Silicone-geranium essential oil blend for long-term antifouling coatings.

This study explores the potential of geranium essential oil as a natural solution for combating marine biofouling, addressing the environmental concerns associated with commercial antifouling coatings. Compounds with bactericidal activities were identified by 13Carbon nuclear magnetic resonance (13C NMR). Thermogravimetric analysis (TGA) revealed minimal impact on film thermal stability, maintaining suitability for antifouling applications. The addition of essential oil induced changes in the morphology of the film and Fourier transform infrared spectroscopy (FTIR) analysis indicated that oil remained within the film. Optical microscopy showed an increase in coating porosity after immersion in a marine environment. A total of 18 bacterial colonies were isolated, with Psychrobacter adeliensis and Shewanella algidipiscicola being the predominant biofilm-forming species. The geranium essential oil-based coating demonstrated the ability to reduce the formation of Psychrobacter adeliensis biofilms and effectively inhibit macrofouling adhesion for a duration of 11 months.

Therapeutic potential of antimicrobial peptides against pathogenic protozoa.

Protozoal infections cause significant morbidity and mortality in humans and animals. The use of several antiprotozoal drugs is associated with serious adverse effects and resistance development, and drugs that are more effective are urgently needed. Microorganisms, mammalian cells and fluids, insects, and reptiles are sources of antimicrobial peptides (AMPs) that act against pathogenic microorganisms; these AMPs have been widely studied as a promising alternative therapeutic option to conventional antibiotics, aiming to treat infections caused by multidrug-resistant pathogens. One advantage of AMP molecules is their adaptability, as they can be easily fine-tuned for broad-spectrum or targeted activity by changing the amino acid residues in their sequence. Consequently, these variations in structural and physicochemical properties can alter the antimicrobial activities of AMPs and decrease resistance development. This article presents an overview of peptide activities against amebiasis, giardiasis, trichomoniasis, Chagas disease, leishmaniasis, malaria, and toxoplasmosis. AMPs and their analogs demonstrate great potential as therapeutics, with potent and selective activity, when compared with commercially available drugs, and hold the potential to act as new scaffolds for the development of novel anti-protozoal drugs.

The genus Anoxybacillus: an emerging and versatile source of valuable biotechnological products.

Thermophilic and alkaliphilic microorganisms are unique organisms that possess remarkable survival strategies, enabling them to thrive on a diverse range of substrates. Anoxybacillus, a genus of thermophilic and alkaliphilic bacteria, encompasses 24 species and 2 subspecies. In recent years, extensive research has unveiled the diverse array of thermostable enzymes within this relatively new genus, holding significant potential for industrial and environmental applications. The biomass of Anoxybacillus has demonstrated promising results in bioremediation techniques, while the recently discovered metabolites have exhibited potential in medicinal experiments. This review aims to provide an overview of the key experimental findings related to the biotechnological applications utilizing bacteria from the Anoxybacillus genus.

Metarhizium anisopliae E6 secretome reveals molecular players in host specificity and toxicity linked to cattle tick infection.

Although Metarhizium anisopliae is one of the most studied fungal biocontrol agents, its infection mechanism is far from being completely understood. Using multidimensional protein identification technology (MudPIT), we evaluated the differential secretome of M. anisopliae E6 induced by the host Rhipicephalus microplus cuticle. The proteomic result showed changes in the expression of 194 proteins after exposure to host cuticle, such as proteins involved in adhesion, penetration, stress and fungal defense. Further, we performed a comparative genomic distribution of differentially expressed proteins of the M. anisopliae secretome against another arthropod pathogen, using the Beauveria bassiana ARSEF2860 protein repertory. Among 47 analyzed protein families, thirty were overexpressed in the M. anisopliae E6 predicted genome compared to B. bassiana. An in vivo toxicity assay using a Galleria mellonella model confirmed that the M. anisopliae E6 secretome was more toxic in cattle tick infections compared to other secretomes, including B. bassiana with cattle ticks and M. anisopliae E6 with the insect Dysdereus peruvianus, which our proteomic results had also suggested. These results help explain molecular aspects associated with host infection specificity due to genetic differences and gene expression control at the protein level in arthropod-pathogenic fungi.

High microbiome variability in pediatric tracheostomy cannulas in patients with similar clinical characteristics

Abstract Objectives: To evaluate the bacterial microbiome found in tracheostomy cannulas of a group of children diagnosed with glossoptosis secondary to Robin Sequence (RS), and its clinical implications. Methods: Pediatric patients were enrolled in the study at the time of the cannula change in the hospital. During this procedure, the removed cannula was collected and stored for amplicon sequencing of 16s rRNA. DNA extraction was performed using DNeasy PowerBiofilm Kit (QIAGEN® - Cat nº 24000-50) while sequencing was performed with the S5 (Ion S5™ System, Thermo Fisher Scientific), following Brazilian Microbiome Project (BMP) protocol. Results: All 12 patients included in the study were using tracheostomy uncuffed cannulas of the same brand, had tracheostomy performed for over 1-year and had used the removed cannula for approximately 3-months. Most abundant genera found were Aggregatibacter, Pseudomonas, Haemophilus, Neisseria, Staphylococcus, Fusobacterium, Moraxella, Streptococcus, Alloiococcus, and Capnocytophaga. Individual microbiome of each individual was highly variable, not correlating to any particular clinical characteristic. Conclusion: The microbiome of tracheostomy cannulas is highly variable, even among patients with similar clinical characteristics, making it challenging to determine a standard for normality. © 2022 Associa¸c˜ao Brasileira de Otorrinolaringologia e Cirurgia C´ervico-Facial. Published by Elsevier Editora Ltda. This is an open access article under the CC BY license (https://creativecommons.org/licenses/by/4.0/).

Metabolomic Strategies to Improve Chemical Information from OSMAC Studies of Endophytic Fungi.

Metabolomics strategies are important tools to get holistic chemical information from a system, but they are scarcely applied to endophytic fungi to understand their chemical profiles of biosynthesized metabolites. Here Penicillium sp. was cultured using One Strain Many Compounds (OSMAC) conditions as a model system to demonstrate how this strategy can help in understanding metabolic profiles and determining bioactive metabolites with the application of metabolomics and statistical analyses, as well as molecular networking. Penicillium sp. was fermented in different culture media and the crude extracts from mycelial biomass (CEm) and broth (CEb) were obtained, evaluated against bacterial strains (Staphylococcus aureus and Pseudomonas aeruginosa), and the metabolomic profiles by LC-DAD-MS were obtained and chemometrics statistical analyses were applied. The CEm and CEb extracts presented different chemical profiles and antibacterial activities; the highest activities observed were against S. aureus from CEm (MIC = 16, 64, and 128 µg/mL). The antibacterial properties from the extracts were impacted for culture media from which the strain was fermented. From the Volcano plot analysis, it was possible to determine statistically the most relevant features for the antibacterial activity, which were also confirmed from biplots of PCA as strong features for the bioactive extracts. These compounds included 75 (13-oxoverruculogen isomer), 78 (austalide P acid), 87 (austalide L or W), 88 (helvamide), 92 (viridicatumtoxin A), 96 (austalide P), 101 (dihydroaustalide K), 106 (austalide k), 110 (spirohexaline), and 112 (pre-viridicatumtoxin). Thus, these features included diketopiperazines, meroterpenoids, and polyketides, such as indole alkaloids, austalides, and viridicatumtoxin A, a rare tetracycline.

High microbiome variability in pediatric tracheostomy cannulas in patients with similar clinical characteristics.

OBJECTIVES: To evaluate the bacterial microbiome found in tracheostomy cannulas of a group of children diagnosed with glossoptosis secondary to Robin Sequence (RS), and its clinical implications. METHODS: Pediatric patients were enrolled in the study at the time of the cannula change in the hospital. During this procedure, the removed cannula was collected and stored for amplicon sequencing of 16s rRNA. DNA extraction was performed using DNeasy PowerBiofilm Kit (QIAGEN® ‒ Cat nº 24000-50) while sequencing was performed with the S5 (Ion S5™ System, Thermo Fisher Scientific), following Brazilian Microbiome Project (BMP) protocol. RESULTS: All 12 patients included in the study were using tracheostomy uncuffed cannulas of the same brand, had tracheostomy performed for over 1-year and had used the removed cannula for approximately 3-months. Most abundant genera found were Aggregatibacter, Pseudomonas, Haemophilus, Neisseria, Staphylococcus, Fusobacterium, Moraxella, Streptococcus, Alloiococcus, and Capnocytophaga. Individual microbiome of each individual was highly variable, not correlating to any particular clinical characteristic. CONCLUSION: The microbiome of tracheostomy cannulas is highly variable, even among patients with similar clinical characteristics, making it challenging to determine a standard for normality.

Antifouling activity of isonitrosoacetanilides against microfouling and macrofouling.

Biofouling is responsible for structural and economic damage to man-made surfaces. Antifouling paints with biocides have been applied to structures to avoid organism adhesion; however, they have high toxicity and are not able to prevent all biofouling processes, necessitating the periodic mechanical removal of organisms and paint reapplication. Thus, there is an urgent demand for novel, effective, and environmentally friendly antifouling alternatives. As isonitrosoacetanilide is the precursor for many compounds with antibacterial activity, we believe that it could have antifouling activity against microfouling and, consequently, against macrofouling. The aim of this work was to investigate the antifouling potential of six isonitrosoacetanilide compounds and their toxicity. The compounds were employed at different concentrations (0.625-1.25-2.5-5-10 µg mL-1) in this study. The biofilm and planktonic bacteria inhibition and biofilm eradication potential were evaluated by crystal violet assay, while Amphibalus amphitrite barnacle settlement was evaluated by cyprid settlement assay. Toxicity evaluation (LC50 and EC50) was performed with A. amphitrite nauplii II and cyprid larvae. At least one of the tested concentrations of 4-Br-INA, 4-CH3-INA, and 2-Br-INA compounds showed nontoxic antifouling activity against microfouling (antibiofilm) and macrofouling (antisettlement). However, only 4-CH3-INA and 2-Br-INA also showed biofilm eradication potential. These compounds with antibiofilm activity and nontoxic effects could be combined with acrylic base paint resin or added directly into commercial paints in place of toxicant biocides to cover artificial structures as friendly antifouling agents.

Endophytic Fungi as a Source of Antibacterial Compounds-A Focus on Gram-Negative Bacteria.

Bacterial resistance has become one of the main motives in the worldwide race for undescribed antibacterial agents. The difficulties in the treatment of bacterial infections are a public health issue that increasingly highlights the need for antimicrobial agents. Endophytic microorganisms are a promising alternative in the search for drugs, due to the vast number of metabolites produced with unique characteristics and bioactive potential. This review highlights the importance of endophytic microorganisms as a source of secondary metabolites in the search for active molecules against bacteria of medical importance, with a special focus on gram-negative species. This fact is supported by the findings raised in this review, which brings an arsenal of 166 molecules with characterized chemical structures and their antibacterial activities. In addition, the low cost, ease of maintenance, and optimization-controlled fermentation conditions favor reproducibility in commercial scale. Given their importance, it is necessary to intensify the search for new molecules from endophytic microorganisms, and to increasingly invest in this very promising font.

Mass Spectrometry Metabolomics Approach Reveals Anti-Trichomonas vaginalis Scaffolds from Marine Fungi.

Trichomoniasis is the most common non-viral sexually transmitted infection (STI) in the world caused by Trichomonas vaginalis. Failures in the treatment with the 5-nitroimidazole class including parasite resistance to metronidazole elicit new alternatives. Marine natural products are sources of several relevant molecules, presenting a variety of metabolites with numerous biological activities. In this work, we evaluated the anti-T. vaginalis activity of fungi associated with marine invertebrates by mass spectrometry-based metabolomics approaches. After screening of six marine fungi, extract from Penicillium citrinum FMPV 15 has shown to be 100% active against T. vaginalis, and the gel permeation column on Sephadex LH-20® yielded twelve organic fractions which five showed to be active. Metabolomics and statistical analyses were performed with all the samples (extract and fractions), and several compounds were suggested to be related to the activity. These components include citrinin, dicitrinin C, citreoisocoumarin, dihydrocitrinone, decarboxycitrinin, penicitrinone C, and others. The minimum inhibitory concentration (MIC) value of anti-T. vaginalis activity of citrinin was 200 µM. The marine fungi metabolites show potential as new alternatives to overcome drug resistance in T. vaginalis infections.

Iron chelation and inhibition of metallopeptidases mediate anti-Trichomonas vaginalis activity by a novel 8-hydroxyquinoline derivative.

Trichomoniasis is a neglected parasitic infection, with no oral therapeutic alternatives to overcome the pitfalls of currently approved drugs. In this context, the search for new anti-Trichomonas vaginalis drugs is imperative. Here we report the selective anti-T. vaginalis activity of a substituted 8-hydroxyquinoline-5-sulfonamide derivative. Six different derivatives were evaluated for anti-T. vaginalis. In vitro and in vivo toxicity methods, association with metal ions, and investigation on the mechanism of action were performed with the most active derivative, PH 152. Cytotoxicity assays showed selectivity for the parasite and the low toxicity was confirmed in G. mellonella larvae model. The mode of action is related to iron chelation by disrupting Fe-S clusters-dependent enzyme activities in the parasite. Proteomic analysis indicated inhibition of metallopeptidases related to T. vaginalis virulence mechanisms and metabolic pathways. PH 152 presented selective trichomonacidal activity through multitarget action.

Anti-Trichomonas vaginalis activity and chemical analysis of metabolites produced by marine-associated fungi.

Trichomoniasis is the most common non-viral sexually transmitted infection worldwide and it may have serious consequences, especially for women. Currently, 5-nitroimidazole drugs are the treatment of choice for trichomoniasis, although presenting adverse effects and reported cases of drug resistance. Metabolites isolated from marine fungi have attracted considerable attention due to their unique chemical structures with diverse biological activities, including antiprotozoal activity. In this study, we showed the anti-Trichomonas vaginalis activity of fractions obtained from marine fungi and the chemical composition of the most active fraction was determined. Ethyl acetate fractions of the fungus Aspergillus niger (EAE03) and Trichoderma harzianum/Hypocrea lixii complex (EAE09) were active against T. vaginalis. These samples, EAE03 and EAE09, were also effective against the fresh clinical isolate metronidazole-resistant TV-LACM2R, presenting MIC values of 2.0 mg/mL and 1.0 mg/mL, respectively. The same MIC values were found against ATCC 30,236 T. vaginalis isolate. In vitro cytotoxicity revealed only the fraction named EAE03 with no cytotoxic effect; however, the active fractions did not promote a significant hemolytic effect after 1-h incubation. Already, the in vivo toxicity evaluation using Galleria mellonella larvae demonstrated that none of the tested samples caused a reduction in animal survival. The fraction EAE03 was followed for purification steps and analyzed by LC-DAD-MS. Eleven compounds were annotated, including butyrolactone, butanolide, and atromentin. Overall, the range of activities reported confirms the potential of marine fungi to produce bioactive molecules.

A new Kunitz trypsin inhibitor from Erythrina poeppigiana exhibits antimicrobial and antibiofilm properties against bacteria.

Erythrina poeppigiana belongs to Fabaceae family (subfamily Papillionoideae) and is commonly found in tropical and subtropical regions in Brazil. Herein, we described the purification and characterization of a new Kunitz-type inhibitor, obtained from E. poeppigiana seeds (EpTI). EpTI is composed by three isoforms of identical amino-terminal sequences with a molecular weight ranging from 17 to 20 kDa. The physicochemical features showed by EpTI are common to Kunitz inhibitors, including the dissociation constant (13.1 nM), stability against thermal (37-100 °C) and pH (2-10) ranging, and the presence of disulfide bonds stabilizing its reactive site. Furthermore, we investigated the antimicrobial, anti-adhesion, and anti-biofilm properties of EpTI against Gram-positive and negative bacteria. The inhibitor showed antimicrobial activity with a minimum inhibitory concentration (MIC, 5-10 µM) and minimum bactericidal concentration (MBC) of 10 µM for Enterobacter aerogenes, Enterobacter cloacae, Klebsiella pneumoniae, Staphylococcus aureus, and Staphylococcus haemolyticus. The combination of EpTI with ciprofloxacin showed a marked synergistic effect, reducing the antibiotic concentration by 150%. The increase in crystal violet uptake for S. aureus and K. pneumoniae strains was approximately 30% and 50%, respectively, suggesting that the bacteria plasma membrane is targeted by EpTI. Treatment with EpTI at 1x and 10 x MIC significantly reduced the biofilm formation and prompted the disruption of a mature biofilm. At MIC/2, EpTI decreased the bacterial adhesion to polystyrene surface within 2 h. Finally, EpTI showed low toxicity in animal model Galleria mellonella. Given its antimicrobial and anti-biofilm properties, the EpTI sequence might be used to design novel drug prototypes.

Capsicumicine, a New Bioinspired Peptide from Red Peppers Prevents Staphylococcal Biofilm In Vitro and In Vivo via a Matrix Anti-Assembly Mechanism of Action.

Staphylococci are pathogenic biofilm-forming bacteria and a source of multidrug resistance and/or tolerance causing a broad spectrum of infections. These bacteria are enclosed in a matrix that allows them to colonize medical devices, such as catheters and tissues, and that protects against antibiotics and immune systems. Advances in antibiofilm strategies for targeting this matrix are therefore extremely relevant. Here, we describe the development of the Capsicum pepper bioinspired peptide "capsicumicine." By using microbiological, microscopic, and nuclear magnetic resonance (NMR) approaches, we demonstrate that capsicumicine strongly prevents methicillin-resistant Staphylococcus epidermidis biofilm via an extracellular "matrix anti-assembly" mechanism of action. The results were confirmed in vivo in a translational preclinical model that mimics medical device-related infection. Since capsicumicine is not cytotoxic, it is a promising candidate for complementary treatment of infectious diseases. IMPORTANCE Pathogenic biofilms are a global health care concern, as they can cause extensive antibiotic resistance, morbidity, mortality, and thereby substantial economic loss. So far, no effective treatments targeting the bacteria in biofilms have been developed. Plants are constantly attacked by a wide range of pathogens and have protective factors, such as peptides, to defend themselves. These peptides are common components in Capsicum baccatum (red pepper). Here, we provide insights into an antibiofilm strategy based on the development of capsicumicine, a natural peptide that strongly controls biofilm formation by Staphylococcus epidermidis, the most prevalent pathogen in device-related infections.

Interfering with cholesterol metabolism impairs tick embryo development and turns eggs susceptible to bacterial colonization.

Cholesterol is a known precursor of arthropod molecules such as the hormone 20-hydroxyecdysone and the antimicrobial boophiline, a component of tick egg wax coat. Because the cholesterol biosynthetic pathway is absent in ticks, it is necessarily obtained from the blood meal, in a still poorly understood process. In contrast, dietary cholesterol absorption is better studied in insects, and many proteins are involved in its metabolism, including Niemann-Pick C (NPC) transporter and acyl-CoA:cholesterol acyltransferase (ACAT), as well as enzymes to convert between free cholesterol and esterified cholesterol. The present work addresses the hypothesis that tick viability can be impaired by interfering with cholesterol metabolism, proposing this route as a target for novel tick control methods. Two drugs, ezetimibe (NPC inhibitor) and avasimibe (ACAT inhibitor) were added to calf blood and used to artificially feed Rhipicephalus microplus females. Results show that, after ingesting avasimibe, tick reproductive ability and egg development are impaired. Also, eggs laid by females fed with avasimibe did not hatch and were susceptible to Pseudomonas aeruginosa adhesion and biofilm formation in their surfaces. The immunoprotective potential of ACAT against ticks was also accessed using two selected ACAT peptides. Antibodies against these peptides were used to artificially feed female ticks, but no deleterious effects were observed. Taken together, data presented here support the hypothesis that enzymes and other proteins involved in cholesterol metabolism are suitable as targets for tick control methods.

Trans-Translation Is an Appealing Target for the Development of New Antimicrobial Compounds.

Because of the ever-increasing multidrug resistance in microorganisms, it is crucial that we find and develop new antibiotics, especially molecules with different targets and mechanisms of action than those of the antibiotics in use today. Translation is a fundamental process that uses a large portion of the cell's energy, and the ribosome is already the target of more than half of the antibiotics in clinical use. However, this process is highly regulated, and its quality control machinery is actively studied as a possible target for new inhibitors. In bacteria, ribosomal stalling is a frequent event that jeopardizes bacterial wellness, and the most severe form occurs when ribosomes stall at the 3'-end of mRNA molecules devoid of a stop codon. Trans-translation is the principal and most sophisticated quality control mechanism for solving this problem, which would otherwise result in inefficient or even toxic protein synthesis. It is based on the complex made by tmRNA and SmpB, and because trans-translation is absent in eukaryotes, but necessary for bacterial fitness or survival, it is an exciting and realistic target for new antibiotics. Here, we describe the current and future prospects for developing what we hope will be a novel generation of trans-translation inhibitors.

Bacteria-invertebrate interactions as an asset in developing new antifouling coatings for man-made aquatic surfaces.

Economic losses can result from biofouling establishment on man-made structures. Macrofouling causes damage to artificial substrates, which justifies the need for its control. However, the antifouling coatings employed nowadays are typically not safe for the environment. Microfouling can affect macrofouling colonization, and thus represents a potential target for alternative antifouling control. From both ecological and economical points of view, information on the ecology and interactions between micro- and macrofouling are crucial to develop successful and safe control strategies, which will prevent biofouling development on man-made structures while preserving water quality and the safety of non-target organisms. This study presents a metabarcoding analysis of biofilm-associated marine bacteria (16S-rRNA-gene) and fungi (ITS-region), with the aim to understand invertebrate settlement over time on hard substrates exposed to natural condition (Control) and two treatments (Antimicrobials and Antifouling Painted). Biofouling composition changed with exposure time (up to 12 days) and showed differences among Control and Antimicrobials and Painted treatments. Antimicrobial treatment influenced more the biofouling composition than traditional antifouling paint (Cu2O-based). Both treatments caused microbial resistance. Macrofouling establishment was strongly influenced by Gram-negative heterotrophic bacteria (mostly Proteobacteria and Bacteroidetes). Nevertheless, each macrofouling taxon settled in response to a specific biofilm bacterial composition, although other factors can also affect the biofouling community as the condition of the substrate. We suggest that proper friendly antifouling technologies should be focused on inhibiting bacterial biofilm adhesion.

The antivirulence compound myricetin possesses remarkable synergistic effect with antibacterials upon multidrug resistant Staphylococcus aureus.

Staphylococcus aureus is an opportunistic pathogen involved in several human diseases and presents ability to produce many virulence factors and resistance to antibacterial agents. One of the current strategies to combat such multidrug resistant bacteria is the antibacterial combination therapy. Myricetin is a flavonoid capable of inhibiting several S. aureus virulence factors without influencing on bacterial growth. Therefore, the combination of antibacterials with the antivirulence compound myricetin may provide a positive interaction to control multidrug resistant-bacteria. This work aims to evaluate the effect of the combination of myricetin with oxacillin and vancomycin against methicillin resistant S. aureus (MRSA) and vancomycin intermediate resistant S. aureus (VISA) strains. Concentrations used in combination assays were determined according to the minimum inhibitory concentration (MIC) for antibacterials and to the biofilm minimum inhibitory concentration (BMIC) for myricetin. Checkerboard evaluations showed reduction in MIC for antibacterials in presence of myricetin and time-kill assays confirmed the synergism for these combinations, except for VISA strain when the flavonoid was combined with vancomycin. Importantly, when myricetin was combined with oxacillin, MRSA strain became susceptible to the antibacterial. Myricetin did not reduce staphyloxanthin production, indicating that the oxacillin susceptibility seems not to be related to this step of functional membrane microdomains. In vivo evaluations using Galleria mellonella confirmed the efficacy of oxacillin plus myricetin in treatment of MRSA infected-larvae when compared to the control groups, increasing in 20% host survival. The present work points out the potential of antibacterial and antivirulence compounds combinations as new alternative to control infections by multidrug resistant-bacteria.

Pseudonajide peptide derived from snake venom alters cell envelope integrity interfering on biofilm formation in Staphylococcus epidermidis.

BACKGROUND: The increase in bacterial resistance phenotype cases is a global health problem. New strategies must be explored by the scientific community in order to create new treatment alternatives. Animal venoms are a good source for antimicrobial peptides (AMPs), which are excellent candidates for new antimicrobial drug development. Cathelicidin-related antimicrobial peptides (CRAMPs) from snake venoms have been studied as a model for the design of new antimicrobial pharmaceuticals against bacterial infections. RESULTS: In this study we present an 11 amino acid-long peptide, named pseudonajide, which is derived from a Pseudonaja textilis venom peptide and has antimicrobial and antibiofilm activity against Staphylococcus epidermidis. Pseudonajide was selected based on the sequence alignments of various snake venom peptides that displayed activity against bacteria. Antibiofilm activity assays with pseudonajide concentrations ranging from 3.12 to 100 µM showed that the lowest concentration to inhibit biofilm formation was 25 µM. Microscopy analysis demonstrated that pseudonajide interacts with the bacterial cell envelope, disrupting the cell walls and membranes, leading to morphological defects in prokaryotes. CONCLUSIONS: Our results suggest that pseudonajide's positives charges interact with negatively charged cell wall components of S. epidermidis, leading to cell damage and inhibiting biofilm formation.

Trichomonas vaginalis NTPDase inhibited by lycorine modulates the parasite-neutrophil interaction.

Lycorine is an Amaryllidaceae alkaloid that presents anti-Trichomonas vaginalis activity. T. vaginalis causes trichomoniasis, the most common non-viral sexually transmitted infection. The modulation of T. vaginalis purinergic signaling through the ectonucleotidases, nucleoside triphosphate diphosphohydrolase (NTPDase), and ecto-5'-nucleotidase represents new targets for combating the parasite. With this knowledge, the aim of this study was to investigate whether NTPDase and ecto-5'-nucleotidase inhibition by lycorine could lead to extracellular ATP accumulation. Moreover, the lycorine effect on the reactive oxygen species (ROS) production by neutrophils and parasites was evaluated as well as the alkaloid toxicity. The metabolism of purines was assessed by HPLC. ROS production was measured by flow cytometry. Cytotoxicity against epithelial vaginal cells and fibroblasts was tested, as well as the hemolytic effect of lycorine and its in vivo toxicity in Galleria mellonella larvae. Our findings showed that lycorine caused ATP accumulation due to NTPDase inhibition. The alkaloid did not affect the ROS production by T. vaginalis; however, it increased ROS levels in neutrophils incubated with lycorine-treated trophozoites. Lycorine was cytotoxic against vaginal epithelial cells and fibroblasts; conversely, it was not hemolytic neither exhibited toxicity against the in vivo model of G. mellonella larvae. Overall, besides having anti-T. vaginalis activity, lycorine modulates ectonucleotidases and stimulates neutrophils to secrete ROS. This mechanism of action exerted by the alkaloid could enhance the susceptibility of T. vaginalis to host immune cell, contributing to protozoan clearance.