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).