Journal of Perinatology (2014), 1–7 © 2014 Nature America, Inc. All rights reserved 0743-8346/14 www.nature.com/jp

ORIGINAL ARTICLE

Impact of lactation stage, gestational age and mode of delivery on breast milk microbiota P Khodayar-Pardo1, L Mira-Pascual2, MC Collado2 and C Martínez-Costa1 OBJECTIVE: There is an increasing evidence of the immunological role of breast milk (BM) microbiota on infant health. This study aims to analyze several determining factors of milk microbiota. STUDY DESIGN: A total of 96 milk samples from 32 healthy mothers (19 preterm vs 13 at term gestations; and 15 vaginal deliveries vs 17 Cesarean sections) were longitudinally collected. Microbiota composition was studied by quantitative PCR and the influence of lactation stage, gestational age and delivery mode was evaluated. RESULT: Globally, Lactobacillus, Streptococcus and Enterococcus spp. were the predominant bacterial groups. Total bacteria, Bifidobacterium and Enterococcus spp. counts increased throughout the lactation period. At all lactation stages, Bifidobacterium spp. concentration was significantly higher in milk samples from at term gestations than in preterm gestations. Higher bacterial concentrations in colostrum and transitional milk were found in Cesarean sections. Nevertheless, Bifidobacterium was detected more frequently in vaginal than in Cesarean deliveries. CONCLUSION: Lactation stage, gestational age and delivery mode all influence the composition of several bacteria inhabiting BM: Bifidobacterium, Lactobacillus, Staphylococcus, Streptococcus and Enterococcus spp., and, consequently, may affect the infant’s early intestinal colonization. Journal of Perinatology advance online publication, 27 March 2014; doi:10.1038/jp.2014.47

INTRODUCTION Breast milk (BM) is considered the optimal source of nutrients and an unmatched supply of essential protective substances for the infant. There is currently an increasing interest on the bioactive components that elicit the protective capacity of BM during the first months of life (immunoglobulin A, lactoferrin, lysozyme, mucine, lactadherin, anti-inflammatory and antioxidant components, oligosaccharides, glycoconjugates and microbial factors).1,2 These benefits are especially relevant in susceptible infants such as low birth weight and/or preterm infants, in whom, compared with those fed with formula, a lower incidence of sepsis and necrotizing enterocolitis has been reported in the context of breastfeeding.3–5 Maternal microbiota is one of the relevant factors in the development of the infant’s immune system, as it represents the infant’s first contact with microorganisms. The mode of delivery has been considered the initial contribution to infant microbiota acquisition,6 although recent studies have reported an earlier microbial contact as bacteria has been detected in amniotic fluid, umbilical cord blood and meconium from neonates born either by vaginal delivery or Cesarean section.7 Presently, it is assumed that BM is the main postnatal source of bacteria for the infant’s intestine, and hence has an important role in microbiota colonization during the first months of life.8 Indeed, there are great differences in fecal microbiota among exclusively breastfed and formula-fed infants.8,9 Furthermore, variations in the composition of the characteristic intestinal microbiota of a healthy breastfed infant have been associated to an increased risk for

immune and inflammatory conditions such as allergic disorders10 and, more recently reported, celiac disease, overweight and obesity.11,12 The composition of BM may vary remarkably even within the same individual, but responsible regulating factors have not yet been thoroughly defined. It has been suggested that maternal lifestyle, dietary habits, nutritional and immunological status, and lactation time influence BM microbiota.13,14 Moreover, specific microbial shifts have been recently linked to maternal body mass index, weight gain and mode of delivery.15 In this context, the aim of our study was to evaluate the influence of specific perinatal factors such as lactation stage, gestational age and mode of delivery on the human milk microbiota. METHODS Subjects and design The study was performed with the collaboration of 32 mothers recruited immediately after delivery at the Maternity Ward of the Hospital Clínico Universitario of Valencia (Valencia, Spain) during a period of time between December 2006 and December 2009. Inclusion criteria were based on early breastfeeding practices, no metabolic or chronic diseases and no probiotic consumption. Clinical data were recorded, including maternal age, parity, gestational age, mode of delivery, antibiotic administration, weight gain during pregnancy, infant anthropometric measurements and clinical assessment (after birth and throughout the subsequent visits). A prospective longitudinal collection of BM samples was performed. The study recruited 13 mothers with gestations at term (GAT) and 19 mothers with preterm gestations. The GAT group comprised infants born at or after

1 Pediatric Gastroenterology and Nutrition Section, Department of Pediatrics, University of Valencia, Hospital Clínico Universitario de Valencia, Valencia, Spain and 2Department of Biotechnology, Institute of Agrochemistry and Food Technology-Spanish National Research Council (IATA-CSIC), Valencia, Spain. Correspondence: Professor C Martínez-Costa, Pediatric Gastroenterology and Nutrition Section, Department of Pediatrics, University of Valencia, Hospital Clínico Universitario de Valencia, Blasco Ibáñez Av., 17, 46010, Valencia, Spain. E-mail: [email protected] Received 18 October 2013; revised 6 February 2014; accepted 14 February 2014

Determining factors of breast milk microbiota P Khodayar-Pardo et al

2 37 weeks of gestation, while the preterm group included neonates born alive before 37 weeks of gestation. The preterm group was divided into the following subcategories according to gestational age: extremely preterm (o28 weeks), moderately preterm ( ⩾28 to o32 weeks) and late preterm ( ⩾32 to o 37 weeks). Samples were collected within the first month of exclusive breastfeeding. BM samples were taken following the three different stages of lactation, obtaining a total of 96 samples: colostrum (1st to 5th day), transitional (6th to 15th day) and mature milk (16th day onwards). Mature milk samples were collected on day 17 (15 to 19) for mothers delivering term and day 18 (16 to 20) for mothers delivering preterm, median (interquartile range). The study protocol was approved by the Hospital’s Ethics Committee and fully complied with the code of ethics of the World Medical Association (Declaration of Helsinki). Recruited mothers received detailed written information of the study and gave their signed informed consent to participate.

BM samples Mothers were received at a time frame between 0900 and 1000 hours at the hospital. Before sample collection, mothers were given written instructions for standardization purposes. After washing their hands with soap and cleaning their nipples with a clorhexidine swab in order to minimize milk contamination, they were asked to use the BM pump to suction milk from the breast opposite to that from which their babies had previously suckled. BM was collected with a sterile automatic BM pump with a vacuum regulator (Medela Symphony, Barr, Switzerland), polystyrene suction funnels and screw-top bottles adapted to suction funnels for the direct milk collection. Bottles and suction funnels were autoclaved before their use. BM samples suctioned with the pump were collected in bottles and immediately aliquoted under sterile conditions. Subsequently, they were frozen and stored at –20 °C for ensuing analysis.

DNA extraction and quantitative PCR BM samples were thawed and centrifuged at 10 000 g for 10 min in order to separate cells and fat from whey. Thereafter, total DNA was isolated from the pellets using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) following the manufacturer's instructions. PCR primers were targeted to total bacteria count and to Bifidobacterium, Lactobacillus, Enterococcus, Staphylococcus and Streptococcus groups (Supplementary Table S1). The quantitative PCRs were conducted as previously described.16 The quantitative PCR amplification and detection were performed by means of the LightCycler 480 Real-Time PCR System (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany). Each reaction mixture of 10 μl was composed of SYBR Green PCR Master Mix (Roche), 0.5 μl of each of the specific primers at a concentration of 0.25 μM and 1 μl of template DNA. Fluorescent products were detected at the last step of each cycle. A melting curve analysis was performed after amplification in order to distinguish the targeted PCR products from the non-targeted. Bacterial concentration in each sample was calculated comparing the Ct (cycle threshold) values obtained from standard curves. These were created using serial 10-fold dilution of pure culture-specific DNA fragments corresponding to 10 to 109 specific fragment copies per ml.

Statistical analysis Statistical analysis was executed with the SPSS 17.0 software package (SPSS, Chicago, IL, USA). Owing to the non-normal distribution of the microbial data, results were expressed in terms of medians with interquartile ranges and non-parametric tests were performed. Friedman's test was used to compare microbial groups throughout time (paired samples). Mann–Whitney U-test was used for comparisons between two groups. Comparisons between more than two groups of infants were performed with the Kruskal–Wallis test, and statistical differences were corrected for a multiple comparison test using the Bonferroni correction. The χ2 test was applied to investigate the differences in bacterial prevalence between the studied groups. A P-value o0.05 was considered statistically significant. Spearman rank test allowed the study of the correlation between variables, and significance was established at a coefficient of 0.5%. Journal of Perinatology (2014), 1 – 7

Table 1. Clinical characteristics of the mothers and their infants (n = 32 mother–infant pairs) At term (n = 13) Preterm (n = 19) Maternal age (years) Parity (%) 1 2 >2

32.4 (29.2–34.6)

28 (23–32.7)

4 (30.8%) 7 (53.8%) 2 (15.4%)

5 (26.3%) 9 (47.4%) 5 (26.3%)

Length of pregnancy (weeks) Mode of delivery Vaginal (%) Elective Cesarean section (%) Non-elective Cesarean section (%)

39.3 (38.2–40.4)

29 (27–31.5)

6 (46.2%) 4 (30.8%) 3 (23.1%)

9 (47.4%) 1 (5.2%) 9 (47.4%)

Maternal antibiotic treatment Before deliverya During deliveryb After delivery No antibiotic treatment Maternal weight (kg)c Maternal height (cm)c Maternal weight (kg) gaina Infant gender (male:female) Birth weight (kg) Birth length (cm) Head circumference (cm)

3 3 0 7

(23.1%) (23.1%) (0%) (53.8%)

6 10 0 4

(30%) (50%) (0%) (20%)

74.8 (69.4–87.8) 66.2 (60.3–77.4) 161 (157–168) 161 (156–169) 13 (10.4–17.5) 8 (5–11) 5:8 16:3 3.22 (2.74–3.23) 1.14 (0.76–1.52) 49.5 (48–50.6) 39 (34.8–39.5) 35 (33.5–35) 26 (24–28.3)

Data are shown as median and interquartile range, or percentage. a During pregnancy until 48 h before delivery. b During the 48 h before delivery and in labor, because of premature rupture of membranes. c Before pregnancy.

RESULTS Clinical characteristics of mothers and infants are shown in Table 1. All mothers were in a healthy condition before delivery. BM microbiota composition according to lactation stage Significant differences between colostrum, transitional and mature milk were detected in terms of total bacteria concentration (P-value = 0.0001), Bifidobacterium spp. (P-value = 0.001) and Enterococcus spp. (P-value = 0.043) counts (Figure 1). Globally, total bacteria, Bifidobacterium spp. and Enterococcus spp. counts increased throughout the lactation period. Total bacteria concentration was significantly lower in colostrum than in transitional (P-value = 0.001) and mature milk (P-value = 0.001). Similarly, Bifidobacterium spp. content was significantly lower in colostrum when compared with mature milk (P-value = 0.002), and in transitional milk when compared with mature milk (P-value = 0.001). The Enterococcus spp. concentration was systematically lower in colostrum than in other stages of milk secretion (P-value = 0.030 in transitional and mature milk). Regarding total bacteria count, a positive correlation was found between colostrum and transitional milk (Rho = 0.715, P-value = 0.0001), and between transitional and mature milk (Rho = 0.662, P-value = 0.001). In particular, Bifidobacterium spp. counts exhibited a positive correlation between colostrum and transitional milk (Rho = 0.763, P-value = 0.0001). The same happened with Lactobacillus spp. counts (Rho = 0.545, P-value = 0.002). Staphylococcus spp. counts showed a positive correlation from colostrum to transitional (Rho = 0.562, P-value = 0.007) and mature milk samples (Rho = 0.526, P-value = 0.010). Finally, higher Enterococcus counts in colostrum were related to higher counts in transitional © 2014 Nature America, Inc.

Determining factors of breast milk microbiota P Khodayar-Pardo et al

Mature

p-value=0.503

Colostrum Log nº Streptococcus group copies/ml

Transitional

Log nº Staphylococcus group copies/ml

Log nº Lactobacillus group copies/ml

Colostrum

Transitional

Mature

p-value=0.420

Colostrum

Log nº Bifidobacterium group copies/ml

p-value=0.0001

Transitional

Mature

p-value=0.001

Colostrum

Transitional

Mature

Transitional

Mature

Transitional

Mature

p-value=0.422

Colostrum Log nº Enterococcus group copies/ml

Log nº Total bacteria copies/ml

3

p-value=0.043

Colostrum

Figure 1. Microbiota composition of colostrum, transitional and mature milk, analyzed by quantitative PCR. P-values were calculated by Friedman’s Test.

(Rho = 0.557, P-value = 0.002) and mature milk (Rho = 0.612, P-value = 0.0001).

gestational age, but these differences did not reach statistical significance (Table 3).

BM microbiota composition according to gestational age Significant differences in BM microbiota composition were found between GAT and preterm groups throughout the different lactation stages (Table 2). The Enterococcus spp. count was lower in the GAT group, but statistical significance was only reached in colostrum (P-value = 0.045). Bifidobacterium spp. counts were significantly higher in GAT than in preterm gestations at all stages of lactation (P-value: colostrum = 0.003, transitional = 0.005, mature milk = 0.014). A direct correlation was found between Bifidobacterium spp. content and gestational age in colostrum (Rho = 0.453, P-value = 0.039), transitional (Rho = 0.483, P-value = 0.011) and mature milk (Rho = 0.470, P-value = 0.012). No correlation was found between gestational age and Lactobacillus, Staphylococcus or Streptococcus spp. concentration. In order to analyze the impact of different degrees of prematurity on BM microbiota composition, mothers were classified according to three subgroups: extremely preterm (n = 7), moderately preterm (n = 7) and late preterm (n = 5). Regarding the analysis of different stages of prematurity, a progressive increase of microbial concentration was observed with increasing

BM microbiota composition in the GAT group according to mode of delivery Higher total bacteria concentrations were found in Cesarean versus vaginal deliveries in colostrum (P-value = 0.033) and transitional milk (P-value = 0.019), whereas similar concentrations were detected in mature milk samples with both modes of delivery (Table 4). Similarly, higher Streptococcus spp. counts were detected in transitional milk (P-value = 0.014) in the context of Cesarean section compared with vaginal delivery. However, colostrum samples were more frequently Streptococcus spp. positive in the vaginal deliveries (P-value = 0.026). The Bifidobacterium group was also more commonly detected in vaginal than in Cesarean section deliveries in colostrum (P-value = 0.026) and in transitional milk (P-value >0.05).

© 2014 Nature America, Inc.

DISCUSSION In addition to providing nutritional support, BM is a fundamental source of bioactive components to infants that directly and indirectly contribute to enhance the intestinal mucosal barrier function and promote the immune development and maturation. Journal of Perinatology (2014), 1 – 7

Determining factors of breast milk microbiota P Khodayar-Pardo et al

4 Table 2.

Microbiota composition of breast milk samples of at term and preterm deliveries by qPCR Log bacterial group (gene copies ml–1) Pr

At term (13)

Mann–Whitney U-test

Pr

Preterm (n = 19)

P-value

Colostrum Total bacteria Bifidobacterium spp. Lactobacillus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

13 9 10 10 9† 13

4.34 2.00 4.33 3.00 3.52 3.50

(4.00–4.58) (1.95–2.10) (4.20–4.60) (2.83–3.08) (2.56–3.80) (2.90–3.90)

19 12 15 13 6 19

4.45 1.85 4.15 3.09 3.30 4.00

(4.04–5.04) (1.55–2.00) (3.90–4.58) (2.76–4.25) (2.50–3.98) (3.58–4.22)

0.308 0.003 0.330 0.208 0.867 0.045

Transition milk Total bacteria Bifidobacterium spp. Lactobacillus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

13 10 13 11 10 13

4.77 2.07 4.32 3.30 3.65 3.96

(4.00–5.55) (1.85–2.25) (4.17–4.45) (3.04–3.38) (3.40–3.80) (3.36–4.20)

19 17 19 14 13 19

4.72 1.65 4.31 3.43 3.71 3.98

(4.11–6.60) (1.40–2.00) (4.08–4.56) (3.00–3.89) (3.07–3.90) (3.70–4.20)

0.335 0.005 0.963 0.412 0.832 0.875

Mature milk Total bacteria Bifidobacterium spp. Lactobacillus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

13 13 13 13 10 13

5.37 2.45 4.31 3.28 3.58 3.95

(4.73–6.06) (2.02–2.60) (4.13–4.42) (3.05–4.00) (3.23–3.88) (3.28–4.34)

19 19 19 19 15 19

5.00 2.00 4.38 3.28 3.42 4.07

(4.01–7.30) (1.90–2.20) (4.08–4.56) (3.00–3.75) (3.35–3.95) (3.75–4.25)

0.769 0.014 0.701 0.520 0.925 0.982

Abbreviations: Pr, prevalence; qPCR, quantitative PCR. Data are shown as median and interquartile range. Statistical analysis was calculated by Mann–Whitney U-test. P-value o0.05 was considered significant. For Pr study (positive samples), statistical analysis was calculated by χ2 test. Significant differences are highlighted in bold.

Table 3.

Microbiota composition of breast milk samples of preterm (n = 19) deliveries according to the degree of prematurity Log bacterial group (gene copies ml–1)

Kruskall-Wallis test

Pr

Extremely preterm (7)

Pr

Moderately preterm (7)

Pr

Late preterm (5)

P-value

Colostrum Total bacteria Bifidobacterium Lactobacillus Staphylococcus Streptococcus Enterococcus

7 3 7 6 2 7

4.47 (3.40–5.08) — 4.12 (3.90–4.37) 3.09 (2.97–4.12) — 3.57 (3.04–3.80)

7 5 7 4 2 7

4.56 (4.34–4.94) 1.86 (1.35–2.00) 4.24 (2.95–4.35) — 4.18 (4.00–4.23)

5 4 5 3 2 5

4.47 (3.36–5.70) 1.80 (1.40–2.00) 4.56 (3.98–4.61) — — 4.09 (3.30–4.44)

0.802 0.382 0.563 0.431 — 0.060

Transition milk Total bacteria Bifidobacterium Lactobacillus Staphylococcus Streptococcus Enterococcus

7 6 7 6 4 7

4.72 1.55 4.31 3.65 3.65 3.76

(3.74–6.92) (1.30–2.01) (4.04–4.46) (2.88–4.10) (3.09–3.95) (3.65–4.10)

7 6 7 4 4 7

4.70 1.62 4.20 3.30 3.60 3.97

(4.42–6.80) (1.35–1.75) (3.80–4.72) (2.85–3.78) (2.57–3.86) (3.82–4.20)

5 5 5 4 5 5

5.55 1.85 4.44 3.46 3.71 4.02

(3.76–7.60) (1.30–2.00) (4.08–4.60) (3.00–3.58) (2.56–3.98) (3.75–4.38)

0.917 0.878 0.842 0.700 0.808 0.275

Mature milk Total bacteria Bifidobacterium Lactobacillus Staphylococcus Streptococcus Enterococcus

7 7 6 7 5 7

5.21 1.95 4.23 3.62 3.55 3.88

(4.01–7.16) (1.88–2.30) (4.08–4.38) (3.07–3.80) (2.88–3.91) (3.69–4.20)

7 7 7 7 5 7

4.64 2.00 4.50 3.44 3.40 4.11

(4.47–7.20) (1.55–2.20) (4.10–4.82) (3.00–4.43) (3.35–4.00) (3.82–4.18)

5 5 5 5 5 5

6.00 2.11 4.53 3.10 3.42 4.06

(3.25–7.46) (2.01–2.20) (3.92–4.80) (2.50–3.35) (3.20–3.88) (3.70–4.22)

0.929 0.307 0.385 0.307 0.970 0.475

Abbreviation: Pr, prevalence. Data are shown as median and interquartile range. Statistical analysis was calculated by Kruskall–Wallis test, and statistical differences were corrected by a multiple comparison test using Bonferroni adjustment. For Pr study (positive samples), statistical analysis was calculated by χ2 test.

Journal of Perinatology (2014), 1 – 7

© 2014 Nature America, Inc.

Determining factors of breast milk microbiota P Khodayar-Pardo et al

5 Table 4.

Microbiota composition of the different stages of term group BM (n = 13) according to type of delivery Log bacterial group (gene copies ml–1) Pr

Vaginal (n = 6)

Mann–Whitney U-test

Pr

C-section (n = 7)

P-value

Colostrum Total bacteria Bifidobacterium spp. Lactobacillus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

6 6 6 4 6 6

4.00 2.00 4.45 2.90 3.46 3.30

(3.47–4.50) (1.90–2.10) (4.23–4.60) (2.45–3.98) (2.00–3.65) (2.90–3.75)

7 3† 4 6 3† 7

4.66 (4.27–4.88) — 4.27 (3.72–4.53) 3.00 (2.86–3.08) 3.86 (3.36–4.00) 3.65 (3.03–4.14)

0.033 0.174 0.522 0.670 0.197 0.394

Transition milk Total bacteria Bifidobacterium spp. Lactobacillus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

6 6 6 4 6 6

4.22 2.00 4.27 3.15 3.45 3.96

(4.00–4.70) (1.60–2.20) (4.05–4.43) (2.80–4.38) (3.37–3.63) (3.33–4.12)

7 4 7 7 4 7

5.90 2.17 4.36 3.30 3.88 4.04

(5.05–6.78) (2.05–2.25) (4.25–4.51) (3.24–3.40) (3.71–34.04) (3.23–4.20)

0.019 0.109 0.394 0.153 0.014 0.831

Mature milk Total bacteria Bifidobacterium spp. Lactobacillus spp. Staphylococcus spp. Streptococcus spp. Enterococcus spp.

6 6 6 6 6 6

5.20 2.50 4.28 3.16 3.65 3.94

(4.52–6.26) (2.03–2.80) (3.88–4.33) (3.00–3.46) (3.15–3.83) (3.18–4.56)

7 7 7 7 4 7

5.55 2.23 4.43 3.90 3.50 4.00

(5.12–6.25) (2.00–2.50) (4.22–4.53) (3.18–4.00) (3.40–3.96) (3.08–4.32)

0.394 0.286 0.088 0.197 0.655 0.670

Abbreviations: BM, breast milk; C-section, Cesarean section; Pr, prevalence. Data are shown as median and interquartile range. Statistical analysis was calculated by Mann–Whitney U-test. P-value o0.05 was considered significant. For Pr study (positive samples), statistical analysis was calculated by χ2 test. Significant differences are highlighted in bold.

Among other components, these bioactive elements present in BM include bacteria,8,15,17 although their significance and activity in relation to infant health remains poorly defined. The origin of milk microbiota is under discussion but the main hypothesis is that certain bacteria present in the maternal gut could reach the mammary gland through an endogenous pathway.18 It is likely that hormonal and physiological changes during late pregnancy and lactogenesis II period may provide the right conditions for immune cells to transport bacteria to the mesenteric lymph nodes and then, to the mammary gland. This could explain differences in microbial composition of BM from preterm and at term deliveries.18 The main bacteria detected in this study were Bifidobacterium spp., Lactobacillus spp., Staphylococcus spp., Streptococcus spp. and Enterococcus spp., as previously described using traditional culture methods and molecular techniques, such as denaturing gradient gel electrophoresis, quantitative PCR and pyrosequencing.8,19-21 These data emphasize the fact that BM constitutes the most relevant postnatal source of a wide range of bacteria for the infant gut. The presence of Bifidobacterium and Lactobacillus spp. in BM may be of utmost importance for the colonization of the infant gut, as they favor the activation of immunoglobulin Aproducing plasma cells in the human neonatal gut, and are enhanced by the fermentation of non-digestible oligosaccharides also present in BM. The levels of Bifidobacterium spp. found in the analyzed milk samples were similar to those ones previously reported.8,15,16,19 Bifidobacteria can be considered as the hallmark of the gut microbiota in healthy breastfed infants, as specific and distinctive species of Bifidobacterium spp. are present in BM.19 Among breastfed infants, Bifidobacterium spp. can reach up 60 to 90% of the total fecal microbiota compared with infants fed with formula, but other compositions are not uncommon.22 Moreover, transmission of specific intestinal Bifidobacterium spp. strains from mothers to infants have been recently reported suggesting that each mother–infant pair may have unique family-specific strains © 2014 Nature America, Inc.

and BM could contribute to the microbial transfer from the mother to the infant and, therefore, impact on the infant gut colonization.23 The longitudinal design of this study permitted an evolutional analysis of BM microbiota composition during lactation with regard to gestational age and mode of delivery. A recent study has described milk microbiota composition changes over time, from colostrum to transitional and mature milk.15 Lactobacillaes comprised the most common bacterial order in colostrum and, although they were the most abundant along lactation, typical inhabitants of the oral cavity such as Veillonella, Leptotrichia and Prevotella spp. were also detected in a significant amount. The results of this study evidenced that the influence of lactation stage was greater in Bifidobacterium and Enterococcus spp. counts, as colostrum samples displayed clear differences with transitional and mature milk, exhibiting a progressive increase of their concentration with lactation time. Moreover, Lactobacillus and Staphylococcus spp. also showed an upward trend throughout the different lactation stages. The finding that these bacteria progressively increase their concentration as lactation period progresses reinforces the belief that the microbiota composition of BM may suit the needs for the infant in each period. Significant additional findings of this study include the fact that BM microbiota composition varies according to gestational age. In this sense, the design of the study included the recruitment of mothers whose pregnancy ended prematurely from 24 weeks onward, and the samples’ categorization into three degrees of prematurity. Indeed, this report is probably the first to show that the milk of mothers who gave birth to preterm babies displays specific microbiota characteristics, and this finding may support that these microorganisms have an important biological role in preterm infants from the earliest stages of premature birth. The entire range of the bacteria groups analyzed in this study (Bifidobacterium, Lactobacillus, Staphylococcus, Streptococcus and Enterococcus spp.) was detected in milk from both at term and Journal of Perinatology (2014), 1 – 7

Determining factors of breast milk microbiota P Khodayar-Pardo et al

6 preterm deliveries. These bacteria were found even in the extremely premature group (o 28 weeks). Nevertheless, Bifidobacterium spp. had lower concentrations in the preterm group than in the GAT one. Taking into consideration that microbiota colonization has a strong potential to promote the development of the premature infant’s immune system, a delayed intestinal bacterial colonization and an immature gastrointestinal tract may significantly increase the infant’s susceptibility to severe infection.5,24,25 In this sense, necrotizing enterocolitis is one of the most devastating diseases in the neonatal period. In particular, extremely low birth weight and premature infants represent the most vulnerable population groups. Large, multicenter, neonatal network databases from the United States and Canada report a mean necrotizing enterocolitis prevalence of 7% in infants with weight below 1500 g and an estimated mortality between 15% and 30%.26 Although ultimate cause of this condition has not been ascertained, infectious, inflammatory and oxidative mechanisms have been suggested.3,27 The implication of BM in the prevention of necrotizing enterocolitis has long been recognized,28 and although the precise components that elicit this defensive capacity are barely known, the involvement of BM bacteria in the microbiota colonization of the infant and, therefore, the development and maturation of the immune system suggests a potential role in its prevention. In this setting, preterm infants who cannot receive BM directly from their mothers call for previously collected and stored milk. Refrigeration up to 96 h29–31 and frozen storage29,32 are the optimal processes for the preservation of BM’s defensive properties. Conversely, thermal processing in the form of pasteurization entails a substantial reduction in nutritional and immunological substances33 and up to 30% in bactericidal capacity.34 Although further investigation is required in order to thoroughly determine the impact of bank BM pasteurization, these findings emphasize the importance of the fact that preterm infants receive their own unpasteurized maternal milk. Regarding the biological role of BM microbiota after the neonatal period, it has been hypothesized that the paradigmatic intestinal microbiota of breastfed infants is associated to mediumand long-term benefits of metabolic, immune and inflammatory nature. In fact, it has been suggested that Bifidobacterium spp. may affect weight gain through mucosal host–microbe crosstalk.11 Recent studies found lower Bifidobacterium spp. counts in milk from overweight mothers in comparison with normal weight mothers,35 thereby indicating a relationship between obesity and microbiota dysbiosis. A similar association was described between obesity and certain immune and inflammatory conditions.36 Finally, BM has demonstrated its potential to influence neonatal microbial recognition by modulating Toll-Like-Receptors-mediated responses,37 as well as its capacity to prevent inflammatory conditions and also, obesity and diabetes.38 Thus, it would be relevant to understand the role of the specific bacterial groups present in BM samples on the infant microbiota configuration and therefore the immune system maturation. Numerous studies have addressed the differences in intestinal microbiota composition among vaginal- and Cesarean-delivered infants,6 but whether the delivery mode impacts on milk microbiota remains undefined. This study found greater total bacteria concentrations, especially Streptococcus spp., and lower counts of Bifidobacterium spp. in Cesarean sections compared with vaginal deliveries. In the last decades, industrialized countries have reported a progressive increase in immune and metabolic diseases, which spread particularly quickly among children. This may be linked to the ever-increasing rates of Cesarean section deliveries, which have long surpassed the recommended barrier of 15% (and in fact almost doubled this percentage during the last decade) especially in high-income countries such as France, Germany, Spain, the United Kingdom and the United States.39 Cesarean section may constitute a disturbance in the infant´s Journal of Perinatology (2014), 1 – 7

microbiota colonization and interfere with the establishment of a balanced immune response. Therefore, differences in BM microbiota in children born by Cesarean section may contribute to the heightened risk for immune and inflammatory conditions.40,41 Regarding the limitations of this study, it should be noted that the sample size is limited. However, the fundamental contribution of this study is its longitudinal design, as longitudinal collection of milk samples is not easy to conduct. In addition, results of preterm milk samples (from 24 weeks onward) are also outstanding. In conclusion, the findings of this study contribute to further characterize the early factors that influence the microbial composition of BM. In particular, lactation stage, gestational age and mode of delivery seem to influence several bacterial groups of BM, and, consequently, may affect the infant’s early intestinal microbiota colonization. These results may help to promote new therapeutic strategies in order to acquire an adequate colonization, especially in those cases where microbial exposure is suboptimal. However, further studies are necessary in order to fully define the biological effects of these microorganisms on the infant’s health. CONFLICT OF INTEREST The author declares no conflict of interest.

ACKNOWLEDGEMENTS The study has ethical adherence in all aspects and received no funding.

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Journal of Perinatology (2014), 1 – 7