The Ability of Bacteriophages to Reduce Biofilms Produced by Pseudomonas aeruginosa Isolated from Corneal Infections

<i>Pseudomonas aeruginosa</i> (<i>P. aeruginosa</i>) is a common antibiotic-resistant pathogen, posing significant public health threats worldwide. It is a major cause of ocular infections, mostly linked to contact lens wear. <i>P. aeruginosa</i> often produces biofilm during infections, and these are also associated with antibiotic resistance. Bacteriophage (phage) therapy is emerging as a promising approach for treating multidrug-resistant <i>P. aeruginosa</i>. <b>Objective</b>: This study aimed to assess the antibiofilm effects of six phages against <i>P. aeruginosa</i> biofilms isolated from patients with corneal infections. <b>Method</b>: This study examined <i>P. aeruginosa</i> strains for their ability to form biofilms using crystal violet assay. Six <i>P. aeruginosa</i> bacteriophages (DiSu1 to DiSu6) were used, which were isolated from sewage water in Melbourne, Australia. Spot tests were used to assess phage sensitivity. The effect of phages against <i>P. aeruginosa</i> strains was determined using time-kill assay and efficiency of plating. The ability of phage to inhibit biofilm formation over 24 h or reduce preformed biofilms was also studied and confirmed using confocal laser scanning microscopy with Live/Dead staining. <b>Result</b>: After 24 h of incubation, all tested <i>P. aeruginosa</i> strains formed moderate to strong biofilms. All <i>P. aeruginosa</i> strains were sensitive to at least four of the six phages. The highest level of bacterial growth inhibition in the liquid infection model was observed when phages were applied at a multiplicity of infection (MOI) of 100. Certain bacteria/phage combinations were able to inhibit biofilm formation over 24 h, with the combination of strain PA235 and phage DiSu3 producing the highest inhibition (83%) at a MOI of 100. This was followed by the combinations of PA223/DiSu3 (56%), and PA225/DiSu5 (52%). For the reduction in preformed biofilms, the best combinations were PA235 (90%), PA221 (61%), and PA213 and PA225 (57% each), all with DiSu3 after 3 h. However, exposing the biofilm with phages for over 24 h appeared to promote phage resistance as there was evidence of biofilm growth, with the only combination still showing a significant reduction being PA221/DiSu3 (58%) at MOI of 100. <b>Conclusions</b>: This study showed that the effect of phages against <i>P. aeruginosa</i> is concentration (MOI) dependent. Phages at higher MOI have the ability to disrupt, inhibit, and reduce <i>P. aeruginosa</i> biofilms. However, prolonged exposure of the biofilm with phages appeared to promote phage resistance. To enhance phage efficacy and address this form of resistance, further studies utilizing phage cocktails or a combination of phages and antibiotics is warranted.