Diversity And Antimicrobial Activity Against Pathogenic MRSA Of Cultivable Commensal Bacteria From Healthy People’s Skin And Nose

Background and objective Staphylococcus aureus is an important and common clinical pathogen causing various kinds of pyogenic infections and toxic diseases.Massive drug-resistant strains have emerged because of the wide use of antibiotics,such as penicillin resistant Staphylococcus aureus and methicillin resistant Staphylococcus aureu(MRSA),even vancomycin resistant Staphylococcus aureus(VRSA).Drug-resistant Staphylococcus aureus is spreading faster than the speed of research and development of new antibiotics,so drugs used for the treatment of Staphylococcus aureus become less and less.It is necessary to excavate new and effective antibacterial molecules as soon as possible.The commensal bacteria play an important role in helping host resist exotic pathogens and maintain normal physiological functions.Studies indicate that healthy people showed more abundant diversity than patients and the commensal bacteria can produce antibacterial molecules against pathogens.Human skin and upper respiratory tract are the first line of defense against foreign pathogenic microorganism invasion.Studies have reported the commensal bacteria diversity of skin and upper respiratory tract and their antibacterial molecules against Staphylococcus aureus.However,the diversity and antibacterial molecules of commensal bacteria from skin and upper respiratory tract were usually region-specific.Staphylococcus aureus mostly confined to the experimental standard strains and individual clinical isolates as test strains screening for antimicrobial molecules.This study was aimed to explore the diversity of commensal bacteria from skin and upper respiratory tract(nose)of Chinese healthy people in Shanghai,and screen for commensal bacteria with obvious antibacterial activity against pathogenic MRSA strains from different countries,infection types and species.Besides,we will identify its antibacterial genes and try to purify antibacterial molecules in order to lay a foundation of studying antibacterial mechanism and antibacterial drugs.Part 1 Diversity of Cultivable Commensal Bacteria from Healthy People’s Skin and NoseObjective: To investigate the genera and species diversity of cultivable commesal bacteria from Chinese healthy people’s skin and nose in Shanghai.Methods: From June to August,2017,18 to 22 year-old healthy people(n = 210)were swabbed on nose and skin and cultured with blood plates at Shanghai University of Medicine&Health Sciences;Species identification was performed using Matrix Assisted Laser Desorption Ionization – Time of Flight-Mass Spectrometry(MALDI-TOF-MS);Statistical analysis was performed using SPSS software and association statistics were tested using Chi-Square tests.Results: In total,2438 cultivable commesalstrains were isolated from 210 healthy people’s nose(1497 isolates)and skin(941 isolates).25 genera were identified of all isolates and Staphylococcus isolates accounted for 81.34%.The proportion of Staphylococcus isolates from skin and nose is 82.03% and 80.23% respectively.Eleven species were identified of all Staphylococcus isolates and coagulase-negative Staphylococci(CoNS)accounted for 93.95%.The CoNS from nose and skin accounted for 90.00% and 99.34% respectively.Furthermore,a variety of other species and differences between men and women were observed.Conclusion: Obvious genera and species diversity was observed on cultivable commesal bacteria from Chinese healthy people’s nose and skin in Shanghai,and CoNS was in the majority.Part 2 Antimicrobial Activity of CoNS against pathogenic MRSAObjective: To screen for healthy people’s CoNS with antibacterial activity against pathogenic MRSA from different countries,infection types and species,and mainly research on the most obvious antibacterial CoNS isolate.Methods: CoNS isolates with antibacterial activity against MRSA were screened by high throughput screening experiment;Concentrated culture supernatant of the most obvious antibacterial CoNS isolate was tested for antibacterial activity which would indicate whether the antibacterial molecules can be secreted;Mixed growth of the most obvious antibacterial CoNS isolate and a selective MRSA strain was performed to elucidate the phase of antibacterial molecules secretion;Representative MRSA strains were cultured using culture supermatant of the most obvious antibacterial CoNS isolate to further clarify the effect of antibacterial molecules;Complete genome sequencing and bioinformatics analysis of the most obvious antibacterial CoNS isolate were performed,and predicted genes for antimicrobial molecules synthesis were inactivated to identify whether the genes synthesize antimicrobial molecules;In addition,transposon mutagenesis was performed to determine genes associated with antimicrobial molecules;Using chemical method to attempt to putify antibacterial molecules in the culture supernatant.Results: Six CoNS isolates could inhibit all sources of MRSA among a total of 19 CoNS isolates with antimicrobial activity from selective 588 CoNS isolates,especially Staphylococcus epidermis N173-2 exhibited the most obvious inhibitory effect.The antimicrobial molecules of Staphylococcus epidermis N173-2 could be secreted because its concentrated culture supernatant demonstrated distinct antimicrobial activity,and Staphylococcus epidermis N173-2 began to secrete the antimicrobial molecules in the middle of logarithmic phase.The culture supernatant of Staphylococcus epidermis N173-2 could obviously inhibit the growth of healthcare-associated MRSA(HA-MRSA)and community-associated MRSA(CA-MRSA)from China and CA-MRSA from North America.The complete sequences of chromosome and two plasmids were assembled by complete genome sequencing.A phylogenetic tree was constructed based on the core genome of Staphylococcus epidermis N173-2 and 9 complete sequenced Staphylococcus epidermis from NCBI_genome,which showed that Staphylococcus epidermis N173-2 was phylogenetically most distinct.The antimicrobial biosynthesis gene cluster of Staphylococcus epidermis N173-2 was verified on plasmid 2 by prediction analysis and experimental confirmation.The antimicrobial biosynthesis gene cluster is a polyketide synthase-nonribosomal peptide synthetase(PKSs-NRPSs)assembly line.Moreover,three genes of Staphylococcus epidermis N173-2 chromosome were screened by transposon mutagenesis,which is related to antibacterial molecules,and two genes is associated with phosphotransferase system(PTS)and one gene encodes cation transporter E1-E2 family ATPase involved in copper export.The crude extract was obtained from the culture supernatant of Staphylococcus epidermis N173-2.The most effective active ingredients were obtained by extracting the crude extract with 30% methanol.Conclusion: Staphylococcus epidermis N173-2 exhibits the most obvious antimicrobial activity against pathogenic MRSA strains from different countries,infection types and species.Its antibacterial molecule can be secreted and is biosynthesized by a PKSs-NRPSs gene cluster on plasmid 2.Three genes on chromosome involved in PTS and copper export may be associated with the synthesis or modification of the antimicrobial molecule.