Emerging Cases of Cat-Transmitted Sporotrichosis Driven by Sporothrix brasiliensis in Northeast Brazil.

Cat-transmitted sporotrichosis is caused by the emerging fungal pathogen Sporothrix brasiliensis and constitutes a significant public health issue that affects people living in resource-poor urban centers in Brazil. The lack of knowledge about transmission dynamics makes it difficult to propose public health policies to contain the advance of sporotrichosis. We describe the recent emergence of 1,176 cases of sporotrichosis in cats (2016 to 2021) in the metropolitan region of Recife, Brazil, leading to significant zoonotic transmission and an overwhelming occurrence of S. brasiliensis as the etiological agent. Most cases were from cats in the cities of Olinda (408/1,176; 34.70%), Jaboatão dos Guararapes (332/1,176; 28.23%), and Recife (237/1,176; 20.15%). Molecular typing using amplified fragment length polymorphism (EcoRI-GA/MseI-AG) revealed low polymorphic information content (PIC = 0.2499) and heterozygosity (H = 0.2928), typical of an outbreak scenario. Dendrogram and multivariate cluster analysis revealed that isolates from Pernambuco are closely related to Rio de Janeiro isolates. We report a substantial occurrence of MAT1-2 idiomorphs in the metropolitan region of Recife (0:60 ratio; χ2 = 60.000, P < 0.0001). The limited population differentiation and genetic diversity of the isolates from Pernambuco suggest a recent introduction, possibly via a founder effect, from the parental population in Rio de Janeiro. Our findings emphasize the critical importance of molecular surveillance of S. brasiliensis for outbreak response. A comprehensive one-health strategy is mandatory to control the spread of cat-transmitted sporotrichosis driven by S. brasiliensis, encompassing sanitary barriers, quick diagnosis, and treatment.

Multifocal Sporotrichosis Associated with Armadillo Hunting in Midwest Brazil: An In-Depth Case Study and Comprehensive Literature Analysis.

Sporotrichosis is a globally distributed subcutaneous mycosis caused by dimorphic Sporothrix species commonly found in soil, mosses, and decaying plant matter. The lymphocutaneous manifestation, historically associated with occupational activities and sapronotic transmission, has recently been observed to also occur through animal contact, particularly notable in Brazil. We describe a rare case of lymphocutaneous sporotrichosis with simultaneous pulmonary complications resulting from the scratching of a southern three-banded armadillo, Tolypeutes matacus, primarily inhabiting the arid forests of South America's central region. Speciation using multiplex quantitative polymerase chain reaction (qPCR) established the etiological agent as S. schenckii s. str., while amplified fragment length polymorphism (AFLP) analysis unveiled a novel genotype circulating in the Midwest of Brazil. The patient received treatment with itraconazole (200 mg/day) for two months, leading to substantial clinical improvement of cutaneous and pulmonary symptoms. This case highlights the critical role of animal-mediated transmission in sporotrichosis epidemiology, particularly within regions with diverse armadillo species. The unusual epidemiology and genetic characteristics of this case emphasize the need for enhanced awareness and diagnostic vigilance in atypical sporotrichosis presentations.

[Genetic diversity of SP1 population of space mutagenized Pueraria montana].

To explore the mutagenic effect of the space environment on Pueraria montana and select the elite germplasm with good growth conditions and high isoflavone content, this study observed the agronomic traits, determined the flower isoflavone content, and labeled amplified fragment length polymorphism(AFLP) fluorescent molecular markers of 79 P. montana plants exposed to space mutagenesis(SP1 group) and 10 control plants of P. montana(CK group). Excel 2019, SPSS 25.0, NTSYSpc-2.11F, and Popgen 32 were employed to analyze the genetic diversity and perform the cluster analysis. The results showed that the SP1 group presented changed leaf hairy attitude and flower structure and higher CV and H' of quantitative traits than the CK group. The cluster analysis screened out five plants in the SP1 group. Ten P. montana plants in the SP1 group had higher content of 6″-O-xylosyl-tectoridin and tectoridin in the flowers than the control group, with the total content of both exceeding 11%. After clustering, 9 plants in the SP1 group were separated. Nine pairs of polymorphic primers were screened out frrom 64 pairs of primers. A total of 1 620 polymorphic loci were detected, with the average percentage of polymorphic loci(PPL) of 83.33%. The average Nei's gene diversity index(H) and Shannon's information index(I) were 0.192 2 and 0.305 2, respectively. After clustering, 4 plants in the SP1 group were screened out. According to the above results, plants No. 30, No. 66, and No. 89 in the SP1 group were subjected to greater mutagenic effect by the space environment and presented better growth and higher flower isoflavone content. Moreover, plant No. 30 showed the flower structure variation and flower weight two times of that in the CK group. These plants can be used as key materials for the subsequent experiments.

Genetic Diversity Analysis of Banana Cultivars (Musa sp.) in Saudi Arabia Based on AFLP Marker.

Banana plantation has been introduced recently to a temperate zone in the southeastern parts of Saudi Arabia (Fifa, Dhamadh, and Beesh, located in Jazan province). The introduced banana cultivars were of a clear origin without a recorded genetic background. In the current study, the genetic variability and structure of five common banana cultivars (i.e., Red, America, Indian, French, and Baladi) were analyzed using the fluorescently labeled AFLP technique. Nine different primer pairs combinations yielded 1468 loci with 88.96% polymorphism. Among all locations, high expected heterozygosity under the Hardy-Weinberg assumption was found (0.249 ± 0.003), where Dhamadh was the highest, followed by Fifa and Beesh, respectively. Based on the PCoA and Structure analysis, the samples were not clustered by location but in pairs in accordance with the cultivar's names. However, the Red banana cultivar was found to be a hybrid between the American and Indian cultivars. Based on ΦST, 162 molecular markers (i.e., loci under selection) were detected among cultivars. Identifying those loci using NGS techniques can reveal the genetic bases and molecular mechanisms involved in the domestication and selection indicators among banana cultivars.

The first established microsatellite markers to distinguish Candida orthopsilosis isolates and detection of a nosocomial outbreak in China.

The infection proportion of Candida orthopsilosis, a member of the C. parapsilosis complex, has increased globally in recent years, and nosocomial outbreaks have been reported in several countries. This study aimed to establish microsatellite loci-based typing method that was able to effectively distinguish among C. orthopsilosis isolates. Three reference C. orthopsilosis genome sequences were analyzed to identify repeat loci. DNA sequences containing over eight bi- or more nucleotide repeats were selected. A total of 51 loci were initially identified, and locus-specific primers were designed and tested with 20 epidemiologically unrelated isolates. Four loci with excellent reproducibility, specificity, and resolution for molecular typing purposes were identified, and the combined discriminatory power (DP, based on 20 epidemiologically unrelated isolates) of these four loci was 1.0. Reproducibility was demonstrated by consistently testing three strains each in triplicate, and stability, demonstrated by testing 10 successive passages. Then, we collected 48 C. orthopsilosis non-duplicate clinical isolates from the China Hospital Invasive Fungal Surveillance Net study to compare the DP of the microsatellite-based typing with internal transcribed spacer (ITS) and amplified fragment length polymorphism (AFLP) typing analyses, using ATCC 96139 as a reference strain. These 49 isolates were subdivided into 12 microsatellite types (COMT1-12), six AFLP types, and three ITS types, while all the isolates with the same COMT belonged to consistent AFLP and ITS type, demonstrating the high DP of our microsatellite-type method. According to our results, COMT12 was found to be the predominant type in China, and COMT5 was the second largest and responsible for causing a nosocomial outbreak. This microsatellite-type method is a valuable tool for the differentiation of C. orthopsilosis and could be vital for epidemiological studies to determine strain relatedness and monitor transmission.

Molecular epidemiology and clinical-laboratory aspects of chromoblastomycosis in Mato Grosso, Brazil.

INTRODUCTION: Chromoblastomycosis is a disease caused by melanized fungi, primarily belonging to the genera Fonsecaea and Cladophialophora, mainly affecting individuals who are occupationally exposed to soil and plant products. This research aimed to determine the clinical, epidemiological and laboratory characteristics of chromoblastomycosis in the state of Mato Grosso, Brazil. MATERIALS AND METHODS: Patients diagnosed with chromoblastomycosis treated at the Júlio Müller University Hospital, Cuiabá, Brazil, from January 2015 to December 2020, whose isolates were preserved in the Research Laboratory of the Faculty of Medicine of the Federal University of Mato Grosso. Isolates were identified by partly sequencing the Internal Transcribed Spacer (ITS) and ß-tubulin (BT2) loci. AFLP fingerprinting was used to explore the genetic diversity. Susceptibility to itraconazole, voriconazole, 5-fluorocytosine, terbinafine and amphotericin B was determined by the broth microdilution technique. RESULTS: Ten patients were included, nine were male (mean age = 64.1 years). Mean disease duration was 8.6 years. Lesions were mainly observed in the lower limbs. Predominant clinical forms were verrucous and scarring. Systemic arterial hypertension and type II diabetes mellitus were the predominant comorbidities. Leprosy was the main concomitant infectious disease. Fonsecaea pedrosoi was the unique aetiological agent identified with moderate genetic diversity (H = 0.3934-0.4527; PIC = 0.3160-0.3502). Antifungal agents with the highest activity were terbinafine, voriconazole and itraconazole. CONCLUSION: Chromoblastomycosis is affecting the poor population in rural and urban areas, mainly related to agricultural activities, with F. pedrosoi being the dominant aetiologic agent. All isolates had low MICs for itraconazole, voriconazole and terbinafine, confirming their importance as therapeutic alternatives for chromoblastomycosis.

AFLP markers based genetic diversity and population structure analysis of Kadaknath: an indigenous black meat poultry breed of India.

The current study is the first worldwide to assess the genetic diversity of Kadaknath poultry using amplified fragment length polymorphism (AFLP) markers. Out of total 96 accessions, four were outliers and 92 were Kadaknath accessions of which 30 were males, 62 were females. Of these, 74 were jet black, 7 penciled and 11 were golden feather color type of Kadaknath. High level of polymorphism (23.94%) was observed in 387 loci amplified using six AFLP primer combinations. The Jaccard's similarity coefficient ranged from 0.211 to 0.754 with an average dissimilarity of 0.517. Based on the neighbor-joining method of clustering, all accessions were clustered into seven major clusters which were not consistent with their respective geographical locations. The mean values of effective multiplex ratio, polymorphic information content, resolving power and marker index were 15.16, 0.38, 9.87 and 5.85 respectively. Further, the high log-likelihood score was produced when the number of populations (K) was set at 7 while carrying out the population STRUCTURE analysis, which was also congruent with clustering analysis based on genetic diversity. The extent of genetic diversity detected in this study could be used for germplasm selection and developing conservation strategies of pure breed of Kadaknath.

Predictors of adverse maternal outcome in jaundiced pregnant women identified as having pregnancy-specific liver disease (P-sLD).

We aimed to study the factors including model for end stage liver disease (MELD) score in predicting mortality in women with pregnancy-specific liver diseases (P-sLD). A total of 154 women with clinical jaundice were studied of which 138 women were diagnosed with P-sLD. The most common P-sLD was HELLP syndrome (51.9%) followed by acute fatty liver of pregnancy (AFLP) (17.5%). The mean age was 26.3 ± 4.7 years and the mean gestational age was 35.1 ± 4.2 weeks. The maternal death rate was 26.8% and the most common cause was coagulopathy followed by sepsis. The mean MELD score among non survivors was 25.98 ± 8.17 compared to 17.29 ± 8.12 among survivors (p value .00). On univariate analysis, gestational age at admission, presence of hypertension, the platelet count, serum creatinine, INR and MELD score were found to significant. The AUC for INR (0.82) and MELD score (0.77) was better than platelet count (0.72) and serum creatinine (0.67). On multivariate analysis, only the INR and presence of AKI were found to be significantly associated with maternal mortality. The performance of INR was better than MELD score in predicting mortality in women with P-sLD. Additional factors like platelet count may be incorporated in to MELD score for the prediction of mortality in pregnant women.IMPACT STATEMENTWhat is already known on this subject? Pregnancy-specific liver disorders (P-sLD) have significant effect on maternal and foetal outcome, often considered as a spectrum of disease with significant overlap of clinical and laboratory parameters. MELD score is used reliably outside the pregnancy to predict mortality may not be good in pregnant women. There are only few studies that looked at the factors predictive of adverse maternal outcome.What do the results of this study add? Though we have demonstrated that MELD score was significantly high among non-survivors, serum bilirubin an important component of MELD score was not found to be significant. The other factors which were found to be significant on univariate analysis include gestational age at admission, hypertension and platelet count. However, international normalised ratio (INR) and acute kidney injury (AKI) were the factors independently associated with mortality.What are the implications for clinical practice and/or further research? The utility of MELD score in P-sLD should be studied prospectively in different populations. Moreover, the feasibility of developing a simple model which incorporates platelet count in addition to other components of MELD score should also be explored in future studies.

Lifetime genealogical divergence within plants leads to epigenetic mosaicism in the shrub Lavandula latifolia (Lamiaceae).

Epigenetic mosaicism is a possible source of within-plant phenotypic heterogeneity, yet its frequency and developmental origin remain unexplored. This study examines whether extant epigenetic heterogeneity within Lavandula latifolia (Lamiaceae) shrubs reflects recent epigenetic modifications experienced independently by different plant parts or, alternatively, it is the cumulative outcome of a steady lifetime process. Leaf samples from different architectural modules (branch tips) were collected from three L. latifolia plants and characterized epigenetically by global DNA cytosine methylation and methylation state of methylation-sensitive amplified fragment-length polymorphism (MS-AFLP) markers. Epigenetic characteristics of modules were then assembled with information on the branching history of plants. Methods borrowed from phylogenetic research were used to assess genealogical signal of extant epigenetic variation and reconstruct within-plant genealogical trajectory of epigenetic traits. Plants were epigenetically heterogeneous, as shown by differences among modules in global DNA methylation and variation in the methylation states of 6 to 8% of MS-AFLP markers. All epigenetic features exhibited significant genealogical signal within plants. Events of epigenetic divergence occurred throughout the lifespan of individuals and were subsequently propagated by branch divisions. Internal epigenetic diversification of L. latifolia individuals took place steadily during their development, a process which eventually led to persistent epigenetic mosaicism.

Strain improvement of Trichoderma spp. through two-step protoplast fusion for cellulase production enhancement.

Fungal protoplast fusion is an approach to introduce novel characteristics into industrially important strains. Cellulases, essential enzymes with a wide range of biotechnological applications, are produced by many species of the filamentous fungi Trichoderma. In this study, a collection of 60 natural isolates were screened for Avicel and carboxymethyl cellulose degradation, and two cellulase producers of Trichoderma virens and Trichoderma harzianum were used for protoplast fusion. One of the resulting hybrids with improved cellulase activity, C1-3, was fused with the hyperproducer Trichoderma reesei Rut-C30. A new selected hybrid, F7, was increased in cellulase activity 1.8 and 5 times in comparison with Rut-C30 and C1-3, respectively. The increases in enzyme activity correlated with an upregulation of the cellulolytic genes cbh1, cbh2, egl3, and bgl1 in the parents. The amount of mRNA of cbh1 and cbh2 in F7 resembled that of Rut-C30 while the bgl1 mRNA level was similar to that of C1-3. AFLP (amplified fragment length polymorphism) fingerprinting and GC-MS (gas chromatography - mass spectrometry) analysis represented variations in parental strains and fusants. In conclusion, the results demonstrate that a 3-interspecific hybrid strain was isolated, with improved characteristics for cellulase degradation and showing genetic polymorphisms and differences in the volatile profile, suggesting reorganizations at the genetic level.

Trends in the molecular epidemiology and population genetics of emerging Sporothrix species.

Sporothrix (Ophiostomatales) comprises species that are pathogenic to humans and other mammals as well as environmental fungi. Developments in molecular phylogeny have changed our perceptions about the epidemiology, host-association, and virulence of Sporothrix. The classical agent of sporotrichosis, Sporothrix schenckii, now comprises several species nested in a clinical clade with S. brasiliensis, S. globosa, and S. luriei. To gain a more precise view of outbreaks dynamics, structure, and origin of genetic variation within and among populations of Sporothrix, we applied three sets of discriminatory AFLP markers (#3 EcoRI-GA/MseI-TT, #5 EcoRI-GA/MseI-AG, and #6 EcoRI-TA/MseI-AA) and mating-type analysis to a large collection of human, animal and environmental isolates spanning the major endemic areas. A total of 451 polymorphic loci were amplified in vitro from 188 samples, and revealed high polymorphism information content (PIC = 0.1765-0.2253), marker index (MI = 0.0001-0.0002), effective multiplex ratio (E = 15.1720-23.5591), resolving power (Rp = 26.1075-40.2795), discriminating power (D = 0.9766-0.9879), expected heterozygosity (H = 0.1957-0.2588), and mean heterozygosity (Havp  = 0.000007-0.000009), demonstrating the effectiveness of AFLP markers to speciate Sporothrix. Analysis using the program structure indicated three genetic clusters matching S. brasiliensis (population 1), S. schenckii (population 2), and S. globosa (population 3), with the presence of patterns of admixture amongst all populations. AMOVA revealed highly structured clusters (PhiPT = 0.458-0.484, P < 0.0001), with roughly equivalent genetic variability within (46-48 %) and between (52-54 %) populations. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for S. schenckii (χ2 = 2.522; P = 0.1122), supporting random mating. In contrast, skewed distributions were found for S. globosa (χ2 = 9.529; P = 0.0020) with a predominance of MAT1-1 isolates, and regional differences were highlighted for S. brasiliensis with the overwhelming occurrence of MAT1-2 in Rio de Janeiro (χ2 = 14.222; P = 0.0002) and Pernambuco (χ2 = 7.364; P = 0.0067), in comparison to a higher prevalence of MAT1-1 in the Rio Grande do Sul (χ2 = 7.364; P = 0.0067). Epidemiological trends reveal the geographic expansion of cat-transmitted sporotrichosis due to S. brasiliensis via founder effect. These data support Rio de Janeiro as the centre of origin that has led to the spread of this disease to other regions in Brazil. Our ability to reconstruct the source, spread, and evolution of the ongoing outbreaks from molecular data provides high-quality information for decision-making aimed at mitigating the progression of the disease. Other uses include surveillance, rapid diagnosis, case connectivity, and guiding access to appropriate antifungal treatment.

Exploring genetic diversity, population structure, and phylogeography in Paracoccidioides species using AFLP markers.

Paracoccidioidomycosis (PCM) is a life-threatening systemic fungal infection acquired after inhalation of Paracoccidioides propagules from the environment. The main agents include members of the P. brasiliensis complex (phylogenetically-defined species S1, PS2, PS3, and PS4) and P. lutzii. DNA-sequencing of protein-coding loci (e.g., GP43, ARF, and TUB1) is the reference method for recognizing Paracoccidioides species due to a lack of robust phenotypic markers. Thus, developing new molecular markers that are informative and cost-effective is key to providing quality information to explore genetic diversity within Paracoccidioides. We report using new amplified fragment length polymorphism (AFLP) markers and mating-type analysis for genotyping Paracoccidioides species. The bioinformatic analysis generated 144 in silico AFLP profiles, highlighting two discriminatory primer pairs combinations (#1 EcoRI-AC/MseI-CT and #2 EcoRI-AT/MseI-CT). The combinations #1 and #2 were used in vitro to genotype 165 Paracoccidioides isolates recovered from across a vast area of South America. Considering the overall scored AFLP markers in vitro (67-87 fragments), the values of polymorphism information content (PIC = 0.3345-0.3456), marker index (MI = 0.0018), effective multiplex ratio (E = 44.6788-60.3818), resolving power (Rp = 22.3152-34.3152), discriminating power (D = 0.5183-0.5553), expected heterozygosity (H = 0.4247-0.4443), and mean heterozygosity (H avp  = 0.00002-0.00004), demonstrated the utility of AFLP markers to speciate Paracoccidioides and to dissect both deep and fine-scale genetic structures. Analysis of molecular variance (AMOVA) revealed that the total genetic variance (65-66 %) was due to variability among P. brasiliensis complex and P. lutzii (PhiPT = 0.651-0.658, P < 0.0001), supporting a highly structured population. Heterothallism was the exclusive mating strategy, and the distributions of MAT1-1 or MAT1-2 idiomorphs were not significantly skewed (1:1 ratio) for P. brasiliensis s. str. (χ2 = 1.025; P = 0.3113), P. venezuelensis (χ2 = 0.692; P = 0.4054), and P. lutzii (χ2 = 0.027; P = 0.8694), supporting random mating within each species. In contrast, skewed distributions were found for P. americana (χ2 = 8.909; P = 0.0028) and P. restrepiensis (χ2 = 4.571; P = 0.0325) with a preponderance of MAT1-1. Geographical distributions confirmed that P. americana, P. restrepiensis, and P. lutzii are more widespread than previously thought. P. brasiliensis s. str. is by far the most widely occurring lineage in Latin America countries, occurring in all regions of Brazil. Our new DNA fingerprint assay proved to be rapid, reproducible, and highly discriminatory, to give insights into the taxonomy, ecology, and epidemiology of Paracoccidioides species, guiding disease-control strategies to mitigate PCM.

Differential DNA methylation and gene expression during development of reproductive and vegetative organs in Ilex species.

Differential epigenetic (DNA cytosine methylation) and gene expression patterns were investigated in reproductive and vegetative organs from Ilex paraguariensis and I. dumosa, at distinct developmental stages. We aimed at contributing towards elucidating major molecular changes underlying the sexual differentiation processes which, in these dioecious species, are completely unknown. Simultaneously, as a first step towards the development of an early sexing system, we searched for promising molecular markers. This was assessed through Methylation Sensitive Amplified Polymorphism (MSAP) and Amplified Fragment Length Polymorphism on cDNA (cDNA-AFLP) techniques, applying discriminant multivariate analyses, and bioinformatic characterization of differential fragments. A significant positive correlation was found between epigenetic and indirect 'genetic' information for both species, indicating influence of the genetic background on the epigenetic variation. Higher epigenetic than genetic diversities were estimated. Our outcomes showed up to 1.86 times more representation of mCG subepiloci than mCCG in all organs sampled. Along the maturing stages of floral buds, the frequency of mCG evidenced an incremental trend, whereas mCCG and unmethylated conditions showed opposite tendencies. Reproductive and vegetative samples tended to cluster apart based on epigenetic patterns; at gene expression level, organs exhibited clear-cut distinctive patterns, nonetheless profiles of young leaves and floral primordia resemble. Epigenetic and expression data allowed discrimination of I. dumosa´s samples according to the gender of the donor; more elusive patterns were observed for I. paraguariensis. In total, 102 differentially methylated and expressed fragments were characterized bioinformatically. Forty-three were annotated in various functional categories; four candidate markers were validated through qPCR, finding statistical differences among organs but not among sexes. The methylation condition of epilocus C13m33 appears as indicative of gender in both species. Thirty-three organ-specific and 34 gender-specific methylated markers were discriminated and deserve further research, particularly those expressed in leaves. Our study contributes concrete candidate markers with potential for practical application.

A pseudo-outbreak of Rhinocladiella similis in a bronchoscopy unit of a tertiary care teaching hospital in London, United Kingdom.

Outbreaks of fungal infections due to emerging and rare species are increasingly reported in healthcare settings. We investigated a pseudo-outbreak of Rhinocladiella similis in a bronchoscopy unit of a tertiary care teaching hospital in London, UK. We aimed to determine route of healthcare-associated transmission and prevent additional infections. From July 2018 through February 2019, we detected a pseudo-outbreak of R. similis isolated from bronchoalveolar lavage (BAL) fluid samples collected from nine patients who had undergone bronchoscopy in a multispecialty teaching hospital, during a period of 8 months. Isolates were identified by MALDI-TOF mass spectrometry. Antifungal susceptibility testing was performed by EUCAST broth microdilution. To determine genetic relatedness among R. similis isolates, we undertook amplified fragment length polymorphism analysis. To determine the potential source of contamination, an epidemiological investigation was carried out. We reviewed patient records retrospectively and audited steps taken during bronchoscopy as well as the subsequent cleaning and decontamination procedures. Fungal cultures were performed on samples collected from bronchoscopes and automated endoscope washer-disinfector systems. No patient was found to have an infection due to R. similis either before or after bronchoscopy. One bronchoscope was identified to be used among all affected patients with positive fungal cultures. Physical damage was found in the index bronchoscope; however, no fungus was recovered after sampling of the affected scope or the rinse water of automated endoscope washer-disinfectors. Use of the scope was halted, and, during the following 12-month period, Rhinocladiella species were not isolated from any BAL specimen. All pseudo-outbreak isolates were identified as R. similis with high genetic relatedness (>90% similarity) on ALFP analysis. The study emphasises the emergence of a rare and uncommon black yeast R. similis, with reduced susceptibility to echinocandins, in a bronchoscope-related pseudo-outbreak with a potential water-related reservoir. Our findings highlight the importance of prolonged fungal culture and species-level identification of melanised yeasts isolated from bronchoscopy samples. Possibility of healthcare-associated transmission should be considered when R. similis is involved in clinical microbiology samples.

Comparative Genomics and Molecular Analysis of Epidermophyton floccosum.

Epidermophyton floccosum is one of the most common agents of human superficial fungal infections, compared with genus Trichophyton and Microsporum, it possesses uniqueness in ecology traits and rarely causing hair infections. E. floccosum is so far the only representative species of genera Epidermophyton, and it is known as anthropophilic dermatophytes. To further reveal the genome sequences and clues of virulence factors, thus in this study, we sequenced the genome of E. floccosum (CGMCC (F) E1d), and performed comparative genomic analysis with other dermatophytes. It is revealed that E. floccosum owns the largest genome size and similar GC content compared with other dermatophytes. A total of 7565 genes are predicted. By comparing with the closest species N. gypseum, our study reveals that number and structure of adhesion factors, secreted proteases and LysM domain might contribute to the pathogenic and ecological traits of E. floccosum. Mating genes is also detected in genome data. Furthermore, we performed AFLP analysis trying to discuss intraspecific differences of E. floccosum, but no significant relationship is found between genotype and geographical distribution. Upon above, our study provides a deeper understanding and strong foundation for future researches about E. floccosum.

Contribution to determination of extracellular DNA origin in the biofilm matrix.

This study is focused on the analysis of extracellular DNA (eDNA) from a biofilm matrix formed by Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. The presence of eDNA in the biofilm of all the studied strains was confirmed by confocal laser scanning microscopy using fluorescent dyes with high affinity to nucleic acid. The protocol for eDNA isolation from the biofilm matrix was established, and subsequent characterization of the eDNA was performed. The purified eDNA obtained from the biofilm matrix of all three microorganisms was compared to the genomic DNA (gDNA) isolated from relevant planktonic grown cells. The process of eDNA isolation consisted of biofilm cultivation, its collection, sonication, membrane filtration, dialysis, lyophilisation, and extraction of DNA separated from the biofilm matrix with cetyltrimethylammonium bromide. An amplified fragment length polymorphism (AFLP) was used for comparing eDNA and gDNA. AFLP profiles showed a significant similarity between eDNA and gDNA at the strain level. The highest similarity, with a profile concordance rate of 94.7% per strain, was observed for S. aureus, L. monocytogenes, and S. enterica exhibited lower profiles similarity (78% and 60%, respectively). The obtained results support the hypothesis that the eDNA of studied bacterial species has its origin in the gDNA.

Comparative Study of the Genetic and Biochemical Variability of Polyscias filicifolia (Araliaceae) Regenerants Obtained by Indirect and Direct Somatic Embryogenesis as a Source of Triterpenes.

Polyscias filicifolia (Araliaceae) is broadly used in traditional medicine in Southeast Asia due to its antimicrobial, immunomodulating and cytotoxic activities. The main groups of compounds responsible for pharmacological effects are believed to be oleanolic triterpene saponins. However, Polyscias plants demonstrate relatively slow growth in natural conditions, which led to applying a developing sustainable source of plant material via primary (PSE), secondary (DSE) and direct somatic embryogenesis from DSE (TSE). The AFLP and metAFLP genotyping resulted in 1277 markers, amplified by a total of 24 pairs of selective primers. Only 3.13% of the markers were polymorphic. The analysis of variance showed that the PSE and TSE regenerants differed only in terms of root number, while the DSE plantlets differed for all studied morphological characteristics. Further, the chemical analysis revealed that oleanolic acid (439.72 µg/g DW), ursolic acid (111.85 µg/g DW) and hederagenin (19.07 µg/g DW) were determined in TSE regenerants. Our results indicate that direct somatic embryogenesis ensures the production of homogeneous plant material, which can serve as a potential source of triterpene compounds. Plants obtained via somatic embryogenesis could also be reintroduced into the natural environment to protect and preserve its biodiversity.

CleanBSequences: an efficient curator of biological sequences in R.

This work presents a new method and tool to solve a common problem of molecular biologists and geneticists who use molecular markers in their scientific research and developments: curation of sequences. Omic studies conducted by molecular biologists and geneticists usually involve the use of molecular markers. AFLP, cDNA-AFLP, and MSAP are examples of markers that render information at the genomics, transcriptomics, and epigenomics levels, respectively. These three types of molecular markers use adaptors that are the template for PCR amplification. The sequences of the adaptors have to be eliminated for the analysis of the results. Since a large number of sequences are usually obtained in these studies, this clean-up of the data could demand long time and work. To automate this work, an R package, named CleanBSequences, was created that allows the sequences to be curated massively, quickly, without errors and can be used offline. The curating is performed by aligning the forward and/or reverse primers or ends of cloning vectors with the sequences to be removed. After the alignment, new subsequences are generated without biological fragments not desired by the user, i.e., sequences needed by the techniques. In conclusion, the CleanBSequences tool facilitates the work of researchers, reducing time, effort, and working errors. Therefore, the present tool would respond to the problems related to the curation of sequences obtained from the use of some types of molecular markers. In addition to the above, being an open source, CleanBSequences is a flexible tool that has the potential to be used in future improvements to respond to new problems.

Development of a specific AFLP-based SCAR marker for Chinese Race 34MKG of Puccinia graminis f. sp. tritici.

Wheat stem rust, caused by Puccinia graminis f. sp. tritici (Pgt), is a fungus that causes the devastating fungalwheat stem rust disease in wheat production. Rapid identification of the physiological races of Pgt are very importance for the prevention of wheat stem rust. In this paper we developed a molecular method to identify the most prevalent race of Pgt, as a supplement for traditionally used host-specific methods. Amplified fragment length polymorphism (AFLP) was employed as a means of analyzing DNA polymorphisms in six common physiological races of Pgt in China and Ug99. In total, 64 pairs of primers were used for AFLP screening of race-specific molecular markers. One primer pair-namely, E7/M7 (5'-GACTGCGTACCAATTCG G-3'/5'-GATGAGTCCTGAGTAACGG-3')-yielded a unique band for the race 34MKG that was purified and cloned into the pGEM-T vector for sequencing. We then designed a new primer pairs (sequence-characterized amplified region marker) to amplify the 171-bp fragment and confirmed that the marker was highly specific for 34MKG. These results provide a new tool for monitoring different races of Pgt for improved control of wheat stem rust in China.

The development of species-specific AFLP-derived SCAR and SSCP markers to identify mantis shrimp species.

Mantis shrimp has become commercially valuable in many countries, while the commercially aquaculture still unsuccessful. The stable supply of the species-specific markers for precise identification can play a key role of foods authentication as well as restoring/enhancing mantis shrimp stocks in future. The aim of this research was to identify species-specific markers for Squillid and Harpiosquillid mantis shrimp taxa using Amplified fragment length polymorphism-Single strand conformation polymorphism (AFLP-SSCP) approaches. Selective amplification would be substituted as a total of 40 primer combinations was performed using either three-base (i.e., EcoRI+3 and MseI+3 in 20 primer combinations) or two-base (i.e., EcoRI+2 and MseI+2 in 20 primer combinations) selective primers. These had been size-fractionated via 6% denaturing polyacrylamide gel electrophoresis, ten AFLP fragments exhibiting species or genus-specific characteristics were cloned, sequenced, and GenBank interrogated. A primer pair was designed and their specificity was tested versus the genomic DNA of various species. Results show that the primer E+2-13/M+2-13Hr158 generated PCR products for just H. harpax, while E+3-14/M+3-2HhHr151 and E+2-13/M+2-13Hh150 generated PCR products for both H. harpax and H. raphidea and not others (i.e., M. nepa, O. oratoria, and E. woodmasoni). SSCP was then applied in order to differentiate between H. harpax and H. raphidea. These SSCP results indicate that species can be differentiated based on polymorphic fragment nucleotides. Indeed, primers E+2-13/M+2-13Hr158, E+3-14/M+3-2HhHr151, and E+2-13/M+2-13Hh150 were all successfully confirmed as present in processed mantis shrimp samples (i.e., saline-preserved and heat-dried). These results provide new species-specific markers for mantis shrimp identification.