Effects Of LysP45 And Zinc Oxide Nanoparticles On Methicillin-resistant Staphylococcus Aureus

Staphylococcus aureus is one of the most important pathogens which cause various infectious diseases in animals and humans, such asbovine mastitis and endometritis, which was main cause of breeding difficulty. The emergence of methicillin-resistant staphylococcus aureus(MRSA) can lead to therapeutic failure and makes oxacillin-resistant staphylococcal infections life-threatening. New therapeutic methods to deal with MRSA are required.Bacteriophages are one type of viruses, using bacteria as the hosts. Bacteriophage could kill bacteria through lysins-mediated cell wall lysis in the lytical growth phase. Thus, both bacteriophage and lysins have a great potential in treatment of infections by MRSA. In addition, zinc oxide has an antimicrobial effect on staphylococcus aureus. Miniaturization of zinc oxide nanoparticles further improves its antimicrobial effect because of its unique physical and chemical properties. Furthermore, nanoparticles are a new kind of enzyme immobilization carriers.Theaimsofthisworkwere:(1)toisolateandscreen a bacteriophage targeting staphylococcus aureus;(2)to express and purify the lysin protein;and(3) to combine the lysin and nano zinc oxide. Itwashopedthattheresultsfromthesethree studies would lead to development of new method to kill MRSA.Firstly, a bacteriophage targeting staphylococcus aureus was isolated, purified and identified. This lytic bacteriophage, P45, had a broad bactericidal spectrum and a strong lysis activity. Other biological characteristics were also determined, such as the optimal multiplicity of infection, one step growth curve and morphological observation. The phage P45 had an isometrically hexagonal head of 50 ± 5 nm in diameter and a contractile tail of approximately 150 ± 5 nm. These morphological characteristics suggested that the phage is a member of the family Myoviridae. The phage titer was the highest when the MOI was0.01 and the concentration of the phage reached 9.9×108 PFU/ml. It showed an eclipse phase of about 20 min and a synthetic phase of 90 min.Secondly, the gene encoding the lysin was cloned from P45 genome and the lysin protein(LysP45) was expressed and purified in vitro. The optimum action conditions, bactericidal activities and bactericidal spectrum were determined. The lysin gene of LysP45 was 1488 bplong and amplified from the genome of phage P45. The pH and temperature for the optimal activity of Lys P45 were 7.0 and 37℃, respectively. The lysin LysP45 displayed a broader lytic spectrum than that of phage P45. Phage P45 was lytic against 12 strains(including 11 S.aureus strains and 1 S. haemolyticus strain) and lysin LysP45 was able to lysis all staphylococcal strains used in our study. The phage P45 and LysP45-mediated bactericidal activities were highly specific to staphylococci, but not to other species, such as Escherichia coli, Lactobacilli and Salmonella bacteria.Thirdly, in order to increase the enzyme activities and stability of lysin LysP45, effects of nano silica, nano titanium dioxide and nano zinc oxide on the activity and stability of LysP45 were determined. Nano zinc oxide significantly inhibited the growth of S. aureus SA45 and the minimal inhibitory concentration was 0.08 mg/mL. In addition, the diameter of inhibition zones of LysP45 increased by 3.1 mm with the addition of nano zinc oxide. Furthermore, we assayed the stability of LysP45, which was stored at 4℃ over a period of 20 days, in the presence or absence of nano zinc oxide. The enzyme in the presence of nano zinc oxide retained more than 80% bactericidal activities against S. aureus SA45 after 20 days. However,the lystic activity of LysP45 in the absence of nano zinc oxide decreased by 50% during the first 10 days, and the activity was totally lost after 18 days.In summary, we obtained lysin LysP45 with a broad bactericidal spectrum and strong bactericidal activities. LysP45 showed a stronger lystic activity to methicillin-resistant staphylococcal strains and a better stability in the presence of zinc oxide nanoparticles.Therefore, nano zinc oxide could be a great adjuvant for phage lysins in treatment of methicillin-resistant staphylococcal infections.