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Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages.

ABSTRACT: In Neisseria gonorrhoeae, the mosaic type of penA, which encodes penicillin-binding protein 2 (PBP 2), is associated with reduced susceptibility to oral cephalosporins. To investigate the relatedness of N. gonorrhoeae clinical isolates with reduced susceptibility, we sequenced the penA genes of 32 isolates. Five different amino acid sequence types of PBP 2 were identified, but all seemed to be derivatives of pattern X of PBP 2 (PBP 2-X). However, multilocus sequence typing of the isolates showed that the isolates belonged to six different sequence types. As PBP 2-X was identified in three different sequence types, horizontal transfer of the penA allele encoding PBP2-X was suggested. We demonstrated that the penA gene could be transferred from an isolate with reduced susceptibility to a sensitive isolate by natural transformation. Comparison of the sequence of the penA-flanking regions of 12 transformants with those of the donor and the recipient suggested that at least a 4-kb DNA segment, including the penA gene, was transferred. During horizontal transfer, some of the penA alleles also acquired variations due to point mutations and genetic exchange within the allele. Our results provide evidence that the capacity for natural transformation in N. gonorrhoeae plays a role in the spread of chromosomal antibiotic resistance genes and the generation of diversity in such genes.

SUBMITTER: Ohnishi M

PROVIDER: S-EPMC2826007 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages.

Ohnishi Makoto M Watanabe Yuko Y Ono Emi E Takahashi Chieko C Oya Hitomi H Kuroki Toshiro T Shimuta Ken K Okazaki Norio N Nakayama Shu-ichi S Watanabe Haruo H

Antimicrobial agents and chemotherapy 20091222 3

In Neisseria gonorrhoeae, the mosaic type of penA, which encodes penicillin-binding protein 2 (PBP 2), is associated with reduced susceptibility to oral cephalosporins. To investigate the relatedness of N. gonorrhoeae clinical isolates with reduced susceptibility, we sequenced the penA genes of 32 isolates. Five different amino acid sequence types of PBP 2 were identified, but all seemed to be derivatives of pattern X of PBP 2 (PBP 2-X). However, multilocus sequence typing of the isolates showed ...[more]

PMID: 20028823

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Project description:Treatment regimens for gonorrhea have limited efficacy worldwide due to the rapid spread of antimicrobial resistance. Cefixime (CFM) is currently not recommended as a first-line treatment for gonorrhea due to the increasing number of resistant strains worldwide. Nonetheless, Neisseria gonorrhoeae strains can be eradicated by CFM at a 400 mg/day dose, provided that the strains are CFM responsive (MIC ≤ 0.064 mg/L). To develop a nonculture test for predicting the CFM responsiveness of N. gonorrhoeae strains, we developed an assay to detect N. gonorrhoeae nonmosaic penA using loop-mediated isothermal amplification (LAMP). To avoid false-positive reactions with commensal Neisseria spp. penA, we amplified specific regions of the N. gonorrhoeae penA (NG-penA-LAMP1) and also the nonmosaic N. gonorrhoeae penA (NG-penA-LAMP3). This assay was validated using isolated N. gonorrhoeae (n = 204) and Neisseria spp. (n = 95) strains. Clinical specimens (n = 95) with confirmed positivity in both culture and real-time PCR were evaluated to validate the system. The combination of the previously described NG-penA-LAMP1 and our new NG-penA-LAMP3 assays had high sensitivity (100%) and specificity (100%) for identifying N. gonorrhoeae carrying the nonmosaic type. To determine whether CFM could be applicable for gonorrhea treatment without culture testing, we developed a LAMP assay that targets penA allele-specific nonmosaic types for use as one of the tools for point-of-care testing of antimicrobial resistance. IMPORTANCE Neisseria gonorrhoeae is among the hot topics of "resistance guided therapy," one of the top 5 urgent antimicrobial threats according to the Centers for Disease Control and Prevention (CDC). There is a need either to develop new agents or to make effective use of existing agents, with the current limited number of therapeutic agents available. Knowing the drug susceptibility information of the target microorganism prior to treating patients is very useful in selecting an effective antibiotic, especially in gonococcal infections where drug resistance is prominent, and is also important in preventing treatment failure. In this study, we developed a new method for obtaining drug susceptibility profiles of Neisseria gonorrhoeae using the loop-mediated isothermal amplification (LAMP) method. The LAMP assay does not require expensive devices. Therefore, this method is expected to be a tool for point-of-care testing of antimicrobial resistance for individualized treatment in the future.

Dataset Information

Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages.

Publications

Spread of a chromosomal cefixime-resistant penA gene among different Neisseria gonorrhoeae lineages.

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