por Variable-Region Typing by DNA Probe Hybridization Is Broadly Applicable to Epidemiologic Studies of Neisseria gonorrhoeae

Boston University, Boston, Massachusetts, United States
Journal of Clinical Microbiology (Impact Factor: 3.99). 05/2005; 43(4):1522-30. DOI: 10.1128/JCM.43.4.1522-1530.2005
Source: PubMed


The porin gene (porB) of Neisseria gonorrhoeae encodes the major outer membrane protein identified as PI or Por. To examine the utility of por variable-region (VR) typing, porB from 206 isolates was characterized by using oligonucleotide probes in a checkerboard hybridization assay that identifies
the sequence types of five VRs of both PIA and PIB porB alleles. The strains represented temporally and geographically distinct isolates, isolates from a large cluster, epidemiologically
linked partner isolates, and a collection of strains from disseminated gonococcal infections. By using rigorous epidemiologic
criteria for transmission of infection between sex partners, por VR typing was more discriminatory than serovar typing in classifying isolates from both members of 43 epidemiologically linked
pairs: 39 of 43 pairs were classified as coinciding by por VR typing compared to 43 of 43 by serovar determination (P = 0.058). porB sequence data confirmed the accuracy of the por VR method. Relationships between VR type and serovar typing monoclonal antibodies were observed for all six PIB and three
of six PIA antibodies. por VR typing is a molecular tool that appears to have broad applicability. This method can be adapted to a wide range of technologies
from simple hybridization to microarray and may allow for typing from noncultured clinical specimens.

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    • "In our study, two novel recombinant mutations formed IB3/6-IB2 and IB2-IB4-IB2 chimeric serotypes, which also indicates a high frequency of recombination among different gonococcal porB1B genes. There have been reports of N. gonorrhoeae strains that react to both PorB1A and PorB1B antibodies, raising the possibility of mosaicism between porB1A and porB1B alleles [44,45]. However, no such mosaicism was found in all the N. gonorrhoeae isolates tested in this study. "
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    ABSTRACT: Variations of porB1A and porB1B genes and their serotypes exist in Neisseria gonorrhoeae isolates from different geographical areas, and some site mutations in the porB1B gene correlate with drug resistance. The β-lactamase production of N. gonorrhoeae isolates was determined by paper acidometric test and nitrocefin discs. The porB1A and porB1B genes of 315 non-penicillinase-producting N. gonorrhoeae (non-PPNG) strains were amplified by PCR for sequencing to determine serotypes and site mutations. A duplex PCR was designed to simultaneously detect both porB1A and porB1B genes. Penicillin and tetracycline resistance was assessed by an in vitro drug sensitivity test. Of the N. gonorrhoeae isolates, 31.1% tested positive for porB1A and 68.9% for porB1B genes. All the 98 porB1A+ isolates belonging to IA6 serotype with either no mutation at the 120 and 121 sites (88.8%) or a D120G (11.2%) mutation and were no resistance to both penicillin and tetracycline. Among the 217 porB1B+ isolates, 26.7%, 22.6% and 11.5% belonged to IB3, IB3/6 and IB4 serotypes, respectively. Particularly, two novel chimeric serotypes, IB3/6-IB2 and IB2-IB4-IB2, were found in 77 and 8 porB1B+ isolates. Two hundred and twelve (97.7%) of the porB1B+ isolates were presented G120 and/or A121 mutations with 163 (76.9%) at both sites. Interestingly, within the 77 porB1B+ isolates belonging to IB3/6-IB2 serotype, 15 were discovered to possess novel deletions at both A121 and N122 sites. All the replacement mutations at these sites in PorB1B were correlated with resistance and the deletion mutation showed the highest resistance. N. gonorrhoeae isolates circulating in Eastern China include a sole PorB1A serotype (IA6) and five PorB1B serotypes. Multiple mutations in porB1B genes, including novel A121 and N122 deletions, are correlated with high levels of penicillin and tetracycline resistance.
    BMC Infectious Diseases 11/2010; 10(1):323. DOI:10.1186/1471-2334-10-323 · 2.61 Impact Factor
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    • "However, these methods have some limitations such as reproducibility and discriminatory power [5] [6] [7] [8] [9]. Therefore, different molecular genetic methods have been developed for the characterization of gonococcal strains include ribotyping [10] [11], pulsed-field gel electrophoresis (PFGE) [12], arbitrarily primed PCR [13], Amplified Fragment length polymorphism (AFLP) [14], opa-typing [15] [16], and sequence typing based on one or more genes [4] [13] [16] [17]. "
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    ABSTRACT: Control and preventive measures for gonococcal infections are based on precise epidemiological characteristics of N. gonorrhoeae isolates. In the present study the potential utility of opa-typing and ribotyping for molecular epidemiological study of consecutive gonococcal strains was determined. Sixty gonococcal isolates were subjected to ribotyping with two restriction enzymes, AvaII and HincII, and opa-typing with TaqI and HpaII for epidemiological characterization of gonococcal population. Ribotyping with AvaII yielded 6 ribotype patterns while twelve RFLP patterns were observed with HincII. Opa-typing of the 60 isolates revealed a total 54 opa-types, which 48 were unique and 6 formed clusters. Fifty-two opa-types were observed with TaqI-digested PCR product while opa-typing with HpaII demonstrated 54 opa-types. The opa-types from isolates that were epidemiologically unrelated were distinct, whereas those from the sexual contacts were identical. The results showed that opa-typing is highly useful for characterizing gonococcal strains from sexual contacts and has more discriminatory than ribotyping that could differentiate between gonococci of the same ribotype. The technique even with a single restriction enzyme has a high level of discrimination (99.9%) between epidemiologically unrelated isolates. In conclusion, the molecular methods such as opa-typing and ribotyping can be used for epidemiological characterization of gonococcal strains.
    International Journal of Microbiology 09/2009; 2009(1687-918X):934823. DOI:10.1155/2009/934823
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    • "MLST, however, has the advantage that isolate information is available alongside genotypic data in an established, publicly accessible database [23,24]. Unlike schemes that rely on antigen gene variation [5,15,29], which is subject to diversifying immune selection, MLST data can also be used to examine the evolutionary relationships among strains. "
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    ABSTRACT: Various typing methods have been developed for Neisseria gonorrhoeae, but none provide the combination of discrimination, reproducibility, portability, and genetic inference that allows the analysis of all aspects of the epidemiology of this pathogen from a single data set. Multilocus sequence typing (MLST) has been used successfully to characterize the related organisms Neisseria meningitidis and Neisseria lactamica. Here, the same seven locus Neisseria scheme was used to characterize a diverse collection of N. gonorrhoeae isolates to investigate whether this method would allow differentiation among isolates, and to distinguish these three species. A total of 149 gonococcal isolates were typed and submitted to the Neisseria MLST database. Although relatively few (27) polymorphisms were detected among the seven MLST loci, a total of 66 unique allele combinations (sequence types, STs), were observed, a number comparable to that seen among isolate collections of the more diverse meningococcus. Patterns of genetic variation were consistent with high levels of recombination generating this diversity. There was no evidence for geographical structuring among the isolates examined, with isolates collected in Liverpool, UK, showing levels of diversity similar to a global collection of isolates. There was, however, evidence that populations of N. meningitidis, N. gonorrhoeae and N. lactamica were distinct, with little support for frequent genetic recombination among these species, with the sequences from the gdh locus alone grouping the species into distinct clusters. The seven loci Neisseria MLST scheme was readily adapted to N. gonorrhoeae isolates, providing a highly discriminatory typing method. In addition, these data permitted phylogenetic and population genetic inferences to be made, including direct comparisons with N. meningitidis and N. lactamica. Examination of these data demonstrated that alleles were rarely shared among the three species. Analysis of variation at a single locus, gdh, provided a rapid means of identifying misclassified isolates and determining whether mixed cultures were present.
    BMC Biology 02/2007; 5(1):35. DOI:10.1186/1741-7007-5-35 · 7.98 Impact Factor
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