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A new study in PLOS Biology highlights the alarming potential of a pandemic clone of wheat blast disease to evolve fungicide-insensitive variants and argues the urgent need for genomic surveillance and preemptive breeding of resistant wheat.

Aspergillus fungi are the cause of an array of diseases affecting humans, animals and plants. The triazole antifungal agents itraconazole, voriconazole, isavuconazole and posaconazole are treatment options against diseases caused by Aspergillus. However, resistance to azoles has recently emerged as a new therapeutic challenge in six continents. Although de novo azole resistance occurs occasionally in patients during azole therapy, the main burden is the aquisition of resistance through the environment. In this setting, the evolution of resistance is attributed to the widespread use of azole-based fungicides. Although ubiquitously distributed, A. fumigatus is not a phytopathogen. However, agricultural fungicides deployed against plant pathogenic moulds such as Fusarium, Mycospaerella and A. flavus also show activity against A. fumigatus in the environment and exposure of non-target fungi is inevitable. Further, similarity in molecule structure between azole fungicides and antifungal drugs results in cross-resistance of A. fumigatus to medical azoles. Clinical studies have shown that two-thirds of patients with azole-resistant infections had no previous history of azole therapy and high mortality rates between 50% and 100% are reported in azole-resistant invasive aspergillosis. The resistance phenotype is associated with key mutations in the cyp51A gene, including TR34/L98H, TR53 and TR46/Y121F/T289A resistance mechanisms. Early detection of resistance is of paramount importance and if demonstrated, either with susceptibility testing or through molecular analysis, azole monotherapy should be avoided. Liposomal amphotericin B or a combination of voriconazole and an echinocandin are recomended for azole-resistant aspergillosis. This article is part of the themed issue ‘Tackling emerging fungal threats to animal health, food security and ecosystem resilience’.

Background Candida auris is a globally emerging multidrug resistant fungal pathogen causing nosocomial transmission. We report an ongoing outbreak of C. auris in a London cardio-thoracic center between April 2015 and July 2016. This is the first report of C. auris in Europe and the largest outbreak so far. We describe the identification, investigation and implementation of control measures. Methods Data on C. auris case demographics, environmental screening, implementation of infection prevention/control measures, and antifungal susceptibility of patient isolates were prospectively recorded then analysed retrospectively. Speciation of C. aur is was performed by MALDI-TOF and typing of outbreak isolates performed by amplified fragment length polymorphism (AFLP). Results This report describes an ongoing outbreak of 50 C. auris cases over the first 16 month (April 2015 to July 2016) within a single Hospital Trust in London. A total of 44 % ( n  = 22/50) patients developed possible or proven C. auris infection with a candidaemia rate of 18 % ( n  = 9/50). Environmental sampling showed persistent presence of the yeast around bed space areas. Implementation of strict infection and prevention control measures included: isolation of cases and their contacts, wearing of personal protective clothing by health care workers, screening of patients on affected wards, skin decontamination with chlorhexidine, environmental cleaning with chorine based reagents and hydrogen peroxide vapour. Genotyping with AFLP demonstrated that C. auris isolates from the same geographic region clustered. Conclusion This ongoing outbreak with genotypically closely related C. auris highlights the importance of appropriate species identification and rapid detection of cases in order to contain hospital acquired transmission.

Invasive fungal infections pose an important threat to public health and are an under-recognized component of antimicrobial resistance, an emerging crisis worldwide. Across a period of profound global environmental change and expanding at-risk populations, human-infecting pathogenic fungi are evolving resistance to all licensed systemic antifungal drugs. In this Review, we highlight the main mechanisms of antifungal resistance and explore the similarities and differences between bacterial and fungal resistance to antimicrobial control. We discuss the research and innovation topics that are needed for risk reduction strategies aimed at minimizing the emergence of resistance in pathogenic fungi. These topics include links between the environment and One Health, surveillance, diagnostics, routes of transmission, novel therapeutics and methods to mitigate hotspots for fungal adaptation. We emphasize the global efforts required to steward our existing antifungal armamentarium, and to direct the research and development of future therapies and interventions.The impacts of fungal infections on human health are of increasing concern, and resistance of pathogenic fungi to all licensed systemic antifungals has been documented. In this Review, Fisher, Verweij and colleagues discuss the research and innovation topics that are needed to understand and minimize the occurrence and impact of antifungal resistance.

The environmental use of azole fungicides has led to selective sweeps across multiple loci in the Aspergillus fumigatus genome causing the rapid global expansion of a genetically distinct cluster of resistant genotypes. Isolates within this cluster are also more likely to be resistant to agricultural antifungals with unrelated modes of action. Here we show that this cluster is not only multi-azole resistant but has increased propensity to develop resistance to next generation antifungals because of variants in the DNA mismatch repair system. A variant in msh6 -G233A is found almost exclusively within azole resistant isolates harbouring the canonical cyp51A azole resistance allelic variant TR 34 /L98H. Naturally occurring isolates with this msh6 variant display up to 5-times higher rate of mutation, leading to an increased likelihood of evolving resistance to other antifungals. Furthermore, unlike hypermutator strains, the G233A variant conveys no measurable fitness cost and has become globally distributed. Our findings further suggest that resistance to next-generation antifungals is more likely to emerge within organisms that are already multi-azole resistant due to close linkage between TR 34 /L98H and msh6 -G233A, posing a major problem due to the prospect of dual use of novel antifungals in clinical and agricultural settings. Here, Bottery et al show that resistance to next generation antifungals is more likely to occur within azole resistant Aspergillus fumigatus due to the close linkage between a globally distributed azole resistance allele and a DNA repair variant which elevates mutation rates.

Respiratory specimens obtained from patients with chronic forms of aspergillosis contain phenotypic variants of azole-resistant Aspergillus fumigatus (ARAF) that co-exist in the airway. Here we aimed to study whether phenotypic variants of ARAF that co-exist in clinical specimens were genetically distinct. A panel of six phenotypic variants of ARAF cultured from two sputum samples collected from two patients with chronic aspergillosis were included. Preliminary identification of all isolates was obtained using MALDI–ToF mass spectrometry and confirmed by AsperGenius ® real-time PCR assay. Antifungal susceptibility testing was determined using EUCAST E.Def 9.3 microbroth dilution. Genomic DNA libraries were constructed with the Illumina TruSeq Nano kit. Prepared whole-genome libraries were sequenced on an Illumina HiSeq 2500. Whole genome data were converted into presence/absence of a SNP with respect to the Af293 reference genome. Colonies of ARAF that co-existed in one respiratory sample demonstrated marked phenotypic diversity. Two cyp51A polymorphisms were found among azole-resistant isolates: TR 34 /L98H/T289A/I364V/G448S was consistently present in four variants with a pan-azole resistant phenotype and TR 34 /L98H was detected in two variants (itraconazole MIC > 16 mg/L). WGS typing showed that despite marked phenotypic variation, each sample contained a population of highly genetically related azole-resistant A. fumigatus variants. Our SNP analysis suggest that mechanisms additional to genetic-based variation are responsible for phenotypic diversity. Our data demonstrate that the phenotypic variants of ARAF that co-exist in clinical specimens are highly clonal and strongly suggest their origination from a single common ancestor.

Cryptococcal meningitis (CM) causes an estimated 180,000 deaths annually, predominantly in sub-Saharan Africa, where most patients receive fluconazole (FLC) monotherapy. While relapse after FLC monotherapy with resistant strains is frequently observed, the mechanisms and impact of emergence of FLC resistance in human CM are poorly understood. Heteroresistance (HetR) - a resistant subpopulation within a susceptible strain - is a recently described phenomenon in Cryptococcus neoformans (Cn) and Cryptococcus gattii (Cg), the significance of which has not previously been studied in humans. A cohort of 20 patients with HIV-associated CM in Tanzania was prospectively observed during therapy with either FLC monotherapy or in combination with flucytosine (5FC). Total and resistant subpopulations of Cryptococcus spp. were quantified directly from patient cerebrospinal fluid (CSF). Stored isolates underwent whole genome sequencing and phenotypic characterization. Heteroresistance was detectable in Cryptococcus spp. in the CSF of all patients at baseline (i.e., prior to initiation of therapy). During FLC monotherapy, the proportion of resistant colonies in the CSF increased during the first 2 weeks of treatment. In contrast, no resistant subpopulation was detectable in CSF by day 14 in those receiving a combination of FLC and 5FC. Genomic analysis revealed high rates of aneuploidy in heteroresistant colonies as well as in relapse isolates, with chromosome 1 (Chr1) disomy predominating. This is apparently due to the presence on Chr1 of ERG11, which is the FLC drug target, and AFR1, which encodes a drug efflux pump. In vitro efflux levels positively correlated with the level of heteroresistance. Our findings demonstrate for what we believe is the first time the presence and emergence of aneuploidy-driven FLC heteroresistance in human CM, association of efflux levels with heteroresistance, and the successful suppression of heteroresistance with 5FC/FLC combination therapy. This work was supported by the Wellcome Trust Strategic Award for Medical Mycology and Fungal Immunology 097377/Z/11/Z and the Daniel Turnberg Travel Fellowship.

Jasmonic acid (JA) is a critical hormonal regulator of plant growth and defense. To advance our understanding of the architecture and dynamic regulation of the JA gene regulatory network, we performed a high-resolution RNA-seq time series of methyl JA-treated Arabidopsis thaliana at 15 time points over a 16-h period. Computational analysis showed that methyl JA (MeJA) induces a burst of transcriptional activity, generating diverse expression patterns over time that partition into distinct sectors of the JA response targeting specific biological processes. The presence of transcription factor (TF) DNA binding motifs correlated with specific TF activity during temporal MeJA-induced transcriptional reprogramming. Insight into the underlying dynamic transcriptional regulation mechanisms was captured in a chronological model of the JA gene regulatory network. Several TFs, including MYB59 and bHLH27, were uncovered as early network components with a role in pathogen and insect resistance. Analysis of subnetworks surrounding the TFs ORA47, RAP2.6L, MYB59, and ANAC055, using transcriptome profiling of overexpressors and mutants, provided insights into their regulatory role in defined modules of the JA network. Collectively, our work illuminates the complexity of the JA gene regulatory network, pinpoints and validates previously unknown regulators, and provides a valuable resource for functional studies on JA signaling components in plant defense and development.

Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection. The emergence of azole resistance in the pathogenic fungus Aspergillus fumigatus has continued to increase, with the dominant resistance mechanisms, consisting of a 34-nucleotide tandem repeat (TR 34 )/L98H and TR 46 /Y121F/T289A, now showing a structured global distribution. Using hierarchical clustering and multivariate analysis of 4,049 A. fumigatus isolates collected worldwide and genotyped at nine microsatellite loci using analysis of short tandem repeats of A. fumigatus (STR Af ), we show that A. fumigatus can be subdivided into two broad clades and that cyp51A alleles TR 34 /L98H and TR 46 /Y121F/T289A are unevenly distributed across these two populations. Diversity indices show that azole-resistant isolates are genetically depauperate compared to their wild-type counterparts, compatible with selective sweeps accompanying the selection of beneficial mutations. Strikingly, we found that azole-resistant clones with identical microsatellite profiles were globally distributed and sourced from both clinical and environmental locations, confirming that azole resistance is an international public health concern. Our work provides a framework for the analysis of A. fumigatus isolates based on their microsatellite profile, which we have incorporated into a freely available, user-friendly R Shiny application (AfumID) that provides clinicians and researchers with a method for the fast, automated characterization of A. fumigatus genetic relatedness. Our study highlights the effect that azole drug resistance is having on the genetic diversity of A. fumigatus and emphasizes its global importance upon this medically important pathogenic fungus. IMPORTANCE Azole drug resistance in the human-pathogenic fungus Aspergillus fumigatus continues to emerge, potentially leading to untreatable aspergillosis in immunosuppressed hosts. Two dominant, environmentally associated resistance mechanisms, which are thought to have evolved through selection by the agricultural application of azole fungicides, are now distributed globally. Understanding the effect that azole resistance is having on the genetic diversity and global population of A. fumigatus will help mitigate drug-resistant aspergillosis and maintain the azole class of fungicides for future use in both medicine and crop protection.

Infections caused by the fungal pathogen Aspergillus fumigatus are increasingly resistant to first-line azole antifungal drugs. However, despite its clinical importance, little is known about how susceptible patients acquire infection from drug-resistant genotypes in the environment. Here, we present a population genomic analysis of 218 A. fumigatus isolates from across the UK and Ireland (comprising 153 clinical isolates from 143 patients and 65 environmental isolates). First, phylogenomic analysis shows strong genetic structuring into two clades (A and B) with little interclade recombination and the majority of environmental azole resistance found within clade A. Second, we show occurrences where azole-resistant isolates of near-identical genotypes were obtained from both environmental and clinical sources, indicating with high confidence the infection of patients with resistant isolates transmitted from the environment. Third, genome-wide scans identified selective sweeps across multiple regions indicating a polygenic basis to the trait in some genetic backgrounds. These signatures of positive selection are seen for loci containing the canonical genes encoding fungicide resistance in the ergosterol biosynthetic pathway, while other regions under selection have no defined function. Lastly, pan-genome analysis identified genes linked to azole resistance and previously unknown resistance mechanisms. Understanding the environmental drivers and genetic basis of evolving fungal drug resistance needs urgent attention, especially in light of increasing numbers of patients with severe viral respiratory tract infections who are susceptible to opportunistic fungal superinfections. Whole-genome sequencing and population genomics of 218 Aspergillus fumigatus environmental and clinical isolates reveals strong genetic clustering and the occurrences of near-identical genotypes, indicating the infection of patients with resistant isolates from the environment.