penA和mtrR基因突变在体外诱导淋球菌对头孢曲松耐药作用的研究

Roles of penA and mtrR gene mutations in in vitro-induced resistance of Neisseria gonorrhoeae to ceftriaxone

目的探讨penA、mtrR基因突变在淋球菌对头孢曲松耐药中的作用。方法分别选取淋球菌标准菌株（ATCC-49226）、临床头孢曲松敏感菌株（2012．4052、2012．15361）及低敏菌株（2012．5616），体外次抑菌浓度诱导对头孢曲松耐药，提取诱导前原代菌株及诱导后子代耐药菌株的DNA，对penA和mtrR基因进行扩增与测序。结果菌株2012—5616和ATCC．49226分别在诱导至第26和28代时获得最低抑菌浓度（MIC）≥1mg／L的耐药菌株，菌株2012—4052和2012．15361分别在诱导至第22和36代时获得MIC≥0．5mg／L的耐药菌株。penA基因测序结果显示，ATCC-49226菌株诱导后出现A501T和G542S突变位点，另3株菌诱导前后penA基因均未出现新的突变位点；4株突变菌株的青霉素结合蛋白2（PBP2）基因序列型都为XⅧ，未检测到penA镶嵌状结构模式。mtrR基因测序结果显示，2012—5616和ATCC-49226菌株在诱导前后均发生A39T突变，2012-4052菌株诱导后在mtrR编码区发生A39T突变。结论penA基因中的A501T和G542S突变与mtrR基因发生的A39T突变可能在头孢曲松耐药中发挥作用。

Objective To investigate the roles of penA and mtrR gene mutations in resistance of Neisseria gonorrhoeae to ceflriaxone. Methods Standard strains of Neisseria gonorrhoeae （ATCC-49226）, clinical strains of Neisseria gonorrhoeae with high sensitivity to ceflriaxone （2012-4052 and 2012-15361） and clinical strains of Neisseria gonorrhoeae with reduced sensitivity to ceftriaxone （2012-5616）were treated with ceftriaxone at subinhibitory concentration （50% MIC）, so as to induce the resistance to ceftriaxone. DNA was extracted from the primary strains before the treatment and daughter strains resistant to ceftriaxone after the treatment, followed by the amplification and DNA sequencing of the penA and mtrR genes. Results For strains 2012-5616 and ATCC-49226, ceftriaxone-resistant strains with MIC ≥ 1 mg/L were obtained after 26 and 28 passages, respectively. For strains 2012-4052 and 2012-15361, eeftriaxone-resistant strains with MIC ≥ 0.5 mg/L were obtained after 22 and 36 passages, respectively. Sequence analysis of the penA gene revealed that A501T and G542S mutations were identified in the induced resistant ATCC- 49226 strains, but no new mutations were observed in the other 3 strains. All the 4 mutant strains showed penicillin- binding protein 2 （PBP2） of gene sequence type ⅩⅧ and no mosaic structure of the penA gene was found in the strains. Sequence analysis of the mtrR gene showed that the A39T mutation was found in the 2012-5616 and ATCC-49226 strains before and after the induction, as well as in the coding region of the mtrR gene in the induced resistant 2012-4052 strains. Conclusion The AS01T and G542S mutations in the penA gene and A39T mutation in the mtrR gene may play a role in the resistance of Neisseria gonorrhoeae to ceftriaxone.