Timmermans Michaël, Latour Samuel, Ceyssens Pieter-Jan, Garcia-Graells Cristina, Kowalewicz Carole, Fretin David, Denis Olivier, Wattiau Pierre, Boland Cécile
Veterinary Bacteriology, Sciensano, Ixelles, Belgium; Faculté de médecine, Université Libre de Bruxelles, Brussels, Belgium.
Veterinary Bacteriology, Sciensano, Ixelles, Belgium.
J Microbiol Methods. 2022 May;196:106472. doi: 10.1016/j.mimet.2022.106472. Epub 2022 Apr 21.
The aim of this study was to develop a highly multiplexed bead array to detect genes and/or mutations frequently associated with resistance to antimicrobials of the β-lactam, (fluoro)quinolone, colistin, macrolide and aminoglycoside families in Enterobacteriaceae such as Escherichia coli, Shigella spp. and Salmonella spp. Ligase Chain Reaction and the Luminex® technology were combined in a 53-plex assay designed to target selected genetic markers with 3 internal controls. The AMR-ARRAY consistently detected resistance determinants as compared to phenotypically expressed resistance for 94.7% (856/904) of the assessed resistances. When compared to resistance profiles inferred from whole genome sequencing results, the AMR-ARRAY showed a selectivity and specificity of 99.3% and 100%, respectively. The strong features of the AMR-ARRAY are (i) its competitive cost, currently 18€/sample (ii) its wide analytical scope, currently 50 markers covering 5 antimicrobial families, (iii) its robust and user-friendly design consisting in a single-tube assay conducted in 4 successive steps (iv) its relatively short turnaround time, less than 8 h (v) its ability to detect allelic variability at critical SNPs (vi) its open access and easily upgradable design, with probes sequences, procedure and software source code freely available. The use of the AMR-ARRAY as a screening method in official antimicrobial resistance monitoring could improve the granularity of the collected data and pinpoint remarkable isolates harbouring unusual resistance determinants thereby enabling fit-for-purpose selection of isolates for Whole Genome analysis.
本研究的目的是开发一种高度多重化的微珠阵列，以检测与肠杆菌科（如大肠杆菌、志贺氏菌属和沙门氏菌属）中β-内酰胺类、（氟）喹诺酮类、黏菌素、大环内酯类和氨基糖苷类抗菌药物耐药性频繁相关的基因和/或突变。连接酶链反应和Luminex®技术被整合到一个53重分析中，该分析旨在靶向选定的遗传标记并带有3个内部对照。与表型表达的耐药性相比，AMR-ARRAY对94.7%（856/904）的评估耐药性一致地检测到了耐药决定因素。与从全基因组测序结果推断的耐药谱相比，AMR-ARRAY的选择性和特异性分别为99.3%和100%。AMR-ARRAY的显著特点包括：（i）具有竞争力的成本，目前为18欧元/样本；（ii）广泛的分析范围，目前有50个标记涵盖5个抗菌药物家族；（iii）其稳健且用户友好的设计，包括在4个连续步骤中进行的单管分析；（iv）相对较短的周转时间，少于8小时；（v）能够检测关键单核苷酸多态性处的等位基因变异；（vi）其开放获取且易于升级的设计，探针序列、程序和软件源代码均可免费获取。将AMR-ARRAY用作官方抗菌药物耐药性监测中的筛查方法，可以提高所收集数据的精细程度，并找出携带异常耐药决定因素的显著分离株，从而能够有针对性地选择分离株进行全基因组分析。
