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1.
Gene ; 764: 145081, 2021 Jan 05.
Artigo em Inglês | MEDLINE | ID: mdl-32860897

RESUMO

Metalaxyl is one of the main fungicides used to control pepper blight caused by Phytophthora capsici. Metalaxyl resistance of P. capsici, caused by the long-term intense use of this fungicide, has become one of the most serious challenges facing pest management. In this study, a conserved domain RPOLA-N of the RPA190 gene of P. capsici (RPA190-pc) was identified from the P. capsici SD1-9 strain. The role of the RPA190-pc underlying the metalaxyl resistance of P. capsici was investigated. Three P. capsici mutants, two with downregulated RPA190-pc (SD1-9C-3 and C-4) expression and one showing upregulation (OESD1-9-1), were obtained by Polyethylene Glycol (PEG) mediated protoplast transformations of P. capsici SD1-9. Quantitative real-time reverse transcription PCR results showed that RPA190-pc was downregulated by more than 60% in SD1-9C-3/C-4 and upregulated 3-fold in OESD1-9-1 compared with that of the control strain SD1-9. Evaluation of the metalaxyl resistance of these three transformants showed that the EC50 values of metalaxyl against SD1-9C-3, SD1-9C-4, and OESD1-9-1 were 120.0 µg·mL-1, 24.4 µg·mL-1, and 15573.0 µg·mL-1, respectively, corresponding to 63.3% decrease, 92.5% decrease, and 47.7-fold increase relative to the EC50 value in SD1-9. Compared with SD1-9, the mycelia of transformants SD1-9C-3, SD1-9C-4, and OESD1-9-1 showed more branches and shorter branches; and the transformants had different pathogenicity to different hosts plants. The expression of the candidate gene RPA190-pc during 10 life-history stages was further studied, the results showed that expression level reached a maximum at the zoospores stage, and it gradually increased with the increase of SD1 and SD1-9 infection time of pepper leaves, indicated that RPA190-pc may be related to the growth and pathogenicity of P. capsici. These results indicate that the expression of RPA190-pc is involved in the regulation of P. capsici resistance to metalaxyl.


Assuntos
Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Fungicidas Industriais/farmacologia , Phytophthora/genética , RNA Polimerase I/genética , Alanina/análogos & derivados , Alanina/farmacologia , Capsicum/microbiologia , Regulação da Expressão Gênica no Desenvolvimento , Regulação Fúngica da Expressão Gênica , Genes Fúngicos , Estágios do Ciclo de Vida/genética , Mutação , Micélio/genética , Micélio/crescimento & desenvolvimento , Phytophthora/efeitos dos fármacos , Phytophthora/crescimento & desenvolvimento , Phytophthora/patogenicidade , Doenças das Plantas/microbiologia , Doenças das Plantas/prevenção & controle , Folhas de Planta/microbiologia , Domínios Proteicos/genética , Esporângios/genética , Esporângios/crescimento & desenvolvimento , Virulência/genética
3.
Sci Total Environ ; 740: 140135, 2020 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-32927573

RESUMO

The increasing human population requires ongoing efforts in food production. This is frequently associated with an increased use of agrochemicals, leading to environmental contamination and altering microbial communities, including human fungal pathogens that reside in the environment. Cryptococcus gattii is an environmental yeast and is one of the etiological agents of cryptococcosis. Benomyl (BEN) is a broad-spectrum fungicide used on several crops. To study the effects of agrochemicals on fungal pathogens, we first evaluated the susceptibility of C. gattii to BEN and the interactions with clinical antifungals. Antagonistic interaction between BEN and fluconazole was seen and was strain- and concentration-dependent. We then induced BEN-resistance by culturing strains in increasing drug concentrations. One strain demonstrated to be more resistant and showed increased multidrug efflux pump gene (MDR1) expression and increased rhodamine 6G efflux, leading to cross-resistance between BEN and fluconazole. Morphologically, BEN-adapted cells had a reduced polysaccharide capsule; an increased surface/volume ratio; increased growth rate in vitro and inside macrophages and also higher ability in crossing an in vitro model of blood-brain-barrier. BEN-adapted strain demonstrated to be hypervirulent in mice, leading to severe symptoms of cryptococcosis, early mortality and higher fungal burden in the organs, particularly the brain. The parental strain was avirulent in murine model. In vivo cross-resistance between BEN and fluconazole was observed, with mice infected with the adapted strain unable to present any improvement in survival and behavior when treated with this antifungal. Furthermore, BEN-adapted cells cultured in drug-free media maintained the hypervirulent and cross-resistant phenotype, suggesting a persistent effect of BEN on C. gattii. In conclusion, exposure to BEN induces cross-resistance with fluconazole and increases the virulence of C. gattii. Altogether, our results indicate that agrochemicals may lead to unintended consequences on non-target species and this could result in severe healthy problems worldwide.


Assuntos
Cryptococcus gattii , Fungicidas Industriais/farmacologia , Animais , Antifúngicos , Farmacorresistência Fúngica , Humanos , Camundongos , Testes de Sensibilidade Microbiana
4.
Plant Dis ; 104(11): 2860-2865, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32946349

RESUMO

The current management of Colletotrichum crown rot (CCR) of strawberry, caused by Colletotrichum gloeosporioides sensu lato, relies on the use of a few fungicide classes, particularly QoI fungicides. Since resistance to QoI fungicides has recently been detected, alternative fungicide groups are needed to control this disease. Our objective was to evaluate the efficacy of succinate-dehydrogenase-inhibitor (SDHI) fungicides in managing CCR. Five SDHI fungicides, fluopyram, isofetamid, penthiopyrad, fluxapyroxad, and benzovindiflupyr, were applied 2 days before or 1 day after inoculation of cultivar Strawberry Festival. SDHI treatments were compared with the most common fungicides used for CCR management, i.e., thiophanate-methyl, pyraclostrobin, and captan. Benzovindiflupyr applied 1 day after inoculation was effective in reducing plant mortality and disease development. The baseline sensitivity of C. gloeosporioides isolates was determined in vitro using a spiral gradient dilution assay. The EC50 for benzovindiflupyr and penthiopyrad varied from 0.08 to 1.11 and 0.45 to 3.17 µg/ml, respectively, whereas the other SDHI fungicides did not inhibit fungal growth. If registered, benzovindiflupyr could serve as an alternative to manage CCR in Florida.


Assuntos
Colletotrichum , Fragaria , Fungicidas Industriais , Farmacorresistência Fúngica , Florida , Fungicidas Industriais/farmacologia , Doenças das Plantas , Ácido Succínico
5.
Plant Dis ; 104(11): 2843-2850, 2020 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-32955405

RESUMO

Despite the resistance problems in Monilinia fructicola, demethylation inhibitor fungicides (DMIs) are still effective for the disease management of brown rot in commercial stone fruit orchards in Brazil. This study aims to investigate the sensitivity of M. fructicola isolates and efficiency of DMIs to reduce brown rot. A set of 93 isolates collected from Brazilian commercial orchards were tested for their sensitivities to tebuconazole, propiconazole, prothioconazole, and myclobutanil. The isolates were analyzed separately according to the presence or absence of the G461S mutation in MfCYP51 gene, determined by allele-specific test. The mean EC50 values for G461S mutants and wild-type isolates were respectively 8.443 and 1.13 µg/ml for myclobutanil, 0.236 and 0.026 µg/ml for propiconazole, 0.115 and 0.002 µg/ml for prothioconazole, and 1.482 and 0.096 µg/ml for tebuconazole. The density distribution curves of DMI sensitivity for both genotypes showed that myclobutanil and prothioconazole curves were mostly shifted toward resistance and sensitivity, respectively. Incomplete cross-resistance was detected among propiconazole and tebuconazole in both wild-type (r = 0.45) and G461S (r = 0.38) populations. No cross-sensitivity was observed among wild-type isolates to prothioconazole and the others DMIs tested. Fungicide treatments on detached fruit inoculated with M. fructicola genotypes showed significant DMI efficacy differences when fruit were inoculated with wild-type and G461S isolates. Protective applications with prothioconazole were more effective for control of both G461S and wild-type isolates compared with tebuconazole. Curative applications with tebuconazole were most effective in reducing the incidence and lesion size of G461S isolates. Sporulation occurred only for G461S isolates treated with tebuconazole under curative and preventative treatments. The differences found among the performance of triazoles against M. fructicola isolates will form the basis for recommendations of rational DMI usage to control brown rot in Brazil.


Assuntos
Fungicidas Industriais , Brasil , Desmetilação , Farmacorresistência Fúngica , Frutas , Fungicidas Industriais/farmacologia
6.
J Med Microbiol ; 69(10): 1221-1227, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32894212

RESUMO

This study evaluated the effect of etomidate against biofilms of Candida spp. and analysed through molecular docking the interaction of this drug with ALS3, an important protein for fungal adhesion. Three fluconazole-resistant fungi were used: Candida albicans, Candida parapsilosis and Candida tropicalis. Growing biofilms were exposed to etomidate at 31.25-500 µg ml-1. Then, an ALS3 adhesive protein from C. albicans was analysed through a molecular mapping technique, composed of a sequence of algorithms to perform molecular mapping simulation based on classic force field theory. Etomidate showed antifungal activity against growing biofilms of resistant C. albicans, C. parapsilosis and C. tropicalis at all concentrations used in the study. The etomidate coupling analysis revealed three interactions with the residues of interest compared to hepta-threonine, which remained at the ALS3 site. In addition, etomidate decreased the expression of mannoproteins on the surface of C. albicans. These results revealed that etomidate inhibited the growth of biofilms.


Assuntos
Candida/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Etomidato/farmacologia , Antifúngicos/metabolismo , Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Etomidato/metabolismo , Fluconazol/farmacologia , Proteínas Fúngicas/metabolismo , Humanos , Glicoproteínas de Membrana/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular/métodos
7.
Epidemiol Mikrobiol Imunol ; 69(2): 57-63, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32819104

RESUMO

OBJECTIVES: Intra-abdominal candidiasis (IAC) is an invasive fungal infection representing the most common type of invasive Candida infection in surgical intensive care units (ICUs). Recently, decreased antifungal susceptibility and progressive shift in the aetiology of invasive candidiasis has been observed worldwide. We explored IAC epidemiology in surgical ICU. MATERIAL AND METHODS: We retrospectively reviewed the records of 64 patients with IAC admitted at our surgical ICU over a 4-year period (2013-2016). IAC incidence, microbiological results, antifungal therapy, and mortality were analysed. RESULTS: The cumulative IAC incidence was 18.4 cases per 1000 admissions (2013: 12.6; 2014: 17.7; 2015: 16.8; 2016: 24.5), including hospital-acquired IAC incidence (2013: 9.8; 2014: 13.3; 2015 10.1; 2016: 13.3) and community-acquired IAC incidence (2013: 2.8; 2014: 4.4; 2015: 6.7; 2016: 11.2). Candida albicans represented the most common species (n = 35, 50.0%) followed by Candida glabrata (n = 15, 21.4%), Candida tropicalis (n = 6, 8.6%) and other yeasts (each < 5.0%). Incidence rate of C. albicans (2013: 7(78%); 2014: 10(59%); 2015: 6(35%); 2016: 12(44%)) and incidence rate of C. non-albicans (2013: 2(22%); 2014: 7(41%); 2015: 9(53%); 2016: 14(52%)) were different in trend. All fungal isolates were susceptible to echinocandins, amphotericin B and voriconazole. Regarding fluconazole susceptibility, C. krusei (n = 3) was resistant and C. glabrata (n = 9) was susceptible-dose dependent (SDD). The ratio of SDD C. glabrata isolates to all isolated C. glabrata strains was 9/15 (60%) (2013: 0/2; 2014: 0/2; 2015: 1/3; 2016: 8/8). Decreased fluconazole susceptibility for C. glabrata isolates was reported in both community-acquired IAC (n = 3) and hospital-acquired IAC (n = 6). Overall 30-day mortality rate was 25.0% (16/64). CONCLUSIONS: We have revealed slowly raising of overall IAC incidence, more increasing trend in incidence of community-acquired IAC compared to rather steady incidence of hospital-acquired IAC. During period 2013-2016 we have observed a significant shift in the aetiology of IAC towards an increased proportion of non-albicans Candida species, particularly C. glabrata. Acquired decreased fluconazole susceptibility was related to C. glabrata isolates exclusively. Emergence of decreased antifungal susceptibility has been preceded by increase of non-albicans Candida isolates.


Assuntos
Unidades de Terapia Intensiva , Antifúngicos/farmacologia , Antifúngicos/uso terapêutico , Candida/efeitos dos fármacos , Cuidados Críticos , Farmacorresistência Fúngica/efeitos dos fármacos , Humanos , Testes de Sensibilidade Microbiana , Estudos Retrospectivos
8.
Sci Total Environ ; 747: 141287, 2020 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-32791412

RESUMO

The potential of Ag-NPs to suppress Monilia fructicola isolates and to broaden the effectiveness of fungicides to overcome resistance was tested in vitro and in vivo. Twenty-three M. fructicola isolates were subjected to fungitoxicity screening with a number of fungicides in vitro, which resulted in the detection of 18 isolates resistant to benzimidazoles (BEN-R) thiophanare methyl (TM) and carbendazim (CARB). DNA sequencing revealed the E198A resistance mutation in the ß-tubulin gene, target site of the benzimidazole fungicides in all resistant isolates. Ag-NPs effectively suppressed mycelial growth in both sensitive (BENS) and resistant isolates. The combination of Ag-NPs with TM led to a significantly enhanced fungitoxic effect compared to the individual treatments regardless resistant phenotype (BEN-R/S) both in vitro and when applied on apple fruit. The above observed additive/synergistic action is probably associated with an enhanced Ag-NPs activity/availability as indicated by the positive correlation between Ag-NPs and TM + Ag-NPs treatments. No correlation was found between AgNO3 and Ag-NPs suggesting that difference(s) exist in the fungitoxic mechanism of action between nanoparticles and their ionic counterparts. Synergy observed between Ag-NPs and the oxidative phosphorylation-uncoupler fluazinam (FM) against both resistance phenotypes indicates a possible role of energy (ATP) metabolism in the mode of action of Ag-NPs. Additionally, the role of released silver ions on the fungitoxic action of Ag-NPs against M. fructicola was found to be limited because the combination with NaCl revealed a synergistic rather than the antagonistic effect that would be expected from silver ion binding with chlorine ions. The results of this study suggested that Ag-NPs can be effectively used against M. fructicola and when used in combination with conventional fungicides they could provide the means for countering benzimidazole resistance and at the same time reduce the environmental impact of synthetic fungicides by reducing doses needed for the control of the pathogen.


Assuntos
Ascomicetos , Fungicidas Industriais , Nanopartículas Metálicas , Farmacorresistência Fúngica , Fungicidas Industriais/toxicidade , Nanopartículas Metálicas/toxicidade , Prata/toxicidade
9.
Int J Food Microbiol ; 334: 108799, 2020 Dec 02.
Artigo em Inglês | MEDLINE | ID: mdl-32799117

RESUMO

Aspergillus flavus is a common and ubiquitous fungal species able to colonize several agricultural commodities, in both pre- and post-harvest conditions. This species represents a very harmful plant pathogen for its ability to synthesize aflatoxin B1, responsible for human primary hepatocellular carcinoma and classified as a group I (human carcinogenic) by the International Agency for Research on Cancer. Several approaches have been proposed to control A. flavus development and related aflatoxin production in field and storage conditions. The Succinate Dehydrogenase Inhibitor (SDHI) fungicide boscalid has been shown to control A. flavus growth and aflatoxin contamination both in vitro and in field experiments. However, this compound is classified as medium-high risk fungicide for triggering fungal resistance and, indeed, resistant strains can occur on crops treated with boscalid. In this paper, we selected laboratory A. flavus strains resistant to boscalid grown on agar medium containing 50 mg/L of boscalid. In order to investigate the molecular mechanism responsible for the resistant phenotype, specific primer pairs were designed to amplify the whole SdhB, SdhC and SdhD genes. By amino acid sequence analysis, two point mutations, Tyrosine replacing Histidine at codon 249 of SdhB (H249Y) and Arginine replacing Glycine at codon 91 of SdhC (G91R), were identified. The effect of SDHI boscalid and isopyrazam on mycelial growth and conidial germination was evaluated. Both resistant genotypes showed high resistance (MIC and EC50 > 1000 mg/L) to boscalid. A positive cross-resistance was found between boscalid and isopyrazam. Specific sub-lethal doses of both fungicides (0.5 mg/L of boscalid and 0.01 mg/L of isopyrazam) interfered with the mechanisms associated to pigmentation of colonies. In particular, fungal colonies appeared depigmented lacking the typical A. flavus green colour shown on un-amended fungicide medium. A strict correlation between lack of pigmentation and increasing aflatoxin production was also observed.


Assuntos
Aflatoxinas/biossíntese , Aspergillus flavus/genética , Farmacorresistência Fúngica/genética , Fungicidas Industriais/farmacologia , Succinato Desidrogenase/antagonistas & inibidores , Aspergillus flavus/efeitos dos fármacos , Aspergillus flavus/crescimento & desenvolvimento , Aspergillus flavus/metabolismo , Compostos de Bifenilo/farmacologia , Farmacorresistência Fúngica/efeitos dos fármacos , Mutação , Niacinamida/análogos & derivados , Niacinamida/farmacologia , Norbornanos/farmacologia , Pigmentação/efeitos dos fármacos , Polimorfismo Genético , Pirazóis/farmacologia , Succinato Desidrogenase/genética
10.
PLoS Negl Trop Dis ; 14(8): e0008493, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32845884

RESUMO

The emergence and spread of cryptococcosis caused by the Cryptococcus gattii species complex has become a major public concern worldwide. C. deuterogattii (VGIIa) outbreaks in the Pacific Northwest region demonstrate the expansion of this fungal infection to temperate climate regions. However, infections due to the C. gattii species complex in China have rarely been reported. In this study, we studied eleven clinical strains of the C. gattii species complex isolated from Guangxi, southern China. The genetic identity and variability of these isolates were analyzed via multi-locus sequence typing (MLST), and the phylogenetic relationships among these isolates and global isolates were evaluated. The mating type, physiological features and antifungal susceptibilities of these isolates were also characterized. Among the eleven isolates, six belonged to C. deuterogattii, while five belonged to C. gattii sensu stricto. The C. deuterogattii strains from Guangxi, southern China were genetically variable and clustered with different clinical isolates from Brazil. All strains were MATα, and three C. deuterogattii isolates (GX0104, GX0105 and GX0147) were able to undergo sexual reproduction. Moreover, most strains had capsule and were capable of melanin production when compared to the outbreak strain from Canada. Most isolates were susceptible to antifungal drugs; yet one of eleven immunocompetent patients died of cryptococcal meningitis caused by C. deuterogattii (GX0147). Our study indicated that the highly pathogenic C. deuterogattii may be emerging in southern China, and effective nationwide surveillance of C. gattii species complex infection is necessary.


Assuntos
Criptococose/epidemiologia , Criptococose/parasitologia , Cryptococcus gattii/genética , Adulto , Antifúngicos/farmacologia , China , Cryptococcus gattii/efeitos dos fármacos , Farmacorresistência Fúngica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Filogenia
11.
Rev Chilena Infectol ; 37(3): 219-230, 2020 Jun.
Artigo em Espanhol | MEDLINE | ID: mdl-32853312

RESUMO

The azoles are drugs that inhibit the 14α-sterol-demethylase enzyme preventing the binding of ergosterol, altering the functionality and structure of the fungal cell wall. Especially the group of triazoles: fluconazole, itraconazole, voriconazole, posaconazole and isavuconazole, are a pharmacological alternative for the treatment of the invasive fungal disease, caused by Aspergillus spp, Candida spp, Cryptococcus spp, by emerging pathogens for example, the Mucoral and finally of endemic mycosis as those caused by Histoplasma spp. and Coccidioides spp. The adverse effects of the triazoles are less frequent compared to those caused by amphotericin B, the main ones being hepatics, gastrointestinals and cardiovasculars, such as the prolongation of the QT interval. The pharmacological interactions are common and occur with molecules that use the substrates of the CYP3A4 cytochrome, for example: antiretroviral, anti-tuberculous and immunomodulators. The history, pharmacological characteristics and clinical trials are reviewed.


Assuntos
Azóis/farmacologia , Antifúngicos , Farmacorresistência Fúngica , Fluconazol , Itraconazol , Testes de Sensibilidade Microbiana , Voriconazol
12.
PLoS Genet ; 16(8): e1009005, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32841236

RESUMO

Azole drugs are the most frequently used antifungal agents. The pathogenic yeast Candida glabrata acquires resistance to azole drugs via single amino acid substitution mutations eliciting a gain-of-function (GOF) hyperactive phenotype in the Pdr1 transcription factor. These GOF mutants constitutively drive high transcription of target genes such as the ATP-binding cassette transporter-encoding CDR1 locus. Previous characterization of Pdr1 has demonstrated that this factor is negatively controlled by the action of a central regulatory domain (CRD) of ~700 amino acids, in which GOF mutations are often found. Our earlier experiments demonstrated that a Pdr1 derivative in which the CRD was deleted gave rise to a transcriptional regulator that could not be maintained as the sole copy of PDR1 in the cell owing to its toxically high activity. Using a set of GOF PDR1 alleles from azole-resistant clinical isolates, we have analyzed the mechanisms acting to repress Pdr1 transcriptional activity. Our data support the view that Pdr1-dependent transactivation is mediated by a complex network of transcriptional coactivators interacting with the extreme C-terminal part of Pdr1. These coactivators include but are not limited to the Mediator component Med15A. Activity of this C-terminal domain is controlled by the CRD and requires multiple regions across the C-terminus for normal function. We also provide genetic evidence for an element within the transactivation domain that mediates the interaction of Pdr1 with coactivators on one hand while restricting Pdr1 activity on the other hand. These data indicate that GOF mutations in PDR1 block nonidentical negative inputs that would otherwise restrain Pdr1 transcriptional activation. The strong C-terminal transactivation domain of Pdr1 uses multiple different protein regions to recruit coactivators.


Assuntos
Candida glabrata/efeitos dos fármacos , Candidíase/tratamento farmacológico , Farmacorresistência Fúngica/genética , Proteínas Fúngicas/genética , Antifúngicos/efeitos adversos , Antifúngicos/farmacologia , Azóis/efeitos adversos , Azóis/farmacologia , Candida glabrata/genética , Candida glabrata/patogenicidade , Candidíase/genética , Candidíase/microbiologia , Proteínas de Ligação a DNA , Farmacorresistência Fúngica/efeitos dos fármacos , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Humanos , Ativação Transcricional/efeitos dos fármacos
13.
PLoS One ; 15(8): e0237046, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32817677

RESUMO

Candida africana is a pathogenic species within the Candida albicans species complex. Due to the limited knowledge concerning its prevalence and antifungal susceptibility profiles, a comprehensive study is overdue. Accordingly, we performed a search of the electronic databases for literature published in the English language between 1 January 2001 and 21 March 2020. Citations were screened, relevant articles were identified, and data were extracted to determine overall intra-C. albicans complex prevalence, geographical distribution, and antifungal susceptibility profiles for C. africana. From a total of 366 articles, 41 were eligible for inclusion in this study. Our results showed that C. africana has a worldwide distribution. The pooled intra-C. albicans complex prevalence of C. africana was 1.67% (95% CI 0.98-2.49). Prevalence data were available for 11 countries from 4 continents. Iran (3.02%, 95%CI 1.51-4.92) and Honduras (3.03%, 95% CI 0.83-10.39) had the highest values and Malaysia (0%) had the lowest prevalence. Vaginal specimens were the most common source of C. africana (92.81%; 155 out of 167 isolates with available data). However, this species has also been isolated from cases of balanitis, from patients with oral lesions, and from respiratory, urine, and cutaneous samples. Data concerning the susceptibility of C. africana to 16 antifungal drugs were available in the literature. Generally, the minimum inhibitory concentrations of antifungal drugs against this species were low. In conclusion, C. africana demonstrates geographical variation in prevalence and high susceptibility to antifungal drugs. However, due to the relative scarcity of existing data concerning this species, further studies will be required to establish more firm conclusions.


Assuntos
Candida/efeitos dos fármacos , Candida/genética , Candida/metabolismo , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candida albicans/patogenicidade , Candidíase Vulvovaginal/microbiologia , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Feminino , Humanos , Testes de Sensibilidade Microbiana , Prevalência , Vagina/microbiologia
14.
Pestic Biochem Physiol ; 169: 104646, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32828365

RESUMO

New succinate dehydrogenase inhibitor fungicides (SDHIs), isopyrazam, pyraziflumid and isofetamid were introduced in the Japanese market in 2017-2018 to control powdery mildew on cucumber. SDHI resistance of the disease fungus (Podosphaera xanthii) was first reported during 2008-2009 against boscalid. Then, penthiopyrad which belongs to SDHIs was introduced in 2010, but subsequent monitoring study was not performed. We investigated the sensitivity of P. xanthii field isolates from Ibaraki Prefecture, Japan, to SDHIs and SdhB, SdhC and SdhD gene mutations, using a leaf disc assay and SDH gene analysis. A total of 19 out of the 22 selected isolates showed resistance to SDHIs. The 19 isolates were phenotypically categorized into three types: Resistant I as moderately and Resistant II as highly resistant to penthiopyrad, isopyrazam and pyraziflumid but sensitive to isofetamid and Resistant III as highly resistant to isofetamid but sensitive to the other three SDHIs. SDH gene analysis revealed that Resistant I and III isolates carried a substitution in PxD-S121P and PxC-A86V, respectively. Resistant II carried three different substitutions: PxC-G151R, PxC-G172D, and PxD-H137R. Among 127 isolates sampled from 16 cucumber greenhouses, 54 exhibited Resistant I phenotype and carried only PxD-S121P. Fifty-six isolates exhibited Resistant II and carried PxC-G151R (four isolates), PxC-G172D (24), and PxD-H137R (28). Only two isolates expressed the Resistant III phenotype carrying PxC-A86V. To the best of our knowledge, this is the first report demonstrating cross-resistance patterns and the molecular characterization of SDHIs in P. xanthii.


Assuntos
Cucumis sativus , Succinato Desidrogenase , Farmacorresistência Fúngica , Japão , Doenças das Plantas
15.
Proc Natl Acad Sci U S A ; 117(36): 22473-22483, 2020 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-32848055

RESUMO

Human fungal infections may fail to respond to contemporary antifungal therapies in vivo despite in vitro fungal isolate drug susceptibility. Such a discrepancy between in vitro antimicrobial susceptibility and in vivo treatment outcomes is partially explained by microbes adopting a drug-resistant biofilm mode of growth during infection. The filamentous fungal pathogen Aspergillus fumigatus forms biofilms in vivo, and during biofilm growth it has reduced susceptibility to all three classes of contemporary antifungal drugs. Specific features of filamentous fungal biofilms that drive antifungal drug resistance remain largely unknown. In this study, we applied a fluorescence microscopy approach coupled with transcriptional bioreporters to define spatial and temporal oxygen gradients and single-cell metabolic activity within A. fumigatus biofilms. Oxygen gradients inevitably arise during A. fumigatus biofilm maturation and are both critical for, and the result of, A. fumigatus late-stage biofilm architecture. We observe that these self-induced hypoxic microenvironments not only contribute to filamentous fungal biofilm maturation but also drive resistance to antifungal treatment. Decreasing oxygen levels toward the base of A. fumigatus biofilms increases antifungal drug resistance. Our results define a previously unknown mechanistic link between filamentous fungal biofilm physiology and contemporary antifungal drug resistance. Moreover, we demonstrate that drug resistance mediated by dynamic oxygen gradients, found in many bacterial biofilms, also extends to the fungal kingdom. The conservation of hypoxic drug-resistant niches in bacterial and fungal biofilms is thus a promising target for improving antimicrobial therapy efficacy.


Assuntos
Antifúngicos/farmacologia , Aspergillus fumigatus , Biofilmes/efeitos dos fármacos , Microambiente Celular , Farmacorresistência Fúngica , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/fisiologia , Hipóxia Celular , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/fisiologia , Oxigênio/farmacologia
16.
Yonsei Med J ; 61(8): 698-704, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32734733

RESUMO

PURPOSE: With changing fungal epidemiology and azole resistance in Aspergillus species, identifying fungal species and susceptibility patterns is crucial to the management of aspergillosis and mucormycosis. The objectives of this study were to evaluate performance of panfungal polymerase chain reaction (PCR) assays on formalin-fixed paraffin embedded (FFPE) samples in the identification of fungal species and in the detection of azole-resistance mutations in the Aspergillus fumigatus cyp51A gene at a South Korean hospital. MATERIALS AND METHODS: A total of 75 FFPE specimens with a histopathological diagnosis of aspergillosis or mucormycosis were identified during the 10-year study period (2006-2015). After deparaffinization and DNA extraction, panfungal PCR assays were conducted on FFPE samples for fungal species identification. The identified fungal species were compared with histopathological diagnosis. On samples identified as A. fumigatus, sequencing to identify frequent mutations in the cyp51A gene [tandem repeat 46 (TR46), L98H, and M220 alterations] that confer azole resistance was performed. RESULTS: Specific fungal DNA was identified in 31 (41.3%) FFPE samples, and of these, 16 samples of specific fungal DNA were in accord with a histopathological diagnosis of aspergillosis or mucormycosis; 15 samples had discordant histopathology and PCR results. No azole-mediating cyp51A gene mutation was noted among nine cases of aspergillosis. Moreover, no cyp51A mutations were identified among three cases with history of prior azole use. CONCLUSION: Panfungal PCR assay with FFPE samples may provide additional information of use to fungal species identification. No azole-resistance mediating mutations in the A. fumigatus cyp51A gene were identified among FFPE samples during study period.


Assuntos
Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Azóis/farmacologia , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica/genética , Hospitais , Mutação/genética , Idoso , Idoso de 80 Anos ou mais , Aspergillus fumigatus/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Feminino , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , República da Coreia
17.
PLoS One ; 15(8): e0237263, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32764812

RESUMO

BACKGROUND: Chronic infected wounds are generally difficult to manage and treatment can be particularly challenging in resource-limited settings where diagnostic testing is not readily available. In this study, the epidemiology of microbial pathogens in chronically infected wounds in rural Ghana was assessed to support therapeutic choices for physicians. METHODS: Culture-based bacterial diagnostics including antimicrobial resistance testing were performed on samples collected from patients with chronic wounds at a hospital in Asante Akim North Municipality, Ghana. Fungal detection was performed by broad-range fungal PCR and sequencing of amplicons. RESULTS: In total, 105 patients were enrolled in the study, from which 207 potential bacterial pathogens were isolated. Enterobacteriaceae (n = 84, 41%) constituted the most frequently isolated group of pathogens. On species level, Pseudomonas aeruginosa (n = 50, 24%) and Staphylococcus aureus (n = 28, 14%) were predominant. High resistance rates were documented, comprising 29% methicillin resistance in S. aureus as well as resistance to 3rd generation cephalosporins and fluoroquinolones in 33% and 58% of Enterobacteriaceae, respectively. One P. aeruginosa strain with carbapenem resistance was identified. The most frequently detected fungi were Candida tropicalis. CONCLUSIONS: The pathogen distribution in chronic wounds in rural Ghana matched the internationally observed patterns with a predominance of P. aeruginosa and S. aureus. Very high resistance rates discourage antibiotic therapy but suggest an urgent need for microbiological diagnostic approaches, including antimicrobial resistance testing to guide the management of patients with chronic wounds in Ghana.


Assuntos
Antibacterianos/farmacologia , Bactérias/isolamento & purificação , Fungos/isolamento & purificação , Infecção dos Ferimentos/tratamento farmacológico , Infecção dos Ferimentos/microbiologia , Adulto , Idoso , Animais , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Candida tropicalis/efeitos dos fármacos , Candida tropicalis/isolamento & purificação , Farmacorresistência Bacteriana , Farmacorresistência Fúngica , Feminino , Fungos/efeitos dos fármacos , Gana/epidemiologia , Hospitais de Distrito , Humanos , Masculino , Testes de Sensibilidade Microbiana , Pessoa de Meia-Idade , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/isolamento & purificação , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/isolamento & purificação , Infecção dos Ferimentos/epidemiologia , Adulto Jovem
18.
Appl Environ Microbiol ; 86(17)2020 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-32631859

RESUMO

Understanding how fungicide application practices affect selection for fungicide resistance is imperative for continued sustainable agriculture. Here, we examined the effect of field applications of the succinate dehydrogenase inhibitor (SDHI) fluxapyroxad at different doses and mixtures on the SDHI sensitivity of Venturia inaequalis, the apple scab pathogen. Fungicide applications were part of selection programs involving different doses (high or low) and mixtures (with a second single-site fungicide or a multisite fungicide). These programs were tested in two apple orchards over 4 years to determine potential cumulative selection effects on resistance. Each year after program applications, apple scab lesions were collected, and relative growth assays were conducted to understand shifts in fluxapyroxad sensitivity. After 4 years, there was a trend toward a reduction in sensitivity to fluxapyroxad for most selection programs in comparison to that in the non-selective-pressure control. In most years, the selection program plots treated with low-dose fluxapyroxad applications resulted in a larger number of isolates with reduced sensitivity, supporting the use of higher doses for disease management. Few significant differences (P < 0.05) in fungicide sensitivity were observed between isolates collected from plots where fungicide mixtures were applied compared to that in untreated plots, supporting the use of multiple modes of action in field applications. In all, appropriate doses and mixtures may contribute to increased longevity of SDHI fungicides used on perennial crops like apples.IMPORTANCE Of much debate is the effect of fungicide application dose on resistance development, as fungicide resistance is a critical barrier to effective disease management in agricultural systems. Our field study in apples investigated the effect of fungicide application dose and mixture on the selection of succinate dehydrogenase inhibitor resistance in Venturia inaequalis, a fungal pathogen that causes the economically important disease apple scab. Understanding how to best delay the development of resistance can result in increased efficacy, fewer applications, and sustainable fungicide use. Results from this study may have relevance to other perennial crops that require multiple fungicide applications and that are impacted by the development of resistance.


Assuntos
Amidas/farmacologia , Ascomicetos/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Proteínas Fúngicas/antagonistas & inibidores , Fungicidas Industriais/farmacologia , Succinato Desidrogenase/antagonistas & inibidores , Ascomicetos/genética , Ascomicetos/fisiologia , Relação Dose-Resposta a Droga , Farmacorresistência Fúngica/genética
19.
PLoS One ; 15(7): e0235746, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32678853

RESUMO

Azole resistant fungal infections remain a health problem for the immune compromised. Current therapies are limited due to rises in new resistance mechanisms. Therefore, it is important to identify new drug targets for drug discovery and novel therapeutics. Arv1 (are1 are2 required for viability 1) function is highly conserved between multiple pathogenic fungal species. Candida albicans (C. albicans) cells lacking CaArv1 are azole hypersusceptible and lack virulence. Saccharomyces cerevisiae (S. cerevisiae) Scarv1 cells are also azole hypersusceptible, a phenotype reversed by expression of CaArv1, indicating conservation in the molecular mechanism for azole susceptibility. To define the relationship between Arv1 function and azole susceptibility, we undertook a structure/function analysis of ScArv1. We identified several conserved amino acids within the ScArv1 homology domain (ScAhd) required for maintaining normal azole susceptibility. Erg11 lanosterol 14-α-demethylase is the rate-limiting enzyme in sterol biosynthesis and is the direct target of azole antifungals, so we used our ScArv1 mutants in order to explore the relationship between ScArv1 and ScErg11. Specific ScArv1 mutants ectopically expressed from a low copy plasmid were unable to restore normal azole susceptibility to Scarv1 cells and had reduced Erg11 protein levels. Erg11 protein stability depended on its ability to form a heterodimeric complex with Arv1. Complex formation was required for maintaining normal azole susceptibility. Scarv1 cells expressing orthologous CaArv1 mutants also had reduced CaErg11 levels, were unable to form a CaArv1-CaErg11 complex, and were azole hypersusceptible. Scarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 could not sustain proper levels of the azole resistant CaErg11Y132F F145L protein. Caarv1/Caarv1 cells expressing CaArv1 mutants unable to interact with CaErg11 were found to lack virulence using a disseminated candidiasis mouse model. Expressing CaErg11Y132F F145L did not reverse the lack of virulence. We hypothesize that the role of Arv1 in Erg11-dependent azole resistance is to stabilize Erg11 protein level. Arv1 inhibition may represent an avenue for treating azole resistance.


Assuntos
Candida albicans/patogenicidade , Candidíase/microbiologia , Sistema Enzimático do Citocromo P-450/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Esterol 14-Desmetilase/metabolismo , Virulência , Sequência de Aminoácidos , Animais , Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Sistema Enzimático do Citocromo P-450/genética , Farmacorresistência Fúngica , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Masculino , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Homologia de Sequência , Esterol 14-Desmetilase/genética
20.
Mikrobiyol Bul ; 54(2): 291-305, 2020 Apr.
Artigo em Turco | MEDLINE | ID: mdl-32723284

RESUMO

Aspergillus fumigatus can cause different clinical manifestations including chronic pulmonary infections, as well as invasive aspergillosis which is highly mortal in the immunocompromised host. Azole antifungal drugs, voriconazole in particular, are the first-line recommended therapeutic regimen. Azoles inhibit 14-α demethylase enzyme encoded by the cyp51A gene. In recent years, increased azole resistance is observed among environmental and clinical A.fumigatus isolates. Two different mechanisms have been proposed for the development of resistance. The first one is the triggering of resistance as a result of long-term clinical azole use. Point mutations in cyp51A gene are generally responsible for this type of azole resistance. The second mechanism is incidental environmental azole exposure due to the use of azoles as agricultural fungicides. Contact with azoles for extended periods and at varying concentrations causes selective pressure and mutations on sporulating A.fumigatus. Since the resistant strains may persist in nature, susceptible individuals may be infected by acquisition of these strains from the environment. When genotypically examined, the cyp51A gene of the resistant isolates of environmental origin specifically presents with a tandem repeat in the promoter region in addition to the point mutation in codon 98 (TR34/L98H). The aim of this study was to investigate azole resistance rates in A.fumigatus strains isolated from clinical specimens and landscaping areas around Hacettepe University Faculty of Medicine Hospital by phenotypical and genotypical methods. Agar screening test was used as the initial test to detect azole resistance in isolates identified as A.fumigatus sensu stricto according to thermotolerance test results. For all strains that grew on any of the azole containing plates in agar screening test, minimum inhibitory concentration (MIC) values were determined by "European Committee on Antimicrobial Susceptibilitiy Testing" reference microdilution method for the confirmation of the resistance. In addition, cyp51A gene sequence was investigated in selected isolates and mutation analysis was performed. A total of 483 clinical and 65 environmental A.fumigatus sensu stricto isolates were included in the study. The first group of clinical isolates consisted of 215 strains isolated in 1997-2015, revived from stock and tested. The second group consisted of 268 strains belonging to the time period of 2016-2018, during which routine azole agar screening tests were performed for A.fumigatus isolates. When all isolates (n= 483) were evaluated together, 11 isolates (1 before 2015 and 10 between 2016-2018), were found to be resistant to itraconazole (2.3%). None of the mutations previously reported to be associated with azole resistance in Aspergillus strains that were detected in cyp51A sequence analysis, However, polymorphisms which are not (yet) fully elucidated in relation to the resistance (Y46F, G89G, V172M, T248N, E255D, L358L, K427E, C454C, Y431D and Q141H in one strain) were shown to exist in resistant isolates. These results have shown that the rate of azole resistance among clinical A.fumigatus isolates was low (2.3%) in our center. Further studies are required to demonstrate the possible role of the detected polymorphisms on azole resistance and to clarify other mechanisms related with high azole MIC values. In addition, since high azole resistance has been reported from one region in our country, it has been concluded that multicenter studies are required to determine the azole resistance status and the range for the azole resistance ratio in different regions and to reveal resistance mutations that may be specific to our country.


Assuntos
Antifúngicos , Aspergilose , Aspergillus fumigatus , Farmacorresistência Fúngica , Antifúngicos/farmacologia , Aspergilose/microbiologia , Aspergillus fumigatus/efeitos dos fármacos , Aspergillus fumigatus/genética , Farmacorresistência Fúngica/genética , Microbiologia Ambiental , Humanos , Testes de Sensibilidade Microbiana
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