ANTIMICROBIAL AGENTS AND CHEMOTHERAPY, Dec. 2002, p. 3744–3749
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 46, No. 12
Mosaic-Like Structure of Penicillin-Binding Protein 2 Gene (penA) in
Clinical Isolates of Neisseria gonorrhoeae with Reduced
Susceptibility to Cefixime
Satoshi Ameyama,1* Shoichi Onodera,2Masahiro Takahata,1Shinzaburo Minami,1Nobuko Maki,3
Katsuhisa Endo,4Hirokazu Goto,2Hiroo Suzuki,5and Yukihiko Oishi2
Research Laboratories, Toyama Chemical Co., Ltd., 2-4-1, Shimookui, Toyama, 930-8508,1Department of Urology, Jikei
University, School of Medicine, 3-19-18, Nishishinbashi, Minato-ku, Tokyo, 105-8741,2Medical Information Center,
Toyama Chemical Co., Ltd., 3-2-5, Nishishinjuku, Shinjuku-ku, Tokyo 160-0023,3Department of Urology,
JR Tokyo General Hospital, 2-1-3, Yoyogi, Shibuya-ku, Tokyo, 151-8528,4and Department of
Urology, Kosei Hospital, 5-25-15, Yayoi-cho, Nakano-ku, Tokyo, 164-8617,5Japan
Received 30 April 2002/Returned for modification 9 July 2002/Accepted 22 August 2002
Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 ?g/ml) were isolated
from male urethritis patients in Tokyo, Japan, in 2000 and 2001. The resistance to cephems including cefixime
and penicillin was transferred to a susceptible recipient, N. gonorrhoeae ATCC 19424, by transformation of the
penicillin-binding protein 2 gene (penA) that had been amplified by PCR from a strain with reduced suscep-
tibility to cefixime (MIC, 0.5 ?g/ml). The sequences of penA in the strains with reduced susceptibilities to
cefixime were different from those of other susceptible isolates and did not correspond to the reported N.
gonorrhoeae penA gene sequences. Some regions in the transpeptidase-encoding domain in this penA gene were
similar to those in the penA genes of Neisseria perflava (N. sicca), Neisseria cinerea, Neisseria flavescens, and
Neisseria meningitidis. These results showed that a mosaic-like structure in the penA gene conferred reductions
in the levels of susceptibility of N. gonorrhoeae to cephems and penicillin in a manner similar to that found for
N. meningitidis and Streptococcus pneumoniae.
Gonococcal infections have existed as sexually transmitted
diseases since early times and have never been regarded as
intractable diseases. In Japan, the numbers of gonococcal in-
fections, including those resistant to antimicrobial therapy,
have gradually increased since the mid-1990s (11).
Penicillins and tetracyclines are used for the treatment of
gonococcal urethritis worldwide. After the emergence and
worldwide spread of penicillin- and tetracycline-resistant Neis-
seria gonorrhoeae strains, fluoroquinolones were recommended
as the primary therapy for uncomplicated gonorrhea in many
countries (24). Fluoroquinolones have been used extensively
for the treatment of gonococcal urethritis due to their high
degrees of efficacy against the disease. Intense selective pres-
sure resulting from the continual exposure of N. gonorrhoeae to
fluoroquinolones resulted in the emergence of resistant strains
with altered GyrA and ParC proteins (3, 6, 21, 22, 23). In
recent years, expanded-spectrum oral cephems have been
widely used instead of fluoroquinolones for the treatment of
gonorrhea in Japan. However, the emergence and spread of
gonococci resistant to oral cephems have been reported (1, 13).
N. gonorrhoeae has three penicillin-binding proteins (PBPs),
denoted PBPs 1, 2, and 3. PBPs 1 and 2 of N. gonorrhoeae are
the major targets of ?-lactam antibiotics. PBP 2, encoded by
the penA gene, has an approximately 10-fold higher affinity for
penicillin than PBP 1 (7). In previous reports, insertion of the
Asp-345A codon into the penA gene has been proved to make
a major contribution to the reduction of the affinity of gono-
coccal PBP 2 to penicillin (5). Other reports showed that C-
terminal amino acid residues of the penA transpeptidase do-
main were also altered in penicillin-resistant N. gonorrhoeae (8,
18, 19). Enhancement of the efflux pump by mutations in the
mtrR and penB loci was reported to be due to ?-lactam resis-
tance (9, 10).
In 2000 we isolated gonococcal strains with reduced suscep-
tibilities to penicillin and cephems including cefixime, which is
recommended as therapy for gonococcal urethritis, during an
investigation into the cause of clinical failure in patients with
gonococcal urethritis treated with oral cephems. This study was
conducted to investigate the susceptibilities to various antimi-
crobials of clinical isolates of N. gonorrhoeae recently isolated
in Japan and to clarify the mechanism of reduced susceptibility
to cefixime in N. gonorrhoeae.
MATERIALS AND METHODS
Bacteria and media. The N. gonorrhoeae strains used in this study were clinical
strains isolated from male urethritis patients at the School of Medicine, Jikei
University, and related hospitals in 2000 (February to July) and 2001 (February
to March). The specimens were directly streaked onto Thayer-Martin selective
agar (Becton Dickinson, Cockeysville, Md.) in the hospitals. The plates were
placed in a Bio-Bag environmental chamber (type C; Becton Dickinson) and
immediately transported to the laboratory, where they were incubated at 35°C
for 48 h in a 5% CO2atmosphere. The organisms were identified by Gram
staining and by oxidase and catalase tests. The identities of isolates cultured on
Chocolate II agar (Becton Dickinson) were further confirmed with a
Gonochek-II kit (EY Laboratories, San Mateo, Calif.). N. gonorrhoeae isolates
were maintained at ?80°C in modified skim milk (15) until antimicrobial sus-
ceptibility testing. The isolates were tested for ?-lactamase production by a
nitrocefin method. In the antibiotic susceptibility test, 53 and 24 strains isolated
in 2000 and 2001, respectively, were used. The penA sequences of six of the
* Corresponding author. Mailing address: Research Laboratories,
Toyama Chemical Co., Ltd., 2-4-1, Shimookui, Toyama, 930-8508,
Japan. Phone: 81-764-31-8268. Fax: 81-764-31-8208. E-mail: SATOSHI
clinical gonococcal isolates described above (strains NG-3, NG-12, NG-25, NG-
46, NG-48, and NG-83) were used. The bacteria were grown at 37°C under a 5%
CO2atmosphere on brain heart infusion agar (Difco Laboratories, Detroit,
Mich.) including 5% sheep defibrinated blood (Nippon Bio-Test Laboratories
Inc., Tokyo, Japan) for 48 h.
Susceptibility testing and antimicrobials. The MICs were determined by an
agar dilution method according to the approved guidelines of the National
Committee for Clinical Laboratory Standards (14). The following reference
antimicrobials were used: penicillin G (Banyu Pharmaceutical Co., Ltd., Tokyo,
Japan); piperacillin, tazobactam-piperacillin, and cefteram (Toyama Chemical
Co., Ltd., Tokyo, Japan); ceftriaxone (Nippon Roche Co., Ltd., Tokyo, Japan);
flomoxef (Shionogi Pharmaceutical Co., Ltd., Osaka, Japan); aztreonam (Eizai
Co., Ltd., Tokyo, Japan); spectinomycin and minocycline (Sigma Chemical Co.,
St. Louis, Mo.); cefixime and cefdinir (Fujisawa Co., Ltd., Osaka, Japan); cef-
podoxime (GlaxoSmithKline Japan, Tokyo, Japan); cefodizime (Kyorin Pharma-
ceutical Co., Ltd., Tokyo, Japan); and levofloxacin (Daiichi Pharmaceutical Co.,
Ltd., Tokyo, Japan). Cefixime, cefdinir, cefpodoxime, and levofloxacin were
extracted from commercially available capsules or tablets. The purities of these
four agents were above 99.8%, as measured by high-performance liquid chro-
Genetic transformation. Genomic DNA was prepared from an N. gonorrhoeae
strain with reduced susceptibility to cefixime (strain NG-3). The penA amplicon
used for transformation was amplified by PCR as follows. Bacteria were sus-
pended in 50 ?l of distilled water, subjected to one freeze-thaw cycle, heated at
100°C for 3 min, and then centrifuged at 10,000 ? g for 5 min. The full-length
gene was amplified by PCR from the supernatant with oligonucleotides NGPA-F
and NGPA-R (Table 1) and Ex Taq polymerase (Takara Shuzo, Kyoto, Japan).
PCR was performed as follows: 5 min of denaturation at 94°C and 35 cycles of
denaturation at 94°C for 1 min, annealing at 60°C for 0.5 min, and extension at
72°C for 2 min, concluding with a final extension at 72°C for 5 min. Transfor-
mation for homologous recombination of the penA gene was done with the PCR
amplicon and by coincubation under static conditions. Transformants were se-
lected on plates containing cefixime at a concentration of 0.0313 ?g/ml.
Nucleotide sequence of N. gonorrhoeae penA gene. The full-length penA gene
was amplified by PCR with oligonucleotides NGPA-F and NGPA-R (Table 1).
The amplicons were purified with a PCR product presequencing kit (Amersham
Pharmacia Biotech, Tokyo, Japan). The cycling reaction was performed with
Thermo Sequenase DNA polymerase (Amersham Pharmacia Biotech) and oli-
gonucleotides Fs1, Fs2, Fs3, Rs1, Rs2, Rs3, and Rs4 (Table 1). Sequencing was
carried out with a DSQ-1000 sequencer (Shimadzu, Kyoto, Japan). Primer Fs1-3
was used for sequencing of the forward sequence, and primer Rs1-4 was used for
sequencing of the reverse sequence (Table 1).
Restriction fragment length polymorphism analysis of penA gene. The ampli-
con obtained by PCR with primers Aat and r1 (Table 1) was digested with the
restriction endonuclease AatII (New England Biolabs, Inc., Beverly, Mass.).
Restriction digests were analyzed by electrophoresis on 4% agarose gels (Aga-
rose X; Nippon Gene, Toyama, Japan). Primer Aat makes a site that is digested
with AatII if the GAC codon Asp-345A is inserted in the penA gene.
TABLE 1. Oligonucleotides used in this study
TABLE 2. Susceptibilities of clinical isolates of N. gonorrhoeae from male urethritis patients in 2000 and 2001
MIC (?g/ml) MIC ratioa
2000 (n ? 53)2001 (n ? 24)
50% 90%50% 90%
aMIC for 2001/MIC for 2000.
bNC, not calculated.
cND, not determined.
TABLE 3. MICs of various antibiotics for N. gonorrhoeae
ATCC 19424, transformant S1-05, and NG-3
aMIC for transformant/MIC for ATCC 19424.
bNC, not calculated.
VOL. 46, 2002MOSAIC-LIKE STRUCTURE OF GONOCOCCAL penA 3745
3746AMEYAMA ET AL.ANTIMICROB. AGENTS CHEMOTHER.
Nucleotide sequence accession number. The penA sequence of N. gonorrhoeae
NG-3 has been deposited in the DDBJ data library under accession number
Antimicrobial susceptibility and ?-lactamase production.
The MICs of various antimicrobials and ?-lactamase produc-
tion were determined for 53 and 24 clinical isolates recovered
in 2000 and 2001, respectively. The MICs at which 50% of
isolates are inhibited (MIC50s) and the MIC90s of various
antimicrobials for the clinical isolates are shown in Table 2.
Nine of 53 strains (17.0%) isolated in 2000 and 4 of 24 strains
(16.7%) isolated in 2001 showed reduced susceptibilities to
cefixime (MICs, 0.25 and 0.5 ?g/ml, respectively). These
strains also exhibited reduced susceptibilities to penicillin and
other ?-lactams, and some of them were cross-resistant to
fluoroquinolones, spectinomycin, and minocycline. There were
no apparent differences in the MIC90s of any antimicrobials for
the strains isolated in 2000 and 2001. However, the MIC50s of
some ?-lactams for the isolates recovered in 2001 were four- to
eightfold higher than those for the isolates recovered in 2000.
?-Lactamase production was not detected in any of the clinical
Antimicrobial susceptibility of the transformant with the
penA gene derived from an N. gonorrhoeae strain with reduced
susceptibility to cefixime. To investigate whether a reason for
the reduced susceptibility to cefixime was alteration of PBP 2,
the penA gene derived from strain NG-3, which had reduced
susceptibility to cefixime, was transformed into N. gonorrhoeae
ATCC 19424 (cefixime MIC, 0.001 ?g/ml). After transforma-
tion of the penA gene, many transformants were obtained on
plates containing 0.0313 ?g of cefixime per ml. These trans-
formants had similar susceptibility profiles. Table 3 shows the
susceptibilities of the recipient (ATCC 19424) and one of the
transformants (S1-05). The MICs of cefixime and ceftriaxone
for the transformant were 0.0625 and 0.002 ?g/ml, respectively.
The susceptibilities of the transformant to penicillin G, ce-
fixime, cefdinir, cefpodoxime, and aztreonam were reduced 64-
to 128-fold, and those to piperacillin and ceftriaxone were
reduced 2- to 8-fold. There were some discrepancies in anti-
microbial susceptibilities between the transformant and a clin-
ical isolate, NG-3, the donor of the resistance gene.
Sequences of penA genes in strains with reduced suscepti-
bilities to cefixime. The full-length penA sequences were de-
termined by using five strains (strains NG-3, NG-25, NG-46,
and NG-48, isolated in 2000, and strain NG-83, isolated in
2001) with reduced susceptibilities to cefixime (MICs, 0.5 and
0.25 ?g/ml for the strains isolated in 2000 and 2001, respec-
tively) and one cefixime-susceptible strain (strain NG-12, iso-
lated in 2000; cefixime MIC, 0.008 ?g/ml). Figure 1 shows the
full-length sequences of the penA genes of NG-3 (Fig. 1B) and
NG-12 (Fig. 1C). In cefixime-susceptible strain NG-12, the
penA gene sequence corresponded to that of penicillin-suscep-
tible N. gonorrhoeae LM306 (GenBank accession no. M320921;
Fig. 1A) except for an extra aspartate codon and an extra 2 bp.
The penA gene of strain NG-3, which had reduced susceptibil-
ity to cefixime, did not have the extra codon (Fig. 1B), and the
sequence was not consistent with the sequence reported in the
database. Of 581 amino acids in the PBP 2 sequence of NG-3,
59 (10.2%) amino acids were different from the sequence of
NG-12, in addition to 1 amino acid insertion and a defect.
As a result of restriction fragment length polymorphism
analysis, it was found that the penA genes of all strains for
which cefixime MICs were below 0.125 ?g/ml had an extra
aspartate (GCA) codon (data not shown). The sequences of
the penA genes of strains NG-25, NG-46, NG-48, and NG-83
(strains with reduced susceptibilities to cefixime) were the
same as that of NG-3 except for a few mutations (data not
shown). These penA genes had a mosaic-like structure that
included regions that were quite similar to each region of the
penA genes of Neisseria perflava (Neisseria sicca) and Neisseria
cinerea (Fig. 2) as well as to those of Neisseria flavescens and
Neisseria meningitidis (data not shown). This mosaic-like struc-
ture was mainly observed in the region of the transpeptidase-
encoding domain of penA. The active-site serine residue (Ser-
X-X-Lys) as well as the Ser-X-Asn and the Lys-Thr-Gly motifs
were conserved in the penA sequence.
FIG. 2. Schematic representation of mosaic-like penA genes of
Neisseria strains. The penA gene of N. gonorrhoeae and the coding
region for PBP 2 are represented in the diagram. The penA genes of
cefixime-susceptible N. gonorrhoeae strain NG-12 (A), an N. gonor-
rhoeae strain with reduced susceptibility to cefixime (strain NG-3) (B),
N. cinerea strain LPN3173 (C), and N. perflava (N. sicca) strain 1654/
1659 (D) are shown. The nucleotide sequence divergences (in percent)
between regions of the N. gonorrhoeae NG-3 penA genes and the
corresponding regions in the penA genes of N. cinerea LPN3173 (u)
and N. perflava (N. sicca) 1654/1659 (o) are shown.
FIG. 1. Nucleotide sequence of penA gene of N. gonorrhoeae. The sequences of the penA genes of penicillin-susceptible strain LM306
(GenBank accession no. M32091) (A), the strain with reduced susceptibility to cefixime (strain NG-3) (B), and cefixime-susceptible strain NG-12
(C) are shown. The insertion of an extra aspartate (Asp-345A) is shown in cefixime-susceptible strain NG-12 but is not shown in the strain with
reduced susceptibility to cefixime, NG-3. Asp?, Asp-345A. The Ser-X-X-Lys, Ser-X-Asn, and Lys-Thr-Gly conserved motifs are indicated by
VOL. 46, 2002 MOSAIC-LIKE STRUCTURE OF GONOCOCCAL penA 3747
In Japan, the emergence of resistance to cephems in N.
gonorrhoeae is a serious concern. A more serious problem,
however, is that these isolates are already resistant to non-?-
lactam antimicrobials (1, 13). N. gonorrhoeae strains with re-
duced susceptibilities to cefixime from male urethritis patients
at hospitals in Tokyo were also resistant to non-?-lactam an-
timicrobials, including fluoroquinolones. From the results of
susceptibility testing with the strains isolated in 2000 and 2001,
it was revealed that the numbers of strains with reduced sus-
ceptibilities to ?-lactams, such as cefixime, cefteram, cefdinir,
cefpodoxime, and aztreonam, had increased. Similar results
were obtained with cefozopran-resistant N. gonorrhoeae strains
isolated in Kitakyushu, Japan, for which the cefixime MICs
were 0.125 to 0.5 ?g/ml (13).
It has been reported that N. gonorrhoeae strains with re-
duced susceptibilities to cephems evolved by the acquisition of
?-lactamases, target modification (alteration of PBPs), alter-
ation of outer membrane transport, or enhancement of
MtrCDE efflux pumps (10). ?-Lactamase production did not
contribute to the resistance in the strains tested in this study
because ?-lactamase activity was not detected in any of the
strains. Transformation of the penA gene from a strain with
reduced susceptibility to cefixime showed that the reduction in
susceptibility to ?-lactams was caused by PBP alterations.
However, the reasons for the differences in the ratios of the
MICs for the transformants to the MICs for the recipients
between some ?-lactams and the differences in susceptibilities
between transformants and clinical isolates have not been
identified. The latter reasons for these differences were con-
sidered enhancement of efflux pumps, alteration of outer
membrane transport, and other PBP mutations.
In previous reports, insertion of the Asp-345A codon into
the penA gene has proved to make a major contribution to the
reduction of the affinity of gonococcal PBP 2 to penicillin (5).
In this study, all strains for which cefixime MICs were below
0.125 ?g/ml had an extra aspartate codon (Asp-345A) and
showed reduced susceptibilities to penicillin, as reported pre-
viously (5). On the other hand, this extra codon was not de-
tected in the strains for which cefixime MICs were 0.25 and 0.5
The sequence of the penA gene of one strain, NG-3, with
reduced susceptibility to cefixime (MIC, 0.5 ?g/ml) was not
completely consistent with the sequence reported in the data-
base and had a mosaic-like structure that included a region
whose sequence was quite similar to the sequences of the penA
genes of N. perflava (N. sicca) and N. cinerea (Fig. 2) as well as
those of N. flavescens and N. meningitidis (data not shown).
Similar results have been reported from studies of the se-
quences of the penA genes of penicillin-resistant strains of N.
meningitidis and Neisseria spp. (2, 4, 12, 16, 18, 20). One of the
donors conferring the penA penicillin resistance gene to N.
meningitidis has been identified as the naturally penicillin-re-
sistant species N. flavescens (20). An N. gonorrhoeae penA gene
with a mosaic-like structure that confers reduced susceptibility
to cefixime might have been constructed by a medley of partial
penA genes from N. perflava (N. sicca), N. cinerea, N. flave-
scens, and N. meningitidis. The reduction of susceptibility to
cephems, including cefixime, in this study might have evolved
by genetic exchange between commensal resistant Neisseria
spp. and the original susceptible gonococci.
N. gonorrhoeae is one of the bacteria isolated from patients
with sexually transmitted diseases. It has recently been re-
ported that, in Japan, N. gonorrhoeae has been isolated from
areas unrelated to the urethra, such as the pharynx (17). In the
present study it was clear that the source of infection was oral
sex for two of four patients from whom N. gonorrhoeae strains
for which the cefixime MIC was 0.5 ?g/ml were isolated. We
speculate that a penA gene with a novel type of mosaic-like
structure might have emerged by the transduction of regions
from the penA genes of Neisseria spp. Due to the diversity of
commercial sex, N. gonorrhoeae can inhabit the pharynx, and
gene transformation between N. gonorrhoeae and other Neis-
seria spp. might proceed.
Our preliminary study with penA genes from isolates with
reduced susceptibilities to cefixime (cefixime MICs, 0.0625 to
0.125 ?g/ml) recovered in 2001 showed that the penA genes of
these strains also had a mosaic-like structure and did not have
the Asp-345A codon insert. This penA gene was different from
that found in strain NG-3 in the present study (data not
shown). The preliminary information presented above and the
results obtained in this study suggest that the complicated
process concerning the evolution of resistance in N. gonor-
rhoeae might be developing, and more attention should be paid
to the emergence of resistance in Neisseria spp., including N.
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3748AMEYAMA ET AL.ANTIMICROB. AGENTS CHEMOTHER.