Elsevier

Anaerobe

Volume 62, April 2020, 102102
Anaerobe

Molecular biology and genetics of anaerobes
Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China

https://doi.org/10.1016/j.anaerobe.2019.102102Get rights and content

Highlights

  • The C. perfringens contamination rate of some duck products in Tai'an retail store was relatively high, ninety-nine C. perfringens isolates were recovered. Most of isolates from duck products and environment exhibited broad-spectrum antimicrobial resistance among which 49.40% of them showed resistant to at least 6 of classes commonly used antibiotics.

  • MLST revealed that cross contamination might occur, animal intestinal contents and retail environment were presumed to be the main causes of carcass contamination.

  • Though all the isolates were identified as type A, considerable genetic diversity was observed in the strains.

  • A part of strains from human and duck was found to be phylogenetically close, indicating that antimicrobial-resistance strains of duck origin pose a potential threat to humans by spreading through the food chain.

Abstract

Clostridium perfringens is an important zoonotic microorganism, which can cause diseases in animal and human under certain conditions. Contamination of C. perfringens in chicken and pork meat has been reported worldwide, but it is rarely reported in duck products. The current study was undertaken to investigate C. perfringens contamination in duck products from a large retail market in Tai'an region, China and the serotype distribution, antimicrobial resistance and genetic relatedness of the isolates. In total, 173 samples of duck products, 10 samples of environmental origins and 7 samples of fresh faeces from healthy shopkeepers were collected between March and November 2018, of which, 58 (31.69%), 10 (100%) and 7 (100%) samples were determined to be positive for C. perfringens, respectively. Ninety-nine isolates of C. perfringens were recovered, all of which were identified as type A. Beta2 (cpb2) toxin gene was found in 54.30% and 33.30% of the isolates from duck products and healthy shopkeepers, respectively. Antimicrobial susceptibility testing revealed that 90.10% of the isolates from duck products and environment showed multiple antibiotic resistance, among which, 49.40% were resistant to at least 6 classes of commonly used antibiotics. Multilocus sequence typing (MLST) showed that 58 representative isolates were divided into 41 sequences types (STs), among which, ST11 (8.60%) was the most common; 37.90% of all isolates were classified into four clonal complexes (CC1-CC4). The most prolific clonal complex (CC1), accounting for 24.13% of all isolates, contained isolates mainly from carcass, animal intestinal contents and environment of four retail stores. A portion of human isolates and duck isolates was distributed in the same CC or ST. In conclusion, C. perfringens contamination in some duck products in Tai'an retail market was relatively high, and most of the isolates exhibited broad-spectrum antimicrobial resistance. Although all the isolates belong to type A, considerable genetic diversity was observed, and a portion of the strains from human and duck was found to be phylogenetically close. The results indicated that antimicrobial-resistance strains of duck origin pose a potential threat to humans by spreading through the food chain.

Introduction

Clostridium perfringens is an anaerobic, spore-forming, Gram-positive pathogen, known as one of the most prevalent food-borne pathogenic organism in the world [1]. This species is widely distributed in various environments and the gastrointestinal tract of healthy animals and human [2], which can cause animal necrotic enteritis, human gas gangrene, food poisoning and non-foodborne gastrointestinal diseases etc. [[3], [4], [5], [6]]. The pathogenicity of C. perfringens is largely attributable to its ability to produces a wide variety of exotoxins and enzymes, among which, toxins alpha, beta, epsilon and iota are the major lethal toxins [7]; these exotoxins and enzymes have been used for pathotyping of C. perfringens [5]. According to the production of these toxins, C. perfringens strains are classified into five pathotypes, including type A (α), B (α, β, ƹ), C (ɑ, β), D (α, ƹ), and E (ɑ, Ɩ) [8].

C. perfringens type A is the most common type that causes food poisoning [1,5]. Pathotyping is a classical method for classification of C. perfringens [8]. However, this method cannot further classify these strains into subtypes. In recent years, new molecular typing methods such as pulsed-field gel electrophoresis and multilocus sequence typing (MLST) have been developed which can be used to track the origin and transmission of pathogens and elucidate the inherent epidemiology of bacteria [[9], [10], [11]]. Maiden et al. used MLST to classify Neisseria meningitis for the first time in 1998 and achieved satisfactory result [11]. A MLST scheme of C. perfringens has been developed by Jost and collaborators [10]. MLST is conducted based on housekeeping genes (generally 7–10) amplified by PCR, after which allele number and sequence type (ST) of each isolate are obtained by sequencing. The difference of allele sequences caused by point mutation or recombinations result in the production of different alleles, and the permutation and combination of different alleles lead to the production of different sequence types [9]. Thus, MLST is highly repeatable and reliable, data sharing and comparison between different laboratories can be realized through the Internet [12]. This approach is of great value for tracking the origin of food poisoning and other diseases caused by this organism.

Toxin typing of C. perfringens including the evaluation of virulence genes, is an important supplement to the evaluation of population phylogenetic characteristics. C. perfringens enterotoxin and beta2 (cpb2) toxin which are encoded by cpe gene and cpb2 gene are considered to be significantly associated with human intestinal -associated diseases such as diarrhea, necrotizing enteritis and food poisoning [1,3,5]. In addition, C. perfringens isolates that produce netB toxin can cause necrotizing enteritis (NE) in chickens [7,12,13]. Enterotoxin and netB toxin that have been shown to be required for specific C. perfringens-mediated diseases are considered to be candidates for future C. perfringens toxinotypes [14].

Antibiotics have been widely used to treat necrotizing enteritis caused by C. perfringens and to promote the development of the poultry industry [15]. With the frequent usage of antibiotics, animal intestinal flora generates high antimicrobial resistance, which not only causes great difficulties in clinical treatment, but also seriously threatens public health. The use of antibiotics varies greatly in different countries and regions [16], and limited information is available on antibiotic resistance of C. perfringens of duck products in China. Hence, it is of great significance to test the antimicrobial susceptibility of C. perfringens of duck products in China for effectively controlling the dissemination of C. perfringens.

Food-borne C. perfringens infections worldwide are related to consumption of C. perfringens-contaminated animal food such as poultry, pork, beef and other food animals [2]. Duck products could also be a reservoir of C. perfringens [1]. Contaminated duck products can be a source of food borne disease, posing a threat to public health. There have been previous reports of C. perfringens contamination in chicken [1,3,4,17,18], but little has been reported on evaluating the risk of C. perfringens contamination in duck products and molecular and epidemiological characteristics of C. perfringens isolated from duck products. China is one of the top duck-consuming countries. It is of great significance to improve the detection of C. perfringens contamination in duck products for the prevention and control of diseases caused by C. perfringens.

This study was undertaken to investigate C. perfringens contamination in retail duck products in Tai'an region, China and serotype distribution, antimicrobial resistance and genetic relationship of isolates from duck products, retail environment and human. Hence, this epidemiological investigation of C. perfringens in duck products can not only provide data for public food security assessment, but also provide an epidemiological reference for animal and human disease associated with this microorganism.

Section snippets

Sample collection

In total, 173 duck product samples and 10 environmental samples were collected from four major stores in the retail market in the downtown of Tai'an from March to November 2018. The duck products at each retail store (store A-store D) came from different farms in the surrounding area of Tai'an. These retail stores were not well ventilated, in the poor facilities and lack of strict implementation of disinfection procedures. After slaughtering, the duck products were transported to these stores

Occurrence of C. perfringens

Among these 183 samples, 58 samples (31.70%) were confirmed to be positive of C. perfringens. Different positive rates of C. perfringens were observed in this study. Carcass (83.30%) was the most common food item to be contaminated by C. perfringens, followed by liver (25%), and intestinal tract (16.50%). The retail environment had the highest positive rate of C. perfringens occurrence (100%). All human faeces samples were positive of C. perfringens. At least one and at most four (C. perfringens

Discussion

Carcasses and cuts after slaughtering can be contaminated by environment and poultry intestinal microorganisms [30]. An incidence level of 30–80% in raw or frozen meat and poultry items has been described in a previous study [17]. In the current study, 27.70% of duck products were contaminated with C. perfringens, which is slightly higher than the contamination rate (22.60%) reported in the live poultry markets of central China [3] and similar to the contamination rate (26%) reported in retail

Conflicts of interest

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Acknowledgments

The authors thank Mr. Zengmin Miao for his guidance and suggestions on experimental ideas and technical help. We also thank Wenping Xu and Huining Zhang for sample collection. Sequence types were kindly donated by Dr. V. Nakano from Anaerobe Laboratory, Department of Microbiology, Institute of Biomedical Science, University of Sao Paulo, Sao Paulo, SP, Brazil. This study was supported by National Key Research and Development Program of China (2018YFD0500500) and Funds of Shandong “Double Tops”

References (42)

  • B. Shojadoost et al.

    The successful experimental induction of necrotic enteritis in chickens by Clostridium perfringens: a critical review

    Vet. Res.

    (2012)
  • T. Yamagishi et al.

    Polymerase chain reaction test for differentiation of five toxin types of Clostridium perfringens

    Microbiol. Immunol.

    (1997)
  • U. Rachel et al.

    Multi-locus sequence typing: a tool for global epidemiology

    Trends Microbiol.

    (2003)
  • M.C. Maiden et al.

    Multilocus sequence typing: a portable approach to the identification of clones within populations of pathogenic microorganisms

    Proc. Natl. Acad. Sci. U.S.A.

    (1998)
  • G. Chalmers et al.

    Multilocus sequence typing analysis of Clostridium perfringens isolates from necrotic enteritis outbreaks in broiler chicken populations

    J. Clin. Microbiol.

    (2008)
  • A.L. Keyburn et al.

    NetB, a new toxin that is associated with avian necrotic enteritis caused by Clostridium perfringens

    PLoS Pathog.

    (2008)
  • J.I. Rood et al.

    Expansion of the Clostridium perfringens toxin-based typing scheme

    Anaerobe

    (2018)
  • K.M. Osman et al.

    Antibiotic resistance of Clostridium perfringens isolates from broiler chickens in Egypt

    Rev. Sci. Tech. O I E

    (2013)
  • A. Martel et al.

    Susceptibility of Clostridium perfringens strains from broiler chickens to antibiotics and anticoccidials

    Avian Pathol.

    (2004)
  • L. Yuan-Tong et al.

    Enterotoxigenicity and genetic relatedness of Clostridium perfringens isolates from retail foods in the United States

    Appl. Environ. Microbiol.

    (2003)
  • GB/T GB 4789.9

    –Microbiological Examination of Food Hygiene-Detection of Campylobacter Jejuni

    (2014)
  • Cited by (12)

    • Prevalence and multilocus sequence typing of Clostridium perfringens isolated from different stages of a duck production chain

      2022, Food Microbiology
      Citation Excerpt :

      The smallest CC (CC4) contained 4.6% (3/65) of the detected isolates, was composed of ST39, ST20 and ST30, which contained strains isolated from cloacae at the age of 12, 27, and 42 days. After adding 7 human origin isolates (ST75 to ST81 in this study) donated by Liu et al. (2019), 7 unique types (ST75 - ST81) were added, among which ST80 enlarged CC3, resulting CC3 include isolates from the breeding stage (cloaca, feed), slaughtering stage (carcass after dehaiding, environmental samples), and human isolates. Viewing the whole phylogenetic trees, the dendrogram was found to be dominated by four large clusters, which contained all CCs, as well as a lots of closely related STs.

    • Isolation, toxinotyping and antimicrobial susceptibility testing of Clostridium perfringens isolated from Pakistan poultry

      2022, Anaerobe
      Citation Excerpt :

      As in some cases we have isolated more than one strain from the same flock of the same farm and there was no significant difference in toxinotyping of these isolates so we considered them as same isolates. Eight housekeeping genes (ddlA, dut, glpK, gmk, plc, recA, sud and tpi) were PCR amplified by using primers and conditions described previously [24,25] PCR reaction mixture (25 μL) was prepared using 12.5 μL 2X PCR master mix 1 μL of each forward and reverse primer, 1 μL template DNA and 14.5 μL PCR grade water. Presence and size of the PCR products were confirmed by 1.5% agarose gel electrophoresis.

    • Prevalence and antimicrobial susceptibility of Clostridium perfringens in chickens and pigs from Beijing and Shanxi, China

      2021, Veterinary Microbiology
      Citation Excerpt :

      cpb2-harbouring C. perfringens have been detected in many different animal species (van Asten et al., 2010) and the C. perfringens producing beta-2 toxin have been associated with enteritis in various animals, particularly in piglets (Revitt-Mills et al., 2019). Our study showed a relatively low prevalence rate of cpb2 (45.7 %, 147/322) compared to previous study (Liu et al., 2019; Ngamwongsatit et al., 2016). The subsequent WGS analysis showed that the toxin genes cpa, colA and nanH, encoding alpha toxin, microbial collagenase and sialidase, respectively, were conserved in all 48 representatives, which supported previous genomic studies (Mahamat Abdelrahim et al., 2019).

    • Prevalence and multilocus sequence typing of Clostridium perfringens isolated from 4 duck farms in Shandong province, China

      2020, Poultry Science
      Citation Excerpt :

      The primers of 8 housekeeping genes ddla, dut, glpk, gmk, plc, sod, recA, and tpiA were synthesized by using the MLST scheme developed by Jost et al. (2006). PCR conditions for the 8 housekeeping genes were as described in previous studies (Liu et al., 2020). The PCR products were submitted to the sequencing company (Tsingke Biological Technology Company, Co., Ltd., Beijing, China) for sample purification and automated nucleotide sequencing in both directions.

    View all citing articles on Scopus
    1

    The authors contributed equally to this work.

    View full text