Genetic Characteristics and Antimicrobial Susceptibility of Arcobacter butzleri Isolates from Raw Chicken Meat and Patients with Diarrhea in China

Arcobacter spp. is a new foodborne pathogen and potential zoonotic agent that can also cause outbreaks of foodborne illness. Arcobacter spp. can cause diarrhea, mastitis, miscarriage and gastroenteritis in animals, and diarrhea, bacteremia, endocarditis and peritonitis in humans^[1]. Arcobacter spp. is prevalent worldwide and has been identified in patients with diarrhea, and in the environment, including in water, animals and foods of animal origin, fish and seafoods, vegetables^[2-4]. Arcobacter spp. can be transmitted through contaminated food and water sources and cause human diseases^[4]. The Arcobacter genus comprises Gram-negative, curved rod-shaped bacteria 0.2–0.5 μm in diameter and 1–3 μm long, growing at temperatures of 15–42 °C, in contrast to the taxonomically related genus Campylobacter^[4]. After 3 days of incubation on blood agar plates, the colonies are round and whitish with diameters of 2–4 mm.

Currently, the Arcobacter genus can be divided into 29 species, among which A. butzleri, A. cryaerophilus and A. skirrowii are considered most closely associated with human diseases, with A. butzleri and A. cryaerophilus considered hazardous to human health by the International Commission on Microbiological Specifications for Foods as early as 2002^[1,2,4]. A. butzleri has been found to be the most prevalent species in a study of Arcobacter in patient feces, and has been reported to cause more long-lasting and aqueous, but less bloody and acute diarrhea, with little presence in the stools, among healthy asymptomatic people. In addition, A. butzleri is widely distributed in animal-derived food. It subsequently colonizes the intestines of animals (cattle, sheep, pigs and poultry) and contaminates the food supply during the slaughter process, thus indicating that these animals may be reservoir hosts^[5]. The pathogenic mechanism of A. butzleri is unclear, but virulence genes have been studied through genome analysis and PCR, including cadF and cjl349, which encode fibronectin-binding proteins; ciaB, which encodes an invasion protein; virulence factors mviN; pldA and tlyA, which encode phospholipases; hecA, which encodes a member of the hemagglutinin family; hecB, which encodes a related hemolysin activation protein; IroE, which encodes an enterobactin hydrolase present in uropathogenic E. coli; and irgA, a homologue of the iron-regulated outer membrane protein in Vibrio cholerae^[4].

In China, the distribution of Arcobacter in raw meat has been reported^[6], but no report has addressed the prevalence of patients with diarrhea. In this study, the distribution and antimicrobial susceptibility of Arcobacter in market raw chicken samples and hospitalized patients with diarrhea were obtained. Next, the genetic characteristics of 18 Arcobacter isolates were investigated by whole genome sequencing to explore potential pathogenicity and drug resistance mechanisms, and to provide data for food safety assessment and Arcobacter disease prevention and treatment. This is the first comprehensive study of Arcobacter in China.

A total of 150 fecal samples from patients with acute diarrhea and 60 fresh raw chicken meat samples, comprising 44 samples from wholesale markets and 16 samples from retail stores, were examined in this study. The patients with diarrhea had three or more bouts of watery, loose, mucus-containing or bloody-stools during a 24 h period in hospitals in the Shunyi district, Beijing, between May 1, 2017 and October 31, 2017. Five milligrams of fresh stool samples were collected from each patient, kept at 4 °C and sent to the laboratory within 24 h. Questionnaire information from each patient was collected by the clinical doctor during hospital visits (Supplementary File S1, available in www.besjournal.com). The unfrozen chicken meat samples were collected from May to July (3 months, 20 samples per month) of 2018.

The Arcobacter isolation was performed with an Arcobacter isolation kit with the membrane filter method (ZC-ARCO-001, Qingdao Sinova Biotechnology Co., Ltd., Qingdao, China). The specific procedures were as follows: 200 mg stool specimens were transferred into 4 mL enrichment buffer which was provided in the kit. The principle component of the enrichment buffer was the modified preston broth which was described in the manual book. The raw chicken meat was fully washed with 500–1,000 mL buffered peptone water for 10 min, and then 2 mL buffered peptone water was added to 4 mL enrichment buffer. The enriched suspension from both stool and chicken samples was incubated for 24 h at 37 °C in a microaerophilic atmosphere consisting of 5% O₂, 10% CO₂, and 85% N₂. Subsequently, 300 μL cultured enrichment suspension was spotted onto Karmali and Columbia agar with a 0.45 μm cellulose membrane filter. After air-drying in a biological safety cabinet for 40 min, the filter membrane was removed, and the plates were incubated in a microaerophilic atmosphere at 37 °C for 48 h.

After incubation, small round and whitish colonies 2 mm in diameter were streaked on plates and confirmed by Gram staining, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and real-time PCR. The mass spectrometry analysis used Flexcontrol software, and the results were interpreted with IVD MALDI Biotyper 2.3 software (Bruker Daltonik GmbH, Bremen, Germany). The criteria for determining the genus and species of bacteria were as follows: 2.300 to 3.000 points indicated reliable species level identification and 2.000 to 2.299 points indicated reliable genus level and possible species level identification. In this study, scores ≥ 2.000 were considered credible. For PCR identification, a loop was used to collect suspicious pure culture colonies, which were re-suspended in 200 μL ultrapure water, boiled for 10 min and centrifuged for 10 min at 8,000 ×g. Subsequently, the supernatant was removed for PCR species identification with a Real-time PCR kit (ZC-ARCO-003, Qingdao Sinova Biotechnology Co., Ltd.). The PCR amplification conditions were as follows: initial denaturation at 94 °C for 5 min, followed by 45 cycles of 94 °C for 15 s and 60 °C for 1 min.

The minimum inhibitory concentrations (MICs) for 11 antibiotics (erythromycin, azithromycin, nalidixic acid, ciprofloxacin, gentamicin, streptomycin, chloramphenicol, florfenicol, tetracycline, telithromycin and clindamycin) were determined with an agar dilution method by using a commercial kit for fastidious bacteria (ZC-AST-010, Qingdao Sinova Biotechnology Co., Ltd.). The MIC was the lowest concentration without visible growth. The cut-offs for resistance used in this study were those with an MIC value greater than or equal to the standards used in the National Antimicrobial Resistance Monitoring System (NARMS-2014) for Campylobacter in the United States: erythromycin (≥ 32 μg/mL), azithromycin (≥ 8 μg/mL), nalidixic acid (≥ 64 μg/mL), ciprofloxacin (≥ 4 μg/mL), gentamicin (≥ 8 μg/mL), streptomycin (≥ 16 μg/mL), chloramphenicol (≥ 32 μg/mL), florfenicol (≥ 8 μg/mL), tetracycline (≥ 16 μg/mL), telithromycin (≥ 16 μg/mL) and clindamycin (≥ 8 μg/mL). C. jejuni ATCC 33560, Staphylococcus aureus ATCC 29213, and Escherichia coli ATCC 25922 were used as controls.

The genomes of 18 Arcobacter isolates were sequenced on the Illumina NovaSeq 6000 platform at the Microbial Genome Research Center (Institute of Microbiology Chinese Academy of Sciences, Beijing, China), and general features of the A. butzleri genome sequences were examined (Table 1). To sequence the genome, we constructed a 500 bp paired-end library and generated 150 bp reads for each isolate. The sequencing reads were filtered with fastp (https://github.com/OpenGene/fastp, version 0.20.0), and low quality reads were removed. The FastQC tool (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, version 0.11.8) was used to evaluate the quality of the raw sequence data. The sequencing reads were trimmed with Trimmomatic (version 0.38) to remove sequencing adapters. High-quality reads were assembled with SPAdes genome assembler software (https://github.com/ablab/spades, version 3.11.0). Annotation was performed with Prokka (https://github.com/tappearann/prokka, version 1.13.3) with the recommended standard settings. Core-genome single nucleotide polymorphisms (SNPs) were called with Snippy4.3.6 software (https://github.com/tappearann/snippy) from the reference (RM4018 complete genome, isolated from a patient with gastroenteritis). Gubbins software (https://github.com/sanger-pathogens/gubbins, version 2.3.4) was used as the recombination-removal tool to determine the pure SNPs without recombination. Phylogeny reconstruction was performed with the maximum likelihood method in raxml software (https://github.com/stamatak/standard-RAxML, version 8.2.12) with 1000 bootstraps. A. cryaerophilus 16CS1285-4 (GenBank accession number CP060693) was used as the outgroup and root of the tree. Resistance genes were predicted with ResFinder (https://cge.cbs.dtu.dk/services/ResFinder/), Abricate (https://github.com/tappearann/abricate, version 0.9.8) and Comprehensive Antibiotic Resistance Database (https://card.mcmaster.ca/?q=CARD/ontology/35506), with an E-value of at least 1 × 10⁻¹⁰ as the cut-off. The identity cut-off and query coverage values were kept > 80% and > 80%, respectively. Virulence factors were identified with VFDB (http://www.mgc.ac.cn/cgi-bin/VFs/v5/main.cgi?func=VFanalyzer).

The detection rate of Arcobacter in raw chicken samples was 26.7% (16/60), and all isolates were identified as A. butzleri. However, one previous study on the contamination status of Arcobacter in retail chicken in Beijing has reported an isolation ratio of 78.21% (61/78), and most of the isolates were identified as A. cryaerophilus (91.20%, 114/125)^[6]. These results indicate that the dominant contaminated species of Arcobacter in retail chicken meat might vary in different markets. In addition, this is the first study on A. butzleri infection in patients with diarrhea in China, and we found two positive cases among the 150 cases of diarrhea (1.3%). The isolation rate was consistent with those reported in studies from Portugal and India, where A. butzleri was detected with an isolation ratio of 1.30% and 2.67%, respectively^[3,7], but was different from those in Belgium (0.7%, 49/6,774), Turkey (0.27%, 9/3,287) and New Zealand (0.50%, 7/1,380)^[2,8,9].

A.butzleri is an opportunistic pathogen that can cause severe and persistent diarrhea and even bacteremia in immunocompromised people^[10]. In our study, the two cases of diarrhea were in immunocompromised people, a post-cancer patient and a 71-year-old male, who were co-infected with other pathogens (one case with V. parahaemolyticus, and another case with both V. parahaemolyticus and enterotoxigenic E. coli). Immuno-compromised and older people with diarrhea should receive more attention for such infections. In the future, additional surveillance studies of patients with diarrhea are needed.

In our study, the resistance ratios of 18 isolates for 11 drugs were 83.33% (15/18) for nalidixic acid, 38.89% (7/18) for ciprofloxacin, 38.89% (7/18) for clindamycin, 33.33% (6/18) for florfenicol, 33.33% (6/18) for chloramphenicol, 22.22% (4/18) for tetracycline, 22.22% (4/18) for telithromycin, 22.22% (4/18) for azithromycin, 11.11% (2/18) for gentamicin and 5.56% (1/18) for streptomycin. In addition, six (33.33%) isolates were multi-drug resistant, and the dominant resistance pattern was nalidixic acid and ciprofloxacin combined resistance (33.33%, 6/18). The resistance ratios to macrolides, aminoglycosides and tetracyclines were similar to those in other reports, but the resistance ratios to nalidixic acid (83.33%) and ciprofloxacin (38.89%) were much higher than those in other reports^[11]. Moreover, genetic determinants of antibiotic resistance were searched through whole genome sequencing analysis, and the C254T mutation, which causes a Thr-85-Ile substitution in the quinolone-resistance determining region of gyrA, was found in some both nalidixic acid and ciprofloxacin resistant isolates (027-Ab, 039-Ab, 051-Ab, and 052-Ab). This result was consistent with those of A. Khalil^[12]. Furthermore, bla_OXA-464 or bla_OXA-491 and tet(H) were identified with both ResFinder and CARD. Fifteen isolates, except 029-Ab, 052-Ab and 059-Ab, had bla_OXA-464 or bla_OXA-491, and only two isolates, 027-Ab and 039-Ab, had tet(H).

The assembled genome size for all 18 strains ranged from 2,068,991 to 2,299,734 bp, with an average of 2,163,707 bp assembled in an average of 39 contigs per genome. An average of 2,152 coding DNA sequences (CDS) and 2,199 genes were obtained from each genome. Two isolates from patients with diarrhea clustered together, apart from the isolates from raw chicken samples, and the chicken strains exhibited genetic diversity (Figure 1). The adhesion associated genes were present in 38.9% (7/18) of the isolates. Analysis of virulence genes indicated that all isolates contained genes involved in motion related flagellin synthesis, such as flgG, flhA, flhB, fliI, fliP and motA. In our study, cadF, cjl349, ciaB, mviN, pldA and tlyA were found in all strains, while hecA, hecB and iroE were found in only some strains, but irgA was not found in any strains. The presence of these virulence genes may play important roles in the pathogenicity of A. butzleri. The heatmap based on virulence gene clusters generated with the pheatmap package is presented in Figure 2.

Figure 1.  Phylogenetic tree of A. butzleri isolates based on the core-Genome SNPs. Phylogenetic tree was constructed based on core-SNPs of A. butzleri using raxml software. The scale bar represents substitution per site. The number before branching represents bootstrap. A. cryaerophilus 16CS1285-4 was used as outgroup and root of the tree. Only bootstrap values greater than 60 were displayed on the tree.

Figure 2.  Heatmap of A. butzleri isolates based on virulence genes. The name of virulence genes is distributed horizontally and the name of A. butzleri isolates is distributed vertically in the heatmap. Gray indicates the presence of the gene and white indicates the absence of the gene.

In summary, we provide the first description of the prevalence and genetic characteristics of Arcobacter in patients with diarrhea in this study. The findings should aid in further investigation of this pathogen in China and worldwide.

Conflicts of Interest　The authors have no conflicts of interest to declare.