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The widespread sulfonamide resistance genes sul1 , sul2 , and sul3 in food and gut bacteria have attracted considerable attention. In this study, we assessed the in vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli , using a murine model. High fitness costs were incurred for sul1 and sul3 gene-dependent E. coli strains in vivo . A fitness advantage was found in three of the eight mice after intragastric administration of sul2 gene-dependent E. coli strains. We isolated three compensatory mutant strains (CMSs) independently from three mice that outcompeted the parent strain P2 in vivo . Whole-genome sequencing revealed seven identical single nucleotide polymorphism (SNP) mutations in the three CMSs compared with strain P2, an additional SNP mutation in strain S2-2, and two additional SNP mutations in strain S2-3. Furthermore, tandem mass tag-based quantitative proteomic analysis revealed abundant differentially expressed proteins (DEPs) in the CMSs compared with P2. Of these, seven key fitness-related DEPs distributed in two-component systems, galactose and tryptophan metabolism pathways, were verified using parallel reaction monitoring analysis. The DEPs in the CMSs influenced bacterial motility, environmental stress tolerance, colonization ability, carbohydrate utilization, cell morphology maintenance, and chemotaxis to restore fitness costs and adapt to the mammalian gut environment.IMPORTANCESulfonamides are traditional synthetic antimicrobial agents used in clinical and veterinary medical settings. Their long-term excessive overuse has resulted in widespread microbial resistance, limiting their application for medical interventions. Resistance to sulfonamides is primarily conferred by the alternative genes sul1 , sul2 , and sul3 encoding dihydropteroate synthase in bacteria. Studying the potential fitness cost of these sul genes is crucial for understanding the evolution and transmission of sulfonamide-resistant bacteria. In vitro studies have been conducted on the fitness cost of sul genes in bacteria. In this study, we provide critical insights into bacterial adaptation and transmission using an in vivo approach., Competing Interests: The authors declare no conflict of interest.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location 1,2 . The extent and mechanisms of spread and vaccine-driven changes in fitness and antimicrobial resistance remain largely unquantified. Here using geolocated genome sequences from South Africa (n = 6,910, collected from 2000 to 2014), we developed models to reconstruct spread, pairing detailed human mobility data and genomic data. Separately, we estimated the population-level changes in fitness of strains that are included (vaccine type (VT)) and not included (non-vaccine type (NVT)) in pneumococcal conjugate vaccines, first implemented in South Africa in 2009. Differences in strain fitness between those that are and are not resistant to penicillin were also evaluated. We found that pneumococci only become homogenously mixed across South Africa after 50 years of transmission, with the slow spread driven by the focal nature of human mobility. Furthermore, in the years following vaccine implementation, the relative fitness of NVT compared with VT strains increased (relative risk of 1.68; 95% confidence interval of 1.59-1.77), with an increasing proportion of these NVT strains becoming resistant to penicillin. Our findings point to highly entrenched, slow transmission and indicate that initial vaccine-linked decreases in antimicrobial resistance may be transient., (© 2024. The Author(s).)

Albendazole (a benzimidazole) and ivermectin (a macrocyclic lactone) are the two most commonly co-administered anthelmintic drugs in mass-drug administration programs worldwide. Despite emerging resistance, we do not fully understand the mechanisms of resistance to these drugs nor the consequences of delivering them in combination. Albendazole resistance has primarily been attributed to variation in the drug target, a beta-tubulin gene. Ivermectin targets glutamate-gated chloride channels (GluCls), but it is unknown whether GluCl genes are involved in ivermectin resistance in nature. Using Caenorhabditis elegans, we defined the fitness costs associated with loss of the drug target genes singly or in combinations of the genes that encode GluCl subunits. We quantified the loss-of-function effects on three traits: (i) multi-generational competitive fitness, (ii) fecundity, and (iii) development. In competitive fitness and development assays, we found that a deletion of the beta-tubulin gene ben-1 conferred albendazole resistance, but ivermectin resistance required the loss of two GluCl genes (avr-14 and avr-15). The fecundity assays revealed that loss of ben-1 did not provide any fitness benefit in albendazole conditions and that no GluCl deletion mutants were resistant to ivermectin. Next, we searched for evidence of multi-drug resistance across the three traits. Loss of ben-1 did not confer resistance to ivermectin, nor did loss of any single GluCl subunit or combination confer resistance to albendazole. Finally, we assessed the development of 124 C. elegans wild strains across six benzimidazoles and seven macrocyclic lactones to identify evidence of multi-drug resistance between the two drug classes and found a strong phenotypic correlation within a drug class but not across drug classes. Because each gene affects various aspects of nematode physiology, these results suggest that it is necessary to assess multiple fitness traits to evaluate how each gene contributes to anthelmintic resistance., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Shaver et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Widespread insecticide resistance in African malaria vectors raises concerns over the potential to compromise malaria vector control interventions. Understanding the evolution of resistance mechanisms, and whether the selective disadvantages are large enough to be useful in resistance management or designing suitable control strategies is crucial. This study assessed whether insecticide resistance to pyrethroids has an effect on the gonotrophic cycle and reproductive potential of malaria vector Anopheles gambiae. Comparative tests were performed with pyrethroid-resistant and susceptible colonies of Anopheles gambiae colonized from the same geographical area, and the reference Kisumu strain was used as a control. Adult females aged 3 days old were given a blood meal and kept separately for individual egg-laying. The number of days taken to lay eggs post-blood-feeding was recorded to determine the length of the gonotrophic cycle. To measure adult longevity and reproduction potential, newly emerged males and females of equal numbers were aspirated into a cage and females allowed to blood feed daily. The number of eggs laid and the surviving mosquitoes were recorded daily to determine fecundity, net reproduction rate, intrinsic growth rate and adult longevity. Overall, the resistant females had a significantly longer (1.8 days) gonotrophic cycle than susceptible females (F 2, 13  = 9. 836, P < 0.01). The proportion of resistant females that laid eggs was lower 31.30% (94/300) compared to 54% (162/300) in the susceptible colony and 65.7% (197/300) in the Kisumu strain. The mean number of eggs laid per female was significantly lower in the resistant colony (88.02 ± 20) compared to the susceptible colony (104.9 ± .28.8) and the Kisumu strain (97.6 ± 34.8). The adult longevity was significantly higher for resistant (39.7 ± 1.6 days) compared to susceptible (29.9 ± 1.7 days) and the Kisumu strain was (29.6 ± 1.1 days) (F 2,8  = 45.05, P < 0.0001). Resistant colony exhibited a lower fecundity (4.3 eggs/females/day) and net reproductive rate (2.6 offsprings/female/generation) compared to the susceptible colony (8.6 eggs/female/day; 4.7 offsprings/female/generation respectively) and Kisumu strain (9.7 eggs/female/day; 4.1 offsprings/female/generation respectively). The study suggests high fitness cost on reproductive parameters of pyrethroid-resistant mosquitoes particularly on the duration of gonotrophic cycle, fecundity and net reproductive rate. These fitness costs are likely associated with maintaining both target site and metabolic mechanisms of resistance to pyrethroids. Despite these costs, resistant mosquitoes had longer longevity. These results give insights to understanding the fitness cost of insecticide resistance and thus are critical when predicting the epidemiological impact of insecticide resistance., (© 2022. The Author(s).)

Background: The efficacy of insecticide-treated nets (ITNs) containing the insect growth regulator pyriproxyfen (PPF) and pyrethroid insecticides (PPF-ITNs) is being assessed in clinical trials to determine whether they provide greater protection from malaria than standard pyrethroid-treated ITNs in areas where mosquitoes are resistant to pyrethroids. Understanding the entomological mode of action of this new ITN class will aide interpretation of the results from these trials., Methods: Anopheles gambiae sensu lato (s.l.) mosquitoes from a susceptible laboratory strain were exposed to PPF-treated netting 24 h, 6 h, and immediately prior to, or 24 h post blood feeding, and the impact on fecundity, fertility and longevity recorded. Pyrethroid-resistant populations were exposed to nets containing permethrin and PPF (PPF-ITNs) in cone bioassays and daily mortality recorded. Mosquitoes were also collected from inside houses pre- and post-distribution of PPF-ITNs in a clinical trial conduced in Burkina Faso; female An. gambiae s.l. were then assessed for fecundity and fertility., Results: PPF exposure reduced the median adult lifespan of insecticide-susceptible mosquitoes by 4 to 5 days in all exposure times (p < 0.05) other than 6 h pre-blood meal and resulted in almost complete lifelong sterilization. The longevity of pyrethroid-resistant mosquitoes was also reduced by at least 5 days after exposure to PPF-ITNs compared to untreated nets, but was unaffected by exposure to standard pyrethroid only ITNs. A total of 386 blood-fed or gravid An. gambiae s.l. females were collected from five villages between 1 and 12 months before distribution of PPF-ITNs. Of these mosquitoes, 75% laid eggs and the remaining 25% appeared to have normal ovaries upon dissection. In contrast, only 8.6% of the 631 blood-fed or gravid An. gambiae s.l. collected post PPF-ITN distribution successfully oviposited; 276 (43.7%) did not oviposit but had apparently normal ovaries upon dissection, and 301 (47.7%) did not oviposit and had abnormal eggs upon dissection. Egg numbers were also significantly lower (average of 138/female prior distribution vs 85 post distribution, p < 0.05)., Conclusion: Exposure to a mixture of PPF and pyrethroids on netting shortens the lifespan of mosquitoes and reduces reproductive output. Sterilization of vectors lasted at least one year under operational conditions. These findings suggest a longer effective lifespan of PPF-pyrethroid nets than reported previously.

Using a validated yeast chemogenomic platform, we characterized the genome-wide effects of several pharmaceutical contaminants, including three N-nitrosamines (NDMA, NDEA and NMBA), two related compounds (DMF and 4NQO) and several of their metabolites. A collection of 4800 non-essential homozygous diploid yeast deletion strains were screened in parallel and the strain abundance was quantified by barcode sequencing. These data were used to rank deletion strains representing genes required for resistance to the compounds to delineate affected cellular pathways and to visualize the global cellular effects of these toxins in an easy-to-use searchable database. Our analysis of the N-nitrosamine screens uncovered genes (via their corresponding homozygous deletion mutants) involved in several evolutionarily conserved pathways, including: arginine biosynthesis, mitochondrial genome integrity, vacuolar protein sorting and DNA damage repair. To investigate why NDMA, NDEA and DMF caused fitness defects in strains lacking genes of the arginine pathway, we tested several N-nitrosamine metabolites (methylamine, ethylamine and formamide), and found they also affected arginine pathway mutants. Notably, each of these metabolites has the potential to produce ammonium ions during their biotransformation. We directly tested the role of ammonium ions in N-nitrosamine toxicity by treatment with ammonium sulfate and we found that ammonium sulfate also caused a growth defect in arginine pathway deletion strains. Formaldehyde, a metabolite produced from NDMA, methylamine and formamide, and which is known to cross-link free amines, perturbed deletion strains involved in chromatin remodeling and DNA repair pathways. Finally, co-administration of N-nitrosamines with ascorbic or ferulic acid did not relieve N-nitrosamine toxicity. In conclusion, we used parallel deletion mutant analysis to characterize the genes and pathways affected by exposure to N-nitrosamines and related compounds, and provide the data in an accessible, queryable database.

Long-term infection of the airways of cystic fibrosis patients with Pseudomonas aeruginosa is often accompanied by a reduction in bacterial growth rate. This reduction has been hypothesised to increase within-patient fitness and overall persistence of the pathogen. Here, we apply adaptive laboratory evolution to revert the slow growth phenotype of P. aeruginosa clinical strains back to a high growth rate. We identify several evolutionary trajectories and mechanisms leading to fast growth caused by transcriptional and mutational changes, which depend on the stage of adaptation of the strain. Return to high growth rate increases antibiotic susceptibility, which is only partially dependent on reversion of mutations or changes in the transcriptional profile of genes known to be linked to antibiotic resistance. We propose that similar mechanisms and evolutionary trajectories, in reverse direction, may be involved in pathogen adaptation and the establishment of chronic infections in the antibiotic-treated airways of cystic fibrosis patients.

Chemical insecticides have been heavily employed as the most effective measure for control of agricultural and medical pests, but evolution of resistance by pests threatens the sustainability of this approach. Resistance-conferring mutations sometimes impose fitness costs, which may drive subsequent evolution of compensatory modifier mutations alleviating the costs of resistance. However, how modifier mutations evolve and function to overcome the fitness cost of resistance still remains unknown. Here we show that overexpression of P450s not only confers imidacloprid resistance in the brown planthopper, Nilaparvata lugens, the most voracious pest of rice, but also leads to elevated production of reactive oxygen species (ROS) through metabolism of imidacloprid and host plant compounds. The inevitable production of ROS incurs a fitness cost to the pest, which drives the increase or fixation of the compensatory modifier allele T65549 within the promoter region of N. lugens peroxiredoxin (NlPrx) in the pest populations. T65549 allele in turn upregulates the expression of NlPrx and thus increases resistant individuals' ability to clear the cost-incurring ROS of any source. The frequent involvement of P450s in insecticide resistance and their capacity to produce ROS while metabolizing their substrates suggest that peroxiredoxin or other ROS-scavenging genes may be among the common modifier genes for alleviating the fitness cost of insecticide resistance., Competing Interests: The authors have declared that no competing interests exist.

Context: Obesity, is a state of chronic inflammation, characterized by elevated lipids, insulin resistance and relative hypogonadotropic hypogonadism. We have defined the accompanying decreased Luteinizing Hormone (LH), Follicle-Stimulating Hormone (FSH), ovarian steroids and reduced pituitary response to Gonadotropin-releasing Hormone (GnRH) as Reprometabolic syndrome, a phenotype that can be induced in healthy normal weight women (NWW) by acute infusion of free fatty acids and insulin., Objective: To identify potential mediators of insulin and lipid-related reproductive endocrine dysfunction., Design, Setting, Participants: Secondary analysis of crossover study of eumenorrheic reproductive aged women of normal Body Mass Index (BMI) (<25 kg/m2) at an academic medical center., Intervention: Participants underwent 6-hour infusions of either saline/heparin or insulin plus fatty acids (Intralipid plus heparin), in the early follicular phase of sequential menstrual cycles, in random order. Euglycemia was maintained by glucose infusion. Frequent blood samples were obtained., Main Outcome Measures: Pooled serum from each woman was analyzed for cytokines, interleukins, chemokines, adipokines, Fibroblast Growth Factor-21 (FGF-21) and markers of endoplasmic reticulum (ER) stress (CHOP and GRP78). Wilcoxon signed-rank tests were used to compare results across experimental conditions., Results: Except for Macrophage Inflammatory Protein-1β (MIP-1β), no significant differences were observed in serum levels of any of the inflammatory signaling or ER stress markers tested., Conclusion: Acute infusion of lipid and insulin, to mimic the metabolic syndrome of obesity, was not associated with an increase in inflammatory markers. These results imply that the endocrine disruption and adverse reproductive outcomes of obesity are not a consequence of the ambient inflammatory environment but may be mediated by direct lipotoxic effects on the hypothalamic-pituitary-ovarian (HPO) axis., Competing Interests: The authors have declared the no competing interests exist.

Several phenotypes that impact the capacity of cancer cells to survive and proliferate are dynamic. Here we used the number of cells in colonies as an assessment of fitness and devised a novel method called Dynamic Fitness Analysis (DynaFit) to measure the dynamics in fitness over the course of colony formation. DynaFit is based on the variance in growth rate of a population of founder cells compared with the variance in growth rate of colonies with different sizes. DynaFit revealed that cell fitness in cancer cell lines, primary cancer cells, and fibroblasts under unhindered growth conditions is dynamic. Key cellular mechanisms such as ERK signaling and cell-cycle synchronization differed significantly among cells in colonies after 2 to 4 generations and became indistinguishable from randomly sampled cells regarding these features. In the presence of cytotoxic agents, colonies reduced their variance in growth rate when compared with their founder cell, indicating a dynamic nature in the capacity to survive and proliferate in the presence of a drug. This finding was supported by measurable differences in DNA damage and induction of senescence among cells of colonies. The presence of epigenetic modulators during the formation of colonies stabilized their fitness for at least four generations. Collectively, these results support the understanding that cancer cell fitness is dynamic and its modulation is a fundamental aspect to be considered in comprehending cancer cell biology and its response to therapeutic interventions. SIGNIFICANCE: Cancer cell fitness is dynamic over the course of the formation of colonies. This dynamic behavior is mediated by asymmetric mitosis, ERK activity, cell-cycle duration, and DNA repair capacity in the absence or presence of a drug., (©2020 American Association for Cancer Research.)

Knowledge about the fitness cost imposed by herbicide resistance in weeds is instrumental in devising integrated management methods. The present study investigated the germination response of ACCase-resistant (R) and susceptible (S) winter wild oat under different environmental conditions. The DNA of the plants was sequenced after being extracted and purified. The segregated F 2 seeds were subjected to various temperatures, water potentials, NaCl concentrations, different pHs, darkness conditions, and burial depths. The results of the sequencing indicated that Ile-2041-Asn mutation is responsible for the evolution of resistance in the studied winter wild oat plants. The seeds were able to germinate over a wide range of temperatures, osmotic potentials, NaCl concentrations, and pHs. Germination percentage of R and S seeds under dark and light conditions was similar and ranged from 86.3 to 88.3%. The highest emergence percentage for both R and S plants was obtained in 0, 1, and 2 cm depths and ranged from 66.6 to 70.3%. In overall, no differences were observed in the germination response between the R and S winter wild oat plants under all studied conditions. No fitness cost at seed level indicates that control of R winter wild oats is more difficult, and it is essential to adopt crop and herbicide rotation to delay the further evolution of resistance.

Drugs against flaviviruses such as dengue (DENV) and Zika (ZIKV) virus are urgently needed. We previously demonstrated that three fluoroquinolones, ciprofloxacin, enoxacin, and difloxacin, suppress replication of six flaviviruses. To investigate the barrier to resistance and mechanism(s) of action of these drugs, DENV-4 was passaged in triplicate in HEK-293 cells in the presence or absence of each drug. Resistance to ciprofloxacin was detected by the seventh passage and to difloxacin by the tenth, whereas resistance to enoxacin did not occur within ten passages. Two putative resistance-conferring mutations were detected in the envelope gene of ciprofloxacin and difloxacin-resistant DENV-4. In the absence of ciprofloxacin, ciprofloxacin-resistant viruses sustained a significantly higher viral titer than control viruses in HEK-293 and HuH-7 cells and resistant viruses were more stable than control viruses at 37 °C. These results suggest that the mechanism of action of ciprofloxacin and difloxacin involves interference with virus binding or entry., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Pericentromeric human satellite II (HSATII) repeats are normally silent but can be actively transcribed in tumor cells, where increased HSATII copy number is associated with a poor prognosis in colon cancer, and in human cytomegalovirus (HCMV)-infected fibroblasts, where the RNA facilitates viral replication. Here, we report that HCMV infection or treatment of ARPE-19 diploid epithelial cells with DNA-damaging agents, etoposide or zeocin, induces HSATII RNA expression, and a kinase-independent function of ATM is required for the induction. Additionally, various breast cancer cell lines growing in adherent, two-dimensional cell culture express HSATII RNA at different levels, and levels are markedly increased when cells are infected with HCMV or treated with zeocin. High levels of HSATII RNA expression correlate with enhanced migration of breast cancer cells, and knockdown of HSATII RNA reduces cell migration and the rate of cell proliferation. Our investigation links high expression of HSATII RNA to the DNA damage response, centered on a noncanonical function of ATM, and demonstrates a role for the satellite RNA in tumor cell proliferation and movement., Competing Interests: Competing interest statement: Icahn School of Medicine at Mt. Sinai and Princeton University have submitted a provisional patent application based on the use of HSATII-specific LNAs for HSATII knockdown, and M.T.N. and T.S. are co-inventors.

Background: In hepatitis C virus (HCV) infection, treatment failure is generally associated with the selection of resistance-associated substitutions (RAS) conferring reduced susceptibility to direct-acting antiviral (DAA) drugs. Resistant variants continue to replicate after the end of treatment with potential for transmission. This may result from the selection of "fitness-associated substitutions"., Aim: To characterise potential "fitness-associated substitutions" in patients infected with genotype 3a failing DAA drugs METHODS: By means of shotgun metagenomics, we sequenced full-length HCV genomes at treatment initiation and at virological relapse in eight patients infected with genotype 3a with cirrhosis failing sofosbuvir and an NS5A inhibitor. The impact of amino acid changes occurring outside of DAA target regions selected in at least two patients were assessed on the in vitro susceptibility to an NS5A inhibitor and replication capacity., Results: At treatment failure, besides selection of known NS5A RASs, especially Y93H, a large number of amino acid changes was observed outside of DAA target regions. We identified four amino acid positions at which observed changes substantially improved in vitro replication capacity without affecting NS5A inhibitor susceptibility., Conclusions: This is the first in vivo observation combined with in vitro confirmation of selection of phenotypically characterised "fitness-associated substitutions" together with RASs at the time of sofosbuvir-NS5A inhibitor treatment failure in patients infected with genotype 3a with cirrhosis. Our findings may explain the persistence of resistant HCV variants after treatment in patients who did not achieve sustained virological remission., (© 2020 John Wiley & Sons Ltd.)

Low success rates during drug development are due, in part, to the difficulty of defining drug mechanism-of-action and molecular markers of therapeutic activity. Here, we integrated 199,219 drug sensitivity measurements for 397 unique anti-cancer drugs with genome-wide CRISPR loss-of-function screens in 484 cell lines to systematically investigate cellular drug mechanism-of-action. We observed an enrichment for positive associations between the profile of drug sensitivity and knockout of a drug's nominal target, and by leveraging protein-protein networks, we identified pathways underpinning drug sensitivity. This revealed an unappreciated positive association between mitochondrial E3 ubiquitin-protein ligase MARCH5 dependency and sensitivity to MCL1 inhibitors in breast cancer cell lines. We also estimated drug on-target and off-target activity, informing on specificity, potency and toxicity. Linking drug and gene dependency together with genomic data sets uncovered contexts in which molecular networks when perturbed mediate cancer cell loss-of-fitness and thereby provide independent and orthogonal evidence of biomarkers for drug development. This study illustrates how integrating cell line drug sensitivity with CRISPR loss-of-function screens can elucidate mechanism-of-action to advance drug development., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

The importance of within-species diversity in determining the evolutionary potential of a population to evolve drug resistance or tolerance is not well understood, including in eukaryotic pathogens. To examine the influence of genetic background, we evolved replicates of 20 different clinical isolates of Candida albicans , a human fungal pathogen, in fluconazole, the commonly used antifungal drug. The isolates hailed from the major C. albicans clades and had different initial levels of drug resistance and tolerance to the drug. The majority of replicates rapidly increased in fitness in the evolutionary environment, with the degree of improvement inversely correlated with parental strain fitness in the drug. Improvement was largely restricted to up to the evolutionary level of drug: only 4% of the evolved replicates increased resistance (MIC) above the evolutionary level of drug. Prevalent changes were altered levels of drug tolerance (slow growth of a subpopulation of cells at drug concentrations above the MIC) and increased diversity of genome size. The prevalence and predominant direction of these changes differed in a strain-specific manner, but neither correlated directly with parental fitness or improvement in fitness. Rather, low parental strain fitness was correlated with high levels of heterogeneity in fitness, tolerance, and genome size among evolved replicates. Thus, parental strain background is an important determinant in mean improvement to the evolutionary environment as well as the diversity of evolved phenotypes, and the range of possible responses of a pathogen to an antimicrobial drug cannot be captured by in-depth study of a single strain background. IMPORTANCE Antimicrobial resistance is an evolutionary phenomenon with clinical implications. We tested how replicates from diverse strains of Candida albicans , a prevalent human fungal pathogen, evolve in the commonly prescribed antifungal drug fluconazole. Replicates on average increased in fitness in the level of drug they were evolved to, with the least fit parental strains improving the most. Very few replicates increased resistance above the drug level they were evolved in. Notably, many replicates increased in genome size and changed in drug tolerance (a drug response where a subpopulation of cells grow slowly in high levels of drug), and variability among replicates in fitness, tolerance, and genome size was higher in strains that initially were more sensitive to the drug. Genetic background influenced the average degree of adaptation and the evolved variability of many phenotypes, highlighting that different strains from the same species may respond and adapt very differently during adaptation., (Copyright © 2020 Gerstein and Berman.)

Insecticide based vector control tools such as insecticide treated bednets and indoor residual spraying represent the cornerstones of malaria control programs. Resistance to chemistries used in these programs is now widespread and represents a significant threat to the gains seen in reducing malaria-related morbidity and mortality. Recently, disruption of the 20-hydroxyecdysone steroid hormone pathway was shown to reduce Plasmodium development and significantly reduce both longevity and egg production in a laboratory susceptible Anopheles gambiae population. Here, we demonstrate that disruption of this pathway by application of the dibenzoylhydrazine, methoxyfenozide (DBH-M), to insecticide resistant An. coluzzii, An. gambiae sl and An. funestus populations significantly reduces egg production in both topical and tarsal application. Moreover, DBH-M reduces adult longevity when applied topically, and tarsally after blood feeding. As the cytochrome p450s elevated in pyrethroid resistant Anopheles only bind DBH-M very weakly, this compound is unlikely to be subject to cross-resistance in a field-based setting. Manipulation of this hormonal signalling pathway therefore represents a potential complementary approach to current malaria control strategies, particularly in areas where high levels of insecticide resistance are compromising existing tools., Competing Interests: Declaration of competing interest None., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Tumor heterogeneity is a large conundrum in cancer medicine, making most therapeutic interventions palliative rather than curative. Here we discuss the implications of how molecularly targeted therapies in solid malignancies that promote limited cancer cell death may in fact make tumors more heterogeneous, increase aggressive phenotypes, and thus worsen patient outcomes., (Published by Elsevier Inc.)

Fitness is closely associated with the development of pesticide resistance in insects, which determines the control strategies employed to target species and the risks of toxicity faced by non-target species. After years of selections with beta-cypermethrin in laboratory, a strain of housefly was developed that was 684,521.62-fold resistant (CRR) compared with the susceptible strain (CSS). By constructing ≤ 21 d and ≤ 30 d life tables, the differences in life history parameters between CSS and CRR were analyzed. The total production numbers of all the detected development stages in CRR were lower than in CSS. Except for the lower mortality of larvae, all the other detected mortalities in CRR were higher than in CSS. ♀:♂ and normal females of CRR were also lower than those of CSS. For CRR, the relative fitness was 0.25 in the ≤ 21 d life table and 0.24 in the ≤ 30 d life table, and a lower intrinsic rate of increase (rm) and net reproductive rate (Ro) were detected. Based on phenotype correlation and structural equation model (SEM) analyses, fecundity and viability were the only directly positive fitness components affecting fitness in CRR and CSS, and the other components played indirect roles in fitness. The variations of the relationships among fitness, fecundity and viability seemed to be the core issue resulting in fitness differences between CRR and CSS. The interactions among all the detected fitness components and the mating frequency-time curves appeared to be distinctly different between CRR and CSS. In summary, fecundity and its related factors separately played direct and indirect roles in the fitness costs of a highly beta-cypermethrin-resistant housefly strain., Competing Interests: The authors have declared that no competing interests exist.

Antibiotics released in the environment exert a selective pressure on the resident microbiota. It is well accepted that the mere measurement of antibiotics does not reflect the actual bioavailability. In fact, antibiotics can be adsorbed or complexed to particles and/or chemicals in water and soil. Bioavailable concentrations of antibiotics in soil and water are subjected to great uncertainty, therefore biological assays are increasingly recognized as that allow an indirect determination of the residual antibiotic activity. Here we propose how a fitness test for bacteria can be used to qualitatively assess the bioavailability of a specific antibiotic in the environment. The findings show that by using a pair of resistant and sensitive bacterial strains, the resulting fitness can indirectly reflect antibiotic bioavailability. Hence, this test can be used as a complementary assay to other biological and chemical tests to assess bioavailability of antibiotics., (Copyright © 2019. Published by Elsevier B.V.)

Background: Sulfoxaflor has been considered as a new tool for Nilaparvata lugens control in the field. In this study, a laboratory-selected resistant strain and a susceptible strain were used to evaluate the inheritance and fitness costs of sulfoxaflor resistance in N. lugens., Results: The resistant strain (SFX-SEL) showed 123.63-fold resistance compared with the susceptible strain (SS). Progenies of reciprocal crosses (F 1 RS and F 1 SR) showed similar concentration-mortality responses (LC 50 ) to sulfoxaflor and also exhibited a similar degree of dominance; -0.16 for F 1 RS and -0.26 for F 1 SR. Significant differences between the observed and expected mortalities of F 2 individuals suggested that sulfoxaflor resistance is associated with multiple genes. The resistant strain had a relative fitness of 0.75 with substantially decreased female adult period, oviposition days, total fecundity, egg hatchability and female adult survival rate, and prolonged pre-adult period and total pre-oviposition period., Conclusion: Sulfoxaflor resistance in N. lugens was inherited as autosomal, incompletely recessive and multigene. The development of resistance may have a significant fitness cost for the resistant population. Current research provides valuable information for researchers to establish management strategies to delay the development of sulfoxaflor resistance and control N. lugens sustainably in the field. © 2019 Society of Chemical Industry., (© 2019 Society of Chemical Industry.)

The soil-dwelling, saprophytic actinomycete Rhodococcus equi is a facultative intracellular pathogen of macrophages and causes severe bronchopneumonia when inhaled by susceptible foals. Standard treatment for R. equi disease is dual-antimicrobial therapy with a macrolide and rifampin. Thoracic ultrasonography and early treatment with antimicrobials prior to the development of clinical signs are used as means of controlling endemic R. equi infection on many farms. Concurrently with the increased use of macrolides and rifampin for chemoprophylaxis and the treatment of subclinically affected foals, a significant increase in the incidence of macrolide- and rifampin-resistant R. equi isolates has been documented. Previously, our laboratory demonstrated decreased fitness of R. equi strains that were resistant to macrolides, rifampin, or both, resulting in impaired in vitro growth in iron-restricted media and in soil. The objective of this study was to examine the effect of macrolide and/or rifampin resistance on intracellular replication of R. equi in equine pulmonary macrophages and in an in vivo mouse infection model in the presence and absence of antibiotics. In equine macrophages, the macrolide-resistant strain did not increase in bacterial numbers over time and the dual macrolide- and rifampin-resistant strain exhibited decreased proliferation compared to the susceptible isolate. In the mouse model, in the absence of antibiotics, the susceptible R. equi isolate outcompeted the macrolide- or rifampin-resistant strains., (Copyright © 2019 American Society for Microbiology.)

Environmental change exposes wildlife to a wide array of environmental stressors that arise from both anthropogenic and natural sources. Many environmental stressors with the ability to alter endocrine function are known as endocrine disruptors, which may impair the hypothalamus-pituitary-thyroid axis resulting in physiological consequences to wildlife. In this study, we investigated how the alteration of thyroid hormone (TH) levels due to exposure to the environmentally relevant endocrine disruptor sodium perchlorate (SP; inhibitory) and exogenous L-thyroxin (T4; stimulatory) affects metabolic costs and energy allocation during and after metamorphosis in a common amphibian (Rana temporaria). We further tested for possible carry-over effects of endocrine disruption during larval stage on juvenile performance. Energy allocated to development was negatively related to metabolic rate and thus, tadpoles exposed to T4 could allocate 24% less energy to development during metamorphic climax than control animals. Therefore, the energy available for metamorphosis was reduced in tadpoles with increased TH level by exposure to T4. We suggest that differences in metabolic rate caused by altered TH levels during metamorphic climax and energy allocation to maintenance costs might have contributed to a reduced energetic efficiency in tadpoles with high TH levels. Differences in size and energetics persisted beyond the metamorphic boundary and impacted on juvenile performance. Performance differences are mainly related to strong size-effects, as altered TH levels by exposure to T4 and SP significantly affected growth and developmental rate. Nevertheless, we assume that juvenile performance is influenced by a size-independent effect of achieved TH. Energetic efficiency varied between treatments due to differences in size allocation of internal macronutrient stores. Altered TH levels as caused by several environmental stressors lead to persisting effects on metamorphic traits and energetics and, thus, caused carry-over effects on performance of froglets. We demonstrate the mechanisms through which alterations in abiotic and biotic environmental factors can alter phenotypes at metamorphosis and reduce lifetime fitness in these and likely other amphibians., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Integrative and Comparative Biology. All rights reserved. For permissions please email: journals.permissions@oup.com.)

This study was designed to evaluate the evolution of antibiotic resistance in Salmonella enterica serovar Typhimurium treated with the combination of antibiotics. The experimental evolution of antibiotic resistance of S. Typhimurium was evaluated either under single antibiotic (kanamycin, KAN; penicillin, PEN; erythromycin, ERY) or in combination of two antibiotics (KAN + PEN or KAN + ERY) as measured by fractional inhibitory concentrations (FICs), stepwise resistance selection, cross-resistance evaluation, resistance fitness and relative gene expression. KAN + PEN and KAN + ERY showed the synergistic effect against S. Typhimurium (FIC index < 0.5). KAN + ERY delayed the induction of de novo mutations in S. Typhimurium. The cross-resistance of S. Typhimurium to all antibiotics except ERY and tetracycline was observed in KAN and PEN alone. The fitness cost was lower in single antibiotic treatments than combinations. The highest relative fitness was 0.91 in PEN, followed by KAN (0.84) and ERY (0.78), indicating the low fitness costs in single antibiotic treatments. The overexpression of efflux pump-related genes (acrA and acrB), outer membrane-related gene (ompC) and adherence-related gene (csgD) were observed in the single antibiotic treatments. Our results suggest that KAN + PEN and KAN + ERY could be used as a potential therapeutic treatment by decreasing the evolution of antibiotic resistance in S. Typhimurium and reusing conventional antibiotics., (© FEMS 2019.)

The diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae), is one of the main pests of Brassica crops worldwide. Management of P. xylostella is particularly challenging, as different field populations have readily acquired resistance to a wide range of insecticides, including Bacillus thuringiensis (Bt) toxins. In this study, a novel strain of P. xyllostela (Fuzhou-R2Ad) with 120-fold resistance to Bt Cry2Ad was selected in the laboratory, after screening for 66 generations from the susceptible strain Fuzhou-S. In the absence of Bt Cry2Ad toxin, the Fuzhou-R2Ad had significantly lower fitness as compared to the susceptible strain, which might be related to induced genetic changes to Bt toxins. We used several models to measure the dominance levels of insecticide resistance among different strains and found an incompletely recessive inheritance pattern of the Fuzhou-R2Ad resistance, which might be controlled by multiple genes. This study constitutes the first report of laboratory-acquired resistance to Cry2Ad toxin in P. xylostella. Our work presents further insights into the mechanism of Bt resistance and has immediate implications for the integrated pest management of P. xylostella globally.

The quasispecies dynamics of viral populations (continuous generation of variant genomes and competition among them) has as one of its frequent consequences variations in overall multiplication capacity, a major component of viral fitness. This parameter has multiple implications for viral pathogenesis and viral disease control, some of them unveiled thanks to deep sequencing of viral populations. Darwinian fitness is an old concept whose quantification dates back to the early developments of population genetics. It was later applied to viruses (mainly to RNA viruses) to quantify relative multiplication capacities of individual mutant clones or complex populations. The present article reviews the fitness concept and its relevance for the understanding of the adaptive dynamics of viruses in constant and changing environments. Many studies have addressed the fitness cost of escape mutations (to antibodies, cytotoxic T cells or inhibitors) as an influence on the efficacy of antiviral interventions. Here, we summarize the evidence that the basal fitness level can be a determinant of inhibitor resistance., (© FEMS 2019.)

There is a growing interest in the influence of vitamin D on ovine non-skeletal health. The aim of this study was to explore the relationship between pre-mating vitamin D status, as assessed by serum concentrations of 25-Hydroxyvitamin D [25(OH)D; comprising D 2 and D 3 ] and subsequent reproductive performance of genetically unimproved Scottish Blackface (UBF), genetically improved Scottish Blackface (IBF) and Lleyn ewes kept under Scottish hill conditions. 25-Hydroxyvitamin D 2 (25(OH)D 2 ) and 25-Hydroxyvitamin D 3 (25(OH)D 3 ) concentrations were determined in serum samples harvested in November from ewes grazed outdoors. There were no significant differences in 25(OH)D 2 concentrations amongst the 3 genotypes. Lleyn ewes had significantly higher 25(OH)D 3 and 25(OH)D concentrations than both Scottish Blackface ewe genotypes, whereas these vitamin D parameters did not differ significantly between the UBF and IBF ewes. Concentrations of 25(OH)D 3 and 25(OH)D were positively associated with subsequent birth weights of singleton and of twin lamb litters. No significant associations between vitamin D status and number of lambs born or weaned per ewe were found. This study demonstrates that concentrations of cutaneously-derived 25(OH)D 3, but not of orally consumed 25(OH)D 2 , differed between breeds. The positive association between ewe vitamin D status and offspring birth weight highlights the need for further investigations.

Objectives: Miltefosine is currently the only oral drug for visceral leishmaniasis, and although deficiency in an aminophospholipid/miltefosine transporter (MT) is sufficient to elicit drug resistance, very few naturally miltefosine-resistant (MIL-R) strains have yet been isolated. This study aimed to make a detailed analysis of the impact of acquired miltefosine resistance and miltefosine treatment on in vivo infection., Methods: Bioluminescent versions of a MIL-R strain and its syngeneic parental line were generated by integration of the red-shifted firefly luciferase PpyRE9. The fitness of both lines was compared in vitro (growth rate, metacyclogenesis and macrophage infectivity) and in BALB/c mice through non-invasive bioluminescence imaging under conditions with and without drug pressure., Results: This study demonstrated a severe fitness loss of MT-deficient parasites, resulting in a complete inability to multiply and cause a typical visceral leishmaniasis infection pattern in BALB/c mice. The observed fitness loss could not be rescued by host immune suppression with cyclophosphamide, whereas episomal reconstitution with a wild-type MT restored parasite virulence, hence linking parasite fitness to MT mutation. Remarkably, in vivo miltefosine treatment or in vitro miltefosine pre-exposure significantly rescued MIL-R parasite virulence. The in vitro pre-exposed MIL-R promastigotes showed a longer and more slender morphology, suggesting an altered membrane composition., Conclusions: The profound fitness loss of MT-deficient parasites most likely explains the low frequency of MIL-R clinical isolates. The observation that miltefosine can reverse this phenotype indicates a drug dependency of the MT-deficient parasites and emphasizes the importance of resistance profiling prior to miltefosine administration.

Background: Artemisinin-based combination therapy (ACT) is the recommended treatment against uncomplicated Plasmodium falciparum infections, and ACT is widely used. It has been shown that gametocytes may be present after ACT and transmission to mosquitoes is still possible. Artemether-lumefantrine (AL) is a broadly used artemisinin-based combination medicine. Here, it is tested whether AL influences behaviour and fitness of Anopheles mosquitoes, which are the main vectors of P. falciparum., Results: Dual-choice olfactometer and screenhouse experiments showed that skin odour of healthy human individuals obtained before, during and after AL-administration was equally attractive to Anopheles coluzzii and Anopheles gambiae sensu stricto, apart from a small (but significant) increase in mosquito response to skin odour collected 3 weeks after AL-administration. Anopheles coluzzii females fed on parasite-free blood supplemented with AL or on control-blood had similar survival, time until oviposition and number of eggs produced., Conclusions: Based on the results, AL does not appear to influence malaria transmission through modification of vector mosquito olfactory behaviour or fitness. Extending these studies to Plasmodium-infected individuals and malaria mosquitoes with parasites are needed to further support this conclusion.

Mitochondrial dysfunction underscores aging and diseases. Mitophagy (mitochondria + autophagy) is a quality control pathway that preserves mitochondrial health by targeting damaged mitochondria for autophagic degradation. Hence, molecules or compounds that can augment mitophagy are therapeutic candidates to mitigate mitochondrial-related diseases. However, mitochondrial stress remains the most effective inducer of mitophagy. Thus, identification of mitophagy-inducing regimes that are clinically relevant is favorable. In this study, pomegranate extract (PE) supplementation is shown to stimulate mitophagy. PE activates transcription factor EB (TFEB) to upregulate the expression of autophagy and lysosomal genes for mitochondrial quality control under basal and stress conditions. Basally, PE alters mitochondrial morphology and promotes recruitment of autophagosomes to the mitochondria (mitophagosome formation). Upon onset of mitochondrial stress, PE further augments mitophagosome formation, and engages PINK1 and Parkin to the mitochondria to potentiate mitophagy. This cellular phenomenon of PE-induced mitophagy helps to negate superfluous mitochondrial reactive oxygen species (ROS) production and mitochondrial impairment. Overall, our study highlights the potential of PE supplementation as a physiological therapy to modulate TFEB activity to alleviate mitochondrial dysfunction in aging and mitochondrial-related diseases.

Antibiotic resistance represents a growing health crisis that necessitates the immediate discovery of novel treatment strategies. One such strategy is the identification of collateral sensitivities, wherein evolution under a first drug induces susceptibility to a second. Here, we report that sequential drug regimens derived from in vitro evolution experiments may have overstated therapeutic benefit, predicting a collaterally sensitive response where cross-resistance ultimately occurs. We quantify the likelihood of this phenomenon by use of a mathematical model parametrised with combinatorially complete fitness landscapes for Escherichia coli. Through experimental evolution we then verify that a second drug can indeed stochastically exhibit either increased susceptibility or increased resistance when following a first. Genetic divergence is confirmed as the driver of this differential response through targeted and whole genome sequencing. Taken together, these results highlight that the success of evolutionarily-informed therapies is predicated on a rigorous probabilistic understanding of the contingencies that arise during the evolution of drug resistance.

In at least some unicellular organisms, mutation rates are temporarily raised upon exposure to environmental stress, potentially contributing to the evolutionary response to stress. Whether this is true for multicellular organisms, however, has received little attention. This study investigated the effects of chronic mild stress, in the form of low-level copper and nickel exposure, on mutational processes in Daphnia pulex using a combination of mutation accumulation, whole genome sequencing and life-history assays. After over 100 generations of mutation accumulation, we found no effects of metal exposure on the rates of single nucleotide mutations and of loss of heterozygosity events, the two mutation classes that occurred in sufficient numbers to allow statistical analysis. Similarly, rates of decline in fitness, as measured by intrinsic rate of population increase and of body size at first reproduction, were negligibly affected by metal exposure. We can reject the possibility that Daphnia were insufficiently stressed to invoke genetic responses as we have previously shown rates of large-scale deletions and duplications are elevated under metal exposure in this experiment. Overall, the mutation accumulation lines did not significantly depart from initial values for phenotypic traits measured, indicating the lineage used was broadly mutationally robust. Taken together, these results indicate that the mutagenic effects of chronic low-level exposure to these metals are restricted to certain mutation classes and that fitness consequences are likely minor and therefore unlikely to be relevant in determining the evolutionary responses of populations exposed to these stressors., (Copyright © 2019 Bull et al.)

The prevalence of antibiotic resistance in drinking water system is pressing public health risk. Antibiotic resistance conferred by chromosomal mutations often produces fitness cost, which may affect its spread and persistence. In this study, the rifampin-resistant strains were competed with their wild-type counterparts at different nutrient levels. It was observed that the ratio of the absolute number between resistant and wild-type cells quickly decreased under rich nutrient conditions, but it slowly reduced or remained stable in the poor nutrient medium. This finding suggested that poor nutrient conditions resulted in the reduction of fitness cost of antibiotic resistance, i.e. the resistant bacteria became more competitive. Implying mechanisms analysis found that the differences of metabolic activity between wild-type and rifampin-resistant strains was significant smaller (P < 0.05) at low nutrient levels. Additionally, distinguishable large colony size rifampin-resistant strains were observed during competition assay. DNA sequencing of RNA polymerase subunit genes further revealed that these colonies could be adaptive mutants from wild-type strain in rpoB gene. To our knowledge, this is the first study to reveal that the oligotrophic conditions facilitate the persistence of antibiotic resistance in drinking water by reducing the fitness cost of the resistant strains., (Copyright © 2018. Published by Elsevier Ltd.)

Background: To improve resistance management strategies for Bacillus thuringiensis (Bt) crops, a better understanding of the relative fitness of pest genotypes with resistance alleles in the absence of Bt toxins is needed. Here, we evaluated the impact of costs of resistance to Bt toxin Cry1Ac on the relative fitness of specific pink bollworm (Pectinophora gossypiella) genotypes. We created two heterogeneous strains with an intermediate frequency of mutant cadherin alleles linked with resistance to Cry1Ac, reared the strains on diet without Bt and tracked the decline in frequency of resistant genotypes for 15-30 generations using polymerase chain reaction amplification. We used a population genetics model and sensitivity analyses to estimate the relative fitness of resistant genotypes., Results: Costs were completely recessive in one strain and almost completely recessive in the other. Estimates of the decline in relative fitness of the resistant homozygotes fed on a diet without Bt were 14-22% in one strain and 21-36% in the other., Conclusion: Our genotype-specific cost estimates and the results of studies discussed herein indicate that costs associated with resistance to Bt are often large enough to significantly delay the evolution of resistance to pyramided Bt crops in pests with recessive inheritance of resistance. © 2018 Society of Chemical Industry., (© 2018 Society of Chemical Industry.)

Viral fitness quantifies the degree of virus adaptation to a given environment. How viral fitness can influence the mutant spectrum complexity of a viral quasispecies subjected to lethal mutagenesis has not been investigated. Here we document that two high fitness hepatitis C virus populations display higher resistance to the mutagenic nucleoside analogues favipiravir and ribavirin than their parental, low fitness HCV. All populations, however, exhibited a mutation transition bias indicative of active mutagenesis. Resistance to the analogues was associated with a limited expansion of mutant spectrum complexity, as evidenced by several diversity indices used to characterize mutant spectra. The results are consistent with a replicative site-drug competition mechanism that was previously proposed for HCV fitness-associated resistance to non-mutagenic inhibitors. Other alternative, non-mutually exclusive mechanisms are considered. The results introduce viral fitness as a relevant parameter to evaluate the response of viruses to lethal mutagenesis, with implications for antiviral designs., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Artemisinin is the most rapidly effective drug for Plasmodium falciparum malaria treatment currently in clinical use. Emerging artemisinin-resistant parasites pose a great global health risk. At present, the level of artemisinin resistance is still relatively low with evidence pointing towards a trade-off between artemisinin resistance and fitness loss. Here we show that artemisinin-resistant P. falciparum isolates from Cambodia manifested fitness loss, showing fewer progenies during the intra-erythrocytic developmental cycle. The loss in fitness was exacerbated under the condition of low exogenous amino acid supply. The resistant parasites failed to undergo maturation, whereas their drug-sensitive counterparts were able to complete the erythrocytic cycle under conditions of amino acid deprivation. The artemisinin-resistant phenotype was not stable, and loss of the phenotype was associated with changes in the expression of a putative target, Exp1, a membrane glutathione transferase. Analysis of SNPs in haemoglobin processing genes revealed associations with parasite clearance times, suggesting changes in haemoglobin catabolism may contribute to artemisinin resistance. These findings on fitness and protein homeostasis could provide clues on how to contain emerging artemisinin-resistant parasites.

VIRIP has been identified as natural HIV-1 inhibitor targeting the gp41 fusion peptide. An optimized analogue (VIR-576) was effective in a phase I/II clinical trial and initial studies showed that HIV-1 resistance to VIRIP-based inhibitors has a high genetic barrier. Partially resistant CXCR4 (X4)-tropic HIV-1 NL4-3 variants could be obtained, however, after more than 15 months of passaging in MT-4 cells in the presence of another derivative (VIR-353). Sequence analyses identified the accumulation of seven mutations across the HIV-1 envelope glycoprotein but outside the gp41 fusion peptide. The authors suggested that the three initial alterations conferred resistance, while subsequent changes restored viral fitness. Here, we introduced these mutations individually and in combination into X4- and CCR5 (R5)-tropic HIV-1 constructs and determined their impact on VIR-353 and VIR-576 susceptibility, viral infectivity, replication fitness, and fusogenicity. We found that essentially all seven mutations contribute to reduced susceptibility to VIRIP-based inhibitors. HIV-1 constructs containing ≥4 changes were substantially more resistant to both VIRIP-based inhibitors and the VRC34.01 antibody targeting the fusion peptide. However, they were also much less infectious and fusogenic than those harboring only the three initial alterations. Furthermore, the additional changes attenuated rather than rescued HIV-1 replication in primary human cells. Thus, the genetic barrier to HIV-1 resistance against VIRIP-based inhibitors is higher than previously suggested, and mutations reducing viral susceptibility come at a severe fitness cost that was not rescued during long-term cell culture passage. IMPORTANCE Many viral pathogens are critically dependent on fusion peptides (FPs) that are inserted into the cellular membrane for infection. Initially, it was thought that FPs cannot be targeted for therapy because they are hardly accessible. However, an optimized derivative (VIR-576) of an endogenous fragment of α1-antitrypsin, named VIRIP, targeting the gp41 FP reduced viral loads in HIV-1-infected individuals. Characterization of HIV-1 variants selected during long-term cell-culture passage in the presence of a VIRIP derivative suggested that just three mutations in the HIV-1 Env protein might be sufficient for VIRIP resistance and that four subsequent changes restored viral fitness. Here, we show that all seven mutations contribute to reduced viral susceptibility to VIRIP-based inhibitors and demonstrate that the additional changes strongly impair rather than rescue HIV-1 infectivity, fusogenicity, and replication fitness. High genetic barrier to resistance and severe fitness cost support further clinical development of this class of antiviral agents., (Copyright © 2018 American Society for Microbiology.)

Background: Fitness costs imposed on bacteria by antibiotic resistance mechanisms are believed to hamper their dissemination. The scale of these costs is highly variable. Some, including resistance of Staphylococcus aureus to the clinically important antibiotic mupirocin, have been reported as being cost-free, which suggests that there are few barriers preventing their global spread. However, this is not supported by surveillance data in healthy communities, which indicate that this resistance mechanism is relatively unsuccessful., Results: Epistasis analysis on two collections of MRSA provides an explanation for this discord, where the mupirocin resistance-conferring mutation of the ileS gene appears to affect the levels of toxins produced by S. aureus when combined with specific polymorphisms at other loci. Proteomic analysis demonstrates that the activity of the secretory apparatus of the PSM family of toxins is affected by mupirocin resistance. As an energetically costly activity, this reduction in toxicity masks the fitness costs associated with this resistance mutation, a cost that becomes apparent when toxin production becomes necessary. This hidden fitness cost provides a likely explanation for why this mupirocin-resistance mechanism is not more prevalent, given the widespread use of this antibiotic., Conclusions: With dwindling pools of antibiotics available for use, information on the fitness consequences of the acquisition of resistance may need to be considered when designing antibiotic prescribing policies. However, this study suggests there are levels of depth that we do not understand, and that holistic, surveillance and functional genomics approaches are required to gain this crucial information.

Neuraminidase (NA) mutations conferring resistance to NA inhibitors (NAIs) are expected to occur at framework or catalytic residues of the NA enzyme. Numerous clinical and in vitro reports already described NAI-resistant A(H1N1)pdm09 variants harboring various framework NA substitutions. By contrast, variants with NA catalytic changes remain poorly documented. Herein, we investigated the effect of R152K and R368K NA catalytic mutations on the NA enzyme properties, in vitro replicative capacity and virulence of A(H1N1)pdm09 recombinant viruses. In NA inhibition assays, the R152K and R368K substitutions resulted in reduced inhibition [10- to 100-fold increases in IC 50 vs the wild-type (WT)] or highly reduced inhibition (>100-fold increases in IC 50 ) to at least 3 approved NAIs (oseltamivir, zanamivir, peramivir and laninamivir). Such resistance phenotype correlated with a significant reduction of affinity observed for the mutants in enzyme kinetics experiments [increased Km from 20 ± 1.77 for the WT to 200.8 ± 10.54 and 565.2 ± 135 μM (P < 0.01) for the R152K and R368K mutants, respectively]. The R152K and R368K variants grew at comparable or even higher titers than the WT in both MDCK and ST6GalI-MDCK cells. In experimentally-infected C57BL/6 mice, the recombinant WT and the R152K and R368K variants induced important signs of infection (weight loss) and resulted in mortality rates of 87.5%, 37.5% and 100%, respectively. The lung viral titers were comparable between the three infected groups. While the NA mutations were stable, an N154I substitution was detected in the HA2 protein of the R152K and R368K variants after in vitro passages as well as in lungs of infected mice. Due to the multi-drug resistance phenotypes and conserved fitness, the emergence of NA catalytic mutations accompanied with potential compensatory HA changes should be carefully monitored in A(H1N1)pdm09 viruses., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background: Rhopalosiphum padi is a destructive insect pest of wheat worldwide. Studies have shown that R. padi has developed resistance to different insecticides, including imidacloprid. We studied the mechanisms conferring resistance to imidacloprid at the biochemical and molecular levels., Results: An R. padi imidacloprid-resistant (IM-R) strain and a susceptible (SS) strain were established. Fitness analysis using life-tables showed that the IM-R strain had obvious disadvantages in several parameters, indicating reduced fitness. Profiles of cross-resistance of IM-R and SS to seven insecticides were detected. Both synergistic and enzyme activity data suggested that P450 plays a role in resistance. Furthermore, the mRNA expression levels of cytochrome P450 (CYP) genes CYP6CY3-1 and CYP6CY3-2 were significantly increased in the IM-R strain. No target-site mutation within the nicotinic acetylcholine receptor (nAChR) subunits was detected in the IM-R strain. Interestingly, the expression levels of the nAChR α1, α2, α3, α7-2, and β1 subunit genes were significantly decreased, suggesting that down-regulation of these subunits may be involved in resistance., Conclusion: Multiple mechanisms confer imidacloprid resistance in R. padi. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Background: The fitness cost of herbicide resistance (HR) in the absence of herbicide selection plays a key role in HR evolution. Quantifying the fitness cost of resistance, however, is challenging, and there exists a knowledge gap in this area. A synthetic (artificially generated) Amaranthus tuberculatus population segregating for five types of HR was subjected to competitive growth conditions in the absence of herbicide selection for six generations. Fitness costs were quantified by using a combination of phenotyping and genotyping to monitor HR frequency changes over generations., Results: In the absence of herbicide selection, a significant fitness cost was observed for resistance to acetolactate synthase-inhibiting herbicides, but not for resistances to atrazine (non-target-site resistance mechanism), protoporphyrinogen oxidase inhibitors, 4-hydroxyphenylpryuvate dioxygenase inhibitors or glyphosate. Glyphosate resistance was conferred by multiple mechanisms in the synthetic population, and further analysis revealed that one mechanism, amplification of the 5-enolypyruvylshikimate-3-phosphate synthase gene, did decrease in frequency., Conclusion: Our results indicate that herbicide-resistance mitigation strategies (e.g. herbicide rotation) that rely on the existence of fitness costs in the absence of herbicide selection likely will be largely ineffective in many cases. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Organismal fitness depends on adaptation to complex niches where chemical compounds and pathogens are omnipresent. These stresses can lead to the fixation of alleles in both xenobiotic responses and proliferative signaling pathways that promote survival in these niches. However, both xenobiotic responses and proliferative pathways vary within and among species. For example, genetic differences can accumulate within populations because xenobiotic exposures are not constant and selection is variable. Additionally, neutral genetic variation can accumulate in conserved proliferative pathway genes because these systems are robust to genetic perturbations given their essential roles in normal cell-fate specification. For these reasons, sensitizing mutations or chemical perturbations can disrupt pathways and reveal cryptic variation. With this fundamental view of how organisms respond to cytotoxic compounds and cryptic variation in conserved signaling pathways, it is not surprising that human patients have highly variable responses to chemotherapeutic compounds. These different responses result in the low FDA-approval rates for chemotherapeutics and underscore the need for new approaches to understand these diseases and therapeutic interventions. Model organisms, especially the classic invertebrate systems of Caenorhabditis elegans and Drosophila melanogaster, can be used to combine studies of natural variation across populations with responses to both xenobiotic compounds and chemotherapeutics targeted to conserved proliferative signaling pathways., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The relationship between bacterial drug resistance and growth fitness is a contentious topic, but some antibiotic resistance mutations clearly have a fitness cost in the laboratory. Whether these costs translate into deleterious effects in natural habitats is less certain however. Previously, fitness effects of resistance mutations were mostly characterized in nutrient-rich, fast-growth conditions, which bacteria rarely encounter in natural habitats. Carbon, phosphate, iron or oxygen limitations are conditions met by bacterial pathogens in various compartments of the human body. Here, we measured the fitness of four different rpoB mutations commonly found in rifampicin-resistant bacterial isolates. The fitness properties and the emergence of these and other alleles were studied in Escherichia coli populations growing under nutrient excess and in four different nutrient-limited states. Consistent with previous findings, all four mutations exhibited deleterious fitness effects under nutrient-rich conditions. In stark contrast, we found positive or neutral fitness effects under nutrient-limited conditions. Two particular rpoB alleles had a remarkable fitness increase under phosphate limitation and these alleles arose to high frequencies specifically under phosphate limitation. These findings suggest that it is not meaningful to draw general conclusions on fitness costs without considering bacterial microenvironments in humans and other animals., (© 2017 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Atrazine (ATZ) is an herbicide most commonly used in China and other regions of the world. It is reported toxic to aquatic organisms, and frequently occurs at relatively high concentrations. Currently, ATZ has been proved to affect reproduction of aquatic species at much lower levels. So it is controversial to perform ecological risk assessment using predicted no-effect concentrations (PENCs) derived from traditional endpoints, which fail to provide adequate protection to aquatic organisms. In this study, PNECs of ATZ were derived based on six endpoints of survival, growth, behavior, biochemistry, genetics and reproduction. The PNEC derived from reproductive lesion was 0.044μg ATZ L -1 , which was obviously lower than that derived from other endpoints. In addition, a tiered ecological risk assessment was conducted in the Taizi River based on six PNECs derived from six categories of toxicity endpoints. Results of these two methods of ecological risk assessment were consistent with each other, and the risk level of ATZ to aquatic organisms reached highest as taking reproductive fitness into account. The joint probability indicated that severe ecological risk rooting in reproduction might exist 93.9% and 99.9% of surface water in the Taizi River, while 5% threshold (HC 5 ) and 1% threshold (HC 1 ) were set up to protect aquatic organisms, respectively. We hope the present work could provide valuable information to manage and control ATZ pollution., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chloroquine, long the default first-line treatment against malaria, is now abandoned in large parts of the world because of widespread drug-resistance in Plasmodium falciparum. In spite of its importance as a cost-effective and efficient drug, a coherent understanding of the cellular mechanisms affected by chloroquine and how they influence the fitness and survival of the parasite remains elusive. Here, we used a systems biology approach to integrate genome-scale transcriptomics to map out the effects of chloroquine, identify targeted metabolic pathways, and translate these findings into mechanistic insights. Specifically, we first developed a method that integrates transcriptomic and metabolomic data, which we independently validated against a recently published set of such data for Krebs-cycle mutants of P. falciparum. We then used the method to calculate the effect of chloroquine treatment on the metabolic flux profiles of P. falciparum during the intraerythrocytic developmental cycle. The model predicted dose-dependent inhibition of DNA replication, in agreement with earlier experimental results for both drug-sensitive and drug-resistant P. falciparum strains. Our simulations also corroborated experimental findings that suggest differences in chloroquine sensitivity between ring- and schizont-stage P. falciparum. Our analysis also suggests that metabolic fluxes that govern reduced thioredoxin and phosphoenolpyruvate synthesis are significantly decreased and are pivotal to chloroquine-based inhibition of P. falciparum DNA replication. The consequences of impaired phosphoenolpyruvate synthesis and redox metabolism are reduced carbon fixation and increased oxidative stress, respectively, both of which eventually facilitate killing of the parasite. Our analysis suggests that a combination of chloroquine (or an analogue) and another drug, which inhibits carbon fixation and/or increases oxidative stress, should increase the clearance of P. falciparum from the host system., (Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Mutations conferring resistance to antibiotics are typically costly in the absence of the drug, but bacteria can reduce this cost by acquiring compensatory mutations. Thus, the rate of acquisition of compensatory mutations and their effects are key for the maintenance and dissemination of antibiotic resistances. While compensation for single resistances has been extensively studied, compensatory evolution of multiresistant bacteria remains unexplored. Importantly, since resistance mutations often interact epistatically, compensation of multiresistant bacteria may significantly differ from that of single-resistant strains. We used experimental evolution, next-generation sequencing, in silico simulations, and genome editing to compare the compensatory process of a streptomycin and rifampicin double-resistant Escherichia coli with those of single-resistant clones. We demonstrate that low-fitness double-resistant bacteria compensate faster than single-resistant strains due to the acquisition of compensatory mutations with larger effects. Strikingly, we identified mutations that only compensate for double resistance, being neutral or deleterious in sensitive or single-resistant backgrounds. Moreover, we show that their beneficial effects strongly decrease or disappear in conditions where the epistatic interaction between resistance alleles is absent, demonstrating that these mutations compensate for the epistasis. In summary, our data indicate that epistatic interactions between antibiotic resistances, leading to large fitness costs, possibly open alternative paths for rapid compensatory evolution, thereby potentially stabilizing costly multiple resistances in bacterial populations.

Gene-gene or gene-drug interactions are typically quantified using fitness as a readout because the data are continuous and easily measured in high throughput. However, to what extent fitness captures the range of other phenotypes that show synergistic effects is usually unknown. Using Saccharomyces cerevisiae and focusing on a matrix of DNA repair mutants and genotoxic drugs, we quantify 76 gene-drug interactions based on both mutation rate and fitness and find that these parameters are not connected. Independent of fitness defects, we identified six cases of synthetic hypermutation, where the combined effect of the drug and mutant on mutation rate was greater than predicted. One example occurred when yeast lacking RA D1 were exposed to cisplatin, and we characterized this interaction using whole-genome sequencing. Our sequencing results indicate mutagenesis by cisplatin in rad1 Δ cells appeared to depend almost entirely on interstrand cross-links at GpCpN motifs. Interestingly, our data suggest that the following base on the template strand dictates the addition of the mutated base. This result differs from cisplatin mutation signatures in XPF-deficient Caenorhabditis elegans and supports a model in which translesion synthesis polymerases perform a slippage and realignment extension across from the damaged base. Accordingly, DNA polymerase ζ activity was essential for mutagenesis in cisplatin-treated rad1 Δ cells. Together these data reveal the potential to gain new mechanistic insights from nonfitness measures of gene-drug interactions and extend the use of mutation accumulation and whole-genome sequencing analysis to define DNA repair mechanisms.

Acquisition of antibiotic resistance is a relevant problem for human health. The selection and spread of antibiotic-resistant organisms not only compromise the treatment of infectious diseases, but also the implementation of different therapeutic procedures as organ transplantation, advanced surgery or chemotherapy, all of which require proficient methods for avoiding infections. It has been generally accepted that the acquisition of antibiotic resistance will produce a general metabolic burden: in the absence of selection, the resistant organisms would be outcompeted by the susceptible ones. If that was always true, discontinuation of antibiotic use would render the disappearance of resistant microorganisms. However, several studies have shown that, once resistance emerges, the recovery of a fully susceptible population even in the absence of antibiotics is not easy. In the present study, we review updated information on the effect of the acquisition of antibiotic resistance in bacterial physiology as well as on the mechanisms that allow the compensation of the fitness costs associated with the acquisition of resistance., (© 2017 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Barnyardgrass (Echinochloa crus-galli) is one of the top 15 herbicide-resistant weeds around the world that interferes with rice growth, resulting in major losses of rice yield. Thus, multi-herbicide resistance in barnyardgrass presents a major threat, with the underlying mechanisms that contribute to resistance requiring elucidation. In an attempt to characterize this multi-herbicide resistance at the proteomic level, comparative analysis of resistant and susceptible barnyardgrasses was performed using iTRAQ, both with and without quinclorac, bispyribac-sodium and penoxsulam herbicidal treatment. A total of 1342 protein species were identified from 2248 unique peptides by searching the UniProt database and conducting data analysis. Approximately 904 protein species with 4774 Gene Ontology (GO) terms were grouped into the categories of biological process, cellular component and molecular function. Among these, 688 protein species were annotated into 1583 KEGG pathways, with 980 protein species relating to metabolism and 93 relating to environmental information processing. A total of 292 protein species showed more than a 1.2-fold change in abundance in the resistant biotype relative to the susceptible biotype. Furthermore, herbicide treatment resulted in 157 protein species that showed more than a 1.2-fold change in the resistant biotype. Moreover, physiological analyses demonstrated an ecological fitness cost in the resistant biotype., Significance: While some studies have shown a fitness cost to be associated with an altered ecological interaction, our understanding of the fitness costs associated with herbicide resistance are limited. Herein, physiological and proteomic analysis demonstrates herbicide resistance associated ecological fitness cost and potential mechanisms of herbicide-resistance in resistant biotypes of E. crus-galli. The results presented herein have revealed differences in ecological adaptation between resistant and susceptible biotypes in E. crus-galli and provide a fundamental basis enabling the development of new strategies for weed control. Lastly, this is the first large-scale proteomics study to examine herbicide stress responses in different barnyardgrass biotypes., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Despite the high prevalence of HIV-1 subtype C (HIV-1C) worldwide, information on HIV-1C viral dynamics and response to antiretroviral therapy (ART) is limited. We sought to measure viral load decay dynamics during treatment and estimate the within-host basic reproductive ratio, R0, and the critical efficacy, εc, for successful treatment of HIV-1C infection., Methods: Individuals initiated on first-line ART in India and monitored for 6 months of treatment were considered. Viral load, CD4 count, and adherence data were collected at baseline, 4, 12, 16 and 24 weeks after ART initiation. Drug resistance genotyping was performed at baseline. R0 and εc were estimated using a mathematical model., Results: Among 257 patients with complete data, mean baseline viral load was 5.7 log10 copies per milliliter and median CD4 count was 165 cells per cubic millimeter. Primary drug resistance was present in 3.1% at baseline. At 6 months, 87.5% had undetectable viral load, indicating excellent response to ART despite high baseline viremia. After excluding those with transmitted resistance, suboptimal adherence and viral rebound, data from 112 patients were analyzed using a mathematical model. We estimated the median R0 to be 5.3. The corresponding εc was ∼0.8., Conclusions: These estimates of R0 and εc are smaller than current estimates for HIV-1B, suggesting that HIV-1C exhibits lower in vivo fitness compared with HIV-1B, which allows successful treatment despite high baseline viral loads. The lower fitness, and potentially lower virulence, together with high viral loads may underlie the heightened transmission potential of HIV-1C and its growing global spread., Competing Interests: The authors have no funding or conflicts of interest to disclose.