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.     

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.     

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     

Compensatory evolution of pbp mutations restores the fitness cost imposed by β-lactam resistance in Streptococcus pneumoniae

The prevalence of antibiotic resistance genes in pathogenic bacteria is a major challenge to treating many infectious diseases. The spread of these genes is driven by the strong selection imposed by the use of antibacterial drugs. However, in the absence of drug selection, antibiotic resistance genes impose a fitness cost, which can be ameliorated by compensatory mutations. In Streptococcus pneumoniae, β-lactam resistance is caused by mutations in three penicillin-binding proteins, PBP1a, PBP2x, and PBP2b, all of which are implicated in cell wall synthesis and the cell division cycle. We found that the fitness cost and cell division defects conferred by pbp2b mutations (as determined by fitness competitive assays in vitro and in vivo and fluorescence microscopy) were fully compensated by the acquisition of pbp2x and pbp1a mutations, apparently by means of an increased stability and a consequent mislocalization of these protein mutants. Thus, these compensatory combinations of pbp mutant alleles resulted in an increase in the level and spectrum of β-lactam resistance. This report describes a direct correlation between antibiotic resistance increase and fitness cost compensation, both caused by the same gene mutations acquired by horizontal transfer. The clinical origin of the pbp mutations suggests that this intergenic compensatory process is involved in the persistence of β-lactam resistance among circulating strains. We propose that this compensatory mechanism is relevant for β-lactam resistance evolution in Streptococcus pneumoniae.     

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.     

Rapid Decline of Ceftazidime Resistance in Antibiotic-Free and Sublethal Environments Is Contingent on Genetic Background

Trade-offs of antibiotic resistance evolution, such as fitness cost and collateral sensitivity (CS), could be exploited to drive evolution toward antibiotic susceptibility. Decline of resistance may occur when resistance to other drug leads to CS to the first one and when compensatory mutations, or genetic reversion of the original ones, reduce fitness cost. Here we describe the impact of antibiotic-free and sublethal environments on declining ceftazidime resistance in different Pseudomonas aeruginosa resistant mutants. We determined that decline of ceftazidime resistance occurs within 450 generations, which is caused by newly acquired mutations and not by reversion of the original ones, and that the original CS of these mutants is preserved. In addition, we observed that the frequency and degree of this decline is contingent on genetic background. Our results are relevant to implement evolution-based therapeutic approaches, as well as to redefine global policies of antibiotic use, such as drug cycling.     

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.     

Bacterial evolution and the cost of antibiotic resistance.

Bacteria clearly benefit from the possession of an antibiotic resistance gene when the corresponding antibiotic is present. But do resistant bacteria suffer a cost of resistance (i.e., a reduction in fitness) when the antibiotic is absent? If so, then one strategy to control the spread of resistance would be to suspend the use of a particular antibiotic until resistant genotypes declined to low frequency. Numerous studies have indeed shown that resistant genotypes are less fit than their sensitive counterparts in the absence of antibiotic, indicating a cost of resistance. But there is an important caveat: these studies have put resistance genes into naive bacteria, which have no evolutionary history of association with the resistance genes. An important question, therefore, is whether bacteria can overcome the cost of resistance by evolving adaptations that counteract the harmful side-effects of resistance genes. In fact, several experiments (in vitro and in vivo) show that the cost of antibiotic resistance can be substantially diminished, even eliminated, by evolutionary changes in bacteria over rather short periods of time. As a consequence, it becomes increasingly difficult to eliminate resistant genotypes simply by suspending the use of antibiotics.     

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.     

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.     

Phenotypic switching of antibiotic resistance circumvents permanent costs in Staphylococcus aureus.

Bacterial antibiotic resistance is often associated with a fitness cost in the absence of the antibiotic [1,2]. We have examined a resistance mechanism in Staphylococcus aureus that negates these costs. Exposure to gentamicin both in vitro and in vivo has been reported to result in the emergence of a gentamicin-resistant small colony variant (SCV)[3-8]. We show that the emergence of SCVs following exposure to gentamicin results from a rapid switch and that bacteria exposed to cycles of gentamicin followed by antibiotic-free medium repeatedly switched between a resistant SCV and a sensitive parental phenotype (revertants). The fitness of revertants relative to S. aureus with stable gentamicin resistance was greater in drug-free media, which suggests that S. aureus has evolved an inducible and reversible resistance mechanism that circumvents a permanent cost to fitness.     

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 of antimicrobial-resistant Campylobacter and Salmonella

Campylobacter and Salmonella are the most commonly reported bacterial causes of human foodborne infections, and increasing proportions of these pathogens become resistant to medically important antimicrobial agents, imposing a burden on public health. Acquisition of resistance to antibiotics affects the adaptation and evolution of Salmonella and Campylobacter in various environments. Many resistance-conferring mutations entail a biological fitness cost, while others (e.g. fluoroquinolone resistance in Campylobacter) have no cost or even enhanced fitness. In Salmonella, the fitness disadvantage due to antimicrobial resistance can be restored by acquired compensatory mutations, which occur both in vitro and in vivo. The compensated or even enhanced fitness associated with antibiotic resistance may facilitate the spread and persistence of antimicrobial-resistant Salmonella and Campylobacter in the absence of selection pressure, creating a significant barrier for controlling antibiotic-resistant foodborne pathogens.     

Enhancement of bacterial competitive fitness by apramycin resistance plasmids from non-pathogenic Escherichia coli

The study of antibiotic resistance has in the past focused on organisms that are pathogenic to humans or animals. However, the development of resistance in commensal organisms is of concern because of possible transfer of resistance genes to zoonotic pathogens. Conjugative plasmids are genetic elements capable of such transfer and are traditionally thought to engender a fitness burden on host bacteria. In this study, conjugative apramycin resistance plasmids isolated from newborn calves were characterized. Calves were raised on a farm that had not used apramycin or related aminoglycoside antibiotics for at least 20 months prior to sampling. Of three apramycin resistance plasmids, one was capable of transfer at very high rates and two were found to confer fitness advantages on new Escherichia coli hosts. This is the first identification of natural plasmids isolated from commensal organisms that are able to confer a fitness advantage on a new host. This work indicates that reservoirs of antibiotic resistance genes in commensal organisms might not decrease if antibiotic usage is halted.     

Restored fitness leads to long-term persistence of resistant Bacteroides strains in the human intestine

Acquired antibiotic resistance typically confers a cost to the bacteria, but these costs can be reduced by genetic compensation over time. The fitness of two Bacteroides thetaiotaomicron clones consecutively isolated in vivo was studied using an in vitro pair-wise competition method. The isolates derived from faecal samples of two clindamycin-exposed healthy volunteers and the two B. thetaiotaomicron clone types could be followed up to 18 months in these two subjects. The two clones were originally susceptible to clindamycin and lacked erm genes; however, after 7 days of clindamycin administration they carried the erm (erythromycin methylase)(G) or (F) gene, respectively, and expressed phenotypic clindamycin resistance. The initial cost of acquired resistance was high as seen in the in vitro pair-wise competition experiments. At 2 weeks post-administration, no growth disadvantage was detected for isolates of either of the two clones in the in vitro experiments and this regained fitness remained for isolates collected up to 18 months. Competition analysis of an in vitro isolated erm(G) positive transconjugant also demonstrated an initial reduction of fitness that was restored over time. The results indicate that the biological cost associated with a resistance gene can rapidly be compensated during in vivo growth. Thus, once the resistant clone has gained its resistance determinant it will be difficult to eliminate.     

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.     

Evolution of antibiotic resistance by human and bacterial niche construction

Antibiotic treatment by humans generates strong viability selection for antibiotic-resistant bacterial strains. The frequency of host antibiotic use often determines the strength of this selection, and changing patterns of antibiotic use can generate many types of behaviors in the population dynamics of resistant and sensitive bacterial populations. In this paper, we present a simple model of hosts dimorphic for their tendency to use/avoid antibiotics and bacterial pathogens dimorphic in their resistance/sensitivity to antibiotic treatment. When a constant fraction of hosts uses antibiotics, the two bacterial strain populations can coexist unless host use-frequency is above a critical value; this critical value is derived as the ratio of the fitness cost of resistance to the fitness cost of undergoing treatment. When strain frequencies can affect host behavior, the dynamics may be analyzed in the light of niche construction. We consider three models underlying changing host behavior: conformism, the avoidance of long infections, and adherence to the advice of public health officials. In the latter two, we find that the pathogen can have quite a strong effect on host behavior. In particular, if antibiotic use is discouraged when resistance levels are high, we observe a classic niche-construction phenomenon of maintaining strain polymorphism even in parameter regions where it would not be expected.