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
penicillin-binding protein 2 that arose by interspecies horizontal gene trans-
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VOL. 46, 2002MOSAIC-LIKE STRUCTURE OF GONOCOCCAL penA 3749