Establishment Of In Vitro Mixed-Biofilm Model Formed By Pseudomonas Aeruginosa And Streptococcus Mitis

Objective To explore the appropriate conditions for cultivating mixed-biofilm and set up a model for further study on the regulation of multispecies bacterial biofilm.Methods Pseudomonas aeruginosa (P.aeruginosa) and Streptococcus mitis (S.mitis) were cultured independently. The density of bacteria was measured by ultraviolet spectrophotometer and cell counting kit-8 under different conditions, including different pH, culture medium, and mix proportion, sequence and timing. The mono-and mixed-biofilm were observed via confocal laser scanning microscopy (CLSM), stained by STYO9/PI and imaged by scanning electron microscopy(SEM).Result Medium with pH7.5 was optimal for both the bacteria to grow (P<0.01). Both P.aeruginosa and S.mitis grew better in brain heart infusion broth medium than tryptone soy broth medium (P<0.01). When the ratio of P.aeruginosa and S.mitis was 1:3 and adding S.mitis after P.aeruginosa, the biofilm grew best among setting groups (P<0.01). Referring to timing, adding at the same time was optimal (P<0.01).The CLSM images showed that the S.mitis alone failed to form biofilm, while P.aeruginosa itself formed mature and compact biofilm. However, the mixed bacterial biofilm was thicker and had higher ratio of live bacteria. The thickness of biofilm formed by P.aeruginosa alone, S.mitis alone and both bacteria was (19.02±1.298) μm, (2.250±0.25) μm and (28.76±3.472) μm, respectively(P<0.05). The images of SEM confirmed that the biofilm structure of multi-species was more solid.Conclusion The multi-species bacterial biofilm model in vitro was successfully established. The model is convenient and has good reliability and reproducibility. It is preliminarily proved that the S.mitis facilitates formation of the biofilm.