Emergence and Spread of Neisseria gonorrhoeae Clinical Isolates Harboring Mosaic-Like Structure of Penicillin-Binding Protein 2 in Central Japan

ABSTRACT Of 150 clinical isolates of Neisseria gonorrhoeae recovered in 2001, we examined 55 clinical isolates of N. gonorrhoeae for which cefixime MICs were ≥0.125 μg/ml and randomly selected 15 isolates for which cefixime MICs were ≤0.06 μg/ml for analysis of alterations in the penicillin-binding protein 2 (PBP 2) gene. We found insertion of an extra codon (Asp-345a) in the transpeptidase domain of PBP 2, and this insertion occurred alone or in conjunction with other amino acid substitutions. We also found a mosaic PBP 2 that was composed of fragments of the PBP 2 proteins from Neisseria cinera and Neisseria perflava. This mosaic PBP 2 was significantly associated with decreased susceptibilities to penicillin and cephalosporins, especially oral cephalosporins. For most of the isolates with a mosaic PBP 2, the cefixime MICs were ≥0.5 μg/ml and the cefdinir MICs were ≥1 μg/ml. Analysis of chromosomal DNA restriction patterns by pulsed-field gel electrophoresis revealed that most isolates with the mosaic PBP 2 were genetically similar. The recombination events that generated the mosaic PBP 2 would likely have contributed to the decreased sensitivities to cephalosporins. Isolates with the mosaic PBP 2 appear to threaten the efficacy of the currently recommended regimen with cefixime. The emergence of such strains may be the result of the in vivo generation of clones in which interspecies recombination occurred between the penA genes of N. gonorrhoeae and commensal Neisseria species.

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Mosaic-Like Structure of Penicillin-Binding Protein 2 Gene (penA) in Clinical Isolates of Neisseria gonorrhoeae with Reduced Susceptibility to Cefixime

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.46.12.3744-3749.2002 ◽

2002 ◽

Vol 46 (12) ◽

pp. 3744-3749 ◽

Cited By ~ 132

Author(s):

Satoshi Ameyama ◽

Shoichi Onodera ◽

Masahiro Takahata ◽

Shinzaburo Minami ◽

Nobuko Maki ◽

...

Keyword(s):

Streptococcus Pneumoniae ◽

Neisseria Gonorrhoeae ◽

Neisseria Meningitidis ◽

Binding Protein ◽

Clinical Isolates ◽

Penicillin Binding Protein ◽

Gene Sequences ◽

Neisseria Flavescens ◽

Neisseria Perflava

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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Analysis of amino acid sequences of penicillin-binding protein 2 in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime and ceftriaxone

Journal of Infection and Chemotherapy ◽

10.1007/s10156-008-0610-7 ◽

2008 ◽

Vol 14 (3) ◽

pp. 195-203 ◽

Cited By ~ 58

Author(s):

Kazuyoshi Osaka ◽

Masahiro Takahata ◽

Tadakazu Takakura ◽

Kayo Narukawa ◽

Katsuhisa Endo ◽

...

Keyword(s):

Amino Acid ◽

Neisseria Gonorrhoeae ◽

Binding Protein ◽

Amino Acid Sequences ◽

Clinical Isolates ◽

Penicillin Binding Protein

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Overproduction of Penicillin-Binding Protein 2 and Its Inactive Variants Causes Morphological Changes and Lysis in Escherichia coli

Journal of Bacteriology ◽

10.1128/jb.00207-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 4975-4983 ◽

Cited By ~ 17

Author(s):

Blaine A. Legaree ◽

Calvin B. Adams ◽

Anthony J. Clarke

Keyword(s):

Escherichia Coli ◽

Signal Sequence ◽

Morphological Changes ◽

Binding Protein ◽

Penicillin Binding Protein ◽

Prolonged Incubation ◽

Lytic Transglycosylases ◽

E Coli ◽

Derivatives Of

ABSTRACT Penicillin-binding protein 2 (PBP 2) has long been known to be essential for rod-shaped morphology in gram-negative bacteria, including Escherichia coli and Pseudomonas aeruginosa. In the course of earlier studies with P. aeruginosa PBP 2, we observed that E. coli was sensitive to the overexpression of its gene, pbpA. In this study, we examined E. coli overproducing both P. aeruginosa and E. coli PBP 2. Growth of cells entered a stationary phase soon after induction of gene expression, and cells began to lyse upon prolonged incubation. Concomitant with the growth retardation, cells were observed to have changed morphologically from typical rods into enlarged spheres. Inactive derivatives of the PBP 2s were engineered, involving site-specific replacement of their catalytic Ser residues with Ala in their transpeptidase module. Overproduction of these inactive PBPs resulted in identical effects. Likewise, overproduction of PBP 2 derivatives possessing only their N-terminal non-penicillin-binding module (i.e., lacking their C-terminal transpeptidase module) produced similar effects. However, E. coli overproducing engineered derivatives of PBP 2 lacking their noncleavable, N-terminal signal sequence and membrane anchor were found to grow and divide at the same rate as control cells. The morphological effects and lysis were also eliminated entirely when overproduction of PBP 2 and variants was conducted with E. coli MHD79, a strain lacking six lytic transglycosylases. A possible interaction between the N-terminal domain of PBP 2 and lytic transglycosylases in vivo through the formation of multienzyme complexes is discussed.

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

Sexually Transmitted Diseases ◽

10.1097/olq.0000000000000645 ◽

2017 ◽

Vol 44 (8) ◽

pp. 492-494 ◽

Cited By ~ 11

Author(s):

A. Jeanine Abrams ◽

Robert D. Kirkcaldy ◽

Kevin Pettus ◽

Jan L. Fox ◽

Grace Kubin ◽

...

Keyword(s):

Neisseria Gonorrhoeae ◽

Binding Protein ◽

Penicillin Binding Protein

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Sequence of the penicillin-binding protein 2-encoding gene (penA) of Neisseria perflava/sicca

Gene ◽

10.1016/0378-1119(94)90839-7 ◽

1994 ◽

Vol 146 (1) ◽

pp. 91-93 ◽

Cited By ~ 4

Author(s):

Amalia Perez-Castillo ◽

Ana M. Perez-Castillo ◽

Juan A. Saez-Nieto

Keyword(s):

Binding Protein ◽

Penicillin Binding Protein ◽

Encoding Gene ◽

Neisseria Perflava

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Pan-β-Lactam Resistance Development in Pseudomonas aeruginosa Clinical Strains: Molecular Mechanisms, Penicillin-Binding Protein Profiles, and Binding Affinities

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00680-12 ◽

2012 ◽

Vol 56 (9) ◽

pp. 4771-4778 ◽

Cited By ~ 83

Author(s):

Bartolomé Moyá ◽

Alejandro Beceiro ◽

Gabriel Cabot ◽

Carlos Juan ◽

Laura Zamorano ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Molecular Mechanisms ◽

Efflux Pump ◽

Binding Protein ◽

Efflux Pumps ◽

Binding Affinities ◽

Penicillin Binding Protein ◽

Content Type ◽

Reverse Transcription Pcr

ABSTRACTWe investigated the mechanisms leading toPseudomonas aeruginosapan-β-lactam resistance (PBLR) development during the treatment of nosocomial infections, with a particular focus on the modification of penicillin-binding protein (PBP) profiles and imipenem, ceftazidime, and ceftolozane (former CXA-101) PBP binding affinities. For this purpose, six clonally related pairs of sequential susceptible-PBLR isolates were studied. The presence ofoprD,ampD, anddacBmutations was explored by PCR followed by sequencing and the expression ofampCand efflux pump genes by real-time reverse transcription-PCR. The fluorescent penicillin Bocillin FL was used to determine PBP profiles in membrane preparations from all pairs, and 50% inhibitory concentrations (IC50s) of ceftolozane, ceftazidime, and imipenem were analyzed in 3 of them. Although a certain increase was noted (0 to 5 2-fold dilutions), the MICs of ceftolozane were ≤4 μg/ml in all PBLR isolates. All 6 PBLR isolates lacked OprD and overexpressedampCand one or several efflux pumps, particularlymexBand/ormexY. Additionally, 5 of them showed modified PBP profiles, including a modified pattern (n= 1) or diminished expression (n= 1) of PBP1a and a lack of PBP4 expression (n= 4), which correlated with AmpC overexpression driven bydacBmutation. Analysis of the essential PBP IC50s revealed significant variation of PBP1a/b binding affinities, both within each susceptible-PBLR pair and across the different pairs. Moreover, despite the absence of significant differences in gene expression or sequence, a clear tendency toward increased PBP2 (imipenem) and PBP3 (ceftazidime, ceftolozane, imipenem) IC50s was noted in PBLR isolates. Thus, our results suggest that in addition to AmpC, efflux pumps, and OprD, the modification of PBP patterns appears to play a role in thein vivoemergence of PBLR strains, which still conserve certain susceptibility to the new antipseudomonal cephalosporin ceftolozane.

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