Quantitative real-time PCR analysis of fecal Lactobacillus species in infants receiving a prebiotic infant formula

Abstract

The developing intestinal microbiota of breast-fed infants is considered to play an important role in the priming of the infants' mucosal and systemic immunity. Generally, Bifidobacterium and Lactobacillus predominate the microbiota of breast-fed infants. In intervention trials it has been shown that lactobacilli can exert beneficial effects on, for example, diarrhea and atopy. However, the Lactobacillus species distribution in breast-fed or formula-fed infants has not yet been determined in great detail. For accurate enumeration of different lactobacilli, duplex 5' nuclease assays, targeted on rRNA intergenic spacer regions, were developed for Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri, and Lactobacillus rhamnosus. The designed and validated assays were used to determine the amounts of different Lactobacillus species in fecal samples of infants receiving a standard formula (SF) or a standard formula supplemented with galacto- and fructo-oligosaccharides in a 9:1 ratio (OSF). A breast-fed group (BF) was studied in parallel as a reference. During the 6-week intervention period a significant increase was shown in total percentage of fecal lactobacilli in the BF group (0.8% +/- 0.3% versus 4.1% +/- 1.5%) and the OSF group (0.8% +/- 0.3% versus 4.4% +/- 1.4%). The Lactobacillus species distribution in the OSF group was comparable to breast-fed infants, with relatively high levels of L. acidophilus, L. paracasei, and L. casei. The SF-fed infants, on the other hand, contained more L. delbrueckii and less L. paracasei compared to breast-fed infants and OSF-fed infants. An infant milk formula containing a specific mixture of prebiotics is able to induce a microbiota that closely resembles the microbiota of BF infants.

FIG. 1.

Comparison of two methods to determine L. acidophilus as percentage of total bacterial load in breast-fed infants (BF) and infants receiving a standard formula supplemented with GOS-FOS (OSF) or a standard formula (SF). Bars represent the standard error. Method A shows a combination of the data of L. acidophilus as a percentage of the total lactobacilli and the genus Lactobacillus as a percentage of the total bacterial load. Method B shows L. acidophilus as a percentage of the total bacterial load.

FIG. 2.

Lactobacilli as a percentage of the total bacterial load in fecal samples of breast-fed infants (BF) and infants who received a standard formula supplemented with GOS-FOS (OSF) or a standard formula (SF). Bars represent SE. ✽, significant difference (P < 0.05) between the BF and SF group or the OSF and SF group at the end of the study; #, significant increase (P < 0.05) during the study period.