Early life events influence whole-of-life metabolic health via gut microflora and gut permeability.

http://informahealthcare.com/mby ISSN: 1040-841X (print), 1549-7828 (electronic) Crit Rev Microbiol, Early Online: 1–15 ! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/1040841X.2013.837863

REVIEW

Early life events influence whole-of-life metabolic health via gut microflora and gut permeability

Critical Reviews in Microbiology Downloaded from informahealthcare.com by McMaster University on 12/29/14 For personal use only.

Caroline A. Kerr1,2, Desma M. Grice1,2, Cuong D. Tran1,4, Denis C. Bauer1,5, Dongmei Li1,2, Phil Hendry2, and Garry N. Hannan1,2 1

Preventative Health Flagship, CSIRO, North Ryde, Australia, 2Division of Animal, Health and Food Sciences, CSIRO, North Ryde, Australia, 3Discipline of Physiology, School of Medical Sciences, The University of Adelaide, Adelaide, Australia, 4Gastroenterology Unit, Women’s and Childrens’s Health Network, North Adelaide, Australia, and 5Division of Mathematical and Information Sciences, CSIRO, North Ryde, Australia

Abstract

Keywords

The capacity of our gut microbial communities to maintain a stable and balanced state, termed ‘resilience’, in spite of perturbations is vital to our achieving and maintaining optimal health. A loss of microbial resilience is observed in a number of diseases including obesity, diabetes and metabolic syndrome. There are large gaps in our understanding of why an individual’s co-evolved microflora consortium fail to develop resilience thereby establishing a trajectory towards poor metabolic health. This review examines the connections between the developing gut microbiota and intestinal barrier function in the neonate, infant and during the first years of life. We propose that the effects of early life events on the gut microflora and permeability, whilst it is in a dynamic and vulnerable state, are fundamental in shaping the microbial consortia’s resilience and that it is the maintenance of resilience that is pivotal for metabolic health throughout life. We review the literature supporting this concept suggesting new potential research directions aimed at developing a greater understanding of the longitudinal effects of the gut microflora on metabolic health and potential interventions to recalibrate the ‘at risk’ infant gut microflora in the direction of enhanced metabolic health.

Development, gut permeability, microbial diversity, microbial resilience, obesity History Received 2 June 2013 Revised 31 July 2013 Accepted 21 August 2013 Published online 19 March 2014

Abbreviations: BMI: body mass index; C. difficile: Clostridium difficile; CRP: C-reactive protein; C. rectus: Campylobacter rectus; C-section: Caesarean section; E. coli: Escherichia coli; GI: gastrointestinal; IBD: inflammatory bowel diseases; Ig: immunoglobulin; IL: interleukins; L/R: lactulose/rhamnose; NAFLD: non-alcoholic fatty liver disease; N. mucosa: Neisseria mucosa; T1DM: type 1 diabetes mellitus; T2DM: type 2 diabetes mellitus; TNF-a: tumor necrosis factor-a; TPN: total parenteral nutrition

Introduction The natural gastrointestinal (GI) microflora is predominantly composed of bacterial taxa, however there are also archaeal, viral and fungal community members present (Nelson et al., 2010). In the GI tract bacterial phyla reach numbers as high as 1014, significantly higher than the 1013 cells that make up the human body (Savage, 1977). During human evolution microbial communities have co-evolved with their human hosts (Ley et al., 2008). Further these communities co-develop with individuals from birth, to become extremely complex in structure (Blaser & Kirschner, 2007). Despite this, the gut microflora community is commonly overlooked as a contributor to the metabolic potential available to the human individual. The extended genome concept states ‘‘that we are the sum of all our genetic contributions: the karyome, the

Address for correspondence: Caroline A Kerr, Division of Preventative Health Flagship, CSIRO, North Ryde 2113, Australia. E-mail: [email protected]

chondriome and the microbiome’’ (Dumas, 2011) and it is the study of our metagenome (the composite of our genome and that of our microbiome) that has rapidly advanced our understanding of the role the gut microflora plays in health and the pathogenesis of disease. The gut microflora is at the intersection between the host genotype and environmental factors, which when combined impact host physiology, in particular, metabolic health status. Dietary studies have suggested that humans and their ancestral relatives have co-evolved with their microflora to live under conditions that require maximal utilization of ingested nutrients, i.e. for nutritional efficiency (Ley et al., 2008). Therefore, the host’s diet influences the gut microbial community structure (Ley et al., 2008). In terms of access to stable food sources, the developed world’s environment differs significantly from that of our ancestors. It is therefore not surprising that an increasing body of literature has implicated the gut microbiome as a factor in the development of metabolic diseases joining the classic factors of host genetics, environmental factors (Blaser & Kirschner, 2007;


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Kaplan & Walker, 2012; Nelson et al., 2010) and more recently intestinal permeability (Teixeira et al., 2012a). An overarching finding from these studies has been that the gut microflora of patients suffering from metabolic diseases, have decreased consortia diversity. Therefore, the consortia are unstable and susceptible to a shift in community structure towards deleterious microflora (Ley et al., 2006). We define this key feature of gut microflora as ‘resilience’ and propose that it is necessary in preventing a long term shift towards dysbiosis and as a consequence, poor disease and health outcomes over a human’s lifetime. We suggest that this may be an indicator of poor health status. Conversely, a stable ‘healthy’ microbial composition resilient to change is a positive trait/hallmark of health (Lozupone et al., 2012; Van den Abbeele et al., 2011). It is therefore important to develop an understanding around how the ‘healthy’ stable balanced microflora can deviate from a state of resilience. This review will unravel the dynamic changes in the gut microflora and intestinal permeability in response to typical perturbations in early life that result in changes across the human lifecycle impacting metabolic health. We briefly examine how disruptions in the gut microbial structure and/ or gut permeability are implicated in a number of adult disease pathologies, in particular those associated with poor metabolic health. We discuss how the resilience of our coevolved gut microflora to perturbations is pivotal for sustained metabolic health and that intestinal permeability may have a significant impact on gut microflora resilience. Table 1 summarizes our current knowledge of changes in gut microbial diversity and intestinal permeability throughout the human lifecycle. We indicate the key environmental stressors and the gaps in our knowledge including the likely effects of genetics and familial association in microbiome development. We examine the effects of life events on the host’s metabolic health starting with those that occur in the pregnant mother and the developing foetus, then the colonization of the newborn and followed by the establishment of the long term ‘adult’ gut microflora in the young child. While beyond the scope of this review, we acknowledge the debates about firstly the hygiene hypothesis and its possible role in metabolic health (see recent reviews: Brooks et al., 2013; Musso et al., 2010b) and secondly probiotic supplementation to improve metabolic health (see recent reviews: Aggarwal et al., 2013; Panwar et al., 2013). This review proposes that the effects of early life events, on the colonization and succession of early microbial consortia and on gut permeability, are pivotal for long term metabolic health and healthy aging (as illustrated in Figure 1).

Microflora optimized for energy harvest: impact on metabolic health The incidence of obesity has risen significantly over the last decades to epidemic proportions affecting all ages and socioeconomic groups in the developed world (Hill, 2006). Of particular concern, childhood and adolescent obesity has reached epidemic levels in developed countries (Dehghan et al., 2005). There is a strong correlation between obesity and the development of type 2 diabetes mellitus (T2DM), metabolic syndrome, cardiovascular disease, gall bladder

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disease, osteoarthritis, sleep and mental disorders and colorectal cancer (Alberti et al., 2005), which are all associated with increased morbidity and mortality. The etiology of human obesity appears to involve interactions between modern environments with individual behavioral, genetic and biological factors that favor energy storage (Alberti et al., 2005; Clement, 2011). One proposed environmental/ biological factor responsible for weight gain and altered energy metabolism is the gut microflora (Harris et al., 2012). The majority of the gut microbial taxa have a symbiotic relationship that is essential and beneficial to the host when conditions are optimal (Flint, 2011). Other species are ‘‘pathobionts’’, commensal in a healthy host, but can become pathogenic when the host is subject to various perturbations such as infection, diet, stress, inflammation and immunosuppression (Chow et al., 2011). An increasing body of literature has indicated that a detrimental gut microflora contributes to the development of metabolic diseases (Musso et al., 2010a). This is presumably because the microflora has the capacity to regulate energy homeostasis, metabolite production and fat disposition (Vrieze et al., 2010). However, whether or not gut microflora actively contribute to change or simply adapt to changes in the host’s health is yet to be ascertained. This topic has recently been extensively reviewed in a number of excellent articles (Angelakis et al., 2012; Holmes et al., 2011; Krajmalnik-Brown et al., 2012; Nicholson et al., 2012). We know that microbial dysbiosis, characterized by shifts in populations and a loss of species diversity, is a feature of chronic disease in adults. This is demonstrated in both animal models (Musso et al., 2010b) and human studies where gut microbiota is different in children with T1DM compared to healthy children (Murri et al., 2013). Therefore, it can be argued that a stable microbial composition is a positive trait or hallmark of health and conversely unstable microbial communities may be an indicator of poor health. Epidemiology and animal model studies have suggested a role for the gut microflora in the ‘‘developmental origins of health and disease’’ (Donnet-Hughes et al., 2010; Van den Abbeele et al., 2011). The microflora in early life has been linked to allergy and autoimmune disease risk (DonnetHughes et al., 2010; Hormannsperger et al., 2012). More recently, it has been suggested that pre- and postnatal effects can influence obesity risk (Kaplan & Walker, 2012). One recent and pivotal, longitudinal epidemiology study has concluded that a combination of early exposures (delivery mode, maternal pre-pregnancy body mass index (BMI) and antibiotics in infancy) influence the risk of being overweight in later childhood (Ajslev et al., 2011; Palmer et al., 2007). One group has concluded that a mother’s gut microflora, in parallel with her metabolic health indices, can ‘‘prime’’ the developing foetus gut prior to the imminent microbial colonization post-birth (Koren et al., 2012). There appears to be an ecological succession of the gut microflora in response to life cycle perturbations (Ley et al., 2008) that combine to potentially impact on long term metabolic health status. Further research is required to define the role that the gut microflora plays in metabolic dysfunction along with other factors such as diet, exercise and genetics. Therefore, tracking dynamic changes in the gut microflora over the

" " " " # #

Formula fed

Nursing home

Inflammatory Bowel Disease

Diabetes

Obesity

Antibiotics

# Diversity

" Bacteroidetes # Firmicutes

"

"

# Diversity

" Compared to breast fed

# Compared to formula fed

Dynamic at early life stages

" Maternal ? Child

"

Dysbiosis

Effect on gut permeability

?Mother ?Child

"

# Diversity

# Diversity

# F. prausnitzii " Fusobacterium nucleatum

# Bacteriodetes " Actinobacteria

E. Coli C. difficles Bacteroides spp Lactobacilli Bifidobacteria Bacteriodes

" Staphylococcus # Bifidobacterium " Akkermansia

Breast fed þ maternal obesity

# Mothers gut and milk diversity against NW mothers

Delay of Bifidobacterium sp & Bacteroides, Anaerobes

" Clostridium species # Bifidobacteria & Bacteroides "Bifidobacteria "Bacteroides " Bifidobacteria lowest numbers of C. difficles and E. coli

Birth delivery (C-section)

Northern European Southern European Breast fed

Normal development trajectory

Placental & foetal inflammation

# Diversity especially in the 3rd trimester Less change compared NW mother

Effect gut microbiome

Lactobacilli

" Proteobacteria & Actinobacteria " Proteobacteria & Actinobacteria (less than NW mother) Viral and bacterial pathogens (unspecified)

Effect on gut microbes: genus or phylum/class

Birth delivery (Vaginal)

Infection during pregnancy

Pregnancy þ maternal obesity

Pregnancy

Environmental factor or stressor

" Glucose, glycine, lipids, inflammatory markers

–

–

–

Antibiotic use risk of overweight later in life – dependent on mothers BMI^

Suggested to increase risk of obesity – Long term consequences? # Newborn growth rates – Long term consequences?

" Growth rates " Adiposity against formula fed

" Twofold childhood obesity

–

? Child

" Blood glucose, " Adiposity

Metabolic consequences

(Claesson et al., 2012)

(Turnbaugh & Gordon, 2009b, Cani et al., 2008)*, (de La Serre et al., 2010)*, (Ferraris & Vinnakota, 1995)* (de Kort et al., 2011)*, (Vaarala et al., 2008a)* (Kang et al., 2010), (Strauss et al., 2011)

(Penders et al., 2005), (Taylor et al., 2009)*, (Weaver et al., 1987)*, (Catassi et al.,1995)* (Bucker et al., 2010), (Mor & Cardenas, 2010)^

(Gronlund et al., 1999)*, (Zhou et al., 2011) (Yatsunenko et al., 2012) (Gronlund et al., 1999), (Neu et al., 2007a), (Zhou et al., 2011) (Fallani et al., 2010) (Fallani et al., 2010) (Rautava et al., 2012), (Smith et al., 2008), (Taylor et al., 2009)*, (Weaver et al., 1987)*, (Catassi et al.,1995)* (Cabrera-Rubio et al., 2012), (Collado et al., 2012)

(Mor & Cardenas, 2010), (Cilieborg et al., 2011)*

(Koren et al., 2012)

(Koren et al., 2012)

Reference

Early life events influence whole-of-life

Grey horizontal shading indicates research areas with significant knowledge gaps where further research is required. *Indicates reference associating gut permeability with the environmental factor or stressor. ^Indicates metabolic changes associated with the environmental factor or stressor. NW ¼ normal weight.

Senescence

Adult

Adolescent

Child

Infant (46 weeks)

Newborn

Pregnant Mother

Human lifecycle stage

Table 1. Summary of current knowledge on the changes that occur in microbial taxa and intestinal permeability across the life stages and what is known about the effects of various external factors.


DOI: 10.3109/1040841X.2013.837863

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Figure 1. Starting early in life, environmental factors influence microbial diversity (blue) and gut permeability (green), affecting the duration of resilience and metabolic health (different shadings). (A) During pregnancy, the developing foetal gut is ‘primed’ by the maternal gut microflora and intestinal permeability (diagonal lines), particularly towards the latter stages of gestation and prior to the imminent microbial colonization post-birth. (B) Following birth, the gut is colonised with bacteria and diversity develops throughout infancy and childhood with a subsequent decrease of the initially very high intestinal permeability. However, early in life, risk factors, such as maternal obesity, chronic intestinal permeability, antibiotics, delivery mode and nutritional regime can influence the development of a diverse population. (C) In adulthood, events in early life have combined with tight intestinal permeability to influence the ‘resilience’ of adult gut’s microflora. This is positively correlated with diversity and determines the health trajectory for the remaining lifespan. (D) In later years, after a period of ‘resilience’, the diversity in the gut microbiome declines and the leakiness increases during normal ageing. A low diversity or high leakiness can lead to the accelerated decline and places the individual at risk of developing a range of chronic metabolic diseases.

human lifecycle is the first step in understanding how a balance between our natural microflora’s co-evolved state and metabolic health can be either maintained or disrupted. The GI tract consists of a continuous simple columnar epithelial cell layer, which is interconnected by junction complexes known as tight junctions (Arrieta et al., 2006). A mucus layer on top of the epithelium, provides the microhabitat and nutrient source for the gut microflora (Garrett et al., 2010). The epithelium and mucus layer are the key components of the mucosal barrier, separating the internal body systems from the external environment (Bjarnason et al., 1995). The ability of the immune system and the gut microflora to co-develop during postnatal life allows the host and microflora to coexist in a mutually beneficial relationship. This process involves the maturation, differentiation and growth of the gut and the development of the innate and adaptive immune systems, which has a profound effect on the mucosal barrier function (Sharma et al., 2010). Therefore, the intestinal mucosa is not only an environment for the microflora to reside, but a physical barrier to prevent toxic luminal content from gaining entry into the host’s circulatory systems (Bjarnason et al., 1995). The mucosa prevents a wide range of environmental pathogens from entering the body, providing protection from infection, inflammation and alteration of normal body functions (de Kort et al., 2011; Hollander, 1999). Maintaining a symbiotic gut microflora protects the host against pathogenic microbes by establishing a competitive barrier to their invasion of the mucosal surface (Groschwitz & Hogan, 2009; Hollander, 1999). One emerging area of important research is around the proposal that a leaky gut (increased intestinal permeability) is associated with a variety of disorders, such as intestinal and liver diseases, autoimmune disorders including T1DM and T2DM (de Kort et al., 2011; Groschwitz & Hogan, 2009; Hollander, 1999).

Maintenance of tight junction integrity and paracellular permeability is especially important for immune system homeostasis (Garrett et al., 2010). Consequently, the integrity of the intestinal barrier can determine health and disease outcomes (Groschwitz & Hogan, 2009). Changes in structure and composition of claudins, a component of the tight junction proteins that forms the paracellular gut barrier, can alter the barrier function (Angelow & Yu, 2007). The expression of claudins is influenced by several factors including disease and infections (Bu¨cker et al., 2010) as well as hormones such as prolactin and steroids (Peixoto & Collares-Buzato, 2006). Therefore, this indicates that there are possible therapies to correct abnormal intestinal permeability as prevention or treatment for some diseases (Arrieta et al., 2006; Duerksen et al., 2005).

Maternal gut microbiome Throughout a healthy pregnancy the mother’s body undergoes substantial hormonal, immunological and metabolic changes (Mor & Cardenas, 2010; Newbern & Freemark, 2011). Recent findings have suggested that the gut microbial community of the mother also undergoes profound changes during pregnancy (Koren et al., 2012). Koren et al. (2012) reported that the mothers’ gut microflora is remodeled to support a healthy pregnancy, with significant compositional changes between the first and third trimesters of pregnancy. Notably, these changes are associated with higher levels of blood glucose and adiposity, increases in Proteobacteria and Actinobacteria, and a reduction in overall microbial diversity (Table 1) (Koren et al., 2012). These changes, in the absence of the alternations in diet and total energy intake, are also observed with obesity (Koren et al., 2012). Further, unlike healthy weight mothers, obese mothers had minimal changes in the microbial structure between trimesters (Koren et al., 2012).

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Obesity during pregnancy is associated with adverse outcomes, including excessive foetal birth weight (macrosomia) as well as low birth-weight, preterm birth, increased risk of caesarean (C-section) delivery, high maternal blood pressure (pre-eclampsia) and gestational diabetes (Smith et al., 2008). It is likely that the mother’s gut microflora produces metabolites that reach the developing fetus through the circulation. The metabolic health of the mother, during pregnancy potentially, impacts on fetal development and the infant gut microflora (Figure 1, Section A ‘Gestation’).


Maternal microbes and metabolites prime the neonates’ gut The effects of events in utero on the subsequent infant gut microflora and life time health status are not understood. As the body surfaces are largely protected from environmental and microbial exposure during fetal life, for a long time it has been considered that the GI tract of a normal fetus is sterile (Mackie et al., 1999) and is then rapidly colonized by a microbial consortium during and after birth (Figure 1, Section B ‘Newborn’ and ‘Infant’). Interestingly, the recent reporting of bacteria in meconium (Jimenez et al., 2008) has caused debate about the likelihood of microbial pre-natal colonization of the fetal gut (Nicholson et al., 2012; Thum et al., 2012). However the consensus view is that this possibility is still to be rigorously proven (Matamoros et al., 2013). Therefore, the neonate may be influenced by the maternal microflora and its metabolite profile. It is therefore conceivable that maternal microbial metabolites and maternal antibodies could prime the neonates developing mucosal immune system for later microbial colonization. To date, there are no reported studies that have investigated the effects of maternal microbial metabolites and obesity on the fetus/ infant. However, there are emerging studies investigating metabolomics signatures in preterm labor (Dessi et al., 2011; Koren et al., 2012; Romero et al., 2010). One study demonstrated that metabolomics signatures representative of bacterial products are associated with preterm labor (Romero et al., 2010). Whilst this study failed to measure levels of gut permeability in the pregnant mother or report on the prepregnancy BMI, coupled with the aforementioned study by Koren (Koren et al., 2012) it at least demonstrates the potential for maternal microbial metabolites to have a significant impact on the developing neonate. Furthermore, a study investigating urine metabolites in newborns with intrauterine growth retardation (small babies) found that these babies had metabolite signatures of metabolic syndrome (i.e. associated with glucose intolerance and insulin resistance in adults (Dessi et al., 2011). Whilst this later study did not mention microbial signatures or include information on the mother’s BMI, the authors suggested that these infants are at risk of obesity later in life. Thus, the effect of potential interactions between maternal microbial metabolites and the developing foetus that the life time metabolic health is worthy of future investigation. During pregnancy, immune and metabolic functions of the foetus are dependent on the mother and these functions are refined in utero and appear to be diet sensitive (Sanz, 2011).


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The aforementioned study (Koren et al., 2012) showed that the gut microflora of children was most similar in composition to that of their mothers’ in early pregnancy (first trimester), despite the mothers microflora changing during pregnancy. Suggesting that the mother’s early, and even prepregnancy microflora, influences the infants very early gut microflora (see Figure 1, Section A ‘‘Gestation’’). Therefore, it is possible that the mother’s gut microflora and metabolic health status may be an important determinant of the colonization of the infant’s gut as well. A growing body of literature suggests that the development of the gut associated mucosal immune system occurs in the neonate and is largely determined by the mother’s metabolite profile, specifically, the gut associated lymphoid tissues, which are developed very early in the foetus by commensal and pathogenic intestinal bacteria, parasites and food components (Pearson et al., 2012). The gut lymphoid tissue inducer cells, which are present in the embryo, are important in the development and organization of prenatal lymphoid tissue (Pearson et al., 2012). Consequently, lymph nodes and Peyer’s patch patterning are pre-programmed in the developing foetus. We propose that the role of the in utero environment, including the mothers’ metabolite profile in the development of the gut-associated lymphoid tissues impacts on the ability of the gut to develop and maintain a healthy resilient microflora in later life with long-term health implications, a crucial area demanding further research (Box 1). Maternal intestinal permeability during pregnancy Changes in intestinal permeability is another factor that plays an important role in determining a resilient microflora during pregnancy, infancy and beyond. It is not known whether the gut barrier function during pregnancy is altered (Box 1), although there is emerging evidence to suggest that there is increased intestinal permeability in pregnant compared to non-pregnant women (Reyes et al., 2006). This is consistent with our preliminary findings (Figure 2) showing that pregnant women (32.8 5.5 years of age and BMI of 27.8 6.2) have a higher intestinal permeability compared to non-pregnant women (29.5 6.1 years of age and BMI of 24.4 4.9) as measured by the timed blood dual sugar lactulose/rhamnose (L/R) ratio intestinal permeability test. It is unclear how pregnancy contributes to increased gut permeability, although a workable hypothesis might be that hormones induce a change in the structure and composition of the tight junction proteins (Peixoto & Collares-Buzato, 2006). The anatomic differentiation of the human fetal gut is completed by 16 week gestation (Neu, 2007a) but complete gut maturation and differentiation is not reached until beyond birth (Lebenthal & Leung, 1987). Maternal changes in gut permeability may affect both the nutrient supply to the fetus (Kobayashi et al., 2009) and may shape the gut microflora community structure and function (Cani et al., 2009). Higher BMI is associated with increased gut permeability in women but the consequences in pregnancy and effect on the fetus are unknown (Teixeira et al., 2012a). The long-term consequences of these changes are unknown and are deserving of investigation as they could potentially affect and regulate

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General questions: How do we define the topology and functionality of a healthy ‘resilient’ gut microbial profile? How can stable gut microbiology be established in general and after a disruption caused by disease/antibiotics? How can gut microflora resilience be targeted for metabolic disturbances including diabetes and metabolic syndrome? Intestinal permeability may be an important part of the link between diet, gut microbial balance, inflammation, and metabolic disorders and improving the barrier function may become a major strategy for metabolic diseases. Are there different stages of the human life cycle amenable to modulation and what approaches are likely to be most successful at each stage? Modulating the infant gut microflora: Breaking the cycle of metabolic dysfunction between mother and infant is crucial and further research is required to determine appropriate methods. Early evidence suggests antibiotics may be used to ‘‘recalibrate’’ the gut microflora of infants born to mothers with a high pre-pregnancy BMI. The body of evidence supporting the mode of delivery in microbial establishment is considerable, suggesting that for caesarean births, inoculation of the foetus with microflora from the birth canal could be incorporated into delivery protocols. Is gut barrier function altered during pregnancy and what is the impact on the developing foetus? Modulating adult microbial resilience: Select for a keystone microflora taxa (diet pre and probiotic) that are beneficial and promote gut health or lost in individuals with metabolic dysfunction to improve metabolic health. Shanahan (2010) suggests ‘rebooting the system’ e.g. by faecal transplantation to treat C. difficile and the appendix (a reservoir of normal microflora), restoring homeostasis, e.g. after antibiotic treatment.

8 L/R ratio (units ± SEM)


Box 1. Future Directions.

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*

4 2 0 Controls

Pregnant

Figure 2. Lactulose/rhamnose (L/R) ratio in 39 healthy non-pregnant and 99 pregnant women (gestational age 25 7.6 weeks) using a timed blood sample. *denotes pregnant women have a significantly higher (p ¼ 0.03) intestinal permeability compared to non-pregnant women.

early microbial colonization of the gut and consequently influence metabolic health status in later life (Box 1).

Early life events on microbial colonization In its early stages the structure and function the intestinal microflora interface of new born is highly plastic and is influenced by the effects of early life events including delivery mode, introduction of solid food and diet, breast/ formula feeding and weaning (Fallani et al., 2010) as well as country of origin (Grzeskowiak et al., 2012) and probably also host genetics (Sanchez et al., 2011). Other factors such as incidental environmental exposures to antibiotics (Rautava et al., 2012) and infection (Jangi and Lamont, 2010) play a major role in the distinctive characteristics of the microbial community in the infant’s first year of life. A Danish epidemiology study (Ajslev et al., 2011) concluded that a combination of early exposures (maternal pre-pregnancy BMI and antibiotics in infancy) influence the risk of being overweight in later childhood (Antibiotics (OR: 1.54, 95% CI: 1.09–2.17) and maternal BMI (OR: 0.85, 95% CI: 0.41– 1.76)). As the infant develops, the gut microflora lose their plasticity and therefore become more resistant to change (Koenig et al., 2011; Lozupone et al., 2012; Palmer et al., 2007; Yatsunenko et al., 2012) and we hypothesize this may ‘‘lock in’’ an ideal or a non-ideal gut microbial consortium

well into adulthood. Low intestinal microbial diversity early in life has been associated with allergies such as atopic eczema later in life in a case control study that followed newborns for 20 months (Abrahamsson et al., 2012). There may be an association of low microbial diversity early in life with the decreased diversity in obese adults (Greenblum et al., 2012). Therefore, it is conceivable that any factors that affect microbial colonization in the infant can potentially have a long-term impact on metabolic health. This is consistent with observations that childhood obesity is a strong determinant of adult obesity (Thompson, 2012). Nonetheless, at this time there is a large gap in knowledge linking the development of the infant gut microflora and the long term effects on adult metabolic health status (Table 1). Overall, this indicates that the relationship between the developing infant microflora and lifelong implications, are very complex and require further investigation (Box 1). However, we suggest that there are advantages in the development and maintenance of a robust stable balanced gut microflora in early life that will have long term health benefits into later years (see Figure 1, Section C ‘‘Resilience’’). The impact of birth delivery mode The first major contributing factor to colonization of the infants gut is the delivery mode. Vaginally born babies are first colonized by microbes from their mother’s birth canal (Penders et al., 2006) (Table 1). The maternal vaginal microflora is decreased in diversity and richness during pregnancy, with dominance of Lactobacillus species (L. iners crispatus, jensenii and johnsonii, and the orders Lactobacillales (and Lactobacillaceae family), Clostridiales, Bacteroidales and Actinomycetales (Aagaard et al., 2012). Bacteria can also originate from the vaginal and fecal microflora through cross-contamination during birth, the mammary glands through breast-feeding, the skin, mouth and the environment (Palmer et al., 2007). In comparison, the timing of intestinal colonization differs between vaginally born infants and infants delivered by C-section (Gronlund et al., 1999). In C-section delivery Bifidobacterium sp. colonization can be delayed by up to one month as they are deprived of contact with the maternal/vaginal microflora and


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lack of strict anaerobes and the presence of facultative anaerobes such as Clostridium species (Gronlund et al., 1999) (Table 1). C-section babies have lower numbers of Bifidobacteria and Bacteroides and were more often colonized with Clostridium difficile (C. difficile), compared with vaginally born infants (Penders et al., 2006) (Table 1). A recent study has demonstrated that children born via C-section are at increased risk of developing obesity later in life (age 3 years) (Huh et al., 2012). This study demonstrated two-fold higher odds of childhood obesity with C-section, even after adjusting for maternal BMI, birth weight and other confounders. These findings were consistent with another earlier study in older children (Zhou et al., 2011). As childhood obesity can be a risk factor for adult obesity, this is a pivotal finding and it is possible that the maternal gut microflora plays a significant role in this relationship. The effect of country of birth A number of studies have indicated that country of birth can have a greater impact than delivery and feeding mode on the infant gut microflora (Penders et al., 2006, Mueller et al., 2006). This is partially an effect of westernization and socio-economic status (Adlerberth et al., 1991). Within Europe, the country of birth has a pronounced effect, with Bifidobacteria dominating in the Northern European countries (Fallani et al., 2010). In comparison, greater early diversification and higher numbers of Bacteroides is observed in Southern European countries (Fallani et al., 2010) (Table 1). This study identified that there are major differences in the gut microflora of 6-month-old infants between low- and high-income countries. This possibly reflects differences in energy harvest from the diet typifying malnutrition and diarrheal diseases in low-income countries and the previously mentioned Western lifestyle diseases in high-income countries (Grzeskowiak et al., 2012). However, there are potentially many other confounding factors, such as climate which can affect rates of metabolism and subsequent energy harvest. Consequently, more detailed epidemiology studies are required to fully elucidate these factors. Another study (Yatsunenko et al., 2012) compared the gut microbiota of healthy children and adults from the Amazonas of Venezuela, rural Malawi and metropolitan areas in the USA and found that ageassociated changes in the microbial genes (an indicator of the composition of the microbial population) involved in vitamin biosynthesis and metabolism were shared across countries. The most prominent differences involved microbial pathways related to vitamin biosynthesis and carbohydrate metabolism. In this study, Malawian and Amerindian babies had higher components of the vitamin B2 (riboflavin) biosynthetic pathway and urease than the Western babies (USA). Overall, the structure of the gut microbiome needs to be considered when evaluating human development, nutritional needs, physiological variations and the impact of westernization. These variables, and factors that are yet to be fully elucidated, could potentially combine to influence the long term resilience of the developing gut microflora in later life (Figure 1, Section D ‘Outcomes’).


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Intestinal permeability in the newborn and the developing gut microflora The developing GI system plays a vital role during infancy as it serves both as a barrier to infectious materials and as a pathway for nutrition. Intestinal permeability needs to be tightly regulated to promote infant growth and avoid severe infant disease (Drozdowski et al., 2010). During the first months of life, newborns are vulnerable to disease as their immune system and mucosal epithelium of the small intestine are anatomically and functionally immature (Rouwet et al., 2002). As the barrier function is still developing at birth, increased gut leakiness is a normal process in the neonate (Beach et al., 1982; Catassi et al., 1995; Weaver et al., 1984). However, both decreases and increases in permeability during the first month after birth have been reported (Beach et al., 1982; Shulman et al., 1998; Weaver et al., 1984). The differences in intestinal permeability may be due to the discrepancies in gestational age, clinical condition, feeding regimen, and postnatal age at the time of assessment (Shulman et al., 1998). Increased permeability can have beneficial effects, such as uptake of nutrients and development of systemic tolerance (van Elburg et al., 2003). However, there are disadvantages, such as increased uptake of microbes and foreign particles leading to the development of infection, inflammation and systemic hypersensitivity (Insoft et al., 1996; van Elburg et al., 2003). It has also been reported that enteropathy, a consequence of chronic or recurrent exposure to microbial pathogens brought about by living in unhygienic and unsanitary conditions, was associated with a compromised barrier function (Campbell et al., 2003). This allowed for translocation of antigenic macromolecules from the gut lumen into the body resulting in systemic immune-stimulation and growth faltering. Furthermore, Monira et al. (2011) reported that in malnourished children, bacterial population of Proteobacteria and Bacteroidetes accounted for 46 and 18%, respectively (Monira et al., 2011). In contrast in healthy children, Proteobacteria and Bacteroidetes account for 5 and 44%, respectively. The authors concluded that the predominance of pathogenic Proteobacteria and minimal level of Bacteroidetes as commensal microbiota may predispose malnourished children to poor metabolic health. These findings differed from Gupta et al., (2011) where authors reported that gut microbiota of children in India in which Bacteroidetes were shown to be more abundant in malnourished than in healthy children (Gupta et al., 2011). This disparity of gut bacteria may presumably be due to low sample size analysed, only one malnourished child compared with a healthy child (Gupta et al., 2011), contrary to Monira et al., (2011) in which gut microbiota of seven children in each group. This inconsistency of the results suggests that more research in this space is warranted. At birth, intestinal permeability is high and then declines rapidly after birth (see Figure 1), leading to a process referred to as ‘‘gut closure’’ (Drozdowski et al., 2010). In humans, the exact timing of gut closure is unknown, however it has been reported that growth factors, hormones, breast milk and changes in the thickness and viscosity of the mucus gel layer play a role in regulating this process (Drozdowski et al., 2010;


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Teichberg et al., 1990; VeeremanWauters et al., 1996). VeeremanWauters et al. (1996) reported that factors like gestational age and enteral nutrition may play an important role in the maturation of the small intestine, thereby decreasing intestinal permeability. Furthermore, it has been suggested that the presence of gut microflora may facilitate mutual growth of the gut (Sharma et al., 2010). This decreases leakiness and promotes survival of the microflora by regulation of intestinal mucosal inflammation. These processes result in the development of a stable core of diverse and native commensal species which is critical and advantageous to the host (Sharma et al., 2010). Observations are consistent with animal studies demonstrating that many morphological intestinal tissue defects appear in germ-free animals compared to their conventionally raised counterparts indicating that the development of the barrier function is inherently connected to presence of microflora (Neu, 2007b). However, the role of the gut microflora in the early plasticity of gut permeability and how these factors contribute to health status later in life is unknown and requires investigation (Box 1). Intestinal permeability transiently decreases during the first week after birth in both healthy term infants and in preterm neonates (Rouwet et al., 2002). Preterm infants have increased intestinal permeability in the first few days of life (van Elburg et al., 2003). However at postnatal age of 4–7 days, intestinal permeability is not different for preterm and term infants suggesting a rapid postnatal adaptation of the small intestine in preterm infants (van Elburg et al., 2003). This was consistent with the findings of Beach et al. (1982) who showed that preterm babies born at 26–29 weeks gestational age, have increased intestinal permeability between 1–2 and 3–4 postnatal weeks which then declined by 4–6 postnatal weeks (i.e. when they would have been close to full term), as demonstrated by the L/R ratio intestinal permeability test (Beach et al., 1982). Similarly, Shulman et al. (1998) reported that 26–30 weeks gestational age infants shown, by L/R ratios, had a mean increase in intestinal permeability from postnatal day 10 to 28 with a decrease at day 50 (Shulman et al., 1998). These findings suggest that preterm infants display a period (between 1 and 4 weeks post-birth) of increased intestinal permeability to promote absorption of larger immunological and growth-promoting molecules from human milk in an attempt to compensate for the shortened gestation (Beach et al., 1982; Insoft et al., 1996; Weaver et al., 1984). Not surprisingly, pre-term babies are highly susceptible to infection by pathogenic microbes and are routinely placed on prophylactic antibiotic treatments (Daley et al., 2004). Besides the immaturity of the immune system, this may be due to increased gut permeability providing a route of infection. Therefore, it is possible that preterm babies are also susceptible to the long term ‘‘obesogenic’’ effects of gut microflora. Recent studies have suggested that diarrhoea, a common early life condition, alters the diversity of bacterial communities colonizing the gut (Monira et al., 2012, 2013). More specifically, a reduction in major commensal bacteria of phyla Bacteroidetes, Firmicutes and Actinobacteria, and an increase in harmful Proteobacteria were observed. This may also explain the prevalent malnutrition in children in the developing countries where diarrheal diseases are endemic


(Monira et al., 2011). Interestingly, Keely et al. revealed that in T84 cells the activation of water movement by epithelial chloride channels significantly diminish E. coli and S. typhimurium internalization and translocation (Keely et al., 2012). Despite these aforementioned studies on infant gut permeability and the rapid increase in publications on the human gut microbiome, there seems to be a substantial gap in understanding the relationship between gut permeability and the developing gut microflora (as highlighted in Table 1). As there are many potential early life factors that affect gut permeability and consequently the developing gut microflora, the role of gut permeability in this setting needs further investigation, particularly as these changes may be a mediator of metabolic health in later life (Box 1). Implications of the nutritional regime Transmission of bacteria from the mother to the neonate through direct contact with maternal microflora during birth and through breast milk during lactation (the milk microflora, see Table 1) plays a role in the microbial colonization of the infant’s gut (Cabrera-Rubio et al., 2012). During lactation, maternal cells from gut-associated lymphoid tissue, containing bacteria and their genetic material, travel to the breast via the lymphatics and peripheral blood system (Donnet-Hughes et al., 2010) which, when ingested by the infant, primes the infants mucosal associated immune system (Greer & O’Keefe, 2011; Mor & Cardenas, 2010). The lymphatic system undergoes pre- and postnatal shaping and requires exposure to intestinal microflora after birth to stimulate lymphatic maturation, development and function (Harvey, 2005). Therefore, there are a number of maternal physiological factors driving the development of the gut and its microflora prenatally, neonatally and in the early months of life (Newbern & Freemark, 2011). There are clear differences in body composition and growth rate between breast and formula fed babies in developed countries, with the latter displaying a higher growth rate and having greater adiposity (reviewed by Thompson (2012)). The microbial structure, particularly the Bifidobacteria component, of a breastfed baby compared to a formula fed baby is controversial (reviewed by Rautava et al. (2012)) with this research confounded by the wide variation in the duration and exclusivity of breastfeeding. Solely formulafed babies tend to be more exclusively colonized with E. coli, C. difficile, Bacteroides spp. and Lactobacilli compared to breastfed babies (Penders et al., 2005). One study has shown that term infants who were born vaginally at home and were exclusively breastfed have the highest numbers of Bifidobacteria and lowest numbers of C. difficile and E. coli (Penders et al., 2005). Whilst it is difficult to determine individual variables, it can be concluded that breastfeeding, natural birth and a lack of hospitalisation correlates with a more beneficial microbial consortium. In breast-fed babies, the presence of Bacteroidetes as pioneer bacteria in the majority of neonates demonstrated that adult-type strict anaerobes may reach adult-like population densities within the first week of life (Jost et al., 2012). Consequently, the switch from facultative to strict anaerobes may occur earlier than previously assumed in breast fed neonates, and the


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establishment of the major butyrate-producing populations may be limited by other factors than the absence of anaerobic conditions (Jost et al., 2012). This is of significance as butyrate producing bacteria (e.g. Faecalibacterium prausnitzii) are considered to be beneficial (Sokol et al., 2008). It would be of great interest to take the mother’s metabolic health status into consideration in future studies as this would help ascertain interactions between feeding mode and BMI. Human milk contains bioactives that have profound effects upon the function and integrity of the GI tract (Goldman, 2000). It has been demonstrated that infants who received the majority of their food as human milk had significantly lower intestinal permeability when compared to infants who received minimal or no human milk at postnatal days 7, 14 and 30 (Taylor et al., 2009). This is consistent with two previous studies (Catassi et al., 1995; Weaver et al., 1987) in which it was demonstrated that lower intestinal permeability occurred in term breastfed infants compared to formula-fed infants. Furthermore, a study of term infants over the first 6 postnatal days showed decreased permeability to lactulose with initiation of human milk but not formula feedings (Insoft et al., 1996). These findings can have significant consequences for microbial colonization and infant exposure to metabolites (Weaver et al., 1987). Importantly, it has been shown that supplying nutritional needs to the body, by way of bypassing the digestive system using total parenteral nutrition (TPN), negatively impacts gut barrier function and gut atrophy in piglets (Kansagra et al., 2003; Shulman et al., 1998). The cellular mechanism for the increase in intestinal permeability during TPN has not been elucidated, especially in the newborn. A potential explanation could be a breakdown in tight junction integrity (Kansagra et al., 2003). Human milk is important to premature babies since in preterm neonates early feeding with human milk reduces intestinal permeability (Shulman et al., 1998). This rapid improvement in gut permeability and hence barrier integrity, may be beneficial to the development of a resilient microflora and consequently reduce the risk of developing metabolic diseases later in life. A mother’s BMI can influence the microbial content of the breast milk, that is the milk microbiome, which in turn inoculates the infant’s gut microflora (Cabrera-Rubio et al., 2012). Maternal obesity and high weight gain during pregnancy has been shown, for at least the first month, to be compositionally less diverse compared to that of normal weight mothers (Cabrera-Rubio et al., 2012). Although these findings require further investigation with larger sample numbers, they suggest that obese mothers may pass on their poor microbial diversity to their infants (see Table 1). Furthermore, overweight mothers reportedly have higher levels of potentially pathogenic Staphylococcus bacteria and lower levels of the beneficial Bifidobacterium compared to normal-weight mothers (Collado et al., 2012). The prevalence of mucous degrading Akkermansia muciniphila bacteria was also higher in overweight mothers, and the numbers of these bacteria were related to the concentration of the inflammatory marker interleukin (IL)-6 in the colostrum (Collado et al., 2012). This in turn is related to lower counts of bacteria of the genus Bifidobacterium in the breast milk of overweight


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women (Collado et al., 2012). The authors suggested that excessive weight gain in mothers continues a cycle of poor metabolic health in the successive generation and contributed to the heightened risk of obesity in their infants (Collado et al., 2012). As this mechanism involves shifting the gut microflora scales towards a loss of resilience, it is therefore conceivable that this microbial associated risk can have a lifelong extension into adulthood (Figure 1, Section C ‘‘Resilience’’). Identifying methods for re-calibrating the gut flora of babies e.g. feeding prebiotics such as insulin and galacto-oligosaccharides (Desbuards et al., 2012), or the radical approach of using antibiotics or modulating the maternal gut microflora towards more beneficial species clearly requires additional research (Box 1). Pathogenic infections and hospitalization To date, no studies have reported a group of ‘keystone’ microbes in infants that may be responsible for obesity later in life. Instead our supposition is that the mechanism involves the loss of microbial consortium resilience as opposed to specific phyla. Despite this, there are a number of examples about how incidental GI infections in infants can have positive long-term effects on GI health. The most convincing example concerns C. difficile infection. Approximately 60–70% of healthy newborns and infants are colonized by the enteric pathogen C. difficile acquired through environmental contamination in the nursery or home environment (Jangi & Lamont, 2010). However, unlike older children and adults who are susceptible to the potent C. difficile exotoxins that cause diarrhoea and pseudomembranous colitis, these infants are unaffected (Hall & Duffett, 1935). In fact, between 12 and 24 months of age C. difficile is eradicated as a commensal (Hafiz & Oakley, 1976). This is presumably due to gradual development of the ‘adult’ colonic microflora and potentially through immunoglobulins (Ig) provided from birth and breastfeeding (Kelly et al., 1992). Furthermore, an IgG response develops during the carrier state which appears to provide lasting protection against subsequent C. difficile infections in later life (Kyne et al., 2000). This finding is highly significant as this microbe is a growing problem in adults exposed to antibiotics (Kelly & LaMont, 2008). As we know microbes can enhance energy harvest (Jumpertz et al., 2011) and are implicated in a number of adult metabolic diseases, it is possible to conclude that the colonization of other microbes in infancy may have life-long impacts (potentially negative and positive) on metabolic health an important area requiring further research (Box 1). The effects of antibiotics of metabolic health status Even though antibiotics have been widely used in the clinical setting for the last 70 years their long term effects on commensal bacterial populations and microbial community resilience are largely unknown (Jernberg et al., 2010). No one has assessed the impact of antibiotic usage on the colonization and succession of early microbial consortia in the developing infant gut the long term implications on metabolic health. The infant gut microflora is more dynamic and less resilient than the adult gut microflora (Cho & Blaser, 2012; Fallani et al., 2010), consequently effects may be more profound in


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infants with long-term consequences for development and microbial resilience. Dethlefsen et al. (2008) examined the short-term effects of a single dose of the antibiotic (ciprofloxacin) in adults and found that it took four weeks for most of the gut microflora composition to recover to its pre-treatment state (Dethlefsen et al., 2008). However, there were still several taxa that failed to recover even after 6 months. In a birth cohort (KOLA) study, infants treated with antibiotic/antifungals (undefined) have been associated with decreased numbers of Bifidobacteria and Bacteroides (Penders et al., 2005) (Table 1). The previously mentioned Danish epidemiology study concluded that a combination of early exposures, including antibiotics (not defined), influence the risk of being overweight in later childhood (Ajslev et al., 2011). This effect could be explained by an impact on the establishment and diversity of the gut microflora. For example, of all children treated with antibiotics in this study, those with mothers with a healthy pre-pregnancy BMI had an increased risk of developing obesity, whereas those of obese mothers were at decreased risk of developing obesity (Ajslev et al., 2011). When the effect of overweight mother’s breast milk is considered (Cabrera-Rubio et al., 2012, Collado et al., 2012), it is possible to conclude that results of antibiotic treatment may be contingent on the mothers’ metabolic health status or on the composition of the pre-treatment gut microflora. Other studies, not focusing on host metabolic health and metabolism, have suggested that antibiotics in early life break tolerance to the gut microflora, altering the microbiome and predisposing individuals to allergies (Madan et al., 2012; Murk et al., 2011). The effects of antibiotics and adiposity have been investigated in mice demonstrating some gut bacteria survive the treatment better than others, shifting digestion towards enhanced energy harvest (Looft et al., 2012). This is not surprising, as in-feed antibiotics have been administered to agricultural animals for disease treatment, disease prevention, and importantly growth promotion for over 50 years. In one study, pigs were raised in a highly controlled environment, with one portion of the littermates receiving a diet containing performance-enhancing antibiotics (chlortetracycline, sulfamethazine and penicillin) and the other portion receiving the same diet but without the antibiotics (Looft et al., 2012). Metagenomic analyses revealed a significant increase in microbial genes relating to energy production and conversion in the antibiotic-fed pigs (Looft et al., 2012). Whilst the antibiotic doses used in this study were low, albeit chronic, compared to typical clinical treatments in human children, they do help shed some light on the growth promoting nature of antibiotics that could also apply to humans. The effects of genetic and familial factors We cannot rule out the likelihood that genetic factors are also involved in the early colonization of the infant gut or that the resilience phenotype itself may be inherited and shared amongst family members. These questions are in need of more research but some support comes from a recent study (Faith et al., 2013) in which family members but not unrelated individuals were found to share strains of gut microflora in


common and that these persisted through adulthood. Also while outside the scope of this review, the debate about the genetic basis of inflammatory bowel diseases (IBD) (Rubin et al., 2012) has pointed to the likelihood of heritability of conditions that alter microbial colonization of the infant gut. IBD may begin as early as the first year of life and recently Mendelian mutations were identified in children with very early-onset Crohn’s disease and ulcerative colitis (Shim et al., 2013). Similarly, along with environmental factors, genetic factors linked to celiac disease have been reported to influence infant gut colonization by Bacteroides species (Sanchez et al., 2011).

Plastic structure of the infant gut and microflora By the first year of life, the infant’s intestines possess a microflora that begins to resemble that of the adult gut microflora, consisting of Bacteroides and Firmicutes, Verrucomicrobia, with low abundance of Proteobacteria and aerobic gram negative bacteria (Palmer et al., 2007). By approximately two and a half (Koenig et al., 2011) to four years of age (Musso et al., 2010a), the gut microflora has fully matured, converging towards the three adult enterotype clusters (Arumugam et al., 2011). Whilst the maternal microflora is thought to have the greatest initial influence on an infant’s initial microbial structure, the factors involved in the transformation into a mature adult microflora remain unclear. It is possible to speculate that it involves diet, host and external factors (e.g. antibiotic treatment) exerting selection pressure towards the ‘fitter’ taxa. The healthy adult microflora then remains relatively stable for approximately 70 years (Biagi et al., 2012). Interestingly, and unlike the dynamic changes observed in the infant’s developing gut microflora (Fallani et al., 2010) the established long term ‘adult’ microflora is considered to be resistant to change in healthy adults (Cho & Blaser, 2012). However, changes in microbial resilience and diversity are observed in disease states (Cani et al., 2008; Turnbaugh & Gordon, 2009; Vaarala et al., 2008). Key questions remain about how the ‘resilience’ of the co-evolved microflora is lost and how that contributes to poor metabolic health, for the host, in later years (Figure 1, Sections C and D). Gut microflora could potentially direct metabolic development early in life and that may then become fixed in place for adult life. Microbial education of the gut mucosal immune system As the infant gut microflora develops, a host/microbial mutualism is also established (Palmer et al., 2007). This symbiosis can influence a number of host physiological processes such as nutrient absorption, development of the gut tissue, the gut associated lymphoid tissue, and the shaping of the innate and adaptive immune system (Sanz, 2011). The main role of the mucosal associated lymphoid immune system is to manage the exposure of potential antigens from food nutrients and the resident microflora (Sjogren et al., 2009). The latter can be considered to be an environmental regulator of mucosal and systemic immune maturation and vice versa. This ability of gut bacteria to ‘educate’ the gut immune system is extremely important as it also involves developing


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tolerance to environmental (nutrients and microbes) antigens (Shanahan, 2010). A fine balance between the tolerance and inflammation is important to prevent the development of food allergies, autoimmune diseases, Crohn’s disease and ulcerative colitis (Kverka & Tlaskalova-Hogenova, 2013; Rubin et al., 2012; Tlaskalova-Hogenova et al., 2011). A balance between T regulatory lymphocytes and T helper 7 lymphocytes is crucial to maintaining tolerance and the ability to respond to pathogens. IL-17 is produced by T helper lymphocytes in response to stimulation of NOD1 and NOD2 by contact with microorganisms. NOD1 and 2 are intracellular sensors of bacterial peptidoglycan and have key roles in host responses to bacteria as part of the innate immune system (Geddes et al., 2011). Crohn’s disease is associated with genetic variation the NOD2 receptor suggesting that host genetics plays a role in the colonization and response to the gut microflora (Hugot et al., 2001; Ogura et al., 2001). For information on the complex interplay between host genomics and the microflora refer to the recent review (Leone et al., 2013). IL-17 has recently been implicated to play a major role in the pathogenesis of IBD via a key role in bacteria initiated inflammation and antimicrobial defence (Geddes et al., 2011). More recent evidence have suggested that polymorphisms in IL-12/23 signalling are likely to be important in IL-17 pathways in these diseases (Griseri et al., 2012; Tang & Iwakura, 2012).

Is the gut microflora ‘‘fine-tuned’’ through adolescence? Whilst the gut microflora in the young child has been thought to resemble that of an adult, recent evidence (Ringel-Kulka et al., 2013) has suggested that the establishment of an adult-like intestinal microbiota occurs at a later age than previously reported. Therefore, there is potentially a period where refinement of the gut microbial diversity and function occurs during adolescence. Unfortunately, to date we know very little about the gut microflora in adolescents (Table 1). Childhood and adolescence obesity has reached epidemic levels in developed countries (Dehghan et al., 2005). For this reason, adolescence is considered to be a critical period in the onset of obesity and related co-morbidities (Alberga et al., 2012). There are marked changes in body composition: insulin sensitivity; physical activity; lifestyle and diet behaviors; and psychological issues that make adolescents at an increased risk of becoming overweight and obese. Furthermore, obese youth are considered to be at high risk to progressing further along a poor growth trajectory into adulthood (Baker et al., 2007; Bibbins-Domingo et al., 2007). It is, likely that the foundations regulating metabolic potential are laid down in early life but that refinement of this process continues throughout adolescence.

Intestinal permeability and metabolic diseases It is also important to consider the emerging concept that a leaky gut (increased intestinal permeability) is associated with a variety of disorders, such as intestinal and liver diseases, autoimmune disorders including T1DM and T2DM (de Kort et al., 2011; Groschwitz & Hogan, 2009; Hollander, 1999). It has been suggested that increased intestinal permeability


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may even be a primary causative factor of disease development and exacerbation (Arrieta et al., 2006). More recently, a leaky gut has been suggested to contribute to systemic malfunctioning and disease development of obesity-related T2DM (de Kort et al., 2011). These observations may also provide an insight into the low level of inflammation that is typically associated with these metabolic disorders, that is the leaky gut allows increased ingress of inflammatory microbial lipopolysaccharide and other microbial components. This could conceivably be due to obesity induced dysbiosis of the small intestinal microbial structure resulting in bacterial overgrowth, in turn promoting altered small intestinal permeability (Sabate´ et al., 2008). A leaky gut results in disturbances between the microflora and the host immune system, ultimately leading to the secretion of proinflammatory cytokines (Cani et al., 2008; de La Serre et al., 2010; Ferraris & Vinnakota, 1995). Consequently, a low degree of inflammation appears to be a common phenomenon in obesity and T2DM results from increased expression and production of cytokines and acute phase reactants such as CRP, ILs, TNF-a or lipopolysaccharides (Hotamisligil & Erbay, 2008). Persistent elevated circulating levels of inflammatory cytokines, may further reinforce intestinal barrier dysfunction by altering structure and localization of the tight junction proteins (Brun et al., 2007). Evidence of tight junction disruption has been reported in an animal model of obesity and metabolic syndrome (Brun et al., 2007). Furthermore, in genetically obese mice, the intestinal barrier function is impaired leading to a consistent leakage of bacterial endotoxins into the portal blood circulation. This is consistent with Miele et al. (2009) showing that nuclear and cytoplasmic expression of zonula occludins-1, tight junction proteins, in the duodenal mucosa of patients with non-alcoholic fatty liver disease was lower than that observed in healthy subjects (Miele et al., 2009). In one of the first human studies, Teixeira et al., demonstrated that obese women have increased paracellular permeability compared to their lean counterparts (Teixeira et al., 2012b). In addition, intestinal permeability parameters in obese women are correlated with anthropometric measurements and metabolic variables (Teixeira et al., 2012b). This suggested that in obese women, intestinal barrier dysfunction might play a role in insulin secretion and immune system activation. Other studies, however, have shown no differences in intestinal permeability between obese and lean subjects (Brignardello et al., 2010). This inconsistency warrants further investigation into the role of intestinal permeability and obesity. Furthermore, there have been limited human studies simultaneously evaluating microbial dysbiosis and intestinal permeability. Teixeria et al. postulated that factors that influence intestinal permeability in obesity include gut microflora, dietary pattern and nutritional deficiencies (Teixeira et al., 2012a). The interactions between gut microflora, immune system, adipose tissue and hormones may be the critical components underlying the altered intestinal permeability in obesity (Teixeira et al., 2012b). More specifically, stimulating the development and maintaining the innate and adaptive immune systems will be critical strategies for improving intestinal permeability

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(Fredrik et al., 2005). Linking these concepts together may provide further insights into the pathophysiology of metabolic diseases.


Conclusions This narrative review has described and drawn conclusions around how the co-evolved gut microbial consortia detour to a trajectory of poor metabolic health status. It has highlighted important gaps in our current knowledge. Up until very recently there have been technological limitations to our understanding of some aspects of the human microbiome. However, with rapid advancements in the genomics field (Schloissnig et al., 2013; Weinstock, 2012) we have concluded that there are a number of key research questions that should now be addressed as outlined in Box 1. The evidence has suggested that adult metabolic health can be promoted through addressing the early life stages that are critical for the development of the microbiome and its resilience. The foetus may be exposed to microbial metabolites (Fujimura et al., 2010) and potentially directly to microbes through elevated intestinal permeability during pregnancy and thus may influence the developing gut and have potentially profound lifelong affects. Alteration to the epithelial cells and tight junctions that form the selective barrier and ensure the regulation of the trafficking of macromolecules between the environment and the host may have effects on the interactions between the mucosal immune system and luminal contents, including dietary antigens, microbes and their products. If the mother’s pre-pregnancy or pregnancy BMI is high, this could influence the infant gut towards enhanced nutrient harvest later in life. As this phenomenon appears to occur at all stages of the lifecycle, combined with the loss of the coevolved gut microflora resilience, to influence poor health outcomes, a leaky gut can therefore further contribute to systemic malfunctioning and disease development. Thus, reinforcing intestinal barrier function may become an important objective to help prevent or counteract pathophysiological mechanisms in patients with metabolic syndrome. It is unclear if the barrier function is a primary causative factor in the predisposition to disease development and the extent to which it contributes to the pathogenesis of obesity remains to be elucidated. Whilst the early gut microflora is considered to be plastic, early life events such as birth mode, mode of feeding, country of birth, infections, nutrition and antibiotic treatment can potentially play a pivotal role in impacting the development a lifelong resilient gut microflora. We concluded that infants and young children at risk of moving towards a trajectory of poor metabolic health would benefit from interventions or preventative strategies to ‘recalibrate’ their gut microflora in the direction of enhanced metabolic health. Whilst we have indicated that there are a number of overarching challenges to be addressed in this space, we can see potential for interventions such as encouraging breast feeding and the strategic use of antibiotics.

Declaration of interest The authors report no conflicts of interest.


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