Open Access

Maternal inheritance of bifidobacterial communities and bifidophages in infants through vertical transmission

  • Sabrina Duranti1,
  • Gabriele Andrea Lugli1,
  • Leonardo Mancabelli1,
  • Federica Armanini2,
  • Francesca Turroni1,
  • Kieran James3,
  • Pamela Ferretti2,
  • Valentina Gorfer4,
  • Chiara Ferrario1,
  • Christian Milani1,
  • Marta Mangifesta5,
  • Rosaria Anzalone1,
  • Moreno Zolfo2,
  • Alice Viappiani5,
  • Edoardo Pasolli2,
  • Ilaria Bariletti4,
  • Rosarita Canto4,
  • Rosanna Clementi4,
  • Marina Cologna4,
  • Tiziana Crifò4,
  • Giuseppina Cusumano4,
  • Sabina Fedi4,
  • Stefania Gottardi4,
  • Claudia Innamorati4,
  • Caterina Masè4,
  • Daniela Postai4,
  • Daniela Savoi4,
  • Massimo Soffiati4,
  • Saverio Tateo4,
  • Anna Pedrotti4,
  • Nicola Segata2,
  • Douwe van Sinderen3 and
  • Marco Ventura1Email author
Contributed equally
Microbiome20175:66

https://doi.org/10.1186/s40168-017-0282-6

Received: 23 March 2017

Accepted: 5 June 2017

Published: 26 June 2017

Abstract

Background

The correct establishment of the human gut microbiota represents a crucial development that commences at birth. Different hypotheses propose that the infant gut microbiota is derived from, among other sources, the mother’s fecal/vaginal microbiota and human milk.

Results

The composition of bifidobacterial communities of 25 mother-infant pairs was investigated based on an internal transcribed spacer (ITS) approach, combined with cultivation-mediated and genomic analyses. We identified bifidobacterial strains/communities that are shared between mothers and their corresponding newborns. Notably, genomic analyses together with growth profiling assays revealed that bifidobacterial strains that had been isolated from human milk are genetically adapted to utilize human milk glycans. In addition, we identified particular bacteriophages specific of bifidobacterial species that are common in the viromes of mother and corresponding child.

Conclusions

This study highlights the transmission of bifidobacterial communities from the mother to her child and implies human milk as a potential vehicle to facilitate this acquisition. Furthermore, these data represent the first example of maternal inheritance of bifidobacterial phages, also known as bifidophages in infants following a vertical transmission route.

Keywords

MicrobiotaViromeMicrobiomeBifidobacteriaVertical transmission

Background

The period immediately following birth is believed to be crucial for the correct establishment of the gut microbiota with possible temporary and long-lasting effects on host health [1]. During this infant stage of (human) life, microorganisms originating from the mother and from environmental microbial communities rapidly colonize the gastrointestinal tract (GIT) of the neonate to form the early infant microbiota whose establishment is also influenced by the feeding method [27]. Bifidobacteria are among the first colonizers of the intestine of newborns and are considered to play pivotal roles in terms of modulation of mucosal physiology and innate immunity of the host [6, 8, 9]. Genomically identical bifidobacterial strains have been isolated from fecal samples of mother and child combinations, as well as the corresponding human milk samples, indicative of a vertical transmission route from maternal GIT to (breastfed) infants [1013]. This has given rise to the hypothesis that microbial colonization of the infant depends on the mother’s fecal/vaginal microbiota as well as on (providing) breast milk [14]. Bacteria may reach the mammary gland of the mother and may be transmitted directly to breastfed infants, although, inversely, bifidobacteria may be introduced into human milk from the infant’s oral cavity during suckling [1417].

Notably, several studies have highlighted the possibility of vertical transmission of (components of) the gut microbiota from mother to child [12, 15, 18]. Furthermore, a recent pilot study involving four mother-child pairs revealed direct transmission of bifidobacterial strains from mothers to their newborns using a novel ITS-based approach [11]. Although intriguing, the biological relevance of such findings in this latter work is limited due to the small number of mother-infant pairs analysed [11].

Another important component of the gut microbiota is represented by virus-like particles in the GIT, which together constitute the gut virome [19, 20]. Currently, very little is known about the infant virome [2123], although the existence and impact of phages specific for bifidobacteria, i.e., bifidophages, in the infant gut has recently been discribed [24].

In the current study, we analyzed the composition of the bifidobacterial communities of 25 mother-infant pairs by means of the ITS profiling approach mentioned above, combined with cultivation and genome investigations. Employing a next-generation sequencing (NGS) approach, we identified bifidobacterial strains that are shared between mothers and their corresponding newborns, while we furthermore obtained evidence for vertical, mother-child transmission of bifido(pro)phages.

Methods

Subject recruitment and sample collection

The study protocol was approved by the Ethical Committee of the “Azienda Provinciale per i Servizi Sanitari” in Trento, Italy, as well as by the Ethical Committee of the University of Parma, Italy, and informed written consent was obtained from all participants or their legal guardians. Twenty-five mother-infant pairs were enrolled in this study, which were selected based on the following criteria: the mother’s age (ranging in age from 18 to 40 years old) and those subjects who were not taking any probiotics or antibiotics. All subjects were considered to be healthy, as based on self-reporting. At the time of delivery, during birth or immediately after it, stool samples were collected of 21 mothers. Stool samples of infants were collected at two different time points, i.e., 7 days and 1 month following birth of the infant. Furthermore, 10 ml of breast milk sample of each mother was collected in sterile tubes (Table 1). For the sample set TVPR_03, we collected stool samples of infants and breast milk samples at 1 month following birth. Stool and milk samples were collected following the “Stool Packaging Instructions” of the “Core Microbiome Sampling Protocol A” reported in the “Manual of Procedures of the Human Microbiome Project” (http://www.hmpdacc.org/doc/HMP_MOP_Version12_0_072910.pdf) [25]. Samples were immediately frozen at −20 °C and shipped under frozen conditions to the laboratory, where they were immediately processed. DNA extractions of stool samples and human milk samples were performed as previously described [11].
Table 1

List of samples included in this study

CoupleID

Samples

TVTR_10005

Mum-T0

2062

Milk-T3

2072

Infant-T3

2074

Milk-T4

2076

Infant-T4

2078

TVTR_10006

Milk-T3

2090

Infant-T3

2092

Milk-T4

2094

Infant-T4

2096

TVTR_10007

Milk-T3

2108

Infant-T3

2110

Milk-T4

2112

Infant-T4

2114

TVTR_10008

Milk-T3

2126

Infant-T3

2128

Milk-T4

2130

Infant-T4

2132

TVTR_10009

Mum-T0

2134

Milk-T3

2144

Infant-T3

2146

Milk-T4

2148

Infant-T4

2150

TVTR_10010

Mum-T0

2152

Milk-T3

2162

Infant-T3

2164

Milk-T4

2166

Infant-T4

2168

TVTR_10017

Mum-T0

2278

Milk-T3

2287

Infant-T3

2290

Milk-T4

2292

Infant-T4

2293

TVTR_10019

Mum-T0

2314

Milk-T3

2324

Infant-T3

2326

Milk-T4

2328

Infant-T4

2330

TVTR_10020

Mum-T0

2332

Milk-T3

2342

Infant-T3

2344

Milk-T4

2346

Infant-T4

2347

TVTR_10021

Mum-T0

2350

Milk-T3

2360

Infant-T3

2362

Milk-T4

2364

Infant-T4

2365

TVTR_10023

Milk-T3

2396

Infant-T3

2398

Milk-T4

2400

Infant-T4

2401

TVTR_10024

Mum-T0

2404

Milk-T3

2414

Infant-T3

2416

Milk-T4

2418

Infant-T4

2419

TVTR_10025

Mum-T0

2422

Milk-T3

2432

Infant-T3

2434

Milk-T4

2436

Infant-T4

2437

TVTR_10028

Mum-T0

2476

Milk-T3

2486

Infant-T3

2487

Milk-T4

2490

Infant-T4

2491

TVTR_10029

Mum-T0

2492

Milk-T3

2502

Infant-T3

2504

Milk-T4

2506

Infant-T4

2508

TVTR_10030

Mum-T0

2509

Milk-T3

2519

Infant-T3

2521

Milk-T4

2523

Infant-T4

2525

TVTR_10031

Mum-T0

2526

Milk-T3

2536

Infant-T3

2538

Milk-T4

2540

Infant-T4

2541

TVTR_10032

Mum-T0

2544

Milk-T3

2554

Infant-T3

2556

Milk-T4

2558

Infant-T4

2559

TVTR_10034

Mum-T0

2580

Milk-T3

2590

Infant-T3

2592

Milk-T4

2594

Infant-T4

2595

TVTR_10035

Mum-T0

2598

Milk-T3

2608

Infant-T3

2610

Milk-T4

2612

Infant-T4

2613

TVTR_10036

Mum-T0

2616

Milk-T3

2626

Infant-T3

2628

Milk-T4

2630

Infant-T4

2632

TVTR_10038

Mum-T0

2652

Milk-T3

2662

Infant-T3

2664

Milk-T4

2666

Infant-T4

2668

TVPR_01

Mum-T0

3000

Milk-T4

3001

Infant-T4

3002

TVPR_02

Mum-T0

3006

Milk-T3

3007

Infant-T3

3008

Milk-T4

3009

Infant-T4

3010

TVPR_03

Mum-T0

3011

Milk-T4

3012

Infant-T4

3013

ITS gene amplification and MiSeq sequencing

Partial ITS sequences were amplified from extracted DNA using the primer pair Probio-bif_Uni/Probio-bif_Rev, which targets the spacer region between the 16S rRNA and the 23S rRNA genes within the ribosomal RNA (rRNA) locus [26]. Illumina adapter overhang nucleotide sequence was then added to the generated ITS amplicons of c. 200 bp. The library of ITS amplicons was prepared following the 16S Metagenomic Sequencing Library Preparation Protocol (Part No. 15044223 Rev. B—Illumina). Sequencing was performed using an Illumina MiSeq sequencer with MiSeq Reagent Kit v3 chemicals.

ITS microbial profiling analysis

Profiling of known bifidobacterial species was performed using the primer pair Probio_bif_uni/Probio_bif_rev, an improved bifidobacterial ITS database encompassing all publicly available bifidobacterial genomes and a custom bioinformatics script, as described previously [26].

Evaluation of the bifidobacterial cell density by qPCR

The presence of bifidobacteria in infant stool samples was evaluated using quantitative real-time PCR (qRT-PCR). The primers used in this study are Probio_bif_uni/Probio_bif_rev to determine numbers of bifidobacteria [26] and Probio_uni/Probio_rev to quantify total bacterial numbers [27]. The quantitative contribution of bifidobacteria to the overall infant gut microbiota of each sample was evaluated by a ratio of the genome copy number ITS gene/16S rRNA gene (the genes targeted were in the same copy per genome). qPCR was performed using SoFast EvaGreen Supermix (Bio-Rad) on a CFX96 system (BioRad, CA, USA) following previously described protocols [28].

Recovery of bifidobacteria on selective media

One gram of each fecal sample or 1 ml of human milk was mixed with 9 ml of phosphate-buffered saline (PBS), pH 6.5. Serial dilutions and subsequent plating were performed using the de Man-Rogosa-Sharpe (MRS) agar (Scharlau Chemie, Barcelona, Spain), supplemented with 0.05% (wt/col) l-cysteine hydrochloride and 50 μg/ml mupirocin (Delchimica, Italy). The agar plates were incubated in an anaerobic atmosphere (2.99% H2, 17.01% CO2, and 80% N2) in a chamber (Concept 400; Ruskin) at 37 °C for 48 h. Ten colonies were taken as a representation of the bacterial strains retrieved from the selective medium. DNA was extracted and subjected to (sub)-species identification as previously described [11].

De novo genome sequencing and bioinformatics analyses

The genome sequence of the new bifidobacterial isolates was determined by GenProbio srl (Parma, Italy) using a MiSeq platform (Illumina, UK). A genome library was generated following the TruSeq Nano DNA library Prep protocol (Part No. 15041110 Rev. D). Library samples were loaded into a Flow Cell V3 600 cycles (Illumina) according to the technical support guide, and generated reads were depleted of adapter sequences, quality-filtered, assembled and protein-encoding open reading frame (ORF) predicted through the MEGAnnotator pipeline [29].

Fermentation profiles on milk glycans

Prior to carrying out growth profile assays, Bifidobacterium cultures were grown from stock in Difco MRS (BD) and sub-cultured twice in the same medium. Subsequently, 5 ml of freshly prepared modified MRS (mMRS) medium [peptone from casein, 10 g/L; meat extract, 10 g/L, and yeast extract, 5 g/L (purchased from Difco); K2HPO4, 3 g/L; KH2PO4, 3 g/L; pyruvic acid, 0.2 g/L; polysorbate 80, 1 mL/L; tri-ammonium citrate, 2 g/L; MgSO4·7H2O, 0.575 g/L; MnSO4·4H2O, 0.12 g/L; cysteine-HCl, 0.3 g/L; and FeSO4·7H2O, 0.034 g/L], supplemented with 0.05% cysteine hydrochloride, and 1% of a particular carbohydrate was inoculated with 50 μl (1%) of a stationary phase culture. All carbohydrates (fucose, lactose, sialic acid, 3′-sialyllactose, 6′-sialyllactose, 2′-fucosyllactose, 3′-fucosyllactose, lacto-N-tetraose, or lacto-N-neotetraose) were dissolved in water and then sterilized by filtration (0.2-μ filter size) and then added to mMRS after sterilization by autoclaving. Uninoculated mMRS medium was used as a negative control. Cultures were incubated under anaerobic conditions in a modular atmosphere-controlled system (Davidson and Hardy, Belfast, Ireland) at 37 °C for 24 h, and the optical density at 600 nm (OD600) was determined manually, using a Shimadzu UV-1280 UV-399 VIS Spectrophotometer. Growth assays were carried out in triplicate, with standard deviation calculated for error values.

Shotgun metagenomic analyses

Fastq files obtained from metagenomic sequencing of infant and mother fecal samples, as well as milk samples, were used as input for the SPAdes assembler (version 3.9, using default parameters and enabling the metagenomic option—meta) for de novo metagenomic assemblies [30]. ORFs were predicted with Prodigal [31] and annotated using NCBI RefSeq databases through RAPSearch2 aligner (cut-off E value of 1 × 10−30) [32]. Each contig was taxonomically classified by means of the gene hit obtained through the NCBI RefSeq databases by the use of the Contig Classifier (CoCla) script [33, 34]. Species-specific contigs were subdivided in different files, obtaining reconstructed genomes of the most abundant (bifido)bacterial taxa within each analysed sample.

Bifidophage analyses

Contigs attributed to bifidobacterial (sub)species, by means of the CoCla script, were screened for the presence of bifidobacterial phages/prophages using a custom database composed of previously identified bifidophages [24]. Identified phage sequences were further manually curated in order to unveil overlapping extremities within the contig sequences. The presence of putatively identified bifidophages was screened in mother and infant fecal samples, and milk samples by PCR based on unique genes of that particular bifidophage.

Results and discussion

Evaluation of the bifidobacterial composition of the gut microbiota of infants

To characterize the bifidobacterial population in the infant gut, we employed a recently developed pipeline based on sequencing of a hypervariable ITS region, here referred to as ITS profiling [26]. We collected fecal samples of 25 infants at two different time points, i.e., 7 days and 1 month following birth (Table 1). ITS profiling of these 50 samples produced a total of 1,703,642 reads, ranging from 1224 to 113,414 reads per sample (Additional file 1: Table S1), which were grouped into clusters of identical sequences (OTUs or operational taxonomic units) and then taxonomically classified. OTU analysis revealed that the dominant bifidobacterial species detected in the investigated fecal samples were Bifidobacterium longum subsp. longum, Bifidobacterium breve, Bifidobacterium bifidum, Bifidobacterium longum subsp. infantis, Bifidobacterium adolescentis, and Bifidobacterium pseudocatenulatum. In addition, ITS-based OTUs display a similarity level below 93% to any of the ITS sequences of the current known bifidobacterial (sub)species (Fig. 1 and Additional file 2: Figure S1). As previously discussed by Milani et al. [26], the comparison between bifidobacterial-type strain ITS sequences reveal that different bifidobacterial (sub)species possess ITS identity above 98%; it is therefore a distinct possibility that identified ITS-based OTUs with a similarity level below 93% represent novel bifidobacterial (sub)species. Notably, and in contrast to what currently is known about the composition of the bifidobacterial communities of the infant gut, we identified that such populations are composed not only by typical infant-gut bifidobacterial species, such as B. breve, B. bifidum, and B. pseudocatenulatum, but also by adult-associated bifidobacterial taxa, like B. adolescentis and B. catenulatum [11, 15]. This finding suggests that an ecological division based on age is not valid or at least not absolute, thus underscoring a possible strain-sharing scenario between adult and infant microbiotas. Furthermore, in seven and six cases, we observed a high abundance of non-common infant gut bifidobacteria such as Bifidobacterium tissieri and Bifidobacterium dentium, respectively (Fig. 1 and Additional file 2: Figure S1). In order to assess the quantitative contribution of bifidobacteria to the overall infant gut microbiota of each sample, the total bacterial load as well as the bifidobacterial cell count were evaluated by qRT-PCR. These analyses indicate that bifidobacteria represent between 0.05 and 96.44% of the overall infant gut microbiota (average of 36.54%), ranging from 2.48E + 03 to 8.22E + 08 genome copy number per gram of feces. No significant differences between the two time points were detected. These findings strengthen the now well-established notion that bifidobacteria represent one of the dominant genera in the infant gut environment (Fig. 1) [10, 3539].
Fig. 1

Profiling of the bifidobacterial communities of infant fecal samples. The heat map representation shows the relative abundance of Bifidobacterium species. On the bottom of the image, the bar plots show qPCR data regarding the proportion (in %) of bifidobacteria relative to other bacteria present in a given sample

Sharing of OTUs between mothers and children

Bacterial transmission from mother to child has been proposed to occur during birth and as a consequence of breastfeeding [40]. In order to explore if a similar scenario is applicable to the bifidobacterial population, we compared bifidobacterial ITS profiles of infants with those of their corresponding mother’s fecal (obtained during or soon after delivery) and milk samples obtained 7 and/or 30 days after delivery (Fig. 2 and Additional file 2: Figure S1). Interestingly, the number of ITS-based OTUs shared by a given sample set ranged from three in TVPR_01 to 273 in TVTR_10032 (Additional file 1: Table S2). These ITS-based OTUs encompass members of the species B. bifidum, B. adolescentis, B. dentium, B. breve, B. longum spp., B. pseudocatenulatum, and B. eulemuris and OTUs displaying a level of similarity below 93% to any of the current known bifidobacterial (sub)species (Fig. 2 and Additional file 2: Figure S1). Furthermore, we highlighted that the most prevalent bifidobacterial species are B. breve, B. longum spp., B. bifidum, B. adolescentis, B. dentium, and B. pseudocatenulatum, all being identified in infant feces. These taxa were also found in the corresponding fecal and milk samples of the mother (Fig. 2 and Additional file 2: Figure S1). Notably, comparison of the ITS-based OTUs identified in the data sets indicated the presence of identical ITS-based OTUs in different sample pairs, implying that these identical sequences correspond to very closely related strains that are present in non-corresponding mother-infant dyads. We therefore decided to verify these findings using a cultivation approach where we isolated such strains followed by de novo genome sequencing of such bifidobacterial isolates.
Fig. 2

Profiling of the bifidobacterial communities of each CoupleID. The heat map represents the relative abundance of bifidobacterial species that were determined to be present in each sample. On the left side, all known and putative novel bifidobacterial species are reported. Sample origin and CoupleID codes are reported on the top side of the heat map

Isolation and genome sequencing of shared mother-child bifidobacterial strains

In order to validate the notion that the identical bifidobacterial ITS-based OTUs that seem to be shared between mother-newborn pairs are the result of vertical transmission, we used 25 infant and mother stool samples, as well as milk samples, to isolate bifidobacteria employing a selective cultivation medium (Table 1). Isolated colonies were identified by PCR amplification of the 16S rRNA gene followed by amplicon sequencing as previously described by Turroni et al. [41]. This cultivation effort resulted in the isolation of 14 bifidobacterial strains, whose taxonomical identity perfectly matched those identified by bifidobacterial ITS profiling described above for the same samples (Fig. 2 and Additional file 2: Figure S1). These 14 isolated strains, representing different bifidobacterial species (Table 2), were then subjected to whole-genome sequencing using an Illumina-MiSeq platform. As outlined in Table 2, the predicted general features of these genomes appear to be similar to those generally observed for bifidobacteria [42]. Moreover, to identify the presence of these strains in stool samples of the corresponding mother, we performed a comparative genome analysis to identify the occurrence of specific, unique genes for each strain. Such analyses allowed the design of strain-specific primer pairs to be used in a PCR-based detection method involving DNA isolated from (mother’s) stool samples as a template (Additional file 1: Table S3). Notably, these experiments resulted in the detection of each of the infant stool-isolated and genome-sequenced strains in the fecal samples of the corresponding mother. In addition, we used isolated DNA from a mother stool sample of a non-corresponding CoupleID to detect false positive results (Fig. 3). Altogether, these data clearly corroborate the notion that these bifidobacterial isolates were obtained by infants via a maternal vertical transmission route (Fig. 3).
Table 2

General genome features of vertical transmitted bifidobacterial strains

 

Couple ID

Biological origin

Contig

Genome length

% GC

Predicted ORFs

tRNA

rRNA

Bifidobacterium longum subsp. longum 1886B

TVTR_10006

Human milk

47

2473746 bp

61.14%

2137

62

3

Bifidobacterium bifidum 1887B

TVTR_10006

Human milk

21

2255543 bp

59.49%

1851

53

3

Bifidobacterium longum subsp. infantis 1888B

TVTR_10020

Infant stool

26

2579732 bp

59.40%

2212

55

3

Bifidobacterium breve 1889B

TVTR_10005

Infant stool

22

2344818 bp

59.66%

2037

53

2

Bifidobacterium longum subsp. longum 1890B

TVTR_10023

Infant stool

109

2341670 bp

59.55%

1955

60

2

Bifidobacterium breve 1891B

TVTR_10008

Infant stool

38

2089648 bp

59.70%

2115

54

2

Bifidobacterium adolescentis 1892B

TVTR_10024

Infant stool

11

2150850 bp

58.65%

1720

55

4

Bifidobacterium dentium 1893B

TVTR_10034

Infant stool

24

2271880 bp

58.74%

2070

56

4

Bifidobacterium breve 1895B

TVPR_01

Infant stool

17

20270860 bp

58.58%

1871

53

2

Bifidobacterium pseudocatenulatum1896B

TVPR_01

Human milk

15

2197471 bp

56.15%

1733

54

5

Bifidobacterium longum subsp. longum 1897B

TVTR_10030

Human milk

55

2453605 bp

59.50%

2143

55

2

Bifidobacterium longum subsp. longum 1898B

TVTR_10008

Infant stool

41

2474386 bp

59.47%

2049

57

3

Bifidobacterium catenulatum 1899B

TVTR_10029

Infant stool

14

2124599 bp

58.90%

1749

55

5

Bifidobacterium breve 1900B

TVTR_10031

Infant stool

22

2287865 bp

59.50%

1910

53

2

Fig. 3

Evaluation of persistence of putative vertically transmitted strains in fecal samples of the infants and mothers as well as in milk samples by PCR assays. Marker lane is the Thermo Scientific GeneRuler 1 kb DNA Ladder. The reference strains used as positive controls for each CoupleID respectively are B. longum 1886B, B. bifidum 1887B, B. breve 1900B, B. adolescentis 1892B, B. longum 1898B, B. longum 1897B, B. dentium 1893B, B. longum subsp. infantis 1888B, B. catenulatum 1899B, B. breve 1891B, B. breve 1895B, B. pseudocatenulatum 1896B, and B. breve 1889B. Primer sequences are reported in Additional file 1: Table S3

Genetic adaptation of early bifidobacterial colonizers to the infant gut

Transmission of (bifido)bacteria from mother to her child may be possible through ingestion of mother’s milk [38, 39, 43, 44]. This human fluid represents a bacterial transmission medium that is ideal not only for microbiota dispersal but also for the provision of nutrients to the initial colonizers of the infant gut [10, 38, 39, 44, 45]. Human milk is a very rich source of glycans, including lactose and human milk oligosaccharides (HMOs) [46], which represent an important carbon and energy source for pioneering saccharolytic members of the infant gut microbiota, in particular bifidobacteria [4749]. However, not all bifidobacteria are genetically adapted to utilize such milk-derived glycans [47, 50].

In order to identify the genomic repertoire involved in the utilization of HMO, lactose, and/or HMO-derived carbohydrates, such as fucose, lactose, sialic acid, fucosyllactose (2-FL and 3-FL), sialyllactose (3-SL and 6-SL), lacto-N-tetraose (LNT), and lacto-N-neotetraose (LNnT), we performed a comparative genomic analysis of all genomes decoded in this study. Our analysis revealed that six strains belonging to B. longum, B. breve, or B. bifidum possess a variety of genes allowing them to metabolize various HMO and HMO-derived glycans [47, 4951] (Fig. 4a). In contrast, these genes are absent in the genomes of B. adolescentis 1892B and B. dentium 1893B, except for the genes coding for β-galactosidase (Fig. 4a). Moreover, this analysis revealed that putative fucosidase- and sialidase-encoding genes are absent in the examined B. longum genomes, except for that of B. longum 1886B, which encompasses an ORF (B1886_0565) specifying a predicted sialidase. Furthermore, no predicted sialidase-encoding genes were identified in the genome of B. pseudocatenulatum 1896B, while no homologs of the gene encoding lacto-N-biose phosphorylase were detected in the chromosomes of B. pseudocatenulatum 1896B, B. longum 1897B, or B. catenulatum 1899B.
Fig. 4

Human milk oligosaccharide degradation capabilities of vertically transmitted bifidobacterial strains. a Heat map representation of the presence/absence of genes encoding enzymes for degradation of milk oligosaccharides and transporters for uptake of the released compounds. Red and black squares represent the presence and absence of genes, respectively. b Growth profile on HMO-derived carbohydrates. Black color indicates that the final OD600 nm is <0.3, yellow indicates the range of final OD600 nm from 0.3 to 0.5, light green indicates the range between >0.5 and 0.8, and green indicates the final OD600 nm >0.8

In order to validate these in silico data, we performed growth experiments involving the bifidobacterial strains that we isolated in this study using an mMRS medium containing various HMOs and HMO-derived glycans, i.e., LNT, LNnT, 3-SL, 6-SL, 2-FL, 3-FL, fucose, sialic acid, and lactose, as the sole carbon source (Fig. 4b). Based on these culturing experiments, we observed that the B. longum strains, B. bifidum 1887B, and B. breve strains were able to grow (OD values greater than 0.3) on LNT, LNnT, and lactose, while B. adolescentis 1892B and B. dentium 1893B were unable to grow on these carbohydrates, except for lactose. Moreover, only B. breve 1889B and B. breve 1895B were able to utilize sialic acid and fucose, while B. bifidum 1887B and B. longum subsp. infantis 1888B were able to utilize 3-SL, 6-SL, and 3-FL. Finally, only B. bifidum 1887B was able to degrade 2-FL. Notably, these data are consistent with current knowledge on bifidobacterial HMO/HMO-derived glycan utilization abilities [47, 49, 5155].

Interestingly, the results of these fermentation profiling experiments, correspond very well with those predicted from the in silico analyses of the genomes decoded in this study.

Genome reconstruction of shared bifidobacteria based on in silico analyses of microbiomes

In order to verify the occurrence of identical bifidobacterial strains in the microbiota of an infant’s fecal sample and in that of the corresponding milk and/or fecal sample of the mother, a shotgun metagenomic analysis was performed of three infant samples (TVPR-01, TVPR-02, and TVPR-03) for which we observed a high number of potentially vertically transmitted bifidobacterial strains. Taxonomic assignments (based on publicly available bifidobacterial genomic data) of the obtained microbiome data (i.e., shotgun metagenomic reads) corresponding to the TVPR-01, TVPR-02, and TVPR-03 infant fecal samples showed that 2.09, 9.02, and 12.16% of these shotgun metagenomic reads were classified as bifidobacterial DNA, respectively (Additional file 1: Table S4). Furthermore, taxonomic classification of these data sets determined that in sample TVPR-01, 1.56% of reads was taxonomically assigned to B. breve, while for TVPR-02, 6.16% of reads were annotated as B. dentium sequences, and finally, in the case of TVPR-03, 8.21% of reads were shown to correspond to B. dentium (Additional file 1: Table S4). Moreover, we reconstructed the complete genome sequences of each of the abovementioned bifidobacterial strains, by an in silico analysis of these three shotgun metagenomics datasets using a previously described, in-house developed pipeline [11, 34].

Genomic comparison of the isolated B. breve 1895B and the reconstructed B. breve TVPR-01 clearly showed the isogenic nature of both genomes, exhibiting an ANI value of 99.99%. Thus, using three different approaches, i.e., ITS profiling, strain isolation from fecal samples, and genome reconstruction from shotgun metagenomic data, we were able to confirm the essentially identical genetic composition of these bifidobacterial strains. Furthermore, we verified, through PCR-amplification of unique gene (Additional file 1: Table S3), that B. breve 1895B/TVPR-01 was shared between mother and child (Fig. 3). Unfortunately, despite several attempts, we were not able to isolate the B. dentium TVPR-02 and B. dentium TVPR-03 strains, most likely because of exigent nutritional requirements of these strains. Nonetheless, PCR amplification attempts targeting specific genes of TVPR-02 and TVPR-03, which were identified by means of comparative genomic analyses of the TVPR-02 and TVPR-03 microbiome-reconstructed genomes against the nine publicly available B. dentium strains, and confirmed the presence of these strains in corresponding mothers and infants (Additional file 3: Figure S2 and Additional file 1: Table S3). In order to avoid false positive results, we used isolated DNA from a mother stool sample of a non-corresponding dataset (Additional file 3: Figure S2). Consequently, these data clearly support the assumption of a vertical maternal inheritance of the most abundant bifidobacterial strains identified in the gut microbiota of children, as previously described in other studies [11, 15].

Bifidoprophages transmission from mother to child

Bacteriophages may also be transmitted from mother to child during the early phases of life, provided that the corresponding hosts for such phages are also passed across. Thus, we searched shotgun metagenomics data, obtained from stool samples of various mother and corresponding child and milk sample sets, for sequences that matched with known bacteriophages infecting bifidobacteria, i.e., bifidophages [24], focusing on datasets in which we had already identified bifidobacterial transmission. In order to identify the presence of both bifidophages as well as bifidoprophages, we scanned all obtained metagenomic datasets using a previously developed bioinformatics pipeline [11, 34]. Putative bifidobacterial prophage sequences were identified by manual examination of all genes belonging to the contigs attributed to bifidobacterial (sub)species [24, 56, 57]. The bifidobacterial phage/prophage screening revealed the presence of 21 putative bifido(pro)phages within nine samples that exhibit integrated or circular phage genome sequences (Table 3). A manually curated screening of the reconstructed phages unveiled the circular status of their genomes for eight bifidophages, highlighting overlapping sequence ends within the assembled contigs (Additional file 4: Figure S3). Interestingly, taxonomical gene classification based on NCBI RefSeq database matches reveal that these phages were predicted to belong to several bifidobacterial (sub)species, i.e., B. adolescentis, B. breve, B. dentium, B. longum, and B. pseudocatenulatum (Additional file 4: Figure S3). Since it was not possible to retrieve sequences of these identified bifidophages from each of the three components of a given metagenomic dataset (i.e., a corresponding mother and infant fecal samples and associated milk sample), we decided to utilize a PCR-based approach involving a PCR primer pair that was designed on unique sequences of a given phage (Additional file 1: Table S5). Furthermore, a second PCR primer pair was designed on the contig edges, in order to determine if the phage DNA was circular/concatameric or integrated into the host chromosome (Additional file 1: Table S5). As shown in Fig. 5, B. longum phage 10029 was identified in the fecal samples T3 and T4 of the same child as well as in the milk samples of the corresponding mother. While the use of the primer pair (P19-P20) results in the amplification of phage DNA in infant and milk samples, the second primer pair (P17-P18) unveiled the presence of a circular phage, yet only in sample T3 of the child. These data clearly indicates that this bifido(pro)phage was vertically transmitted by the mother through breastfeeding as part of the bifidobacterial host and was then apparently induced in the gut of the newborn. Furthermore, for samples Mum_2598, Infant_T3_2610, and Infant_T4_2613, the PCR amplification of both primer sets of the B. longum phage 10035 produced an amplicon (Fig. 5). This finding suggests the presence of the same bifidophage in both mother and infant microbiome, expanding the previous view of bifidobacterial phages multiplying in the infant gut thereby perhaps limiting overgrowth by their bifidobacterial host [24].
Table 3

Identified bifidophage/bifidoprophages within metagenomes

Samples

Species assignment

Genome status

TVPR3B

B. adolescentis

Integrated

B. adolescentis

Integrated

B. adolescentis

Circular

C10009IS2149FEt5

B. bifidum

Integrated

B. bifidum

Unknown

B. breve

Integrated

B. breve

Integrated

C10009MS2134FEt0

B. adolescentis

Circular

C10019IS2329FEt5

B. breve

Circular

C10029IS2503FEt3

B. longum subsp. longum

Unknown

B. longum subsp. longum

Unknown

B. longum subsp. longum

Unknown

B. longum subsp. longum

Unknown

B. longum subsp. longum

Circular

n.d.

Circular

C10029MS2492FEt0

B. pseudolongum

Circular

C10035IS2609FEt3

B. longum subsp. longum

Integrated

B. longum subsp. longum a

Circular

C10035IS2913FEt4

B. breve

Circular

B. longum subsp. longum a

Circular

C10035MS2598FEt0

B. longum subsp. longum

Unknown

n.d. Bifidobacterial species not detected, Unknown genome-status unknown

a B. longum phages that share the same genomic sequences between samples

Fig. 5

Vertically transmitted bifidophages from mothers to newborns. Indicated at the top of the figure are the genomic maps of the identified B. longum phage 10029 and 10035. The modular genomic structure is indicated by different patterns, which specifies their predicted function (violet, lysogeny module; blue, DNA replication; red, DNA packaging and head; green, tail and tail fiber; yellow, lysis module; black arrows, hypothetical protein; grey arrows, similar to bacterial protein). On the bottom are reported the presence of the phage within the mother’s and infant’s metagenomic samples through PCR analysis. Primer sequences are reported in Additional file 1: Table S5

Conclusions

Passage through the birth canal during delivery and subsequent breastfeeding are considered important events that affect microbial colonization of the gastrointestinal tract of the newborn [15, 45, 58, 59]. In this study, an NGS approach was employed to confirm the existence of a vertical transmission route of bifidobacterial communities in 25 mother and newborn pairs. Furthermore, our analyses highlight how bifidobacteria, being one of the dominant members of the infant gut microbiota, are inherited from the mother through a vertical transmission route, and imply human milk as a potential vehicle to facilitate this acquisition. Milk-mediated transmission of certain bifidobacterial strains may be supported by their ability to utilize HMOs and/or HMO-derived glycans. It is plausible that the first microbial colonizers of the human gut include commensals that are able to metabolize such milk glycans, such as the bifidobacterial strains belonging to B. bifidum species. However, these initial bifidobacterial colonizers may be important to promote the establishment of bifidobacterial colonizers that are not by themselves able to degrade and thus utilize milk carbohydrates. These bifidobacterial colonizers can access HMO degradation products, such as sialic acid and fucose, employing a cross-feeding behavior, which has been recently described [54, 55, 60, 61].

Another crucial microbial player involved in the establishment and maintenance of a so-called climax gut microbiota encompasses bacteriophages [19, 62]. Very recently, bifidophages have been discovered and their phage particles have been morphologically characterized [24]. However, their inheritance in the infant gut from the gut virome of the respective mother was only hypothesized but never experimentally demonstrated. Our data clearly highlight the existence of a vertical transfer route for bifidophages, thereby facilitating their spread from mother to newborn.

Abbreviations

2-FL: 

2-Fucosyllactose

3-FL: 

3-Fucosyllactose

3-SL: 

3-Sialyllactose

6-SL: 

6-Sialyllactose

GIT: 

Gastrointestinal tract

HMO: 

Human milk oligosaccharides

ITS: 

Internal transcribed spacer

LNnT: 

Lacto-N-neotetraose

LNT: 

Lacto-N-tetraose

MRS: 

de Man-Rogosa-Sharpe medium

OTUs: 

Operational taxonomic units

PBS: 

Phosphate-buffered saline

qRT-PCR: 

Quantitative real-time PCR

Declarations

Acknowledgements

We thank the operative units of Obstetrics and Gynecology and of Neonatology of the S. Chiara Hospital (Trento, Italy). Part of this research is conducted using the High Performance Computing (HPC) facility of the University of Parma.

Funding

This work was financially supported by Fondazione Caritro, Trento, Italy; GenProbio srl for the financial support of the Laboratory of Probiogenomics; and EU Joint Programming Initiative—A Healthy Diet for a Healthy Life (JPI HDHL, http://www.healthydietforhealthylife.eu/) to MV and DvS (Grant No. 15/JP/HDHL/3280) and MIUR to MV. SD is supported by Fondazione Caritro, Trento, Italy. LM is supported by Fondazione Cariparma, Parma, Italy. DvS is a member of The APC Microbiome Institute funded by Science Foundation Ireland (SFI), through the Irish Government’s National Development Plan (Grant No. SFI/12/RC/2273).

Availability of data and materials

Bifidobacterial ITS profiling data for all the sample sets are accessible through the accession number SRP102222. The bifidobacterial sequences reported in this article have been deposited in the GenBank database under accession numbers NAPZ00000000, NAQA00000000, NAQB00000000, NAQC00000000, NAQD00000000, NAQE00000000, NAQF00000000, NAQG00000000, NAQH00000000, NAQI00000000, NAQJ00000000, NAQK00000000, and NAQL00000000. The versions described in this paper are NAPZ01000000, NAQA01000000, NAQB01000000, NAQC01000000, NAQD01000000, NAQE01000000, NAQF01000000, NAQG01000000, NAQH01000000, NAQI01000000, NAQJ01000000, NAQK01000000, and NAQL01000000. Shotgun metagenomics data have been deposited in GenBank under the following accession numbers SRP102273.

Authors’ contributions

SD and GAL designed and performed the experiments and wrote the manuscript. LM, GAL, CM, EP, and MZ performed the bioinformatics analyses. FT, KJ, CF, MM, RS, and AV performed the experiments. FA, PF, VG, IL, RC, RC, MC, TC, GC, SF, SG, CI, CM, DP, DS, MS, ST, and AP provided the samples. MV, FT, and DVS conceived the study and revised and approved the manuscript. All authors reviewed the manuscript. All authors read and approved the final manuscript.

Competing interests

The authors declare that they have no competing interests.

Consent for publication

Not applicable.

Ethics approval and consent to participate

Stool and milk samples were collected in accordance with the protocols approved by the Ethical Committee of the “Azienda Provinciale per i Servizi Sanitari” in Trento, Italy, and Ethical Committee of the University of Parma, Italy.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors’ Affiliations

(1)
Laboratory of Probiogenomics, Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma
(2)
Centre for Integrative Biology, University of Trento
(3)
APC Microbiome Institute and School of Microbiology, Bioscience Institute, National University of Ireland
(4)
Azienda Provinciale per i Servizi Sanitari
(5)
GenProbio srl

References

  1. Sim K, Powell E, Shaw AG, McClure Z, Bangham M, Kroll JS. The neonatal gastrointestinal microbiota: the foundation of future health? Arch Dis Child Fetal Neonatal Ed. 2013;98:F362–4.View ArticlePubMedGoogle Scholar
  2. Azad MB, Konya T, Maughan H, Guttman DS, Field CJ, Chari RS, Sears MR, Becker AB, Scott JA, Kozyrskyj AL, Investigators CS. Gut microbiota of healthy Canadian infants: profiles by mode of delivery and infant diet at 4 months. Can Med Assoc J. 2013;185:385–94.View ArticleGoogle Scholar
  3. La Rosa PS, Warner BB, Zhou Y, Weinstock GM, Sodergren E, Hall-Moore CM, Stevens HJ, Bennett Jr WE, Shaikh N, Linneman LA, et al. Patterned progression of bacterial populations in the premature infant gut. Proc Natl Acad Sci U S A. 2014;111:12522–7.View ArticlePubMedPubMed CentralGoogle Scholar
  4. Dominguez-Bello MG, Costello EK, Contreras M, Magris M, Hidalgo G, Fierer N, Knight R. Delivery mode shapes the acquisition and structure of the initial microbiota across multiple body habitats in newborns. Proc Natl Acad Sci U S A. 2010;107:11971–5.View ArticlePubMedPubMed CentralGoogle Scholar
  5. Backhed F, Ley RE, Sonnenburg JL, Peterson DA, Gordon JI. Host-bacterial mutualism in the human intestine. Science. 2005;307:1915–20.View ArticlePubMedGoogle Scholar
  6. Turroni F, Taverniti V, Ruas-Madiedo P, Duranti S, Guglielmetti S, Lugli GA, Gioiosa L, Palanza P, Margolles A, van Sinderen D, Ventura M. Bifidobacterium bifidum PRL2010 modulates the host innate immune response. Appl Environ Microbiol. 2014;80(2):730–40. doi:10.1128/AEM.03313-13. Epub 2013 Nov 15.
  7. Zhang CH, Derrien M, Levenez F, Brazeilles R, Ballal SA, Kim J, Degivry MC, Quere G, Garault P, Vlieg JETV, et al. Ecological robustness of the gut microbiota in response to ingestion of transient food-borne microbes. ISME J. 2016;10:2235–45.View ArticlePubMedPubMed CentralGoogle Scholar
  8. Duranti S, Gaiani F, Mancabelli L, Milani C, Grandi A, Bolchi A, Santoni A, Lugli GA, Ferrario C, Mangifesta M, et al. Elucidating the gut microbiome of ulcerative colitis: bifidobacteria as novel microbial biomarkers. FEMS Microbiol Ecol. 2016;92(fiw191):1–12.Google Scholar
  9. Round JL, Mazmanian SK. The gut microbiota shapes intestinal immune responses during health and disease. Nat Rev Immunol. 2009;9:313–23.View ArticlePubMedPubMed CentralGoogle Scholar
  10. Martin R, Jimenez E, Heilig H, Fernandez L, Marin ML, Zoetendal EG, Rodriguez JM. Isolation of bifidobacteria from breast milk and assessment of the bifidobacterial population by PCR-denaturing gradient gel electrophoresis and quantitative real-time PCR. Appl Environ Microbiol. 2009;75:965–9.View ArticlePubMedGoogle Scholar
  11. Milani C, Mancabelli L, Lugli GA, Duranti S, Turroni F, Ferrario C, Mangifesta M, Viappiani A, Ferretti P, Gorfer V, et al. Exploring vertical transmission of bifidobacteria from mother to child. Appl Environ Microbiol. 2015;81:7078–87.View ArticlePubMedPubMed CentralGoogle Scholar
  12. Makino H, Kushiro A, Ishikawa E, Muylaert D, Kubota H, Sakai T, Oishi K, Martin R, Ben Amor K, Oozeer R, et al. Transmission of intestinal Bifidobacterium longum subsp. longum strains from mother to infant, determined by multilocus sequencing typing and amplified fragment length polymorphism. Appl Environ Microbiol. 2011;77:6788–93.View ArticlePubMedPubMed CentralGoogle Scholar
  13. Jost T, Lacroix C, Braegger CP, Rochat F, Chassard C. Vertical mother-neonate transfer of maternal gut bacteria via breastfeeding. Environ Microbiol. 2014;16:2891–904.View ArticlePubMedGoogle Scholar
  14. O’Sullivan A, Farver M, Smilowitz JT. The influence of early infant-feeding practices on the intestinal microbiome and body composition in infants. Nutr Metab Insights. 2015;8(Suppl 1):1–9.Google Scholar
  15. Makino H, Kushiro A, Ishikawa E, Kubota H, Gawad A, Sakai T, Oishi K, Martin R, Ben-Amor K, Knol J, Tanaka R. Mother-to-infant transmission of intestinal bifidobacterial strains has an impact on the early development of vaginally delivered infant’s microbiota. PLoS One. 2013;8:e78331.View ArticlePubMedPubMed CentralGoogle Scholar
  16. Scarpellini E, Ianiro G, Attili F, Bassanelli C, De Santis A, Gasbarrini A. The human gut microbiota and virome: Potential therapeutic implications. Dig Liver Dis. 2015;47(12):1007–12. doi:10.1016/j.dld.2015.07.008.
  17. Rodriguez JM. The origin of human milk bacteria: is there a bacterial entero-mammary pathway during late pregnancy and lactation? Adv Nutr. 2014;5:779–84.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Vaishampayan PA, Kuehl JV, Froula JL, Morgan JL, Ochman H, Francino MP. Comparative metagenomics and population dynamics of the gut microbiota in mother and infant. Genome Biol Evol. 2010;2:53–66.View ArticlePubMedPubMed CentralGoogle Scholar
  19. Ogilvie LA, Jones BV. The human gut virome: a multifaceted majority. Front Microbiol. 2015;6(918):1-12.Google Scholar
  20. Minot S, Sinha R, Chen J, Li HZ, Keilbaugh SA, Wu GD, Lewis JD, Bushman FD. The human gut virome: inter-individual variation and dynamic response to diet. Genome Res. 2011;21:1616–25.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Lim ES, Zhou YJ, Zhao GY, Bauer IK, Droit L, Ndao IM, Warner BB, Tarr PI, Wang D, Holtz LR. Early life dynamics of the human gut virome and bacterial microbiome in infants. Nat Med. 2015;21:1228-+.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Lim ES, Wang D, Holtz LR. The bacterial microbiome and virome milestones of infant development. Trends Microbiol. 2016;24:801–10.View ArticlePubMedGoogle Scholar
  23. Kramna L, Kolarova K, Oikarinen S, Pursiheimo JP, Ilonen J, Simell O, Knip M, Veijola R, Hyoty H, Cinek O. Gut virome sequencing in children with early islet autoimmunity. Diabetes Care. 2015;38:930–3.View ArticlePubMedGoogle Scholar
  24. Lugli GA, Milani C, Turroni F, Tremblay D, Ferrario C, Mancabelli L, Duranti S, Ward DV, Ossiprandi MC, Moineau S, et al. Prophages of the genus Bifidobacterium as modulating agents of the infant gut microbiota. Environ Microbiol. 2016;18:2196–213.View ArticlePubMedGoogle Scholar
  25. Human Microbiome Project C. Structure, function and diversity of the healthy human microbiome. Nature. 2012;486:207–14.View ArticleGoogle Scholar
  26. Milani C, Lugli GA, Turroni F, Mancabelli L, Duranti S, Viappiani A, Mangifesta M, Segata N, van Sinderen D, Ventura M. Evaluation of bifidobacterial community composition in the human gut by means of a targeted amplicon sequencing (ITS) protocol. FEMS Microbiol Ecol. 2014;90:493–503.PubMedGoogle Scholar
  27. Milani C, Hevia A, Foroni E, Duranti S, Turroni F, Lugli GA, Sanchez B, Martin R, Gueimonde M, van Sinderen D, et al. Assessing the fecal microbiota: an optimized ion torrent 16S rRNA gene-based analysis protocol. PLoS One. 2013;8:e68739.View ArticlePubMedPubMed CentralGoogle Scholar
  28. Milani C, Lugli GA, Duranti S, Turroni F, Mancabelli L, Ferrario C, Mangifesta M, Hevia A, Viappiani A, Scholz M, Arioli S, Sanchez B, Lane J, Ward DV, Hickey R, Mora D, Segata N, Margolles A, van Sinderen D, Ventura M. Bifidobacteria exhibit social behavior through carbohydrate resource sharing in the gut. Sci Rep. 2015;5:15782.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Lugli GA, Milani C, Mancabelli L, van Sinderen D, Ventura M. MEGAnnotator: a user-friendly pipeline for microbial genomes assembly and annotation. FEMS Microbiol Lett. 2016;363(fnw049):1–8.Google Scholar
  30. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, Lesin VM, Nikolenko SI, Pham S, Prjibelski AD, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol. 2012;19:455–77.View ArticlePubMedPubMed CentralGoogle Scholar
  31. Hyatt D, Chen GL, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. Bmc Bioinformatics. 2010;11(119):1–11.Google Scholar
  32. Zhao Y, Tang H, Ye Y. RAPSearch2: a fast and memory-efficient protein similarity search tool for next-generation sequencing data. Bioinformatics. 2012;28:125–6.View ArticlePubMedGoogle Scholar
  33. Lugli GA, Milani C, Mancabelli L, Turroni F, Ferrario C, Duranti S, van Sinderen D, Ventura M. Ancient bacteria of the Ötzi’s microbiome: a genomic tale from the Copper Age. Microbiome. 2017;5:5.View ArticlePubMedPubMed CentralGoogle Scholar
  34. Lugli GA, Milani C, Mancabelli L, Turroni F, Ferrario C, Duranti S, van Sinderen D, Ventura M. Erratum to: ancient bacteria of the Otzi’s microbiome: a genomic tale from the Copper Age. Microbiome. 2017;5:23.View ArticlePubMedPubMed CentralGoogle Scholar
  35. Turroni F, Peano C, Pass DA, Foroni E, Severgnini M, Claesson MJ, Kerr C, Hourihane J, Murray D, Fuligni F, et al. Diversity of Bifidobacteria within the infant gut microbiota. PLoS One. 2012;7(5/e36957):1–12.Google Scholar
  36. Arboleya S, Watkins C, Stanton C, Ross RP. Gut Bifidobacteria populations in human health and aging. Front Microbiol. 2016;7(1204):1–9.Google Scholar
  37. Heavey MP, Rowland RI. The gut microflora of the developing infant: microbiology and metabolism. Microb Ecol Health Dis. 1999;11:75–83.View ArticleGoogle Scholar
  38. Makino H, Martin R, Ishikawa E, Gawad A, Kubota H, Sakai T, Oishi K, Tanaka R, Ben-Amor K, Knol J, Kushiro A. Multilocus sequence typing of bifidobacterial strains from infant’s faeces and human milk: are bifidobacteria being sustainably shared during breastfeeding? Benef Microbes. 2015;6:563–72.View ArticlePubMedGoogle Scholar
  39. Jost T, Lacroix C, Braegger C, Chassard C. Assessment of bacterial diversity in breast milk using culture-dependent and culture-independent approaches. Br J Nutr. 2013;110:1253–62.View ArticlePubMedGoogle Scholar
  40. Jiménez E, María Marín L, Martín R, Odriozola JM, Olivares M, Xaus J, Fernández L, Rodríguez JM. Is meconium from healthy newborns actually sterile? Res Microbiol. 2008;159(3):187–93.Google Scholar
  41. Turroni F, Foroni E, Pizzetti P, Giubellini V, Ribbera A, Merusi P, Cagnasso P, Bizzarri B, de’Angelis GL, Shanahan F, et al. Exploring the diversity of the bifidobacterial population in the human intestinal tract. Appl Environ Microbiol. 2009;75:1534–45.View ArticlePubMedPubMed CentralGoogle Scholar
  42. Milani C, Lugli GA, Duranti S, Turroni F, Bottacini F, Mangifesta M, Sanchez B, Viappiani A, Mancabelli L, Taminiau B, et al. Genomic encyclopedia of type strains of the genus Bifidobacterium. Appl Environ Microbiol. 2014;80:6290–302.View ArticlePubMedPubMed CentralGoogle Scholar
  43. Rodriguez JM, Murphy K, Stanton C, Ross RP, Kober OI, Juge N, Avershina E, Rudi K, Narbad A, Jenmalm MC, et al. The composition of the gut microbiota throughout life, with an emphasis on early life. Microb Ecol Health Dis. 2015;26:26050.PubMedGoogle Scholar
  44. Fernandez L, Langa S, Martin V, Maldonado A, Jimenez E, Martin R, Rodriguez JM. The human milk microbiota: origin and potential roles in health and disease. Pharmacol Res. 2013;69:1–10.View ArticlePubMedGoogle Scholar
  45. Funkhouser LJ, Bordenstein SR. Mom knows best: the universality of maternal microbial transmission. PLoS Biol. 2013;11(8):1–9. e1001631.Google Scholar
  46. Smilowitz JT, Lebrilla CB, Mills DA, German JB, Freeman SL. Breast milk oligosaccharides: structure-function relationships in the neonate. Annu Rev Nutr. 2014;34:143–69.View ArticlePubMedPubMed CentralGoogle Scholar
  47. Sela DA, Chapman J, Adeuya A, Kim JH, Chen F, Whitehead TR, Lapidus A, Rokhsar DS, Lebrilla CB, German JB, et al. The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proc Natl Acad Sci U S A. 2008;105:18964–9.View ArticlePubMedPubMed CentralGoogle Scholar
  48. Turroni F, Foroni E, Serafini F, Viappiani A, Montanini B, Bottacini F, Ferrarini A, Bacchini PL, Rota C, Delledonne M, et al. Ability of Bifidobacterium breve to grow on different types of milk: exploring the metabolism of milk through genome analysis. Appl Environ Microbiol. 2011;77:7408–17.View ArticlePubMedPubMed CentralGoogle Scholar
  49. Asakuma S, Hatakeyama E, Urashima T, Yoshida E, Katayama T, Yamamoto K, Kumagai H, Ashida H, Hirose J, Kitaoka M. Physiology of consumption of human milk oligosaccharides by infant gut-associated bifidobacteria. J Biol Chem. 2011;286:34583–92.View ArticlePubMedPubMed CentralGoogle Scholar
  50. Sela DA. Bifidobacterial utilization of human milk oligosaccharides. Int J Food Microbiol. 2011;149:58–64.View ArticlePubMedGoogle Scholar
  51. Katayama T. Host-derived glycans serve as selected nutrients for the gut microbe: human milk oligosaccharides and bifidobacteria. Biosci Biotechnol Biochem. 2016;80:621–32.View ArticlePubMedGoogle Scholar
  52. Turroni F, Bottacini F, Foroni E, Mulder I, Kim JH, Zomer A, Sanchez B, Bidossi A, Ferrarini A, Giubellini V, et al. Genome analysis of Bifidobacterium bifidum PRL2010 reveals metabolic pathways for host-derived glycan foraging. Proc Natl Acad Sci U S A. 2010;107:19514–9.View ArticlePubMedPubMed CentralGoogle Scholar
  53. Turroni F, Strati F, Foroni E, Serafini F, Duranti S, van Sinderen D, Ventura M. Analysis of predicted carbohydrate transport systems encoded by Bifidobacterium bifidum PRL2010. Appl Environ Microbiol. 2012;78:5002–12.View ArticlePubMedPubMed CentralGoogle Scholar
  54. Egan M, Motherway MO, Kilcoyne M, Kane M, Joshi L, Ventura M, van Sinderen D. Cross-feeding by Bifidobacterium breve UCC2003 during co-cultivation with Bifidobacterium bifidum PRL2010 in a mucin-based medium. BMC Microbiol. 2014;14:282.View ArticlePubMedPubMed CentralGoogle Scholar
  55. Egan M, O’Connell Motherway M, Ventura M, van Sinderen D. Metabolism of sialic acid by Bifidobacterium breve UCC2003. Appl Environ Microbiol. 2014;80:4414–26.View ArticlePubMedPubMed CentralGoogle Scholar
  56. Ventura M, Turroni F, Lima-Mendez G, Foroni E, Zomer A, Duranti S, Giubellini V, Bottacini F, Horvath P, Barrangou R, et al. Comparative analyses of prophage-like elements present in bifidobacterial genomes. Appl Environ Microbiol. 2009;75:6929–36.View ArticlePubMedPubMed CentralGoogle Scholar
  57. Ventura M, Lee JH, Canchaya C, Zink R, Leahy S, Moreno-Munoz JA, O’Connell-Motherway M, Higgins D, Fitzgerald GF, O’Sullivan DJ, van Sinderen D. Prophage-like elements in bifidobacteria: insights from genomics, transcription, integration, distribution, and phylogenetic analysis. Appl Environ Microbiol. 2005;71:8692–705.View ArticlePubMedPubMed CentralGoogle Scholar
  58. Tsuji H, Oozeer R, Matsuda K, Matsuki T, Ohta T, Nomoto K, Tanaka R, Kawashima M, Kawashima K, Nagata S, Yamashiro Y. Molecular monitoring of the development of intestinal microbiota in Japanese infants. Benef Microbes. 2012;3:113–25.View ArticlePubMedGoogle Scholar
  59. Martin R, Makino H, Cetinyurek Yavuz A, Ben-Amor K, Roelofs M, Ishikawa E, Kubota H, Swinkels S, Sakai T, Oishi K, et al. Early-Life events, including mode of delivery and type of feeding, siblings and gender, shape the developing gut microbiota. PLoS One. 2016;11:e0158498.View ArticlePubMedPubMed CentralGoogle Scholar
  60. Turroni F, Ozcan E, Milani C, Mancabelli L, Viappiani A, van Sinderen D, Sela DA, Ventura M. Glycan cross-feeding activities between bifidobacteria under in vitro conditions. Front Microbiol. 2015;6(1030):1–8.Google Scholar
  61. Turroni F, Milani C, Duranti S, Mancabelli L, Mangifesta M, Viappiani A, Lugli GA, Ferrario C, Gioiosa L, Ferrarini A, et al. Deciphering bifidobacterial-mediated metabolic interactions and their impact on gut microbiota by a multi-omics approach. ISME J. 2016;10:1656–68.View ArticlePubMedGoogle Scholar
  62. Lozupone CA, Stombaugh JI, Gordon JI, Jansson JK, Knight R. Diversity, stability and resilience of the human gut microbiota. Nature. 2012;489:220–30.View ArticlePubMedPubMed CentralGoogle Scholar

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