Rapid detection of the mosaic structure of the Neisseria gonorrhoeae penA Gene, which is associated with decreased susceptibilities to oral cephalosporins

Abstract

In Neisseria gonorrhoeae, the mosaic structure of the penA gene (encoding penicillin-binding protein 2 [PBP 2]), which is composed of fragments of the penA genes from Neisseria cinerea and Neisseria perflava, has been significantly associated with decreased susceptibility to cephalosporins, particularly oral cephalosporins. The aim of this study was to develop a rapid assay for the detection of mosaic PBP 2 of N. gonorrhoeae by real-time PCR. This assay successfully detected the mosaic penA gene of N. gonorrhoeae, and its sensitivity was >or=10(1) copies/reaction. Six hundred twenty-one clinical strains were examined by this assay for the presence of mosaic PBP 2, which was detected in 85 (39.4%) of 216 strains from 2002, 69 (40.6%) of 170 strains from 2003, 71 (44.4%) of 160 strains from 2004, and 31 (41.3%) of 75 strains from 2005. The MICs of cephalosporins for strains with the mosaic PBP 2 detected by the assay were statistically higher than those for strains without the mosaic PBP 2. One hundred sixty-six (64.8%) of 256 strains with the mosaic PBP 2 exhibited cefixime MICs of >or=0.5 microg/ml. The emergence and spread of strains with mosaic PBP 2 could be a threat to the cefixime treatment of gonorrhea. This real-time PCR assay for the detection of mosaic PBP 2 of N. gonorrhoeae is thus useful in the prediction of decreased susceptibilities to oral cephalosporins.

FIG. 1.

Locations of the primers and probe in the penA gene and the sequence of the penA genes from bases 801 to 1020. The light-gray arrows indicate the positions of the primers; the dark-gray shading indicates the position of the probe.

FIG. 2.

(A) Fluorescence profiles of 10-fold serially diluted penA/pAcHLT-A DNA from 1 × 10¹ to 1 × 10⁷ copies/reaction analyzed by real-time PCR using an NG89-P1 probe. ΔRn was calculated by subtracting the baseline fluorescence from the reporter fluorescence, which was normalized by an internal reference. (B) A standard curve plot of the 10-fold serial dilution of penA/pAcHLT-A. Linearity is observed throughout the range from 1 × 10¹ to 1 × 10⁷ copies/reaction.

FIG. 3.

Distributions of the MICs of cefozopran, cefdinir, cefixime, and ceftriaxone for the clinical strains of N. gonorrhoeae from 2002 to 2005. The black bars indicate the number of strains with the mosaic PBP 2; the white bars indicate the number of strains without the mosaic PBP 2. Mann-Whitney U tests were used to compare the MICs for the strains with the mosaic PBP 2 with those for the strains without the mosaic PBP 2. The MICs of each cephalosporin for the strains with the mosaic PBP 2 were statistically higher than those for the strains without the mosaic PBP 2.

FIG. 3.

Distributions of the MICs of cefozopran, cefdinir, cefixime, and ceftriaxone for the clinical strains of N. gonorrhoeae from 2002 to 2005. The black bars indicate the number of strains with the mosaic PBP 2; the white bars indicate the number of strains without the mosaic PBP 2. Mann-Whitney U tests were used to compare the MICs for the strains with the mosaic PBP 2 with those for the strains without the mosaic PBP 2. The MICs of each cephalosporin for the strains with the mosaic PBP 2 were statistically higher than those for the strains without the mosaic PBP 2.