Background: Neisseria gonorrhoeae, the etiological agent of the sexually transmitted infection (STI) gonorrhoea, has established itself as a challenging pathogen in terms of antimicrobial therapy. The bacterium has exhibited a remarkable ability to develop resistance to a wide array of antibiotics including the extended spectrum cephalosporins (ESCs), which form the mainstay of anti-gonococcal therapy around the world, and may ultimately become untreatable in certain circumstances in the future.
Paucity of ideal alternative therapies coupled with escalating drug resistance puts the onus on antimicrobial resistance (AMR) surveillance methods for the addressing the issue of gonorrhoea disease control. Understanding the genetic basis of resistance to antibiotics, in tandem with epidemiological studies has the potential to provide valuable information regarding the emergence and spread of resistant strains and guide antimicrobial therapy. However, the genetic markers associated with reduced susceptibility to ESCs are yet to be fully elucidated, making AMR tracking especially for the ESCs challenging.
Moreover, countries like Australia are witnessing a paradigm shift in diagnosis of gonorrhoea; from culture based techniques to nucleic acid analysis tests (NAATs), thereby severely affecting the number of cultured isolates available for epidemiological and AMR studies. This limits the ability of reference laboratories to pursue traditional culture-based AMR surveillance practices and is especially alarming in light of emergence and spread of novel resistance phenotypes including the H041 and F89 ceftriaxone-resistant strains.
This Doctor of Philosophy (PhD) project aimed at addressing the above problems by development and application of novel molecular tools to (a) characterise isolates with reduced susceptibility to ESCs and identifying genetic markers associated with this phenomenon, and (b) explore AMR characterisation and genotyping directly from non-cultured clinical specimens.
Methods: A range of gonococcal isolates selected on the basis of their AMR profile and of diverse geographical and temporal origins were investigated in this study. The emphasis was on the AMR mechanisms against the beta-lactam antibiotics ceftriaxone and penicillin, two antibacterials of most relevance in the Australian population. In addition, nucleic acid extracts of clinical specimens from various geographical locations of Australia that were submitted to pathology laboratories for STI screening by NAATs were also investigated. The main premise of the study was as follows-
• Characterisation of novel AMR markers in gonococcal isolates exhibiting reduced susceptibility to ESCs.
• Investigation into genetic lineages among gonococcal isolates exhibiting reduced susceptibility to ESCs.
• Adaptation of a popular gonococcal genotyping platform, the N. gonorrhoeae multi-antigen sequence typing (NG-MAST) for use on non-cultured clinical specimens.
• Development and validation of several real-time polymerase chain reaction (PCR) assays targeting a range of gonococcal AMR mechanisms for use on non-cultured clinical specimens.
Results: This PhD project resulted in the successful development of several real-time NAATs capable of characterising N. gonorrhoeae resistance markers to beta lactam antibiotics directly from clinical samples. The main achievements of this project include-
• Discovery of novel AMR markers associated with reduced susceptibility to ESCs in N. gonorrhoeae, viz. substitutions at position 542 and 551 of the penicillin binding protein 2 (PBP2).
• Adaptation of NG-MAST for direct use on clinical specimens.
• Development of a real-time PCR assay for detecting a conserved region of the beta-lactamase producing plasmid in N. gonorrhoeae directly from clinical specimens and its successful use for epidemiological studies in the Northern Territory of Australia.
• Development of a real-time PCR assay for direct detection of the first ceftriaxone-resistant gonococcal strain (H041) from non-cultured clinical specimens.
• Development of real-time PCR assays for direct detection AMR associated with reduced susceptibility to ESCs such the modified mtrR C-D-E efflux pump, the mosaic PBP2 and the A501V mutation on PBP2.
The results of this PhD project highlight the feasibility of rapid development and application of PCR methods when faced with emergence of newer forms of resistance in gonococci. However, for the time being, replacement of phenotypic AMR detection methods with genotypic methods entirely, is not conceivable.
Conclusion: It is likely that the future of gonorrhoea disease control and treatment will be greatly influenced by strong AMR surveillance practices where nucleic acid amplification based methods successfully pin-point the incursion and spread of resistant strains while detailed investigation of newer resistance mechanisms is possible via cultured isolates. Control of international spread of extensively drug-resistant gonococcal strains will require concerted effort from public health authorities around the world. Maintaining a vigilant surveillance network, re-assessing therapeutic treatment guidelines and further research into gonococcal resistance remain the key strategies for combating a potential public health disaster posed by deteriorating drug resistance in the gonococcus.