作者投稿
专家审稿
编辑办公
邮件订阅
RSS
In Vitro Drug Sensitivity of Ureaplasma Species and Mycoplasma Hominis and the Mechanism of Resistance to Quinolones: A Single-center Retrospective Study
-
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。 Abstract: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. Methods The 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. 利益冲突 无刘亚丽、张文娟、王洁对本文同等贡献 -
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
-
[1] Jorgensen JH, Pfaller MA. ISBN: 9781555817374. Manual of clinical microbiology[S]. 11th ed. Washington DC: ASM Press, 2015. [2] Garcia LS. ISBN: 9781555815271. Clinical Microbiology Procedures Handbook[S]. 3rd ed. Washington DC: ASM Press, 2007. [3] Zeng XY, Xin N, Tong XN, et al. Prevalence and antibiotic susceptibility of Ureaplasma urealyticum and Mycoplasma hominis in Xi'an, China[J]. Eur J Clin Microbiol Infect Dis, 2016, 35:1941-1947. doi: 10.1007/s10096-016-2745-2 [4] Beeton ML, Chalker VJ, Kotecha S, et al. Comparison of full gyrA, gyrB, parC and parE gene sequences between all Ureaplasma parvum and Ureaplasma urealyticum serovars to separate true fluoroquinolones antibiotic resistance mutations from non-resistance polymorphism[J]. J Antimicrob Chemother, 2009, 64:529-538. doi: 10.1093/jac/dkp218 [5] Beeton ML, Chalker VJ, Maxwell NC, et al. Concurrent titration and determination of antibiotic resistance in Ureaplasma species with identification of novel point mutations in genes associated with resistance[J]. Antimicrob Agents Chemother, 2009, 53:2020-2027. doi: 10.1128/AAC.01349-08 [6] Lee MY, Kim MH, Lee WI, et al. Prevalence and Antibiotic Susceptibility of Mycoplasma hominis and Ureaplasma urealyticum in Pregnant Women[J]. Yonsei Med J, 2016, 57:1271-1275. doi: 10.3349/ymj.2016.57.5.1271 [7] Redelinghuys MJ, Ehlers MM, Dreyer AW, et al. Antimicrobial susceptibility patterns of Ureaplasma species and Mycoplasma hominis in pregnant women[J]. BMC Infect Dis, 2014, 14:171. doi: 10.1186/1471-2334-14-171 [8] 张睿, 叶阿里, 孔令君, 等.临床患者三种性传播疾病分子生物学检测分析[J].现代检验医学杂志, 2015, 30:107-110. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=sxyxjy201503031 [9] Song T, Ye A, Xie X, et al. Epidemiological investigation and antimicrobial susceptibility analysis of Ureaplasma species and Mycoplasma hominis in outpatients with genital manifestations[J]. J Clin Pathol, 2014, 67:817-820. doi: 10.1136/jclinpath-2014-202248 [10] Piccinelli G, Gargiulo F, Biscaro V, et al. Analysis of mutations in DNA gyrase and topoisomerase Ⅳ of Ureaplasma urealyticum and Ureaplasma parvum serovars resistant to fluoroquinolones[J]. Infect Genet Evol, 2017, 47:64-67. doi: 10.1016/j.meegid.2016.11.019 [11] Xiao L, Crabb DM, Duffy LB, et al. Chromosomal mutations responsible for fluoroquinolones resistance in Ureaplasma species in the United States[J]. Antimicrob Agents Chemother, 2012, 56:2780-2783. doi: 10.1128/AAC.06342-11 [12] Bébéar CM, Renaudin H, Charron A, et al. DNA gyrase and topoisomerase Ⅳ mutations in clinical isolates of Ureaplasma spp. and Mycoplasma hominis resistant to fluoroquinolones[J]. Antimicrob Agents Chemother, 2003, 47:3323-3325. doi: 10.1128/AAC.47.10.3323-3325.2003 [13] Duffy L, Glass J, Hall G, et al. Fluoroquinolones resistance in Ureaplasma parvum in the United States[J]. J Clin Microbiol, 2006, 44:1590-1591. doi: 10.1128/JCM.44.4.1590-1591.2006 [14] Song J, Qiao Y, Kong Y, et al.Frequent topoisomerase Ⅳ mutations associated with fluoroquinolones resistance in Ureaplasma species[J]. J Med Microbiol, 2015, 64:1315-1320. doi: 10.1099/jmm.0.000153 [15] Fernández J, Karau MJ, Cunningham SA, et al.Antimi-crobial Susceptibility and Clonality of Clinical Ureaplasma Isolates in the United States[J]. Antimicrob Agents Chemother, 2016, 60:4793-4798. doi: 10.1128/AAC.00671-16 [16] Kamiya Y, Shimada Y, Ito S, et al. Analysis of the quinolones-resistance determining region of the gyrA gene and the analogous region of the parC gene in Ureaplasma parvum and Ureaplasma urealyticum detected in first-void urine of men with non-gonococcal urethritis[J]. J Antimicrob Chemother, 2013, 68:480-482. doi: 10.1093/jac/dks417 [17] Kawai Y, Nakura Y, Wakimoto T, et al. In vitro activity of five quinolones and analysis of the quinolones resistance-determining regions of gyrA, gyrB, parC, and parE in Ureaplasma parvum and Ureaplasma urealyticum clinical isolates from perinatal patients in Japan[J]. Antimicrob Agents Chemother, 2015, 59:2358-2364. doi: 10.1128/AAC.04262-14 [18] Schneider SC, Tinguely R, Droz S, et al. Antibiotic susceptibility and sequence type distribution of Ureaplasma species isolated from genital samples in Switzerland[J]. Antimicrob Agents Chemother, 2015, 59:6026-6031. doi: 10.1128/AAC.00895-15 [19] 朱小飞, 彭红新, 李岷.喹诺酮抗性决定区域位点突变诱导解脲脲原体耐喹诺酮类药物的系统评价[J].临床检验杂志, 2014, 32:46-48. http://www.wanfangdata.com.cn/details/detail.do?_type=perio&id=lcjyzz201501013 [20] Jin H, Qi C, Zou Y, et al. Biochanin A partially restores the activity of ofloxacin and ciprofloxacin against topoisomerase IV mutation-associated fluoroquinolones-resistant Ureaplasma species[J]. J Med Microbiol, 2017, 66:1545-1553. doi: 10.1099/jmm.0.000598 [21] Robicsek AJ. Strahilevitz GA, Jacoby M, et al. Fluoroquinolones-modifying enzyme:a new adaptation of a common aminoglycoside acetyltransferase[J]. Nat Med, 2006, 12:83-88. doi: 10.1038/nm1347 -
点击查看大图
图(2) / 表(4)
计量
- 文章访问数: 414
- HTML全文浏览量: 71
- PDF下载量: 12
- 被引次数: 0
