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<p>We investigated the genetic and biochemical determinants that reduce the activity of several β-lactam/β-lactamase inhibitor combinations against <i>Serratia marcescens</i> clinical isolates after carbapenem exposure.&#xa0;We analyzed paired clinical <i>S. marcescens</i> 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 <i>bla</i><sub>FOX−14</sub> gene was cloned into <i>Escherichia coli</i>, to assess the phenotype in wild-type and low-permeability backgrounds. FOX-14 was purified to determine steady-state kinetics and avibactam IC<sub>50</sub>.&#xa0;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 <i>ompF</i>, loss of an additional putative porin, and overexpressed of <i>bla</i><sub>FOX−14</sub> due to triplication of the <i>bla</i><sub>FOX−14</sub> surrounding region. Cloning of <i>bla</i><sub>FOX−14</sub> conferred an extended-spectrum AmpC phenotype in <i>E. coli</i>, 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 <i>K</i><sub>m</sub> values, while negligible turnover for aztreonam and ertapenem and a low avibactam IC<sub>50</sub>.&#xa0;In <i>S. marcescens</i>, 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 <i>S. marcescens</i> infections.</p>

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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

  • 期刊论文

  • 发表日期：

    2025年11月11日

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  • Salud Rodríguez-Pallares,
  • Lucía González-Pinto,
  • María Tarriño-León,
  • Pablo Aja-Macaya,
  • Lucía Sánchez-Peña,
  • Miriam Moscoso,
  • Gloria Pérez-Rodríguez,
  • Tania Blanco-Martín,
  • Alejandro Beceiro,
  • Germán Bou,
  • Jorge Arca-Suárez

摘要

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.