微生物学

Endophytic actinomycetes, particularly Streptomyces species, have gained significant attention due to their potential to produce novel bioactive compounds. In this study, we isolated and characterized an endophytic Streptomyces sp. VITGV100 from the tomato plant (Lycopersicon esculentum), employing the direct streak method and whole-genome sequencing. A genome analysis was done to uncover its biosynthetic potential and identify indole-type compounds. The strain's secondary metabolite production was evaluated through GC–MS analysis, and its antimicrobial activity was tested against selected human pathogenic bacteria. Our protocol outlines a comprehensive approach, describing the isolation and extraction of metabolites and genome mining for indole-type compounds. This isolate has potential pharmaceutical applications, accelerating the discovery of novel indole-type bioactive compounds.

Since the creation of the Global Polio Eradication Initiative (GPEI) in 1988, significant progress has been made toward attaining a poliovirus-free world. This has resulted in the eradication of wild poliovirus (WPV) serotypes two (WPV2) and three (WPV3) and limited transmission of serotype one (WPV1) in Pakistan and Afghanistan. However, the increased emergence of circulating vaccine-derived poliovirus (cVDPV) and the continued circulation of WPV1, although limited to two countries, pose a continuous threat of international spread of poliovirus. These challenges highlight the need to further strengthen surveillance and outbreak responses, particularly in the African Region (AFRO). Phylogeographic visualization tools may provide insights into changes in poliovirus epidemiology, which can in turn guide the implementation of more strategic and effective supplementary immunization activities and improved outbreak response and surveillance. We created a comprehensive protocol for the phylogeographic analysis of polioviruses using Nextstrain, a powerful open-source tool for real-time interactive visualization of virus sequencing data. It is expected that this protocol will support poliovirus elimination strategies in AFRO and contribute significantly to global eradication strategies. These tools have been utilized for other pathogens of public health importance, for example, SARS-CoV-2, human influenza, Ebola, and Mpox, among others, through real-time tracking of pathogen evolution (https://nextstrain.org), harnessing the scientific and public health potential of pathogen genome data.

This manuscript details two modified protocols for the isolation of long-stranded or high molecular weight (HMW) DNA from Magnaporthaceae (Ascomycota) fungal mycelium intended for whole genome sequencing. The Cytiva Nucleon PhytoPure and the Macherey-Nagel NucleoBond HMW DNA kits were selected because the former requires lower amounts of starting material and the latter utilizes gentler methods to maximize DNA length, albeit at a higher requirement for input material. The Cytiva Nucleon PhytoPure kit successfully recovered HMW DNA for half of our fungal species by increasing the amount of RNase A treatment and adding in a proteinase K treatment. To reduce the impact of pigmentation development, which occurs toward later stages of culturing, extractions were run in quadruplicate to increase overall DNA concentration. We also adapted the Macherey-Nagel NucleoBond HMW DNA kit for high-quality HMW DNA by grinding the sample to a fine powder, overnight lysis, and splitting the sample before washing the precipitated DNA. For both kits, precipitated DNA was spooled out pre-washing, ensuring a higher percentage of high-integrity long strands. The Macherey-Nagel protocol offers advantages over the first through the utilization of gravity columns that provide gentler treatment, yielding >50% of high-purity DNA strands exceeding 40 kbp. The limitation of this method is the requirement for a large quantity of starting material (1 g). By triaging samples based on the rate of growth relative to the accumulation of secondary metabolites, our methodologies hold promise for yielding reliable and high-quality HMW DNA from a variety of fungal samples, improving sequencing outcomes.