In a recent study, we used in vitro microbial communities isolated from different parts of the GI tract of mice (oral cavity and intestines) as a model system to examine the interflora interactions. We demonstrated that an in vitro community composed of bacterial members isolated from the same natural niche exhibited invasion resistance and prevented the integration of bacterium of foreign origin (accompanying manuscript). This study was designed to further understand the underlying mechanisms of the species restriction phenomenon between microbial communities of different origin through molecular analysis using E. coli as a model intestinal bacterium.
was one of the bacterial species frequently isolated from the intestinal tract of mice, but not the oral cavity. As an intestinal bacterium, E. coli
suffered great loss in viability when encountering an established oral microbial community (Fig. ). This is in agreement with the result of our accompanying study in which all tested microbial isolates from the GI tract of mice displayed a striking community preference, and they only thrived in the community of their origin and were excluded from the community of foreign origin, suggesting that an existing microbial community could develop invasion resistance and impose selective pressure on incoming foreign bacterial species independent of host selection (“community selection” effect). From an ecological point of view, the invasion resistance is an important feature of established communities and is regarded as one of the key mechanisms in maintaining community stability [5
]. The invasion resistance has been demonstrated in a variety of ecosystems, such as grassland [12
], a marine ecosystem [36
], and a stream fish community [3
]. The fact that E. coli
was unable to establish itself in the oral community suggested a similar invasion resistance effect against E. coli
Although most of the current invasion resistance concepts were derived from nonmicrobial ecosystem, the striking similarity between the resistance phenomenon observed in the oral microbial community and nonmicrobial systems suggested that the similar principle could apply to the microbial world. First, we observed that the whole cultivable microbial communities are more effective than a community composed of fewer microbial species in excluding the integration of bacterium of different origin (accompanying manuscript). This is consistent with the observation that species-rich communities are more resistant to invasion by exotics than their species-poor counterparts [8
]. Furthermore, our observation is corroborated by similar phenomena that were reported in other microbial community. Burmølle et al. demonstrated that a microbial community inhabiting the surface of the marine algae Ulva australis
can inhibit the settlement of fouling organisms, and species isolated from the same epiphytic bacterial community can interact synergistically in biofilms and resist foreign bacterial invasion to a greater extent than single-species biofilms [4
]. More relevantly, it has been shown that the GI tract-associated indigenous microbiota can prevent the colonization of pathogens [35
]. However, although these phenomena are well documented and several studies indicated that commensal bacterial isolates can produce antimicrobial compounds which could inhibit the growth of certain pathogens, no detailed analysis has been performed to further study the molecular mechanism(s) underlying this invasion resistance phenomenon between established microbial communities and invading species.
Using our established in vitro system, we demonstrated that the oral microbial community produced hydrogen peroxide in response to the presence of an intestinal bacterial species (E. coli
) as an effective weapon to kill this foreign invader. Hydrogen peroxide is an effective bactericidal agent that is naturally produced by some organisms as a byproduct of oxygen metabolism. Commensal oral bacterial species, such as Streptococci
spp. and Lactobacillus
spp., are able to produce hydrogen peroxide [14
]; their protective role in defending their natural niches and protecting against infectious diseases has been suggested. For example, Uehara et al. demonstrated that viridans group streptococci may prevent methicillin-resistant Staphylococcus aureus
colonization of the oral cavities of newborns via the production of hydrogen peroxide [38
From the community point of view, the production of hydrogen peroxide could be a double-edged sword, while it kills the intruder, and it might also have negative effects on the residents of the community. However, our data suggested that the oral flora has a few mechanisms to keep this weapon in check. First, hydrogen peroxide is only produced when the oral community encounters intestinal invaders—E. coli
cells (Fig. )—which ensures that the “weapon” is activated only when needed. Second, while the viability of E. coli
suffered a drastic reduction during cocultivation, the viable count of oral microbial community remained quite constant (Fig. ), and the profile of oral flora showed no significant change even after 4 days of cocultivation (Fig. ), indicating that, compared to oral bacteria, E. coli
might have a lower H2
tolerance. This is supported by the MIC data showing a MIC for hydrogen peroxide of less than 1 mM for E. coli
, while most oral isolates displayed higher resistance to H2
up to 3 mM (Supplementary Table 1
The fact that exogenously added vitamin C was able to rescue E. coli
from being killed when cocultivated with the oral community further confirmed that hydrogen peroxide could be the main factor involved in killing intestinal species. Vitamin C—or ascorbic acid—is a reducing agent and well known for its antioxidant activity [26
]. By providing electrons to reactive oxygen species, such as hydroxyl radicals formed from hydrogen peroxide, ascorbate is oxidized to dehydroascorbate, which is relatively stable and does not cause cell damage, while the reactive oxygen species can be reduced to water.
The most intriguing finding of our study is that the oral microbial community is able to detect the presence of the intestinal bacterial species E. coli
and trigger the hydrogen peroxide production defensive response by recognizing the E. coli
LPS. LPS is a major component of the outer membrane of gram-negative bacteria, contributing to their structural integrity [29
]. However, due to its exposure to the very outer surface of the bacterial cell, LPS is also an easy target for recognition by host antibodies and can be detected by an ancient receptor—toll-like receptor 4—of the innate immune system present on immune cells, such as macrophages and neutrophils [28
] and induce a strong response from normal animal immune systems [1
]. Upon recognizing bacterial LPS, one of the effectors produced by immune cells in an effort to eliminate and prevent the invasion of foreign bacteria is hydrogen peroxide [18
], the same toxic chemical we found to be produced by oral bacterial cells when physically interacting with E. coli
The recognition of E. coli LPS by O-mix cells was correlated with the ability of these cells to kill E. coli since the E. coli LPS mutant (waaL) exhibited greatly reduced induction of oxygen-free radicals (Fig. , spot c1; and thus killing) than wild type; however, this triggering ability was restored when wild-type E. coli LPS was added to the medium (Fig. ). Taken together, these results show that bacteria in the O-mix have the ability to recognize the LPS of E. coli and then trigger the production of oxygen-free radicals to kill E. coli, a mechanism similar to that employed by the body’s immune response. Further work is needed to identity the sensor component for the E. coli LPS and the corresponding hydrogen peroxide producer within the oral community.
In our in vitro system, a significant number of the intestinal microflora members belong to the Enterobacteriaceae family, including E. coli, Enterobacter spp., and Shigella spp., which are gram-negative species carrying LPS on their outer membrane, while the oral community is mainly comprised of gram-positive species, such as Streptococcus spp., Staphylococcus spp., and Lactobacillus spp. In the oral cavity, these gram-positive microbes are the major species detected in saliva and supragingival plaque where often the first encounter between the introduced intestinal bacteria and oral microbes takes place. It would be interesting to know whether the presence of LPS on the surface of other intestinal bacterial species can also trigger hydrogen peroxide production and if the LPS-induced H2O2 production is a more generalized mechanism employed by the oral community to prevent the integration of certain intestinal bacteria.
Due to the limitation of culture-dependent methods, the results we obtained using the in vitro system cannot entirely represent the real situation in native conditions. Information regarding the contributions of host selection and noncultivable subpopulation to the shaping and maintenance of the microbial community could not be obtained using in vitro system, and it is possible that factors other than induced oxidative stress could also be contributing to the killing of E. coli. Nevertheless, the results presented here provide important molecular insights as to the mechanism by which oral bacteria restrict colonization of the oral cavity by one of intestinal bacteria—E. coli. Understanding the mechanism of interfloral relationships is important as it will enhance our understanding of the data derived from the human microbiome project and assist in the development of new therapeutic strategies for modulating the protective/probiotic effects of different microbial communities against different pathogens.