PMMB 2020, 4, 1; a0000175. doi: 10.36877/pmmb.a0000175 http://journals.hh-publisher.com/index.php/pmmb Review The chemistry of gut microbiome in health and diseases Agnes Wei Yin Lau 1 , Loh Teng-Hern Tan 1 , Nurul-Syakima Ab Mutalib 1,2 , Sunny Hei Wong 3 , Vengadesh Letchumanan 1* , Learn-Han Lee 1* Article History 1Novel Bacteria and Drug Discovery (NBDD) Research Group, Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, Monash University Malaysia, 47500, Selangor Darul Ehsan, Malaysia. weiyin.agnes.0816@gmail.com (AW-Y.L); loh.teng.hern@monash.edu (LT- HT); vengadesh.letchumanan1@monash.edu (VL); lee.learn.han@monash.edu (L-H.L) 2UKM Medical Molecular Biology Institute (UMBI), UKM Medical Centre, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia; syakima@ppukm.ukm.edu.my (N.-S.A.M) 3Li Ka Shing Institute of Health Sciences, Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Shatin, Hong Kong, China; wonghei@cuhk.edu.hk (S.H.W) *Corresponding author: Vengadesh Letchumanan, vengadesh.letchumanan1@monash.edu; Learn-Han Lee, lee.learn.han@monash.edu Received: 30 November 2020; Received in Revised Form: 31 January 2021; Accepted: 2 February 2021; Available Online: 8 February 2021 Abstract: There are trillions of microbes residing in our body, with their collective genomes much more than human genomes. They have been living in a close relationship with us and play a role in various biological functions. The human microbes begin to build up in utero, accumulate, and fluctuate until a set point is achieved around three years of age. The gut microbiome is altered by several factors, which include age, diet, and antibiotic use. After the exposure, the microbe may shift back to retain its balance, but some factors may leave a permanent footprint on the gut flora. This may be significant as our review has shown the relationships between microbes and diseases. When the homeostasis of gut-microbes is disrupted, multiple mechanisms have been shown to contribute to diseases' development. The balance between protective and pathogenic microbes must be kept in check to prevent disease onset. With a better understanding of this relationship, we will investigate the potential methods to modify the gut flora as the background of developing therapeutic options. There are already some therapeutic options such as prebiotics, probiotics, and fecal transplantation, but their clinical use is limited and restricted. Therefore, there is still a need to investigate the characteristic microbiome association with gut-dysbiosis-related diseases, which may help manage the disease and develop diagnostic and monitoring tools. This review aims to discuss our gut microbiome and its association with human health and diseases. Keywords: Microbes, gut microbiome, homeostasis, gut-dysbiosis, diseases. mailto:syakima@ppukm.ukm.edu.my mailto:wonghei@cuhk.edu.hk PMMB 2020, 4, 1; a0000175 2 of 40 1. Introduction It has been a chaotic start to the New Year 2021, with a rising number of Covid-19 cases worldwide [1-4]. People are still suffering from this disease, exhibiting asymptomatic to severe symptoms, and losing their loved ones to the disease [5-7]. The Covid-19 pandemic has brought in many changes to our daily and professional life. People around the globe adopted the “new norm” – together, we fight against this virus. These days homes are our new offices – where everything is held virtually, and our social life has all been altered. The work from home concept has brought new challenges to our professional, personal, and family life. In whatever circumstances, we should always devote our time to a healthy lifestyle and fitness. Foremost, we should understand our body before we step into changing our life. A range of microorganisms colonizes our body, comprising bacteria, viruses, eukaryotic, and archaea, collectively forming the microbiome community [8]. It is a vast active population consisting of nearly 100 trillion microbes and weighing as heavy as our human brain [9]. Every organ has distinct microbial communities that vary in composition and function, with its aggregate function and metabolic capacity [8]. They live in a symbiotic relationship with the host and responsible for various roles, including protecting against pathogens, digestion of nutrients, and metabolism of drugs and substances [10]. These microbiotas are incredibly dynamic. They continuously fluctuate around a set point and vary from site to site [11, 12]. Also, the composition of the microbiome is determined by multiple environmental and local factors. For instance, the organ site's pH and substrate determine microbes suitable for surviving and living in the community [13, 14]. Among the different microbiomes on different human body sites, the gastrointestinal tract has the most affluent microbes’ community. So, what makes a good gut microbiome community and how they play in health and diseases. There are hundreds to thousands of distinct bacterial species in our gut, some can be beneficial or pathogenic. The Bacteroidetes and Firmicutes phyla dominates the main pool of community in the gut [13]. Our healthy gut microbiome produces short-chain fatty acids (SCFA), which is crucial in regulating the gut's functional and structural integrity [15, 16]. The gut community plays a vital role in developing gastrointestinal mucosal immunity, an essential part of our immune defense system [17-19]. Any disruption of this balanced ecosystem has been influenced and implicated by the gut microbiome. These include cancer, autoimmune disorders, inflammatory bowel disorders, diabetes, psoriasis, eczema, asthma, and autistic spectrum disorder (ASD) [20-27]. The evidence is mounting on gut microbiome association in health and diseases. Our biologists are exploring and studying the gut ecosystem, in order to prevent the development of diseases. There is an growing interest in potential novel treatments, preventive and diagnostic methods for the diseases using homeostasis of the gut microbiome as the stepping stone [28, 29]. This review aims to discuss human health, particularly examining the association between the gut microbiome and health and diseases (Figure 1). PMMB 2020, 4, 1; a0000175 3 of 40 Figure 1. The association of gut microbiome with health and disease. Probiotics, prebiotics, dietary changes, and fecal transplantation are potential or current therapeutic options for gut-dysbiosis associated diseases. 2. Origins of our gut microbiome We have always thought humans are born with sterile guts, not until recent contradicting evidence supporting in-utero transmission of microbes [30]. The advancement of genetic sequencing has given us the privilege to investigate the microorganisms in maternal tissues better than before [31]. Studies of the meconium microbiome have enabled us to understand that gut colonization occurs before birth [32]. This is evident by the similarity in meconium microbiota among newborns even if delivered via different methods (vaginal delivery and cesarean delivery) [33]. Also, the endometrium and placenta are not wholly sterile [34]. They may act as possible sources for gut colonization to occur in the fetus. However, it is still unclear how and when exactly colonization of the fetus’ gut occurs. Meanwhile, ingestion of amniotic fluid has been suggested as one of the possible routes [35]. When the infant is being delivered, the process allows colonization of the microbiome to occur. Thus, arises the controversy of whether the mode of delivery can affect human microbiota. Newborns delivered via cesarean section instead of vaginal delivery have been observed to lack the composition of maternal vaginal and fecal bacteria (e.g., Lactobacillus and Prevotella) [36]. However, recent studies have shown that by two months of age, the delivery mode subsides effects. Other confounders may cause the discrepancy of gut flora between infants delivered vaginally or via cesarean section [37, 38]. For instance, maternal comorbidities are implicated in cesarean delivery and lower exclusive breastfeeding rates in this group of mothers [39]. Interesting to know that breastfed infants have a lower diversity of gut flora before six months of age than their non-exclusively breastfed counterparts. PMMB 2020, 4, 1; a0000175 4 of 40 However, exclusively breastfed seems to be a protective factor against bowel diseases [40]. Thus, the author postulates that a less diverse gut microbiome may be required for the first few months to develop a healthy gut [40]. Perhaps, more studies are needed to investigate the significance of differences in gut microbiome between these two groups of infants. Infants will gradually build up their unique microbiome via exposure to many factors [8]. Biological and environmental factors can all leave footprints on the composition of the human microbiome [41]. When infants start a solid diet around six months of age, the initial diversity gut microbiome begins flourishing with different microbes. This diet contributes to forming a complex and diversifying gut microbiome, which achieves its stability at around three years of age [31, 42]. Besides, a study has shown that our diet can affect our gut microbiome [43, 44]. For instance, a high fiber diet has increased Bacteroidales and Firmicutes [45] population, potentially commensal to a healthy gut. However, as part of aging, dysbiosis of the gut could occur [46]. Aging is a physiological process associated with diminished gut microbiome diversity and slowly weakened gut functional integrity [46]. Today, our dependency on antibiotics has played a vital role in forming our gut diversity and community. Dethlefsen and Relman's study revealed an alteration of the gut community after using ciprofloxacin for 3 to 4 days [47]. However, despite the gut microbiome's tendency to shift back to its initial composition, the recovery was observed to be incomplete [47]. Thus, the effect of repeated or long-term use of antibiotics on gut flora remains unknown. Further research is required to understand the role of antibiotics in gut dysbiosis completely. 3. Gut microbes and diseases In the early days, people believe in Galenism, which explains diseases resulting from an imbalance in bodily fluids or humor [48]. Not until the 16th century, with the development of a microscope, the model by Parcelsians which regards tiny inorganic particles as the culprit of diseases are justified [48]. However, this exciting concept submerged for decades until Louis Pasteur and Robert Koch successfully demonstrated germ theory's validity. They proved that microorganisms could be the causative agents of diseases [49]. Scientists have observed the symbiotic relationship between the host-microbe system in fecal and oral microbiota [8]. In addition, environmental microbes have been seen to live in an involved community [50]. Human microbes are a community of microorganisms that interact with one another and the host so delicately that its disruption may lead to various health diseases and mental health disorders. There are blooming studies on the association between human microbes and many diseases. These studies have demonstrated gut dysbiosis's relationship in human health [20, 51-54]. 3.1. Gastrointestinal diseases 3.1.1. Inflammatory bowel diseases PMMB 2020, 4, 1; a0000175 5 of 40 Inflammatory bowel disease (IBD) is the chronic inflammation of the gastrointestinal tract, consisting of ulcerative colitis and Crohn’s disease. The pro-inflammatory state in IBD can be attributed to the dysregulated host’s inflammatory response in genetically susceptible individuals towards the gut microbiome [55]. The environmental factors are then responsible for the disease's onset and recurrences [56, 57]. When comparing the gut microbiota of individuals with IBD and healthy counterparts, there is a significant discrepancy between them [58-60]. It is observed that individuals with IBD have a higher population of pro-inflammatory microbes (Proteobacteria, Escherichia spp., Fusobacterium spp.) and a lower population of protective microbes (Bacteroidetes, Firmicutes, butyrate-producing spp.) [40, 61, 62]. By reducing butyrate-producing species responsible for short-chain fatty acids (SCFA) production, it is not surprising that individuals with IBD have a lower concentration of these molecules [63]. The significance of lacking these molecules and reduced population of protective microbes [64] is that they have been shown to play an anti-inflammatory role in immune responses. The deficit in these microbes can lead to a pro-inflammatory state, parallel with the disease state in individuals with IBD. When discussing the differences between the gut microbiome of IBD individuals and healthy counterparts, it is unsure whether it is a cause of the disease or its result. However, recent studies have been able to show that genetic factors such as nucleotide oligomerization domain (NOD 2), caspase recruitment domain-containing protein 9 (CARD 9), and autophagy-related 16-like 1 (ATG16L1) [65-67] may be attributed to the differences. The expression of these genes is responsible for the anti-inflammatory and antimicrobial effect in the gastrointestinal tract. Besides, they also help to regulate the homeostasis of the gut microbiome by determining its composition. Studies revealed that Nod-2 deficient mice have a higher gut commensal population and a higher vulnerability to be colonized by pathogenic microbes [68, 69]. On the other hand, ATG16L1 mice have been observed to have a higher Bacteroides species population with an anti-inflammatory role [70]. Thus, altering this gene expression can disrupt the gut's colonization and its inflammatory response and defense against pathogens. When extrapolating this to the human host, it explains the vulnerability of genetically susceptible individuals to disruption of gut homeostasis, leading to inflammatory bowel disease [71]. The terminal ileum is the most affected segment of the gut in Crohn’s disease and the colon segment in ulcerative colitis. This corresponds to the gastrointestinal tract sites in individuals with IBD with a higher microbes concentration than healthy individuals [72]. The parallel distribution of microbes and affected gut segments in IBD illustrates the effect of gut dysbiosis in the disease activity of IBD. Moreover, mice that have received fecal microbiome from mice with colitis have been observed to develop colitis [73]. Besides that, improvement of IBD symptoms by using antibiotics, probiotics, and fecal transplantation, suggest that PMMB 2020, 4, 1; a0000175 6 of 40 dysbiosis of gut flora contributes to the pathogenesis of the disease [74]. The disease activity of IBD is correlated to the biodiversity of the gut microbiome [75]. They have half the gut microbiome's diversity compared to healthy individuals [76]. Interestingly, Swidsinski et al. have identified the characteristic spatial composition of fecal flora in Crohn’s and ulcerative colitis [77]. The difference in abundance of mucosa- associated or fecal Faecalibacterium prausnitzii can be potentially used as a biomarker to differentiate between the two diseases [78]. It is observed that Crohn’s disease has depleted F. prausnitzii with normal leukocyte count. In contrast, ulcerative colitis has a higher abundance of F. prausnitzii with increased leukocytes in the fecal mucus transition zone [77]. However, compared to healthy individuals, individuals with IBD have less diversity in subtypes of F. prausnitzii, and there is a shift in the phylotype distribution of F. prausnitzii species [79]. Some F. prausnitzii subtypes are disease-specific, allowing discrimination between individuals with IBD and healthy counterparts [79]. Perhaps we may consider analyzing punched fecal cylinders obtained from individuals with IBD to diagnose and monitor the disease, given the characteristic composition and spatial distribution of gut microbes in individuals with IBD [77, 78]. 3.1.2. Gastrointestinal malignancies a) Colorectal cancer Colorectal cancer is one of the most common forms of cancer that affects both genders [80]. It is known that chemoresistance is a hurdle in colon cancer therapies. Hence, there is an interest to study the anti-colon cancer potential of novel streptomycetes as an effective treatment for colon cancer [81-85]. Recently, studies revealed the role of colon microbes in the carcinogenesis of colorectal cancer [69]. These studies' evidence is supported by the positive correlation between microbes' concentration and cancer cells' distribution. Cancers have been linked with our immune and genetic dysregulation. The gut microbes are known to involve themselves in cancer development by altering the gut into pro-inflammatory and pro- carcinogenic states. Some of the microbes can act as ‘bacteria drivers’ which damage the gut epithelial DNA. This can lead to hyperproliferative epithelial cells, the cells' ability to evade apoptosis, and so on, which are the cancer hallmarks [86, 87]. Besides, the ‘bacteria drivers’ can disrupt the gut integrity, favoring the proliferation of pathogenic ‘passenger microbes’, which plays a synergistic effect with the ‘bacteria driver’ [86, 87]. Wu et al. have shown specific gut commensal mechanisms in activating the host’s immune system, creating a favorable pro- inflammatory state for cancer development [88]. This may be postulated as the disruption of gut homeostasis, allowing the overgrowth of pathogenic commensal, leading to the development of colorectal cancer. PMMB 2020, 4, 1; a0000175 7 of 40 Fusobacterium nucleatum is a causative effect in the development of colorectal cancer [89, 90]. This microbe has been observed to have a negative relationship with the number of CD3+ T cells in individuals with CRC [89, 91]. They can bind via the bacteria-derived protein Fap2 on exclusively human T cell immunoglobulin and ITIM domain (TIGIT) molecules thus inhibiting natural killer cells in removing cancer cells [89]. This evades the tumour suppression process, thus contributing to the development of tumors [89]. Also, F. nucleatum spp. can recruit CD11b+ to promote a pro-inflammatory and oncogenic environment, which further supports its virulence in causing CRC [92, 93]. Interesting to note that, with the use of metronidazole, mice study has shown that the population of F. nucleatum is reduced and the retarded tumor growth and proliferation of cancer cell. Perhaps further investigation down this path can allow for the potential use of antimicrobial in managing colorectal cancer [90]. There is also a discrepancy between the gut microbiome in individuals with colorectal cancer and healthy individuals. Individuals with colorectal cancer have higher population of Streptococcus bovis [94], Bacteroides fragilis [95, 96], Clostridium septicum [97], Fusobacterium spp. [92], Enterococcus faecalis [98] and Escherichia coli [99]. Studies have observed that these microbes are associated with carcinogenesis via different virulence factors [96, 100, 101]. They can alter host metabolism, immune system, and production of genotoxins, which can align the gut environment towards pro-carcinogenic state [102]. Arthur et al. have shown the mechanisms of virulence factors and metabolites of certain microbes in the progression of colorectal cancer [103, 104]. This further supports that the gut microbes do play a vital role in developing colorectal cancer, and more research is needed to identify the exact microbiota associated with the disease. In addition, recent studies have shown that microbes can form biofilms that can hold them together forming a shield against external agents [105]. They proliferate within the biofilms and damaging the E-cadherin in the colon[106]. This increases intestinal permeability, activates the local inflammatory response, and induces gut dysbiosis [106]. This has been seen in mice studies in which biofilms formation has been an imperative factor in promoting the oncogenic potential of microbiome in CRC development [107, 108]. Meta-analysis studies have shown an increase in protein, choline, and mucin [109, 110]. Moreover, it is associated with the enhanced fermentation process, gluconeogenesis, and secondary bile acids production with reduced carbohydrate metabolism [109, 110]. This portrays a shift from the microbiome that utilizes carbohydrate metabolism in healthy gut to use amino acids and fats. Furthermore, individuals with CRC have a higher concentration of amino acids in the feces and gastrointestinal epithelium, suggesting the association between meat-rich high-fat diets with colorectal cancer [109, 110]. This is plausible as diet is known to be one of the factors that can modify the gut microbiome [92, 93], which influences our health. PMMB 2020, 4, 1; a0000175 8 of 40 b) Gastric cancer With the emergence of Louis Pasteur and Robert Koch's germ theory, there is a bloom in research on the microbiome's role in diseases. In the late 19th century, the relationship between gastric microbes and gastric cancer is proposed despite the fact that the gastric organism's predominant organism is yet to be identified [111]. This was later denied by Heinemann and Ecker, who observed that the microbes suggested by the previous study were Lactobacillus. Reduction in gastric acidity has allowed the proliferation of this species, and it is not causative of gastric cancer [112]. It is not until the late 19th century, with the discovery of Helicobacter pylori, researchers begin to relate the association between gut microbiome with gastric cancer [113]. The H. pylori affect approximately 50% to 60% of the world’s population [114, 115]. It is known to cause gastric cancer in 1-3% of those with H. pylori infection [116] and contributes to around three-quarters of all gastric cancer cases [117]. They are multiple strains of H. pylori, and each possesses different virulence factors that can cause genetic mutation, thus interfering with gastric epithelial cellular division, function, and immune responses [118, 119]. One of the proposed mechanisms is that H. pylori can produce reactive oxygen species that can lead to DNA mutations, aberrant methylation, and oxidative damage, leading to loss of functioning tumor suppressor genes [120]. Another virulence factor worth noting is the CagA protein. CagA is associated with a higher risk of premalignant and malignant gastric lesions [121]. The H. pylori utilize its adhesion molecules for prolonged colonization of gastric epithelium and injecting its oncogenic proteins, including CagA, into the gastric epithelial cells. This oncogenic protein then leads to alteration of the morphology of epithelial cells and disrupts the epithelial intercellular junction. In addition, these molecules trigger metaplasia and dysplasia of the epithelial lining which leads to the development of gastric cancer [122- 124]. The H. pylori possess vacA toxin, which can disrupt the mitochondrial membranes, creating vacuoles in the cytoplasm, causing apoptosis of the gastric epithelium. This is aligned with the mechanism of H. pylori-related atrophic gastritis, which subsequently undergoes metaplasia and dysplasia of the epithelium, leading to the development of gastric cancer [120]. Moreover, these virulence factors promote inflammation and create an environment favorable for tumorigenesis [118, 125]. The disrupted gut epithelial cells will begin to increase uncontrollably, leading to gastric cancer. Saenz et al. have proposed that gastric epithelium can respond to injury by cellular plasticity and reprogramming. However, the mechanism of dedifferentiation and differentiation is vulnerable to the accumulation of genetic mutations, which can further predispose the host to gastric cancer [126]. This is supported by the stem cell-like cells observed in the transition of epithelial to mesenchymal cells in the process of transitioning to gastric malignancy [127]. PMMB 2020, 4, 1; a0000175 9 of 40 Besides H. pylori, other microbes may also contribute to gastric cancer development with or without the aid of H. pylori infection. When observing individuals with H. pylori infection's gut microbiome, they have a higher proportion of Actinobacteria, Firmicutes, and Bacteroidetes. [128]. This discrepancy is significant because Bacteroidetes has been shown to promote gastric cancer in genetically predisposed germ-free mice to a similar extent as genetically predisposed specific pathogen-free mice [129]. This indicates that microbes can play a part in carcinogenesis. Gen genetically predisposed specific pathogen-free mice to appear to have more severe and vigorous gastric cancer progression than genetically predisposed germ-free mice [130].This illustrates that H. pylori can alter the gut microbiome by disrupting commensal, which confer protection and promotes pathogens' growth, enhancing its carcinogenic effect. It also shows that pathogenic microbes can alter the gut microbiome into a pro-carcinogenic environment that can favour more severe disease development. Fortunately, with the availability of treatment option for H. pylori infection, we can eradication this pathogen before it causes detrimental harm to our body. 3.2. Infectious diseases 3.2.1. Clostridium difficile infection Clostridium difficile infection beautifully demonstrates gut dysbiosis as the pathogenesis of the disease. The infection is due to pathological overgrowth of an organism following antibiotic use [131]. C. difficile is part of the gut flora, and its population is regulated by the homeostasis of the gut microbiome [131]. Antibiotics are used to disrupt this equilibrium, reduce microbe diversity, and cause a significant shift in composition [131, 132]. Gu et al. have shown that antibiotic use is associated with a diminished number of putative butyrate- producing anaerobes and increased endotoxin-producing opportunistic pathogens [131]. This may explain the opportunistic overgrowth of C. difficile in patients with recent antibiotic use. The C. difficile infection is caused by the toxin released by the microbes following germination of the C. difficile spores [133, 134] and bile acids are critical for the germination of these spores. The liver produces bile acids that are secreted into the gastrointestinal tract. Some of these acids tracked down the colon, which is subsequently bio-transformed by the gut flora into secondary bile acids [135, 136]. The biotransformation happens via the catalyst activity of 7α‐dehydroxylation and some other enzymes [134, 135]. This balance may be disrupted by the use of antibiotics, which results in the accumulation of primary bile acids (cholic acid, chenodeoxycholic acid) and reduction in secondary bile acids (deoxycholic acid, lithocholic acid), thus favoring the germination of C. difficile spores [137]. Kang et al. have reported that some of the commensal possessing bile acid 7α‐ dehydroxylating property, for instance, Clostridium scindens and Clostridium sordellii can inhibit the in vivo growth of C. difficile [135]. They secrete tryptophan derived endogenous antibiotics to outcompete other microbes in the complex gut ecosystem [135]. Interestingly, the secondary bile acids produced by the 7α‐dehydroxylating bacteria themself can further PMMB 2020, 4, 1; a0000175 10 of 40 enhance the endogenous antimicrobial effect [135]. Thus, any disruption in this regulating mechanism of the gut will reduce the protective microbes’ population, which favors the growth of C. difficile. 3.2.2. Human immunodeficiency virus infection Human immunodeficiency virus (HIV) is an on-going global public health issue. With the development of highly active anti-retroviral therapy (HAART), the incidence of acquired immunodeficiency syndrome (AIDS) has significantly reduced. Mortality of HIV has dropped tremendously from 80% to 50% within ten years since its discovery [138]. However, HAART treatment does not seem to revert the chronic inflammatory status in individuals with HIV [139]. This pro-inflammatory state in HIV individuals has been shown to drive the disease progression [140, 141], on top of its associated high morbidity and mortality [142, 143] Researchers investigated the chronic inflammatory state's etiology in individuals with HIV, including individuals treated with HAART with undetectable viral load. They noticed a massive reduction of CD4 T cells in the gut, especially those responsible for enhancing gut barrier integrity [144, 145]. Vujkovic-Cvijin et al. took a step further to show that altering the gut immune system in HIV individuals can be linked to disruption of the gut microbiome with its associated local inflammation [146]. It is still inconclusive on the findings regarding the shift of gut microbiome in individuals with HIV. There is a consensus on reducing gut microbiome diversity, but the gut microbiome's specific signature in individuals with HIV is yet to be identified [147-149]. Some common findings include the increase in Prevotella spp. and decrease in a decrease in Bacteroides spp. These microbes are proposed to have different gut immunology roles, and the alteration in their abundance can affect gut diversity. For instance, Prevotella spp. can create an inflammatory response by activating myeloid dendritic cells, colonic T cells, expression of CD40, and some other inflammatory mediators [147]. Meanwhile, the Bacteroides spp. is responsible for protecting gut health by expression polysaccharide-A (PSA). This molecule inhibits the activation of Th17, which is a subset of CD4 + T cells [150, 151]. The species also stimulates the invariant natural killer T in the gut-associated lymphoid tissue [152-154]. Together with the Th17 cells, they are responsible for controlling the gut microbiome's homeostasis and regulating the microbiome's translocation. Reduction in Th17 cells indicates the loss of security of the gut barrier, in which the tight intercellular junctions are disrupted, thus allowing gut microbes to enter the systemic circulation. This creates a persistent inflammatory state in individuals with HIV [155, 156], even under HAART treatment [151, 157]. When isolating the fecal microbiome from individuals with HIV individuals, it is observed to have higher levels of activated T cells and monocytes, which is positively correlated with the host's viral load [158]. Even in individuals receiving antiretroviral therapy (ART), there is still activation of adaptive immunity despite a lesser extent than individuals not receiving HRT [158]. Neff et al. also suggested that tumor necrosis factor α and Toll-like PMMB 2020, 4, 1; a0000175 11 of 40 receptor-2 are the immune mediators that activate the inflammatory response in individuals with HIV. Besides the ‘leaky’ gut hypothesis, the gut microbiome's pro-inflammatory properties in HIV individuals can lead to a chronic inflammatory state [158]. There are still gaps to be filled in terms of the gut microbes' involvement in the progression of HIV disease so that adjuvant therapies that may restore gut homeostasis can be developed [159], thus reducing HIV- associated morbidity and mortality. 3.3. Metabolic disorders 3.3.1. Obesity Microbes have many potential roles in our gut, ranging from digestion, regulation of absorption to metabolism of drugs and substances. They can also synthesize molecules such as short-chain fatty acids, affecting our immune and metabolic systems [160]. Unsurprisingly obesity is part of metabolic disorders [161]. The disruption of gut homeostasis can affect the balance energy uptake from diet and its expenditure [162]. This can be explained by the role of microbes in nutrient handling. They can break down indigestible diet and convert them into short-chain fatty acids (SCFA), for instance, acetate, butyrate, and propionate. These substances are then involved in different functions and activities in various organs. Butyrate, acetate, and propionate are required to produce glucose and lipids in the liver [163]. They stimulate the enteroendocrine L cells to release glucagon-like neuropeptided-1 and release local factor peptide YY, regulating lipid digestion and lipid metabolism on top of deposition of fatty acids in the liver. Meanwhile, butyrate also acts as a significant energy source for the colon's epithelial cells [163]. The concentration of these end products is dependent on the gut flora of the host as different gut microbiome are associated with varying properties of metabolism. Turnbaugh et al. have shown that genetically obese mice have higher acetate and butyrate levels in the gastrointestinal tract and lower energy content in their feces than genetically lean mice [164]. This illustrates that the gut community in genetically obese mice are better in energy extraction than their lean counterparts. On the other hand, some of these SCFA end products can have a beneficial effect on health. For instance, butyrate and propionate have been seen to enhance satiety [148, 149]. Furthermore, butyrate enhances mitochondrial function and oxidation of fats, increasing the energy expenditure [101]. Thus, it is reasonable to suggest that different gut microbiome compositions can have other metabolic effects that can affect the hosts' metabolic profile. It is also observed that obese host has reduced gut microbes biodiversity [164] and higher Firmicutes to Bacteroidetes ratio versus healthy individuals [165, 166]. The Firmicutes are associated with the production of SCFA and utilized in lipogenesis [167]. Interestingly, this discrepancy in Firmicutes to Bacteroidetes ratio can be reversed via weight loss demonstrated in mice studies [168] and in individuals who have undergone bariatric surgery [169, 170]. This characteristic of gut microbiota and its reversibility illustrate that specific microbe are more favorable to a specific metabolic phenotype in nutrient handling and metabolism. Meanwhile, PMMB 2020, 4, 1; a0000175 12 of 40 Bacteroidetes are responsible for the breakdown of branched-chain amino acids (BCAAs) which are strongly associated with obesity [171]. Thus, reduction of the species in obese individuals can explain the higher circulating levels of BCAAs [171], which results in hyperphagia [172]. However, other study results on the composition of the obesity-related gut microbiome are contradicting. To date, the data is still inconclusive on the exact composition of the obesity-related gut microbiome, other than the common findings on reduced diversity of gut flora in obese individuals [165]. Even so, animal studies on gut microbes and obesity are still quite promising. When fecal microbes from obese or lean twins were transplanted to germ-free mice, they showed similar metabolic phenotypes and adiposity as their host-source [173]. When microbes from lean co- twins were transplanted to obese mice fed with a regular diet, they successfully prevented the obese mice from further gaining adipose tissue source [173]. This illustrates the possibility of transmitting the lean and obese phenotype by transplantation of gut microbes and the influence of different gut microbiome ecosystems on individuals' metabolic profile. Furthermore, microbes play a significant role in energy harvesting, which can impact the host’s metabolic profile. It is observed that transplanting gut microbes from conventionally raised mice to germ-free mice can cause significant gain in body fat despite reduced food consumption [174]. This can be explained by microorganisms' ability to ferment indigestible dietary carbohydrates, which can then be absorbed and converted into lipids [174]. These lipids are then further promoted to be stored as adipose tissues [174, 175]. This can be extrapolated on obese individuals such that they may possess characteristic gut microbiota that has enhanced efficacy in extracting energy from the diet, thus contributing to their excess storage. 3.3.2. Type 2 diabetes Type 2 diabetes (T2DM) is a chronic disease characterized by increased blood sugar level, relative insulin deficit, and insulin resistance [26, 176]. It is also associated with other metabolic abnormalities such as incretin deficiency, raised glucagon level, and increased lipids breakdown [177, 178]. T2DM, as with other chronic diseases, has been attributed to gut dysbiosis. This has been illustrated by Backhead et al. in which different gut microbiota has demonstrated different glucose metabolism profile, even when the mice are of similar genotype and controlled for their diet pattern [174]. This shows that a diverse gut microbiome with different glucose metabolism capabilities and lower glucose metabolism can predispose the host to T2DM. It is also long known that T2DM is associated with low-grade systemic inflammation, which leads to insulin resistance of the host [179]. This pro-inflammatory state can be led back to the host’s microbiome which can play a role in metabolism and immunology. As in other chronic diseases, reducing gut microbial diversity has been observed in individuals with T2DM [180]. This allows the overgrowth of pathogenic microbes to promote local inflammation by activating innate immunity [180]. The inflammatory mediators from the PMMB 2020, 4, 1; a0000175 13 of 40 immune response or microbes’ toxins disrupt the intestinal barrier, allowing gut content, including microbes, to enter the systemic circulation [180]. The immune system is then activated by the bacteria and toxin in the blood, which creates a low-grade systemic inflammation that is pro-insulin resistant [180]. Overall, Gurung et al. have reported a consistent increase in Fusobacterium, Ruminococcus, and Blautia genera, decreasing Bacteroides, Bifidobacterium Akkermansia, Faecalibacterium, and Roseburia genera in individuals with T2DM [181]. Among these protective microbes, Bifidobacterium genus is the strongest protective factor against T2DM [182, 183]. Animal studies have successfully demonstrated the reduction in blood glucose level when introduced to Bifidobacterium spp. [184, 185]. Also, losing these protective gut microbes can contribute to the chronic inflammatory state in T2DM individuals. These protective microbes can inhibit pro-inflammatory mediators. For instances, TNF-α, IL-8,IL-1β, CD36, Monocyte Chemoattractant Protein-1, Intercellular adhesion molecule-1, and C-reactive protein [186, 187]. Other butyrate-producing microbes also produce metabolites that suppress NF-kB [188]. Without them, it is not surprising that the inflammatory pathway will be dominant, which causes chronic inflammation in the host. Interesting to note that individuals with T2DM have a lower population of Roseburia spp. [189]. The Roseburia spp. has been inversely associated with the plasma glucose level in individuals with T2DM [190]. This shows that different microbes have different metabolic properties, which may either confer towards or against the disease activity of T2DM. As discussed, some gut commensal, especially Clostridium spp. is involved in converting primary into secondary bile acids [191]. These bile acids can then affect glucose metabolism by activating membrane-bound, G-protein-coupled receptor TGR 5 and nuclear farnesoid X receptor (FXR). These receptors can be activated to inhibit the enzymes involved in gluconeogenesis [192]. Also, TGR 5 membrane receptor on the enteroendocrine L cells has been suggested to increase the secretion of glucagon-like peptide- 1 (GLP-1). GLP-1 is an incretin hormone that enhances the insulin effect and increases satiety [193]. On the other hand, FXR is shown to improve glucose tolerance and increase insulin sensitivity [191]. Thus, gut dysbiosis that interferes with bile acids' biotransformation can have a ripple effect on glucose metabolism and glucose level regulation, thus contributing to T2DM. In addition, it is known that the gut microbiome can metabolize nutrients into SCFAs [194]. Most of these end products are then absorbed and utilized by the host for various functions [194]. The SCFAs include butyrate and propionate, beneficial to the host's energy balance and metabolism [194]. For example, propionate stimulates GLP-1 and peptide YY secretion, which reduces energy intake [195]. Sanna et al. have shown that the gut microbiome that produces butyrate can enhance beta cells' function. In contrast, those with reduced propionate absorption can confer an increased risk of T2DM [194]. In short, various mechanisms can implicate the development of T2DM, and maintenance of the homeostasis of gut flora is the key to prevention. PMMB 2020, 4, 1; a0000175 14 of 40 3.4. Pulmonary health and asthma The lower airways were once thought to be sterile until recent years; studies have demonstrated microbes in the lungs, even in healthy individuals [196, 197]. Lungs microbes are detectable right after birth [198], and their composition is enriched by the upper airway [199]and oropharyngeal microbes [200]. The gut-lung axis concept was also suggested in parallel to the increased attention given to the correlation between human microbes and diseases. This has been demonstrated by Schuijt et al., in which gut microbes depleted mice have a more disseminated disease, complications, and higher mortality when infected with Streptococcus pneumonia [201]. When fecal microbes were transplanted into pneumococcal infected mice with depleted gut microbes, the lungs' inflammatory response was reduced [201]. This shows that gut microbiota can enhance host defense against lung infection. Asthma is an atopy related respiratory disease characterized by chronic inflammation of the smaller airways [202]. When comparing the microbiome of asthmatic to healthy individuals, it is found that asthmatic patients have a higher population and diversity of microbes [197, 203]. The colonization of an infant’s oropharyngeal by Moraxella, Haemophilus, and Streptococcus before one year of age has been shown to predict the risk of developing asthma later on [204]. While moving to the comparison of the gut microbiome, a study showed a reduction in the population of genera Faecalibacterium, Lachnospira, Veillonella and Rothia in infants with increased risk of developing asthma in the first hundred days of life [205]. Meanwhile, another study showed an increase in Clostridium neonatale and Lachnospira in the infant's gut flora, which is predictive of asthma risk at the pre-school age [206]. Recently, there is increased research on the gut-lung axis concept, a bi-directional relationship between the gut and the lungs [207]. They are proposed to be involved in regulating a ‘common mucosal immunological system’ [208]. There is increasing evidence that one mucosal compartment's inflammation can impact the distal mucosal site [208]. This is in line with the dysbiosis of both lungs and gut microbiome observed in individuals with asthma. In addition, childhood use of broad-spectrum antibiotics has also been associated with increased asthma development later [209, 210]. This further illustrates that disruption of the homeostasis of the gut microbiome can affect the lungs' immunity. However, the exact mechanism is still unknown, and more studies are needed to understand this mucosal immunological system and the bi-directional relationship between compartments [208]. If it is indeed evident, the concept can be beneficial in explaining various diseases, including asthma, associated with airway mucosa inflammation. Besides that, the composition of the lung microbiome is related to its treatment responsiveness. Corticosteroid-resistant patients are populated by Proteobacteria (Haemophilus, Neisseria), which produces endotoxin triggering chronic inflammation [211]. In contrast, Fusobacterium and Bradyrhizobium, which have lower endotoxicity, were found PMMB 2020, 4, 1; a0000175 15 of 40 in treatment sensitive patients [211]. Moreover, the severity can also influence the gut flora's alignment with the gut-lung axis concept. Bisgaard et al. demonstrated that the gut microbiome could be modified by viruses [212]. This allows the overgrowth of specific pathogens that are associated with severe asthma. There are evidence implying a high fiber diet can reduce asthmatic symptoms by modifying the gut microbiome's composition [213]. It is shown that gut microbes can ferment soluble high fiber diets to produce short-chain fatty acids with an immunomodulatory effect [213]. The molecules can counteract the pro- inflammatory state in asthmatic individuals' airways, modifying the disease activity. 3.5. Mental health The gut-brain axis concept can be traced back to the early 20th century, where the gut's functional integrity and microbes were attributed as part of the pathophysiology of mental health disorders [214]. Researchers back then have believed that autointoxication can affect one’s mental health [214]. They suggested that toxic gut content can have a profound impact on the brain [214]. In recent years, there are increasing studies supporting this bi-directional relationship between the brain and the gut [215, 216]. The gut microbes produce metabolites and molecules that can modulate the immune system, which interacts with the brain; vice versa, the brain can alter the composition of gut microbiome via neural signaling [217]. Homeostasis of the gut microbiome has been shown to protect intestinal and blood-brain barrier integrity. Disruption of this balance allows pathogenic microbes to overgrow and induce local inflammation. They then increase the gut permeability, allowing translocation of gut content into the systemic circulation, which triggers a systemic immune response [218]. A pro-inflammatory state is shown to alter the integrity of the blood-brain barrier [219]. This allows immunomodulatory substances and pathogenic microbes to reach the brain [219]. On the other hand, some gut microbes produce metabolites that indirectly modify the brain function via the neural signaling pathway [220]. In short, this illustrates the imbalance in the gut ecosystem can affect brain function, which predisposes individuals to the development of mental health disorders. Various studies have supported this view where a pro-inflammatory state has been observed in individuals with mental health disorders. For instances, raised inflammatory mediators, inflammatory markers, and antibodies specific to gut commensal is observed in individuals with major depressive disorders [221, 222], higher activated level of c1q in individuals with schizophrenia, and increased intestinal permeability [223]and inflammatory state in individuals with autistic spectrum disorders [224]. Besides that, some gut microbes are known to secrete substances that can act as neuromodulators [225]. For example, Lactobacilli spp. produces gamma-aminobutyric acid; Bacillus, Escherichia and Saccharomyces spp. produce noradrenaline; Streptococcus, Enterococcus, and Candida spp. produce serotonin (5-HT), and Bacillus produces dopamine [226]. The balance of these neuronal signaling messengers is crucial in the functioning of PMMB 2020, 4, 1; a0000175 16 of 40 cognition and behavior of individuals. Matsumo et al. exhibited that dopamine concentrations were two fold higher in germ-free (GF) mice than in ex-germ-free mice [227]. Meanwhile, another study showed that GF mice have a higher level of 5-HT and their metabolite in the hippocampus. This can occur without alteration in the expression of genes responsible for their production [228]. A ‘healthy gut flora’ is essential in the balance of neurotransmitter levels of the brain. The alteration in neurotransmitters has been attributed as the mechanism behind various mental diseases. It can be extrapolated that the homeostasis of the gut ecosystem is important in preserving healthy mental states. 3.6. Autism spectrum disorder DSM-5 defines autism spectrum disorder (ASD) as a neurodevelopmental disorder characterized by impaired communication and interactions with repetitive and/or restricted behaviors [229]. The disease is considerably associated with various comorbidities, including epilepsy, bowel disorders, and sleep disorders [230]. The exact cause of autism spectrum disorder is still unknown despite strong genetic evidence [231, 232]and environmental factors. Genetic factors have been estimated to contribute to half of the ASD cases [231, 232], with the remaining contributed by ecological and other risk factors. Considering the gut-brain axis concept, studies have explored the possible association between the gut microbiome and ASD. ASD children were found to have more bowel disorders, including abdominal pain, constipation, diarrhea, and gastric reflux [233, 234]. It will be worth finding a causal relationship between gut dysbiosis, which contributes to bowel disorders, and autistic behavior in ASD individuals. Exciting to note that not until recently, the effect of microbiota transfer therapy has been shown to improve the behavioral symptoms of ASD for at least 8 weeks [235]. This further supports the direction of investigating the link between the gut microbiome and ASD as it shows the possibility of reversing autistic behavior with alteration of gut flora. It is found that children with ASD have a deficit in gut integrity [236] and a discrepancy in the gut microbiome's n composition instead of healthy individuals. Studies over the years have noted the differences in gut microbiota between healthy versus ASD individuals, with some common findings including Clostridium spp., Prevotella spp., Bifidobacterium spp., Candida spp., and Firmicutes spp., to be greater in population and lacking anaerobic bacteria in individuals with ASD [237-241]. The significance of gut flora composition is that there were events where children treated with antibiotics developed chronic diarrhea, followed by regressive symptoms [237, 240, 242, 243]. These clinical progressions may be due to the altered gut microbiome's antibiotics' consequences [244]. Clostridium spp., especially C. bolteae, C. perfringens , C. difficile [237] and C. histolyticum [239], is abundant gut flora of ASD children. It is suggested that they play a role in developing autistic behavior via neurotoxin ρ-cresol production [243, 245], which is evident PMMB 2020, 4, 1; a0000175 17 of 40 by the raised serum level of p-cresol chemically similar metabolites in ASD children [245]. The toxin produced by the gut microbes can be transported by the vagus nerve directly into the brain [243]. The toxin then affects the cleaving of synaptic vesicle membrane protein, affecting the release of neurotransmitters [243]. This lowered neurotransmitter level causes behavioral changes, which are observed to be in parallel with the characteristic behavior of ASD children [243]. In addition, a higher concentration of Clostridium spp. is associated with more severe autistic symptoms [246]. The notion is further supported by the transient improvement of ASD symptoms in individuals with regressive autism treated with vancomycin, which is an antibiotic with activity against Clostridium spp. [247]. This suggest that Clostridium spp. may be a potential cause of regressive autism, but subsequent controlled studies have yet to replicate the results. Besides that, Candida spp. has also been recently found to be associated with ASD [246]. Gut dysbiosis in individuals with ASD has allowed this commensal's overgrowth, producing propionic acids and ammonia [248]. These metabolites have been shown to react and result in beta-alanine production, chemically like inhibitory neurotransmitter gamma-aminobutyric acid (GABA) found in the brain. Assuming if the beta-alanine can cross the blood-brain barrier [249], they can occupy the GABA receptors [250] in human brain tissues, thus stimulating the compensatory production of GABA [251]. This excess in GABA is in keeping with the increased GABA concentration observed in children with ASD [251]. Further studies can be done to determine whether the raised level of beta-alanine observed in individuals with ASD does function similarly as suggested and subsequently triggering the remaining chain of reaction, leading to the development of ASD [252]. Other organisms are being studied for their association with ASD, but no significant results have been found, or the mechanisms of them causing ASD is yet to be explained [253]. As discussed, gut dysbiosis is a possible mechanism that leads to the development of ASD. Factors that may alter the gut flora include antibiotics [253], diets, variation in genetic and medical heterogeneity, and comorbidities. Pinpointing the exact cause of gut dysbiosis, which subsequently leads to ASD impossible [254]. Finally, increased intestinal permeability in ASD children allows the escape of gut content into the systemic circulation, thus leading to a systemic pro-inflammatory state. This corresponds with the chronically raised cytokine levels observed in individuals with ASD, and some of these inflammatory mediators (IL-1β, IL-6, IL-8, and IL-12p40) are linked explicitly to low social interaction and communication [255, 256]. In short, further studies are needed to identify the exact organisms and their mechanisms in the development of ASD and recognize factors that may alter the homeostasis and integrity of the gut, leading to the development of ASD. PMMB 2020, 4, 1; a0000175 18 of 40 3.7. Alzheimer’s disease Alzheimer’s disease (AD) is a neurodegenerative disease characterized by progressive cognitive function loss [257]. The most widely accepted mechanism that leads to AD is the deposition of amyloid plaques and neurofibrillary tangles [257]. However, the exact cause of this deposition is still unknown and is believed, to date, to be multifactorial [258]. With the emergence of the gut-brain axis, AD has also been attributed to gut dysbiosis [257, 259]. Disease progression of AD has been hypothesized to be due to chronic inflammation following deposition of amyloid plaques [260]. However, recent studies have shown that these plaques possess antimicrobial activities, suggesting that neuroinflammation may cause the deposition of these plaques instead of vice versa [258]. Moreover, a study suggests that amyloid plaques and neuroinflammation deposition is a vicious cycle [261]. Thus, there is now a chronic inflammatory state in the brain tissue of individuals with AD. More studies are needed to fill the gap of causal effect relationships between amyloid plaques and neuroinflammation. When considering the possibility of the gut microbiome affecting neuronal functions, animal studies have shown a significant reduction of amyloid plaques in germ-free mice [262]. Nonetheless, when gut microbiota is reintroduced, the mice demonstrated an increase in cerebral amyloid plaques pathology [262]. This is further supported by several studies that showed that gut infection is associated with AD development [258]. For example, individuals infected by Borrelia burgdorferi and H. pylori have been observed to have raised serum Aβ40 and Aβ42, which is a biomarker of AD [263]. These pathogens may also contribute to AD's development by inducing an inflammatory response in the brain [264]. AD individuals have an abundance of pro-inflammatory microbiome and reduced anti-inflammatory gut microbes [265]. Some of these pathogenic microbes can secrete pro-inflammatory neurotoxins, which can affect the neurofunction [266]. Another pathology observed in AD is the reduction of BDNF proteins in the hippocampus [258]. It is showed that probiotics have been able to reverse this in the mice model, suggesting that specific gut microbiome are protective and needed for the brain's normal functioning [267]. Recent breakthroughs in discovering the superior temporal lobe and hippocampus lysates of individuals with AD are saturated with lipopolysaccharide [266]. This could be due to the gram-negative gut microbes, which can stimulate local inflammation, disrupting the gut barrier, allowing the translocation of gut microbes, and endotoxins into the systemic circulation [268]. When these molecules such as lipopolysaccharides reach the brain, it can induce local inflammation or affect the nerve functions directly or indirectly [269]. On the other hand, some gut microbes can produce neuroprotective molecules such as short-chain fatty acids, and losing these protective microbes can alter brain tissue's normal function [258]. For instance, Li et al. have shown that Clostridium butyricum species can enhance the intestinal and blood brain barrier structural integrity and improve neurodegeneration symptoms [270]. PMMB 2020, 4, 1; a0000175 19 of 40 Is it also known that our gut microbiome changes physiologically as we age [258]. There is an increase in Proteobacteria and reduction of probiotics (Bifidobacteria) and SCFA producing microbes [271]. The significant decrease in functioning microbiome, especially those with SCFA is their deficit associated with raised inflammatory mediators [272]. This aging-related pro-inflammatory state is known as inflamm-aging. It is the common pathology for a wide range of age-related diseases, including degeneration of cognitive functions in AD [273] and increased AD prevalence with age. 4. Emerging treatments Probiotics Probiotics are live microorganisms that have numerous benefits for our health when consumed [274]. The story of probiotics begins about a century ago, where the process of fermentation was initially used as a means of food preservation [275]. With people starting to notice the health benefits of consuming fermented food, this food was adapted for various uses. For example, treatment of diseases and consumption for energy and strength [275]. Elie Metchnikoff, an immunologist, suggested that probiotics found in fermented food can improve health and increase lives [274]. This, later, created a ripple effect, encouraging more scientists to venture into the discovery of probiotics. They believe that not all bacteria are pathogenic, and some of them can replace the pathogenic flora, creating a healthy gut microbiome. With the blooming studies on the gut health axis, therapies to create a ‘healthy’ gut flora are always searching. With the known effect of probiotics, it is not surprising that probiotics are proposed as one of the therapeutic options. Probiotics are suggested to confer its effect by preventing pathogenic microbes' overgrowth, enhancing the gut's structural and functional integrity, and modulating the gut immune system [18]. This effect is conferred by the probiotics' cell-surface architecture, such as their surface proteins and capsule. This can be done by various mechanisms that include enhancing natural killer cells, immune phagocytes, or upregulation of antibodies [276, 277]. To date, there are numerous studies on various health diseases and probiotics use, with some showing promising results. For instance, probiotics use has been shown to reduce the duration of diarrhea in acute gastroenteritis and C. difficile infection [278, 279], inducing more prolonged remission in IBD [280, 281]and modulating the immune response in patients with allergy [282, 283]. Some probiotics strains can increase anti- inflammatory mediators' concentration, such as tumor necrosis factors, which can abate diseases [276, 284]. Traditionally, the most widely used yeast strain is the Saccharomyces cerevisiae, whereas the most common bacterial probiotics are the Bifidobacterium and Lactobacillus species [285]. For instance, L. plantarum, L. rhamnosus, B. bifidum, and B. breve [286]. There is a variation of the functions between different probiotics strains, even if there are of the same species [285]. For example, strains that initially fail to prevent necrotizing enterocolitis PMMB 2020, 4, 1; a0000175 20 of 40 in infants [287], will successfully prevent sepsis when replaced with another formulation [288]. Thus, it will be imperative to evaluate the functions and efficacy of each of these strains of probiotics for disease treatment [285]. Another issue with probiotics is worth noting because its overall effectiveness is also influenced by the host factors, including the baseline gut microbiome [28]. Maldonado et al. have shown that the efficacy of probiotic Bifidobacterium longum subsp. Longum AH1206 is the highest if the host has low abundance of B. longum and carbohydrate metabolizing genes [289] However, disappointing results contradict the positive outcomes with probiotics use [290- 292]. Also, there were reported events of adverse effects associated with probiotics consumption [293]. The significant side effects are particularly concerned in individuals with an immunodeficiency. They are at risk of sepsis, bacteremia, or endocarditis with probiotics consumption [285]. This makes the current recommendation of probiotics as a therapeutic option impossible. The studies cannot conclude as there is always discrepancy on the type of probiotics, criteria of the selected population, and the outcomes [293]. Thus, we cannot know which particular strains are beneficial and which populations will benefit the most from probiotics consumption [293]. Besides that, there are also insufficient studies to identify people at risk of consuming probiotics [293]. This issue needs to be addressed in view that probiotics may be a potential therapeutic option in the future. Furthermore, there is still difficulty explaining probiotics' outcomes on human health via mechanisms found in laboratory studies [290]. This makes it difficult to extrapolate the effect of probiotics on human diseases [290]. It is known that alteration of gut microbes can affect the gut's functional and structural integrity, which can affect other host’s organ systems. With this strong relationship between gut dysbiosis and various health diseases, it is still worth continuing the search for the effect of probiotics on human health. The probiotic use has been recommended in some clinical guidelines for children in antibiotic-associated diarrhea, acute infectious diarrhea, necrotizing enterocolitis, infantile colic, ulcerative colitis, and irritable bowel disease [294, 295]. However, there is still no clinical recommendations for the clinical use of probiotics in adults [28]. Current research has been exploring different human commensals other than the traditional genera Bifidobacterium and Lactobacillus in view that different strains have distinctive effects on the host [28]. This research also needs to be gradually moved on to human trials after confirming its safety profile and efficacy on animal experiments [296]. It is also essential to understand that the outcomes depend on the formulations, participants, doses, and clinical endpoint [28]. Perhaps, with more standardized trials done, we will be able to come out with a more definite conclusion on the clinical use of probiotics [290]. Prebiotics and dietary fibers Fiber is plant-derived food that cannot be digested by the human gastrointestinal tract [297]. Instead, they need to be fermented by gut microbes to extract their nutrients [298]. They are various kind of fibers which can be derived from different plants [299, 300]. Each of them PMMB 2020, 4, 1; a0000175 21 of 40 possesses specific properties and has other effects on the host when consumed [300]. If the fibers can modify the gut microbiome in such a way that confers health benefits, they can be considered prebiotics [301]. This modification includes altering the microbiome's composition, regulating the activities and metabolites produced by the microbes beneficial to the host [302]. A diet low in fibers has been shown to shift the gut microbiome drastically. Human trials have demonstrated that a switch from a high fiber plant-based diet to a fiber-deprived meat- based diet can cause significant changes within as short as 24 hours [303]. Also, fibers deprived of fiber is associated with reduced biodiversity of the gut microbiome [304]. This is supported by the depleted diversity of gut microbiota observed in mice fed with fiber depleted diet [305]. However, this deprivation is reversible by re-introducing a high fiber diet [306]. This reversibility of gut flora biodiversity may shine a light on various diseases that have been associated with the low diversity of gut microbes. This suggests that a diet high in fibers can help maintain the gut microbiome's homeostasis, preventing pathogenic microbes from causing various physical and mental diseases. The fermentation end products are mainly short-chain fatty acids (SCFA), which confers invaluable benefits to human health [303]. For instance, Lactobacilli spp. produces metabolites, including SCFA, via fermentation of fibers with anti-carcinogenic effect [307]. They have been shown to prevent colorectal cancer by reducing the pH of colorectal cancer [307]. On the other hand, lacking of SCFA has been observed in individuals with IBD due to their depletion of SCFA producing bacteria [308]. Depriving SCFA means the host has lost its ability to inhibit pro-inflammatory state activation, as in individuals with IBD. This is supported by the reduction of inflammatory markers via the consumption of prebiotics [309]. Moreover, specific prebiotics such as psyllium, beta-glucans, and pectins are classified as high glycemic index food [299]. They possess unique properties of slowing glucose absorption, which can benefit patients with metabolic disorders [299, 310]. Interestingly, despite the unknown mechanism, prebiotics has been shown to suppress type 2 T helper responses, which can have an immunomodulatory effect on individuals with atopy diseases [311, 312]. Arslanoglu et al. have shown that infants fed with prebiotic- supplemented hypoallergenic formula had a lower risk of allergies in pre-school age [313]. Moreover, animal studies have shown that prebiotics that can promote the growth of protective gut microbes, in which intestinal permeability is reduced, the concentration of endotoxin is decreased, thus reducing the prevalence of metabolic disorders [314] When extrapolating prebiotic use into clinical use, the evidence is still insufficient compared to probiotics [28]. There are currently no clinical guidelines recommending prebiotic use in neither the pediatric nor adult population [28]. As with probiotics, studies have shown that prebiotics' effectiveness depends on the baseline gut flora, which can be influenced by multiple factors such as age, diet, and lifestyle [315, 316]. Besides that, formulations, participants, doses, and clinical endpoints must be considered when planning PMMB 2020, 4, 1; a0000175 22 of 40 future research. All these factors can influence prebiotic use's effectiveness compared with probiotics [28]. When discussing therapies' safety profile, prebiotics seems to have minimal life- threatening side effects compared to probiotics [317]. They are mostly harmless and just indigestible fibers. Thus, the most probable side effects are the osmotic functions of the prebiotics' fermented product [317]. For example, prebiotic consumption can cause osmotic diarrhea, flatulence, and abdominal cramps [317]. Interestingly, the safety profile is dose- dependent, and the therapeutic dose is usually low. This implicates that if consumed appropriately, the side effects of prebiotics are usually mild [318]. A further advantage of prebiotics is that it’s convenient in production, storage, and transportation compared to probiotics and fecal transplantation [317]. It does not need to be preserved via a cold chain makes the whole process of making prebiotics available to the patients much easier [317]. Overall, a diet high in fibers and prebiotics has multiple benefits for our health and can play a disease-modifying role in various diseases. With more human trials done, perhaps it can be a potential intervention and recommended therapeutic option for many conditions. Fecal microbiota transplantation Fecal microbiota transplantation (FMT) transfers gut microbes from a donor to a recipient, either by infusion into the colon by colonoscopy or delivered via upper gastrointestinal tract such as capsule ingestion, by gastroenteric tube or endoscope [303]. This procedure was reported as early as 1958 by Eiseman et al., where few patients with pseudomembranous colitis recovered after receiving FMT [319]. Fischer et al., also showed the beneficial effect of FMT on individuals with severe and severe-complicated Clostridium difficile infection [320]. They have shown promising results with a significant cure rate in all the participants [320]. The increasing studies on the association between gut microbes and health diseases have created ripples to modify the gut microbiota as a therapeutic option for gut dysbiosis related health diseases [268, 321]. With the success of FMT in the treatment of Clostridium difficile infection, FMT seems to be a plausible method to help modify other diseases related to disruption of the gut microbiome. Currently, FMT has been adopted by the U.S guidelines second-line treatment for recurrent Clostridium difficile infection [322]. At the same time, the European proposed FMT guidelines after the first episode of severe and refractory C. difficile infection [323]. It is also showing promising experimental results in gut dysbiosis related diseases such as IBD [324, 325], metabolic syndrome [326], and autism spectrum disorder[235] but all of them are still in the experimental phase [23]. The donor stools for FMT can be obtained from either universal donors via stool banks or patient-directed donors [327]. Patient-directed donors have been less frequent as it is more time-consuming to collect, screen, and process, leading to treatment delay [328]. Also, the donor chosen may feel vulnerable for their confidential information [329]. Thus, the most preferred option to date is the universal donor FMT. Stools are collected from PMMB 2020, 4, 1; a0000175 23 of 40 young adults who have passed the screening test [330]. The advantage of universal donors is that recipients can receive microbiome from multiple donors who may provide a greater diversity of microbes instead of receiving only the gut flora from a single donor. This is further supported by an RCT which showed that multidoor recipients showed improvement in the disease progression of ulcerative colitis [331]. With the increase in popularity and evidence on the efficacy of FMT, stool banks such as OpenBiome have emerged [332]. They are usually strict criteria to screen donors, standardized protocols for stool handling, processing, and delivering. However, there is a concern that some infections may go undetectable, which may risk infecting the recipients [332]. To note that a few years back, the FDA has been alerting health practitioners and patients on the potentially severe and life-threatening infections due to FMT. However, in these cases, pathogens have been identified from the fecal transplantation product [333]. Thus, a regulated protocol on FMT should be detailed enough to maximize reproducible outcomes and minimize adverse events to patients [334]. Other than the risk of infection, typical side effects with FMT are loose stools and bloating, which usually resolve spontaneously within a day [335]. Also, the long-term impact and risks of FMT are still unknown. Longer-term follow-ups must fill this gap. Lastly, it is still unknown what constitutes a ‘healthy’ gut microbiome, which can be the ‘ideal’ fecal microbiota for transplantation. We are assuming healthy individuals have ‘healthy’ microbiota. More research on the genomes of microbes required for good health is crucial for understanding the mechanism behind FMT [336]. 5. Conclusion From this review, we know a strong association between the gut microbiome and various diseases, regardless of whether it is a physical or mental disorder. The structural and functional integrity of the gastrointestinal tract is imperative in the maintenance of good health. Disruption of gut homeostasis has been demonstrated to contribute to the development of diseases. Meanwhile, the microbes can be categorized into protective and pathogenic subgroups. Different species have their mechanism in disrupting or protecting the gut flora. If the gut's homeostasis is lost, local inflammation will lead to gut microbes' translocation, leading to a pro-inflammatory state observed in various diseases. Some toxins or beneficial SCFA molecules can also influence other systems such as metabolic and neurofunction by several pathways. Moreover, we have now known that we may reverse or alter gut-dysbiosis-related diseases by modifying the gut microbiome [337]. We have reviewed that probiotics, prebiotics, and fecal transplantation are potential or current therapeutic options for gut-dysbiosis associated diseases. Despite supporting data, more standardized studies are still needed to obtain a definitive outcome for their safety and beneficial use in humans. If it is proven to be effective, studies can even move on to investigate their use in disease prevention, especially with prebiotics that has minimal side effects in human studies. In conclusion, there is a strong PMMB 2020, 4, 1; a0000175 24 of 40 association between the human microbiome and diseases. This is a potential field further to investigate the characteristic microbiome for every gut-dysbiosis related disease. With that, it may be possible to modify illnesses that have never been possible before, such as autistic spectrum disorder, which will be life changing. Author Contributions: AW-YL performed the literature search, critical data analysis as well as manuscript writing. LT-HT, N-SAM, SHW, L-HL and VL provided review, editing, and proofreading for this manuscript. L-HL and VL conceptualize this review writing project. Funding: The SEED Funding funded this work from Microbiome and Bioresource Research Strength (MBRS), Jeffrey Cheah School of Medicine and Health Sciences, (Vote Number: MBRS/JCSMHS/02/2020) awarded to VL. 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Author(s) shall retain the copyright of their work and grant the Journal/Publisher right for the first publication with the work simultaneously licensed under: Creative Commons Attribution-NonCommercial 4.0 International (CC BY-NC 4.0). This license allows for the copying, distribution and transmission of the work, provided the correct attribution of the original creator is stated. Adaptation and remixing are also permitted. 1. Introduction 2. Origins of our gut microbiome 3. Gut microbes and diseases 3.1. Gastrointestinal diseases 3.1.1. Inflammatory bowel diseases 3.1.2. Gastrointestinal malignancies a) Colorectal cancer b) Gastric cancer 3.2. Infectious diseases 3.2.1. Clostridium difficile infection 3.2.2. Human immunodeficiency virus infection 3.3. Metabolic disorders 3.3.1. Obesity 3.3.2. Type 2 diabetes 3.4. Pulmonary health and asthma 3.5. Mental health 3.6. Autism spectrum disorder 3.7. Alzheimer’s disease 4. Emerging treatments 5. Conclusion References