Establishment of PCR assays and genetic polymorphism analysis of genes encoding Clostridium perfringens β2 toxin from different sources / 中华流行病学杂志

Objective: To establish and optimize PCR methods for the gene encoding of Clostridium perfringens β2 toxin (cpb2) and atypical-cpb2 (aty-cpb2), analyze the epidemiological characteristics and genetic polymorphism of the cpb2 of Clostridium perfringens in 9 Chinese areas from 2016 to 2021. Methods: The cpb2 of 188 Clostridium perfringens strains were examined by PCR; the cpb2 sequences were acquired by whole-genome sequencing to analyze the genetic polymorphism. Using Mega 11 and the Makeblastdb tool, a phylogenetic tree, and cpb2-library based on 110 strains carrying the cpb2 were produced. Using the Blastn technique, a comparison was made to discover sequence similarity between consensus-cpb2 (con-cpb2) and aty-cpb2. Results: The specificity of PCR assay for the cpb2 and aty-cpb2 was verified. The PCR results for cpb2 amplification were highly consistent with the whole-genome sequencing approach (Kappa=0.946, P<0.001). A total of 107 strains from nine regions in China carried cpb2, 94 types A strains carried aty-cpb2, 6 types A strains carried con-cpb2, and 7 types F strains carried aty-cpb2. The nucleotide sequence similarity between the two coding genes was 68.97%-70.97%, and the similarity between the same coding genes was 98.00%-100.00%. Conclusions: In this study, a specific PCR method for cpb2 toxin was developed, and the previous PCR method for detecting aty-cpb2 was improved. aty-cpb2 is the primary gene encoding of β2 toxin. There is a significant nucleotide sequence variance between the various cpb2 genotypes.

Determination of multidrug resistance mechanisms in Clostridium perfringens type A isolates using RNA sequencing and 2D-electrophoresis

In this study, we screened differentially expressed genes in a multidrug-resistant isolate strain of Clostridium perfringens by RNA sequencing. We also separated and identified differentially expressed proteins (DEPs) in the isolate strain by two-dimensional electrophoresis (2-DE) and mass spectrometry (MS). The RNA sequencing results showed that, compared with the control strain, 1128 genes were differentially expressed in the isolate strain, and these included 227 up-regulated genes and 901 down-regulated genes. Bioinformatics analysis identified the following genes and gene categories that are potentially involved in multidrug resistance (MDR) in the isolate strain: drug transport, drug response, hydrolase activity, transmembrane transporter, transferase activity, amidase transmembrane transporter, efflux transmembrane transporter, bacterial chemotaxis, ABC transporter, and others. The results of the 2-DE showed that 70 proteins were differentially expressed in the isolate strain, 45 of which were up-regulated and 25 down-regulated. Twenty-seven DEPs were identified by MS and these included the following protein categories: ribosome, antimicrobial peptide resistance, and ABC transporter, all of which may be involved in MDR in the isolate strain of C. perfringens. The results provide reference data for further investigations on the drug resistant molecular mechanisms of C. perfringens.

Adhesion and invasion of Clostridium perfringens type A into epithelial cells

ABSTRACT Clostridium perfringens is the causative agent for necrotic enteritis. It secretes the major virulence factors, and α- and NetB-toxins that are responsible for intestinal lesions. The TpeL toxin affects cell morphology by producing myonecrosis, but its role in the pathogenesis of necrotic enteritis is unclear. In this study, the presence of netB and tpeL genes in C. perfringens type A strains isolated from chickens with necrotic enteritis, their cytotoxic effects and role in adhesion and invasion of epithelial cells were evaluated. Six (27.3%) of the 22 C. perfringens type A strains were harboring the tpeL gene and produced morphological alterations in Vero cells after 6 h of incubation. Strains tpeL (-) induced strong cell rounding after 6 h of incubation and produced cell enlargement. None of the 22 strains harbored netB gene. All the six tpeL (+) gene strains were able to adhere to HEp-2 cells; however, only four of them (66.6%) were invasive. Thus, these results suggest that the presence of tpeL gene or TpeL toxin might be required for the adherence of bacteria to HEp-2 cells; however, it could not have any role in the invasion process.

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

Abstract 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.

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

Molecular characterization and antimicrobial resistance of Clostridium perfringens isolates of different origins from Costa Rica. Clostridium perfringens, a Gram positive, spore-forming anaerobe, is widely distributed in nature. Based upon their production of four major toxins α, β, ε and ι, 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°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 α 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. Rev. Biol. Trop. 59 (4): 1479-1485. Epub 2011 December 01.

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

An enrichment broth culture-duplex PCR combination assay for the rapid detection of enterotoxigenic Clostridium perfringens in fecal specimens.

An enrichment broth culture-duplex PCR combination assay was devised to identify Clostridium perfringens directly from fecal samples. The method consists of a combination of short enrichment of samples in selective media, DNA isolation, and performing duplex PCR using two pairs of primers which identify C. perfringens strains that harbor the virulence enterotoxin gene. Comparison of two selective enrichment media and two incubation temperatures showed that the reinforced clostridial medium with neomycin was better than the fluid thioglycollate medium with neomycin (p<0.001); and incubation at 37 degrees C vs 45 degrees C showed no statistically significant difference (p=0.238). The optimal short time for pre-enrichment culture was 4 hours. The developed assay was applied to detect phospholipase C (plc) and enterotoxin (cpe) genes for C. perfringens in feces inoculated artificially with enterotoxigenic C. perfringens. The method could detect both gene products in samples inoculated with a minimum of 10(4) CFU per ml. When the method was applied to detect enterotoxigenic C. perfringens in 198 diarrhea patients, C. perfringens was found in 121 samples; 7 out of 121 samples were positive for both plc and cpe (prevalence of 5.8%). These results indicate that the developed assay was a suitable method for the rapid and specific detection of enterotoxigenic C. perfringens directly in fecal specimens of diarrhea patients, which will assist epidemiological investigations of food poisoning outbreaks and quality control of food products.

Evaluation of synthetic DNA probes for confirmation of Clostridium perfringens enterotoxin gene PCR products.

A new diagnostic reagent was developed that is capable of detecting the presence of Clostridium perfringens rapidly and accurately compared to the conventional methods. C. perfringens enterotoxin (cpe) gene is the gene of interest since it encodes the enterotoxin responsible for food poisoning. Two new cpe-specific labeled DNA probes were evaluated using Southern and dot blot hybridization. Bacterial DNA was amplified by a duplex PCR procedure. The results showed that 40 enterotoxin producing C. perfringens strains generated two bands of amplicons with sizes of 420 and 280 bp, whereas 40 non-enterotoxin producing strains produced a single band of 280 bp on agarose gel-electrophoresis. No bands were observed from 32 strains of Clostridium spp and other bacteria. Southern blot analysis using either cpe-specific DNA or oligonucleotide probe showed hybridization specifically to the 420 bp band in enterotoxin-positive C. perfringens. On the dot blot membrane, both cpe-specific DNA and oligonucleotide probes were able to hybridize specifically with the corresponding DNA templates but with different efficacy (100% vs 91.1%).

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

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.

Development of duplex PCR assay for rapid detection of enterotoxigenic isolates of Clostridium perfringens.

A duplex PCR assay was developed for the rapid and specific amplification of the alpha-toxin (phospholipase C, plc) gene and the enterotoxin (cpe) gene from Clostridium perfringens. Two pairs of primers were newly designed for the species identification and also for the differentiation between enterotoxin-positive and enterotoxin-negative C. perfringens strains in a single reaction. The detection by agarose gel electrophoresis yielded 2 bands of 280-bp of plc and 420-bp of cpe for all four enterotoxin-positive reference strains tested without the need for further hybridization, and one band of 280-bp of plc for all seven enterotoxin-negative reference strains. While 50 strains of other Clostridium species and other bacteria tested by PCR were negative for both genes. The detection limit, as measured with purified DNA was 10 fg or as few as 4 organisms could be detected. This assay was used to identify primary fecal spore isolates from 244 fecal specimens of patients with diarrhea. Of total 432 colonies from 144 positive growth cultures determined, 21 revealed both plc and cpe genes and 411 were positive for plc gene only. This suggested a prevalent of 5% of all C. perfringens strains that carry the enterotoxin gene. The results indicate the duplex PCR as a simple, sensitive, specific, cost-effective and time saving assay for detection of potentially enterotoxigenic isolates of C. perfringens, and has potential application for epidemiological investigations of food poisoning outbreaks and quality control of food products for humans and animal feeds.