Change in Human Gut Flora and in Immune Functions Following Probiotic Administration

Study Subjects The study included 14 healthy non-pregnant women. All subjects provided a written informed consent. The subjects had to (i) be free of systemic (diabetes, autoimmune disease, cancer), gastrointestinal or liver diseases that are known to be associated with alterations in intestinal flora, (ii) be non-obese (body mass index in the range of 20 to 25 Kg/m2), and (iii) not have taken any anti-microbial agent, probiotics, gastric acid suppressant drugs or drugs that alter gastrointestinal motility, in the previous 6 weeks.

Study design Each subject was studied at 3 time points: (i) baseline (enrolment), (ii) after administration of a probiotic in usual dose for four weeks, and (iii) four weeks after discontinuation of probiotic administration. Each subject received Cap VSL#3, 2 capsules daily (each capsule contains 112.5 billion bacteria -- a mixture of 8 bacteria -- Streptococcus thermophilus, Bifido-bacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus delbrueckii). At each time point, gut microbiota profile and immune responses were studied.

Metagenomic study for analysis of gut flora Analysis for identification and profiling of gut microflora was done using sequencing of V3 region of the 16S ribosomal ribonucleic acid gene. This gene contains nine variable regions flanked by conserved stretches in all bacteria. Amplification and sequencing of any hypervariable region using specific primers can be used to determine the nature of the bacterium (phylum, family, genus, species, etc). The most widely used regions are V3, V4 and V6; we used V3 region, due to its higher taxonomic resolution.

Stool specimen were collected from subjects at 3 time points as indicated above by asking the subject to pass stool into a clean sterile receptacle; the receptacle was immediately frozen and transported to the laboratory. DNA was isolated from each specimen using standard protocols, quantified, normalised and stored frozen until further use.

Polymerase chain reaction amplification of V3 region was done. Gel-purified amplicons (with different adapter sequences so that data for each sample can be separated at analysis stage) were quantified, normalised and pooled in equimolar quantities (multiplexing). The multiplexed library was subjected to quality control using an Agilent Bioanalyser DNA Chip.