FIG 2.

Acquisition of carbapenem resistance by natural transformation in a mixed culture. (A) Kinetics of emergence of carbapenem- and rifampicin-resistant recombinants in a mixed culture of M2 Rif^r and 40288 (Imi^r). Bacterial suspensions of M2 Rif^r at an OD₆₀₀ of 0.01 were mixed with either an equal volume of a suspension of 40288 at an OD₆₀₀ of 0.01 or gDNA extracted from 40288 (at 200 ng/μL). The mixture (2.5 μL) was deposited on the surface of tryptone-NaCl medium and incubated overnight at 37°C. Recombinant frequencies represent the ratio of Rif^r and Imi^r CFU over the total. The limit of detection (10⁻⁸) is indicated by the gray area. (B) Sensitivity of recombinant frequencies to DNase I treatment under the same conditions as for panel A, with a mixed culture of 6 h. (C) Recombinants emerge by the ComEC-dependent acquisition of carbapenem resistance by susceptible isolates. Rif^r isolates of M2, AYE, 27024, or their comEC mutants were mixed with the carbapenem-resistant (Imi^r) clinical isolates 40288 and AB5075 or their comEC mutants. Rif^r and Imi^r recombinants were determined after 24 h of mixed culture. The boxplots represent the distributions of Rif^r and Imi^r recombinants divided by the total CFU count (recombinant frequencies). A horizontal line represents the median. The limit of detection (10⁻⁸) is indicated by the gray area. In panel B, statistical analysis was conducted using the nonparametric Mann-Whitney-Wilcoxon test (two tailed). P values are indicated.