Synergistic anti-virulence efficacy of citral and carvacrol against mixed vaginitis causing Candida albicans and Gardnerella vaginalis: An in vitro and in vivo study.

Mixed vaginitis due to bacterial vaginosis (BV) and vulvovaginal candidiasis (VVC) is the most prevalent form and presents a significant therapeutic challenge globally. Since, the administration of monotherapy leads to subsequent recurrent infections, synergistic therapy that completely eradicates both pathogens is of dire need to manage mixed vaginities scenario and to prevent its recurrence. The current investigation was focused on exploring the synergistic inhibitory efficacy of phytochemicals against the virulence traits of individual and mixed species of C. albicans and G. vaginalis in vitro and in vivo (Galleria mellonella). Out of five phytochemicals (carvacrol, thymol, cinnamaldehyde, eugenol, and borneol) screened for synergism with citral [(Ct) as the prime molecule owing to its myriad therapeutic potential], carvacrol (Ca) in combination with citral exhibited promising synergistic effect. Time-kill kinetics and one-minute contact-killing assays demonstrated the phenomenal microbicidal effect of Ct-Ca combination against both mono and dual-species within 30 min and one-minute time intervals, respectively. Furthermore, the sub-CMICs (synergistic combinatorial MIC) of Ct-Ca have significantly eradicated the mature biofilms and remarkably reduced the virulence attributes of both C. albicans and G. vaginalis (viz., yeast to hyphae transition, filamentation, protease production, and hydrophobicity index), in single and dual species states. The non-toxic nature of Ct-Ca combination was authenticated using in vitro (human erythrocyte cells) and in vivo (Galleria mellonella) models. In addition, the in vivo efficacy evaluation and subsequent histopathological investigation was done using the invertebrate model system G. mellonella, which further ascertained the effectiveness of Ct-Ca combination in fighting off the infection caused by individual and mixed species of C. albicans and G. vaginalis. Concomitantly, the current work is the first of its kind to delineate the in vitro interaction of C. albicans and G. vaginalis mixed species at their growth and biofilm states, together emphasizes the promising therapeutic potential of acclaimed phytochemicals as combinatorial synergistic therapy against mixed vaginitis.

Untargeted metabolomics uncovers prime pathways linked to antibacterial action of citral against bacterial vaginosis-causing Gardnerella vaginalis: An in vitro and in vivo study.

Global increase in recurrence of bacterial vaginosis (BV) and worrisome rise in antimicrobial resistance pose an urgent call for new/novel antibacterial agents. In light of the circumstance, the present study demonstrates the in vitro and in vivo antibacterial activity of a phytochemical citral, with a particular emphasis to elucidate its mechanistic action against Gardnerella vaginalis -a potential cause of BV. Out of 21 phytochemicals screened initially against G. vaginalis, citral was envisaged to be a phenomenal antibacterial agent showing MIC and MBC at 128 µg/mL. Citral's rapid killing ability was revealed by a time-killing kinetics assay supported by CFU, signifying that it completely killed the given inoculum of planktonic G. vaginalis cells within 60 min. Further, citral was found to exhibit 1 min contact-killing efficacy together with mature-biofilm disintegrating ability at increasing MICs. To further understand the molecular action of citral, in vitro investigations such as ROS estimation, PI staining and intracellular protein release assay were performed, which demonstrated that citral deteriorated the membrane integrity of G. vaginalis. Galleria mellonella, a simple invertebrate model used to evaluate citral's non-toxic and antibacterial activity in vivo, demonstrates that citral completely restored the larvae from G. vaginalis infection. The metabolite level investigation using LC-MS revealed that citral had negative impact on biotin metabolism (via., biotin), spermidine metabolism (via., 5'-methylthioadenosine and spermidine) and nucleotide metabolism (via., guanine, adenine and uridine). Since that biotin is associated with seven different metabolic pathways, it is conceivable that citral could target biotin biosynthesis or its metabolism and as a result, disrupt other metabolic pathways, such as lipid and fatty acid synthesis, which is essential for the creation of cell membranes. Thus, the current study is the first of its kind to delineate the promising in vitro and in vivo antibacterial efficacy of citral and decipher its plausible antibacterial action mechanism through metabolomic approach, which concomitantly emphasizes citral as a viable natural therapeutic alternative to manage and control BV.

ROS mediated anticandidal efficacy of 3-Bromopyruvate prevents vulvovaginal candidiasis in mice model.

Candidal infections, particularly vulvovaginal candidiasis (VVC), necessitate effective therapeutic interventions in clinical settings owing to their intricate clinical nature and elusive understanding of their etiological mechanisms. Given the challenges in developing effective antifungal therapies, the strategy of repurposing existing pharmaceuticals has emerged as a promising approach to combat drug-resistant fungi. In this regard, the current study investigates molecular insights on the anti-candidal efficacy of a well-proven anticancer small molecule -3-bromopyruvate (3BP) against three clinically significant VVC causing Candida species viz., C. albicans, C. tropicalis and C. glabrata. Furthermore, the study validates 3BP's therapeutic application by developing it as a vaginal cream for the treatment of VVC. 3BP exhibited phenomenal antifungal efficacy (killing >99%) with minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) of 256 µg/mL against all tested Candida spp. Time killing kinetics experiment revealed 20 min as the minimum time required for 3BP at 2XMIC to achieve complete-killing (99.9%) in all Candida strains. Moreover, the ergosterol or sorbitol experiment explicated that the antifungal activity of 3BP does not stem from targeting the cell wall or the membrane component ergosterol. Instead, 3BP was observed to instigate a sequence of pre-apoptotic cascade events, such as phosphatidylserine (PS) externalization, nuclear condensation and ROS accumulations, as evidenced by PI, DAPI and DCFH-DA staining methods. Furthermore, 3BP demonstrated a remarkable efficacy in eradicating mature biofilms of Candida spp., achieving a maximum eradication level of 90%. Toxicity/safety profiling in both in vitro erythrocyte lysis and in vivo Galleria mellonella survival assay authenticated the non-toxic nature of 3BP up to 512 µg/mL. Finally, a vaginal cream formulated with 3BP was found to be effective in VVC-induced female mice model, as it significantly decreasing fungal load and protecting vaginal mucosa. Concomitantly, the present study serves as a clear demonstration of antifungal mechanistic action of anticancer drug -3BP, against Candida species. This finding holds significant potential for mitigating candidal infections, particularly VVC, within healthcare environments.

In silico analysis unravels the promising anticariogenic efficacy of fatty acids against dental caries causing Streptococcus mutans.

Globally, dental caries is a prevalent oral disease caused by cariogenic bacteria, primarily Streptococcus mutans. It establishes caries either through sucrose-dependent (via glycosyltransferases) or through sucrose-independent (via surface adhesins Antigen I/II) mechanism. Sortase A (srtA) attaches virulence-associated adhesins to host tissues. Because of their importance in the formation of caries, targeting these proteins is decisive in the development of new anticariogenic drugs. High-throughput virtual screening with LIPID MAPS -a fatty acid database was performed. The selected protein-ligand complexes were subjected to molecular dynamics simulation (MDs). The Binding Free Energy of complexes was predicted using MM/PBSA. Further, the drug-likeness and pharmacokinetic properties of ligands were also analyzed. Out of 46,200 FAs scrutinized virtually against the three protein targets (viz., GtfC, Ag I/II and srtA), top 5 FAs for each protein were identified as the best hit based on interaction energies viz., hydrogen bond numbers and hydrophobic interaction. Further, two common FAs (LMFA01050418 and LMFA01040045) that showed high binding affinity against Ag I/II and srtA were selected for MDs analysis. A 100ns MDs unveiled a stable conformation. Results of Rg signified that FAs does not induce significant structural & conformational changes. SASA indicated that the complexes maintain higher thermodynamic stability during MDs. The predicted binding free energy (MM/PBSA) of complexes elucidated their stable binding interaction. ADME analysis suggested the FAs are biologically feasible as therapeutic candidates. Overall, the presented in silico data is the first of its kind in delineating FAs as promising anticaries agents of future.Communicated by Ramaswamy H. Sarma.

Deciphering the Mechanism of MRSA Targeting Copper(II) Complexes of NN2 Pincer-Type Ligands.

WHO lists AMR as one of the top ten global public health issues. Therefore, constant effort is needed to develop more efficient antimicrobial drugs. As a result, earth-abundant transition-metal complexes have emerged as an excellent solution. In this regard, new aminoquinoline-based copper(II) pincer complexes 1-3 were designed, synthesized, and characterized by modern spectroscopic techniques. It is worth mentioning that, at the highest concentration (1024 µg/mL) of complexes (1-3), the hemolysis was found to be <15%, implying their less toxicity. Further, the complexes effectively interfered with the growth of Gram positive MRSA and the fungus Candida albicans. Among them, complex 2 was promising (MIC = 16 µg/mL) against MRSA, which was better than the known antibacterial drug kanamycin (64 µg/mL) under identical conditions. The Alamar blue cell viability test and the MBC/MFC identified by spot assay were in accordance with MIC values. Moreover, the insilico studies explained the most probable mechanism of action as inhibition of cell wall biosynthesis and dysfunction of antibiotic sensing proteins. Similarly, the antifungal action might be due to the cell surface adhesion protein dysfunction by the complexes. Furthermore, we are expecting to draw these compounds for clinical applications.

Two novel phages PSPa and APPa inhibit planktonic, sessile and persister populations of Pseudomonas aeruginosa, and mitigate its virulence in Zebrafish model.

The present study explores the avenue of phage therapy as an alternative antimicrobial therapeutic approach to counter multidrug-resistant (MDR) Pseudomonas aeruginosa infection. Our study investigated two novel virulent phages PSPa and APPa, specific to P. aeruginosa, in which in vitro evaluations were carried out to assess the therapeutic potential of phages. Both the identified phages exhibited host specificity by showing antagonistic activity of about 96.43% (27/28) and 92.85% (26/28) towards the 28 MDR clinical isolates of P. aeruginosa. The PSPa phage was found to have linear dsDNA with a sequence length of 66,368 bp and 92 ORFs, of which 32 were encoded for known functions of the phage life cycle and the remaining 60 were hypothetical functions. The APPa phage was found to have linear dsDNA with 59,591 bp of genome length and 79 ORFs, of which 15 were found to have known phage functions and the remaining 64 were found to be hypothetical proteins. Notably, the genome of both the phages lacks genes coding for tRNA, rRNA, and tmRNA. The phylogenetic analysis revealed that PSPa and APPa share > 95% sequence similarity with previously sequenced Pseudomonas viruses of their respective families. Further, the in vivo efficacy evaluation using the zebrafish model revealed that the treatment with PSPa and APPa has remarkably improved the survival rate of bacterial-infected zebrafish, reinforcing the anti-infective potential of the isolated phages PSPa and APPa against P. aeruginosa infection.

Antibacterial activity of 2-hydroxy-4-methoxybenzaldehyde and its possible mechanism against Staphylococcus aureus.

AIM: Staphylococcus aureus causes several complicated infections. Despite decades of research on developing new antimicrobials, methicillin-resistant S. aureus (MRSA) remains a global health problem. Hence, there is a dire need to identify potent natural antibacterial compounds as an alternative to antimicrobials. In this light, the present work divulges the antibacterial efficacy and the action mechanism of 2-hydroxy-4-methoxybenzaldehyde (HMB) isolated from Hemidesmus indicus against S. aureus. METHODS AND RESULTS: Antimicrobial activity of HMB was assessed. HMB exhibited 1024 µg ml-1 as the minimum inhibitory concentration (MIC) and 2 × MIC as the minimum bactericidal concentration against S. aureus. The results were validated by spot assay, time kill, and growth curve analysis. In addition, HMB treatment increased the release of intracellular proteins and nucleic acid contents from MRSA. Additional experiments assessing the structural morphology of bacterial cells using SEM analysis, ß-galactosidase enzyme activity, and the fluorescence intensities of propidium iodide and rhodamine123 dye divulged that the cell membrane as one of the targets of HMB to hinder S. aureus growth. Moreover, the mature biofilm eradication assay revealed that HMB dislodged nearly 80% of the preformed biofilms of MRSA at the tested concentrations. Further, HMB treatment was found to sensitize MRSA cells upon combining tetracycline treatment. CONCLUSIONS: The present study suggests that HMB is a promising compound with antibacterial and antibiofilm activities and could act as a lead structure for developing new antibacterial drugs against MRSA.

Alteration of Cell Membrane Permeability by Cetyltrimethylammonium Chloride Induces Cell Death in Clinically Important Candida Species.

The increased incidence of healthcare-related Candida infection has necessitated the use of effective disinfectants/antiseptics in healthcare settings as a preventive measure to decontaminate the hospital environment and stop the persistent colonization of the offending pathogens. Quanternary ammonium surfactants (QASs), with their promising antimicrobial efficacy, are considered as intriguing and appealing candidates for disinfectants. From this perspective, the present study investigated the antifungal efficacy and action mechanism of the QAS cetyltrimethylammonium chloride (CTAC) against three clinically important Candida species: C. albicans, C. tropicalis, and C. glabrata. CTAC exhibited phenomenal antifungal activity against all tested Candida spp., with minimum inhibitory concentrations (MIC) and minimum fungicidal concentrations (MFC) between 2 and 8 µg/mL. The time−kill kinetics of CTAC (at 2XMIC) demonstrated that an exposure time of 2 h was required to kill 99.9% of the inoculums in all tested strains. An important observation was that CTAC treatment did not influence intracellular reactive oxygen species (ROS), signifying that its phenomenal anticandidal efficacy was not mediated via oxidative stress. In addition, sorbitol supplementation increased CTAC's MIC values against all tested Candida strains by three times (8−32 µg/mL), indicating that CTAC's possible antifungal activity involves fungus cell membrane destruction. Interestingly, the increased fluorescence intensity of CTAC-treated cells in both propidium iodide (PI) and DAPI staining assays indicated the impairment of cell plasma membrane and nuclear membrane integrity by CTAC, respectively. Additionally, CTAC at MIC and 2XMIC was sufficient (>80%) to disrupt the mature biofilms of all tested spp., and it inhibited the yeast-to-hyphae transition at sub-MIC in C. albicans. Finally, the non-hemolytic activity of CTAC (upto 32 µg/mL) in human blood cells and HBECs signified its non-toxic nature at the investigated concentrations. Furthermore, thymol and citral, two phytocompounds, together with CTAC, showed synergistic fungicidal effectiveness against C. albicans planktonic cells. Altogether, the data of the present study appreciably broaden our understanding of the antifungal action mechanism of CTAC and support its future translation as a potential disinfectant against Candida-associated healthcare infections.

Heteroleptic pincer palladium(II) complex coated orthopedic implants impede the AbaI/AbaR quorum sensing system and biofilm development by Acinetobacter baumannii.

Implant-associated infections mediated by Acinetobacter baumannii biofilms have become a major concern in the healthcare sector. As biofilm formation by this important pathogen is mediated by quorum sensing, quorum sensing inhibitors (QSI) have gained much attention. The present study confirms that novel thiazolinyl-picolinamide based palladium(II) complexes had good biofilm disruptive and QSI properties against A. baumannii. Key QS-mediated virulence factors like pili mediated surface motility and polysaccharide production were inhibited by the best Pd(II) complex (E). This also showed potent inhibitory activity against both the standard and clinical strains of A. baumannii. Molecular docking analysis also proved the potent binding affinity of Pd(II)-E with the virulence targets. The Pd(II) complex also disrupted preformed biofilms and down-regulated the expression of QS mediated virulence genes in the biofilms established on implant material (titanium plates). As a whole, the present study showed that the novel thiazolinyl-picolinamide based Pd(II) complexes offer a promising anti-infective strategy to combat biofilm-mediated implant infections.

Bacterial Quorum-Sensing Molecules as Promising Natural Inhibitors of Candida albicans Virulence Dimorphism: An In Silico and In Vitro Study.

Farnesol, a self-secreted quorum-sensing molecule (QSM) of Candida albicans, has been known to limit yeast-to-hyphal transition by blocking the RAS1-cAMP-PKA pathway. In a similar fashion, certain bacterial QSMs have also been reported to be successful in attenuating C. albicans biofilm and hyphal formation at relatively high cell density. This prompted us to investigate the antihyphal efficacy of certain bacterial QSMs through virtual docking against seminal drug targets, viz., CYCc and RAS1, that have been reported to be the hallmark players in C. albicans dimorphic virulence cascade. Against this backdrop, 64 QSMs belonging to five different bacterial QS signaling systems were subjected to initial virtual screening with farnesol as reference. Data of the virtual screening unveiled QSMs belonging to diketopiperazines (DKPs), i.e., 3-benzyl-6-isobutylidene-2,5-piperazinedione (QSSM 1157) and cyclo(l-Pro-l-Leu) (QSSM 1112), as potential inhibitors of CYCc and RAS1 with binding energies of -8.2 and -7.3 kcal mol-1, respectively. Further, the molecular dynamics simulations (for 50 ns) of CYCc-QSSM 1157 and RAS1-QSSM 1112 complexes revealed the mean ligand root mean square deviation (RMSD) values of 0.35 and 0.27 Å, respectively, which endorsed the rigid nature, less fluctuation in binding stiffness, and conformation of binding complexes. Furthermore, the identified two QSMs were found to be good in solubility, absorption, and permeation and less toxic in nature, as revealed by pharmacokinetics and toxicity analyses. In addition, the in vitro antihyphal assays using liquid and solid media, germ-tube experiment, and microscopic analysis strongly validated DKP-QSSM 1112 as a promising inhibitor of hyphal transition. Taken together, the present study unequivocally proves that DKPs can be used as potent inhibitors of C. albicans virulence dimorphism.

Catechol thwarts virulent dimorphism in Candida albicans and potentiates the antifungal efficacy of azoles and polyenes.

The present study was deliberately focused to explore the antivirulence efficacy of a plant allelochemical-catechol against Candida albicans, and attempts were made to elucidate the underlying mechanisms as well. Catechol at its sub-MIC concentrations (2-256 µg/mL) exhibited a dose dependent biofilm as well as hyphal inhibitory efficacies, which were ascertained through both light and fluorescence microscopic analyses. Further, sub-MICs of catechol displayed remarkable antivirulence efficacy, as it substantially inhibited C. albicans' virulence enzymes i.e. secreted hydrolases. Notably, FTIR analysis divulged the potency of catechol in effective loosening of C. albicans' exopolymeric matrix, which was further reinforced using EPS quantification assay. Although, catechol at BIC (256 µg/mL) did not disrupt the mature biofilms of C. albicans, their initial adherence was significantly impeded by reducing their hydrophobic nature. Besides, FTIR analysis also unveiled the ability of catechol in enhancing the production of farnesol-a metabolite of C. albicans, whose accumulation naturally blocks yeast-hyphal transition. The qPCR data showed significant down-regulation of candidate genes viz., RAS1, HWP1 and ALS3 which are the key targets of Ras-cAMP-PKA pathway -the pathway that contribute for C. albicans' pathogenesis. Interestingly, the up-regulation of TUP1 (a gene responsible for farnesol-mediated hyphal inhibition) during catechol exposure strengthen the speculation of catechol triggered farnesol-mediated hyphal inhibition. Furthermore, catechol profusely enhanced the fungicidal efficacy of certain known antifungal agent's viz., azoles (ketoconazole and miconazole) and polyenes (amphotericin-B and nystatin).

Indian Ethnomedicinal Phytochemicals as Promising Inhibitors of RNA-Binding Domain of SARS-CoV-2 Nucleocapsid Phosphoprotein: An In Silico Study.

SARS-CoV-2, an etiological agent of COVID-19, has been the reason for the unexpected global pandemic, causing severe mortality and imposing devastative effects on public health. Despite extensive research work put forward by scientist around globe, so far, no suitable drug or vaccine (safe, affordable, and efficacious) has been identified to treat SARS-CoV-2. As an alternative way of improvising the COVID-19 treatment strategy, that is, strengthening of host immune system, a great deal of attention has been given to phytocompounds from medicinal herbs worldwide. In a similar fashion, the present study deliberately focuses on the phytochemicals of three Indian herbal medicinal plants viz., Mentha arvensis, Coriandrum sativum, and Ocimum sanctum for their efficacy to target well-recognized viral receptor protein through molecular docking and dynamic analyses. Nucleocapsid phosphoprotein (N) of SARS-CoV-2, being a pivotal player in replication, transcription, and viral genome assembly, has been recognized as one of the most attractive viral receptor protein targets for controlling the viral multiplication in the host. Out of 127 phytochemicals screened, nine (linarin, eudesmol, cadinene, geranyl acetate, alpha-thujene, germacrene A, kaempferol-3-O-glucuronide, kaempferide, and baicalin) were found to be phenomenal in terms of exhibiting high binding affinity toward the catalytic pocket of target N-protein. Further, the ADMET prediction analysis unveiled the non-tumorigenic, noncarcinogenic, nontoxic, non-mutagenic, and nonreproductive nature of the identified bioactive molecules. Furthermore, the data of molecular dynamic simulation validated the conformational and dynamic stability of the docked complexes. Concomitantly, the data of the present study validated the anti-COVID efficacy of the bioactives from selected medicinal plants of Indian origin.

CRISPR based development of RNA editing and the diagnostic platform.

Clustered Regularly Interspersed Short Palindromic Repeat-CRISPR-Associated (CRISPR-Cas) system has improved the ability to edit and control gene expression as desired. Genome editing approaches are currently leading the biomedical research with improved focus on direct nuclease dependent editing. So far, the research was predominantly intended on genome editing over the DNA level, recent adapted techniques are initiating to secure momentum through their proficiency to provoke modifications in RNA sequence. Integration of this system besides to lateral flow method allows reliable, quick, sensitive, precise and inexpensive diagnostic. These interesting methods illustrate only a small proportion of what is technically possible for this novel technology, but several technological obstacles need to be overcome prior to the CRISPR-Cas genome editing system can meet its full ability. This chapter covers the particulars on recent advances in CRISPR-Cas9 genome editing technology including diagnosis and technical advancements, followed by molecular mechanism of CRISPR-based RNA editing and diagnostic tools and types, and CRISPR-Cas-based biosensors.

CRISPR based bacterial genome editing and removal of pathogens.

Engineering nucleases to achieve targeted genome editing has turned out to be a revolutionary means for manipulating the genetic content in diversified living organisms. For targeted genome editing, till to date, only three engineered nucleases exist viz. zinc finger nucleases, transcription activator-like effector nucleases and RNA-mediated nucleases (RGNs) (Cas nucleases) from the clustered regularly interspaced short palindromic repeat (CRISPR). Among, Cas9 nuclease has been considered as a simplest tool for efficient modification of endogenous genes in an extensive stretch of organisms, owing to its amenability to design guide RNA compatible to the sequence of new targets. Moreover, CRISPR/Cas system delivers a multipurpose RNA-guided DNA-targeting platform called as CRISPR interference (CRISPRi), as well as epigenetic modifications and high throughput screening in diverse organism including bacteria, all in a sequence explicit way. With these entire advancements, the present chapter illustrates the deployment of CRISPR/Cas9 in bacterial genome editing and removal of pathogens.

Promising phytochemicals of traditional Indian herbal steam inhalation therapy to combat COVID-19 - An in silico study.

BACKGROUND: COVID-19, the presently prevailing global public health emergency has culminated in international instability in economy. This unprecedented pandemic outbreak pressingly necessitated the trans-disciplinary approach in developing novel/new anti-COVID-19 drugs especially, small molecule inhibitors targeting the seminal proteins of viral etiological agent, SARS-CoV-2. METHODS: Based on the traditional medicinal knowledge, we made an attempt through molecular docking analysis to explore the phytochemical constituents of three most commonly used Indian herbs in 'steam inhalation therapy' against well recognized viral receptor proteins. RESULTS: A total of 57 phytochemicals were scrutinized virtually against four structural protein targets of SARS-CoV-2 viz. 3CLpro, ACE-2, spike glycoprotein and RdRp. Providentially, two bioactives from each of the three plants i.e. apigenin-o-7-glucuronide and ellagic acid from Eucalyptus globulus; eudesmol and viridiflorene from Vitex negundo and; vasicolinone and anisotine from Justicia adhatoda were identified to be the best hit lead molecules based on interaction energies, conventional hydrogen bonding numbers and other non-covalent interactions. On comparison with the known SARS-CoV-2 protease inhibitor -lopinavir and RdRp inhibitor -remdesivir, apigenin-o-7-glucuronide was found to be a phenomenal inhibitor of both protease and polymerase, as it strongly interacts with their active sites and exhibited remarkably high binding affinity. Furthermore, in silico drug-likeness and ADMET prediction analyses clearly evidenced the usability of the identified bioactives to develop as drug against COVID-19. CONCLUSION: Overall, the data of the present study exemplifies that the phytochemicals from selected traditional herbs having significance in steam inhalation therapy would be promising in combating COVID-19.

Global multi-omics and systems pharmacological strategy unravel the multi-targeted therapeutic potential of natural bioactive molecules against COVID-19: An in silico approach.

Understanding the immunological behavior of COVID-19 cases at molecular level is essential for therapeutic development. In this study, multi-omics and systems pharmacology analyses were performed to unravel the multi-targeted mechanisms of novel bioactives to combat COVID-19. Immuno-transcriptomic dataset of healthy controls and COVID-19 cases was retrieved from ArrayExpress. Phytocompounds from ethnobotanical plants were collected from PubChem. Differentially expressed 98 immune genes associated with COVID-19 were derived through NetworkAnalyst 3.0. Among 259 plant derived compounds, 154 compounds were targeting 13 COVID-19 immune genes involved in diverse signaling pathways. In addition, pharmacological properties of these phytocompounds were compared with COVID-19 drugs prescribed by WHO, and 25 novel phytocompounds were found to be more efficient with higher bioactive scores. The current study unravels the virogenomic signatures which can serve as therapeutic targets and identified phytocompounds with anti-COVID-19 efficacy. However, further experimental validation is essential to bring out these molecules as commercial drug candidates.

Marine bacterial DNase curtails virulence and disrupts biofilms of Candida albicans and non-albicans Candida species.

Candida is one of the most prevalent fungal pathogens in clinical settings which form antibiotic-resistant biofilms on biomedical devices. Hence, there is a need for non-antimicrobial alternatives to combat these infections. The present study investigates the anti-biofilm effect of marine bacterial DNase by targeting the eDNA present in the biofilms of Candida spp. A strain of Vibrio alginolyticus (AMSII) which showed enhanced DNase activity was isolated from marine sediment. Treatment of young and mature Candida biofilms with purified marine bacterial DNase (MBD) caused a 60-80% reduction in biofilm biomass, similar to treatment with DNase I from Bovine pancreas. Scanning electron microscopy showed that MBD significantly reduced the formation of biofilms on urinary catheters and more importantly prevented the virulent yeast to hyphae dimorphic switch in C. albicans. The present study identified a potential non-antibiotic alternative therapy to eradicate Candida biofilms and can be used to develop enzyme fabricated antifouling indwelling medical devices.

Extracted chitosan disrupts quorum sensing mediated virulence factors in Urinary tract infection causing pathogens.

Quorum sensing (QS) plays an important role during the aetiology of urinary tract infection (UTI), as several virulence factors are under the regulation of QS. Pseudomonas aeruginosa and Serratia marcescens, the primary causative agents of UTI, employ acyl homoserine lactone (AHL) as signal molecules to coordinate various virulence factors. In this present study, chitosan extracted from the marine crab Portunus sanguinolentus was screened for its ability to inhibit the QS-signaling molecules of P. aeruginosa (PA01) and few clinical isolates of P. aeruginosa and S. marcescens. The extracted chitosan on comparison with a commercial chitosan showed significant inhibition of several QS-dependent virulence factors in P. aeruginosa and S. marscenes. Furthermore, qPCR analysis was carried out to confirm the down-regulation of fimA, fimC and flhD genes involved in adhesion and pathogenesis of S. marcescens and lasI and rhlI genes that governs the P. aeruginosa quorum sensing system. Moreover, the chitosan when coated on a catheter was also able to disrupt the mature biofilms which was revealed by scanning electron microscopy. Collectively, the present study showcases the QS inhibitory property of extracted chitosan from crab shells which is being discarded as a recalcitrant biowaste.

Effects of a traditional Thai polyherbal medicine 'Ya-Samarn-Phlae' as a natural anti-biofilm agent against Pseudomonas aeruginosa.

Ya-Samarn-Phlae (YaSP) is a traditional Thai polyherbal formula for the treatment of chronic wounds. Although its ethanol extract has been proven to possess several wound-related biological activities, there is no scientific information available for the infused oil of YaSP, which is its traditionally prepared form. This present study therefore aimed to evaluate the efficacy of different infused oils obtained from either fresh or oven-dried herbal parts of YaSP (F-YaSP and D-YaSP) against biofilms of Pseudomonas aeruginosa, which reside in chronic wounds. Its main active herbal component, Garcinia mangostana (F-GM and D-GM), as well as α-mangostin were also tested in this study. All infused oils significantly inhibited the biofilm formation of P. aeruginosa with a percentage of reduction ranging from 50 to 90%. Visualization of the inhibition of biofilm development was confirmed using scanning electron and atomic force microscopes. All tested agents resulted in a reduction in the mean average roughness of the biofilm, whereas only treating with D-YaSP, D-GM, and α-mangostin led to a decrease in both peak height and peak-valley height. MTT reduction assays revealed that the metabolic activity of P. aeruginosa mature biofilms decreased considerably up to 50% after only 3 h of incubation and after only 9 h of exposure to D-YaSP. Confocal laser scanning micrographs illustrated that a maximum biofilm eradication was found when treated with the extracts for 3 h, whereas the biomass, the average thickness, maximum thickness, and the surface to volume ratio of the treated biofilm was reduced after up to 18 h of contact time. It can be concluded that D-YaSP can effectively inhibit biofilm formation and eradicate mature biofilms of P. aeruginosa. It should be noted that G. mangostana and α-mangostin contribute in YaSP as principle active agents for anti-biofilm efficacy.