A NOVEL METHOD FOR IN - VITRO BETA-LACTAMASE QUANTIFICATION: PROOF-OF-CONCEPT

Abstract

Intro: Extended – spectrum beta-lactamase (ESBL) producing Enterobacteriaceae pose a therapeutic challenge across all ages and demographics. ESBL infections are associated with increased antimicrobial resistance and greater morbidity and mortality worldwide. There are limited methods to quantify the production of ESBL enzymes as part of phenotypic antimicrobial susceptibility testing (AST). With widespread empiric broad- spectrum β-lactam use creating selective pressure, quantification of ESBL produced by organisms could improve the precision with which we select and prescribe antimicrobial therapy. We aim to develop a method to quantify ESBL production in-vitro and correlate this with minimum inhibitory concentrations and clinical outcomes. Methods: Gold electrodes were coated with iridium oxide to create a pH sensitive sensor. A range of beta lactamase concentrations (0-10mg/mL) were prepared using ESBL powder (Sekisui Diagnostics) diluted in Mueller Hinton broth (ThermoFisher). Sensors were placed in each enzyme concentration and allowed to stabilise before adding 5ml Penicillin G (512mg/L). After 5 minutes, open circuit potentials were recorded. Data was analysed, plotting enzyme concentration against potential. Triplet repeats of experiments were performed and sensors were calibrated in PBS between each run to check durability and stability over time. Findings: Sensors remained stable throughout the experiments with calibrations measured in PBS solutions of different pH between 4.5 and 8. The slope of the line was Nernstian. Across 3 repeats the sensors measured a positive correlation between enzyme concentration and potential. The sensors were able to measure mean absolute changes of 0.01323V. Conclusion: The data suggests that an iridium oxide-based pH sensor can detect a change in ESBL concentration in standard in-vitro experimental conditions. This could provide a novel method of ESBL quantification as part of in-vitro AST. Future work will apply this methodology to in-vitro assessment of Enterobacteriaceae phenotype.