Investigation Of Resistance And Plasmid Profiles In Neisseria Gonorrhoeae In Chengdu From The Years Of 2004 To 2008

Objective:Gonorrhea is a common sexually transmitted diseases(STD).As the traditional antibiotics,penicillin and tetracycline are no longer used to treat gonorrhea because of the prevalence of drug resistance.Some documents show that the plasmid-mediated high-level TRNG(tetracycline resistance of Neisseria gonorrhoeae) and chromosome-mediated TRNG's were increased,but PPNG's(penicillin resistant of Neisseria gonorrhoeae ) rate have the downward trend in recent years which may be related to penicillin as a treatment for gonorrhea is no longer first-line drugs.Therefore,the study of drug-resistant strains of Neisseria gonorrhoeae(NG) is still the epidemiological surveillance of gonococcal resistance in one of the important indicators.The resistance of NG is relatead to chromosome-mediated,plasmid-mediated,multi-drug-resistant system that can transmit mtr efflux system,fatty acid resistance system that is far efflux systems and efflux system of the norm.The study found that plasmid-mediated resistance in drug-resistant strains accounted for about 91%.It show a different stripe which have different molecular weight through electrophoresis of the plasmid.Molecular biology techniques were used plasmid profile.The techniques analysis can be more accurate monitoring of changes in resistance.In the studies of Chengdu Neisseria gonorrhea drug resistance from the years of 2004 to 2008,the auther provided more detailed basis for the treatment of gonorrhea,through analysis of the past five years of NG's plasmid profiles changes and gonorrhea drug resistance monitoring in the region. Method:1.Collect 150 NG strain from2004 to2008,and through the bacterial morphology,oxidase and glucose fermentation experiments further confirmed;2.Made the collection of NG bacteria 10⁸CFU/ml suspension,adding lysis buffer,100℃heating for 15 minutes, 10000r/min centrifugation for 15 minutes,take the supernatant from 2ul as a PCR template amplified in the micro-centrifuge tube followed by adding 10×buffer(containing Mgcl₂ 1.5mmol/l) 3ul;10pmol/ l-specific pros and cons of each primer 1ul;10mmol/l dNTP 0.25ul;Taq DNA polymerase 1.5U;template DNA 2ul;adding 30ul double distilled water and liquid paraffin after 10ul reaction conditions in accordance with the PCR amplification,micro-centrifuge tube after the addition of 0.25%bromophenol blue 5ul,mixing after standing by adding 6ul get 2% horizontal agarose gel electrophoresis sample hole slot and hole in the head and tail increases to 6ul DNA marker for reference,the electrophoresis voltage regulator to 75V,electrophoresis power off after two hours,remove the flat-panel under the instrument in the UV observations and photography,drawing record.To collate the data on,we conclude the average of PPNG resistance rate from the years of 2004 to 2008.So we can have the data after compare this two rate.To the same,we can get the data of TRNG and CPNG.We get the plasmid profiles of NG from the years of 2004 to 2008 after analysis the NG plasmid size and type.Results:1.We do experiment with 30 strains of NG every year,and get the data of PPNG strains as follows:27,3,0,6,18;The rates of CPNG resistance every year as follows:90.00%,10.00%,0.00%,20.00%, 60.00%.The average rate of resistance of CPNG from the years of 2004 to 2008 is 36.00%.2.The types of PPNG resistance:In 2004 and 2008 to the chromosome-mediated PPNG mainly for the size of 340bp of penA gene(resistant to penicillin-binding protein 2 coding sequence);in 2005 and 2007 mainly to plasmid-mediated mainly a product of 535bp length of the TEM gene.3.We do experiment with 30 strains of NG every year,and get the data of TRNG strains as follows:12,20,13,23,15;The rates of CPNG resistance every year as follows:40.00%,66.67%,43.33%, 76.66%,50.00%.The average rate of resistance of CPNG from the years of 2004 to 2008 is 55.33%.4.The types of TRNG:a plasmid-mediated mainly for the size of 501bp gene tetM(tetracycline resistance ribosomal protection protein coding sequence).5.We do experiment with 30 strains of NG every year,and get the data of CPNG strains as follows: 29,21,2,9,25;The rates of CPNG resistance every year as follows: 96.67%,70.00%,10.00%,30.00%,83.33%.The average rate of resistance of CPNG from the years of 2004 to 2008 is 58.00%.6.CPNG Type: chromosome are mediated for the size of 380bp of gyrA gene(DNA gyrase A subunit coding sequence).Conclusion:1 The average resistance rate of PPNG fell 58.07%,which from the past years(years from 2004 to 2008)(36.00%) to the over past years(years from 2000 to 2004)(94.07%).Through statistical analysis,the difference was significant (X²=372.001,P＜0.05).This data show that there is a upgrade tendency to the penicillin sensitivity of NG,when we didn't use penicillin to treat gonorrhea.2 The average resistance rate of TRNG fell 2.24%,which from the past years(years from 2004 to 2008)(53.33%) to the over past years (years from 2000 to 2004)(57.57%).Through statistical analysis,there is no significant difference(X²=0.265,P＞0.05).3 The average resistance rate of CPNG fell 18.79%,which from the past years(years from 2004 to 2008)(58.00%) to the over past years(years from 2000 to 2004)(76.79%).Through statistical analysis,the difference was significant (X²=249.197,P＜0.05).This data show that there is a upgrade tendency to the ciprofloxacin sensitivity of NG,when we didn't use ciprofloxacin to treat gonorrhea.4 In the years of 2004 and 2008 of Chengdu,the drug resistances of PPNG were mediated by penA gene,which is one of the chromosomes and it's size is 340bp.In the years of 2005 and 2007,the drug resistances of PPNG were mediated by TEM gene,which is one of the plasmids and it's size is 535bp.5 In the years of 2004 and 2008 of Chengdu,the drug resistances of TRNG were mediated by tetM gene, which is one of the plasmids and it's size is 501bp.6 In the years of 2004 and 2008 of Chengdu,the drug resistances of CPNG were mediated by gyrA gene,which is one of the chromosomes and it's size is 380bp.7 From the years of 2004 to 2008,the epidemic plasmid profiles of PPNG in Chengdu is 535bp.The epidemic plasmid profiles of TRNG in Chengdu are 501bp +700 bp and 501bp.8 From the years of 2004 to 2008,all of the PPNG strain belongs to Asian type.9 This experiment confirmed that the CPNG resistance mechanisms are mainly mediated by chromosome.This report is basically the same as internal and abroad.