Department of Health Science, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Department of Clinical Chemistry and Laboratory Medicine, Graduate School of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
J Antimicrob Chemother. 2021 Jan 19;76(2):362-369. doi: 10.1093/jac/dkaa439.
A patient repeatedly developed bacteraemia despite the continuous use of antibiotics. We obtained two Klebsiella pneumoniae isolates from the patient's blood on Days 72 and 105 after hospitalization. Each of the two isolates belonged to ST45, but while the first isolate was susceptible to most antibiotics, the second one was resistant to multiple drugs including carbapenems.
To identify the genetic differences between the two isolates and uncover alterations formed by the within-host bacterial evolution leading to the antimicrobial resistance.
Whole-genome comparison of the two isolates was carried out to identify their genetic differences. We then profiled their outer membrane proteins related to membrane permeability to drugs. To characterize a ramR gene mutation found in the MDR isolate, its WT and mutant genes were cloned and expressed in the MDR isolate.
The two isolates showed only three genomic differences, located in mdoH, ramR and upstream of ompK36. In the MDR isolate, a single nucleotide substitution in the ompK36 upstream region attenuated OmpK36 expression. A single amino acid residue insertion in RamR in the MDR isolate impaired its function, leading to the down-regulation of OmpK35 and the subsequent up-regulation of the AcrAB-TolC transporter, which may contribute to the MDR.
We identified very limited genomic changes in the second K. pneumoniae clone during within-host evolution, but two of the three identified mutations conferred the MDR phenotype on the clone by modulating drug permeability.
一名患者在连续使用抗生素的情况下仍反复发生菌血症。我们从患者住院后第 72 天和第 105 天的血液中获得了两株肺炎克雷伯菌分离株。这两株分离株均属于 ST45，但第一株对大多数抗生素敏感，而第二株对包括碳青霉烯类在内的多种药物耐药。
确定两株分离株之间的遗传差异，并揭示导致抗菌药物耐药性的细菌在体内进化形成的改变。
对两株分离株进行全基因组比较，以确定其遗传差异。然后，我们分析了与药物膜通透性相关的外膜蛋白。为了研究在多药耐药（MDR）分离株中发现的 ramR 基因突变，我们克隆并在 MDR 分离株中表达了其 WT 和突变基因。
两株分离株仅显示出三个基因组差异，位于 mdoH、ramR 和 ompK36 上游。在 MDR 分离株中，ompK36 上游区域的单个核苷酸取代削弱了 OmpK36 的表达。MDR 分离株中 RamR 的单个氨基酸残基插入破坏了其功能，导致 OmpK35 的下调和随后 AcrAB-TolC 转运蛋白的上调，这可能导致 MDR。
我们在宿主内进化过程中发现第二株肺炎克雷伯菌克隆的基因组变化非常有限，但三个鉴定出的突变中有两个通过调节药物通透性赋予了克隆 MDR 表型。
