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Corresponding author: Cecilia Dahlberg, Solna, S 171 82, Sweden., cecilia.dahlberg{at}smi.ki.se (E-mail)
Communicating editor: H. OCHMAN
 | ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Although plasmids can provide beneficial functions to their host bacteria, they might confer a physiological or energetic cost. This study examines how natural selection may reduce the cost of carrying conjugative plasmids with drug-resistance markers in the absence of antibiotic selection. We studied two plasmids, R1 and RP4, both of which carry multiple drug resistance genes and were shown to impose an initial fitness cost on Escherichia coli. To determine if and how the cost could be reduced, we subjected plasmid-containing bacteria to 1100 generations of evolution in batch cultures. Analysis of the evolved populations revealed that plasmid loss never occurred, but that the cost was reduced through genetic changes in both the plasmids and the bacteria. Changes in the plasmids were inferred by the demonstration that evolved plasmids no longer imposed a cost on their hosts when transferred to a plasmid-free clone of the ancestral E. coli. Changes in the bacteria were shown by the lowered cost when the ancestral plasmids were introduced into evolved bacteria that had been cured of their (evolved) plasmids. Additionally, changes in the bacteria were inferred because conjugative transfer rates of evolved R1 plasmids were lower in the evolved host than in the ancestral host. Our results suggest that once a conjugative bacterial plasmid has invaded a bacterial population it will remain even if the original selection is discontinued.
OUR capacity to decrease antibiotic resistance among clinically important bacteria depends in part on the fitness costs associated with the resistances. If a cost is associated with a resistance phenotype, sensitive strains should be able to invade and outcompete resistant bacterial populations when the use of antibiotics is reduced. However, several studies examining the effects of reducing antibiotic use have shown that for chromosomally encoded resistances the cost is often ameliorated by compensatory mutations without loss of resistance (
The potential cost to a bacterial host of plasmid-borne antibiotic resistance may be associated with the resistance function itself or with plasmid regulation. For instance, plasmid-borne antibiotic resistances often act through alteration or efflux of the antibiotic and these mechanisms may disturb bacterial growth. In addition, plasmid replication and gene expression may interfere with bacterial growth in ways independent of any resistance functions carried by that plasmid. These costs can be expected to be specific for each plasmid/bacteria combination.
In the absence of the selecting antibiotics, a cost associated with plasmid carriage has been observed for nonconjugative plasmids bearing antibiotic-resistance genes (
Since plasmids contribute greatly to antibiotic-resistance development in bacteria, it is fundamentally important to understand how plasmid-borne resistances may respond to a decreased use of antibiotics. This report focuses on two well-characterized plasmids with different replication systems, R1 (
100 kb) and RP4 (60 kb). We examine how the costs of these plasmids on an Escherichia coli host can be ameliorated by natural selection in the absence of antibiotics.
| MATERIALS AND METHODS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Strains and designations:
Table 1 lists the E. coli K12 strains and plasmids used in this study. The plasmids R1 and RP4 were selected for this study on the basis of their differences in replication control and host range. Plasmid R1 has an inhibitor-target mechanism of replication control and has a narrow host range whereas RP4 has an iteron-based control of replication and a very broad host range. A notation where b refers to the bacterial host and p to the plasmid it carries was used for this study. The clones used to found long-term serial cultures were hence designated b0p0 or b0 and clones isolated at the end of the experiment were designated b1p1 or b1* (b1* was used for clones that had evolved in the absence of plasmids to distinguish them from constructions where b1p1 were turned into b1 by curing). Further strain constructions made from these isolates were designated as shown in Fig 1 and Table 1. Strain J53-1 was used as the host in all long-term selection experiments. Strains CD104 and CD105 are derivatives of J53-1 where plasmid R1 or plasmid RP4 was, respectively, introduced by conjugation. Strain CD100 is a spontaneous mutant of J53-1 resistant to bacteriophage T5 and was used as a common competitor in all the pairwise competition experiments (see below).
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Growth media:
The bacterial populations were propagated in Davis minimal (DM) medium (5 x 108 cells/ml. DM agar with a glucose concentration of 1000 µg/ml was used for plating. Final concentrations of antibiotics in Luria agar were 100 µg/ml nalidixic acid, 25 µg/ml kanamycin sulfate, 10 µg/ml tetracycline, 25 µg/ml chloramphenicol, 20 µg/ml streptomycin, and 100 µg/ml ampicillin. For phage resistance selection, a lysate of phage T5 corresponding to 109 phages was spread onto DM plates prior to spreading of the bacteria.
Long-term serial cultures:
A schematic presentation of the experimental evolution and the construction of new plasmid-host combinations are shown in Fig 1. Three replicate populations 1, 2, and 3 of CD104 and three replicate populations 4, 5, and 6 of CD105 were initiated from stationary-phase cultures grown in LB with selective antibiotics for the respective plasmid. Following the notation developed for this study (see Strains and designations), the bacteria and plasmid used to start either of these triplicate populations were all designated b0p0. Population 7 was started with the plasmid-free J53-1 and the starting clone was given the designation b0. The populations were serially propagated every 24 hr with 40 µl of culture into 4 ml of fresh DM without selecting antibiotics in 16-mm tubes. The cultures were incubated at 37° in a slanted rack, shaking at 250 rpm. Aerosol-resistant filter tips were used in all work associated with transfers of the evolving populations. The transfers continued for 166 days corresponding to 1100 generations. About every 50 generations the presence of the plasmids was determined on the appropriate antibiotic plates for 100 colonies from each population. Plasmid isolation was used to verify the presence of plasmids if resistance markers were absent. One random clone from each end-population was isolated for further analysis [CD111–116 (b1p1) and CD117 (b1*) (Table 1)]. Clones with new resistance phenotypes that had evolved during the experiment were also isolated (CD139 and CD142–143) and further analyzed when they represented >5% of the total population. All clones isolated at the end of the experiment are referred to as evolved in the following text.
Curing and construction of new plasmid-host combinations from evolved clones:
The plasmids from the evolved clones were transferred into the ancestral host J53-1. Transfer was done either by conjugation in two steps, where E. coli LE392-1 served as an intermediate host, or by transformation using the CaCl2 procedure (
Estimates of fitness:
Pairwise competition experiments were performed against a common competitor, CD100. The competing clones were separately preconditioned in DM for one 24-hr cycle. For each competition experiment cultures were inoculated with the preconditioned cultures of the competitors at a 1:1 ratio and were then propagated for one 24-hr cycle as described above. The initial and final ratios of the two competitors were assayed on DM plates with and without phage T5. The relative fitness (see method of fitness estimation below) of the T5-resistant common competitor was 0.992 ± 0.014 relative to the J53-1 ancestor. At least 10 independent competitions were conducted for each strain. An increased sensitivity can also be obtained by measuring fitness over several cycles but this method was avoided to minimize potential effects of plasmid transfer between the competitors.
Calculation of Malthusian parameter:
Relative fitness (W) was determined as described by
where Ni(0) and Nj(0) are the initial densities of the tested clone and the common competitor, respectively, and Ni(1) and Nj(1) are their corresponding final densities.
Determination of relative copy number changes:
Minimal inhibitory concentrations of ampicillin were determined for the ancestral and evolved strains as an indirect measure of plasmid copy number (
Phenotypic and molecular analysis of evolved plasmids:
The resistance phenotype of 100 isolates from each end population was determined by replica plating on antibiotic-containing media. Restriction enzyme analysis was performed on plasmid DNA that was isolated by alkaline lysis (
| RESULTS |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Plasmid stability and general fitness changes:
Plasmid-free cells were never detected in the populations founded by R1- or RP4-bearing clones over the 1100 generations of selection in an antibiotic-free environment. The plasmids initially decreased the fitness of E. coli J53-1, with 6% (t-test, P < 0.002) for R1 (CD104, b0p0) and 21% (P < 0.001) for RP4 (CD105, b0p0; Table 2). The random clones isolated at the end of the experiment (b1p1) had increased their fitness, with up to 70% as compared to their plasmid-bearing ancestors (b0p0; Table 2).
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Compensatory evolution:
The respective contributions of host- and plasmid-encoded mutations to the general fitness increase observed in the evolved clones (b1p1) were measured on combinations of ancestral and evolved hosts and plasmids.
The costs of the evolved plasmids were tested in both the evolved and ancestral host backgrounds. With one exception (see below, clone CD122) there were no significant differences in fitness between the evolved clones (b1p1) and the evolved clones cured of their plasmids (b1). The cost of the evolved plasmids on the ancestral host (b0p1) was further tested and showed that five of the six evolved plasmids had a lower cost than the corresponding ancestral plasmid (b0p0). In contrast, one of the evolved plasmids, the R1 derivative in clone CD112, showed an increased cost on the ancestral host (CD131, 17%, P < 0.001).
The effect of host evolution was assessed by measuring the cost of the ancestral plasmids on the evolved hosts. Where the ancestral plasmids were transferred to the evolved hosts that were cured of their evolved plasmids (b1p0) we found no significant costs of the plasmids. For the host that had evolved in the absence of plasmids reduced costs of the ancestral plasmids (b1*p0) were demonstrated, as compared to the costs on the ancestral host (b0p0). For the ancestral plasmid R1 on this host (CD148, b1*p0), we found no significant cost and the ancestral plasmid RP4 (CD148, b1*p0) had reduced its cost to 9% (P < 0.002; Table 2).
Control experiments where the evolved plasmid was reintroduced into the cured host (i.e., reconstruction of b1p1 from b1) showed that the curing procedure did not affect fitness (data not shown) with one exception. This cured clone CD122 showed a higher fitness when its plasmid had been reintroduced compared to the fitness of the plasmid-bearing clone before curing.
A second problem we encountered when curing the evolved clones was observed in the evolved RP4-bearing clone CD116 (b1p1). During curing when this clone was transformed with the plasmid pFF1ts97 that carries the same basic replicon as the ancestral RP4, the expected loss of the resident evolved RP4 plasmid was not seen. Both plasmids were still present, as determined by plasmid isolation, after several successive platings on media selective for pFF1ts97.
Phenotypic evolution:
Clones that had lost one or more antibiotic resistances emerged in all R1 populations but represented a minority of the cells (Table 3). The plasmids from representatives of these minority phenotypes were further analyzed by restriction endonuclease digestion and showed loss of restriction fragments of the size corresponding to the respective antibiotic-resistance genes (Fig 2A;
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Several changes in the evolved plasmids from the randomly drawn clones were also revealed. The RP4 isolates CD114 and CD115 (b1p1) differed from the ancestral plasmid in their restriction endonuclease digestion patterns (Fig 2B) and showed several novel restriction sites in the transfer and replication regions (data not shown). These changes were probably associated with transposition of chromosomally located IS elements into the plasmids. For the plasmids in both CD114 and CD115 possible insertions into the trbE region that is involved in pilus production were found (
The evolved RP4 plasmids in clones CD114 and CD115 were transfer deficient in both the evolved and the ancestral host. The evolved RP4 plasmid in clone CD116 had retained its transferability but the transfer rates of this plasmid and of the ancestral RP4 could not be determined for the liquid culture conditions used in these experiments, as they did not exceed the transfer that occurred after plating on the selective media. The evolved R1 clones (b1p1) showed lower transfer rates compared to the ancestral R1 clone (b0p0). The reduction in transfer rate of the evolved plasmids was not seen in the ancestral host (b0p1; Table 4). Transfer of the ancestral plasmid R1 was also measured from the host that had evolved in the absence of plasmids, CD148 (b1*p0). This transfer rate was of the same magnitude as transfer from the ancestral host (b0p0; Table 4).
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The values obtained by the two different methods used for copy number measurements did not fully correlate. Only when both methods showed a similar deviation in copy number was an average of these values used to assign changes in copy number. The RP4 derivative in clone CD116 (b1p1) showed an increase in copy number of 70% and the R1 clone CD111 (b1p1) showed a 20% reduction in copy number (Table 5).
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| DISCUSSION |
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| TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Many antibiotic-resistance genes are carried by conjugative plasmids but studies to date on compensatory evolution of antibiotic resistance have focused on genes encoded either on chromosomes or on nonconjugative plasmids (1100 generations by serial transfer. The cost associated with plasmid carriage before and after experimental evolution was determined in competition experiments using a plasmid-free common competitor. Both plasmids R1 and RP4 imposed fitness costs on E. coli J53-1 at the start of the experiment (Table 2). At the end of the experiment all tested clones showed an increased fitness compared to the respective ancestral plasmid-host combination (Table 2). Genetic adaptation to the general growth conditions was expected to account for part of this fitness increase but changes in response to the cost of plasmid carriage were also anticipated.
Reduction of the cost of plasmid carriage can be accomplished through two main processes: segregation and compensatory mutation. For plasmid-borne antibiotic resistance segregation will lead to the loss of resistance by the formation of plasmid-free cells whereas compensatory mutations may or may not abolish resistance. Both segregation and compensatory mutation are likely to occur in a large population, resulting in a race between the two. Under conditions of antibiotic-resistance selection, compensatory mutation is primarily available, but under conditions where antibiotic selection is released it is unclear which of these processes will dominate.
Like most natural conjugative plasmids, the plasmids used in this study have stability systems such as postsegregational killing and partitioning that make their segregation rates very low (
Since segregation was not detected in these experiments we directed our efforts to examine the effect of compensatory evolution. The first indication of compensatory mutations was seen when the fitness of plasmid-free clones (b1) constructed from evolved plasmid-bearing clones (b1p1) was measured. These measurements showed no difference in fitness between the pairs of plasmid-carrying (b1p1) and plasmid-free (b1) clones with one exception (see below, clone CD122; Table 2). Compensatory mutations in either the chromosome or the plasmid could explain these results. The costs of five of the six tested evolved plasmids on the ancestral host (b0p1) were lower than the costs of the corresponding ancestral plasmid (b0p0; Table 2), suggesting that these evolved plasmids had acquired compensatory mutations. The fact that four of six evolved plasmids had no cost at all suggests that compensatory mutations on the plasmid may prevent selection for segregants and hence plasmid loss from the population.
If compensatory mutations on the evolved plasmids were alone responsible for the decreased cost one would expect that the ancestral plasmids would still impose a similar cost on the evolved hosts as on the ancestral host. This was, however, not seen; instead we found that the ancestral plasmids had no significant cost on the evolved hosts that were cured of their evolved plasmids (b1p0; Table 2). This result indicates that additional compensatory mutations had occurred in the chromosome. The surprising aspect of these results lies in the apparent redundancy of the compensatory mutations shown by the compensatory mutations occurring in both the plasmid and the host. When a mutation compensatory for the cost of plasmid carriage occurs on either the host or the plasmid replicon, selection for a second compensatory mutation on the second replicon should be abolished.
The host that evolved in the absence of plasmids (b1*) expectedly demonstrated an increase in fitness in response to the growth conditions. However, a second surprising result showed that this well-adapted host naive to plasmid carriage was more tolerant to the cost of a plasmid. For the ancestral plasmid R1 on this host we found no significant cost and the ancestral plasmid RP4 had greatly reduced its cost (Table 2).
As an explanation for the results on compensatory evolution we suggest that during the experiment both the host and the plasmids acquire mutations that are selected but for different purposes. The plasmids evolve compensatory mutations that are selected because they enhance fitness of the host whereas the host evolves toward the general growth conditions with a side effect of decreasing the cost of plasmid carriage. If the plasmid evolves compensatory mutations more rapidly than the host evolves mutations with compensatory side effects this would result in the apparent redundancy of compensatory mutations. It would also explain why a host that has evolved in the absence of plasmids suffers a lower cost of an ancestral plasmid than an ancestral host does. This explanation is also consistent with the results that an evolved plasmid does better than its ancestor in the ancestral host.
The cured clone CD122 showed a higher fitness when its plasmid had been reintroduced compared to the fitness of the plasmid-bearing clone before curing in the control experiment. This suggests that chromosomal genetic changes occurred during the curing procedure and the fitness values measured for the cured state of this clone are uncertain. A reasonable explanation for this result is that a mutualistic association resulting in strong plasmid dependency had evolved in this clone. We believe that the strong selection applied when the resident plasmid was forced out by an incompatible replicon during the curing procedure selected for a mutation compensatory for the loss of the plasmid.
What was the basis for the decreased costs of the evolved plasmids from five out of six independent clones? First one may consider conjugation. Plasmid RP4 has a constitutively expressed transfer function whereas R1 is repressed for transfer. One part of the cost may be associated with pilus production. We found that two of the evolved RP4 isolates were transfer deficient and that this deficiency correlated with changes in their restriction endonuclease digestion patterns in genes involved in pilus production. The evolved R1 clones (b1p1) showed lower transfer rates than the ancestral R1 clone (b0p0). This reduction in transfer rate was likely due to host evolution resulting in a host-directed suppression of transfer since the reduced transfer of these plasmids was observed only in the evolved host (b1p1) and not in the ancestral host (b0p1; Table 4). The transfer rate of the ancestral R1 from the host that had evolved in the absence of plasmids (b1*p0) was of the same magnitude as transfer from the ancestral host (b0p0). This result supports the idea that the mechanism responsible for the reduced transfer rates in the former case evolved in direct response to plasmid carriage (Table 4). We did not attempt to localize the chromosomal mutations responsible for this effect but several host-encoded factors that influence plasmid transfer have been identified in previous studies. For example, the ArcA protein, involved in cellular redox sensing, affects R1 transfer (
Copy number reduction is another possible route for cost reduction (4–5/cell for plasmid R1 and 4–6 for plasmid RP4 (
Antibiotic-resistance expression is a third type of plasmid function that can be altered to reduce cost. Most of the clones from the evolved populations had retained all their antibiotic resistances and had obviously reduced their costs by other means. However, R1-carrying populations did contain minor fractions of clones that had lost resistances. The antibiotic sensitivities of these evolved clones correlated with deletions of the resistance genes and included loss of all the plasmid-borne resistances. Similar results have previously been presented for the conjugative plasmid TP120, where loss of resistances was observed after selection in chemostats (
Conclusions:
These results demonstrate that the fitness cost of conjugative resistance plasmid carriage can be reduced and is often completely ameliorated after selection in an antibiotic-free environment. Reversion to sensitivity by plasmid segregation was not observed in these experiments. Instead, compensatory evolution was the major pathway. As previously suggested a decreased usage of antibiotics may hence not be sufficient for dealing with the antibiotic-resistance problem as sensitive bacteria are prevented from invading resistant bacterial populations after compensatory evolution has occurred. However, the emergence of plasmids with deletion of all antibiotic-resistance markers from the basic replicon in some of our experiments shows that there is still a potential for selection of antibiotic-sensitive populations. A consequence of the plasmid-encoded compensatory mutations is that the resistance genes associated with these plasmids may have an enhanced potential to invade new populations by horizontal transfer as compared to the more limited chromosomal compensatory mutations that still are confined to one population. It remains to be elucidated if plasmid compensatory mutations are specific for the strain they were selected in or if they also reduce the cost of the plasmid carriage when it transfers to other strains or species.
| ACKNOWLEDGMENTS |
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We thank Dan Andersson, Diarmaid Hughes, Bruce Levin, Sophie Maisnier-Patin, and two anonymous reviewers for helpful comments and discussions; Kenn Gerdes, Donald Helinski, Daniel Dykhuizen, and Michael Travisano for the gifts of plasmids and phage; and Kamal Gandhi for excellent technical assistance. This work was funded by grants from the Swedish Research Council and The Royal Swedish Academy of Sciences to C.D and from the National Institutes of General Medical Sciences and National Institutes of Health to L.C.
Manuscript received May 23, 2003; Accepted for publication July 25, 2003.
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