Clostridium Perfringens Isolates Research Articles

Isolation and genotyping of Clostridium perfringens from goats in Minas Gerais, Brazil

ABSTRACT: Despite the known importance of Clostridium perfringens as an enteropathogen in small ruminants, little is known about the role of its additional virulence factors or the frequency of the various C. perfringens genotypes in healthy goats; this complicates the laboratory diagnosis of the infections caused by this microorganism. In light of this, the aim of the present study was to isolate and genotype C. perfringens from stool samples from healthy goats in Brazil. Stool samples from 250 apparently healthy adult goats from 17 different herds in Minas Gerais, Brazil were collected, and isolation and genotyping of C. perfringens was performed. C. perfringens type A was isolated from 189 (75.6%) goats, whereas C. perfringens types C and D were each detected in one goat (0.4%). All isolates were negative for enterotoxin-, NetB-, NetE-, and NetF-encoding genes. These results confirmed C. perfringens type A as part of the microbiota in these animals, and they suggested that C. perfringens type C and D are rarely isolated from healthy goats.

Isolation, molecular characterization and prevalence of Clostridium perfringens in sheep and goats of Kashmir Himalayas, India.

Aim:The study was conducted to report the occurrence of the Clostridium perfringens in sheep and goats of the Kashmir valley for the 1st time and to characterize them molecularly with respect to toxin genes to determine the prevalence of the various toxinotypes.Materials and Methods:A total of 177 samples (152 from sheep and 25 from goats) collected from healthy, diarrheic animals, and morbid material of animals suspected to have died of enterotoxaemia were screened for C. perfringens toxinotypes. The presumptive positive isolates were confirmed using 16S rRNA gene-based polymerase chain reaction (PCR). All the confirmed isolates were screened for six toxin genes, namely; cpa, cpb, etx, cpi, cpb2, and cpe using a multiplex PCR.Results:The PCR amplification of 16S rRNA gene revealed that out of 177 samples collected, 125 (70.62%) were found positive for C. perfringens, of which 110 (72.36%) were from sheep and 15 (60%) were from goats. The highest prevalence of C. perfringens toxinotype D was observed in lambs (56.16%) and kids (46.16%) followed by 3.84% in adult sheep while it was absent in samples obtained from adult goats. The multiplex PCR revealed that 67 (60.90%) isolates from sheep and 8 (53.33%) isolates from goats belonged to toxinotype A, while 43 (39.09%) isolates from sheep and 7 (46.66%) isolates from goats were detected as toxinotype D. None of the isolates was found to be toxinotype B, C, or E. All the C. perfringens toxinotype A isolates from sheep were negative for both cpb2 and cpe genes, however, 27.90% toxinotype D isolates from sheep carried cpb2 gene, and 6.97% possessed cpe gene. In contrast, 12.50% C. perfringens toxinotype A isolates from goats harbored cpb2 and cpe genes while 14.28% isolates belonging to toxinotype D carried cpb2 and cpe genes, respectively.Conclusion:The high prevalence of C. perfringens was observed, even in day-old lambs. The toxinotypes A and D are prevalent in both sheep and goats. The severity of disease and mortality may be associated with the presence of minor toxins in both the detected toxinotypes.

Characterization of enterotoxin producing Clostridium perfringens isolated from foods of animal origin

Screening of 105 samples comprising of milk, meat and their products for isolation of Clostridium perfringens revealed a total of 39 (37.14%) samples to be positive for C. perfringens, yielding an equal number of isolates. Among the total isolates, 11 (20.0%) belonged to milk and milk products, of which 10 were isolated from raw cow’s milk. The remaining isolate was from paneer sample. Similarly, 28 (56.0%) C. perfringens isolates were recovered from meat and meat products. Majority (37) of the isolates belonged to toxin type A while two isolates recovered each from raw beef and chicken meats were of toxin type C. Among the 39 isolates of C. perfringens, only 5(12.82%) isolates could reveal the presence of cpe (enterotoxin) gene. The protein profile (SDS-PAGE) of both the crude and partially purified enterotoxins prepared from cpe positive C. perfringens isolates (type A and C) revealed a very little difference. Both the partially purified enterotoxins of type A and C of C. perfringens displayed almost similar type of cytopathic effects (CPE) and death of cells in Vero cell line.

Characterization of enterotoxin producing Clostridium perfringens isolated from foods of animal origin

Screening of 105 samples comprising of milk, meat and their products for isolation of Clostridium perfringens revealed a total of 39 (37.14%) samples to be positive for C. perfringens, yielding an equal number of isolates. Among the total isolates, 11 (20.0%) belonged to milk and milk products, of which 10 were isolated from raw cow’s milk. The remaining isolate was from paneer sample. Similarly, 28 (56.0%) C. perfringens isolates were recovered from meat and meat products. Majority (37) of the isolates belonged to toxin type A while two isolates recovered each from raw beef and chicken meats were of toxin type C. Among the 39 isolates of C. perfringens, only 5(12.82%) isolates could reveal the presence of cpe (enterotoxin) gene. The protein profile (SDS-PAGE) of both the crude and partially purified enterotoxins prepared from cpe positive C. perfringens isolates (type A and C) revealed a very little difference. Both the partially purified enterotoxins of type A and C of C. perfringens displayed almost similar type of cytopathic effects (CPE) and death of cells in Vero cell line.

Isolation and Identification of Clostridium perfringens and its Enterotoxin in Food poisoning Patients

Background: Clostridium perfringens enterotoxin (CPE), which is one of the most common cause’s foodborne illnesses and contribute to diarrhea that is associated with broadspectrum antibiotic treatment.Objectives: This study focuses on diagnosis of Clostridium perfringens enterotoxin (CPE) from patients suffering from food poisoning and diarrhea associated with antibiotic treatment cases in stool samples and to determine the resistance of isolated against antibiotics.Methods: Samples were taken during the period of first of June 2015 until the end of April 2016 from Baghdad hospitals. Enzyme Linked Immunosorbent Assay (ELISA) was used to detect Clostridium perfringens enterotoxin in stool samples. Api 20A kit and culture to confirm isolation and identification was used, disk diffusion was performed for antibiotic resistance.Results: The infection cases increased among old adult age group, were (8.7%) and their age range was (64≥) years old,and children (5.3%) their age range was(15≤) years old. Overall positivity was (23%) in present studied groups and infection increased with causes of food poisoning (61.5%).Conclusion: This study revealed that the majority percent from age ≥64year (8.7%) and this percent decreased under this age. The future advances research should explain the epidemiology of enterotoxigenic C. perfringens and also participate to the prevention of C. perfringens food poisoning outbreaks and other CPE-associated human diseases.

Clostridium perfringens Type A Isolates of Animal Origin with Decreased Susceptibility to Metronidazole Show Extensive Genetic Diversity.

Metronidazole (MTZ) is a first-line drug for the treatment of human infections caused by anaerobic bacteria. Although most Clostridium perfringens isolates are susceptible to metronidazole, a number of strains with decreased susceptibility have been obtained in clinical and environmental surveys. However, little is known yet about the toxinotype and genetic diversity of metronidazole-resistant C. perfringens strains. We tested for susceptibility to metronidazole and assessed the toxigenic status and amplified fragment length polymorphism (AFLP) diversity of a collection of 80 C. perfringens isolates of animal origin. In vitro susceptibility testing by the standard agar dilution technique revealed that 22 isolates (27.5% of total) were susceptible to metronidazole, 54 (67.5%) intermediate susceptible, and four (5%) resistant. All isolates belonged to toxinotype A, and two, 41 and 17, of them yielded a positive PCR result for presence of the gene encoding for enterotoxin (cpe), the consensus form of β2 toxin (cpb2) and the atypical form of β2 toxin, respectively. AFLP fingerprinting classified isolates into 65 genotypes indicating a high molecular diversity among isolates in this study, and this diversity was reflected among isolates from each susceptibility category. In conclusion, the results of this study revealed extensive genetic diversity among C. perfringens type A isolates regardless of their susceptibility to metronidazole.

Incidence and antibiogram of Clostridium perfringens isolated from herbs and spices widely distributed in the Egyptian market

The present study was designed for isolation and identification of Clostridium perfringens (C. perfringens) from herbsand spices that are widely distributed in the Egyptian markets; and determination of the drug of choice for its control basedon the Antimicrobial Susceptibility Test (AST). A total of 392 samples of herbs and spices were randomly andanonymously selected from the enormous number of samples obtained from the Egyptian markets. An overall occurrenceof C. perfringens was 8.42% (33 out of 392). Separately, occurrence was higher in herbs than spices being 10.5 and 6.25%,respectively. C. perfringens isolates were identified and confirmed with macroscopical, microscopical and biochemicalanalysis. AST results revealed that isolates of C. perfringens from the tested herbs and spices samples were found mostsusceptible to ampicillin-sulbactam and Penicillin-G with inhibition zones of 19.4 ± 0.98 and 16.6 ± 1.16 mm, respectively.On the other hand, the isolates were found nearly resistant to clindamycin and vancomycin with inhibition zones of 6.28± 0.63 and 8.78 ± 0.41 mm, respectively. Meanwhile, the isolates were intermediary susceptible to trimethoprimsulphamethoxazolecombination and chloramphenicol. Data of the present study indicate occurrence of C. perfringens inherbs and spices with a considerable rate and the antimicrobials of choice for its control are penicillins and ceftazidime.higher sanitation procedures in herbs and spices industry before its distribution to the Egyptian markets have beenrecommended

The safe enterocin DD14 is a leaderless two-peptide bacteriocin with anti-Clostridium perfringens activity

Enterococcus faecalis 14, a strain previously isolated from meconium, displayed activity against four Clostridium perfringens isolates when co-cultured on agar plates. The anti-Clostridium activity was ascribed to the production of enterocin DD14, which was subsequently purified. The minimum inhibitory concentration (MIC) of enterocin DD14 against one collection strain and one clinical C. perfringens strain was determined at 50 µg/mL. Furthermore, using the intestinal epithelial cell line IPEC-1, it was shown that E. faecalis 14 was not cytotoxic after 24 h of contact, and no cytotoxicity was observed when IPEC-1 cells were incubated with pure enterocin DD14 for 4 h. Enterocin DD14 was characterised using mass spectrometry and was shown to consist of two small proteins of 5200.74 Da and 5206.41 Da, respectively. The two peptides (DD14A and DD14B) have highly similar amino acid sequences and no signal peptide, which classifies enterocin DD14 as a class IIb leaderless two-peptide bacteriocin. The genes encoding DD14A and DD14B were sequenced and were shown to be 100% identical to other previously described enterocins MR10A and MR10B, in contrast to the producing strains, which are different. Consequently, the present in vitro study supports the potential of this E. faecalis 14 strain and/or its purified enterocin DD14 as putative anti-C. perfringens compounds in chickens.

Isolation of Clostridium perfringens from Fecal Sample of Zoo Animals in Iraq

In this study the isolated Clostridium perfringens from fecal samples of zoo animals of Al-zawraa Zoo of Baghdad city. A total of 170 fecal samples was aseptically collected from different mammalian species of apparently healthy animals for the isolation and identification of bacterial flora. All the collected samples were inoculated in sheep blood agar media and incubated anaerobically in an anaerobic jar with the gas bag for the study of their cultural properties. Gram’s staining, motility test and biochemical tests were also performed for the proper diagnosis of the isolated bacteria.

Isolation, Genotyping and Antibiogram Profile of Clostridium perfringens Isolates Recovered from Freshwater Fish and Fish Products from Kolkata Region

Isolation, Genotyping and Antibiogram Profile of Clostridium perfringens Isolates Recovered from Freshwater Fish and Fish Products from Kolkata Region

A chicken intestinal ligated loop model to study the virulence of Clostridium perfringens isolates recovered from antibiotic-free chicken flocks

ABSTRACTNecrotic enteritis (NE) is a major problem in antibiotic-free (ABF) chicken flocks and specific strains of Clostridium perfringens are known to induce NE. The objective of this study was to develop a chicken intestinal ligated loop model in order to compare the virulence of various C. perfringens strains recovered from consecutive ABF flocks with and without NE. Intestinal loops were surgically prepared in 10 anaesthetized specific-pathogen-free chickens and alternately inoculated with C. perfringens isolates or brain heart infusion (BHI) media. Histological lesion scoring was performed for each loop. All strains from NE-affected flocks induced histological lesions compatible with NE whereas inoculation of loops with a commensal C. perfringens strain or BHI did not. Among inoculated strains, CP0994 (netB-positive and cpb2-positive) and CP-2003-1256 (netB-positive) demonstrated mean histological lesion scores significantly higher (P < 0.01) than those obtained with a commensal strain or BHI whereas strain CP1073 (netB-negative and cpb2-positive) induced intestinal lesions without significantly higher scores. In loops where villi were colonized by Gram-positive rods, significantly higher (P < 0.01) mean histological lesion scores were observed. This result supports the hypothesis that colonization of the intestinal mucosa by C. perfringens is a critical step in the pathogenesis of NE. Finally, we demonstrated the importance of controlling virulent C. perfringens strains in ABF chicken flocks as a highly virulent strain can be present in consecutive flocks with NE and possibly affect multiple flocks.

Isolation and molecular characterization of Clostridium perfringens from healthy Merino lambs in Patagonia region, Argentina

The presence and molecular characterization of Clostridium perfringens in healthy Merino lambs over a six-month period was investigated in this study. Overall, a high prevalence of C. perfringens was detected, even in day-old lambs. Even though the majority of the isolates were characterized as being of type A, types C and D were also isolated. Furthermore, a high genetic diversity was observed by PFGE among the type A isolates.

Identification of enterototxin harboring gene among Clostridium perfringens isolates with different toxin types in Iran

Background: Clostridium perfringens is known as the most widely distributed pathogenic microorganism in nature. It is an extremely important pathogen of human and domestic animals. In a commonly used classification scheme, C. perfringens is divided into five toxinotypes (A to E) based on the production of four major toxins (alpha, beta, epsilon, and iota). Enterotoxin is not usually used for C. perfringens typing but it is a fatal toxin with necrotic activity. Based on our knowledge there is no published scientific report regarding identification of enterotoxin positive C. perfringens isolates from animals in Iran. OBJECTIVES: To study the presence and frequency of enterotoxin gene among C. perfringence isolates with different types. METHODS: A specific single PCR assay was developed and used for detection of cpe gene to identify the entrerotoxin harboring isolates among different types of C. perfringens isolated from animal enteric diseases in Iran. RESULTS: It was found that cpe gene presents among C.perfringens isolates types A, B, C and D with 63.6% (7/11), 25% ( 5/20), 21.4% (3/14), 53.3% (8/15), respectively. Totally 23 of 60 ( 38.3%) isolates screened by PCR were cpe-positive. CONCLUSIONS: This is the first report of cpe- positive isolates of C.perfringens causing enterototoxemia in animals in Iran. Further studies to investigate the synergistic effect of CPE toxin in pathogenesis of enteric diseases in animals is suggested.

Isolation and genotyping of Clostridium perfringens isolated from healthy and diarrheic dogs

Clostridium perfringens has been known as a cause of diarrhoea in dogs. The aim of this research was isolation C. perfringens by culturing and toxinotyping by PCR molecular method. In this research 151 dogs’ faecal samples were collected from northwest of Iran, 131 of which were apparently healthy, and 20 of which were diarrheic. These faecal samples were cultured on 5% sheep blood agar; the suspected colonies with double homolysis that using multiplex and single polymerase chain reaction (PCR) assay were admitted to detect toxinotypes of the isolates by specific primers. C. perfringens strains were isolated from 5/20 (25%) the diarrheic group and 31/131(23.8%) the non-diarrheic group. All isolates (36/151) were classified as C. perfringens type A (cpa+). Fourteen isolates (38.8%) with cpa+cpb2+netB-tpeL- profile and one isolate (2.8%) had cpa+cpe+netB-tpeL- toxin’s profile. More studies are needed to elucidate the epidemiology of C. perfringens in dogs and its role as a zoonotic agent and public health hazard. Based on author’s knowledge, this is the first study was performed in order to isolation C. perfringens and genotyping from dogs in Iran. The cpa+cpe+ gene was reported from one C. perfringens isolated from healthy dogs.

Typing Toxigenic Clostridium perfringens Strains from the Ruminants of Yazd Province by Multiplex Polymerase Chain Reaction

Background: Anaerobic bacterial infections, especially enterotoxemia, are common ruminant disorders. Objectives: The purpose of this study was to identify the toxigenic isolates of Clostridium perfringens in the ruminants of Yazd province. Materials and Methods: In total, 485 fecal and intestinal samples were obtained and analyzed for typing C. perfringens toxovars by multiplex polymerase chain reaction (PCR). Only 179 bacterial strains passed the biochemical tests and 87 C. perfringens strains were confirmed by multiplex PCR. Results: Interestingly, the predominant C. perfringens toxovar was type A (89.7%), while type D (9.2%) was also identified as a pathogen in the ruminants of Yazd province. Conclusions: Detection of toxigenic C. perfringens isolates with multiplex PCR was performed for the first time in this field. The multiplex PCR used in this study provides a useful and reliable tool for C. perfringens genotyping in routine veterinary diagnostics,a nd epidemiological studies of the prevalent types of C. perfringens in Iran are possible by this technique. Genotyping of C. perfringens is recommended before starting vaccination programs.

Clostridium perfringens: Comparative effects of heat and osmotic stress on non-enterotoxigenic and enterotoxigenic strains

Clostridium perfringens isolates associated with food poisoning carries a chromosomal cpe gene, while non-foodborne human gastrointestinal disease isolates carry a plasmid cpe gene. The enterotoxigenic strains tested produced vegetative cells and spores with significantly higher resistance than non-enterotoxigenic strains. These results suggest that the vegetative cells and spores have a competitive advantage over non-enterotoxigenic strains. However, no explanation has been provided for the significant associations between chromosomal cpe genotypes with the high resistance, which could explain the strong relationship between chromosomal cpe isolates and C. perfringens type A food poisoning. Here, we analyse the action of physical and chemical agent on non-enterotoxigenic and enterotoxigenic regional strains. And this study tested the relationship between the sensitivities of spores and their levels SASPs (small acid soluble proteins) production in the same strains examined.

Phenotypic Identification of Clostridium Perfringens Associated with Necrotic Enteritis of Broiler Chickens in Sharkia Governorate

A total of 314 samples of liver and intestinal loop were collected from broiler chickens suffering from diarrhea during 2013-2014 .The tissue samples were examined for the prevelance of C.perfringens revealed 91 (66.93%) of them were toxinogenic and 44(31.06%) non toxinogenic isolates .One hundered and thirty two samples were positive for Clostridium perfringens typing by intradermal inoculation in guinea pig. The higher incidence of C.perfringens wasrecorded in winter (53.12%-57.5%) followed by autumn (50%) then summer (32.60%-37.93%) and the lower incidence was recorded in spring (30%-31.25%).The incidence of C.perfringens from intestinal samples was(65.85%-58.66%)and liver samples were(21.95%-21.33%). The most effective antibiotic was recommended for treatment of necrotic enteritis were amoxicillin, Ampicillin, Piperacillin, ceftraxione, cefruxime, Cefotaxime which was applied by sensitivity test to 91 isolates of toxinogenic Clostridium perfringens. Our study recorded that there is the isolation rate of Clostridium perfringens from intestine was exteremly higher than liver samples and ranged from (58.66%-65.85%) and (21.95 %-21.33), respectively that subtyped into type A 76 (57.57%) and typeD 15 (11.36%).

Diversity of Clostridium perfringens isolates from various sources and prevalence of conjugative plasmids

Clostridium perfringens is an important pathogen, causing food poisoning and other mild to severe infections in humans and animals. Some strains of C. perfringens contain conjugative plasmids, which may carry antimicrobial resistance and toxin genes. We studied genomic and plasmid diversity of 145 C. perfringens type A strains isolated from soils, foods, chickens, clinical samples, and domestic animals (porcine, bovine and canine), from different geographic areas in the United States between 1994 and 2006, using multiple-locus variable-number tandem repeat analysis (MLVA) and/or pulsed-field gel electrophoresis (PFGE). MLVA detected the genetic diversity in a majority of the isolates. PFGE, using SmaI and KspI, confirmed the MLVA results but also detected differences among the strains that could not be differentiated by MLVA. All of the PFGE profiles of the strains were different, except for a few of the epidemiologically related strains, which were identical. The PFGE profiles of strains isolated from the same domestic animal species were clustered more closely with each other than with other strains. However, a variety of C. perfringens strains with distinct genetic backgrounds were found among the clinical isolates. Variation was also observed in the size and number of plasmids in the strains. Primers for the internal fragment of a conjugative tcpH gene of C. perfringens plasmid pCPF4969 amplified identical size fragments from a majority of strains tested; and this gene hybridized to the various-sized plasmids of these strains. The sequences of the PCR-amplified tcpH genes from 12 strains showed diversity among the tcpH genes. Regardless of the sources of the isolates, the genetic diversity of C. perfringens extended to the plasmids carrying conjugative genes.

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

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.

Pathological and bacteriological characterization of neonatal porcine diarrhoea of uncertain aetiology.

Neonatal porcine diarrhoea of uncertain aetiology has been reported from a number of countries. This study investigated 50 diarrhoeic and 19 healthy piglets from 10 affected Swedish herds. The piglets were blood-sampled for analysis of serum γ-globulin and necropsied, and the intestines were sampled for histopathology and cultured for Escherichia coli, Clostridium perfringens and Clostridium difficile. Escherichia coli isolates (n = 276) were examined by PCR for virulence genes encoding LT, STa, STb, EAST1, VT2e, F4, F5, F6, F18, F41, AIDA-I, intimin, and for the genes aaiC and aggR. Selected isolates were analysed for additional virulence genes by a microarray and subjected to O-typing. Clostridium perfringens isolates (n = 152) were examined by PCR for genes encoding major toxins, enterotoxin and beta2-toxin. There was no difference in serum γ-globulin concentration between diarrhoeic and non-diarrhoeic piglets, and pathological lesions in the intestines were generally mild. Porcine enterotoxigenic Escherichia coli, a common cause of piglet diarrhoea, was only found in two piglets. Further, the virulence gene profiling did not suggest involvement of other diarrhoeogenic pathotypes of Escherichia coli. Growth of Clostridium perfringens did not differ between diarrhoeic and non-diarrhoeic piglets. All isolates were type A, all were negative for enterotoxin, and 151 of 152 isolates were beta2-toxin positive. In pigs ≥ 2 days old, moderate to profuse growth of Clostridium difficile was more common in the controls. In conclusion, it was not possible to relate Escherichia coli, Clostridium perfringens type A and C or Clostridium difficile to neonatal porcine diarrhoea in any of the investigated herds.