肉鸡屠宰环节中金黄色葡萄球菌的流行及分子特征和耐药性

Prevalence,Molecular Characteristics and Antimicrobial Susceptibility of Staphylococcus aureus in Broiler Slaughter and Processing Chain

为探究肉鸡屠宰加工环节中金黄色葡萄球菌的污染关键环节及菌株的分子特征和耐药性,对陕西省某肉鸡屠宰场5个屠宰环节(活鸡肛拭、浸烫、预冷、分割和贮存)进行4次样本收集。通过选择培养和聚合酶链式反应(polymerase chain reaction,PCR)扩增nuc基因对金黄色葡萄球菌进行分离鉴定。然后对分离株进行23种毒素编码基因、27种耐药基因、16种抗生素耐药性及葡萄球菌蛋白A(staphylococcal protein A,SPA)、多位点序列分型(multilocus sequence typing,MLST)和肠杆菌科基因间重复序列PCR(enterobacterial repetitive intergenic consensus-PCR,ERIC-PCR)分型检测。结果表明,收集的144份样本中有32份样本被污染(22.2%,32/144)。除活鸡肛拭样本无检出外,其余环节样本均有检出。其中浸烫褪毛环节污染率最高(40.0%,12/30),其次为分割环节(26.7%,8/30)、预冷环节(21.9%,7/32)和贮存环节(15.6%,5/32)。23种被检毒素编码基因中16种毒素编码基因被检出,其中pvl、seb、sek、seg、sei、sem、sen、seo、seu、sep和seq基因常被检出,且96.9%(31/32)的分离株携带新型肠毒素编码基因。27种被检耐药基因中仅有4种耐药基因被检出,包括blaZ、tet(K)、erm(B)和aac(6’)/aph(2’’)。16种所测试抗生素中,菌株对7种抗生素表现为耐药,其中对甲氧苄啶/磺胺甲恶唑耐药最为普遍,其次为氨苄西林、青霉素、四环素、环丙沙星、红霉素和庆大霉素。所有分离株共有5种克隆型(ST7-t091、ST5-t010/t002和ST59-t437/t441)和4种ERIC-PCR聚类簇(I1~I4)。不同批次样品分离株中存在相同ERIC-PCR图谱、分子型、耐药谱、毒素和耐药基因。结果表明,肉鸡屠宰过程中普遍存在金黄色葡萄球菌污染。且不同环节存在交叉污染现象,其中浸烫被认为是受污染的关键环节。研究还发现,分离株携带肠毒素编码基因与分子型有显著相关性(P<0.05)。通过对不同环节的监测,有助于识别加工环节污染的关键控制点,可以有效预防及控制食源性疾病的暴发。

To investigate the key links of Staphylococcus aureus pollution in the broiler slaughtering process and the molecular characteristics and antimicrobial susceptibility of S. aureus isolates, we collected microbial samples from different production stages(anal swabs from live chicken, soaking, pre-cooling, segmentation, and storage) in a broiler slaughterhouse in Shaanxi Province four times. S. aureus isolates were isolated and identified by selective culture and polymerase chain reaction(PCR) amplification of the nuc gene. Twenty-three virulence genes, 27 drug resistance genes, resistance to 16common antibiotics, staphylococcal protein A(SPA), multi-locus sequence typing(MLST), and enterobacterial repetitive intergenic consensus-polymerase chain reaction(ERIC-PCR) typing of the isolates were investigated. The results showed that 32 of the 144 samples collected(22.2%) were contaminated by S. aureus. The S. aureus contamination rate in the defeathering process was the highest(40.0%, 12/30), followed by the segmentation(26.7%, 8/30), pre-cooling(21.9%, 7/32),and storage processes(15.6%, 5/32). S. aureus was not detected in the anal swabs. Sixteen virulence genes were detected.Among them, the pvl, seb, sek, seg, sei, sem, sen, seo, seu, sep, and seq genes were detected frequently. Additionally, 96.9%(31/32) of the isolates carried novel enterotoxin genes. Only four drug resistant genes were detected, including blaZ, tet(K),erm(B), and aac(6′)/aph(2′′). The isolates showed resistance to seven of the 16 antibiotics tested, and were most commonly resistant to trimethoprim/sulfamethoxazole, followed by ampicillin, penicillin, tetracycline, ciprofloxacin, erythromycin,and gentamicin. Five clonal types(ST7-t091, ST5-t010/t002 and ST59-t437/t441) and 4 ERIC-PCR clusters(I1-I4) were detected in all the isolates. The isolates from different stages of the broiler slaughtering process had the same ERIC-PCR map, molecular type, drug-resistance profile, virulence gene profile, and antibiotic resistance gene profile. The results showed that S. aureus contamination was common in broiler slaughtering process. Cross-contamination could be observed among different processing stages, among which scalding was considered to be the key link of contamination. It was also found that the virulence genes carried by the isolates had a significant correlation with the molecular type(P < 0.05). The monitoring of different slaughter steps is helpful to identify the critical control points of S. aureus pollution, which can effectively prevent and control the outbreak of food-borne diseases.