The antibiotic susceptibility testing of indistinguishable isolates from the 37 domestic cases involving young heterosexuals showed similar MIC values; in 33 of the 37 cases, the difference was within ±1 log2. The 37 isolates were all fully susceptible to ciprofloxacin, and none produced β-lactamase.
Including patterns with one-band to three-band differences increased the major cluster to 69 isolates and the percentage of isolates from domestic cases involving young heterosexuals to 77%.
Among the 84 IB-3 isolates, there were also four presumed clusters of isolates (n = 2, n = 2, n = 3, and n = 6) from young heterosexuals with epidemiologic connections, all exposed in one small town in Sweden. Three of these clusters (belonging to the major cluster of the IB-3 isolates) were confirmed by indistinguishable genetic fingerprints and similar antibiograms. In the fourth cluster (n = 2), one of the isolates belonged to the major cluster, but the other isolate differed in the fingerprint by five bands with use of Spe I. The isolates could not be distinguished by PFGE with Bgl II or by the antibiograms.
PFGE of Isolates of the 25 Other Represented Serovars
A total of 79 clinical isolates of 25 different serovars yielded 50 distinguishable fingerprints with Spe I and 63 with Bgl II.
With use of Spe I, PFGE revealed 33 isolates with unique patterns, seven pairs of indistinguishable isolates of the same serovars, and 32 isolates with fingerprints that were indistinguishable from fingerprints of isolates of some other serovar. Consequently, 10 different fingerprint patterns were seen among 32 isolates belonging to different serovars.
With use of Bgl II, 51 isolates were considered unique, five pairs and one triplet of indistinguishable isolates were of the same serovar, and 15 isolates were indistinguishable from isolates of some other serovar. Thus, six fingerprint patterns were identified among 15 isolates of different serovars.
In addition, six of the Spe I fingerprint patterns identified among the IB-2 isolates and one of the IB-3 isolates were also seen among the isolates of the 25 other represented serovars.
Four of the Bgl II fingerprints of the IB-2 isolates were indistinguishable from some of the fingerprints of the isolates of the 25 other represented serovars.
PFGE of Multiple Isolates from the Same Patient
For 21 of the 25 patients from whom more than one isolate was obtained, all isolates were genetically indistinguishable by PFGE with either of the two enzymes. In 20 cases this finding was in congruence with the identical results for the isolates in phenotypic characterization (serotyping and antibiotic susceptibility testing), and in one case it was in such congruence except that one β-lactamase-producing and one β-lactamase-negative isolate were preserved on the same occasion.
From four patients, two different N gonorrhoeae strains were isolated. One of the patients was infected with one strain of the serovar IB-6 but also a strain of the serovar IB-33, preserved on the same occasion. These strains had different genetic fingerprints (more than seven-band differences, independent of enzyme used) but similar antibiograms. From three of the patients, two strains of the same serovar were isolated. The isolates from two of these patients were preserved on the same occasion but from different specimens, and the isolates from one of the patients were preserved on different occasions. The two isolates of each patient showed separately indistinguishable fingerprints with use of Bgl II and similar antibiograms. However, with use of Spe I, the fingerprints of the two isolates from each patient were only closely related, with two-band or three-band differences.
The results of the current study, together with background epidemiologic and microbiologic data, indicate a spread of one N gonorrhoeae clone of each of the serovars IB-2 and IB-3, accounting for most of the cases in the two identified domestic core groups. None of these N gonorrhoeae strains showed any decreased susceptibility to the antibiotics tested. Since the study period, the domestic core group of young heterosexuals infected with N gonorrhoeae IB-3 strains has practically disappeared (data not included), probably because of greater awareness of the core group, accurate contact-tracing, effective antibiotic treatment, and dissemination of information about risk behaviors to the affected core group. However, in 2000, there was still an increase in domestic homosexual cases due to serovars other than IB-2, predominantly in the Stockholm area.
The current study also identified high genetic diversity within the two preponderant serovars of N gonorrhoeae, IB-2 and IB-3. This heterogeneity within serovars was also noted among the 25 other serovars included. According to the results, PFGE fingerprinting with use of Spe I or Bgl II is a sharper and more discriminating epidemiologic tool than the routinely used serological typing. This observation is in full agreement with the findings of previous studies. 4–6 However, characterization by PFGE is a new system of classification and not congruent with serological typing because it offers greater discrimination; on the contrary, for some isolates of different serovars, PFGE showed indistinguishable genetic fingerprints, as seen in the current study and others. 4
Serological typing is important as a primary epidemiologic marker, but PFGE could, when required, be used for greater discrimination within serovars.
Thus, by PFGE it is possible to confirm presumed epidemiologic connections or discriminate isolates of suspected clusters. In the current study, one suspected cluster comprising four IB-2 isolates from domestic homosexual cases and one comprising two IB-3 isolates from young heterosexuals with presumed epidemiologic connections were further discriminated by PFGE. In addition, three IB-3 clusters were confirmed.
Four patients infected with two different strains of N gonorrhoeae were identified. In three of these cases the different isolates were preserved on the same occasion, and these findings indicate double infections with different N gonorrhoeae strains. In one case different strains were preserved on different occasions, perhaps indicating reinfection with another N gonorrhoeae strain. The isolates from one patient with presumed double infection were of the serovars IB-6 and IB-33. These serovars differ only by the reaction of two monoclonal antibodies in serological characterization; there have been problems with these monoclonal antibodies (2D6 and 2G2) in terms of reproducibility of coagglutination reactions in previous studies. 3 PFGE, however, confirmed the serological difference. In three of the cases with different strains, neither phenotypic characterization nor PFGE with Bgl II could distinguish the isolates. PFGE fingerprinting with Spe I, however, distinguished the isolates with two-band to three-band differences, consistent with the occurrence of one genetic event. One possibility is that it is the same strain but that a minor genetic change in the bacterial genome has occurred in one of the strains.
Thus, the presumed diverse strains of these three patients could not be recognized with phenotypic characterization only by PFGE with Spe I. These results show the benefit of a typing technique that has greater discriminatory ability than serotyping. However, even minor genetic changes of the bacterial chromosome, i.e., point mutations, can cause large differences in the genetic fingerprints of isolates. This fact makes PFGE unsuitable for use as the one and only method of strain characterization. One of the patients from whom multiple isolates were recovered also was infected with a strain that, as noted in one isolate, possibly had lost the β-lactamase plasmid.
Overall, PFGE, independent of the restriction enzyme used, showed a high discriminatory ability and typeability (all 237 isolates were typeable).
In the comparison between the two restriction enzymes used in PFGE, Bgl II showed greater discriminatory ability than Spe I, especially for IB-2 isolates and the majority of the other represented serovars. For IB-3 isolates the major cluster was further discriminated with use of Bgl II, but overall the two restriction enzymes had an approximately equal discriminatory ability for the IB-3 isolates. Overall, Bgl II seems to be the best restriction enzyme to use in PFGE on N gonorrhoeae isolates, as noted in a previous study. 16 However, the Bgl II fingerprints contained more bands and in a few cases were more difficult to interpret.
It would be interesting to further refine the characterization of the isolates used in the current study by means of a genetic method, analyzing a single genetic locus. This approach would complement the great discriminatory ability of PFGE which can potentially index the whole genome. One possible genetic locus to study is the porB gene–encoding PorB, which forms the basis for the serological typing. In the future, it would also be of great interest to reproduce the current study with material collected over 1 year or several years. This would make it possible to examine genetic relatedness in strain populations in the community, temporal and geographic changes, and perhaps evolutionary changes. The results of such studies would certainly resolve questions about emergence and spread of existing and newly introduced N gonorrhoeae strains, information which would improve development of effective prevention and control measures.
Because of the great variety of N gonorrhoeae isolates identified by PFGE and with phenotypic characterization, as well as the low prevalence of gonorrhea in Sweden, it is no longer acceptable to use direct microscopy or DNA amplification techniques as the sole methods for diagnosis. Today these methods are unable to detect any antibiotic susceptibility patterns or give important epidemiologic information about strain populations, clonal relationships between strains, or the emergence and spread of different strains. This information, together with epidemiologic data from questionnaires, is the basis for identifying core groups and risk behaviors and accomplishing accurate contact-tracing, effective antibiotic treatment, and all kinds of preventive measures essential to stemming the increase of gonorrhea in the Swedish community.
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