Mini-Multilocus Sequence Typing Scheme for the Global Population of Neisseria gonorrhoeae.

The increasing problem of antimicrobial resistance in N. gonorrhoeae necessitates the development of molecular typing schemes that are suitable for rapid and mass screening. The objective of this study was to design and validate a mini-MLST scheme for N. gonorrhoeae based on global pathogen population data. Using sequences of seven housekeeping genes of 21,402 isolates with known MLSTs from the PubMLST database, we identified eighteen informative polymorphisms and obtained mini-MLST nucleotide profiles to predict MLSTs of isolates. We proposed a new MLST grouping system for N. gonorrhoeae based on mini-MLST profiles. Phylogenetic analysis revealed that MLST genogroups are a stable characteristic of the N. gonorrhoeae global population. The proposed grouping system has been shown to bring together isolates with similar antimicrobial susceptibility, as demonstrated by the characteristics of major genogroups. Established MLST prediction algorithms based on nucleotide profiles are now publicly available. The mini-MLST scheme was evaluated using a MLST detection/prediction method based on the original hydrogel DNA microarray. The results confirmed a high predictive ability up to the MLST genogroup. The proposed holistic approach to gonococcal population analysis can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

Emergence of Azithromycin-Resistant Neisseria gonorrhoeae Isolates Belonging to the NG-MAST Genogroup 12302 in Russia.

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.

Azithromycin Susceptibility Testing and Molecular Investigation of Neisseria gonorrhoeae Isolates Collected in Russia, 2020-2021.

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.

Genomic Diversity and Chromosomal Rearrangements in Neisseria gonorrhoeae and Neisseria meningitidis.

Chromosomal rearrangements in N. gonorrhoeae and N. meningitidis were studied with the determination of mobile elements and their role in rearrangements. The results of whole-genome sequencing and de novo genome assembly for 50 N. gonorrhoeae isolates collected in Russia were compared with 96 genomes of N. gonorrhoeae and 138 genomes of N. meningitidis from the databases. Rearrangement events with the determination of the coordinates of syntenic blocks were analyzed using the SibeliaZ software v.1.2.5, the minimum number of events that allow one genome to pass into another was calculated using the DCJ-indel model using the UniMoG program v.1.0. Population-level analysis revealed a stronger correlation between changes in the gene order and phylogenetic proximity for N. meningitidis in contrast to N. gonorrhoeae. Mobile elements were identified, including Correa elements; Spencer-Smith elements (in N. gonorrhoeae); Neisserial intergenic mosaic elements; IS elements of IS5, IS30, IS110, IS1595 groups; Nf1-Nf3 prophages; NgoФ1-NgoФ9 prophages; and Mu-like prophages Pnm1, Pnm2, MuMenB (in N. meningitidis). More than 44% of the observed rearrangements most likely occurred with the participation of mobile elements, including prophages. No differences were found between the Russian and global N. gonorrhoeae population both in terms of rearrangement events and in the number of transposable elements in genomes.

Antibiotic Resistance in Neisseria gonorrhoeae: Challenges in Research and Treatment.

Gonococcal infection caused by the Gram-negative bacteria Neisseria gonorrhoeae is one of the most common sexually transmitted infections (STIs) worldwide [...].

In vitro Susceptibility to ß-Lactam Antibiotics and Viability of Neisseria gonorrhoeae Strains Producing Plasmid-Mediated Broad- and Extended-Spectrum ß-Lactamases.

Neisseria gonorrhoeae plasmids can mediate high-level antimicrobial resistance. The emergence of clinical isolates producing plasmid ß-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 ß-lactamases (ESBLs) were obtained in vitro, and their viability and ß-lactam antibiotic susceptibility were studied. Artificial pbla TEM-1 and pbla TEM-20 plasmids were constructed by site-directed mutagenesis from a pbla TEM-135 plasmid isolated from a clinical isolate. Minimum inhibitory concentration (MIC) values for a series of ß-lactam antibiotics, including benzylpenicillin, ampicillin, cefuroxime, ceftriaxone, cefixime, cefotaxime, cefepime, meropenem, imipenem, and doripenem, were determined. The N. gonorrhoeae strain carrying the pbla TEM-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 pbla TEM-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 pbla TEM-20 plasmid reflected several processes: the hydrolysis of ceftriaxone by the TEM-20 ß-lactamase and the growth and gradual death of cells. The demonstrated reduction in the viability of N. gonorrhoeae strains carrying the pbla TEM-20 plasmid probably explains the absence of clinical isolates of ESBL-producing N. gonorrhoeae.

Comparative Whole-Genome Analysis of Neisseria gonorrhoeae Isolates Revealed Changes in the Gonococcal Genetic Island and Specific Genes as a Link to Antimicrobial Resistance.

Comparative whole-genome analysis was performed for Neisseria gonorrhoeae isolates belonging to the Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) types predominant worldwide - 225, 1407, 2400, 2992, and 4186 - and to genogroup 807, the most common genogroup in the Russian Federation. Here, for the first time, the complete genomes of 25 N. gonorrhoeae isolates from genogroup 807 were obtained. For NG-MAST types 225, 1407, 2400, 2992, and 4186, genomes from the Pathogenwatch database were used. The phylogenetic network constructed for 150 genomes showed that the clustering according to NG-MAST type corresponded to the clustering according to genome. Comparisons of genomes of the six sequence types revealed 8-20 genes specific to each sequence type, including the loci for phase variations and genetic components of the gonococcal genetic island (GGI). NG-MAST type 2992 and 4186 isolates either lacked the GGI or carried critical mutations in genes essential for DNA secretion. In all analyzed genogroup 807 isolates, substitution of the essential atlA gene with the eppA gene was found, accompanied by a change in the traG allele, replacement of the ych gene with ych1, and the absence of the exp1 gene, which is likely to result in loss of GGI functionality. For the NG-MAST type 225, 1407 and 2400 isolates, no premature stop codons or reading frameshifts were found in the genes essential for GGI function. A relationship between isolate susceptibility to ciprofloxacin, penicillin, tetracycline and the presence of lesions in GGI genes necessary for DNA secretion was established. The N. gonorrhoeae evolutionary pathways, which allow a particular sequence type to maintain long-term predominance in the population, may include changes in genes responsible for adhesion and virulence, changes in the GGI structure, preservation of genes carrying drug resistance determinants, and changes in genes associated with host adaptation or encoding enzymes of biochemical pathways.

Hydrogel Droplet Microarray for Genotyping Antimicrobial Resistance Determinants in Neisseria gonorrhoeae Isolates.

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.

Prediction of ceftriaxone MIC in Neisseria gonorrhoeae using DNA microarray technology and regression analysis.

BACKGROUND: Decreased susceptibility of Neisseria gonorrhoeae to extended-spectrum cephalosporins is a major concern. Elucidation of the phenotypic and genetic characteristics of such isolates is a priority task. METHODS: We developed a method for predicting the N. gonorrhoeae ceftriaxone susceptibility level (MICcro) by identifying genetic determinants of resistance using low-density hydrogel microarrays and a regression equation. A training dataset, containing 5631 isolates from the Pathogenwatch database and 181 isolates obtained in the Russian Federation during 2018-19, was used to build a regression model. The regression equation was tested on 14 WHO reference strains. Ceftriaxone resistance determinants for the 448 evaluated clinical isolates collected in Russia were identified using microarray analysis, and MICcro values were calculated using the regression equation and compared with those measured by the serial dilution method. RESULTS: The regression equation for calculating MICcro values included 20 chromosomal resistance determinants. The greatest contributions to the increase in MICcro were shown to be PBP2: Ala-501→Pro, Ala-311→Val, Gly-545→Ser substitutions, Asp(345-346) insertion; and PorB: Gly-120→Arg substitution. The substitutions PBP2: Ala-501→Thr/Val, PorB: Gly-120→Asn/Asp/Lys and PBP1: Leu-421→Pro had weaker effects. For 94.4% of the isolates in the evaluation set, the predicted MICcro was within one doubling dilution of the experimentally determined MICcro. No ceftriaxone-resistant isolates were identified in the analysed samples from Russia, and no interpretative errors were detected in the MICcro calculations. CONCLUSIONS: The developed strategy for predicting ceftriaxone MIC can be used for the continuous surveillance of known and emerging resistant N. gonorrhoeae isolates.

Molecular Typing of Neisseria gonorrhoeae Clinical Isolates in Russia, 2018-2019: A Link Between penA Alleles and NG-MAST Types.

This work aimed to study penA gene polymorphisms in clinical isolates of Neisseria gonorrhoeae collected in Russia in 2018-2019 and the contribution of the penA allele type to susceptibility to ß-lactam antibiotics. A total of 182 isolates were analyzed. penA allele types were determined by sequencing, and the minimum inhibitory concentrations (MICs) of benzylpenicillin and ceftriaxone were measured. The influence of genetic factors on MICs was evaluated by regression analysis. All isolates were susceptible to ceftriaxone, and 40.1% of isolates were susceptible to penicillin. Eleven penA allele types were identified. The mosaic type XXXIV penA allele and the Gly120Lys substitution in PorB made the greatest contributions to increasing the ceftriaxone MIC; the presence of the blaTEM plasmid, Gly120Asp, Ala121Gly/Asn substitutions in PorB, and the adenine deletion in the promoter region of the mtrR gene caused an increase in the penicillin MIC. Among 61 NG-MAST types identified, the most frequent were types 228, 807, 9486, 1993, and 6226. A link between penA alleles and Neisseria gonorrhoeae multi-antigen sequence typing (NG-MAST) types was established. Resistance to two groups of ß-lactam antibiotics was associated with non-identical changes in penA alleles. To prevent the emergence of ceftriaxone resistance in Russia, NG-MAST genotyping must be supplemented with penA allele analysis.

Genetic diversity of Neisseria gonorrhoeae multi-antigen sequence types in Russia and Europe.

OBJECTIVES: The goal of this work was to assess the genetic diversity of Neisseria gonorrhoeae isolates in Russia and Europe and to compare the distribution of the N. gonorrhoeae multi-antigen sequencing types (NG-MAST) of Russian isolates with that of isolates from European countries. METHODS: NG-MAST typing was performed for 804 N. gonorrhoeae isolates collected in Russia in 2013-2018. For isolates from European countries, data from the https://pathogen.watch/collection/eurogasp2013 database were used. RESULTS: Among the isolates from Russia, 296 NG-MAST types were found. A maximum likelihood phylogenetic tree was constructed. Phylogenetic analysis revealed seven major genogroups uniting the most frequent Russian sequence types: G807, G1993, G9476, G14942, G1152, G9486, and G12531. CONCLUSIONS: The NG-MAST type distribution in Russia differed from that in European countries. Most of the Russian isolates had sequence types that were not found in Europe. Only 33% of the Russian isolates belonged to genogroups established for European countries, and the widespread European genogroup G1407 was represented by only nine isolates. Analysis of the Russian isolates belonging to phylogenetically close European genogroups indicated similarities in drug resistance, although no epidemically dangerous drug-resistant clones were found among the Russian isolates.

Resistance of Neisseria gonorrhoeae isolates to beta-lactam antibiotics (benzylpenicillin and ceftriaxone) in Russia, 2015-2017.

The goal of this work was to study the phenotypic susceptibility and resistance determinants of N. gonorrhoeae isolates to beta-lactam antimicrobials (benzylpenicillin and ceftriaxone). A total of 522 clinical isolates collected in Russia in 2015-2017 were analysed for susceptibility using the agar dilution method. DNA loci involved in antimicrobial resistance were identified using DNA microarray analysis and sequencing. Resistance to benzylpenicillin remained high, with 7.7% of isolates resistant (MICpen > 1 mg/L) and 47.5% of isolates showing intermediate susceptibility (MICpen = 0.12-1 mg/L). The most frequent resistance determinant (72.4% isolates) was the Asp345 insertion in penA, both as a single mutation and in combination with other mutations, particularly with the substitution Leu421Pro in ponA (39.0%). Mutations affecting the influx and efflux of drugs were also found, including amino acid substitutions in PorB (26.8% isolates) and delA in the promoter region of mtrR (22.8%). The accumulation of mutations in chromosomal genes (penA, pon, porA, and mtrR) led to a stepwise increase in MICpen to values characteristic of intermediate resistance. The presence of blaTEM plasmids was found in 25 isolates (4.8%), resulting in a strong increase in resistance to penicillin (MICpen > 16 mg/L) compared with the chromosomal mutations; 23 plasmids were of the African type with TEM-1 beta-lactamase, and two plasmids were of the Toronto/Rio type with TEM-135 beta-lactamase. Only three isolates were found with reduced susceptibility to ceftriaxone, with MICcef = 0.12-0.25 mg/L. Sequencing of penA did not reveal mutations associated with resistance to third-generation cephalosporins, and the gene structure was non-mosaic. The majority of isolates (21 of 25) carrying the blaTEM plasmid also contained the conjugative plasmid with tetM (resistance to tetracyclines), consistent with previously reported data that the presence of the conjugative plasmid facilitates the transfer of other plasmids associated with antimicrobial resistance.