Phage Delivery Strategies for Biocontrolling Human, Animal, and Plant Bacterial Infections: State of the Art.

This review aims at presenting the main strategies that are currently available for the delivery of bacteriophages to combat bacterial infections in humans, animals, and plants. It can be seen that the main routes for phage delivery are topical, oral, systemic, and airways for humans. In animals, the topical and oral routes are the most used. To combat infections in plant species, spraying the plant's phyllosphere or drenching the soil are the most commonly used methods. In both phage therapy and biocontrol using phages, very promising results have been obtained so far. However, more experiments are needed to establish forms of treatment and phage doses, among other parameters. Furthermore, in general, there is a lack of specific standards for the use of phages to combat bacterial infections.

An Edible Antibacterial Coating Integrating Lytic Bacteriophage Particles for the Potential Biocontrol of Salmonella enterica in Ripened Cheese.

The goal of this research was to create an antibacterial biopolymeric coating integrating lytic bacteriophages against Salmonella enterica for use in ripened cheese. Salmonella enterica is the main pathogen that contaminates food products and the food industry. The food sector still uses costly and non-selective decontamination and disease control methods. Therefore, it is necessary to look for novel pathogen biocontrol technologies. Bacteriophage-based biocontrol seems like a viable option in this situation. The results obtained show promise for food applications since the edible packaging developed (EdiPhage) was successful in maintaining lytic phage viability while preventing the contamination of foodstuff with the aforementioned bacterial pathogen.

Molecular Characterization and Genome Mechanical Features of Two Newly Isolated Polyvalent Bacteriophages Infecting Pseudomonas syringae pv. garcae.

Coffee plants have been targeted by a devastating bacterial disease, a condition known as bacterial blight, caused by the phytopathogen Pseudomonas syringae pv. garcae (Psg). Conventional treatments of coffee plantations affected by the disease involve frequent spraying with copper- and kasugamycin-derived compounds, but they are both highly toxic to the environment and stimulate the appearance of bacterial resistance. Herein, we report the molecular characterization and mechanical features of the genome of two newly isolated (putative polyvalent) lytic phages for Psg. The isolated phages belong to class Caudoviricetes and present a myovirus-like morphotype belonging to the genuses Tequatrovirus (PsgM02F) and Phapecoctavirus (PsgM04F) of the subfamilies Straboviridae (PsgM02F) and Stephanstirmvirinae (PsgM04F), according to recent bacterial viruses' taxonomy, based on their complete genome sequences. The 165,282 bp (PsgM02F) and 151,205 bp (PsgM04F) genomes do not feature any lysogenic-related (integrase) genes and, hence, can safely be assumed to follow a lytic lifestyle. While phage PsgM02F produced a morphogenesis yield of 124 virions per host cell, phage PsgM04F produced only 12 virions per host cell, indicating that they replicate well in Psg with a 50 min latency period. Genome mechanical analyses established a relationship between genome bendability and virion morphogenesis yield within infected host cells.

The Role of Bacteriophages in the Gut Microbiota: Implications for Human Health.

Bacteriophages (phages) are nano-sized viruses characterized by their inherent ability to live off bacteria. They utilize diverse mechanisms to absorb and gain entry into the bacterial cell wall via the release of viral genetic material, which uses the replication mechanisms of the host bacteria to produce and release daughter progeny virions that attack the surrounding host cells. They possess specific characteristics, including specificity for particular or closely related bacterial species. They have many applications, including as potential alternatives to antibiotics against multi-resistant bacterial pathogens and as control agents in bacteria-contaminated environments. They are ubiquitously abundant in nature and have diverse biota, including in the gut. Gut microbiota describes the community and interactions of microorganisms within the intestine. As with bacteria, parasitic bacteriophages constantly interact with the host bacterial cells within the gut system and have obvious implications for human health. However, it is imperative to understand these interactions as they open up possible applicable techniques to control gut-implicated bacterial diseases. Thus, this review aims to explore the interactions of bacteriophages with bacterial communities in the gut and their current and potential impacts on human health.

Newly isolated phages preying on Pseudomonas syringae pv. garcae: In vitro and ex vivo inactivation studies in coffee plant leafs.

Coffee canker, or bacterial halo blight (BHB) of coffee, is a disease caused by the phytopathogenic bacterium Pseudomonas syringae pv. garcae (Psg), having been found for the first time in 1955, in the Garça region (State of São Paulo), and which has stood out in the Brazilian coffee plantations in recent years, leading to severe economic losses that seriously affect coffee trade. The treatments available are still scarce, involving frequent spraying of coffee plantations with either copper derivatives or the antibiotic kasugamycin. However, these compounds should be avoided due to environmental toxicity and the development of bacterial resistances. Herein we report the isolation and physical/biological characterisation of two novel lytic phages and their efficacy in the control of Psg. Phages ph002F and ph004F were isolated from coffee plant leaves in Brazil (Sorocaba/SP and Itu/SP cities), using Psg IBSBF-158 as the host. According to the transmission electron microscopy analyses, both phages belong to the class Caudoviricetes and present myovirus-like morphotypes. Phages ph002F and ph004F showed eclipse times of 5 min and 20 min, respectively, and a burst size of 123 PFU/host cell and 12 PFU/host cell, respectively, allowing to conclude they replicate well in Psg IBSBF-158 with latency periods of 50 min. Phage ph002F (reduction of 4.59 log CFU/mL, compared to uninfected culture) was more effective in inactivating Psg than phage ph004F (reduction of 3.85 log CFU/mL) after 10 h of incubation at a MOI of 10. As a cocktail, the two phages were highly effective in reducing the bacterial load (reduction of 5.26 log CFU/mL at a MOI of 0.1 or reduction of 5.03 log CFU/mL at a MOI of 10, relative to untreated culture), after 12 h of treatment. This study provides evidence that the isolated phages are promising candidates against the causative agent of BHB in coffee plants.

Therapeutic Potential of Marine Probiotics: A Survey on the Anticancer and Antibacterial Effects of Pseudoalteromonas spp.

Due to the increasing limitations and negative impacts of the current options for preventing and managing diseases, including chemotherapeutic drugs and radiation, alternative therapies are needed, especially ones utilizing and maximizing natural products (NPs). NPs abound with diverse bioactive primary and secondary metabolites and compounds with therapeutic properties. Marine probiotics are beneficial microorganisms that inhabit marine environments and can benefit their hosts by improving health, growth, and disease resistance. Several studies have shown they possess potential bioactive and therapeutic actions against diverse disease conditions, thus opening the way for possible exploitation of their benefits through their application. Pseudoalteromonas spp. are a widely distributed heterotrophic, flagellated, non-spore-forming, rod-shaped, and gram-negative marine probiotic bacteria species with reported therapeutic capabilities, including anti-cancer and -bacterial effects. This review discusses the basic concepts of marine probiotics and their therapeutic effects. Additionally, a survey of the anticancer and antibacterial effects of Pseudoalteromonas spp. is presented. Finally, marine probiotic production, advances, prospects, and future perspectives is presented.

An Edible Biopolymeric Microcapsular Wrapping Integrating Lytic Bacteriophage Particles for Salmonella enterica: Potential for Integration into Poultry Feed.

This research work aimed at developing an edible biopolymeric microcapsular wrapping (EBMW) integrating lytic bacteriophage particles for Salmonella enterica, with potential application in poultry feed for biocontrol of that pathogen. This pathogen is known as one of the main microorganisms responsible for contamination in the food industry and in foodstuff. The current techniques for decontamination and pathogen control in the food industry can be very expensive, not very selective, and even outdated, such as the use of broad-spectrum antibiotics that end up selecting resistant bacteria. Hence, there is a need for new technologies for pathogen biocontrol. In this context, bacteriophage-based biocontrol appears as a potential alternative. As a cocktail, both phages were able to significantly reduce the bacterial load after 12 h of treatment, at either multiplicity of infection (MOI) 1 and 10, by 84.3% and 87.6%, respectively. Entrapment of the phage virions within the EBMW matrix did not exert any deleterious effect upon their lytic activity. The results obtained showed high promise for integration in poultry feed aiming at controlling Salmonella enterica, since the edible biopolymeric microcapsular wrapping integrating lytic bacteriophage particles developed was successful in maintaining lytic phage viability while fully stabilizing the phage particles.

Evaluation of urban tree barks as bioindicators of environmental pollution using the X-ray fluorescence technique.

Some chemical elements released in nature due to anthropogenic actions are harmful to living beings, and finding efficient and low-cost ways to measure their presence is a challenge. The major goal of this work was to use the barks of urban trees as bioindicators of the presence of these elements. For this purpose, tree barks of sixteen individual trees were collected, including Ipê (Bignoniaceae Family); Sibipiruna (Fabaceae Family); Pine (Pinaceae Family), in the city of Sorocaba, SP, Brazil, in three different districts. Two samples, one of Ipê and another of Sibipiruna, collected in the Mata Atlântica forest in Juquitiba, SP, Brazil, were used as control samples. They were also analyzed; six soil samples were collected in the same places as the tree barks in Sorocaba. The samples were analyzed using the Energy Dispersion X-Ray Fluorescence Spectroscopy technique. The elements studied ranged from Al to Bi. The results were submitted to univariate and multivariate statistical analysis showing that Sibipiruna presented a high concentration of the element Ca. At the same time, Ipê and Pine showed high concentrations of K. In the identified elements, the probable sources of contamination were pointed out, such as elements from the dust of braking automobiles (Al, Si, S, Ti, Fe, Cu, and Ba), elements from the paint used to paint the asphalt (Si, Ca, Cr and Pb) and elements from the tire tread wear (Al, S, Ca and Zn). From the analysis of soil samples and trees, it was found that there was high pollution by the element Pb in the specimens collected in front of the old Saturnia battery factory, located in the district of Éden in the city of Sorocaba, SP, Brazil (Coordinates: Lat 23K253141 m E; Long 23K7405583 m S).

Isolation and Molecular Characterization of Two Novel Lytic Bacteriophages for the Biocontrol of Escherichia coli in Uterine Infections: In Vitro and Ex Vivo Preliminary Studies in Veterinary Medicine.

E. coli is one of the etiological agents responsible for pyometra in female dogs, with conventional treatment involving ovariohysterectomy. Here, we report the isolation and full characterization of two novel lytic phages, viz. vB_EcoM_Uniso11 (ph0011) and vB_EcoM_Uniso21 (ph0021). Both phages belong to the order Caudovirales and present myovirus-like morphotypes, with phage ph0011 being classified as Myoviridae genus Asteriusvirus and phage ph0021 being classified as Myoviridae genus Tequatrovirus, based on their complete genome sequences. The 348,288 bp phage ph0011 and 165,222 bp phage ph0021 genomes do not encode toxins, integrases or antimicrobial resistance genes neither depolymerases related sequences. Both phages were shown to be effective against at least twelve E. coli clinical isolates in in vitro antibacterial activity assays. Based on their features, both phages have potential for controlling pyometra infections caused by E. coli. Phage ph0011 (reduction of 4.24 log CFU/mL) was more effective than phage ph0021 (reduction of 1.90 log CFU/mL) after 12 h of incubation at MOI 1000. As a cocktail, the two phages were highly effective in reducing the bacterial load (reduction of 5.57 log CFU/mL) at MOI 100, after 12 h of treatment. Both phages were structurally and functionally stabilized in vaginal egg formulations.

Isolation and Molecular Characterization of a Novel Lytic Bacteriophage That Inactivates MDR Klebsiella pneumoniae Strains.

The worldwide increase in serious infections caused by multidrug-resistant (MDR) K. pneumoniae emphasizes the urgent need of new therapeutic strategies for the control of this pathogen. There is growing interest in the use of bacteriophages (or phages) to treat K. pneumoniae infections, and newly isolated phages are needed. Here, we report the isolation and physical/biological/molecular characterization of a novel lytic phage and its efficacy in the control of MDR K. pneumoniae. The phage vB_KpnS_Uniso31, referred to hereafter as phage Kpn31, was isolated from hospital wastewater using K. pneumoniae CCCD-K001 as the host. Phage Kpn31 presents a siphovirus-like morphotype and was classified as Demerecviridae; Sugarlandvirus based on its complete genome sequence. The 113,444 bp Kpn31 genome does not encode known toxins or antimicrobial resistance genes, nor does it encode depolymerases related sequences. Phage Kpn31 showed an eclipse time of 15 min and a burst size of 9.12 PFU/host cell, allowing us to conclude it replicates well in K. pneumoniae CCCD-K001 with a latency period of 30 min. Phage Kpn31 was shown to be effective against at least six MDR K. pneumoniae clinical isolates in in vitro antibacterial activity assays. Based on its features, phage Kpn31 has potential for controlling infections caused by MDR K. pneumoniae.

Pseudomonas-tailed lytic phages: genome mechanical analysis and putative correlation with virion morphogenesis yield.

Aim: To unveil a putative correlation between phage genome flexibility and virion morphogenesis yield. Materials & methods: A deeper analysis of the mechanical properties of three Pseudomonas aeruginosa lytic phage genomes was undertaken, together with full genome cyclizability calculations. Results & conclusion: A putative correlation was established among phage genome flexibility, eclipse timeframe and virion particle morphogenesis yield, with a more flexible phage genome leading to a higher burst size and a more rigid phage genome leading to lower burst sizes. The results obtained are highly relevant to understand the influence of the phage genome plasticity on the virion morphogenesis yield inside the infected bacterial host cells and assumes particular relevance in the actual context of bacterial resistance to antibiotics.

Transdermal permeation of curcumin promoted by choline geranate ionic liquid: Potential for the treatment of skin diseases.

The transdermal permeation of curcumin aided by choline and geranic acid ionic liquid (CAGE-IL) was addressed as a potential treatment for skin diseases. An in-depth analysis of the effect of CAGE-IL concentration in the enhancement of transdermal permeation of curcumin was performed, and the results were modelled via nonlinear regression analysis. The results obtained showed that a low percentage of CAGE-IL (viz. 2.0%, w/w) was effective in disrupting the skin structure in a transient fashion, facilitating the passage of curcumin dissolved in it.

Transdermal Permeation Assays of Curcumin Aided by CAGE-IL: In Vivo Control of Psoriasis.

Psoriasis is a clinically heterogeneous skin disease with an important genetic component, whose pathophysiology is not yet fully understood and for which there is still no cure. Hence, alternative therapies have been evaluated, using plant species such as turmeric (Curcuma longa Linn.) in topical preparations. However, the stratum corneum is a barrier to be overcome, and ionic liquids have emerged as potential substances that promote skin permeation. Thus, the main objective of this research was to evaluate a biopolysaccharide hydrogel formulation integrating curcumin with choline and geranic acid ionic liquid (CAGE-IL) as a facilitator of skin transdermal permeation, in the treatment of chemically induced psoriasis in mice. The developed gel containing curcumin and CAGE-IL showed a high potential for applications in the treatment of psoriasis, reversing the histological manifestations of psoriasis to a state very close to that of normal skin.

Characterization and in vitro testing of newly isolated lytic bacteriophages for the biocontrol of Pseudomonas aeruginosa.

Aim: Two lytic phages were isolated using P. aeruginosa DSM19880 as host and fully characterized. Materials & methods: Phages were characterized physicochemically, biologically and genomically. Results & conclusion: Host range analysis revealed that the phages also infect some multidrug-resistant (MDR) P. aeruginosa clinical isolates. Increasing MOI from 1 to 1000 significantly increased phage efficiency and retarded bacteria regrowth, but phage ph0034 (reduction of 7.5 log CFU/ml) was more effective than phage ph0031 (reduction of 5.1 log CFU/ml) after 24 h. Both phages belong to Myoviridae family. Genome sequencing of phages ph0031 and ph0034 showed that they do not carry toxin, virulence, antibiotic resistance and integrase genes. The results obtained are highly relevant in the actual context of bacterial resistance to antibiotics.

Transdermal Permeation of Caffeine Aided by Ionic Liquids: Potential for Enhanced Treatment of Cellulitis.

Ginoid hydrolipodystrophy (HDLG) or "cellulite" involves alteration of the cutaneous relief and occurs in 80-90% of the female population. Several topical treatments are available with the use of substances capable of stimulating lipolysis, such as caffeine. However, the effectiveness of topical therapy is related to the processes of release and permeation of the active in skin cells. In this sense, ionic liquids, such as choline geranate, are considered to facilitate topical permeation agents. In this way, the aim of this research was to develop and evaluation of the effectiveness of a cosmetic product for topical treatment of cellulite with caffeine in association with choline geranate. The choline geranate was synthesized by the reaction between geranic acid and choline hydroxide [1: 2]. The gel was prepared using 2% Carpobol 940®, 5% caffeine, and 1% choline geranate. Preliminary and accelerated stability tests were performed by checking pH, spreadability, and organoleptic characteristics. The transdermal permeation capacity of caffeine in vitro was evaluated by the Franz cell permeation assay, and the gel cytotoxicity by the MTS method. To prove the efficacy in the treatment of cellulite, a pilot type 1 clinical trial was carried out. The formulation was considered stable and the product maintained your characteristics during 180 days of storage. The product showed moderate cytotoxicity and high skin permeation capacity. In the clinical trial, it showed results superior to the caffeine gel without ionic liquid. The developed gel favored the cutaneous permeation of caffeine, showing a promising product in the treatment of cellulite.

Bacteriophage-Based Biosensing of Pseudomonas aeruginosa: An Integrated Approach for the Putative Real-Time Detection of Multi-Drug-Resistant Strains.

During the last decennium, it has become widely accepted that ubiquitous bacterial viruses, or bacteriophages, exert enormous influences on our planet's biosphere, killing between 4-50% of the daily produced bacteria and constituting the largest genetic diversity pool on our planet. Currently, bacterial infections linked to healthcare services are widespread, which, when associated with the increasing surge of antibiotic-resistant microorganisms, play a major role in patient morbidity and mortality. In this scenario, Pseudomonas aeruginosa alone is responsible for ca. 13-15% of all hospital-acquired infections. The pathogen P. aeruginosa is an opportunistic one, being endowed with metabolic versatility and high (both intrinsic and acquired) resistance to antibiotics. Bacteriophages (or phages) have been recognized as a tool with high potential for the detection of bacterial infections since these metabolically inert entities specifically attach to, and lyse, bacterial host cells, thus, allowing confirmation of the presence of viable cells. In the research effort described herein, three different phages with broad lytic spectrum capable of infecting P. aeruginosa were isolated from environmental sources. The isolated phages were elected on the basis of their ability to form clear and distinctive plaques, which is a hallmark characteristic of virulent phages. Next, their structural and functional stabilization was achieved via entrapment within the matrix of porous alginate, biopolymeric, and bio-reactive, chromogenic hydrogels aiming at their use as sensitive matrices producing both color changes and/or light emissions evolving from a reaction with (released) cytoplasmic moieties, as a bio-detection kit for P. aeruginosa cells. Full physicochemical and biological characterization of the isolated bacteriophages was the subject of a previous research paper.

Performance of Choline Geranate Deep Eutectic Solvent as Transdermal Permeation Enhancer: An In Vitro Skin Histological Study.

In the present research work, we addressed the changes in skin by which deep eutectic solvents (DES) enhanced transdermal permeation of bioactive compounds and propose a rationale for this mechanism. Several studies showed that these unusual liquids were ideal solvents for transdermal delivery of biomolecules, but to date, no histological studies relating the action of DES to changes in the structure of the outer skin barrier have been reported. In the research effort described herein, we presented an in-depth analysis of the changes induced in the skin by choline geranate DES, a compound with proven capabilities of enhancing transdermal permeation without deleterious impacts on the cells. The results obtained showed that a low percentage of DES acted as a transient disruptor of the skin structure, facilitating the passage of bioactive compounds dissolved in it.

Kiwifruit bacterial canker: an integrative view focused on biocontrol strategies.

MAIN CONCLUSION: Phage-based biocontrol strategies can be an effective alternative to control Psa-induced bacterial canker of kiwifruit. The global production of kiwifruit has been seriously affected by Pseudomonas syringae pv. actinidiae (Psa) over the last decade. Psa damages both Actinidia chinensis var. deliciosa (green kiwifruit) but specially the susceptible Actinidia chinensis var. chinensis (gold kiwifruit), resulting in severe economic losses. Treatments for Psa infections currently available are scarce, involving frequent spraying of the kiwifruit plant orchards with copper products. However, copper products should be avoided since they are highly toxic and lead to the development of bacterial resistance to this metal. Antibiotics are also used in some countries, but bacterial resistance to antibiotics is a serious worldwide problem. Therefore, it is essential to develop new approaches for sustainable agriculture production, avoiding the emergence of resistant Psa bacterial strains. Attempts to develop and establish highly accurate approaches to combat and prevent the occurrence of bacterial canker in kiwifruit plants are currently under study, using specific viruses of bacteria (bacteriophages, or phages) to eliminate the Psa. This review discusses the characteristics of Psa-induced kiwifruit canker, Psa transmission pathways, prevention and control, phage-based biocontrol strategies as a new approach to control Psa in kiwifruit orchards and its advantages over other therapies, together with potential ways to bypass phage inactivation by abiotic factors.