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

Transfer of a gonococcal β-lactamase plasmid into Neisseria gonorrhoeae belonging to the... Sir, Neisseria gonorrhoeae isolates belonging to multi-antigen ST (NG-MAST)1 ST1407 have been detected globally,2 with the proportion ranging from 16.1% in the Far East3 to 23% in Europe.4 ST1407 gonococcal isolates are associated with cefixime and ciprofloxacin resistance, show raised MICs of ceftriaxone and azithromycin4 and are responsible for many cefixime or ceftriaxone treatment failures.2 Despite occurring in great numbers and having global distribution, ST1407 isolates have not been reported to harbour the β-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 β-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 procedure5 with strain WHO-M,6 which contains an ‘African’ β-lactamase plasmid, as the donor. As a ‘control’ recipient, we used strain WHO-K (NG-MAST ST1424),6 which is β-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). Table 1. N. gonorrhoeae ST1407 isolates selected for conjugational transfer of β-lactamase plasmids and subsequent number of ST1407 transconjugants and conjugation frequency Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA NA, not applicable. a Conjugation frequency was calculated as the number of transconjugants obtained per recipient cell exposed to donor cells. b Mean conjugation frequency. Table 1. N. gonorrhoeae ST1407 isolates selected for conjugational transfer of β-lactamase plasmids and subsequent number of ST1407 transconjugants and conjugation frequency Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA NA, not applicable. a Conjugation frequency was calculated as the number of transconjugants obtained per recipient cell exposed to donor cells. b Mean conjugation frequency. Suspected transconjugants were confirmed to be Gram-negative cocci and oxidase positive. Nitrocefin (Oxoid, Basingstoke, UK) was used to detect β-lactamase production and thus confirm the acquisition of the β-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.7 Transconjugants were archived at −80°C and the stability of the β-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 β-lactamase negative (Table 1). Optimum selection of transconjugants was achieved using 4 mg/L for both penicillin and ciprofloxacin (Table 1). The presence of β-lactamase and the plasmid profiles of the transconjugants confirmed that the 3.2 MDa African β-lactamase plasmid was successfully transferred to all seven ST1407 isolates. The ST1407 transconjugants still harboured the β-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 β-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 β-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.5 However the ST1407 isolates studied here all contained the cryptic plasmid (by comparisons using agarose gel electrophoresis) and readily acquired an African β-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 β-lactamase plasmids. For example, in England the ST1407 clone has been associated primarily with MSM,8 whereas contemporaneous PPNG were significantly associated with heterosexuals.9 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 heterosexuals4 and an association between heterosexuals and PPNG has been established over some years (2011–14)10 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 β-lactamase plasmid in the absence of selection may be elucidated by whole-genome sequence analysis and maintenance may be dependent upon plasmid type. There seems to be no biological barriers to prevent natural acquisition of high-level plasmid-mediated penicillin resistance, with this successful multiresistant clone needing only to overcome the ‘problem’ of stable maintenance and potentially harbour an additional resistance mechanism to its already extensive repertoire. Acknowledgements Many thanks to Dr Stephanie Chisholm (previously at PHE) and Dr Darren Mernagh and Professor Graham Mills (University of Portsmouth) for their support and advice in relation to the study. We would also like to acknowledge all those involved in the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP), from which some isolates were obtained. Funding The work was supported by Public Health England. Transparency declarations None to declare. References 1 Martin IM , Ison CA , Aanensen DM et al. Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area . J Infect Dis 2004 ; 189 : 1497 – 505 . Google Scholar CrossRef Search ADS PubMed 2 Unemo M , Nicholas RA. Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea . Future Microbiol 2012 ; 7 : 1401 – 22 . Google Scholar CrossRef Search ADS PubMed 3 Shimuta K , Unemo M , Nakayama S et al. Antimicrobial resistance and molecular typing of Neisseria gonorrhoeae isolates in Kyoto and Osaka, Japan, 2010 to 2012: intensified surveillance after identification of the first strain (H041) with high-level ceftriaxone resistance . Antimicrob Agents Chemother 2013 ; 57 : 5225 – 32 . Google Scholar CrossRef Search ADS PubMed 4 Chisholm SA , Unemo M , Quaye N et al. Molecular epidemiological typing within the European Gonococcal Antimicrobial Resistance Surveillance Programme reveals predominance of a multidrug-resistant clone . Euro Surveill 2013 ; 18 : pii=20358 . 5 Ison CA , Gill MJ , Woodford N. Transfer of β-lactamase plasmids by conjugation in Neisseria gonorrhoeae . Genitourin Med 1990 ; 66 : 82 – 6 . Google Scholar PubMed 6 Unemo M , Fasth O , Fredlund H et al. Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes . J Antimicrob Chemother 2009 ; 63 : 1142 – 51 . Google Scholar CrossRef Search ADS PubMed 7 Roberts M , Falkow S. Conjugal transfer of R plasmids in Neisseria gonorrhoeae . Nature 1977 ; 266 : 630 – 1 . Google Scholar CrossRef Search ADS PubMed 8 Ison CA , Town K , Obi C et al. Decreased susceptibility to cephalosporins among gonococci: data from the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales, 2007-2011 . Lancet Infect Dis 2013 ; 13 : 762 – 8 . Google Scholar CrossRef Search ADS PubMed 9 HPA . GRASP 2011 Report: The Gonococcal Resistance to Antimicrobials Surveillance Programme. http://webarchive.nationalarchives.gov.uk/20140714113628/http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317136030908. 10 ECDC . Gonococcal Antimicrobial Susceptibility Surveillance in Europe 2014. http://ecdc.europa.eu/en/publications/Publications/gonococcal-antimicrobial-susceptibility-surveillance-Europe-2014.pdf. © Crown copyright 2018. This article contains public sector information licensed under the Open Government Licence v3.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Antimicrobial Chemotherapy Oxford University Press

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

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Abstract

Sir, Neisseria gonorrhoeae isolates belonging to multi-antigen ST (NG-MAST)1 ST1407 have been detected globally,2 with the proportion ranging from 16.1% in the Far East3 to 23% in Europe.4 ST1407 gonococcal isolates are associated with cefixime and ciprofloxacin resistance, show raised MICs of ceftriaxone and azithromycin4 and are responsible for many cefixime or ceftriaxone treatment failures.2 Despite occurring in great numbers and having global distribution, ST1407 isolates have not been reported to harbour the β-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 β-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 procedure5 with strain WHO-M,6 which contains an ‘African’ β-lactamase plasmid, as the donor. As a ‘control’ recipient, we used strain WHO-K (NG-MAST ST1424),6 which is β-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). Table 1. N. gonorrhoeae ST1407 isolates selected for conjugational transfer of β-lactamase plasmids and subsequent number of ST1407 transconjugants and conjugation frequency Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA NA, not applicable. a Conjugation frequency was calculated as the number of transconjugants obtained per recipient cell exposed to donor cells. b Mean conjugation frequency. Table 1. N. gonorrhoeae ST1407 isolates selected for conjugational transfer of β-lactamase plasmids and subsequent number of ST1407 transconjugants and conjugation frequency Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA Recipient strain no. MIC (mg/L) No. of transconjugants (conjugation frequency)a at varying levels of antimicrobial selection Transconjugant β-lactamase status penicillin (β-lactamase status) ciprofloxacin cefixime ceftriaxone 8 mg/L penicillin and 16 mg/L ciprofloxacin 8 mg/L penicillin and 8 mg/L ciprofloxacin 4 mg/L penicillin and 4 mg/L ciprofloxacin G10-1836 2 (negative) >32 0.12 0.03 0 34 (6.3 × 10−7) 38 (7.0 × 10−7) positive G11-1015 1 (negative) >32 0.12 0.015 0 5 (2.2 × 10−7) 6 (2.6 × 10−7) positive G11-1052 2 (negative) >32 0.12 0.03 0 116 (2.4 × 10−6) 170 (3.5 × 10−6) positive G12-0410 2 (negative) >32 0.25 0.03 0 100 (2.0 × 10−6) 100 (2.0 × 10−6) positive G12-1225 2 (negative) >32 0.12 0.03 0 25 (6.0 × 10−7) 30 (7.1 × 10−7) positive G12-1360 2 (negative) >32 0.25 0.03 1 30 (6.5 × 10−7) 38 (8.3 × 10−7) positive G12-1682 1 (negative) >32 0.12 0.03 0 20 (3.7 × 10−7) 24 (4.4 × 10−7) positive WHO-K (control; ST1424) 2 (negative) >32 0.25 0.06 7.3 × 10−7b NA NA positive WHO-M (PPNG donor strain; ST3304) 8 (positive) 2 ≤0.016 0.016 NA NA NA NA NA, not applicable. a Conjugation frequency was calculated as the number of transconjugants obtained per recipient cell exposed to donor cells. b Mean conjugation frequency. Suspected transconjugants were confirmed to be Gram-negative cocci and oxidase positive. Nitrocefin (Oxoid, Basingstoke, UK) was used to detect β-lactamase production and thus confirm the acquisition of the β-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.7 Transconjugants were archived at −80°C and the stability of the β-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 β-lactamase negative (Table 1). Optimum selection of transconjugants was achieved using 4 mg/L for both penicillin and ciprofloxacin (Table 1). The presence of β-lactamase and the plasmid profiles of the transconjugants confirmed that the 3.2 MDa African β-lactamase plasmid was successfully transferred to all seven ST1407 isolates. The ST1407 transconjugants still harboured the β-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 β-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 β-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.5 However the ST1407 isolates studied here all contained the cryptic plasmid (by comparisons using agarose gel electrophoresis) and readily acquired an African β-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 β-lactamase plasmids. For example, in England the ST1407 clone has been associated primarily with MSM,8 whereas contemporaneous PPNG were significantly associated with heterosexuals.9 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 heterosexuals4 and an association between heterosexuals and PPNG has been established over some years (2011–14)10 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 β-lactamase plasmid in the absence of selection may be elucidated by whole-genome sequence analysis and maintenance may be dependent upon plasmid type. There seems to be no biological barriers to prevent natural acquisition of high-level plasmid-mediated penicillin resistance, with this successful multiresistant clone needing only to overcome the ‘problem’ of stable maintenance and potentially harbour an additional resistance mechanism to its already extensive repertoire. Acknowledgements Many thanks to Dr Stephanie Chisholm (previously at PHE) and Dr Darren Mernagh and Professor Graham Mills (University of Portsmouth) for their support and advice in relation to the study. We would also like to acknowledge all those involved in the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP), from which some isolates were obtained. Funding The work was supported by Public Health England. Transparency declarations None to declare. References 1 Martin IM , Ison CA , Aanensen DM et al. Rapid sequence-based identification of gonococcal transmission clusters in a large metropolitan area . J Infect Dis 2004 ; 189 : 1497 – 505 . Google Scholar CrossRef Search ADS PubMed 2 Unemo M , Nicholas RA. Emergence of multidrug-resistant, extensively drug-resistant and untreatable gonorrhea . Future Microbiol 2012 ; 7 : 1401 – 22 . Google Scholar CrossRef Search ADS PubMed 3 Shimuta K , Unemo M , Nakayama S et al. Antimicrobial resistance and molecular typing of Neisseria gonorrhoeae isolates in Kyoto and Osaka, Japan, 2010 to 2012: intensified surveillance after identification of the first strain (H041) with high-level ceftriaxone resistance . Antimicrob Agents Chemother 2013 ; 57 : 5225 – 32 . Google Scholar CrossRef Search ADS PubMed 4 Chisholm SA , Unemo M , Quaye N et al. Molecular epidemiological typing within the European Gonococcal Antimicrobial Resistance Surveillance Programme reveals predominance of a multidrug-resistant clone . Euro Surveill 2013 ; 18 : pii=20358 . 5 Ison CA , Gill MJ , Woodford N. Transfer of β-lactamase plasmids by conjugation in Neisseria gonorrhoeae . Genitourin Med 1990 ; 66 : 82 – 6 . Google Scholar PubMed 6 Unemo M , Fasth O , Fredlund H et al. Phenotypic and genetic characterization of the 2008 WHO Neisseria gonorrhoeae reference strain panel intended for global quality assurance and quality control of gonococcal antimicrobial resistance surveillance for public health purposes . J Antimicrob Chemother 2009 ; 63 : 1142 – 51 . Google Scholar CrossRef Search ADS PubMed 7 Roberts M , Falkow S. Conjugal transfer of R plasmids in Neisseria gonorrhoeae . Nature 1977 ; 266 : 630 – 1 . Google Scholar CrossRef Search ADS PubMed 8 Ison CA , Town K , Obi C et al. Decreased susceptibility to cephalosporins among gonococci: data from the Gonococcal Resistance to Antimicrobials Surveillance Programme (GRASP) in England and Wales, 2007-2011 . Lancet Infect Dis 2013 ; 13 : 762 – 8 . Google Scholar CrossRef Search ADS PubMed 9 HPA . GRASP 2011 Report: The Gonococcal Resistance to Antimicrobials Surveillance Programme. http://webarchive.nationalarchives.gov.uk/20140714113628/http://www.hpa.org.uk/webc/HPAwebFile/HPAweb_C/1317136030908. 10 ECDC . Gonococcal Antimicrobial Susceptibility Surveillance in Europe 2014. http://ecdc.europa.eu/en/publications/Publications/gonococcal-antimicrobial-susceptibility-surveillance-Europe-2014.pdf. © Crown copyright 2018. This article contains public sector information licensed under the Open Government Licence v3.0 (http://www.nationalarchives.gov.uk/doc/open-government-licence/version/3/)

Journal

Journal of Antimicrobial ChemotherapyOxford University Press

Published: Jun 3, 2018

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