Most bacteria in nature exist in biofilms, which are inherently tolerant to antibiotics. There is currently very limited understanding of how biofilms evolve in response to sub-lethal concentrations of antimicrobials. In this study, we use a biofilm evolution model to study the effects of sub-inhibitory concentrations of three antibiotics on Salmonella Typhimurium biofilms. We show that biofilms rapidly evolve resistance to each antibiotic they are exposed to, demonstrating a strong selective pressure on biofilms from low antibiotic concentrations. While all antibiotics tested select for clinical resistance, there is no common mechanism. Adaptation to antimicrobials, however, has a marked cost for other clinically important phenotypes, including biofilm formation and virulence. Cefotaxime selects mutants with the greatest deficit in biofilm formation followed by azithromycin and then ciprofloxacin. Understanding the impacts of exposure of biofilms to antibiotics will help understand evolutionary trajectories and may help guide how best to use antibiotics in a biofilm context. Experimental evolution in combination with whole-genome sequencing is a powerful tool for the prediction of evolution trajectories associated with antibiotic resistance in biofilms.

To survive and adapt to changing environments, bacteria have evolved mechanisms to express appropriate genes at appropriate times. Exposure to antimicrobials triggers a global stress response in Enterobacteriaceae, underpinned by activation of a family of transcriptional regulators, including MarA, RamA, and SoxS. These control a program of altered gene expression allowing a rapid and measured response to improve fitness in the presence of toxic drugs. Increased expression of marA, ramA, and soxS up regulates efflux activity to allow detoxification of the cell. However, this also results in trade-offs in other phenotypes, such as impaired growth rates, biofilm formation and virulence. Here, we review the current knowledge regarding the trade-offs that exist between drug survival and other phenotypes that result from induction of marA, ramA, and soxS. Additionally, we present some new findings linking expression of these regulators and biofilm formation in Enterobacteriaceae, thereby demonstrating the interconnected nature of regulatory networks within the cell and explaining how trade-offs can exist between important phenotypes. This has important implications for our understanding of how bacterial virulence and biofilms can be influenced by exposure to antimicrobials.

Most bacteria can form biofilms, which typically have a life cycle from cells initially attaching to a surface before aggregation and growth produces biomass and an extracellular matrix before finally cells disperse. To maximize fitness at each stage of this life cycle and given the different events taking place within a biofilm, temporal regulation of gene expression is essential. We recently described the genes required for optimal fitness over time during biofilm formation in Escherichia coli using a massively parallel transposon mutagenesis approach called TraDIS-Xpress. We have now repeated this study in Salmonella enterica serovar Typhimurium to determine the similarities and differences in biofilm formation through time between these species. A core set of pathways involved in biofilm formation in both species included matrix production, nucleotide biosynthesis, flagella assembly and LPS biosynthesis. We also identified several differences between the species, including a divergent impact of the antitoxin TomB on biofilm formation in each species. We observed deletion of tomB to be detrimental throughout the development of the E. coli biofilms but increased biofilm biomass in S. Typhimurium. We also found a more pronounced role for genes involved in respiration, specifically the electron transport chain, on the fitness of mature biofilms in S. Typhimurium than in E. coli and this was linked to matrix production. This work deepens understanding of the core requirements for biofilm formation in the Enterobacteriaceae whilst also identifying some genes with specialised roles in biofilm formation in each species.

Biofilms complete a life cycle where cells aggregate, grow and produce a structured community before dispersing to colonize new environments. Progression through this life cycle requires temporally controlled gene expression to maximize fitness at each stage. Previous studies have largely focused on identifying genes essential for the formation of a mature biofilm; here, we present an insight into the genes involved at different stages of biofilm formation. We used TraDIS-Xpress, a massively parallel transposon mutagenesis approach using transposon-located promoters to assay the impact of disruption or altered expression of all genes in the genome on biofilm formation. We identified 48 genes that affected the fitness of cells growing in a biofilm, including genes with known roles and those not previously implicated in biofilm formation. Regulation of type 1 fimbriae and motility were important at all time points, adhesion and motility were important for the early biofilm, whereas matrix production and purine biosynthesis were only important as the biofilm matured. We found strong temporal contributions to biofilm fitness for some genes, including some where expression changed between being beneficial or detrimental depending on the stage at which they are expressed, including dksA and dsbA. Novel genes implicated in biofilm formation included zapE and truA involved in cell division, maoP in chromosome organization, and yigZ and ykgJ of unknown function. This work provides new insights into the requirements for successful biofilm formation through the biofilm life cycle and demonstrates the importance of understanding expression and fitness through time.