INTRODUCTION
Gonorrhea is among the most common sexually transmitted diseases worldwide. According to a report published by the European Centre for Disease Prevention and Control (ECDC) in 2025, the incidence of gonorrhea in Europe, based on reported cases between 2014 and 2023, increased by 321%. A similar epidemiological trend has been observed in Poland, where data from the National Institute of Public Health indicate that the number of reported cases rose from 455 in 2014 to 1,372 in 2023.
The etiological agent of the disease is Neisseria gonorrhoeae. This bacterium can be detected and identified directly in clinical specimens using real-time polymerase chain reaction (PCR). However, assessment of antimicrobial susceptibility requires bacterial culture. For successful cultivation, clinical material must be inoculated immediately after collection onto an appropriate solid medium and incubated under microaerophilic conditions. N. gonorrhoeae is highly sensitive to external factors and usually does not survive routine transport from the collection site to the laboratory. This limitation may be particularly pronounced in multidrug-resistant strains, which often exhibit reduced viability compared with wild-type isolates due to the accumulation of chromosomal mutations. The bacterium does not grow in liquid media, and the lowest transport-related losses have been associated with the use of Nutritive Transport Systems [1]. Larger laboratories commonly identify N. gonorrhoeae using mass spectrometry. Although molecular methods, including whole-genome sequencing, are increasingly used, they cannot reliably determine minimum inhibitory concentrations (MICs) for most antibiotics.
Within the framework of the European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP), funded by the ECDC, antimicrobial susceptibility of N. gonorrhoeae in European Union countries is continuously monitored. Each year, most European Union countries submit approximately 50–100 cultured N. gonorrhoeae isolates, together with epidemiological data, to Euro-GASP reference laboratories. Between 2014 and 2024, isolates from Poland were submitted from Warsaw and evaluated phenotypically and molecularly in Euro-GASP laboratories at Public Health England (during the period when the United Kingdom was a member of the European Union) and at Örebro University Hospital, Sweden [2–5]. As part of its External Quality Assessment (EQA) programme, Euro-GASP conducts annual audits of N. gonorrhoeae culture laboratories in participating European Union countries [2]. According to the 2024 Euro-GASP report, resistance to cefixime, azithromycin, and ciprofloxacin, as defined by EUCAST criteria, was observed in 0.3%, 25.6%, and 65.9% of N. gonorrhoeae strains tested in the European Union/European Economic Area in 2022, respectively. Two isolates were resistant to both ceftriaxone (MIC 0.25 mg/l) and cefixime (MIC 1 mg/l). Additionally, separately published data indicate that 63.4% of strains were resistant to tetracyclines according to EUCAST criteria.
The aim of this article is to discuss diagnostic challenges and the spread of drug-resistant N. gonorrhoeae strains, to describe the mechanisms underlying antimicrobial resistance, and to review emerging therapeutic options.
CRITERIA FOR THE ANTIBIOTIC SUSCEPTIBILITY AND RESISTANCE OF NEISSERIA GONORRHOEAE
There are significant differences between Europe – European Committee on Antimicrobial Susceptibility Testing (EUCAST, v. 2025) [6] and US Clinical and Laboratory Standards Institute (CLSI) criteria for susceptibility and resistance to most antibiotics. Some strains identified as resistant in Europe will not be identified as susceptible in the USA.
It is also noteworthy that CLSI recommends the use of the twofold agar dilution method for determination of minimum inhibitory concentrations (MICs), whereas EUCAST allows the use of intermediate concentrations, including gradient diffusion methods such as E-tests. For example, according to EUCAST, an azithromycin MIC of 1.1 mg/l is interpreted as resistance, while under CLSI criteria, only an MIC ≥ 2 mg/l is classified as non-susceptible. These differences are summarized in Table 1 [7].
Table 1
Criteria for susceptibility testing of N. gonorrhoeae to antibiotics according to EUCAST (Europe) and CLSI (USA)
| Antimicrobial agents | EUCAST 2025 | CLSI 2025 | |||
|---|---|---|---|---|---|
| MIC [mg/l] | |||||
| S | R | S | I | R | |
| Benzylpenicillin/penicillin** | ≤ 0.06 | > 1 | ≤ 0.06 | 0.12–1.0 | ≥ 2 |
| Ceftriaxone | ≤ 0.125 | > 0.125 | ≤ 0.25 | – | – |
| Cefixime | ≤ 0.125 | > 0.125 | ≤ 0.25 | – | – |
| Azithromycin | * | * | ≤ 1.0 | – | – |
| Tetracycline | ≤ 0.5 | > 0.5 | ≤ 0.25 | 0.5–1.0 | ≥ 2 |
| Ciprofloxacin | ≤ 0.03 | > 0.06 | ≤ 0.06 | 0.12–0.5 | ≥ 1 |
| Spectinomycin | ≤ 64 | > 64 | ≤ 32 | 64 | ≥ 128 |
MECHANISMS OF ANTIBIOTIC RESISTANCE IN NEISSERIA GONORRHOEAE
Resistance to oxyimino-cephalosporins (ceftriaxone and cefixime)
In European Union countries, resistance to ceftriaxone and cefixime was reported in 0.1% and 0.5% of N. gonorrhoeae strains isolated in 2020, respectively. In contrast, no resistant strains were identified in Poland during the same period [8].
Mutations in the penA gene, which encodes penicillin-binding protein 2 (PBP2) transpeptidase, are considered the primary mechanism underlying resistance to ceftriaxone and cefixime in N. gonorrhoeae. Based on sequencing of an approximately 1,750-bp fragment of the penA gene, alleles have been classified into 338 major types and numerous subtypes. Individual alleles may differ by as few as one to as many as approximately 60 amino acids. Mutation-induced alterations in the amino acid sequence of PBP2 including substitutions, single amino acid deletions, and insertions give rise to so-called mosaic, semi-mosaic, or non-mosaic PBP2 patterns [9, 10].
Elevated MICs of ceftriaxone and cefixime may also result from overexpression of the MtrCDE efflux pump, most commonly caused by deletion of the −35A nucleotide in the mtrR promoter region or by amino acid substitutions in the MtrR repressor protein (e.g., G45D). In addition, amino acid substitutions in the PorB1b porin protein at positions 120 and 121 have been associated with reduced susceptibility. However, none of these mechanisms alone is sufficient to confer clinically significant cephalosporin resistance [11].
Dozens of N. gonorrhoeae strains have been described with ceftriaxone MICs ranging from 0.5 to 2.0 mg/l. Approximately 80% of these isolates carried the penA 60.001 allele, which has been reported to be most prevalent among strains isolated in Asia [12–14].
Certain mutations in the rpoB and rpoD genes, encoding subunits of DNA-dependent RNA polymerase, have also been shown to influence changes in MICs and the emergence of resistance to ceftriaxone and cefixime in N. gonorrhoeae. Substitution of G158V or P157L in the RpoB protein increases the ceftriaxone MIC from 0.023 mg/l to 0.5 or 0.75 mg/l, respectively [15]. The precise mechanism underlying this phenomenon remains unclear. It has been hypothesised that mutations in RNA polymerase may affect the relative expression levels of penicillin-binding proteins (PBPs), particularly PBP2. In Gram-positive bacteria, resistance to β-lactam antibiotics resulting from overproduction of PBPs has been described previously [16]. Alternatively, mutant polymerase activity may introduce transcriptional errors affecting the penA gene encoding the PBP2 transpeptidase.
Resistance of N. gonorrhoeae to cephalosporins may also be mediated by the production of extended-spectrum β-lactamases. Recently, a 5,154-bp pbla<sub>TEM-20</sub> plasmid - a modified variant of the Toronto/Rio plasmid, was introduced into the N. gonorrhoeae ATCC 49226 model strain via transformation, resulting in ceftriaxone resistance (MIC 4 mg/l) and cefixime resistance (MIC 16 mg/l) [17]. TEM-20 is classified as a class A extended-spectrum β-lactamase (ESBL), characterised by the M182T and G238S amino acid substitutions.
To date, TEM β-lactamase production in N. gonorrhoeae has been associated primarily with penicillin resistance. Among Gram-negative bacilli, 214 TEM β-lactamase variants have been described (TEM-1 to TEM-262), including 86 ESBLs and 9 inhibitor-resistant ESBLs (IRESBLs) [18]. ESBL-type TEM β-lactamases, such as TEM-191, have already been detected in clinical N. gonorrhoeae isolates; however, their presence has not been associated with high MICs for oxyimino-cephalosporins [19].
It is assumed that expression of these β-lactamases is subject to inducible regulation, with penicillin acting as the primary inducer, whereas ceftriaxone and cefixime do not induce enzyme production. Notably, a single mutation within the regulatory region may result in constitutive β-lactamase expression, independent of antibiotic induction.
Penicillin resistance and intermediate susceptibility
Intermediate susceptibility to penicillins in N. gonorrhoeae results from mutations in chromosomal genes encoding penicillin-binding protein 2 (PBP2; penA), the PorB1b porin, and penicillin-binding protein 1 (ponA), as well as from mutations leading to overexpression of the MtrCDE efflux pump. In contrast, high-level resistance to penicillins is mediated by the production of TEM β-lactamases, the corresponding genes of which are located on plasmids.
More than a dozen TEM β-lactamase variants have been described in N. gonorrhoeae, with TEM-1 being the most prevalent. In addition, seven types of penicillinase plasmids have been identified, the most common being the Asia, Africa, and Toronto/Rio plasmids [19, 20]. The prevalence of β-lactamase-producing N. gonorrhoeae strains varies geographically and is substantially higher in Asian countries (> 80%) than in European countries (approximately 20%). In Poland, the proportion of β-lactamase-producing isolates ranged from 1% to 19% between 2010 and 2019 and declined to 0% in 2020 [9].
Azithromycin resistance
In European Union countries, azithromycin resistance in N. gonorrhoeae was reported in 9.4% of strains isolated in 2020, with the highest prevalence observed in Poland (35%) [8]. Resistance is primarily mediated by mutations in the 23S rRNA gene and by overexpression of the MtrCDE efflux pump, which actively expels azithromycin from the bacterial cell.
An A2059G transition present in all four alleles of the 23S rRNA gene confers high-level resistance, with azithromycin MICs exceeding 256 mg/l. In contrast, C2611T transitions or C2611G transversions in all four 23S rRNA alleles are associated with lower-level resistance, resulting in azithromycin MICs ranging from 2 to 32 mg/l. In addition, some N. gonorrhoeae strains exhibit azithromycin resistance (MICs 2–8 mg/l) due to amino acid substitutions in the MtrR repressor protein, including G45S, A86T, and Y105H [21]. Increases in azithromycin MICs, which are insufficient on their own to confer resistance, may be associated with mutations in the rplV and rplD genes encoding the ribosomal proteins L22 and L4, respectively, as well as with alterations in the mtrR promoter region - particularly the −35A deletion and amino acid substitutions in the MtrR protein (e.g., G45D, D79N, A39T, L99G, and H variants). In addition, mosaic alterations in mtrR and/or mtrD, resulting from transformation and recombination with DNA from other Neisseria species, have also been implicated [9, 22].
Resistance to fluoroquinolones (ciprofloxacin)
In European Union countries, resistance to ciprofloxacin in N. gonorrhoeae has been reported in 57.9% of strains, whereas in Poland it affects approximately 50% of isolates [8]. Fluoroquinolone resistance is primarily mediated by amino acid substitutions in the quinolone resistance–determining regions of DNA topoisomerase II and IV, encoded by the gyrA and parC genes, respectively.
The most commonly identified substitutions in GyrA include S91F or S91T and D95A, D95G, D95N, or D95Y. In ParC, resistance-associated substitutions include D86N; S87C, S87I, S87K, S87N, S87R, or S87Y; S88A or S88P; and E91A, E91G, E91K, or E91Q [9, 23].
Tetracycline resistance
Tetracycline resistance in N. gonorrhoeae is primarily mediated by the TetM protein, which is encoded on conjugative plasmids and confers active ribosomal protection against tetracycline. Two major types of TetM-encoding plasmids have been identified: the Dutch and the American types. Expression of TetM is associated with tetracycline MICs ranging from 16 to 64 mg/l.
In addition, reduced susceptibility to tetracycline may result from overexpression of the MtrCDE efflux pump. Among N. gonorrhoeae strains isolated in 2022 in European Union countries, tetracycline resistance was reported in 63.4% of isolates according to EUCAST criteria and in 38.6% according to CLSI criteria. In Poland, the corresponding resistance rates were 53.3% and 13.3%, respectively [9, 24].
Spectinomycin resistance
Spectinomycin, an aminocyclitol antibiotic, inhibits protein synthesis by binding to the 16S rRNA within the 30S ribosomal subunit and interfering with translocation mediated by elongation factor G (EF-G). Resistance to spectinomycin in N. gonorrhoeae has been associated with a C1192U substitution in the 16S rRNA gene, as well as with mutations in the rpsE gene encoding ribosomal protein S5, including deletion of codon 27 (valine) and the K28E amino acid substitution [25].
In the European Union, only sporadic spectinomycin-resistant N. gonorrhoeae strains were identified between 2014 and 2020, while no spectinomycin resistance was reported among strains isolated in Poland during this period.
New drugs active against N. gonorrhoeae
N. gonorrhoeae resistant to second-generation cephalosporins and/or fluoroquinolones is included on the World Health Organization’s 2024 list of 15 priority bacterial pathogens posing the greatest threat to public health and requiring the development of new therapeutic options.
One of the novel agents with activity against gonorrhea, including drug-resistant strains, is gepotidacin, a triazaacenaphthylene compound that inhibits bacterial topoisomerase IV. In 2025, gepotidacin was approved by the U.S. Food and Drug Administration (FDA) for the treatment of uncomplicated urinary tract infections [26]. Another promising agent is zoliflodacin, a spiropyrimidinetrione antibiotic that inhibits bacterial DNA topoisomerase II, which was approved by the FDA in 2025 for the treatment of uncomplicated gonorrhea. Both agents have demonstrated efficacy comparable to ceftriaxone and may retain activity against ceftriaxone-resistant N. gonorrhoeae strains [27, 28].
Solithromycin, a ketolide antibiotic that inhibits protein synthesis and is used in the treatment of community-acquired pneumonia, has also been evaluated for gonorrhea. However, comparative clinical trials demonstrated lower efficacy than the combination of ceftriaxone and azithromycin, making it unlikely to be recommended as a first-line treatment for gonorrhea.
In addition, two fluoroquinolones sitafloxacin and delafloxacin, which inhibit bacterial DNA gyrase and topoisomerase IV, have shown activity against many ciprofloxacin-resistant N. gonorrhoeae strains. Nevertheless, clinical trial data indicate that delafloxacin is less effective than ceftriaxone in the treatment of gonorrhea, limiting its potential role in routine therapy [9].
Molecular typing of N. gonorrhoeae
Molecular typing of N. gonorrhoeae is most commonly performed using four methods based on whole-genome sequencing or sequencing of selected genomic fragments: whole-genome sequencing (WGS), N. gonorrhoeae multi-antigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR).
Whole-genome sequencing does not rely on a dedicated epidemiological typing database and is most often used as a data source for downstream typing approaches such as NG-MAST, MLST, and NG-STAR.
The NG-MAST version 2.0 (NG-MAST v2.0) method is currently the most widely used and highly discriminatory typing system for N. gonorrhoeae. It is based on sequencing a 430–511 bp fragment of the porB gene encoding porin B and a 367–416 bp fragment of the tbpB gene encoding the B subunit of transferrin-binding protein. Between 2009 and 2013, genogroup G1407 was dominant in the European Union (23.3–16.5%); however, its prevalence declined to 2.1% by 2018, when genogroups G12302 (5.6%), G5441 (5.6%), and G11461 (5.4%) predominated. In Poland, genogroup G1407 was first detected in 2012 [29].
The multilocus sequence typing (MLST) method, widely applied across bacterial species, involves sequencing 450–500 bp fragments of seven housekeeping genes (abcZ, adk, aroE, fumC, gdh, pdhC, and pgm). Typing of N. gonorrhoeae using MLST yields a relatively limited number of sequence types [30]. In European Union countries, the most frequently reported MLST sequence types include ST1901, ST9363, and ST7363 [14]. Greater discriminatory power is provided by core genome MLST (cgMLST), which analyses 1,659 conserved genes [31].
The NG-STAR method focuses on antimicrobial resistance determinants and involves sequencing fragments of seven genes: penA (encoding PBP2), mtrR (encoding the repressor of the MtrCDE efflux pump), porB (encoding porin B), ponA (encoding PBP1), gyrA (encoding DNA gyrase subunit), parC (encoding topoisomerase IV subunit), and the 23S rRNA gene, which contains the target site for azithromycin. Although mutations in the 23S rRNA gene are detected, NG-STAR does not distinguish the number of mutated alleles present, which may limit interpretation of azithromycin resistance levels. In addition, the NG-STAR scheme does not include determinants of tetracycline or spectinomycin resistance, nor does it account for β-lactamase production. Among N. gonorrhoeae isolates collected from 26 European Union countries in 2018, the most frequently identified NG-STAR type was ST442 [14].
CONCLUSIONS
Given the risk of dissemination of multidrug-resistant strains, continuous monitoring of antimicrobial susceptibility of N. gonorrhoeae across different geographic regions, including Europe and Poland, is essential. Epidemiological genotyping represents a valuable tool for tracking the spread of resistant strains. Among emerging therapeutic options for gonorrhea, zoliflodacin and gepotidacin appear to be the most promising.
