Sierra JM, Ruiz J, Jimenez De Anta MT, Vila J. for purifying, neutral, and positive; species) have been identified as the leading global cause of multidrug-resistant bacterial infections, and overexpression of multidrug efflux (MEX) transport systems has been identified as one of the most critical mechanisms facilitating the evolution of multidrug resistance in ESKAPE pathogens. Despite efforts to develop efflux pump inhibitors to combat antibiotic resistance, the need persists to identify additional targets for future investigations. We evaluated evolutionary pressures on 110 MEX-encoding genes from all annotated ESKAPE organism genomes. We identify several MEX genes under stabilizing selectionrepresenting targets which can facilitate BMS-1166 broad-spectrum treatments with evolutionary constraints limiting the potential emergence of escape mutants. We also examine MEX systems being evaluated as drug targets, demonstrating that FJX1 divergent selection may underlie some of the problems encountered in the development of effective treatmentsspecifically in relation to the NorA system in spp.), noted for their propensity to escape the inhibitory action of traditional antibiotic drugs, cause a significant proportion of nosocomial and biofilm-mediated infections (4,C6). Upregulated expression of multidrug efflux (MEX) pumps is one BMS-1166 mechanism by which the ESKAPE pathogens become resistant. These MEX pumps act by expelling compounds, including BMS-1166 antibiotics, from the intracellular compartment/intermembrane space at a high rate, preventing drug concentrations from reaching inhibitory concentrations (7,C12). Six superfamilies of MEX pumps have been identified: the ATP-binding cassette (ABC) family, the small multidrug resistance (SMR) family, the major facilitator superfamily (MFS), the resistance-nodulation-division (RND) family, the multidrug and toxic compound extrusion (MATE) family, and the recently recognized sixth superfamily, the proteobacterial antimicrobial compound efflux (PACE) family, although this group of MEX pumps is known only to efflux cationic biocides (13, 14). As MEX pump upregulation is critical to the evolution of MDR phenotypes across a broad array of pathogens, MEX pumps are attractive targets for drug development and discovery (15). Efflux pump inhibitors (EPIs) can be combined with antimicrobial agents in order to treat otherwise MDR infections (16), rendering previously resistant bacteria susceptible to multiple classes of antibiotic agents (17). While many potential EPIs have been described in the literature (16,C19), challenges associated with EPIs as therapeutic compounds, including toxicity to patients, have prevented their widespread use. Despite these challenges, there has been a renewed interest in these compounds to treat MDR infections (15). Studies have taken steps to address substrate specificity (20, 21), but few studies have considered the conservation of genes encoding efflux pumps at the strain level (18, 22, 23). Examining how these genes are conserved prior to EPI development can ensure that potential EPIs will target the widest range of pathogenic strains and minimize the likelihood of promoting escape mutants. In this study, we take an evolutionary approach to evaluate selection across MEX systems in the ESKAPE pathogens to identify genes described by models of purifying selection (i.e., evolution that eliminates deleterious alleles from a population), stabilizing selection (i.e., selection favoring intermediate rather than extreme variants), neutral selection (i.e., stochastic evolution), and diversifying selection (i.e., selection favoring the tails of a trait distribution resulting in adaptive divergence). In doing so, we examine the breadth of likely applications for potential EPIs targeting each of these specific MEX systems. This identification of conservation provides a comprehensive evolutionary context to all MEX pumps in the ESKAPE pathogens, along with insights into the evolutionary origins of these important structures. RESULTS We took an approach to identify genes encoding ESKAPE pathogen efflux pumps undergoing evolutionary pressure. Using only those MEX genes with previously demonstrated involvement in antibiotic resistance, chromosomal genes encoding complete or partial efflux pumps were screened for evidence of selection by calculating the ratio of nonsynonymous to synonymous mutations (and had the highest evidence for selection, with all sites classified as under selection. Moderate selection was observed for and MDR efflux pump genes, with six of the nine genes having and were of the RND BMS-1166 superfamily of efflux pumps. Purifying selection was identified for.