Fitness Costs Of Resistance Mutations Research Articles

Fitness effects for Ace insecticide resistance mutations are determined by ambient temperature

BackgroundInsect pest control programs often use periods of insecticide treatment with intermittent breaks, to prevent fixing of mutations conferring insecticide resistance. Such mutations are typically costly in an insecticide-free environment, and their frequency is determined by the balance between insecticide treatment and cost of resistance. Ace, a key gene in neuronal signaling, is a prominent target of many insecticides and across several species, three amino acid replacements (I161V, G265A, and F330Y) provide resistance against several insecticides. Because temperature disturbs neuronal signaling homeostasis, we reasoned that the cost of insecticide resistance could be modulated by ambient temperature.ResultsExperimental evolution of a natural Drosophila simulans population at hot and cold temperature regimes uncovered a surprisingly strong effect of ambient temperature. In the cold temperature regime, the resistance mutations were strongly counter selected (s = − 0.055), but in a hot environment, the fitness costs of resistance mutations were reduced by almost 50% (s = − 0.031). We attribute this unexpected observation to the advantage of the reduced enzymatic activity of resistance mutations in hot environments.ConclusionWe show that fitness costs of insecticide resistance genes are temperature-dependent and suggest that the duration of insecticide-free periods need to be adjusted for different climatic regions to reflect these costs. We suggest that such environment-dependent fitness effects may be more common than previously assumed and pose a major challenge for modeling climate change.

The effect of antibiotic and nutrient limitation to antibiotic resistant bacteria in single-cell level

Heterogeneity in bacterial populations can manifest in various ways, such as resistant cells, which can be observed in harsh environments after the use of antibiotics. Many studies have looked at the evolution of resistance and the effect of inhibitory and sub-inhibitory concentrations of antibiotics by batch culture measurements without considering the heterogeneity of bacterial populations. But antibiotic susceptibility and fitness costs of resistance mutations or plasmids are affected by the growth rate and physiology of individual cells. Single-cell analysis in microfluidic systems has opened up new possibilities enabling us to investigate the various putative mechanisms behind the persistence phenomenon required direct observation under the microscope. In this study, we use a gradient mixer and a novel micro-chemostat, to create concentration gradients of growth substrates and/or antibiotics to study the effect of nutrient and antibiotic concentration on individual cells growing under constant and defined conditions in cell-sized channels. The single-cell elongation, morphology and growth rate of ribosome-targeting antibiotics resistant E. coli was tracked by combining the microfluidics, microscope phase contrast imaging and fluorescent tag in high throughput mode. A mechanistic cellular model was used to describe the reaction between antibiotics and ribosome and the resulting effects on bacterial growth; then we linked the intracellular chemical-reaction kinetics processes to the population level and predicted the behaviour of population responses. Our approach has enabled the investigation of single-cell individuality and predictions of population dynamics under different environment.

Quantifying the fitness cost of HIV-1 drug resistance mutations through phylodynamics.

Drug resistant HIV is a major threat to the long-term efficacy of antiretroviral treatment. Around 10% of ART-naïve patients in Europe are infected with drug-resistant HIV type 1. Hence it is important to understand the dynamics of transmitted drug resistance evolution. Thanks to routinely performed drug resistance tests, HIV sequence data is increasingly available and can be used to reconstruct the phylogenetic relationship among viral lineages. In this study we employ a phylodynamic approach to quantify the fitness costs of major resistance mutations in the Swiss HIV cohort. The viral phylogeny reflects the transmission tree, which we model using stochastic birth–death-sampling processes with two types: hosts infected by a sensitive or resistant strain. This allows quantification of fitness cost as the ratio between transmission rates of hosts infected by drug resistant strains and transmission rates of hosts infected by drug sensitive strains. The resistance mutations 41L, 67N, 70R, 184V, 210W, 215D, 215S and 219Q (nRTI-related) and 103N, 108I, 138A, 181C, 190A (NNRTI-related) in the reverse trancriptase and the 90M mutation in the protease gene are included in this study. Among the considered resistance mutations, only the 90M mutation in the protease gene was found to have significantly higher fitness than the drug sensitive strains. The following mutations associated with resistance to reverse transcriptase inhibitors were found to be less fit than the sensitive strains: 67N, 70R, 184V, 219Q. The highest posterior density intervals of the transmission ratios for the remaining resistance mutations included in this study all included 1, suggesting that these mutations do not have a significant effect on viral transmissibility within the Swiss HIV cohort. These patterns are consistent with alternative measures of the fitness cost of resistance mutations. Overall, we have developed and validated a novel phylodynamic approach to estimate the transmission fitness cost of drug resistance mutations.

Abstract PR07: The strength of drug selection determines the maximum fitness cost of resistance mutations in culture and xenografts

Abstract Cancer cells often acquire resistance to therapy due to genetic mutations. However, without drug selection, these mutations would likely have a fitness cost to cancer cells and we hypothesize that the severity of this fitness cost is correlated with the effectiveness of drug treatment, with more effective treatments leading to more drastic genetic lesions. The size of a genetic lesion often correlates with its fitness cost - point mutation having little cost, aneuploidy having a large cost. However, aneuploidy is fast because the search space is only 46 gains or losses, while point mutations are slow with billions of base pairs to search before finding the right mutations. We designed experimental and computational systems to understand the selection pressures that would lead to resistance by these different genetic mechanisms. Mice were implanted with xenograft tumors from the HT1080 fibrosarcoma cell line that were heterozygous for DR5 (TNFRSF10B) and extremely sensitive to treatment with a DR5 specific monoclonal antibody, which induced extrinsic apoptosis. Treatment with three doses of anti-DR5 cured 3/11 mice, while 8/11 tumors relapsed. Isolation and culture of the relapse tumors showed they were completely resistant to DR5-mediated killing and had lost the functional copy of DR5. Assaying copy number variation using next generation sequencing (CNVSeq) showed that the relapse lines had lost one copy of chromosome 8p, where the functional DR5 gene was located. Treatment with two doses of anti-DR5 lead to relapses in all mice with loss of 8p. Finally, treatment with a single dose resulted in 3/4 tumors with loss of 8p. However 1/4 tumors became more resistant to anti-DR5 but did not lose the DR5 gene, presumably becoming resistant by smaller genetic changes or by non-genetic means that altered DR5 signaling. Thus, in most cases, treatment of xenograft tumors led to resistance by the loss of a chromosome arm, while resistance was rarely induced by other mechanisms. In contrast, cultured HT1080 cells treated with three doses of anti-DR5 became resistant but never lost 8p. In culture, treatment with anti-DR5 only kills a maximum of 80% of the cells and was therefore weaker than in mice where anti-DR5 could be curative. Thus, we observed an association between the strength of drug selection and the genetic mechanism by which cells became resistant. Experiments to further decrease selective pressure in xenografts and increase it in culture are ongoing. Next, we developed a computational model of tumor evolution to further explore the interplay between strength of selection and type of adaptation. In this simulation, cells were under two selective pressures, drug selection and competition for nutrients. Drug sensitive tumor cells could become resistant by two mechanisms analogous to point mutations or loss of 8p. Point mutations in this model caused resistance incrementally thereby taking several generations to acquire resistance, but they had no fitness cost in terms of competing for nutrients. Loss of 8p in this model caused complete resistance but had a fitness cost. Simulations with strong selective pressure led to the emergence of tumors that had completely lost 8p, even when the fitness cost was high. This result occurred because cells that did not lose 8p were eliminated by the drug. In contrast, simulations with selective pressure similar to culture (80% kill) led to tumors that became resistant without loss of 8p. In this case, sensitive cells incrementally acquired resistance mutations and then out competed cells that lost 8p for nutrients. Thus, simulation showed that resistance mutations with a large fitness cost will only arise under strong drug selection. In conclusion, our data suggest that the closer a therapy is to curing a tumor, the more extreme and costly resistance mutations will be. Citation Format: Lee A. Albacker, Rainer Kohler, Ralph Weissleder, Peter K. Sorger. The strength of drug selection determines the maximum fitness cost of resistance mutations in culture and xenografts. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr PR07.

Abstract B35: The strength of drug selection determines the maximum fitness cost of resistance mutations in culture and xenografts

Abstract This abstract is being presented as a short talk. A full abstract is printed in the Proffered Abstracts section (PR07) of the Conference Proceedings. Citation Format: Lee A. Albacker, Rainer Kohler, Ralph Weissleder, Peter K. Sorger. The strength of drug selection determines the maximum fitness cost of resistance mutations in culture and xenografts. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Drug Sensitivity and Resistance: Improving Cancer Therapy; Jun 18-21, 2014; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(4 Suppl): Abstract nr B35.

Management of MDR-TB household contacts: how difficult is it to climb the mountain? [Editorial

THE ISSUE of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB) is still hot in many perspectives. Policy makers and public health offi cers, in addition to clinicians managing index patients, require evidence supporting targeted interventions among household contacts of MDR-TB cases. The yield of secondary cases among contacts of drug-susceptible TB is known to have important i nter-country variations (from 1% to 8%), although these estimates are not clearly time-bound. The question as to whether the transmission rates of drugr esistant TB are similar remains unanswered, given the longer period of infectiousness, often attributed to delays in diagnosis, and to the diffi culties in achieving bacteriological conversion in such cases. Conversely, the reduced transmissibility of drugresistant strains due to the potential fi tness cost of resistance mutations has often been discussed but has never been demonstrated. Two very interesting contributions on this question are presented in this issue of the Journal , from pivotal countries in the fi ght against MDR- and XDRTB: South Africa and Peru. Both studies demonstrate very high TB incidence among household contacts. In the South African study, the incidence of MDRand XDR-TB cases among contacts was respectively 1765 and 1952 per 100 000 population, while the study from Peru shows that 5% of the contacts developed TB in a 3-year follow-up period (2360 per 100 000 contact follow-up years). Among those with available drug susceptibility test (DST) results, 80% had MDR-TB strains. Therefore, MDR-TB incidence among Peruvian contacts could be estimated at 1888/100 000 contact follow-up years.

Nonoptimal DNA Topoisomerases Allow Maintenance of Supercoiling Levels and Improve Fitness of Streptococcus pneumoniae

Fluoroquinolones, which target gyrase and topoisomerase IV, are used for treating Streptococcus pneumoniae infections. Fluoroquinolone resistance in this bacterium can arise via point mutation or interspecific recombination with genetically related streptococci. Our previous study on the fitness cost of resistance mutations and recombinant topoisomerases identified GyrAE85K as a high-cost change. However, this cost was compensated for by the presence of a recombinant topoisomerase IV (parC and parE recombinant genes) in strain T14. In this study, we purified wild-type and mutant topoisomerases and compared their enzymatic activities. In strain T14, both gyrase carrying GyrAE85K and recombinant topoisomerase IV showed lower activities (from 2.0- to 3.7-fold) than the wild-type enzymes. These variations of in vitro activity corresponded to changes of in vivo supercoiling levels that were analyzed by two-dimensional electrophoresis of an internal plasmid. Strains carrying GyrAE85K and nonrecombinant topoisomerases had lower (11.1% to 14.3%) supercoiling density (σ) values than the wild type. Those carrying GyrAE85K and recombinant topoisomerases showed either partial or total supercoiling level restoration, with σ values being 7.9% (recombinant ParC) and 1.6% (recombinant ParC and recombinant ParE) lower than those for the wild type. These data suggested that changes acquired by interspecific recombination might be selected because they reduce the fitness cost associated with fluoroquinolone resistance mutations. An increase in the incidence of fluoroquinolone resistance, even in the absence of further antibiotic exposure, is envisaged.

Compensation of Fitness Costs and Reversibility of Antibiotic Resistance Mutations

Strains of bacterial pathogens that have acquired mutations conferring antibiotic resistance often have a lower growth rate and are less invasive or transmissible initially than their susceptible counterparts. However, fitness costs of resistance mutations can be ameliorated by secondary site mutations. These so-called compensatory mutations may restore fitness in the absence and/or presence of antimicrobials. We review literature data and show that the fitness gains in the absence and presence of antibiotic treatment need not be correlated. The aim of this study is to gain a better conceptual grasp of how compensatory mutations with different fitness gains affect evolutionary trajectories, in particular reversibility. To this end, we developed a theoretical model with which we consider both a resistance and a compensation locus. We propose an intuitively understandable parameterization for the fitness values of the four resulting genotypes (wild type, resistance mutation only, compensatory mutation only, and both mutations) in the absence and presence of treatment. The differential fitness gains, together with the turnover rate and the mutation rate, strongly affected the success of antibacterial treatment, reversibility, and long-term abundance of resistant strains. We therefore propose that experimental studies of compensatory mutations should include fitness measurements of all possible genotypes in both the absence and presence of an antibiotic.

The Fitness Cost of Mutations Associated with Human Immunodeficiency Virus Type 1 Drug Resistance Is Modulated by Mutational Interactions

It is generally accepted that the fitness cost of resistance mutations plays a role in the persistence of transmitted drug-resistant human immunodeficiency virus type 1 and that mutations that confer a high fitness cost are less able to persist in the absence of drug pressure. Here, we show that the fitness cost of reverse transcriptase (RT) mutations can vary within a 72-fold range. We also demonstrate that the fitness cost of M184V and K70R can be decreased or enhanced by other resistance mutations such as D67N and K219Q. We conclude that the persistence of transmitted RT mutants might range widely on the basis of fitness and that the modulation of fitness cost by mutational interactions will be a critical determinant of persistence.

A genetic model to investigate drug–target interactions at the ribosomal decoding site

Recent advances in X-ray crystallography have greatly contributed to the understanding of the structural interactions between aminoglycosides and the ribosomal decoding site. Efforts to genetically probe the functional relevance of proposed drug–nucleotide contacts have in part been hampered by the presence of multiple rRNA operons in most bacteria. A derivative of the Gram-positive Mycobacterium smegmatis was rendered single rRNA operon allelic by means of gene inactivation techniques. In this system, genetic manipulation of the single chromosomal rRNA operon results in cells carrying homogeneous populations of mutant ribosomes. An exhaustive mutagenesis study of the ribosomal A site has been performed to define the importance of individual drug–nucleotide contacts. Mutational alterations in the M. smegmatis decoding site are discussed here, comparing the results with those obtained in other organisms. Implications for the selectivity of antimicrobial agents and for the fitness cost of resistance mutations are addressed.