Isolation and Characterization of Bacteriophage SAP4 and Its Endolysin LysSAP4 targeting Staphylococcus aureus

Abstract

Staphylococcus aureus is one of the most common foodborne pathogens that cause serious threats to human and animals. Prevalence of antibiotic-resistant S. aureus, such as methicillin-resistant S. aureus (MRSA) and vancomycin-resistant S. aureus (VRSA), increases the necessity of alternative antibacterial agents those can replace traditional antibiotics. Bacteriophage and endolysin have been regarded as new bio-control agents against S. aureus. In this study, bacteriophage SAP4 targeting S. aureus was isolated and characterized. SAP4 is a prophage obtained from S. aureus clinical isolate. It belongs to Siphoviridae family with non-contractile tail and prolonged head, while most staphylococcal phages had icosahedral head. SAP4 had strong lytic activity against S. aureus and its activity was maintained over 29 h. It could infect 7 S. aureus strains including one MRSA and one additional staphylococcal strain (S. haemolyticus) among 18 staphylococcal strains tested. Genome analysis showed that phage SAP4 genome contains 43,120-bp double stranded DNA, encoding 61 open reading frames (ORFs) with no tRNA. Among 61 genes, a putative endolysin gene, which was highly homologous to N-acetyl-muramoyl-L-alanine amidase, was identified. LysSAP4, the endolysin of SAP4, contains two enzymatically active domains (EADs) such as cysteine, histidine-dependent aminohydrolase/peptidase (CHAP) and N-acetyl-muramoyl-L-alanine amidase (MurNAc-LAA, amidase_3) and one cell wall binding domain (CBD) homologous to the SH3b domain. LysSAP4 was highly active between pH 8.0 and 37℃ in broad salt concentration conditions. Moreover, lytic activity was dependent on divalent metal ions, especially Zn2+. The antimicrobial spectrum of LysSAP4 was broader than that of phage SAP4 that it could specifically kill all staphylococcal strains tested. Also the biofilm was reduced by about 70% with LysSAP4 treatment. CBD of LysSAP4 showed specific binding to staphylococcal strains. Taken together, LysSAP4 is a good candidate as a bio-control agent for S. aureus.