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Comparative Study
. 2010 Jun;76(12):3948-58.
doi: 10.1128/AEM.00109-10. Epub 2010 Apr 23.

Oral bacteria as potential probiotics for the pharyngeal mucosa

Simone Guglielmetti  1 , Valentina Taverniti, Mario Minuzzo, Stefania Arioli, Milda Stuknyte, Matti Karp, Diego Mora
Affiliations
Comparative Study

Oral bacteria as potential probiotics for the pharyngeal mucosa

Simone Guglielmetti et al. Appl Environ Microbiol. 2010 Jun.

Abstract

The research described here was aimed at the selection of oral bacteria that displayed properties compatible with their potential use as probiotics for the pharyngeal mucosa. We included in the study 56 bacteria newly isolated from the pharynges of healthy donors, which were identified at the intraspecies level and characterized in vitro for their probiotic potential. The experiments led us to select two potential probiotic bacterial strains (Streptococcus salivarius RS1 and ST3) and to compare them with the prototype oral probiotic S. salivarius strain K12. All three strains efficiently bound to FaDu human epithelial pharyngeal cells and thereby antagonized Streptococcus pyogenes adhesion and growth. All were sensitive to a variety of antibiotics routinely used for the control of upper respiratory tract infections. Immunological in vitro testing on a FaDu layer revealed different responses to RS1, ST3, and K12. RS1 and ST3 modulated NF-kappaB activation and biased proinflammatory cytokines at baseline and after interleukin-1beta (IL-1beta) induction. In conclusion, we suggest that the selected commensal streptococci represent potential pharyngeal probiotic candidates. They could display a good degree of adaptation to the host and possess potential immunomodulatory and anti-inflammatory properties.

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Figures

FIG. 1.
FIG. 1.
Unweighted-pair group method using average linkages (UPGMA) dendrogram derived from similarity coefficients calculated by the Jaccard method (simple Jaccard [Sj] coefficients; shown on the scale at the bottom), showing the relationship among Streptococcus salivarius pharyngeal isolates, analyzed by BOX-PCR and RAPD analysis using primers M13, OPI02mod, OPI17mod, and PedAF. Samples with a similarity coefficient higher than 0.9 have been included in the same genotype. Selected bacterial isolates included in antagonism experiments are indicated in boldface.
FIG. 2.
FIG. 2.
Adhesion of bacterial strains to the FaDu epithelial cell layer according to their adhesion indexes (ADI; number of bacteria/100 FaDu cells). (A) +++, strong adhesion (ADI of >2,500); ++, good adhesion (ADI of between 2,500 and 500); +, weak adhesion (ADI of between 500 and 100); −, no adhesion (ADI of <100). (B) Adhesion to FaDu cell monolayers, as observed with Giemsa staining under a light microscope. Bars, 8 μm. One FaDu nucleus for each layer is indicated with the letter N.
FIG. 3.
FIG. 3.
Antagonistic exclusion activity of bacterial pharyngeal isolates against bioluminescent Streptococcus pyogenes C11LucFF on FaDu hypopharyngeal carcinoma cells (A) and HaCaT keratinocytes (B). Data reported as percent variation of light emission, which referred to the cell layer treated with only PBS buffer before incubation with S. pyogenes. Numerical results are given as arithmetic means ± standard deviations. Each sample was processed in triplicate in at least two independent experiments. Strains belonging to species S. salivarius are indicated in boldface. Statistically significant differences compared to strain K12 were calculated according to an unpaired Student's t test (**, P < 0.001; *, P < 0.05).
FIG. 4.
FIG. 4.
Antagonistic competition activity of bacterial pharyngeal isolates against bioluminescent Streptococcus pyogenes C11LucFF on FaDu hypopharyngeal carcinoma cells. Data reported as percent variation of light emission, which referred to the cell layer treated with only S. pyogenes cells. Numerical results are given as arithmetic means ± standard deviations. All samples resulted as being significantly different compared to the control (P < 0.001, according to an unpaired Student's t test).
FIG. 5.
FIG. 5.
Cytokine secretions that changed significantly after treatment of the FaDu layer with bacterial cells, as determined by using the Bio-Plex assay. The same results are also included in Table 2. FaDu layers were incubated overnight with bacterial cells (2 × 108 cells ml−1) without (A) and in the presence of (B) 2 ng ml−1 of IL-1β. The values are the means from two experiments conducted in duplicate. The vertical bars indicate standard deviations.
FIG. 6.
FIG. 6.
Effects of selected bacterial strains on FaDu cells stably transfected with an NF-κB/luciferase reporter vector, without (A) or with (B) stimulation with IL-1β (2 ng ml−1). Luciferase activity is expressed as percent change of relative luminescence units (RLU), assuming the control as 100%. Control, FaDu layers incubated without bacterial cells. The values are the means (± standard deviations) from at least three independent experiments conducted in duplicate. Asterisks indicate statistically significant differences compared to the control. MOI, multiplicity of infection (bacterial cells per FaDu cell).
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