[Identification and antifungal activity of halophilic bacteria isolated from saline soils in Campeche, México]. / Identificación y actividad antifúngica de bacterias halófilas aisladas de suelos salinos en Campeche, México.

Phytopathogenic fungi Alternaria alternata and Colletotrichum gloeosporioides cause diseases in plant tissues as well as significant postharvest losses. The use of chemical fungicides for their control has negative effects on health and the environment. Secondary metabolites from halophilic bacteria are a promising alternative for new antifungal compounds. In the present study, halophilic bacteria were isolated and characterized from two sites with saline soils called branquizales in Campeche, Mexico. A total of 64 bacteria were isolated. Agrobacterium, Bacillus, Inquilinus, Gracilibacillus, Metabacillus, Neobacillus, Paenibacillus, Priestia, Staphylococcus, Streptomyces and Virgibacillus were among the identified genera. The antifungal potential of the culture supernatant (CS) of 39 halophilic bacteria was investigated against C. gloeosporioides and A. alternata. The bacteria showing the greatest inhibition of mycelial growth corresponded to Bacillus subtilis CPO 4292, Metabacillus sp. CPO 4266, Bacillus sp. CPO 4295 and Bacillus sp. CPO 4279. The CS of Bacillus sp. CPO 4279 exhibited the highest activity and its ethyl acetate extract (AcOEt) inhibited the germination of C. gloeosporioides, with IC50 values of 8,630µg/ml and IC90 of 10,720µg/ml. The organic partition of the AcOEt extract led to three fractions, with acetonitrile (FAcB9) showing the highest antifungal activity, with values exceeding 66%. Halophilic bacteria from 'blanquizales' soils of the genus Bacillus sp. produce metabolites with antifungal properties that inhibit the phytopathogenic fungus C. gloeosporioides.

Establishment of a screening platform based on human coronavirus OC43 for the identification of microbial natural products with antiviral activity.

IMPORTANCE: The COVID-19 pandemic has revealed the lack of effective treatments against betacoronaviruses and the urgent need for new broad-spectrum antivirals. Natural products are a valuable source of bioactive compounds with pharmaceutical potential that may lead to the discovery of new antiviral agents. Specifically, compared to conventional synthetic molecules, microbial natural extracts possess a unique and vast chemical diversity and are amenable to large-scale production. The implementation of a high-throughput screening platform using the betacoronavirus OC43 in a human cell line infection model has provided proof of concept of the approach and has allowed for the rapid and efficient evaluation of 1,280 microbial extracts. The identification of several active compounds validates the potential of the platform for the search for new compounds with antiviral capacity.

Pigmented Microbial Extract (PMB) from Exiguobacterium Species MB2 Strain (PMB1) and Bacillus subtilis Strain MB1 (PMB2) Inhibited Breast Cancer Cells Growth In Vivo and In Vitro.

Breast cancer (BC) continues to be one of the major causes of cancer deaths in women. Progress has been made in targeting hormone and growth factor receptor-positive BCs with clinical efficacy and success. However, little progress has been made to develop a clinically viable treatment for the triple-negative BC cases (TNBCs). The current study aims to identify potent agents that can target TNBCs. Extracts from microbial sources have been reported to contain pharmacological agents that can selectively inhibit cancer cell growth. We have screened and identified pigmented microbial extracts (PMBs) that can inhibit BC cell proliferation by targeting legumain (LGMN). LGMN is an oncogenic protein expressed not only in malignant cells but also in tumor microenvironment cells, including tumor-associated macrophages. An LGMN inhibition assay was performed, and microbial extracts were evaluated for in vitro anticancer activity in BC cell lines, angiogenesis assay with chick chorioallantoic membrane (CAM), and tumor xenograft models in Swiss albino mice. We have identified that PMB from the Exiguobacterium (PMB1), inhibits BC growth more potently than PMB2, from the Bacillus subtilis strain. The analysis of PMB1 by GC-MS showed the presence of a variety of fatty acids and fatty-acid derivatives, small molecule phenolics, and aldehydes. PMB1 inhibited the activity of oncogenic legumain in BC cells and induced cell cycle arrest and apoptosis. PMB1 reduced the angiogenesis and inhibited BC cell migration. In mice, intraperitoneal administration of PMB1 retarded the growth of xenografted Ehrlich ascites mammary tumors and mitigated the proliferation of tumor cells in the peritoneal cavity in vivo. In summary, our findings demonstrate the high antitumor potential of PMB1.

A Simple Antifungal assay for Testing Actinomycetes and Other Microbial Extracts.

Antifungal assay in vitro is a useful tool for the characterization of biological activity of microbial extracts. Here we describe a simple in vitro test at two final extract concentrations that allows long-term storage of the plates containing dry extracts before using. The assay protocol is described for two fungal strains, a unicellular yeast, with clinical interest (Candida albicans), and a sporulated and phytopathogenic filamentous fungus (Botrytis cinerea). They could serve as models for adapting other filamentous/yeast-like fungi.Plates are prepared by placing 100 and 10 µg, respectively, of the organic extracts in microtiter 96-well plates, where the test will be performed. The assay develops by adding 200 µL of a spore suspension 104 spores/mL for B. cinerea and 106 cells/mL for C. albicans in Sabouraud medium.After the incubation of the plates at 25 °C, for 2 days for C. albicans and 5 days for B. cinerea , the growth of the fungal targets is evaluated in a plate reader for unicellular yeast , or visually under the microscope for filamentous fungi. If visually evaluated, observed growth can be assigned to different categories by comparison with growth control and inhibition control. Inhibition effect on C. albicans at eight concentrations of amphotericin B (8-4-2-1-0.5-0.25-0.125-0.00625 µg/mL) or B. cinerea exposed of eight concentrations of iprodione (100-50-10-5-1-0.5-0.1-0.05 µg/mL) are used as inhibition controls, respectively.

A Novel Fluorescence-Based Screen for Inhibitors of the Initiation of DNA Replication in Bacteria.

BACKGROUND: One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited. MATERIALS AND METHODS: Using various genetic tools, we constructed a novel high throughput, cellbased, fluorescence screen for inhibitors of chromosome replication initiation in bacteria. RESULTS: The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA. We also demonstrated that neither tetracycline nor ciprofloxacin triggers a false positive result. Finally, 400 extracts isolated mainly from filamentous actinomycetes were subjected to the screen. CONCLUSION: We concluded that the presented screen is applicable for identifying putative inhibitors of DNA replication initiation in a high throughput setup.

High-throughput screening platform for natural product-based drug discovery against 3 neglected tropical diseases: human African trypanosomiasis, leishmaniasis, and Chagas disease.

African trypanosomiasis, leishmaniasis, and Chagas disease are 3 neglected tropical diseases for which current therapeutic interventions are inadequate or toxic. There is an urgent need to find new lead compounds against these diseases. Most drug discovery strategies rely on high-throughput screening (HTS) of synthetic chemical libraries using phenotypic and target-based approaches. Combinatorial chemistry libraries contain hundreds of thousands of compounds; however, they lack the structural diversity required to find entirely novel chemotypes. Natural products, in contrast, are a highly underexplored pool of unique chemical diversity that can serve as excellent templates for the synthesis of novel, biologically active molecules. We report here a validated HTS platform for the screening of microbial extracts against the 3 diseases. We have used this platform in a pilot project to screen a subset (5976) of microbial extracts from the MEDINA Natural Products library. Tandem liquid chromatography-mass spectrometry showed that 48 extracts contain potentially new compounds that are currently undergoing de-replication for future isolation and characterization. Known active components included actinomycin D, bafilomycin B1, chromomycin A3, echinomycin, hygrolidin, and nonactins, among others. The report here is, to our knowledge, the first HTS of microbial natural product extracts against the above-mentioned kinetoplastid parasites.

In vitro anticancer activity of microbial isolates from diverse habitats / In vitro atividade anticancerígena de isolados microbianos de diversos habitats

Extracts from natural products, especially microorganisms, have served as a valuable source of diverse molecules in many drug discovery efforts and led to the discovery of several important drugs. Identification of microbial strains having promising biological activities and purifying the bio-molecules responsible for the activities, have led to the discovery of many bioactive molecules. Extracellular, as well as intracellular, extracts of the metabolites of thirty-six bacterial and twenty-four fungal isolates, grown under unusual conditions such as high temperature, high salt and low sugar concentrations, were in vitro tested for their cytotoxic potential on various cancer cell lines. The extracts were screened on HeLa and MCF-7 cell lines to study the cytotoxic potential. Nuclear staining and flow cytometric studies were carried out to assess the potential of the extracts in arresting the cell cycle. The crude ethylacetate extract of isolate F-21 showed promising results by MTT assay with IC50 as low as 20.37±0.36 µg/mL on HeLa, and 44.75±0.81 µg/mL on MCF-7 cells, comparable with Cisplatin. The isolate F-21 was identified as Aspergillus sp. Promising results were also obtained with B-2C and B-4E strains. Morphological studies, biochemical tests and preliminary chemical investigation of the extracts were also carried out.

Extratos de produtos naturais, especialmente de microrganismos, constituíram-se em fonte valiosa de diversas moléculas em muitas descobertas de fármacos e levaram à descoberta de fármacos importantes. A identificação de espécies microbianas que apresentam atividade biológica e a purificação de biomoléculas responsáveis pelas atividades levou à descoberta de muitas moléculas bioativas. Extratos extracelulares tanto quanto intracelulares de metabólitos de 36 isolados de bactérias e 24 isolados de fungos, que cresceram sob condições não usuais, como alta temperatura, alta concentração de sal e baixa concentração de açúcar, foram testados in vitro quanto ao seu potencial citotóxico em várias linhagens de câncer. Os extratos foram ensaiados em células HeLa e MCF-7 para o estudo do potencial citotóxico. A coloração nuclear e os estudos de citometria de fluxo foram realizados para avaliar o potencial dos extratos em bloquear o ciclo celular. O extrato bruto em acetato de etila do isolado F-21 mostrou resultados promissores no ensaio de MTT, com IC50 de 20,37±0,36 µg/mL em células HeLa e 44,.75±0,81 µg/mL em células MCF-7, comparativamente à cisplatina. O isolado F-21 foi identificado como Aspergillus sp. Resultados promissores foram obtidos com cepas B-2C e B-4E. Realizaram-se, também, estudos morfológicos, testes bioquímicos e investigação química preliminar dos extratos.