Bacteriological and molecular typing of Clostridium perfringens strains isolated in retail beef in Beijing, China.
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ABSTRACT:
SUBMITTER: Jiang H
PROVIDER: S-EPMC8569878 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Clostridium perfringens type A, is both a ubiquitous environmental bacterium and a major cause of human gastrointestinal disease, which usually involves strains producing C. perfringens enterotoxin (CPE). The gene (cpe) encoding this toxin can be carried on the chromosome or a large plasmid. Interestingly, strains carrying cpe on the chromosome and strains carrying cpe on a plasmid often exhibit different biological characteristics, such as resistance properties against heat. In this study, we investigated the genetic properties of C. perfringens by PCR-surveying 21 housekeeping genes and genes on representative plasmids and then confirmed those results by Southern blot assay (SB) of five genes. Furthermore, sequencing analysis of eight housekeeping genes and multilocus sequence typing (MLST) analysis were also performed. Fifty-eight C. perfringens strains were examined, including isolates from: food poisoning cases, human gastrointestinal disease cases, foods in Japan or the USA, or feces of healthy humans. In the PCR survey, eight of eleven housekeeping genes amplified positive reactions in all strains tested. However, by PCR survey and SB assay, one representative virulence gene, pfoA, was not detected in any strains carrying cpe on the chromosome. Genes involved in conjugative transfer of the cpe plasmid were also absent from almost all chromosomal cpe strains. MLST showed that, regardless of their geographic origin, date of isolation, or isolation source, chromosomal cpe isolates, i) assemble into one definitive cluster ii) lack pfoA and iii) lack a plasmid related to the cpe plasmid. Similarly, independent of their origin, strains carrying a cpe plasmid also appear to be related, but are more variable than chromosomal cpe strains, possibly because of the instability of cpe-borne plasmid(s) and/or the conjugative transfer of cpe-plasmid(s) into unrelated C. perfringens strains.
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Project description:Clostridium perfringens toxinotype D, toxinotype E, and gastroenteritis-linked BEC/CPILE-positive strains have never been reported in healthy children. We isolated, whole-genome sequenced and bioinformatically characterised three C. perfringens isolates-type D (IQ1), type E (IQ2) and BEC/CPILE-positive (IQ3), recovered from the stools of three healthy two-year-olds, which were further compared to 128 C. perfringens genomes available from NCBI. The analysis uncovered a previously under-described putative toxin gene alv (alveolysin) encoded by isolates IQ2 and IQ3, which appeared to be a clade-specific trait associated with strains from domestic animals. A plasmid analysis indicated that the iota-toxin was encoded on a near-intact previously described plasmid pCPPB-1 in type E strain IQ2. The BEC genes becA and becB were carried on a near-identical pCPOS-1 plasmid previously associated with Japanese gastroenteritis outbreaks. Furthermore, a close phylogenetic relatedness was inferred between the French C. perfringens type E isolates cp515.17 and newly sequenced IQ2, suggesting geographical links. This study describes novel C. perfringens isolates from healthy individuals which encode important toxin genes, indicating the potential spread of these veterinary and clinically important strains and mobile genetic elements, and highlights areas for future research.
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Project description: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.
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Project description: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 one strains and most genes controlled in one or two strains only.
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Project description:Background and aimClostridium perfringens can cause enteritis in ostriches. The toxin release is believed to play a major role in determining pathogenesis properties of these pathogenic bacteria. This study was conducted to isolate and characterize C. perfringens strains from ostriches in Vietnam for identifying if particular virulence factors of these pathogenic bacteria are associated with enteritis progress in ostriches.Materials and methodsThe prevalence of cpa, cpb, iA, etx, cpe, and cpb2 genes among C. perfringens isolates was determined by a multiplex polymerase chain reaction (PCR) method. The NetB toxin-encoding gene was detected by PCR and then sequenced to observe their variation. The expression of NetB toxin was checked by SDS-PAGE.ResultsA total of 116 C. perfringens isolates were obtained from 318 fecal samples and 105 intestinal organs. Of 80 isolates from fecal samples, 33 isolates were from healthy and 47 isolates were from diseased ostriches. The results of multiplex PCR showed that all 116 C. perfringens strains from healthy and enteric disordered ostriches were positive for the alpha toxin-encoding gene (cpa). The cpe and cpb2 genes were found in only one and five diseased ostriches, respectively. The netB gene was detected in 1/33 (3.03%) C. perfringens isolates from healthy ostriches, in 8/47 (17.05%) isolates from feces, and in 7/36 (19.44%) intestinal contents of diseased ostriches. The full-length sequences of 5 out of 15 netB-positive isolates from diseased ostriches showed 100% identity to each other as well as to the netB sequences available in GenBank. All of these five isolates produced NetB toxin in vitro.ConclusionType A is the most prevalent among C. perfringens isolates from ostriches in Vietnam. Especially, the study provides data emphasizing the role of NetB toxin in causing necrotic enteritis by C. perfringens in ostriches.
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