Use of critically important antimicrobial classes early in life may adversely impact bacterial resistance profiles during adult years: potential co-selection for plasmid-borne fluoroquinolone and macrolide resistance via extended-spectrum beta-lactam use in dairy cattle.

The transfer of antimicrobial resistance genes commonly occurs via vertical and horizontal gene transfer, as such genes are often found on the same mobile genetic element. This occurrence can lead to the co-selection of resistance to antimicrobials without their application. Dairy cattle located in the south-western United States were enrolled in a matched-pair longitudinal study to evaluate the effects of a two-dose ceftiofur treatment for metritis on levels of third-generation cephalosporin resistance among faecal Escherichia coli temporally. Escherichia coli chosen for further investigation were isolated on selective media, harboured extended-spectrum beta-lactam, fluoroquinolone and macrolide resistance genes. This combination has previously been unreported; importantly, it included genes encoding for resistance to antibiotics that can only be used in dairy cattle less than 20 months of age. Fluoroquinolones, macrolides and third and higher generation cephalosporins are considered critically important and highest priority for human medicine by the World Health Organization.

Artemisinin Resistance and the Unique Selection Pressure of a Short-acting Antimalarial.

Resistance to the artemisinin derivatives, our most effective antimalarial drugs, has not manifest as a classical resistance phenotype in which parasites can tolerate higher drug concentrations. Instead, resistant parasites have an altered maturation. We hypothesize that the short half-life of artemisinin concentrations is an unanticipated driver of this novel resistance phenotype.

Highly parallel lab evolution reveals that epistasis can curb the evolution of antibiotic resistance.

Genetic perturbations that affect bacterial resistance to antibiotics have been characterized genome-wide, but how do such perturbations interact with subsequent evolutionary adaptation to the drug? Here, we show that strong epistasis between resistance mutations and systematically identified genes can be exploited to control spontaneous resistance evolution. We evolved hundreds of Escherichia coli K-12 mutant populations in parallel, using a robotic platform that tightly controls population size and selection pressure. We find a global diminishing-returns epistasis pattern: strains that are initially more sensitive generally undergo larger resistance gains. However, some gene deletion strains deviate from this general trend and curtail the evolvability of resistance, including deletions of genes for membrane transport, LPS biosynthesis, and chaperones. Deletions of efflux pump genes force evolution on inferior mutational paths, not explored in the wild type, and some of these essentially block resistance evolution. This effect is due to strong negative epistasis with resistance mutations. The identified genes and cellular functions provide potential targets for development of adjuvants that may block spontaneous resistance evolution when combined with antibiotics.

A Quinolinone Compound Inhibiting the Oligomerization of Nucleoprotein of Influenza A Virus Prevents the Selection of Escape Mutants.

The emergence of resistance to currently available anti-influenza drugs has heightened the need for antivirals with novel mechanisms of action. The influenza A virus (IAV) nucleoprotein (NP) is highly conserved and essential for the formation of viral ribonucleoprotein (vRNP), which serves as the template for replication and transcription. Recently, using in silico screening, we identified an antiviral compound designated NUD-1 (a 4-hydroxyquinolinone derivative) as a potential inhibitor of NP. In this study, we further analyzed the interaction between NUD-1 and NP and found that the compound interferes with the oligomerization of NP, which is required for vRNP formation, leading to the suppression of viral transcription, protein synthesis, and nuclear export of NP. We further assessed the selection of resistant variants by serially passaging a clinical isolate of the 2009 H1N1 pandemic influenza virus in the presence of NUD-1 or oseltamivir. NUD-1 did not select for resistant variants after nine passages, whereas oseltamivir selected for resistant variants after five passages. Our data demonstrate that NUD-1 interferes with the oligomerization of NP and less likely induces drug-resistant variants than oseltamivir; hence, it is a potential lead compound for the development of novel anti-influenza drugs.

Glucocorticoids, male sexual signals, and mate choice by females: Implications for sexual selection.

We review work relating glucocorticoids (GCs), male sexual signals, and mate choice by females to understand the potential for GCs to modulate the expression of sexually selected traits and how sexual selection potentially feeds back on GC regulation. Our review reveals that the relationship between GC concentrations and the quality of male sexual traits is mixed, regardless of whether studies focused on structural traits (e.g., coloration) or behavioral traits (e.g., vocalizations) or were examined in developmental or activational frameworks. In contrast, the few mate choice experiments that have been done consistently show that females prefer males with low GCs, suggesting that mate choice by females favors males that maintain low levels of GCs. We point out, however, that just as sexual selection can drive the evolution of diverse reproductive strategies, it may also promote diversity in GC regulation. We then shift the focus to females where we highlight evidence indicating that stressors or high GCs can dampen female sexual proceptivity and the strength of preferences for male courtship signals. Hence, even in cases where GCs are tightly coupled with male sexual signals, the strength of sexual selection on aspects of GC physiology can vary depending on the endocrine status of females. Studies examining how GCs relate to sexual selection may shed light on how variation in stress physiology, sexual signals, and mate choice are maintained in natural populations and may be important in understanding context-dependent relationships between GC regulation and fitness.

Bacterial communities associated with the midgut microbiota of wild Anopheles gambiae complex in Burkina Faso.

Plasmodium falciparum is transmitted by mosquitoes from the Anopheles gambiae sensu lato (s.l) species complex and is responsible for severe forms of malaria. The composition of the mosquitoes' microbiota plays a role in P. falciparum transmission, so we studied midgut bacterial communities of An. gambiae s.l from Burkina Faso. DNA was extracted from 17 pools of midgut of mosquitoes from the Anopheles gambiae complex from six localities in three climatic areas, including cotton-growing and cotton-free localities to include potential differences in insecticide selection pressure. The v3-v4 region of the 16S rRNA gene was targeted and sequenced using Illumina Miseq (2 × 250 nt). Diversity analysis was performed using QIIME and R software programs. The major bacterial phylum was Proteobacteria (97.2%) in all samples. The most abundant genera were Enterobacter (32.8%) and Aeromonas (29.8%), followed by Pseudomonas (11.8%), Acinetobacter (5.9%) and Thorsellia (2.2%). No statistical difference in operational taxonomic units (OTUs) was found (Kruskal-Wallis FDR-p > 0.05) among the different areas, fields or localities. Richness and diversity indexes (observed OTUs, Chao1, Simpson and Shannon indexes) showed significant differences in the cotton-growing fields and in the agroclimatic zones, mainly in the Sudano-Sahelian area. OTUs from seven bacterial species that mediate refractoriness to Plasmodium infection in An. gambiae s.l were detected. The beta diversity analysis did not show any significant difference. Therefore, a same control strategy of using bacterial species refractoriness to Plasmodium to target mosquito midgut bacterial community and affect their fitness in malaria transmission may be valuable tool for future malaria control efforts in Burkina Faso.

Evidence of insecticide resistance selection in wild Anopheles coluzzii mosquitoes due to agricultural pesticide use.

BACKGROUND: The wetlands used for some agricultural activities constitute productive breeding sites for many mosquito species. Thus, the agricultural use of insecticide targeting other pests may select for insecticide resistance in malaria mosquitoes. The purpose of this study is to clarify some knowledge gaps on the role of agrochemicals in the development of insecticide resistance in malaria vectors is of utmost importance for vector control. METHODS: Using the CDC bottle test and the log-probit analysis, we investigated for the first time the resistance levels of Anopheles coluzzii mosquitoes to neonicotinoids, insecticides used exclusively for crop protection in Côte d'Ivoire. The study was conducted in two agricultural regions (Tiassale and Gagnoa) and one non-agricultural region (Vitre) between June and August 2017 using clothianidin, acetamiprid and imidacloprid. RESULTS: Mosquito populations from Tiassale and Gagnoa (agricultural settings) were determined to be resistant to acetamiprid with mortality rates being < 85% at 24 h post-exposure. In Vitre (non-agricultural area) however, the mosquito population was susceptible to acetamiprid. In all three localities, mosquito populations were resistant to imidacloprid (mortality rates were 60% in Vitre, 37% in Tiassale, and 13% in Gagnoa) and completely susceptible to clothianidin (100% mortality). An. coluzzii represented 100% of mosquito collected in Gagnoa, 86% in Tiassale and 96% in Vitre. CONCLUSIONS: This study provides strong evidence that agricultural use of insecticides can cause insecticide resistance in malaria vector populations. Insecticide resistance driven by agrochemical usage should be considered when vector control strategies are developed.

Mapping the drivers of within-host pathogen evolution using massive data sets.

Differences among hosts, resulting from genetic variation in the immune system or heterogeneity in drug treatment, can impact within-host pathogen evolution. Genetic association studies can potentially identify such interactions. However, extensive and correlated genetic population structure in hosts and pathogens presents a substantial risk of confounding analyses. Moreover, the multiple testing burden of interaction scanning can potentially limit power. We present a Bayesian approach for detecting host influences on pathogen evolution that exploits vast existing data sets of pathogen diversity to improve power and control for stratification. The approach models key processes, including recombination and selection, and identifies regions of the pathogen genome affected by host factors. Our simulations and empirical analysis of drug-induced selection on the HIV-1 genome show that the method recovers known associations and has superior precision-recall characteristics compared to other approaches. We build a high-resolution map of HLA-induced selection in the HIV-1 genome, identifying novel epitope-allele combinations.

Genomic signatures of heterokaryosis in the oomycete pathogen Bremia lactucae.

Lettuce downy mildew caused by Bremia lactucae is the most important disease of lettuce globally. This oomycete is highly variable and rapidly overcomes resistance genes and fungicides. The use of multiple read types results in a high-quality, near-chromosome-scale, consensus assembly. Flow cytometry plus resequencing of 30 field isolates, 37 sexual offspring, and 19 asexual derivatives from single multinucleate sporangia demonstrates a high incidence of heterokaryosis in B. lactucae. Heterokaryosis has phenotypic consequences on fitness that may include an increased sporulation rate and qualitative differences in virulence. Therefore, selection should be considered as acting on a population of nuclei within coenocytic mycelia. This provides evolutionary flexibility to the pathogen enabling rapid adaptation to different repertoires of host resistance genes and other challenges. The advantages of asexual persistence of heterokaryons may have been one of the drivers of selection that resulted in the loss of uninucleate zoospores in multiple downy mildews.

Heavy metal-induced co-selection of antibiotic resistance genes in the gut microbiota of collembolans.

Heavy metal induced co-selection of antibiotic resistance genes (ARGs) has become an emerging environmental issue. The guts of soil fauna offer a unique habitat in the terrestrial ecosystem and harbor a variety of microorganisms. However, the effects of heavy metals on the gut-associated ARGs of soil fauna are poorly understood. In the present study, collembolans were cultivated with four types of heavy metals (Zn, Cu, Cd, and Cr) and one antibiotic (oxytetracycline), to investigate their impact on the gut-associated ARGs. High-throughput quantitative PCR and 16S rRNA gene amplicon sequencing were used to examine changes in the gut-associated ARGs and microbial composition caused by the metals and antibiotic. The results showed that heavy metals alone induced co-selection of ARGs in the collembolan gut, but the effects were weaker than selection by oxytetracycline. When Zn or Cu was present together with oxytetracycline, there was a strong synergistic effect between the compounds, which increased the selection of ARGs in the collembolan guts. Furthermore, redundancy analysis revealed that the gut microbiota and mobile genetic elements (MGEs) were significantly correlated with the ARG composition. These results extend our understanding on effects of heavy metals on the dispersal of ARGs in the soil food web.

Does nano silver promote the selection of antibiotic resistance genes in soil and plant?

Growing evidences have demonstrated that heavy metal contamination can promote the proliferation of antibiotic resistance genes (ARGs) via co-selection. However, effects of nano-metal-materials on the occurrence and level of ARGs in the soil and plant, have not been fully explored. To gain insights into this impact, we conducted a pot experiment by adding nano­silver particles (AgNPs) as a stimuli and Ag ion (AgNO3) and tetracycline as a comparison. By using high throughput quantitative PCR, our results indicated that application of AgNPs (~20 nm and ~50 nm) at a concentration of 100 ppm resulted in no significant changes in the abundance of ARGs in either soil or phyllosphere (P > 0.05). Nevertheless, the overall pattern of resistome, especially in soil, was shifted following AgNPs application, with a significance increase in the relative abundance of efflux pumps genes, which is an important mechanism for co-selection of ARGs by heavy metals. By comparison, Ag ion at an equivalent Ag mass of AgNPs markedly increased ARGs abundance and shifted ARGs profile in soil, indicating that free Ag ion had a stronger impact on ARGs than AgNPs. These findings provide new insights in assessing the risks of manufactured nanomaterials accumulated in the environment.

Evolutionary Toxicogenomics of the Striped Killifish (Fundulus majalis) in the New Bedford Harbor (Massachusetts, USA).

In this paper, we used a Genotyping-by-Sequencing (GBS) approach to find and genotype more than 4000 genome-wide SNPs (Single Nucleotide Polymorphisms) from striped killifish exposed to a variety of polychlorinated biphenyls (PCBs) and other aromatic pollutants in New Bedford Harbor (NBH, Massachusetts, USA). The aims of this study were to identify the genetic consequences of exposure to aquatic pollutants and detect genes that may be under selection. Low genetic diversity (HE and π) was found in the site exposed to the highest pollution level, but the pattern of genetic diversity did not match the pollution levels. Extensive connectivity was detected among sampling sites, which suggests that balanced gene flow may explain the lack of genetic variation in response to pollution levels. Tests for selection identified 539 candidate outliers, but many of the candidate outliers were not shared among tests. Differences among test results likely reflect different test assumptions and the complex pollutant mixture. Potentially, selectively important loci are associated with 151 SNPs, and enrichment analysis suggests a likely involvement of these genes with pollutants that occur in NBH. This result suggests that selective processes at genes targeted by pollutants may be occurring, even at a small geographical scale, and may allow the local striped killifish to resist the high pollution levels.

Mycoplasmas under experimental antimicrobial selection: The unpredicted contribution of horizontal chromosomal transfer.

Horizontal Gene Transfer was long thought to be marginal in Mycoplasma a large group of wall-less bacteria often portrayed as minimal cells because of their reduced genomes (ca. 0.5 to 2.0 Mb) and their limited metabolic pathways. This view was recently challenged by the discovery of conjugative exchanges of large chromosomal fragments that equally affected all parts of the chromosome via an unconventional mechanism, so that the whole mycoplasma genome is potentially mobile. By combining next generation sequencing to classical mating and evolutionary experiments, the current study further explored the contribution and impact of this phenomenon on mycoplasma evolution and adaptation using the fluoroquinolone enrofloxacin (Enro), for selective pressure and the ruminant pathogen Mycoplasma agalactiae, as a model organism. For this purpose, we generated isogenic lineages that displayed different combination of spontaneous mutations in Enro target genes (gyrA, gyrB, parC and parE) in association to gradual level of resistance to Enro. We then tested whether these mutations can be acquired by a susceptible population via conjugative chromosomal transfer knowing that, in our model organism, the 4 target genes are scattered in three distinct and distant loci. Our data show that under antibiotic selective pressure, the time scale of the mutational pathway leading to high-level of Enro resistance can be readily compressed into a single conjugative step, in which several EnroR alleles were transferred from resistant to susceptible mycoplasma cells. In addition to acting as an accelerator for antimicrobial dissemination, mycoplasma chromosomal transfer reshuffled genomes beyond expectations and created a mosaic of resistant sub-populations with unpredicted and unrelated features. Our findings provide insights into the process that may drive evolution and adaptability of several pathogenic Mycoplasma spp. via an unconventional conjugative mechanism.

Evidence for rapid adaptive evolution of tolerance to chemical treatments in Phytophthora species and its practical implications.

Chemical treatments are used widely in agricultural and natural settings to protect plants from diseases; however, they may exert an important selection pressure on plant pathogens, promoting the development of tolerant isolates through adaptive evolution. Phosphite is used to manage diseases caused by Phytophthora species which include a large number of the most economically damaging plant pathogens worldwide. Phosphite controls the growth of Phytophthora species in planta without killing it; as a result, isolates can develop tolerance to phosphite after prolonged exposure. We investigated the inter- and intra-specific variability in phosphite tolerance of eleven Phytophthora species, including P. ramorum, an internationally important, highly regulated pathogen. Phytophthora ramorum is a good model system because it is comprised of multiple genetically homogeneous lineages. Seven species were found to be consistently sensitive to phosphite based on the low Effective Concentration (EC) 50 values of all isolates tested (amount of phosphite required to inhibit mycelial growth by 50% relative to growth in the absence of phosphite). However, P. ramorum, P. lateralis, P. crassamura and P. cambivora showed intraspecific variability in sensitivity to phosphite, with at least one isolate showing significantly higher tolerance than the other isolates. Within the three P. ramorum evolutionarily divergent lineages tested, NA1 was the most susceptible to phosphite, the NA1 and EU1 lineages showed intralineage variability and the NA2 lineage showed a decreased sensitivity to phosphite overall as all isolates were relatively tolerant. This finding is relevant because NA1 is dominant in the wild and can be controlled using phosphite, while the EU1 lineage has recently been identified in the wild and is phosphite-tolerant, making the treatment approach potentially less effective. Phytophthora ramorum, P. lateralis and P. crassamura are either selfing, homothallic species, or are known to reproduce exclusively clonally, indicating tolerance to phosphite can emerge even in the absence of sexual recombination.

Antibiotic-Induced Genetic Variation: How It Arises and How It Can Be Prevented.

By targeting essential cellular processes, antibiotics provoke metabolic perturbations and induce stress responses and genetic variation in bacteria. Here we review current knowledge of the mechanisms by which these molecules generate genetic instability. They include production of reactive oxygen species, as well as induction of the stress response regulons, which lead to enhancement of mutation and recombination rates and modulation of horizontal gene transfer. All these phenomena influence the evolution and spread of antibiotic resistance. The use of strategies to stop or decrease the generation of resistant variants is also discussed.

Effect of Selective Antibiotic Pressure on the MLS-B Phenotype in Methicillin-Resistant Staphylococcus aureus Strains Originating From Patients From Transplantation Wards: 24 Years of Observations.

INTRODUCTION: Staphylococcus aureus infection, and health care-associated-methicillin resistant S aureus (HA-MRSA) in particular, is a serious risk for patients treated with organ transplantation. The frequent combined resistance of these bacteria to macrolides, lincosamides, and streptogramin-B (MLS-B) limits the use of these drugs in therapy. AIM: Evaluation of the mechanism of MLS-B resistance among HA-MRSA strains derived from patients treated in surgical-transplantation wards, over a 24-year period, and assessment of correlation of clindamycin use and resistance phenotype. MATERIALS AND METHODS: One hundred and twelve HA-MRSA strains from patients in surgical-transplantation wards (clinical hospital, Warsaw), hospitalized in the period from 1991 to 2014. Methicillin-resistance was determined using phenotypic and genetic methods by detecting the mecA gene. Erythromycin/clindamycin resistance was determined by E-test, the iMLS-B (inductive) and cMLS-B (constitutive) phenotypes by the D-test method. The number of defined daily doses (DDD), statistically per 1000 person-days, was calculated in accordance with the WHO guidelines. RESULTS: Resistance to erythromycin/clindamycin in MRSA strains increased from 1991 to 2004-2007 from 64.7/11.8% to 100/76.9%, respectively. The frequency of the cMLS-B phenotype in the years 1991/2010-2011/2012 was 5.9%/76.9%/69.7%, respectively, and correlated with the increased use of clindamycin in the examined wards. In 2012, the percentage of MLS-B-sensitive isolates increased from 3.9 to 21.7%, while constitutive resistance decreased to 69.7%, which correlated with a decrease in the use of clindamycin. CONCLUSIONS: The proportion of cMLS-B to iMLS-B phenotypes in HA-MRSA is related to the amount of clindamycin used in hospital wards. Limiting the selection pressure of antibiotics can lead to complete loss of resistance or return to the inductive mechanism of its regulation.

Evolution of high-level resistance during low-level antibiotic exposure.

It has become increasingly clear that low levels of antibiotics present in many environments can select for resistant bacteria, yet the evolutionary pathways for resistance development during exposure to low amounts of antibiotics remain poorly defined. Here we show that Salmonella enterica exposed to sub-MIC levels of streptomycin evolved high-level resistance via novel mechanisms that are different from those observed during lethal selections. During lethal selection only rpsL mutations are found, whereas at sub-MIC selection resistance is generated by several small-effect resistance mutations that combined confer high-level resistance via three different mechanisms: (i) alteration of the ribosomal RNA target (gidB mutations), (ii) reduction in aminoglycoside uptake (cyoB, nuoG, and trkH mutations), and (iii) induction of the aminoglycoside-modifying enzyme AadA (znuA mutations). These results demonstrate how the strength of the selective pressure influences evolutionary trajectories and that even weak selective pressures can cause evolution of high-level resistance.

Rapid selection of sulphadoxine-resistant Plasmodium falciparum and its effect on within-population genetic diversity in Papua New Guinea.

The ability of the human malarial parasite Plasmodium falciparum to adapt to environmental changes depends considerably on its ability to maintain within-population genetic variation. Strong selection, consequent to widespread antimalarial drug usage, occasionally elicits a rapid expansion of drug-resistant isolates, which can act as founders. To investigate whether this phenomenon induces a loss of within-population genetic variation, we performed a population genetic analysis on 302 P. falciparum cases detected during two cross-sectional surveys in 2002/2003, just after the official introduction of sulphadoxine/pyrimethamine as a first-line treatment, and again in 2010/2011, in highly endemic areas in Papua New Guinea. We found that a single-origin sulphadoxine-resistant parasite isolate rapidly increased from 0% in 2002/2003 to 54% in 2010 and 84% in 2011. However, a considerable number of pairs exhibited random associations among 10 neutral microsatellite markers located in various chromosomes, suggesting that outcrossing effectively reduced non-random associations, albeit at a low average multiplicity of infection (1.35-1.52). Within-population genetic diversity was maintained throughout the study period. This indicates that the parasites maintained within-population variation, even after a clonal expansion of drug-resistant parasites. Outcrossing played a role in the preservation of within-population genetic diversity despite low levels of multiplicity of infection.

Impact of the HIV-1 genetic background and HIV-1 population size on the evolution of raltegravir resistance.

BACKGROUND: Emergence of resistance against integrase inhibitor raltegravir in human immunodeficiency virus type 1 (HIV-1) patients is generally associated with selection of one of three signature mutations: Y143C/R, Q148K/H/R or N155H, representing three distinct resistance pathways. The mechanisms that drive selection of a specific pathway are still poorly understood. We investigated the impact of the HIV-1 genetic background and population dynamics on the emergence of raltegravir resistance. Using deep sequencing we analyzed the integrase coding sequence (CDS) in longitudinal samples from five patients who initiated raltegravir plus optimized background therapy at viral loads > 5000 copies/ml. To investigate the role of the HIV-1 genetic background we created recombinant viruses containing the viral integrase coding region from pre-raltegravir samples from two patients in whom raltegravir resistance developed through different pathways. The in vitro selections performed with these recombinant viruses were designed to mimic natural population bottlenecks. RESULTS: Deep sequencing analysis of the viral integrase CDS revealed that the virological response to raltegravir containing therapy inversely correlated with the relative amount of unique sequence variants that emerged suggesting diversifying selection during drug pressure. In 4/5 patients multiple signature mutations representing different resistance pathways were observed. Interestingly, the resistant population can consist of a single resistant variant that completely dominates the population but also of multiple variants from different resistance pathways that coexist in the viral population. We also found evidence for increased diversification after stronger bottlenecks. In vitro selections with low viral titers, mimicking population bottlenecks, revealed that both recombinant viruses and HXB2 reference virus were able to select mutations from different resistance pathways, although typically only one resistance pathway emerged in each individual culture. CONCLUSIONS: The generation of a specific raltegravir resistant variant is not predisposed in the genetic background of the viral integrase CDS. Typically, in the early phases of therapy failure the sequence space is explored and multiple resistance pathways emerge and then compete for dominance which frequently results in a switch of the dominant population over time towards the fittest variant or even multiple variants of similar fitness that can coexist in the viral population.

Relating High Insecticide Residues in Larval Breeding Habitats in Urban Residential Areas to the Selection of Pyrethroid Resistance in Anopheles gambiae s.l. (Diptera: Culicidae) in Akim Oda, Ghana.

The main objective of this study was to assess insecticide contamination in Anopheles breeding habitats in urban residential areas and pyrethroid susceptibility status of mosquitoes found in the habitats. A larval survey was conducted in Akim Oda between July and October 2016. The larvae that were reared to adult were used for susceptibility test against four different pyrethroid insecticides (deltamethrin 0.05%, permethrin 0.75%, cyfluthrin 0.15%, and etofenprox 0.5%). Gas chromatography was used to analyze pesticide residues in water collected from the breeding habitats. High levels of permethrin and deltamethrin plus traces of several organochlorine and organophosphate insecticides were detected in the larval-breeding habitats. None of the four pyrethroid insecticides caused more than 10% mortality. Anopheles coluzzii Coetzee & Wilkerson dominated in the study area with high frequency of kdr mutation (93.5%). We report for the first time in Ghana, high levels of pyrethroid insecticides contamination in Anopheles breeding habitats in urban residential areas where there are no major agricultural activities. The contamination is suspected to be the major cause of pyrethroid resistance in the Anopheles species. Improper disposal of old insecticide-treated net and other domestic insecticides and the use of herbicides are suspected to be the source of insecticide contamination.