Shanghai Neisseria gonorrhoeae Isolates Exhibit Resistance to Extended-Spectrum Cephalosporins and Clonal Distribution

Abstract

The emergence of Neisseria gonorrhoeae strains with resistance (R) to extended-spectrum cephalosporins (ESCs^R) represents a public health threat of untreatable gonococcal infections. This study was designed to determine the prevalence and molecular mechanisms of ESC^R of Shanghai N. gonorrhoeae isolates. A total of 366 N. gonorrhoeae isolates were collected in 2017 in Shanghai. Susceptibility to ceftriaxone (CRO), cefixime (CFM), azithromycin (AZM), ciprofloxacin (CIP), spectinomycin, penicillin, and tetracycline was determined using the agar dilution method. A subset of 124 isolates was subjected to phylogenetic analysis for nine antimicrobial resistance-associated genes, i.e., penA, porB, ponA, mtrR, 23S rRNA, gyrA, parC, 16S rRNA, and rpsE. Approximately 20.0% of the isolates exhibited CFM^R [minimum inhibitory concentration (MIC) >0.125 mg/L], and 5.5% were CRO^R (MIC > 0.125 mg/L). In total, 72.7% of ESC^R isolates were clonal and associated with mosaic penA 10 and 60 alleles. Non-mosaic penA 18 allele and substitutions of PenA A501T, G542S, and PorB1b G213S/Y were observed in non-clonal ESC^R. Approximately 6.8% of the isolates showed AZM MIC above the epidemiological cutoff (ECOFF, 1 mg/L), were associated with 23S rRNA A2059G mutation, and did not exhibit clonal distribution. Almost all isolates were CIP^R (resistance to ciprofloxacin) and associated with GyrA-91/92 and ParC-85/86/87/88/89/91 alterations. Isolates with ParC S88P substitution were clustered into the ESC^R clade. The Shanghai isolates exhibited a high level of ESC^R and distinct resistant patterns.

Keywords: Neisseria gonorrhoeae; extended-spectrum cephalosporins; multidrug resistance; phylogenetic analysis; resistance determinants.

FIGURE 1

Phylogenetic reconstruction of nine genes, patterns of antimicrobial resistance, and genetic polymorphisms in 124 N. gonorrhoeae isolates. Left: Phylogenetic reconstruction of 124 isolates based on maximum likelihood. The red arrow indicates the reference strain FA1090. Heatmap. Columns 1 and 2: MIC values of CRO and CFM. Columns 3–5: Susceptible/resistant categories according to the EUCAST MIC breakpoints of CRO, CFM, and ESCs. Column 6: NG-STAR genotype (white band indicates NG-STAR genotypes new to the NG-STAR database). Columns 7–10: A specific NG-STAR genotype. Column 11: Any mosaic penA allele. Columns 12–15: A specific mosaic penA allele. Columns 16–21: Non-synonymous amino acid changes from wild type in PorB1b. Columns 22 and 23: Non-synonymous amino acid changes from wild type in PonA. Column 24: MIC values of AZM. Column 25: Susceptible/resistant categories according to the EUCAST MIC breakpoints of AZM. Column 26: A2059G mutation in 23S rRNA. Columns 27–31: Non-synonymous amino acid changes from wild type in MtrR. Column 32: The –35A deletion in the mtrR promoter. Column 33: MIC values of SPT. Column 34: Susceptible/resistant categories according to the EUCAST MIC breakpoints of SPT. Column 35: C1192U mutation in 16S rRNA. Column 36: MIC values of CIP. Column 37: Susceptible/resistant categories according to the EUCAST MIC breakpoint of CIP. Columns 38–40: Non-synonymous amino acid changes from wild type in GyrA. Columns 41–46: Non-synonymous amino acid changes from wild type in ParC. The purple and orange rectangles indicate clades A and B described in the text, respectively. Sequence names are colored by cluster.

FIGURE 2

penA gene phylogeny, patterns of antimicrobial resistance, and genetic polymorphisms of 124 N. gonorrhoeae isolates. Left: Phylogenetic reconstruction of 124 isolates based on the penA gene and using the maximum likelihood. The red arrow indicates reference strain FA1090. Heatmap. Columns 1 and 2: MIC values of CRO and CFM. Columns 3–5: Susceptible/resistant categories according to the EUCAST MIC breakpoints of CRO, CFM, and ESCs. Column 6: Any mosaic penA allele. Column 7: penA alleles. Columns 8–22: Non-synonymous amino acid changes from wild type in PenA. The purple rectangle indicates clade C as described in the text. The yellow rectangle indicates substitutions in the mosaic penA coding region. Sequence names are colored by cluster.

FIGURE 3

Heat map visualization of CIP MICs with ParC and GyrA substitutions in N. gonorrhoeae. MICs of CIP are indicated on the left panel. ParC and GyrA substitutions are indicated on the middle and right panels, respectively.