A. Oliveira

The First Paenibacillus larvae Bacteriophage Endolysin (PlyPl23) with High Potential to Control American Foulbrood

Endolysins, which are peptidoglycan-degrading enzymes expressed during the terminal stage of the reproduction cycle of bacteriophages, have great potential to control Gram-positive pathogens. This work describes the characterization of a novel endolysin (PlyPl23) encoded on the genome of Paenibacillus larvae phage phiIBB_Pl23 with high potential to control American foulbrood. This bacterial disease, caused by P. larvae, is widespread in North America and Europe and causes important economic losses in apiculture. The restriction to antibiotic residues in honey imposed by the EU legislation hinders its therapeutic use to combat American foulbrood and enforces the development of alternative antimicrobial methods. The new endolysin described herein has an N-acetylmuramoyl-L-alanine amidase catalytic domain and exhibits a broad-spectrum activity against common P. larvae genotypes. Moreover, the enzyme displays high antimicrobial activity in a range of pH that matches environmental conditions (pH between 5.0 and 7.0), showing its feasible application in the field. At pH 7.0, a concentration of 0.2 μM of enzyme was enough to lyse 104 CFU.mL-1 of P. larvae in no more than 2 h. The presence of sucrose and of the substances present in the larvae gut content did not affect the enzyme activity. Interestingly, an increase of activity was observed when PlyPl23 was previously incubated in royal jelly. Furthermore, in vivo safety evaluation assays demonstrated that this enzyme is not toxic to the bee larvae. The present work describes for the first time an endolysin encoded in a P. larvae phage that presents high potential to integrate a commercial product to control the problematic American foulbrood.

Isolation and characterization of bacteriophages for avian pathogenic E. coli strains

Aims:  To isolate and characterize bacteriophages, and to evaluate its lytic performance against avian pathogenic Escherichia coli (APEC) strains with high patterns of antibiotic resistance, in order to select phages for a therapeutic product to treat colibacillosis in chickens.

Complete genome sequence of the broad-host-range Paenibacillus larvae phage phiIBB_Pl23

ABSTRACT Paenibacillus larvae is a Gram-positive bacterium that causes American foulbrood, an important disease in apiculture. We report the first complete genome sequence of a P. larvae phage, phiIBB_Pl23, isolated from a hive in northern Portugal. This phage belongs to the family Siphoviridae.

The influence of the mode of administration in the dissemination of three coliphages in chickens

Escherichia coli can cause severe respiratory and systemic infections in chickens, and it is often associated with significant economic losses in the poultry industry. Bacteriophages (phages) have been shown to be potential alternatives to the antibiotics in the treatment of bacterial infections. To accomplish that, phage particles must be able to reach and remain active in the infected organs. The present work aims at evaluating the effect of the route of administration and the dosage in the dissemination of 3 coliphages in the chickens organs. In vivo trials were conducted by infecting chickens orally, spray, and i.m. with 10(6), 10(7), and 10(8) plaque-forming units/mL suspensions of 3 lytic phages: phi F78E (Myoviridae), phi F258E (Siphoviridae), and phi F61E (Myoviridae). Birds were killed 3, 10, and 24 h after challenge and the phage titer was measured in lungs and air sacs membranes, liver, duodenum, and spleen. When administered by spray, the 3 phages reached the respiratory tract within 3 h. Oral administration also allowed all phages to be recovered in lungs, but only phi F78E was recovered from the duodenum, the liver, and the spleen. These differences can be explained by the possible replication of phi F78E in commensal E. coli strains present in the chicken gut, thus leading to a higher concentration of this phage in the intestines that resulted in systemic circulation of phage with consequent phage in organs. When phages were administered i.m., they were found in all of the collected organs. Despite this better response, i.m. administration is a nonpracticable way of protecting a large number of birds in a poultry unit. In general, the results suggest that oral administration and spray allowed phages to reach and to remain active in the respiratory tract and can, therefore, be considered promising administration routes to treat respiratory E. coli infections in the poultry industry.

Bacteriophages and their derivatives for the treatment and control of food-producing animal infections

Abstract Nowadays, the world is facing an increasing emergence of antibiotic resistant bacteria. Simultaneously, the banning of some existing antibiotics and the lack of development of new antimicrobials have created an urgent need to find new alternatives against animal infections. Bacteriophages (phages) are naturally occurring predators of bacteria, ubiquitous in the environment, with high host specificity and harmless to animals. For these reasons, phages and their derivatives are being considered valuable antimicrobial alternatives and an opportunity to reduce the current use of antibiotics in agri-food production, increasing animal productivity and providing environmental protection. Furthermore, the possibility of combining phage genetic material with foreign genes encoding peptides of interest has enabled their use as vaccine delivery tools. In this case, besides bacterial infections, they might be used to prevent viral infections. This review explores current data regarding advances on the use of phages and phage-encoded proteins, such as endolysins, exolysins and depolymerases, either for therapeutic or prophylactic applications, in animal husbandry. The use of recombinant phage-derived particles or genetically modified phages, including phage vaccines, will also be reviewed.

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.

In vivo toxicity study of phage lysate in chickens

1. Bacteriophage (phage) crude lysate of Gram-negative bacteria often contains bacterial debris, including lipopolysaccharides found in the outer membrane of the cell wall, which are potentially toxic. 2. In this study, an in vivo evaluation of the toxicity of a suspension of three phages to control pathogenic Escherichia coli strains in poultry was performed. 3. Eighteen commercial layers, 7 weeks old, were intramuscularly injected with phage lysate (8.21 × 104 Endotoxin Units/dose). The control group was injected with sterile Luria Bertani (LB) broth. 4. Bird prostration and decrease in body weight gain and water intake per gram of body weight were observed only on the day of the inoculation in the challenged group. Over the following 6 d, no differences were observed in the chickens’ activity. 5. These results support the view that phage crude lysate carrying endotoxins are not toxic for chickens.

Characterization and genomic analyses of two newly isolated Morganella phages define distant members among Tevenvirinae and Autographivirinae subfamilies

Morganella morganii is a common but frequent neglected environmental opportunistic pathogen which can cause deadly nosocomial infections. The increased number of multidrug-resistant M. morganii isolates motivates the search for alternative and effective antibacterials. We have isolated two novel obligatorily lytic M. morganii bacteriophages (vB_MmoM_MP1, vB_MmoP_MP2) and characterized them with respect to specificity, morphology, genome organization and phylogenetic relationships. MP1’s dsDNA genome consists of 163,095 bp and encodes 271 proteins, exhibiting low DNA (<40%) and protein (<70%) homology to other members of the Tevenvirinae. Its unique property is a >10 kb chromosomal inversion that encompass the baseplate assembly and head outer capsid synthesis genes when compared to other T-even bacteriophages. MP2 has a dsDNA molecule with 39,394 bp and encodes 55 proteins, presenting significant genomic (70%) and proteomic identity (86%) but only to Morganella bacteriophage MmP1. MP1 and MP2 are then novel members of Tevenvirinae and Autographivirinae, respectively, but differ significantly from other tailed bacteriophages of these subfamilies to warrant proposing new genera. Both bacteriophages together could propagate in 23 of 27 M. morganii clinical isolates of different origin and antibiotic resistance profiles, making them suitable for further studies on a development of bacteriophage cocktail for potential therapeutic applications.

New Vibratory Device for Wrist Rehabilitation

Wrist injuries are very common in most of the population, specially bone fractures, but also other pathologies such as tendinitis and neurological diseases. When the wrist is injured, their flexion-extension and radial-ulnar deviation and pronation-supination movements of the forearm are compromised and interdict most daily activities. Therefore, it is important to recover the normal functionality of the wrist, namely, the range of motion. In this paper, a new vibratory device for wrist rehabilitation is proposed. This device aims to overcome the main problems of the existing technologies, such as their price and complexity. For this purpose, two models were developed. In this work numerical simulations were performed, as well as experimental tests on patients and on the device itself. The devices promoted an increase of range of motion of the wrist and forearm at appropriate frequencies. Thus, they constitute an important complement to the rehabilitation process of patients with wrist injuries.

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 honeys capacity to damage the bacterial cell membrane and also to its ability to penetrate the biofilm matrix, promoting and enhancing the subsequent phage infection.