Phylogenomic analysis reveals persistence of gonococcal strains with reduced-susceptibility to extended-spectrum cephalosporins and mosaic penA-34

Abstract

The recent emergence of strains of Neisseria gonorrhoeae associated with treatment failures to ceftriaxone, the foundation of current treatment options, has raised concerns over a future of untreatable gonorrhea. Current global data on gonococcal strains suggest that several lineages, predominately characterized by mosaic penA alleles, are associated with elevated minimum inhibitory concentrations (MICs) to extended spectrum cephalosporins (ESCs). Here we report on whole genome sequences of 813 N. gonorrhoeae isolates collected through the Gonococcal Isolate Surveillance Project in the United States. Phylogenomic analysis revealed that one persisting lineage (Clade A, multi-locus sequence type [MLST] ST1901) with mosaic penA-34 alleles, contained the majority of isolates with elevated MICs to ESCs. We provide evidence that an ancestor to the globally circulating MLST ST1901 clones potentially emerged around the early to mid-20th century (1944, credibility intervals [CI]: 1935-1953), predating the introduction of cephalosporins, but coinciding with the use of penicillin. Such results indicate that drugs with novel mechanisms of action are needed as these strains continue to persist and disseminate globally.

Fig. 1. Maximum likelihood tree based on core genome SNPs of 813 Neisseria gonorrhoeae strains collected from 2005 to 2017 in the United States, including clade/fastbaps cluster, year, and sex of sex partner.

MSW men that have sex with women, MSM men that have sex with men, MSMW men that have sex with men and women, HHS Health and Human Services region, MIC minimum inhibitory concentration with respect to azithromycin (AZM), ciprofloxacin (CIP), penicillin (PEN), tetracycline (TET), cefixime (CFM), and ceftriaxone (CRO). Highlighted clades contain isolates where we identified the majority of elevated cephalosporin MICs. The tree also includes several reference genomes: FA19 (root, ǂ), international MLST ST1903 strains possessing the mosaic penA-60 allele (*) including strains from Japan (FC428, FC460) and Canada (47707); WHO reference strains WHO-X (H041, Japan) and WHO-Z (A8806, Australia) marked by (ɤ); and WHO-Y (F89, France) marked by (⁂). A summary of antimicrobial susceptibility to each respective antibiotic is shown with colors: susceptible (light orange), elevated MIC range (teal), or high-level MICs (dark green). Alleles for penA are colored based on their amino acid sequence homology to penA-34 (≥98% is a derivative allele; teal), differing mosaic penA allele (dark green), or absence of a mosaic penA allele.

Fig. 2. Time-scaled (strict model) BEAST phylogeny of 805 isolates, including 340 GISP isolates identified in Clade A-B of this study and 465 international MLST ST1901 strains.

Clade AB1 and Clade AB2 represent the divergent sub-lineages that we identified in the phylogeny. The oldest sequenced isolate is from the Philippines collected in 1992. The phylogeny includes epidemiological details and antimicrobial susceptibility. Sex of sex partners are colored [MSW men that have sex with women (red), MSM men that have sex with men (light orange), MSMW men that have sex with men and women (light blue)]. Antimicrobial susceptibility is based on minimum inhibitory concentration (MIC) to each respective antibiotic and is shown with colors: susceptible (light orange), elevated MIC range (teal), or high-level MICs (dark green). Alleles for penA are colored based on their amino acid sequence homology to penA-34 (≥98% is a derivative allele; teal), differing mosaic penA allele (dark green), or absence of a mosaic penA allele. Amino acid mutations in PBP2 associated with elevated cephalosporin MICs are shown at the top of figure. The bottom scale bar represents the time to the most recent common ancestor (tMRCA) of the internal node of the phylogenetic tree.  Numbers highlighted in red are the ancestral nodes of the time-scaled phylogenetic tree and represent estimated years and credibility intervals based on tMRCA: 1) Root; 1818 [1781 - 1865], 2) 1925 [1913 - 1951], 3) 1944 [1935 - 1953], 4) 1952 [1942 - 1964], 5) 1970 [1964 - 1977], 6) 1972 [1966 -1979], 7) 1985 [1980 - 1990], 8) 1987 [1983 - 1992],  9) 1990 [1986 - 1994].