Antimicrobial effects of commensal oral species are regulated by environmental factors

Abstract

Objectives

The objectives of this study are to identify oral commensal species which can inhibit the growth of the main periodontopathogens, to determine the antimicrobial substances involved in these inhibitory activities and to evaluate the influence of environmental factors on the magnitude of these inhibitions.

Methods

The spotting technique was used to quantify the capacity of 13 commensal species to inhibit the growth of Aggregatibacter actinomycetemcomitans , Porphyromonas gingivalis and Prevotella intermedia . By altering experimental conditions (distance between spots and size of spots and concentration of commensal and pathogen) as well as environmental factors (inoculation sequence, oxygen and nutrition availability) the influence of these factors was evaluated. Additionally, the mechanism of inhibition was elucidated by performing inhibition experiments in the presence of peroxidase, trypsin and pepsin and by evaluating acid production.

Results

Streptococcus sanguinis , Streptococcus cristatus , Streptococcus gordonii , Streptococcus parasanguinis , Streptococcus mitis and Streptococcus oralis significantly inhibit the growth of all pathogens. The volume of the spots and concentration of the commensal have a significant positive correlation with the amount of inhibition whereas distance between the spots and concentration of the pathogen reduced the amount of inhibition. Inhibition is only observed when the commensal species are inoculated 24 h before the pathogen and is more pronounced under aerobic conditions. Hydrogen peroxide production by the commensal is the main mechanism of inhibition.

Conclusion

Bacterial antagonism is species specific and depending on experimental as well as environmental conditions. Blocking hydrogen peroxide production neutralizes the inhibitory effect.

Clinical significance

Identifying beneficial oral bacteria and understanding how they inhibit pathogens might help to unravel the mechanisms behind dysbiotic oral diseases. In this context, this study points towards an important role for hydrogen peroxide. The latter might lead in the future to novel preventive strategies for oral health based on improving the antimicrobial properties of commensal oral bacteria.

Introduction

The oral cavity is a complex microbial system which harbors more than 700 different bacterial species . These complex bacterial communities form biofilms which reside on the soft and hard tissues and are covered with saliva and crevicular fluid . Oral biofilms are often called dental plaque . They are typically associated with oral diseases such as caries, gingivitis or periodontitis. However, it has been suggested that they also can have a beneficial role by protecting the host against infectious diseases such as tooth decay and periodontitis . The reason for this apparent duality might reside in bacterial interactions that influence the composition and the pathogenicity of the biofilm community . In healthy persons, the indigenous microbiota plays an important role in limiting the growth and the colonization of pathogenic bacteria . Streptococci are a major part of this commensal microbiota and have an important function in microbial colonization of the oral cavity . They influence oral microbial ecology development by their ability to adhere to soft and hard tissues , their capacity to metabolize a wide range of carbohydrates and the production of antimicrobial substances , which all can influence biofilm formation and oral pathogens.

The prevalence of some species, primarily streptococci, has been used as an indicator for oral health . Several studies have attempted to characterize the interaction between oral commensal bacteria and pathogens . For instance, Streptococcus sanguinis and Streptococcus gordonii can inhibit the growth of cariogenic strains . It is also known that streptococcus species can produce different antimicrobial substances to inhibit the growth of pathogenic oral bacteria. For example, S. sanguinis produces H 2 O 2 that can kill Aggregatibacter actinomycetemcomitans . Some streptococus species such as Streptococcus salivarius K12 can synthesize bacteriocins that are specific proteinaceous antibiotics to repress the growth of oral pathogens . Additionally streptococci can produce significant amounts of acids which decrease the pH and by this prevent the growth of pathogens .

These inhibitory properties have primarily been described for a limited number of bacterial species and shown primarily on cariogenic species. Additionally, the main mechanism by which oral pathogens are inhibited (H 2 O 2 , acids or bacteriocins) is currently not well known. Furthermore, these inhibitory effects are most likely regulated by environmental conditions such as the timing of the colonization, depletion of the nutrients, absence of oxygen and pH. However, the influence of these factors on the inhibitory properties of commensal oral bacteria is unknown.

The objectives of this study were to identify oral beneficial bacterial species that can antagonize the growth of the main periodontopathogens, to determine the production of antimicrobial substances which are involved in these inhibitory activities and to evaluate the influence of environmental factors on the magnitude of these inhibitions.

Material and methods

Bacterial strains and media

Oral commensal bacterial species ( Streptococcus sanguinis LMG14657, Streptococcus cristatus ATCC 49999, Streptococcus gordonii ATCC 49818, Streptococcus parasanguinis DSM 6778, Streptococcus mitis DSM 12643, Streptococcus oralis DSM 20627, Streptococcus intermedius DSM 20573, Actinomyces naeslundii ATCC 51655, Actinomyces viscosus DSM 43327 , Actinomyces massiliensis DSM 23047 , Capnocytophaga sputigena DSM 7273 , Veillonella parvula DSM 2007 , Gemella morbillorum DSM 20572 and Granulicatella adiacens DSM 9848) and periodontopathogens ( Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 43718 and Prevotella intermedia ATCC 25611) were maintained on blood agar (Blood agar Base No.2, Oxoid, Basingstoke, UK) supplemented with hemin (5 mg/ml) (Sigma Chemical Co., St. Louis, MO), menadione (1 mg/ml) (Calbiochem-Novabiochem, La Jolla, CA) and 5% sterile horse blood (Defibrinated horse blood, E&O Laboratories Limited, Bonnybridge, Scotland). Liquid cultures were made in Brain Hearth Infusion (BHI) broth (Difco, Sparks, MD). The agar plate inhibition experiments were performed on Brain Hearth Infusion 2 agar (BHI-2) . The bacteria were cultured under aerobic (5% CO 2 ) or anaerobic (80% N 2 , 10% H 2 and 10% CO 2 ) conditions as described in the experiments. Cell densities were determined via spectrophotometry (OD600, GeneQuant 100 Spectrophotometer, GE Healthcare, Buckinghamshire, UK).

Antagonistic experiments on agar plates

The spotting technique was used to detect and quantify the amount of inhibition between commensal and pathogenic bacteria. This technique is a competitive assay which consists of inoculating two spots containing different bacterial species next to each other on an agar plate. Afterwards, the capacity of one to inhibit the growth of the other could be evaluated. Unless otherwise stated, each spot contained 7 μl of an 10 9 CFU/ml overnight bacterial culture. After 24 or 48 h the area of inhibition was inspected and a calibrated (ruler) standardized (distance between agar plate and photo camera) photograph was taken from the agar plate. The amount of inhibition was determined by measuring (in mm) the distance between the border of the inhibitor spot to the border of the inhibited spot ( Fig. 1 ) using ImageJ (Imaging processing and Analysis in Java). Each experiment was repeated on 3 different days.

Fig. 1
Amount of inhibition (AI) was determined by measuring the distance between the border of the inhibitor spot to the border of the inhibited spot using ImageJ.

Screening for inhibitory species

Agar plates were spotted with an overnight culture of one of the commensal species, as described above. After 24 h of aerobic incubation (anaerobic for G. morbillorum , G. adiacens , C. sputigena, V. parvula ) the agar plates were spotted again with an overnight culture of a pathogenic species, next to the spot containing the commensal species ( Fig. 1 ). The agar plates were incubated for 24 ( A. actinomycetemcomitans , P. intermedia ) or 48 ( P. gingivalis ) hours under anaerobic ( P. intermedia, P. gingivalis ) or aerobic ( A. actinomycetemcomitans ) conditions after which they were analysed as described above.

Characterization of the inhibition influencing factors

To evaluate the effect of spot size, different volumes (7, 10, 15 and 20 μl) of inhibitor ( S. oralis , S. gordonii , S. cristatus ) were spotted on the agar plates. After 24 h of aerobic incubation, the pathogens ( A. actinomycetemcomitans P. intermedia, P. gingivalis ) (7 μl) were spotted next to the inhibitor spots.

To evaluate the effect of spotting distance, 7 μl of inhibitor ( S. oralis , S. gordonii , S. cristatus ) was spotted on the agar plate and after overnight aerobic incubation, 7 μl of pathogen ( A. actinomycetemcomitans P. intermedia, P. gingivalis ) was spotted at different distances (2, 4, 6, 8 and 10 mm) from the edges of the inhibitor spot.

The influence of cell density was investigated by using different inoculum concentrations of inhibitor and pathogens. In one set of experiments, the concentration of an overnight culture of was adjusted to 1 × 10 9 CFU/ml and decimal dilutions (10 9 –10 5 CFU/ml, 7 μl) were spotted on the agar plates. After overnight aerobic incubation, 7 μl of pathogen at a concentration of 1 × 10 9 CFU/ml was spotted next to the inhibitor spots. In a second set of experiments, 7 μl of a 1 × 10 9 CFU/ml overnight culture of inhibitor was spotted on the agar plates. After 24 h of aerobic incubation, 7 μl of pathogen and decimal dilutions (10 9 –10 5 CFU/ml) thereof were spotted next to the inhibitor spots.

Unless otherwise mentioned, all these experiments were executed and analyzed as described above. Each experiment was repeated on 3 different days.

Characterization of the antagonistic action

The antagonistic effects were evaluated in perspective of colonization sequence, oxygen availability and nutrition availability using the spotting technique as reference technique.

The impact of the colonization sequence of inhibitor species ( S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis ) and the pathogens was evaluated by: (1) Spotting the inhibitors 24 h before the pathogen, as described above. (2) Spotting the inhibitor and the pathogen at the same time and incubating them anaerobically for 24 h. (3) Spotting the pathogen first on the agar plate and incubating the plate for 24 h under anaerobic conditions. Afterwards the inhibitor was spotted next to the pathogen spot and the plates were incubated for 24 h under anaerobic conditions.

The impact of oxygen availability on the magnitude of the inhibition was evaluated by spotting the different inhibitor strains and incubating them for 24 h under aerobic (5% CO 2 ) or anaerobic (80% N 2 ,10% CO 2 and 10% H 2 ) conditions. After 24 h, the pathogenic strains were spotted next to the inhibitors as described above.

The influence of nutrient depletion on the antagonistic activity of the inhibitors was investigated by performing the above described antagonistic experiments on full strength and diluted (1 in 2, 1 in 5, 1 in 10, 1 in 20 and 1 in 50) BHI-2 agar medium using Phosphate Buffered Saline (PBS).

Unless otherwise mentioned, all experiments were executed and analyzed as described above. Each experiment was repeated on 3 different days.

Identification of antimicrobial substances

Unless otherwise mentioned, all experiments were executed and analyzed as described above. Each experiment was repeated on 3 different days.

H 2 O 2 production

The contribution of H 2 O 2 production to the inhibitory activity was evaluated on agar plates. Inhibitor strains ( S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis ) were spotted on the agar plates. After 24 h of aerobic growth, 7 μl of horseradish peroxidase (40 μg/μl, Sigma–Aldrich) was spotted next to the inhibitor spot, at the left side. After 5 min of drying, 7 μl of pathogen was spotted at the left side of the inhibitor spot (overlaying the horseradish peroxidase spot) and 7 μl of pathogen was spotted at the right side of the inhibitor spot. Further incubation and analysis was performed as described above. To determine the amount of hydrogen peroxide production by the inhibitor strains, the production of the H 2 O 2 in liquid cultures was determined using an enzymatic kit (Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit, Life technologies). Briefly, overnight cultures of the inhibitors were centrifuged (7970 × g , 10 min) and the supernatant was discarded. The obtained pellets were resuspended in BHI-2 broth and the concentration was adjusted to 1 × 10 9 CFU/ml. 1 ml of this suspension was added to 9 ml of BHI-2 broth and incubated once under aerobic and once under anaerobic conditions. After 24 h, the samples were centrifuged as described above and the supernatant was subsequently filter sterilized (0.8 and 0.2 μm Acrodisc syringe filter, Pall corporation, Life Sciences). The amount of H 2 O 2 in the samples was determined by the Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit, according to the manufacturer’s instructions.

Acid production

Bromocresol purple indicator (0.12 g/l) (Merck) was added to the BHI-2 agar plates as a pH indicator. Inhibitor strains ( S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis ) were spotted on the bromocresol containing BHI-2 agar plates and incubated during 24 h under aerobic or anaerobic conditions. The relative amount of acid production was evaluated by the change of color (from purple (pH 7) to different range of yellowish colors (pH between 5–6)) underneath the inhibitor spots and in the surrounding areas. Standardized pictures were taken.

Bacteriocin production

The contribution of bacteriocins on the inhibitory activity was evaluated on agar plates. Inhibitor strains were spotted on the agar plates. After 24 h of aerobic or anaerobic incubation, 7 μl of protease was spotted next to the inhibitor spot, at the left side. After 5 min of drying, 7 μl of pathogen was spotted at the left side of the inhibitor spot (overlaying the protease spot) and 7 μl of pathogen was spotted at the right side of the inhibitor spot. Further incubation and analysis was performed as described above. As proteases solutions, 0,05% Trypsin-EDTA (Gibco by Life Technologies, Paisley, UK) or 64 μg/μl Pepsin from porcine gastric mucosa (Sigma–Aldrich, USA) in PBS were used.

Statistical analysis

A four-factor Analysis of Variance (ANOVA) model was applied with pathogen, oxygen condition, inhibitor and dilution as fixed factors. Comparisons between the levels of each factor were carried out for every combination of the other factors. Corrections for simultaneous hypothesis testing were performed according to Sidak. Residual plots and normal quantile plots of residuals were used to validate the basic assumptions of the Anova model.

The relation between concentration of pathogens or inhibitors, distance between pathogens and inhibitors or volume of inhibitors or pathogens and inhibition were modeled via linear mixed models with concentration, distance or volume as continuous fixed variables and experiment as random factor. The sign of the regression coefficient, if significant, pointed to a positive or negative relation between concentration and inhibition. Residual values were tested for normality by means of a normal quantile plot.

Additionally, a two-way Anova model with inhibitor and oxygen condition as fixed factors was built. Comparisons between the levels of the fixed factors were made per level of the other factor and corrections for simultaneous hypothesis testing were performed according to Sidak. Residual plots and normal quantile plots of residuals were used to validate the basic assumptions of the Anova model.

Material and methods

Bacterial strains and media

Oral commensal bacterial species ( Streptococcus sanguinis LMG14657, Streptococcus cristatus ATCC 49999, Streptococcus gordonii ATCC 49818, Streptococcus parasanguinis DSM 6778, Streptococcus mitis DSM 12643, Streptococcus oralis DSM 20627, Streptococcus intermedius DSM 20573, Actinomyces naeslundii ATCC 51655, Actinomyces viscosus DSM 43327 , Actinomyces massiliensis DSM 23047 , Capnocytophaga sputigena DSM 7273 , Veillonella parvula DSM 2007 , Gemella morbillorum DSM 20572 and Granulicatella adiacens DSM 9848) and periodontopathogens ( Porphyromonas gingivalis ATCC 33277, Aggregatibacter actinomycetemcomitans ATCC 43718 and Prevotella intermedia ATCC 25611) were maintained on blood agar (Blood agar Base No.2, Oxoid, Basingstoke, UK) supplemented with hemin (5 mg/ml) (Sigma Chemical Co., St. Louis, MO), menadione (1 mg/ml) (Calbiochem-Novabiochem, La Jolla, CA) and 5% sterile horse blood (Defibrinated horse blood, E&O Laboratories Limited, Bonnybridge, Scotland). Liquid cultures were made in Brain Hearth Infusion (BHI) broth (Difco, Sparks, MD). The agar plate inhibition experiments were performed on Brain Hearth Infusion 2 agar (BHI-2) . The bacteria were cultured under aerobic (5% CO 2 ) or anaerobic (80% N 2 , 10% H 2 and 10% CO 2 ) conditions as described in the experiments. Cell densities were determined via spectrophotometry (OD600, GeneQuant 100 Spectrophotometer, GE Healthcare, Buckinghamshire, UK).

Antagonistic experiments on agar plates

The spotting technique was used to detect and quantify the amount of inhibition between commensal and pathogenic bacteria. This technique is a competitive assay which consists of inoculating two spots containing different bacterial species next to each other on an agar plate. Afterwards, the capacity of one to inhibit the growth of the other could be evaluated. Unless otherwise stated, each spot contained 7 μl of an 10 9 CFU/ml overnight bacterial culture. After 24 or 48 h the area of inhibition was inspected and a calibrated (ruler) standardized (distance between agar plate and photo camera) photograph was taken from the agar plate. The amount of inhibition was determined by measuring (in mm) the distance between the border of the inhibitor spot to the border of the inhibited spot ( Fig. 1 ) using ImageJ (Imaging processing and Analysis in Java). Each experiment was repeated on 3 different days.

Fig. 1
Amount of inhibition (AI) was determined by measuring the distance between the border of the inhibitor spot to the border of the inhibited spot using ImageJ.

Screening for inhibitory species

Agar plates were spotted with an overnight culture of one of the commensal species, as described above. After 24 h of aerobic incubation (anaerobic for G. morbillorum , G. adiacens , C. sputigena, V. parvula ) the agar plates were spotted again with an overnight culture of a pathogenic species, next to the spot containing the commensal species ( Fig. 1 ). The agar plates were incubated for 24 ( A. actinomycetemcomitans , P. intermedia ) or 48 ( P. gingivalis ) hours under anaerobic ( P. intermedia, P. gingivalis ) or aerobic ( A. actinomycetemcomitans ) conditions after which they were analysed as described above.

Characterization of the inhibition influencing factors

To evaluate the effect of spot size, different volumes (7, 10, 15 and 20 μl) of inhibitor ( S. oralis , S. gordonii , S. cristatus ) were spotted on the agar plates. After 24 h of aerobic incubation, the pathogens ( A. actinomycetemcomitans P. intermedia, P. gingivalis ) (7 μl) were spotted next to the inhibitor spots.

To evaluate the effect of spotting distance, 7 μl of inhibitor ( S. oralis , S. gordonii , S. cristatus ) was spotted on the agar plate and after overnight aerobic incubation, 7 μl of pathogen ( A. actinomycetemcomitans P. intermedia, P. gingivalis ) was spotted at different distances (2, 4, 6, 8 and 10 mm) from the edges of the inhibitor spot.

The influence of cell density was investigated by using different inoculum concentrations of inhibitor and pathogens. In one set of experiments, the concentration of an overnight culture of was adjusted to 1 × 10 9 CFU/ml and decimal dilutions (10 9 –10 5 CFU/ml, 7 μl) were spotted on the agar plates. After overnight aerobic incubation, 7 μl of pathogen at a concentration of 1 × 10 9 CFU/ml was spotted next to the inhibitor spots. In a second set of experiments, 7 μl of a 1 × 10 9 CFU/ml overnight culture of inhibitor was spotted on the agar plates. After 24 h of aerobic incubation, 7 μl of pathogen and decimal dilutions (10 9 –10 5 CFU/ml) thereof were spotted next to the inhibitor spots.

Unless otherwise mentioned, all these experiments were executed and analyzed as described above. Each experiment was repeated on 3 different days.

Characterization of the antagonistic action

The antagonistic effects were evaluated in perspective of colonization sequence, oxygen availability and nutrition availability using the spotting technique as reference technique.

The impact of the colonization sequence of inhibitor species ( S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis ) and the pathogens was evaluated by: (1) Spotting the inhibitors 24 h before the pathogen, as described above. (2) Spotting the inhibitor and the pathogen at the same time and incubating them anaerobically for 24 h. (3) Spotting the pathogen first on the agar plate and incubating the plate for 24 h under anaerobic conditions. Afterwards the inhibitor was spotted next to the pathogen spot and the plates were incubated for 24 h under anaerobic conditions.

The impact of oxygen availability on the magnitude of the inhibition was evaluated by spotting the different inhibitor strains and incubating them for 24 h under aerobic (5% CO 2 ) or anaerobic (80% N 2 ,10% CO 2 and 10% H 2 ) conditions. After 24 h, the pathogenic strains were spotted next to the inhibitors as described above.

The influence of nutrient depletion on the antagonistic activity of the inhibitors was investigated by performing the above described antagonistic experiments on full strength and diluted (1 in 2, 1 in 5, 1 in 10, 1 in 20 and 1 in 50) BHI-2 agar medium using Phosphate Buffered Saline (PBS).

Unless otherwise mentioned, all experiments were executed and analyzed as described above. Each experiment was repeated on 3 different days.

Identification of antimicrobial substances

Unless otherwise mentioned, all experiments were executed and analyzed as described above. Each experiment was repeated on 3 different days.

H 2 O 2 production

The contribution of H 2 O 2 production to the inhibitory activity was evaluated on agar plates. Inhibitor strains ( S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis ) were spotted on the agar plates. After 24 h of aerobic growth, 7 μl of horseradish peroxidase (40 μg/μl, Sigma–Aldrich) was spotted next to the inhibitor spot, at the left side. After 5 min of drying, 7 μl of pathogen was spotted at the left side of the inhibitor spot (overlaying the horseradish peroxidase spot) and 7 μl of pathogen was spotted at the right side of the inhibitor spot. Further incubation and analysis was performed as described above. To determine the amount of hydrogen peroxide production by the inhibitor strains, the production of the H 2 O 2 in liquid cultures was determined using an enzymatic kit (Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit, Life technologies). Briefly, overnight cultures of the inhibitors were centrifuged (7970 × g , 10 min) and the supernatant was discarded. The obtained pellets were resuspended in BHI-2 broth and the concentration was adjusted to 1 × 10 9 CFU/ml. 1 ml of this suspension was added to 9 ml of BHI-2 broth and incubated once under aerobic and once under anaerobic conditions. After 24 h, the samples were centrifuged as described above and the supernatant was subsequently filter sterilized (0.8 and 0.2 μm Acrodisc syringe filter, Pall corporation, Life Sciences). The amount of H 2 O 2 in the samples was determined by the Amplex ® Red Hydrogen Peroxide/Peroxidase Assay Kit, according to the manufacturer’s instructions.

Acid production

Bromocresol purple indicator (0.12 g/l) (Merck) was added to the BHI-2 agar plates as a pH indicator. Inhibitor strains ( S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis ) were spotted on the bromocresol containing BHI-2 agar plates and incubated during 24 h under aerobic or anaerobic conditions. The relative amount of acid production was evaluated by the change of color (from purple (pH 7) to different range of yellowish colors (pH between 5–6)) underneath the inhibitor spots and in the surrounding areas. Standardized pictures were taken.

Bacteriocin production

The contribution of bacteriocins on the inhibitory activity was evaluated on agar plates. Inhibitor strains were spotted on the agar plates. After 24 h of aerobic or anaerobic incubation, 7 μl of protease was spotted next to the inhibitor spot, at the left side. After 5 min of drying, 7 μl of pathogen was spotted at the left side of the inhibitor spot (overlaying the protease spot) and 7 μl of pathogen was spotted at the right side of the inhibitor spot. Further incubation and analysis was performed as described above. As proteases solutions, 0,05% Trypsin-EDTA (Gibco by Life Technologies, Paisley, UK) or 64 μg/μl Pepsin from porcine gastric mucosa (Sigma–Aldrich, USA) in PBS were used.

Statistical analysis

A four-factor Analysis of Variance (ANOVA) model was applied with pathogen, oxygen condition, inhibitor and dilution as fixed factors. Comparisons between the levels of each factor were carried out for every combination of the other factors. Corrections for simultaneous hypothesis testing were performed according to Sidak. Residual plots and normal quantile plots of residuals were used to validate the basic assumptions of the Anova model.

The relation between concentration of pathogens or inhibitors, distance between pathogens and inhibitors or volume of inhibitors or pathogens and inhibition were modeled via linear mixed models with concentration, distance or volume as continuous fixed variables and experiment as random factor. The sign of the regression coefficient, if significant, pointed to a positive or negative relation between concentration and inhibition. Residual values were tested for normality by means of a normal quantile plot.

Additionally, a two-way Anova model with inhibitor and oxygen condition as fixed factors was built. Comparisons between the levels of the fixed factors were made per level of the other factor and corrections for simultaneous hypothesis testing were performed according to Sidak. Residual plots and normal quantile plots of residuals were used to validate the basic assumptions of the Anova model.

Results

Selection of commensal and pathogenic species

The selection of the commensal species used in this study was based on screening the scientific literature for oral commensal bacteria with either a high prevalence in periodontal health and low prevalence in periodontal disease ( S. sanguinis , S. cristatus , S. oralis , S. parasanguinis , S. gordonii , S. mitis , S. intermedius , A. viscosus , A. naeslundii , A. massiliensis , G. morbillorum , G. adiacens , C. sputigena , V. parvula ) or with a known inhibitory activity against oral pathogens ( S. sanguinis , S. oralis , S. parasanguinis , S. gordonii , S. mitis , S. cristatus , S. intermedius , A. viscosus , A. naeslundii ). The selected pathogens are well known for their involvement in periodontal diseases . Additionally, whereas P. gingivalis and P. intermedia are strict anaerobes, A. actinomycetemcomitans is able to grow under aerobic and anaerobic conditions.

Commensal species that can inhibit periodontopathogens

As shown in Table 1 , species specific differences could be observed in terms of inhibitory potential as well as in terms of susceptibility towards inhibition. Of all selected commensal bacteria, only S. sanguinis , S. cristatus , S. gordonii , S. parasanguinis , S. mitis and S. oralis could inhibit the pathogens. S. mitis inhibited the pathogens significantly more than the other commensal species ( p < 0.05) whereas S. parasanguinis showed the lowest inhibitory potential of all inhibiting species. Additionally, P. gingivalis and P. intermedia were significantly more susceptible to inhibition than A. actinomycetemcomitans ( p < 0.05).

Table 1
Amount of inhibition (mean ± standard deviation, N = 3) induced by commensal species on A . actinomycetemcomitans , P . intermedia and P . gingivalis . Data are expressed in mm.
A. actinomycetemcomitans (1) P. intermedia (2) P. gingivalis (3)
S. sanguinis A 3.59 ± 0.52 (BCDF23) 5.62 ± 0.22 (CDF1) 4.72 ± 0.07 (DF1)
S. oralis B 2.36 ± 0.16 (AF23) 5.96 ± 0.11 (CDF1) 5.21 ± 0.50 (CDF1)
S. cristatus C 2.39 ± 0.16 (AF23) 4.28 ± 0.18 (ABF1) 3.79 ± 0.08 (BEF1)
S. parasanguinis D 1.87 ± 0.42 (AEF23) 3.78 ± 0.33 (ABEF1) 2.90 ± 0.53 (ABEF1)
S. gordonii E 3.26 ± 0.16 (DF23) 5.30 ± 0.37 (DF1) 5.19 ± 0.23 (CDF1)
S. mitis F 6.22 ± 0.50 (ABCDE23) 9.37 ± 0.47 (ABCDE1) 9.55 ± 0.12 (ABCDE1)
S. intermedius 0 ± 0 0 ± 0 0 ± 0
A. naeslundii 0 ± 0 0 ± 0 0 ± 0
A. massiliensis 0 ± 0 0 ± 0 0 ± 0
G. adiaciens 0 ± 0 0 ± 0 0 ± 0
G. morbillorium 0 ± 0 0 ± 0 0 ± 0
C. sputigena 0 ± 0 0 ± 0 0 ± 0
V. parvula 0 ± 0 0 ± 0 0 ± 0
ABCDE designate statistically significant ( p < 0.05) differences between the intra-group rows. 123 designates statistically significant ( p < 0.05) inter-group differences.

Characterization of the inhibition influencing factors

Since different experimental variables could potentially affect the measured amount of inhibition, the volume of the spot, the distance between spots and the initial cell density of the inoculum were analyzed.

The volume of the inhibitor spots was gradually increased by using different volumes (7, 10, 15, 20 μl) of commensal species ( Table 2 ). With the exception of the interaction between S. cristatus and P. gingivalis, there was a statistically significant ( p < 0.05) positive correlation between the size of the spot of the inhibitor and the magnitude of inhibition. This indicates that the larger the spot size of the inhibitor, the more inhibition of the pathogen.

Table 2
Amount of inhibition (mean ± standard deviation, N = 3) and regression coefficient induced by different spot sizes of commensal species against A. actinomycetemcomitans , P. intermedia and P. gingivalis . Spot sizes were expressed as the volume of the initial inoculum (7, 10, 15 and 20 μl).
Volume of commensal species and corresponding inhibition of pathogenic species (mean ± standard deviation, N = 3) Regression coefficient
A. actinomycetemcomitans 7 μl 10 μl 15 μl 20 μl
S. cristatus 2.21 ± 0.03 2.27 ± 0.02 2.33 ± 0.09 2.59 ± 0.29 0.028*
S. gordonii 2.83 ± 0.16 2.98 ± 0.24 3.06 ± 0.21 3.18 ± 0.30 0.025*
S. oralis 1.85 ± 0.15 2.06 ± 0.28 2.07 ± 0.12 2.32 ± 0.97 0.032*
P. gingivalis 7 μl 10 μl 15 μl 20 μl
S. cristatus 3.89 ± 0.05 4.04 ± 0.53 3.49 ± 0.26 4.16 ± 0.36 0.006
S. gordonii 2.59 ± 0.25 3.12 ± 0.30 3.38 ± 0.39 3.09 ± 0.32 0.035*
S. oralis 2.90 ± 0.06 2.91 ± 0.07 2.95 ± 0.03 2.96 ± 0.01 0.005*
P. intermedia 7 μl 10 μl 15 μl 20 μl
S. cristatus 4.58 ± 0.25 5.24 ± 0.32 5.20 ± 0.44 5.62 ± 0.53 0.006*
S. gordonii 5.45 ± 0.29 5.55 ± 0.23 5.82 ± 0.09 6.18 ± 0.37 0.057*
S. oralis 5.30 ± 0.37 5.39 ± 0.28 5.66 ± 0.26 5.80 ± 0.28 0.039*
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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Antimicrobial effects of commensal oral species are regulated by environmental factors
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