Laboratory of Bacteriology, Hellenic Pasteur Institute, Athens, Greece.
Microbiol Spectr. 2022 Aug 31;10(4):e0093822. doi: 10.1128/spectrum.00938-22. Epub 2022 Jul 19.
In the absence of a molecule that would collectively inhibit both metallo-β-lactamases and serine-reactive carbapenemases, containment of their genes is the main weapon currently available for confronting carbapenem resistance in hospitals. Cost-effective methodologies rapidly detecting carbapenemase-producing enterobacteria (CPE) would facilitate such measures. Herein, a low-cost CPE detection method was developed that was based on the direct colorimetry of the yellow shift caused by the accumulation of diketopiperazines-products of the acid-catalyzed imipenem oligomerization-induced by carbapenemase action on dense solutions of imipenem/cilastatin. The reactions were studied by spectrophotometry in the visible spectrum using preparations of β-lactamases from the four molecular classes. The effects of various buffers on reaction mixtures containing the potent carbapenemases NDM-1 and NMC-A were monitored at 405 nm. Optimal conditions were used for the analysis of cell suspensions, and the assay was evaluated using 66 selected enterobacteria, including 50 CPE as well as 16 carbapenemase-negative strains overexpressing other β-lactamases. The development of the yellow color was specific for carbapenemase-containing enzyme preparations, and the maximum intensity was achieved in acidic or unbuffered conditions in the presence of zinc. When applied on bacterial cell suspensions, the assay could detect CPE with 98% sensitivity and 100% specificity, with results being comparable to those obtained with the Carba NP technique. Direct colorimetry of carbapenemase-induced imipenem decomposition required minimum reagents while exhibiting high accuracy in detecting CPE. Therefore, it should be considered for screening purposes after further clinical evaluation. Currently, the spread of multidrug-resistant (MDR) carbapenemase-producing enterobacteria (CPE), mostly in the clinical setting, is among the most pressing public health problems worldwide. In order to effectively control CPE, use of reliable and affordable methods detecting carbapenemase genes or the respective β-lactamases is of vital importance. Herein, we developed a novel method, based on a previously undescribed phenomenon, that can detect CPE with few reagents by direct colorimetry of bacterial suspensions and imipenem/cilastatin mixtures.
在缺乏能够同时抑制金属β-内酰胺酶和丝氨酸反应性碳青霉烯酶的分子的情况下，遏制其基因是目前应对医院碳青霉烯类耐药性的主要手段。快速检测产碳青霉烯酶肠杆菌（CPE）的具有成本效益的方法将有助于采取这些措施。在此，开发了一种低成本的 CPE 检测方法，该方法基于在酸性条件下，由碳青霉烯酶作用于密集的亚胺培南/西司他丁溶液导致的碳青霉烯类化合物聚合诱导的二酮哌嗪（二酮哌嗪是由β-内酰胺酶催化的碳青霉烯类化合物的酸催化聚合产生的）积累引起的黄色位移的直接比色法。使用来自四个分子类别的β-内酰胺酶制剂在可见光谱中通过分光光度法研究了这些反应。在 405nm 处监测了含有强力碳青霉烯酶 NDM-1 和 NMC-A 的反应混合物中各种缓冲液的影响。使用 66 种选定的肠杆菌，包括 50 种 CPE 以及 16 种过表达其他β-内酰胺酶的碳青霉烯酶阴性菌株，对细胞悬浮液进行了分析。该方法发展了针对含碳青霉烯酶酶制剂的黄色，并且在存在锌的酸性或无缓冲条件下达到最大强度。当应用于细菌细胞悬浮液时，该测定法可以检测到 98％的 CPE ，具有 100％的灵敏度和特异性，其结果与 Carba NP 技术的结果相当。碳青霉烯酶诱导的亚胺培南分解的直接比色法所需的试剂最少，同时在检测 CPE 方面具有很高的准确性。因此，在进一步的临床评估后，应考虑将其用于筛选目的。目前，在全球范围内，主要在临床环境中，耐多药（MDR）产碳青霉烯酶肠杆菌（CPE）的传播是最紧迫的公共卫生问题之一。为了有效控制 CPE，使用可靠且负担得起的方法检测碳青霉烯酶基因或相应的β-内酰胺酶至关重要。在此，我们开发了一种新方法，该方法基于以前未描述的现象，通过直接比色法对细菌悬浮液和亚胺培南/西司他丁混合物进行检测，仅用少量试剂即可检测 CPE。
