Brazilian Journal of Microbiology最新文献

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.

Lignocellulosic materials used in the food industry as containers or packaging can suffer fungal biodeterioration. For this reason, the aim of this study was to evaluate the superficial growth of Aspergillus ellipticus on lignocellulosic materials of food containers and packaging. A strain of A. ellipticus was selected for its high productivity of lignocellulolytic enzymes. Fragments the diameter of a 110 mm Petri dish were cut from six types of lignocellulosic materials used as supports: coated cardboard, uncoated cardboard, corrugated cardboard, kraft paper, cupcake wrapper and Whatman 5 filter paper (positive control). The thickness (µm) and initial pH were measured for each material by contacting 2 g of material with 100 mL of distilled water at 15 °C and 40 °C and 10 µL of a spore suspension (10⁶ spores/mL) was inoculated and incubated at 32 °C and 87% relative humidity in a desiccator with saturated barium solution. For 21 days, the diameter of the colony (mm) was measured growing on lignocellulosic materials. The pH of the materials was between 7.2 and 7.4, considering them neutral materials. The growth of the fungal colony was between 2 mm and 38.6 mm, being higher on filter paper, followed by kraft paper, corrugated cardboard, uncoated cardboard, coated cardboard and cupcake wrapper. It is concluded that fungal biodeterioration of lignocellulosic packaging material for food can occur in conditions similar to those of tropical climates.

The present research was intended to identify the bioactive molecules from the ethanolic extract of T. arjuna leaf against a diverse array of Vibrio species by means of a bioassay-guided fractionation. The antibacterial efficacy of compounds was assessed by the microdilution technique, while the brine shrimp mortality assay was employed to determine their toxicity. Following an initial screening, the ethanol extract underwent silica gel chromatography, succeeded by reversed phase HPLC, to identify the most potent fraction towards V. parahaemolyticus. Through further UHPLC-orbitrap-ion trap mass spectrometry, three likely compounds were identified, demonstrating broad-spectrum efficiency against several Vibrio species, including V. parahaemolyticus, V. harveyi, V. alginolyticus, V. vulnificus, and V. anguillarum. Among the bacteria, V. vulnificus was the most sensitive species (IC₅₀ ranged from 17 to 25 µg/mL) while V. harveyi was the most resistant species (IC₅₀ > 200 µg/mL) for the compounds. The toxicity test findings indicated that the compounds tested were not detrimental to shrimp. This is one of the most important reports identifying these specific fatty acid derivatives with anti-Vibrio activity in shrimp. The findings of the current study suggest that the leaf extract of T. arjuna may include bioactive compounds that might serve as potential complementary or prophylactic agents for the treatment of pathogenic Vibrio species in shrimp aquaculture.

Bovine Viral Diarrhea (BVD) significantly impacts cattle production and reproduction, causing substantial economic losses in the livestock industry. The etiological agent is a virus from the Flaviviridae family, genus Pestivirus, which is globally distributed with a prevalence of 60-85% in South American cattle herds. A relevant characteristic of this disesase is to produce immunotolerant animals (persistently infected - PI) that would not detected and eliminate a large amount of viruses in the herd, being the primary source of viral transmission. Early viral detection in the herd would allow a more effective control program. Here, we compared traditional molecular techniques (conventional PCR and Real-time PCR) to one of the latest generation (droplet digital PCR, ddPCR) which has shown significant advantages over the aforementioned ones. Serum samples from 46 animals, previously tested for BVD using conventional PCR and including both positive and negative results, were used for this comparison. Additionally, the NADL BVDV reference strain and a synthetic plasmid were used as positive controls, while non-template controls were used as negative controls. ddPCR showed higher sensitivity and precision for the detection and quantification of BVDV. ddPCR Limit of Detection was 0.24 copies/µL (286.3 × 10^-6 ηg/µL) for NADL BVDV reference strain, outperforming previous methods by an order of magnitude. The results showed that droplet digital PCR is a robust tool, with higher sensitivity, specificity and reproducibility, and it can be helpful when used in BVD control programs using pools of serum or bulk milk tanks.

In this study, we characterized 86 plant growth-promoting bacterial strains belonging to the genus Nitrospirillum, isolated from diverse host plants and geographic regions. We investigated their evolutionary relationships through phylogenetic analyses of the 16S rRNA and recA genes, complemented by phylogenomic approaches incorporating genomic similarity metrics, such as ANI and dDDH. The classification of type strains was further supported by in silico analyses of chemotaxonomic markers, particularly genes involved in fatty acid biosynthesis and elongation, phospholipid and quinone production, and nitrogen fixation (nifHDK operon). Phenotypic and chemotaxonomic characterization was performed using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry, fatty acid methyl ester (FAME) profiling, and physiological assays. These included evaluations of nitrogen fixation capacity, antibiotic resistance, carbon source utilization, and enzymatic activities. This integrative approach provided detailed insight into the characteristics and diversity of the studied strains. Phylogenetic and genomic analyses revealed six novel taxa within the genus Nitrospirillum, in addition to the previously described species N. amazonense, N. iridis, and N. viridazoti. The distinctiveness of these new lineages was supported by both genomic metrics and phenotypic traits. All novel strains also exhibited activity of nitrogenase enzyme, confirming their nitrogen-fixing ability under in vitro conditions. Based on these findings, we propose the formal description of six novel species: Nitrospirillum bahiense sp. nov. (= BR 11865^T, = UCCCB 233^T), Nitrospirillum guanabarense sp. nov. (= BR 11163^T, = UCCCB 228^T), Nitrospirillum guaranorum sp. nov. (= BR 11164^T, = UCCCB 229^T), Nitrospirillum karajorum sp. nov. (= BR 11752^T, = UCCCB 231^T), Nitrospirillum goiasense sp. nov. (= BR 11828^T, = UCCCB 232^T), and Nitrospirillum pindoramense sp. nov. (= BR 11622^T, = UCCCB 230^T).

The global spread of multi-drug-resistant bacteria, including strains resistant to both polymyxin and carbapenems, poses a significant threat to public health, highlighting the need to develop new antimicrobial agents. This study assessed the antimicrobial potential of cinnamaldehyde, alone and in combination with antibiotics, against carbapenem-polymyxin-resistant K. pneumoniae strains (CPR-Kp). The antimicrobial activity was assessed through minimum inhibitory concentration (MIC), checkerboard assay, and survival curve analysis. Cinnamaldehyde showed inhibitory effects (MIC 281 µg/mL), and when combined with polymyxin B, resulted in synergistic effects, effectively overcoming resistance to both polymyxin and carbapenem. Notably, cinnamaldehyde (70 µg/mL) combined with polymyxin B (1 µg/mL) led to a significant reduction in the MIC of polymyxin B, from 64 µg/mL to 1 µg/mL, with a fractional inhibitory concentration index (FICI) of 0.26, indicating synergy. The ZIP synergy score analysis further corroborated these findings, revealing a global synergy score of 32.728, with the highest values observed at cinnamaldehyde concentrations of 70-140 µg/mL in combination with polymyxin B. Similarly, in vivo the combination of cinnamaldehyde (30 or 100 mg/kg) with polymyxin B (2 mg/kg) significantly reduced bacterial loads in blood and peritoneal lavage (p < 0.0001) and improved survival rates. These findings underscore the efficacy of cinnamaldehyde as an adjuvant to polymyxin B in treating infections caused by CPR-Kp. The observed synergistic effect suggests that cinnamaldehyde as a pivotal component in future therapeutic formulations, providing a promising avenue for further research in combating antimicrobial resistance.