Phenotypic and genotypic characterisation of Neisseria gohorrhoeae isolates from New Zealand with reduced susceptibility to ceftriaxone : a thesis submitted to the College of Health in partial fulfilment of the requirements for the Master of Science in Microbiology at Massey University, New Zealand

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Objectives Currently, ceftriaxone is the last remaining drug recommended for empirical treatment of gonorrhoea. Neisseria gonorrhoeae with reduced susceptibility to ceftriaxone have been isolated worldwide in countries such as Japan, France, Spain, Slovenia, Australia and Sweden. These have led to treatment failures and the emergence of ceftriaxone-resistant N. gonorrhoeae. Various mutations in penA (mosaic and nonmosaic), which encodes the penicillin-binding protein 2 (PBP2), have been reported to be the primary reason for reduced ceftriaxone susceptibility, but it can be reduced further by mutations in mtrR, porBIB and ponA. In this study, we aimed to determine the antimicrobial resistance patterns of New Zealand isolates of N. gonorrhoeae with reduced susceptibility to ceftriaxone and to characterise the penA, mtrR, porBIB and ponA in the isolates. Methods A total of 28 N. gonorrhoeae isolates with elevated ceftriaxone MIC (0.03 to 0.12 mg/L), collected from 2012 to 2015 and obtained from the Institute of Environmental Science and Research (ESR), were examined in this study. Samples came from laboratories in Auckland (26), Wellington (1) and Taranaki (1). The antimicrobial resistance of penicillin G, tetracycline, ciprofloxacin, azithromycin and ceftriaxone were determined through antimicrobial susceptibility test, using minimum inhibitory concentration (MIC) test strips. Polymerase chain reactions (PCRs) and sequencing to identify specific mutations in penA, mtrR, porBIB and ponA, that are associated with elevated minimum inhibitory concentrations (MICs) to ceftriaxone, were undertaken. The association between the phenotypic and genotypic results was investigated by comparing the presence of the number of mutated genes and the MIC level of ceftriaxone. Results Based on the AST results using MIC test strips and interpreted using The European Committee on Antimicrobial Susceptibility Testing (EUCAST) criteria, 23 out of 28 isolates (82%) showed reduced susceptibility to ceftriaxone, with MICs of 0.03 to 0.06 mg/L. All of the isolates were resistant to ciprofloxacin, while 36%, 25% and 7% were resistant to penicillin G, tetracycline and azithromycin, respectively. Two azithromycin-resistant N. gonorrhoeae isolates were observed, and isolate 264 (azithromycin MIC: 4mg/L) also exhibited reduced susceptibility to ceftriaxone (MIC: 0.03 mg/L). A total of 21% (6/28) of the isolates produced ß- lactamase. The 23 isolates that conveyed reduced ceftriaxone susceptibility were found to harbour three or four mutated genes (penA, mtrR and/or porBIB and ponA). Reduced susceptibility to ceftriaxone among N. gonorrhoeae isolates in this study was associated with mosaic PBP2 (encoded by penA) with G545S/A501V mutations, with nonmosaic PBP2 with an A501V mutation, plus the presence of mutation in mtrR promoter with G120 and A121 alterations in PorBIB. A total of 65% (15/23) of the N. gonorrhoeae isolates with reduced susceptibility to ceftriaxone harboured mosaic PBP2 XXXIV, a pattern found in N. gonorrhoeae associated with ceftriaxone treatment failures in Europe and Australia. The current study also revealed that the partial sequences of four mosaic PBP2 (M-2, M-3, M-4, M-5) were different from the common mosaic PBP2 sequences reported in various studies. Conclusion There is an association between the phenotypic and genotypic character of N. gonorrhoeae isolates expressing reduced susceptibility to ceftriaxone in this study population. Furthermore, the presence of important mosaic PBP2 that link to ceftriaxone treatment failure might be circulating among N. gonorrhoeae isolates in New Zealand . Keywords: Neisseria gonorrhoeae, ceftriaxone, reduced susceptibility, New Zealand
Neisseria gonorrhoeae, Gonorrhea, Genetic aspects, New Zealand, Research Subject Categories::NATURAL SCIENCES::Biology::Cell and molecular biology::Genetics, Ceftriaxone