Aspartic acid unveils as antibiofilm agent and tobramycin adjuvant against mucoid and small colony variants of Pseudomonas aeruginosa isolates in vitro within cystic fibrosis airway mucus

Antibiotics are central to managing airway infections in cystic fibrosis (CF), yet current treatments often fail due to the presence of <i>Pseudomonas aeruginosa</i> biofilms, settling down the need for seeking therapies targeting biofilms. This study aimed to investigate the antibiofilm activity of aspartic acid and its potential as an adjuvant to tobramycin against <i>P. aeruginosa</i> biofilms formed by mucoid and small colony variant (SCV) tobramycin tolerant strain. We assessed the effect of aspartic acid on both surface-attached and suspended <i>P. aeruginosa</i> biofilms within CF artificial mucus and investigated the synergistic impact of combining it with non-lethal tobramycin concentrations. Our findings showed that aspartic acid inhibited planktonic <i>P. aeruginosa</i> without affecting its viability and prevented biofilm formation by hindering bacterial adhesion or interfering with EPS production, depending on the experimental conditions. In CF mucus, aspartic acid significantly reduced bacterial growth, with the highest inhibition observed when combined with tobramycin, showing notable effects against the mucoid and tolerant SCV strain. Despite these reductions, <i>P. aeruginosa</i> repopulated the mucus within 24 h of stress withdrawal. Additional strategies, including delayed tobramycin application and a second dose of co-application of aspartic acid and tobramycin were explored to address bacterial survival and recovery. Although none of the strategies eradicated <i>P. aeruginosa</i>, the second co-application resulted in slower bacterial recovery rates. In conclusion, this study highlighted aspartic acid as an effective antibiofilm agent and demonstrated for the first time its potential as an adjuvant to tobramycin. The combined use of aspartic acid and tobramycin offers a promising advancement in CF therapeutics, particularly against <i>P. aeruginosa</i> biofilms formed by mucoid and SCV strains, mitigating their antibiotic resistance.