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Background: Quinolone-resistant Neisseria gonorrhoeae (QRNG) arise from mutations in gyrA (intermediate resistance) or gyrA and parC (resistance). Here we tested the consequence of commonly isolated gyrA(91/95) and parC86 mutations on gonococcal fitness., Methods: Mutant gyrA(91/95) and parC86 alleles were introduced into wild-type gonococci or an isogenic mutant that is resistant to macrolides due to an mtrR(-79) mutation. Wild-type and mutant bacteria were compared for growth in vitro and in competitive murine infection., Results: In vitro growth was reduced with increasing numbers of mutations. Interestingly, the gyrA(91/95) mutation conferred an in vivo fitness benefit to wild-type and mtrR(-79) mutant gonococci. The gyrA(91/95), parC86 mutant, in contrast, showed a slight fitness defect in vivo, and the gyrA(91/95), parC86, mtrR(-79) mutant was markedly less fit relative to the parent strains. A ciprofloxacin-resistant (Cip(R)) mutant was selected during infection with the gyrA(91/95), parC86, mtrR(-79) mutant in which the mtrR(-79) mutation was repaired and the gyrA(91) mutation was altered. This in vivo-selected mutant grew as well as the wild-type strain in vitro., Conclusions: gyrA(91/95) mutations may contribute to the spread of QRNG. Further acquisition of a parC86 mutation abrogates this fitness advantage; however, compensatory mutations can occur that restore in vivo fitness and maintain Cip(R).

Typing of the porB variable region (VR) is an epidemiological tool that classifies gonococcal strains based on sequence differences in regions of the porB gene that encode surface-exposed loops. The frequent isolation of certain porB VR types suggests that some porin sequences confer a selective advantage during infection and/or transmission. Alternatively, certain porin types may be markers of strains that are successful due to factors unrelated to porin. In support of the first hypothesis, here we show urogenital tract isolates representing the most common PIA VR types identified in an urban clinic in Baltimore, MD, over a 10-year period belonged to several different clonal types, as determined by pulsed-field gel electrophoresis (PFGE). Serum resistance, which was confirmed by factor H and C4b-binding protein binding studies, was more often associated with gonococcal the most common VR types. In contrast, three porin-independent phenotypes, namely, lactoferrin utilization, beta-lactamase production, and multiple transferable resistance (Mtr), were segregated with the PFGE cluster and not with the VR type. Data combined with another PIA strain collection showed a strong correlation between serum resistance and the most common VR types. A comparison of VR typing hybridization patterns and nucleotide sequences of 12 porB1a genes suggests that certain porin loop 1, 3, 6, and/or 7 sequences may play a role in the serum resistance phenotype. We conclude that some PorB PIA sequences confer a survival or transmission advantage in the urogenital tract, perhaps via increased resistance to complement-mediated killing. The capacity of some porin types to evade a porin-specific adaptive immune response must also be considered.

Background: Distinguishing the recent introduction of quinolone resistant gonococci into a population from diversification of resistant strains already in the population is important for planning effective infection control strategies. We applied molecular evolutionary analyses to DNA sequences from 9 housekeeping genes and gyrA, parC and porB of 24 quinolone resistant N. gonorrhoeae (QRNG) and 24 quinolone sensitive isolates collected in Israel during 2000-2001., Results: Phylogenetic and eBURST analyses and estimates of divergence time indicated QRNG were introduced on 3 separate occasions and underwent limited diversification by mutation, deletion and horizontal gene transfer. Reconstruction of N. gonorrhoeae demography showed a slowly declining effective strain population size from 1976 to 1993, rapid decline between 1994 and 1999, and an increase from 1999 to 2001. This is partially attributable to declining gonorrhea case rates from 1973 to 1994. Additional contributing factors are selective sweeps of antibiotic resistant gonococci and increased transmission from sex workers. The abrupt decline in the mid-1990s heralded an increased incidence of gonorrhea from 1997 to the present. The subsequent increase in effective strain population size since 1999 reflects the increased gonococcal census population and introduction of quinolone resistance strains., Conclusion: Our study demonstrates the effective use of population genetic approaches to assess recent and historical population dynamics of N. gonorrhoeae.

A polymerase chain reaction (PCR)-based enzyme-linked immunosorbent assay (ELISA) methodology was developed to characterize Neisseria gonorrhoeae porB gene variable regions (VR); the methodology was evaluated in comparison to porB VR typing by checkerboard hybridization. Clinical noncultured samples from 35 men who have sex with men (MSM), positive by nucleic amplification assays for N. gonorrhoeae, were typed using a panel of 40 oligonucleotide probes to porB VRs and compared to checkerboard hybridization. Complete concordance was observed between the two methods at PIB VRs 1, 3, and 7. At the more degenerate VRs 5 and 6, PCR ELISA resulted in obtaining more typeable VRs than checkerboard hybridization due to single nucleotide mismatches. By PCR ELISA, two predominant PIB porB types were identified in 58% of the samples and the remaining 16 samples had one of six other porB types. Both PCR ELISA and checkerboard hybridization methods of porB VR typing allowed characterization of N. gonorrhoeae from noncultured clinical samples including throat and rectal swabs and discriminated N. gonorrhoeae from N. meningitidis present in some of the samples. PCR ELISA is a rapid, relatively inexpensive and alternative molecular typing method for N. gonorrhoeae, suitable for use in conjunction with molecular diagnostic tests., (Copyright (c) 2007 Wiley-Liss, Inc.)

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.

Molecular methods that characterize the Neisseria gonorrhoeae porin protein Por are needed to study gonococcal pathogenesis in the natural host and to classify strains from direct clinical samples used with nucleic acid amplification-based tests. We have defined the capabilities of por variable region (VR) typing and determined suitable conditions to apply the method to direct clinical specimens. Nested PCR from spiked urine samples detected 1 to 10 copies of template DNA; freezing spiked whole urine greatly reduced the ability to amplify porB. In a laboratory model of mixed gonococcal infections, the por type of one strain could be determined in the presence of a 100-fold excess of another. por VR typing was used to examine clinical samples from women enrolled in studies conducted in Baltimore, Md., and Madagascar. por type was determined from 100% of paired cervical swab and wick samples from 20 culture-positive women from Baltimore; results for eight individuals (40%) suggested infection with more than one strain. In frozen urine samples from Madagascar, porB was amplified and typed from 60 of 126 samples from ligase chain reaction (LCR)-positive women and 3 samples from LCR-negative women. The por VR types of 13 samples (21%) suggested the presence of more than one gonococcal strain. Five por types, identified in >45% of women with typed samples, were common to both geographic areas. Molecular typing is an important adjunct to nucleic acid amplification-based diagnostics. Methods that utilize direct clinical samples and can identify mixed infections may contribute significantly to studies of host immunity, gonococcal epidemiology, and pathogenesis.

Quinolone resistance is increasing rapidly in Neisseria gonorrhoeae and is a significant public health problem that requires ongoing surveillance. To examine the feasibility of molecular surveillance of quinolone resistance, and to further characterize an outbreak of resistant N. gonorrhoeae in Israel, the quinolone resistance-determining region (QRDR) sequences and the por types of 80 N. gonorrhoeae isolates were determined using molecular techniques. QRDRs of gyrA and parC were amplified by polymerase chain reaction and were sequenced directly. The por type was determined by checkerboard hybridizations performed using oligonucleotide probes to regions encoding 5 variable loops of the porin protein. All 42 ciprofloxacin-resistant (CipR) isolates had mutations in QRDRs of both gyrA and parC, and identical mutations were found in 93% of these isolates. One intermediately resistant isolate had 1 mutation in gyrA, and susceptible isolates showed no mutations. Forty isolates had 1 of 2 por types that differed only by an in-frame deletion in variable region 5; all but 1 of these isolates were CipR. QRDR sequencing and por type determination showed that the outbreak of CipR N. gonorrhoeae in Israel was clonal. QRDR mutations were consistent with those previously characterized; this indicates that DNA probes can be developed for rapid detection and surveillance of quinolone-resistant N. gonorrhoeae in settings in which nonculture diagnostic methods are used.

The Neisseria gonorrhoeae porin protein (Por) is a potential vaccine target and is the antigenic determinant for serovar typing. Two classes of Por, PIA and PIB, and antigenically distinct variants within each class result from sequence variations in the por gene variable regions (VRs) encoding surface-exposed loops. Oligonucleotide probes to 5 VRs of each class were used in checkerboard hybridizations to type 282 clinical gonococcal isolates selected from strains collected over the course of 10 years. PIA strains (n=63) showed limited por diversity, with 90% having 1 of 4 por types. PIB strains (n=219) were more diverse, although several common por types were identified that persisted over time. Variation within individual VRs was found to be limited. The present study provides information about the diversity of Por in strains circulating in a single geographic region over time, illustrates the utility of a novel por typing method, and has implications for vaccine development.

The porin proteins PIA and PIB of Neisseria gonorrhoeae are serotyping antigens for the serovar classification system and leading candidates for gonococcal vaccine development. Although serotyping has been a useful tool, this method can be insensitive to critical sequence changes in the por gene, including those in surface-exposed variable regions (VRs). A sensitive and specific typing system for N. gonorrhoeae has been developed that uses biotin-labeled oligonucleotide probes with chemiluminescence detection to type PIB gene VRs. The PIB VR types of geographically and temporally diverse gonococcal strains and sexual contact isolates were determined. por VR typing discriminated between most unrelated isolates and provided information about individual VR type that was not apparent from serovar designations. PIB VR typing avoids limited monoclonal antibody availability, interlaboratory variation, and the requirement for culture-based surveillance associated with gonococcal serotyping, and provides useful information about the molecular epidemiology of individual por gene VRs.