Strain variability of the biofilm-forming ability of Salmonella enterica under various environmental conditions

The biofilm-forming ability of 60 Salmonella enterica strains was assessed at different pH values (3.8–7.0), NaCl concentrations (0.5–8.0%) and temperatures (4–37 °C). A total of 4320 biofilm formation tests (60 strains × 12 different environmental conditions × 6 replicates) were carried out. Biofilm formation was evaluated in tryptone soy broth after 48 h of incubation in polystyrene microtiter plates using crystal violet staining, and its quantification was based on the difference between the optical density measurements of the test and negative control (uninoculated) samples (ΔOD580 nm). The tested strains formed biofilms under a wide range of environmental conditions, while extensive strain variability was observed with the mean ΔOD580 nm values ranging from 0 to 2.388 depending on the strain and the condition evaluated. The strain variability of biofilm formation was affected by all three of the environmental parameters tested, and appeared to increase as the environmental conditions became less favorable for the organism. In addition, the increase in the strain variability caused by pH was found to be much greater than that caused by NaCl or temperature. For example, the coefficient of variation (CV = standard deviation / mean ∗ 100) of ΔOD580 nm among the tested strains at pH 7.0–0.5% NaCl-37 °C was 104%, while at pH 3.8–0.5% NaCl-37 °C, pH 7.0–8.0% NaCl-37 °C and pH 7.0–0.5% NaCl-8 °C was 351.5%, 204.1% and 175.6%, respectively. The optimum conditions for biofilm formation, providing the maximum ΔOD580 nm, varied significantly among the tested strains. Among the evaluated conditions, most of the S. enterica strains were clustered as forming their highest amount of biofilm at pH 5.5 (35 strains; 58.3%), at 0.5% NaCl (29 strains; 48.3%) and at 25 °C (32 strains; 53.3%). No relationships were observed between the biofilm-forming ability of the strains and their serotype or their growth kinetic behavior as this was evaluated in a previous study. The findings of this study provide useful information in advancing the current understanding of strain variability, as well as in strain selection for the evaluation of the efficacy of disinfection/sanitation procedures against biofilm formation.