Transfer of a gonococcal &bgr;-lactamase plasmid into Neisseria gonorrhoeae belonging to the globally distributed ST1407 lineage

Sir, Neisseria gonorrhoeae isolates belonging to multi-antigen ST (NG-MAST) ST1407 have been detected globally, with the proportion ranging from 16.1% in the Far East to 23% in Europe. ST1407 gonococcal isolates are associated with cefixime and ciprofloxacin resistance, show raised MICs of ceftriaxone and azithromycin and are responsible for many cefixime or ceftriaxone treatment failures. Despite occurring in great numbers and having global distribution, ST1407 isolates have not been reported to harbour the b-lactamase-encoding plasmids that confer high-level penicillin resistance in N. gonorrhoeae and the reasons for this are not known. We sought to establish whether a panel of ST1407 isolates could acquire, express and stably maintain a gonococcal b-lactamase plasmid via conjugation. We selected seven ST1407 gonococci isolated from patients attending UK clinics during 2010–12 (Table 1). Conjugation experiments were performed using a filter-mating procedure with strain WHO-M, which contains an ‘African’ b-lactamase plasmid, as the donor. As a ‘control’ recipient, we used strain WHO-K (NG-MAST ST1424), which is b-lactamase negative and also has ciprofloxacin and cefixime resistance, similar to that of the ST1407 isolates. For matings between the WHO-K recipient and WHO-M, GC base (BD, Oxford, UK) supplemented with 1% Vitox (Oxoid, Basingstoke, UK) and both penicillin (8 mg/L) and ciprofloxacin (16 mg/L) was used for selection of transconjugants. For conjugations between ST1407 isolates and WHO-M, a range of penicillin and ciprofloxacin concentrations was used to establish optimal selection (Table 1). Suspected transconjugants were confirmed to be Gramnegative cocci and oxidase positive. Nitrocefin (Oxoid, Basingstoke, UK) was used to detect b-lactamase production and thus confirm the acquisition of the b-lactamase plasmids. Conjugation frequencies were calculated as the number of transconjugants obtained per input recipient cell exposed to donor cells. The plasmid profiles of the control strains, the ST1407 isolates and the transconjugants were established by extracting the plasmids using the QIAprep Spin Miniprep Kit (Qiagen, Manchester, UK) using the manufacturer’s standard protocol and growth from two culture plates. Extracted plasmids were run on 0.8% agarose gels incorporating GelRed dye (Biotium, Hayward, USA) for 5 h at 100 V. Plasmids were sized by comparison with those from isolates defined previously. Transconjugants were archived at #80 C and the stability of the b-lactamase plasmids was assessed after retrieval from#80 C and sub-culture without penicillin selection. The seven ST1407 recipients and strain WHO-K were all confirmed to be highly resistant to ciprofloxacin (MICs .32 mg/L) and, despite resistance to penicillin (MICs 1–2 mg/L), were b-lactamase negative (Table 1). Optimum selection of transconjugants was achieved using 4 mg/L for both penicillin and ciprofloxacin (Table 1). The presence of b-lactamase and the plasmid profiles of the transconjugants confirmed that the 3.2 MDa African b-lactamase plasmid was successfully transferred to all seven ST1407 isolates. The ST1407 transconjugants still harboured the b-lactamase plasmid after retrieval from #80 C, as indicated via the Nitrocefin test, but lost it after one sub-culture without penicillin selection. By contrast, WHO-K-derived transconjugants stably maintained the b-lactamase plasmid in the absence of penicillin selection for at least seven sub-cultures. N. gonorrhoeae isolates of a particular auxotype [one that requires proline, arginine and uracil and does not utilize ornithine (PAOU requiring)] do not acquire b-lactamase plasmids for reasons that are not fully elucidated, although the lack of the 2.6 MDa cryptic plasmid in these isolates may be a factor. However the ST1407 isolates studied here all contained the cryptic plasmid (by comparisons using agarose gel electrophoresis) and readily acquired an African b-lactamase plasmid via conjugation, so seem not to have mechanisms that prevent plasmid acquisition. The apparent absence of clinical ST1407 penicillinase-producing N. gonorrhoeae (PPNG) isolates may be due to an inability of the ST to maintain the plasmids stably in the absence of selection or due to a lack of exposure to other gonococci that harbour b-lactamase plasmids. For example, in England the ST1407 clone has been associated primarily with MSM, whereas contemporaneous PPNG were significantly associated with heterosexuals. A lack of bridging between heterosexual and MSM networks might provide an epidemiological explanation for why PPNG has never been detected in ST1407, rather than there being any underlying microbiological explanation. Nevertheless, in Europe ST1407 isolates were initially detected in both MSM and heterosexuals and an association between heterosexuals and PPNG has been established over some years (2011– 14) so there may have been more opportunity for ST1407 and PPNG isolates to infect the same host. The reason why the ST1407 isolates in this study could not maintain the African b-lactamase plasmid in the absence of selection may be elucidated by whole-genome sequence analysis and