产生肺炎克雷伯菌碳青霉烯酶(KPC)变体的肺炎克雷伯菌菌株对头孢他啶-阿维巴坦(CZA)具有抗性,通常对美罗培南(MEM)敏感。提示这种碳青霉烯抗生素的潜在治疗用途。然而,体外研究表明,这些菌株可以突变成为MEM抗性,引起人们对碳青霉烯类抗生素作为治疗选择的有效性的担忧。我们已经研究了在属于ST11的CZA抗性肺炎克雷伯菌中,从MEM敏感性KPC-114向MEM抗性KPC-2的逆转发生的突变率。进行两步波动测定(FA)。简而言之,将显示1µg/mLMEMMIC的产生KPC-114的肺炎克雷伯菌的初始培养物涂在含有2-8µg/mLMEM的Mueller-Hinton琼脂平板上。FA的第二步,在4-16µg/mL时,从2µg/mLMEM获得的突变菌落进行MEM。使用最大似然估计计算突变率。亲本和突变菌株由IlluminaNextSeq测序,并通过变异识别分析预测突变。在8微克/毫升MEM,来自亲本CZA抗性(MIC≥64µg/mL)/MEM敏感(MIC=1µg/mL)KPC-114阳性肺炎克雷伯菌的突变体表现出3.05×10-19突变/细胞/代的累积突变率,而在16µg/mLMEM时,1.33×10-19个突变/细胞/代的累积突变率导致KPC-114(S181_P182缺失)恢复为KPC-2.这些发现突出表明,在CZA耐药肺炎克雷伯菌ST11中,MEM敏感型KPC-114向MEM耐药型KPC-2的逆转与低突变率相关,这表明治疗失败的风险较低。体内研究对于证实MEM对抗CZA抗性KPC变体的临床潜力是必要的。重要性由于治疗选择有限,耐碳青霉烯类肺炎克雷伯菌中头孢他啶-阿维巴坦(CZA)耐药性的出现是一个主要问题。引人注目的是,介导CZA耐药的KPC突变通常与美罗培南易感性相关,提示这种碳青霉烯抗生素的潜在治疗用途。然而,美罗培南易感,美罗培南耐药的逆转是可以预期的。因此,了解与该遗传事件相关的突变率对于评估美罗培南对CZA耐药的产生KPC的肺炎克雷伯菌的潜在用途至关重要.在这项研究中,我们展示,在体外,在高浓度的美罗培南下,在属于全球高风险ST11的CZA耐药/美罗培南易感肺炎克雷伯菌中,KPC-114向KPC-2的逆转与低突变率有关。
Klebsiella pneumoniae strains that produce Klebsiella pneumoniae Carbapenemase (KPC) variants displaying resistance to ceftazidime-avibactam (CZA) often remain susceptible to meropenem (MEM), suggesting a potential therapeutic use of this carbapenem antibiotic. However, in vitro studies indicate that these sorts of strains can mutate becoming MEM-resistant, raising concerns about the effectiveness of carbapenems as treatment option. We have studied mutation rates occurring from the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae belonging to ST11. Two-step fluctuation assays (FAs) were conducted. In brief, initial cultures of KPC-114-producing K. pneumoniae showing 1 µg/mL MEM MIC were spread on Mueller-Hinton agar plates containing 2-8 µg/mL MEM. A second step of FA, at 4-16 µg/mL MEM was performed from a mutant colony obtained at 2 µg/mL MEM. Mutation rates were calculated using maximum likelihood estimation. Parental and mutant strains were sequenced by Illumina NextSeq, and mutations were predicted by variant-calling analysis. At 8 µg/mL MEM, mutants derived from parental CZA-resistant (MIC ≥ 64 µg/mL)/MEM-susceptible (MIC = 1 µg/mL) KPC-114-positive K. pneumoniae exhibited an accumulative mutation rate of 3.05 × 10-19 mutations/cell/generation, whereas at 16 µg/mL MEM an accumulative mutation rate of 1.33 × 10-19 mutations/cell/generation resulted in the reversion of KPC-114 (S181_P182 deletion) to KPC-2. These findings highlight that the reversion of MEM-susceptible KPC-114 to MEM-resistant KPC-2, in CZA-resistant K. pneumoniae ST11 is related to low mutation rates suggesting a low risk of therapeutic failure. In vivo investigations are necessary to confirm the clinical potential of MEM against CZA-resistant KPC variants.IMPORTANCEThe emergence of ceftazidime-avibactam (CZA) resistance among carbapenem-resistant Klebsiella pneumoniae is a major concern due to the limited therapeutic options. Strikingly, KPC mutations mediating CZA resistance are generally associated with meropenem susceptibility, suggesting a potential therapeutic use of this carbapenem antibiotic. However, the reversion of meropenem-susceptible to meropenem-resistant could be expected. Therefore, knowing the mutation rate related to this genetic event is essential to estimate the potential use of meropenem against CZA-resistant KPC-producing K. pneumoniae. In this study, we demonstrate, in vitro, that under high concentrations of meropenem, reversion of KPC-114 to KPC-2 in CZA-resistant/meropenem-susceptible K. pneumoniae belonging to the global high-risk ST11 is related to low mutation rates.