食物中毒来源金黄色葡萄球菌ST6和ST7菌株分子特征研究

Molecular characterization of Staphylococcus aureus ST6 and ST7 isolates from food-borne illness outbreaks

目的对2020年8月21日和9月27日广州市2起食物中毒事件中分离的41株金黄色葡萄球菌(金葡菌)进行肠毒素、肠毒素基因、耐药性、耐药基因情况以及分子分型分析。方法对2起食物中毒样本分离的41株金黄色葡萄球菌,通过多位点序列分型(MLST)和spa基因分型方法进行分子分型;ELISA检测菌株产肠毒素型别;PCR检测肠毒素基因;采用纸片扩散法对菌株进行药敏检测。选取21株代表性菌株进行全基因组测序,分析其耐药性和毒力基因,运用Snippy进行同源性分析。结果2起食物中毒的41株菌经MLST和spa基因分型方法分为ST6-t701和ST7-t091。耐药性分析显示,27株ST7-t091中有2株鉴定为耐甲氧西林金黄色葡萄球菌(MRSA),其余25株ST7-t091为甲氧西林敏感金色葡萄球菌(MSSA),对7种抗菌药物耐药;14株ST6-t701菌株的耐药谱完全一致,对6种抗菌药物耐药。PCR结果显示,所有菌株都携带sea基因。全基因组分析显示,21株金葡菌具有相同毒力基因谱scn、sak、sea、hla、hld、hlgA、hlgB、hlgC、lukD,携带的scn-sak-sea属于人类免疫逃避基因簇D型,位于前噬菌体ϕSa3上。单核苷酸多态性同源分析显示,2株MRSA ST7-t091高度同源,而12株MSSA ST7-t091和7株MSSA ST6-t701分别聚为一簇。结论2020年8月21日和9月27日广州市2起食物中毒事件分别为MRSA ST7-t091和MSSA ST7-t091混合感染以及MSSA ST6-t701感染引起,菌株耐药性较强且具有高致病力。

Objective To analyze the Staphylococcal enterotoxins,Staphylococcal enterotoxin genes,drug resistance and molecular typing of 41 Staphylococcus aureus isolates from 2 food-borne illness outbreaks on 21 August and 27 September 2020 in Guangzhou.Methods A total of 41 Staphylococcus aureus isolates from 2 outbreaks were analyzed by multilocus sequence typing(MLST)and spa typing.The Staphylococcal enterotoxins typing and the Staphylococcal enterotoxin genes of the isolates were analyzed by ELISA and PCR,respectively.The antimicrobial susceptibility of the isolates was performed by disc diffusion.21 Staphylococcus aureus isolates were characterized using whole genome sequencing(WGS).Based on the whole genome single nucleotide polymorphism(SNP),the phylogenetic tree was constructed by Snippy.Results 41 Staphylococcus aureus isolates were divided into 2 types by MLST and spa typing:ST6-t701 and ST7-t091.2 ST7-t091 isolates were identified as methicillin-resistant Staphylococcus aureus(MRSA).25 ST7-t091 isolates and 14 ST6-t701 isolates were methicillin-sensitive Staphylococcus aureus(MSSA),and were resistant to 7 and 6 antibiotics,respectively.All isolates were positive for sea by PCR.WGS revealed all 21 isolates carried scn,sak,sea,hla,hld,hlgA,hlgB,hlgC,lukD virulence genes.The results showed the isolates contained an immune evasion cluster type D which located in bacteriophageϕSa3.The SNP phylogenetic tree showed 2 MRSA ST7-t091 were constituted a separate clade from the 12 MSSA ST7-t091 isolates and 7 ST6-t701 isolates showed high similarity to each other.Conclusion Base on the results of phylogenetic analysis,the 2 food-borne illness outbreaks occurred on 21 August and 27 September 2020 are caused by the combination of the MRSA ST7-t091 strain and the MSSA ST7-t091 strain,and the MSSA ST6-t701 strain,respectively.All isolates have high level of antibiotic resistance and carry high virulent genes.