目的 了解贵州省微小扇头蜱体内微生物群落和抗生素抗性基因（ARGs）的携带情况。方法 2022—2023年4、5月使用动物体表检蜱法采集贵州省道真、紫云和修文县以及盘州市牛体表微小扇头蜱,将收集到的蜱标本按地区分组,每个地区随机选取15只雌蜱,分为3管进行宏基因组测序。测序结果经数据质控、拼接组装等操作后与美国国立生物技术信息中心（NCBI）非冗余蛋白库和抗性基因数据库（SARG）比对,获得各样本物种和ARGs注释信息。然后通过R 3.6.3软件和GraPhlAn 1.1.3软件进行可视化分析,包括物种组成、ARGs组成和组间相似性分析等。结果 共采集到550只吸血微小扇头蜱,其中雌蜱404只,雄蜱85只,若蜱61只。微小扇头蜱的优势菌门为变形菌门,平均相对丰度为60.01%,其次为厚壁菌门（36.76%）；优势菌属为克雷伯菌属（38.58%）,其次为葡萄球菌属（22.00%）；优势菌种为肺炎克雷伯菌（38.58%）,其次为金黄色葡萄球菌（21.90%）,此外还检出了嗜吞噬细胞无形体、福尼尔立克次体、康氏立克次体、日本立克次体、伽氏疏螺旋体和贝氏柯克斯体等蜱携病原体,分别占3.08%、3.04%、0.76%、0.70%、0.01%和0.01%。组间相似性分析结果显示,组间差异大于组内差异（R=0.586,P=0.002）。ARGs注释发现与β-内酰胺类（54.22%）、氨基糖苷类（20.18%）、氯霉素类（13.61%）和多药类（11.99%）抗生素抗性相关的3 316个基因,各地区ARGs种类和丰度不同。结论 贵州省微小扇头蜱体内微生物菌群组成丰富,体内病原体种类较多,同时携带大量ARGs,应加强蜱微生物群及其抗性基因的研究,指导合理用药和预防蜱传疾病的发生。
Objective To understand the microbial community and antibiotic resistance genes (ARGs) in Rhipicephalus microplus in Guizhou Province, China. Methods From April to May in 2022 and 2023, R. microplus was collected from cattle surface in Daozhen, Ziyun, Xiuwen counties, and Panzhou City of Guizhou Province using the method of examining ticks on animal body surface. The collected tick specimens were grouped by region, and 15 female ticks were randomly selected from each region and divided into 3 tubes for metagenomic sequencing. The sequencing data were subjected to quality control, splicing, and assembly, followed by comparison against the structured antibiotic resistance gene database and the non-redundant protein library of the National Center for Biotechnology Information to obtain the annotation information of species and ARGs from each specimen. Then visual analysis was performed using R 3.6.3 and GraPhlAn 1.1.3, including taxonomical composition, ARG composition, and group similarities. Results A total of 550 blood-sucking R. microplus were collected, including 404 females, 85 males, and 61 nymphs. The dominant phylum of bacteria in R. microplus was Proteobacteria, with an average relative abundance of 60.01%, followed by Firmicutes (36.76%). The dominant genus was Klebsiella (38.58%), followed by Staphylococcus (22.00%). The dominant species was K. pneumoniae (38.58%), followed by Sta. aureus (22.00%). In addition, tick-borne pathogens such as Anaplasma phagocytophilum, Rickettsia fournieri, R. conorii, R. japonica, Borrelia garinii, and Coxiella burnetii were also detected, accounting for 3.08%, 3.04%, 0.76%, 0.70%, 0.01%, and 0.01%, respectively. The analysis of group similarities showed that the difference between groups was greater than that within groups (R=0.586, P=0.002). ARG annotation identified 3 316 genes related to resistance against β-lactams (54.22%), aminoglycosides (20.18%), chloramphenicols (13.61%), and multidrugs (11.99%). The types and abundance of ARGs were different in different regions. Conclusions The microflora in R. microplus in Guizhou Province is diverse, and there are many types of pathogens in R. microplus. Moreover, R. microplus carries a large number of ARGs. Therefore, it is necessary to strengthen the study of tick microflora and resistance genes, so as to guide the rational use of drugs and prevent the occurrence of tick-borne diseases.
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