Interception of Epoxide ring to quorum sensing system in Enterococcus faecalis and Staphylococcus aureus.

Quorum sensing inhibitor (QSI) has been attracting attention as anti-virulence agent which disarms pathogens of their virulence rather than killing them. QSI marking cyclic peptide-mediated QS in Gram-positive bacteria is an effective tool to overcome the crisis of antibiotic-dependent chemotherapy due to the emergence of drug resistance strain, e.g., methicillin resistant Staphylococcus aureus (MRSA) and Vancomycin resistant Enterococci (VRE). From a semi-large-scale screening thus far carried out, two Epoxide compounds, Ambuic acid and Synerazol, have been found to efficiently block agr and fsr QS systems, suggesting that the Epoxide group is involved in the mode of action of these QSIs. To address this notion, known natural Epoxide compounds, Cerulenin and Fosfomycin were examined for QSI activity for the agr and fsr systems in addition to in silico and SAR studies. As a result, most of investigated Epoxide containing antibiotics correlatively interfere with QSI activity for the agr and fsr systems under sublethal concentrations.

Characterizations of highly efficient moderately halophilic toluene degrading exiguobacterium mexicanum M7 strain isolated from Egyptian saline sediments.

Toluene and other monoaromatic compounds are released into the environment particularly saline habitats due to the inappropriate disposal methods of petroleum products. Studying the bio-removal strategy is required to clean up these hazardous hydrocarbons that threaten all ecosystem life using halophilic bacteria with higher biodegradation efficiency of monoaromatic compounds as a sole carbon and energy source. Therefore, sixteen pure halophilic bacterial isolates were obtained from saline soil of Wadi An Natrun, Egypt, which have the ability to degrade toluene and consume it as the only source of carbon and energy. Amongst these isolates, isolate M7 exhibited the best growth with considerable properties. This isolate was selected as the most potent strain and identified based on phenotypic and genotypic characterizations. The strain M7 was belonging to Exiguobacterium genus and founded to be closely matched to the Exiguobacterium mexicanum with a similarity of 99%. Using toluene as sole carbon source, strain M7 showed good growth at a wide range temperature degree (20-40ºC), pH (5-9), and salt concentrations (2.5-10%, w/v) with optimal growth conditions at 35ºC, pH 8, and 5%, respectively. The biodegradation ratio of toluene was estimated at above optimal conditions and analyzed using Purge-Trap GC-MS. The results showed that strain M7 has the potentiality to degraded 88.32% of toluene within greatly short time (48 h). The current study findings support the potential ability to use strain M7 as a biotechnological tool in many applications such as effluent treatment and toluene waste management.

Dual Inoculation of Plant Growth-Promoting Bacillus endophyticus and Funneliformis mosseae Improves Plant Growth and Soil Properties in Ginger.

Co-inoculation with beneficial microbes has been suggested as a useful practice for the enhancement of plant growth, nutrient uptake, and soil nutrients. For the first time in Uzbekistan the role of plant-growth-promoting Bacillus endophyticus IGPEB 33 and arbuscular mycorrhizal fungi (AMF) on plant growth, the physiological properties of ginger (Zingiber officinale), and soil enzymatic activities was studied. Moreover, the coinoculation of B. endophyticus IGPEB 33 and AMF treatment significantly increased the plant height by 81%, leaf number by 70%, leaf length by 82%, and leaf width by 40% compared to the control. B. endophyticus IGPEB 33 individually increased plant height significantly by 51%, leaf number by 56%, leaf length by 67%, and leaf width by 27% as compared to the control treatment. Compared to the control, B. endophyticus IGPEB 33 and AMF individually significantly increased chlorophyll a by 81-58%, chlorophyll b by 68-37%, total chlorophyll by 74-53%, and carotenoid content by 67-55%. However, combination of B. endophyticus IGPEB 33 and AMF significantly increased chlorophyll a by 86%, chlorophyll b by 72%, total chlorophyll by 82%, and carotenoid content by 83% compared to the control. Additionally, plant-growth-promoting B. endophyticus IGPEB 33 and AMF inoculation improved soil nutrients and soil enzyme activities compared to the all treatments. Co-inoculation with plant-growth-promoting B. endophyticus and AMF could be an alternative for the production of ginger that is more beneficial to soil nutrient deficiencies. We suggest that a combination of plant-growth-promoting B. endophyticus and AMF inoculation could be a more sustainable and eco-friendly approach in a nutrient-deficient soil.

Fighting multidrug-resistant Enterococcus faecalis via interfering with virulence factors using green synthesized nanoparticles.

This study aims to synthesize silver nanoparticles by the green method and test it against specific virulence factors in multi-drug resistant Enterococcus faecalis bacteria. virulence factors of E. faecalis clinical isolates were determined and the most potent isolate was selected for further investigations. The prepared Ag-NPs were characterized using UV spectroscopy, FTIR spectroscopy, dynamic light scattering, and transmission electron microscopy (TEM). The result revealed the concentration of 0.0625 mg/ml Ag-NPs was significantly reducing virulence factors in multidrug-resistant E. faecalis without affecting planktonic cell growth. UV-Visible spectroscopy characterization revealed a characteristic surface Plasmon band [SPR] at a wavelength ranging from 256 to 345 and 510 nm in the prepared Ag-NPs. dynamic light scattering indicated it tended to an electrostatic attraction between nanoparticles in the prepared solution. TEM images revealed the average size of Ag-NPs were prepared to be 28.8 nm and the shape was spherical. Green synthesized Ag-NPs have the ability to combat multi-drug-resistant E. faecalis via reducing virulence factors, which is considered a good approach toward resolving the multidrug resistance crisis.

Secondary Metabolites of Actinomycetales as Potent Quorum Sensing Inhibitors Targeting Gram-Positive Pathogens: In Vitro and In Silico Study.

Anti-virulence agents are non-bacteriostatic and non-bactericidal emerging therapeutic options which hamper the production of virulence factors in pathogenic flora. In Staphylococcus aureus and Enterococcus faecalis, regulation of virulence genes' expression occurs through the cyclic peptide-mediated accessory gene regulator (agr) and its ortholog fsr quorum sensing systems, respectively. In the present study, we screened a set of 54 actinomycetales secondary metabolites as novel anti-virulence compounds targeting quorum sensing system of the Gram-positive bacteria. The results indicated that four compounds, Phenalinolactones A-D, BU-4664LMe, 4,5-dehydrogeldamycin, and Questinomycin A, potentially inhibit the agr quorum sensing system and hemolytic activity of S. aureus. On the other hand, Decatromicin A and B, Okilactomycin, Rishirilide A, Abyssomicin I, and Rebeccamycin selectively blocked the fsr quorum sensing system and the gelatinase production in E. faecalis at sub-lethal concentrations. Interestingly, Synerazol uniquely showed the capability to inhibit both fsr and agr quorum sensing systems. Further, in silico molecular docking studies were performed which provided closer insights into the mode of action of these compounds and proposed that the inhibitory activity of these compounds could be attributed to their potential ability to bind to the ATP-active site of S. aureus AgrA. Taken together, our study highlights the potential of actinomycetales secondary metabolites with diverse structures as anti-virulence quorum sensing inhibitors.

Correlation between phenotypic virulence traits and antibiotic resistance in Pseudomonas aeruginosa clinical isolates.

Pseudomonas aeruginosa is a ubiquitous pathogen capable of infecting virtually all tissues and its one of the standout amongst the most hazardous microorganisms of high morbidity and mortality rates especially in debilitated patients with few successful antibiotic choices available. This pathogen regulating most virulence traits by that so-called quorum sensing (QS), a cell to cell communication system. the present study was intended to phenotypically evaluate the activity of specific virulence traits (including swarming and swimming motility, protease, pyocyanin, and biofilm production) in Pseudomonas aeruginosa clinical isolates and assess the statistical correlation between these traits and antibiotic resistance. One hundred and thirteen bacterial isolates were obtained from different clinical samples and identified as P. aeruginosa, among them, 73.4% have the ability to forming biofilm with different degrees; 59.2% were able to produce pyocyanin pigment while all isolates having the ability to make swarming and swimming motility and able to produce protease enzyme with different degrees. The isolates that produce the higher levels of the virulence traits were identified by both biochemical using Vitek2 automated system and genetically via 16s rRNA gene analysis. The statistical analysis results indicate that a positive significant correlation was found between biofilm formation and other studied virulence traits except for protease (r = 0.584: 0.324, P < 0.05) while a non-significant correlation was found between biofilm formation and protease activity (r = 0.105, P ˃ 0.05). Swimming and swarming motility have a positive significant correlation with other studied virulence traits (r = 0.613: 0.297, P < 0.05) except for protease. Pyocyanin pigment production have a positive significant correlation with other studied virulence traits (r = 0.33: 0.297, P < 0.05) except for protease. on the other hand, negative significant correlations were found between biofilm formation, swimming; and swarming motility, Pyocyanin pigment production, and the susceptibility of antibiotics (r = -0.512: -0.281, P < 0.05). Detection of such correlations in P. aeruginosa is useful for study the behavior of this pathogen and may be provide a new target for the treatment of MDR infections.

Statistical optimization of chromium (VI) reduction using response surface methodology (RSM) by newly isolated Stenotrophomonas sp. (a novel strain).

Isolation of Microorganisms capable of reducing toxic chromium (VI) into less toxic one (Cr (III)) has been given attention due to their significance in bioremediation of the contaminated sites. In the present study, Stenotrophomonas sp. Crt94-4A an isolated strain from tannery wastewater and identified genetically by 16s rRNA gene sequencing was able to grow at concentrations up to 354 mg/L of Cr (VI). The results revealed 1% (w/v) NaCl, 2% (v/v) (2 × 106 CFU) inoculum size, and PH 7 in culture containing glucose and peptone as carbon and nitrogen sources respectively were the best conditions for Cr (VI) reduction. Statistical optimization was performed using Plackett-Burman design where peptone, inoculum size, and NaCl had significant effects on Cr (VI) reduction which were tested by three factors Box-Behnken design (BBD) to determine their correlation. The reduction capacity of Cr (VI) by Stenotrophomonas Sp. Crt94-4A was increased from 82, 55, and 23 to 96, 76, and 45% at 88.5, 177 and 354 mg/L of Cr (VI) respectively, which make this strain a good candidate for bioremediation of Cr (VI).

Genetic and Histopathological Alterations in Caco-2 and HuH-7 Cells Treated with Secondary Metabolites of Marine fungi.

The present work aimed to study the activity of naturally derived fungal secondary metabolites as anticancer agents concerning their cytotoxicity, apoptotic, genetic, and histopathological profile. It was noticed that Aspergillus terreus, Aspergillus flavus, and Aspergillus fumigatus induced variable toxic potential that was cell type, secondary metabolite type, and concentration dependent. Human colonic adenocarcinoma cells (Caco-2) showed less sensitivity than hepatocyte-derived cellular carcinoma cells (HuH-7), and in turn, the half-maximal inhibitory concentration (IC50) was variable. Also, the apoptotic potential of Aspergillus species-derived fungal secondary metabolites was proven via detection of up-regulated pro-apoptotic genes and down-regulation of anti-apoptotic genes. The expression level was cell type dependent. Concurrently, apoptotic profile was accompanied with cellular DNA accumulation at the G2/M phase, as well as an elevation in Pre-G1 phase but not during G0/G1 and S phases. Also, there were characteristic apoptotic features of treated cells presented as abnormal intra-nuclear eosinophilic structures, dead cells with mixed euchromatin and heterochromatin, ruptured cell membranes, apoptotic cells with irregular cellular and nuclear membranes, as well as peripheral chromatin condensation. It can be concluded that Aspergillus secondary metabolites are promising agents that can be used as supplementary agents to the currently applied anti-cancer drug regimen.

Hydrazonoyl bromide precursors as DHFR inhibitors for the synthesis of bis-thiazolyl pyrazole derivatives; antimicrobial activities, antibiofilm, and drug combination studies against MRSA.

Microbial resistance is a big concern worldwide, making the development of new antimicrobial drugs difficult. The thiazole and pyrazole rings are important heterocyclic compounds utilized to produce a variety of antimicrobial medications. As a result, a series of new bis-thiazolyl-pyrazole derivatives 3, 4a-c, 5a, b, and 6a-c was synthesized by reacting bis hydrazonoyl bromide with several active methylene reagents in a one-pot reaction. The assigned structure was characterized entirely based on elemental and spectral analyses. The antimicrobial activity represented by MIC was performed using a resazurin-based turbidimetric (TB) assay. The results exhibited good antimicrobial activity against gram-positive strains, especially S. aureus (ATCC6538) while showing poor to moderate activity against gram-negative and fungal strains. Furthermore, the most active derivatives 3, 4a, 4c, and 5b were evaluated for MIC, MBC, antibiofilm, hemolytic assay, and drug combination testing against two S. aureus (ATCC6538) and MRSA (ACL18) strains. Additionally, bis-thiazolyl pyrazole 3, 4c, and 5b exhibited more potent inhibitory activity for DHFR with IC50 values (6.34 ± 0.26, 7.49 ± 0.28, and 3.81 ± 0.16 µM), respectively, compared with Trimethoprim (8.34 ± 0.11 µM). The bis-1-(substituted-thiazol-2-yl)-1H-pyrazole-4-carbonitrile derivative 5b was the most active member with MIC values ranging from (0.12-0.25 µM) compared to Vancomycin (1-2 µM), and MBC values ranging from (0.5-1 µM) for S. aureus (ATCC6538) and MRSA (ACL18). Surprisingly, compound 5b displayed bactericidal behavior, synergistic effect with three commercial antibiotics, and inhibited DHFR with 2.1 folds higher than Trimethoprim. Finally, good findings were obtained from in silico investigations incorporating toxicity prediction and molecular docking simulation.

Quorum Quenching Strategy Targeting Gram-Positive Pathogenic Bacteria.

Quorum sensing (QS) is a cell density-dependent regulatory system that orchestrates the group behavior of unicellular organisms by synchronizing the expression of certain gene(s) within the clonal community of same species. Bacterial pathogens often employ QS system to establish efficiently an infection. A large part of low GC Gram-positive bacteria belonging to phylum Firmicutes use thiolactone/lactone peptides as communication signals so-called autoinducing peptides (AIPs) to coordinate QS circuit. In particular, QS of staphylococci, enterococci, and clostridia have been intensively studied in terms of alternative target of anti-pathogenic chemotherapy independent of bactericidal antibiotics. Thus far, a number of quorum quenching (QQ) agents that targeting the QS circuit of these Gram-positive pathogens have been developed by random screening of natural compounds or rationale design of AIP antagonists. This review summarizes those QQ agents and previews their potential as post-antibiotic drugs.

Cyclodepsipeptides produced by actinomycetes inhibit cyclic-peptide-mediated quorum sensing in Gram-positive bacteria.

Cyclic peptides are commonly used as quorum-sensing autoinducers in Gram-positive Firmicutes bacteria. Well-studied examples of such molecules are thiolactone and lactone, used to regulate the expression of a series of virulence genes in the agr system of Staphylococcus aureus and the fsr system of Enterococcus faecalis, respectively. Three cyclodepsipeptides WS9326A, WS9326B and cochinmicin II/III were identified as a result of screening actinomycetes culture extracts for activity against the agr/fsr system. These molecules are already known as receptor antagonists, the first two for tachykinin and the last one for endothelin. WS9326A also inhibited the transcription of pfoA regulated by the VirSR two-component system in Clostridium perfringens. Receptor-binding assays using a fluorescence-labeled autoinducer (FITC-GBAP) showed that WS9326A and WS9326B act as receptor antagonists in this system. In addition, an ex vivo assay showed that WS9326B substantially attenuated the toxicity of S. aureus for human corneal epithelial cells. These results suggest that these three natural cyclodepsipeptides have therapeutic potential for targeting the cyclic peptide-mediated quorum sensing of Gram-positive pathogens.

High-throughput screening of inhibitors targeting Agr/Fsr quorum sensing in Staphylococcus aureus and Enterococcus faecalis.

Staphylococcus aureus and Enterococcus faecalis employ cyclic peptide-mediated quorum sensing (QS) systems, termed agr and fsr respectively, to regulate the expression of a series of virulence genes. To identify quorum sensing inhibitors (QSIs) that target agr/fsr systems, an efficient screening system was established. In addition to the gelatinase-induction assay to examine E. faecalis fsr QS, the use of an S. aureus agr reporter strain that carries luciferase and green fluorescence protein genes under the agr P3 promoter facilitated the development of a high-throughput screen (HTS) for QSIs. As a result of screening of 906 actinomycetes culture extracts, four showed QSI activity against the agr and fsr systems without growth inhibitory activity. The extracts were purified on a small scale, and three HPLC peaks were obtained with obvious QSI activity. In sum, the established HTS system is a promising strategy for the discovery of anti-pathogenic agents targeting cyclic peptide-mediated QS in Gram-positive pathogens.