耐药鲍曼不动杆菌不同年份分离株耐药元件基因比较研究

目的调查两组相隔7年的鲍曼不动杆菌菌株可能存在的耐药基因状况及菌株间的亲缘关系。方法分别收集南京医科大学附属淮安第一医院2008年20株鲍曼不动杆菌,2016年20株鲍曼不动杆菌。菌种鉴定为鲍曼不动杆菌种特异mdfA基因与blaoyA基因PCR双重检测为阳性方确认为鲍曼不动杆菌。再用PCR方法检测32种β-内酰胺类药物获得性耐药基因、15种氨基糖苷类药物获得性耐药基因、10种可移动遗传元件遗传标记,最后对检测结果作样本聚类分析(UPGMA法)。结果2016年分离株对第三代头孢类药物、碳青霉烯类药物、氨基糖苷类药物、喹诺酮药物均为耐药。2008年分离株仅对第三代头孢类药物均为耐药,而对碳青霉烯类药物均为敏感,对喹诺酮药物、氨基糖苷类药物分别有50.00%和60.00%的敏感性。2008和2016年分离株均检出blaADC和blaOXA-1,但bldOXA2册、blaOXA-23群只有2016年分离株均被检出。2008年分离株对第三代头孢类药物耐药主要与bldspc相关,2016年分离株对第三代头孢类药物、碳青霉烯类药物耐药主要与blaADC、blaOXA-2和blaOXA-23群相关。2016年分离株均测出aac(2)-I b、aph(3)-I、armA等3种氨基糖苷类药物耐药相关基因,而2008年分离株均无检出。可移动遗传元件遗传标记tmpU、tmp513、IS26和ISabal等4种也只有2016年分离株被全部检出。菌株亲缘关系分析可见2008和2016年不同年份分离株分成两个独立的簇群(A簇群和B簇群),2016年分离株聚集性更强相差系数更小。A和B族群均可分为两个亚簇群。A-1亚簇群有两个克隆播散,A-2、B-1和B2亚簇群各有一个克隆播散。其中2016年分离株B-簇群一个有14株菌构成的大克隆播散。分离株耐药元件检测阳性模式显示,2008年分离株仅携带2~7种耐药元件基因;2016年分离株则携带14种~16种耐药元件基因。结论多种β-内酰胺类药物耐药基因、氨基糖苷类药物耐药基因和可移动遗传元件是导致鲍曼不动杆菌对抗菌药物耐药的重要原因。在同一家医院的对相隔7年的鲍曼不动杆菌临床分离株进行耐药元件基因检测与分析是国内首次报道。不同年份分离株的耐药性和耐药元件基因携带状况差异明显:2016年分离株比2008年分离株耐药性更强,携带的耐药元件基因更多。2008年和2016年分离株均有克隆播散,提示有医院感染的存在。

Investigation of resistant determinants in drug-resistant Acinetobacter baumannii isolated in different years

Objective To investigate the distribution of resistant determinants in two groups of drug-resistant Acinetobacter baumannii (A. baumannii) isolated seven years apart, and their relationships among strains. Methods 20 strains of A. baumannii in 2008 and 20 strains in 2016 were collected in the Affiliated Huaian No.1 People's Hospital of Nanjing Medical University. Double positive of mdfA and blaOXA-51 were identified as A. baumannii. 32 kinds ofacquired beta-lactamase genes, 15 kinds of acquired resistant genes to aminoglycosides, and 10 kinds of genetic markers of mobile genetic elements were analyzed by PCR. At last, sample cluster analysis was performed using the UPGMA method. Results Isolates in 2016 were all resistant to the third generation cephalosporins, carbapenems, aminoglycosides, and quinolones. While isolates in 2008 were all resistant to the third generation cephalosporins, but all susceptible to carbapenems. The susceptible rates were 50% and 60% to quinolones and aminoglycosides, respectively. The genes blaADC and blaOXA-51 were both positive in 2008 and 2016, but blaOXA-2 and blaOXA-23 group were only positive in 2016. The resistance of isolates in 2008 to the third generation cephalosporins was mainly related to blaADC, and the resistance of isolates in 2016 to the third generation cephalosporins and carbapenems was mainly related to blaADC, blaOXA-2 group, and blaOXA-23 group. In addition, three kinds of resistant genes to aminoglycosides, aac(2')-Ⅰb, aph(3')-Ⅰ, and armA, were all positive in isolates in 2016, while three genes were all negative in isolates in 2008. Four kinds of genetic markers of mobile genetic elements, tnpU, tnp513, IS26, and ISaba1, were all positive only in 2016. Phylogenetic relationship analysis showed that isolates in 2008 to and 2016 were divided into two independent clusters (cluster A and cluster B). Clustering in isolates in 2016 was stronger, and the difference coefficient was smaller than isolates in 2008. Cluster A and cluster B could be divided into 2 subclusters. Subcluster A-1 had two transmitted clones, and subclusters A-2, B-1, and B2 had a transmitted clone, respectively. Moreover, cluster B in 2016 had a large transmitted clone of 14 strains. Positive mode of resistant determinants showed that isolates in 2008 only harbored two to seven kinds of resistant determinants, while isolates in 2016 harbored 14 to 16 kinds of resistant determinants. Conclusion A variety of beta-lactamase genes, aminoglycoside resistance genes, and mobile genetic elements played a key role in resistance to antimicrobial agents. It’s the first report in China to detect and analyze resistant determinants of A. baumannii isolated in the same hospital seven years apart. The resistance and distribution of resistant determinants in two groups had significant difference: isolates in 2016 were more resistant, and harbored more resistant determinants than isolates in 2008. Transmitted clones existed in 2008 and 2016, which suggested the presence of nosocomial