DNA is extracted directly from faecal samples. Hypervariable gene sequences (most often 16S rRNA) are amplified using PCR primers that anneal with conserved sequences that span the selected hypervariable regions. One of the PCR primers has a GC-rich end (GC clamp) to prevent complete denaturation of the DNA fragments during gradient gel electrophoresis. Using 16S rRNA gene sequences as example, amplified fragments from different types of bacteria and present in the PCR product are separated using polyacrylamide gel electrophoresis. In DGGE (denaturing gradient gel electrophoresis), the double-stranded 16S fragments migrate through a polyacrylamide gel containing a gradient of urea and formamide until they are partially denatured by the chemical conditions. The fragments do not completely denature because of the GC clamp, and migration is radically slowed when partial denaturation occurs. Because of the variation in the 16S sequences of different bacterial species, chemical stability is also different; therefore, different 16S “species” can be differentiated by this electrophoretic method. Similarly, in TTGE (temporal temperature gradient electrophoresis), the 16S sequences can be separated by gradually increasing the temperature of the polyacrylamide gel during electrophoresis. Separation is achieved on the basis of differing temperature stability of the 16S fragments. These methods generate a profile of the numerically predominant members of the bacterial community. Individual fragments of DNA can be cut from DGGE/TTGE gels, further amplified and cloned, then sequenced. The sequence can be compared to those in gene databanks in order to obtain identification of the bacterium from which the 16S sequence originated. Depending on the length of the sequence, identification to at least bacterial genus can be made.
Fluorescent in situ hybridization/fluorescence-activated cell sorting (FISH/FC)
DNA (oligonucleotide) probes target specific rRNA sequences (16S or 23S) within ribosomes to which they hybridize. The probes are labelled with a fluorescent dye which permits both detection and quantification of specific bacterial populations. Bacterial cells within which hybridisation with a probe has occurred fluoresce and hence can be detected and counted by epifluorescence microscopy (preferably automated) or fluorescence-activated flow cytometry. Continual reassessment of the specificity and coverage of FISH probes is essential in order to update and confirm their continuing specificity and hence reliability. This is because new 16S rRNA gene sequences are constantly added to databases. Epifluoresence microscopic detection is laborious and time consuming, and manual microscopic enumeration requires careful attention by the operator. A lower detection limit of about bacteria per gram of faeces can be achieved. An automated method of counting fluorescent bacterial cells has been developed by coupling fluorescence microscopy to a computerized system of image analysis. Using this automated counting device, the lower detection threshold has been estimated to be bacteria per gram of faeces. Therefore, only the more numerous members of the bacterial community can be detected. Nevertheless, identification of individual bacterial cells, as well as morphological and topographical information are valuable characteristics of fluorescence microscopy. Combined with flow cytometry, FISH provides a high throughput quantitative and qualitative method of analysis. Flow cytometry combines quantitative and multiparametrics analysis (size, internal granularity, fluorescence signal). A lower threshold of detection of 0.4% relative to the total number of bacteria determined with the universal bacterial probe EUB338 has been demonstrated.
Quantitative PCR
PCR primers and fluorescent probes targeting nucleic acid sequences, usually 16S rRNA gene sequences, which are unique to particular bacterial species, are used to quantify the specific sequences in DNA extracted from faeces. Real-time quantitative PCR can be used to quantify specific populations or phylogenetic clusters using specific PCR primers and fluorescent probes. Target sequences in DNA are amplified and simultaneously quantified (as absolute number of copies, or relative amount when normalized to DNA input, or by reference to additional normalizing genes). The procedure follows the general principle of PCR but its key feature is that the amplified DNA is detected as the reaction progresses in real time in contrast to standard PCR where the product of the reaction is detected at its end. Two common methods for detection of products in real-time PCR are: non-specific fluorescent dyes that intercalate with any double-stranded DNA, or (2) sequence-specific DNA probes consisting of oligonucleotides that are labelled with a fluorescent reporter which permits detection only after hybridization of the probe with its complementary DNA target.
16S rRNA gene phylogeny
Older studies utilised PCR amplification of 16S rRNA genes from bulk DNA extracted from faeces followed by cloning the 16S rRNA gene sequences in a plasmid vector in an Escherichia coli host, prior to sequencing. More recently, high throughput bead/emulsion-based sequencing of PCR-amplified DNA or random sequencing of DNA fragments derived from bacterial communities in faeces (metagenome) has been used. These approaches provide catalogues of the constituent bacterial types (usually broad phylogenetic groups) of the community when analysed in relation to a databank of 16S rRNA gene sequences (such as the Ribosomal Database Project pyrosequencing pipeline tools).
Metagenomics
A microbial community is studied in terms of its collective genomes. Nowadays, this approach involves shotgun genome methods to sequence random fragments of DNA from microbes in a sample collected from a biome of interest. DNA is directly extracted from the sample, is broken into small fragments, and portions of these fragments are sequenced. Searches of DNA sequence databases permit collation of the sequencing information in terms of 16S rRNA genes (biodiversity), genes associated with metabolic pathways including their potential regulation, and cell structural molecules. This methodology can reveal novel and fundamental insights about the biodiversity and metabolic impacts of microbial life in biomes.
Metatranscriptomics
Measuring the transcriptomics (gene expression) of microbial communities in the wild. RNA (which includes mRNA) is extracted directly from samples. Ribosomal RNA, which forms the major portion of the total RNA is removed. Then, remaining RNA is converted to cDNA by reverse-transcription PCR. Random sequencing of the cDNA reveals the transcripts produced in the ecosystem. This approach has mostly been used with oceanic samples but application of the methodology to bowel samples is possible.
