Center for Infectious Diseases, School of Public Health, University of Texas Health Science Center, Houston, TX 77030, USA.
Center for Antimicrobial Resistance and Microbial Genomics, Division of Infectious Diseases, University of Texas Health Science Center at Houston McGovern Medical School, Houston, TX 77030, USA.
J Antimicrob Chemother. 2021 Jan 19;76(2):385-395. doi: 10.1093/jac/dkaa447.
Approximately half of clinical carbapenem-resistant Enterobacterales (CRE) isolates lack carbapenem-hydrolysing enzymes and develop carbapenem resistance through alternative mechanisms.
To elucidate development of carbapenem resistance mechanisms from clonal, recurrent ESBL-positive Enterobacterales (ESBL-E) bacteraemia isolates in a vulnerable patient population.
This study investigated a cohort of ESBL-E bacteraemia cases in Houston, TX, USA. Oxford Nanopore Technologies long-read and Illumina short-read sequencing data were used for comparative genomic analysis. Serial passaging experiments were performed on a set of clinical ST131 Escherichia coli isolates to recapitulate in vivo observations. Quantitative PCR (qPCR) and qRT-PCR were used to determine copy number and transcript levels of β-lactamase genes, respectively.
Non-carbapenemase-producing CRE (non-CP-CRE) clinical isolates emerged from an ESBL-E background through a concurrence of primarily IS26-mediated amplifications of blaOXA-1 and blaCTX-M-1 group genes coupled with porin inactivation. The discrete, modular translocatable units (TUs) that carried and amplified β-lactamase genes mobilized intracellularly from a chromosomal, IS26-bound transposon and inserted within porin genes, thereby increasing β-lactamase gene copy number and inactivating porins concurrently. The carbapenem resistance phenotype and TU-mediated β-lactamase gene amplification were recapitulated by passaging a clinical ESBL-E isolate in the presence of ertapenem. Clinical non-CP-CRE isolates had stable carbapenem resistance phenotypes in the absence of ertapenem exposure.
These data demonstrate IS26-mediated mechanisms underlying β-lactamase gene amplification with concurrent outer membrane porin disruption driving emergence of clinical non-CP-CRE. Furthermore, these amplifications were stable in the absence of antimicrobial pressure. Long-read sequencing can be utilized to identify unique mobile genetic element mechanisms that drive antimicrobial resistance.
大约一半的临床碳青霉烯类耐药肠杆菌科（CRE）分离株缺乏碳青霉烯水解酶，并通过替代机制产生碳青霉烯耐药性。
阐明在易感染人群中克隆、反复出现的产超广谱β-内酰胺酶（ESBL）肠杆菌科（ESBL-E）菌血症分离株中碳青霉烯耐药机制的发展情况。
本研究调查了美国德克萨斯州休斯顿的一组 ESBL-E 菌血症病例。使用牛津纳米孔技术长读长和 Illumina 短读长测序数据进行比较基因组分析。对一组临床 ST131 大肠杆菌分离株进行连续传代实验，以重现体内观察结果。使用定量 PCR（qPCR）和 qRT-PCR 分别测定β-内酰胺酶基因的拷贝数和转录水平。
非碳青霉烯酶产生的 CRE（非-CP-CRE）临床分离株从 ESBL-E 背景中出现，主要是通过 IS26 介导的 blaOXA-1 和 blaCTX-M-1 组基因的扩增以及孔蛋白失活同时发生。携带和扩增β-内酰胺酶基因的离散、模块化可移动单元（TU）从染色体上 IS26 结合的转座子中发生细胞内转移，并插入孔蛋白基因中，从而同时增加β-内酰胺酶基因的拷贝数并失活孔蛋白。在厄他培南存在的情况下，通过传代临床 ESBL-E 分离株可重现碳青霉烯耐药表型和 TU 介导的β-内酰胺酶基因扩增。在没有厄他培南暴露的情况下，临床非-CP-CRE 分离株具有稳定的碳青霉烯耐药表型。
这些数据表明，IS26 介导的机制是β-内酰胺酶基因扩增的基础，同时伴有外膜孔蛋白破坏，导致临床非-CP-CRE 的出现。此外，这些扩增在没有抗菌压力的情况下是稳定的。长读测序可用于鉴定驱动抗菌药物耐药性的独特移动遗传元件机制。
