Strategies to potentiate bactericidal antimicrobial activity based on the suppression of bacterial stress response systems

Abstract

One of the major threats to human health is the emergence of antimicrobial resistance. Since the discovery of antimicrobials, new mechanisms of resistance have emerged. Various strategies are being studied to combat the emergence of resistance including the development of new antimicrobial, use of adjuvants to enhance susceptibility, genome-editing technologies, and phage therapy. However, since microorganisms can evolve rapidly to ensure their survival, new resistance mechanisms appear frequently. Although resistance mechanisms in bacteria are usually expressed phenotypically, this expression may not affect the whole bacterial population equally. This phenomenon is known as heteroresistance. Heteroresistance is defined as the presence of a heterogeneous population of bacteria with one or multiple subpopulations showing higher levels of antimicrobial resistance relative to the main population. This makes it more difficult to categorize clinical isolates according to the level of resistance and can lead to treatment failure. Different strategies are therefore needed to combat resistance and heteroresistance. The SOS response is a coordinated cellular response to genotoxic damage that may contribute to the evolution of antimicrobial resistance, since genes related to DNA repair and recombination processes are transcribed during the SOS response. Certain antimicrobials, such as quinolones and β-lactams, induce the SOS response. Suppression of the SOS response has been shown to enhance the bactericidal activity of antimicrobials such as quinolones. Furthermore, bactericidal antimicrobials such as quinolones are also associated with the accumulation of reactive oxygen species (ROS) under aerobic conditions, inducing reactions that can contribute to cellular damage. Microorganisms contain detoxification systems able to detoxify ROS. The objectives of this thesis were threefold: (i) to evaluate the synergistic effect of suppression of the SOS response (by suppressing the recA gene) and overproduction of ROS (by suppressing detoxification systems or stress regulatory systems) in increasing the activity and lethality of fluoroquinolones; (ii) to evaluate the interplay between the SOS response (DNA repair and recombination processes) and detoxification systems in the evolution of ciprofloxacin resistance under gradual or sudden antimicrobial exposure; and (iii) to determine the impact of deletion of the recA gene (the SOS response activator, also required for homologous recombination in E. coli) on the reversal of heteroresistance. For the first and second objectives, a set of E. coli mutants was prepared. In E. coli BW25113, these included single mutants with suppression of the SOS response (ΔrecA), detoxification systems (ΔkatG, ΔkatE, ΔsodA, ΔsodB, ΔahpC) or stress regulatory systems (ΔoxyR, ΔrpoS); double deletion mutants with both recA and a detoxification system suppressed (ΔkatG/ΔrecA, ΔkatE/ΔrecA, ΔsodA/ΔrecA, ΔsodB/ΔrecA, ΔahpC/ΔrecA, ΔoxyR/ΔrecA, ΔrpoS/ΔrecA); double deletion mutants with two detoxification systems suppressed (ΔkatG/ΔkatE, ΔsodA/ΔsodB) and triple deletion mutants (ΔkatG/ΔkatE/ΔrecA).In E. coli BW15, single (ΔkatG, ΔsodA, ΔrecA) and double deletion (ΔkatG/ΔrecA, ΔsodA/ΔrecA) mutants were prepared. The BW15 strain has quinolone resistance mechanisms, carrying amino acid changes in GyrA (D87G), GyrB (E465D), and ParE (K390N), as well as a deletion in the marR gene. For the third objective, 23 clinical isolates of E. coli of bacteremic or urinary origin were used, among them eight isolates belonging to the high-risk clone ST131. A synergistic sensitization effect was found for ciprofloxacin when the SOS response (recA gene) and detoxification or stress regulatory systems were suppressed. However, this effect was not always replicated for other fluoroquinolones. Unexpectedly, no further sensitization was found for BW15 strain mutants. Suppression of the recA gene with detoxification system genes or stress regulatory system genes prevented bacterial growth in the presence of sublethal concentrations of ciprofloxacin and had an enhanced bactericidal effect on E. coli. After sudden exposure to ciprofloxacin, suppression of the SOS response, through deletion of the recA gene, and detoxification systems helps to reduce the evolution of resistance in E. coli. After gradual exposure to ciprofloxacin, suppression of the SOS response helps to reduce the evolution of resistance. Under the latter condition, however, suppression of detoxification systems, alone or in combination with the SOS response, may favour mutagenesis and the evolution of resistance to ciprofloxacin. In terms of heteroresistance, the percentages of clinical isolates expressing heteroresistance by disk diffusion varied from 30% to 100%, depending on the antimicrobial tested. Deletion of the recA gene resulted in reversal of heteroresistance to antimicrobials such as quinolones and β-lactams. Finally, heteroresistance was associated with tandem amplification of resistance genes, such as qnrA1, or an increase in the copy number of plasmid-borne resistance genes such as blaTEM-1B.