Recently, there has been a marked decrease in the susceptibility of N. gonorrhoeae
strains to cefixime throughout Japan. These bacterial strains were previously shown to possess a mosaic-like structure of PBP2. Although earlier investigations have reported mutations of either PBP1 or PBP2 for numerous isolates (7
), there has been no systematic study of PBP sequences from various clinical isolates. In this study, the complete sequence of the genes encoding PBP1, PBP2, and porin were determined from 58 isolates. We found that the presence of mosaic PBP2 was strongly correlated with reduced susceptibility to cefixime and other cephems. Interestingly, all the strains with a mosaic PBP2 carried the L421P mutation in PBP1. The L421P substitution in PBP1, together with overexpression of MtrCDE and mutations in porin and PilQ, are reported to be involved in high-level resistance to penicillin (19
). Our transformation experiments demonstrate that recombination of full-length mosaic PBP2 could not fully explain the observed resistance of the clinical isolates (MSC02236) to cephem antibiotics, such as cefotiam, ceftriaxone, and cefpodoxime (Table ). Therefore, the amino acid substitution in PBP1 could be involved not only in the high-level resistance to penicillin (13
) but also in resistance to the cephem antibiotics. Indeed, the PBP1(L421P) mutation was found in all the strains with reduced susceptibility to cefixime, suggesting that it might partly contribute to the increase in MIC. However, further study is needed to clarify the association of the PBP1(L421P) mutation and resistance to cephems. As reported previously (6
), extensive sequence variability was identified for the porin, with 42 different amino acid patterns determined from 58 strains. Although most of the strains possess mutations at positions 120 and 121 of the porin protein, there is no clear correlation with increased resistance to cefixime.
Four different mosaic PBP2 sequences, designated mosaic-1 to mosaic-4, were detected from 28 isolates. Mosaic-1 sequence, the most frequently identified in this study, was identical in the isolates from Kitakyushu, Japan (strains SNG32, SNG33, SNG46 and SNG50; GenBank accession no. AY146782 to 146785), and also from the central region of Japan (47 strains of pattern X) (7
). Twelve out of 22 isolates with reduced susceptibility to cefixime (MIC of ≥0.25 μg/ml) from the Kinki area of Japan also possessed the mosaic-1 mutation in PBP2 (unpublished data). These observations suggest that isolates with mosaic-1 are spreading nationwide and comprise the strains with reduced susceptibility to cefixime.
We have identified three amino acid substitutions in mosaic-1 (G545S, I312M, and V316T) associated with reduced susceptibility to cefixime and other oral cephems. While full-length recombination of mosaic-1 led to a 16-fold reduction in susceptibility to cefixime, the mutation G545S plus either I312M or V316T conferred an eightfold reduction, suggesting that these substitutions are important for the resistance mechanism. The primary mutation G545S is located downstream of the conserved K497TG motif. The mutations conferring additional resistance to cephems, I312M and V316T, are associated with the conserved S310AIK motif. Mosaic-2 and mosaic-3 sequences differ from the sequence of mosaic-1 by a single amino acid change at position 101 or 214, respectively. Because these amino acid substitutions are not located in the transpeptidase domain, which constitutes the β-lactam binding site, the same three mutations found in mosaic-1 are likely to be crucial for conferring resistance to the mosaic-2 and -3 isolates. By contrast, the mosaic-4 sequence does not contain the G545S alteration. However, during the transformation experiments a spontaneous mutant with a single A501V mutation in PBP2, which was found in mosaic-4 sequence, was selected with cephalexin. Analysis of this transformant revealed that the A501V mutation in PBP2 led to a two- to fourfold increase in the MICs of cefixime and other cephems. This increase in resistance is similar to that observed for the PBP2(G545S) mutation. We assume that A501V complements the G545S substitution and is the primary mutation in mosaic-4 isolates.
It has been proposed that horizontal genetic exchange of the penA
genes between commensal Neisseria
species, such as Neisseria cinerea
, Neisseria perflava
and Neisseria flavescens
, resulted in the mosaic-like structure of PBP2 in N. gonorrhoeae
and Neisseria meningitidis
). Analysis of the amino acid sequences of PBP2 from various Neisseria
species indicated that PBP2 from N. perflava
1654/1659 (GenBank accession no. X76422) and N. flavescens
NCTC8263 (GenBank accession no. M26645) had methionine 312 and threonine 316, as does mosaic PBP2 in N. gonorrhoeae
. These observations suggest that the I312M and V316T substitutions originated from N. perflava
or N. flavescens
through horizontal gene transfer. However, no commensal Neisseria
species was found to possess serine 545. Therefore, the G545S alteration is probably the result of antibiotic selective pressure.
The results of our study suggest that the extensive use of cefixime, or other cephems with reduced affinity to mosaic PBP2, will select the resistant N. gonorrhoeae strains harboring mosaic PBP2. The antibacterial activity of ceftriaxone and cefditoren was only slightly reduced for strains with mosaic PBP2. Both these cephalosporins possess a long side chain at the C-3 position of the cephem skeleton, which might result in a strong affinity for the altered PBP2. Continued surveillance of antimicrobial susceptibility and genetic studies to identify the mechanisms of resistance will be needed to establish appropriate treatments. The results from such studies may also assist in the development of new antibiotics of therapeutic utility.