Characterization of a new Staphylococcus aureus Kayvirus harboring a lysin active against biofilms

Staphylococcus aureus is one of the most relevant opportunistic pathogens involved in many biofilm-associated diseases, and is a major cause of nosocomial infections, mainly due to the increasing prevalence of multidrug-resistant strains. Consequently, alternative methods to eradicate the pathogen are urgent. It has been previously shown that polyvalent staphylococcal kayviruses and their derived endolysins are excellent candidates for therapy. Here we present the characterization of a new bacteriophage: vB_SauM-LM12 (LM12). LM12 has a broad host range (>90%; 56 strains tested), and is active against several MRSA strains. The genome of LM12 is composed of a dsDNA molecule with 143,625 bp, with average GC content of 30.25% and codes for 227 Coding Sequences (CDSs). Bioinformatics analysis did not identify any gene encoding virulence factors, toxins, or antibiotic resistance determinants. Antibiofilm assays have shown that this phage significantly reduced the number of viable cells (less than one order of magnitude). Moreover, the encoded endolysin also showed activity against biofilms, with a consistent biomass reduction during prolonged periods of treatment (of about one order of magnitude). Interestingly, the endolysin was shown to be much more active against stationary-phase cells and suspended biofilm cells than against intact and scraped biofilms, suggesting that cellular aggregates protected by the biofilm matrix reduced protein activity. Both phage LM12 and its endolysin seem to have a strong antimicrobial effect and broad host range against S. aureus, suggesting their potential to treat S. aureus biofilm infections.This study was supported by Lisando GmbH and by the Portuguese Foundation for Science and Technology (FCT), under the scope of the scope of the project the Project PTDC/BBB-BSS/6471/2014 (POCI-01-0145-FEDER-016678), the strategic funding of UID/BIO/04469/2013 unit, COMPETE 2020 (POCI-01-0145-FEDER-006684), and BioTecNorte operation (NORTE-01-0145-FEDER-000004), funded by the European Regional Development Fund under the scope of Norte2020—Programa Operacional Regional do Norte. Ana Brandão and Ergun Akturk acknowledge FCT for grants SFRH/BD/133193/2017 and PD/BD/13524/2017, respectively. The authors declare that they have no competing financial interests.info:eu-repo/semantics/publishedVersio

The use of antibiotics to improve phage detection and enumeration by the double-layer agar technique

<p>Abstract</p> <p>Background</p> <p>The Double-Layer Agar (DLA) technique is extensively used in phage research to enumerate and identify phages and to isolate mutants and new phages. Many phages form large and well-defined plaques that are easily observed so that they can be enumerated when plated by the DLA technique. However, some give rise to small and turbid plaques that are very difficult to detect and count. To overcome these problems, some authors have suggested the use of dyes to improve the contrast between the plaques and the turbid host lawns. It has been reported that some antibiotics stimulate bacteria to produce phages, resulting in an increase in final titer. Thus, antibiotics might contribute to increasing plaque size in solid media.</p> <p>Results</p> <p>Antibiotics with different mechanisms of action were tested for their ability to enhance plaque morphology without suppressing phage development. Some antibiotics increased the phage plaque surface by up to 50-fold.</p> <p>Conclusion</p> <p>This work presents a modification of the DLA technique that can be used routinely in the laboratory, leading to a more accurate enumeration of phages that would be difficult or even impossible otherwise.</p

Isolation and preliminary characterization of a new bacteriophage against Sphaerotilus natans

Activated sludge process, the most commonly used system for biological wastewater treatment, is driven by a community of microorganisms that play a key role in the conversion of the organic matter and removal of nutrients from wastewater. In this artificial ecosystem, filamentous and floc-forming bacteria co-exist in a dynamic equilibrium. However, filamentous bacteria overgrowth lead to a decrease of performance of the plant by causing two well-known phenomena: bulking and/or foaming. Sphaerotilus natans is a filamentous bacterium that can cause or contribute to the malfunction of these systems by leading to bulking occurrence. Bacteriophages are regarded as possible novel treatment against the filamentous overgrowth. In this work, the isolation and preliminary characterization of a new lytic bacteriophage against S. natans are described. Results clearly indicate the potential effect of this bacteriophage for future wastewater treatment system management.Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of UID/BIO/04469 unit and COMPETE 2020 (POCI-01-0145-FEDER-006684), the project INNOVCONTROLFIL (PTDC/AAG-TEC/3331/2014), the Grant SFRH/BPD/73720/2010 to Susana Costez and BioTecNorte operation (NORTE-01-0145-FEDER-000004) funded by the European Regional Development Fund under the scope of Norte2020 - Programa Operacional Regional do Norteinfo:eu-repo/semantics/publishedVersio

Adaptability and Stability of Productive Characteristics on the Selection of Pennisetum purpureum Schum. Clones Grazed by Sheep

Elephant grass (Pennisetum purpureum Schum.) is one of the most widely used forages in Brazil, due to its high potential productivity, forage quality, palatability, vigour and persistence. Dwarf types of elephant grass are the object of selection programs for their higher leaf/stem ratio and lower grazing height, resulting in more efficient pasture management. They have high potential for ruminant production (Almeida et al. 2000), but have markedly different morphological and productive characteristics (Cunha et al. 2011) to tall varieties of the same species. Adaptability and stability analyses are selection tools which allow identification of plant responses to different environments (Cruz and Regazzi 2001) and can identify stable productive clones across a range of environments. This study evaluated the genotype x environment interaction for productive characteristics of P. purpureum clones grazed by sheep

Safety issues concerning phage therapy for veterinary applications

Phages have been proposed as natural antimicrobial agents to fight bacterial infections in animals. This is supported by several scientific evidences of the efficacy of phage therapy for veterinary applications. However, there are important safety issues that should be taken into consideration when developing a phage product for veterinary applications. For example it is of utmost importance to guarantee that phages are non‐temperate and do not encode bacterial toxins. The phage administration strategy and timing should also be adequate in order to reduce the development of phage‐resistant mutants. The present work describes the main strategies used to ensure a safe phage product for veterinary application, based on the results obtained on the scope of the European Project Phagevet‐P (Veterinary Phage Therapies as Alternatives to Antibiotics in Poultry Production FP6‐2003‐Food‐2‐A:007224)

Bacteriophage-receptor binding proteins for multiplex detection of Staphylococcus and Enterococcus in blood

Health care-associated infections (HCAIs) affect hundreds of millions of patients, representing a significant burden for public health. They are usually associated to multidrug resistant bacteria, which increases their incidence and severity. Bloodstream infections (BSIs) are among the most frequent and life-threatening HCAIs, with Enterococcus and Staphylococcus among the most common isolated pathogens. The correct and fast identification of the etiological agents is crucial for clinical decision-making, allowing to rapidly select the appropriate antimicrobial and to prevent from overuse and misuse of antibiotics and the consequent increase in antimicrobial resistance. Conventional culture methods are still the gold standard to identify these pathogens, however are time-consuming and may lead to erroneous diagnosis, which compromises an efficient treatment. (Bacterio)phage receptor binding proteins (RBPs) are the structures responsible for the high specificity conferred to phages against bacteria and thus are very attractive biorecognition elements with high potential for specific detection and identification of pathogens. Taking into account all these facts, we have designed and developed a new, fast, accurate, reliable and unskilled diagnostic method based on newly identified phage RBPs and spectrofluorometric techniques that allows the multiplex detection of Enterococcus and Staphylococcus in blood samples in less than 1.5 hours after an enrichment step.This study was supported by the Portuguese Foundation for Science and Technology (FCT) under the scope of the project “Phages‐on‐chip” PTDC/BTM‐SAL/32442/2017 (POCI‐01‐0145‐FEDER‐032442) and the strategic funding of UIDB/04469/2020 unit and BioTecNorte operation (NORTE‐01‐0145‐FEDER‐000004) funded by the European Regional Development Fund under the scope of Norte2020 − Programa Operacional Regional do Norte. Catarina Nogueira, Ana Brandão and Susana Costa were supported by the FCT grants PD/BD/143037/2018, SFRH/BD/133193/2017 and SFRH/BD/130098/2017, respectively. We would also like to acknowledge Professor Hermínia de Lencastre, Doctor Carina Almeida and Doctor Nuno Cerca for gently providing some of the strains used in this study. We acknowledge Professor Paulo Freitas for providing some of the infrastructures to perform the experiments.info:eu-repo/semantics/publishedVersio