Mechanism Of The Horizontal Transfer Of Antibiotic Resistance In Staphylococci From Bovine Mastitis

Staphylococcus aureus,a dangerous bacterial pathogen,causes many severe infectious diseases including bovine mastitis,significantly threatening the health of animals and the sustainability of the dairy industry.Currently,more than 70%of the antibiotics producted annually in worldwide have been used in anminal production.The overuse and abuse of antibiotics cause the rise of multi-drug resistant bacteria,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 livestock-associated MRSA.MRSA is generated when a methicillin-susceptible S.aureus?MSSA?exogenously acquires a mobile genetic element Staphylococcal cassette chromosome mec?SCCmec?.The mecA gene as the cargo in the SCCmec,encodes the penicillin binding protein 2a?PBP2a?and functions as an alternative transpeptidase in cell-wall biosynthesis.This confers staphylococci resistance to methicillin and almost all?-lactam antibiotics,and extremely narrows the therapeutic options.Consequently,to stop the horizontal transfer of mecA gene,we must understand the molecular mechanism of the transfer of methicillin resistance between staphylococci.To determine the prevalence of methicillin resistance in staphylococcal strains in Northwest China,145 staphylococcal strains were isolated from 200 milk samples of 53 dairy cows which were suspected to have mastitis or already had mastitis.In this collection,37Staphylococcus aureus strains were identified by 16S rRNA sequencing,interestingly,35 of these strains were coagulase negative.PCRs and agar dilution assays indicated that 18 strains were mecA positive.Moreover,the main type of SCCmec were type IV and type V.But in a multi-drug resistant MRSA strain BA01611,we could not identify any known ccr recmbinase gene.Using high-throughput sequencing,we have revealed the detail of the genome of MRSA BA01611.It possessed a genome and a plasmid with lengths of 2,885,865 bp and 7,440 bp,carrying 2,964 and 6 ORFs,respectively.Comparative genomics indicated this strain carried14 antibiotic resistant genes,23 adhesion factor genes and 14 genes involved in the tolerance of heavy metals,but lacked genes for toxins and virulence factors.These features conferred this strain the tolerance to many environmental stresses.We first identified and reported a new ccr recombinase gene,ccrC2,in the SCCmec of MRSA BA01611,which showed 62.6%-69.4%sequence identities to all published ccrC1 sequences.The ccr gene complex harboring the ccrC2 gene was designated a type 9 complex and the SCCmec of BA01611 was considered as a novel type designated as type XII?9C2?.These findings enriched the knowledge in the diversity of SCCmec elements.To uncover the detailed mechanism of CcrC2-mediated horizontal transfer of SCCmec,multiple molecular approaches were used.We found CcrC2 could efficiently exert the excision and integration of many SCC elements including SCCmec,?SCC and Composite SCC.Moreover,overexpression of CcrC2 efficiently removed the SCCmec from the genome and transformed methicillin-resistant S.aureus?MRSA?to methicillin-susceptible S.aureus?MSSA?.Furthermore,two nucleotide residues?G₅C₆?in the direct repeat sequence within an att site were found to be critical for excision and acquisition efficiencies.In addition,any domain truncation in the C-terminal of CcrC2 completely inactivated the enzyme,while S12,R84 and K149 in the N-terminal were necessary for the catalytic activity of CcrC2.The“cut and paste”working manner of CcrC2 could transform MRSA to MSSA effiently,but without altering the abundance of SCCmec in the staphylococcal population.To intervene the horizontal transfer of methicillin resistance,a SCCmec killer system was developed by combining the CcrC2-mediated SCCmec excision and the mecA-targeting CRISPR-Cas9 machinery.The SCCmec killer transformed MRSA to MSSA and disrupted the mecA-carrying SCCmec intermediate,thereby eliminating methicillin resistant determinant mecA gene inside a MRSA and blocking the horizontal transfer of SCCmec.In conclusion,the prevalence and the antibiotic resistance profile of the staphylococcal isolates in bovine mastitis from Northwest China have been comprehensively investigated.The genetic features of a typical MRSA strain have been demonstrated by high-throughput sequencing and comparative genomic analyses.In this study,we revealed the mechanism of CcrC2-mediated horizontal transfer of SCCmec in pathogenic S.aureus.Finally,a SCCmec killer system has been developed to eliminate the methicillin resistance.This work improved the understanding the functions of Ccr recombinases and laid the groundwork for identifying ways to stop the rapid spread of methicillin resistance.