PHENOTYPIC AND GENOTYPIC CHARACTERIZATION OF NEISSERIA GONORRHOEAE ISOLATES IN SINGAPORE

Abstract

A study was carried out on 65 clinical isolates of Neisseria gonorrhoeae collected from Singapore in 1984. The strains were previously typed serologically by coagglutination as belonging to WI, WII and WIII (Sandstrom & Danielsson, 1980) and were further classified into serovar using monoclonal antibodies directed against epitopes of protein I. They were characterized in terms of their auxotypes, plasmid content and sensitivity to 10 antibiotics. Each isolate was also tested for »-lactamase production. The W-serogrouped isolates were further subdivided into 14 serovars. Serovar Aehih prevailed among the WI serogroups, Bacjk for WII and Beghjk for WIII. An expected correlation among the PPNG strains to possess the 4. 4-Md (Asian+) plasmid and high MIC for penicillin derivatives (penicillin-G and ampicillin) were found. Three plasmid profiles were evident among the 40 non-PPNG while 2 profiles for the 25 PPNG isolates were observed. The 24.5-Md conjugative plasmid were found in both PPNG and non-PPNG strains. Forty-nine percent of the isolates were proline (Pro-) requiring, followed by 29% wild type strains (Proto). The remaining 9 strains were comprised of three other auxotypes. Five non-PPNG strains did not grow on any of the auxotyping medium. When serovar, auxotyping and B-lactamase production were correlated, 36 combinations were observed. Chloramphenicol, tetracycline and erythromycin seemed to be the drug of choice for the treatment for all the isolates, since they demonstrated MICs between 0.0312 mg/1 and 8 mg/J. which were relatively low compared to the other antibiotics tested, although rifampicin can be administered for the treatment of the non-PPNG strains as revealed by their invitro susceptibility testing. A method for endonuclease analysis DNA was preparation for restriction developed for the genotypic differentiation of N. gonorrhoeae. Different protocols of DNA extraction were employed to isolate the total genomic DNA. Gonococcal DNA from 26 isolates were cleaved with eleven different restriction enzymes and electrophoresed in horizontal agarose gels· to determine their genomic "fingerprints". The in-situ extraction method ( Dawson et al., 1986) which makes use of entrapped bacterial cells in low melting point agarose, followed by in-situ lysis and restriction enzyme cleavage, was found to be a simple and fast method. The restriction endonuclease patterns were reproducible and stable after subsequent in-vitro passages. Other techniques such as pulse-field electrophoresis and reverse-field electrophoresis were also applied to improve the separation of the tight DNA bands generated by restriction enzymes ClaI, Sau3AI, EcoRI, HindIII and AvaII. None was found to resolve the restriction patterns in a way comparable to the conventional horizontal agarose gel method, which has always been a rapid and simple set-up. BglII and HinfI were found suitable for the analysis as they produced restriction endonuclease patterns that were easily read. on the basis of densitometric scanning of electrophoretograms generated by HinfI digestion, the 26 isolates representing 12 serovars were divided into 7 groups. BgIII was found to be more descriminative and 15 RE patterns were established among the 26 isolates. RE patterns generated by both restriction enzymes showed that there was no correlation between a particular RE pattern and a serovar as strains with identical RE pattern were from different serovars. With the exception of two strains (D3 and D14) which demonstrated positive correlation using both enzymes, the rest with identical serovar pattern had different RE patterns. Multilocus enzyme electrophoresis technique was also applied to differentiate strains and to determine the genetic relatioships of the 65 isolates. Electrophoretic mobilities of 9 metabolic enzymes were analysed. Isolates with the same electrophoretic enzyme profile were classified as belonging to the same electrophoretic type (ET). There were 10 enzyme loci and 16 ETs detected. The average heterogeneity (genetic diversity) per enzyme loci was found to be 0.2118. The 14 serovars were further subdivided into 45 ETs. Ali the isolates were assigned to ETs including the non-auxotypable strains. This technique may be suitable as an epidemiologic marker as well as in the characterization of Neisseria gonorrhoeae.