Results
In vitro effect of CFS on HT-29 cells
The use of CFS generated by L. fermentum 5.2 resulted in a decrease in cell viability at concentrations of 40 and 20 mg/mL, with a viability of 8.61 ± 0.44% and 11.32 ± 7.52%, respectively, when compared to the control group. Concentrations from 10 to 0.625 mg/mL promoted cell proliferation. The concentrations of 10, 5, 2.5, 1.25, and 0.625 mg/mL resulted in viability of 324.7 ± 2.54%, 364.7 ± 4.39%, 362.7 ± 2.93%, 360.1 ± 0.73%, and 356.1 ± 4.76% compared to the control group, respectively (Figure 1).
The use of CFS generated by L. plantarum 6.2 promoted a decrease in viability at concentrations of 40, 20, 10 and 5 mg/mL, with viability of 6.41 ± 0.34%, 6.57 ± 0.53%, 7.25 ± 0.74% and 31.57 ± 16.54%, when compared to the control group, respectively, while concentrations of 2.5, 1.25 and 0.624 mg/mL did not significantly alter cell viability, with viability of 103.70 ± 11.50%, 89.85 ± 9.55%, and 94.15 ± 7.98% in relation to the control group, respectively (Figure 2).
The use of CFS generated by L. plantarum 7.1 resulted in a decrease in cell viability at concentrations of 40 and 20 mg/mL, with viability of 6.50 ± 0.37% and 10.18 ± 5.47% when compared to the group control, respectively, while concentrations of 10, 5, 2.5, 1.25 and 0.624 mg/mL did not significantly alter cell viability, maintaining the viability of 103.3 ± 10.68%, 101.7 ± 6.85 %, 92.9 ± 18.40%, 100.2 ± 5.72% and 103.9 ± 3.08% compared to the control group, respectively (Figure 3).
Autoaggregation and coaggregation of BAL and E. coli F4
Over the 5 hours of analysis, the bacterium L. fermentum 5.2 showed, autoaggregation of 2.95 ± 2.16%, 11.62 ± 3.35%, 15.89 ± 1.54%, 17.87 ± 1.71% and 21.42 ± 1.96% (Figure 4A); the bacteriumL. plantarum 6.2 showed autoaggregation of 12.72 ± 0.28%, 11.34 ± 1.36%, 16.50 ± 0.86%, 24.93 ± 1.78% and 30.85 ± 1.76% (Figure 4B); the bacterium L. plantarum 7.1 showed autoaggregation of 4.06 ± 1.21%, 5.52 ± 0.84%, 11.16 ± 0.66%, 15.33 ± 1.01% and 21.65 ± 0.37% (Figure 4C); and E. coli F4 bacteria showed autoaggregation of 10.89 ± 0.08%, 15.39 ± 1.39%, 33.54 ± 2.10%, 41.17 ± 1.25% and 45.06 ± 0.27% (Figure 4D).
When cultivated in association, the culture of E. coli F4 andL. fermentum 5.2 showed, over the 5 hours, coaggregation of 0%, 5.65 ± 1.26%, 16.03 ± 0.39, 24.02 ± 0.07% and 26.01 ± 1.11% (Figure 5A); the association of E. coli F4 and L. plantarum 6.2 showed coaggregation of 1.69 ± 1.35%, 21.53 ± 1.08%, 26.30 ± 0.62%, 31.32 ± 1.01% and 39.98 ± 1.09% (Figure 5B); and the association between E. coli F4 and L. plantarum 7.1 showed coaggregation of 0.12 ± 0.20%, 10.44 ± 0.58%, 21.71 ± 0.71%, 30.37 ± 0.22%, and 34.64 ± 0.82% (Figure 5C). The bacterium L. plantarum 6.2 showed faster coaggregation capacity compared to other bacteria, maintaining this high coaggregative capacity over the analysis times (Figure 6).
Inhibition of E. coli F4 in MH agar
The evaluated CFS were not able to inhibit the in vitro growth ofE. coli F4 in MH agar regardless of the concentration of supernatant used (data not shown).
Degradation of biofilms produced by E. coli F4
Despite the cytotoxic effect in HT-29 culture, a concentration of 40 mg/mL of CFS was used in this assay to optimize the conditions found inin vivo tests and standardize the assay for future determination of the MIC. Among the evaluated CFS, only the CFS generated by L. plantarum 7.1 resulted in a significant reduction in E. coli F4 biofilm. The BFI of the CFS generated by L. fermentum 5.2,L. plantarum 6.2 and L. plantarum 7.1 were 48.64 ± 1.01, 50.38 ± 6.12 and 34.26 ± 4.50, respectively; the BFI of the positive control, assessed by biofilm formation of E. coli F4 without the presence of CFS, was 55.0 ± 1.95 (Figure 7).