Abstract
Non-heritable, phenotypic drug resistance toward antibiotics challenges antibiotic therapies. Characteristics of such phenotypic resistance have implications for the evolution of heritable resistance. Diverse forms of phenotypic resistance have been described, but phenotypic resistance characteristics remain less explored than genetic resistance. Here, we add novel combinations of single-cell characteristics of phenotypic resistant E. coli cells and compare those to characteristics of susceptible cells of the parental population by exposure to different levels of recurrent ampicillin antibiotic. Contrasting expectations, we did not find commonly described characteristics of phenotypic resistant cells that arrest growth or near growth. We find that under ampicillin exposure, phenotypic resistant cells reduced their growth rate by about 50% compared to growth rates prior to antibiotic exposure. The growth reduction is a delayed alteration to antibiotic exposure, suggesting an induced response and not a stochastic switch or caused by a predetermined state as frequently described. Phenotypic resistant cells exhibiting constant slowed growth survived best under ampicillin exposure and, contrary to expectations, not only fast-growing cells suffered high mortality triggered by ampicillin but also growth-arrested cells. Our findings support diverse modes of phenotypic resistance, and we revealed resistant cell characteristics that have been associated with enhanced genetically fixed resistance evolution, which supports claims of an underappreciated role of phenotypic resistant cells toward genetic resistance evolution. A better understanding of phenotypic resistance will benefit combatting genetic resistance by developing and engulfing effective anti-phenotypic resistance strategies. IMPORTANCE: Antibiotic resistance is a major challenge for modern medicine. Aside from genetic resistance to antibiotics, phenotypic resistance that is not heritable might play a crucial role for the evolution of antibiotic resistance. Using a highly controlled microfluidic system, we characterize single cells under recurrent exposure to antibiotics. Fluctuating antibiotic exposure is likely experienced under common antibiotic therapies. These phenotypic resistant cell characteristics differ from previously described phenotypic resistance, highlighting the diversity of modes of resistance. The phenotypic characteristics of resistant cells we identify also imply that such cells might provide a stepping stone toward genetic resistance, thereby causing treatment failure.