Gonorrhoeae Clinical Isolates Research Articles

Low Oxygen Concentration Reduces Neisseria gonorrhoeae Susceptibility to Resazurin.

Neisseria gonorrhoeae has developed resistance to every antibiotic currently approved for the treatment of gonorrhea, prompting the development of new therapies. The phenoxazine dye resazurin exhibits robust antimicrobial activity against N. gonorrhoeae in vitro but fails to limit vaginal colonization by N. gonorrhoeae in a mouse model. The lack of in vivo efficacy may be due to oxygen limitation as in vitro susceptibility assays with resazurin are conducted under atmospheric oxygen while a microaerophilic environment is present in the vagina. Here, we utilized broth microdilution assays to determine the susceptibility of N. gonorrhoeae to resazurin under low and atmospheric oxygen conditions. The minimal inhibitory concentration of resazurin for multiple N. gonorrhoeae clinical isolates was significantly higher under low oxygen. This effect was specific to resazurin as N. gonorrhoeae was equally susceptible to other antibiotics under low and atmospheric oxygen conditions. The reduced susceptibility of N. gonorrhoeae to resazurin under low oxygen was largely attributed to reduced oxidative stress, as the addition of antioxidants under atmospheric oxygen mimicked the reduced susceptibility to resazurin observed under low oxygen. Together, these data suggest oxygen concentration is an important factor to consider when evaluating the efficacy of new antibiotics against N. gonorrhoeae in vitro.

Essential oils of Pinus sylvestris, Citrus limon and Origanum vulgare exhibit high bactericidal and anti-biofilm activities against Neisseria gonorrhoeae and Streptococcus suis

Antimicrobial resistance is a worldwide problem that urges novel alternatives to treat infections. In attempts to find novel molecules, we assess the antimicrobial potential of seven essential oils (EO) of different plants (Pinus sylvestris, Citrus limon, Origanum vulgare, Cymbopogon martini, Cinnamomum cassia, Melaleuca alternifolia and Eucalyptus globulus) against two multidrug-resistant bacteria species, i.e. Neisseria gonorrhoeae and Streptococcus suis. EOs of P. sylvestris and C. limon revealed higher bactericidal activity (MIC ≤ 0.5 mg/mL) and capacity to rapidly disperse biofilms of several N. gonorrhoeae clinical isolates than other EOs. Examination of biofilms exposed to both EO by electron microscopy revealed a reduction of bacterial aggregates, high production of extracellular vesicles, and alteration of cell integrity. This activity was dose-dependent and was enhanced in DNase I-treated biofilms. Antibiotic susceptibility studies confirmed that both EOs affected the outer membrane permeability, and analysis of EO- susceptibility of an LPS-deficient mutant suggested that both EO target the LPS bilayer. Further analysis revealed that α- and β-pinene and d-limonene, components of both EO, contribute to such activity. EO of C. martini, C. cassia, and O. vulgare exhibited promising antimicrobial activity (MIC ≤ 0.5 mg/mL) against S. suis, but only EO of O. vulgare exhibited a high biofilm dispersal activity, which was also confirmed by electron microscopy studies. To conclude, the EO of P. sylvestris, C. limon and O. vulgare studied in this work exhibit bactericidal and anti-biofilm activities against gonococcus and streptococcus, respectively.

Neisseria gonorrhoeae diagnostic escape from a gyrA-based test for ciprofloxacin susceptibility and the effect on zoliflodacin resistance: a bacterial genetics and experimental evolution study.

The aetiological bacterial agent of gonorrhoea, Neisseria gonorrhoeae, has become resistant to each of the first-line antibiotics used to treat it, including ciprofloxacin. One diagnostic approach to identify ciprofloxacin-susceptible isolates is to determine codon 91 in the gene encoding the A subunit of DNA gyrase, gyrA, where coding for the wild-type serine (gyrA91S) is associated with ciprofloxacin susceptibility and phenylalanine (gyrA91F) with resistance. The aim of this study was to investigate the possibility of diagnostic escape from gyrA susceptibility testing. We used bacterial genetics to introduce pairwise substitutions in GyrA positions 91 (S or F) and 95 (D, G, or N), which is a second site in GyrA associated with ciprofloxacin resistance, into five clinical isolates of N gonorrhoeae. All five isolates encoded GyrA S91F, an additional substitution in GyrA at position 95, substitutions in ParC that are known to cause an increased minimum inhibitory concentration (MIC) to ciprofloxacin, and GyrB 429D, which is associated with susceptibility to zoliflodacin (a spiropyrimidinetrione-class antibiotic in phase 3 trials for treatment of gonorrhoea). We evolved these isolates to assess for the existence of pathways to ciprofloxacin resistance (MIC ≥1 μg/mL) and measured MICs for ciprofloxacin and zoliflodacin. In parallel, we searched metagenomic data for 11 355 N gonorrhoeae clinical isolates with reported ciprofloxacin MICs that were publicly available from the European Nucleotide Archive for strains that would be identified as susceptible by gyrA codon 91-based assays. Three clinical isolates of N gonorrhoeae with substitutions in GyrA position 95 associated with resistance (G or N) maintained intermediate ciprofloxacin MICs (0·125-0·5 μg/mL), which has been associated with treatment failure, despite reversion of GyrA position 91 from phenylalanine to serine. From an in-silico analysis of the 11 355 genomes from N gonorrhoeae clinical isolates, we identified 30 isolates with gyrA codon 91 encoding a serine and a ciprofloxacin resistance-associated mutation at codon 95. The reported MICs for these isolates varied from 0·023 μg/mL to 0·25 μg/mL, including four with intermediate ciprofloxacin MICs (associated with substantially increased risk of treatment failure). Finally, through experimental evolution, one clinical isolate of N gonorrhoeae bearing GyrA 91S acquired ciprofloxacin resistance through mutations in the gene encoding for the B subunit of DNA gyrase (gyrB) that also conferred reduced susceptibility to zoliflodacin (ie, MIC ≥2 μg/mL). Diagnostic escape from gyrA codon 91 diagnostics could occur through either reversion of the gyrA allele or expansion of circulating lineages. N gonorrhoeae genomic surveillance efforts might benefit from including gyrB, given its potential for contributing to ciprofloxacin and zoliflodacin resistance, and diagnostic strategies that reduce the likelihood of escape, such as the incorporation of multiple target sites, should be investigated. Diagnostics that guide antibiotic therapy can have unintended consequences, including novel resistance determinants and antibiotic cross-resistance. US National Institutes of Health National Institute of Allergy and Infectious Diseases, National Institute of General Medical Sciences, and the Smith Family Foundation.

Biochemical Atypia in Russian Neisseria gonorrhoeae Clinical Isolates Belonging to the G807 NG-MAST Genogroup/ST1594 MLST.

Many current gonococcal clinical isolates in Russia show atypical taxonomically significant biochemical activity, which leads to species misidentification. Molecular typing of such cultures according Neisseria gonorrhoeae multiantigen sequence typing (NG-MAST) and multilocus sequence typing (MLST) protocols assigned them to the G807 NG-MAST GENOGROUP/ST1594 MLST that has been predominant in Russia in recent years. The goal of the study was to analyze the molecular mechanisms of biochemical atypia in N. gonorrhoeae clinical isolates characterized as the members of G807 NG-MAST GENOGROUP/ST1594 MLST. Sixteen isolates of this genogroup were included in the study, eight showed defective amino acid metabolism or loss of D-glucose fermentation. Comparative bioinformatic analysis based on WGS data divided these isolates into two clusters strictly associated with typical or atypical biochemical activity. Cultures with defective amino acid metabolism had a 5-nucleotide insertion in the pip-gene that caused a stop codon and led to synthesis of the non-functional enzyme. Comparison of the sequenced genomes with publicly available N. gonorrhoeae genomes showed the rarity of this insertion. In the global N. gonorrhoeae phylogenetic tree the G807 NG-MAST GENOGROUP/ST1594 MLST forms a distinct branch characterized by 170 SNPs, most of which are non-synonymous. We hypothesized a unique strategy for G807 NG-MAST GENOGROUP/ST1594 MLST clone persistence in the global N. gonorrhoeae population via escape of antimicrobial therapy due to diagnostic misidentification.

Whole-genome sequence analysis of high-level penicillin-resistant strains and antimicrobial susceptibility of Neisseria gonorrhoeae clinical isolates from Thailand.

The increasing rate of antimicrobial-resistant Neisseria gonorrhoeae poses a considerable public health threat due to the difficulty in treating gonococcal infections. This study examined antimicrobial resistance (AMR) to drugs recommended for gonorrhea treatment between 2015 and 2017, and the AMR determinants and genetic compositions of plasmids in 3 gonococcal strains with high-level penicillin resistance. We collected 117 N. gonorrhoeae isolates from patients with gonococcal infections who attended Siriraj Hospital, Bangkok, Thailand, between 2015 and 2017. Minimum inhibitory concentrations (MICs) of penicillin, tetracycline, ciprofloxacin, azithromycin, spectinomycin, cefixime, and ceftriaxone were determined by the agar dilution method. PCR amplification and sequencing of 23S rRNA and mtrR (a negative regulator of MtrCDE efflux pump) were performed. Whole genomes of 3 PPNG strains with high-level penicillin resistance (MIC ≥ 128 μg/ml) were sequenced using Illumina and Nanopore sequencing platforms. The proportions of N. gonorrhoeae isolates with resistance were 84.6% for penicillin, 91.5% for tetracycline, and 96.6% for ciprofloxacin. All isolates were susceptible to spectinomycin, azithromycin, cefixime, and ceftriaxone. An adenine deletion within a 13 bp inverted repeat sequence in the mtrR promoter and an H105Y mutation in the mtrR coding region were found in the N. gonorrhoeae isolate with the highest azithromycin MIC value (1 μg/ml). Three high-level penicillin-resistant isolates contained nonmosaic type II penA and had mutations in penB and the mtrR coding region. All isolates with high-level penicillin resistance carried the conjugative plasmids with or without the Dutch type tetM determinant, the beta-lactamase plasmid (Rio/Toronto), and the cryptic plasmid. The gonococcal population in Thailand showed high susceptibility to ceftriaxone and azithromycin, current dual therapy recommended for gonorrhea treatment. As elevated MIC of azithromycin has been observed in 1 strain of N. gonorrhoeae, expanded and enhanced surveillance of antimicrobial susceptibility and study of genetic resistance determinants are essential to improve treatment guidelines.

High-Resolution Melting Analysis to Detect Antimicrobial Resistance Determinants in South African Neisseria gonorrhoeae Clinical Isolates and Specimens.

Background Antimicrobial resistance is limiting treatment options for Neisseria gonorrhoeae infections. To aid or replace culture and the syndromic management approach, molecular assays are required for antimicrobial susceptibility testing to guide appropriate and rapid treatment. Objective We aimed to detect single-nucleotide polymorphisms and plasmids associated with antimicrobial resistance from N. gonorrhoeae isolates from a clinic population in South Africa, using real-time PCR as a rapid test for AMR detection. Methods N. gonorrhoeae isolates, from female and male patients presenting for care at a sexually transmitted infections clinic in Durban, South Africa, were analysed using phenotypic and genotypic methods for identification and antibiotic susceptibility testing (AST). Real-time PCR and high-resolution melting analysis were used to detect porA pseudogene (species-specific marker) and resistance-associated targets. Whole-genome sequencing was used as the gold standard for the presence of point mutations. Results The real-time porA pseudogene assay identified all N. gonorrhoeae-positive isolates and specimens. Concordance between molecular detection (real-time PCR and HRM) and resistance phenotype was ≥92% for blaTEM (HLR penicillin), rpsJ_V57M (tetracycline), tetM (tetracycline), and gyrA_S91F (ciprofloxacin). Resistance determinants 16SrRNA_C1192U (spectinomycin), mtrR_G45D (azithromycin), and penA_D545S, penA_mosaic (cefixime/ceftriaxone) correlated with the WHO control isolates. Conclusions Eight resistance-associated targets correlated with phenotypic culture results. The porA pseudogene reliably detected N. gonorrhoeae. Larger cohorts are required to validate the utility of these targets as a convenient culture-free diagnostic tool, to guide STI management in a South African population.

1355. Cluster of Multi-drug Resistance Neisseria gonorrhoeae Isolates with Reduce Ceftriaxone Susceptibility, First Report in Thailand

Abstract Background Rapid emergence of global azithromycin resistance, with ceftriaxone reduce susceptibility/resistant strains, threatens the current recommends dual therapy with ceftriaxone and azithromycin for gonorrhea to ensure effective treatment. Thailand is a major risk area for gonorrhea because it as a key destination for sex tourism industry, where antibiotic resistance gonorrhea can spread easily and quickly across the region. Here, we identified a first cluster of Neisseria gonorrhoeae isolates with decreased ceftriaxone susceptibility in Thailand. Methods A 134 N. gonorrhoeae isolates collected from Thai Red Cross Anonymous Clinic, Bangkok, during 2016–2018 were studied. Broth micro-dilution and plate dilution methods to define resistance or decreased susceptibilityaccording to CLSI guidelines. Select isolates were subjected to whole genome sequencing with an Illumina MiSeq V2 instrument and analysis using CLCBio Genomics Workbench v6.5. Molecular docking studies were carried out to understand binding mode analysis and orientation of ceftriaxone in the active site of PBP2. Biofilm formation measured by confocal laser scanning microscopy. Results Among the N. gonorrhoeae clinical isolates, two isolates had significant reduced susceptibility to ceftriaxone (MICs of 0.125 mg/L), which were isolated from urethral swab in male heterosexual patients. Both were multidrug resistance and strong biofilm producers with ceftriaxone tolerance (MBEC > 128 mg/L). One isolate was resistant to azithromycin (MIC of 1 mg/L), and other one remained susceptible (MIC of 0.5 mg/L). Reduced susceptibility to ceftriaxone associated with alterations in PBP2, PBP1, PorB, MtrR, and mtrR promoter region with one belonged to ST7235 and second one had new allele number of tbpB with new sequence type (ST). Ceftriaxone weakly occupy the active site of mosaic XXXIV penicillin-binding protein 2 (PBP2) variant in both. Molecular epidemiology results reviled that both isolates display similarities with isolates from UK, USA, and Netherlands. Conclusion This first cluster of genetically related gonococcal isolates with decreased ceftriaxone susceptibility may bring threat of treatment failure in Thailand. Also highlighted the importance of maintaining surveillance for antimicrobial resistance. Disclosures All Authors: No reported disclosures

Is gentamicin a viable therapeutic option for treating resistant Neisseria gonorrhoeae in New South Wales?

The key issues with Neisseria gonorrhoeae infections, in Australia and elsewhere, are coincident increases in disease rates and in antimicrobial resistance (AMR), although these factors have not been shown to be correlated. Despite advances in diagnosis, control of this disease remains elusive, and incidence in Australia continues to increase. Of the Australian jurisdictions, New South Wales (NSW) has the highest N. gonorrhoeae notifications, and over the five-year period 2015-2019, notifications in NSW have increased above the national average (by 116% versus 85%, respectively). Gonococcal disease control is reliant on effective antibiotic regimens. However, escalating AMR in N. gonorrhoeae is a global health priority, as the collateral injury of untreated infections has substantive impacts on sexual and newborn health. Currently, our first-line therapy for gonorrhoea is also our last line, with no ideal alternative identified. Despite some limitations, gentamicin is licensed and readily available in Australia, and is proposed for treatment of resistant N. gonorrhoeae in national guidelines; however, supportive published microbiological data are lacking. Analysis of gonococcal resistance patterns within Australia for the period 1991-2019, including 35,000 clinical isolates from NSW, illustrates the establishment and spread of population-level resistance to all contemporaneous therapies. An analysis of gentamicin susceptibility on 2,768 N. gonorrhoeae clinical isolates from NSW, for the period 2015-2020, demonstrates that the median minimum inhibitory concentration (MIC) for gentamicin in NSW has remained low, at 4.0 mg/L, and resistance was not detected in any isolate. There has been no demonstration of MIC drift over time (p = 0.91, Kruskal-Wallis test), nor differences in MIC distributions according to patients' sex or site of specimen collection. This is the first large-scale evaluation of gentamicin susceptibility in N. gonorrhoeae in Australia. No gentamicin resistance was detected in clinical isolates, 2015-2020, hence this is likely to be an available treatment option for resistant gonococcal infections in NSW.

Trends in antimicrobial resistance in Neisseria gonorrhoeae in Hanoi, Vietnam, 2017\u20132019

BackgroundAntimicrobial resistance (AMR) in Neisseria gonorrhoeae is an emerging global health threat. Surveillance of AMR in N. gonorrhoeae in the Western Pacific Region is important, as resistant strains have typically emerged from this region. There are sparse data regarding antibiotic susceptibility of N. gonorrhoeae from Vietnam. This study aimed to provide updated data on antibiotic susceptibilities in N. gonorrhoeae isolates from Hanoi, Vietnam.MethodsFrom 2017 to 2019, 409 N. gonorrhoeae clinical isolates were collected at the National Hospital for Venereology and Dermatology in Hanoi, Vietnam. Antibiotic susceptibility testing was performed by disk diffusion method according to the Clinical and Laboratory Standards Institute (CLSI) protocol. The zone diameters of inhibition were recorded and interpreted according to standard CLSI criteria, except for azithromycin, due to the absence of CLSI interpretation. Categorical variables were analyzed by Chi-square and Fisher’s exact tests. Linear regression was used to evaluate zones of inhibition by year.ResultsAmong the 409 isolates, no isolates were susceptible to penicillin, 98.3% were resistant to ciprofloxacin, and all isolates were susceptible to spectinomycin. There were 122/407 (30.0%) isolates resistant to azithromycin and there was an association between resistance and year (p < 0.01), ranging from 15.3% of isolates in 2017 to 46.7% of the isolates in 2018. Resistance to cefixime was found in 13/406 (3.2%) of isolates and there was no association by year (p = 0.30). Resistance to ceftriaxone occurred in 3/408 (0.7%) of isolates. Linear regression indicated the zone of inhibition diameters decreased by 0.83 mm each year for ceftriaxone (95% CI: − 1.3, − 0.4; p < 0.01) and decreased by 0.83 mm each year (95% CI: − 1.33, − 0.33; p < 0.01) for azithromycin; the association was not significant for cefixime (p = 0.07).ConclusionsWe found decreasing susceptibility of N. gonorrhoeae to ceftriaxone and azithromycin, as well as a high prevalence of resistance to azithromycin, among isolates in Hanoi, Vietnam from 2017 to 2019. The trends of decreasing susceptibility to first-line treatments are concerning and highlight the urgency of addressing antimicrobial resistance in N. gonorrhoeae. Expanded surveillance efforts within the Western Pacific Region are critical to monitoring trends and informing treatment guidelines.

Multiplex High-Resolution Melting Assay for Simultaneous Identification of Molecular Markers Associated with Extended-Spectrum Cephalosporins and Azithromycin Resistance in Neisseria gonorrhoeae

Antimicrobial resistance in Neisseria gonorrhoeae persists as a major public health concern globally. We developed and evaluated a multiplex assay that relied on high-resolution melting (HRM) technology as a rapid, simple, and cost-effective method for simultaneously detecting and identifying different molecular markers associated with extended-spectrum cephalosporins (ESCs) and azithromycin (AZM) resistance in N. gonorrhoeae. Forty-eight well-characterized N. gonorrhoeae clinical isolates were selected for initial assay establishment. The multiplex HRM assays were able to accurately identify different nucleotide variations of the antimicrobial resistance determinants related to ESCs and AZM resistance. Specificity and cross-reactivity were assessed by testing 15 nongonococcal strains. Then, the assay was validated on 218 archived DNA specimens that had been sequenced using whole-genome sequencing technology. Compared with whole-genome sequencing, these assays had a sensitivity of 98.6%, with a specificity of 99.2%. For further validation of the assay's performance, a total of 338 samples (156 clinical isolates and 182 clinical specimens) were screened using the multiplex HRM assay. The results showed good concordance with the results of PCR sequencing. Given its rapidity (within 90 minutes), ease of performing, and low cost (<$1.00 per sample), this method may be applied to large-scale epidemiologic programs for increasing surveillance of ESCs and AZM resistance in N. gonorrhoeae.

Australian Gonococcal Surveillance Programme Annual Report, 2019.

The Australian Gonococcal Surveillance Programme (AGSP) has continuously monitored antimicrobial resistance in clinical isolates of Neisseria gonorrhoeae since 1981. In 2019, a total of 9,668 clinical isolates of gonococci from the public and private sector in all jurisdictions were tested for in vitro antimicrobial susceptibility by standardised methods. The current treatment recommendation for gonorrhoea, for the majority of Australia, continues to be dual therapy with ceftriaxone and azithromycin. Decreased susceptibility (DS) to ceftriaxone (minimum inhibitory concentration [MIC] value ≥ 0.06 mg/L) was found nationally in 1.3% of isolates. Five N. gonorrhoeae clinical isolates were ceftriaxone-resistant (MIC value ≥ 0.25 mg/L), and therefore also resistant to penicillin; all were resistant to ciprofloxacin but susceptible to azithromycin. These isolates were reported from Victoria (3), non-remote Western Australia (1) and New South Wales (1). Resistance to azithromycin (MIC value ≥ 1.0 mg/L) was found nationally in 4.6% of N. gonorrhoeae isolates, continuing a downward trend observed and reported since 2017. Isolates with high-level resistance to azithromycin (MIC value ≥ 256 mg/L) continue to be reported sporadically in Australia, with eight detected in 2019: two each from New South Wales, Queensland, and Victoria, and one each from Tasmania and non-remote Western Australia. In 2019, in Australia, 2,136 gonococcal isolates (22.1%) were penicillin resistant; however, there remains considerable variation by jurisdiction, and in some remote settings there is little resistance and this drug is recommended empiric therapy. In 2019, in the remote Northern Territory, no penicillin resistance was reported, however in remote Western Australia six out of 85 isolates (7.1%) were penicillin resistant. There was no ciprofloxacin resistance reported from isolates tested from remote regions of the Northern Territory, and ciprofloxacin resistance rates remain comparatively low (7/85; 8.2%) in remote Western Australia.

Comparative phylogenetic analysis of Neisseria gonorrhoeae clinical isolates in Russia, European Union, and Japan

Surveillance of multidrug-resistant infections is a priority task for contemporary epidemiology. The aim of this study was to genotype modern clinical isolates of N. gonorrhoeae using the NG-MAST technique (Neisseria gonorrhoeae multi-antigen sequence typing) and to compare the phylogeny of the gonococcal pathogens coming from Russia, European Union and Japan. We studied a total of 822 isolates collected in Russia from 2013 through 2018. We also used NG-MAST data from the following databases: PathogenWatch (European Union, 1,071 isolates) and PubMLST (Japan, 206 isolates). Russian isolates represented 301 different NG-MAST types. The most common were types 807, 228, 1993, 5714, and 9476 (8.3%, 3.3%, 3.2%, 3.2%, and 2.7%, respectively). There were only 3 isolates (0.4%) from Russia that represented the epidemiologically significant sequence type 1407 prevailing in many countries and characterized by multiple determinants of antimicrobial resistance. A phylogenetic tree for the NG-MAST types found in Russia and European countries was constructed. The cluster analysis of the proportion of isolates belonging to unique sequence types and the country population size allowed us to identify 2 clusters (significance level — 0.01): the first cluster included Russia and Japan, the second, European countries. A distribution pattern was identified for unique sequence types: the greater is the population size, the higher is their proportion. The phylogenetic analysis demonstrated a genetic distance between the most common Russian, European and Japanese sequence types, suggesting that the Russian population of N. gonorrhoeae has been evolving relatively locally.

Comparative phylogenetic analysis of Neisseria gonorrhoeae clinical isolates in Russia, European Union, and Japan

Мониторинг возбудителей инфекционных заболеваний, развивающих множественную устойчивость к антимикробным препаратам, является важной и актуальной задачей. Целью работы являлось генотипирование современных российских клинических изолятов N. gonorrhoeae по протоколу NG-MAST (Neisseria gonorrhoeae multi-antigen sequence types) и сравнительный филогенетический анализ возбудителя гонококковой инфекции в России, странах ЕС и Японии. Всего исследовано 822 изолята, собранных в РФ в период с 2013 по 2018 гг. Использовали также данные NG-MAST-типирования из баз данных «PathogenWatch» (страны ЕС, 1071 образец) и «PubMLST» (Япония, 206 образцов). Изоляты РФ принадлежали к 301 различному NG-MAST типу, наиболее распространенными являлись 807, 228, 1993, 5714, 9476 (8,3%; 3,3%; 3,2%; 3,2%; 2,7% соответственно). В РФ обнаружено только 3 изолята (0,4%) пандемически значимого NG-MAST 1407, характеризующегося множественными детерминантами резистентности к антимикробным препаратам и доминирующего во многих странах мира. Построено филогенетическое древо NG-MAST типов, найденных в России и в европейских странах. Кластерный анализ данных по доле изолятов с уникальными сиквенс-типами и численности населения показал существование двух кластеров (уровень значимости 0,01): первый составили Россия и Япония, второй — европейские страны. Показана тенденция в распределении уникальных сиквенс-типов — их доля тем выше, чем больше численность населения страны. Филогенетический анализ показал генетическую отдаленность наиболее распространенных российских, европейских и японских сиквенс-типов, что указывает на локальный характер формирования и эволюции российской популяции N. gonorrhoeae.

Comparative Proteomics of Extended-Spectrum Cephalosporin-Resistant Neisseria gonorrhoeae Isolates Demonstrates Altered Protein Synthesis, Metabolism, Substance Transport, and Membrane Permeability.

Neisseria gonorrhoeae isolates exhibit resistance to extended-spectrum cephalosporins (ESCs), the last remaining option for first-line empirical monotherapy. Here, we investigated the proteomic profiles of N. gonorrhoeae clinical isolates with ESC-resistance to support exploration of the antimicrobial resistance mechanisms for N. gonorrhoeae. We used comparative iTRAQ quantitative proteomics to investigate differential protein expression of three ESC-resistant N. gonorrhoeae clinical isolates using N. gonorrhoeae ATCC49226 as a reference strain. The expression of 40 proteins was downregulated and expression of 56 proteins was upregulated in all three ESC-resistant N. gonorrhoeae isolates. Proteins with predicted function of translation, ribosomal structure and biogenesis, as well as components of the Type IV secretory systems, were significantly upregulated. Two differentially expressed proteins of ABC transporters were also reported by other teams in proteomics studies of N. gonorrhoeae isolates under antimicrobial stress conditions. Differentially expressed proteins are involved in energy production and metabolism of carbohydrates and amino acids. Our results indicated that amino acid and carbohydrate metabolism, cell membrane structure, interbacterial DNA transfer, and ribosome components might be involved in mediating ESC-resistance in N. gonorrhoeae. These findings facilitate a better understanding of the mechanisms of ESC-resistance in N. gonorrhoeae and provide useful information for identifying novel targets in the development of antimicrobials against N. gonorrhoeae.

Biological microchip for assessing tetracycline-resistance in &lt;i&gt;Neisseria gonorrhoeae&lt;/i&gt; clinical isolates in Russian Federation

A total of 399 Neisseria gonorrhoeae clinical isolates collected in different regions of the Russian Federation in 20152017 were analyzed for tetracycline susceptibility and genetic markers of resistance. Drug susceptibility testing was performed by serial dilution method in agar and minimum inhibitory concentration (MIC) was measured according to the Russian “Guidelines for microbial susceptibility testing for antibacterial agents No. 4.2.1890-04”. Tetracycline resistance determinants were studied by using hydrogel microarray with immobilized oligonucleotide probes able to identify a series of chromosomal mutations and detect plasmid tetM gene. Different resistance determinants were found in 193 isolates (48.4%). Mutation in codon 57 in the rpsJ gene (41.2%) was most common that decreases tetracycline affinity to ribosome 30S subunit, mainly due to Val57Met substitution both as a point mutation as well as in combination with others. Mutations in the rpsJ gene were found in strains with the intermediate tetracycline susceptibility. Mutations in the porB gene (lower tetracycline influx) held the se cond place in prevalence pattern (23.1%); the Gly120Lys substitution usually led to emergence of tetracycline resistance either as a point mutation or in combination with other substitutions. Substitutions of Gly120 for other residues (Asp, Asn, and Thr) and Ala121 for Asp, Asn, and Gly had much less effect on resistance level. The –35 delA deletion in the promoter region of mtrR gene (increased expression of MtrC-MtrD-MtrE efflux pump) was observed in 11.3% strains. The tetM gene was found in 27 strains including 17 American and 10 Dutch type tetM determinants. Evolutionary tree was constructed for the tetM genes with the estimation of their homology with similar genes in genera Streptococcus, Enterococcus and Mycoplasma. Mutations in chromosomal genes resulted in increase of tetracycline MIC up to 2–4 mg/L; 4 mg/L MIC was observed in case of simultaneous presence of several mutations. Strains bearing tetM gene-containing plasmid showed extremely high resistance level: MIC ≥ 8 mg/L (64 mg/L for the two samples). Thus, long-lasting withdrawal of tetracycline use for treatment of gonococcal infections in Russia (since 2003) resulted in decreased percentage of resistant strains (including strains with intermediate susceptibility) from 75% down to 45.4%. However, currently tetracycline resistance in Russia remains elevated that is explained by the presence of different resistance determinants in the half of isolates under study.

Trends in Antimicrobial Resistance Patterns in Neisseria Gonorrhoeae in Australia and New Zealand: A Meta-analysis and Systematic Review.

(1) Background: The widespread development of resistance among Neisseria gonorrhoeae (NG) clinical isolates has been reported by surveillance systems around the world. This meta-analysis estimated the changes in susceptibility patterns among antibiotics under surveillance in Australia and New Zealand. (2) Methods: Articles published in English from 1980–2018, from Australia or New Zealand, that met the selection criteria were included. The meta-analysis was carried out using the R statistical software. (3) Results: In Australia, there has been decreasing susceptibility of gonococcal isolates to selected antimicrobials over time. Azithromycin (Odds Ratio (OR): 0.73; 95% Confidence Interval (CI) 0.64–0.82) and ceftriaxone (OR: 0.69; 95% CI 0.59–0.80) showed decreasing levels of susceptibility each year. Western Australia (OR: 0.76; 95% CI 0.60–0.96) and Victoria (OR: 0.74; 95% CI 0.60–0.90) also had decreasing levels of susceptibility to ceftriaxone over time compared with other states and territories. (4) Conclusions: The results highlight the need for the development of new approaches for managing cases of gonorrhoea. Improved antimicrobial stewardship, enhanced surveillance and contact tracing are needed to identify and respond to changes in antibiotic resistance in a timely manner. Increasing awareness and public health follow-up of cases can help to interrupt the cycle of infection and limit transmission.

Antimicrobial Resistance And Molecular Characteristics Among Neisseria gonorrhoeae Clinical Isolates In A Chinese Tertiary Hospital.

PurposeThe resistance of N. gonorrhoeae to antimicrobial agents has been increasing year by year due to the overuse of antibiotics. The primary aims of the present study were to investigate the molecular characteristics of the clinical isolates of Neisseria gonorrhoeae and the resistance to azithromycin in a Chinese tertiary hospital.MethodsFrom January 2014 to May 2017, a total of 55 clinical isolates of N. gonorrhoeae were collected. Genes associated with azithromycin resistance (AZM-R), including mutations in 23S rRNA alleles, the mtrR promoter and coding regions, and rplD and rplV were evaluated by PCR and DNA sequencing. All clinical isolates were subjected to N. gonorrhoeae multiantigen sequence typing (NG-MAST), while the AZM-R isolates were further characterized by multilocus sequence typing (MLST).ResultsThe AZM-R rate in this study was 23.64% (13/55), and a single (A)-nucleotide deletion mutation in the mtrR promoter region, a G45D mutation in the mtrR coding region, a point mutation in rplD, and an A2047G mutation in 23S rRNA alleles were detected in 13, 4, 3 and 4 isolates, respectively; no mutations were found in rplV. There was no significant difference in the mtrR coding region mutation rate between the azithromycin-sensitive and AZM-R groups (P > 0.05); however, there was a significant difference in the mutation rate of the mtrR promoter region (P < 0.05). Among the 55 isolates studied, 43 distinct NG-MAST were determined, while the AZM-R isolates were allocated into 10 distinct MLST/NG-MAST combinations. All three isolates with high-level AZM-R belonged to the sequence types (STs) NG-MAST ST1866 and MLST ST10899.ConclusionN. gonorrhoeae clinical isolates from Wenzhou, eastern China, showed considerable genetic diversity. Measures should be implemented to monitor the spread of the NG-MAST ST1866 and MLST ST10899 N. gonorrhoeae clones, which exhibit high-level AZM-R in eastern China.

Evaluation of Susceptibilities to Carbapenems and Faropenem Against Cephalosporin-Resistant Neisseria gonorrhoeae Clinical Isolates with penA Mosaic Alleles.

Neisseria gonorrhoeae is a principal pathogen for sexually transmitted infections, especially for male urethritis. Currently, the prevalence of multidrug resistance is increasing. Carbapenems are broad-spectrum antimicrobials that are widely used in the clinical setting, especially for multidrug-resistant Gram-negative bacteria. However, susceptibility to carbapenems has not been well evaluated for cephalosporin-resistant N. gonorrhoeae isolates. In this study, we determined the susceptibility to a series of carbapenems (meropenem, imipenem, doripenem, and biapenem) and faropenem against cephalosporin-resistant (resistant to cefixime, but susceptible to ceftriaxone) and cephalosporin-susceptible N. gonorrhoeae clinical isolates. The gene mutations associated with β-lactam resistance were evaluated. All cephalosporin-resistant N. gonorrhoeae isolates possessed mosaic mutation alleles in penA (NG-STAR penA-10.001, 27.001, or 108.001). They exhibited a low minimum inhibitory concentration (MIC) (≤0.125 mg/L) for meropenem and markedly high MICs (0.5-2 mg/L) for other carbapenems and faropenem. The strongest association was observed between the mosaic alleles in penA and decreased susceptibility to carbapenems and faropenem compared with mutations in mtrR, porB, and ponA. These results suggest that meropenem may serve as an alternative therapeutic agent for cephalosporin-resistant N. gonorrhoeae with a mosaic allele in penA, whereas other carbapenems and faropenem may be ineffective.

Temporal evolution of antimicrobial resistance among Neisseria gonorrhoeae clinical isolates in the most populated South American Metropolitan Region.

A total of 124 Neisseria gonorrhoeae isolates recovered during a 12-year period (2003-2015) from outpatients assisted at Centro de Referência e Treinamento DST/AIDS-CRT of São Paulo city, Brazil, were analysed. The following resistance rates were observed: penicillin-59.6%, ciprofloxacin-15.3%, and azithromycin-6.7%. Although reduced susceptibility to these drugs was observed since 2003, no ceftriaxone-resistant isolates were detected. Ciprofloxacin- and azithromycin non-susceptible isolates were grouped in 11 clusters. Mutations were detected in GyrA and ParC of isolates 124 and 260, and a C2611T substitution on 23S rRNA alleles was also observed in isolate 260. Both isolates belonged to ST1901/ST6210 (MSLT/NG-MAST schemes).

Complete ciprofloxacin resistance in gonococcal isolates in an urban Ugandan clinic: findings from a cross-sectional study.

Antimicrobial resistance (AMR) to gonorrhoea is a threat to global health security. There have been concerns expressed that countries with high rates of disease have poor surveillance. The objectives of the study were to determine the AMR patterns of Neisseria gonorrhoeae clinical isolates to antimicrobial agents in patients with HIV or high risk of HIV acquisition, to compare the concordance of disk diffusion and agar dilution as methods for determining AMR to N. gonorrhoeae, and to describe methodological challenges to carrying out AMR testing. The study was conducted at an HIV outpatient service for at-risk populations and an outreach clinic for commercial sex workers in Kampala. Patients were offered a sexually transmitted infection screen using a polymerase chain reaction (PCR)-based assay. Samples positive for gonorrhoea were cultured. Antimicrobial susceptibility testing was performed using disk diffusion and isolates were sent to a reference laboratory for agar dilution direct susceptibility testing. Five hundred and seventy-five patients were screened. There were 33 (5.7%) patients with gonorrhoea detected by PCR. Of the 16 viable N. gonorrhoeae isolates, 100% were resistant to ciprofloxacin and tetracycline by disk diffusion and 31% exhibited reduced susceptibility to ceftriaxone and cefixime. By agar dilution, 100% of isolates were resistant to ciprofloxacin and all isolates were susceptible to ceftriaxone and cefixime. There was concordance between disk diffusion and agar dilution for ciprofloxacin and tetracycline resistance and a significant discordance for third-generation cephalosporins. More than half the women with gonorrhoea were asymptomatic and represent a potential reservoir for ongoing transmission. AMR testing of N. gonorrhoeae isolates is needed to ensure optimal treatment and prevention of antibiotic resistance progression.