青海省果洛藏族自治州鼠疫耶尔森菌病原学分析及耐药相关基因检测

Etiological analysis and determination of antibiotic resistance-related genes of Yersinia pestis in Guoluo Tibetan autonomous prefecture,Qinghai province, China

目的对青海省果洛藏族自治州(果洛州)鼠疫耶尔森菌(鼠疫菌)进行病原学分析及耐药相关基因检测,为该地区的鼠疫防治提供科学依据。方法对1978-2016年间青海省果洛州分离的13株鼠疫菌进行糖醇类酵解试验、毒力测定、毒力因子检测、质粒分析及鼠疫菌基因差异区段(DFR)分型等研究,并根据美国国立生物信息中心(NCBI)公布的耐氨基糖苷类链霉素strA、strB基因、耐β-内酰胺类抗菌药物tem、ctx-m基因和耐磺胺类药物sul1、sul2基因序列,在每个基因上设计1对引物,以该13株鼠疫菌DNA为模板分别进行上述6个耐药基因的PCR扩增,以检测其是否携带耐药相关基因。结果根据生化分型指标,13株被试菌株中9株为青藏高原型,4株为祁连山型。毒力测定结果显示,13株被试菌株均为强毒级别的鼠疫菌,13株菌中有11株具备鼠疫菌特有的4个毒力因子,被试菌株携带相对分子质量分别为6×106、45×106、65×106和6×106、45×106、52×106的2种质粒谱。DFR分为4个基因组型,5型(7株)、8型(4株)、36型和01b型(各1株)。耐药基因检测结果显示,阴性对照和阳性对照均成立的情况下,所测13株鼠疫菌的PCR检测结果均为阴性,未发现耐链霉素、耐β-内酰胺类抗菌药物及耐磺胺类药物的菌株。结论青海省果洛州分离的鼠疫菌具备青藏高原鼠疫病原体特征,鼠疫菌的毒力强,但尚未出现耐抗菌药物菌株,但仍需加强鼠疫监测和鼠疫防治知识宣传力度,严防动物间鼠疫波及人间。

Objective To provide a scientific basis for plague prevention and control in Guoluo Tibetan autonomous prefecture, Qinghai province, China(hereinafter referred to as Guoluo prefecture), by etiological analysis and determination of the antibiotic resistance-related genes of Yersinia pestis in that area. Methods A series of experiments including sugar alcohol fermentation test, toxicity test, virulence factor assay, plasmid analysis, and different region(DFR) genotyping were conducted on 13 Y. pestis strains isolated from Guoluo prefecture from 1978 to 2016. According to the gene sequences(aminoglycosides streptomycin-resistant strA and strB;beta-lactam antibiotics-resistant tem and ctx-m;and sulfonamidesresistant sul1 and sul2) published by the National Center for Biotechnology Information(U.S.), a pair of primers were designed for each gene. Polymerase chain reaction(PCR) amplification of the above 6 antibiotic resistance-related genes was carried out using the DNAs from the above 13 Y. pestis strains as templates, to detect whether they carried antibiotic resistance-related genes. Results According to the biochemical grouping, of the 13 test strains, 9 belonged to Qing-Tibet Plateau ecotype, and 4 belonged to Qilian Mountain ecotype. As shown by the results of the toxicity test, all the 13 strains were velogenic strains and 11 contained four Y. pestis-specific virulence factors. The test strains contained 2 types of plasmids with a molecular weight of 6×106, 45×106, and 65×106 versus 6×106, 45×106, and 52×106, respectively. The DFRs were divided into 4 genomovars, i.e., genomovar 5(7 strains), genomovar 8(4 strains), genomovar 36(1 strain), and genomovar 01 b(1 strain). In case of valid positive and negative controls, PCR assay of the antibiotic-resistant genes revealed a negative result for all the above 13 Y. pestis strains, and no strain was found to be resistant to streptomycin, betalactam antibiotics, or sulfonamides. Conclusion The strains isolated from Guoluo prefecture show the characteristics of Y.pestis from Qinghai-Tibet Plateau. Despite the fact that no antibiotic-resistant strains were found, which demonstrate high toxicity;therefore, it is necessary to enhance plague surveillance and health promotion to prevent plague spreading from animals to humans.