Study On Resistance Genes And Molecular Epidemiology Of Treponema Pallidum

Chapter 1Detection of macrolide and tetracycline resistance genes in Treponema pallidumObjective:Due to the shortage of penicillin and the high allergy rate,macrolide and tetracycline antibiotics have been used as the main second-line oral treatment drugs for syphilis for a long time.The purpose of this study is to conduct a preliminary study on the macrolide and tetracycline antibiotic resistance genes in Treponema pallidum which collected in Nanjing.Methods:From 2013 to 2020,skin lesion secretions of 240 early syphilis patients were obtained from the Department of Venereology,Institute of Dermatology,Chinese Academy of Medical Sciences.After DNA extraction,the 23S rRNA gene?16S rRN A regions and tetB gene were amplified.We analysed the sequences to find out macrolide and tetracycline resistance gene mutations.Results:Among 240 samples,a total of 226 samples were T.pallidum positive for detecting the T.pallidum specific gene PolA.Macrolide resistance-related gene mutations A2058G and A2059G were99%(224/226)and 1%(2/226),respectively.There are no mutations in the 16S rRNA gene region which related to tetracycline resistance.The positive rate of tetB gene which related to tetracycline resistance was 8.3%(7/84).Conclusion:Our study showed that macrolide resistance mutations of T.pallidum was widespread.No mutations related to tetracycline resistance were found in the 16S rRNA region;the tetB gene was detected in T.pallidum positive clinical samples,and it is necessary to be alert to the possibility of obtaining tetracycline resistance gene tetB in T.pallidum.Chapter 2Molecular epidemiology Research of Treponema pallidum Section ? Study on the ECDCT genotype of Treponema pallidumObjective:The ECDCT genotype of Treponema pallidum plays an important role in scientific research and clinical prevention and control of syphilis.It can reflect the spread and prevalence of different strains of T.pallidum in different regions or populations.In order to provide a theoretical basis for clinical treatment and prevention,this part of the research aims to study the ECDCT(Enhanced-CDC Typing)of T.pallidum which collected in Nanjing.Methods:From 2013 to 2020,skin lesion secretions were obtained from 240 early syphilis patients in the Department of Venereology,Institute of Dermatology,Chinese.After DNA extraction,226 positive samples of T.pallidum were screened out(T.pallidum-specific PolA gene was amplified),then arp,tpr and tp0548 gene were amplified.According to the results of PCR electrophoresis and sequencing,the ECDCT genotype was carried out.Results:A total of 143 specimens were successfully fully typed according to the results of PCR electrophoresis and sequencing.A total of 11 ECDCT gene types(8d/f?9d/f?13d/f?13a/f?14a/f?14b/f?14d/f?14d/Qn?15a/f?15d/f?16d/f)were identified.In addition,there are 6 arp types(8,9,13,14,15 and 16),4 tpr types(b,d,j and a)and 4 tp0548 genotypes(f,d,c,Qn)were identified.Most strains(78/143;54.5%)belong to the 14d/f type.Conclusion:Our research indicates that there is a complex genetic diversity of T.pallidum in Nanjing,China.The dominant strain is 14d/f.The discovery of the new 14d/Qn genotype further expands the database of ECDCT typing.Section ? Identification and analysis of a novel tp0548 genotypeObjective:The tp0548 gene which encode the hypothetical outer membrane protein of Treponema pallidum(T.pallidum).It is not only an important gene locus for a variety of T.pallidum molecular typing methods,but also has been applied to distinguish different T.pallidum subspecies.Therefore,it plays an important role in the molecular epidemiology of T.pallidum.The purpose of this part is to identify and analyze a T.pallidum strain which carries a variant tp0548 gene.Methods:In the ECDCT study of T.pallidum which collected in Nanjing,we found a strain with tp0548 genotype mutation.Further sequencing and aligment was conducted to identify and analysis it.PCR,first-generation sequencing and construction of evolutionary trees were used to identify and analysis the T.pallidum strain which carries a mutant tp0548 gene.Results:Based on the sequencing alignment result,a novel tp0548 genotype was first identified.With a mutation at the T158C locus,the new sequence is very similar to the previous report tp0548 genotype "Q".Therefore,the novel tp0548 genotype was named as "Qn".Conclusion:The novel tp0548 sequence type of T.pallidum not only expands the tp0548 genotype database to 27 different genotypes,but also indicates the existence of complex genetic diversity in the tp0548 gene sequence.We found that the T pallidum strain which carrying the variant tp0548 gene was TP SS14 strain and the ECDCT genotype was 14d/Qn.