| Staphylococcus aureus, a kind of Gram-positive bacterium, which is one of themost important sources of foodborne diseases, widely exists in air, soil, seawater, andtableware. It can cause systemic nonspecific inflammation such as nausea, vomiting,evil cold, stomach cramps, pneumonia, and pericarditis pneumonia, seriously leadingto septicemia and systemic infections. Consequently, Staphylococcus aureus-inducedfood poisoning has brounght greatly potential threat to human health. Phage as a newkind of biological agent, with many adventages such as strong specificity, high safetyperformance, less resistance characteristics, can be used for biological prevention andcontrol of staphylococcus aureus.In order to explore an effective method of controlling Staphylococcus aureus,phage qdsa001was isolated and identified from sewage water with Staphylococcusaureus (ATCC6538) as host cell, and its physiological characteristics and preliminaryapplication were also studied. It laid the foundation for biological control with phage.1．Phage qdsa001was isolated, identified and purified by double-layer agar platemethod with Staphylococcus aureus (ATCC6538) as host cell. Its titer could reach to109pfu/ml after liquid, solid and PEG20000proliferation. The plaques of the phageqdsa001were round and clear. It was a strong lysis bacteriophage and good materialfor the next study.2.Transmission electron microscopic observation showed that the phageqdsa001with an iosahedral head and a180nm tail， which whole length is about220nm, belonging to the Siphoviridae family. Nucleic acid genome extraction andgenome identification displayed the genotypes of phage qdsa001is DNA.3. The biological characteristics of qdsa001indicated that the phage had a strongendurance of temperature below60℃, and sensitive to temperature above70℃.Moveover, qdsa001remained high lysis activities in a range of pH5to9and theoptimum pH value was7. The optimum MOI value of qdsa001was0.1, the latentperiod and lytic period was10min and90min, respectively. In addition, the averageburst size of phage qdsa001is32.3. 4. Phage qdsa001showed favorable inhibition effect to Staphylococcus aureusin milk. The concentration of Staphylococcus aureus could decrease from105cfu/mLto the limit of detection after adding phage qdsa00112h at37℃. Meanwhile, afteradding phage qdsa001for24h, the concentration of Staphylococcus aureus wasreduced2.5log compared with the host control at4℃，at the same time, theconcentration of Staphylococcus aureus was declined from102cfu/mL to the limit ofdetection after72h at4℃.5. In this study, sodium alginate was used as the coating material to improvemicroencapsulation of phage by extrusion. On the basis of single-factor experiments,the optimizations of microencapsulation technological parameters were sodiumalginate2.2%, calcium chloride200mmol/L, and skim milk4.0%. Under theseconditions, the encapsulation rate was about50%. Moreover the microencapsulationconditions presented in this study could effectively protect phage qdsa001fromadverse gastric condition.6. The phage qdsa001showed a wide spectrum of lytic activity against bothmethicillin-susceptible S. aureus ATCC6538and methicillin-resistant S. aureusATCC43300. Furthermore, phage qdsa001showed a strong biofilm removal effect.The biofilm bacteria of ATCC6538could be killed approximately by90%after24h.Meanwhile, the biofilm bacteria of ATCC43300could also be inhibited by77%after24h. In additon, SEM analysis visually showed that phage qdsa001destroyed thebiofilm matrix and killed bacterial cells.In conclusion, we had separated a new kind of phage, it was a strong lysisbacteriophage particularly which could lyse MRSA and had strong tolerance tophysical and chemical factors. Expecilally, the tolerance to heavily acidic conditionhad been improved significantly by microencapsulation. In addition, it had remarkableinhibition to Staphylococcus aureus in both milk and biofilm bacterial cell. |
PDF Full Text Request