Practical Considerations

Methods for characterization of the intestinal microbiota are based on cultivation techniques or molecular tools (Fig. 2-1). The selection of the best approach depends on the study problem (e.g., detection of specific pathogens in clinical specimens, or general characterization of the intestinal ecosystem), the cost, and the availability of technologies. Each method has strengths but also limitations (outlined below). For general ecologic surveys of microbial communities, molecular high-throughput sequencing techniques yield the most information as they allow in-depth identification of microorganisms. For screening of specific pathogens, culture techniques and species specific polymerase chain reaction (PCR) assays may be most useful. Both of these methods are sensitive to sample handling and processing. Detailed instructions for sample collection and shipping for each particular assay should be acquired before sample submission as many laboratories use their own in-house culture or PCR assays. In the case of molecular methods, there is usually no standardization of DNA extraction or PCR protocols among laboratories, and such factors impact on the sensitivity and specificity of the assays. Improper DNA extraction, especially from fecal samples, may result in the presence of residual PCR inhibitors that cause false negative results. Because of the high sensitivity of PCR assays (theoretically a single target copy can be amplified), any DNA contamination can lead to false-positive results. A laboratory should be chosen that has expertise in molecular analysis, and that has validated each assay in the target specimen.

Figure 2-1 Molecular methods for characterization of the intestinal microbiome. PCR amplification of the 16S rRNA (ribosomal RNA) gene allows either direct identification of bacterial phylotypes or the creation of a molecular fingerprint representing the bacterial diversity in a sample. New metagenomic and transcriptomic approaches, based on high-throughput sequencing of DNA or messenger RNA (mRNA) without prior amplification of a specific gene, yield an overview over the gene content of the sample and therefore the functional properties of the intestinal microbiome.

Bacterial Culture

Traditional evaluation of the composition of the canine and feline intestinal microbiota has been obtained using culture techniques. Bacterial culture is useful for assessing the viability of organisms, determination of an active infection, and antibiotic susceptibility testing in clinical specimens. Individual isolates can be typed for epidemiologic surveys of specific strains and their virulence factors. Culture is also valuable for understanding the metabolic properties of individual microbes. The value of bacterial culture is greatest when a clinical sample is evaluated for the presence of specific pathogens (e.g., Salmonella, Campylobacter jejuni).

Several limitations are associated with culture methods, especially if they are used to survey for the presence of unknown microorganisms in intestinal samples. Bacterial culture underestimates total bacterial numbers in the intestine, as microscopic counts (especially when using fluorescent dyes) are typically higher than the total viable counts obtained from culture. Although the majority of intestinal bacteria cannot be cultured, this does not necessarily mean that they are uncultivable, but rather, that insufficient information is currently available about their optimal growth requirements. Furthermore, many microbes depend on mutualistic interactions with other bacteria and the host, hindering their successful isolation in vitro. While recent advances have increased the cultivable fraction,³ it is estimated that less than 10% of intestinal bacteria can be cultured, and an even smaller fraction can be correctly classified. Therefore studies of the intestinal ecosystem may exhibit bias toward the minor cultivable portion of the gut microbiota. The analytical sensitivity of bacterial culture depends on the organism and its growth requirements. Bacterial culture is associated with difficulties in handling, storing, and shipping of clinical specimens. Ideally, samples should be processed immediately to preserve anaerobic species. Many selective culture media lack sufficient specificity, and often organisms other than the target are enumerated.⁴ Phenotypic and biochemical identification systems often fail to accurately classify many microorganisms, requiring DNA sequencing of isolates.

Molecular Techniques

Because bacterial culture underestimates microbial diversity, the use of molecular tools has now become the standard approach in microbial ecology.^5–7 The principle of these methods is that DNA or RNA is extracted from intestinal samples, and a specific gene is amplified with universal primers that target conserved regions (located up- and downstream of variable regions within the gene).⁸ This approach allows in theory the amplification of DNA from all known and unknown bacterial species in a sample (see Fig. 2-1). The mixture of sequences can then be separated by subcloning and identified by sequencing, or they can be separated by methods that yield a “fingerprint” of the bacterial community.^8,9 The 16S ribosomal RNA (rRNA) gene is most commonly targeted as more than 1.6 million unique sequences are available in public databases (Ribosomal Database Project; http://rdp.cme.msu.edu/). Other more rarely used genomic targets include the 16S to 23S internal transcribed spacer (ITS) region or the chaperonin (cpn60) sequences.¹⁰ If the sequence for a particular phylotype is known, specific PCR assays can be designed for its detection. Real-time PCR assays (with universal-, group-, or species-specific primers) can be used for quantitative analysis. Novel techniques analyze total genomic DNA or messenger RNA (mRNA) without prior amplification of specific genes and yield information about the gene content (metagenomics) or the expressed genes (transcriptomics) of the intestinal microbiome.

Molecular Fingerprinting

Molecular fingerprinting techniques are used for simultaneous analysis and comparison of microbial communities in multiple samples. These techniques provide information on microbial changes over time and in response to treatment. Available techniques include denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE), and terminal restriction fragment length polymorphism (T-RFLP). The goal is to separate the mixture of PCR amplicons that were generated with broad range primers (universal or group specific) to yield a “fingerprint” of the bacterial community. This is achieved as each bacterial phylotype has a unique nucleotide composition (i.e., guanine+cytosine content). These differences in nucleotide composition result in unique melting behaviors of PCR amplicons. Each PCR amplicon reaches a specific melting point in a different position in a polyacrylamide gel, where it will denature and slow its migration. This banding pattern illustrates the bacterial diversity in the sample (Fig. 2-2). In DGGE, a gel containing a linear gradient of DNA denaturants is used, whereas in TGGE a temperature gradient is used for separation. Bands of interest can be excised and sequenced. DGGE and TGGE are inexpensive and rapidly performed. However, because DGGE/TGGE bands are usually short, only limited resolution of PCR amplicons can be achieved, and many bacterial phylotypes will have similar or the same melting behaviors. Therefore, these techniques yield typically only 20 to 40 bands, capturing only changes in the predominant bacterial groups. The use of T-RFLP allows profiling, but also quantification of microbial communities. Bacteria are amplified in PCR assays containing a fluorescent labeled primer. The PCR products are then fragmented by size with sequence specific restriction enzymes. The fragments are separated by capillary electrophoresis with subsequent quantitative measurement of the fluorescence.

Figure 2-2 Denaturing gradient gel electrophoresis (DGGE) as an example for the use of molecular fingerprinting for profiling and comparison of microbial diversity between intestinal samples. This figure illustrates the differences in bacterial diversity in the various segments of the canine GI tract and the differences in bacterial communities between individual dogs (samples analyzed in duplicate). (C, colon; D, duodenum; I, ileum; J, jejunum; unlabeled lanes represent gel markers).

Quantification of Bacterial Groups

Commonly used methods include quantitative real-time PCR assays,^10,12 fluorescent in-situ hybridization (FISH),¹³ RNA dot blot hybridization,^14,15 and flow cytometry of fluorescent-labeled probes. Universal-, group-, or species-specific primers can be utilized. FISH allows for quantification of bacterial groups (Fig. 2-3), and this method also permits visualization of the location of bacteria in relation to the epithelium (i.e., intracellular, adherent, or invasive).

Figure 2-3 Fluorescent in-situ hybridization (FISH). The use of fluorescence-labeled FISH probes allows quantification and visualization of bacteria in relation to the epithelial mucosa (i.e., mucosa-adherent or invasive). This figure shows normal intestinal epithelium. Bacteria (green) are located in the mucus adherent to the epithelial cells. (Green, eubacterial probe labeled with 6-FAM; blue, nuclei of epithelial cells labeled with DAPI.)

(Courtesy of Kenneth W. Simpson, Cornell University.)

There is an inherent bias in use of the 16S rRNA gene for the purpose of absolute quantification. The 16S rRNA genes of bacteria are organized into “operons” that vary in number from 1 to 15 among individual phylotypes. The operon number may also change during the growth phase and altered activity of cells.^16,17 Consequently, molecular results should be related to absolute cell counts with caution. It is more appropriate to express quantitative results as relative proportions to either total bacteria or to other bacterial groups.

Metagenomics and Transcriptomics

Analysis of 16S rRNA genes has provided new information about the phylogenetic diversity of the intestinal microbiota. However, to understand the impact of the microbiota on GI health it is necessary to (a) identify members of the intestinal ecosystem, and (b) explore the functionality of the microbial community. Metagenomics and transcriptomics are emerging fields in microbiology that are based on high-throughput sequencing techniques or the use of microarrays (see Fig. 2-1). In metagenomics, DNA extracted from a sample is sequenced without prior amplification of specific genes. This results in a snapshot of the gene pool and functional potential of the microbiome. For example, metagenomic approaches have revealed the existence of a “core microbiome” in the intestine, because despite obvious differences in bacterial composition between individuals, these individuals share common microbial genes and metabolic pathways.¹⁸ In transcriptomics, mRNA is analyzed to provide a measure of gene expression within the intestinal microbiome. These techniques are expected to yield more in-depth understanding of microbial-host interactions in health and disease.

Presence of a Core Microbiome and Functional Redundancy

There are marked differences in the composition of the microbiota between individuals, and even between monozygotic twins. However, the metabolic end products are similar between individuals. Additionally, although some environmental influences lead to significant changes in bacterial groups, these changes are not immediately associated with any obvious changes in gut function in healthy animals. New metagenomic studies have evaluated the gene content of the intestinal microbiota and suggest that the intestine harbors a “core microbiome,” because despite observed differences in bacterial phylotypes among individuals, the microbiome of each individual appears to have similar gene content and therefore similar functions.¹⁸ Furthermore, a functional redundancy exists in the GI tract. Several members of the community can perform similar functions, and if one group is displaced because of perturbations (e.g., antibiotic therapy), other members of the community are able to maintain a stable ecosystem. These findings highlight the need to evaluate the intestinal microbiome as an entity, including phylogenetic relationships and metabolic functions (i.e., metagenome, transcriptome, and metabolome).