Epidemiological Research Of Gonorrhea And Antibiotic-resistance Of Neisseria Gonorrhoeae

Gonorrhea is one of the classical sexually transmitted diseases (STDs). Human is the only host for its pathogen, bacterium Neisseria gonorrhoeae. Patients with gonorrhea are usually treated with antibiotics; however, drug-resistant N.gonorrhoeae has been increasingly common due to abuse and irregular use of antibiotics, which has come an important public health issue in China. In this study, we investigated the susceptibility to antibiotics of the clinical N.gonorrhoeae isolates, and explored possibilities of drug-resistant mechanisms for N.gonorrhoeae by using molecular epidemiological approaches. The study provided useful iniormation in the prevention and control of gonorrhea in China.Part One. Epidemiological Study of Gonorrhea and Antibiotic-Resistance of Clinical N.gonorrhoeae IsolatesI. Characteristics of Gonorrhea Patients and their Health Care-Seeking BehaviorEighty-eight outpatients who had attended the designated STD clinics in Shanghai were selected, and eighty were participated in the study. Of the participants, 87.5% were male, 93.6% were aged 20-49 years old, and 65.1% were either industry-workers, or agriculture-workers or office-workers. The main reason for infection with N.gonorrhoea was outmarriage sex (76.3%). Only 18.7% used condom consistently. Approximately 28.8% of patients remained sexually active after appearance of STD symptoms. Only 32.5% of patients had sought for health-care services within three days after symptoms appeared, and 17.5% of patients were inclined to attend a private clinic. One fifth patients had an history of antibiotic-treatment during the past two weeks. The data indicated that outmarriage sex was a main reason for gonorrhea infection in Shanghai and unprotected sex after infections were common. Patients should be counseled and encouraged for safe sex and timely health care seeking. Irregular treatment is likely a risk factor for the transmission of gonorrhea and may results in resistant strains to spread.II. Determining the Antibiotics-Susceptibility of 80 Clinical N.gonorrhoeae Isolates in Shanghai.The agar-dilution method was used to determine the minimum inhibitory concentration (MIC) of nine antibiotics including Penicillin-G, Tetracycline, Ceftriaxone, Doxycycline, Ofloxacin, Lomefloxacin, Ciprofloxacin, Azithromycin and Spectinomycin. Penicillinase-producing N. gonorrhoeae (PPNG) was determined by using the papyrus-acidic method through the detection of β-lactamase. All these clinical isolates were susceptible to Spectinomycin and Ceftriaxone. For other antibiotics, 87.5% of the isolates were resistant to Penicillin, while 45% were penicillinase-producing N.gonorrhoeae (PPNG); 88.7% of them were resistant to Tetracycline, while 18.8% were plasmid-mediated Tetracycline-resistant N.gonorrhoeae (TRNG); and 94.7% of these isolates were resistant to Azithromycin. The proportion of N.gonorrhoeae resistant to Ofloxacin, Lomefloxacin and Ciprofloxacin were 95.5%, 97.5% and 95.0%, respectively. Cluster analysis was applied to classify the N.gonorrhoeae isolates according to their antibiotic-resistant type and the susceptibility level of individual antibiotics. These eighty isolates were grouped into four antibiotic-resistant types, and three susceptibility levels of antibiotics. We concluded that resistance to antibiotics of clinical isolates was very serious in Shanghai area.Part Two. Correlation Between Genotype and Antibiotic-resistance among Neisseria gonorrhoeae Isolates.I. RAPD (Random Amplified Polymorphism DNA) Method for Genotyping N. gonorrhoeae, and its Application in Tracing Infectious Pathogens.First, four sample-pretreatment methods for extracting genomic DNA from N.gonorrhoeae were compared. Second, 10-mer arbitrary primers were used to amplify the polymorphic DNA products from genomic DNA to generate the RAPD fingerprint maps. Then, the RAPD maps were compared for the conservation between strains isolated from a patient and his/her sex-partner. Results showed that CTAB-method (cetyltrimethylammonium bromide) can extract integrate genomic DNA, suitable for generate better RAPD maps. Fingerprints from different N.gonorrhoeae strains were distinctive. RAPD maps from a pair of sex-partners were similar. Based on these findings, choose the best extracting method and suitable RAPD primer, The RAPD fingerprint maps could type Neisseria gonorrhoeaeeffectively, and could be used as an additional approach in molecular epidemiology for tracing infection source.II. Exploring the Relationship Between Genotypes and Antibiotic-resistance among N.gonorrhoeae Isolated in Shanghai.The RAPD method was used to differentiate the clinical isolated N.gonorrhoeae strains. The susceptibility level of the 80 N.gonorrhoeae isolates to nine antibiotics was tested. Optimally selected RAPD primers can generate a group of bands for PCR products. Some bands were common in all N.gonorrhoeae isolates, others varied. These N.gonorrhoeae isolates were classified into three groups (I, II and III) according to their RAPD genotypes. These isolates were also grouped into four phenotypes (A, B, C and D) according to their drug-resistant profiles. Genotypes of N.gonorrhoeae were closely related to drug-resistant phenotypes (x~2=27.59, P< 0.05).Part Three. Relationship Between Plasmid Profiles and Antibiotic-Resistance among N.gonorrhoeae IsolatesI. Research on the Plasmid Profiles of N.gonorrhoeae isolatesThe plasmid profiles were obtained by using alkaline-lysis plasmid extraction method. A total of four types of plasmid were detected (42.5kb, 39.5kb, 7.4kb and 4.2kb), among them, the percentages of presence of 7.4kb and 4.2kb plasmids were 75.0% and 96.3%, respectively. A total of ten types of plasmid profiles were observed. Four main types were 42.5kb +39.5kb +7.4kb +4.2kb type, 39.5kb +7.4kb +4.2kb type, 7.4kb +4.2kb type and 4.2kb type, accounting for 86.25% of total isolates. All the PPNG strains carried a 7.4-kb plasmid; all the TRNG strains carried both 42.5-kb and 39.5-kb plasmids, while the plasmid profile of non PPNG or TRNG varied greatly. The results indicated the 4.2kb plasmids could be detected in most isolates, while its function in resistance to antibiotics was not clear. The 7.4kb might mediate high level penicillin resistance, the 42.5kb and 39.5kb plasmids might have relationship with tetracycline resistance.II. Relationship Between Plasmid Profiles and Antibiotic-Resistance among N.gonorrhoeae IsolatesWe removed the plasmids hosted in one N.gonorrhoeae isolate, and transformedthe removed plasmids into competent E.coli strain DH5α. The antibiotic-resistant levels were compared before and after the removining of plasmids harbored in the N.gonorrhoeae isolate or the introducing of the plasmids into E.coli strain. The result showed that N.gonorrhoeae lost its plasmid either totally or partially under the SDS treatment in sub-lethal concentration. After removing the plasmid, the antibiotic-resistant level of the N.gonorrhoeae isolate was decreased. After introducing the plasmid into E.coli strain, the antibiotic-resistant level of E.coli strain was increased. These plasmids had conferred the antibiotic-resistant ability to the N.gonorrhoeae strains. The data suggests that plasmid plays an important role in mediating drug resistance of Neisseria gonorrhoeae. The isolates could exchange their plasmids in different ways, which results in the drug-resistance.Part Four. Relationship of Chromosome Bearing Drug-Resistant Genes and the Antibiotic-Resistance among N.gonorrhoeae isolatesI. Relationship Between Mutations of gyrA and parC Genes and Quinolone-Resistance among N.gonorrhoeae Isolates.Quinolone-resistant determining regions (QRDR) of gyrA and parC genes were amplified by polymerase-chain-reaction (PCR). Some of PCR products were sequenced. The sensitive strain had only one mutation in the QRDR of gyrA(Asp95) refer to the prototype, while at least two mutations were detected in QRDR of gyrA (S91F, Ser92 or Asp95 mutation) and in ParC genes (Asp86, Ser87 and Glu91) in the intermediate and high Quinolone-resistant strains.Four pairs of primers were designed to include a restriction enzyme digestion site into the amplified PCR products. The RFLP of PCR products were analyzed by digestion with restriction enzymes HinfI, SalI and PstI. Sequence and RFLP analysis of the N.gonorrhoeae isolates showed that the mutated gyrA and parC genes had a close relationship with Quinolone-resistance of N.gonorrhoeae. Mutations in gyrA may be a main reason for Quinolone resistance, especially the Ser-91→Phe mutation, while the alteration of parC is likely to play a complementary role.II. Exploring the Function of mtr System in Mediating Azithromycin Resistance of N.gonorrhoeae and the Negative Expressing Regulation of the mtr Genes in N.gonorrhoeaeMtrR and mtrR promoter regions were amplified and sequenced. Real-time PCR wasused to measure the expression level of mtrC, for which the expression was controlled by mtrR. Results showed that all the Azithromycin-intermediate and high-resistant strains had an H105Y mutation detected in mtrR coding region and a base "A" deletion in its promoter region. There was no mutation detected in mtrR promoter region among Azithromycin-sensitive strains of the prototype mtrR gene. Those strains had mutations detected in mtrR and mtrR promoter region, and had a high expression level of mtrC gene as compared with the strains without mutations detected (x~2=9.525, P＜0.05). The difference in the mtrC expression level among three resistant groups (S-sensitive, I-intermediate and R-resistant) was significant (x~2 =15.94, P<0.01). All the results indicated that mtrR could negatively regulate the expression of upstream genes. When mutations happened in the mtrR region or its promoter region, its regulation function might decrease and cause an over-expression of mtrCDE, causing resistance to antibiotics.III. Mutations in penA and ponA Genes Associated with Penicillin-resistance among N.gonorrhoeae Isolates.PCR-SSCP and PCR-RFLP were used to detect the mutations in ponA and penA genes, which encode the Penicillin Binding Protein-1 and Penicillin Binding Protein-2 (PBP1 and PBP2), respectively. All the 80 N.gonorrhoeae isolates had a D345 insertion detected in penA, which reduced the PBP2 affinity to Penicillin. We detected 93.7% of N.gonorrhoeae isolates having a point mutation Leu421—>Pro in ponA, which reduced the PBP1 affinity to Penicillin. Most of PPNG strains possessed the mutations in ponA and penA. We concluded that the plasmid and chromosome mediated Penicillin-resistance conjugately increased the resistant level.