Characterization And Application Of Methicillin-Resistant Staphylococcus Aureus Phages

Staphylococcus aureus is a common foodborne pathogen that can contaminate food through colonisation of food processing equipment,transport surfaces and livestock farming environments,and can multiply rapidly and secrete a variety of toxins under suitable conditions,resulting in food poisoning incidents.With the emergence of methicillin-resistant Staphylococcus aureus,the control of Staphylococcus aureus is under great pressure and poses a serious threat to consumer health and food safety.Therefore,new countermeasures to control drug-resistant bacteria need to be found as soon as possible.Phage is a natural antibacterial virus that specifically lyses bacteria and has the potential to become a novel antibacterial agent due to its wide source,specificity and self-proliferation.In this study,virulence and antibiotic resistance analysis of laboratory milk-derived Staphylococcus aureus was performed to clarify strain information,and methicillin-resistant Staphylococcus aureus was used as an indicator bacterium.Lytic phages were isolated and purified from meat,aquatic products and sewage samplesand the phages with a broad lysis spectrum were screened;observed the phage morphology and the optimal infection complex.The whole genome was sequenced and analysed.Finally,the potential application for S.aureus phage was assessed by inhibition in LB broth,inhibition of biofilm formation,removal of mature biofilm and simulated environmental sterilization assays,providing new antibacterial ideas of the prevention and control of multi-drug resistant S.aureus.The main results are as follows.1.Drug resistance and virulence analysis of Staphylococcus aureusThis study investigated the antibiotic resistance and virulence of milk-derived Staphylococcus aureus.Seven isolates were tested for resistance to eight antibiotics in five major groups by micro-broth dilution.The results showed that all strains tested were resistant to at least two antibiotics,two of which were multi-resistant methicillin-resistant S.aureus.In addition,all strains tested were resistant to sulfamethoxazole,followed by 71.4% and 57.1% for chloramphenicol and ampicillin,respectively.By PCR amplification of virulence gene primers,the results showed that staphylococcal flavins(crt N),haemolysins(Hla,Hlb)and enterotoxins(Seg,Seh,Sei)were detected in 28 strains of S.aureus at a high rate of 96.4%,71.4% and 25.0%,respectively,;toxic shock syndrome toxin(TSST-1)and leukocyte-killing toxin(TSST-1)and leukocyte-killing toxin(PVL)were not detected.Therefore,milk-derived S.aureus has a high pathogenic potential.The pathogenicity of S.aureus carrying more virulence genes was investigated by subcutaneous injection in mice and the results showed that five strains could cause subcutaneous abscesses in mice,with SA028 being the most pathogenic.Therefore,S.aureus of milk origin had a high potential for treatment,and some strains have been shown to be pathogenic.2.Isolation and purification of Staphylococcus aureus phages and its biological propertiesIn this study,methicillin-resistant Staphylococcus aureus phages was isolated and purified from clams and chicken purchased from Shanghai farmers’ markets using a spotting method.vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 were used to determine their lysis ability against 30 strains of Staphylococcus aureus using a spotting method.vB＿SauH＿SPJ2 lysis rates were 93.3% and 43.3%,respectively,with a broad host spectrum.Using transmission electron microscopy,the phage vB＿SauS＿SPG4 was observed to have an elongated polyhedral head with a non-contractible tail;vB＿SauH＿SPJ2 had an icosahedral head and a contractible tail and belonged to the myxoviridae family of the order Caudovirales.The results showed that the optimal number of infection complexes for vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 were 1 and 10 respectively.vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 had incubation periods of 10 min and15 min respectively,lysis periods of 30 min and 20 min respectively,and lysis volumes of 1 and 2.The lysis amounts were 94 PFU/cell and 99 PFU/cell respectively,indicating that the phage could be amplified in large quantities within a short period of time.The results of phages stability experiments showed that vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 had good tolerance to both temperature and pH,and were tolerant to temperatures below 50 ℃.The optimum pH ranges are 4～11 and 3～12 respectively,which meet the future practical application needs of phages.3.Genome analysis of the phagesThe whole genome of the phage was sequenced and the results showed that vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 are both double-stranded linear DNA phages.vB＿SauS＿SPG4 has a genome length of 46,416 bp and encodes 68 ORFs.vB＿SauH＿SPJ2 has a whole genome size of 142,874 bp and encodes 203 ORFs.No t RNA genes,lysogenic genes or antibiotic resistance genes were found for either.A comparative paired gene analysis of the phages with the highest homology on NCBI using Easyfig v.2.2.5 software revealed that vB＿SauS＿SPG4 had the highest homology with S.aureus phage SMSAP5,but differed more from the corresponding SMSAP5 protein in DNA replication and regulatory-related proteins;phage vB＿SauH＿SPJ2encodes most proteins with high homology to the corresponding proteins of LSA2308,but some structural proteins are different from the LSA2308.Thus,both of phages are novel.The phylogenetic tree was constructed based on the sequence of the Terminase large subunit and the DNA polymerase sequence,and the analysis of the results showed that phage vB＿SauS＿SPG4 was classified in the genus Triavirus of the order Caudovirales,and phage vB＿SauH＿SPJ2 belonged to the subfamily Twortvirinae of the family Herelleviridae Silviavirus genus.4.Phages against Staphylococcus aureusThe results showed that both the phage vB＿SauS＿SPG4 and vB＿SauH＿SPJ2significantly inhibited the growth of methicillin-resistant S.aureus and MDR S.aureus at 24 h.However,vB＿SauS＿SPG4 showed a tendency to increase at around 10 h of co-culture with the bacteria.The phage vB＿SauH＿SPJ2 was able to completely inhibit the growth of the bacteria at MOI of 100 and 1000,demonstrating a strong bacterial inhibitory potential.The biofilm was quantified using crystalline violet staining to determine the ability of the phage to prevent and control the biofilm.The results showed that the phages vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 significantly inhibited the formation of biofilms of the three strains of S.aureus in a concentration-dependent manner,with the phage vB＿SauH＿SPJ2 having a better inhibitory effect.vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 had a significant removal effect on the mature biofilms of the three S.aureus isolates at different concentrations.The vB＿SauS＿SPG4and the vB＿SauH＿SPJ2 showed significant scavenging effects on the mature biofilms of the S.aureus at different concentrations and were concentration dependent.Among them,vB＿SauH＿SPJ2 reduced the biofilm of SA008,SA009 and CMCC26003 by approximately 87.67%,92.31% and 90.65%,respectively.Thus vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 had strong potential for application in biofilm prevention and control on the surface of food processing machinery.Simulating the bactericidal effect of phages against Staphylococcus aureus in a closed environment,the results showed that both phages vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 exhibited good bactericidal effects.With the increase of treatment time,the inhibition effect of phage vB＿SauS＿SPG4 and vB＿SauH＿SPJ2 treatment groups became better,up to 99.6% and 100%,so they had the potential to become new environmental bactericides.In conclusion,in this study,two methicillin-resistant Staphylococcus aureus phages were isolated and purified from clams and chicken,which were able to lyse multiple methicillin-resistant or multi-drug-resistant Staphylococcus aureus with strong inhibition ability and have the potential to become a new type of antimicrobial agent.