我国淋病奈瑟菌头孢曲松耐药趋势及运用比较蛋白质组学研究其部分耐药机制
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摘要
淋病奈瑟菌(Neisseria gonorrhoeae,Ng)是淋病的病原菌,主要经性途径传播。据估计,全世界每年新增6000万淋病患者,这无疑对公共卫生构成严重威胁。淋病主要引起黏膜感染,如不及时、有效的治疗,感染可扩散至邻近器官,或经血播散。妇女和儿童感染淋病可产生较为严重的并发症,如不孕、失明等。此外,淋病患者感染HIV的机会可增加近5倍。
据全国流行病学资料显示,从上世纪80～90年代,淋病呈上升趋势;至2000年,淋病开始缓慢下降。但是,由于抗生素的滥用,Ng耐药株特别是多重耐药株的出现,使淋病疫情复杂化。头孢曲松(ceftriaxone,CRO)是各国推荐治疗淋病的一线药物,但敏感性呈下降趋势。可以预见,一旦Ng对第三代头孢类抗生素产生耐药,有效治疗手段将面临枯竭的窘境,发病率将急剧增加,淋病的流行也会更为广泛。有鉴于此,研究Ng对CRO的耐药机制刻不容缓。迄今为止,国内外分离出耐CRO Ng临床株非常罕见,这直接制约着对其耐药机制的研究。为此,我们拟采用人工诱导的方法,在实验室诱导出耐CRO Ng株,在此基础上,运用比较蛋白质组学技术寻找Ng对CRO耐药的差异表达的蛋白质分子。可以预见,本研究的结果对我国淋病的预防和治疗可能会产生一定积极的作用。全文分为4个部分,各章的主要内容简述如下:
第一部分我国淋病奈瑟菌头孢曲松耐药监测及趋势的系统分析
检索中国期刊全文数据库、PubMed、Cochrane数据库收集近期国内外发表的有关中国大陆地区Ng CRO耐药监测文献。筛选合格文献进行系统评价,分析文献的质量及Ng的耐药趋势。入选46篇文献,共报道10163株Ng CRO药敏情况;23篇文献主要来自华南地区(50%);26篇提供的标准菌株为WHO Ng分离株(56.52%);32篇文献药敏试验方法为WHO推荐的琼脂糖稀释法(69.57%);Ng的CRO耐药率仍较低但有增加趋势。由此可见,应该进一步提高Ng耐药监测研究的质量,使之能更好地反映Ng耐药状况;此外,要加强有关耐药机制的前瞻性研究,防范于末然。
第二部分淋病奈瑟菌临床头孢曲松敏感株体外诱导耐药试验
采用次抑菌浓度法对1株Ng CRO敏感株体外经CRO反复诱导。诱导前Ng临床株CRO的MIC值为0.0156μg/ml,经过120代传代,诱导后菌株对CRO的MIC值为1.0μg/ml;耐药子代经过30d的无药传代后MIC值无明显变化。可见,体外人工诱导虽然过程漫长,但仍可获得Ng耐CRO株,且该获得性耐药稳定性较好。体外诱导耐药的成功使后续的比较蛋白质组学研究成为可能,同时也说明,如果临床继续滥用CRO治疗Ng,耐CRO Ng迟早会产生。
第三部分淋病奈瑟菌头孢曲松敏感株和耐药株双向凝胶电泳试验
本研究的目的是分离Ng CRO临床敏感株和诱导耐药株差异表达的蛋白质分子,为进一步研究Ng的蛋白质组学打下基础。分别提取母代、子代两种细菌的总蛋白,进行等电点聚焦和SDS-聚丙烯胺凝胶电泳,对所得胶图利用ImageMaster 5.0软件进行分析。最好得到了分辨率和重复性均较好的双向凝胶电泳图谱,通过统计分析确定了差异表达＞1.3倍的蛋白斑点24个。该结果证明在Ng CRO临床敏感株和诱导耐药株中存在差异表达的蛋白质分子,双向凝胶电泳技术能对这些分了进行较有效地分离。
第四部分运用比较蛋白质组技术研究淋病奈瑟菌头孢曲松敏感株和耐药株之间差异表达的蛋白质
本研究的目的是筛选与Ng产生CRO耐药相关的蛋白质分子,揭示其部分耐药机制。对蛋白质表达差异＞1.3倍的24个差异蛋白质斑点进行切取,其中有两个蛋白质斑点(795、1014)没有切到相应的蛋白质,最后共有22个蛋白质斑点进行了LC-MS/MS分析。将原始质谱文件输入SEQUEST软什并查寻NCBI数据库,搜索后匹配到的蛋白质点有21个,每个蛋白质点有1～多个蛋白群组成。其中第1127蛋白质斑点未检索到相应的蛋白质,可能为一种新的蛋白质分子,需要进一步鉴定。已鉴定的蛋白质与细菌的运动、新陈代谢、信号转导、分裂、基因合成和甲基化、β-内酰胺水解等有关,可为进一步研究Ng耐CRO机制提供线索。
Neisseria gonorrhoeae is responsible for the sexually transmitted disease gonorrhoea.The estimated 60 million new cases of gonorrhoeae occurring globally each year constitute a major public health problem.These are mainly mucosal infections,but if left untreated or improperly treated,may be complicated by spread to nearby organs, dissemination via the blood stream.Infection of the genitals in females with N. gonorrhoeae can result in pelvic inflammatory diseases if left untreated,which can result in infertility.Gonorrhoea may be added the amplication of spread of sexually transmitted HIV-up to five fold-that occurs in those with gonorrhoea while exposed to HIV.
According to the national epidemiology data,the incidence of gonorhoea increased in our country from the 1980s to the 1990s,it has been decreased again since 2000.But the emergence of antibiotic-resistance of N.gonorrhoeae,especially the multidrug resistant strain,has aggravated deeply to this trend.More importantly,some strains of N. gonorrhoeae with decreased susceptibility to ceftriaxone have been reported in many countries.Since the ceftriaxone is the first line drug recommended by CDC,WHO and the Health of Ministry of our country in the treatment of gonococcal infections,the emergence of ceftriaxone-resistant N.gonorrhoeae will make the management and prevention of gonorrhea become more difficult.So,to carry out the researches on the molecular mechanism of ceftriaxone-resistant N.gonorrhoeae in vitro ahead of the emergence of clinical isolate of ceftriaxone-resistant N.gonorrhoeae is very useful and urgent.Because few clinical strains of ceftriaxone-resistant N.gonorrhoeae isolated from clinical specimens so far,there are rare papers related to this issue published at present.A plan is developed to induce ceftriaxone-resistant N.gonorrhoeae strains in vitro and to investigate its molecular mechanism of ceftriaxone-resistance by comparative proteomics. The results will be help in making decisions to the treatment and prevention of gonococcal infections.The current research is divided into four parts as follow:
Chapter 1
Surveillance of ceftriaxone resistance in N.gonorrhoeae in mainland China:a systematic review
Firstly,to collect all recent references with regard to surveillance of ceftriaxone resistance in N.gonorrhoeae in mainland China,then to evaluate its quality and infer the tendency of ceftriaxone resistance by it.References were searched by CNKI,PubMed and Cochrane database,and eligible references were included for systematic review. Forty-six references entered the systematic review in which 10 163 strains were reported. Twenty three references were from South China(50%);the control strains in 26 references were WHO isolates of N.gonorrhoeae(56.52%);antimicrobial susceptibility testing was performed by agar dilution using GC agar base according to WHO in 32 references(50%);the rate of ceftriaxone resistance in N.gonorrhoeae in mainland China was low but it showed an increasing tendency.The quality of surveillance of antibiotic resistance in N.gonorrhoeae in mainland China should be improved to better reveal the situation of ceftriaxone resistance in it.
Chapter 2
Study on in vitro induced ceftriaxone Resistance of N.gonorrhoeae
Our objective is to induce ceftriaxone resistant N.gonorrhoeae strain from a clinical isolated strain by sub-MIC culture.The clinical strain of neisseria gonorrheae was induced successfully with ceftriaxone after cultured for 120 passages and whose MICs increased from 0.0156μg/ml to 1.00μg/ml.The strain was transferred 30 passages on ceftriaxone-free medium and the MICs was the same as those after induced.From the results,we make a conclusion that the ceftrixone induction experiment could make N. gonorrhoeae acquire stable resistance to ceftriaxone.
Chapter 3
Separation of differential proteins from ceftriaxone sensitive strain to resistant strain of N.gonorrhoeae by two-dimensional polyacrylamide gel electrophoresis
These two different strains of N.gonorrhoeae were cultured,and total proteins were extracted from these bacteria.After preparation of the total proteins,solubilized proteins were separated in the first molecular in IPG(immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis.Those differential protein spots were visualized by image analysis software of ImageMaster 5.0.The repeatability and resolution of Gel electrophoresis were high.2-DE was performed to find expression-altered proteins(＞1.3 fold) between sensitive and resistant strains.The results indicate that there are differential proteins between ceftriaxone sensitive and resistant strains of N.gonorrhoeae,and further research on the cerftriaxone resistant mechanism of N.gonorrhoeae can be based on it.
Chapter 4
Research of differential proteins in ceftriaxone sensitive and in vitro-induced ceftriaxone resistant N.gonorrhoeae by comparative proteome analysis
To screen the differential express proteins related to ceftriaxone resistant mechanism in N.gonorrhoeae.First,we induced ceftriaxone resistant N.gonorrhoeae strain from a clinical isolated by sub-MIC culture and total proteins were separated by SDS-polyacylamide gel electrophoresis.Those differential protein spots were identified by mass spectrometry and the results indexed in database.Twenty one differential express proteins(＞1.3 fold) were identified successfully,which were constituted by one or more protein.But the spot of 1127 was not be matched in NCBI databases and it maybe a new protein of N.gonorrhoeae.Further research is needed on whether these proteins could be used as diagnostic markers or therapeutic targets.
据全国流行病学资料显示,从上世纪80～90年代,淋病呈上升趋势;至2000年,淋病开始缓慢下降。但是,由于抗生素的滥用,Ng耐药株特别是多重耐药株的出现,使淋病疫情复杂化。头孢曲松(ceftriaxone,CRO)是各国推荐治疗淋病的一线药物,但敏感性呈下降趋势。可以预见,一旦Ng对第三代头孢类抗生素产生耐药,有效治疗手段将面临枯竭的窘境,发病率将急剧增加,淋病的流行也会更为广泛。有鉴于此,研究Ng对CRO的耐药机制刻不容缓。迄今为止,国内外分离出耐CRO Ng临床株非常罕见,这直接制约着对其耐药机制的研究。为此,我们拟采用人工诱导的方法,在实验室诱导出耐CRO Ng株,在此基础上,运用比较蛋白质组学技术寻找Ng对CRO耐药的差异表达的蛋白质分子。可以预见,本研究的结果对我国淋病的预防和治疗可能会产生一定积极的作用。全文分为4个部分,各章的主要内容简述如下:
第一部分我国淋病奈瑟菌头孢曲松耐药监测及趋势的系统分析
检索中国期刊全文数据库、PubMed、Cochrane数据库收集近期国内外发表的有关中国大陆地区Ng CRO耐药监测文献。筛选合格文献进行系统评价,分析文献的质量及Ng的耐药趋势。入选46篇文献,共报道10163株Ng CRO药敏情况;23篇文献主要来自华南地区(50%);26篇提供的标准菌株为WHO Ng分离株(56.52%);32篇文献药敏试验方法为WHO推荐的琼脂糖稀释法(69.57%);Ng的CRO耐药率仍较低但有增加趋势。由此可见,应该进一步提高Ng耐药监测研究的质量,使之能更好地反映Ng耐药状况;此外,要加强有关耐药机制的前瞻性研究,防范于末然。
第二部分淋病奈瑟菌临床头孢曲松敏感株体外诱导耐药试验
采用次抑菌浓度法对1株Ng CRO敏感株体外经CRO反复诱导。诱导前Ng临床株CRO的MIC值为0.0156μg/ml,经过120代传代,诱导后菌株对CRO的MIC值为1.0μg/ml;耐药子代经过30d的无药传代后MIC值无明显变化。可见,体外人工诱导虽然过程漫长,但仍可获得Ng耐CRO株,且该获得性耐药稳定性较好。体外诱导耐药的成功使后续的比较蛋白质组学研究成为可能,同时也说明,如果临床继续滥用CRO治疗Ng,耐CRO Ng迟早会产生。
第三部分淋病奈瑟菌头孢曲松敏感株和耐药株双向凝胶电泳试验
本研究的目的是分离Ng CRO临床敏感株和诱导耐药株差异表达的蛋白质分子,为进一步研究Ng的蛋白质组学打下基础。分别提取母代、子代两种细菌的总蛋白,进行等电点聚焦和SDS-聚丙烯胺凝胶电泳,对所得胶图利用ImageMaster 5.0软件进行分析。最好得到了分辨率和重复性均较好的双向凝胶电泳图谱,通过统计分析确定了差异表达＞1.3倍的蛋白斑点24个。该结果证明在Ng CRO临床敏感株和诱导耐药株中存在差异表达的蛋白质分子,双向凝胶电泳技术能对这些分了进行较有效地分离。
第四部分运用比较蛋白质组技术研究淋病奈瑟菌头孢曲松敏感株和耐药株之间差异表达的蛋白质
本研究的目的是筛选与Ng产生CRO耐药相关的蛋白质分子,揭示其部分耐药机制。对蛋白质表达差异＞1.3倍的24个差异蛋白质斑点进行切取,其中有两个蛋白质斑点(795、1014)没有切到相应的蛋白质,最后共有22个蛋白质斑点进行了LC-MS/MS分析。将原始质谱文件输入SEQUEST软什并查寻NCBI数据库,搜索后匹配到的蛋白质点有21个,每个蛋白质点有1～多个蛋白群组成。其中第1127蛋白质斑点未检索到相应的蛋白质,可能为一种新的蛋白质分子,需要进一步鉴定。已鉴定的蛋白质与细菌的运动、新陈代谢、信号转导、分裂、基因合成和甲基化、β-内酰胺水解等有关,可为进一步研究Ng耐CRO机制提供线索。
Neisseria gonorrhoeae is responsible for the sexually transmitted disease gonorrhoea.The estimated 60 million new cases of gonorrhoeae occurring globally each year constitute a major public health problem.These are mainly mucosal infections,but if left untreated or improperly treated,may be complicated by spread to nearby organs, dissemination via the blood stream.Infection of the genitals in females with N. gonorrhoeae can result in pelvic inflammatory diseases if left untreated,which can result in infertility.Gonorrhoea may be added the amplication of spread of sexually transmitted HIV-up to five fold-that occurs in those with gonorrhoea while exposed to HIV.
According to the national epidemiology data,the incidence of gonorhoea increased in our country from the 1980s to the 1990s,it has been decreased again since 2000.But the emergence of antibiotic-resistance of N.gonorrhoeae,especially the multidrug resistant strain,has aggravated deeply to this trend.More importantly,some strains of N. gonorrhoeae with decreased susceptibility to ceftriaxone have been reported in many countries.Since the ceftriaxone is the first line drug recommended by CDC,WHO and the Health of Ministry of our country in the treatment of gonococcal infections,the emergence of ceftriaxone-resistant N.gonorrhoeae will make the management and prevention of gonorrhea become more difficult.So,to carry out the researches on the molecular mechanism of ceftriaxone-resistant N.gonorrhoeae in vitro ahead of the emergence of clinical isolate of ceftriaxone-resistant N.gonorrhoeae is very useful and urgent.Because few clinical strains of ceftriaxone-resistant N.gonorrhoeae isolated from clinical specimens so far,there are rare papers related to this issue published at present.A plan is developed to induce ceftriaxone-resistant N.gonorrhoeae strains in vitro and to investigate its molecular mechanism of ceftriaxone-resistance by comparative proteomics. The results will be help in making decisions to the treatment and prevention of gonococcal infections.The current research is divided into four parts as follow:
Chapter 1
Surveillance of ceftriaxone resistance in N.gonorrhoeae in mainland China:a systematic review
Firstly,to collect all recent references with regard to surveillance of ceftriaxone resistance in N.gonorrhoeae in mainland China,then to evaluate its quality and infer the tendency of ceftriaxone resistance by it.References were searched by CNKI,PubMed and Cochrane database,and eligible references were included for systematic review. Forty-six references entered the systematic review in which 10 163 strains were reported. Twenty three references were from South China(50%);the control strains in 26 references were WHO isolates of N.gonorrhoeae(56.52%);antimicrobial susceptibility testing was performed by agar dilution using GC agar base according to WHO in 32 references(50%);the rate of ceftriaxone resistance in N.gonorrhoeae in mainland China was low but it showed an increasing tendency.The quality of surveillance of antibiotic resistance in N.gonorrhoeae in mainland China should be improved to better reveal the situation of ceftriaxone resistance in it.
Chapter 2
Study on in vitro induced ceftriaxone Resistance of N.gonorrhoeae
Our objective is to induce ceftriaxone resistant N.gonorrhoeae strain from a clinical isolated strain by sub-MIC culture.The clinical strain of neisseria gonorrheae was induced successfully with ceftriaxone after cultured for 120 passages and whose MICs increased from 0.0156μg/ml to 1.00μg/ml.The strain was transferred 30 passages on ceftriaxone-free medium and the MICs was the same as those after induced.From the results,we make a conclusion that the ceftrixone induction experiment could make N. gonorrhoeae acquire stable resistance to ceftriaxone.
Chapter 3
Separation of differential proteins from ceftriaxone sensitive strain to resistant strain of N.gonorrhoeae by two-dimensional polyacrylamide gel electrophoresis
These two different strains of N.gonorrhoeae were cultured,and total proteins were extracted from these bacteria.After preparation of the total proteins,solubilized proteins were separated in the first molecular in IPG(immobilized pH gradient) strips depending on their pI and subsequently in the second dimension according to their molecular weight by SDS-polyacrylamide gel electrophoresis.Those differential protein spots were visualized by image analysis software of ImageMaster 5.0.The repeatability and resolution of Gel electrophoresis were high.2-DE was performed to find expression-altered proteins(＞1.3 fold) between sensitive and resistant strains.The results indicate that there are differential proteins between ceftriaxone sensitive and resistant strains of N.gonorrhoeae,and further research on the cerftriaxone resistant mechanism of N.gonorrhoeae can be based on it.
Chapter 4
Research of differential proteins in ceftriaxone sensitive and in vitro-induced ceftriaxone resistant N.gonorrhoeae by comparative proteome analysis
To screen the differential express proteins related to ceftriaxone resistant mechanism in N.gonorrhoeae.First,we induced ceftriaxone resistant N.gonorrhoeae strain from a clinical isolated by sub-MIC culture and total proteins were separated by SDS-polyacylamide gel electrophoresis.Those differential protein spots were identified by mass spectrometry and the results indexed in database.Twenty one differential express proteins(＞1.3 fold) were identified successfully,which were constituted by one or more protein.But the spot of 1127 was not be matched in NCBI databases and it maybe a new protein of N.gonorrhoeae.Further research is needed on whether these proteins could be used as diagnostic markers or therapeutic targets.
引文
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