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Open Access

Peer-reviewed

Research Article

Molecular typing and antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli Isolates from Patients and raw meat in Huzhou, China, 2021–2022

  • Xiaofang Wu,

    Roles Conceptualization, Funding acquisition, Investigation, Methodology, Writing – original draft

    Affiliation Huzhou Center for Disease Control and Prevention, Huzhou, China

    ⨯
  • Chen Liping,

    Roles Methodology

    Affiliation Huzhou Center for Disease Control and Prevention, Huzhou, China

    ⨯
  • Fenfen Dong,

    Roles Software

    Affiliation Huzhou Center for Disease Control and Prevention, Huzhou, China

    ⨯
  • Wei Yan,

    Roles Software

    Affiliation Huzhou Center for Disease Control and Prevention, Huzhou, China

    ⨯
  • Yuehua Shen,

    Roles Investigation, Methodology

    Affiliation Huzhou Center for Disease Control and Prevention, Huzhou, China

    ⨯
  • Lei Ji

    Roles Data curation, Funding acquisition, Writing – review & editing

    jileichn@163.com

    Affiliation Huzhou Center for Disease Control and Prevention, Huzhou, China

    ⨯

Abstract

Background

Campylobacter species are zoonotic pathogens, and are considered to be the major foodborne pathogen that causes outbreaks and sporadic gastrointestinal illnesses both in developed and developing countries. In this study, the molecular typing and antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli isolates from patients and raw meat between 2021 and 2022 in Huzhou were analyzed by using pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and antimicrobial susceptibility testing.

Methods

From September 1, 2021 to December 31, 2022, a total of 342 fecal specimens from diarrheal patients at a sentinel hospital in Huzhou and 168 samples of raw meat products collected from farmers’ markets and supermarkets, were subjected to Campylobacter isolation and identification. The agar dilution method was used to determine resistance of the Campylobacter isolates to eleven antibiotics. In addition, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were performed to compare their genetic relationships.

Results

78 Campylobacter isolates were recovered, comprising 58 isolates (74.36%, 58/78) of Campylobacter jejuni (34 patient isolates and 16 food isolates) and 20 isolates (25.64%, 20/78) of Campylobacter coli (6 patient isolates and 14 food isolates). Campylobacter has emerged as a predominant foodborne pathogen in the local region, with detection rate reached 11.70% among 342 diarrhea samples. The Campylobacter isolation rate in 168 raw meat was 22.62% (38/168), all originating from poultry meat, with chicken been the major source of infection (86.84%, 33/38). Both PGFE type and MLST data confirmed that Campylobacter stains circulating in Huzhou are genetically diverse, with Campylobacter jejuni isolates being more diverse than Campylobacter Coli. PFGE typing revealed 45 band patterns among 54 Campylobacter jejuni strains and 17 band patterns among 19 Campylobacter Coli strains. 50 Campylobacter jejuni strains from different sources were classified into 37 ST types, showing a dispersed distribution and encompassing over 12 clonal complexes (CCs), with CC-21 being the most prevalent CC (22.00%, 11/50). The distribution of ST types in the 18 Campylobacter Coli strains was relatively concentrated, with 83.33% (15/18) of isolates belonging to the CC-828. In this study, 2 groups of Campylobacter jejuni strains (PFGE J2-ST464 and PFGE J9-ST-2328) originated from humans and chickens showed high genetic homologies by comparing PFGE and MLST results. Besides, some disagreement between PFGE and MLST was observed for certain ST, indicating a weak correlation between PFGE and MLST for certain Campylobacter strains. Most of the Campylobacter isolates were highly resistant to nalidixic-acid, ciprofloxacin and tetracycline. The multiple antibiotic resistance of Campylobacter Coli (89.47%) is higher than Campylobacter jejuni (29.63%).

Conclusion

Campylobacter is an important foodborne pathogen in both diarrheal patients and raw meat products in Huzhou City, exhibiting multiple antibiotic resistance and high level of genetic diversity.

Citation: Wu X, Liping C, Dong F, Yan W, Shen Y, Ji L (2024) Molecular typing and antimicrobial susceptibility profiles of Campylobacter jejuni and Campylobacter coli Isolates from Patients and raw meat in Huzhou, China, 2021–2022. PLoS ONE 19(12): e0311769. https://doi.org/10.1371/journal.pone.0311769

Editor: Dwij Raj Bhatta, Tribhuvan University, NEPAL

Received: February 6, 2024; Accepted: September 24, 2024; Published: December 11, 2024

Copyright: © 2024 Wu et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the article and its Supporting Information files.

Funding: This work was supported by the Huzhou Science and Technology Bureau (Grant Number: 2021GYB26), the Special Support Plan for Local High-Level Talents in South Taihu Lake (Grant Number: 202011019), and the Key Laboratory of Emergency Detection for Public Health of Huzhou. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Campylobacter, a zoonotic pathogen, can cause symptoms such as diarrhea, fever, and abdominal pain. Additionally, it can lead to severe complications like Guillain-Barré syndrome and reactive arthritis, posing a significant threat to human health [1]. In developed countries, Campylobacter infection has become more prevalent than infections caused by pathogens such as Salmonella, E. coli and, Vibrio parahaemolyticus, establishing it as the leading cause of bacterial diarrhea worldwide [2]. Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli) are the main Campylobacter species that cause gastroenteritis in humans and responsible for approximately 95% of all Campylobacter infections in developing countries [3]. It has been reported that globally, there are 400 to 500 million cases of diarrhea annually caused by C. jejuni, making it a serious public health concern [4]. Campylobacter widely inhabits the human and animal intestines [5]. Poultry is a significant source of contamination in the human food chain. During poultry farming, poultry infected with Campylobacter do not exhibit any clinical symptoms but can continuously shed the bacteria into the environment and carry it for life [6]. This can easily lead to cross-contamination between poultry and livestock and its products during slaughter, processing, and retail stages [7]. Epidemiological studies have shown that up to 30% of human Campylobacter infections are caused by handling, preparing, and consuming raw or undercooked poultry. Poultry meat—especially chicken meat—is the most common source of infection in humans, along with other insufficiently heated meat, raw milk, and contaminated water [8–11].

With the increasing awareness of the major public health importance of Campylobacter, studies about the prevalence of Campylobacter isolated from clinical cases in China have been carried out in the recent years. However, only a few studies have investigated the isolation rate and molecular characterization of Campylobacter spp. from both food and human clinical sources in China. The link between foodborne and human clinical isolates of Campylobacter has remained largely uncharacterized. The aim of this study was to investigate the molecular typing and antimicrobial susceptibility profiles of C. jejuni and C. coli isolates from patients and raw meat in Huzhou and to evaluate the phylogenetic relationships of Campylobacter strains from human patients and raw meat products using PFGE and MLST methods.

Materials and methods

Ethics statement

The protocol was approved by the ethics committee of Huzhou Center for Disease Control and Prevention (approval number: HZ2021005). The only human material used in this study is fecal specimen from outpatient with acute diarrhea for local foodborne disease surveillance project, data records and collected clinical specimens were deidentified and anonymous. Patient consent was not required as the research results will not be used as a basis for any auxiliary diagnosis or for any commercial purposes. Furthermore, any identifiers related to participants will be removed from the research results to ensure that personal privacy is not compromised. Therefore, there is no objective risk to the participants.

Sample collection and strain isolation

According to the guidelines of the local foodborne disease surveillance project in Huzhou, from September 1, 2021 to December 31, 2022, a total of 342 fecal specimens from outpatients with acute diarrhea at the sentinel hospital and 168 samples of raw meat products (50 samples of livestork meat products and 118 samples of raw poultry products) purchased from farmers’ markets and supermarkets, were subjected to Campylobacter isolation. 168 raw meat products mainly refer to fresh poultry, frozen poultry, and fresh livestork meat, including 25 portions of raw pork and 25 portions of raw beef, 30 ducks, and 88 chickens.

For Campylobacter spp. isolation, raw meat was first placed in sterile self-sealing bags containing 500 ml of BPW culture medium, followed by vigorous rubbing for 5 minutes. Then a Campylobacter isolation kit incorporating a membrane filter method (ZC-CAMPY-001 for specimens and ZC-CAMPY-002 for meat, Qingdao Sinova Biotechnology Co., Ltd., Qingdao, China) was used to isolate Campylobacter. Briefly, 2 mL of meat suspension and suitable amount of fecal specimen was transferred to 4 mL of growth-promoting enrichment Preston broth provided in the kit. The enrichment broth was then incubated at 42°C under microaerobic conditions (5% O2, 10% CO2, and 85% N2) for 24 hours. Three hundred microliters drop of the enrichment broth were applied to the 0.45-μm pore-size filter and left on the surface of Karmali and Columbia blood agar plates. After 30 minutes, the filters were removed, and these plates were further incubated at 42°C under microaerobic condition. Suspicious colonies were subcultured, and identified using matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (VITEK MS).

PFGE molecular typing

Pulsed-field gel electrophoresis (PFGE) molecular typing was performed according to the PulseNet standardized protocol for C. jejuni (Available online: https://www.cdc.gov/pulsenet/PDF/campylobacter-pfge-protocol-508c.pdf). In brief, genomic DNA was digested with SmaI (Takara, Dalian, China), and run on a CHEF Mapper PFGE system (Bio-Rad Laboratories, Hercules, CA) for 16 h on SeaKem gold agarose (Lonza, Rockland, MD, USA) in 0.5×Tris-borate-EDTA. XbaI-digested DNA from Salmonella enterica serovar Braenderup H9812 was used as the standard size. Salmonella enterica serovar Braenderup digested with XbaI (Takara) was used as the molecular reference marker. The gel images were stored electronically as TIFF files and bands were analyzed by using BioNumerics software v. 7.6 (Applied Maths, Kortrijk, Belgium). The similarity between chromosomal fingerprints was scored using the Dice coefficient.

MLST molecular typing

Multilocus sequence typing (MLST) was performed by sequencing seven housekeeping loci (aspA, glnA, gltA, glyA, pgm, tkt, and uncA) according to previously described primers for C. jejuni and C. coli (https://pubmlst.org/organisms/Campylobacter-jejunicoli/primers). The nucleotide sequences of the amplicons were submitted to the pubMLST database (https://pubmlst.org/) for online data analysis, resulting in corresponding Sequence Types (ST) and Clonal Complexes (CC). For ST types not found in the database, new ST types were applied for based on strain sequences using blast. A minimum spanning tree (MST) and dendrogram of MLST data was created using BioNumerics v.7.6 (Applied Maths, Kortrijk, Belgium).

Antibiotic susceptibility testing

Antibiotic susceptibility testing was conducted using the agar dilution method recommended by the Clinical and Laboratory Standards Institute (CLSI) using a commercial kit (ZC-AST-001, Zhongchuang Biotechnology Ltd. Corp., Qingdao, China). The antibiotics tested included macrolides: erythromycin (ERY) and azithromycin (AZI); quinolones and fluoroquinolones: nalidixic acid (NAL) and ciprofloxacin (CIP); aminoglycosides: gentamicin (GEN) and streptomycin (STR); chloramphenicol: chloramphenicol (CHL) and florfenicol (FLO); tetracyclines: tetracycline (TET); ketolides: telithromycin (TEL); lincosamides: clindamycin (CLI). MICs were interpreted in accordance with the standard of National Antimicrobial Resistance Monitoring System (NARMS-2014). The breakpoints for resistance were as follows: ERY ≥ 32 μg/mL, AZI ≥ 1 μg/mL, NAL ≥ 32 μg/mL, CIP ≥ 4 μg/mL, GEN ≥ 4 μg/mL, STR ≥ 16 μg/mL, CHL ≥ 32 μg/mL, FLO ≥ 8 μg/mL, TET ≥ 16 μg/mL, TEL ≥ 8 μg/mL, CLI ≥ 1 μg/mL. Quality control was performed with C. jejuni ATCC 33560. The multiple antibiotic resistance index (MARI) was used to quantify the multi-resistance of Campylobacter isolates. MAR index = a/b. In this formula, “a” indicated the number of antibiotics to which the isolate was resistant and “b” indicated the total number of antibiotics to which the isolate was tested. Multi-drug resistance (MDR) was defined as resistance to three or more classes of antimicrobials in this study.

Statistical analysis

Statistical analysis was performed using SPSS 19.0 software. The χ2 test was employed, and significance was determined at P < 0.05.

Results

Prevalence of Campylobacter spp. in Huzhou

We collected 342 fecal specimens from diarrheal patients and 168 samples of raw meat products from farmers’ markets and supermarkets during 2021 and 2022. Seventy eight Campylobacter isolates were recovered, comprising 58 isolates of C. jejuni (74.36%, 58/78) and 20 isolates of C.coli (25.64%, 20/78). The isolation rate of Campylobacter in diarrhea patients was 11.70% (40/342), with detection rates of C. jejuni and C.coli at 9.94% (34/342) and 1.75% (6/342), respectively. The isolation rate of Campylobacter in raw meat products was 22.62% (38/168), with all strains isolated from poultry meat (5 from duck and 33 from chicken). A significant higher percentage (P = 0.035,χ2 = 4.4476) of Campylobacter spp. isolates was observed in chicken samples (37.50%, 33/88) as compared to those in duck samples (16.67%, 5/30). The detection rate for C. jejuni in raw meat was 14.29% (24/168), while C.coli had a detection rate of 1.75% (6/342). The comparison of detection rates between C. jejuni and C.coli in samples from diarrhea patients showed statistically significant differences (χ2 = 20.81, P < 0.0001), see Table 1.

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Table 1. Detection rate of Campylobacter in raw meat and diarrheal samples in Huzhou.

https://doi.org/10.1371/journal.pone.0311769.t001

PFGE clustering

A total of 78 Campylobacter strains were subjected to PFGE after digestion with the restriction enzyme Sma I and 73 valid profiles were obtained, comprising 54 C. jejuni strains (34 from patients and 20 from food samples) and 19 C.coli strains (6 from patients and 13 from food samples). Cluster analysis revealing a band pattern similarity of 26.8% to 100% among the 73 Campylobacter isolates, see Fig 1.

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Fig 1. Dendrogram of 73 Campylobacter strains based on SmaI-mediated PFGE profiles.

Sample ID, strain source, isolation year, MLST type are depicted on the right. Asterisk indicates newly designated ST in this study. Fifteen groups of closely related C. jejuni isolates (band pattern similarity greater than 85.0%) were assigned as J1-J15, while five groups of closely related C. coli isolates (band pattern similarity greater than 85.0%) were assigned as C1-C5. Two groups (PFGE J2-ST464 and PFGE J9-ST-2328) of C. jejuni strains originated from humans and chickens which were confirmed to be clonally related by comparing PFGE and MLST results are marked with red rectangle.

https://doi.org/10.1371/journal.pone.0311769.g001

Among the 54 C. jejuni strains, a total of 45 band patterns were obtained, with 15 groups of closely related isolates (greater than 85.0% similarity in banding patterns) which were assigned a profile group number (J1–J15). The most common profile group was J1, which included 6 isolates from diarrhoea patient. 4 groups had both patient and food isolates (J2, J4, J6, J9) and only 1 group (J9) each of patient isolate (ID 2022636) and food isolate (ID 2022756) had identical profiles. 17 band patterns were obtained from the 19 C.coli isolates by SmaI digestion analysis. 5 groups were identified that had isolates possessing over 85% similarities with each other and were designated C1–C5. The most common profile group was C3, which included 6 food isolates. Only one group (C2) comprised both patient and food isolates and shared 85.7% similarities with each other. No isolates from patients formed identical PFGE patterns with chicken or duck among C. coli strains.

MLST typing

50 C. jejuni strains (34 patient isolates and 16 food isolates) and 18 C. coli strains (6 patient isolates and 12 food isolates) were selected for MLST molecular typing, with the results presented in Table 2. We identified 37 STs of 12 CCs among the 50 C. jejuni isolates, including 5 STs (ST-11775, ST-11822, ST-12371, ST-12391, ST-12392) were newly designated in this study. 13 STs from 17 isolates did not assign to any known CCs. The most commonly isolated CC was CC-21 (22.00%, 11/50), followed by CC-353(6.00%, 3/50), CC-464(6.00%, 3/50) and CC-574 (6.00%, 3/50). The most common STs in patients and food were ST-298(4 strains) and ST-2328 (3 strains), respectively. STs overlapped in both patients and food isolates included ST-6500, ST-464, ST-9621 and ST-2328. MLST analysis for the 18 C.coli isolates resulted in 11 STs, of which 1 ST (ST12390) was new. Except for 3 unclassified STs (ST-1145, ST-11932 and ST-12337), all other STs were classified into the same clonal complex, CC-828. STs overlapped in both patients and food isolates for C.coli in this study was ST-825. MLST data summary of 50 C. jejuni strains and 18 C. coli strains in this study were presented in S1 Table.

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Table 2. MLST typing results of Campylobacter isolates from patient and raw meat in Huzhou.

https://doi.org/10.1371/journal.pone.0311769.t002

The minimum spanning trees based on MLST data for the cluster analysis of C. jejuni and C.coli isolates are depicted in Figs 2 and 3. The results revealed close genetic relationships among strains within the same CCs, see Fig 2. For instance, ST-11822, ST-298, and ST-6500 from the CC-21 were positioned on the same small branch. Similarly, within the CC-828, ST-829, ST-825, ST-1563 and ST-5511 displayed close genetic relationships. Strains from different CCs exhibited relatively distant genetic relationships. C. jejuni and C.coli from diarrheal patients and raw meat products demonstrated a dispersed distribution, with no significant clustering observed, See Fig 3.

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Fig 2. Minimum spanning tree of 50 C. jejuni strains and 18 C. coli strains.

The isolates are featured by different color according to the corresponding CCs. Different circles correspond to different STs. Size of circle indicates number of isolates within the same ST, branches and numbers represent allelic differences between isolates.

https://doi.org/10.1371/journal.pone.0311769.g002

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Fig 3. Minimum spanning tree of 50 C. jejuni strains and 18 C. coli strains.

The isolates are featured by different color according to isolated sources. Different circles correspond to different STs. Size of circle indicates number of isolates within the same ST, branches and numbers represent allelic differences between isolates.

https://doi.org/10.1371/journal.pone.0311769.g003

Comparison of genetic relatedness between PFGE and MLST revealed that not all clustered PFGE types with 100.0% similarity in banding pattern showed identical ST types, while not all the isolates with the same STs shared 100.0% similarity in PFGE profile type, see Figs 1 and 2 groups (PFGE J2-ST464 and PFGE J9-ST-2328) of C. jejuni strains originated from humans and chickens were confirmed to be clonally related by comparing PFGE and MLST results.

Antibacterial susceptibility of C. jejuni and C. coli isolates

Antibiotic susceptibility testing was conducted on 73 Campylobacter isolates, including 54 C. jejuni (34 from patient isolates and 20 from food isolates) and 19 C.coli strains (6 from patient isolates and 13 from food isolates), see Table 3. C. jejuni showed resistance most frequently to NAL (94.44%), followed by TET (88.89%), CIP (87.04%), CLI (16.67%), ERY /GEN/FLO (12.96%), AZM/TEL (11.11%), STR (9.26) and CHL (5.56%), while C. coli displayed highest resistance rate to NAL/CIP (94.74%), followed by TET (84.21%), ERY (63.16%), AZM (52.63%), TEL/CLI (42.11%), GEN (31.58%), STR (26.32), FLO (10.53) and CHL (5.26%). Compared with C. jejuni, statistical higher resistant rates were observed in C. coli for ERY (χ2 = 18.39, p < 0.0001), AMZ (χ2 = 14.16, p = 0.0001) and TEL (χ2 = 8.71, p = 0.003). Specifically, ERY resistance in C. coli (63.16%) is much more prevalent than that in C. jejuni (12.96%). The MARI of the tested C. jejuni and C. coli isolates from the current study are presented in Tables 4 and 5, respectively. A total of 18 different antibiotic resistance patterns with MARI ranging from 0.18 to 0.91 were observed in 54 C. jejuni isolates, while a total of 12 different antibiotic resistance patterns with MARI ranging from 0.09 to 0.73 were observed in 19 C. coli isolates. The MDR rate were 29.63% (16/54) for C. jejuni and 89.47% (17/19) for C. coli. The difference in MDR rates between the two groups was statistically significant (χ2 = 20.321, P < 0.01).

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Table 3. Antimicrobial resistant rate of 54 C. jejuni and 19 C. coli isolates from different sources against 11 antimicrobials.

https://doi.org/10.1371/journal.pone.0311769.t003

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Table 4. Resistance spectra of 54 C. jejuni isolates to various antibiotic combinations.

https://doi.org/10.1371/journal.pone.0311769.t004

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Table 5. Resistance spectra of 19 C. coli isolates to various antibiotic combinations.

https://doi.org/10.1371/journal.pone.0311769.t005

Discussion

Bacterial diarrhea poses a serious global public health challenge, with Campylobacter infection considered one of the primary culprits [12]. In recent years, Campylobacter infections have been on the rise worldwide [13]. Conventional methods for the isolation and identification of Campylobacter include enrichment culturing and selective isolation. The enrichment with double membrane filtration method was recognized as a more effective isolation method for Campylobacter and several studies of Campylobacter prevalence in diarrhea cases have been conducted using this method previously, with isolation ratio from 7.0%-12.1% [14–18]. Zhang et al. reported the Campylobacter isolation rate in diarrhea patients in Beijing was 7.81% [14]. Another study in Wenzhou (Southeast China) in patients with diarrhea showed that prevalence of Campylobacter infection was 10.5% [17]. Yan et al. revealed a C. jejuni prevalence of 4.0% in children and 5.8% in adults with diarrhea in Shenzhen (South China) [15]. The discrepancy in prevalence may be caused by regional differences or variations in sample size. Besides, picking out the suspected Campylobacter colonies on the selective medium was laboratory experience depending, and this might be another reason why there has been such variations in prevalence reported for diarrheal patients [16]. In this study, the double membrane filtration method was employed for the first time starting from September 2021 to detect Campylobacter in fecal specimens from diarrhea patients in Huzhou. The detection rate reached 11.70% among 342 diarrhea samples, surpassing the detection rates of other causative agents such as enteropathogenic Escherichia coli (6.49%), Vibrio parahaemolyticus (5.36%), and Salmonella (2.85%) in recent years [19,20]. Campylobacter has emerged as a predominant foodborne pathogen in the local region. Notably, the detection rate of C. jejuni in diarrheal patients was significantly higher than that of C. coli (χ2 = 20.81, P < 0.0001), consistent with previous study in other region of China [17,18]. Poultry has long been identified as the primary vehicle for sporadic Campylobacteriosis and the most common cause of Campylobacter outbreaks [17,21]. A meta-analysis by Zbrunab et al. on the global prevalence of Campylobacter in animal products also highlighted chickens as the primary source of Campylobacter transmission [22]. Similar results were also found in this study, the double membrane filtration method was applied to test 168 raw meat products (50 samples of livestork meat products and 118 samples of raw poultry products), and 38 Campylobacter isolates were detected, all originating from poultry meat, with chicken been the major source of infection (86.84%, 33/38).

The MLST and other genotyping approaches, including PFGE technology, shows that Campylobacter is not a genetically monomorphic organism, but includes highly diverse assemblies with an array of dierent phenotypes [17,23–25]. Consistent with previous reports, both PGFE type and MLST data confirmed that Campylobacter stains circulating in Huzhou are genetically diverse, with C. jejuni isolates being more diverse than C. coli based on MLST analysis. PFGE typing revealed 45 band patterns among 54 C. jejuni strains and 17 band patterns among 19 C. coli strains. Our findings demonstrated that the 50 C. jejuni strains from different sources were classified into 37 ST types, showing a dispersed distribution and encompassing over 12 CCs. The distribution of ST types in the 18 C. coli strains was relatively concentrated, with 83.33% (15/18) of isolates belonging to the CC-828, consistent with previous reports [26,27]. We also identified seven C. jejuni strains and one C. coli strain with novel ST types, enriching the global MLST database.

Numerous studies have reported varying major CCs of C. jejuni in different countries and regions, but CC-21, CC-45, CC-353, and CC-574 are consistently the predominant CCs among isolates in many investigations [17,24,28–30], with CC-21 considered closely associated with human infections [31] and representing 17.9% of all C. jejuni strains submitted to the PubMLST database. Our study reveals that the most prevalent CC among different sources of C. jejuni is CC-21 (22.00%, 11/50), with 9 strains from diarrhea patients and 2 strains from raw meat products. The CC-464, reported as a dominant clone in domestic settings [17], was detected in 2 strains of patients isolate and 1 strain of food isolates.

Recent phylogenetic studies using relatedness between PFGE and MLST have revealed that the two methods have effective discriminatory power in evaluating the genetic homology among Campylobacter strains [17,21,32]. In this study, 2 groups of C. jejuni strains (PFGE J2-ST464 and PFGE J9-ST-2328) originated from humans and chickens showed high genetic homologies by comparing PFGE and MLST results. Besides, some disagreement between PFGE and MLST was observed for certain ST, indicating a weak correlation between PFGE and MLST for certain Campylobacter strains. As the sequencing cost continues to decrease, next-generation sequencing (NGS) technology, which has the advantages of high throughput, high precision, and rich genetic information, may be more suitable for evaluating the genetic homology among Campylobacter strains from different sources.

In recent years, the widespread and sometimes inappropriate use, even misuse, of antibiotics in clinical practice and extensive long-term use of antibiotic drugs in animal husbandry have led to the emergence of antibiotic-resistant Campylobacter strains. According to literature reports, Campylobacter exhibits high resistance to quinolones and tetracycline antibiotics, with fluoroquinolone resistance rates ranging from 75% to 90% in Campylobacter strains from different countries [33,34]. The use of fluoroquinolones in food-producing animals has resulted in fluoroquinolone-resistant Campylobacter strains worldwide [35]. In this study, C. jejuni showed resistance most frequently to NAL (94.44%), followed by TET (88.89%), CIP (87.04%), CLI (16.67%), while C. coli displayed highest resistance rate to NAL/CIP (94.74%), followed by TET (84.21%). Overall, C. coli showed higher resistance rates to all antimicrobials than C. jejuni did except for CHL, FLO and TET, with MDR rate significantly higher than that of C. jejuni. Similar findings have been reported in Southeast [17] and North China [14], as well as other countries. Additionally, we observed ERY resistance in C. coli (63.16%) is much more prevalent than that in C. jejuni (63.16% vs 12.96%), in accordance with other studies [26,36–39]. The widely observed higher rate of macrolide resistance in C. coli than in C. jejuni may be associated with fitness costs impacts of certain antibiotic-resistant mutants, with the underlying mechanisms remain to be further elucidated [26,40]. In addition, over 90% of C. jejuni and C. coli clinical samples were susceptible to chloramphenicol (CHL), indicating that chloramphenicol remains effective for the treatment of C. jejuni and C. coli infection in Huzhou area.

Furthermore, it is worth mentioning that research regarding zoonotic diseases often focuses on infectious diseases animals have given to humans. However, an increasing number of reports indicate that bacteria expressing resistance to critically important antimicrobials were likely introduced along pathways involving reverse zoonosis (human-animal transmission) [41]. This included emergence of human pandemic O25:H4-ST131 CTX-M-15 extended-spectrum-beta-lactamase-producing Escherichia coli among companion animals [42] and community-associated methicillin-resistant Staphylococcus aureus, in dairy cow [43]. Recent reports from New Zealand demonstrated that fluoroquinolone resistance detected there among poultry was attributable to the emergence of a new clone of C. jejuni (ST6964) and it has been hypothesized that this clone was potentially introduced via exposure to other species (human or other livestock) because fluoroquinolones are not registered for use in poultry in New Zealand [44]. Therefore, the risk of antibiotic-resistant Campylobacter being transmitted from humans, including raw meat handlers, to poultry should not be overlooked.

Conclusions

In conclusion, this study provides a preliminary understanding of the molecular genetic features and antibiotic resistance characteristics of Campylobacter spp. from raw meat products and diarrhea cases in the Huzhou area. Campylobacter is an important foodborne pathogen in both diarrheal patients and raw meat products in Huzhou City, exhibiting multiple antibiotic resistance and high level of genetic diversity. Two groups of C. jejuni strains originated from humans and chickens were confirmed to be clonally related by comparing PFGE and MLST results. More comprehensive study based on the genetic correlation between isolates from humans and food animals is needed to prevent and control diseases caused by them. Considering the advantages of NGS, future work is warranted to integrate NGS-based typing methods into routine foodborne pathogen surveillance to elucidate the molecular characteristics of Campylobacter spp. isolates.

Supporting information

S1 Table. MLST data summary of 50 C. jejuni strains and 18 C. coli strains in this study.

https://doi.org/10.1371/journal.pone.0311769.s001

(DOCX)

Acknowledgments

We thank the staff of the First People’s Hospital in Huzhou for collecting the samples.

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