Effectiveness of colistin-antimicrobial peptides combinations against Pseudomonas aeruginosa and Staphylococcus aureus single and double-species biofilms

Antimicrobial research is increasingly being focused on the problem of microbial resistance and biofilm formation. Alternative mechanisms of action, as those of antimicrobial peptides (AMP), and synergic antimicrobial combinations can lead to more effective therapeutic strategies for this ever-growing world-wide problem. Hence, this work aimed to evaluate the antimicrobial and cytotoxic potential of combinations of colistin (CST) with other AMP against planktonic and biofilm cultures of two major pathogens, Pseudomonas aeruginosa and Staphylococcus aureus. CST was combined with the AMP temporin A (TEMP-A), citropin 1.1 (CIT-1.1) and a linear analogue of tachyplesin I (TP-1-L) and tested against planktonic, single- and double-species biofilm cultures of P. aeruginosa and S. aureus, comprising a reference and a clinical isolated strain for each bacteria. The methods applied encompassed checkerboard assays, statistical and biological significance analysis of biofilm-cell viability and cytotoxicity evaluation. Results showed overall synergy for planktonic P. aeruginosa and synergy (CST + TP-1-L) and additiveness for planktonic S. aureus. Biofilm growth was successfully prevented with overall synergic and additive effects for both bacteria. The eradication of already pre-established 24-h biofilms proved challenging, especially for S. aureus and for the double-species biofilms. Interestingly, CST revealed low toxicity against mammalian cells but its combinations were toxic for high concentrations. In summary, CST-AMP combinations showed promising results and other strategies are being investigated to improve effectiveness and reduce toxicity against pre-established biofilms, notably by adding a third antimicrobial agent.