Insights into cucumber (Cucumis sativus) genetics: Genome-wide discovery and computational analysis of the Calreticulin Domain-Encoding gene (CDEG) family.

Cucumber is an essential vegetable crop throughout the world. Cucumber development is vital for accomplishing both quality and productivity requirements. Meanwhile, numerous factors have resulted in substantial cucumber losses. However, the calreticulin domain-encoding genes (CDEGs) in cucumber were not well-characterized and had little function. In the genome-wide association study (GWAS), we recognized and characterized the CDEGs in Cucumis sativus (cucumber). Through a comprehensive study of C. sativus, our research has unveiled the presence of three unique genes, denoted as CsCRTb, CsCRT3, and CsCNX1, unevenly distributed on three chromosomes in the genome of C. sativus. In accordance to the phylogenetic investigation, these genes may be categorized into three subfamilies. Based on the resemblance with AtCDE genes, we reorganized the all CsCDE genes in accordance with international nomenclature. The expression analysis and cis-acting components revealed that each of CsCDE gene promoter region enclosed number of cis-elements connected with hormone and stress response. According to subcellular localization studies demonstrated that, they were found in deferent locations of the cell such as endoplasmic reticulum, plasma membrane, golgi apparatus, and vacuole, according to subcellular localization studies. Chromosomal distribution analysis and synteny analysis demonstrated the probability of segmental or tandem duplications within the cucumber CDEG gene family. Additionally, miRNAs displayed diverse modes of action, including mRNA cleavage and translational inhibition. We used the RNA seq data to analyze the expression of CDEG genes in response to cold stress and also improved cold tolerance, which was brought on by treating cucumber plants to an exogenous chitosan oligosaccharide spray. Our investigation revealed that these genes responded to this stress in a variety of ways, demonstrating that they may adapt quickly to environmental changes in cucumber plants. This study provides a base for further understanding in reference to CDE gene family and reveals that genes play significant functions in cucumber stress responses.

Microbial Biofilms: Applications, Clinical Consequences, and Alternative Therapies.

Biofilms are complex communities of microorganisms that grow on surfaces and are embedded in a matrix of extracellular polymeric substances. These are prevalent in various natural and man-made environments, ranging from industrial settings to medical devices, where they can have both positive and negative impacts. This review explores the diverse applications of microbial biofilms, their clinical consequences, and alternative therapies targeting these resilient structures. We have discussed beneficial applications of microbial biofilms, including their role in wastewater treatment, bioremediation, food industries, agriculture, and biotechnology. Additionally, we have highlighted the mechanisms of biofilm formation and clinical consequences of biofilms in the context of human health. We have also focused on the association of biofilms with antibiotic resistance, chronic infections, and medical device-related infections. To overcome these challenges, alternative therapeutic strategies are explored. The review examines the potential of various antimicrobial agents, such as antimicrobial peptides, quorum-sensing inhibitors, phytoextracts, and nanoparticles, in targeting biofilms. Furthermore, we highlight the future directions for research in this area and the potential of phytotherapy for the prevention and treatment of biofilm-related infections in clinical settings.

Myriad applications of bacteriophages beyond phage therapy.

Bacteriophages are the most abundant biological entity on the planet, having pivotal roles in bacterial ecology, animal and plant health, and in the biogeochemical cycles. Although, in principle, phages are simple entities that replicate at the expense of their bacterial hosts, due the importance of bacteria in all aspects of nature, they have the potential to influence and modify diverse processes, either in subtle or profound ways. Traditionally, the main application of bacteriophages is phage therapy, which is their utilization to combat and help to clear bacterial infections, from enteric diseases, to skin infections, chronic infections, sepsis, etc. Nevertheless, phages can also be potentially used for several other tasks, including food preservation, disinfection of surfaces, treatment of several dysbioses, and modulation of microbiomes. Phages may also be used as tools for the treatment of non-bacterial infections and pest control in agriculture; moreover, they can be used to decrease bacterial virulence and antibiotic resistance and even to combat global warming. In this review manuscript we discuss these possible applications and promote their implementation.

In vitro inhibition of biofilm and virulence factor production in azole-resistant strains of Candida albicans isolated from diabetic foot by Artemisia vulgaris stabilized tin (IV) oxide nanoparticles.

The advent of nanotechnology has been instrumental in the development of new drugs with novel targets. Recently, metallic nanoparticles have emerged as potential candidates to combat the threat of drug-resistant infections. Diabetic foot ulcers (DFUs) are one of the dreadful complications of diabetes mellitus due to the colonization of numerous drug-resistant pathogenic microbes leading to biofilm formation. Biofilms are difficult to treat due to limited penetration and non-specificity of drugs. Therefore, in the current investigation, SnO2 nanoparticles were biosynthesized using Artemisia vulgaris (AvTO-NPs) as a stabilizing agent and were characterized using ultraviolet-visible (UV-vis) spectroscopy, Fourier transform infrared spectroscopy (FT-IR), X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy-dispersive X-ray spectroscopy (EDX). Furthermore, the efficacy of AvTO-NPs against biofilms and virulence factors of drug-resistant Candida albicans strains isolated from DFUs was assessed. AvTO-NPs displayed minimum inhibitory concentrations (MICs) ranging from 1 mg/mL to 2 mg/mL against four strains of C. albicans. AvTO-NPs significantly inhibited biofilm formation by 54.8%-87%, germ tube formation by 72%-90%, cell surface hydrophobicity by 68.2%-82.8%, and exopolysaccharide (EPS) production by 69%-86.3% in the test strains at respective 1/2xMIC. Biosynthesized NPs were effective in disrupting established mature biofilms of test strains significantly. Elevated levels of reactive oxygen species (ROS) generation in the AvTO-NPs-treated C. albicans could be the possible cause of cell death leading to biofilm inhibition. The useful insights of the present study could be exploited in the current line of treatment to mitigate the threat of biofilm-related persistent DFUs and expedite wound healing.

Phytomicrobiome communications: Novel implications for stress resistance in plants.

The agricultural sector is a foremost contributing factor in supplying food at the global scale. There are plethora of biotic as well as abiotic stressors that act as major constraints for the agricultural sector in terms of global food demand, quality, and security. Stresses affect rhizosphere and their communities, root growth, plant health, and productivity. They also alter numerous plant physiological and metabolic processes. Moreover, they impact transcriptomic and metabolomic changes, causing alteration in root exudates and affecting microbial communities. Since the evolution of hazardous pesticides and fertilizers, productivity has experienced elevation but at the cost of impeding soil fertility thereby causing environmental pollution. Therefore, it is crucial to develop sustainable and safe means for crop production. The emergence of various pieces of evidence depicting the alterations and abundance of microbes under stressed conditions proved to be beneficial and outstanding for maintaining plant legacy and stimulating their survival. Beneficial microbes offer a great potential for plant growth during stresses in an economical manner. Moreover, they promote plant growth with regulating phytohormones, nutrient acquisition, siderophore synthesis, and induce antioxidant system. Besides, acquired or induced systemic resistance also counteracts biotic stresses. The phytomicrobiome exploration is crucial to determine the growth-promoting traits, colonization, and protection of plants from adversities caused by stresses. Further, the intercommunications among rhizosphere through a direct/indirect manner facilitate growth and form complex network. The phytomicrobiome communications are essential for promoting sustainable agriculture where microbes act as ecological engineers for environment. In this review, we have reviewed our building knowledge about the role of microbes in plant defense and stress-mediated alterations within the phytomicrobiomes. We have depicted the defense biome concept that infers the design of phytomicrobiome communities and their fundamental knowledge about plant-microbe interactions for developing plant probiotics.

Assessment of Foodborne Bacterial Pathogens in Buffalo Raw Milk Using Polymerase Chain Reaction Based Assay.

Milk is a putrescible commodity that is extremely prone to microbial contamination. Primarily, milk and dairy products are believed to be easily contaminated by pathogenic microorganisms, including Listeria monocytogenes, Salmonella spp., and Staphylococcus aureus. The microbiological quality of raw milk and dairy products regarding foodborne pathogens is of paramount importance due to concern of human health. In this study 400 buffalo raw milk samples were screened for assessing the prevalence of L. monocytogenes, Salmonella spp., and S. aureus. This study implemented uniplex-polymerase chain reaction (u-PCR) and multiplex-polymerase chain reaction (m-PCR) assays for the fast simultaneous detection of these pathogens comparing to the conventional culturing methods. Raw milk samples were found contaminated with the prevalence of 2.2%, 4.0%, and 14.2% for L. monocytogenes, Salmonella spp., and S. aureus, respectively. These pathogens were detected with the optimized polymerase chain reaction assays after 6 h of enrichment. u-PCR and m-PCR demonstrated the limit of detection as 104, 102, and 10 cells/mL after 6, 12, 18, and 24 h for each culture of the pathogens. A high sensitivity (10 colony-forming unit [CFU]/mL) of the m-PCR protocol was noted. The developed protocol is a cost-effective and rapid method for the simultaneous detection of pathogens associated with raw milk and dairy industries.

Prevalence and Molecular Characterization of Metallo ß-Lactamase Producing Gram-Negative Pathogens Causing Eye Infections.

Purpose: Metallo ß-lactamases (MßL) production is a worldwide problem, particularly in gram-negative bacteria. As scanty data is available on the prevalence of MBL, the present study is being undertaken to determine the prevalence, antibacterial sensitivity patterns, and molecular characterization of MßL associated resistant genes in gram-negative bacteria isolated from ocular infections. Material and Methods: At a tertiary eye care center in south India, 359 gram-negative pathogens, 200 isolates from eye infections, and 159 isolates from normal flora of the eye were studied. A gold standard microbiology method was used to identify the isolates. An antibiotic double disc synergy test and a combination disc test were used to detect MßL production. Multiplex PCR was used to investigate the molecular characteristics of the MßL encoding genes blaVIM, blaIMP, and blaNDM. Results: Of the 359 gram-negative bacterial pathogens, Pseudomonas aeruginosa 108 (30.1%) and Enterobacter agglomerans 46 (12.8%) were commonly isolated. High prevalence of P. aeruginosa 81% (17 strains) was detected as an MßL producer and it shows 100% resistance to 2nd and 3rd generation cephalosporins and meropenem. Multiplex PCR detected only the blaVIM gene in 56 (28%) of various eye infections and 27 (17%) of normal flora of the gram-negative bacteria (GNB). The blaVIM gene is detected predominantly in 51.8% of keratitis and 21.4% of postoperative endophthalmitis. High prevalence of the gene was detected in P. aeruginosa 42.9% (24 of 56) and Alcaligens denitrificans 10.7% (6 of 56) from eye infections. Whereas, in the control group, P. aeruginosa and E. coli each had 14.8% (4 of 27) that were shown positive. Conclusion: The emerging MßLs mediated resistance among P. aeruginosa is a challenging task for ophthalmologists, especially in patients with endophthalmitis and bacterial keratitis. This local knowledge will aid in advising appropriate antibiotic use and avoiding unnecessary antibiotic prescriptions, which are highly warranted.

Broad-Spectrum Antimicrobial, Antioxidant, and Anticancer Studies of Leaf Extract of Simarouba glauca DC In Vitro.

The current study aimed to screen the preliminary phytochemicals in the leaf extract of the medicinal plant Simarouba glauca and to analyze its potential antimicrobial, antioxidant and anticancer properties. The phytochemical profile of the methanol extract was analyzed, and bioactive compounds were identified using chromatography, FTIR and GCMS. Antimicrobial activity and Minimum Inhibitory Concentration (MIC) were determined against 14 bacterial and 6 fungal strains. Moreover, the synergistic effect of a plant extract with commercially available antibiotics was also evaluated using the checkerboard method. The ethanolic and methanolic extracts showed exclusive activity against S. aureus and profound activity against E. coli and S. marcescens. Upon comparing breakpoints, methanolic extract demonstrated higher antimicrobial activity with a MIC value of 3.2 mg/mL against the test pathogens. Furthermore, the extracts demonstrated potential antioxidant activity; methanol extract had higher antioxidant potential compared to the ethanol extract. The major proactive bioactive compound with maximum antioxidant capacity was observed to be terpenoids. The methanol extract of S. glauca showed significant cytotoxicity against the MCF-7 breast cancer cell line with an IC50 value of 16.12 µg/mL. The overall results of our work provide significant evidence for the usage of methanolic extract of S. glauca as an efficient ethnomedicinal agent and a potential candidate for relieving many human ailments.

Interference of quorum sensing regulated bacterial virulence factors and biofilms by Plumbago zeylanica extract.

There has been tremendous spread of antimicrobial resistance globally, mainly due to the excessive and unnecessary use of antibiotics, making the situation alarming. This has created a need for the development of alternative strategies to selectively target the bacterial pathogenicity without exerting selection pressure for the development of antimicrobial resistance. Targeting quorum sensing (QS)-mediated virulence and biofilms by nontoxic natural products is gaining importance as new control strategy to combat the virulence and biofilms of pathogenic bacteria. In this study, the crude extract of Plumbago zeylanica was fractioned in different solvents using liquid-liquid partitioning to obtain the most bioactive fraction. The inhibitory effect of the bioactive extract of P. zeylanica on QS at sub-minimum inhibitory concentrations (MICs) was studied against Chromobacterium violaceum 12472, Pseudomonas aeruginosa PAO1, and Serratia marcescens MTCC 97. Biofilm inhibition was studied using microtiter plate assay, scanning electron microscopy, and confocal laser scanning microscopy. Major phytocompounds detected were cinnamaldehyde dimethyl acetal, plumbagin, asarone, 4-chromanol, phthalic acid, palmitic acid, ergost-5-en-3-ol, stigmasterol, and ß-sitosterol. The violacein production in C. violaceum 12472 was reduced by >80% in the presence of P. zeylanica hexane fraction (PZHF; 200 µg/ml). The most active PZHF inhibited QS-mediated virulence factors of P. aeruginosa PAO1 such as pyocyanin, pyoverdin, rhamnolipid production, motility, etc., significantly at sub-MICs. Similarly, PZHF showed 59 to 76% inhibition of biofilm formation of above test pathogens. The findings revealed that active fraction of P. zeylanica was effective against the QS-regulated functions and biofilms development of Gram -ve pathogenic bacteria.

Prescribed burning reduces species richness of wood-decay fungi in the forests of northwest Arkansas.

The present study, which was carried out at three localities in the Ozark Mountains of northwest Arkansas, investigated the effects of prescribed burning on wood-decomposing fungi using samples of decaying woody debris (DWD) placed in plastic incubation chambers. One of the localities had not been subjected to recent prescribed burning, whereas the other localities contained both an unburned area as well as an area recently subjected to burning. In all three localities, small pieces of decaying woody debris (DWD) were collected, placed in the incubation chambers and the latter kept moist for any extended period of time. Pieces of DWD collected in the areas subjected to burning typically displayed evidence of considerable charring. Fruiting bodies appearing in the incubation chambers were removed and identified by sequencing of ribosomal DNA region. A total of 101 specimens representing 80 different taxa were recorded in the entire investigation, but the numbers of both specimens and taxa were appreciably higher for the unburned collecting sites. As such, the data obtained indicate that prescribed burning lowers the species richness of the wood-decomposing fungi associated with DWD at a particular locality. The unique aspect of the present study was the use of incubation chambers to characterize the taxa of fungi associated with CWD.

Phyto-Mediated Synthesis of Porous Titanium Dioxide Nanoparticles From Withania somnifera Root Extract: Broad-Spectrum Attenuation of Biofilm and Cytotoxic Properties Against HepG2 Cell Lines.

There is grave necessity to counter the menace of drug-resistant biofilms of pathogens using nanomaterials. Moreover, we need to produce nanoparticles (NPs) using inexpensive clean biological approaches that demonstrate broad-spectrum inhibition of microbial biofilms and cytotoxicity against HepG2 cell lines. In the current research work, titanium dioxide (TiO2) NPs were fabricated through an environmentally friendly green process using the root extract of Withania somnifera as the stabilizing and reducing agent to examine its antibiofilm and anticancer potential. Further, X-ray diffraction (XRD), Fourier transform infrared (FTIR), scanning electron microscopy (SEM), transmission electron micrograph (TEM), energy-dispersive X-ray spectroscopy (EDS), dynamic light scattering (DLS), thermogravimetric analysis (TGA), and Brunauer-Emmett-Teller (BET) techniques were used for determining the crystallinity, functional groups involved, shape, size, thermal behavior, surface area, and porosity measurement, respectively, of the synthesized TiO2 NPs. Antimicrobial potential of the TiO2 NPs was determined by evaluating the minimum inhibitory concentration (MIC) against Escherichia coli, Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus, Listeria monocytogenes, Serratia marcescens, and Candida albicans. Furthermore, at levels below the MIC (0.5 × MIC), TiO2 NPs demonstrated significant inhibition of biofilm formation (43-71%) and mature biofilms (24-64%) in all test pathogens. Cell death due to enhanced reactive oxygen species (ROS) production could be responsible for the impaired biofilm production in TiO2 NP-treated pathogens. The synthesized NPs induced considerable reduction in the viability of HepG2 in vitro and could prove effective in controlling liver cancer. In summary, the green synthesized TiO2 NPs demonstrate multifarious biological properties and could be used as an anti-infective agent to treat biofilm-based infections and cancer.

Food color 'Azorubine' interferes with quorum sensing regulated functions and obliterates biofilm formed by food associated bacteria: An in vitro and in silico approach.

Quorum sensing (QS) plays a crucial role in different stages of biofilm development, virulence production, and subsequently to the growth of bacteria in food environments. Biofilm mediated spoilage of food is one of the ongoing challenge faced by the food industry worldwide as it incurs substantial economic losses and leads to various health issues. In the present investigation, we studied the interference of quorum sensing, its regulated virulence functions, and biofilm in food-associated bacteria by colorant azorubine. In vitro bioassays demonstrated significant inhibition of QS and its coordinated virulence functions in Chromobacterium violaceum 12472 (violacein) and Pseudomonas aeruginosa PAO1 (elastase, protease, pyocyanin, and alginate). Further, the decrease in the production EPS (49-63%) and swarming motility (61-83%) of the pathogens was also recorded at sub-MICs. Azorubine demonstrated broad-spectrum biofilm inhibitory potency (50-65%) against Chromobacterium violaceum, Pseudomonas aeruginosa, E. coli O157:H7, Serratia marcescens, and Listeria monocytogenes. ROS generation due to the interaction between bacteria and azorubine could be responsible for the biofilm inhibitory action of the food colorant. Findings of the in vitro studies were well supported by molecular docking and simulation analysis of azorubine and QS virulence proteins. Azorubine showed strong binding to PqsA as compared to other virulent proteins (LasR, Vfr, and QscR). Thus, it is concluded that azorubine is a promising candidate to ensure food safety by curbing the menace of bacterial QS and biofilm-based spoilage of food and reduce economic losses.

Bio-inspired facile fabrication of silver nanoparticles from in vitro grown shoots of Tamarix nilotica: explication of its potential in impeding growth and biofilms of Listeria monocytogenes and assessment of wound healing ability.

Novel, safe, and effective antilisterial agents are required in order to prevent Listeria monocytogenes infections and maintain food safety. This study synthesized silver nanoparticles (AgNPs) from the shoot extract of in vitro-grown Tamarix nilotica (TN) and characterized them using X-ray diffraction, Fourier transform infrared spectroscopy, UV-visible spectroscopy, dynamic light scattering, scanning electron microscopy (SEM), energy-dispersive X-ray spectroscopy (EDXS), and transmission electron microscopy (TEM). We also assessed the antilisterial potential of the synthesized TN-AgNPs by determining the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) against two strains of L. monocytogenes and L. innocua. TN-AgNPs (2×MICs) showed a significant decrease in growth in all Listeria test strains. Release of cellular content and cell morphology analysis of TN-AgNP-treated bacterial cells demonstrated the mechanism of bactericidal activity of AgNPs. In addition, TN-AgNPs induced a significant decrease in swimming motility (62-71%), biofilm formation (57-64%), and preformed biofilms (48-58%) in all Listeria test strains at sub-inhibitory concentrations. Microtitre plate assay results for biofilm inhibition were confirmed by SEM and CLSM visualization of TN-AgNP-treated and TN-AgNP-untreated Listeria test strains. TN-AgNPs also showed wound-healing activity in MCF-7 cells by inhibiting cell migration in a scratch plate assay. TN-AgNP-induced enhanced reactive oxygen species generation in treated cells could be a plausible reason for the biofilm inhibitory activity of AgNPs. TN-AgNPs having antilisterial, antibiofilm, and wound-healing properties can be effectively used to prevent L. monocytogenes infections in the food industry and healthcare.

Mitigation of acyl-homoserine lactone (AHL) based bacterial quorum sensing, virulence functions, and biofilm formation by yttrium oxide core/shell nanospheres: Novel approach to combat drug resistance.

The present study evaluated the efficacy of Y2O3:Tb (core) and Y2O3:Tb@SiO2 nanospheres (core/shell NSs) against virulence functions regulated by quorum sensing (QS) and biofilm formation in pathogenic bacteria. Scanning electron microscope (SEM) images were used to study the size, shape, and morphology. The images clearly displayed spherical shaped, mono-dispersed particles with narrow size distribution and an average grain size of 110-130 nm. The chemical composition of the samples was determined by using energy dispersive X-ray (EDX) and X-ray photoelectron spectroscopy (XPS). We determined the impact of core and core/shell NSs on QS using sensor strains of Chromobacterium violaceum CVO26 and Pseudomonas aeruginosa PAO1 in a comparative study. Sub-MICs of core and core/shell NSs substantially suppressed QS-controlled violacein production in C. violaceum. Similar concentration-dependent effect of sub-MICs of synthesized core and core/shell NSs was observed in the QS-regulated virulence functions (elastase, total protease, pyocyanin production, swarming motility, and exopolysaccharide production) in PAO1. A concentration-dependent decrease (14-60%) was recorded in the biofilm forming capability of PAO1, upon treatment with core and core/shell NSs. Moreover, core/shell NSs were more effective in inhibiting biofilm at higher tested concentrations as compared to core-NSs. The synthesized NSs demonstrated significantly impaired attachment of cells to the microtiter plate indicating that NSs target biofilm inhibition at the attachment stage. Based on these results, we predict that core and core/shell NSs may be an alternative to combat the threat of drug-resistant pathogenic bacteria.

24-Epibrassinolide (EBR) Confers Tolerance against NaCl Stress in Soybean Plants by Up-Regulating Antioxidant System, Ascorbate-Glutathione Cycle, and Glyoxalase System.

: The present research was performed to assess the effect of 24-epibrassinolide (EBR) on salt-stressed soybean plants. Salt stress suppressed growth, biomass yield, gas exchange parameters, pigment content, and chlorophyll fluorescence, but all these parameters were up-regulated by EBR supply. Moreover, salt stress increased hydrogen peroxide, malondialdehyde, and electrolyte leakage. EBR supplementation reduced the accumulation of oxidative stress biomarkers. The activities of superoxide dismutase and catalase, and the accumulation of proline, glycinebetaine, total phenols, and total flavonoids increased with NaCl stress, but these attributes further increased with EBR supplementation. The activities of enzymes and the levels of non-enzymatic antioxidants involved in the Asc-Glu cycle also increased with NaCl stress, and further enhancement in these attributes was recorded by EBR supplementation. Salinity elevated the methylglyoxal content, but it was decreased by the EBR supplementation accompanying with up-regulation of the glyoxalase cycle (GlyI and GlyII). Salinity enhanced the Na+ uptake in root and shoot coupled with a decrease in uptake of Ca2+, K+, and P. However, EBR supplementation declined Na+ accumulation and promoted the uptake of the aforementioned nutrients. Overall, EBR supplementation regulated the salt tolerance mechanism in soybean plants by modulating osmolytes, activities of key enzymes, and the levels of non-enzymatic antioxidants.