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

Blair Lawley; Karen Munro; Alan Hughes; Alison J Hodgkinson; Colin G Prosser; Dianne Lowry; Shao J Zhou; Maria Makrides; Robert A Gibson; Christophe Lay; Charmaine Chew; Pheng Soon Lee; Khai Hong Wong; Gerald W Tannock

doi:10.7717/peerj.3375

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

PeerJ. 2017 May 25:5:e3375. doi: 10.7717/peerj.3375. eCollection 2017.

Authors

Blair Lawley¹, Karen Munro¹, Alan Hughes¹, Alison J Hodgkinson², Colin G Prosser³, Dianne Lowry³, Shao J Zhou^{4

5}, Maria Makrides⁶, Robert A Gibson⁵, Christophe Lay⁷, Charmaine Chew⁷, Pheng Soon Lee⁸, Khai Hong Wong⁸, Gerald W Tannock^{1

9

10}

Affiliations

¹ Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
² AgResearch, Ruakura, Hamilton, New Zealand.
³ Dairy Goat Cooperative (NZ) Ltd., Hamilton, New Zealand.
⁴ Women's and Children's Health Research Institute, Adelaide, Australia.
⁵ School of Agriculture, Food and Wine, University of Adelaide, Adelaide, Australia.
⁶ Healthy Mothers, Babies and Children, South Australian Health and Medical Research Institute, Adelaide, Australia.
⁷ Danone Nutricia Research, Biopolis, Singapore.
⁸ Mead Johnson Nutrition, Marina Bay, Singapore.
⁹ Riddet Institute Centre of Research Excellence, Palmerston North, New Zealand.
¹⁰ Microbiome Otago, University of Otago, Dunedin, New Zealand.

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.

Keywords: Bifidobacterium; Functional gene; Infantis; Longum; qPCR.

Grants and funding

The work was supported by MBIE contract UOOX1405. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.