Colonization is the initial step in the pathogenesis of many enteric bacterial pathogens. A great deal of insight has been gained on the genetic adaptations that enteric pathogens have evolved to permit successful colonization and the eventual development of disease (12
). A key emphasis has been placed on examining the interaction between pathogenic bacteria and host cells. An aspect of bacterial pathogenesis that has been less studied is the interaction between pathogenic bacteria and the preexisting microbiota that inhabits a particular ecologic niche within the host. The indigenous microbiota of the host have been postulated to interfere with the invasion by pathogenic organisms, so-called “colonization resistance” (6
). It has been observed that certain bacteria residing in the GI tract, whether naturally occurring or experimentally administered, can protect the host from pathogenic bacteria (1
). It is likely that a number of mechanisms, including nutrient depletion, competition for binding sites on the mucosal epithelium, and the production of inhibitory substances contribute to colonization resistance (7
One of the reasons that the interaction between pathogenic bacteria and the indigenous host microbiota has not been studied in detail is that the study of complex microbial communities has been difficult. The intestinal tract of mammals is inhabited by a large and phylogenetically diverse community of microorganisms, many of them obligate anaerobes. Over the past decade, molecular-based approaches have revealed enormous phylogenetic diversity in the microbial world that is not yet represented in culture (34
). These non-culture-based techniques, which generally involve the retrieval of the DNA sequence of the small subunit rRNA gene (16S rRNA-encoding gene in the case of bacteria), were initially developed to examine microbial diversity in soil and aquatic environments. More recently, these techniques have been used to examine the indigenous microbiota of mammals. Earlier culture-based examinations of the biota of the mammalian gastrointestinal tract suggested that the majority of morphotypes seen by microscopic examination could be cultivated in the laboratory (33
). More recent culture-independent analysis of the microbial ecology of the gastrointestinal tract has suggests that the overall diversity is greater than previously estimated (4
). This is due in large part to the fact that sequence-based methodologies can discriminate between bacterial isolates that may have identical morphologies and similar in vitro characteristics.
Initially, culture-independent studies on the mammalian gastrointestinal tract cataloged the species richness that is encountered in that environment (29
). More recently, these techniques have also been used to follow changes in the intestinal microbiota over time and to compare the resident microbiota between individuals. These studies have revealed that individuals posses a community of microbes that can vary extensively from individual to individual, and within an individual, it can vary with anatomic location (11
). We have used these techniques to monitor the changes in the fecal microbiota that can occur in the setting of antibiotic-associated diarrhea (50
is a murine pathogen that has been found to be widespread in research mouse colonies. Depending on the strain of mouse, H. hepaticus
infection is associated with biliary tract or lower gastrointestinal tract disease. In many strains of mice colonized with H. hepaticus
, hepatic disease is subclinical and may be accompanied by subclinical enteritis (generally typhlitis or colitis) (41
). However, in mice with altered immune function, the typhlitis/colitis can be severe, leading to rectal prolapse, weight loss, and death. This murine typhlitis/colitis in the setting of altered immune function has been employed as a model for inflammatory bowel disease (14
In the experiments presented here, we used culture-independent community analysis to monitor changes in the mucosa-associated microbiota of the cecum during murine infection with H. hepaticus. Wild-type animals were chosen for this infection study to avoid any changes in the microbial community structure secondary to the development of typhlocolitis. In this way, any alterations in the mucosa-associated microbiota could be attributed to colonization by H. hepaticus alone and not by changes in the mucosal environment due to the development of an active inflammatory response.
T-RFLP analysis proved to be a useful method for estimating the abundance of H. hepaticus
in the mucosa-associated community. Previous studies using culture (16
) and a quantitative PCR assay targeting the cytolethal distending toxin of H. hepaticus
) suggest that the organism is encountered in high numbers in the cecum of colonized mice. Although there are potential biases that result from using PCR to interrogate an entire community (46
), the T-RFLP data presented here suggest that H. hepaticus
becomes the dominant member of the mucosa-associated microbiota of the cecum in infected animals. This conclusion is based on the assumption that peak height by TRF is proportional to the abundance of a particular 16S rRNA-encoding gene species in the community and thus the relative abundance of that particular bacterial species. Clone library analysis also confirmed that H. hepaticus
readily colonizes the cecal mucosa and becomes the predominant bacterial species present.
It has been previously suggested that T-RFLP analysis is appropriate for monitoring changes in a given community (28
). The use of T-RFLP also revealed that the process of colonization of the cecal mucosa by H. hepaticus
was associated with reproducible shifts in the overall structure of the microbial community. In the time course experiment, the kinetics by which H. hepaticus
increased as a component of the community were similar from animal to animal, as were the changes in the non-H. hepaticus
TRFs. Additionally, the C57BL/6 mice in the first experiment came from a different colony than the C57BL/6 mice used in the time course experiment. There were detectable differences in the TRF profiles between uninfected animals from each of these colonies (data not shown). However, the final shifts in the community resulting from H. hepaticus
colonization were very similar, suggesting that H. hepaticus
challenge applies a reproducible ecologic stress on the existing microbiota.
We used indices of community diversity, traditionally applied to macroecologic communities such as wetlands and forests, to quantify the changes observed using T-RLFP analysis in the community structure of the mucosa-associated microbiota following colonization with H. hepaticus. H. hepaticus colonization resulted in a significant decrease in the diversity of this community. This effect appears to be primarily due to the relative dominance that H. hepaticus assumes within the community. Reanalysis of the T-RFLP profiles following removal of H. hepaticus from the mucosal communities of infected animals revealed that the diversity of the remainder of the community was not significantly different from that seen in uninfected animals. This suggests that H. hepaticus has minimal interactions with other members of the indigenous microbiota, at least not interactions detectable by analysis of the entire community by T-RFLP profiling.
Reanalysis of the clone libraries with suppression of H. hepaticus
-specific clones in general supports the findings of the repeat T-RFLP analysis. However, the finer-scale resolution of taxonomic information provided by 16S rRNA-encoding gene sequence analysis supplies some additional insights into the community dynamics encountered in animals infected with H. hepaticus
. LIBSHUFF analysis provides a measure of the difference between two 16S rRNA-encoding gene clone libraries based on the ability of one library to “cover” the diversity seen in a second library (40
). LIBSHUFF analysis of the libraries when H. hepaticus
16S rRNA-encoding gene clones are included indicates that the library from the uninfected animal is unable to provide coverage of the library from the H. hepaticus
-infected animal (P
= 0.002), but in the reverse case, the library from the infected animal provides at least partial coverage of the library from the uninfected animal (P
= 0.076). These results are consistent with the idea that the library from the uninfected animal is a subset of the library from the infected animal (40
), and this is supported by visual inspection of the phylogenetic tree. However, LIBSHUFF analysis following the removal of 16S rRNA-encoding gene clones with homology to H. hepaticus
provides additional information regarding the diversity of the indigenous microbiota in H. hepaticus
-infected animals. This reanalysis shows that the library from the uninfected animal easily provides coverage of the library from the infected animal (P
= 0.31) when H. hepaticus
is removed. However, there is a trend (P
= 0.051) towards inadequate coverage of the library from the uninfected animal by the library from the infected animal. Therefore, while the most obvious changes in diversity, as measured by T-RFLP and clone library analysis, are due to the dominance that H. hepaticus
assumes within the community, clone library analysis suggests that more subtle perturbations of the indigenous microbiota may also be occurring.
At first glance, it is somewhat surprising that H. hepaticus
was able to “invade” an established, diverse ecosystem with such ease. However, H. hepaticus
, a bona fide murine pathogen, is excluded from most specific-pathogen-free mouse colonies (47
). Thus, experimentally introduced H. hepaticus
may be filling an underutilized or possibly “empty” ecologic niche in the gastrointestinal tract. In part, this can explain the apparent lack of interaction (e.g., direct competition) between H. hepaticus
and other members of the mucosal microbiota. It has been proposed that successful introduction of an “invasive species” to an established ecosystem is a function of how different an invader is from established species (44
To our knowledge, this is the first time that culture-independent techniques have been used to monitor the invasion of a complex mammal-associated microbial community by a pathogen. Our results demonstrate the power of this type of analysis on revealing details of the relationship between a pathogen and the existing microbiota of the gut. Given the importance of the indigenous GI microbiota in both health and disease, the development and use of methods by which this complex population can be monitored will aid in the study of a wide variety of gastrointestinal conditions including gastroenteritis, antibiotic-associated diarrhea, and inflammatory bowel disease.