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Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets.

ABSTRACT: BACKGROUND:Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many studies, so that their prevalence and relative abundances are not accurately known. This may largely be due to difficulty in assigning subspecies identity using DNA sequences of 16S rRNA or tuf genes that are commonly used in bacterial taxonomy. METHODS:We developed a qPCR method targeting the sialidase gene (subsp. infantis) and sugar kinase gene (subsp. longum) to differentiate the subspecies using specific primers and probes. Specificity of the primers/probes was tested by in silico, pangenomic search, and using DNA from standard cultures of bifidobacterial species. The utility of the method was further examined using DNA from feces that had been collected from infants inhabiting various geographical regions. RESULTS:A pangenomic search of the NCBI genomic database showed that the PCR primers/probes targeted only the respective genes of the two subspecies. The primers/probes showed total specificity when tested against DNA extracted from the gold standard strains (type cultures) of bifidobacterial species detected in infant feces. Use of the qPCR method with DNA extracted from the feces of infants of different ages, delivery method and nutrition, showed that subsp. infantis was detectable (0-32.4% prevalence) in the feces of Australian (n = 90), South-East Asian (n = 24), and Chinese babies (n = 91), but in all cases at low abundance (<0.01-4.6%) compared to subsp. longum (0.1-33.7% abundance; 21.4-100% prevalence). DISCUSSION:Our qPCR method differentiates B. longum subspecies longum and infantis using characteristic functional genes. It can be used as an identification aid for isolates of bifidobacteria, as well as in determining prevalence and abundance of the subspecies in feces. The method should thus be useful in ecological studies of the infant gut microbiota during early life where an understanding of the ecology of bifidobacterial species may be important in developing interventions to promote infant health.

SUBMITTER: Lawley B

PROVIDER: S-EPMC5446769 | biostudies-literature | 2017

REPOSITORIES: biostudies-literature

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Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets.

Lawley Blair B Munro Karen K Hughes Alan A Hodgkinson Alison J AJ Prosser Colin G CG Lowry Dianne D Zhou Shao J SJ Makrides Maria M Gibson Robert A RA Lay Christophe C Chew Charmaine C Lee Pheng Soon PS Wong Khai Hong KH Tannock Gerald W GW

PeerJ 20170525

<h4>Background</h4>Members of the genus Bifidobacterium are abundant in the feces of babies during the exclusively-milk-diet period of life. Bifidobacterium longum is reported to be a common member of the infant fecal microbiota. However, B. longum is composed of three subspecies, two of which are represented in the bowel microbiota (B. longum subsp. longum; B. longum subsp. infantis). B. longum subspecies are not differentiated in many stu ...[more]

PMID: 28560114

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Project description:BackgroundHistorically, bifidobacteria were the dominant intestinal bacteria in breastfed infants. Still abundant in infants in developing nations, levels of intestinal bifidobacteria are low among infants in developed nations. Recent studies have described an intimate relationship between human milk and a specific subspecies of Bifidobacterium, B. longum subsp. infantis (B. infantis), yet supplementation of breastfed, healthy, term infants with this organism, has not been reported. The IMPRINT Study, a Phase I clinical trial, was initiated to determine the safety and tolerability of supplementing breastfed infants with B. infantis (EVC001).MethodsEighty mother-infant dyads were enrolled in either lactation support plus B. infantis supplementation (BiLS) or lactation support alone (LS). Starting with Day 7 postnatal, BiLS infants were fed 1.8-2.8 × 1010 CFU B. infantis EVC001 daily in breast milk for 21 days. Mothers collected fecal samples, filled out health questionnaires, and kept daily logs about their infants' feeding and gastrointestinal symptoms from birth until Day 61 postnatal. Safety and tolerability were determined from maternal reports.ResultsThere were no differences in the mean gestational age at birth, weight 1 and 2 months postnatal, and breast milk intake between groups. The mean Log10 change in fecal Bifidobacterium from Day 6 to Day 28 was higher (p = 0.0002) for BiLS (6.6 ± 2.8 SD) than for LS infants (3.5 ± 3.5 SD). Daily stool number was higher (p < 0.005) for LS and lower (p < 0.05) for BiLS infants during supplementation than at Baseline. During supplementation, watery stools decreased and soft stools increased by 36% over baseline in BiLS infants (p < 0.05) with no significant changes in stool consistency for the LS infants. None of the safety and tolerability endpoints, including flatulence, bloody stool, body temperature, ratings of gastrointestinal symptoms, use of antibiotics or gas-relieving medications, infant colic, jaundice, number of illnesses, sick doctor visits, or diagnoses of eczema were different for the groups at any point.ConclusionsThe B. infantis EVC001 supplement was safely consumed and well-tolerated. Stools were fewer and better formed in infants in the BiLS group compared with LS group. Adverse events were those expected in healthy infants and not different between groups.Trial registrationClinicalTrials.gov NCT02457338 . Registered May 27, 2015.

Project description:Attempts to alter intestinal dysbiosis via administration of probiotics have consistently shown that colonization with the administered microbes is transient. This study sought to determine whether provision of an initial course of Bifidobacterium longum subsp. infantis (B. infantis) would lead to persistent colonization of the probiotic organism in breastfed infants. Mothers intending to breastfeed were recruited and provided with lactation support. One group of mothers fed B. infantis EVC001 to their infants from day 7 to day 28 of life (n = 34), and the second group did not administer any probiotic (n = 32). Fecal samples were collected during the first 60 postnatal days in both groups. Fecal samples were assessed by 16S rRNA gene sequencing, quantitative PCR, mass spectrometry, and endotoxin measurement. B. infantis-fed infants had significantly higher populations of fecal Bifidobacteriaceae, in particular B. infantis, while EVC001 was fed, and this difference persisted more than 30 days after EVC001 supplementation ceased. Fecal milk oligosaccharides were significantly lower in B. infantis EVC001-fed infants, demonstrating higher consumption of human milk oligosaccharides by B. infantis EVC001. Concentrations of acetate and lactate were significantly higher and fecal pH was significantly lower in infants fed EVC001, demonstrating alterations in intestinal fermentation. Infants colonized by Bifidobacteriaceae at high levels had 4-fold-lower fecal endotoxin levels, consistent with observed lower levels of Gram-negative Proteobacteria and Bacteroidetes. IMPORTANCE The gut microbiome in early life plays an important role for long-term health and is shaped in large part by diet. Probiotics may contribute to improvements in health, but they have not been shown to alter the community composition of the gut microbiome. Here, we found that breastfed infants could be stably colonized at high levels by provision of B. infantis EVC001, with significant changes to the overall microbiome composition persisting more than a month later, whether the infants were born vaginally or by caesarean section. This observation is consistent with previous studies demonstrating the capacity of this subspecies to utilize human milk glycans as a nutrient and underscores the importance of pairing a probiotic organism with a specific substrate. Colonization by B. infantis EVC001 resulted in significant changes to fecal microbiome composition and was associated with improvements in fecal biochemistry. The combination of human milk and an infant-associated Bifidobacterium sp. shows, for the first time, that durable changes to the human gut microbiome are possible and are associated with improved gut function.

Dataset Information

Differentiation of Bifidobacterium longum subspecies longum and infantis by quantitative PCR using functional gene targets.

Publications

Differentiation of <i>Bifidobacterium longum</i> subspecies <i>longum</i> and <i>infantis</i> by quantitative PCR using functional gene targets.

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