温州口岸截获蜱体内微生物群落结构、抗生素抗性基因及毒力因子的宏基因组分析

Metagenomic analysis of microbial community structure,antibiotic resistance genes,and virulence factors of ticks captured at Wenzhou port,Zhejiang province,China

目的以宏基因组学为技术手段,了解温州口岸在进口南非盐湿牛皮上检疫发现的无色扇头蜱体内微生物群落结构特征,及抗生素抗性基因(ARGs)、毒力因子携带情况。方法将截获的蜱随机分成5份,经HiPure Bacterial DNA Kits提取DNA后,采用高通量测序的方法,分析其微生物群落结构、致病菌、抗生素抗性基因及毒力因子。结果5份样本微生物组成相似。在门水平上,均以厚壁菌门、变形菌门为主要菌门。其中厚壁菌门丰度最高,占全部门的66.00%以上。在属水平上,均以链球菌属(>55.30%)、芽孢杆菌属(>11.20%)为优势菌属。在全部蜱样本中均检测到变形链球菌、蜡样芽孢杆菌、肠沙门菌、肺炎链球菌等常见条件致病菌,占注释总水平的71.00%以上。抗性基因分析发现蜱样本含有Acr,bacA,mdtK,arnA,mdtH等16种抗性基因,其中Acr丰度最高(30.19%),其次是bacA(20.75%)。这些抗性基因分别介导对大环内酯类、氨基糖苷类、β-内酰胺酶、甘氨酰环素、吖啶黄素等多种抗菌药物的耐药。细菌毒力因子数据库(VFDB)注释分析发现蜱样本共有3923个毒力基因,主要与黏附(22.66%)、分泌系统(13.97%)、侵袭(13.56%)等毒力因子相关。结论无色扇头蜱体内病原体种类较多,检出肠沙门菌等危害较大的致病菌,同时携带大量抗生素抗性基因及毒力因子,因此相关部门及一线从业人员应注意防控跨境蜱传疾病传入,防止对我国公共卫生造成影响。

Objective To investigate the microbial community structure,antibiotic resistance genes(ARGs),and virulence factors by the metagenomic technique in ticks captured from wet-salted cow hides imported from South Africa at a port in Wenzhou,Zhejiang province,China.Methods We randomly divided the captured ticks into 5 groups,extracted DNA using HiPure Bacterial DNA Kits,and used high-throughput sequencing to analyze the microbial community structure,pathogenic bacteria,ARGs,and virulence factors of the ticks.Results The 5 samples had similar microbial community structure.At the phylum level,Firmicutes and Proteobacteria were the dominant phyla,and Firmicutes was the most abundant,accounting for more than 66.00%of the total phyla.At the genus level,Streptococcus(>55.30%)and Bacillus(>11.20%)were the dominant genera.All the tick samples were positive for St.mutans,Ba.cereus,Salmonella enterica,and St.pneumoniae,accounting for more than 71.00%of the total species annotated.Sixteen ARGs were identified in the tick samples,including Acr,bacA,mdtK,arnA,and mdtH,and Acr had the highest abundance(30.19%),followed by bacA(20.75%).These ARGs mediated resistance to antibiotics such as macrolides,aminoglycosides,β-lactamases,glycylcycline,and acriflavine.A total of 3923 virulence genes were found in the tick samples through annotation with the virulence factor database,which were mainly related to adherence(22.66%),secretion system(13.97%),and invasion(13.56%).Conclusion The imported ticks carry various species of pathogens,including highly harmful pathogens such as Sa.enterica,and harbor numerous ARGs and virulence factors.Relevant authorities and frontline workers should pay attention to preventing imported tick-borne diseases and taking effective treatment measures to avoid affecting public health in China.