Genome-based epidemiology and antimicrobial resistance of Neisseria gonorrhoeae in Spain: A prospective multicentre study.

BACKGROUND: Whole-genome sequencing (WGS) of Neisseria gonorrhoeae isolates combined with epidemiological and phenotypic data provides better understanding of population dynamics. AIM: The objective of this study was to investigate the molecular epidemiology of N. gonorrhoeae isolates from three centres in Spain and determine associations of antimicrobial resistance. METHODS: Genetic characterization was performed in 170 N. gonorrhoeae isolates. WGS was carried out with the HiSeq platform (Illumina). Genome assemblies were submitted to the PubMLST Neisseria database website to determine NG-MAST, MLST and NG-STAR. Antimicrobial resistance genes and point mutations were identified with PubMLST. Phylogenomic comparison was based on whole-genome single nucleotide polymorphism analysis. RESULTS: Twenty-six MLST, 49 NG-MAST and 41 NG-STAR sequence types were detected, the most prevalent being MLST-ST9363 (27.1%), NG-MAST ST569 (12.4%) and NG-STAR ST193 (14.7%). Phylogenetic analysis identified 13 clusters comprising 69% of the isolates, with two of note: one involved cefixime-resistant isolates from Barcelona presenting a mosaic penA X and belonging to MLST-ST7363 and the other involved azithromycin-resistant isolates from Mallorca that possessed the C2611T mutation in the four 23S rRNA alleles belonging to MLST-ST1901. CONCLUSION: The population of N. gonorrhoeae is quite heterogeneous in Spain. Our results agree with previous data published in Europe, albeit with some differences in distribution between regions. This study describes the circulation of two gonococcal populations with a specific resistance profile and sequence type in a specific geographic area. WGS is an effective tool for epidemiological surveillance of gonococcal infection and detection of resistance genes.

Emergence and dissemination of three mild outbreaks of Neisseria gonorrhoeae with high-level resistance to azithromycin in Barcelona, 2016-18.

BACKGROUND: Neisseria gonorrhoeae (NG) isolates with high-level azithromycin resistance (HL-AziR) have emerged worldwide in recent decades, threatening the sustainability of current dual-antimicrobial therapy. OBJECTIVES: This study aimed to characterize the first 16 NG isolates with HL-AziR in Barcelona between 2016 and 2018. METHODS: WGS was used to identify the mechanisms of antimicrobial resistance, to establish the MLST ST, NG multiantigen sequence typing (NG-MAST) ST and NG sequence typing for antimicrobial resistance (NG-STAR) ST and to identify the clonal relatedness of the isolates with other closely related NG previously described in other countries based on a whole-genome SNP analysis approach. The sociodemographic characteristics of the patients included in the study were collected by comprehensive review of their medical records. RESULTS: Twelve out of 16 HL-AziR isolates belonged to the MLST ST7823/NG-MAST ST5309 genotype and 4 to MLST ST9363/NG-MAST ST3935. All presented the A2059G mutation in all four alleles of the 23S rRNA gene. MLST ST7823/NG-MAST ST5309 isolates were only identified in men who have sex with women and MLST ST9363/NG-MAST ST3935 were found in MSM. Phylogenomic analysis revealed the presence of three transmission clusters of three different NG strains independently associated with sexual behaviour. CONCLUSIONS: Our findings support the first appearance of three mild outbreaks of NG with HL-AziR in Spain. These results highlight the continuous capacity of NG to develop antimicrobial resistance and spread among sexual networks. The enhanced resolution of WGS provides valuable information for outbreak investigation, complementing the implementation of public health measures focused on the prevention and dissemination of MDR NG.

Differences in Neisseria gonorrhoeae population structure and antimicrobial resistance pattern between men who have sex with men and heterosexuals.

This study compared the antimicrobial susceptibility and genotypes of strains of Neisseria gonorrhoeae isolated from men who have sex with men (MSM) and from heterosexuals. One hundred and eleven strains were characterized from 107 patients, comprising 57 strains from 54 heterosexuals and 54 strains from 53 MSM. Antimicrobial resistance rates were higher in strains from heterosexual patients, with resistance to cefixime (P = 0·0159) and ciprofloxacin (P = 0·002) being significantly higher. Typing by N. gonorrhoeae multi-antigen sequence typing (NG-MAST) showed that the most prevalent sequence types (ST) and genogroups (G) respectively were ST2400, ST2992, and ST5793, and G1407, G2992, and G2400. A statistically significant association was observed for MSM and genogroups G2400 (P = 0·0005) and G2992 (P = 0·0488), and G1407 with heterosexuals (P = 0·0002). We conclude that in our region distinct populations of gonococci are circulating among subjects with different sexual practices, with their corresponding transmission patterns. Furthermore, the high prevalence of genotype G2400 in MSM, has not to our knowledge been previously described.

Neisseria gonorrhoeae antimicrobial susceptibility in Barcelona: penA, ponA, mtrR, and porB mutations and NG-MAST sequence types associated with decreased susceptibility to cephalosporins.

The aims of this study were to determine the antimicrobial susceptibility of Neisseria gonorrhoeae (NG) in our area, to analyze the molecular mechanisms involved in cephalosporins resistance, and to undertake molecular typing of our NG strains. Antimicrobial susceptibility was determined using the Etest. The genes penA, mtrR, penB, and ponA were studied. Molecular typing was performed by N. gonorrhoeae multiantigen sequence typing. Of 329 strains analyzed in 2013, none showed high-level cephalosporin resistance, but 8.2 % had resistance to cefixime [minimum inhibitory concentration (MIC) > 0.125 µg/mL] and 0.6 % to ceftriaxone (MIC > 0.125 µg/mL). Azithromycin resistance was documented in 4.3 % and ciprofloxacin resistance in 49.2 %. Among 48 strains with an MIC ≥ 0.125 µg/mL to cefixime, 58.3 % showed the penA mosaic pattern XXXIV, 98 % a Leu → Pro substitution at position 421 of the ponA gene, 100 % amino acid changes at positions 101 and 102 of the PorB1b porin, and 87.5 % of strains an adenine deletion in the promoter region of the MtrC-D-E efflux pump. A significant difference between strains with and without decreased cephalosporin susceptibility (MIC ≥ 0.125 µg/mL) was observed for these four genes. Of the 48 strains with an MIC ≥ 0.125 µg/mL to cefixime, 43.8 % belonged to the genogroup G1407 and 27.1 % belonged to the genogroup G2400. A significant association of G1407 with decreased susceptibility (MIC ≥ 0.125 µg/mL) and G2992 with susceptibility was found, and also between G1407 and mosaic pattern XXXIV and between G2400 and A501T substitution in penA. The NG resistance rate in our area is higher than the median of Europe. We have detected the emergence of G2400, which may be a source of antimicrobial resistance.

Comparison between conventional culture and NAATs for the microbiological diagnosis in gonococcal infection.

Gonorrhea is a public health problem. Fast diagnostics is necessary. The aim of this study was to compare the diagnostic yield of culture with that of polymerase chain reaction (PCR) in gonococcal infection. This is a study comparing the results of Neisseria gonorrhoeae detection by culture versus PCR from July to December 2012. Molecular diagnosis was performed by real-time PCR using the Versant CT/GC DNA 1.0 assay. In the 768 specimens, 96.9% the results were concordant. In 3.1%, the results were discordant, being PCR-positive and culture-negative in 21 cases and PCR-negative and culture-positive in 3. The sensitivity, specificity, positive predictive value, and negative predictive value for culture were 86.2%, 99.8%, 99.2%, and 96.7%, and for PCR, 98.7%, 100%, 100% and 99.7%, respectively. In laboratories where antimicrobial susceptibility is monitored, an effective approach would be to perform culture in addition to PCR in symptomatic patients.