte membranes were incubated with the toxin and [gamma-32P]ATP in the presence or absence of ethanol, [32P]PA formation was maximal within 30 s, then sharply decreased, and began again after 5 min of incubation. Ethanol had no effect on the early appearance (at approximately 5 min) of PA formation induced by the toxin but significantly inhibited formation of PA over 10 min of incubation. Treatment of erythrocyte membranes with alpha-toxin resulted in the biphasic formation of 1,2-diacylglycerol and PA as well as an increase of inositol-1,4,5-trisphosphate (IP3) and decrease of phosphatidylinositol-4,5-bisphosphate (PIP2) within 30 s. Neomycin inhibited the toxin-induced increase in turbidity of egg yolk suspensions but did not inhibit the toxin-induced hemolysis of intact erythrocytes. On the other hand, neomycin inhibited the toxin-induced hemolysis of saponin-treated erythrocytes. In addition, neomycin inhibited PA formation induced by the toxin in erythrocyte membranes. IP3 was released by incubation of PIP2 with erythrocyte membranes but not by incubation of PIP2 with the toxin. The toxin stimulated the membrane-induced release of IP3 from PIP2. These data suggest that the toxin-induced hemolysis is dependent on the action of phospholipase C in erythrocyte membranes. PMID:8395469

  14. Diverse modes of galacto-specific carbohydrate recognition by a family 31 glycoside hydrolase from Clostridium perfringens

    PubMed Central

    Grondin, Julie M.; Duan, Da; Kirlin, Alyssa C.; Abe, Kento T.; Chitayat, Seth; Spencer, Holly L.; Spencer, Craig; Campigotto, Alisha; Houliston, Scott; Arrowsmith, Cheryl H.; Allingham, John S.; Boraston, Alisdair B.; Smith, Steven P.

    2017-01-01

    Clostridium perfringens is a commensal member of the human gut microbiome and an opportunistic pathogen whose genome encodes a suite of putative large, multi-modular carbohydrate-active enzymes that appears to play a role in the interaction of the bacterium with mucin-based carbohydrates. Among the most complex of these is an enzyme that contains a presumed catalytic module belonging to glycoside hydrolase family 31 (GH31). This large enzyme, which based on its possession of a GH31 module is a predicted α-glucosidase, contains a variety of non-catalytic ancillary modules, including three CBM32 modules that to date have not been characterized. NMR-based experiments demonstrated a preference of each module for galacto-configured sugars, including the ability of all three CBM32s to recognize the common mucin monosaccharide GalNAc. X-ray crystal structures of the CpGH31 CBM32s, both in apo form and bound to GalNAc, revealed the finely-tuned molecular strategies employed by these sequentially variable CBM32s in coordinating a common ligand. The data highlight that sequence similarities to previously characterized CBMs alone are insufficient for identifying the molecular mechanism of ligand binding by individual CBMs. Furthermore, the overlapping ligand binding profiles of the three CBMs provide a fail-safe mechanism for the recognition of GalNAc among the dense eukaryotic carbohydrate networks of the colonic mucosa. These findings expand our understanding of ligand targeting by large, multi-modular carbohydrate-active enzymes, and offer unique insights into of the expanding ligand-binding preferences and binding site topologies observed in CBM32s. PMID:28158290

  15. In vivo antimicrobial potentials of garlic against Clostridium perfringens and its promotant effects on performance of broiler chickens.

    PubMed

    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.

  16. Removal and fate of Cryptosporidium parvum, Clostridium perfringens and small-sized centric diatoms (Stephanodiscus hantzschii) in slow sand filters.

    PubMed

    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.

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

    PubMed

    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.

  18. A Fatal Spontaneous Gas Gangrene due to Clostridium perfringens during Neutropenia of Allogeneic Stem Cell Transplantation: Case Report and Literature Review.

    PubMed

    Lee, Hae-Lim; Cho, Sung-Yeon; Lee, Dong-Gun; Ko, Yumi; Hyun, Ji In; Kim, Bo Kyoung; Seo, Jae Hyun; Lee, Jung Woo; Lee, Seok

    2014-09-01

    Most cases of gas gangrene caused by Clostridium species begin with trauma-related injuries but in rare cases, spontaneous gas gangrene (SGG) can occur when patients have conditions such as advanced malignancy, diabetes, or immunosuppression. Clostridium perfringens, a rare cause of SGG, exists as normal flora of skin and intestines of human. Adequate antibiotics with surgical debridement of infected tissue is the only curative therapeutic management. Mortality rate among adults is reported range of 67-100% and majority of deaths are occurred within 24 hours of onset. We experienced a case of SGG on the trunk, buttock and thigh in a neutropenic patient with acute lymphoblastic leukemia. His clinical course was rapid and fatal during pre-engraftment neutropenic period of allogeneic stem cell transplantation.

  19. Analysis of genetic similarities between Clostridium perfringens isolates isolated from patients with gas gangrene and from hospital environment conducted with the use of the PFGE method.

    PubMed

    Brzychczy-Włoch, Monika; Bulanda, Małgorzata

    2014-03-01

    The objective of the study was to perform a comparative analysis of genetic similarity, with the use of pulsed field gel electrophoresis (PFGE), of Clostridium perfringens isolates originating from patients with gas gangrene and from the hospital environment. The study encompassed two patients with a clinical and microbiological diagnosis of gas gangrene, who were hospitalized in one of the hospitals of the Małopolska province in the time period between 31st March 2012 and 18th May 2012. Clostridium perfringens isolates genotyping indicated that the isolates originating from the two studied patients did not display genetic similarity and represented two different PFGE types, which corresponded to two different clones (clone A and B). Whereas the strains isolated from the hospital environment were genetically identical with the strain coming from the second patient and represented one PFGE type, which corresponded to one clone (clone A). As a result of the study, it is possible to conclude that both patients developed endogenous infection. Even so, the examination of the hospital environment indicates the possibility of the appearance of exogenous infections. It prompts recommending and following the exact regulations of sanitary regime in the ward and the operating theater if a patient is diagnosed with gas gangrene.

  20. 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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  1. Development of an integrated model for heat transfer and dynamic growth of Clostridium perfringens during the cooling of cooked boneless ham.

    PubMed

    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

  2. Functional analysis of a bacitracin resistance determinant located on ICECp1, a novel Tn916-like element from a conjugative plasmid in Clostridium perfringens.

    PubMed

    Han, Xiaoyan; Du, Xiang-Dang; Southey, Luke; Bulach, Dieter M; Seemann, Torsten; Yan, Xu-Xia; Bannam, Trudi L; Rood, Julian I

    2015-11-01

    Bacitracins are mixtures of structurally related cyclic polypeptides with antibiotic properties. They act by interfering with the biosynthesis of the bacterial cell wall. In this study, we analyzed an avian necrotic enteritis strain of Clostridium perfringens that was resistant to bacitracin and produced NetB toxin. We identified a bacitracin resistance locus that resembled a bacitracin resistance determinant from Enterococcus faecalis. It contained the structural genes bcrABD and a putative regulatory gene, bcrR. Mutagenesis studies provided evidence that both bcrA and bcrB are essential for bacitracin resistance, and that evidence was supported by the results of experiments in which the introduction of both the bcrA and bcrB genes into a bacitracin-susceptible C. perfringens strain was required to confer bacitracin resistance. The wild-type strain was shown to contain at least three large, putatively conjugative plasmids, and the bcrRABD locus was localized to an 89.7-kb plasmid, pJIR4150. This plasmid was experimentally shown to be conjugative and was sequenced. The sequence revealed that it also carries a tpeL toxin gene and is related to the pCW3 family of conjugative antibiotic resistance and toxin plasmids from C. perfringens. The bcr genes were located on a genetic element, ICECp1, which is related to the Tn916 family of integrative conjugative elements (ICEs). ICECp1 appears to be the first Tn916-like element shown to confer bacitracin resistance. In summary, we identified in a toxin-producing C. perfringens strain a novel mobile bacitracin resistance element which was experimentally shown to be essential for bacitracin resistance and is carried by a putative ICE located on a conjugative plasmid.

  3. Differential outgrowth potential of Clostridium perfringens food-borne isolates with various cpe-genotypes in vacuum-packed ground beef during storage at 12°C.

    PubMed

    Xiao, Yinghua; Wagendorp, Arjen; Abee, Tjakko; Wells-Bennik, Marjon H J

    2015-02-02

    In the current study, the outgrowth of spores of 15 different food isolates of Clostridium perfringens was evaluated in vacuum-packed ground beef during storage at 12°C and 25°C. This included enterotoxic strains carrying the gene encoding the CPE enterotoxin on the chromosome (C-cpe), on a plasmid (P-cpe) and cpe-negative strains. The 15 strains were selected from a larger group of strains that were first evaluated for their ability to sporulate in modified Duncan-Strong sporulating medium. Sporulation ability varied greatly between strains but was not associated with a particular cpe genotype. In line with previous studies, the tested C-cpe strains produced spores with significantly higher heat resistance than the cpe-negative and P-cpe strains (both IS1151 and IS1470-like) with the exception of strain VWA009. Following inoculation of vacuum-packed cooked ground beef with spores, the heat-resistant C-cpe strains showed lower outgrowth potential in this model food stored at 12°C than the P-cpe and cpe-negative strains, while no significant differences were observed at 25°C. These results suggest that the latter strains may have a competitive advantage over C-cpe strains at reduced temperatures during storage of foods that support the growth of C. perfringens. While spores of P-cpe strains are readily inactivated by heat processing, post-processing contamination by food handlers who may carry P-cpe strains that have a better growth potential at lower temperatures must be avoided. The varying responses of C. perfringens spores to heat and the differences in outgrowth capacity at different temperatures are factors to be considered in strain selection for challenge tests, and for predictive modelling of C. perfringens.

  4. Haemorrhagic bowel syndrome in dairy cattle: possible role of Clostridium perfringens type A in the disease complex.

    PubMed

    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.

  5. Experimental induction of abdominal tympany, abomasitis, and abomasal ulceration by intraruminal inoculation of Clostridium perfringens type A in neonatal calves.

    PubMed

    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.

  6. Amplified fragment length polymorphism (AFLP) analysis of Clostridium novyi, C. perfringens and Bacillus cereus isolated from injecting drug users during 2000.

    PubMed

    McLauchlin, J; Salmon, J E; Ahmed, S; Brazier, J S; Brett, M M; George, R C; Hood, J

    2002-11-01

    As part of the follow-up investigations associated with an outbreak of severe illness and death among illegal injecting drug users during 2000, 43 cultures of Clostridium novyi type A, 40 C. perfringens type A and 6 isolates of Bacillus cereus were characterised by amplified fragment length polymorphism (AFLP) analysis. Among the 43 C. novyi isolates, 23 different AFLP profiles were detected. The same AFLP profile was detected in isolates from 18 drug users investigated during 2000 from Scotland, England, the Republic of Ireland and Norway and a wound from a patient in 2000 who was not identified as a drug user. Unique AFLP profiles were obtained from four drug users from England and the Republic of Ireland, 10 historical isolates from culture collections, an isolate from food (1989) and three isolates from wounds (1995, 1991, 1988). The 40 C. perfringens isolates were from 13 drug users, the contents of one syringe and two samples of heroin. Sixteen AFLP types of C. perfringens were distinguished and there was little evidence for commonality among the isolates. The AFLP types of C. perfringens from heroin differed and were unique. Six isolates of B. cereus were from four drug users and two samples of heroin. Four different AFLP patterns were distinguished. Three AFLP types were isolated from four drug users. B. cereus isolates from an aspirate and a heroin sample collected from the same drug user were identical, and were also indistinguishable from an isolate from a groin infection in a second drug user. The AFLP type of the isolate from a second and unrelated heroin sample was unique. The AFLP results showed no or very limited evidence for commonality between the different isolates of B. cereus and C. perfringens. In marked contrast, the C. novyi isolates from the majority of the drug users during 2000 were homogeneous, suggesting a common source or clonal selection of a C. novyi type, or both, which either had an adaptive advantage in spore germination, survival

  7. Behavior and Inactivation of Enterotoxin-Positive Clostridium perfringens in Pork Picadillo and Tamales Filled with Pork Picadillo under Different Cooking, Storage, and Reheating Conditions.

    PubMed

    Villarruel-López, A; Ruíz-Quezada, S L; Castro-Rosas, J; Gomez-Aldapa, C A; Olea-Rodríguez, M A; Nuño, K; Navarro-Hidalgo, V; Torres-Vitela, M R

    2016-05-01

    This study analyzed the behavior of Clostridium perfringens in individual ingredients and tamales containing different pathogen concentrations upon exposure to different temperatures and methods of cooking, storage, and reheating. In ground pork, C. perfringens cells were inactivated when exposed to 95°C for 30 min. Three lots of picadillo inoculated with 0, 3, and 5 log CFU/g C. perfringens cells, respectively, were exposed to different storage temperatures. At 20°C, cell counts increased 1 log in all lots, whereas at 8°C, counts decreased by 2 log. Four lots of tamales prepared with picadillo inoculated with 0, 2, 3, and 7 log CFU/g prior to the final cooking step exhibited no surviving cells (91°C for 90, 45, or 35 min). Four lots of tamales were inoculated after cooking with concentrations of 0, 0.6, 4, and 6 log CFU/g of the pathogen and then stored at different temperatures. In these preparations, after 24 h at 20°C, the count increased by 1.4, 1.7, and 1.8 log in the tamales inoculated with 0.6, 4, and 6 log inoculum, respectively. When they were stored at 8°C for 24 h, enumerations decreased to <1, 2.5, and 1.9 log in the tamales inoculated with 0.6, 4, and 6 log of C. perfringens cells, respectively. However, when the lots were exposed to 20°C and then 8°C, 0.8, 1.8, and 2.4 log changes were observed for the tamales inoculated with 0.6, 4, and 6 log, respectively. Microwaving, steaming, and frying to reheat tamales inoculated with 6 log CFU/g C. perfringens cells showed that the pathogen was inactivated after 2 min of exposure in the microwave and after 5 min of exposure to steam. In contrast, no inactivation was observed after 5 min of frying. The tamales inoculated with spores (7 log most probable number [MPN]/g) showed a decrease of 2 log after steaming or frying, and no survival was observed after microwaving. Tamales inoculated with spores (7 log MPN/g) after cooking were susceptible to microwaves, but 2.4 and 255 MPN/g remained after frying

  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.

    PubMed

    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. Comparative neuropathology of ovine enterotoxemia produced by Clostridium perfringens type D wild-type strain CN1020 and its genetically modified derivatives.

    PubMed

    Garcia, J P; Giannitti, F; Finnie, J W; Manavis, J; Beingesser, J; Adams, V; Rood, J I; Uzal, F A

    2015-05-01

    Clostridium perfringens type D causes enterotoxemia in sheep and goats. The disease is mediated by epsilon toxin (ETX), which affects the cerebrovascular endothelium, increasing vascular permeability and leading to cerebral edema. In the present study, we compared the distribution and severity of the cerebrovascular changes induced in lambs by C. perfringens type D strain CN1020, its isogenic etx null mutant, and the ETX-producing complemented mutant. We also applied histochemical and immunohistochemical markers to further characterize the brain lesions induced by ETX. Both ETX-producing strains induced extensive cerebrovascular damage that did not differ significantly between each other in nature, neuroanatomic distribution, or severity. By contrast, lambs inoculated with the etx mutant or sterile, nontoxic culture medium did not develop detectable brain lesions, confirming that the neuropathologic effects observed in these infections are dependent on ETX production. Lambs treated with the wild-type and complemented strains showed perivascular and mural vascular edema, as well as serum albumin extravasation, particularly severe in the cerebral white matter, midbrain, medulla oblongata, and cerebellum. Brains of animals inoculated with the ETX-producing strains showed decreased expression of glial fibrillary acidic protein and increased expression of aquaporin-4 in the end-feet processes of the astrocytes around blood vessels. Early axonal injury was demonstrated with anti-amyloid precursor protein immunohistochemistry. Perivascular accumulation of macrophages/microglia with intracytoplasmic albumin globules was also observed in these animals. This study demonstrates that ETX is responsible for the major cerebrovascular changes in C. perfringens type D-induced disease.

  10. Toxin-associated and other genes in Clostridium perfringens type A isolates from bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS).

    PubMed

    Schlegel, Benjamin J; Nowell, Victoria J; Parreira, Valeria R; Soltes, Glenn; Prescott, John F

    2012-10-01

    This study examined known or possible virulence-associated genes in type A Clostridium perfringens from cases of both bovine clostridial abomasitis (BCA) and jejunal hemorrhage syndrome (JHS) and compared these to isolates from calves that were healthy or had undifferentiated diarrheal illness. A real-time polymerase chain reaction (PCR) assay was used to genotype the 218 C. perfringens isolates. Isolates were sourced from healthy and diarrheic young and mature cattle (n = 191), from calves with confirmed or suspected BCA (n = 22), and from mature cattle with JHS (n = 5). Of 216 isolates (96%), 208 were positive for the cpa gene and 13% (29/218) were positive for atypical cpb2. Three of 8 (37.5%) confirmed BCA isolates, 2 of 13 (15.4%) suspected BCA isolates, and no JHS isolates tested positive for atypical cpb2. As all isolates were negative for cpb, cpb2, cpe, etx, netB, and tpeL, the results of the present study do not support a role for these genes in BCA or JHS. A subset of unique genes identified in 1 bovine clostridial abomasitis isolate (F262), for which a genome sequence is available, was searched for in 8 BCA isolates by PCR. None of the 10 genes was consistently present in all or even in a majority of BCA isolates. Many of these genes were also variably and inconsistently present in type A isolates from calves that did not have BCA. Although a virulence signature to aid in the diagnosis of BCA caused by C. perfringens type A was not identified, further work may discover a gene or group of genes that would constitute such a signature.

  11. Occurrence of microbial indicators and Clostridium perfringens in wastewater, water column samples, sediments, drinking water, and Weddell seal feces collected at McMurdo Station, Antarctica

    USGS Publications Warehouse

    Lisle, J.T.; Smith, J.J.; Edwards, D.D.; McFeters, G.A.

    2004-01-01

    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.

  12. Crystallization and preliminary X-ray diffraction studies of θ-toxin (perfringolysin O), a pore-forming cytolysin of Clostridium perfringens

    NASA Astrophysics Data System (ADS)

    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.

  13. Effect of antibiotic growth promoters and anticoccidials on growth of Clostridium perfringens in the caeca and on performance of broiler chickens.

    PubMed

    Elwinger, K; Berndtson, E; Engström, B; Fossum, O; Waldenstedt, L

    1998-01-01

    The effects of the growth promoters avoparcin and avilamycin and the ionophore anticoccidials maduramicin, narasin and monensin on the growth of Clostridium perfringens (Cp) in the caeca and on performance of broiler chickens were tested in 2 experiments. The supplements were fed as single feed additives or in some combinations. No clinical signs or lesions caused by coccidia were observed in any of the studies. All supplements had an antibacterial effect on Cp and improved growth rate significantly. Carcass yield of birds fed growth promoters avilamycin or avoparcin was significantly higher compared with birds fed anticoccidials. These data indicate that, what concerns bird performance, during good hygienic conditions supplementation with antibiotic growth promoters may not be necessary when the diet is supplemented with an anticoccidial with antibacterial effects.

  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.

    PubMed

    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. Viability of Clostridium perfringens, Escherichia coli, and Listeria monocytogenes surviving mild heat or aqueous ozone treatment on beef followed by heat, alkali, or salt stress.

    PubMed

    Novak, John S; Yuan, James T C

    2003-03-01

    The threat of pathogen survival following ozone treatment of meat necessitates careful evaluation of the microorganisms surviving under such circumstances. The objective of this study was to determine whether sublethal aqueous ozone treatment (3 ppm of O3 for 5 min) of microorganisms on beef surfaces would result in increased or decreased survival with respect to subsequent heat, alkali, or NaCl stress. A mild heat treatment (55 degrees C for 30 min) was used for comparison. Reductions in three-strain cocktails of Clostridium perfringens, Escherichia coli O157:H7, and Listeria monocytogenes on beef following the heat treatment were 0.14, 0.77, and 1.47 log10 CFU/g, respectively, whereas reductions following ozone treatment were 1.28, 0.85, and 1.09 log10 CFU/g, respectively. C. perfringens cells exhibited elevated heat resistance at 60 degrees C (D60 [time at 60 degrees C required to reduce the viable cell population by 1 log10 units or 90%] = 17.76 min) following heat treatment of beef (55 degrees C for 30 min) but exhibited reduced viability at 60 degrees C following ozone treatment (D60 = 7.64 min) compared with the viability of untreated control cells (D60 = 13.84 min). The D60-values for L. monocytogenes and E. coli O157:H7 following heat and ozone exposures were not significantly different (P > 0.05). C. perfringens cells that survived ozone treatment did not exhibit increased resistance to pH (pH 6 to 12) relative to non-ozone-treated cells when grown at 37 degrees C for 24 h. The heat treatment also resulted in decreased numbers of surviving cells above and below neutral pH values for both E. coli O157:H7 and L. monocytogenes relative to those of non-heat-treated cells grown at 37 degrees C for 24 h. There were significant differences (P < 0.05) in C. perfringens reductions with increasing NaCl concentrations. The effects of NaCl were less apparent for E. coli and L. monocytogenes survivors. It is concluded that pathogens surviving ozone treatment of beef

  16. 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.

    PubMed

    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.

  17. Genome wide transcriptomic analysis identifies pathways affected by the infusion of Clostridium perfringens culture supernatant in the duodenum of broilers in situ.

    PubMed

    Athanasiadou, S; Russell, K M; Kaiser, P; Kanellos, T; Burgess, S T G; Mitchell, M; Clutton, E; Naylor, S W; Low, C J; Hutchings, M R; Sparks, N

    2015-06-01

    Clostridium perfringens type A is the main etiological factor for necrotic enteritis, a multifactorial enteric disease that penalizes performance, health, and welfare of poultry. Lack of knowledge of host responses and disease pathogenesis is slowing down progress on developing therapies for disease control. A combined genomewide and targeted gene approach was used to investigate pathways and biological functions affected by the infusion of C. perfringens culture supernatant in the duodenum of broilers in 2 experiments. An in situ isolated loop of duodenum was prepared in anesthetized broilers of 3 wk of age (Exp. 1) and was infused either with crude C. perfringens culture supernatant (n = 7; treated), positive for necrotic enteritis B-like toxin (NetB) as determined by a cytotoxicity assay, or with a control preparation (n = 6; control). Birds were maintained alive for 1 h and then euthanized for tissue recovery. The use of the Affymetrix chicken genome array on RNA samples from loop tissue showed top biological functions affected by culture supernatant infusion included cell morphology, immune cell trafficking, and cell death; pathways affected included death receptor signaling, inflammatory response, and nuclear factor (NF)-κB signaling. In a second in situ study (Exp. 2), broilers were maintained alive for 4 h to monitor temporal expression patterns of targeted genes. Duodenal tissue was removed at 0.5, 1, 2, and 4 h post-infusion with culture supernatant (n = 9) or a control preparation (n = 5) for histology and gene expression analysis. Genes encoding proinflammatory cytokines, such as interferon γ (IFNγ), cell trafficking, such as neuroblastoma 1 (NBL1) and B cell CLL/Lymphoma 6 (BCL6), and cell death, such as Fas cell surface death receptor (FAS) and GTPase IMAP family member 8 (GIMAP8), were differentially expressed in the duodenum of treated and control broilers (P < 0.05). We have demonstrated that C. perfringens culture supernatant (NetB positive

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

    PubMed

    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

  19. Direct dynamic kinetic analysis and computer simulation of growth of Clostridium perfringens in cooked turkey during cooling

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This research applied a new one-step methodology to directly construct a tertiary model for describing the growth of C. perfringens in cooked turkey meat under dynamically cooling conditions. The kinetic parameters of the growth models were determined by numerical analysis and optimization using mu...

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

    PubMed Central

    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. PMID:25853427

  1. Contributions of NanI sialidase to Caco-2 cell adherence by Clostridium perfringens type A and C strains causing human intestinal disease.

    PubMed

    Li, Jihong; McClane, Bruce A

    2014-11-01

    Previous studies showed that Clostridium perfringens type D animal disease strain CN3718 uses NanI sialidase for adhering to enterocyte-like Caco-2 cells. The current study analyzed whether NanI is similarly important when type A and C human intestinal disease strains attach to Caco-2 cells. A PCR survey determined that the nanI gene was absent from typical type A food poisoning (FP) strains carrying a chromosomal enterotoxin (CPE) gene or the genetically related type C Darmbrand (Db) strains. However, the nanI gene was present in type A strains from healthy humans, type A strains causing CPE-associated antibiotic-associated diarrhea (AAD) or sporadic diarrhea (SD), and type C Pig-Bel strains. Consistent with NanI sialidase being the major C. perfringens sialidase when produced, FP and Db strains had little supernatant sialidase activity compared to other type A or C human intestinal strains. All type A and C human intestinal strains bound to Caco-2 cells, but NanI-producing strains had higher attachment levels. When produced, NanI can contribute to host cell attachment of human intestinal disease strains, since a nanI null mutant constructed in type A SD strain F4969 had lower Caco-2 cell adhesion than wild-type F4969 or a complemented strain. Further supporting a role for NanI in host cell attachment, sialidase inhibitors reduced F4969 adhesion to Caco-2 cells. Collectively, these results suggest that NanI may contribute to the intestinal attachment and colonization needed for the chronic diarrhea of CPE-associated AAD and SD, but this sialidase appears to be dispensable for the acute pathogenesis of type A FP or type C enteritis necroticans.

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

    PubMed

    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.

  3. Ruminococcin C, a new anti-Clostridium perfringens bacteriocin produced in the gut by the commensal bacterium Ruminococcus gnavus E1.

    PubMed

    Crost, E H; Ajandouz, E H; Villard, C; Geraert, P A; Puigserver, A; Fons, M

    2011-09-01

    When colonizing the digestive tract of mono-associated rats, Ruminococcus gnavus E1 - a bacterium isolated from human faeces - produced a trypsin-dependent anti-Clostridium perfringens substance collectively named Ruminococcin C (RumC). RumC was isolated from the caecal contents of E1-monocontaminated rats and found to consist of two antimicrobial fractions: a single peptide (RumCsp) of 4235 Da, and a mixture of two other peptides (RumCdp) with distinct molecular masses of 4324 Da and 4456 Da. Both RumCsp and RumCdp were as effective as metronidazole in combating C. perfringens and their activity spectra against different pathogens were established. Even if devoid of synergistic activity, the combination of RumCsp and RumCdp was observed to be much more resistant to acidic pH and high temperature than each fraction tested individually. N-terminal sequence analysis showed that the primary structures of these three peptides shared a high degree of homology, but were clearly distinct from previously reported amino acid sequences. Amino acid composition of the three RumC peptides did not highlight the presence of any Lanthionine residue. However, Edman degradation could not run beyond the 11th amino acid residue. Five genes encoding putative pre-RumC-like peptides were identified in the genome of strain E1, confirming that RumC was a bacteriocin. This is the first time that a bacteriocin produced in vivo by a human commensal bacterium was purified and characterized.

  4. Both epsilon-toxin and beta-toxin are important for the lethal properties of Clostridium perfringens type B isolates in the mouse intravenous injection model.

    PubMed

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

    2007-03-01

    Clostridium perfringens is capable of producing up to 15 toxins, including alpha-toxin (CPA), beta-toxin (CPB), epsilon-toxin (ETX), enterotoxin, beta2-toxin (CPB2), and perfringolysin O. Type B isolates, which must produce CPA, CPB, and ETX, are associated with animal illnesses characterized by sudden death or acute neurological signs, with or without intestinal damage. Type B pathogenesis in ruminants is poorly understood, with some animals showing lesions and clinical signs similar to those caused by either type C or type D infections. It is unknown whether host or environmental conditions are dominant for determining the outcome of type B disease or if disease outcomes are determined by variable characteristics of type B isolates. To help clarify this issue, 19 type B isolates were evaluated for toxin production during late-log-phase growth via quantitative Western blotting and by biological activity assays. Most type B isolates produced CPB levels similar to those produced by type C isolates in vitro and have the potential to produce genotype C-like disease. The lethality of type B isolate supernatants administered intravenously to mice was evaluated with or without prior trypsin treatment, and monoclonal antibody neutralization studies also were performed. Correlation analyses comparing toxin levels in type B supernatants versus lethality and neutralization studies both found that the main contributor to lethality without pretreatment with trypsin was CPB, whereas neutralization studies indicated that CPB and ETX were both important after trypsin pretreatment. At least part of the CPB produced by type B isolates remained active after trypsin treatment. However, the overall lethalities of most supernatants were lower after trypsin pretreatment. Also, there was a significant association between ETX, CPB2, and CPA production in vitro among type B isolates. However, our results suggest that both CPB and ETX are likely the most important contributors to the

  5. Immunization with recombinant bivalent chimera r-Cpae confers protection against alpha toxin and enterotoxin of Clostridium perfringens type A in murine model.

    PubMed

    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.

  6. NanI Sialidase, CcpA, and CodY Work Together To Regulate Epsilon Toxin Production by Clostridium perfringens Type D Strain CN3718

    PubMed Central

    Li, Jihong; Freedman, John C.

    2015-01-01

    ABSTRACT Clostridium perfringens type D strains are usually associated with diseases of livestock, and their virulence requires the production of epsilon toxin (ETX). We previously showed (J. Li, S. Sayeed, S. Robertson, J. Chen, and B. A. McClane, PLoS Pathog 7:e1002429, 2011, http://dx.doi.org/10.1371/journal.ppat.1002429) that BMC202, a nanI null mutant of type D strain CN3718, produces less ETX than wild-type CN3718 does. The current study proved that the lower ETX production by strain BMC202 is due to nanI gene disruption, since both genetic and physical (NanI or sialic acid) complementation increased ETX production by BMC202. Furthermore, a sialidase inhibitor that interfered with NanI activity also reduced ETX production by wild-type CN3718. The NanI effect on ETX production was shown to involve reductions in codY and ccpA gene transcription levels in BMC202 versus wild-type CN3718. Similar to CodY, CcpA was found to positively control ETX production. A double codY ccpA null mutant produced even less ETX than a codY or ccpA single null mutant. CcpA bound directly to sequences upstream of the etx or codY start codon, and bioinformatics identified putative CcpA-binding cre sites immediately upstream of both the codY and etx start codons, suggesting possible direct CcpA regulatory effects. A ccpA mutation also decreased codY transcription, suggesting that CcpA effects on ETX production can be both direct and indirect, including effects on codY transcription. Collectively, these results suggest that NanI, CcpA, and CodY work together to regulate ETX production, with NanI-generated sialic acid from the intestines possibly signaling type D strains to upregulate their ETX production and induce disease. IMPORTANCE Clostridium perfringens NanI was previously shown to increase ETX binding to, and cytotoxicity for, MDCK host cells. The current study demonstrates that NanI also regulates ETX production via increased transcription of genes encoding the CodY and Ccp

  7. Clostridium perfringens enterotoxin fragment removes specific claudins from tight junction strands: Evidence for direct involvement of claudins in tight junction barrier.

    PubMed

    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.

  8. A low-toxic site-directed mutant of Clostridium perfringens ε-toxin as a potential candidate vaccine against enterotoxemia

    PubMed Central

    Li, Qing; Xin, Wenwen; Gao, Shan; Kang, Lin; Wang, Jinglin

    2013-01-01

    Clostridium perfringens epsilon toxin (ETX), one of the most potent toxins known, is a potential biological weapon; therefore, the development of an effective vaccine is important for preventing intoxication or disease by ETX. In this study, genetically detoxified epsilon toxin mutants were developed as candidate vaccines. We used site-directed mutagenesis to mutate the essential amino acid residues (His106, Ser111 and Phe199). Six site-directed mutants of ETX (mETXH106P, mETXS111H, mETXS111Y, mETXF199H, mETXF199E, mETXS111YF199E) were generated and then expressed in Escherichia coli. Both mETXF199E and mETXH106P with low or non-cytotoxicity that retained their immunogenicity were selected to immunize mice 3 times, and the mouse survival data were recorded after challenging with recombinant wild-type ETX. mETXF199E induces the same protection as mETXH106P, which was reported previously as a promising toxin mutant for vaccine, and both of them could protect immunized mice against a 100× LD50 dose of active wild-type recombinant ETX. This work showed that mETXF199E is another promising candidate vaccine against enterotoxemia and other diseases caused by ETX. PMID:23835363

  9. Prevention and treatment of Clostridium perfringens epsilon toxin intoxication in mice with a neutralizing monoclonal antibody (c4D7) produced in Nicotiana benthamiana

    PubMed Central

    Garcia, J.P.; Beingesser, J.; Bohorov, O.; Bohorova, N.; Goodman, C.; Kim, D.; Pauly, M.; Velasco, J.; Whaley, K.; Zeitlin, L.; Roy, C.J.; Uzal, F.A.

    2014-01-01

    Epsilon toxin (ETX), produced by Clostridium perfringens types B and D, is among the most lethal toxins known. ETX is a potential bioterrorism threat that was listed as a Category B agent by the U.S. Centers for Disease Control until 2012 and it still remains a toxin of interest for several government agencies. We produced a monoclonal antibody (MAb) against ETX (ETX MAb c4D7) in Nicotiana benthamiana and characterized its preventive and therapeutic efficacy in mice. The ETX preparation used was highly lethal for mice (LD50 =1.6 μg/kg) and resulted in a mean time from inoculation to death of 18 and 180 minutes when administered intravenously or intraperitoneally, respectively. High lethal challenge resulted in dramatic increases of a variety of pro-inflammatory cytokines in serum, while lower, but still lethal doses, did not elicit such responses. ETX MAb c4D7 was highly effective prophylactically (ED50 = 0.3 mg/kg; ED100 = 0.8 mg/kg) and also provided protection when delivered 15-30 minutes post-ETX intoxication. These data suggest that ETX MAb c4D7 may have use as a pre- and post-exposure treatment for ETX intoxication. PMID:24950050

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

    PubMed Central

    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 bovine enterotoxemia. The aim of this study was to evaluate whether strains originating from bovine enterotoxemia cases were superior in in vitro production of virulence factors (alpha toxin, perfringolysin O, mucinase, collagenase) that are potentially involved in enterotoxemia. To approach this, a collection of strains originating from enterotoxemia cases was compared to bovine strains isolated from healthy animals and to strains isolated from other animal species. Results Strains originating from bovine enterotoxemia cases produced variable levels of alpha toxin and perfringolysin O that were not significantly different from levels produced by strains isolated from healthy calves and other animal species. All tested strains exhibited similar mucinolytic activity independent of the isolation source. A high variability in collagenase activity between strains could be observed, and no higher collagenase levels were produced in vitro by strains isolated from enterotoxemia cases. Conclusions Bovine enterotoxemia strains do not produce higher levels of alpha toxin, perfringolysin O, mucinase and collagenase, as compared to strains derived from healthy calves and other animal species in vitro. PMID:24479821

  11. Crystal structure of the catalytic domain of Clostridium perfringens neuraminidase in complex with a non-carbohydrate-based inhibitor, 2-(cyclohexylamino)ethanesulfonic acid.

    PubMed

    Lee, Youngjin; Youn, Hyung-Seop; Lee, Jung-Gyu; An, Jun Yop; Park, Kyoung Ryoung; Kang, Jung Youn; Ryu, Young Bae; Jin, Mi Sun; Park, Ki Hun; Eom, Soo Hyun

    2017-03-16

    Anti-bacterial and anti-viral neuraminidase agents inhibit neuraminidase activity catalyzing the hydrolysis of terminal N-acetylneuraminic acid (Neu5Ac) from glycoconjugates and help to prevent the host pathogenesis that lead to fatal infectious diseases including influenza, bacteremia, sepsis, and cholera. Emerging antibiotic and drug resistances to commonly used anti-neuraminidase agents such as oseltamivir (Tamiflu) and zanamivir (Relenza) have highlighted the need to develop new anti-neuraminidase drugs. We obtained a serendipitous complex crystal of the catalytic domain of Clostridium perfringens neuraminidase (CpNanICD) with 2-(cyclohexylamino)ethanesulfonic acid (CHES) as a buffer. Here, we report the crystal structure of CpNanICD in complex with CHES at 1.24 Å resolution. Amphipathic CHES binds to the catalytic site of CpNanICD similar to the substrate (Neu5Ac) binding site. The 2-aminoethanesulfonic acid moiety and cyclohexyl groups of CHES interact with the cluster of three arginine residues and with the hydrophobic pocket of the CpNanICD catalytic site. In addition, a structural comparison with other bacterial and human neuraminidases suggests that CHES could serve as a scaffold for the development of new anti-neuraminidase agents targeting CpNanI.

  12. Clostridium perfringens enterotoxin elicits rapid and specific cytolysis of breast carcinoma cells mediated through tight junction proteins claudin 3 and 4.

    PubMed

    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.

  13. Clostridium perfringens Enterotoxin Elicits Rapid and Specific Cytolysis of Breast Carcinoma Cells Mediated through Tight Junction Proteins Claudin 3 and 4

    PubMed Central

    Kominsky, Scott L.; Vali, Mustafa; Korz, Dorian; Gabig, Theodore G.; Weitzman, Sigmund A.; Argani, Pedram; Sukumar, Saraswati

    2004-01-01

    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. PMID:15111309

  14. Variable protection against experimental broiler necrotic enteritis after immunization with the C-terminal fragment of Clostridium perfringens alpha-toxin and a non-toxic NetB variant

    PubMed Central

    Fernandes da Costa, Sérgio P.; Mot, Dorien; Geeraerts, Sofie; Bokori-Brown, Monika; Van Immerseel, Filip; Titball, Richard W.

    2016-01-01

    ABSTRACT Necrotic enteritis toxin B (NetB) is a pore-forming toxin produced by Clostridium perfringens and has been shown to play a key role in avian necrotic enteritis, a disease causing significant costs to the poultry production industry worldwide. The aim of this work was to determine whether immunization with a non-toxic variant of NetB (NetB W262A) and the C-terminal fragment of C. perfringens alpha-toxin (CPA247–370) would provide protection against experimental necrotic enteritis. Immunized birds with either antigen or a combination of antigens developed serum antibody levels against NetB and CPA. When CPA247–370 and NetB W262A were used in combination as immunogens, an increased protection was observed after oral challenge by individual dosing, but not after in-feed-challenge. PMID:26743457

  15. 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

    PubMed Central

    Latorre, Juan D.; Hernandez-Velasco, Xochitl; Kuttappan, Vivek A.; Wolfenden, Ross E.; Vicente, Jose L.; Wolfenden, Amanda D.; Bielke, Lisa R.; Prado-Rebolledo, Omar F.; Morales, Eduardo; Hargis, Billy M.; Tellez, Guillermo

    2015-01-01

    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 Bacillus amyloliquefaciens and one of the strains as Bacillus subtilis. The DFM was included at a concentration of 108 spores/g of feed in five 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 ~2log10, suggesting a full-life cycle development. Further studies to evaluate in vivo necrotic enteritis effects are in

  16. Discrimination Efficacy of Fecal Pollution Detection in Different Aquatic Habitats of a High-Altitude Tropical Country, Using Presumptive Coliforms, Escherichia coli, and Clostridium perfringens Spores

    PubMed Central

    Byamukama, Denis; Mach, Robert L.; Kansiime, Frank; Manafi, Mohamad; Farnleitner, Andreas H.

    2005-01-01

    The performance of rapid and practicable techniques that presumptively identify total coliforms (TC), fecal coliforms (FC), Escherichia coli, and Clostridium perfringens spores (CP) by testing them on a pollution gradient in differing aquatic habitats in a high-altitude tropical country was evaluated during a 12-month period. Site selection was based on high and low anthropogenic influence criteria of paired sites including six spring, six stream, and four lakeshore sites spread over central and eastern parts of Uganda. Unlike the chemophysical water quality, which was water source type dependent (i.e., spring, lake, or stream), fecal indicators were associated with the anthropogenic influence status of the respective sites. A total of 79% of the total variability, including all the determined four bacteriological and five chemophysical parameters, could be assigned to either a pollution, a habitat, or a metabolic activity component by principal-component analysis. Bacteriological indicators revealed significant correlations to the pollution component, reflecting that anthropogenic contamination gradients were followed. Discrimination sensitivity analysis revealed high ability of E. coli to differentiate between high and low levels of anthropogenic influence. CP also showed a reasonable level of discrimination, although FC and TC were found to have worse discrimination efficacy. Nonpoint influence by soil erosion could not be detected during the study period by correlation analysis, although a theoretical contamination potential existed, as investigated soils in the immediate surroundings often contained relevant concentrations of fecal indicators. The outcome of this study indicates that rapid techniques for presumptive E. coli and CP determination may be reliable for fecal pollution monitoring in high-altitude tropical developing countries such as those of Eastern Africa. PMID:15640171

  17. A synthetic peptide corresponding to the extracellular loop 2 region of claudin-4 protects against Clostridium perfringens enterotoxin in vitro and in vivo.

    PubMed

    Shrestha, Archana; Robertson, Susan L; Garcia, Jorge; Beingasser, Juliann; McClane, Bruce A; Uzal, Francisco A

    2014-11-01

    Clostridium perfringens enterotoxin (CPE) action starts when the toxin binds to claudin receptors. Claudins contain two extracellular loop domains, with the second loop (ECL-2) being slightly smaller than the first. CPE has been shown to bind to ECL-2 in receptor claudins. We recently demonstrated that Caco-2 cells (a naturally CPE-sensitive enterocyte-like cell line) can be protected from CPE-induced cytotoxicity by preincubating the enterotoxin with soluble full-length recombinant claudin-4 (rclaudin-4), which is a CPE receptor, but not with recombinant nonreceptor claudins, such as rclaudin-1. The current study evaluated whether a synthetic peptide corresponding to the claudin-4 ECL-2 sequence can similarly inhibit CPE action in vitro and in vivo. Significant protection of Caco-2 cells was also observed using either rclaudin-4 or the claudin-4 ECL-2 peptide in both a preincubation assay and a coincubation assay. This inhibitory effect was specific, since rclaudin-1 and a synthetic peptide based on the claudin-1 ECL-2 offered no protection to Caco-2 cells. However, the claudin-4 ECL-2 peptide was unable to neutralize cytotoxicity if CPE had already bound to Caco-2 cells. When the study was repeated in vivo using a rabbit small intestinal loop assay, preincubation or coincubation of CPE with the claudin-4 ECL-2 peptide significantly and specifically inhibited the development of CPE-induced luminal fluid accumulation and histologic lesions in rabbit small intestinal loops. No similar in vivo protection from CPE was afforded by the claudin-1 ECL-2 peptide. These results suggest that claudin-4 ECL-2 peptides should be further investigated for their potential therapeutic application against CPE-associated disease.

  18. In Silico, In Vitro and In Vivo Analysis of Binding Affinity between N and C-Domains of Clostridium perfringens Alpha Toxin

    PubMed Central

    Uppalapati, Siva Ramakrishna; Kingston, Joseph Jeyabalaji; Qureshi, Insaf Ahmed; Murali, Harishchandra Sripathy; Batra, Harsh Vardhan

    2013-01-01

    Clostridium perfringens alpha toxin/phospholipase C (CP-PLC) is one of the most potent bacterial toxins known to cause soft tissue infections like gas gangrene in humans and animals. It is the first bacterial toxin demonstrated to be an enzyme with phospholipase, sphingomyelinase and lecithinase activities. The toxin is comprised of an enzymatic N-domain and a binding C-domain interconnected by a flexible linker. The N-domain alone is non-toxic to mammalian cells, but incubation with C-domain restores the toxicity, the mechanism of which is still not elucidated. The objectives of the current study were to investigate the formation of a stable N and C-domain complex, to determine possible interactions between the two domains in silico and to characterize the in vitro and in vivo correlates of the interaction. To establish the existence of a stable N and C-domain hybrid, in vitro pull down assay and dot-Far Western blotting assays were employed, where it was clearly revealed that the two domains bound to each other to form an intermediate. Using bioinformatics tools like MetaPPISP, PatchDock and FireDock, we predicted that the two domains may interact with each other through electrostatic interactions between at least six pairs of amino acids. This N and C-domains interacted with each other in 1:1 ratio and the hybrid lysed mouse erythrocytes in a slower kinetics when compared with wild type native Cp-PLC. BALB/c mice when challenged with N and C-domain hybrid demonstrated severe myonecrosis at the site of injection while no death was observed. Our results provide further insight into better understanding the mechanism for the toxicity of Cp-PLC N and C-domain mixture. PMID:24349173

  19. Immunization with a novel Clostridium perfringens epsilon toxin mutant rETXY196E-C confers strong protection in mice

    PubMed Central

    Yao, Wenwu; Kang, Jingjing; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Xin, Wenwen; Wang, Jinglin

    2016-01-01

    Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETXY196E-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:105 in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 μg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETXY196E-C is a potential vaccine candidate for future applied researches. PMID:27048879

  20. Immunization with a novel Clostridium perfringens epsilon toxin mutant rETX(Y196E)-C confers strong protection in mice.

    PubMed

    Yao, Wenwu; Kang, Jingjing; Kang, Lin; Gao, Shan; Yang, Hao; Ji, Bin; Li, Ping; Liu, Jing; Xin, Wenwen; Wang, Jinglin

    2016-04-06

    Epsilon toxin (ETX) is produced by toxinotypes B and D of Clostridium perfringens. It can induce lethal enterotoxemia in domestic animals, mainly in sheep, goats and cattle, causing serious economic losses to global animal husbandry. In this study, a novel and stable epsilon toxin mutant rETX(Y196E)-C, obtained by substituting the 196th tyrosine (Y196) with glutamic acid (E) and introducing of 23 amino acids long C-terminal peptide, was determined as a promising recombinant vaccine candidate against enterotoxemia. After the third vaccination, the antibody titers against recombinant wild type (rETX) could reach 1:10(5) in each immunized group, and the mice were completely protected from 100 × LD50 (50% lethal dose) of rETX challenge. The mice in 15 μg subcutaneously immunized group fully survived at the dose of 500 × LD50 of rETX challenge and 80% of mice survived at 180 μg (1000 × LD50) of rETX administration. In vitro, immune sera from 15 μg subcutaneously immunized group could completely protect MDCK cells from 16 × CT50 (50% lethal dose of cells) of rETX challenge and protect against 10 × LD50 dose (1.8 μg) of rETX challenge in mice. These data suggest that recombinant protein rETX(Y196E)-C is a potential vaccine candidate for future applied researches.

  1. Comparison of four techniques for the detection of Clostridium perfringens type D epsilon toxin in intestinal contents and other body fluids of sheep and goats.

    PubMed

    Uzal, F A; Kelly, W R; Thomas, R; Hornitzky, M; Galea, F

    2003-03-01

    Polyclonal capture enzyme-linked immunosorbent assay (PC-ELISA), monoclonal capture ELISA (MC-ELISA), mouse neutralization test (MNT), and counterimmunoelectrophoresis (CIEP), were compared for their ability to detect epsilon toxin in intestinal contents and body fluids of sheep and goats. When used to evaluate intestinal contents of sheep artificially spiked with epsilon prototoxin, PC-ELISA detected 0.075 mouse lethal dose (MLD)50/ml, whereas the MNT, MC-ELISA, and CIEP detected 6, 25, and 50 MLD50/ml, respectively. Amounts of epsilon toxin detected by PC-ELISA, MC-ELISA, MNT, and CIEP in sheep pericardial fluid artificially spiked with epsilon prototoxin were 0.075, 0.75, 6, and 200 MLD50/ml, respectively. For assaying epsilon toxin in aqueous humor, PC-ELISA and MC-ELISA detected 0.075 MLD50/ml, whereas CIEP detected 200 MLD50/ml (MNT was not evaluated). When 51 samples of intestinal contents of sheep and goats (32 positive and 19 negative to MNT) were analyzed by the other 3 techniques, the relative sensitivity of PC-ELISA, MC-ELISA, and CIEP was 93.75, 84.37, and 37.50%, respectively. The specificity of PC-ELISA, MC-ELISA, and CIEP was 31.57, 57.89, and 84.21%, respectively. The absolute sensitivity of PC-ELISA, MC-ELISA, CIEP, and MNT was 90.90, 69.69, 15.15, and 54.54%. The absolute specificity of the 4 techniques was 100%. These results show that there is a marked inconsistency among techniques routinely used to detect Clostridium perfringens epsilon toxin. Until more consistent results are achieved, the diagnosis of enterotoxemia should not only be based solely on epsilon toxin detection, but also on clinical and pathological data.

  2. Discrimination efficacy of fecal pollution detection in different aquatic habitats of a high-altitude tropical country, using presumptive coliforms, Escherichia coli, and Clostridium perfringens spores.

    PubMed

    Byamukama, Denis; Mach, Robert L; Kansiime, Frank; Manafi, Mohamad; Farnleitner, Andreas H

    2005-01-01

    The performance of rapid and practicable techniques that presumptively identify total coliforms (TC), fecal coliforms (FC), Escherichia coli, and Clostridium perfringens spores (CP) by testing them on a pollution gradient in differing aquatic habitats in a high-altitude tropical country was evaluated during a 12-month period. Site selection was based on high and low anthropogenic influence criteria of paired sites including six spring, six stream, and four lakeshore sites spread over central and eastern parts of Uganda. Unlike the chemophysical water quality, which was water source type dependent (i.e., spring, lake, or stream), fecal indicators were associated with the anthropogenic influence status of the respective sites. A total of 79% of the total variability, including all the determined four bacteriological and five chemophysical parameters, could be assigned to either a pollution, a habitat, or a metabolic activity component by principal-component analysis. Bacteriological indicators revealed significant correlations to the pollution component, reflecting that anthropogenic contamination gradients were followed. Discrimination sensitivity analysis revealed high ability of E. coli to differentiate between high and low levels of anthropogenic influence. CP also showed a reasonable level of discrimination, although FC and TC were found to have worse discrimination efficacy. Nonpoint influence by soil erosion could not be detected during the study period by correlation analysis, although a theoretical contamination potential existed, as investigated soils in the immediate surroundings often contained relevant concentrations of fecal indicators. The outcome of this study indicates that rapid techniques for presumptive E. coli and CP determination may be reliable for fecal pollution monitoring in high-altitude tropical developing countries such as those of Eastern Africa.

  3. Gene expression profiling within the spleen of Clostridium perfringens-challenged Broilers fed antibiotic-medicated and non-medicated diets

    PubMed Central

    Sarson, Aimie J; Wang, Ying; Kang, Zhumei; Dowd, Scot E; Lu, Yang; Yu, Hai; Han, Yanming; Zhou, Huaijun; Gong, Joshua

    2009-01-01

    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. The significance in host

  4. 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.

    PubMed

    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.

  5. Epsilon-Toxin Production by Clostridium perfringens Type D Strain CN3718 Is Dependent upon the agr Operon but Not the VirS/VirR Two-Component Regulatory System

    PubMed Central

    Chen, Jianming; Rood, Julian I.; McClane, Bruce A.

    2011-01-01

    ABSTRACT Clostridium perfringens type B and D strains cause enterotoxemias and enteritis in livestock after proliferating in the intestines and producing epsilon-toxin (ETX), alpha-toxin (CPA), and, usually, perfringolysin O (PFO). Although ETX is one of the most potent bacterial toxins, the regulation of ETX production by type B or D strains remains poorly understood. The present work determined that the type D strain CN3718 upregulates production of ETX upon close contact with enterocyte-like Caco-2 cells. This host cell-induced upregulation of ETX expression was mediated at the transcriptional level. Using an isogenic agrB null mutant and complemented strain, the agr operon was shown to be required when CN3718 produces ETX in broth culture or, via a secreted signal consistent with a quorum-sensing (QS) effect, upregulates ETX production upon contact with host cells. These findings provide the first insights into the regulation of ETX production, as well as additional evidence that the Agr-like QS system functions as a global regulator of C. perfringens toxin production. Since it was proposed previously that the Agr-like QS system regulates C. perfringens gene expression via the VirS/VirR two-component regulatory system, an isogenic virR null mutant of CN3718 was constructed to evaluate the importance of VirS/VirR for CN3718 toxin production. This mutation affected production of CPA and PFO, but not ETX, by CN3718. These results provide the first indication that C. perfringens toxin expression regulation by the Agr-like quorum-sensing system may not always act via the VirS/VirR two-component system. PMID:22167225

  6. Predictive model for growth of Clostridium perfringens during cooling of cooked beef supplemented with NaCl, sodium nitrite and sodium pyrophosphate

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This paper presents a model for predicting relative growth of C. perfringens in ground beef products at different percentages of salt (0%, 1%, 2% and 3%) and nitrite (0 and 200 ppm). Included in the experiments were different levels of sodium pyrophosphate (SPP). The results of the experiments indic...

  7. Purification of Clostridium toxoids.

    PubMed

    Buchowicz, I; Hay, M; Schiller, B; Korbecki, M; Sochańska, R

    1977-01-01

    A two-step fractionation procedure was applied for purification and concentration of the individual Clostridium toxoids. The toxoids were precipitated with hydrochloric acid in the presence of sodium sextametaphosphate, then antigenic fractions were separated from inactive contaminants by Sephadex G-75 filtration. Specific activity of the preparations thus obtained, as determined by Mancini radial immunodiffusion, was 150--565 binding units per mg of protein nitrogen for Clostridium perfringens toxoid, 204--352 binding units for Clostridium oedematiens toxoid and 26.6 -- 51.2 binding units for Clostridium septicum toxoid.

  8. Application of quantitative microbial risk assessments for estimation of risk management metrics: Clostridium perfringens in ready-to-eat and partially cooked meat and poultry products as an example.

    PubMed

    Crouch, Edmund A; Labarre, David; Golden, Neal J; Kause, Janell R; Dearfield, Kerry L

    2009-10-01

    The U.S. Department of Agriculture, Food Safety and Inspection Service is exploring quantitative risk assessment methodologies to incorporate the use of the Codex Alimentarius' newly adopted risk management metrics (e.g., food safety objectives and performance objectives). It is suggested that use of these metrics would more closely tie the results of quantitative microbial risk assessments (QMRAs) to public health outcomes. By estimating the food safety objective (the maximum frequency and/or concentration of a hazard in a food at the time of consumption) and the performance objective (the maximum frequency and/or concentration of a hazard in a food at a specified step in the food chain before the time of consumption), risk managers will have a better understanding of the appropriate level of protection (ALOP) from microbial hazards for public health protection. We here demonstrate a general methodology that allows identification of an ALOP and evaluation of corresponding metrics at appropriate points in the food chain. It requires a two-dimensional probabilistic risk assessment, the example used being the Monte Carlo QMRA for Clostridium perfringens in ready-to eat and partially cooked meat and poultry products, with minor modifications to evaluate and abstract required measures. For demonstration purposes, the QMRA model was applied specifically to hot dogs produced and consumed in the United States. Evaluation of the cumulative uncertainty distribution for illness rate allows a specification of an ALOP that, with defined confidence, corresponds to current industry practices.

  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 Enterotoxin1

    PubMed Central

    Litkouhi, Babak; Kwong, Joseph; Lo, Chun-Min; Smedley, James G; McClane, Bruce A; Aponte, Margarita; Gao, Zhijian; Sarno, Jennifer L; Hinners, Jennifer; Welch, William R; Berkowitz, Ross S; Mok, Samuel C; Garner, Elizabeth I O

    2007-01-01

    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-4-expressing 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 Claudin-4 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. PMID:17460774

  10. Biofilms of Clostridium species.

    PubMed

    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.

  11. A Randomized Controlled Trial Assessing Infectious Disease Risks from Bathing in Fresh Recreational Waters in Relation to the Concentration of Escherichia coli, Intestinal Enterococci, Clostridium perfringens, and Somatic Coliphages

    PubMed Central

    Wiedenmann, Albrecht; Krüger, Petra; Dietz, Klaus; López-Pila, Juan M.; Szewzyk, Regine; Botzenhart, Konrad

    2006-01-01

    We performed epidemiologic studies at public freshwater bathing sites in Germany to provide a better scientific basis for the definition of recreational water quality standards. A total of 2,196 participants were recruited from the local population and randomized into bathers and non-bathers. Bathers were exposed for 10 min and had to immerse their head at least three times. Water samples for microbiological analysis were collected at 20-min intervals. Unbiased concentration–response effects with no-observed-adverse-effect levels (NOAELs) were demonstrated for three different definitions of gastroenteritis and four fecal indicator organisms. Relative risks for bathing in waters with levels above NOAELs compared with nonbathing ranged from 1.8 (95% CI, 1.2–2.6) to 4.6 (95% CI, 2.1–10.1), depending on the definition of gastroenteritis. The effect of swallowing water provided additional evidence for true dose–response relationships. Based on the NOAELs, the following guide values for water quality are suggested: 100 Escherichia coli, 25 intestinal enterococci, 10 somatic coliphages, or 10 Clostridium perfringens per 100 mL. Recreational water quality standards are intended to protect the health of those consumers who are not already immune or resistant to pathogens that may be associated with indicator organisms. In contrast to current World Health Organization recommendations, we concluded that standards should be based on rates of compliance with NOAELs rather than on attributable risks determined above NOAELs, because these risks depend mainly on the unpredictable susceptibility of the cohorts. Although in theory there is no threshold in real concentration–response relationships, we demonstrated that a NOAEL approach would be a more robust and practical solution to the complex problem of setting standards. PMID:16451859

  12. Acute Hemolysis with Renal Failure due to Clostridium Bacteremia in a Patient with AML

    PubMed Central

    Medrano-Juarez, R. M.; Sotello, D.; D'Cuhna, L.; Payne, J. D.

    2016-01-01

    We present a case of acute hemolytic anemia, renal failure, and Clostridium perfringens bacteremia in a patient with acute myelogenous leukemia. The high fatality of C. perfringens bacteremia requires that clinicians recognize and rapidly treat patients at risk for this infection. Although other hemolytic processes are in the differential diagnosis of these events, the presence of high fever, chills, and rapidly positive blood cultures may help narrow the diagnosis. Most cases of C. perfringens bacteremia have a concomitant coinfection, which makes broad spectrum empiric therapy essential. There is a high mortality rate of C. perfringens infections associated with leukemia. PMID:27774325

  13. ClosTron-mediated engineering of Clostridium.

    PubMed

    Kuehne, Sarah A; Heap, John T; Cooksley, Clare M; Cartman, Stephen T; Minton, Nigel P

    2011-01-01

    The genus Clostridium is a diverse assemblage of Gram positive, anaerobic, endospore-forming bacteria. Whilst certain species have achieved notoriety as important animal and human pathogens (e.g. Clostridium difficile, Clostridium botulinum, Clostridium tetani, and Clostridium perfringens), the vast majority of the genus are entirely benign, and are able to undertake all manner of useful biotransformations. Prominent amongst them are those species able to produce the biofuels, butanol and ethanol from biomass-derived residues, such as Clostridium acetobutylicum, Clostridium beijerinkii, Clostridium thermocellum, and Clostridium phytofermentans. The prominence of the genus in disease and biotechnology has led to the need for more effective means of genetic modification. The historical absence of methods based on conventional strategies for "knock-in" and "knock-out" in Clostridium has led to the adoption of recombination-independent procedures, typified by ClosTron technology. The ClosTron uses a retargeted group II intron and a retro-transposition-activated marker to selectively insert DNA into defined sites within the genome, to bring about gene inactivation and/or cargo DNA delivery. The procedure is extremely efficient, rapid, and requires minimal effort by the operator.

  14. Phylogenetic positions of Clostridium chauvoei and Clostridium septicum based on 16S rRNA gene sequences.

    PubMed

    Kuhnert, P; Capaul, S E; Nicolet, J; Frey, J

    1996-10-01

    The sequences of the 16S rRNA genes (rrs genes) of Clostridium chauvoei, the causative agent of blackleg in cattle, and the phenotypically related organism Clostridium septicum were determined. After amplification of 1,507-bp PCR fragments from the corresponding rrs genes, the sequences were determined in a single round of sequencing by using conserved region primers. A sequence similarity analysis of the sequences revealed the close phylogenetic relationship of C. chauvoei and C. septicum in Clostridium cluster I (M. D. Collins, P. A. Lawson, A. Willems, J. J. Cordoba, J. Fernandez-Garayzabal, P. Garcia, J. Cai, H. Hippe, and J. A. E. Farrow, Int. J. Syst. Bacteriol. 44:812-826, 1994), which includes Clostridium carnis, Clostridium perfringens, Clostridium botulinum, and Clostridium tetani. We found that 99.3% of the nucleotides in the genes of C. chauvoei and C. septicum are identical.

  15. Monitoring of anti-C. perfringens bacteriophage CpV1 persistence in gastrointestinal tracts of broilers.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A factor limiting promotion of poultry products to the world market is any contamination of birds with pathogens, including Clostridium perfringens. The latter is often accountable for significant economical losses in production of commercial birds because of a possibility of the development of necr...

  16. Isolation of Clostridium absonum and its cultural and biochemical properties.

    PubMed

    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.

  17. A case of Clostridium septicum spontaneous gas gangrene.

    PubMed

    Dylewski, Joe; Drummond, Robert; Rowen, John

    2007-03-01

    Severe skin and soft tissue infections (SSTIs) are often life-threatening emergencies that require a rapid diagnosis. Gas gangrene is one of the most fulminant types of SSTI and is usually caused by Clostridium perfringens' contamination of an open wound. Although gas gangrene is usually associated with fecally contaminated wounds, "spontaneous" cases occur and are most commonly caused by Clostridium (C.) septicum. We report a case of spontaneous gas gangrene caused by C. septicum that only became manifest while the patient was being monitored in the emergency department. We also review the diagnosis and treatment aspects of this entity.

  18. Quality assurance of C. perfringens epsilon toxoid vaccines--ELISA versus mouse neutralisation test.

    PubMed

    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

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

    DTIC Science & Technology

    1983-01-01

    occurred with, for example, urea/mercaptoethanol (UME) treatment of Bacillus megaterium (Vary, 1973). alkaline dithioerythritol/sodium dodecyl sulphate...cetylmuremides of Bacillus subtilis YT 25. Agr. Biol. Chem. 38 2357-65. Accepted 15 June 1979 91 Germination of C perfringns spores Journal qf General Microbiology...spore-lytic enzymes of Bacillus cereus have been isolated and extensively studied by Strange & Dark (1957). Gould et al. (1966), Warth (1972) and Brown et

  20. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... cause sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice... until injections of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall...

  1. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... cause sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice... until injections of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall...

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

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard... of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for...

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... cause sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice... until injections of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall...

  4. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2012 CFR

    2012-01-01

    ... sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard... of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for...

  5. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard... of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for...

  6. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2014 CFR

    2014-01-01

    ... cause sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice... until injections of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall...

  7. 9 CFR 113.454 - Clostridium Perfringens Type C Antitoxin.

    Code of Federal Regulations, 2013 CFR

    2013-01-01

    ... sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard... of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for...

  8. 9 CFR 113.455 - Clostridium Perfringens Type D Antitoxin.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... cause sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one-tenth unit of Standard Antitoxin and cause death in at least 80 percent of injected mice... until injections of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall...

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

    Code of Federal Regulations, 2011 CFR

    2011-01-01

    ... sickness or death in injected mice. (iii) L + dose. The smallest quantity of toxin which can be mixed with one unit of Standard Antitoxin and cause death in at least 80 percent of injected mice. (iv) Standard... of mice can be made. (vi) Five Swiss white mice, each weighing 16-20 grams, shall be used for...

  10. Clostridium and Bacillus Binary Enterotoxins: Bad for the Bowels, and Eukaryotic Being

    PubMed Central

    Stiles, Bradley G.; Pradhan, Kisha; Fleming, Jodie M.; Samy, Ramar Perumal; Barth, Holger; Popoff, Michel R.

    2014-01-01

    Some pathogenic spore-forming bacilli employ a binary protein mechanism for intoxicating the intestinal tracts of insects, animals, and humans. These Gram-positive bacteria and their toxins include Clostridium botulinum (C2 toxin), Clostridium difficile (C. difficile toxin or CDT), Clostridium perfringens (ι-toxin and binary enterotoxin, or BEC), Clostridium spiroforme (C. spiroforme toxin or CST), as well as Bacillus cereus (vegetative insecticidal protein or VIP). These gut-acting proteins form an AB complex composed of ADP-ribosyl transferase (A) and cell-binding (B) components that intoxicate cells via receptor-mediated endocytosis and endosomal trafficking. Once inside the cytosol, the A components inhibit normal cell functions by mono-ADP-ribosylation of globular actin, which induces cytoskeletal disarray and death. Important aspects of each bacterium and binary enterotoxin will be highlighted in this review, with particular focus upon the disease process involving the biochemistry and modes of action for each toxin. PMID:25198129

  11. Nosocomial diarrhea: evaluation and treatment of causes other than Clostridium difficile.

    PubMed

    Polage, Christopher R; Solnick, Jay V; Cohen, Stuart H

    2012-10-01

    Diarrhea is common among hospitalized patients but the causes are distinct from those of diarrhea in the community. We review existing data about the epidemiology of nosocomial diarrhea and summarize recent progress in understanding the mechanisms of diarrhea. Clinicians should recognize that most cases of nosocomial diarrhea have a noninfectious etiology, including medications, underlying illness, and enteral feeding. Apart from Clostridium difficile, the frequency of infectious causes such as norovirus and toxigenic strains of Clostridium perfringens, Klebsiella oxytoca, Staphylococcus aureus, and Bacteroides fragilis remains largely undefined and test availability is limited. Here we provide a practical approach to the evaluation and management of nosocomial diarrhea when tests for C. difficile are negative.

  12. Physiology and Sporulation in Clostridium.

    PubMed

    Dürre, Peter

    2014-08-01

    Clostridia are Gram-positive, anaerobic, endospore-forming bacteria, incapable of dissimilatory sulfate reduction. Comprising approximately 180 species, the genus Clostridium is one of the largest bacterial genera. Physiology is mostly devoted to acid production. Numerous pathways are known, such as the homoacetate fermentation by acetogens, the propionate fermentation by Clostridium propionicum, and the butyrate/butanol fermentation by C. acetobutylicum, a well-known solvent producer. Clostridia degrade sugars, alcohols, amino acids, purines, pyrimidines, and polymers such as starch and cellulose. Energy conservation can be performed by substrate-level phosphorylation as well as by the generation of ion gradients. Endospore formation resembles the mechanism elucidated in Bacillus. Morphology, contents, and properties of spores are very similar to bacilli endospores. Sporulating clostridia usually form swollen mother cells and accumulate the storage substance granulose. However, clostridial sporulation differs by not employing the so-called phosphorelay. Initiation starts by direct phosphorylation of the master regulator Spo0A. The cascade of sporulation-specific sigma factors is again identical to what is known from Bacillus. The onset of sporulation is coupled in some species to either solvent (acetone, butanol) or toxin (e.g., C. perfringens enterotoxin) formation. The germination of spores is often induced by various amino acids, often in combination with phosphate and sodium ions. In medical applications, C. butyricum spores are used as a C. difficile prophylaxis and as treatment against diarrhea. Recombinant spores are currently under investigation and testing as antitumor agents, because they germinate only in hypoxic tissues (i.e., tumor tissue), allowing precise targeting and direct killing of tumor cells.

  13. Effects of High-Pressure Treatment on Spores of Clostridium Species

    PubMed Central

    Doona, Christopher J.; Feeherry, Florence E.; Setlow, Barbara; Wang, Shiwei; Li, William; Nichols, Frank C.; Talukdar, Prabhat K.; Sarker, Mahfuzur R.; Li, Yong-Qing; Shen, Aimee

    2016-01-01

    ABSTRACT This work analyzes the high-pressure (HP) germination of spores of the food-borne pathogen Clostridium perfringens (with inner membrane [IM] germinant receptors [GRs]) and the opportunistic pathogen Clostridium difficile (with no IM GRs), which has growing implications as an emerging food safety threat. In contrast to those of spores of Bacillus species, mechanisms of HP germination of clostridial spores have not been well studied. HP treatments trigger Bacillus spore germination through spores' IM GRs at ∼150 MPa or through SpoVA channels for release of spores' dipicolinic acid (DPA) at ≥400 MPa, and DPA-less spores have lower wet heat resistance than dormant spores. We found that C. difficile spores exhibited no germination events upon 150-MPa treatment and were not heat sensitized. In contrast, 150-MPa-treated unactivated C. perfringens spores released DPA and became heat sensitive, although most spores did not complete germination by fully rehydrating the spore core, but this treatment of heat-activated spores led to almost complete germination and greater heat sensitization. Spores of both clostridial organisms released DPA during 550-MPa treatment, but C. difficile spores did not complete germination and remained heat resistant. Heat-activated 550-MPa-HP-treated C. perfringens spores germinated almost completely and became heat sensitive. However, unactivated 550-MPa-treated C. perfringens spores did not germinate completely and were less heat sensitive than spores that completed germination. Since C. difficile and C. perfringens spores use different mechanisms for sensing germinants, our results may allow refinement of HP methods for their inactivation in foods and other applications and may guide the development of commercially sterile low-acid foods. IMPORTANCE Spores of various clostridial organisms cause human disease, sometimes due to food contamination by spores. Because of these spores' resistance to normal decontamination regimens, there

  14. Small RNAs in the genus Clostridium.

    PubMed

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

    2011-01-25

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

  15. Myonecrosis by Clostridium septicum in a dog, diagnosed by a new multiplex-PCR.

    PubMed

    Ribeiro, Márcio Garcia; Silva, Rodrigo Otávio Silveira; Pires, Prhiscylla Sadanã; Martinho, Anna Paula Vitirito; Lucas, Thays Mizuki; Teixeira, Ana Izabel Passarela; Paes, Antonio Carlos; Barros, Claudenice Batista; Lobato, Francisco Carlos Faria

    2012-10-01

    Clostridial myositis is an acute, generally fatal toxemia that is considered to be rare in pet animals. The present report describes an unusual canine clostridial myositis that was diagnosed by a new multiplex-PCR (mPCR) designed for simultaneous identification of Clostridium sordellii, Clostridium septicum, Clostridium perfringens type A, Clostridium chauvoei, and Clostridium novyi type A. A ten-month-old male Rottweiler dog, that had displayed lameness and swelling of the left limb for 12 h, was admitted to a veterinary hospital. The animal was weak, dyspneic and hyperthermic, and a clinical examination indicated the presence of gas and edema in the limb. Despite emergency treatment, the animal died in only a few minutes. Samples of muscular tissue from the necrotic area were aseptically collected and plated onto defibrinated sheep blood agar (5%) in anaerobic conditions. Colonies suggestive of Clostridium spp. were submitted to testing by multiplex-PCR. Impression smears of the tissues, visualized with Gram and also with panoptic stains, revealed long rod-shaped organisms, and specimens also tested positive using the fluorescent antibody technique (FAT). The FAT and mPCR tests enabled a diagnosis of C. septicum myonecrosis in the dog.

  16. Organization and regulation of the neurotoxin genes in Clostridium botulinum and Clostridium tetani.

    PubMed

    Raffestin, Stéphanie; Marvaud, Jean Christophe; Cerrato, Rosario; Dupuy, Bruno; Popoff, Michel R

    2004-04-01

    Botulinum and tetanus neurotoxins are structurally and functionally related 150 kDa proteins that are potent inhibitors of neuroexocytosis. Botulinum neurotoxin associates with non-toxic proteins to form complexes of various sizes. The botulinum neurotoxin and non-toxic protein genes are clustered in a DNA segment called the botulinum locus. This locus is probably located on a mobile or degenerate mobile element, which accounts for the various genomic localizations (chromosome, plasmid, phage) in different Clostridium botulinum types. The botulinum neurotoxin and non-toxic protein genes are organized in two polycistronic operons (ntnh-bont and ha operons) transcribed in opposite orientations. The gene that separates the two operons of the botulinum locus in C. botulinum A encodes a 21 kDa protein BotR/A, which is a positive regulator of the expression of the botulinum locus genes. Similarly, in Clostridium tetani, the gene located immediately upstream of the tetanus toxin gene, encodes a positive regulatory protein, TetR. BotR and TetR are possibly alternative sigma factors related to TxeR and UviA, which regulate C. difficile toxin and C. perfringens bacteriocin production, respectively. TxeR and UviA define a new sub-group of the sigma(70) family of RNA polymerase initiation factors. In addition, the C. botulinum genome contains predicted two-component system genes, some of which are possibly involved in regulation of toxinogenesis.

  17. PCR detection of Clostridium chauvoei in pure cultures and in formalin-fixed, paraffin-embedded tissues.

    PubMed

    Uzal, F A; Hugenholtz, P; Blackall, L L; Petray, S; Moss, S; Assis, R A; Fernandez Miyakawa, M; Carloni, G

    2003-02-02

    The polymerase chain reaction (PCR) was used to amplify specific segments of the 16S ribosomal RNA gene of Clostridium chauvoei, a major pathogen of ruminants. Three sets of primers were used to produce amplicons of 159, 836 and 959 base pairs (bp), respectively. The PCR was evaluated by testing clinically important strains of Clostridium, including 21 strains of C. chauvoei, five strains each of Clostridium septicum and Clostridium perfringens and two strains each of Clostridium novyi, Clostridium histolyticum and Clostridium sordellii. Both purified DNA and biomass from pure cultures of each of these microorganisms were evaluated as templates in the PCR. In addition, extracts of formalin-fixed, paraffin-embedded tissues of eight sheep experimentally inoculated with C. chauvoei or C. septicum (four animals each) were also tested by the PCR using the three sets of primers. Purified DNA template of all C. chauvoei strains produced PCR amplicons of the expected size for all three primer pairs. However, when biomass from pure cultures of C. chauvoei or tissue extracts were used as templates, only the primer pair designed to produce the 159bp amplicon gave consistently positive results. No positive results were obtained with any primer pair when purified DNA or biomass from pure cultures of non-target clostridial species were used as templates. Therefore, the PCR primer sets appear to be very specific for identifying C. chauvoei in both cultures and tissues.

  18. Necrotic Enteritis in Chickens Associated with Clostridium sordellii.

    PubMed

    Rimoldi, Guillermo; Uzal, Francisco; Chin, R P; Palombo, Enzo A; Awad, Milena; Lyras, Dena; Shivaprasad, H L

    2015-09-01

    Three outbreaks of necrotic enteritis-like disease associated with Clostridium sordelii were diagnosed in commercial broiler chicken flocks with 18,000 to 31,000 birds between 18 and 26 days old. Clinical signs in the affected flocks included high mortality up to 2% a day, depression, and diarrhea. The main gross changes included segmental dilation of the small intestine with watery contents, gas, mucoid exudate, and roughened and uneven mucosa, occasionally covered with a pseudomembrane. Microscopic lesions in the small intestine were characterized by extensive areas of coagulative necrosis of the villi, fibrinous exudate in the lumen, and high numbers of large, Gram-positive rods, occasionally containing subterminal spores, seen in the necrotic tissue and lumen. These rods were identified as C. sordellii by immunohistochemistry. Clostridium sordellii was isolated in an almost pure culture from the intestine of affected birds. A retrospective study of commercial broiler chicken and turkey submissions to the California Animal Health and Food Safety Laboratory System revealed that C. sordellii had been isolated from intestinal lesions in outbreaks of necrotic enteritis-like disease in 8 of 39 cases, 5 times together with Clostridium perfringens and 3 times alone. The latter three cases are reported here.

  19. Structure of a C. perfringens enterotoxin mutant in complex with a modified Claudin-2 extracellular loop 2.

    PubMed

    Yelland, Tamas S; Naylor, Claire E; Bagoban, Tannya; Savva, Christos G; Moss, David S; McClane, Bruce A; Blasig, Ingolf E; Popoff, M; Basak, Ajit K

    2014-09-09

    CPE (Clostridium perfringens enterotoxin) is the major virulence determinant for C. perfringens type-A food poisoning, the second most common bacterial food-borne illness in the UK and USA. After binding to its receptors, which include particular human claudins, the toxin forms pores in the cell membrane. The mature pore apparently contains a hexamer of CPE, claudin and, possibly, occludin. The combination of high binding specificity with cytotoxicity has resulted in CPE being investigated, with some success, as a targeted cytotoxic agent for oncotherapy. In this paper, we present the X-ray crystallographic structure of CPE in complex with a peptide derived from extracellular loop 2 of a modified, CPE-binding Claudin-2, together with high-resolution native and pore-formation mutant structures. Our structure provides the first atomic-resolution data on any part of a claudin molecule and reveals that claudin's CPE-binding fingerprint (NPLVP) is in a tight turn conformation and binds, as expected, in CPE's C-terminal claudin-binding groove. The leucine and valine residues insert into the binding groove while the first residue, asparagine, tethers the peptide via an interaction with CPE's aspartate 225 and the two prolines are required to maintain the tight turn conformation. Understanding the structural basis of the contribution these residues make to binding will aid in engineering CPE to target tumor cells.

  20. Quantitative real-time PCR assay for Clostridium septicum in poultry gangrenous dermatitis associated samples.

    PubMed

    Neumann, A P; Dunham, S M; Rehberger, T G; Siragusa, G R

    2010-08-01

    Clostridium septicum is a spore-forming anaerobe frequently implicated in cases of gangrenous dermatitis (GD) and other spontaneously occurring myonecrotic infections of poultry. Although C. septicum is readily cultured from diseased tissues it can be difficult to enumerate due to its tendency to swarm over the surface of agar plates. In this study a quantitative real-time PCR assay was developed in order to more accurately measure the levels of C. septicum in healthy as well as GD associated poultry samples. The assay was specifically designed to target the C. septicum alpha toxin gene, csa, which is, to our knowledge, carried by all strains of C. septicum and has been shown to be essential for virulence. Genomic DNAs from a diverse collection of bacterial species, including closely related Clostridium chauvoei, Clostridium carnis, Clostridium tertium as well as several strains of Clostridium perfringens, all failed to produce a positive reaction. An approximate reproducible limit of detection in spiked extracts of at least 10(3) cfu/g of C. septicum was observed for a variety of different sample types. C. septicum levels in broiler chicken field samples estimated from the results of qPCR were statistically correlated to culture based enumerations obtained from those same tissues.

  1. Clostridium botulinum type E occurs and grows in the alga Cladophora glomerata

    USGS Publications Warehouse

    Byappanahalli, M.N.; Whitman, R.L.

    2009-01-01

    In recent years, massive avian die-offs from Clostridium botulinum type E infection have occurred in the Sleeping Bear Dunes National Lakeshore (SLBE) area of Lake Michigan. These outbreaks have been coincidental with massive blooms of the green algae Cladophora, mostly Cladophora glomerata. We tested the hypothesis that Clostridium botulinum type E can grow under suitable conditions in these algal mats. In a lab mesocosm study, Cladophora from four outbreak-impacted beaches from SLBE were compared with four unimpacted beaches in the Milwaukee–Racine area for bontE gene of Clostridium botulinum. Frequency of the bontE gene was higher after incubation (25 °C for up to 6 weeks) of Cladophora from impacted vs. the unimpacted area. Since no type E gene was detected initially in Cladophora from any of the eight locations, we infer that the increased occurrence of type E gene arose from spore germination or vegetative Clostridium growth within the existing algal mats of SLBE. Moreover, we found that the congener Clostridium perfringens readily grows in mesocosms containing Cladophora.

  2. Clostridium difficile infection in horses: a review.

    PubMed

    Diab, S S; Songer, G; Uzal, F A

    2013-11-29

    Clostridium difficile is considered one of the most important causes of diarrhea and enterocolitis in horses. Foals and adult horses are equally susceptible to the infection. The highly resistant spore of C. difficile is the infectious unit of transmission, which occurs primarily via the fecal-oral route, with sources of infection including equine feces, contaminated soil, animal hospitals, and feces of other animals. Two major risk factors for the development of C. difficile associated disease (CDAD) in adult horses are hospitalization and antimicrobial treatment, although sporadically, cases of CDAD can occur in horses that have not received antimicrobials or been hospitalized. The most common antibiotics associated with CDAD in horses are erythromycin, trimethoprim/sulfonamides, β-lactam antimicrobials, clindamycin, rifampicin, and gentamicin. Clinical signs and intestinal lesions of CDAD infection are not specific and they cannot be used to distinguish infections by C. difficile from infections by other agents, such as Clostridium perfringens or Salmonella sp. The distribution of lesions throughout the intestinal tract seems to be age-dependent. Small intestine is invariably affected, and colon and cecum may or may not have lesions in foals<1-month old. Naturally acquired disease in older foals and adult horses has a more aboral distribution, affecting colon and sometimes cecum, but rarely the small intestine. Detection of toxin A, toxin B or both in intestinal contents or feces is considered the most reliable diagnostic criterion for CDAD in horses. Isolation of toxigenic strains of C. difficile from horses with intestinal disease is highly suggestive of CDAD. A better understanding of pathogenesis, reservoirs of infection, and vaccines and other methods of control is needed. Also further studies are recommended to investigate other possible predisposing factors and/or etiological agents of enteric diseases of horses.

  3. Regulation of toxin and bacteriocin synthesis in Clostridium species by a new subgroup of RNA polymerase sigma-factors.

    PubMed

    Dupuy, Bruno; Matamouros, Susana

    2006-04-01

    Many Clostridium species are pathogenic for humans and animals, and most of the resulting diseases, such as tetanus, botulism, gas gangrene and pseudomembranous colitis, are due to the production of potent extracellular toxins. The biochemical mechanisms of action of Clostridium toxins have been extensively studied in the past ten years. However, detailed information about the regulation of toxin gene expression has only recently emerged. TcdR, BotR, TetR and UviA are now known to be related alternative RNA polymerase sigma factors that drive transcription of toxin A and toxin B genes in C. difficile, the neurotoxin genes in C. botulinum and C. tetani, and a bacteriocin gene in C. perfringens. Although the Clostridium sigma factors have some similarity to members of the ECF sigma factor group, they differ sufficiently in structure and function so that they have been assigned to a new group within the sigma(70)-family.

  4. Detection of Clostridium septicum hemolysin gene by polymerase chain reaction.

    PubMed

    Takeuchi, S; Hashizume, N; Kinoshita, T; Kaidoh, T; Tamura, Y

    1997-09-01

    A polymerase chain reaction (PCR) was developed for the detection of the hemolysin (alpha toxin) gene of Clostridium septicum. The PCR primers were designed from the sequence of the hemolysin gene and synthesized. A DNA fragment of 270 bp was amplified from 10 strains of C. septicum, but was not from strains of C. chauvoei, C. perfringens, C. novyi, or C. haemolyticum. When the PCR product was digested with Sau3AI, two DNA fragments of the expected 148 bp and 122 bp were recognized. The lowest detectable threshold of PCR for the hemolysin gene was 3.8 x 10(3) cells/ml. The PCR technique may be useful for rapid detection or identification of C. septicum associated with malignant edema.

  5. Detection and characterization of Clostridium species in soil of Zambia.

    PubMed

    Hang'ombe, B M; Isogai, E; Lungu, J; Mubita, C; Nambota, A; Kirisawa, R; Kimura, K; Isogai, H

    2000-10-01

    In the retrospective study of soil-borne diseases of cattle in Zambia, malignant edema and blackquarter were widespread. One hundred and sixty-five cases with malignant edema and 103 cases with blackquarter were reported between 1985 and 1997. It was found that specific soil-conditions associate the emergence of the soil-borne diseases. Soil samples from five areas in Zambia were examined for the presence of genus Clostridium. Direct immunofluorescent assay (IFA) examination showed that C. septicum, C. novyi and C. chauvoei were detected in the soil of specific areas in Zambia, respectively. Causal organisms such as C. perfringens were isolated from the soil samples. The information of area-specific distribution of Clositridium species may give an efficient program in protecting cattle and man.

  6. Collagenase Clostridium Histolyticum Injection

    MedlinePlus

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

  7. Clostridium Difficile Infections

    MedlinePlus

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