Environmental Microbiology (EM) | Microbial Ecology and Bioremediation
Microbiol. Biotechnol. Lett. 2021; 49(1): 1-8
https://doi.org/10.48022/mbl.2010.10011
Heetae Lee1, Chong-Kil Lee2 and Kyungjae Kim1*
1College of Pharmacy, Sahmyook University, Seoul 01795, Republic of Korea 2College of Pharmacy, Chungbuk National University, Cheongju 28644, Republic of Korea
Correspondence to :
Kyungjae Kim,
kimkj@syu.ac.kr
Human breast milk is a potential source of bacteria for the development of the intestinal microbiota of infants. Several species within the genera Lactobacillus and Bifidobacterium were demonstrated to shape the gut microbiota of infants. In this study, the bacterial diversity was investigated in the breast milk and feces of a mother-infant pair, and probiotic candidates were identified. Importantly, the novel L. gasseri EJL and B. breve JTL strains were isolated from breast milk and infant feces samples, respectively; their completed genome was resolved using de novo sequencing. In addition, the bacterial composition in the infant’s feces at 1 week revealed the prevalence of Bifidobacterium and Streptococcus; a higher diversity was observed after 3 weeks. In particular, the abundance of Akkermansia was sharply increased at 7 weeks, further increasing thereafter, up to 15 weeks. Our results suggest that human breast milk and infant’s feces are a source of probiotic candidates.
Keywords: Human breast milk, Lactobacillus gasseri, Bifidobacterium breve, gut microbiota, probiotics
Probiotics are defined as ‘live micro-organisms which when administered in adequate amounts confer a health benefit on the host’ [1]. An increasing number of studies have supported the health benefits of probiotics and highlighted the significant role of probiotics in the modulation of gut microbiota composition [2]. Dysbiosis of gut microbiota can lead to several diseases associated with host energy metabolism and immune system. Recent studies have highlighted the function of probiotics for health improvement [3, 4].
Human breast milk is the ultimate source of nutrition for newborns, and provides not only essential nutrition but also the micro-organisms for the development of initial intestinal microbiota [5, 6]. Several isolated bacterial species belonging to
Human breast milk and infant feces contain a variety of bacterial species. However, the potential candidates for use as beneficial probiotics have not been fully identified. This study aims to 1) identify the isolated potential probiotic candidates of
One mother-baby pair was recruited in this longitudinal study. The mother was a 35-year-old Korean (BMI: 22.4) and had no underlying diseases. Male infant was delivered at 39 weeks’ gestation by spontaneous vaginal delivery. Mother was administered a probiotic formulation (Duolac-Gold, Cell Biotech Co. Ltd.) daily 20 weeks before delivery and the administration was discontinued after delivery. Probiotics formulation included
Fecal samples and milk were collected from motherinfant pair at 1, 3, 5, 7, 9, 11, and 15 weeks. Prior to obtaining the milk samples using breast pump (after UV sterilization), the nipple and areola of the mother were cleaned with 70% isopropyl alcohol swabs. Fecal samples were collected in sterile containers and all samples were stored at -20℃ until shifting them to -70℃ in the laboratory. The study was approved by the Sahmyook University Institutional Review Board (2-104781-AB-N-012016115BR).
Bacterial total DNA was extracted using ExgeneTM Cell SV mini (GeneAll Biotechnology Co., Ltd.), according to manufacturer’s instructions. 16S rRNA gene was amplified using 27f/1492r primer set (forward: 5′-AGAGTTTGATCCTGGCTCAG-3′, reverse: 5′-GGTTACCTTGTTACGACTT-3′). Each 25 μl PCR reaction mixture contained 2.5 μl 10X IP-Taq buffer I, 0.2 μl forward primer (50 uM), 0.2 μl reverse primer (50 uM), 0.5 μl IP - Taq DNA polymerase (2.5 u/ul), 1 μl dNTPs (each 2.5 mM), 1 μl DNA template, and 19.6 μl distilled water. PCR was performed in a T100TM thermal cycler (Bio-Rad Laboratories, Inc.) using the following cycling conditions: initial denaturation at 95℃ for 5 min, 35 cycles of denaturation at 95℃ for 30 s, annealing at 50℃ for 30 s, and extension at 72℃ for 30 s. A final extension was performed at 72℃ for 10 min. Amplified PCR products were separated by electrophoresis on a 1% agarose gel, extracted using FavorPrepTM GEL/PCR Purification Mini Kit (Favorgen Biotech Co.). Sequence analysis of the products was contracted out to a Cosmo Genetech, South Korea. The sequences were compared to those in the GenBank database using the NCBI BLAST search program. Phylogenetic analysis was performed to confirm the taxonomy of the isolated genus
Library preparation and sequencing were conducted by Theragen Bio Itex, South Korea. Briefly, sequences of quality control-passed genomic DNA from isolates were produced by PacBio RS II sequencing system. Whole genome
Amplified PCR products of 16S rRNA gene from milk and infant feces were identified as novel strains of
The genomic attributes of
Table 1 . Genome features of
Attributes | Strains | |
---|---|---|
No. of bases | 2,289,039 | 2,289,549 |
G + C content (%) | 35.0 | 58.5 |
No. of contigs | 3 | 1 |
Coding genes (CDS) | 2,325 | 1,874 |
rRNA | 14 | 6 |
tRNA | 65 | 54 |
Infant’s fecal samples (n = 7) at 1, 3, 5, 7, 9, 11, and 15 weeks and mother’s fecal (n = 2) and milk (n = 2) samples at 1 and 15 weeks were analyzed for bacterial community. A total of 1,168,314 sequence reads were generated from 11 samples. An average of 97,360 ± 33,681 reads were covered per sample. The lowest Shannon diversity index was observed in infant’s feces at 1 week, which increased after 3 weeks. The Shannon diversity index of mother’s feces and milk were higher than that of infant’s feces (Fig. S1).
In mother’s fecal samples, the most abundant bacterial phyla at 1 and 15 weeks were
At the genus level,
The relative abundance of genus
Breast milk was identified as a source of abundant bacteria, using culture-independent genome sequencing. Two novel strains, namely
Breast milk is the potential source of bacteria for infant intestinal track (estimated as 1 × 105 to 1 × 107 bacteria daily in 800 ml/day of breast milk) [15]. Specific bacterial strains such as
Moreover, the composition of microbiota in breast milk varies within individuals and at different time periods, which in turn may be affected by the type of birth and maternal clinical factors [20, 21]. A recent study reported that
In this study,
Among the bacterial strains in probiotics formulation that the mother was administered during pregnancy,
In conclusion, novel strains of
This work was supported by the Sahmyook University Research Fund [RI12020003 (2020)].
The authors have no financial conflicts of interest to declare.
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