The aim of this work was to study the resistance to macrolides (azithromycin) in the modern Russian population of N. gonorrhoeae with the analysis of genetic resistance determinants. Azithromycin is not used to treat gonococcal infection in Russia. However, among 162 isolates collected in 2020–2021, 22 isolates (13.6%) were phenotypically resistant to azithromycin. Mutations in 23S rRNA genes were found only in two isolates; erm and mefA genes were absent. Azithromycin resistance was shown to be predominantly associated with mutations in the mtrR and mtrD genes of the MtrCDE efflux pump and their mosaic alleles which may have formed due to a horizontal transfer from N. meningitidis. A total of 30 types of mtrR alleles and 10 types of mtrD alleles were identified including mosaic variants. Matching between the mtrR and mtrD alleles was revealed to indicate the cooperative molecular evolution of these genes. A link between the mtrR and mtrD alleles and NG-MAST types was found only for NG-MAST 228 and 807, typical of N. gonorrhoeae in Russia. The high level of resistance to azithromycin in Russia may be related to the spread of multiple transferable resistance to antimicrobials regardless of their use in the treatment of gonococcal infection.

A multiplex assay based on a low-density hydrogel microarray was developed to identify genomic substitutions in N. gonorrhoeae that determine resistance to the currently recommended treatment agents ceftriaxone and azithromycin and the previously used drugs penicillin, tetracycline, and ciprofloxacin. The microarray identifies 74 drug resistance determinants in the N. gonorrhoeae penA, ponA, porB, gyrA, parC, rpsJ, mtrR, blaTEM, tetM, and 23S rRNA genes. The hydrogel elements were formed by automated dispensing of nanoliter-volume droplets followed by UV-induced copolymerization of NH2-containing oligonucleotides with gel-forming monomers. Polybutylene terephthalate plates without special modifications were used as microarray substrates. Sequences and concentrations of immobilized oligonucleotides, gel composition, and hybridization conditions were carefully selected, and the median discrimination ratio ranged from 2.8 to 29.4, allowing unambiguous identification of single-nucleotide substitutions. The mutation identification results in a control sample of 180 N. gonorrhoeae isolates were completely consistent with the Sanger sequencing results. In total, 648 clinical N. gonorrhoeae isolates obtained in Russia during the last 5 years were analyzed and genotyped using these microarrays. The results allowed us to draw conclusions about the present situation with antimicrobial susceptibility of N. gonorrhoeae in Russia and demonstrated the possibility of using hydrogel microarrays to control the spread of antibiotic resistance.

The goal of this work was to determine the factors affecting the emergence of azithromycin-resistant Neisseria gonorrhoeae isolates in Russia, where azithromycin was never recommended for the treatment of gonococcal infections. Clinical N. gonorrhoeae isolates collected in 2018–2021 (428 isolates) were analyzed. No azithromycin-resistant isolates were found in 2018–2019, but in 2020–2021, a significant increase in the ratio of azithromycin-resistant isolates was observed: 16.8% and 9.3%, respectively. A hydrogel DNA microarray was developed for the analysis of resistance determinants: mutations in the genes encoding the mtrCDE efflux system and in all four copies of the 23S rRNA gene (position 2611). A majority of the azithromycin-resistant Russian isolates belonged to the NG-MAST G12302 genogroup, and the resistance was associated with the presence of a mosaic structure of the mtrR gene promoter region with the −35 delA deletion, an Ala86Thr mutation in the mtrR gene, and a mosaic structure of the mtrD gene. A comparative phylogenetic study of modern Russian and European N. gonorrhoeae populations allowed us to conclude that the emergence of azithromycin resistance in Russia in 2020 was the result of the appearance and spread of European N. gonorrhoeae strains belonging to the G12302 genogroup due to possible cross-border transfer.

Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance. The emergence of clinical isolates producing plasmid β-lactamases that can hydrolyze cephalosporins, the mainstay treatment for gonorrhea, may be a serious threat. In this work, N. gonorrhoeae strains producing plasmid-mediated broad- and extended-spectrum β-lactamases (ESBLs) were obtained in vitro, and their viability and β-lactam antibiotic susceptibility were studied. Artificial pblaTEM-1 and pblaTEM-20 plasmids were constructed by site-directed mutagenesis from a pblaTEM-135 plasmid isolated from a clinical isolate. Minimum inhibitory concentration (MIC) values for a series of β-lactam antibiotics, including benzylpenicillin, ampicillin, cefuroxime, ceftriaxone, cefixime, cefotaxime, cefepime, meropenem, imipenem, and doripenem, were determined. The N. gonorrhoeae strain carrying the pblaTEM-20 plasmid exhibited a high level of resistance to penicillins and second–fourth-generation cephalosporins (MIC ≥2 mg/L) but not to carbapenems (MIC ≤0.008 mg/L). However, this strain stopped growing after 6 h of culture. The reduction in viability was not associated with loss of the plasmid but can be explained by the presence of the plasmid itself, which requires additional reproduction costs, and to the expression of ESBLs, which can affect the structure of the peptidoglycan layer in the cell membrane. Cell growth was mathematically modeled using the generalized Verhulst equation, and the reduced viability of the plasmid-carrying strains compared to the non-plasmid-carrying strains was confirmed. The cell death kinetics of N. gonorrhoeae strains without the pblaTEM-20 plasmid in the presence of ceftriaxone can be described by a modified Chick–Watson law. The corresponding kinetics of the N. gonorrhoeae strain carrying the pblaTEM-20 plasmid reflected several processes: the hydrolysis of ceftriaxone by the TEM-20 β-lactamase and the growth and gradual death of cells. The demonstrated reduction in the viability of N. gonorrhoeae strains carrying the pblaTEM-20 plasmid probably explains the absence of clinical isolates of ESBL-producing N. gonorrhoeae.

N. gonorrhoeae strains producing plasmid-mediated broad- and extended-spectrum beta-lactamases (ESBLs) have been obtained in vitro and studied for their viability and susceptibility to beta-lactam antibiotics. The artificial pblaTEM-1 and pblaTEM-20 plasmids were constructed by site-directed mutagenesis from a pblaTEM-135 plasmid of the Toronto/Rio type, which was extracted from the clinical isolate. MIC values were determined for a series of beta-lactam antibiotics, including benzylpenicillin, ampicillin, cefuroxime, ceftriaxone, cefixime, cefotaxime, cefepime, meropenem, imipenem, and doripenem. The N. gonorrhoeae strain carrying the pblaTEM-20 plasmid exhibited a high level of resistance to penicillins and II-IV generation cephalosporins (MIC ≥ 2 mg/L) but not to carbapenems (MIC ≤ 0.008 mg/L). However, this strain stopped growing after 6 hours of cultivation. The reduced viability was not associated with the plasmid loss but can be explained by the following factors: the presence of the plasmid itself, which requires additional costs for its reproduction, and the expression of ESBL, which can affect the structure of the peptidoglycan layer in the cell membrane. The cell growth was mathematically modeled using the generalized Verhulst equation. The parameter, characterizing the maximum possible number of cells grown under given conditions, decreased within the wild type (without plasmids) - pblaTEM-135 - pblaTEM-1 - pblaTEM-20 series, i.e., the plasmid-bearing strains had reduced viability compared to that of the wild-type strain. The kinetics for the cell death of N. gonorrhoeae strains without the pblaTEM-20 plasmid in the presence of ceftriaxone can be described by a modified Chick-Watson law. For the N. gonorrhoeae strain that harbors the pblaTEM-20 plasmid, the kinetics for cell number changes with time reflected several processes: the hydrolysis of ceftriaxone by TEM-20 beta-lactamase, growth and gradual death of cells. The demonstrated reduced viability of N. gonorrhoeae strains with the pblaTEM-20 plasmid probably explains the absence of clinical isolates of N. gonorrhoeae that produce ESBL.