Introduction
The gastrointestinal microbiota is a complex and dynamic ecosystem consisting of several
hundreds of different microbes, mainly bacteria (1011–12 bacteria/g of colonic content,
forming 60% of total fecal mass; Eckburg et al., 2005; O’Hara and Shanahan, 2006).
Total number of bacteria exceeds 10 times the number of human cells, and the collection
of microbial genome (microbiome) contains 100 times more genes than the human genome
(Vael and Desager, 2009). Gut microbiota influence the growth and differentiation
of gut epithelial cells, and play pivotal nutritive, metabolic, immunological, and
protective functions (O’Hara and Shanahan, 2006). Its deregulation is involved in
the pathogenesis of immunological, cardiovascular, and metabolic diseases (Hammer,
2011; Maslowski and MacKay, 2011; Harris et al., 2012).
The investigation on microbiota composition started in 1900 (Tissier, 1900) and has
been performed by culturing methods since the recent advent of DNA sequence-based
methods, that, thanks to their ability to identify a large number of species that
cannot be cultivated, have allowed a more complete and rapid assessment of the gastrointestinal
ecosystem (Palmer et al., 2007; Adlerberth and Wold, 2009). On the basis of 16S ribosomial
– RNA encoding gene, more than 7000 distinct phylotypes have been detected in the
human distal gut (Vael and Desager, 2009), with high inter-individual and age variability,
but belonging to a limited number of broad taxonomic divisions (mainly the anaerobes
Bacteroides, Eubacterium, Clostridium; Hayashi et al., 2002; Eckburg et al., 2005;
Zoetendal et al., 2008). In a very recent study, Arumugam et al. (2011), by combining
fecal metagenomes of individuals from different countries, identified three different
enterotypes (with the prevalence of Bacteroides, Prevotella, and Ruminococcus species,
respectively) that are not nation or continent specific, and showed that intestinal
microbiota variation is stratified, not continuous, indicating further the existence
of a limited number of well-balanced host-microbial symbiotic states. These enterotypes
do not seem to differ in functional richness and apparently do not correlate with
nationality, gender, age, or body mass index; at the same time, they seem to characterize
and be quite stable in individuals, so that they can be restored after perturbations.
Gut microbiota composition and concentration physiologically varies throughout the
gastrointestinal tract (increasing gradient from the stomach to the colon and characteristic
gut-compartment distribution of microflora) and life stages, progressing from the
newborn sterility to the extremely variable and dense colonization of adult gut, under
the influence of various internal host-related and external factors (Mackie et al.,
1999; Palmer et al., 2007).
Establishment and Development of Intestinal Microflora
The fetal intestine is sterile and bathed by amniotic fluid. The establishment of
the gut microbial population is a continuous and complex process which starts at delivery
and proceeds for several years through successive stages under the influence of several
internal and external factors (Mackie et al., 1999; Fanaro et al., 2003; Adlerberth
and Wold, 2009; Arumugam et al., 2011). Due to the abundance of oxygen in the neonatal
gut, facultative aerobes (mainly Enterobacteriaceae, Enterococcus, and Streptococcus
species) represent the first colonizers. Escherichia coli, Enterococcus fecalis, and
faecium are the most represented, followed by Klebsiella and Enterobacter, and, more
rarely and transiently, Aeromonas, Pseudomonas, Acinetobacter, alpha-haemolyticus
Streptococci, and coagulase-negative Staphylococci (Penders et al., 2006; Adlerberth
and Wold, 2009; Vael and Desager, 2009). Their expansion leads to a gradual consumption
of oxygen, so to a more reduced environment, which favors the proliferation of obligately
anaerobic bacteria, with the dominance of Bifidobacterium, Bacteroides, and Clostridium,
followed by Veillonella, Eubacterium, and Ruminococcus species (Penders et al., 2006;
Adlerberth and Wold, 2009; Vael and Desager, 2009). With time, anaerobic species will
expand and outnumber facultative bacteria (Penders et al., 2006; Adlerberth and Wold,
2009; Vael and Desager, 2009), toward an adult-like microbiota profile, characterized
by the preponderance of Bacteroides and Firmicutes, common occurrence of Verrucomicrobia
and very low abundance of Proteobacteria and aerobic Gram negative bacteria (Palmer
et al., 2007).
Colonizing bacteria derive from the mother (mainly vaginal and intestinal microflora),
breast milk (for breast-fed babies), and surrounding environment (which includes equipment,
air, other infants, and nursing staff). The pattern and level of exposure during the
neonatal period is likely to influence the microbial succession and colonization in
the GI tract. Factors influencing microbial colonization can be grouped in two main
categories: extrinsic, which include geographic area, maternal, and surrounding environment
bacteria, mode of delivery, hygiene measures, and feeding habits, and drug therapies;
and intrinsic, which include the neonatal genetics, bacterial mucosal receptors, and
interactions, intestinal pH and secretions, peristalsis, and immune response (Mackie
et al., 1999; Penders et al., 2006; Adlerberth and Wold, 2009; Fallani et al., 2010).
Diet has a dominant role over other possible variables such ethnicity, sanitation,
hygiene, geography, and climate, in shaping the gut microbiota (De Filippo et al.,
2010).
The Impact of Breast-Feeding on Microbiota Composition
Human milk presents a complex and dynamic composition, influenced by gestational age
at parturition, lactation period, and woman’s diet, which differs from formula feeding
for nutrients concentrations and composition, and, more importantly, for the exclusive
presence of growth factors, cytokines, immunoglobulins, and digestion enzymes (Le
Huerou-Luron et al., 2010; Roncada et al., 2012).
Feeding type has been demonstrated to influence microbiota composition directly, by
providing the substrates for bacterial proliferation and function (Le Huerou-Luron
et al., 2010) and sources of bacterial contamination (originating from the nipple
and surrounding skin, and milk ducts for breast milk; from the dried powder, the equipment
used for preparation and the water used for suspension for formula milk; Mackie et
al., 1999), and indirectly, by modulating the morphology, cell composition and physiology
of the intestinal mucosa, and the pancreatic function (Le Huerou-Luron et al., 2010).
Studies performed in the last two decades on large populations of neonates aged ≥4 weeks,
using both culturing and molecular methods, demonstrated that Bifidobacteria were
the most represented species in both breast- and formula-fed infants (Balmer and Wharton,
1989; Mackie et al., 1999; Harmsen et al., 2000; Fanaro et al., 2003; Bezirtzoglou
et al., 2011; Fallani et al., 2011). In most of the cases, no significant count differences
were found between breast- and formula-fed infants (Mackie et al., 1999; Harmsen et
al., 2000; Fanaro et al., 2003; Fallani et al., 2011). Conversely, Bezirtzoglou et
al. (2011) observed more than two times increased numbers of bacteria cells in breast-fed
infants, compared to formula-fed ones. Among Bifidobacteria, Bifidobacterium breve,
B. adolescentis, B. longum, and B. bifidum are isolated in both formula- and breast-fed
infants, whereas B. infantis is typical of breast-feds, B. fragilis of formula-fed
infants (Mackie et al., 1999; Penders et al., 2006). In most of the studies, Bacteroides
and Enterobacteria represent the two most frequently found species after Bifidobacteria
(Balmer and Wharton, 1989; Mackie et al., 1999; Harmsen et al., 2000; Fanaro et al.,
2003; Fallani et al., 2011). Palmer et al. (2007) and Favier et al. (2002) failed
to demonstrate Bacteroides as part of the dominant microbiota; this finding could
be due to the interfering action of other environmental factors (Penders et al., 2006).
Breast-fed newborns have been demonstrated to carry a more stable and uniform population
when compared to the formula-fed ones (Bezirtzoglou et al., 2011). Relatively small
amounts of formula supplementation of breast-fed infants will result in shifts from
a breast-fed to a formula-fed pattern (Mackie et al., 1999), characterized by a wider
microbiota spectrum. In particular, the counts and incidences and counts of Clostridium
(C. paraputrificum, C. perfringens, C. clostridiiforme, C. difficile, and C. tertium)
and Streptococcus (S. bovis, S. faecalis, and S. faecium) species, Bacillus subtilis,
Bacteroides vulgatus, Veillonella parvula, Lactobacillus acidophilus, Escherichia
coli, Pseudomonas aeruginosa (Benno et al., 1984; Mackie et al., 1999; Penders et
al., 2006; Adlerberth and Wold, 2009; Fallani et al., 2010; Bezirtzoglou et al., 2011),
Enterococcus faecalis (Jimenez et al., 2008; Adlerberth and Wold, 2009), and Atopobium
(Bezirtzoglou et al., 2011) in the bottle-fed infants were significantly higher than
those in the breast-fed infants. On the other hand, L. rhamnosus and Staphylococci
prevailed in breast-fed infants (Adlerberth and Wold, 2009), with Staphylococcus epidermidis
representing the distinctive tract of the feces of lactating woman and their infant,
while it was almost absent in samples from feces of formula-fed infants.
The introduction of solid food profoundly impacts on the microbial ecology of breast-fed
infants (Stark and Lee, 1982; Mackie et al., 1999; Adlerberth and Wold, 2009). Once
dietary supplementation begins, microbiota profile of breast-fed infants changes toward
formula-fed-infants profile, with the significant increase in the count of Enterococci
and Enterobacteria, and the appearance of Bacteroides, Clostridia, and other anaerobic
Streptococci (Stark and Lee, 1982; Mackie et al., 1999; Adlerberth and Wold, 2009).
Between the first and the second year of life, differences between breast- and formula-fed
infants are lost, and the microbiota profile resembles that of the adult for composition
and microbiota counts (Stark and Lee, 1982; Mackie et al., 1999; Adlerberth and Wold,
2009).
The Impact of Breast-Feeding on Immediate and Long-Term Health-Effects
Numerous studies have been performed in the last decades with the aim to define short-
and long-term effects related to the initial microbial gut colonization.
The nature of mucosal microflora acquired in early infancy has been proven to be critical
in the determination of mucosal immune response and tolerance, so that alterations
of gut environment are directly responsible for mucosal inflammation and disease,
autoimmunity, and allergic disorders in childhood and adulthood (Gronlund et al.,
2000; Ogra and Welliver, 2008). The type of feeding, through its selective action
on bacterial colonization and growth, which, in turn, induce specific T cell responses
and modulates substrates oxidation and consumption, has a major impact on the development
of immune functions and oral tolerance (Palma et al., 2012). Systematic revisions
of available data, pointed out the protective role of breast-feeding against the development
of diarrhoea and necrotizing enterocolitis in the newborn (Mackie et al., 1999), and
allergic and autoimmune diseases in childhood, including coeliac disease (Akobeng
et al., 2006; Palma et al., 2012), type I diabetes and atopic dermatitis, whereas
no clear risk reduction was evident in relation with asthma or allergic rhinitis (Bjorksten,
2005; Kramer, 2011). Later in life, breast-feeding has been associated to a reduced
risk of inflammatory bowel diseases, cardiovascular diseases, obesity, and type-2
diabetes.
Potential Protective Role Related to the Addiction of Prebiotics and Probiotics to
Formula Food
Because of the recognized healthy properties, exclusive breast-feeding has been recommended
by the World Health Organization for the first 6 months of life and supplemental breast-feeding
up to 2 years and beyond (Le Huerou-Luron et al., 2010). According to a recent analysis
by Le Huerou-Luron et al. (2010), the prevalence of exclusive breast-feeding in the
world between 2000 and 2005 was 90% in the early postpartum, but only 41% at 4–6 months
of age, with the highest percentages in Africa, followed by East and South Asia, Latin
America and The Pacific, and, finally, Europe.
Considerable efforts have been made to mimic the composition of human milk by the
addition to formula feeding of living bacteria (probiotics), non-digestible fibers,
nucleotides and oligosaccharides (prebiotics), and bovine lactoferrin in order to
induce a breast-fed-similar microbiota colonization in formula-fed infants, with the
final aim to stimulate the maturation and proper function of the immune system (Fanaro
et al., 2003; Rinne et al., 2005; Singhal et al., 2008; Vael and Desager, 2009). Overall,
the implementation of formula food with prebiotics and probiotics has been demonstrated
to be effective in changing microflora composition toward the desired breast-feeding
pattern and stimulating immune response (Rinne et al., 2005; Sherman et al., 2009).
No definitive results are available regarding the real health improvement related
to their use (Bjorksten, 2005; Sherman et al., 2009; Vael and Desager, 2009) although
in preterm infants their supplementation is associated with a reduced incidence of
necrotizing enterocolitis and sepsis (Mackie et al., 1999; Lee, 2011).
Conclusions
Several studies performed in the past decades have clearly demonstrated the complexity
of gut microbiota composition and the modulatory effect played by several endogenous
and exogenous factors on it. Type of feeding in the first months of life appears as
one of the most important determinants of the child and adult well-being, and its
protective action seems to rely mainly on its ability to modulate intestinal microflora
composition at early stages of life. In recent years, the implementation of milk formula
with prebiotics, probiotics, and lactoferrin has been demonstrated to change newborns’
microflora composition toward breast-feeding pattern and stimulate immune response.
At the same time, no definitive results are available regarding the real health improvement,
so that breast milk, whose beneficial health-effects are undoubtedly unique, has to
be considered the food of choice for infants in the first 6 months of life.
For the same reasons, breast-feeding should be encouraged and, at the same time, new
researches are advised in order to better define the composition of intestinal microbial
ecosystem and the specific interactions amongst diet, microbiota composition, and
children health.