Synergistic efficacy of Bisbenzimidazole and Carbonyl Cyanide 3-Chlorophenylhydrazone combination against MDR bacterial strains

Devapriya Sinha,Vinod Tiwari,Kirti Sad,Vibha Tandon,Stuti Pandey,Raja Singh

doi:10.1038/srep44419

Devapriya Sinha, Vinod Tiwari + Show 4 more

Open Access

https://doi.org/10.1038/srep44419

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Abstract

Activation of efflux systems and the formation of biofilm are majorly adapted by microbes to resist antimicrobial agents. PPEF (bisbenzimidazole) targeting topoisomerase IA is observed to be an effective bactericidal agent against both Gram-positive and Gram-negative bacterial strains and thus can be developed as potent broad-spectrum antibiotic against MDR strains. PPEF treatment did not cause target specific mutation instead it leads to up-regulation of efflux gene in E. coli K12 as a mechanism of resistance. Microscopy, fluorescence spectroscopy and flow cytometry result demonstrate higher accumulation of PPEF in efflux gene deleted E. coli K12 mutants, and also suggest that Carbonyl Cyanide 3-Chlorophenylhydrazone (CCCP), resist the efflux of PPEF, and thus increases efficacy of PPEF. Herein, we report, PPEF and CCCP synergistically killed the persistent bacterial cells, which are not killed by PPEF alone. The above two compounds together inhibited biofilm formation, eradicate preformed biofilms and kills the biofilm cells of P. aeruginosa. PPEF and CCCP together reduced bacterial load of E. coli ATCC25922 by 6 log10 in neutropenic thigh infection model of balb/c mice. Present study suggests that combination therapy could be a promising antimicrobial strategy to handle MDR pathogenic strains.

Highlights

The emergence of multi-drug resistant (MDR) bacterial strains and their rapid world-wide spread are a threat to human health[1,2]
6 potent BBZs were assessed for their antibacterial activity against the common MDR human pathogenic strains Klebsiella sp., A. baumannii, S. typhimurium, Enterococcus sp., S. aureus, S. flexineri, P. aeruginosa, Enterobacter sp. and Providencia sp. (Tables 1 and 2)
The results indicates, that BBZs used in the study are not targeting Gram-negative bacteria and show significantly good antibacterial activity against Gram-positive bacterial strains which include pathogenic Enterococcus sp., S. aureus and the Methicillin-resistant Staphylococcus aureus (MRSA) strains (Table 2)

Summary

Introduction

The emergence of multi-drug resistant (MDR) bacterial strains and their rapid world-wide spread are a threat to human health[1,2]. Studies have recognized activation of efflux pumps as one of the major cause of resistance to many classes of antibiotics[7,8]. Association of efflux mechanisms to antibiotic resistance, suggest efflux pump inhibitors (EPIs) as adjuvants could potentiate the activities of antibacterial agent. This hypothesis leads us to study the effect of bisbenzimidazole in combination with EPIs to target the MDR bacterial strains, the persistent population and the sessile cells forming biofilm. An important aspect of synergism between PPEF and efflux pump inhibitor CCCP was studied and the effect of these molecules in combination and individually on the persistent population, the sessile cells and their vivo efficacy were carried out. An important aspect of synergism between PPEF and CCCP has been demonstratedand validated by the different in vitro and in vivo studies

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