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
strains that had been previously serotyped on multiple oc-
casions (C. Ison, personal communication) and nine por VR
typing control strains (Table 2 and Table 3). Associations
observed between VR type, or clusters of closely related VR
types, and MAb reactivities were further examined by com-
paring the VR sequences and serovar designation of porB
genes in the GenBank database (Fig. 1A to C). MAb 1F5
was associated with PIB1-1 and some PIB1-3. MAb 3C8 was
associated with PIB5-1, PIB5-2, PIB5-4, PIB5-7, and PIB5-8,
and MAb 2G2 was associated with probes PIB5-3, PIB5-5,
PIB5-6, and some PIB5-9. These two groups of VR5 types
can be seen in the dendrogram in Fig. 1B and differ by a
6-bp deletion in the 2G2 group. Importantly, these two
MAbs distinguish common serovars IB-1 and IB-2 from
serovars IB-5 and IB-7. MAb 2D6 was associated with
PIB6-4 or PIB6-4,6 and, in some instances, PIB6-2. MAb
2D4 was associated with PIB6-5. This MAb is one of three
that define the common serovar IB-4.
PIA serovar MAb 6D9 corresponded to PIA6-1,4. Antibod-
ies 2F12 and 4G5 were bound by all strains in Table 3, sug-
gesting an association with PIA VRs 1, 3, or 7, which were not
sufficiently diverse to determine the antibody epitopes. MAbs
4A12, 5G9, and 5D1 did not appear to correspond with any
TABLE 1. PIA and PIB probe characteristics
Por class and
Sequence (5?33?) Accession no.b
aProbes marked with an asterisk (*) have minor modifications from the previously reported sequence (26) or are new.
bGenBank accession numbers for porB sequences of control strains.
cControl strains used in hybridizations.
dDiffers from probe by one base pair.
eDiffers from probe by two base pairs.
f—, no control strain available.
gNA, not available.
hThe porB VR5 region of this strain was identical to that of the strain with GenBank sequence accession no. NGU17235. Other VRs of this strain have not been
1524 BASH ET AL.J. CLIN. MICROBIOL.
Sheffield cluster strains. Strains identified by opa type as
part of a large epidemiologically linked cluster (41) as well as
four A/S-matched unrelated control strains were por VR typed
in a blinded fashion. The 14 cluster strains and 1 control strain
(2491) had identical por types, except for 1 cluster strain (S32)
that had decreased hybridization signal of the PIB5-2 probe
(Fig. 2). This was consistent with porB sequence data showing
that strain S32 had a single base pair mutation in VR5. Of the
three controls that had different porB sequences but the same
A/S type, differences were identified by por VR type in VR1 for
all three strains and in VR5 for two strains.
Boston partner strains. The por types of 109 strains col-
lected as part of a study of gonococcal transmission were de-
termined. Among these strains, 28 were PIA and 81 were PIB.
Four PIA por types were identified; the most common type was
A1;2;1;1;1 (64% of PIA strains). Twenty-three PIB por types
were identified; the three most common were B2;2;4;4;2
(18.5% of PIB strains), B2;1;6;5;3 (16% of PIB strains), and
B2;2;7;4;2 (8.6% of PIB strains). In addition, eight strains
(9.9%) were similar to one of these three PIB por types, dif-
fering only in the strength of the hybridization signal for one
VR, which suggests that there are single nucleotide differences
in those regions. No strains were completely nontypeable.
Among PIB isolates, 14 strains did not bind a VR5 probe, 9
strains did not bind a VR7 probe, 4 strains did not bind a VR1
probe, and 2 strains did not bind a VR6 probe.
Among the 107 strains in which both por type and A/S type
were available, there were 26 different por types, 28 different
A/S types, and 15 serovar types. Although the level of discrim-
ination was similar overall, por VR typing and A/S typing
differed dramatically among PIB strains in terms of which
strains were identified as having the same type (Table 4). Four
different serovars (seven A/S types) were assigned to strains
with the common PIB por type B2;2;4;4;2. Conversely, there
were seven different por types among 18 IB1 strains: four por
types among 8 IB1 proline-requiring strains and three por types
among 8 IB1 prototrophic strains. Two IB1 proline- and hy-
poxanthine-requiring strains had a por type found among IB1
prototrophic strains. The por gene was sequenced for 14 strains
and in all cases confirmed the accuracy of the por VR type. The
TABLE 2. PIB por VR types and serovars of gonococcal por VR and serovar control strains
Isolate group and strain
Result of hybridization with probe(s) targeting
sequence encoding loopa:
Binding by MAbb:
13567 3C8 1F52D6 2G2 2D42H1
por VR typing controls
aResults are given in the shortcut form VR-probe (see “por VR typing nomenclature” in the text for details). Where more than one probe was bound, probe numbers
are separated by a comma. Parentheses indicate decreased signal intensity. NT, nontypeable.
bX, strain was bound.
cSerovars were previously determined (3, 17, 38).
TABLE 3. PIA por VR types and serovars of gonococcal por VR and serovar control strains
Isolate group and strain
Result of hybridization with probe(s) targeting
sequence encoding loopa:
Binding by MAbb:
por VR typing controls
aResults are given in the shortcut form VR-probe (see “por VR typing nomenclature” in the text for details). Where more than one probe was bound, probe numbers
are separated by a comma. Parentheses indicate decreased signal intensity.
b?, binding result was equivocal; X, strain was bound.
cSerovars were previously determined.
VOL. 43, 2005GONOCOCCAL por VR TYPING 1525
accuracy of either typing method in distinguishing unrelated
partnership pairs or in identifying extended networks cannot
be accurately assessed without further analysis such as multilo-
cus sequence typing, since extended epidemiologic data to
identify links between groups are not available.
Among the 109 strains, 81 were from individuals who met
epidemiologic criteria for the partner transmission study: 68
strains from 34 male-female partnerships, 9 strains from three
groups of one male with two identified female partners, and 4
strains from a group of one male with three female partners.
Concordance of por type was found among strains from 39 of 43
defined partnerships (?90%). Of the four discordant females,
FIG. 1. Dendrograms from multiple sequence alignments of PIB VR 1 (A), 5 (B), and 6 (C) were generated by using GCG PILEUP (Genetics
Computer Group GCG 10.2-Unix).MAb binding is derived from the serovar stated in the GenBank database or the associated reference. ?,
serovar includes the identified MAb antibody; ?, serovar does not include the identified antibody; n.a., a serovar designation was not available.
1526 BASH ET AL.J. CLIN. MICROBIOL.
two were from one partnership group in which the strain isolated
from the male was different, while the strains from both women
had identical por types. A third discordant female occurred in
another group of one male with two identified partners, and the
fourth occurred in one of the 34 male-female partnerships. In
each case the A/S types were the same.
DGI strains. The por types of 53 DGI strains were determined
(Table 5). A total of 42 strains expressed PIA (79%), and among
these, three PIA por types were identified: A1;3;1;1,4;1 (36 of 42,
or 85.7%), A3;1;2;2;2 (4 of 42, or 9.5%), and A1;2;1;1;1 (2 of 42,
PIB por types were identified. The por type of 10 strains was
confirmed by porB sequencing, and 10 had RFLP analysis of the
tbpB gene. Five of the 10 DGI strains analyzed by tbpB RFLP
were selected from the group of strains with the common A1;3;
1;1,4;1 por type; the other 5 DGI strains had a variety of por types.
The A1;3;1;1,4;1 strains had identical RFLP patterns with all four
enzymes. Of the other 5 DGI strains, and 18 other unrelated N.
gonorrhoeae strains that were not part of this study, all had unique
RFLP patterns (data not shown).
por 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 accu-
rately identify epidemiologically linked isolates; no strains
were completely nontypeable, and the accuracy of por VR
typing was confirmed by porB sequencing.
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 anti-
body 2D4 with probe PIB6-5 are consistent with these reports;
and probes PIB5-1, PIB5-2, PIB5-4, PIB5-7, and PIB5-8, cor-
responding to MAb 3C8, all identify sequences encoding the
sequence YS(I/M)PS in the loop 5 region. Epitopes for anti-
bodies 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 se-
quences that hybridize PIB5-3, PIB5-5, PIB5-6, and some se-
quences that hybridize PIB5-9 while antibody 2D6 corresponds
to sequences binding probes PIB6-4 and PIB6-6 and some that
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 DAKLT
WRND region of strain FA19, MAb 4G5 binds the loop 3
sequence IAQPEE, and 2F12 binds the N-terminal region of
strain FA19 (3, 27, 37). In this study, the PIA1-1 probe iden-
tified 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.
VOL. 43, 2005 GONOCOCCAL por VR TYPING1527
The 4G5 epitope corresponds to PIA3-1 and PIA3-2, consis-
tent 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 dif-
ferent serovars were seen within por type groups, consistent
with previous reports of discrepancies between serovar deter-
minations and porB sequence (20, 30, 37). 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 part-
nerships 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 concor-
dance 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 dissemi-
nated infections (2, 28), 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 deter-
mination 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.
FIG. 2. Checkerboard hybridization of PIB probes to serovar control strains (D4 to S12) and 12 of 14 Sheffield cluster strains (S14 to S324).
The porB sequences of cluster strains S32 and S140 differed by a single base pair in VR5. S32 and S140 porB sequences were otherwise identical
and differed from probes B1-1, B1-3, B 6-3, and B7-3 each by 1 bp and from B7-1 by 2 bp.
1528 BASH ET AL.J. CLIN. MICROBIOL.
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, 26).
Structural, functional, or immunologic restrictions on Por di-
versity 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, 17, 20, 27, 34,
37). 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.
por 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, sug-
gesting single base pair differences, cannot be distinguished as
synonymous versus nonsynonymous without subsequent se-
quence 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 al-
low for the development of rapid, accurate, and widely avail-
able typing tools. Hybridization can be utilized in a variety of
methods ranging in level of technical sophistication from sim-
ple 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 ap-
proximation of sequence can be obtained from probe hybrid-
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 char-
acterization 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 cul-
tivated 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
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 interven-
tions. 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 com-
patible with porB sequencing methods, and it has the potential
to be applied to nonculture-based clinical samples in conjunc-
tion 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.
We are grateful to Cathy Ison and Iona Martin for thoughtful
contributions during the development of this work, for providing
strains, and for review of the manuscript, and to Carl E. Frasch, Wendy
Carr, and M. S. Blake for critical comments on the manuscript.
D. McKnew was supported in part by the FDA Office of Women’s
Health through a postdoctoral fellowship administered by the Oak
Ridge Institute for Science and Education.
TABLE 4. por type of Boston strains
B2; 2; 4; 4; 215IB1
B2; 1; 6; 5; 3 13
B 2; 2; 7; 4; 27
Other PIB por typesc
Other (7 types)
A 1; 2; 1; 1; 1 18
A 3; 1; 2; 2; 26
A 3; 1; 1-2; 2-3; 23 IB323
A 2; 4; 3; 3; 31 IB241
aAmong strains with common por types, serovar designations that are consis-
tent with the associations between serovar MAb and VR probes are shown in
bNA, serovar not available.
cOther PIB por types totaled 19.
TABLE 5. por VR type of 53 disseminated gonococcal
por typen (%)
A 1; 3; 1; 1; 4; 1..............................................................................36 (68)
A 1; 2; 1; 1; 1................................................................................... 2 (4)
A 3; 1; 2; 2; 3; 2.............................................................................. 4 (7)
B (7 different por types).................................................................11 (21)
VOL. 43, 2005GONOCOCCAL por VR TYPING 1529
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