Genome sequencing of Cladophialophora exuberans, a novel candidate for bioremediation of hydrocarbon and heavy metal polluted habitats.

Cladophialophora exuberans is a filamentous fungus related to black yeasts in the order Chaetothyriales. These melanized fungi are known for their 'dual ecology', often occurring in toxic environments and also being frequently involved in human infection. Particularly Cladophialophora exuberans, C. immunda, C. psammophila, and Exophiala mesophila have been described with a pronounced ability to degrade aromatic compounds and xenobiotic volatiles, such as benzene, toluene, ethyl-benzene, and xylene, and are candidates for bioremediation applications. The objective of the present study is the sequencing, assembly, and description of the whole genome of C. exuberans focusing on genes and pathways related to carbon and toxin management, assessing the tolerance and bioremediation of lead and copper, and verifying the presence of genes for metal homeostasis. Genomic evaluations were carried out through a comparison with sibling species including clinical and environmental strains. Tolerance of metals was evaluated via a microdilution method establishing minimum inhibitory (MIC) and fungicidal concentrations (MFC), and agar diffusion assays. Heavy metal bioremediation was evaluated via graphite furnace atomic absorption spectroscopy (GFAAS). The final assembly of C. exuberans comprised 661 contigs, with genome size of 38.10 Mb, coverage of 89.9X and a GC content of 50.8%. In addition, inhibition of growth was shown at concentrations of 1250 ppm for copper and at 625 ppm for lead, using the MIC method. In the agar tests, the strain grew at 2500 ppm of copper and lead. In GFAAS tests, uptake capacities were observed of 89.2% and 95.7% for copper and lead, respectively, after 21 experimental days. This study enabled the annotation of genes involved in heavy metal homeostasis and also contributed to a better understanding of the mechanisms used in tolerance of and adaptation to extreme conditions.

Vaccuuming method as a successful strategy in the diagnosis of active infestation by Pediculus humanus capitis.

Most human epidemiological and clinical studies use visual inspection of the hair and scalp to diagnose Pediculus humanus capitis , however this method has low sensitivity to diagnose active infestations (presence of nymphs and adult lice). Vacuuming the hair and scalp has been used as a diagnostic method, but there are no previous data comparing its effectiveness with visual inspection. The aim of this study was to determine the prevalence of overall infestation (nits and trophic stages), of active infestation by Pediculus humanus capitis , and to evaluate the effectiveness of vacuuming in comparison with the visual inspection. Visual inspection was performed by three examiners and vacuuming of the scalp by one investigator, with an adapted vacuum cleaner. A total of 166 children aged 4 to 10 years old were randomly selected from public schools in Southern Brazil. Considering the positive results obtained by both methods, the prevalence of overall infestation was 63.3%, whereas active infestation was 18.7%. The visual inspection was more effective on diagnosing overall infestation, however, its effectiveness to detect active infestation was lower, ranging from 0.6% (RR=3%, p<0.001) to 6.6% (RR=35%, p=0.001), depending on the number of examiners. The effectiveness of vacuuming to diagnose active infestation was higher than the one of visual inspection, with a prevalence rate of 16.3% (RR=87%, p=0.332). As presented in our study, the vacuuming method was 2.74 to 7.87 times most likely to detect active infestation, thus it could be adopted as a more accurate method to diagnose active pediculosis.

Environmental Screening of Fonsecaea Agents of Chromoblastomycosis Using Rolling Circle Amplification.

Chromoblastomycosis is a chronic, cutaneous or subcutaneous mycosis characterized by the presence of muriform cells in host tissue. Implantation disease is caused by melanized fungi related to black yeasts, which, in humid tropical climates, are mainly members of the genus Fonsecaea. In endemic areas of Brazil, F. pedrosoi and F. monophora are the prevalent species. The current hypothesis of infection is traumatic introduction via plant materials, especially by plant thorns. However, isolation studies have demonstrated a low frequency of the agents in environmental substrates. The present study aimed to detect F. pedrosoi and F. monophora in shells of babassu coconuts, soil, plant debris, and thorns from endemic areas of chromoblastomycosis in Maranhão state, northern Brazil, using Rolling Circle Amplification (RCA) with padlock probes as a new environmental screening tool for agents of chromoblastomycosis. In addition to molecular screening, the environmental samples were analyzed by fungal isolation using mineral oil flotation. The limit of detection of the RCA method was 2.88 × 107 copies of DNA per sample for the used padlock probes, indicating that this represents an efficient and sensitive molecular tool for the environmental screening of Fonsecaea agents. In contrast, with isolation from the same samples using several selective methods, no agents of chromoblastomycosis were recovered.

Selective isolation of agents of chromoblastomycosis from insect-associated environmental sources.

Chromoblastomycosis is a neglected disease characterized by cutaneous, subcutaneous or disseminated lesions. It is considered an occupational infectious disease that affects mostly rural workers exposed to contaminated soil and vegetal matter. Lesions mostly arise after a traumatic inoculation of herpotrichiellaceous fungi from the Chaetothyriales order. However, the environmental niche of the agents of the disease remains obscure. Its association with insects has been predicted in a few studies. Therefore, the present work aimed to analyze if social insects, specifically ants, bees, and termites, provide a suitable habitat for the fungi concerned. The mineral oil flotation method was used to isolate the microorganisms. Nine isolates were recovered and phylogenetic analysis identified two strains as potential agents of chromoblastomycosis, i.e., Fonsecaea pedrosoi CMRP 3076, obtained from a termite nest (n = 1) and Rhinocladiella similis CMRP 3079 from an ant exoskeleton (n = 1). In addition, we also identified Fonsecaea brasiliensis CMRP 3445 from termites (n = 1), Exophiala xenobiotica CMRP 3077 from ant exoskeleton (n = 1), Cyphellophoraceae CMRP 3103 from bees (n = 1), Cladosporium sp. CMRP 3119 from bees (n = 1), Hawksworthiomyces sp. CMRP 3102 from termites (n = 1), and Cryptendoxyla sp. from termites (n = 2). The environmental isolate of F. pedrosoi CMRP 3076 was tested in two animal models, Tenebrio molitor and Wistar rat, for its pathogenic potential with fungal retention in T. molitor tissue. In the Wistar rat, the cells resembling muriform cells were observed 30 d after inoculation.

Scalp microbiota alterations in children with pediculosis.

Pediculosis is a disease caused by the insect Pediculus humanus capitis that mainly occurs in childhood. A comparative study was carried out evaluating groups of schoolchildren with (group A) and without pediculosis (group B) to analyse the characteristics of the scalp microbiota. Samples were collected by swab using Stuart transport medium and incubate in Sabouraud dextrose agar with tetracycline to analyse the fungal microbiota and in blood agar to assess the bacterial microbiota. The isolates identity was confirmed by sequencing of the 16S and 18S regions of the ribosomal DNA gene for bacteria and fungi, respectively. The analysis of the 186 isolates led to the identification of 35 bacteria and 40 fungi in group A and 47 bacteria and 64 fungi in group B. The results indicate differences in bacterial and fungal species in the groups analysed. In the observed bacterial microbiota, Staphylococcus capitis occurred more frequently than Staphylococcus epidermidis in group A vs B. Among fungal isolates, Debaryomyces sp. was more frequent in group B vs A. Our findings showed scalp microbiota alterations in children with pediculosis, meriting future studies to analyse the relationship between these agents and their impact on human health.

A Model for Trans-Kingdom Pathogenicity in Fonsecaea Agents of Human Chromoblastomycosis.

The fungal genus Fonsecaea comprises etiological agents of human chromoblastomycosis, a chronic implantation skin disease. The current hypothesis is that patients acquire the infection through an injury from plant material. The present study aimed to evaluate a model of infection in plant and animal hosts to understand the parameters of trans-kingdom pathogenicity. Clinical strains of causative agents of chromoblastomycosis (Fonsecaea pedrosoi and Fonsecaea monophora) were compared with a strain of Fonsecaea erecta isolated from a living plant. The clinical strains of F. monophora and F. pedrosoi remained concentrated near the epidermis, whereas F. erecta colonized deeper plant tissues, resembling an endophytic behavior. In an invertebrate infection model with larvae of a beetle, Tenebrio molitor, F. erecta exhibited the lowest survival rates. However, F. pedrosoi produced dark, spherical to ovoidal cells that resembled muriform cells, the invasive form of human chromoblastomycosis confirming the role of muriform cells as a pathogenic adaptation in animal tissues. An immunologic assay in BALB/c mice demonstrated the high virulence of saprobic species in animal models was subsequently controlled via host higher immune response.