Small molecule adjuvants potentiate colistin activity and attenuate resistance development in escherichia coli by affecting pmrab system

Abstract

Background: Colistin is one of the last-resort antibiotics to treat multi-drug resistant (MDR) Gram-negative bacterial infections in humans. Further, colistin has been also used to prevent and treat Enterobacteriaceae infections in food animals. However, chromosomal mutations and mobile colistin resistance (mcr) genes, which confer resistance to colistin, have been detected in bacterial isolates from food animals and humans worldwide; thus, limiting the use of colistin. Therefore, strategies that could aid in ameliorating colistin resistance are critically needed. Objective: Investigate the adjuvant potential of novel small molecules (SMs) on colistin. Materials and Methods: Previously, we identified 11 membrane-affecting SMs with bactericidal activity against avian pathogenic Escherichia coli (APEC). Here, we investigated the potentiation effect of those SMs on colistin using checkerboard assays and wax moth (Galleria mellonella) larval model. The impact of the SM combination on colistin resistance evolution was also investigated by analyzing whole genome sequences of APEC isolates passaged with colistin alone or in combination with SMs followed by quantitating pmrCAB and pmrH expression in those isolates. Results: The SM combination synergistically reduced the minimum bactericidal concentra-tion of colistin by at least 10-fold. In larvae, the SM combination increased the efficacy of colistin by two-fold with enhanced (>50%) survival and reduced (>4 logs) APEC load. Further, the SM combination decreased the frequency (5/6 to 1/6) of colistin resistance evolution and downregulated the pmrCAB and pmrH expression. Previously unknown mutations in pmrB (L14Q, T92P) and pmrA (A80V), which were predicted deleterious, were identified in the colistin-resistant (ColR) APEC isolates when passaged with colistin alone but not in combination with SMs. Our study also identified mutations in hypothetical and several phage-related proteins in ColR APEC isolates in concurrent with pmrAB mutations. Conclusion: Our study identified two SMs (SM2 and SM3) that potentiated the colistin activity and attenuated the development of colistin resistance in APEC. These SMs can be developed as anti-evolution drugs that can slow down colistin resistance development.