Perfringens Toxin Research Articles

Contact with enterocyte-like Caco-2 cells induces rapid upregulation of toxin production byClostridium perfringenstype C isolates

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 versus in vitro differences in C. perfringens toxin expression, and implicate VirS/VirR as a possible contributor to some C. perfringens enteric diseases.

Ulcerative Enterocolitis in Two Goats Associated with Enterotoxin- and beta2 Toxin–Positive Clostridium Perfringens Type D

Enterotoxemia caused by Clostridium perfringens type D in sheep is believed to result from the action of epsilon toxin (ETX). However, the sole role of ETX in the intestinal changes of the acute and chronic forms of enterotoxemia in goats remains controversial, and the synergistic action of other C. perfringens toxins has been suggested previously. The current study examined 2 goats that were found dead without premonitory clinical signs. Gross lesions at necropsy consisted of multifocal fibrinonecrotic enterocolitis, edematous lungs, and excess pleural fluid. Histologically, there were multifocal fibrinonecrotic and ulcerative ileitis and colitis, edema of the colonic serosa, and proteinaceous interstitial edema of the lungs. Clostridium perfringens type D carrying the genes for enterotoxin (CPE) and beta2 toxin (CPB2) was cultured from intestinal content and feces of 1 of 2 goats, while C. perfringens type D CPB2-positive was isolated from the other animal. When multiple colonies of the primary isolations from both animals were tested by Western blot, most of the isolates expressed CPB2, and only a few isolates from the first case expressed CPE. Alpha toxin and ETX were detected in ileal and colonic contents and feces of both animals by antigen capture enzyme-linked immunosorbent assay. CPB2, but not CPE, was identified in the small and large intestines of both goats by immunohistochemistry. These findings indicate that CPB2 may have contributed to the necrotic changes observed in the intestine, possibly assisting ETX transit across the intestinal mucosa.

Diagnosis of Clostridium Perfringens Intestinal Infections in Sheep and Goats

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.

Clostridium perfringens toxin genotypes in the feces of healthy North Americans

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 ∼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.

Toxin genotyping of Clostridium perfringens field strains isolated from healthy and diseased chickens

Clostridium perfringens is well known as the aetiological agent of necrotic enteritis in chicken. Type A and type c are considered the C. perfringens toxin types responsible for this disease. The aim of this study was to determine the presence of genes coding for α, β, ε, ι, β2 and enterotoxin in C. perfringens field strains collected from healthy and diseased chickens. Thirty-seven C. perfringens field strains were toxin typed: all strains resulted to be toxin type A and 3 of these tested positive for the presence of the toxin β2 coding gene. Four isolates showed the cpa gene with the insertion of a group II intron. Our findings confirm the most recent results reported from different countries and the data suggest that the role of C. perfringens type c should be revaluated in the etiopathogenesis of necrotic enteritis.

Real-time multiplex PCR assay for rapid detection and toxintyping of Clostridium perfringens toxin producing strains in feces of dairy cattle

Clostridium perfringens is an anaerobic, gram-positive, spore-forming bacterium associated with a wide variety of diseases in domestic animals and humans. We have developed dual-labeled fluorescence hybridization probe (TaqMan ®)-based real-time multiplex PCR assay for detection of toxin genes alpha ( cpa), beta ( cpb), iota ( ia), epsilon ( etx), beta2 ( cpb2) and enterotoxin ( cpe) of C. perfringens directly from cattle feces. The assay was standardized using ATCC reference strains of C. perfringens producing alpha, beta, iota, epsilon and enterotoxin, respectively. The assay for detection of beta2 toxin gene was standardized using a field strain of C. perfringens producing beta2 toxin. The minimum detection limit for the real time PCR assay ranged from 5 to 70 pg of DNA for the six toxin genes. A total of 307 fecal samples collected from seven dairy herds in Pennsylvania were analyzed using the multiplex assay. The real-time PCR assay revealed that cpa, cpb, ia, etx, cpb2 and cpe were detected in 68 (28.2%), 6 (2.5%), 6 (2.5%), 4 (1.6%), 164 (68%) and 11 (4.5%) of 241 PCR positive samples, respectively. The findings of the study revealed that C. perfringens beta2 toxin producing strains were widely prevalent in lactating cows in Pennsylvania and they may play an important role in C. perfringens associated diarrheal diseases.

Real-time multiplex PCR assays for reliable detection of Clostridium perfringens toxin genes in animal isolates

Typing of Clostridium perfringens strains by PCR-based determination of toxin genes proved to be a reliable method for diagnosis of enterotoxaemia in various animal species. We report the establishment and validation of three real-time fluorogenic (TaqMan ®) multiplex PCRs for the detection of C. perfringens α-, β-, β2-, ɛ-, entero- and ι-toxin genes. The composition of the PCRs was chosen with regard to robustness of the assays and in order to increase sensitivity compared to the conventional simplex PCRs. The combination of probe dyes selected for the real-time assays (FAM/TAMRA, Cy-5/BHQ-2 and VIC/TAMRA) as well as the designation of the chromosome-borne α-toxin as internal positive control allowed the creation of highly specific and sensitive, as well as time and cost effective PCRs. One hundred and three strains of C. perfringens isolated in Switzerland derived from clinical or suspected cases of enterotoxaemia in 10 different animal species were tested. The toxin genotypes were in agreement in both the conventional PCRs and the newly designed multiplex PCRs. Furthermore, the real-time PCR carried out as simplex allows to quantitate the copy numbers of plasmid-borne toxin genes in relation to the chromosomally located α-toxin gene.

Clostridium perfringens and its toxins in minced meat from Kars, Turkey

The aim of this study was to determine the prevalence of Clostridium perfringens and its toxins in minced meat. A total of 96 minced meat samples were collected from local markets (16) and small butcher's shops (80) in Kars (Turkey). Samples were analysed for the presence of C. perfringens and its toxins using a commercially available ELISA kit. A total of 31 (32%) Clostridium spp. strains were isolated and 17 (55%) of these isolates were identified as C. perfringens. Four (25%) of the samples from local markets and 27 (34%) from small butcher's shops were contaminated with Clostridium spp. Furthermore, C. perfringens was isolated from two (12%) and 15 (19%) samples from local markets and small butcher's shops, respectively. Mean counts of Clostridium spp. were 2.2 ± 0.83 × 102 CFU g−1 for local markets and 4.35 ± 8.53 × 102 CFU g−1 for small butcher's shops; mean counts for C. porringers were 2.75 ± 0.21 × 102 and 6.82 ± 10.96 × 102 CFU g−1 from markets and butcher's shops, respectively. The number of samples contaminated with both Clostridium spp. and C. perfringens was higher in small butcher's shops than in local markets. Moreover, higher numbers of Clostridium spp. and C. perfringens were isolated in samples from small butcher's shops than from local markets. A total of 13 (13%) samples were also positive for toxins produced by the organism, as detected by ELISA. Eleven samples from small butcher's shops and two samples from local markets were positive for the C. perfringens toxins tested. Moreover, two (12%), one (1%), four (4%) and two (2%) samples were contaminated with C. perfringens types A, B, C and D, respectively. In conclusion, some meat samples collected from local markets and small butcher's shops contained C. perfringens and its toxins and, therefore, present a potential risk of food poisoning.

Laboratory diagnosis of antibiotic-associated diarrhea: a Polish pilot study into the clinical relevance of Clostridium difficile and Clostridium perfringens toxins

In the present study, we investigated the prevalence of the Clostridium perfringens enterotoxin (CPEnt) in stool samples originally submitted for detection of Clostridium difficile toxins. Fifty-two fecal samples from inpatients were screened simultaneously for C. difficile and C. perfringens toxins: 75% of the specimens were positive for TcdA/TcdB toxins, 40% were positive for CPEnt, and 31% gave positive test results for both. It is interesting to note that only a relatively small number of C. perfringens isolates were positive for the cpe gene. All C. difficile strains were susceptible to metronidazole, but intermediate metronidazole resistance was documented for the C. perfringens isolates, which decreased upon in vitro passaging in the absence of metronidazole. We recommend that CPEnt detection should be included when diagnosing patients with presumed antibiotic-associated diarrhea.

Diet- and Colonization-Dependent Intestinal Dysfunction Predisposes to Necrotizing Enterocolitis in Preterm Pigs

Preterm birth and formula feeding are key risk factors associated with necrotizing enterocolitis (NEC) in infants, but little is known about intestinal conditions that predispose to disease. Thus, structural, functional, and microbiologic indices were used to investigate the etiology of spontaneous NEC development in preterm pigs. Piglets were delivered by cesarean section at 92% gestation, reared in infant incubators, and fed infant formula or colostrum every 3 hours (n = 120) until tissue collection at 1-2 days of age. Clinical and histopathologic signs of NEC were observed in 57% of pigs fed FORMULA (26/46) and in 5% of pigs fed COLOSTRUM (2/38) (P < .05). Relative to COLOSTRUM, both healthy and sick FORMULA pigs had reduced intestinal villous heights, enzyme activities, nutrient absorption, and antioxidant levels and higher inducible nitric oxide synthetase activity (P < .05). In healthy pigs, mucosal microbial diversity remained low and diet independent. NEC pigs showed bacterial overgrowth, and a high mucosal density of Clostridium perfringens was detected in some but not all pigs. Germ-free conditions and antiserum against Clostridium perfringens toxin prevented intestinal dysfunction and NEC in formula-fed pigs, whereas the gut trophic factors, epidermal growth factor, and glucagon-like peptide 2 had limited effects. A subclinical, formula-induced mucosal atrophy and dysfunction predispose to NEC and bacterial overgrowth. The adverse feeding effects are colonization dependent and may be reduced by factors in colostrum that include antibodies against aggressive toxins such as those of Clostridium perfringens.

いわゆる嫌気性菌が関与する感染症に関する最近の話題

Considerable information has been accumulated in the field of anaerobic bacteria and anaerobic infections in the last ten years. Here we tried to briefly introduce several selected topics of clinical importance in this field: Proposal of the term "Nanaerobe", Changes of classification and nomenclature of anaerobes, Anaerobic bacteremia, Lemierre's syndrome as a revival anaerobic infection, Atopobium vaginae as Bacterial Vaginosis-associated bacteria, and new actions of the Clostridium perfringens toxins.

Prevalence and characteristics of bacteria and host factors in an outbreak situation of antibiotic-associated diarrhoea

Antibiotic-associated diarrhoea (AAD) represents a clinical entity leading to prolonged hospital stays and diagnostic and therapeutic procedures, and results in additional costs. The aim of the present study was to assess the prevalence and characteristics of different bacteria in stools of patients with AAD. The reliability of diagnostic procedures under routine conditions was evaluated. Host factors were also analysed. From June 2002 to April 2003 89 cases of diarrhoea were reported at a hospital unit for internal medicine. Clostridium difficile and Clostridium perfringens toxin enzyme-immunoassays (EIAs), and culture for C. difficile, C. perfringens and Staphylococcus aureus were performed on stool samples from all patients. Toxin production was determined in isolated S. aureus strains. In vitro susceptibility of S. aureus for oxacillin and of C. difficile for vancomycin, metronidazole, linezolid, fusidic acid and tetracycline was tested. Host factors, such as age, comorbidities, antibiotic exposure and contact with other patients, were evaluated. Twenty-six stools were positive for C. difficile toxins by an EIA technique, while C. difficile was cultured from 39. C. difficile was isolated from 21 stools that were EIA negative. Additionally, from 28 stools S. aureus and/or C. perfringens could be isolated. Nine samples contained only S. aureus and/or C. perfringens. Thirty-one stools were negative in all tests. All C. difficile isolates were susceptible to vancomycin and metronidazole. Age >60 years, and diseases of the vascular system, the heart, the kidneys and the lungs were identified as risk factors for acquiring C. difficile in this setting (P values < 0.05). Stool culture for C. difficile was shown to be more sensitive than toxin EIA in this study. Risk factors for the acquisition of C. difficile in outbreak situations seem to differ from risk factors in the normal hospital setting. The role of toxin-producing S. aureus in cases of AAD needs further investigation.

Multipathogen oligonucleotide microarray for environmental and biodefense applications

Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

Genotyping of Clostridium perfringens toxins using multiple oligonucleotide microarray hybridization

A microarray-based method for characterization of six Clostridium perfringens toxin genes: iA (iota toxin), cpa (alpha toxin), cpe (enterotoxin E), etxD (epsilon toxin), cpb1 (beta toxin 1),and cpb2 (beta toxin 2) was developed and evaluated using 17 C. perfringens isolates. Three individual oligonucleotide probes (oligoprobes), complementary to the unique sequences of each toxin gene, were designed and immobilized on a surface of aldehyde-coated glass slides. Multiplex PCR was used to simultaneously amplify DNA target regions of all six genes. Single-stranded DNA (ssDNA) samples for microarray analysis were prepared by following a primer extension of amplicons in the presence of one primer. Fluorescent moieties (Cy3) were incorporated into the ssDNA by chemical modification of guanine bases. The presence of toxin genes in C. perfringens was established by hybridization of the fluorescently labeled ssDNA representing different samples to the microarray gene-specific oligoprobes. Results of the study showed sensitivity and specificity of genotyping C. perfringens using multiple microarray-based assays.

Evaluation of a Predictive Model for Clostridium perfringens Growth during Cooling

Proper temperature control is essential in minimizing Clostridium perfringens germination, growth, and toxin production. The U.S. Department of Agriculture Food Safety and Inspection Service offers two options for the cooling of meat products: follow a standard time-temperature schedule or validate that alternative cooling regimes result in no more than a 1-log CFU/g increase of C. perfringens and no growth of Clostridium botulinum. The Juneja 1999 model for C. perfringens growth during cooling may be helpful in determining whether the C. perfringens performance standard has been achieved, but this model has not been extensively validated. The objective of this study was to validate the Juneja 1999 model under a variety of temperature situations. The Juneja 1999 model for C. perfringens growth during cooling is fail safe when low (<1 log CFU/ml) or high (>3 log CFU/ml) observed increases occur during exponential cooling. The Juneja 1999 model consistently underpredicted growth at intermediate observed increases (1 to 3 log CFU/ml). The Juneja 1999 model also underpredicted growth whenever exponential cooling took place at two different rates in the first and second portions of the cooling process. This error may be due to faster than predicted growth of C. perfringens cells during cooling or to an inaccuracy in the Juneja 1999 model.

Large scale purification of Clostridium perfringens toxins: a review

Clostridium perfringens, a Gram-positive anaerobic bacterium, is widespread in the environment and commonly found in the intestines of animals, including humans. C. perfringens strains are classified into five toxinotypes (A, B, C, D and E) based on the production of four major toxins (±, ², µ, ¹). However the toxins (theta, delta, lambda and enterotoxin) are also synthesized by C. perfringens strain. Many attempts to purify the toxins produced by C. perfringens have been proposed. In this review we discuss the purification methods used to isolate toxins from C. perfringens reported in last four decades.

Detection of bovine Clostridium perfringens by polymerase chain reaction

A polymerase chain reaction (PCR) assay to detect Clostridium perfringens alpha toxin gene (cpa) was used to identify eighty-nine C. perfringens strains obtained from bovine clinical material. The strains were biochemically characterized as C. perfringens. The isolated strains were cultured on plates containing brain heart infusion agar with 5% sheep blood under anaerobic conditions. DNA extraction was performed by boiling. The 324 bp amplification product of cpa was observed in all isolates. C. sordellii, C. botulinum, C. novyi, and C. septicum were also tested but did not produce any alpha toxin gene amplification. These findings suggest that PCR is a useful assay in identifying C. perfringens toxin types.

Genotypic and Phenotypic Characterization ofClostridium perfringensandClostridium difficilein Diarrheic and Healthy Dogs

The objectives of this study were to examine the potential roles ofClostridium difficileand enterotoxigenicClostridium perfringensin diarrhea in dogs by comparison of isolation, determination of toxin status via enzyme‐linked immunosorbent assay (ELISA), and application of multiplex polymerase chain reaction (PCR). These techniques were used to evaluate fecal specimens in 132 healthy and diarrheic dogs. These dogs were prospectively evaluated by grouping them into the following 3 categories: hospitalized dogs with diarrhea (n = 32), hospitalized dogs without diarrhea (n = 42), and apparently healthy outpatient dogs without diarrhea (n = 58). All fecal specimens were cultured using selective media forC difficile, Salmonellaspp., andCampylobacterspp. and selective media after heat shock forC perfringens.No significant difference was found in the isolation ofC perfringensorC difficileamong the 3 groups. A significant association was found between the presence of diarrhea and detection ofC perfringensenterotoxin (CPE) or toxin A via ELISA for bothC perfringensandC difficile, respectively. PCR performed onC difficileisolates for toxin A and toxin B genes revealed no significant differences among the 3 groups, but diarrheic dogs were significantly more likely to be positive for the enterotoxin gene ofC perfringens.Based on the results of this study, the use of ELISA for detection of CPE in feces combined with the detection of enterotoxigenic fecal isolates obtained via heat shock provides the strongest evidence for the presence ofC perfrigences‐associated diarrhea.

Toxin types of Clostridium perfringens isolated from free-ranging, semidomesticated reindeer in Norway

Samples of faeces were taken from 166 healthy domesticated reindeer (Rangifer tarandus tarandus) from three flocks in different reindeer husbandry districts in northern Norway and examined bacteriologically for the presence...

Clostridium perfringens toxin types from wild-caught Atlantic cod (Gadus morhua L.), determined by PCR and ELISA.

Ninety-five fecal samples from Atlantic cod (Gadus morhua L.), caught along the northern Norwegian coast, were examined bacteriologically for occurrence of C. perfringens. Isolates were examined by polymerase chain reaction (PCR) for genes encoding the four lethal toxins (alpha, beta, epsilon, and iota) for classification into toxin types and for genes encoding enterotoxin and the novel beta2 toxin for further subclassification. In addition, a commercial enzyme-linked immunosorbent assay (ELISA) kit for detection of C. perfringens alpha, beta, and epsilon toxin was used. Clostridium perfringens could be isolated in 37 fecal samples (38.9%) from cod. All isolates were C. perfringens toxin type A (alpha toxin positive) as determined by PCR and also ELISA. In addition, in isolates from two cod (2.1%) the gene encoding for beta2 toxin was found (A, beta2) by PCR. Genes encoding for beta, epsilon, and iota toxins and enterotoxin were not found. This is the first detection of C. perfringens alpha and beta2 toxin in cod and of beta2 toxin in fish in general. The origin of this bacterium in cod is discussed.