The myriad interactions between the indigenous gastrointestinal microbiota and its mammalian host have been a focus of considerable recent scientific investigation. Studies of human subjects have the advantage of directly examining the natural community responsible for specific diseases. However, due to technical and ethical constraints on examining the human microbiota, a great deal of effort has been applied to studying model systems, in particular murine models.
Two main lines of research have provided insights about host-microbiota interactions in murine models. Studies with germ-free and gnotobiotic mice have demonstrated that gut bacteria can transmit signals that influence host responses (20
). However, these are highly simplified systems where community complexity is orders of magnitude lower than that of the naturally occurring murine microbiota. An alternative approach has been to study how ecological stressors shape complex communities in murine model systems. In many cases, antibiotics are employed to alter the indigenous microbiota, thus disturbing the normal, baseline host-microbe interactions. Such an approach has demonstrated a role for the microbiota in genetic models of murine inflammatory bowel disease (26
) and in the modulation of glucose tolerance in mouse models of insulin resistance (37
). Antibiotic treatment studies have shown that antibiotic-resistant bacterial pathogens can exploit innate immune deficits triggered by antibiotic administration (5
). Antibiotic regimens have been used to demonstrate a role for the indigenous microbiota in shaping physiological responses of the gut mucosa, including mediating protective responses to direct epithelial injury (43
) and directing the differentiation of IL-17-producing T-helper cells in the mucosa of the small intestine (24
). Antibiotic-treated mice demonstrate altered susceptibility to experimental infection with pathogenic bacteria. Treatment of mice with streptomycin increases susceptibility to oral infection with Salmonella enterica
serovar Typhimurium (52
). A recently described murine model of Clostridium difficile
-associated colitis employed pretreatment with a mixture of five antibiotics followed by a single dose of clindamycin a day prior to oral challenge with C. difficile
These studies have generally focused on the host response to the alteration in the indigenous microbiota. In most cases, the nature of the antibiotic-induced changes in the microbiota was not investigated. Some studies measured changes in total aerobic and anaerobic culturable bacteria following antibiotic administration, and in a few cases, limited culture-independent investigation of the microbiota was performed. An implicit assumption of these studies is that genetically identical mice harbor a consistent baseline microbiota. A further assumption is that the use of antibiotics would result in reproducible changes in the microbiota that would be responsible for the altered host responses observed. These critical assumptions have never been formally addressed in detail until the current study.
It has been proposed that an adult mammal harbors a stable “climax community” in each anatomic area of the gastrointestinal tract (48
). Although there can be individual variation in the composition of shallow phylogenetic lineages within the gut microbiota, there are relatively few deep lineages, with Firmicutes
generally dominant in most surveys (11
). These observations most likely reflect the influence of a variety of ecological and evolutionary constraints on the gut microbial community (28
). Our results, demonstrating marked similarity between the gut microbiotas from individual animals, albeit among individuals with identical genetic backgrounds maintained in a tightly controlled environment, provide strong evidence that the gut microbial community represents a stable ecosystem. This high degree of similarity also provides evidence for the existence of community “assembly rules” that govern the establishment and stability of these microbial consortia.
Perhaps the more critical assumption in experiments that involve antibiotic manipulation of the indigenous gut microbiota is that drug treatment results in reproducible alterations of the microbial community structure. The relative stability of the indigenous microbiota has been debated. From an ecological standpoint, the term “stability” (also commonly referred to as “robustness”) encompasses a number of components (2
). One aspect is temporal stability, which is the constancy of community structure over time. In addition, the term “resistance” refers to the ability of a community to maintain a given structure in the setting of a perturbation, while “resilience” is the ability of a community to return to its baseline structure following a perturbation in community structure. In this regard, if a community exhibits temporal stability, this implies the presence of resistance and resilience in the community structure, since one assumes that most communities will experience ecological stress at some point.
A number of studies have indicated that an individual's gut microbiota can have a relatively stable community composition over a period of months to years (36
). These observations have led to the conclusion that the community of microbes in the gut is relatively resistant to perturbation by various ecological stressors. Subsequent environmental influences, including diet, host genetics, medication use, and exposure to infectious agents, can all influence the resultant microbial community (11
). It has been reported that short-term administration of antibiotics could result in long-term changes in the structure of the fecal microbiota of humans (12
). In all of these human studies, there was considerable individual baseline variation in the microbiotas, which made it difficult to make interindividual comparisons in the microbiota responses.
Although human studies such as these are important, one advantage of conducting murine experiments as described here is the ability to conduct true controlled, replicate experiments. Our replicate experiments allowed us to detect consistent shifts in the gut microbial community in response to antibiotic administration. The reproducibility of these changes indicates that even if the influences on microbial community structure are complex and numerous, the community will exhibit stereotypic responses if ecological stressors are consistently applied. We observed reproducible shifts in the community structure of the gut microbiota following antibiotic treatment, including significant alterations in both the richness and the distribution of 16S V6 phylotypes. The power of a deep survey of diversity allowed us to demonstrate that certain low-abundance phylotypes present at baseline could become dominant in response to the shift in environmental conditions brought about by antibiotic administration. In control animals, 16S V6 tag sequences corresponding to members of the family Enterobacteriaceae made up only a small fraction of the population (1%). During AMB administration, this group of organisms became the dominant phylotype, indicating that this antibiotic regimen created an environment that somehow favored this taxonomic group of organisms. Simple resistance to the antibiotics cannot entirely explain this observation, because other phylotypes were unchanged in relative abundance following AMB administration and did not undergo the remarkable relative expansion during drug treatment exhibited by the Enterobacteriaceae.
In this case, the gut microbial community exhibited resilience as the community structure shifted back toward the baseline state following cessation of the AMB treatment. However, the ability of this community to recover following antibiotic disturbance was not absolute. The administration of cefoperazone also caused dramatic shifts in community structure, but in this case, diversity did not recover even 6 weeks after the discontinuation of the drug. Rarefaction analysis revealed a persistent, significant decrease in overall species richness in the gut community following cefoperazone administration. However, the addition of an untreated mouse to cages of cefoperazone-treated animals during the recovery phase allowed complete restoration of diversity, presumably through natural coprophagic activity. This observation indicates that cefoperazone administration did not change host physiology, since exposure to a baseline microbiota resulted in normalization of the community structure. Additionally, we infer that the baseline community structure is “preferred,” since all four animals in the cage possessed a community that we cannot distinguish from that in untreated control animals. Since the donor animals were exposed to the altered communities present in the cefoperazone-treated animals, it is possible that the resultant communities would possess the antibiotic-altered community structure or an intermediate structure.
The reasons for the differences observed in community resilience are not entirely clear. The ecological disturbance mediated by cefoperazone appears to have overcome community resilience, potentially due to different spectra of antimicrobial activity. Regardless of the underlying reasons for the differences in observed community resilience, from an experimental standpoint it is important to understand that manipulation of the indigenous gut microbiota by various antibiotic regimens may result in altered community structures that persist even after the antibiotic is discontinued. Whether or not the gut community returns to the baseline state after perturbation can influence the conclusions that can be drawn from a particular experiment.
The implications of long-lasting changes in community diversity following antibiotic administration are severalfold. Even though the microbial composition of the animals that recovered from cefoperazone treatment remained altered from the baseline, overall bacterial biomass returned to the level observed in control mice. It has been postulated that functional redundancy in complex microbial communities can allow an altered community to perform ecosystem functions equivalent to those of the original community (2
). Studies are ongoing to determine if the specific alterations in the gut microbiota result in any significant changes in gut ecosystem functioning.
One function of the gut microbiota that has captured our attention is “colonization resistance,” the ability of the indigenous microbiota to prevent the ingress of pathogens into the gut community (16
). Antibiotic-associated colitis resulting from Clostridium difficile
infection may result from a loss of the intrinsic colonization resistance of the gut microbiota (4
). Theoretically, the administration of antibiotics could disturb the indigenous microbiota, allowing C. difficile
spores encountered in the environment to germinate and successfully colonize the gut (4
). Although C. difficile
infection responds to the administration of specific antimicrobial therapy, including metronidazole or vancomycin, recurrence following the end of C. difficile
therapy has become an increasing problem (35
). In a previous study, we provided evidence that recurrent C. difficile
infection is associated with a decrease in fecal microbial diversity (7
). This observation is in line with the fact that the administration of stool from healthy individuals to patients with recurrent C. difficile
can break the cycle of recurrence (1
). The data presented here indicate that antibiotic therapy of sufficient magnitude can result in an altered microbial community. It remains to be determined if this can be directly correlated with a loss of colonization resistance, but our findings provide evidence that antibiotic administration can result in long-term decreases in gut microbial diversity, which in turn are associated with recurrent C. difficile
As we learn more about the intricate relationship between the gut microbiota and its host, we may find that unintended disturbance of this microbial community will have significant deleterious health effects. A more complete understanding of the ecological forces that determine the formation and maintenance of microbial community structures could lead to novel ways to prevent or treat diseases that result from disruptions of the gut microbiota.