临床表葡菌gyrA、gyrB、grlA和grlB基因突变与其对环丙沙星耐药性的关系

Relationship of grlA,grlB,gyrA and gyrB mutations and ciprofloxacin resistance in clinical isolates of Staphylococcus epidermidis

用平皿二倍稀释法测定18株表葡菌对环丙沙星的MIC(0.125～128μg/ml)。分别针对表葡菌中gyrA、gyrB、grlA、grlB等四种基因进行引物设计与合成,提取基因组DNA,PCR扩增,对PCR产物进行克隆、鉴定并测序,用DNASIS软件分析测序结果。在表葡菌的GrlA中,只有Ser80→Phe、Ser80→Tyr或Asp84→Asn、Asp84→Ala、Asp84→Tyr的改变。在GyrA中,存在Ser84→Tyr、ser84→Phe、Glu88→Lys的改变;在任一株表葡菌中未见有gyrB或grlB的突变。在被检测的18株临床分离表葡菌中有3个分离株对环丙沙星的MICs≤0.5μg/ml,在GyrA或GrlA的决定区没有变化。对环丙沙星的MICs≥2μg/ml的15个分离株中,GyrA中有Ser84和Glu88及GrlA中的Ser80和Asp84的氨基酸改变。1株MIC为2μg/ml的GrlA中发生一个氨基酸改变,而GyrA中无变化。说明拓朴异构酶IV可能是环丙沙星作用于表葡菌的首要靶位酶。在高水平耐药株中,在GrlA和GyrA中均存在多位点的突变,提示耐药水平的高低与GrlA、GyrA位点的突变直接相关。

Agar dilution test was used to detect the MICs against ciprofloxacin of clinical isolates of S. epidermidis. Their MICs were in the range of O. 125 - 128μg/ml. The primers for gyrA, gyrB, grlA and grlB gene were designed and synthesized. Genome DNA were extracted. The fragments of gyrA, gyrB, grlA and grlB gene from 18 S. epidermidis was gotten respectively by PCR. The products were cloned and identified. Nucleotides sequences were determined and analysied by DNASIS. The results of sequencing showed that there were changes in Ser80→Phe, Ser80→Tyr or Asp84→Asn, Asp84→Ala, Asp84→Tyr of GrlA and Ser84→Tyr, Ser84→Phe, Glu88→Lys of GyrA, but there weren＇t any changes in GyrB and GrlB in any S. epidermidis isolates. In three isolates （MICs≤〈0.5μg/ml）, there were no changes in GyrA and GrlA. In other fifteen isolates （MICs ≥2μg/ml）, there were changes in Sero84, Glu-88 of GyrA and GrlA Ser-80, Asp-84 of GrlA. One isolate （MIC 2μg/ml） only had one change in GrlA and no change in GyrA. Its suggested that the primary target enzyme to S. epidermidis of ciprofloxaxin was topoisomerase IV. In high-level resistant isolates, there were changes in multi-site of GrlA and GyrA. These suggested that the resistant level had a direct correlation with mutations of GrlA and GyrA.