Sub-inhibitory tobramycin concentration suppresses ToxA and LipA in single- and dual-species biofilms

Pseudomonas aeruginosa causes persistent infections largely through biofilm formation. In polymicrobial environments, it forms dual-species biofilms in which microbial interactions influence structure, virulence, and antibiotic response. Sub-inhibitory antibiotic concentrations may modulate these behaviors differently in single- versus dual-species settings. This study evaluated the effect of Tobramycin at ½ minimal inhibitory concentration (MIC) on biofilm formation and virulence gene expression (toxA, lipA) in P. aeruginosa under single- and dual-species biofilm conditions. Clinical isolates were tested for Tobramycin susceptibility, MIC determination, and biofilm production. Biofilm biomass was quantified by microtiter assays, and gene expression was assessed by qRT-PCR. Overall, 78% of isolates were resistant to Tobramycin, with most MICs at 32 µg/ml (37.3%) and 1024 µg/ml (11.8%). All isolates formed biofilms, predominantly at moderate levels (63.08%). Tobramycin at ½ MIC significantly reduced biofilm biomass in both single-species (median OD₆₃₀: 0.128 ± 0.08 to 0.077 ± 0.09; p < 0.0001) and dual-species biofilms (0.158 ± 0.037 to 0.106 ± 0.041; p = 0.0005). toxA expression decreased markedly in dual-species biofilms compared to single-species (0.003 ± 0.0002 vs. 0.05 ± 0.009; p = 0.0003), while lipA expression showed a nonsignificant decline. In conclusion, sub-MIC Tobramycin reduces P. aeruginosa biofilm biomass and downregulates virulence genes, with more pronounced effects in dual-species biofilms. These findings highlight the potential impact of low-level antibiotic exposure on biofilm-associated pathogenicity and may inform optimized dosing strategies for chronic polymicrobial infections.