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
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.
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.
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.
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