Laboratório de Pesquisa em Resistência Bacteriana (LABRESIS), Centro de Pesquisa Experimental, Hospital de Clínicas de Porto Alegre (HCPA), Porto Alegre, Brazil.
Programa de Pós-Graduação em Ciências Farmacêuticas (PPGCF), Faculdade de Farmácia, Universidade Federal do Rio Grande do Sul (UFRGS), Porto Alegre, Brazil.
Braz J Microbiol. 2020 Sep;51(3):1029-1035. doi: 10.1007/s42770-019-00222-y. Epub 2020 Jan 27.
Carbapenem-resistant Enterobacterales (CREs) have been recognized as an important threat to global health. CRE cause the majority of the difficult-to-treat infections in health-care settings and are associated with high mortality. Klebsiella pneumoniae carbapenemase (KPC)-producing CREs, in particular Klebsiella pneumoniae, are globally disseminated and responsible for a large number of outbreaks. Development of rapid methods for KPC detection can provide great clinical and epidemiological benefits to prevent KPC dissemination. The aim of this study was to standardize and validate a LC-MS/MS method to detect KPC. This method was also tested against a broad variety of species, including CRE with other carbapenemase genes and the recently reported mcr-1. For validation, 111 isolates with reduced susceptibility to carbapenems were selected (49 KPC-positive and 62 KPC-negative). The presence of four tryptic peptides related to the KPC enzyme was evaluated, and the identification of at least two of them classified the isolate as "KPC-positive." The LTLGSALAAPQR and LALEGLGVNGQ peptides were both detected in 47 of 49 isolates with the bla gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the bla gene, respectively. The method correctly classified 47 of 49 KPC-positive and all KPC-negative isolates yielding 96.07% of sensitivity and 100% of specificity. In conclusion, our results demonstrate that the KPC peptide markers were robustly detected by the method which presented high sensitivity and full specificity and therefore can be used as a reliable method to identify this resistance mechanism.
碳青霉烯类耐药肠杆菌科（CRE）已被认为是对全球健康的一个重要威胁。CRE 导致了医疗机构中大多数难以治疗的感染，并与高死亡率相关。产碳青霉烯酶肠杆菌科（CRE），特别是肺炎克雷伯菌，已在全球传播，并导致了大量的暴发。开发快速检测 KPC 的方法可以为预防 KPC 传播提供重要的临床和流行病学效益。本研究的目的是标准化和验证一种用于检测 KPC 的 LC-MS/MS 方法。该方法还针对多种不同的物种进行了测试，包括具有其他碳青霉烯酶基因的 CRE 和最近报道的 mcr-1。为了验证，选择了 111 株对碳青霉烯类药物敏感性降低的分离株（49 株 KPC 阳性和 62 株 KPC 阴性）。评估了与 KPC 酶相关的四个胰蛋白酶肽的存在情况，并且鉴定出至少两种肽将分离株分类为“KPC 阳性”。LTLGSALAAPQR 和 LALEGLGVNGQ 肽均在 49 株bla 基因阳性的分离株中检测到。另外两种肽，GFLAAAVLAR 和 APIVLAVYTR，分别在 46 株和 19 株 bla 基因阳性的分离株中检测到。该方法正确分类了 47 株 KPC 阳性和所有 KPC 阴性分离株，敏感性为 96.07%，特异性为 100%。总之，我们的结果表明，该方法可以可靠地检测到 KPC 肽标志物，具有较高的敏感性和完全的特异性，因此可以作为一种可靠的方法来识别这种耐药机制。
