coli strains were isolated from the intestinal microflora of 1181 patients living in South Moravia, Czech Republic. A set of 183 E. coli strains was isolated at St. Anne’s University Hospital, Brno, CZ, and 998 E. coli strains at the University Hospital, Brno, CZ. E. coli strains were isolated between July 2007 and April 2010. 565 E. coli strains were isolated from female patients and 616 E. coli strains from males. All clinical Fer-1 cost samples were collected after patients gave informed consent. For children under the age of 18, consent was

obtained from parents. The study was approved by the ethics committee of the Faculty of Medicine, Masaryk University, Brno, CZ. A single isolate of E. coli was collected from each patient. Testing with ENTEROtest16 (Erba Lachema, Czech Republic) was used for bacterial identification. Indicator strains used for screening of bacteriocin production and the control bacteriocin producers used for PCR detection

of bacteriocin genes, were previously described in detail [21]. Screening of bacteriocin production Bacteriocin production was detected using the method described by Šmajs et al. (2010) [21]. Briefly, each of 1181 E. coli strains were simultaneously cultivated (37°C for 48 hours) in parallel on four different agar plates containing (i) TY (Trypton-yeast) agar (HiMedia, Mumbai, India) (1.5%, w/v, solid agar), (ii) TPCA-1 Difco™ Nutrient broth (Difco Laboratories, Sparks, MD, USA), (iii) TY agar supplemented with mitomycin C, and (iv) TY agar supplemented with trypsin. Macrocolonies were then killed using chloroform vapors and overlaid with a top TY agar layer (0.7%, w/v, soft agar) containing 107 cells from one of 6 indicator strains (E. coli K12-Row, E. coli C6 (φ), E. coli 5 K, E. coli P400, E. coli S40 and Shigella sonnei 17). The plates were subsequently incubated at 37°C for 24 hours and bacteriocin producers were identified. PCR detection

of genes encoding bacteriocins Detection of the 24 colicin and 7 microcin genes was carried out using the method described by Šmajs et Edoxaban al. (2010) [21]. Briefly, genomic DNA was isolated using DNAzol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. Template DNA was diluted 100-fold in sterile distilled water. All Selleckchem Verubecestat producer strains were tested, in parallel, using the colony PCR method (one bacterial colony from each strain was resuspended in 100 μl of sterile distilled water; then 1 μl of this suspension was added to the PCR mix). PCR reactions were performed using the primers described by Šmajs et al. (2010) [21]; for colicins E1, L and microcin M additional primer pairs were used (Additional file 2: Table S2). The following protocol was used for PCR amplification: 94°C (2 minutes); 94°C (30 seconds), 60°C (30 seconds), 72°C (1 minute), 30 cycles; 72°C (7 minutes). For colony PCR, the initial step was 5 minutes. Microcins H47 and M are sensitive to chloroform vapors [19], therefore all 539 bacteriocin-nonproducing E.