Molecular Mechanisms of Drug Resistance and Epidemiology of Multidrug-Resistant Variants of Neisseria gonorrhoeae

The paper presents various issues related to the increasing drug resistance of Neisseria gonorrhoeae and the occurrence and spread of multidrug-resistant clones. One of the most important is the incidence and evolution of resistance mechanisms of N. gonorrhoeae to beta-lactam antibiotics. Chromosomal resistance to penicillins and oxyimino-cephalosporins and plasmid resistance to penicillins are discussed. Chromosomal resistance is associated with the presence of mutations in the PBP2 protein, containing mosaic variants and nonmosaic amino acid substitutions in the transpeptidase domain, and their correlation with mutations in the mtrR gene and its promoter regions (the MtrCDE membrane pump repressor) and in several other genes, which together determine reduced sensitivity or resistance to ceftriaxone and cefixime. Plasmid resistance to penicillins results from the production of beta-lactamases. There are different types of beta-lactamases as well as penicillinase plasmids. In addition to resistance to beta-lactam antibiotics, the paper covers the mechanisms and occurrence of resistance to macrolides (azithromycin), fluoroquinolones and some other antibiotics. Moreover, the most important epidemiological types of multidrug-resistant N. gonorrhoeae, prevalent in specific years and regions, are discussed. Epidemiological types are defined as sequence types, clonal complexes and genogroups obtained by various typing systems such as NG-STAR, NG-MAST and MLST. New perspectives on the treatment of N. gonorrhoeae infections are also presented, including new drugs active against multidrug-resistant strains.


Introduction
The increasing antimicrobial drug resistance of Neisseria gonorrhoeae, the etiologic agent of gonorrhea, is becoming a global problem. Gonorrhea is among the most common bacterial sexually transmitted infections, with the WHO estimating in 2020 that 82,400,000 cases of the disease occurred worldwide [1]. In addition, the incidence of reported cases of gonorrhea in the European Union is increasing. Per 100,000 people in the population, an increase was observed from 6.6 in 2009 to 26.9 in 2018 [2,3]. According to the Centers for Disease Control and Prevention (CDC) in the United States, the incidence of gonorrhea increased by 111% from 2009 to 2020. In 2019, the number of cases per 100,000 people was 187.8 and in 2020, 206.5 [4]. The susceptibility of N. gonorrhoeae to drugs (ceftriaxone, cefixime, azithromycin, ciprofloxacin, spectinomycin and gentamicin) in EU countries and the UK is monitored on an ongoing basis as part of the European Gonococcal Antimicrobial Susceptibility Programme (Euro-GASP), which is funded by the European Centre for Disease Prevention and Control (ECDC) [5][6][7][8]. The antibiotic resistance of N. gonorrhoeae is monitored in the United States as part of the GISP (Gonococcal Isolate Surveillance Project), funded by the CDC.
In 2017, N. gonorrhoeae was included in a WHO-created list of 12 pathogens whose drug resistance poses a global health threat and which most urgently require the development of In 2017, N. gonorrhoeae was included in a WHO-created list of 12 pathogens whose drug resistance poses a global health threat and which most urgently require the development of new antibiotics due to the worldwide emergence of multidrug-resistant strains including those resistant to beta-lactams (ceftriaxone, cefixime and penicillin) and simultaneously resistant to oxyimino-cephalosporins (ceftriaxone, cefixime) and azithromycin [9]. In recent years, N. gonorrhoeae strains collected from various EU countries, including Poland, have been characterized by the Euro-GASP for antibiotic susceptibility, which was studied using conventional methods, and their genomes were sequenced using the WGS (whole-genome sequencing) method. The WGS method makes it possible to specify the genetic determinants of resistance and replaces other methods of bacteria molecular typing, allowing simultaneous determination of membership in such sequence types as NG-MAST, NG-STAR and MLST. The results of these studies, depicting the epidemiological situation in the countries studied, have been published [5,6,10]. Sequences of newly described determinants of resistance to antibiotics and new epidemiological types are constantly updated in the worldwide NG-STAR, NG-MAST and MLST databases.
Crucial in the development of N. gonorrhoeae resistance to ceftriaxone and cefixime are mutations in the penA gene, especially in the domain encoding PBP2 transpeptidase. An increase in the MICs of these drugs can also result from the overproduction of MtrCDE membrane pump proteins (most commonly a deletion of −35A) in the mtrR promoter region, and substitutions in the MtrR protein (G45D) and amino acid substitutions in the PorB1b protein, but both of these mechanisms alone do not determine resistance [47]. Although the link between N. gonorrhoeae resistance to oxyimino-cephalosporins and the above-mentioned mutations is indisputable, strains with identical or very similar genetic characteristics but drastically different MICs of cephalosporins have been isolated [48][49][50][51][52]. The implication is that some factor determining resistance to ceftriaxone and cefixime is being missed, and it is impossible to predict from DNA sequencing alone whether N. gonorrhoeae is resistant to cefixime and ceftriaxone. This may involve regulatory mechanisms regarding PBP2 protein expression, which translates into the amount of PBP2 protein in the N. gonorrhoeae cell. Mechanisms of resistance to beta-lactams resulting from the overproduction of PBP proteins have been described in Gram-positive bacteria [53]. The effect of certain mutations in the rpoB and rpoD genes (they encode DNA-dependent RNA polymerase subunits) on changes in the MIC and the development of resistance to ceftriaxone and cefixime is presented in Table 2 [54].However, the mechanism of this resistance is not currently understood. Mutations in the penA gene encode PBP2 transpeptidase. Based on sequencing of a 1745-1752 bp fragment of the penA gene, about 489 alleles of the PBP2 protein have been described, which are divided into 234 major types (I-V, VII, IX-XIX, XXI-XXII, XXVII, XXXIV-XXXV, 37-234) and many subtypes (Tables 3 and 4).The proteins encoded by the individual alleles contain from 1 to over 60 amino acid mutations compared to ancestral wild-type alleles. Mutation-induced changes in the amino acid pattern of PBP2 transpeptidase (mostly substitutions and single amino acid deletions and insertions), are referred to as mosaic, semimosaic or nonmosaic patterns of PBP2. The mosaic-like structure of the penA gene in N. gonorrhoeae has evolved by intragenic recombination with penA genes of other Neisseria species, which are mainly commensal: Neisseria perflava, Neisseria sicca, Neisseria cinerea, Neisseria flavescens and Neisseria meningitidis. Mosaic alleles of PBP2 have approximately 60 amino acid alterations from the PBP2 of wild-type strains and most often the D346a insertion is absent [55,56]. Semimosaic alleles usually have 20-30 amino acid alterations and some have the D346a insertion [57]. The nonmosaic alleles usually have a D346a insertion and between 1 and 13 (most often [4][5][6][7][8] mutations in the C-terminus [57]. In mosaic patterns, the most important substitutions responsible for ceftriaxone and cefixime resistance (MIC > 0.125 mg/L) are A311V, I312M, V316P/T, T483S, A501P/V/T/T, F504L, N512Y A516G and G545S [40,47,[57][58][59][60], and in nonmosaic patterns, the substitutions are A501P/V/T/R/S, F504L, N512Y, A516G, G542S and P551S [47,[57][58][59]61,62]. Moreover, A516G does not appear to alter resistance very much [62]. The main types of nonmosaic, semimosaic and mosaic PenA are shown in Tables 3 and 4 [30,[40][41][42][43][44]52,54,57,[63][64][65]. There are differences in the numbering of some amino acids (usually by 1) between the source papers and the NG-STAR databases.      Tomberg et al. showed that the transformation of FA19 (an antibiotic-susceptible strain) with the WHO X penA allele is sufficient to confer resistance to ceftriaxone and cefixime that surpasses the EUCAST-and CDC-defined resistance (MICs for ceftriaxone = 0.31 mg/L and for cefixime = 1.6 mg/L) [59]. However, the presence of a mosaic penA allele alone in most cases is not sufficient for the resistance to these antibiotics and other determinants, as substitutions in the proteins PonA (L421P) and PorB, mutations in the promoter region of the mtrR gene (most often deletion −35A) and mtrCDE, and/or substitutions in the MtrR protein (G45D) are needed [20,23,66,67].
The MtrCDE membrane pump removes beta-lactams, macrolides, tetracyclines, rifampicin and detergents. The expression of mtrCDE in wild-type gonococci is repressed by MtrR and, in the presence of an inductor, is activated by MtrA. MtrA and MtrR bind to regions within a 250 bp sequence that contains overlapping, divergent promoters for the transcription of mtrR and mtrCDE. The overproduction of membrane pump proteins is determined by substitutions in the MtrR pump repressor protein (G45D and A39T) and mutations in the region upstream of the mtrR gene (e.g., deletion of adenine at the −35 position preceding the mtrR gene and mutation conditioning the formation of a new mtrCDE promoter, the so-called mtr120, which is not regulated by MtrR and MtrA) [68][69][70][71][72][73]. The expression and efficiency of the MtrCDE pump are also significantly influenced by mosaic changes within the mtrR and mtrCDE genes, probably caused by the transformation and recombination of DNA from saprophytic Neisseria spp. (e.g., N. lactamica) and N. meningitidis. Mosaic sequences within the gene encoding the inner membrane transporter protein mtrD have shown a strong linkage and epistatic effects that likely enhance the efflux pump activity of MtrCDE [70,73]. Alterations within the mtrR and mtrCDE genes, especially mosaic alterations within mtrD, are one of the most important determinants in N. gonorrhoeae of a novel azithromycin resistance phenotype (MIC of 2 to 4 mg/L), which is not associated with 23S rRNA mutations (for details, see Section 3) [70,73].
Amino acid substitutions of the PorB1b protein at positions 120 and 121 result in changes in the MIC of penicillins, cephalosporins and tetracyclines. Based on the sequencing of a 30 bp fragment of PorB1b, the amino acid substitutions of the PorB1b protein were determined to be G120C, D, E, K, N, P, Q, R, S or T and A121D, G, H, N, P, S or V [71]. Mutations in the porB gene determining substitutions in the PorB1b protein (G120K, G120D/A121D, G120P/A121P and G120R/A121H) are named penB mutations [73][74][75]. PorB mutations reduce antibiotic diffusion into the periplasmic space only in the presence of mtrR or mtr promoter mutations [73]. The combined occurrence of mutations in the porB and mtr genes in strain FA19penA4 (penA4 being the allele from FA6140, a pen-resistant isolate) increased the MIC of penicillin from 0.12 mg/L to 1.0 mg/L and the MIC of tetracycline from 0.15 mg/L to 1.0 mg/L. The combined presence of mutations in the porB and mtr genes and the pilQ mutation (named the penC mutation) in this strain increased the MIC of penicillin to 2.0 mg/L and the MIC of tetracycline to 2.0 mg/L, whereas the simultaneous occurrence of four mutations in the porB, mtr, ponA (L429P in the PonA protein) and pilQ genes increased the MIC of penicillin to 4.0 mg/L [74].Together, the PilQ protein and type IV pili form an SDS-resistant multimeric complex that acts as a pore for antibiotics and small molecules. A mutation causing a G666L substitution in the PilQ protein, known as the pilQ2 or penC mutation, hinders the formation of the PilQ multimer, disrupting pore formation and thereby reducing penicillin influx [76]. The pilQ mutations in these studies arise spontaneously and at a high frequency in the laboratory. However, no study has found that pilQ mutations are present in clinical isolates, likely because the bacteria require an intact type IV pilus machinery for infection [77].
TEM beta-lactamases are common in bacteria. In total, 197 (numbering from 1 to 246, some numbers removed) types of these enzymes have been described, and enzymes with an extended spectrum that hydrolyze oxyimino-cephalosporins, i.e., ceftriaxone and cefixime, among others, are commonly used in the treatment of gonorrhea [95,96]. It is only a matter of time before such TEM variants appear in N. gonorrhoeae. It is difficult to predict whether these will be sporadic incidents or whether a strain with such an enzyme variant is likely to spread endemically or pandemically in the population.

Macrolide (Azithromycin) Resistance
Azithromycin binds to a four-nucleotide fragment of rRNA (in the peptidyltransferase region) within the V domain of the 23S rRNA in the 50S subunit of the ribosome, which results in the inhibition of protein synthesis (Figure 1 (Figure 1).
Mutations in the rplV genes that condition tandem duplications of ARAK at position 90 or KGPSLK at position 83 in ribosomal protein L22 [102] and mutations in the rplD gene that condition a G70D substitution in ribosomal protein L4 may also be the cause of the azithromycin MIC increase. This mutation causes an increase in the MIC of azithromycin 2.5-4-fold to a level of 0.4-0.5 mg/L, which does not cause azithromycin resistance according to current criteria [103].
Mutations in the promoter region of the macA-macB efflux system may also be the cause of the azithromycin MIC increase [104].
ErmB and ErmF 23S rRNA methylases were also detected in N. gonorrhoeae (Figure 1). The MIC of azithromycin in these strains had values ranging from 1 to 4 mg/L [105]. The lack of complete molecular characterization of these strains makes it impossible to determine what caused azithromycin resistance in thes e strains. The presence of ermB, ermC, ermF and mefA genes (encoding a membrane pump protein categorized as MFS) in many N. gonorrhoeae strains isolated between 1940 and 1987 has also been described [106]. The occurrence of different erm and mefA genes is characteristic for Gram-positive cocci and for Gram-negative anaerobic bacilli Bacteroides spp. [106,107].
In recent years, a successive increase in the percentage of azithromycin-resistant N. gonorrhoeae strains has been observed in many European countries [108].

Resistance to Fluoroquinolones (Ciprofloxacin)
Fluoroquinolones are classified as bactericidal drugs. They inhibit the activity of topoisomerase II (gyrase) and topoisomerase IV enzymes responsible for DNA supercoiling and relaxation (Figure 1

Resistance to Tetracyclines
Tetracyclines are antibiotics that inhibit protein synthesis by interfering with the 30S subunit of the ribosome (Figure 1). The criterion for tetracycline resistance according to the EUCAST is an MIC > 1.0 mg/L, and according to the CLSI, is an MIC ≥ 2.0 mg/L [11,12].
The TetM (ribosomal protection) protein is encoded by conjugative plasmids. Two variants of the tetM gene have been described, which have been named Dutch and American (MIC tetracycline 16-64 mg/L), and were found in 41,998-42,003 bp conjugative plasmids (GenBank acc. CP068762, CP045833). Two types of plasmids of 25.2 Mda have also been described, which have also been named Dutch (pOZ101; GenBank L12242) and American (pOZ100; GenBank L12241). In Poland, as in many other European countries, the Dutch TetM type was more common than the American type [111]. The most common correspondence is between gene type and plasmid type [112,113].
Other mechanisms determine the MIC of tetracycline 2-4 mg/L. Individually, the MIC can be >4 mg/L. Out of 1479 isolates, 8 such strains have been described [101]. These include mutations in the chromosomal gene mtrR and its promoter regions, causing overproduction of the membrane pump protein MtrCDE, and mutations in the genes porB, pilQ and rpsJ (V57M substitution in the ribosomal protein S10) [114] (Figure 1). However, alterations of the pilQ gene in N. gonorrhoeae are unlikely contributors to decreased susceptibility to tetracyclines in clinical gonococcal strains because the bacteria require an intact type IV pilus machinery for infection [77].
The criterion for spectinomycin resistance according to the EUCAST is an MIC > 64 mg/L, and according to the CLSI, is an MIC ≥ 128 mg/L [11,12]. The cause of N. gonorrhoeae resistance to spectinomycin may be due to a C1192U transition in 16SrRNA and mutations in the rpsE gene encoding the 5S ribosomal protein (deletion of codon 27 (valine) and a K28E substitution) [11].

Resistance to Gentamicin
Gentamycin is classified as an aminoglycoside. Aminoglycosides are bactericidal antibiotics that inhibit protein synthesis by interfering with the 30S subunit of the ribosome (Figure 1). There are no criteria for the resistance of N. gonorrhoeae to gentamicin according to the EUCAST and CLSI.
A missense mutation in the fusA gene, which encodes elongation factor G (EF-G) and causes an A563V substitution in the IV domain of EF-G, has been described (Figure 1  .004-0.5 mg/L, respectively. Resistance to ciprofloxacin, azithromycin, cefixime and ceftriaxone was 49.9%, 6.7%, 1.6% and 0.2%, respectively [122]. A study of 873 N. gonorrhoeae strains isolated in 21 European countries between 2012 and 2014 showed the MIC range, modal MIC, MIC50 and MIC90 were ≤0.002 to 0.25 mg/L, 0.125 mg/L, 0.064 mg/L and 0.125 mg/L, respectively [123]. Zoliflodacin showed no cross-resistance with the other antimicrobial agents tested. GyrB was highly conserved, and no gyrB mutations conditioning zoliflodacin resistance were found. None of the fluoroquinolone-target resistance mutations, GyrA or ParC, or mutations causing overexpression of the efflux pump MtrCDE had a significant effect on the MIC of zoliflodacin [122]. In a phase II clinical trial (RCT), a single 3 g dose of zoliflodacin resulted in a 100% cure rate for uncomplicated genitourinary (47/47) and rectal (6/6) gonorrhea, and the cure rate for pharyngeal gonorrhea was 78% (7/9). Zoliflodacin was well tolerated, with limited transient gastrointestinal side effects [124]. To date, no clinical isolates of zoliflodacin-resistant gonococci have been identified, but in vitro mutants with GyrB D429A/N or K450N/T substitutions have been selected that had zoliflodacin MICs of 1-2 mg/L [122,125].

Sitafloxacin
Sitafloxacin (DNA gyrase and topoisomerase IV inhibitor) belongs to a new generation of fluoroquinolones. The activity of sitafloxacin was studied against 250 strains of N. gonorrhoeae isolated between 1991 and 2013. Sitafloxacin had bactericidal activity, with MIC, MIC50 and MIC90 ranges of ≤0.001-1, 0.125 and 0.25 mg/L, respectively. There was a high correlation between the MICs of sitafloxacin and ciprofloxacin, but the MIC50 and MIC90 of sitafloxacin were 6-fold and >6-fold lower, respectively [126]. The MIC of sitafloxacin was tested in 35 ciprofloxacin-resistant N. gonorrhoeae isolates (ciprofloxacin MIC from 2 to 32 mg/L). The MIC of sitafloxacin ranged from 0.03 to 0.5 mg/L. None of the identified substitutions in the QRDR of GyrA and ParC increased the MIC of sitafloxacin above 0.5 mg/L [127].

Delafloxacin
Delafloxacin is an inhibitor of DNA gyrase and topoisomerase IV belonging to the new generation of fluoroquinolones. [128]. The activity of delafloxacin was tested against 117 strains of N. gonorrhoeae isolated between 2012 and 2015. The MIC50, MIC90 and MIC range of delafloxacin were 0.06 mg/L, 0.125 mg/L and ≤0.001 to 0.25 mg/L, respectively. The frequency of spontaneous mutation ranged from 10 −7 to <10 −9 [128]. The mutant had an S91Y substitution in GyrA and a delafloxacin MIC of 1 mg/L [128].

Gepotidacin
Gepotidacin (GSK2140944) is a novel, first-in-class triazaacenaphthylene, DNA gyrase and topoisomerase IV inhibitor [129][130][131] (Figure 1). Phase II clinical trials (RCTs) evaluating the use of gepotidacin in a single oral dose of 1.5 and 3 g for the treatment of uncomplicated gonorrhea showed 97% and 95% efficacy, respectively. The activity of gepotidacin against 252 strains of N. gonorrhoeae was tested. The modal MIC, MIC50, MIC90 and MIC range of gepotidacin were 0.5, 0.5, 1 and 0.032-4 mg/L, respectively [129]. Inactivation of the efflux pump MtrCDE was shown to lower the MIC of gepotidacin. A D86N substitution in ParC and A92T substitution in GyrA were found to be associated with high MIC values of gepotidacin [129]. In another study, the MIC50, MIC90 and MIC range of gepotidacin were 0.12-0.25, 0.5 and 0.06-1 mg/L, respectively. Two N. gonorrhoeae strains with MICs of 1 mg/L achieved treatment failure after a single dose of gepotidacin and the MICs of N. gonorrhoeae strains increased from 1 to ≥32 mg/L [131]. The PAEs (postantibiotic effects) for gepotidacin against the wild-type strain ranged from 0.5 to >2.5 h, and the PAE-SMEs (subinhibitory effects) were >2.5 h [130].

Solithromycin
Solithromycin (CEM-101) is a novel oral fluoroketolide antimicrobial with substantial in vitro activity against N. gonorrhoeae.
It also shows activity against Chlamydia trachomatis and Mycoplasma genitalium. Solithromycin binds to the 23S rRNA in the 50S subunit of the ribosome, which causes inhibition of bacterial protein synthesis [132,133] (Figure 1). The drug is currently in phase III clinical trials. Solithromycin displayed a MIC50 and MIC90 of 0.0625 and 0.125 mg/L for clinical strains of N. gonorrhoeae [134]. Two groups of gonorrhea patients were studied. One group received oral solithromycin 1000 mg and the other group received intramuscular ceftriaxone 500 mg plus oral azithromycin 1000 mg. Eradication of N. gonorrhoeae was achieved in 80% of patients receiving solithromycin and in 84% of patients receiving ceftriaxone plus azithromycin. The incidence of adverse events was higher in the solithromycin group than in the ceftriaxone plus azithromycin group (53% and 34%), most commonly, diarrhea (24% and 15%) and nausea (21% and 11%) [135].

Ertapenem
Ertapenem is a beta-lactam antibiotic from the carbapenem group and is used to treat infections with certain Gram-negative bacilli.

Aminoethyl Spectinomycins
A series of aminoethyl spectinomycins (AmSPCs) obtained by modifying the 3 keto group in the C ring of spectinomycin were studied. AmSPC preparations were shown to have slightly higher in vitro activity against N. gonorrhoeae and Chlamydiatrachomatis than spectinomycins. N. gonorrhoeae strains resistant to spectinomycin were also resistant to AmSPC [140].

Fosfomycin
The fosfomycin phosphoenolpyruvate analogue antibiotic is used in the treatment of cystitis. It acts as a bactericide by binding to the enolpyruvate transferase (MurA), and consequently, inhibiting cell wall synthesis (Figure 1). The susceptibility of 89 N. gonorrhoeae isolates to fosfomycin was tested. The MIC50, MIC90 and MIC range were 8 mg/L, 16 mg/L and ≤1 to 32 mg/L, respectively [141].

TP0480066 and Other Antimicrobials
TP0480066 is a novel 8-(methylamino)-2-oxo-1,2-dihydroquinoline (MAOQ) derivative (DNA gyrase and topoisomerase IV inhibitor). The TP0480066 MIC of fourteen N. gonorrhoeae reference strains (including strains resistant to ciprofloxacin and levofloxacin, as well as to other drug groups) was determined. The MIC range obtained for these strains was from ≤0.00012 to 0.0005 mg/L [142].

Epidemiological Typing of N. gonorrhoeae
Closely related to the acquisition and presence of resistance genes in N. gonorrhoeae is molecular typing, a fundamental method in the study of many bacterial species. Molecular typing makes it possible to trace the occurrence, spread and evolution of multidrug-resistant strains globally. Various molecular typing systems exist. In N. gonorrhoeae, in some typing systems (e.g., NG-STAR), sequence types are determined solely on the basis of mutations in genes associated with resistance or reduced sensitivity to beta-lactams, macrolides and fluoroquinolones.
The typing of N. gonorrhoeae is usually carried out based on four methods related to the sequencing of the whole genome or its fragments: WGS (whole-genome sequencing), NG-MAST (Neisseria gonorrhoeae multi-antigen sequence typing), MLST (multi-locus sequence typing) and NG-STAR (Neisseria gonorrhoeae sequence typing for antimicrobial resistance).

Neisseria gonorrhoeae Multi-Antigen Sequence Typing (NG-MAST)
The NG-MAST method has been the most widely used method for typing N. gonorrhoeae since 2004. It involves sequencing a 490 bp fragment of the porB gene (430-511 bp as of mid-2021), encoding porin B and a 390 bp fragment of the tbpB gene (367-416 bp as of mid-2021) and encoding the B subunit of the transferrin-binding protein. The allele number of each gene under study is being determined in the worldwide NG-MAST database [144]. By the end of 2020, about 12,800 alleles of the porB gene and about 3200 alleles of the tbpB gene had been described. [71]. In mid-2021, the NG-MAST database was reorganized (current name NG-MASTv2.0). porB sequences with a number greater than 11,028 and tbpB sequences with a number greater than 2868 were removed. Currently (20 July 2022), the database contains 11911 porB alleles (numbering from 1 to 12059) and 2994 tbpB alleles (numbering from 1 to 3087). Based on the combination of porB and tbpB alleles, the NG-MAST sequence type is determined. By the end of 2020, about 22,000 NG-MAST sequence types had been described [71]. As a result of the reorganization of the database in mid-2021, sequence types with numbers higher than 11028 were removed. About 100 removed STs were re-entered into the database, but under different numbers. Currently (20 July 2022), the base contains 20218 STs (numbering from 1 to 20,729). Using the WGS method, NGMASTER is a tool for rapidly determining NG-MAST types in silico from N. gonorrhoeae genomes [145]. Sequenced NG-MAST types are grouped within several hundred genogroups. Genogroup membership is determined by comparing the porB and tbpB sequences of the sequence type from which the genogroup is named (e.g., ST-A) with the porB and tbpB sequences of another ST (e.g., ST-B and ST-C). If porB differs by ≤5 bp and tbpB is the same, tbpB differs by ≤4 bp and porB is the same, or the sum of differences in porB and tbpB in ST-A and ST-B and ST-A and ST-C is ≤5 bp (substitutions, insertions, deletions, inversions are treated as a change of 1 bp), then we can include ST-B and ST-C in genogroup GST-A. From this definition, it follows that between ST-B and ST-C, they can differ by 1 to 10 bp. The choice of genogroup name (traditional or referring to the most common ST in a particular area or time period) is also debatable. The largest genogroup, G1407, includes more than 500 sequence types, and each of the other large genogroups such as G21, G51, G225, G2992, G4822 and G10799 contains more than 200 sequence types. Among the N. gonorrhoeae isolated in 21 EU countries in 2013 and characterized by the Euro-GASP, the most frequently isolated were ST1407 (7.6%), ST2992 (6.6%), ST2400 (3.9%), ST4995 (3. 0%) and ST21 (2.4%). The most frequently isolated genogroups were G1407 (14.8%), G2992 (7.7%), G21 (6.2%), G2400 (5.6%), G51 (5.1%), G225 (4.0%), G4995 (3.2%) and G387 (2.5%) [146]. Among the N. gonorrhoeae isolated in 26 EU countries and the UK in 2018 and characterized by the Euro-GASP, the most frequently isolated were ST11461 (4.7%), ST5441(3.7%), ST12302 (3.4%), ST14994 (3.2%) and 14769 (3.0%) [27]. The most frequently isolated NG-MAST genogroups in the EU in 2018 were: G12302 (5.6%), G5441 (5.6%) and G11461 (5.4%). The incidence of the G1407 genogroup, which was dominant in the EU in 2009-10 (23.3%) and 2013 (16.5%), decreased to 2.1% in 2018. G12302 is most often NG-STAR CC168 and CC63, and MLST ST9363. G5441 is most often NG-STAR CC442. G11461 is most often NG-STAR CC42 [27]. The most common NG-MAST sequence types found in selected countries are shown in Table 5 [26,27,42,[50][51][52]101,[146][147][148][149][150][151]. Table 5. Prevalence of NG-MAST sequence types in Europe and selected non-European countries.

Neisseria gonorrhoeae Sequence Typing for Antimicrobial Resistance (NG-STAR)
The NG-STAR method, used since 2017 [64,65], involves sequencing fragments of seven genes of N. gonorrhoeae: penA (encodes PBP2; 524 alleles), mtrR (encodes membrane pump repressor protein mtrCDE; 515 alleles), porB (encodes porin B; 64 alleles), ponA (encodes PBP1; 19 alleles, numbered 1-18 and 100), gyrA (encodes gyrase subunit; 58 alleles), parC (encodes topoisomerase IV subunit; 175 alleles) and 23S rRNA (contains target site for azithromycin; 71 alleles). Some mutations in the above-mentioned genes condition resistance to beta-lactam antibiotics, fluoroquinolones and macrolides. It is unfortunate that in the case of mutations in the 23S rRNA, no account was taken of how many alleles of the gene (the 23S rRNA gene always occurs in four copies) the mutation occurs in, which would translate directly into azithromycin resistance or lack thereof.
There are two publicly available global databases: the Canadian NG-STAR v. 2.0 [153] and the European Pub MLST NG-STAR [64]. Based on the sequence (usually using the WGS method), the allele number of each gene under study is determined, and based on the combination of the seven alleles, the NG-STAR sequence type is determined. As of May 2022, 4340 ST NG-STAR types have been described (numbering up to ST4464). Among the N. gonorrhoeae isolated in 26 EU countries in 2018 and surveyed by the Euro-GASP, the most frequently isolated NG-STAR type was ST442 [18]. Among the 1479 N. gonorrhoeae isolated in the US in 2018, the most frequently isolated NG-STAR type was ST436 (6%), ST63 (4.9%) and ST520 (3.9%) [101]. In 2021, Golparian et al. [39] divided 1-2602 ST NG-MAST into 317 clonal complexes (CC) and 169 sequence types that (ST) NG-MAST defined as ungroupable.

Conclusions
The most serious problem regarding N. gonorrhoeae drug resistance is the emergence and spread of clones resistant to ceftriaxone and azithromycin. Many strains of N. gonorrhoeae with an MIC ≥ 0.5 mg/L of ceftriaxone have been described, but most of them have not spread worldwide. The first such clone, FC428, was described in Japan in 2015 and has spread in Asian countries, but is not observed in Europe and the US. A second possible option for acquisition of ceftriaxone resistance is the emergence and spread of extended-spectrum TEM beta-lactamase variants (many such variants have been described in Gram-negative bacilli, but the spectrum of none incules carbapenems). This event is also more likely in Asian countries where there is a high percentage of beta-lactamase production by N. gonorrhoeae. Additionally, of concern is the increase in the number of azithromycin-resistant N. gonorrhoeae strains observed in Europe in 2016-19. In light of these risks, it is expedient to search for new drugs active against N. gonorrhoeae.