3.2 Persister eradication approach
In A. baumannii , the first bactericidal agent used to eradicate persister cells was Art-175 from the Artilysine family [136]. Art-175 is a phagic endolysin which degrades peptidoglycan, fused with specific peptides that destabilize the outer membrane [136]. The specificity between peptidoglycan and endolysin results from the long and intense coevolution between phages and bacteria. Art-175 (60 µg/ml) was highly effective at 1 h and eradicated all strains after 24 h of treatment. Persister selected with tobramycin and treated with 30 × MIC Art-157 on the RUH134 strain led to a drastic population reduction in just 20 min. It took only 120 min to reach the limit of bacteria detection.
The effect of molecules with an aromatic core (1-BC or 2-NC), an L-lysine moiety (K), and a variable lipophilic chain (8, 10, 12 carbon chain) were also tested on E. coli , Klebsiella pneumoniae, P. aeruginosa and A. baumannii persister cells [137]. NCK-10 and BCK-12 activity on A. baumannii clinical isolates (MTCC 1425, R676 and R674) showed lytic activity between 1.5 and 9 μM. NCK-10 (8 × MIC) did not induce resistance even after 20 cycles in E. coli . NCK-10 appears to depolarized and permeabilize the membranes of E. coli persister cells (induced by 300 μg/mL of ampicillin for 3 h). NCK-10 was burn-tested on female Balb/c mice infected with A. baumannii at concentrations of 17.5 mg/kg and did not induce any toxic effects. Moreover, this compound significantly reduced the bacterial load after a 7-day application.
Squalamine, a polycationic aminosterol isolated from the sharkSqualus acanthias with antibacterial properties, was tested on persister cells generated by ciprofloxacin [36]. This molecule contains a polyamine tail, which is known to be able to lead to a fast membrane depolarization and disruption [138]. In this study, unlike those presented above, the methodology is slightly different. The authors generated persister cells using an antibiotic (ciprofloxacin 1000 × MIC). They first characterized the selected population to determine the proportion of persister cells and VNBCs. Then the authors tested squalamine, the molecule of interest, with colistin as the control for cell lysis. Squalamine and colistin were able to eradicate persister cells at 100 mg/L (50 × MIC) and 500 mg/L (100 × MIC) within 9 and 12 h respectively. Colistin concentrations (100 × MIC) are higher than those of squalamine (50 × MIC), and require more time to eradicate the persistent population, which underlines the efficacy of squalamine. It was mentioned that squalamine was used at concentrations below the minimum hemolytic concentration (IC50 > 66 mg/L [139]) demonstrating a poor toxicity.
Carvacrol, Eugenol, and Thymol are compounds approved by the US Food and Drug Administration (FDA), having a GRAS (generally recognized as safe) status [125]. They can be used as food additives. These compounds were tested on persister cells selected with meropenem (100 × MIC). Thymol used alone at its MIC concentration lead to a complete eradication of meropenem induced persisters. In combination with meropenem, it completely eradicated persisters at lower concentration (0.5 × MIC). It allowed also the total eradication of 48 h mature biofilm at concentration 1 × MIC. Concerning eugenol and carvacrol, they eradicated biofilm persisters but at much higher concentrations than thymol. Thymol treatment significantly inhibited meropenem persisters of MDR A. baumannii isolates, both as monotherapy and co-therapy. Thymol was also effective on rifampicin, tigecycline and polymyxin B persisters at concentrations equal to or less than 1 × MIC. Thymol was demonstrated to induce a membrane depolarization and permeability, as well as an increased ROS production. It inhibited proton motive force (PMF) and multiple drug efflux pumps.
Recently, it has been shown that phages can have an effect on dormant cells [140]. The action of the phage Paride on dormant cells ofPseudomonas aeruginosa has been studied. This study showed that this phage has an active lytic cycle on P. aeruginosa cells in deep dormancy [135]. To our knowledge, phages with an effective lytic cycle on persistent A. baumannii cells have not yet been studied.
Table 2 : Therapeutic investigation for anti- persister cells in A. baumannii