A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.     

Estimating fitness by competition assays between drug susceptible and resistant Mycobacterium tuberculosis of predominant lineages in Mumbai, India

Background

Multi Drug Resistant Tuberculosis (MDR TB) is a threat to global tuberculosis control. A significant fitness cost has been associated with DR strains from specific lineages. Evaluation of the influence of the competing drug susceptible strains on fitness of drug resistant strains may have an important bearing on understanding the spread of MDR TB. The aim of this study was to evaluate the fitness of MDR TB strains, from a TB endemic region of western India: Mumbai, belonging to 3 predominant lineages namely CAS, Beijing and MANU in the presence of drug susceptible strains from the same lineages.

Methodology

Drug susceptible strains from a single lineage were mixed with drug resistant strain, bearing particular non synonymous mutation (rpoB D516V; inhA, A16G; katG, S315T1/T2) from the same or different lineages. Fitness of M.tuberculosis (M.tb) strains was evaluated using the difference in growth rates obtained by using the CFU assay system.

Conclusion/Significance

While MANU were most fit amongst the drug susceptible strains of the 3 lineages, only Beijing MDR strains were found to grow in the presence of any of the competing drug susceptible strains. A disproportionate increase in Beijing MDR could be an alarm for an impending epidemic in this locale. In addition to particular non synonymous substitutions, the competing strains in an environment may impact the fitness of circulating drug resistant strains.     

Susceptibility of Agrobacterium tumefaciens Strains to Two Agrocin-Producing Agrobacterium Strains

Sixty-five strains and isolates of Agrobacterium tumefaciens representing each of the known biotypes, were tested for in vitro and in vivo susceptibility to the agrocin-producing strains Agrobacterium radiobacter 84 and A. tumefaciens D286. No biotype 3 strain was susceptible to the effects of either of the agrocinogenic strains in vitro. On datura and tobacco, the best inhibition of tumor formation was obtained when the agrocinogenic strains were applied to wounds 24 h before the pathogens and by the concomitant application of agrocin producer and pathogen at cell ratios of 10:1 or 3:1; inhibition of infection tended to decrease progressively as the cell ratio decreased from 10:1 to 3:1 to 1:1. Generally, strain 84 was superior to D286 in inhibiting tumor formation. A combined cell suspension of 84 and D286 was as effective as 84 alone. The overall pattern of inhibition of tumor formation by biotype 1 and 2 pathogens resistant to the agrocinogenic strains in vitro was similar to that obtained with strains that were susceptible in vitro.     

Vermiculite as a culture substrate greatly improves the viability of frozen cultures of ectomycorrhizal basidiomycetes

We assessed a new cryopreservation protocol that uses vermiculite as a culture substrate, called the vermiculite protocol (VP), by assessing the viability, recovery time of hyphae after revival, and colony diameter of cryosensitive ectomycorrhizal basidiomycete strains after storage for 2 weeks or 1 year in a vapour-phase liquid nitrogen tank. Twelve difficult-to-preserve strains of nine species (Amanita citrina, A. pantherina, A. rubescens, A. spissa, Kobayasia nipponica, Lactarius akahatsu, L. hatsudake, Sarcodon aspratus, and Tricholoma flavovirens) that did not achieve good revival after cryopreservation with our previous Homolka’s perlite protocol and modified perlite protocol (MPP) experiments were used to assess the new methodology. Vermiculite and liquid medium were put into a cryotube and inoculated with an agar plug containing mycelia. The cryotube was cultured for various incubation times. After adequate mycelial growth, a mixture of cryoprotectants (5% dimethyl sulfoxide and 10% trehalose [5D10T] or 5% glycerol and 10% trehalose [5G10T]) was placed into the cryotube. The cryotube was frozen in a freezing container in a –80 °C freezer and then stored in vapour-phase liquid nitrogen. In the recovery test, 10 of 12 strains showed 100% revival after 2 weeks of storage in the 5G10T cryoprotectant, and all 12 strains showed 100% revival after 2 weeks of storage in the 5D10T cryoprotectant. Furthermore, all strains were viable after 1 year of storage in a vapour-phase liquid nitrogen tank. Thus, the VP is applicable to a wide range of ectomycorrhizal basidiomycete cultures, including highly cryosensitive strains.     

Evaluation of Genetic Diversity among Pseudomonas citronellolis Strains Isolated from Oily Sludge-Contaminated Sites

The diversity among a set of bacterial strains that have the capacity to degrade total petroleum hydrocarbons (TPH) in soil contaminated with oily sludge (hazardous hydrocarbon waste from oil refineries) was determined. TPH is composed of alkane, aromatics, nitrogen-, sulfur-, and oxygen-containing compound, and asphaltene fractions of crude oil. The 150 bacterial isolates which could degrade TPH were isolated from soil samples obtained from diverse geoclimatic regions of India. All the isolates were biochemically characterized and identified with a Biolog microbial identification system and by 16S rDNA sequencing. Pseudomonas citronellolis predominated among the 150 isolates obtained from six different geographically diverse samplings. Of the isolates, 29 strains of P. citronellolis were selected for evaluating their genetic diversity. This was performed by molecular typing with repetitive sequence (Rep)-based PCR with primer sets ERIC (enterobacterial repetitive intergenic consensus), REP (repetitive extragenic palindromes), and BOXAIR and PCR-based ribotyping. Strain-specific and unique genotypic fingerprints were distinguished by these molecular typing strategies. The 29 strains of P. citronellolis were separated into 12 distinguishable genotypic groups by Rep-PCR and into seven genomic patterns by PCR-based ribotyping. The genetic diversity of the strains was related to the different geoclimatic isolation sites, type of oily sludge, and age of contamination of the sites. These results indicate that a combination of Rep-PCR fingerprinting and PCR-based ribotyping can be used as a high-resolution genomic fingerprinting method for elucidating intraspecies diversity among strains of P. citronellolis.     

A new shuttle vector for gene expression in biopolymer-producing Ralstonia eutropha

Ralstonia eutropha (formerly Alcaligenes eutrophus) is a fascinating microorganism with a great scientific importance and an immense commercial potential. A new genetic transformation system for the organism would greatly facilitate the biological study and molecular engineering of this organism. We report here a versatile gene expression method for the genetic engineering of R. eutropha. This method, based on a simplified electroporation protocol, uses a recombinant plasmid, pBS29-P2, containing a Pseudomonas syringae promoter (P2) and two antibiotic-resistance markers (i.e., genes coding for kanamycin (Km)- and tetracycline (Tc)-resistance). Using this method, we successfully achieved transformation of wild-type R. eutropha and its poly(hydroxyalkanoate)-negative mutant, R. eutropha PHB⁻4, with various pBS29-P2-based recombinants. A transformation frequency as high as 4 × 10³ Km-resistance colonies/μg DNA was obtained per electroporation experiment. We further demonstrated the successful expression of a heterologous gene coding for green-fluorescent-protein by fluorescence measurement. In addition, our results indicated the expression of a truncated but active Streptomyces coelicolor α-galactosidase in R. eutropha.     

Tomato plant growth promotion and antibacterial related-mechanisms of four rhizobacterial Bacillus strains against Ralstonia solanacearum

Bacillus strains are extensively studied for their beneficial role in plant growth and biological control of tomato bacterial wilt (TBW), however their underlying mechanisms remained unexplored. In this study, four rhizobacterial strains, Bacillus amyloliquefaciens D29, B. amyloliquefaciens Am1, B. subtilis D16 and B. methylotrophicus H8 were investigated for their antibacterial activity against (TBW) pathogen and their ability to stimulate Tomato growth. Results revealed that all four strains were able to form robust biofilm, produce Indole acetic acid (IAA) and siderophores, while only D29, Am1 and H8 have capability to solubilize phosphate. The culture filtrate of each strain significantly suppressed the growth and biofilm of Ralstonia solanacearum, where, the cell wall was severely disrupted, which resulted into cell lysis and subsequent leakage of intracellular cytosolic contents. PCR analysis revealed that all four strains are harboring the antimicrobial associated genes for biosynthesis of Bacyllomicin, Fengycin, Iturin, Surfactin and Bacylisin. Subsequent real-time qPCR analysis revealed that the expression of ituC and srfAA genes in Am1 and D16 was remarkably up-regulated during in vitro interaction with R. solanacearum. This suggest that the potential antibacterial and anti-biofilm related mechanisms are associated to their ability to secret the corresponding lipopeptides in surrounding niche. In greenhouse, a positive correlation (0.777 and 0.686) was noted between the IAA amount produced by Bacillus strains and fresh/dry weight of bacterized tomato plants. This the first report demonstrated the mode of antibacterial effect of Bacillus strains against R. solanacearum, moreover this study will help in understanding the mode of action of Bacillus strains during biological management of TBW and promoting the growth of tomato plants.     

Requirement of DNA Repair Mechanisms for Survival of Burkholderia cepacia G4 upon Degradation of Trichloroethylene

A Tn5-based mutagenesis strategy was used to generate a collection of trichloroethylene (TCE)-sensitive (TCS) mutants in order to identify repair systems or protective mechanisms that shield Burkholderia cepacia G4 from the toxic effects associated with TCE oxidation. Single Tn5 insertion sites were mapped within open reading frames putatively encoding enzymes involved in DNA repair (UvrB, RuvB, RecA, and RecG) in 7 of the 11 TCS strains obtained (4 of the TCS strains had a single Tn5 insertion within a uvrB homolog). The data revealed that the uvrB-disrupted strains were exceptionally susceptible to killing by TCE oxidation, followed by the recA strain, while the ruvB and recG strains were just slightly more sensitive to TCE than the wild type. The uvrB and recA strains were also extremely sensitive to UV light and, to a lesser extent, to exposure to mitomycin C and H₂O₂. The data from this study establishes that there is a link between DNA repair and the ability of B. cepacia G4 cells to survive following TCE transformation. A possible role for nucleotide excision repair and recombination repair activities in TCE-damaged cells is discussed.     

Genetic Linkage between the AH Locus and a Major Gene That Inhibits Susceptibility to Audiogenic Seizures in Mice

Mice of the DBA/2 (D2) strain are highly susceptible to sound-induced seizures at 21 days of age; whereas, mice of the C57BL/6 (B6) strain are resistant to these seizures. Although the difference in susceptibility to audiogenic seizures (ASs) between these two strains is inherited as a multiple-factor trait, an association was observed between susceptibility to ASs and the Ah locus. The Ah locus controls the inducibility of aryl hydrocarbon hydroxylase (AHH) activity by a number of aromatic hydrocarbons. B6 mice carry the Ah^b allele and have inducible AHH activity; whereas, D2 mice carry the Ah^d allele and have noninducible activity. Inducibility is inherited as a Mendelian dominant trait in crosses between these strains. Mice carrying the Ah^b allele are generally less susceptible to ASs at 21 days of age than are mice carrying the Ah^d allele. The combined results from B6 x D2 recombinant inbred strains, congenic strains (where the Ah^b allele was placed into the D2 genome and the Ah^d allele placed into the B6 genome), the B6D2F₁ x D2 backcross generation, and a random survey of various inbred strains, suggest that the association between these two traits is due to genetic linkage, rather than to pleiotrophy or to chance. A major gene that inhibits susceptibility to ASs appears to be closely linked to the Ah locus. This gene has been designated Ias, for inhibition of ASs. A large portion of the genetic variability of AS susceptibility may be due to the segregation of Ias.     

Dysoxylum binectariferum bark as a new source of anticancer drug camptothecin: Bioactivity-guided isolation and LCMS-based quantification

Camptothecin (CPT, 1) is a potent anticancer natural product which led to the discovery of two clinically used anticancer drugs topotecan and irinotecan. These two drugs are semisynthetic analogs of CPT, and thus the commercial production of CPT as a raw material from various plant sources and tissue culture methods is highly demanding. In the present study, the Dysoxylum binectariferum bark, was identified as an alternative source of CPT, through bioassay-guided isolation. The barks showed presence of CPT (1) and its 9-methoxy analog 2, whereas CPT alkaloids were not present in seeds and leaves. This is the first report on isolation of CPT alkaloids from Meliaceae family. An efficient chromatography-free protocol for enrichment and isolation of CPT from D. binectariferum has been established, which was able to enrich CPT up to 21% in the crude extract. The LCMS (MRM)-based quantification method revealed the presence of 0.105% of CPT in dry barks of D. binectariferum. The discovery of CPT from D. binectariferum bark will certainly create a global interest in cultivation of this plant as a new crop for commercial production of CPT. Isolation of anticancer drug CPT from this plant, indicates that along with rohitukine, CPT and 9-methoxy CPT also contributes significantly to the cytotoxicity of D. binectariferum.     

Towards Molecular Tagging of Karnal Bunt Resistance Gene(s) in Wheat

We describe preliminary results of cosegregation of a DNA probe with Karnal bunt resistance genets) in wheat. The tagging strategy utilized 4 uniformly resistant and 13 uniformly susceptible F³ families from a cross of Karnal bunt resistant (HD29) and susceptible (WL711) lines of Triticum aestivum. Bulked DNA of 8–10 plants in each family were tested by gel blot DNA hybridization. Thirty three per cent of the probes (11/33) detected restriction fragment length polymorphism (RFLP) between Karnal bunt resistant and susceptible lines. Probe Cxp1 from chromosome group 6 detected the resistant parent (HD29) polymorphic band in all the resistant family bulks but was missing in 6 uniformly susceptible family bulks. A similar pattern was observed with probe XKsu G34 from 1DL. The intensity of the resistant parent band in the susceptible family bulks was less as compared to the resistant family bulks. Probe Xksu H8 which mapped on chromosomes 2, 3, 5 and 7D on the T. tauschii map did not differentiate resistant family bulks from susceptible family bulks. Our results indicated that one or more resistance genets) are located on chromosome groups 1 and 6 of wheat.     

Adaptation of Musca domestica L. Field Population to Laboratory Breeding Causes Transcriptional Alterations

Background

The housefly, Musca domestica, has developed resistance to most insecticides applied for its control. Expression of genes coding for detoxification enzymes play a role in the response of the housefly when encountered by a xenobiotic. The highest level of constitutive gene expression of nine P450 genes was previously found in a newly-collected susceptible field population in comparison to three insecticide-resistant laboratory strains and a laboratory reference strain.

Results

We compared gene expression of five P450s by qPCR as well as global gene expression by RNAseq in the newly-acquired field population (845b) in generation F₁, F₁₃ and F₂₉ to test how gene expression changes following laboratory adaption. Four (CYP6A1, CYP6A36, CYP6D3, CYP6G4) of five investigated P450 genes adapted to breeding by decreasing expression. CYP6D1 showed higher female expression in F₂₉ than in F₁. For males, about half of the genes accessed in the global gene expression were up-regulated in F₁₃ and F₂₉ in comparison with the F₁ population. In females, 60% of the genes were up-regulated in F₁₃ in comparison with F₁, while 33% were up-regulated in F₂₉. Forty potential P450 genes were identified. In most cases, P450 gene expression was decreased in F₁₃ flies in comparison with F₁. Gene expression then increased from F₁₃ to F₂₉ in males and decreased further in females.

Conclusion

The global gene expression changes massively during adaptation to laboratory breeding. In general, global expression decreased as a result of laboratory adaption in males, while female expression was not unidirectional. Expression of P450 genes was in general down-regulated as a result of laboratory adaption. Expression of hexamerin, coding for a storage protein was increased, while gene expression of genes coding for amylases decreased. This suggests a major impact of the surrounding environment on gene response to xenobiotics and genetic composition of housefly strains.     

In vitro Activity of Moxifloxacin Against 179 Strains of Anaerobic Bacteria Found in Pulmonary Infections

The activity of moxifloxacin (BAY 12-8039), a new 8-methoxyquinolone, was determined using the NCCLS-approved Wadsworth brucella laked blood agar method and compared to the activities of metronidazole, penicillin G, piperacillin/tazobactam and trovafloxacin. Breakpoints used to define susceptible and resistant categories were, respectively: ≤ 8 and ≥ 32 μg/mL for metronidazole, ≤ 2 and ≥ 8 μg/mL for moxifloxacin and trovafloxacin, ≤ 0.5 and ≥ 2 μg/mL for penicillin G and ≤ 32 and ≥128 μg/mL for piperacillin/tazobactam. A total of 179 anaerobic isolates from pulmonary infections were tested. Piperacillin/tazobactam was the most active antimicrobial, inhibiting 99% of strains at the susceptible breakpoint. Ninety-seven percent of these isolates were susceptible to moxifloxacin; 96% to trovafloxacin, 89% to metronidazole and 43% to penicillin G. Geometric mean moxifloxacin MIC values forBacteroides fragilis and the B. fragilis group were 0.5 and 0.8 μg/mL, respectively. Eighty-eight percent of B. fragilis and 100% of other B. fragilis group species were susceptible to both moxifloxacin and trovafloxacin. All of the strains of B. fragilis and most of the other B. fragilis group species were resistant to penicillin G. At least 99% of other Bacteroides species, Prevotella, and Fusobacterium strains were susceptible to moxifloxacin, metronidazole, piperacillin/tazobactam and trovafloxacin (88% were susceptible to trovafloxacin at 2 μg/mL and all were susceptible at 4 μg/mL). The strains of Clostridium difficile andClostridium ramosum found in these specimens were both resistant to penicillin G but susceptible to the other agents. All strains of Peptostreptococcus species were susceptible to all of the agents except penicillin G. Activities of the agents against non-spore-forming Gram-positive rods at the intermediate breakpoint were, respectively, moxifloxacin-100%, metronidazole-49%, penicillin G-86%, piperacillin/tazobactam-100%, and trovafloxacin-97%. The promising in vitro activity of moxifloxacin against anaerobic pulmonary isolates warrants further investigation, including clinical correlation studies.     

Accord insertion in the 5′ flanking region of CYP6G1 confers nicotine resistance in Drosophila melanogaster

What has driven the sweep of the Accord retrotransposon insertion allele of CYP6G1 in the natural populations of Drosophila melanogaster is unknown. Previous studies on the DDT selection hypothesis produced conflicting data. To reexamine the DDT selection hypothesis and search for alternative explanations, we conducted a series of correlation and genetic linkage experiments with eight D. melanogaster natural populations collected from California (CM1, CM2, CM3, and CM7) and Africa (AM2, AM3, AM4, AM7). Diagnostic PCR showed that CM1, CM2, CM7, and AM3 have the Accord insertion in the CYP6G1 locus, whereas the other four strains do not. RT-PCR analysis exhibits a 100% correlation between Accord insertion and CYP6G1 overexpression. However, among the four strains with Accord-mediated CYP6G1 overexpression only CM1 and CM7 are resistant to DDT, and the other two strains (CM2 and AM3), like the four Accord-free strains, are susceptible to DDT. By contrast, all the four strains with Accord-mediated CYP6G1 overexpression are resistant to nicotine, a plant allelochemical. Genetic crosses between DDT resistant and susceptible Accord-insertion strains, as well as crosses between Accord-insertion and Accord-free strains demonstrated that Accord insertion and CYP6G1 overexpression are genetically linked to nicotine resistance rather than DDT resistance. These results suggest that naturally-occurring allelochemicals such as nicotine are the initial driving force for the worldwide prevalence of the Accord insertion allele of CYP6G1 in D. melanogaster natural populations.     

Clonal Population Structure and Specific Genotypes of Multidrug-Resistant Campylobacter coli from Turkeys

Commercial turkey flocks in North Carolina have been found to be colonized frequently with Campylobacter coli strains that are resistant to several antimicrobials (tetracycline, streptomycin, erythromycin, kanamycin, and ciprofloxacin/nalidixic acid). Such strains have been designated multidrug resistant (MDR). However, the population structure of MDR C. coli from turkeys remains poorly characterized. In this study, an analysis of multilocus sequence typing (MLST)-based sequence types (STs) of 59 MDR strains from turkeys revealed that the majority of these strains corresponded to one of 14 different STs, with three STs accounting for 41 (69%) of the strains. The major STs were turkey specific, and most (87%) of the strains with these STs were resistant to the entire panel of antibiotics mentioned above. Some (13%) of the strains with these STs were susceptible to just one or two of the antibiotics in this panel. Further subtyping using fla typing and pulsed-field gel electrophoresis with SmaI and KpnI revealed that the major MDR STs corresponded to strains of related but distinct subtypes, providing evidence for genomic diversification within these STs. These findings suggest that MDR strains of C. coli from turkeys have a clonal population structure characterized by the presence of a relatively small number of clonal groups that appear to be disseminated in the turkey production system. In addition, the observed correlation between STs and the MDR profiles of the microbes indicates that MLST-based typing holds potential for source-tracking applications specific to the animal source (turkeys) and the antimicrobial resistance profile (MDR status) of C. coli.     

Comparative Genome of GK and Wistar Rats Reveals Genetic Basis of Type 2 Diabetes

The Goto-Kakizaki (GK) rat, which has been developed by repeated inbreeding of glucose-intolerant Wistar rats, is the most widely studied rat model for Type 2 diabetes (T2D). However, the detailed genetic background of T2D phenotype in GK rats is still largely unknown. We report a survey of T2D susceptible variations based on high-quality whole genome sequencing of GK and Wistar rats, which have generated a list of GK-specific variations (228 structural variations, 2660 CNV amplification and 2834 CNV deletion, 1796 protein affecting SNVs or indels) by comparative genome analysis and identified 192 potential T2D-associated genes. The genes with variants are further refined with prior knowledge and public resource including variant polymorphism of rat strains, protein-protein interactions and differential gene expression. Finally we have identified 15 genetic mutant genes which include seven known T2D related genes (Tnfrsf1b, Scg5, Fgb, Sell, Dpp4, Icam1, and Pkd2l1) and eight high-confidence new candidate genes (Ldlr, Ccl2, Erbb3, Akr1b1, Pik3c2a, Cd5, Eef2k, and Cpd). Our result reveals that the T2D phenotype may be caused by the accumulation of multiple variations in GK rat, and that the mutated genes may affect biological functions including adipocytokine signaling, glycerolipid metabolism, PPAR signaling, T cell receptor signaling and insulin signaling pathways. We present the genomic difference between two closely related rat strains (GK and Wistar) and narrow down the scope of susceptible loci. It also requires further experimental study to understand and validate the relationship between our candidate variants and T2D phenotype. Our findings highlight the importance of sequenced-based comparative genomics for investigating disease susceptibility loci in inbreeding animal models.     

Effect of temperature and water activity on the production of fumonisins by Aspergillus niger and different Fusariumspecies

Background

Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing.

Results

The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96%) isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78%) fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates), G448E (n = 3), G307S (n = 3), K143R (n = 3) and Y123H, S405F and R467K (each n = 1). DNA sequencing revealed a novel substitution, G450V, in one isolate.

Conclusion

The sensitive RCA assay described here is a simple, robust and rapid (2 h) method for the detection of ERG11 polymorphisms. It showed excellent concordance with ERG11 sequencing and is a potentially valuable tool to track the emergence and spread of azole-resistant C. albicans and to study the epidemiology of ERG11 mutations. The RCA method is applicable to the study of azole resistance in other fungi.     

Expression of Xenobiotic Metabolizing Cytochrome P450 Genes in a Spinosad-Resistant Musca domestica L. Strain

Background

Spinosad is important in pest management strategies of multiple insect pests. However, spinosad resistance is emerging in various pest species. Resistance has in some species been associated with alterations of the target-site receptor, but in others P450s seems to be involved. We test the possible importance of nine cytochrome P450 genes in the spinosad-resistant housefly strain 791spin and investigate the influence of spinosad on P450 expression in four other housefly strains.

Results

Significant differences in P450 expression of the nine P450 genes in the four strains after spinosad treatment were identified in 40% of cases, most of these as induction. The highly expressed CYP4G2 was induced 6.6-fold in the insecticide susceptible WHO-SRS females, but decreased 2-fold in resistant 791spin males. CYP6G4 was constitutively higher expressed in the resistant strain compared to the susceptible strain. Furthermore, CYP6G4 gene expression was increased in susceptible WHO-SRS flies by spinosad while the expression level did not alter significantly in resistant fly strains. Expression of CYP6A1 and male CYP6D3 was constitutively higher in the resistant strain compared to the susceptible. However, in both cases male expression was higher than female expression.

Conclusion

CYP4G2, CYP6A1, CYP6D3 and CYP6G4 have expressions patterns approaching the expectations of a hypothesized sex specific spinosad resistance gene. CYP4G2 fit requirements of a spinosad resistance gene best, making it the most likely candidate. The overall high expression level of CYP4G2 throughout the strains also indicates importance of this gene. However, the data on 791spin are not conclusive concerning spinosad resistance and small contributions from multiple P450s with different enzymatic capabilities could be speculated to do the job in 791spin. Differential expression of P450s between sexes is more a rule than an exception. Noteworthy differences between spinosad influenced expression of P450 genes between a field population and established laboratory strains were shown.