The Mammalian Microbiome and Its Importance in Laboratory Animal Research

Abstract

In this issue are assembled 10 fascinating, well-researched papers that describe the emerging field centered on the microbiome of vertebrate animals and how these complex microbial populations play a fundamental role in shaping homeostasis of the host. The content of the papers will deal with bacteria and, because of relative paucity of information on these organisms, will not include discussions on viruses, fungus, protozoa, and parasites that colonize various animals. Dissecting the number and interactions of the 500–1000 bacterial species that can inhabit the intestines of animals is made possible by advanced DNA sequencing methods, which do not depend on whether the organism can be cultured or not. Laboratory animals, particularly rodents, have proven to be an indispensable component in not only understanding how the microbiome aids in digestion and protects the host against pathogens, but also in understanding the relationship of various species of bacteria to development of the immune system. Importantly, this research elucidates purported mechanisms for how the microbiome can profoundly affect initiation and progression of diseases such as type 1 diabetes, metabolic syndromes, obesity, autoimmune arthritis, inflammatory bowel disease, and irritable bowel syndrome. The strengths and limitations of the use of germfree mice colonized with single species of bacteria, a restricted flora, or most recently the use of human-derived microbiota are also discussed.

Defining the Microbial Landscape

The microbiome of hosts, also known as microflora or microbiota, is routinely defined as all the microorganisms inhabiting a specific environment, and these terms are often used interchangeably. The term microbiota has been used historically, and, most likely, the suffix “-biota” was used to define “living organisms in an ecosystem.” The term microbiome was coined in the “-ome” and “-omics” era by Lederberg and McCray “to signify the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share our body space and have been all but ignored as determinants of health and disease” (2001). Although “-om” and “-ome” surely have meanings in linguistics and biology, the authors proposed that “the -ome idea is borrowed from the multitude of terms already ensconced into English or the scientific lingua franca,” rather than being derivations from Greek or Sanskrit (Lederberg and McCray 2001). In contrast, others proposed to define microbiota as the microbial taxa and the term microbiome as the catalog of these microbes and their genes (Ursell et al. 2012). Either term can be used to describe microbial communities, and the holistic “-ome” approach also includes their genetic information.

Research in this field has attracted considerable interest in the scientific community over the last decade (Figure 1). Reasons for this are the increasing realization that the microbiota has an enormous impact on the phenotype of various animal models and, secondly, that molecular methods have become available, allowing the analysis of complex microbial communities to a degree that was not obtainable using traditional culture methods. Importantly, guidelines for analyses of microbiota via bacterial culture or sequencing platforms are reviewed in this issue by Hiergeist and colleagues (2015).

Figure 1.

PubMed Search using terms microbiome or microflora, total hits per year.

The Indigenous Microbiota

It is well established that mammals are colonized by microorganisms that outnumber the host in respect to human cells by a factor of 10–100. For example, the human intestine contains 10¹⁴ bacteria with approximately 10⁶ microbial genes. Both microbes and the host benefit in a mutualistic way, the first from specific habitats, the last from the microbial activity (e.g., degradation of xenobiotics, epithelial homeostasis, protection against pathogens) (Eberl 2010). At birth, humans are colonized by approximately 100 bacterial species, increasing to 700 at weaning and 1000 in adulthood.

In humans, several factors influence microbiota composition, including age, method of delivery, breast versus bottle feeding, as well as environmental factors like medication, diet, and stress. As an example, the effect of xenobiotics and their effect on intestinal microbiota and resulting metabolomic profile are reviewed in this issue by Lu and colleagues (2015). Compositional changes in the microflora may lead to dysbiosis, possibly contributing to development of various diseases like inflammatory bowel disease, diabetes, asthma, or obesity, to name a few (reviewed by Nicholson et al. 2012).

Composition of the Microbiota

On the phylum level, the gut bacteria are similar in mammals, for example, in humans and mice. However, this does not apply to the species level (Figure 2). Presence and importance of species-specific microbiota, even within rodent species, was demonstrated three decades ago and was also shown more recently (Boot et al. 1985, 1989; Chung et al. 2012; Heidt et al. 1990; Koopman et al. 1984). Of the more than 50 bacterial phyla (29 of which have cultured representatives) (Youssef et al. 2015), humans and mice are colonized mainly by Firmicutes (a phylum containing bacteria like clostridia, lactobacilli, streptococci and staphylococci), Bacteriodetes (like Bacteroides, Porphyromonas), Actinobacteria (like Actinomyces, Streptomyces), and Proteobacteria (which contain Enterobacteriaceae like E. coli or Helicobacter spp.). Composition and complexity vary at different regions of the body, with the highest number of species being found in the colon (mainly Firmicutes and Bacteroidetes in mice and humans) and only a few species in the acid-secreting stomach or the genital tract (Dethlefsen et al. 2007; Sheh and Fox 2013).

Figure 2.

Taxonomic classification of bacteria. Descriptions of the gastrointestinal microbiota focus on the levels of phylum and genus (modified from Sheh and Fox 2013).

In mice, considerable variation has been detected in microbiota composition between mice housed in different facilities, different barriers within a given facility, and even different cages and strains within a barrier in which the mice are housed (Büchler et al. 2012; Hufeldt et al. 2010; Yang et al. 2013). Gut microbiota composition in mice mainly depends on the type of barrier (degree of protection from environmental microbes) and how the barrier was established. For example, the use of germfree mice, or mice colonized with known bacteria like the altered Schaedler's flora (ASF), which is reviewed in this issue by Brand and colleagues (Brand et al. 2015). Further, acquisition and use of mice from commercial vendors and how mice are imported to a facility (quarantine or embryo derivation) is discussed. Importantly, variables such as diet and treatment of water (e.g., chlorination, acidification) is covered (Bleich and Hansen 2012; Wolf et al. 2014). Each of these issues is emphasized and reviewed in this issue by Hansen and colleagues (2015).

Impact of the Microbiota on Animal Models

The impact of the microbiota on host physiology is readily observed when comparing conventional to germfree mice. The enlarged cecum in germfree animals is a characteristic finding evident to even a casual observer. The enlarged cecum occurs from osmosis due to nondegraded mucopolysaccharides that bind sodium and enhance intestinal atonia. A variety of mucosal parameters differ in germfree mice like decreased epithelial renewal, enzyme production, and mucosa thickness. Microbiota play a role in bile salt metabolism, production of short-chain fatty acids, or vitamin K and B-complex vitamins in the large intestine. Immunological changes in germfree animals, such as reduced populations of various innate and adaptive immune cells and reduced or altered cell-specific activities, have been reviewed (Round and Mazmanian 2009). Germfree mice are very susceptible to intestinal pathogens, such as Salmonella spp., and are easily colonized with Escherichia coli; even the probiotic bacterium E. coli Nissle can induce significant disease in mice of certain genetic backgrounds (Bleich et al. 2008).

Given these variables, it becomes clear that absence of the microbiota, as in germfree rodents, reviewed in this issue by Nicklas and colleagues, has a considerable impact on animal models as well (2015). While effects on gut inflammatory phenotypes and intestinal disease models could be expected from absence of gut microbiota (Bleich and Mähler 2005), other conditions such as diabetes, obesity, arthritis, allergy, inflammatory pain, or atopic dermatitis can be affected as well (reviewed in Bleich and Hansen 2012). Even changes in microbiota more subtle than presence or absence lead to phenotypic variation. Limited microbial diversity might be of concern in newly established barrier mouse colonies that were initiated using gnotobiotic mice (usually mice colonized with known bacterial species like the ASF). It has been shown that ASF-colonized mice share more similarities to germfree than to conventionally colonized mice with respect to a number of metabolistic characteristics (Norin and Midtvedt 2010). Therefore, it is possible that a limited diversity could lead to artificial or even a loss of phenotypes in widely used animal models. Further, variation of complex microbiota might lead to phenotypic alterations. A well-known example is the interleukin-10-deficient mouse model of inflammatory bowel disease, which, when maintained at different institutions, develops either spontaneous or Helicobacter-induced disease at differing rates and severity (Keubler et al. 2015; Mähler and Leiter 2002; Yang et al. 2013). Intestinal polyp development was recently described as being reduced in HB-EGFR-transgenic mice after antibiotic treatment (Bongers et al. 2014), and diabetes incidence was reduced in nonobese diabetic (NOD) mice after they received acidified water (Wolf et al. 2014). In principle, all of these models are influenced by variation in the microflora, and the number of published models being affected by microbial variation continues to grow. These models and the importance of the microbiome in the etiopathogenesis of immune-mediated diseases are reviewed in this issue by Hörmannsperger and colleagues and Becker and colleagues (Becker et al. 2015; Hörmannsperger et al. 2015).

Analyzing the Effects of Microbiota

Different strategies are used to identify relevant bacteria as well as mechanisms of microbiota-related phenotype variation. Approaches range from analyzing the effect of single species, simplified communities, or even complex microbiota (e.g., in gnotobiotic animals, reviewed in this issue by Ericsson and Franklin [2015]). Using models of intestinal inflammation, protective effects have been observed using Lactobacillus spp. (Madsen et al. 1999; Schultz et al. 2002; Ukena et al. 2007), Bifidobacterium spp., or E. coli Nissle (Ukena et al. 2007), which are considered to be probiotic bacteria, whereas proinflammatory effects were observed using Enterococcus faecalis, E. coli (Balish and Warner 2002; Kim et al. 2005), Bacteroides vulgatus (Rath et al. 1999), Helicobacter spp. (Fox et al. 1994, 1995, 2011), and murine norovirus (Basic et al. 2014; Cadwell et al. 2010). Segmented filamentous bacteria (SFB) are now known to induce Th17 cells in the intestine, and mice from barriers without SFB may have low Th17 responses compared with SFB-positive, barrier-maintained mice that have strong Th17 responses; and the effect is transferrable with transfer of SFB (Gaboriau-Routhiau et al. 2009; Ivanov et al. 2009). Simplified bacterial communities maintained in gnotobiotic mice, or even complex microbiota administered to germfree animals, revealed pathophysiological effects that vary with the microbial communities (Eun et al. 2014; Faith et al. 2014; Slezak et al. 2014; Wos-Oxley et al. 2012). Molecular analysis of microbiomes using sequencing approaches is increasingly used to elucidate the effect of complex communities on the phenotype expressed in different animal models (Yang et al. 2013).

What is the Relevance of the Microbiome for Scientists Using Animals?

Scientists should be aware of the importance of the intestinal microbiota on the pathophysiology of the host and on the development of various diseases. Alterations in the microbiota can lead to variations even more dramatic than pathogenic organisms. Furthermore, the microbiota will be increasingly important when evaluating the health status of various mouse colonies. Health profiles traditionally and continuously focus on pathogens or potential pathogens (Figure 3a). Screening for specific microorganisms in health-monitoring programs depends on their possible detrimental effect on animal or human health or certain types of research (Mähler et al. 2014; Pritchett-Corning et al. 2014; Shek in press). New concepts in the broader appreciation of microbe–host interactions are reviewed in this issue by Hornef (2015). The future of microbial surveillance and standardization may include some characterization of the complexity and diversity of the microbiota and the presence or absence of phenotypically relevant (but not pathogenic) agents such as the SFB (Figure 3b). Technologies for such analyses are available and are becoming increasingly feasible in rodent health and surveillance programs.

Figure 3.

Concepts for microbial surveillance and standardization in laboratory animals. (a) Traditional and current focus of health monitoring is based on virulence of microorganisms (symbiotic, opportunistic, pathogenic) and aims to exclude or report pathogens (and potential pathogens), depending on the barrier level and specified excluded agents. (b) Microbial relevance (other than pathogenicity) to the host is increasingly recognized, as is the complexity and diversity of the microbiota.

Institutional Animal Care and Use Committee (IACUC) Considerations

For most, if not all, IACUCs, there are currently no requirements for detailing the microbiome of animals being used for research or testing, other than perhaps a list of pathogenic organisms excluded from specific pathogen-free colonies. The issue of the microbiome regarding specified research questions is clearly part of an increasingly popular research theme and is considered in the context of individual research protocols, where principal investigators have the expertise to probe a particular question regarding the microbiome. To contemplate IACUC review and approval of microbiome profiles in animal models is premature and fraught with unforeseen problems.

Organization of the Current Issue

This issue of ILAR Journal offers 10 current, concise, and informative reviews on the microbiota's impact in shaping the host's immune response, in the central role of the microbiota in determining susceptibility to a variety of diseases, and on research considerations in animal models. The issue starts with descriptive information and definitions, followed by reviews of the function of microbiota, and it concludes with technical aspects. In the first review, Hornef provides information on the definition of microorganisms according to their virulence potential (2015). This is followed by a description of the microbiota of mammals, summarized in the informative paper by Nelson (2015). Defined floras are a valuable tool to model complex microbiota–host interactions, which is elaborated upon by Brand and colleagues, who review the history, composition, and use of the ASF (2015). Effects of the intestinal microbiota on the host are reviewed by Becker and colleagues and Hörmannsperger and colleagues (Becker et al. 2015; Hörmannsperger et al. 2015). Ericsson and Franklin elaborate on approaches to manipulate the gut microbiota of research animals (2015). The interaction of xenobiotics with the gut microbiota is reviewed by Lu and colleagues (2015). Technical aspects are covered by Hiergeist and coworkers, who provide standards for analyzing the microbiome (2015), and Nicklas and colleagues, who describe the maintenance and monitoring of gnotobiotic rodents (2015). Finally, Hansen and colleagues conclude with applied veterinary aspects with regard to the impact of the gut microbiota on rodent models (2015).