The use of quorum-quenching phage to combat biofouling

Biofouling caused by bacterial biofilm growth is a pervasive challenge in industrial and medical settings with enormous economic and health impacts. Phages naturally lyse host bacteria, a method that can be used as an antifouling approach. In addition, quorum sensing, bacterial cell-cell communication, is known to regulate biofilm formation. Any disruption of such communication (quorum quenching) could mitigate this biofilm formation. The proposed research combines quorum quenching and phage treatment into a single entity of synthetic quorum-quenching phage, an engineered T7 phage expressing an enzyme lactonase upon lysing the host bacterial BL21 strain. Results showed that, in a mixed-species biofilm consisted of P. aeruginosa PAO1, E. coli TG1 and E. coli BL21, both T7aiiA and T7wt (wild type) inhibited biofilm formation compared to no phage control. The T7aiiA phage caused significant reduction of 74.9% and 65.9% for 4 and 8 h post-plating while T7wt showed a reduction of 23.8% and 31.7%. In addition, the antibiofilm efficacy of the T7aiiA phage is dose dependent. An increase of the concentration of host bacteria E. coli BL21 during plating, from OD 600nm 0.05 to 0.9, caused a statistically significant increase of biofilm inhibition. The antibiofilm effect T7aiiA phage is 2.5 times more effective than the quorum-quenching E. coli. The inhibition of biofilm formation by phages was also accompanied by phage multiplication, which suggests that the synthetic phage would persist in microbial communities and steadily release the quorum-quenching enzymes. The dosage of phages in the experiments also played a role. A PFU of 103 show higher values of infectivity to both single and mixed species biofilm. Experiments also considered the applicability, the specificity and stability of the genetically engineered phage. A single culture containing E. coli BL21 resulted in stable T7 phages even after 27h of experiment. Host cells were added to the system every 3 hours and phage multiplication was given by PFUs. When combined both T7wt and T7aiiA phages, PCR results from PFU samples showed an increase in T7aiiA over time. Thus, the engineered phage showed better conditions to lyse in the media than T7wt. In surface water samples, both phages were kept constant up to 15 days when media with a 20% carbon source was added to the system (at the beginning of the experiment). Better results were found (more infectivity given by number of PFUs) with glucose as a carbon source and periodic addition of host cells and a carbon source (after the carbon source and PFU number was reduced to about 50%).