The ionophore oxyclozanide enhances tobramycin killing of Pseudomonas aeruginosa biofilms by permeabilizing cells and depolarizing the membrane potential

Abstract

Objectives To assess the ability of oxyclozanide to enhance tobramycin killing of Pseudomonas aeruginosa biofilms and elucidate its mechanism of action. Methods Twenty-four hour biofilms formed by the P. aeruginosa strain PAO1 and cystic fibrosis (CF) isolates were tested for susceptibility to oxyclozanide and tobramycin killing using BacTiter-Glo™ and cfu. Biofilm dispersal was measured using crystal violet staining. Membrane potential and permeabilization were quantified using DiOC2(3) and TO-PRO-3, respectively. Results Here we show that the ionophore anthelmintic oxyclozanide, combined with tobramycin, significantly increased killing of P. aeruginosa biofilms over each treatment alone. This combination also significantly accelerated the killing of cells within biofilms and stationary phase cultures and it was effective against 4/6 CF clinical isolates tested, including a tobramycin-resistant strain. Oxyclozanide enhanced the ability of additional aminoglycosides and tetracycline to kill P. aeruginosa biofilms. Finally, oxyclozanide permeabilized cells within the biofilm, reduced the membrane potential and increased tobramycin accumulation within cells of mature P. aeruginosa biofilms. Conclusions Oxyclozanide enhances aminoglycoside and tetracycline activity against P. aeruginosa biofilms by reducing membrane potential, permeabilizing cells and enhancing tobramycin accumulation within biofilms. We propose that oxyclozanide counteracts the adaptive resistance response of P. aeruginosa to aminoglycosides, increasing both their maximum activity and rate of killing. As oxyclozanide is widely used in veterinary medicine for the treatment of parasitic worm infections, this combination could offer a new approach for the treatment of biofilm-based P. aeruginosa infections, repurposing oxyclozanide as an anti-biofilm agent.

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

“…This observation became clear by using sensitive genetically encoded fluorescent proteins, and these tools open up a new avenue to study the longterm effects of antibiotic treatment on cells and mixed cultures. Another curious corollary is the observation that protonophores enhance aminoglycoside killing in Pseudomonas biofilms 55,56 , which stands in opposition to our observation that protonophores protect planktonic E. coli. The differences driven by these species specific and context dependent observations will hopefully add to a more complete picture of aminoglycoside activity in multiple bacterial species.…”

Membrane voltage dysregulation driven by metabolic dysfunction underlies bactericidal activity of aminoglycosides

“…This observation became clear by using sensitive genetically encoded fluorescent proteins, and these tools open up a new avenue to study the long-term effects of antibiotic treatment on cells and mixed cultures. Another curious corollary is the observation that protonophores enhance aminoglycoside killing in Pseudomonas biofilms ( Maiden et al, 2018 ; Maiden et al, 2019 ), which stands in opposition to our observation that protonophores protect planktonic E. coli . The differences driven by these species specific and context-dependent observations will hopefully add to a more complete picture of aminoglycoside activity in multiple bacterial species.…”

Drugs Repurposing for Multi-Drug Resistant Bacterial Infections

“…Different agents are promising both in vitro and in vivo candidates to be repositioned as antimicrobial agents to treat infections caused by MDR Gram-negative bacilli. A variety of drugs with different mechanisms of action and targets have been selected including: DNA, RNA and proteins inhibitors [16][17][18][19][20], QS regulators [15,17,[21][22][23][24][25], biofilm formation inhibitors and disruptors [26,27], drugs that interact with cell membrane [28][29][30], drugs that interact with iron metabolism [31][32][33][34][35], and host immune system modulators [36][37][38][39]. These drugs and their mechanisms of action against critical-priority pathogens (A. baumannii, P. aeruginosa and Enterobacterales) are summarized in Figure 1 and Table 1.…”

“…Niclosamide and rafoxanide were discovered to increase the negative surface charge of bacterial membrane in A. baumannii and K. pneumoniae clinical strains in vitro [30,46]. In turn, oxyclozanide has enhanced the activity of additional tobramycin against P. aeruginosa by reducing the membrane potential and increasing tobramycin accumulation [28]. This increase in the negative surface charges allow to restore the activity of colistin in colistin-resistant (Col-R) A. baumannii and K. pneumoniae, and the activity tobramycin in tobramycin-resistant P. aeruginosa [30,46].…”

“…The anthelmintic drugs niclosamide, oxyclozanide, rafoxanide and ivermectin have been shown to restore the activity of colistin against a collection of Col-R P. aeruginosa in vitro [46][47][48]57]. Not only in combination with colistin, oxyclozanide has presented synergy with tobramycin to destruct the biofilm formation, permeabilizing the cells membrane and depolarizing the membrane potential of P. aeruginosa strains resistant to tobramycin in vitro [28]. In the murine model of peritoneal sepsis by Col-R P. aeruginosa clinical isolate, rafoxanide plus CMS compared with CMS alone, increased mice survival to 73.3%, and reduced bacterial loads in tissues and blood between 3 and 5 log10 cfu/g or mL, respectively [46].…”