Filamentous Pseudomonas Phage Pf4 in the Context of Therapy-Inducibility, Infectivity, Lysogenic Conversion, and Potential Application

Abstract

More than 20% of all Pseudomonas aeruginosa are infected with Pf4-related filamentous phage and although their role in virulence of P. aeruginosa strain PAO1 is well documented, its properties related to therapy are not elucidated in detail. The aim of this study was to determine how phage and antibiotic therapy induce Pf4, whether the released virions can infect other strains and how the phage influences the phenotype of new hosts. The subinhibitory concentrations of ciprofloxacin and mitomycin C increased Pf4 production for more than 50% during the first and sixth hour of exposure, respectively, while mutants appearing after infection with obligatory lytic phage at low MOI produced Pf4 more than four times after 12–24 h of treatment. This indicates that production of Pf4 is enhanced during therapy with these agents. The released virions can infect new P. aeruginosa strains, as confirmed for models UCBPP-PA14 (PA14) and LESB58, existing both episomally and in a form of a prophage, as confirmed by PCR, RFLP, and sequencing. The differences in properties of Pf4-infected, and uninfected PA14 and LESB58 strains were obvious, as infection with Pf4 significantly decreased cell autoaggregation, pyoverdine, and pyocyanin production, while significantly increased swimming motility and biofilm production in both strains. In addition, in strain PA14, Pf4 increased cell surface hydrophobicity and small colony variants’ appearance, but also decreased twitching and swarming motility. This indicates that released Pf4 during therapy can infect new strains and cause lysogenic conversion. The infection with Pf4 increased LESB58 sensitivity to ciprofloxacin, gentamicin, ceftazidime, tetracycline, and streptomycin, and PA14 to ciprofloxacin and ceftazidime. Moreover, the Pf4-infected LESB58 was re-sensitized to ceftazidime and tetracycline, with changes from resistant to intermediate resistant and sensitive, respectively. The obtained results open a new field in phage therapy—treatment with selected filamentous phages in order to re-sensitize pathogenic bacteria to certain antibiotics. However, this approach should be considered with precautions, taking into account potential lysogenic conversion.

Pf4 Phage Variant Infection Reduces Virulence-Associated Traits in Pseudomonas aeruginosa

“…Indeed, when Pf4* was added to P. aeruginosa cultures, the bacteria showed lower cytotoxicity and virulence in mice ( 24 ), as well as reduced production of the siderophore pyoverdine ( 35 ). Interestingly, a recent study shows that Pf4 phages influence many virulence factors of newly infected P. aeruginosa strains, with the exception of swimming motility and biofilm production ( 36 ). In addition, it was recently shown that the superinfection exclusion protein PfsE binds to PilC to avoid extension of the of type IV pili, hence affecting twitching motility ( 23 ).…”

“…In addition, acute infection of P. aeruginosa by the Pf4 bacteriophage inhibited the production of the virulence factor pyoverdine ( 35 , 40 ). Recently, it was shown that Pf4 phages were produced in larger amounts upon exposure to sublethal concentrations of ciprofloxacin and mitomycin C ( 36 ). Interestingly, the released Pf4 virions were able to successfully infect new strains of P. aeruginosa , establishing very complex interactions with other indigenous filamentous (pro)phages ( 36 ).…”

“…Recently, it was shown that Pf4 phages were produced in larger amounts upon exposure to sublethal concentrations of ciprofloxacin and mitomycin C ( 36 ). Interestingly, the released Pf4 virions were able to successfully infect new strains of P. aeruginosa , establishing very complex interactions with other indigenous filamentous (pro)phages ( 36 ). Infections by these phages reduced pyocyanin and pyoverdine production of lysogenic strains, suggesting that Pf4 decreased the toxicity of P. aeruginosa strains ( 36 ).…”

“…Interestingly, the released Pf4 virions were able to successfully infect new strains of P. aeruginosa , establishing very complex interactions with other indigenous filamentous (pro)phages ( 36 ). Infections by these phages reduced pyocyanin and pyoverdine production of lysogenic strains, suggesting that Pf4 decreased the toxicity of P. aeruginosa strains ( 36 ). In other bacteria, such as Ralstonia solanacearum , infection by the ϕRSS1 filamentous phage increases virulence through enhancement of expression of virulence factors encoding genes ( 41 ), while ϕRSM3 filamentous phage infection leads to a decreased virulence ( 42 ), thereby confirming the relationship between virulence and filamentous phage infection.…”

Engineered superinfective Pf phage prevents dissemination ofPseudomonas aeruginosain a mouse burn model

“…SI-Pf variants emerge spontaneously under stress conditions. This is due to mutations in the phage genome that result in excision from the bacterial chromosome with subsequent dysregulation of replication (23, 24). Along such lines, we have reported that a P. aeruginosa mutant lacking the mismatch repair enzyme MutS spontaneously produced SI-Pf at a high frequency (25).…”