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Antibiotic resistance mediated by beta-lactamase enzyme expression is a serious and growing threat. The typical workflow in a clinical microbiology lab leading to identification of beta-lactamase expressing organisms typically requires 48 to 72 hours and involves multiple manual steps. Empiric treatment with potentially ineffective antibiotics often occurs before susceptibility testing results are available. Objective: The objective of our study was to determine if beta-lactamase activity could be detected in clinical urine samples to provide same-day rule-out of select empiric treatment regimens for urinary tract infections. Methods: We acquired urine samples from 40 patients with extended spectrum beta-lactamase expressing (ESBL) infections and 100 patients without ESBL infection as determined by standard culture and sensitivity methods. These samples were incubated with spiked beta-lactam antibiotics susceptible to ESBL degradation (ceftriaxone, cefazolin, and oxacillin) or resistant to ESBL degradation (meropenem). Results: Hydrolysis of spiked drug was detected after a 4 hour incubation with either liquid-chromatography mass-spectrometry (LC-MS) or matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) methods. A 100 to 1000 fold reductions in measured concentrations of spiked ceftriaxone, cefazolin, or oxacillin were observed with LC-MS or MALDI-TOF methods for all 40 ESBL containing specimens and unchanged concentrations of spiked drug were observed in all 100 non-ESBL containing specimens. Concentration of spiked meropenem (resistant to ESBL degradation) were unchanged in all specimens after incubation regardless of ESBL status. Addition of a beta-lactamase inhibitor prevented ESBL degradation of ceftriaxone, cefazolin, and oxacillin demonstrating that beta-lactamase activity (and not another method of drug degradation or signal masking) is being detected. Incubation times as long as 24 hours (to determine method specificity) and as short as 30 minutes (to determine method sensitivity) all showed robust discrimination between ESBL-containing and non-ESBL containing specimens. Conclusion: Our results demonstrate that beta-lactamase activity can be robustly detected in urine samples days before culture and susceptibility results could be available. Clinical utilization of the assay described would enable clinicians to reduce ineffective empiric treatment of ESBL urinary infections.

Direct Detection of Beta-Lactamase Mediated Antibiotic Resistance In Clinical Specimens