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Pulsed-Field Gel Electrophoresis as an Epidemiologic Tool for Neisseria gonorrhoeae: Identification of Clusters Within Serovars

Unemo, Magnus MSc,*; Berglund, Torsten BSc,†; OlcEn, Per MD, PhD,* AND; Fredlund, Hans MD, PhD*‡

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From the *National Reference Laboratory for Pathogenic Neisseria and ‡Unit for Infectious Disease Control, Department of Clinical Microbiology and Immunology, Örebro Medical Centre Hospital, Örebro, Sweden; and the †Units for Infectious Disease Epidemiology, Microbiology and Tumour Biology Centre, Karolinska Institutet, Stockholm, and Swedish Institute for Infectious Disease Control, Solna, Sweden

The authors thank the staff of all the Swedish clinical microbiologic laboratories, clinicians, and counselors for the isolates and epidemiologic reports. They are also grateful for the technical assistance of Birgitta Sjöberg and Karin Bäsén (antibiotic susceptibility testing), Angela Steen and Margareta Jurstrand (serologic characterization), Lotta Fransson (collection of isolates), Lisbeth Henriksson (collection of epidemiologic data), and Catherine Ison and Peter Kohl (monoclonal antibodies).

Supported by grants from the Research Committee of Örebro County, the Örebro Medical Centre Research Foundation, and the National Institute for Public Health, Sweden.

Correspondence: Magnus Unemo, MSc, Department of Clinical Microbiology and Immunology, Örebro Medical Centre Hospital, SE-701 85 Örebro, Sweden. E-mail: magnus.unemo@orebroll.se

Received for publication February 6, 2001,

revised June 26, 2001 and accepted July 5, 2001.

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Abstract

Background: The increasing incidence of gonorrhea in Sweden in 1998 was due to mostly domestic cases. Among these, two core groups were identified: homosexual men with serovar IB-2 and young heterosexuals with serovar IB-3.

Goals: To explore the genetic homogeneity/heterogeneity within the predominant serovars, IB-2 and IB-3, of Neisseria gonorrhoeae in Sweden by pulsed-field gel electrophoresis (PFGE) and to compare these results to epidemiologic information, as well as examine the genetic diversity within and between the 25 other represented serovars of N gonorrhoeae.

Study Design: By PFGE, 237 N gonorrhoeae isolates were examined, and the results were compared with epidemiologic data for the IB-2 and IB-3 isolates.

Results: In 79% of the domestic IB-2 cases involving homosexuals and 66% of the domestic IB-3 cases involving young heterosexuals, the isolates were genetically indistinguishable by PFGE. A high genetic diversity was identified within and between the 27 included serovars.

Conclusions: Examination by means of PFGE indicated that one N gonorrhoeae clone each of the serovars IB-2 and IB-3 created the majority of the two core groups of domestic cases.

GONORRHEA, including its complications, remains a major public health problem worldwide, 1 and its epidemiologic characteristics have changed over time. Precise characterization of Neisseria gonorrhoeae can provide valuable information on populations of particular strains in the community, temporal changes, and the emergence and spread of certain strains. In the absence of a vaccine, better knowledge of the molecular epidemiology of N gonorrhoeae infection will be useful for the development of improved control measures. The most widely used methods for differentiation of N gonorrhoeae strains are based on auxotyping and serologic characterization. However, these systems have inherent limitations, 2,3 and there is skepticism about whether these systems provide sufficient discrimination. 4–6 More powerful typing methods have therefore been developed; these are based on DNA characterization. 5 Digestion of genomic DNA by rarely cutting restriction endonucleases, followed by separation with pulsed-field gel electrophoresis (PFGE) of the obtained DNA fragments, is one frequently used technique. PFGE potentially indexes the whole genome and is highly discriminatory. 4–6

After many years of declining incidence of gonorrhea, Sweden reached an all-time low of 2.4 cases per 100,000 inhabitants in 1996, 7 with the majority of cases imported, just as in preceding years. 8 Since 1997, however, the incidence has increased, mostly because of a significant increase in domestic cases. 9 In a 12-month period during this increase, clinical and epidemiologic data from all cases of gonorrhea reported in Sweden were prospectively obtained, and all N gonorrhoeae isolates were phenotypically characterized by means of serotyping and antibiograms. The serovars IB-2 and IB-3 represented the predominant serovars of the N gonorrhoeae isolates. Two core groups of domestic cases were identified: those involving homosexual men with serovar IB-2 as the most prevalent N gonorrhoeae isolate, and those involving young heterosexuals of both sexes, with IB-3 as the most prevalent serological variant. 9 Subsequently, by means of such epidemiologic and microbiologic studies, the epidemiologic characteristics of gonorrhea could be better understood and the detection of core groups was made possible, as has been noted by other investigators. 10,11

The aims of the current study were to explore the genetic homogeneity/heterogeneity within the predominant serovars (IB-2 and IB-3) of N gonorrhoeae in Sweden by PFGE and to compare these results to epidemiologic information, as well as examine the genetic diversity within and between the 25 other represented serovars of N gonorrhoeae.

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Materials and Methods

One-Year Swedish N gonorrhoeae Prevalence Study

In the Swedish Communicable Diseases Act it is stated that each case of gonorrhea must be reported to the Swedish Institute for Infectious Disease Control and to the County Medical Officer of Communicable Disease Control. All cases of gonorrhea diagnosed in Sweden from February 1998 through January 1999 were included in the study. The clinical microbiologic laboratories in Sweden (n = 29) reported all the positive findings, as well as the total number of processed samples, to the institute. During the 12-month study, samples were obtained from 33,000 of the 8.8 million inhabitants of Sweden. N gonorrhoeae isolates were sent to the Swedish National Reference Laboratory for Pathogenic Neisseria (Department of Clinical Microbiology and Immunology, Örebro Medical Centre Hospital, Örebro). In addition, a questionnaire was sent to the clinician reporting each case, seeking the following information: route of transmission (homosexual or heterosexual); geographic site of exposure (town or country); partner's place (country) of residence; and laboratory number of the N gonorrhoeae strain, in order to link the epidemiologic data to the N gonorrhoeae strain isolated. At the reference laboratory all isolates were cultured for 18 hours to 20 hours at 37 °C in 5% CO2 on GCSPP agar, which is a 3% GC medium base (Difco Laboratories, Detroit) with 1% supplements (40% D-glucose, 1% L-glutamine, 0.01% cocarboxylase, and 0.05% ferric nitrate) and enrichment with 0.5% IsoVitaleX (BBL, Becton-Dickinson, Cockeysville, MD). All isolates from each patient were serotyped by the coagglutination technique with use of monoclonal antibodies 12,13 and were preserved at −70 °C.

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Gonococcal Isolates Linked with Complete Epidemiologic Data

During the study period 375 culture-proven cases of gonorrhea and five cases diagnosed by direct microscopy were reported to the Swedish Institute for Infectious Disease Control. Isolates from 357 of these patients were received at the national reference laboratory. In addition, N gonorrhoeae isolates from 13 nonreported cases were received. For the 357 reported cases with isolates, epidemiologic data and strain characteristics could be linked. In six cases the place of exposure was unknown, however, and in three other cases the route of transmission was unclear. Consequently, N gonorrhoeae isolates of 27 different serovars from 348 cases (i.e., 89% of the 393 cases of gonorrhea diagnosed during the study period) with complete epidemiologic data were received. Heterosexual exposure was predominant (262 cases, with N gonorrhoeae isolates of 27 serovars, versus 86 cases of homosexual exposure, with isolates of 12 serovars).

In the current study, we analyzed isolates of all cases involving the two predominant serovars, IB-2 (n = 44) and IB-3 (n = 84), as well as one to six isolates of the 25 remaining serovars (n = 79). In 15 of the cases, one or two additional isolates were preserved on the same occasion but from different specimens, and in 10 of the cases one to three additional strains isolated at different times were preserved. These multiple isolates (n = 30) were also included. Consequently, a total of 237 N gonorrhoeae isolates were analyzed.

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Antibiotic Susceptibility Testing

β-Lactamase production was analyzed by means of a chromogenic cephalosporin test with Nitrocefin discs (AB Biodisk, Solna, Sweden). The susceptibility to cefuroxime, erythromycin, and ciprofloxacin was determined with the E-test (AB Biodisk) on GC II agar (BBL, Becton Dickinson) supplemented with 1% hemoglobin and 1% IsoVitaleX enrichment.

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Pulsed-Field Gel Electrophoresis

N gonorrhoeae isolates were cultured on GCSPP agar for 18 hours to 20 hours at 37 °C in a 5% CO2 atmosphere. The bacteria were suspended in sterile NaCl (0.15 mol/l) to a final concentration of approximately 2 × 109 cells/ml. From each suspension, 300 μl was used for preparation and digestion of the genomic DNA of the bacteria (GenePath Group 3 Reagent Kit, Bio-Rad Laboratories, Hercules, CA), according to the manufacturer's protocol. Two different restriction enzymes were separately used:Spe I, recommended by Bio-Rad and used in previous studies, 4,6,14–16 and Bgl II, also used in previous studies. 5,16 PFGE fingerprints were obtained by separation of the digested DNA on 1% agarose gels (molecular biology certified agarose, Bio-Rad) in a contour-clamped homogenous electrical field apparatus with a hexagonal electrode array (GenePath system). The electrophoresis was performed in a 0.5× TBE buffer (45 mmol/l Tris-borate [45 mmol/l Tris base, 45 mmol/l boric acid] and 1 mmol/l EDTA), equilibrated at 14 °C with a constant voltage of 6 V/cm, with a pulse-time ramping of 2 seconds to 17 seconds for 17 hours at a 120° angle. Gels were stained with ethidium bromide, after which the separated DNA fragments were visualized under ultraviolet transillumination and digitized with Gel Doc 2000 system (Bio-Rad). The fingerprints were normalized, aligned, and compared with use of Molecular Analyst Fingerprinting software (version 1.6; Bio-Rad) and visual inspection. An international reference strain, WHO A (CCUG 15821), was included in triplicates on each gel for normalization of the gels by the software, to reduce inter-gel and intra-gel variations.

Isolates were considered genetically indistinguishable if no bands differed and distinguishable if any band difference was documented. Isolates were designated as closely related if their fingerprints differed by no more than three bands. 17

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Results

PFGE of IB-2 Isolates

There were 12 distinguishable fingerprints identified among the IB-2 isolates (n = 44) with use of Spe I and 19 identified with Bgl II (Fig. 1). Visual inspection of the restriction enzyme patterns of the Spe I-digested DNA identified one major cluster consisting of 25 indistinguishable isolates, one cluster of 6, 3 pairs of indistinguishable isolates, and seven isolates with unique patterns (Fig. 2). With use of Bgl II, the major cluster was reduced to 21 isolates. Five pairs of indistinguishable isolates and 13 with unique fingerprints were also identified. Thus, eight of the isolates were further discriminated with use of Bgl II instead of Spe I (Fig. 2).

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The major cluster of isolates that could not be distinguished with use of either of the enzymes (n = 21) comprised mainly isolates from domestic cases involving homosexuals (n = 15, or 71%). The remaining six isolates were from homosexuals exposed abroad (n = 4) and heterosexuals exposed in Sweden (n = 2). Accordingly, in 79% of all domestic IB-2 cases involving homosexuals (n = 19), the isolates were indistinguishable.

The antibiotic susceptibility testing of the 21 indistinguishable cluster isolates showed similar MICs, with differences within ±1 log2. The 21 isolates were all fully susceptible to ciprofloxacin (MIC, ≤0.064 mg/l), and none were β-lactamase-producing.

Including patterns with differences of one band to three bands increased the major cluster to 26 isolates (5 from domestic cases involving heterosexuals were included).

One suspected cluster with epidemiologic connections comprising four isolates from domestic homosexual IB-2 cases was included in the data. This collection was discriminated as two pairs of indistinguishable isolates, with four-band differences in the fingerprints with use of Spe I and more than seven-band differences with use of Bgl II. Thus, the two pairs were regarded as different. The results of PFGE were supported by differences in the antibiograms of the four isolates. One pair was fully susceptible to cefuroxime and erythromycin, whereas the other pair showed a decreased susceptibility to these antibiotics. All isolates were fully susceptible to ciprofloxacin, and none were β-lactamase-producing.

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PFGE of IB-3 Isolates

The IB-3 isolates (n = 84) yielded 15 distinguishable fingerprints with use of either of the restriction enzymes (Fig. 3). PFGE of Spe I-digested DNA identified one major cluster of 57 isolates, one minor cluster of 12, two pairs of indistinguishable isolates, and 11 isolates with unique patterns (Fig. 4). With use of Bgl II, the major cluster consisted of 52 isolates and the minor cluster comprised the same indistinguishable 12 isolates as with Spe I, as well as three other isolates (Fig. 4). Of the IB-3 isolates, 61% (51 of 84) consequently showed the same indistinguishable restriction enzyme patterns when both enzymes were used separately (Fig. 4). Altogether, 37 of 51 indistinguishable isolates (73%) were recovered in domestic cases involving young (<25 years of age) heterosexuals of both sexes. The rest of the major cluster (27%) consisted of heterosexuals exposed abroad (n = 3) and domestic cases involving heterosexuals ≥25 years of age (n = 11). Consequently, of all domestic IB-3 cases involving young heterosexuals, 66% (37 of 56) yielded indistinguishable isolates.

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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.

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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.

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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.

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Discussion

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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