24 and 48 h after inoculation, bacterial cells were collected and thoroughly resuspended by vortexing in phosphate-buffered saline (PBS). Thereafter, Lactobacillus and coliform concentrations in the co-cultures and in the controls was determined on MRS agar plates additioned with vancomycin (0.2% w/v) and MacConkey agar plates,

which are selective for Lactobacillus spp. and coliforms, respectively. Antimicrobial activity was calculated by comparing the coliform growth in the co-culture and control [8]. Results were expressed as log10 CFU/ml. The experiment was performed in triplicate. Statistical Analyses Sample size was calculated based on a difference between groups of 1.5 MK-4827 log10 CFU/g faeces. Using α = 0.05, β = 0.20 and an estimated standard deviation within groups of 2 log10 CFU/g faeces, 30 patients were needed in each group. Counts (log10 CFU/g) of the total amount of coliform bacteria were calculated for each stool sample. Data are summarized by counts

and median and range for categorical and continuous variables respectively. Differences between groups were evaluated with Mann-Whitney’s U-test for continuous variables, whereas associations between categorical variables were evaluated with Fisher’s exact test. Differences between colicky infants and controls in total amount of each species detected were evaluated with Mann-Whitney’s this website test with Bonferroni correction. Statistical significance was set at a p-value < 0.05. All statistical calculations were performed with commercially available software

(SPSS for Windows release 15Æ0 SPSS Inc., Chicago, IL, USA). Results Isolation and identification of coliforms from colicky infants Thalidomide Coliform colonies were obtained on MacConkey agar plates from faeces of all the 45 colicky infants and 42 controls. The average count of total coliforms in the 45 faecal samples of colicky infants was 5.98 (2.00-8.76) log10 CFU/g of faeces, whereas total coliforms in the control group were 3.90 (2.50-7.10) log10 CFU/g of faeces. The difference between the two groups was statistically significant (p = 0.015). A total of 145 colonies was randomly picked up from the higher dilutions agar plates (10-6-10-8) and, only from colicky infants after sub-culturing in LB agar, each purified strain was examined for gas production and characterized at species level by DNA sequencing and carbohydrate fermentation profiling. All isolated strains were found to produce gas from lactose according to the method described above and the BBL™ Enterotube™ II system. They were ascribed to six different species (Escherichia coli, Klebsiella oxytoca, Klebsiella pneumoniae, Enterococcus faecalis, Enterobacter aerogenes, and Enterobacter cloacae), as described in Table 3. The percentage of detection of each species in the faecal samples examined was reported in descending order (Table 3). The same taxonomic identification was obtained with the two methods employed.