Effect of amikacin-humic acid combination on Acinetobacter baumannii biofilm: an in vitro and in silico study

Aim:Acinetobacter baumannii (AB) is a clinically important bacterial pathogen responsible for nosocomial infections. The biofilm-forming capability of these pathogens reduces the antibiotic penetration and its efficacy, thereby complicating the treatment. The current work aims to isolate the most potent biofilm-forming Acinetobacter species from clinical isolates of the patient samples and to evaluate the efficacy of the amikacin-humic acid combination against it. Methods: The combination effect of Amikacin-Humic (AMK-HUM) acid against the highest biofilm-producing A. baumannii SLMK001 was studied via in-vitro (microscopic analysis) and in-silico (Network Pharmacology) analysis. Results: The amikacin-humic acid combination significantly inhibited both the biofilm formation and cell viability of A. baumannii SLMK001. The images observed via Scanning Electron Microscope (SEM) showed a significant decrease in the biofilm matrix. Confocal Laser Scanning Microscope (CLSM) confirmed a reduction of the Z value of its three-dimensional structure. Further, the Network Pharmacology approach supported these experimental findings by identifying the key targets of the amikacin-humic acid combination against the biofilm pathways of A. baumannii. Conclusion: The in-vitro results aligned with the in-silico findings, indicating that the AMK-HUM combination is a promising treatment that significantly activates the key proteins against A. baumannii biofilm formation and pathogenesis. Acinetobacter baumannii is a type of bacteria that forms protective sticky layers and causes an illness that is difficult to treat. This study looked at 15 Acinetobacter baumannii isolates from sick patients. We identified one that was particularly good at forming the protective layer and was difficult to kill. We tried two drugs in the study: one called Amikacin (AMK), and a natural substance called Humic Acid (HUM). Both were used alone and as a mixture. The AMK and HUM mixture worked well, stopping the formation of the protective sticky layer and killing the bug. This might be a potential treatment for difficult Acinetobacter baumannii infections. The manuscript explores the biofilm-forming properties of Acinetobacter spp. clinical isolates, with a focus on Acinetobacter baumannii. The study employs various assays, including slime synthesis, in-vitro adherence on glass surfaces, hydrophobicity index and antibiotic susceptibility testing, to assess the biofilm-forming capabilities of clinical isolates. Out of 15 isolates, strain AB-4 is identified as a strong biofilm former and further analyzed using genotypic identification (16s rRNA sequencing). Antibiogram analysis using Vitek-2 provides information on the antibiotic susceptibility profile of A. baumannii SLMK001. Amikacin is identified as a potential drug of choice due to its sensitivity. The study explores the antimicrobial activity of amikacin, humic acid (HUM) and their combination (AMK-HUM). Sub-MIC concentrations are used to evaluate their impact on biofilm biomass inhibition and cell viability. AMK-HUM showed significant effectiveness in inhibiting biofilm formation and reducing cell viability compared with individual treatments. The microscopic analysis demonstrates the disruptive effect of AMK-HUM on preformed biofilms, supported by reduced biofilm thickness and altered 3D structure observed through CLSM and SEM. The network pharmacology results of the AMK-HUM combination revealed a favorable interaction with key proteins that play a crucial role in A. baumannii pathogenesis. The findings suggest potential strategies for mitigating biofilm-associated A. baumannii infections and improving treatment outcomes.