金黄色葡萄球菌的耐药性及其耐氟喹诺酮机制的研究

目的了解金黄色葡萄球菌(金葡菌)的耐药性及其耐氟喹诺酮的机制。方法用纸片扩散法测定200株金葡菌对12种抗生素的耐药性，琼脂稀释法测定52株耐环丙沙星金葡菌对3种氟喹诺酮的最低抑菌浓度（MIC）。聚合酶链反应-限制性长度多态性分析及利血平逆转实验分别检测耐环丙沙星金葡菌gyrA和grlA基因突变及norA基因表达。结果耐甲氧西林金葡菌（MRSA）占34%，并对多种抗菌药物耐药，但对万古霉素均敏感。MRSA对环丙沙星的耐药率高达79.4%,且对其他氟喹诺酮交叉耐药。52株耐环丙沙星金葡菌中42株（80.8%）检出gyrA基因84位点突变，grlA基因80位点和84位点突变分别有10株（19.2%）和14株（26.9%），gyrA或双基因突变株对环丙沙星的MIC显著高于单独grlA突变株或不存在gyrA84位点突变株(P<0.01)。利血平逆转后52株耐药菌对3种氟喹诺酮的MIC显著降低(P<0.01)。结论金葡菌的耐药性日趋严重，特别是MRSA仅对万古霉素等少数抗菌药物敏感，目前氟喹诺酮不适宜治疗MRSA；临床分离金葡菌耐氟喹诺酮的机制大多涉及药物作用靶位gyrA和grlA的基因突变以及主动外排泵增强，耐药菌株grlA突变位点可能有地区差异。

A study on the resistance of Staphylococcus aureus and the mechanisms of its resistance to fluoroquinolone

OBJECTIVE: To investigate the resistance of Staphylococcus aureus(SA) and the mechanisms of its resistance to fluoroquinolones (FQ). METHODS: The susceptibility of SA (200 strains) to 12 antibiotics was detected by disc diffusion, The minimal inhibitory concentrations (MICs) of 52 strains to three FQ were determined by agar dilution method. 52 strains resistant to ciprofloxacin (MIC> or =4 mg/L) were studied for the presence of point mutations in the gyrA gene and grlA gene by polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) method and for the expression of norA gene by reserpine reverse test respectively. RESULTS: 34% of the strains were resistant to oxacillin(methicillin resistant Staphylococcus aureus, MRSA) and other antimicrobials as well, but no vancomycin resistant strain was found. The resistance rate of MRSA to ciprofloxacin was 79.4% and cross-resistance existed. It was found that 42 strains (80.8%) had a mutation at gyrA codon 84 (TCA-->TTA or GCA). Mutations at grlA codon 80 (TCC-->TAC or TTC) and codon 84 (GAA-->AAA)were observed in 10 (19.2%) and 14 strains(26.9%) respectively.Strains containing mutations in gyrA or both gyrA and grlA gene showed a higher level of ciprofloxacin resistance than those with alternation in grlA gene but with wild type gyrA or non-gyrA mutants (P < 0.01). Decreased MICs to ciprofloxacin, norfloxacin and levofloxacin in reserpine reverse test indicated the presence of norA phenotype. CONCLUSIONS: It is clear that emergence of resistant SA strains will continue to be a problem, especially in MRSA which was resistant to most of the antibiotics. Fluoroquinolones are not the choice for MRSA now. The resistance to fluoroquinolones in clinical isolates of SA are due to the mutations of the gyrA and grlA gene encoding the target enzyme of fluoroquinolones and cell membrane resistance.Mutations of grlA gene may differ in different districts.