• bacteriophages
• batteriofagi
• caratterizzazione
• characterization
• coevolution
• coevoluzione
• direct evolution
• evoluzione diretta
• infezioni antibiotico-resistenti
• multidrug-resistant bacteria infections
• Pseudomonas aeruginosa

Nowadays, antimicrobial resistance (AMR) is an urgent global public health threat caused mainly by the misuse and overuse of antibiotics, which has led to the selection of multidrug-resistant (MDR) bacteria that cause infections that are not eradicable with traditional treatments.
One of the most relevant opportunistic Gram-negative pathogens is Pseudomonas aeruginosa, which is responsible for pneumonia, infections at the level of the urinary tract, wounds, burn injuries, bones and joints. Additionally, systemic and nosocomial infections, especially in immunocompromised and cystic fibrosis patients, could lead to increased morbidity and mortality. P. aeruginosa has many mechanisms of resistance to antibiotics, including the capacity to modify antimicrobials or their targets, to degrade antibiotics, efflux pumps to extrude drugs and it is capable to form biofilms that are very difficult to treat.
The threat of AMR revitalized research in alternative solutions to antibiotics and phage therapy is a promising therapeutic strategy based on the use of bacteriophages, that are naturally occurring viruses, that specifically infect and kill bacteria. Moreover, phage-bacterium interactions induce in both mutations in their genomes which, on the one hand will produce phages more efficient in infecting the bacterial host that are useful for clinical use, on the other hand will produce bacterial clones resistant to phage infection.
In this context, five phages were genotypically characterised: three of them, Φ2L2Pa3iii PP, Φ2L2Pa3iii PA and Φ3CPa1i, were before isolated from phages mixture while the others two phages, Φ2EPa3i and Φ3BPa3i, were selected even for phenotypical analysis on their host, Pa3 host, carrying out One-Step Growth Curves to evaluate phages latent period and burst size, Temperature and pH Stability Assays to evaluate phages stability at different temperatures (from 4 to 70°C) and pH values (from 3 to 12).
Then, a phage protocol adaptation of 15 rounds of 5 days was used to increase phages lytic activity of Φ2EPa3i and Φ3BPa3i on a clinical isolate of P. aeruginosa, Pa3.Gr.Pv. strain. Subsequently, the lytic activity of 24 hours of evolved phages from two rounds (round 10 and round 15) were tested on planktonic cells of Pa3.Gr.Pv. to assess the increase in the duration of bacterial growth inhibition and decrease of levels of bacterial load compared to the growth control and to the wild-type phages. Then, mutant phages Φ2EPa3i and Φ3BPa3i obtained from round 10 and 15 of evolution were genotypically analysed to evaluate the presence of mutations in genes encoding for proteins involved in the bacterial infection.
Moreover, phage Φ2EPa3i, having lower lytic activity than phage Φ3BPa3i, was selected to improve its infectious efficiency through the Coevolution Assay of 10 rounds of 24 hours against Pa3.Gr.Pv. strain, that simulate in vitro the in vivo phage administration in an infected patient and that allowed to study the coevolution dynamics phage-bacterium also observable in nature. The lytic activity of 24 hours and genotypic analysis, to look for mutations advantageous for phage lytic activity, were performed also for the three replicates of coevolved phages of round 10.
Subsequently, the Efficiency of Plating of Φ2EPa3i wild-type and its mutants obtained from the Evolution and Coevolution assays were performed against a panel of 12 P. aeruginosa clinical isolates to evaluate the lytic host-spectrum and the eventual presence of differences in the host-spectrum between mutants and the wild-type phage.
Results showed the lytic nature for phages Φ2EPa3i, Φ3BPa3i and Φ2L2Pa3iii PP and temperate nature for Φ2L2Pa3iii PA and Φ3CPa1i. All the five phages showed the absence of virulence factors and antibiotic resistance genes. Phenotypic analysis showed that Φ2EPa3i resulted to have a latent period of 5 minutes and a burst from 10^5 to 10^7 from 5 to 40 minutes and to be stable up to 60°C with a significant drop of 1log10 at 70°C but it was stable at each pH value tested. Phage Φ3BPa3i had a latent period of 10 minutes and the burst from 10^7 to 10^10 from 10 to 70 minutes and resulted stable up to 60°C and from pH 4 to 12.
About lytic activity, the mutant of round 15 for Φ2EPa3i and the mutant of round 10 for Φ3BPa3i of Evolution Assay displayed the higher lytic activity compared to other mutants and wild-type phages against the P. aeruginosa clinical isolate. Then, evolved phages mutations analysis showed the presence of mutations in genes encoding for structural proteins involved in phage adsorption as tail protein for Φ2EPa3i and tail fiber protein and tail sheath for Φ3BPa3i.
Regarding Coevolution Assay results, since no zero reduction in phage or bacterial loads has been observed in any of the three replicates of Coevolution Assay, this suggested that coexistence occurred, despite the short-term experiment of 10 days.
Regarding lytic activity, all three coevolved phage replicates of round 10 showed a longer lasting bacterial growth inhibition than wild-type phage and a significant reduction of bacterial load compared to the growth control. Additionally, coevolved phages of round 10 showed 3 different mutations, of which one common to all three phage replicates, all occurring in the same sequence annotated as a tail protein.
The Efficiency of plating of Φ2EPa3i wild-type was narrow and for its mutants intermediate because, interestingly, phage mutants were active against two clinical isolates against which the wild-type was not, suggesting the presence in mutants’ genomes of important mutations in genes encoding for a tail protein involved in phage adsorption.