Evolutionary Trajectories toward High-Level β-Lactam/β-Lactamase Inhibitor Resistance in the Presence of Multiple β-Lactamases

Abstract

Beta-Lactam antibiotics are the first choice for the treatment of most bacterial infections. However, the increased prevalence of beta-lactamases, in particular extended-spectrum beta-lactamases, in pathogenic bacteria has severely limited the possibility of using beta-lactam treatments. Combining beta-lactam antibiotics with beta-lactamase inhibitors can restore treatment efficacy by negating the effect of the beta-lactamase and has become increasingly important against infections caused by beta-lactamase-producing strains. Not surprisingly, bacteria with resistance to even these combinations have been found in patients. Studies on the development of bacterial resistance to beta-lactam/beta-lactamase inhibitor combinations have focused mainly on the effects of single, chromosomal or plasmidborne, beta-lactamases. However, clinical isolates often carry more than one beta-lactamase in addition to multiple other resistance genes. Here, we investigate how the evolutionary trajectories of the development of resistance to three commonly used beta-lactam/beta-lactamase inhibitor combinations, ampicillin-sulbactam, piperacillin-tazobactam, and ceftazidime-avibactam, were affected by the presence of three common beta-lactamases, TEM-1, CTX-M-15, and OXA-1. First-step resistance was due mainly to extensive gene amplifications of one or several of the beta-lactamase genes where the amplification pattern directly depended on the respective drug combination. Amplifications also served as a stepping-stone for high-level resistance in combination with additional mutations that reduced drug influx or mutations in the beta-lactamase gene bla(CTX-M-15). This illustrates that the evolutionary trajectories of resistance to beta-lactam/beta-lactamase inhibitor combinations are strongly influenced by the frequent and transient nature of gene amplifications and how the presence of multiple beta-lactamases shapes the evolution to higher-level resistance.