Prevalence, toxin-typing, and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul, Korea

doi:10.1016/j.anaerobe.2020.102235

Anaerobe

Volume 64, August 2020, 102235

https://doi.org/10.1016/j.anaerobe.2020.102235 Get rights and content

Highlights

•
We investigated Clostridium perfringens in beef, chicken, and pork meat.
•
All isolates were cpe-negative C. perfringnes type A.
•
Thirty of 38 isolates (78.9%) were resistant to multiple drugs.

Abstract

Clostridium perfringens is a ubiquitous, Gram-positive, spore-forming bacterium. It can contaminate many types of retail meat products and cause food poisoning by producing enterotoxins in the small intestines of humans and domestic animals. We investigated the prevalence, toxin-encoding gene profile, and antimicrobial resistance of C. perfringens in beef, chicken, and pork meat purchased from retail markets in Seoul, Korea. C. perfringens was detected according to the International Organization for Standardization 7937, with some modifications, and confirmed using the Vitek 2 system. In total, 38 C. perfringens strains were isolated from 200 meat samples (38/200, 19%; thirty-three from chicken, and five from beef). Among the six toxins evaluated, including alpha, beta, epsilon, iota, enterotoxin (encoded in the cpe gene), and netB, only the cpa gene was detected in all isolates by polymerase chain reaction (PCR) amplification. The antimicrobial resistance of the isolates was evaluated using the agar dilution method and resistance to ampicillin (12/38, 31.6%), tetracycline (38/38, 100%), chloramphenicol (26/38, 68.4%), metronidazole (13/38, 34.2%), and imipenem (27/38, 71%) was observed. Interestingly, 30 of the 38 isolates (78.9%) were multiple-drug resistant, showing resistance to more than three different antimicrobial classes.

Introduction

Clostridium perfringens is one of the most prevalent spore-forming, rapidly growing pathogenic bacteria in the world [1]. It is widely distributed in the environment and is present in soil, sewage, food, and dust [2]. C. perfringens has been recognized as a major public health risk, causing various human, and veterinary diseases [3]. Thus, food authorities in several countries have established the tolerance limit for the existence of C. perfringens in raw meat products; Korea has zero tolerance policy for meat products that are consumed raw and the USA has a performance standard of no more than one log growth during a stabilization step (cooling) after heat treatment [4,5].

Most diseases caused by C. perfringens are mediated by one or more toxins [6]. C. perfringens is classified into seven toxigenic types, A to G, based on the production of six major toxins: alpha (α), beta (β), epsilon (ε), iota (ι), enterotoxin (CPE), and NetB [7]. All toxigenic types of C. perfringens produce the α toxin that is encoded in the cpa gene. Additionally, type B produces β and ε toxin, type C produces β toxin, type D produces ε toxin, type E produces ι toxin, type F produces CPE, and type G produces NetB [7]. C. perfringens food-borne illness is caused by type F strains that were formerly called CPE-positive C. perfringens type A strains [8]. Thus, determining the toxin type of C. perfringens isolates is critical to better describe the potential risk of the isolates and to trace the source of contamination in various food production steps [9].

C. perfringens causes a toxico-infectious food-borne illness, in which both infection by viable bacterial cells as well as their toxins plays an important role in causing gastroenteritis in the host [10]. C. perfringens infections cause gas gangrene and food poisoning in humans and necrotizing enteritis in animals [11]. To minimize the economic losses caused by these infections, many antimicrobials, such as ampicillin, tetracycline, chloramphenicol, metronidazole, and imipenem, have been used preemptively in the livestock industry in several countries [12]. As a result, antimicrobial resistance of C. perfringens to tetracycline, lincomycin, and erythromycin has increased significantly over the past three decades [13]. Despite increasing awareness of the importance of combating antimicrobial resistance to improve public health, resistance of C. perfringens from various sources to antimicrobial agents has not been actively investigated over the past decades, even in the most common source of its infection: the meat samples [14]. In this study, the prevalence and toxin type of C. perfringens in retail meat samples in Korea was investigated and the antimicrobial resistance of C. perfringens isolates to the most frequently employed antimicrobial agents (ampicillin, tetracycline, chloramphenicol, metronidazole, and imipenem) was evaluated using agar dilution method.

Section snippets

Samples

Two hundred retail meat samples, including 50 beef, 100 chicken, and 50 pork meats, were purchased from department stores (large stores with complex distribution structures selling various goods including foods), wholesale markets (groups of wholesale establishments selling meat samples directly from manufacturers), and online markets (selling various foods, including meat products, online) in Seoul, Korea, from June to August in 2018. All meat samples were either raw or processed (chopped).

Prevalence of C. perfringens in retail meat samples

In total, 38 C. perfringens strains were isolated from the 200 meat samples. The highest prevalence was observed in chicken meat (33/100, 33%), followed by beef (5/50, 10%); C. perfringens was not detected in any of the pork samples in this study.

Toxin type of the C. perfringens isolates from retail meat samples

Toxin genotyping of the 38 meat isolates revealed that all isolates were type A (Table 1), showing positive results only for the cpa gene and were negative for the cpe, cpb, etx, iap, and netB genes (Fig. 1).

Antimicrobial resistance of the C. perfringens isolates from meat samples

The MIC values and antimicrobial

Discussion

In the present study, we investigated the prevalence of C. perfringens in beef, chicken, and pork meat samples purchased from retail meat markets in Seoul, Korea and evaluated the antimicrobial resistance of the bacteria. Several studies have investigated the prevalence and distribution of C. perfringens in retail food samples, mostly in western countries [12, 26, 27]. The present study is one of the few studies to identify the prevalence of this bacterium in Korea [14, 16, 19, 21]. Our data

Declaration of competing interest

None.

Acknowledgements

This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (NRF-2018R1A2A2A14021671), and by the Korea Institute of Planning and Evaluation for Technology in Food, Agriculture, Forestry and Fisheries (IPET), through the Agriculture, Food and Rural Affairs Research Center Support Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (119055-2). This paper was also supported by Konkuk University Researcher Fund in 2019.

References (41)

M.A. Khan et al.
Distribution and antimicrobial resistance patterns of Clostridium perfringens isolated from vaccinated and unvaccinated goats
Small Rumin. Res.
(2019)
L. Petit et al.
Clostridium perfringens: toxinotype and genotype
Trends Microbiol.
(1999)
C.G. Baums et al.
Diagnostic multiplex PCR for toxin genotyping of Clostridium perfringens isolates
Vet. Microbiol.
(2004)
J.P. Yadav et al.
Molecular characterization and antimicrobial resistance profile of Clostridium perfringens type A isolates from humans, animals, fish and their environment
Anaerobe
(2017)
A. Johansson et al.
Antimicrobial susceptibility of Swedish, Norwegian and Danish isolates of Clostridium perfringens from poultry, and distribution of tetracycline resistance genes
Vet. Microbiol.
(2004)
D.W. Hecht
Anaerobes: antibiotic resistance, clinical significance, and the role of susceptibility testing
Anaerobe
(2006)
S. Gharaibeh et al.
Molecular typing and antimicrobial susceptibility of Clostridium perfringens from broiler chickens
Anaerobe
(2010)
W.S. Hu et al.
Molecular genotyping, biofilm formation and antibiotic resistance of enterotoxigenic Clostridium perfringens isolated from meat supplied to school cafeterias in South Korea
Anaerobe
(2018)
S. Brynestad et al.
Clostridium perfringens and foodborne infections
Int. J. Food Microbiol.
(2002)
J.I. Rood et al.
Expansion of the Clostridium perfringens toxin-based typing scheme
Anaerobe
(2018)

D.A. Rowe-Magnus et al.

The role of integrons in antibiotic resistance gene capture

Int. J. Med. Microbiol.

(2002)

B. Ngamwongsatit et al.

Multidrug resistance in Clostridium perfringens isolated from diarrheal neonatal piglets in Thailand

Anaerobe

(2016)

S.M. Gubash et al.

Comparison of a new, bismuth-iron-sulfite-cycloserine agar for isolation of Clostridium perfringens with the tryptose-sulfite-cycloserine and blood agars

Zentralbl. Bakteriol.

(1997)

S. Hosseinzadeh et al.

Molecular characterization of Clostridium perfringens isolated from cattle and sheep carcasses and its antibiotic resistance patterns in Shiraz slaughterhouse, southern Iran

Vet. Arh.

(2018)

U. Tansuphasiri et al.

Antimicrobial resistance among Clostridium perfringens isolated from various sources in Thailand

Southeast Asian J. Trop. Med. Publ. Health

(2005)

Ministry of Food and Drug Safety

Processing Standards and Ingredient Specifications for Livestock Products

(2019)

New 2017 food code section bandages, finger cots, or finger stalls

L.A. Llanco et al.

Toxinotyping and antimicrobial susceptibility of Clostridium perfringens isolated from broiler chickens with necrotic enteritis

Int. J. Microbiol. Res.

(2012)

M.A. Navarro et al.

NanI sialidase is an important contributor to Clostridium perfringens type F strain F4969 intestinal colonization in mice

Infect. Immun.

(2018)

K.R. Matthews et al.

Food Microbiology: an Introduction

(2017)

Cited by (23)

Prevalence, toxin-genotype distribution, and transmission of Clostridium perfringens from the breeding and milking process of dairy farms
2024, Food Microbiology
This study aimed to elucidate the distribution, transmission, and cross-contamination of Clostridium perfringens during the breeding and milking process from dairy farms. The prevalence of 22.3% (301/1351) yielded 494 C. perfringens isolates; all isolates were type A, except for one type D, and 69.8% (345/494) of the isolates carried atyp. cpb2 and only 0.6% (3/494) of the isolates carried cons. cpb2. C. perfringens detected throughout the whole process but without type F. 150 isolates were classified into 94 pulsed-field gel electrophoresis (PFGE) genotypes; among them, six clusters contained 34 PFGE genotypes with 58.0% isolates which revealed epidemic correlation and genetic diversity; four PFGE genotypes (PT57, PT9, PT61, and PT8) were the predominant genotypes. The isolates from different farms demonstrated high homology. Our study confirmed that C. perfringens demonstrated broad cross-contamination from nipples and hides of dairy cattle, followed by personnel and tools and air-introduced raw milk during the milking process. In conclusion, raw milk could serve as a medium for the transmission of C. perfringens, which could result in human food poisoning. Monitoring and controlling several points of cross-contamination during the milking process are essential as is implementing stringent hygiene measures to prevent further spread and reduce the risk of C. perfringens infection.
Comparative evaluation of two molecular multiplex syndromic panels with acute gastroenteritis: Multiplex Syndromic Tests for Gastroenteritis
2024, Diagnostic Microbiology and Infectious Disease
We compared the Allplex Gastrointestinal V/B1/B2 Assays and Seeplex Diarrhea V/B1/B2 ACE Detection Assays in patients with acute gastroenteritis (AGE). Of the total 432 specimens, 48.8% and 54.9% samples were positive for any bacterial or viral target using Seeplex and Allplex, respectively (P = 0.002). The overall percent agreement (OPA) between the two panels was >95% and the lowest OPA was 95.4% for CdB. Allplex identified 40 samples positive for Salmonella spp., while Seeplex and OBC identified only 27 (67.5%) and 8 (20%), respectively. Shigella spp. were detected by assays in six samples, but none were identified using culture. Clostridium perfringens with Seeplex was detected in 70 (16.2%). It remained an informative species in identifying AGE although cpe gene showed only 9.8% positivity. Pathogenic Escherichia coli with Allplex could be detected in 40 (9.3%) samples, which could provide valuable information for the diagnosis of AGE.
Efficacy of Bacillus subtilis to replace in-feed antibiotics of broiler chickens under necrotic enteritis-challenged experiments: a systematic review and meta-analysis
2023, Poultry Science
Necrotic enteritis (NE) and coccidiosis are among the most prevalent infectious diseases in broiler chickens, contributing to large profitability losses. Bacillus subtilis is a promising direct-fed probiotic to counter various pathogens infection in broiler chickens. Here, we performed a meta-analysis to investigate the effects of B. subtilis on broiler chickens performance. A total of 28 studies were selected according to a PRISMA checklist. Random-effect model and mixed-effect model of meta-analysis were fitted to estimate the overall effects of B. subtilis (BS) treatment compared to either the control group (CON) or NE-infected group (NE_inf) as a baseline. Hedges' g effect size and its variance were used as estimators of standardized mean difference (SMD) calculation where the results were presented at a 95% confidence interval (95% CI) of the SMD. Overall, NE_inf broiler chickens depressed (P < 0.01) body weight (BW), average daily gain (ADG), and feed intake, and elevated (P < 0.01) feed conversion ratio (FCR). Treatment with BS improved ADG and final BW of NE_inf with no difference (P = 0.15) between BS and antibiotics (AB), indicating that they had comparable efficacy to treat NE in broiler chickens. BS supplemented to uninfected CON (BSS) improved (P < 0.01) final BW, ADG, and FCR. Compared to CON, BS, and AB failed to recover the FCR but these treatments decreased (P < 0.01) FCR when compared to the NE_inf group with similar efficacy (P = 0.97). As expected, NE_inf birds had a higher mortality rate (P < 0.01) and higher lesion score (P < 0.01) compared to CON, and treatment using AB and BS successfully decreased (P < 0.01) the mortality rate and lesion score. Compared to BS, AB was more effective to lower (P = 0.01) mortality rate, but comparable (P = 0.65) to minimize lesion score. To conclude, B. subtilis could be an effective natural additive to replace in-feed antibiotics in broiler chickens challenged with C. perfringens. However, the efficacy to reduce mortality rate was better with antibiotics treatment.
Prevalence, toxinotyping, antimicrobial susceptibility and biofilm-forming ability of Clostridium perfringens isolated from free-living rodents and shrews
2022, Anaerobe
Citation Excerpt :
Of the 27 isolates, 9 (33.3%) were found to be harbouring cpb2 gene, which has the potential for giving rise to antibiotic-associated diarrhoea (AAD) & sporadic diarrhoea (SD) and aggravating the diarrhoeal symptoms in infected patients [26]. The absence of cpe, iap, etx and NetB toxin genes are in concordance with some previous C. perfringens studies conducted in India and other parts of the world [7,28,36]. Usually, all C. perfringens strains possess a cpa gene that is located on the chromosome; nevertheless, the additional toxin genes, cpb, iap, etx, and, NetB are constantly positioned on plasmids, excluding cpe, which may be present either on the plasmids or chromosome [9,37,38].
Clostridium perfringens (C. perfringens), is a spore-forming and toxin-producing pathogenic Gram-positive rod-shaped bacterium with immense public health/zoonotic concern. Rodents are well-known reservoirs and vectors for a large number of zoonoses and strong links have been recognized between synanthropic rodents and foodborne disease outbreaks throughout the world. To date, no study has been conducted for studying the prevalence of C. perfringens in rodents and shrews. In this study, we investigated faecal samples from free-living rodents and shrews trapped in Meghalaya, a North-eastern hill state of India for the presence of virulent and antimicrobial-resistant C. perfringens.
A total of 122 animals comprising six species of rodents and one species of shrews were trapped: Mus musculus (n = 15), Mus booduga (n = 7), Rattus rattus (n = 9), Rattus norvegicus (n = 3), Bandicota indica (n = 30), Bandicota bengalensis (n = 32) and Suncus murinus (n = 26). The faecal swabs were collected and processed for the isolation of C. perfringens. Toxinotyping was done using PCR. Antimicrobial susceptibility testing and biofilm forming ability testing were done using Kirby Bauer disc diffusion method and crystal violet assay.
C. perfringens was isolated from 27 of the 122 faecal swabs (22.1%), from six species of rodents and shrews. Five of the host species were rodents, Bandicota bengalensis (25%), Bandicota indica (16.7%), Rattus norvegicus (33.3%), Mus musculus (13.3%), Mus booduga (42.8%) and Suncus murinus (shrew) (29.6%). The common toxinotype was type A (59.2%) followed by Type A with beta2 toxin (33.3%), Type C (3.7%) and Type C with beta2 toxin (3.7%). None of the isolates harboured cpe, etx, iap, and NetB genes and therefore none was typed as either B, D, E, F, or G. Nine isolates (33.3%) turned out to be multi-drug resistant (MDR), displaying resistance to three or more categories of antibiotics tested. Twenty-three out of twenty-seven isolates (85.2%) were forming biofilms.
Globally, this is the first study to report the prevalence of C. perfringens and its virulence profile and antimicrobial resistance in free-living rodents and shrews. The rodents and shrews can potentially contaminate the food and environment and can infect humans and livestock with multi-drug resistant/virulent Type A and Type C C. perfringens.
Tracing Clostridium perfringens strains from beef processing of slaughter house by pulsed-field gel electrophoresis, and the distribution and toxinotype of isolates in Shaanxi province, China
2022, Food Microbiology
Citation Excerpt :
A similar rate (4%) of cpe-positive isolates from with cpe locating on plasmid was reported from Japan (Miki et al., 2008). Jang et al. (2020) found all C. perfringens isolates were type A and negative for the cpe gene in retail beef in Seoul. A recent study showed 33.3% (6/18) C. perfringens isolates carried chromosomal cpe and 27.8% (5/18) carried plasmid cpe gene from bovine (Park and Rafii, 2019).
The purpose of this study was to investigate the distribution and specify the transmission and cross-contamination of Clostridium perfringens (C. perfringens) in the beef slaughtering and butchering process. The prevalence of 21.2% (150/708) yielded 208 isolates of C. perfringens, including 80.8% type A and 19.2% type D, 0.4% (3/708) samples carried both type A and D strains, and 72.5% type D isolates carried both cpe and atyp.cpb2 genes. C. perfringens were identified through the whole slaughtering process but no type F (cpe and cpa isolates) was found. 69 isolates were further analyzed and classified into 28 PFGE genotypes and clade I contained 94.2% isolates and 24 PFGE genotypes, which showed the genetic diversity and epidemic correlation. Our study traced C. perfringens contamination along the handling processes and showed a gradually ascending contamination rate during the whole process, revealing widespread cross-contamination from the feces and hides of slaughtered cattle to the carcass in the slaughtering workshop, so as from tools and personnel to meat of the cutting workshops. Strains from different slaughterhouses (regions) have high homology, and type A is the predominant toxinotype. It is necessary to monitor and control several key points of cross-contamination during slaughtering process to reduce a risk of C. perfringens infection.
Prevalence and characterization of Clostridium perfringens isolated from different chicken farms in China
2021, Anaerobe
Citation Excerpt :
Poultry feed ingredients contaminated by microorganisms may be considered in relation to the performance of birds and also with the public health significance [22]. Genotyping results showed that 98.72% of the isolates were identified as type A, and the other isolates were Type F, which was consistent with previous reports [23,24]. Considerable evidence implicates cpe as the toxin responsible for the gastrointestinal symptoms of C. perfringens type F food poisoning [25].
Clostridium perfringens (C. perfringens) is a common pathogenic microorganism present in nature, which can cause animal and human diseases, such as necrotizing enteritis (NE) in poultry. Little is known about the current prevalence status of C. perfringens from poultry farms of different types and regions in China. From December 2018 to August 2019, we investigated the prevalence, genotype distribution and drug resistance of C. perfringens from Guangdong, Pingyin, Tai'an and Weifang. A total of 622 samples were collected and processed for C. perfringens isolation, among which 239 (38.42%) samples were determined to be positive for C. perfringens. A total of 312 isolates of C. perfringens were recovered (1–5 strains were isolated for each positive sample), and 98.72% of the isolates were identified as type A, while the others were type F. Antimicrobial susceptibility testing revealed that 47.71% of the isolates were resistant to at least five classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 74 representative isolates were divided into 63 sequence types (STs), and the Simpson's diversity index (Ds) of the STs for the five farms was 0.9799. 37.84% of the isolates were classified into seven clonal complexes (CC1–CC7), and the isolates from the same farm were more concentrated in the minimum spanning tree. In addition, some cloaca isolates and feed isolates were distributed in the same ST or CC; this result indicates that the C. perfringens in chicken can come from the environment (feed etc.).

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Anaerobes in animal diseasePrevalence, toxin-typing, and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul, Korea

Highlights

Abstract

Introduction

Section snippets

Samples

Prevalence of C. perfringens in retail meat samples

Toxin type of the C. perfringens isolates from retail meat samples

Antimicrobial resistance of the C. perfringens isolates from meat samples

Discussion

Declaration of competing interest

Acknowledgements

Small Rumin. Res.

Trends Microbiol.

Vet. Microbiol.

Anaerobe

Vet. Microbiol.

Anaerobe

Anaerobe

Anaerobe

Int. J. Food Microbiol.

Anaerobe

Int. J. Med. Microbiol.

Anaerobe

Zentralbl. Bakteriol.

Molecular characterization of Clostridium perfringens isolated from cattle and sheep carcasses and its antibiotic resistance patterns in Shiraz slaughterhouse, southern Iran

Vet. Arh.

Antimicrobial resistance among Clostridium perfringens isolated from various sources in Thailand

Southeast Asian J. Trop. Med. Publ. Health

Processing Standards and Ingredient Specifications for Livestock Products

New 2017 food code section bandages, finger cots, or finger stalls

Toxinotyping and antimicrobial susceptibility of Clostridium perfringens isolated from broiler chickens with necrotic enteritis

Int. J. Microbiol. Res.

NanI sialidase is an important contributor to Clostridium perfringens type F strain F4969 intestinal colonization in mice

Infect. Immun.

Food Microbiology: an Introduction

Anaerobes in animal disease
Prevalence, toxin-typing, and antimicrobial susceptibility of Clostridium perfringens from retail meats in Seoul, Korea