Meningococcal Quinolone Resistance Originated from Several Commensal <i>Neisseria</i> Species

Abstract

Quinolone resistance is increasing in <i>Neisseria meningitidis</i>, 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 <i>gyrA</i> alleles in <i>N. meningitidis</i> A total of 198 <i>N. meningitidis</i> isolates and 293 commensal <i>Neisseria</i> 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 <i>gyrA</i> and <i>parC</i> were sequenced for all isolates, while a few isolates were sequenced on the Illumina platform. The prevalences of quinolone resistance in the <i>N. meningitidis</i> and commensal <i>Neisseria</i> isolates were 67.7% (134/198) and 99.3% (291/293), respectively. All 134 quinolone-resistant <i>N. meningitidis</i> isolates possessed mutations in T91 (<i>n</i> = 123) and/or D95 (<i>n</i> = 12) of GyrA, with 7 isolates also harboring ParC mutations and exhibiting higher MICs. Phylogenetic analysis of the <i>gyrA</i> sequence identified six clusters. Among the 71 mutation-harboring <i>gyrA</i> alleles found in 221 <i>N. meningitidis</i> isolates and genomes (<i>n</i> = 221), 12 alleles (<i>n</i> = 103, 46.6%) were included in the <i>N. meningitidis</i> cluster, while 20 alleles (<i>n</i> = 56) were included in the <i>N. lactamica</i> cluster, 27 alleles (<i>n</i> = 49) were included in the <i>N. cinerea</i> cluster, and 9 alleles (<i>n</i> = 10) were included in the <i>N. subflava</i> cluster. Genomic analyses identified the exact <i>N. lactamica</i> donors of seven mutation-harboring <i>gyrA</i> alleles (<i>gyrA92</i>, <i>gyrA97</i>, <i>gyrA98</i>, <i>gyrA114</i>, <i>gyrA116</i>, <i>gyrA151</i>, and <i>gyrA230</i>) and the <i>N. subflava</i> donor isolate of <i>gyrA171</i>, with the sizes of the recombinant fragments ranging from 634 to 7,499 bp. Transformation of <i>gyrA</i> 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 <i>N. meningitidis</i> isolates acquired resistance by horizontal gene transfer from three commensal <i>Neisseria</i> species. Quinolone resistance in <i>N. meningitidis</i> increases in a stepwise manner.