Biofilm Formation and Resistance to Fungicides in Clinically Relevant Members of the Fungal Genus Fusarium

Clinically relevant members of the fungal genus, Fusarium, exhibit an extraordinary genetic diversity and cause a wide spectrum of infections in both healthy individuals and immunocompromised patients. Generally, Fusarium species are intrinsically resistant to all systemic antifungals. We investigated whether the presence or absence of the ability to produce biofilms across and within Fusarium species complexes is linked to higher resistance against antifungals. A collection of 41 Fusarium strains, obtained from 38 patients with superficial and systemic infections, and three infected crops, were tested, including 25 species within the Fusarium fujikuroi species complex, 14 from the Fusarium solani species complex (FSSC), one Fusarium dimerum species complex, and one Fusarium oxysporum species complex isolate. Of all isolates tested, only seven strains from two species of FSSC, five F. petroliphilum and two F. keratoplasticum strains, recovered from blood, nail scrapings, and nasal biopsy samples, could produce biofilms under the tested conditions. In the liquid culture tested, sessile biofilm-forming Fusarium strains exhibited elevated minimum inhibitory concentrations (MICs) for amphotericin B, voriconazole, and posaconazole, compared to their planktonic counterparts, indicating that the ability to form biofilm may significantly increase resistance. Collectively, this suggests that once a surface adherent biofilm has been established, therapies designed to kill planktonic cells of Fusarium are ineffective.

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title: Challenges in Fusarium, a Trans-Kingdom Pathogen
abstract: Fusarium species are emerging human pathogens, next to being plant pathogens. Problems with Fusarium are in their diagnostics and in their difficult treatment, but also in what are actual Fusarium species or rather Fusarium-like species. In this issue Guevara-Suarez et al. (Mycopathologia.  2016) characterized 89 isolates of Fusarium from Colombia showing especially lineages within the Fusarium solani and oxysporum species complexes to be responsible for onychomycosis.

title: Spectrum of Fusarium infections in tropical dermatology evidenced by multilocus sequencing typing diagnostics
abstract: Fusarium species are emerging causative agents of superficial, cutaneous and systemic human infections. In a study of the prevalence and genetic diversity of 464 fungal isolates from a dermatological ward in Thailand, 44 strains (9.5%) proved to belong to the genus Fusarium. Species identification was based on sequencing a portion of translation elongation factor 1‐alpha (tef1‐α), rDNA internal transcribed spacer and RNA‐dependent polymerase subunit II (rpb2). Our results revealed that 37 isolates (84%) belonged to the Fusarium solani species complex (FSSC), one strain matched with Fusarium oxysporum (FOSC) complex 33, while six others belonged to the Fusarium incarnatum‐equiseti species complex. Within the FSSC two predominant clusters represented Fusarium falciforme and recently described F. keratoplasticum. No gender differences in susceptibility to Fusarium were noted, but infections on the right side of the body prevailed. Eighty‐nine per cent of the Fusarium isolates were involved in onychomycosis, while the remaining ones caused paronychia or severe tinea pedis. Comparing literature data, superficial infections by FSSC appear to be prevalent in Asia and Latin America, whereas FOSC is more common in Europe. The available data suggest that Fusarium is a common opportunistic human pathogens in tropical areas and has significant genetic variation worldwide.

title: Fusariosis, a complex infection caused by a high diversity of fungal species refractory to treatment
abstract: In recent years the number of opportunistic invasive fusariosis has increased significantly, the main factors involved in these infections being reviewed here. In spite of the extensive literature published the advances in the management of disseminated fusariosis have been very poor and it remains a severe infection, refractory to treatment and with a high mortality rate. There are no ideal therapies and the presence of neutropenia has a critical part to play in the outcome of the infection. At least 70 species have been involved in fusariosis. Fusarium solani species complex is responsible for nearly 60 % of the cases and F. oxysporum species complex for approximately 20 % of them. Most of the infections are caused by four species, i.e. F. petroliphilum, F. keratoplasticum and other two unnamed phylogenetic species. The efficacy of amphotericin B and voriconazole, the most used antifungal drugs, for treating invasive fusariosis are controversial but in general the percentage of patients cured in the different clinical trials is low. Infections by Fusarium verticillioides seem to have the best prognosis. The recent release of complete genome sequences of the most clinically relevant species and the emergence of fungal genomics offer excellent opportunities for examining the multifactorial processes of Fusarium pathogenicity. Using knockout mutants of genes encoding sequence-specific proteins, several virulence factors have been characterized.

title: Emergence of fusarioses in a university hospital in Turkey during a 20-year period
abstract: Fusarium species have started appearing increasingly as the main cause of infections, particularly in immunocompromised patients. In this study, we aimed to present the first epidemiological data from Turkey, analyze fusariosis cases that have been monitored in a university hospital during the past 20 years, identify the responsible Fusarium species, and determine antifungal susceptibilities. A total of 47 cases of fusariosis was included in the study. Fusarium isolates were identified by multilocus sequence typing (MLST). Antifungal susceptibility was tested by the broth microdilution method according to the Clinical and Laboratory Standards Institute (CLSI) methodology. Of the Fusarium infections, 23.4 % were superficial, 44.7 % were locally invasive, and 31.9 % were disseminated. A significant increase was observed over the years. The Fusarium fujikuroi species complex (FFSC) proved to be the most frequent agent group (17 cases; 51.5 %), followed by the Fusarium solani species complex (FSSC) (14 cases; 42.4 %), the Fusarium dimerum species complex (FDSC), and the Fusarium oxysporum species complexes (FOSC) (one case each). Amphotericin B had the highest in vitro activity against all species. Voriconazole and posaconazole showed interspecies variability across and within Fusarium species complexes. In conclusion, our data support the fact that regional differences exist in the distribution of the Fusarium species and that species-specific differences are observed in antifungal susceptibility patterns. The monitoring of local epidemiological data by determining fungal identity and susceptibility are of importance in guiding the clinical follow-up of patients.

title: Molecular Phylogenetic Diversity of Dermatologic and Other Human Pathogenic Fusarial Isolates from Hospitals in Northern and Central Italy
abstract: Fifty-eight fusaria isolated from 50 Italian patients between 2004 and 2007 were subject to multilocus DNA sequence typing to characterize the spectrum of species and circulating sequence types (STs) associated with dermatological infections, especially onychomycoses and paronychia, and other fusarioses in northern and central Italy. Sequence typing revealed that the isolates were nearly evenly divided among the Fusarium solani species complex (FSSC; n = 18), the F. oxysporum species complex (FOSC; n = 20), and the Gibberella (Fusarium) fujikuroi species complex (GFSC; n = 20). The three-locus typing scheme used for members of the FSSC identified 18 novel STs distributed among six phylogenetically distinct species, yielding an index of discrimination of 1.0. Phylogenetic analysis of the FOSC two-locus data set identified nine STs, including four which were novel, and nine isolates of ST 33, the previously described widespread clonal lineage. With the inclusion of eight epidemiologically unrelated ST 33 isolates, the FOSC typing scheme scored a discrimination index of 0.787. The two-locus GFSC typing scheme, which was primarily designed to identify species, received the lowest discrimination index, with a score of 0.492. The GFSC scheme, however, was used to successfully identify 17 isolates as F. verticillioides, 2 as F. sacchari, and 1 as F. guttiforme. This is the first report that F. guttiforme causes a human mycotic infection, which was supported by detailed morphological analysis. In addition, the results of a pathogenicity experiment revealed that the human isolate of F. guttiforme was able to induce fusariosis of pineapple, heretofore its only known host.

title: In vitro resistance of clinical Fusarium species to amphotericin B and voriconazole using the EUCAST antifungal susceptibility method.
abstract: Susceptibility testing using the EUCAST-AFST method against 39 clinical Fusarium strains consecutively collected from local and invasive infections during the last 10years assessed the in vitro activities of amphotericin B (AmB) and triazole antifungal agents. In addition, the susceptibility pattern of 12 reference strains from the CBS-KNAW Fungal Biodiversity Centre (CBS) was evaluated. In particular Fusarium petroliphilum and F. solani sensu lato were involved in disseminated infections and known for treatment failure. AmB displayed the lowest MICs followed by voriconazole VRC, posaconazole (POC). Itraconazole (ITC) showed high MIC values, displaying in vitro resistance. Clinical isolates were significantly (P <0.05) more resistant to AmB, VRC, and POC, than the CBS reference isolates probably due to previous exposure to antifungal therapy. Resistant profiles to AmB and VRC, which are the currently recommended agents in the guidelines for treatments, and a late diagnosis may be associated with high mortality rate in immunocompromised patients. The present antifungal susceptibility profiles showed that species- and strain-specific differences in antifungal susceptibility exist within Fusarium and that susceptibility testing is important and may improve the prognosis of these infections.

title: In vitro combinations of natamycin with voriconazole, itraconazole and micafungin against clinical Fusarium strains causing keratitis.
abstract: Fusarium species cause a broad spectrum of infections, from superficial to disseminated disease. Because Fusarium species are intrinsically resistant to most antifungal drugs, new approaches are needed. The aim of the present study was to evaluate the in vitro combination of natamycin with currently used antifungal drugs.



The in vitro interactions of combinations between natamycin and voriconazole, itraconazole and micafungin applied to 20 clinical Fusarium strains (members of Fusarium falciforme, Fusarium napiforme, Fusarium petroliphilum, Fusarium proliferatum, Fusarium pseudensiforme and Fusarium sacchari) were evaluated using a chequerboard microdilution method. The MICs of all drugs alone and in combination were determined visually after 48 h and interactions were assessed using fractional inhibitory concentration index (FICI) analysis.



MICs of voriconazole and natamycin alone were 4 to >16 and 4-8 mg/L, respectively. Values were reduced 3.5-10-fold to 0.02-0.5 mg/L and 0.5-5-fold to 0.13-2 mg/L in combination, for the currently used antifungals and natamycin, respectively, demonstrating additive to synergistic interactions. The combinations natamycin/voriconazole, natamycin/itraconazole and natamycin/micafungin were synergistic (FICI ≤0.5) for 70%, 15% and 5% of the strains, respectively. No antagonism was found.



The combination of natamycin with voriconazole was strongly synergistic at clinically achievable serum concentrations.

title: Genotyping of Fusarium Isolates from Onychomycoses in Colombia: Detection of Two New Species Within the Fusarium solani Species Complex and In Vitro Antifungal Susceptibility Testing
abstract: Fusariosis have been increasing in Colombia in recent years, but its epidemiology is poorly known. We have morphologically and molecularly characterized 89 isolates of Fusarium obtained between 2010 and 2012 in the cities of Bogotá and Medellín. Using a multi-locus sequence analysis of rDNA internal transcribed spacer, a fragment of the translation elongation factor 1-alpha (Tef-1α) and of the RNA-dependent polymerase subunit II (Rpb2) genes, we identified the phylogenetic species and circulating haplotypes. Since most of the isolates studied were from onychomycoses (nearly 90 %), we carried out an epidemiological study to determine the risk factors associated with such infections. Five phylogenetic species of the Fusarium solani species complex (FSSC), i.e., F. falciforme, F. keratoplasticum, F. lichenicola, F. petroliphilum, and FSSC 6 as well as two of the Fusarium oxysporum species complex (FOSC), i.e., FOSC 3 and FOSC 4, were identified. The most prevalent species were FOSC 3 (38.2 %) followed by F. keratoplasticum (33.7 %). In addition, our isolates were distributed into 23 haplotypes (14 into FOSC and nine into FSSC). Two of the FSSC phylogenetic species and two haplotypes of FSSC were not described before. Our results demonstrate that recipients of pedicure treatments have a lower probability of acquiring onychomycosis than those not receiving such treatments. The antifungal susceptibility of all the isolates to five clinically available agents showed that amphotericin B was the most active drug, while the azoles exhibited lower in vitro activity.

title: Head-to-Head Comparison of the Activities of Currently Available Antifungal Agents against 3,378 Spanish Clinical Isolates of Yeasts and Filamentous Fungi
abstract: We have compared the activities of posaconazole and other currently available antifungal agents against a collection of 3,378 clinical isolates of yeasts and filamentous fungi. A total of 1,997 clinical isolates of Candida spp., 359 of other yeast species, 697 strains of Aspergillus spp., and 325 nondermatophyte non-Aspergillus spp. were included. The average geometric means of the MICs of agents that were tested against Candida spp. were 0.23 μg/ml for amphotericin B, 0.29 μg/ml for flucytosine, 0.97 μg/ml for fluconazole, 0.07 μg/ml for itraconazole, 0.04 μg/ml for voriconazole, 0.15 μg/ml for caspofungin, and 0.03 μg/ml for posaconazole. Voriconazole and posaconazole were active in vitro against the majority of isolates, with resistance to fluconazole and itraconazole, and against Cryptococcus neoformans and other Basidiomycota yeasts. Posaconazole was the most active of antifungal agents tested against Aspergillus spp., with an average geometric mean of 0.10 μg/ml. It was active against Paecilomyces spp., Penicillium spp., Scedosporium apiospermum, and some black fungi, such as Alternaria spp. Multiresistant filamentous fungi, such as Scedosporium prolificans, Scopulariopsis brevicaulis, and Fusarium solani, were also resistant to voriconazole, caspofungin, and posaconazole. Amphotericin B and posaconazole were found to be active against most of the Mucorales strains tested. Posaconazole and currently available antifungal agents exhibit a potent activity in vitro against the majority of pathogenic fungal species.

title: Antifungal susceptibility for common pathogens of fungal keratitis in Shandong Province, China.
abstract: To analyze common pathogens of fungal keratitis and results of antifungal drug sensitivity test in Shandong Province, China and provide guidance for appropriate choice of antifungal drugs in clinic.



Retrospective, noncomparative study.



The pathogens isolated from 674 fungal keratitis patients between January 1, 2001 and December 31, 2006 were cultured and identified in Shandong Eye Institute, of which some common strains were tested for sensitivity to antifungal drugs.



Fungi were positively cultured in 549 (81.5%) patients, in which the dominating pathogen was genus Fusarium (77.6%), with F. solani (37.3%), F. moniliforme (30.0%), and F. oxysporum (27.9%) being common species; Fusarium was mostly sensitive to natamycin, next to amphotericin B, and then to terbinafin. The second common pathogen was genus Aspergillus (10.8%), in which the main species were A. flavus (49.2%) and A. fumigatus (35.6%); Aspergillus was mostly sensitive to natamycin, next to terbinafin, and then to amphotericin B. Relatively, both Fusarium and Aspergillus were insensitive to ketoconazole, miconazole, itraconazole, fluconazole, and fluorocytosine.



Fusarium is the most common pathogen of fungal keratitis, followed by Aspergillus, in Shandong Province, China. Natamycin is still the first choice in the treatment of hyphomycetic keratitis. Fusarium and Aspergillus are also sensitive to amphotericin B and terbinafin. Early diagnosis and treatments are vital to good prognosis in the treatment of fungal keratitis.

title: Genotyping and In Vitro Antifungal Susceptibility Testing of Fusarium Isolates from Onychomycosis in India
abstract: Onychomycosis refers to fungal infection of the nail and is commonly caused by dermatophytes, while yeasts and non-dermatophytic molds (NDM) are increasingly recognized as pathogens in nail infections. The present study was done to delineate molecular epidemiology of Fusarium onychomycosis in India. Five hundred nail samples of Indian patients clinically suspected of onychomycosis were subjected to direct microscopy and fungal culture. Representative Fusarium isolates were further identified to species level by multi-locus sequencing for internal transcribed spacer, translation elongation factor 1 alpha (tef1-α) and RNA polymerase II subunit (rpb2) regions (primer pairs: ITS1/ITS4, EF1/EF2, 5f2/7cr, respectively). These representative strains were also tested for in vitro antifungal susceptibility by the broth microdilution method. Members of the genus Fusarium proved to be the most common NDM responsible for onychomycosis. The Fusarium spp. responsible for onychomycosis belonged to the Fusarium solani species complex (F. keratoplasticum and F. falciforme) and Fusarium fujikuroi species complex (F. proliferatum, F. acutatum and F. sacchari). Antifungal susceptibility results indicated that amphotericin B was the most effective antifungal across all isolates (MIC ranging 0.5–2 mg/L), followed by voriconazole (MIC ranging 1–8 µg/ml). However, a large variation was shown in susceptibility to posaconazole (MIC ranging 0.5 to >16 µg/ml). To conclude, we identified different Fusarium spp. responsible for onychomycosis in India with variation within species in susceptibility to antifungal agents, showing that fusariosis requires correct and prompt diagnosis as well as antifungal susceptibility testing.

title: Mechanism of Fluconazole Resistance in Candida albicans Biofilms: Phase-Specific Role of Efflux Pumps and Membrane Sterols
abstract: Candida albicans biofilms are formed through three distinct developmental phases and are associated with high fluconazole (FLU) resistance. In the present study, we used a set of isogenic Candida strains lacking one or more of the drug efflux pumps Cdr1p, Cdr2p, and Mdr1p to determine their role in FLU resistance of biofilms. Additionally, variation in sterol profile as a possible mechanism of drug resistance was investigated. Our results indicate that parent and mutant strains formed similar biofilms. However, biofilms formed by double and triple mutants were more susceptible to FLU at 6 h (MIC = 64 and 16 μg/ml, respectively) than the wild-type strain (MIC > 256 μg/ml). At later time points (12 and 48 h), all the strains became resistant to this azole (MIC ≥ 256 μg/ml), indicating lack of involvement of efflux pumps in resistance at late stages of biofilm formation. Northern blot analyses revealed that Candida biofilms expressed CDR and MDR1 genes in all the developmental phases, while planktonic cells expressed these genes only at the 12- and 48-h time points. Functionality of efflux pumps was assayed by rhodamine (Rh123) efflux assays, which revealed significant differences in Rh123 retention between biofilm and planktonic cells at the early phase (P = 0.0006) but not at later stages (12 and 48 h). Sterol analyses showed that ergosterol levels were significantly decreased (P < 0.001) at intermediate and mature phases, compared to those in early-phase biofilms. These studies suggest that multicomponent, phase-specific mechanisms are operative in antifungal resistance of fungal biofilms.

title: Aspergillus fumigatus Forms Biofilms with Reduced Antifungal Drug Susceptibility on Bronchial Epithelial Cells
abstract: Aspergillus fumigatus is a leading cause of death in immunocompromised patients and a frequent colonizer of the respiratory tracts of asthma and cystic fibrosis (CF) patients. Biofilms enable bacteria and yeasts to persist in infections and can contribute to antimicrobial resistance. We investigated the ability of A. fumigatus to form biofilms on polystyrene (PS) and human bronchial epithelial (HBE) and CF bronchial epithelial (CFBE) cells. We developed a novel in vitro coculture model of A. fumigatus biofilm formation on HBE and CFBE cells. Biofilm formation was documented by dry weight, scanning electron microscopy (SEM), and confocal scanning laser microscopy (CSLM). The in vitro antifungal activities of seven antifungal drugs were tested by comparing planktonic and sessile A. fumigatus strains. A. fumigatus formed an extracellular matrix on PS and HBE and CFBE cells as evidenced by increased dry weight, SEM, and CSLM. These biofilms exhibited decreased antifungal drug susceptibility and were adherent to the epithelial cells, with fungi remaining viable throughout 3 days. These observations might have implications for treatment of A. fumigatus colonization in chronic lung diseases and for its potential impact on airway inflammation, damage, and infection.

title: Aspergillus flavus Keratitis: Experience of a Tertiary Eye Clinic in Turkey
abstract: We investigated the clinical and mycological characteristics of four cases of mycotic keratitis caused by Aspergillus flavus that occurred from July 2014 to May 2015 at Çukurova University Hospital, Adana, Turkey. In a 10-month period, a total of 64 corneal smear/scrapings were examined from patients with suspected mycotic keratitis. Fungal cultures were positive in six of these patients, indicating a 9.4% incidence of mycotic keratitis in this region, including four cases of A. flavus and two cases of Fusarium spp. The predisposing factors, clinical presentation, and success of the therapeutic approaches were further evaluated. For all cases, topical voriconazole was the first choice of treatment. Surgical procedures were required to control infection in 3 of the 4 cases, including intrastromal voriconazole injection for two cases and keratoplasty for one case. Predisposing factors included trauma (two cases, 50%), contact lens use (one case, 25%), and previous ocular surgery (one case, 25%). The clinical presentations also differed, including a well-limited ulcer (one case), an ulcer with an irregular feathery margin (one case), and ulcers with satellite lesions (two cases). The mean duration between the time of presentation and definitive diagnosis by culture was 14 days (8–25 days). We observed that A. flavus keratitis can present with different underlying factors and clinical conditions. A combination of antifungal therapy and supportive surgical intervention may resolve infections caused by A. flavus in the cornea.

title: Biofilm Formation by the Fungal PathogenCandida albicans: Development, Architecture, and Drug Resistance
abstract: Biofilms are a protected niche for microorganisms, where they are safe from antibiotic treatment and can create a source of persistent infection. Using two clinically relevant Candida albicans biofilm models formed on bioprosthetic materials, we demonstrated that biofilm formation proceeds through three distinct developmental phases. These growth phases transform adherent blastospores to well-defined cellular communities encased in a polysaccharide matrix. Fluorescence and confocal scanning laser microscopy revealed that C. albicans biofilms have a highly heterogeneous architecture composed of cellular and noncellular elements. In both models, antifungal resistance of biofilm-grown cells increased in conjunction with biofilm formation. The expression of agglutinin-like (ALS) genes, which encode a family of proteins implicated in adhesion to host surfaces, was differentially regulated between planktonic and biofilm-grown cells. The ability ofC. albicans to form biofilms contrasts sharply with that of Saccharomyces cerevisiae, which adhered to bioprosthetic surfaces but failed to form a mature biofilm. The studies described here form the basis for investigations into the molecular mechanisms of Candida biofilm biology and antifungal resistance and provide the means to design novel therapies for biofilm-based infections.

title: Biofilm formation by Aspergillus fumigatus.
abstract: Aspergillus fumigatus is a well adapted, opportunistic fungus that causes a severe and commonly fatal disease, invasive pulmonary aspergillosis (IPA), in highly immunocompromised patients, aspergilloma in patients with lung cavities and allergic bronchopulmonary aspergillosis (ABPA) in hypersensitive individuals. Recent studies have suggested that biofilm formation by A. fumigatus may be one of the most important virulence factors in IPA and aspergilloma. Several fungal constituents may contribute to the formation of biofilm structures on host cells, including cell wall components, secondary metabolites and drug transporters. The biofilm phenotype of the fungus is refractory to most conventional antifungal treatment options. Thus, an in-depth analysis and understanding of A. fumigatus biofilms is necessary to devise newer and better antifungal targets for treating complex A. fumigatus biofilm-associated diseases.

title: Keratitis-associated fungi form biofilms with reduced antifungal drug susceptibility.
abstract: To investigate the biofilm-forming capacity of Fusarium solani, Cladosporium sphaerospermum, and Acremonium implicatum, and the activities of antifungal agents against the three keratitis-associated fungi.



The architecture of biofilms was analyzed using scanning electron microscopy and confocal scanning laser microscopy (CSLM). Susceptibility against six antifungal drugs was measured using the CLSI M38-A method and XTT reduction assay.



Time course analyses of CSLM revealed that biofilm formation occurred in an organized fashion through four distinct developmental phases: adhesion, germling formation, microcolony formation, and biofilm maturation. Scanning electron microscopy revealed that mature biofilms displayed a complex three-dimensional structure, consisting of coordinated network of hyphal structures glued by the extracellular matrix (ECM). The antifungal susceptibility testing demonstrated a time-dependent decrease in efficacy for all six antifungal agents as the complexity of fungal hyphal structures developed. Natamycin (NAT), amphotericin B (AMB), and NAT were the most effective against F. solani, C. sphaerospermum, and A. implicatum biofilm, respectively.



Corneal isolates of F. solani, C. sphaerospermum, and A. implicatum could produce biofilms that were resistant to antifungal agents in vitro.

title: Characterization of fusarium keratitis outbreak isolates: contribution of biofilms to antimicrobial resistance and pathogenesis.
abstract: Fusarium is a major cause of microbial keratitis, and its ability to form biofilms was suggested as a contributing factor in recent outbreaks. We investigated the ability of outbreak Fusarium isolates (F. solani species complex [FSSC] and F. oxysporum species complex [FOSC]) to form biofilms in vitro and in vivo, and evaluated their antifungal susceptibilities.



Biofilm formation was assessed using our in vitro contact lens model and in vivo murine model. Biofilm architecture was assessed using confocal laser scanning microscopy (CLSM). Susceptibility against amphotericin B (AmB), voriconazole (VCZ), and natamycin (NAT) was determined using the CLSI-M38-A2 method and XTT metabolic assay.



FSSC strains formed more biofilms than FOSC, in a strain- and clade-dependent manner. CLSM analyses revealed that "high biofilm forming" (HBF) strains had denser and thicker biofilms than "low biofilm forming" (LBF) strains of both species (thickness 51 vs. 41 μm for FSSC and 61 vs. 45 μm for FOSC strains, P < 0.05 for both comparisons). Fusarium biofilms exhibited species-dependent antifungal susceptibilities (e.g., FSSC biofilms AmB minimal inhibitory concentrations [MIC] ≥16 μg/mL, while NAT or VCZ MICs were 2-8 μg/mL). FSSC-infected mice had severe corneal opacification independent of biofilm thickness, while FOSC infection resulted in moderate corneal opacification. Corneal fungal burden of mice infected with HBF strains was higher than those of the LBF strains. In contrast, the reference ATCC isolate was unable to cause infection.



The ability to form biofilms is a key pathogenicity determinant of Fusarium, irrespective of the thickness of these biofilms. Further studies are warranted to explore this association in greater detail.

title: Fusarium and Candida albicans Biofilms on Soft Contact Lenses: Model Development, Influence of Lens Type, and Susceptibility to Lens Care Solutions
abstract: Fungal keratitis is commonly caused by Fusarium species and less commonly by Candida species. Recent outbreaks of Fusarium keratitis were associated with contact lens wear and with ReNu with MoistureLoc contact lens care solution, and biofilm formation on contact lens/lens cases was proposed to play a role in this outbreak. However, no in vitro model for contact lens-associated fungal biofilm has been developed. In this study, we developed and characterized in vitro models of biofilm formation on various soft contact lenses using three species of Fusarium and Candida albicans. The contact lenses tested were etafilcon A, galyfilcon A, lotrafilcon A, balafilcon A, alphafilcon A, and polymacon. Our results showed that clinical isolates of Fusarium and C. albicans formed biofilms on all types of lenses tested and that the biofilm architecture varied with the lens type. Moreover, differences in hyphal content and architecture were found between the biofilms formed by these fungi. We also found that two recently isolated keratitis-associated fusaria formed robust biofilms, while the reference ATCC 36031 strain (recommended by the International Organization for Standardization guidelines for testing of disinfectants) failed to form biofilm. Furthermore, using the developed in vitro biofilm model, we showed that phylogenetically diverse planktonic fusaria and Candida were susceptible to MoistureLoc and MultiPlus. However, Fusarium biofilms exhibited reduced susceptibility against these solutions in a species- and time-dependent manner. This in vitro model should provide a better understanding of the biology and pathogenesis of lens-related fungal keratitis.

title: Virulence Attributes and Antifungal Susceptibility Profile of Opportunistic Fungi Isolated from Ophthalmic Infections
abstract: Investigations of both virulence factors and antifungal susceptibility profiles are crucial for understanding the pathogenesis and prognosis of ophthalmic mycoses. In this study, we investigated the in vitro antifungal susceptibility of amphotericin B (AMB), voriconazole (VRC), and natamycin (NAT) against a set of 50 fungal isolates obtained from patients with ocular mycoses using the Clinical and Laboratory Standards Institute broth microdilution method. In addition, putative virulence factor, such as secretory phospholipases and proteinases, and biofilm formation activity were analyzed. The geometric means (GMs) of the minimum inhibitory concentrations (MICs) of the antifungals across all isolates were the following (in increasing order): VRC (0.70 μg/mL), AMB (0.81 μg/mL), and NAT (1.05 μg/mL). The highest activity against 14 Aspergillus strains was exhibited by VRC (GM MIC: 0.10 μg/mL), followed by AMB and NAT (GM MICs: 0.21 and 0.27 μg/mL), respectively. However, for 12 Fusarium spp., the GM MIC of VRC (2.66) was higher than those of NAT and AMB (GM MICs 1.3 and 0.8 μg/mL, respectively). Proteinase and phospholipase activity were observed in 30 % and 42 % of the isolates, respectively, whereas only 8 % of the isolates were able to produce biofilms. Phospholipase activity was observed in all Fusarium isolates, but not in any of the Aspergillus isolates. In contrast, biofilm-forming capability was detected in 25 % of the Fusarium isolates, but none of the Aspergillus isolates. The differences in the MICs of AMB, VRC, and NAT, biofilm-forming ability and proteinase and phospholipase activities among the isolates were not significant (p > 0.05). Overall, our study suggests no significant correlation between the antifungal susceptibility profiles and virulence attributes of ocular fungal isolates.

title: Multistate outbreak of Fusarium keratitis associated with use of a contact lens solution
abstract: ContextFusarium keratitis is a serious corneal infection, most commonly associated with corneal injury. Beginning in March 2006, the Centers for Disease Control and Prevention received multiple reports of Fusarium keratitis among contact lens wearers.ObjectiveTo define the specific activities, contact lens hygiene practices, or products associated with this outbreak. A confirmed case was defined as keratitis with illness onset after June 1, 2005, with no history of recent ocular trauma and a corneal culture growing Fusarium species. Data were obtained by patient and ophthalmologist interviews for case patients and neighborhood-matched controls by trained personnel. Available Fusarium isolates from patients' clinical and environmental specimens were genotyped by multilocus sequence typing. Environmental sampling for Fusarium was conducted at a contact lens solution manufacturing plant.Main Outcome MeasuresKeratitis infection with Fusarium species.ResultsAs of June 30, 2006, we identified 164 confirmed case patients in 33 states and 1 US territory. Median age was 41 years (range, 12-83 years). Corneal transplantation was required or planned in 55 (34%). One hundred fifty-four (94%) of the confirmed case patients wore soft contact lenses. Forty-five case patients and 78 controls were included in the case-control study. Case patients were significantly more likely than controls to report using a specific contact lens solution, ReNu with MoistureLoc (69% vs 15%; odds ratio, 13.3; 95% confidence interval, 3.1-119.5). The prevalence of reported use of ReNu MultiPlus solution was similar between case patients and controls (18% vs 20%; odds ratio, 0.7; 95% confidence interval, 0.2-2.8). Fusarium was not recovered from the factory, warehouse, solution filtrate, or unopened solution bottles; production of implicated lots was not clustered in time. Among 39 isolates tested, at least 10 different Fusarium species were identified, comprising 19 unique multilocus genotypes.ConclusionsThe findings from this investigation indicate that this outbreak of Fusarium keratitis was associated with use of ReNu with MoistureLoc contact lens solution. Contact lens users should not use ReNu with MoistureLoc.

title: The Enterococcal Surface Protein, Esp, Is Involved in Enterococcus faecalis Biofilm Formation
abstract: The enterococcal surface protein, Esp, is a high-molecular-weight surface protein of unknown function whose frequency is significantly increased among infection-derived Enterococcus faecalisisolates. In this work, a global structural similarity was found between Bap, a biofilm-associated protein of Staphylococcus aureus, and Esp. Analysis of the relationship between the presence of the Esp-encoding gene (esp) and the biofilm formation capacity in E. faecalis demonstrated that the presence of the esp gene is highly associated (P < 0.0001) with the capacity of E. faecalis to form a biofilm on a polystyrene surface, since 93.5% of the E. faecalis esp-positive isolates were capable of forming a biofilm. Moreover, none of the E. faecalis esp-deficient isolates were biofilm producers. Depending on theE. faecalis isolate, insertional mutagenesis ofesp caused either a complete loss of the biofilm formation phenotype or no apparent phenotypic defect. Complementation studies revealed that Esp expression in an E. faecalis esp-deficient strain promoted primary attachment and biofilm formation on polystyrene and polyvinyl chloride plastic from urine collection bags. Together, these results demonstrate that (i) biofilm formation capacity is widespread among clinical E. faecalis isolates, (ii) the biofilm formation capacity is restricted to the E. faecalis strains harboringesp, and (iii) Esp promotes primary attachment and biofilm formation of E. faecalis on abiotic surfaces.

title: Inhibitory activity of hinokitiol against biofilm formation in fluconazole-resistant Candida species
abstract: The aim of this study was to investigate the ability of hinokitiol to inhibit the formation of Candida biofilms. Biofilm inhibition was evaluated by quantification of the biofilm metabolic activity with XTT assay. Hinokitiol efficiently prevented biofilm formation in both fluconazole-susceptible and fluconazole-resistant strains of Candida species. We determined the expression levels of specific genes previously implicated in biofilm development of C. albicans cells by real-time RT-PCR. The expression levels of genes associated with adhesion process, HWP1 and ALS3, were downregulated by hinokitiol. Transcript levels of UME6 and HGC1, responsible for long-term hyphal maintenance, were also decreased by hinokitiol. The expression level of CYR1, which encodes the component of signaling pathway of hyphal formation-cAMP-PKA was suppressed by hinokitiol. Its upstream general regulator RAS1 was also suppressed by hinokitiol. These results indicate that hinokitiol may have therapeutic potential in the treatment and prevention of biofilm-associated Candida infections.

title: In Vitro Interaction of Voriconazole and Anidulafungin against Triazole-Resistant Aspergillus fumigatus
abstract: Voriconazole is the recommended drug of first choice to treat infections caused by Aspergillus fumigatus. The efficacy of voriconazole might be hampered by the emergence of azole resistance. However, the combination of voriconazole with anidulafungin could improve therapeutic outcomes in azole-resistant invasive aspergillosis (IA). The in vitro interaction between voriconazole and anidulafungin was determined against voriconazole-susceptible and voriconazole-resistant (substitutions in the cyp51A gene, including single point [M220I and G54W] and tandem repeat [34-bp tandem repeat in the promoter region of the cyp51A gene in combination with substitutions at codon L98 and 46-bp tandem repeat in the promoter region of the cyp51A gene in combination with mutation at codons Y121 and T289] mutations) clinical A. fumigatus isolates using a checkerboard microdilution method with spectrophotometric analysis and a viability-based XTT {2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide} assay within 2 h of exposure after 24 and 48 h of incubation at 35°C to 37°C. Fractional inhibitory concentration (FIC) indexes (FICis) were determined using different MIC endpoints and Bliss independence analysis performed based on the response surface calculation of the no-drug interaction. Significant synergistic interactions obtained based on measuring the FIC index were dependent on the MIC endpoint, in which FICs were inversely related to voriconazole and anidulafungin MICs and were influenced by the CYP51A genotype. A statistically significant difference was observed between FIC indexes of isolates harboring tandem repeat mutations and wild-type controls (P = 0.006 by one-way analysis of variance [ANOVA]), indicating that synergy is decreased in azole-resistant strains. Our results indicated that a combination of voriconazole and anidulafungin might be effective against infections caused by both azole-susceptible and azole-resistant A. fumigatus isolates, but the combination could possibly be less effective in voriconazole-resistant strains with high MICs. Studies in vivo and in vitro-in vivo correlation investigations are required to validate the potential synergy of voriconazole and anidulafungin.

title: Cell Density and Cell Aging as Factors Modulating Antifungal Resistance of Candida albicans Biofilms
abstract: Biofilm formation is a major virulence attribute of Candida pathogenicity which contributes to higher antifungal resistance. We investigated the roles of cell density and cellular aging on the relative antifungal susceptibility of planktonic, biofilm, and biofilm-derived planktonic modes of Candida. A reference and a wild-type strain of Candida albicans were used to evaluate the MICs of caspofungin (CAS), amphotericin B (AMB), nystatin (NYT), ketoconazole (KTC), and flucytosine (5FC). Standard, NCCLS, and European Committee on Antibiotic Susceptibility Testing methods were used for planktonic MIC determination. Candida biofilms were then developed on polystyrene wells, and MICs were determined with a standard 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium hydroxide assay. Subsequently, antifungal susceptibility testing was performed for greater inoculum concentrations and 24- and 48-h-old cultures of planktonic Candida. Furthermore, Candida biofilm-derived planktonic cells (BDPC) were also subjected to antifungal susceptibility testing. The MICs for both C. albicans strains in the planktonic mode were low, although on increasing the inoculum concentration (up to 1 × 108 cells/ml), a variable MIC was noted. On the contrary, for Candida biofilms, the MICs of antifungals were 15- to >1,000-fold higher. Interestingly, the MICs for BDPC were lower and were similar to those for planktonic-mode cells, particularly those of CAS and AMB. Our data indicate that higher antifungal resistance of Candida biofilms is an intrinsic feature possibly related to the biofilm architecture rather than cellular density or cellular aging.

title: Biofilm Formation by Pseudallescheria/Scedosporium Species: A Comparative Study
abstract: Pseudallescheria/Scedosporium species are medically important fungi that are present in soil and human impacted areas and capable of causing a wide spectrum of diseases in humans. Although little is known about their pathogenesis, their growth process and infection routes are very similar to those of Aspergillus species, which grow as biofilms in invasive infections. All nine strains tested here displayed the ability to grow as biofilms in vitro and to produce a dense network of interconnected hyphae on both polystyrene and the surfaces of central venous catheters, but with different characteristics. Scedosporium boydii and S. aurantiacum clinical isolates were able to form biofilms faster than the corresponding environmental strains, as evidenced in kinetic assays for S. boydii and CLSM for S. aurantiacum. Biofilms formed by Pseudallescheria/Scedosporium species had significantly higher resistance to the class of antifungal azole than was observed in planktonic cells, indicating a protective role for this structure. In addition, the clinical S. aurantiacum isolate that formed the most robust biofilms was also more virulent in a larvae Galleria mellonella infection model, suggesting that the ability to form biofilms enhances virulence in Pseudallescheria/Scedosporium species.

title: Effect of alkylphospholipids on Candida albicans biofilm formation and maturation.
abstract: The aim of this study was to evaluate miltefosine and four synthetic compounds (TCAN26, TC19, TC106 and TC117) for their in vitro inhibitory activity against Candida albicans planktonic and biofilm cells and investigate whether these compounds are able to inhibit the biofilm formation and to reduce the viability of mature C. albicans biofilm cells.



The XTT reduction assay and transmission and scanning electron microscopy were employed to determine the inhibitory effects of the test compounds in comparison with amphotericin B and fluconazole against both planktonic cells and sessile cells in biofilms.



C. albicans planktonic cells were susceptible to miltefosine, TCAN26 and TC19, all alkylphospholipid compounds. Miltefosine and TCAN26 present a fungicidal activity with similar values of MIC and minimum fungicidal concentration (MFC), ranging from 2 to 8 mg/L. Cell treatment with sub-inhibitory concentrations of alkylphospholipids induced several ultrastructural alterations. In relation to biofilms, miltefosine reduced formation (38%-71%) and mature biofilms viability (32%-44%), at concentrations of 64 mg/L. TCAN26 also reduced biofilm formation (24%-30%) and mature biofilm viability (15%-20%), at concentrations of 64 mg/L. Although amphotericin B reduced biofilm formation similarly to miltefosine (51%-74%), its activity was lower on mature biofilms (24%-30%). Miltefosine antibiofilm activity was significantly higher than amphotericin B, on both formation and mature biofilms (P<0.05 and P<0.0001, respectively). Fluconazole was the least effective compound tested.



Promising antibiofilm activity was displayed by miltefosine and other alkylphosphocholine compounds, which could be considered a putative option for future treatment of candidaemia associated with biofilm formation, although further evaluation in in vivo systems is required.

title: How to build a biofilm: a fungal perspective.
abstract: Biofilms are differentiated masses of microbes that form on surfaces and are surrounded by an extracellular matrix. Fungal biofilms, especially those of the pathogen Candida albicans, are a cause of infections associated with medical devices. Such infections are particularly serious because biofilm cells are relatively resistant to many common antifungal agents. Several in vitro models have been used to elucidate the developmental stages and processes required for C. albicans biofilm formation, and recent studies have begun to define biofilm genetic control. It is clear that cell-substrate and cell-cell interactions, hyphal differentiation and extracellular matrix production are key steps in biofilm development. Drug resistance is acquired early in biofilm formation, and appears to be governed by different mechanisms in early and late biofilms. Quorum sensing might be an important factor in dispersal of biofilm cells. The past two years have seen the emergence of several genomic strategies to uncover global events in biofilm formation and directed studies to understand more specific events, such as hyphal formation, in the biofilm setting.

title: Widespread Occurrence of Diverse Human Pathogenic Types of the Fungus Fusarium Detected in Plumbing Drains
abstract: It has been proposed that plumbing systems might serve as a significant environmental reservoir of human-pathogenic isolates of Fusarium. We tested this hypothesis by performing the first extensive multilocus sequence typing (MLST) survey of plumbing drain-associated Fusarium isolates and comparing the diversity observed to the known diversity of clinical Fusarium isolates. We sampled 471 drains, mostly in bathroom sinks, from 131 buildings in the United States using a swabbing method. We found that 66% of sinks and 80% of buildings surveyed yielded at least one Fusarium culture. A total of 297 isolates of Fusarium collected were subjected to MLST to identify the phylogenetic species and sequence types (STs) of these isolates. Our survey revealed that the six most common STs in sinks were identical to the six most frequently associated with human infections. We speculate that the most prevalent STs, by virtue of their ability to form and grow in biofilms, are well adapted to plumbing systems. Six major Fusarium STs were frequently isolated from plumbing drains within a broad geographic area and were identical to STs frequently associated with human infections.

title: Development of a novel multiplex DNA microarray for Fusarium graminearum and analysis of azole fungicide responses
abstract: BackgroundThe toxigenic fungal plant pathogen Fusarium graminearum compromises wheat production worldwide. Azole fungicides play a prominent role in controlling this pathogen. Sequencing of its genome stimulated the development of high-throughput technologies to study mechanisms of coping with fungicide stress and adaptation to fungicides at a previously unprecedented precision. DNA-microarrays have been used to analyze genome-wide gene expression patterns and uncovered complex transcriptional responses. A recently developed one-color multiplex array format allowed flexible, effective, and parallel examinations of eight RNA samples.ResultsWe took advantage of the 8 × 15 k Agilent format to design, evaluate, and apply a novel microarray covering the whole F. graminearum genome to analyze transcriptional responses to azole fungicide treatment. Comparative statistical analysis of expression profiles uncovered 1058 genes that were significantly differentially expressed after azole-treatment. Quantitative RT-PCR analysis for 31 selected genes indicated high conformity to results from the microarray hybridization. Among the 596 genes with significantly increased transcript levels, analyses using GeneOntology and FunCat annotations detected the ergosterol-biosynthesis pathway genes as the category most significantly responding, confirming the mode-of-action of azole fungicides. Cyp51A, which is one of the three F. graminearum paralogs of Cyp51 encoding the target of azoles, was the most consistently differentially expressed gene of the entire study. A molecular phylogeny analyzing the relationships of the three CYP51 proteins in the context of 38 fungal genomes belonging to the Pezizomycotina indicated that CYP51C (FGSG_11024) groups with a new clade of CYP51 proteins. The transcriptional profiles for genes encoding ABC transporters and transcription factors suggested several involved in mechanisms alleviating the impact of the fungicide. Comparative analyses with published microarray experiments obtained from two different nutritional stress conditions identified subsets of genes responding to different types of stress. Some of the genes that responded only to tebuconazole treatment appeared to be unique to the F. graminearum genome.ConclusionsThe novel F. graminearum 8 × 15 k microarray is a reliable and efficient high-throughput tool for genome-wide expression profiling experiments in fungicide research, and beyond, as shown by our data obtained for azole responses. The array data contribute to understanding mechanisms of fungicide resistance and allow identifying fungicide targets.

title: Transcription Factor CCG-8 as a New Regulator in the Adaptation to Antifungal Azole Stress
abstract: Antifungal azoles are widely used for controlling fungal infections. Fungi are able to change the expression of many genes when they adapt to azole stress, and increased expression of some of these genes can elevate resistance to azoles. However, the regulatory mechanisms behind transcriptional adaption to azoles in filamentous fungi are poorly understood. In this study, we found that deletion of the transcription factor gene ccg-8, which is known to be a clock-controlled gene, made Neurospora crassa hypersensitive to azoles. A comparative genome-wide analysis of the responses to ketoconazole of the wild type and the ccg-8 mutant revealed that the transcriptional responses to ketoconazole of 78 of the 488 transcriptionally ketoconazole-upregulated genes and the 427 transcriptionally ketoconazole-downregulated genes in the wild type were regulated by CCG-8. Ketoconazole sensitivity testing of all available knockout mutants for CCG-8-regulated genes revealed that CCG-8 contributed to azole adaption by regulating the ketoconazole responses of many genes, including the target gene (erg11), an azole transporter gene (cdr4), a hexose transporter gene (hxt13), a stress response gene (locus number NCU06317, named kts-1), two transcription factor genes (NCU01386 [named kts-2] and fsd-1/ndt80), four enzyme-encoding genes, and six unknown-function genes. CCG-8 also regulated phospholipid synthesis in N. crassa in a manner similar to that of its homolog in Saccharomyces cerevisiae, Opi1p. However, there was no cross talk between phospholipid synthesis and azole resistance in N. crassa. CCG-8 homologs are conserved and are common in filamentous fungi. Deletion of the CCG-8 homolog-encoding gene in Fusarium verticillioides (Fvccg-8) also made this fungus hypersensitive to antifungal azoles.

title: Fungal Biofilm Resistance
abstract: Fungal biofilm infections have become increasingly recognised as a significant clinical problem. One of the major reasons behind this is the impact that these have upon treatment, as antifungal therapy often fails and surgical intervention is required. This places a large financial burden on health care providers. This paper aims to illustrate the importance of fungal biofilms, particularly Candida albicans, and discusses some of the key fungal biofilm resistance mechanisms that include, extracellular matrix (ECM), efflux pump activity, persisters, cell density, overexpression of drug targets, stress responses, and the general physiology of the cell. The paper demonstrates the multifaceted nature of fungal biofilm resistance, which encompasses some of the newest data and ideas in the field.

title: Matrix polymers of Candida biofilms and their possible role in biofilm resistance to antifungal agents.
abstract: Extracellular polymeric material (EP), comprising the matrix of Candida albicans biofilms, was isolated and its composition was compared with that of EP obtained from culture supernatants of planktonically grown (suspended) organisms. Both preparations consisted of carbohydrate, protein, phosphorus and hexosamine, but biofilm EP contained significantly less total carbohydrate (41%) and protein (5%) than planktonic EP. It also had a higher proportion of glucose (16%) and contained galactose, suggesting that it might possess components unique to biofilms. To investigate whether the EP matrix plays a role in the resistance of biofilms to antifungal agents, susceptibility profiles of biofilms incubated statically (which have relatively little matrix) were compared with those for biofilms incubated with gentle shaking (which produce much more matrix material). Biofilms grown with or without shaking did not exhibit significant differences in susceptibility to any of the drugs tested, indicating that drug resistance is unrelated to the extent of matrix formation. However, biofilms formed on two different types of polyvinyl chloride catheter, obtained from different manufacturers, showed differences in susceptibility to amphotericin B, suggesting that drug resistance may arise as a result of highly specific, surface-induced gene expression.

title: Candida albicans Biofilms Produce Antifungal-Tolerant Persister Cells
abstract: Fungal pathogens form biofilms that are highly recalcitrant to antimicrobial therapy. The expression of multidrug resistance pumps in young biofilms has been linked to increased resistance to azoles, but this mechanism does not seem to underlie the resistance of mature biofilms that is a model of in vivo infection. The mechanism of drug resistance of mature biofilms remains largely unknown. We report that biofilms formed by the major human pathogen Candida albicans exhibited a strikingly biphasic killing pattern in response to two microbicidal agents, amphotericin B, a polyene antifungal, and chlorhexidine, an antiseptic, indicating that a subpopulation of highly tolerant cells, termed persisters, existed. The extent of killing with a combination of amphotericin B and chlorhexidine was similar to that observed with individually added antimicrobials. Thus, surviving persisters form a multidrug-tolerant subpopulation. Interestingly, surviving C. albicans persisters were detected only in biofilms and not in exponentially growing or stationary-phase planktonic populations. Reinoculation of cells that survived killing of the biofilm by amphotericin B produced a new biofilm with a new subpopulation of persisters. This suggests that C. albicans persisters are not mutants but phenotypic variants of the wild type. Using a stain for dead cells, rare dark cells were visible in a biofilm after amphotericin B treatment, and a bright and a dim population were physically sorted from this biofilm. Only the dim cells produced colonies, showing that this method allows the isolation of yeast persisters. Given that persisters formed only in biofilms, mutants defective in biofilm formation were examined for tolerance of amphotericin B. All of the known mutants affected in biofilm formation were able to produce normal levels of persisters. This finding indicates that attachment rather than formation of a complex biofilm architecture initiates persister formation. Bacteria produce multidrug-tolerant persister cells in both planktonic and biofilm populations, and it appears that yeasts and bacteria have evolved analogous strategies that assign the function of survival to a small part of the population. In bacteria, persisters are dormant cells. It remains to be seen whether attachment initiates dormancy that leads to the formation of fungal persisters. This study suggests that persisters may be largely responsible for the multidrug tolerance of fungal biofilms.

title: Extracellular DNA Release Acts as an Antifungal Resistance Mechanism in Mature Aspergillus fumigatus Biofilms
abstract: Aspergillus fumigatus has been shown to form biofilms that are associated with adaptive antifungal resistance mechanisms. These include multidrug efflux pumps, heat shock proteins, and extracellular matrix (ECM). ECM is a key structural and protective component of microbial biofilms and in bacteria has been shown to contain extracellular DNA (eDNA). We therefore hypothesized that A. fumigatus biofilms also possess eDNA as part of the ECM, conferring a functional role. Fluorescence microscopy and quantitative PCR analyses demonstrated the presence of eDNA, which was released phase dependently (8 < 12 < 24 < 48 h). Random amplification of polymorphic DNA (RAPD) PCR showed that eDNA was identical to genomic DNA. Biofilm architectural integrity was destabilized by DNase treatment. Biochemical and transcriptional analyses showed that chitinase activity and mRNA levels of chitinase, a marker of autolysis, were significantly upregulated as the biofilm matured and that inhibition of chitinases affected biofilm growth and stability, indicating mechanistically that autolysis was possibly involved. Finally, using checkerboard assays, it was shown that combinational treatment of biofilms with DNase plus amphotericin B and caspofungin significantly improved antifungal susceptibility. Collectively, these data show that eDNA is an important structural component of A. fumigatus ECM that is released through autolysis, which is important for protection from environmental stresses, including antifungal therapy.