Abstract
High levels of antibiotic tolerance are a hallmark of bacterial biofilms. In contrast to well-characterized inherited antibiotic resistance, molecular mechanisms leading to reversible and transient antibiotic tolerance displayed by biofilm bacteria are still poorly understood. The physiological heterogeneity of biofilms influences the formation of transient specialized subpopulations that may be more tolerant to antibiotics. In this study, we used random transposon mutagenesis to identify biofilm-specific tolerant mutants normally exhibited by subpopulations located in specialized niches of heterogeneous biofilms. Using Escherichia coli as a model organism, we demonstrated, through identification of amino acid auxotroph mutants, that starved biofilms exhibited significantly greater tolerance towards fluoroquinolone ofloxacin than their planktonic counterparts. We demonstrated that the biofilm-associated tolerance to ofloxacin was fully dependent on a functional SOS response upon starvation to both amino acids and carbon source and partially dependent on the stringent response upon leucine starvation. However, the biofilm-specific ofloxacin increased tolerance did not involve any of the SOS-induced toxin–antitoxin systems previously associated with formation of highly tolerant persisters. We further demonstrated that ofloxacin tolerance was induced as a function of biofilm age, which was dependent on the SOS response. Our results therefore show that the SOS stress response induced in heterogeneous and nutrient-deprived biofilm microenvironments is a molecular mechanism leading to biofilm-specific high tolerance to the fluoroquinolone ofloxacin.
Highlights
Formation of bacterial biofilms on medical implants is a major health threat due to their high levels of tolerance to multiple antibiotics [1]
Biofilm surface-attached communities have the capacity to tolerate high concentrations of antibiotics, and bacterial biofilms formed on indwelling medical devices are difficult to eradicate and often lead to the onset of chronic or systemic infections
We found that biofilms formed by amino acid auxotroph mutants display high antibiotic tolerance and we demonstrated that amino acid starvation strongly increases the antibiotic tolerance of biofilm bacteria
Summary
Formation of bacterial biofilms on medical implants is a major health threat due to their high levels of tolerance to multiple antibiotics [1]. Biofilm-associated antibiotic tolerance is mainly attributed to two distinct processes: persistence and drug indifference, which both characteristically disappear once multicellular conditions subside [2,3]. Persistence occurs in subpopulations of slow or non-growing bacteria, whereas drug indifference is exhibited by the entire population [4,5]. The molecular bases of persistence are under active investigation [5,6], drug indifference is far less well understood and is hypothesized to be multifactorial and to result from reduced antibiotic diffusion to slow growth rate of many cells within biofilms [7]. Biofilm heterogeneity creates specialized niches in which bacteria respond to local cues, leading to genetically and metabolically distinct subpopulations exhibiting high tolerance to extracellular stresses such as antibiotics
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