Isolation and characterization of phage AHP-1 and its combined effect with chloramphenicol to control Aeromonas hydrophila.

To develop an alternative bio-control measure for multi-drug resistant pathogenic Aeromonas hydrophila, which causes motile Aeromonas septicemia in fish, novel virulent phage (AHP-1) was isolated from carp tissues. Morphological analysis by transmission electron microscopy revealed that AHP-1 belongs to Myoviridae family. AHP-1 displayed 81% of moderate adsorption by 25 min, and latent period of 40 min with burst size of 97 PFU mL-1 at an optimal multiplicity of infection (MOI) 0.1. AHP-1 was stable over a broad range of pH (4-11), temperature (4-50 °C), and salinity (0.1-3.5%). Both time and MOI dependent in vitro A. hydrophila growth inhibition was observed with AHP-1. AHP-1 (10 MOI) showed higher growth inhibition against A. hydrophila than chloramphenicol (5 µg mL-1), and combined treatment was more promising than individuals. Immune gene expression analysis of zebrafish upon continuous bath exposure to AHP-1 resulted significantly higher (il-6 and sod-1) or slight induction (tnf-α, il1-ß, il-10, and cxcl-8a) than controls at beginning of the phage exposure, but those lowered to basal level by day 12 post-phage exposure. It suggests no adverse immune responses have occurred for the AHP-1 dose that used, and have potential for the phage therapy. Further detailed in vivo studies are needed to confirm the protective efficacy of newly isolated AHP-1 against A. hydrophila infection.

Structure of the three N-terminal immunoglobulin domains of the highly immunogenic outer capsid protein from a T4-like bacteriophage.

The head of bacteriophage T4 is decorated with 155 copies of the highly antigenic outer capsid protein (Hoc). One Hoc molecule binds near the center of each hexameric capsomer. Hoc is dispensable for capsid assembly and has been used to display pathogenic antigens on the surface of T4. Here we report the crystal structure of a protein containing the first three of four domains of Hoc from bacteriophage RB49, a close relative of T4. The structure shows an approximately linear arrangement of the protein domains. Each of these domains has an immunoglobulin-like fold, frequently found in cell attachment molecules. In addition, we report biochemical data suggesting that Hoc can bind to Escherichia coli, supporting the hypothesis that Hoc could attach the phage capsids to bacterial surfaces and perhaps also to other organisms. The capacity for such reversible adhesion probably provides survival advantages to the bacteriophage.

PEGylation of bacteriophages increases blood circulation time and reduces T-helper type 1 immune response.

The increasing occurrence of antibiotic-resistant pathogens is of growing concern, and must be counteracted by alternative antimicrobial treatments. Bacteriophages represent the natural enemies of bacteria. However, the strong immune response following application of phages and rapid clearance from the blood stream are hurdles which need to be overcome. Towards our goal to render phages less immunogenic and prolong blood circulation time, we have chemically modified intact bacteriophages by conjugation of the non-immunogenic polymer monomethoxy-polyethylene glycol (mPEG) to virus proteins. As a proof of concept, we have used two different polyvalent and strictly virulent phages of the Myoviridae, representing typical candidates for therapeutical approaches: Felix-O1 (infects Salmonella) and A511 (infects Listeria). Loss of phage infectivity after PEGylation was found to be proportional to the degree of modification, and could be conveniently controlled by adjusting the PEG concentration. When injected into naïve mice, PEGylated phages showed a strong increase in circulation half-life, whereas challenge of immunized mice did not reveal a significant difference. Our results suggest that the prolonged half-life is due to decreased susceptibility to innate immunity as well as avoidance of cellular defence mechanisms. PEGylated viruses elicited significantly reduced levels of T-helper type 1-associated cytokine release (IFN-γ and IL-6), in both naïve and immunized mice. This is the first study demonstrating that PEGylation can increases survival of infective phage by delaying immune responses, and indicates that this approach can increase efficacy of bacteriophage therapy.