Extensive studies have been performed to reveal the microbial diversity in human oral cavity and intestinal tract, two important microbial habitats along the human GI tract [1
]. So far, more than a thousand distinct species or phylotypes have been recovered from the human intestinal tract [31
], while over 700 species have been identified from human oral cavity based on traditional cultural studies and culture-independent molecular studies [1
]. Even though both the oral cavity and the intestinal tract are part of the human GI tract and most intestinal microbes may initially enter through the oral cavity, recent microbial community analysis showed that the two habitats share surprisingly few bacterial species [25
]. This raised an interesting and fundamental question: How are these peculiar microbial communities established and what are the underlying mechanisms in maintaining the existing microflora?
A critical role for host factors in shaping the GI tract-associated endogenous microbial communities has previously been implicated [19
]. Reciprocal gut microflora transplants from Zebrafish and mice to germ-free recipients by Rawls et al. established that the transplanted microfloras are similar to the community of origin [33
]. However, the relative richness of the lineages shifted toward the composition of the normal intestinal microflora in the recipient, thus convincingly demonstrating the host habitat selection on the intestinal tract-associated microbial community [33
]. The same principle could apply to the microbiota associated with different parts of the human GI tract. Different micro-environments along the GI tract (local anatomical structures, nutrient availability, and host immune reaction) could exert selective pressure on the microbes which result in the distinct microbial communities.
While the local host and nutritional factors could contribute significantly to the microbial compositions in different parts of the GI tract, we hypothesized that the existing microbial flora plays a role in selecting the bacterial species that can be integrated into the community independent of host factors. To test this hypothesis, we established an in vitro system comprised of cultivable microbial flora isolated from two distinct niches along the GI tract of mice, the oral cavity and intestinal tract. One of the limitations of studying microbial community using culture-based methods is the often under-represented microbial composition in cultivable microbes compared to the original samples [38
]. Due to this limitation, the in vitro system we established in this study using BHI-cultivable microbial species can only represent small subsets of the two distinct microbiotas along the mice GI tract. Nevertheless, culture-dependent methods are indispensable in providing valuable information, including both phenotypic and genetic characterization of individual bacteria, as well as new physiological functions prompted by the interactions among different microbial community inhabitants. Our in vitro study using two cultivable microbial sub-communities allowed a glimpse into the intriguing dynamic interactions between the microbial floras associated with oral cavity and intestinal tract of mice.
16S rDNA-based PCR-DGGE analysis revealed distinct microbial patterns within the two cultivable communities (Fig. ). Staphylococcus
spp., and Lactobacillus
spp. are among the most dominant genera recovered from the oral samples of mice, while Enterococcus
spp., E. coli
, and Lactobacillus
spp. were most frequently cultivated from the corresponding intestinal tracts. These results correlated well with previous cultivation-based microbial community analyses of the GI tract-associated microbiota of mice [4
The in vitro community competition assay revealed an intriguing antagonistic action between cultivable oral and intestinal sub-floras. Depending on the sequence of inoculation, the established pre-existing microbial community always exhibited a survival advantage and exerted inhibitory effects on the members of the incoming flora of different origin (Fig. ). In environmental ecosystems, community level competition is a relatively rare event and only occurs when natural barriers break down [40
]. However, when this happens, one community may overtake a second community, causing all or most of its members to go extinct [40
]. Furthermore, pre-existing communities develop invasion resistance over time, which enhances their ability to defend their habitats [6
]. The behavior of the two distinct communities tested in this study is consistent with these observations. The original isolates and their closely related strains tested persisted well in their original community and were inhibited when grown together with the microbial community of different origin (Figs. and ). In environmental ecology, it has been shown that isolated communities often interact by sending “invasion propagules” between one another. Such small-scale probes, however, often had no permanent effect on the communities due to the invasion resistance developed in the communities and were regarded as one of the mechanisms in maintaining the stability of the community [6
]. Although most of the current invasion resistance concepts are derived from non-microbial ecosystem, the same principle could apply to the microbial world. Our in vitro data are in agreement with above ecological phenomenon, suggesting that the dynamics among members from the same community could contribute to the invasion resistance, thus play an important role in maintaining the structure and stability of the community.
The maximum inhibitory effect toward foreign bacteria was achieved when the whole cultivable community was used, suggesting a community-based antagonistic action (Figs. and ). This is in agreement with observation in other ecosystems where species-rich communities are more resistant to invasion by exotics than species-poor communities [13
]. Our in vitro oral and intestinal community comprised of at least 20 and 18 different bacterial species, respectively. The members derived from the same microflora are likely to have co-evolved and therefore have experienced extensive interactions with each other and built certain coordinated relationships among themselves [17
]. This might contribute to the community invasion resistance by initiating a series of defense responses through synergistic interactions among the endogenous residents when foreign bacterial invaders are encountered. Our observation is corroborated by similar phenomena that have been reported for other microbial communities. The microbial community isolated from the surface of the marine alga Ulva australis
was demonstrated to interact synergistically in biofilms and resist foreign bacterial invasion to a greater extent than single-species biofilms [5
]. Furthermore, the result of the spent medium assay indicates that the antagonistic effects toward foreign bacteria are unlikely due to the accumulation of toxic metabolites from the respective communities (Supplementary Fig. S1
) but rather the result of more active processes involving interactions between foreign bacteria and community members.
Clearly, this newly established inter-flora interaction system is just a starting point and far from being a perfect system. Since only a small sub-population of oral and intestinal communities can be recovered by cultivation, the data obtained in this study cannot entirely represent the real situation in the host. Nevertheless, we were able to consistently observe community-based antagonistic actions toward foreign bacteria even under the same nutritional conditions independent of host environment. Similar results were obtained for another major intestinal representative species E. coli (see the accompanying manuscript He et al. 2010). Taking advantage of the well-understood genetic background and pre-established mutant collections in E. coli, we were able to reveal the molecular mechanism underlying the observed phenomenon.
From an evolutionary and ecological point of view, the GI tract represents a complex ecosystem with several distinct microbial habitats, such as the oral cavity and intestinal tract. The microbial diversity within each habitat is the result of co-evolution between microbial communities and their specific host factors, and the actual events of community establishment and maintenance often involve a series of microbial intra-, interspecies, and microbe–host interactions. While host factors could be the key determinants for establishing the GI tract-associated microbial community structure, our in vitro analyses of cultivable mice oral and intestinal microbial communities suggested an important role of the dynamics within a given microbial flora in shaping and maintaining the existing communities. The observed inhibitory effect toward foreign bacteria could contribute to the protective/probiotic effect of established microbial communities within the GI tract and play a significant role in fending off potential pathogens which would be recognized as foreign.