Rapid assay to assess colonization patterns following in-vivo probiotic ingestion
© Tobin et al.; licensee BioMed Central Ltd. 2013
Received: 6 November 2012
Accepted: 3 July 2013
Published: 5 July 2013
Colonization of the intestine with some microorganisms has been shown to have beneficial health effects. The association of bacteria with its human host starts soon after birth; however in infants born prematurely establishment of normal intestinal flora is interrupted with colonization with potential pathogenic organisms Probiotic supplementation may therefore be beneficial to the health of preterm infants. As most probiotic organisms are difficult to culture, confirmation of their colonization after supplementation is difficult. In this study, rapid qPCR assays for detection of presence of probiotic species in the intestine by faecal sampling is described in both preterm infant and adult participants.
Probiotic colonization was determined using qPCR directed at amplification of organisms present in the ingested probiotic Streptococcus thermophilus, Bifidobacterium animalis subsp. lactis and B. longum subsp. infantis. Overall, differential detection of probiotic strains in faeces were found between adult and preterm infants, with 50% of infants continuing to shed at least two probiotic strains three weeks after probiotic ingestion had ceased.
This study demonstrated rapid assessment of the preterm infant gut for colonization with probiotic strains using real-time PCR. This method would be of great importance in studies of probiotics in prevention of diseases and adverse clinical outcomes.
The intestinal micro biota contains a diverse range of bacterial species . The bacterial community has also been shown to have a symbiotic relationship with their human host and is beneficial to human health [2, 3]. This association starts soon after birth . Bifidobacterium species (B. spp), commonly found in the intestine of healthy infants, may influence postnatal immune development, including susceptibility to sepsis and allergic diseases . In preterm infants, the development of the intestinal micro biome is affected by the neonatal intensive care unit environment, with decreased microbial diversity, including colonization with potential pathogens and association with late-onset sepsis and necrotizing enterocolitis [6, 7]. Supplementation with appropriate probiotic strains including B.spp may therefore be beneficial to the health of preterm infants, subject to confirmation of detection in fecal output and possible colonization. However, probiotic organisms are mostly fastidious anaerobes and difficult to culture using conventional techniques, as well as difficult to speciate, being closely related to other naturally occurring species . Rapid, sensitive, molecular tests can provide a faster and more accurate method to confirm micro biota colonization [9, 10]. In this study, changes in detection of in-vivo probiotic ingested organisms were measured using quantitative real-time PCR directed to specific detection of the three organisms present in the formulation.
Following approval by the Royal Women’s Hospital Human Research and Ethics Committees, 12 preterm infants born below 32 weeks gestation, weighing less than 1500 g and seven healthy adult volunteers were recruited to participate. Written informed consent for participation in the study was obtained from participants or, in cases of infants from their parent or guardian. The probiotic formulation, ABC Dophilus for Infants®, (Solgar, New York, USA) containing 2 × 108Bifidobacterium longum subsp. infantis, 2.3 × 108B. animalis subsp. lactis and 2.3 × 108Streptococcus thermophilus per gram was ingested by adult participants (15 g or 1010 organisms) and 6 preterm infants (1.5 g or 109 organisms) for seven days, while six control infants were fed routinely with their mother’s expressed breast milk.
Specimens for assay were collected by rotating a flocked swab in feces, and then rotated in 400 μL of phosphate buffered saline (PBS). Fecal samples were collected prior to and at completion of probiotic ingestion, then weekly up to 4 weeks and stored at −80°C until analyzed. Two hundred μl of cell suspensions were extracted using MagNA Pure LC system (Roche Diagnostics, Branchburg, NJ) with the associated DNA Isolation Kit I protocol. DNA was eluted in a final volume of 100 μL of MagNA Pure Elution Buffer (Roche Diagnostics). Extracted DNA was tested by three quantitative real-time PCR (qPCR) assays for detection of B. longum subsp. infantis, S. thermophilus and B. lactis. These assays are not strain specific and will only detect the respective species within the sample being tested.
PCR primer and probes sequences utilized in the study
B. animalis subsp. lactis
P: FAM-TTCACAGGTGGTGCATGGTCGT BHQ1
B. longum subsp. infantis
P: CY5-TCAAgCCCAggTAAggTTCTTCgC BHQ3
P: CY5-ACTACAAGATGGACCTGCGT BHQ3
In the adult group, S. thermophilus was present before and throughout the intervention in all participants, due to presence of this organism in readily available cheese and yoghurts. B. longum subsp. infantis was detected in feces of all 7 adult participants at Week 1, but not four weeks after probiotic ingestion had ceased, whereas B. animalis subsp. lactis was detectable in up to 40% of adults, 4 weeks post probiotic ingestion (Figure 1B).
It is important to test a pre-supplementation sample to ensure the subject is not positive for the probiotic organisms prior to its administration, as it is possible for gut micro biota to already contain the organism . This study demonstrated rapid assessment using real-time PCR of preterm infant gut for colonization of ingested probiotic strains. This method would be a valuable tool in studies evaluating probiotics in prevention of diseases and adverse clinical outcomes .
- Eckburg PB, Bik EM, Bernstein CN, Purdom E, Dethlefsen L, Sargent M, Gill SR, Nelson KE, Relman DA: Diversity of the human intestinal microbial flora. Science. 2005, 308 (5728): 1635-1638. 10.1126/science.1110591.PubMedPubMed CentralView ArticleGoogle Scholar
- Hooper LV, Bry L, Falk PG, Gordon JI: Host-microbial symbiosis in the mammalian intestine: exploring an internal ecosystem. Bioessays. 1998, 20 (4): 336-343. 10.1002/(SICI)1521-1878(199804)20:4<336::AID-BIES10>3.0.CO;2-3.PubMedView ArticleGoogle Scholar
- Mazmanian SK, Liu CH, Tzianabos AO, Kasper DL: An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell. 2005, 122 (1): 107-118. 10.1016/j.cell.2005.05.007.PubMedView ArticleGoogle Scholar
- Huurre A, Kalliomaki M, Rautava S, Rinne M, Salminen S, Isolauri E: Mode of delivery - effects on gut micro biota and humoral immunity. Neonatology. 2008, 93 (4): 236-240. 10.1159/000111102.PubMedView ArticleGoogle Scholar
- Prescott SL, Bjorksten B: Probiotics for the prevention or treatment of allergic diseases. J Allergy Clin Immunol. 2007, 120 (2): 255-262. 10.1016/j.jaci.2007.04.027.PubMedView ArticleGoogle Scholar
- Penders J, Thijs C, Vink C, Stelma FF, Snijders B, Kummeling I, van den Brandt PA, Stobberingh EE: Factors influencing the composition of the intestinal micro biota in early infancy. Pediatrics. 2006, 118 (2): 511-521. 10.1542/peds.2005-2824.PubMedView ArticleGoogle Scholar
- Schwiertz A, Gruhl B, Lobnitz M, Michel P, Radke M, Blaut M: Development of the intestinal bacterial composition in hospitalized preterm infants in comparison with breast-fed, full-term infants. Pediatr Res. 2003, 54 (3): 393-399. 10.1203/01.PDR.0000078274.74607.7A.PubMedView ArticleGoogle Scholar
- Léké A, Romond MB, Mullié C: Insights in the Human Bifidobacterial Flora Through Culture-Dependent and Independent Techniques. Communicating Current Research and Educational Topics and Trends in Applied Microbiology, Volume 2. Edited by: Méndez-Vilas A. 2007, Badajoz, Spain: Formatex, 758-765.Google Scholar
- Pammi M, Flores A, Leeflang M, Versalovic J: Molecular assays in the diagnosis of neonatal sepsis: a systematic review and meta-analysis. Pediatrics. 2011, 128 (4): e973-e985. 10.1542/peds.2011-1208.PubMedView ArticleGoogle Scholar
- Venkatesh M, Flores A, Luna RA, Versalovic J: Molecular microbiological methods in the diagnosis of neonatal sepsis. Expert Rev Anti Infect Ther. 2010, 8 (9): 1037-1048. 10.1586/eri.10.89.PubMedPubMed CentralView ArticleGoogle Scholar
- Furet JP, Quenee P, Tailliez P: Molecular quantification of lactic acid bacteria in fermented milk products using real-time quantitative PCR. Int J Food Microbiol. 2004, 97 (2): 197-207. 10.1016/j.ijfoodmicro.2004.04.020.PubMedView ArticleGoogle Scholar
- Matsuki T, Watanabe K, Tanaka R, Fukuda M, Oyaizu H: Distribution of bifidobacterial species in human intestinal microflora examined with 16S rRNA-gene-targeted species-specific primers. Appl Environ Microbiol. 1999, 65 (10): 4506-4512.PubMedPubMed CentralGoogle Scholar
- Ventura M, Reniero R, Zink R: Specific identification and targeted characterization of Bifidobacterium lactis from different environmental isolates by a combined multiplex-PCR approach. Appl Environ Microbiol. 2001, 67 (6): 2760-2765. 10.1128/AEM.67.6.2760-2765.2001.PubMedPubMed CentralView ArticleGoogle Scholar
- 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 guts micro biota. PLoS One. 2012, 7 (5): e36957-10.1371/journal.pone.0036957.PubMedPubMed CentralView ArticleGoogle Scholar
- Garland S, Tobin J, Pirotta M, Tabrizi S, Opie G, Donath S, Tang M, Morley C, Hickey L, Ung L, et al: The ProPrems trial: investigating the effects of probiotics on late onset sepsis in very preterm infants. BMC Infect Dis. 2011, 11 (1): 210-10.1186/1471-2334-11-210.PubMedPubMed CentralView ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.