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.

FIG. 1.

Relationships of 33 PBP 2 types. A neighbor-joining tree was constructed from the PBP 2 amino acid sequences. The tree contains the LM306, PBP 2, and the PBP 2 types reported by Ito et al. (10), Whiley et al. (27), Takahata et al. (25), and Lindberg et al. (14) and in this study. The PBP 2 types that resulted in the reduced susceptibility of N. gonorrhoeae to cefixime are shaded in gray. Black dots indicate the PBP 2 types found in this study.

FIG. 2.

PFGE patterns of clones obtained by in vitro penA-X transfer. NG0202 (Cef^s of ST1901) and NG0003 (Cef^Rs of ST7363) were cocultivated overnight, and then colonies that were resistant to both cefixime (Cef) and ciprofloxacin (Cip) were identified by using GC agar plates containing 0.031 μg/ml of cefixime and 2 μg/ml of ciprofloxacin. SpeI-digested genomic DNA from 12 of the clones obtained was analyzed by PFGE. Lanes M, size marker consisting of SpeI-digested Salmonella enterica serovar Braendecup strain H9812 genomic DNA.

FIG. 3.

Comparison of sequences from nucleotides 241 to 720 among penA-X, penA-V, and minor variants of the transformants. Dashes indicate the same nucleotide as penA-X of strain NG0003 (shown on the first line). Shaded boxes in penA Tf-3 and NG0202 (positions 241 to 417) indicate regions where the sequences between them are identical. The underlined region indicates the possible junction site of Tf-3.

FIG. 4.

Sequence diversity in penA-flanking regions (6,299 bp) among strain NG0003, strain NG0202, and the transformants generated by in vitro cocultivation. (A) Sequence identity of each 100 bp between NG0003 and NG0202. (B) Boxes indicate the five open reading frames in this region, mraW, NGO1543, penA, murE, and dcaA. Gray boxes, the highly variable region; dark gray boxes, sequences that are identical to the sequence of the penA-X-flanking region of NG0003; bright gray boxes, sequences identical to the sequence of the penA-V-flanking region of NG0202; fine vertical lines (white and black), polymorphic sites that match the nucleotide bases of NG0202. (C) Sequence diversity in the penA-murE-dcaA regions of additional clinical isolates of ST1901.