Before use, transconjugants were kept in buffered pepton containing 30% glycerol at -80°C. The donor E38.27 contained a second plasmid IncHI1, which was not transferred to the transconjugant T38.27. Resistance phenotypes of D, R and T were used in the experiments to select for D, R or T on selective plates, for quantification purpose. The IncI1 plasmid of E38.27 contains

two addiction factors pndAC and yacAC coding for Class II toxin-antitoxin (TA) systems (Dr Hilde Smith, personal ABT-737 in vivo communication). The antitoxins bind to toxins by protein-protein complex formation [17]. The antitoxins are less stable than the toxins, hence plasmid-free daughter cells will be killed after cell division. Experimental set up Three experiments were carried out. Firstly

selleck D, R and T were grown as single learn more populations from which growth parameters were determined. From the growth experiment with T, we also estimated plasmid loss. Secondly, experiments were done to estimate the conjugation coefficient and growth parameters in the presence of other bacterial populations. Thirdly, long-term dynamics were studied during a 3-months experiment. All experiments were conducted in static liquid cultures. Experiment 1 was conducted in 100 ml Erlenmeyer flasks and Experiments 2 and 3 in glass culture tubes. Start concentrations were determined by taking a sample directly after adding and mixing the inoculum in the medium.

Below we describe the experiment and an overview is listed in Additional file 2. Experiment 1 Single Celecoxib population experiments In experiment 1 growth curves of single populations of D, R and T were constructed from liquid cultures with two different start concentrations: 102 and 106 cfu/ml made in 25 ml Luria Bertani (LB) broth. Start concentrations were determined directly at the start of incubation by a colony count. The flasks were incubated at 37°C. Enumerations of D (experiment 1a,b,c,d), R (experiment 1e,f,g) and T (experiment 1h,i,j) were done by serial dilutions on selective plates. For the experiments with start concentration 102 cfu/ml this was done at 0, 2, 4, 6, 8, 24, 30 and 48 h after the start of the experiment, whereas for the experiments with start concentration 106 cfu/ml at 0, 1, 2, 3, 4, 6, 8, 24, 30 and 48 h after the start of the experiment. The growth rate, maximum density and lag-phase parameters were estimated from these data as described below in the section on the parameter estimation. Plasmid loss was determined along with the growth experiment of T (experiment 1i). At 4, 8 and 24 h, 94 colonies taken from the colony count plates of T, were each suspended in a single well of a 96 well microtitre plate (one colony per well) in LB broth. In the two remaining wells control isolates were suspended (T and D).