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” Evolutionary paths tohigh-level ampicillin resistance “” Thewidespread use of beta-lactams has resulted in a growing prevalence of highlyresistant pathogens.Indeed, clinical isolates can have very high resistance to beta-lactams, mostprominently to ampicillin where resistance level could even exceed 1000 µg/ml. Yet, instriking contrast, bacteria evolved in laboratory settings, typically plateauon much lower levels of resistance. Here, evolving Escherichiacoli onthe Microbial Evolution and Growth Arena (MEGA) plate, we foundthat large population sizes circumvent the previously observed saturation ofresistance under ampicillin selection, selecting for mutants resistant toampicillin at over 5000 µg/mg. Whole-genome sequencing of resistant isolatesrevealed that this high ampicillin resistance was acquired via a combination ofsingle-point mutations (SNP) and increasingly focused gene amplification ofresistant genes, most notably the beta-lactamase enzyme AmpC. Importantlythough, blocking AmpC-mediated resistance only slightly reduced the adaptivepotential: strains deleted for ampC were able to evolve high-level resistancethrough combinations of genetic changes in genes involved in multidrugresistance such as efflux pumps, transcriptional regulators, and porins. Ourresults reveal that combinations of distinct genetic mutations, accessible atlarge population sizes, can drive high-level resistance to ampicillin evenindependently of beta-lactamases. “
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