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).