Evaluation of gut microbiota
Conventional stool culture methods were used to evaluate the predominant
bacterial flora in the stool samples. Fresh stool samples were
immediately transported to the laboratory for evaluation of microflora.
MacConkey agar and Blood Agar were used for isolation of aerobic
bacteria, whereas Robertson Cooked Meat medium (RCM), brain heart
infusion blood agar (BHIBA), Bacteriodes Bile Esculin agar (BBEA),
cefoxitin-cycloserine fructose agar (CCFA) and deManRogosa agar (MRSA)
were used for isolation of anaerobic bacteria. BHIBA is a general
enriched medium that supports the growth a wide range of anaerobic
bacteria. CCFA medium is a selective medium for Clostridiodes
difficile , with the addition of antibiotics- cycloserine and cefoxitin,
which inhibit the growth of other aerobic and anaerobic bacteria. To
detect spore bearing Clostridia spp. the stool samples were
subjected to alcohol shock treatment before culturing on CCFA, wherein
about O.5 ml. of the sample was first treated with equal amounts of
100% ethanol for 60 min. at 37 0C (alcohol treatment)
prior to plating. BBE medium is used for isolation of Bacteroidesspecies, the medium is rendered selective by addition of oxgall and
gentamicin, which inhibit growth of other bacteria. MRS medium is a
selective medium for Lactobacillus species. Further species
identification was done using matrix assisted laser desorption
ionization-time of flight mass spectrometry (MALDI-TOF MS bioMerieux).