Joshua Hayden (Presenter)
Weill Cornell Medical College
Bio: Joshua Hayden, PhD DABCC FABC is an assistant professor of pathology and laboratory medicine at Weill Cornell Medical College where he serves as the director of the Toxicology and Therapeutic Drug Monitoring laboratory. He received his PhD from Carnegie Mellon University, conducted postdoctoral research at the Massachusetts Institute of Technology and completed a clinical chemistry fellowship at the University of Washington in Seattle. His research focuses on the shortcoming of traditional diagnostic assays and the development and advancement of new diagnostics, specifically those utilizing tandem mass spectrometry.
Authorship: Chase Mazur(1), Kathy Fauntleroy(1), Stephen Jenkins (1), Lars Westblade (1), Joshua Hayden (1)
(1) Weill Cornell Medical College, New York, NY
Our objective was to develop a rapid, functional assay for carbapenemase activity that could potentially differentiate the various classes (A, B and D). The assay uses dilute-and-shoot liquid chromatography tandem mass spectrometry (LCMS) to monitor meropenem degradation in tryptic soy broth inoculated with bacterial isolates. The method showed excellent agreement with the modified carbapenemase inactivation method (mCIM) on 74 clinical isolates. Ethylenediaminetetraacetic acid inhibited the class B zinc-dependent carbapenemases (IMP, NMD and VIM), allowing segregation of this class. The class D (OXA-type) carbapenemases exhibited slower meropenem degradation at early time points, differentiating them from class A serine (IMI and KPC) enzymes. This method represents a rapid, functional assay that can offer valuable insight into carbapenem resistance mechanisms in clinical isolates.
Antimicrobial resistance is one of the most serious public health threats of our time, with an estimated two million people infected and over 23,000 dying annually in the United States from resistant organisms (1). In particular, the rise and spread of carbapenem-resistant Enterobacteriaceae (CRE) has become a major public health concern. Addressing this public health issue requires active detection of carbapenem resistance by the clinical laboratory. A number of genotypic and phenotypic methods exist for this purpose (2). One simple to perform method is the carbapenem inactivation method (mCIM) which represents a functional assessment, allowing for detection of unusual resistance genes (3). A drawback is that the mCIM requires incubation anywhere from 6 to 24 h, delaying determination of carbapenem resistance. In addition, the CIM method is only able to detect the presence or absence of resistance; it does not give information on the type of resistance genes, such as class (A vs B vs D). Here we sought to develop a rapid, functional assay of carbapenemase activity that could differentiate the various classes (A, B and D).
A modified CIM (mCIM) was performed with slight modifications. Briefly, 2 mL of tryptic soy broth (TSB) was inoculated with one 10 microliter loop of isolate, vortexed, and then a 10 microgram meropenem disk was added. The cultures were then allowed to incubate at 35°C for 4 after which the meropenem disk was placed onto a Mueller–Hinton agar plate inoculated with a suspension of E. coli ATCC 29522 (a meropenem-susceptible strain) equivalent in turbidity to that of a 0.5 McFarland standard. The plates were allowed to incubate for 24 h after which the zones of inhibition were read. This assay was also performed using 5 mM EDTA in TSB.
For LCMSMS analysis, 100 microliters of the inoculated TSB were taken at time points 20, 40, 60 and 240 minutes. Aliquots were frozen immediately after removal. These frozen aliquots were thawed and diluted 1:1,000 with LC/MS-grade H2O. The diluted aliquots were used for analysis. Separation and sample analysis was performed on an Agilent 1290 Infinity II LC coupled to an Agilent 6495 Triple Quad LC/MS/MS with Agilent MassHunter software. The LC separation column used was an Agilent Zorbax Eclipse Plus C18 RRHD (2.1x50mm, 1.8um) heated to 40C. Mobile phases used were A: 5mM ammonium formate in H2O and B: 5mM ammonium formate in methanol. The MRM transitions used: meropenem 384.1-141.1 and 384.1-68.2 m/z; meropenem-decarboxylate 358.2-140 and 358.2-121.9 m/z. No calibration curve was utilized in this assay and results were reported as relative quantifications by comparing peak areas to those of control samples.
A total of 74 clinical isolates were tested by both the mCIM and LCMSMS method. These isolates included 34 non-carbapenem resistant isolates which harbored various beta-lactamase resistance mechanisms and 38 carbapenemase producing strains encompassing a range of resistance genes including KPC (-1, -2 and -3), IMI, OXA (-48, -181 and -232), IMP, NDM and VIM. All strains tested had been characterized genetically.
The mCIM and LCMSMS method both correctly identified the carbapenemase producing strains. The LCMSMS method was able to yield this information an hour after inoculation of the TSB with the isolate (as opposed to the mCIM which required analysis of the plates up to 24 h later). In addition, the LCMSMS method allowed detection of both meropenem degradation and formation of a putative meropenem degradation product at 358.2->140 m/z (decarboxylated product). The detection of this degradation product provides the first evidence of the long-suspected mechanism of the CIM method (meropenem degradation). EDTA was found to effectively inhibit meropenem degradation, allowing for characterization of class B carbapenemases (as the EDTA mCIM approach was able to do).
Kinetic analysis showed that the average rates for the non-OXA strains were substantially faster, allowing differentiation of the OXA enzymes based on their slower kinetics.
Conclusions & Discussion
The ability to rapidly monitor meropenem loss utilizing the LCMSMS method described here allowed for rapid functional assessment of carbapenemase activity and differentiation of the class (A versus B versus D). The presence of carbapenemase activity in clinical isolates was evident from the 20-minute time period, reducing the time for detection of resistance. Detection of class B carbapenemases was rapidly facilitated through detection of EDTA inhibited meropenem degradation. Further, on average, the class D (OXA-type) carbapenemases showed noticeably slower rates of meropenem degradation. This differentiation of class A and class D carbapenemases is not possible with existing functional assays.
References & Acknowledgements:
1. Centers for Disease Control and Prevention, Office of Infectious Disease Antibiotic resistance threats in the United States, 2013. Apr, 2013. Available at: http://www.cdc.gov/drugresistance/threat-report-2013. Accessed October 8, 2017.
2. Evaluation of Genotypic and Phenotypic Methods to Detect Carbapenemase Production in Gram-Negative Bacilli. Clinical Chemistry Mar 2017, 63 (3) 723-730; DOI: 10.1373/clinchem.2016.264804
3. Modified Carbapenem Inactivation Method for Phenotypic Detection of Carbapenemase Production among Enterobacteriaceae. J. Clin. Microbiol. August 2017 vol. 55 no. 8 2321-2333.
|Grants||yes||Agilent Technologies, Roche, Binding Site|
IP Royalty: no
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