鸡蛋壳蜡样芽孢杆菌的分离鉴定及耐药性分析

【目的】 分析不同环境下鸡蛋外壳携带蜡样芽孢杆菌的情况和分离菌株的基本特征。【方法】 从养殖场和超市各采集9种新鲜鸡蛋，对鸡蛋表面携带的蜡样芽孢杆菌进行分离鉴定、基因分型、毒力基因筛查和耐药性分析。采用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）技术对分离菌株进行初步鉴定，全基因组测序后进行菌种鉴定并确定基因分型、筛选毒力基因和耐药基因，同时采用微量肉汤稀释法测定分离株对头孢曲松、红霉素、万古霉素等12种抗菌药物的耐药性。【结果】 经MALDI-TOF MS初步鉴定和基因组菌种鉴定，确定从养殖场3种未经清洁加工的鲜蛋中分离出的6个菌株为蜡样芽孢杆菌，其序列分型（ST）较为多样，其中有2株新的ST型。在毒力基因筛查方面，6株蜡样芽孢杆菌均检出了非溶血型肠毒素nheB基因、腹泻型毒力因子hlyⅡ基因和侵袭免疫系统的inhA基因；nheA和nheC基因携带率为83.3%，编码溶血型肠毒素HBL的hblA、hblC和hblD基因携带率均为66.7%；cytK和hlyⅢ基因携带率分别为33.3%和50.0%；肠毒素基因BceT、entFM和致吐毒素基因ces均未检出。耐药性方面，6株菌均对庆大霉素、四环素、氯霉素、利福平、利奈唑胺和环丙沙星敏感，对氨苄西林、头孢曲松和泰妙菌素耐药；对红霉素、克林霉素的中介率分别为83.3%和100%；33.3%的菌株对万古霉素耐药。6株菌均携带有磷霉素耐药基因FosB和万古霉素耐药基因簇vanA的部分基因vanRSYZ，其中5株菌携带大环内酯类药物耐药基因mphL，部分菌株携带β-内酰胺类药物耐药基因或核苷类药物耐药基因。【结论】 本试验从养殖场未经消毒处理的鸡蛋外壳上成功分离到了蜡样芽孢杆菌，并发现其携带有多种毒力基因，存在多重耐药性，有潜在致病性和公共安全风险。

Isolation,Identification and Antibiotic Resistance Analysis of Bacillus cereus on Eggshell

【Objective】 This study was aimed to analyze the presence of Bacillus cereus on eggs in different environments and the basic characteristics of these isolated strains.【Method】 9 kinds fresh eggs from different farms and supermarkets were collected.Isolation and identification, genotyping, virulence gene screening and drug resistance analysis of Bacillus cereus carried on the surface of eggs were carried out.Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used for preliminary identification of isolated strains.The strains were identified, genotyped and screened for virulence genes and resistance genes after whole genome sequencing.The resistance of the strains to 12 antibiotics including ceftriaxone, erythromycin, vancomycin, etc., was tested by the microbroth method.【Result】 After the preliminary identification by MALDI-TOF MS and genomic strain identification, it was determined that 6 strains isolated from 3 kinds of unsterilized fresh eggs in the farms were all Bacillus cereus. The sequence typing (ST) of 6 strains were various, and two new ST types appeared.Non hemolytic enterotoxin gene nheB, diarrheal type virulence factor gene HlyⅡ and immune system invader gene inhA were detected in all 6 strains of Bacillus cereus.The carrying rates of nheA and nheC genes were both 83.3%, and the carrying rates of hblA, hblC and hblD genes which encoded hemolysin BL (HBL) were all 66.7%.The carrying rates of cytK and hlyⅢ genes were 33.3% and 50.0%, respectively.While the enterotoxin genes BceT and entFM and cereulide gene ces were not detected.All of 6 strains were sensitive to gentamicin, tetracycline, chloramphenicol, rifampicin, linezolid and ciprofloxacin, and they were resistant to ampicillin, ceftriaxone and tiamulin.The mediations to erythromycin and clindamycin were 83.3% and 100%, respectively, and 33.3% of strains were resistant to vancomycin.All 6 strains carried the fosfomycin resistance genes FosB and vanRSYZ, part of the vancomycin resistance gene cluster vanA.5 strains carried the macrolide drug resistance gene mphL, and some strains carried the β-lactam drug resistance gene or nucleoside drug resistance gene.【Conclusion】 In this experiment, Bacillus cereus strains were successfully isolated from the shells of unsterilized eggs in the farms.It was found that the isolated strains carried a variety of virulence genes and had multiple drug resistance, with potential pathogenicity and public safety risks.