Neisseria gonorrhoeae Infection Models

Neisseria gonorrhoeae (N. gonorrhoeae) is a Gram-negative diplococcus and the cause of gonorrhea, the second most commonly reported notifiable sexually transmitted infection. It colonizes the mucosal surfaces of the genital tract, rectum, throat, and conjunctiva; infection is frequently asymptomatic, particularly in women, which allows it to spread and to ascend to pelvic inflammatory disease, a leading cause of infertility and ectopic pregnancy, and it increases the transmission of HIV. Young people aged 15–24 are at highest risk.

What makes N. gonorrhoeae a priority for drug development is not the acute infection but the near-exhaustion of the drugs that treat it: the organism has developed resistance to every antibiotic class deployed against it in turn, and ceftriaxone is now the last reliably effective option in most of the world. The emergence of ceftriaxone-resistant and extensively drug-resistant lineages – “super gonorrhea” – raises the prospect of untreatable infection. The CDC classifies drug-resistant N. gonorrhoeae as an urgent threat, and the WHO lists third-generation cephalosporin- and fluoroquinolone-resistant N. gonorrhoeae as a high-priority pathogen on its 2024 Bacterial Priority Pathogens List.

TransPharm Preclinical Solutions evaluates candidates against N. gonorrhoeae in a genital-tract infection model and can develop resistant-strain challenges to a program’s specifications.

The scientific problem in an N. gonorrhoeae study starts with the fact that it is a human-specific pathogen with no natural animal host, which shapes how it can be modeled. The established in vivo approach is the murine female genital-tract colonization model, in which sustained colonization is made possible by hormonal (estradiol) treatment and by suppression of the competing vaginal flora; the readout is gonococcal colonization of, and clearance from, the genital tract over time. Strain choice is the decisive variable for this pathogen more than most: because the development question is almost always about resistance, a susceptible reference strain and a ceftriaxone-resistant or penA-mosaic clinical isolate answer very different questions, and a resistance-breaker candidate needs the resistant challenge to make its case. TransPharm Preclinical Solutions maintains an on-site library of hundreds of validated pathogens and can source or validate characterized N. gonorrhoeae isolates, including defined resistant phenotypes, to a program’s specifications.

Study parameters are configurable to the scientific question: challenge strain, host, hormonal and flora-suppression conditioning, route of administration, comparator agents, and dosing schedule. The core readout is quantitative bacterial burden – gonococcal recovery from the genital tract over the course of infection – with the supporting measures a program requires.

The following are available as add-ons to any study:

• Pharmacokinetic profiling and PK/PD analysis
• Blood chemistry
• Additional tissue harvests
• Histopathology

The vaginal (genital-tract) model reproduces mucosal N. gonorrhoeae infection, the setting in which the organism causes disease and in which a candidate must clear it. Consistent with the established murine gonococcal model, colonization is supported by hormonal conditioning and suppression of competing flora, and the infection is followed by quantifying gonococcal burden recovered from the genital tract over time to measure colonization and clearance. The model suits candidates aimed at treating or preventing mucosal infection, including agents developed against resistant strains. Model is validated in BALBc mice using strain ATCC 700825; however, this model has proven to be difficult to reproduce, and sustained colonization is elusive. We are currently working on standardizing the model to maximize colonization.

In vitro susceptibility testing complements the in vivo model. N. gonorrhoeae is tested by the agar dilution reference method on gonococcal medium, which characterizes a candidate against susceptible and resistant isolates and situates it against the shifting breakpoints that define this pathogen’s resistance.

N. gonorrhoeae is the textbook case of an organism outrunning its treatments. Over decades it has acquired clinically significant resistance to sulfonamides, penicillins, tetracyclines, and fluoroquinolones in succession, then to the oral cephalosporin cefixime, and it has eroded the azithromycin half of the dual therapy that was standard until recently – leaving ceftriaxone as the last reliably effective agent, now itself threatened.

The mechanisms are well characterized: mosaic penA alleles that remodel penicillin-binding protein 2 and drive cephalosporin resistance (the penA-60 alleles behind the FC428 lineage and recent ceftriaxone-resistant cases), overexpression of the MtrCDE efflux pump via mtrR changes, porB alterations that reduce drug entry, 23S rRNA mutations conferring azithromycin resistance, gyrA and parC mutations for fluoroquinolones, and plasmid-borne β-lactamase and tetracycline determinants. Extensively drug-resistant strains combining ceftriaxone resistance with high-level azithromycin resistance have now been reported on several continents.