Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

BACKGROUND: Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. METHODS: We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. RESULTS: Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3')-IIIa, aph(2'')-If, catA, lnu(C), blaOXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. CONCLUSIONS: The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety.

Monitoring Corynespora cassiicola Resistance to Boscalid, Trifloxystrobin, and Carbendazim in China.

Cucumber leaf spot (CLS), caused by Corynespora cassiicola, is a serious disease of greenhouse cucumbers. With frequent use of existing fungicides, C. cassiicola has developed resistance to some of them, with serious implications for the control of CLS in the field. With a lack of new fungicides, it is necessary to use existing fungicides for effective control. Therefore, this study monitored the resistance of C. cassiicola to three commonly used and effective fungicides, boscalid, trifloxystrobin, and carbendazim, from 2017 to 2021. The frequency of resistance to boscalid showed an increasing trend, and the highest frequency was 85.85% in 2020. The frequency of resistance to trifloxystrobin was greater than 85%, and resistance to carbendazim was maintained at 100%. Among these fungicides, strains with multiple resistance to boscalid, trifloxystrobin, and carbendazim were found, accounting for 32.00, 25.25, 33.33, 43.06, and 37.24%, respectively. Of the strains that were resistant to boscalid, 87% had CcSdh mutations, including seven genotypes: B-H278L/Y, B-I280V, C-N75S, C-S73P, D-D95E, and D-G109V. Also, six mutation patterns of the Ccß-tubulin gene were detected: E198A, F167Y, E198A&M163I, E198A&F167Y, M163I&F167Y, and E198A&F200C. Detection of mutations of the CcCytb gene in resistant strains showed that 98.8% were found to have only the G143A mutation. A total of 27 mutation combinations were found and divided into 14 groups for analysis. The resistance levels differed according to genotype. The development of genotypes showed a complex trend, increasing from 4 in 2017 to 13 in 2021 and varying by region. Multiple fungicide resistance is gradually increasing. Therefore, it is necessary to understand the types of mutations and the trend of resistance to guide the use of fungicides to achieve disease control.

Entomological monitoring data driving decision-making for appropriate and sustainable malaria vector control in Côte d'Ivoire.

BACKGROUND: Entomological surveillance provides critical information on vectors for appropriate malaria vector control and strategic decision-making. The widely documented insecticide resistance of malaria vectors in Côte d'Ivoire requires that any vector control intervention deployment be driven by entomological data to optimize its effectiveness and appropriate resource allocations. To achieve this goal, this study documents the results of monthly vector surveillance and insecticide susceptibility tests conducted in 2019 and a review of all previous entomological monitoring data used to guide vector control decision making. Furthermore, susceptibility to pirimiphos-methyl and clothianidin was assessed in addition to chlorfenapyr and pyrethroids (intensity and piperonyl butoxide (PBO) synergism) tests previously reported. Vector bionomic data were conducted monthly in four sites (Sakassou, Béoumi, Dabakala and Nassian) that were selected based on their reported high malaria incidence. Adult mosquitoes were collected using human landing catches (HLCs), pyrethrum spray catches (PSCs), and human-baited CDC light traps to assess vector density, behaviour, species composition and sporozoite infectivity. RESULTS: Pirimiphos-methyl and clothianidin susceptibility was observed in 8 and 10 sites, respectively, while previous data reported chlorfenapyr (200 µg/bottle) susceptibility in 13 of the sites, high pyrethroid resistance intensity and increased mortality with PBO pre-exposure at all 17 tested sites. Anopheles gambiae sensu lato was the predominant malaria vector collected in all four bionomic sites. Vector density was relatively higher in Sakassou throughout the year with mean biting rates of 278.2 bites per person per night (b/p/n) compared to Béoumi, Dabakala and Nassian (mean of 48.5, 81.4 and 26.6 b/p/n, respectively). The mean entomological inoculation rate (EIR) was 4.44 infective bites per person per night (ib/p/n) in Sakassou, 0.34 ib/p/n in Beoumi, 1.17 ib/p/n in Dabakala and 1.02 ib/p/n in Nassian. The highest EIRs were recorded in October in Béoumi (1.71 ib/p/n) and Nassian (3.22 ib/p/n), in July in Dabakala (4.46 ib/p/n) and in May in Sakassou (15.6 ib/p/n). CONCLUSION: Based on all results and data review, the National Malaria Control Programme developed and implemented a stratified insecticide-treated net (ITN) mass distribution in 2021 considering new generation ITNs. These results also supported the selection of clothianidin-based products and an optimal spraying time for the first indoor residual spraying (IRS) campaign in Sakassou and Nassian in 2020.

Fungicide Resistance: Progress in Understanding Mechanism, Monitoring, and Management.

Fungicide treatments are often essential for maintaining healthy crops and to achieve reliable and high-quality yields. However, continued use of fungicides with the same modes of action can lead to development of fungicide resistance, which has emerged in various plant pathogens and is a serious threat to effective crop protection. Exploration of resistance mechanisms is critical for resistance monitoring and management. This brief review summarizes advances during the past five decades in understanding the molecular resistance mechanisms of plant pathogenic fungi and oomycetes to major classes of fungicides, including benzimidazoles, myosin inhibitors, sterol demethylation inhibitors, quinone outside inhibitors, succinate dehydrogenase inhibitors, anilinopyrimidines, carboxylic acid amides, and oxysterol-binding protein homolog inhibitors. Based on known resistance mechanisms, PCR- and loop-mediated isothermal amplification-based approaches have been developed to allow high-throughput monitoring and early/rapid detection of emerging resistance. Classical principles in fungicide resistance management are also summarized, including using different modes of action of fungicides, limiting the number of applications of the chemicals with site-specific modes of action, and avoidance of their eradicant use. Future studies on novel strategies of disease management, including development of epigenetics- and RNA-based fungicides, will provide valuable knowledge for management of fungicide resistance.

Long-term survey and characterization of cyflumetofen resistance in Tetranychus urticae populations from Turkey.

The two-spotted spider mite, Tetranychus urticae Koch (Acari: Tetranychidae) is the most economically important mite pest in agricultural areas and chemical acaricides are widely used to control T. urticae populations. Cyflumetofen is a recently introduced acaricide that inhibits the mitochondrial electron transport chain at complex II (succinate dehydrogenase, SDH), which represents the most recently developed mode of action for mite control worldwide. In the present study, started upon the launch of cyflumetofen in Turkey, a five-year survey was performed to monitor cyflumetofen susceptibility in 28 T. urticae populations collected from agricultural fields across the country. The first resistance case that might cause control failure in practical field conditions was uncovered in 2019, three years after the registration of cyflumetofen. In addition, an extremely resistant population (1722-fold resistance) was also detected towards the end of 2019. Cyflumetofen resistance did not decrease in the laboratory after relaxation of selection pressure for over one year in field-collected populations, suggesting the absence of a fitness cost associated with resistance in these populations. Next to phenotypic resistance, metabolic and physiological mechanisms underlying the decreased susceptibility were also investigated. Synergism assays showed the involvement of P450 monooxygenases in cyflumetofen resistance. Downregulation of carboxylesterases as resistance mechanism, is underpinned by the fact that pre-treatment with esterase inhibitor DEF decreased cyflumetofen toxicity in field-collected strains. Furthermore, a novel H258L substitution in the subunit B of complex II was uncovered in a field population. In silico modeling of the new mutation suggested that the mutation might indeed influence toxicity to complex II inhibitors cyenopyrafen and pyflubumide, but most likely not cyflumetofen. However, further studies are needed to uncover the exact role of this mutation in resistance to this new class of complex II inhibitors.

SDH mutations confer complex cross-resistance patterns to SDHIs in Corynespora cassiicola.

Succinate dehydrogenase inhibitors (SDHIs) are one of the most frequently used fungicides in cucumber fields in China. Our previous studies indicated that the sensitivity profile of Corynespora cassiicola, the causal agent of Corynespora leaf spot, to different SDHIs varied greatly; however, the underlying mechanism remains unclear. The 50% effective concentration (EC50) values of boscalid, fluopyram, fluxapyroxad and isopyrazam in C. cassiicola collected from 2017 to 2020 shifted, with resistance frequencies of 79.83%, 78.43%, 83.19% and 49.86%, respectively. The sequence alignment of sdhB/C/D of resistant strains revealed that eight single amino acid mutations (B-H278Y/L, B-I280V, C-S73P, C-N75S, C-H134R, D-D95E and D-G109V), and three dual-mutations (B-I280V&C-S73P, B-I280V&C-N75S and C-S73P&C-N75S) conferred various SDHI resistance levels and cross-resistance profiles. The expression level of the sdhB/C/D gene and succinate dehydrogenase (SDH) activity in the mutants were significantly altered by the presence of SDHIs, compared with the wild type strain. Additionally, molecular docking results suggested that the missense mutation influenced the crystal structure of SDH and subsequently interfered with the interaction bonds and bond distances among the target protein and chemicals. In brief, amino acid mutations altered the fungicide response of target gene expression, SDH activity and the binding features of SDH-ligand complexes and subsequently conferred multiple resistance levels and complex cross-resistance patterns to SDHIs in C. cassiicola. The evaluation of C. cassiicola resistance to SDHIs provided a significant foundation for efficient chemical development and integrated CLS management strategies.

Monitoring and characterization of prochloraz resistance in Fusarium fujikuroi in China.

Rice bakanae disease, caused by Fusarium fujikuroi, is a destructive seed-borne disease throughout the world. Prochloraz, a DMI (C-14α-demethylase inhibitor) fungicide, has been registered in China for >20 years. Prochloraz resistance in F. fujikuroi was severe in China with resistance frequencies of 34.56%, 45.33%, and 48.45% from 2019 to 2021. The fitness of prochloraz-resistant population was lower than that of sensitive population, with an average CFI of 2.86 × 106 and 4.56 × 106, respectively. No cross-resistance was detected between prochloraz and tebuconazole or hexaconazole, and the prochloraz-resistant isolates were still sensitive to fludioxonil, phenamacril, and pydiflumetofen. S312T mutation in Ffcyp51b or overexpression of Ffcyp51a and Ffcyp51b was detected in the highly resistant isolates. AS-PCR primers were designed to detect the prochloraz-resistant isolates with S312T mutation in the field. Resistant isolates carrying S312T mutation were the dominant group in prochloraz-resistant population with frequencies of 43.26%, 23.59%, and 71.20% from 2019 to 2021, which indicated that more attention should be paid to this genotype when monitoring and managing the prochloraz resistance in F. fujikuroi.

Resistance to Beta-cypermethrin, Azadirachtin, and Matrine, and Biochemical Characterization of Field Populations of Oedaleus asiaticus (Bey-Bienko) in Inner Mongolia, Northern China.

Oedaleus asiaticus (Bey-Bienko) is an economically devastating locust species found in grassland and pastoral areas of the Inner Mongolia region of northern China. In this study, resistance to three frequently used insecticides (beta-cypermethrin, matrine, and azadirachtin) was investigated in six field populations of O. asiaticus using the leaf-dip bioassay method. The inhibitory effects of synergists and the activities of detoxification enzyme activities in the different populations were determined to explore potential biochemical resistance mechanisms. The results showed that the field populations SB (resistance ratio [RR] = 7.85), ZB (RR = 5.64), and DB (RR = 6.75) had developed low levels of resistance to beta-cypermethrin compared with a susceptible control strain. Both the SB (RR = 5.92) and XC (RR = 6.38) populations had also developed low levels of resistance against matrine, with the other populations remaining susceptible to both beta-cypermethrin and matrine. All field populations were susceptible to azadirachtin. Synergism analysis showed that triphenyl phosphate (TPP) and diethyl-maleate (DEM) increased the toxicity of beta-cypermethrin significantly in the SB population, while the synergistic effects of TPP, piperonyl butoxide (PBO), and DEM on the toxicity of matrine were higher in SB (SR 3.86, 4.18, and 3.07, respectively) than in SS (SR 2.24, 2.86, and 2.29, respectively), but no synergistic effects of TPP, PBO, and DEM on azadirachtin were found. Biochemical assays showed that the activities of carboxylesterases (CarEs) and glutathione-S-transferases (GSTs) were significantly raised in all field populations of O. asiaticus, with a significant positive correlation observed between beta-cypermethrin resistance and CarE activity. The activities of cytochrome P450 monooxygenases (P450) and multi-function oxidases (MFO) were elevated in all six field populations, and P450 activity displayed strong positive correlations with the three insecticides. Our findings suggest that resistance to beta-cypermethrin in O. asiaticus may be mainly attributed to elevated CarE and GST activities, while P450 plays an important role in metabolizing matrine and azadirachtin. Our study provides insights that will help improve insecticide resistance management strategies.

Management of insecticides for use in disease vector control: a global survey.

BACKGROUND: Vector control plays a critical role in the prevention, control and elimination of vector-borne diseases, and interventions of vector control continue to depend largely on the action of chemical insecticides. A global survey was conducted on the management practices of vector control insecticides at country level to identify gaps to inform future strategies on pesticide management, seeking to improve efficacy of interventions and reduce the side-effects of chemicals used on health and the environment. METHODS: A survey by questionnaire on the management practices of vector control insecticides was disseminated among all WHO Member States. Data were analysed using descriptive statistics in MS Excel. RESULTS: Responses were received from 94 countries, or a 48% response rate. Capacity for insecticide resistance monitoring was established in 68-80% of the countries in most regions, often with external support; however, this capacity was largely lacking from the European & Others Region (i.e. Western & Eastern Europe, North America, Australia and New Zealand). Procurement of vector control insecticides was in 50-75% of countries taking place by agencies other than the central-level procuring agency, over which the central authorities lacked control, for example, to select the product or assure its quality, highlighting the importance of post-market monitoring. Moreover, some countries experienced problems with estimating the correct amounts for procurement, especially for emergency purposes. Large fractions (29-78%) of countries across regions showed shortcomings in worker safety, pesticide storage practices and pesticide waste disposal. Shortcomings were most pronounced in countries of the European & Others Region, which has long been relatively free from mosquito-borne diseases but has recently faced challenges of re-emerging vector-borne diseases. CONCLUSIONS: Critical shortcomings in the management of vector control insecticides are common in countries across regions, with risks of adverse pesticide effects on health and the environment. Advocacy and resource mobilization are needed at regional and country levels to address these challenges.

Spatial Variation and Temporal Dynamics of Fungicide Sensitivity in Venturia effusa Within a Pecan Orchard.

An 18-ha commercial pecan orchard was sampled over 3 years to study the spatial and temporal variation in fungicide sensitivity of Venturia effusa, cause of pecan scab. The orchard was divided into a two-dimensional, 8 × 8 grid of 64 quadrats, each containing nine trees (unless there were missing trees), and samples were collected once per year from each quadrat to be tested for sensitivity to fentin hydroxide, propiconazole, and thiophanate-methyl. Averaged across the orchard, insensitivity to all three fungicides was significantly lower in 2016 compared with 2015, but significantly greater for fentin hydroxide and thiophanate-methyl in 2017. Although significant spatial autocorrelation was observed for sensitivity to propiconazole in 2017 and for thiophanate-methyl in 2015 and 2017, indicating clustering, all other fungicide-by-year combinations were not significant. Omnidirectional spatial dependence was observed for sensitivity to propiconazole and thiophanate-methyl in 2017. In both instances, the semivariance increased linearly with lag distance; however, the range of spatial dependence was >276.5 m and could not be estimated accurately. Additionally, a separate sampling was conducted in all 3 years to identify an appropriate sampling size and pattern for fungicide sensitivity screening. A leaflet sample size of 165 in 11 groups of 15 allowed for accurate sensitivity testing for the three fungicides in all 3 years; however, a sample size of 45 leaflets in three groups of 15 was sufficient for quantifying sensitivity for propiconazole and thiophanate-methyl, in most cases. These results indicate that considerable biological variation in fungicide sensitivity exists in orchard-scale populations of V. effusa and that the spatial characteristics of those populations may differ in two-dimensional space depending on the growing season.

Monitoring of field-evolved resistance to flonicamid, neonicotinoid, and conventional insecticides in the Oxycarenus hyalinipennis costa.

Oxycarenus hyalinipennis Costa is a polyphagous insect pest and can develop insecticide resistance. The resistance of O. hyalinipennis to neonicotinoids (clothianidin and dinotefuran), flonicamid, and conventional insecticides; carbamates (methomyl and carbosulfon), organophosphates (chlorpyrifos and malathion), and pyrethroids (cypermethrin and zeta-cypermethrin) was evaluated. The O. hyalinipennis populations were sampled from four locations in Pakistan and performed bioassays against the insecticides by leaf dip protocol. The O. hyalinipennis' populations showed low resistance to carbosulfan (resistance ratio (RR) = 2.06-6.34) and methomyl (RR = 2.78-7.27), moderate to high resistance to chlorpyrifos (RR = 30-45), malathion (RR = 20.29-88.19), and flonicamid (RR = 14.24-46.97), in comparison with the susceptible strain. Susceptibility to low resistance against cypermethrin (RR = 1.27-2.82), zeta-cypermethrin (RR = 2.62-3.38), and clothianidin (RR = 1.74-3.40), and low to moderate resistance to dinotefuran (RR = 3.84-13.43) in the field populations, was observed compared to the susceptible strain. A rotational usage of carbamates and pyrethroids with an integrated pest management tool should be considered to deal with O. hyalinipennis' insecticide resistance.

Using full chloroplast genomes of 'red' and 'yellow' Bixa orellana (achiote) for kmer based identification and phylogenetic inference.

BACKGROUND: Full chloroplast genomes provide high resolution taxonomic discrimination between closely related plant species and are quickly replacing single and multi-locus barcoding regions as reference materials of choice for DNA based taxonomic annotation of plants. Bixa orellana, commonly known as "achiote" and "annatto" is a plant used for both human and animal foods and was thus identified for full chloroplast sequencing for the Center for Veterinary Medicine (CVM) Complete Chloroplast Animal Feed database. This work was conducted in collaboration with the Instituto de Medicina Tradicional (IMET) in Iquitos, Peru. There is a wide range of color variation in pods of Bixa orellana for which genetic loci that distinguish phenotypes have not yet been identified. Here we apply whole chloroplast genome sequencing of "red" and "yellow" individuals of Bixa orellana to provide high quality reference genomes to support kmer database development for use identifying this plant from complex mixtures using shotgun data. Additionally, we describe chloroplast gene content, synteny and phylogeny, and identify an indel and snp that may be associated with seed pod color. RESULTS: Fully assembled chloroplast genomes were produced for both red and yellow Bixa orellana accessions (158,918 and 158,823 bp respectively). Synteny and gene content was identical to the only other previously reported full chloroplast genome of Bixa orellana (NC_041550). We observed a 17 base pair deletion at position 58,399-58,415 in both accessions, relative to NC_041550 and a 6 bp deletion at position 75,531-75,526 and a snp at position 86,493 in red Bixa orellana. CONCLUSIONS: Our data provide high quality reference genomes of individuals of red and yellow Bixa orellana to support kmer based identity markers for use with shotgun sequencing approaches for rapid, precise identification of Bixa orellana from complex mixtures. Kmer based phylogeny of full chloroplast genomes supports monophylly of Bixaceae consistent with alignment based approaches. A potentially discriminatory indel and snp were identified that may be correlated with the red phenotype.

Multiple insecticide resistance in onion thrips populations from Western USA.

Thrips tabaci is a key pest of onions, especially in the Pacific Northwestern USA. Management of T. tabaci is dominated by the application of various insecticides. However, T. tabaci is known to develop insecticide resistance which possibly leads to control failures, crop loss, and environmental concern. Here, we evaluated resistance status of T. tabaci populations from conventional and organic commercial onion fields to three widely used insecticides: oxamyl, methomyl, and abamectin with on-field concentration-mortality bioassays. The biochemistry and molecular mechanisms underlying resistance to these insecticides were also investigated by using enzymatic assays and detecting resistance-associated mutations. Field-evolved resistance to oxamyl, methomyl and abamectin were detected in most of the T. tabaci populations collected from conventional onion farms. At the labeled field rate, all the tested insecticides, particularly methomyl and oxamyl, had significantly reduced efficacy. Enzymatic assays of insecticide target and detoxification enzymes indicated that T. tabaci populations in Western USA onions harbor multiple mechanisms of resistance including enhanced activities of detoxification enzymes and target site insensitivity. Our results provide new information in understanding the dynamics of T. tabaci adaptation to multiple insecticides, which will help to design sustainable insecticide resistance management strategies for T. tabaci. Furthermore, this study provides the foundation for future research in identifying the biochemical and molecular markers associated with insecticide resistance in T. tabaci.

Occurrence and Extent of Boscalid Resistance in Populations of Alternaria alternata from California Pistachio Orchards.

Alternaria late blight (ALB) caused by Alternaria spp. is an annual disease problem in California pistachio and requires repeated applications of fungicides to prevent significant losses of pistachio foliage and nut quality. From 2003 onward, the succinate dehydrogenase inhibiting fungicide boscalid has played a key role in ALB management. The development of boscalid resistance in A. alternata populations was monitored from 2005 to 2012 in pistachio producing areas in California. A total of 1,765 single-spore isolates, collected from commercial and experimental pistachio orchards with or without a history of boscalid exposure, were tested in a radial growth assay in agar media amended with the discriminatory dose of 10 µg/ml of boscalid. The frequency of boscalid-resistant isolates in 2005 was 12% but increased significantly and remained stable toward the end of the survey period. Most of the resistant isolates exhibited a high level of resistance (R) to boscalid with percent of mycelial growth inhibition (PGI) values between 0 and 50%, whereas significantly fewer isolates had an intermediate level (IR) of resistance (50 < PGI < 75%). The frequency of sensitive (S) isolates (75 < PGI < 100%) was generally the highest in orchards with no history of boscalid usage, whereas mean incidences of boscalid-resistant populations of Alternaria were 81, 92.4, 80.2, and 98%, in 2006, 2007, 2011, and 2012, respectively, in orchards that received a high number (at least three per season) of boscalid spray applications. In comparison, none to relatively low frequencies (0 to 12%) of resistance were observed in populations with no or limited exposure to Pristine, suggesting an air-movement of resistant spores through wind from treated to nontreated areas. In 2012, boscalid-resistant isolates were found practically in every sampled location in all counties, with the orchards in Fresno, Madera, Tulare, and King Counties being the locations with the highest frequencies of resistance (100%). Monitoring of A. alternata AaSDHB, AaSDHC, and AaSDHD mutations in 286 boscalid-resistant phenotypes identified 11 mutations, leading to amino acid substitutions in AaSDHB (seven mutations: H277Y/R/L, P230A/R, N235D/T), AaSDHC (one mutation: H134R), and AaSDHD (three mutations: D123E, H133R/P), with AaSDHB mutations being the most prevalent (80%) ones throughout the survey period. The majority of isolates carrying these mutations exhibited the R phenotype toward boscalid. The increased prevalence of boscalid resistance in populations of A. alternata is a likely contributing factor to the inability of pistachio farmers to successfully control ALB with Pristine. Other factors implicated in the rapid and widespread occurrence of A. alternata boscalid-resistant populations in California pistachios are further discussed.

A new method to obtain isofemale lines for monitoring Chilo suppressalis resistance to Bt toxin.

Cost-effective Bt resistance monitoring programmes exist to manage insect resistance to Bt crops. F2 screening is widely used in detecting rare resistant alleles. One way to establish numerous isofemale lines for F2 bioassays is acquiring many field-trapped copulated females. However, it is difficult to obtain sufficient Chilo suppressalis isofemale lines because females have low mating rates and fecundity. We developed a new method to establish C. suppressalis isofemale lines with field-collected egg masses. No significant difference in detection ability was observed in the F2 screen between the old and new methods. Moreover, the new method is economical, convenient and efficient.

Pyrethroid and carbamate resistance in Australian Helicoverpa armigera (Lepidoptera: Noctuidae) from 2008 to 2015: what has changed since the introduction of Bt cotton?

Pyrethroid and carbamate resistance was evaluated in Helicoverpa armigera from 2008 to 2015. Insects were collected as eggs primarily from cultivated hosts in the major cropping areas of New South Wales and Queensland, Australia. Larvae reared from eggs were tested for resistance to fenvalerate, bifenthrin or methomyl in the F0 generation using a topical application of a discriminating dose of insecticide. In 2008-2009, resistance to fenvalerate was 71% and no resistance to bifenthrin was recorded. In the following two seasons, resistance to pyrethroids was relatively stable with fenvalerate resistance ranging from 63% to 67% and bifenthrin resistance ranging from 5.6% and 6.4% in 2009-2010 and 2010-2011, respectively. However, in 2011-2012, pyrethroid resistance had increased to 91% and 36% for fenvalerate and bifenthrin, respectively. Resistance remained above 90% for fenvalerate and above 35% for bifenthrin in the following three seasons from 2012 to 2015. In 2008-2009, methomyl resistance was 33% and declined to 22% and 15% in 2009-2010 and 2010-2011, respectively. Methomyl resistance remained at moderate levels from 2011-12 to 2014-15, ranging from 21% to 40%. Factors that influenced selection pressure of pyrethroid and carbamate insecticides and impacted resistance frequency in H. armigera may have been associated with changes in the composition of the cropping landscape. The rapid expansion of the pulse industry and the commensurate increased use of insecticide may have played a role in reselection of high-level pyrethroid resistance, and highlights the need for an urgent and strategic response to insecticide resistance management in the Australian grains industry.

[Characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli in animal feces, from Shandong Province].

Objective: To understand the infection status, characteristics and drug resistance of non-O157 Shiga toxin-producing E. coli (STEC) in animal feces in Shandong Province. Methods: From 2015 to 2016, convient sampling method was used to collect 1 022 fresh feces of animals in Weishan county and Laizhou city, and 24 non-O157 STEC were isolated. The serotypes of non-O157 STEC strains were confirmed through serum agglutination test. The susceptibility was explored through the antimicrobial sensitivity experiments. ESBLs activity was confirmed by double-disc diffusion. PCR method was used to detect the resistance genes. PFGE typing was operated to assess the relatedness and variability of the strains. The multi-locus sequence typing (MLST) was adopted to get the allelic profile and ST sequence of strains. Analysis was made on the evolutionary relationship between different ST groups was made through CLC Sequence Viewer and Counting Express. Results: A total of 24 non-O157 STEC were isolated from animal feces. 23 strains were from pig feces, and 1 strain was from cow feces, and the serotypes were more dispersed. All of the 24 strains carried stx2 genes. The highest resistance rate was sulfamethoxazole(22 strains), the mount of cotrimoxazole and nalidixic acid was 18 strains, chloramphenicol was 13 strains, tetracycline was 19, and there was a phenomenon of multiple drug resistance. The drug resistance spectrum was sulfamethoxazole tetracycline-compound novammin-naphthidine-chloramphenicol. All strains were sensitive to cefepime and imipenem. The ESBLs confirmatory test showed that 4 strains of non O157 STEC produced beta lactamase. PCR detected 7 resistance genes, and 4 tetracycline resistance genes (Tet A, Tet B, tetC and tetD) were detected. The beta lactamase resistance genes (blaSHV-1, bla CTX-M, bla TEM) were all negative. 24 strains were divided into 15 PFGE types, and their clustering results were more dispersed and no dominant PFGE type. There were 11 kinds of MLST types, most of them are ST540 and ST5133 types, each of which was 4 strains, and clustered into 1 MLST genomes. Conclusion: The serotypes of non-O157 STEC in animal feces O157 STEC were dispersed, and the resistant rate to common antibiotic was high. MLST typing results presents obvious polymorphism. Surveillance and manage ment of these strains should be strengthened.

Prevalence of Colistin Resistance Gene mcr-1 and Absence of mcr-2 in Escherichia coli Isolated from Healthy Food-Producing Animals in Japan.

We screened mcr-1 and mcr-2 genes in 9,306 Escherichia coli strains isolated from healthy animals in the Japanese Veterinary Antimicrobial Resistance Monitoring (JVARM) system. mcr-1 was detected in 39 strains (5, 20, and 14 strains isolated from cattle, swine, and broilers, respectively), whereas mcr-2 was not detected. mcr-2 was also not detected with the investigation sequence homology search against our curated GenEpid-J database.

An assessment of the relationship between the World Health Organization HIV drug resistance early warning indicators and HIV drug resistance acquisition.

OBJECTIVES: The World Health Organization (WHO)'s HIV drug resistance (HIVDR) early warning indicators (EWIs) measure antiretroviral therapy (ART)-site factors associated with HIVDR prevention, without HIVDR laboratory testing. We assessed the relationship between EWIs and HIVDR acquisition using data from British Columbia, Canada. METHODS: Eligible patients were ART-naïve, were ≥ 19 years old, had initiated ART between 1 January 2000 and 31 December 2012, had ≥ 15 months of follow-up, and were without transmitted HIVDR. Patients were followed for acquired HIVDR until 31 March 2014, the last contact date, or death. We built logistic regression models to assess the associations and predictive ability of individual indicators and of the EWI Score (the number of indicators for which a patient did not meet the criteria) on HIVDR acquisition (to any class of HIVDR, lamivudine (3TC)/emtricitabine (FTC), nonnucleoside reverse transcriptase inhibitors (NNRTIs), nucleoside reverse transcriptase inhibitors (NRTIs) or protease inhibitors (PIs)]). RESULTS: All explored EWIs were associated with at least one class of HIVDR, with the exception of 'ART prescribing practices'. We observed a dose-response relationship between acquiring HIVDR to any antiretroviral class and an increasing EWI score in our predictive logistic regression model. The area under the curve was 0.848 (excellent discrimination). The adjusted odds ratios for acquiring any class of HIVDR for an EWI score of 1, 2 and ≥ 3 versus 0 were 2.30 [95% confidence Interval (CI) 1.21-4.38], 3.35 (95% CI: 1.86-6.03) and 7.26 (95% CI: 4.18-12.61), respectively. CONCLUSIONS: Several EWIs were associated with and predictive of HIVDR, supporting the WHO EWIs as a component of the HIVDR prevention method in settings where HIVDR testing is not routinely or widely available.

A methodology based on insecticide impregnated filter paper for monitoring resistance to deltamethrin in Triatoma infestans field populations.

The domiciliary presence of Triatoma infestans (Klug) (Hemiptera: Reduviidae) after control interventions was reported in recent years. Toxicological studies showed high levels of resistance to pyrethroids suggesting resistance as one of the main causes of deficient control. The aim of the present study was to develop a protocol to test resistance to deltamethrin in T. infestans collected from the field by discriminate concentration. To evaluate field insects, the effect of age (early vs. later) and nutritional state (starved vs. fed) on the deltamethrin susceptibility of each developmental stage was studied. Topical and insecticide impregnated paper bioassays were used. Using the impregnated paper, the susceptibility to deltamethrin was not affected by the age of the stadium and the nutritional states, and varied with the post-exposure time and with the different developmental stages. A discriminant concentration of deltamethrin (0.36% w/v) impregnated in filter paper was established for all developmental stages. Finally, the methodology and the discriminant concentration were evaluated in the laboratory showing high sensitivity in the discrimination of resistance. The present study developed a methodology of exposure to insecticide impregnated papers and proposes a protocol to test T. infestans in field populations with the aim to detect early evolution of resistance to deltamethrin.