Perfringens Food Poisoning Research Articles

Identification of a new Clostridium perfringens variant with a chromosomally encoded enterotoxin gene in a suspected persistent food poisoning outbreak in Eritrea.

Clostridium perfringens is a causative agent of various human and animal enteric diseases including food poisoning. In this study, we describe an interesting case of a persistent food poisoning outbreak among Finnish peacekeepers in Eritrea, possibly caused by Clostridium perfringens carrying a new variant of the chromosomally encoded enterotoxin gene. C. perfringens strains causing food poisoning carry the enterotoxin gene, cpe, in its chromosome (c-cpe) or on a plasmid (p-cpe). PCR assays are widely used for toxinotype C. perfringens strains. The integration sites for the cpe gene are highly conserved, and PCR assays targeting the cpe gene and the adjacent IS elements (the IS1470 in c-cpe and the IS1470-like or IS1151 in p-cpe strains) are used to further determine the genetic location of the cpe gene. We sequenced nine enteropathogenic C. perfringens strains related to a persistent food poisoning outbreak among Finnish peacekeepers in Eritrea. Six of these strains produced non-typeable cpe results in the standard PCR assay due to divergence in the enterotoxin integration site. The gene order of the new variant of the chromosomal cpe insertion site with an additional IS1470 element impairing genotyping PCR assay for the location of cpe is described. In addition, variant c-cpe strains carried 58-81 copies of IS1470 in their genomes, compared to 9-23 copies found in previously described c-cpe strains. Thus, the present study represents an untraditional type of C. perfringens food poisoning caused by variant c-cpe strains, and the sequenced strains bring geographic variation to the existing strain collection of sequenced C. perfringens.

Types A and F Clostridium perfringens in healthcare wastewater from Ghana.

Clostridium perfringens causes gas gangrene and food poisoning in humans, and monitoring this bacterium is important for public health. Although whole-genome sequencing is useful to comprehensively understand the virulence, resistome, and global genetic relatedness of bacteria, limited genomic data from environmental sources and developing countries hamper our understanding of the richness of the intrinsic genomic diversity of this pathogen. Here, we successfully accumulated the genetic data on C. perfringens strains isolated from hospital effluent and provided the first evidence that predicted pathogenic C. perfringens may be disseminated in the clinical environment in Ghana. Our findings suggest the importance of risk assessment in the environment as well as the clinical setting to mitigate the potential outbreak of C. perfringens food poisoning in Ghana.

Clostridium perfringens food poisoning: An update on toxins, occurrence, diseases and detection techniques

Clostridium perfringens food poisoning: An update on toxins, occurrence, diseases and detection techniques

A cohort study investigating a point source outbreak of Clostridium perfringens associated with consumption of roasted meat and gravy at a buffet on Mothering Sunday 2018, South West, England

In March 2018, Public Health England was alerted to an outbreak of gastrointestinal illness in persons who had eaten at a restaurant in the South West of England on Mothering Sunday. We aimed to determine the source of infection and control the outbreak. A retrospective cohort study was undertaken among all individuals who ate at the restaurant on March 11, 2018. Cases were defined as any person who consumed food from the restaurant and had diarrhoea or vomiting between 12:00 March 11, 2018 and 23:59 March 15, 2018. Data were collected using a secure electronic questionnaire. Descriptive, univariable and multivariable analyses were undertaken with odds ratios (OR) and 95% confidence intervals (CI) calculated for exposures. Clinical and food samples were collected for microbiological testing and molecular typing. The restaurant reported 293 people attending, 176 (60%) completed the survey and 146 (50%) provided sufficient information for inclusion in the analysis. Eighty-one persons met the case definition (attack rate 55.5%); eating from the roast meat buffet was associated with illness (OR: 7.80, 95% CI: 2.03–43.56). No single food item explained all the cases. Independent associations between consumption of gravy (adjusted OR (aOR): 5.35, 95% CI:2.21–12.93), lamb (aOR: 2.51, 95% CI:1.06–5.96), and eating during the later 16:00 sitting (aOR: 5.23, 95% CI:1.76–15.54) were identified. Clostridium perfringens enterotoxin was detected in clinical samples and the same strain of enterotoxigenic C. perfringens that was isolated from clinical samples was also detected in roasted lamb, beef and turkey. We found strong evidence for an outbreak of C. perfringens food poisoning associated with gravy and/or meat consumption. The data suggests cross-contamination between items, which increased over time. We recommend that health agencies ensure that restaurants serving roasted meat buffets adhere to minimum oven temperatures and take special measures to prevent food poisoning and cross contamination at the preparation and serving stage.

Dangers of Clostridium perfringens food poisoning in psychiatric patients.

Clostridium perfringens food poisoning can be fatal in patients with chronic constipation. We report the investigation and management of a probable outbreak of C. perfringens food poisoning among psychiatric patients in Cape Town, South Africa, in 2013.

The inhibitory effects of essential oil constituents against germination, outgrowth and vegetative growth of spores of Clostridium perfringens type A in laboratory medium and chicken meat

C. perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and non-food-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly heat-resistant spores, it is of great interest to develop strategies alternative to thermal processing to inactivate C. perfrinegens. Thus, in this study we evaluated the inhibitory effects of essential oil constituents (EOCs) (cinnamaldehyde, eugenol, allyl isothiocyanate (AITC), and carvacrol) against germination, outgrowth and vegetative growth of spores of C. perfringens FP and NFB disease isolates in laboratory medium and chicken meat. The cinnamaldehyde, eugenol and carvacrol, but not AITC, all at 0.05–0.1%, inhibited the germination of spores of all tested C. perfringens isolates in Tripticase-glucose-yeast extract (TGY) medium. Furthermore, all tested EOCs at 0.05–0.1% arrested the outgrowth and vegetative growth of C. perfringens spores in TGY, with AITC and carvacrol being the most effective. However, among four tested EOCs, only AITC (at 0.5%–2.0%) was able to inhibit the growth of C. perfringens spores in chicken meat and no such inhibitory effect was observed even with a 10-fold higher concentration (5%) of carvacrol. In conclusion, our current work identified AITC as an effective EOC to control spores and vegetative cells of C. perfringens isolates in laboratory medium and chicken meat. Further studies on evaluating the effectiveness of different combination of EOCs against C. perfringens spore growth in different meat products should establish an effective use of EOCs to control the risk of C. perfringens-mediated illnesses.

Epidemiological studies on Clostridium perfringens food poisoning in retail foods.

Clostridium perfringens is an important anaerobic pathogen causing food-borne gastrointestinal (GI) diseases in humans and animals. Meat and meat products are the most common vehicles of C. perfringens type A food poisoning. Contamination of meat by the intestinal contents of slaughtered animals may serve as an important source of this pathogen to the food supply. One hundred and fifty-five non-outbreak food samples were obtained from meat and retail food and examined for the presence of C. perfringens. Multiplex polymerase chain reaction assay to determine the toxin genotype of C. perfringens isolates, and extraction and purification of C. perfringens enterotoxin from enterotoxin gene (cpe)-positive isolates were carried out. The homogeneity of the purified enterotoxin was demonstrated by polyacrylamide gel electrophoresis. In addition, stool samples were collected from 150 persons who had been in contact with animals, and enzyme-linked immunosorbent assays were carried out for the qualitative determination of C. perfringens enterotoxin in the stool samples. The results demonstrated that approximately 2.6% of the tested meat and retail meat samples were contaminated with cpe-positive C. perfringens. The recommended laboratory criteria used to implicate C. perfringens in food-borne disease should involve the detection of C. perfringens enterotoxin production or the presence of the cpe gene in foods or faeces, or in the suspected C. perfringens isolates. In the present study some isolates such as tuna contained the enterotoxin gene although they had a low count of C. perfringens.

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.

عزل وتشخيص جرثومة Clostridium perfringens وسمومها المعوية من مرضى التسمم الغذائي

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.

Preventing Foodborne Illness: Clostridium perfringens

The bacterium Clostridium perfringens causes of one of the most common type of foodborne gastroenteritis, often referred to as perfringens food poisoning, in the US (FDA 2012). It is associated with consuming contaminated food that contains great numbers of vegetative cells and spores that will produce toxin inside the intestine. There are two forms of disease caused by C. perfringens: gastroenteritis and enteritis necroticans. The latter disease, also known as pig-bel disease, is not common in the US. It is often associated with contaminated pork (FDA 2012) and can be very severe.

Chitosan inhibits enterotoxigenic Clostridium perfringens type A in growth medium and chicken meat

Clostridium perfringens is a spore-forming bacterium and a major cause of bacterial food-borne illness. In this study, we evaluated the inhibitory effects of chitosan against spore germination, spore outgrowth and vegetative growth of C. perfringens food poisoning (FP) isolates. Chitosan of differing molecular weights inhibited germination of spores of all tested FP isolates in a KCl germinant solution containing 0.1 mg/ml chitosan at pH 4.5. However, higher level (0.25 mg/ml) of chitosan was required to effectively arrest outgrowth of the germinated C. perfringens spores in Tripticase-yeast extract-glucose (TGY) medium. Furthermore, chitosan (1.0 mg/ml) was bacteriostatic against vegetative cells of C. perfringens in TGY medium. Although chitosan showed strong inhibitory activities against C. perfringens in laboratory medium, higher levels (2.0 mg/g) were required to achieve similar inhibition of spores inoculated into chicken meat. In summary, the inhibitory effects of chitosan against C. perfringens FP isolates was concentration dependent, and no major difference was observed when using different molecule weight chitosan as an inhibitor. Our results contribute to a better understanding on the potential application of chitosan in cooked meat products to control C. perfringens-associated disease.

Identification and Characterization of a New Enterotoxin Produced by Clostridium perfringens Isolated from Food Poisoning Outbreaks.

There is a strain of Clostridium perfringens, W5052, which does not produce a known enterotoxin. We herein report that the strain W5052 expressed a homologue of the iota-like toxin components sa and sb of C. spiroforme, named Clostridium perfringens iota-like enterotoxin, CPILE-a and CPILE-b, respectively, based on the results of a genome sequencing analysis and a systematic protein screening. In the nicotinamide glyco-hydrolase (NADase) assay the hydrolysis activity was dose-dependently increased by the concentration of rCPILE-a, as judged by the mass spectrometry analysis. In addition, the actin monomer of the lysates of Vero and L929 cells were radiolabeled in the presence of [32P]NAD and rCPILE-a. These findings indicated that CPILE-a possesses ADP-ribosylation activity. The culture supernatant of W5052 facilitated the rounding and killing of Vero and L929 cells, but the rCPILE-a or a non-proteolyzed rCPILE-b did not. However, a trypsin-treated rCPILE-b did. Moreover, a mixture of rCPILE-a and the trypsin-treated rCPILE-b enhanced the cell rounding and killing activities, compared with that induced by the trypsin-treated rCPILE-b alone. The injection of the mixture of rCPILE-a and the trypsin-treated rCPILE-b into an ileum loop of rabbits evoked the swelling of the loop and accumulation of the fluid dose-dependently, suggesting that CPILE possesses enterotoxic activity. The evidence presented in this communication will facilitate the epidemiological, etiological, and toxicological studies of C. perfringens food poisoning, and also stimulate studies on the transfer of the toxins’ gene(s) among the Genus Clostridium.

Location and stoichiometry of the protease CspB and the cortex-lytic enzyme SleC in Clostridium perfringens spores

The protease CspB and the cortex-lytic enzyme SleC are essential for peptoglycan cortex hydrolysis during germination of spores of the Clostridium perfringens food poisoning isolate SM101. In this study, Western blot analyses were used to demonstrate that CspB and SleC are present exclusively in the C. perfringens SM101 spore coat layer fraction and absent in the lysate from decoated spores and from the purified inner spore membrane. These results indicate why decoating treatments greatly reduce both germination and apparent viability of C. perfringens spores in the absence of an exogenous lytic enzyme. In addition, quantitative Western blot analyses showed that there are approximately 2000 and 130,000 molecules of CspB and pro-SleC, respectively, per C. perfringens SM101 spore, consistent with CspB's role in acting catalytically on pro-SleC to convert this zymogen to the active enzyme.

Molecular Epidemiology ofClostridium perfringensIsolated from Food Poisoning in Seoul, 2013

Clostridium perfringens food poisoning ranks among the most common gastrointestinal diseases in developed countries. In Korea, C. perfringens food poisoning gradually increases. Using PCR, 72 strains of C. perfringens isolated in Seoul, 2013 were tested for the presence of toxin genes. Of the tested strains, 32 isolates carried the cpe gene, 37 isolates carried the cpb2 gene and 3 isolates carried the cpe and cpb2 genes, respectively. 32 cpe- positive strains were isolated from the food poisoning patient, whereas among 37 cpb2-positive strains, 22 strains were isolated from asymptomatic person. To investigate epidemiological relationship between the isolates, Pulsed-filed gel electrophoresis (PFGE) was performed. The genetic relatedness of the isolates ranged from 55.9% to 100% and 47 distinct PFGE profiles were observed. The results show that the cpe- positive outbreak strains showed close genetic relation, whereas the cpb2-positive isolates revealed a wide genetic diversity.

Inactivation strategy for Clostridium perfringens spores adhered to food contact surfaces

The contamination of enterotoxigenic Clostridium perfringens spores on food contact surfaces posses a serious concern to food industry due to their high resistance to various preservation methods typically applied to control foodborne pathogens. In this study, we aimed to develop an strategy to inactivate C. perfringens spores on stainless steel (SS) surfaces by inducing spore germination and killing of germinated spores with commonly used disinfectants. The mixture of l-Asparagine and KCl (AK) induced maximum spore germination for all tested C. perfringens food poisoning (FP) and non-foodborne (NFB) isolates. Incubation temperature had a major impact on C. perfringens spore germination, with 40 °C induced higher germination than room temperature (RT) (20 ± 2 °C). In spore suspension, the implementation of AK-induced germination step prior to treatment with disinfectants significantly (p < 0.05) enhanced the inactivation of spores of FP strain SM101. However, under similar conditions, no significant spore inactivation was observed with NFB strain NB16. Interestingly, while the spores of FP isolates were able to germinate with AK upon their adhesion to SS chips, no significant germination was observed with spores of NFB isolates. Consequently, the incorporation of AK-induced germination step prior to decontamination of SS chips with disinfectants significantly (p < 0.05) inactivated the spores of FP isolates. Collectively, our current results showed that triggering spore germination considerably increased sporicidal activity of the commonly used disinfectants against C. perfringens FP spores attached to SS chips. These findings should help in developing an effective strategy to inactivate C. perfringens spores adhered to food contact surfaces.

Detection, cloning, and sequencing of the enterotoxin gene of Clostridium perfringens type C isolated from goat

Clostridium perfringens enterotoxin (CPE) is an important virulence factor involved in C. perfringens food poisoning and other gastrointestinal illnesses. In our experiment a Clostridium perfringens enterotoxin gene (cpe) positive type C strain was obtained by multiplex PCR from 34 C. perfringens isolates from goat in Guizhou Province, China. Then a pair of specific primers was designed and synthesized according to the published cpe gene, and the complete cpe gene was amplified from the genomic DNA of the cpe-positive C. perfringens isolate. The PCR product was cloned into pMD18-T vectors and transformed into competent cell DH5a. The recombinant plasmid was identified by PCR and restriction enzyme then sequenced and analyzed. The results showed that the cloned gene was 960 bp in length; the gene coded 319 amino acids, which shared 99.4~99.8% sequence identity and 99.1~-99.7% amino acid sequence identity with reference strains in GenBank. These results provide a basis for further research on the structure, properties, and biological activities of Clostridium perfringens enterotoxin. It also set the groundwork for further investigation of the mechanism of disease caused by C. perfringens enterotoxin.

Enterotoxigenic &lt;i&gt;Clostridium perfringens&lt;/i&gt;: Detection and Identification

Recent advances in understanding the genetics of enterotoxigenic Clostridium perfringens, including whole genome sequencing of a chromosomal cpe strain and sequencing of several cpe-carrying large plasmids, have led to the development of molecular approaches to more precisely investigate isolates involved in human gastrointestinal diseases and isolates present in the environment. Sequence-based PCR genotyping of the cpe locus (cpe genotyping PCR assays) has provided new information about cpe-positive type A C. perfringens including: 1) Foodborne C. perfringens outbreaks can be caused not only by chromosomal cpe type A strains with extremely heat-resistant spores, but also less commonly by less heat-resistant spore-forming plasmid cpe type A strains; 2) Both chromosomal cpe and plasmid cpe C. perfringens type A strains can be found in retail foods, healthy human feces and the environment, such as in sewage; 3) Most environmental cpe-positive C. perfringens type A strains carry their cpe gene on plasmids. Moreover, recent studies indicated that the cpe loci of type C, D, and E strains differ from the cpe loci of type A strains and from the cpe loci of each other, indicating that the cpe loci of C. perfringens have remarkable diversity. Multi-locus sequence typing (MLST) indicated that the chromosomal cpe strains responsible for most food poisoning cases have distinct genetic characteristics that provide unique biological properties, such as the formation of highly heat-resistant spores. These and future advances should help elucidate the epidemiology of enterotoxigenic C. perfringens and also contribute to the prevention of C. perfringens food poisoning outbreaks and other CPE-associated human diseases.

Inhibitory Effects of Nisin Against Clostridium perfringens Food Poisoning and Nonfood‐Borne Isolates

The enterotoxigenic Clostridium perfringens type A is the causative agent of C. perfringens type A food poisoning (FP) and nonfood-borne (NFB) human gastrointestinal diseases. Due to its ability to form highly resistant endospores, it has become a great concern to the meat industry to produce meat free of C. perfringens. In this study, we evaluated the antimicrobial effect of nisin against C. perfringens FP and NFB isolates. No inhibitory effect of nisin was observed against germination of spores of both FP and NFB isolates in laboratory medium. However, nisin effectively arrested outgrowth of germinated spores of C. perfringens in rich medium. Interestingly, germinated spores of NFB isolates possessed higher resistance to nisin than that of FP isolates. Furthermore, nisin exhibited inhibitory effect against vegetative growth of both FP and NFB isolates in laboratory medium, with vegetative cells of NFB isolates showing higher resistance than that of FP isolates. However, the antimicrobial activity of nisin against C. perfringens was significantly decreased in a meat model system. In conclusion, although nisin showed inhibitory effect against spore outgrowth and vegetative cells of C. perfringens FP and NFB isolates in laboratory conditions, no such effect was observed against C. perfringens spores inoculated into a meat model system.

A Mild Outbreak of Gastroenteritis in Long-Term Care Facility Residents Due toClostridium perfringens, Australia 2009

Clostridium perfringens food poisoning is a commonly cited cause of gastroenteritis outbreaks among elderly long-term care facility (LTCF) residents, yet little is known about the natural history of disease in this vulnerable group. In July 2009, an investigation into diarrheal illness among LTCF residents was commenced. An environmental health investigation and retrospective cohort study were undertaken to confirm the outbreak, to identify a source and mode of transmission, and to implement public health measures to prevent further cases. Menu listings and food safety program details were obtained and food-handling practices were observed. Clinical notes of all residents were reviewed. A possible case was defined as any resident developing one or more acute loose stool episodes between the evenings of 23 July and 27 July. Fifty-two residents (41%) had been ill with diarrhea, and eight residents had fecal samples positive for C. perfringens enterotoxin. LTCF staff failed to perform routine temperature checks on hot foods before the outbreak. A sweet-and-sour pork lunch served on 23 July was implicated in causing residents' illness, but no residual food remained for microbiological testing. Independent associations with illness were demonstrated among residents living in two wings of the facility that received a standard level of service, whereas an inverse association with illness was shown among residents living in an "extra service" wing. Male residents were also more likely to become ill. Illness was mild with case patients reporting a median of two loose stools (range 1-12). C. perfringens is an important cause of both foodborne and nonfoodborne gastroenteritis outbreaks in LTCF, but may be missed due to the often mild nature of illness. This investigation highlights the potential burden of C. perfringens disease among vulnerable LTCF populations. To prevent C. perfringens outbreaks, facilities must adhere to food safety plans and ensure high standards of infection control practice.

Clostridium perfringens in London, July 2009: two weddings and an outbreak

Food poisoning outbreaks caused by Clostridium perfringens enterotoxin occur occasionally in Europe but have become less common in recent years. This paper presents the microbiological and epidemiological results of a large C. perfringens outbreak occurring simultaneously at two weddings that used the same caterer. The outbreak involved several London locations and required coordination across multiple agencies. A case-control study (n=134) was carried out to analyse possible associations between the food consumed and becoming ill. Food, environmental and stool samples were tested for common causative agents, including enterotoxigenic C. perfringens. The clinical presentation and the epidemiological findings were compatible with C. perfringens food poisoning and C. perfringens enterotoxin was detected in stool samples from two cases. The case-control study found statistically significant associations between becoming ill and eating either a specific chicken or lamb dish prepared by the same food handler of the implicated catering company. A rapid outbreak investigation with preliminary real-time results and the successful collaboration between the agencies and the caterer led to timely identification and rectification of the failures in the food handling practices.