Validating bifidobacterial species and subspecies identity in commercial probiotic products

Journal name:
Pediatric Research
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The ingestion of probiotics to attempt to improve health is increasingly common; however, quality control of some commercial products can be limited. Clinical practice is shifting toward the routine use of probiotics to aid in prevention of necrotizing enterocolitis in premature infants, and probiotic administration to term infants is increasingly common to treat colic and/or prevent atopic disease. Since bifidobacteria dominate the feces of healthy breast-fed infants, they are often included in infant-targeted probiotics.


We evaluated 16 probiotic products to determine how well their label claims describe the species of detectable bifidobacteria in the product. Recently developed DNA-based methods were used as a primary means of identification, and were confirmed using culture-based techniques.


We found that the contents of many bifidobacterial probiotic products differ from the ingredient list, sometimes at a subspecies level. Only 1 of the 16 probiotics perfectly matched its bifidobacterial label claims in all samples tested, and both pill-to-pill and lot-to-lot variation were observed.


Given the known differences between various bifidobacterial species and subspecies in metabolic capacity and colonization abilities, the prevalence of misidentified bifidobacteria in these products is cause for concern for those involved in clinical trials and consumers of probiotic products.

At a glance


  1. Figure 1:

    Mock community composition and measurement. The expected values (as defined by the ratios of input DNA initially measured by A260) and observed values for each of the 20 different mock communities assayed are shown here.

  2. Figure 2:

    Bifidobacterial composition of probiotic products by polymerase chain reaction–based methods. Each product was assayed four times, shown here in order grouped by product, lot 1 pill 1, lot 1 pill 2, lot 2 pill 1, lot 2 pill 2. Blank plot area not between two different products indicates no amplicon was detected.


  1. Fasoli S, Marzotto M, Rizzotti L, Rossi F, Dellaglio F, Torriani S. Bacterial composition of commercial probiotic products as evaluated by PCR-DGGE analysis. Int J Food Microbiol 2003;82:5970.
  2. Canganella F, Paganini S, Ovidi M, et al. A microbiology investigation on probiotic pharmaceutical products used for human health. Microbiol Res 1997;152:1719.
  3. Angelakis E, Million M, Henry M, Raoult D. Rapid and accurate bacterial identification in probiotics and yoghurts by MALDI-TOF mass spectrometry. J Food Sci 2011;76:M56872.
  4. Goldstein EJ, Citron DM, Claros MC, Tyrrell KL. Bacterial counts from five over-the-counter probiotics: are you getting what you paid for? Anaerobe 2014;25:14.
  5. Marcobal A, Underwood MA, Mills DA. Rapid determination of the bacterial composition of commercial probiotic products by terminal restriction fragment length polymorphism analysis. J Pediatr Gastroenterol Nutr 2008;46:60811.
  6. Patro JN, Ramachandran P, Lewis JL, et al. Development and utility of the FDA ‘GutProbe’ DNA microarray for identification, genotyping and metagenomic analysis of commercially available probiotics. J Appl Microbiol 2015;118:147888.
  7. Temmerman R, Pot B, Huys G, Swings J. Identification and antibiotic susceptibility of bacterial isolates from probiotic products. Int J Food Microbiol 2003;81:110.
  8. Huys G, Vancanneyt M, D’Haene K, Vankerckhoven V, Goossens H, Swings J. Accuracy of species identity of commercial bacterial cultures intended for probiotic or nutritional use. Res Microbiol 2006;157:80310.
  9. Madan JC, Farzan SF, Hibberd PL, Karagas MR. Normal neonatal microbiome variation in relation to environmental factors, infection and allergy. Curr Opin Pediatr 2012;24:7539.
  10. Zivkovic AM, Lewis ZT, German JB, Mills DA. Establishment of a Milk-Oriented Microbiota (MOM) in early life : How Babies Meet Their MOMs. Funct Food Rev 2013;5:312.
  11. La Rosa PS, Warner BB, Zhou Y, et al. Patterned progression of bacterial populations in the premature infant gut. Proc Natl Acad Sci USA 2014;111:125227.
  12. AlFaleh K, Anabrees J. Probiotics for prevention of necrotizing enterocolitis in preterm infants. Evid Based Child Health 2014;9:584671.
  13. Bokulich NA, Mills DA, Underwood MA. Surface microbes in the neonatal intensive care unit: changes with routine cleaning and over time. J Clin Microbiol 2013;51:261724.
  14. Brooks B, Firek BA, Miller CS, et al. Microbes in the neonatal intensive care unit resemble those found in the gut of premature infants. Microbiome 2014;2:1.
  15. Ohishi A, Takahashi S, Ito Y, et al. Bifidobacterium septicemia associated with postoperative probiotic therapy in a neonate with omphalocele. J Pediatr 2010;156:67981.
  16. Jenke A, Ruf EM, Hoppe T, Heldmann M, Wirth S. Bifidobacterium septicaemia in an extremely low-birthweight infant under probiotic therapy. Arch Dis Child Fetal Neonatal Ed 2012;97:F2178.
  17. Yatsunenko T, Rey FE, Manary MJ, et al. Human gut microbiome viewed across age and geography. Nature 2012;486:2227.
  18. Turroni F, Peano C, Pass DA, et al. Diversity of bifidobacteria within the infant gut microbiota. PLoS One 2012;7:e36957.
  19. Fukuda S, Toh H, Hase K, et al. Bifidobacteria can protect from enteropathogenic infection through production of acetate. Nature 2011;469:5437.
  20. Huda MN, Lewis Z, Kalanetra KM, et al. Stool microbiota and vaccine responses of infants. Pediatrics 2014;134:e36272.
  21. Romond MB, Colavizza M, Mullié C, et al. Does the intestinal bifidobacterial colonisation affect bacterial translocation? Anaerobe 2008;14:438.
  22. Chichlowski M, De Lartigue G, German JB, Raybould HE, Mills DA. Bifidobacteria isolated from infants and cultured on human milk oligosaccharides affect intestinal epithelial function. J Pediatr Gastroenterol Nutr 2012;55:3217.
  23. Sheil B, MacSharry J, O’Callaghan L, et al. Role of interleukin (IL-10) in probiotic-mediated immune modulation: an assessment in wild-type and IL-10 knock-out mice. Clin Exp Immunol 2006;144:27380.
  24. Tanabe S, Kinuta Y, Saito Y. Bifidobacterium infantis suppresses proinflammatory interleukin-17 production in murine splenocytes and dextran sodium sulfate-induced intestinal inflammation. Int J Mol Med 2008;22:1815.
  25. Preising J, Philippe D, Gleinser M, et al. Selection of bifidobacteria based on adhesion and anti-inflammatory capacity in vitro for amelioration of murine colitis. Appl Environ Microbiol 2010;76:304851.
  26. Underwood MA, Kalanetra KM, Bokulich NA, et al. A comparison of two probiotic strains of bifidobacteria in premature infants. J Pediatr 2013;163:15851591.e9.
  27. Sela DA, Chapman J, Adeuya A, et al. The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proc Natl Acad Sci USA 2008;105:189649.
  28. LoCascio RG, Ninonuevo MR, Freeman SL, et al. Glycoprofiling of bifidobacterial consumption of human milk oligosaccharides demonstrates strain specific, preferential consumption of small chain glycans secreted in early human lactation. J Agric Food Chem 2007;55:89149.
  29. LoCascio RG, Desai P, Sela DA, Weimer B, Mills DA. Broad conservation of milk utilization genes in Bifidobacterium longum subsp. infantis as revealed by comparative genomic hybridization. Appl Environ Microbiol 2010;76:737381.
  30. Garrido D, Kim JH, German JB, Raybould HE, Mills DA. Oligosaccharide binding proteins from Bifidobacterium longum subsp. infantis reveal a preference for host glycans. PLoS One 2011;6:e17315.
  31. Ganguli K, Meng D, Rautava S, Lu L, Walker WA, Nanthakumar N. Probiotics prevent necrotizing enterocolitis by modulating enterocyte genes that regulate innate immune-mediated inflammation. Am J Physiol Gastrointest Liver Physiol 2013;304:G13241.
  32. Schell MA, Karmirantzou M, Snel B, et al. The genome sequence of Bifidobacterium longum reflects its adaptation to the human gastrointestinal tract. Proc Natl Acad Sci USA 2002;99:144227.
  33. Lewis ZT, Bokulich NA, Kalanetra KM, Ruiz-Moyano S, Underwood MA, Mills DA. Use of bifidobacterial specific terminal restriction fragment length polymorphisms to complement next generation sequence profiling of infant gut communities. Anaerobe 2013;19:629.
  34. Ewaschuk JB, Diaz H, Meddings L, et al. Secreted bioactive factors from Bifidobacterium infantis enhance epithelial cell barrier function. Am J Physiol Gastrointest Liver Physiol 2008;295:G102534.
  35. Mattareli P, Bonaparte C, Pot B, et al. Proposal to reclassify the three biotypes of Bifidobacterium longum as three subspecies: Bifidobacterium longum subsp. longum subsp. nov., Bifidobacterium longum subsp. infantis comb. nov. and Bifidobacterium longum subsp. suis comb. nov. Int J Syst Evol Microbiol 2008;58:767772.
  36. Lewis ZT, Totten SM, Smilowitz JT, et al. Maternal fucosyltransferase 2 status affects the gut bifidobacterial communities of breastfed infants. Microbiome 2015;3:13.
  37. Lee JH, Karamychev VN, Kozyavkin SA, et al. Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth. BMC Genomics 2008;9:247.
  38. Ofek Shlomai N, Deshpande G, Rao S, Patole S. Probiotics for preterm neonates: what will it take to change clinical practice? Neonatology 2014;105:6470.
  39. Janvier A, Malo J, Barrington KJ. Cohort study of probiotics in a North American neonatal intensive care unit. J Pediatr 2014;164:9805.
  40. Donovan SM. Promoting bifidobacteria in the human infant intestine: why, how, and which one? J Pediatr Gastroenterol Nutr 2011;52:6489.
  41. Ruiz-Moyano S, Totten SM, Garrido DA, et al. Variation in consumption of human milk oligosaccharides by infant gut-associated strains of Bifidobacterium breve. Appl Environ Microbiol 2013;79:60409.

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Author information


  1. Department of Food Science and Technology, University of California, Davis, California

    • Zachery T. Lewis,
    • Guy Shani,
    • Chad F. Masarweh,
    • Steve A. Frese &
    • David A. Mills
  2. Foods for Health Institute, University of California, Davis, California

    • Zachery T. Lewis,
    • Guy Shani,
    • Chad F. Masarweh,
    • Steve A. Frese,
    • Mark A. Underwood &
    • David A. Mills
  3. School in Science and Technologies for Health Products, University of Modena and Reggio Emilia, Italy

    • Mina Popovic
  4. Department of Food Science, University of Massachusetts, Amherst, Massachusetts

    • David A. Sela
  5. Department of Pediatrics, University of California, Davis, California

    • Mark A. Underwood

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