Impacts of dual active-ingredient bed nets on the behavioural responses of pyrethroid resistant Anopheles gambiae determined by room-scale infrared video tracking.

BACKGROUND: The success of insecticide treated bed nets (ITNs) for malaria vector control in Africa relies on the behaviour of various species of Anopheles. Previous research has described mosquito behavioural alterations resulting from widespread ITN coverage, which could result in a decrease in net efficacy. Here, behaviours were compared including timings of net contact, willingness to refeed and longevity post-exposure to two next-generation nets, PermaNet® 3.0 (P3 net) and Interceptor® G2 (IG2 net) in comparison with a standard pyrethroid-only net (Olyset Net™ (OL net)) and an untreated net. METHODS: Susceptible and resistant Anopheles gambiae mosquitoes were exposed to the nets with a human volunteer host in a room-scale assay. Mosquito movements were tracked for 2 h using an infrared video system, collecting flight trajectory, spatial position and net contact data. Post-assay, mosquitoes were monitored for a range of sublethal insecticide effects. RESULTS: Mosquito net contact was focused predominantly on the roof for all four bed nets. A steep decay in activity was observed for both susceptible strains when P3 net and OL net were present and with IG2 net for one of the two susceptible strains. Total mosquito activity was higher around untreated nets than ITNs. There was no difference in total activity, the number, or duration, of net contact, between any mosquito strain, with similar behaviours recorded in susceptible and resistant strains at all ITNs. OL net, P3 net and IG2 net all killed over 90% of susceptible mosquitoes 24 h after exposure, but this effect was not seen with resistant mosquitoes where mortality ranged from 16 to 72%. All treated nets reduced the willingness of resistant strains to re-feed when offered blood 1-h post-exposure, with a more pronounced effect seen with P3 net and OL net than IG2 net. CONCLUSION: These are the first results to provide an in-depth description of the behaviour of susceptible and resistant Anopheles gambiae strains around next-generation bed nets using a room-scale tracking system to capture multiple behaviours. These results indicate that there is no major difference in behavioural responses between mosquito strains of differing pyrethroid susceptibility when exposed to these new ITNs under the experimental conditions used.

Human Infection Challenge with Serotype 3 Pneumococcus.

Rationale: Streptococcus pneumoniae serotype 3 (SPN3) is a cause of invasive pneumococcal disease and associated with low carriage rates. Following the introduction of pediatric 13-valent pneumococcal conjugate vaccine (PCV13) programs, SPN3 declines are less than other vaccine serotypes and incidence has increased in some populations coincident with a shift in predominant circulating SPN3 clade, from I to II. A human challenge model provides an effective means for assessing the impact of PCV13 on SPN3 in the upper airway. Objectives: To establish SPN3's ability to colonize the nasopharynx using different inoculum clades and doses, and the safety of an SPN3 challenge model. Methods: In a human challenge study involving three well-characterized and antibiotic-sensitive SPN3 isolates (PFESP306 [clade Ia], PFESP231 [no clade], and PFESP505 [clade II]), inoculum doses (10,000, 20,000, 80,000, and 160,000 cfu/100 µl) were escalated until maximal colonization rates were achieved, with concurrent acceptable safety. Measurement and Main Results: Presence and density of experimental SPN3 nasopharyngeal colonization in nasal wash samples, assessed using microbiological culture and molecular methods, on Days 2, 7, and 14 postinoculation. A total of 96 healthy participants (median age 21, interquartile range 19-25) were inoculated (n = 6-10 per dose group, 10 groups). Colonization rates ranged from 30.0-70.0% varying with dose and isolate. 30.0% (29/96) reported mild symptoms (82.8% [24/29] developed a sore throat); one developed otitis media requiring antibiotics. No serious adverse events occurred. Conclusions: An SPN3 human challenge model is feasible and safe with comparable carriage rates to an established Serotype 6B human challenge model. SPN3 carriage may cause mild upper respiratory symptoms.

Nutrition-related outcomes and dietary intake in non-mechanically ventilated critically ill adult patients: A pilot observational descriptive study.

BACKGROUND: Critically ill patients who do not receive invasive mechanical ventilation (IMV) are a growing population, experiencing complex interventions that may impair dietary intake and nutrition-related outcomes. OBJECTIVES: The objectives of this study were to quantify intake and nutrition-related outcomes of non-IMV critically ill patients and to establish feasibility of methods to measure nutrition-related outcomes in this population. METHODS: Non-IMV adult patients expected to remain in the intensive care unit (ICU) for ≥24 h were eligible. Nutrition-related outcomes were assessed at baseline by subjective global assessment (SGA); on alternate study days by mid-upper arm circumference (MUAC), calf circumference (CC), and ultrasound of quadriceps muscle layer thickness (QMLT); and daily by body weight and bioelectrical impedance analysis (BIA). Data were censored at day 5 or ICU discharge. Dietary intake from all sources, including oral intake via investigator-led weighed food records, was quantified on days 1-3. Feasibility was defined as data completion rate ≥70%. Data are expressed as mean (standard deviation) or median [interquartile range (IQR)]. RESULTS: Twenty-three patients consented (50% male; 53 [42-64] y; ICU stay: 2.8 [1.9-4.0] d). Nutrition-related outcomes at baseline and ICU discharge were as follows: MUAC: 33.2 (8.6) cm (n = 18) and 29.3 (5.4) cm (n = 6); CC: 39.5 (7.4) cm (n = 16) and 37.5 (6.2) cm (n = 6); body weight: 95.3 (34.8) kg (n = 19) and 95.6 (41.0) kg (n = 10); and QMLT: 2.6 (0.8) cm (n = 15) and 2.5 (0.3) cm (n = 5), respectively. Oral intake provided 3155 [1942-5580] kJ and 32 [20-53] g protein, with poor appetite identified as a major barrier. MUAC, CC, QMLT, and SGA were feasible, while BIA and body weight were not. CONCLUSIONS: Oral intake in critically ill patients not requiring IMV is below estimated requirements, largely because of poor appetite. The small sample and short study duration were not sufficient to quantify changes in nutrition-related outcomes. MUAC, CC, QMLT, and SGA are feasible methods to assess nutrition-related outcomes at a single time point in this population.

Macrodiolide formation by the thioesterase of a modular polyketide synthase.

Elaiophylin is an unusual C2 -symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2 -symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.

Biosynthesis of mupirocin by Pseudomonas fluorescens NCIMB 10586 involves parallel pathways.

Mupirocin, a clinically important antibiotic produced via a trans-AT Type I polyketide synthase (PKS) in Pseudomonas fluorescens, consists of a mixture of mainly pseudomonic acids A, B, and C. Detailed metabolic profiling of mutant strains produced by systematic inactivation of PKS and tailoring genes, along with re-feeding of isolated metabolites to mutant stains, has allowed the isolation of a large number of novel metabolites, identification of the 10,11-epoxidase, and full characterization of the mupirocin biosynthetic pathway, which proceeds via major (10,11-epoxide) and minor (10,11-alkene) parallel pathways.

Comparative analysis of the Potter Tower and a new Track Sprayer for the application of residual sprays in the laboratory.

BACKGROUND: Efforts to evaluate the residual efficacy of new indoor residual spraying (IRS) formulations have identified limitations with the industry standard laboratory sprayer, the Potter Spray Tower (PT). Calibrating the PT can be time-consuming, and the dosing of surfaces may not be as accurate or uniform as previously assumed. METHODS: To address these limitations, the Micron Horizontal Track Sprayer with Spray Cabinet (TS) was developed to provide higher efficiency, ease of operation and deposition uniformity equal to or better than the PT. A series of studies were performed using a fluorescent tracer and three IRS formulations (Actellic® 300CS, K-Othrine WG250 and Suspend PolyZone) sprayed onto surfaces using either the PT or the TS. RESULTS: Deposition volumes could be accurately calibrated for both spray systems. However, the uniformity of spray deposits was higher for the TS compared to the PT. Less than 12% of the volume sprayed using the PT reaches the target surface, with the remaining 88% unaccounted for, presumably vented out of the fume hood or coating the internal surfaces of the tower. In contrast, the TS deposits most of the spray on the floor of the spray chamber, with the rest contained therein. The total sprayed surface area in one run of the TS is 1.2 m2, and the operational zone for spray target placement is 0.7 m2, meaning that 58% of the applied volume deposits onto the targets. The TS can treat multiple surfaces (18 standard 15 × 15 cm tiles) in a single application, whereas the PT treats one surface at a time and a maximum area of around 0.0225 m2. An assessment of the time taken to perform spraying, including the setup, calibration and cleaning, showed that the cost of application using the TS was around 25-35 × less per tile sprayed. Standard operating procedures (SOPs) for calibration and use of both the Potter Tower and Track Sprayer have been developed. CONCLUSIONS: Overall, the TS represents a significant improvement over the PT in terms of the efficiency and accuracy of IRS formulation applications onto test substrates and offers a useful additional tool for researchers and manufacturers wanting to screen new active ingredients or evaluate the efficacy of IRS or other sprayable formulations for insect control.

Biosynthesis of Antifungal Solanimycin May Involve an Iterative Nonribosomal Peptide Synthetase Module.

Dickeya solani, a plant-pathogenic bacterium, produces solanimycin, a potent hybrid polyketide/nonribosomal peptide (PKS/NRPS) anti-fungal compound. The biosynthetic gene cluster responsible for synthesis of this compound has been identified. Because of instability, the complete structure of the compound has not yet been elucidated, but LC-MS2 identified that the cluster produces two main compounds, solanimycin A and B, differing by a single hydroxyl group. The fragmentation pattern revealed that the central part of solanimycin A is a hexapeptide, Gly-Dha-Dha-Dha-Dha-Dha (where Dha is dehydroalanine). This is supported by isotopic labeling studies using labeled serine and glycine. The N-terminal group is a polyketide-derived C16 acyl group containing a conjugated hexaene, a hydroxyl, and an amino group. The additional hydroxyl group in solanimycin B is on the α-carbon of the glycine residue. The incorporation of five sequential Dha residues is unprecedented because there is only one NRPS module in the cluster that is predicted to activate and attach serine (which is subsequently dehydrated to Dha), meaning that this NRPS module must act iteratively. While a few other iterative NRPS modules are known, they all involve iteration of two or three modules. We believe that the repetitive use of a single module makes the solanimycin biosynthetic pathway unique among NRPSs so far reported.

Solanimycin: Biosynthesis and Distribution of a New Antifungal Antibiotic Regulated by Two Quorum-Sensing Systems.

The increasing emergence of drug-resistant fungal infections has necessitated a search for new compounds capable of combating fungal pathogens of plants, animals, and humans. Microorganisms represent the main source of antibiotics with applicability in agriculture and in the clinic, but many aspects of their metabolic potential remain to be explored. This report describes the discovery and characterization of a new antifungal compound, solanimycin, produced by a hybrid polyketide/nonribosomal peptide (PKS/NRPS) system in Dickeya solani, the enterobacterial pathogen of potato. Solanimycin was active against a broad range of plant-pathogenic fungi of global economic concern and the human pathogen Candida albicans. The genomic cluster responsible for solanimycin production was defined and analyzed to identify the corresponding biosynthetic proteins, which include four multimodular PKS/NRPS proteins and several tailoring enzymes. Antifungal production in D. solani was enhanced in response to experimental conditions found in infected potato tubers and high-density fungal cultures. Solanimycin biosynthesis was cell density dependent in D. solani and was controlled by both the ExpIR acyl-homoserine lactone and Vfm quorum-sensing systems of the bacterial phytopathogen. The expression of the solanimycin cluster was also regulated at the post-transcriptional level, with the regulator RsmA playing a major role. The solanimycin biosynthetic cluster was conserved across phylogenetically distant bacterial genera, and multiple pieces of evidence support that the corresponding gene clusters were acquired by horizontal gene transfer. Given its potent broad-range antifungal properties, this study suggests that solanimycin and related molecules may have potential utility for agricultural and clinical exploitation. IMPORTANCE Fungal infections represent a major clinical, agricultural, and food security threat worldwide, which is accentuated due to the difficult treatment of these infections. Microorganisms represent a prolific source of antibiotics, and current data support that this enormous biosynthetic potential has been scarcely explored. To improve the performance in the discovery of novel antimicrobials, there is a need to diversify the isolation niches for new antibiotic-producing microorganisms as well as to scrutinize novel phylogenetic positions. With the identification of the antifungal antibiotic solanimycin in a broad diversity of phytopathogenic Dickeya spp., we provide further support for the potential of plant-associated bacteria for the biosynthesis of novel antimicrobials. The complex regulatory networks involved in solanimycin production reflect the high metabolic cost of bacterial secondary metabolism. This metabolic regulatory control makes many antibiotics cryptic under standard laboratory conditions, and mimicking environmental conditions, as shown here, is a strategy to activate cryptic antibiotic clusters.

Metabolic engineering is key to a sustainable chemical industry.

The depletion of fossil fuel stocks will prohibit their use as the main feedstock of future industrial processes. Biocatalysis is being increasingly used to reduce fossil fuel reliance and to improve the sustainability, efficiency and cost of chemical production. Even with their current small market share, biocatalyzed processes already generate approximately US$50 billion and it has been estimated that they could be used to produce up to 20% of fine chemicals by 2020. Until the advent of molecular biological technologies, the compounds that were readily accessible from renewable biomass were restricted to naturally-occurring metabolites. However, metabolic engineering has considerably broadened the range of compounds now accessible, providing access to compounds that cannot be otherwise reliably sourced, as well as replacing established chemical processes. This review presents the case for continued efforts to promote the adoption of biocatalyzed processes, highlighting successful examples of industrial chemical production from biomass and/or via biocatalyzed processes. A selection of emerging technologies that may further extend the potential and sustainability of biocatalysis are also presented. As the field matures, metabolic engineering will be increasingly crucial in maintaining our quality of life into a future where our current resources and feedstocks cannot be relied upon.

Manipulation of quorum sensing regulation in Pseudomonas fluorescens NCIMB 10586 to increase mupirocin production.

Transcription of the 74 kb Pseudomonas fluorescens mupirocin [pseudomonic acid (PA)] biosynthesis cluster depends on quorum sensing-dependent regulation via the LuxI/LuxR homologues MupI/MupR. To facilitate analysis of novel PAs from pathway mutants, we investigated factors that affect mup gene expression. First, the signal produced by MupI was identified as N-(3-oxodecanoyl)homoserine lactone, but exogenous addition of this molecule did not activate mupirocin production prematurely nor did expression of mupI in trans increase metabolite production. Second, we confirmed that mupX, encoding an amidase/hydrolase that can degrade N-acylhomoserine lactones, is also required for efficient expression, consistent with its occurrence in a regulatory module linked to unrelated genes in P. fluorescens. Third, and most significantly, mupR expression in trans to wild type and mutants can increase production of antibiotic and novel intermediates up to 17-fold.

Enantioselective syntheses of alpha-Fmoc-Pbf-[2-(13)C]-L-arginine and Fmoc-[1,3-(13)C2]-L-proline and Incorporation into the neurotensin receptor 1 ligand, NT(8-13).

Enantioselective syntheses of selectively labeled, orthogonally protected [2-(13)C]-L-arginine and [1,3-(13)C(2)]-L-proline are described from the commercially available precursors [2-(13)C]bromoacetic acid and potassium [(13)C]cyanide. Interestingly the enhanced signal assigned to C-2 in the (13)C NMR spectrum of alpha-Fmoc-Pbf-[2-(13)C]-L-arginine was very broad at room temperature. The two Fmoc-labeled amino acids were used to prepare [2-(13)C]-Arg9 and [1,3-(13)C(2)]-Pro10 labeled ligand (NT(8-13)) by manual Fmoc-SPSS.

Development and application of a tri-allelic PCR assay for screening Vgsc-L1014F kdr mutations associated with pyrethroid and organochlorine resistance in the mosquito Culex quinquefasciatus.

BACKGROUND: Culex quinquefasciatus has a widespread distribution across tropical and sub-tropical regions, and plays an important role in the transmission of vector-borne diseases of public health importance, including lymphatic filariasis (LF) and multiple arboviruses. Increased resistance to insecticides threatens the efficacy and sustainability of insecticide-based anti-vector interventions which mitigate the burden of mosquito transmitted diseases in endemic regions. In C. quinquefasciatus two non-synonymous voltage gated sodium channel (Vgsc) variants, both resulting in a leucine to phenylalanine change at codon 1014, are associated with resistance to pyrethroids and DDT. This tri-allelic variation has compromised the ability to perform high-throughput single-assay screening. To facilitate the detection and monitoring of the Vgsc-1014 locus in field-caught mosquitoes, an Engineered-Tail Allele-Specific-PCR (ETAS-PCR) diagnostic assay was developed and applied to wild mosquitoes from Brazil, Tanzania and Uganda. RESULTS: This new cost-effective, single-tube assay was compared to two, well-established, genotyping approaches, pyrosequencing and TaqMan. The ETAS-PCR assay showed high specificity for discriminating the three alleles at Vgsc-L1014F, with genotyping results strongly correlated with pyrosequencing and TaqMan results (98.64% and 100% respectively). CONCLUSIONS: Our results support the utility of the ETAS-PCR/Vgsc-1014 diagnostic assay, which stands as an effective alternative for genotyping tri-allelic variants.

Synthetic and biological studies on the spiro-mamakone system.

An exploration of the chemistry of the spiro-mamakone system, exemplified by the cytotoxic, fungal metabolite spiro-mamakone A, is presented. The first reported synthesis of the spiro-mamakone carbon skeleton was achieved, as well as the synthesis of a variety of closely related analogues of the natural product. Biological testing of the synthetic analogues generated a structure-activity profile for the natural product, establishing the importance of the enedione moiety to biological activity.

Evolving trends in the dereplication of natural product extracts. 2. The isolation of chrysaibol, an antibiotic peptaibol from a New Zealand sample of the mycoparasitic fungus Sepedonium chrysospermum.

By the application of an HPLC bioactivity profiling/microtiter technique in conjunction with capillary NMR instrumentation and access to the AntiMarin database the conventional evaluation/isolation dereplication/characterization procedures can be dramatically truncated. This approach is illustrated using the isolation of a new peptaibol, chrysaibol (1), from a New Zealand isolate of the mycoparasitic fungus Sepedonium chrysospermum. The unique nature of chrysaibol was recognized by bioactivity-guided fractionation using HPLC bioactivity profiling/microtiter plate analysis in conjunction with capillary NMR instrumentation and the AntiMarin database. 2D NMR techniques, in combination with MS fragmentation experiments, determined the planar structure of chrysaibol (1), while the absolute configurations of the amino acid residues were defined by Marfey's method. Chrysaibol (1) was cytotoxic against the P388 murine leukemia cell line (IC50 6.61 microM) and showed notable activity against Bacillus subtilis (IC50 1.54 microM).

Broadening substrate specificity of a chain-extending ketosynthase through a single active-site mutation.

An in vitro model system based on a ketosynthase domain of the erythromycin polyketide synthase was used to probe the apparent substrate tolerance of ketosynthase domains of the mycolactone polyketide synthase. A specific residue change was identified that led to an emphatic increase in turnover of a range of substrates.

Iterative Mechanism of Macrodiolide Formation in the Anticancer Compound Conglobatin.

Conglobatin is an unusual C2-symmetrical macrodiolide from the bacterium Streptomyces conglobatus with promising antitumor activity. Insights into the genes and enzymes that govern both the assembly-line production of the conglobatin polyketide and its dimerization are essential to allow rational alterations to be made to the conglobatin structure. We have used a rapid, direct in vitro cloning method to obtain the entire cluster on a 41-kbp fragment, encoding a modular polyketide synthase assembly line. The cloned cluster directs conglobatin biosynthesis in a heterologous host strain. Using a model substrate to mimic the conglobatin monomer, we also show that the conglobatin cyclase/thioesterase acts iteratively, ligating two monomers head-to-tail then re-binding the dimer product and cyclizing it. Incubation of two different monomers with the cyclase produces hybrid dimers and trimers, providing the first evidence that conglobatin analogs may in future become accessible through engineering of the polyketide synthase.

Macrodiolide Formation by the Thioesterase of a Modular Polyketide Synthase.

Elaiophylin is an unusual C2-symmetric antibiotic macrodiolide produced on a bacterial modular polyketide synthase assembly line. To probe the mechanism and selectivity of diolide formation, we sought to reconstitute ring formation in vitro by using a non-natural substrate. Incubation of recombinant elaiophylin thioesterase/cyclase with a synthetic pentaketide analogue of the presumed monomeric polyketide precursor of elaiophylin, specifically its N-acetylcysteamine thioester, produced a novel 16-membered C2-symmetric macrodiolide. A linear dimeric thioester is an intermediate in ring formation, which indicates iterative use of the thioesterase active site in ligation and subsequent cyclization. Furthermore, the elaiophylin thioesterase acts on a mixture of pentaketide and tetraketide thioesters to give both the symmetric decaketide diolide and the novel asymmetric hybrid nonaketide diolide. Such thioesterases have potential as tools for the in vitro construction of novel diolides.

A natural plasmid uniquely encodes two biosynthetic pathways creating a potent anti-MRSA antibiotic.

BACKGROUND: Understanding how complex antibiotics are synthesised by their producer bacteria is essential for creation of new families of bioactive compounds. Thiomarinols, produced by marine bacteria belonging to the genus Pseudoalteromonas, are hybrids of two independently active species: the pseudomonic acid mixture, mupirocin, which is used clinically against MRSA, and the pyrrothine core of holomycin. METHODOLOGY/PRINCIPAL FINDINGS: High throughput DNA sequencing of the complete genome of the producer bacterium revealed a novel 97 kb plasmid, pTML1, consisting almost entirely of two distinct gene clusters. Targeted gene knockouts confirmed the role of these clusters in biosynthesis of the two separate components, pseudomonic acid and the pyrrothine, and identified a putative amide synthetase that joins them together. Feeding mupirocin to a mutant unable to make the endogenous pseudomonic acid created a novel hybrid with the pyrrothine via "mutasynthesis" that allows inhibition of mupirocin-resistant isoleucyl-tRNA synthetase, the mupirocin target. A mutant defective in pyrrothine biosynthesis was also able to incorporate alternative amine substrates. CONCLUSIONS/SIGNIFICANCE: Plasmid pTML1 provides a paradigm for combining independent antibiotic biosynthetic pathways or using mutasynthesis to develop a new family of hybrid derivatives that may extend the effective use of mupirocin against MRSA.

Concise, stereoselective route to the four diastereoisomers of 4-methylproline.

The full stereochemical characterization of 4-methylproline, a rare amino acid found in a number of peptidic secondary metabolites, has often been hindered by long reaction sequences or low stereoselectivity in the synthesis leading to reference samples. The preparation of the four diastereoisomers of 4-methylproline by a concise and stereoselective route is presented and features a six-step route with late-stage stereodivergence, good stereoselectivity for both cis- and trans-series (75% and 88% de, respectively), and good overall yields (cumulative yields of 30-40%). Additional data on the Marfey's derivatives of the stereoisomers are also presented.