Effect of Probiotic Administration on Gut Flora Composition

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.

The sequencing library containing V3 amplicons from an equi-amount mixture of various clinical samples was sequenced using an Illumina machine in both directions. The sequence reads were binned according to index sequences, subjected to quality control and sequences in the two directions were fused together to obtain a single read. The sequence data were analysed to determine the profile of gut flora.

Immunological studies Collection of blood specimens Venous blood (6 ml) was collected in lithium heparin/EDTA, at (i) baseline (before starting probiotic administration), (ii) at the end of probiotic treatment (at 4 weeks), and (iii) at 4 weeks after discontinuation of probiotic intake. From 2.5 ml of blood, plasma was separated and stored at -70 degree centigrade. The remaining heparinized blood was used for whole blood culture and for measurement of frequencies of Th17 and Treg cells.

Heparinized blood was used and anti-CD28 (1 ug/ml) for stimulation of T cells and lipopolysaccharide for stimulation of macrophages, in separate wells. Culture supernatants were harvested after 72 hours and stored at -70 degree centigrade. Levels of cytokines (TNF-alpha, IL-10, IFN-gamma, IL-12p70, IL-6 and IL-4) were measured in culture supernatant and plasma using sandwich ELISAs.

Th1, Th2 and Th17 frequencies were determined by stimulation of whole blood with PMA and ionomycin, followed by staining of cells for CD4 and intracellular IFN, IL-4 and IL-1L-17. For Treg enumeration, dual staining for CD4 and Fox-P3 was done.

Ethics considerations The study involves administration of probiotics to healthy subjects. However, these contain bacteria that are a part of the normal gut flora in healthy persons and hence free of any adverse events. In fact, several healthy persons consume these as 'health supplements'. Hence, the administration of these agents should not carry more than minimal risk. The only specimens proposed to be collected are stool specimens and small volumes of blood. No clinical outcomes was collected.