Analysis of Biofilm Formation and Small Colony Variant Development under Colistin Pressure in Different Clones of Acinetobacter spp

Acinetobacter baumannii is a Gram-negative bacterium frequently associated with healthcare-associated infections (HAIs) and represents a growing public health concern due to its resistance to multiple antimicrobials. A. baumannii is particularly notable for its ability to form biofilms, complex cellular structures adhered to surfaces, and for its clonal diversity, which complicates infection control. Additionally, the formation of small colony variants (SCVs) has been associated with increased virulence and resistance. This study aimed to analyze biofilm formation and SCV occurrence among A. baumannii clones, as well as to investigate the presence of the bap gene, which is associated with biofilm production. Biofilms were developed under static conditions in 6-well plates at 37°C for 24 hours. Results showed that colistin-resistant A. baumannii strains produced significantly higher biofilm cell densities (5.02 × 10¹¹ CFU/mL) compared to susceptible strains (4.9 × 10¹⁰ CFU/mL; p = 0.0173). Moreover, resistant strains exhibited a higher frequency of SCV generation (5.4 × 10⁻²) than susceptible isolates (1 × 10⁻²; p = 0.0226). Three distinct Acinetobacter baumannii and Acinetobacter calcoaceticus–A. baumannii (ACB) complex clones were identified using Random Amplified Polymorphic DNA (RAPD) analysis. Clone 2 produced the highest biofilm cell density under subinhibitory colistin concentration (1.29 × 10¹⁴ CFU/mL), which was significantly greater than that of Clone 1 (5.22 × 10¹¹ CFU/mL; p = 0.033) and Clone 3 (1.79 × 10¹¹ CFU/mL; p = 0.021). Additionally, the bap gene was detected in all isolates, suggesting its important role in adhesion and persistence. In conclusion, the ability of Acinetobacter baumannii to form dense biofilms, together with the emergence of small colony variants (SCVs), represents a major challenge for the effective treatment and control of healthcare-associated infections (HAIs). These adaptive traits contribute to increased persistence in clinical environments, enhanced tolerance to antimicrobial agents, and difficulties in eradication, highlighting the need for improved strategies to prevent and manage these infections.