Brazilian Journal of Microbiology最新文献

Influenza A virus (IAV) is present in most swine-producing countries causing production losses and concerns on public health. In Brazil, influenza is endemic in pig herds, and a great genetic diversity has been described in swine IAVs due to multiple introductions of pre-2009 human-seasonal IAVs followed by reassortment events with 2009 pandemic H1N1 (H1N1pdm) virus. Here, we compile 14 years of IAV monitoring data and describe the subtypes and major lineages of H1 and H3 viruses co-circulating in Brazilian pigs. Using multiplex RT-qPCR and sequencing, we identified H1N1pdm as the most frequently detected virus, accounting for 41.3% of the subtyped samples (165/399), followed by H1huN2 (108/399), H3N2 (77/399), and H1N1hu (9/399). The three dominant subtypes were detected co-circulating annually and consistently in seven of the nine states sampled, as well as among pigs at different production phases. Other reassortants were found sporadically and included H1pdmN2 (22/399) and H1huN1pdm (4/399). The high diversity observed indicates that IAVs from distinct lineages are widely disseminated across the country. These findings strongly suggest substantial movement of pigs between regions and states, which may have implications for vaccine design, disease control, and updating of diagnostic tests. Continuous efforts to monitor IAV are crucial to better understand their ecology and to generate relevant data for pandemic preparedness.

The bovine uterus hosts a diverse microbiome whose role in reproductive physiology and pathology is increasingly recognized. While Bacillus species have been occasionally isolated from the uterus, their biofilm and exopolysaccharide (EPS) forming capabilities have not been systematically characterized. In this study, four Bacillus strains (BU13, BU14, BU15, and BU16) were isolated from the bovine uteri and examined for their taxonomic affiliation, phenotypic characteristics, EPS production, biofilm formation, and antibiotic susceptibility. Phylogenetic analyses based on nearly full-length 16S rRNA gene sequences revealed that all isolates belonged to the B. subtilis group, with BU13, BU14, and BU16 closely related to B. licheniformis, and BU15 related to B. amyloliquefaciens and B. siamensis. BU13 and BU16 demonstrated high levels of EPS and biofilm production, especially in sucrose-supplemented media and under nutrient-rich conditions. Notably, these strains also exhibited relatively smaller inhibition zones against β-lactam antibiotics, which may be associated with their robust EPS-biofilm phenotypes. In contrast, larger inhibition zones were observed with gentamicin, enrofloxacin, and trimethoprim. These findings underscore the importance of characterizing commensal Bacillus spp. in the uterus and highlight that certain strains may possess traits that facilitate persistence and reduce antimicrobial responsiveness. This is the first study to comprehensively evaluate the biofilm-forming potential of uterine Bacillus isolates and provides a foundation for future investigations into their role in reproductive health and disease.

Candida parapsilosis is a common cause of candidiasis worldwide, with biofilm formation and secretion of aspartic proteases (Saps) as key virulence factors. Conversely, serine protease secretion by this fungus is poorly understood. In this study, we investigated the secretion of serine-type proteases by planktonic- and biofilm-forming cells of C. parapsilosis cultured in brain heart infusion (BHI) medium. Cell-free supernatant from the reference strain (ATCC 22019) was screened against various serine protease substrates, revealing pronounced activity toward N-benzoyl-Phe-Val-Arg-pNa (0.74 nmol pNA.mg^- 1.min^- 1), with optimal activity at pH 9.0 and temperatures between 32 °C and 40 °C. Proteolytic activity was significantly reduced by serine protease inhibitors PMSF (32.8%), TLCK (40.2%) and benzamidine (50.7%), while inhibitors of other protease classes had no effect, confirming its serine-type specificity. Notably, serine protease activity was detected in supernatants from cells grown in BHI but absent in those cultured in albumin-supplemented yeast carbon base medium, a known inducer of Saps, suggesting culture-dependent regulation of protease expression. Serine protease activity also increased over time, rising from 0.36 pNA.mg^- 1.min^- 1 at 24-hour to 1.14 pNA.mg^- 1.min^- 1 at 72-hour. Clinical isolates of C. parapsilosis exhibited significantly higher serine protease activity than the reference strain under optimal conditions. Serine-type protease activity was also detected in the supernatant of mature biofilms, showing a correlation with metabolic activity and biomass. Infection of Galleria mellonella larvae with C. parapsilosis isolates revealed no correlation between larval mortality and serine protease production. These findings suggest that C. parapsilosis serine proteases contribute to fungal growth and biofilm development, representing potential targets for antifungal intervention.

This study investigates the immobilization of crude cellulase from Aspergillus foetidus, produced through solid-state fermentation by different methods, including entrapment in agar cube, adsorption on agar xerogel, and adsorption on agar xerogel pretreated with glutaraldehyde. The immobilization efficiency achieved was 90.80%, 84.08%, and 84.23%, while enzyme activity obtained was 1.12 IU/g, 1.52 IU/g, and 1.48 IU/g for crude cellulase immobilized by entrapment in agar cube, adsorption on agar xerogel, and adsorption on agar xerogel pretreated with glutaraldehyde respectively. The immobilized crude cellulase was characterized, revealing an optimal temperature shift from 50 °C to 60 °C for agar xerogel-adsorbed crude cellulase, which improved its thermal stability. The pH optimum for free and all immobilized crude cellulase was observed at pH 4. Also, all immobilized crude cellulases retained significant activity even after multiple cycles, depicting their reusability. The enzyme kinetics observed were K_m of 11.71 mg/mL, 57.0 mg/mL, 14.86 mg/mL, 62.32 mg/mL, while V_max was 4.20 µmol/mL/min, 6.58 µmol/mL/min, 3.25 µmol/mL/min, and 5.78 µmol/mL/min for free crude cellulase, crude cellulase immobilized by entrapment in agar cube, crude cellulase adsorbed on agar xerogel, and crude cellulase adsorbed on agar xerogel pretreated with glutaraldehyde respectively. The free and immobilized crude cellulases were applied for carrot juice extraction which showed increased juice yield, clarity, and reducing sugar content while reduced viscosity compared to untreated samples. These findings highlight agar xerogel as an encouraging support for cellulase immobilization, presenting a sustainable and economical method for enzyme reuse in food processing.

Campylobacter fetus is an important etiological agent of reproductive infections in cattle, which can be managed through vaccination. Nevertheless, the efficacy of bovine reproductive vaccines against C. fetus remains uncertain due to the absence of standardized guidelines and variable vaccine formulations. Thus, this study aimed to quantify and compare C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) in six commercial reproductive vaccines current available in Brazil in 2022. We observed significant differences in the estimated number of genome copies of Cff and Cfv among the tested vaccines. Analysis of vaccine cost and antigen concentration revealed no direct correlation between price and C. fetus antigen concentration. These findings underscore the discrepancies in antigen titration among bovine reproductive vaccines and highlight the lack of informative labeling in current commercial products. Altogether, there is a pressing need for standardized guidelines for C. fetus vaccinal doses to ensure consistent bacterial loads and efficient immunization of the herds. Nevertheless, more studies regarding vaccine efficiency are required to ensure an appropriate immune response in cattle herds.

Paratuberculosis (PTB) is a chronic intestinal disease affecting ruminants and somenon-ruminants, caused by Mycobacterium avium subsp. paratuberculosis (MAP). In the Sudan, published data on the incidence and prevalence of PTB are Limited. we detected MAP in human patients with gastrointestinal complaints highlights its zoonotic potential and raises public health concerns. This study aimed at assessing PTB prevalence in cattle and identifying risk factors for MAP infection as well as investigating the phylogeny of MAP circulating in the Sudan. Both serum and faecal samples were collected from the same individual animals of 810 cattle in 153 herds in five states spanning three regions (Southern, Northern, and Central) of the country. ELISA was used to detect MAP antibodies in sera, while faecal samples were tested for MAP DNA using a recombinase aided amplification (RAA) assay and cultured for MAP isolation followed by partial sequencing of MAP insertion sequence 1311 with subsequent phylogeny analysis. At the animal level, the apparent prevalence was 5.0% for ELISA and 4.2% for RAA, with true prevalence estimates of 8.5% and 4.8%, respectively. At the herd level, apparent prevalence was 28.2% for ELISA and 22.3% for RAA, while true prevalence reached 54.2% for ELISA and 24.9% for RAA. Significant (P < 0.05) risk factors for MAP infection included exposure to wild animals and high rainfall. Phylogenetic analysis of the Sudanese MAP isolates revealed close relatedness to type S (I/III) strains worldwide suggesting a shared evolutionary origin. The present study provides baseline data on PTB prevalence and risk factors in Sudanese cattle, emphasising the role of environmental and management factors in disease dynamics. These findings highlight the necessity of adopting targeted control strategies to reduce MAP impact on cattle and other animals as well as to prevent its potential public health hazard.

Sabinene is a type of monoterpene that is widely used in flavors, fragrances and pharmaceuticals. Though sabinene biosynthesis has been investigated in a variety of microorganisms, application of sabinene is still limited due to its high production cost and lesser yielding strains. The baker's yeast Saccharomyces cerevisiae, which is generally recognized as safe (GRAS), is a suitable cell factory for the food and beverage industries. In this study, we aimed to enhance the production of sabinene from corn hydrolysates by employing genetic engineering techniques on S. cerevisiae. Here, we engineered S. cerevisiae for the production of sabinene by overexpressing sabinene synthase (SabS) and geranyl diphosphate synthase (GPPS) via CRISPR-Cas9, which is a simple and efficient tool for targeted and marker-free genome engineering. Subsequently, the culture medium and process conditions were optimized to enhance sabinene production and achieve ~ 23.6 mg/L under flask fermentation conditions. Based on the optimized culture conditions, we further investigated the production of sabinene from corn hydrolysates, which is a major source of dietary nutrients worldwide and an inexpensive source of sugars, and a high-level production of 60.0 mg/L was achieved in shake-flask fermentation. Our results implied that corn hydrolysates was a suitable medium for sabinene production and that CRISPR-Cas9 could boost the marker-free engineered yeast strain, which was more suitable for the food and beverage industry. Altogether, our work represents the progress in the bioproduction of food-grade sabinene from an inexpensive raw material.

The emergence of SARS-CoV-2 marked the onset of the COVID-19 pandemic, which has challenged public health worldwide. Mass testing performed by different assays represents an essential strategy to control virus spread, especially in low-income regions. This study aimed to standardize and validate RT-LAMP as an alternative tool for SARS-CoV-2 detection. Different sets of primers were assessed in silico and in vitro, and the N2 target was selected for the validation stage. The extracted RNA from the clinical samples was used to standardize the RT-LAMP fluorometric assay with BST 3.0 and RT enzymes, and the colorimetric assay was performed with WarmStart^® Colorimetric LAMP Master Mix. Clinical samples were also subjected to the Wondfo 2019-nCoV antigen test. The proposed protocols showed robust diagnostic accuracy in high-viral-load samples (C_T ≤ 30), 98.25% sensitivity and 90.91% specificity for fluorometric assay, and 92.98% sensitivity and 100% specificity for colorimetric assay. Considering the two visualization techniques, the fluorometric technique yielded more accordant results. In contrast, the rapid antigen test presented a lower performance, with 82.46% sensitivity and 100% specificity in samples with a C_T score ≤ 30. RT-LAMP visualization with a simple ultraviolet light source showed high sensitivity in active infection patient samples. The faster and simplest execution, cost-effectiveness, and accessible interpretation of the results are favorable points for improving this diagnostic tool as a potential screening tool for active transmission of COVID-19, especially in regions with limited financial resources.

To adapt to cold environments, such as the Arctic and Antarctic regions, high mountain peaks, cold soils, deserts, caverns, and cryopegs, fungi employ various strategies. Crucial aspect of this adaptation is maintaining the functions of their cell membranes. To study the mechanisms of cell membrane protection, we analyzed the composition of osmolytes and membrane lipids and their fatty acids in a submerged culture of the psychrotolerant fungus Psychrophilomyces antarcticus during growth at temperatures of 20 °C and 8 °C. The object of the study exhibits a broad growth range from - 3 °C to 27 °C, with an optimal temperature of 20-22.5 °C. For adaptation to cold (8 °C), the fungus significantly increases the degree of unsaturation of phospholipids by enhancing the proportion of α-linolenic acid (40% of the total), compared to the optimal temperature (15% of the total). Additionally, the proportion of sterols in the membrane lipids is twice as low, and the amount of arabitol in the composition of osmolytes is twice as high, compared to the optimal temperature. At both temperatures, the fungus is characterized by the dominance of two osmolytes in the cytosol: trehalose and arabitol. It also shows a predominance of non-bilayer phospholipids, specifically phosphatidic acids (35-42% of the total) and phosphatidylethanolamines (~ 20% of the total), in its membrane lipid composition, along with trace amounts of sterol esters and the formation of mucus in the culture fluid. The combined adjustments in osmolyte and membrane lipid profiles contribute to the fungus' adaptation to a wide range of temperatures. These findings provide insights into the molecular basis of psychrotolerance and open up opportunities for research into potential biotechnological applications.

Among the 2,692 orchid species endemic to Brazil, Laelia tenebrosa has significant economic value because of the beauty of its flowers and the size of the plant. Despite being listed as endangered, it is one of the most cultivated Laelia species. Anthracnose is one of the main diseases affecting orchids. The fungus responsible for the disease, Colletotrichum sp., can spread efficiently in orchid collections in Brazil owing to favorable temperature and humidity conditions. To date, 46 species of Colletotrichum have been identified as the causal agents of anthracnose in various orchid species worldwide. In December 2018, flowers of Laelia tenebrosa (Orchidaceae) with necrotic spots were collected from the orchidarium maintained at the coffee nursery of the Universidade Federal de Viçosa (UFV), State of Minas Gerais, Brazil. The fungus was identified based on morphological characteristics and phylogenetic analyses using Bayesian inference, and the maximum likelihood method was performed using sequences from each region (ACT, CHS, GAPDH, ITS, and TUB2) and concatenated sequences. The disease is caused by Colletotrichum orchidophilum, a species known to infect Bletilla striata in China, Cycnoches aureum in Panama, Dendrobium sp. in Thailand and the United States, Phalaenopsis sp. in the United Kingdom, x Ascocenda sp. in the United States, and Reunion Island as the causal agent of black spots on Vanilla planifolia. For the first time, C. orchidophilum was reported in Brazil and was the first to cause anthracnose in Laelia worldwide.