Correlation between lactoferrin and beneficial microbiota in breast milk and infant’s feces Paola Mastromarino, Daniela Capobianco, Giuseppe Campagna, Nicola Laforgia, Pietro Drimaco, Alessandra Dileone & Maria E. Baldassarre BioMetals An International Journal on the Role of Metal Ions in Biology, Biochemistry and Medicine ISSN 0966-0844 Biometals DOI 10.1007/s10534-014-9762-3
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Author's personal copy Biometals DOI 10.1007/s10534-014-9762-3
Correlation between lactoferrin and beneficial microbiota in breast milk and infant’s feces Paola Mastromarino • Daniela Capobianco • Giuseppe Campagna • Nicola Laforgia • Pietro Drimaco • Alessandra Dileone • Maria E. Baldassarre
Received: 16 March 2014 / Accepted: 6 June 2014 Ó Springer Science+Business Media New York 2014
Abstract Lactoferrin (LF) is a natural component of human milk with antimicrobial, immunostimulatory and immunomodulatory properties. Several in vitro studies suggest that LF could promote an environment in the gut of neonates that favors colonization with beneficial bacteria. However, clinical studies on the correlation between the concentration of LF in breast milk and feces of infants and the gut microbiota in infants are lacking. In our study we analyzed the content of LF and the microbiota of breast milk and feces of infants of 48 mother–infant pairs (34 full-term and 14 pre-term infants) at birth and 30 days after delivery. In the term group, a significant decrease of mean LF concentration between colostrum (7.0 ± 5.1 mg/ml) and mature milk (2.3 ± 0.4 mg/ml) was observed. In pre-term group, breast milk LF levels were similar to those observed in full-term group. Fecal LF concentration of healthy infants
P. Mastromarino (&) D. Capobianco Section of Microbiology, Department of Public Health and Infectious Diseases, Sapienza University, Piazzale Aldo Moro 5, 00185 Rome, Italy e-mail:
[email protected] G. Campagna Department of Experimental Medicine, Sapienza University, Rome, Italy N. Laforgia P. Drimaco A. Dileone M. E. Baldassarre Section of Neonatology and NICU, Department of Medical Science and Oncology, University of Bari, Bari, Italy
was extremely high both in term and pre-term infants, higher than the amount reported in healthy children and adults. In term infants mean fecal LF levels significantly increased from birth (994 ± 1,828 lg/ml) to 1 month of age (3,052 ± 4,323 lg/ml). The amount of LF in the feces of 30 day-old term infants was significantly associated with maternal mature milk LF concentration (p = 0.030) confirming that breast milk represents the main source of LF found in the gut of infants. A linear positive correlation between colostrum and mature milk LF concentration was observed (p = 0.008) indicating that milk LF levels reflect individual characteristics. In pre-term infants higher mean concentrations of fecal LF at birth (1,631 ± 2,206 lg/ml) and 30 days after delivery (7,633 ± 9,960 lg/ml) were observed in comparison to full-term infants. The amount of fecal bifidobacteria and lactobacilli resulted associated with the concentration of fecal LF 3 days after delivery (p = 0.017 and p = 0.026, respectively). These results suggest that high levels of fecal LF in neonates, particularly in the first days of life, could represent an important factor in the initiation, development and/or composition of the neonatal gut microbiota. Since early host–microbe interaction is a crucial component of healthy immune and metabolic programming, high levels of fecal LF in neonates may beneficially contribute to the immunologic maturation and well-being of the newborn, especially in pre-term infants. Keywords Lactoferrin Newborn Beneficial microbiota Feces Breast milk
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Introduction Human milk is a complex mixture of fat, proteins, enzymes, cytokines, antibodies and nutrients that provides energy and immunological protection to infants and contributes to the development of neonatal defence mechanisms. Breastfeeding is considered the ‘‘second step of immunological education’’, in fact breast milk provides several bioactive compounds which influence the growth, the modulation and maturation of the immune system, the protection from pathogens and toxins and, finally, the establishment of the intestinal microbiota (Bollard and Marrow 2013). In addition, breast milk contains viable beneficial microorganisms such as lactobacilli and bifidobacteria (Collado et al. 2009). It is now well known that gut microbiota has a crucial role in the development and functionality of innate and adaptive immune responses, and in regulating intestinal barrier homeostasis, nutrient absorption and fat distribution (Hooper and Macpherson 2010). In infants of 1.5–5 months of age the most abundant classes of microorganisms present in the fecal samples are Bifidobacteriales, being present at 80.6 %, while second and third most abundant classes are Lactobacillales and Clostridiales being present at 7.2 and 3.1 %, respectively (Turroni et al. 2012). Bifidobacteria and lactobacilli are the main microorganisms of the ‘‘healthy’’ microbiota observed in the feces of breast-fed infants. The infant’s microbiota is acquired during the perinatal period. Although the initial transmission of bacteria from the mother to the newborn occurs through direct contact with the maternal microbiota during delivery, the human milk is suggested to be an important factor in the initiation, development and/or composition of the neonatal gut microbiota during lactation. Several components of breast milk are able to modulate the developing microbiota within the infant gastrointestinal tract, including complex oligosaccharides, immunoglobulins (IgA), lactoferrin (LF), lysozyme and cytokines (Zivkovic et al. 2011; Newburg 2005). Lactoferrin is a major whey protein present in breast milk, particularly in colostrum. Many biological functions of LF have been reported, including antimicrobial activity, immunostimulatory and immunomodulatory effects (Farnaud and Evans 2003; Actor et al. 2009). LF at high concentrations has a known ability to promote growth and differentiation of the immature gut by enhancing proliferation of
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enterocytes and closure of enteric gap junctions while at lower concentrations LF stimulates differentiation of enterocytes and expression of intestinal digestive enzymes (Buccigrossi et al. 2007). Finally, LF is considered as a growth promoter for bifidobacteria, the predominant beneficial microorganisms of human intestine. Several in vitro studies have shown that human LF is able to stimulate the growth of bifidobacteria (Miller-Catchpole et al. 1997; Petschow et al. 1999; Kim et al. 2004; Liepke et al. 2002) however this effect is differentially exerted on different species and strains of bifidobacteria (Kim et al. 2004; Tomita et al. 1994). In vivo a bifidogenic effect of LF has also been suggested, but conflicting results have been reported. In a study of human infant flora-associated mice, bovine LF-fortified milk increased the amount of gut bifidobacteria (Hentges et al. 1992), but in another study the intestinal flora of human infants fed with LFsupplemented formula (Balmer et al. 1989) was not modified in comparison to babies fed with not supplemented formula. On the contrary, Roberts et al. (1992) have found a bifidobacteria-predominant microflora in one half of the infants after 3 months of treatment with a LF enriched formula. Recently, bovine transgenic milk containing recombinant human LF has been shown to modulate the intestinal flora in the neonatal pig as an animal model for the human infant (Hu et al. 2012). In conclusion, administration of LF or some peptides deriving from its digestion seem to have a possible prebiotic effect. However, data emerging from clinical trials are not yet conclusive and further studies are necessary to reach a final conclusion. To our knowledge, there has not been a study evaluating the correlation between the concentration of LF in breast milk and feces of infants and the gut microbiota in infants. Therefore, the aim of our study was to investigate mother–infant pairs analyzing the content of LF and the microbiota of breast milk and feces of term and pre-term infants at birth and 30 days after delivery.
Materials and methods Study design and subjects This study was designed to examine the correlation between the concentration of LF in breast milk and feces of infants and to verify if LF levels in breast milk
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and infant feces was correlated with maternal milk microbiota and/or the gut microbiota of infants. Moreover, our purpose was to determine whether amounts of LF and microbiota in breast milk and feces of infants are significantly different between the mother–child pairs with birth at term compared to pre-term birth. Forty-eight healthy women (mean age 32.1 ± 5.1; range 20–41) were enrolled after delivery in the Department of Medical Science and Oncology, Section of Neonatology and NICU, of Policlinico Hospital (University of Bari). Thirty-four of them gave birth at term, while 14 have given birth pre-term (i.e. before 37 completed weeks of gestation). Exclusion criteria were twin pregnancy, pregnancy diseases, diabetes and other chronic illness. The study was reviewed and approved by the Institutional Review Board of the University of Bari, Medical School, Italy. Participation was voluntary and informed written consent was obtained from all participating mothers. Specimen collection Breast milk samples were collected after birth (colostrum) and 1 month after delivery (mature milk) in sterile plastic tubes, using a manual breast-pump after cleaning the nipples and areola by wiping with a swab soaked in sterile water, and immediately frozen at -80 °C. Stool samples from each baby were collected after birth (meconium) and 1 month after delivery and placed in sterile plastic tubes. Immediately after collection, samples were transported in ice and stored at -80 °C prior to processing. In the full-term group, one mother was unable to provide any milk sample, one did not provide colostrum and one did not provide the mature milk. In the pre-term group, three mothers were unable to provide any milk sample, one did not provide colostrum and five did not provide the mature milk. Lactoferrin assays Fecal LF was quantified from frozen stool specimens by use of a commercially available polyclonal-based enzyme-linked immunosorbent assay (ELISA) according to the manufacturer’s instructions (IBD-SCAN TechLab, Inc., Blacksburg, VA, USA). Stool specimens were serially ten fold diluted up to 1,000,000
times until the absorbance value fell within the LF standard curve. Lactoferrin concentration in milk samples was measured by ELISA using the Human LF ELISA kit (Bethyl Laboratories, Inc., Montgomery, TX, USA). Because milk samples generated values higher than the highest standard, we diluted colostrum 300,000 times and mature milk 100,000 times. Bacterial DNA isolation from milk samples A fraction of the breast milk samples (1 ml) was centrifuged at 18,4009g for 30 min at 4 °C and the upper fat phase and the supernatant were removed. After three washes with PBS, the pellet was resuspended in 180 ll of 20 mM Tris–HCl pH 8.0, 2 mM sodium EDTA, 1.2 % Triton X-100 added with 20 mg/ml lysozyme (Sigma-Aldrich, USA), 200 IU/ ml penicillin and 200 lg/ml streptomycin (PAA, Pasching, Austria). After 2 h incubation at 37 °C, a volume of glass beads roughly similar to the pellet (150–212 lm Sigma-Aldrich, USA), 25 ll of proteinase K and 200 ll of AL buffer were added and samples were vortexed for 5 min. DNA was extracted using the QIAGEN DNeasy Blood and Tissue kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. DNA was eluted in 200 ll of AE buffer (provided in the kit) and stored at -20 °C. Bacterial DNA isolation from fecal samples Bacterial DNA from fecal samples was extracted using the QIAamp DNA Stool Kit (Qiagen, Hilden, Germany). Approximately 200 mg of feces were cut from frozen samples using sterile disposable scalpel, resuspended in 1.4 ml of ASL lysis buffer from the stool mini kit, added with glass beads (150–212 lm Sigma-Aldrich, USA) and homogenized thoroughly. The suspension was incubated at 95 °C for 5 min and DNA was purified according to the manufacturer’s instructions. Purified DNA aliquots were stored at -20 °C until analysis. Real-time PCR assays Real-time PCR was used to quantify bifidobacteria and lactobacilli using genus-specific primers as described by Matsuki et al. (2004) and Heilig et al. (2002), respectively. PCR amplification and detection
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were performed on optical-grade 96-well plates using the Applied Biosystems 7500 Real-Time PCR System. The reaction mixture (25 ll) was composed of Power SYBR GREEN PCR Master Mix (Applied Biosystems, Foster City, Calif.), 200 nM of each primer for Bifidobacterium genus and 900 nM for Lactobacillus genus and 2.5 ll of template DNA. The fluorescent products were detected at the last step of each of 40 cycles. A melting curve analysis was made after amplification to distinguish the targeted PCR product from the non targeted PCR products. Standard curves were created using serial ten-fold dilutions of bacterial DNA extracted from pure cultures in mid-logarithmic growth phase of Lactobacillus brevis and Bifidobacterium breve. Microorganisms, from which the DNA was extracted, were quantified by plating serial tenfold dilutions on MRS-agar. All samples were analysed in duplicate in two independent real-time PCR assays. Statistical analysis Statistical analysis was performed using SAS v. 9.3. Shapiro–Wilk test was used to verify the normality of distribution of continuous variables. When data were not normally distributed, logarithmic transformation was performed. The paired t test was used when data were normally distributed, while Wilcoxon test was used for not normally distributed data. A p value of \0.05 was considered to be statistically significant. Pearson (normal distribution) and Spearman (non normal distribution) correlation analysis were used to assess the correlations between the continuous parameters. Bivariate model of linear regression analysis was performed to identify the possible role of breast milk or feces LF as an independent predictor of breast milk and feces bifidobacteria and lactobacilli content at birth and 30 days after delivery.
Results Study population Descriptive data of the population under investigation are shown in Table 1. Causes of pre-term delivery were pre-eclampsia (50 %), placenta previa (14.3 %),
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maternal hypertension (14.3 %), others (21.4 %). In the pre-term group 21 % of births occurred at less than 28 weeks (extreme prematurity), 50 % at 28 to \32 weeks (severe prematurity), 29 % at 32 to \37 weeks (moderate to late prematurity). There was a significant difference between the two groups in particular in the proportion of infants born by vaginal delivery. Breast milk LF levels Milk LF concentration decreased during the first month of lactation in both full-term and pre-term groups (Fig. 1a). A significant decrease of LF concentration between colostrum (mean 7.0 ± 5.1 mg/ml) and mature milk (mean 2.3 ± 0.8 mg/ml) was observed in full-term group (p = 0.001). In the pre-term group, LF levels in colostrum (mean 7.3 ± 3.2 mg/ml) and mature milk (mean 2.3 ± 0.4 mg/ml) were similar to those observed in full-term group; however there was no statistically significant difference in the pre-term group longitudinally, probably due to the low number of samples. Milk concentrations of LF between the preterm and full-term groups were not significantly different. A linear positive correlation between colostrum and mature milk LF concentration was observed in full-
Table 1 Demographic data and clinical characteristics of the infants in the study population Characteristics
Full-term
Pre-term
No. of infants
34
14
Male sex, n (%)
20 (58.8)
9 (64.3)
Gestational age, mean (SD) [range], wk
39.0 (1.1) [36–41]
30.5 (3.4) [26–36]
Vaginal delivery, n (%)
23 (67.6)
1 (7.1)
Birth weight, mean (SD) [range], g
3,415 (493.5) [2,760–4,730]
1,402.5 (666) [725–3,050]
One-minute Apgar score, mean (SD)
8.8 (0.56)
6.2 (1.7)
Five-minute Apgar score, mean (SD)
10 (0)
8.6 (1.4)
Exclusive breast feeding, n (%)
31(91.2)
9 (64.3)
Exclusive formula feeding, n (%)
0
2 (14.3)
Mixed breast and formula feeding, n (%)
3 (8.8)
3 (21.4)
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Fig. 2 Levels of fecal LF in full-term and pre-term infants. The black bars represent the median
Fig. 1 a Lactoferrin concentrations in human colostrum (C) and mature milk of full-term and pre-term groups. The black bars represent the median. b Linear positive correlation between C and mature milk (MM) LF concentration in full-term group (R = 0.49, p = 0.008)
term group (R = 0.49, p = 0.008, Fig. 1b) and this was confirmed in bivariate model linear regression analysis (b ± SE = 1,617.3 ± 564.8, p = 0.0082; R2 = 0.24). No correlation between colostrum and mature milk LF concentration was observed in preterm group. Fecal LF levels Fecal LF concentration of healthy infants was extremely high both in term and pre-term infants (Fig. 2). Fecal LF level increased from birth to 1 month age in neonates. Mean LF concentrations
Fig. 3 Linear positive correlation between fecal LF of 1 month old infants and MM LF concentration in full-term group (R = 0.44, p = 0.030)
were significantly higher (p = 0.0001) in 1 month-old healthy term infants (3,052 ± 4,323 lg/ml) in comparison to 3-days old (994 ± 1,828 lg/ml). In preterm infants higher mean concentrations of fecal LF at birth (1,631 ± 2,206 lg/ml) and 30 days after delivery (7,633 ± 9,960 lg/ml) were observed in comparison to full-term infants, however, as for breast milk LF, there was no statistically significant difference in the pre-term group longitudinally. At birth fecal LF levels in pre-term newborns was not correlated with gestational age or cause of pre-term delivery.
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compared with milk from women delivering at term (p = 0.02). Fecal neonatal beneficial microbiota
Fig. 4 Lactobacilli and bifidobacteria concentrations in colostrum (C) and MM of full-term and pre-term groups. Box and whisker plots based on log10 16S rRNA gene copies per ml milk obtained from women who delivered at term (open square) and prematurely (filed square). The horizontal line in the middle of each box represents the median, while the top and bottom borders of the box represent the 75 and 25 % percentiles, respectively. The outliers are represented as individual points outside the boxes. In MM of women who delivered prematurely concentrations of lactobacilli were significantly greater compared with milk from women delivering at term *(p = 0.02)
The amount of LF in the feces of 30 day-old term infants was significantly associated with maternal mature milk LF concentration (R = 0.44, p = 0.030, Fig. 3) as confirmed by bivariate model linear regression analysis (b ± SE = 5 9 10-4 ± 2.0 9 10-4, p = 0.03; R2 = 0.20). Breast milk beneficial microbiota Using a qPCR approach, lactobacilli were detected in all the breast milk samples (Fig. 4), whereas all but one sample had bifidobacteria: a colostrum sample obtained from a woman in the pre-term group. The mean concentration of lactobacilli in colostrum of women who delivered at term did not show significant difference in comparison to mature milk (4.3 ± 16 9 102 vs 1.8 ± 6.9 9 103 cells/ml). Similar behaviour was observed for bifidobacteria amount (2.7 ± 6.2 9 103 vs 2.6 ± 2.9 9 103 cells/ml). Also in women delivering prematurely, similar amounts of lactobacilli or bifidobacteria were detected in colostrum and mature milk (lactobacilli, 2.1 ± 5.1 9 103 vs 1.1 ± 0.9 9 103 cells/ml and bifidobacteria, 1.2 ± 2.1 9 104 vs 2.4 ± 2.9 9 103 cells/ml). In mature milk of women who delivered prematurely concentrations of lactobacilli were significantly higher
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Lactobacilli were absent in two fecal samples collected at birth, one from term and one from pre-term group, whereas bifidobacteria were not detected only in a sample of meconium from term group. The amount of fecal lactobacilli and bifidobacteria both in full-term and pre-term infants significantly increased from birth to 1 month of age (Fig. 5a). Levels of fecal bifidobacteria at birth and 1 month of age of term neonates (mean 1.4 ± 7.3 9 107 and 1.2 ± 2.5 9 108 cells/g feces, respectively) were not significantly different compared with the corresponding data of pre-term neonates (mean 1.8 ± 5.0 9 106 and 3.3 ± 6.9 9 107 cells/g feces, respectively). However, the amounts of fecal lactobacilli at one month of age in pre-term infants (mean 2.5 ± 4.9 9 105 cells/g feces) were significantly lower compared with values from term infants (mean 1.2 ± 2.4 9 106 cells/g feces) (p = 0.0004). Of note, in term group fecal concentration of lactobacilli and bifidobacteria at birth and 1 month of age did not show significant differences according to the mode of delivery. There was a positive relationship between milk and infant feces bifidobacteria levels at birth in full-term group (R = 0.39, p = 0.035). There were no significant relationships detected between the concentration of LF in breast milk and feces of term infants with milk or feces microbiota. However, as illustrated in Fig. 5b, c, fecal levels of lactobacilli and bifidobacteria correlated directly with the concentration of fecal LF at birth in pre-term infants (R = 0.64, p = 0.026 and R = 0.70, p = 0.017, respectively), as confirmed by bivariate model linear regression analysis (b ± SE = 0.44 ± 0.17, p = 0.26; R2 = 0.40 and b ± SE = 0.59 ± 0.20, p = 0.017; R2 = 0.49). A linear positive correlation between lactobacilli and bifidobacteria levels in feces of pre-term infants was observed at birth (R = 0.78, p = 0.008, Fig. 5d), as confirmed by bivariate model linear regression analysis (b ± SE = 0.93 ± 0.27, p = 0.0081; R2 = 0.60).
Discussion Lactoferrin is present in breast milk, with particularly high concentrations in colostrum. During breastfeeding
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Fig. 5 a Fecal lactobacilli and bifidobacteria concentrations in full-term and pre-term infants. Box and whisker plots based on log10 16S rRNA gene copies per g of feces obtained from fullterm (filled square) and pre-term infants (open square). The horizontal line in the middle of each box represents the median, while the top and bottom borders of the box represent the 75 and 25 % percentiles, respectively. The outliers are represented as individual points outside the boxes. The amounts of fecal lactobacilli at 1 month of age in pre-term infants were
significantly lower compared with values from term infants *(p = 0.0004). b Linear positive correlation between concentration of fecal LF and fecal levels of lactobacilli at birth in preterm infants (R = 0.64, p = 0.026). c Linear positive correlation between concentration of fecal LF and fecal levels of bifidobacteria at birth in pre-term infants (R = 0.70, p = 0.017). d Linear positive correlation between lactobacilli and bifidobacteria levels in feces of pre-term infants at birth (R = 0.78, p = 0.008)
high amounts of LF are transferred to the intestine of the newborn. We performed a study on the correlation of LF levels in breast milk and feces of neonates in a cohort of 48 mother–infant pairs (34 term infants and 14 pre-term infants). To our knowledge, this is the first study which shows that concentration of LF in the feces of breastfed neonates at 1 month of age is significantly associated with LF levels in breast milk, confirming that breast milk represents the main source of LF found in the gut of infants.
Fecal LF concentration of healthy neonates was extremely high both in term and pre-term infants, particularly in infants 1 month of age (mean 3,052 ± 4,323 lg/ml and 7,633 ± 9,960 lg/ml, respectively) much greater than the mean amount reported in healthy children (1.17 ± 0.47 lg/ml) and in children with inflammatory bowel disease (1,880 ± 565 lg/ml) (Walker et al. 2007). The concentration of fecal LF increases in newborns over time from birth to 1 month of age, whereas breast milk LF decreases longitudinally. It
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must be considered, however, that a baby, on the basis of the different amount of milk that consumes daily during the first month of life, ingests in this period by feeding the same daily amount of LF, i.e. approximately 2 grams per day. Therefore, it is not surprising that the amount of LF in the gut of breastfed infants at 1 month of age is closely related to that found in the breast milk of their respective mothers. The LF level we found in fecal samples of neonates at birth was not correlated with the amount of LF present in colostrum. Most of the fecal samples collected at birth are represented by the first meconium, thus the content of LF in these samples in part reflects the LF acquired during the prenatal period. Indeed, during pregnancy, the fetus swallows amniotic fluid that contains LF (Otsuki et al. 1999) which passes through the gastrointestinal tract. It is worthy to note (Fig. 1a) that the distribution of milk LF values was clustered in mature milk of women from both term and pre-term groups, whereas values were more dispersed in colostrum samples, suggesting inter-individual variability probably related to clinical events that occur during late pregnancy or at birth, as well as genetic characteristics. Indeed, the positive correlation between colostrum and mature milk LF concentrations (Fig. 1b) seems to indicate that milk LF levels also reflect individual characteristics. Our data did not show an association between breast milk’s and infant feces’s LF concentration and milk or feces healthy microbiota in term group, while in pre-term group we observed a positive correlation between LF values and bifidobacteria and lactobacilli amounts in the feces of newborns at birth. In pre-term infants median fecal LF concentrations at birth were higher in comparison to term infants (202 vs 92 lg/ ml), therefore it is possible to hypothesize that higher amounts of LF are transferred from the mother to the fetus through the amniotic fluid, and this fact could be responsible for the association. To our knowledge, only one study evaluated LF concentration in amniotic fluid at different stages of pregnancy (Niemela¨ et al. 1989). No detectable concentration of LF was found in amniotic fluid before week 20 of pregnancy. A significant increase in the LF concentration was observed around week 30 and it remained high until term. Our results demonstrated no association between levels of fecal LF at birth and gestational age. Indeed, 4 out of 8 very premature newborns (gestational age 26–30 weeks) showed high fecal LF concentration (1,260–3,580 lg/ml), while the others had lower
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levels (16–171 lg/ml) (data not shown). On the basis of the results reported here, it would be interesting to verify the LF concentrations in the amniotic fluid of women who deliver pre-term in a larger clinical trial to evaluate the potential role of LF on the establishment of the initial intestinal microbiota of pre-term infants. Indeed, a recent study demonstrated that administration to pre-term infants of bovine LF in combination with Lactobacillus rhamnosus GG reduced the incidence of late-onset sepsis, a common and severe complication in premature neonates (Manzoni et al. 2009). Interestingly, we observed a significant reduction in the amount of fecal lactobacilli in pre-term infants at 1 month of age in comparison to term infants and the difference, although not significant, was already present at birth, indicating that the shift in bacterial composition had a long-term effect. It should be noted that ten of the 14 preterm infants with a birth weight \1,500 g enrolled in our study were treated with parenteral antibiotics in the first week of life and all pre-term infants, except one, were born by cesarean section. Both factors may be responsible for the observed reduction in the amount of fecal lactobacilli. Normal vaginal delivery exposes the infant to the vaginal microbiota of the mother and the mode of delivery is known to influence the neonatal gut microbiota composition (Dominguez-Bello et al. 2010; Huurre et al. 2008). The low amount of fecal lactobacilli we observed in pre-term infants, suggests that administration of Lactobacillus containing probiotic products could restore a healthy intestinal microbiota in pre-term newborns, thus reducing the risk of late-onset sepsis. Nevertheless, due to the fact that only thirteen fecal samples from pre-term newborns were analyzed in our study, we should apply caution in drawing conclusions as regards to the potential role of LF in the establishment of the pre-term infant intestinal microbiota at birth. As the initial intestinal microbiota in the neonate may have a role in the prevention of development of neonatal sepsis and necrotizing enterocolitis (Mai et al. 2013; Jakaitis and Denning 2014; Torrazza et al. 2013), it will be extremely important to confirm, through a study involving a larger number of mother-infant pairs, the correlation between the levels of LF and the healthy microbiota in the feces of infants at birth, as well as LF concentrations in the amniotic fluid. It seems that nature has provided the ability to maintain high levels of LF in the gut of the infant. In
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the period from the first days of life until the age of 1 month similar daily amounts of LF are taken in by the infant through breast milk. The presence of such high levels of LF suggests that this protein plays a crucial role in neonatal life. It has been suggested that breast milk could deliver beneficial microorganisms in the gut of neonates in a tolerogenic milieu for the presence of bioactive molecules that could substantially modulate interactions between the host immune system and the developing gut microbiota (Rautava et al. 2012). We hypothesize that LF thanks to its antimicrobial, immunostimulatory and immunomodulatory effects could represent an important component in this network of bioactive compounds central to the infant’s intestinal and immune development.
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