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 blaKPC gene. The other two peptides, GFLAAAVLAR and APIVLAVYTR, were detected in 46 and 19 isolates with the blaKPC 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.

中文翻译：

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LC-MS/MS 高效检测肠杆菌中肺炎克雷伯菌碳青霉烯酶


耐碳青霉烯类肠杆菌（CRE）已被认为是对全球健康的重要威胁。 CRE 导致医疗机构中大多数难以治疗的感染，并与高死亡率相关。产生肺炎克雷伯菌碳青霉烯酶 (KPC) 的 CRE，特别是肺炎克雷伯菌，在全球范围内传播并导致大量疫情暴发。开发 KPC 快速检测方法可以为防止 KPC 传播提供巨大的临床和流行病学益处。本研究的目的是标准化和验证用于检测 KPC 的 LC-MS/MS 方法。该方法还针对多种物种进行了测试，包括具有其他碳青霉烯酶基因的 CRE 和最近报道的 mcr-1。为了进行验证，选择了 111 个对碳青霉烯类药物敏感性降低的分离株（49 个 KPC 阳性和 62 个 KPC 阴性）。对与 KPC 酶相关的四种胰蛋白酶肽的存在进行了评估，并且至少其中两种的鉴定将分离物分类为“KPC 阳性”。在 49 个带有 blaKPC 基因的分离株中，有 47 个分离株中检测到了 LTLGSALAAPQR 和 LALEGLGVNGQ 肽。另外两种肽 GFLAAAVLAR 和 APIVLAVYTR 分别在 46 个和 19 个带有 blaKPC 基因的分离株中检测到。该方法对 49 个 KPC 阳性分离株中的 47 个和所有 KPC 阴性分离株进行了正确分类，灵敏度为 96.07%，特异性为 100%。总之，我们的结果表明，该方法可以稳健地检测到 KPC 肽标记，具有高灵敏度和完全特异性，因此可以用作识别这种耐药机制的可靠方法。