Study Of The Antibiotic Resistance Of Staphylococci And The Function And Machanism Of Bacterial Polysaccharide Ligase Lcpc From Bovine Mastitis

Staphylococcus aureus is a dangerous bacterial pathogen that can cause many severe infectious diseases,including bovine mastitis,significantly threatening the health of animals and the dairy industry.Over the years,continued selective pressure by overuse or misuse of different antimicrobials has resulted in microorganisms with multi-drug resistance,such as the Methicillin-Resistant Staphylococcus aureus(MRSA).MRSA can also spread to humans directly from animals or animal products,thus,attracting more public concerns about the live-associated MRSA.Multi-drug resistant S.aureus poses a significant threat to public healthcare and animal production.It is challenging to cure bovine mastitis for two reasons:(1)the emergence of MRSA narrowed the therapeutic options;(2)S.aureus is capable of evading antimicrobial chemotherapy and host immune defense via internalization into host cells.Both antimicrobial resistance and adhesion ability of S.aureus are significantly affected by the outer cell wall architecture.To determine the antibioctic resistance of staphylococcal strains in Northwest China,144 staphylococcal strains was isolated from 200 milk samples of 53 dairy cows which were suspected to have mastitis or already had mastitis.The antibiotic resistance of β-lactams,glycopeptide,macrolides and chloramphenicol in these 144 staphylococcal strains is dermined.In this study,7.6% of these strains are resistant to teicoplanin;14.6% of these strains are resistant to cefazolin;50% of these strains are resistant to ciprofloxacin and 11.1% of these strains are resistant to chloramphenicol.Among these 144 staphylococcal strains,12.5% of these strains are resistant to two of these antibiotics and 6.9% of thsese strains are resistant to three of these antibiotics.Additionally,one Staphylococcus epidermidis which is resistant to all of these four antibiotics was found.To study the unknown genes associated with antibiotic resistance and pathogenicity of S.aureus,we created the regulator-knockout library in BA01611,which is an MRSA isolated from dairy cows by CRISPR-Cas9.In this study,we found five unknown genes related to antibiotic resistance and one unknown gene is both related to antibiotics and biofilm formation.Among these unknown genes,we focused on lcp C,which encodes the protein belonged to the number of Lyt R-Cps A-Psr family protein(LCP).Peptidoglycans(PGs)are a major component of the cell wall in S.aureus,which is heavily decorated with wall teichoic acids(WTAs)and capsular polysaccharides(CPs).The ligation of WTAs and CPs to PGs is catalyzed by LCP.However,the involvement of Lcp C in antimicrobial resistance of S.aureus and its infection to host cells remains unknown.By creating the Lcp C-knockout strains,we showed that the deficiency in Lcp C decreased the antimicrobial resistance to β-lactams and glycopeptides and impeded the binding to various epithelial cells.These changes were accompanied by the morphological changes in the bacterial cell wall.More importantly,the knockout of Lcp C significantly reduced the pathogenicity of MRSA in mice.Our results suggest that Lcp C might be an appealing target for developing a therapeutic approach against MRSA infections.In conclusion,our work suggests that it is essential for avoiding the abuse of antibiotics in animal production and might help the development of new treatment strategies for MRSA-associated infections.