The Prevalence Of Staphylococcus Aureus Isolated From Milk And The Pharmacodynamics Study Of Its Phage

Staphylococcus aureus（S.aureus）is one of the most common pathogens that cause mastitis in dairy cows.The abuse of antibiotics not only leads to serious multi-drug resistance,but also increases the residue of antibiotics in milk,Threatening public health safety.Bacteriophage have the potential to become an ideal substitute for antibiotics due to its stong specificity,low toxicity,and anti-bacterial property.In this study,we first used metagenomic sequencing technology to understand the microbial diversity and community structure in fresh milk in Shandong Province.S.aureus and its specific lytic phages were isolated and identified.Through epidemiological analysis of S.aureus,and the biological characteristics,genomics and pharmacodynamic analysis of phages,to explore the law of the combined antibacterial effect of phages and antibiotics,and providea theoretical basis for the study of S.aureus phages and the subsequent treatment and control.1.Metagenomic sequencing technology was used to analyze the microbial flora of 18 milk samples from cows with mastitis,subclinical mastitis and normal cows.The results showed that a total of 845574 sequences and 5465 OTUs were obtained.At the genus level,the dominant bacterial groups and their proportions are different between milk samples from different sources,Halomonas accounted for the highest proportion of 9.0%in mastitis milk samples,Staphylococcus was only detected in subclinical mastitis milk samples,accounting for 13.5%.In normal milk samples,Lactobacillus is the dominant bacteria,and accounting for 33.9%.This study provided a theoretical basis for the accurate evaluation of the microbial community in fresh milk and the prevention and treatment of cow mastitis.2.121 S.aureus strains were isolated from 418 fresh milk samples of dairy farms in different regions of Shandong Province,with the separation rate was 28.9%,and the sparation rate of methicillin-resistant S.aureus（MRSA）was 0.7%.The multi-drug resistance rate of S.aureus was 55.4%.The most predominant virulence genes were sed and sec accounted for13.2%and 8.3%.18 different ST types were identified using the multilocus sequence typing（MLST）,the predominant STs were ST50 and ST398,accounting for 13.2%and 12.4%respectively.Staphylococcal protein A gene（spa）typing identifed spa types t034（12.4%）and t189（9.9%）most frequently among S.aureus isolates.SCCmec genotyping found that MRSA carrying mec A gene was SCCmec III.This study can provide a reference for the monitoring and control of S.aureus in fresh milk in Shandong Province,and the rational selection of high-quality antibiotics.3.The lysis profile of 96 phages was determined,and found that the lysis range of 121 S.aureus was 3%-82.5%.Two phages RPCSa20091 and RPCSa20062 with high lysis rate were selected for biological characteristics and genome analysis.The two phages were observed by electron microscopy as phages with tails,and the length was about 194-294nm.The two phages were not resistant to high temperature and have certain resistance to acid and alkali.The optimal multiplicity of infection for RPCSa20091 was 0.1,and the optimal multiplicity of infection for RPCSa20062 was 0.01.Further analysis of the genome sequence revealed that the genome size of RPCSa20091 was 42829bp and the GC content was 34.64%,64 ORFs were predicted with an average length of 210 bp.The genome size of RPCSa20062 was 43274 bp with a GC content of34.09%.63 ORFs were predicted with an average length of 214 bp.This study showed that phages has potential application value in the prevention and treatment of S.aureus infection.4.In this study,we initially established the phage pharmacology system and research methods.The results showed that the MIC of ceftiofur,doxycycline and gentamicin decreased significantly after phage treatment.In the pharmacodynamics of phage and bacteria,the titer of RPCSa20091 can reached 10¹¹pfu·mL^-1,which can inhibit the number of host bacteria in a short time.The combined effect of enrofloxacin and RPCSa20091 resuced the MIC from 4μg·mL^-1to 2μg·mL^-1.indicating that enrofloxacin and phage have a synergistic effect.This study provided a theoretical basis for the application of phages and antibiotics in the treatment of cow mastitis.