An In Vitro Co-Culture System for Rapid Differential Response to Fusarium oxysporum f. sp. vasinfectum Race 4 in Three Cotton Cultivars.

Fusarium oxysporum f. sp. vasinfectum race 4 (FOV4) is a devastating fungus pathogen that causes Fusarium wilt in both domesticated cotton species, Gossypium hirsutum (Upland) and G. barbadense (Pima). Greenhouse and field-based pathogenicity assays can be a challenge because of nonuniform inoculum levels, the presence of endophytes, and varying environmental factors. Therefore, an in vitro coculture system was designed to support the growth of both domesticated cotton species and FOV4 via an inert polyphenolic foam substrate with a liquid medium. A Fusarium wilt-susceptible Pima cotton cultivar, G. barbadense 'GB1031'; a highly resistant Pima cotton cultivar, G. barbadense 'DP348RF'; and a susceptible Upland cotton cultivar, G. hirsutum 'TM-1', were evaluated for 30 days during coculture with FOV4 in this foam-based system. Thirty days after inoculation, disease symptoms were more severe in both susceptible cultivars, which displayed higher percentages of foliar damage, and greater plant mortality than observed in 'DP348RF', the resistant Pima cotton cultivar. This foam-based in vitro system may be useful for screening cotton germplasm for resistance to a variety of fungus pathogens and may facilitate the study of biotic interactions in domesticated cotton species under controlled environmental conditions.

Phylogenomic Analysis of a 55.1-kb 19-Gene Dataset Resolves a Monophyletic Fusarium that Includes the Fusarium solani Species Complex.

Scientific communication is facilitated by a data-driven, scientifically sound taxonomy that considers the end-user's needs and established successful practice. In 2013, the Fusarium community voiced near unanimous support for a concept of Fusarium that represented a clade comprising all agriculturally and clinically important Fusarium species, including the F. solani species complex (FSSC). Subsequently, this concept was challenged in 2015 by one research group who proposed dividing the genus Fusarium into seven genera, including the FSSC described as members of the genus Neocosmospora, with subsequent justification in 2018 based on claims that the 2013 concept of Fusarium is polyphyletic. Here, we test this claim and provide a phylogeny based on exonic nucleotide sequences of 19 orthologous protein-coding genes that strongly support the monophyly of Fusarium including the FSSC. We reassert the practical and scientific argument in support of a genus Fusarium that includes the FSSC and several other basal lineages, consistent with the longstanding use of this name among plant pathologists, medical mycologists, quarantine officials, regulatory agencies, students, and researchers with a stake in its taxonomy. In recognition of this monophyly, 40 species described as genus Neocosmospora were recombined in genus Fusarium, and nine others were renamed Fusarium. Here the global Fusarium community voices strong support for the inclusion of the FSSC in Fusarium, as it remains the best scientific, nomenclatural, and practical taxonomic option available.

The genome of opportunistic fungal pathogen Fusarium oxysporum carries a unique set of lineage-specific chromosomes.

Fusarium oxysporum is a cross-kingdom fungal pathogen that infects plants and humans. Horizontally transferred lineage-specific (LS) chromosomes were reported to determine host-specific pathogenicity among phytopathogenic F. oxysporum. However, the existence and functional importance of LS chromosomes among human pathogenic isolates are unknown. Here we report four unique LS chromosomes in a human pathogenic strain NRRL 32931, isolated from a leukemia patient. These LS chromosomes were devoid of housekeeping genes, but were significantly enriched in genes encoding metal ion transporters and cation transporters. Homologs of NRRL 32931 LS genes, including a homolog of ceruloplasmin and the genes that contribute to the expansion of the alkaline pH-responsive transcription factor PacC/Rim1p, were also present in the genome of NRRL 47514, a strain associated with Fusarium keratitis outbreak. This study provides the first evidence, to our knowledge, for genomic compartmentalization in two human pathogenic fungal genomes and suggests an important role of LS chromosomes in niche adaptation.

Characterization of a Francisella tularensis-Caenorhabditis elegans Pathosystem for the Evaluation of Therapeutic Compounds.

Francisella tularensis is a highly infectious Gram-negative intracellular pathogen that causes tularemia. Because of its potential as a bioterrorism agent, there is a need for new therapeutic agents. We therefore developed a whole-animal Caenorhabditis elegans-F. tularensis pathosystem for high-throughput screening to identify and characterize potential therapeutic compounds. We found that the C. elegans p38 mitogen-activate protein (MAP) kinase cascade is involved in the immune response to F. tularensis, and we developed a robust F. tularensis-mediated C. elegans killing assay with a Z' factor consistently of >0.5, which was then utilized to screen a library of FDA-approved compounds that included 1,760 small molecules. In addition to clinically used antibiotics, five FDA-approved drugs were also identified as potential hits, including the anti-inflammatory drug diflunisal that showed anti-F. tularensis activity in vitro Moreover, the nonsteroidal anti-inflammatory drug (NSAID) diflunisal, at 4× MIC, blocked the replication of an F. tularensis live vaccine strain (LVS) in primary human macrophages and nonphagocytic cells. Diflunisal was nontoxic to human erythrocytes and HepG2 human liver cells at concentrations of ≥32 µg/ml. Finally, diflunisal exhibited synergetic activity with the antibiotic ciprofloxacin in both a checkerboard assay and a macrophage infection assay. In conclusion, the liquid C. elegans-F. tularensis LVS assay described here allows screening for anti-F. tularensis compounds and suggests that diflunisal could potentially be repurposed for the management of tularemia.

Fusarium infection: report of 26 cases and review of 97 cases from the literature.

Fusarium species is a ubiquitous fungus that causes opportunistic infections. We present 26 cases of invasive fusariosis categorized according to the European Organization for Research and Treatment of Cancer/Mycoses Study Group (EORTC/MSG) criteria of fungal infections. All cases (20 proven and 6 probable) were treated from January 2000 until January 2010. We also review 97 cases reported since 2000. The most important risk factors for invasive fusariosis in our patients were compromised immune system, specifically lung transplantation (n = 6) and hematologic malignancies (n = 5), and burns (n = 7 patients with skin fusariosis), while the most commonly infected site was the skin in 11 of 26 patients. The mortality rates among our patients with disseminated, skin, and pulmonary fusariosis were 50%, 40%, and 37.5%, respectively. Fusarium solani was the most frequent species, isolated from 49% of literature cases. Blood cultures were positive in 82% of both current study and literature patients with disseminated fusariosis, while the remaining 16% had 2 noncontiguous sites of infection but negative blood cultures. Surgical removal of focal lesions was effective in both current study and literature cases. Skin lesions in immunocompromised patients should raise the suspicion for skin or disseminated fusariosis. The combination of medical monotherapy with voriconazole or amphotericin B and surgery in such cases is highly suggested.

Concepts and principles of photodynamic therapy as an alternative antifungal discovery platform.

Opportunistic fungal pathogens may cause superficial or serious invasive infections, especially in immunocompromised and debilitated patients. Invasive mycoses represent an exponentially growing threat for human health due to a combination of slow diagnosis and the existence of relatively few classes of available and effective antifungal drugs. Therefore systemic fungal infections result in high attributable mortality. There is an urgent need to pursue and deploy novel and effective alternative antifungal countermeasures. Photodynamic therapy (PDT) was established as a successful modality for malignancies and age-related macular degeneration but photodynamic inactivation has only recently been intensively investigated as an alternative antimicrobial discovery and development platform. The concept of photodynamic inactivation requires microbial exposure to either exogenous or endogenous photosensitizer molecules, followed by visible light energy, typically wavelengths in the red/near infrared region that cause the excitation of the photosensitizers resulting in the production of singlet oxygen and other reactive oxygen species that react with intracellular components, and consequently produce cell inactivation and death. Antifungal PDT is an area of increasing interest, as research is advancing (i) to identify the photochemical and photophysical mechanisms involved in photoinactivation; (ii) to develop potent and clinically compatible photosensitizers; (iii) to understand how photoinactivation is affected by key microbial phenotypic elements multidrug resistance and efflux, virulence and pathogenesis determinants, and formation of biofilms; (iv) to explore novel photosensitizer delivery platforms; and (v) to identify photoinactivation applications beyond the clinical setting such as environmental disinfectants.

Polymerase chain reaction-based assays for the diagnosis of invasive fungal infections.

Currently accepted fungal diagnostic techniques, such as culture, biopsy, and serology, lack rapidity and efficiency. Newer diagnostic methods, such as polymerase chain reaction (PCR)-based assays, have the potential to improve fungal diagnostics in a faster, more sensitive, and specific manner. Preliminary data indicate that, when PCR-based fungal diagnostic assays guide antifungal therapy, they may lower patient mortality and decrease unnecessary antifungal treatment, improving treatment-associated costs and avoiding toxicity. Moreover, newer PCR techniques can identify antifungal resistance DNA loci, but the clinical correlation between those loci and clinical failure has to be studied further. In addition, future studies need to focus on the implementation of PCR techniques in clinical decision making and on combining them with other diagnostic tests. A consensus on the standardization of PCR techniques, along with validation from large prospective studies, is necessary to allow widespread adoption of these assays.

The effect of cumulative length of hospital stay on the antifungal resistance of Candida strains isolated from critically ill surgical patients.

Fluconazole is the first line of therapy for the management of candidiasis. However, fluconazole-resistant strains pose an emerging challenge in everyday clinical practice. In this study, we sought to determine whether cumulative length of hospital stay (CLOS) is a predictive factor for the acquisition of non-susceptible Candida strains to fluconazole. Thirty-three critically ill emergency surgery patients with 56 Candida isolates were enrolled in this prospective study. We divided our isolates according to their minimum inhibitory concentration (MIC) to fluconazole using 8 mcg/ml as a cutoff. We then compared the two groups with respect to basic demographics, antifungal agents prescribed, number of wide-spectrum antibiotics, duration of central venous catheter placement, elapsed time to positive culture, duration of prior hospital stay, and length of hospital stay. Non-susceptible fluconazole samples belonged to patients with a significantly longer prior hospital stay and a longer CLOS (P = 0.02 and 0.01, respectively). The difference between the 2 groups regarding non-albicans strains was statistically significant (P < 0.001). By fitting a non-parametric receiver-operating characteristics (ROC) curve into our analysis, a CLOS ≥ 29 days predicted the occurrence of non-susceptible strains with 90% sensitivity and 79.6% specificity (correct classification 81.5%). A CLOS ≥ 29 days is a strong predictor for the isolation of non-susceptible Candida isolates to fluconazole among critically ill emergency surgery patients. Clinicians should consider the duration of previous hospital stay when deciding on empiric antifungal therapy.

Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.