Sequence-Typing Method to Assess Strain Diversity
and Antibiotic Resistance in San Francisco, California
Neisseria gonorrhoeae Multiantigen
Sean Buono,1Abel Wu,1David C. Hess,2Jonathan S. Carlson,1Leah Rauch,1Susan S. Philip,3
Pennan Barry,3Kyle Bernstein,3Jeffrey D. Klausner,3and Mark W. Pandori1
Urogenital Neisseria gonorrhoeae isolates (266) collected in San Francisco, CA, in 2009 were analyzed for anti-
microbial susceptibility and were subsequently genotyped by N. gonorrhoeae multiantigen sequence typing (NG-
MAST). Isolates of identical or closely related sequence types were found to possess highly similar phenotypes
with regard to drug susceptibility. Isolates containing decreased susceptibility to oral cephalosporins were
detected in 2009 and were found to contain the mosaic penA allele (XXXIV) found previously to be associated
with decreased susceptibility to cephalosporins. A better understanding of the relationships between phenotypic
and genotypic markers for antimicrobial resistance may be helpful to the development of effective surveillance
systems for drug-resistant N. gonorrhoeae.
wide, despite ongoing public health efforts. Continuous
evolution of this organism has resulted in strains capable of
resisting antimicrobial treatment, including strains that pos-
sess a reduced susceptibility to multiple agents. Serial emer-
gence of drug resistance in the United States has resulted in a
severely reduced pharmacological arsenal for use against this
organism.3,11Current treatment guidance in the United States
recommends the use of third-generation cephalosporins (ei-
ther injected or orally administered).4However, even this
treatment regime is under a potential threat, as multiple re-
ports indicate that resistance to cephalosporins may now be
spreading internationally.2,5,10,11,14,18,19,21Within the United
States, drug resistance of N. gonorrhoeae is monitored by the
Gonococcal Isolate Surveillance Project (GISP). GISP is a
Centers for Disease Control and Prevention’s (CDC) project
separated throughout the United States for the gathering and
testing of Gonococcal isolates. Through this program, sentinel
laboratories obtain antimicrobial susceptibility data on uro-
genital isolates located within their jurisdictions on a monthly
basis. Analyses include assessments of the organisms’ ability
to grow in the presence of varying amounts of penicillin,
tetracycline, spectinomycin, cefixime, ceftriaxone, cefpodox-
ime, ciprofloxacin, and azithromycin. Data gathered through
eisseria gonorrhoeae infections continue world-
the GISP are phenotypic. Hence, while the phenotypic di-
characterized, the genetic diversity of such isolates is cur-
rently unknown within the United States.
We sought to characterize the relatedness of all GISP iso-
lates collected at the San Francisco, CA, municipal STD clinic
during 2009. Isolates were genotyped by the N. gonorrhoeae
genotyping method, N. gonorrhoeae multiantigen sequence
typing (NG-MAST).12Isolates considered identical or closely
related according to NG-MAST were compared to one an-
other with regard to their antimicrobial susceptibility phe-
notypes. Such an analysis allowed a determination of
whether phenotypic susceptibility patterns of isolates segre-
gated according to the NG-MAST sequence type. Moreover,
during the course of this analysis, isolates from 2009 with
reduced susceptibility to oral cephalosporins were detected
and their genotypes subsequently analyzed.
Materials and Methods
Two hundred sixty-six isolates of urogenital N. gonorrhoeae
were collected from symptomatic men at San Francisco City
Clinic, the municipal STD clinic, during 2009. All cultures
were initially isolated on Modified Thayer Martin Selective
Agar (Becton Dickinson) and immediately incubated for 24
hours in 10% CO2at 35?C. Plates were transferred to the San
1San Francisco Department of Public Health Laboratory, San Francisco, California.
2Department of Biology, Santa Clara University, Santa Clara, California.
3Division of STD Control and Prevention, San Francisco Department of Public Health, San Francisco, California.
MICROBIAL DRUG RESISTANCE
Volume 00, Number 0, 2012
ª Mary Ann Liebert, Inc.
MDR-2011-0229-ver9-Buono_1P.3d05/23/123:18pm Page 1
Francisco Public Health Laboratory and identified via Gram
stain and oxidase tests. Cultures were purified on chocolate
agar and incubated for 24 hours in 10% CO2at 35?C. Pure
cultures were frozen as paired samples in a Tryptic Soy Broth
with 1% Glycerol. One set of samples was shipped to a CDC-
sponsored GISP laboratory at the University of Washington,
Seattle. The second set was stored at -30?C.
Gonococcal Isolate Surveillance Project
Isolates sent to the University of Washington GISP Labora-
tory were subjected to antibiotic susceptibility testing using the
agar dilution method as outlined by the CDC GISP protocol.9
For standardization and to facilitate execution, this protocol
includes the collection of urogenital specimens from men.
Isolates were examined for minimum inhibitory concentra-
tion (MIC) against the following eight drugs: penicillin, tetra-
cycline, spectinomycin, cefixime, ceftriaxone, ciprofloxacin,
cefpodoxime, and azithromycin (only MIC for ciprofloxacin,
cefpodoxime, and azithromycin are shown). The CDC assigns
the following alert MIC values to the following compounds:
thromycin, ‡2.0mg/ml; ceftriaxone: 0.125mg/ml.
N. gonorrhoeae multiantigen sequence typing
N. gonorrhoeae DNA for genetic analysis was prepared by
MagNA pure instrument (Roche Diagnostics). NG-MAST se-
quence typing was performed by amplifying regions of the
porB and tbpB genes using a published polymerase chain re-
on a 1% agarose gel; porB products were visible around 700bp
and tbpB products at the around 500bp after electrophoresis
for 60 minutes. The PCR products were purified using an
automated PCR-Cleanup protocol (Qiagen). All strains that
2009 were subject to analysis and are shown in this work. This
correlates to a prevalence of 2%. Primers were obtained from
Integrated DNA Technologies, San Diego, CA.
Maximum likelihood tree generation
POR alleles and TBP alleles were combined into a single
sequence for all NG-MAST sequence types in this study were
assembled into a standard FASTA file. These sequences were
aligned using Bioedit standard CLUSTAL-W alignment.8
The aligned sequences were assembled into a relatedness
tree using DNAml DNA maximum likelihood method.7
Purified samples were subjected to DNA sequencing for
penA, tbpB, or porB. Sequencing reactions for the por gene
were carried out by using Bigdye 3.1 on the ABI Genetic
Analyzer and separated by matrix (NoanoPOP 7) on 3730xl
(Applied Biosystems Inc.). Each sample was subjected to 30
cycles of 98?C for 10 seconds, 50?C for 5 seconds, and 60?C
for 2.30 minutes; reactions for the tbpB gene were run with
the same conditions, but with an annealing temperature of
58?C. The resulting DNA sequences were entered into the
NG-MAST online database (www.ng-mast.net) and assigned
allele/sequence type identities. Primers were obtained from
Integrated DNA Technologies, San Diego, CA.
Mosaic penA identification
The mosaic penA allele was screened using a real-time
PCR assay previously described by Ochiai et al.13penA se-
quencing was performed as previously described.16
In total, 266 N. gonorrhoeae isolates were collected evenly
from January through December 2009 and were analyzed
with regard to antimicrobial susceptibility by the agar dilu-
tion method within 3 months of collection. These 266 isolates
constituted 40% of all positive gonorrhea cases in men over
the course of 2009 at the clinical collection site. Antimicrobial
agents tested included penicillin, tetracycline, spectinomycin,
ceftriaxone, cefixime, cefpodoxime, ciprofloxacin, and azi-
thromycin. All isolates were subsequently subjected to gen-
otyping by way of the NG-MAST method. This included
amplification and sequencing of the porB and tbpB alleles
before submission of the allelic sequences to the NG-MAST
database for sequence-type assignment.
Application of NG-MAST to all 266 isolates revealed 117
different sequence types. The majority of the isolates (150/
266, 56.4%) belonged to 15 sequence types, whereas the re-
maining isolates (116, 43.6%) belonged to 102 distinct se-
quence types. One sequence type, ST 2992, accounted for
20% of all isolates. Of the 117 different sequence types de-
tected, 82 were novel to the NG-MAST database and as such
were first-time contributions to the database.
To determine the relatedness of the eight most common
sequence types in our dataset, we generated a relatedness
Fig. 1) using the DNA maximum likelihood method-
ology on the porB and tbpB sequences for each sequence type.
Two features were apparent in this relatedness tree. First was
that two porB and tbpB alleles were common among our set
of isolates. Both ST3935 and ST1407 contain the porB 908
allele, while sequence types 3935, 730, and 2992 all contain
tbpB 29. Overall, 119 of 266 isolates (45%) possessed the tbpB
29 allele. These shared allele types suggest possible related-
ness among the strains surveyed in our collection. Ad-
ditionally, it was found that ST4254 and ST589 are identical
in their tbpB sequence and nearly identical in their porB se-
quence (only two nucleotides different). By comparison,
ST589 and ST2992 (the next closest sequence type in this
study, by relatedness) have 74 nucleotide differences and a
number of insertions and deletions when aligned with each
other. The near identity between ST4254 and ST589 suggests
that they are likely descendant from a common ancestor
strain in the very near past. Both sequence types were found
also to possess identical sensitivity patterns to the antimi-
These results led us to investigate whether the other major
sequence types in our study were closely related to any of the
minority sequence types. We found that each sequence type
had at least one additional isolate that was a near identical
(seven or fewer nucleotide differences, which constitutes a
1% overall variability in sequence divergence for the se-
quences of tbpB and porB combined) sequence match for
NG-MAST. These sequence types are listed in
example of the close relatedness of these additional sequence
types is portrayed in
Fig. 2. For this figure, we analyzed the
relatedness of the eight most common sequence types and
the five sequence types closely related to ST2992. Expanding
Table 1. An
2BUONO ET AL.
the grouping of sequence types to include these other, very
closely related sequence types (using the same criteria of
seven or fewer nucleotide differences) creates an ST2992-like
group with a membership of 60 isolates (60/266, 22.5%).
When we compare the antimicrobial sensitivities for these
additional strains that are nearly identical to one of our eight
most common sequence types, we find that in all but one
case, the sensitivity profiles are identical (data not shown).
The single exception is seen for ST28 (nine total isolates) and
ST1440 (four total isolates). These sequence types have
identical tbpB sequences and only one nucleotide difference
in their porB sequence. However, all nine isolates of ST28
show sensitivity to the lowest concentrations of ciprofloxacin
(MIC £0.030). In contrast, all four isolates of ST1440 pos-
sessed alert levels for ciprofloxacin MIC (MIC levels of 0.25,
4.0, 4.0, and 16.0 for these four isolates). While these two
sequence types are clearly related to a common ancestor in
the recent past, divergence has occurred. Two possibilities
for this include either that ST1440 has acquired a decreased
susceptibility to ciprofloxacin or that ST28 has lost decreased
susceptibility to ciprofloxacin.
We next determined that whether isolates of the same NG-
MAST sequence type possessed comparable phenotypic pro-
files with regard to drug susceptibility. Inspection of anti-
biograms for isolates of the same NG-MAST type revealed
highly comparable MIC values for most antimicrobial agents.
For each sequence type that occurred with a frequency >2%
(i.e., sequence types for which more than 5 isolates were avail-
able for comparison), we constructed histograms to illustrate
the distributions of MIC that occurred for cefpodoxime, cipro-
floxacin, and azithromycin. (
Fig. 3) Histograms describing cef-
triaxone were not constructed because of the complete
2/266 instances of MIC >0.03). Cefixime is likewise not shown
since another oral cephalosporin, cefpodoxime, is shown.
Within sequence types, MIC values for most antimicrobial
agents were highly similar. A summary of the variability in
drug susceptibility seen for each of the major genotypes is
Table 2. Across the evaluated sequence types
and weighted for the number of isolates, the NG-MAST se-
quence type was found to be predictive of MIC within a
twofold range from the modal MIC: for cefpodoxime, this
occurred 91% of the time, for ciprofloxacin 98% of the time,
and for azithromycin 81% of the time. A twofold differential
from a given MIC value is considered as an acceptable level
Table 1. Close Relatives of Popular Sequence
Types, San Francisco, 2009
4246 (2) 42423391 4397 4661
4414 (2) 4667
The number in parentheses indicates that the number of isolates of
that strain type in our study. No number indicates that only a single
isolate of that strain type was found.
maximum likelihood on the por and tbp sequences for the
seven most common strain types in 2009. Each terminal
branch is labeled with the Neisseria gonorrhoeae multiantigen
sequence-typing (NG-MAST) strain type represented on that
branch. The blue-shaded region indicates all strain types
with the TBP allele 29. The red-shaded area indicates all
strain types with the POR allele 908.
Unrooted relatedness tree generated using DNA
maximum likelihood on the por and tbp sequences for the
seven most common strain types in 2009 and the six strain
types closely related to the strain type 2992. Each terminal
branch is labeled with the NG-MAST strain type represented
on that branch. The purple-shaded region indicates all strain
types in the 2992 strain type family.
Unrooted relatedness tree generated using DNA
DRUG RESISTANCE IN N. GONORRHOEAE
MDR-2011-0229-ver9-Buono_1P.3d05/23/123:18pm Page 3
type for each drug shown. CPD, cefpodoxime; CIP, ciprofloxacin; AZI, azithromycin.
(A–G) Histograms showing the frequencies of minimum inhibitory concentration (MIC) values for each major strain
MDR-2011-0229-ver9-Buono_1P.3d05/23/123:18pm Page 4
of variation for proficiency testing in the performance of the
GISP.9A notable variation was seen in the cefpodoxime MIC
for ST1407. This sequence type has been shown, previously,
both to possess a decreased susceptibility for and to be as-
sociated with treatment failures to oral cephalosporins.1,14,16
We noted that some of the isolates from 2009 were found
to possess alert values (MIC ‡0.250mg/L) for cefpodoxime
(5 of 266 isolates, 1.9%). Previous studies in the same geo-
graphic area had revealed the presence of N. gonorrhoeae
isolates that possessed a reduced susceptibility to oral
cephalosporins.16Such isolates had been shown to possess
highly altered mosaic penA alleles. For that reason, we ana-
lyzed all 266 of the 2009 isolates described herein for the
presence of mosaic penA alleles using a real-time PCR
screening assay. This PCR assay detects mosaic penA alleles,
including those previously associated with reduced suscep-
tibility to oral cephalosporins in vitro, or with actual treat-
ment failures.10,13,16Screening by this method revealed that 9
of 266 (3.8%) isolates possessed PCR-reactive penA alleles.
All nine isolates were subsequently subjected to DNA se-
quencing of the entire penA gene to confirm the PCR results
and to determine specifically what the penA allele possessed.
As shown in
Table 3, all nine PCR-reactive isolates possessed
a mosaic penA allele that was previously described and de-
tected in San Francisco. Four of the isolates possessed the
penA allele XXXIV (previously referred to as SF-A16), which
contains a high degree of similarity with that of the mosaic
allele X previously described and associated with oral
cephalosporin treatment failures in Europe and Asia.14Five
of the isolates contained the penA allele XXXVIII (previously
referred to as SF-B16), which possesses a markedly different
sequence, and lacks certain of the mutations shown
MDR-2011-0229-ver9-Buono_1P.3d05/23/12 3:18pmPage 5
DRUG RESISTANCE IN N. GONORRHOEAE
previously to be required for conference of oral cephalo-
All seven of the ST 1407 isolates detected during this study
possessed mosaic penA alleles. Those ST1407 isolates that
contained the XXXIV penA allele possessed MIC of 0.5 and
2.0 for cefpodoxime. One isolate whose sequence
found singly (ST 4533) possessed a mosaic penA allele
(XXXIV) and an MIC of 1.0 for cefpodoxime. Another isolate,
ST 3431, also contained the XXXIV allele and possessed an
MIC of 1.0 for cefpodoxime. This sequence type had been
previously described, as an isolate of this sequence type was
associated with a treatment failure to cefixime in England.10
All isolates that contained the XXXVIII penA allele possessed
MIC of 0.125 or below for cefpodoxime. All isolates con-
taining either XXXIV or XXXVIII penA alleles also possessed
allele 110 for tbpB.
A previous study has shown that isolates of the same NG-
MAST sequence type were found to possess highly similar
phenotypic patterns of drug susceptibility.15This is perhaps
not surprising, given that isolates of the same NG-MAST
type are either identical or closely related. Larger NG-MAST
families in our study unsurprisingly tended to possess a
greater variability of MIC. For most NG-MAST types in-
vestigated, variability was particularly high among the MIC
for azithromycin. A previous study investigating isolates
from San Diego County with unusually a high MIC for azi-
thromycin (MIC of 8.0–16.0) showed that those isolates were
found to have the NG-MAST type ST 2992.20In this study,
ST 2992 isolates were found to have azithromycin MIC
ranging from 0.03 to 1.0 (nonalert value). Possibly, the loci
6BUONO ET AL.
that control the phenotype of azithromycin susceptibility are
polymorphic and change on a time scale that may be much
faster than that of either the por or tbpB gene, which dictates
the NG-MAST-typing method. This indicates that while NG-
MAST typing may provide valuable information about a
particular isolate’s antimicrobial susceptibilities, it would by
no means be definitive. This caution is further supported by
an example such as that seen for ST 730, for which one of
nine isolates possessed an MIC for ciprofloxacin 533 times
higher than that of the other eight ST 730 isolates.
susceptibility to oral cephalosporins in San Francisco in 2009.
Seven of 266 isolates (2.6%) possessed the ST1407 sequence
type, which has previously been associated with treatment
failures to oral cephalosporins. All ST1407 isolates were found
to possess mosaic penA alleles, previously described in a study
of local Gonococcal isolates (XXXIV and XXXVIII). Previously,
and G545S mutations in penA, which are each vital in contrib-
allele was shown to possess I312M and V316T with the wild-
type residue (glycine) at position 545.16
Isolates of sequence types ST1407 and ST 3431 that were
found to possess the XXXIV mosaic allele also possessed
markedly elevated MIC to oral cephalosporin, whereas those
possessing the XXXVIII mosaic allele did not. Of note was the
fact that all mosaic penA-containing isolates also possessed
the 110 tbpB allele. The explanation for this may be rooted in
the fact that penA and tbpB are very close to one another
on the N. gonorrhoeae chromosome.6The exchange of genetic
Table 3. Isolates from 2009 Containing Mosaic 1 penA Alleles
Isolate no.PENTETCFXCROCIP CPDAZIMosaica
aMosaic penA allele nomenclature as per Ref.11
b1 base-pair difference with the allele 908.
PEN, penicillin; TET, tetracycline; CFX, cefixime; CRO, ceftriaxone.
Table 2. Summary of Minimum Inhibitory Concentration Values for Major Sequence Types
NG-MAST sequence type
(no. of isolates found)
and antibiotic evaluated
Range of MIC
observed (mg/ml)Fold range
% of isolates with MIC
of the mode
ST2992 (53) CPD
ST3935 (24) CPD
ST589 (11) CPD
ST28 (9) CPD
ST730 (9) CPD
ST4254 (8) CPD
ST1407 (7) CPD
0.015, 0.03, 0.06a
aST730 was multimodal for CPD.
bAn MIC of 8.0 occurred, n=1. All other isolates (n=8) occurred with MIC of 0.015.
NG-MAST, Neisseria gonorrhoeae multiantigen sequence typing; MIC, minimum inhibitory concentration; CPD, cefpodoxime; CIP,
ciprofloxacin; AZI, azithromycin.
DRUG RESISTANCE IN N. GONORRHOEAE
MDR-2011-0229-ver9-Buono_1P.3d05/23/123:18pm Page 7
material responsible for acquisition of a penA allele may also
involve incorporation of a portion of the 110 tbpB locus.
The relatively low frequency (10.2%) of isolates with alert
value MIC for ciprofloxacin found in 2009 in this study was
of interest. In previous years, in San Francisco, particularly
2005–2007, frequencies of isolates with alert value cipro-
floxacin MIC ranged from 30% to 45% annually. It is un-
known whether the drop in frequency seen for alert value
MIC for ciprofloxacin is due to a shift in sequence types
located in San Francisco, or whether sequence types have
persisted over time, but have lost the mutations required for
resistance due to a lack of selective pressure. Experiments are
in progress to answer this question.
The data herein support that phenotypic markers of anti-
microbial resistance are associated with specific N. gonor-
rhoeae sequence types. These methods hold promise in
improving our ability to better monitor potential increases in
local transmission and the importation of resistant strains of
N. gonorrhoeae. Further characterization of the phenotypic,
genotypic, and epidemiologic characteristics of NG from
diverse geographic areas may be needed.
We thank the San Francisco Department of Public Health
STD Control branch and City Clinic for the acquisition of
GISP isolates. Antimicrobial phenotype testing was provided
by the CDC through the University of Washington Regional
GISP Laboratory in Seattle, WA. This research was sup-
ported in part by the Emerging Infectious Diseases (EID)
Fellowship Program administered by the Association of
Public Health Laboratories (APHL) and funded by the CDC.
No competing financial interests exist.
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Address correspondence to:
Mark W. Pandori, Ph.D.
San Francisco Department of Public Health Laboratory
101 Grove St. Room 419
San Francisco, CA 94102
8BUONO ET AL.
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