Specialist Antimicrobial and Chemotherapy Unit, Public Health Wales, University Hospital of Wales, Cardiff, UK.
Centre for Genomic Pathogen Surveillance, Wellcome Genome Campus, Cambridge, UK.
J Hosp Infect. 2021 Apr;110:148-155. doi: 10.1016/j.jhin.2021.01.010. Epub 2021 Jan 22.
The emergence of multi-drug-resistant (MDR) micro-organisms prompted new interest in older antibiotics, such as colistin, that had been abandoned previously due to limited efficacy or high toxicity. Over the years, several chromosomal-encoded colistin resistance mechanisms have been described; more recently, 10 plasmid-mediated mobile colistin resistance (mcr) genes have been identified. Spread of these genes among MDR Gram-negative bacteria is a matter of serious concern; therefore, reliable and timely mcr detection is paramount.
To design and validate a multiplex real-time polymerase chain reaction (PCR) assay for detection and differentiation of mcr genes.
All available mcr alleles were downloaded from the National Center for Biotechnology Information Reference Gene Catalogue, aligned with Clustal Omega and primers designed using Primer-BLAST. Real-time PCR monoplexes were optimized and validated using a panel of 120 characterized Gram-negative strains carrying a wide range of resistance genes, often in combination. Melt-curve analysis was used to confirm positive results.
In-silico analysis enabled the design of a 'screening' assay for detection of mcr-1/2/6, mcr-3, mcr-4, mcr-5, mcr-7, mcr-8 and mcr-9/10, paired with an internal control assay to discount inhibition. A 'supplementary' assay was subsequently designed to differentiate mcr-1, mcr-2, mcr-6, mcr-9 and mcr-10. Expected results were obtained for all strains (100% sensitivity and specificity). Melt-curve analysis showed consistent melting temperature results. Inhibition was not observed.
The assay is rapid and easy to perform, enabling unequivocal mcr detection and differentiation even when more than one variant is present. Adoption by clinical and veterinary microbiology laboratories would aid the surveillance of mcr genes amongst Gram-negative bacteria.
多药耐药（MDR）微生物的出现促使人们重新关注以前由于疗效有限或毒性较高而被弃用的老抗生素，如黏菌素。多年来，已经描述了几种染色体编码的黏菌素耐药机制；最近，已经确定了 10 种质粒介导的移动黏菌素耐药（mcr）基因。这些基因在 MDR 革兰氏阴性菌中的传播令人严重关切；因此，可靠和及时的 mcr 检测至关重要。
设计和验证用于检测和区分 mcr 基因的多重实时聚合酶链反应（PCR）检测。
从国家生物技术信息中心参考基因目录中下载所有可用的 mcr 等位基因，使用 Clustal Omega 进行比对，并使用 Primer-BLAST 设计引物。使用携带广泛耐药基因（通常是组合）的 120 种特征革兰氏阴性菌株进行单重实时 PCR 优化和验证。使用熔解曲线分析来确认阳性结果。
在计算机分析的帮助下，可以设计一种用于检测 mcr-1/2/6、mcr-3、mcr-4、mcr-5、mcr-7、mcr-8 和 mcr-9/10 的“筛选”检测，以及配对的内部对照检测以排除抑制。随后设计了一种“补充”检测来区分 mcr-1、mcr-2、mcr-6、mcr-9 和 mcr-10。所有菌株均获得预期结果（100%的灵敏度和特异性）。熔解曲线分析显示出一致的熔点结果。没有观察到抑制。
该检测方法快速且易于操作，即使存在多种变体，也能明确检测和区分 mcr。临床和兽医微生物学实验室的采用将有助于监测革兰氏阴性菌中的 mcr 基因。
