Resistance to Ceftazidime/Avibactam plus Meropenem/Vaborbactam When Both Are Used Together Is Achieved in Four Steps in Metallo-β-Lactamase-Negative Klebsiella pneumoniae

Abstract

Serine β-lactamases are dominant causes of β-lactam resistance in Klebsiella pneumoniae isolates. Recently, this has driven clinical deployment of the β-lactam-β-lactamase inhibitor pairs ceftazidime/avibactam and meropenem/vaborbactam. We show that four steps, i.e., ompK36 and ramR mutation plus carriage of OXA-232 and KPC-3-D178Y variant β-lactamases, confer ceftazidime/avibactam and meropenem/vaborbactam resistance when both pairs are used together. These findings have implications for decision making about sequential and combinatorial use of these β-lactam-β-lactamase inhibitor pairs to treat K. pneumoniae infections.

Keywords: KPC; OXA-48; porin; β-lactamase.

FIG 1

Checkerboard assays for ceftazidime and meropenem in the presence of avibactam and vaborbactam. Each image represents duplicate assays for an 8-by-8 array of wells in a 96-well plate. All wells contained CA-MHB, including avibactam (4 μg · ml⁻¹) and vaborbactam (8 μg · ml⁻¹). A serial dilution of meropenem (MEM; x axis) and ceftazidime (CAZ, y axis) was created from 32 μg · ml⁻¹ in each plate as recorded. All wells were inoculated with a suspension of bacteria, made according to CLSI microtiter MIC guidelines (18), and the plate was incubated at 37°C for 20 h. Growth was recorded by measuring OD₆₀₀, and growth above background (broth) is shown as a yellow block. Growth at 8 μg · ml⁻¹ ceftazidime and 8 μg · ml⁻¹ meropenem (indicated in red) in the presence of vaborbactam and avibactam defined resistance based on CLSI breakpoints (19). Bacterial suspensions used were as follows: top row, KP21[ramR] ompK36; second row, KP21[ramR] ompK36(pOXA-232); third row, KP21[ramR] M[ompK36](pOXA-232); fourth row, KP21[ramR] M[ompK36] ompK35(pOXA-232); and fifth row, KP47 ompK36(pOXA-232). In each case, bacteria also carried the following plasmids (where tested): first column, pCTX-M-14 P170S; second column, pKPC-3; third column, pKPC-3-D178Y; and fourth column, pKPC-3-V239G.