The cost of multiple drug resistance in Pseudomonas aeruginosa

The spread of bacterial antibiotic resistance mutations is thought to be constrained by their pleiotropic fitness costs. Here we investigate the fitness costs of resistance in the context of the evolution of multiple drug resistance (MDR), by measuring the cost of acquiring streptomycin resistance mutations (StrepR) in independent strains of the bacterium Pseudomonas aeruginosa carrying different rifampicin resistance (RifR) mutations. In the absence of antibiotics, StrepR mutations are associated with similar fitness costs in different RifR genetic backgrounds. The cost of StrepR mutations is greater in a rifampicin‐sensitive (RifS) background, directly demonstrating antagonistic epistasis between resistance mutations. In the presence of rifampicin, StrepR mutations have contrasting effects in different RifR backgrounds: StrepR mutations have no detectable costs in some RifR backgrounds and massive fitness costs in others. Our results clearly demonstrate the importance of epistasis and genotype‐by‐environment interactions for the evolution of MDR.     

Decreased thermal niche breadth as a trade-off of antibiotic resistance

Evolutionary theory predicts that adaptations, including antibiotic resistance, should come with associated fitness costs; yet, many resistance mutations seemingly contradict this prediction by inducing no growth rate deficit. However, most growth assays comparing sensitive and resistant strains have been performed under a narrow range of environmental conditions, which do not reflect the variety of contexts that a pathogenic bacterium might encounter when causing infection. We hypothesized that reduced niche breadth, defined as diminished growth across a diversity of environments, can be a cost of antibiotic resistance. Specifically, we test whether chloramphenicol-resistant Escherichia coli incur disproportionate growth deficits in novel thermal conditions. Here we show that chloramphenicol-resistant bacteria have greater fitness costs at novel temperatures than their antibiotic-sensitive ancestors. In several cases, we observed no resistance cost in growth rate at the historic temperature but saw diminished growth at warmer and colder temperatures. These results were consistent across various genetic mechanisms of resistance. Thus, we propose that decreased thermal niche breadth is an under-documented fitness cost of antibiotic resistance. Furthermore, these results demonstrate that the cost of antibiotic resistance shifts rapidly as the environment changes; these context-dependent resistance costs should select for the rapid gain and loss of resistance as an evolutionary strategy.Subject terms: Bacterial evolution, Microbial ecology, Antibiotics     

The in vitro fitness cost of antimicrobial resistance in Escherichia coli varies with the growth conditions

The objective of this study was to investigate the influence of stressful growth conditions on the fitness cost of antimicrobial resistance in Escherichia coli BJ4 caused by chromosomal mutations and plasmid acquisition. The fitness cost of chromosomal streptomycin resistance increased significantly when the bacteria were grown under all stress conditions tested, while the cost in 1/3 Luria–Bertani was not significantly changed in a streptomycin+rifampicin mutant. The increase in the fitness cost depended in a nonregular manner on the strain/stress combination. The fitness cost of plasmid-encoded resistance on R751 did not differ significantly, and was generally less under stressful growth conditions than in rich media. The fitness cost associated with R751 with the multiple drug resistance cassette from Salmonella Typhimurium DT104 increased significantly only under stressful conditions at low pH and at high-salt concentrations. Strains with an impaired rpoS demonstrated a reduced fitness only during growth in a high-salt concentration. In conclusion, it was demonstrated that bacterial fitness cost in association with antimicrobial resistance generally increases under stressful growth conditions. However, the growth potential of bacteria with antimicrobial resistances did not increase in a straightforward manner in these in vitro experiments and is therefore probably even more difficult to predict in vivo .     

Genotype‐by‐environment interactions due to antibiotic resistance and adaptation in Escherichia coli

Mutations that are beneficial in one environment can have different fitness effects in other environments. In the context of antibiotic resistance, the resulting genotype‐by‐environment interactions potentially make selection on resistance unpredictable in heterogeneous environments. Furthermore, resistant bacteria frequently fix additional mutations during evolution in the absence of antibiotics. How do these two types of mutations interact to determine the bacterial phenotype across different environments? To address this, I used Escherichia coli as a model system, measuring the effects of nine different rifampicin resistance mutations on bacterial growth in 31 antibiotic‐free environments. I did this both before and after approximately 200 generations of experimental evolution in antibiotic‐free conditions (LB medium), and did the same for the antibiotic‐sensitive wild type after adaptation to the same environment. The following results were observed: (i) bacteria with and without costly resistance mutations adapted to experimental conditions and reached similar levels of competitive fitness; (ii) rifampicin resistance mutations and adaptation to LB both indirectly altered growth in other environments; and (iii) resistant‐evolved genotypes were more phenotypically different from the ancestor and from each other than resistant‐nonevolved and sensitive‐evolved genotypes. This suggests genotype‐by‐environment interactions generated by antibiotic resistance mutations, observed previously in short‐term experiments, are more pronounced after adaptation to other types of environmental variation, making it difficult to predict long‐term selection on resistance mutations from fitness effects in a single environment.     

Environmental variation alters the fitness effects of rifampicin resistance mutations in Pseudomonas aeruginosa

The fitness effects of antibiotic resistance mutations in antibiotic‐free conditions play a key role in determining the long‐term maintenance of resistance. Although resistance is usually associated with a cost, the impact of environmental variation on the cost of resistance is poorly understood. Here, we test the impact of heterogeneity in temperature and resource availability on the fitness effects of antibiotic resistance using strains of the pathogenic bacterium Pseudomonas aeruginosa carrying clinically important rifampicin resistance mutations. Although the rank order of fitness was generally maintained across environments, fitness effects relative to the wild type differed significantly. Changes in temperature had a profound impact on the fitness effects of resistance, whereas changes in carbon substrate had only a weak impact. This suggests that environmental heterogeneity may influence whether the costs of resistance are likely to be ameliorated by second‐site compensatory mutations or by reversion to wild‐type rpoB. Our results highlight the need to consider environmental heterogeneity and genotype‐by‐environment interactions for fitness in models of resistance evolution.     

AMELIORATION OF THE DELETERIOUS PLEIOTROPIC EFFECTS OF AN ADAPTIVE MUTATION IN BACILLUS SUBTILIS

The deleterious pleiotropic effects of an adaptive mutation may be ameliorated by one of two modes of evolution: (1) by replacement, in which an adaptive mutation with harmful pleiotropic effects is replaced by one that confers an equal benefit but at less cost; or (2) by compensatory evolution, in which natural selection favors modifiers at other loci that compensate for the deleterious effects of the mutant allele. In this study, we have measured the potential of these two modes of evolution to ameliorate the deleterious pleiotropic effects of resistance to the antibiotic rifampicin in the soil bacterium Bacillus subtilis. One approach was to measure the fitness cost of a series of spontaneous rifampicin-resistance mutations from each of several strains. The potential for amelioration by the replacement mode was estimated by the variation in fitness cost among the mutants of a single strain. Another approach was to introduce a series of different rifampicin-resistance alleles into a diversity of strains, and to measure the fitness cost of rifampicin resistance for each allele-by-strain combination. The potential for amelioration by the replacement mode was estimated by the variation in fitness costs among rifampicin-resistance alleles; the potential for compensatory evolution was estimated by variation in the fitness cost of rifampicin resistance among strains. This study has shown that the cost of rifampicin resistance may be ameliorated by both the compensatory and replacement modes.     

Elevated Mutagenesis Does Not Explain the Increased Frequency of Antibiotic Resistant Mutants in Starved Aging Colonies

The frequency of mutants resistant to the antibiotic rifampicin has been shown to increase in aging (starved), compared to young colonies of Eschierchia coli. These increases in resistance frequency occur in the absence of any antibiotic exposure, and similar increases have also been observed in response to additional growth limiting conditions. Understanding the causes of such increases in the frequency of resistance is important for understanding the dynamics of antibiotic resistance emergence and spread. Increased frequency of rifampicin resistant mutants in aging colonies is cited widely as evidence of stress-induced mutagenesis (SIM), a mechanism thought to allow bacteria to increase mutation rates upon exposure to growth-limiting stresses. At the same time it has been demonstrated that some rifampicin resistant mutants are relatively fitter in aging compared to young colonies, indicating that natural selection may also contribute to increased frequency of rifampicin resistance in aging colonies. Here, we demonstrate that the frequency of mutants resistant to both rifampicin and an additional antibiotic (nalidixic-acid) significantly increases in aging compared to young colonies of a lab strain of Escherichia coli. We then use whole genome sequencing to demonstrate conclusively that SIM cannot explain the observed magnitude of increased frequency of resistance to these two antibiotics. We further demonstrate that, as was previously shown for rifampicin resistance mutations, mutations conferring nalidixic acid resistance can also increase fitness in aging compared to young colonies. Our results show that increases in the frequency of antibiotic resistant mutants in aging colonies cannot be seen as evidence of SIM. Furthermore, they demonstrate that natural selection likely contributes to increases in the frequency of certain antibiotic resistance mutations, even when no selection is exerted due to the presence of antibiotics.     

Epistasis buffers the fitness effects of rifampicin- resistance mutations in Pseudomonas aeruginosa

Epistatic interactions between resistance mutations in antibiotic-free environments potentially play a crucial role in the spread of resistance in pathogen populations by determining the fitness cost associated with resistance. We used an experimental evolution approach to test for epistatic interactions between 14 different pairs of rifampicin mutations in the pathogenic bacterium Pseudomonas aeruginosa in 42 different rifampicin-free environments. First, we show that epistasis between rifampicin-resistance mutations tends to be antagonistic: the fitness effect of having two mutations is generally smaller than that predicted from the effects of individual mutations on the wild-type. Second, we show that sign epistasis between resistance mutations is both common and strong; most notably, pairs of deleterious resistance mutations often partially or completely compensate for each others' costs, revealing a novel mechanism for compensatory adaptation. These results suggest that antagonistic epistasis between intragenic resistance mutations may be a key determinant of the cost of antibiotic resistance and compensatory adaptation in pathogen populations.     

Host plant and population determine the fitness costs of resistance to Bacillus thuringiensis

Novel adaptations often cause pleiotropic reductions in fitness. Under optimal conditions individual organisms may be able to compensate for, or reduce, these fitness costs. Declining environmental quality may therefore lead to larger costs. We investigated whether reduced plant quality would increase the fitness costs associated with resistance to Bacillus thuringiensis in two populations of the diamondback moth Plutella xylostella. We also measured the rate of decline in resistance on two host-plant (Brassica) species for one insect population (Karak). Population X plant species interactions determined the fitness costs in this study. Poor plant quality increased the fitness costs in terms of development time for both populations. However, fitness costs seen in larval survival did not always increase as plant quality declined. Both the fitness and the stability experiment indicated that fitness costs were higher on the most suitable plant for one population. Theoretically, if the fitness cost of a mutation interacts additively with environmental factors, the relative fitness of resistant insects will decrease with environmental quality. However, multiplicative costs do not necessarily increase with declining quality and may be harder to detect when fitness parameters are more subject to variation in poorer environments.     

EVOLUTIONARY REVERSALS OF ANTIBIOTIC RESISTANCE IN EXPERIMENTAL POPULATIONS OF PSEUDOMONAS AERUGINOSA

Antibiotic resistance mutations are accompanied by a fitness cost, and two mechanisms allow bacteria to adapt to this cost once antibiotic use is halted. First, it is possible for resistance to revert; second, it is possible for bacteria to adapt to the cost of resistance by compensatory mutations. Unfortunately, reversion to antibiotic sensitivity is rare, but the underlying factors that prevent reversion remain obscure. Here, we directly study the evolutionary dynamics of reversion by experimentally mimicking reversion mutations—sensitives—in populations of rifampicin‐resistant Pseudomonas aeruginosa. We show that, in our populations, most sensitives are lost due to genetic drift when they are rare. However, clonal interference from lineages carrying compensatory mutations causes a dramatic increase in the time to fixation of sensitives that escape genetic drift, and mutations surpassing the sensitives’ fitness are capable of driving transiently common sensitive lineages to extinction. Crucially, we show that the constraints on reversion arising from clonal interference are determined by the potential for compensatory adaptation of the resistant population. Although the cost of resistance provides the incentive for reversion, our study demonstrates that both the cost of resistance and the intrinsic evolvability of resistant populations interact to determine the rate and likelihood of reversion.     

Antibacterial activity of rifampicin against the causative agents of suppurative, septic diseases]

The antibacterial activity of rifampicin was studied in comparison with other antibiotics with respect to clinical strains isolated from cases with various purulent inflammatory processes caused by Staphylococcus, E. coli, Ps. aeruginose, Proteus. The aim of the study was to define the role of rifampicin in the treatment of the above infections. No rifampicin resistant strains were found among staphylococci belonging to the phenotype carrying the determinants of resistance to 2-8 antibiotics. Rifampicin was less active against gramnegative organisms. High heterogeneity of the microbial population of rifampicin was shown with respect to all microbial strains tested. The rate of the spontaneous mutants was high. The average rate of the mutants was 1-7.7-10-8. The studies on the dynamics of the rifampicin resistance increase in the strains of Staphylococci, E. Coli, Ps. aeruginosa and Proteus showed that the resistance increased after 1-2 passages, which means that one-stage mutation was characteristic rifampicin.     

Fitness-compensatory mutations in rifampicin-resistant RNA polymerase

Mutations in rpoB (RNA polymerase β-subunit) can cause high-level resistance to rifampicin, an important first-line drug against tuberculosis. Most rifampicin-resistant (Rif(R)) mutants selected in vitro have reduced fitness, and resistant clinical isolates of M. tuberculosis frequently carry multiple mutations in RNA polymerase genes. This supports a role for compensatory evolution in global epidemics of drug-resistant tuberculosis but the significance of secondary mutations outside rpoB has not been demonstrated or quantified. Using Salmonella as a model organism, and a previously characterized Rif(R) mutation (rpoB R529C) as a starting point, independent lineages were evolved with selection for improved growth in the presence and absence of rifampicin. Compensatory mutations were identified in every lineage and were distributed between rpoA, rpoB and rpoC. Resistance was maintained in all strains showing that increased fitness by compensatory mutation was more likely than reversion. Genetic reconstructions demonstrated that the secondary mutations were responsible for increasing growth rate. Many of the compensatory mutations in rpoA and rpoC individually caused small but significant reductions in susceptibility to rifampicin, and some compensatory mutations in rpoB individually caused high-level resistance. These findings show that mutations in different components of RNA polymerase are responsible for fitness compensation of a Rif(R) mutant.     

The biological cost of mutational antibiotic resistance: any practical conclusions?

A key parameter influencing the rate and trajectory of the evolution of antibiotic resistance is the fitness cost of resistance. Recent studies have demonstrated that antibiotic resistance, whether caused by target alteration or by other mechanisms, generally confers a reduction in fitness expressed as reduced growth, virulence or transmission. These findings imply that resistance might be reversible, provided antibiotic use is reduced. However, several processes act to stabilize resistance, including compensatory evolution where the fitness cost is ameliorated by additional mutation without loss of resistance, the rare occurrence of cost-free resistance mechanisms and genetic linkage or co-selection between the resistance markers and other selected markers. Conceivably we can use this knowledge to rationally choose and design targets and drugs where the costs of resistance are the highest, and where the likelihood of compensation is the lowest.     

Fisher's geometrical model and the mutational patterns of antibiotic resistance across dose gradients

Fisher's geometrical model (FGM) has been widely used to depict the fitness effects of mutations. It is a general model with few underlying assumptions that gives a large and comprehensive view of adaptive processes. It is thus attractive in several situations, for example adaptation to antibiotics, but comes with limitations, so that more mechanistic approaches are often preferred to interpret experimental data. It might be possible however to extend FGM assumptions to better account for mutational data. This is theoretically challenging in the context of antibiotic resistance because resistance mutations are assumed to be rare. In this article, we show with Escherichia coli how the fitness effects of resistance mutations screened at different doses of nalidixic acid vary across a dose‐gradient. We found experimental patterns qualitatively consistent with the basic FGM (rate of resistance across doses, gamma distributed costs) but also unexpected patterns such as a decreasing mean cost of resistance with increasing screen dose. We show how different extensions involving mutational modules and variations in trait covariance across environments, can be discriminated based on these data. Overall, simple extensions of the FGM accounted well for complex mutational effects of resistance mutations across antibiotic doses.     

Fitness Consequences of Genetically Engineered Herbicide and Antibiotic Resistance in Arabidopsis Thaliana

Researchers have often invoked the concept of metabolic drain to explain the lower growth rates of bacteria containing plasmids that confer antibiotic resistance. This idea posits that the energetic input needed to produce detoxifying enzymes diverts resources from clonal reproduction. In this paper we examine whether the concept of metabolic drain can be applied successfully to plants that differ from bacteria in several key aspects including their relative genome size and reproductive rate. We have conducted a field experiment using mutant and transgenic Arabidopsis thaliana that allows the comparison of genotypes differing by a single gene conferring resistance to either the herbicide chlorsulfuron or the antibiotic kanamycin. In addition to testing whether these traits reduce fitness, this experiment was conducted at two levels of resource availability to examine whether costs of resistance are sensitive to environmental quality. We found that herbicide-resistant individuals produced 26% fewer seeds than susceptible counterparts. However, contrasting published results in bacterial systems, the fecundity of individuals was completely unaffected by the expression of an introduced antibiotic resistance gene. The fitness cost associated with chlorsulfuron resistance was greater in nutrient-poor conditions relative to nutrient-rich conditions for comparisons involving mutant, but not transgenic, genotypes.     

Source-sink dynamics shape the evolution of antibiotic resistance and its pleiotropic fitness cost

Understanding the conditions that favour the evolution and maintenance of antibiotic resistance is the central goal of epidemiology. A crucial feature explaining the adaptation to harsh, or 'sink', environments is the supply of beneficial mutations via migration from a 'source' population. Given that antibiotic resistance is frequently associated with antagonistic pleiotropic fitness costs, increased migration rate is predicted not only to increase the rate of resistance evolution but also to increase the probability of fixation of resistance mutations with minimal fitness costs. Here we report in vitro experiments using the nosocomial pathogenic bacterium Pseudomonas aeruginosa that support these predictions: increasing rate of migration into environments containing antibiotics increased the rate of resistance evolution and decreased the associated costs of resistance. Consistent with previous theoretical work, we found that resistance evolution arose more rapidly in the presence of a single antibiotic than two. Evolution of resistance was also more rapid when bacteria were subjected to sequential exposure with two antibiotics (cycling therapy) compared with simultaneous exposure (bi-therapy). Furthermore, pleiotropic fitness costs of resistance to two antibiotics were higher than for one antibiotic, and were also higher under bi-therapy than cycling therapy, although the cost of resistance depended on the order of the antibiotics through time. These results may be relevant to the clinical setting where immigration is known to be important between chemotherapeutically treated patients, and demonstrate the importance of ecological and evolutionary dynamics in the control of antibiotic resistance.     

Constitutive versus responsive gene expression strategies for growth in changing environments

Microbes respond to changing environments by adjusting gene expression levels to the demand for the corresponding proteins. Adjusting protein levels is slow, consequently cells may reach the optimal protein level only by a time when the demand changed again. It is therefore not a priori clear whether expression “on demand” is always the optimal strategy. Indeed, many genes are constitutively expressed at intermediate levels, which represents a permanent cost but provides an immediate benefit when the protein is needed. Which are the conditions that select for a responsive or a constitutive expression strategy, what determines the optimal constitutive expression level in a changing environment, and how is the fitness of the two strategies affected by gene expression noise? Based on an established model of the lac- and gal-operon expression dynamics, we study the fitness of a constitutive and a responsive expression strategy in time-varying environments. We find that the optimal constitutive expression level differs from the average demand for the gene product and from the average optimal expression level; depending on the shape of the growth rate function, the optimal expression level either provides intermediate fitness in all environments, or maximizes fitness in only one of them. We find that constitutive expression can provide higher fitness than responsive expression even when regulatory machinery comes at no cost, and we determine the minimal response rate necessary for “expression on demand” to confer a benefit. Environmental and inter-cellular noise favor the responsive strategy while reducing fitness of the constitutive one. Our results show the interplay between the demand-frequency for a gene product, the genetic response rate, and the fitness, and address important questions on the evolution of gene regulation. Some of our predictions agree with recent yeast high throughput data, for others we propose the experiments that are needed to verify them.     

Lytic phages obscure the cost of antibiotic resistance in Escherichia coli

The long-term persistence of antibiotic-resistant bacteria depends on their fitness relative to other genotypes in the absence of drugs. Outside the laboratory, viruses that parasitize bacteria (phages) are ubiquitous, but costs of antibiotic resistance are typically studied in phage-free experimental conditions. We used a mathematical model and experiments with Escherichia coli to show that lytic phages strongly affect the incidence of antibiotic resistance in drug-free conditions. Under phage parasitism, the likelihood that antibiotic-resistant genetic backgrounds spread depends on their initial frequency, mutation rate and intrinsic growth rate relative to drug-susceptible genotypes, because these parameters determine relative rates of phage-resistance evolution on different genetic backgrounds. Moreover, the average cost of antibiotic resistance in terms of intrinsic growth in the antibiotic-free experimental environment was small relative to the benefits of an increased mutation rate in the presence of phages. This is consistent with our theoretical work indicating that, under phage selection, typical costs of antibiotic resistance can be outweighed by realistic increases in mutability if drug resistance and hypermutability are genetically linked, as is frequently observed in clinical isolates. This suggests the long-term distribution of antibiotic resistance depends on the relative rates at which different lineages adapt to other types of selection, which in the case of phage parasitism is probably extremely common, as well as costs of resistance inferred by classical in vitro methods.