Effect of Probiotic Administration on Gut Flora Composition
Condition: Probiotics
Study type: Interventional

Summary: 
      Healthy human gut contains a large number of bacteria, which belong to several different
      species. Some genes in these bacteria encode enzymes that the human body cannot produce.
      These enzymes can catalyze metabolic reactions in the distal small bowel. For instance,
      bacterial enzymes can breakdown indigestible dietary constituents, making available extra
      energy to the host. The current paradigm treats the human body as a 'metagenome', i.e. a
      composite of Homo sapiens genes and genes in the genomes of the colonizing bacteria.

      Till recently, accurate determination of bacterial gut flora was not possible. Recent
      development of multi-parallel sequencing techniques has allowed unbiased determination of
      profile of gut flora. These techniques have revealed changes in gut flora in several disease
      conditions, including those of the gastrointestinal tract and liver. This has prompted the
      use of drugs, such as probiotics to restore the gut flora.

      Probiotics contain living microorganisms, and are administered in an attempt to obtain health
      benefits by restoring normal gut flora. These preparations provide benefit to patients with
      several diseases, including childhood diarrhea, antibiotic-associated diarrhea, inflammatory
      bowel disease, vaginitis, etc. However, the mechanisms of their beneficial effects remains
      unclear. Gut microbiota appear to modulate the development of immune system and maintain a
      balance between Th17 and T regulatory cells in animals. However, it is not known whether
      administration of probiotics changes the profile (nature and relative density of various
      species) of gut flora, and whether these changes are short-lasted or persistent.

      This proposal aimed to study whether probiotic administration influences the gut bacterial
      profile and host immune responses. In addition, we wished to determine whether the changes in
      gut flora and immune responses persist after probiotic administration is stopped.
    

Detailed description: 
      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.
    

Intervention name(s): Probiotic (VSL#3)
Intervention type(s): Dietary Supplement
Intervention description(s): Each subject provided morning stool and venous blood ubes) specimens at three time-points, i.e. at baseline (before probiotic administration), after probiotic administration (VSL#3®, one capsule twice a day) for 4 weeks, and at 4 weeks after stopping the probiotic intake. Each capsule contained approximately 112.5 billion live freeze-dried bacteria (a mixture of eight species -- Streptococcus thermophilus, Bifidobacterium breve, Bifidobacterium longum, Bifidobacterium infantis, Lactobacillus acidophilus, Lactobacillus plantarum, Lactobacillus paracasei, and Lactobacillus delbrueckii), which had been stored at 2-4ºC till ingestion.
Intervention model: Single Group Assignment
Intervention model description: Healthy young women will be given VSL#3 for 4 weeks
Masking: None (Open Label)
Primary purpose: Basic Science
Keywords: Probiotic; Gut microbiome; VSL#3