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.

Fatty acid amide hydrolase drives adult mammary gland development by promoting luminal cell differentiation.

Mammary gland development occurs primarily in adulthood, undergoing extensive expansion during puberty followed by cycles of functional specialization and regression with every round of pregnancy/lactation/involution. This process is ultimately driven by the coordinated proliferation and differentiation of mammary epithelial cells. However, the endogenous molecular factors regulating these developmental dynamics are still poorly defined. Endocannabinoid signaling is known to determine cell fate-related events during the development of different organs in the central nervous system and the periphery. Here, we report that the endocannabinoid-degrading enzyme fatty acid amide hydrolase (FAAH) plays a pivotal role in adult mammary gland development. Specifically, it is required for luminal lineage specification in the mammary gland, and it promotes hormone-driven secretory differentiation of mammary epithelial cells by controlling the endogenous levels of anandamide and the subsequent activation of cannabinoid CB1 receptors. Together, our findings shed light on the role of the endocannabinoid system in breast development and point to FAAH as a therapeutic target in milk-production deficits.

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.

Neuronal Cannabinoid CB1 Receptors Suppress the Growth of Melanoma Brain Metastases by Inhibiting Glutamatergic Signalling.

Melanoma is one of the deadliest forms of cancer. Most melanoma deaths are caused by distant metastases in several organs, especially the brain, the so-called melanoma brain metastases (MBMs). However, the precise mechanisms that sustain the growth of MBMs remain elusive. Recently, the excitatory neurotransmitter glutamate has been proposed as a brain-specific, pro-tumorigenic signal for various types of cancers, but how neuronal glutamate shuttling onto metastases is regulated remains unknown. Here, we show that the cannabinoid CB1 receptor (CB1R), a master regulator of glutamate output from nerve terminals, controls MBM proliferation. First, in silico transcriptomic analysis of cancer-genome atlases indicated an aberrant expression of glutamate receptors in human metastatic melanoma samples. Second, in vitro experiments conducted on three different melanoma cell lines showed that the selective blockade of glutamatergic NMDA receptors, but not AMPA or metabotropic receptors, reduces cell proliferation. Third, in vivo grafting of melanoma cells in the brain of mice selectively devoid of CB1Rs in glutamatergic neurons increased tumour cell proliferation in concert with NMDA receptor activation, whereas melanoma cell growth in other tissue locations was not affected. Taken together, our findings demonstrate an unprecedented regulatory role of neuronal CB1Rs in the MBM tumour microenvironment.

Identification of fatty acid amide hydrolase as a metastasis suppressor in breast cancer.

Clinical management of breast cancer (BC) metastasis remains an unmet need as it accounts for 90% of BC-associated mortality. Although the luminal subtype, which represents >70% of BC cases, is generally associated with a favorable outcome, it is susceptible to metastatic relapse as late as 15 years after treatment discontinuation. Seeking therapeutic approaches as well as screening tools to properly identify those patients with a higher risk of recurrence is therefore essential. Here, we report that the lipid-degrading enzyme fatty acid amide hydrolase (FAAH) is a predictor of long-term survival in patients with luminal BC, and that it blocks tumor progression and lung metastasis in cell and mouse models of BC. Together, our findings highlight the potential of FAAH as a biomarker with prognostic value in luminal BC and as a therapeutic target in metastatic disease.

Genome-wide association reveals host-specific genomic traits in Escherichia coli.

BACKGROUND: Escherichia coli is an opportunistic pathogen which colonizes various host species. However, to what extent genetic lineages of E. coli are adapted or restricted to specific hosts and the genomic determinants of such adaptation or restriction is poorly understood. RESULTS: We randomly sampled E. coli isolates from four countries (Germany, UK, Spain, and Vietnam), obtained from five host species (human, pig, cattle, chicken, and wild boar) over 16 years, from both healthy and diseased hosts, to construct a collection of 1198 whole-genome sequenced E. coli isolates. We identified associations between specific E. coli lineages and the host from which they were isolated. A genome-wide association study (GWAS) identified several E. coli genes that were associated with human, cattle, or chicken hosts, whereas no genes associated with the pig host could be found. In silico characterization of nine contiguous genes (collectively designated as nan-9) associated with the human host indicated that these genes are involved in the metabolism of sialic acids (Sia). In contrast, the previously described sialic acid regulon known as sialoregulon (i.e. nanRATEK-yhcH, nanXY, and nanCMS) was not associated with any host species. In vitro growth experiments with a Δnan-9 E. coli mutant strain, using the sialic acids 5-N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc) as sole carbon source, showed impaired growth behaviour compared to the wild-type. CONCLUSIONS: This study provides an extensive analysis of genetic determinants which may contribute to host specificity in E. coli. Our findings should inform risk analysis and epidemiological monitoring of (antimicrobial resistant) E. coli.

A novel biocompatible polymer derived from D-mannitol used as a vector in the field of genetic engineering of eukaryotic cells.

The design and preparation of new vectors to transport genetic material and increase the transfection efficiency continue being an important research line. Here, a novel biocompatible sugar-based polymer derived from D-mannitol has been synthesized to be used as a gene material nanocarrier in human (gene transfection) and microalga cells (transformation process). Its low toxicity allows its use in processes with both medical and industrial applications. A multidisciplinary study about the formation of polymer/p-DNA polyplexes has been carried out using techniques such as gel electrophoresis, zeta potential, dynamic light scattering, atomic force microscopy, and circular dichroism spectroscopy. The nucleic acids used were the eukaryotic expression plasmid pEGFP-C1 and the microalgal expression plasmid Phyco69, which showed different behaviors. The importance of DNA supercoiling in both transfection and transformation processes was demonstrated. Better results were obtained in microalga cells nuclear transformation than in human cells gene transfection. This was related to the plasmid's conformational changes, in particular to their superhelical structure. It is noteworthy that the same nanocarrier has been used with eukaryotic cells from both human and microalga.

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).

The effect of probiotics on functional constipation in adults: A randomized, double-blind controlled trial.

BACKGROUND: Two formulations were developed in the form of an oral sachet containing probiotics, and their efficacy and safety were evaluated in adults with functional constipation. METHODS: One formulation with Lactobacillus acidophilus, Bifidobacterium bifidum and Lactobacillus rhamnosus (3 billion Colony Forming Units - CFU); and another with Lactobacillus acidophilus, Bifidobacterium bifidum, Lactobacillus rhamnosus, Lactobacillus paracasei, Bifidobacterium longum, Bifidobacterium lactis, Lactobacillus casei, Bifidobacterium animallis (8 billion CFU). The participants were randomized in a 3-arm parallel study and one oral sachet was auto-administered once a day for 30 days. RESULTS: Primary outcomes were improvement in increasing the frequency of weekly bowel movements and improvement in stool quality. Secondary outcomes were number of adverse events. In the first week one observed an increase in stool frequency and in the quality of stools, showing an improvement in constipation. No statistically significant differences were observed between the three treatment groups in relation to these outcomes (P ≥ .05). Only one adverse event was observed in a patient of group 2, related to abdominal pain. CONCLUSION: The two probiotic cocktails were effective in improving the symptoms of functional constipation, by increasing both the weekly frequency of evacuation and stool quality, and were deemed safe. Clinicaltrials.gov number: NCT04437147.

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.

Single-Cell Transcriptome Profiling Reveals Intratumoral Heterogeneity and Molecular Features of Ductal Carcinoma In Situ.

Ductal carcinoma in situ (DCIS) is a precursor to invasive breast cancer. The frequency of DCIS is increasing because of routine mammography; however, the biological features and intratumoral heterogeneity of DCIS remain obscure. To address this deficiency, we performed single-cell transcriptomic profiling of DCIS and invasive ductal carcinoma (IDC). DCIS was found to be composed of several transcriptionally distinct subpopulations of cancer cells with specific functions. Several transcripts, including long noncoding RNAs, were highly expressed in IDC compared with DCIS and might be related to the invasive phenotype. Closeness centrality analysis revealed extensive heterogeneity in DCIS, and the prediction model for cell-to-cell interactions implied that the interaction network among luminal cells and immune cells in DCIS was comparable with that in IDC. In addition, transcriptomic profiling of HER2+ luminal DCIS indicated HER2 genomic amplification at the DCIS stage. These data provide novel insight into the intratumoral heterogeneity and molecular features of DCIS, which exhibit properties similar to IDC. SIGNIFICANCE: Investigation of the molecular features of ductal carcinoma in situ at single cell resolution provides new insights into breast cancer biology and identifies candidate therapeutic targets and diagnostic biomarkers.

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.

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.

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.