Study On The Tendency Of Ceftriaxone Resistance Of Neisseria Gonorrhoeae In Mainland China And Its Partly Associated Mechanism By Comparative Proteomics

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 1Surveillance of ceftriaxone resistance in N.gonorrhoeae in mainland China:a systematic reviewFirstly,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 2Study on in vitro induced ceftriaxone Resistance of N.gonorrhoeaeOur 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 3Separation of differential proteins from ceftriaxone sensitive strain to resistant strain of N.gonorrhoeae by two-dimensional polyacrylamide gel electrophoresisThese 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 4Research of differential proteins in ceftriaxone sensitive and in vitro-induced ceftriaxone resistant N.gonorrhoeae by comparative proteome analysisTo 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.