Meningococcal Quinolone Resistance Originated from Several Commensal Neisseria Species

Abstract

Quinolone resistance is increasing in Neisseria meningitidis, with its prevalence in China being high (>70%), but its origin remains unknown. The aim of this study was to investigate the donors of mutation-harboring gyrA alleles in N. meningitidis A total of 198 N. meningitidis isolates and 293 commensal Neisseria isolates were collected between 2005 and 2018 in Shanghai, China. The MICs of ciprofloxacin were determined using the agar dilution method. The resistance-associated genes gyrA and parC were sequenced for all isolates, while a few isolates were sequenced on the Illumina platform. The prevalences of quinolone resistance in the N. meningitidis and commensal Neisseria isolates were 67.7% (134/198) and 99.3% (291/293), respectively. All 134 quinolone-resistant N. meningitidis isolates possessed mutations in T91 (n = 123) and/or D95 (n = 12) of GyrA, with 7 isolates also harboring ParC mutations and exhibiting higher MICs. Phylogenetic analysis of the gyrA sequence identified six clusters. Among the 71 mutation-harboring gyrA alleles found in 221 N. meningitidis isolates and genomes (n = 221), 12 alleles (n = 103, 46.6%) were included in the N. meningitidis cluster, while 20 alleles (n = 56) were included in the N. lactamica cluster, 27 alleles (n = 49) were included in the N. cinerea cluster, and 9 alleles (n = 10) were included in the N. subflava cluster. Genomic analyses identified the exact N. lactamica donors of seven mutation-harboring gyrA alleles (gyrA92, gyrA97, gyrA98, gyrA114, gyrA116, gyrA151, and gyrA230) and the N. subflava donor isolate of gyrA171, with the sizes of the recombinant fragments ranging from 634 to 7,499 bp. Transformation of gyrA fragments from these donor strains into a meningococcal isolate increased its ciprofloxacin MIC from 0.004 μg/ml to 0.125 or 0.19 μg/ml and to 0.5 μg/ml with further transformation of an additional ParC mutation. Over half of the quinolone-resistant N. meningitidis isolates acquired resistance by horizontal gene transfer from three commensal Neisseria species. Quinolone resistance in N. meningitidis increases in a stepwise manner.

Keywords: Neisseria meningitidis; commensal Neisseria; gyrA; horizontal gene transfer; parC; quinolone resistance.

FIG 1

Phylogenetic analysis of the gyrA quinolone resistance-determining region of the Neisseria isolates and genomes. Phylogenetic analysis of the nucleotide sequences of 263 gyrA alleles (nucleotides 115 to 639) from N. meningitidis (n = 12,771), N. gonorrhoeae (n = 4,118), N. lactamica (n = 562), N. subflava (n = 30), N. cinerea (n = 21), N. mucosa (n = 22), and other commensal Neisseria (n = 63) isolates and genomes collected in this study and from the Neisseria PubMLST database was conducted with the MEGA (version 5) program using the unweighted pair group method with arithmetic mean averages (UPGMA). Clusters were identified if bootstrap values were >70% in the bootstrap test with 1,000 replicates.

FIG 2

Recombination breakage points within gyrA92-harboring N. meningitidis isolate CR24. (A) Sequence alignment of the upstream sequences of the gyrA gene showing the recombination breakage point at position 5040 upstream of the gyrA gene of serogroup C strain 053442. (B) Sequence alignment of gyrA genes showing the recombination breakage point within the gene at position 2459 relative to the sequence of strain 053442. (C) Recombination breakage points detected by at least one of seven methods included in RDP (version 4.97). The topmost horizontal bar represents the CR24 sequence; the blue and red arrows correspond to the locations of the breakage points identified in panels A and B.