Bacteriophages (phages) are obligate intracellular parasites of bacteria that usually kill the bacterial host. Bacteriophage therapy is a recently revived approach for treating bacterial infection that relies on the traits of the phage lytic cycle. A lot of attention has been given to phage therapy with new research being published weekly and international conferences organised every year, bringing together the academic and industrial phage communities. However, despite this huge effort and considerable scientific interest there is still a great lack of understanding on how to use phage effectively and overcome the many obstacles in the near future. One of the main triggers for such interest was the increasing evidence of antibiotic resistance among human bacterial pathogens, which were once efficiently eliminated by drugs but are now causing alarmingly high levels of morbidity and mortality. Also, bacteria when causing a disease are able to produce highly protective biofilm communities. Biofilms are major causes of impairment of wound healing and two of the most common and aggressive wound pathogens are Staphylococcus aureus (Gram-positive) and Pseudomonas aeruginosa (Gram-negative), both displaying a large repertoire of virulence factors and reduced susceptibility to antibiotics. This work reports and explores the use of phages to target both S. aureus and P. aeruginosa pathogen biofilm producers. Firstly, isolation of promising phage candidates was performed and cocktails were established. Two phages (DRA88 and phage K) formed the cocktail to target S. aureus and six phages (DL52, DL54, DL60, DL62, DL64 and DL68) formed a cocktail to target P. aeruginosa. A thorough characterisation of each of the selected phages was performed, including their range of host infectivity and their genome sequences were analysed. The phage’s ability to infect and kill planktonic cultures was successfully studied and afterwards such ability was assayed on biofilms using an in vitro static biofilm system (microtitre-plate), followed by an in vitro dynamic biofilm system (The Modified Robbins Device). Both cocktails were shown to be effective in reducing and dispersing biofilms formed by the clinical strains showing them to be promising not only to combat topical bacterial infections (related to biofilm production), but also to control biofilms produced on the surfaces of medical devices, such as catheters. Finally, the phage cocktail’s ability to treat systemic infections caused by the two pathogens was assessed in an in vivo G. mellonella infection model. In the case of the P. aeruginosa infection, although the phages were not able to fully treat the larvae, the cocktail allowed a delay of larval death, caused by the infection. For the S. aureus infection, the cocktail did not show the same trend, but most likely the high bacterial cell numbers involved in the experiment interfered with a successful study on the phage cocktail. The phage mixture may form the basis of an effective treatment for infections caused by S. aureus and P. aeruginosa biofilms.

This study examined the use of phage therapy against Pseudomonas aeruginosa strain PA14 biofilms. Part 1: 24 and 96h PA14 biofilms grown in microplates were phage treated and bacterial biomass was quantified using crystal violet staining. Part 2: 24 and 96h PA14 biofilms grown in prepared root canals of human mandibular incisors were treated with phages and intra-canal samples using paper points and round burs were taken to assess phage and bacterial counts. Part 1: Two phages (JBD4 and JBD44a) were used. Treatment with phages produced significant reduction in the mean percentage of biomass in 24h (p<0.05) and 96h (p=0.08) biofilms. Part 2: In 24 and 96h biofilms in a root canal model, no significant difference was found in colony forming units after phage treatment (p>0.05). Phage application significantly reduced the biomass of 24 and 96h PA14 biofilms grown on microplates, but did not in the extracted tooth models.

This study examined the use of phage therapy against Pseudomonas aeruginosa strain PA14 biofilms. Part 1: 24 and 96h PA14 biofilms grown in microplates were phage treated and bacterial biomass was quantified using crystal violet staining. Part 2: 24 and 96h PA14 biofilms grown in prepared root canals of human mandibular incisors were treated with phages and intra-canal samples using paper points and round burs were taken to assess phage and bacterial counts. Part 1: Two phages (JBD4 and JBD44a) were used. Treatment with phages produced significant reduction in the mean percentage of biomass in 24h (p<0.05) and 96h (p=0.08) biofilms. Part 2: In 24 and 96h biofilms in a root canal model, no significant difference was found in colony forming units after phage treatment (p>0.05). Phage application significantly reduced the biomass of 24 and 96h PA14 biofilms grown on microplates, but did not in the extracted tooth models.

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