Genotypic antimicrobial resistance assays for use on <i>E</i>. <i>coli</i> isolates and stool specimens

Antimicrobial resistance (AMR) is an emerging public health problem and methods for surveillance are needed. We designed 85 sequence-specific PCR reactions to detect 79 genes or mutations associated with resistance across 10 major antimicrobial classes, with a focus on E. coli. The 85 qPCR assays demonstrated >99.9% concordance with sequencing. We evaluated the correlation between genotypic resistance markers and phenotypic susceptibility results on 239 E. coli isolates. Both sensitivity and specificity exceeded 90% for ampicillin, ceftriaxone, cefepime, imipenem, ciprofloxacin, azithromycin, gentamicin, amikacin, trimethoprim/sulfamethoxazole, tetracycline, and chloramphenicol phenotypic susceptibility results. We then evaluated the assays on direct stool specimens and observed a sensitivity of 97% ± 5 but, as expected, a lower specificity of 75% ± 31 versus the genotype of the E. coli cultured from stool. Finally, the assays were incorporated into a convenient TaqMan Array Card (TAC) format. These assays may be useful for tracking AMR in E. coli isolates or directly in stool for targeted testing of the fecal antibiotic resistome.抗菌素耐药性（Antimicrobial resistance, AMR）是一类日益严峻的公共卫生问题，亟需建立配套的监测手段。本研究设计了85项序列特异性聚合酶链式反应（sequence-specific PCR），用于检测10大类主要抗菌素类别下共79个与耐药性相关的基因或突变，研究重点聚焦于大肠杆菌（Escherichia coli, E. coli）。该85项实时定量聚合酶链式反应（quantitative real-time PCR, qPCR）检测体系与测序结果的一致性超过99.9%。我们针对239株大肠杆菌分离株，分析了基因型耐药标记与表型药敏结果之间的相关性。针对氨苄西林、头孢曲松、头孢吡肟、亚胺培南、环丙沙星、阿奇霉素、庆大霉素、阿米卡星、甲氧苄啶/磺胺甲恶唑、四环素以及氯霉素的表型药敏检测，该体系的灵敏度与特异度均超过90%。随后我们在直接粪便标本中验证了该检测方法，以粪便培养得到的大肠杆菌基因型作为参照，结果显示其灵敏度为97%±5，但正如预期的那样，特异度有所降低，为75%±31。最终，我们将该检测体系整合至便捷的TaqMan阵列卡（TaqMan Array Card, TAC）格式中。本套检测方法可用于大肠杆菌分离株的抗菌素耐药性追踪，或直接应用于粪便标本的靶向肠道抗生素耐药组检测。