Proven anti-virulence therapies in combating methicillin- and vancomycin-resistant Staphylococcus aureus infections.

Introduction: Despite years of efforts to develop new antibiotics for eradicating multidrug-resistant (MDR) and multi-virulent Methicillin-Resistant Staphylococcus aureus (MRSA) and Vancomycin-Resistant Staphylococcus aureus (VRSA) infections, treatment failures and poor prognoses in most cases have been common. Therefore, there is an urgent need for new therapeutic approaches targeting virulence arrays. Our aim is to discover new anti-virulence therapies targeting MRSA and VRSA virulence arrays. Methodology: We employed phenotypic, molecular docking, and genetic studies to screen for anti-virulence activities among selected promising compounds: Coumarin, Simvastatin, and Ibuprofen. Results: We found that nearly all detected MRSA and VRSA strains exhibited MDR and multi-virulent profiles. The molecular docking results aligned with the phenotypic and genetic assessments of virulence production. Biofilm and hemolysin productions were inhibited, and all virulence genes were downregulated upon treatment with sub-minimum inhibitory concentration (sub-MIC) of these promising compounds. Ibuprofen was the most active compound, exhibiting the highest inhibition and downregulation of virulence gene products. Moreover, in vivo and histopathological studies confirmed these results. Interestingly, we observed a significant decrease in wound area and improvements in re-epithelialization and tissue organization in the Ibuprofen and antimicrobial treated group compared with the group treated with antimicrobial alone. These findings support the idea that a combination of Ibuprofen and antimicrobial drugs may offer a promising new therapy for MRSA and VRSA infections. Conclusion: We hope that our findings can be implemented in clinical practice to assist physicians in making the most suitable treatment decisions.

Anti-cancer and antimicrobial potential of five soil Streptomycetes: a metabolomics-based study.

Lack of new anti-cancer and anti-infective agents directed the pharmaceutical research to natural products' discovery especially from actinomycetes as one of the major sources of bioactive compounds. Metabolomics- and dereplication-guided approach has been used successfully in chemical profiling of bioactive actinomycetes. We aimed to study the metabolomic profile of five bioactive actinomycetes to investigate the interesting metabolites responsible for their antimicrobial and anti-cancer activities. Three actinomycetes, namely, Streptomyces sp. SH8, SH10 and SH13, were found to exhibit broad spectrum of antimicrobial activities, whereas isolate SH4 showed the broadest antimicrobial activity against all tested strains. In addition, isolates SH8, SH10 and SH12 displayed potent cytotoxicity against the breast cancer cell line Michigan Cancer Foundation-7 (MCF-7), whereas isolates SH4 and SH12 exhibited potent anti-cancer activity against the hepatoma cell line hepatoma G2 (HepG2) compared with their weak inhibitory properties on the normal breast cells MCF-10A and normal liver cells transformed human liver epithelial-2 (THLE2), respectively. All bioactive isolates were molecularly identified as Streptomyces sp. via 16S rRNA gene sequencing. Our actinobacterial dereplication analysis revealed putative identification of several bioactive metabolites including tetracycline, oxytetracycline and a macrolide antibiotic, novamethymycin. Together, chemical profiling of bioactive Streptomycetes via dereplication and metabolomics helped in assigning their unique metabolites and predicting the bioactive compounds instigating their diverse bioactivities.

The inhibitory effect of dextranases from Bacillus velezensis and Pseudomonas stutzeri on Streptococcus mutans biofilm.

Background and Objectives: Dental caries is a breakdown of the teeth enamel due to harmful bacteria, lack of oral hygiene, and sugar consumption. The acid-producing bacterium Streptococcus mutans is the leading cause of dental caries. Dextranase is an enzyme that can degrade dextran to low molecular weight fractions, which have many therapeutic and industrial applications. The purpose of the present study was to isolate a novel dextranase-producing bacteria from a source (molasses). The cell-free extracts containing dextranases were tested as antibiofilm agents. Materials and Methods: Dextranase-producing bacteria were identified using phenotypic and genotypic methods such as 16S rRNA gene sequencing and enzymatic characterization. Results: The highest six dextranase-producing bacterial isolates were Bacillus species. The best conditions for dextranase productivity were obtained after 72 hours of culture time at pH 7. The addition of glucose to the medium enhanced the production of the enzymes. The cell-free extract of the six most active isolates showed remarkable activity against biofilm formation by Streptococcus mutans ATCC 25175. The highest inhibition activities reached 60% and 80% for Bacillus velezensis and Pseudomonas stutzeri, respectively. Conclusion: Therefore, our study added to the current dextranase-producing bacteria with potential as a source of dextranases.

Microbial etiologies of ventilator-associated pneumonia (VAP) in intensive care unit of Beni-Suef University's Hospital.

BACKGROUND: Ventilator-associated pneumonia (VAP) is a major health problem for people intubated in intensive care units (ICUs), leading to increased mortality rates, hospital stay, and treatment costs. In the present study, the core pathogens causing VAP in Beni-Suef University's Hospital, Egypt, was investigated over a study period of 2 years (2017-2019). RESULTS: Of a total of 213 patients subjected to mechanical ventilation, 60 have developed VAP during their stay in the ICU. The mortality rate reached 41.7% among VAP patients. Sixty bacteria were isolated from an endotracheal aspirate of hospitalized patients. The different isolates were cultured followed by running biochemical tests, sensitivity assays, and automated VITEK®2 System analysis. Unexpectedly, all the isolates were Gram-negative bacteria. Klebsiella pneumoniae were the main pathogen encountered (27/60 isolates) followed by Acientobacter baumannnii (7/60) and other microorganisms belonging to the genera Moraxella, Escherichia, and Pseudomonas (11/60). Antibiotic sensitivity testing was performed via the VITEK®2 System using up to 16 different antibiotics representing 8 different antibiotic classes and subclasses (aminoglycosides, carbapenems, fluoroquinolones, penicillin/ß-lactamase inhibitor, extended-spectrum cephalosporins, aminopenicillins, aminopenicillins/ß-lactamase inhibitor, folic acid synthesis inhibitor). Majority of the isolates (28/60) showed a remarkable extensive drug resistance (XDR) pattern, while 15 isolates were multi-drug resistant (MDR) and only 6 were pan-drug resistant (PDR) with regard to antibiotics under evaluation. CONCLUSION: The association of VAP with multi-drug-resistant bacteria is alarming, and rapid management is crucial. Identification of core pathogens is essential for identifying the most appropriate technique for infection control.

A metabolomic approach to target antimalarial metabolites in the Artemisia annua fungal endophytes.

Fungal endophytes are a major source of anti-infective agents and other medically relevant compounds. However, their classical blinded-chemical investigation is a challenging process due to their highly complex chemical makeup. Thus, utilizing cheminformatics tools such as metabolomics and computer-aided modelling is of great help deal with such complexity and select the most probable bioactive candidates. In the present study, we have explored the fungal endophytes associated with the well-known antimalarial medicinal plant Artemisia annua for their production of further antimalarial agents. Based on the preliminary antimalarial screening of these endophytes and using LC-HRMS-based metabolomics and multivariate analyses, we suggested different potentially active metabolites (compounds 1-8). Further in silico investigation using the neural-network-based prediction software PASS led to the selection of a group of quinone derivatives (compounds 1-5) as the most possible active hits. Subsequent in vitro validation revealed emodin (1) and physcion (2) to be potent antimalarial candidates with IC50 values of 0.9 and 1.9 µM, respectively. Our approach in the present investigation therefore can be applied as a preliminary evaluation step in the natural products drug discovery, which in turn can facilitate the isolation of selected metabolites notably the biologically active ones.

Exploration of Chemical Diversity and Antitrypanosomal Activity of Some Red Sea-Derived Actinomycetes Using the OSMAC Approach Supported by LC-MS-Based Metabolomics and Molecular Modelling.

In the present study, we investigated the actinomycetes associated with the Red Sea-derived soft coral Sarcophyton glaucum in terms of biological and chemical diversity. Three strains were cultivated and identified to be members of genera Micromonospora, Streptomyces, and Nocardiopsis; out of them, Micromonospora sp. UR17 was putatively characterized as a new species. In order to explore the chemical diversity of these actinobacteria as far as possible, they were subjected to a series of fermentation experiments under altering conditions, that is, solid and liquid fermentation along with co-fermentation with a mycolic acid-containing strain, namely Nocardia sp. UR23. Each treatment was found to affect these actinomycetes differently in terms of biological activity (i.e., antitrypanosomal activity) and chemical profiles evidenced by LC-HRES-MS-based metabolomics and multivariate analysis. Thereafter, orthogonal projections to latent structures discriminant analysis (OPLS-DA) suggested a number of metabolites to be associated with the antitrypanosomal activity of the active extracts. The subsequent in silico screenings (neural networking-based and docking-based) further supported the OPLS-DA results and prioritized desferrioxamine B (3), bafilomycin D (10), and bafilomycin A1 (11) as possible antitrypanosomal agents. Our approach in this study can be applied as a primary step in the exploration of bioactive natural products, particularly those from actinomycetes.

Discovery of Two Brominated Oxindole Alkaloids as Staphylococcal DNA Gyrase and Pyruvate Kinase Inhibitors via Inverse Virtual Screening.

In the present study, a small marine-derived natural products library was assessed for antibacterial potential. Among 36 isolated compounds, a number of bis-indole derivatives exhibited growth-inhibitory activity towards Gram-positive strains (Bacillus subtilis and multidrug-resistant Staphylococcus aureus). 5- and 6-trisindoline (5-Tris and 6-Tris) were the most active derivatives (minimum inhibitory concentration, MIC, 4-8 µM) that were subsequently selected for anti-biofilm activity evaluation. Only 5-Tris was able to inhibit the staphylococcal biofilm formation starting at a 5 µM concentration. In order to investigate their possible molecular targets, both natural products were subjected to in silico inverse virtual screening. Among 20 target proteins, DNA gyrase and pyruvate kinase were the most likely to be involved in the observed antibacterial and anti-biofilm activities of both selected natural products. The in vitro validation and in silico binding mode studies revealed that 5-Tris could act as a dual enzyme inhibitor (IC50 11.4 ± 0.03 and 6.6 ± 0.05 µM, respectively), while 6-Tris was a low micromolar gyrase-B inhibitor (IC50 2.1 ± 0.08 µM), indicating that the bromine position plays a crucial role in the determination of the antibacterial lead compound inhibitory activity.

Correction: Bioassay- and metabolomics-guided screening of bioactive soil actinomycetes from the ancient city of Ihnasia, Egypt.

[This corrects the article DOI: 10.1371/journal.pone.0226959.].

Enantioselective sulfoxidation using Streptomyces glaucescens GLA.0.

Asymmetric oxidation of prochiral sulfides is a direct means for production of enantiopure sulfoxides which are important in organic synthesis and the pharmaceutical industry. In the present study, Streptomyces glaucescens GLA.0 was employed for stereoselective oxidation of prochiral sulfides. Growing cells selectively catalyzed the oxidation of phenyl methyl sulfide to the corresponding sulfoxide. Only very little overoxidation was observed, resulting in minor amounts of the unwanted sulfone. Addition of isopropyl alcohol as a co-solvent, time of substrate addition and composition of the reaction media resulted in enhanced phenyl methyl sulfide biotransformation. The concentration of the undesired by-product (sulfone) was as low as 4% through the reaction course under optimal reaction conditions. The results show that S. glaucescens GLA.0 is a promising whole-cell biocatalyst for preparing highly enantiopure (R)-phenyl methyl sulfoxide in high yield (90%) with an enantiomeric excess (ee) exceeding 99%.

Bioassay- and metabolomics-guided screening of bioactive soil actinomycetes from the ancient city of Ihnasia, Egypt.

Literature surveys, taxonomical differences, and bioassay results have been utilized in the discovery of new natural products to aid in Actinomycetes isolate-selection. However, no or less investigation have been done on establishing the differences in metabolomic profiles of the isolated microorganisms. The study aims to utilise bioassay- and metabolomics-guided tools that included dereplication study and multivariate analysis of the NMR and mass spectral data of microbial extracts to assist the selection of isolates for scaling-up the production of antimicrobial natural products. A total of 58 actinomycetes were isolated from different soil samples collected from Ihnasia City, Egypt and screened for their antimicrobial activities against indicator strains that included Bacillus subtilis, Escherichia coli, methicillin-resistant Staphylococcus aureus and Candida albicans. A number of 25 isolates were found to be active against B. subtilis and/or to at least one of the tested indicator strains. Principal component analyses showed chemical uniqueness for four outlying bioactive actinomycetes extracts. In addition, Orthogonal Projections to Latent Structures Discriminant Analysis (OPLS-DA) and dereplication study led us to further select two outlying anti-MRSA active isolates MS.REE.13 and 22 for scale-up work. MS.REE.13 and 22 exhibited zones of inhibition at 19 and 13 mm against MRSA, respectively. A metabolomics-guided approach provided the steer to target the bioactive metabolites (P<0.01) present in a crude extract or fraction even at nanogram levels but it was a challenge that such low-yielding bioactive natural products would be feasible to isolate. Validated to occur only on the active side of OPLS-DA loadings plot, the isolated compounds exhibited medium to weak antibiotic activity with MIC values between 250 and 800 µM. Two new compounds, P_24306 (C10H13N2) and N_12799 (C18H32O3) with MICs of 795 and 432 µM, were afforded from the scale-up of MS.REE. 13 and 22, respectively.

Comparative Structural Analysis of Different Mycobacteriophage-Derived Mycolylarabinogalactan Esterases (Lysin B).

Mycobacteriophage endolysins have emerged as a potential alternative to the current antimycobacterial agents. This study focuses on mycolylarabinogalactan hydrolase (LysB) enzymes of the α/ß-hydrolase family, which disrupt the unique mycolic acid layer of mycobacterium cell wall. Multiple sequence alignment and structural analysis studies showed LysB-D29, the only enzyme with a solved three-dimensional structure, to share several common features with esterases (lacking lid domain) and lipases (acting on long chain lipids). Sequence and structural comparisons of 30 LysB homology models showed great variation in domain organizations and total protein length with major differences in the loop-5 motif harboring the catalytic histidine residue. Docking of different p-nitrophenyl ligands (C4-C18) to LysB-3D models revealed that the differences in length and residues of loop-5 contributed towards wide diversity of active site conformations (long tunnels, deep and superficial funnels, shallow bowls, and a narrow buried cave) resembling that of lipases, cutinases, and esterases. A set of seven LysB enzymes were recombinantly produced; their activity against p-nitrophenyl esters could be related to their active site conformation and acyl binding site. LysB-D29 (long tunnel) showed the highest activity with long chain p-nitrophenyl palmitate followed by LysB-Omega (shallow bowl) and LysB-Saal (deep funnel).

Secondary metabolites from fungal endophytes of Solanum nigrum.

Three endophytic fungi, Aspergillus sp. (SNFSt), Aspergillus sp. (SNFL) and Lasiodiplodia theobromae (SNFF) were isolated from stems, leaves and fruits of Solanum nigrum L, respectively. The static fermentation of the three fungal strains led to the characterization of nine known metabolites (1-9) using HRESIMS and NMR analyses.

Heterologous expression of the avirulence gene ACE1 from the fungal rice pathogen Magnaporthe oryzae.

The ACE1 and RAP1 genes from the avirulence signalling gene cluster of the rice blast fungus Magnaporthe oryzae were expressed in Aspergillus oryzae and M. oryzae itself. Expression of ACE1 alone produced a polyenyl pyrone (magnaporthepyrone), which is regioselectively epoxidised and hydrolysed to give different diols, 6 and 7, in the two host organisms. Analysis of the three introns present in ACE1 determined that A. oryzae does not process intron 2 correctly, while M. oryzae processes all introns correctly in both appressoria and mycelia. Co-expression of ACE1 and RAP1 in A. oryzae produced an amide 8 which is similar to the PKS-NRPS derived backbone of the cytochalasans. Biological testing on rice leaves showed that neither the diols 6 and 7, nor amide 8 was responsible for the observed ACE1 mediated avirulence, however, gene cluster analysis suggests that the true avirulence signalling compound may be a tyrosine-derived cytochalasan compound.

Rational domain swaps decipher programming in fungal highly reducing polyketide synthases and resurrect an extinct metabolite.

The mechanism of programming of iterative highly reducing polyketide synthases remains one of the key unsolved problems of secondary metabolism. We conducted rational domain swaps between the polyketide synthases encoding the biosynthesis of the closely related compounds tenellin and desmethylbassianin. Expression of the hybrid synthetases in Aspergillus oryzae led to the production of reprogrammed compounds in which the changes to the methylation pattern and chain length could be mapped to the domain swaps. These experiments reveal for the first time the origin of programming in these systems. Domain swaps combined with coexpression of two cytochrome P450 encoding genes from the tenellin biosynthetic gene cluster led to the resurrection of the extinct metabolite bassianin.