Shielding the Next Generation: Symbiotic Bacteria from a Reproductive Organ Protect Bobtail Squid Eggs from Fungal Fouling.

The importance of defensive symbioses, whereby microbes protect hosts through the production of specific compounds, is becoming increasingly evident. Although defining the partners in these associations has become easier, assigning function to these relationships often presents a significant challenge. Here, we describe a functional role for a bacterial consortium in a female reproductive organ in the Hawaiian bobtail squid, Euprymna scolopes Bacteria from the accessory nidamental gland (ANG) are deposited into the egg jelly coat (JC), where they are hypothesized to play a defensive role during embryogenesis. Eggs treated with an antibiotic cocktail developed a microbial biomass primarily composed of the pathogenic fungus Fusarium keratoplasticum that infiltrated the JC, resulting in severely reduced hatch rates. Experimental manipulation of the eggs demonstrated that the JC was protective against this fungal fouling. A large proportion of the bacterial strains isolated from the ANG or JC inhibited F. keratoplasticum in culture (87.5%), while a similar proportion of extracts from these strains also exhibited antifungal activity against F. keratoplasticum and/or the human-pathogenic yeast Candida albicans (72.7%). Mass spectral network analyses of active extracts from bacterial isolates and egg clutches revealed compounds that may be involved in preventing microbial overgrowth. Several secondary metabolites were identified from ANG/JC bacteria and egg clutches, including the known antimicrobial lincomycin as well as a suite of glycerophosphocholines and mycinamicin-like compounds. These results shed light on a widely distributed but poorly understood symbiosis in cephalopods and offer a new source for exploring bacterial secondary metabolites with antimicrobial activity.IMPORTANCE Organisms must have strategies to ensure successful reproduction. Some animals that deposit eggs protect their embryos from fouling/disease with the help of microorganisms. Although beneficial bacteria are hypothesized to contribute to egg defense in some organisms, the mechanisms of this protection remain largely unknown, with the exception of a few recently described systems. Using both experimental and analytical approaches, we demonstrate that symbiotic bacteria associated with a cephalopod reproductive gland and eggs inhibit fungi. Chemical analyses suggest that these bacteria produce antimicrobial compounds that may prevent overgrowth from fungi and other microorganisms. Given the distribution of these symbiotic glands among many cephalopods, similar defensive relationships may be more common in aquatic environments than previously realized. Such defensive symbioses may also be a rich source for the discovery of new antimicrobial compounds.

Novel Paranannizziopsis species in a Wagler's viper (Tropidolaemus wagleri), tentacled snakes (Erpeton tentaculatum), and a rhinoceros snake (Rhynchophis boulengeri) in a zoological collection.

We report several cases of fungal infections in snakes associated with a new species within the genus Paranannizziopsis. Three juvenile Wagler's vipers (Tropidolaemus wagleri) presented with skin abnormalities or ulcerative dermatitis, and two snakes died. Histologic examination of skin from the living viper revealed hyperplastic, hyperkeratotic, and crusting epidermitis with intralesional fungal elements. The terrestrial Wagler's vipers were housed in a room with fully aquatic tentacled snakes (Erpeton tentaculatum), among which there had been a history of intermittent skin lesions. Approximately 2 months after the biopsy of the viper, a skin sample was collected from one tentacled snake (TS1) with skin abnormalities and revealed a fungal infection with a similar histologic appearance. Fungal isolates were obtained via culture from the Wagler's viper and TS1 and revealed a novel species, Paranannizziopsis tardicrescens, based on phenotypic characterization and molecular analysis. P. tardicrescens was cultured and identified by DNA sequence analysis 8 months later from a dead tentacled snake in an exhibit in an adjacent hallway and 13 months later from a living rhinoceros snake (Rhynchophis boulengeri) with two focal skin lesions. Antifungal susceptibility testing on three of four cultured isolates demonstrated potent in vitro activity for terbinafine and voriconazole.

Species of Aspergillus section Aspergillus from clinical samples in the United States.

The diversity of Aspergillus species in clinical samples is continuously increasing. Species under the former name Eurotium, currently accommodated in section Aspergillus of the genus Aspergillus, are xerophilic fungi widely found in the human environment and able to grow on substrates with low water activity. However, their prevalence in the clinical setting is poorly known. We have studied the presence of these species in a set of clinical samples from the United States using a multilocus sequence analysis based on the internal transcribed spacer (ITS) region of the rRNA, and fragments of the genes ß-tubulin (BenA), calmodulin (CaM), and polymerase II second largest subunit (RPB2). A total of 25 isolates were studied and identified as follows: A. montevidensis (44%), A. chevalieri (36%), A. pseudoglaucus (8%), and A. costiformis (4%). A new species Aspergillus microperforatus is also proposed, which represented 8% of the isolates studied and is characterized by uniseriate conidial heads, subglobose to pyriform vesicles, rough conidia, globose to subglobose cleistothecia, and lenticular and smooth ascospores. The in vitro antifungal activity of eight clinically available antifungals was also determined against these isolates, with the echinocandins and posaconazole having the most potent activity.

New Species Spiromastigoides albida from a Lung Biopsy.

The new species Spiromastigoides albida (Onygenales, Eurotiomycetes, Ascomycota), from a lung biopsy in USA, is proposed and described based on morphological data and the analysis of rRNA, and fragments of actin and ß-tubulin gene sequences. This species is characterized by white colonies and a malbranchea-like asexual morph with profusely branching curved conidiophores forming sporodochia-like structures. Moreover, new combinations for Gymnoascus alatosporus, and for some new species recently described under the generic name Spiromastix, are provided.

[A case of coccidioidomycosis in Turkey imported from the United States of America]. / Türkiye'de Amerika Birlesik Devletleri kaynakli bir koksidiyoidomikoz olgusu.

Coccidioidomycosis caused by Coccidioides immitis or Coccidioides posadasii is a rare infectious disease except in endemic regions. In this report the third documented imported case of coccidioidomycosis in Turkey was presented. A thirty-year-old male patient was admitted to our hospital with fever and purulent drainage from his chest tube. He had worked in Arizona, USA, until 4 months before this presentation. While in Arizona, he experienced cough and hemoptysis and was diagnosed as pulmonary coccidioidomycosis. He was treated with itraconazole for two months and he had no symptoms for 3 years. He then returned to Turkey and 2 months after his return to Turkey, he was admitted to another hospital in Istanbul with dyspnea and diagnosed as hydro-pneumothorax, and pleural fluid obtained from the inserted chest tube was found to be purulent. One gram of BID amoxicillin-clavulanate was given. Physical examination on admission revealed a purulent drainage on the right side chest tube, a temperature of 38.5°C and decreased breath sounds on the right lung. Piperacillin-tazobactam 3 x 4.5 g intravenous and fluconazole 400 mg intravenous once daily were started. Human immunodeficiency virus test was negative. Gram-negative diplococci and rods, gram-positive cocci and septate hyphae were seen in the Gram stain of his pleural fluid. Pleural fluid culture revealed Moraxella catarrhalis after 24 hours incubation and a mold after 72 hours of incubation. Anti-coccidioidal antibodies were found positive in a titer of 1/2. Hydro-pneumothorax, atelectasis and a 3 mm nodules in the right lung were seen in his thorax CT. The patient's pleural fluid and the culture plates were sent to the Public Health Institute of Turkey, Mycology Reference Laboratory (PHIT-MRL), with a clinical suspicion of coccidioidomycosis. The specimen and plates were submitted to the PHIT-MRL Bio Safety Level-3 laboratory for mycological evaluation. The microscopic examination of 15% KOH preparations of pleural fluid specimens revealed septate hyphae which appear to be in the early stages of forming arthroconidia. The pleural fluid culture grew buff-white coloured colonies with aerial hyphae, which were suspected of being a Coccidioides spp. The strain was identified as C.immitis/posadasii by direct microscopy and culture, and subsequently confirmed by the FDA-approved DNA probe. DNA sequence analysis of the ITS and D1/D2 rDNA regions confirmed the isolate to be C.posadasii species [ITS 100% match to GenBank Accession No. AB232901 (630/630 base pair match), and D1/D2 100% match to GenBank Accession No. AB232884 (617/617 base pair match)]. ITS1 and ITS2 barcode analysis also confirmed the species to be C.posadasii, which is the species endemic in Arizona. Susceptibility testing was performed according to Clinical and Laboratory Standards Institute M38-A2 guidelines in the Fungus Testing Laboratory of the University of Texas Health Science Center at San Antonio and minimal inhibitory concentration values were; 0.125 µg/ml for amphotericin B, posaconazole and voriconazole, 0.5 µg/ml for itraconazole and 8 µg/ml for fluconazole. He had decortication of the pleura and was discharged from hospital after six weeks treatment with intravenous fluconazole which was continued orally for one year. Anti-coccidioidal antibodies were negative after two months of treatment. The patient is currently asymptomatic. The presented case is the third case reported from Turkey and provides additional contribution to the existing literature with regard to the appearance of arthroconidium, which is the unusual hyphal form, instead of the expected spherules in the infected tissue.

Four new species of Talaromyces from clinical sources.

The genus Talaromyces constitutes an important group of molds with species that are mainly found in soil, indoor environments and food products. Traditionally, it has been considered, together with Eupenicillium, the teleomorphic state of Penicillium. However, the taxonomy of these fungi has changed considerably, and Talaromyces currently includes sexually and asexually reproducing species. In a previous study of the occurrence of penicillium-like fungi from clinical samples in the USA, we used the combined phylogeny of the internal transcribed spacer (ITS) region of the rDNA and ß-tubulin (BenA) gene to identify 31 isolates of Talaromyces, 85 of Penicillium and two of Rasamsonia. However, seven isolates of Talaromyces were assigned to the corresponding sections but not to any particular species. In this study, we have resolved the taxonomy of these isolates through a multilocus sequence analysis of the ITS, fragments of the BenA, calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2) genes, and a detailed phenotypic study. As a result, four new species are described and illustrated, ie Talaromyces alveolaris, T. georgiensis, T. minnesotensis and T. rapidus.

Fungal Keratitis Secondary to Trametes betulina: A Case Report and Review of Literature.

PURPOSE: We report the first case of human infection and keratitis secondary to Trametes betulina, a rare filamentous fungus. METHODS: Clinical examination including external and slit-lamp examination and corneal scrapings with microbiologic evaluation were performed on a patient with chronic allergic conjunctivitis, entropion and a long-standing corneal ulcer resistant to treatment. RESULTS: The culture from the corneal scraping revealed a basidiomycetous fungus which was submitted for identification. DNA extraction with sequencing and analysis of the ITS and D1/D2 regions were performed on the isolate and demonstrated 100% similarity to Lenzites betulina/Trametes betulina. Susceptibility testing demonstrated potent in vitro activity of voriconazole (MIC < 0.03 µg/ml). The patient was treated with voriconazole, and the corneal ulcer and infiltrate resolved. The infection resulted in corneal thinning and a dense central corneal scar. Penetrating keratoplasty was performed 5 months after diagnosis and treatment and revealed stromal scarring without fungal elements. CONCLUSION: This is the first reported case of keratitis caused by Trametes betulina. This organism should be considered in the differential diagnosis for rare filamentous fungal keratitis and its treatment with voriconazole also noted.

Fatal disseminated Rasamsonia infection in cystic fibrosis post-lung transplantation.

BACKGROUND: Disseminated fungal infections are a known serious complication in individuals with cystic fibrosis (CF) following orthotopic lung transplantation. Aspergillus fumigatus and Scedosporium species are among the more common causes of invasive fungal infection in this population. However, it is also important for clinicians to be aware of other emerging fungal species which may require markedly different antifungal therapies. CASE SUMMARY: We describe the first laboratory-documented case of a fatal disseminated fungal infection caused by Rasamsonia aegroticola in a 21-year-old female CF patient status post-bilateral lung transplantation, which was only identified post-mortem. Molecular analysis revealed the presence of the identical Rasamsonia strains in the patient's respiratory cultures preceding transplantation. DISCUSSION: We propose that the patient's disseminated fungal disease and death occurred as a result of recrudescence of Rasamsonia infection from her native respiratory system in the setting of profound immunosuppression post-operatively. Since Rasamsonia species have been increasingly recovered from the respiratory tract of CF patients, we further review the literature on these fungi and discuss their association with invasive fungal infections in the CF lung transplant host. CONCLUSION: Our report suggests Rasamsonia species may be important fungal pathogens that may have fatal consequences in immunosuppressed CF patients after solid organ transplantation.

Coelomycetous Fungi in the Clinical Setting: Morphological Convergence and Cryptic Diversity.

Human infections by coelomycetous fungi are becoming more frequent and range from superficial to systemic dissemination. Traumatic implantation of contaminated plant material is the most common cause. The typical morphological feature of these fungi is the production of asexual spores (conidia) within fruiting bodies called conidiomata. This study aimed to determine the distribution of the coelomycetes in clinical samples by a phenotypic and molecular study of a large set of isolates received from a U.S. reference mycological institution and by obtaining the in vitro antifungal susceptibility pattern of nine antifungals against a selected group of isolates. A total of 230 isolates were identified by sequencing the D1 and D2 domains of the large subunit (LSU) nuclear ribosomal RNA (nrRNA) gene and by morphological characterization. Eleven orders of the phylum Ascomycota were identified: Pleosporales (the largest group; 66.1%), Botryosphaeriales (19.57%), Glomerellales (4.35%), Diaporthales (3.48%), Xylariales (2.17%), Hysteriales and Valsariales (0.87%), and Capnodiales, Helotiales, Hypocreales and Magnaporthales (0.43% each). The most prevalent species were Neoscytalidium dimidiatum, Paraconiothyrium spp., Phoma herbarum, Didymella heteroderae, and Epicoccum sorghinum The most common anatomical site of isolation was superficial tissue (66.5%), followed by the respiratory tract (17.4%). Most of the isolates tested were susceptible to the majority of antifungals, and only flucytosine showed poor antifungal activity.

Species diversity of Aspergillus section Versicolores in clinical samples and antifungal susceptibility.

Aspergillus section Versicolores includes species of clinical relevance and many others that have been poorly studied but are occasionally found in clinical samples. The aim of this study was to investigate, using a multilocus phylogenetic approach, the spectrum of species of the section Versicolores and to determine their in vitro antifungal susceptibility. The study was based on a set of 77 clinical isolates from different USA medical centres, which had been previously identified as belonging to this section. The genetic markers used were internal transcribed spacer (ITS), ß-tubulin (BenA), calmodulin (CaM), and RNA polymerase II second largest subunit (RPB2), and the drugs tested, following the CLSI guidelines, were amphotericin B (AMB), itraconazole, posaconazole, voriconazole, anidulafungin, caspofungin, micafungin, terbinafine (TBF), and flucytosine (5FC). The most frequent species were Aspergillus sydowii (26 %), Aspergillus creber (22 %), and Aspergillus amoenus (18.2 %), followed by Aspergillus protuberus (13 %), Aspergillus jensenii (10.4 %), and Aspergillus tabacinus (5.2 %); while Aspergillus cvjetkovicii, Aspergillus fructus, Aspergillus puulaauensis, and Aspergillus versicolor were represented by only one isolate each (1.3 %). This is the first time that A. jensenii and A. puulaauensis have been reported from clinical samples. Considering the high number of isolates identified as belonging to this fungal group in this study, its clinical relevance should not be overlooked. Aspergillus versicolor, traditionally considered one of the most common species in this section in a clinical setting, was only rarely recovered in our study. The in vitro antifungal results showed that echinocandins and TBF were the most potent drugs, the azoles showed variable results, AMB was poorly active, and 5FC was the less active.

Veterinary Fusarioses within the United States.

Multilocus DNA sequence data were used to assess the genetic diversity and evolutionary relationships of 67 Fusarium strains from veterinary sources, most of which were from the United States. Molecular phylogenetic analyses revealed that the strains comprised 23 phylogenetically distinct species, all but two of which were previously known to infect humans, distributed among eight species complexes. The majority of the veterinary isolates (47/67 = 70.1%) were nested within the Fusarium solani species complex (FSSC), and these included 8 phylospecies and 33 unique 3-locus sequence types (STs). Three of the FSSC species (Fusarium falciforme, Fusarium keratoplasticum, and Fusarium sp. FSSC 12) accounted for four-fifths of the veterinary strains (38/47) and STs (27/33) within this clade. Most of the F. falciforme strains (12/15) were recovered from equine keratitis infections; however, strains of F. keratoplasticum and Fusarium sp. FSSC 12 were mostly (25/27) isolated from marine vertebrates and invertebrates. Our sampling suggests that the Fusarium incarnatum-equiseti species complex (FIESC), with eight mycoses-associated species, may represent the second most important clade of veterinary relevance within Fusarium Six of the multilocus STs within the FSSC (3+4-eee, 1-b, 12-a, 12-b, 12-f, and 12-h) and one each within the FIESC (1-a) and the Fusarium oxysporum species complex (ST-33) were widespread geographically, including three STs with transoceanic disjunctions. In conclusion, fusaria associated with veterinary mycoses are phylogenetically diverse and typically can only be identified to the species level using DNA sequence data from portions of one or more informative genes.

Efficacy of echinocandins against murine infections by Diutina (Candida) rugosa.

Echinocandins are recommended as a first-line therapy for invasive candidiasis. Candida rugosa was recently transferred to the new genus Diutina. We have determined the in vitro killing kinetics of two echinocandins, anidulafungin, and caspofungin and their in vivo efficacy, administering doses of 5 or 10 mg/kg, and 1 or 5 mg/kg, respectively against 2 clinical strains of D. rugosa. Both drugs showed a fungicidal concentration-dependent activity and, in a neutropenic murine model of disseminated infection, were able to reduce tissue burden and to prolong survival of mice. These results suggest that both echinocandins could be useful to treat infections by this fungus when isolates show minimal inhibitory concentrations within the range of susceptibility for both drugs.

Identification and Antifungal Susceptibility of Penicillium-Like Fungi from Clinical Samples in the United States.

Penicillium species are some of the most common fungi observed worldwide and have an important economic impact as well as being occasional agents of human and animal mycoses. A total of 118 isolates thought to belong to the genus Penicillium based on morphological features were obtained from the Fungus Testing Laboratory at the University of Texas Health Science Center in San Antonio (United States). The isolates were studied phenotypically using standard growth conditions. Molecular identification was made using two genetic markers, the internal transcribed spacer (ITS) and a fragment of the ß-tubulin gene. In order to assess phylogenetic relationships, maximum likelihood and Bayesian inference assessments were used. Antifungal susceptibility testing was performed according to CLSI document M38-A2 for nine antifungal drugs. The isolates were identified within three genera, i.e., Penicillium, Talaromyces, and Rasamsonia The most frequent species in our study were Penicillium rubens, P. citrinum, and Talaromyces amestolkiae The potent in vitro activity of amphotericin B (AMB) and terbinafine (TRB) and of the echinocandins against Penicillium and Talaromyces species might offer a good therapeutic alternative for the treatment of infections caused by these fungi.

Voriconazole minimum inhibitory concentrations are predictive of treatment outcome in experimental murine infections by Candida glabrata.

In this study, 27 clinical isolates of Candida glabrata with voriconazole (VRC) minimum inhibitory concentrations (MICs) ranging from ≤0.03 µg/mL to 8 µg/mL were tested to determine whether in vitro data are predictive of in vivo efficacy. The efficacy of VRC administered at 40 mg/kg was assayed in a neutropenic murine model of disseminated infection by C. glabrata. The reduction in fungal tissue burden in the kidneys was used as a marker of treatment efficacy. VRC reduced the fungal tissue burden in mice infected with strains that had MICs below the epidemiological cut-off value (ECV) of 0.25 µg/mL. Variable efficacy of VRC was obtained when the MIC equalled the ECV, and VRC was ineffective when the MIC exceeded the ECV. These results suggest that the use of in vitro data could be useful to predict the outcome for infections by this fungus.

Update from the Laboratory: Clinical Identification and Susceptibility Testing of Fungi and Trends in Antifungal Resistance.

Despite the availability of new diagnostic assays and broad-spectrum antifungal agents, invasive fungal infections remain a significant challenge to clinicians and are associated with marked morbidity and mortality. In addition, the number of etiologic agents of invasive mycoses has increased accompanied by an expansion in the immunocompromised patient populations, and the use of molecular tools for fungal identification and characterization has resulted in the discovery of several cryptic species. This article reviews various methods used to identify fungi and perform antifungal susceptibility testing in the clinical laboratory. Recent developments in antifungal resistance are also discussed.

Ustilago echinata: Infection in a Mixed Martial Artist Following an Open Fracture.

Ustilago, a common fungal parasite of grains, is infrequently isolated as a pathogen in humans. We describe a case of Ustilago echinata infection following an open distal tibia fracture, review the current literature of this genus as a cause of invasive fungal infection in humans, and discuss management issues.

Voriconazole and posaconazole therapy for experimental Candida lusitaniae infection.

The in vitro activity of posaconazole (PSC) and voriconazole (VRC) was tested by using time-kill studies against 3 strains of Candida lusitaniae. Both drugs showed fungistatic activity against all strains. The efficacy of those compounds was evaluated by reducing kidney fungal burden and by determining (1→3)-ß-d-glucan serum levels in a murine model of invasive infection of C. lusitaniae. The therapies tested were VRC at 10, 25, or 40 mg/kg/day and PSC at 5, 12.5, or 20 mg/kg/twice a day. All the dosages showed efficacy in a dose-dependant manner being high doses of both antifungals able to sterilize some kidneys after 10 days. With the exception of the strain FMR 9474, against which PSC was more effective than VRC, no differences in reducing tissue burden were found between the treatments. All doses of both antifungals were able to significantly reduce (1→3)-ß-d-glucan serum levels with no significant differences between treatments and between the same doses of both drugs.

First Detection of TR34 L98H and TR46 Y121F T289A Cyp51 Mutations in Aspergillus fumigatus Isolates in the United States.

Azole resistance in Aspergillus fumigatus is an increasing problem. The TR34 L98H and TR46 Y121F T289A mutations that can occur in patients without previous azole exposure have been reported in Europe, Asia, the Middle East, Africa, and Australia. Here, we report the detection of both the TR34 L98H and TR46 Y121F T289A mutations in confirmed A. fumigatus isolates collected in institutions in the United States. These mutations, other mutations known to cause azole resistance, and azole MICs are reported here.

Invasive Fusariosis in the Voriconazole Era: Single-Center 13-Year Experience.

Background. Invasive fusariosis remains an aggressive, albeit infrequent infection in immunocompromised patients. Methods. We identified all cases of invasive fusariosis between January 2002 and December 2014. We recorded patient characteristics including clinical presentation, treatment, and outcomes at 6 and 12 weeks after diagnosis, as well as species identification and antifungal drug susceptibilities. Results. Fifteen patients were diagnosed with proven (12, 80%) or probable (3, 20%) fusariosis. Median age was 60 years (range, 26-78), and 10 patients were male. Underlying conditions included hematological malignancies (13, 87%), juvenile idiopathic arthritis (1, 7%), and third-degree burns (1, 7%). Five patients underwent hematopoietic stem-cell transplantation before diagnosis. Six patients (40%) received systemic glucocorticoids, and 11 patients (73%) had prolonged neutropenia at the time of diagnosis. Clinical presentations included the following: skin/soft tissue infection (8, 53%), febrile neutropenia (4, 27%), respiratory tract infection (2, 13%), and septic arthritis (1, 7%). Twelve patients were treated with voriconazole: 6 (40%) with voriconazole alone, 4 (27%) with voriconazole and terbinafine, and 2 (13%) with voriconazole, terbinafine, and amphotericin. One patient (7%) was treated with terbinafine alone, and another with micafungin alone. Four patients underwent surgical debridement (4, 27%). Susceptibility testing was performed on 9 isolates; 8 demonstrated voriconazole minimum inhibitory concentrations ≥4 µg/mL. The cumulative probability of survival was 66.7% and 53.3% at 6 and 12 weeks after diagnosis. Conclusions. Mortality associated with invasive fusariosis remains high. Cumulative mortality at our center was lower than previous reports despite elevated voriconazole minimum inhibitory concentrations. Combination therapy should be studied systematically for fusariosis.

Cladosporium Species Recovered from Clinical Samples in the United States.

Cladosporium species are ubiquitous, saprobic, dematiaceous fungi, only infrequently associated with human and animal opportunistic infections. We have studied a large set of Cladosporium isolates recovered from clinical samples in the United States to ascertain the predominant species there in light of recent taxonomic changes in this genus and to determine whether some could possibly be rare potential pathogens. A total of 92 isolates were identified using phenotypic and molecular methods, which included sequence analysis of the internal transcribed spacer (ITS) region and a fragment of the large subunit (LSU) of the nuclear ribosomal DNA (rDNA), as well as fragments of the translation elongation factor 1 alpha (EF-1α) and actin (Act) genes. The most frequent species was Cladosporium halotolerans (14.8%), followed by C. tenuissimum (10.2%), C. subuliforme (5.7%), and C. pseudocladosporioides (4.5%). However, 39.8% of the isolates did not correspond to any known species and were deemed to comprise at least 17 new lineages for Cladosporium. The most frequent anatomic site of isolation was the respiratory tract (54.5%), followed by superficial (28.4%) and deep tissues and fluids (14.7%). Species of the two recently described cladosporiumlike genera Toxicocladosporium and Penidiella are reported for the first time from clinical samples. In vitro susceptibility testing of 92 isolates against nine antifungal drugs showed a variety of results but high activity overall for the azoles, echinocandins, and terbinafine.