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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

刘亚丽 张文娟 王洁 叶莎 赵颖 王贺 窦红涛 郭莉娜 王瑶 孙宏莉 刘文静 张小江 谢秀丽 原英 杨启文 徐英春

刘亚丽, 张文娟, 王洁, 叶莎, 赵颖, 王贺, 窦红涛, 郭莉娜, 王瑶, 孙宏莉, 刘文静, 张小江, 谢秀丽, 原英, 杨启文, 徐英春. 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[J]. 协和医学杂志, 2019, 10(3): 249-256. doi: 10.3969/j.issn.1674-9081.2019.03.011
引用本文: 刘亚丽, 张文娟, 王洁, 叶莎, 赵颖, 王贺, 窦红涛, 郭莉娜, 王瑶, 孙宏莉, 刘文静, 张小江, 谢秀丽, 原英, 杨启文, 徐英春. 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[J]. 协和医学杂志, 2019, 10(3): 249-256. doi: 10.3969/j.issn.1674-9081.2019.03.011
Ya-li LIU, Wen-juan ZHANG, Jie WANG, Sha YE, Ying ZHAO, He WANG, Hong-tao DOU, Li-na GUO, Yao WANG, Hong-li SUN, Wen-jing LIU, Xiao-jiang ZHANG, Xiu-li XIE, Ying YUAN, Qi-wen YANG, Ying-chun XU. In Vitro Drug Sensitivity of Ureaplasma Species and Mycoplasma Hominis and the Mechanism of Resistance to Quinolones: A Single-center Retrospective Study[J]. Medical Journal of Peking Union Medical College Hospital, 2019, 10(3): 249-256. doi: 10.3969/j.issn.1674-9081.2019.03.011
Citation: Ya-li LIU, Wen-juan ZHANG, Jie WANG, Sha YE, Ying ZHAO, He WANG, Hong-tao DOU, Li-na GUO, Yao WANG, Hong-li SUN, Wen-jing LIU, Xiao-jiang ZHANG, Xiu-li XIE, Ying YUAN, Qi-wen YANG, Ying-chun XU. In Vitro Drug Sensitivity of Ureaplasma Species and Mycoplasma Hominis and the Mechanism of Resistance to Quinolones: A Single-center Retrospective Study[J]. Medical Journal of Peking Union Medical College Hospital, 2019, 10(3): 249-256. doi: 10.3969/j.issn.1674-9081.2019.03.011

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

doi: 10.3969/j.issn.1674-9081.2019.03.011
基金项目: 

中国医学科学院医学与健康科技创新工程重大协同创新项目 2016-I2M-1-014

详细信息
    通讯作者:

    徐英春 电话:010-69159766, E-mail:xycpumch@139.com

    刘亚丽、张文娟、王洁对本文同等贡献

    刘亚丽、张文娟、王洁对本文同等贡献

  • 中图分类号: R372;R375

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

More Information
    Corresponding author: XU Ying-chun Tel: 86-10-69159766, E-mail: xycpumch@139.com
  • 摘要:   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。
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    刘亚丽、张文娟、王洁对本文同等贡献
  • 图  1  9种抗菌药物对脲原体属和人型支原体体外抗菌活性比较

    图  2  脲原体属与人型支原体对9种抗菌药物体外敏感性比较

    表  1  喹诺酮类药物耐药相关基因的引物信息[4-5]

    引物名称 引物序列 退火温度(℃) 目的基因长度(bp)
    gyrA-1 5′-TTGCTGCTTTCGAAAACGG-3′ 50 336
    gyrA-2 5′-CTGATGGTAAAACACTTGG-3′
    gyrB-1 5′-GAAAATAGACGTGGTCG-3′ 55 1668
    gyrB-2 5′-TTCGAATATGACTGCCATC-3′
    gyrB-3 5′-CCTGGTAAATTAGCTGACTG-3′ 55 1249
    gyrB-4 5′-CTGATGGTAAAACACTTGG-3′
    parC-1 5′-ACGCAATGAGTGAATTAGG-3′ 55 309
    parC-2 5′-CACTATCATCAAAGTTTGGAC-3′
    parC-3 5′-GCTAAAACGCTACAAGAACG-3′ 55(UP)/48(UU) 1318(UP)/1324(UU)
    parC-4 5′-CAACGTTGGCATAAATTGG-3′
    parE-1 5′-ATGGGCGGAAAATTAACGC-3′ 55 313
    parE-2 5′-CTTGGATGTGACTACCATCG-3′
    UP:微小脲原体;UU:解脲脲原体
    下载: 导出CSV

    表  2  不同性别间脲原体属对9种抗菌药物体外敏感性差异比较 (%, N=661)

    抗菌药物 男性(n=71) 女性(n=590)
    敏感 中介 耐药 敏感 中介 耐药
    强力霉素 98.57 1.43 0.00 98.64 0.68 0.68
    交沙霉素 97.14 1.43 1.43 97.11 2.55 0.34
    氧氟沙星 12.86 44.29 42.86 5.93 23.05 71.02
    红霉素 60.00 30.00 10.00 38.03 44.48 17.49
    四环素 90.00 7.14 2.86 83.70 14.09 2.21
    环丙沙星 2.86 18.57 78.57 2.71 15.76 81.53
    阿奇霉素 62.86 28.57 8.57 42.69 51.02 6.29
    克拉霉素 84.29 8.57 7.14 74.83 17.52 7.65
    原始霉素 100.00 0.00 0.00 98.98 0.17 0.85
    下载: 导出CSV

    表  3  微小脲原体与解脲脲原体体外药物敏感性比较 (%)

    抗菌药物 微小脲原体(n= 66) 解脲脲原体(n=11)
    敏感 中介 耐药 敏感 中介 耐药
    强力霉素 98.48 1.52 0.00 90.91 0.00 9.09
    交沙霉素 98.48 1.52 0.00 100.0 0.00 0.00
    氧氟沙星 10.61 33.33 56.06 27.27 9.09 63.64
    红霉素 65.15 30.30 4.55 72.73 18.18 9.09
    四环素 98.48 1.52 0.00 72.73 18.18 9.09
    环丙沙星 3.03 16.67 80.30 0.00 9.09 90.91
    阿奇霉素 69.70 25.76 4.55 63.64 27.27 9.09
    克拉霉素 95.45 0.00 4.55 81.82 9.09 9.09
    原始霉素 98.48 1.52 0.00 100.00 0.00 0.00
    下载: 导出CSV

    表  4  喹诺酮类药物耐药相关突变筛查结果

    标本编号 氧氟沙星
    (μg/ml)
    环丙沙星
    (μg/ml)
    氨基酸序列改变
    GyrA ParC ParE
    微小脲原体
     支3/4/9/31/35/38/43/44/45/46/47/53/54/55/57/59/60/62/66/67/80/87/90/92/98/101;尿20/55/64/96(n=30) R(≥4) R(≥2) S83L
     支28/33/41/49/63/95/100;尿10/38
    (n=9)
    I(>1且<4) R(≥2) S83L
     支34(n=1) R(≥4) R(≥2) S83L, M105I
     支56;尿56(n=2) S(≤1) R(≥2) S83L
     支71;尿28(n=2) R(≥4) R(≥2) S83L, R718W
     支81(n=1) I(>1且<4) R(≥2) R448K
     支32(n=1) R(≥4) R(≥2) S83L, D530N, L553I, T570A, I702V, A735S, I743M, S789N, I790M, D794N, D799N, I831T
     支42(n=1) I(>1且<4) R(≥2) S83L, D530N, L553I, T570A, I702V, A735S, I743M, S789N, I790M, D794N, D799N, I831T
     支73(n=1) I(>1且<4) R(≥2) A136T, D530N, T570A, I702V, A734T, A735S, I743M, Q767E, S789N, I790M, D794N, D799N, M828I, I831T
     支76(n=1) R(≥4) R(≥2) L176F S83L, D530N, L553I, T570A, I702V, A735S, I743M, S789N, I790M, D794N, D799N, I831T
     支84/86(n=2) I(>1且<4) R(≥2)
     尿17;支88/30(n=3) S(≤1) I(>1且<2)
     支83/74/72(n=3) I(>1且<4) I(>1且<2)
     支27(n=1) S(≤1) I(>1且<2) D530N, T570A, A735S, I743M, Q767E, S789N, I790M, D794N, D799N, I831T
     支99(n=1) I(>1且<4) I(>1且<2) L540F
    解脲脲原体
     支6/39/50/91(n=4) R(≥4) R(≥2) S83L
     支94(n=1) I(>1且<4) R(≥2)
     支102/70;尿97(n=3) R(≥4) R(≥2)
     尿36/40(n=2) S(≤1) R(≥2)
     支37(n=1) S(≤1) I(>1且<2)
    A:丙氨酸Ala; R:精氨酸Arg; D:天冬氨酸Asp; C:半胱氨酸Cys; Q:谷氨酰胺Gln; E:谷氨酸Glu; H:组氨酸His; I:异亮氨酸Ile; G:甘氨酸Gly; N:天冬酰胺Asn; L:亮氨酸Leu; K:赖氨酸Lys; M:甲硫氨酸Met; F:苯丙氨酸Phe; P:脯氨酸Pro; S:丝氨酸Ser; T:苏氨酸Thr; W:色氨酸Trp; Y:酪氨酸Tyr; V:缬氨酸Val; R:耐药; I:中介; S:敏感; 下划线标注者为新发现突变
    下载: 导出CSV
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  • 收稿日期:  2017-10-10
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