RESUMO
OBJECTIVES: Molecular analysis of meropenem-resistant mechanisms in mutants emerging from long-term in vitro meropenem exposure to borderline meropenem-susceptible carbapenemase-producing Enterobacterales (CPE) and non-CPE. METHODS: Escherichia coli TUM13867 harbouring both blaIMP-6- and blaCTX-M-2-carrying IncN plasmid and Citrobacter koseri TUM13189 with blaCTX-M-2-carrying chromosome were used. Meropenem MIC was 1â mg/L against both strains. Each strain was cultured in the hollow-fibre infection model (HFIM) to approximately 1â×â106 colony formation unit (cfu)/mL, and meropenem 1â g q8h treatment was initiated. Then, changes in total and meropenem-resistant populations were observed for 124â h. Meropenem resistance mechanisms were analysed using full-length whole-genome sequencing (WGS), reverse-transcription quantitative PCR and digital PCR. RESULTS: Meropenem reduced TUM13867 and TUM13189 to approximately 5 and 2 log10â cfu/mL, respectively, at 2â h after initiation, but regrowth was observed at 24â h. The meropenem-resistant mutant emergence frequency at 120 and 124â h was 4.4â×â10-4 for TUM13867 and 7.6â×â10-1 for TUM13189. Meropenem MIC of the mutants derived from TUM13867 (TUM20902) and TUM13189 (TUM20903) increased 4- and 16-fold, respectively. TUM20902, which harboured pMTY20902_IncN plasmid with a 27 505-bp deletion that included blaCTX-M-2, and blaIMP-6 showed 4.21-fold higher levels of transcription than the parental strain. TUM20903 had a 49 316-bp deletion that included ompC and a replicative increase of blaCTX-M-2 to three copies. CONCLUSIONS: Molecular analysis including full-length WGS revealed that the resistance mechanisms of meropenem-resistant mutants that emerged during long-term in vitro meropenem exposure were increased blaIMP-6 transcripts in CPE and increased blaCTX-M-2 transcripts due to gene triplication and OmpC loss resulting from ompC deletion in non-CPE.