Characterization of newly-isolated bacteriophages targeting Staphylococcus aureus and application of phages on bioactive food packaging films

Abstract

Staphylococcus aureus is a gram-positive, nosocomial pathogen that is associated with multidrug resistance. Methicillin-resistant S. aureus and vancomycin-resistant S. aureus are of particular concern as these superbugs can no longer be treated with routine antibiotics. These concerns have led to continued efforts for discovering novel antibiotic-alternatives and bacteriophages (phages), viruses targeting specific host bacteria, have been actively studied. Phages have high potential as antibiotic alternatives for their abundance in the environment and no reported side effects. The present research isolated four S. aureus phages and characterized two phages with the highest lytic activities, HSA30 and HSA84, to evaluate their ability to control foodborne pathogens. Belonging to the Myoviridae family, HSA30 possessed a long-contractile tail with an icosahedral head. It demonstrated a broad host range against 22 out of 29 S. aureus strains tested. Moreover, it showed a strong lytic activity that was sustained for 19 hours upon divalent cation supplementation, highlighting its potential as an antibiotics alternative. Genomic analysis revealed that HSA30 genome had 140,358 bp-long nucleotides that encoded 224 coding DNA sequences and 3 tRNAs. Although no virulence factors nor drug resistance genes were identified in the genome, presence of recombinases suggest HSA30s potential to form lysogens. HSA84 was identified as a Siphoviridae and its genome encoded of cro- and cI- like repressors, implying that HSA84 may be a temperate phage. However, HSA84s ability to control 16 out of 29 tested S. aureus strains and strong host infectivity suggest its potential as an antimicrobial agent. Development of phage-immobilized food packaging materials suggested an alternative method to apply phages on food. Two virulent phages, SA11 (Myoviridae) and SA13m (Siphoviridae), were immobilized on two different cellulose media (cellulose membrane and nitrocellulose [NC] membranes). For enhanced immobilization efficiency, polyvinylamine (PVAm) was used to modify surface charge of the cellulose membrane. The amount of phages retained by the PVAm-coated cellulose membrane (PVAm membrane) was 4 log higher than that of the original membrane. Moreover, NC membrane was also evaluated as an immobilization medium and it achieved comparable immobilization efficiency as that of the PVAm membrane. Loss of infectivity was apparent in the membranes upon drying. To overcome this hurdle, Gum Arabic was coated on the bioactive packaging material. The overall result suggested an improved infectivity of the SA11-immobilized NC and PVAm membranes, and SA13m-immobilized PVAm membrane, but no bioactivity was observed in SA13m-immobilized NC membrane. When analyzed the functional properties (pH and storage stability) of the phage-immobilized membranes, SA11-immobilized NC membrane exhibited the highest stability against both pH stress and 2-day storage. It was able to achieve 3 log reduction of the host bacteria at all pH conditions. Moreover, this phage-membrane combination maintained its antimicrobial activity for 2 days upon storage at RH 85%.