Clostridium Perfringens Isolates Research Articles

A Wide Variety of Clostridium perfringens Type A Food-Borne Isolates That Carry a ChromosomalcpeGene Belong to One Multilocus Sequence Typing Cluster

Of 98 suspected food-borne Clostridium perfringens isolates obtained from a nationwide survey by the Food and Consumer Product Safety Authority in The Netherlands, 59 strains were identified as C. perfringens type A. Using PCR-based techniques, the cpe gene encoding enterotoxin was detected in eight isolates, showing a chromosomal location for seven isolates and a plasmid location for one isolate. Further characterization of these strains by using (GTG)(5) fingerprint repetitive sequence-based PCR analysis distinguished C. perfringens from other sulfite-reducing clostridia but did not allow for differentiation between various types of C. perfringens strains. To characterize the C. perfringens strains further, multilocus sequence typing (MLST) analysis was performed on eight housekeeping genes of both enterotoxic and non-cpe isolates, and the data were combined with a previous global survey covering strains associated with food poisoning, gas gangrene, and isolates from food or healthy individuals. This revealed that the chromosomal cpe strains (food strains and isolates from food poisoning cases) belong to a distinct cluster that is significantly distant from all the other cpe plasmid-carrying and cpe-negative strains. These results suggest that different groups of C. perfringens have undergone niche specialization and that a distinct group of food isolates has specific core genome sequences. Such findings have epidemiological and evolutionary significance. Better understanding of the origin and reservoir of enterotoxic C. perfringens may allow for improved control of this organism in foods.

Molecullar Typing, Prevalence of netB and Antimicrobial Susceptibility among Clinical Isolates of Clostridium perfringens from Chickens in Southern China

Molecullar Typing, Prevalence of netB and Antimicrobial Susceptibility among Clinical Isolates of Clostridium perfringens from Chickens in Southern China

LyophilizedCarnobacterium divergens AS7 bacteriocin preparation improves performance of broiler chickens challenged withClostridium perfringens

The present study aimed to investigate the effects of Carnobacterium divergens AS7 bacteriocin (divercin AS7) on growth performance, digestibility, fermentation processes, selected microbial populations, and histomorphology in broiler chickens challenged with a mixture of 3 Clostridium perfringens isolates. In total, 480 one-day-old male Ross 308 chicks were randomly assigned to 4 experimental groups (12 replicate pens of 10 birds per treatment). The diets were either nonsupplemented or supplemented with a lyophilized preparation of divercin AS7. On d 18, 19, and 20, half of the birds were challenged twice a day with the C. perfringens mixture. The C. perfringens challenge did not influence broiler BW gain but impaired feed conversion ratio from d 29 to 42 (P=0.023) and throughout the experimental period (P=0.038). Moreover, the C. perfringens challenge resulted in decreased pH levels of crop, gizzard, and ileum contents (P<0.05) and reduced the numbers of lactic acid bacteria in the ceca (P=0.01). Divercin supplementation decreased broiler feed intake from d 14 to 28 (P=0.001) but increased BW gain from d 29 to 42 (P=0.048). The divercin supplementation increased the AMEn level (P=0.015) and reduced digesta pH in crop and ileum (P=0.004 and P=0.042, respectively), but of nonchallenged birds only. Divercin supplementation, moreover, increased gizzard lactate concentrations (P=0.003). The crop concentrations of lactate and succinate and the ileum concentration of lactate were increased by divercin supplementation (P=0.005, P=0.027, and P=0.002, respectively) and C. perfringens challenge (P=0.034, P=0.053, and P=0.0002, respectively). Divercin supplementation decreased villus heights (P=0.0006) and crypt depths (P=0.044) in noninfected birds, whereas in challenged birds, villus heights (P<0.0001) were increased. In conclusion, the present study demonstrated a very complex response pattern of broilers exposed to C. perfringens challenge and dietary divercin AS7 supplementation, but it indicated that divercin AS7 may partly counterbalance the negative effects associated with C. perfringens.

The majority of atypical cpb2 genes in Clostridium perfringens isolates of different domestic animal origin are expressed

This study examined the prevalence and expression of the “consensus” and the “atypical”cpb2 genes in Clostridium perfringens isolates from cattle, chickens, dogs, goats, horses, pigs and sheep using polymerase chain reaction (PCR), sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting. Almost all porcine isolates (12/14) carried and expressed the consensus form of cpb2 but, when present in 108 non-porcine isolates, the gene was usually the atypical form (40 atypical versus 9 consensus). Western blotting showed expression in 30 of 40 (75%) atypical cpb2-positive isolates, considerably more frequently than reported previously. CPB2 was expressed by almost all (20/21) the consensus cpb2-positive isolates, regardless of source.

Genetic relatedness and netB prevalence among environmental Clostridium perfringens strains associated with a broiler flock affected by mild necrotic enteritis

In a previous study we investigated pulsed-field gel electrophoresis (PFGE) genotype diversity and prevalence of the netB toxin gene in Clostridium perfringens (CP) isolates recovered from a broiler flock (flock 1) affected by necrotic enteritis (NE). In this follow-up work, we examined samples collected before placement of flock 1, to see if NE during rearing could be traced back to the cleaned and empty building or the day-old chicks. Litter from the next flock in the same building (flock 2) was also examined. We detected 25 different PFGE genotypes, five of which were found only in litter from flock 2. Six genotypes which had been found in flock 1 during rearing were detected in samples collected before placement. NetB positive isolates belonging to two of these genotypes had been recovered from NE lesions during rearing, suggesting that virulent strains were transmitted from the cleaned and disinfected broiler house. NetB frequency among isolates from the empty building was 45%, indicating that netB positive strains were prevalent in a building that previously had housed a healthy flock offered in-feed narasin (flock 0). NetB frequency among isolates from litter used by flock 2 was 22%, indicating that netB positive strains were present in the environment of a 14-days-old healthy flock offered in-feed narasin. Two prevalent genotypes were consistently either netB negative or netB positive. However, the presence of genotypes represented by both negative and positive isolates may suggest that the gene can spread horizontally among different CP strains.

Typing of toxigenic isolates of Clostridium perfringens by ELISA in ostrich

Toxigenic types of Clostridium perfringens are significant causative agents of enteric disease in domestic animals, although type E is presumably rare, appearing as an uncommon cause of enterotoxemia of lambs, calves and rabbits. In 100 feces samples collected from ostrich farm, 40 strains of C. Perfringens were isolated by biochemical test, of these, 4 strains were toxigenic strains type A and any toxigenic strains of type B and type C and type D are not fond. Enzyme-linked immunosorbent assay (ELISA) kit for detection of type E not exist and isolation of type E not purpose. The technique of isolation and ELISA test are described in this paper. Key words: Toxigenic strains, Clostridium perfringens, Enzyme-linked immunosorbent assay (ELISA).

Complete genome sequence of the podoviral bacteriophage ΦCP24R, which is virulent for Clostridium perfringens

Bacteriophage ΦCP24R was isolated from raw sewage from a waste treatment plant, and lytic activity was observed against a type A Clostridium perfringens isolate. Electron microscopy revealed a small virion (44-nm-diameter icosahedral capsid) with a short, non-contractile tail, indicative of a member of the family Podoviridae. The phage had a linear, double-stranded DNA genome of 18,919 base pairs (bp) with 41 bp inverted terminal repeats and a type B DNA polymerase, which are characteristics of members of the subfamily Picovirinae. Out of 22 predicted genes in the genome, ten had significant sequence similarity to proteins of known function. Three distinct genes with lytic domains were identified, including a zinc carboxypeptidase domain that has not been previously reported in viruses. The ΦCP24R genome described herein is only the second Clostridium perfringens podovirus genome reported to date.

Alpha toxin specific PCR for detection of toxigenic strains of Clostridium perfringens in Poultry

Aim : Isolation of Clostridium perfringens from necrotic enteritis cases in poultry and confirmation by alpha toxin specific PCR Materials and methods: Robertson cooked meat medium with Brain Heart Infusion broth was used for isolation of C. perfringens from intestinal contents of necrotic enteritis suspected birds. Positive cultures from perfringens agar were further confirmed by biochemical tests and subjected to alpha toxin specific PCR. Results and Discussion: Twenty Clostridium perfringens isolates were isolated from intestinal contents of thirty five NE suspected birds. Out of the twenty isolates, fourteen were isolated from commercial broilers of 2 to 6 wk of age and six from commercial layers of 9 to 15 wk of age. Frequency of isolation of C. perfringens was more with Robertson cooked meat medium with BHI broth than thioglycollate broth alone. When positive cultures were streaked on to clostridial agar appreciable luxuriant growths were obtained and the selective streaking of these colonies on perfringens agar with supplements revealed rough and black colonies with sulphate reduction. The isolates produced rough and black colonies with sulphate reduction on perfringens agar, double zone haemolysis on sheep blood agar, stormy clot fermentation on milk medium and opalescence on egg yolk medium. The isolates were found negative for oxidase, catalase, liquefied gelatin, fermented glucose, maltose, lactose and sucrose except mannitol. All the fourteen isolates obtained from commercial broilers proved the alpha toxin producing strains of C. perfringens when they were subjected to alpha toxin specific PCR. Conclusion : This study revealed alpha toxin specific PCR is highly useful for detection of toxigenic strains of Clostridium perfringens in poultry

Toxinotyping of Clostridium perfringens isolates by ELISA and PCR from lambs suspected of enterotoxemia

Clostridium perfringens is a gram-positive, anaerobic bacterium that causes a wide range of diseases in humans and animals. The purposes of this study were to determine, using the enzyme-linked immunosorbent assay (ELISA), the clostridial toxins in intestinal samples of lambs with suspected enterotoxemia; to conduct molecular typing of C. perfringens isolates; and to investigate the presence of the genes of 4 major toxins (\alpha, \beta, \epsilon, and \iota) in the isolates by polymerase chain reaction (PCR). According to the ELISA results, clostridial toxins were determined in 32 intestinal samples. Of the samples, 13 (40.62%) were C. perfringens type A, 1 (3.125%) was C. perfringens type B, 9 (28.125%) were C. perfringens type C, and 9 (28.125%) were C. perfringens type D. C. perfringens was isolated from 13 (8.66%) out of 150 intestinal samples. A total of 13 C. perfringens isolates from lambs were genotyped by PCR. Of the isolates, 10 (76.92%) were type A, 2 (%15.38) were type D, and 1 (7.69%) was type C. Type B and type E were not identified. The results showed that C. perfringens type A was the most common type by both ELISA and PCR in lambs with enterotoxemia.

Molecular detection and characterization of cpb2 gene in Clostridium perfringens isolates from healthy and diseased chickens

Clostridium perfringens is an important pathogen in both human and veterinary medicine. Necrotic enteritis (NE) is the most clinically dramatic bacterial enteric disease of poultry induced by C. perfringens. The pathogenicity of this bacterium is associated with the production of extracellular toxins produced by some of its strains, such as beta2 toxin. The exact role of beta2 toxin in NE pathogenesis is still controversial. In the present study, C. perfringens isolates from healthy and diseased poultry flocks from different parts of Iran were analyzed by PCR assay to determine the presence of all variants of the beta2 toxin gene (cpb2). The products of two positive cpb2 PCR reactions were sequenced, compared to each other and to the cpb2 sequences published in GenBank (by multiple alignment and phylogenetic analysis). The current work represents the first study of cpb2 in poultry C. perfringens isolates in Asia, and reports the highest percentage of cpb2-positive isolates in both apparently healthy chickens (97.7%) and those afflicted with NE (94.4 %). The sequenced isolates were classified as atypical. This study did not show a direct correlation between NE occurrence and cpb2 presence.

Toxinotyping of necrotic enteritis-producing and commensal isolates of Clostridium perfringens from chickens fed organic diets

The present study determined the effect of Clostridium perfringens isolates taken from necrotic enteritis (NE) outbreaks on organic farms in a NE virulence testing model. Thirteen strains were isolated in the course of the study. Six C. perfringens field isolates were taken from a naturally occurring NE outbreak on an organic farm. Polymerase chain reaction toxinotyping was used to establish C. perfringens strains, as well as to create a toxin profile. All field isolates were found to be type A and positive for alpha, beta-2 and netB toxin genes. During the NE virulence model, digesta samples were collected before oral inoculation to define the C. perfringens found as part of the natural flora. Three of the five natural flora isolates were found to be C. perfringens type E while the other two isolates were type A; only four of five isolates were positive for either netB or beta-2 toxin genes. Two isolates collected after inoculation were C. perfringens type A positive for cpb2 and netB. All isolates were tested positive for the quorum-sensing-related gene luxS, regardless of the strain source. The presence of luxS, alpha, netB and beta-2 toxin genes seems not to be a determinant of the disease as they were present in isolates from both outbreak birds as well as healthy and pre-inoculated birds. The C. perfringens field isolates induced mild NE lesions in one-half of the birds during the challenge study. Other mechanisms must play a role in the development of the disease beyond toxinotype, potentially including intestinal ecology and health, which would account for acute disease as seen in the field outbreak.

Genotyping of isolates of Clostridium perfringens from vaccinated and unvaccinated sheep

Enterotoxaemia vaccine is a polyvalence vaccine from different types of Clostridium perfringens, and C. septicum toxins that prevents various diseases, the most important one being enterotoxaemia in sheep. The aim of the present study was to evaluate the enterotoxaemia vaccine effects in reducing isolates of intestinal clostridia genus specifically C. perfringens. Sheep dung samples were randomly collected from 10 places in Kerman, Iran. The samples were taken from 90 vaccinated Kermani sheep against enterotoxaemia and 50 unvaccinated sheep of same age from flocks with similar management. Following processing and culture of the samples, colonies were identified applying morphological, gram stain and biochemical tests. Using these tests C. perfringens were isolated from 27 out of 50 unvaccinated sheep (54.0%) and from 2 out of 90 vaccinated sheep (2.2%). All of the clostridia isolates were analyzed by multiplex PCR. Genotyping of 2 strains isolated from the vaccinated sheep indicated that these strains were type D, while the strains isolated from the unvaccinated sheep were types A, B, C and D; 14.8% (4 out of 27), 22.2% (6 out of 27), 40.7% (11 out of 27) and 22.2% (6 out of 27), respectively. However, no isolate containing the iota gene (type E) was detected. Vaccination against enterotoxaemia had a significant effect ( P < 0.01) on reducing C. perfringens isolates. Occurrence of the disease in the vaccinated and unvaccinated groups was 3.3% and 64.0% ( P < 0.01), respectively.

Isolation, identification, and antimicrobial susceptibility of Clostridium perfringens isolates from acute necrotic enteritis of broiler chickens

The aim of this study was to isolate, identify and determine the antimicrobial susceptibility of Clostridium perfringens (CP) isolates from acute necrotic enteritis of broiler chickens. All broiler carcasses diagnosed as necrotic enteritis (NE) were sampled, subjected to microbial tests and 40 isolates were identified according to standard procedures. The antimicrobial susceptibility of CP isolates to 20 antibacterial agents was then determined. The results show widespread resistance among CP isolates. The most frequent resistance was observed to neomycin sulfate (87.5%), and then to lincomycin and tetracycline (both 80%). No isolate was resistant to chloramphenicol and the least frequency of resistance was observed to vancomycin (10%), sulfamethoxazole+trimethoprim (17.5%), and penicillin (20%). All isolates were multiple drug resistant types. There were 39 resistant patterns among the CP isolates, 95% of which were distributed in 38 resistant patterns. These multiple and variable resistance patterns observed among the CP isolates, even among different isolates from one farm, demonstrate a challenge for veterinarians in the field to choose the correct compound to combat the occurrence of NE.

Clostridium perfringens in retail chicken

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

Association between avian necrotic enteritis andClostridium perfringensstrains expressing NetB toxin

A novel toxin, NetB, has recently been identified in virulent avian Clostridium perfringens isolates and shown to be an essential virulence factor in a clinical necrotic enteritis isolate. To assess whether NetB is more generally associated with avian necrotic enteritis isolates we have screened a range of C. perfringens strains from geographically diverse locations for both the presence and expression of the netB gene. Forty-four isolates were derived from necrotic enteritis disease cases from Australia, Belgium, Denmark and Canada and 55 isolates from healthy chickens from Australia and Belgium. The majority of strains isolated from necrotic enteritis-affected birds were netB positive (70%) and there was an absolute correlation between the presence of netB and in vitro expression of the NetB protein. Only two of the C. perfringens isolates from healthy chickens carried netB. Sequencing of the netB gene from 23 positive isolates showed that NetB is highly conserved, with only one predicted amino acid (A168T) difference, in six isolates, compared to the published sequence. This change did not alter the in vitro activity of the NetB toxin. The gene encoding the recently discovered TpeL toxin was also screened using PCR and only found in a small proportion of NetB-positive isolates from diseased birds. A selection of NetB-negative isolates, originating from diseased birds, was unable to cause disease in a necrotic enteritis induction model. This study provides further evidence that NetB is important in pathogenesis and advances our current understanding of C. perfringens virulence factors in avian necrotic enteritis.

Hemorrhagic Gastritis Associated with Renal Failure, Hemoglobinuria, and Isolation of Clostridium perfringens in a Horse

A 3-year-old Quarter Horse mare was presented for acute colic with bright red-black foul-smelling gastric reflux containing long rod-shaped bacteria consistent with Clostridium sp. and red-black urine. The serum creatinine concentration was 5.5 mg/dL (N = 0.9–1.7), and blood urea nitrogen was 41 mg/dL (N = 9–20). At necropsy, the stomach wall was diffusely thickened, hemorrhagic, and edematous. Histopathologically, hemorrhagic necrosis was evident, with numerous colonies of spore-forming rods within the submucosa. Clostridium perfringens was cultured from the stomach contents. Polymerase chain reaction (PCR) genotyping was consistent with type A C. perfringens. Bilaterally, the kidneys were grossly enlarged, diffusely dark red-brown, and congested. The renal tubular epithelium was diffusely, acutely necrotic, with interstitial hemorrhage and massive accumulation of intratubular granular and proteinaceous casts. A diagnosis of massive hemolysis with hemoglobinuria and renal failure due to C. perfringens, type A infection was made. Alpha-toxin–induced intravascular hemolysis occurs rarely in humans and sheep. To our knowledge, this has not been described in horses with clostridial enterocolitis nor in equine clostridial gastritis.

Antimicrobial resistance in Clostridium perfringens isolates from broilers in Belgium

The antimicrobial susceptibility of Clostridium perfringens strains isolated from Belgian broilers between May and September 2007 was investigated. All 39 tested isolates were sensitive to enrofloxacin, erythromycin, tylosin, florfenicol and bacitracin. Twenty-six (66%) and 24 (61%) out of the 39 tested isolates showed acquired resistance to tetracycline and lincomycin, respectively. The C. perfringens isolates were also screened by PCR for the presence of the resistance genes tet(K), tet(L), tet(M), tetB(P), tet(O), tet(W), lnu(A) and lnu (B). In 22/26 tetracycline resistant strains and 7/24 lincomycin resistant strains, resistance could be attributed to one or more of these genes. An extended frequency distribution range of MICs was seen for ampicillin. These data are consistent with data derived from studies carried out in 1980 and in 2004, indicating that no changes in antimicrobial resistance patterns have taken place during time in C. perfringens isolates from broilers in Belgium.

Prevalence of netB among some clinical isolates of Clostridium perfringens from animals in the United States

A previously unknown pore forming toxin, called NetB toxin, which is produced by some Australian strains of Clostridium perfringens has recently been reported. This toxin was reported to be critical to the development of the disease necrotic enteritis, in chickens. To investigate the occurrence of the toxin gene ( netB) in non-Australian C. perfringens strains, one hundred and six American isolates of C. perfringens were examined. Ninety-two isolates were from chickens, and 14 were from cattle. The netB gene was found in 14 isolates from chickens (7 from chickens with necrotic enteritis, and 7 from unrelated chickens with no evidence of necrotic enteritis). The netB gene was also detected in an isolate recovered from a 3-year-old cow with liver abscesses. The products of all positive netB PCR reactions were sequenced, and these showed 100% nucleotide identity to the netB sequence published in GenBank. Five isolates which had been recovered from five chickens with necrotic enteritis (from four flocks) were netB negative. An additional 24 isolates recovered from one of these lesioned chickens were also netB negative. The present study represents the first study of C. perfringens isolates outside Australia for netB, and the first identification of netB in an isolate from a species other than chickens. The results indicate that the role of NetB in the induction of necrotic enteritis needs to be further investigated, by determining the disease producing capability of both netB positive strains recovered from normal chickens, and netB negative strains recovered from chickens with necrotic enteritis.

Origin of Clostridium perfringens isolates determines the ability to induce necrotic enteritis in broilers

Since the ban on growth-promoting antibiotics in animal feed in the European Union, necrotic enteritis has become a major cause of mortality in broiler chickens. Despite the importance of the disease, the pathogenesis is still not completely understood. In the current study, Clostridium perfringens strains isolated from healthy flocks and isolates from outbreaks of necrotic enteritis were evaluated for the ability to cause gut necrosis in an intestinal loop model in laying hens and in an experimental infection model in broilers. High, intermediate and low alpha toxin producing strains were chosen from each isolation source. Only the isolates from field outbreaks induced necrotic gut lesions, independent of the amount of alpha toxin produced in vitro. It was also shown that alpha toxin producing isolates from calf hemorrhagic enteritis cases were not able to induce necrotic enteritis in poultry. These results suggest the presence of host specific virulence factors in C. perfringens strains, isolated from chickens with intestinal necrotic enteritis lesions.

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

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