3.1 Preventing approach: combination of treatments
Combining antibiotics to prevent the formation of persister cells is a strategy that generally uses antibiotics with radically different targets, the membrane being one of the preferred targets. Thus, most of the studies combine the use of the polymyxin B or colistin with β-lactams.
Recently, it has been shown that meropenem or carbenicillin treatments resulted in the appearance of a particular cell phenotype that survive: round, metabolically active cells that have lost their cell wall [126]. A modification of their envelope leads to a susceptibility to treatment with β-lactam (ampicillin and carbenicillin) with an increase in the killing power from 100 to 1000 fold [126]. These results suggest that antibiotic treatment can cause a modification of the envelope, making the bacteria more sensitive to another antibiotic targeting the membrane. Another study focused on three clinical strains (Acb-1, Acb-8 and Acb-2) and determined the level of persister cells generated by polymyxin B and meropenem, tested at different concentrations alone or in combination [130]. Mono-treatment with these antibiotics at 5, 10 or 15 mg/L, induced between 0.0031 to 0.9743 % of persisters depending on the choice of antibiotics. However, these antibiotics in combination did not produce persister cells. This study highlighted and confirmed the useful effects of combined therapy.
Synergistic effects were also observed with other antibiotic combinations: 30 μg/mL carbenicillin with 2 μg/mL colistin or 200 μg/mL ampicillin with 2 μg/mL colistin. They increased the killing efficiency on two clinical isolates [126]. These combinations provide a two-log increase in bacterial eradication.
Colistin has also been tested in combination with other antibiotics,e.g . colistin plus tobramycin, rifampicin or colistin [131]. These authors show that these combinations eradicate all cells. Sequential antibiotic combinations were also examined [132]. It was demonstrated that treatment by colistin followed by amikacin eradicated the entire population, when the reverse sequence did not. Even though no high antibiotic concentration was used to select persisters, this study clearly shows that sensitization by a membranolytic agent is highly effective. Colistin in combination with the econazole, an imidazole derivative, was also tested [133]. This combination showed a synergic effect with a reduction of MIC colistin by 128 fold with 10 µg/mL econazole.
The combination of an antimicrobial agent with a phage cocktail to control or limit biofilm formation has also been investigated [134]. Combinations of four temperate bacteriophages (SA1, Eve, Ftm and Gln) and different antibiotics (ampicillin/sulbactam, meropenem and colistin) enhanced biofilm inhibition and degradation on thirty extensively drug-resistant (XDR) strains of A. baumannii . Moreover, it has been shown that a combination of Paride and meropenem can sterilize deep-dormant cultures in vitro and greatly reduce a resilient bacterial infection of a tissue cage implant in mice [135]. In this context, it would be interesting to test these phage/antibiotic combinations on persister cells.