What Is Known
- Infants born via cesarean-section present altered gut microbiota colonization and an associated risk of immune-related disease.
- Emerging evidence suggests that probiotic bacteria could reduce immune-related disease risk in cesarean-section infants.
- The effect of probiotic interventions on cesarean-section microbiota colonization is poorly understood.
What Is New
- Feeding with formula containing Lactobacillus reuteri DSM 17938 appears to specifically increase Lactobacillus abundance in vaginally born infants and to strongly modulate the microbiota in cesarean-section babies, by inducing a shift of global profile and taxa abundance toward the vaginal delivery composition.
Many infants receive probiotic bacteria either in infant formula or as dietary supplements during their first months of life. Health benefits associated with the administration of probiotics during this period range from the management of gastroenteritis or functional gastrointestinal disorders to risk reduction of immune-mediated diseases such as allergy or necrotizing enterocolitis (1). The mechanisms involved in the positive impact of probiotic strains on infant health are generally unknown but could be related to either a direct interaction of the bacteria with the host or to an indirect effect via the modulation of the resident microbiota.
Infant microbiota is remarkably unstable and can be affected by a number of environmental factors such as the mode of delivery or the type of feeding. This early microbiota is expected to be particularly sensitive to a potential modulation by probiotic administration, especially in infants born by cesarean (C)-section (2). C-section infants present altered microbiota colonization patterns and suffer from an associated risk of immune-related diseases (3,4). Accordingly, Kuitunen et al (5) showed that early probiotic administration reduced the risk of immunoglobulin E–associated allergic disease in children born via C-section but not in those born by the vaginal route. Also the immune response to vaccination was particularly increased in C-section infants fed a Bifidobacterium lactis–enriched formula (6). To our knowledge, only 1 study has compared the effect of a probiotic on C-section and vaginally delivered infant microbiota, and reported a bifidogenic effect of a B lactis–containing formula in C-section but not in vaginally delivered infants (7). This study, however, used a technique specific for Bifidobacterium quantification, which provided only a partial understanding of the microbiota changes.
Lactobacillus reuteri, which is an indigenous component of the human microbiota and has been isolated from breast milk (8), is one of the best studied probiotic bacteria. Dietary supplementation with L reuteri Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSM) 17938 (L reuteri) or with its parental strain L reuteri ATCC 55730 during early life has been shown to alleviate and/or prevent multiple infant disorders (9–15). L reuteri is now commercially available for infant supplementation in the form of probiotic drops and also in infant formula. Although L reuteri seems able to temporary colonize the infant gut (16,17), it is not clear whether it affects the resident microbiota, as studies available show either significant modulation of some bacterial groups (17,18) or no effect (16,19). Most of these studies used analytical techniques targeting specific groups of bacteria; thus, only offering a restricted view of the potential microbiota changes. In addition, none of them controlled for the impact of the mode of delivery on the probiotic effect. The objective of our study was to perform a comprehensive assessment of the effect of feeding with an L reuteri–containing formula on the early microbiota composition of vaginally and C-section–born infants.
Patients and Trial Design
The infants included in this study were part of a larger multicentric, prospective, randomized, and controlled, double-blind safety trial conducted in Athens, Greece, from May 2010 to July 2011 and investigating the impact on d-lactate production of exclusive feeding with formula containing L reuteri DSM 17938 (L reuteri) (18). The study protocol is described in details elsewhere (18). Only infants whose parents/caregivers had decided to exclusively feed them with formula at the time of recruitment were assessed for eligibility to enter the trial. Inclusion criteria included being healthy, born at term (≥37 weeks) and being 72 hours old or younger at the time of enrollment. Exclusion criteria included having any chromosomal or any other major congenital abnormality, having any significant prenatal or postnatal disease, having been treated with antibiotics, and participation in another study. In addition, infants were excluded from the study if their mothers had taken probiotic supplements during the last trimester of pregnancy or had been treated with antibiotics during the last 14 days of pregnancy.
Infants were assigned to receive either a starter formula manufactured to contain L reuteri (1.2 × 109 colony-forming unit per liter) or a control formula with identical composition but without L reuteri until 6 months of age. The concentration of the probiotic in the study formula was estimated to provide a daily dose close to the one used in previous trials demonstrating safety and efficacy of L reuteri in infants (9,11,17,18). During the study, formulas were stored at 4°C in a dry environment. L reuteri was stable during the study period in these conditions (data not shown). To preserve the probiotic viability, parents were instructed to reconstitute the formulas by using water at a temperature <40°C. None of the study formulas contained prebiotics.
Block randomization (block sizes of 2, 4, 6, or 8) with stratification by sex and delivery mode (vaginal or cesarean) was performed using R version 2.12.0 (R Foundation for Statistical Computing, Vienna, Austria). Supplementary Figure 1 (http://links.lww.com/MPG/A641) shows the CONSORT flow diagram of the trial. The enrollment process and drop-out reasons are described in details in a previous publication (18). The number of adverse events was low in this study and not significantly different between both groups (18).
Parents or legal representatives signed an informed consent before infant randomization into the study groups. The study was reviewed and approved by the institutional review board/institutional ethical committee from the 2 study centers: Helena Venizelou Maternity Hospital (Athens, Greece) and Alexandra General Hospital (Athens, Greece). The trial was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. It is registered at www.clinicaltrials.gov (ID NCT01119170).
Fecal DNA Extraction, Amplification, and 16S rRNA Pyrosequencing
Among the 62 infants who completed the trial, fecal samples from 40 infants (n = 20/formula group) were used for microbiota analysis by pyrosequencing. The infant's donor selection was aimed at having a balanced distribution of sex and delivery mode, and was performed randomly and blind to the type of formula. To study the early evolution of the microbiota colonization during the exclusive formula feeding period, without interference of complementary foods, stool samples were collected at 2 weeks (14 ± 3 days) and at 4 months (112 ± 3 days) of infant age. Samples were collected from the diaper by using a sterile spatula, introduced in a sterile pot, refrigerated at 4°C for a maximum of 10 hours after emission, and kept frozen at −40°C until the microbiota analysis was carried out. DNA was extracted using the QIAamp DNA Stool Mini Kit (Qiagen, Hombrechtikon, Switzerland) following the manufacturer's instructions, except for the addition of a series of mechanical disruption steps (11 × 45 seconds) using a FastPrep apparatus and Lysing Matrix B tubes (MP Biochemicals, Santa Ana, CA) to improve the extraction efficiency from bifidobacteria. For each sample, the 16S rRNA variable regions V1 to V3 were polymerase chain reaction (PCR) amplified with a barcoded mixture of primers ensuring an optimal coverage of bacterial phylogenetic diversity (20), including the Bifidobacterium taxon and single-end 454 pyrosequenced as previously described (21).
Raw sequence data were analyzed using the QIIME version 1.7.0 pipeline (http://qiime.org/index.html) with default parameters except when specified (22). Sequences were demultiplexed using the barcode of the PCR primers and quality filtered by discarding reads <200 nt, >650 nt, and with an average quality index <25. Because the infant microbiota of this geographical region (southeastern Europe) was never analyzed by 16S rRNA sequencing before, operational taxonomic units (OTUs) were defined using de novo OTU picking at 97% identity, and chimera check were performed using UCHIME (http://drive5.com/uchime/uchime_download.html) (23). The α diversity was computed using the “alpha_diversity.py” script from QIIME 1.7.0 and selecting “shannon” and “chao1” as “–metrics” option. Singleton OTUs were discarded because they were present in only 1 subject and were most likely because of sequencing errors. The taxonomy assignment was performed on representative sequences using the Ribosomal Database Project Classifier (https://rdp.cme.msu.edu/classifier/classifier.jsp) with confidence threshold of 0.6 (24). All the OTU representative sequences were aligned using the Python Nearest Alignment Space Termination (PyNast) method (25) and Uclust as pairwise alignment method. The resulting multiple alignments were then filtered and used to build a phylogenetic tree with the FastTree method (26). Phylogenetic distances between all the samples were computed as weighted UniFrac distances, and significant differences of distances between groups have been assessed using the Adonis function, available in QIIME (27).
To evaluate the proportion of Lactobacilli sequences associated with L reuteri, a more specific analysis was carried out on representative sequences of OTUs annotated as Lactobacillus genus. These representative sequences were matched using BlastN (National Center for Biotechnology Information, https://www.ncbi.nlm.nih.gov/) to a reference collection of 16S rRNA sequences belonging to type strain species from the Lactobacillus genus (downloaded from the Ribosomal Database Project database) including 3 L reuteri 16s rRNA sequences (EU394679, AY735406, and NR_075063). Results were filtered by keeping only representative sequences that had matches with minimum 97% identity on L reuteri 16S rRNA reference sequences only and covering ≥99% of the representative sequence length. From the 21 Lactobacillus OTU representative sequences, 10 could be annotated as L reuteri based on the mentioned criteria. The combined relative abundance of these OTUs was calculated for each sample.
Microbiota composition was a secondary outcome of the clinical study, the primary objective of which was to assess the safety profile of the L reuteri formula. Hence, an exploratory approach has been followed, and no power computation has been performed on this specific outcome.
The differences of gestational age and birth weight between L reuteri and control formula groups were assessed with the Student t test, whereas differences in the proportions of males versus females and cesarean versus vaginal delivery were tested using Fisher exact test. Microbiota alpha diversity index (ie, Chao1 and Shannon diversity) of the different groups was globally compared using the Kruskal–Wallis test.
The effect of the mode of delivery on the microbiota of each formula group and at each infant age was assessed using a non-parametric multivariate analysis of variance (NP MANOVA) test (28) on all taxa in the 16S rRNA gene region. Differences between groups in the relative abundance of individual taxonomic groups were assessed using 2-sided Wilcoxon tests. Based on the average number of reads per sample (ie, about 1000), biologically relevant observations were defined for each comparison as taxa detected with an abundance of >2% (ie, 20 sequences) in ≥1 sample and showing a median value >0.5% (ie, 5 sequences) in ≥1 group. Significance level was set up at 5%, although the data presented in this report shall be considered exploratory, as microbiota was a secondary outcome of the original trial.
No significant differences were observed in the baseline characteristics of the formula groups in the subpopulation included in the microbiota study (Table 1). None of the infants received antibiotics, probiotics other than L reuteri, or weaning foods during the study period.
The microbiota analysis of the fecal samples collected at 2 weeks and 4 months of infant age yield a raw data set composed of 99,353 sequences (median size 473 bp). Data processing allowed the clustering of 85,334 sequences into 1868 OTUs at 97% identity, classified at all taxonomic levels (from phylum to genus), and reported in Figure 1. A total of 67% of OTUs had ≤10 sequences. On average, 133 OTUs and 1067 sequences described the microbiota of each infant fecal sample.
Effect of the Mode of Delivery on the Microbiota Response to the Type Formula
The NP MANOVA test revealed that the delivery mode had a significant impact on the microbiota composition of the control formula group at week 2 (P = 0.012). Therefore, the subsequent analyses were stratified according to the delivery mode, leading to 4 groups of comparison: infants born via vagina, fed control (VCt) or L reuteri (VLr) formula, and infants born via C-section fed control (CCt) or L reuteri (CLr) formula.
Alpha Diversity Analysis
A rarefaction curve analysis of the α diversity (Shannon index) showed that 510 sequences per sample were sufficient to describe the microbial communities of the infant fecal samples (Supplementary Fig. 2, http://links.lww.com/MPG/A642). The mode of delivery and the type of formula did not significantly affect the microbial richness and diversity at any age, as measured by Chao1 (Supplementary Table 4, http://links.lww.com/MPG/A646) and Shannon (Supplementary Table 5, http://links.lww.com/MPG/A647) diversity indexes respectively.
To provide a global view of the difference in microbiota composition of the 4 groups of comparison, the community phylogenetic distances between samples were calculated by weighted UniFrac analysis and averaged for each group versus the other groups. The similarities between the phylogenetic distances of each group are shown in Figure 2. At week 2, VLr, CLr, and VCt groups clustered together, and no significant differences were found between them. By contrast, all the 3 groups were significantly distant from CCt (P = 0.009 vs VCt, P = 0.010 vs VLr, and P = 0.013 vs CLr). At month 4, all the 4 groups clustered together, and no significant difference appeared anymore.
From the OTUs classified at phylum, family, and genus levels (Fig. 1), the effect of the mode of delivery and type of formula on the microbiota composition at 2 weeks (Supplementary Table 1, http://links.lww.com/MPG/A643) and 4 months (Supplementary Table 2, http://links.lww.com/MPG/A644) was statistically assessed. For each comparison, a criterion of biological relevance was defined to prevent an excessive interpretation of taxa differences close to the detection limit of our profiling approach (see Methods). Only these relevant observations are discussed.
Effect of the Mode of Delivery on the Control Formula Samples
At week 2, the median relative abundance of the phylum Actinobacteria was largely lower in CCt than in VCt (0.5% vs 44.5%, P = 0.008). Bifidobacterium was the genus contributing the most to this difference (0.0% vs 28.6%, P = 0.039). In contrast, CCt showed higher Proteobacteria (27.6% vs 2.5%, P = 0.011) than VCt, in particular Enterobacter (0.6% vs 0.0%, P = 0.043) and unclassified bacteria from the Enterobacteriaceae family (2.3% vs 0.2%, P = 0.004). Concerning the phylum Firmicutes, the CCt group was characterized by higher proportions of Enterococci (8.9% vs 1.7%, P = 0.011) and of 2 genera of the Clostridiaceae–Clostridium (3.1% vs 0.1%, P = 0.001), and unclassified Clostridiaceae (3.0% vs 0.0%, P < 0.001) than the VCt group.
At month 4, lower Actinobacteria abundance was still observed in CCt but was a result of a strong reduction of the Coriobacteriaceae Collinsella (0.0% vs 20.9%, P = 0.018) and not any longer to the Bifidobacteria level. The Enterococcus taxa was still overrepresented in CCt compared with VCt (7.7% vs 0.7%, P = 0.035). Increased levels of Coprococci in CCt were also observed at this age (3.1% vs 0.0%, P = 0.026).
Effect of the L reuteri Formula on the C-Section Delivery Samples
The effect of the probiotic formula on the C-section microbiota was assessed by comparing CLr with CCt and by benchmarking CLr with the vaginal-delivery groups VCt and VLr. At 2 weeks of age, Actinobacteria were strongly increased in CLr compared with CCt (25.4% vs 0.5%, P = 0.015) toward vaginal delivery values (P = 0.423 vs VCt; P = 0.683 vs VLr). Specifically, Bifidobacterium was undetectable in 50% of infants of the CCt group (median = 0.0%) but was increased to 24.8% (P = 0.008) and was detected in 91% of the infants in the CLr group to reach a level not significantly different to what was observed in VCt (28.6%, P = 0.863) and VLr (35.1%, P = 0.795) (Fig. 3). In addition, the abundance of unclassified bacteria from the Enterobacteriaceae family was reduced in the CLr group compared with CCt (0.4% vs 2.3%, P = 0.010) and was not significantly different from VCt (P = 0.217) and VLr (P = 0.175). By contrast, the levels of Clostridia and Enterococcus were similarly high in both CCt and CLr when compared with the groups born by the vaginal route. Enterobacter in CLr was not significantly different from CCt or from the vaginal delivery groups.
At 4 months, Coprococcus was similarly high in both C-section groups when compared with the vaginal delivery groups. By contrast, neither Collinsella nor Enterococcus was significantly different between CLr and VCt or VLr. Lactobacillus was consistently higher in CLr than in CCt infants at both 2 weeks (4.4% vs 0.0%, P = 0.027) and 4 months (0.3% vs 0.0%, P = 0.051).
Effect of the L reuteri Formula on the Vaginal Delivery Samples
Similar to the observation in C-section samples, the genus Lactobacillus was present in more infants and was significantly more abundant in VLr than in VCt at both week 2 (2.8% vs 0.1%, P = 0.045) and month 4 (0.6% vs 0.0%, P = 0.012). Coprococcus abundance was also higher in VLr than in CCt but only at 4 months (4.5% vs 0.0%, P = 0.005). No other significant difference was observed between these 2 groups.
Effect of the L reuteri Formula on Lactobacillus spp and L reuteri Abundance
Although the pyrosequencing reads did not cover the full 16S rRNA gene, the 16S rRNA sequences corresponding to the L reuteri species were unambiguously identified among the Lactobacilli OTUs (Supplementary Table 3, http://links.lww.com/MPG/A645). Some infants receiving the control formula had detectable levels of L reuteri, although, as expected, abundance of this species was significantly higher in the infants receiving the probiotic formula, independently of the mode of delivery and of infant age. In the infants fed the L reuteri formula, the occurrence of the Lactobacillus genus was always associated with the cooccurrence of L reuteri, indicating that L reuteri contributed to the increased Lactobacillus levels. The abundance of L reuteri, however, was always lower than that of Lactobacillus, suggesting that the presence of L reuteri promoted the growth of other Lactobacillus species.
To our knowledge, this is the first comprehensive report comparing the effect of a probiotic on the global microbiota composition in vaginal- and cesarean-delivered infants. The most remarkable finding of our study has been that whereas the L reuteri–supplemented formula had a limited effect on the microbiota of vaginally born infants, this formula appeared to strongly modulate the microbiota in babies delivered by C-section, by inducing a shift of the global profile and of the taxa abundance toward the vaginal delivery composition, with a strong effect observed already at 2 weeks of age.
The only consistent diet-associated microbiota change observed in both vaginal- and C-section delivery samples at the 2 study ages was increased Lactobacillus in the groups fed with the probiotic formula. These data confirm previous results of our trial using a targeted technique of Lactobacilli identification (18) and from Savino et al (17) in colicky infants fed drops containing the same probiotic. As proposed by Savino et al (17), promotion of Lactobacillus could contribute to the efficacy of this probiotic on infantile colic management, as this condition is associated with depressed Lactobacilli levels, which even precede the onset of colic symptoms (29,30). Our results suggest that the increase in Lactobacillus is explained by the presence of the probiotic itself in the fecal samples concomitantly with the stimulation of other commensal Lactobacillus strains.
Despite the increase of Lactobacillus, the phylogenetic distance analysis of the vaginally born infant data indicated no significant differences in the global microbiota composition between control and L reuteri formula groups at any of the ages studied. These results are in agreement with the findings of a recent trial where breast-fed colicky infants were supplemented with the same L reuteri strain in the form of oily drops (30).
We confirm in our study earlier data showing strong perturbations of the early stages of gut colonization in infants born via C-section. Like others, we observe a delayed colonization by Actinobacteria, in particular by the genus Bifidobacterium(31–37) and Collinsella(38), and increased levels of Enterobacteriaceae(37), Clostridium(3,37), and Enterococcus(39) in the early life microbiota of C-section infants fed the control formula. Inspired by recent extensive characterizations of the developing infant fecal microbiota (38,40), a schematic view of its progression across the first year of life was proposed, including the effect of the mode of delivery and formula feeding (41). Our results concur with this model for the effect of Caesarean delivery proposed to modify the progression by delaying the transition from a Proteobacteria- to an Actinobacteria-dominated microbiota. It is believed that aberrant colonization is a major factor contributing to the increased risk of immune-related disease in infants born by this mode of delivery (3). Notably, low levels of Bifidobacteria and high abundance of Enterobacteria during the first weeks of life have been associated with poor vaccine response (42), and increased susceptibility to a range of disorders (30,38,43–45). As previously shown with the probiotic B lactis(7), Bifidobacterium abundance and occurrence were remarkably increased in the C-section group fed the L reuteri formula, whereas Enterobacteriaceae abundance was largely decreased, reaching values not significantly different from the vaginal groups. Furthermore, the phylogenetic profile of the microbiota of the C-section group fed the probiotic formula was not significantly distant from that of the vaginal groups. These findings suggest that an early intervention with L reuteri can induce a fast recovery of the early microbiota dysbiosis induced by C-section delivery, which in turn may decrease the risk of the disorders associated with this condition. It has to be noted, though, that in the 14-day C-section samples, the L reuteri formula failed to reduce the abundance of Clostridium, a genus that includes potential pathogenic species. Nevertheless, gut colonization with Clostridium strains is very frequent during the neonatal period and is usually asymptomatic (46). Accordingly, no infection symptoms were observed in these babies, and Clostridium abundance was similarly low in all the groups at 4 months, independently of the mode of delivery.
The strong modulatory effect of the L reuteri formula on the early C-section infant microbiota may be explained by the absence of bacterial groups normally transmitted during vaginal delivery. The infants in our study started to receive the study formula in an early phase of microbiota development. Upon vaginal delivery, bacterial taxa transferred from the vagina to the infant gut include Lactobacillus, although it remains at relatively low abundance in this new environment. This taxon is virtually absent in the early C-section microbiota, which in turn is dominated by skin-resident bacteria (47). In our treated C-section group, L reuteri possibly occupied the ecological niche of the natural Lactobacilli population. Our results show that, despite its relatively low abundance, L reuteri had a profound effect on the structure of the microbial community. This indicates that L reuteri plays the role of keystone species (48), in place of the Lactobacilli from vaginal origin. Similar to other Lactobacilli strains, L reuteri is able to produce lactate by carbohydrate fermentation. The subsequent acidification of the intestinal milieu may have promoted the development of Bifidobacteria and other low abundant Lactobacilli strains in detriment of the acid-sensitive Enterobacteria(49). Our observation of transiently increased urinary lactate levels in the infants who received the L reuteri formula at 1 and 2 weeks of age supports this hypothesis (18). The capacity of this probiotic to produce the bacteriocin reuterin (50) may have also played a role on its microbiota modulatory effect.
An important limitation in the study is that the original trial (18) was not powered for microbiota assessment. Thus, our data, even if consistent with previous reports, shall be confirmed by a properly designed trial. To describe the impact of the probiotic intervention on the microbiota, the present study is also technologically restricted to the observation of compositional changes of the microbial community, whereas a modification in the gene expression of the intestinal microbiota cannot be excluded.
In summary, our results are consistent with previous reports of delayed gut microbiota colonization in infants born via C-section. Feeding with a formula containing L reuteri DSM 17938 does not appear to essentially modify the microbiota composition if the infant is born via the natural route, but it seems to promote a fast recovery of the microbiota dysbiosis induced by C-section delivery. These findings suggest that an early intervention with this probiotic can exert a substantial and possibly beneficial modulation of the gut microbial community in the absence of a normal pattern of colonization. Nevertheless, further studies are required to confirm these results.
The authors thank Dr Enea Rezzonico for his suggestions during data analysis, Dr Lionel Philippe for his support to the logistics of the study, and Ms Fiona Paratte for English edits.
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