The Roles Of Staphylococcus Sciuri MecA And Staphylococcus Aureus MecA In Oxacillin Resistance And Cell Wall Biosynthesis Of Staphylococcus Sciuri

Staphylococcus aureus (S. aureus) is a versatile and dangerous pathogen in humans. In the past,β-lactam antibiotics were powerful weapons to treat infections caused by staphylococci. However, treatment of these infections had stumbled since the emergence of methicillin-resistant S. aureus (MRSA). The genetic determinant of methicillin resistance in MRSA is mecA, which encodes the low-affinity penicillin-binding protein 2A (PBP2A). PBP2A takes over the transpeptidase role of the normal penicillin-binding proteins (PBPs) in the presence of oxacillin, thus continuing the process of cell wall synthesis. The mecA gene that is carried by SCCmec vehicle is not an original gene of MRSA and speculated to be evolved from the species of Staphylococcus sciuri (S. sciuri) in staphylococcal genus. S. sciuri belongs to coagulase-negative staphylococci (CNS) and is generally considered one of the most primitive staphylococcal species. It is distantly related to S. aureus along taxonomic lines. One hundred thirty four independent and genetically diverse S. sciuri isolates were each found to carry the mecA homologue, yet most of the mecA homologues do not confer resistance to antibiotics. Further study showed the laboratory S. sciuri mutant selected by methicillin was highly resistant to methicillin duo to a point mutation in the mecA promoter. Introduction of the activated S. sciuri mecA into S. aureus led to the increase of antibiotic resistance. The imported S. sciuri mecA was involved in the process of cell wall synthesis of the host strain of S. aureus. Recently, a clinical S. sciuri isolate K3 harboring both S. sciuri mecA and S. aureus mecA was isolated and showed resistance to methicillin. K3 strain was unstable in the absence of drug selection and tended to segregate into K3w accompanied by the loss of S. aureus mecA and K3y which had the same phenotypic and genetic characters as parental strain. This result promotes us to characterize the roles of S. sciuri mecA and S. aureus mecA in theβ-lactam antibiotic resistance and cell wall synthesis. Objective: To identify biological and genetic characteristics of S. sciuri strains and give insights into the evolution of the mecA gene in the MRSA strain as well as the mechanism(s) of methicillin resistance in staphylococci; To clarify the molecular mechanisms of antibiotic resistance among the increasingly isolated multiple-drug resistant strains of S. sciuri, which would provide useful information for clinical researches. Method: K3 containing both S. sciuri mecA and S. aureus mecA was the main strain used in this thesis. Isolates representing three subspecies of S. sciuri were also included in different experiments. Homo* derivatives of K3 were obtained after antibiotic selection. Antibiotic susceptibility profiles of the strains were analyzed by E-test and population analysis profiles (PAP). PCR was used to amplify two mecA genes and prepare all the probes; SCCmec typing was determined by multiple PCR. Expression of mecA genes were studied by Northern blot and Western blot; The genetic organization of orfX region was identified by cloning and sequencing; The putative function of the newly-identified genes was revealed by comparing with the homologues in GenBank; Detection of variations of orfX and mecA genes in chromosomal location was performed by pulse field with Sma I digestion and Southern blot; Penicillin-binding proteins were identified and characterized by PBP flurographic assay; High-pressure liquid chromatography was used to analyze the compositions of cell wall. Result: K3 strain carrying two copies of mecA genes confered heterogenous resistance to oxacillin. Segregation of antibiotic-susceptible cells K3w containing one copy of S. sciuri mecA was inhibited under oxaicllin pressure. Strains K3HO, K3HW, and K3HWO which were derived from K3 strain showed homogenous resistance to oxacillin. Expression of S. aureus mecA was highly induced in K3 strain with oxacillin and expression of S. sciuri mecA was not detectable by Northern blot. Slightly increase was detected in K3 homo* derivatives. K3 belonged to SCCmecⅢB and its SCCmec was precisely integrated in the orfX region. The genetic locations of S. sciuri mecA and orfX gene varied strain by strain. Six PBPs were identified in S. sciuri strains. PBP4 was only detected in oxacillin resistant S. sciuri strains and it was the genetic product of S. sciuri rnecA and showed low-affinity to oxacillin. These observations implied that PBP4 may be responsible for the resistance in oxacillin resistant S. sciuri strains. Furthermore, PBP6 was also determined to be a major PBP in conferring oxacillin resistance since the enhancement of PBP6 in oxacillin resistant strain K1M200 and the loss of PBP6 in K3w along with the decreased MIC comparing with parental strain K3. Cross-linking degree of cell wall in oxacillin-susceptible S. sciuri strains was 27.9% for K1 strain and 29.1% for K3w strain whereas the cross-linking rate was increased to 36.6% in K3, 36.7% in strain K3HO, 37.7% in strain SS-37 and 59.8% in strain K1M200. There was no proportional relationship between cross-linking degree and MIC value. In the presence of oxacillin, S. aureus mecA was expressed dominantly in K3 strain and the product serving as transpeptidase can use the cell wall precursor of S. sciuri to synthesize the S. sciuri type of cell wall. Conclusion: S. aureus mecA was more likely to respond to antibiotic pressure and confered resistance to the S. sciuri host. Nevertheless, S. sciuri mecA was not so silent as we thought, orfX and S. sciuri mecA genes were located at hotspots for genetic recombination, which might favor the bacteria for adoption of SCCmec and donation of S. sciuri mecA. The species of S. sciuri would have more options in development of resistance toβ-lactam antibiotics depending on the special relationship with the two mecA genes. Cross-linking degree was higher in oxacillin resistant S. sciuri strains than oxacillin susceptible S. sciuri strains. S. aureus mecA and S. sciuri mecA were functionally interchangeable in the cell wall synthesis.