Spread of a Chromosomal Cefixime-Resistant penA Gene among Different Neisseria gonorrhoeae Lineages

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.Neisseria gonorrhoeae is one of the most common sexually transmissible infective agents. Humans are the only natural host for N. gonorrhoeae, and transmission is restricted to direct person-to-person sexual contact. As there is no vaccine for gonorrhea, the control of dissemination depends on timely identification and initiation of an appropriate antibiotic treatment for the infected person in order to prevent transmission.N. gonorrhoeae strains that are resistant to various types of antibiotics have emerged, causing critical concern for public health around the world. Resistance to oral cephalosporins, such as cefixime, is emerging (2, 3, 10, 18), and approximately 30% of N. gonorrhoeae isolates in Japan now show reduced susceptibility to cefixime (20). The molecular mechanism of resistance has been elucidated as the formation of a mosaic structure of penA-encoded penicillin-binding protein 2 (PBP 2). The mosaic penA was generated by interspecies recombination with other neisserial species (3, 10), which is the same mechanism for chromosomally mediated penicillin resistance in N. gonorrhoeae (23). However, the precise junctions of recombination have not been fully elucidated.penA-encoded PBP 2 proteins of N. gonorrhoeae are divided into several types on the basis of the amino acid sequence, and some of these types are associated with reduced susceptibility to cefixime (10, 14, 25, 27). Among these, the most common PBP 2 type is pattern X (PBP 2-X), implying the expansion of a single clone. According to the spread of isolates with reduced susceptibility to cefixime, the expansion of a single clone, which emerged at an early phase, is suggested (18). However, another possibility is that recombination of the penA gene occurred several times independently, followed by multiclonal expansion. Understanding of the mode of spread of antibiotic-resistant clones could help us construct a public health strategy for preventing the further spread of resistant clones.To investigate the mode of dissemination of the newly emerged antibiotic-resistant N. gonorrhoeae isolates, we retrospectively characterized isolates with reduced susceptibility to cefixime (cefixime MIC ≥ 0.25 μg/ml, referred to hereafter as Cef^Rs isolates), using penA sequencing and multilocus sequence typing (MLST) with seven housekeeping genes. We also examined whether the horizontal transfer of penA occurred in vitro, resulting in the one-step emergence of Cef^Rs isolates from the susceptible isolate.