Paving the way forward: Escherichia coli bacteriophages in a One Health approach.

Escherichia coli is one of the most notorious pathogens for its ability to adapt, colonize, and proliferate in different habitats through a multitude of acquired virulence factors. Its presence affects the food-processing industry and causes food poisoning, being also a major economic burden for the food, agriculture, and health sectors. Bacteriophages are emerging as an appealing strategy to mitigate bacterial pathogens, including specific E. coli pathovars, without exerting a deleterious effect on humans and animals. This review globally analyzes the applied research on E. coli phages for veterinary, food, and human use. It starts by describing the pathogenic E. coli pathotypes and their relevance in human and animal context. The idea that phages can be used as a One Health approach to control and interrupt the transmission routes of pathogenic E. coli is sustained through an exhaustive revision of the recent literature. The emerging phage formulations, genetic engineering and encapsulation technologies are also discussed as a means of improving phage-based control strategies, with a particular focus on E. coli pathogens.

Complete genome sequence of a novel bacteriophage vB_Pci_PCMW57 infecting phytobacteria pseudomonas cichorii.

BACKGROUND: A novel virulent bacteriophage infecting phytobacteria Pseudomonas cichorii (P. cichorii) was isolated from leafy vegetables in Brazil. P. cichorii is a Gram-negative soil phytobacterium, the causal agent of a number of economically important plant diseases worldwide. METHODS AND RESULTS: In this study, a new phage specific for P. cichorii was isolated from solid samples (lettuce, chicory and cabbage), designated vB_Pci_PCMW57. Electron microscopy revealed a small virion (~ 50-nm-diameter icosahedral capsid) with a short, non-contractile tail. The genome of vB_Pci_PCMW57 is 40,117 bp in size, with a GC content of 57.6% and encodes 49 open reading frames. The phage is genetically similar to P. syringae phages Pst_GM1 and Pst_GIL1, and the P. fluorescens phages WRT and KNP. According to electron microscopy and whole-genome sequence analysis, vB_Pci_PCMW57 should be classified as a Caudoviticetes, family Autographiviridae, subfamily Studiervirinae. CONCLUSIONS: The complete phage genome was annotated, and the sequence identity of the virus with other Pseudomonas viruses was higher than 95%. To our knowledge, this is the first report of a bacteriophage infecting Pseudomonas cichorii.

Suggestion for a new bacteriophage genus for the Klebsiella pneumoniae phage vB_KpnS-Carvaje.

This work describes the newly isolated Klebsiella pneumoniae phage vB_KpnS-Carvaje that presents unique features in relation to other phages reported to date. These findings provide new insights into the diversity and evolutionary pathways of Klebsiella phages. The genome characterization of the Carvaje phage revealed that its genome length is approximately 57 kb with 99 open reading frames (ORFs), 33 of which have assigned functions while 66 are unknown. This phage differs from other sequenced Klebsiella phages, showing the closest resemblance (up to 65.32%) with Salmonella phages belonging to the Nonanavirus and Sashavirus genera. Comparisons at the amino acid level and phylogeny analysis among homologous genomes indicate that the Klebsiella Carvaje phage forms a novel sister taxon within the node of the Nonanaviruses and Sashaviruses cluster. Due to the unique features of the Carvaje phage, we propose the constitution of a new genus within the Caudoviricetes class. Further studies include the exploitation of this phage and its identified proteins for the control of Klebsiella infections and as recognition molecules in diagnostic methods.

Otitis media pathogens - A life entrapped in biofilm communities.

Otitis media is a group of inflammatory diseases of the middle ear with great impact on children worldwide. The most common reported bacterial pathogens are Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis. Over the last years, the role of biofilms formed by otopathogens that contribute to otitis media recurrence and chronicity has been established. An improved understanding of the properties of biofilms formed by these bacteria, which factors influence them, and how these affect the host inflammatory response is important for the development of novel strategies for the treatment of otitis media. This review focuses on the biofilm nature that the most prevalent otopathogens adopt in otitis media infections. In addition, new treatment approaches targeting biofilms are highlighted.

Control of Salmonella Enteritidis on food contact surfaces with bacteriophage PVP-SE2.

Salmonella is one of the worldwide leading foodborne pathogens responsible for illnesses and hospitalizations, and its capacity to form biofilms is one of its many virulence factors. This work evaluated (bacterio)phage control of adhered and biofilm cells of Salmonella Enteritidis on three different substrata at refrigerated and room temperatures, and also a preventive approach in poultry skin. PVP-SE2 phage was efficient in reducing both 24- and 48-h old Salmonella biofilms from polystyrene and stainless steel causing 2 to 5 log CFU cm-2 reductions with a higher killing efficiency at room temperature. PVP-SE2 phage application on poultry skins reduced levels of Salmonella. Freezing phage-pretreated poultry skin samples had no influence on the viability of phage PVP-SE2 and their in vitro contamination with S. Enteritidis provided evidence that phages prevented their further growth. Although not all conditions favor phage treatment, this study endorses their use to prevent and control foodborne pathogen colonization of surfaces.

Ability of phages to infect Acinetobacter calcoaceticus-Acinetobacter baumannii complex species through acquisition of different pectate lyase depolymerase domains.

Bacteriophages are ubiquitous in nature and represent a vast repository of genetic diversity, which is driven by the endless coevolution cycle with a diversified group of bacterial hosts. Studying phage-host interactions is important to gain novel insights into their dynamic adaptation. In this study, we isolated 12 phages infecting species of the Acinetobacter baumannii-Acinetobacter calcoaceticus complex which exhibited a narrow host range and similar morphological features (podoviruses with short tails of 9-12 nm and isometric heads of 50-60 nm). Notably, the alignment of the newly sequenced phage genomes (40-41 kb of DNA length) and all Acinetobacter podoviruses deposited in Genbank has shown high synteny, regardless of the date and source of isolation that spans from America to Europe and Asia. Interestingly, the C-terminal pectate lyase domain of these phage tail fibres is often the only difference found among these viral genomes, demonstrating a very specific genomic variation during the course of their evolution. We proved that the pectate lyase domain is responsible for phage depolymerase activity and binding to specific Acinetobacter bacterial capsules. We discuss how this mechanism of phage-host co-evolution impacts the tail specificity apparatus of Acinetobacter podoviruses.

Phage Therapy: a Step Forward in the Treatment of Pseudomonas aeruginosa Infections.

Antimicrobial resistance constitutes one of the major worldwide public health concerns. Bacteria are becoming resistant to the vast majority of antibiotics, and nowadays, a common infection can be fatal. To address this situation, the use of phages for the treatment of bacterial infections has been extensively studied as an alternative therapeutic strategy. Since Pseudomonas aeruginosa is one of the most common causes of health care-associated infections, many studies have reported the in vitro and in vivo antibacterial efficacy of phage therapy against this bacterium. This review collects data of all the P. aeruginosa phages sequenced to date, providing a better understanding about their biodiversity. This review further addresses the in vitro and in vivo results obtained by using phages to treat or prevent P. aeruginosa infections as well as the major hurdles associated with this therapy.

Bacteriophage-encoded depolymerases: their diversity and biotechnological applications.

Bacteriophages (phages), natural enemies of bacteria, can encode enzymes able to degrade polymeric substances. These substances can be found in the bacterial cell surface, such as polysaccharides, or are produced by bacteria when they are living in biofilm communities, the most common bacterial lifestyle. Consequently, phages with depolymerase activity have a facilitated access to the host receptors, by degrading the capsular polysaccharides, and are believed to have a better performance against bacterial biofilms, since the degradation of extracellular polymeric substances by depolymerases might facilitate the access of phages to the cells within different biofilm layers. Since the diversity of phage depolymerases is not yet fully explored, this is the first review gathering information about all the depolymerases encoded by fully sequenced phages. Overall, in this study, 160 putative depolymerases, including sialidases, levanases, xylosidases, dextranases, hyaluronidases, peptidases as well as pectate/pectin lyases, were found in 143 phages (43 Myoviridae, 47 Siphoviridae, 37 Podoviridae, and 16 unclassified) infecting 24 genera of bacteria. We further provide information about the main applications of phage depolymerases, which can comprise areas as diverse as medical, chemical, or food-processing industry.

Discrimination of bacteriophage infected cells using locked nucleic acid fluorescent in situ hybridization (LNA-FISH).

Bacteriophage-host interaction studies in biofilm structures are still challenging due to the technical limitations of traditional methods. The aim of this study was to provide a direct fluorescence in situ hybridization (FISH) method based on locked nucleic acid (LNA) probes, which targets the phage replication phase, allowing the study of population dynamics during infection. Bacteriophages specific for two biofilm-forming bacteria, Pseudomonas aeruginosa and Acinetobacter, were selected. Four LNA probes were designed and optimized for phage-specific detection and for bacterial counterstaining. To validate the method, LNA-FISH counts were compared with the traditional plaque forming unit (PFU) technique. To visualize the progression of phage infection within a biofilm, colony-biofilms were formed and infected with bacteriophages. A good correlation (r = 0.707) was observed between LNA-FISH and PFU techniques. In biofilm structures, LNA-FISH provided a good discrimination of the infected cells and also allowed the assessment of the spatial distribution of infected and non-infected populations.

Isolation and characterization of a new Staphylococcus epidermidis broad-spectrum bacteriophage.

Staphylococcus epidermidis is considered an important nosocomial pathogen, being very tolerant to the host immune system and antibiotherapy, particularly when in biofilms. Due to its high resistance, alternative antimicrobial strategies are under development. The use of bacteriophages is seen as an important strategy to combat pathogenic organisms. In this study, a S. epidermidis myovirus, SEP1, was isolated and characterized. The genome of this phage was sequenced and shown to be related peripherally to the genus Twortlikevirus. However, when compared with other phages of this genus, it showed DNA sequence identities no greater than 58.2 %. As opposed to other polyvalent viruses of the genus Twortlikevirus, SEP1 is highly specific to S. epidermidis strains. The good infectivity shown by this phage as well as its high lytic spectrum suggested that it might be a good candidate for therapeutic studies.

Isolation of Bacteriophages for Clinically Relevant Bacteria.

The isolation of bacteriophages targeting most clinically relevant bacteria is reasonably straightforward as long as its targeted host does not have complex chemical, physical, and environmental requirements. Often, sewage, soil, feces, and different body fluids are used for bacteriophage isolation procedures, and following enrichment, it is common to obtain more than a single phage in a sample. This chapter describes a simple method for the enrichment and isolation of bacteriophages from liquid and solid samples that can be adapted for different clinically important aerobic bacteria.

Bacteriophage Control of Infectious Biofilms.

Biofilm formation, a strategy of bacterial survival, is a significant concern in different areas, including health, where infectious biofilms are very difficult to combat with conventional antimicrobial therapies. Bacteriophages, the viruses that infect bacteria, are promising agents to prevent and control biofilm-related infections. This chapter describes a series of standard procedures that can be used to study the potential of bacteriophages for biofilm control, from biofilm formation to bacteriophage treatment and evaluation of its efficacy.

Polymeric Microneedles for Health Care Monitoring: An Emerging Trend.

Bioanalyte collection by blood draw is a painful process, prone to needle phobia and injuries. Microneedles can be engineered to penetrate the epidermal skin barrier and collect analytes from the interstitial fluid, arising as a safe, painless, and effective alternative to hypodermic needles. Although there are plenty of reviews on the various types of microneedles and their use as drug delivery systems, there is a lack of systematization on the application of polymeric microneedles for diagnosis. In this review, we focus on the current state of the art of this field, while providing information on safety, preclinical and clinical trials, and market distribution, to outline what we believe will be the future of health monitoring.

Designing an antimicrobial film for wound applications incorporating bacteriophages and ε-poly-l-lysine.

Alginate-based dressings have been shown to promote wound healing, leveraging the unique properties of alginate. This work aimed to develop and characterize flexible individual and bilayered films to deliver bacteriophages (phages) and ε-Poly-l-lysine (ε-PLL). Films varied in different properties. The moisture content, swelling and solubility increased with higher alginate concentrations. The water vapour permeability, crucial in biomedical films to balance moisture levels for effective wound healing, reached optimal levels in bilayer films, indicating these will be able to sustain an ideal moist environment. The bilayer films showed improved ductility (lower tensile strength and increased elongation at break) compared to individual films. The incorporated phages maintained viability for 12 weeks under vacuum and refrigerated conditions, and their release was sustained and gradual. Antibacterial immersion tests showed that films with phages and ε-PLL significantly inhibited Pseudomonas aeruginosa PAO1 growth (>3.1 Log CFU/cm2). Particle release was influenced by the swelling degree and diffusional processes within the polymer network, providing insights into controlled release mechanisms for particles of varying size (50 nm to 6 µm) and charge. The films developed, demonstrated modulated release capabilities for active agents, and may show potential as controlled delivery systems for phages and wound healing adjuvants.

Genome sequence of the broad-host-range Pseudomonas phage Φ-S1.

The broad-host-range lytic Pseudomonas phage Φ-S1 possess a 40,192 bp double-stranded DNA (dsDNA) genome of 47 open reading frames (ORFs) and belongs to the family Podoviridae, subfamily Autographivirinae, genus T7likevirus.

Evaluation of the ability of C. albicans to form biofilm in the presence of phage-resistant phenotypes of P. aeruginosa.

Pseudomonas aeruginosa and Candida albicans are disparate microbial species, but both are known to be opportunistic pathogens frequently associated with nosocomial infections. The aim of this study was to provide a better understanding of the interactions between these microorganisms in dual-species biofilms. Several bacteriophage-resistant P. aeruginosa phenotypes have been isolated and were used in dual-species mixed-biofilm studies. Twenty-four and 48 h mixed-biofilms were formed using the isolated phenotypes of phage-resistant P. aeruginosa and these were compared with similar experiments using other P. aeruginosa strains with a defined lipopolysaccharide (LPS) deficiency based on chromosomal knockout of specific LPS biosynthetic genes. Overall, the results showed that the variants of phage-resistant P. aeruginosa and LPS mutants were both less effective in inhibiting the growth of C. albicans in mixed-biofilms compared to the wild-type strains of P. aeruginosa. Conversely, the proliferation of P. aeruginosa was not influenced by the presence of C. albicans. In conclusion, the ability of strains of P. aeruginosa to inhibit the formation of a biofilm of C. albicans appears to be correlated with the LPS chain lengths of phenotypes of P. aeruginosa, suggesting that LPS has a suppressive effect on the growth of C. albicans.

Bacteriophage Delivery Systems for Food Applications: Opportunities and Perspectives.

Currently, one-third of all food produced worldwide is wasted or lost, and bacterial contamination is one of the main reasons. Moreover, foodborne diseases are a severe problem, causing more than 420,000 deaths and nearly 600 million illnesses yearly, demanding more attention to food safety. Thus, new solutions need to be explored to tackle these problems. A possible solution for bacterial contamination is using bacteriophages (phages), which are harmless to humans; these natural viruses can be used to prevent or reduce food contamination by foodborne pathogens. In this regard, several studies showed the effectiveness of phages against bacteria. However, when used in their free form, phages can lose infectivity, decreasing the application in foods. To overcome this problem, new delivery systems are being studied to incorporate phages and ensure prolonged activity and controlled release in food systems. This review focuses on the existent and new phage delivery systems applied in the food industry to promote food safety. Initially, an overview of phages, their main advantages, and challenges is presented, followed by the different delivery systems, focused in methodologies, and biomaterials that can be used. In the end, examples of phage applications in foods are disclosed and future perspectives are approached.

Antibiofilm Efficacy of the Pseudomonas aeruginosa Pbunavirus vB_PaeM-SMS29 Loaded onto Dissolving Polyvinyl Alcohol Microneedles

Resistant bacteria prevail in most chronic skin wounds and other biofilm-related topical skin infections. Bacteriophages (phages) have proven their antimicrobial effectiveness for treating different antibiotic-resistant and multidrug-resistant bacterial infections, but not all phages are effective against biofilms. Phages possessing depolymerases can reach different biofilm layers; however, those that do not have depolymerase activity struggle to penetrate and navigate in the intricate 3D biofilm structure and mainly infect bacteria lodged in the outer biofilm layers. To address this, Pseudomonas aeruginosa phage vB_PaeM-SMS29, a phage with poor antibiofilm properties, was incorporated into polyvinyl alcohol (PVA, Mowiol 4:88) supplemented with 0.1% (v/v) of glycerol, and cast onto two different microneedle arrays varying in geometry. The dissolving microneedles were thoroughly characterized by microscopy, force-displacement, swelling, phage release and stability. Furthermore, 48 h-old biofilms were formed using the colony biofilm procedure (absence of broth), and the antibiofilm efficacy of the phage-loaded microneedles was evaluated by viable cell counts and microscopy and compared to free phages. The phages in microneedles were fairly stable for six months when stored at 4 °C, with minor decreases in phage titers observed. The geometry of the microneedles influenced the penetration and force-displacement characteristics but not the antimicrobial efficacy against biofilms. The two PVA microneedles loaded with phages reduced P. aeruginosa PAO1 biofilms by 2.44 to 2.76 log10 CFU·cm-2 at 24 h. These values are significantly higher than the result obtained after the treatment with the free phage (1.09 log10 CFU·cm-2). Overall, this study shows that the distribution of phages caused by the mechanical disruption of biofilms using dissolving microneedles can be an effective delivery method against topical biofilm-related skin infections.

Ex vivo transtympanic permeation of the liposome encapsulated S. pneumoniae endolysin MSlys.

An increase in bacterial resistance to systemic antibiotics has sparked interest into alternative antimicrobial compounds as well as methods for effective local, non-invasive drug delivery. Topical treatments, however, may be hindered by the presence of biological barriers, such as the tympanic membrane in the case of otitis media. Herein, the transtympanic permeation ability of liposomes loaded with the pneumococcal endolysin MSlys and of free MSlys was evaluated ex vivo. MSlys loaded in PEGylated liposomes showed an increased permeation across human tympanic membranes, as compared to its free form, being able to reduce the pneumococcal cell load after 2 h of permeation. However, antipneumococcal activity was no longer detected after 4 h of permeation and hydrolysis of the endolysin was observed after an extended incubation time (≥48 h). This work provides a first assessment of a successful, non-invasive delivery method for endolysins across an intact tympanic membrane. Findings have implications for non-systemic, local treatment of otitis media.