Using experimental evolution to identify druggable targets that could inhibit the evolution of antimicrobial resistance

Abstract

With multi-drug and pan-drug resistant bacteria becoming increasingly common in hospitals, antibiotic resistance has threatened to return us to a pre-antibiotic era that would completely undermine modern medicine. There is an urgent need to develop new antibiotics and strategies to combat resistance that are substantially different from earlier drug discovery efforts. One such strategy that would complement current and future antibiotics would be a class of co-drugs that target the evolution of resistance and thereby extend the efficacy of specific classes of antibiotics. A critical step in the development of such strategies lies in understanding the critical evolutionary trajectories responsible for resistance and which proteins or biochemical pathways within those trajectories would be good candidates for co-drug discovery. We identify the most important steps in the evolution of resistance for a specific pathogen and antibiotic combination by evolving highly polymorphic populations of pathogens to resistance in a novel bioreactor that favors biofilm development. As the populations evolve to increasing drug concentrations, we use deep sequencing to elucidate the network of genetic changes responsible for resistance and subsequent in vitro biochemistry and often structure determination to determine how the adaptive mutations produce resistance. Importantly, the identification of the molecular steps, their frequency within the populations and their chronology within the evolutionary trajectory toward resistance is critical to assessing their relative importance. In this work, we discuss findings from the evolution of the ESKAPE pathogen, Pseudomonas aeruginosa to the drug of last resort, colistin to illustrate the power of this approach.

Environment Shapes the Accessible Daptomycin Resistance Mechanisms inEnterococcus faecium

“…1A, bioreactor-derived isolates produced up to 10-fold more biofilm than HOU503 and the flask-transfer isolates, consistent with growing in different adaptive environments. While the bioreactor-derived isolates typically formed strong biofilms, isolate R2P29 produced little biofilm, consistent with our previous studies showing that bioreactors can favor highly polymorphic populations and maintain planktonic sub-populations (25, 30–33). This isolate is discussed further in the Supplementary Text.…”

“…Using methods described previously (25, 30–33) and more extensively in the Supplementary Text, two independent HOU503 populations were evolved to DAP resistance within 12 and 10 days (Population 1 and Population 2, respectively) in a bioreactor system that favors polymorphic populations and biofilm formation (25, 30, 31, 33). To identify the frequency of each mutation over time and the likely order of mutations, a polymorphic sample was taken daily for metagenomic deep sequencing (Fig.…”

“…Because mutations that arise early and at high frequency are those more likely to contribute to DAP resistance (25, 31–33), we identified divIVA and oatA as candidate genes for further study (Fig. 4).…”

“…However, due to the prevalence of biofilm in enterococcal cultures, optical density (OD) probes were rendered useless. To circumvent this problem, CO 2 was measured by a Magellan Tandem Pro Gas Analyzer and used as a proxy to monitor cell density and maintain logarithmic growth as described previously (25, 30, 31, 33). Manual OD measurements were taken periodically to ensure appropriate cell density.…”

The Essential Role of Hypermutation in Rapid Adaptation to Antibiotic Stress

“…Experimental evolution is a powerful approach to understanding the genetic and biochemical basis for antibiotic resistance (16,(20)(21)(22)(23)(24)(25)(26). In this work, P. aeruginosa PAO1 was evolved to colistin resistance as a continuous culture in a bioreactor where the population was constantly maintained at mid-exponential phase in the presence of subinhibitory drug concentrations (24). The observation of hypermutation, accompanied by a dramatic increase in mutation rate among the evolving PAO1 population, presented both challenges and opportunities for understanding the evolution of colistin resistance.…”