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The use and misuse of antibiotics have resulted in the selection of difficult-to-treat resistant bacteria. Two key parameters that influence the selection of resistant bacteria are the minimal selective concentration (MSC) and the fitness cost of resistance, both of which have been measured during planktonic growth in several studies. However, bacterial growth most often occurs in biofilms, and it is unclear if and how these parameters differ under these two growth conditions. To address this knowledge gap, we compared a selection of several types of antibiotic-resistant Escherichia coli mutants during planktonic and biofilm growth to determine the fitness costs and MSCs. Biofilm-forming Escherichia coli strains are commonly found in catheter-associated and recurrent urinary tract infections. Isogenic strains of a biofilm-forming E. coli strain, differing only in the resistance mechanisms and the fluorescent markers, were constructed, and susceptible and resistant bacteria were grown in head-to-head competitions at various concentrations of antibiotics under planktonic and biofilm conditions. Mutants with resistance to five different antibiotics were studied. The results show that during both planktonic and biofilm growth, selection for the resistant mutants occurred for all antibiotics at sub-MICs far below the MIC of the antibiotic. Even though differences were seen, the MSC values and the fitness costs did not differ systematically between planktonic and biofilm growth, implying that despite the different growth modes, the basic selection parameters are similar. These findings highlight the risk that resistant mutants may, similarly to planktonic growth, also be selected at sub-MICs of antibiotics in biofilms.

IMPORTANCE Our understanding of how and where antibiotic resistance is selected in response to antibiotic exposure is still limited, and this is particularly true for selective processes when bacteria are growing in biofilms, arguably the most significant mode of growth of bacteria in human and animal infections as well as in other settings. In this study, we compared how different types of resistant E. coli strains were selected in response to antibiotic exposure during planktonic and biofilm growth. Determination of the minimal selective concentrations (MSCs) and fitness costs of resistance showed that they were comparable under these two different conditions, even though some differences were observed. Importantly, the MSCs were far below the MICs for all mutants under both planktonic and biofilm growth, emphasizing the significance of low antibiotic concentrations in driving the emergence and enrichment of resistant bacteria. Our understanding of how and where antibiotic resistance is selected in response to antibiotic exposure is still limited, and this is particularly true for selective processes when bacteria are growing in biofilms, arguably the most significant mode of growth of bacteria in human and animal infections as well as in other settings. In this study, we compared how different types of resistant E. coli strains were selected in response to antibiotic exposure during planktonic and biofilm growth.