脲原体属和人型支原体体外药物敏感性及其对喹诺酮类药物耐药机制分析:单中心回顾性研究

In Vitro Drug Sensitivity of Ureaplasma Species and Mycoplasma Hominis and the Mechanism of Resistance to Quinolones:A Single-center Retrospective Study

目的分析脲原体属和人型支原体体外药物敏感性特点,并分析其对喹诺酮类药物耐药的相关机制。方法回顾性总结北京协和医院2012年9月至2017年4月通过体外培养方法检测出的脲原体属和人型支原体标本,结合患者临床资料和菌种鉴定结果分析其体外药物敏感性特点。针对喹诺酮类药物耐药特点,采用普通PCR法扩增目的基因并测序,将测序结果翻译成蛋白质序列,与NCBI数据库中参考序列进行比对,检测DNA促旋酶(GyrA/GyrB)和拓扑异构酶Ⅳ(ParC/ParE)序列的突变情况。结果脲原体属混合人型支原体的体外敏感性总体低于脲原体属或人型支原体单独体外敏感性;除大环内酯类外,脲原体属对喹诺酮类、四环素类、交沙霉素、原始霉素、强力霉素的敏感性普遍低于人型支原体,但差异无统计学意义(P均>0. 05)。女性患者体内分离得到的脲原体属对阿奇霉素、红霉素、克拉霉素、氧氟沙星的体外敏感性较男性低(P均<0. 05)。微小脲原体对大部分抗菌药物的敏感性高于解脲脲原体,尤以对四环素的敏感率差异最大(98. 48%比72. 73%,差值25. 8%,P<0. 05)。在GyrA、 GyrB、 ParC和ParE的序列比对中,共发现21个突变位点,其中ParC S83L突变占96. 22%(51/53),为最主要突变位点;其余为ParC A136T突变、ParE R448K突变、GyrA L176F和ParC S83L联合突变等以及6个目前尚未报道过的突变位点,即ParC的L540F、R718W、Q767E、S789N、M828I和I831T突变。结论脲原体属和人型支原体的体外药物敏感性与感染种属有关,脲原体属的体外敏感性还与菌种、患者性别有关。对脲原体属喹诺酮类耐药的机制研究表明,单独ParC S83L突变是脲原体属对喹诺酮类耐药的主要机制,而新发现6个突变位点可能的作用机制仍有待深入研究。

Objective The aim of this study was to summarize and analyze the in vitro antimicrobial susceptibility of Ureaplasma species and Mycoplasma hominis and the mechanisms responsible for resistance to quinolones. MethodsThe clinical data of Ureaplasma species and Mycoplasma hominis detected by in vitro culture from September 2012 to April 2017 in Peking Union Medical College Hospital were retrospectively collected and analyzed;their characteristics of antimicrobial susceptibility were also analyzed combined with the information about the patients and species identification. According to the drug-resistance characteristics of quinolone, the target genes were amplified and sequenced by PCR, and the sequencing results were translated into protein sequences. The mutations in DNA gyrase(GyrA/GyrB) and topoisomerase Ⅳ(ParC/ParE) that were related to quinolones resistance were detected by comparing sequences in the NCBI database. Results In vitro sensitivity to antibiotics of Ureaplasma species mixed with Mycoplasma hominis was significantly lower than that of Ureaplasma species or Mycoplasma hominis alone. Compared to Mycoplasma hominis , Ureaplasma species was less susceptible to quinolones, tetracycline, josamycin, and primycin, except for macrolides, but the difference was not statistically significant (all P >0.05). In addition, the susceptibility of Ureaplasma species to azithromycin, erythromycin, clarithromycin, and ofloxacin in female patients was lower than that in male patients (all P <0.05). Ureaplasma parvum were more susceptible than Ureaplasma urealyticum to most antibiotics, especially tetracycline (98.48% vs. 72.73%;25.75% discrepancy, P <0.05). Moreover, twenty-one mutations from sequences of GyrA, GyrB, ParC, and ParE were determined. The mutation in ParC with S83L substitution was the most frequent, 96.22%(51/53);mutations of A136T substitution in ParC, R448K substitution in ParE, and L176F in GyrA combined with S83L in ParC were also detected. This study also found six novel mutations that have not been reported: L540F , R718W, Q767E, S789N, M828I, and I831T amino acid substitutions in ParC protein. Conclusions The in vitro antimicrobial susceptibility of Ureaplasma species or Mycoplasma hominis is associated with genus, and the in vitro sensitivity of Ureaplasma species to antibiotics is correlated with species and genders of patients. Sole S83L substitution in ParC might be the major mechanism of resistance to quinolones of Ureaplasma species, while the possible function of the six novel mutations remains further studies.