Virulence and antimicrobial resistance gene profiling in multidrug-resistant Pseudomonas aeruginosa with evaluation of levofloxacin and amikacin therapy.

Multidrug-resistant (MDR) Pseudomonas aeruginosa is an opportunistic pathogen responsible for severe oral and hospital-acquired infections due to its strong biofilm-forming ability and multidrug resistance (MDR). This study investigated the virulence and resistance of P. aeruginosa isolated from subgingival biofilms and evaluated the antibiofilm activity of levofloxacin (LVX) and amikacin (AMK), individually and in combination. From 157 subgingival samples, 14 isolates were confirmed as P. aeruginosa, all resistant to antibiotic classes, indicating MDR status. Susceptibility testing revealed complete resistance to amoxiclav (100 %) and high resistance to ceftazidime (85.7 %), with moderate resistance to ciprofloxacin and gentamicin. The isolate with the highest biofilm-forming ability was used for further analysis. Crystal violet quantification demonstrated that LVX and AMK inhibited biofilm biomass by 39.9 % and 59.9 % at 24 h and by 70 % and 75.1 % at 48 h, respectively, whereas their combination achieved 96.2 % inhibition at 48 h. Live cell inhibition assessed by acridine orange staining showed that the LVX-AMK combination resulted in 97 ± 1.2 % reduction of viable biofilm cells at 48 h (p < 0.001). qRT-PCR analysis revealed 0.2-0.4-fold downregulation of virulence genes (fadA, toxA, lasB, algD, exoS) in the combination group. Structural modeling and Ramachandran analysis showed conserved and stable protein regions. These findings suggest that combined LVX-AMK therapy exhibits synergistic antibiofilm and anti-virulence effects against MDR P. aeruginosa, shows potential for treating biofilm-associated infections.