Genomic Background Governs Opposing Responses to Nalidixic Acid upon Megaplasmid Acquisition in Pseudomonas

Abstract

Horizontal gene transfer is a significant driver of evolutionary dynamics across microbial populations. Although the benefits of the acquisition of new genetic material are often quite clear, experiments across systems have demonstrated that gene transfer events can cause significant phenotypic changes and entail fitness costs in a way that is dependent on the genomic and environmental context. Here, we test for the generality of one previously identified cost, sensitization of cells to the antibiotic nalidixic acid after acquisition of an ∼1-Mb megaplasmid, across Pseudomonas strains and species. Overall, we find that the presence of this megaplasmid sensitizes many different Pseudomonas strains to nalidixic acid but that this same horizontal gene transfer event increases resistance of Pseudomonas putida KT2440 to nalidixic acid across assays as well as to ciprofloxacin under competitive conditions. These phenotypic results are not easily explained away as secondary consequences of overall fitness effects and appear to occur independently of another cost associated with this megaplasmid, sensitization to higher temperatures. Lastly, we draw parallels between these reported results and the phenomenon of sign epistasis for de novo mutations and explore how context dependence of effects of plasmid acquisition could impact overall evolutionary dynamics and the evolution of antimicrobial resistance.IMPORTANCE Numerous studies have demonstrated that gene transfer events (e.g., plasmid acquisition) can entail a variety of costs that arise as by-products of the incorporation of foreign DNA into established physiological and genetic systems. These costs can be ameliorated through evolutionary time by the occurrence of compensatory mutations, which stabilize the presence of a horizontally transferred region within the genome but which also may skew future adaptive possibilities for these lineages. Here, we demonstrate another possible outcome, that phenotypic changes arising as a consequence of the same horizontal gene transfer (HGT) event are costly to some strains but may actually be beneficial in other genomic backgrounds under the right conditions. These results provide a new viewpoint for considering conditions that promote plasmid maintenance and highlight the influence of genomic and environmental contexts when considering amelioration of fitness costs after HGT events.

Keywords: antibiotic resistance; drug resistance evolution; horizontal gene transfer; megaplasmids; quinolones.

FIG 1

Kirby-Bauer diffusion assays recapitulate a nalidixic acid sensitivity phenotype in Pseudomonas stutzeri. We have previously demonstrated that acquisition of megaplasmid pMPPla107 increases sensitivity to nalidixic acid in two independent rifampin-resistant derivatives of P. stutzeri 28a24, DBL332 and DBL386. Here, we show that assays based on diffusion of nalidixic acid out of a disc recapitulate this phenotype. (A) Representative scans from a disc diffusion assay using (from left to right) a strain that either lacks (DBL386) or contains (DBL408) megaplasmid pMPPla107. Image areas covered by each of these two representative pictures (gray) and the sensitivity halo around the disc containing nalidixic acid and strain DBL386 (black) or strain DBL408 (white) are overlaid onto each other at the far right of this image. The larger the inhibition halo, the greater the sensitivity to nalidixic acid. (B) Data from disc diffusion overlay assays comparing P. stutzeri strains DBL332 and DBL386 with derivatives which have acquired megaplasmid pMPPla107. Each strain was assayed at least three independent times, with at least 2 (but usually 6) replicates per strain per assay. Halo size was normalized to the relevant “wild-type” strain within each assay (DBL332 for DBL453, DBL386 for DBL408) and plotted on the y axis. Individual data points are shown for all assays, with box hinges corresponding to first and third quartiles and means plotted as horizontal lines in the center of the boxes. Strains containing the megaplasmid are plotted in the gray boxes and are more sensitive to inhibition by nalidixic acid (F_1,36 = 132.143, P < 0.0001) than paired parental strains lacking the megaplasmid (plotted in white boxes). Additionally, there are clear statistical differences between the areas of the inhibition halos for each pair of megaplasmid^−/+ strains (DBL332/DBL453, t test, P < 0.001; DBL386/DBL408, t test, P < 0.001).

FIG 2

Acquisition of megaplasmid pMPPla107 sensitizes numerous Pseudomonas strains to nalidixic acid. Data are shown for disc diffusion overlay assays comparing P. stutzeri strains DBL880, DAB282, DBL883, and DBL885 with derivatives which have acquired megaplasmid pMPPla107 (DBL907, DBL287, DBL910, and DBL912, respectively). Data are also shown for derivatives of P. syringae pv. lachrymans strain 8003 and P. fluorescens Pf0-1 which either lack (DBL462 and DBL163, respectively) or contain (DBL895 and DBL877, respectively) megaplasmid pMPPla107. Strain names of the original isolates of genomic backgrounds lacking the megaplasmid are shown in gray headers. Data for “wild-type” strains are plotted in white boxes, while data for strains containing the megaplasmid are plotted in gray boxes. Each strain was assayed at least three independent times, with at least 4 (but usually 6) replicates per strain per assay. Two additional assays were performed for P. syringae strains. Halo size was normalized to the relevant “wild-type” strain within each assay and plotted on the y axis. Individual data points are shown for all assays, with box hinges corresponding to first and third quartiles and means plotted as horizontal lines in the center of the boxes. In each case, the presence of megaplasmid pMPPla107 sensitizes strains to nalidixic acid as shown by increased halos of inhibition. Asterisks indicate statistical significance by t test: ns (not significant) = >0.05, * = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001. All intrastrain comparisons between parent strains that lack the megaplasmid and derivative strains containing the megaplasmid are significantly different (P < 0.001) by t test with the exception of the Pf0-1 comparison, which was P < 0.05 by t test.

FIG 3

Acquisition of megaplasmid pMPPla107 by P. putida increases resistance to nalidixic acid but not ciprofloxacin in disc diffusion assays. Data are shown for disc diffusion overlay assays comparing P. putida strains DBL305 (KT2440) and DBL1604 (KT2442) with derivatives which have acquired megaplasmid pMPPla107 (DBL759 and DBL1620, respectively). Each strain was assayed at least three independent times for each antibiotic, with 6 replicates per strain per assay. Halo size was normalized to the relevant “wild-type” strain within each assay and plotted on the y axis. Individual data points are shown for all assays, with box hinges corresponding to first and third quartiles and means plotted as horizontal lines in the center of the boxes. Data for “wild-type” strains are plotted in white boxes, while data for strains containing the megaplasmid are plotted in gray boxes. (A) Presence of megaplasmid pMPPla107 increases resistance of P. putida strains to nalidixic acid as shown by decreased halos of inhibition (F_1,85 = 147.57, P < 0.0001). Moreover, there are clear statistical differences between the areas of the inhibition halos for each pair of megaplasmid^−/+ strains (DBL305/DBL759, t test, P < 0.001; DBL1604/DBL1620, t test, P < 0.001). (B) Presence of megaplasmid pMPPla107 appears to sensitize P. putida strains to ciprofloxacin as shown by increased halos of inhibition (F_1,80 = 12.99, P < 0.001). However, although ciprofloxacin halo inhibition areas statistically differ between strains DBL305 and DBL759 (t test, P < 0.001), there is no clear difference in the inhibition halos between strains DBL1604 and DBL1620 (t test, P = 0.29).

FIG 4

Contrasting effects of acquisition of megaplasmid pMPPla107 in P. putida across different environments as measured by competitive fitness assays. Competitive fitness assays in liquid KB medium were carried out between strain DBL305 (P. putida KT2440) and lacZ⁺ strains that either lack (DBL1604) or contain (DBL1620) megaplasmid pMPPla107. At least two competitive fitness assays were carried out for each pairing with 4 replicates per assay in all but one case (one assay at 40 μg/ml nalidixic acid had only 3 replicates). Individual data points are shown for all assays, with box hinges corresponding to first and third quartiles and means plotted as horizontal lines in the center of the boxes. Asterisks indicate statistical significance by t test: ns = >0.05, * = <0.05, ** = <0.01, *** = <0.001, **** = <0.0001. (A) Although there is no measurable difference in competitive fitness due to the lacZ⁺ marker (assays between DBL305 and DBL1604), the presence of the megaplasmid leads to an ∼10% fitness cost during growth in KB medium (F_1,20 = 600.3, P < 0.0001). (B and C) Although the ∼10% fitness cost of the megaplasmid is again apparent during competition between DBL305 and DBL1620 in unsupplemented KB medium, strain 1620 (containing megaplasmid pMPPla107) displays increasingly higher relative fitness than strain DBL305 when medium is supplemented with nalidixic acid at either 20 or 40 μg/ml (F_2,17 = 264.82, P < 0.0001; all comparisons significant at P < 0.05 by Tukey’s HSD) (B) or with ciprofloxacin at 0.5 μg/ml (F_1,18 = 178.89 P < 0.0001) (C). (D) The fitness cost of megaplasmid carriage greatly increases, to >50%, when competitions are carried out at higher temperatures of 37°C (F_1,18 = 319.38, P < 0.0001).