Šmarda), E coli pCol5 and E coli pCol10 (H Pilsl) As microcin

Šmarda), E. coli pCol5 and E. coli pCol10 (H. Pilsl). As microcin control BAY 80-6946 clinical trial producers, the following bacterial strains were used: E. coli 449/82 pColX (microcin B17); E. coli 313/66 pColG (microcin H47); E. coli 363/79 pColV (microcin V, original source: H. Lhotová); E. coli TOP10F’

pDS601 (microcin C7); E. coli D55/1 (microcin J25); E. coli B1239 (microcin L, D. Šmajs). Cultivation conditions The ability to produce bacteriocins of all the strains was GF120918 cost tested in parallel on 4 different agar plates containing (i) TY medium, (ii) nutrient broth, (iii) TY medium supplemented with mitomycin C, and (iv) TY medium supplemented with trypsin. The rich TY medium consisted of yeast extract (Hi-Media, Mumbai, India) 5 gl-1, tryptone (Hi-Media) 8 gl-1, sodium chloride 5 gl-1; the TY agar consisted of a base layer (1.5%, w/v, solid agar) and a top layer (0.7%, w/v, soft agar). As a relatively unenriched medium, a Difco™nutrient broth (Difco Laboratories, Sparks, MD) 8 gl-1, NaCl 5 gl-1, was used for 1.5% (w/v) agar

plates. For induction of colicin production, the base agar layer was supplemented with 0.01% find more (w/v) mitomycin C. To test protease sensitivity of the inhibitive agents, 0.1% (w/v) trypsin was added to the base layer of agar. Detection of colicin producers The agar plates were inoculated by needle stab with fresh broth cultures and the plates were incubated at 37°C for 48 hours. The bacteria were then killed using chloroform vapors and each plate was then overlaid with

a thin layer of soft agar containing 107 cells ml-1 of an indicator strain. The plates were then incubated at 37°C overnight. All 772 E. coli strains of clinical origin were tested on four parallel plates against all 6 indicators, i.e. each strain underwent 24 individual tests. Identification tetracosactide of colicin and microcin types and determination of E. coli phylogenetic group Identification of individual colicin types (colicins A, B, D, E1-E9, Ia, Ib, Js, K, M, N, S4, U, Y, 5 and 10) was performed using PCR with primers designed using the Primer3 program [42] or with previously published primers [26]. The list of primer pairs and the corresponding length of PCR products are listed in Additional file 1. Total bacterial DNA was isolated using DNAzol (Invitrogen, Carlsbad, CA) reagent according to the manufacturer’s protocol. After 100-fold dilution, this DNA was used as a template for PCR reactions. Alternatively, all producer strains were tested with colony PCR. A bacterial colony was picked with a sterile inoculation loop and resuspended in 100 μl of autoclaved water. For each individual PCR reaction, 1 μl of cell suspension was added to the reaction. The PCR detection protocol was as follows: 94°C (2 minutes); 94°C (30 seconds), 60°C (30 seconds), 72°C (1 minute), 30 cycles; 72°C (7 minutes). For DNA amplification directly performed from lysed whole cells (colony PCR), the initial step was extended to 5 minutes (94°C, 5 minutes).

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