Isolation Of A Novel Bacteriophage Against Staphylococcus Aureus And Its Lytic Properties’ Study

Staphylococcus aureus is a widely-spread gram-positive pathogen causing endocarditis,osteomyelitis, pneumonia and food poisoning. Although it can be inhibited by a variety ofphysical and chemical methods, as well as by antibiotics, the inhibition effect of thesemethods has been weaker and weaker because it has acquired resistance to most antibioticsthese years. In this study, a novel phage against S. aureus was isolated from the sewages,using S. aureus ATCC25923and ATCC6538as the host strains. Then, the characterization,biological properties, decontamination of S. aureus in food systems and adsorption site on thehost cells were analyzed. The main research work and results were listed below.Firstly, a novel bacteriophage against S. aureus was isolated from the sewages, using S.aureus ATCC25923and ATCC6538as the host strains, and named as JS01. The transmissionelectron microscope showed that phage JS01belonged to the Siphoviridae family. Thesequencing of the partial sequence of the JS01genome indicated that JS01DNA is identicalto the DNA of several Staphyloccal phages of Siphoviridae family, with the highest identityof73.21%, thus demonstrating it as a novel Staphyloccal phage of Siphoviridae family. Thebiological properties of JS01were similar with that of most other Staphyloccal phages, but itshowed the much higher thermal stability and narrow pH stability. JS01could adsorb to thehost cells wthin5min. The latent period was40min and the burst size was86pfu/cfu. Afterchallenged with JS01, the viable cell numbers of S. aureus in culture reduced4.8log unitswhin1h.Secondly, the biocontrol of JS01against the contaminated S. aureus in three ready-to-eatfoods was analyzed. The protection effect of JS01against the contaminated S. aureus in milkwas great. When the mulitiplicity of infection (MOI) was more than10at4℃and22℃, theviable cell number of S. aureus in contaminated milk reduced to0cfu/mL. Phage JS01could also control the S. aureus contamination in hot dogs. At the MOI of1.0, the viable cellnumber of S. aureus in contaminated hot dogs reduced to0cfu/mL and continued for2-3dafter adding phage. However, the JS01could not inhibit the S. aureus contamination in yogurt,partially due to the sensitivity of JS01to the acid condition.Finally, a bacteriophage-insensitive mutant (BIM) strain called ATCC25923R wasisolated from the culture of S. aureus ATCC25923challenged with phage JS01. Thestructures of teichoic acids in ATCC25923and ATCC25923R were analysed, then theadsorption site of JS01on S. aureus was determined. The gas chromatography-combinedmass spectrometry (GC-MS) of teichoic acids of ATCC25923and ATCC25923R showed alittle difference between them. A pentose component appeared in the extracted teichoic acid ofATCC25923R, but not in ATCC25923. The influence of glucose, N-acetylglucosamine(GlcNAc), teichoic acid, peptidoglycan and capsule polysaccharide from ATCC25923andATCC25923R on the phage lysis were analysed. The results indicated that GlcNAc was thephage adsorption site on ATCC25923.Here a novel phage of high efficiency and specificity for controlling S. aureus wasisolated. Phage JS01had a good protective effect in several redy-to-eat foods. Although the BIM strain appeared after phage JS01challenge, phage JS01could reduce the contaminated S.aureus effectively. Therefore, phage JS01could be used as an efficient and safe biocontrolagent against the pathogen S. aureus.