Antimicrobial and Antibiofilm Activities of Weissella cibaria against Pathogens of Upper Respiratory Tract Infections

Abstract

Recently discovered preventive effects of probiotics on oral health have attracted interest to their use for the prevention and treatment of various diseases. This study aimed to evaluate the antimicrobial and antibiofilm properties of Weissella cibaria against Streptococcus pyogenes, Staphylococcus aureus, S. pneumoniae, and Moraxella catarrhalis, the major pathogens of upper respiratory tract infections (URTIs). The antimicrobial activities of W. cibaria were compared with those of other oral probiotics using a competitive inhibition assay and the determination of the minimum inhibitory concentrations (MICs). In addition, a time-kill assay, spectrophotometry, and confocal laser scanning microscopy were used to confirm the antimicrobial and antibiofilm abilities of W. cibaria CMU (oraCMU) and CMS1 (oraCMS1). Both live cells and cell-free supernatants of all tested probiotics, except Streptococcus salivarius, showed excellent antimicrobial activities. All target pathogens were killed within 4 to 24 h at twice the MIC of oraCMU and oraCMS1, which showed the highest antimicrobial activities against M. catarrhalis. The antimicrobial substances that affected different target pathogens were different. Both oraCMU and oraCMS1 showed excellent abilities to inhibit biofilm formation and remove preformed biofilms. Our results suggest that the W. cibaria probiotics offer new possibilities for the prevention and treatment of bacterial URTIs.

Keywords: Weissella cibaria; antibiofilm; antimicrobial; probiotic; upper respiratory tract.

Figure 1

Competitive indexes (CIs) in coculture of oral care probiotics and major pathogens of URTIs. (a) Streptococcus pyogenes, (b) Staphylococcus aureus, (c) Streptococcus pneumoniae, and (d) Moraxella catarrhalis. Different letters (a and b) indicate significant differences at p < 0.05. CMU, Weissella cibaria CMU; CMS1, W. cibaria CMS1; L. reu, Limosilactobacillus reuteri; L. sal, Ligilactobacillus salivarius; S. sal, Streptococcus salivarius.

Figure 2

Time-kill curves for cell-free supernatants of Weissella cibaria strains against (a) Streptococcus pyogenes, (b) Staphylococcus aureus, (c) S. pneumoniae, and (d) Moraxella catarrhalis, at different minimum inhibitory concentration (MIC) increments: ▲, 2 × MIC; ■, 1 × MIC; ●, untreated control. Solid line, W. cibaria CMU (oraCMU); broken line, W. cibaria CMS1 (oraCMS1). Different letters (A–F) indicate significant differences among oraCMU treatment groups (p < 0.05). Different letters (a–f) indicate significant differences among oraCMS1 treatment groups (p < 0.05). OD₆₀₀, absorbance at 600 nm; CFU, colony-forming units.

Figure 3

Dose-dependent effects of organic acids, H₂O₂, and bacteriocin-like compounds (BLCs) in cell-free supernatants (CFSs) of Weissella cibaria strains against (a) Streptococcus pyogenes, (b) Staphylococcus aureus, (c) S. pneumoniae, and (d) Moraxella catarrhalis. ●, CFS; ▲, organic acids; ■, H₂O₂; X, BLCs. Solid line, W. cibaria CMU (oraCMU); broken line, W. cibaria CMS1 (oraCMS1). Different letters (A–C) indicate significant differences among oraCMU treatment groups (p < 0.05). Different letters (a–c) indicate significant differences among oraCMS1 treatment groups (p < 0.05). OD₆₀₀, absorbance at 600 nm.

Figure 4

Inhibitory effects of Weissella cibaria strains on biofilm formation by (a) Streptococcus pyogenes, (b) Staphylococcus aureus, (c) S. pneumoniae, and (d) Moraxella catarrhalis. Black bar, 24 h; gray bar, 48 h. CMU, W. cibaria CMU; CMS1, W. cibaria CMS1; CMU sup, cell-free supernatant (CFS) of W. cibaria CMU; CMS1 sup, CFS of W. cibaria CMS1. Different letters (a–e) indicate significant differences at p < 0.05.

Figure 5

Removal effects of Weissella cibaria strains on preformed biofilms of (a) Streptococcus pyogenes, (b) Staphylococcus aureus, (c) S. pneumoniae, and (d) Moraxella catarrhalis. Black bar, 24 h; gray bar, 48 h. CMU, W. cibaria CMU; CMS1, W. cibaria CMS1; CMU sup, cell-free supernatant (CFS) of W. cibaria CMU; CMS1 sup, CFS of W. cibaria CMS1. Different letters (a–e) indicate significant differences at p < 0.05.

Figure 5

Removal effects of Weissella cibaria strains on preformed biofilms of (a) Streptococcus pyogenes, (b) Staphylococcus aureus, (c) S. pneumoniae, and (d) Moraxella catarrhalis. Black bar, 24 h; gray bar, 48 h. CMU, W. cibaria CMU; CMS1, W. cibaria CMS1; CMU sup, cell-free supernatant (CFS) of W. cibaria CMU; CMS1 sup, CFS of W. cibaria CMS1. Different letters (a–e) indicate significant differences at p < 0.05.

Figure 6

Results of confocal scanning microscopy analysis. (a) Overall biofilm volumes; (b) live/dead cell ratios. Black bar, live cells; gray bar, dead cells. CMU, Weissella cibaria CMU; CMS1, W. cibaria CMS1; CMU sup, cell-free supernatant (CFS) of W. cibaria CMU; CMS1 sup, CFS of W. cibaria CMS1.

Figure 7

Confocal laser scanning microscopy images of Streptococcus pyogenes biofilms. (a) Untreated control; (b) treatment with live cells of Weissella cibaria CMU (oraCMU); (c) treatment with live cells of W. cibaria CMS1 (oraCMS1); (d) treatment with a cell-free supernatant (CFS) of oraCMU; (e) treatment with a CFS of oraCMS1. Green, live cells; red, dead cells (magnification, 40×).