What Is Known
- Preterm birth is a high-priority public health concern, due to increasing rates and associated health problems.
- Preterm birth is frequently associated to infections and microbial dysbiosis during pregnancy.
- Gut microbiota establishment is altered in preterms, what increases their risk of experiencing severe infections.
What Is New
- Dietary strategies can modulate maternal and/or infant microbiomes.
- Early feeding with colostrum/human milk benefits microbiota establishment, improving preterm neonates’ health outcomes.
- Probiotics appear to be safe and may reduce risk of experiencing severe infections in preterms.
- It is essential to ascertain whether microbiome-based strategies may reduce preterm birth incidence.
PRETERM BIRTH, A PUBLIC HEALTH PRIORITY
Preterm births (PTB), which are defined by WHO as those occurring before 37 completed weeks of gestation, can be further classified based on gestational age in extremely PTB (<28 weeks), very PTB (>28–<32 weeks), moderate PTB (>32–<34 weeks), and late PTBs (>34–<37 weeks) (Fig. 1). Globally, >10% of all babies born worldwide in 2010 were preterm, with 15 million estimated PTBs and >1 million fatalities as a consequence of infant prematurity (1). In addition, prematurity is rising in most countries in which data are available, what highlights that PTB is truly a global problem.
Over the last few decades the survival gap between babies born in high-income countries and those born in the poorest countries has widened dramatically. Although in the first settings even extremely preterm babies can survive, deaths of any premature baby are still perceived as inevitable in the second ones (2). Prematurity is now the second leading cause of death in children younger than 5 years of age, and the most important cause of death in the critical first month of life (2). Indeed, being born preterm increases a baby's risk of dying from neonatal infections. Overall PTB is estimated to be a risk factor in at least 50% of all neonatal deaths (3). In addition, PTB sequelae may continue throughout life, impairing neurological development, increasing the risk of cerebral palsy, learning impairment and visual disorders, and affecting long-term physical health with a higher risk of noncommunicable diseases, such as diabetes and hypertension (3). These conditions represent a heavy burden for families, society, and the health system. Hence, PTB is one of the conditions included in the Global Burden of Disease analysis given its high mortality and considerable risk of life-long impairment (4). Therefore, PTBs have been rated as a high public health priority in low-, medium-, and high-income countries (1).
PATHOGENESIS OF PRETERM DELIVERY: PROTECTING AND PREDISPOSING FACTORS
Multiple mechanisms are thought to increase the risk of spontaneous PTB, although triggering factors remain unknown in, at least, 50% of the cases (5). Therefore, understanding of environmental and genetically inherited predisposing factors (Fig. 2A), and early identification of risk markers are crucial to allow the implementation of appropriate preventive measures.
Among the known predisposing factors, a personal or family history of PTB, spontaneous abortions, and/or fetal death in previous pregnancies are recognized as important risk factors. This fact suggests the existence of genetic and epigenetic inheritable predisposing factors (6). Indeed, racial predisposition is also observed, with African black women experiencing twice the rate of PTB than Caucasian women even when the socioeconomic biases are controlled (7). The genetic determinants behind this predisposition are, however, not fully understood and only some polymorphisms in genes involved in the biosynthesis of cytokines, toll-like receptors, or other immune molecules have been described (8).
Other factors that correlate with significantly higher PTB incidence include multiple pregnancies, maternity at ages younger than 18 years or older than 35 years, obesity, nutritional deficiencies (eg, those in vitamins B6, B12, and D, folates, zinc, or iron), stressful lifestyle, anxiety, and exposure to toxic substances (eg, those derived from smoking) (9–11). This evidences that preconception and pregnancy care are key steps in PTB control and prevention.
Bacterial, viral, and fungal infections in pregnant women have been long considered a leading cause of intrauterine growth restriction and PTB. Certain infectious agents can reach the amniotic sac, establish an intra-amniotic infection and initiate an inflammatory response at the maternal and fetal tissues, which promotes prelabor rupture of membranes and PTB (12,13). It is worth highlighting that culture-independent technologies have detected bacterial DNA even at placentas and umbilical cords of women subjected to elective cesarean delivery at term. This challenges the traditional view of fetal development in a sterile uterine environment and points toward the existence of a naturally occurring microbiome at maternal-fetal tissues even in healthy pregnancies (14–18). Preliminary results suggest that alterations in the composition of the microbial populations transmitted from mother to fetus may promote proinflammatory states at maternal-fetal tissues during pregnancy, eventually initiating prelabor rupture of membranes and PTB (12–16,19–21).
Infectious agents can reach fetal tissues through fallopian tubes, by ascension through the lower urogenital tract and/or through hematogenous transplacental translocation. The particular contribution of each of these routes has not been studied yet in detail, but genitourinary infections, bacterial vaginosis, and sexually transmitted diseases, such as those caused by Gardnerella vaginalis, Prevotella spp., Candida spp., Chlamydia trachomatis, Gonorrhea spp., Treponema spp., HIV, Mycoplasma hominis, or Ureaplasma urealyticum, have been frequently associated with PTB episodes (22–23). Bacteria causing infections at distant locations in the mother's body can also reach fetal tissues through hematogenous transplacental translocation (18,21). In fact, periodontal infections have been associated with PTB and some bacterial species from the oral cavity, such as Fusobacterium nucleatum or Porphyromonas gingivalis, have also been isolated from intrauterine infections (13,24,25). Interestingly, a recent meta-analysis has reported that root teeth scaling in periodontitis-experiencing pregnant women significantly reduced PTB incidence (26). Unanimous agreement has, however, not been observed among studies testing different periodontitis treatments and, therefore, further studies are still required to clarify the actual role of periodontal infections in PTB. A remarkable similarity between maternal oral and placental microbiomes has also been described in healthy full-term pregnancies, although further research is needed to shed light on the mechanisms and health consequences of a translocation of maternal oral microorganisms to the fetus (14).
It is worth highlighting that for many of these microbial-associated risks there are no effective preventive programs in place yet. As an example, despite the frequent association of certain infections with PTB, prophylactic antibiotic treatments have not been successful so far (27). In fact, antibiotic administration during preconception or pregnancy leads to alterations in the digestive, vaginal, and mammary ecosystems before birth (28,29), with potential morbidity and long-term effects on the early microbial colonization of the neonate (30).
An increasing body of knowledge also points toward the fact that PTB can result from an asymptomatic dysbiosis state in pregnant women. For instance, although the vaginal microbiota of healthy pregnant women is rich in Lactobacillus species, their absence early during pregnancy has been associated with increased risk of vaginal infection, which could then lead to PTB (31). Indeed, vaginal microbiome diversity has been correlated with PTB incidence, although independent studies have reported contradictory results. DiGiulio and colleagues (32) found a positive correlation between Lactobacillus-poor vaginal microbial communities of high diversity and PTB risk. Another study, however, found no correlation between Lactobacillus scarcity/absence and PTB, and in this latter case vaginal microbiome diversity was inversely correlated to PTB risk in Caucasian women (Shannon Diversity Index of 0.088 ± 0.082 in preterm group [n = 7] vs 0.578 ± 0.608 in the control group [n = 30]) (33). Ethnic differences may explain these apparently contradictory results. Nevertheless, further studies are required to ascertain the specific bacteria or metabolic routes responsible for the augmented PTB risk of well-defined populations. Despite the association encountered between vaginal dysbiosis and PTB incidence, clinical trials using vaginal microbiome modulation as a target for PTB prevention have not been executed yet. Future studies should address the potential of such strategies (31). Regarding the gut microbiome during pregnancy, reduced Clostridium and Bacteroides populations have been found in mothers delivering preterm neonates as compared to those delivering at full term (34). Further research is required to discern how these changes in the gut may condition PTB and the future neonate's health.
All these facts emphasize the need for more research on the inter-relationships between infection, microbiomes, immune response, and the cascade of events resulting in PTB (5,12,21). Such research can lead to the development of novel screening, prevention, and/or treatment protocols targeting underlying microbiota-associated risk factors (eg, asymptomatic bacteriuria, bacterial vaginosis, and periodontal disease), which should be taken into consideration at pregnancy care to reduce PTB rate and sequelae (26,27,31). Interestingly, dietary supplementation with probiotics during the last trimester of pregnancy has been associated with a modulation of the vaginal microbiota and cytokine secretion, resulting in an immunomodulatory effect at fetal membrane tissues (35,36). Although these effects may protect against PTB, further studies are required to confirm probiotics efficacy in PTB prevention. Clinical trials including sufficient numbers of PTB patients are desirable with this aim. Recently, it has been shown that the supernatant of a probiotic culture prevents lipopolysaccharide-induced PTB and reduces inflammation in pregnant CD-1 mice (37). Also, increasing scientific evidence suggests that transmission of maternal microbes to offspring is a common phenomenon that determines epigenetic marks of the newborn baby and can condition its future health (18). Therefore, promoting appropriate maternal microbiomes not only may prevent PTB but also can favor the transmission of healthy maternal microbes to the newborn, what may have a critical impact in the healthy development of the (preterm) infant (20,21,38).
IMPAIRED EARLY GUT COLONIZATION: A KEY HEALTH PROBLEM IN PRETERM NEONATES
Premature neonates show numerous signs of organ immaturity responsible for their inability to appropriately face postnatal life challenges. Immediately after PTB, the immature gut is exposed to a plethora of microbiological, immunological, and nutritional-related challenges difficult to cope with due to deficiencies in structural integrity, digestive and absorptive capacity, immunity, and blood flow at this organ. A compromised gut barrier may render the mucosa susceptible to invasion by gut bacteria, leading to the production of proinflammatory cytokines, which further compromises the intestinal defense mechanisms (Fig. 3A). This leads to a vicious cycle of maldigestion, bacterial invasion, immune activation, and uncontrolled inflammation with negative short- and long-term health consequences (38).
In contrast, it is remarkable that the fecal microbiota of premature infants is different from that of term infants and their gut colonization has been described as delayed and aberrant. It is generally characterized by lower diversity, more fluctuations, a higher representation of potential pathogenic microorganisms typically encountered in hospital environments and a reduced representation of health-related commensal microorganisms (39–41). Indeed, many factors usually associated to PTB are known to affect the infant gut microbiota establishment, what exacerbates these differences. Preterm neonates are frequently born by C-section, are minimally exposed to maternal microbes, receive frequent antibiotic treatments and delayed enteral feeding, and are exposed to hospital environments for long periods of time (Fig. 2) (39–41). In conjunction with their immaturity, all these factors increase a baby's risk of experiencing severe health conditions, sometimes with fatal outcomes.
Furthermore, the gut microbial ecosystem is known to play crucial roles in the digestive function and general well-being of the individuals. Recent animal studies have demonstrated that the early neonatal period is the most important moment for reaching the microbiota-induced host-homeostasis. Indeed, absent or abnormal microbiota during this critical period may have life-long effects (42–44). Microbiota provides stimuli necessary for an adequate developmental programming of epithelial barrier function, gut homeostasis, angiogenesis, and innate and host adaptive immune functions (45–47). In addition, it can affect the development of other organs with deep consequences at systemic level (48,49). The functional development of the brain is of particular interest because it has been shown to be greatly susceptible to modulation during perinatal life (50,51). As an example, there seems to be an association between common neurodevelopmental disorders, such as autism and schizophrenia, and infections during the perinatal period (52).
Globally, it is becoming apparent that an impaired gut microbiota functionality is directly linked to many of the health conditions and sequelae typically associated with preterm infants. A better understanding of the factors that cause these deficiencies will aid the identification of means to improve the infant gastrointestinal tract maturation, reducing gut inflammation, and improving health outcomes, with subsequent reductions in morbidity and mortality associated to PTB.
EARLY GUT COLONIZATION IN PRETERM NEONATES
In the healthy full-term vaginally delivered newborn, gut microbial colonization starts with facultative anaerobic microorganisms which contribute, by lowering the intestinal redox potential, to the subsequent establishment of strict anaerobic microorganisms, such as Bifidobacterium, Bacteroides, or Clostridium(39,41). During the first months of life there is a significant presence of Actinobacteria and, in many cases, Proteobacteria(30,41,53,54), which is in contrast to the dominance of Bacteroidetes and Firmicutes during adulthood (55). As stated before, several factors influence the early gut microbiota and its further development in the infant. Among these, gestational age at birth (30,41), mode of delivery (56–58), use of perinatal antibiotics (30,59,60), or feeding habits (58,61) have been studied to some extent. To date, little is, however, known on the effect of other factors on the microbiota establishment in the neonate.
By using traditional culture-dependent methods, reduced levels of symbiotic microorganisms and increased levels of potential pathogens have been repeatedly reported in preterm neonates as compared to term babies. Culture-independent techniques have confirmed these observations in that they allowed the identification of clear alterations in the process of microbiota establishment in preterm neonates when compared to full-term babies (30,40,41). As previously stated, microbiota alterations in the gut of preterm neonates are associated with serious health conditions such as necrotizing enterocolitis (NEC) or sepsis, which can be fatal. There is no single microbiological or environmental factor triggering NEC, but a low gut microbiota diversity coupled to overgrowth of one or several opportunistic pathogens is a common characteristic (62). Recent data on the preterm infant microbiota have also indicated that microbiota alterations may precede the development of diseases, such as sepsis or NEC (63–65). In preterm infants, up to 3 weeks before NEC diagnosis gut microbiota shifts toward an increased presence of potential pathogens, with members of Corynebacterium, Klebsiella, Clostridium, or Enterobacter species being among the most frequently identified microorganisms (64), what opens new avenues to identify markers of NEC that would aid in the establishment of early therapeutic approaches.
During the first months of life, an increased abundance of Enterobacteriaceae, a delayed colonization by symbiotic bacteria, including Bifidobacterium and Bacteroides, and a higher colonization by pathogens, such as Klebsiella, is usually observed when the microbiota of preterm babies is compared with that of term infants (30,41). Other noticeable difference is the reduced number of total bacteria during the first days of life in preterm neonates. Some factors often related to premature delivery seem to further increase these differences. Although these factors are not fully understood yet, perinatal exposure to antibiotics appears to play a critical role (30). In general, preterm neonates harbor lower levels of strict anaerobes but increased levels of facultative anaerobes and aerotolerant microorganisms, in comparison with full-term babies. These alterations in the intestinal microbiota composition lead to differences in the main microbial metabolites in the intestine, the short chain fatty acids, which concentration in feces is lower in preterm neonates than in infants delivered at full term (41,61)).
To sum up, available data indicate the existence of specific alterations in the microbiota establishment process in preterm infants, what allows the identification of potential targets for microbiota modulation during early days after birth with the aim of reducing PTB-associated morbidity and mortality. These early phases of life may represent a unique window of opportunity for microbiota modulation in the infant. Later on, after weaning, the microbiota complexity and diversity increases rapidly during the first years of childhood (54).
DIETARY STRATEGIES TO MODULATE GUT COLONIZATION AND MATURATION IN PRETERM NEONATES
The type of enteral diet (mother's colostrum and milk, donor milk, and formula), dietetic supplements (immunonutrients, amniotic fluid, and probiotics), and mode of feeding (parenteral, enteral, or minimal enteral) play significant roles in neonatal development and resistance to disease. Feeding strategies recommended in preterm neonates and their role in gut microbiota establishment and maintenance and immune system maturation are summarized below.
During the last trimester of pregnancy, fetuses swallow approximately 750 mL of amniotic fluid daily (66). Amniotic fluid intake has a relevant role in fetal nutrition and in normal development of the fetal intestine, particularly during mid- to late-gestation because of the presence of a variety of factors with putative functions in cellular growth, proliferation, and cell-to-cell signaling, including the epidermal growth factor, the insulin-like growth factor 1, bioactive peptides, and hormones (38,67,68). In addition, it contains antimicrobial peptides that provide protection against bacterial infection (69). In fact, postnatal administration of porcine amniotic fluid to preterm pigs reduced bacterial density and NEC severity, and induced downregulation of genes involved in gut inflammatory responses (CD55, IFN-γ, IL-1α, IL-2 receptor, IL-4 receptor, iNOS, TLR-3, tumor necrosis factor, and tumor necrosis factor receptor) and upregulation of factors associated with immunomodulation (toll-interacting protein and interferon regulatory factor) (70). Other studies revealed that amniotic fluid–fed preterm pigs showed slower gastric emptying, reduced meal-induced release of gastric inhibitory peptide and glucagon-like peptide 2, changed gut microbiota and immune profiles, and reduced intestinal permeability (71). Furthermore, a simulated human amniotic fluid–like solution, containing the enterocyte growth factors erythropoietin and granulocyte-colony stimulating factor, has been suggested to improve tolerance to milk feeding in preterm neonates (72). Globally, such observations suggest that amniotic fluid may provide protection against intestinal lesions in preterm infants through suppression of inflammatory pathways, what would contribute to maintain an appropriate intestinal barrier, and therefore limiting pathogens translocation and proliferation (38). Moreover, preliminary data suggest that amniotic fluid may be the first source of commensal bacteria to the fetus, albeit at low concentrations (73).
Further studies are required to assess whether the provision of concentrated human amniotic fluid to preterm infants as a supplement, during parenteral nutrition or together with enteral milk feeding, can exert the desired synergistic maturational, antimicrobial, and immunological effects on the immature gut. Similarly, the identification of the particular amniotic fluid components with potential beneficial effects on the preterm neonate may help to design better nutritional supplements specifically tailored for the particular needs of the preterm infants.
Infant formulas fail to replicate the important maturational and protective effects of the own mother's milk (OMM), which may be due to the lack of bioactive constituents and an inappropriate nutrient composition. The mother's colostrum and milk promote intestinal growth, immune modulation, and maintenance of a healthy microbial environment. This is particularly important for preterm neonates, in which deficiencies in intestinal integrity, barrier function, digestive capacities, and intestinal immunity lead to increased susceptibility to inflammatory diseases.
Colostrum is secreted during the early days post-birth when the paracellular pathways between the mammary epithelial cells are opened, what permits the translocation of high molecular weight compounds into the fluid. As a result, colostrum contains a larger range of growth factors, anti-inflammatory components, and anti-infective components than mature milk, which facilitates the transition from intrauterine to extrauterine infant nutrition (67). Indeed, early administration of colostrum in extremely premature infants seems to be critical to compensate for the shortened period of in utero maturation (74). Moreover, prolonged secretion of colostrum following extremely premature birth may be a specific protective mechanism for the compromised infant (75).
It is worth noting that human colostrum is different from mature milk, presenting higher concentrations of regulatory and anti-inflammatory cytokines, chemokines, growth factors, immunoglobulins, lactoferrin, oligosaccharides, soluble CD14, antioxidants, and other protective components with potential anti-infectious, anti-inflammatory, and immunomodulatory roles. Recent studies suggest an inverse relationship between the duration of pregnancy and the concentration of these agents in maternal colostrum. Thus, the mothers of the least mature infants produce the most protective colostrum (75,76). Indeed, human colostrum is rich in compounds, such as inositol, that may play a critical role in fetal and early neonatal life through promotion of lungs maturation. Interestingly, colostrum from mothers who deliver prematurely has significantly higher inositol concentrations than colostrum from mothers who deliver at term (77). Inositol administration has been related to statistically significant and clinically important reductions in short-term adverse neonatal outcomes including bronchopulmonary dysplasia and retinopathy of prematurity (78). This justifies the need for multicenter controlled trials to confirm such findings and fully discern the role of own mother's colostrum in preterm neonates health.
Human colostrum is known to be a source of commensal bacteria (73) and prebiotic human milk oligosaccharides (HMOs), which favor the growth of beneficial commensal microbes at intestinal level. In particular, human colostrum presents higher concentrations of α-1,2 fucosylated oligosaccharides (79), and a higher bacterial diversity than mature milk (80,81), what may represent particular adaptations for the preterm neonate requirements. It is worth highlighting that specific HMOs have been linked to newborn protection from NEC-related pathogens (82). The specific effect that these differences in HMOs composition have on the gut development in the preterm newborn, however, remains to be elucidated and deserves further attention.
A recent study has demonstrated the safety and feasibility of oropharyngeal administration of colostrum before the introduction of trophic feedings in extremely low-birth-weight infants, which led to systemic immunomodulation, local interference with microbe attachment to the digestive mucous membranes, trophic effects, and a specific role in protection from ventilator-associated pneumonia (83). Therefore, it would be greatly desirable to start, as soon as possible, trophic feedings using the own mother's colostrum in very preterm or extremely preterm infants because it is an easy and inexpensive procedure well-tolerated by even the smallest and sickest very preterm infants (74,84).
A second critical feeding period is the first 14 to 28 days post-birth. Several studies have demonstrated at this period a dose-response relationship between the amount of OMM received by very low-birth-weight and extremely low-birth-weight infants and the reduction in the risk of short- and long-term morbidities, including enteral feeding intolerance, nosocomial infections, NEC, chronic lung disease, retinopathy of prematurity, total number of morbidities during the neonatal intensive care unit (NICU) stay, developmental and neurocognitive delay, and rehospitalization after NICU discharge (74). Feeding with high doses of human milk appears to program or promote many health benefits, which may be linked to structural and functional changes in the gastrointestinal environment of the preterm neonate (85). The mechanisms by which human milk provides this protection are varied and synergic, involving specific human milk components that are absent or found at low concentrations in other mammals’ milk. Among those, the type and amount of long-chain polyunsaturated fatty acids and digestible proteins, the extraordinary diversity and complexity of the HMOs, and the commensal bacteria found in human milk are considered relevant (Fig. 3) (80,83).
During the first days of life the gastrointestinal tract is colonized almost immediately with an array of commensal and potentially pathogenic bacteria. As stated above, many factors surrounding the birth of a premature or NICU infant, such as cesarean birth, antibiotic use, and delayed enteral feeding predispose the intestine to episodes of dysbiosis and increase the risk of disease. Human milk, which contains both probiotic bacteria and prebiotic carbohydrates, however, favors the predominant presence of commensal microorganisms in the gut and promotes bacteria-enterocyte crosstalk in the developing intestine, aiding infant intestine development and maturation (43,47). Another protective mechanism associated with OMM feeding is the progressive closure of paracellular pathways through the formation of tight junctions between the enterocytes in the infant's intestine, which prevents the translocation of high-molecular-weight bacteria and toxins from the gut lumen to the bowel wall, avoiding the establishment of inflammatory processes (86).
Kangaroo Mother Care (KMC), based in promoting early and frequent skin-to-skin contact between the newborn and the mother, is associated with a range of health benefits to the preterm neonate. Indeed, KMC started at the first week post-birth is associated with a 51% reduction in neonatal mortality for babies weighing <2000 g, compared to incubator care (3). An updated Cochrane review also reported a 40% reduction in risk of post-discharge mortality, approximately a 60% reduction in neonatal infections and an almost 80% reduction in hypothermia. Other benefits included increased breastfeeding rates, weight gain, mother-baby bonding, and developmental outcomes (87). In addition, KMC may represent a mean to promote transmission of maternal commensal microbes to the preterm neonate, and thus accelerating the establishment of healthy microbiomes (88). Further research is envisaged in this area, especially considering that KMC is a friendly care system that reduces hospital stay and nursing load, and therefore leading to cost savings. KMC was endorsed by the program implementation guide developed by WHO in 2003 (89).
At present, all relevant health organizations recognize OMM as the first choice in preterm infant feeding, because its peculiar properties are met neither by donor human milk nor by commercial infant formula (89,90). Therefore, OMM feeding should be an NICU priority and dedicated efforts should be made to promote its implementation. When breastfeeding is not possible and OMM is not available, donor milk should be the first choice for preterm infants. Beneficial bacteria present in human milk, HMOs, and immunological components are believed to play a crucial role guiding the infant microbiome establishment (85). Donor milk is, however, subjected to pasteurization and storage processes that result in total or partial inactivation of some microorganisms and bioactive compounds. Active bacteria are killed during pasteurization, HMOs stability during heat treatments has not been well established yet, and some cytokines are likely to be inactivated during the process (91,92). For these reasons, further research is required to select appropriate preservation technologies and/or to design specific supplements to be used in combination with donor milk. Globally, donor milk, however, provides a wider array of benefits for the infant than preterm formulas (83–88,93).
Probiotics, Prebiotics, and Synbiotics
As previously stated, the microbiota establishment in the preterm neonate constitutes a challenging process, which results in an abnormal pattern of intestinal colonization that may contribute to the pathogenesis of different PTB-related diseases. Therefore, there is an increasing interest in the management of the microbial colonization process in the preterm infant. Among the strategies that can be used with this objective, the administration of selected probiotic bacteria or prebiotic compounds, with appropriate microbiota modulation capabilities, constitute a promising approach (94). Probiotics and prebiotics may influence the preterm infant health, reducing the risk of disease through numerous potential mechanisms (95). Among them, the maintenance of the intestinal barrier is of particular interest. The efficacy of this barrier depends on several factors, including the integrity of tight junctions, intestinal peristalsis, secretory immunoglobulin A, gastric acid production, T-cell immunity or macrophage phagocytosis, all of which are normally impaired in preterm neonates (94). Therefore, preterm infants may benefit more from the use of probiotics and prebiotics during the early life's “window period”, when the intestinal microbiota and immune system are becoming established. Indeed, certain probiotics and prebiotics, and combinations of both of them (synbiotics), have demonstrated benefits for preterm infants in human clinical randomized controlled trials (RCTs).
The reported beneficial effects of probiotics and prebiotics in infants include prevention and treatment of acute diarrhea and atopic eczema (as one of the best established effects on allergic symptoms) (96–100). Further studies are required to determine the efficacy of probiotics and prebiotics for the prevention of other allergic conditions frequently associated with PTB, such as asthma (100). Among other effects from which premature newborns may benefit the most, NEC prevention by probiotics is likely the best studied outcome. Results of a systematic review and meta-analysis of 20 RCTs (total sample size = 3816) confirmed that probiotic supplementation significantly reduces the risk of NEC and all-cause mortality without any significant adverse effects, such as probiotic sepsis, in preterm very low-birth-weight (<1500 g) infants (101). Experts agree that probiotics reduce the risk of NEC and the associated mortality, and the evidence for mortality reduction is as conclusive as that existing for other well-established interventions such as use of antenatal corticosteroids, surfactants, and cooling (95). In fact, probiotics have the best effect size (risk ratio 0.33; 95% confidence interval 0.24–0.46; P < 0.00001) compared with other tested dietary strategies, including lactoferrin, arginine, or prebiotic administration, for reducing severity and incidence of NEC (92,101). Ofek Shlomai et al (102) reviewed the Cochrane systematic reviews database for primary prophylactic strategies (n = 49) for preterm infants and selected 25 strategies that have been incorporated in clinical practice. Sixteen of them have been reported as beneficial in improving the primary outcome, with only 4 reducing mortality. Compared with other strategies that reduce mortality, probiotics show the largest effect size (risk ratio 0.56; 95% confidence interval 0.43–0.73; P < 0.0001) (101). All these RCTs and meta-analyses indicate that specific probiotics reduce the severity and incidence of NEC in preterm neonates. There is, however, still a lot of reluctance in adopting probiotic prophylaxis to reduce the risk of NEC and death in preterm infants because of the heterogeneity in strains and conditions used in the different RCTs (103). Furthermore, the existence of important methodological limitations in several trials, due to limited sample size or to the lack of double blinds, limits drawing conclusions regarding probiotics efficacy (104). Rigorous design of RCTs according to current guidelines and recommendations is therefore essential to evaluate probiotics efficacy. In addition, the specific properties of the probiotic strains for preterm infants are not well defined yet and the best performing strains and conditions need to be identified (105). For instance, although most studies indicate the benefit of probiotics to reduce NEC incidence and severity, this effect is greatly strain dependent, since a recent large trial demonstrated no effect of a bifidobacterial strain (106). Indeed, different probiotic strains may have different mechanisms of action and their effects are strain-specific for a particular health condition. Meta-analyses provide a simplified view of probiotics efficacy on a specific health condition, by comparing a variety of different probiotic strains. Nevertheless, a meta-analysis concluding that probiotics are effective to improve a certain health condition does not mean that all analyzed probiotics exert that particular effect. Indeed, when sufficient trials are available, it is advisable to limit meta-analyses to specific probiotic strains. There are also some studies, which report the beneficial effects of some prebiotics in reducing NEC incidence (107), but comparative studies suggest a higher potential for probiotic- or synbiotic-based approaches (108).
On the contrary, probiotics and prebiotics have globally failed to reduce late-onset sepsis in the PTB infant population (95,107,109,110), although some positive results have been published as well (108). Moreover, a preventive effect of probiotics and prebiotics administration for preterm infants on viral respiratory tract infections has been recently reported (111).
Another issue of special relevance for premature babies is the establishment of enteral tolerance, which facilitates weight gain and infant growth. Enteral tolerance is often delayed in preterm newborns due to their intestinal immaturity. RCTs have shown that prebiotics improve the establishment of enteral tolerance (112,113) and a recent systematic review concludes that probiotics reduce the time to full enteral feeding in preterm newborns (114). Nevertheless, additional studies are needed to define the optimal strains and conditions.
It is important to underline that, so far, the probiotic strains being used have not been selected on the basis of their target-specific characteristics. Therefore, the identification of the best suited strains and conditions is still a pending task. During recent years, efforts have been focused on the isolation and identification of the best suited probiotic strains and prebiotic compounds for the specific needs of premature newborns in terms of intestinal microbiota modulation (115). Human milk has often been considered as an ideal source of potential probiotic strains for neonates (116,117), and strains isolated from human milk have been reported to reduce inflammatory markers in preterm infants (118).
Taking into account the special vulnerability of this population a careful evaluation of the potential risks associated with administration of probiotics and prebiotics in preterm infants is required. The potential benefits and risks of external interventions favoring the establishment of a gut microbiota resembling that of term babies are not well understood yet. It can be assumed that the microbiota composition of term, vaginally delivered, exclusively breastfed infants, harboring high levels of nonpathogenic bacteria and low levels of pathogens, would result beneficial for preterm newborns. Especially because preterm neonates harbor increased levels of pathogens such as Klebsiella pneumoniae or Clostridium difficile(41,119), which may result in an increased risk of infection by these microorganisms, the former microorganism being among the most common causes of nosocomial infection in preterm infants. Interestingly, colonization by C. difficile has been related to the microbiota composition. Infants colonized by C. difficile are more frequently colonized by K. pneumoniae and show lower levels of Bifidobacterium longum and Staphylococcus epidermidis(120). This provides a rationale for microbiota modulation in preterm newborns to reduce the risk of infectious disease.
The most commonly used probiotic strains are from the genera Lactobacillus and Bifidobacterium. These are microorganisms with a long history of safe use. Several probiotic and prebiotic trials have been conducted in neonates and low-birth-weight infants without observing serious adverse effects (95,97). Indeed, the extensive use of probiotics in neonatal units along the world without observing negative effects, together with the long-term safety of probiotics administration (103,120–121), constitute a good evidence of their safety. Nevertheless, some rare cases of infection during probiotic therapy of preterm infants have been reported (122,123) underlining the importance of performing safety studies and ensuring the proper manipulation of the administered products.
Recently, a stool substitute preparation, made from purified intestinal bacterial cultures derived from stool samples of a single healthy donor, has proved successful for the treatment of recurrent C. difficile infections that had repeatedly failed to standard antibiotics treatment (124). In comparison to fecal transplant approaches, the advantages of synthetic or minimal microbial communities are clear as the composition of the synthetic mixtures can be controlled and tested extensively for the absence of undesired pathogens and viruses. Moreover, such synthetic mixtures can be reproducibly manufactured (125) and their viability can be controlled and optimized. Implementation of these synthetic microbial communities in next-generation therapies would greatly benefit the patients, further advancing our understanding of the intestinal microbiome. Research efforts are needed to define the required composition and mechanisms of action of synthetic or minimal microbiomes specifically tailored to prevent or treat infections in the preterm neonate. In this framework, ecosystem therapeutics, or repopulation, of the preterm infants’ bowel with defined bacterial communities from human milk or breastfed infant feces, represents an attractive approach for the prevention and treatment of infections in the preterm population in the context of the increasing emergence of multidrug-resistant bacterial pathogens.
CONCLUDING REMARKS AND FUTURE PERSPECTIVES
Recent scientific advances are revealing that human-associated microbiomes exert critical roles in perinatal health, which may also have a significant impact in future adulthood health. On the one hand, dysbiosis in different body locations (ie, oral, vaginal, intestinal) during pregnancy may be the subjacent cause behind early birth initiation, although the particular molecular mechanisms have not been fully discerned yet. Indeed, both bacterial cells and bacterial DNA have been recently found in placental tissue, even after full-term healthy pregnancies occurring without membranes rupture. These facts open exciting new avenues for research on early PTB detection and prevention through microbiome-targeted therapies and on further understanding the precise mechanisms behind the infection-PTB correlation. On the other hand, preterm neonates are a particularly sensitive human subpopulation group. Their organs immaturity, long hospitalization periods and alterations in normal gut microbiota establishment, result in a series of developmental problems and in an increased risk of diseases that may lead to life-threatening conditions, such as NEC and sepsis, and to life-long impairment conditions, including cognitive and metabolic syndromes. It remains to be determined whether microbiomes in pregnant women may have an early effect on the future microbiota establishment in the neonate and, therefore, whether probiotic administration in these women may reduce PTB incidence and positively influence early gut colonization in the neonate.
In the last few years, important scientific efforts have been devoted to understand the gut microbiota establishment process in the preterm neonate, as compared to that of full-term babies; thus, revealing delayed and aberrant microbiota patterns. The gut microbiota of the preterm neonate is enriched in potentially pathogenic microorganisms, probably due to the long stay in hospital environments, and is poor in commensal and beneficial bacteria, due to the delayed/absent breastfeeding, which is known to provide an essential array of immunological factors, prebiotic HMOs, and beneficial microorganisms. Several preliminary approaches have proved successful to ameliorate the aberrancies in the establishment of the gut microbiota of the preterm neonate. These include early feeding with colostrum, OMM, or donor's milk, but also dietary supplementation with probiotics and prebiotics (section Dietary Strategies to Modulate Gut Colonization and Maturation in Preterm Neonates), which may be capable of balancing the proportion of commensal and pathogenic bacteria in the preterm neonate intestine in favor of a nonproinflammatory microbiome, with a significant reduction in NEC rates. Despite promising preliminary results, some neonatologists are still reluctant to accept probiotic interventions as a routine in NICUs. Further research in the field is essential to address the knowledge gaps concerning microbial roles in health protection of preterm neonates and to overcome the resilience to routine probiotics use in clinical practice.
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