**5. Omics in microbiota**

The advancement in understanding the interaction between gut microbial community and its host is only possible with recent microbial genomics. The main omics techniques, including metagenomics, metataxonomics, metatranscriptomics, and metabolomics allows the exploration of this area of research. At the initial stages of research, bacterial gene analysis was allow relying on the 16S rRNA sequencing method. Scientists were targeting the conserved region of the nucleotide sequence and compared that with other reference sequences to identify the type of bacterial species in the gut [52, 53]. However, sequencing with 16S provides less information about the functional microbial community in the gut which did not allow the studies to make a correlation between microbes and its potential effects causing failures in experiments [53]. This method was mostly targeting the gut bacterial community but not the other type of microbes such as archea and viruses.

Later, the sequencing method was complemented by the metagenomics approach, where the whole genomic content was accessed using the microbial DNA. Reference genes were used to compare the similarities against the newly available genomic data to identify the functions of the genes coding for the new microbial community [54]. This approach could provide important information on all types of microorganism including archaea, fungi, and viruses at their strain level [55, 56]. However, this approach was not sufficient to understand the functional microbial community at the DNA level, it was needed to translate into functional proteins. Thus, metagenomics was accompanied by metatranscriptomic analysis by translating microbial DNA into RNA [57]. The RNA was later translated into proteins and analysis on microbial functions was continued using metaproteomics. This approach was found to be more comprehensive as it could differentiate between metabolically active microbes in the gut [58]. Mass spectrometry is being used in metaproteomics to measure the expressed proteins which is the important for most biological processes. This information is vital when studying the *in vivo* host-associated microbiomes interactions [57].

In addition, to shed light on the identification of microbial activities in dense, microbial metabolites were targeted by using a tool known as metabolomics. Metabolomic uses techniques such as nuclear magnetic resonance (NMR) spectroscopy or mass spectroscopy (MS) to measure the metabolites present in the gut. Studies has shown that MS is more sensitive in identification of metabolites compared to NMR [59]. The metabolites act as the signaling markers in the communication between the host and its microbiome. As such, imbalance in the intestinal metabolites can be a factor towards development of disease in the host [60]. The various omics technologies explained earlier has been summarized in **Figure 1**.

In the presence of all these omics technologies, scientist believe that they could identify the correlation of microbiome with important human diseases. The gut microbiome influences health, due to the interactions with the immune system.

#### **Figure 1.**

*The use of various omics technologies in revealing the gut microbiota and brain axis relationship.*

Understanding the microbial signals will allow new ways to tackle disease. But it is not as easy as it sounds, where more than 50% of the human gut microbiome is yet to be elucidated.

### **6. Future directions: therapeutic interventions**

It is important to identify the key gaps and needs in gut microbiota-brain axis research to plan the future directions and therapeutic interventions. Scientists are only beginning to understand the network between gut microbiota-brain axis. Unraveling the modulation of gut microbiota on the brain health, increases the potential for improving the quality of human life and well-being. The gut microbiome responses to the external factors such as diet and drugs. Drugs are able to modulate the gut microbiome. An integrated understanding on the interaction between the drugs and gut microbiome using the meta-omics technologies can be a major approach towards drug treatment and usage of drugs on certain diseases. There should be rapidly growing studies towards the drug-microbiome interactions targeting available drugs in the markets [61].

Most studies in this field has only attempted using animal models. This method is time consuming, expensive and the findings are difficult to be translated on the human subjects. Culturing the human microbiome by ex vivo culturing together with the meta-omics approach allows development microbiome assays for rapid testing on drug microbiome interactions [57]. Future studies should be focused on understanding the immunological effects of human gut microbes and their role in brain disorders, mapping of neurotransmitters produced by gut microbiota and effect of microbes in early brain development by using human subjects [9]. These interventions are focused in providing nutritional and therapeutic strategies and likely to improve the human quality of life. In most cases when it comes to brain disorders, it is unlikely that these finding provide a permanent cure, however by having the knowledge of these bidirectional communication between the gut microbiota and the brain axis, early predictions or strategies in altering the microbiome to slow down the process would be definitely possible [9].

Nutritional strategies can also be another great practice and are even already on the market, including foods and supplements which help to improve mood, sleep and stress. For instance, altering the diet plan for a child with ASD, could influence the gut microbiota in providing a comfort to gastrointestinal irritation and calm anxiety and hyperactivity. It could be even possible to use probiotics as a complement to drug and therapy for disorders such as schizophrenia. So far many successful trials have achieved by showing the efficacy of probiotics in both strain-specific and diseasespecific clinical cases [62]. Studies showed that probiotic supplements are able to benefit the host by producing high bacterial count and the antibiotic therapy could cause a reestablishment of the host microbiome [63, 64].

## **7. Conclusions**

Many advance technologies and animal studies have revealed many interesting facts in elucidating the communication between the gut microbiota and brain axis. However, the fact that most of the studies failed to show the translation of their research finding into human subject is the major gap to be filled in area of research. Thus, future direction of gut microbiota-brain axis research should focus on the mapping of human gut microbes and they byproducts and finding the immunological effects on the brain disorders. There should be more intervention and preclinical studies focusing on human subjects. The direct link between the human gut microbiota and brain can be only achieved if the bidirectional pathways are revealed from researches focusing on human population.
