Fig. 4
Changes in biofilm characteristics of S. aureus strains associated with mastitis after treatment with lactic acid bacteria strains. This figure illustrates the changes in three key biofilm parameters: biovolume (µm³) (left), maximum thickness (µm) (middle), and cellular viability (%) (right) in S. aureus SA4 a and SA55 b biofilms after treatment with active cultures and CFS of L. fermentum I7, L. reuteri 7SNG3-30 and L. salivarius 22SNG3-30. Biovolume, maximum thickness, and cellular viability data were analyzed using IMARIS software. The biovolume and maximum thickness were assessed through the processing of confocal laser scanning microscopy (CLSM) images, while cellular viability was calculated based on the segmentation of live and dead cell populations, with the MeasurementPro module of IMARIS used for quantification (assisted by the Filmtracer™ LIVE/DEAD™ Biofilm Viability Kit). The boxplots display the biovolume and maximum thickness, with the center line representing the median, box limits indicating the upper and lower quartiles, whiskers showing extreme values, and circles denoting outliers. Comparisons between biovolume and maximum thickness for treated and untreated 24-hour control biofilms were performed using one-way ANOVA. Statistical significance is marked as *(p < 0.05), **(p < 0.01), and ***(p < 0.001). For each condition, the following number of samples were analyzed: SA4: n = 4 and SA55: n = 4 for S. aureus biofilm 24 h; SA4: n = 6 and SA55: n = 5 for treatment with L. fermentum I7 active culture; SA4: n = 4 and SA55: n = 4 for treatment with L. fermentum I7 CFS; SA4: n = 7 and SA55: n = 3 for treatment with L. reuteri 7SNG3-30 active culture; SA4: n = 5 and SA55: n = 4 for treatment with L. reuteri CFS; SA4: n = 6 and SA55: n = 3 for treatment with L. salivarius 22SNG3-30 active culture; and, SA4: n = 4 and SA55: n = 5 for treatment with L. salivarius 22SNG3-30 CFS.
