JOURNAL OF CLINICAL MICROBIOLOGY, Apr. 2005, p. 1522–1530
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 43, No. 4
por Variable-Region Typing by DNA Probe Hybridization Is Broadly
Applicable to Epidemiologic Studies of Neisseria gonorrhoeae
Margaret C. Bash,1,2* Peixuan Zhu,1† Sunita Gulati,3Durrie McKnew,1,4Peter A. Rice,3
and Freyja Lynn1
Division of Bacterial, Parasitic and Allergenic Products, Center for Biologics Evaluation and Research,1and Department of
Pediatrics, Uniformed Services University of the Health Sciences,2Bethesda, Maryland; Evans Biomedical Research Center,
Department of Medicine and Section of Infectious Diseases, Boston University Medical Center, Boston, Massachusetts3;
and Division of Infectious Diseases, Children’s National Medical Center, Washington, D.C.4
Received 5 May 2004/Returned for modification 11 August 2004/Accepted 8 November 2004
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.
Neisseria gonorrhoeae is one of the most common commu-
nicable diseases worldwide. In the United States, 351,852 cases
were reported to the Centers for Disease Control and Preven-
tion in 2001, over six times the Healthy People 2010 objective
(5). The World Health Organization estimated that in 1999,
62.35 million cases occurred worldwide (43). Reported cases
vastly underestimate the prevalence of this disease (6, 36).
Gonococcal infections are usually uncomplicated genitouri-
nary infections or are asymptomatic; however, they can result
in serious medical and public health consequences such as
disseminated gonococcal infection (DGI), pelvic inflammatory
disease with subsequent complications of ectopic pregnancy
and infertility, and increased transmission of human immuno-
deficiency virus infection (11). Additionally, antibiotic resis-
tance develops rapidly, as illustrated by the spread of high-level
fluoroquinolone resistance, loss of this antibiotic as first-line
therapy, and the need for revised treatment guidelines (7, 44,
Although N. gonorrhoeae has been described as a panmictic
organism, clonal outbreaks have been described in association
with disease presentation (16, 18) or antibiotic resistance (47).
Within defined temporal periods and geographic regions, iso-
late typing can be used to examine transmission patterns, dis-
ease clusters, and antibiotic resistance outbreaks. Strain typing
can assist in identifying high-prevalence, high-transmission
subgroups known as core groups and in guiding focused public
Methods available for gonococcal strain characterization in-
clude phenotypic typing such as auxotype and serovar (A/S)
determination and antibiotic resistance testing. Genotypic
methods include pulsed-field gel electrophoresis (45) and opa
typing (29) based on restriction fragment length polymor-
phisms (RFLPs) and sequence typing based on one or more
genes (17, 39, 40). Serovar determination is the classic pheno-
typic characterization of the gonococcus based on the reaction
of strains with a panel of monoclonal antibodies (MAbs) di-
rected against the porin protein (Por), historically referred to
as PI (19). Serovar determination is technologically simple and
has been widely performed, but problems with reproducibility
and MAb availability have hampered its utility.
Characterization of the two classes of Por, PIA and PIB, has
been expanded and refined by investigations of the sequence
variability of the two mutually exclusive genes porB.1A and
porB.1B (12, 15, 17, 31, 37). The porB gene encodes a protein
that forms a homotrimer consisting of three identical barrel-
shaped channels (25). Sequence diversity of porB is primarily
localized to regions encoding the predicted surface-exposed
loops (12). Por is essential for cell viability, does not undergo
phase variation (1), and is of interest in relation to both the
pathogenicity and the immunogenicity of N. gonorrhoeae (10,
14, 24, 33, 42), making Por an attractive typing target.
We have previously characterized the variability of porB by
using oligonucleotide probe hybridizations to identify variable
regions (VRs) (26, 34). However, this method has not been
evaluated for use in diverse epidemiologic studies. In this
study, we refined our method, examined the relationship be-
* Corresponding author. Mailing address: Division of Bacterial, Par-
asitic and Allergenic Products, HFM-428, Center for Biologics Evalu-
ation and Research, 1401 Rockville Pike, Rockville, MD 20852. Phone:
(301) 496-2044. Fax: (301) 402-2776. E-mail: firstname.lastname@example.org.
† Present address: Creatv MicroTech, Inc., Potomac, MD 20854.
tween serovar, VR types and porB sequence, and applied our
method to a variety of previously well-characterized strain col-
lections to examine the potential utility of this method as a
molecular epidemiologic tool.
(Part of this research was presented at the 13th International
Pathogenic Neisseria Conference, 1 to 6 September 2002, Oslo,
Norway, and at Diagnostic Approaches for Infectious Disease:
Future Promises and Impact on Clinical Management [IDSA],
Orlando, Fla., 29 April to 1 May 2001.)
MATERIALS AND METHODS
Bacterial isolates and description of infected populations. A total of 206 study
isolates and 14 control strains were characterized by por VR typing. Study
isolates included the following: a panel of 18 temporally and geographically
diverse strains developed to evaluate gonococcal typing methods (38); 8 strains
(T13, F6, F62, N10, S12, 7122, D4, and G7) commonly used as controls in serovar
determinations (kindly provided by C. Ison, Health Protection Agency, London,
United Kingdom); a cluster of 14 epidemiologically linked strains (41) with 4
strains of the same serovar or auxotype; 53 DGI strains collected at Boston City
and University Hospitals between 1975 and 1982 (28); and 109 previously A/S
typed strains collected in the 2-year period following September 1988 in Boston,
Mass. Among the 109 strains, there were 69 strains from 37 partners enrolled in
a study of N. gonorrhoeae and Chlamydia transmission (21) and 12 strains from
6 partners who were classified by the same strict epidemiologic criteria but were
not enrolled in the published study. The por VR typing control strains 1861, 5441,
5589, 9299, 3744, FA19, 2432, W062, PI83, S62, 256, MS11, 909, 7, 133, and
911007 have been previously described (26); strain PU186 was provided by C.
DNA preparation and porB amplification. Genomic DNA was isolated from
cultured N. gonorrhoeae cells or 200 ?l of frozen culture stock by using a Wizard
genomic DNA purification kit (Promega Corp., Madison, Wis.) according to the
manufacturer’s instructions. Amplification of the porB gene was performed as
described (26) by using an Expand High-Fidelity PCR System containing Taq
DNA polymerase and Tgo DNA polymerase with proofreading activity (Roche
Molecular Biochemicals, Indianapolis, Ind.) and primers PIB.Fpr (5?-ATTGCC
CTGACTTTGGCAGCCCTTCCT) and PIB.Rpr (5?TTGCAACCAGCCGGC
AGAAACCAAGGC), complementary to the signal peptide and loop 8 coding
regions, respectively. Amplifications performed equally well with either PIA or
PIB porB alleles. PCR products were analyzed for size and concentration with an
Agilent 2100 Bioanalyzer (Rockville, Md.).
Sequence analysis and oligonucleotide probe design. A multiple sequence
alignment analysis, using the GCG PILEUP program from the Genetics Com-
puter Group package (GCG10.2-Unix; University of Wisconsin), was conducted
by using 36 full-length PIA sequences identified in the GenBank database (ac-
cession no. AF044782 to AF044783 , AF090808 to AF090824 , AF015117
to AF015120, AF015122 , L19958 to L19966 , J03029, X58073 , and
Z69259 ). Multiple sequence alignment was followed by individual align-
ments of the gene segments 161 to 250, 328 to 380, 437 to 530, 768 to 852, and
888 to 929 (based on J03029 ) encoding the predicted surface-exposed loops
1, 2, 3, 6, and 7 of the mature PIA protein. A PIB alignment (34) was followed
by multiple sequence alignments of regions 251 to 316, 500 to 620, 737 to 866, 901
to 952, and 1021 to 1090 (based on M21289 ) encoding the predicted surface-
exposed loops 1, 3, 5, 6, and 7 of the mature PIB protein. Dendrograms were
generated by GCG PILEUP for regions 1, 5, and 6. Biotin 5? end-labeled
oligonucleotide probes were designed with similar melting temperatures (range,
56.6 to 61.0°C) to match the sequence variants identified for the VRs encoding
the predicted surface-exposed loops. Adjustments to previously reported probes
(26) (indicated in Table 1) have not altered the results, based on comparison with
control strains, but improved their interpretation by increasing specific signals
and/or decreasing cross-reactivity with closely related sequences.
Checkerboard hybridization and signal detection. Checkerboard hybridiza-
tions were conducted as previously described (26). Briefly, 400 ng of denatured
PCR-amplified porB DNA was applied to Zeta-Probe-GT nylon membranes
(Bio-Rad, Hercules, Calif.) by using a 30-slot vacuum apparatus (Immunetics,
Cambridge, Mass.). Hybridizations were conducted by using a 45-channel vac-
uum apparatus (Immunetics) at 59°C for 3 h, and bound probes were visualized
by using streptavidin-horseradish peroxidase conjugate (Roche Molecular Bio-
chemicals) and ECL chemiluminescent substrate (Amersham Pharmacia Bio-
tech, Piscataway, N.J.). Hybridization signals were compared to those of control
strains (see Fig. 2).
DNA sequencing. PCR-amplified porB DNA, generated with primers GC-
PorBF.outer and GCPorBR.outer, was purified by QIAquick spin-columns
(QIAGEN). DNA sequences were determined from both strands for each strain
by using an ABI PRISM dye terminator sequencing kit with AmpliTaq DNA
polymerase FS (Perkin Elmer) on a model 377 automated sequencer (Applied
Biosystems). Sequences were collated and analyzed by using SEQED,
(GCG10.2-Unix). The porB gene sequences of strains 280044, 280, 280042, 177,
177007, 192, 192014, 252, 255, 255034, 271, 271536, 163, 163006, DGI 17, DGI
18, DGI 19, DGI 27, DGI 29, DGI 34, DGI 37, DGI 40, DGI 43, DGI 61, DGI
70, S32, and S140 were determined. The porB sequences of seven Sheffield
cluster strains were previously determined and submitted to the GenBank data-
base under accession numbers AY297697 to AY297703.
Restriction analysis. The transferrin-binding protein B gene (tbpB) from se-
lected DGI strains was amplified by using primers T1 (5?ATGAACAATCCAT
TGGTGA) and T2 (5?TGGCGTTTCGCACCGAATAC). Amplified DNA frag-
ments were digested with restriction endonucleases AluI, HaeIII, RsaI, and MspI
(Roche Molecular Biochemicals) by using 8 ?l of PCR product, 1.0 ?l of 10?
restriction endonuclease buffer, 10 U of restriction endonuclease, and sterile
distilled water to a final volume of 10 ?l. The mixture was incubated at 37°C for
1 h and then separated on 1.5% agarose gel and stained with ethidium bromide.
por VR typing nomenclature. Hybridization results for a single VR are referred
to as the “VR type,” designated by the Por class, the VR, and the probe, e.g.,
PIB1-1. When more than one probe is bound for a single VR, each probe is
listed, separated by a comma (e.g., PIB6-4,6), and decreased signal intensity
compared to homologous controls is indicated by parentheses. The “por type”
includes results for all VRs tested, listed sequentially and separated by a semi-
colon; for example, B3;1;4;4,6;2 refers to a strain hybridizing PIB1-3, PIB3-1,
PIB5-4, PIB6-4 and 6-6, and PIB7-2 probes.
Nucleotide sequence accession numbers. The porB gene sequences of strains
280044, 280, 280042, 177, 177007, 192, 192014, 252, 255, 255034, 271, 271536,
163, 163006, DGI 17, DGI 18, DGI 19, DGI 27, DGI 29, DGI 34, DGI 37, DGI
40, DGI 43, DGI 61, DGI 70, S32, and S140 were submitted to the GenBank
database under accession numbers AY765435 to AY765461.
The applicability of por VR typing was examined by using
diverse and well-characterized collections of strains. These in-
cluded temporally and geographically diverse isolates (38), se-
rovar typing control isolates, epidemiologically linked isolates
(41) with several unrelated control strains of the same A/S
type, a large collection of partner isolates (21), and isolates
from DGIs (28). In total, 206 isolates were por VR typed by
using probes to five VRs of both PIA and PIB porB alleles.
Among these isolates, 54 different por types were identified.
The accuracy of por VR typing was confirmed by using 27 porB
sequences determined in this study and published porB se-
quences of 10 control strains. The relationships between sero-
var and por VR type were examined by using the MAb binding
patterns of strains that have been serovar typed on multiple
occasions. These relationships were further investigated by ex-
amining the VR sequences of porB from PIB and PIA strains
in the GenBank database that had an identified serovar.
por VR type of a panel of diverse strains. A panel of 18
diverse strains was developed by van Looveren et al. for the
purpose of evaluating gonococcal typing methods (38). The 18
strains of the panel were discriminated by por VR typing al-
though two PIB strains (3790 and 855) differed only by the
strength of the hybridization signal of probe B1-2. por VR
typing was as discriminating among these strains as opa and
A/S typing and was more discriminatory than serovar typing
Relationship between VR type and serovar MAb binding.
Relationships between serovar MAb binding and VR sequence
were determined by examining the por VR types of the panel
of strains described above, as well as eight serovar control
VOL. 43, 2005 GONOCOCCAL por VR TYPING1523
1. Blake, M. S., and E. C. Gotschlich. 1987. Functional and immunologic
properties of pathogenic Neisseria surface proteins, p. 377-400. In M. Inouye
(ed.), Bacterial outer membranes as model systems. John Wiley & Sons, Inc.,
New York, N.Y.
2. Brunham, R. C., F. Plummer, L. Slaney, F. Rand, and W. DeWitt. 1985.
Correlation of auxotype and protein I type with expression of disease due to
Neisseria gonorrhoeae. J. Infect. Dis. 152:339–343.
3. Carbonetti, N. H., V. I. Simnad, H. S. Seifert, M. So, and P. F. Sparling.
1988. Genetics of protein I of Neisseria gonorrhoeae: construction of hybrid
porins. Proc. Natl. Acad. Sci. USA 85:6841–6845.
4. Carbonetti, N. H., and P. F. Sparling. 1987. Molecular cloning and charac-
terization of the structural gene for protein I, the major outer membrane
protein of Neisseria gonorrhoeae. Proc. Natl. Acad. Sci. USA 84:9084–9088.
5. Centers for Disease Control and Prevention. 2003. Sexually transmitted
disease surveillance, 2002. U.S. Department of Health and Human Services,
Centers for Disease Control and Prevention, Atlanta, Ga.
6. Centers for Disease Control and Prevention. 2002. Sexually transmitted
disease surveillance, 2001. U.S. Department of Health and Human Services,
Centers for Disease Control and Prevention, Atlanta, Ga.
7. Centers for Disease Control and Prevention. 2000. Fluoroquinolone resis-
tance in Neisseria gonorrhoeae, Hawaii, 1999, and decreased susceptibility to
azithromycin in N. gonorrhoeae, Missouri, 1999. JAMA 284:1917–1919.
8. Cooke, S. J., H. de la Paz, C. Lapoh, C. A. Ison, and J. E. Heckels. 1997.
Variation within serovars of Neisseria gonorrhoeae detected by structural
analysis of outer-membrane protein PIB and by pulsed-field gel electro-
phoresis. Microbiology 143:1415–1422.
9. Cooke, S. J., K. Jolley, C. A. Ison, H. Young, and J. E. Heckels. 1998.
Naturally occurring isolates of Neisseria gonorrhoeae, which display anoma-
lous serovar properties, express PIA/PIB hybrid porins, deletions in PIE or
novel PIA molecules. FEMS Microbiol. Lett. 162:75–82.
10. Edwards, J. L., E. J. Brown, S. Uk-Nham, J. G. Cannon, M. S. Blake, and
M. A. Apicella. 2002. A co-operative interaction between Neisseria gonor-
rhoeae and complement receptor 3 mediates infection of primary cervical
epithelial cells. Cell Microbiol. 4:571–584.
11. Fleming, D. T., and J. N. Wasserheit. 1999. From epidemiological synergy to
public health policy and practice: the contribution of other sexually trans-
mitted diseases to sexual transmission of HIV infection. Sex Transm. Infect.
12. Fudyk, T. C., I. W. Maclean, J. N. Simonsen, E. N. Njagi, J. Kimani, R. C.
Brunham, and F. A. Plummer. 1999. Genetic diversity and mosaicism at the
por locus of Neisseria gonorrhoeae. J. Bacteriol. 181:5591–5599.
13. Giles, J. A., J. Falconio, J. D. Yuenger, J. M. Zenilman, M. dan, and M. C.
Bash. 2004. Quinolone resistance-determining region mutations and por
type of Neisseria gonorrhoeae isolates: resistance surveillance and typing by
molecular methodologies. J. Infect. Dis. 189:2085–2093.
14. Gorby, G. L., A. F. Ehrhardt, M. A. Apicella, and C. Elkins. 2001. Invasion
of human fallopian tube epithelium by Escherichia coli expressing combina-
tions of a gonococcal porin, opacity-associated protein, and chimeric lipo-
oligosaccharide. J. Infect. Dis. 184:460–472.
15. Gotschlich, E. C., M. E. Seiff, M. S. Blake, and M. Koomey. 1987. Porin
protein of Neisseria gonorrhoeae: cloning and gene structure. Proc. Natl.
Acad. Sci. USA 84:8135–8139.
16. Gutjahr, T. S., M. O’Rourke, C. A. Ison, and B. G. Spratt. 1997. Arginine-,
hypoxanthine-, uracil-requiring isolates of Neisseria gonorrhoeae are a clonal
lineage within a non-clonal population. Microbiology 143:633–640.
17. Hobbs, M. M., T. M. Alcorn, R. H. Davis, W. Fischer, J. C. Thomas, I.
Martin, C. Ison, P. F. Sparling, and M. S. Cohen. 1999. Molecular typing of
Neisseria gonorrhoeae causing repeated infections: evolution of porin during
passage within a community. J. Infect. Dis. 179:371–381.
18. Ison, C. A., J. Pepin, N. S. Roope, E. Demba, O. Secka, and C. S. F. Easmon.
1992. The dominance of a multiresistant strain of Neisseria gonorrhoeae
among prostitutes and STD patients in The Gambia. Genitourin. Med.
19. Knapp, J. S., M. R. Tam, R. C. Nowinski, K. K. Holmes, and E. G. Sand-
strom. 1984. Serological classification of Neisseria gonorrhoeae with use of
monoclonal antibodies to gonococcal outer membrane protein I. J. Infect.
20. Lau, Q. C., V. T. Chow, and C. L. Poh. 1993. Polymerase chain reaction and
direct sequencing of Neisseria gonorrhoeae protein IB gene: partial nucleo-
tide and amino acid sequence analysis of strains S4, S11, S48 (serovar IB4)
and S34 (serovar IB5). Med. Microbiol. Immunol. 182:137–145.
21. Lin, J. S., S. P. Donegan, T. C. Heeren, M. Greenberg, E. E. Flaherty, R.
Haivanis, X. H. Su, D. Dean, W. J. Newhall, J. S. Knapp, S. K. Sarafian, R. J.
Rice, S. A. Morse, and P. A. Rice. 1998. Transmission of Chlamydia tracho-
matis and Neisseria gonorrhoeae among men with urethritis and their female
sex partners. J. Infect. Dis. 178:1707–1712.
22. Lynn, F., M. M. Hobbs, J. M. Zenilman, F. M. T. F. Behets, K. Van Damme,
A. Rasamindrakotroka, and M. C. Bash. 2005. Genetic typing of the porin
protein of Neisseria gonorrhoeae from clinical noncultured samples: strain
characterization and identification of mixed gonococcal infections. J. Clin.
23. Martin, I. M. C., and C. A. Ison. 2003. Detection of mixed infection of
Neisseria gonorrhoeae. Sex. Transm. Infect. 79:56–58.
24. Massari, P., S. Ram, H. Macleod, and L. M. Wetzler. 2003. The role of
porins in neisserial pathogenesis and immunity. Trends Microbiol. 11:87–93.
25. Mauro, A., M. Blake, and P. Labarca. 1988. Voltage gating of conductance
in lipid bilayers induced by porin from outer membrane of Neisseria gonor-
rhoeae. Proc. Natl. Acad. Sci. USA 85:1071–1075.
26. Mcknew, D. L., F. Lynn, J. M. Zenilman, and M. C. Bash. 2003. Porin
variation among clinical isolates of Neisseria gonorrhoeae over a 10-year
period, as determined by Por variable region typing. J. Infect. Dis. 187:1213–
27. Mee, B. J., H. Thomas, S. J. Cooke, P. R. Lambden, and J. E. Heckels. 1993.
Structural comparison and epitope analysis of outer-membrane protein PIA
from strains of Neisseria gonorrhoeae with differing serovar specificities.
J. Gen. Microbiol. 139:2613–2620.
28. O’Brien, J. P., D. L. Goldenberg, and P. A. Rice. 1983. Disseminated gono-
coccal infection: a prospective analysis of 49 patients and a review of patho-
physiology and immune mechanisms. Medicine (Baltimore) 62:395–406.
29. O’Rourke, M., C. A. Ison, A. M. Renton, and B. G. Spratt. 1995. Opa-typing:
a high-resolution tool for studying the epidemiology of gonorrhea. Mol.
30. Poh, C. L., Q. C. Lau, and V. T. Chow. 1995. Differentiation of Neisseria
gonorrhoeae IB-3 and IB-7 serovars by direct sequencing of protein IB gene
and pulsed-field gel electrophoresis. J. Med. Microbiol. 43:201–207.
31. Posada, D., K. A. Crandall, M. Nguyen, J. C. Demma, and R. P. Viscidi.
2000. Population genetics of the porB gene of Neisseria gonorrhoeae: different
dynamics in different homology groups. Mol. Biol. Evol. 17:423–436.
32. Rudel, T., A. Schmid, R. Benz, H. A. Kolb, F. Lang, and T. F. Meyer. 1996.
Modulation of Neisseria porin (PorB) by cytosolic ATP/GTP of target cells:
parallels between pathogen accommodation and mitochondrial endosymbio-
sis. Cell 85:391–402.
33. Simpson, S. D., Y. Ho, P. A. Rice, and L. M. Wetzler. 1999. T lymphocyte
response to Neisseria gonorrhoeae porin in individuals with mucosal gono-
coccal infections. J. Infect. Dis. 180:762–773.
34. Thompson, D. K., C. D. Deal, C. A. Ison, J. M. Zenilman, and M. C. Bash.
2000. A typing system for Neisseria gonorrhoeae based on biotinylated oligo-
nucleotide probes to PIB gene variable regions. J. Infect. Dis. 181:1652–
35. Tompkins, J. R., and J. M. Zenilman. 2001. Quinolone resistance in Neisseria
gonorrhoeae. Curr. Infect. Dis. Rep. 3:156–161.
36. Turner, C. F., S. M. Rogers, H. G. Miller, W. C. Miller, J. N. Gribble, J. R.
Chromy, P. A. Leone, P. C. Cooley, T. C. Quinn, and J. M. Zenilman. 2002.
Untreated gonococcal and chlamydial infection in a probability sample of
adults. JAMA 287:726–733.
37. Unemo, M., P. Olcen, J. Albert, and H. Fredlund. 2003. Comparison of
serologic and genetic porB-based typing of Neisseria gonorrhoeae: conse-
quences for future characterization. J. Clin. Microbiol. 41:4141–4147.
38. van Looveren, M., C. A. Ison, M. Ieven, P. Vandamme, I. M. Martin, K.
Vermeulen, A. Renton, and H. Goossens. 1999. Evaluation of the discrimi-
natory power of typing methods for Neisseria gonorrhoeae. J. Clin. Microbiol.
39. Viscidi, R. P., and J. C. Demma. 2003. Genetic diversity of Neisseria gonor-
rhoeae housekeeping genes. J. Clin. Microbiol. 41:197–204.
40. Viscidi, R. P., J. C. Demma, J. Gu, and J. Zenilman. 2000. Comparison of
sequencing of the por gene and typing of the opa gene for discrimination of
Neisseria gonorrhoeae strains from sexual contacts. J. Clin. Microbiol. 38:
41. Ward, H., C. A. Ison, S. E. Day, I. Martin, A. C. Ghani, G. P. Garnett, G.
Bell, G. Kinghorn, and J. N. Weber. 2000. A prospective social and molec-
ular investigation of gonococcal transmission. Lancet 356:1812–1817.
42. Wen, K. K., P. C. Giardina, M. S. Blake, J. Edwards, M. A. Apicella, and
P. A. Rubenstein. 2000. Interaction of the gonococcal porin P.IB with G- and
F-actin. Biochemistry 39:8638–8647.
43. World Health Organization. 2001. Global prevalence and incidence of se-
lected curable sexually transmitted infections: overview and estimates. World
Health Organization, Geneva, Switzerland.
44. World Health Organization.2002. Surveillance of antibiotic resistance in
Neisseria gonorrhoeae in the WHO Western Pacific Region, 2001. Commun.
Dis. Intell. 26:541–545.
45. Xia, M., W. L. Whittington, K. K. Holmes, F. A. Plummer, and M. C.
Roberts. 1995. Pulsed-field gel electrophoresis for genomic analysis of Neis-
seria gonorrhoeae. J. Infect. Dis. 171:455–458.
46. Xia, M., W. L. Whittington, K. K. Holmes, and M. C. Roberts. 1997.
Genomic homogeneity of the AHU/IA-1,2 phenotype of Neisseria gonor-
rhoeae during its disappearance from an urban population. Sex. Transm. Dis.
47. Yagupsky, P., A. Schahar, N. Peled, N. Porat, R. Trefler, M. Dan, Y. Keness,
and C. Block. 2002. Increasing incidence of gonorrhea in Israel associated
with countrywide dissemination of a ciprofloxacin-resistant strain. Eur.
J. Clin. Microbiol. Infect. Dis. 21:368–372.
1530 BASH ET AL.J. CLIN. MICROBIOL.