The effect of bacterial growth conditions on biofilm formation

Escherichia coli is known as one of the predominant species among facultative anaerobic bacteria found in gastrointestinal tract and it is known as most common causative agent of urinary tract infections. In most cases, these infections are associated with biofilm formation. The aim of this study was to analyse biofilm formation by clinical Escherichia coli isolates under different growth conditions and determine how these conditions affect the formation of biofilm. A total of 177 clinical Escherichia coli isolates were obtained from biobank at RSU that were isolated from chronic kidney disease patients with UTI with several antimicrobial resistance patterns (including ESBL). Bacterial cultures were grown in LB medium for 24 h at 37oC, than transfered into 96-well plates and incubated in different conditions to obtain biofilm production. Grow conditions that were changed was temperature (39oC, 37oC, 32oC, 28oC), incubation time (24 h, 36 h, 48 h, 60 h, 72 h) and glucose concentration in media (2%, 3%, 4%). Detection of biofilm formation of the bacterial strains was carried out in 96-well microtiter plate using crystal violet assay. Results were obtained measuring optical density at 570 nm wavelength. All experiments were done in triplicate. 35% of tested samples showed biofilm formation under standard conditions (48h, 37oC). Addition of glucose to the medium reduced biofilm formation activity by ca. 20%. Culturing bacteria at reduced temperature (28oC) increased the number of biofilm producers by 30%, while cultivation at elevated temperature, reduced the number of biofilm producers. At 28oC 73% of the samples showed biofilm forming activity, while at 39oC only 9% were biofilm positives. The optimal cultivation time for biofilm formation was 48 hours. If the cultivation time was shortened or extended, the biofilm formation activity decreased. Bacterial culturing conditions affect their ability to form biofilm.