A Case of Decreased Susceptibility to Ceftriaxone in Neisseria gonorrhoeae in the Absence of a Mosaic Penicillin-Binding Protein 2 (penA) Allele

ABSTRACT Neisseria gonorrhoeae strains with reduced susceptibility to cefixime (MICs, 0.25 to 0.5 μg/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 susceptibility to cefixime (MIC, 0.5 μg/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.

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Amino Acid Substitutions in Mosaic Penicillin-Binding Protein 2 Associated with Reduced Susceptibility to Cefixime in Clinical Isolates of Neisseria gonorrhoeae

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00626-06 ◽

2006 ◽

Vol 50 (11) ◽

pp. 3638-3645 ◽

Cited By ~ 68

Author(s):

Sho Takahata ◽

Nami Senju ◽

Yumi Osaki ◽

Takuji Yoshida ◽

Takashi Ida

Keyword(s):

Amino Acid ◽

Neisseria Gonorrhoeae ◽

Molecular Mechanisms ◽

Binding Protein ◽

Complete Sequence ◽

Clinical Isolates ◽

Amino Acid Substitutions ◽

Penicillin Binding Protein ◽

Fourfold Increase ◽

Genes Encoding

ABSTRACT The molecular mechanisms of reduced susceptibility to cefixime in clinical isolates of Neisseria gonorrhoeae, particularly amino acid substitutions in mosaic penicillin-binding protein 2 (PBP2), were examined. The complete sequence of ponA, penA, and por genes, encoding, respectively, PBP1, PBP2, and porin, were determined for 58 strains isolated in 2002 from Japan. Replacement of leucine 421 by proline in PBP1 and the mosaic-like structure of PBP2 were detected in 48 strains (82.8%) and 28 strains (48.3%), respectively. The presence of mosaic PBP2 was the main cause of the elevated cefixime MIC (4- to 64-fold). In order to identify the mutations responsible for the reduced susceptibility to cefixime in isolates with mosaic PBP2, penA genes with various mutations were transferred to a susceptible strain by genetic transformation. The susceptibility of partial recombinants and site-directed mutants revealed that the replacement of glycine 545 by serine (G545S) was the primary mutation, which led to a two- to fourfold increase in resistance to cephems. Replacement of isoleucine 312 by methionine (I312M) and valine 316 by threonine (V316T), in the presence of the G545S mutation, reduced susceptibility to cefixime, ceftibuten, and cefpodoxime by an additional fourfold. Therefore, three mutations (G545S, I312M, and V316T) in mosaic PBP2 were identified as the amino acid substitutions responsible for reduced susceptibility to cefixime in N. gonorrhoeae.

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Penicillin-binding protein 2 genes of chromosomally-mediated penicillin-resistant Neisseria gonorrhoeae from Greece: screening for codon Asp-345A

Journal of Antimicrobial Chemotherapy ◽

10.1093/jac/32.5.677 ◽

1993 ◽

Vol 32 (5) ◽

pp. 677-684 ◽

Cited By ~ 4

Author(s):

I. Tirodimos ◽

E. Tzelepi ◽

V. C. Katsougiannopoulos

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Protein ◽

Penicillin Binding Protein

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Penicillin-binding protein 2 genes of non-?-lactamase-producing, penicillin-resistant strains of Neisseria gonorrhoeae

Molecular Microbiology ◽

10.1111/j.1365-2958.1989.tb00101.x ◽

1989 ◽

Vol 3 (1) ◽

pp. 35-41 ◽

Cited By ~ 43

Author(s):

C. G. Dowson ◽

A. E. Jephcott ◽

K. R. Gough ◽

B. G. Spratt

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Resistant Strains

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Mutations in penicillin-binding protein 2 from cephalosporin-resistant Neisseria gonorrhoeae hinder ceftriaxone acylation by restricting protein dynamics

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012617 ◽

2020 ◽

Vol 295 (21) ◽

pp. 7529-7543

Author(s):

Avinash Singh ◽

Jonathan M. Turner ◽

Joshua Tomberg ◽

Alena Fedarovich ◽

Magnus Unemo ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Protein Dynamics ◽

Conformational Changes ◽

Active Site ◽

Molecular Mechanisms ◽

Binding Protein ◽

Transmitted Disease ◽

Penicillin Binding Protein ◽

Sexually Transmitted ◽

Diminished Capacity

The global incidence of the sexually transmitted disease gonorrhea is expected to rise due to the spread of Neisseria gonorrhoeae strains with decreased susceptibility to extended-spectrum cephalosporins (ESCs). ESC resistance is conferred by mosaic variants of penicillin-binding protein 2 (PBP2) that have diminished capacity to form acylated adducts with cephalosporins. To elucidate the molecular mechanisms of ESC resistance, we conducted a biochemical and high-resolution structural analysis of PBP2 variants derived from the decreased-susceptibility N. gonorrhoeae strain 35/02 and ESC-resistant strain H041. Our data reveal that mutations both lower affinity of PBP2 for ceftriaxone and restrict conformational changes that normally accompany acylation. Specifically, we observe that a G545S substitution hinders rotation of the β3 strand necessary to form the oxyanion hole for acylation and also traps ceftriaxone in a noncanonical configuration. In addition, F504L and N512Y substitutions appear to prevent bending of the β3–β4 loop that is required to contact the R1 group of ceftriaxone in the active site. Other mutations also appear to act by reducing flexibility in the protein. Overall, our findings reveal that restriction of protein dynamics in PBP2 underpins the ESC resistance of N. gonorrhoeae.

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The role of penicillin-binding protein 2 (PBP2) in the cephalosporin susceptibility of Neisseria gonorrhoeae and the need for consensus in naming of PBP2

Journal of the Formosan Medical Association ◽

10.1016/j.jfma.2012.03.015 ◽

2012 ◽

Vol 111 (12) ◽

pp. 665-666 ◽

Cited By ~ 6

Author(s):

Chun-Chen Chen ◽

Kun-Yen Lin ◽

Shu-Ying Li

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Protein ◽

Penicillin Binding Protein

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Antimicrobial Resistance and Molecular Typing of Neisseria gonorrhoeae Isolates in Kyoto and Osaka, Japan, 2010 to 2012: Intensified Surveillance after Identification of the First Strain (H041) with High-Level Ceftriaxone Resistance

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01295-13 ◽

2013 ◽

Vol 57 (11) ◽

pp. 5225-5232 ◽

Cited By ~ 71

Author(s):

Ken Shimuta ◽

Magnus Unemo ◽

Shu-ichi Nakayama ◽

Tomoko Morita-Ishihara ◽

Misato Dorin ◽

...

Keyword(s):

Antimicrobial Resistance ◽

Neisseria Gonorrhoeae ◽

Molecular Typing ◽

Multilocus Sequence Typing ◽

Binding Protein ◽

Sporadic Case ◽

Penicillin G ◽

Penicillin Binding Protein ◽

Content Type ◽

High Level

ABSTRACTIn 2009, the first high-level ceftriaxone-resistantNeisseria gonorrhoeaestrain (H041) was isolated in Kyoto, Japan. The present study describes an intensified surveillance (antimicrobial resistance and molecular typing) ofNeisseria gonorrhoeaeisolates in Kyoto and its neighboring prefecture Osaka, Japan, in 2010 to 2012, which was initiated after the identification of H041. From April 2010 to March 2012, 193N. gonorrhoeaeisolates were collected and the MICs (μg/ml) to six antimicrobials, including ceftriaxone, were determined. All isolates showed susceptibility to ceftriaxone and cefixime (MIC values, <0.5 μg/ml), and spectinomycin. The rates of resistance (intermediate susceptibility) to azithromycin, penicillin G, and ciprofloxacin were 3.6% (19.7%), 24.4% (71.0%), and 78.2% (0.5%), respectively. Multilocus sequence typing (MLST) showed that 40.9%, 19.2%, and 17.1% of isolates belonged to ST1901, ST7359, and ST7363, respectively. Furthermore,N. gonorrhoeaemultiantigen sequence typing (NG-MAST) revealed that 12 (63%) of the 19 isolates with decreased susceptibility to ceftriaxone (MIC > 0.064 μg/ml) were of ST1407. NG-MAST ST1407 was also the most prevalent ST (16.1%; 31 of 193 isolates). In those NG-MAST ST1407 strains, several mosaic typepenAalleles were found, including SF-A type (penicillin binding protein 2 allele XXXIV) and its derivatives. These were confirmed using transformation of thepenAmosaic alleles as critical determinants for enhanced cefixime and ceftriaxone MICs. The intensified surveillance in Kyoto and Osaka, Japan, did not identify any dissemination of the high-level ceftriaxone-resistantN. gonorrhoeaestrain H041, suggesting that H041 might have caused only a sporadic case and has not spread further.

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