沙眼衣原体临床分离株药物敏感性测定及耐药基因检测

Susceptibility of Clinical Isolates of Chlamydia Trachomatis to Antimicrobials and Its Resistant Gene in Vitro

目的检测本地区泌尿生殖道沙眼衣原体(CT)对四环素类、大环内酯类、喹诺酮类抗生素的体外药物敏感性,并从基因水平上探讨其耐药机制。方法将42株经细胞培养法证实有CT感染的临床株进行McCoy细胞培养,传代至感染率达90%以上,收集标本进行药敏试验。分别检测四环素耐药质粒tetM基因;大环内酯类耐药相关的23S核蛋白体RNA基因、核糖体蛋白基因L4,通过产物测序检测基因突变;限制性片段分析(RFLP)检测gyrA喹诺酮耐药决定区(QRDR)常见的基因点突变。结果药敏MIC(单位均为μg/mL)分别为红霉素0.5～2.0,克拉霉素0.008～0.032,阿奇霉素0.125～0.50,四环素0.157～0.625,多西环素0.063～0.125,米诺环素0.032～0.128,左氧氟沙星0.50～1.00,莫西沙星0.060～0.120,司帕沙星0.064～0.128,其中发现2株红霉素耐药株(MIC值2μg/mL),其23srRNA基因有C2452A,T2611C的突变(大肠杆菌序列编号),红霉素耐药株及敏感株L4基因均发现脯氨酸113(CCG)→亮氨酸(CTG)、脯氨酸156(CCC)→丙氨酸(GCC)两个位点的突变(GenBankNC000117.1),25株临床分离株中检测到tetM基因,未发现gyrA-QRDR基因中Ser83→Ile点突变。结论发现2株红霉素耐药株,未发现其它八种抗菌药的耐药株。克拉霉素显示了较高的体外抗CT活性。C2452A,T2611C的突变导致红霉素低水平耐药,L4基因的点突变可能与红霉素耐药无关。

Objective To test the activity of tetracyclines, macrolides and quinolones against of urogenital Chlamydia trachomatis isolated from clinical lately in vitro and explore the mechanism of resistance in gene molecular level. Methods Forty-two clinical strains confirmed CT infection by the cell culture method were cultured and passaged. Plasmids containing TetM associated with tetracyelines-resistant were detected by PCR amplification. After 23S ribosomal RNA and ribosomal protein IA genes amplified by RT-PCR and PCR respectively, the products were sequenced to detect mutations caused by macrolide-resistant. The common gene mutation of gyrA QRDR （Quinolone-Resistance Determining Region） associated with quinolone resistance was detected by restriction fragment analysis （RFLP）. Results The MIC of erythromycin ronged from 0.5 to 2.0mcg/mL and of clarithromycin, azithromycin, tetracycline, doxycycline, minoeyeline, levofloxaein, moxifloxaein and sparfloxacin was 0.008 - 0.032 mcg/mL, 0. 125 - 0.500mcg/mL, 0. 157 - 0.625 mcg/mL, 0. 063 - 0. 125 mcg/ mL, 0.032 - 0. 128 mcg/mL. 0.500 - 1. 000mcg/mL, 0. 060 - 0. 120mcg/mL and 0.064 - 0. 128 mcg/mL respectively. Two isolates which had resistance to erythromycin had the mutations of C2452A and T2611C （ Escherichia coli numbering） in the 23S rRNA gene. No isolates had resistance to other 8 antimierobial . The studied region of gene L4 in resistant isolates showed no difference from that of sensitive isolates. All isolates had the double mutations found： Pro Pll3（CCG→Leu L（CTG） and Pro P156 （CCC）→AIa A （GCC） published in GenBank sequence （ NC000117.1 ）. Plasmids containing TetM were detected in 25 clinical isolates of Chlamydia trachomatis and there was no one point mutation（G→T） found which caused Ser83-to-Ile change. Conclusions 2 strains resistant to erythromyein were found, and no strain was observed resistant to other 8 antimierobial. Clarithromycin showed best activities against CT in vitro. The low-level erythromyein resistance is associated with the mutations of C2452A and T2611A/Cin the 23S rRNA gene. Double mutations in the L4 gene are not responsible for erythromycin resistance.