DEC isolates were further characterised for their antimicrobial susceptibility and extended spectrum β-lactamase (ESBL) production. In addition, the EPEC isolates were characterised for their serotypes and intimin subtypes . Methods Subjects The subjects included 537 consecutive children hospitalised with acute diarrhoea (defined as three or more loose stools during a 24 h period with CP-690550 supplier duration of diarrhoea ≤ 14 days) and 113 control children without diarrhoea. The diarrhoeal children were hospitalised because of dehydration. The children
were up to five years of age and were recruited from Al-Adan Hospital (AH) or Al-Farwaniya Hospital (FH), Kuwait, during August 2005 to March 2007. Control children were admitted for non-gastrointestinal illnesses, but were matched for corresponding age of the diarrhoeal children. The children had not taken antibiotics prior to hospital admission and there was no follow-up of them after stool sample collection. Informed oral consent was given by the parents or guardians of children for the study as per local institutional guidelines. Stool samples A fresh stool specimen was collected from children with diarrhoea, and from control children without diarrhoea, as soon as after admission. It was promptly sent to the Microbiology Laboratory of each hospital where it was
cultured on Selleckchem AZD0156 MacConkey agar (Oxoid, Basingstoke, UK). The plate was incubated at 37°C for 24 h. The next day, the MacConkey plate (Oxoid) and the stool specimen were sent in a refrigerated box to Department of Microbiology, Faculty of Medicine, Kuwait University. Detection of DEC Entire E. LY2835219 price coli growth from MacConkey plate (including both about lactose fermenting and non-lactose fermenting colonies) was transferred to Luria broth (Becton Dickinson, Franklin Lakes, NJ, USA) containing 30% (vol/vol) glycerol, which was then frozen at -70°C until studied for detection of ETEC, EPEC, EIEC, EHEC and EAEC by PCR assays as described by Robins-Browne et al . For detection of these DEC, a loopful of the frozen culture was grown in 2.5 ml of MacConkey broth (Oxoid) in a shaker incubator at 37°C overnight. The pelleted bacterial
growth was washed in 1 ml of phosphate buffered saline (PBS)(pH, 7.2), resuspended in 200 μl sterile distilled water, and boiled for 10 min. After cooling on ice, bacteria were pelleted by centrifugation and supernatant stored for ≤ 1 week at -20°C before use. PCR reaction was carried out in a total volume of 25 μl using 5 μl of thawed supernatant diluted 1: 5 in PBS (pH, 7.2) as the template in all PCR reactions. Initially, the presence of E. coli was checked by PCR reaction for lacZ gene . If positive, then PCR assays for DEC were carried out. The primers and the PCR conditions corresponded to lac Z gene , eltA and estA genes (for ETEC), bfpA and eaeA genes (for EPEC), stx1and stx2 genes (for EHEC), and AggA gene (for EAEC)  and ipH gene (for EIEC) .