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

<b>Aim:</b><i>Acinetobacter baumannii</i> (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 <i>Acinetobacter</i> species from clinical isolates of the patient samples and to evaluate the efficacy of the amikacin-humic acid combination against it. <b>Methods:</b> The combination effect of Amikacin-Humic (AMK-HUM) acid against the highest biofilm-producing <i>A. baumannii</i> SLMK001 was studied via <i>in</i>-<i>vitro</i> (microscopic analysis) and <i>in</i>-<i>silico</i> (Network Pharmacology) analysis. <b>Results:</b> The amikacin-humic acid combination significantly inhibited both the biofilm formation and cell viability of <i>A. baumannii</i> 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 <i>A. baumannii</i>. <b>Conclusion:</b> The <i>in-vitro</i> results aligned with the <i>in-silico</i> findings, indicating that the AMK-HUM combination is a promising treatment that significantly activates the key proteins against <i>A. baumannii</i> biofilm formation and pathogenesis. <i>Acinetobacter baumannii</i> is a type of bacteria that forms protective sticky layers and causes an illness that is difficult to treat. This study looked at 15 <i>Acinetobacter baumannii</i> 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 <i>Acinetobacter baumannii</i> infections. The manuscript explores the biofilm-forming properties of <i>Acinetobacter spp.</i> clinical isolates, with a focus on <i>Acinetobacter baumannii</i>. 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 <i>A. baumannii</i> 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 <i>A. baumannii</i> pathogenesis. The findings suggest potential strategies for mitigating biofilm-associated <i>A. baumannii</i> infections and improving treatment outcomes.