- Email: [email protected]
Prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolated from retail food in China
Accepted Manuscript Prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolated from retail food in China Xian Zhong, Qingping Wu, Jumei Zhang, Shuxian Shen PII:
S0956-7135(15)30211-5
DOI:
10.1016/j.foodcont.2015.09.032
Reference:
JFCO 4666
To appear in:
Food Control
Received Date: 25 June 2015 Revised Date:
16 September 2015
Accepted Date: 24 September 2015
Please cite this article as: Zhong X., Wu Q., Zhang J. & Shen S., Prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolated from retail food in China, Food Control (2015), doi: 10.1016/j.foodcont.2015.09.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT 1
Prevalence, genetic diversity and antimicrobial susceptibility of
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Campylobacter jejuni isolated from retail food in China
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Xian Zhong 1, 2, Qingping Wu 1*, Jumei Zhang 1, Shuxian Shen1 1. Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology
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Southern China; Guangdong Provincial Key Laboratory of Microbial Culture Collection
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and Application, Guangdong Open Laboratory of Applied Microbiology, 510070, PR
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China
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2. Department of Chemical Engineering, School of Chemical Engineering and Light Industry,
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Guangdong University of Technology, Guangzhou, Guangdong 510006, PR China
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*Corresponding author:
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Qingping Wu
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Guangdong Institute of Microbiology, No. 100 Central Xianlie Road, Guangzhou 510070,
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China
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Tel: +86-20-87688132; fax: +86-20-87688132
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E-mail: [email protected] 1
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ABSTRACT
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The aim of this study was to determine the prevalence, antimicrobial resistance and molecular
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epidemiology of Campylobacter jejuni isolated from retail food samples in China and assess
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the potential risk to consumer’ health. A total of 34 strains of Campylobacter jejuni isolated
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from 1798 retail food samples in China were identified using conventional methods and
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duplex PCR aimed 16S and hipo genes. There are 59 positive samples by conventional
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method, and the prevalence of C.jejuni was 3.30% (59/1798). Using flaA RFLP-PCR (flaA
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Restriction Fragment Length Polymorphism) typing with a relative similarity coefficient of
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0.81, the 34 isolates and 1 reference strains were grouped into 4 clusters and 2 singletons.
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Antibiotic resistance profiles determined using the disk diffusion method showed that a high
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proportion of C.jejuni isolates were resistant to the tested antibiotics, particularly for
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ciprofloxacin (88.5%) and nalidixic acid (88.5%). Most isolates were multidrug resistant,
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with 20 strains showing resistance to 5-8 antibiotics and 2 strains showing resistance to 9
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antibiotics. Moreover, analysis using MAMA-PCR to determine the gyrA mutation status
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(C257T) indicated that approximately 30 strains had mutations, and these findings were
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consistent with disk diffusion assay results. Examination for the presence of 11putative
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virulence genes showed that the majority of these genes were detected in all C.jejuni strains.
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There was no direct evidence to suggest a connection between antibiotic resistance and
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virulence genes. This study is the first to explore the potential sources of C.jejuni
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contamination in retail foods in China and provide comprehensive surveillance on its
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incidence in retail foods. These data will ensure more accurate treatment of human
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campylobacteriosis using effective antibiotics.
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Keyword: Campylobacter jejuni; Prevalence; Antibiotic resistance; flaA RFLP-PCR;
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Virulence-related gene
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1. Introduction Campylobacter jejuni is a major food-borne pathogen that cause bacterial
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gastroenteritis worldwide (Qin, et al., 2011). As a zoonotic pathogen, it is transmitted to
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human via the consumption of contaminated food, especially poultry. The sources and
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transmission routes of campylobacter in staple foods are not fully understood, and the
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often contradictory epidemiological evidence described to date is partly attributable to
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the asymptomatic nature of C. jejuni infection and high levels of genetic diversity (Frost,
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2001). Several molecular typing methods have been used to study the epidemiology of
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campylobacter infections, such as multilocus sequence typing (MLST) (Kovac, et al.,
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2014), Pulsed Field Gel Electrophoresis (PFGE) (Zhang, et al., 2014), and flaA Short
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Variable Region Sequence (flaA- SVR) (Zhang, et al., 2014). In this study, we used flaA
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Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (flaA PCR-
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RFLP) subtyping method because it is not only a standard technique but is also rapid
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and cost effective (Ahmed, Dunn, & Ivanova, 2012; Bednarski, Wieliczko, &
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Mazurkiewicz, 2011).
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In the United States, an estimated 2.4 million cases of human campylobacteriosis occur
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each year (W. Bae, et al., 2005), and the most common species (approximately 80-90%)
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causing infection is C.jejuni. Most cases of campylobacteriosis do not require treatment, as
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they are of short duration, clinically mild and self-limiting. Antimicrobial treatment is
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necessary for systemic Campylobacter infections, infections in immune-suppressed patients
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and severe or long-lasting infections (Gibreel, et al., 2004). However, people sometimes 3
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veterinary medicine, the excessive use of these drugs is common. Currently, the increasing
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prevalence of campylobacter isolates that are resistant to clinical quinolone antibiotics,
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resulting in serious cases of human campylobacteriosis, warrants concern. An increase in the
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number of ciprofloxacin- resistant Campylobacter isolates has been reported in several
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countries (Agunos, et al., 2013; Nobile, Costantino, Bianco, Pileggi, & Pavia, 2013), and the
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rising levels of resistance to these antibiotics among Campylobacter spp. has been recognized
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as an emerging public health problem (J. Bae, Oh, & Jeon, 2014; Englen, Fedorka-Cray,
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Ladely, & Dargatz, 2005).
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ACCEPTED MANUSCRIPT improperly use antibiotics for the treatment of infections that tend to be self-limiting; in
Because little information has been published regarding the prevelance and antimicrobial
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susceptibility of C.jejuni isolated from food in the retail trade in China, the objective of this
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report was to determine the prevalence and antibiotic resistance of C.jejuni in staple foods
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while evaluating the risks to consumer health.
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2 Materials and methods
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2.1Sample collection
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A total of 1798 food samples representing seven different types of food, including dairy
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products, vegetables, cooked food, edible fungi, flash-frozen food, sea food and meat
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products, were collected from July 2013 to June 2014. The meat products including chicken,
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duck, pigeon, pork, beef, and bacon, except bacon, other meat was fresh, all meat products
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were raw. Sea food contains two types, saltwater fish and freshwater fish. The dairy product
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mainly was pasteurised milk. The cooked food includes baked food and cold food, such as 4
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character, and flash-frozen food were mainly dumplings and stuffed bun. The samples were
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obtained from supermarkets or farmers’ markets in 24 cities, representing most of provincial
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capitals of China. After collection, samples were tightly sealed in sterile plastic wrap and
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transported to the laboratory on ice in an insulated container.
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2.2 Isolation and identification of Campylobacter jejuni
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ACCEPTED MANUSCRIPT roast duck and cold noodles. The sample acquisition of vegetable and fungi according to local
The enrichment and isolation of C.jejuni was performed according to GB/T4789.9-2008
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for the microbiological examination of food hygiene (National Food Safety Standards of
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China) with slight Modifications. In brief, shears were used to cut the food samples under
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aseptic operation, and 25g of sample was mixed with 225ml of Bolton enrichment broth
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(Huankai, Guangzhou, China) and homogenized in a stomacher bag for1-2 min, followed by
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incubation under Microaerophilic conditions (5% O2, 10% CO2, 85% N2) at 42°C for 48 h. A
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loopful of broth enrichement culture was streaked onto Skirrow agar Plates (Huankai,
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Guangzhou, China) and incubated at 42°C for 48 h under microaerophilic conditions. Three
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or four presumptive colonies, which had similar colony morphologies that were moist and
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grey, were selected to inoculate onto Columbia sheep Blood Agar (Huankai, Guangzhou,
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China). After 48h of incubation under microaerophilic conditions, colonies were picked for
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further identification via Gram staining and an oxidase test. Confirmation of C.jejuni was
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performed using the API Campy system (bioMérieux, France) according to the
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manufacturer’s instructions, and all isolates identified as C.jejuni were further analysed by
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duplex PCR targeting the 16S and hipO genes (Denis, et al., 1999). The primers are shown
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inTable 1. We used C.jejuni CICC 22936 as a standard strain.
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2.3 flaA PCR-RFLP typing flaA PCR–RFLP typing is one of the simplest and most cost-effective genotyping methods
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for the investigation of large numbers of Campylobacter spp. The technique involves PCR
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amplification of the flaA gene and restriction enzyme digestion to generate simple restriction
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patterns (Harrington, Moran, Ridley, Newell, & Madden, 2003). In this study, DdeⅠ(Thermo
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Scientific, Waltham, US) was chosen as the digestion enzyme. The Primers FlaA-F and
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FlaA-R (Table 1) were used to amplify the flaA gene in C.jejuni. The PCR reaction mixture
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and procedure were as follows: 12.5µl of PCR mix (Dongsheng Biotech, Guangzhou, China),
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10mmol/µl primer 1µl, 2µl DNA (40-60ng/µl), add pure water up to final volum (25µl); initial
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denaturation at 94°C for 30 s, primer annealing at 55°C for 40 s, extension at 72°C for 40s,
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and a final extension step at 72°C for 4 min. The PCR products were purified using a DNA
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extraction kit (Magen, Guangzhou, China) prior to enzyme digestion. The fragments were
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separated on 2% agarose gels at 90 V for 1 h and visualized under a UV transilluminator gel
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imaging system (GE Healthcare, WI, USA). The images were saved as TIFF files for
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analysis.
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2.4Antibiotic susceptibility tests
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Antibiotic susceptibility tests were performed using the standard disk diffusion method on
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Muller-Hinton agar contained 5% defibrinated sheep's blood, following by BSAC
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standardized disc susceptibility testing method (version 11)(Howe, Andrews, & Party, 2012).
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Twelve antibiotic disks (OXIOD, UK) were used to perform the resistance test. The disc
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included ampicillin (AMP, 10µg), tetracycline (TE, 30µg), ciprofloxacin (CIP, 5µg),
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gentamicin (CN, 10µg), streptomycin (S, 25µg), erythromycin (E, 15µg), vancomycin (VA, 6
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ACCEPTED MANUSCRIPT 30µg), clindamycin (DA, 2µg), nalidixic acid (NA, 30µg), amoxicillin (AML, 20µg),
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cefoperazone (CFP, 75µg), and kanamycin (K, 30µg).
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2.5Detection of genetic determinants of antimicrobial resistance Isolates were subjected to MAMA-PCR to detect mutation of the quinolone (similar to
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ciprofloxacin and nalidixic acid) resistance-determining region (gyrase A gene,C257T)
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described by Zirnstein et al.(Zirnstein, et al., 1999) .PCR primers are shown in Table 1.
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2.6Determination of virulence-related genes
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Six PCR tests were performed to detect the presence of eleven virulence-related genes;
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specifically, five duplex PCRs were designed to detect rpoN, CdtA, CdtB, CdtC, flig, racR,
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ciaB, pldA, imaA and CadF genes, and an individual PCR was performed for cjaB gene. The
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primers were designed by primer premier 5.0.The specific genes and primer sequences are
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shown in Table 2.
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2.7Statistical analysis of antimicrobial resistance rates
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The frequencies of antimicrobial resistance patterns from different isolates were
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compared using a v2-test and Fisher’s exact two-tailed test with SPSS19.0 software. Values of
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p < 0.05 were considered statistically significant.
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3 Results
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3.1Contamination by Campylobacter jejuni
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A total of 1798 food samples were examined by conventional methods and duplex PCR 7
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C.jejuni was 3.30% (59/1798); for the most commonly contaminated food, i.e. meat, the
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positive rate was up to14.81% (57/385). The distribution represented in Table 3.
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Contamination of vegetable and sea food was rare (only two positive samples), and no
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contamination was detected in the other types of food. In terms of the geographic distribution,
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the prevalence in south China was higher than north China (3.7%/0.6%). The identification
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results for C.jejuni isolates using the API campy system (bioMérieux, France) were in
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agreement with those obtaining using duplex PCR.
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3.2 Antibiotic susceptibility tests and mutations of the gyrA QRDR.
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ACCEPTED MANUSCRIPT (see Fig.S1 in supporting information), and it was determined that the average incidence of
The antibiotic susceptibility results for 34 isolates are shown in Table 4. Isolates of
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C.jejuni were tested for different levels of antibiotic resistance. Resistance to AMP, TE, CIP,
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CN, S, E,VA, DA, NA, AML, CFP and K was found in 29.4%, 55.9%, 88.2%, 14.7%, 5.9%,
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2.94%, 85.3%, 11.8%, 88.2%, 38.2%, 82.4% and 23.5% of isolates, respectively. In general,
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the proportions of resistant C.jejuni against the tested antibiotics were severity, particularly
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for CIP and NA. Most isolates were multidrug resistant, 20 strains were resistant to 5-8
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antibiotics, and 2 strains were resistant to 9 antibiotics. Moreover, we found that 30 strains
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were resistant to both CIP and NA. The quinolone-resistant isolates had a change (from C to
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T) in codon 86 of the gyrA gene, resulting in a threonine-to-isoleucine substitution in the
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functional protein. MAMA-PCR results confirmed the presence of this mutation (see Fig.S2
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in supporting information).
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3.3Detection of virulence-related genes in C.jejuni isolates
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and were then examined for the presence of 11 virulence genes by PCR (see Fig.S3 in
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supporting information). The prevalence of virulence genes is shown in Fig.1. All strains
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harboured the fliG sequence. The rpoN and cadF sequences were found in 97.1% (33/34).
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CjaB was found in only two strains, and pldA and imaA were found in only three strains. With
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the exception of cjaB, pldA and imaA, the other genes tested were present in more than 67.6%
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of isolates. Additionally, almost every isolate carried 7-8types of virulence-associate genes.
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3.4 flaA PCR-RFLP typing of C.jejuni
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ACCEPTED MANUSCRIPT All 34 isolates obtained in this study were identified by biochemical tests and duplex PCR
The results of the PCR-RFLP analysis for 34 C.jejuni isolates and 1 reference strain are
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shown in Fig. 2. In this study, PCR-RFLP allowed for perfect discrimination using the
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Simpson’s index of diversity (D=0.95). The DNA profiles consisted of four to seven bands
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ranging from 100 to 3700bp, and the 150, 300, 900bp fragments were the main bands in most
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strains (see Fig.S4 in supporting information). At a relative similarly coefficient of 0.81, 21
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types of 34 isolates were placed into six clusters (designatedⅠ,Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ).
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ClusterⅠcontained all strains that had no mutations in gyrA gene, including 2523A, 2523-2B,
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330B, and 110B. These strains were more susceptible to antibiotics than other isolates and
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were only resistant to two types of antibiotics. Moreover, only 2523A and 110B had the
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virulence-related gene imaA. Cluster Ⅱcontained two strains that they carried the same
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virulence genes. In cluster Ⅲ, 2678 and 2274-2A shared the same type and also had the same
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virulence genes. It is possible that cross contamination occurred, as seen with the
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identification of 178-2A, 178-2B and178-2C in one samples, although they belonged to
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different clusters. Cluster Ⅳ and Ⅴwere singletons. The last cluster contained five stains
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4Discussion
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4.1Risky analysis
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ACCEPTED MANUSCRIPT that were isolated from Guangzhou.
To our knowledge, this is first study to examine the prevalence and antibiotic resistance of
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C.jejuni from retail foods in China. According to the findings of this study, the meat and its
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products are the main foods contaminated by C.jejuni that correlates with what we already
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know about source of C.jejuni. In terms of the geographic distribution, the prevalence in
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south China was higher than in north China (3.7%/0.6%), likely because C.jejuni is a
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thermopilic bacterium. The people of south china should take more effective action to control
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contamination. There was a low level of contamination by C.jejuni in retail foods in China.
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The average incidence of C.jejuni was only3.30%, which is far below other reports, although
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similar contamination levels were noted in fresh meat and poultry in particular (Jamali, et al.,
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2015; Mattheus, Botteldoorn, Heylen, Pochet, & Dierick, 2012; Ramonaite, et al., 2013). In
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November 2011 to August 2012, our laboratory have investigated the contamination of
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C.jejuni in retail foods in parts of south China, and had the similar positive rate of meat
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product and the average contamination, 13.2% and 2.51%, respectively(Zheng, et al., 2014).
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As part of the normal intestinal flora of poultry (Mackiw, Korsak, Rzewuska, Tomczuk, &
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Rozynek, 2012), the prevalence of C.jejuni in meat products, especially poultry, was high. To
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comprehensively evaluate the risk of this bacterium in our daily life, other types of food were
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tested, including dairy products, vegetables, cooked food, edible fungi, and flash-frozen food.
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Contamination in these types of food is rarely detected given the harsh living conditions of
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cross-contaminated from contaminated raw food (Mackiw, et al., 2012). Highly virulent C.
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jejuni strains may cause disease similar to those caused by Listeria monocytogenes (Bras,
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Chatterjee, Wren, Newell, & Ketley, 1999), Salmonella spp. (Williams, et al., 2015) and
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others. C.jejuni has several putative virulence genes that are associated with the cytolethal
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distending toxin (CDT) and certain stages of the infection cycle including adhesion(Konkel,
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Gray, Kim, Garvis, & Yoon, 1999) , intestinal colonization (Bras, et al., 1999) and invasion
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(Muller, Schulze, Muller, & Hanel, 2006) , and may be potentially pathogenic for consumers .
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Our results showed that greater than 70% of isolates harbored adhesion- and intestinal-
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associated genes(cadF, fliG, racR, rpoN), although invasion- associated genes(cjaB, pldA,
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imaA) were found in less than 8.8% of isolates, indicating that most of the strains demonstrate
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strong colonization but weak invasion ability, may because the sample is food, not clinic
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(Fonseca, et al., 2014). The CDT gene was detected at a prevalence of up to 80%, indicating
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that most of the isolates may possess full pathogenic potential. However, the presence of
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these virulence- related genes in a C.jejuni isolate does not always imply that it is capable of
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causing disease (Cho, Kim, Min, Ku, & Kim, 2014). Therefore, further studies are required to
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examine whether the strains isolated in this study are pathogenic. This is the first time
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C.jejuni risk has been analysed for retail food in China.
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4.2 Antibiotic susceptibility
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ACCEPTED MANUSCRIPT this bacterium; only two positive samples were detected and may have been
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C.jejuni is generally susceptible to a wide range of antibiotics as determined in the 1990s
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(Jacobsreitsma, Koenraad, Bolder, & Mulder, 1994; Li, Chiu, Wu, Huang, & Lin, 1998).
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Antimicrobial resistance has become a major public health concern in both developed and 11
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including the World Health Organization (WHO) (Moore, et al., 2006; Padungton & Kaneene,
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2003). A report on annual trends in resistance for different antibiotics, including ampicillin,
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ciprofloxacin, nalidixic acid, tetracycline, gentamicin, and erythromycin, from 2004-2009
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(Mattheus, et al., 2012) found that resistance for almost antibiotics has increased over time. In
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2009, the CIP, NA, and TE resistance reached 40% respectively, and in this current study, the
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data indicated that resistance had considerably increased for CIP (88.2%), NA (88.2%) , and
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TE (55.9%), which was similar to the results of Mackiw (Mackiw, et al., 2012).
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Fluoroquinolones are normally considered the first choice for the treatment of Campylobacter
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enteritis, although high-level resistance to fluoroquinolones has been reported throughout the
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world (Mackiw, et al., 2012; Mattheus, et al., 2012; Saenz, et al., 2000). Resistance to FQs is
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mainly attributable to a single point mutation in the DNA gyrase gene gyrA, particularly a
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Thr-86-Ile change mediated by the C257T mutation (Alfredson & Korolik, 2007). Almost
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88% (30/34) of strains had this mutation, and other reported mutations of gyrA in C.jejuni
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included Thr-86-Ala, although rarely occurred (Kinana, et al., 2006). Given the low number
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of strains in this study, it was not surprising that no strain appeared to have other mutations in
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gyrA genes. An increasing in ciprofloxacin-resistant C.jejuni may translate to greater
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prevalence of gastroenteritis in humans caused by the consumption of chicken meat
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contaminated by ciprofloxacin-resistant campylobacter. This naturally renders effective
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therapy more difficult (Mackiw, et al., 2012). These results represent a warning to take
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effective action to reduce the occurrence of resistance in C.jejuni quickly as possible
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4.3 flaA RFLP-PCR
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ACCEPTED MANUSCRIPT developing countries in recent years and has been recognized by various national authorities
12
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campylobacters due to its rapidity and simplicity, a highly gene diversity was identified in the
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C.jejuni isolated from retail food in China. We obtained a high percentage of unique strains,
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similar to the genetic heterogeneity found in numerous studies of human, animal and
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environmental isolates (Denis, et al., 2011; O'Leary, et al., 2011). There were 21flaA types
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identified among 35strains, which was in accordance with the results of Ramonaite
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(Ramonaite, et al., 2013) who found a similarly high genetic diversity. At a similarity
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coefficient of 0.81, a total of 34 isolates and 1 reference strain were divided into 4 clusters
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and 2 singletons. As shown in Fig.2, we observed four strains that were susceptible to CIP
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and NA belonging to the same cluster, and these similar antibiotic profiles represented greater
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susceptibility to the tested antibiotics than the other isolates. Isolates 2523A and 2523-2B
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were from the same sample but represented different types. Isolates 330B and 2523-2B were
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from different samples with different geographical distribution but shared the same type. This
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phenomenon implies that cross-contamination may have occurred, which is common with
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other food-borne pathogens, such as L. monocytogenes (Wu, Wu, Zhang, Chen, & Yan, 2015)
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and salmonella spp. (Wang, et al., 2015). In our study, we could not identify a direct
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relationship between antibiotic profiles and virulence-related genes.
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5. Conclusions
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ACCEPTED MANUSCRIPT By applying the PCR-RFLP method, which has been widely used for the genotyping of
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In conclusion, this study is the first systematical investigation of the prevalence and
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contamination levels of C.jejuni in retail foods in China. The average contamination (3.30%)
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was lower than other reports, although a high prevalence (14.81%) of C.jejuni in meat
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product was observed, especially for poultry, which showed that this food might be a major 13
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belonged to six clusters with a relative similarly coefficient of 0.81. High genetic diversity
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was observed based on PCR-RFLP subtyping, with 21 types identified among 34 isolates.
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The high antibiotic resistance observed in this research is alarming and raising public health
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concern. There was little relationship between antibiotic resistance and virulence genes.
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Additionally, mutations detected in the quinolone resistant determination region are caused
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for concerned. Thus, it is necessary to remind consumers, sellers, producers, carriers and
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Chinese food safety regulators to increase awareness regarding the importance of controlling
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antibiotics use. Effective measures should be implemented in food processing to control
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contamination.
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ACCEPTED MANUSCRIPT carrier of C.jejuni in retail foods in China. The isolates of C.jejuni from positive samples
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Acknowledgment
This work was supported by the Science and Technology Projects of Guangdong, China
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(No.2012B050800007) and the Science and Technology Projects of Guangzhou, China
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(No.2014J4100214).
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genetic diversity of Listeria monocytogenes isolated from retail ready-to-eat foods in
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Subtyping and Antimicrobial Susceptibilities of Campylobacter coli Isolates from
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Disease, 11(8), 610-619.
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Zheng, Y., Wu, Q., Wu, K., Zhang, J., Guo, W., & Wu, K. (2014). Virulence-associated gene
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detection and ERIC-PCR typing of Campylobacter jejuni strains isolated from foods in
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four Southern Chinese provinces. Acta Microbiologica Sinica, 54(1), 14-23. Zirnstein, G., Li, Y., Swaminathan, B., & Angulo, F. (1999). Ciprofloxacin resistance in
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Campylobacter jejuni isolates: Detection of gyrA resistance mutations by mismatch
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amplification mutation assay PCR and DNA sequence analysis. Journal of Clinical
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Table
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Table 1 Primers used and the expected amplicon sizes
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438
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Table 2 Virulence-related gene sequence
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Table 3 Distribution of C. jejuni in seven kinds of retail food in China
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“C”: “ conventional method”, “D”: ”duplex PCR”.
442 20
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Table 4 Results of antibiotic susceptibility tests of C.jejuni isolates from retail foods in China
445
*R: resistant; I: intermediate resistance; S: susceptibility.
447
Figure
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Fig.1 The eleven virulence-related genes in isolates
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Fig. 2 flaA RFLP- PCR DNA finger prints analyses of C.jejuni from retail foods in China. ND:
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not detected, SUS-CIP-NA: susceptibility to ciprofloxacin and nalidixic acid. I to VI refer to
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clusters 1 to 6 of C.jejuni according to RFLP-PCR. The numbers represent the type of
453
antibiotic resistance. “-’’: PldA, cjaB, and ImaA were not detected.
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Primers used and the expected amplicon sizes Target gene
Primer name
Oligo nucleotide sequence (5’-3’)
FlaA
FlaA-F FlaA-R
GGATTTCGTATTAACACAAATGGTGC
16s-F 16s-R
ATCTAATGGCTTAACCATTAAAC
Hipo-F Hipo-R
GTACTGCAAAATTAGTGGCG
Gyra-F Gyra-R
TTT TTA GCA AAG ATT CTG AT
(Denis, et al., 1999)
GCAAAGGCAAAGCATCCATA
CAA AGC ATC ATA AAC TGC AA
149
This study
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(Zirnstein, Li, Swaminathan, & Angulo, 1999)
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Thr-86-Ile mutations gyrA
857
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HipO
(Ramonaite, et al., 2013)
CTGTAGTAA TCTTAAAACATT TTG
GGACGGTAACTAGTTTAGTATT
Reference
1725
SC
16s
Sizes(in bp)
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Size (bp)
rpon
150
cdtA
403
cadF
158
Cjab
381
plda
913
cdtC
348
flig
469
cdtB
144
racR
338
imaA
518
ciab
986
Nucleotides sequence(5’-3’)
Annealing Temperature(°C) 52
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52
52
52
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GAACAGATTCTTCCTCCTT TGCTCTTACTCGCTCAA ACATTTGTGCGTGATTG AGCGGTGCTGATTTAGT TTTCTCACCCACATCAATA GTGTTAGACTTGGTTATCATTTT ACCTCGGCTTTATGGA GGAGCAGCATCCTACC AAGCTTATGCGTTTTT TATAAGGCTTTCTCCA ATAACTTTAGCCTTTGC AGTTGTTGTTGGCATT GTGGTGGTTTGGAATA TCTGCTAAGCGTTCAT TAATGTTGGCACTTGG GTGGCTGTTCTTGGTA AAGGCTATGATTGTCTG TTCAGCAGGAGTAAGTG GCACAAAATATATCATTACA TTCACGACTACTACTATGAGG TTTTTATCAGTCCTTA TTTCGGTATCATTAGC
45
45
45 45 50 50 45
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Detection method
Positive samples
Positive rate (%)
Meat product Vegetables Seafood Flash-frozen food Edible fungi Cooked food Dairy product Total
385 161 330 231 262 330 99 1798
C and D C and D C and D C and D C and D C and D C and D C and D
57 1 1 0 0 0 0 59
14.81 0.60 0.30 0 0 0 0 3.30
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“C”: “ Conventional method”, “D”:”Duplex PCR”.
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Types of products
ACCEPTED MANUSCRIPT Table 4 Results of antibiotic susceptibility tests of C.jejuni isolates from retail foods in China C.jejuni(n=34)
R
I
10 (29.4) 19 (55.9) 30 (88.2) 5 (14.7) 2 (5.9) 1 (2.94) 29 (85.3) 4 (11.8) 30 (88.2) 13 (38.2) 28 (82.4) 8 (23.5)
≦13 ≦11 ≦15 ≦12 ≦10 ≦13 ≦14 ≦14 ≦13 ≦13 ≦15 ≦14
13-16 12-14 16-20 13-14 11-12 14-22 15-16 15-20 14-18 14-17 16-20 15-17
4 (11.8) 2 (5.9) 0 (0) 1 (2.9) 1 (2.9) 6 (17.6) 1 (2.9) 3 (8.8) 0 (0) 10 (29.4) 1 (2.9) 2 (5.9)
20 (58.8) 13 (38.2) 4 (11.8) 28 (82.4) 31 (91.2) 27 (79.4) 4 (11.8) 27 (79.4) 4 (11.8) 11 (32.4) 5(14.7) 24 (70.6)
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*R: resistant; I: intermediate resistance; S: susceptibility.
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S
≧17 ≧15 ≧21 ≧15 ≧13 ≧23 ≧17 ≧21 ≧19 ≧18 ≧21 ≧18
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No.(%)of R No.(%)of I No.(%)of S
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Ampicillin(AMP) Tetracycline(TE) Ciprofloxacin(CIP) Gentamicin(CN) Streptomycin(S) Erythromycin(E) Vancomycin(VA) Clindamycin(DA) Nalidixicacid(NA) Amoxicillin(AML) Cefoperazone(CFP) Kanamycin(K)
Zone diameter(mm)
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Highlights C.jejuni was primarily determined in meat and meat products in retail foods in China
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C.jejuni isolates was showed high antibiotic resistance, especially quinolone
Virulence-related gene cjaB, pldA, imaA rarely contained in isolates.
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flaA RFLP-PCR subtyping were used for genetic diversity analyses.
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Supporting Information
Prevalence, genetic diversity and antimicrobial
retail food in China
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susceptibility of Campylobacter jejuni isolated from
Xian Zhong 1, 2, Qingping Wu 1*, Jumei Zhang 1, Shuxian Shen1
SC
1. Guangdong Institute of Microbiology, State Key Laboratory of Applied
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Microbiology Southern China; Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, 510070, PR China 2. Department of Chemical Engineering, School of Chemical Engineering
TE D
and Light Industry, Guangdong University of Technology, Guangzhou,
AC C
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Guangdong 510006, PR China
*Corresponding author: Qingping Wu
Guangdong Institute of Microbiology, No. 100 Central Xianlie Road, Guangzhou 510070, China Tel: +86-20-87688132; fax: +86-20-87688132 E-mail: [email protected]
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Fig.S1-1
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Fig.S1-2
Fig.S1 The gel photographs of duplex PCR to detect C.jejuni, aimed at 16S (857bp) and hipo (149bp) genes. The maker is 2000bp, have 6 band, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp. In gel image (Fig.S1-1), there are 26 positive isolates contained 2 positive DNA bands, and others
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are negative isolates only 16S DNA band. In Fig.S1-2, all of 8 strains have two positive bands, and the first position on the right of maker is
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standard strain CICC22936.
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Fig.S2-2
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FigS2-1
Fig.S2 The gel image of MAMA-PCR, there are 22 positive DNA band at 265bp in Fig.S2-1, and 8 strains have this mutation in Fig.S2-2.the box
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labeled lane is negative control, the positive band at 265bp represents mutation in gyrA region. The maker is 2000bp, have 6 bands, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp in Fig.S2-1, and the marker is
2000bp, 3000bp, 5000bp in Fig.S2-2.
AC C
Fig.S3-2
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Fig.S3-1
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5000bp, have the bands 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp,
Fig.S3-3
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AC C
EP
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Fig.S3-4
Fig.S3-5
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Fig.S3-6
Fig.S3 The gel images of virulence-related genes. There are six
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photographs contained 34 isolates. The maker is 2000bp, have 6 band, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp. Every isolate detects 11
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virulence genes. The gene fragments are list in Table.2.
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Fig.S4-1
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Fig.S4-2
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Fig.S4The gel photograph of flaA RFLP- PCR, there are two images contain 34sisolates and standard strain CICC22936, and standard strain is
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last position of positive band in Fig.S4-1. The marker has six band, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp.
Table S1 The numbers represent the strains on gel photograph
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Strains
No.
Strains
1
CJ302
19
2523-2B
2
126A
20
178-2A
3
109
21
330B
4
CJ300
22
5
103B
23
6
2678
24
7
325
25
8
2674
9
178-2C
10
178-2B
11
109-3B
12 13
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No.
2691
2324B
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2523A
110B
27
6B
28
6A
29
3C
148B
30
6C
318
31
4A
1474B
32
2
2274-2B
33
6A-1
16
105C
34
7B
17
CJ2574
S
CICC22936
18
1473A
AC C
15
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14
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26
S0956-7135(15)30211-5
DOI:
10.1016/j.foodcont.2015.09.032
Reference:
JFCO 4666
To appear in:
Food Control
Received Date: 25 June 2015 Revised Date:
16 September 2015
Accepted Date: 24 September 2015
Please cite this article as: Zhong X., Wu Q., Zhang J. & Shen S., Prevalence, genetic diversity and antimicrobial susceptibility of Campylobacter jejuni isolated from retail food in China, Food Control (2015), doi: 10.1016/j.foodcont.2015.09.032. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Prevalence, genetic diversity and antimicrobial susceptibility of
2
Campylobacter jejuni isolated from retail food in China
3
Xian Zhong 1, 2, Qingping Wu 1*, Jumei Zhang 1, Shuxian Shen1 1. Guangdong Institute of Microbiology, State Key Laboratory of Applied Microbiology
5
Southern China; Guangdong Provincial Key Laboratory of Microbial Culture Collection
6
and Application, Guangdong Open Laboratory of Applied Microbiology, 510070, PR
7
China
9
SC
2. Department of Chemical Engineering, School of Chemical Engineering and Light Industry,
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Guangdong University of Technology, Guangzhou, Guangdong 510006, PR China
10 11
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12 13
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16 17
*Corresponding author:
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Qingping Wu
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Guangdong Institute of Microbiology, No. 100 Central Xianlie Road, Guangzhou 510070,
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China
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Tel: +86-20-87688132; fax: +86-20-87688132
22
E-mail: [email protected] 1
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ABSTRACT
24
The aim of this study was to determine the prevalence, antimicrobial resistance and molecular
25
epidemiology of Campylobacter jejuni isolated from retail food samples in China and assess
26
the potential risk to consumer’ health. A total of 34 strains of Campylobacter jejuni isolated
27
from 1798 retail food samples in China were identified using conventional methods and
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duplex PCR aimed 16S and hipo genes. There are 59 positive samples by conventional
29
method, and the prevalence of C.jejuni was 3.30% (59/1798). Using flaA RFLP-PCR (flaA
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Restriction Fragment Length Polymorphism) typing with a relative similarity coefficient of
31
0.81, the 34 isolates and 1 reference strains were grouped into 4 clusters and 2 singletons.
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Antibiotic resistance profiles determined using the disk diffusion method showed that a high
33
proportion of C.jejuni isolates were resistant to the tested antibiotics, particularly for
34
ciprofloxacin (88.5%) and nalidixic acid (88.5%). Most isolates were multidrug resistant,
35
with 20 strains showing resistance to 5-8 antibiotics and 2 strains showing resistance to 9
36
antibiotics. Moreover, analysis using MAMA-PCR to determine the gyrA mutation status
37
(C257T) indicated that approximately 30 strains had mutations, and these findings were
38
consistent with disk diffusion assay results. Examination for the presence of 11putative
39
virulence genes showed that the majority of these genes were detected in all C.jejuni strains.
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There was no direct evidence to suggest a connection between antibiotic resistance and
41
virulence genes. This study is the first to explore the potential sources of C.jejuni
42
contamination in retail foods in China and provide comprehensive surveillance on its
43
incidence in retail foods. These data will ensure more accurate treatment of human
44
campylobacteriosis using effective antibiotics.
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Keyword: Campylobacter jejuni; Prevalence; Antibiotic resistance; flaA RFLP-PCR;
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2
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Virulence-related gene
47
1. Introduction Campylobacter jejuni is a major food-borne pathogen that cause bacterial
49
gastroenteritis worldwide (Qin, et al., 2011). As a zoonotic pathogen, it is transmitted to
50
human via the consumption of contaminated food, especially poultry. The sources and
51
transmission routes of campylobacter in staple foods are not fully understood, and the
52
often contradictory epidemiological evidence described to date is partly attributable to
53
the asymptomatic nature of C. jejuni infection and high levels of genetic diversity (Frost,
54
2001). Several molecular typing methods have been used to study the epidemiology of
55
campylobacter infections, such as multilocus sequence typing (MLST) (Kovac, et al.,
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2014), Pulsed Field Gel Electrophoresis (PFGE) (Zhang, et al., 2014), and flaA Short
57
Variable Region Sequence (flaA- SVR) (Zhang, et al., 2014). In this study, we used flaA
58
Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (flaA PCR-
59
RFLP) subtyping method because it is not only a standard technique but is also rapid
60
and cost effective (Ahmed, Dunn, & Ivanova, 2012; Bednarski, Wieliczko, &
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Mazurkiewicz, 2011).
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In the United States, an estimated 2.4 million cases of human campylobacteriosis occur
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each year (W. Bae, et al., 2005), and the most common species (approximately 80-90%)
64
causing infection is C.jejuni. Most cases of campylobacteriosis do not require treatment, as
65
they are of short duration, clinically mild and self-limiting. Antimicrobial treatment is
66
necessary for systemic Campylobacter infections, infections in immune-suppressed patients
67
and severe or long-lasting infections (Gibreel, et al., 2004). However, people sometimes 3
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veterinary medicine, the excessive use of these drugs is common. Currently, the increasing
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prevalence of campylobacter isolates that are resistant to clinical quinolone antibiotics,
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resulting in serious cases of human campylobacteriosis, warrants concern. An increase in the
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number of ciprofloxacin- resistant Campylobacter isolates has been reported in several
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countries (Agunos, et al., 2013; Nobile, Costantino, Bianco, Pileggi, & Pavia, 2013), and the
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rising levels of resistance to these antibiotics among Campylobacter spp. has been recognized
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as an emerging public health problem (J. Bae, Oh, & Jeon, 2014; Englen, Fedorka-Cray,
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Ladely, & Dargatz, 2005).
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Because little information has been published regarding the prevelance and antimicrobial
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susceptibility of C.jejuni isolated from food in the retail trade in China, the objective of this
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report was to determine the prevalence and antibiotic resistance of C.jejuni in staple foods
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while evaluating the risks to consumer health.
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2 Materials and methods
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2.1Sample collection
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A total of 1798 food samples representing seven different types of food, including dairy
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products, vegetables, cooked food, edible fungi, flash-frozen food, sea food and meat
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products, were collected from July 2013 to June 2014. The meat products including chicken,
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duck, pigeon, pork, beef, and bacon, except bacon, other meat was fresh, all meat products
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were raw. Sea food contains two types, saltwater fish and freshwater fish. The dairy product
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mainly was pasteurised milk. The cooked food includes baked food and cold food, such as 4
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character, and flash-frozen food were mainly dumplings and stuffed bun. The samples were
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obtained from supermarkets or farmers’ markets in 24 cities, representing most of provincial
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capitals of China. After collection, samples were tightly sealed in sterile plastic wrap and
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transported to the laboratory on ice in an insulated container.
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2.2 Isolation and identification of Campylobacter jejuni
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ACCEPTED MANUSCRIPT roast duck and cold noodles. The sample acquisition of vegetable and fungi according to local
The enrichment and isolation of C.jejuni was performed according to GB/T4789.9-2008
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for the microbiological examination of food hygiene (National Food Safety Standards of
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China) with slight Modifications. In brief, shears were used to cut the food samples under
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aseptic operation, and 25g of sample was mixed with 225ml of Bolton enrichment broth
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(Huankai, Guangzhou, China) and homogenized in a stomacher bag for1-2 min, followed by
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incubation under Microaerophilic conditions (5% O2, 10% CO2, 85% N2) at 42°C for 48 h. A
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loopful of broth enrichement culture was streaked onto Skirrow agar Plates (Huankai,
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Guangzhou, China) and incubated at 42°C for 48 h under microaerophilic conditions. Three
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or four presumptive colonies, which had similar colony morphologies that were moist and
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grey, were selected to inoculate onto Columbia sheep Blood Agar (Huankai, Guangzhou,
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China). After 48h of incubation under microaerophilic conditions, colonies were picked for
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further identification via Gram staining and an oxidase test. Confirmation of C.jejuni was
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performed using the API Campy system (bioMérieux, France) according to the
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manufacturer’s instructions, and all isolates identified as C.jejuni were further analysed by
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duplex PCR targeting the 16S and hipO genes (Denis, et al., 1999). The primers are shown
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inTable 1. We used C.jejuni CICC 22936 as a standard strain.
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2.3 flaA PCR-RFLP typing flaA PCR–RFLP typing is one of the simplest and most cost-effective genotyping methods
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for the investigation of large numbers of Campylobacter spp. The technique involves PCR
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amplification of the flaA gene and restriction enzyme digestion to generate simple restriction
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patterns (Harrington, Moran, Ridley, Newell, & Madden, 2003). In this study, DdeⅠ(Thermo
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Scientific, Waltham, US) was chosen as the digestion enzyme. The Primers FlaA-F and
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FlaA-R (Table 1) were used to amplify the flaA gene in C.jejuni. The PCR reaction mixture
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and procedure were as follows: 12.5µl of PCR mix (Dongsheng Biotech, Guangzhou, China),
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10mmol/µl primer 1µl, 2µl DNA (40-60ng/µl), add pure water up to final volum (25µl); initial
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denaturation at 94°C for 30 s, primer annealing at 55°C for 40 s, extension at 72°C for 40s,
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and a final extension step at 72°C for 4 min. The PCR products were purified using a DNA
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extraction kit (Magen, Guangzhou, China) prior to enzyme digestion. The fragments were
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separated on 2% agarose gels at 90 V for 1 h and visualized under a UV transilluminator gel
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imaging system (GE Healthcare, WI, USA). The images were saved as TIFF files for
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analysis.
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2.4Antibiotic susceptibility tests
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Antibiotic susceptibility tests were performed using the standard disk diffusion method on
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Muller-Hinton agar contained 5% defibrinated sheep's blood, following by BSAC
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standardized disc susceptibility testing method (version 11)(Howe, Andrews, & Party, 2012).
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Twelve antibiotic disks (OXIOD, UK) were used to perform the resistance test. The disc
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included ampicillin (AMP, 10µg), tetracycline (TE, 30µg), ciprofloxacin (CIP, 5µg),
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gentamicin (CN, 10µg), streptomycin (S, 25µg), erythromycin (E, 15µg), vancomycin (VA, 6
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cefoperazone (CFP, 75µg), and kanamycin (K, 30µg).
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2.5Detection of genetic determinants of antimicrobial resistance Isolates were subjected to MAMA-PCR to detect mutation of the quinolone (similar to
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ciprofloxacin and nalidixic acid) resistance-determining region (gyrase A gene,C257T)
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described by Zirnstein et al.(Zirnstein, et al., 1999) .PCR primers are shown in Table 1.
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2.6Determination of virulence-related genes
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Six PCR tests were performed to detect the presence of eleven virulence-related genes;
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specifically, five duplex PCRs were designed to detect rpoN, CdtA, CdtB, CdtC, flig, racR,
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ciaB, pldA, imaA and CadF genes, and an individual PCR was performed for cjaB gene. The
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primers were designed by primer premier 5.0.The specific genes and primer sequences are
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shown in Table 2.
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2.7Statistical analysis of antimicrobial resistance rates
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The frequencies of antimicrobial resistance patterns from different isolates were
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compared using a v2-test and Fisher’s exact two-tailed test with SPSS19.0 software. Values of
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p < 0.05 were considered statistically significant.
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3 Results
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3.1Contamination by Campylobacter jejuni
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A total of 1798 food samples were examined by conventional methods and duplex PCR 7
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C.jejuni was 3.30% (59/1798); for the most commonly contaminated food, i.e. meat, the
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positive rate was up to14.81% (57/385). The distribution represented in Table 3.
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Contamination of vegetable and sea food was rare (only two positive samples), and no
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contamination was detected in the other types of food. In terms of the geographic distribution,
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the prevalence in south China was higher than north China (3.7%/0.6%). The identification
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results for C.jejuni isolates using the API campy system (bioMérieux, France) were in
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agreement with those obtaining using duplex PCR.
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3.2 Antibiotic susceptibility tests and mutations of the gyrA QRDR.
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The antibiotic susceptibility results for 34 isolates are shown in Table 4. Isolates of
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C.jejuni were tested for different levels of antibiotic resistance. Resistance to AMP, TE, CIP,
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CN, S, E,VA, DA, NA, AML, CFP and K was found in 29.4%, 55.9%, 88.2%, 14.7%, 5.9%,
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2.94%, 85.3%, 11.8%, 88.2%, 38.2%, 82.4% and 23.5% of isolates, respectively. In general,
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the proportions of resistant C.jejuni against the tested antibiotics were severity, particularly
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for CIP and NA. Most isolates were multidrug resistant, 20 strains were resistant to 5-8
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antibiotics, and 2 strains were resistant to 9 antibiotics. Moreover, we found that 30 strains
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were resistant to both CIP and NA. The quinolone-resistant isolates had a change (from C to
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T) in codon 86 of the gyrA gene, resulting in a threonine-to-isoleucine substitution in the
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functional protein. MAMA-PCR results confirmed the presence of this mutation (see Fig.S2
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in supporting information).
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3.3Detection of virulence-related genes in C.jejuni isolates
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8
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and were then examined for the presence of 11 virulence genes by PCR (see Fig.S3 in
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supporting information). The prevalence of virulence genes is shown in Fig.1. All strains
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harboured the fliG sequence. The rpoN and cadF sequences were found in 97.1% (33/34).
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CjaB was found in only two strains, and pldA and imaA were found in only three strains. With
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the exception of cjaB, pldA and imaA, the other genes tested were present in more than 67.6%
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of isolates. Additionally, almost every isolate carried 7-8types of virulence-associate genes.
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3.4 flaA PCR-RFLP typing of C.jejuni
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ACCEPTED MANUSCRIPT All 34 isolates obtained in this study were identified by biochemical tests and duplex PCR
The results of the PCR-RFLP analysis for 34 C.jejuni isolates and 1 reference strain are
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shown in Fig. 2. In this study, PCR-RFLP allowed for perfect discrimination using the
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Simpson’s index of diversity (D=0.95). The DNA profiles consisted of four to seven bands
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ranging from 100 to 3700bp, and the 150, 300, 900bp fragments were the main bands in most
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strains (see Fig.S4 in supporting information). At a relative similarly coefficient of 0.81, 21
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types of 34 isolates were placed into six clusters (designatedⅠ,Ⅱ, Ⅲ, Ⅳ, Ⅴ, and Ⅵ).
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ClusterⅠcontained all strains that had no mutations in gyrA gene, including 2523A, 2523-2B,
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330B, and 110B. These strains were more susceptible to antibiotics than other isolates and
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were only resistant to two types of antibiotics. Moreover, only 2523A and 110B had the
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virulence-related gene imaA. Cluster Ⅱcontained two strains that they carried the same
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virulence genes. In cluster Ⅲ, 2678 and 2274-2A shared the same type and also had the same
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virulence genes. It is possible that cross contamination occurred, as seen with the
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identification of 178-2A, 178-2B and178-2C in one samples, although they belonged to
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different clusters. Cluster Ⅳ and Ⅴwere singletons. The last cluster contained five stains
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4Discussion
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4.1Risky analysis
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ACCEPTED MANUSCRIPT that were isolated from Guangzhou.
To our knowledge, this is first study to examine the prevalence and antibiotic resistance of
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C.jejuni from retail foods in China. According to the findings of this study, the meat and its
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products are the main foods contaminated by C.jejuni that correlates with what we already
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know about source of C.jejuni. In terms of the geographic distribution, the prevalence in
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south China was higher than in north China (3.7%/0.6%), likely because C.jejuni is a
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thermopilic bacterium. The people of south china should take more effective action to control
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contamination. There was a low level of contamination by C.jejuni in retail foods in China.
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The average incidence of C.jejuni was only3.30%, which is far below other reports, although
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similar contamination levels were noted in fresh meat and poultry in particular (Jamali, et al.,
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2015; Mattheus, Botteldoorn, Heylen, Pochet, & Dierick, 2012; Ramonaite, et al., 2013). In
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November 2011 to August 2012, our laboratory have investigated the contamination of
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C.jejuni in retail foods in parts of south China, and had the similar positive rate of meat
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product and the average contamination, 13.2% and 2.51%, respectively(Zheng, et al., 2014).
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As part of the normal intestinal flora of poultry (Mackiw, Korsak, Rzewuska, Tomczuk, &
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Rozynek, 2012), the prevalence of C.jejuni in meat products, especially poultry, was high. To
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comprehensively evaluate the risk of this bacterium in our daily life, other types of food were
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tested, including dairy products, vegetables, cooked food, edible fungi, and flash-frozen food.
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Contamination in these types of food is rarely detected given the harsh living conditions of
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cross-contaminated from contaminated raw food (Mackiw, et al., 2012). Highly virulent C.
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jejuni strains may cause disease similar to those caused by Listeria monocytogenes (Bras,
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Chatterjee, Wren, Newell, & Ketley, 1999), Salmonella spp. (Williams, et al., 2015) and
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others. C.jejuni has several putative virulence genes that are associated with the cytolethal
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distending toxin (CDT) and certain stages of the infection cycle including adhesion(Konkel,
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Gray, Kim, Garvis, & Yoon, 1999) , intestinal colonization (Bras, et al., 1999) and invasion
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(Muller, Schulze, Muller, & Hanel, 2006) , and may be potentially pathogenic for consumers .
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Our results showed that greater than 70% of isolates harbored adhesion- and intestinal-
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associated genes(cadF, fliG, racR, rpoN), although invasion- associated genes(cjaB, pldA,
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imaA) were found in less than 8.8% of isolates, indicating that most of the strains demonstrate
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strong colonization but weak invasion ability, may because the sample is food, not clinic
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(Fonseca, et al., 2014). The CDT gene was detected at a prevalence of up to 80%, indicating
229
that most of the isolates may possess full pathogenic potential. However, the presence of
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these virulence- related genes in a C.jejuni isolate does not always imply that it is capable of
231
causing disease (Cho, Kim, Min, Ku, & Kim, 2014). Therefore, further studies are required to
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examine whether the strains isolated in this study are pathogenic. This is the first time
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C.jejuni risk has been analysed for retail food in China.
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4.2 Antibiotic susceptibility
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ACCEPTED MANUSCRIPT this bacterium; only two positive samples were detected and may have been
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C.jejuni is generally susceptible to a wide range of antibiotics as determined in the 1990s
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(Jacobsreitsma, Koenraad, Bolder, & Mulder, 1994; Li, Chiu, Wu, Huang, & Lin, 1998).
237
Antimicrobial resistance has become a major public health concern in both developed and 11
239
including the World Health Organization (WHO) (Moore, et al., 2006; Padungton & Kaneene,
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2003). A report on annual trends in resistance for different antibiotics, including ampicillin,
241
ciprofloxacin, nalidixic acid, tetracycline, gentamicin, and erythromycin, from 2004-2009
242
(Mattheus, et al., 2012) found that resistance for almost antibiotics has increased over time. In
243
2009, the CIP, NA, and TE resistance reached 40% respectively, and in this current study, the
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data indicated that resistance had considerably increased for CIP (88.2%), NA (88.2%) , and
245
TE (55.9%), which was similar to the results of Mackiw (Mackiw, et al., 2012).
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Fluoroquinolones are normally considered the first choice for the treatment of Campylobacter
247
enteritis, although high-level resistance to fluoroquinolones has been reported throughout the
248
world (Mackiw, et al., 2012; Mattheus, et al., 2012; Saenz, et al., 2000). Resistance to FQs is
249
mainly attributable to a single point mutation in the DNA gyrase gene gyrA, particularly a
250
Thr-86-Ile change mediated by the C257T mutation (Alfredson & Korolik, 2007). Almost
251
88% (30/34) of strains had this mutation, and other reported mutations of gyrA in C.jejuni
252
included Thr-86-Ala, although rarely occurred (Kinana, et al., 2006). Given the low number
253
of strains in this study, it was not surprising that no strain appeared to have other mutations in
254
gyrA genes. An increasing in ciprofloxacin-resistant C.jejuni may translate to greater
255
prevalence of gastroenteritis in humans caused by the consumption of chicken meat
256
contaminated by ciprofloxacin-resistant campylobacter. This naturally renders effective
257
therapy more difficult (Mackiw, et al., 2012). These results represent a warning to take
258
effective action to reduce the occurrence of resistance in C.jejuni quickly as possible
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4.3 flaA RFLP-PCR
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ACCEPTED MANUSCRIPT developing countries in recent years and has been recognized by various national authorities
12
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campylobacters due to its rapidity and simplicity, a highly gene diversity was identified in the
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C.jejuni isolated from retail food in China. We obtained a high percentage of unique strains,
263
similar to the genetic heterogeneity found in numerous studies of human, animal and
264
environmental isolates (Denis, et al., 2011; O'Leary, et al., 2011). There were 21flaA types
265
identified among 35strains, which was in accordance with the results of Ramonaite
266
(Ramonaite, et al., 2013) who found a similarly high genetic diversity. At a similarity
267
coefficient of 0.81, a total of 34 isolates and 1 reference strain were divided into 4 clusters
268
and 2 singletons. As shown in Fig.2, we observed four strains that were susceptible to CIP
269
and NA belonging to the same cluster, and these similar antibiotic profiles represented greater
270
susceptibility to the tested antibiotics than the other isolates. Isolates 2523A and 2523-2B
271
were from the same sample but represented different types. Isolates 330B and 2523-2B were
272
from different samples with different geographical distribution but shared the same type. This
273
phenomenon implies that cross-contamination may have occurred, which is common with
274
other food-borne pathogens, such as L. monocytogenes (Wu, Wu, Zhang, Chen, & Yan, 2015)
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and salmonella spp. (Wang, et al., 2015). In our study, we could not identify a direct
276
relationship between antibiotic profiles and virulence-related genes.
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5. Conclusions
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ACCEPTED MANUSCRIPT By applying the PCR-RFLP method, which has been widely used for the genotyping of
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In conclusion, this study is the first systematical investigation of the prevalence and
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contamination levels of C.jejuni in retail foods in China. The average contamination (3.30%)
280
was lower than other reports, although a high prevalence (14.81%) of C.jejuni in meat
281
product was observed, especially for poultry, which showed that this food might be a major 13
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belonged to six clusters with a relative similarly coefficient of 0.81. High genetic diversity
284
was observed based on PCR-RFLP subtyping, with 21 types identified among 34 isolates.
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The high antibiotic resistance observed in this research is alarming and raising public health
286
concern. There was little relationship between antibiotic resistance and virulence genes.
287
Additionally, mutations detected in the quinolone resistant determination region are caused
288
for concerned. Thus, it is necessary to remind consumers, sellers, producers, carriers and
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Chinese food safety regulators to increase awareness regarding the importance of controlling
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antibiotics use. Effective measures should be implemented in food processing to control
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contamination.
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ACCEPTED MANUSCRIPT carrier of C.jejuni in retail foods in China. The isolates of C.jejuni from positive samples
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Acknowledgment
This work was supported by the Science and Technology Projects of Guangdong, China
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(No.2012B050800007) and the Science and Technology Projects of Guangzhou, China
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(No.2014J4100214).
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Antibiotic resistance in Campylobacter strains isolated from animals: Foods, and humans
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Zheng, Y., Wu, Q., Wu, K., Zhang, J., Guo, W., & Wu, K. (2014). Virulence-associated gene
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Table
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Table 1 Primers used and the expected amplicon sizes
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Table 2 Virulence-related gene sequence
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Table 3 Distribution of C. jejuni in seven kinds of retail food in China
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“C”: “ conventional method”, “D”: ”duplex PCR”.
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Table 4 Results of antibiotic susceptibility tests of C.jejuni isolates from retail foods in China
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*R: resistant; I: intermediate resistance; S: susceptibility.
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Figure
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Fig.1 The eleven virulence-related genes in isolates
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Fig. 2 flaA RFLP- PCR DNA finger prints analyses of C.jejuni from retail foods in China. ND:
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not detected, SUS-CIP-NA: susceptibility to ciprofloxacin and nalidixic acid. I to VI refer to
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clusters 1 to 6 of C.jejuni according to RFLP-PCR. The numbers represent the type of
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antibiotic resistance. “-’’: PldA, cjaB, and ImaA were not detected.
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Primers used and the expected amplicon sizes Target gene
Primer name
Oligo nucleotide sequence (5’-3’)
FlaA
FlaA-F FlaA-R
GGATTTCGTATTAACACAAATGGTGC
16s-F 16s-R
ATCTAATGGCTTAACCATTAAAC
Hipo-F Hipo-R
GTACTGCAAAATTAGTGGCG
Gyra-F Gyra-R
TTT TTA GCA AAG ATT CTG AT
(Denis, et al., 1999)
GCAAAGGCAAAGCATCCATA
CAA AGC ATC ATA AAC TGC AA
149
This study
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(Zirnstein, Li, Swaminathan, & Angulo, 1999)
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Thr-86-Ile mutations gyrA
857
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HipO
(Ramonaite, et al., 2013)
CTGTAGTAA TCTTAAAACATT TTG
GGACGGTAACTAGTTTAGTATT
Reference
1725
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16s
Sizes(in bp)
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Size (bp)
rpon
150
cdtA
403
cadF
158
Cjab
381
plda
913
cdtC
348
flig
469
cdtB
144
racR
338
imaA
518
ciab
986
Nucleotides sequence(5’-3’)
Annealing Temperature(°C) 52
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52
52
52
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GAACAGATTCTTCCTCCTT TGCTCTTACTCGCTCAA ACATTTGTGCGTGATTG AGCGGTGCTGATTTAGT TTTCTCACCCACATCAATA GTGTTAGACTTGGTTATCATTTT ACCTCGGCTTTATGGA GGAGCAGCATCCTACC AAGCTTATGCGTTTTT TATAAGGCTTTCTCCA ATAACTTTAGCCTTTGC AGTTGTTGTTGGCATT GTGGTGGTTTGGAATA TCTGCTAAGCGTTCAT TAATGTTGGCACTTGG GTGGCTGTTCTTGGTA AAGGCTATGATTGTCTG TTCAGCAGGAGTAAGTG GCACAAAATATATCATTACA TTCACGACTACTACTATGAGG TTTTTATCAGTCCTTA TTTCGGTATCATTAGC
45
45
45 45 50 50 45
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Detection method
Positive samples
Positive rate (%)
Meat product Vegetables Seafood Flash-frozen food Edible fungi Cooked food Dairy product Total
385 161 330 231 262 330 99 1798
C and D C and D C and D C and D C and D C and D C and D C and D
57 1 1 0 0 0 0 59
14.81 0.60 0.30 0 0 0 0 3.30
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Types of products
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R
I
10 (29.4) 19 (55.9) 30 (88.2) 5 (14.7) 2 (5.9) 1 (2.94) 29 (85.3) 4 (11.8) 30 (88.2) 13 (38.2) 28 (82.4) 8 (23.5)
≦13 ≦11 ≦15 ≦12 ≦10 ≦13 ≦14 ≦14 ≦13 ≦13 ≦15 ≦14
13-16 12-14 16-20 13-14 11-12 14-22 15-16 15-20 14-18 14-17 16-20 15-17
4 (11.8) 2 (5.9) 0 (0) 1 (2.9) 1 (2.9) 6 (17.6) 1 (2.9) 3 (8.8) 0 (0) 10 (29.4) 1 (2.9) 2 (5.9)
20 (58.8) 13 (38.2) 4 (11.8) 28 (82.4) 31 (91.2) 27 (79.4) 4 (11.8) 27 (79.4) 4 (11.8) 11 (32.4) 5(14.7) 24 (70.6)
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S
≧17 ≧15 ≧21 ≧15 ≧13 ≧23 ≧17 ≧21 ≧19 ≧18 ≧21 ≧18
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No.(%)of R No.(%)of I No.(%)of S
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Ampicillin(AMP) Tetracycline(TE) Ciprofloxacin(CIP) Gentamicin(CN) Streptomycin(S) Erythromycin(E) Vancomycin(VA) Clindamycin(DA) Nalidixicacid(NA) Amoxicillin(AML) Cefoperazone(CFP) Kanamycin(K)
Zone diameter(mm)
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Highlights C.jejuni was primarily determined in meat and meat products in retail foods in China
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C.jejuni isolates was showed high antibiotic resistance, especially quinolone
Virulence-related gene cjaB, pldA, imaA rarely contained in isolates.
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flaA RFLP-PCR subtyping were used for genetic diversity analyses.
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Supporting Information
Prevalence, genetic diversity and antimicrobial
retail food in China
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susceptibility of Campylobacter jejuni isolated from
Xian Zhong 1, 2, Qingping Wu 1*, Jumei Zhang 1, Shuxian Shen1
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1. Guangdong Institute of Microbiology, State Key Laboratory of Applied
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Microbiology Southern China; Guangdong Provincial Key Laboratory of Microbial Culture Collection and Application, Guangdong Open Laboratory of Applied Microbiology, 510070, PR China 2. Department of Chemical Engineering, School of Chemical Engineering
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and Light Industry, Guangdong University of Technology, Guangzhou,
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Guangdong 510006, PR China
*Corresponding author: Qingping Wu
Guangdong Institute of Microbiology, No. 100 Central Xianlie Road, Guangzhou 510070, China Tel: +86-20-87688132; fax: +86-20-87688132 E-mail: [email protected]
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Fig.S1-1
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Fig.S1-2
Fig.S1 The gel photographs of duplex PCR to detect C.jejuni, aimed at 16S (857bp) and hipo (149bp) genes. The maker is 2000bp, have 6 band, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp. In gel image (Fig.S1-1), there are 26 positive isolates contained 2 positive DNA bands, and others
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are negative isolates only 16S DNA band. In Fig.S1-2, all of 8 strains have two positive bands, and the first position on the right of maker is
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standard strain CICC22936.
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FigS2-1
Fig.S2 The gel image of MAMA-PCR, there are 22 positive DNA band at 265bp in Fig.S2-1, and 8 strains have this mutation in Fig.S2-2.the box
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labeled lane is negative control, the positive band at 265bp represents mutation in gyrA region. The maker is 2000bp, have 6 bands, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp in Fig.S2-1, and the marker is
2000bp, 3000bp, 5000bp in Fig.S2-2.
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Fig.S3-1
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5000bp, have the bands 100bp, 250bp, 500bp, 750bp, 1000bp, 1500bp,
Fig.S3-3
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Fig.S3-5
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Fig.S3 The gel images of virulence-related genes. There are six
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photographs contained 34 isolates. The maker is 2000bp, have 6 band, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp. Every isolate detects 11
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virulence genes. The gene fragments are list in Table.2.
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Fig.S4-2
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Fig.S4The gel photograph of flaA RFLP- PCR, there are two images contain 34sisolates and standard strain CICC22936, and standard strain is
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last position of positive band in Fig.S4-1. The marker has six band, 100bp, 250bp, 500bp, 750bp, 1000bp, 2000bp.
Table S1 The numbers represent the strains on gel photograph
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Strains
No.
Strains
1
CJ302
19
2523-2B
2
126A
20
178-2A
3
109
21
330B
4
CJ300
22
5
103B
23
6
2678
24
7
325
25
8
2674
9
178-2C
10
178-2B
11
109-3B
12 13
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No.
2691
2324B
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2523A
110B
27
6B
28
6A
29
3C
148B
30
6C
318
31
4A
1474B
32
2
2274-2B
33
6A-1
16
105C
34
7B
17
CJ2574
S
CICC22936
18
1473A
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15
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