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Abstract
Biofilm formation, one of the most important virulence factors of pathogenic bacteria, protects bacteria against desiccation, antibiotics, phages and host immune responses. However, phage-derived depolymerases show antibiofilm activity and demonstrate great potential to treat infections caused by biofilm-forming bacteria. In this study, the Escherichia coli phage vB_EcoM_ECOO78 was isolated and characterised, and we observed its ability to lyse five out of 34 tested E. coli clinical isolates. The highest phage titre was observed at a multiplicity of infection of 10-5 and a burst size of approximately 74 plaque forming units (PFU)/infection. Electron micrographs indicated that vB_EcoM_ECOO78 belongs to the family Myoviridae. The presence of increasing halos surrounding the lysis plaques formed by vB_EcoM_ECOO78 indicated that this phage may encode a depolymerase. Based on a sequencing analysis, the complete genome of vB_EcoM_ECOO78 was found to be 41,289 bp in size, with a GC content of 53.07%. Additionally, vB_EcoM_ECOO78 has 56 predicted open reading frames, 51 (91.07%) of which are assumed to be functional. A BLAST analysis indicated that ORF42 of vB_EcoM_ECOO78 (Dpo42) has low identity with other reported phage-associated depolymerases. Dpo42 was expressed and purified as a soluble protein using E. coli BL21. The biofilm formation ability of E. coli isolates and the antibiofilm activity of Dpo42 were tested by performing spot assays and using a 96-well micro-titre plate method. Dpo42 degraded the capsular polysaccharides surrounding E. coli and exhibited dose-dependent biofilm-formation prevention activity. Based on these results, Dpo42 appears to be a novel phage-derived depolymerase that represents a new potential strategy for preventing E. coli biofilm formation.
Highlights
Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is a common Gram-negative pathogen in China (Zhang et al, 2016) that causes community- and hospital-acquired infections
The structure and composition of bacterial biofilms were first described in Zobell (1943), and vary between species or strains based on biofilm age and environmental conditions (Harper et al, 2014)
Biofilms are microbial communities attached to a surface and encased in an extracellular matrix mainly composed by extracellular polymeric substances (EPSs), and by proteins, nucleic acids, lipids, water and mineral ions (Costerton, 1995; O’Toole et al, 2000; Flemming and Wingender, 2010)
Summary
Extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli is a common Gram-negative pathogen in China (Zhang et al, 2016) that causes community- and hospital-acquired infections. The number of ESBL-positive E. coli strains has significantly increased in recent years (Canton et al, 2008). One of the most important virulence factors of E. coli is the ability to form biofilms. Biofilms are microbial communities attached to a surface and encased in an extracellular matrix mainly composed by extracellular polymeric substances (EPSs), and by proteins, nucleic acids, lipids, water and mineral ions (Costerton, 1995; O’Toole et al, 2000; Flemming and Wingender, 2010). Three major EPSs have been reported, including β-1,6-N-acetyl-D-glucosamine polymer (PGA), cellulose and colanic acid, that comprise the E. coli biofilm matrix (Sharma et al, 2016). The ability of bacteria to form biofilms represents a serious medical challenge given the increasing prevalence of ESBL-positive E. coli strains. There is a need to identify and develop new therapeutic strategies to eradicate E. coli biofilms (Stewart and Costerton, 2001)
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