In vivo evolution of resistance to novel β-lactam/β-lactamase inhibitor combinations through overproduction of the horizontally acquired extended-spectrum AmpC β-lactamase FOX-14 and porin disruption in Serratia marcescens.

We investigated the genetic and biochemical determinants that reduce the activity of several β-lactam/β-lactamase inhibitor combinations against Serratia marcescens clinical isolates after carbapenem exposure. We analyzed paired clinical S. marcescens isolates recovered before and after treatment of a prolonged infection with ertapenem. We tested the susceptibility of the isolates to ceftazidime/avibactam, aztreonam/avibactam, imipenem/relebactam and meropenem/vaborbactam. Mechanisms of resistance were investigated using whole-genome sequencing and RNA expression analysis. The blaFOX-14 gene was cloned into Escherichia coli, to assess the phenotype in wild-type and low-permeability backgrounds. FOX-14 was purified to determine steady-state kinetics and avibactam IC50. The baseline isolate was susceptible to ceftazidime/avibactam, aztreonam/avibactam, imipenem/relebactam and meropenem/vaborbactam, whereas the post-therapy isolate displayed high-level resistance. The post-therapy isolate harboured a premature stop in ompF, loss of an additional putative porin, and overexpressed of blaFOX-14 due to triplication of the blaFOX-14 surrounding region. Cloning of blaFOX-14 conferred an extended-spectrum AmpC phenotype in E. coli, which was further accentuated under reduced permeability. Complementation of the post-therapy resistant clinical isolate with functional OmpF restored susceptibility to new β-lactam/β-lactamase inhibitor combinations. Purified FOX-14 displayed marked cephalosporinase activity against ceftazidime and cefepime, with low Km values, while negligible turnover for aztreonam and ertapenem and a low avibactam IC50. In S. marcescens, in vivo resistance in clinical isolates emerged from FOX-14 overproduction combined with reduced outer-membrane permeability, predominantly due to OmpF disruption. These findings elucidate resistance to newer β-lactam/β-lactamase inhibitor combinations and warrant close monitoring of antimicrobial activity during carbapenem-based therapy for S. marcescens infections.