In vivo fitness of sul gene-dependent sulfonamide-resistant Escherichia coli in the mammalian gut.

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.

Implications of cypermethrin exposure on population dynamics and fitness traits of laboratory-selected resistant and susceptible populations of Spodoptera litura (Fabricius).

The tobacco cutworm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is an economically important agricultural polyphagous pest worldwide. It has shown high resistance to several insecticides, including cypermethrin, a synthetic pyrethroid that is used in large-scale commercial agricultural applications. The present study investigated the development of selection-induced resistance to cypermethrin and associated fitness costs in S. litura. After continuous exposure to cypermethrin for consecutive fifteen generations, the cypermethrin-selected population (CYP-Sel) of S. litura developed a 21.2-fold resistance. The CYP-Sel strain had a relative fitness of 0.16 when treated with LC50, prolonged larval duration, and development time. Meanwhile, the strain also showed shorter adult duration, lower fecundity, and hatchability compared with the Unsel-Lab population. CYP-Sel population showed a significant disadvantage in intrinsic rate of natural increase (rm), net reproductive rate (Ro), and finite rate of increase (λ) when compared to the Unsel-Lab population. This knowledge could help to design resistance management strategies against this particular pest, along with potential management strategies to overcome the development of resistance.

Geographical migration and fitness dynamics of Streptococcus pneumoniae.

Streptococcus pneumoniae is a leading cause of pneumonia and meningitis worldwide. Many different serotypes co-circulate endemically in any one location1,2. 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.

Quantifying the fitness effects of resistance alleles with and without anthelmintic selection pressure using Caenorhabditis elegans.

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.

Insecticide resistant Anopheles gambiae have enhanced longevity but reduced reproductive fitness and a longer first gonotrophic cycle.

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 (F2, 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) (F2,8 = 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.

A genome-wide portrait of pervasive drug contaminants.

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.

Pyriproxyfen-treated bed nets reduce reproductive fitness and longevity of pyrethroid-resistant Anopheles gambiae under laboratory and field conditions.

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.

Compensatory evolution of Pseudomonas aeruginosa's slow growth phenotype suggests mechanisms of adaptation in cystic fibrosis.

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.

Peroxiredoxin alleviates the fitness costs of imidacloprid resistance in an insect pest of rice.

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.

A randomised trial examining inflammatory signaling in acutely induced hyperinsulinemia and hyperlipidemia in normal weight women-the reprometabolic syndrome.

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-1ß (MIP-1ß), 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.

No fitness cost associated with Asn-2041-Ile mutation in winter wild oat (Avena ludoviciana) seed germination under various environmental conditions.

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 F2 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.

Evolution of resistance to fluoroquinolones by dengue virus serotype 4 provides insight into mechanism of action and consequences for viral fitness.

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.

Cancer Cell Fitness Is Dynamic.

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.

HSATII RNA is induced via a noncanonical ATM-regulated DNA damage response pathway and promotes tumor cell proliferation and movement.

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.

Fitness-associated substitutions following failure of direct-acting antivirals assessed by deep sequencing of full-length hepatitis C virus genomes.

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.

Drug mechanism-of-action discovery through the integration of pharmacological and CRISPR screens.

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.

Candida albicans Genetic Background Influences Mean and Heterogeneity of Drug Responses and Genome Stability during Evolution in Fluconazole.

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.

A steroid hormone agonist reduces female fitness in insecticide-resistant Anopheles populations.

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.

Understanding the Cause and Consequence of Tumor Heterogeneity.

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.

Role transformation of fecundity and viability: The leading cause of fitness costs associated with beta-cypermethrin resistance in Musca domestica.

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.