Microbiome: Paediatricians’ perspective

Introduction

Humans inhabit a whole community of microorganisms in and on their body1. Almost 100 trillion symbiotic microbes inhabit a single human body which is almost 10 times the number of cells present in an adult human. It amounts to almost 1-3 per cent of the body weight2. The term “microbiome” was first used by Joshua Lederberg3 for “the ecological community of commensal, symbiotic, and pathogenic microorganisms that literally share the human body”. He suggested that these microorganisms should be included as part of the human genome because of their influence on human physiology. Research in the field of metagenomics suggests that the brigade of microorganisms living in close proximity, both in and on, human beings is indispensible for human survival. This has led to the hypothesis that the human being must be regarded as a super-organism in whom symbiotic microorganisms perform multiple tasks ranging from digestion of food to angiogenesis45. The disruption in this flora is associated with many disease conditions ranging from diarrhoea to neoplasia.

Basic terminology

The more commonly used term ‘microflora’ or microbial ‘flora’ is actually a misnomer as it technically means the plant or vegetable microbial community of a region. Recently, the term ‘micobiota’ has become prevalent which literally means all the living organisms of a region. Considering the fact that these organisms are an integral part of the human genetic landscape, the term ‘microbiome’ seems to be best suited as it symbolises all the microorganisms along with their genetic make-up as well as their interactions in a particular environment3. Conventionally, the major part of the research relevant to human microbiome has focussed on identification and study of the inhabiting bacteria, however, there also exists a ‘virome’ i.e. the viruses inhabiting the body, that are probably as important6. ‘Phylotypes’ or ‘operational taxonomic units’ (OTUs) are a group of microbes generally defined by the level of sequence similarity (percentage) between the 16S rRNA genes (e.g. ≥ 98% for a ‘species’-level phylotype)7.

The recent advent in the knowledge of human microbiome is undoubtedly attributed to the preceding advances in the field of metagenomics. ‘Metagenomics’ is the science of directly analysing the genome (the complete set of DNA present in a single cell) contained in an environmental sample8. Metagenomics has opened up the avenues for obtaining the genetic information on potential biocatalysts, genomic links between function and phylogeny along with the evolutionary profile of the microbial community8. Three closely related fields that are coming up are ‘metatranscriptomics’ i.e. the study of the transcriptomes (reverse-transcribed RNA transcripts) of a group of interacting microbes9, ‘metaproteomics’ i.e. the study of the entire protein complement of the microbial community10 and ‘metabolomics’ i.e. the study of small-molecule metabolites of the microorganisms11.

Studies carried out in ‘germ-free animals’ i.e., the ones born and reared in sterile environments free of any microbiota have provided useful insight into the complex interactions taking place between the microbiome and its host12. Likewise, ‘gnotobiotic systems’ have been developed to get animals with desired microbiome by transferring or synthetically implanting the desired microbes from another host into the germ-free animals13. Researchers have developed gnotobiotic systems with humanized microbiota to evaluate the effects of controlled interventions14.

Mapping the microbiome

The foundation of present day microbiology was laid down in the nineteenth century after Robert Koch developed the technique of selectively growing and isolating bacteria in culture medium. Until recently we were dependent on the conventional methods of ‘cultivating’ bacterial ‘pure cultures’ for isolating the microorganisms in a sample and then depending on growth on a particular type of medium, colony morphology and consumption or production of a particular metabolite to finally identify it. But the observation that only a few of the microbes out of the many visualised microscopically could be isolated by culture prompted scientists to develop the newer ‘culture-independent’ techniques to recognise these yet unidentified microbes15. Escherichia coli that was considered as the major component of gut microbiota actually constitutes much less than 1 per cent of gut bacteria, but as it grows easily in culture, it can be detected even at low abundance16. It is estimated that almost 20-60 per cent of our microbiome, depending on the region of the body, cannot be cultured by the conventional methods17.

The prokaryotic cell contains 16S RNA gene in its ribosome that has about 1500 nucleotide; it is highly conserved between different species of bacteria but has several hypervariable regions that can allow identification at genus or species level13. Sequence analysis of 16S rRNA gene is thus utilised in many phylogenetic studies to simultaneously amplify the genetic regions of clusters of diverse bacteria by PCR. Initial DNA sequencing techniques were based on tedious fingerprinting methods for the separation of 16S RNA like denaturing gradient gel electrophoresis (DGGE) and restriction fragment length polymorphism (RFLP)12. Sanger technique which was developed in 1977 was used for more than two decades for sequencing of the amplified and cloned 16S RNA. It was based on the classical chain termination methods which could be used only on short strands (100-1000 base pairs). Cloning longer DNA strands became possible with the advent of Shot-gun sequencing by which longer sequences could be subdivided into smaller fragments, and subsequently re-assembled to give the overall sequence18. With the recent technological explosion, many low-cost, high-throughput sequencing technologies have been developed that parallelize the sequencing process and can produce millions of sequences concurrently. These ‘highly-parallelized’ or ‘next-generation’ sequencing techniques like 454 Pyrosequencing, Sequencing by Synthesis (Illumina) And Sequencing by Ligation (SOLiD) are much more accurate and less time consuming19. Whole-Genome Shotgun (WGS) metagenomic sequencing has emerged as an important strategy enabling scientists to analyze the DNA extracted directly from a sample and evaluate not only its composition (taxonomic diversity), but also the metabolic tasks (functional metagenomics) performed by the microbial community1213. The sequenced clones are utilised for the purpose of identification by finding the closest match in the existing gene database bank if available; and the novel sequences are added to the database to facilitate future research.

Virus identification is a relatively tougher task as the viruses lack this 16S RNA gene. Probably that is the reason why the researchers believe that virome research has lagged much behind than that has occurred for their bacterial counterparts6. But with the advent of shotgun sequencing that enables deciphering each and every sequence of DNA in the sample, viral metagenomics is being utilised to discover the role of human virome in health and disease2021.

The task of mapping the human microbiome has been taken up by scientists around the world through large scale projects like Human Microbiome Project (HMP) and Metagenomics of the Human Intestinal Tract (MetaHIT). The United States National Institutes of Health (NIH) launched the HMP in 2008 with the goal of characterizing healthy human microbiome using the culture-independent methods, to determine whether perturbations in the microbiome affect health/disease status, to provide a standardized data resource and new technological approaches to enable further research, and to evaluate the ethical, legal, and social implications of the same17. The European Commission initiated the MetaHIT in 2008 to decipher the intestinal metagenome and analyze their association with human phenotypes22. These projects have helped scientists to discover an enormous database comprising thousands of taxonomic profiles that constitute more than trillion bytes of data of metagenomic sequences2223.

Composition and biodiversity of healthy human microbiome

The human body is home for taxonomically diverse classes of friendly microbes ranging from eukaryotes, archaea, bacteria and even viruses23. The gastrointestinal tract is the largest reservoir of commensals in the body and hence has been studied most extensively121424252627. Other sites which have been sampled and analysed include skin, oral cavity, nasal cavity, lower respiratory tract, and vagina. Costello et al26 in their study of multiple body sites were able to detect members of 22 bacterial phyla, of which > 90 per cent was contributed by four predominant phyla, viz., Actinobacteria (36.6%), Firmicutes (34.3%), Proteobacteria (11.9%), and Bacteroidetes (9.5%)26282930. Though the number of phyla is limited, the biodiversity increases at the level of class, family, genus and becomes enormous at the level of species24. Each body habitat is dominated by certain signature microbes like Propionibacterium on the skin and Lactobacillus in the vagina173132.

Bacteroidetes and Firmicutes are the predominant phyla inhabiting the gut amounting to more than 95 per cent of the adult gut microbiome25262730. Family members share more similar gut microbiome as compared to unrelated individuals733. Each adult individual has a unique microbiome whose composition tends to remain stable over a period of time33. Neonates are born with an almost sterile gastrointestinal tract. Environmental exposures during infancy lead to dense colonization of the gut that is highly variable over time but by the end of infancy the microbiome converges to resemble almost like an adult33. The amount and variety of microorganisms inhabiting the gut during this time have a significant impact on the development of a person's immune system with life-long consequences34. Multiple factors affect the timing and composition of this ecosystem in the neonatal gut. Penders et al35 observed that infants delivered by caesarean section had lower colonization rates and counts of bifidobacteria and Bacteroides fragilis but higher prevalence and counts of Clostridium difficile and E. coli. Breastfed infants at the age of one month were predominantly colonized with bifidobacteria whereas formula-fed infants were colonized with E. coli, C. difficile, B. fragilis, and lactobacilli. Hospitalization and prematurity were associated with higher rates of colonization with C. difficile. Use of oral antibiotic decreased the levels of obligate anaerobes like bifidobacteria and Bacteroides. Infants having an older sibling demonstrated higher bifidobacterial counts. They concluded that term infants, delivered vaginally at home and exclusively breastfed have the most beneficial gut microbial composition, i.e. having large number of bifidobacteria and less of C. difficile and E. coli35. Antibiotics are known to inhibit the healthy microbiota allowing pathogenic microbes like C. difficile to proliferate. Antibiotic exposure, direct as well as indirectly through breast milk in neonates has been shown to result in significant alterations in the gut microbiome that may last for days to months3536.

The predominant phyla present on the skin are Actinobacteria, Firmicutes, and Proteobacteria whereas Bacteroidetes which is predominant in the gut is a minor component of the skin3738. In comparison to adults, infant skin shows Firmicutes predominance39. Akin to gut microbiome, the cutaneous microbiome also evolves during infancy with staphylococci dominance during the initial period giving way to a more diverse ecosystem by the end of the first year39. Skin is an indispensible physical and immune barrier for humans hence its early microbial colonization is an important determinant of body's defence against pathogens. The microbial composition of infants as well as adults is highly variable but site specific depending upon local anatomy, lipid content, pH, sweat, and sebum secretion at the site40. This could be the underlying reason as to why certain diseases of the skin that have been linked with microbial causation have predilection for specific skin sites like acne, atopic dermatitis, psoriasis and seborrhoeic dermatitis40.

Majority of the bacteria sampled at the nostrils belong to Firmicutes and Actinobacteria; but Proteobacteria are very few unlike the skin elsewhere41. Oropharynx is known to inhabit microbes belonging to Firmicutes, Proteobacteria, and Bacteroidetes41. Till date, very few researchers have been able to characterize the healthy lower respiratory tract microbiome as obtaining pure lung derived samples not contaminated by upper airway microbes is a relatively difficult task. Charlson et al42 have observed that the lung microbiome is similar in composition to upper respiratory tract but has a lesser biomass and hypothesised that it probably originates by micro-aspiration from upper airways. Pyrosequencing techniques have established that the female genital microbiome is dominated by lactobacilli that belong to phylum Firmicutes32.

It is not only the taxonomic composition, but also the taxonomic diversity that has been implicated in causation of several diseases. Two parameters that are routinely employed for this purpose are alpha diversity, i.e. how many kinds of taxa or lineages are within a sample and beta diversity, i.e. how many kinds of taxa or lineages are shared among samples from same habitat among different subjects43. Data from NIH-HMP demonstrated that saliva had the maximum variety of bacteria (highest alpha diversity) but different individuals had similar microorganisms (lowest beta diversity) in their saliva43. On the other hand, skin microbiome showed intermediate alpha diversity but highest beta diversity, whereas vaginal samples demonstrated the lowest alpha as well as beta diversity at the genus level. Temporal analysis has revealed that each adult harbours a unique microbiome that stays more or less stable over time as compared to the population as a whole both in terms of microbial composition as well as metabolic functions43.

Studies evaluating the metagenomics have proved that in spite of variations in the taxonomic profile, the metabolic functions carried out by the microbiome of a particular body site were similar among different individuals. This has prompted scientists to hypothesise about the possible existence of a ‘Core Microbiome’ that might be sharing a set of genes and/or metabolic capabilities7. Tap and colleagues30 observed that inspite of presence of a large number of species in the gut a limited number of OTUs were shared amongst many individuals and hence these might represent the phylogenetic core of human gut microbiome. Recently, Li and colleagues44 attempted to characterize the core microbiome of different body habitats using two parameters viz. ubiquity and abundance. These have been represented in the Figure.

Close

Figure

Representation of the core taxa at different human microbial habitats.

Export to PPT

Microbiome and diseases of childhood

Gut microbiome plays a vital role in several body functions like nutrient processing and assimilation; defence against pathogenic microbes; and even stimulation of angiogenesis454546. Alteration in this ecosystem has been associated with causation of several diseases of the gut in children, like infantile colic and necrotizing enterocolitis (NEC)4748495051. Imbalances in intestinal microbiome have been implicated in causation of many non-gastrointestinal disorders as well, like asthma, atopy, obesity, type -1 diabetes, and autism as depicted in the Table727474849505152535455565758596061626364.

Table Paediatric diseases implicated to result from dysbiosis in human microbiome

Several skin disorders like atopic dermatitis and psoriasis have been associated with changes in the local cutaneous microbiome575859. Similarly alterations in respiratory microbiome during infancy have been linked with wheezing as well as future development of bronchial asthma6061. Researchers have demonstrated the role of altered microbiome in many oronasal diseases like caries, periodontitis, and chronic rhinosinusitis626364.

Virome

Majority of the viruses that inhabit humans are the bacteriophages, i.e. viruses that infect bacteria. Hence the human virome apart from influencing the cellular processes directly could be acting indirectly by altering the symbiotic bacterial functioning, composition, or abundance6. Lysholm et al21 analysed the respiratory secretions of children with severe lower respiratory tract infection by metagenomic sequencing and observed that three of the RNA virus families were responsible for more than 90 per cent of these infections. These were Paramyxoviridae - human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV) and human parainfluenza virus (hPIV); Orthomyxoviridae - influenza virus; and Picornaviridae - human rhinovirus (HRV)21. It has been observed that viruses that may be non-pathogenic otherwise, interact with various susceptibility genes in predisposed individuals and result in diseases like Crohn's disease, type-I diabetes and bronchial asthma which would not have manifested if either the susceptibility allele or the virus was absent6566. On the other hand, certain pathogenic viruses like herpes viruses tend to adapt to the host body causing lifelong infection, are at times considered a part of the human virome67. EBV and other herpes viruses have been implicated in causation of allergic and atopic diseases like asthma and eczema by immunomodulatory mechanisms67. Wylie et al68 carried out sequence analysis of the human virome in nasal swabs from febrile and afebrile children and observed that children with unexplained fevers had more viruses than healthy kids hinting towards a viral aetiology. This knowledge could be utilized in developing tests that would help in rapid identification of the virus and thus avoid unnecessary antibiotic usage.

Microbiome research in children in the Indian context

Very few paediatric studies have been carried out in India to evaluate the role of microbiome in health and disease. A study from Pune observed that the intestinal flora of infants born by caesarean section was more diverse as compared to infants delivered vaginally69. The most abundant bacterial species present in vaginally delivered infants were Acinetobacter spp., Bifidobacterium spp. and Staphylococcus spp. whereas caesarean delivered infants’ faecal microbiota was dominated by Citrobacter spp., E. coli and C. difficile but lacked in Bifidobacterium spp. In another study published from Vellore, it was observed that one species of Bifidobacterium i.e. B. longum subspecies infantis colonized and predominated the neonatal gut70. They also observed that asymptomatic rotavirus infection in neonates did not alter the development of the intestinal microbiota in terms of bifidobacterial diversity or colonization.

Future implications

The observed gut microbiome dysbiosis in diseases like gastroenteritis, necrotizing enterocolitis, inflammatory bowel disease, malabsorption, obesity and atopy can open up the avenues for prevention and management of these diseases by several ways. Animal studies have demonstrated that many gut bacteria produce immunomodulatory, anti-inflammatory, and growth promoting molecules717273. These microorganisms also produce antibacterial substances like bacteriocin and lacticin that inhibit the growth of pathogens like C. difficile7475. Further characterization of human microbiome might help to utilize these bacterial derivatives as therapeutic agents to control various disease states. The term “pharmabiotic” has thus been used to denote any material that has been obtained from the intestinal microbiome and can be utilized for health promotion, be it a molecular by- product or a microorganism itself.

Microbiological modification of gut microbiome in a desired manner can be attempted by means of administering various prebiotics and probiotics, separately or in combination as synbiotics. These “functional food ingredients” are being utilized to enrich the gut microbiome with bacteria like Bifidobacterium and Lactobacillus that are believed to be health promoting76. The role of prebiotics and probiotics has been well established in acute gastroenteritis as well as antibiotic associated diarrhoea. In a meta-analysis by Deshpande et al77, it was observed that probiotic supplementation in preterm neonates not only reduced the risk of necrotizing enterocolitis but also the risk of mortality. Similarly in a meta-analysis by Brenner et al78, probiotic usage in patients of irritable bowel syndrome has been found to be efficacious.

Faecal microbiota transplantation (FMT) has been tried by occasional researchers in the last century also as a therapeutic intervention for antibiotic associated diarrhoea caused by C. difficile. A systematic review found out its efficacy to the tune of 92 per cent but further research is required to standardize the procedure; evaluate its safety and suitability before it is approved as a treatment modality by the regulatory agencies7980.

de Weerth et al47 in their study on infants with colic, observed the consistent presence of a few Proteobacteria linked to Escherichia, Klebsiella, Serratia, Vibrio, Yersinia, and Pseudomonas with the colic phenotype. Saulnier et al81 have been able to link specific “signature” microbes with several diseases like irritable bowel syndrome. These disease specific signature phylotypes might serve to devise diagnostic as well as therapeutic strategies in the future.

Recent advances in pharmacogenomics that focus on the role of genetics in an individual's response to a drug have prompted researchers to explore other environmental influences that affect drug metabolism. Gut microbiome is one such important determinant. Personalized drug therapy to improve efficacy and reduce adverse effects might become feasible with an approach utilizing pre-dose metabolite profiling to predict an individual's response to a drug. This novel approach has been termed as pharmaco-metabonomic approach82.

The ultimate objective of the microbiome research is to utilize this knowledge to predict the risk of disease development, develop newer techniques to diagnose related diseases and evolve therapeutic approaches for manipulation of the human microbiome for betterment of mankind.