Investigation of the Effect of Antibiotic Resistance Selected via Disk Diffusion and Cross-Resistance in Klebsiella pneumoniae on Biofilm Formation

Objective: Antibiotic resistance has become a global health issue, making it difficult to manage treatable infections. This study aimed to investigate the development of resistance in Klebsiella pneumoniae exposure to antibiotic diffusion gradients generated by disk diffusion. The emergence of cross-resistance and the impact of this resistance on biofilm formation are further evaluated.Materials and Methods: K. pneumoniae was exposed to antibiotic diffusion gradients using Oxoid disks (36 µg) to select resistant mutants of piperacillin-tazobactam (TZP), leading to the development of a resistant strain (P₃). Comparative analyses of antibiotic susceptibility (TZP, amoxicillin/clavulanic acid [AMC], cefixime [CFM], and trimethoprim-sulfamethoxazole [SXT]) and biofilm formation were conducted between the parental (P₀) and resistant (P₃) strains. Biofilm production was evaluated under varying temperatures (37°C and 45°C) and glucose concentrations (0%-2.5%).Results: Resistance to TZP increased significantly in P₃, with moderate cross-resistance observed against AMC and SXT (p<0.05). Although the strain was susceptible to most antibiotics at baseline (P0), it exhibited primary resistance to CFM in both P0 and P3 stages. Biofilm formation was higher at 37°C than at 45°C under the tested conditions. Increasing the glucose concentration from 0 % to 2.5% reduced biofilm production by nearly 40%. However, no direct correlation was found between antibiotic resistance development and biofilm-forming capacity.Conclusion: Selection through antibiotic gradients can induce resistance and cross-resistance in K. pneumoniae, but does not necessarily enhance biofilm production. Environmental parameters, particularly temperature and nutrient availability, and particularly glucose concentration, are more influential in modulating biofilm formation. Future molecular characterisation of β-lactamase and biofilm-associated genes are essential for elucidating the underlying mechanisms.