Chen Xin, Sun Zhewei, Xu Xiaogang, Jiang Jianping, Su Jiachun
Institute of Antibiotics, Huashan Hospital, Fudan University, Shanghai, PR China.
Key Laboratory of Clinical Pharmacology of Antibiotics, National Health and Family Planning Commission, Shanghai, PR China.
J Med Microbiol. 2023 Jan;72(1). doi: 10.1099/jmm.0.001647.
Carbapenem-resistant (CRKP) has become a serious threat to global public health. Colistin is regarded as the last-resort antibiotic for CRKP infections, but colistin resistance among CRKP is increasingly being reported, making clinical treatment for CRKP infections more difficult. The molecular mechanisms of colistin resistance in spp. under the pressure of colistin have not been fully investigated. We aimed to investigate the phenotypic and genetic variation in two colistin-susceptible spp. strains under selective pressure of colistin. One hundred microlitres of overnight cultures of the CRKP clinical strain CRKP12-130 and of ATCC 700603 was spread on five Mueller-Hinton Agar (MHA) plates with colistin concentrations of 2, 4, 8, 16 and 32 µg ml, and growth of colonies was observed for five consecutive days. Colonies collected from plates were passaged daily for 10 days on MHA plates without colistin and susceptibility testing of colistin was performed by broth microdilution. Thirty-four colistin-resistant strains randomly selected were submitted to whole genome sequencing (WGS). Transcriptional levels of genes involved in colistin resistance (, , , , , , and ) were measured by quantitative real-time PCR. A total of 114 and 119 colistin-resistant colonies were obtained from CRKP12-130 and ATCC 700603 in this study, among which 16 and 18 colonies were submitted to WGS, respectively. Among these 34 sequenced isolates, mutation in (13/16, 81.25 %) was the main genetic factor mediating colistin resistance in strains from CRKP12-130, while for strains from ATCC 700603, mutation associated with (8/18, 44.44 %) was found to be the commonest. Mutation of led to a significant increase in the MIC for colistin (from 64 to >128 µg ml), and a novel mutation C28R in was first reported in this study. Colistin-resistant spp. could be easily selected under pressure of different concentrations of colistin. Mutations of , , and genes were the main mechanisms leading to chromosomally mediated colistin resistance in spp.
耐碳青霉烯类肺炎克雷伯菌（CRKP）已成为全球公共卫生的严重威胁。黏菌素被视为治疗CRKP感染的最后一道抗生素防线，但CRKP对黏菌素的耐药性报道日益增多，使得CRKP感染的临床治疗更加困难。在黏菌素压力下，肺炎克雷伯菌对黏菌素耐药的分子机制尚未得到充分研究。我们旨在研究在黏菌素选择压力下，两株对黏菌素敏感的肺炎克雷伯菌菌株的表型和基因变异情况。将CRKP临床菌株CRKP12 - 130和ATCC 700603的100微升过夜培养物分别接种在5个含黏菌素浓度为2、4、8、16和32μg/ml的 Mueller - Hinton琼脂（MHA）平板上，连续5天观察菌落生长情况。从平板上收集的菌落每天转接至不含黏菌素的MHA平板上，持续10天，并通过肉汤微量稀释法进行黏菌素敏感性测试。随机选取34株耐黏菌素菌株进行全基因组测序（WGS）。通过定量实时PCR检测与黏菌素耐药相关基因（、、、、、、和）的转录水平。本研究中，从CRKP12 - 130和ATCC 700603分别获得了114个和119个耐黏菌素菌落，其中分别有16个和18个菌落进行了WGS。在这34株测序分离株中，（13/16，81.25%）突变是介导CRKP12 - 130菌株黏菌素耐药的主要遗传因素，而对于ATCC 700603菌株，与（8/18，44.44%）相关的突变是最常见的。的突变导致黏菌素的最低抑菌浓度（MIC）显著增加（从64增加到>128μg/ml），并且本研究首次报道了中一个新的突变C28R。在不同浓度黏菌素压力下，很容易筛选出耐黏菌素的肺炎克雷伯菌。、、和基因的突变是导致肺炎克雷伯菌染色体介导的黏菌素耐药的主要机制。
