Spatial structure facilitates the accumulation and persistence of antibiotic-resistant mutants in biofilms

The emergence and spread of antibiotic resistance in bacterial pathogens are a global crisis. Because many bacterial infections are caused by pathogens that reside in biofilms, we sought to investigate how biofilms influence the evolution of antibiotic resistance. We hypothesize that the inherent spatial structure of biofilms facilitates the accumulation and persistence of spontaneously evolved antibiotic-resistant mutants. To test this, we tracked the frequency of mutants resistant to kanamycin and rifampicin in biofilm populations of Escherichia coli before, during, and after an antibiotic treatment regimen. Our results show that biofilms accumulate resistant mutants even in the absence of antibiotics. This resistance was found to be heritable and thus unlike the phenotypic plasticity of so-called "persister cells" that have been shown to occur in biofilms. Upon exposure to an antibiotic, resistant mutants swept to high frequency. Following the conclusion of treatment, these resistant mutants remained at unexpectedly high frequencies in the biofilms for over 45 days. In contrast, when samples from kanamycin-treated biofilms were used to found well-mixed liquid cultures and propagated by serial transfer, the frequency of resistant cells dramatically decreased as they were outcompeted by sensitive clones. These observations suggest that the emergence of antibiotic resistance through spontaneous mutations in spatially structured biofilms may significantly contribute to the emergence and persistence of mutants that are resistant to antibiotics used to treat bacterial infections.