Characterization of a Novel Neisseria gonorrhoeae Penicillinase-Producing Plasmid Isolated in Australia in 2012

LETTER

Neisseria gonorrhoeae plasmids harboring a penicillinase gene are responsible for dissemination of high-level penicillin resistance among gonococci worldwide. Penicillinase-producing N. gonorrhoeae (PPNG) strains are believed to have originally acquired such plasmids via horizontal genetic transfer from Haemophilus species (1). The “ancestral” N. gonorrhoeae plasmid harboring a bla_TEM-1 gene, the “Asian” plasmid, was first isolated in southeast Asia and is 7,426 bp in size (2). Several other variants, with various deletions and insertions (3), have since been described and named on the basis of their epidemiological origin; these include the African (5,599 bp) (4, 5), Rio-de-Janerio/Toronto (5,154 bp) (6, 7), Nimes (6,798 bp) (8), New-Zealand (9,309 bp) (9), and Johannesburg (4,865 bp) (10) plasmids. Here, we report a novel N. gonorrhoeae plasmid isolated in Australia.

This study was conducted as a part of the Gonorrhoea Resistance Assessment by Nucleic Acid Detection (GRAND) study (11, 12), an Australian study aimed at developing molecular methods of tracking N. gonorrhoeae antimicrobial resistance (AMR). As part of these investigations, we aimed to examine the conservation of sequence targets utilized by a previously described PPNG real-time PCR assay (13), a method that is now routinely used to detect PPNG strains in noncultured clinical samples in the Australian state of Western Australia (14). The assay acts as an indirect marker of penicillinase activity by amplifying a region of sequence predicted to be conserved across all known PPNG plasmid types while not specifically targeting the bla coding sequence (13).

In this study, we used PPNG-PCR to screen 342 PPNG isolates, identified by standard phenotypic procedures, obtained from reference laboratories throughout Australia for the first 6 months of 2012; these comprised approximately 98% of all PPNG strains isolated in Australia in this time period (12). In total, 341/342 isolates were positive whereas one isolate from the state of South Australia was negative. The isolate exhibited resistance to penicillin (MIC > 2 mg/liter), tetracycline (MIC > 16 mg/liter), and ciprofloxacin (MIC = 2 mg/liter) and was susceptible to ceftriaxone (MIC ≤ 0.004 mg/liter). Single nucleotide polymorphism-based multilocus sequence typing (MLST) (11) showed that the isolate was of MLST type 8784. An isolate of this sequence type was previously observed in Thailand and was found to a harbor a Rio-de-Janerio/Toronto plasmid with a bla_TEM-135 allele, considered to be a precursor to an extended-spectrum β-lactamase gene (15).

Complete DNA sequencing of the plasmid sequence from the South Australian isolate was achieved by PCR and Sanger sequencing using several overlapping primer pairs to span the entire plasmid. This sequencing revealed a plasmid of 3,269 bp (GenBank accession number KJ842484), the smallest such gonococcal plasmid described to date. When aligned with the “Asian” plasmid sequence (see Fig. S1 in the supplemental material), it was found to resemble the Rio-de-Janerio/Toronto plasmid by having a 2,273-bp deletion at positions 3795 to 6066 but exhibited a further 1,885-bp deletion corresponding to nucleotides 502 to 2385 (Asian plasmid numbering; GenBank accession number U20374). This 1,885-bp deletion comprised the region targeted by the PPNG-PCR, hence accounting for the false-negative result by the PPNG-PCR. The deletion did not, however, have an impact on the coding region of the bla_TEM-135 gene. Of further note was that the 1,885-bp deletion was flanked by two 92-bp “DR-2” direct repeat regions (see Fig. S1). Such repeat regions have been implicated in the formation of gonococcal deletion variant plasmids (16), and so these DR-2 sequences likely played a key role in the observed deletion.

Genetic variation within the chromosome and plasmids of the gonococcus is well recognized. This novel plasmid variant, henceforth called the Australian plasmid, further highlights the challenges of using molecular methods for N. gonorrhoeae diagnostics and characterization, particularly, as in this case, molecular tracking of high-level penicillin resistance using the “universal” PPNG-PCR assay. This sequence variant also serves as yet another reminder of the need for quality assurance testing to ensure the reliability of molecular AMR assays.