Accepted Article

The human microbiome: opportunities and challenges for clinical care1

A/Prof Geraint B. Rogers Director, Microbiome Research SAHMRI Infection and Immunity Theme, School of Medicine, Flinders University, Adelaide, South Australia Tel: (0)8 8204 7614 Fax: (0)8 8204 4733 Email: [email protected]

Word count: 3664 Short title: Human microbiome and infection

This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process, which may lead to differences between this version and the Version of Record. Please cite this article as doi: 10.1111/imj.12650

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Accepted Article Abstract

There is a growing appreciation of the importance of the human microbiome to our normal physiology. This complex microbial ecosystem plays a range of roles, including influencing the development and function of our immune systems, providing essential nutrients, regulating metabolism, and protecting us from opportunistic infections. Our increasing understanding of these processes is due, to a large extent, to the development of high throughput sequencing technologies, providing for the first time a means by which complex microbial dynamics can be detailed. There is also a growing recognition that disruption of commensal microbiota, a phenomenon known as dysbiosis, is associated with a number of common disorders, including inflammatory bowel disease, type 2 diabetes, and oncogenesis. Further, where innate immunity fails to protect us, the microbial communities that colonise the external surfaces of our bodies represent a ready source of infection. This reviews discusses the mechanisms that govern our interaction with our resident microbiota, both in health and disease, the technological advances that allow us to gain insight into these relationships, and the way in which our growing understanding can inform clinical practice.

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We commonly think of the human body as an independent entity, however we each carry with us throughout our lives a complex microbial ecosystem. This ecosystem, referred to as the “human microbiome”, is comprised of bacteria, archaea, viruses, fungi and protozoa, of which the bacterial component alone outnumbers our own cells by a factor of 10 to 1. 1 Excepting the surface of the eye, the upper genitourinary tract, and distal airways, these microbial populations colonise our entire external surface, with a range of host defence mechanisms preventing their translocation from our skin and mucosae into sterile internal regions of the body. Despite the ease with which the ‘human microbiota’ (see glossary) can be overlooked during health, there is an increasing appreciation of the contribution it makes to our normal physiology. Further, where there is suppression or dysfunction of innate immunity, these same commensal microbes can quickly become a source of infection, or a driver of disease. Our ability to understand the roles that our microbiota play, both in health and disease, have been transformed by recent analytical advances. In particular, developments in DNA sequencing technologies have provided a rapid and relatively inexpensive means to determine the composition of complex microbial communities in high detail. However, these analytical gains can only be translated into improvements in patient care if clinicians are aware of their capabilities and limitations, and are able to interpret the data that they generate in a way that is clinically informative.

The human microbiome; from cradle to grave The composition of the human microbiota change greatly over the course of our lives, in part, reflecting our own growth and development.2,3 Recent studies, in which bacteria have

been cultured successfully from the first meconium of full-term, healthy neonates4 suggest

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that our colonisation by microbes may begin even before birth. Our entry into the world, whether by vaginal or caesarean delivery, further influences our initial exposure to bacteria5, with breast feeding also influencing early colonisation.6 As we progress through early life, our neonatal microbiota differentiates into distinct, site-specific, bacterial communities across the body.5

Perhaps our most important interface with microbial communities is in the gastrointestinal tract (GIT). In neonates, bacterial colonisation of this niche is linked intimately with the development of the nascent immune system,7 gut anatomy8 and the nervous system.9 The functionality of this early GIT microbiota is also important in neonatal nutrient acquisition, with differences between the microbiome in infants and adults reflecting its vital contribution to host metabolism. For example, the GIT microbiome in babies is enriched in genes involved in the de novo biosynthesis of folate, while those of adults have a significantly higher representation of genes that metabolise folate acquired through diet.10 By about three years of age, our GIT microbiome is starting to approach an adult-like state.10

The composition of the intestinal microbiota is now relatively stable and is only altered transiently by external disturbances, such as antibiotic exposure,11 changes in diet,12 or

illness.13

Once established, the mature GIT microbiota represents a highly complex and diverse microbial system, comprised of many thousands of different bacterial species.14 The combined genomes of these bacteria contains more than 5 million genes, thus outnumbering our own genetic potential by two orders of magnitude.14 The scale and composition of the microbiota varies along the gastrointestinal tract,15 peaking in the distal gut, where the concentration of bacterial cells can reach 1011 per gram in the colon.16 Here, the dominant phyla are Firmicutes and Bacteroidetes, followed distantly by Actinobacteria and

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Verrucomicrobia.17 The relative abundance of taxa varies considerably between individuals,18 with these differences linked to health and disease.19 While the concentration and diversity of commensal bacteria is greatest in the colon, microbiota analogous to those in the gut develop across the external surfaces of our body. The skin microbiota of adults, for example, contains as least 19 different bacterial phyla,19 of which Actinobacteria, Firmicutes, Proteobacteria, and Bacteroidetes are the most prevalent. Microbiota composition is determined by the physical and chemical characteristics of the niche being colonised, regardless of where in the body this is. For example, in the case of skin microbiota, key selective factors include local humidity and the production of sebum and sweat.19,20 By selecting for species best adapted to these conditions, pathogenic species are effectively excluded, a phenomenon referred to as ‘colonisation resistance’. As we age, the stability of our commensal microbiome starts to decrease. In later life, considerable variation can be seen in the composition of the GIT microbiota21 with falling diversity and functionality22 and greater inter-individual variation.23 Much of this variance appears to be diet-driven, and correlates strongly with indicators of relative health, including measures of frailty and inflammation.23 An analogous process occurs in the respiratory tract, with a shift in microbiota composition away from that seen in middle age, characterised by a loss of differentiation between different airway niches.24 Finally, upon death, our commensal microbes overcome our external defences and, by translocating through compromised mucosae, begin the process of putrefaction.25

The human microbiome in health Not only are the bacterial populations that colonise the colon the largest and most diverse of the human associated microbiota, they also make the greatest beneficial contribution to our physiology; the GIT microbiome contributes substantially to our nutrition,26 influences

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metabolic function,27 shapes the regulation of our immune system,28 and even affects our thought processes.9

Given the highly complex nature both of gastrointestinal physiology and the GIT microbiome, it is unsurprising that we are only just beginning to understand the mechanisms that govern their interactions. However, an important contribution to this interface may be the production by the gut microbiota of short-chain fatty acids (SCFAs). SCFAs are a subset of fatty acids produced during the bacterial fermentation of dietary fibre, which are both used locally by enterocytes and transported across the gut epithelium into the bloodstream. SCFA levels impact on the host beneficially and are known to be important in relation to nutrition, adipose tissue deposition, immunity and cancer amongst other conditions.29,30 Different

SCFAs affect specific physiological processes. For example, butyrate is largely used as an energy source by the colonic epithelium, propionate is primarily utilised by the liver, whereas a significant proportion of acetate reaches peripheral tissues,31 while all can cross the bloodbrain barrier and modulate CNS functions, brain development and behaviour.32 Importantly, variation in GIT microbiota composition is associated with changes in the composition of SCFA output.33 The importance of the GIT microbiota is illustrated clearly by animal models that are germfree or have a substantially ablated microbiota. Here, such derangement is associated with abnormal immune regulation, intestinal physiology, and enhanced allergic reactions (reviewed Kamada, 201434).

The human microbiome in disease Where there is suppression or dysfunction of innate immunity, our commensal microbiota can become important sources of infective agents. These infections can be divided into three broad categories.

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i) Acute infection of internal sterile sites Unlike external body surfaces, internal regions are thought to be free from substantial microbial colonisation during health. However, polymicrobial infections of internal sites can occur where mucosal defences are breached. Such breaches can result from trauma, such as a puncture wound, or from structural or physiological change, such as loss of tight junctions in gut epithelia.35 Failure to kill or remove microbes that translocate into internal regions results in the development of acute infection. For example, spontaneous bacterial peritonitis (SBP) characteristic of end-stage alcoholic liver disease36 is thought to be the result of such a process. Here, a loss of integrity in the intestinal epithelial barrier, bacterial overgrowth within the colonic microbiota, and reduced hepatic clearance, may all contribute to the occurrence of SBP (reviewed by Yan, 201237). Translocation of bacteria, particularly those associated with the periodontium, can also occur through oral epithelia. This movement results in transient bacteraemia, and can give rise to septic emboli and infection at remote sites. Cases of endocarditis, for example, have been shown to involve bacteria that have originated in the oral cavity38 and a range of processes,

including distant abscesses and failed joint prostheses, may occur through the same process. Importantly, the movement of bacteria into the bloodstream from the oral cavity is not limited to context of periodontal disease, but is associated with a range of normal daily activities, including eating, brushing the teeth, or using toothpicks.39

ii) Acute infection of external sites normally free from microbial growth The body has a number of external or externally contiguous surfaces (such as the distal airways and ocular surface) that are maintained free from substantial microbial colonisation

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in healthy individuals. In the distal airways, for example, epithelial colonisation is prevented not by physical barriers, but through processes that remove microbes that enter from adjoining regions or the external environment. These mechanisms include the mucociliary escalator, the action of alveolar macrophages, and the sequestration of iron (required by many pathogenic bacteria for proliferation and virulence).40 The balance between the entry of microbes into the lower airways and their removal can become tipped towards colonisation, either through increased microbial influx, or through disruption of clearance mechanisms.41 An example is intubation, where the presence of an endotracheal tube (ETT) can lead to the impairment of mucociliary motility and cough reflex, the pooling of contaminated oropharyngeal secretions over the ETT cuff, and its subsequent movement into the lungs through a hydrostatic gradient.42 Together, these factors present an

increased risk of ventilator-associated pneumonia (VAP). The importance of mucosal microbiota in early-onset VAP is reflected by the common aetiological agents, which include residents of the upper airways, such as Staphylococcus aureus, Streptococcus pneumoniae, and Haemophilus influenzae.43

iii) Chronic infection of external sites normally free from microbial growth In addition to acute disruption, the mechanisms that maintain external sites free from microbial colonisation can be suppressed chronically as a result of genetic mutation or sustained inflammation. For example, in the lung, conditions such as cystic fibrosis, non-CF bronchiectasis, and primary ciliary dyskinesia are characterised by chronic bacterial colonisation that stems from combinations of impaired ciliary function, abnormal airway secretions, and chronic inflammation.44-46 In these contexts, stable and complex infective microbiota become established that are comparable in complexity to commensal communities associated with other mucosal surfaces.47 The infective microbiota that characterise these

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contexts represent a number of particular challenges, which include apparent increased stability and resilience to therapy compared with single species infection.48-50

Infective microbiota versus individual pathogens From a traditional diagnostic microbiology perspective, whether an infection involves a single species or a diverse infective microbiota is usually considered to be of little consequence. Rather, it is the presence of particular pathogens, recognised to be associated with poor clinical outcome, which is of primary concern. The techniques used in the analysis of clinical samples are therefore designed to selectively isolate such microbes, most commonly through culture under specific conditions. There is, however, an increasing realisation that the clinical properties of an infective microbiota can be quite distinct from that of its constitutive members, particularly in the case of complex, chronic infections. For example, many non-pathogenic bacterial species detectable in CF lung infections have been shown to increase the virulence of Pseudomonas aeruginosa when present together.51 In the development of human dental caries, the expression of certain virulence factors by Streptococcus mutans, an important aetiological agent, is inhibited by other species of oral bacteria through interference with their cell–cell signalling mechanism.52 Considering the clinical significance of any one member of an infective microbiota in isolation is therefore potentially misleading.

Dysbiosis

In addition to the infective contexts outlined above, the human microbiota can also contribute to pathogenesis in the absence of infection. Disruption of commensal microbiota in a way that impacts on host homeostasis, a situation referred to as dysbiosis, can have significant clinical implications. Commonly precipitated by external factors, such as exposure to

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antibiotics,53 dysbiosis can lead to normally benign microbial communities becoming proinflammatory,54 invasive,55 or being overgrown by pathogens.56 In the gastrointestinal tract for example, dysbiosis has been implicated in a range of disorders, including inflammatory bowel disease,54 metabolic disorders,57 and oncogenesis.58

Disease resulting from the disruption of commensal populations is not limited to the GIT. Bacterial vaginosis (BV), the most common vaginal disorder in women of reproductive age,59 is believed to occur not as the result of any single aetiological agent, but as the result of a shift in the composition of vaginal microbiota. This process involves the depletion of lactobacillus populations, and an overgrowth of commensal anaerobic bacteria.60 While

asymptomatic in most instances, BV is associated with a range of adverse outcomes, including preterm birth,61 pelvic inflammatory diseases,62 and the acquisition and transmission of sexually transmitted infections.63

The sequencing revolution Recognising the clinical importance of understanding infective microbiota is a substantial advance. However, obtaining detailed characterisations of polymicrobial samples, and interpreting these data appropriately, still represents a major challenge. Even at the beginning of the 20th century it was understood that certain infections were

polymicrobial in nature.64 Despite this, the basis for diagnostic microbiology has remained

largely unchanged since the end of the 19th century; isolation of specific aetiological agents through enrichment culture. Attempting to use such an approach to comprehensively characterise complex bacterial microbiota would be both prohibitively laborious, and likely to isolate only a fraction of the species present.65 However, recent sweeping advances in culture-independent analytical techniques have transformed our ability to characterise such microbial systems. In broad terms, these techniques are based on the analysis of nucleic acids

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extracted directly from clinical samples without prior cultivation, resulting in comprehensive bacterial characterisation. In providing a basis for detailed, high-throughput analysis, culture-independent approaches have made investigation of even the highly complex microbiota present in the human colon now tractable. The advances in molecular microbiology that have made this possible have occurred in three steps. The first was the discovery of the polymerase chain reaction (PCR) in 1983,66 a process that allows the amplification of a single or a few copies of a DNA sequence across several orders of magnitude. The second was the realisation that, in having both highly conserved regions that allow PCR amplification from any bacterial species and highly variable regions that allow differentiation and identification, the 16S ribosomal RNA gene could be used as a basis for analysis of polymicrobial systems.67 The third was the development of Next Generation Sequencing (NGS). NGS allows the huge numbers of 16S rRNA genes copies that can be amplified by PCR from clinical samples to be sequenced, and the species they were derived from identified through comparison with a sequence database. Since the introduction of NGS-based approaches, the field of human microbiome analysis has expanded rapidly; in part, the result of a dramatic fall in the costs associated with this type of analysis.68 The establishment of investigative consortia, such as the Human Microbiome

Project69 and METAHit (Metagenomics of the Human Intestinal Tract consortium)70 has further contributed to this expansion. While 16S rRNA gene sequencing can be hugely informative, it does have a number of limitations that should be considered. These include biases associated with PCR amplification that can distort the relative levels at which particular microbes are reported,71 the susceptibility of PCR reactions to contamination with bacterial DNA, 72 and the need to cluster similar sequences together for identification in what are referred to as operational taxonomic units (OTUs). This latter process typically occurs at a level of 97% sequence

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similarity, a level at which many species cannot be differentiated, meaning that identities can only be reported at a genus or family level.73 A number of reviews have now been published

that provide practical guidance for the application of sequencing technologies to clinical contexts.73,74

How can microbiome analysis inform clinical practice? The development of sophisticated, culture-independent techniques for the analysis of clinical samples provides an ability to detect many more species than can be reported through traditional microbiology, including those refractory to culture under standard conditions. However, to view these advances as merely an extension of current analytical approaches would be wrong. Rather than simply improving our ability to detect particular pathogens, the highly detailed characterisations that such techniques generate also allow us to investigate how the behaviour of microbial communities can differ from that of their individual members, viewed in isolation. Further, these approaches allow us to probe the subtle interplay between these microbial systems and factors such as host inflammatory response or therapeutic intervention. These research efforts can therefore be divided into three main themes.

i) Defining normal microbiota in niches across the body, and characterising dysbiosis Defining what constitutes ‘normal’ microbiota at different body sites provides a basis for assessing changes that occur during dysbiosis. While projects such as HMP and MetaHIT are attempts to do this in a substantial and systematic way, comprehensive data are only likely to emerge over time. A particular challenge is that ‘healthy’ microbiota conformations may be stratified, rather than continuous,75 and subject to substantial inter-personal variation relating to age, lifestyle, genetics, and diet.75

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In addition to defining variation in microbiota composition between individuals, it is also important to understand how these microbial systems develop within individuals and change over time. The ordered establishment of the GIT microbiota in neonates is intimately linked to immune and metabolic programming.76,77 As discussed above, disruption of this process is

associated with long-lasting disease risks. For example, caesarean section delivery appears to be associated with an increased risk of celiac disease, type 1 diabetes, and asthma, which is generally associated with excessive or aberrant T-helper responses.78 In addition, lower numbers of Bifidobacterium during early infancy (6 and 12 months) is correlated with obesity at 7 years of age.79 It is only through the careful characterisation of these processes that such risk factors can be identified and addressed.

ii) Needles in a microbiological haystack – identifying the unknown pathogens within a microbiota

Conventional culture-based diagnostics are used currently to detect specific pathogens that have associations with clinical conditions. However, given the huge complexity that exists within the human microbiome, it is certain that many clinically important host-microbe interactions are yet to be discovered. Detailed characterisation across a range of conditions will be important for identifying pathogens or pathogenic consortia. Associations between particular species and disease have now been reported using sequencing approaches in contexts such as colorectal cancer,80 periodontal disease,81 and chronic lung infection.46 However, demonstrating pathogenic causality remains a greater, and ongoing, challenge.

iii) Understanding the wider impact of antimicrobial therapy There is a growing understanding that the impact of systemic antimicrobial therapies is not limited to their intended target, but can exert an effect across host-associated microbiota.

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Such disruption means that treatment of one condition can predispose us to another. Perhaps the most common example of this is antibiotic-associated diarrhoea (AAD). While AAD may only be an unpleasant side-effect of therapy, the results of GIT microbiota disruption can be much more serious. For example, antibiotic treatment is probably the most significant risk factor in the development of Clostridium difficile-associated diarrhoea (CDAD)82 and enteric Salmonella typhimurium infection.83 This predisposition to infection is thought to result from the breaking of colonisation resistance. Indeed, 16S rRNA gene sequencing has been used to show that antibiotic therapy is associated with major changes in GIT microbiota composition, some of which appear irreversible, even following a single course of treatment.53 As above, the human-associated microbiota is critically involved in the postnatal maturation of mucosal and systemic immunity. Antibiotic perturbation of the commensal microbiota during the developmental period in early life may result in persistent alterations in host immunity and an increased occurrence of immune-mediated diseases, as mouse studies have demonstrated.84 Here, the implications of GIT microbiota perturbation in neonates,

particularly resulting from antibiotic therapy85 is a source of increasing concern.

Antimicrobial therapy can also have clinically important side-effects through the impairment of microbiota function. For example, the GIT microbiota in CF patients is substantially different to that in healthy individuals,86 the likely result, in part, of CFTR dysfunction in the gut epithelium.87 However, in addition to the underlying causes of GIT dysbiosis in this patient group, repeated courses of antibiotic therapy for acute exacerbations of chronic lung infections are also likely to be a substantial contributory factor. Importantly, this dysbiosis is likely to contribute to fat malabsorption, an important contributor to the failure of CF patients to thrive, with nutritional status associated with the underlying pulmonary pathophysiology88

and long term disease progression.89

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In addition to antimicrobial therapies, there are many other types of interventions that are likely to have a substantial impact on the microbiome. For example, significant GIT dysbiosis may also result from chemotherapy90 and nutritional supplementation.91 Given the close links between microbiota function and host nutrition, such effects may substantially worsen clinical outcomes. The association between GIT dysbiosis and a range of maladies has resulted in increased interest in the therapeutic potential of probiotics. For example, probiotic formulations have been shown to be effective in limiting antibiotic-associated diarrhoea92 and may represent an

inexpensive adjunct to antimicrobial therapy.

Beyond DNA sequencing 16S rRNA gene sequencing is a highly effective tool for determining the identity and relative abundance of the bacteria in clinical samples, which has resulted in a vast expansion of our knowledge of human associated bacteria. However, it may be useful to assess not only which species are present, but also how they are behaving, or what potential they have to express pathogenic traits. A range of different “omic” approaches have now been developed that complement 16S rRNA amplicon based microbiomic analysis and provide insight into functional potential and detailed

phylogeny

(metagenomics),

microbial

behaviour

(metatransciptomics

and

proteomics), and the impact that a microbiota has on its site of colonisation (metabolomics). These techniques are increasingly being applied to the analysis of human systems, such as the gut.74,93-95

Future directions

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Microbiome research is an emerging field, and the extent to which it can inform clinical understanding will only become clear with time. Further, computational systems that allow us to integrate complex bacterial community data (let alone, information on viral or fungal populations, not discussed here) with clinical and demographic data, remain in development. However, the role played by the microbes associated with the human body in health and disease is undeniable. The prospect of tools that enable us to understand these interactions better is therefore an exciting one.

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Accepted Article References

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Table 1. Glossary Amplicon sequencing – sequencing approaches that involve the prior PCR amplification of a specific gene region, such as a 16S rRNA gene fragment Colonisation resistance – the ability of a commensal microbiota to inhibit niche colonisation

by potentially pathogenic microbes Diversity – a measure of both the number of different taxa present in a microbiota, and their relative abundance Dysbiosis – an imbalance in the microbes present in a particular niche due to a change in

conditions

Metabolomics - analysis of all metabolites within a sample Metagenome – the total genomic material within a particular niche, sample, or community.

Metagenomics – analysis of the metagenome composition, usually obtained by shotgun sequencing. This term is sometimes used to descibe all high-throughput approaches to the analysis of microbiota Metaproteomics – analysis of protein production and function within a particular niche, sample, or community Metatransciptomics – shotgun sequencing of reverse-transcribed RNA transcripts, to provide

information on gene expression patterns by a community or within a sample Microbiome – the totality of the microbes, their genetic information, and their gene products, within a colonised site Microbiota – the totality of the microbes that colonise a particular site

Microflora – a now defunct term, broadly equivalent to ‘microbiota’

Niche – a site of potential microbial colonisation, defined by its physical and chemical characteristics

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Resilience – the rate at which a microbiota returns to its original composition following perturbation

Shotgun metagenomics sequencing - sequencing of all DNA fragments from a microbiota metagenome, without prior amplification of a specific region Taxa – a taxonomic category, such as species or genus

Table 1. Glossary Amplicon sequencing – sequencing approaches that involve the prior PCR amplification of a specific gene region, such as a 16S rRNA gene fragment Colonisation resistance – the ability of a commensal microbiota to inhibit niche colonisation

by potentially pathogenic microbes Diversity – a measure of both the number of different taxa present in a microbiota, and their relative abundance Dysbiosis – an imbalance in the microbes present in a particular niche due to a change in

conditions

Metabolomics - analysis of all metabolites within a sample Metagenome – the total genomic material within a particular niche, sample, or community.

Metagenomics – analysis of the metagenome composition, usually obtained by shotgun sequencing. This term is sometimes used to descibe all high-throughput approaches to the analysis of microbiota Metaproteomics – analysis of protein production and function within a particular niche, sample, or community Metatransciptomics – shotgun sequencing of reverse-transcribed RNA transcripts, to provide information on gene expression patterns by a community or within a sample

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Accepted Article

Microbiome – the totality of the microbes, their genetic information, and their gene products, within a colonised site Microbiota – the totality of the microbes that colonise a particular site

Microflora – a now defunct term, broadly equivalent to ‘microbiota’

Niche – a site of potential microbial colonisation, defined by its physical and chemical characteristics Resilience – the rate at which a microbiota returns to its original composition following perturbation

Shotgun metagenomics sequencing - sequencing of all DNA fragments from a microbiota metagenome, without prior amplification of a specific region Taxa – a taxonomic category, such as species or genus

This article is protected by copyright. All rights reserved.

The human microbiome: opportunities and challenges for clinical care.

There is a growing appreciation of the importance of the human microbiome to our normal physiology. This complex microbial ecosystem plays a range of ...
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