Lu Xuedong, Nie Shuping, Xia Chengjing, Huang Lie, He Ying, Wu Runxiang, Zhang Li
Department of Laboratory Medicine, Futian Hospital, Guangdong Medical College, Shenzhen 518033, China.
Department of Laboratory Medicine, Futian Hospital, Guangdong Medical College, Shenzhen 518033, China.
J Microbiol Methods. 2014 Jul;102:26-31. doi: 10.1016/j.mimet.2014.04.006. Epub 2014 Apr 24.
Aiming to identify macrolide and beta-lactam resistance in clinical bacterial isolates rapidly and accurately, a two-step algorithm was developed based on detection of eight antibiotic resistance genes.
Targeting at genes linked to bacterial macrolide (msrA, ermA, ermB, and ermC) and beta-lactam (blaTEM, blaSHV, blaCTX-M-1, blaCTX-M-9) antibiotic resistances, this method includes a multiplex real-time PCR, a melting temperature profile analysis as well as a liquid bead microarray assay. Liquid bead microarray assay is applied only when indistinguishable Tm profile is observed.
The clinical validity of this method was assessed on clinical bacterial isolates. Among the total 580 isolates that were determined by our diagnostic method, 75% of them were identified by the multiplex real-time PCR with melting temperature analysis alone, while the remaining 25% required both multiplex real-time PCR with melting temperature analysis and liquid bead microarray assay for identification. Compared with the traditional phenotypic antibiotic susceptibility test, an overall agreement of 81.2% (kappa=0.614, 95% CI=0.550-0.679) was observed, with a sensitivity and specificity of 87.7% and 73% respectively. Besides, the average test turnaround time is 3.9h, which is much shorter in comparison with more than 24h for the traditional phenotypic tests.
Having the advantages of the shorter operating time and comparable high sensitivity and specificity with the traditional phenotypic test, our two-step algorithm provides an efficient tool for rapid determination of macrolide and beta-lactam antibiotic resistances in clinical bacterial isolates.
为了快速、准确地鉴定临床分离细菌中的大环内酯类和β-内酰胺类耐药性，基于对8个抗生素耐药基因的检测开发了一种两步算法。
针对与细菌大环内酯类（msrA、ermA、ermB和ermC）和β-内酰胺类（blaTEM、blaSHV、blaCTX-M-1、blaCTX-M-9）抗生素耐药性相关的基因，该方法包括多重实时PCR、熔解温度曲线分析以及液珠微阵列检测。仅在观察到难以区分的熔解温度曲线时才应用液珠微阵列检测。
在临床分离细菌上评估了该方法的临床有效性。在通过我们的诊断方法测定的总共580株分离株中，75%仅通过多重实时PCR和熔解温度分析即可鉴定，而其余25%则需要多重实时PCR、熔解温度分析和液珠微阵列检测才能鉴定。与传统的表型抗生素敏感性试验相比，总体一致性为81.2%（kappa = 0.614，95%CI = 0.550 - 0.679），敏感性和特异性分别为87.7%和73%。此外，平均检测周转时间为3.9小时，与传统表型试验的超过24小时相比要短得多。
我们的两步算法具有操作时间短以及与传统表型试验相当的高敏感性和特异性的优点，为快速测定临床分离细菌中的大环内酯类和β-内酰胺类抗生素耐药性提供了一种有效的工具。
