Fitness Costs of Plasmids: a Limit to Plasmid Transmission.

Plasmids mediate the horizontal transmission of genetic information between bacteria, facilitating their adaptation to multiple environmental conditions. An especially important example of the ability of plasmids to catalyze bacterial adaptation and evolution is their instrumental role in the global spread of antibiotic resistance, which constitutes a major threat to public health. Plasmids provide bacteria with new adaptive tools, but they also entail a metabolic burden that, in the absence of selection for plasmid-encoded traits, reduces the competitiveness of the plasmid-carrying clone. Although this fitness reduction can be alleviated over time through compensatory evolution, the initial cost associated with plasmid carriage is the main constraint on the vertical and horizontal replication of these genetic elements. The fitness effects of plasmids therefore have a crucial influence on their ability to associate with new bacterial hosts and consequently on the evolution of plasmid-mediated antibiotic resistance. However, the molecular mechanisms underlying plasmid fitness cost remain poorly understood. Here, we analyze the literature in the field and examine the potential fitness effects produced by plasmids throughout their life cycle in the host bacterium. We also explore the various mechanisms evolved by plasmids and bacteria to minimize the cost entailed by these mobile genetic elements. Finally, we discuss potential future research directions in the field.

The genomic basis of adaptation to the fitness cost of rifampicin resistance in Pseudomonas aeruginosa.

Antibiotic resistance carries a fitness cost that must be overcome in order for resistance to persist over the long term. Compensatory mutations that recover the functional defects associated with resistance mutations have been argued to play a key role in overcoming the cost of resistance, but compensatory mutations are expected to be rare relative to generally beneficial mutations that increase fitness, irrespective of antibiotic resistance. Given this asymmetry, population genetics theory predicts that populations should adapt by compensatory mutations when the cost of resistance is large, whereas generally beneficial mutations should drive adaptation when the cost of resistance is small. We tested this prediction by determining the genomic mechanisms underpinning adaptation to antibiotic-free conditions in populations of the pathogenic bacterium Pseudomonas aeruginosa that carry costly antibiotic resistance mutations. Whole-genome sequencing revealed that populations founded by high-cost rifampicin-resistant mutants adapted via compensatory mutations in three genes of the RNA polymerase core enzyme, whereas populations founded by low-cost mutants adapted by generally beneficial mutations, predominantly in the quorum-sensing transcriptional regulator gene lasR. Even though the importance of compensatory evolution in maintaining resistance has been widely recognized, our study shows that the roles of general adaptation in maintaining resistance should not be underestimated and highlights the need to understand how selection at other sites in the genome influences the dynamics of resistance alleles in clinical settings.

Interactions between horizontally acquired genes create a fitness cost in Pseudomonas aeruginosa.

Horizontal gene transfer (HGT) plays a key role in bacterial evolution, especially with respect to antibiotic resistance. Fitness costs associated with mobile genetic elements (MGEs) are thought to constrain HGT, but our understanding of these costs remains fragmentary, making it difficult to predict the success of HGT events. Here we use the interaction between P. aeruginosa and a costly plasmid (pNUK73) to investigate the molecular basis of the cost of HGT. Using RNA-Seq, we show that the acquisition of pNUK73 results in a profound alteration of the transcriptional profile of chromosomal genes. Mutations that inactivate two genes encoded on chromosomally integrated MGEs recover these fitness costs and transcriptional changes by decreasing the expression of the pNUK73 replication gene. Our study demonstrates that interactions between MGEs can compromise bacterial fitness via altered gene expression, and we argue that conflicts between mobile elements impose a general constraint on evolution by HGT.

The genetic basis of the fitness costs of antimicrobial resistance: a meta-analysis approach.

The evolution of antibiotic resistance carries a fitness cost, expressed in terms of reduced competitive ability in the absence of antibiotics. This cost plays a key role in the dynamics of resistance by generating selection against resistance when bacteria encounter an antibiotic-free environment. Previous work has shown that the cost of resistance is highly variable, but the underlying causes remain poorly understood. Here, we use a meta-analysis of the published resistance literature to determine how the genetic basis of resistance influences its cost. We find that on average chromosomal resistance mutations carry a larger cost than acquiring resistance via a plasmid. This may explain why resistance often evolves by plasmid acquisition. Second, we find that the cost of plasmid acquisition increases with the breadth of its resistance range. This suggests a potentially important limit on the evolution of extensive multidrug resistance via plasmids. We also find that epistasis can significantly alter the cost of mutational resistance. Overall, our study shows that the cost of antimicrobial resistance can be partially explained by its genetic basis. It also highlights both the danger associated with plasmidborne resistance and the need to understand why resistance plasmids carry a relatively low cost.

Linking system-wide impacts of RNA polymerase mutations to the fitness cost of rifampin resistance in Pseudomonas aeruginosa.

UNLABELLED: Fitness costs play a key role in the evolutionary dynamics of antibiotic resistance in bacteria by generating selection against resistance in the absence of antibiotics. Although the genetic basis of antibiotic resistance is well understood, the precise molecular mechanisms linking the genetic basis of resistance to its fitness cost remain poorly characterized. Here, we examine how the system-wide impacts of mutations in the RNA polymerase (RNAP) gene rpoB shape the fitness cost of rifampin resistance in Pseudomonas aeruginosa. Rifampin resistance mutations reduce transcriptional efficiency, and this explains 76% of the variation in fitness among rpoB mutants. The pleiotropic consequence of rpoB mutations is that mutants show altered relative transcript levels of essential genes. We find no evidence that global transcriptional responses have an impact on the fitness cost of rifampin resistance as revealed by transcriptome sequencing (RNA-Seq). Global changes in the transcriptional profiles of rpoB mutants compared to the transcriptional profile of the rifampin-sensitive ancestral strain are subtle, demonstrating that the transcriptional regulatory network of P. aeruginosa is robust to the decreased transcriptional efficiency associated with rpoB mutations. On a smaller scale, we find that rifampin resistance mutations increase the expression of RNAP due to decreased termination at an attenuator upstream from rpoB, and we argue that this helps to minimize the cost of rifampin resistance by buffering against reduced RNAP activity. In summary, our study shows that it is possible to dissect the molecular mechanisms underpinning variation in the cost of rifampin resistance and highlights the importance of genome-wide buffering of relative transcript levels in providing robustness against resistance mutations. IMPORTANCE: Antibiotic resistance mutations carry fitness costs. Relative to the characteristics of their antibiotic-sensitive ancestors, resistant mutants show reduced growth rates and competitive abilities. Fitness cost plays an important role in the evolution of antibiotic resistance in the absence of antibiotics; however, the molecular mechanisms underlying these fitness costs is not well understood. We applied a systems-level approach to dissect the molecular underpinnings of the fitness costs associated with rifampin resistance in P. aeruginosa and showed that most of the variation in fitness cost can be explained by the direct effect of resistance mutations on the enzymatic activity of the mutated gene. Pleiotropic changes in transcriptional profiles are subtle at a genome-wide scale, suggesting that the gene regulatory network of P. aeruginosa is robust in the face of the direct effects of resistance mutations.

The fitness cost of rifampicin resistance in Pseudomonas aeruginosa depends on demand for RNA polymerase.

Bacterial resistance to antibiotics usually incurs a fitness cost in the absence of selecting drugs, and this cost of resistance plays a key role in the spread of antibiotic resistance in pathogen populations. Costs of resistance have been shown to vary with environmental conditions, but the causes of this variability remain obscure. In this article, we show that the average cost of rifampicin resistance in the pathogenic bacterium Pseudomonas aeruginosa is reduced by the addition of ribosome inhibitors (chloramphenicol or streptomycin) that indirectly constrain transcription rate and therefore reduce demand for RNA polymerase activity. This effect is consistent with predictions from metabolic control theory. We also tested the alternative hypothesis that the observed trend was due to a general effect of environmental quality on the cost of resistance. To do this we measured the fitness of resistant mutants in the presence of other antibiotics (ciprofloxacin and carbenicillin) that have similar effects on bacterial growth rate but bind to different target enzymes (DNA gyrase and penicillin-binding proteins, respectively) and in 41 single-carbon source environments of varying quality. We find no consistent effect of environmental quality on the average cost of resistance in these treatments. These results show that the cost of rifampicin resistance varies with demand for the mutated target enzyme, rather than as a simple function of bacterial growth rate or stress.