Genomic evolution of Neisseria gonorrhoeae since the preantibiotic era (1928-2013): antimicrobial use/misuse selects for resistance and drives evolution

Abstract

Background: Multidrug-resistant Neisseria gonorrhoeae strains are prevalent, threatening gonorrhoea treatment globally, and understanding of emergence, evolution, and spread of antimicrobial resistance (AMR) in gonococci remains limited. We describe the genomic evolution of gonococci and their AMR, related to the introduction of antimicrobial therapies, examining isolates from 1928 (preantibiotic era) to 2013 in Denmark. This is, to our knowledge, the oldest gonococcal collection globally.

Methods: Lyophilised isolates were revived and examined using Etest (18 antimicrobials) and whole-genome sequencing (WGS). Quality-assured genome sequences were obtained for 191 viable and 40 non-viable isolates and analysed with multiple phylogenomic approaches.

Results: Gonococcal AMR, including an accumulation of multiple AMR determinants, started to emerge particularly in the 1950s-1970s. By the twenty-first century, resistance to most antimicrobials was common. Despite that some AMR determinants affect many physiological functions and fitness, AMR determinants were mainly selected by the use/misuse of gonorrhoea therapeutic antimicrobials. Most AMR developed in strains belonging to one multidrug-resistant (MDR) clade with close to three times higher genomic mutation rate. Modern N. gonorrhoeae was inferred to have emerged in the late-1500s and its genome became increasingly conserved over time.

Conclusions: WGS of gonococci from 1928 to 2013 showed that no AMR determinants, except penB, were in detectable frequency before the introduction of gonorrhoea therapeutic antimicrobials. The modern gonococcus is substantially younger than previously hypothesized and has been evolving into a more clonal species, driven by the use/misuse of antimicrobials. The MDR gonococcal clade should be further investigated for early detection of strains with predispositions to develop and maintain MDR and for initiation of public health interventions.

Keywords: Antimicrobial resistance; Evolution; Genomic epidemiology; Neisseria gonorrhoeae; Temporal analysis; Whole-genome sequencing.

Fig. 1

Treatment guidelines and antimicrobial resistance development in Denmark since the 1920s. a Coloured arrows indicate the time period the different antimicrobials were recommended and used for treatment of gonorrhoea in Denmark. b Mean MIC of antimicrobials previously and currently used for treatment of gonorrhoea (2–4 antimicrobials per box) and first identification of antimicrobial resistance determinants for the specific antimicrobials in the examined isolates from Denmark (black arrow)

Fig. 2

Core single nucleotide polymorphisms (SNPs) in 232 Neisseria gonorrhoeae genomes was initially determined using Gubbins to exclude the recombinant regions and a maximum likelihood tree was constructed using 28,196 SNPs. The columns next to the tree describe the antimicrobial resistance determinants in each isolate. The reference WHO O genome [19] is not annotated and is shown in black. More than 100 different N. gonorrhoeae multi-antigen sequence typing (NG-MAST), multilocus sequence typing (MLST), and N. gonorrhoeae sequence typing antimicrobial resistance (NG-STAR) sequence types were found. The pie charts below the tree reflect the proportion of each antimicrobial resistance determinant per era

Fig. 3

Core single nucleotide polymorphisms (SNPs) in 192 Neisseria gonorrhoeae genomes was initially obtained using Gubbins to exclude the recombinant regions and maximum likelihood tree was reconstructed using 26,744 SNPs. The columns next to the tree describe the antimicrobial susceptibility of each isolate. The antimicrobial susceptible clade is coloured in green and the multidrug-resistant clade in red. The pie charts below the tree reflect the proportion of isolates resistant to each antimicrobial per era. Reference strain WHO O [19] is included in the tree

Fig. 4

Phylogenomic tree with the size of the core genome for the three separate eras. a Phylogenomic tree including the size of the core genome for the preantibiotic, golden, and postmodern era consisting of 1016, 1401, and 1542 genes, respectively, using the Roary pan genome pipeline. b The proportion of the genes for each era is divided into core, soft-core, shell, and cloud genes, which are changing over time and the core genome length is increasing from 0.91 Mbp to 1.4 Mbp. Core = ≥99–100% of isolates share genes. Soft-core = ≥95- < 99% of isolates share genes. Shell = ≥15- < 95% of isolates share genes. Cloud = 0- < 15% of isolates share genes

Fig. 5

Maximum likelihood tree including internal node labels for viable Neisseria gonorrhoeae isolates (n = 191) from 1928 to 2013 dated using least-squares dating (LSD) software v0.3 and 18,779 SNPs, to estimate the rate and the dates of the input phylogeny given temporal constraints. The relationship in the phylogeny shows that the isolates from the postmodern era belonging to the multidrug-resistant clade alone have a most recent common ancestor relatively soon after the inferred emergence of the whole collection, within the late 17th – early eighteenth century

Fig. 6

Phylogeny and recombination landscape for viable Neisseria gonorrhoeae isolates (n = 191). The phylogeny (left), with associated level of recombination and era of isolation, is displayed alongside the linearized chromosome of WHO O reference strain (top). Line graph (bottom) shows the recombination profile of all isolates as well as separately for the antimicrobial susceptible and multidrug-resistant clade. The number of recombination nucleotide sites ranged from 213,780–539,280 sites, with a mean number of 388,811 sites and median of 393,987 sites