In Situ Monitoring and Quantitative Determination of R27 Plasmid Conjugation

Abstract

Horizontal gene transfer (HGT) by plasmid conjugation is a major driving force in the spread of antibiotic resistance among Enterobacteriaceae. Most of the conjugation studies are based on calculation of conjugation ratios (number of transconjugants/number of donors) after viable counting of transconjugant and donor cells. The development of robust, fast and reliable techniques for in situ monitoring and quantification of conjugation ratios might accelerate progress in understanding the impact of this cellular process in the HGT. The IncHI1 plasmids, involved in multiresistance phenotypes of relevant pathogens such as Salmonella and E. coli, are distinguished by the thermosensitivity of their conjugative transfer. Conjugation mediated by IncHI1 plasmids is more efficient at temperatures lower than 30 °C, suggesting that the transfer process takes place during the environmental transit of the bacteria. In this report, we described a methodology to monitor in situ the conjugation process during agar surface matings of the IncHI1 plasmid R27 and its derepressed derivative drR27 at different temperatures. A three-color-labeling strategy was used to visualize the spatial distribution of transconjugants within the heterogeneous environment by epifluorescence and confocal microscopy. Moreover, the fluorescent labelling was also used to quantify conjugation frequencies in liquid media by flow cytometry.

Keywords: IncHI; R27; flow cytometry; fluorophores; in situ monitoring; plasmid conjugation.

Figure 1

In situ monitoring of the R27 and drR27 transfer on solid surfaces using epifluorescence microscopy. Columns represent bright field (BF), epifluorescence (YFP* for recipient cells and CFP* for transconjugants), and overlay images of representative areas of agar surfaces inoculated with strain SAR08/pAR179Km carrying the R27 plasmid (upper row) or the drR27 plasmid (lower row) as a donor cells, and SAR20 as a recipient, after 36 h of incubation at 25 °C.

Figure 2

Confocal microscopy reveals the single cell level spatial organization of R27 (A) and drR27 transconjugants (B) emerging during mating on agar surfaces. The epifluorescence channels: CFP*, corresponding to the transconjugants (A1,B1), YFP*, corresponding to the recipient cells (A2,B2) and Dsred2.T3, corresponding to the donor cells (A3,B3), are shown. The representative mating areas are also displayed as overlay images (A4,B4). Images were taken on representative areas of agar surfaces inoculated with E. coli SAR20 as a recipient and SAR08/pAR179Km donor cells carrying plasmids R27 or the drR27, after 36 h of incubation at 25 °C. The scale bars represent 100 µm. (C) Frequency of conjugation of R27 and drR27 plasmids determined in liquid conjugation assays or on agar surface (plate) mating assays at 25 °C. Mean values of three independent experiments with standard deviations are plotted. Statistical differences between average values were tested by performing an unpaired, two-tailed Student’s t test. The resulting p-values are presented by the following symbols: n.s. = non-significant (p > 0.05); * p < 0.05.

Figure 3

In situ monitoring of the R27 and drR27 transfer on solid surfaces at 30 °C and 37 °C using epifluorescence microscopy. Columns represent bright field (BF), epifluorescence (YFP* for recipient cells and CFP* for transconjugants), and overlay images of representative areas of agar surfaces inoculated with E. coli SAR20 recipient and SAR08/pAR179Km donor cells carrying the R27 plasmid (upper row) or the drR27 plasmid (lower row) after 36 h of incubation at 30 °C or 18 h of incubation at 37 °C.

Figure 4

Confocal laser scanning microscopy (CLSM) of agar surface matings performed at 30 °C of R27 (A) and drR27 transconjugants (B). The epifluorescence channels: CFP*, corresponding to the transconjugants (A1,B1); YFP*, corresponding to the recipient cells (A2,B2) and Dsred2.T3, corresponding to the donor cells (A3,B3), are shown. The representative mating areas are also displayed as overlay images (A4,B4). Images were taken on representative areas of agar surfaces inoculated with E. coli SAR20 recipient and SAR08/pAR179Km donor cells carrying the R27 or the drR27 plasmids after 36 h of incubation at 30 °C. The scale bars represent 50.0 µm (A) and 50.2 µm (B), respectively.

Figure 5

CLSM of agar surface mating performed at 37 °C. The epifluorescence channels CFP*, corresponding to the transconjugants (1); YFP*, corresponding to the recipient cells (2) and Dsred2.T3, corresponding to the donor cells (3), are shown. The representative mating areas are also displayed as overlay images (4). Images were taken of a representative area of an agar surface inoculated with E. coli SAR20 recipient and SAR08/pAR179Km donor cells carrying the drR27 plasmids, after 18 h of incubation at 37 °C.

Figure 6

Using flow cytometer to quantify R27 transfer. Detected fluorescence by the cytometer using pure cultures of the donor strain (SAR08/pAR179Km), where no fluorescence is detected (A); the recipient strain (SAR20), emitting yellow fluorescence (B) and the strain DY330Nal carrying the plasmid drR27-cfp* (a ΔlacI strain which therefore allows the expression of cfp* present in the conjugative plasmid and that, dissimilar from the recipient strain, is not marked with YFP*) (C). Analysis of the conjugation frequency at 25 °C obtained using stationary phase donor cultures of SAR08/pAR179Km carrying R27 (D) or drR27 (E) plasmids, respectively. G3 quadrant shows the quantified donor cells, G4 quadrant shows the recipient cells and G2 quadrant shows the transconjugants. Selection within the H region was performed due to the low frequency of conjugation or the R27 plasmid, in order to ensure recipient cells are not been wrongly selected as transconjugants in that case. (F). Frequency of conjugation of R27 and drR27 plasmids in liquid medium at 25 °C, calculated by flow cytometry. Mean values of three independent experiments with standard deviations are plotted. Statistical differences between average values were tested by performing an unpaired, two-tailed Student’s t test. The resulting p-value is presented by the following symbol: ** p < 0.005. All bacterial cultures were grown in PB at 25 °C under static conditions.

Figure 7

Conjugation frequencies of the plasmid drR27 calculated by flow cytometry assay and traditional plate-based conjugation experiments, using donor cells at different time points during the growth curve (A). The OD_600nm of the cultures at 25 °C of the donor cells are indicated. O/N indicate cultures in late stationary phase (16 h incubation). Statistical differences between average values calculated by flow cytometry were tested by performing an unpaired, two-tailed Student’s t test. The resulting p-values are presented by the following symbols: * p < 0.05; ** p < 0.005. Growth curve of donor strain (B), a representative experiment is shown. All bacterial cultures were grown in PB at 25 °C under shaking conditions.