The Antibacterial Activity of Novel Bacteriophages and the Emergence of Bacterial Resistance to Phage Infections: An In Vitro Study
Authors: Moda-Silva Letícia de Souza , Oliveira Viviane de Cássia , da Cruz Tatiana Areas , da Rocha Amanda Carolina Souza Delfino , Watanabe Evandro 
Published: April 2024

The emergence of bacteria resistant to bacteriophage (phage) infection may compromise the success and effectiveness of phage therapy. The aim of this study was to evaluate the in vitro antibacterial activity of five novel phages, as well as the emergence of bacterial resistance to phage infections. The antibacterial activity of lytic phages was evaluated against standard strains of Pseudomonas aeruginosa (ATCC 27853), Escherichia coli (ATCC 25927), Enterococcus faecalis (ATCC 29212) and Staphylococcus aureus (ATCC 6538). Phages were initially grown in the presence of host bacteria in an exponential growth phase, then purified and titrated. In a second exposure, 20 μL of each phage was inoculated with 106 CFU/mL of P. aeruginosa/E. coli/E. faecalis/S. aureus, separately. In a third exposure, resistant colonies were isolated, cultivated and exposed again to the phages. Bacterial colonies resistant to phage infection after the third exposure were evaluated for their susceptibility profile to different antibiotics via the diffusion disk technique. The diameters of the inhibition halos were evaluated with Image J software (version 1.54g) and the definition of the susceptibility profile to antibiotics was determined according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria. In addition, fourteen cocktails with different phages were formulated to evaluate the emergence of a bacterial resistance to phage infections. The phages exhibited specificity for P. aeruginosa and did not infect E. coli, E. faecalis and S. aureus. The presence of bacterial colonies resistant to phage infection in the three successive exposures was identified, and the bacterial resistance to phage infection was confirmed in all phages titrated at 108 PFU/mL, in four phages titrated at 1010 PFU/mL and in one phage titrated at 1013 PFU/mL. The development of a resistance to infection by phages (~108 PFU/mL) did not change the susceptibility profile of P. aeruginosa to antibiotics and, when evaluating the emergence of a resistance to infection by phage cocktails (~108 PFU/mL, ~1010 PFU/mL, ~1013 PFU/mL), bacterial resistance to phage infection was confirmed in all cocktails with phages titrated at 108 PFU/mL, in ten cocktails with phages titrated at 1010 PFU/mL and in seven cocktails with phages titrated at 1013 PFU/mL. In conclusion, the presence of resistant P. aeruginosa colonies to phage infection after successive exposures was evidenced, although some phages at title ~1010 PFU/mL and ~1013 PFU/mL were effective in inhibiting the growth of resistant colonies. The development of resistance did not change the susceptibility profile of P. aeruginosa to antibiotics. Variants of P. aeruginosa that were resistant to phage infection were isolated and their resistance to infection via the phage cocktail was demonstrated regardless of the viral titer, although some cocktails at title ~1010 PFU/mL and ~1013 PFU/mL were effective in inhibiting the growth of resistant colonies. Despite the emergence of bacterial variants resistant to phage infection, new studies involving the applicability of phages in the control of infections must be conducted.