When the Clap Hits Back Part III: Molecular Mechanisms of Resistance in Neisseria gonorrhoeae and How Molecular Tests May Save Us All

Oct. 22, 2018

Despite national and global surveillance efforts, the prevalence of antimicrobial resistance among Neisseria gonorrhoeae isolates may be underestimated due to the lack of capacity for performing phenotypic antimicrobial susceptibility testing (AST) in clinical laboratories. Additionally, the abandonment of routine culture and the increased use of nucleic acid amplification tests (NAATs) as a standard of care for the diagnosis of gonococcal infections has resulted in a lack of susceptibility data for antibiotics commonly used to treat infections caused by N. gonorrhoeae. In my previous blog posts, I talked about how quickly N. gonorrhoeae has acquired antimicrobial resistance throughout the antibiotic era, and how a threat once thought to be imminent is now a frightening reality with the discovery of a truly untreatable gonococcal infection. Without a more efficient way to track antimicrobial resistance in this organism, in the next few years we could face global dissemination of N. gonorrhoeae infections that are resistant to all available treatment options.

To address this problem and enhance the capabilities of clinical and public health laboratories to detect and track the spread of antimicrobial resistance in N. gonorrhoeae, researchers around the world have been developing rapid molecular tests to predict N. gonorrhoeae antimicrobial resistance profiles. These assays may be performed on pure clinical isolates or directly from clinical specimens and provide an accurate prediction of the organism’s susceptibility to one or multiple antibiotics without having to wait for phenotypic susceptibility results. Although confirmatory phenotypic antimicrobial susceptibility testing is still required, these rapid molecular tests can deliver preliminary, actionable results to physicians to determine the most effective treatment regimen for each patient. Additionally, these results allow for real-time surveillance of antimicrobial resistance in N. gonorrhoeae, and may potentially extend the life of our current empirical regimen.

Molecular Mechanisms of Antimicrobial Resistance In N. gonorrhoeae: a Moving Target

As of 2016, 44.1% of N. gonorrhoeae isolates collected from Gonococcal Isolate Surveillance Project (GISP) sites in the US were resistant to penicillin, tetracycline, ciprofloxacin, or some combination of these, each of which was once very effective against gonococcal infections. As we move from drug to drug in a quest to overcome resistance problems in N. gonorrhoeae, molecular characterization of gonococcal isolates allows researchers to identify the mechanism for the emergence of antimicrobial resistance in N. gonorrhoeae. Several genetic alterations have been associated with certain antibiotic resistance phenotypes. These changes can result in drug target modification that decrease affinity to antimicrobials, overexpression of existing antibiotic resistance genes, enzymatic degradation of antibiotics, and alteration in activity of influx and efflux pump that decreases the intracellular concentration of antibiotics. Targeted molecular tests predicting antimicrobial susceptibility are designed based on these known mutations.

Resistance to sulfonamides, a competitive substrate to dihydropteroate synthetase (DHPS) enzyme, were due to increased production of p-aminobenzoic acid (a natural substrate to DHPS) or mutations in the folP gene, which encodes the enzyme DHPS itself. These events led to treatment failure of up to 85% among all cases within the first few decades of use. Not long after the emergence of sulfonamides resistance, N. gonorrhoeae started developing resistance to penicillin, which was caused by mutations in genes involved in cell wall biosynthesis pathway such as penA and ponA1, or other mutations in genes involved in regulation of periplasmic drug concentration (penB, penC and mtrR). In the 1970s, penicillin was no longer part of the empirical regimen after a plasmid-mediated β-lactamase gene type TEM was identified in gonococcal isolates with high-level penicillin resistance (penicillin MIC of up to 128 μg/mL). Tetracycline, once used in a regimen for patients with penicillin allergy, was no longer considered part of an empirical regimen after the worldwide spread of highly tetracycline-resistant isolates harboring the tetM gene, which is a plasmid-mediated genetic resistance determinant potentially derived from streptococci.

Ciprofloxacin became the first-line treatment for uncomplicated gonococcal infections shortly after resistance to penicillin and tetracycline became widespread. Unfortunately, the success of ciprofloxacin as an empirical treatment did not last long, as high-level resistance among gonococcal isolates has been observed globally since the early 1990s. Resistance to ciprofloxacin is mostly due to a decreased affinity between the drug and its targets, DNA gyrase enzyme (GyrA) and topoisomerase IV (ParC). Point mutations within genes for these targets can result in intermediate- to high-level resistance to ciprofloxacin. The steady increase in the prevalence of ciprofloxacin resistance in the US led to the Centers for Disease Control and Prevention (CDC)’s decision to remove ciprofloxacin from its sexually transmitted diseases treatment guidelines in 2007. Spectinomycin is an aminocyclitol antibiotic invented in the 1960s specifically for treatment of N. gonorrhoeae infection especially isolates with high-level penicillin MICs. Although resistance to spectinomycin is exceedingly rare, production of this antibiotic has been discontinued and the drug became unavailable in the US since 2006, leading to removal of spectinomycin from CDC’s practice guidelines.

Currently, CDC’s recommendation for empirical treatment of uncomplicated gonococcal infections includes one-time dosing of the extended-spectrum cephalosporin (ESC) ceftriaxone 250 mg intramuscularly and azithromycin 1 g orally. Although this regimen appears to be very safe and effective, each of the drugs in this dual regimen is suffering from the same fate as many of their predecessors. Resistance to azithromycin is multifactorial, and has been associated with a few different genetic aberrations. These include overexpression of the mtrCDE efflux pump caused by disruption of the promoter region of the gene encoding repressor MtrR, decreased affinity between the drug and its target due to mutations in 23S rRNA gene, methylation within the 50S subunit of the bacterial ribosome, or a combination of these mechanisms. GISP began testing isolates for azithromycin susceptibility since 1992, leading to the identification of an N. gonorrhoeae isolate in Hawaii with high-level resistance to azithromycin (>512 μg/mL) in 2011. During 2014–2016, the percentage of gonococcal isolate in the US with decreased susceptibility to azithromycin, now no longer recommended as monotherapy, alarmingly increased from 2.4 to 3.6%, raising concern about the future of azithromycin as part of the empirical regimen.

Mechanisms of decreased susceptibility to ESCs are more complicated than those of other antibiotics. The increased ESC MICs are mostly mediated by multiple point mutations in the penA gene (PBP2). These mutations are thought to a be the product of horizontal gene transfer with commensal Neisseria species. A unique pattern of mutation across the penA gene is designated a “Mosaic Type”. There are over 80 different mosaic types distributed around the world. Other potential causes of increased ESC MICs are other mutations in the penA (non-mosaic), mtrR, and penB genes. It has not yet been determined whether the truly ceftriaxone-resistant N. gonorrhoeae isolate from England contains a particular penA mosaic type or mutations in genes that could result in the isolate’s resistance to ceftriaxone.

What Are the Benefits of Performing Molecular Susceptibility Prediction Assays?

Molecular susceptibility testing on N. gonorrhoeae directly from clinical specimens allows for the course of treatment to be more individualized. With a rapid turnaround time compared to conventional susceptibility testing, molecular tests predicting antimicrobial resistance will deliver actionable results to physicians to avoid prescribing antibiotics predicted to be resistant, and determine the most effective treatment regimen for each patient. Microfluidics and nanotechnology allow researchers to develop prototypes of rapid molecular point-of-care tests that can be run while the patient is still at the clinic and concurrently detect the presence of N. gonorrhoeae and genetic markers of antimicrobial resistance. These point-of-care diagnostics will be especially helpful in low-resource regions where the diagnosis of gonococcal infections may be suboptimal and dependent upon syndromic management. Although these molecular tests are still in development and not commercially available for in vitro diagnostics purposes, many studies have reported satisfactory performance in some of these prototype assays.

N. gonorrhoeae surveillance programs such as GISP may benefit from results of molecular assays predicting antimicrobial resistant profiles. As previously mentioned, a regimen containing ceftriaxone and azithromycin is the only reliable empirical treatment for uncomplicated gonorrhea. However, with the ongoing ESC MIC creep and a jump in the prevalence of isolates with high-level azithromycin resistance, a continuous monitoring of antimicrobial susceptibility among gonococcal isolates is desperately needed. Molecular testing allows for a high-throughput screening to identify N. gonorrhoeae isolates that may have elevated MICs to the antibiotic of interest, on which confirmatory phenotypic susceptibility testing will be performed. An active surveillance strategy coupled with rapid molecular testing will help local or national public health programs identify a critical increase of MICs in clinical isolates before they get a chance to develop full resistance and spread into the community.

To address a concern about elevated ESC MICs among gonococcal isolates, many molecular assays targeting penA mosaic patterns and other genetic elements conferring resistance to ESCs have been published (nicely summarized up until 2017 in Table 1 in this article).  These assays mostly rely on real-time PCR and may be performed on pure isolates or clinical specimens. However, the sensitivity of the PCR assay, which is considered a targeted approach, would depend on the prevalence of penA mosaic types among isolates with elevated ESC MIC in a geographical area in which the assay would be performed. For example, a previous study on N. gonorrhoeae isolates from California demonstrated the presence of mosaic XXXIV in all isolates with elevated ESC MICs. Using this observation, an assay designed to detect mosaic XXXIV (shameless plug) was validated using a panel of 150 clinical isolates. The assay was 97% sensitive and 100% specific in predicting whether an isolate had elevated ESC MICs. However, it failed to detect two isolates in the panel with decreased susceptibility to ESCs. Further investigation revealed that these isolates harbored different penA mosaic types (IX and XII), both of which cannot be detected by the XXXIV assay. These finding suggested that this assay would not be useful in regions where mosaic types other than XXXIV contribute to the increased ESC MICs among local isolate.

In addition to the potential benefits discussed above, molecular assays predicting antimicrobial susceptibility may indirectly help extend the clinical usefulness of the currently recommended empirical regimen for uncomplicated gonorrhoeae. This is based on an assumption that there is a fitness cost for bacteria to harbor antimicrobial resistance mutations and the level of resistance should decrease if the antibiotic is no longer being used. Currently, the overall prevalence of N. gonorrhoeae resistant to ciprofloxacin (which was removed from CDC’s treatment guidelines in 2007) is around 19.2%. This observation suggests that more than 80% of cases could have been effectively treated with ciprofloxacin. A molecular test to predict ciprofloxacin susceptibility can determine whether this antibiotic can be used in lieu of our only empirical regimen of ceftriaxone and azithromycin. By providing results rapidly to physicians and limiting the use of the empirical treatment only for ciprofloxacin-resistant cases, we may be able to slow down the MIC creep and be able to keep the empirical regimen a bit longer until we have a new solution for the resistance problem.

Next-Generation Sequencing Studies on N. gonorrhoeae

Currently available technologies have made sequencing of the whole bacteria genome possible, allowing researchers to fully explore genetic elements of interest in a genomics context. Whole-genome sequencing (WGS) data could be used as an input to several typing tools publicly available, such as NG-MAST and MLST. Results from these analyses could provide information on disease transmission inside or outside of an outbreak setting, and may allow for early detection of emerging antimicrobial-resistant isolates that may be circling in the community. Information acquired from genomics data also provide a basis for the development of targeted molecular tests such as PCRs. The CDC also has an initiative to perform WGS and use the data to develop novel antimicrobials and diagnostic tests.

This concludes my series of articles on N. gonorrhoeae. Now that a threat once thought to be imminent has finally arrived, it is crucial that we, clinical and public health microbiologists, have a good understanding of the dynamics of antimicrobial resistance among gonococcal isolates. By being vigilant and keeping our fingers on the pulse of emerging resistance, we will be able to identify antimicrobial resistance in N. gonorrhoeae before the community is affected and provide support for physicians, infection preventionists, and local or state public health authorities in a coordinated effort to limit the spread of this superbug.
Catch up with the first 2 posts in this series:

When the Clap Hits Back: Antimicrobial Resistance Threats in Neisseria gonorrhoeae

When the Clap Hits Back Part II: What Clinical Labs Can Do to Prevent the Spread of Antimicrobial-Resistant Neisseria gonorrhoeae

The above represents the opinions of the author and does not necessarily reflect those of the American Society for Microbiology.


Author: Peera Hemarajata

Peera Hemarajata
Peera Hemarajata is a diplomate of the American Board of Medical Microbiology and an Assistant Director at Los Angeles County Public Health Laboratories. His research interests include emerging infectious diseases, molecular assay development, antimicrobial resistance, and microbial genomics.