scholarly journals Evidence for the agricultural origin of antimicrobial resistance in a fungal pathogen of humans

2020 ◽  
Author(s):  
S. Earl Kang ◽  
Leilani G. Sumabat ◽  
Tina Melie ◽  
Brandon Mangum ◽  
Michelle Momany ◽  
...  
Keyword(s):  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Azole Resistance ◽  
Biosynthetic Gene ◽  
Urgent Problem ◽  
Qoi Fungicides ◽  
Azole Antifungals ◽  
Quinone Outside Inhibitor ◽  
Ergosterol Synthesis ◽  
Life Threatening

AbstractResistance to clinical antimicrobials is an urgent problem, reducing our ability to combat deadly pathogens of humans. Azole antimicrobials target ergosterol synthesis and are highly effective against fungal pathogens of both humans and plants leading to their widespread use in clinical and agricultural settings1,2. The fungus Aspergillus fumigatus causes 300,000 life-threatening infections in susceptible human hosts annually and azoles are the most effective treatment3. Resistance to clinical azole antifungals has become a major problem in Europe and India over the last decade, where identical mutations in cyp51A, an ergosterol biosynthetic gene, have been found in strains from both clinical and agricultural settings4. Shared cyp51A genotypes suggest that clinical azole resistance might have had an agricultural origin; however, until now, independent origins of clinical and agricultural mutations could not be ruled out. Here we show that azole-resistant isolates of A. fumigatus from clinical and agricultural settings also carry mutations conferring resistance to quinone outside inhibitor (QoI) fungicides, which are used exclusively in agricultural settings. This is the first report of a clear marker for the agricultural origin of resistance to a clinical antifungal. We anticipate that our work will increase the understanding of interactions between pathogens of plants and pathogens of humans.

scholarly journals TheAspergillus fumigatusDamage Resistance Protein Family Coordinately Regulates Ergosterol Biosynthesis and Azole Susceptibility

mBio ◽  
2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Jinxing Song ◽  
Pengfei Zhai ◽  
Yuanwei Zhang ◽  
Caiyun Zhang ◽  
Hong Sang ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Fungal Pathogens ◽  
Regulatory System ◽  
Ergosterol Biosynthesis ◽  
Heme Binding ◽  
Cytochrome P450 Enzymes ◽  
Azole Antifungals ◽  
Ergosterol Synthesis ◽  
Resistance Protein ◽  
Cytochrome P450 Protein

ABSTRACTErgosterol is a major and specific component of the fungal plasma membrane, and thus, the cytochrome P450 enzymes (Erg proteins) that catalyze ergosterol synthesis have been selected as valuable targets of azole antifungals. However, the opportunistic pathogenAspergillus fumigatushas developed worldwide resistance to azoles largely through mutations in the cytochrome P450 enzyme Cyp51 (Erg11). In this study, we demonstrate that a cytochromeb5-like heme-binding damage resistance protein (Dap) family, comprised of DapA, DapB, and DapC, coordinately regulates the functionality of cytochrome P450 enzymes Erg5 and Erg11 and oppositely affects susceptibility to azoles. The expression of all three genes is induced in an azole concentration-dependent way, and the decreased susceptibility to azoles requires DapA stabilization of cytochrome P450 protein activity. In contrast, overexpression of DapB and DapC causes dysfunction of Erg5 and Erg11, resulting in abnormal accumulation of sterol intermediates and further accentuating the sensitivity of ΔdapAstrains to azoles. The results of exogenous-hemin rescue and heme-binding-site mutagenesis experiments demonstrate that the heme binding of DapA contributes the decreased azole susceptibility, while DapB and -C are capable of reducing the activities of Erg5 and Erg11 through depletion of heme.In vivodata demonstrate that inactivated DapA combined with activated DapB yields anA. fumigatusmutant that is easily treatable with azoles in an immunocompromised mouse model of invasive pulmonary aspergillosis. Compared to the single Dap proteins found inSaccharomyces cerevisiaeandSchizosaccharomyces pombe, we suggest that this complex Dap family regulatory system emerged during the evolution of fungi as an adaptive means to regulate ergosterol synthesis in response to environmental stimuli.IMPORTANCEKnowledge of the ergosterol biosynthesis route in fungal pathogens is useful in the design of new antifungal drugs and could aid in the study of antifungal-drug resistance mechanisms. In this study, we demonstrate that three cytochromeb5-like Dap proteins coordinately regulate the azole resistance and ergosterol biosynthesis catalyzed by cytochrome P450 proteins. Our new insights into the Dap regulatory system in fungal pathogens may have broad therapeutic ramifications beyond their usefulness for classic azole antifungals. Moreover, our elucidation of the molecular mechanism of Dap regulation of cytochrome P450 protein functionality through heme-binding activity may extend beyond the Kingdom Fungi with applicability toward Dap protein regulation of mammalian sterol synthesis.

scholarly journals Downy Mildew from Lake Erie Vineyards is Diverse for the G143A SNP Conferring Resistance to Quinone Outside Inhibitor Fungicides

2013 ◽  
Vol 14 (1) ◽  
pp. 23
Author(s):  
Christopher T. Gee ◽  
Stephanie Chestnut ◽  
Eilene Duberow ◽  
Andrea Collins ◽  
Michael A. Shields
Keyword(s):  
Downy Mildew ◽  
Fungal Pathogens ◽  
Lake Erie ◽  
Single Mode ◽  
Plasmopara Viticola ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Development Of Resistance ◽  
Qualitative Level ◽  
Qoi Resistance

Downy mildew (Plasmopara viticola) is a significant problem in grape vineyards throughout the growing season. Control of downy mildew is carried out with a combination of host tolerance and chemical applications. Even in vineyards planted with very tolerant varieties (e.g., Concord), control is important in years with ideal pathogen conditions. Fungicides with a single mode of action possess a very high potential for the development of resistance. Resistance has been observed often in the Quinone outside inhibitor (QoI) fungicides, such as strobilurins. We ascertained the levels of QoI resistance in downy mildew colonies on diseased leaves using CAPS-PCR to detect the glycine to alanine mutation (G143A) known to confer a qualitative level of resistance in fungal pathogens. Our data uncovered a small percentage of samples that contain G143A, suggesting an overall low level of QoI resistance. The low prevalence of the resistant single nucleotide polymorphism (SNP) suggests that QoI fungicides should remain a viable control mechanism in Lake Erie vineyards. Additionally, it appears that a viticultural region where tolerant hosts predominant and QoI use is minimal, resistance buildup in the pathogen population will be minimal. Accepted for publication 15 January 2013. Published 22 April 2013.

scholarly journals Genetic Analysis of Candida auris Implicates Hsp90 in Morphogenesis and Azole Tolerance and Cdr1 in Azole Resistance

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
pp. e02529-18 ◽  
Author(s):  
Sang Hu Kim ◽  
Kali R. Iyer ◽  
Lakhansing Pardeshi ◽  
José F. Muñoz ◽  
Nicole Robbins ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Filamentous Growth ◽  
Antifungal Drugs ◽  
Clinical Isolates ◽  
Azole Resistance ◽  
Candida Auris ◽  
Content Type ◽  
Insight Into

ABSTRACT Candida auris is an emerging fungal pathogen and a serious global health threat as the majority of clinical isolates display elevated resistance to currently available antifungal drugs. Despite the increased prevalence of C. auris infections, the mechanisms governing drug resistance remain largely elusive. In diverse fungi, the evolution of drug resistance is enabled by the essential molecular chaperone Hsp90, which stabilizes key regulators of cellular responses to drug-induced stress. Hsp90 also orchestrates temperature-dependent morphogenesis in Candida albicans, a key virulence trait. However, the role of Hsp90 in the pathobiology of C. auris remains unknown. In order to study regulatory functions of Hsp90 in C. auris, we placed HSP90 under the control of a doxycycline-repressible promoter to enable transcriptional repression. We found that Hsp90 is essential for growth in C. auris and that it enables tolerance of clinical isolates with respect to the azoles, which inhibit biosynthesis of the membrane sterol ergosterol. High-level azole resistance was independent of Hsp90 but dependent on the ABC transporter CDR1, deletion of which resulted in abrogated resistance. Strikingly, we discovered that C. auris undergoes a morphogenetic transition from yeast to filamentous growth in response to HSP90 depletion or cell cycle arrest but not in response to other cues that induce C. albicans filamentation. Finally, we observed that this developmental transition is associated with global transcriptional changes, including the induction of cell wall-related genes. Overall, this report provides a novel insight into mechanisms of drug tolerance and resistance in C. auris and describes a developmental transition in response to perturbation of a core regulator of protein homeostasis. IMPORTANCE Fungal pathogens pose a serious threat to public health. Candida auris is an emerging fungal pathogen that is often resistant to commonly used antifungal drugs. However, the mechanisms governing drug resistance and virulence in this organism remain largely unexplored. In this study, we adapted a conditional expression system to modulate the transcription of an essential gene, HSP90, which regulates antifungal resistance and virulence in diverse fungal pathogens. We showed that Hsp90 is essential for growth in C. auris and is important for tolerance of the clinically important azole antifungals, which block ergosterol biosynthesis. Further, we established that the Cdr1 efflux transporter regulates azole resistance. Finally, we discovered that C. auris transitions from yeast to filamentous growth in response to Hsp90 inhibition, accompanied by global transcriptional remodeling. Overall, this work provides a novel insight into mechanisms regulating azole resistance in C. auris and uncovers a distinct developmental program regulated by Hsp90.

scholarly journals Mixed Infections and In Vivo Evolution in the Human Fungal Pathogen Cryptococcus neoformans

mBio ◽  
2010 ◽  
Vol 1 (1) ◽  
Author(s):  
Marie Desnos-Ollivier ◽  
Sweta Patel ◽  
Adam R. Spaulding ◽  
Caroline Charlier ◽  
Dea Garcia-Hermoso ◽  
...  
Keyword(s):  
Cryptococcus Neoformans ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Molecular Diversity ◽  
Therapeutic Strategies ◽  
Mixed Infections ◽  
Content Type ◽  
Human Fungal Pathogen ◽  
Life Threatening

ABSTRACTKoch’s postulates are criteria establishing a causal relationship between a microbe and a disease that lead to the assumption that diseases are caused by a single strain or its evolved forms.Cryptococcus neoformansis a life-threatening human fungal pathogen responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis. To assess the molecular diversity of clinical isolates and gain knowledge ofC. neoformansbiology in the host, we analyzed clinical cultures collected during the prospective CryptoA/D study. Using molecular analysis of unpurified isolates, we demonstrated that mixed infections in humans are more common than previously thought, occurring in almost 20% of patients diagnosed with cryptococcosis. These mixed infections are composed of different mating types, serotypes, and/or genotypes. We also identified genetically related haploid and diploid strains in the same patients. Experimental infections and quantitative PCR show that these ploidy changes can result from endoreplication (duplication of DNA content) and that shuttling between haploid and diploid states can occur, suggestingin vivoevolution. Thus, the concept of one strain/one infection does not hold true forC. neoformansand may apply to other environmentally acquired fungal pathogens. Furthermore, the possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.IMPORTANCECryptococcus  neoformansis a life-threatening human fungal pathogen that is present in the environment and is responsible for an estimated 1 million cases of cryptococcosis/year, predominantly meningoencephalitis in HIV-infected patients. To assess the molecular diversity of clinical isolates and gain knowledge ofC. neoformansbiology in the host, we analyzed clinical cultures collected during a prospective study on cryptococcosis. Using molecular analysis of unpurified isolates, we uncovered an unexpectedly high frequency (almost 20%) of mixed infections. We further demonstrated that these mixed infections could result from infestation by multiple strains acquired from the environment. We also made the serendipitous discovery ofin vivoevolution leading to endoreplication of the yeasts within the host. Thus, the concept of one strain causing one infection does not hold true forC. neoformansand potentially for other environmentally acquired fungal pathogens. The possibility of mixed and/or evolving infections should be taken into account when developing therapeutic strategies against these pathogens.

Sensitivity of C. fructicola and C. siamense of peach in China to multiple classes of fungicides and characterization of pyraclostrobin-resistant isolates

Plant Disease ◽  
2021 ◽  
Author(s):  
Hafiz Muhammad Usman ◽  
Qin Tan ◽  
Mohammad Mazharul Karim ◽  
Muhammad Adnan ◽  
Weixiao Yin ◽  
...  
Keyword(s):  
High Resistance ◽  
Mycelial Growth ◽  
Management Strategies ◽  
Cross Resistance ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Dmi Fungicides ◽  
Colletotrichum Gloeosporioides Species Complex ◽  
Cyt B Gene

Anthracnose, mainly caused by Colletotrichum gloeosporioides species complex including C. fructicola and C. siamense, is a devastating disease of peach. The chemical control has been widely used for years and management failures have increased towards commonly used fungicides. Therefore, screening of sensitivity of Colletotrichum spp. to fungicides with different modes of action is needed to make proper management strategies for peach anthracnose. In this study, sensitivity of 80 isolates of C. fructicola and C. siamense was screened for pyraclostrobin, procymidone, prochloraz and fludioxonil based on mycelial growth inhibition at discriminatory doses. Results showed that C. fructicola and C. siamense isolates were highly resistant to procymidone and fludioxonil with 100% resistance frequencies to both fungicides, but sensitive to prochloraz, i.e., no resistant isolates were found. For pyraclostrobin, 74% of C. fructicola isolates showed high resistance and 26 % were low resistant, all of the C. siamense isolates were low resistant. No positive cross-resistance was observed between pyraclostrobin and azoxystrobin, even they are members of the same quinone outside inhibitor (QoI) fungicide group, and between pyraclostrobin and non-QoIs. Resistant isolates to QoI fungicides were evaluated for the fitness penalty. Results showed that no significant differences except for mycelial growth rates were detected between highly resistant and low-resistant isolates of C. fructicola. Molecular characterization of Cyt b gene revealed that the G143A point mutation was the determinant of the high resistance in C. fructicola. This study demonstrated the current resistance status of C. fructicola and C. siamense to different fungicides and their future perspectives. Demethylation inhibitor (DMI) fungicides are the best option among different chemicals to control peach anthracnose in China.

Nanoscale adhesion forces between the fungal pathogen Candida albicans and macrophages

2016 ◽  
Vol 1 (1) ◽  
pp. 69-74 ◽  
Author(s):  
Sofiane El-Kirat-Chatel ◽  
Yves F. Dufrêne
Keyword(s):  
Atomic Force Microscopy ◽  
Candida Albicans ◽  
Immune Cells ◽  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Adhesion Forces ◽  
Force Microscopy ◽  
Atomic Force

We establish atomic force microscopy as a new nanoscopy platform for quantifying the forces between fungal pathogens and immune cells.

Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae

Plant Disease ◽  
2020 ◽  
Author(s):  
Hua Li ◽  
William Barlow ◽  
Ed Dixon ◽  
Bernadette F. Amsden ◽  
Robert Hirsch ◽  
...  
Keyword(s):  
Characteristic Curve ◽  
Point Mutations ◽  
Receiver Operator Characteristic Curve ◽  
Resistance Mutations ◽  
Pcr Assay ◽  
Cytb Sequence ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Tobacco Production ◽  
Moderately Resistant

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for over a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC50). Based on azoxystrobin EC50, isolates were categorized into three discrete groups: high azoxystrobin sensitivity (< 0.08 µg/ml), moderate azoxystrobin sensitivity (0.14 to 0.64 µg/ml) and low azoxystrobin sensitivity (> 1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC50group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1161 nucleotides encoding 386 amino acids. Cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a PCR assay was developed to screen for mutations. Using this assay, 80% (n=99) of testedC. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n=9) carried the G143A mutation and were highly resistant. A receiver operator characteristic curve analysis suggested the PCR assay is a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly-occurring cytb mutation.

scholarly journals Characterization of Quinone Outside Inhibitor Fungicide Resistance in Cercospora sojina and Development of Diagnostic Tools for its Identification

Plant Disease ◽  
2015 ◽  
Vol 99 (4) ◽  
pp. 544-550 ◽  
Author(s):  
F. Zeng ◽  
E. Arnao ◽  
G. Zhang ◽  
G. Olaya ◽  
J. Wullschleger ◽  
...  
Keyword(s):  
Cytochrome B ◽  
Leaf Spot ◽  
Fungicide Resistance ◽  
Cytochrome B Gene ◽  
Diagnostic Tools ◽  
Polymerase Chain Reaction Primer ◽  
Cercospora Sojina ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Frogeye Leaf Spot

Frogeye leaf spot of soybean, caused by the fungus Cercospora sojina, reduces soybean yields in most of the top-producing countries around the world. Control strategies for frogeye leaf spot can rely heavily on quinone outside inhibitor (QoI) fungicides. In 2010, QoI fungicide-resistant C. sojina isolates were identified in Tennessee for the first time. As the target of QoI fungicides, the cytochrome b gene present in fungal mitochondria has played a key role in the development of resistance to this fungicide class. The cytochrome b genes from three QoI-sensitive and three QoI-resistant C. sojina isolates were cloned and sequenced. The complete coding sequence of the cytochrome b gene was identified and found to encode 396 amino acids. The QoI-resistant C. sojina isolates contained the G143A mutation in the cytochrome b gene, a guanidine to cytosine transversion at the second position in codon 143 that causes an amino acid substitution of alanine for glycine. C. sojina-specific polymerase chain reaction primer sets and TaqMan probes were developed to efficiently discriminate QoI-resistant and -sensitive isolates. The molecular basis of QoI fungicide resistance in field isolates of C. sojina was identified as the G143A mutation, and specific molecular approaches were developed to discriminate and to track QoI-resistant and -sensitive isolates of C. sojina.

scholarly journals Prevalence of QoI resistance and mtDNA diversity in the Irish Zymoseptoria tritici population

2019 ◽  
Vol 58 (1) ◽  
pp. 27-33
Author(s):  
S. Kildea ◽  
D.E. Bucar ◽  
F. Hutton ◽  
S. de la Rosa ◽  
T.E. Welch ◽  
...  
Keyword(s):  
Septoria Tritici Blotch ◽  
Septoria Tritici ◽  
Entire Group ◽  
Zymoseptoria Tritici ◽  
Qoi Fungicides ◽  
Quinone Outside Inhibitor ◽  
Strain Collection ◽  
Increased Sensitivity ◽  
Mtdna Diversity

Abstract The emergence and spread of Quinone outside Inhibitor (QoI) fungicide resistance in the Irish Zymoseptoria tritici population in the early 2000s had immediate impacts on the efficacy of the entire group of fungicides for the control of septoria tritici blotch. As a result, a dramatic reduction in the quantities applied to winter wheat occurred in the following seasons. Even in the absence of these fungicides, the frequency of the resistance allele, G143A in the pathogens mtDNA has remained exceptionally high (>97%), and as such, it can be anticipated that continued poor efficacy of current QoI fungicides will be observed. Amongst the isolates with G143A, differences in sensitivity to the QoI pyraclostrobin were observed in vitro. The addition of the alternative oxidase (AOX) inhibitor salicylhydroxamic acid increased sensitivity in these isolates, suggesting some continued impairment of respiration by the QoI fungicides, albeit weak. Interestingly, amongst those tested, the strains from a site with a high frequency of inserts in the MFS1 transporter gene known to enhance QoI efflux did not exhibit this increase in sensitivity. A total of 19 mtDNA haplotypes were detected amongst the 2017 strain collection. Phylogenetic analysis confirmed the suggestion of a common ancestry of all the haplotypes, even though three of the haplotypes contained at least one sensitive strain.

scholarly journals IDENTIFIKASI DAN UJI PATOGENISITAS CENDAWAN ENTOMOPATOGEN LOKAL TERHADAP Leptocorisa oratorius

EUGENIA ◽  
2011 ◽  
Vol 17 (3) ◽  
Author(s):  
Emmy Senewe ◽  
Guntur Manengkey
Keyword(s):  
Fungal Pathogen ◽  
Fungal Pathogens ◽  
Pathogenic Fungus ◽  
Pathogenic Fungi ◽  
North Sulawesi ◽  
Pathogenicity Tests ◽  
Major Pest ◽  
White Mycelium ◽  
Fusarium Sp ◽  
Leptocorisa Oratorius

ABSTRACT Leptocorisa oratorius is one major pest of rice in North Sulawesi. Hence, it is necessary to control the pest. The research objective was to identify and to test pathogenicity of local  entomopathogen fungi which infected  Leptocorisa oratorius. The pathogens were collected through sampling of L. oratorius which had been infected by the fungi in the field. The pathogenic fungi was isolated using PDA medium, identified followed by inoculation for pathogenecity test.  During several sampling pest, it was found that  L. oratorius was attacked by fungal pathogens in the field. The identification revelead that the fungal pathogens were Beauveria sp and Fusarium sp. Both the fungal pathogen produced white mycelium and could only be distinguished using microscope in the laboratory. Result of pathogenicity tests showed that the two fungal pathogens caused different mortality of the L. oratorius. Mortality of  L. oratorius caused by pathogenic fungus Beauveria sp was  30.3% . Whereas, mortality of  L. oratorius caused by Fusarium sp was only 3.33%. Keywords : pathogenic fungi, entomopathogen, pathogenicity tests, L. oratorius

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