Antimicrobial Discovery Research Articles

The Effect of Cytotoxicity and Antimicrobial of Synthesized CuO NPs from Propolis on HEK-293 Cells and Lactobacillus acidophilus.

Drug resistance is currently possible anywhere in the world. Due to the discovery of antimicrobials, medicine, and health have made tremendous advances over the past several decades. This research evaluated the antimicrobial and cytotoxicity effects of green synthesis of copper oxide nanoparticles (CuO NPs) on Lactobacillus acidophilus and human embryonic kidney 293 cells (HEK). Method and Materials. Propolis was sampled and extracted. Green synthesis of CuO NPs was synthesized and characterized using SEM, TEM, DLS, BET, and zeta potential methods. L. acidophilus (ATCC 4356) was used, and the antimicrobial tests were carried out at different concentrations (10≥ mg/ml). Moreover, the cytotoxicity was evaluated using an MTT assay on human embryonic kidney 293 cells (HEK). Synthesized CuO NPs using propolis extracts from Khalkhal (sample 1) and Gillan (sample 2) showed -13.2 and -14.4 mV, respectively. The hydrodynamic sizes of well-dispersed samples 1 and 2 were 3124.9 nm and 1726.7 nm, respectively. According to BET analysis, samples 1 and 2 had 5.37 and 8.45 m2/g surface area, respectively. The surface area was decreased due to the addition of propolis extract, and the pore size was increased. CuO NPs of samples 1 and 2 were visible on SEM images with diameters ranging from 75 to 145 nm and 120 to 155 nm, respectively. Based on TEM analysis, the size of CuO particles was increased in samples 1 and 2. CuO NPs particles had narrow size distributions with evenly dispersed NPs on all sides. The cell viability of the CuO NPs of samples 1 and 2 after 24, 48, and 72 hours was greater than 50%. As a result of the MIC and MBC tests, it was determined that samples 1 and 2 had the same effect against L. acidophilus (0.0024 mg/ml). Biofilm formation and degradation of sample 1 were more efficient against L. acidophilus. There was no evidence of cytotoxicity in the samples. In addition, results showed that the green synthesized CuO NPs from Khalkhal propolis were effective against L. acidophilus. Thus, the green synthesized CuO NPs from Khalkhal propolis were the best candidates for clinical application.

Evaluation of some o-benzenedisulfonimido–sulfonamide derivatives as potent antimicrobial agents

Background and Aims: The discovery of new antimicrobials to overcome antimicrobial resistance has always been an important topic for sustainable world health. Since the sulfonamide carrying heterocyclic compounds present a number of advantages as biologically active compounds, in our work reported herein, a small collection of previously synthesized o-benzenedisulfonimido-sulfonamide derivatives were assayed to determine antimicrobial profiles against ten different microorganisms in search of finding promising new antibacterial/antifungal agents. Methods: Eight compounds and their standards were tested against seven bacterial and three fungal strains, including members of Gram-positive, Gram-negative bacteria, and Candida spp., using the microbroth dilution method to measure their MIC (minimum inhibitory concentration) values. Results: All assayed molecules showed different inhibitory effects on ten different targets, with considerable MIC values. Particularly, compound 2 exhibited better antimicrobial activity against the largest number of assayed microorganisms. Conclusion: Further modification and development of o-benzenedisulfonimido-sulfonamide derivatives and additional in vitro studies against putative targets may result in new antimicrobial drug candidates in the near future.

Evaluation of antimicrobial activity of Streptomyces pactum isolated from paddy soils and identification of bioactive volatile compounds by GC-MS analysis.

The objective of this study was to isolate Streptomyces sp. from north paddy soils of Iran and investigation and identification of the bioactive compounds by carrying out GC-MS analysis. Antimicrobial activity was investigated by well diffusion agar technique against the pathogenic microorganisms including Enterococcus faecalis ATCC 29212, Micrococcus luteus ATCC 4698, Bacillus cereus ATCC 11778, Staphylococcus aureus ATCC 9144, Pseudomonas aeruginosa ATCC 27853, Proteus mirabilis ATCC 43071. Based on the results of gene sequencing of gene 16S rRNA and phylogenetic analysis, the isolated sample belongs to the genus Streptomyces with the highest degree of resemblance (99/87%) to the Streptomyces pactum strain ACT12. The isolate showed a broad spectrum of antibacterial activity against test microorganisms. This isolate showed maximum antibacterial activity against M. luteus (25 ± 0.5 mm) and the most resistant microorganism against antibacterial activity of this isolate was P. aeruginosa (9 ± 0.5 mm). The contact bioautography method was used to detect compounds that were responsible for antimicrobial activity and showed the active compounds with Rf values of 0.8-0.9. The identification of bioactive metabolites were performed using gas chromatography-mass spectrometry (GC-MS). GC-MS analysis of the extract showed the presence of 15 volatile compounds. The main compounds were Methyl-3-(3,5-ditertbutyl-4-hydroxyphenyl) (10.88%) and Dibutyl phthalate (8.34%) in comparison with other bioactive compounds. The results showed that north paddy soils of Iran are a rich source of microbial flora for the production of antimicrobial compounds and useful for antimicrobial compounds discovery from Streptomyces sp.

In vitro antimicrobial activity of Bixa orellana L . Leaves extract against anaerobic bacteria associated to bacterial vaginosis and Lactobacillus spp.

Motivation for the study: bacterial vaginosis is a bacterial infection that frequently affects women of reproductive age. The treatment is based on synthetic antimicrobials. Bixa orellana L. possesses antimicrobial properties and could represent a potential non-synthetic therapeutic alternative. Main findings: in vitro results suggest that, methanolic extract of Bixa orellana L. leaves possesses potential antimicrobial properties against bacteria associated to bacterial vaginosis. Implications: to identify new sources with therapeutic potential, and to promote research, discovery, and characterization of non-synthetic antimicrobials. To describe the in vitro antimicrobial activity of the methanolic extract of Bixa orellana L. leaves against anaerobic bacteria associated to bacterial vaginosis and Lactobacillus spp. Eight ATCC reference strains; Gardnerella vaginalis, Prevotella bivia, Peptococcus niger, Peptostreptococcus anaerobius, Mobiluncus curtisii, Atopobium vaginae, Veillonella parvula, and Lactobacillus crispatus, and twenty-two clinical isolates; eleven Gardnerella vaginalis and eleven Lactobacillus strains, were included in the study. The antimicrobial susceptibility was determined by the agar diffusion method. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) were determined by using agar dilution and a modified dilution plating method, respectively. All ATCC reference strains showed high levels of susceptibility to the extract, except P. vibia, V. parvula and L. crispatus. Interestingly, all G. vaginalis clinical isolates and the G. vaginalis ATTC strain were the most susceptible to the extract, given their low MIC (1.0 - 2.0 mg/mL) and MBC (1.0 - 4.0 mg/mL) values, whereas, the Lactobacillus spp. clinical isolates and the L. crispatus ATCC strain were the least susceptible bacteria given their high MIC (32.0 mg/mL) and MBC (≥ 32.0 mg/mL) values. In vitro experiments suggest that the extract possesses selective antimicrobial properties given its high activity against bacterial vaginosis-associated anaerobic bacteria and low activity against Lactobacillus species.

A Dual Detergent Strategy to Capture a Bacterial Outer Membrane Proteome in Peptidiscs for Characterization by Mass Spectrometry and Binding Assays.

The outer membrane of Gram-negative bacteria plays a critical role in protecting the cell against external stressors, including antibiotics, and therefore is a prime target for antimicrobial discovery. To facilitate the discovery efforts, a precise knowledge of the outer membrane proteome, and possible variations during pathogenesis, is important. Characterization of the bacterial outer membrane remain challenging, however, and low throughput, due to the high hydrophobicity and relatively low abundance of this cell compartment. Here we adapt our peptidisc-based method to selectively isolate the outer membrane proteome before analysis by mass spectrometry. Using a dual detergent membrane solubilization approach, followed by protein purification in peptidiscs, we capture over 70 outer membrane proteins, including 26 integral β-barrels and 26 lipoproteins. Many of these proteins are present at high peptide intensities, indicative of a high abundance in the library sample. We further show that the isolated outer membrane proteome can be employed in downstream ligand-binding assays. This peptidisc library made of outer membrane proteins may therefore be useful to systematically survey other bacterial outer membrane proteomes, but also as a nanoparticle format able to support the discovery of next-generation antimicrobials. Data are available via ProteomeXchange identifier PXD036749.

β-Lactamase Suppression as a Strategy to Target Methicillin-Resistant Staphylococcus aureus: Proof of Concept.

β-Lactamase (penicillinase) renders early, natural β-lactams like penicillin G useless against methicillin-resistant Staphylococcus aureus (MRSA), which also expresses PBP2a, responsible for resistance to semisynthetic, penicillinase-insensitive β-lactams like oxacillin. Antimicrobial discovery is difficult, and resistance exists against most treatment options. Enhancing β-lactams against MRSA would revive its clinical utility. Most research on antimicrobial enhancement against MRSA focuses on oxacillin due to β-lactamase expression. Yet, Moreillon and others have demonstrated that penicillin G is as potent against a β-lactamase gene knockout strain, as vancomycin is against wild-type MRSA. Penicillin G overcame PBP2a because β-lactamase activity was blocked. Additionally, animals treated with a combination of direct β-lactamase inhibitors like sulbactam and clavulanate with penicillin G developed resistant infections, clearly demonstrating that direct inhibition of β-lactamase is not a good strategy. Here, we show that 50 μM pyrimidine-2-amines (P2As) reduce the minimum inhibitory concentration (MIC) of penicillin G against MRSA strains by up to 16-fold by reducing β-lactamase activity but not by direct inhibition of the enzyme. Oxacillin was not enhanced due to PBP2a expression, demonstrating the advantage of penicillin G over penicillinase-insensitive β-lactams. P2As modulate an unknown global regulator but not established antimicrobial-enhancement targets Stk1 and VraS. P2As are a practical implementation of Moreillon's principle of suppressing β-lactamase activity to make penicillin G useful against MRSA, without employing direct enzyme inhibitors.

Antibiotic discovery in the artificial intelligence era.

As the global burden of antibiotic resistance continues to grow, creative approaches to antibiotic discovery are needed to accelerate the development of novel medicines. A rapidly progressing computational revolution-artificial intelligence-offers an optimistic path forward due to its ability to alleviate bottlenecks in the antibiotic discovery pipeline. In this review, we discuss how advancements in artificial intelligence are reinvigorating the adoption of past antibiotic discovery models-namely natural product exploration and small molecule screening. We then explore the application of contemporary machine learning approaches to emerging areas of antibiotic discovery, including antibacterial systems biology, drug combination development, antimicrobial peptide discovery, and mechanism of action prediction. Lastly, we propose a call to action for open access of high-quality screening datasets and interdisciplinary collaboration to accelerate the rate at which machine learning models can be trained and new antibiotic drugs can be developed.

Identification of novel inhibitors against Escherichia coli utilizing HisC as a target from histidine biosynthesis pathway

Escherichia coli is a gram-negative bacterial pathogen that poses a significant challenge both clinically and epidemiologically. Large numbers of multi-drug resistant E. coli have emerged in the last decade, because of the selection pressure generated by the inadequate use of antibiotics. Although research to combat antibiotic resistance has been going on extensively but still lags in the rate of development of newer antibiotics. Therefore, newer approaches are required to speed up the rate of discovery of antibiotics. Computational methods for screening of inhibitors have made a significant contribution to the discovery of novel antimicrobials. The present study utilized histidinol-phospho aminotransferase (HisC) as a target. HisC is an enzyme that plays a crucial role in the biosynthesis of histidine and its absence in mammals makes it an attractive drug target. A ZINC library of 5000 natural compounds was screened against HisC (PDB ID: 1FG7) using PyRx and the first 500 hits were selected for secondary screening after sorting the result on the basis of binding score. Fifteen compounds passed the secondary filter ADME and out of these five passed toxicity filters; the best among five hits was selected on the basis of its binding score and inhibition constants. Further, molecular dynamics simulations and free binding were computed of selected five compounds and two natural compounds ZINC402598829 and ZINC31157928 complexed with HisC were found as highly stable. Overall, our results indicated that these natural sources could be used as potential HisC inhibitors. Communicated by Ramaswamy H. Sarma

Lipid-A-dependent and cholesterol-dependent dynamics properties of liposomes from gram-negative bacteria in ESKAPE

AntiMicrobial Resistance (AMR) is a worldwide health emergency. ESKAPE pathogens include the most relevant AMR bacterial families. In particular, Gram-negative bacteria stand out due to their cell envelope complexity which exhibits strong resistance to antimicrobials. A key element for AMR is the chemical structure of lipid A, modulating the physico-chemical properties of the membrane and permeability to antibiotics. Liposomes are used as models of bacterial membrane infective vesicles. In this work, coarse-grained molecular dynamics simulations were used to model liposomes from ESKAPE Gram-negative bacteria (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa). We captured the role of lipid A, cardiolipin and cholesterol on liposome morphology and physico-chemical properties. Additionally, the reported antimicrobial peptides Cecropin B1, JB95, and PTCDA1-kf, were used to unveil their implications on membrane disruption. This study opens a promising starting point to understand molecular keys of bacterial membranes and to promote the discovery of new antimicrobials to overcome AMR.

Direct Detection of Antibacterial-Producing Soil Isolates Utilizing a Novel High-Throughput Screening Assay.

The ever-increasing global threat of common infections developing resistance to current therapeutics is rapidly accelerating the onset of a primitive post-antibiotic era in medicine. The prevention of further antimicrobial resistance development is unlikely due to the continued misuse of antibiotics, augmented by the lack of discovery of novel antibiotics. Screening large libraries of synthetic compounds have yet to offer effective replacements for current antibiotics. Due to historical successes, discovery from large and diverse natural sources and, more specifically, environmental bacteria, may still yield novel alternative antibiotics. However, the process of antibiotic discovery from natural sources is laborious and time-consuming as a result of outdated methodologies. Therefore, we have developed a simple and rapid preliminary screening assay to identify antibacterial-producing bacteria from natural sources. In brief, the assay utilizes the presence or absence of luminescence in bioluminescent reporter bacteria and test bacterium co-cultures in a 96-well plate format to determine the absence or presence of antibacterial compound production. Our assay, called the bioluminescent simultaneous antagonism (BSLA) assay, can accurately distinguish between known antibacterial-producing and non-producing test bacteria. The BSLA assay was validated by screening 264 unknown soil isolates which resulted in the identification of 10 antibacterial-producing isolates, effectively decreasing the pool of isolates for downstream analysis by 96%. By design, the assay is simple and requires only general laboratory equipment; however, we have shown that the assay can be scaled to automated high-throughput screening systems. Taken together, the BSLA assay allows for the rapid pre-screening of unknown bacterial isolates which, when coupled with innovative downstream dereplication and identification technologies, can effectively fast-track antimicrobial discovery.

Antibody-Based Immunotherapies as a Tool for Tackling Multidrug-Resistant Bacterial Infections.

The discovery of antimicrobials is an outstanding achievement of mankind that led to the development of modern medicine. However, increasing antimicrobial resistance observed worldwide is rendering commercially available antimicrobials ineffective. This problem results from the bacterial ability to adapt to selective pressure, leading to the development or acquisition of multiple types of resistance mechanisms that can severely affect the efficacy of antimicrobials. The misuse, over-prescription, and poor treatment adherence by patients are factors strongly aggravating this issue, with an epidemic of infections untreatable by first-line therapies occurring over decades. Alternatives are required to tackle this problem, and immunotherapies are emerging as pathogen-specific and nonresistance-generating alternatives to antimicrobials. In this work, four types of antibody formats and their potential for the development of antibody-based immunotherapies against bacteria are discussed. These antibody isotypes include conventional mammalian polyclonal antibodies that are used for the neutralization of toxins; conventional mammalian monoclonal antibodies that currently have 100 IgG mAbs approved for therapeutic use; immunoglobulin Y found in birds and an excellent source of high-quality polyclonal antibodies able to be purified noninvasively from egg yolks; and single domain antibodies (also known as nanobodies), a recently discovered antibody format (found in camelids and nurse sharks) that allows for a low-cost synthesis in microbial systems, access to hidden or hard-to-reach epitopes, and exhibits a high modularity for the development of complex structures.

From drugs to targets: Reverse engineering the virtual screening process on a proteomic scale

Phenotypic screening is a powerful technique that allowed the discovery of antimicrobials to fight infectious diseases considered deadly less than a century ago. In high throughput phenotypic screening assays, thousands of compounds are tested for their capacity to inhibit microbial growth in-vitro. After an active compound is found, identifying the molecular target is the next step. Knowing the specific target is key for understanding its mechanism of action, and essential for future drug development. Moreover, this knowledge allows drug developers to design new generations of drugs with increased efficacy and reduced side effects. However, target identification for a known active compound is usually a very difficult task. In the present work, we present a powerful reverse virtual screening strategy, that can help researchers working in the drug discovery field, to predict a set of putative targets for a compound known to exhibit antimicrobial effects. The strategy combines chemical similarity methods, with target prioritization based on essentiality data, and molecular-docking. These steps can be tailored according to the researchers’ needs and pathogen’s available information. Our results show that using only the chemical similarity approach, this method is capable of retrieving potential targets for half of tested compounds. The results show that even for a low chemical similarity threshold whenever domains are retrieved, the correct domain is among those retrieved in more than 80% of the queries. Prioritizing targets by an essentiality criteria allows us to further reduce, up to 3–4 times, the number of putative targets. Lastly, docking is able to identify the correct domain ranked in the top two in about two thirds of cases. Bias docking improves predictive capacity only slightly in this scenario. We expect to integrate the presented strategy in the context of Target Pathogen database to make it available for the wide community of researchers working in antimicrobials discovery.

Biosynthesis Investigations of Terpenoid, Alkaloid, and Flavonoid Antimicrobial Agents Derived from Medicinal Plants.

The overuse of antibiotics in the past decades has led to the emergence of a large number of drug-resistant microorganisms. In recent years, the infection rate caused by multidrug-resistant microorganisms has been increasing, which has become one of the most challenging problems in modern medicine. Plant-derived secondary metabolites and their derivatives have been identified to display significant antimicrobial abilities with good tolerance and less adverse side effects, potentially having different action mechanisms with antibiotics of microbial origin. Thus, these phyto-antimicrobials have a good prospect in the treatment of multidrug-resistant microorganisms. Terpenoids, alkaloids, and flavonoids made up the predominant part of the currently reported phytochemicals with antimicrobial activities. Synthetic biology research around these compounds is one of the hotspot fields in recent years, which not only has illuminated the biosynthesis pathways of these phyto-antimicrobials but has also offered new methods for their production. In this review, we discuss the biosynthesis investigations of terpenoid, alkaloid, and flavonoid antimicrobial agents—using artemisinin and oleanolic acid (terpenoids), berberine and colchicine (alkaloids), and baicalin (flavonoids) as examples—around their antimicrobial action mechanisms, biosynthesis pathway elucidation, key enzyme identification, and heterologous production, in order to provide useful hints for plant-derived antimicrobial agent discovery and development.

Antibiofilm and Antimicrobial Properties of 1-allyl-3-(2-diisopropylaminoethyl) Benzimidazolium Chloride and its Silver(I)-NHC Complex

Today, the number of antimicrobials used in treatment has decreased, especially due to drug resistance. We need new antimicrobials. Biofilms are an important cause of antimicrobial resistance. In this study, the antimicrobial and antibiofilm properties of the salt and silver complex of benzimidazolium-based NHC compound, which were previously synthesized and characterized, were evaluated. The antimicrobial properties were tested using the broth microdilution method, while their antibiofilm potential was determined by microtiter plate assay. Salt of the NHC compound (1a) showed antimicrobial activity on microorganisms at concentrations between 31.25-125 µg/mL. The silver complex (2a) of the NHC compound showed higher antimicrobial and antibiofilm activity than the salt compound. This activity was highest on Candida albicans yeast (MIC 3.9 µg/mL). Compound 2a reduced the biofilm structure of C. albicans yeast by 86.1% compared to the control. In addition, compound 2a showed 76.4-80.6% antibiofilm activity on gram-negative bacteria. NHC compounds are seen as a promising resource for the development of new generation antimicrobials. The NHC compound evaluated in this study was found to have significant antimicrobial and antibiofilm activity. These compounds could be an important resource for the discovery of future biofilm-acting antimicrobials.

MgrB Mutations and Altered Cell Permeability in Colistin Resistance in Klebsiella pneumoniae.

There has been a resurgence in the clinical use of polymyxin antibiotics such as colistin due to the limited treatment options for infections caused by carbapenem-resistant Enterobacterales (CRE). However, this last-resort antibiotic is currently confronted with challenges which include the emergence of chromosomal and plasmid-borne colistin resistance. Colistin resistance in Klebsiella pneumoniae is commonly caused by the mutations in the chromosomal gene mgrB. MgrB spans the inner membrane and negatively regulates PhoP phosphorylation, which is essential for bacterial outer membrane lipid biosynthesis. The present review intends to draw attention to the role of mgrB chromosomal mutations in membrane permeability in K. pneumoniae that confer colistin resistance. With growing concern regarding the global emergence of colistin resistance, deciphering physical changes of the resistant membrane mediated by mgrB inactivation may provide new insights for the discovery of novel antimicrobials that are highly effective at membrane penetration, in addition to finding out how this can help in alleviating the resistance situation.

Quercetin Is a Novel Inhibitor of the Choline Kinase of Streptococcus pneumoniae.

The effectiveness of current antimicrobial methods for addressing for food-borne Gram-positive pathogens has dropped with the emergence of resistant strains. Consequently, new methods for addressing Gram-positive strains have to be developed continuously. This includes establishing novel targets for antimicrobial discovery efforts. Eukaryotic choline kinases have been highly developed as drug targets for the treatment of cancer, rheumatoid arthritis, malaria and many other conditions and diseases. Recently, choline kinase (ChoK) has been proposed as a drug target for Gram-positive species generally. The aim of this work was to discover novel, natural sources of inhibitors for bacterial ChoK from tea extracts. We report the first natural bacterial ChoK inhibitor with antimicrobial activity against Streptococcus pneumoniae: quercetin.

Streptomyces peucetius M1 and Streptomyces lavendulae M3 Soil Isolates as a Promising Source for Antimicrobials Discovery

The ever-increasing fatality due to the surge of drug resistance has demanded the development of therapeutic metabolites with novel modes of mechanism. Despite the discovery of new antimicrobials, antibiotic resistance is increasing at an alarming rate. The emergence of novel molecules is necessary for targeting the Multidrug (MDR) and Pan-Drug resistant (PDR) pathogens which are the principal cause of life-threatening infections. Streptomyces strains commonly found in the soil produce secondary metabolites which have outstanding potential for being an antifungal and antibacterial agent. This study aims to isolate, identify, and characterize Streptomyces strains isolated from soil and extract their crude metabolites to determine their hidden therapeutic property. Out of four isolates identified, Streptomyces peucetius and Streptomyces lavendulae have shown significant antimicrobial activity against test organisms. To further determine the activity of their extracts GC-MS analysis, and MIC, as well as radical scavenging activity, were determined. GC-MS analysis revealed the presence of compounds 2,3-butanediol, 1-methyl-2-pyrrolidone-4-carboxamide, and L-Lactic acid which are frequently associated with potent antimicrobial nature. The most vulnerable pathogen to M1 extract was Candida albicans with a MIC value of 7.8125 μg/mL while the susceptible bacteria to M3 extract included Staphylococcus aureus with a MIC value of 3.90 μg/mL. The data obtained for antioxidant activity in the case of metabolic extracts M1 and M3 had an IC50 value of 43.17 µg/mL and 22.65 µg/mL respectively. The above findings hint at the unrealized potential of Streptomyces strains.

A Novel High-Throughput Ex Vivo Ovine Skin Wound Model for Testing Emerging Antibiotics.

The development of antimicrobials is an expensive process with increasingly low success rates, which makes further investment in antimicrobial discovery research less attractive. Antimicrobial drug discovery and subsequent commercialization can be made more lucrative if a fail-fast-and-fail-cheap approach can be implemented within the lead optimization stages where researchers have greater control over drug design and formulation. In this article, the setup of an ex vivo ovine wounded skin model infected with Staphylococcus aureusis described, which is simple, cost-effective, high throughput, and reproducible. The bacterial physiology in the model mimics that during infection as bacterial proliferation is dependent on the pathogen's ability to damage the tissue. The establishment of wound infection is verified by an increase in viable bacterial counts compared to the inoculum. This model can be used as a platform to test the efficacy of emerging antimicrobials in the lead optimization stage. It can be contended that the availability of this model will provide researchers developing antimicrobials with a fail-fast-and-fail-cheap model, which will help increase success rates in subsequent animal trials. The model will also facilitate the reduction and refinement of animal use for research and ultimately enable faster and more cost-effective translation of novel antimicrobials for skin and soft tissue infections to the clinic.

Antimicrobial activity: potential of Spondias purpurea (Anacardiaceae) against bacterial and fungal species.

Introduction. Plants have been used as medicines for centuries to treat human diseases. Studies with plants are extremely important for the development of future drugs that can benefit the human population.Hypothesis/Gap Statement. With the emergence of pathogens resistant to antimicrobial agents, there is an urgent need to direct research towards the discovery of new antimicrobials.Aim. In this study, Spondias purpurea L. (Anacardiaceae) was evaluated for its antimicrobial activity, antioxidant activity and cytotoxicity.Methodology. Antimicrobial activity was evaluated by the MIC using the 96-well plate microdilution technique of ethanolic, hexanic and dicloromethanic extracts of dried S. purpurea leaves against bacteria, yeast and filamentous fungi. The antioxidant activity of extracts was evaluated by the 2,2-diphenyl-1-picrylhydrazine (DPPH) method. To evaluate the safety of extracts, a cytotoxicity study against HaCat, J774 and HepG2 cells was performed.Results. The extracts had no activity against the bacteria at the maximum concentration of 5.0 mg ml-1, but showed fungistatic action against Candida species and dermatophytes. The ethanolic extract showed 88 % antioxidant activity and showed no significant cytotoxicity against the previously mentioned cells.Conclusion. This study showed that the 100 % ethanolic (EtOH) extract was favourable for antifungal and antioxidant activities and did not present significant cytotoxicity against the three studied cell lines, indicating that S. purpurea leaves are promising for the development of new antimicrobials.

The Rare Actinobacterium Crossiella sp. Is a Potential Source of New Bioactive Compounds with Activity against Bacteria and Fungi.

Antimicrobial resistance has become a global problem in recent decades. A gradual reduction in drug discoveries has led to the current antimicrobial resistance crisis. Caves and other subsurface environments are underexplored thus far, and they represent indispensable ecological niches that could offer new molecules of interest to medicine and biotechnology. We explored Spanish show caves to test the bioactivity of the bacteria dwelling in the walls and ceilings, as well as airborne bacteria. We reported the isolation of two strains of the genus Crossiella, likely representing a new species, isolated from Altamira Cave, Spain. In vitro and in silico analyses showed the inhibition of pathogenic Gram-positive and Gram-negative bacteria, and fungi, as well as the taxonomical distance of both strains from their closest relative, Crossiella cryophila. The presence of an exclusive combination of gene clusters involved in the synthesis of lanthipeptides, lasso peptides, nonribosomal peptides and polyketides indicates that species of this genus could represent a source of new compounds. Overall, there is promising evidence for antimicrobial discovery in subterranean environments, which increases the possibility of identifying new bioactive molecules.