Combined effects of low incubation temperature, minimal growth medium, and low hydrodynamics optimize acinetobacter baumannii biofilm formation

Abstract

Background: Biofilm formation is an important virulence factor expressed by Acinetobacter baumannii. It shields and protects microbial cells from host immune responses, antibiotics, and other anti-infectives. Its effects on Acinetobacter baumannii infection treatments notwithstanding, important environmental factors that influence its formation have not been fully investigated. Methods: Biofilm formation was assessed using the qualitative modified Congo red assay and quantitative microtiter plate methods. The combined effect of temperature, medium and shear force was determined by measuring adherence (OD570 nm) in microtiter plate after incubation at 26°C, 30°C, and 37°C when biofilm-grown cells were cultured in the presence of minimal nutrient medium (EAOB) and nutrient-rich medium (TSB) without or with agitation at 50 rpm. Antibiotics susceptibility of meropenem, imipenem, and ciprofloxacin were tested with Kirby-Bauer disc method. P<0.05 was considered statistically significant in all the tests. Results: A noticeable variation in adherence was observed among the isolates cultured with both media. Biofilm forming capacity of the isolates range from 0.09–0.33. The majority of the isolates had their relative biofilm-forming capacity significantly (p<0.05) higher than the positive control, Acinetobacter baumannii ATCC 19606. The biofilm biomass during growth in nutrient-rich medium (TSB) without shaking was significantly different (p<0.05; Tukey’s test) among the three temperatures tested compared with when it was cultured in EAOB without shaking. A positive correlation was observed between biofilm formation and resistance to imipenem (r=0.2889; p=0.05). There was a statistically significant difference among the median of the three source groups (p<0.05) compared with the median between the source groups. Conclusion: This observation extended further the view that A. baumannii biofilm formation is enhanced when nutrient-poor medium is used at room temperature (26°C) with or without agitation compared to growth at 37°C.