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

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Molecular biology and genetics of anaerobes Molecular biology and genetics of anaerobes

Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China Yu Liu a, b, 1, Li Xiu a, b, 1, Zengmin Miao c, Hairong Wang a, b, * a

Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, Tai'an, Shandong, 271018, China Shandong Provincial Key Laboratory of Animal Biotechnology and Disease Control and Prevention, Shandong Agricultural University, Tai'an, Shandong, 271018, China c Taishan Medical College, Tai'an, Shandong, 271000, China b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 May 2019 Received in revised form 18 August 2019 Accepted 11 September 2019 Available online xxx

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. © 2019 Elsevier Ltd. All rights reserved.

Keywords: Duck products Clostridium perfringens Antimicrobial resistance Multilocus sequence typing Phylogenetics

1. Introduction Clostridium perfringens is an anaerobic, spore-forming, Gram-

* Corresponding author. Shandong Provincial Engineering Technology Research Center of Animal Disease Control and Prevention, Shandong Agricultural University, 61 Daizong Road, Tai'an, Shandong, 271018, China. E-mail address: [email protected] (H. Wang). 1 The authors contributed equally to this work.

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. [3e6]. 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

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Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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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 (a), B (a, b, ƹ), C (ɑ, b), D (a, 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 [9e11]. 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 7e10) 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. 2. Materials and methods 2.1. 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 and directly placed on shelves for sale without being refrigerated or packaged. Three types of duck products including intestines, livers, and carcasses were randomly collected. Samples from containers, water, cutter and chopping board etc of two retail stores as well as fresh faeces from healthy shopkeepers and employees (adults without diarrhea) of these four stores were also collected. The intestines and livers were rapidly transferred into sterile sampling bags after purchase, the carcass and environmental samples were wiped with cotton balls containing 2 mL sterile PBS. Each sample of carcass was swiped on areas (at least 100 cm2) [19] including the inner surface and the outer surface and immediately transferred into the sterile sample bags after the wipe was completed. Samples were transported to the laboratory within 1 h in a freezer box. Other isolates of C. perfringens were obtained from a suspected NE-affected chicken (farm1, n ¼ 4), two healthy chickens (farm1 and farm2, n ¼ 2), and a duck with enteritis, which belonged to a farm (farm3, n ¼ 1) around Tai'an. 2.2. Isolation and identification of C. perfringens The intestine segments of about 20 mm, 3 g liver tissue, carcass and environmental samples in PBS (0.5e1 mL) was placed in fluid thioglycollate medium (FTG) broth and incubated in anaerobic condition (90% N2, 10% CO2) for 8 h at 42  C with shaking at 180 rpm. Subsequently, the pre-enriched FTG broth was cultured on tryptose-sulfite-cycloserine (TSC) agar (Qingdao Hope BioTechnology Co., Ltd), and then purified on a 5% defibrinated sheep blood agar (Qingdao Hope Bio-Technology Co., Ltd) and incubated at 37  C for 24 h under anaerobic condition. AnaeroPackAnaero (MITSUBISHI GAS CHEMICALCO., INC, Japan), a disposable oxygen-absorbing and carbon dioxide-generating agent, was used in the process of cultivation. C. perfringens was identified by colony morphology, cell morphology and hemolytic characteristics (grampositive bacterium under a microscope, black colonies on TSC agar, dual hemolysis on sheep blood agar). Two to five colonies suspected to be C. perfringens were plated on agar plates for purification. All positive colonies were used for antimicrobial susceptibility tests, pathotyping and toxin genes detection, and duck origin strains for MLST were selected according to the antibiotic resistance profiles and origins (Fig. 1). At least one strain was selected from each antibiotic resistance profile for typing. All chicken origin strains were used for MLST and only one strain of every person was selected from each C. perfringens-positive sample at random. 2.3. DNA extraction Genomic DNA from fresh bacterial solution was extracted by one-step bacterial genome extraction kit (Beijing Norbelai Biotechnology company, China), dissolved in double distilled water of 50 mL and stored at 20  C.

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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C. perfringens isolate, and isolates were also assayed for the presence of cpb2, cpe, tpeL, and netB genes [1,22]. C. perfringens reference strains, including C. perfringens type A, National Collection of Type Culture (NCTC) 528 (cpa); type C, NCTC3180 (cpb), NCTC 4989 (cpb, cpb2); type D, NCTC 8346 (etx) and type E, NCTC8084 (iap, cpe), were used as positive controls of toxin typing. 2.6. Sequencing of housekeeping genes The primers of eight housekeeping genes ddla (D-Ala-D-Ala ligase), dut (dUTP nucleotidohydrolase), glpk (Glycerol kinase), gmk (Deoxyguanylate kinase), plc (Phospholipase C (alpha toxin)), sod (Superoxide dismutase), recA (DNA repair), and tpiA (riosephosphate isomerase) were synthesized by using the MLST scheme developed by JOST et al. [10]. PCR assays were performed in final volumes of 25 mL, containing 10  Buffer (1.5 mM MgCl2) 2.5 mL, dNTP (2.5 mM) 4 mL, each primer (20 mM) 0.5 mL, Taq enzyme (2.5 U/ mL) 0.25 mL, and DNA template (50 ng/mL) 2 mL, and the remainder was supplemented with double distilled water. Reactions were performed with an initial denaturation at 95  C for 5 min, followed by 35 cycles at 94  C for 30 s, 50  C for 30 s, and 72  C for 40 s, and a final extension at 72  C for 7 min. To correct the size of the PCR amplicon, PCR markers (2000 bp) were used as the standards. Then, the PCR products were submitted to the sequencing company (Beijing ruiboxing technology company, China) for sample purification and automated nucleotide sequencing in both directions. 2.7. Multilocus sequence typing and evolutionary relationship analysis

Fig. 1. Heat map representation of antibiotic resistance profiles of the 99 C. perfringens isolated from duck products, retail environment and healthy human. The left vertical axis lists the different retails or hosts and the horizontal axis is labeled with the antibiotics (AeL). The color scale bar represents the range of inhibition zone between 0 and 40 mm. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.) Notes: A for penicillin; B for cefotaxime; C for cefepime; D for ciprofloxacin; E for flufenicol; F for lincomycin; G for bacitracin; H for erythromycin; I for tetracycline; J for sulfamethoxazole; K for gentamicin.

2.4. Antimicrobial susceptibility test The Kirby-Bauer disk diffusion method was applied for antimicrobial susceptibility test [15], in accordance with the guidelines of British Society for Antimicrobial Chemotherapy (BSAC). Antibiotics were selected based on their use in poultry and human. These antibiotics include penicillin (1 UI), cefotaxime (30 mg), cefepime (30 mg), lincomycin (20 mg), ciprofloxacin (5 mg), florfenicol (30 mg), bacitracin (10 mg), erythromycin (15 mg), tetracycline (30 mg), sulfamethoxazole (300 mg), and gentamicin (10 mg). C. perfringens reference strain ATCC13124 was used as a quality control strain for antimicrobial susceptibility test. The inhibition zone was measured according to BSAC methods for antimicrobial susceptibility [20]. Standards that are not listed can be found from the instructions by the manufacturer (Hangzhou microbial reagent co. LTD). Resistance of bacitracin was defined as bacitracin  9 mm. 2.5. Toxin gene detection Toxin genes including plc, cpb, etx, and iap were detected by using a previously published multiple PCR assay [21] for each

Genetic relationship of 58 C. perfringens was analyzed using MLST. These isolates were selected based on their origin (different stores and hosts) and antimicrobial resistance profiles according to a previous study [23]. Eight housekeeping genes successfully sequenced by bidirectional sequencing were assembled by the DNASTAR 8.0 software package (available at http://www.dna star. com), and ambiguities were resolved during assembling, after which, all examined genes were aligned and trimmed to an equal length by the BioEdit software (available at http://bioedit.software. informer.com) according to the reference sequence of each allele. At the same time, the nucleotide sequence of C. perfringens ATCC13124 (accession number: NC-008261.1), Strain13 (accession number: NC_003366.1) [24] and SM101 [25] were obtained from NCBI repository for comparison. After assembling, data of all examined genes (FASTA files) were imported into Bionumerics software (Bionumerics, version 7.6 (3); Applied Maths, Inc., Austin, TX) to create an allele database. After imported, sequences of all 58 isolates were first compared by alleles to obtain the allele numbers and then by alleles and profiles to obtain the sequence types (STs) using BioNumerics. Based on one representative of each ST, linkage disequilibrium between all examined genes was calculated in START2 software package (http://pubmlst.org/software/analysis/ start2), the Maynard-Smith index of association (IA) was calculated to test for recombination, and the ratio of synonymous to nonsynonymous mutation (dN/dS) was computed by the NeiGojobori method as a measure of selection [26]. The eBURST tool (http://eburst.mlst.net/) for MLST analysis was used to group STs into clonal complex (CC) (identified as groups of independent isolates that share identical alleles at seven or more of the eight loci in this study). Single ST and a possible ancestor of each clonal complex were also identified [27]. Both STs and CCs were considered to be C. perfringens MLST subtypes [25]. The phylogenetic relationship of all examined strains and the allelic differences among different STs were identified, and a minimum spanning tree was drawn by Bionumerics using the MST

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3. Results

cefepime was less than 5% (Table 3). All isolates were resistant to 2 to 9 antibiotics. Strains which are resistant to three or more classes of antibiotics are defined as multidrug resistant, the proportion of multidrug resistant isolates was 90.10% (73/81). And the proportion of strains which showed resistance to six classes of antibiotics was the highest with a rate of 21% (17/81). Resistance to eight and nine classes of antibiotics accounted for 11.1% (9/81), but no isolates showed resistance to all antibiotics used in this study (Table 3). Different antibiotic resistance profiles were observed in human isolates. For example, resistance against erythromycin, sulfamethoxazole, tetracycline, and penicillin was 88.90%, 27.80%, 27.80%, and 5.60%, respectively. Based on the heat map (https://www.graphpad. com/register/confirmation/), we can intuitively see the differences in the similarity of antibiotic resistance of strains from different sources. We also found that the resistance profiles of stains from the same store had high similarity, and the resistance profiles were significantly different in strains from different stores or hosts (Fig. 1).

3.1. Occurrence of C. perfringens

3.4. Allelic analysis

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) isolates from each positive sample were identified. A total of 99 isolates were obtained in all positive samples (Table 1).

The diversity of MLST loci in 58 strains of C. perfringens is shown in Table 4. Polymorphism of gmk gene is the lowest with only four different alleles, and the highest polymorphism was observed in glpk gene with 21 alleles, followed by plc gene with 20 alleles. The average number of alleles for all loci is 13.9. The polymorphism index was determined by the percentage of polymorphic sites. The percentage of polymorphism for plc gene was the highest. The mutation site accounted for 8.60% of all sites, while the percentage of polymorphism for gmk and tpi gene was the lowest, with 3.70% and 3.40% of all sites, respectively (Table 4). All allelic sequences examined in this study were coding sequences; thus, the ratio of nonsynonymous to synonymous mutations was used as a measure of selective pressure on each allele. Based on this analysis, all genes possessed a dN/dS ratio of <1, indicating purifying selection. The recA gene possessed the minimum dN/dS value of 0.0063. A significant linkage disequilibrium was detected between the genes examined, as determined by classical-Maynard-Smith IA value of 1.1511 (P ¼ 0.000, based on one representative of each ST).

method. Sources of strains were indicated by different colors in the minimum spanning tree. For comparison, 20 strains (ST1-ST20 listed in Nakano's publication, and numbered as ST45-ST64 in this study) of C. perfringens isolated from healthy children reported by Nakano et al. [24] were also used for MLST in the minimum spanning tree. To better examine ST relatedness at the sequence-level revolution, the optimum inferred phylogenetic tree was generated by the neighbor-joining (NJ) and maximum composite likelihood (MCL) methods based on eight housekeeping genes which were concatenated together to form a sequence of 2449 bp, which was imported in MEGA7.0 to estimate evolutionary distances. The topology was validated by bootstrapping (1500 replicates) [28,29]. To display antibiotic resistance profiles of examined isolates, each evolutionary cluster was attached to the corresponding resistance profile (heat map), which was constructed by an online software (https://evolgenius.info/evolview-v2/).

3.2. Toxin gene screening All isolates (n ¼ 106) of different origins were identified as C. perfringens type A, which means that cpb, etx, and iap genes were not detected in all isolates. The cpb2 toxin gene was detected in 52 of 81 (64.20%) and 6 of 18 (33.33%) C. perfringens isolates from the retail stores (duck isolates and environmental isolates) and healthy adults, respectively. And the cpe gene was only identified in one strain isolated from healthy adults. The netB and Tpel toxin gene were not detected in all strains (Table 2, only the strains used for MLST are displayed). 3.3. Antibiotic resistance profiles Antimicrobial susceptibility testing showed that broad antibiotic resistance was observed in strains isolated from duck products and environment (Table 3). Resistance against sulfasoxazole was the most prevalent (95.10%), followed by gentamicin (88.90%), bacitracin (85.20%), lincomycin (74.10%), tetracycline (67.90%), erythromycin (55.60%), penicillin (32.10%), ciprofloxacin (23.50%), and florfenicol (8.60%). Resistance against cefotaxime, and Table 1 Positive rate of Clostridium perfringens from different samples. Source

No. of samples

Positive rate (%)

No. of isolatesa

Intestinal tract Liver Carcass Environment Total Human faeces

109 40 24 10 183 7

18/109 (16.50) 10/40 (25) 20/24 (83.30) 10/10 (100) 58/183 (31.70) 7/7 (100)

24 13 34 10 81 18

a

At least one C. perfringens was collected from each positive sample.

3.5. Sequence types and eBURST analysis Fifty-eight strains of C. perfringens from chicken, duck products, environment and healthy adults were successfully divided into 41 sequence types. The reference strains ATCC13124, Strain13 and SM101 segregated into individual sequence type containing a single isolate each (ST42-ST44), bringing the total number of STs to 44 (Table 2). Among 44 duck or environment origin strains, 33 unique STs were identified, and 6 isolates from NE and healthy chicken were divided into 3 STs [ST34 (n ¼ 1), ST35 (n ¼ 4), ST36 (n ¼ 1)]. Since none of the seven strains isolated from adults belonged to a single ST, they were divided into 7 different STs. Among the 44 STs, the most prolific ST was ST11 (n ¼ 5), followed by ST6 (n ¼ 4), ST35 (n ¼ 4), ST26 (n ¼ 3), and ST27 (n ¼ 3). Surprisingly, ST6 contained four strains from different hosts (adults, healthy chickens and ducks). Among the poultry and environment origin strains, 8.60% (3/35) of STs contained strains from different stores. ST11 contained isolates from four stores (store A, store B, store C, and store D), ST26 contained strains from two stores (store C and store D), and ST24 contained strains from two stores (store C and store D). ST35 contained four strains from a single suspected NE chicken (farm1), and ST34 contained only one strain from a healthy chicken (farm2). ST33 only contained a single strain from a diseased duck (farm3) (Table 2). BURST (eBURST implementation) analysis defined a CC as

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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Table 2 Strain number, host (source), associated disease, sequence type, and toxin genes. Strains

Host

Source

Stores (Farms)

Associated Disease

ST

Toxin genes cpa

a

etx

iap

cpb2

Cpe

e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e

e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e

þ þ e þ þ þ þ þ þ þ þ þ þ þ þ þ þ þ e e þ þ e þ þ e e þ þ þ þ

e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e e

ST26 ST26 ST27 ST26 ST27 ST28 ST27 ST29 ST11 ST30 ST31 ST24 ST32 ST33 ST34 ST35 ST35 ST35 ST35 ST6 ST36 ST37 ST38 ST39 ST40 ST6 ST41

þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e Toxin genes cpa Cpb þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e þ e

etx e e e e e e e e e e e e e e e e e e e e e e e e e e e

iap e e e e e e e e e e e e e e e e e e e e e e e e e e e

cpb2 e þ þ þ e e e þ þ þ þ þ þ þ e e e e e e e þ e e e þ e

Cpe e e e e e e e e e e e e e e e e e e e e e e e e e e þ

ST42 ST43 ST44

þ þ þ

e e e

e e e

e e þ

e þ e

N01 N02a N03 N04 N05 N06 N07 N08 N09 N10 N11 N12 N13 N14 N15b N16 N17 N18b N19 N20 N21 N22 N23 N24 N25 N26 N27 N28 N29 N30 N31 Strains

Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying Laying e e e Host

duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck duck

Ileum Jejunum Ileum Ileum Ileum Ileum Jejunum Jejunum Ileum Ileum Jejunum Ileum Jejunum Liver Liver Liver Liver Liver Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Carcass Cutter Shelf Floor Source

A A A A A A A A B B B C C B B B C D C C C C C C C C C C C C C Stores

Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy e e e AssociatedDisease

ST1 ST2 ST3 ST4 ST5 ST5 ST6 ST7 ST8 ST9 ST6 ST10 ST11 ST12 ST13 ST11 ST14 ST15 ST16 ST17 ST11 ST18 ST19 ST20 ST21 ST11 ST22 ST17 ST23 ST24 ST25 ST

N32 N33 N34 N35c N36c N37 N38 N39 N40 N41 N42 N43 N44 N45 N46 N47d N48d N49d N50d N51 1P 2P 3P 4P 5P 6P 7P ATCC13124d ATCC13124d SM101d Strain13d

e e Laying duck Laying duck Laying duck Laying duck Laying duck Laying duck Laying duck e e e e Shelduck Chicken Chicken Chicken Chicken Chicken Chicken Human Human Human Human Human Human Human

Water Water Carcass Carcass Carcass Carcass Carcass Carcass Carcass Floor Cutter Bucket Shelf liver Liver Liver Jejunum Caecum Ileum Jejunum Faeces Faeces Faeces Faeces Faeces Faeces Faeces

C C D D D D D D D D D D D Farm Farm Farm Farm Farm Farm Farm A B B C D A D

e e Healthy Healthy Healthy Healthy Healthy Healthy Healthy e e e e Enteritis Healthy NE NE NE NE Healthy Healthy Healthy Healthy Healthy Healthy Healthy Healthy

Human e e

e FP Soil

e e e

G e e

3 1 1 1 1 1 2

cpb

e e e

þ: presence of the gene; -: absence of the gene; NE: necrotizing enteritis; G: gas gangrene; FP: food poisoning. a Strains isolated from the same intestinal sample. b Strains isolated from the same liver sample. c Strains isolated from the same carcass sample. d Clostridium perfringens reference strain.

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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Table 3 Prevalence (%) of antibiotic resistance in 99 strains of Clostridium perfringens isolated from Poultry Market and healthy adults in Tai'an. Antibiotics

Cefotaxime Cefepime Penicillin Lincomycin Ciprofloxacin Florfenicol Bacitracin Tetracycline Sulfasoxazole Gentamicin Erythromycin a

No. (%) of antibiotic resistance isolates

No. of antimicrobial resistance classes of environmental and duck isolates and their dominent antibiotic resisrance profiles (a)

Human isolates

2R (n ¼ 8)

0 (0) 0 (0) 1/18 (5.60) 17/18 (94.40) 0 (0) 1/18 (5.60) 12/18 (66.70) 5/18 (27.80) 5/18 (27.80) 18/18 (100) 16/18 (88.90)

environmental and duck isolates

3e5R (n ¼ 33)

6R (n ¼ 17)

7R (n ¼ 14)

8R (n ¼ 7)

9R (n ¼ 2)

a

4/81 (4.90) 0/81 (0) 26/81 (32.10) 60/81 (74.10) 19/81 (23.50) 7/81 (8.60) 69/81 (85.20) 55/81 (67.90) 77/81 (95.10) 72/81 (88.90) 45/81 (55.60)

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

a

Isolates in corresponding antibiotic resistance profiles showed resistance to this antibiotic.

Table 4 Diversity at the Clostridium perfringens MLST loci. Genes

Sequences (bp)

No. of alleles

(%) of allelesa

No. (%) of polymorphic locib

dN/dSc

ddla dut glpK gmk plc recA sod tpiA

265 259 446 321 327 298 265 268

16 11 21 4 20 11 15 10

14.80 10.20 19.40 3.70 18.50 10.10 13.90 9.30

21 (7.90) 17 (6.60) 25 (5.60) 12 (3.70) 28 (8.60) 25 (8.40) 22 (8.30) 9 (3.40)

0.0505 0.0466 0.1186 0.0467 0.1262 0.0063 0.0126 0.0851

a b c

Percentage of alleles to all isolated strains (n ¼ 58). Percentage of polymorphic loci to all alleles. Calculated in the START2 software package by the method of Nei-Gojobori.

comprising isolates in which seven of eight alleles were identical. In total, four CC subtypes, containing 37.95% (22 of 58) of the 58 isolates, were identified. Three reference strains do not belong to any clonal complex groups. Thirty-six STs were identified as singletons with no observed CC associations (Fig. 2). CC1, the largest CC, contained carcass isolates, intestinal isolates and environmental isolates from four stores and five STs (ST1, ST11, ST24, ST26, and ST27), with a total of 14 strains which accounted for 24.13% (14 of 58) of all examined strains. CC2 grouped strains from duck products and the cutter (ST10, ST22, ST28, and ST31). CC3 contained strains from intestine and mesa (ST8, and ST32). CC4 only contained intestinal strains (ST7, and ST9). All other STs were clustered singly by the eBURST analysis. After adding STs kindly donated by Nakano et al., three new clonal complexes (CC5-CC7) were generated. CC5 contained a retail environmental isolate and an intestinal isolate of a healthy child (ST30, and ST59). CC6 contained duck intestinal isolates and two isolates of a healthy child intestinal tract (ST4, ST47, and ST58) (Fig. 2). Based on ratios of single and multiple-locus variants, eBURST analysis was able to identify potential founding or ancestral genotypes for CCs with more than two members. eBURST identified ST27 and ST4 as the potential ancestral genotypes for CC1 and CC6, respectively. Whereas multiple candidates (ST10, and ST31) for the ancestral genotype were identified for CC2, no ancestral genotype was predicted for CC3, CC4, and CC5 (Fig. 2).

contained at least one and at most four strains. Based on the differences of alleles, the evolutionary relationship between CC2 (this study) and CC5 (this study and Nakano's ST) is relatively close, and only two alleles are different from each other; except for the difference of six alleles between CC3 and CC2, the evolutionary relationships between other clonal complexes were far apart, with a difference of eight alleles. The evolutionary relationship between CC4 and other clonal complexes was the furthest, as CC4 distributed in the periphery of the whole minimal spanning tree. A special STST6 had only two alleles different from CC7 (human strains from V. Nakano). The evolutionary relationship between reference strains SM101, Strain.13 (ST43, ST44) and human strains 1P (ST36), 4P (ST39) was the closest, with a difference of two and three alleles, respectively. However, ATCC13124 (ST42) had the closest evolutionary relationship with the environmental strain N42 (ST31, CC2), and there was also a difference of two alleles (Fig. 3). The results of phylogenetic trees are basically consistent with the minimum spanning trees, but not completely (Figs. 3, and Fig. 4). Strains in the same clonal complex were usually clustered together. Strains in CC1, CC3 and CC4 were clustered together, but there were exceptions. For example, four strains in CC2 were assigned to different clusters of the tree (Fig. 4).

3.6. Phylogenetic analysis

Carcasses and cuts after slaughtering can be contaminated by environment and poultry intestinal microorganisms [30]. An incidence level of 30e80% 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

The minimal spanning tree of 81 C. perfringens strains was drawn using the MST method in Bionumerics based on alleles and STs (Fig. 3). The minimum spanning tree was mainly composed of seven clonal complexes (CC1-CC7), accounting for 27.30% of all strains, and accompanied by the distribution of single ST which

4. Discussion

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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Fig. 2. Distribution of STs and clone complex groups of strains (n ¼ 81). The dendrogram was generated using the eBURST tool.

contamination rate (26%) reported in retail poultry in the United States [17]; the contamination rate of carcass was 83.30%, which is higher than that of 15.10% reported in central China [3], 65.90% of fresh and frozen chickens in Canada [18] and 42.90% of duck meat reported in South Korea [1]. The duck intestinal tract showed the lowest positive rate of C. perfringens occurrence (16.5%), which is slightly lower than that (23.1%) of chicken cloacal swab samples reported in central China [3]. Although the intestinal tract is the main habitat of C. perfringens [2], the low positive rate may be related to the fact that C. perfringens is not the dominant flora in the intestinal tract of ducks under many circumstances [7]. Overall, this study showed a relatively high occurrence rate of C. perfringens. All environmental samples were positive of C. perfringens. Although C. perfringens can exist as part of commensal flora, retail environment such as cutters and containers should be kept as clean as possible to prevent contamination. Environmental sampling can not only help us assess the sanitary conditions of the retail store, but also support us to trace the source of contamination in the duck carcass or liver. Otherwise, a previous study showed that bacteria from the air and the environment can contaminate broiler meat [31], the poor ventilation of the store which may lead to enrichment of C. perfringens spores in the air also may increase the contamination of duck products. It was speculated that the high contamination rate of C. perfringens in duck carcass and the environment was mainly related to the poor sanitary condition and lack of a disinfection procedure. This viewpoint was supported by the eBURST analysis that a portion of the isolates from duck products and environment were in the same CC or ST, and phylogenetically close. If the duck products from retail stores are uncleaned and uncooked, there will be a potential risk to public health. Therefore, reasonable cleaning and refrigeration in the sales processes as well as proper slaughtering and transport processes should be taken to effectively reduce the contamination of C. perfringens. In recent years, the abuse of antibiotic feed additives has led to the increase of antimicrobial resistance of some intestinal flora [15], and some zoonotic pathogens have developed multiple antimicrobial resistance. Previous studies have reported antibiotic resistance of C. perfrigens in food animals [1,12,15,16,23,32]. In Egypt [15], C. perfringens isolated from NE chickens were resistant to

gentamicin, lincomycin, erythromycin and ciprofloxacin; in south Korea [1], C. perfringens strains recovered from food supplied to school cafeterias show resistance to penicillin (3%), lincomycin (80%), bacitracin (3%), gentamicin (90%), tetracycline (7%), and erythromycin (17%). In Belgium [32], C. perfringens strains recovered from broilers were sensitive to bacitracin, enrofloxacin, erythromycin, and flufenicol, which showed lower antibiotic resistance compared to those in this study. This phenomenon may be related to the ban of antibiotics in animal feed by the European Union since 2006 [23]. In this study, the isolates from the poultry markets showed a relatively high antibiotic resistance, all isolates from duck products and environment showed resistance to at least two classes of antibiotics. A total of 49.40% of the isolates were resistant to at least 6 classes of commonly used antibiotics (Table 3), which may be associated with the fact that multiple antibiotics have been constantly used in the poultry industry of Mainland China. Multidrug-resistant C. perfringens was observed among these isolates in this study with a multidrug-resistant rate of 90.10%, which is higher than that of 53% reported by Mwangi et al. [23]. Fortunately, all isolates in this study were highly sensitive to cefotaxime or cefepime which can be used as the first choice for the treatment of C. perfringens related diseases in this region. Moreover, according to the heat map, the antibiotic resistance profiles showed relatedness with store sources which might be related to specific antibiotic usage in different farms (Fig. 1), as the same batch of duck products came from a single farm. In our study, based on the MLST scheme previously published by JOST [10], C. perfringens strains of different origins were genotyped at multiple loci. Through the polymorphism of alleles, we realized that considerable genetic diversity existed in the core genome of isolates in this study. MLST had been successfully applied to classify these isolates and compare the evolutionary relationships of C. perfringens from animal, environment and human. There were 13.5 alleles of the loci examined in this study on average. A total of 41 STs and four CCs were identified among C. perfringens strains from animal, environment and human. In comparison, JOST et al. [10] divided 132 strains of C. perfringens from different host species and toxinotypes into 80 STs and three CCs, with an average allele number of 24.4. Nakano et al. [24] identified an average of 10.25

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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Fig. 3. (1) (2): 58 strains of Clostridium perfringens from different sources were analyzed by MLST - minimal spanning tree, and 20 groups of human sequences from V. Nakano et al. were also used for analysis. Part of retail duck products isolates and environmental isolates were clustered together to form CC1, CC2 and CC3 (green circle shadow part), and two duck intestinal isolates were clustered together to form CC4 (green circle shadow part). A retail environmental isolate was clustered with a healthy adult (stall) isolate to form a CC5 (green circle red circle shaded part), a duck intestinal isolate and two human strains of V. Nakano's human isolates were clustered together to form CC6 (green circle red circle shaded part) and ST6 (three color circles) contained three different strains (healthy shopkeeper, healthy chicken and duck products). The minimum spanning tree was constructed by Bionumerics software (Bionumerics, version 7.6 (3); Applied Maths, Inc.,Austin, TX). The area of the circle represents the number of strains, different colors represent different sources, and the number on the branch represents the difference of alleles. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

alleles, 30 STs and three CCs among 40 strains from children and chicken. Chambers et al. [12] analyzed 61 C. perfringen isolates from NE and healthy chicken and an average of 5.9 alleles, 22 STs and six CCs were identified. Hibberd et al. [25] evaluated 139 isolates from poultry affected by necrotic enteritis (NE) and poultry gangrene (PG), and found an average of 12.2 alleles, 41 STs and six CCs. Though the average number of alleles in this study is not the highest compared to previous studies, the index of diversity in STs was the highest, as determined by Simpson's index of diversity (0.9665), which indicates that our isolates had considerable genetic diversity. Although considerable polymorphism was observed in the loci analyzed, four clonal complexes (CC1-CC4) were identified from 58 isolates in this study, mainly coming from strains recovered from duck products and the retail environment. A significant

linkage disequilibrium was observed among all genes examined, as determined by classical-Maynard-Smith IA (1.1511), indicating a low recombination rate in the genomes of the C. perfringens isolates examined. This hypothesis is substantiated by the observation that 22 of the 58 isolates (37.90%) were partitioned into four clonal complexes. Contamination of C. perfringens in raw meat from the retail market may occur in the slaughtering process or come from the retail environment [30,33,34]. In this study, CC1 and CC2 harbored five STs (ST1, ST11, ST24, ST26, and ST27) and four STs (ST10, ST22, ST28, and ST31), respectively, which mainly contained strains from carcass, intestine and retail environment. In CC2, ST10 (intestinal isolate) or ST31 (cutter isolate) were identified as the most probable ancestors (Fig. 2), indicating fecal material or retail environment

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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Fig. 4. Dendrogram of sequence types from 58 C. perfringens from human (P) and animal (N). The dendrogram was generated using the Neighbor-Joining and maximum compositelikelihood (MCL) method (1500 replicates) by MEGA 7.0. Antibiotic resistance profiles of these isolates are also shown in this graph (0e40 stands for inhibition zone). Notes: PG: for penicillin; CTX: for cefotaxime; CPM: for cefepime; CIP: for ciprofloxacin; FON: for flufenicol; LIN: for lincomycin; BAC: for bacitracin; ERY: for erythromycin; TET: for tetracycline; SLZ: for sulfamethoxazole; GEN: for gentamicin.

might be the sources of contamination. ST27 (carcass isolates) was the most probable ancestor of CC1, which indicates that cross contamination might have occurred. Moreover, our strains (of duck or environment origin) which had considerable genetic diversity, were not clustered within the STs described by Chalmers et al. [12] or Hibberd et al. [25] but clustered within STs (of children origin) reported by Nakano et al. [24] as well as STs (of adult origin) in this study. For example, CC6 contained strains of different sources (duck products and children), and ST4 (duck intestinal isolate) was identified as the most probable ancestor of CC6. Also, ST6 contained strains of adult and duck intestinal tract origin. The above results indicated that C. perfringens recovered from duck products poses a potential risk of transmission through the food chain to healthy humans. In the future, more methods can be used to further verify this conclusion. Little has been reported on multilocus sequence typing of duckderived C. perfringens, therefore, it is quite difficult to find a control (healthy or disease duck) on the website. We are interested in the differences of genetic diversity between healthy and diseased individuals caused by C. perfringens, and the ducks sold in the retail stores are presumably healthy ducks, so we added strains newly isolated from suspected NE chicken and healthy chicken for control. In our study, strain N01 (ST1) and N02 (ST2) recovered from the

intestine of a single duck belonged to different STs. A similar result was observed in N15 (ST13) and N18 (ST15), which were recovered from a single liver sample. Four strains recovered from the liver, jejunum, caecum and ileum of a single suspected NE-affected chicken showed no genetic differences in all alleles among them and were assigned to the same ST (ST35) (Fig. 3). In this way, strains isolated from healthy birds seemed to have higher genetic diversity compared to those isolated from NE-affected birds, which is consistent with the previous study reported by Chalmers et al. [12]. Pathotyping results showed that all isolates were identified as C. perfringens type A which is a major globe category associated with food poisoning [1,5], which is consistent with that in China and other countries [1,3,24]. Beta2 toxin, which can be produced by all types of C. perfringens [1], has been found in many animal species [35]. In this study, toxin gene cpb2 was detected in strains from duck products and human with a positive rate of 54.30% and 33.33%, respectively. In a previous study, 38.5% of C. perfringens strains from chicken meat in Japan contained the cpb2 gene [36]. In South Korea, approximately 50.0% of isolates from animal meat were positive for the cpb2 gene [1]. In the strains of animal and children origin reported by Nakano et al. [24], cpb2 was not detected. Our study showed a relatively high positive rate of cpb2 gene, and cpb2 in C. perfringens type A has been shown to be

Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102

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associated with gastrointestinal diseases in human and animal [1,10]. Among all the isolates used for MLST, 77.30% of the strains harbored cpb2 gene, and the presence of cpb2 was observed in CC1CC6. Enterotoxin gene, which was not only closely associated with antibiotic-associated diarrhea but also outbreaks of food poisoning [37e40], was not detected in duck isolates. However, C. perfringens isolates involved in many cases of food poisoning are cpe negative. In a recent research of France, the cpe gene was not detected in strains related to 13 outbreaks [6], 43.26% (61/141) of strains implicated in foodborne outbreaks were cpe-negative; in Japan, four foodborne outbreaks caused by a new type of enterotoxin producing C. perfringens were described [41]; and C. perfringens can also cause hemolysis-associated C. perfringens septicemia without clear infection source [42]. Therefore, the risks to public health cannot be underestimated. And cpe gene was detected in human strains in this study with a positive rate of 14.20%, which is close to that (18%) of cpe gene in faeces of food handlers reported by Annamari et al. [38]. Annamari described that food poisoning caused by cpe-positive strains is mediated by the people who carry these strains and the people who handle the food or raw materials. Shopkeepers who handle duck products may cause contamination in duck products and even mediate food poisoning if a cpe-positive strain is introduced by them. The netB and Tpel toxin gene were not detected in all isolates, as the netB and Tpel toxin gene were closely associated with NE-affected chickens [7,12,13,24,25]. Our study described the high antibiotic resistance in the isolates from duck products, environment and human, and genetic relatedness were also observed in these isolates. We found that the strains from the same clonal complex were usually clustered together (not completely), which is related to the numbers of point mutation in all alleles. Therefore, the combination of minimum spanning tree and phylogenetic tree can help us better analyze the genetic relationship of isolates (Fig. 4). Antibiotic resistance profiles of the strains in the same CC seem to be more similar than the strains in different CCs (Fig. 4). In summary, this is the first report showing a MLST scheme of C. perfringens of duck origin. We found that the C. perfringens contamination rate in some duck products in Tai'an retail market was relatively high, and these isolates exhibited broad-spectrum antimicrobial resistance. Although all the isolates belong to type A, considerable genetic diversity was observed. MLST showed that cross contamination might occur, animal intestinal contents and retail environment were presumed to be the main causes of contamination, and a portion of strains from healthy humans and duck products was found to be phylogenetically close, indicating that antimicrobial-resistance strains of duck origin pose a potential risk to humans by spreading through the food chain. Thus, adequate measures should be taken to reduce the contamination of duck products. 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.

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Please cite this article as: Y. Liu et al., Occurrence and multilocus sequence typing of Clostridium perfringens isolated from retail duck products in Tai'an region, China, Anaerobe, https://doi.org/10.1016/j.anaerobe.2019.102102