摘要:
目的 了解近年来杭州地区甲型副伤寒沙门菌的基因组流行病学特征.方法 采用二代测序技术(NGS)对2002—2013年杭州地区分离的60株甲型副伤寒沙门菌代表株进行测序并下载公共数据库中的391株甲型副伤寒沙门菌基因组数据.以ATCC9150基因组为参考序列,鉴定所有基因组的单核苷酸多态性(SNP)位点并去除重组,构建基于SNP位点的系统发育树.用SRST2和多位点序列分型(MLST)工具扫描获得MLST型别,用SISTR扫描获得核心基因组多位点序列分型(cgMLST)型别,用SRST2和BLASTN扫描获得耐药基因.选择7种抗生素对60株杭州菌株进行药物敏感试验.结果 451株甲型副伤寒沙门菌基因组序列去重组后共鉴定得到19258个SNP位点,菌株平均距离为0.0070,距离小于0.05的占96.73%,提示451株甲型副伤寒沙门菌基因组差异较小.58株杭州ST85型分离株基因组序列高度相似,提示2002—2013年杭州地区甲型副伤寒疫情主要由ST85型菌株克隆传播引起.杭州菌株与5株国内5省菌株遗传距离较远(平均距离为0.057),与15株云南菌株距离较近(平均距离为0.0032),与柬埔寨菌株的距离最近(平均距离为0.0018),提示ST85型菌株存在跨国传播的可能性.2株杭州ST129型分离株中,HZ333与分离自江苏的两株菌近源(平均距离为0.0097),提示ST129型部分菌株存在国内传播的可能性.60株杭州菌株除2株未分型外,其余58株菌被分为9个cgMLST型别;公共数据库中391株菌除57株未分型外,其余334株菌被分为165个cgMLST型别.60株杭州菌株均携带耐药基因,其中56株菌携带aac6-Iy耐药基因,4株菌携带aac6-Iaa耐药基因;公共数据库中的391株甲型副伤寒沙门菌除了13株菌未携带耐药基因,其他378株菌均携带aac6-Iy耐药基因.60株杭州菌株中56株菌对7种抗生素均敏感;3株菌对复方新诺明耐药;1株菌对氨苄西林、四环素耐药.结论 2002—2013年杭州地区甲型副伤寒疫情主要由ST85型菌株克隆传播引起,该型菌株存在跨国传播的可能性;ST129型部分菌株存在国内传播的可能性.SNP位点分析技术分辨力高于脉冲场凝胶电泳(PFGE)技术,能够帮助我们更为精准的对疫情进行溯源.aac6-Iy是全球范围内甲型副伤寒沙门菌普遍携带的一种耐药基因.二代测序技术在传染病防控中有着良好的应用前景.%Objective To understand the genomic epidemiology of Salmonella paratyphi A strains circulating in Hangzhou area in recent years. Methods Next generation sequencing(NGS) technology was used to obtain genomes of 60 Salmonella paratyphi A strains isolated in Hangzhou area from 2002 to 2013. Genomes of 391 Salmonella paratyphi A strains were downloaded from the Sequence Read Archive (SRA) and Assembly database. After removing recombinations, the phylogenetic tree of all 451 genomes based on single nucleotide polymorphisms(SNP) was constructed using ATCC9150 as the reference. SRST2 and mul-tilocus sequence typing (MLST) were used to analyze sequence types(ST). The Salmonella In Silico Typ-ing Resource (SISTR) was used for core genome multilocus sequence typing (cgMLST). Resistant genes were screened out with SRST2 and BLASTN. Seven kinds of antibiotics were selected to conduct drug sus-ceptibility test in the 60 strains isolated in Hangzhou. Results A total of 19 258 SNP loci were found in 451 genomes. The average distance in the phylogenetic tree between strains was 0.007 0 and the distance less than 0.05 accounted for 96.73%, indicating a little difference in the 451 Salmonella paratyphi A ge-nomes. Fifty-eight Hangzhou strains of ST85 were highly similar in genomic sequences,which suggested that the clonal spread of ST85 strains caused the epidemic of paratyphoid A in Hangzhou area during 2002-2013. Salmonella paratyphi A strains isolated in Hangzhou were distantly related to five domestic strains (average distance:0.057),but close to 15 Yunnan strains(average distance:0.003 2) and closest to strains isola-ted in Cambodia (average distance: 0.001 8), suggesting the possibility of transnational spread of ST85 strains. Among two Hangzhou strains of ST129,HZ333 was closely related to two strains isolated in Jiangsu Province(average distance:0.009 7),suggesting the possibility of domestic transmission of ST129 strains. Except for two untyped strains,the other 58 strains were divided into nine cgMLST types. Except for 57 un-typed strains,the other 334 strains obtained from public databases were classified into 165 cgMLST types. Fifty-six out of the 60 Hangzhou strains carried the resistant gene aac6-Iy and the other four carried aac6-Iaa gene. Thirteen out of the 391 strains obtained from public databases didn′t carry resistant genes, while the other 378 strains carried the resistant gene aac6-Iy. Among the 60 Hangzhou strains,56 were sensitive to all of the seven kinds of antibiotics;three were resistant to cotrimoxazole and one was resistant to ampicillin and tetracycline. Conclusion The epidemic of paratyphoid A fever in Hangzhou from 2002 to 2013 was mainly caused by the clonal spread of ST85 strains that had the possibility of transnational spread. Some Hangzhou strains of ST129 had the possibility of domestic transmission. SNPs analysis had an advantage over pulse field gel electrophoresis(PFGE) technology in resolution and could be used to accurately trace the cause of paratyphoid A fever. Resistant gene aac6-Iy was generally carried. NGS technology had a promising prospect in the control and prevention of infectious diseases.