Keywords
intestinal microflora - necrotizing enterocolitis - preterm infants
Introduction
Despite overall decreased mortality and morbidity in preterm very low-birth-weight
(VLBW: <1,500 g) infants over the past decades, necrotizing enterocolitis (NEC), a
devastating emergency intestinal disease in preterm VLBW infants, remains 5 to 6%
and even increase despite much effort on peri-postnatal care.[1] More worrisome, approximately 20 to 30% of VLBW infants with NEC will die[2] and the remaining survivors will be at substantial risk for long-term complications,
including impaired neurodevelopment and short bowel syndrome.[3] Around 10% of infants with NEC are term or near-term neonates; the risk factors
of NEC in term neonates include congenital heart diseases, prenatal asphyxia, and
patent ductus arteriosus requiring indomethacin therapy.[4] Thus, pathophysiology of NEC should be different between term and preterm infants.
We thus review the role of intestinal microflora on NEC in preterm infants only.
The pathogenesis of NEC in preterm infants is multifactorial; the immaturity of the
gastrointestinal (GI) tract in terms of motility, digestive function, circulatory
regulation, barrier function, and immune defense are the important underlying risk
factors. The widely accepted hypothesis at present is that enteral feeding (providing
substrate) in the presence of intestinal colonization by pathogens provokes an inappropriately
accentuated inflammatory response by immature intestinal epithelial cells of preterm
neonates; these result in a disruption of the intestinal barrier and translocation
of bacterial endotoxins.[5]
[6]
Unique Features of the Preterm Intestinal Microbiome
Unique Features of the Preterm Intestinal Microbiome
Intestinal microfloras are composed of approximately 1014 bacteria, which represent approximately 10 times the number of cells in the human
body.
Upon delivery, the neonate is exposed to microbes from a variety of sources, including
maternal vaginal, fecal, and skin bacteria. Initial colonization of the infant gut
is highly influenced by the mother's vaginal and fecal bacterial communities, which
include facultative anaerobes such as streptococci and Enterobacteriaceae. Indeed,
the first and most important phase of normal colonization occurs when the newborn
fetus passes through the birth canal and ingests maternal vaginal and colonic microorganisms.
These bacteria further proliferate when oral feeding is initiated. After 48 hours
of birth, the number of bacteria is already as high as approximately 104 to 106 colony-forming units per milliliter of intestinal contents. Many factors may influence
this process, including gestational age (preterm or full term), mode of delivery (vaginal
or cesarean section), infant diet (breastfeeding or formula), birth environment of
neonatal intensive care unit (NICU), and use of drugs such as antibiotics or proton
pump inhibitors.[7]
Preterm infants are very unique in regard to sudden interruption of the in utero environment;
many preterm infants are born via cesarean section and could not have own maternal
nipple feeding. This makes preterm infants nearly no chance to get maternal generic
microflora colorization in the GI tract. Furthermore, preterm infants are mandated
to develop within the NICU and therefore they acquire their GI tract microbiota within
the confines of the NICU where colonization is significantly influenced by iatrogenic
manipulations such as the frequent administrations of diverse drugs (broad-spectrum
antibiotics, opioids, and histamine-2 receptor antagonists) or numerous instrumentations
(endotracheal tubes, feeding tubes, and suctioning tubes).
An environment is not only the factor that affects the microflora colorization of
the GI tract in preterm infants. Studies have demonstrated that immature intestinal
epitheliums are decreased in intestinal motility, and are characterized by the alterations
of surface glycoconjugate glycosylation patterns, intestinal mucus densities, and
carbohydrate contents: All of these characteristics may alter bacterial binding.[8]
[9] It is reasonable inference that interactions between the immaturity of the GI tract
and specific environmental factors of NICU alter the preterm infant's endogenous microbiota
and its susceptibility to NEC.
On the other hand, it is also suggested that genetic factors play an important role
on microflora colorization of the GI tract in infants. Studies have demonstrated that
related preterm twins share similar gut microbiome development, even within the complex
environment of NICU. This finding appears to be a result of shared genetic and immunomodulatory
factors as well as exposures to the same maternal microbiome during birth, the same
skin contact, and the same expressed breast milk.[10] However, many very preterm infants are born via caesarean section and could not
have own maternal nipple feeding. This suggests that shared factors (genetic and environmental)
are important in determining the gut microbiome even in an environment with many complex
variable factors that also affect microbial community development.[11]
[12]
Intestinal Microbial Ecology Affecting Necrotizing Enterocolitis
Intestinal Microbial Ecology Affecting Necrotizing Enterocolitis
Almost four decades ago, Sántulli et al observed that bacterial colonization was a
critical factor in NEC pathogenesis in early descriptions of the disease.[13] After that, numerous clinical evidence and laboratory data supporting this assertion
have accumulated. These include the following: changes in the microbial community
have been reported to be associated with NEC and sepsis in unrelated preterm neonates[14]
[15]; specific pathogens caused numerous NEC outbreaks[16]; gut colonization in preterm infants is often delayed[17]; abnormal gut microbiota such as decreased diversity was found particularly in infants
who developed NEC.[18]
[19]
[20]
Studies reporting specific bacterial associations with NEC suggest a role for Proteobacteria,
commonly Enterobacteriaceae.[14]
[21] Other evidence supporting the role of gut microbiota is that an early antibiotic
exposure can be a risk factor for NEC because of its effect on gut microbial colonization.[22]
[23] In fact, more recently, investigators have utilized culture-independent assays in
piglet NEC models and showed that NEC is associated with disturbances in the intestinal
microbiota.[24]
[25]
Recent study by Torrazza et al further disclosed that infants who develop NEC did
exhibit a different pattern of microbial colonization compared with controls as following[26]: there was a higher proportion of Proteobacteria (61%) 2 weeks and of Actinobacteria
(3%) 1 week before diagnosis of NEC compared with controls (19 and 0.4%, respectively);
it was characterized by the lower numbers of Bifidobacteria counts and Bacteroidetes
proportions in the weeks before NEC diagnosis; in the first fecal samples obtained
during week 1 of life, a novel signature sequence, distinct from but matching closest
to Klebsiella pneumoniae, that was strongly associated with NEC development later in life, was detected.
Meanwhile, three recent studies by 16S rRNA gene analysis have identified an abundance
of organisms from the phylum Proteobacteria (which includes many commonly observed
gram-negative pathogens) in fecal samples of babies with NEC.[14]
[15]
[18] Additional findings included a loss of gut microbial diversity[18] and depletion of enterococcal populations in NEC samples.[15]
Because fecal microbes do not necessarily correlate with the organisms found within
intestinal mucosa, studying microbes within intestinal tissue removed from preterm
babies might provide more information of the cross talk between microflora and epithelium
in the pathogenesis of NEC. In this regard, a study of microbes present in formalin-fixed
intestinal samples resected from 24 infants with NEC demonstrated an abundance of
organisms from the phylum Proteobacteria and the genus Ralstonia. Interestingly, a
significant correlation was found between the clinical finding of pneumatosis intestinalis
and the presence of clostridial species (Clostridium butyricum and Clostridium paraputrificum).[27]
These studies suggest that NEC is associated with both unusual intestinal microbial
species and an overall reduction in the diversity of microbiota.[14]
[18]
[28] Taken together, the variability of these results suggests that NEC may not result
from a single causative species but more likely from a currently undefined dysbiosis.
Further evidence also comes from the findings that NEC was not observed in germ-free
animals[29] and NEC did not occur in the intrauterine environment, despite ingestion of 150
mL/kg/day of amniotic fluid that contains proteins, carbohydrates, fat, immunoglobulin,
and electrolytes.[30] All of these findings strongly suggest that bacterial colonization is an important
factor in the pathogenesis of this disease.
New evidence disclosed that the majority of the differentially abundant genes in the
NEC patient were associated with carbohydrate metabolism and mapped to members of
the family Enterobacteriaceae.[31] This may indicate an adaptation of the community to an altered profile of substrate
availability for specific members as a first step toward the development of NEC.
Although dysbiosis of the gut microbiome is implicated in disease development, no
single causative agent has been convincingly identified yet as a biomarker of NEC.
This is because the data in this regard are limited in extremely low-birth-weight
infants who carry the highest risk for NEC. The association between gut microbiome
and development of NEC needs further investigation to provide detailed pathophysiology
and most logic approach to prevent NEC in vulnerable infants.
Clinical Observations and Experimental Models Support the Critical Role of Microbiome
in Necrotizing Enterocolitis
Clinical Observations and Experimental Models Support the Critical Role of Microbiome
in Necrotizing Enterocolitis
Numerous experimental models and clinical observations have confirmed the critical
role of gut microbiota in NEC, for example, preterm neonates show delayed colonization
by “healthy commensal” organisms, especially bifidobacteria and lactobacilli.[32]
[33]
[34] These data suggest that low colonization of Bifidobacterium and Lactobacillus in
VLBW infants may serve as a predisposing factor in microbial infection and NEC. Based
on these findings, it is speculated that probiotics could be potentially used to prevent
NEC in prematurity.
Using animal model, Caplan et al[35] and Butel et al[36] showed that bifidobacterial supplement in rat and quail model resulted in intestinal
colonization and subsequent reduction in NEC-like lesion. Researcher began to examine
whether probiotics could prevent the occurrence of NEC. Kitajima et al started the
first trial of early administration of Bifidobacterium breve to preterm infants, but
failed to prove the hypothesis because of inadequate sample size.[37]
Subsequently, Lactobacillus acidophilus
[38] and Bifidobacterium infantis
[39] had been used as probiotics to reduce the incidence of NEC in Italy and Columbia,
respectively. They reported the contradicting results, that is, the former was negative,[38] while the latter was positive.[39] Meanwhile, our randomized controlled trials either with single or multicenter center
was the first to prove that administration of probiotics that contain Lactobacillus
and Bifidobacterium in VLBW infants reduces the incidence and severity of NEC.[40]
[41]
After our study, there were 25 randomized controlled trials and 7 recent meta-analyses
that enrolled a total of 6,655 preterm infants and confirmed that oral probiotics
effectively prevent NEC and death[42]
[43]
[44]
[45]
[46]
[47]
[48]: the use of different probiotics in all studies has made the selection of an optimal
probiotic regimen difficult, and therefore, clinicians are facing challenges in assessing
which probiotics are most effective to prevent NEC in preterm VLBW infants.
Our premature rat model showed that administration of a mixture of probiotic strains
with Bifidobacterium bifidum and Bifidobacterium longum was most effective in preventing death and NEC in this animal model[49]; an updated meta-analysis showed that combined probiotics strains resulted in a
marked reduction in the incidence of NEC with a pooled OR of 0.37 and improved mortality
with a pooled OR of 0.58 in preterm infants (≤ 34 weeks' gestation) or those of a
birth weight ≤ 1,500 g (Chang HY et al, 218 Congress of Taiwan Pediatrics Society
2015 April; Taipei, Taiwan: accepted by the European Clinical Nutrition 2015).
Though there is no clinical randomized controlled trial examining the protective effect
of B. longum subspecies infantis (B. infantis) on NEC, B. infantis is unique in its prodigious capacity to digest and consume any human milk oligosaccharide
structure. It is also characterized that B. infantis grows better than other bacterial strains in the presence of human milk oligosaccharides,
and displays anti-inflammatory activity in premature intestinal cells, and decreases
intestinal permeability.[50] In addition, a large repertoire of bacterial genes encoding an array of glycosidases
and oligosaccharide transporters are not found in other bacterial species. Further
study is needed to investigate the protective effect on NEC of single B. infantis compared with combined probiotics or synbiotics.
Future Direction
We believe that identification of simple species may not be a robust enough measure
to identify the links between NEC and gut microbes. The advanced deep sequencing by
the currently available sequencing platforms makes it possible to characterize the
potentially important organisms that are present at a low abundance. Available data
suggest that the colonization pattern of the healthy full-term infants who are breast
fed may represent optimal colonization for the preterm infant as well. Future study
should focus on the head-to-head control trial on different probiotics regimen and
possible use of the microbiome of breast-fed preterm infants for total fecal transplantation
to eradicate NEC.
