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.

Chestnut Honey and Bacteriophage Application to Control Pseudomonas aeruginosa and Escherichia coli Biofilms: Evaluation in an ex vivo Wound Model.

Chronic skin wounds represent a major burn both economically and socially. Pseudomonas aeruginosa and Escherichia coli are among the most common colonizers of infected wounds and are prolific biofilm formers. Biofilms are a major problem in infections due to their increasingly difficult control and eradication, and tolerance to multiple prescribed drugs. As so, alternative methods are necessary. Bacteriophages (phages) and honey are both seen as a promising approach for biofilm related infections. Phages have specificity toward a bacterial genus, species or even strain, self-replicating nature, and avoid dysbiosis. Honey has gained acknowledgment due to its antibacterial, antioxidant and anti-inflammatory and wound healing properties. In this work, the effect of E. coli and P. aeruginosa phages vB_EcoS_CEB_EC3a and vB_PaeP_PAO1-D and chestnut honey, alone and combined, were tested using in vitro (polystyrene) and ex vivo (porcine skin) models and against mono and dual-species biofilms of these bacteria. In general, colonization was higher in the porcine skins and the presence of a second microorganism in a consortium of species did not affect the effectiveness of the treatments. The antibacterial effect of combined therapy against dual-species biofilms led to bacterial reductions that were greater for biofilms formed on polystyrene than on skin. Monospecies biofilms of E. coli were better destroyed with phages and honey than P. aeruginosa monospecies biofilms. Overall, the combined phage-honey formulations resulted in higher efficacies possibly due to honey's capacity to damage the bacterial cell membrane and also to its ability to penetrate the biofilm matrix, promoting and enhancing the subsequent phage infection.

Synergistic Antimicrobial Interaction between Honey and Phage against Escherichia coli Biofilms.

Chronic wounds afford a hostile environment of damaged tissues that allow bacterial proliferation and further wound colonization. Escherichia coli is among the most common colonizers of infected wounds and it is a prolific biofilm former. Living in biofilm communities, cells are protected, become more difficult to control and eradicate, and less susceptible to antibiotic therapy. This work presents insights into the proceedings triggering E. coli biofilm control with phage, honey, and their combination, achieved through standard antimicrobial activity assays, zeta potential and flow cytometry studies and further visual insights sought by scanning electron microscopy and transmission electron microscopy. Two Portuguese honeys (PF2 and U3) with different floral origin and an E. coli-specific phage (EC3a), possessing depolymerase activity, were tested against 24- and 48-h-old biofilms. Synergic and additive effects were perceived in some phage-honey experiments. Combined therapy prompted similar phenomena in biofilm cells, visualized by electron microscopy, as the individual treatments. Honey caused minor membrane perturbations to complete collapse and consequent discharge of cytoplasmic content, and phage completely destroyed cells leaving only vesicle-like structures and debris. Our experiments show that the addition of phage to low honey concentrations is advantageous, and that even fourfold diluted honey combined with phage, presents no loss of antibacterial activity toward E. coli. Portuguese honeys possess excellent antibiofilm activity and may be potential alternative therapeutic agents in biofilm-related wound infection. Furthermore, to our knowledge this is the first study that assessed the impacts of phage-honey combinations in bacterial cells. The synergistic effect obtained was shown to be promising, since the antiviral effect of honey limits the emergence of phage resistant phenotypes.