Rapid screening of cefotaxime resistance in Escherichia coli isolates by liquid chromatography with absorbance detection.

Abstract

The clinical outcome of bacterial infection relies on proper and timely diagnosis of antibiotic susceptibility. We demonstrate that liquid chromatography (LC) with diode-array absorbance detection (DAD) accurately diagnoses antimicrobial resistance by measuring cefotaxime recovery following incubation with bacterial isolates either positive or negative for the cefotaximase (CTX-M) family of serine-β-lactamases. Reversed phase, high-performance liquid chromatography with absorbance detection was employed for cefotaxime analysis. A total of 43 bla_CTX-M-producing and 5 bla_CTX-M-negative Escherichia coli isolates were incubated with 0.5 mg/mL of cefotaxime at 37 °C for 1 and 2 h. Remarkably, after 2 h of incubation, the median ± median absolute deviation percentage of cefotaxime recovery was zero (0 %) for bla_CTX-M producing and cefotaxime-resistant E. coli isolates compared to the cefotaxime recovery in bla_CTX-M negative (61 ± 5.38 %) and cefotaxime-susceptible (70.68 ± 6.25 %) E. coli isolates. This result allows facile sorting of organism resistance status after only 1–2 h with near-perfect performance. The diagnostic sensitivity (Se) and specificity (Sp) of the chromatographic approach were comparable to widely used phenotypic (Epsilometer test, E-test) and genotypic assays (Whole Genome Sequencing, WGS), but chromatography reduces diagnostic time by >10-fold. Additionally, the optical absorption measurement is fully compatible with microfluidic platforms, suggesting the development of low-cost, high-throughput sensors based on this measurement principle is possible. We conclude LC-DAD is suitable and reliable to determine the cefotaxime resistance status of E. coli isolates with a turn-around time of only 1–2 h.