淋病奈瑟氏菌中国流行株gyrA、praC和mtrR-CDE基因与耐药性形成的关系
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摘要
从1993～2001年，在我国的西安、湛江和长春三个地区共收集604株淋病奈瑟氏菌(NG)，对耐药性表型、质粒谱表型、营养型、PEN／TET耐药表型以及β-内酰胺酶表型进行了流行病学研究。用琼脂稀释法测定了其对8种抗生素的最小抑菌浓度(MIC)。通过质粒抽提和琼脂糖凝胶电泳确定NG耐药株的质粒谱，并用PCR方法鉴定了部分耐药株。使用直线相关与回归分析、t检验和χ~2检验进行耐药相关性分析。结果显示，全部604株中，产青霉素酶淋球菌(PPNG)有6株(1.0％)；116(19.2％)属于产青霉素酶-质粒介导四环素耐药淋球菌(PP／TRNG)；168株(27.8％)为质粒介导四环素耐药株(TRNG)。三种喹诺酮类药物中，环丙沙星(CIP)耐药率最低，62％(417／604)菌株对其敏感；13.2％(80／604)的NG对壮观霉素(SPE0耐药；13.4％(81／604)的分离株出现了对头孢三嗪(CRO)的敏感性降低，2.2％(13／604)表现完全耐药。耐药性最小抑菌浓度(MIC)相关性分析表明，CIP和氧氟沙星(OFL)、诺氟沙星(NOR)耐药性呈正相关P＜0.05；非PPNG株，青
第四军医大学博士学位论文
霉素（＊＊N）和四环素（拈T）呈负相关P＜O．0卜 阿奇霉素（A二1）与
＊RO呈负相关P＜队05。不同地区耐药性分析表明，我国湛江地区对喳
诺酮类的敏感性明显底于西安和长春地区（P＜0．05）；西安地区分离的菌
株对TET和AZI的耐药性检出率高于长春和湛江地区；对其他药物的
敏感性则没有显著性差异。我们还分析了质粒谱表型、营养型、PEN／TET
耐药表型以及p－内酞胺酶表型与耐药性的关系。我们认为过去曾广泛使
用的某些抗生素己经不再适用于淋病的治疗：临床常用的头抱曲松、钟E
及AZI的耐药株的出应引起重视。
为了探讨DNA螺旋酶A亚单位基因（gyTA）和拓扑异构酶IVA亚
单位基因中raC）与喳诺酮耐药性形成的关系，我们筛选出76株对CIP
高水平耐药的 QMG株，通过 PCR扩增了其中的 18株 NG的 gyTA和
praC基因喳诺酮耐药决定区（QRDR人并对扩增子直接测序。通过Whel
酶切、脉冲电场凝胶电泳（PFEG）分析了这些菌株的遗传关系。结果表
明，对照组中的 18株敏感菌只有两株发生了 gyTA的单一突变，未发
现 praC变异。而耐药组中 89％门）菌株的 gytA发生了 Ser习－Phe
的单一突变株和／或praC的双突变。88．9％门5）的菌株的gyTA发生了
Ser习—Phe和 Asp95—Gly的突变。在发生 praC突变中 72％属于
Asp86—Asn突变丁FEG分析发现10株具有完全一致的gyTA和PraC
突变模式。同时也发现来自不同地区的菌株的PFEG指纹图是不同的。
这个研究表明，NG中国流行株对喳诺酮产生耐药性与gpA和praC基
因的单一或双重突变有着密切的关系。
NG对红霉素和 Triton X－100的抗性，是由多重传递耐药性调控基
因（mtrR）基因介导的、多重耐药基因复合体（m汀CDE）编码的外排泵
的过表达有关htrR编码可调节m汀CDE操纵子表达的转录抑制剂）。
6
第四军医大学博土学位论文
我们为了确定临床分离株对AZI敏感性的降低是否与mtrR的变异有关。
我们选择了 4株AZI耐药的NG株，提取染色体DNA，PCR扩增 mtrR
基因，在此基础上进行序列分析。结果我们发现在被检菌3株NG在
mtrR 45位氨基酸 出现甘氨酸一天冬氨酸置换和 mtrR启动子中 13hp
反向重复序列内有一个单碱基缺失，成功地把其中的1株染色体DNA
转化到野生型标准株，使后者出现mtrR启动子突变，同时其阿奇霉素
敏感性降低了9倍；l株在mtrR启动子中，含有一个迄今为止尚未描述
过的突变，这个突变表现为在13hp反向重复序列内有一个双核昔酸的
插入（TT）。通过转化实验的方法成功地造成mud基因的插入突变，致
使 mrtDE转录失活，转化子对 AZI的敏感性提高了 10倍。由此我们
认为，mdDE转录阻遏物的基因（mrR）的转录失活的突变或抑制编
码基因失去功能的突变，可导致NG过度表达mndDE外排泵，从而增
加其泵出HAs的能力。这些疏水性试剂包括染料（如结晶紫），清洁剂
（如 Tition X．100）和药物（如红霉素和 AZI）我们认为，mtheDE编码
的外排泵可识别AZI，对AZI敏感性降低的原因，至少有一部分可以被
mtrR的变异解释。
604 strains of Neisseria gonorrhoeae which have been separated in three cities of china, changchun, zhanjiang and xi'an, from 1993 to 2001. The following phenotypes have been researched, there are antimicriobia resistance, plamid profile, auxiotype, PEN/TET resistance and 3 -lactamase.We have assayed minimal inhibitory concentration (MIC) of 8 antibiotics to these strains using agar dilution method. Plasmid profile of drug resistant strains was determined by extraction of plasmid and agarose gel electrophoresis. The frequency and patterns of mutations within the gyrA and praC genes were investigated part of resistant strains were appraised through PCR methods. Drug resistant correlation was analyzed using linear correlation and regression analysis, t test and X2 test. Results shows that of all the 604 strains, 6 strains (1.0%) are classified into PPNG strain and 116 strains (19.2%) belong to PP/TRNG . Of the three most useful quinolone kind drugs, cinrofloxacin has the best effect, 62% (417/604) separated strains are sensitive to it. 13.2% (80/604) separated strains are resistant to actinospectacin. 13.4% (81/604) separated strains show decrease of sensitivity to ceftriazone and 2.2% (13/604) strains are totally resistant to it.
Tolerance correlation analysis demonstrates that cinrofloxacin, ofloxacin and norfloxacin are positively correlate to drug tolerance (r=0.9,P<0.05). In PPNG strains, penicillin and economycin are negatively correlated to drug tolerance (r=0.6 P<0.05). Ceftriaxone and Azithromycin are negatively correlated to drug resistance, either (r=0.6, P <0.05). Drug resistance analysis in different areas show that sensitivity of quinolone kinds of drugs in Zhanjiang is lower than that of north China(Xi'an and Changchun) (P<0.05). Drug resistance ratio checked out in Xi'an area is higher than that in Chuangchun and Zhanjing). Sensitivity to other kinds of drugs has no obvious difference. We also have analyzed the relationship between drug resistance and plamid profile, auxotype, phenotype of PEN/TET drug resistance and P -lactamase. We suggest that Many kinds of extensively used drugs before are no longer suitable to the treatment of gonorrhea. Much attention should be paid to the appearance of drug resistant strains to Ceftriaxone, Actinospectacin and Azithromycin.
in order to investigate gyrA,praC gene of Neisseria gonorrhoeae in relation to acquired resistance, 76 QRNG strains that have high level CIP resistance are selected.selected 18 N. gonorrhoeae strains were investigated for the frequency and patterns of mutations within the gyrA and praC genes. To identify mutations in the gyrA and praC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and praC genes were amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced. The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE) and Nhel enzyme digest. The result shows that only 2 strains in the controlled 18 sensitive strains have single gyrA mutation. There is no praC mutation. But in drug resistance
group, 89%(16/18) strains developed single mutation from Ser-91 to Phe or double mutation including praC. gyrA in 88.9% (15) strains have mutation from Ser-91 to Phe and Asp-95 to Gly. 72% praC mutations are from Asp-86 to Asn. The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). PFEG analysis shows that ten strains have the same mutation pattern in the gyrA and praC genes, and the PFEG fingerprint maps in the strains from different areas are different. Results from this study suggest that gyrA mutations plus a praC mutation(s) play an important role in the development of high-level fluoroquinolone resistance in N. gonorrhoeae .
Resistance of NG to erythromycin and Triton X-100 is due to overexpression of the mtrCDE-encoded efflux pump mediated by mtrR gene, which encodes a transcriptional represser that modulates expression of the mtrCDE operon. Accordingly, we questioned whether clinical
第四军医大学博士学位论文
霉素（＊＊N）和四环素（拈T）呈负相关P＜O．0卜 阿奇霉素（A二1）与
＊RO呈负相关P＜队05。不同地区耐药性分析表明，我国湛江地区对喳
诺酮类的敏感性明显底于西安和长春地区（P＜0．05）；西安地区分离的菌
株对TET和AZI的耐药性检出率高于长春和湛江地区；对其他药物的
敏感性则没有显著性差异。我们还分析了质粒谱表型、营养型、PEN／TET
耐药表型以及p－内酞胺酶表型与耐药性的关系。我们认为过去曾广泛使
用的某些抗生素己经不再适用于淋病的治疗：临床常用的头抱曲松、钟E
及AZI的耐药株的出应引起重视。
为了探讨DNA螺旋酶A亚单位基因（gyTA）和拓扑异构酶IVA亚
单位基因中raC）与喳诺酮耐药性形成的关系，我们筛选出76株对CIP
高水平耐药的 QMG株，通过 PCR扩增了其中的 18株 NG的 gyTA和
praC基因喳诺酮耐药决定区（QRDR人并对扩增子直接测序。通过Whel
酶切、脉冲电场凝胶电泳（PFEG）分析了这些菌株的遗传关系。结果表
明，对照组中的 18株敏感菌只有两株发生了 gyTA的单一突变，未发
现 praC变异。而耐药组中 89％门）菌株的 gytA发生了 Ser习－Phe
的单一突变株和／或praC的双突变。88．9％门5）的菌株的gyTA发生了
Ser习—Phe和 Asp95—Gly的突变。在发生 praC突变中 72％属于
Asp86—Asn突变丁FEG分析发现10株具有完全一致的gyTA和PraC
突变模式。同时也发现来自不同地区的菌株的PFEG指纹图是不同的。
这个研究表明，NG中国流行株对喳诺酮产生耐药性与gpA和praC基
因的单一或双重突变有着密切的关系。
NG对红霉素和 Triton X－100的抗性，是由多重传递耐药性调控基
因（mtrR）基因介导的、多重耐药基因复合体（m汀CDE）编码的外排泵
的过表达有关htrR编码可调节m汀CDE操纵子表达的转录抑制剂）。
6
第四军医大学博土学位论文
我们为了确定临床分离株对AZI敏感性的降低是否与mtrR的变异有关。
我们选择了 4株AZI耐药的NG株，提取染色体DNA，PCR扩增 mtrR
基因，在此基础上进行序列分析。结果我们发现在被检菌3株NG在
mtrR 45位氨基酸 出现甘氨酸一天冬氨酸置换和 mtrR启动子中 13hp
反向重复序列内有一个单碱基缺失，成功地把其中的1株染色体DNA
转化到野生型标准株，使后者出现mtrR启动子突变，同时其阿奇霉素
敏感性降低了9倍；l株在mtrR启动子中，含有一个迄今为止尚未描述
过的突变，这个突变表现为在13hp反向重复序列内有一个双核昔酸的
插入（TT）。通过转化实验的方法成功地造成mud基因的插入突变，致
使 mrtDE转录失活，转化子对 AZI的敏感性提高了 10倍。由此我们
认为，mdDE转录阻遏物的基因（mrR）的转录失活的突变或抑制编
码基因失去功能的突变，可导致NG过度表达mndDE外排泵，从而增
加其泵出HAs的能力。这些疏水性试剂包括染料（如结晶紫），清洁剂
（如 Tition X．100）和药物（如红霉素和 AZI）我们认为，mtheDE编码
的外排泵可识别AZI，对AZI敏感性降低的原因，至少有一部分可以被
mtrR的变异解释。
604 strains of Neisseria gonorrhoeae which have been separated in three cities of china, changchun, zhanjiang and xi'an, from 1993 to 2001. The following phenotypes have been researched, there are antimicriobia resistance, plamid profile, auxiotype, PEN/TET resistance and 3 -lactamase.We have assayed minimal inhibitory concentration (MIC) of 8 antibiotics to these strains using agar dilution method. Plasmid profile of drug resistant strains was determined by extraction of plasmid and agarose gel electrophoresis. The frequency and patterns of mutations within the gyrA and praC genes were investigated part of resistant strains were appraised through PCR methods. Drug resistant correlation was analyzed using linear correlation and regression analysis, t test and X2 test. Results shows that of all the 604 strains, 6 strains (1.0%) are classified into PPNG strain and 116 strains (19.2%) belong to PP/TRNG . Of the three most useful quinolone kind drugs, cinrofloxacin has the best effect, 62% (417/604) separated strains are sensitive to it. 13.2% (80/604) separated strains are resistant to actinospectacin. 13.4% (81/604) separated strains show decrease of sensitivity to ceftriazone and 2.2% (13/604) strains are totally resistant to it.
Tolerance correlation analysis demonstrates that cinrofloxacin, ofloxacin and norfloxacin are positively correlate to drug tolerance (r=0.9,P<0.05). In PPNG strains, penicillin and economycin are negatively correlated to drug tolerance (r=0.6 P<0.05). Ceftriaxone and Azithromycin are negatively correlated to drug resistance, either (r=0.6, P <0.05). Drug resistance analysis in different areas show that sensitivity of quinolone kinds of drugs in Zhanjiang is lower than that of north China(Xi'an and Changchun) (P<0.05). Drug resistance ratio checked out in Xi'an area is higher than that in Chuangchun and Zhanjing). Sensitivity to other kinds of drugs has no obvious difference. We also have analyzed the relationship between drug resistance and plamid profile, auxotype, phenotype of PEN/TET drug resistance and P -lactamase. We suggest that Many kinds of extensively used drugs before are no longer suitable to the treatment of gonorrhea. Much attention should be paid to the appearance of drug resistant strains to Ceftriaxone, Actinospectacin and Azithromycin.
in order to investigate gyrA,praC gene of Neisseria gonorrhoeae in relation to acquired resistance, 76 QRNG strains that have high level CIP resistance are selected.selected 18 N. gonorrhoeae strains were investigated for the frequency and patterns of mutations within the gyrA and praC genes. To identify mutations in the gyrA and praC genes of the gonococcal mutants, the quinolone resistance determining regions of the gyrA and praC genes were amplified by polymerase chain reaction (PCR) and the PCR products were directly sequenced. The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE) and Nhel enzyme digest. The result shows that only 2 strains in the controlled 18 sensitive strains have single gyrA mutation. There is no praC mutation. But in drug resistance
group, 89%(16/18) strains developed single mutation from Ser-91 to Phe or double mutation including praC. gyrA in 88.9% (15) strains have mutation from Ser-91 to Phe and Asp-95 to Gly. 72% praC mutations are from Asp-86 to Asn. The strains were analyzed for genetic relationship by pulsed-field gel electrophoresis (PFGE). PFEG analysis shows that ten strains have the same mutation pattern in the gyrA and praC genes, and the PFEG fingerprint maps in the strains from different areas are different. Results from this study suggest that gyrA mutations plus a praC mutation(s) play an important role in the development of high-level fluoroquinolone resistance in N. gonorrhoeae .
Resistance of NG to erythromycin and Triton X-100 is due to overexpression of the mtrCDE-encoded efflux pump mediated by mtrR gene, which encodes a transcriptional represser that modulates expression of the mtrCDE operon. Accordingly, we questioned whether clinical
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