FIG 3.

Contribution of contact-dependent T6SS-killing activity and contact-independent DNA release to the acquisition of the carbapenem resistance in mixed cultures. (A) Contribution of the T6SS to the frequency of recombinants generated in a mixed culture of 40288 and M2 Rif^r. 40288 was mixed with the M2 Rif^r strain or the M2 Rif^r Δhcp mutant, defective for T6SS activity. Imipenem-resistant transformants of the M2 strain were determined after 4 h or 24 h of mixed culture. Transformation frequencies represent the ratio of M2 Rif^r Imi^r CFU over the total count of M2 Rif^r CFU. (B) Contribution of contact-independent DNA release by 40288 to the acquisition of the imipenem resistance by M2 in mixed culture. Bacterial suspensions of M2 Rif^r at an OD₆₀₀ of 0.01 were mixed with an equal volume of either genomic DNA extracted from 40288 (at 200 ng/μL), a suspension of 40288 at an OD₆₀₀ of 0.01, or filtered spent medium of a 4-h culture in tryptone-NaCl medium of 40288. The mixture (2.5 μL) was deposited on the surface of tryptone-NaCl medium and incubated for 6 h at 37°C. Recombinant frequencies represent the ratio of Rif^r and Imi^r CFU over the total. (C) Contact-independent DNA release promotes both intra- and interstrain recombination. The frequency of intrastrain recombinants from M2 Rif^r and M2 Apra^r strains was determined in mixed culture or when M2 Rif^r was exposed to the spent medium of the M2 Apra^r strain. To test for interstrain recombination, M2 Rif^r was mixed with 40288 Apra^r or with the spent medium from that strain. Recombinant frequencies represent the ratio of Rif^r and Apra^r CFU over the total. (D) Fluorescence-based quantification of DNA present in the spent medium of strains M2 and 40288. Samples treated with DNase I represent the detection limit of the assay (∼0.4 ng). When displayed, P values of statistical analysis were obtained using the nonparametric Mann-Whitney-Wilcoxon test (two tailed). The limit of detection (10⁻⁸) is indicated by the gray area.