Packaging Covered with Antiviral and Antibacterial Coatings Based on ZnO Nanoparticles Supplemented with Geraniol and Carvacrol.

The purpose of the study was to obtain an external coating based on nanoparticles of ZnO, carvacrol, and geraniol that could be active against viruses such as SARS-Co-V2. Additionally, the synergistic effect of the chosen substances in coatings was analyzed. The goal of the study was to measure the possible antibacterial activity of the coatings obtained. Testing antiviral activity with human pathogen viruses, such as SARS-Co-V2, requires immense safety measures. Bacteriophages such as phi 6 phage represent good surrogates for the study of airborne viruses. The results of the study indicated that the ZC1 and ZG1 coatings containing an increased amount of geraniol or carvacrol and a very small amount of nanoZnO were found to be active against Gram-positive and Gram-negative bacteria. It is also important that a synergistic effect between these active substances was noted. This explains why polyethylene (PE) films covered with the ZC1 or ZG1 coatings (as internal coatings) were found to be the best packaging materials to extend the quality and freshness of food products. The same coatings may be used as the external coatings with antiviral properties. The ZC1 and ZG1 coatings showed moderate activity against the phi 6 phage that has been selected as a surrogate for viruses such as coronaviruses. It can be assumed that coatings ZG1 and ZC1 will also be active against SARS-CoV-2 that is transmitted via respiratory droplets.

Biochemical and Molecular Characteristics of Pc1 Virulent Phage Isolate Infecting Pectobacterium carotovorum.

BACKGROUND AND OBJECTIVE: Pectobacterium carotovorum subsp. carotovorum is a plant-pathogenic bacterium. It is a post-harvest pathogen and causes soft rot diseases in infected plants. Different virulent bacteriophages have been isolated from different regions in the world. These bacteriophages were tolerant to high concentrations of calcium chloride and magnesium chloride. Whereas, the high concentrations of zinc chloride and aluminum chloride decreased the activity and stability of phages. Therefore, the present research aimed to study the biology of P. carotovorum phage (Pc1) by using a one-step growth experiment, its stability to different concentrations of some chemicals and molecular characteristics of this phage isolate. MATERIALS AND METHODS: One step growth experiment, chemical stability, and molecular characteristics by using RAPD-PCR of P. carotovorum phage (Pc1) were studied. RESULTS: The P. carotovorum phage (Pc1) isolate was found to have a latent period of 20 min and its burst size is about 92 pfu cell-1. Calcium chloride, magnesium chloride, and copper sulphate (from 0.1-0.5 mM) increased the infectivity of Pc1 phage, while, zinc chloride in the same concentrations reduced its infectivity. RAPD-PCR amplification was indicated that the total amplified products were 32 bands with size ranged from 0.179-2.365 Kbp. CONCLUSION: Since, zinc chloride (at concentrations of 0.1-0.5 mM) reduced infectivity of Pc1 phage isolate, therefore, any chemical compounds containing zinc must be avoided in designing biocontrol strategy by using phages against soft rot bacterium (P. carotovorum) in potatoes.

Bacteriophages, a New Therapeutic Solution for Inhibiting Multidrug-Resistant Bacteria Causing Wound Infection: Lesson from Animal Models and Clinical Trials.

Wound infection kills a large number of patients worldwide each year. Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, and Pseudomonas aeruginosa are the most important colonizing pathogens of wounds that, with various virulence factors and impaired immune system, causes extensive tissue damage and nonhealing wounds. Furthermore, the septicemia caused by these pathogens increases the mortality rate due to wound infections. Because of the prevalence of antibiotic resistance in recent years, the use of antibiotics to inhibit these pathogens has been restricted, and the topical application of antibiotics in wound infections increases antibiotic resistance. Therefore, finding a new therapeutic strategy against wound infections is so essential since these infections have a destructive effect on the patient's mental health and high medical costs. In this review, we discussed the use of phages for the prevention of multidrug-resistant (MDR) bacteria, causing wound infection and their role in wound healing in animal models and clinical trials. The results showed that phages have a high ability to inhibit different wound infections caused by MDR bacteria, heal the wound faster, have lower side effects and toxicity, destroy bacterial biofilm, and they are useful in controlling immune responses. Many studies have used animal models to evaluate the function of phages, and this study appears to have a positive impact on the use of phages in clinical practice and the development of a new therapeutic approach to control wound infections, although there are still many limitations.

Defeating Antibiotic-Resistant Bacteria: Exploring Alternative Therapies for a Post-Antibiotic Era.

Antibiotics are one of the greatest medical advances of the 20th century, however, they are quickly becoming useless due to antibiotic resistance that has been augmented by poor antibiotic stewardship and a void in novel antibiotic discovery. Few novel classes of antibiotics have been discovered since 1960, and the pipeline of antibiotics under development is limited. We therefore are heading for a post-antibiotic era in which common infections become untreatable and once again deadly. There is thus an emergent need for both novel classes of antibiotics and novel approaches to treatment, including the repurposing of existing drugs or preclinical compounds and expanded implementation of combination therapies. In this review, we highlight to utilize alternative drug targets/therapies such as combinational therapy, anti-regulator, anti-signal transduction, anti-virulence, anti-toxin, engineered bacteriophages, and microbiome, to defeat antibiotic-resistant bacteria.

In vitro inactivation of Mycobacterium avium subsp. paratuberculosis (MAP) by use of copper ions.

BACKGROUND: Mycobacterium avium subsp. paratuberculosis (MAP) is the causative agent of paratuberculosis, a contagious infectious disease that affects domestic and wild ruminants causing chronic inflammation of the intestine. MAP has proven to be very resistant to both physical and chemical processes, making it difficult to control this pathogen. Based on the recognized antimicrobial properties of copper, the objective of this study was to evaluate the effectiveness of copper ions to reduce MAP numbers and/or MAP viability in a fluid matrix. Besides, methicillin-resistant Staphylococcus aureus (MRSA), and Escherichia coli were used as controls of the effectiveness of copper ions. MAP-spiked PBS was subjected to copper ions treatment at 24 V for 5 min and the PBS suspensions were sampled before and after treatment. MAP viability and quantification were determined using three complementary techniques: a phage amplification assay, MGIT culture and qPCR. RESULTS: Moderate numbers (103 CFU ml-1) of the two control bacteria were completely eliminated by treatment with copper ions. For MAP, copper ions treatment reduced both the viability and numbers of this pathogen. Phage assay information quickly showed that copper ions (24 V for 5 min) resulted in a significant reduction in viable MAP. MGIT culture results over time showed statistically significant differences in time-to-detection (TTD) values between PRE and POST treatment. MAP genome equivalent estimates for PBS suspensions indicated that MAP numbers were lower in samples POST-treatment with copper ions than PRE-treatment. CONCLUSIONS: The use of copper ions resulted in a significant reduction of MAP in a liquid matrix, although some MAP survival on some occasions was observed.

The viability of lytic bacteriophage ΦD5 in potato-associated environments and its effect on Dickeya solani in potato (Solanum tuberosum L.) plants.

Dickeya solani is one of the most important pectinolytic phytopathogens responsible for high losses in potato, especially in seed potato production in Europe. Lytic bacteriophages can affect the structure of the host population and may influence spread, survival and virulence of the pathogen and in consequence, infection of the plant. In this study, we aimed to acquire information on the viability of the broad host lytic bacteriophage ΦD5 on potato, as well as to apprehend the specific effect of this bacteriophage on its host D. solani type-strain in different settings, as a preliminary step to target co-adaptation of phages and host bacteria in plant environment. Viability of the ΦD5 phage in tuber extract, on tuber surface, in potting compost, in rainwater and on the leaf surface, as well as the effect of copper sulfate, were examined under laboratory conditions. Also, the interaction of ΦD5 with the target host D. solani in vitro and in compost-grown potato plants was evaluated. ΦD5 remained infectious in potato tuber extract and rain water for up to 72 h but was inactivated in solutions containing 50 mM of copper. The phage population was stable for up to 28 days on potato tuber surface and in potting compost. In both, tissue culture and compost-grown potato plants, ΦD5 reduced infection by D. solani by more than 50%. The implications of these findings are discussed.

Phenolic Compounds of Potato Peel Extracts: Their Antioxidant Activity and Protection against Human Enteric Viruses.

Potato peels (PP) contain several bioactive compounds. These compounds are known to provide human health benefits, including antioxidant and antimicrobial properties. In addition, these compounds could have effects on human enteric viruses that have not yet been reported. The objective of the present study was to evaluate the phenolic composition, antioxidant properties in the acidified ethanol extract (AEE) and water extract of PP, and the antiviral effects on the inhibition of Av-05 and MS2 bacteriophages, which were used as human enteric viral surrogates. The AEE showed the highest phenolic content and antioxidant activity. Chlorogenic and caffeic acids were the major phenolic acids. In vitro analysis indicated that PP had a strong antioxidant activity. A 3 h incubation with AEE at a concentration of 5 mg/ml was needed to reduce the PFU/ml (plaque-forming unit per unit volume) of Av-05 and MS2 by 2.8 and 3.9 log10, respectively, in a dose-dependent manner. Our data suggest that PP has potential to be a source of natural antioxidants against enteric viruses.

The effects of a thermophile metabolite, tryptophol, upon protecting shrimp against white spot syndrome virus.

White spot syndrome virus (WSSV) is a shrimp pathogen responsible for significant economic loss in commercial shrimp farms and until now, there has been no effective approach to control this disease. In this study, tryptophol (indole-3-ethanol) was identified as a metabolite involved in bacteriophage-thermophile interactions. The dietary addition of tryptophol reduced the mortality in shrimp Marsupenaeus japonicus when orally challenged with WSSV. Our results revealed that 50 mg/kg tryptophol has a better protective effect in shrimp than 10 or 100 mg/kg tryptophol. WSSV copies in shrimp were reduced significantly (P < 0.01) when supplemented with 50 mg/kg tryptophol, indicating that virus replication was inhibited by tryptophol. Consequently, tryptophol represents an effective antiviral dietary supplement for shrimp, and thus holds significant promise as a novel and efficient therapeutic approach to control WSSV in shrimp aquaculture.

Microbiological study of bacteriophage induction in the presence of chemical stress factors in enhanced biological phosphorus removal (EBPR).

Polyphosphate accumulating organisms (PAOs) are responsible for carrying the enhanced biological phosphorus removal (EBPR). Although the EBPR process is well studied, the failure of EBPR performance at both laboratory and full-scale plants has revealed a lack of knowledge about the ecological and microbiological aspects of EBPR processes. Bacteriophages are viruses that infect bacteria as their sole host. Bacteriophage infection of polyphosphate accumulating organisms (PAOs) has not been considered as a main contributor to biological phosphorus removal upsets. This study examined the effects of different stress factors on the dynamics of bacteriophages and the corresponding effects on the phosphorus removal performance in a lab-scale EBPR system. The results showed that copper (heavy metal), cyanide (toxic chemical), and ciprofloxacin (antibiotic), as three different anthropogenic stress factors, can induce phages integrated onto bacterial genomes (i.e. prophages) in an enriched EBPR sequencing batch reactor, resulting in a decrease in the polyphosphate kinase gene ppk1 clades copy number, phosphorus accumulation capacity, and phosphorus removal performance. This study opens opportunities for further research on the effects of bacteriophages in nutrient cycles both in controlled systems such as wastewater treatment plants and natural ecosystems.

The large scale antibacterial, antifungal and anti-phage efficiency of Petamcin-A: new multicomponent preparation for skin diseases treatment.

BACKGROUND: Human and animal skin diseases of bacterial, fungal and viral nature and their complications are widespread and globally cause a serious trouble. Their prevalence is increasing mainly due to drug resistance. Consequently, demand has increased for new effective antimicrobial drugs, which also should be less toxic, possess a wider spectrum of action and be economically more beneficial. The goal was to investigate antibacterial, antifungal and anti-phage activity of Petamcin-A-a new multicomponent preparation. It contains acetic acid and hexamethylenetetramine as main active antimicrobial components, as well as phosphatidylcholine, tocopheryl acetate and glycerol as excipients. METHODS: Bacteriostatic activity and minimal inhibitory concentrations of the preparation against various test-organisms were determined by agar well diffusion assay. Antifungal activity was tested by agar dilution assay. To explore anti-phage activity double agar overlay plaque assay was used. Nystatin, chlorhexidine and acetic acid were used as control agents for comparative analysis. Statistical analysis was done with GraphPad Prism 5.03 or R 3.1.0 software. RESULTS: The results showed a higher activity of Petamcin-A against all bacterial and fungal test strains compared with its components or control agents. The preparation was more effective against tested gram-positive bacteria than gram-negative ones. Petamcin-A expressed bactericidal activity against almost all test strains. In addition, the preparation demonstrated high activity against T4 phage of Escherichia coli C-T4 completely inhibiting its growth. 5-fold diluted Petamcin-A also exhibited considerable activity reducing phage concentration by 2.6 Log10. CONCLUSIONS: Petamcin-A has a high antimicrobial activity against all tested strains of bacteria, yeasts and moulds. The preparation also exhibited high anti-phage activity. Moreover, taking into account that Petamcin-A has no observable toxicity on skin and its components are not expensive, it can be advantageous for management of various skin medical conditions.

Chemical constitution and effect of extracts of tomato plants byproducts on the enteric viral surrogates.

Byproducts of tomato are known to include phenolic compounds but have not been studied in depth. In this study, the phenolic compositions of (stem, leaf, root, and whole plant) of two tomato cultivars, Pitenza and Floradade, were analyzed by HPLC-DAD. In parallel, the antiviral effects of crude extracts on viral surrogates, the bacteriophages MS2 and Av-05 were evaluated. The leaf extracts from the two varieties showed the highest concentration of phenolic compounds. The compounds identified were gallic acid, chlorogenic acid, ferulic acid, cafeic acid, rutin, and quercetin, and they represented 3174.3 and 1057.9 mg/100 g dried weight of the Pitenza and Floradade cultivars, respectively. MS2 and Av-05 titers at 5 mg/mL were reduced by 3.47 and 5.78 log10 PFU/mL and 3.78 and 4.93 log10 PFU/mL by Pitenza and Floradade cultivar leaf extract, respectively. These results show that tomato extracts are natural sources of bioactive substances with antiviral activity.

Underlying mechanism of antimicrobial activity of chitosan microparticles and implications for the treatment of infectious diseases.

The emergence of antibiotic resistant microorganisms is a great public health concern and has triggered an urgent need to develop alternative antibiotics. Chitosan microparticles (CM), derived from chitosan, have been shown to reduce E. coli O157:H7 shedding in a cattle model, indicating potential use as an alternative antimicrobial agent. However, the underlying mechanism of CM on reducing the shedding of this pathogen remains unclear. To understand the mode of action, we studied molecular mechanisms of antimicrobial activity of CM using in vitro and in vivo methods. We report that CM are an effective bactericidal agent with capability to disrupt cell membranes. Binding assays and genetic studies with an ompA mutant strain demonstrated that outer membrane protein OmpA of E. coli O157:H7 is critical for CM binding, and this binding activity is coupled with a bactericidal effect of CM. This activity was also demonstrated in an animal model using cows with uterine diseases. CM treatment effectively reduced shedding of intrauterine pathogenic E. coli (IUPEC) in the uterus compared to antibiotic treatment. Since Shiga-toxins encoded in the genome of bacteriophage is often overexpressed during antibiotic treatment, antibiotic therapy is generally not recommended because of high risk of hemolytic uremic syndrome. However, CM treatment did not induce bacteriophage or Shiga-toxins in E. coli O157:H7; suggesting that CM can be a potential candidate to treat infections caused by this pathogen. This work establishes an underlying mechanism whereby CM exert antimicrobial activity in vitro and in vivo, providing significant insight for the treatment of diseases caused by a broad spectrum of pathogens including antibiotic resistant microorganisms.

Antibiotic treatment expands the resistance reservoir and ecological network of the phage metagenome.

The mammalian gut ecosystem has considerable influence on host physiology, but the mechanisms that sustain this complex environment in the face of different stresses remain obscure. Perturbations to the gut ecosystem, such as through antibiotic treatment or diet, are at present interpreted at the level of bacterial phylogeny. Less is known about the contributions of the abundant population of phages to this ecological network. Here we explore the phageome as a potential genetic reservoir for bacterial adaptation by sequencing murine faecal phage populations following antibiotic perturbation. We show that antibiotic treatment leads to the enrichment of phage-encoded genes that confer resistance via disparate mechanisms to the administered drug, as well as genes that confer resistance to antibiotics unrelated to the administered drug, and we demonstrate experimentally that phages from treated mice provide aerobically cultured naive microbiota with increased resistance. Systems-wide analyses uncovered post-treatment phage-encoded processes related to host colonization and growth adaptation, indicating that the phageome becomes broadly enriched for functionally beneficial genes under stress-related conditions. We also show that antibiotic treatment expands the interactions between phage and bacterial species, leading to a more highly connected phage-bacterial network for gene exchange. Our work implicates the phageome in the emergence of multidrug resistance, and indicates that the adaptive capacity of the phageome may represent a community-based mechanism for protecting the gut microflora, preserving its functional robustness during antibiotic stress.

Evaluation of bacteriophage therapy to control Clostridium difficile and toxin production in an in vitro human colon model system.

Clostridium difficile is a leading cause of hospital-acquired diarrhoea and represents a major challenge for healthcare providers. Due to the decreasing efficacy and associated problems of antibiotic therapy there is a need for synergistic and alternative treatments. In this study we investigated the use of a specific bacteriophage, ΦCD27, in a human colon model of C. difficile infection. Our findings demonstrate a significant reduction in the burden of C. difficile cells and toxin production with phage treatment relative to an untreated control, with no detrimental effect on commensal bacterial populations. The results demonstrate the potential of phage therapy, and highlight the limitations of using phages that have lysogenic capacity.

Examining the interactome of huperzine A by magnetic biopanning.

Huperzine A is a bioactive compound derived from traditional Chinese medicine plant Qian Ceng Ta (Huperzia serrata), and was found to have multiple neuroprotective effects. In addition to being a potent acetylcholinesterase inhibitor, it was thought to act through other mechanisms such as antioxidation, antiapoptosis, etc. However, the molecular targets involved with these mechanisms were not identified. In this study, we attempted to exam the interactome of Huperzine A using a cDNA phage display library and also mammalian brain tissue extracts. The drugs were chemically linked on the surface of magnetic particles and the interactive phages or proteins were collected and analyzed. Among the various cDNA expressing phages selected, one was identified to encode the mitochondria NADH dehydrogenase subunit 1. Specific bindings between the drug and the target phages and target proteins were confirmed. Another enriched phage clone was identified as mitochondria ATP synthase, which was also panned out from the proteome of mouse brain tissue lysate. These data indicated the possible involvement of mitochondrial respiratory chain matrix enzymes in Huperzine A's pharmacological effects. Such involvement had been suggested by previous studies based on enzyme activity changes. Our data supported the new mechanism. Overall we demonstrated the feasibility of using magnetic biopanning as a simple and viable method for investigating the complex molecular mechanisms of bioactive molecules.

Virucidal properties of metal oxide nanoparticles and their halogen adducts.

Selected metal oxide nanoparticles are capable of strongly adsorbing large amounts of halogens (Cl(2), Br, I(2)) and mixed halogens. These solid adducts are relatively stable thermally, and they can be stored for long periods. However, in the open environment, they are potent biocides. Herein are described studies with a number of bacteriophage MS2, phiX174, and PRD-1 (virus examples). PRD-1 is generally more resistant to chemical disinfection, but in this paper it is shown to be very susceptible to selected interhalogen and iodine adducts of CeO(2), Al(2)O(3), and TiO(2) nanoparticles. Overall, the halogen adducts of TiO(2) and Al(2)O(3) were most effective. The mechanism of disinfection by these nanoparticles is not completely clear, but could include abrasive properties, as well as oxidative powers. A hypothesis that nanoparticles damage virons or stick to them and prevent binding to the host cell is a consideration that needs to be explored. Herein are reported comparative biocidal activities of a series of adducts and electron microscope images of before and after treatment.

Engineered bacteriophage targeting gene networks as adjuvants for antibiotic therapy.

Antimicrobial drug development is increasingly lagging behind the evolution of antibiotic resistance, and as a result, there is a pressing need for new antibacterial therapies that can be readily designed and implemented. In this work, we engineered bacteriophage to overexpress proteins and attack gene networks that are not directly targeted by antibiotics. We show that suppressing the SOS network in Escherichia coli with engineered bacteriophage enhances killing by quinolones by several orders of magnitude in vitro and significantly increases survival of infected mice in vivo. In addition, we demonstrate that engineered bacteriophage can enhance the killing of antibiotic-resistant bacteria, persister cells, and biofilm cells, reduce the number of antibiotic-resistant bacteria that arise from an antibiotic-treated population, and act as a strong adjuvant for other bactericidal antibiotics (e.g., aminoglycosides and beta-lactams). Furthermore, we show that engineering bacteriophage to target non-SOS gene networks and to overexpress multiple factors also can produce effective antibiotic adjuvants. This work establishes a synthetic biology platform for the rapid translation and integration of identified targets into effective antibiotic adjuvants.

Antiviral effects on bacteriophages and rotavirus by cranberry juice.

Studies were undertaken to investigate the antiviral effects of comestible juices, especially cranberry juice, on non-related viral species. After exposure of bacteriophage T2 to a commercially available cranberry (Vaccinium macrocarpon) juice cocktail (CJ), virus infectivity titer was no longer detectible. After a 60-min exposure to orange (OJ) and grapefruit juices (GJ), phage infectivity was reduced to 25-35% of control, respectively. Similar data were observed for the bacteriophage T4. CJ inactivation of phage T4 was rapid, dose-dependent, and occurred at either 4 or 23 degrees C. Neither pH nor differences in sugar/carbohydrate levels among the juices may be ascribed to the recognized antiviral effects. Further studies were performed to identify the occurrence of antiviral activity by CJ to a mammalian enteric virus. The treatment of the simian rotavirus SA-11 with a 20% CJ suspension was sufficient to inhibit hemagglutination. Under scanning and transmission electron microscopy, CJ was observed to inhibit the adsorption of phage T4 to its bacterial host cells and prevented the replication of rotavirus in its monkey kidney (MA-104) host cells, respectively. The data suggest, for the first time, a non-specific antiviral effect towards unrelated viral species (viz., bacteriophages T2 and T4 and the simian rotavirus SA-11) by a commercially available cranberry fruit juice drink.

Novel angular furo and thieno-quinolinones: synthesis and preliminary photobiological studies.

A number of new furo and thienoquinolinones carrying an electron-withdrawing function or unsubstituted at the position 3 were synthesized in order to obtain new potential photochemotherapeutic agents with increased antiproliferative activity and decreased toxic side effects. Our interest in studying the SAR of these derivatives also prompted us to investigate the influence of N-methylation on biological activity, by preparing N-methyl derivatives. The antiproliferative activity of all the newly synthesized compounds was evaluated and compared to 8-methoxypsoralen (8-MOP), the drug widely used in PUVA-therapy. The 3-unsubstituted thienoquinolinones were generally the most potent derivatives, followed by the furo-analogues. In particular, the unsubstituted thieno[2,3-h]quinoline-2(1H)one showed the highest activity in T2 bacteriophage, HeLa cells and Ehrlich cells tests. All the compounds, assayed on Escherichia coli WP2 TM9, showed a similar mutagenic activity, very close to that of 8-MOP. Except for 2-oxo-1,2-dihydrothieno[2,3-h]quinoline-3-carboxylic acid, which appeared to be very effective, all compounds generated singlet oxygen to slightly larger amounts when compared to 8-MOP. The N-methyl analogues only induced moderate skin erythemas on albino guinea pigs, while all other derivatives appeared to be entirely inactive. On the basis of these results, the unsubstituted thieno[2,3h]quinoline 2(1H)one seems to be the most interesting potential drug for PUVA photochemotherapy and photopheresis.

[Antimicrobial properties of pectins and their effects on antibiotics]. / Antimikrobnye svolstva pektinov i ikh vliianie na aktivnost' antibiotikov.

The influence of food fibres and plant proteins on microorganisms, bacteriophages, antibiotics and penicillinase was studied in vitro. It was shown that pectin was the only agent that had a bactericidal effect on the most widely distributed pathogenic and opportunistic microorganisms and did not influence indigenic microflora. High concentrations of pectin (> 2 per cent) had an inactivating effect on therapeutic bacteriophages. There was also a decrease in the antimicrobial activity of penicillins. The other agents tested i.e. wheat bran, soya isolate and soybean flour had no influence on microorganisms, bacteriophages and antibiotics. No sorption activity of the food fibres and plant proteins with respect to microorganisms and antibiotics or their effect on penicillinase was observed.