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Shrinkage of the human core microbiome and a proposal for launching microbiome biobanks Abolfazl Barzegari1,2,3, Nazli Saeedi1 & Amir Ata Saei*,1

ABSTRACT: The Human Microbiome Project (HMP) revealed the significance of the gut microbiome in promoting health. Disruptions in microbiome composition are associated with the pathogenesis of numerous diseases. The indigenous microflora has co-evolved with humans for millions of years and humans have preserved the inherited microbiomes through consumption of fermented foods and interactions with environmental microbes. Through modernization, traditional foods were abandoned, native food starters were substituted with industrial products, vaccines and antibiotics were used, extreme hygiene measures were taken, the rate of cesarean section increased, and breast feeding changed into formula. These factors have reduced human exposure to microbial symbionts and led to shrinkage of the core microbiome. Reduction in microbiome biodiversity can compromise the human immune system and predispose individuals to several modern diseases. This article suggests launching microbiome biobanks for archiving native microbiomes, supervising antibiotic use, probiotic design and native starter production, as well as advertising a revisit to native lifestyles. The human intestinal tract harbors one of the most biodiverse microbial communities ever discovered. In the human supraorganism microbial cells outnumber Homo sapiens cells by a factor of 10 [1] . The microbiome describes the totality of microorganisms (MOs) and their genomes that a human acquires from the environment during labor and after birth. HMP (http://commonfund. nih.gov/hmp) and European Metagenomics of the Human Intestinal Tract (MetaHIT; www. metahit.eu) were launched to characterize the microbial signature of 18 body sites from diseasefree individuals and to describe, if possible, a core microbiome shared among all humans [1,2] . The HMP has produced more than seven terabases of sequence data, and research has been accelerated by technological advances in metagenomics, metabolomics, data handling and high-throughput next generation sequencing [3–7] . The importance of human gut microflora has been known for a long time; however, no mechanistic view was available on the functions of human microbiota. Although in ancient times no one knew exactly how microbiota interacted with the human body, people had discovered the benefits of consuming dairy products containing microbial species to maintain their welfare. However, in the past few years intensive research on the human gut microbiome has shed light on the nature, composition and interactions of the human microbiome with diet, antibiotics, human physiology and the immune system. Gut microbiome functions in nutrient digestion, mucosal and systemic tolerance, immunomodulation, developmental regulation of intestinal angiogenesis, detoxification of harmful substances, defense against potential pathogens, and, finally, signaling from the periphery to the

KEYWORDS

• antibiotic • biodiversity • biopreservation • biorepository • cryopreservation • food starter • gut • native microbiome • personalized medicine • probiotic

Research Center for Pharmaceutical Nanotechnology, Tabriz University of Medical Sciences, Tabriz, Iran Nutrition Research Center, Tabriz University of Medical Sciences, Tabriz, Iran 3 The School of Advanced Biomedical Sciences (SABS), Tabriz University of Medical Sciences, Tabriz, Iran *Author for correspondence: Tel.: +98 914 1192320; Fax: +98 411 3367929; [email protected] 1 2

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Figure 1. Interactions of gut microbiota with diet, the human immune system and its physiological functions. (A) Microbiota-derived molecules such as 5-HT, GABA, SCFAs, melatonin and cytokines are transmitted to the nervous system and may be involved in mood, emotion and cognition. (B) Microbiota contribute to digestion of foods and produce SCFAs, vitamins such as B12, folate and immunomodulins, each of which have specific functions and benefits for humans. (C) Microbiota interact with the human epithelial barrier functions, as well as the adaptive and innate immune system through Peyer’s patches, lymphocytes and dendritic cells. (D) Microbiota can engage in the production of proinflammatory or anti-inflammatory responses, and (E) in turn, Homo sapiens cells dictate the composition and diversity of the gut microbiome by releasing neurotransmitters such as noradrenaline and inhibitory molecules such as defensins. 5-HT: Serotonin; ACTH: Adrenocorticotropic hormone; Ag: Antigen; DC: Dendritic cell; SCFA: Short-chain fatty acid; SFB: Segmentous filamentous bacteria; Th: T-helper cell.

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Shrinkage of the human core microbiome & a proposal for launching microbiome biobanks brain [8–11] . The human microbiome can influence human immunophysiological, inflammatory, metabolic and neurological functions [12] . The microbiome manipulates anxiety, depression and behavior through the gut–brain axis [13] . Clades and metabolism of the microbiota have been reported to be partially associated with host properties and phenotypes such as age, gender and BMI [14] . A few important interactions of the microbiota with the human diet, immune system and physiology have been depicted in Figure 1. The fundamental role of the gut microbiome is to provide a physiochemical barrier against external microbial intruders through colonization resistance and/or competitive exclusion [15] . In the large surface area of the GI tract, healthy and pathogenic bacteria compete for dominance [16] and both classes are required for maintenance of a healthy core microbiome [17] . In spite of the significance of the gut micro biome in maintaining health and consideration of probiotics (which originally belong to the microbiome) as a countermeasure for treatment of a variety of diseases [18] , modern lifestyles unfortunately result in shrinkage of the core microbiome [19] . This article highlights the inter-relationship of gut microbiome ecology (bacteria in particular), the human immune system, diet, antibiotics and lifestyle, and advises about the reduction of microbiome biodiversity, which can be associated with the risk of modern disease emergence. Finally, this article suggests launching national and international micro biome biobanks to save native microbiomes from environmental samples, foods and fermented products, to supervise antibiotic use, probiotic design and starter production, and to encourage people to choose native lifestyles. Biobanks are repositories with cryogenic storage facilities for collection, processing, storage and protection of biological samples (cells, biological fluids, tissues and plant seeds) and associated data for use in research, diagnosis and treatment of diseases, especially towards personalized medicine. Microbiota & the human immune system Human gut microbiota are essential for the development and functionality of the immune system [20] . For example, SFB that colonize the rodent intestine at the time of weaning stimulate maturation of the IgA antibody response and induce Th17 intestinal cells in the mouse gut [21] . Animals lacking SFB in their microbiome cannot control colonization by the invasive pathogen


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Citrobacter rodentium [21] . Compared with conventional animals, germ-free (GF) animals possess smaller spleens containing fewer lympho cytes [22] and Th2-skewed immune responses [23] . Furthermore, GF animals have smaller organized immune structures (e.g., Peyer’s patches and mesenteric lymph nodes) [24] , lower numbers of dendritic cells, CD41 T cells and IgA-producing B cells in the lamina propria, and decreased numbers of intraepithelial T cells [25–27] . The composition of the microbial community determines the ratio of beneficial to pathogenic bacteria, the level of different microbial metabolites and the relative production of proinflammatory to anti-inflammatory signals from the immune system. Although the development of gut-associated lymphoid tissue occurs before birth, its maturation and the recruitment of IgA secreting plasma cells and activated T cells to mucosal sites are largely dependent on signals derived from microbiota [28] . After birth, both genetic and microbial factors contribute to a healthy intestine with a dynamic balance. Not only is there bacterial–epithelial cross-talk (such as eukaryotic cell Toll-like receptor [TLR] recognition) [29] , but gut microbiota can also protect humans against autoimmune and inflammatory diseases by antigenic competition and immunoregulation [30] . Reciprocally, host immune response in the gut epithelial cells influences the composition of microbiome by restricting microbial growth and secretions [31] . Microbiome studies on mice have been successfully used as a tool to link immunity and the microbiota. For instance, deficiency in MyD88 – an adaptor molecule that senses microbial sensors by many TLRs – is associated with protection from Type 1 diabetes (T1D) [32] . Pathogen-free non-obese diabetic mice lacking MyD88 protein do not develop T1D [32] . This effect is deemed to be dependent on gut microbes, since GF MyD88-negative NOD mice develop severe diabetes, while colonization of these GF MyD88-negative NOD mice with human gut-like microbiota attenuates diabetes [32] . Exposure to the microbiota of specific pathogen-free MyD88-negative non-obese diabetic donors attenuates T1D in GF non-obese diabetic recipients [32] . Obesity and metabolic syndrome have also been linked to innate immune alterations associated with gut microbiota. Mice lacking TLR5 – an immunosensory receptor for microbial flagellin present on the surface of intestinal

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Perspective Barzegari, Saeedi & Saei and immune cells – have an altered microbiota makeup [33] . Hypotheses indicate that disturbed microbiota may induce proinflammatory cytokines, which can subsequently lead to desensitization of insulin receptor signaling [33] . Diet, gut microbiome & human health Nutrition has an extraordinary impact on the composition of the gut microbiome [34] . Wen et al. reported that diet can rapidly affect gut microbial enterotypes and that even small amounts of specific compounds such as aspartame can modify bacterial communities in the gut [35] . If the composition of microbiome is under partial dietary control, then diet has the potential to substantially affect human physiology and the immune system [36] . Indeed, specific diets have been shown to predispose animals to metabolic syndrome by transformation of the gut microbiome [37] . The results of a case–control study among residents in Hawaii demonstrated that diet may play a role in the etiology of noncardia gastric cancer among individuals with Helicobacter pylori infection [38] . On the contrary, dietary intervention has been shown to induce metabolic improvements in high-density lipoprotein (HDL) profile in a hamster model of hypercholesterolemia, which was strongly linked to alterations of gut microbiota manifested by an increase in the population of Bifidobacteria and decrease in the number of Coriobacteriaceae family members [39] . Dietary components have also been associated with susceptibility or resistance to other health conditions. The mice that were on diets with a higher protein-to-carbohydrate ratio survived better following inoculation with Salmonella typhimurium [40] . Furthermore, epidemiological studies indicate that dietary intake of fiber is associated with lower risk of certain diseases such as colorectal cancer, while a higher risk of colorectal cancer has been reported with diets containing red meat [41] . Among different dietary fibers, resistant starch can protect against DNA damage induced by red meat diets and colorectal cancer induced by carcinogens [42] . The amount of protection is proportional to the production of SCFAs especially butyrate. Therefore, changes in dietary habits can significantly influence the composition of the human microbiome and, consequently, health. Antibiotics & gut biodiversity After World War II, antibiotics found extensive applications, and shortly after agricultural

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scientists discovered that farm animals gain weight by consuming subtherapeutic doses of antibiotics [43] . In fact, application of antibiotics for promoting growth in farm animals accounts for more than 50% of total antibiotic sales in USA [44] . By the age of 18 years, the average child in developed countries has received between 10 and 20 courses of antibiotics [45] . Although antibiotics help treat infections, they have several collateral consequences. Upon antibiotic therapy, beneficial bacteria in the gut are also affected [46] . It has been demonstrated that after antibiotic intake, some of the commensal gut microbiota do not fully recover [47] or may be replaced by resistant organisms [48] . Even 6 months after termination of oral ciprofloxacin, several microbial taxa remained depleted [49] . Taking a short-term clarithromycin, metronidazole and omeprazole regimen commonly used for H. pylori infections significantly perturbs the gut microbiota such that some microbial species are affected up to 4 years post-treatment [50] . Amoxicillin induced the depletion of Lactobacillus genus rat pup microbial populations and significantly altered the expression of nearly one-third of developmentally regulated genes [51] . Mice treated with vancomycin also show depletion of Gram-positive bacteria and disruption in the fermentation of carbohydrates [52] . In animal models, streptomycin and vancomycin increase the susceptibility to invasive salmonellosis [53] . Since in this study antibiotics did not diminish the total number of bacteria in the gut, it can be inferred that microbiome biodiversity is important in mediating host resistance to invading pathogens [53] . Antibiotic-induced alterations in the gut microbiome may impact host health. For example, Oxalobacter formigenes is sensitive to commonly prescribed antibiotics [54] and vanishes after antibiotic treatment [55] . Since O. formigenes degrades oxalate, people who lose this microbe are hypothetically at increasing risk of kidney stone disease or hyperoxaluria [56] . Even in case of H. pylori, which is a risk factor for peptic ulcers and gastric cancer, studies indicate that individuals without this bacterium are more likely to develop asthma or skin allergies in childhood [57] . Interestingly, young mice infected with H. pylori are protected against experimental asthma (experimental asthma is produced in model animals by initial intraperitoneal sensitization to ovalbumin during days 1–13, and subsequently challenging with OA aerosols on 8 following days) [58] . Indeed, other


Shrinkage of the human core microbiome & a proposal for launching microbiome biobanks MO-derived agents might have a role in the development of the immune system, which can subsequently lead to protective effects for asthma and atopy [59] . Endotoxins from Gram-negative bacteria [60] and fungal β(1→3)glucans and extracellular polysaccharides [61] are associated with reduced risk of allergy and asthma separately in rural and urban populations. A 2011 systematic review reported that antibiotics seem to slightly increase the risk of childhood asthma [62] . Besides, antibiotic therapy in children with Escherichia coli strain O157:H7 infection can increase the risk of hemolytic–uremic syndrome [63] . An altered intestinal flora in infancy is thought to contribute, as a predisposing factor, to the etio pathogenesis of inflammatory bowel diseases (IBD). Shaw et al. demonstrated that subjects diagnosed with IBD in childhood are more likely to have used antibiotics in their first year of life [64] . Another recent retrospective cohort study on a total of 1,072,426 subjects has also confirmed that childhood antianaerobic antibiotic exposure is associated with IBD development [65] . Subjects diagnosed with IBD are more likely to have been prescribed antibiotics 2–5 years before their diagnosis [66] . Long-term changes in the diversity of beneficial bacteria in the human gut may also increase the susceptibility to infection [67,68] . Pattern-recognition receptors (PRRs) sense microbiota-associated molecules and initiate inflammatory responses during infections [69] . Ligands from bacterial microbiota utilize signaling through PRRs to induce development of the host immune system. A hypothesis suggests that PRRs have evolved to mediate the human microbiome cross-talk and to protect against disease [69] . Antibiotics may also increase the risk of immune-allergic and metabolic diseases [70] . Subtherapeutic antibiotic treatment of young mice alters the murine colonic microbiome and leads to higher adiposity, dietary energy harvest, bone mineral density and SCFA levels in treated mice versus control [71] . Lower exposure to microbes early in life may have even been responsible for the overall height increase in developed nations during the 20th century [72] . Evolution of human diet: possible impacts on human health It has been suggested that the integration of components (such as ω-3 fatty acids, polyphenols, fiber and plant sterols) from Paleolithic diets into


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modern functional foods can be used to prevent chronic diseases [73] . Over the past 10,000 years, after the beginning of the Neolithic revolution and the advent of agriculture and animal husbandry, our feeding behavior has substantially changed from a Paleolithic diet into the current modern pattern. This change has adversely affected certain dietary parameters including glycemic load, fatty acid composition, acid/base load, sodium/potassium ratio and fiber content [74] . Nowadays, we are mostly consuming refined, cooked and processed foods that are high in sodium and hydrogenated fats, and low in fiber. This has brought about an increase in the incidence of chronic conditions and disorders such as obesity, cardiovascular diseases, diabetes and cancer. “For many in the developed world, eating has become a leisure pursuit, and cooking has changed to a hobby. But our bodies are still hard-wired for a tougher world where food means survival.” [75] . It has been postulated that modern metabolic and cardiovascular diseases partially result from a diet and lifestyle not in agreement with our Paleolithic genome [76] . These chronic conditions seem not to have existed in the Paleolithic era [77] . A typical American diet contains 24% fats and oils, 24% grain products, 17% sugar and sweeteners, 13% meat and poultry, 9% dairy products, 8% fruits and vegetables, 3% legumes, nuts and soy, 1% eggs and 1% fish, while food groups in a typical Paleolithic diet consisted of 65% fruits, vegetables, nuts and honey, as well as 35% of lean game, eggs, fish and shellfish [78,79] . Paleolithic diet has been shown to improve the health status of patients with Type 2 diabetes (T2D) [80] , improve glucose tolerance in individuals with ischemic heart disease [81] , reduce the risk for cardiovascular diseases [82] , and exert metabolic and physiologic improvements [83] . A traditional African diet has 450 kJ/100 g of energy density, while the average energy density of three fast foods in Britain was determined to be 1100 kJ/100g [84] . Sweet snacks such as cookies and ice cream possess a higher energy density of 1500–2000 kJ/100 g and savory snacks such as potato chips have an energy density of 2200 kJ/100 g [85] . Fruit and vegetables have an average energy density of 100 kJ/100 g [85] . In an epidemiologic study Lindeberg et al. noted the absence of ischemic heart disease, stroke or markers of the metabolic syndrome


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Figure 2. What shrinkage of the human core microbiome means.

in the traditional Pacific Islanders of Kitava in New Guinea, possibly owing to their traditional lifestyle [86] . In the Republic of Korea, people have mostly maintained their traditional high-vegetable diet despite social and economic changes [87] , and this has resulted in a lower incidence of chronic diseases and obesity than other industrialized countries [88] . There is general consensus that traditional Japanese food and the so-called ‘Mediterranean’ diet are healthier than western fast-food diets and are more similar to the prehistoric human diets [89] .

E. coli bacteria [95,96] in patients with IBD. Interestingly, human metabolic markers (urinary formate and hipporate) are associated with diet and blood pressure, a major risk factor for coronary heart disease and stroke [97] . Indeed, a very recent study by Pluznick et al. has shown that SCFAs, end products of bacterial fermentation in the gut, have the potential to modulate blood pressure and renin secretion via Olfr78 and G-protein-coupled receptor 41 [98] . Propionate, a SCFA, was shown to induce vasodilation ex vivo and to subsequently produce an acute hypotensive response in wild-type mice. Authors also Reduction in biodiversity of gut reported that antibiotic treatment elevates blood microbiome through modernization pressure in Olfr78 knockout mice by reducing & its consequences the biomass of the gut microbiota [98] . Human core microbiome is subject to disappearUntil now, microbiome dysbiosis is implicated ance or shrinkage (Figure 2) [19] upon exposure in the pathogenesis of IBD [99] , obesity and meta to environmental factors including changes in bolic syndrome [100] , irritable bowel syndrome dietary habits (e.g., consuming pasteurized and (IBS) [101] , nonalcoholic fatty liver disease [102] , processed foods with additives, high fat and asthma and allergies [103] , cardiovascular diseases high sugar, as well as popular drinks instead [97] , bacterial vaginosis and preterm birth [104] . of functional foods containing co-evolutionary Microbiome dysbiosis may also be implicated in probiotics), selection of only a few species as food rheumatoid arthritis [105] , autism spectrum disstarters in industrial products (instead of native orders [106] , esophagitis and Barrett’s esophagus and versatile starters), excessive and/or irrational [107] , colon cancer [108,109] , and obesity-related antibiotic use, cleaner water, provision of robust hepatocellular carcinoma [110] . Gut microbiome sanitary measures, transformation of the native has also been implicated as a causal factor in lifestyles into urbanism, and reduced interaction kwashiorkor (Figure 3) [111] . A decrease in Gramwith farm animals in addition to daily stresses, positive bacteria and increase in Gram-negative cesarean section and replacing maternal breast bacteria, lipopolysaccharide (LPS)-mediated milk with formula [90,91] . This overall change upregulation of proinflammatory cytokines, in lifestyle can potentially lead to reduction in relaxation of the lower esophageal sphincter the biodiversity and/or homogenization of the via inducible nitric oxide synthase (iNOS), and human gut microbiome. delaying gastric emptying via cyclooxygenase-2 Any perturbations in the integrity of flora can cause esophageal adenocarcinoma. Excess (even in small subpopulations) can potentially hygiene, an increase in Clostridia and decrease in alter human health status [92] . For example, Bifidobacteria, a decrease in pylori, and decreased several studies have demonstrated an under- exposure to fungal β(1→3)glucans and Gramrepresentation of Faecalibacterium prausnitzii positive endotoxins are associated with asthma, [93,94] and an increase in the number of adherent allergy and atopic disorders. Furthermore, in

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Figure 3. Alterations of the human microbiome are associated with the risk of emergence of many health conditions.

T1D reduction in the number of SFB – which prevent pancreatic islet cell damage through promoting the differentiation of Th cells into Th17 cells – is observed. In T2D, decrease in Bifidobacteria loosens tight junctions and LPS leaks through the gut wall, leading to metabolic endotoxemia, which results in low-grade inflammation and finally insulin resistance. In the case of metabolic syndrome and obesity, a decrease in Bacteroidetes and increase in Firmicutes are observed. In addition, ATP, serum amyloid A,


or CC-chemokine ligand 5 signaling induced by potentially harmful SFB or Lactobacilli results in local expansion of proinflammatory Th17 cells and Th1 cells. These autoreactive T cells turn B cells into autoantibody-producing plasma cells. After migration of plasma cells to synovial tissue, the inflammatory cascade is amplified through the activation of macrophages, fibroblasts, osteoclasts, cytokines and proteinases. This process can lead to arthritis and formation of pannus. In individuals with autism spectrum disorders,


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Perspective Barzegari, Saeedi & Saei an increase in Clostridium bolteae spores as well as increase in gut biodiversity and dysbiosis have been noted. A high red meat diet, low SCFAs, increase in 7-alpha dehydroxylating bacteria, and thus higher conversion of cholic acid to deoxycholic acid (a co-carcinogen), low number of H2Smetabolizing bacteria in colon and low vitamin absorption are associated with colorectal cancer. Industrialization has changed the type of food we eat. According to Hannley, “In 1955, with the opening of the first McDonald’s in Des Plaines, Illinois, post-war America took a quantum leap into the Fast Food Era” [112] . Traditional plant-based nutrition gave way to high-fat, energy-dense diets and modern marketing practices largely replaced local or ethnic dietary patterns [113] . Antibiotics are driving important microbial species of core microbiome into extinction [114] . Cleaner water reduces our exposure to fecal organisms, which might be our evolutionary symbionts. Although clean water is generally seen as a critical component of public health in that it reduces risk of serious infections, the hygiene hypothesis suggests that reduction in microbial exposure in developed countries as a result of improved health measures can bring about immunological imbalance in the gut microbiome and contribute to the incidence of autoimmune diseases such as IBD [115] . Furthermore, exposure to social stressors can alter the composition of the gut microbiome [116] . The increase in the circulatory levels of IL-6 and monocyte chemoattractant protein-1 (MCP-1) were significantly and positively correlated to the changes in three bacterial genera of Coprococcus, Pseudobutyrivibrio and Dorea. In follow-up experiments in which mice were treated with an antibiotic cocktail before exposure to stress, no changes in circulatory IL-6 or MCP-1 levels were noted [116] . In comparison to conventionally raised animals, GF mice exhibit an exaggerated corticosterone and adrenocorticotrophin (ACTH) response to restraint stress [117] . Cesarean section also hinders the vertical transmission of maternal vaginal flora to newborns, the gut microbiome of whom is foundational for lifelong health and disease susceptibility [118] . Transition from maternal breast milk to formula can also affect the colonization of the newborn gut [119] . Reductions in diversity of the gut could affect microbiome resilience to various disturbances and the host immune system [120] . Since species-rich habitats use limited resources more

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efficiently, with different species specialized to each potentially limiting resource, they are less prone to invasion by intruders [121] . In a study by Manichanh et al. the diversity of the rat intestinal microbiome surpassed that of the human gut microbiome by two to three times [46] . The authors suggested that laboratory-raised rats may establish a higher biodiversity in their gut microbiome to harvest more nutrients from their basic diet [46] . Not only diversity, but also the type and functionality of present bacteria are important. The basic diet may simply exert a reduction in biodiversity of the rat microbiome, followed by reduction in resilience of the gut to hinder entrance of nonindigenous opportunistic intruders, which may finally result in a false increase in biodiversity. The homogenization of human food, elimination of functional components from diets (e.g., probiotics), and availability of many vitamin and mineral supplements can diminish the need Homo sapiens feel to keep their microbial symbionts. Interestingly, it has been hypothesized that the shift in the dietary habits of Americans within the past 30 years has caused a general reduction in the phylo genetic and functional complexity of the human intestinal microbiota [122] . De Filippo et al. studied the impact of diet in shaping gut microbiota using a comparative study in children from Italy and the rural village of Boulpon in Burkina Faso [123] . Diet in this rural setting has maintained a high fiber content and is similar to the diet of early human settlements in the Neolithic era. Rural children showed a significant enrichment of Bacteroidetes and depletion in Firmicutes and Enterobacteriaceae, while harboring a unique abundance of Prevotella and Xylanibacter completely absent in the European children. The latter bacteria are known to contain a set of genes for cellulose and xylan hydrolysis, and may enhance the ability to extract calories from indigestible polysaccharides. The authors hypothesized that gut microbiota may have evolved with response to the polysaccharide-rich diet of rural individuals and indicated the importance of preserving this treasure of microbial diversity from ancient rural communities worldwide. Besides, higher amount of SCFA produced in rural children is believed to be critically important for immunoregulation [124] . Bacteria of the Bacteroidetes phylum are believed to suppress the development of diabetes, presumably through the production of immunomodulatory SCFAs. SCFAs have


Shrinkage of the human core microbiome & a proposal for launching microbiome biobanks many other benefits on human fitness, which is reviewed in detail by Maslowski et al. [124] . The shrinkage of the human gut microbiome can have many unwanted consequences yet to be realized in the future. The gut microbiome has co-evolved with H. sapiens through generations [125] . Evolution selected those surviving microbial populations that conferred conserved benefits to humans and maintained the fitness of both individual hosts and the group as a whole [19,126] . In this mutual relationship, the human microbiome has protected man from dangerous chronic and infectious diseases. For example, H. pylori has dominated the human gastric niche for more than 58,000 years [127] . No other single species is so dominant in the human body. Through a very intricate co-evolutionary relationship (e.g., H. pylori injects the CagA protein into its epithelial cells in the gut), H. pylori has escaped from elimination by the human immune system [128] . In the industrialized world, H. pylori is being eliminated from the human gut – at least in affluent countries [129] . Although H. pylori is associated with an enhanced risk of gastric cancer [130] , it has been hypothesized that “the anti apoptotic effect of EGFR up-regulation promoted by H. pylori may be useful to the host early in life, during the reproductive life span” [131] . In this complex interaction, it is the degree of EGFR upregulation and the context in which it occurs that may decide whether H. pylori is beneficial or harmful. In the wrong niche, mutualists can become pathogens, while in a restricted right niche, pathogens can become mutualists [17] . It has been predicted that decrease in H. pylori population in human gut will have consequences such as increased rates of esophageal adenocarcinomas [132] , metabolic diseases [133] , as well as asthma and allergies [134] . Universalization of dietary habits and elimination of functional native probiotics from diets may also lead to homogenization of the gut microbiome in human species. Results of MetaHIT confirmed the existence of only three enterotypes including Bacteroides, Prevotella and Ruminococcus, each of which are characterized by a predominant bacterial population [135] . The results indicated the existence of a limited number of well-balanced host–microbial symbiotic states [135] . Enterotypes are significantly associated with long-term dietary patterns, particularly protein and animal fat versus carbohydrates [35] . Thus, the observed convergence of human microbiota into only three enterotypes could have resulted


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from convergence of our foods and lifestyles. Gordon et al. have also indicated that “Changes in human ecology may lead to the homogenization of human-associated microbial communities, with resulting erasure of key features of the evolutionary histories of our microbiotas. Therefore, it is imperative that our human microbiome be sampled as thoroughly and as rapidly as possible, particularly in societies that are undergoing dramatic cultural, socioeconomic and technological transformations” [136] . Capturing and maintaining a collection of microbial cultures from people living in the developing world and more specifically from agrarian cultures has been already proposed to help preserve potentially important and beneficial members of human microbiota [137] . In future, individuals – at least those with the same enterotypes or those in the same population – can be hypothesized to have the same amount of resistance and susceptibility to infectious agents, which may result from homo genization and shrinkage of microbiomes. If the resilience and resistance of the human micro biome against even one pathogenic species is compromised by the long-term changes in microbiome composition, human populations will be faced with the great danger of infectious epidemics. Although highly speculative, such a claim requires attention owing to its potential importance in human health in future. Furthermore, alterations in the composition and shrinkage of the gut microbiome might influence human health. Thus, saving native endangered microbiomes can be one of the ultimate purposes for launching microbiome biobanks. Preserving biodiversity of the gut and native foods can lead to environmental security and sustainability of the human immune system. Future perspective: a proposal for establishing microbiome biobanks Biodiversity guarantees the stability of living communities. Loss of microbial biodiversity is deemed to be possible in the foreseeable future, at least in some biomes. Reconstitution of the human biome (in the case of helminthes) has been regarded as the most reasonable solution for epidemics of allergic and autoimmune diseases [138] . Preservation of microbial DNA from a range of endangered environments has been called for [139] . In addition, it has been proposed that MOs should be high on the DNA preservation list and researchers have started to save biological specimens [140] .


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Perspective Barzegari, Saeedi & Saei The Norwegian Svalbard Global Seed Vault on the remote Spitsbergen Island in Svalbard, a permafrost archipelago in the Arctic Ocean, is a seed bank of food crops [141] . Opened officially in February 2008, the vault has the capacity to conserve 4.5 million seed samples, with the mission to preserve them against regional or global crises, as well as against accidental loss of diversity in traditional gene banks. The vault now harbors more than 750,000 distinct samples from all over the world and has been established to ensure global food security. If the future human food security necessitates the establishment of such large facilities for storing seed samples, it is very surprising that despite the high significance of microflora as food starters, thickeners, stabilizers, as agents responsible for providing food flavor, and especially as constituents of human gut microbiome – our co-evolutionary symbionts [142] – no such attempt has been made for banking microflora. The existence of meristem and seed biobanks, national genetic resources centers, animal, insect, plant and flower collections, selected culture collections of industrial and infectious microbial agents, as well as human stem cell and blood biobanks refer to the fact that the sustainability of

human species in the future may depend on the storage of biospecimens associated with human welfare. These biospecimens – microbiomes in this case – may be used in disease diagnosis and therapy, and increase our knowledge of disease mechanisms. The shrinkage of human gut microbiome diversity can be regarded as a great danger threatening human immune security. This is even exacerbated by the emergence of antibioticresistant pathogens [143] . Thus, countries must engage in preservation of the biodiversity of the human gut microbiome. National and international microbiome biobanks can serve multiple functions (Figure 4) . Once established, they must encourage the public to revert to the consumption of native and traditional foods, and initiate the sampling, isolation, characterization and storage of traditional food microflora as well as the gut microbiome of individuals. One important strategy would be to advertise the health benefits of traditional foods to promote people’s interest. However, it should also be noted that public health agencies have been advocating particular diets for decades with minimal success; thus, persuading the public to consume traditional diets will be a time-consuming

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Figure 4. Possible functions of microbiome biobanks. Central photograph from UK biobank has been kindly provided by Stephen McGowan from the University of Manchester (Manchester, UK).

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Shrinkage of the human core microbiome & a proposal for launching microbiome biobanks challenge. Preservation of native foods, especially from local and agrarian habitats, is a necessity. One cannot exactly determine the content of the universal Paleolithic diet. Since native and local diets in agrarian regions may have preserved some food trends from past, they could provide us with a good starting point to coming close to the desired Paleolithic diet. With the development of modern pasteurized, sterilized and fast foods, the microflora responsible for food taste and flavor seem to be eliminated to some extent or replaced with a number of defined commercial species. One of the major tasks of a biobank is to repopularize native fermented foods in every country or region. The texture, stability and sensorial qualities of fermented dairy products, especially cheese and yogurt, is highly dependent on the diversity of constituent MOs. Nowadays, most companies use commercial starters for production of fermented products. This will drive the native microflora into extinction. One mission of the biobank would thus be the sampling, isolation and banking of indigenous microflora from traditional fermented food starters. Local microbiota can be best identified and preserved by national biobanks, while international biobanks may engage in following food consumption trends and genetic association studies, as well as providing holistic views on linking diet, microbiome composition, health and frequency of different diseases in different countries. A variety of native and population-specific starters can be used in the production of foods. Biobanks may directly engage in the production and distribution of starters, or may have a supervisory role. The current rate of antibiotic use can possibly lead to the crisis of antibiotic resistance in the future. New antibiotics will be needed to treat futuristic superpathogens. Therefore, biobanks will fight against an antibiotic resistance threat. Biobanks may also become involved with isolating, designing and large-scale production of probiotics, which could be potentially used for prevention of infectious diseases [144] . In a recent article, we proposed that probiotics isolated from the native microbiome can be more effective in native populations [17] . Probiotics are highly integrated with human physiology; for example, in a recent study, probiotic ingestion in a fermented milk product for 4 weeks was shown to modulate brain function in humans, as assessed by functional MRI. In healthy women, this intervention was reported to alter intrinsic connectivity of brain or responses to emotional attention tasks [145] .


Perspective

Yet another futuristic possibility would be to use microbiome snapshots as fecal microbiota transplants to treat health conditions. The human gut microbiota can be readily cultured, manipulated and archived [146] . Goodman et al. demonstrated that isolates from a donor can be clonally archived and taxonomically mapped in multiwell format to create personalized microbiota collections, part of which can then be used to assemble a reunited, defined microbiota in gnotobiotic mice for studying disease and host biology [147] . Taken together, one may be able to snapshot and cryopreserve an individual’s microbiome in his/her best health status, to be used for autografting or allografting in case of disease or major alterations in the microbiome during their lifetime. Since many health conditions are (or will be) attributed to alterations in human microbiota, microbiome transplantation – which is now being employed for treatment of a number of diseases [148] – may emerge as a new therapeutic modality for a large gamut of disease. Clearly, biobanks will provide researchers with the opportunity to use the collected specimens for research purposes. Finally, research will increase our knowledge of the sophisticated relationship between microbiota, environmental players, diet, biodiversity, human immunophysiology and disease mechanisms. Simultaneous collection of human blood samples can create the opportunity to analyze human genomic data and to conduct genomic–metagenomic correlation studies. A complementary strategy would be to collect additional microbiome and blood samples from a fraction of subjects (in subsequent years) for validation of preservation strategies, and for intermediate and long-term epidemiological studies. ●●Organization & implementation

of microbiome biobanks

National biobanks may be funded by a combination of public and private partnerships, from various resources such as national research councils and charities, as well as pharmaceutical and biotech companies who may seek long-term benefits in the utilization of research output for the production of diagnostic, prognostic and therapeutic platforms (e.g., microbiome research may lead to finding complementary treatments for alleviating obesity and diabetes). In the case of human microbiome samples, some costs may be covered by patients or subjects. Furthermore, microbiome biobanks may acquire the necessary funding by production of probiotic-based supplements and native industrial starters.


649

Perspective Barzegari, Saeedi & Saei Comprehensive protocols and standard operating procedures (SOPs) must be developed for collection, processing and archiving of samples in the biobanks, while also including built-in quality assurance and control procedures for prevention and detection of errors. Preferably, samples must be immediately processed and cryopreserved to prevent damage to any highly labile microbial entities and molecules. Furthermore, a robust laboratory information management system (LIMS) must be used for storing sample-associated information, specific processing and archiving conditions, and personal and medical details about the subjects. LIMS will also be used in tracking, annotation and retrieval of samples and/or data. Cryopreservation and freeze-drying are among the most common methods for preservation of biological specimens. While cryopreservation is the storage of living organism at ultralow temperatures of colder than -130°C, freeze-drying or lyophilization refers to controlled dehydrating of labile products through vacuum desiccation. Liquid nitrogen is theoretically the method of choice in long-term storage, because it provides the lowest storage temperature. On the other hand, freeze-drying is a cheap technique that may permit to establish biobanks in the setting of limited resources; however, not all bacteria can tolerate the freeze-drying process [149] . Depending on the nature and importance of sample and storage end point, one of the above methods will be used. Furthermore, the presence of viruses and yeasts in the microbiome should be noted. Ideally, the largest possible number of fermented food and starter samples must be collected

by field trip to rural and agrarian regions. However, collection of human microbiomes can be performed in centralized locations. Samples may be broadly classified based on type and if necessary further within subgroups. Several professional workgroups must be engaged to work in different modules of the biobanks. ●●Public impacts of microbiome biobanks

Public awareness of the importance of the gut microbiome in human health and disease will alter people’s perception of the microbes that live on and within our body [150] . Microbial biobanking will raise concerns about hygiene, microbes and antibiotics, among others. Addressing the population’s concerns and questions with proper education will gain their trust in the forthrightness of the biobank rationale. International workgroups can be established to define roadmaps and SOPs for the practical implementation of large-scale microbiome biobanking. For example, guidelines can be adapted from other national and international efforts; for example, from the International Society for Biological and Environmental Repositories (ISBER) and UK biobank. Financial & competing interests disclosure The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, grants or patents received or pending, or royalties. No writing assistance was utilized in the production of this manuscript.

EXECUTIVE SUMMARY Functions of the human microbiome ●●

The microbiome refers to the totality of microorganisms and their genomes that an individual human acquires from the environment during labor and after birth.

●●

The gut microbiome is highly integrated with human health and the immune system.

Diet, gut microbiome & human health ●●

Diet has an extraordinary impact on the composition of the gut microbiome and affects gut microbial enterotypes.

●●

Thus, diet has the potential to substantially affect human physiology and the immune system.

●●

Dietary components are associated with susceptibility or resistance to many health conditions.

Antibiotics & microbiota biodiversity ●●

650

Antibiotics influence the biodiversity of microbiota in the gut.



Shrinkage of the human core microbiome & a proposal for launching microbiome biobanks

Perspective

EXECUTIVE SUMMARY (CONT.) Antibiotics & microbiota biodiversity (cont.) ●●

After antibiotic intake, some of the commensal gut microbiota do not fully recover.

●●

Antibiotic intake and subsequent perturbation of microbiota can lead to significant alterations in the expression of developmentally regulated genes, fermentation of carbohydrates in the gut, susceptibility to infections including invasive salmonellosis, and vulnerability to other diseases including hyperoxaluria, asthma, atopy, obesity and inflammatory bowel disease.

Evolution of human diet: possible impacts on human health

By the advent of agriculture and animal husbandry, our feeding behavior transformed from a Paleolithic diet into the

●●

current modern pattern.

This transformation has been adversely affected the dietary glycemic load, fatty acid and micronutrient composition,

●●

acid/base load, sodium/potassium ratio, and fiber content.

Modern metabolic and cardiovascular diseases result from a modern diet and lifestyle, which are not in harmony with

●●

our Paleolithic genome.

Paleolithic diet has been shown to improve the health status of patients with Type 2 diabetes, to improve glucose

●●

tolerance in individuals with ischemic heart disease, to reduce the risk of cardiovascular diseases, and to exert metabolic and physiologic improvements.

Furthermore, epidemiologic studies have noted the absence of ischemic heart disease, as well as chronic and

●●

metabolic conditions in the traditional communities who have preserved their traditional lifestyles and diets. Reduction in biodiversity of the gut microbiome through modernization & its consequences

Human core gut microbiota are subject to disappearance or shrinkage upon exposure to environmental factors

●●

throughout industrialization.

Changes in dietary habits, selection of only a few non-native species as food starters in industrial products, antibiotic

●●

use, clean water, provision of robust sanitary measures, transformation of the native lifestyle, reduced interaction with farm animals, daily stresses, cesarean section and replacing maternal breast milk with formula are bringing about the reduction of gut biodiversity and/or homogenization of the human gut microbiome, defective human immune system and, subsequently, reduction of human resilience against infectious epidemics, immune-related disorders and chronic conditions.

Alterations in the composition of the gut microbiome has been implicated in the pathogenesis of several conditions

●●

including metabolic disorders, inflammatory bowel disease, irritable bowel syndrome, nonalcoholic fatty liver disease, asthma, allergies, cardiovascular diseases, bacterial vaginosis and preterm birth.

Microbiome dysbiosis is also possibly implicated in rheumatoid arthritis, autism spectrum disorders, esophagitis and

●●

Barrett’s esophagus, colorectal cancer and obesity-related hepatocellular carcinoma. ●●

The gut microbiome has also been implicated as a causal factor in kwashiorkor.

●●

The shrinkage of the human gut microbiota can have a plethora unwanted consequences to be realized in the future.

Future perspective: a proposal for establishing microbiome biobanks ●●

The indigenous microflora has coevolved with humans for millions of years.

●●

Saving native microbiomes can lead to human immune security.

●●

Capturing the biodiversity of gut and native foods is the primary aim of the proposed human microbiome biobanks.

●●

National or international microbiome biobanks may serve a variety of functions: --

Preservation of native foods;

--

Recording native lifestyles;



651

Perspective Barzegari, Saeedi & Saei EXECUTIVE SUMMARY (CONT.) Future perspective: a proposal for establishing microbiome biobanks (cont.) --

Sampling and preservation of native food starters;

--

Designing native probiotic;

--

Popularization of native foods and lifestyles;

--

Fighting the crisis of antibiotic resistance;

--

Allo- or auto-transplantation of cryopreserved fecal microbiome snapshots in personalized medicine;

--

Providing platforms and funding for extending future research about microbiome–human interactions, disease mechanisms, identification of novel drug targets and diseases biomarkers, drug discovery, and maintenance of human health.

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