Heteroresistance of KPC-producing Klebsiella pneumoniae results of three subpopulations associated with a broad landscape of mutations. Heteroresistance

Carbapenemase-producing Klebsiella pneumoniae (CPKp) have disseminated globally and represent a major threat in hospitals with few therapeutic options and high mortality rates. K. pneumoniae isolates producing the carbapenemase KPC (KPC-Kp) might be misclassified as susceptible by antibiotic susceptibility testing (AST), allowing the use of imipenem or meropenem for treatment of infections, but with a high chance of failure. KPC-Kp frequently show heteroresistance (HR), with colonies growing in the inhibition halo of agar-based AST. By analysing a set of 82 CPKp, we showed that HR is indeed frequent among KPC-Kp and specific to these isolates compared to those producing other carbapenemases (VIM, NDM or OXA-48). By monitoring the single colony appearance at 8-fold the imipenem minimal inhibitory concentration using the ScanLag setup, we discriminated two surviving subpopulations: a Gaussian-like distribution of early-appearing colonies, with a delayed emergence compared to colonies arising in the absence of antibiotics, and a long tail of late-appearing colonies. A subset of colonies showed a reduced growth rate, likely resulting from costly mutations. For in-depth characterization of survival dynamics, 333 colonies were subjected to whole-genome sequencing. We identified a broad landscape of genetic alterations in 28% of these colonies, including gene loss, DNA amplification and point mutations. hypothesis of an heterogeneous population encompassing tolerant and persistent cells, but also resistant bacteria showing genetic alterations. Altogether, these data revealed the complexity of imipenem HR among KPC-Kp isolates.

产碳青霉烯酶肺炎克雷伯菌（Carbapenemase-producing Klebsiella pneumoniae, CPKp）已在全球范围内播散，是医院感染防控领域的重大威胁，其临床治疗选择有限且病死率极高。产KPC型碳青霉烯酶的肺炎克雷伯菌（KPC-Kp）可能在抗菌药物敏感性试验（Antibiotic Susceptibility Testing, AST）中被误判为敏感菌株，进而导致临床选用亚胺培南或美罗培南进行抗感染治疗，但治疗失败风险极高。KPC-Kp常表现出异质性耐药（Heteroresistance, HR），即菌落可在基于琼脂的AST试验的抑菌圈内生长。本研究通过分析82株CPKp菌株，证实异质性耐药在KPC-Kp中确实高发，且相较于产其他碳青霉烯酶（VIM、NDM或OXA-48）的菌株，该耐药表型具有菌株特异性。借助ScanLag检测体系，在8倍亚胺培南最低抑菌浓度（minimal inhibitory concentration, MIC）条件下监测单菌落的出现情况，研究人员成功区分出两类存活亚群：一类为早期出现的类高斯分布菌落，其出现时间晚于无抗生素环境中生长的菌落；另一类为晚期出现的长尾分布菌落。其中部分菌落的生长速率显著降低，推测这一现象源于代价高昂的基因突变。为深入解析存活动态，研究人员对333株分离菌落开展全基因组测序。结果显示，28%的菌落存在广泛的遗传改变，涵盖基因丢失、DNA扩增以及点突变。研究提出异质性种群假说：该种群既包含耐受菌与持留菌，也存在携带遗传改变的耐药菌。综上，本研究数据揭示了KPC-Kp菌株中亚胺培南异质性耐药的复杂机制。