The objective of this study was to assess the impact of subminimum inhibitory concentrations (sub-MICs) of chosen antimicrobial agents (Amikacin, imipenem, benzalkonium chloride, and chlorhexidine) and natural product (garlic) on biofilm formation ability, bacterial adherence and invasion. Susceptibility profiles of 50 non-repetitive Acinetobacter baumannii clinical isolates to eight antibiotics were investigated. MIC of various antibiotics, antiseptics and garlic were measured by the broth microdilution method. Quantification of biofilm formation was carried out using a microtiter plate assay. The ability of test compounds to affect the bacterial adherence and invasion was investigated using Type II pneumocyte cell line (A549) and the bacterial cells count was determined using flow cytometer. Screening for the presence of antiseptic resistant gene qacA/B was done using PCR. Ten isolates exhibited variable susceptibilities toward both amikacin and imipenem. The sub-MICs of benzalkonium chloride (BZC) markedly increased the biofilm formation. Additionally, amikacin, applied at sub minimal inhibitory concentrations, showed the highest induction in the bacterial adherence post treatment. Significant increase in bacterial invasion 3 h post treatment was detected upon applying BZC (616%) and imipenem (324%). BZC showed the greatest effect on the bacterial invasion. The highest impact on the bacterial invasion in case of 5 h post treatment was evident with the use of both BZC and Garlic. Results demonstrated that antibiotics, antiseptics and natural product at sub-MICs increased significantly the biofilm formation ability, bacterial adherence and invasion of A. baumanni clinical isolates, therefore careful consideration of sub-MIC effects is mandatory before their use.

Keywords: A. baumannii, Sub-MICs, biofilm formation, bacterial adherence and invasion, qac A/B