Genomic Epidemiology and Molecular Resistance Mechanisms of Azithromycin-Resistant Neisseria gonorrhoeae in Canada from 1997 to 2014

Abstract

The emergence of Neisseria gonorrhoeae strains with decreased susceptibility to cephalosporins and azithromycin (AZM) resistance (AZM(r)) represents a public health threat of untreatable gonorrhea infections. Genomic epidemiology through whole-genome sequencing was used to describe the emergence, dissemination, and spread of AZM(r) strains. The genomes of 213 AZM(r) and 23 AZM-susceptible N. gonorrhoeae isolates collected in Canada from 1989 to 2014 were sequenced. Core single nucleotide polymorphism (SNP) phylogenomic analysis resolved 246 isolates into 13 lineages. High-level AZM(r) (MICs ≥ 256 μg/ml) was found in 5 phylogenetically diverse isolates, all of which possessed the A2059G mutation (Escherichia coli numbering) in all four 23S rRNA alleles. One isolate with high-level AZM(r) collected in 2009 concurrently had decreased susceptibility to ceftriaxone (MIC = 0.125 μg/ml). An increase in the number of 23S rRNA alleles with the C2611T mutations (E. coli numbering) conferred low to moderate levels of AZM(r) (MICs = 2 to 4 and 8 to 32 μg/ml, respectively). Low-level AZM(r) was also associated with mtrR promoter mutations, including the -35A deletion and the presence of Neisseria meningitidis-like sequences. Geographic and temporal phylogenetic clustering indicates that emergent AZM(r) strains arise independently and can then rapidly expand clonally in a region through local sexual networks.

FIG 1

Whole-genome core SNP maximum likelihood phylogenetic tree of 236 Neisseria gonorrhoeae strains collected from 1989 to 2014 in Canada, including 10 international reference strains. The length of the scale bar in the maximum likelihood tree represents the estimated evolutionary divergence between isolates on the basis of the average pairwise distance between strains (estimated number of substitutions in the sample/total number of high-quality SNPs). NG-MAST and MLST relatedness groups determined by goeBURST analysis and the year and region of collection are indicated. Susceptibility to AZM, CRO, and CFM is shown. Green segments in the molecular marker columns indicate the absence of the particular marker (wild type). The colors of the segments in columns 23S 2059 and 23S 2611 represent the number of alleles with the A2059G or C2611T mutation (E. coli numbering), respectively, as indicated in the key. The mtrR p column depicts disruptions to the mtrR promoter region, including the −35A deletion (red), N. meningitidis-like sequences (light red), and WHO-P-like disruptions (orange).

FIG 2

Genetic relatedness of NG-MAST STs determined by goeBURST minimum spanning tree analysis of Neisseria gonorrhoeae concatenated and aligned NG-MAST porB and tbpB sequences. The number labels on the branches indicate the number of SNP differences between sequence types; branch lengths are not to scale. Node sizes are proportional to the number of isolates in the node. Colored nodes indicate relatedness groups defined by ≤5 SNPs. An asterisk on a node indicates a sequence type of Canadian isolates (ST1948 [n = 2], ST4980, ST5343, ST8690) with HL-AZM^r (MIC ≥ 256 μg/ml). Black nodes correspond to HL-AZM^r sequence types reported in China (ST1866); Scotland (ST470, ST649); England, Wales, and the United States (ST649); Ireland (ST649, ST3311); Italy (ST835, ST1392, ST2142, ST2386); Sweden (ST285, ST8727); Australia (ST649, ST5309, ST8917, ST10133, ST10572); and Argentina (ST696). ST285 also represents one low-level AZM^r Canadian strain grouping into NG-MAST relatedness group 1. Further details on the associated phylogenetic clades are presented in Fig. 1.

FIG 3

Genetic relatedness of MLSTs determined by goeBURST minimum spanning tree analysis using the Neisseria gonorrhoeae MLST allelic profiles of seven MLST housekeeping genes. Number labels on the branches indicate the number of allelic variations between sequence types; branch lengths are not to scale. Colored nodes indicate relatedness groups defined by a single-locus variation from a founding sequence type. An asterisk on a node indicates a sequence type of Canadian isolates (ST1579, ST1901, ST7823, ST10899, ST11426) with HL-AZM^r (MIC ≥ 256 μg/ml). Further details on the associated phylogenetic clades are presented in Fig. 1.

FIG 4

Alignment of mtrR-mtrC intergenic DNA sequences of Neisseria gonorrhoeae strains and N. meningitidis M01-240355. Sequence position 1 corresponds to a location −86 bp upstream from the start codon of mtrC. The start codon of mtrR is indicated. The sequences of FA1090 and NCCP11945 were derived from N. gonorrhoeae complete genomes (GenBank accession no. NC_002946 and NC_011035, respectively), and the M01-240355 sequence was derived from the complete genome of N. meningitidis M01-240355 (GenBank accession no. CP002422). Other sequences are those of the following isolates (whose phylogenomic clades are indicated in parentheses in the figure and whose GenBank accession numbers appear in parentheses after the isolate designation): 38202 (KT954111), 38155 (KT954112), WHO-N (KT954113), WHO-L (KT954114), 30978 (KT954115), 29360 (KT954116), WHO-K (KT954117), 38132 (KT954118), WHO-F (KT954119), ATCC 49226 (KT954120), 34106 (KT954121), 36952 (KT954122), and WHO-P (KT954123). The locations of mutations associated with macrolide resistance, the −35A deletion in the mtrR promoter region and MtrR A39T and G45D amino acid substitutions, are indicated. The promoter regions of the sequences of isolates 37624, 38197, and 37088 (GenBank accession no. KT954124, KT954125, and KT954126, respectively) most closely align to the promoter region of the sequence of N. meningitidis M01-240355 with sequence identities of 94%, 98%, and 98%, respectively, using the NCBI BLASTN Suite program. The sequence of isolate 37624 is identical to that of an N. meningitidis-like sequence previously reported by Trembizki et al. (49).