Genetically engineered phages to tackle polymicrobial biofilm-related infections

The presence of structured communities of bacteria embedded in a protective extracellular matrix, known as biofilms, is a major hallmark of chronic infections, promoting infection persistence and high levels of morbidity. In the search for effective therapeutic strategies, (bacterio)phages, the viruses of bacteria, are considered promising candidates, although it is known that biofilms can limit their efficacy. In this work, the potential of phages for biofilm control was assessed, and two different strategies were implemented to improve it, namely the adaptive evolution of phages to biofilms and the engineering of phages with heterologous genes. The first step of this work consisted of an analysis of 605 published experiments using phages for in vitro biofilm control, which revealed that phages with higher burst sizes, shorter latent periods, and higher titers, lead to more pronounced biofilm reduction. Then, the published cases of phage therapy against biofilm-related infections were screened and revealed that chronic wounds and chronic lung infections are the leading infections addressed, with Pseudomonas aeruginosa and Staphylococcus aureus being the main target bacteria. For most of these cases, phage therapy resulted in wound resolution and improvement of lung infections. In this work, phage therapy was monitored in a Portuguese patient with cystic fibrosis (CF) and P. aeruginosa lung infection, revealing the potential of phages for clinical improvement, even without bacterial eradication. However, the decreased effectiveness of later phage therapy cycles highlighted that bacteria adapted to the CF lung environment pose significant challenges to phage treatments. Targeted adaptation of phage PE1 to biofilms formed by a P. aeruginosa CF isolate resulted in improved biofilm control in conditions mimicking the CF lung environment, due to increased activity against the heterogeneous biofilm population, as a consequence of mutations in tail fiber and baseplate genes. Genetic manipulation of P. aeruginosa phage PE3 with the staphylococcal lysin CHAPk-Sh3blys, resulted in reduction of P. aeruginosa and S. aureus liquid co-cultures and polymicrobial biofilms, but the effect of the phage against both strains was lower in an in vitro artificial wound model. Taken together, this work supports the use of phages in biofilm control and proposes novel strategies for improved therapeutic outcomes, while stressing the need of using experimental conditions that mimic the infection environment.