Xu Juan, Zhu Zhongliang, Chen Yanmin, Wang Weizhong, He Fang
School of Public Health, Hangzhou Medical College, Hangzhou, China.
Department of Clinical Laboratory, Zhejiang Provincial People's Hospital, People's Hospital of Hangzhou Medical College, Hangzhou, China.
Front Microbiol. 2021 Feb 24;12:644949. doi: 10.3389/fmicb.2021.644949. eCollection 2021.
The emergence and prevalence of tigecycline-resistant have seriously compromised the effectiveness of antimicrobial agents in the treatment of infections. To explore the role of the plasmid-borne (A) gene in tigecycline resistance in carbapenem-resistant (CRKP), a total of 63 CRKP isolates were collected from a tertiary hospital in Hangzhou, China. The minimum inhibitory concentration (MIC) of tigecycline, mutation rate of (A) gene, genetic surroundings of (A)-carrying transmissible plasmid and the contribution of (A) mutation to tigecycline resistance were analyzed using antimicrobial susceptibility test, whole-genome sequencing, tigecycline resistance evolution experiment, and plasmid conjugation experiment. Our results showed that 52.4% (33 isolates) of the test isolates carried the (A) gene; among them, 75.8% (25 isolates) exhibited a tigecycline non-susceptible phenotype (MIC = 4 mg/L). Three clonal groups (cluster I, cluster II, and cluster III) were identified in these (A)-bearing isolates. All 17 isolates belonged to serotype KL21 (cluster I), which differed by only 13 SNPs, suggesting a clonal spread of (A)-positive ST11 with serotype KL21 occurred in the sampling hospital. The induction of tigecycline resistance experiments showed that 71.4% of strains evolved (A) mutations and developed a high-level tigecycline resistance. Eight amino acid substitutions were identified in these mutants. The most common amino acid substitution was A370V, followed by S251A and G300E. Twelve isolates carrying (A) mutants succeeded in the filter mating experiment with a conjugation efficiency of 10-10. Tigecycline MICs in EC600 transconjugants with a mutated (A) were 2 to 8-fold higher than those in EC600 transconjugants with a wild-type (A). One ColRNAI/IncFII type and two IncFII type (A)-bearing conjugative plasmids were identified in this study, including a class 1 integron containing multiple antibiotic resistance genes, i.e., (A), , , , , and . Our study revealed the wide-spread situation of plasmid-borne (A) gene in clinical CRKP, and mutation of (A) is a potential driven force that lead to tigecycline resistance.
替加环素耐药菌的出现和流行严重影响了抗菌药物在感染治疗中的有效性。为探讨质粒携带的（A）基因在耐碳青霉烯类肺炎克雷伯菌（CRKP）对替加环素耐药中的作用，从中国杭州一家三级医院收集了63株CRKP分离株。采用药敏试验、全基因组测序、替加环素耐药进化实验和质粒接合实验，分析了替加环素的最低抑菌浓度（MIC）、（A）基因的突变率、携带（A）的可传播质粒的遗传环境以及（A）突变对替加环素耐药的贡献。我们的结果显示，52.4%（33株）的受试分离株携带（A）基因；其中，75.8%（25株）表现出对替加环素不敏感的表型（MIC = 4 mg/L）。在这些携带（A）的分离株中鉴定出三个克隆群（克隆群I、克隆群II和克隆群III）。所有17株分离株均属于血清型KL21（克隆群I），仅相差13个单核苷酸多态性（SNP），表明在采样医院中发生了携带（A）的ST11且血清型为KL21的克隆传播。替加环素耐药诱导实验表明，71.4%的菌株发生了（A）突变并产生了高水平的替加环素耐药。在这些突变体中鉴定出8个氨基酸替换。最常见的氨基酸替换是A370V，其次是S251A和G300E。12株携带（A）突变体的分离株在滤膜接合实验中成功，接合效率为10-10。携带突变（A）的EC600接合子中的替加环素MIC比携带野生型（A）的EC600接合子中的高2至8倍。本研究鉴定出一种ColRNAI/IncFII型和两种IncFII型携带（A）的接合质粒，包括一个含有多个抗生素耐药基因的1类整合子，即（A）、、、、、和。我们的研究揭示了质粒携带的（A）基因在临床CRKP中的广泛存在情况，并且（A）突变是导致替加环素耐药的潜在驱动力。
