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目的研究结核分枝杆菌(Mycobacterium tuberculosis,MTB)对左氧氟沙星(levofloxacin,LVF)与莫西沙星(moxifloxacin,MXF)的交叉耐药性,分析gyrA和gyrB基因突变位点分布及突变位点与耐药水平的关系。方法采用改良罗氏培养基检测MTB标准菌株H37Rv、66株LVF耐药和55株LVF敏感的MTB临床分离菌株LVF和MXF的最低抑菌浓度(minimal inhibition concentra-tion,MIC)。通过PCR直接测序法测定gyrA和gyrB耐药基因片段。结果 MXF的MIC比LVF低2~4倍,MXF抗MTB菌株的活性是LVF的2~4倍。MTB标准菌株H37Rv未见gyrA和gyrB基因突变。66株LVF耐药和55株LVF敏感菌株均存在gyrA AGC95ACC(Ser→Thr)突变;55株LVF敏感菌株gyrA和gyrB基因未见其他突变。66株LVF耐药菌株中,40株(60.6%)gyrAGAC94(AAC或GGC或GCC或CAC或TAC)(Asp→Asn或Gly或Ala或His或Tyr)突变;19株(28.8%)gyrA GCG90GTG(Ala→Val)和1株(1.5%)gyrA GCG90AAG(Ala→Lys)(未见报导)双碱基突变;3株(4.6%)gyrA TCG91CCG(Ser→Pro)突变;1株(1.5%)gyrB GAC500AAC(Asp→Asn)突变;2株(3.0%)呈gyrA GAC94(AAC或GCC)(Asp→Asn或Ala)与gyrB GGG551AGG(Gly→Arg)(未见报导)双位点突变。gyrA GAC94(AAC或GGC)(Asp→Asn或Gly)突变引起FQs药物较高水平耐药;GAC94GCC(Asp→Ala)和GCG90GTG(Ala→Val)突变引起FQs药物较低水平耐药。结论 LVF与MXF之间存在交叉耐药,MXF MIC随LVF MIC增高而增高,但耐药水平不同。GyrA基因突变位点可能与耐药水平有关,有可能根据基因突变的位点分析耐药水平。
Objective To study the cross resistance of Mycobacterium tuberculosis (MTB) to levofloxacin (LVF) and moxifloxacin (MXF), and to analyze the distribution of gyrA and gyrB gene mutation sites, Horizontal relationship. Methods Minimal inhibitory concentration (MIC) of MTB standard strains H37Rv, 66 strains of LVF and 55 LVF sensitive MTB isolates of clinical isolates LVF and MXF were detected by modified Roche media. The gyrA and gyrB resistance gene fragments were determined by PCR direct sequencing. As a result, the MIC of MXF was 2 to 4 times lower than that of LVF, and the activity of MXF anti-MTB strain was 2 to 4 times that of LVF. No mutation of gyrA and gyrB gene was found in MTB standard strain H37Rv. There were gyrA AGC95ACC (Ser → Thr) mutations in 66 strains of LVF-resistant and 55 strains of LVF-sensitive strains. No other mutations were found in the gyrA and gyrB genes of 55 strains of LVF-susceptible strains. Among 66 strains of LVF resistant strains, 40 (60.6%) gyrAGAC94 (AAC or GGC or GCC or CAC or TAC) (Asp → Asn or Gly or Ala or His or Tyr) were mutated; 19 (28.8%) gyrA GCG90GTG Ala → Val) and 1 (1.5%) gyrA GCG90AAG (Ala → Lys) (unobserved) double base mutation; 3 (4.6%) gyrA TCG91CCG (Ser → Pro) GAC500AAC (Asp → Asn). Two strains (3.0%) showed double-site mutation of gyrA GAC94 (AAC or GCC) (Asp → Asn or Ala) and gyrB GGG551AGG (Gly → Arg) Mutations of gyrA GAC94 (AAC or GGC) (Asp → Asn or Gly) caused higher levels of FQs resistance; mutations in GAC94GCC (Asp → Ala) and GCG90GTG (Ala → Val) resulted in lower drug resistance of FQs. Conclusion There is cross resistance between LVF and MXF. MXF MIC increases with the increase of LVF MIC, but the drug resistance is different. The site of GyrA gene mutation may be related to the drug resistance level, and it is possible to analyze the drug resistance level based on the gene mutation site.