Species Composition, Phenotypic and Genotypic Resistance Levels in Major Malaria Vectors in Teso North and Teso South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Knockdown resistance (kdr) is strongly linked to pyrethroid insecticide resistance in Anopheles gambiae in Africa, which may have vital significance to the current increased use of pyrethroid-treated bed net programmes. The study is aimed at determining species composition, levels of insecticide resistance, and knockdown patterns in Anopheles gambiae sensu lato in areas with and areas without insecticide resistance in Teso North and Teso South subcounties, Western Kenya. MATERIALS AND METHODS: For WHO vulnerability tests, mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes (4944 at 100 mosquitoes per insecticide) which were exposed to 0.75% permethrin, 0.05% deltamethrin, and 0.1% bendiocarb using the WHO tube assay method. Species identification and kdr East gene PCRs were also performed on randomly selected mosquitoes from the collections; including adult mosquitoes (3448) sampled using standard collection methods. RESULTS: Anopheles gambiae sensu stricto were the majority in terms of species composition at 78.9%. Bendiocarb caused 100% mortality while deltamethrin had higher insecticidal effects (77%) on female mosquitoes than permethrin (71%). Susceptible Kengatunyi cluster had higher proportion of An. arabiensis (20.9%) than resistant Rwatama (10.7%). Kengatunyi mosquitoes exposed to deltamethrin had the highest KDT50 R of 8.2. Both Anopheles gambiae sensu stricto and Anopheles arabiensis had equal S allelic frequency of 0.84. Indoor resting mosquitoes had 100% mortality rate after 24 h since exposure. Overall SS genotypic frequency in Teso North and Teso South subcounties was 79.4% against 13.7% homozygous LL genotype and 6.9% heterozygous LS genotype. There was a significant difference (ρ < 0.05) in S allele frequencies between Kengatunyi (0.61) and Rwatama (0.95). Mosquito samples collected in 2013 had the highest S allelic frequency of 0.87. Discussion. Most likely, the higher the selection pressure exerted indoors by insecticidal nets, the higher were the resistance alleles. Use of pyrethroid impregnated nets and agrochemicals may have caused female mosquitoes to select for pyrethroid resistance. Different modes of action and chemical properties in different types of pyrethroids aggravated by a variety of edaphic and climatic factors may have caused different levels of susceptibility in both indoor and outdoor vectors to pyrethroids and carbamate. Species composition and populations in each collection method may have been influenced by insecticide resistance capacity in different species. Conclusions and Recommendations. Both phenotypic and genotypic insecticide resistance levels have been confirmed in Teso North and Teso South subcounties in Western Kenya. Insecticide resistance management practices in Kenya should be fast tracked and harmonized with agricultural sector agrochemical-based activities and legislation, and possibly switch to carbamate use in order to ease selection pressure on pyrethroids which are useable in insecticidal nets and indoor residual spray due to their low human toxicity. The implication of such high resistance levels in mosquitoes collected in Teso subcounties is that resistance is likely to persist and or even increase if monomolecules of permethrin and deltamethrin or both continue to be used in all net- and nonnet-based mosquito control purposes. Usage of mutually reinforcing piperonyl butoxide (PBO) that prohibits particular enzymes vital in metabolic activities inside mosquito systems and has been integrated into pyrethroid-LLINs to create pyrethroid-PBO nets is an extremely viable option.

Impact of Insecticide Resistance on P. falciparum Vectors' Biting, Feeding, and Resting Behaviour in Selected Clusters in Teso North and South Subcounties in Busia County, Western Kenya.

INTRODUCTION: Behavioural resistance to insecticides restrains the efficacy of vector control tools against mosquito-transmitted diseases. The current study is aimed at determining the impact of insecticide resistance on major malaria vectors' biting, feeding, and resting behaviour in areas with and areas without insecticide resistance in Teso North and Teso South, Busia County, Western Kenya. METHODS: Mosquito larvae were sampled using a dipper, reared into 3-5-day-old female mosquitoes [4944] which were exposed to 0.75% permethrin and 0.05% deltamethrin using World Health Organization tube assay method. Blood meal, species identification, and kdr Eastgene PCRs were also performed on adult mosquitoes sampled using mosquito collection methods [3448]. Biting, feeding, resting, and exiting behaviours of field-collected mosquitoes from five selected clusters were analysed. RESULTS: The lowest Kdr genotypic frequency (SS) proportion was found in female Anophelines collected in Kengatunyi at 58% while Rwatama had the highest genotypic frequency at 93%, thus susceptible and resistant clusters, respectively. The peak hour for mosquito seeking a human bite was between 0300 and 0400 hrs in the resistant cluster and 0400-0500 hrs in the susceptible cluster. The heterozygous mosquitoes maintained the known 2100-2200 hrs peak hour. There was a higher proportion of homozygous susceptible vectors (86.4%) seeking humans indoor than outdoor bitters (78.3%). Mosquito blood meals of human origin were 60% and 87% in susceptible Kengatunyi and resistant Rwatama cluster, respectively. There was significant difference between homozygous-resistant vectors feeding on human blood compared to homozygous susceptible mosquitoes (p ≤ 0.05). The proportion of bovine blood was highest in the susceptible cluster. A higher proportion of homozygous-resistant anophelines were feeding and resting indoors. No heterozygous mosquito was found resting indoor while 4.2% of the mosquitoes were caught while exiting the house through the window. Discussion. A shift in resistant Anopheles gambiae sl highest peak hour of aggressiveness from 2100-2200 hrs to 0300-0400 hrs is a key change in its biting pattern. Due to the development of resistance, mosquitoes no longer have to compete against the time the human host enters into the formerly lethal chemical and or physical barrier in the form of long-lasting insecticide-treated net. No heterozygous LS mosquito rested indoors possibly due to disadvantages of heterozygosity which could have increased their fitness costs as well as energy costs in the presence of the insecticidal agents in the treated nets. Conclusions and recommendations. Out of bed biting by female mosquitoes and partial susceptibility may contribute to residual malaria transmission. Insecticide-resistant vectors have become more endophagic and anthropophillic. Hence, insecticidal nets, zooprophylaxis, and novel repellents are still useful chemical, biological, and physical barriers against human blood questing female mosquitoes. Further studies should be done on genetic changes in mosquitoes and their effects on changing mosquito behaviour.

A rheostat mechanism governs the bifurcation of carbon flux in mycobacteria.

Fatty acid metabolism is an important feature of the pathogenicity of Mycobacterium tuberculosis during infection. Consumption of fatty acids requires regulation of carbon flux bifurcation between the oxidative TCA cycle and the glyoxylate shunt. In Escherichia coli, flux bifurcation is regulated by phosphorylation-mediated inhibition of isocitrate dehydrogenase (ICD), a paradigmatic example of post-translational mechanisms governing metabolic fluxes. Here, we demonstrate that, in contrast to E. coli, carbon flux bifurcation in mycobacteria is regulated not by phosphorylation but through metabolic cross-activation of ICD by glyoxylate, which is produced by the glyoxylate shunt enzyme isocitrate lyase (ICL). This regulatory circuit maintains stable partitioning of fluxes, thus ensuring a balance between anaplerosis, energy production, and precursor biosynthesis. The rheostat-like mechanism of metabolite-mediated control of flux partitioning demonstrates the importance of allosteric regulation during metabolic steady-state. The sensitivity of this regulatory mechanism to perturbations presents a potentially attractive target for chemotherapy.

Next-generation antimicrobials: from chemical biology to first-in-class drugs.

The global emergence of multi-drug resistant bacteria invokes an urgent and imperative necessity for the identification of novel antimicrobials. The general lack of success in progressing novel chemical entities from target-based drug screens have prompted calls for radical and innovative approaches for drug discovery. Recent developments in chemical biology and target deconvolution strategies have revived interests in the utilization of whole-cell phenotypic screens and resulted in several success stories for the discovery and development novel drug candidates and target pathways. In this review, we present and discuss recent chemical biology approaches focusing on the discovery of novel targets and new lead molecules for the treatment of human bacterial and protozoan infections.

Targeting bacterial central metabolism for drug development.

Current antibiotics, derived mainly from natural sources, inhibit a narrow spectrum of cellular processes, namely DNA replication, protein synthesis, and cell wall biosynthesis. With the worldwide explosion of drug resistance, there is renewed interest in the investigation of alternate essential cellular processes, including bacterial central metabolic pathways, as a drug target space for the next generation of antibiotics. However, the validation of targets in central metabolism is more complex, as essentiality of such targets can be conditional and/or contextual. Bearing in mind our enhanced understanding of prokaryotic central metabolism, a key question arises: can central metabolism be bacteria's Achilles' heel and a therapeutic target for the development of new classes of antibiotics? In this review, we draw lessons from oncology and attempt to address some of the open questions related to feasibility of targeting bacterial central metabolism as a strategy for developing new antibacterial drugs.

Exploring the mode of action of bioactive compounds by microfluidic transcriptional profiling in mycobacteria.

Most candidate anti-bacterials are identified on the basis of their whole cell anti-bacterial activity. A critical bottleneck in the early discovery of novel anti-bacterials is tracking the structure activity relationship (SAR) of the novel compounds synthesized during the hit to lead and lead optimization stage. It is often very difficult for medicinal chemists to visualize if the novel compounds synthesized for understanding SAR of a particular scaffold have similar molecular mechanism of action (MoA) as that of the initial hit. The elucidation of the molecular MoA of bioactive inhibitors is critical. Here, a new strategy and routine assay for MoA de-convolution, using a microfluidic platform for transcriptional profiling of bacterial response to inhibitors with whole cell activity has been presented. First a reference transcriptome compendium of Mycobacterial response to various clinical and investigational drugs was built. Using feature reduction, it was demonstrated that subsets of biomarker genes representative of the whole genome are sufficient for MoA classification and deconvolution in a medium-throughput microfluidic format ultimately leading to a cost effective and rapid tool for routine antibacterial drug-discovery programs.