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Abstract：
The emergence of nosocomial infections caused by hypervirulent and carbapenem-resistant K. pneumoniae (hv-CRKP) has become a significant public health challenge. The genetic traits of virulence and resistance plasmids in hv-CRKP have been extensively studied; however, research on the adaptive evolution strategies of clinical strains inside the host was scarce. This study aimed to understand the effects of antibiotic treatment on the phenotype and genotype characteristics of hv-CRKP. We investigated the evolution of hv-CRKP strains isolated from the same patient to elucidate the transition between hospital invasion and colonization. A comparative genomics analysis was performed to identify single nucleotide polymorphisms in the rmpA promoter. Subsequent validation through RNA-seq and gene deletion confirmed that distinct rmpA promoter sequences exert control over the mucoid phenotype. Additionally, biofilm experiments, cell adhesion assays, and animal infection models were conducted to illuminate the influence of rmpA promoter diversity on virulence changes. We demonstrated that the P12T and P11T promoters of rmpA possess strong activity, which leads to the evolution of CRKP into infectious and virulent strains. Meanwhile, the specific sequence of polyT motifs in the rmpA promoter led to a decrease in the lethality of hv-CRKP and enhanced cell adhesion and colonization. To summarize, the rmpA promoter of hv-CRKP is utilized to control capsule production, thereby modifying pathogenicity to better suit the host\'s ecological environment.IMPORTANCEThe prevalence of hospital-acquired illness caused by hypervirulent carbapenem-resistant Klebsiella pneumoniae (hv-CRKP) is significant, leading to prolonged antibiotic treatment. However, there are few reports on the phenotypic changes of hv-CRKP in patients undergoing antibiotic treatment. We performed a comprehensive examination of the genetic evolutionary traits of hv-CRKP obtained from the same patient and observed variations in the promoter sequences of the virulence factor rmpA. The strong activity of the promoter sequences P11T and P12T enhances the consistent production of capsule polysaccharides, resulting in an invasive strain. Conversely, weak promoter activity of P9T and P10T is advantageous for exposing pili, hence improving bacterial cell attachment ability and facilitating bacterial colonization. This finding also explains the confusion of some clinical strains carrying wild-type rmpA but exhibiting a low mucoid phenotype. This adaptive alteration facilitates the dissemination of K. pneumoniae within the hospital setting.
摘要：
高毒力和耐碳青霉烯类肺炎克雷伯菌（hv-CRKP）引起的医院感染的出现已成为重大的公共卫生挑战。hv-CRKP中的毒力和抗性质粒的遗传性状已被广泛研究；然而，临床菌株在宿主内部的适应性进化策略研究很少。本研究旨在了解抗生素治疗对hv-CRKP表型和基因型特征的影响。我们调查了从同一患者分离出的hv-CRKP菌株的演变，以阐明医院入侵和定植之间的过渡。进行比较基因组学分析以鉴定rmpA启动子中的单核苷酸多态性。随后通过RNA-seq和基因缺失的验证证实了不同的rmpA启动子序列对粘液表型发挥控制作用。此外,生物膜实验，细胞粘附试验，并建立了动物感染模型来阐明rmpA启动子多样性对毒力变化的影响。我们证明了rmpA的P12T和P11T启动子具有很强的活性，这导致CRKP进化为传染性和毒性菌株。同时,rmpA启动子中polyT基序的特定序列导致hv-CRKP的致死性降低，并增强细胞粘附和定植。总结一下,hv-CRKP的rmpA启动子用于控制胶囊生产，从而改变致病性以更好地适应宿主的生态环境。重要性由高毒力耐碳青霉烯类肺炎克雷伯菌（hv-CRKP）引起的医院获得性疾病的患病率显着，导致长期的抗生素治疗。然而,关于接受抗生素治疗的患者hv-CRKP表型变化的报道很少。我们对从同一患者获得的hv-CRKP的遗传进化特征进行了全面检查，并观察到毒力因子rmpA启动子序列的变化。启动子序列P11T和P12T的强活性增强了胶囊多糖的一致生产，导致侵入性应变。相反，P9T和P10T的弱启动子活性有利于暴露菌毛，从而提高细菌细胞附着能力和促进细菌定植。这一发现也解释了一些携带野生型rmpA但表现出低粘液表型的临床菌株的混淆。这种适应性改变有利于肺炎克雷伯菌在医院环境中的传播。
