The aim of our investigation was to verify whether phage therapy (PT) can induce antiphage antibodies. The antiphage activity was determined in sera from 122 patients from the Phage Therapy Unit in Wroc1aw with bacterial infections before and during PT, and in sera from 30 healthy volunteers using a neutralization test. Furthermore, levels of antiphage antibodies were investigated in sera of 19 patients receiving staphylococcal phages and sera of 20 healthy volunteers using enzyme-linked immunosorbent assay. The phages were administered orally, locally, orally/locally, intrarectally, or orally/intrarectally. The rate of phage inactivation (K) estimated the level of phages' neutralization by human sera. Low K rates were found in sera of healthy volunteers (K £ 1.73). Low K rates were detected before PT (K £ 1.64). High antiphage activity of sera K > 18 was observed in 12.3% of examined patients (n = 15) treated with phages locally (n = 13) or locally/orally (n = 2) from 15 to 60 days of PT. High K rates were found in patients treated with some Staphylococcus aureus, Pseudomonas aeruginosa, and Enterococcus faecalis phages. Low K rates were observed during PT in sera of patients using phages orally (K £ 1.04). Increased inactivation of phages by sera of patients receiving PT decreased after therapy. These results suggest that the antiphage activity in patients' sera depends on the route of phage administration and phage type. The induction of antiphage activity of sera during or after PT does not exclude a favorable result of PT.

BackgroundMembers of the genus Klebsiella are among the leading microbial pathogens associated with nosocomial infection. The increased incidence of antimicrobial resistance in these species has propelled the need for alternate/combination therapeutic regimens to aid clinical treatment. Bacteriophage therapy forms one of these alternate strategies.MethodsElectron microscopy, burst size, host range, sensitivity of phage particles to temperature, chloroform, pH, and restriction digestion of phage DNA were used to characterize Klebsiella phages.Results and conclusionsOf the 32 isolated phages eight belonged to the family Myoviridae, eight to the Siphoviridae whilst the remaining 16 belonged to the Podoviridae. The host range of these phages was characterised against 254 clinical Enterobacteriaceae strains including multidrug resistant Klebsiella isolates producing extended-spectrum beta-lactamases (ESBLs). Based on their lytic potential, six of the phages were further characterised for burst size, physicochemical properties and sensitivity to restriction endonuclease digestion. In addition, five were fully sequenced. Multiple phage-encoded host resistance mechanisms were identified. The Siphoviridae phage genomes (KP16 and KP36) contained low numbers of host restriction sites similar to the strategy found in T7-like phages (KP32). In addition, phage KP36 encoded its own DNA adenine methyltransferase. The φKMV-like KP34 phage was sensitive to all endonucleases used in this study. Dam methylation of KP34 DNA was detected although this was in the absence of an identifiable phage encoded methyltransferase. The Myoviridae phages KP15 and KP27 both carried Dam and Dcm methyltransferase genes and other anti-restriction mechanisms elucidated in previous studies. No other anti-restriction mechanisms were found, e.g. atypical nucleotides (hmC or glucosyl hmC), although Myoviridae phage KP27 encodes an unknown anti-restriction mechanism that needs further investigation.

Bacteriophages (phages), discovered 100 years ago, are able to infect and destroy only bacterial cells. In the current crisis of antibiotic efficacy, phage therapy is considered as a supplementary or even alternative therapeutic approach. Evolution of multidrug-resistant and pandrug-resistant bacterial strains poses a real threat, so it is extremely important to have the possibility to isolate new phages for therapeutic purposes. Our phage laboratory and therapy center has extensive experience with phage isolation, characterization, and therapeutic application. In this article we present current progress in bacteriophages isolation and use for therapeutic purposes, our experience in this field and its practical implications for phage therapy. We attempt to summarize the state of the art: properties of phages, the methods for their isolation, criteria of phage selection for therapeutic purposes and limitations of their use. Perspectives for the use of genetically engineered phages to specifically target bacterial virulence-associated genes are also briefly presented.