Combining phages and antibiotic to enhance antibiofilm efficacy against an in vitro dual species wound biofilm.

Chronic wound management is extremely challenging because of the persistence of biofilm-forming pathogens, such as Pseudomonas aeruginosa and Staphylococcus aureus, which are the prevailing bacterial species that co-infect chronic wounds. Phage therapy has gained an increased interest to treat biofilm-associated infections, namely when combined with antibiotics. Here, we tested the effect of gentamicin as a co-adjuvant of phages in a dual species-biofilm wound model formed on artificial dermis. The biofilm-killing capacity of the tested treatments was significantly increased when phages were combined with gentamicin and applied multiple times as multiple dose (three doses, every 8 h). Our results suggest that gentamycin is an effective adjuvant of phage therapy particularly when applied simultaneously with phages and in three consecutive doses. The multiple and simultaneous dose treatment seems to be essential to avoid bacterial resistance development to each of the antimicrobial agents.

A Tailspike with Exopolysaccharide Depolymerase Activity from a New Providencia stuartii Phage Makes Multidrug-Resistant Bacteria Susceptible to Serum-Mediated Killing.

Providencia stuartii is emerging as a significant drug-resistant nosocomial pathogen, which encourages the search for alternative therapies. Here, we have isolated Providencia stuartii phage Stuart, a novel podovirus infecting multidrug-resistant hospital isolates of this bacterium. Phage Stuart is a proposed member of a new Autographivirinae subfamily genus, with a 41,218-bp genome, direct 345-bp repeats at virion DNA ends, and limited sequence similarity of proteins to proteins in databases. Twelve out of the 52 predicted Stuart proteins are virion components. We found one to be a tailspike with depolymerase activity. The tailspike could form a highly thermostable oligomeric ß-structure migrating close to the expected trimer in a nondenaturing gel. It appeared to be essential for the infection of three out of four P. stuartii hosts infected by phage Stuart. Moreover, it degraded the exopolysaccharide of relevant phage Stuart hosts, making the bacteria susceptible to serum killing. Prolonged exposure of a sensitive host to the tailspike did not cause the emergence of bacteria resistant to the phage or to serum killing, opposite to the prolonged exposure to the phage. This indicates that phage tail-associated depolymerases are attractive antivirulence agents that could complement the immune system in the fight with P. stuartiiIMPORTANCE The pace at which multidrug-resistant strains emerge has been alarming. P. stuartii is an infrequent but relevant drug-resistant nosocomial pathogen causing local to systemic life-threatening infections. We propose an alternative approach to fight this bacterium based on the properties of phage tailspikes with depolymerase activity that degrade the surface bacterial polymers, making the bacteria susceptible to the immune system. Unlike antibiotics, phage tailspikes have narrow and specific substrate spectra, and by acting as antivirulent but not bactericidal agents they do not cause the selection of resistant bacteria.

Synergistic Action of Phage and Antibiotics: Parameters to Enhance the Killing Efficacy Against Mono and Dual-Species Biofilms.

Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and are commonly found in polymicrobial biofilm-associated diseases, namely chronic wounds. Their co-existence in a biofilm contributes to an increased tolerance of the biofilm to antibiotics. Combined treatments of bacteriophages and antibiotics have shown a promising antibiofilm activity, due to the profound differences in their mechanisms of action. In this study, 48 h old mono and dual-species biofilms were treated with a newly isolated P. aeruginosa infecting phage (EPA1) and seven different antibiotics (gentamicin, kanamycin, tetracycline, chloramphenicol, erythromycin, ciprofloxacin, and meropenem), alone and in simultaneous or sequential combinations. The therapeutic efficacy of the tested antimicrobials was determined. Phage or antibiotics alone had a modest effect in reducing biofilm bacteria. However, when applied simultaneously, a profound improvement in the killing effect was observed. Moreover, an impressive biofilm reduction (below the detection limit) was observed when gentamicin or ciprofloxacin were added sequentially after 6 h of phage treatment. The effect observed does not depend on the type of antibiotic but is influenced by its concentration. Moreover, in dual-species biofilms it was necessary to increase gentamicin concentration to obtain a similar killing effect as occurs in mono-species. Overall, combining phages with antibiotics can be synergistic in reducing the bacterial density in biofilms. However, the concentration of antibiotic and the time of antibiotic application are essential factors that need to be considered in the combined treatments.

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.

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.