Reduced susceptibility to ceftriaxone in Neisseria gonorrhoeae is associated with mutations G542S, P551S and P551L in the gonococcal penicillin-binding protein 2.

OBJECTIVES: Reduced susceptibility to extended-spectrum cephalosporins in Neisseria gonorrhoeae has, to date, been associated with three alterations: a mosaic penA allele encoding the penicillin-binding protein 2 (PBP2); A-del-mtrR, an adenine deletion in the mtrR promoter; and penB, comprising mutated alleles of PorBIb. In this study, we examined an association between reduced susceptibility to ceftriaxone and additional mutations in gonococcal PBP2. METHODS: N. gonorrhoeae isolates (n = 76) exhibiting reduced susceptibility to ceftriaxone but lacking the mosaic penA sequence were investigated for A-del-mtrR and penB as well as substitutions at PBP2 501, 542 and 551 using a previously described real-time PCR approach. To further investigate PBP2 542 and 551 substitutions, we reanalysed penA sequence data from a previous study of 98 gonococci exhibiting a range of ceftriaxone MICs. RESULTS: Of 76 N. gonorrhoeae isolates exhibiting reduced susceptibility to ceftriaxone and lacking the mosaic penA sequence, a 501 (A501V or A501T) substitution was present in 9/76, a 542 substitution in 39/76 and a 551 substitution in 26/76 isolates. Reanalysis of 98 gonococcal isolates from a previous study showed that substitutions at PBP2 542 (G542S) and 551 (P551S or P551L) were significantly associated with raised MICs to ceftriaxone (P = 0.0186 and 0.001, respectively) and penicillin (P = 0.0231 and 0.0007, respectively). CONCLUSIONS: Our findings provide strong evidence for the involvement of PBP2 G542S and P551S/P551L in reduced susceptibility to ceftriaxone and to penicillin. Further studies are needed to investigate the precise and relative roles played by these mutations.

Evidence that the gonococcal porA pseudogene is present in a broad range of Neisseria gonorrhoeae strains; suitability as a diagnostic target.

AIMS: The primary aim of the study was to determine if the gonococcal porA pseudogene is a stable sequence target for the detection of Neisseria gonorrhoeae by PCR. METHODS: A total of 240 gonococcal strains from various geographic locations were tested by porA pseudogene PCR. In addition, porA pseudogene PCR positivity rates were compared with established gonococcal assays in three Australian states. RESULTS: All N. gonorrhoeae isolates provided positive results in the porA pseudogene PCR. Positivity rates compared favourably with established gonococcal assays, with increased N. gonorrhoeae detection in the Northern Territory and Western Australia. CONCLUSIONS: The results of this multicentre study provide further evidence that the porA pseudogene is highly conserved across a diverse range N. gonorrhoeae strains and is a suitable PCR target for routine detection of N. gonorrhoeae.

One confirmed and one suspected case of pharyngeal gonorrhoea treatment failure following 500mg ceftriaxone in Sydney, Australia.

Emerging antimicrobial resistance within Neisseria gonorrhoeae (NG) is a significant global public health threat. Detection and investigation of treatment failures is a crucial component of the World Health Organisation's response to this challenge. We report the cases of two homosexual men, both treated for pharyngeal NG with 500mg intramuscular ceftriaxone, in whom a test of cure 1 week after treatment showed persisting infection. Both men denied further sexual activity. In the first case, treatment failure was confirmed, since the isolates before and after treatment were identical by auxotype, antibiogram, multilocus sequence type (MLST) and multi-antigen sequence type (NG-MAST). In the second case, the MLSTs before and after treatment were identical, but NG-MAST results were similar but not indistinguishable. These cases underline the importance of test-of-cure and molecular investigations in identifying treatment failure, but also highlight the complexity of distinguishing treatment failure from reinfection when relying on highly variable molecular targets that may be subject to drug pressure.

Diversity of penA alterations and subtypes in Neisseria gonorrhoeae strains from Sydney, Australia, that are less susceptible to ceftriaxone.

Increasing numbers of Neisseria gonorrhoeae strains with decreased susceptibilities to ceftriaxone and other oral cephalosporins widely used for the treatment of gonorrhea have been isolated in Sydney, Australia, over several years. In this study, we examined the complete penicillin-binding protein 2 (PBP 2) amino acid sequences of 109 gonococci, selected on the basis of their diverse temporal and geographic origins and because they exhibited a range of ceftriaxone MICs: < OR =0.03 microg/ml (n = 59), 0.06 microg/ml (n = 43), and 0.125 microg/ml (n = 7). Auxotyping, serotyping, and genotyping by N. gonorrhoeae multiantigen sequence typing sequence-based analysis was also performed. In total, 20 different amino acid sequence patterns were identified, indicating considerable variation in the PBP 2 sequences in this study sample. Only some of the N. gonorrhoeae isolates with significantly higher ceftriaxone MICs contained a mosaic PBP 2 pattern, while more isolates exhibited a nonmosaic PBP 2 pattern containing an A501V substitution. Although particular N. gonorrhoeae genotypes in our sample were shown to be less susceptible to ceftriaxone, the reduced susceptibility to ceftriaxone was not specific to any particular genotype and was observed in a broad range of auxotypes, serotypes, and genotypes. Overall, the results of our study show that N. gonorrhoeae strains exhibiting reduced sensitivity to ceftriaxone are not of a particular subtype and that a number of different mutations in PBP 2 may contribute to this phenomenon.

Dynamics of appearance and expansion of a prolyliminopeptidase- negative subtype among Neisseria gonorrhoeae isolates collected in Sydney, Australia, from 2002 to 2005.

Recent studies have demonstrated a wide geographic circulation of isolates of Neisseria gonorrhoeae that fail to produce prolyliminopeptidase (PIP). Tests based on the production of this enzyme are important elements of a number of identification systems for gonococci. We documented the appearance, expansion, and contraction of subtypes of 165 PIP-negative gonococci detected in an extended and systematic sample of the 3,926 N. gonorrhoeae isolates collected in Sydney, Australia, from July 2002 to September 2005. Their appearance, peak, and decline followed an "epidemic" curve. At the peak of their prevalence in 2003, PIP-negative gonococci comprised 22% of all isolates. Closely related phenotypes accounted for 162/165 of the PIP-negative gonococci. Algorithms for confirmation of N. gonorrhoeae should take account of the temporal and geographic variability of gonococci by utilizing two or more distinct confirmatory methods.