Ciprofloxacin resistance in Campylobacter jejuni isolates: detection of gyrA resistance mutations by mismatch amplification mutation assay PCR and DNA sequence analysis

Abstract

The gyrA quinolone resistance determining region was sequenced from 13 ciprofloxacin-resistant and 20 ciprofloxacin-susceptible Campylobacter jejuni isolates. All isolates resistant to ciprofloxacin had Thr-86-to-Ile mutations, a mutation frequently associated with the acquisition of resistance to fluoroquinolones. A mismatch amplification mutation assay (MAMA) PCR protocol was developed that detects this gyrA mutation in quinolone-resistant isolates. The MAMA PCR provides a means for routine detection of the gyrA mutation without the need for sequencing the gyrA gene.

FIG. 1

Comparison of C. jejuni gyrA QRDR DNA sequences. Sequence L04566 is a portion of the gyrA QRDR of C. jejuni UA580 (GenBank no. L04566). 1997 NARMS isolates CS5, CS34, and CS50 (bold type) have a Thr-86-to-Ile (ACA→ATA) gyrA mutation frequently associated with fluoroquinolone resistance. Isolate CS50 has an additional Pro-104-to-Ser (CCA→TCA) mutation. Silent mutations were noted at amino acid positions 78, 81, 82, 92, 110, 117, 119, and 120. ∗, C. jejuni subsp. jejuni ATCC 33560 type strain; ∗∗, C. jejuni subsp. doylei ATCC 49349 type strain.

FIG. 2

Agarose gel of C. jejuni MAMA PCR products. Lanes contain the following: A, 0.8 μg of a 100-bp DNA ladder (Bethesda Research Laboratories, Inc., Gaithersburg, Md.); B (control lane), no DNA plus CampyMAMAgyrA1 and GZgyrA4 conserved primers; C through E, DNAs from wild-type, ciprofloxacin-sensitive C. jejuni subsp. jejuni ATCC 33560, C. jejuni subsp. doylei ATCC 49349, and CS18, respectively, with conserved primer CampyMAMAgyrA1 and mutation primer CampyMAMAgyrA5; F (positive control lane), C. jejuni subsp. jejuni ATCC 33560 DNA and conserved primers GZgyrA4 and CampyMAMAgyrA1 (a 368-bp PCR product is generated); and G through K, DNAs from Thr-86-to-Ile (ACA→ATA) gyrA ciprofloxacin-resistant mutants CS5, CS8, CS34, CS143, and CS166, respectively, with conserved primer CampyMAMAgyrA1 and mutation primer CampyMAMAgyrA5 (a 265-bp PCR product is generated).