DNA gyrase-mediated natural resistance to fluoroquinolones in Ehrlichia spp

Abstract

Fluoroquinolone susceptibility heterogeneity between various Ehrlichia species has been previously demonstrated. In gram-negative bacteria, resistance to fluoroquinolones most often corresponds to specific amino acid variations in a portion of the protein sequence of the A subunit of DNA gyrase (GyrA), referred to as the quinolone resistance-determining region (QRDR). We suspected a similar mechanism to be responsible for natural resistance in some Ehrlichia species. To verify this hypothesis, we sequenced the entire gyrA gene of the quinolone-susceptible species Ehrlichia sennetsu and designed specific primers to amplify and sequence the QRDR of four other Ehrlichia species as well as the closely related species Cowdria ruminantium. We identified in the fluoroquinolone-resistant species Ehrlichia chaffeensis and Ehrlichia canis a specific GyrA QRDR amino acid sequence, also present in C. ruminantium (whose susceptibility to fluoroquinolones remains unknown). These three species belong to a single phylogenetic cluster referred to as the E. canis genogroup. A different GyrA QRDR pattern, shared by the Ehrlichia species representatives of the E. sennetsu and Ehrlichia phagocytophila genogroups, was identified. Three of the four species tested are known to be susceptible to fluoroquinolones. A serine residue in position 83 (Escherichia coli numbering) in the susceptible species is replaced by an alanine residue in fluoroquinolone-resistant species. These results are consistent with the current knowledge on fluoroquinolone resistance in other gram-negative bacteria. They are indicative of a natural gyrase-mediated resistance to fluoroquinolones in the E. canis genogroup.

FIG. 1

DNA and amino acid sequences of E. sennetsu gyrA. The putative −10 and −35 promoter regions, the putative ribosome binding sites, the ATG start codon, the HPHGD conserved motif, and the active-site tyrosine residue (Y118) are underlined.

FIG. 2

DNA sequence alignment of the QRDR of the six Ehrlichia species studied.

FIG. 3

Alignment of the putative amino acid sequences of the QRDR of the six Ehrlichia species studied, as well as E. coli GyrA and ParC and R. prowazekii GyrA.

FIG. 4

Stereoview of a ribbon diagram of the E. coli DNA gyrase A subunit (protein database accession code 1AB4). The blue arrows represent beta strands, the red ribbons represent alpha helices, and turns and loops are yellow. Residues involved in mutations associated with resistance phenotype (S83, A84, and D87) and the tyrosine residue (Y122) are marked on the picture. The figure was prepared using TURBO-FRODO (25).

FIG. 5

Phylogenetic tree based on 16S rRNA gene sequences, and correlation between quinolone susceptibility and gyrA sequences in studied Ehrlichia species. S, susceptible (MIC ≤ 2 μg/ml); R, resistant (MIC > 2 μg/ml).