Bacterial Antagonism Against Periodontopathogens
For figures, tables and references we refer the reader to the original paper.
The composition of the oral microbiota is determined by a variety of synergistic interactions, such as
food webs and intergeneric coaggregation, that facilitate their persistence in a dynamic
environment.1 Likewise, interbacterial antagonism is an evolutionary mechanism that arms certain
bacterial populations against elimination by other microorganisms. The summation of the
antagonistic effects caused by so-called beneficial oral microbiota presents a substantial prevention
of colonization by exogenous and opportunistic endogenous pathogens. Any disruption of this
harmonic relationship between the host and commensal microorganisms is therefore considered an
important factor for the development of oral pathologies, such as tooth decay and periodontal
diseases.2,3 Some in vitro studies have already been performed characterizing potential probiotic
bacteria in the oral cavity.4,5 Conversely, comparison of the prevalence of commensal species with
beneficial properties for periodontal health between healthy and diseased individuals is only sparsely
investigated.
Clear differences were observed between healthy and diseased individuals in the prevalence of
bacterial strains that are antagonistic toward oral pathogens.6,7 Subgingival plaque samples of
healthy patients contained organisms that could inhibit the growth of the—at that time—presumed
periodontopathogens.6,7 Subgingival plaque samples from diseased sites from patients with
aggressive periodontitis and refractory periodontitis almost invariably lacked such inhibitory bacteria.
Interestingly, subgingival plaque samples from clinically healthy sites in these patients with
periodontitis contained inhibitory bacteria in proportions similar to those in subgingival plaque taken
from healthy control patients.6,7 This observation was also validated for other potential beneficial
species, such as Lactobacillus gasseri,8 Bifidobacteria,9 and Streptococcus sanguinis.10
With the emergence of widespread antibiotic resistance, an alternative for conventional periodontal
therapy is necessary. The importance of beneficial commensal bacteria has already been shown with
regard to the immune response and periodontal colonization.11,12 Because the presence of these
commensal bacteria in healthy compared with diseased individuals has only been investigated for a
small study population and with a limited study design, no firm conclusions can be drawn. The aim of
the current study is to investigate whether oral bacteria can cause antagonism toward
periodontopathogens using a larger study population and comparing different assay techniques.
Additionally, the inhibitory effect of some commercial dietary probiotics on periodontopathogens
was evaluated and compared with the inhibitory effect of orally derived beneficial bacteria toward a
panel of periodontopathogens.
Bacterial Strains and Culturing Conditions
The periodontopathogens Aggregatibacter actinomycetemcomitans,‡ Porphyromonas gingivalis,§
Prevotella intermedia,‖ and Fusobacterium nucleatum¶ were maintained on blood agar,#
supplemented with hemin (5 mg/mL), menadione (1 mg/mL), and 5% sterile horse blood.** The
probiotic Lactobacillus strains Lactobacillus rhamnosus,††‡‡ Lactobacillus casei,§§‖‖¶¶ Lactobacillus
fermentum,## and Lactobacillus paracasei L07-21*** were maintained on agar plates.††† All
incubations of broth cultures‡‡‡ were performed in anaerobic conditions (80% N2, 10% H2, and 10%
CO2) at 37°C unless stated otherwise.
Participant Selection and Sample Collection
Subgingival plaque samples were obtained from 35 patients attending the Department of
Periodontology of the University Hospital Leuven. The project received full approval from the Ethical
Committee of the University Hospital of the Catholic University of Leuven, Leuven, Belgium. Patients
were well informed of the study protocol and objectives and gave their written consent before
participation. The patients were included if they fulfilled the following inclusion and exclusion
criteria: 1) ≥35 years of age; 2) ≥14 teeth with ≥3 teeth in every quadrant; 3) no dental implants or
prosthetic devices of any kind present in the mouth; 4) no medication usage (including antibiotics); 5)
no antiseptic mouthrinse usage or professional dental cleaning within 6 months before sample
taking; 6) no systemic diseases in the past and no common diseases (e.g., flu or cold) within 6 months
before sampling; 7) no oral lesions or signs of necrotizing oral diseases at the time of examination; 8)
no pregnancy; 9) no smoking; 10) no periodontal treatment (root planing) in the past; and 11) a
Turesky plaque index > 1. This population was divided into a group of patients with periodontitis
(later referred to as “periodontitis”) and a group of individuals that did not have clinical symptoms of
periodontitis (later referred to as “healthy”). Periodontal health or disease was evaluated by clinical
examination. Patients with periodontitis exhibited ≥8 teeth with a probing depth (PD) and clinical
attachment level (CAL) ≥ 6 mm with ≥1 of these teeth in each quadrant. Patients without
periodontitis (healthy) exhibited no teeth with a PD >3 mm and a CAL >4 mm. Clinical examination
and sample taking were performed by one single calibrated examiner (WT). Two weeks after baseline
examination, plaque samples were taken with eight sterile paper points§§§ in a gingival sulcus
(healthy volunteers) or from the apex of the deepest periodontal pocket (patients with periodontitis)
in each quadrant after having removed all supragingival plaque with a sterile curet and having
isolated and dried the sampling area with cotton rolls and airflow. In each individual, the four
quadrants were sampled, and the paper points were pooled in 1.5 mL of reduced transport fluid until
processing. Processing was initiated within a 2-hour timeframe.
Bacterial Isolation Procedure
Plaque samples were dispersed by vortexing and used for anaerobic culturing on blood agar plates as
described previously.13 After 7 days of incubation, 50 random colonies of each patient were checked
for their ability to produce growth inhibition of the periodontal pathogens P. gingivalis, P. intermedia,
F. nucleatum, and A. actinomycetemcomitans. For this purpose, the agar overlay technique was used
as described previously.14 Plates were incubated at 37°C in an anaerobic atmosphere. Test strains
that exhibited growth inhibition of the indicator strain were selected and retrieved from the backup
culture plate and streaked on blood agar plates until pure cultures were obtained. Strains were
stored at −80°C until further use.
Quantification of Inhibition Mechanisms of Oral Isolates and Probiotic Strains
The quantification of the inhibitory mechanism was performed with the agar well diffusion
method.15 Next to the test strains, broth cultures of the test strains were filter sterilized by passing
them through 0.2-μm syringe filters‖‖‖ to obtain a cell-free supernatant. A fraction of the cell-free
supernatant was, before filtration, neutralized with 1N NaOH to pH 7.0. A blank was also included in
each assay, which was 50 μL sterile broth.¶¶¶ After application of the four different fractions of the
test culture to the respective blood agar plates, the plates were incubated anaerobically at 37°C. The
zones of inhibition (the distance between the edge of the well and the edge of the lawn of the
indicator strains) were measured when the indicator bacteria formed a confluent lawn on the plates.
Triplicate experiments were performed on separate days.
Identification of Oral Isolates
The 15 oral isolates that on average demonstrated the most pronounced inhibition of the indicator
strains were identified with a combination of phenotypical and molecular tests. The phenotypical
identification included Gram staining, colony morphology, and biochemical tests.### Molecular
identification was performed on purified DNA that was extracted**** from liquid†††† overnight
cultures of the isolates. According to the results of the phenotypic identification tests, a procedure
was selected for 16S ribosomal RNA (rRNA) gene sequence analysis.
Amplification of Streptococcus 16S rRNA gene sequences.
The majority of the variation between 16S rRNA gene sequences of streptococcal species is found in
the 5′ terminal part of the gene, especially in the v1 and v2 regions corresponding to bases 63 to 92
and 193 to 222 of the Streptococcus pneumoniae R6 16S rRNA gene (GenBank accession no.
NC_003098).16 This region was amplified by polymerase chain reaction (PCR) using primers
Streptococcus v1 forward (5′-AGTTTGATCCTGGCTCAGGACG-3′ and Streptococcus v2 reverse (5′CAACTAGCTAATACAACGCAGGTC-3′).17
Amplification of coryneform 16S rRNA gene sequences.
The 16S rRNA gene sequence of the coryneform bacteria was amplified using primers TPU-1 (5AGAGTTTGATCMTGGCTCAG-3′) and 1492RPL (5-GGTTACCTTGTTACGACTT-3′).18 PCRs (25 μL)
contained 0.4 μM each primer,‡‡‡‡ 0.025 U/μL DNA polymerase,§§§§ 1× PCR buffer, 1.5 mM MgCl2,
0.2 mM concentrations of deoxynucleoside triphosphates, and 2.5 μL DNA. Thermal cycling consisted
of an initial denaturation at 95°C for 2 minutes, followed by 35 cycles of 45 seconds at 95°C, 30
seconds at 50°C, and 2 minutes at 72°C. A final elongation step of 10 minutes at 72°C was included.
The presence of the PCR product was verified on a 1% agarose gel.
Cloning and sequencing of the amplified 16S sequences.
Cloning was performed as described previously.19 The insert was cycle sequenced‖‖‖‖ according to
the instructions of the manufacturer. Sequencing primers T7 (5′- TAATACGACTCACTATAGGG-3′) and
SP6 (5′-TATTTAGGTGACACTATAG-3′) flanking the multiple cloning site of the vector produced
sequencing reads in two directions. After purification¶¶¶¶ of the sequencing product, the samples
were loaded on a capillary electrophoresis system. The obtained sequence reads were verified and
assembled.#### The consensus sequences were used for homology search with the basic local
alignment search tool (BLAST) and for comparison to the Ribosomal Database Project.20 Strains were
identified to species level if the 16S rRNA gene sequence of the individual strain was ≥97.0%
homologous to the type strain of a species20 and when phenotypic testing did not indicate any
aberrant reactions regarding the published data for this particular species.
Statistical Analyses
The difference in the prevalence of the inhibiting strains among healthy and diseased individuals was
evaluated by performing a χ2 test. To compare the extent of the growth inhibition caused by the
isolated strains, a mixed linear model was built. The growth inhibition of the different isolated strains
could be compared for each periodontopathogen. Also an evaluation between the extent of growth
inhibition between different periodontopathogens was made for the isolated strain. The agreement
between the results obtained with the agar overlay technique and the agar well diffusion assay was
evaluated with a Fisher exact test. All statistical analyses for this study were performed using
software***** with the level of significance set at P <0.05.
Isolation of Antagonistic Strains From Subgingival Plaque Samples: Agar Overlay Assay
Subgingival plaque samples from 16 healthy volunteers and 19 patients with periodontitis were
collected. Patient characteristics are shown in Table 1. In total, 1,750 individual colonies were
screened with the agar overlay assay for their ability to induce growth inhibition of A.
actinomycetemcomitans, P. gingivalis, P. intermedia, and F. nucleatum. With this technique,
inhibitory colonies could be isolated from nine of the 35 sampled individuals with no significant
difference (P >0.05) between healthy patients and patients with periodontitis (Table 1). From these,
74 inhibitory colonies could be isolated, which represent 4.23% of the total number of isolated
colonies.
P. intermedia was the most frequently inhibited periodontopathogen, followed by P. gingivalis, A.
actinomycetemcomitans, and F. nucleatum. Of the 39 colonies that inhibited P. intermedia, only 15
were from healthy volunteers. In contrast, of the 23 colonies that inhibited P. gingivalis, 20 were
from healthy volunteers. The number of colonies that inhibited either P. intermedia or P. gingivalis
was significantly different between healthy volunteers and patients with periodontitis (P <0.05).
Although tendencies were observed, no significant differences (P >0.05) were found in the number of
colonies that inhibited A. actinomycetemcomitans or F. nucleatum between patients with
periodontitis and healthy individuals.
Agar Well Diffusion Assay: Spectrum of Antagonistic Activity
Although the agar well diffusion assay (Fig. 1) was used to quantify the amount of growth inhibition
on each periodontopathogen for the 74 strains that were isolated with the agar overlay assay (see
below), this assay also provided data on the antagonistic spectrum of these strains, albeit analyzed
with a different technique. Of the 74 isolates that showed antagonistic activity toward ≥1
periodontopathogen via the agar overlay assay, only 48 isolates also showed antagonistic activity
toward ≥1 periodontopathogen via the agar well diffusion assay. These 48 strains were derived in
equal proportions from healthy individuals and patients with periodontitis (Table 1).
The agar well diffusion assay showed that P. gingivalis was the most frequently inhibited
periodontopathogen, followed by F. nucleatum, P. intermedia, and A. actinomycetemcomitans. No
significant differences (P >0.05) were observed in the number of colonies that inhibited P. gingivalis,
P. intermedia, A. actinomycetemcomitans, or F. nucleatum between patients with periodontitis and
healthy individuals. When looking at the seven commercially available probiotic bacteria (Fig. 2), they
all inhibited the growth of P. gingivalis and P. intermedia, whereas A. actinomycetemcomitans was
only inhibited by five strains and F. nucleatum only by two strains.
Agar Well Diffusion Assay: Quantification of Antagonistic Activity
Using the agar well diffusion assay (Fig. 3), the amount of growth inhibition toward each
periodontopathogen was evaluated for the 74 isolates. With this assay, 21 isolates demonstrated an
inhibition zone of ≥2 mm toward P. gingivalis. For the periodontopathogens F. nucleatum and A.
actinomycetemcomitans, six oral isolates demonstrated an inhibition zone of ≥2 mm. Inhibition of
the growth of P. intermedia by >2 mm could not be recorded with this assay. As shown in Table 2, P.
gingivalis was quantitatively most clearly inhibited with an average inhibition zone for all 74 oral
isolates of 1.15 ± 0.73 mm. The average inhibition zones for A. actinomycetemcomitans, F.
nucleatum, and P. intermedia were 0.39 ± 0.41, 0.38 ± 0.36, and 0.30 ± 0.24 mm, respectively. No
significant differences (P >0.05) were found for oral isolates originating from healthy patients versus
patients with periodontitis.
For the commercial probiotic bacteria, P. gingivalis (average zone of inhibition, 4.07 ± 0.84 mm) and
P. intermedia (average zone of inhibition, 1.71 ± 0.39 mm) were much more inhibited in their growth
than A. actinomycetemcomitans (average zone of inhibition, 0.47 ± 0.24 mm) and F. nucleatum
(average zone of inhibition, 0.14 ± 0.15 mm). Neutralizing the pH of the cell-free supernatant with
NaOH completely eliminated the inhibition of periodontopathogenic growth.
If these results are compared with the average antagonistic activity of the isolated strains, the
following observations are made: the probiotic strains cause 1) a 3.54 times stronger inhibition of P.
gingivalis, 2) a 5.70 times stronger inhibition of P. intermedia, and 3) a 1.21 times stronger inhibition
of A. actinomycetemcomitans. The inverse was observed for F. nucleatum, in which it was observed
that the isolated strains on average caused a 2.71 times stronger inhibition compared with the
probiotic strains.
The 15 isolated strains that on average caused the most pronounced inhibition were identified as
follows: 1) one strain of Streptococcus salivarius; 2) three strains of Streptococcus mitis; 3) two
strains of Streptococcus oralis; 4) three strains of Bifidobacterium dentium; 5) one strain of
Actinomyces viscosus; and 6) five strains of Actinomyces naeslundii (Table 3). The growth inhibition
on the four periodontopathogens for these strains was only present if the complete overnight
cultures were used (Fig. 3). No growth inhibition could be observed in the negative control, with cellfree supernatants or with the pH neutralized, cell-free supernatants.
If the results from the seven commercially available probiotic bacteria are compared with the results
from the 15 strains just mentioned, the differences between the groups were much less pronounced
(Figs. 2 and 3). However, the commercially available probiotics still showed a stronger inhibition of P.
gingivalis and P. intermedia, and the oral isolated strains showed a clearly stronger inhibition of F.
nucleatum and A. actinomycetemcomitans.
Discussion
Certain indigenous bacterial species and their products are beneficial for a healthy periodontium.21
When the homeostasis within the oral microbiota is disrupted, diseases such as dental caries and
periodontitis can develop. These indigenous bacterial species are equipped with a number of specific
or non-specific mechanisms to antagonize pathogenic species. Mechanisms, such as competition for
and exclusion of adhesion receptors, competition for nutrients, and production of antimicrobials or
surfactants, account for the protective effects of beneficial bacteria.11,22 Hillman et al.6,7
demonstrated that the prevalence of species capable of antagonizing oral pathogens is higher in
periodontally healthy individuals. Especially antagonistic strains that inhibited the growth of P.
intermedia were more prevalent in healthy individuals than in those with periodontitis (0% versus
37%, respectively). Similar observations were made for other periodontopathogens by this group of
researchers. In the current study, the study of Hillman et al.6,7 is repeated, although a substantially
larger study population was sampled and screened for the prevalence of antagonistic strains by
means of the agar overlay assay. P. gingivalis, A. actinomycetemcomitans, F. nucleatum, and P.
intermedia were selected as the model periodontopathogens, representing key pathogens from the
red and orange complexes.23
When comparing the results of both studies, some similarities were observed. Although not always
statistically significant in the present study, the prevalence of strains antagonistic toward P.
gingivalis, A. actinomycetemcomitans, and F. nucleatum was found to be higher in healthy individuals
than in individuals with periodontitis. This suggests the involvement of the beneficial microbiota in
the protection of oral health. An exception to this finding is that, in the current study, the prevalence
of strains that inhibit P. intermedia is lower in the healthy population (2.67% in the diseased
population compared with 1.76% in the healthy population). At this time, to the best of our
knowledge, a conclusive explanation for this finding is unavailable. However, because the study
included 35 patients, a chance factor can still be involved.
The substantially higher concentration of antagonizing strains by Hillman et al.7 compared with our
observations brings into focus the importance of the study methodology used. The observed
discrepancy with our results could at least partially be explained by the fact that the incubations in
the study of Hillman et al. were all performed in a microaerophilic atmosphere. This is in contrast to
our methodology, in which all incubations were performed in complete absence of oxygen.7 In
anaerobiosis, it has been shown that there is a strong downregulation of peroxidogenesis by oral
streptococci.24,25 Eliminating the effect of hydrogen peroxide from the antagonistic interactions
between beneficial and pathogenic oral species could (partially) account for the substantial
difference between the detection rates of antagonizing strains in this study compared with the
former study. However, it has to be stressed that the study of Hillman et al.7 sampled only one
healthy individual.
The 74 isolates that inhibited growth of the periodontopathogens in the agar overlay assay were
subsequently investigated with the agar well diffusion assay. The comparison between the two
antagonism assays generally yielded a poor agreement for these 74 strains. This poor agreement
between the two tests shows that, for the evaluation of microbial antagonisms among dental plaque
bacteria, study results obtained by different techniques are incomparable. A factor that can be
involved in the discrepancy between the two assays is the fact that the sequence of inoculation of
test and indicator strains differs in the two assays: in the agar overlay assay, the test bacteria are
allowed to grow to a stationary phase before being exposed to the pathogen as indicator strain.
Conversely, in the agar well diffusion assay, both test and indicator strains are inoculated
simultaneously on the assay culture plate. This aspect has been shown to considerably influence
antagonistic interactions among oral bacteria in vitro.26 It seems most likely that the earlier
inoculated strain has an evident advantage toward the later introduced strain because of an
abundance of nutrients, although the authors speculate that interspecies communications are
involved in this interaction. This is analogous with our observations because the total number of
colonies that were able to antagonize ≥1 pathogen was found to be lower in the agar well diffusion
assay than in the agar overlay assay (Table 1).
Our results suggest that the mechanism by which the isolated strains suppress growth of the
periodontopathogens was caused by the competition for nutrients: although the overnight cultures
of most of these strains induced growth inhibition, no detectable inhibitory substance was present in
the supernatants of any of the isolates. The 15 strains that on average caused the most explicit
growth inhibition were identified as typical representatives of the oral microbiota. All of these strains
are species of viridans streptococci, Actinomyces, or Bifidobacteria. Although competition for
nutrients seems the most probable antagonistic mechanism observed in our experiments, these
genera have also been shown to be capable of causing broad-spectrum antagonism toward other
strains by the production of organic acids, such as formic, lactic, and succinic acids.22,27-29 Lactic
acid is also known to function as a permeabilizer of the Gram-negative bacterial membrane30 and
even to have some iron-chelating capacity.31 Although in most interbacterial relationships the
concomitant lowering of the environment pH32 is considered to be of negligible importance
compared with the direct cellular effect of the organic acids, this may not be so in respect to the
periodontopathogens that prefer basic environments. This is especially true for P. gingivalis, which
stops growing if the environmental pH drops below 6.5.33 Although this is a suitable explanation for
the fact that P. gingivalis is the most strongly inhibited strain among the four tested
periodontopathogens, this clearly only partially accounts for the antagonistic activity because the
amount of growth inhibition in the wells containing cell-free supernatants was below the detection
limit.
A wide range of bacterial species have been shown to produce bacteriocins, narrow-spectrum
antimicrobial substances that are produced to withstand competition by similar strains. The absence
of such bacteriocin-producing strains in our collection of 1,750 examined strains is in contrast to a
number of publications that describe the discovery of bacteriocin-producing Gram-negative oral
bacteria. Again, this discrepancy is most likely caused by a different study methodology because, in
some studies, the antimicrobial compounds can only be isolated from the concentrated intracellular
fraction of bacterial cultures.34-36 The physiologic function of an intracellular antimicrobial
substance is at least peculiar, and the poor characterization of the antagonistic compounds cannot
exclude a biologic function different from bacteriocin.35
The microbial antagonism of probiotic Lactobacillus strains toward oral pathogens has been
described recently.8 The authors describe a slightly different antagonistic spectrum compared with
that of the probiotic strains that are used in this study. Although the four tested pathogens were all
susceptible to antagonisms of the Lactobacillus strains, in our observation, P. gingivalis is
substantially more inhibited than A. actinomycetemcomitans, P. intermedia, and F. nucleatum. KõllKlais et al.,8 however, found that A. actinomycetemcomitans was most pronouncedly inhibited.
Because in the latter study two different assays were used for the evaluation of the antagonisms of
A. actinomycetemcomitans and P. gingivalis, respectively, the comparison between antagonistic
potentials may not be appropriate.
Our data suggest that the growth inhibition caused by the lactic acid bacteria toward the pathogens
was mainly caused by the production of large amounts of organic acids because the neutralization of
supernatants pH completely eliminated the antagonistic activity. This effect is in line with
observations in other reports in which it has been shown that Lactobacillus strains antagonize many
Gram-negative bacteria, mostly by the antimicrobial effect of lactic acid.37
In a study by Stamatova et al.,38 the adhesion of Lactobacillus strains to saliva-coated surfaces was
evaluated in vitro. The results showed significant variations in the adhesion capacity of the studied
Lactobacillus strains. Adhesion to oral surfaces is of primary importance for colonization in the oral
cavity, and, in that aspect, the use of the commensal oral microbiota as beneficial strains circumvents
this problem.38
In general, it can be concluded from the current study that the investigation of microbial
antagonisms among dental plaque bacteria can be performed using different approaches. However,
different methodologies yield results that are difficult to compare. Results suggested that some oral
bacteria can cause antagonism toward periodontopathogens by means of competition for nutrients.
The effects described here, however, probably only represent part of the entire beneficial effect of
such strains on oral health: it is important to mention the involvement of hydrogen peroxide as an
antimicrobial substance that was not included in our tests. It is generally accepted that hydrogen
peroxide is produced by viridans streptococci25 and Lactobacillus species,39 and it is therefore likely
that this augments their antimicrobial effect in vivo.
Conclusions
The commensal oral microbiota is considered to induce a beneficial oral immune response11or to
interfere with periodontopathogen colonization.40 The interference with pathogen recolonization
was confirmed in vivo as an addition to the standard therapy of periodontitis.12 These observations
underline the therapeutic and prophylactic potential of applications that stimulate oral health by the
application of beneficial effector strains.
Acknowledgments
This study was supported by Catholic University of Leuven Grant OT 07/057 and Research Foundation
Flanders Grants G.0772.09 and 1.5.153.10. Dr. Teughels was also supported as a postdoctoral fellow
of the Research Foundation Flanders. The authors report no conflicts of interest related to this study.