Original Article
Rapid detection method of carbapenemase-producing Enterobacteriaceae by MALDI-TOF MS with imipenem/cilastatin (KB) disc and zinc sulfate solution

https://doi.org/10.1016/j.jiac.2020.09.013Get rights and content

Abstract

Introduction

Carbapenemase-producing Enterobacteriaceae (CPE) is a major global health threat, and development of rapid detection methods is desired. Here, we established a cost-effective and relatively rapid CPE detection method using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS).

Methods

We examined 134 CPE strains (IMP-type, NDM-type, VIM-type, KPC-type, OXA-48-like-type, and GES-type) and 107 non-CPE strains, previously confirmed by genetic tests. The proposed MALDI-TOF MS method involves mixing of a carbapenem drug [here, the commercially available imipenem (IPM) KB disc] and the bacterial strains to be tested, and the consequent drug hydrolysis owing to bacterial carbapenemase activity is confirmed by a waveform spectrum before and after 2 h of the mixing. As metallo-beta-lactamases require zinc in their active site, the false-negatives obtained from our method were cultured in presence of zinc sulfate solution and tested again.

Results

Based on the presence or absence of the IPM (+cyano-4-hydroxy-cinnamic acid)-specific waveform peak near 489.45 m/z (±500 ppm), the detection sensitivity and specificity of our method for CPE were determined to be 94.8% and 91.6%, respectively. Seven false-negatives of IMP-type (4), VIM-type (2), and GES-type (1) were found, of which the IMP- and VIM-types tested positive as CPE after culture with zinc sulfate solution. Thus, the overall detection sensitivity improved to 99.3%.

Conclusion

Our study proposes a new approach for CPE detection using MALDI-TOF MS. Moreover, we propose cultivation of test strains with zinc sulfate solution for efficient detection of IMP-type CPE, not only for MALDI-TOF MS, but also for other detection methods.

Introduction

In 2016, an action plan for antimicrobial resistance was announced in Japan, and by 2020, countries worldwide were required to reduce the prevalence of drug-resistant bacteria centering on carbapenemase-producing Enterobacteriaceae (CPE). Drug susceptibility tests are used to detect CPE in microbial laboratories, and for strains suspected of being a CPE, further confirmation methods include detecting the presence of drug resistance genes (bla IMP, bla NDM, bla VIM, bla KPC, bla OXA-48, and bla GES) [1,2], and modified carbapenem inactivation method (mCIM) [[3], [4]] based on Clinical and Laboratory Standards Institute M100-S27 [5]. In addition, some facilities use the Carba NP test [[4], [6], [7], [8]], which detects carbapenemase, and the immunochromatography method [[9], [10]]. In recent years, the MBT STAR-Carba® [6] kit (Bruker Japan, Tokyo, Japan) has been marketed to rapidly detect CPE in combination with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), a technique that can be used for bacterial identification. However, MBT STAR-Carba® kit has the disadvantages that the target antibacterial drug used for detecting CPE is limited and the assay is expensive. Therefore, we applied MBT STAR-Carba analysis method and tried to detect CPE rapidly using imipenem/cilastatin (IPM/CS) of the KB disc (Eiken Chemical, Tokyo, Japan), which is used for commercial drug sensitivity test and in vitro diagnostics. The establishment of this method can allow the measurement of antibacterial activity of many agents using a commercially available KB disc, as well as the detection of extended-spectrum beta-lactamase (ESBL)-producing strains other than carbapenemase. In addition, the method would be relatively inexpensive.

Currently, Japan shows high prevalence of the imipenemase metallo-beta-lactamase type (IMP-type) of CPE, while the Klebsiella pneumoniae carbapenemase (KPC-type) and oxacillin carbapenemase-48-like (OXA-type) types are more prevalent in Europe and America. The New Delhi metallo-beta-lactamase type (NDM-type) occurs more frequently in Southeast Asian countries and the VIM-type is observed frequently in Italy [11]. The mainstream IPM type in Japan belongs to Class B of the Ambler classification. Beta-lactamase belonging to this class B requires zinc at the active center and is called a metallo-beta-lactamase. To detect IMP-type beta-lactamase activity accurately, it is necessary to have a sufficient amount of zinc in the active center of the enzyme. However, in reality, there may be cases where the zinc is insufficient, leading to poor detection of the enzyme. Yong et al. reported the restoration of metallo-beta-lactamase activity in Bacteroides fragilis when zinc was added [12]. Therefore, we attempted to improve the detection sensitivity of metallo-beta-lactamase belonging to class B (IMP-type, NDM-type, and VIM-type) of the Ambler classification by providing sufficient amounts of zinc to the active center of beta-lactamase.

Section snippets

Bacterial strains

A total of 134 CPE strains [IMP-type, NDM-type, VIM-type, KPC-type, OXA-48-like-type, and Guiana extended-spectrum-beta-lactamase (GES)-type] and 107 non-CPE strains, which had been confirmed by genetic testing (Cica Geneus® Carbapenemase Genotype Detection Kit 2 (Kanto Chemical Co., Ltd., Tokyo) and Loop-Mediated Isothermal Amplification (LAMP) [2]), were stored at the Laboratory of Saga University Hospital and the Center for Clinical Microbial & Genetic Testing, School of Health Sciences at

CPE detection sensitivity and specificity of MALDI-TOF MS

Table 2 shows the results of the rapid detection of CPE by MALDI-TOF MS using the KB disc of IPM. Among the 134 strains confirmed as CPE by genetic testing, only 7 strains retained the specific IPM waveform near 489.45 m/z (±500 ppm), indicating that the remaining 127 were CPE. Thus, the detection sensitivity of CPE was 94.8%. In case of the 107 non-CPE strains tested, 98 retained the specific IPM waveform; thus, the detection specificity of CPE was 91.6%. In addition, the positive predictive

Discussion

The detection rate of CPE has been increasing every year worldwide, and in the case of bloodstream infections, the mortality rate is 26–44% higher when compared with infections caused by carbapenem-susceptible bacteria [16]; reports have termed this as “Nightmare Bacteria” [17]. Further, the drug resistance gene of CPE is classified into chromosome-derived type and plasmid-derived type, but since the plasmid type has a characteristic of being easily transmitted to other strains, there is a high

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

Authorship statement

Oho M: Designed the research, Acquired and interpreted the data, Drafted the manuscript. Funashima Y: Acquired and interpreted the data. Nagasawa Z: Designed the research, Acquired and interpreted the data, Miyamoto H: Designed the research, interpreted the data, Revised the manuscript critically. Sueoka E: Designed the research and interpreted the data, Approval of the submitted and final versions. All co-authors contributed to the writing of the final manuscript, and read, approved the final

Declaration of Competing Interest

The authors declare no conflict of interests.

Acknowledgments

We would like to express our gratitude to Mr. Kyohei Kato (Beckman Coulter, Japan) for his cooperation in this research.

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