Table_1.DOCX

Tigecycline is one of the last resort treatments for carbapenem-resistant Klebsiella pneumoniae (CRKP) infections. Tigecycline resistance often occurs during the clinical treatment of CRKP, yet its mechanism has still not been clearly elucidated. This study presents an analysis of a tigecycline resistance mechanism that developed in clinical isolates from a 56-year-old female patient infected with CRKP during tigecycline treatment. Consecutive clonal consistent K. pneumoniae isolates were obtained during tigecycline treatment. Whole genome sequencing of the isolates was performed, and putative single nucleotide polymorphisms and insertion and deletion mutations were analyzed in susceptible and resistant isolates. The identified gene of interest was examined through experiments involving transformations and conjugations. Four isolates, two of which were susceptible and two resistant, were collected from the patient. All of the isolates belonged to Sequence Type 11 (ST11) and were classified as extensively drug resistant (XDR). One amino acid substitution S251A in TetA was identified in the tigecycline-resistant isolates. Subsequent transformation experiments confirmed the contribution of the TetA variant (S251A) to tigecycline resistance. The transfer capacity of tigecycline resistance via this mutation was confirmed by conjugation experiments. Using southern blot hybridization and PCR assays, we further proved that the tetA gene was located on a transferable plasmid of ca. 65 kb in an Escherichia coli EC600 transconjugant. Our results provide direct in vivo evidence that evolution in the tetA gene can lead to tigecycline treatment failure in CRKP clinical strains that carry tetA. Moreover, the transfer capacity of tigecycline resistance mediated by mutated tetA is a threat.

替加环素（Tigecycline）是治疗碳青霉烯类耐药肺炎克雷伯菌（carbapenem-resistant Klebsiella pneumoniae, CRKP）感染的最后一线治疗方案之一。临床治疗CRKP感染的过程中常出现替加环素耐药性，但该耐药性的具体分子机制尚未完全阐明。本研究针对1例56岁女性CRKP感染患者在替加环素治疗期间分离得到的临床菌株，分析其出现的替加环素耐药机制。治疗期间连续分离得到克隆型一致的肺炎克雷伯菌分离株，对这些菌株开展全基因组测序，对比分析敏感株与耐药株之间的潜在单核苷酸多态性及插入缺失突变。通过转化实验与接合实验对目标候选基因进行功能验证。本研究从该患者体内共分离得到4株菌株，其中2株为替加环素敏感株、2株为耐药株；所有菌株均属于序列型11（Sequence Type 11, ST11），且均为广泛耐药（extensively drug resistant, XDR）表型。在替加环素耐药株中，鉴定到TetA蛋白存在一处氨基酸替换S251A。后续转化实验证实了该TetA变异体（S251A）可介导替加环素耐药性。接合实验进一步验证了该突变介导的替加环素耐药性的转移能力。通过Southern印迹杂交（Southern blot hybridization）与聚合酶链式反应（PCR）实验，本研究证实tetA基因位于大肠杆菌EC600接合子中约65 kb的可转移质粒上。本研究结果为携带tetA基因的CRKP临床菌株中，tetA基因的进化可导致替加环素治疗失败提供了直接的体内实验证据。此外，突变型tetA介导的替加环素耐药性的转移能力亦存在潜在临床威胁。