VR typing is an attractive approach for gonococcal strain typing. As a molecular method, it can be applied to nonviable bacterial cell samples (13
); the reagents are easily synthesized, and the method is easy to use and highly reproducible. In order to examine the utility of por
VR typing as a microbiologic and epidemiologic tool, we determined the por
types of strains from several well-characterized collections. The results of this study suggest that por
VR typing has broad applicability when used with the present set of 40 oligonucleotide probes. por
VR typing discriminated unrelated strains and was able to accurately identify epidemiologically linked isolates; no strains were completely nontypeable, and the accuracy of por
VR typing was confirmed by porB
We explored the relationship between typing MAbs (19
) and por
VR types. Previous studies have identified specific binding epitopes or suggested binding regions for several antibodies. Carbonetti et al. localized MAb 1F5 to the N-terminal 60 residues and 3C8, 2H1, and 2D4 to the loop 5 or 6 regions of the PIB strain MS11 by using constructed PIA/PIB hybrids (3
). Cooke et al. further determined that antibody 2D4 bound an epitope of PIB loop 6 encoded by the sequence K(L/Y)YQNQLVRD and suggested the loop 5 sequence YSIPS as the epitope for 3C8 (8
). The porB
sequences of naturally occurring PIA/PIB hybrids and a PIB VR5 deletion strain supported these observations and indicated that the 2H1 antibody bound a loop 5 region that was common to most PIB strains (9
). Unemo et al. recently expanded and refined the comparison of the porB
sequence and serovar (37
). In our study, the associations of antibody 1F5 with PIB1-1 and antibody 2D4 with probe PIB6-5 are consistent with these reports; and probes PIB5-1, PIB5-2, PIB5-4, PIB5-7, and PIB5-8, corresponding to MAb 3C8, all identify sequences encoding the sequence YS(I/M)PS in the loop 5 region. Epitopes for antibodies 2G2 and 2D6 have not been previously identified, but sequence and serovar data from the GenBank and the data presented here suggest that antibody 2G2 corresponds to sequences that hybridize PIB5-3, PIB5-5, PIB5-6, and some sequences that hybridize PIB5-9 while antibody 2D6 corresponds to sequences binding probes PIB6-4 and PIB6-6 and some that bind PIB6-2.
Previous studies indicate that PIA MAbs 4A12, 5G9, and 5D1 recognize complex epitopes involving both the N- and C-terminal regions, MAb 6D9 binds the loop 6 DAKLTWRND region of strain FA19, MAb 4G5 binds the loop 3 sequence IAQPEE, and 2F12 binds the N-terminal region of strain FA19 (3
). In this study, the PIA1-1 probe identified the N-terminal sequence required for 5D1 binding. The VR6 porB
sequence of FA19 (GenBank accession number J03029
]) differs from the PIA6-1 probe by 1 nucleotide and from the PIA6-4 probe by 2 nucleotides, explaining the FA19 hybridization pattern PIA6-1,4 that corresponds to MAb 6D9. The 4G5 epitope corresponds to PIA3-1 and PIA3-2, consistent with the sequence analysis by Unemo et al. (37
). 2F12 binding was associated with hybridization of probes PIA1-1, PIA3-1, and PIA7-1, but in the context of earlier work by Carbonetti et al. (3
), the epitope corresponds to PIA1-1.
Among partner strains overall, common serovars within each por
type group were consistent with the relationships described above. In many instances, though, a number of different serovars were seen within por
type groups, consistent with previous reports of discrepancies between serovar determinations and porB
). Interestingly, based on the relationships between serovar MAbs and the previous polyclonal typing system (19
), the four most common por
types among these strains would also be predicted to correspond to WI (A 1;2;1;1;1), WII (B 2;2;4;4;2 and B 2;2;7;4;2), and WIII (B 2;1;6;5;3) isolates. Regardless of the differences observed between serovar and por
VR type, concordance was very high among partners that met epidemiologic criteria. The few partnerships that were identified as discordant by por
VR typing may have occurred because among partners that were both infected, it is possible that not all identified strains had been exchanged between partners. By using rigorous epidemiologic criteria (21
), concordance rates of 97% among single male-female partnerships and >90% including males with multiple identified partners were observed. Confirmations of concordance and the ability to accurately identify strains with the same or very similar Por proteins are important for studies of transmission, acquired immunity, or pathogenesis.
PIA-expressing strains have been associated with disseminated infections (2
), so the predominance of PIA por
types within the DGI collection was not surprising. Of interest was the identification of a single por
type in 68% of the DGI strains (85.7% of PIA strains) collected over a 7-year period, as well as the suggestion that these strains represent a clonal population based on tbpB
RFLP analysis. Genetic characterization of strains of the PIA-1,2 arginine-, hypoxanthine-, and uracil-requiring A/S type associated with DGI strains in Seattle also showed little genetic diversity over time (46
). Further determination of the clonal nature of the DGI strains examined here, such as by multilocus sequence typing (39
), is warranted in light of the potential use of this collection for studies to identify genes associated with disseminated disease.
We have previously used por
VR typing to examine the diversity of Por over 10 years in a large urban community. This study and others suggest that Por diversity is restricted (12
). Structural, functional, or immunologic restrictions on Por diversity require further investigation, and genotypic analyses of porB
will have several advantages over present serologic typing schemes. Studies have shown that strains of the same serovar may have differences in sequences encoding surface-exposed regions of Por, and, conversely, strains with different serovars may have many antigenic regions in common (12
). The propensity for recombination, resulting in mosaic porins, and a lack of specific antibodies for each antigenic region complicate characterization of the role that specific epitopes may play in pathogenicity or protection from disease. By identifying each VR independently, por
VR typing is well suited to examining this mosaic gene.
VR typing has some limitations. porB
variation occurs through both point mutations and horizontal genetic exchange and is subject to selective pressure (12
). Over time, or between geographic regions, por
type may not accurately reflect strain relatedness. por
VR typing may not be as discriminatory as sequencing, and minor variations in hybridization signal, suggesting single base pair differences, cannot be distinguished as synonymous versus nonsynonymous without subsequent sequence analysis. Additionally, since antigenic differences may result from single amino acid changes or may not accompany much larger changes, immunologic reactivity with sera or MAbs remains necessary for some applications.
Regardless of these limitations, molecular methods will allow for the development of rapid, accurate, and widely available typing tools. Hybridization can be utilized in a variety of methods ranging in level of technical sophistication from simple colony or dot blots to microarray or fluorescent-labeled probe detection. Analysis and interpretation of results from probe hybridization assays are technically simple. Sequencing and hybridization methods are complementary, since an approximation of sequence can be obtained from probe hybridization patterns.
Hybridization-based methods may provide an advantage in detecting mixed gonococcal infections. Coinfection with more than one gonococcal strain was recently identified in 20% of males by comparison of the opa
type obtained from urethral swab specimens to the opa
type of the primary culture (23
). One of the goals of molecular typing is to allow for the characterization of strains directly from nucleic acid amplification test samples. We have identified mixed infections in direct clinical specimens by using por
VR typing (22
), and in the present study, unrecognized coinfection with more than one gonococcal strain may have contributed to the overall 9.3% (4 of 43 pairs) discrepancy observed in partners where only cultivated strains were tested rather than infected secretions. Identifying the presence of and distinguishing mixed infections in direct clinical samples may be difficult with sequence-based typing.
Rapid and widely available typing systems have the potential to provide information useful to the control and prevention of N. gonorrhoeae infections and to guide public health interventions. In this study we have shown that por VR typing is a molecular tool that is applicable to a wide variety of strain collections and is both discriminatory and accurate. It is compatible with porB sequencing methods, and it has the potential to be applied to nonculture-based clinical samples in conjunction with nucleic acid amplification diagnostic tests. por VR typing holds promise as an epidemiological tool and as a means to increase our understanding of the role of Por in neisserial pathogenicity and human immunity.