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Genotypic and phenotypic characterisation of Neisseria gonorrhoeae
Linköping University, Department of Molecular and Clinical Medicine, Clinical Microbiology. Linköping University, Faculty of Health Sciences.
2003 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The bacterium Neisseria gonorrhoeae (the gonococcus) is the aetiological agent of gonorrhoea, which remains a major sexually transmitted infection/disease (STI/STD) worldwide. The incidence of gonorrhoea was previously high in many countries, Sweden included. The incidence in Sweden culminated in 1970 with 487 cases per 100,000 inhabitants. After that, the incidence declined almost every year until an all-time low of 2.4 was observed in 1996. ln 1997 the incidence of gonorrhoea began to significantly increase in Sweden, due mainly to a larger number of domestic cases of young heterosexuals of both sexes and homosexual men. This observation, in combination with the widespread use of suboptimal methods for characterisation and, in some countries, for diagnosis of the bacterium, as well as the rapid increase of resistance to several antibiotics, has intensified the research in the field of N. gonorrhoeae.

In the present thesis (paper 1), a high prevalence of decreased susceptibility or resistance to several of the traditionally used gonorrhoea antibiotics was identified and correlated to the geographic area of exposure, especially Asia. Nevertheless, effective antibiotics for treating gonorrhoea are at hand. A substantial genetic heterogeneity within identical serological variants (serovars), i.e. intraserovar, as well as interserovar of N. gonorrhoeae strains circulating within the community was revealed, emphasising the importance of using highly discriminative (DNA-based) epidemiological characterisation methods for the bacteria (papers II-V). Effective DNA-based characterisation methods, i.e. pulsed-field gel electrophmesis (PFGE) of genomic DNA digested with rarely cutting restriction endonucleases and porB gene sequencing, were adapted, optimised and evaluated against conventional phenotypic methods, as epidemiological tools on Swedish N. gonorrhoeae isolates. These molecular techniques showed a significantly higher discriminatory ability, reproducibility, and objectivity than the serovar determinations using the Genetic Systems (OS) or the Pharmacia panel (Ph) ofmonoclonal antibodies (MAbs) (papers II & III). According to a comparison of serologic and genetic parB-based typing of N. gonorrhoeae, the precise amino acid residues of porB, critical for the reactivity of several of the GS MAbs, were difficult to identity (paper IV). In papers IV & V, a determination of genetic group (genogroup) and genetic variant (genovar) was developed based on real-time PCR of the entire porB gene with subsequent sequence analysis in real-time by synthesis, i.e. pyrosequencing technology, of short, highly polymorphic porB gene segments. This method provides a rapid, high-throughput, objective, highly discriminative, typeable, portable for interlaboratory comparisons, and reproducible molecular characterisation for N. gonorrhoeae. Genogroup and genovar determination can complement or even replace the internationally established serovar determination in routine use for following the transmission of individual strains in the community and confirming presumed epidemiological connections or discriminating isolates of suspected clusters of gonorrhoea cases.

Abstract [sv]

Bakterien Neisseria gonorrhoeae (gonokocken) orsakar den sexuellt överförbara infektionen gonorré som med eller utan allvarliga komplikationer, som exempelvis infertilitet och utomkvedshavandeskap, är ett globalt folkhälsoproblem. Infektionen var tidigare mycket vanlig i flertalet länder, så även i Sverige där incidensen kulminerade år 1970 varefter den sjönk i stort sett varje år fram till och med år 1996. Från och med 1997 började infektionens incidens öka igen i Sverige, vilket också kunde noteras i flera andra länder. Framförallt orsakades den svenska ökningen av en ökad inhemsk smittspridning bland yngre heterosexuella kvinnor och män samt homosexuella män. En ökad resistens hos bakterien mot flertalet antibiotika är ett känt problem sedan decennier. De senaste åren har en nationell och internationell intensifiering skett av forskningen. Fokus ligger på effektiva diagnostiska tekniker, optimala karakteriseringsmetoder, epidemiologiska studier samt identifiering och övervakning av begynnande eller ökande antibiotikaresistens. Även patogenes, virulens och vaccinutveckling studeras.

I denna avhandlings arbete I identifierades, bland svenska N. gonorrhoeae isolat, en hög prevalens av nedsatt känslighet eller resistens mot flertalet av de traditionella antibiotika för behandling av gonorré. En korrelation mellan nedsatt känslighet/resistens och geografisk smittort, framförallt Asien, kunde fastställas. Flera olika antibiotika för effektiv behandling finns dock fortfarande tillgängliga. I arbete I-V identifierades en stor genetisk heterogenitet inom och mellan olika serologiska varianter (serovarer) av N. gonorrhoeae stammar. Detta belyser behovet av att använda högdiskriminerande (DNA-baserade) metoder för epidemiologisk karakterisering av bakterien. Effektiva DNA-baserade molekylärgenetiska tekniker, som pulsfältgelelektrofores (PFGE) av genomiskt DNA efter klyvning med restriktionsenzym och sekvenseling av porB genen, adapterades, optimerades och evaluerades mot traditionella fenatypiska metoder, som epidemiologiska verktyg för svenska N. gonorrhoeae isolat. Båda metoderna visade en signifikant högre diskriminemnde förmåga, reproducerbarhet och objektivitet än traditionell karakterisering (arbete II & III). En jämförelse mellan serologisk och genetisk porB-baserad typning av N. gonorrhoeae visade på stora svårigheter att komplett identifiera de använda monoklonala antikropparnas antigena epitoper hos porB (arbete IV). I arbete IV & V utvecklades en bestämning av genetisk grupp (genogrupp) och genetisk variant (genovar) baserad på realtids-PCR av hela porB genen med efterföljande sekvensering i realtidsformat, med hjälp av pyrosequencing teknologi, av korta högvariabla segment av porB genen. Metoden är mycket snabb, högdiskriminerande, reproducerbar, objektiv samt har en hög kapacitet. Metoden skulle kunna ersätta alternativt komplettera den bristfalliga men internationellt etablerade och rutinmässigt använda serovarbestämningen av N. gonorrhoeae för att identifiera spridningen av individuella stammar i samhället, karakterisera isolat i misstänkta kluster av gonorréfall och för rutinmässig partnerspårning.

Place, publisher, year, edition, pages
Linköping: Linköpings universitet , 2003. , 103 p.
Series
Linköping University Medical Dissertations, ISSN 0345-0082 ; 828
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:liu:diva-26673Local ID: 11240ISBN: 91-7373-517-5 (print)OAI: oai:DiVA.org:liu-26673DiVA: diva2:247222
Public defence
2003-12-19, Wilandersalen, Universitetssjukhuset, Örebro, 13:00 (Swedish)
Opponent
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2012-10-04Bibliographically approved
List of papers
1. One year of Neisseria gonorrhoeae isolates in Sweden: the prevalence study of antibiotic susceptibility shows relation to the geographic area of exposure
Open this publication in new window or tab >>One year of Neisseria gonorrhoeae isolates in Sweden: the prevalence study of antibiotic susceptibility shows relation to the geographic area of exposure
Show others...
2002 (English)In: International Journal of STD and AIDS (London), ISSN 0956-4624, E-ISSN 1758-1052, Vol. 13, no 2, 109-114 p.Article in journal (Refereed) Published
Abstract [en]

The aim of this study was to compare epidemiological data with antibiotic susceptibility patterns, so as to characterize the risk of infection with a highly resistant Neisseria gonorrhoeae strain. N. gonorrhoeae strains isolated in Sweden from February 1998 through January 1999 were tested for antibiotic susceptibility. Epidemiological data were received from each clinician reporting a case of gonorrhoea and these data were linked to the N. gonorrhoeae strains. A total of 348 N. gonorrhoeae isolates, representing 89% of all Swedish cases diagnosed during the 12-month period, were tested for antibiotic susceptibility. Of all isolates, 24% were β-lactamase-producing, and 18% had decreased susceptibility to ciprofloxacin (MIC>0.064 mg/l). All isolates were fully susceptible to ceftriaxone and spectinomycin. More than 99% of the isolates were fully susceptible to azithromycin. The antibiotic susceptibility varied with the places where patients were exposed to infection. When exposed in Asia, 63% of the isolates showed reduced susceptibility to ciprofloxacin, compared with 0-8.5% of the isolates from patients exposed in other places (RR=8.5, P<0.001). Ciprofloxacin cannot be recommended as the first choice of treatment if the place of exposure was in Asia.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26456 (URN)10.1258/0956462021924730 (DOI)11004 (Local ID)11004 (Archive number)11004 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
2. Pulsed-field gel electrophoresis as an epidemiologic tool for Neisseria gonorrhoeae: Identification of clusters within serovars
Open this publication in new window or tab >>Pulsed-field gel electrophoresis as an epidemiologic tool for Neisseria gonorrhoeae: Identification of clusters within serovars
2002 (English)In: Sexually Transmitted Diseases, ISSN 0148-5717, E-ISSN 1537-4521, Vol. 29, no 1, 25-31 p.Article in journal (Refereed) Published
Abstract [en]

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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26457 (URN)11773875 (PubMedID)11005 (Local ID)11005 (Archive number)11005 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
3. Molecular epidemiology of Neisseria gonorrhoeae: sequence analysis of the porB gene confirms presence of two circulating strains
Open this publication in new window or tab >>Molecular epidemiology of Neisseria gonorrhoeae: sequence analysis of the porB gene confirms presence of two circulating strains
Show others...
2002 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 40, no 10, 3741-3749 p.Article in journal (Refereed) Published
Abstract [en]

The phenotypic and genotypic characteristics of Neisseria gonorrhoeae strains fluctuate over time both locally and globally, and highly discriminative and precise characterization of the strains is essential. Conventional characterization of N. gonorrhoeae strains for epidemiological purposes is mostly based on phenotypic methods, which have some inherent limitations. In the present study sequence analysis of porB1b gene sequences was used for examination of the genetic relationships among N. gonorrhoeae strains. Substantial genetic heterogeneity was identified in the porB genes of serovar IB-2 isolates (8.1% of the nucleotide sites were polymorphic) and serovar IB-3 isolates (5.2% of the nucleotide sites were polymorphic) as well as between isolates of different serovars. The highest degree of diversity was identified in the gene segments encoding the surface-exposed loops of the mature PorB protein. Phylogenetic analysis of the porB1b gene sequences confirmed previous findings that have indicated the circulation of one N. gonorrhoeae strain each of serovar IB-2 and serovar IB-3 in the Swedish community. These strains caused the majority of the cases in two domestic core groups comprising homosexual men and young heterosexuals, respectively, and were also detected in other patients. The phylogenetic analyses of porB gene sequences in the present study showed congruence, but not complete identity, with previous results obtained by pulsed-field gel electrophoresis of the same isolates. In conclusion, porB gene sequencing can be used as a molecular epidemiological tool for examination of genetic relationships among emerging and circulating N. gonorrhoeae strains, as well as for confirmation or discrimination of clusters of gonorrhea cases.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26455 (URN)10.1128/​JCM.40.10.3741-3749.2002 (DOI)11003 (Local ID)11003 (Archive number)11003 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
4. Comparison of serologic and genetic porB-based typing of Neisseria gonorrhoeae: consequences for future characterization
Open this publication in new window or tab >>Comparison of serologic and genetic porB-based typing of Neisseria gonorrhoeae: consequences for future characterization
2003 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 41, no 9, 4141-4147 p.Article in journal (Refereed) Published
Abstract [en]

Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of Neisseria gonorrhoeae is essential. In the present study genetic heterogeneity in the porB genes of N. gonorrhoeae was examined, and serovar determination was compared to porB gene sequencing. Among 108 N. gonorrhoeae isolates, phylogenetic analysis of the entire porB alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous porB gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the porB gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of N. gonorrhoeae. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108 N. gonorrhoeae isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the porB gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of N. gonorrhoeae strains within the community.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-26458 (URN)10.1128/​JCM.41.9.4141-4147.2003 (DOI)11006 (Local ID)11006 (Archive number)11006 (OAI)
Available from: 2009-10-08 Created: 2009-10-08 Last updated: 2017-12-13Bibliographically approved
5. Genetic variant (genovar) determination of Neisseria gonorrhoeae by pyrosequencing of highly polymorphic segments of the porB gene
Open this publication in new window or tab >>Genetic variant (genovar) determination of Neisseria gonorrhoeae by pyrosequencing of highly polymorphic segments of the porB gene
(English)Manuscript (preprint) (Other academic)
Abstract [en]

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in the pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The DNA sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy-sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each one of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface exposed amino acid loops of the mature porB protein) ranged from 5 to 11 and 8 to 39, respectively. Among porB1a isolates (n=22) and porB1b isolates (n=65), 22 and 64 unique genovars were identified. All isolates were typeable. The current results provide evidence of a high discriminatory ability, practically the same as sequencing of the entire porB gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of N. gonorrhoeae.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:liu:diva-84322 (URN)
Available from: 2012-10-04 Created: 2012-10-04 Last updated: 2012-10-04Bibliographically approved

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