江苏省部分地区畜禽源弯曲菌耐药基因、毒力基因及遗传多样性分析

Antimicrobial resistance genes, virulence genes, and genetic diversity of Campylobacter isolated from chicken and swine farms in Jiangsu Province

摘要:

【背景】 弯曲菌(Campylobacter)是引起食源性疾病的主要病原菌之一，家禽和猪是其常见和重要宿主，畜禽在屠宰加工过程中易污染胴体导致人类感染，对食品安全和公共健康造成巨大威胁。【目的】 通过应用全基因组测序(whole genome sequencing, WGS)分析52株畜禽来源弯曲菌的耐药基因、毒力基因及遗传多样性。【方法】 采用琼脂稀释法对其最小抑菌浓度(minimal inhibitory concentration, MIC)进行测定，通过二代测序平台进行全基因组测序，应用基因组流行病学数据平台ResFinder数据库分析其耐药基因，通过毒力因子数据库(virulence factor database, VFDB)筛选毒力基因，应用PubMLST数据库分析菌株序列型(sequence type, ST)和克隆复合体(clonal complex, CC)。【结果】 52株弯曲菌对环丙沙星、四环素、萘啶酸耐药明显，对氟苯尼考敏感，多重耐药率为57.69%。WGS分析发现52株弯曲菌共有6大类16种获得性耐药基因及与喹诺酮类耐药相关的gryA基因点突变和与大环内酯类耐药相关的23S rRNA点突变。除检出常见的氨基糖苷类、β-内酰胺类、四环素类(tetO)耐药基因外，还发现ermB、ermA、fexA、optrA、tetL和lnuC等在弯曲菌新流行的耐药基因；52株弯曲菌携带120种毒力相关基因，其中细菌黏附、荚膜合成、侵袭、鞭毛及趋化蛋白等相关基因携带率较高，空肠弯曲菌(Campylobacter jejuni)携带毒力基因的数量普遍多于结肠弯曲菌(Campylobacter coli)；多位点序列分析(multilocus sequencing typing, MLST)结果显示共得到28个ST型和14个CCs (包括4个已知的CCs和10个未知CCs，以CC-828克隆复合体为主)，分离株之间具有较高的遗传多样性。【结论】 江苏畜禽源弯曲菌多重耐药现象严重，耐药基因复杂多样，毒力基因分布广泛，以克隆复合体CC-828为主，具有较高的遗传多样性。该研究结果为我国动物源性弯曲菌耐药性监测提供了基础数据。

Abstract:

[Background] Campylobacter spp. are a genus of the main pathogens causing foodborne diseases. They colonize the caeca of broilers and swine and can contaminate carcasses after slaughter during the processing, posing a risk of exposure to consumers and a serious threat to food safety and public health. [Objective] To investigate the antimicrobial resistance genes, virulence genes, and genetic diversity of 52 Campylobacter strains isolated from chicken and swine farms in Jiangsu Province by whole genome sequencing (WGS). [Methods] The agar dilution method was used to determine the minimal inhibitory concentrations (MICs) of Campylobacter isolates against eight antimicrobial agents. WGS was performed for the 52 Campylobacter strains. ResFinder of the Center for Genomic Epidemiology (CGE) was used to analyze the antimicrobial resistance genes. Virulence genes were identified from the core sequences downloaded from the virulence factor database (VFDB). Sequence type (ST) was determined in silico from the WGS data and isolates were assigned into clonal complex (CC) in PubMLST. [Results] Fifty-two Campylobacter strains were significantly resistant to ciprofloxacin, tetracycline, and nalidixic acid and sensitive to florfenicol, with the multidrug resistance rate of 57.69%. Sixteen acquired resistance genes belonging to six major categories were detected among with gyrA mutation associated with quinolones resistance and 23S rRNA gene mutation associated with macrolides resistance. In addition to the previously reported resistance genes to aminoglycosides, β-lactams, and tetracyclines (tetO), several new antimicrobial resistance genes emerging in Campylobacter in recent years were detected in this study, including ermB, ermA, fexA, optrA, tetL, and lnuC. A total of 120 virulence genes were detected in 52 strains of Campylobacter, among which the genes involved in adhesion, capsule synthesis, invasion, flagella, and chemotactic proteins had higher carrying rates. Campylobacter jejuni exhibited higher abundance of virulence genes than C. coli. MLST assigned 28 STs and 14 CCs (including 4 known CCs and 10 unknown CCs, among which CC-828 was the main one), indicating high genetic diversity among the isolates. [Conclusion] The Campylobacter isolated from chicken and swine farms in Jiangsu Province presented severe multidrug resistance. The resistance and virulence genes were complex, diverse, and widely distributed in Campylobacter strains. CC-828 was the main clonal complex, and the Campylobacter strains exhibited high genetic diversity. The results provide basic data for the monitoring of antimicrobial resistance of animal-derived Campylobacter in China.