We describe in vivo evolution of carbapenem and ceftazidime-avibactam resistance by analyzing four longitudinal Klebsiella pneumoniae clinical isolates from a patient with pneumonia following antimicrobial treatment. The patient had fever, cough associated with expectoration, and new infiltration was found on the chest CT. Antimicrobial susceptibility was determined, and whole genome sequencing (WGS) was performed to investigate its dynamic change of resistance phenotype. Population analysis profile was performed to investigate the population of Klebsiella pneumoniae. The infection started with a KPC-2-producing K. pneumoniae (ZRKP01, ceftazidime-avibactam-S/carbapenem-R). Then, after ceftazidime-avibactam treatment, the strain switched to D179Y mutant that is KPC-33 (ZRKP02, ceftazidime-avibactam-R/carbapenem-S), which restored carbapenem susceptibility. However, the restored carbapenem susceptibility in vivo was not stable and the subsequent use of imipenem against KPC-33-producing K. pneumoniae infection resulted in a reversion of KPC-2 producers (ZRKP03 and ZRKP04, ceftazidime-avibactam-S/carbapenem-R). Genetic analysis demonstrated that all four K. pneumoniae isolates belonged to sequence type 11and had identical capsular polysaccharide (KL47), identical porin genes, and same plasmid replicon types. Phylogenetic analysis indicated that four K. pneumoniae isolates showed a high degree of relatedness. Single nucleotide polymorphisms analysis indicated that the number of mutations observed in the KPC-33 isolate was more than in the wild-type KPC-2 isolates and the four KPC-Kp isolates evolved from a longitudinal evolution of K. pneumoniae harboring bla_KPC-2 gene. This is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. Through WGS, we demonstrated the role of selective pressure of antibiotic in the mutation and reversion of bla_KPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems.

Statement: Recently, studies reported the emergence of ceftazidime-avibactam-resistant strains. The KPC mutations mediating ceftazidime-avibactam resistance are generally associated with the restoration of carbapenem susceptibility. However, clinical significance of this observation is unclear. In this manuscript, we demonstrate the role of selective pressure of antibiotic in the mutation and reversion of bla_KPC genes, which leading to the dynamic change of KPC enzymes and the dynamic emergence of resistance to ceftazidime-avibactam and carbapenems. To the best of our knowledge, this is the first report to observe the in vivo evolution of wild-type KPC-2 to KPC-33 and then the reversion to its original wild-type KPC-2. It should be noted that understanding the clinical significance of this observation is of critical importance, and reversion to carbapenem susceptibility would not imply a potential role for carbapenems monotherapy. We hope our study will draw attention to clinicians, so that this agent can be used most effectively for the longest period of time.

我们描述 体内 碳青霉烯类和头孢他啶-阿维巴坦耐药性的四个纵向分析 肺炎克雷伯菌抗生素治疗后肺炎患者的临床分离物。患者发热、咳嗽伴咳痰，胸部CT发现新浸润。确定抗生素敏感性，并进行全基因组测序（WGS）以研究其耐药表型的动态变化。进行人口分析概况以调查人口肺炎克雷伯菌. 感染始于产生 KPC-2肺炎克雷伯菌（ZRKP01，头孢他啶-阿维巴坦-S/碳青霉烯-R）。然后，在头孢他啶-阿维巴坦处理后，菌株切换到 D179Y 突变体，即 KPC-33（ZRKP02，头孢他啶-阿维巴坦-R/碳青霉烯-S），这恢复了碳青霉烯的敏感性。然而，恢复的碳青霉烯敏感性体内 不稳定，随后使用亚胺培南对抗产生 KPC-33 肺炎克雷伯菌感染导致 KPC-2 生产者（ZRKP03 和 ZRKP04、头孢他啶-阿维巴坦-S/碳青霉烯-R）的逆转。遗传分析表明，所有四个肺炎克雷伯菌分离株属于序列类型 11，具有相同的荚膜多糖 (KL47)、相同的孔蛋白基因和相同的质粒复制子类型。系统发育分析表明，四肺炎克雷伯菌分离株表现出高度的相关性。单核苷酸多态性分析表明，在 KPC-33 分离株中观察到的突变数量多于野生型 KPC-2 分离株，四个 KPC-Kp 分离株从纵向进化进化而来。肺炎克雷伯菌 窝藏 布拉_KPC-2基因。这是第一份观察到的报告体内野生型 KPC-2 进化为 KPC-33，然后恢复为其原始野生型 KPC-2。通过WGS，我们证明了抗生素的选择压力在突变和逆转中的作用布拉_KPC基因，导致 KPC 酶的动态变化和对头孢他啶-阿维巴坦和碳青霉烯类耐药性的动态出现。

陈述：最近，研究报告了头孢他啶-阿维巴坦耐药菌株的出现。介导头孢他啶-阿维巴坦耐药性的 KPC 突变通常与碳青霉烯敏感性的恢复有关。然而，这一观察结果的临床意义尚不清楚。在这份手稿中，我们证明了抗生素的选择压力在突变和逆转中的作用布拉_KPC基因，导致 KPC 酶的动态变化和对头孢他啶-阿维巴坦和碳青霉烯类耐药性的动态出现。据我们所知，这是第一份观察到体内野生型 KPC-2 进化为 KPC-33，然后恢复为其原始野生型 KPC-2。应该注意的是，了解这一观察结果的临床意义至关重要，碳青霉烯类药物敏感性恢复并不意味着碳青霉烯类单药治疗的潜在作用。我们希望我们的研究能引起临床医生的注意，使这种药物可以最有效地使用最长时间。