Dutta Enakshy, Loy John Dustin, Deal Caitlyn A, Wynn Emily L, Clawson Michael L, Clarke Jennifer, Wang Bing
Department of Statistics, University of Nebraska-Lincoln, Lincoln, NE 68583, USA.
School of Veterinary Medicine and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583, USA.
Pathogens. 2021 Jan 13;10(1):64. doi: 10.3390/pathogens10010064.
Antimicrobial resistance (AMR) in bovine respiratory disease (BRD) is an emerging concern that may threaten both animal and public health. Rapid and accurate detection of AMR is essential for prudent drug therapy selection during BRD outbreaks. This study aimed to develop a multiplex quantitative real-time polymerase chain reaction assay (qPCR) to provide culture-independent information regarding the phenotypic AMR status of BRD cases and an alternative to the gold-standard, culture-dependent test. Bovine clinical samples (297 lung and 111 nasal) collected in Nebraska were subjected to qPCR quantification of macrolide (MAC) and tetracycline (TET) resistance genes and gold-standard determinations of AMR of BRD pathogens. Receiver operating characteristic curve analysis was used to classify AMR based on the qPCR results. For lung tissues, the qPCR method showed good agreement with the gold-standard test for both MACs and TETs, with a sensitivity of 67-81% and a specificity higher than 80%. For nasal swabs, qPCR results passed validation criteria only for TET resistance detection, with a sensitivity of 88%, a specificity of 80% and moderate agreement. The culture-independent assay developed here provides the potential for more rapid AMR characterization of BRD cases directly from clinical samples at equivalent accuracy and higher time efficiency compared with the gold-standard, culture-based test.
牛呼吸道疾病（BRD）中的抗菌药物耐药性（AMR）是一个新出现的问题，可能会威胁动物和公众健康。在BRD疫情期间，快速准确地检测AMR对于谨慎选择药物治疗至关重要。本研究旨在开发一种多重定量实时聚合酶链反应检测法（qPCR），以提供与BRD病例表型AMR状态相关的非培养信息，并作为金标准培养依赖性检测的替代方法。对内布拉斯加州收集的牛临床样本（297份肺样本和111份鼻样本）进行了大环内酯类（MAC）和四环素（TET）耐药基因的qPCR定量以及BRD病原体AMR的金标准测定。采用受试者工作特征曲线分析根据qPCR结果对AMR进行分类。对于肺组织，qPCR方法在MAC和TET检测方面与金标准检测显示出良好的一致性，敏感性为67%-81%，特异性高于80%。对于鼻拭子，qPCR结果仅在TET耐药性检测方面通过了验证标准，敏感性为88%，特异性为80%，一致性中等。与基于培养的金标准检测相比，这里开发的非培养检测法有可能以同等的准确性和更高的时间效率直接从临床样本中更快速地对BRD病例进行AMR特征分析。
