Biofilm Control in Flow-Through Systems Using Polyvalent Phages Delivered by Peptide-Modified M13 Coliphages with Enhanced Polysaccharide Affinity

Abstract

Eradication of biofilms that may harbor pathogens in water distribution systems is an elusive goal due to limited penetration of residual disinfectants. Here, we explore the use of engineered filamentous coliphage M13 for enhanced biofilm affinity and precise delivery of lytic polyvalent phages (i.e., broad-host-range phages lysing multiple host strains after infection). To promote biofilm attachment, we modified the M13 major coat protein (pVIII) by inserting a peptide sequence with high affinity for Pseudomonas aeruginosa (P. aeruginosa) extracellular polysaccharides (commonly present on the surface of biofilms in natural and engineered systems). Additionally, we engineered the M13 tail fiber protein (pIII) to contain a peptide sequence capable of binding a specific polyvalent lytic phage. The modified M13 had 102- and 5-fold higher affinity for P. aeruginosa-dominated mixed-species biofilms than wildtype M13 and unconjugated polyvalent phage, respectively. When applied to a simulated water distribution system, the resulting phage conjugates achieved targeted phage delivery to the biofilm and were more effective than polyvalent phages alone in reducing live bacterial biomass (84 vs 34%) and biofilm surface coverage (81 vs 22%). Biofilm regrowth was also mitigated as high phage concentrations induced residual bacteria to downregulate genes associated with quorum sensing and extracellular polymeric substance secretion. Overall, we demonstrate that engineered M13 can enable more accurate delivery of polyvalent phages to biofilms in flow-through systems for enhanced biofilm control.

Synergistic action of phages and lytic proteins with antibiotics: a combination strategy to target bacteria and biofilms

“…Sobhy et al [ 30 ] isolated three novel lytic phages from sewage samples; their cocktail completely inhibited the growth of multidrug-resistant S. Enteritidis in vitro. Sun et al [ 31 ] modified phage M13 by inserting a peptide sequence with a high affinity for Pseudomonas aeruginosa to enhance biofilm attachment and remove biofilm-containing pathogens more effectively. Phage cocktails have promising applications in combating multi-drug resistant bacteria and biofilms, with a focus on the species diversity of phages and their rational modification.…”

Strategies and materials for the prevention and treatment of biofilms

“…This is a significant attempt of phages to move from the experimental to the clinical stage. Engineering bacteriophages may be promising candidates for the inhibition of pathogenic bacteria and biofilms in the future [ 109 , 110 ].…”