Characterization of interspecies competition between S. mutans and other oral streptococcal species. To get a global view of how prevalent interspecies competition is between S. mutans and other oral streptococci, we analyzed the inhibitory spectrum of S. mutans strain UA140 against 11 streptococcal species, including members of the mitis, mutans, viridans, and pyogenic groups: S. gordonii ATCC 10558, S. oralis ATCC 10557, S. mitis ATCC 33399, S. mitis ATCC 903, S. pneumoniae, S. parasanguinis ATCC 15911, S. sanguinis ATCC 10556, S. sanguinis NY101, S. sobrinus OMZ176. S. cristatus ATCC 49999, and S. pyogenes. UA140 was inoculated onto BHI plates and grown for 24 h before the other species were inoculated nearby. As shown in Fig. , S. mutans could inhibit the growth of all tested strains; however, the growth inhibition was less severe against S. sobrinus, a member of the mutans group. Based on this result, S. sanguinis was chosen for further analysis because of its well-known history of antagonism toward S. mutans.
FIG. 1. Inhibition of oral streptococcal species by S. mutans UA140. 1, S. gordonii; 2, S. pyogenes; 3, S. oralis; 4, S. mitis ATCC 33399; 5, S. mitis ATCC 903; 6, S. pneumoniae; 7, S. cristatus; 8, S. parasanguinis; 9, S. sanguinis ATCC 10556; 10, S. sanguinis (more ...) Competition between S. mutans and S. sanguinis in time and space.
A simple competition assay was developed to test the antagonistic interactions between S. mutans and S. sanguinis. Overnight cultures of S. mutans UA140 and S. sanguinis ATCC 10556 were inoculated on half-strength BHI plates. Three tests were conducted: (i) S. mutans was inoculated first and allowed to grow overnight (as the early colonizer) before S. sanguinis was inoculated nearby (as the later colonizer), (ii) vice versa, and (iii) both species were inoculated at the same time. As shown in Fig. , the early colonizer always inhibited the growth of the later colonizer regardless of the bacterial species (left and middle). This competitive exclusion was reduced to a negligible level when both species were inoculated at the same time (right). This suggested that the sequence of inoculation determined the competition outcome.
FIG. 2. Competition assays between S. mutans and S. sanguinis. (A) Competition assay on half-strength BHI plate. (B) Confocal laser scanning microscopy analysis of competition in biofilms. Green cells, S. mutans (green fluorescent protein); red cells, S. sanguinis (more ...)
Since competitive exclusion could result from either nutritional deprivation by the growth of the early colonizer or production of inhibitory substances by the early colonizer, we decided to test the first possibility by performing the same competition assay described above but using different strains of the same species. We reasoned that nutritional deprivation would be more severe within the same species because the bacteria have the same nutrient requirements. We used another S. mutans
strain, UA159 (1
), in the competition assay with UA140 and another S. sanguinis
strain, NY101, in the competition assay with ATCC 10556. No growth inhibition was observed in either competing pair regardless of the sequence of inoculation (data not shown). This result suggested that some diffusible substance produced by S. mutans
and S. sanguinis
rather than nutrient deprivation was responsible for the observed competitive exclusion.
To see if this competitive exclusion also occurred in space, such as in biofilms, we constructed an S. mutans
green fluorescent protein (gfp
) reporter strain, UA140::Φ(ldhp-gfp
) (see Materials and Methods). UA140::Φ(ldhp-gfp
) carries a gfp
fusion to the lactate dehydrogenase (ldh
) promoter on the chromosome. Since the ldh
promoter is constitutively expressed (24
) cells continually exhibit green fluorescence throughout growth. This property made it easier to distinguish S. mutans
from S. sanguinis
, which was labeled with red fluorescence using a cell tracker dye (CellTracker Orange) 2 h prior to microscopy. UA140::Φ(ldhp-gfp
) and S. sanguinis
were then subjected to the previously described competition assays (see Materials and Methods). As shown in Fig. , when S. mutans
attached first, almost no S. sanguinis
bacteria could attach and grow in the biofilm (left). The same was true for S. sanguinis
when it attached first (middle). However, when both were inoculated at the same time, a mixed-species biofilm could form (right). This result was reminiscent of the observations made by Mikx et al. nearly 30 years ago in the germ-free-rat experiment (25
), suggesting that the competition between S. mutans
and S. sanguinis
observed in this in vitro assay may also occur in vivo.
Environmental conditions modulate competition and coexistence between S. mutans and S. sanguinis.
Since the dental biofilm in nature is continually challenged by adverse conditions, such as cycles of feast and famine and fluctuations of pH, we were interested to see whether the competition between S. mutans and S. sanguinis was influenced by these environmental conditions. We performed a plate assay similar to that shown in Fig. under three conditions: a “nutrient-rich” growth condition in which sucrose was added to BHI and the medium was buffered to pH 7.0 with phosphate buffer, a “stress” condition in which the pH of BHI was lowered to 5.5, and a “nutrient-limiting” condition in which BHI was diluted to half strength, as in Fig. . As expected, the “nutrient-limiting” condition resulted in the same pattern of inhibition shown in Fig. . Surprisingly, under “nutrient-rich” or “stress” conditions, there was negligible or no inhibition between the species regardless of the sequence of inoculation (Fig. ); the lesser growth of S. sanguinis under “stress” conditions is due to the growth inhibition of S. sanguinis by acidic pH. These results suggested that environmental conditions modulated competition/coexistence between bacterial species.
Investigation of possible inhibitory substances produced by S. mutans and S. sanguinis.
The results presented in Fig. suggested that both S. mutans
and S. sanguinis
produced diffusible substances that inhibited the growth of the other species. To identify the possible inhibitory substances, we grew S. mutans
and S. sanguinis
on a half-strength BHI plate for 24 h and applied peroxidase (40 μg), peptidase (64 μg), or phosphate-buffered saline beside each colony for 10 min before the other species was inoculated at the same spot. The two enzymes (peptidase and peroxidase) were chosen based on previous knowledge that proteinaceous inhibitory substances (8
) and H2
were produced by oral streptococci (34
). As shown in Fig. , addition of peroxidase abolished the inhibitory effect of S. sanguinis
toward S. mutans
(Fig. , left), while addition of peptidase diminished the inhibitory effect of S. mutans
toward S. sanguinis
(Fig. , middle). Given the fact that the inhibitory substance(s) produced by S. mutans
is proteinaceous, one logical candidate would be a peptide antibiotic, e.g., bacteriocin, since S. mutans
is known to produce multiple bacteriocins called mutacins (8
). Strain UA140, used in this study, was known to produce two major mutacins, mutacin I and mutacin IV (32
). To determine whether the mutacins were responsible for inhibiting the growth of S. sanguinis
, we constructed a mutacin-defective isogenic strain, UA140I−
, in which the production of both mutacins was eliminated by inactivation of the mutacin-biosynthetic genes (see Materials and Methods). This mutant strain was tested in competition assays with S. sanguinis
on the plate, as well as in the biofilm. As shown in Fig. , UA140I−
could no longer inhibit the growth of S. sanguinis
on the plate or in the biofilm even when it was inoculated first. Similar results were obtained with all 11 oral streptococci used in the initial screen (data not shown). To test which mutacin was responsible for the inhibitory effect, UA140 derivative strains defective in either mutacin I or mutacin IV were constructed (see Materials and Methods). Competition assays using these strains showed that they were still able to inhibit the growth of S. sanguinis
(data not shown). These results demonstrate that both mutacins serve as inhibitory substances and that either mutacin is sufficient to inhibit the growth of S. sanguinis
and other streptococcal strains.
FIG. 3. Identification of inhibitory substances produced by S. mutans and S. sanguinis. (A) S. sanguinis (Ss) was inoculated first. (B) S. mutans (Sm) was inoculated first. After 24-h growth on half-strength BHI plates, 40 μg of peroxidase (left), 64 (more ...)
Since the inhibitory substance(s) produced by S. sanguinis was sensitive to peroxidase (Fig. ), hydrogen peroxide (H2O2), became the likely candidate. To test this hypothesis, we used a leuco crystal violet assay (see Materials and Methods) to measure H2O2 production by S. sanguinis and found that under high-cell-density conditions, approximately 120 μM H2O2 was produced by S. sanguinis, which would be sufficient to affect the growth of S. mutans. Although a direct quantification of H2O2 production on the plate was not technically feasible, we did observe considerable H2O2 production by S. sanguinis grown on plates (see Fig. ). These data suggested that H2O2 produced by S. sanguinis could be one of the diffusible inhibitory substances responsible for preventing the growth of S. mutans.
FIG. 5. Effects of growth conditions on mutacin I gene expression (A), mutacin production (B), and H2O2 production (C). Mutacin I gene expression (mutAp-luc) was measured as relative light units (RLU) per OD600 unit; mutacin production was measured by diameters (more ...)
To get more direct evidence that these compounds (mutacins and H2O2) indeed can carry out the inhibitory effects on S. sanguinis and S. mutans, respectively, we conducted direct growth inhibition studies. S. mutans was challenged with different concentrations of H2O2, and growth inhibition was measured (Fig. ). The lowest concentration that could inhibit the growth of S. mutans was 0.0005% (142 μM), which was in the same range as the H2O2 produced by S. sanguinis in the cell pellet (see Fig. ). Purified mutacin I and mutacin IV were both able to inhibit the growth of S. sanguinis in an overlay assay up to an eightfold dilution (Fig. ). In addition, we conducted overlay assays with the different mutacin mutants. These experiments showed that both mutacins are involved in the S. sanguinis growth inhibition and that the double mutant had a dramatically reduced ability to inhibit the growth of S. sanguinis (Fig. ). These results demonstrate the ability of H2O2 and mutacin to inhibit the growth of S. mutans and S. sanguinis, respectively.
FIG. 6. Effects of juxtaposition between S. mutans and S. sanguinis on mutacin gene expression and H2O2 production. (A) Mutacin I gene expression of strain UA140::Φ(mutAp-luc). (B) H2O2 production by S. sanguinis. Bars 1, single-species planktonic culture; (more ...) Mutacin gene expression and H2O2 production are regulated by growth conditions.
To determine the effect of mutacin and H2O2 production on the competition outcome between S. mutans and S. sanguinis, we studied the effect of medium conditions on the production of mutacin and H2O2. To quantify mutacin gene expression, we constructed reporter strains in which the promoterless firefly luciferase gene (luc) was fused to the mutacin I (mutA) and the mutacin IV (nlmA) promoters on the chromosome. The reporter strains were inoculated on the three conditioned plates as described in Fig. . After 24 h of incubation, the cells were scraped from the plate and measured for luciferase activity and OD600. The spent plates were overlaid with an indicator strain to measure mutacin production. Both mutacin I (mutA) and mutacin IV (nlmA) promoters exhibited the same pattern of expression under these conditions; shown in Fig. are the results of the mutacin I promoter expression (mutAp-luc) and mutacin I production. Compared to the “nutrient-limiting” plate (bar 1), mutacin I promoter expression was reduced ~10-fold on both “nutrient-rich” (bar 2) and “stress” (bar 3) condition plates (Fig. ). Consequently, the inhibition zone on the “nutrient-rich” plate (Fig. , bar 2) was reduced >5-fold compared to that on the “nutrient-limiting” plate (bar 1), and no inhibition zone was observed on the “stress” condition plate (bar 3).
The effect of environmental conditions on H2O2 production by S. sanguinis was measured on the plate by a modified peroxidase assay (see Materials and Methods). Darker color on and around the colony indicated the presence of larger amounts of H2O2. As shown in Fig. , the amounts of H2O2 on the “nutrient-rich” (plate 2) and “stress” (plate 3) condition plates were conspicuously less than that on the “nutrient-limiting” (plate 1) plate. Taken together, these results correlated well with the phenotypic observations depicted in Fig. .
Mutacin gene expression and H2O2 production are both inhibited by juxtaposition between S. mutans and S. sanguinis.
The results presented in Fig. demonstrated that despite competitive exclusion between S. mutans and S. sanguinis, they can coexist under certain circumstances, such as when both species are inoculated at the same time (right). To determine whether close cell-cell proximity between the two species could result in mutual inhibition of inhibitory-substance production by the competing species, we developed a mixed-culture pelleting assay that would create an environment for cell-cell contact but without complications of extensive cell growth. Overnight cultures were diluted and grown to early log phase (OD600, ~0.1), and the two species were mixed in a 1:1 ratio and centrifuged. The mixed cultures were incubated for 2 h as cell pellets before luciferase activity, H2O2 production, and OD600 were measured. As controls, single-species cultures of S. mutans and S. sanguinis in planktonic and pelleted conditions were used. Since mutacin I and IV promoters behaved similarly, only the results of mutacin I promoter expression are presented here (Fig. ). In the single-species culture, mutacin I gene expression increased 10-fold in the cell pellet (bar 2) compared with the planktonic culture (bar 1) (Fig. ). Similarly, H2O2 production by S. sanguinis increased twofold in the cell pellet (bar 2) compared with the planktonic culture (bar 1) (Fig. ). These results suggested that high cell density enhanced mutacin gene expression by S. mutans and H2O2 production by S. sanguinis. Surprisingly, in the mixed-species cell pellet, mutacin gene expression by S. mutans was reduced fivefold (Fig. , bar 3) and H2O2 production by S. sanguinis was reduced threefold (Fig. , bar 3) compared with their respective single-species cell pellets.
To further confirm that this inhibition of mutacin gene expression by juxtaposition with S. sanguinis
happens only between different species, not within the same species, we performed the same pelleting assay with two S. mutans
strains carrying different fluorescent protein reporters. UA140::Φ(mutAp-mrfp
) carries a red fluorescent protein fused to the mutacin I promoter, and UA159::Φ(ldhp-gfp
) carries a green fluorescent protein fused to the ldh
). Pelleting assays were performed with either UA140::Φ(mutAp-mrfp
) alone (Fig. ), UA140::Φ(mutAp-mrfp
) plus UA159::Φ(ldhp-gfp
) (Fig. ), or UA140::Φ(mutAp-mrfp
) plus S. sanguinis
(Fig. ). After 2 h of incubation, the cell pellet was analyzed by confocal microscopy. UA140::Φ(mutAp-mrfp
) cells alone or in a mixture with UA159::Φ(ldhp-gfp
) exhibited bright-red fluorescence, indicating a high level of mutacin I gene expression; in contrast, the same UA140::Φ(mutAp-mrfp
) cells displayed almost no fluorescence in the mixed culture with S. sanguinis
(Fig. ). To exclude the possibility that the diminished fluorescence of UA140::Φ(mutAp-mrfp
) in the mixed-species culture was due to fewer S. mutans
cells in the cell aggregates, a fluorescein isothiocyanate-conjugated monoclonal antibody specific to S. mutans
) was used to label cells in the mixed-species cell aggregates. As shown in Fig. , similar amounts of UA140::Φ(mutAp-mrfp
) cells existed in the mixed-species cell aggregates and in the single-species cell aggregates.
To test whether the reduced mutacin gene expression and H2O2 production in the mixed-species cell pellet was due to inhibition of cell growth, cells in the single-species and mixed-species cell pellets were plated at the beginning and the end of the experiment. No difference was observed between the single-species and mixed-species cultures, suggesting that the reduced mutacin gene expression and H2O2 production in the mixed-species cell pellet was not due to inhibition of cell growth of either species during the 2-h coculturing period (data not shown). To test further whether live cells were required to exert this inhibitory effect, S. mutans cells were mixed with UV-killed S. sanguinis cells or vice versa, and pelleting assays were performed. Mutacin gene expression or H2O2 production was not inhibited when dead cells of the other species were present (data not shown).
Since it could be argued that the pelleting assay created an artificial high-cell-density environment, which may not represent the natural dental biofilm situation, we did another experiment under biofilm conditions. We inoculated UA140::Φ(mutAp-luc
) as a single-species culture and as a mixed-species culture with S. sanguinis
in a 1:1 ratio on a BHI plate and incubated the cells for 6 h. Under this biofilm condition, both bacterial species could grow on a surface with an air interface, as could be found in the dental biofilm. The cells were scraped from the plate, and luciferase activity was determined. After normalization with the number of viable cells, we found a 15-fold reduction of the luciferase activity in the mixed-species culture compared to UA140::Φ(mutAp-luc
) alone (Fig. ). As a control, UA140::Φ(ldhp-luc
) was used to monitor the expression of the housekeeping gene ldh
(lactate dehydrogenase), which would reflect the metabolic status of the cells (24
). As shown in Fig. , the expression of the ldh
gene remained the same in the mixed-species biofilm as in the single-species biofilm. The increase in the change from 5-fold (in the pellet) to 15-fold (on the plate biofilm) could be explained by the longer incubation time of S. mutans
in the presence of S. sanguinis
. The longer incubation time was necessary to yield visible cell growth on the plate. This result further confirmed the observations made in the pelleting assays (Fig. ), suggesting that in the dental biofilm, the presence of S. sanguinis
could inhibit mutA
gene expression of S. mutans
FIG. 7. Relative mutacin I (mutAp-luc) and lactate-dehydrogense (ldhp-luc) gene expression in single- and mixed-species surface biofilms. Overnight cultures of all strains were adjusted to an OD600 of 1. Ten microliters of strain UA140::Φ(mutAp-luc) or (more ...)