Fully Characterized Effective Bacteriophages Specific against Antibiotic-Resistant Enterococcus faecalis, the Causative Agent of Dental Abscess.

Background and Objectives: Enterococcus faecalis (E. faecalis) is a primary pathogen responsible for dental abscesses, which cause inflammation and pain when trapped between the crown and soft tissues of an erupted tooth. Therefore, this study aims to use specific phages as an alternative method instead of classical treatments based on antibiotics to destroy multidrug-resistant E. faecalis bacteria for treating dental issues. Materials and Methods: In the current study, twenty-five bacterial isolates were obtained from infected dental specimens; only five had the ability to grow on bile esculin agar, and among these five, only two were described to be extensive multidrug-resistant isolates. Results: Two bacterial isolates, Enterococcus faecalis A.R.A.01 [ON797462.1] and Enterococcus faecalis A.R.A.02, were identified biochemically and through 16S rDNA, which were used as hosts for isolating specific phages. Two isolated phages were characterized through TEM imaging, which indicated that E. faecalis_phage-01 had a long and flexible tail, belonging to the family Siphoviridae, while E. faecalis_phage-02 had a contractile tail, belonging to the family Myoviridae. Genetically, two phages were identified through the PCR amplification and sequencing of the RNA ligase of Enterococcus phage vB_EfaS_HEf13, through which our phages shared 97.2% similarity with Enterococcus phage vB-EfaS-HEf13 based on BLAST analysis. Furthermore, through in silico analysis and annotations of the two phages' genomes, it was determined that a total of 69 open reading frames (ORFs) were found to be involved in various functions related to integration excision, replication recombination, repair, stability, and defense. In phage optimization, the two isolated phages exhibited a high specific host range with Enterococcus faecalis among six different bacterial hosts, where E. faecalis_phage-01 had a latent period of 30 min with 115.76 PFU/mL, while E. faecalis_phage-02 had a latent period of 25 min with 80.6 PFU/mL. They were also characterized with stability at wide ranges of pH (4-11) and temperature (10-60 °C), with a low cytotoxic effect on the oral epithelial cell line at different concentrations (1000-31.25 PFU/mL). Conclusions: The findings highlight the promise of phage therapy in dental medicine, offering a novel approach to combating antibiotic resistance and enhancing patient outcomes. Further research and clinical trials will be essential to fully understand the therapeutic potential and safety profile of these bacteriophages in human populations.

An obligate microsporidian parasite modulates defense against opportunistic bacterial infection in the yellow fever mosquito, Aedes aegypti.

The ability of Aedes aegypti mosquitoes to transmit vertebrate pathogens depends on multiple factors, including the mosquitoes' life history traits, immune response, and microbiota (i.e., the microbes associated with the mosquito throughout its life). The microsporidium Edhazardia aedis is an obligate intracellular parasite that specifically infects Ae. aegypti mosquitoes and severely affects mosquito survival and other life history traits critical for pathogen transmission. In this work, we investigated how E. aedis impacts bacterial infection with Serratia marcescens in Ae. aegypti mosquitoes. We measured development, survival, and bacterial load in both larval and adult stages of mosquitoes. In larvae, E. aedis exposure was either horizontal or vertical and S. marcescens was introduced orally. Regardless of the route of transmission, E. aedis exposure resulted in significantly higher S. marcescens loads in larvae. E. aedis exposure also significantly reduced larval survival but subsequent exposure to S. marcescens had no effect. In adult females, E. aedis exposure was only horizontal and S. marcescens was introduced orally or via intrathoracic injection. In both cases, E. aedis infection significantly increased S. marcescens bacterial loads in adult female mosquitoes. In addition, females infected with E. aedis and subsequently injected with S. marcescens suffered 100% mortality which corresponded with a rapid increase in bacterial load. These findings suggest that exposure to E. aedis can influence the establishment and/or replication of other microbes in the mosquito. This has implications for understanding the ecology of mosquito immune defense and potentially disease transmission by mosquito vector species. IMPORTANCE: The microsporidium Edhazardia aedis is a parasite of the yellow fever mosquito, Aedes aegypti. This mosquito transmits multiple viruses to humans in the United States and around the world, including dengue, yellow fever, and Zika viruses. Hundreds of millions of people worldwide will become infected with one of these viruses each year. E. aedis infection significantly reduces the lifespan of Ae. aegypti and is therefore a promising novel biocontrol agent. Here, we show that when the mosquito is infected with this parasite, it is also significantly more susceptible to infection by an opportunistic bacterial pathogen, Serratia marcescens. This novel discovery suggests the mosquito's ability to control infection by other microbes is impacted by the presence of the parasite.

Sugar restriction and blood ingestion shape divergent immune defense trajectories in the mosquito Aedes aegypti.

Immune defense is comprised of (1) resistance: the ability to reduce pathogen load, and (2) tolerance: the ability to limit the disease severity induced by a given pathogen load. The study of tolerance in the field of animal immunity is fairly nascent in comparison to resistance. Consequently, studies which examine immune defense comprehensively (i.e. considering both resistance and tolerance in conjunction) are uncommon, despite their exigency in achieving a thorough understanding of immune defense. Furthermore, understanding tolerance in arthropod disease vectors is uniquely relevant, as tolerance is essential to the cyclical transmission of pathogens by arthropods. Here, we tested the effect(s) of dietary sucrose concentration and blood ingestion on resistance and tolerance to Escherichia coli infection in the yellow fever mosquito Aedes aegypti. Resistance and tolerance were measured concurrently and at multiple timepoints. We found that mosquitoes from the restricted sugar treatment displayed enhanced resistance at all timepoints post-infection compared to those from the laboratory standard sugar treatment. Blood also improved resistance, but only early post-infection. While sucrose restriction had no effect on tolerance, we show that consuming blood prior to bacterial infection ameliorates a temporal decline in tolerance that mosquitoes experience when provided with only sugar meals. Taken together, our findings indicate that different dietary components can have unique and sometimes temporally dynamic impacts on resistance and tolerance.

Genome Sequence of an Okra Leaf Curl Virus from Egypt.

A complete Okra leaf curl virus DNA-A was sequenced from okra in Egypt. Here, we report the complete genome sequence of this monopartite virus, comprising 2,764 bp and encoding 6 open reading frames (ORFs) with a GC content of 44.6% and 88.3% similarity to a virus reported earlier from Cameroon.

Ameliorating the Adverse Effects of Tomato mosaic tobamovirus Infecting Tomato Plants in Egypt by Boosting Immunity in Tomato Plants Using Zinc Oxide Nanoparticles.

Tomato mosaic virus (ToMV) is one of the economically damageable Tobamovirus infecting the tomato in Egypt that has caused significant losses. It is therefore of great interest to trigger systemic resistance to ToMV. In this endeavor, we aimed to explore the capacity of ZnO-NPs (zinc oxide nanoparticles) to trigger tomato plant resistance against ToMV. Effects of ZnO-NPs on tomato (Solanum lycopersicum L.) growth indices and antioxidant defense system activity under ToMV stress were investigated. Noticeably that treatment with ZnO-NPs showed remarkably increased growth indices, photosynthetic attributes, and enzymatic and non-enzymatic antioxidants compared to the challenge control. Interestingly, oxidative damage caused by ToMV was reduced by reducing malondialdehyde, H2O2, and O2 levels. Overall, ZnO-NPs offer a safe and economic antiviral agent against ToMV.

Characterization and Full Genome Sequence of Novel KPP-5 Lytic Phage against Klebsiella pneumoniae Responsible for Recalcitrant Infection.

Klebsiella pneumoniae is a hazardous opportunistic pathogen that is involved in many serious human diseases and is considered to be an important foodborne pathogen found in many food types. Multidrug resistance (MDR) K. pneumoniae strains have recently spread and increased, making bacteriophage therapy an effective alternative to multiple drug-resistant pathogens. As a consequence, this research was conducted to describe the genome and basic biological characteristics of a novel phage capable of lysing MDR K. pneumoniae isolated from food samples in Egypt. The host range revealed that KPP-5 phage had potent lytic activity and was able to infect all selected MDR K. pneumoniae strains from different sources. Electron microscopy images showed that KPP-5 lytic phage was a podovirus morphology. The one-step growth curve exhibited that KPP-5 phage had a relatively short latent period of 25 min, and the burst size was about 236 PFU/infected cells. In addition, KPP-5 phage showed high stability at different temperatures and pH levels. KPP-5 phage has a linear dsDNA genome with a length of 38,245 bp with a GC content of 50.8% and 40 predicted open reading frames (ORFs). Comparative genomics and phylogenetic analyses showed that KPP-5 is most closely associated with the Teetrevirus genus in the Autographviridae family. No tRNA genes have been identified in the KPP-5 phage genome. In addition, phage-borne virulence genes or drug resistance genes were not present, suggesting that KPP-5 could be used safely as a phage biocontrol agent.

Molecular Characterization of the Alfalfa mosaic virus Infecting Solanum melongena in Egypt and the Control of Its Deleterious Effects with Melatonin and Salicylic Acid.

During the spring of 2019, distinct virus-like symptoms were observed in the Kafr El-Sheikh Governorate in Egypt in naturally infected eggplants. Leaves of affected plants showed interveinal leaf chlorosis, net yellow, chlorotic sectors, mottling, blisters, vein enation, necrotic intervention, and narrowing symptoms. The Alfalfa mosaic virus (AMV) was suspected of to be involved in this disease. Forty plant samples from symptomatic eggplants and 10 leaf samples with no symptoms were collected. The samples were tested by double antibody sandwich ELISA (DAS-ELISA) using AMV-IgG. Six of the 40 symptomatic leaf samples tested positive for AMV, while, DAS-ELISA found no AMV in the 10 leaf samples without symptoms. The AMV Egyptian isolate (AMV-Eggplant-EG) was biologically isolated from the six positive samples tested by DAS-ELISA and from the similar local lesions induced on Chenopodium amaranticolor and then re-inoculated in healthy Solanum melongena as a source of AMV-Eggplant-EG and confirmed by DAS-ELISA. Reverse transcription polymerase chain reaction (RT-PCR) assay with a pair of primers specific for coat protein (CP) encoding RNA 3 of AMV yielded an amplicon of 666 bp from infected plants of Solanum melongena with AMV-Eggplant-EG. The amplified PCR product was cloned and sequenced. Analysis of the AMV-Eggplant-EG sequence revealed 666 nucleotides (nt) of the complete CP gene (translating 221 amino acid (aa) residues). Analysis of phylogeny for nt and deduced aa sequences of the CP gene using the maximum parsimony method clustered AMV-Eggplant-EG in the lineage of Egyptian isolates (shark-EG, mans-EG, CP2-EG, and FRE-EG) with a high bootstrap value of 88% and 92%, respectively. In addition to molecular studies, melatonin (MTL) and salicylic acid (SA) (100 µM) were used to increase the resistance of eggplant to AMV- infection. Foliar spray with MLT and SA caused a significant increase in the morphological criteria (shoot, root length, number of leaves, leaf area, and leaf biomass), chlorophyll and carotenoid content, antioxidant enzymes, and gene expression of some enzymes compared to the infected plants. On the other hand, treatment with MLT and SA reduced the oxidative damage caused by AMV through the reduction of hydrogen peroxide, superoxide anions, hydroxyl radicals, and malondialdehyde. In conclusion, MLT and SA are eco-friendly compounds and can be used as antiviral compounds.

Comparative genomics of the gut commensal Bifidobacterium bifidum reveals adaptation to carbohydrate utilization.

Bifidobacterium bifidum is one of the most abundant members of the gut microbiota at the early stage of life. The established association of the bacterium with the human gut confers health benefits. Such successful persistence of B. bifidum necessitates metabolic adaptation to the host-derived carbohydrates, a process which is poorly understood. The current study focuses on revealing the genomic-based phylogeny (phylogenomics) of B. bifidum and utilizing comparative genomics to decipher the glycolytic abilities of bifidobacterial strains isolated from different human body niches (feces, human gut, vagina, and breast milk). When the phylogenomic analysis was performed on 95 B. bifidum strains, currently available on the RefSeq database, the bacterium was clearly distinguished from other members of the Bifidobacterium genus. Furthermore, a pairwise genomic comparison indicated that a large proportion of orthologous gene families were shared among the B. bifidum strains. These findings highlight the notion that the B. bifidum species is genetically similar and may perform similar functions in their host. When 15 B. bifidum genomes representing strains from different human body niches were annotated, the resulting functional profile showed the presence of enriched proteins involved in carbohydrate utilization. Moreover, mining the 15 B. bifidum genomes for the presence of Carbohydrate-Active Enzyme (CAZY) systems, the analysis found the existence of diverse protein families which include glycosyl hydrolases, glycosyl transferases, carbohydrate-binding modules, and carbohydrate esterases. Collectively, these CAZY systems enables B. bifidum to utilize host-derived glycans (e.g., mucin) and diet-derived carbohydrates (e.g., starch). In contrast, a correlation analysis revealed that B. bifidum strains isolated from the different body niches were indistinguishable in the context of presence-absence of CAZY systems. These findings emphasize the valuable use of comparative genomics in deciphering the glycolytic abilities of B. bifidum and consequently its adaptation to carbohydrate utilization in the human gut environment.

Controlling foodborne pathogens with natural antimicrobials by biological control and antivirulence strategies.

Foodborne diseases represent a global health threat besides the great economic losses encountered by the food industry. These hazards necessitate the implementation of food preservation methods to control foodborne pathogens, the causal agents of human illnesses. Until now, most control methods rely on inhibiting the microbial growth or eliminating the pathogens by applying lethal treatments. Natural antimicrobials, which inhibit microbial growth, include traditional chemicals, naturally occurring antimicrobials, or biological preservation (e.g. beneficial microbes, bacteriocins, or bacteriophages). Although having great antimicrobial effectiveness, challenges due to the adaptation of foodborne pathogens to such control methods are becoming apparent. Such adaptation enables the survival of the pathogens in foods or food-contact environments. This imperative concern inspires contemporary research and food industry sector to develop technologies which do not target microbial growth but disarming microbial virulence factors. These technologies, referred to as "antivirulence", render the microbe non-capable of causing the disease with very limited or no opportunities for the pathogenic microorganisms to develop resistance. For the sake of safer and fresh-like foods, with no effect on the sensory properties of foods, a combination of two or more natural antimicrobials or with other stressors, is now widespread, to preserve foods. This review introduces and critically describes the traditional versus the emerging uses of natural antimicrobials for controlling foodborne pathogens in foods. Development of biological control strategies using natural antimicrobials proved to be effective in inhibiting microbial growth in foods and allowing improved food safety. In the meanwhile, discovery of new antivirulence agents could be a transformative strategy in food preservation in the far future.

Improving Regulation of Enzymatic and Non-Enzymatic Antioxidants and Stress-Related Gene Stimulation in Cucumber mosaic cucumovirus-Infected Cucumber Plants Treated with Glycine Betaine, Chitosan and Combination.

Cucumber mosaic cucumovirus (CMV) is a deadly plant virus that results in crop-yield losses with serious economic consequences. In recent years, environmentally friendly components have been developed to manage crop diseases as alternatives to chemical pesticides, including the use of natural compounds such as glycine betaine (GB) and chitosan (CHT), either alone or in combination. In the present study, the leaves of the cucumber plants were foliar-sprayed with GB and CHT-either alone or in combination-to evaluate their ability to induce resistance against CMV. The results showed a significant reduction in disease severity and CMV accumulation in plants treated with GB and CHT, either alone or in combination, compared to untreated plants (challenge control). In every treatment, growth indices, leaf chlorophylls content, phytohormones (i.e., indole acetic acid, gibberellic acid, salicylic acid and jasmonic acid), endogenous osmoprotectants (i.e., proline, soluble sugars and glycine betaine), non-enzymatic antioxidants (i.e., ascorbic acid, glutathione and phenols) and enzymatic antioxidants (i.e., superoxide dismutase, peroxidase, polyphenol oxidase, catalase, lipoxygenase, ascorbate peroxidase, glutathione reductase, chitinase and ß-1,3 glucanase) of virus-infected plants were significantly increased. On the other hand, malondialdehyde and abscisic acid contents have been significantly reduced. Based on a gene expression study, all treated plants exhibited increased expression levels of some regulatory defense genes such as PR1 and PAL1. In conclusion, the combination of GB and CHT is the most effective treatment in alleviated virus infection. To our knowledge, this is the first report to demonstrate the induction of systemic resistance against CMV by using GB.

Probiotic Lactobacillus and Bifidobacterium strains possess safety characteristics, antiviral activities and host adherence factors revealed by genome mining.

BACKGROUND: Probiotics belonging to Lactobacillus and Bifidobacterium spp. have been exploited for their health benefits in treatment and prevention of many pathological conditions and promoting human health. Recent advances in understanding probiotics-human interaction through microbiome research in the context of various medical conditions suggest their provisional role in preventive, personalized, and predictive medicine. To streamline their application in disease prevention, development of personalized-based treatments, or their use as biomarkers for predictive diagnosis, in vitro screening for strains with potential probiotic properties should be performed. In this work, we aimed to emphasize the probiotic features of four Lactobacillus and two Bifidobacterium probiotic strains which showed antagonistic properties against microbial pathogens. METHODS: Firstly, cytotoxicity assessment of cell-free preparations from these strains was performed using a baby hamster kidney (BHK) cells and cell viability was measured by means of sulfo-rhodamine B stain. Secondly, Newcastle disease (ND) and infectious bursal disease (IBD) viruses which pose a great threat in infected poultry were used for assessing antiviral activity of probiotics. Thirdly, the genomes of six probiotic strains were used to identify genes encoding host adherence factors that mediate interaction with human tissues. RESULTS: Probiotic preparations exhibited insignificant toxicity as indicated by the high survival rate of BHK cells (surviving fraction varied from 0.82 to 0.99) as compared to the untreated control. Cell-free preparations of probiotics mixed with equal volume of ND and IBD viruses (106 and 104 Tissue Culture Infectious Dose 50, respectively) reduced the titer of ND and IBD viruses on chicken embryo fibroblast cells. Genome mining analysis revealed that the draft genomes of these strains were predicted to encode LPXTG-containing proteins, surface layer proteins, tight adherence pili, sortase-dependent pili, fibronectin, or collagen binding proteins and other factors that adhere to human tissues such as mucus. Such adherence factors enable probiotic bacteria to interact and colonize the host. CONCLUSION: Taken together, safety privileges, antiviral activities, and genomically encoded host interaction factors confirmed probiotic features of the six probiotic strains and their potential in promoting human health.

Print to detect: a rapid and ultrasensitive phage-based dipstick assay for foodborne pathogens.

Foodborne pathogens are a burden to the economy and a constant threat to public health. The ability to rapidly detect the presence of foodborne pathogens is a vital component of any strategy towards establishing a safe and secure food supply chain. Bacteriophages (phages) are viruses capable of infecting and replicating within bacteria in a strain-specific manner. The ubiquitous and selective nature of phages makes them ideal for the detection and biocontrol of bacteria. Therefore, the objective of this research was to develop and test a phage-based paper dipstick biosensor for the detection of various foodborne pathogens in food matrices. The first step was to identify the best method for immobilizing phages on paper such that their biological activity (infectivity) was preserved. It was found that piezoelectric inkjet printing resulted in lower loss of phage infectivity when compared with other printing methods (namely gravure and blade coating) and that ColorLok paper was ideally suited to create functional sensors. The phage-based bioactive papers developed with use of piezoelectric inkjet printing actively lysed their target bacteria and retained this antibacterial activity for up to 1 week when stored at room temperature and 80% relative humidity. These bioactive paper strips in combination with quantitative real-time PCR were used for quantitative determination of target bacteria in broth and food matrices. A phage dipstick was used to capture and infect Escherichia coli O157:H7, E. coli O45:H2, and Salmonella Newport in spinach, ground beef and chicken homogenates, respectively, and quantitative real-time PCR was used to detect the progeny phages. A detection limit of 10-50 colony-forming units per millilitre was demonstrated with a total assay time of 8 h, which was the duration of a typical work shift in an industrial setting. This detection method is rapid and cost-effective, and may potentially be applied to a broad range of bacterial foodborne pathogens. Graphical abstract ᅟ.