TECHNOLOGY FEATURE Methods in microbiome research Namrata Iyer From the freezing lakes of Antarctica to satisfied most of these requirements. The metametabolomics are more technically the thermal vents deep within the oceans, most widely used method in microbiome challenging, given the sheer diversity of microbes have managed to survive and analysis is 16s rRNA sequencing2. The proteins and metabolites that each microbe thrive in the most inhospitable of con- 16s rRNA gene is highly conserved in all can produce. While these techniques are ditions. Unsurprisingly, the relatively bacteria and sequencing of its regions of still in their infancy, they promise to reveal luxurious abode of the human body is hyper- variability allows the identification unique insights into the communication chockablock with microbial life. We have of different bacterial species. But this tech- and cooperation between the different coevolved over centuries with these micro- nique suffers from inaccuracies at species members of the microbiota, and also host- organisms, collectively referred to as our level classification2. Furthermore, it limits microbiota interactions. microbiota, resulting in a relationship the analysis to only bacterial species while that is mutually beneficial. However, it has ignoring other members in the community. Microbiota structure only been in the past few decades that the Platforms are now being developed that can Community structure is a function of who importance of the microbiota to human couple this technique with other genetic is present in the microbiota community health has been uncovered. It is now well- markers, allowing detection of eukaryotic and how these members interact with each known that imbalance in the microbiota— members of the microbiota as well3. other. The behavior of each microbe is or dysbiosis—is strongly linked with a While 16s rRNA sequencing looks at determined by the microbes and host cells number of pathologies, such as Crohn’s a single gene in every bacterium, whole in its immediate vicinity. Several patholo- disease, inflammatory bowel disease, can- metagenome sequencing looks at the entire gies, such as irritable bowel syndrome cer and many others1. In order to under- genomic content of the microbiota in an (IBD) and Crohn’s disease, are now known stand the link between these microbes and unbiased manner. This provides accurate to arise not only from an imbalance in the Nature America, Inc. All rights reserved. America, Inc. Nature various diseases, it becomes imperative to species-level identity of the microbes as microbiota, but also in their spatial orga- 6 find the answers to several important ques- well as their complete genome. Analysis nization within the host. Visualization © 201 tions: What are the specific microbes that can reveal the metabolic potential of these of this spatial organization is performed inhabit our body? What functions do these communities, giving a better idea of their using FISH (fluorescent in situ hybridiza- microbes perform? Where are they located function as a whole. tion). Using fluorescent probes against 16s npg with respect to the host and each other? rRNA sequences, FISH can specifically And finally, how do changes in microbiota Community function label various bacterial communities in lead to disease? Community composition has been the host tissues and reveal how these different Recent times have seen vast leaps in primary tool for microbiome research microbes are organized within their niche modern technology that allow us to begin in the past decades. As more is revealed (Fig. 1)6. These imaging techniques can answering some of these key questions. about the microbiota in different dis- further be coupled to mass spectrometry ease states, researchers are now trying to imaging to reveal the spatial location of a The diversity of microbes understand how these communities con- bacterium as well as its metabolic status Any site in the body can be considered as an tribute to the physiological state of the at the time. ecosystem, inhabited by different microbial host. Metagenome information does not species, ranging from bacteria and archaea tell us which subsets of genes are expressed Mechanistic link between microbiota to fungi and viruses. To accurately define at any given time. Keeping this in mind, and host health this ecosystem, assigning an identity to whole metagenome sequencing is now Studies have uncovered startling correla- its members is of paramount importance. being coupled with metatranscriptome tions between changes in the microbiome The methods employed for this purpose sequencing to reveal the community gene and diseases ranging from diabetes to must be accurate, capable of detecting expression profile4. This dynamic picture autism. While most of these studies reveal even rare species, fast and cost-effective. of community function can help identify correlation, causation is yet to be compre- ‘Next Generation’ sequencing has largely the consequences of dysbiosis. Studies are hensively established. now being taken to the next level, ana- Answers to these puzzles are now being Department of Molecular Microbiology and lyzing not only transcriptomes but also found using in vitro models of microbiota Immunology, Brown University, Providence, RI. Correspondence should be addressed to N.I. proteomes and metabolomes of the com- research. These models simulate condi- ([email protected]) munity as a whole5. Metaproteomics and tions in the specific niche to dissect out LAB ANIMAL Volume 45, No. 9 | SEPTEMBER 2016 323 TECHNOLOGY FEATURE host-microbe and microbe-microbe 7 Probe ­interactions . These models could provide valuable insight into the physico-chemical Sample processes that occur at the gut interface. Fixation A comprehensive understanding of the Target (ribosomal RNA) link between microbiota and health, how- ever, requires the use of live animals. The + – gold standard for such studies is gnotobiot- + + ic mice (mice with defined microbiological Epifluorescence – + Fixed cells are microscopy – status). Germ-free mice, born and bred in permeabilized + Ribosome – + completely sterile isolators (Fig. 2), reveal how the complete absence of microbiota affects host physiology. The lack of micro- Fluorescently labelled Flow cytometry oligonucleotides (probes) biota can have systemic effects on these Hybridization Quantification animals, ranging from an inability to effi- ciently digest food to an under-developed Washing immune system. Hybridized cells These mice can be further selectively colonized with defined microbiota to assess their effect. Such studies have made FIGURE 1 | Basic steps of fluorescence in situ hybridization (FISH). The sample is first fixed to stabilize important revelations about the influence the cells and to permeabilize the cell membranes. The labeled oligonucleotide probe is then added and allowed to hybridize to its intracellular targets before the excess probe is washed away. The sample is of our microbiota on our metabolic sta- then ready for single-cell identification and quantification by either epifluorescence microscopy or flow tus. Obese and lean body types are now cytometry. From Nat. Rev. Microbiol. 6, 339–348 (2008). known to be transmissible between mice via a transfer of just their microbiota8. microbiota function and host response. of linked reactors that mimic the human Gnotobiotic mice have also been instru- Many systems recreating the human intes- gut, starting from the stomach to the colon. mental in disclosing the complex relation- tine are now available. Examples such A recent model, known as ‘Gut-on-a-chip’, ship between our microbiota, diet and as the SHIME (Simulator of the Human is a microfluidic system that co-cultures health. Diet is now thought to be a factor Nature America, Inc. All rights reserved. America, Inc. Nature Intestinal Microbial Ecosystem) make use gut cells with microbes to study ­various that dominates over our genome in deter- 6 mining which microbes colonize our gut9. © 201 The translational impact of these stud- ies has further improved with the use of humanized mice (mice colonized with npg microbes from the human gut). Research integrating gnotobiotic mice, dietary vari- ations and mathematical modeling have allowed scientists to predict the effects of diet on the composition of our microbiota and thereby our health10. This has wide implications for the growing fields of pre- biotics and probiotics. The dynamic nature of the microbiota, however, also means that minor changes in diet and other environmental conditions can significantly affect the microbiome, causing groups of mice that are genetically identical to display different phenotypes. The variability in husbandry practices at different animal care facilities can, there- fore, cause problems with the reproducibil- ity of these studies11. Greater uniformity in animal maintenance and comprehensive FIGURE 2 | Example of a typical gnotobiotic facility with sterile isolators. All components (such as reporting of these conditions are required food and bedding) have to be sterilized before being placed in each isolator, creating a strict set of to generate a stronger foundation for requirements for maintaining gnotobiotic colonies. Image from NIAID. microbiota research. LAB ANIMAL Volume 45, No. 9 | SEPTEMBER 2016 325 TECHNOLOGY FEATURE Future perspectives the key to breakthroughs in therapeutics omic approach. Gut 62, 1591–1601 (2013). The Human Genome Project started with for a gamut of diseases. 6. Amann, R.